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Anatomical, biomechanical and behavioural characteristics of the human masseter muscle Tonndorf, Monica de Lorenzo 1993

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ANATOMICAL, BIOMECHANICAL AND BEHAVIOURAL CHARACTERISTICSOF THE HUMAN MASSETER MUSCLEbyMoNICA DE LORENZO TONNDORFD.D.S., Federal University of Rio de Janeiro, 1986A THESIS SUBMHTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Department of Oral Biology)We accept this thesis as conformingTHE UNIVERSITY OF BRITISH COLUMBIAOctober 1993©Mônica L. Tonndorf, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)______________________Department of Oral BiologyThe University of British ColumbiaVancouver, CanadaDate November 8, 1993DE-6 (2/88)ABSThACThere is little information on the anatomical and functional organization ofthe human jaw muscles, despite their importance to the masticatory system and disorderswhich affect it. The current studies examined the anatomical, biomechanical andfunctional characteristics in the multipennate human masseter muscle as a model forunderstanding function in the human jaw.Masseter anatomy was investigated in fetuses, adult cadavers, and livingsubjects by histology, gross anatomical dissection and Magnetic-Resonance (MR)imaging. Nerve pathways and muscle-fibre arrangements suggested that both fetal andadult muscles could be divided into at least four neuromuscular compartments. Althoughsome fetal specimens had developed peimation, internal aponeuroses were seldompresent. Structural variations were also found in the cadaveric material and livingsubjects. As the number of aponeuroses increased with development, and their thicknessvaried between individuals, individualized contraction strategies are likely to occurduring function.Movements of masseter insertion sites during jaw function were modelled indry skulls. Muscle origins and insertions were measured three-dimensionally at differentgapes and simulated masticatory positions. Their movements varied with craniofacialdimensions and their locations on the mandible. For some parts of the muscle, thebalancing-side and the incisal-contact positions provided the most advantageous lines ofmuscle fibre action, while for others, the working-side task was most favourable.Separate portions of the muscle are thus uniquely placed to perform specific tasks.11Movements of four insertion sites were also recorded in four living subjects. Insertionareas were identified on MR-derived reconstructions, and movements recorded with ajaw-tracking device. Ranges of insertion displacement were similar to these estimatedfor dry skulls, and varied between individuals.Interference electromyography, and single-motor-unit (MU) techniques wereused to study physiological responses in eight subjects. The behaviour of low-thresholdMUs was investigated relative to changes in bite side and experimental paradigm whilesubjects were biting on a force transducer. For each unit, the recruitment threshold,sustained-bite forces, the rate and regularity of sustained firing, and the coefficient ofvariation, were measured. Assessments were also made of the accuracy with which thetarget rates were attained, and of the contribution of each MU’s firing rate to bite force.These measures of behaviour frequently differed between tasks, but not reproducibly.The highest reproducibility and firing-rate accuracy were achieved when visual andauditory MU feedback was provided. A tendency for increased firing rate, and decreaseddischarge variability was found when subjects had no feedback. As approximately 50%of the units did not show reproducible behavioural characteristics, it seems thatdifferences in intramuscular activation, differential activation of other muscles, and theinherent variability seen in low-threshold MU studies generally make quantitativecomparisons of focal activity in the masseter implausible.Finally, a method was developed for locating the positions of moving needleelectrodes relative to internal aponeuroses. It combined scanning electromyography,optical tracking of electrodes, MR imaging, and three-dimensional reconstruction. The111territorial sizes of 162 MUs were then assessed in the muscles of four subjects. Theirmean width was 3.7 ± 2.3 mm. Most MU territories were confined between tendons,although 10% of the units clearly extended across at least one tendon. This focaldispersion of most territories provides a firm anatomical basis for selective activation ofthe muscle.The findings collectively indicate an anatomical, biomechanical andphysiological basis for differential motor control of at least four neuromuscularcompartments in the human masseter muscle. The extent to which the central nervoussystem selectively activates these compartments, or coactivates them, remains to bedemonstrated under functional conditions.ivTABLE OF CONTENTSPAGEABSTRACT iiTABLE OF CONTENTS vLISTOFFIGURES xiLIST OF TABLES xviACKNOWLEDGEMENTS xvili1. INTRODUCTION 11.0 Introduction to the Thesis 1Review of the Literature1.1 Skeletal Muscle Development 21.2 Muscle Architecture and Mechanics 101.3 Motor-Unit Organization 181.3.1 Motor-Unit Arrangement 181.3.2 Fibre Characteristics 191.4 Motor-Unit Activity 201.4.1 Recruitment 211.4.2 Firing Rate 211.4.3 Behaviour 221.4.4 Summary 251.5 Partitioning 25V1.6 Jaw Muscle Organization.1.6.1 Structure1.6.2 Fibre Composition1.6.3 Jaw Biomechanics1.6.4 Summary1.7 Anatomical Design of the Masseter.1.7.1 Muscle Organization1.7.2 Innervation Pattern1.7.3 Muscle Fibre Characteristics1.7.4 Spindle Distribution1.7.5 Summary1.8 Functional Design of the Masseter1.8.1 Motor-unit Territory1.8.2 Functional Differentiation1.8.3 Motor-unit Activity1.8.3 Conclusion2. STATEMENT OF THE PROBLEM3. STUDIES3.1 Masseter Morphology282933353638394345464747474952545660613.1.1 Exploratory Experiments on Fetal Masseter Anatomy 613.1.1.1 Materials and Methods3. Nerve Staining6265vi3. Connective-Tissue and Muscle-Fibre Staining. 683. Muscle-Fibre Staining and OrientationAssessment 683. Muscle Reconstruction 683.1.1.2 Results 743. Nerve Distribution 743. Connective-Tissue Development 763. Muscle-Fibre Orientation 763.1.1.3 Discussion 833.1.2 Adult Masseter Anatomy 873.1.2.1 Materials and Methods 883. Gross Anatomical Dissection 883. Chemical Dissection 893.1.2.2 Results 903.1.2.3 Discussion 983.1.3 Morphological Reconstruction in Living Subjects 1013.1.3.1 Methods 1013. Magnetic-Resonance Imaging 1013. Imaging 1033. Reconstruction 1073. Error of Method 1113.1.3.2 Results 111vii3.1.3.3 Discussion.1173.2 Movement of Masseter Insertions at different Jaw Positions 1253.2.1 Simulated Function in Dry Skulls 1263.2.1.1 Methods 1263.2.1.2 Results 1333. Cephalometric Analysis of the Skull Sample.. 1333. Sample Variation in Insertion Site Locationduring Dental Intercuspation 1363. Effect of Jaw Position on Insertion SiteLocation 1363. Differences within Putative Muscle Layers ... 1413. Differences between Putative Muscle Layers . 1433. Orientation of Masseter Insertion relative toOrigin 1453. Discussion 1473.2.2 Function in Living Subjects 1573.2.2.1 Methods 1583.2.2.2 Results 1633.2.2.3 Discussion 1693.3 Evaluation of the Masseter’s Functional Performance 1753.3.1 Electromyographic Recording Techniques 1753.3.2 Preliminary Experiments using Single-Wire EMO RecordingviiiTechnique 1763.3.2.1 Materials and Methods 1773.3.2.2 Results 1833.3.2.3 Discussion 1873.3.3 Motor-Unit Behaviour 1923.3.3.1 General Methods 1923. Motor-Unit Recording 1923. Bite-Force Measurement 1963. Sampling and Data Analysis 2013.3.3.2 Effect of Bite Side on MU Behaviour 2023. Methods 2043. Results 2083. Discussion 2173.3.3.3 Effect of Experimental Paradigm 2223. Methods 2223. Results 2243. Discussion 2273.4 Motor-Unit Territory Relative to the Masseter’s Internal Architecture . 2303.4.1 Methods 2323.4.1.1 Stereotactic Location of EMG Needle Electrode Scans 2323. Morphologic Reconstruction 2323. Motor-Unit Recording 233ix• . 234• . 236• . 239• . 243• . 245• . 250• . 260• . 265• . 269• . 2993. Needle Location3. Scan Location3. Methodological Errors3.4.1.2 Motor-Unit Territory3.4.2 Results3.4.3 Discussion4. GENERAL DISCUSSION AN]) CONCLUSIONS5. FUTURE DIRECTIONS6. BIBLIOGRAPHY7. APPENDIXxLIST OF FIGURESPAGEFigure 1 Fetal peripheral-nerve staining 66Figure 2 Fetal connective-tissue staining 69Figure 3 Cross-sectional optical scan (Z-scan) through a 10-jim fetal-musclesection 72Figure 4 Superimposition of two horizontal optical sections situated at bothextremes of a 1O-m-thick fetal muscle section 73Figure 5 Masseteric nerve pathway in the human fetal masseter 75Figure 6 Development of aponeurotic layers in the fetal masseter and medialpterygoid muscles 77Figure 7 Masseter muscle fibres in the 18 weeks old human fetus 78Figure 8 Characteristics of fetal muscle tissues 79Figure 9 Parasagittal sections from two 18-week human fetuses showing fibreorientation complexity 80Figure 10 Masseter-muscie dissection showing the superficial and intermediatemuscle layers 92Figure 11 Masseter-muscie dissection showing its layered arrangementposteriorlyand the ascending attachment sites of muscle fibres as the layersbecome deeper 93Figure 12 Masseter-muscie cross-section showing its internal aponeuroses . . . 95xiFigure 13 Masseter-muscie fibre endings.96Figure 14 Reference L-shaped grid attached to a plastic eyeglass frame 105Figure 15 Coronal spin-echo Magnetic Resonance image 106Figure 16 Antero-lateral view of the sectional outlines of a reference grid andright masseter muscle with its internal tendons 109Figure 17 Three-dimensional reconstruction of the reference grid, massetermuscle and internal tendons 110Figure 18 Linear and angular measures used to compare the reconstructedagainst the true reference grid 112Figure 19 MR coronal sections of two subjects showing differences in internalmuscle architecture 115Figure 20 Computer reconstruction of interstitial tissues in the right massetermuscle, viewed frontally 116Figure 21 Schematic illustration of MR data acquisition 122Figure 22 Masseter attachment points represented by six origin and twelveinsertion points 128Figure 23 Orientation of the three planes of the skuil’s coordinate system . . . 130Figure 24 Illustration of the seven representative jaw positions 132Figure 25 Mean displacements of the twelve insertion points at six different jawpositions 142Figure 26 Mean functional displacements of four putative muscle attachmentsites in the human masseter 146xliFigure 27 Orientation of origin and insertion sites 148Figure 28 Orientation of origin and insertion sites at three jaw positions .... 150Figure 29 Anterior view of the sectional outlines of a reference grid, rightmandibular ramus and condyle, and selected right-masseter-tendinousinsertions 159Figure 30 Schematic illustration of the global setup for a jaw tracking run ... 161Figure 31 Diagram illustrating the displacement trajectories of insertions ofaponeuroses II and IV in living subjects 168Figure 32 Rectified, digitized data from three muscle sites, obtained during avertically oriented maximum tooth clench 180Figure 33 Illustration of the right masseter muscle in the lateral and frontalviews, showing wire electrode location 182Figure 34 Rectified, digitized data from three electrode sites 184Figure 35 Smoothed, normalized data from three electrode sites plotted againsteach other for three representative tasks 185Figure 36 Raw data from four muscle sites, obtained during different clenchingtasks 186Figure 37 Raw data from four muscle sites, obtained during two distinct grindingmovements 188Figure 38 Motor-unit recording technique in the masseter muscle 193Figure 39 Effects of different bandpass-fflters on a single MU waveform shape 195Figure 40 Sagittal and frontal profiles of the skull showing location andxliiorientation of bite force measurements 197Figure 41 Calibration of the force transducer 199Figure 42 Illustration of the Bite Force Transducer in situ 200Figure 43 Recruitment and firing pattern of a masseter MU at different biteforce levels 205Figure 44 Distribution of recruitment thresholds by task 209Figure 45 Recruitment thresholds arranged according to task for 10 MUs fromfive subjects 210Figure 46 Changes in bite force when attempted MU firing at 10 Hz is increasedtol5Hz 212Figure 47 Distribution of Coefficients of Variation arranged by task forattempted MU firing rates of 10 Hz and 15 Hz 213Figure 48 Two-dimensional plot of the ISI standard deviations of the first againstthe second right-sided biting task, and against the left side 214Figure 49 Distribution of “Accuracy Indices” arranged by task for attempts at twodifferent rates of MU firing 216Figure 50 Schematic illustration of the global setup for a needle tracking run 235Figure 51 Schematic representation of the merge of the three datasets 237Figure 52 Right antero-lateral view of the final reconstruction 238Figure 53 Typical EMG records obtained during two separate needle scans . 240Figure 54 Coronal view of the 3D reconstruction of part of the right massetermuscle 241xivFigure 55 Needle movement velocity during two separate needle scans 247Figure 56 Distribution histogram of the territorial widths of 162 single MUs inthe right masseter muscle 248Figure 57 Relative sizes and frequencies of confined and extended MUterritories 249Figure 58 Distribution of MU territories with widths greater than 3 mm,according to muscle region 251xvLIST OF TABLESPAGETable I Orientation of muscle-fibre subgroups relative to the mandibularramus on the horizontal plane (Angle I) and relative to the plane ofcut (Angle II) 82Table II Linear and angular dimensions measured from homologous points onthe reference grids of six subjects and their reconstructedcounterparts 113Table III Facial dimensions of female and male skulls 134Table IV Craniometric measurements in fourteen skulls 135Table V Mean location of putative muscle insertion sites for 14 skulls 137Table VI Effect of jaw position on displacement of insertions expressed by meanorthogonal distances 138Table VII Effect of jaw position on displacement of insertions expressed by meanlinear distances 144Table VIII Linear regression analyses for points representing attachment sites 149Table IX Anteroposterior dimensions of the mandibular insertions ofAponeuroses II and IV in four subjects 164Table X Displacements of one insertion point of one subject at different jawpositions 165Table XI Mean displacements and standard deviations of four insertion pointsxviof four subjects at different jaw positions 166Table XII Mean displacements and standard deviations of four insertions of foursubjects during right-sided chewing 170Table XIII Mean displacements and standard deviations of four subjects duringleft-sided chewing 171Table XIV Discharge patterns and sustained-bite force levels of MUs activatedduring four tasks 226Table XV Muscle width and mediolateral territorial dimensions from MUs in theright masseter muscle of four subjects 252Table XVI Displacements of four insertion points of subject I at different jawpositions 300Table XVII Displacements of four insertion points of subject II at different jawpositions 301Table XVIII Displacements of four insertion points of subject III at different jawpositions 302Table XIX Displacements of four insertion points of subject IV at different jawpositions 303xiACKNOWLEDGEMENTSI wish to express my gratitude to my supervisor Dr Alan G Hannam, whosescientific guidance and enthusiasm made this project possible.I would like to thank Drs JM Gosline, JA Pearson and VM Diewert for theirinvaluable comments, suggestions and encouragement throughout the development ofthis thesis; Mr JP Sweeney, Ms B Tait, L Weston, C Vadgama and 3 Scott for theirtechnical advice and assistance, Ms C Louie for the grammatical corrections of themanuscript, Mr B McCaughey for the photography, and Mr P Ma for his statisticaladvice. I am indebted to Dr K Sasaki for his guidance in the early stages of this work,Dr C Price who enabled my access to the Magnetic Resonance Unit, and Dr T Koriothwho introduced and guided me through the I-DEAS package. Special thanks for all myfriends and experimental subjects for their continuous support and enthusiasm.I am especially grateful to Drs JD Waterfield and DM Brunette for theircontinuous support and advice, which enabled the fulfilment of this thesis.This thesis is dedicated to my husband, Emilio, and parents, Maria Lucia,Manfred and Nib. Their unfailing support, love and strength gave me the endurance toovercome these difficult years and to accomplish this project.xviii1. INTRODUCI1ON1.0 Introduction to the ThesisThe human masseter is a powerful muscle, consisting of several muscle layersand interleaving internal aponeuroses arranged in a multipennate pattern, whichbecomes more complex with the development of function. Together with othermasticatory muscles, it displaces the mandible, creates interocclusal and articular forces,and induces stress in the mandible and cranium. Contraction of the masticatory musclesaffects craniofacial development, is commonly associated with craniomandibulardisorders, and can interfere with clinical, orthodontic corrections made to thecraniofacial skeleton.Although this multipennate arrangement of the masseter provides ananatomical substrate for complex internal muscle mechanics, little is known on how itcontracts. In most jaw models, its function is represented as a simple force vector, whenin fact muscle tension acts over a broad attachment area with diverse regionalmagnitudes of force.Contrary to the early assumption that the masseter is composed of ahomogeneous, whole-muscle motoneuron pool, recent studies have revealed that MUsare activated in separate task-groups. In some occasions, task-groups can be inactive,depending on the performed jaw effort, however most often they are active throughoutvarious tasks, showing only changes in their patterns of behaviour. Whether theseseparate motoneuron pools are related to internal muscle architecture, however, remains1uncertain.It is necessary to develop new experimental techniques for use in living humansubjects which can record both the internal muscle architecture and the three-dimensional location of the jaw during various tasks, and which can also stereotacticallylocate muscle-activity recording electrodes. Only when such techniques are developedwill the structural, biomechanical and functional interactions within the muscle be betterunderstood.Review of the Literature1.1 Skeletal Muscle DevelopmentThe craniofacial muscles are the first to develop in the body, in keeping withthe cephalocaudal sequence of fetal development (Sperber, 1989; Noden, 1991). Thesemuscles develop from paraxial mesoderm that condenses rostrally as incompletelysegmented somitomeres and fully-segmented somites. The myomeres of the somitomeresand of the somites form primitive mononucleated myogenic cells, termed myoblasts;these undergo repeated mitotic division and subsequently fuse with each other to formmultinucleated myotubes. Once this has occurred further nuclear division ceases, andthus myocytes (muscle fibres) are formed (Sperber, 1989; Mastaglia, 1981). Fewmyotubes remain after the 20th week, at which time most fibres are packed withmyofibrils and have peripheral nuclei (Fenichel, 1966). Myotubes showing degenerativechanges have been found in muscles of 10-16 week old human fetuses, and it has been2suggested that cell death is part of the normal process of myogenesis during intrauterinelife (Webb, 1977).While the formation of primary myotubes is well known, there is stilluncertainty about the source of subsequent generations of myotubes and the mechanismwhereby the number of muscle fibres increases during late fetal development (Mastaglia,1981). Initially, it was suggested that fibre numbers increase by a process of budding orby longitudinal splitting of the first-formed fibres (Cuajunco, 1942). Later, a more likelyalternative view was provided: that secondary and subsequent generations of myotubesdevelop from a persisting population of undifferentiated mononucleated cells lying inclose proximity to the primary myotubes (Kelly and Zacks, 1969; Ontell, 1977). Aproportion of these undifferentiated “fibroblast-like” cells are thought to remain in thiscondition as sateffite cells which are seen in muscle fibres in late fetal (Ishikawa, 1966)and early adult life (Mauro, 1961).Whereas the myogenic lineages of the masticatory muscles are traceable toparaxial mesoderm, the connective tissues associated with these muscles originate fromthe neural crest (Noden, 1991). During formation, the myoblast populations destined toform branchial-arch skeletal muscles move from the paraxial mesoderm into the neuralcrest, which provides their surrounding connective tissue: epimysium, perimysium andendomysium (Sperber, 1989). Initially, most of the myoblast populations are compact andmorphologically homogeneous, but as they near their terminal locations the myogeniccells become interspersed with the nonmyogenic cells (Noden, 1991).During the embryonic period, a definitive architecture for each muscle3emerges as tendons and connective tissue septa become distinct and muscle fibresbecome arranged in specific configurations. This arrangement of individual musclesfollows distinct scenarios (McClearn and Noden, 1988; Noden, 1991). In the simplestcase, a myogenic condensation forms within a single somitomere and then shifts to itsterminal site giving rise to a single muscle. In other cases, a common premuscle masswill give rise to several muscles. Individual muscles may segregate from the originalcondensation either prior to or following the appearance of primary myotubes.Subsequently, the developing myofibres elongate and align in precise orientations thatprecede and predict the later segregation of individual muscles. Although the actualorientation of muscle fibres often changes during later stages of craniofacial growth,these initial differences in alignment between compartments remain constant. All theseevents occur prior to the formation of definitive attachments, suggesting that cues formyofibre orientation are present in the mesenchymal environment of the fusingmyoblasts prior to differentiation of connective tissues (McClearn and Noden, 1988).This notion that myogenic cells have little role in regulating gross spatialpatterning in voluntary muscles has been confirmed by experiments involvingtransplantation of trunk somites into the head, with subsequent formation of typicalmasticatory muscles (Noden, 1986). Hence, myoblast populations are not preprogrammedwith respect to the geometry of the tissue they will form; rather, after migration, theyparticipate in muscle development according to the dictates of the local environment. Incontrast, the connective tissue precursors are known to contain detailed instructionsregarding their ultimate shape and relation to other elements (Noden, 1991).4The stage at which attachments with skeletal connective tissues form is uniquefor each muscle and is independent of the other aspects of muscle maturation.Differential growth of individual muscles and gross changes in head morphology causethe relations both among muscles and between muscles and skeletal elements to changedramatically (Noden, 1991). The masticatory muscles differentiate as individual entitiesfrom first-arch mesenchyme, migrate, and gain attachment to bone some time after theirdifferentiation. The migration of muscles and ligaments relative to their bonyattachments is a consequence of periosteal growth. The masseter has little distance tomigrate, but its growth and attachment are closely associated with the mandibular ramus,which undergoes remodelling throughout the early, active growth period. As aconsequence, these muscles must continually readjust their insertions and, to a muchlesser extent, their sites of origin (Sperber, 1989; Herring et al, 1993; Covell and Herring,1993).Although the actual orientation of muscle fibres often changes during laterstages of craniofacial growth, the initial orientation of myotubes presages the adultpatterns (Noden, 1991). This is also observed in the fetal pig masseter, in which the basicarrangement of internal tendons and disposition of muscle fibres is already present(Herring and Wineski, 1986). The relative size of tendons, however, varies between fetaland adult specimens. With age, the length of fasciculi decreases relative to muscleweight, while the variance in length among different parts of the muscle increases(Herring and Wineski, 1986). Likewise, a discrepancy between the growth velocities ofthe entire muscle and the muscle fibres is found in the rabbit, where the origin-insertion5length increases relatively more quickly than the fibre length (Langenbach and Weijs,1990).During the formation of the trigeminal nervous system, an increase in motorneuron production occurs in focal zones known as rhombomeres (Källen, 1956, 1962).Nuclei of the trigeminal (Vth) cranial nerve are found to span two rhombomeres(Keynes and Lumsden, 1990), and the generation of this efferent population ofteninvolves several waves of neuron production across the intervening inter-rhombomericboundary (Altman and Bayer, 1982; Covell and Noden, 1989). In contrast, neurons of thecranial sensory ganglia derive from the neural crest and from neurogenic placodes, whichare focal thickenings of the embryonic surface ectoderm (Hamburger, 1961; Johnstonand Hazelton, 1972; D’Amico-Martel and Noden, 1983).Whereas the early stages of myogenesis may proceed normally in the absenceof a nerve supply, the process of innervation enhances muscle development and isimportant for the complete differentiation of the muscle fibre (Mastaglia, 1981). Thehistogenesis and morphogenesis of muscles and nerves influence each other as themuscle masses form and the nerve and blood supply to them differentiates. Motor nervesestablish contact with the myocytes, stimulating their activity and further growth byhypertrophy; failure of nerve contact or activity results in muscle atrophy. Nerve andvascular connections, established at this time, persist even after the muscles havemigrated from their site of origin. This explains the long and sometimes tortuous pathsthat nerves and arteries may follow in adult anatomy (Sperber, 1989).In the m. biceps brachii of the human fetus primitive nerve branches ramify6among the developing muscle cells during the 10th week, and myoneural junctions beginto form during the 11th week of intrauterine life (Cuajunco, 1942). At the same time,the myoblasts show more abundant and better-developed myofibrils, and at the peripheiyof most of these cells, all the characteristic striations of adult human voluntary musclefibres may be distinguished. The earliest stage of the neuromuscular spindle formationis also found in this period (Cuajunco, 1940), and at 15 weeks, contacts between axonsand muscle cells are seen frequently (Gamble, Fenton and Allsopp, 1978).Since myoneural junctions have formed in the limb muscles by the 10th weekof fetal life, they must have formed earlier in the head and neck, following the cephalocaudal sequence. Indirect evidence for this is that the earliest reflex movement elicitablein the human fetus occurs at 7½ weeks’ menstrual age1 (Hamilton, Boyd & Mossman,1962), when some neck muscles contract in response to cutaneous stimuli applied to thelips. This indicates that the trigeminal nerve is the first cranial nerve to become active.Soon after the initial reflex muscle activity, spontaneous muscle movements can also beidentified. These are believed to begin at 9½ weeks’ menstrual age, followed by activemouth closure at 11 weeks, and intrauterine thumb sucking as early as 18 weeks.Swallowing occurs at 12 weeks, as the fetus starts drinking amniotic fluid (Humphrey,1970; Sperber, 1989). At the same time, myelinization, which is an indicator of nerveactivity, appears first in the trigeminal nerve but is not complete until the age of 1-2years (Sperber, 1989).1 Development events are commonly dated from the last menstrual period.Therefore, the menstrual age of the developing human is 2 weeks greater than the true age(i.e. when fertilization occurs) of the fetus (Moore, 1983).7During the early stages of development, several motor axons with differentthresholds innervate a single muscle fibre (“polyneural innervation”). Studies at differentpostnatal ages show that the elimination of the redundant axon terminals occursgradually, the mature one-on-one pattern being established a few weeks after birth inmost muscles (Purves and Lichtman, 1980; Redfern, 1970; Bagust et a!, 1973). Theelimination of some of these initial connections results from a competitive withdrawalof axonal branches, and the course of this competitive rearrangement is influenced byactivity (Lichtman and Purves, 1983). It has been postulated that this selective loss ofcertain axon branches is in order to refine the topographic projection of the neuronsgiving rise to these axons (Tolbert, 1987). Another possible role for this process ofsynapse elimination is that it could shape the adult innervation territory in a complexmuscle from a less precise innervation pattern (Brown and Booth, 1983; Bennett andLavidis, 1984). In a recent study however, it has been shown that, at least for the ratlateral gastrocnemius muscle, this is not true. This muscle is innervated in acompartment-specific manner at birth, and postnatal synapse elimination generally doesnot serve to establish “neuromuscular compartments2”.Essentially, these are establishedby a process occurring before the time of birth; the innervation pattern being fullyconsistent with the infrastructures of the muscles (Donahue and English, 1989).Subsequently, as the muscle gradually develops, a denser innervation forms withoutmodifying the basic structure of the original pattern (Schumacher, 1989).2 Each neuromuscular compartment is defined as the innervation territory of asingle naturally occurring primary muscle nerve branch and has been shown to be composedof an aggregation of single motor units (English and Weeks, 1984; Janun and English, 1986).8All muscles undergo a period of secondary myogenesis that begins late in fetallife (Donahue et at, 1991). This process is nearly complete at birth in some muscles, sothat most muscle fibres increase in size but not in number early in infancy (Ontell andDunn, 1978). Secondary myogenesis continues postnatally in other muscles, increasingthe number of muscle fibres up to 60% (Donahue et a!, 1991). Satellite cells are believedto play a role in this postnatal growth (Mastaglia, 1981); moreover the discontinuous andgradual increase in muscle fibre size can be accounted for mainly by an increase in themyofibrillar material within the fibre (Goldspink, 1980). With age there is a change inthe proportions of the fibre types (Sjöström et a!, 1992), and with exercise, hypertrophyof the individual fibres is observed. The converse, muscle disuse, results in fibre atrophy.Although it is commonly assumed that there is no increase in the number of fibres withage or training, it was recently reported that the mean number of fibres in a musclefascicle increases from childhood to adult age, and thereafter (middle and old age)reduces (Sjöström et a!, 1992). In addition to hypertrophy, muscle fibres also increasein length during postnatal growth. This increase in length is associated with an increasein the number of sarcomeres in series along the myofibrils, and hence along the lengthof the fibres (Goldspink, 1980).At birth, the suckling muscles of the lips and cheeks are relatively moredeveloped than the muscles of mastication (Gasser, 1967). Indeed, at birth, mobility ofthe face is limited. Later, a more rapid growth of the muscles of mastication is observed,as there is a conversion from suckling toward mastication, and later into adultmastication. Hence, the facial muscles increase fourfold in weight and the muscles of9mastication increase sevenfold, between birth and adulthood (Sperber, 1989).In summary, there is no definite ontological data regarding the fetaldevelopment of the jaw muscles in humans, but the available evidence suggest that mostmajor structural elements are formed at an early stage. The rate of growth of jawmuscles and their attachments have been studied in humans, sub-human primates, rats,goats, capybara and rabbits, as increasing demands (such as dietary changes) are madeon masticatory function. A common theme is the increase in the diversity of fibredirections, pennation and whole-muscle orientations. In humans, qualitative data implythat although the muscle attachments migrate with growth, their number and relativedisposition remain fairly constant from birth (Gaspard, 1987). And with the exceptionof Schumacher’s (1962) account of the increased pennation complexity from newborn toold age cadavers, there are no detailed studies on aponeuroses and muscle fibre lengths,angulation and attachment location according to fetal age.1.2 Muscle Architecture and MechanicsMany muscle fibres are grouped into bundles (fascicles) of varying size andpattern, each of which is surrounded by “perimysium”, and several of which form anindividual muscle. Fascicles vary in length from a few millimetres to many centimetres(15-30 cm in sartorius; Williams et a!, 1989), and may be composed of multiple fibres inseries (Loeb et al, 1987). For example, in the semitendinosus muscle in goats, fasciclesare 16 cm long, and are composed of short fibres, which overlap each other by at least30% of their length, and are arranged into groups of narrow bands that run end-to-end.10In separate parts of the muscle, bands are composed of a different number ofoverlapping fibres. These fibres also vary in length according to region (Gans et a!, 1989;Thomson et a!, 1991). In addition, muscle fibres are not uniform in type and diameter(ranging from 10-60 am), and may vary between whole muscles, sometimes betweenmuscle subvolumes or even within a single subvolume (Williams et al, 1989; Hopf, 1934).Their attachment to bone can be either “musculous”, in which case the fibresattach directly to periosteum, or “aponeurotic11in which case the attachment is throughan intervening collagenous structure, which can be a fascia, tendon or aponeurosis. Inaddition, there are two categories for an aponeurotic attachment: internal where musclefibres insert into both sides, or external where fibres insert into one side only(Dullemeijer, 1974; Van der Klaauw, 1963).To understand the mechanics of a muscle, it is important to know thefascicular orientation, which may be parallel or oblique relative to the muscle’s directionof pull. Muscles which have their fascicles arranged parallel to their line of traction arereferred to as parallel-fibred muscles. These produce translational motion exclusively.Muscles with obliquely arranged fascicles may be triangular (fan-shaped) or pennate(feather-like) in construction, and this enables the fibres to rotate about their origins,increasing the angle of pennation as they shorten (Gans, 1982), while the insertiontranslates along the desired direction (Otten, 1988). Pennate muscles may vary in theircomplexity and can be classified as unipennate, bipennate or multipennate structures(Gans and Bock, 1965; Williams et a!, 1989).When compared against pennate structures, parallel-fibred muscles have the11largest number of sarcomeres in series. Consequently, during contraction their insertionshave the greatest excursions and achieve higher movement velocities, while theirsarcomeres simultaneously maintain an advantageous position on the length-tensioncurve, which is broader in parallel-fibred muscles. The role of this type of muscle is toenable large and rapid movements (Gans and Bock, 1965; Gans, 1982).In contrast, pennate-muscle fibres run roughly parallel to each other, but atan angle towards their insertion along which the muscle force is exerted. Here, individualfibres are shorter, and the range of excursion (in which maximum tension can beproduced) is reduced. The absolute distance that individual fibres can act is smaller,even though the resultant movement of the tendon is slightly more than that of any fibre.The advantage of permation is that it allows more fibres to be packed into availablespaces, increasing the muscle’s cross-sectional area and therefore overall forceproduction. Another advantage is that it provides a structural basis for the nervoussystem to control portions of a muscle independently, perhaps to produce force vectorsin various directions. Similarly, if activated differentially, fibres may move their insertionlaterally as well as toward its origin (Gans, 1982).Theoretical models of permate muscles are generally drafted with fibresattached to parallel tendinous insertions at both ends. In many of these muscles, theaponeuroses are unconstrained and may show local deformation (such as bent ends),as a unilateral contraction induces moments near the attachment site (Gans and Gaunt,1991). That is, fibres induce couples, which are a pair of forces of the same magnitude,with parallel lines of action (along the origin and insertion tendons) and opposite12directions. The sum of the components of these two forces cancel each other, and theequivalent rotational moments will tend to twist the set of muscles inward, toward theline of action. In such cases, only mechanical stops, such as bones, fascia and other activemuscles can keep the lines of origin and insertion from rotating towards the same plane(Gans, 1982). Provided that this rotation is avoided, the resultant vector of the forcegenerated by the fibres will then contribute to translation of the tendinous insertions,that will slide past each other, maintaining the distance between them and the musclevolume unchanged (Benninghoff and Rollhäuser, 1952; Gans and Bock, 1965; Gans,1982; Hatze, 1978; Otten, 1988). Since muscle fibres are angled relative to the tendon,some of the force will be lost in the static condition. In the dynamic condition, however,an increased excursion of the central tendon is achieved which partially compensates forsome of this force loss (Muhl, 1982; Gans and de Vree, 1987).Other designs were proposed for different types of muscle architecture. Oneexample was introduced by Woittiez and colleagues (1984), in which the distancebetween tendinous sheets decreases upon muscle contraction, due to their initial nonparallel arrangement (Woittiez et a!, 1984); and another example (Benninghoff andRollhäuser, 1952; Otten, 1988), in which muscle fibres have slightly different orientationsand therefore different pennation angles relative to their insertion. Here, provided thatthe aponeuroses move parallel to their original orientations, these fibres mustconsequently shorten by different amounts.Most often, investigators have dealt with unipemiate muscles, which have abony attachment at their origin, and a parallel tendinous sheet at their insertion. As13noted earlier, a rotational moment can be maintained at the bony insertion, and theforce of muscle pull transmitted along the aponeurosis (Otten, 1988). In some muscles,however, two tendinous sheets are arranged at an angle relative to the point-likeattachments to bone or external tendons. In this case, no moment can be sustained atthe transition of aponeurosis to external tendon; therefore, the most likely direction ofmuscle pull is a straight line through the external tendons (Huijing and Woittiez, 1984;Woittiez et al, 1984; Otten, 1988; Heslinga and Huijing, 1990; Scott and Winter, 1991).This difference in design affects the magnitude of the resultant force and the tendondisplacement. Relative to the former case, the amount of shortening and volumedecreases in the latter system, indicating that this type of muscle should have bulging ofcurved fibres (Otten, 1988).The force generated by a muscle fibre depends on its physiological cross-section (Weber, 1846; Weijs and Hillen, 1985; Gans and de Vree, 1987) and itsphysiological characteristics. The latter will be reviewed in subsequent sections. Inaddition, the force production is affected by the position of the fibres’ sarcomeres on thelength-tension curve, by a change of length during isometric contraction (shorteningproduces less force; lengthening, produces greater forces), and by the overlap of actinand myosin filaments (Gans, 1982). Since force generation is also affected by thedistance and velocity of the myofibre displacement, the positions of its attachment sitesare critical (Gans and Gaunt, 1991).It is commonly believed that the force generated by a whole muscle is thescaled-up version of the force generated by a single motor unit, which in turn is a scaled14up version of the force generated by a single muscle fibre. This assumption seemedplausible for simple, parallel-fibred muscles, in which fibres extend the entire length ofthe fascicles, from origin to insertion. However, this assumption was found not to be truefor muscles with more complex architecture, such as the anterior sartorius muscle of cats(Scott et a!, 1992). The anterior sartorius is a parallel-fibred muscle composed of short,in series fibres, arranged into separate neuromuscular compartments (Thomson et a!,1991). When these fibres are independently stimulated, it causes the muscle to shortenat the end closest to the nerve branch and lengthen at the neighbouring site. At short,whole-muscle lengths, stimulation of one compartment generates only a small fractionof the total muscle-force output, while at increased whole-muscle lengths, an increasedfraction of total muscle force can be generated. This relationship between active andpassive muscle subvolumes allows complex modulation of whole-muscle force production(Scott et a!, 1992). Moreover, it could be suggested that differential hypertrophy ofmuscle fibres occurs within a muscle as a consequence of intramuscular forceheterogeneity. Hence, it is possible that fibre angulation may change differently inseparate regions of a muscle.Hypertrophy is responsible for the increase in length of pennate musclesduring exercise and especially growth (e.g. gastrocnemius medialis; Woittiez et a!, 1986;1989; Heslinga and Huijing, 1990), while it has been suggested that hypertrophy playsa minor role in the increase of the length of parallel-fibred muscles (e.g. semitendinosuslateralis; Willems and Huijing, 1992). The latter type of muscle increases its length byincreasing the number of sarcomeres within the fibres. There are functional differences15between the two types of muscles; parallel-fibred muscles maintain a greater range oflength in which active force can be generated, while pennate muscles have this rangereduced. For both types of muscle, the increase in muscle fibre diameter isaccommodated by an increase in length of the aponeurosis and muscle fibres and by theenlargement of the bony attachment area. Although the fibre angles relative to the bonyattachment remain constant, changes of the aponeurosis angle with the line of pull canbe observed and may differ within different regions of a muscle (Willems and Huijing,1992).Consequently, non-homogeneous behaviour of separate muscle portionsintroduces new difficulties for understanding the design of muscles, especially muscleswith complex structures. Differential contraction of distinct muscle subvolumes maycause internal shearing forces and regional changes in intramuscular pressure, affectingoverall muscle behaviour in complex ways. In addition, fibres which attach on one sideto tendinous sheets and on the other to bony surfaces may intrinsically shorten bydifferent amounts and over different angles due to changes in the length of theattachment tendon (Otten, 1988).Finally, we can note that the architectural design of a muscle has a significanteffect on its mechanical action. The arrangement of both muscle fibre and connectivetissue influences muscle function and can introduce differences in compliance3(Borgand Caulfield, 1980). Within a muscle, connective tissue is present in the sarcolemma,between fibres, and as tendinous inscriptions, aponeuroses and attachment tendonsCompliance is the ratio of change in length to change in force.16(Partridge and Benton, 1981), each of which has inherent elastic properties. Theyoperate in conjunction with the contractile muscle components (Hill, 1950; Partridge andBenton, 1981; Zajac, 1989), and are of importance in the generation of musclestiffness4, especially in muscles with long tendons. For instance, increased tendonstiffness will limit the stretch of the tendon, therefore allowing the muscle fibres toremain in the desired portion of the length tension range (Peterson et a!, 1982, 1984).Because of the difficulty in linking the physiology of single muscle fibres with that ofwhole bone-muscle-connective tissue structures, models have been comnioiily used tooffer functional and mechanical explanations of muscle design. One way of producingsuch a model is to incorporate detailed structural and functional properties of aparticular muscle to produce a realistic mechanical result. Once these models are testedagainst measurable functional characteristics, they can be used to assess functionalproperties which are difficult to measure in vivo. Additionally, it is clear that individualmuscles have their own characteristics, making it necessary to match function to thestructural system that it belongs to (Otten, 1988). However, this is a formidableundertaking for pennate muscles, and it has not yet been achieved for any complexmuscle in a proper manner (Hannam and McMillan, 1993).Stiffness is the ratio of change in force and the change of length causing theforce.171.3 Motor-Unit Organization1.3.1 Motor-Unit ArrangementSeveral skeletal muscle fibres are innervated by a single alpha motoneuron;together they form a “motor-unit” (MU), which is the basic functional unit of the motorsystem (Sherrington, 1929). It has been commonly assumed that all fibres of a MU areof the same histochemical type (Henneman and Olson, 1965; Brandstater and Lambert,1973), although recent studies have questioned this (Larsson, 1992). There is bothhistochemical and electrophysiological evidence that muscle fibres of a single MU arediffusely scattered within the MU’s territory and intermingled with fibres of other units(Ekstedt, 1964; Edström and Kugelberg, 1968; Stálberg et a!, 1976; Buchthal andSchmalbruch, 1980). This wide distribution with extensive MU overlap is partlyresponsible for the evenness and smoothness obtained during light voluntary musclecontraction, resulting in a more or less uniform tension at the muscle tendon, even whenvery few motor units are active (Buchthal and Schmalbruch, 1970).Both the number of MUs and the number of muscle fibres belonging to a MUvary greatly within a particular muscle and especially between different muscles. Forexample, muscles which are used in high-precision tasks show considerably fewer fibresper MU (Stâlberg et a!, 1986). It has been estimated that for the human extra-ocularmuscles there are 2970 MUs and 9 fibres per MU (Feinstein et a!, 1955), while for themasseter muscle there are 1452 MUs and 640 fibres per unit (CarlsöO, 1958).181.3.2 Fibre CharacteristicsIdentification of fibre types was originally achieved by the differentiation ofthe metabolic characteristics of the muscle fibres. These were analysed by histochemicalstaining procedures to reveal the activity of their metabolic enzymes. In one of the earlystudies, Ranvier (1873) related differences in muscle function to colour, classifyingmuscles as red or white. Later, the scale for rating the fibre’s oxidative potentials wasfurther expanded to include intermediate staining intensities. In recent years, moresensitive immunocytochemical techniques have been developed for use with bothfluorescence and electron microscopy to reveal muscle fibre’s structural proteins(Goilnick and Hodgson, 1986; Miller, 1991).Although histochemical methods yield valuable information concerning theproperties of muscle fibres, ultimately the most important aspect of the typing of fibresrelates to what role the fibres play during muscle contraction. To study this, physiologicaltechniques have been developed to activate MUs independently (Burke, 1981; Buchthaland Schmalbruch, 1980). With the advancement of these techniques, the initialclassification of red, intermediate and white became synonymous with slow-contracting(S), fast-contracting fatigue-resistant (FR), and fast-contracting fatigue-susceptiblemuscles (FF), respectively (Burke et a!, 1971; Goilnick and Hodgson, 1986).Subsequently, other nomenclatures have been introduced to characterize fibre types (asreviewed by Close, 1973; Burke, 1981; and Mao et al, 1992); type I, hA, IIB fibres(Brooke and Kaiser, 1970); slow twitch oxidative (SO), fast twitch oxidative glycolytic(FOG), and fast twitch glycolytic (FG) fibres (Barnard et a!, 1971); and B, C, A fibres19(Heimeman and Olson, 1965). Historically, this diversity of nomenclature created greatconfusion, making comparisons of data from different studies difficult. The histochemicalproperties of types of muscle fibre differ between muscles (Luschei and Goldberg, 1981;Taylor et a!, 1973) and between species (Gollnick and Hodgson, 1986); they can alsochange with age (Sjöström et a!, 1992) and gender (Miller, 1991).Muscle fibre diameter is frequently related to the histochemical fibre type. Inmany mammalian muscles, the diameters of type IIB fibres are larger than those of hAfibres, which in turn are larger than those of type I fibres (Burke, 1981). However,exceptions to this pattern are found in some human muscles, where type I fibres arelarger than type H fibres. This inconsistency might be the result of fibre size changesfollowing exercise or inactivity, and again different types of fibres may be affecteddifferently. Training increases the cross-sectional muscle fibre area, while inactivitydecreases it (MacDougall et a!, 1980; Salmons and Henriksson, 1981). Furthermore, ithas been conclusively demonstrated that muscle fibres adapt to increased demands forendurance by changing their metabolic profile. This is brought about by increasing theiroxidative potential (Edström and Grimby, 1986).1.4 Motor-Unit ActivityGiven the arrangement of MUs described above, a description of howmuscular force is attained by the nervous system is appropriate. The nervous systemproduces graded increases in force in two primary ways: (1) Through the progressive“recruitment” of MUs, it increases the number of active MUs. (2) Through “rate coding”,20it modulates the firing frequency of individual motor neurons (Henneman, 1979; Freund,1983; De Luca, 1985; Carew and Ghez, 1985). A brief description follows.1.4.1 RecruitmentDuring a graded skeletal muscle contraction, MUs are normally activated inan orderly sequence, which is related to the size of their innervating motoneurons(reviewed in Burke, 1981). Neurons with the smallest cell bodies are the first to beactivated, and as the synaptic input increases in strength, larger motoneurons areprogressively recruited. This recruitment order is known as the “size principle”(Herineman, 1981).Human motor-unit recruitment thresholds (RTs), i.e. the forces at which unitsare first activated for a given task, are generally reproducible under controlled conditionsbut they can vary (Tanji and Kato, 1973a; Freund et a!, 1975; Miles et a!, 1986). RTsdepend on the rate of isometric contraction of the muscles involved, decreasing the unit’sthreshold dramatically when ballistic contractions are used (Budingen and Freund, 1976),and upon muscle length (Miles et a!, 1986). In addition, RTs of biceps-brachui MUs werealso found to be reduced by successive voluntary isometric contractions (Suzuki et a!,1990).1.42 Firing RateIt is well-known that small units are the first to be activated, and as their firingrates increase, muscle force output increases proportionally (Freund et a!, 1975; Tanji21and Kato, 1973b; Monster and Chan, 1977; Mimer-Brown et a!, 1973; De Luca, 1985).Small, low-threshold units discharge regularly and with a far more pronounced firing ratemodulation than units recruited at higher muscle forces (Freund et a!, 1975). The lowestpossible firing rate of a unit does not seem to differ significantly between MUs recruitedat different thresholds or between muscles (for review see Freund, 1983), and this firingrate is characterized by an irregular pattern of discharge (Kranz and Baumgartner, 1974;Person and Kudina, 1972) which becomes more regular as the firing rate increases abovea critical level.Thus, the interaction of progressive MU recruitment and the modulation ofMU firing rates is responsible for the transition from a series of motor-unit twitches toa tetanic contraction, with consequent smooth increase in muscle force (Buchthal andSchmalbruch, 1980; Burke, 1981; Clamann, 1970; De Luca, 1985).1.4.3 BehaviourMost evidence on the patterns of motor control has been obtained fromanimal studies, in which global electromyographic activity is commonly recorded withimplanted electrodes in awake animals, and single MU characteristics sampled duringhighly-invasive surgical procedures. While the latter type of experimental approach iswell controlled, the observed behaviour of MU activity is however stereotyped, and theeffect of decerebration on the pattern of motor control is uncertain (Burke, 1981). Inturn, the use of human subjects offers many advantages in MU behaviour studies, dueto their ability to perform highly-skilled, voluntary motor tasks (Freund, 1983; Miles et22a!, 1986; Hannam and McMfflan, 1993).A consistent pattern of MU behaviour has been conventionally reported forseveral limb muscles. Small units are recruited at low thresholds. They have highresistance to fatigue, long contraction time (Edström and Grimby, 1986), and their lowerfiring rate limit is maintained between 5 and 8 Hz, producing lower twitch tensions thanlarger units. However, this lower rate limit is not precisely fixed and seems to vary forfunctionally different muscles (Tanji and Kato, 1981; Person and Kudina, 1972).In addition, there has been abundant documentation of the fact that duringslow ramp contractions, consistent ordering of MU recruitment is generally found withina particular collection of units belonging to a single anatomically defined muscle whichis known as a motor pooi (Desmedt, 1980; Burke, 1981; Schmidt and Thomas, 1981;Thomas et a!, 1978; 1987). These findings introduced the idea that the RT of a unit,within the spectrum of thresholds in a motor pool, is a stable property, and that thethreshold level remains unchanged in different motor acts (Henneman et a!, 1974; Burke,1981). Contrary to this classical concept, it has been occasionally observed that unit RTsmay vary significantly during the performance of a given movement (Petajan, 1981).When repeated, movements may appear to be identical, but can be accomplished byvarious strategies (Freund, 1983), such as by varying degrees of muscle contractiondepending on the level of coactivation of antagonistic muscles (Carew and Ghez, 1985;Petajan, 1981). The recruitment order can be also altered under different postures(Person, 1974), or by contracting multifunctional muscles in different directions (Thomaset a!, 1978; Desmedt and Godaux, 1981; ter Haar Romeny et a!, 1982). This alteration23in MU recruitment order has been generally observed between units with similarthresholds (Tanji and Kato, 1973b), and slight changes in synaptic efficiency or theactivation of sensory afferents with an uneven distribution of the terminals to themotoneuron pool have been implicated in RT reversals (Kanda et a!, 1977; Lflscher eta!, 1979). It has been also observed in a multi-functional muscle that as the musclechanges the direction of effort, the resultant muscle force produced also changes.However, when the ratios of RT relative to muscle force for the different tasks arecompared, they apparently remain identical (Thomas, Ross and Stein, 1986). This arearemains quite controversial (Enoka and Stuart, 1984), however evidence suggests thatat least in a single function muscle, the recruitment pattern is highly stereotyped, withlittle chance for flexibility in MU recruitment order (Schmidt and Thomas, 1981).Generally, in the past, investigators analysed randomly distributed MUsactivated by simple, whole-muscle contractions. As seen earlier, however, in somemuscles a MU may perform multiple tasks (ter Haar Romeny et a!, 1982), and in othermuscles distinct tasks may recruit separate parts of the classically defined motoneuronpool, that is separate groups of MUs. This has been observed for the human bicepsbrachii (ter Haar Romeny et a!, 1982) and extensor digitorum commmunis (Thomas eta!, 1978; Riek and Bawa, 1992). In the latter muscle, the overlapping subpopulations ofmotoneurons were reported to maintain an orderly recruitment pattern, despite ofchanges in task (Riek and Bawa, 1992).241.4.4 SummaryThe MU represents the functional quantum by which the nervous systemregulates the development of muscle force. This regulation occurs by concurrentvariation in the number of active MUs and their rate of firing. The relationship betweenMU recruitment and firing rate modulation varies between muscles, but the result isalways an ability to finely grade muscle force.The strategy by which the nervous system controls MUs to generate andmodulate muscle force is, however, not fully understood. It is believed that there isuniform behaviour of recruitment and firing rates of a collection of MUs belonging toa motoneuron pool; this property is termed the “common drive”. However, the behaviourof a motoneuron pool can be altered depending on the task performed by the muscle.For example, a motoneuron pool may or may not be activated, or alternatively it mayhave its properties altered upon task change.The concept of “task group” was therefore proposed, in which within a muscleseparate functional groups of motor and sensory neurons are differentially activated toachieve optimal control over restricted parts of a movement.1.5 Partitioning“Neuromuscular partitioning” refers to features of neuromuscular organizationthat provide a morphological basis for subdividing a muscle into recognizablesubdivisions (Windhorst et al, 1989). A number of recent studies have shown thatmammalian muscles are partitioned with respect to both their motor and sensory25innervation. These partitions are organized about the primary branches of the respectivemuscle nerves and are known as “neuromuscular compartments” (English and Letbetter,1982a). As each primary branch of a nerve enters a muscle it innervates fibres in adiscrete subvolume of the muscle and contains an unique group of motor axons. Thatis, branches of individual motor axons course exclusively in only one primary musclenerve branch (English and Weeks, 1984; Janun and English, 1986; English, 1990),although in one single case in a newborn animal an axon branched in two directions ata nerve branch point (English, 1990). In the adult these innervation territories do notoverlap (English and Weeks, 1984; English, 1990).In some muscles, the neuromuscular compartments may be partially separatedby internal tendon sheets (English and Letbetter, 1982a; Richmond et a!, 1985;Armstrong et a!, 1988). These were thought to either act as physical barriers to axongrowth or to serve as axon guidance cues during development, resulting in the restrictionof axons to a particular compartment. In the gluteus maximus of the rat, however, nosuch tendons are found, although the muscle is compartmentalized by the primarybranches of both muscle nerves. This finding suggests that the neonatal compartment-specific pattern of innervation for this muscle must be established independently of thearrangements of tendons (English, 1990). The same has been observed in the rat anteriordigastric, which, despite its simple muscle architecture, consists of three neuromuscularcompartments (English and Tiiumis, 1991).These compartments may differ in both their composition of muscle fibretypes (English and Letbetter, 1982b; Bennett et a!, 1988) and fibre architecture. Within26each neuromuscular compartment of the pig masseter, fibre type is relativelyhomogeneous, but there are marked differences between them (Herring et a!, 1979).MUs are also generally confined to small volumes within a single compartment (Englishand Weeks, 1984). This pattern was confirmed in the pig masseter, and territories werefound to be smaller than those in most limb muscles. This may be due to the length ofmasseter fibres, which are shorter than these in limb (Herring et a!, 1989).Afferent axons supplying muscle spindles within a single intramuscularcompartment are found in the same nerve branches as the motor axons innervating thesurrounding extrafusal muscle fibres (Farina and Letbetter, 1977). When these motoraxons are stimulated, a more potent response of muscle receptors is observed withintheir own intramuscular compartment than in adjacent and more distant regions of themuscle (Cameron et al, 1981). This preferential response of muscle receptors to localizedperturbations within a muscle is known as “sensory partitioning”, and has beendemonstrated in both cat (Binder and Stuart, 1980) and human subjects (McKeon andBurke, 1983).Analysis of the anatomical organization of motor nuclei has shown that spinalmotoneurons that supply a neuromuscular compartment can be organized into groupsforming a “compartment nucleus”. That is, a motor nucleus can be arrangedtopographically (Weeks and English, 1985). In addition, different compartment nucleicontain motoneurons of different sizes, which are highly correlated with the types ofmuscle fibres they innervate (Weeks and English, 1987). Similar topographic organizationhas been observed for afferent axons that project to the spinal cord within a relatively27discrete portion of the muscle’s afferent entry zone (Cameron et a!, 1981). Thispartitioning of segmental connections within a nucleus is known as “central partitioning”(Windhorst et a!, 1989).Collectively, it appears that the role of partitioning is to provide a basis forthe nervous system to control structurally complex muscles differentially. On the onehand, partitioning may moderate the effects of mechanical heterogeneity. In somecomplex muscles, variations have been found in the fibre length and in the angle ofpennation. In this type of muscle, differential motor control may enable distinct regionsto undergo different relative amounts of lengthening and shortening, in order tocontribute uniformly to motion at the muscle’s common tendon. On the other hand,partitioning may provide the substrate for differential control of muscular regions thatare specialized for diverse functional capabilities (Windhorst et a!, 1989). The latterpostulate has been confirmed in some complex limb muscles, where compartments couldbe activated independently of other compartmental subdivisions within the muscle duringcontrolled activity (English, 1984; English and Weeks, 1987).1.6 Jaw Muscle OrganizationThe masticatory system comprises five muscles: temporalis, medial pterygoidand masseter, which are jaw-closing muscles, and digastric and lateral pterygoid. Eachof these latter two muscles are formed by two heads: the anterior and posterior beffiesof the digastric, and the superior and inferior heads of the lateral pterygoid. Althoughthese muscles have multiple functions, they are normally classified as jaw openers28(Miller, 1991).As stated earlier, a muscle may be classified as parallel-fibred, unipennate,bipemiate or multipemiate, depending on the complexity of its structure. In the jawmuscles, examples of all four types of structure can be found.1.6.1 StructureThe digastric muscle consists of two parallel-fibred bellies separated by anintermediate tendon, which is attached to the hyoid bone. The posterior belly arises fromthe digastric notch on the mastoid process, projects anteriorly and inferiorly, connectingwith the anterior belly which projects anteriorly and superiorly to the mandible (Williamset al, 1989; Miller, 1991; Hannam and McMillan, 1993).Traditionally, it has been thought that both bellies contract simultaneously toachieve jaw opening, retrusion or hyoid elevation. The posterior and anterior beffies are,however, innervated separately by trigeminal and facial motoneurons and, at least in rats,are divided into separate neuromuscular compartments (English and Timmis, 1991). Thisstructure offers a basis for differential activation strategies. Non-synchronous activity hasbeen occasionally observed (Munro, 1974; Widmaim et al, 1987a), neverthelesselectromyographic data suggest that most often there is synchronized activity in the twobeffies during all mandibular movements, including chewing and swallowing (Munro,1972; 1974; Yemm, 1977).Conventionally, the lateral ptervgoid muscle is considered to be one muscle,consisting of two heads which have different orientations. The superior head originates29from the infratemporal crest and roof of the infratemporal fossa and convergesposteriorly, laterally and caudally, while the almost three times larger inferior headoriginates from the lateral surface of the lateral pterygoid plate of the sphenoid boneand converges laterally and posteriorly towards the neck of the condyle. Both fan-likemuscle heads converge towards the fovea of the mandibular condyle and attach to fovea,articular capsule, and articular disc. Despite several intramuscular tendons at origin andinsertion, the lateral pterygoid has a very simple anatomy (Schumacher, 1961c; Widmaimet a!, 198Th; Wilkinson, 1988; Meyenberg et a!, 1986; Carpentier et a!, 1988; Miller, 1991;Hannam and McMillan, 1993).The lateral pterygoid’s two bellies are believed to function mostlyindependently; the inferior head is more efficient in jaw opening, and the superior headmore inclined to facilitate jaw closing and tooth clenching (Miller, 1991). An alternativeview is that all fibres act as one muscle, with varying amounts of activity that couldgradually change the whole-muscle line of pull according to the biomechanical demandsof the task (Widmaim et a!, 198Th). Although there is no direct evidence for thispostulate, it is consistent with previous observations that fibres belonging to the inferiorhead are very active during tooth-clenching in eccentric jaw positions, even though theyare normally considered to be active only in the balancing side during jaw laterotrusion(Wood et a!, 1986) and in mandibular retrusion (Widmalm et a!, 198Th; Hannam andMcMillan, 1993).When viewed from the lateral aspect, the temporalis muscle is a broad fanshaped muscle originating from the temporal fossa and converging into a flat, also fan30shaped tendon that inserts onto the anterior, medial and posterior surfaces of thecoronoid process, as well as the anterior border of the ascending mandibular ramus. Theinsertion tendon extends upwards as an internal aponeuroses, separating the muscle intotwo layers. When viewed frontally, the anterior part of the muscle has a typicalbipennate structure, with the fibres diverging from the central aponeurosis at increasingpennation angles in the infero-superior direction. Seen from the lateral view, fibres runvertically in the anterior part of the muscle, radiating to become almost horizontalposteriorly. At this level, the muscle is thinner mediolaterally and has no obviousbipennate arrangement (Schumacher, 1961c; Hannam and McMillan, 1993).Anatomically, the more complex structure of the temporalis suggest thatgroups of fibres may be separately activated, depending on mechanical demand. It hasbeen known for some time that graded, task-sensitive activation can occur in theanterior, middle and posterior fibres, during the elevation, retrusion and lateraldisplacement of the jaw (MØller, 1966; Miller, 1991; Blanlcsma and van Eijden, 1990;Hannam and McMillan, 1993). Mediolaterally, the superficial and deep muscle layers oneither side of the central aponeurosis can be also selectively activated (Wood, 1986).Together, this behavioural flexibility and structural composition of regions of the muscle,which have different cross-sectional areas (anteriorly the muscle has a much larger crosssection than posteriorly), implies not only differential muscle usage, but also that thedevelopment of tensions throughout the muscle is not homogeneous (Blanksma and vanEijden, 1990; Hannam and McMillan, 1993).The medial ptervgoid is a rectangular, bulky muscle situated on the medial31side of the mandibular ramus, running downwards, backwards and outwards from itsorigin in the pterygoid fossa to insert onto a triangular area on the inside of themandibular angle. The internal structure is multipennate, consisting from six to eightinterleaving aponeuroses, which can extend about two thirds of the way into the muscle,attaching to bone above and below in an alternating fashion (Schumacher, 1961c). In thefrontal view, the orientation and closeness of the interleaving aponeuroses offer littlegeometry with which to vary the line of muscle pull. When viewed laterally, the musclefibres have a slightly fanned arrangement. When fibres located anteriorly are comparedagainst the others located posteriorly, their alignment is more vertical, (i.e. they areoriented upwards, inwards, but with a lesser forward angulation (Hannam and McMillan,1993).Electromyographic studies in the medial pterygoid have shown that this muscleis highly active during intercuspal clenching, protrusion and contralateral laterotrusion(Gibbs et a!, 1984; MØller, 1966; Vitti and Basmajian, 1977). During clenching in theintercuspal position, the level of muscle activity can be modulated by varying the biteforce direction (Wood, 1986), suggesting that a task-dependent gradation of differentmuscle regions is possible. Information on differential contraction, however, is difficultto obtain, partly because of the difficult physical access to the muscle for electricalrecordings and partly because individual subjects use the muscles differently (Hannamand Wood, 1981; Hannam and McMillan, 1993).The masseter is a rectangular-shaped, multipennate muscle, which has thesecond largest cross-sectional area of all masticatory muscles (Weijs and Hiilen, 1985;32Hannam and Wood, 1989). It can be divided anatomically into two or three partiallyseparate layers, which originate from the zygomatic arch and extend downwards,backwards and inwards to attach to the lateral surface of the mandibular ramus. Likethe medial pterygoid, internally it consists of three to five interleaving and alternatingaponeuroses, which extend into the muscle by approximately two thirds of the whole-muscle length (Ebert, 1939; Schumacher, 1961c).The overall role of this muscle is to elevate, protrude and move the mandiblelaterally, however, increasing evidence that different parts of this muscle contractdifferentially depending on the jaw task indicates that this muscle can also produce fine-skilled movements (Greenfield and Wyke, 1956; Belser and Hannam, 1986; Blanksmaet al, 1992).1.6.2 Fibre CompositionIn general, the fibre-type properties of intrafusal fibres in the masseter of rat,guinea-pig, rabbit, cat and macaque monkey resemble those in limb muscle spindles(Rowlerson et a!, 1988), but there are significant differences between jaw and limbextrafusal muscle fibre types. Firstly, larger fibre diameters can be found in limbs(Eriksson and Thornell, 1983) where different types of fibres are distributed in a mosaic-like pattern. In contrast, jaw muscles contain many fibres of the same histochemical type(Stãlberg et a!, 1986). Based on this histological appearance of densely-packed fibres ofthe same type, the speculation of fibre-clumping was raised (Eriksson and Thornell,1983). This appearance would be considered pathological if present in the limb or trunk,33where it would indicate denervation and reinervation (Schwartz et a!, 1976). To addressthis, the fibre concentration (i.e. the mean number of fibres per MU within the smalluptake area of the single-fibre electrode, Stâlberg and Thiele, 1975) of the masseter wascompared against that of the first dorsal interosseus. The fibre concentration in bothmuscles was found to be similar; thus, the masseter does not show any sign of groupingof fibres from the same MU (Stálberg et a!, 1986).In addition, large numbers of ATPase intermediate (IM) fibres and type IICfibres are found in the jaws, but they are scarce in the limbs (Ringqvist et a!, 1982). Thecomposition of myosin isozymes is different between jaw and limb muscles. The humanmasseter contains neonatal myosin heavy chains and embryonic myosin light chains thatare characteristic of developing muscles (Butler-Browne et a!, 1988), and cardiac-specificmyosin-heavy-chain is also exclusively found in skeletal muscles which originate from thecranial part of the embryo (Bredman et a!, 1991). These differences between jaw andlimb muscles suggest that finer gradation of contraction speeds in jaw muscle fibrescould be possible.In the jaw muscles, regions are composed of different histochemical fibre typesin varying proportions, implying a functionally-related distribution. In all muscles, typeI fibres predominate, especially in the anterior and deeper regions, while there is a trendtowards equal proportions of type I and II fibres posteriorly and superficially (Miller,1991; Hannam and McMillan, 1993). A similar pattern is observed for jaw muscles inpigs and primates (Herring et a!, 1979; Clark and Luschei, 1981). Here, MUs areconfined to regions, which were previously defined histocheniically.341.6.3 Jaw BiomechanicsDuring mastication, a complex contraction pattern of the masticatory musclesinduces spatial rotations and translations of the mandible, simultaneously producingforces to the food bolus and articular joints. While these forces should ideally bemeasured directly from individual subjects, this is not always feasible, and insteadmathematical models have been used to calculate them.In this approach, it has been commonly assumed that the mandible is a rigidbeam which behaves according to static equilibrium principles. Here, muscle forcevectors are applied at their appropriate two or three-dimensional sites, and bite forcesand joint reaction forces are then derived (Osborn and Baragar, 1985; Throckmorton andThrockmorton, 1985; Nelson, 1986; Smith et a!, 1986; Faulkner et a!, 1987; Koolstra etal, 1988a; Korioth and Harmam, 1990). The orientation of these muscle lines of action(including the point of application and direction of muscle force) are generallydetermined by dissection (Smith et a!, 1986; Koolstra et a!, 1988a), estimated from driedbone (Baron and Debussy, 1979) or radiographs (Throckmorton and Throckmorton,1985), or by defining a line through the centroids of contiguous muscle slices (An et a!,1984; Koolstra et a!, 1989; Hannam and Wood, 1989). The maximum magnitude that anindividual muscle force can reach is determined by its physiological cross-sectional area(Weijs and Hillen, 1984a; Hannam and Wood, 1989), while its degree of activationcontributing to a particular jaw task is determined electromyographically (MØller, 1966;MacDonald and Harinam, 1984a,b).From these models, it was found that the resultant forces produced at the35teeth and joints depend on several factors. Firstly, they depend on the bite point location(Pruim et a!, 1980; Nelson, 1986), on mandibular position (Koolstra et a!, 1988a) and onthe orientation of the jaw muscle vectors, rather than only on the muscle’s intrinsicstrengths (Throckmorton and Throckmorton, 1985). In addition, depending on themuscles contraction patterns, bite and joint forces can be developed in a wide range ofdirections (Nelson, 1986), in which the vectors together form a force envelope (Koolstraet a!, 1988a). The shape of these envelopes, in turn, is influenced by changes in thedirection of masticatory muscle vectors, especially vectors of the masseter muscle(Koolstra et al, 1988b).While useful, these models have several limitations. Aside from the fact thatthey are based on static and rigid beam theories, interactions between the structure andfunction of the jaw muscles are normally limited by the incorporation of muscleproperties which are umnatched to the morphology of the model under study, andrestricted by simple, single vector muscle representation. Consequently, in recent yearsa different modelling approach, involving finite-element modelling, has been used toreveal internal structural stresses, as well as local deformations (Korioth et a!, 1992). Sofar, this method has been successfully used to study deformations within a humanmandible upon simulated static tooth-clenching, utilizing, however, average physiologicaldata which was not specific to the morphology under investigation.1.6.4 SummaryDespite the importance of masticatory muscles to craniofacial growth and36mastication, our understanding of their design, functional characteristics andbiomechanics is limited. In this system, examples of both parallel-fibred andmultipermate arrangements can be found, each of which has its unique characteristics.And, as the result of their coordinated contractions, they induce complex dental, articularand cranial tensions, as well as movements of the mandible.The diverse arrangement of muscle fascicles, coupled with the regionaldistribution of different biochemical fibre types, offers a substantial anatomical basis fordifferential contraction. This may subsequently produce heterogeneous tensionsthroughout the muscle, and therefore produce resultant force vectors in a variety ofdirections, which are known to affect reaction forces at the condyles and bite points.Among the masticatory muscles, the masseter is the most complex andpowerful, with the potential of influencing the bite force envelope most extensively. Itis an important muscle during the power stroke in chewing, it is multipennate, and it hascomplex histochemical properties. In addition, its peripheral and central connectivitiesare better understood than most jaw muscles, possibly because it is easily accessible forelectromyographic recordings.In humans, the masseter is commonly involved in disorders of the orofacialarea. Despite a universal structural principle of the same muscles among differentspecies, muscle properties are unique to the species. Therefore, a better understandingof the characteristics of the human masseter’s contraction is desirable, since they aredirectly applicable to the understanding of the structural and functional organization ofhuman masticatory apparatus.37There are considerable advantages and disadvantages of using human subjects.Human subjects, unlike animals, are able to comply with complex instructions. Thedisadvantages, however, include the technical restrictions imposed by handling livingtissues, as opposed to more invasive methods (including post mortem material) whichare possible in animal experiments. Despite the limited kind of experiments in humans,promising techniques are presently being developed, which could be applied to the jawmuscles.Based on the above outlined characteristics, the human masseter muscle is agood study model to further understand the contraction patterns of the masticatorysystem. Therefore, a more detailed description of the masseter’s anatomical andfunctional design will be introduced in the next sections.1.7 Anatomical Design of the MasseterMammalian masseter muscles arise from the lower border of the zygomaticarch as far anteriorly as the zygomatic process, and extend downwards and backwardsto insert over a wide area of the mandibular ramus, extending from the mandibularangle to the level of the coronoid process. The masseter incorporates a complex systemof internal aponeuroses (tendinous sheets) which divide the muscle into subvolumes withdiffering fibre orientations and pennation angles. In each subvolume, muscle fibres runcontinuously from one aponeurosis to the other, and at least in the rat masseter, notendinous inscriptions between the muscle fibres occur (Matsumoto and Katsura, 1985).Common to all mammals is the size of the aponeuroses which cover large areas of each38muscle layer antero-posteriorly. In contrast, the orientation and position of theaponeuroses vary between species, due to their individual functional needs. Sinceinternal architecture is important for the motor performance of a muscle, a detaileddescription of the masseter muscle anatomy will be presented and compared againstother mammals in the following sections.1.7.1 Muscle OrganizationThe rat masseter muscle has been described as having anywhere from two tofour parts (Romer, 1939; Becht, 1954). In order to reconcile this lack of descriptiveconsistency, Hiiemae (1967) proposed that the separation of a muscle into distinct partswhere no clear anatomical division exists (as occurs in the masseter) is justified onlywhen there are large differences in fibre orientation and position, enabling the inferencethat the parts so defined have a different action. On this functional basis, Hliemae andHouston (1971) divided the rat (Rattus noivegius L.) masseter into four: one superficial,urilpennate part (essentially regarded as a separate muscle), and three deep portions. Ina more recent report by Matsumoto and Katsura (1985), the masseter muscle was furthersubdivided. Eleven compartments were described on the basis of fibre architecture alone.However, potential errors may arise using this classification, which may divide the muscleinto too many compartments. It is possible to separate the muscle into different putativeparts erroneously, influenced solely by the gradual changes in orientation that the fibresundergo from the muscle’s origin to insertion. Nevertheless, irrespective of the quantityof separate muscle portions, dissections by Schumacher (1961a) revealed a system of39three interleaving aponeuroses.In contrast, Herring and Scapino (1973) were unable to describe discrete,anatomically separate divisions in the masseter muscles of the pig (Sus Scrofa dom.).Macroscopic examination of this muscle (Herring et a!, 1979) revealed gradual changesin fibre angulation dorso-ventrally at its surface. These observations lead Herring et a!(1989) to the conclusion that it was not possible to identify strict anatomicalcompartments in the muscle on the basis of fibre orientation alone since the angulationof fibres was not constant through the same muscle layer. Also dissimilar to the rat,Schumacher (1961b) identified four internal aponeuroses. In the anterior part of themuscle, the aponeuroses are obliquely oriented, interdigitating septa, while in theposterior part they become broad and parasagittal. Among them, the fasciculi are longand vertical (relative to the tooth row) anteriorly, while the posterior fasciculi are shortand near-horizontal, extending antero-posteriorly (Herring, 1980).A number of detailed accounts on the human masseter anatomy are availablein the literature. However, reports are inconsistent, describing the human masseter asconsisting of two to eight portions of muscle. The simplest description divides the muscleinto two parts, one “superficial” and one “deep” (Sicher, 1960; Schumacher, 1961c;DuBrul, 1980). In agreement with Sicher’s original definition, Last (1966), Mosolov(1972), Gaspard et a! (1973), and Williams et a! (1989) also reported superficial anddeep portions, but expanded the description by adding a third “intermediate” portion. Incontrast, Ebert (1939) identified a third, “anterior” portion, situated laterally andseparately from the superficial portion. This author’s rationale was that although this40small portion was difficult to identify initially, it deserved to be described as separate,due to its simple parallel-fibred arrangement. Additional anatomical subdivisions werelater introduced by Gaspard et a! (1973), who separated the superficial part into twosubsidiary layers, the 11laniina prima et secunda”. To further confuse the issue, Yoshikawaand Suzuki (1962) divided the masseter into eight parts. In their report, the superficialmasseter is divided into two layers (lamina prima et secunda), consistent with Gaspard’sanatomical account. They also identified one intermediate and three deep portions. Theanterior deep has a simple structure, while the deep posterior portion is divided into fourparts arranged medio-laterally, the deepest of which constitutes a separate portion calledthe M. maxillo-mandibularis. Despite the lack of agreement among previous reports, allabove mentioned authors agree that irrespective of the number of muscle portions orlayers, the various muscle portions are fused together on the zygomatic arch. The variousportions also fuse anteriorly at their insertions on the mandible, but they diverge fromeach other posteriorly to allow passage of the masseteric artery and nerve.Dissections performed by Ebert (1939) and Schumacher (1961c) revealedseveral internal aponeuroses which extend into the muscle body, alternating from thezygomatic arch and the lateral surface of the mandibular ramus. These reports howeverdiffered with respect to the number of aponeuroses. At least four of them could bevisualized by magnetic resonance imaging in living subjects (Lam et a!, 1991). Inconcurrence with Schumacher’s (1961c) nomenclature, Aponeurosis I was the mostlateral of the four and anchored to the zygomatic arch. Aponeurosis II attached to themandibular ramus and extended superiorly into the muscle. Aponeurosis ifi attached to41the zygomatic arch and extended inferiorly into the muscle, more medial thanaponeurosis II. The most medial aponeurosis, IV, was located on the deep surface of themuscle. Each aponeurosis had an unique three-dimensional orientation determined bythe relative orientations of the zygomatic arch and mandibular ramus, both of whichvaried with craniofacial morphology (Lam et a!, 1991). Because of morphologicalvariations, widths between adjacent aponeurosis varied among subjects, and it wassuggested that muscle fibre orientations between aponeuroses may result in unequalpennation angles at each end of the fibres (Lam et a!, 1991).Besides these differently-graded angles of pennation, the muscle fibres alsodiffer in length, averaging 26 mm, but ranging from 14 to 39 mm (Schumacher, 1961c;Van Eijden and Raadsheer, 1992). In the muscle, these differences in length occuranteroposteriorly. Anteriorly they range from 24-30 mm; in the middle of the musclethey range from 19-26 mm and posteriorly from 14-19 mm. In the coronal view, themuscle fibres are arranged in various orientations between the contiguous aponeuroses.Whereas their angles of pennation vary between 12° and 14° in the anterior portion ofthe muscle (decreasing posteriorly to average 11° in the main, superficial portion),muscle fibres have identical lengths throughout a given coronal section (Ebert, 1939).The masseter’s origin and insertion has consistently been described asconsisting of mixed musculous and aponeurotic attachments (Ebert, 1939; Schumacher,1961c; Gaspard, 1987). Dissections and osteological studies revealed that besides themain aponeuroses, there are other, possibly smaller, tendinous attachments inserting intothe lateral surface of the mandibular ramus (Baron and Debussy, 1979; Gaspard, 1987).42Collectively, this muscle has a highly complex architecture, which varies amongspecies, among individuals, and with functional demand. Its multipennate arrangementallows more fibres to be packed into a small volume, resulting in a large physiologicalcross-sectional area, which may also vary significantly in humans depending on theindividual’s craniofacial type, being greatest in subjects with brachiocephalic skulls, shortfacial heights and small gonial angles (Weijs and Hillen, 1984b). In addition, the fibreorientation, relative to the functional occiusal plane, allows this muscle to produce largeforces at the teeth (Hannam and Wood, 1989).1.72 Innervation PatternThe masseter muscle is innervated by a branch of the mandibular division ofthe trigeminal nerve. This branch, termed the masseteric nerve, enters the muscle at itsmedial and posterior aspect, after passing through the mandibular notch (Williams et a!,1989). Immediately before entering the muscle, the nerve divides into different trunks,which then diverge rostrally, ventrally and caudally (Lau, 1972; Xiguang et a!, 1986) toramify into the muscle layers situated between the aponeuroses (Schumacher, 1989).In pigs, three separate nerve branches enter the masseter and distributearound the internal aponeuroses. Two rostral branches, which also supply thezygomatico-mandibularis, are small nerves and supply the dorsorostral aspect of themasseter. A third, more caudal main branch, divides into four terminal nerves withvariable distributions. It innervates all of the muscle including at least part of thedorsorostral corner (Herring et a!, 1989). In dogs and sheep however, only two separate43branches of the masseteric nerve enter the muscle (Lau, 1972). One supplies theproximal third of the muscle and the zygomatico-mandibularis, being equivalent to thefirst branch found in the pig. The second branch innervates a large portion of themuscle, extending across the muscle’s medial and distal thirds. Despite the fact that thesedifferences in innervation patterns between species were observed, they were consideredto be minor and not fundamental (Schumacher, 1989).In humans, the masseteric nerve may divide into two (Lau, 1972) or three(Xiguang et al, 1986) main branches just before entering the muscle. The first runs justinferiorly and along the zygomatic arch innervating the proximal, deep part of themuscle. The second runs on an oblique trajectory across the entire muscle and suppliesits medial and distal thirds. In a few cases the second main branch splits into two trunksjust before entering the muscle (Xiguang et a!, 1986). The first trunk runs itsconventional oblique trajectory, while the second trunk runs straight downwards, towardsthe angle of the mandible, innervating the posterior aspect of the superficial masseter.Further ramifications of the first branch also innervate a muscle portion situatedbetween the masseter and temporalis. This portion is conimonly described in the pig andrat, as well as in other mammals, as the zygomatico-mandibularis muscle (Eisler, 1912;Lau, 1972). In humans, this portion of the muscle was classified as the maxillomandibularis muscle by Yoshikawa and Suzuki (1962), a surprising nomenclature giventhe muscle’s zygomatic origin.441.7.3 Muscle Fibre CharacteristicsWith the exception of the posterior superficial part where there areapproximately equal proportions of type I and II fibres (mostly type IIB), the adulthuman masseter consist mainly of type I fibres, which make up 62-72% of the muscle’stotal fibre content (Eriksson and Thornell, 1983; Vignon et a!, 1980). Type I fibresoccupy 88% of the cross-sectional area of the muscle’s anterior deep part, but only 70%of the posterior superficial part. In contrast, type IIB fibres constitute only 7-21% ofthese subdivisions. Most of the type IIB fibres are found in the posterior superficial part(20.5%), and posterior deep part (15.2%). ATPase IM and type IIC fibres represent onlyabout 9% of the total fibre population. Most type I fibres are larger than the type IIBfibres except in the intermediate part of the muscle (Eriksson and Thornell, 1983), andmuscle fibre diameter differs between muscle subvolumes. In the superficial portion,diameters are larger than in the deep portion (Hopf, 1934). The significance of this isunclear, but like the distribution of fibre types, it implies that some kind of functionaldifferentiation may occur. The masseter’s type I fibres contain slow myosin, and its typeII fibres contain mainly fast myosin, similar to the limb muscles (Thornell et a!, 1984).However, the adult masseter also contains a neonatal myosin heavy chain (MHC)isozyme which has not been described in normal limb muscles (Butler-Browne et at,1988). This neonatal MHC is expressed in ATPase IM and IIC fibres. In addition anembryonic isoform of myosin light chain (Butler-Browne et a!, 1988; Soussi-Yanicostaset a!, 1990) and a cardiac-specific myosin heavy chain have been found in the adultmasseter, although its location is uncertain at present (Bredman et a!, 1991).451.7.4 Spindle DistributionTo further elucidate mechanisms in mandibular motor control, Eriksson andThornell (1987) examined the muscle-spindle density and size in relation to extrafusalfibre-type composition in different portions of the human masseter by enzymehistochemistry. In contrast to findings by Kubota and Masegi (1977) who found similarspindle concentrations throughout the muscle of a new-born, Eriksson and Thornell(1987) found that spindles were distributed non-uniformly in the adult masseter muscle.In all subjects, spindle density had intramuscular differences. Most spindles were locatedin the deep portion, and no difference within this portion (antero-posteriorly) was found.The spindles in the deep portion were distinctive in that they contained the largestnumber of intrafusal fibres and had the largest diameters. Seventy-two percent of theintrafusal muscle fibres were contained in the spindles occupying the deep portion.Seventy-four percent of spindles were in the deep portion which is predominantlycomposed of type I fibres. These results are in concordance with Freimann’s (1954) andRowlerson and colleagues’ (1988) findings that spindle density is greater in the deepmasseter than in the superficial. Voss (1935) and Freimann (1954) also observedespecially large spindles in the deep masseter. As in the case of fibre-typing studies,these findings also imply that individual portions of the masseter have specializedfunctions. In general, there are more spindles in anti-gravity, postural muscles, and inmuscles controlling finely-graded movements, than in those used for coarse movements(Cooper and Daniel, 1963; Voss, 1971). Based on this knowledge, Eriksson and Thornell(1987) suggested considerable capacity for segmental proprioceptive reflexes in the adult46human masseter, especially in its deep portion.1.7.5 SummaryAlthough muscle organization seems to be based on mechanical demands andefficiency, quantification of fibre orientation, the spatial location of muscle fibres,intramuscular septa and attachment sites, is rare. Although jaw muscle attachment sitesin adult skulls have been described for one mandibular position, the large areas ofattachment of many jaw muscles and the trajectories of functional jaw movements meanthat various parts of these muscle’s insertions must move differently; yet there are nodata regarding such displacements as have been described in the rabbit (Weijs et a!,1989a,b).1.8 Functional Design of the Masseter1.8.1 Motor-unit TerritoryMotor-unit territories have been investigated extensively in animal models(Burke, 1981), but are often analysed as a two-dimensional map, when in reality it is athree-dimensional volume. In addition, the total number of fibres in a MU is notrepresented in a single cross-section (Burke and Tsairis, 1973), therefore multiplesections along the muscle are necessary to reconstruct MU territory properly (Hannamand McMillan, 1993). In humans, where histological techniques are not possible,electrophysiological techniques are used to determine the volume in which fibres of aMU are distributed (Buchthal and Schmalbruch, 1970; Stálberg et a!, 1976; McMillan47and Hannam, 1989a). Using this technique, MU territories have been estimated in thehuman masseter to be focal, spherically-shaped, and 3.7 mm long (Stâlberg and Eriksson,1987), although a small number of units reached 9.1-12.5 mm and were thought toextend over the whole muscle cross-section. In a different approach, measuring the lineardistance between paired recording sites for the same MU, McMillan and Hannam (1991)reported average MU territory size of 8.8 mm for the masseter. These authors reportedthe territories as elliptical, oriented in an anteroposterior direction, and apparentlyarranged in layers throughout the muscle. These studies, nevertheless, have not beenable to reveal the location of the MU territories relative to the intramuscularaponeuroses.From studies on experimental animals, it is known that in at least somecomplex muscles, the innervation of primary branches divide the muscle intoneuromuscular compartments (English and Letbetter, 1982a; English and Weeks, 1984),that may be defined by an internal tendinous network, and that muscle fibres pertainingto a single MU are contained within the boundaries of one of the compartments (Englishand Weeks, 1984). In this case, an individual motor axon may branch either distally tothe primary branching of the muscle nerve, or proximally with all branches entering asingle primary branch.Alternatively, the possibility that the muscle fibres in a single MU aredistributed over more than one neuromuscular compartment stifi exists for other muscles.Such a situation could arise if branching of individual motor axons occur proximally tothe primary muscle nerve branch point, and the branches do not all enter a single48primary branch. Herring et a! (1989) believes that this alternative innervation pattern isunlikely for the masseter muscle in pig, although in their study two or more widelyseparated regions in several instances, showed glycogen depletion. The authors’ argumentfor rejecting this alternative explanation was that first, very few axons seemed to haveproximal branching points, second, that multiple-area MUs have never been reportedwithin any other muscle, and third, that multiple-area MUs would obscure any patternof differential muscle activity, which are distinguished in the pig masseter (Herring et a!,1979).It was then concluded that MU territories in the pig masseter are confined tomuscle fascicles, and that the spatial separation of depleted regions were the result ofstimulating nerve filaments containing adjacent nerve axons which supply different partsof the muscle. This was supported by their findings that, in contrast to other muscles, thenerve trunk in the pig masseter is not ordered somatotopically (Herring et a!, 1989).Similar lack of organization was found for the facial nerve, although both the motornuclei and the extratemporal portion of the nerve do show a somatotopic arrangement(Radpour and Gacek, 1985).1.82 Functional DifferentiationThe question of functional heterogeneity, that is whether the masseter alwayscontracts as a unit or whether regional differences occur, has been posed for the pig(Herring et a!, 1979), rabbit (Weijs and Dantuma, 1981) and man (Greenfield and Wyke,1956). In pig, the electromyographic evidence shows that the different parts of the49masseter sometimes contract simultaneously with similar amplitude, during crushing onthe balancing side and the power stroke on the working side, but more often there areregional differences (Herring et a!, 1979). Seven to eight functional compartments withdifferent electromyographic patterns could be described, but these compartments do notlie within strict anatomical boundaries, nor do they have any particular fibre orientation(Herring et a!, 1989).In the rabbit, on the other hand, differences in firing patterns of the anteriorand posterior parts of the superficial masseter are negligible, while the well markedanterior and posterior portions of the deep masseter show slightly different firingpatterns. Mediolaterally, five different compartments situated between aponeuroses fireat different times during chewing. On the balancing side, the superficial portions startfiring, followed in a gradual transition by the deeper portions. The reversed firingsequence is observed in the working side muscle (Weijs and Dantuma, 1981). Theseauthors suggested that because of the continuity of the firing pattern any grouping ofcompartments is arbitrary.As reviewed earlier, anatomically the human masseter consists of at least twoportions (superficial and deep). With respect to the Frankfort horizontal plane5, thedeep muscle fibres run almost vertically downward, differentiating from those of thesuperficial muscle portion which are directed downward and posteriorly. Consequently,Carlsöö (1952) concluded that the deep portion of the masseter muscle could haveThe Frankfort horizontal plane is defined by three points: the centre of theexternal auditory meatus on both sides, and the lowest point on the right infraorbital notch.50closing and retractive functions.Many studies have attempted to separate the human masseter into separateportions on a functional basis. With surface electrodes, Greenfield and Wyke (1956)demonstrated differences in muscle activity between the superficial and deep portionsof the human masseter, reporting greater activity in the deep portion during jawretrusion and ipsilateral clenching. Conversely, the superficial portion was more activeduring protrusion, contralateral molar and incisor biting, and during protrusion withouttooth contact. Belser and Hannam (1986), did not demonstrate any significant differencein electromyographic activity between surface and fine-wire electrodes when recordingfrom the deep masseter, and decided to use the noninvasive surface electrodes forfurther investigation. In accordance with Greenfield and Wyke (1956), the authors founddistinct separation of activity when intercuspal clenching was directed retrusively, andwhen subjects were clenching on the ipsilateral side. During these tasks, the deep portionof the muscle was more active. Their results indicated that for most clenching tasks, themasseter contracts differentially, nevertheless both parts of the muscle are active, evenduring incisal clenching. This led them to the speculation that despite some differentialcontraction, the muscle has normally a relatively restricted range of action because somuch of it appears to contract so much of the time (Belser and Hannam, 1986).In a more recent study, six fine-wire electrodes were used in the masseter;three in the deep and three in the superficial portions (Blanksma et at, 1992). Althoughno statistically significant differences between different muscle regions were found, asignificant interaction between region and bite-force direction was present. By far the51largest deviation from the rest of the muscle was found for the deep posterior region;that is, its activity was the highest during postero-ipsilateral bites and lowest in oppositelydirected efforts. The same pattern, although less striking and more variable, was foundfor the posterior superficial masseter. In the deep muscle, the activity pattern of theanterior and middle portions were very similar, but differed with higher activity from thedeep posterior part during anteriorly- and anteromedially- directed bites. During theseefforts, differential contraction was nearly absent but became more obvious during otherdirected tasks, unveiling a functional partitioning of the masseter in at least three parts:anterior deep, posterior deep and superficial.Similar to the rabbit, in humans during chewing cycles, the masseter on theworking-side was always more active than the masseter on the balancing-side; andmaximum activity appeared earlier at the balancing side than at the working side. Inaddition, mediolateral differences within the muscle were found. On the balancing side,the peak muscular activity shifted from the superficial to the deep portion; the patternis inversed on the working side (Van Eijden et a!, 1993). On this latter side, the deepfibres of the masseter muscle contribute with relatively more activity to the final phaseof the chewing stroke than does the superficial portion of the muscle, even though bothare active bilaterally (Belser and Hannam, 1986).1.8.3 Motor-unit ActivitySimilar to what occurs in limbs, an orderly recruitment pattern of MUs hasbeen reported in the jaw muscles of human and non-human primates (Desmedt and52Godaux, 1975, 1979; Goldberg and Derfier, 1977; Yemm, 1977; Clark et at, 1978), withouly rare occurrences of activation order reversals (Desmedt and Godaux, 1979). In themasseter, recruitment threshold (RT) and force output of MTJs are also correlatedthroughout force development (Goldberg and Derfier, 1977; Nordstrom and Miles, 1990;Yemm, 1977), although RTs could vary depending on task, prolonged muscle contraction(Nordstrom and Miles, 1991b), and with jaw opening (Miles et a!, 1986). On the lattercase, as the jaw gape increases, the RT also increases.In earlier studies in the jaw muscles, MUs were assumed to belong to ahomogeneous, whole-muscle motoneuron pool. The human masseter and temporalismuscles are, however, complex muscles, and are composed of a mixture of uni- andmultifunctional units (Eriksson et a!, 1984), the latter showing regional differences inbehaviour (McMillan and Harinam, 1992). That is, most units located in the posterior,superficial region of the masseter contribute to tasks involving tooth contact, whereas inthe anterior, inferior and superficial portion MIJs were active in non-dental tasks,apparently contributing to jaw posture (McMillan and Hannam, 1992). In addition,masseter MUs were found to have their RTs changed with the direction of bite force;their “preferred” direction of effort was reflected in their RTs (Hattori et a!, 1991). Theseobservations infer that the masseter is organized into separate motoneuron task groups,which may be arranged to discrete regions at the motor nucleus and restricted toanatomical subvolumes at the periphery.Contrary to large limb muscles in which MUs are continuously recruited untilat least 90% of the maximum voluntary contraction (MVC) is reached (De Luca, 1985),53smaller muscles (such as the temporalis and masseter) recruit 50% of their MUs within10-20% of MVC. To further increase their force output between 20-100% MVC, thesemuscles rely predominately on firing rate modulation (Clark et al, 1978; Derfier andGoldberg, 1978).In the jaw muscles, the majority of MUs increase their mean firing rate withincreases in bite force, and although spike train characteristics are similar to thoserecorded in limb muscles (Derfier and Goldberg, 1978), the lowest sustainable firingfrequency (LSFF) of jaw muscles (10 Hz; Nordstrom et a!, 1989) appear to be higherthan in limb (6 Hz; Petajan, 1981). In the masseter and temporalis, this LSFF alsodepends on the jaw task performed (Erilcsson et al, 1984; McMillan and Hannam, 1992),and on the anatomical site (McMillan and Hannam,, 1992).1.8.3 ConclusionThe localization of MUs to anatomical subvolumes offers the potential formore specific motor control rather than simple contraction of the muscle as a whole. Itcould permit the activation of muscle regions relatively independently of one another asneeded, with the regional interactions dependent on the particular task and movementstrategy involved (English, 1985). In the human masseter, MU territories are mostlysmall, and therefore appear to be restricted to muscle fascicles just as MU territories arein the pig masseter (Herring et a!, 1989). However, the wide distribution of some MUterritories (Stâlberg and Eriksson, 1987) and the continuity in firing pattern observedduring chewing (Van Eijden et a!, 1993) suggests that in humans at least, muscle54compartments may not be defined entirely by internal muscle boundaries, and it ispossible that functional distinct regions in the masseter may be fewer than those inferredby muscle architecture alone.552. STATEMENT OF THE PROBLEMThe jaw muscles are responsible for the generation of forces within dental,articular and cranial tissues. Their functions promote jaw movements, have importantimplications in craniofacial growth and bone stresses, and can also be involved indisorders of the musculoskeletal system. Despite their importance in peripheral motorcontrol, the structural, biomechanical and functional organization of the masticatorymuscles are not well understood. It is known that each muscle has unique characteristics,including complex structural and histochemical properties, but we still cannot adequatelyexplain how these muscles contract.Little is known about human orofacial muscle development other thandifferentiation times and changes in size or weight. While the morphology andbiomechanics of adult jaw muscles have received attention, there is virtually noinformation regarding their fetal counterparts, and although muscle organization seemsto be based on mechanical and functional demands, quantification of intramusculartendon sheets and attachment sites is scarce. Attachment sites in adult skulls have beendescribed as extending into large bone areas, which suggest that during functional jawmovements different parts of these muscles’ insertions must move differently; yet thereare no data regarding such displacements. In addition, muscle activity appears to varyaccording to muscle region and task, suggesting that the human jaw muscles arepartitioned in a manner roughly similar to the divisions of the same muscles in rabbits,rats and pigs. However, little is known on how reproducible are the firing characteristicsof MUs, or how these are arranged relative to internal tendinous structures. The human56masseter muscle would be a good model to further basic knowledge in this area,because it exemplifies a complex muscular structure, its peripheral and centralconnectivities are better understood than most jaw muscles, and its location facilitatesfunctional investigation.To study muscle biomechanics and function relative to internal architecturein living humans, the following requirements should be met:a. Anatomical and biomechanical investigations of muscle structures should preferablybe made in living subjects, instead of relying solely on gross anatomical andhistological studies of cadaver material, which may have different dispositions ofinternal structures. In addition, non-invasive methods enable the evaluation ofmuscle mechanics during natural tasks.b. Appropriate intramuscular EMG recording techniques must be used to measurefocal muscle activity with minimum discomfort to the subject.c. The reproducibility of activation in a muscle subvolume should be ascertained whenthe activity of a focal muscle region is to be analysed according to task, or ifdifferent muscle regions are to be compared.d. EMO recording-site locations should be related to internal muscle structures if thebiomechanical role of muscle subvolumes is to be evaluated.In this thesis, the following specific hypotheses and objectives are proposed:571) It is hypothesized that in the masseter, the major structural boundaries that separategroups of muscle fibres with different orientations, are already present at an earlyfetal stage. Confirmation of this hypothesis would determine whether developmentof the human masseter is consistent with current views of muscle development ingeneral, and add insight to our understanding of the formation of its structuralmuscle compartments. The aim would be to compare the internal structures of thefetal masseter muscle against their adult analogues.2) It is hypothesized that portions of the masseter’s insertion have differentdisplacement characteristics, depending on the jaw movement performed, and thatthese differences reflect local biomechanical demands. Information regardingattachment displacement would be needed for any model of muscle mechanics, andcould be useful for evaluating the muscle’s length-tension characteristics underfunctional conditions. The goal would be to investigate spatial changes imposedupon the masseter’s insertions as a result of the rotational and translatorymovements of the human jaw.3) It is hypothesized that the human masseter is divided into separate muscle portionswhich are activated differentially, depending on the task performed. Confirmationof this hypothesis would clarify our understanding of peripheral motor control in themasseter. The hypothesis could be examined by testing regional electrical muscleactivity. The aim would be to assess the functional performance of a sample of MUsin one specific region of the masseter muscle, by testing both the effect of task on58MU activity and its reproducibility.4) It is hypothesized that MU territories in the masseter are small and confined bytendinous boundaries. Confirmation of this hypothesis would signify that ananatomical basis for differential muscle contraction is possible in the humanmasseter, possibly enabling the generation of internal force vectors with differentdirections. The aim would be to combine imaging of the muscle with three-dimensional verification of MU territories revealed by MU recordings in themasseter.Collectively, these experiments could be used to refine biomechanical modelsof the masticatory musculoskeletal system. They could provide new and fundamentalinsights into interactions between neuromuscular compartments and they could alsoadvance the understanding of skeletal muscle function, so important in the etiology ofmuscular disorders.593. STUDIESIn order to test the proposed hypotheses, experiments were carried outinvolving both structural and physiological studies as well as a combination of these typesof investigations.Anatomical studies were performed with different methodologies, includinghistological serial sectioning, conventional gross anatomical dissections, and MagneticResonance (MR) derived reconstructions of the masseter muscle. The use of histologicalsections of human fetal material was a practical means (due to its smaller size) ofdeveloping a methodology to study internal muscle architecture in the adult. The grossanatomical dissections were conducted to enhance familiarization with the muscle’sanatomy for better interpretation of the MR images.Experiments designed to study muscle activity were initially conductedexclusively with physiological methods, but in later projects both anatomical andphysiological methodologies were used in conjunction. Here, new technical approacheshad to be developed, i.e. a method for imaging the masseter internal architecture, a focalEMG recording technique, and a stereotactic method for locating the needle electroderecording site.In this chapter, each experiment is presented in a separate section, whichcontains that study’s specific material and methods, its related results, and theirdiscussion.603.1 Masseter Morphology3.1.1 Exploratory Experiments on Fetal Masseter AnatomyDuring post-natal development, from suckling to chewing, the masticatoryapparatus undergoes dramatic conversion in function (Herring, 1985). Masticatorybehaviour changes are gradual and occur during the transition through the deciduous,mixed and permanent dentitions. Concomitant and considerable changes in humanmasticatory movement patterns can be observed (Wickwire et a!, 1981). While sucklingmainly requires strenuous activity of the facial and lingual muscles, mastication involvesstrong activity in the jaw-closing muscles.Whereas the early stages of myogenesis may proceed normally in the absenceof a nerve supply (Mastaglia, 1981), the process of innervation enhances muscledevelopment. Myoneural junctions begin to form in the human limb during the 11thweek of intrauterine life (Cuajunco, 1942), a time in which jaw closure commences(Humphrey, 1971). Considerable mandibular development and muscular differentiationtake place at this time (Furstman, 1963; Diewert et a!, 1991). Of particular interest arethe nature and destination of major nerve divisions, and the extent to which a patternof connectivity is established at this early fetal age.Considerable work has been performed in non-human animals with respectto the growth of the jaw muscles (Zey, 1939; Gagnot et a!, 1977; Cachel, 1984; Herringand Wineski, 1986; Weijs et a!, 1987; Hurov et a!, 1988; Dechow and Carison, 1990;Langenbach, 1992). Despite a marked change in skull shape, and concurrent change inthe jaw muscles, the basic arrangement of muscle fibres and tendinous aponeuroses61seems to be relatively constant for pigs (Herring and Wineski, 1986) and rabbits (Weijset a!, 1987). Generally, the rabbit masseter has a positive allometric growth and itscontribution to total muscle weight increases strongly (Langenbach and Weijs, 1990). Theratio of muscle-fibre to muscle-length decreases and a higher variance in length indifferent parts of the masseter muscle are observed during its development (Weijs et a!,1987; Herring and Wineski, 1986). It is also believed that pennation in the rabbitmasseter increases with growth (Langenbach and Weijs, 1990).Besides some data regarding attachment sites (Gaspard, 1987) andaponeuroses (Schumacher, 1962), in neonatal and adolescent human masseter musclethere is little research on human masticatory muscle development. Knowledge of theinternal architecture from the fetal to the adult stages is desirable for a betterunderstanding of the relationship between structure and changing function of the muscle.In this section of the thesis, it is hypothesized that in the human massetermuscle major structural boundaries are found early in fetal life, and that these form thebasis for later regional organization in the more complex adult muscle. In addition, it isspeculated that fibre collections and major intramuscular tissue boundaries seen in thefetus have morphological and functional analogues in the adult. Thus the specific aimof this study was to divide the muscle into anatomical portions, and compare itsorganization with that of the adult muscle. Materials and MethodsSix intact formalin-fixed fetal masseter muscles were obtained from human62specimens at 17-20 weeks. These fetal stages were selected on the basis of muscle tissuedevelopment. Based on previous reports, it was expected that both muscle fibres andmyotubes would be present in fetuses older than 15 weeks (Mastaglia 1981; Gamble eta!, 1978). In addition, the relationship between fibre orientation, aponeuroses and theramus of the mandible are relatively constant throughout the peak human fetal growthperiod (Burdi and Spyropoulus, 1978).The heads were obtained from the collection of the Department of ClinicalDental Sciences, University of British Columbia. They were hemissected, and individualmasseter muscles were removed by cutting through the zygomatic arch anteriorly andposteriorly to the muscle’s origin, through the body of the mandible anteriorly to themuscle’s insertion, and through the tissue posterior to the mandibular ramus andcondyle. In addition, horizontal cuts were made superior to the zygomatic arch andbelow the inferior border of the mandible. Because the developing skeletal elements offetuses of this age are partly calcified, decalcification with 5% formic acid wasundertaken over a period of two weeks. The specimens were placed in the decalcificationmedium, which was subjected to constant motion on a histokinette at room temperature,and the solution was changed every other day to prevent calcium build-up. Radiographswere taken at the end of each week to monitor the decalcification process. Afterdecalcification was completed, the specimens were washed in running tap water for 24hours.To reconstruct three-dimensional (3D) shapes of tissues obtained from twodimensional serial sections, external reference markers have been commonly used63(Gaunt, 1971; Meyer and Domanico, 1988). These markers can be natural objects (e.g.chives, thread or piece of paper) introduced into the embedding material along with thespecimen, or tissue fiducials which can be either small holes drilled through the plasticembedding block peripheral to the biological specimen, or paint brushed along theblock’s sides (Brändle, 1989; Meyer and Domanico, 1988). The advantage of usingnatural objects as external markers is that they can be embedded in paraffin and can beeasily sectioned, while artificial markers require the use of plastic embedding materials.The disadvantages of the use of natural markers, however, are that the introducedobjects do not lie exactly perpendicular to the plane of section, and that they couldtheoretically undergo considerable distortion. If artificial reference markers are utilized,deformations caused by compression or stretching during sectioning can be correctedmathematically by the determination of correction factors. That is, if it is assumed thatthe deformation of a histological section is linear, affecting all points of that sectionequally, then all coordinate points of all reference markers and contour points shouldbe shifted in the x and y directions by the same factors relative to their originallocations. By calculating the distances between fiducial points and the angles formedbetween these points and by determining the change in positions of the referencemarkers relative to the reference system, distortions can be subsequently corrected.Since the masseter muscles are fairly large (approximately 6 x 8 mm) at 17weeks, they present a technical difficulty in terms of size. Cutting knives commonly usedin sectioning plastic blocks have short edges, meant for sectioning smaller tissues. Inaddition, only thin sections can be obtained from plastic blocks. Stain infiltration into the64tissues embedded in plastic can also be reduced. To accommodate the large sampledimensions, and to obtain thicker sections of the tissue for silver staining techniques,paraffin was chosen as the embedding material. Natural markers (earthworms) were usedas external references, which were useful as a coarse guide for matching the consecutiveserial sections. After embedding, specimens were sectioned at 10 jim and sampledserially every 100 jim through the entire length of each block. The six specimens weredivided into three groups, which were sectioned in the transverse, parasagittal and axialplanes. Alternate sections were stained for nerve, connective tissue and muscle fibres(where present), with a modified Bielshowsky’s silver impregnation method, picrosiriusconnective tissue stain, and haematoxylin and eosin, respectively. Nerve StainingNumerous impregnation methods for the demonstration of nerves in paraffinsections have been described (Samuel, 1953a,b; Peters, 1955a,b; Rowles, 1960). In thepresent study, three different techniques which can be applied to paraffin sections andperipheral nerve fibres were used: Bielschowsky (Culling, 1963), a modification ofPalmgren’s (Goshgarian, 1977), and the Marsland, Glees and Erikson’s (Drury andWallington, 1980) silver impregnation methods. However, none of the methods wasspecific, showing impregnation of nuclei and of connective tissue in addition to nerves.The specificity of silver impregnation depends largely upon an interrelationship betweenpH, time, temperature and silver concentration of the impregnating solution (Fernheadand Linder, 1956). The methods of fixation and tissue preparation, type of silver65Figure 1 Fetal peripheral-nerve staining. Histological feature of peripheral nerveshowing neurofilament immunohistochenijcal reactivity on the axons. The calibration barrepresents 30 jim.4 -—. -1._lt•.—66compound used, buffer type, incubation time and the characteristics of the developingsolution also play important roles in the specificity, reliability and reproducibility of silverimpregnation (Linder, 1978). Although most of these variables could be carefullycontrolled during staining of the fetal specimens, the fixation process could not, becauseit had been carried out earlier, prior to the obtainment of the fetal material. If tissuesare not fixed thoroughly immediately after the specimens are initially sampled, it wouldbe expected that the nerve fibres would show a reduced affinity for silver, possibly dueto loss of lipid, and that the finer nerve fibres and nerve endings could remain unstained(Linder, 1978). Apparently this was the case with the specimens used in this study, sincein the attempt to stain nerve bundles, sections were developed beyond the ideal timewhen a high contrast between nervous and non-nervous tissue is expected. At longerdevelopment periods, the intensity of the background started to increase becauseconnective tissue started to take up silver, thus compromising staining for nervespecificity.Immunohistochemistry staining of the nerve fibres was therefore performedas an alternative method. The technique was identical to that described by McGeer etal (1989), in which specimens were processed with anti-human neurofilament antibody.Figure 1 shows the results of this technique in a peripheral nerve in a fetal masseter.Successful staining of the axons was obtained, while the surrounding tissues were stainedonly very weakly.673.1.1.12 Connective-Tissue and Muscle-Fibre StainingThe combination of picrosirius (sirius red) staining (Junqueira et a!, 1979a,b)and polarization microscopy allows the specific detection of collagen (Figure 2). It isknown that Sirius Red stains collagen by reacting via the dye’s sulphonic acid groupswith the basic groups present in the collagen molecule. In addition, it is believed that thebirefringency enhancement is achieved by the attachment of the elongated dye moleculesto the collagen fibre so that the axes of both collagen and dye are parallel (Junqueiraet al, 1979b).3.1. 1.1.3 Muscle-Fibre Staining and Orientation AssessmentHaematoxylin and eosin staining (Culling, 1963) is commonly used to revealthe histological structure of tissues. A further advantage of eosin is that it fluoresces,enabling the detection of stained structures with confocal microscopy examinationsemploying Argon 488 laser beams. Muscle ReconstructionTo reconstruct the fetal muscles, individual histological sections werephotographed at low magnification. Since the specimens were large, each section wasphotographed over several fields. Negatives were printed and several of them were usedto form a montage of each histological section. Major tissue boundaries, that is,tendinous sheets and connective-tissue outlines separating muscle fibres with markeddifferences in fibre orientation (Hiiemae, 1967; Matsumoto and Katsura, 1987) were68Figure 2 Fetal connective-tissue staining. Polarized light micrograph of a tissuesection of human fetal masseter muscle cut in the parasagittal plane. The tissue wasstained by the picrosirius method. Tendon and connective tissue are seen as whitestructures within the black muscle. The front of the muscle is oriented towards the upperleft corner of the picture. The zygomatic arch and the mandibular ramus are seen aboveand below the muscle. The calibration bar represents 1 mm.69then traced from the montages. The initial goal was to code major muscle subdivisionsas outlines, by measuring muscle-fibre orientation by the method described byMatsumoto and Katsura (1987). Essentially this consisted of measuring the axial ratios(minimum/maximum) of ten randomly chosen sectioned fibres of each homogeneousgroup, then using these ratios to classify fibre cross-sectional profiles into various classes(i.e. 0.21-0.30; 0.3 1-0.40, etc). Neighbouring outlines with ratios that fell within the samerange were merged, and those with ratios that belonged to different classes were codedas separate muscle subdivisions. These were numbered 1-5, starting from the mostanterior region of the muscle and progressing posteriorly. Therefore, neighbouringoutlines always consisted of muscle fibres with distinct orientations, but it was possiblethat distant outlines were composed of fibres with similar axial ratios. Coded outlineswere then digitized and assembled into profile stacks.An extension of this technique was to measure the angle of the long axis often representative muscle fibres belonging to each group. To achieve this reproducibly,each histological slice was oriented on the microscope stage by aligning the lateralsurface of the mandibular ramus with an eyepiece grid. In one specimen, the angles weremeasured relative to a common reference line (lateral surface of the mandibular ramus)in the plane of section (horizontal section) using a confocal microscope (Laser ScanMicroscope, Carl Zeiss mc, Oberkochen, Germany). The mandibular ramus was usedas the plane of reference because it was found that the measurement of fibre angulationwas more reproducible than when fibres were measured relative to the median plane.In addition, the angle which the muscle fibres made to the plane of section70were measured. Again, each histological slice was kept oriented on the microscope stagealigned to the lateral surface of the mandibular ramus. A perpendicular optical scan wasperformed on the area of interest (Figure 3). The orientation of a given muscle fibre wasthen measured with the available confocal software by measuring the angle of the lineextending between the centroids of both fibre endings relative to the plane of cut. Sincethe muscle-fibre extremes were often imaged as diffuse endings, this method did notprove to be reproducible. An alternative, more reliable method of measuring this anglewas therefore pursued. It consisted of optically sectioning the specimen at two horizontaldepth levels and subsequently calculating the fibre-orientation angle from depth and shiftmeasurements. Each ten-J.Lm-thick section was optically sectioned every 0.5 m. At bothdepth extremes, the first optical section which resulted in sharp images were selected asthe topmost and inferiormost levels. These were colour-coded and stored in themicrocomputer. Sections were then superimposed, and the distance between the centroidof a single muscle fibre at one level was measured relative to the centroid of the samemuscle fibre at the second level (Figure 4). The angle was then calculated as thearctangent of the ratio of the distance between centroids over the distance between thetwo levels. Ideally, these measurements should have been performed in thicker tissues;however, technically it proved too difficult to obtain paraffin sections which were thickenough (40 jm).The two angles, that is fibre angulation relative to mandibular ramus and fibreangulation relative to the plane of the section, were then used to assign 3D orientationsto the different fibre collections. The 3D orientation of each representative fibre71Figure 3 Cross-sectional optical scan (Z-scan) through a 1O-JLm fetal-muscle section.Confocal-microscope scan of a horizontal section through a fetal masseter muscle. Thetissue was stained with haematoxylin and eosin and the muscle fibres were cut obliquelyto the cross section (below). The white horizontal line represents the XY location atwhich the cross-sectional optical scan was performed. The tissue thickness is shownabove. Two cursors (crosses) were placed at the centres of both ends of a selectedmuscle fibre, and the angle relative to the plane of cut was measured. In this example,the muscle fibre was oriented at 77° to the plane of cut.phL:7?.2Z:X2 BZ:18.6572Figure 4 Superimposition of two horizontal optical sections situated at bothextremes of a 10-pm-thick fetal muscle section. Confocal-microscope scan of a horizontalsection through a fetal masseter muscle. The tissue was stained with haematoxylin andeosin and as before, the muscle fibres were cut obliquely to the cross section. The topsection is colour-coded with red, while the bottom section with green. Cursors (crosses)are placed on the centroids of the muscle-fibre cross-section. Distance between thecursors is measured on the XY-plane and is 4.5 pm long.—73measured was then displayed graphically as a constant length vector passing through thesection profile at the respective location of measurement. By stacking section profiles,and with the aid of graphics rotation, it was possible to visualize changes in fibreorientation within groups as the level of section changed, and to do so in relationshipto major septal divisions. -The angles of muscle fibre orientation relative to the mandibular ramus andrelative to the plane of section were measured in one fetal masseter muscle sectionedin the horizontal plane. Five sections 400 m apart were chosen for this analysis. Inaddition, two sets of two sections 100 m apart were also measured to control for theaccuracy of the method. Each muscle section was divided into muscle subportionsdefined according to fibre minimum/maximum ratios, as described above. Fibre angleswere measured for a sample of ten fibres within each subgroup. Mean and standarddeviation values were calculated for each group in each selected section.3.1.12 Results3.1.12.1 Nerve DistributionAs in the adult masseter muscle (Lau, 1972; Xiguang et a!, 1986), in four fetalspecimens the masseteric nerve was found to branch into two trunks before entering themuscle (Figure 5A). As previously described, trunk A divided into two main branches,which supply the posterior portion of the deep masseter. Trunk B divided into two mainbranches in the posterior third (Figure 5A) and in the middle (Figure 5B) of the muscle.These innervated the anterior and posterior portions of the superficial masseter, as well74Figure 5 Masseteric nerve pathway in the human fetal masseter. Tissues werestained with haematoxylin and eosin. (a) Medial parasagittal section of a massetermuscle. The nerve enters the muscle in two separate branches (A and B) from themedio-posterior aspect. Branch A innervates the deep posterior muscle portion. BranchB innervates the anterior and superficial muscle portions. (b) Horizontal section of amasseter muscle half-way through the muscle’s longitudinal axis. The masseteric nerveruns parallel to the mandibular ramus forward and splits into two branches. One branchinnervates the anterior deep portion. The second branch divides again into two; the firstof which innervates the anterior superficial, while the second supplies the posteriorsuperficial muscle portion (dots and arrows). Anterior is to the left. The calibration barrepresents 1 mm. (P) Parotid gland; (R) Mandibular ramus; (Z) Zygomatic arch.7, 4,_. .‘‘75as the anterior portion of the deep masseter.3.1.122 Connective-Tissue DevelopmentDense regular connective tissue was found in all specimens, and formed thefetal analogue of Aponeurosis I. With the exception of one specimen in which theprimordial of Aponeuroses II and ifi (Figure 2) were also seen, other major aponeurosesin the masseter muscle were not reliably identifiable at this fetal stage, and clearseparation of the masseter muscle into recognizable compartments by the identificationof collagenous boundaries proved difficult in all investigated specimens. In contrasthowever, the medial pterygoid muscle showed well-formed dense connective-tissue layersin all sectioned specimens (Figure 6). Muscle-Fibre OrientationAs expected, human fetuses at 17-20 weeks menstrual age comprised bothmyotubes and muscle fibres in mixed proportions (Figure 7). The embryonic musclefibres consisted of centrally placed nuclei and peripherally disposed myofibrils (Figure8). An increased number of blood cells was also commonly observed in specimens at thisfetal age (Figure 8B).Although the pennation pattern was not expected to be fully developed, therelationship between masseter muscle-fibre orientation and the ramus of the mandible,and also with occlusal plane, has been shown to be relatively constant throughout thepeak human fetal growth period (Burdi and Spyropoulus, 1978). In the present study,76Figure 6 Development of aponeurotic layers in the fetal masseter and medialpterygoid muscles. Tissue was stained with Sirius Red and visualized with lightmicroscopy. Note that only Aponeurosis I (arrows) is well formed in the massetermuscle. In contrast, in the medial pterygoid muscle several aponeuroses are alreadypresent (arrows). Anterior is to the left. (M) Masseter muscle; (MPt) Medial Pterygoid;(R) Mandibular ramus.77...1mm-Figure 7 Masseter muscle fibres in the 18 weeks old human fetus. Muscle fibres arearranged within abundant loose connective tissue. Note the striation pattern alreadypresent at this developmental stage. Tissue was stained with haematoxylin and eosin andvisualized with the confocal microscope.78Figure 8 Characteristics of fetal muscle tissues. Both figures are different segmentsof the same histological section of masseter cut along the horizontal plane and stainedwith haematoxylin and eosin. Above, muscle fibres are cross-sectioned and are arrangedin fascicles between loose connective tissue. The nuclei are centrally placed and at thisstage myoblast fusions are still expected. Below, muscle fibres were cut obliquely. Thisregion is also rich in blood cells. The calibration bar represents 25 ,m.Iiq’i.-.. FjI ‘‘‘4I79Figure 9 Parasagittal sections from two 18-week human fetuses showing fibreorientation complexity. (A) Medial section through the deep masseter region; (B) and(C) lateral sections through two different the superficial masseters. [A and B areobtained from the same specimen.] Note the different morphological pattern betweenB and C. Anterior is to the left..-o_f; :-;•... Fi_ -,80perination varied in complexity between specimens. Muscle fibres could run parallel inone case, while they formed arcades in similar regions of a different specimen (Figure9). These differences in appearance could not be accounted for by differences in theplanes of section.Table I shows the muscle subdivisions per section, which were classifiedaccording to Matsumoto and Katsura’s (1987) method of measuring muscle fibre cross-sectional minimum/maximum ratio (e.g. 0.21-0.30; 0.3 1-0.40, etc). In this horizontallysectioned specimen, muscle subdivision 1 is situated in the most anterior portion of eachmuscle slice, while the other subdivisions were progressively assigned towards theposterior muscle portion. Mean and standard deviation values for both angles, that is,fibre direction relative to the mandibular ramus (Angle I) and fibre direction relative tothe plane of section (Angle II) were measured with confocal microscopy and are listedunder the various muscle-section subdivisions. No consistent number of subdivisions werefound when different sections were compared, and muscle fibre angulation did not followa constant pattern for subsequent sections. Since the methods used were based partly onan already-established approach, and partly upon confocal microscopy which is knownto have a higher resolution, these results were unexpected. They could be explained bythe fact that either the muscle at this age was poorly organized, consisting of myotubesand/or short immature fibres aligned in various directions, or that there were flaws inthe methodology used.To confirm whether the methodological approach was reliable, the technicalerror of the measurement (Knapp, 1992) was evaluated by calculating the difference in81TableIOrientationofmuscle-fibresubgroupsrelativetothemandibularramusonthehorizontalplane(AngleI)andrelativetotheplaneof cut(AngleII).Musclesubdivisionspersectionweredefinedrelativetotheminimum/maximumratioof themusclefibres.Meanandstandarddeviationvaluesareexpressedindegrees.Thevaluesfor thetechnicalerrorof themeasurementareshowninbracketswhereapplicable.ANGLEI00MuscleSubdivisionsSection#123458022±5-25±744±1012072±1346±854±223±245±14160-42±3345±519058±1764±1326±11-31±1686±1720055±14[1.5]78±6[7]48±6[11]80±16[56]240-23±14-41±1776±6-80±355±1225025±4[24]longitudinaltransversal57±8[69]109±7[27]ANGLEIIMuscleSubdivisionsSection#123458019±237±458±1712033±725±629±822±349±1016030±861±219033±1565±8-57±20-20±5-17±420026±6[3.5]42±7[12]56±26[57]20±48[20]240-15±5-21±5-39±11-70±34-27±7250-63±20[24]longitudinaltransversal-51±9[10]-41±8[7]angulation of muscle fibres belonging to the equivalent subdivision in two consecutivesections 100 m apart, (see Methods). The squared value of the difference was thendivided by 2n (n is the sample size). The square root of the ratio resulted in the estimateof the technical error of the method. This was estimated for all muscle subdivisions(when applicable) for two pairs of sections (i.e. section 190 and 200; 240 and 250), andit was found that it varied from 1.5 to 69, suggesting that the approach was unreliableat this stage of fibre maturation. DiscussionA primary muscle nerve branch has been defined as one of the branches ofa muscle nerve as it enters the muscle at its hilus (English and Weeks, 1984). Glycogendepletion studies have demonstrated that the primary branches of muscle nerves supplydiscrete subvolumes of the muscle (English, 1985) in a fascicle-specific pattern (Herringet a!, 1989). Muscle fibres supplied by primary muscle nerve branches formneuromuscular compartments which have been postulated as the anatomical substratefor motor control (English and Weeks, 1984; English and Letbetter, 1982a). Thesecompartments are known to be established before birth (Donahue and English, 1989).In the present study, two primary nerve branches were found, innervating the deep andsuperficial masseter. One of the main branches divided into three terminal branchesintramuscularly, supplying the anterior deep, and anterior and posterior superficialmuscle portions. Therefore, based upon its fetal development, it is possible that thehuman masseter muscle is divided into at least four neuromuscular compartments, each83of which could theoretically be activated differentially according to need.Studies of postnatal muscle growth indicate that the weight of the massetermuscle has a positive allometric growth in rats (Hurov et a!, 1988) and rabbits(Langenbach and Weijs, 1990), while it is isometric in primates (Cachel, 1984). Sincemasseter growth in rabbits is characterized by an overall increase of muscle length whichis much smaller than the increase in muscle weight, the predominant muscle growthlikely occurs by the increase of tendinous material and by the increase in muscle protein(actin and myosin). Increase in tendinous material was confirmed in the rat massetermuscle, in which tendon cross-sectional areas showed an isometric growth patternrelative to that of the muscle weight (Hurov et a!, 1988). While the relative size of thesurface area of the aponeurosis in the pig masseter remained unchanged during growth,the thickness increased disproportionally faster than the muscle mass growth (Richterand Herring, 1993). Since no clearly defined internal aponeuroses were found in the 20-week-old human masseter muscles, it seems that the connective tissue pattern presentat this fetal stage evolves into tendinous sheets, in a similar fashion to the developmentof the muscle in rats and pigs. The formation of a dense collagen network would bestimulated by functional demand, increasing predominantly in thickness rather than inlength. This proposition is partially supported by Schumacher’s (1962) findings on themasseter muscles from the neonatal to old age. In contrast, the medial pterygoid has welldeveloped internal tendinous sheets, at least as early as at 17 weeks fetal stage. Thisfinding is in accordance with Schumacher’s (1962) report that at birth this musclepresents the most complex system of aponeuroses of all masticatory muscles.84Based on the literature and on the present findings, it appears that in humansmuscle fibres are arranged in a complex, pennate pattern from the early fetal stages, andthat they may differ between individuals. Muscle fibres seem to be initially attached atboth ends to loose connective tissue; this tissue presumably differentiates into tendinouslayers as functional demands increase. Since it is known that in pigs the proportion ofmuscle length and aponeurosis length remains constant during growth (Richter andHerring, 1993), and that changes in masseter orientation in rabbits (Langenbach, 1992)and humans (Burdi and Spyropoulos, 1978) are minimal, it is further suggested that thearrangement of muscle fibre in the human masseter does not change drastically withgrowth. Thus, despite some changes in the pennation angles with the increase in musclefibre length and jaw growth, the pennation pattern probably remains fairly unchangedthroughout life.In animal experiments, muscle fibres belonging to a single motor unit (MU)can be quite easily followed through several histological sections if the fibres have beenglycogen-depleted by stimulating their functionally-isolated axons (Herring et a!, 1989;Ounjian et a!, 1991). Two-dimensional outlines of the MU fibres can then be digitizedat a number of levels along the longitudinal axis of the muscle for subsequent 3Dreconstruction (Ounjian et a!, 1991). The glycogen-depletion method cannot, however,be employed in human experimentation, and it is clear that alternative techniques offollowing nerve pathways and their associated muscle fibres in human material have notyet been fully developed. Furthermore, as shown in this study, it is difficult to follow MUfibre contours in different histological sections of fetal human muscles, and very difficult85to classify their orientation reliably and objectively. The muscle fibres are relativelyimmature, compounding the problem of analysis, and serial cross-sections of the wholefetal muscle offer a poor basis for muscle reconstruction, because the microtome knifefrequently intersects the fibres at odd angles, and fibre architecture can be extremelydifficult to interpret. It is possible that careful dissection of fetal muscles may prove tobe a better way to indicate fibre placement and muscle subdivisions.In summary, the identification of peripheral nerve pathways and of complexmuscle fibre arrangements suggest that the fetal masseter muscle can be divided intofour neuromuscular compartments, which present fairly developed pennation patterns.Since major structural boundaries were not found in early fetal life, and themeasurement of fibre angulation proved difficult, it was not possible to compare regionalorganization of the human fetus directly with the adult counterpart.863.12 Adult Masseter AnatomyThe performance of a muscle is determined by its internal architecture(Edgerton, Roy and Apor, 1986), which is characterized by fibre length and angle andthe division of muscle mass by tendons (Bodine et a!, 1982). Muscle fibre and tendonlengths should be considered separately, in a functioning muscle, for tendon compliancecan affect internal mechanics and force output (Zajac, 1989).Although the architectural design and the biomechanical properties of askeletal muscle are highly correlated with its physiological characteristics, little is knownabout how the architectural features affect the physiological properties of motor units(Edgerton et a!, 1987; Gans, 1982; Muhl, 1982; Ounjian et a!, 1991). Numerous reportson muscle fibre lengths indicate that these may vary among and within muscles, withinas well as across species (Huber, 1916; Sacks and Roy, 1982; Loeb et a!, 1987;Schumacher, 1961c; Van Eijden and Raadsheer, 1992). Huber (1916) reported that inthe rabbit gastrocnemius, a pennate muscle, the majority of the muscle fibres extendedbetween tendinous insertions, on both sides. In contrast, it has been demonstrated thatin long, parallel-fibred muscles, fibres may terminate in the middle of a fascicle and maytaper at either their proximal or distal end, or both (Loeb et a!, 1987). Ounjian et a!(1991) also showed that within the cat tibialis anterior muscle, single MUs of the fasttype did not extend the entire distance between musculotendinous planes of origin andinsertion. Their fibres had slightly different lengths, although in some cases where fibresshared an origin at one end of a fascicle, they tended to be of similar lengths.Intrafascicular fibre endings were tapered. In contrast, fibres from slow units extended87the entire distance between both musculotendinous planes, and terminated at the tendonas blunt endings (Ounjian et a!, 1991). Yet another type of ending (jartia1 tapering) wasfound for slow units in the tibialis anterior, indicating another mode for MU force relayto tendon (Eldred et a!, 1993). Finally, despite the fact that fibres had varied lengths,subgroups of fibres of a unit tended to end at about the same level whether the endingswere midfascicular or at a tendon (Ounjian et a!, 1991). Although muscle fibrearchitecture is important in the transfer of muscle force to tendon and bone, especiallyin a powerful muscle like the masseter, the fibre orientation, MU architecture andlocation relative to tendons and type of fibre endings are not known for this muscle.The aims of the present study were to determine the muscles’ innervationpattern relative to muscle portions, whether the muscle could be classified intoanatomically discrete compartments on which physiological analyses could be performedin living subjects, and to identify the type of fibre endings present in the humanmasseter. These data could also be used in the future as an anatomical basis formodelling contraction mechanics.3.12.1 Materiais and Methods3.12.1.1 Gross Anatomical DissectionSeven adult masseter muscles (six male and one female) were obtained fromthe Department of Anatomy, University of British Columbia. Most of the muscles wereobtained from partially-dentate cadavers. One muscle was obtained from a fully-dentate,while another from an edentulous cadaver. Ages varied between 50 and 70 years. The88muscles were excised from their attachments by separating the periosteum from themandibular ramus and by cutting through the origin tendons at the zygoma and throughthe interdigitating fibres of the temporalis. All muscles were fixed by immersion in 10%formalin. Three specimens were then placed in 20% nitric acid for 24 hours and washedin tap water before dissection. The nitric acid treatment was used to facilitate dissectionby loosening connective tissues and staining muscle fibres yellow while leaving nervesand connective tissues white. From an additional masseter, small blocks of muscle wereexcised from representative areas, and embedded in paraffin. Twenty j.m transversesections were sampled serially every 100 j.m through the entire length of each block.Alternate sections were stained for muscle fibres and connective tissue with haematoxylinand eosin and picrosirius collagen stain. The histological sections were used as an aidfor the conventional dissection in order to clarify regional muscle structure. Anadditional three muscles were washed in running tap water for 24 hours and embeddedin alginate. The muscles were oriented within the alginate block, so that the superior andanterior borders were roughly aligned with the edges of the block. This block was thensliced into 3 nun sections in the frontal plane. Photographs were taken from thedissections and from each muscle slice. Chemical DissectionTo evaluate muscle fibre endings, one adult human masseter muscle waschemically dissected with 30% nitric acid, which was gradually replaced with glycerol(Loeb and Gans, 1986). The masseter was dissected out with the origins and insertions89of all fibres included, it was placed into the centre of a dish, and then 30% nitric acidin saline poured over it. The dish was covered and the specimen was initially checkedevery hour for the first 8 hours. Thereafter the specimen was checked every 12 hours.After 4 days, fibres began to fall apart of their own accord, separating into sets offascicles that were easily moved about. The nitric acid was then replaced with a 50%glycerin/30% nitric acid mixture for two days, to slow down the digestion. After this, themixture was finally replaced by 50% glycerin in water, to arrest further breakdown. Oncethe major portions had been separated, fascicles were gently transferred into a Petri dish,and portions of the specimen were removed until only a few undamaged fibres remained.This process was checked under a dissecting microscope. Selected, undamaged fibreswere then gently floated onto a standard microscope slide onto which a coverslip wasmounted with glycerin and sealed with nail polish. The fibres were then examined undera light microscope.3.122 ResultsIn one specimen, the nerve trunk divided into three primary branches, whilein the other specimens it divided into only two. The superior and middle primarybranches in the first specimen, and the superior primary branch in the other specimens,innervated the deep masseter region. They entered the muscle through the mandibularnotch just anterior to the condylar region, and extended anteriorly running parallel tothe zygomatic arch, just medially to Aponeurosis IV. The inferior primary branch in allspecimens appeared to be the continuation of the main trunk and started fairly parallel90to the other primary branches. It initially divided into two intramuscular branches, theoffspring continuing to run parallel to the two primary branches, innervating the deepmasseter muscle. The main trunk continued its path laterally, running obliquely from thesupero-posterior corner towards the anterior, superficial masseter. Several smallerintramuscular branches spread off the main trunk, innervating the intermediate masseter.The nerve distribution supplied the more lateral regions by running along theaponeuroses and crossing through the muscular layers. The “Nervenknoten” describedby Lau (1972) was observed in all specimens.Most muscle fibres and aponeuroses extended into a thick periosteum layerwhich covered a wide area of the mandibular angle and ramus. This periosteum layerextended approximately three-quarters of the length and three-quarters of the height ofthe muscle, measured from the mandibular angle. Other muscle fibres and tendonsinserted into a thinner periosteum layer (anterior region), and directly into bone (deepposterior masseter portion).The findings from the dissections performed for this thesis (Figure 10) areconsistent with Gaspard et al’s (1973) and Baron and Debussy’s (1979) reports. Themasseter was found to be formed by four incompletely separate muscle sheets: massetersuperficialis (lamina prima and lamina secunda), masseter intermedius and masseterprofundus (pars anterior and pars posterior). The simple, parallel-fibred anterior portiondescribed by Ebert (1939) was also identified in all specimens, being most prominent inthe edentulous individual.It has been observed in this study, as well as by others, that the deepest part91Figure 10 Masseter-muscie dissection showing the superficial and intermediatemuscle layers. Parasagittal view of the lateral side of a left adult human masseter.Anterior is to the left, and superior is to the top. The laniina prima (left) and laminasecunda (right) of the Masseter superficialis are shown reflected. The Masseterintermedius is shown to the right, below the lamina secunda of the Masseter superficialis.92Figure 11 Masseter-muscie dissectionshowingits layered arrangementposteriorlyandthe ascending attachment sites of muscle fibres as the layers become deeper. Parasagittalview of the lateral side of a left adult human masseter. Anterior is to the left andsuperior is to the top. Masseter superficialis and Masseter intermedius are reflected.Masseter profundus fibres are seen at the top right, including their aponeuroticattachment, which is somewhat like that of the temporalis muscle.93of the masseter is often inseparably fused with the most superficial fibres of the temporalmuscle. This group of muscle fibres has been previously described as a separate muscleunit, termed the zygomaticomandibular muscle. It has been reported that identifying thedistinction of this muscle unit in many animals is easier than in man (Sicher, 1960; Lau,1972). In the present study, a separate portion of the deep masseter, situated mediallyto Aponeurosis V (Figure 12), originated from the lower border and medial surface ofthe zygomatic arch and inserted into the basal part of the coronoid process and theadjacent parts of the mandibular ramus. This deep masseter portion could also bedescribed as the zygomaticomandibular muscle. The fibres of both the deep masseterand of the zygomaticomandibular muscle were arranged in a fan shape, resembling thetemporalis when viewed laterally (Figure 11).Initially, an attempt was made to identify the intramuscular aponeurosesaccording to Schumacher’s (1961c) classification. Although the masseters all hadnumerous aponeuroses, they were not continuous through the muscles, and did notdivide the muscles into distinct compartments. Interleaving aponeuroses started eitherat the muscle origin or at the insertion, extending into the muscle mass downwards orupwards. In most cases, during dissection, aponeuroses started near bone with thickcross-sectional area, tapering towards the other extremity. Numerous differences werefound between subjects in terms of quantity, thickness and distribution of aponeuroses,and it was not possible to identify Schumacher’s aponeuroses in all specimens. Themasseter from the female edentulous cadaver had the smallest cross-sectional size of thesample, and the smallest number of aponeuroses, which were thicker than these of other94._IIII’;If4,:17Figure12Masseter-musciecross-sectionshowingitsinternalaponeuroses.Thelevelofcross-sectionisapproximatelyhalfwaythroughthemuscleinbothfigures.(a)Inthisspecimen,fiveaponeurosessimilartothosedescribedbySchumacher(1961c)canbedepicted.Here,superficial(betweenApIandII),intermediate(betweenApIIandifi),anddeepmasseterscanbeseen.VascularizationandinnervationareparalleltothemedialaspectofAponeurosisIV(*).(b)Inthisspecimen,aponeurosesII,IVandVcanbeseen.Additionaltendonsheetsarealsopresent.Inbothfigures,themainaponeurosesareindicatedbynumbers.Medialistotheleft.UiC71Z*:I—4Figure 13 Masseter-muscie fibre endings. Both, blunt (A) and partial tapered (B)fibre endings are found. A group of fibres were intentionally damaged, and their endsshown for comparison (C). The calibration bar represents 25 j&m.C—96specimens from partially dentate individuals. In this case, only three aponeuroses wereidentified, namely Aponeurosis I, II and III. One partially-dentate individual had thelargest number of aponeuroses. These were thin, and in many cases would disappear inthe next 3 mm slice, or after two slices. Basically, this muscle possessed all fiveaponeuroses as described by Schumacher (1961c), and a few other tendon sheets situatedpredominantely in the posterior two thirds of the muscle (Figure 12B). The largestmuscle of all was found in the fully dentate individual. This muscle showed the thickestaponeuroses, and was almost identical to Schumacher’s (1961c) description. The onlyexception was Aponeurosis V, which inserted into the mandibular ramus, rather thanoriginating from the zygomatic arch. No additional aponeurosis was found in this case(Figure 12A). The aponeuroses were qualitatively longer in the superficial muscle regionthan in the deep muscle region. A slight antero-posterior size difference was also noted.In some cases, the aponeuroses presented an antero-posterior “zig-zagged” edge withinthe muscle mass. This pattern was predominantly found in Aponeurosis I, and has beenillustrated before by Ebert (1939), Schumacher (1961c) and Lam (1991). Although theywere not measured, relative tendon length to muscle size did not seem to vary betweenindividual specimens.Chemical dissection revealed that fibres from the masseter muscle had twotypes of endings, blunt and partially tapered (Figure 13). The fibres could be divided intothree groups. In one group fibres had both ends tapered, in another, fibres had bluntends at both poles, and in the third group, fibres were blunt at one end, and tapered atthe other end.973.12.3 DiscussionThe present description of the masseteric nerve agrees with that illustrated byLau (1972) and Xiguang et a! (1986). The general pattern of the first primary branchsupplying the deep masseter and the main branch innervating the entire muscle whilerunning in a lateral, forwards and downwards direction is similar to that found in pig(Herring et a!, 1989), sheep and dog (Lau, 1972). As previously discussed by Herring eta! (1989), although the nerve trunks may be common to several muscles, and in this casemay be common to several muscle anatomical compartments, individual axons areprobably not, innervating motor unit territories which may be confined to tendinousboundaries.The results of the present study, showing that tendon lengths vary with muscleregion, are in accordance with Van Eijden and Raadsheer’s (1992) report. The inter-individual variation in number of aponeuroses might be explained by differences infunctional demands on the masseter between different individuals. This notion would beconsistent with Schumacher’s (1962) report on increased internal tendinous complexitywith increased functional demand. This individual variance in aponeurotic design mayalso account for the lack of consistency in the description of the masseter in theliterature.Eldred et a! (1993) observed that even fibres which had blunt ends at bothpoles were not strictly uniform in calibre throughout their lengths, and had their shapedescribed as a tenuous spindle with truncated ends. In these MUs, the mean values forareas along the poles were 16% smaller than areas along the central segment. The98functional consequence of this finding is that along an individual fibre of varying calibrethe production of force should be greatest where the cross-sectional area is maximal, andalong a tapering termination the capacity for production of force decreases until it isfinally lost at the fibre tip. A concomitant premise is that along each of these fibres, theforce production is larger at the fibre’s wide segment than at its thinner endings. Theexcess of force produced at the wide segment must therefore be transmitted to the fibresurroundings, otherwise it may stretch the tapered fibre terminals (Street, 1983).During the contraction of a MU, the produced force is transferred to thetendon via the connective tissue (Schmalbruch, 1974; Swatland, 1975; Rowe, 1981) andneighbouring, passive muscle fibres (Demiêville and Partridge, 1980). If we assume thatin the masseter muscle all muscle fibres end on tendons, most of the unit’s force shouldbe directly transferred to the tendon via the specialized surface features (Tidball andDaniel, 1986) of the blunt type of ending. However, in the case of fibres with thepartially tapered ends, force should be still relayed via the surrounding tissue (Street,1983). In the masseter, where both blunt and partially tapered fibres apparently projectonto a tendinous insertion, both types of force transfer, direct and indirect, can beexpected.In an analysis of three-dimensional tendon plane orientation, Lam et a! (1991)suggested that muscle fibre orientations between tendon planes may not have equalpennation angles at their proximal and distal ends, since the tendons are not parallel toeach other. It is possible that the partially tapered muscle fibre endings observed in thepresent study play a role in compensating for the non-parallelism between aponeuroses,99ensuring that the angle of pennation is equivalent on either side of the fibre, and/or oneither side of the resultant force applied to the tendon.1003.1.3 Morphological Reconstruction in living SubjectsTo study muscle biomechanics and function relative to internal architecture,anatomical and physiological investigations of muscle structures should ideally beperformed in living subjects. This obviates sole reliance on gross anatomical andhistological studies of cadaver material, which may have different dispositions of internalstructures. In addition, non-invasive methods enable the evaluation of muscle mechanicsduring natural tasks. For this purpose, Magnetic Resonance (MR) imaging offers asignificant tool in the visualization of internal muscle architecture, and efforts were madeto determine whether it could be applied to the human masseter. Methods3. Magnetic-Resonance ImagingBecause of the importance of MR imaging in living-subject studies, a briefreview on the technique will be presented. Proton MR imaging is a noninvasiveresonance measurement technique, which utilizes a high strength static magnetic field,pulsed radiowaves, and switched gradient magnetic fields to generate images of bothhard and soft tissues (Lam et al, 1989). This imaging system probes the nuclear magneticproperties of the hydrogen atom to generate the MR signal (Selzer, 1986; Nixon, 1987).A hydrogen atom has a nucleus consisting of one proton. This has a minuscule amountof intrinsic magnetism, which is proportional to another intrinsic entity of elementaryparticles called “spin”. In this case, spin does not represent a physical rotation, but itmeans that a proton may be likened to a tiny bar magnet (Young, 1984). These magnetic101dipoles are randomly oriented within a tissue (Harms and Kramer, 1985). Magnets usedin MR imaging possess stationary magnetic field strengths from 0.15-2.25 Tesla. Whena tissue is placed into one of these magnetic fields, the randomly oriented magneticdipoles respond to the force of the field by trying to orient parallel to it. Under theseconditions, each proton begins to precess at the same rate, but at a random phase.Together these individual magnetic moments create a net magnetic moment, pointingalong the axis of the external magnetic field. Once this is achieved, a radiofrequency (rf)field is activated, acting in a direction perpendicular to the main field. When the rf is inphase with the precessing spins, it induces rotation of the net magnetic moment by 90degrees. This transition into transverse magnetization is called resonance, where thenuclear magnets tend to spin inphase with one another. Without the creation ofresonance there would be no detectable signal to create an image. After the if field hasbeen turned off the magnetic moment can induce voltage in the receiver coil, situatedin the transverse plane. Once the rf field is removed the transverse magnetizationgradually decays; the resulting loss of signal intensity as a function of time is known asa “free induction decay” (Fm). The processes involved with signal loss are generallycalled “relaxation”. Spin-lattice relaxation or “T1” refers to the return of the nuclearmagnets to their original alignment with the static magnetic field. Spin-spin relaxationor “T21’ refers to the loss of the inphase spinning of the nuclear magnets (Harms andKramer, 1985; Fullerton, 1982). Because the initial signal amplitude is proportional tothe transverse magnetization, which is itself proportional to the number of nuclei excitedin a particular voxel of tissue, differences in hydrogen density become discernible in the102MR image.Contrast between areas of-differing-protondensity can be enhanced if a MRscan is biased towards T1 or T2 characteristics. This effect can be achieved by using ifpulse sequences such as “inversion recovery” or “spin-echo”; the latter being the mostversatile sequence producing an image biased towards T2 (Thornton, 1987). The contrastin a spin-echo image is determined by the relative decay of MR signal emitted fromdifferent tissues at the time of sampling. Signal intensities of musculoskeletal tissues ata given time vary considerably, being of highest intensity for muscle and medullary bone,of intermediate intensity for fat and of low intensity for blood vessels, tendons andcortical bone (Thornton, 1987; Berquest, 1987; Scholz et at, 1989).Materials which contain significant amounts of iron, cobalt, nickel, andchromium may exhibit ferromagnetic properties due to their high magneticpermeabilities (Lam et a!, 1989). These materials are commonly used in metallicimplants, dental fillings, appliances and surgical prosthesis, producing artifacts on MRimages, which usually appear as low-intensity, black regions (Lam et a!, 1989; Fache eta!, 1987; Seltzer and Wang, 1987). ImagingSix subjects were imaged; muscles and internal tendons were traced, digitizedand reconstructed according to the method described below. The muscle reconstructionswere then used for subsequent studies in this thesis.To permit merging the MR images of the orofacial region with other datasets103in future experiments, an L-shaped fiducial reference grid, consisting of thermoplasticmaterial filled with 5 mM copper sulphate at each corner, was attached rigidly to aplastic eyeglass frame, so that it was positioned just posterior to the right massetermuscle. This concentration of aqueous copper-sulphate solution was chosen from animaging trial, in which several different concentrations were tested. The 5 mM solutionyielded the best image of the reference grid, that is it yielded high-intensity signal withthe smallest amount of “blur”. Silicone liners were used to custom-fit the frame to thenose and ears for stability and reproducibility of placement (Figure 14). Each subjectwore this device during imaging, and later, during EMG recording sessions.A MR 1.5-Tesla system (Signa; General Electric Medical Systems, Milwaukee,WI) was used to obtain conventional T2-weighted images of the muscle. Each subjectwas asked to lie in a supine position on the unit’s gantry bed, and a 5 cm surfacereceiving-coil was placed over the grid and the masseter muscle. The subject’s head wasthen aligned with the Frankfort horizontal plane at a right angle to the gantry’s bed withthe aid of a laser-light referencing system. During imaging, each subject was asked tobite lightly at the intercuspal position, while a series of contiguous, 3 mm sections in thecoronal plane were obtained. This plane was chosen according to previousrecommendations by Lam (1991) and Koolstra et a! (1990).Signal averaging hinders tendon plane visualization in the sagittal plane, anddoes not permit the differentiation between the deep masseter and the temporalismuscle in the axial plane. Therefore, coronal sectioning was carried out to resolve theinternal architecture of the masseter muscle optimally. To obtain the best MR signal104Figure 14 Reference L-shaped grid attached to a plastic eyeglass frame. Right lateralview showing the eyeglass lenses (right) and the sfficone-lined earpieces (left). Thereference grid is built of thermoplastic material and filled with copper-sulphate, whichresonates during imaging.105Figure 15 Coronal spin-echo Magnetic Resonance imige. The masseter muscle (M),internal tendons (thin arrows), mandibular ramus (R), and reference marker (arrow) aredepicted. The image was obtained with a 5 cm surface coil (TR: 2000 ms, TE: 25 ms).The scale bar is 5 cm.106contrast between muscle and tendon, without the interference of adipose tissue, spin-echo sequences with a repetition time (TR) of 2000 ms and with echo times (TB) of 25and 80 ms were used. Two excitations were utilized with a 12 cm field of view and a 256x 192 matrix (Figure 15). With this pulse sequence, the external reference markerproduced the highest intensity signal, seen in the image as white structures. In contrast,cortical bone and the internal tendons produced low-intensity signals, appearing blackagainst the intermediate light-grey image of the muscular tissue. Although higher tissuedifferentiation is expected from longer TB images, higher contrast between tissue wasobtained from the 25 ms TB, which produced a more anatomically defined image. Hardcopies of all coronal sections were then produced, and a 5 cm scale bar was included oneach image. ReconstructionSectional outlines of the muscle, its internal tendons, bone and the coppersulphate markers were traced onto acetate overlays and digitized, (Model HP9874ADigitizer and HP350 computer, Hewlett Packard Canada, Vancouver, B.C.). Calibrationbars on each section were used to scale and reference these sections to a common origin.Profile coordinates at every 0.3 mm were stored, and each section was assigned itsappropriate coronal depth (Hannam and Wood, 1989). From the individual proffle data,50 equally-spaced points were selected to describe the boundary of each tissue outline.This number of points has previously been found to be adequate in rendering satisfactoryrepresentation of complex shapes such as intramuscular tendon contours (Lam, 1991).107Three-dimensional reconstruction of the data was then carried out with anengineering solid-object modelling package (1-DEAS version 6.0, SDRC, Milford, Ohio)and a graphics workstation (HP Turbo SRX, Hewlett-Packard, Canada). Each group of50 profile coordinates was transformed into closed third-order splines6, forming a“wireframe7”(Figure 16). Several contiguous planar outlines were then “skinned” by theconnection between corresponding points of adjacent profiles to create 3D surfaces orsolids (Figure 17). Since the equally-spaced 50 points in each profile had been arrangedin a clock-wise sequence starting from the top-most position, minimal surface distortion(twist) was observed in the final objects. Accentuated twists were only found in thecondylar region of the mandibular reconstruction of two subjects, due to the suddenchange in shape from one contiguous profile to the next. Similar distortions have beenalso previously reported in solid models constructed using different approaches (Sinclairet a!, 1989; Korioth, 1992). To improve one of the reconstructions of the mandible, whichpresented accentuated twists in the condylar area, an alternative approach was tried. Thismethod treated the outlines’ raw data as a matrix, consisting of 50 points per outlinetimes the number of contiguous sections of the given tissue. The matrix was then fittedwith a surface skin by interpolation (Korioth, 1992). However this did not render betterresults; on the contrary, the twists were even more accentuated. Since little or no twistwas present at the areas of interest; that is, at the tendons and at the area of attachmentA spline is a closed or open curve whose shape is defined by a higher orderpolynomial.‘ A wireframe is a collection of points and curves.108Figure 16 Antero-lateral view of the sectional outlines of a reference grid and rightmasseter musde with its internal tendons. Masseter muscle contours are plotted withstippled lines. These outlines are closed third-order splines, which form a “wireframe”in IDEASTM.1 \‘IIII I1 II II,I IIII IIIiiII(\109Figure 17 Three-dimensional reconstruction of the reference grid, masseter muscleand internal tendons. Antero-lateral view of the solid objects in 1-DEAS.110of the masseter into the mandibular ramus no further alternative reconstructiontechnique was pursued. Error of MethodTo determine geometrical differences between the original structures and thereconstructed model, three linear dimensions, and an angle of the 11true” L-shapedreference grid, were compared against their modelled counterparts. Spatial coordinatesof the centroids on the lateral surfaces of the fiducial-grid-corner cylinders were recordedwith an optical three-dimensional measuring system (Reflex Metrograph, HF Ross,Salisbury, Wilts, U.K.). Linear distances between the centroids and the angle betweentwo of these lines were then calculated (Figure 18A). In addition, the thickness of thecylinders were measured with calipers (Figure 18B). Distances were then comparedagainst measurements taken from homologous locations on the grid’s reconstructedmodel. Measurements were made for each custom L-shaped fiducial grid. The linear andangular values for both “true” and “MR-derived reconstructions” of the grids are shownin Table II. The error is defined as the length or angular difference, expressed as apercentage of the true value. From this table, most cases had estimated errors below 3%,with the exception of one case along the vertical bar (7.3%), and two cases along thehorizontal bar (19.6% and 3.5%).3.1.32 ResultsIn all subjects, a high-intensity signal (white) was observed running diagonally111Figure 18 linear and angular measures used to compare the reconstructed againstthe true reference grid. (A) Sagittal view of two linear distances measured between thecentroids on the lateral surface of the L-shaped grid cylinders, representing thehorizontal (H) and the vertical (V) bar dimensions. Between the intersecting lines theangle (a) is calculated. (B) represents the thickness of the corner cylinder, which ismeasured along the medio-lateral direction.cEb112Table II linear and angular dimensions measured from homologous points on thereference grids of six subjects and their reconstructed counterparts. The distances aregiven in millimetres and the angles in degrees. Differences are expressed in percentagesrelative to the true grids. Representation of the linear and angular measures are shownin Figure 18.SUBJECTS ANGLE VERflCAL BAR HORIZONTAL BAR THICKNESSTrue MRI Error True MRI Error True MRI Error True MRI Error1Mt D,tJ Il-DEASI (%I IMdro 0.5.1 fl-DEASJ L%J IM,tro 0.5.1 11-DEASI (%J IC.Iip..,1 Il-DEAS) (%J1 90.35 91.57 -1.35 54.37 53.15 2.24 24.30 24.37 -0.29 7.65 7.76 .1.442 91.01 90.00 1.11 59.08 54.78 7.28 22.91 18.43 19.55 6.55 6.42 1.983 90.15 90.22 -0.07 54.74 53.75 1.81 23.03 22.69 1.50 6.90 7.00 -1.454 87.36 87.35 0.01 52.61 52.04 1.08 25.87 24.96 3.52 7.90 7.89 0.135 88.58 88.55 0.04 56.11 54.63 2.63 26.06 25.52 2.06 6.90 7.06 -2.326 90.50 90.26 0.26 54.70 54.36 0.61 23.23 23.16 0.32 8.06 8.01 0.50113from the lateral aspect of the muscle to the mandibular ramus, separating the superficialportion from the deep posterior portion of the masseter (Figure 19A). This is the imageof the connective-tissue layer, which surrounds nerves and blood vessels (seen on theimage slice, close to the mandibular ramus, as small dark structures) which is presentinside the posterior muscle “pocket”, frequently cited in anatomical reports of themasseter. The delineation between the masseter and temporal muscles was arbitrary inall cases, since no space nor tendon exists between these two muscles. In reality theyform an anatomic unit (Sicher, 1960).In all imaged individuals, the five aponeuroses described by Schumacher(1961c) were present. In three individuals, the muscle structure was simple, with only afew small additional tendinous layers. Examples of two individuals are shown in Figure15 and Figure 19A. However, in the remainder of the subjects, internal architecture wasmore complex. In these, the basic five aponeuroses could be depicted, but severaladditional layers added to the internal architectural complexity. Figure 19B shows anMR coronal section in one of these individuals, while Figure 20 shows the entire musclereconstruction of another subject.Similar to the findings from the muscle dissections, aponeuroses thickness andnumber varied according to subject. The three subjects having the largest masseter crosssectional sizes presented an increased number of aponeuroses, which were thicker thanin other individuals. The subject with the narrowest muscle, however, had slightly thickeraponeuroses than another subject with also small muscle cross-sectional size.114Figure19MRcoronalsectionsoftwosubjectsshowingdifferencesininternalmusclearchitecture.(a)Thisimagesliceshowstheconnectivetissueseparatingthesuperficialanddeepmasseterportions,bloodvessels(*),andAponeurosesI,II,ifi,IVandV.(b)Imagefromasecondsubjectwithamorecomplexinternalmusclearchitecture.AponeurosesI,II,ifi,IVandVareseenwithadditionalothers.Calibrationbarsrepresent5cm.(R)Mandibularramus;(Z)Zygomaticarch.ISI.S.4—vFigure 20 Computer reconstruction of interstitial tissues in the right masseter muscle,viewed frontally. Frontal MR-images were digitized, stacked, and “skinned” to representsolid objects. Frontal software cuts divided the muscle and mandibular ramus andzygoma in three segments, shown here separately. (A) Anterior; (B) central; and (C)posterior segments. On the left side of each picture, the tissues are seen from the front,while on the right side they are seen from the back. As in the anatomical specimen(Figure 12B), the classical aponeuroses [labelled according to Schumacher’s (1961c)definition] can be seen together with additional ones. In (A), Aponeuroses I and IIappear to be continuous when seen from the front. Note that when seen from the back,it is clear that they are separate structures.1163.1.3.3 DiscussionIn vitro studies have shown that 12 relaxation times depend on collagen fibresorientation in the magnetic field. When the fibres are oriented at 00, 90° and 180° inrelation to the constant magnetic induction field, the echo nearly disappears, primarilydue to the structural anisotropy of collagen (Berendsen, 1962; Migchelsen andBerendsen, 1973). An increase in tendinous signal intensity is however observed at the“magic angle” of 550 (Fullerton et al, 1985). In the masseter muscle, the tendons arealigned along the long axis of the body, thus are oriented at small angles relative to themagnet. Fullerton et a! (1985) argued that in such cases, tendons would be rarelyobserved in clinical MR imaging, on the one hand because the “magic angle” would beseldom encountered, and on the other, due to the relative long TEs which are usedclinically. Therefore, there should be a lack of echogeneity from the tendons, whichshould appear black in all spin-echo images, yielding high contrast against the proton-rich background of the surrounding muscle. More recent reports have shown however,that although angle-dependent signal intensity should be most prominent at short TEsof 10 msec, it is still sometimes observed at TEs of 25 msec. This is in part due to theimproved signal-to-noise ratio of stronger (1.5T) magnets, partly because parts oftendons may be oriented at 55° in relation to the magnetic field and partly because ofthe blending of the tendon with muscle fibres at the musculotendinous insertion(Erickson et a!, 1991). It is assumed that the “magic angle” effect is quite rare duringimaging of the masseter, due to the 15° angulation (Schumacher, 1961c) of the internaltendons relative to the medial plane. Furthermore, the internal anatomy of our117reconstructions is consistent with anatomical reports.In the creation of a MR image, the magnetic field is mapped in such a mannerthat it becomes position-dependent. Signals from contiguous, predefined tissue volumes(voxels) are detected simultaneously, and then converted into a two-dimensional (pixel)image. Since spatial accuracy is essential for most imaging purposes, MR systems havebeen previously examined for inherent non-linearities caused by inhomogeneities in thestatic magnetic and radiofrequency fields. It has been established that in a large volumeat the center of the magnet coil, no distortions occur, producing linear images (Stewart,1987). In addition, the use of a fixed receiver coil, previously used in the orofacial region(Seltzer and Wang, 1987; van Spronsen et a!, 1987; Hannam and Wood, 1989; Koolstraet a!, 1990; Schellhas, 1989) produces signals of even distribution throughout an entireslice. The signal-to-noise ratio of these signals are further improved with a surface coiland the number of repetitive signal acquisitions. The latter, the spatial resolution alongone axis of the specimen, and the TR determine the length of imaging time (Lam, 1991).Since it is required that subjects remain motionless during the imaging sessions to avoidmovement artifacts, the need to keep imaging time short becomes evident. Therefore,the image matrix was reduced to 256 x 192 and only two signal averages were chosenthroughout image acquisition. The pixel dimension of the images obtained can bededuced by dividing the field of view by the number of pixels in the matrix (Price et a!,1992). In the present study, the resolution of the plane of image was 0.5 x 0.6 mm, witha slice thickness of 3 mm. While it is possible to obtain images with thinner slices,disadvantages include reduction in the signal-to-noise ratio, and requirement of greater118acquisition time.Three-dimensional morphological reconstructions are commonly achieved fromtwo-dimensional planar images (Hannam and Wood, 1989; Koolstra et at, 1990; Lam,1991; Korioth, 1992). The fidelity of these reconstructions depend on minimum headmovement during image acquisition, slice thickness and tracing accuracy (Koolstra et a!,1990). Minor head movements from breathing and swallowing are common during theimage acquisition process, and they may distort or blur tissue boundaries (Lam, 1991).Slice thickness may affect the images similarly, especially where the amount of a giventissue for signal averaging is minimal; for example at musculotendinous junctions.Additionally, slice thickness is of great concern when a surface skin between sectional,planar contours is to be produced. Here, the problem of approximating surfaces betweensuccessive slice contours is greatly reduced when their separation approaches zero,particularly when adjacent contours are irregular or dissimilar in shape (Sinclair et at,1989). Lam (1991) expressed some concern regarding the surface generation betweenadjacent tendon contours obtained from 5 mm MR images. Surface formation fromthinner, 3 mm MR slices have, however, greatly improved. As previously discussed byKoolstra et at (1990) and Lam (1991), muscle outlines were not always clearlydistinguishable. The interdigitation of fibres from the deep masseter and temporalismuscles does not permit the boundary between these two muscles to be sharply defined.In addition, a low-intensity signal depicting the masseter fascia, which is tightly attachedto the superficial tendon of the masseter (Sicher, 1960), made the distinction in certainregions between fascia and the superficial-masseter Aponeurosis I difficult.119It is also possible to generate volumetric images. That is, from such a data set,transverse, sagittal and coronal images across any point within the imaged volume canbe displayed (Chakeres et a!, 1992). Usually the MR information is directly transferredinto computer-reconstruction programs avoiding the need for tracing and digitizing.Software can be written to assign boundaries automatically between tissues, by detectingdifferences in the grey-shade scale. This approach has produced quite successful resultsin the reconstruction of bony structures. However, in the case of the masseteric region,operator interpretation of the obtained image is still necessary, since muscle tissueboundaries are less well-defined.The quantification of the error between a complex three-dimensional form andits reconstruction is not trivial, since it is difficult to obtain homologous points in bothstructures. The criterion for homology is that points must be consistently and reliablylocated with a measurable degree of accuracy on both considered forms (Lele andRichtsmeier, 1991). Form of an object involves both size and shape. Landmark andoutline data are two types of data commonly used to compare distinct forms (Lele,1991). With this purpose, several different morphometric analyses have been developed,which involve rigorous statistical methodologies and which are based on the descriptionof outline or landmark data in two- (Rohlf and Bookstein, 1990) or three dimensions(Lele, 1991; Lele and Richtsmeier, 1991).Since landmarks on the biological object were non-existent and a series ofmeasurements made up of external dimensions such as maximum breadth and length ofthe structure proved to be difficult, homologous points on the external reference grid in120this study, were used to assess the error between the “true” object and its reconstruction.In this assessment, any errors which occurred during all steps of the methodology werecombined.The relatively large errors found in the horizontal-bar reconstructions, that isin the antero-posterior direction, may be explained as the result of a combination of twofactors: the image-plane of section, and the averaging process by which the MR sliceswere obtained. Theoretically, a well-defined line should be pictured at the interface ofthe high-intensity signal from the copper-sulphate-filled cylinder against the darkbackground. This should be observed medio-laterally with a resolution of 0.5 mm (asdiscussed above) and supero-inferiorly with a resolution of 0.6 mm, in the coronal planeof section. This decreased resolution in the supero-inferior axis explains the larger errorsfound in this dimension. In the antero-posterior direction, each slice is the result ofsignal averages through 3 mm of tissue. Let us assume that the copper-sulphate-filledcylinder ended half-way through the 3 mm depth on both sides. In this case, only half ofthe signal intensity (intermediate grey) would be depicted in the first and last imagesections in which the copper sulphate appears, when compared against an image sectionright across the grid. Despite this apparent loss in signal, the true length of the cylinderis detected. In another example, consider that the cylinder ended at only one quarter ofthe 3 mm depth, on both sides. Here, the detected signal is even weaker on bothextremes, but the sections are still included in the reconstruction. In this situation, anantero-posterior “shift” of the boundaries occurs (Figure 21). Here, the potential foroverestimating the sizes of structures is apparent, again explaining the results.121Figure 21 Schematic ifiustration ofMR data acquisition. Examples of data acquisitionfrom copper-sulphate-filled horizontal bars placed at different positions relative to theplanes of section. Contiguous 3 mm MR slices are taken in the coronal plane (illustratedby solid lines). Stippled lines represent averaged data, which form the image. Shortvertical lines (below) represent traced contours of the imaged bars viewed from thesagittal aspect. Arrows indicate the reconstructed length of the imaged object. In (a) thehorizontal bar ends half way through each 3 mm averaged section. The true dimensionof the bar is reproduced from the images. In (b) the horizontal bar ends at both sides,at one quarter of the thickness of the respective slices. Error of overestimating the bar’slength in this case is possible.a bI I I “ 4 I I I122In a study of the validation of three-dimensional reconstructions of the kneeanatomy, measurements between external reference markers and biological loci weretaken from milled slices of a cadaveric specimen, and from the MR-basedreconstructions. It was found that the accuracies of intermarker distances and of theknee-condylar dimensional measurements on the reconstructions averaged 97.5% and93% of the milled slices, respectively (Smith et al, 1989). Therefore, it is expected thatthe error of the reconstruction of the masseter muscle is also slightly larger than theerror of the reconstruction of the fiducial grid. This assumption is based on the fact thatsignal average processes are more complex on the biological tissue than on the copper-sulphate - air interface, contributing to interpretation errors during tracing.Though it may be desirable to validate muscle reconstruction in livingindividuals against their true muscle morphology, it is impractical. Validation could notbe carried out in the present study, and it was assumed that the muscle reconstructionsaccurately represented true individual muscle morphology in accordance with previousMR studies of the masseter in fresh human cadavers (Lam, 1991) and rabbits (Ralph etal, 1991), and the knowledge obtained from direct muscle dissections. Similar to thefindings in one dissected cadaver (Figure 12B), all reconstructed muscles had the basicaponeuroses described by Schumacher (1961c) as well as other smaller tendinous sheets.The internal architecture varied among individuals. Some muscles had more internaltendinous sheets than others, perhaps due to the different functional strategies used byeach individual. The findings suggest that it is important to study morphology andfunction in the same individual, and to compare the results only against matched123anatomical and physiological data obtained similarly from other subjects.12432 Movement of Masseter Insertions at different Jaw PositionsA complex system of internal aponeurotic septa separating groups of musclefibres with varying lines of action is common in many mammalian jaw muscles (Hliemaeand Houston, 1971; Herring et a!, 1979; Weijs and Dantuma, 1981; English, 1985; Weijset a!, 1987). The human masseter is typical, since it contains wide anteroposterioraponeurotic septa which extend into the muscle mass alternately from the zygomatic archand from the lateral surface of the mandible (Ebert, 1939; Schumacher, 1961c; Lam eta!, 1991). Interleaving aponeuroses enable muscle fibres to be packed into amultipennate arrangement (Gans and Bock, 1965; Herring, 1980; Wineski and Gans,1984), allowing the fibres to attach either to an insertion tendon or directly toperiosteum. As a result, masseter origin and insertion sites cover broad areas of thezygomatic arch and mandibular ramus (Schumacher, 1961c; Gaspard, 1987).To characterize the functional capabilities of a muscle, knowledge of its three-dimensional (3D) architectural design is necessary (Gans and deVree, 1987). Within thehuman masseter, 3D orientation of muscle fibres and tendons have been estimated(Baron and Debussy, 1979; Lam et a!, 1991), as well as the size and placement ofsarcomeres (Van Eijden and Raadsheer, 1992). Sarcomere length vary in separatemuscle regions and are known to be affected differentially with computer simulations ofsimplified, arbitrary jaw displacements (Van Eijden and Raadsheer, 1992). Since thedisplacement of the adult human jaw is complex, involving a combination of rotationsand translations about instantaneous centres of rotation (Grant, 1973; Brown, 1975), itis expected that the movement of the masseter insertion area is of equal complexity. It125is also expected that insertion site displacements vary according to region.The aims of the present study were to determine how masseter muscleinsertions are affected three-dimensionally by jaw movements, and to relate the findingsto regional muscle function.32.1 Simulated Function in Dry Skulls32.1.1 MethodsFourteen adult human East Indian skulls (seven female and seven male), fromthe collection of the Department of Anatomy at the University of British Columbia wereused. To describe the sample, and to reveal any differences in the craniofacialdimensions, a lateral cephalometric radiograph was taken of each specimen at a tube-film distance of 165 cm and an object-film distance of 14 cm. Thirteen selectedconventional linear and angular cephalometric variables were then measured accordingto criteria and definitions described by Lowe (1980). In conformity with hisabbreviations, the anterior position of the maxilla was defined by the angle SNA,occlusal plane by the angle SN-OP, palatal plane by the lines ANS-PNS and SN,maxillary length by the linear distance from the midcondylar point to the inferior pointof the anterior maxilla, anterior position of the mandible by the angle SNB, mandibularplane angle by the lower border of the mandible and the line SN, gonial angle by thelower border of the mandible and the posterior border of the ramus, mandibular lengthby the linear distance from the midcondylar point to pogonion,, relative mandibularprognathism by the angle ANB, maxillary-mandibular length difference calculated from126the mandibular length minus the maxillary length, upper face height by the lineardistance between nasion and a projection from ANS along a perpendicular to the nasionmenton line, and the same projection of ANS, and ramus height by the distance fromthe superior condylar point to gonion measured perpendicular to the line SN. Inaddition, transverse distances were measured directly on the skull by means of calipers.Bizygomatic width was measured as the largest distance between the zygomatic arches;intercondylar width as the distance between the lateral poles of the condyles; andbigonial width as the distance between the gonions.The masseter muscle attachment sites were represented by six origin andtwelve insertion points. The locations of these followed osteological criteria reportedpreviously (Baron and Debussy, 1979; Gaspard, 1987), but were also guided by knownmuscle morphology (Ebert, 1939; Schumacher, 1961c; Gaspard, 1987). The location ofthese attachment points (Figure 22) are as follows: ol) is the most anterior point of themasseteric attachment, and occupies the top of an eminence immediately anterior to thezygomaticomaxillary suture; o2) is the top of an eminence which is the Paturet’s subjugal eminence to which Ziabek’s tendon attaches (Paturet, 1951); o3) is situated at thejunction of the anterior and middle thirds of the inferior border of the zygomatic bone,and is located at the top of an eminence posteriorly to the Paturet’s sub-jugal eminence;o4) is at the small bony spur just anterior to the squamoso-zygomatic suture at theposterior border of the superficial masseter; o5) is at the geometric centre of thelongitudinal fossa of the inferior border of the zygomatic process of the squama; o6) isthe most posterior point of the masseteric attachment, just anterior to the articular127Figure 22 Masseter attachment points represented by six origin and twelveinsertion points. Points 1-8 represent the superficial masseter attachment area, andpoints 9-12 the deep masseter insertion. Similarly, ol-o4 represent superficial, and o4-o6deep-masseter origins.31128eminence; (1) is at the pre-angular bony projection which corresponds to the anteriorlimit of Cihak and Vlcek’s masseteric tuberosity (1962); (2) is at the centre of theangular bony projection; (3) is at the post-angular bony projection which corresponds tothe posterior limit of Cihak and Vlcek’s masseteric tuberosity (1962); (4) is the anteriorborder of the attachment of “Sehnenspiegel 2”; (5) is the middle part of the attachmentof “Sehnenspiegel 2”; (6) is the posterior part of the attachment of “Sehnenspiegel 2”; (7)is at the geometric centre of a rhomboidal area corresponding to Weidenreich’smasseteric fossa; (8) is the most anterior point of the erista to which the anterior borderof the superficial masseter attaches; (9) is at the Lenhossék’s crista ectocondyloidea; (10)is at the tuberculum musculi zygomaticomandibularis, which is the inferior limit of thefovea musculi zygomaticomandibularis, situated between the coronoid process and thecondylar process (Cihak and Vicek’s, 1962); (11) is at the geometric centre of the “V’shaped crista musculi zygomaticomandibularis; (12) is at the geometric centre of apentagon centred on the body of the coronoid process. Insertion sites 1-3, 4-6, 7-8, 9-10and 11-12 were selected to represent the muscle layers I and II of the superficial, ifi ofthe intermediate, and IV and V of the deep masseter, respectively.For each skull, all anatomical reference landmarks and attachment points weredigitized three-dimensionally at different jaw positions with an optical system (ReflexMetrograph, I{F Ross, England) capable of 0.1 mm resolution (Takada et a!, 1983). Themetrograph coordinates of the muscle attachment sites were transformed into skull-referenced coordinates by translating the metrograph origin to a skull origin, then bymathematically rotating the data. The skull’s references were defined as follows: origin:129Figure 23 Orientation of the three planes of the skull’s coordinate system. Origin(0); Frankfort horizontal (XY); mid-sagittal (YZ); and coronal (XZ) planes.xzxz130a point halfway between the centre of the two external auditory meati; yz plane: the midsagittal plane; xz plane: the coronal plane; and xy plane: a plane parallel to theFrankfort horizontal plane (Figure 23).In order to analyze the degree of movement that the muscle attachmentsunderwent during simulated functional jaw movements, seven representative jawpositions were selected (Figure 24). They included the intercuspal position (IP), midlinejaw openings at three gapes (10, 20 and 35 mm measured between the incisors),simulated masticatory positions on the working and balancing sides (10 mm incisalopening with 5 mm incisal lateral deviation to the respective side), and incisal edge-to-edge contact. With the exception of the intercuspal position, these positions weremaintained by silicon bite blocks placed between the incisors and molars. The simulatedmasticatory positions were obtained by moving the mandibles from the maximumintercuspation to the various functional jaw positions, which were determined by dentaland condylar guidance coupled with the predetermined jaw openings, based on previousreports on chewing cycles by Lundeen and Gibbs (1982) and Hamiam et al (1977). Theaverage thicknesses of the articular soft tissue layers and disks were simulated with waxinserts according to data published by Hansson et al (1977) and Christiansen et a! (1987).Vertical lines drawn on the upper and lower arches at the central incisors, and at thetemporomandibular joints with the teeth in maximal intercuspation were used as a guideto measure the amount of distance moved by the mandible. All vertical, transverse andantero-posterior distances were measured on or between these lines with calipers toconfirm the deviation of the mandible from the intercuspal position.131Figure 24 Illustration of the seven representative jaw positions. Frontal andhorizontal views of the mandible indicating the five midline gapes, and two simulatedmasticatory positions. Positions include intercuspal position (IP); incisal contact (I); threedifferent jaw openings at (10); (20); and (35) mm; working (W); and balancing sides (B).Mandibular movements were controlled by dental- and articular-guidance paths.Calibration bar represents 10 mm.I I. 35132The orthogonal distances that the muscle attachment landmarks moved werethen calculated from the coordinates of each landmark for each jaw position, andexpressed relative to their common coordinates at maximal tooth intercuspation.To test for any statistically significant difference between female and malecephalometric measures, and to express the differences between the various distancestravelled by each insertion at different jaw positions, multiple one-way analysis ofvariance followed by Tukey tests were used. In the latter case, jaw position wasconsidered as the factor and the three orthogonal directions (x, y, z distances) asdependent variables. Here, the tests were not used to determine whether the insertionpoints had significantly different movement patterns with varying jaw positions onindividual specimens, because all insertion points were located on the same rigid bone(mandible), and their displacements were dependent and predictably different from eachother. However, the differences between the various distances travelled by each insertionat different jaw positions were used as descriptors of sample behaviour, since this wasnot readily predictable given the anatomical variations in the specimens. Results32.12.1 Cephalometric Analysis of the Skull SampleComparisons between female and male skeletal data showed that sexdifferences were negligible for most measures (p > 0.05). Exceptions were the mandibularand maxillary length, upper face height and bigonial width, which had slightly largervalues in males (see Table ifi). Because the variation in craniofacial dimensions were133Table ifi Facial dimensions of female and male skulls. Means and standarddeviations are expressed in mm1.f/rn *Measure2 Female (f) Male(m) 100%LengthMandibular 111.5 ± 4.7 119.1 ± 5.2 93.6Maxillary 89.2 ± 3.4 93.0 ± 3.3 95.9Difference btwMand. and Max. 22.2 ± 3.1 26.1 ±3.1 85.1HeightFace (total) 116.3 ± 6.4 124.8 ± 8.0 93.2Upper face 48.6 ± 2.5 52.9 ± 3.9 91.9WidthBigonial 88.9 ± 6.5 99.3 ± 3.9 89.51No. of observations: 7 in each group2For definitions, see text.134TableWCraniometricmeasurementsinfourteenskulls.Unitsareexpressedinmmanddegrees.Coefficientsofvariation(CV)arereportedinpercentages(SD/Mean*100%).RelationshipMeasurementMeanSDMaximumMinimumCV(%)MaxillatoAnteriorpositionofmaxilla(SNA)85.54.392.278.6-craniumOcciusalplane(SN-OP) mandible(SNB)78.63.785.673.1-craniumMandibularplane(SN-MP)35.24.744.926.5-Gonialangle126.34.3133.5118.7-Mandibularlength115.36.2126.0107.05.4(I’MaxillatoRelativemandibularprognathism(ANB) width109.94.4117.0104.04.0Bigonial width94.17.5104.077.08.0small, no further gender distinction was made in subsequent analysis. The group means,standard deviations, ranges and coefficient of variations derived from the cephalometricradiography, as well as mean bizygomatic, bigonial and intercondylar width, are shownin Table IV. The cephalometric characteristics of our sample appeared to be typical ofa modern population and fairly comparable to the subjects from other studies (Weijs andHillen, 1986; Hannam and Wood, 1989; van Spronsen et a!, 1991; Lam et a!, 1991). Afew variables, however, differed from those in the previous studies. Mandibular planeangle, mandibular length and relative mandibular prognathism were larger, whilebizygomatic and bigonial widths were smaller. These differences are probably explainedby race and perhaps gender differences between this sample and those previouslystudied.32.1.22 Sample Variation in Insertion Site Location during DentalIntercuspationVariations in the location of the twelve insertion sites are presented in TableV. Mean values of the coordinate points and their standard deviations are shown for thefourteen skulls, with their mandibles in the intercuspal position. The largest variationsin the sample occurred in the superficial masseter insertion sites, especially in thevertical dimension (z), reflecting differences in size and shape of the lower jaws.32.12.3 Effect of Jaw Position on Insertion Site LocationTable VI describes the amount of displacement of masseter insertions at the136Table V Mean location of putative muscle insertion sites for 14 skulls.Insertion x y z1 43.2 ± 4.5 21.1 ± 4.0 59.8 ± 5.92 45.3 ± 5.2 14.6 ± 3.5 54.9 ± 6.23 44.9 ± 4.5 10.1 ± 2.9 46.3 ± 5.54 43.1 ± 3.5 24.5 ± 4.3 56.1 ± 5.45 44.7 ± 3.9 18.1 ± 3.7 51.4 ± 5.36 45.1 ± 3.8 14.3 ± 3.2 44.0 ± 4.97 43.9 ± 2.8 24.7 ± 2.6 43.5 ± 4.08 39.8 ± 2.9 35.4 ± 2.8 54.8 ± 4.69 43.2 ± 2.3 31.5 ± 3.5 32.7 ± 4.510 45.6 ± 2.2 25.4 ± 2.9 24.2 ± 2.811 45.0 ± 2.5 30.8 ± 2.9 18.9 ± 2.712 45.2 ± 2.7 37.8 ± 3.6 14.2 ± 3.0Note: Means and standard deviations of the coordinate points are in mm.All measurements are made in the intercuspal position.For descriptions of insertion codes, axes and origins, see Figs. 22, 23 and text.137TableVIEffectof jawpositionondisplacementofinsertionsexpressedbymeanorthogonal distances.Alldisplacementsarerelativetotheintercuspalposition. Meanandstandarddeviationsareexpressedinmm1.I w 00INo.ofobservations:14ineachcase.2Forinsertionsitedescriptions(1-12)seetext.JawpositionInsertion2WorkingsideBalancingsideIncisatbitexyzxyzxyx1-2.1±0.87.2±1.3-0.8±0.62.0±1.04.3±1.1-3.2±1.1-0.3±0.6-1.8±1.3-1.9±0.72-1.5±0.76.5±0.90.0±0.72.0±0.83.8±1.2-3.2±1.0-0.1±0.8-1.7±1.0-1.8±0.73-1.1±0.55.3±1.10.4±0.51.6±0.92.8±1.2-2.1±0.90.0±0.8-1.9±1.1-1.7±0.74-2.1±0.86.7±1.1-1.1±0.82.2±1.34.2±1.2-3.6±1.0-0.2±0.8-1.6±1.4-1.8±0.85-1.5±0.96.2±1.2-0.4±0.72.1±0.83.4±1.2-2.7±1.30.1±0.8-1.6±1.1-1.6±0.76-1.4±0.65.6±1.1-0.1±0.51.6±0.82.6±1.1-2.4±1.10.0±0.8-1.6±1.1-1.7±0.87-L7±0.75.4±1.2-1.2±0.42.1±0.62.6±1.2-3.6±1.1-0.1±0.9-L6±1.O-1.7±0.88-2.3±0.66.7±0.9-2.6±0.52.8±1.14.1±1.2-5.0±0.80.1±0.9-1.7±1.1-2.1±0.99-2.1±0.64.1±1.2-2.3±0.61.6±0.71.3±1.1-4.6±1.1-0.4±0.8-1.6±1.2-1.7±0.810-1.5±0.53.2±0.7-1.6±0.51.5±0.804±10-3.9±0.7-0.3±0.7-1.6±0.9-1.6±0.811-1.5±0.52.4±0.6-2.4±0.51.6±0.7-0.4±1.2-4.8±1.0-0.1±0.6-1.9±1.0-1.7±0.912-1.4±0.61.6±0.7-31±0.51.8±1.0-1.1±1.1-5.5±1.20.0±0.9-1.9±1.1-1.6±1.0Note:Positivedirectionofthex-,y-andx-axesaretotheleft,posteriorlyandupwards,respectively.TableVIContinuation.I ScJawpositionInsertion10mmopen20mmopen35mmopenxyzxyzxyz1-0.2±0.94.9±1.3-2.6±0.70.0±1.012.0±1.2-2.6±0.70.1±1.222.9±2.9-1.4±1.62-0.1±0.84.3±1.5-1.9±0.70.2±0.910.6±1.3-1.6±1.10.5±1.220.4±2.71.0±1.430.0±0.93.2±1.3-1.2±0.90.2±0.98.4±1.3-0.8±0.80.4±0.816.4±2.82.0±1.54-0.1±0.84.7±1.2-2.9±0.80.1±1.111.2±1.1-3.7±1.00.2±1.121.7±2.4-2.9±1.950.0±1.04.1±1.3-2.3±0.90.3±1.110.1±1.1-2.3±1.10.4±1.219.4±2.3-0.7±1.76-0.1±0.93.1±1.2-1.9±0.80.1±1.28.4±1.4-1.7±0.80.4±1.216.0±2.30.1±1.270.0±0.93.3±1.0-3.1±0.50.1±0.98.3±1.2-4.1±0.80.2±1.116.8±2.1-4.1±1.280.2±1.04.6±1.6-4.4±0.50.2±1.311.5±1.5-6.7±1.00.4±1.222.4±2.6-7.7±1.49-0.1±0.71.9±1.3-4.0±0.8-0.2±1.05.9±1.5-6.2±1.00.3±1.013.0±2.3-7.9±1.110-0.1±0.91.0±1.1-3.4±0.50.1±0.93.9±0.9-5.2±1.00.1±0.98.9±1.4-6.3±1.311-0.1±0.50.1±1.2-4.1±0.8-0.1±0.82.5±1.0-6.6±0.90.3±0.87.1±1.0-9.0±1.2120.0±0.7-0.4±0.9-4.7±0.60.1±1.11.4±1.2-8.4±0.80.4±1.05.5±1.0-12.1±1.4different jaw positions. These data are expressed as means and standard deviations fordistances between the maximum intercuspation and the different jaw positions in thethree orthogonal directions.Deviations from sample means were generally less than 1 mm, except formean displacements in the anteroposterior (y) direction. The deviation was mostnoticeable on the balancing side and during midline tasks, and probably reflects bothdifferences in the size of the ramus and in the variables responsible for anteriormovement during lateral excursion and jaw opening.For every insertion site, as expected, the one-way analysis of variance revealedsignificant differences in transverse, antero-posterior and vertical muscle insertiondisplacement as the jaw positions changed (p < 0.001). To test for the effects of jawdisplacement on the movement of putative muscle layers, five representative muscleinsertions were chosen. These represented the center of the attachment of theaponeurotic sheet I (2), of aponeurotic sheet 11(5), and of the muscular attachment tothe mandible of aponeurotic sheet III (7), as well as the deep posterior (10) and deepanterior muscle portions (11). Multiple comparisons were made between the 3Ddistances travelled by these insertions at the working- and balancing-side positions, andat a 10 mm midline jaw opening. Midline incisal edge-to-edge, 20 mm and 35 mm jawopenings were not included in this analysis, because for these tasks all insertion sitespredictably move in a single plane by increasing amounts, relative to the increasing jawopening.When the working side position was compared against the balancing side, and140against a 10 mm jaw opening, the analysis of variance revealed statistically-significantdifferent amounts of movement for insertion 2 (p <0.05). Significantly different distanceswere also found for the displacement of insertion 5, when the working side wascompared against a 10 mm jaw opening (p < 0.01). Finally, when the balancing andworking sides were compared, significant differences in displacement were found forinsertion 11 (p<0.01).32.12.4 Differences within Putative Muscle LayersIn order to analyze the displacement that the various muscle layers aresubjected to during normal mandibular movements, the different insertion sites weregrouped into layers I (1-2-3), 11(4-5-6), III (7-8), IV (9-10) and V (11-12). A qualitativedisplacement analysis was performed within the individual muscle layers by comparinganterior against posterior insertions. Each jaw position was considered separately.Generally, anterior insertion sites moved consistently longer distances relativeto posterior insertions, showing differences in displacement ranging from 0.1 to 7.4 mmwithin the same layer. If incisal bite and a 35 mm jaw opening are not taken into theanalysis, displacement differences ranged from 0.5 to 4.2 mm. For the incisal bitehowever, all insertions displaced by equal amounts, maintaining the same direction ofmovement (Figure 25).141Figure 25 Mean displacements of the twelve insertion points at six different jawpositions. These include working (A) and balancing sides (B), and the four midlinegapes: incisal bite (C), 10 mm (D), 20 mm (E) and 35 mm (F) jaw openings. For eachjaw position, the coronal projection of attachment side movements is shown on the left,and the corresponding sagittal projection is shown on the right. Continuous-mandibleoutlines illustrate the jaw at the intercuspal position. Stippled outlines illustrate the jawat the various gapes and eccentric jaw positions. All insertion displacements are shownas lines connecting the location of each insertion point at the different jaw positionsrelative to the location of the same insertion point at the intercuspal position (topmostpoint of each line). The 12 insertion points are indicated by numbers in (A), and are alsoillustrated for the other jaw positions (B-F).0AUI10/V6,.7‘—51/I ‘BEIii11111’I“1FCI1423.2.12.5 Differences between Putative Muscle LayersComparisons between the various muscle layers and between thecorresponding anterior and posterior insertions of a single layer were also madequalitatively for each jaw position. Considered three dimensionally, the displacementscovered a wide range. The shortest mean distance was 2.5 mm for the deep-posterior(10) portion at the incisal contact position, while the longest distance was 23.8 mm forthe superficial-anterior (8) muscle at maximum gape. Collectively, the more superficiallayers moved longer distances than did the deeper layers for all jaw positions, except forthe incisal edge-to-edge bite. Variations in the amount of insertion displacement betweenthe different skulls were more prominent in symmetric than in asymmetric jaw positions.In the asymmetric jaw positions, they were smaller for the balancing than for the workingside (Table VII).Sagittal and coronal projections of attachment site movements are shown inFigure 25, and infer fascicle lengthening and rotation. They reveal that for thesuperficial-anterior (8), superficial-posterior (3), and deep-posterior (10) masseter, thebalancing-side masticatory position and the incisal contact position provided the mostadvantageous lines of action, for muscle fibres which contribute mostly to jawdisplacement by changing their length. For the deep-anterior (9) masseter, the workingside task was most advantageous in this respect. Superficial-anterior and superficial-posterior masseter had also an advantageous line of action at 10 mm jaw opening, whilethe deep-posterior and deep-anterior masseters were well aligned at 20 and 35 mm jawopenings.143TableVIIEffectofjawpositionondisplacementof insertionsexpressedbymeanlineardistances.Alldisplacementsarerelativetotheintercuspalpositionandareexpressedasthree-dimensionalresultants.Meanandstandarddeviationsareexpressedinmm.JawpositionInsertion2WorkingsideBalancingsideIncisal bite10mmopen20mmopen35mmopen17.6±1.25.9±0.82.9±1.05.7±1.012.5±1.323.0±2.926.7±0.95.1±0.82.7±0.94.9±1.110.8±1.220.5±2.735.5±1.14.1±1.12.8±1.03.7±0.88.5±1.316.6±2.847.2±1.06.2±0.92.7±1.25.7±0.811.9±1.022.0±2.456.5±1.15.1±0.92.6±0.94.9±1.010.4±1.119.6±2.365.6±1.14.3±0.82.4±1.03.9±0.78.5±1.216.0±2.375.8±1.05.2±0.82.7±1.04.7±0.69.4±1.017.4±1.987.6±0.77.2±0.83.0±0.96.6±0.913.4±1.223.8±2.595.2±1.05.3±1.02.7±1.14.7±0.78.7±0.815.3±2.1104.0±0.54.4±0.82.5±1.03.8±0.66.6±0.711.0±1.2113.7±0.45.2±1.12.7±1.14.3±0.87.2±0.911.6±1.0123.9±0.66.0±1.32.7±1.24.8±0.68.6±0.813.4±1.2IS1No.ofobservations:14ineachcase.2Forinsertionsitedescriptions(1-12)seetext.When the three jaw openings (10, 20 and 35 mm; Figure 25 D-F) werecompared, a striking feature was the change in direction of movement of insertionsbelonging to the superficial and deep muscle portions. It is suggested that the fibres ofthe superficial masseter (1-6) are well-aligned to contribute to closing movements atsmall gapes (around 10 mm; Figure 25 D) but less well-aligned to contribute tomovements at large gapes (35 mm; Figure 25 F), where fibres in the deep part (9-12)of the muscle may have a better line of action. As expected, from the incisal contactposition (Figure 25 C) all insertions displaced similarly.For the simulated functional jaw movements, there are considerabledifferences in the movements of attachment sites in both projections when the workingside (Figure 25 A) is compared with the balancing side (Figure 25 B). On the workingside, all attachment sites tend to move upwards, forwards, and medially. The superficialsites (1-6) move much more horizontally than do the deep sites (9-12) in bothprojections. These movement patterns are not consistent with the probable orientationsof the muscle fibres in either part of the muscle. When insertions are moved towards thebalancing side the majority of points move upwards, forwards, and laterally. In contrast,these directions are more consistent with probable muscle fibre orientations (Figure 26).32.12.6 Orientation of Masseter Insertion relative to OriginTo evaluate the orientations of masseter muscle attachment sites relative toeach other when the teeth were in the intercuspal position, both origin and insertionpoints of the superficial and deep muscle portions were plotted in the (horizontal) XY145Figure 26 Mean functional displacements offour putative muscle attachment sitesin the human masseter. Movements between four jaw positions are simulated. Thepositions include incisal, edge-to-edge tooth contact (I); 10 mm jaw opening (10); theturnaround between opening and closing for a chewing stroke on the ipsilateral workingside (W); and on the balancing side (B). The apices of the movement vectors representthe dental intercuspal position. Data are shown in both frontal and lateral projection,and are derived from simulations in 14 adult skulls. Calibration bar represents 10 mm.I I1 cm10 BWI146plane. Origin points ol-o4 and insertion points 1-3 and 8 were used to represent thesuperficial masseter attachment areas, while points o4-o6 and 9-11 were used torepresent attachment areas of the deep-masseter portion (Figure 27). For each skull,linear-regression lines were fitted through individual sets of points, and their intercept,slope and R-values recorded. Mean and standard deviations were subsequentlycalculated for the intercepts and slopes of regressions that had R-values larger than 0.45(Table VIII). The resultant four mean regression lines were then plotted, and theirangles measured relative to the midsagittal axis. For the superficial and deep masseterportions, the origin and insertion attachment sites were angled at 25°, 15°, -4° and 19°relative to the midsagittal plane, respectively. During working-side-mandibularmovements, the insertion lines of the deep masseter approached parallelism with theirrespective origin attachment sites. However, the insertion lines of the superficial portiondeviated even further from parallelism relative to their origin. During balancing-sidemovements, the inverse was true. The attachment sites of the superficial portionsapproached parallelism, while the attachment sites of the deep masseter deviated evenfurther from each other (Figure 28).32.12.7 DiscussionIdeally, the displacement of muscle attachment areas should be measuredduring normal jaw function. This requires muscles to be imaged in living subjects,computer reconstruction of attachment landmarks, and recordings of movements ofidentified sites to be made in three dimensions with a transducer system having six147Figure 27 Orientation of origin and insertion sites. Insertion points are plottedin the XY-plane relative to their origin counterparts. The jaw is at the intercuspalposition in both graphs. Superficial (A), and deep (B) masseter insertions (filledsymbols) are shown relative to their origins (open symbols) for six skulls. Each skull isrepresented by a different symbol.A —30.00—36.67 —.::::—56.67—63.33—70.00 I I IB —30.00—36.67—43.33—50.00-56.67—63.33—70.00 I I0 16 32 48 64 80ANTZRO-POS2’IRIOR DINENSION148TableVifiLinearregressionanalysesforpointsrepresentingattachment sites.Linearregressionthroughthepointsattheorigin(0)andinsertion(I)ofthesuperficialanddeepmassetermusclesareexpressedinmm.Meanandstandarddeviationsof theinterceptsandslopeswerecalculatedonlyforlinearregressionswithR-valuesgreaterthan0.45.SuperficialportionDeepportionZygomaticarch(0)Mandible(I)Zygomaticarch(0)Mandible(I)SkufiInterceptSlopeR-valInterceptSlopeR-valInterceptSlopeR-valInterceptSlopeR-val1-76.40.510.91-52.80.320.98-56.4-0.060.50-55.10.330.892-76.40.480.98-50.20.321.00-53.8-0.060.87- 28 Orientation of origin and insertion sites at three jaw positions. In (A),the superficial-masseter insertion is illustrated in the balancing side (B), intercuspalposition (IP) and working side (W), represented by linear-regression lines in the XYplane relative to their origin in the zygomatic arch (ZA). In (B), the deep-masseterinsertion is illustrated in the same jaw positions relative to its origin. Regression lineswere fitted through insertion and origin points of all 14 skulls.AB—36.67—43.33—50.00—56.67—63.33—70.00-30.00—36.67—43.33—50.00—56.67—63.33—70.000 16 32 41 64 *0100 16 32 4* 64ANTIRO-POSFERIOR DIMFNJION150degrees of freedom. While theoretically possible, such an experiment poses formidabletechnical problems.A second approach could have been to use a computer model to predictmultiple trajectories, at multiple sites, for simulated jaw movements made for manydifferent craniofacial configurations. As the mandible is a rigid body, the displacementsof all points on it are predictable provided the displacement of any three are known.Thus, a computer model could be used to predict the displacement of attachment sitesfor any combinations of skeletal and of movement variables, provided that these areknown. Such a model could have provided multiple predictive curves, but it would bedifficult to decide which parts of the displacement curves should be considered importantunless specific morphological dimensions and related movement patterns were available.Even having accomplished this, one would be left with the problem of specifying whichcombinations best represent those likely to be found in a sample of living humansubjects.The third approach, which was the method of choice here, involved themeasurement of putative muscle attachment sites on mandibles which were subjected tomovements simulated according to the condylar and dental guidances likely to be presentin a series of differently-shaped, dry human skulls. Using this approach it was possibleto investigate a larger number of specimens (as opposed to a study in living subjects, inwhich the number of participants is even more restricted). It had, however, somelimitations. Although the landmark locations were dfrectly visualized based onosteological criteria, they were arbitrary and only approximated the “true” attachment151locations in each skull since muscles were not present. Also, the assumption of tasks withcommon dental arch positions may not have been correct for the different crariiofacialshapes used in the sample, and the simulation of articular movements with publishedrecords and artificial articular disks may not have duplicated the real jaw movement thatoccurred for each specimen in life. Although these errors were present, the data werereasonably homogeneous and indicated the probable order of displacement expected ina living sample with comparable skeletal morphology. They also provided a realisticestimate of the demands placed upon the average masseter muscle. In addition, thesedata may be used in future models of the masseter and encourage further experimentsto reveal individual differences in living human subjects.It is known that the human masseter muscle has a complex internalarchitecture. Its muscle fibres are arranged on a multipennate pattern between ampleaponeurotic sheets, and together with their sarcomeres show moderate length differencesanteroposteriorly (30%) and mediolaterally (5%), (Ebert, 1939; Schumacher, 1961c; VanEijden and Raadsheer, 1992). Lam et a! (1991) also suggested that the muscle fibreorientations between aponeurotic sheets may have different pennation angles at theirproximal and distal ends. This, added to regional variation of sarcomere length, suggestsa substrate for differential contraction according to task, and suggest that differentialtension may be produced within the muscle.Despite the possibility of dissimilar contraction patterns of the muscle masseson either side of the aponeurotic sheets, it may be assumed that these muscle fibresmostly co-activate, and that for any degree of activation, the resultant line of action152would be along the planes of orientation of the aponeurosis (Gans and Bock, 1965;Otten, 1988). If so, the orientation of aponeuroses and the displacement of any of theirmandibular attachment sites during function have an extreme impact on suitability forparticipation in a particular task. On the other hand, the line of contraction followed bymasseter muscle fibres may or may not coincide with the direction of movement of theirattachment to the mandible, since the latter is determined by the combined action ofseveral muscles. It is therefore possible that particular groups of fibres are moreefficiently aligned for some tasks than for others, independent of the fibre’s line ofaction.In this study, the extent and direction of movement of the various aponeuroticattachment sites were evaluated, and the relationships between the various insertionmovement trajectories and the probable orientation of the aponeurotic sheet planes wereanalyzed. The results suggest that contraction of muscle fibres in the masseter would bestcontribute to displacement of the mandible when jaw closing takes place in the midlineand when the muscle is on the balancing side at the beginning of the closing strokeduring chewing. In these cases, fibre alignment approximates the lines of movement ofthe attachment sites. However masseter fibres are poorly aligned to shorten with jawdisplacement during movement from an edge-to-edge incisal position towards fullintercuspation in incision and on the working side during chewing, (note Figure 26-(I)in the sagittal projection and Figure 26-(W) in the coronal projection). Here the actionsof the fibres are actually perpendicular to the movement of the attachments. Becausethe masseter muscle is known to be active during these functions, it is assumed that the153produced forces are not used for generating jaw movement, but are used for developingforward and lateral compression between the teeth when the jaw moves into theintercuspal position.The analysis is in agreement with physiological findings. It has been known foryears that the deep and superficial masseters are capable of differential contractionaccording to task. Recently, it has been suggested that it can be even further divided intoat least three parts: anterior deep, posterior deep and superficial (Blanksma et al, 1992),although the results of her study were obtained under isometric conditions. Duringlateral, posterolateral and posterior directed tooth-clenching the deep portion is moreactive than the superficial portion of the muscle. The same activity pattern is presentduring ipsilateral tooth-clenching at eccentric jaw position. Distinctions however must bemade between static and dynamic acts with regard to the muscle’s internal behaviour.During chewing at the ipsilateral side, both muscle portions were equally active, whileduring contralateral chewing the deep portion was more active than the superficialmuscle portion. Furthermore, during the act of incision the masseter muscle is as activeas during chewing (Hylander and Johnson, 1985), which is not the case during staticincisal biting (Belser and Hannam, 1986).This study also shows that there can be considerable differences betweensubjects. Even though this sample was small and from a single ethnic group, the skeletaldifferences were sufficient to show quite large differences in the movement ofattachment sites when similar functional acts were simulated. This implies differentmechanical and functional activation strategies, which could explain the great inter154subject variability known to exist in muscle activation patterns and frequentlydemonstrated in electromyographic studies. This variabifity makes pooling of functionaldata difficult.Recently, Van Eijden and Raadsheer (1992) have provided a comprehensivedescription of regional muscle fibre, tendon and sarcomere length in the humanmasseter. In their study, parameters were initially measured with the mandible in aclosed position, then changes in length were calculated with a computer modelsimulating a series of tasks similar to those used in the present study. Sarcomereexcursions were estimated to be relatively small in the deep-posterior region and largein the superficial-anterior part when the jaw was rotated open and closed about atransverse axis, but the reverse was true when the jaw was rotated contralaterally abouta vertical axis. In this situation, sarcomere lengths shortened initially, but started tolengthen as the movement continued. The angle at which this transition betweensarcomere lengthening and shortening occurred was determined by the tilt of themuscle’s line of action relative to the vertical axis. It was argued that a correlation existsbetween the muscle fibre tilt and the jaw-deviation angle at which this transition occurs(i.e. the larger the tilt, the larger is the angle), and that insertions were displaced froma posterior to a anterior location relative to their point of origin. In the skull sample,however, this situation occurred only 14% of the time. In the remaining cases, theinsertion remained posterior to the origin, suggesting that in this skull sample thetransition of sarcomere shortening to lengthening would occur only at large balancingside mandibular deviations. Although the location of insertion points varied in both155studies, that is, in the present sample the mandibles were shorter and narrower, bothstudies complement each other in the sense that while the former provides anatomicalinformation of fibre, sarcomere and tendon lengths, the skull sample providesinformation on the full, natural range of attachment movement.In conclusion, the human masseter is clearly not a simple muscle. It consistsof different structural compartments with varying fibre, tendon and sarcomere lengths,which are also angled differently in 3D. These structural characteristics and functionaldemands place very different constraints upon regional displacement of different partsof the muscle, and probably vary significantly between individuals. This leads to thesuggestion that intramuscular morphology, electromyographic activity and attachmentdisplacement in 3D during functional movements should probably be studied togetherin individual living subjects. In this way, the functional role of separate muscle portionscould be better assessed.156322 Function in Living SubjectsMeasurements of the displacement of the human mandible during function arecommon and are referred to as jaw tracking (Lundeen and Gibbs, 1982; Jemt, 1984;Mohi et at, 1990; Hannam, 1992). This measurement is obtained by recording thetrajectory of movement of sensors (magnetic, light-emitting or reflective) attached tospecific points of interest, normally anatomical. Most jaw tracking devices measure onlythree degrees of freedom, usually expressing motion of the incisor point. The limitationof these types of devices is that they are only capable of tracking the displacementpatterns of the region where the sensor is attached. Whenever there is the interest ofrecording any other site remote from the moving sensor, a tracking device with sixdegrees of freedom must be used. This type of tracking device allows an investigator tomeasure simultaneous motion of other anatomical or reference points based on theassumption that the mandible and attachments are a rigid unit, and mathematical dataconversion. These more elaborate sensing systems have been used to quantify the three-dimensional motion of distant condylar and tooth cusp points (Lundeen and Gibbs, 1982;Merlini and Palla, 1988; Hagiwara et a!, 1993b), and could be used to measure themotion of any other site including the attachment areas of the masticatory muscles. Thus,the availability of this new type of jaw-tracking system enables the recording of thedisplacement of masticatory muscles’ insertions during natural jaw movements.Improvements in MR imaging techniques enable the visualization of internal musclearchitecture, and therefore, the determination of the actual location of the attachmentsites. In addition, modern engineering software (i.e. solid-object modelling techniques)157facilitates the three-dimensional reconstruction of the muscle architecture. Theavailability of these techniques inspired the development of a method which combinesthe muscle reconstructions and jaw-tracking records through a common reference system,and allows the measurement of the actual displacements of the masseter muscleinsertions in four living subjects. Thus the goal in this part of the study was to developan experimental approach that might be useful in future studies of muscle mechanics,and to determine whether estimates of attachment site displacement made previously indry skulls were representative of those in living subjects.322.1 MethodsDuring each jaw movement recording session a customized plastic eyeglassframe was worn by the subject to provide a fixed spatial reference (details on page 103).Each subject wore this device during a separate imaging session in which a 1.5 Tesla unit(Signa, GE Medical Systems, Milwaukee) was used to obtain T2-weighted images of themuscle and the reference grid. Spin-echo sequences with TR of 2000 ms and TE of 25and 80 ms were used to obtain a series of contiguous, 3 mm sections in the coronalplane. From each coronal section, outlines from the muscle, internal tendons, bones andcopper-sulphate markers were traced and digitized (Model HP9874A Digitizer, HewlettPackard, Canada). The profile coordinates were then used in an engineering solid-objectmodelling package (1-DEAS, SDRC, Milford) to reconstruct the muscle and its internalstructures in 3D, as shown in Figure 29.An optical motion-analysis device (MacReflex System, Qualisys AB, Partille,158Figure 29 Anterior view of the sectional outlines of a reference grid, rightmandibular ramus and condyle, and selected right-masseter-tendinous insertions. Theseoutlines are closed third-order splines, which form a “wireframe” in I-DEAS. (C)Condyle; (R) Mandibular ramus; (RG) Reference grid; (T) Tendinous insertions.RG159Sweden) and two marker triads were used to record the 3D movement of the mandible.One triad was fixed to the front of the eyeglass frame, while the other was attached toa dental clutch cemented to the labial surfaces of the mandibular front teeth (canine tocanine). Each triad consisted of three reflective markers. The optical system was usedto measure the 3D coordinates of the mandible’s triangle relative to the reflectivemarkers on the front of the frame (Figure 30). The tracking system was calibrated sothat markers could be detected within a cube of 130 mm (x axis), by 150 mm (y axis),by 100 mm (z axis), with a static resolution better than 0.1 mm in any dimension(Hagiwara et al, 1993a).Each subject was asked to perform four separate gentle, static biting tasks.These included biting at the intercuspal position, with the jaw in left and rightlaterotrusion, and incisal biting. All biting tasks were performed on a plastic suction tipwith a diameter of 10 mm. To estimate reproducibility, one subject was asked to repeatthese tasks five times. For each jaw position, the coordinates were sampled by two videocameras every 20 ms for 0.5 s, then stored and converted into 3D spatial coordinates ina microcomputer (Macintosh Ilsi, Apple, Cupertino, California, and MacReflex software,Qualysis AB, Partille, Sweden). The reflective-markers’ coordinates were sampled 25times and averaged for further analysis.In addition to the biting tasks, all subjects were asked to chew gumunilaterally, on both the right and left sides. For chewing, the coordinates were sampledevery 20 ms for 15 s. With this sampling rate, at least 13 complete envelopes wererecorded for each unilateral chew of each subject. To simplify analysis, only one atypical”160Figure 30 Schematic illustration of the global setup for a jaw tracking run. Acopper-sulphate-filled, L-shaped grid attached to an eyeglass frame (A). Three reflectivemarkers are mounted on the front of the frame (B) and three on a triangle cementedto the anterior teeth (C).ABC161chewing envelope of each task was selected from those available.To express attachment site displacement relative to the same coordinatesystem used in the skull study, a modified dental earbow with reflective markers on eachearbar and on an infraorbital pointer, was used. Each subject was instructed to bitegently in the intercuspal position, while the location of the mandibular-clutch-markertriangle was recorded relative to the earbow. These data were then used in 1-DEAS toorientate the reference grid, masseter muscle and bone reconstructions to the desiredcoordinate system, that is, relative to the Frankfort Horizontal plane, with the originsituated halfway between the meatal landmarks (Hagiwara et a!, 1993a).To relate the jaw-motion data to the MR images, a separate optical measuringsystem (Reflex Metrograph, HF Ross, Salisbury, UK) was used to measure the 3Dcoordinates of the markers on the front of the frame relative to those of the L-shapedgrid visualized during imaging. All data were then transferred to the solid-objectmodelling package (1-DEAS, SDRC Corp. software), in which coordinate conversionswere performed. Conversions involved superimposition of like markers by a series oftranslations and triaxial rotations.Four muscle attachment points were selected for each subject. These weredefined as the most anterior and posterior points of the mandibular insertions ofaponeuroses II and IV, as described by Schumacher (1961c) and Lam (1991). Theorthogonal distances that these landmarks had to move were then calculated from thejaw-motion data at each jaw position. For the chewing data, only four selected datapoints per envelope were analyzed. These were defined as the most left lateral, right162lateral, inferior and superior points of the representative chewing cycle. Displacementsof the attachment points were then calculated relative to their common coordinates atmaximal tooth intercuspation.Due to the small sample size, and for reasons discussed above, no statisticaltest to determine the significance of differences between the various distances travelledby each insertion at different jaw positions were used.3222 ResultsDistances between the anterior and posterior insertion points of bothaponeurosis (II and IV) are shown in Table IX. The antero-posterior length ofaponeurosis II varied greatly between subjects, but tended to be larger than aponeurosisIV, which showed small inter-subject variability. The standard error of the measurementwas estimated to be 0.07 mm. This was obtained by sampling the position of oneinsertion site, with the teeth in the intercuspal position, on three separate occasions.When the subject repeated the static tasks five times, the largest variation wasfound to occur in the supero-inferior (y) direction (Table X). It was also observed thatduring the working side task, insertion displacement in the lateral (x) direction wassmaller than expected, and in two cases it deviated to the opposite side (medially). Apossible explanation for this finding is the presence of a cross-bite in the right canineregion of this subject.Table Xl shows, for all four subjects, the mean and standard deviation valuesof the four insertion points, namely, posterior (1) and anterior (2) insertions of163Table IX Anteroposterior dimensions of the mandibular insertions of Aponeuroses IIand IV in four subjects. All values are expressed in mm.SubjectAponeurosis I II III IV Mean ± SDII 26.01 12.50 15.23 10.94 16.17 ± 5.88IV 11.64 13.19 9.27 11.43 11.38 ± 1.40164TableXDisplacementsof oneinsertionpoint ofonesubjectatdifferentjawpositions.Eachpositionwasrepeatedfivetimes.Displacementsareexpressedasorthogonaldistances(inmm)calculatedfromtheinsertion’scoordinatepointatmaximumintercuspation.RepeatsTaskIIIifiIVVMean±SD0.46-1.10-0.170.04-0.19-0.19±0.51WorkingSidey-2.451.69-0.04-1.82-1.70-0.86±1.51z-7.46-9.72-9.35-7.35-8.19-8.41±0.97I.ONx2.030.730.620.621.341.07±0.55BalancingSidey-4.28-1.06-0.53-0.75-2.26-1.78±1.39z-4.93-6.95-6.69-7.33-6.52-6.48±0.82x0.31-0.150.32-0.50-0.32-0.07±0.33Openy-3.39-1.07-1.97-1.51-2.45-2.08±0.80z-6.76-5.61-5.98-5.96-6.25-6.11±0.38TableXlMeandisplacementsandstandarddeviationsoffourinsertionpointsoffoursubjectsatdifferentjawpositions. Displacementsareexpressedasorthogonal distances(inmm)calculatedfromtheinsertion’scoordinatepointatmaximumintercuspation.I. ONInsertionPointsTask1234x-0.80±0.75-0.94±0.92-0.57±0.68-0.67±0.73WorkingSidey-1.88±1.28-3.28±1.39-2.96±1.10-3.90±1.16z-4.79±0.81-5.76±0.58-2.27±1.79-3.10±1.10x1.54±0.321.95±0.501.51±0.301.78±0.48BalancingSidey-4.34±0.69-5.63±0.34-5.37±0.53-6.25±0.38z-2.07±1.90-3.07±1.640.49±2.18-0.40±1.90x0.41±0.380.35±0.390.60±0.330.45±0.27Openy-3.99±1.04-5.44±0.64-5.12±0.40-6.13±0.48z-3.44±1.43-4.47±1.30-0.79±2.21-1.62±1.80aponeurosis II, and posterior (3) and anterior (4) insertions of aponeurosis IV, duringthe working, balancing and incisal bites with jaw opening. As in the skull study,displacement of the insertions in the antero-posterior (z) direction varied the most,reflecting differences in mandibular ramus lengths. Additionally, when compared withthe skull data, the living subjects showed a tendency to displace the insertions oversmaller distances in the medio-lateral direction during the working side bite. Largermean distances were observed, however, in the supero-inferior direction at all three tasksand in the medio-lateral direction at the incisal bite.When the displacements of the anterior and posterior insertions werecompared, there was a tendency for the anteriorly located insertions to displace largerdistances than their posterior counterparts. The exception to this pattern was theinsertion displacements of aponeurosis IV during the balancing side bite. The meanvalue of the posterior insertion (3), antero-posterior (z) movement was larger than thatof the anterior attachment, and in addition it was oppositely directed (anteriorly), [TableXI, Figure 31]. When the data for each subject were individually analyzed (seeAppendix), it was found that for subject Ill (Figure 31 C), insertion 3 moved anteriorlyduring all three tasks, while insertion 4 moved anteriorly during balancing and incisalbite. In addition, for these two tasks insertion 4 moved smaller antero-posterior (z)distances than 3. For subject IV (Figure 31 D), the insertions of aponeurosis IV movedanteriorly only during the balancing side bite. In this subject insertion 3 moved largerdistances in the antero-posterior direction during the working and incisal bite tasks.When the static-bite-tasks were compared against the chewing data, it was167Figure 31 Diagram illustrating the displacement trajectories of insertions ofaponeuroses II and IV in living subjects. Diagram in (A) illustrates masseter muscleorientation. (B) illustrates displacements of the anterior (2) and posterior (1) insertionpoints of aponeurosis II, and anterior (4) and posterior (3) points of aponeurosis IV. Theaverage trajectory movements of the insertions in four subjects are represented by two-dimensional vectors in the parasagittal plane. Displacements of insertions of subject III(C), and of subject IV (D) are shown separately. Afi trajectories are relative to theintercuspal position (point of convergence), and represent working (W), balancing (B)and incisal bites (0) with the jaw opened by 10 mm.ACBD168found that all insertions had similar displacements at both incisal bite and at maximumopening in the chewing cycle. In contrast, at the maximum medial and lateral deviationsof the mandible in the chewing cycle displacements were smaller than during static bites.During chewing, similar findings of the deep masseter (aponeurosis IV) insertiondisplacements were found (Tables XII and XIII). When compared against the skull datait was found that jaw movements in living subjects displaced greater distances in thesupero-inferior direction and smaller distances medio-laterally.322.3 DiscussionAlthough MR imaging has been used previously for muscle visualization(Koolstra et al, 1990; Lam et al, 1991; Ralph et a!, 1991) and tracking devices have beenused to measure jaw movements (Lundeen and Gibbs, 1982; Merilni and Palla, 1988;Hagiwara et a!, 1993b), there have been no attempts to combine these techniques tostudy structure and function. Despite some limitations in the visualization of internalstructures, especially muscle fibre orientation as discussed in an earlier section of thisthesis, the method presented here should be useful for future work where the structureand function of muscles in individual subjects are to be evaluated. It could also proveto be extremely valuable in experiments in which regional electromyographic activity issimultaneously recorded, and the electrodes located relative to the internal musclestructure.A limitation of the method, however, is that it does not take tissue (bone andmuscle) deformation into account. It is known that the mandible deforms by different169TableXIIMeandisplacementsandstandarddeviationsoffourinsertionpointsoffoursubjectsduringright-sidedchewing.Representativepointswerechosenfromonetypical chewingstrokefromeachsubject.Thatis,themostlateral,medialandinferiorpointsoftheenvelope.Displacementsaremeasuredrelativetotheintercuspalpositionandareexpressedasorthogonaldistances(inmm).CInsertionPointsTask1234x-0.80±0.95-0.84±1.12-0.65±0.82-0.68±0.89Medialy-2.59±2.25-3.45±2.56-3.23±2.22-3.73±2.46z-1.39±1.46-1.88±1.42-0.04±0.70-0.41±0.66x-0.91±0.70-1.04±0.71-0.78±0.65-0.84±0.65Lateraly-1.49±1.48-2.30±1.96-2.08±1.72-2.50±1.90z-2.11±1.12-2.69±2.00-1.08±0.97-1.42±1.46x-1.11±0.98-1.37±1.12-1.08±0.79-1.19±0.73Openy-4.02±2.45-5.81±3.41-5.51±3.11-6.51±3.39z-3.52±1.30-4.77±2.22-0.87±0.61-1.68±1.34TableXfflMeandisplacementsandstandarddeviationsof fourinsertionpointsoffoursubjectsduringleft-sidedchewing.Representativepointswerechosenfromonetypical chewingstrokefromeachsubject.Thatis,themostlateral,medialandinferiorpointsoftheenvelope.Displacementsaremeasuredrelativetotheintercuspalpositionandareexpressedasorthogonal distances(inmm).ISInsertionPointsTask1234x0.97±0.961.26±1.080.89±0.841.06±0.93Medialy-1.97±1.19-2.52±1.71-2.42±1.59-2.67±1.75z-0.74±0.72-1.28±1.55-0.06±0.86-0.39±1.10x0.73±1280.96±1.530.74±1.190.91±1.25Lateraly-2.99±2.23-3.68±2.51-3.55±2.31-3.93±2.47z-0.49±1.05-0.99±0.910.61±1.020.25±0.77x0.65±1.221.08±1.730.78±1.271.01±1.43Openy-4.79±3.22-6.70±4.36-6.47±4.16-7.52±4.45z-3.18±1.20-4.80±1.17-0.34±1.67-1.33±1.03amounts during maximum isometric clenching, depending on the task performed(Korioth, 1992). The method presented in this segment of the thesis, however, is basedon the assumption that the mandible is a rigid structure, and does not correct formandibular deformations that might occur during function. Mandibular deformations are,however, small relative to movements (approximately 1 mm at the gonial angle duringunilateral maximum clenching at the molar teeth region; i.e. approximately one fifth ofthe attachment movement). Although it is possible that the reduced amount of insertiondisplacement observed during chewing is the result of an estimation error rather thana different strategy used by the subjects to perform the different tasks, it is not likely. Inaddition, it can be argued that muscle activity seldom achieves maximum levels duringgum chewing, and that deformations at submaximum levels should be minimal (less than1 mm at the mandibular angle).During function, it is possible that the intramuscular structures are deformeddifferently according to task. A possible approach to test for this potential deformationwould be to image each subject repeatedly with the mandible at different positions. Thedrawbacks to this approach are the length of imaging time, which precludes imaging themuscle at sustained maximum contraction, and imaging costs. An alternative approachmight be to image the muscle at different jaw positions with ultrasound. Here it remainsto be seen whether the resolution is adequate for the recognition of internal musclestructures.When compared against the skull data, there was a tendency for the insertionsites to displace greater distances in the infero-superior direction and smaller distances172medio-laterally. This might be explained by the need for more vertical-directed, bite-force vectors during natural acts performed by living subjects than during those simulatedin the skull sample. An alternative explanation for these differences could be the samplepopulations (i.e. East Indian skulls versus the Mongoloid morphology of the livingsubjects, who were Japanese). Additionally, if a distinct “bolus hardness” had been usedinstead of the chewing gum, the dimensions of the chewing envelope could have beendifferent, possibly approaching those in the skull study.As previously discussed by Van Eijden and Raadsheer (1992) and consistentwith the skull study, the deep masseter insertion sites behave differently from the othermuscle attachment areas. When the mean data from the living subjects are analyzed(Table XI), it can be noted that while the posterior insertion point of the deep massetermoves forwards, the anterior insertion displaces backwards. If the fibres in this muscleportion are arranged parallel to each other in the sagittal view, it would seem that thismuscle portion might twist longitudinally during function. It is known, however, fromanatomical dissections (Figure 11) that this portion of the muscle is fan-shaped. It couldbe argued therefore that insertion displacement runs parallel to muscle fibre orientation,which differs from the anterior to the posterior portion of the deep masseter. It couldbe also proposed that the deep muscle portion is structurally and functionallycompartmentalized into anterior and posterior regions. Additionally, when individualsubjects are analyzed (see Appendix), muscle fibre orientation may vary betweensubjects, being more vertical in some subjects than others. These propositions however,cannot be proven with the present technology, since imaging methods with sufficient173resolution to identify muscle fibres are unavailable.In conclusion, the data obtained from the skull sample generally match theinsertion displacement measured in the small sample of living subjects. While they areonly an approximation, they offer the advantage of estimating probable insertionmovements from a large sample. On the other hand, measurement of attachmentmovement in living subjects as demonstrated in this thesis opens up the possibility ofevaluating “true” insertion displacements relative to regional muscle activity. From bothstudies, it can be suggested that the masseter is a multifunctional muscle having regionswhich are best suited to certain tasks. For example, it is proposed that the massetercontributes mostly to jaw movement when the muscle is on the balancing side duringchewing, while it generates interocclusal, crushing forces when it is on the working side.In addition, the deep masseter has different lines of action, varying biomechanicallyantero-posteriorly. Finally, the study also confirms the feasibility of making highresolution muscle reconstructions and of locating the tendinous insertion sites on themandible which can then be tracked with six degrees of freedom. This method could beused to estimate insertion displacements in other muscles and in any motor controlstudies where it is important to measure insertion displacement and muscle activitysimultaneously.1743.3 Evaluation of the Masseter’s Functional Performance3.3.1 Electromyographic Recording TechniquesElectromyography (EMG) is a method commonly used to study the electricalactivity of skeletal muscles arising from voluntary or involuntary contractions. Themyoelectric signal originates in the depolarization of the muscle-cell membrane to thethreshold level resulting in an action potential (AP), which is generated and propagatedalong the muscle fibre.Conventional needle EMG techniques do not record single-muscle-fibre APs,but rather potentials generated by the near synchronous depolarization of many musclefibres in a single-motor unit (MU) which summate to form a compound AP. Thecharacteristics of the compound APs may vary significantly depending on the anatomicaland physiological properties of the MU muscle fibres, the control scheme of theperipheral nervous system, the temperature of the muscle, the age of the subject, thedistance between the active muscle fibre and the recording sites, the chemical propertiesof the metal-electrolyte interface, as well as the characteristics of the instrumentationthat it is used to detect and observe them (Daube, 1978; Gath and Stâlberg, 1975;Lenman and Ritchie, 1987; Stãlberg and Trontelj, 1979). Various characteristics of theMU compound AP can be described both qualitatively or quantitatively. These includepeak-to-peak amplitude, number of phases, duration, number of turns, area, and patternof firing (Nandedkar and Sanders, 1989; Stálberg and Trontelj, 1979), and are useful inthe diagnosis of a number of muscle disorders (Daube, 1978; Stálberg and Trontelj,1979).175When several MUs are firing, the random temporal and spatial superpositionof their APs produce a complex myoelectric signal, known as interference pattern EMG.This type of myoelectric activity is commonly recorded in kinesiological studies whichhave utilized surface or bipolar fine-wire electrodes based on the original designdescribed by Basmajian and Stecko (1962). To evaluate these complex signals, variousanalytical techniques are available. These include integration, frequency analyses, turnscounting, zero-crossing analysis and analyses of the degree of superimposition (Richfieldet a!, 1981).332 Preliminary Experiments using Single-Wire EMG Recording TechniqueSince the human masseter muscle can be divided into at least four anatomicalcompartments innervated by separate main branches of the masseteric nerve (theanterior and posterior regions of the superficial masseter, and the anterior and posteriorregions of the deep masseter), it is possible that at least some functional differentiationoccurs between them. It has long been known that selective contraction of fibre groupsis found in the masseter muscles of pig (Herring et a!, 1979), rabbit (Weijs and Dantuma,198 1) and to a certain extent in man (Belser and Hannam, 1986). Although surfaceelectrodes are commonly used to record activity in human masticatory muscles, they arenot selective enough to sample regions within a muscle. Concentric-needle electrodes orbipolar-wire electrodes are more appropriate for this purpose but have their limitations.Needle electrodes are impractical in mobile jaw muscles which are activated duringmastication and tooth grinding, and paired-wire electrodes rarely maintain a consistent176spacing between the bared electrode tips (Jonsson and Bagge, 1968; Loeb and Gans,1986). It has been suggested that offset twist hooks are suitable for pennate muscles,reducing the possibility that the contacts will short and providing a more predictableinterelectrode distance (Loeb and Gans, 1986). However, the insertion of bipolar wireelectrodes in a muscle does not always guarantee an interference signal of predictablequality or range of pick-up, and variations in the sample volume from which electricalactivity is recorded may result in poor reproducibility (Kadaba et a!, 1985; Komi andBuskirk, 1970; Perry et a!, 1981). This has led to the proposal that unipolar wireelectrodes should be considered as an alternative (Kadaba et al, 1985).The aim of this study was to provide focal multiunit EMG at data collectionrates which would be manageable for data storage, and which would be reliable for thefuture investigation of functional masseter subdivisions. The long-term goal was to testthe hypothesis that the human masseter muscle is divided into at least threeneuromuscular compartments, based upon the assumption that differences between theanterior and posterior portions of the superficial masseter were unlikely due to theircommon fibre orientations.3.32.1 Materials and MethodsEach single-wire electrode was constructed from Teflon-coated stainless-steelwire, 0.075 mm in diameter (Medwire Corp, Mount Vernon, NY). The recording areawas obtained by carefully stripping the Teflon coat off the tip of the wire strands toavoid nicking the wire, then warming the remaining teflon against a soldering iron to177produce a smooth, continuous border between the bared tip and the insulated part ofthe wire, and cutting off the bared tip so that it was 1 mm long (Loeb and Gans, 1986).This end was then bent to form a 2 mm hook, which should anchor the wire in position.The wire was inserted percutaneously into the muscle with the aid of a 27 gaugehypodermic needle. The electrode is designed to combine symmetrical, omni-directionalproperties with a decreased shunting effect (due to its large tip size) in order to recordhigh-amplitude, high-frequency, intramuscular signals. A conventional skin-patchelectrode, 30 x 15 mm (Lec Tec Corp, Minnetonka, MN) was fixed to the skin overlyingthe muscle and was used as the indifferent. The signals from both electrodes weredifferentially amplified using a bandpass filter with -3 dB roll-offs at 300 and 3000 Hz.At maximal muscle contraction, the spectral peak of surface EMG recorded from themasseter muscle is about 78 Hz, although with lesser degrees of activation a second peakmay appear around 130 Hz (Palla and Ash, 1987). These components are, therefore,removed by bandpass filtering. The myoelectric signals are then sampled digitally.To evaluate whether this system would be useful for quantifying regionalactivity in the jaw muscles, the following protocol was used. Two single-wire electrodeswere inserted into a subject’s right masseter muscle. One was placed approximately 10mm into the superficial body of the muscle and the other about 12 mm into thepreauricular trigonum to reach the muscle’s deeper, posterior fibres. The determinationof the wire location was based on known differences in the anatomical sites of superficialand deep parts of the masseter muscle and direct palpation. Individual patch surfaceelectrodes were placed on the overlying skin in each case. Although a common178indifferent surface electrode could have been used, it was found that separate electrodesyielded improved signal to noise ratios. For comparative purposes, a pair of silver surfaceelectrodes (Grass Instrument Co, Quincy, MA) were placed 20 mm apart over the centreof the muscle. A ground electrode was fixed to the back of the neck. All signals wereamplified differentially (Al 2130 amplifiers, Axon Instruments mc, Burlingame, CA)using bandpasses from 300-3000 Hz (wire electrodes) and 30-1000 Hz (surfaceelectrodes).The subject was asked to carry out a series of brief tooth-clenching tasksdesigned to differentially activate the two regions of the muscle. These tasks consistedof maximum clenching with the teeth in full intercuspation, with efforts directedvertically (perpendicular) to the plane of dental occlusion, anteriorly (protrusively) andtowards the left and right sides.The signals were sampled every 0.1 msec by a data acquisition system andmicrocomputer (HP 3852 A and HP 350, Hewlett-Packard Canada, Vancouver, Canada)over manually triggered sampling periods of 2 seconds. For each electrode, the datarecorded for each task carried out were rectified and smoothed with a low-passButterworth digital filter at 10 Hz cut-off frequency as shown in Figure 32 (Scott et a!,1983). These data were then normalized to the maximum value recordable from thatelectrode irrespective of task. One hundred data values representing the task epoch(between 400 and 1500 msec of the sampling period) were downloaded to amicrocomputer for graphical display and statistical analysis. Data from the threedifferent muscle sites were displayed in an X,Y plot in order to test their relationship179Figure 32 Rectified, digitized data from three muscle sites, obtained during avertically oriented maximum tooth clench. Smoothed waveforms resulting from a 10 Hzdigital Butterworth ifiter have been superimposed in each case. The task epoch isdefined by the horizontal bar. The horizontal calibration bar represents 200 msec, andthe vertical bar represents 360 JW, 510 V and 500 jV for surface, deep and superficialmuscle regions respectively.SURFACEDEEPSUPERFICIAL180by means of linear regression. This test was chosen since regions of the muscle whichbehaved identically would be expected to show linearly related responses for the sametask, and a regression line with a zero intercept and a slope of unity.To test the hypothesis that the human masseter is divided into at least threefunctionally separate regions, three single-wire electrodes were inserted into the subject’sright masseter muscle during a separate recording session. One was placed approximately5 mm into the deep portion of the muscle via an intra-oral approach. The loose end ofthe wire was then fixed against the upper canine tooth to avoid the possibility of thesubject biting on it. The second wire was placed approximately 12 mm into thepreauricular trigonum to reach the muscle’s deep posterior portion, while the third wirewas placed about 10 mm into the centre of the inferior bulge of the superficial masseter(Figure 33). Individual reference patch surface electrodes were placed on the overlyingskin, and a ground electrode was fixed to the back of the neck. Surface electrodes wereplaced 20 mm apart over the centre of the muscle. All signals were amplified andbandpass-filtered in the same fashion as described above.The subject was again asked to carry out a series of brief tooth-clenching tasksdesigned to differentially activate the three regions of the muscle. These tasks consistedof maximum clenching with the teeth in full intercuspation, and with efforts directedvertically to the occiusal plane, posteriorly, anteriorly, towards the left and right sides,and towards the right antero-lateral side. In addition, the subject was asked to grind theteeth from the right canine edge-to-edge position, past the intercuspal position, all theway to the left canine edge-to-edge position, and then to grind again in the opposite181Figure 33 Diustration of the right masseter muscle in the lateral and frontal views,showing wire electrode location.Coronal section182direction. Recorded signals were sampled and stored as previously described.3.322 ResultsRectified, digitized data from the first test are shown in Figure 34 for all threeelectrodes both during rest and during three clenching tasks. Each record represents asingle clench. The greatest amount of activity for both wire and surface electrodes wasrecorded when clenching was carried out perpendicular to the dental occiusal plane. Amarked separation between superficial and deep masseter intramuscular responses wasseen when efforts were directed towards the left and right sides. These responses wereconsistent with previous reports of masseter muscle behaviour (Belser and Hannam,1986).In Figure 35, X,Y piots of normalized muscle activity are shown. Although thedeep and superficial muscle portions behaved similarly during the intercuspal toothclench, the deep masseter muscle was almost exclusively active during a right-directedclench (on the ipsilateral side) while it was less active during a left canine tooth clench.The surface electrode preferentially selected activity from the superficial fibres of themuscle at low levels of contraction, but appeared to reflect activity from another sourceas contraction efforts increased (Figure 35), (Guid et a!, 1973).Figure 36 shows oscilloscope traces of muscle activity recorded from theanterior deep, posterior deep and superficial masseter regions, as well as activityrecorded with a surface electrode during static clenches. As shown before, the greatestamount of activity for the wire and surface electrodes was recorded when clenching was183Figure 34 Rectified, digitized data from three electrode sites. The superficial anddeep recordings were obtained from single-wire electrodes, and the surface record frombipolar silver discs. Rest activity 1) and maximum clenches were directed towards theright side 2), anteriorly 3) and vertically 4). Responses recorded from the skin surfaceand superficial parts of the masseter muscle have an increasing pattern of activity from1-4, while the deep masseter muscle behaves differently. The horizontal calibration barrepresents 400 msec, and the vertical bar represents 720 J.N, 1030 V and 1000 jV forsurface, deep and superficial regions respectively.SURFACE 1SUPERFICIAL3u1k1Jw.Ji1iiLiL JilL.DEEP2L. I4184Figure 35 Smoothed, normalized data from three electrode sites plotted against eachother for three representative tasks. In each case, the stippled line represents the idealrelationship between the two variables, assuming that they behave identically. The solidline represents a regression line fitted through 100 data points (p <0.001). The graph in4 represents the relationship between surface EMG activity and superficial masseteractivity. Spm: superficial masseter; Dnim: deep masseter muscle; Sfm: surface masseter.100INTERCUSPAL. CLENCH60100RIGHT MOLAR CLENCH40so2060>0EE1304°0 20 40 60 so too4-4-0EE>.4-0EE24Spm Activity (%)LEFT CANINE CLENCH01000 20 40 60 5020100100Spm Activity (%)LEFT CANINE CLENCH6040/20////—. so> 60 . F.:.,4••0 20 40 60 50 100Spm Activity (%)00 20 40 60 50 1008pm Activity (%)185Figure 36 Raw data from four muscle sites, obtained during different clenching tasks.The horizontal calibration bar represents 200 msec, and the vertical bar represents 500jV. Tasks are intercuspal position (ICP), posterior (P), left (L), anterior (A), anteroright (A-R), and right (R) clenches. aDM: anterior deep masseter; pDM: posterior deepmasseter; SM: superficial masseter; Surf: surface electrode recording.aDMpDMSMSurfaDMpDMSMSurf186carried out in the intercuspal position. Both anterior and posterior deep masseter regionswere active during posterior- and right- directed clenching, while the superficial masseterwas almost silent. In contrast, during left- and anteriorly- directed clenching, thesuperficial masseter was more active than the other regions. During the right anterolateral clench, the distinction between muscle regions was not so obvious. During thistask, and to a lesser extent during the right lateral clench, the anterior deep region wasmore active than the posterior deep masseter.Figure 37 shows oscilloscope traces of muscle activity recorded with all threewire and surface electrodes during two dynamic tasks. Maximum myoelectrical activityis obtained at the centre of each burst, approximately when the teeth are in theintercuspal position. At the beginning and end of a left- directed grind, the superficialmasseter is more active than the other muscle portions, especially when the teethapproach the contralateral (left) canine edge-to-edge position. Initially during the right-directed grind, rapidly increasing muscle activity is observed in the superficial masseter.At the interocclusal position, slightly more activity is seen in the posterior than in theanterior deep masseter. Towards the end of the grind (ipsilateral side), the myoelectricactivity pattern reverses. The posterior deep masseter shows the highest level of activityfollowed by the anterior deep portion. Almost no muscle activity can be seen in thesuperficial masseter at this point in the movement.3.32.3 DiscussionThe single-wire approach for recording jaw muscle activity avoids the187Figure 37 Raw data from four muscle sites, obtained during two distinct grindingmovements. The ho±ontal calibration bar represents 200 msec, and the vertical barrepresents 500 j.V. Tasks are two grinding strokes from the right canine edge-to-edgeposition to the left canine edge-to-edge position, and from the left canine edge-to-edgeposition to the right canine edge-to-edge position. Abbreviations are the same as inFigure 36.aDMpDMSMSurf188reproducibility problem posed by variable interelectrode distances commonly associatedwith conventional bipolar wire electrodes. While the regional selectivity heredemonstrated is theoretically less than that for paired-wire systems, it seems to besufficiently focal to distinguish major muscle subgroups. The signals consist mainly ofmultiunit interference patterns, although single MUs can occasionally be identified. Thepeculiarity of the technique permits several different analytical procedures, e.g. turnanalyses and estimations of integrated activity, in addition to the low-pass digital filterdemonstrated here. Although the approach described produces interference signals notunlike those of surface electromyograms and can be analyzed in a similar fashion, it doesnot share their wide-field sampling characteristics, and the signal-to-noise ratio obtainedis consistently good due in part to the low-frequency cut-off. At low levels of musclecontraction however, single-wire electrodes, like bipolar indwelling electrodes, tend toproduce the clumped discharges of a few MUs and sometimes single MU spikes. Burstswith high frequency components and large interdischarge intervals are difficult toquantify reliably by the filtering and integrative techniques commonly used ininterference EMG.It has been often assumed that when bipolar fine-wire electrodes areselectively placed and closely spaced, APs are recorded from only a small population ofactive muscle fibres. In addition, when recordings are made differentially with highquality amplifiers, it is expected that the EMG records do not contain significantelectrical activity from other sources, such as surrounding muscles (Loeb and Gans,1986). It is known, however, that when muscle fibres contract, they produce action189currents which spread by volume conduction (resistive and capacitive) through alladjacent tissues (Gydikow et a!, 1982; Loeb and Gans, 1986). Therefore, any indwellingEMG electrode may record APs from muscle regions other than the region under study.Although such “cross-talk” has been found to be minimal in some studies (Fetz andCheney, 1980) recently it has been suggested that it could be quite significant, especiallywhen records are made from small muscles or from small animals (Mangun et al, 1986;O’Donovan et a!, 1985; Bawa et a!, 1986). In an attempt to examine the extent of crosstalk in the cat lateral gastrocnemius, English and Weeks (1989) found that when aneuromuscular compartment was stimulated, small potentials resulting from passiveconduction could be recorded in adjacent muscle compartments. Sometimes, theamplitude of the cross-talk signals recorded from the activation of large MUs could beas large as small units present in the compartment of residence. In addition, whenelectrodes were placed at compartment boundaries, no clear compartment selectivity wasfound.In humans, it is very difficult to evaluate the extent of signal contaminationby cross-talk. While direct neural stimulation of the masseteric nerve (Dao et a!, 1988)and concomitant recording of different muscle regions are theoretically possible, it ispresently difficult to stimulate a nerve branch which clearly innervates a single musclecompartment. Another approach could be to stimulate a group of muscle fibres directly,and record from different muscle areas in order to demonstrate “cross-talk”. In thisthesis, however, such lines of investigation were not pursued.In conclusion, even though fine-wire electrodes may be extremely useful in190kinesiological studies, they have limitations. Their advantages include the fact that theseelectrodes are extremely fine and therefore almost painless, they remain in place duringjaw movements and strong muscle contractions, and their electrode tip can be adaptedfor broad or focal pick-up area (i.e. interference-pattern or single-MU EMG studies)within a specific muscle region. In addition, if Teflon-coated silver wires are used theycould be located three-dimensionally when detected by a combination of frontal andlateral radiographs. However, precautions need to be taken in the design of experimentsin which wire electrodes are used to study various masseter regions simultaneously, dueto the potential for cross-talk.During the course of this investigation, an extensive interference EMG studyof regional activity in the human masseter was published by Blanksma et a! (1992).These workers used multiple paired-wire electrodes inserted into six regions of themuscle, and concluded that the masseter can certainly be divided functionally intosuperficial, anterior deep and posterior deep parts, and possibly additional ones. Thelimitations of the paired-wire approach and its analysis however have already beendiscussed. Similar limitations disclosed by the present study argue for the use of a morediscriminatory approach for recording focal muscle activity in the masseter. Since thesingle MU represents the most discrete functional entity within the muscle, it wasdecided to pursue further physiological studies at this level, and to avoid the quantitativeproblems associated with the analysis of multiunit discharges.1913.3.3 Motor-Unit BehaviourThe goals of these investigations were to develop methods for recording MUactivity associated with different voluntary oral tasks, and to establish the reproducibilityof the measurements under controlled experimental conditions. Since the long-term aimwas to compare the behaviour of a population of units selected from different parts ofthe masseter during the performance of various physiological tasks, it was consideredimportant to confirm that stable measurements of MU performance were possible. General Methods3. Motor-Unit RecordingActivity from multiple low-threshold motor units (MUs) located in the centralpart of the superficial right masseter muscle was recorded with monopolar needleelectrodes (MF37, TECA Corp, Pleasantville, NY) from eight healthy adults. Theseneedles were made of stainless steel, measuring 0.35 mm in diameter (28 gauge) and 37mm in length. With the exception of the electrode’s conically-sharpened recording tip(area = 0.12 mm2 ), the needle was insulated with Teflon. A surface reference electrode(SynCor, Eec Tec Corp, Mimietonka, MN) was placed over the masseter muscle. Asecond surface patch-electrode was fixed to the back of the neck to act as a ground.During each session, the needle was inserted percutaneously at right anglesto the skin surface, and its hub supported by an extraoral framework to stabilize theelectrode’s position (Figure 38). Subjects were asked to lightly activate the masseter (lessthan 10% of maximum voluntary contraction), while the needle was gently moved192Figure 38 Motor-unit recording technique in the masseter muscle. A surface patch-electrode with a hole in its centre is attached to the skin overlying the right massetermuscle to act as a reference. A perforated metal platform is positioned over the patch-electrode and fixed to the skin by adhesive tape. A monopolar needle is then insertedinto the muscle through the reference electrode’s perforation, and stabilized in situ bythe platform.193inwards and outwards along a medio-lateral trajectory until an action potential wasobtained. These were amplified and bandpass-filtered between 300 Hz and 5 kHz(Model Al 2010, Axon Instruments, Burlingame, CA). Furthermore, the signals weremodulated with a variable gain control to allow continuous adjustment of spikeamplitude prior to high-speed sampling.Although it has been postulated that physiological signals may have high-frequency components up to 30 kHz, spectrum analysis of the action potential generatedwhen a single cell depolarizes indicates that essentially all of the signal is producedwithin the frequency domain from DC to 10 kHz. Since compound-action potentials(CAPs) are the sum of several single-cell action potentials, the frequency domain ofextra-cellular physiological signals should be equal or less than the frequency domain ofthe single-cell action potential. Hence, monitoring CAPs with amplifiers at thebandwidth of DC to 10 kHz should be adequate. The signal components of a single MUCAP is also determined by the type of EMG electrode being used to record them, by thedistance between the recording area and the active muscle fibres, and by volumeconduction. Because of this latter property, slow-frequency components on an EMGrecord may have in fact been originated in quite distant regions (Stálberg and Trontelj,1979). For this reason, low-cut filters have been successfully used to improve theselectivity of EMG recordings, while still preserving important waveform components ofthe CAPs (Gath and Stãlberg, 1976; Stâlberg and Trontelj, 1979).In the masseter muscle, an amplifier bandwidth of 300 Hz to 10 kHz has beenconsidered potentially useful when monopolar needle electrodes are used in experiments194Figure 39 Effects of different bandpass-fflters on a single MU waveform shape. CAPsof a masseter muscle unit were averaged (n = 16) for the display of waveform changesdue to different amplifier bandpass-filter settings. In (a) the bandwidth was 10 Hz- 10kHz, in (b) 10 Hz - 5 kHz, and in (c) 300 Hz- 5 kHz.0.4 mV5 msa b C195requiring quantification of unit firing patterns (McMiIlan, 1989). This filtering procedureyields a good signal-to-noise ratio and preserves an adequate definition of waveformsingularities for spike interval analysis. In the present work, digital sampling rates wererestricted to a maximum of 12 kHz. Hence, the high-cut ifiters were lowered to 5 kHzto avoid the problem of aliasing during sampling.Changes in the waveform shape of a CAP, recorded from a continuously firingmasseter single MU in a normal subject when the filter settings were changed, are shownin Figure 39. When the high-cut filter setting was altered from 10 kHz to 5 kHz with thelow-cut filter remaining constant at 10 Hz, the amplitude of the signal did not lowersubstantially, nor did high frequency components change dramatically. When the low-cutfilter was changed from 10 Hz to 300 Hz, while the high-cut ifiter was maintained at 5kHz, almost no change in signal amplitude was observed although slower frequencycomponents were reduced. These findings are in agreement with previous reports(Stâlberg and Trontelj, 1979; McMillan, 1989), and the change to a lower high-cut filterwas not considered to affect either the signal-to-noise ratio or the signal waveformseriously. Bite-Force MeasurementStudy casts were obtained for each subject and mounted on a Denar Mark IIsemi-adjustable articulator (Denar Corporation, Anaheim, CA). These were used toconstruct a bite force device which could be inserted between the teeth with minimumjaw separation. The device included a strain-gauge transducer, which fitted a slide-in196Figure 40 Sagittal and frontal proffles of the skull showing location and orientationofbite force measurements. The MU recording site is indicated by the monopolar needleelectrode inserted into the right masseter muscle, portrayed in the sagittal and frontalviews. The functional occiusal plane is illustrated as a straight line in the sagittal view,and the location and orientation of the force measurements are pictured in arrows. Notethat the mandible is rotated in a minimal jaw opening at the midline, and that the bitingforces are applied perpendicular to the functional occlusal plane.197acrylic housing cemented to the mandibular canine tooth, and an opposing acrylicplatform cemented to the maxillary canine. The device was customized to each subject,so that isometric biting forces could be recorded perpendicular to the occiusal plane atthe right and left canine teeth with the jaw positioned in the midline (Figure 40).The strain-gauge transducer was built with two parallel stainless steel bars,measuring 1.5 mm in thickness and 13 mm in width. An overload stopper wasincorporated between the bars, separating them by 3 mm and creating a 30 mm longcantilever. Two strain-gauges with 120 Ohm resistance were mounted on opposite sidesof one of the bars (beam) at the portion that is subject to bending strain. Under theseconditions, when force is applied, one strain gauge is subjected to tensile strain while theother experiences compressive strain. Since both gauges are affected by intra-oraltemperatures in the same way, the bridge remains balanced for temperature variationsand the gauges provide twice the sensitivity of one gauge for beam deflections.To ensure a uni-directional force application through a constant point on thetransducer, a spherical load point was permanently fixed to the sensor. Calibrationstudies were performed to determine the reproducibility and linearity of the transducer.A bench-mounted calibration system permitted various loads to be applied to thetransducer (Universal Testing Machine, Instron Model 4301, Canton, Mass). Thetransducer was capable to record forces up to at least 30 N, although the working rangeof canine bite forces sufficient to activate single MUs was consistently less than 10 N(Figure 41).All forces were recorded with the strain-gauge transducer, which could be198Figure 41 Calibration of the force transducer. Force in Newtons (N) plotted againstvoltage (V) for a series of loads applied perpendicular to the force transducer. A linearregression line was fitted through the plotted values.4030zC)0LL100I I0 10 20 30 40Voltage (V)199Figure 42 Illustration of the Bite Force Transducer in situ. The device was slid intoa metal sleeve within the cantilevered support of the mandibular canine tooth housing.The position of the load button was constant with respect to the opposing maxillaryhousing. Matched copings were made for the canine teeth on the right and left sides.Maxilla CanineMandibular Canine —— Bite Force_— Load ButtonCantilever Support200housed in either of two customized, labially-cantilevered supports temporarily cementedbilaterally on the mandibular canine teeth at minimum occiusal separation (5 mmmeasured at the incisors). Its spherical load point opposed a flat platform on matchingsupports on the respective opposing teeth (Figure 42). Thus it was possible to change thelaterality of contact while still using the same sensor. Force signals were amplified witha carrier amplifier (Tektronix mc, Beaverton, Ore) and bandpass-filtered between DCand 300 Hz. Sampling and Data AnalysisMU activity and bite force were initially sampled simultaneously onto tape(Unitrade, Data Acquisition and Storage System) at 48 kHz, and then sampled off-lineat 12 and 1 kHz, respectively, by a data acquisition and microcomputer system designedfor this purpose (Discovery, Brainwave Systems Corp, Denver, Co). Individual recordswere analyzed off-line to determine the firing pattern, recruitment threshold andsustained force levels of all the single units recorded during the separate tasks. For thispurpose a combination of commercial software (Discovery, Brainwave Systems Corp,Denver, Co) and custom-written software was used. Motor-unit CAPs in each recordwere discriminated by the identification and discrimination of waveforms with differentshapes. The method is based on extracting waveform parameters (i.e. peak amplitude,valley amplitude, spike height and width, template match, etc.) for each waveform. Thesecan then be used to describe the difference between various groups of waveforms bydisplaying various combinations of waveform parameters in X/Y point plots. Waveforms201which share similar features form “clusters” of points on these X/Y point plots. Theseclusters are then assigned into different groups (or MUs) for further analysis. Spiketrains were then examined visually for reliability. Data were rejected if more than 5spikes were unclassified due to recognition errors. Additionally, for each unit, interspikeintervals (ISIs) smaller than 20 ms and exceeding 200 ms were excluded from thecalculation of the mean ISI and standard deviation, to avoid the inclusion of falsepositive spikes, or errors of spike recognition. This last procedure minimized the effectof recognition errors on the firing rate statistics (Eriksson et a!, 1984; Nordstrom andMiles, 1991a). In single MU records, this method allowed reliable recognition of theunit’s waveforms with only occasional discrimination errors. In multiple unit records,however, some additional spikes were missed due to occasional waveformsuperimposition of synchronous CAPs of different units.For each unit, the mean ISI and standard deviation were calculated from 200intervals obtained from 201 consecutively occurring CAPs in the middle of each EMGrecord (MeMillan and Hannam, 1992). The mean bite force and standard deviation werealso calculated over this period of time.3.3.32 Effect of Bite Side on MU BehaviourThe complexity of the masseter muscle, and the bilateral tasks to which itcontributes suggest that MU properties such as recruitment threshold and the regularityof sustained rates of firing might alter according to task. In previous studies, it has beenassumed that the twitch tension produced by a motor unit (MU) in the masseter muscle202is directly transferred to the teeth (Nordstrom et at, 1989). Based on this assumption,when a MU in the right masseter activates, its tension should be identically transferredto the right or left canine teeth, provided that the location and angulation of therecorded bite force are the same when viewed sagittally. However, for tasks which arecarried out by more than one muscle, and for muscles which are compartmentalized,differences in the activation of separate muscles and in regional activation of themasseter may severely affect this coupling from unit through intramuscular aponeurosesto the endpoint at the tooth. For example, reduced or no forces may be produced at theteeth during coactivation of elevators and depressors even though elevator MUs areactive; and in the case of regional muscle activation, the transfer of MU twitch tensioncould be affected by differential intramuscular forces produced by nearby compartments.Thus, heterogeneity may result in different forces produced at the level of the teeth asbite strategy changes. If this is the case, the contribution of a given unit to bite force maydiffer due to any muscle length change (Miles et a!, 1986), a change in location of thebite point with respect to the unit (due to different lever arms), and as a consequenceof altered inter- and intra- muscular mechanics upon differential muscle activation. Inan experimental situation in which bite-force-lever arms are identical in the parasagittalplane, it is possible that during slow increasing followed by sustained MU firing,recruitment thresholds and sustained bite forces may differ, because of dissimilarintramuscular unit recruitment patterns which change with task. In the present study, thispossibility was addressed by analyzing the functional performance of a sample of low-threshold MLJs in the superficial region of the masseter muscle when they contributed203to two different, symmetrical, unilateral biting tasks. Of special interest was thequantification of the effect of sidedness, as well as the reproducibility of MU behaviourwhen the same task was repeated. The study required a more rigorous analysis of jawmuscle MU behaviour than has been usual in previous investigations. MethodsTo test for the effect of bite side on MU behaviour, the subjects wereinstructed to carry out three tasks: two separate sustained biting tasks with thetransducer placed on the right side, and one sustained biting task with the transducer onthe left side. The order in which tasks were performed was changed randomly. For eachtask, the subject was asked to alter the force of his or her bite slowly through MUrecruitment threshold, and then with the aid of auditory and visual feedback, to attemptsteady MU firing for approximately 30 s first at 10 Hz, then at 15 Hz (Figure 43). Thevisual feedback target consisted of triggering the spikes on an oscilloscope andinstructing the subject to maintain the MU firing at such a rate, that spikes would fallwithin a marked bandwidth on the monitor (100 ± 10 ms; 67 ± 7 ms). During multi-unitrecordings, a window discriminator was used to separate the “target unit” from thebackground units. During changeovers, the maxilla-jaw relationship was kept unchangedwith the aid of a bite block and the MU was driven without significant pauses. On a fewoccasions, the needle was gently repositioned to retain maximum amplitude of the CAP.To ensure that tasks were performed by the same unit, the waveform shapes of the CAPswere always compared.204Figure 43 Recruitment and firing pattern of a masseter MU at different bite forcelevels. Recruitment of a masseter MU, (centre traces). Five levels of bite forcemeasured on the right side of one subject are shown, (lower traces). These increase fromleft to right, passing from rest, through the recruitment threshold, and include efforts tomaintain unit firing at 10 Hz and 15 Hz. Variable gain was used to modulate spikeamplitude (arrows) and facilitate MU discrimination, (centre and upper traces). Thespike components shown were discriminated by cluster-cutting mixed-unit responses.$1’!H H4NIvRES1 ThRESHOLD 10Hz 15Hz205For each task, five functional properties of the MUs were assessed:recruitment threshold, coefficient of variation of the firing rate, accuracy index (i.e.difference between the target ISI and the performed mean ISI), mean sustained biteforce, and a sensitivity index (i.e. defined as the slope of the curve describing therelationship between mean ISI and bite force).To test for the reproducibility between both right-sided bites, and to test forthe difference between right and left-sided bites, repeated measures analyses of variancewere carried out for the entire data sample of the following measures: recruitmentthresholds, mean sustained bite forces, coefficient of variation of the firing rate, and thesensitivity index. Linear regression analysis was also performed to test for significantrelationships between the variables.Although subjects were instructed to control the mean ISI of the feedbackunits at 100 ms, their ability to control the MU firing was not precise. To compare thevariability of MU discharge for different tasks, it is therefore necessary to normalize thevariance measure to a mean ISI of 100 ms for each unit performing each task. Toachieve this, an adaptation of a previous method suggested by Eriksson et a! (1984) andNordstrom and Miles (1991a) was employed. For each unit, all three datasets containing200 intervals were used. Mean and standard deviation (SD) were calculated for a slidingdata window defined at a width of 11 intervals and a slide increment of 1 interval.Resulting mean values were then grouped into equally sized bins (5 ms each). The meanvalue of the SD values in each of these classes was then calculated. The median valuesof each bin and the mean SD values were plotted against each other, and a linear206regression performed. The equation of the linear regression was then used to estimatethe value of the SD at a mean ISI of 100 ms. Provided that the 100 ms was within therange of mean ISI values used in the calculation of the linear regression, this finalmeasure of variability of the unit, the estimated SD, was considered reliable. This wasthe case for 23 units which were included in the analysis. The estimated SDs from allunits were grouped by task and linear regression analysis performed to test for therelationship between MU firing variability in the different tasks.To compare MU firing rates between tasks, separate one-way analyses ofvariance were used. Since it is known that the distribution of ISIs in a slow firing unitis skewed to the right (Person and Kudina, 1972; Derfier and Goldberg, 1978), thedatabase was normalized by natural logarithmic transformations (McMillan and Hannam,1992). Each unit was initially tested for the reproducibility of MU characteristics duringright-sided biting (which were presumed to be identical), and for differences between theright and left-sided biting by analysis of variance followed by Tukey’s test. Statisticallysignificant differences were then analyzed for their practical implications. For thispurpose, the differences between the median values of the ISIs of the tasks werecalculated for each unit. A difference of ± 10 ms (i.e. between 9 and 11 Hz) and of ±7 ms (i.e. between 14 and 17 Hz) was not considered important (Samuels, 1989). Inaddition, the median value of the ISI of each unit was subtracted from the “target value”(i.e. from 100 ms [10 Hz], and from 67 ms [15 Hz]) for all tasks. Again, differences of± 10 ms and ± 7 ms were considered practically insignificant.2073.3.32.2 ResultsFifty-one MUs were included in the analysis. Figure 43 shows the typicalcontribution of a MU to bite force. It was recruited at 1.2 N and as its firing rate wasincreased, a proportional increase in bite force was obtained. In most experiments theMUs clearly participated in the different tasks, but their behaviour could not always beassessed due to non-discriminable summation between units. In some experimentscompletely different units became active when the bite side was changed; and in fivecases, units unequivocally fired on one side only. Therefore, the total number of unitsvaried in the different population samples.During right- and left- sided bites, the recruitment thresholds of 29% and 44%of the units, respectively, were too low to measure (i.e. these units did not contributedirectly to these particular bite forces). Recorded threshold values ranged from 0.3 N to12 N, and were neither reproducible between the right-side bite repetitions nor showedconsistent task-related differences when the right- and left- side bites were compared(Figures 44 and 45). This was confirmed by statistical analysis of the entire data sample.Repeated-measures analysis of variance did not reveal significant differences betweenthe mean recruitment thresholds for each task (p = 0.5), and regression analysis revealedthat the highest correlation occurred between the two right-sided tasks (R = 0.73;R2 =0.53; p <0.0001), accounting for the reproducibility in only 53% of the sample.Between the recruitment thresholds of the right and left sided bites, the Pearsonregression analysis revealed a lower correlation (R = 0.67; R2 = 0.45; p <0.0001).Similar to the behaviour of recruitment thresholds, repeated-measures analysis208Figure 44 Distribution of recruitment thresholds by task. The data are shown as boxplots. In each case, the centre horizontal line represents the median of the measuredthresholds. The central box spans the interquartile range. The whiskers include valueswhich fall within 1.5 times of this range. Outside values are plotted as asterisks andempty circles. The numbers below each box indicate the sample sizes for the respectivetasks, which include two right-sided biting acts (Ri and its repeated version, R2), anda left-sided one (L).8 I I06z..*40)I-.fl_LL 1____41 52—2 IRi R2 LTask209z0a)3-ia)j3-4C)a)Figure 45 Recruitment thresholds arranged according to task for 10 MUs from fivesubjects. Abbreviations as for Figure 44. No consistent trend is evident as the taskchanges.765432In=1Os= 50Ri R2 LTask210of variance did not reveal significant differences in mean bite forces with task for unitsdriven at 10 Hz (p = 0.49) and at 15 Hz (p = 0.80). Regression analysis revealed that whenthe right side bite was repeated with units firing at 10 Hz and 15 Hz, mean sustainedbite forces were correlated at R=0.59 (R2 =0.34; p<O.0001) and R0.42 (R2 =0.18;p <0.01), respectively. When the right bite was compared with the left bite, it was foundthat at 10 Hz the data was correlated with R = 0.39 (R2 = 0.15; p = 0.03), and at 15 Hz themean bite forces were not correlated (R = 0.22; R2 = 0.05; p = 0.23). The relationshipbetween mean bite force and mean ISI when the target rate for one task changed from10 Hz to 15 Hz, is shown in Figure 46. Here, 14 MUs were selected from one subjectwho was biting on the left side. In most cases, bite force increased as the ISIs shortened,i.e. as the firing rate increased, but in two units there was an actual decrease in biteforce. The majority of units made a positive and roughly similar contribution to biteforce, irrespective of task. The overall mean sensitivity index (slope) was 60 mN/ms. Thehighest number of small, unmeasurable changes in bite force occurred on the left side.Variations in the patterns of discharge (expressed by the coefficient ofvariation, and arranged according to task) are shown in Figure 47. Consistently, the ISIsvaried less for tasks performed at 15 Hz than for those at 10 Hz. Repeated-measuresanalysis of variance of the entire data sample failed to reveal significant differencesbetween the mean coefficient of variations of the three tasks when attempts were madeat either target rate (p >0.05). Regression analysis revealed that the discharge variabilityin a unit performing different tasks was correlated. When the right side repeats and theleft versus right side variability were plotted against each other, correlation coefficients211Figure 46 Changes in bite force when attempted MU firing at 10 Hz is increased to15 Hz. Data are shown for 14 units from the same subject. The horizontal axis representsthe duration of the inter-spike interval (ISI) which is the reciprocal of the instantaneousfiring rate. The open and closed circles are paired measurements from the same units.In each case, the lines connecting them show the relationship between the change in biteforce and the change in ISI.I I I In=1 40 10 Hz‘I- •l5Hz40 60 80 100 120 140 160Mean Inter—spike Interval (ms)212Figure 47 Distribution of Coefficients of Variation arranged by task for attemptedMU firing rates of 10 Hz and 15 Hz. All abbreviations and conventions for box plots asfor Figure 44.I I**050 -40 II *ci>•_ 30-Ri-i 0 Ri-i 5 R24 0 R2-l 5 L-i 0 L-1 5Task213Figure 48 Two-dimensional plot of the IS! standard deviations of the first against thesecond right-sided biting task, and against the left side. The data for the right sidecomparison is presented by filled circles, and data for the right-left side comparison byopen circles. The straight line represents the optimum linear relationship if tasks wereto be identical.0 10 20 30 40 50IS/SD (n,s)214were R = 0.82 (R2 = 0.68; p <0.0001) and R = 0.83 (R2 = 0.69; p <0.0001), respectively (seealso Figure 48).When individual units were tested for reproducibility of firing (n=37), theanalysis of variance revealed statistically significant differences between both right-sidedbites for all units activated while the subjects attempted to drive them at 10 Hz(p <0.05). When the difference between the median firing rates were compared, 45% ofthe units were reproducible. From these reproducible units, 64% were driven at thesame firing rate during both, left- and right- sided tasks, while 23% were faster and 12%were slower on the left-sided task than on the right bite. When the difference betweenthe median firing rates of units being driven at approximately 15 Hz were compared,80% of the units were reproducible during both right-side bites. From these, 84% of theunits had the same firing rate at the left and right bites, while 16% were faster at theleft side. No unit fired at lower rates at the left side. In addition, EMG records duringleft-sided clenching frequently included more units than their right-sided counterpart.When the subjects attempted to drive the units at the two target rates, therewas an equal distribution of mean ISIs above and below the target rates (Figure 49).Subjects frequently reported that it was difficult to drive MUs steadily at 10 Hz, and twosubjects could only maintain steady firing at 13 Hz. The evaluation of unit accuracy indexby task (difference between observed median firing rate and “target°), revealed thatsubjects asked to activate the units at 15 Hz did so more accurately (i.e. with lessvariation) than at 10 Hz. When the subjects were asked to activate their units at 10 Hz,only 29% of the units were accurate irrespective of task, while when they were activating215E000Figure 49 Distribution of “Accuracy Indices” arranged by task for attempts at twodifferent rates of MU firing. The data are presented as box plots. The open boxesrepresent attempted firing at 10 Hz, while the filled boxes represent attempted firing at15 Hz. All other abbreviations as for Figure 44.I - 1 I I*100500—500 037 38 35 37 42 41—100Ri-i 0 Ri-i 5 R2-1 0 R24 5TaskL-10 L-15216units at 15 Hz, 60% and 72% of the units were accurate on the left- and right- sidedtasks, respectively.33323 DiscussionAs for the limb, it is believed that the size principle provides a generaldescription of MU recruitment in the masseter muscle (Goldberg and Derfier, 1977). Ifthe muscle length is kept constant and if the rate of increase in isometric contraction iscontrolled (Miles et a!, 1986), the threshold force for activation of a unit is assumed tobe reproducible. On rare occasions, however, alterations in recruitment order occur(Desmedt and Godaux, 1979). The general acceptance that MU recruitment thresholdsin the limb muscles are quite reproducible when measured under controlled conditions(Freund, 1983) has been questioned. For example, it has been reported that they mayvary with the complexity of the motor task (ter Haar Romeny et a!, 1982), and theexperimental situation (Romaiguère et a!, 1989). Variability in recruitment threshold iscommonly found between different tasks performed by a multifunctional muscle (terHaar Romeny et a!, 1982), especially when other muscles also contribute to the producedforce (Thomas et a!, 1986). Recruitment threshold has been shown to vary with thedirection of application of bite force (Hattori et a!, 1991), and coactivation of other jawmuscles have been shown to alter masseter MU spike-triggered bite force (McMillan eta!, 1990). The present findings that MU recruitment thresholds were neitherreproducible, nor varied consistently with task, are therefore similar to the observationsby Romaiguêre et a! (1989) regarding fluctuations in MU recruitment thresholds. These217authors suggested that motoneuron pooi excitability was greatly influenced by thesubject’s emotional, attentional and physiological state. It is also possible that the presentfindings are the result of co-activation of other jaw muscles. It has been reported thatthe increase of isometric force, controlled by conventional methods of the kind used inthe present study, is never a completely smooth ramp, but incorporate small, rapidfluctuations which are significant determinants of the variation of recruitment threshold(Miles et a!, 1986). This assumption might be especially critical for jaw muscles whichare known to be less developed for pursuit-tracking experiments than for limb muscles(Van Steenberghe et a!, 1991), possibly because the jaw muscles are not designed forlong sustained bite force tasks during natural function. Hence, the input to the variousmotoneuron pools may fluctuate, so that units belonging to different muscles and todifferent groups within a muscle may together achieve the proposed task. It istheoretically possible that the transducer system itself contributed to recruitmentthreshold variability, but this is unlikely. Care was taken to ensure that only one sensorwas used, and that it always sensed forces at the same load point. The device’s locationand orientation were fixed relative to the opposing teeth, and ensured by its assemblyon articulated dental casts.The majority of units made a positive and roughly similar contribution to biteforce, irrespective of task. It is known that recruitment of MUs is not the solemechanism contributing to the total force production by a muscle, but that increase inMU firing rate also contributes to force increase. Similar mechanisms are used by MUsin the masseter muscle, where there is the tendency for MUs with the lowest recruitment218thresholds to show the largest rate changes per kilogram of interocciusal force change(Derfier and Goldberg, 1978). Thus, during small inter-occlusal force production, it isexpected that several MUs are recruited simultaneously to achieve significant forceincreases. The present study showed that the highest number of small changes inrecruitment threshold and sustained bite force, the largest number of MU recruitment,and the largest tendency for MUs to be driven faster occurred during left-sidedclenching. Provided that the original assumption is correct (that the lever arms for bothbite force points are identical), it is proposed that a common strategy to achieve left-sided bite forces is to recruit a larger number of units. Concurrently, small units willshow larger increases in firing rate because of the increased excitatory input onto themotoneuron pooi. That is, in this task the excitatory drive, responsible for therecruitment and frequency coding of MUs in the right superficial masseter muscle, hasa different weighting factor than when a subject performs a right-sided bite. Thissuggestion is in accordance with Ter Haar Romeny’s et a! (1982) report for the bicepsmuscle.The majority of MUs studied were polymodal, whereas a few units wereselectively activated depending on the task being carried out. This finding concurs withprevious reports on multitask units in limbs (Thomas et a!, 1986; 1987; Ter HaarRomeny et a!, 1982) and in the human masseter (Eriksson et a!, 1984; McMillan andHannam, 1992). However, the presence of task-specific units, while also reported byEriksson et a! (1984), was not confirmed by McMillan and Hannam (1992). This lack ofagreement might be explained by the fact that while in the present study subjects were219able to activate units at two very specific bite points, in the latter work they wereencouraged to attempt an array of intraoral tasks until MU firing was resumed.The lowest steady firing rate (LSFR) at which subjects are able to drivemasseter MUs seems to be greater than the LSFR at which subjects commonly drivetheir units in limb muscles (Petajan, 1981). It has been indicated that it is difficult todrive MUs consistently, without significant pauses below 8 Hz (Nordstrom et a!, 1989;Eriksson et a!, 1984), and that the LSFR may also vary with intraoral task (McMillan andHannam, 1992). Better maintenance of consistent ISIs is thus expected at higher ratesof firing, since the variability of spike discharge increases exponentially when the ratedemanded falls below a critical value (Freund, 1983). For these reasons, the target firingfrequencies of 10 and 15 Hz were chosen to increase the likelihood of dischargereproducibility, and because they were similar to those often used in jaw muscle MUstudies (Miles and Türker, 1986; McMillan and Hannam, 1989b). In the presentpopulation of MUs, the firing rate of units for most subjects was actually 10 Hz or less,whereas for two subjects it was reported to be 13 Hz or more. The lack of any systematicchange in the ability of subjects to drive MUs at either rate when the bite side alterssuggests that most low threshold units in the superficial region of the muscle contributeroughly equally to right and left-sided biting, at least at the low firing rates tested.The regularity and rate of MU firing are determined by the intrinsic propertiesof the motoneuron and by the excitatory drive to the motoneuron pooi, which changeswith variations in central and peripheral inputs (Henneman and Mandell, 1981). In thetrigeminal system, peripheral sensory receptors include muscle spindles, cutaneous, joint220and periodontal mechanoreceptors, and free nerve endings (Dubner et a!, 1978).Periodontal receptors have been located in higher numbers in the anterior region of thedental arch, and are known to be directionally sensitive (Hannam, 1982). They appearto be involved in inhibitory feedback mechanisms to jaw closing muscles (Kidokoro eta!, 1968), and therefore are likely to have played a role in the input modulation to thestudied motoneuron pool.There are several possible outcomes for MU firing behaviour during theperformance of different biting tasks. A given MU may fire for one task but not another;its recruitment threshold may be different from one task to another; the firing rate ata controlled frequency may vary more for one task than another; and the MU maycontribute more vigorously to one task than to another. Properties like these requiresome form of quantifiable measurement if comparisons of MU behaviour between tasks,or between regions during the performance of a standard task, are needed. In addition,the results suggest that when muscle regions are surveyed for task specificity, MUsshould be driven at 15 Hz instead of 10 Hz, and tested for firing reproducibility.Presently, conventional physiological measures of MU behaviour, while perhaps usefulfor qualitative comparisons, are so variable that useful comparisons are impossible oncethe task is changed.In conclusion, very large sample sizes may be necessary to establish any effectof sidedness (or for that matter, task), at least for low threshold MUs. The results alsosuggest that care should be exercised when recruitment threshold or lower rate limit areused as dependent variables to classify jaw muscle low threshold MUs. Examples here221include jaw MU recruitment thresholds, putative unit twitch tensions, and taskcomparisons. The study infers that not only a change in task, but also the repetition ofthe same task after an interval, could produce very different results than those reportedin many previous studies. These conclusions however do not necessarily apply to mediumor high threshold MUs in the masseter muscle, which may have different functionalproperties and which remain relatively unstudied. Effect of Experimental ParaligmSince it has been shown that the experimental paradigm itself can affectrecruitment threshold of MUs in the wrist extensor muscles (Romaiguêre et al, 1989),it was hypothesized that MU firing properties and resultant bite force might have beenaffected by the kind of feedback available to the subjects. The extent to which theexperimental paradigm might have affected the measurements reported above wasinvestigated in the following manner. MethodsAll subjects were instructed to activate a single MU and maintain a steadyfiring rate for approximately 30 s while biting on the force transducer placed betweenthe right canines. Subjects repeated this task four times. The first time, subjects wereinstructed to maintain steady MU firing at 10 Hz with the aid of auditory and visualfeedback (as described in section During this segment of the experiment, the222mean bite-force value was evaluated on-line with the aid of a digital voltmeter. After ashort pause, during which the jaw relationship was maintained, the subject was againasked to reactivate the unit. The second time, instead of auditory and visual MUfeedback, the force record was displayed on the oscifioscope in a fast trace so that onlya horizontal line crossed the entire screen. The subjects were requested to maintain avertical level bite-force based on the previous voltmeter reading. After another shortpause, the subjects were asked to repeat the same task for a third time, with no feedbackwhatsoever. Here, the subjects could only rely on memory. Finally, the subjects wereasked to repeat the task once more, again with visual and auditory MU feedback. Withthe exception of the first nrn, the order at which subsequent tasks were carried out wasrandom. In addition, CAP shapes were always compared for all four tasks to ensure thatthe same unit was retained throughout the experiment.Motor-unit mean firing frequencies and their ‘target” accuracy were estimatedfor each trial as before. Differences smaller than 10 ms were considered insignificant.Discharge variability per task was assessed by adding the standard deviations of ISIs ofeach unit into a population sample and then comparing them by repeated-measuresanalysis of variance, followed by Tukey’s test.Since it was expected that the amplitude of sustained bite forces between tasksmight vary, the mean bite force values over the 20 s recording period, during which eachunit’s ISIs were analyzed, were also compared. Here, in each case, the mean bite forcefor the first task was normalized to unity, and bite forces for the other tasks were thenexpressed relative to this value. To test for differences in sustained bite-force values223between tasks, repeated-measures analysis of variance was carried out for the entire datasample, followed by Tukey’s test. All normalized values were then averaged accordingto group, and compared as a population. Differences less than 0.10 were consideredinsignificant. ResultsThe responses of 38 units were analyzed. Although most were active for allfour tasks, occasionally some units stopped firing, or could not be reliably discriminated.Therefore, the total number of units in a given sample varied according to task.The subjects had little difficulty performing the task when visual and auditoryunit feedback was given. When force feedback was provided instead of unit feedback,20% of the MUs under investigation were silent, although bite-force levels wereadequately maintained. In these cases, the experimental run was interrupted, unitfeedback was provided, then withdrawn, and force feedback given for another 30 s.When no feedback of any kind was provided, force levels were usually slightly higher andadditional MUs were activated.When the difference between the median firing rates (i.e. ISIs) of units firingwith visual and auditory feedback, and with force feedback were compared (n=28), 43%of the MUs had reproducible firing rates. When the difference between the medianfiring rates of units firing with visual and auditory feedback, and with no feedback werecompared (n=27), 26% of the units had reproducible responses. Finally, when thedifference between the median ISI values of units firing between both runs with visual224and auditory feedback were compared (n=32), it was found that 54% of units hadreproducible firing rates.Variations in the patterns of discharge are shown in Table XIV. Althoughdifferences in discharge variability between tasks were found to be statisticallyinsignificant (p >0.5), there was a tendency for freely firing MUs (without feedback) tohave lower SDs, as opposed to when subjects were target-tracking.When the unit-accuracy index (difference between observed median firing rateand 100 ms) was investigated for both tasks in which subjects were given auditory andvisual feedback, 61% and 62% of the units, respectively, were driven at accurate rates.In contrast, 39% and 38% of the units, respectively, were driven faster than thesuggested target rate. When the subjects were given force feedback, 36% of the unitsfired accurately, while 57% were driven faster. When no feedback was provided, subjectsactivated only 33% of the units at 10 Hz, while 63% were driven above that rate.Mean sustained-bite forces ranged from 1.4 N to 17.7 N. For all tasks, themean bite-force values and standard deviations, and their normalized values andstandard deviations are shown in Table XIV. When bite-force levels were comparedbetween the initial visual and auditory feedback and force feedback, it was found thatonly 30% of bite-force levels of the entire sample were reproducible (i.e. varied less than0.1 of the control value). From these units, 50% had reproducible bite-force levels whenalso compared with the task where no feedback was provided. The vast majority of units,however, did not show reproducible bite-force levels when different types of feedbackwere provided.225Table XIV Discharge patterns and sustained-bite force levels of MUs activated duringfour tasks. Discharge patterns are expressed in ISIs and in coefficients of variation (CV).Force levels are expressed in Newtons and in normalized values. Sample sizes (n) arereported in the text.MU Discharge PatternFeedback Type Mean ± SD CV (%)Visual and Auditory 96 ± 29 30Force 90 ± 28 31None 81 ± 25 31Visual and Auditory 97 ± 30 31Bite-Force LevelFeedback Type Mean ± SD Norm ± SDVisual and Auditory 6.9 ± 3.7 1.00 ± 0.00Force 6.2 ± 3.6 1.07 ± 0.47None 6.9 ± 4.0 1.11 ± 0.55Visual and Auditory 7.1 ± 4.7 1.14 ± 0.58226Thus the main finding in the present study was that although auditory and unitfeedback ensured the highest reproducibility of MU firing pattern, the mean firing ratesof 46% of active MUs in the superficial portion of the human masseter muscle were notsimilar when the same task was performed twice. Although there was a tendency for thefiring variance to improve when the task was performed freely (with no feedback), thiscould not be confirmed statistically. As expected, the unit-firing “accuracy’ was thehighest when unit feedback was used, while units were driven at faster rates during theforce-tracking run, and were especially fast when no feedback was provided. On severaloccasions units would stop firing when bite-force feedback was used. Finally, the meansustained bite forces were not reproducible, but fluctuated between tasks. DiscussionFluctuation of MU firing within a motoneuron pool has been reported byNordstrom and Miles (1991b), who found that 58% of active background units changedtheir mean firing rate with time, when a “feedback unit” was driven at a fixed frequency.These observations, and the present study, imply that even within a small region ofmuscle, the behaviour of MUs during the performance of a constant task is not stableover time. The non-reproducibility of many units’ mean firing rates when a task isrepeated indicates that there is a differential change in the net excitation of a proportionof MUs in the muscle with prolonged activity.It is commonly accepted that features such as size-related recruitment order,proportional increase in firing frequency of active units as the force increases (Mimer227Brown et a!, 1973; Monster and Chan, 1977; Goldberg and Derfler, 1977), and thecommon modulation of firing of simultaneously active units during a constant-forceisometric contraction is brought about by the uniform descending signal to themotoneurons which receive a large number of common inputs. However, if input signalsare unequally distributed amongst motoneurons in a pooi, they would mediatedifferential changes in MU firing pattern. An additional physiological mechanism whichhas been implicated in the maintenance of consistent firing patterns of spinalmotoneurons is recurrent inhibition (Freund, 1983). However, no Renshaw-typerecurrent collaterals have been identified in the jaw muscles (Derfier and Goldberg,1978).A single classically defined motoneuron pool and its associated MUs are,however, rarely activated independently during a contraction. Rather, a contraction mayinvolve a group of synergists (for example the temporalis, masseter and medial pterygoidmuscles) acting as a unit, or it may involve only a part of a complex muscle. The groupof MUs activated for a particular task is defined as a “task group” (Loeb, 1985). Sincethe essence of the size principle is to increase force smoothly and minimize fatigue(Henneman and Olson, 1965), then the orderly recruitment should hold within every taskgroup, which could comprise a group of synergists, a single muscle, or only a part of amuscle (Riek and Bawa, 1992). If it is assumed that during the chosen biting task themotoneuron task group was activated according to the notions of the size principle, novariability would have been expected. However, most fibres in the human masseter arehomogeneous in terms of size and type (Eriksson and Thornell, 1983). Therefore, it is228possible that fluctuations in the excitatoly input of the MU pool caused the variabilityof the physiological features. Similarly, it has been argued that it is excessive to expecta perfect recruitment order of hundreds of units, and that occasional errors may beeither imperceptible or inconsequential (Cope and Clark, 1991). An alternative notionto this variability is that MU recordings were made from a group of MUs, which was notthe principal task group responsible for force production. Rather, it was in the fringe ofwhere the main activity was occurring therefore explaining the variability in MU firing.In summary, regardless of the source of differential changes in MU activitywith task repetition, the present results demonstrate that jaw muscle MU firing patternsmay be modified by facifitation or suppression of some units during a continuousisometric contraction.It seems clear that even when the source of feedback for an individualattempting to drive MUs in the masseter is optimized, approximately half the units willshow behavioural characteristics which preclude the statistical confirmation ofreproducibility in performance when the same task is repeated. When these results aretaken together with those of MU behaviour in the previous study, it is concluded thatconventional measurements of behaviour in low-threshold masseteric MUs are unsuitedfor quantitative comparisons between MUs in different parts of this muscle.2293.4 Motor-Unit Territory Relative to the Masseter’s Internal ArchitectureThe relationship between human masseter MU territories and the muscle’sinternal anatomy is unknown. In experimental animals it is possible to use glycogendepletion to map MU territories, as has been done in several skeletal muscles (Englishand Letbetter, 1982a; English and Weeks, 1984; Richmond et a!, 1985), including the pigmasseter (Herring et a!, 1989). However, this method is impossible in humans, andelectrophysiological techniques which scan muscles cross-sectionally are used instead todetermine the volume in which fibres of a MU are distributed (Buchthal andSchmalbruch, 1970; Stâlberg et a!, 1976).Two physiological techniques have been used to measure MU territories inthe human masseter. In one, scanning electromyography with a roving needle disclosedthe temporal and spatial distribution of electrical activity along a uniaxial cross-sectionof the MU (Stâlberg and Antoni, 1980; Stâlberg and Eriksson, 1987). In the other, singleMU activity was sampled from two muscle sites simultaneously. Here, the recording tipsof two stationary needle electrodes were located stereotaxically, and the three-dimensional distance between the recording sites was calculated (McMillan and Hannam,1989a). Both techniques have limitations. Scanning EMG does not measure theanteroposterior nor the superior-inferior dimensions of the territory, although it doesreveal quite accurately the limits of MU activity in the axis of the scan. The twin-needlemethod has the advantage of providing a three-dimensional estimate of the territory’sdimension, but it can systematically underestimate the limits of unit activity because bothneedles are required to record discernible action potentials.230Recently, a different approach, which included the stereotactic location of electroderecording sites within the human masseter, has been used (McMiflan and Hannam,1989a). This technique utilized MR imaging, which has become a useful technique forthe three-dimensional reconstruction of human anatomical structures (Hannam andWood, 1989; Schelihas, 1989; Sasaki et a!, 1989). However, in the McMillan and Hannamstudy the recording sites were located stereotaxically in whole-muscle reconstructions,and their position relative to internal aponeurotic boundaries was unknown. Anexperimental approach with the ability to image internal aponeurotic boundaries wouldpermit the question of anatomical compartmentalization of MUs to be addressed.Improvements in MR imaging now enable the visualisation of internal musclearchitecture, and solid-object modelling techniques make three dimensionalreconstruction of muscle architecture feasible in living subjects (Lam et a!, 1991). Inaddition, the development of new, highly accurate, three-dimensional tracking devicesmakes it possible to monitor the orientation and translational displacement of the jawand any other object, including needle electrodes, in real time during recording sessions.These improvements encouraged the development of a method for mapping MUterritory in relation to internal muscle anatomy in order to gain insight into functionalcompartmentalization in the human masseter.In this part of the study, the aim was to test the hypothesis that MU territoriesare small and confined to aponeurotic boundaries, and that any large territories arelocated in the anterior, fused part of the muscle. Support for this hypothesis would implythat the human masseter is capable of differential contraction exerting internal force231vectors with different directions.3.4.1 Methods3.4.1.1 Stereotactic Location of EMG Needle Electrode ScansTo map MU territory in relation to the internal aponeurotic network of thehuman masseter a method was developed that combined data from three separatesources: magnetic resonance imaging of the muscle, three-dimensional tracking of theneedle electrode, and EMG scanning technique. For clarity, each component of themethod will be described in separate sections.3.4. 1.1.1 Morphologic ReconstructionDuring the imaging session, each subject wore a customized eyeglass frame,lined with silicone impression material to fit to the nose and ears of each subject andensure stability and reproducibility during placement. Each frame incorporated a fiducialL-shaped reference grid filled with 5 mM copper sulphate, rigidly fixed to a position justposterior to the right masseter muscle. A 1.5 Tesla MR unit (Signa, GE MedicalSystems, Milwaukee) was used to obtain T2-weighted images of the muscle and thereference grid. Spin-echo sequences with repetition time (TR) of 2000 ins and echotimes (TE) of 25 and 80 ins were used to obtain a series of contiguous, 3 mm sectionsin the coronal plane. As described in more detail in an earlier section of this thesis,outlines from the muscle, internal aponeuroses and copper-sulphate markers were tracedand digitized from each coronal section (Model HP9874A Digitizer, Hewlett Packard,232Canada). The profile coordinates were then used in an engineering solid-objectmodelling package (1-DEAS, SDRC, Milford) to reconstruct the muscle and its internalstructures in three dimensions. Motor-Unit RecordingDuring each recording session, the customized plastic eyeglass frame was wornby the subject to provide a fixed spatial reference. In each case, an insulated, monopolarneedle electrode (MF-37, TECA, Pleasantville, NY) with a surface reference electrodeoverlying the muscle recorded activity from a MU. This method was selected becausethe smaller diameter of the monopolar needle causes less discomfort during insertion (anadvantage during multiple recording sessions, especially with facial muscles) and hasbeen shown to be sufficiently selective for macro MU recording if combined withbandpass-filtering to remove low frequency components of the EMG signal (Gath andStãlberg, 1976; McMillan and Harinam, 1989a; McMillan and Hannam, 1991). Theneedle was introduced percutaneously, perpendicular to the skin surface. Muscle fibreactivity was amplified (Model Al 2130, Axon Instruments, Burlingame, CA), andbandpass-filtered between 300 Hz and 10 kHz (Gath and Stãlberg, 1976). A separatesurface electrode was attached to the back of the neck as a ground. The needle electrodewas attached to a needle holder carrying three reflective markers 10 mm in diameter,which were arranged in a triangle when viewed coronally.The subject was asked to activate the MU by gently biting with the teethtogether in the dental intercuspal position, and to maintain a continuous low rate of233firing (12-15 Hz) with the aid of auditory feedback. The compound action potentials(APs) obtained from the MU were monitored on a digital storage oscilloscope (Model2232, Tektronix Canada, Vancouver, BC). When a clearly defined AP was obtained, thesubject was asked to maintain a constant effort. The needle electrode was advancedslowly into the muscle until the activity disappeared, then withdrawn gently along amediolateral trajectory so that steady unit activity reappeared, increased in amplitude,decreased again, and was finally lost. Each complete electromyographic scan wassampled on digital tape at 48 kHz (DAS System, Unitrade, Minneapolis, MN). Needle LocationThe three dimensional movement of the roving needle was recordedcontinuously with an optical motion analysis device (MacReflex System, Qualisys AB,Partille, Sweden). This had a precision of 0.03 nun and dynamic accuracy of 0.3 mm, andit was used to measure the three-dimensional (3D) coordinates of the centroids of theneedle’s markers with respect to three markers located on the front of the frame (Figure50). The coordinates recorded by the two video cameras were sampled every 20 ms overa period of 15 s, stored into a microcomputer (Macintosh Ilsi, Vancouver, B.C.) andconverted into 3D spatial coordinates by means of the optical system’s software(MacReflex, Qualisys AB, Partille, Sweden).At the end of each recording session, while the needle electrode was stillattached to its holder, a separate optical system (Reflex Metrograph, HF Ross, Salisbury,Wilts, U.K.) with a resolution of 0.1 mm (Takada et a!, 1983) was used to measure the234Figure 50 Schematic illustration of the global setup for a needle tracking run. Acopper-sulphate-filled L-shaped Grid attached to an eyeglass frame (A). Three reflectivemarkers are mounted on the front of the frame (B) and three on the needle electrodeholder (C). The LED used for timing is also fixed to the front of the frame (D).AC2353D coordinates of the centroids of the needle holder’s markers and the needle’s tip. Thecoordinates of the centroids on all markers on the front of the frame and those of theL-shaped grid were also measured with the same instrument.The location of the needle tip relative to the anatomical landmarks wascalculated in 1-DEAS with an algorithm that permitted coordinate rotation andsuperimposition. Initially, the three centroids of the L-shaped grid were determined fromthe solid object model. Then, sequential superimpositions of the locations of markersdetermined from the MacReflex and Metrograph systems were performed. In this way,the needle electrode tip position was calculated relative to the solid object origin (Figure51) and displayed graphically within the reconstructed muscle (Figure 52). Scan LocationTo synchronize the time bases of the MU recording with the needle tip’slocation, an infrared light emitting diode (LED) one mm in diameter was fixed to theframe (Figure 50). At the beginning of the MacReflex sample, a single LED flash (1 msduration, and synchronous with the camera sample onset) was hand-triggered.Simultaneously, a 1 ms square wave pulse was embedded onto the digital tape. After therecording session, the electromyographic record was replayed, sampled at 32 kHz andstored with the aid of commercially available software (BrainWave Systems Corp,Thornton, Colorado) for off-line analysis in a microcomputer (Premium, AST Research,mc, Irvine, California). Sequential spike-by-spike visual analysis of the regularly firingMU ensured that the same unit was sampled as its amplitude and fibre content changed236Figure 51 Schematic representation of the merge of the three datasets. (I)represents the reconstructed muscle and the reference L-shaped grid obtained from theMR images. (II) represents the spatial relationship between the same reference grid andthe reflective-marker triangle on the eyeglass frame. Their 3D coordinates weremeasured with the Reflex Metrograph. (III) represents the same eyeglass frame triangle(right), and the needle-holder triangle with the attached electrode (left). Data from onetriangle relative to the other was obtained with the MacReflex system, while therelationship of the electrode to the triangle was obtained with the Reflex Metrograph.Data superimposition of the same triangles from these separate datasets is performedin 1-DEAS, to enable the display of the needle tip location relative to the muscle asshown on the far right.IIII237Figure 52 Right antero-lateral view of the final reconstruction. The right massetermuscle, eyeglass frame triangle, needle-holder triangle and needle electrode aregraphically displayed. Similar data of electrode tip position are available for every 20msec of a needle scan through the muscle.238uniformly during electrode movement (Figure 53A, top), and determined the beginningand end of MU activity (Figure 53A, bottom). Time stamps were used to mark the limitsof the recorded spike train, when the spike amplitude became so small that thedifferentiation of the MU from the background activity was no longer possible. Thescanning record was rejected when the start or end of the unit activity was uncertain dueto contamination by other units or baseline noise. The time stamps were referenced tothe calculated needle tip locations at corresponding times in the tracking record. Thelinear distances between the limits of recordable single MU activity for each subjectwere then displayed graphically as bars within the reconstructed masseter muscle. Forexample, typical mediolateral electromyographic scans for two MU5 are shown in Figure53A (center) and 53B. These had linear scans of 3.2 mm and 4.3 mm respectively, andare ifiustrated in Figure 54. Together, they provide examples of an average-sized MUwhich lies well within identified aponeurotic boundaries, and a larger MU which clearlycrosses a tendinous sheet.3.4. 1.1.5 Methodological ErrorsTo assess the reproducibifity with which the frame could be repositioned, weattached a plastic reference triangle, with three reflective markers, to an indented acrylicbite-fork held firmly between the teeth. The frame, with its three markers, was thenplaced on the subject, and the three-dimensional location of all six markers wererecorded with the MacReflex system. Three linear distances were calculated betweencorresponding pairs of reference and frame markers. The frame was then removed, and239Figure 53 Typical EMG records obtained during two separate needle scans. In(A) the middle trace shows the response of a single MU as the needle is moved throughthe muscle. The unit increases in amplitude, then decreases again, as the needle ismoved. An expanded version of selected spikes of the unit (not consecutive, for reasonsof clarity) is shown in the top trace. The bottom trace diagrammatically represents theneedle movement, recorded with the tracking system. S and E indicate the start and endof MU activity. The square wave pulse and LED flash are illustrated on each trace onthe left. In (B) the response of a second MU is shown for comparison as the needle ismoved through the muscle.4msO.2mV1.5 st’IIiI IB.BLi IBIWW hllhIIIhIIh.O.2mV1.5s240Figure 54 Coronal view of the 3D reconstruction of part of the right massetermuscle. The mediolateral territorial dimensions of two different MUs are representedby bars. The top one lies between two aponeuroses (Ap.I [left] and Ap.llI), and thebottom one passes through an additional tendon sheet (insert above and magnificationbelow). Only part of the reconstruction is shown for clarity. (Z=zygomatic arch;M=ramus of the mandible) Scale bars represent 1 cm (top) and 1.8 mm (bottom).241the procedure repeated five times. Since the average linear distances between the twotriangles were estimated to be 113.70, 76.45 and 68.23 mm, and their standard deviationswere 0.52, 0.53 and 0.41 mm, respectively, the coefficients of variation were 0.5%, 0.7%and 0.6%, respectively.The accuracy with which the needle tip position could be predicted was testedin the following manner. A subject was imaged according to the protocol describedabove, and the MR images were traced, digitized, and reconstructed. The centroids ofthe L-shaped grid were determined and the frame markers added to the reconstruction.In a separate recording session, the tip of a needle electrode was placed against themarked centroid of the lateral surface of the copper-sulphate-filled marker at the cornerof the L-shaped grid, instead of inside the muscle. This location was also recorded withthe MacReflex system. Both the needle tip and needle holder markers’ centroids werethen measured with the Metrograph, permitting the location of the needle tip to becalculated and compared with the corresponding position in the grid reconstruction. Thisprocedure incorporated all inherent errors in the system, including those incurred duringthe imaging, digitizing and reconstruction procedures, the use of the MacReflex andMetrograph, and operator error. The difference between the two positions was estimatedto be 0.07 mm in the mediolateral dimension, 1.02 mm in the vertical dimension, and0.45 mm in the anteroposterior dimension. Mediolaterally, the error of the method isminimal in relation to the average tendon sheet thickness of approximately 1 mm andthe average MU territory width of approximately 3.5 mm.The method employed to estimate MU territory size is based on the242measurement of the distance between the beginning and end of unit activity, whichshould represent the diameter of the MU. The error of this measurement is howeverdifficult to determine in humans. MU territory size can be underestimated if thetrajectory of movement of the needle electrode is through the periphery of the unit’sthree-dimensional volume. Alternatively, it can be overestimated depending on thevolume conduction of the unit’s action potentials, and/or depending on the electrode’suptake radius. Considering the electrode used in the present study, an error of up to 1mm at both ends of the unit’s cross-section could be expected. Since the averagethickness of an aponeurosis is 1 mm, only a territory overestimation greater than 1 mmat one end of the mediolateral dimension (the end closest to an aponeurosis) would havebeen sufficient to erroneously affect the relationship of a unit’s territory for crossing aboundary.3.4.12 Motor-Unit TerritoryExperiments were carried out on four adult male subjects. Their ages rangedfrom 29-36 years, and all had a complete natural dentition and no history of jawdysfunction. The subjects were selected because of their skill in activating single MUs.The experiment was approved by the Human Experimentation Committee at theUniversity of British Columbia, and each subject gave informed consent.In each case, a monopolar needle electrode (MF-37, TECA, Pleasantville, NY)was inserted into the right masseter muscle and referenced to a surface patch electrodeattached to the overlying skin. In some cases, two monopolar electrodes were inserted243simultaneously to record synchronous activity. An additional surface electrode wasattached to the back of the neck and used as a ground. At the beginning of eachrecording session, the subject was asked to activate a single MU by gently biting with theteeth in full dental intercuspation, thus producing a continuous low rate of firing (12-15Hz) with the aid of auditory feedback. MU activity was amplified, band-pass-filtered, andsampled according to the previous description. Scanning recordings were randomlyobtained from different parts of the right masseter muscle on all subjects. Multiplerecording sessions were used to enable different parts of each muscle to be searchedsystematically.Following the analysis of each spike train, time stamps representing the limitsof MU activity were referenced to the calculated needle tip locations at correspondingtimes in the needle movement record, and subsequently located relative to itscorresponding internal muscle architecture. Graphical representations of themediolateral scans were then produced and their relationship to aponeuroses wasclassified into two groups, one where the mediolateral territorial dimension was confinedto tendinous boundaries, and the second where it passed through an aponeurosis bymore than 0.5 mm. This paradigm was chosen to minimize the influence of any technicalerror in the determination of MU territory location, thereby enabling territoryclassification to be ascertained. Mean mediolateral dimensions for both groups werecompared statistically, by means of an independent t-test, to determine whether themediolateral territorial dimensions of MUs that extended across an aponeurosis werelarger. Then, their spatial distribution relative to gross anatomical portions was244investigated. Each muscle was divided into three antero-posterior and two medio-lateralregions on the basis of gross anatomical subdivisions (Ebert, 1939; Schumacher, 1961c).The width of the muscle was also assessed for each subject. The mean valuewas obtained by measuring the distance along a transverse axis between the most lateralpoint and the medial contour of the five widest, contiguous muscle coronal outlines. Totest for the dependence of the mediolateral territorial dimension on the muscle’s widthin each subject, both analysis of variance and regression analysis were used.3.42 ResultsSingle-MU recordings were obtained for 162 units. In most recordings theelectrical activity increased and decreased quite smoothly during the electrode scan, asfound in previous studies (Stâlberg and Antoni, 1980; Stâlberg and Eriksson, 1987). Noabrupt changes in amplitude due to the passage of the electrode through a tendon wereobserved, although the shape of the spike train in a scan was not always symmetrical. Ina few cases, a reduction in spike amplitude was observed at the centre of a scan, withsubsequent increase as the electrode moved further through the unit territory, and inother cases the amplitude slowly increased to maximum and then rapidly declined, untilactivity was lost. The formation of these complex patterns suggested that they could havebeen the result of a change in velocity, or even a change in the direction of the electrodemovement during the particular scan. To determine whether this assumption was correct,plots of the needle movement velocity were made for two asymmetrical scans. These areshown in Figure 55, in which time stamps at every 180 ms within the start and end of the245respective MU activities are plotted against the linear distances of electrodedisplacement which occurred between consecutive time intervals. Contrary to what wasexpected, in both cases the velocity of the needle movement was constant throughout thescan. An alternative explanation for this type of appearance is that as the recording tipmoves across the unit territory it samples a different combination of action potentialsfrom the scattered muscle fibres belonging to the same MU. This assumption is howeververy difficult to prove, requiring complex computer simulations of the number and of thedistribution of active muscle fibres relative to the electrode’s field of pick-up, and ofother variables, such as volume conduction and filtering systems.The mediolateral territorial dimensions of the single MUs varied between 0.4and 13.1 mm. Seven units in three muscles showed values between 9.1 and 13.1 mm, andare comparable to the larger units reported by Stâlberg and Eriksson (1987). Theaverage width and standard deviation for 162 units from the four muscles was 3.7 ± 2.3mm. Their distribution is shown in Figure 56.Most MU territories (145/162) were confined to subvolumes within tendinoussheets (Figure 57). A small number of these spanned the entire muscle layer, reachingthe tendon sheets on either side of the layer. Only 17 territories (approximately 10% ofthe sample) crossed aponeuroses. These MUs ranged in width from 3.0 to 13.1 mm anddid not include any of the smaller MU sizes (<3 mm) found in the aponeurosis-confinedgroup. The relative distributions of confined and extended territories are shown in Figure57. A one-tailed t-test performed on logarithmic transformations of the data revealedthat the mean sizes of extended MUs were greater than those of confined MUs246Figure 55 Needle movement velocity during two separate needle scans. In (A) thetop trace shows an asymmetric response of a single MU as the needle is moved throughthe muscle. The bottom diagram represents the needle movement velocity plot. In (B)the response of a second MU as the needle is moved through the muscle is shown withthe record’s needle movement velocity. This is an example of a spike train in which theamplitude decreased and increased again in the centre of the MU territory. Note thatin both diagrams the velocity of the needle scan is constant, indicated by the slopes ofthe two graphs.A1BOH1920 3840 0Time (msec)11920 3840 5760 7680 9600 11520Time (msec)247Figure 56 Distribution histogram of the territorial widths of 162 single MUs inthe right masseter muscle. MU territories are grouped into classes with individual widthsof 1 rmTL. Every second bin has been labelled at the center of the class boundary. Themean value for this distribution is 3.7 mm.40Ci)D20G)2:5zo FTOTAL162<1 2.5 4.5 6.5 8.5 10.5 12.5MU Territory Width (mm)248Figure 57 Relative sizes and frequencies of confined and extended MU territories.Distribution histogram of territorial widths of MUs which are confined by tendinousboundaries (above), and those which extend across tendon sheets (below). MU territorieswith widths greater than 3 mm are shown to the right of the dotted line (shaded bins).Numbers indicate relative sample sizes.(0D0a)0EDz4020040200<1 2.5 4.5 6.5 8.5 10.5 12.5MU Territory Width (mm)EXTENDED17/162249(p<O.OO1)The distribution by region of 88 MUs with dimensions larger than 3 nun isshown in Figure 58. The location of the territories which extended across aponeuroticlayers did not appear to be specific to any muscle region, although there was a tendencyfor them to be found in the middle region in the para-sagittal plane and in the centreand deep regions in the coronal plane (Figure 58).Analysis of variance revealed that the mean mediolateral territorialdimensions between subjects were significantly different at the 99% confidence limit(p <0.01). Additionally, the individual mediolateral dimensions were shown to dependon that individual’s muscle width (Linear regression: r = 0.98). The subject with thesmallest muscle had the smallest territories, while larger individuals had larger territories(Table XV).3.4.3 DiscussionMagnetic resonance imaging reliably visualizes the intramuscular tendons,which anatomically subdivide the masseter muscle in rabbit (Ralph et a!, 1991) and inhumans (Lam et a!, 1991). Because few mobile protons are available in tendons, theyproduce a low intensity MR signal, and are seen as black structures adjacent to thesurrounding proton-rich muscle (Berendsen, 1962; Migchelsen and Berendsen, 1973). Onoccasion, normal tendons may demonstrate increased tendinous signal intensity. Thisoccurs when tendons are oriented at approximately 55° relative to the magnetic field(Fullerton et a!, 1985) and is most prominent on Ti-weighted, imaging sequences250Figure 58 Distribution of MU territories with widths greater than 3 mm,according to muscle region. (A) Lateral and frontal diagrams of the masseter showingthe six different regions. (P=posterior, Mi=niiddle, A=anterior, S=superficial,C = central and D = deep) (B) Distribution of MUs with territories confined toaponeuroses (above), and extended across tendon sheets (below).ALateral FrontalB(1)D4-0-E:5z40•0ci4-C0C-)0400U)Ca)4-.xw03Ii,ir12 I7jmIm1.rP Mi A S C D251Table XV Muscle width and mediolateral territorial dimensions from M(Js in theright masseter muscle of four subjects. All values are expressed in mm.Muscle Width (mm) MU Territory Width (mm)SubjectsMean SD Mean SD Mm. Max. n1 21.5 1.2 4.4 2.4 1.1 11.2 402 19.7 1.6 3.8 2.8 0.6 13.1 483 16.4 1.3 3.4 2.2 0.4 11.6 404 14.8 1.1 2.9 1.2 0.9 5.3 34252(Erickson et a!, 1991). In the present study, coronal sections through the masseter wereobtained. In this plane, low intensity signals from the tendinous sheets were expectedbecause of their orientation of 15° relative to the midsagittal axis (Schumacher, 1961c).To further ensure high contrast differentiation between the tendinous structures and themuscle, we selected T2-weighted images. Using this imaging sequence, the possibility ofobtaining higher angle-dependent signals from tendons was reduced. Reliable internalanatomy identification was then confirmed from our reconstructions, which are consistentwith previous anatomical reports.Electromyographic scanning does not indicate the total territory of a MU, butonly its extent along the recorded cross-sectional area. The scan, on occasions, may wellbe at the periphery of the MU territory. Other factors that have to be taken intoconsideration when evaluating MU territory are the pickup area of the recordingelectrode and its possible bending, especially when a fine needle is used. The uptakeradius has been estimated previously to be about 1 mm for a concentric needle electrode(Thiele and Boehle, 1975), and should be approximately the same for the electrode usedin this study given the similarity of the intramuscular recording surface areas, and thefiltering procedure used (Ekstedt and Stâlberg, 1973; Gath and Stâlberg, 1976; StAlbergand Trontelj, 1979). Theoretically, a 10 degree deflection of the shaft at the needle hubcould result in a 6 mm deflection of the needle tip in the anteroposterior or superiorinferior direction relative to the recording cameras. However, this would only result ina 0.6 mm error in the estimation of needle tip location along the mediolateral axis in themuscle. Thus the error in locating MU territories relative to the tendon sheets is253minimal in the coronal plane of interest used in this study.The transverse width of MU territories measured with this method correspondclosely with previously published figures for the human masseter muscle (Stâlberg andEriksson, 1987; McMillan and Hannam, 1991). With the present technique however,observations can be made of both the number of territories which are large enough tocross tendinous boundaries and those which are restricted to small muscle subvolumes.The study shows that the identification of regional morphology by MR maybe correlated with electromyographic assessments of muscle function in human subjects.This method can be used to estimate the relationship between neuromuscularpartitioning and intramuscular architecture in the different orofacial muscles, and it canbe easily adapted to verify unit locations in studies of human motor control.The recording method was a modification of the EMG scanning techniqueintroduced by Stalberg and Antoni (1980). Originally, the technique employed twoneedle electrodes. One, a single-fibre EMG electrode, was stabilized in a muscle site torecord a single-fibre action potential which acted as a trigger. A second concentricelectrode was then moved through the muscle to record motor-unit activity, synchronouswith the single-fibre action potentials. In the present study, however, a single monopolarneedle was used because it is more comfortable in the facial region, especially duringmultiple insertions at repeated recording sessions. This type of electrode, when used inconjunction with a high lowcut-filter, has a similar selectivity to that of a concentricelectrode. If the radius of electrical pick-up had been wide, the results would haverevealed large MU territories for most units. In contrast, the mean territorial width (3.7254± 2.3 mm) in the experiment was highly consistent with previous reports. Stâlberg andErilcsson (1987) reported the mean scan length for 32 single motor-units in the inferiorpart of the masseter to be 3.7 ± 0.6 mm, and McMillan and Hannam (1991) reported themean distances measured along a medio-lateral axis for 32 paired recording sites to be3.2 ± 2.3 mm. Confirmation of the selectivity of the single-needle approach was alsoobtained by the preliminary experiments in which two needles recorded activity from thesame MU, one stationary electrode being used to trigger the display of activity recordedfrom a second, roving electrode in a manner comparable to that described by Stâlbergand Antoni (1980). When the width of the MU was estimated from the triggeredrecordings and also from the roving needle alone (as was done in this experiment), theresults were identical.In Stâlberg and Eriksson’s (1987) work, the twin needle technique was alsobelieved to reduce the potential error of overestimating territory size that might occurshould the electrode be moved obliquely to the muscle-fibre direction. Their means ofdetecting a skewed scan was to observe a continuous change in latency between thetriggering-action potential and the recorded motor-unit potential. In the presentexperimental design, any such error in orientation could be easily detected on thegraphical display of the reconstruction.The human masseter muscle can be divided into three anteroposterior groupsbased on fibre length (Schumacher, 1961c), which varies from 25-39 mm. Medio-laterally,the muscle fibres are equally long within a given anteroposterior group (Ebert, 1939).The muscle fibres which comprise a MU are considered to be of uniform length and255histochemical type (Zajac, 1989; Brandstater and Lambert, 1973). From the presentexperiment, it seems reasonable to assume that typical MU territories in the humanmasseter occupy volumes whose boundaries are defined supero-inferiorly by the lengthof the muscle fibres and by the internal tendon sheets. In the horizontal plane, theirmediolateral dimensions are about 3-4 mm. Since the muscle is arranged in strips offibres arranged anteroposteriorly, masseter MU territories are most likely arranged instrips of varying lengths, and have elliptical cross sections with their long axes orientedanteroposteriorly. This organization has been previously suggested by Herring et a!(1991) for the pig masseter, in which MUs are restricted to small volumes of a musclefascicle.Earlier studies have suggested that most MUs are small, occupying discretemuscle volumes, while few units are large, occupying larger volumes (Stâlberg andEriksson, 1987; McMillan and Hannam, 1991). In glycogen-depletion studies in the pigmasseter, depleted fibres, belonging to small MUs, have been found occasionallyoccupying large volumes in several discontinuous, widely-separated muscle regions,separated by aponeuroses (Herring et a!, 1989). Theoretically, this broad innervationareas may result from axon branching. If an axon divides into two nerve branches,multiple muscle areas can be innervated (Pfeifer and Friede, 1985; Herring et a!, 1989;English, 1990). This possibility however has been considered unlikely, and the presenceof widely-distributed, depleted fibres was attributed to the technical difficulty in isolatinga single axon for electrical stimulation (Herring et a!, 1989). Similar patterns ofdiscontinuous MU territories were not found in scanning EMG studies, however, large256units were found to cross aponeuroses. This finding may be interpreted as the result ofthe technical procedure itself. For example, wide-field electrode pick-up as aconsequence of volume conduction may occur within the muscle (Stãlberg and Trontelj,1979). While this is a possible explanation for the results seen in the present study, it isunlikely, and although not proven, axon branching, is another possible explanation forthis type of structural arrangement.The correlation between MU territory size and overall muscle width is alsorelevant here. The same tendency was observed in the infant pig by Herring et a! (1991),who reported the two largest territories in the masseters of older (and larger) piglets.Stâlberg and Eriksson (1987) postulated that these units belonged to especially widecompartments. If their interpretation is correct, the largest units should be found in thesuperficial, anterior part of the muscle, where the deep and superficial portions fuse.This idea is only partially supported in the present study, for while a few large, confinedunits (± 9 mm) were encountered in the wide superficial muscle region, some largeterritories were found in other muscle regions, where they passed through aponeuroses.In the masseter, units with the lowest thresholds are located preferentially in the deep,medial part of the muscle, while higher threshold units seem to predominate in thesuperficial posterior region (Eriksson, 1982). Since the recordings were mostly from lowthreshold MUs, they probably represent the type-I fibre population. This might explainthe relatively larger number of MUs recorded in the middle and deep regions of themuscle.The functional significance of widely-dispersed innervation territories is257uncertain. Stálberg and Eriksson (1987) suggested that large MU territories might beadvantageous in motor tasks where there is less need for finely-graded control of jawmovements, and where the recruitment of such units may favour force development instatic contraction, as for example in biting in the intercuspal position. It is also possiblethat these territories ensure internal tendon stiffness in situations where only onecompartment is active, avoiding extreme internal tendon deformation and permitting finevector gradations. The regional restriction of most MUs provides the basis for the ideathat the central nervous system might control the contraction of discrete muscle regionsindependently, as has been previously proposed for the human masseter (Stâlberg andEriksson, 1987). This arrangement would allow differential contraction of MUs on eitherside of a tendon sheet to develop forces in several directions, and thereby explain thefunctional heterogeneity frequently reported in this muscle (Belser and Hannam, 1986;Tonndorf et at, 1989; Blanksma et at, 1992). Alternatively, these units could be simplyavailable for developmental plasticity, when muscle adaptation to new conditions suchas change or loss of dentition is required.In conclusion, the human masseter seems to have a highly compartmentalizedneuromuscular organization. The presence of territories between intramuscular tendonssuggests that differential contraction may be possible on either side of central tendonsprovided the nervous system utilizes this structural substrate. The presence of a few largeterritories which extend across aponeuroses suggests that in these units, tensions mustalways be produced on either side of the aponeurosis whenever these units are active.The findings are thus generally consistent with the idea that there is at least some form258of mechanical heterogeneity in the masseter. The study also confirms the feasibility ofmaking high-resolution reconstructions and of locating either stationary or roving EMGneedles stereotactically relative to internal muscle compartments. This method could beused to estimate territories in other muscles and also be used in motor-control studieswhen it is important to locate recording sites.2594. GENERAL DISCUSSION AND CONCLUSIONSThe present studies of anatomical, biomechanical and physiologicalbehaviour in the human masseter aimed to extend the knowledge of the functionalorganization of this muscle. The primary goal was to establish the presence ofneuromuscular compartments within the muscle, their behaviour relative to intraoraltasks, and their relationship with the muscle’s internal architecture. For many years, ithas been assumed that the motor system would control the various jaw muscles assimple, whole structures, which would contract differentially to grade inter-occiusal forcesand jaw movements during natural function. However, now it seems clear that in fact (atleast in the masseter) a more refined foundation for the coordination of intramuscularactivity exists.Anatomical and physiological findings indicate that the human massetermuscle may be divided into at least three (possibly four) neuromuscular compartments.These form a multipennate muscle, which may vary in internal structural complexitybetween individuals, both in fetal and adult specimens. In an attempt to perform adetailed quantification of muscle fibre orientation in humans, it was found, however, thatthere are some major technical difficulties. Human specimens are not readily available,and the large sizes of human specimens complicate a thorough investigation with currenthistological methods. In addition, muscle fibres in young fetuses are not totallydeveloped, which contribute to the difficulty in measuring fibre angulation and inidentifying internal tendinous structures.In contrast, the development of a Magnetic-Resonance imaging and three260dimensional modelling technique used in the present experiments permitted thereconstruction of the complex internal structure of the human masseter muscle andmandible. Although the orientation of muscle fibres could not be visualized, thetechnique was found to be reliable, as confirmed by comparisons between thereconstructed muscles and the conventional anatomical dissections. An additional valueof this technique is that it permits the investigation of the internal tendinous musclestructure in living subjects.During jaw movements, insertions of muscle fibres in distinct regions ofthe human masseter displace by different amounts and directions, which suggests thatits muscle fascicles lengthen and rotate differentially. Regional differences in thesephysical attributes vary with task being performed, and suggest that some muscleportions have more advantageous lines of action for certain tasks than for others, andthat they vary between individuals. In this study, the ability to measure jaw movementswith six degrees of freedom, added to the method for combining the orofacial tissuereconstruction with jaw-movement records, allowed the three-dimensional measurementof masseter-insertion displacement in living subjects. This has not been possible,previously.During function, recruitment and rate-coding of motor units (MUs) in thehuman masseter by the central nervous system seem to follow the general principlesregarding “size” and “common-drive” found in the motoneuron pools of other skeletalmuscles (De Luca and Mambrito, 1987). Recruitment thresholds are however not stable,and the order of recruitment can change. Both are influenced by the type of task261performed, the manner in which it is approached, and its duration. Masseter MUs areusually polymodal, in that they can contribute to more than one intra-oral task, and theyare difficult to drive at rates below 10 Hz. To produce bite forces, most MUs arerecruited quite early in the performance of a task. The motor system relies mainly uponrate-coding over a wide range of voluntary contraction, although later recruitment ofadditional higher-threshold units does also contribute to the increase in bite force toreach the maximum level (Derfier and Goldberg, 1978). Since most masseter MUs areof similar size and type (Eriksson and Thornell, 1983) and since they are recruitedconcomitantly during light bite forces, it is possible that random fluctuations in themembrane potentials of motoneurons may affect the mechanism by which synapticcurrent is converted into spike trains (Calvin and Stevens, 1968). Thus, it is suggestedthat non-static behaviour of low-threshold masseter MU firing patterns is the result ofa constant modification of the properties of some units by facifitation or suppressionduring a continuous isometric contraction. The modified low-threshold MU behaviouris further complicated by the effects of task, which may affect the interaction betweendescending, corticobulbar drive and peripheral input from orofacial and musclereceptors.The present results support the notion that coactivation within andbetween the jaw muscles is a possible factor influencing the fluctuations in recruitmentthreshold, sustained-bite force and MU firing properties. In addition, there is enoughcircumstantial evidence to suggest that regional differences in MU behaviour also exist.However, no direct links between MU behaviour and internal muscle structure have262been established so far. It is important to emphasize that this, and most other, studieshave been restricted to low-threshold MUs, which may behave differently than theirhigh-threshold counterparts. The present results suggest that efforts to demonstrateregional differences in low-threshold MU behaviour with changes in task are difficult tovalidate statistically. Thus caution should be exercised when variables similar to theseare used to classify MU type in human jaw muscles.Previous electromyographic investigations of motor-unit (MU) sizes(Stâlberg and Eriksson, 1987; McMillan and Hamiam, 1991) in the human masseter havefailed to correlate them with the muscle’s internal architecture. The development of amethod to locate electromyographic-needle-electrode scans stereotactically relative tointernal tendinous boundaries, enabled the investigation of MU territories within thehuman masseter. Masseter MU mapping revealed that the mean territorial width was 3.7± 2.3 nmi and varied between 0.4 mm and 13.1 mm. The widths were comparable tothose of previous reports (Stálberg and Eriksson, 1987; McMillan and Hannam, 1991),and were related to the subject’s muscle size as also noted in pig (Herring et a!, 1991).The fact that most territories were distributed within discrete tendon-boundedcompartments in the masseter provides an anatomical basis for selective activation of themuscle. However, it is also possible that this arrangement provides a flexible means forensuring tendon stiffness and mechanical adaptation of the multipennate masseter duringgrowth and development, whether or not the muscle is activated selectively. Since themajority of MU territories are dispersed within tendinous boundaries, motoneurons maybe organized into separate task groups, which alter their contribution to bite force263depending on the intraoral task and experimental paradigm.Collectively, the fmdings of the present study underline the complexity ofthe human stomatognathic system. The unique structural, MU-territorial organization,and task-related functional properties of the MUs in the masseter apparently provide aperipheral system which can be constantly adjusted by descending control and peripheralinputs to shade muscle activity to perform the intended task. Inter-individual variabilitywith respect to anatomical, biomechanical and functional properties suggest that infuture studies, it will be important to combine morphological and physiologicalinformation within a given human subject, and that care should be exercised wheninterpreting data from pooled samples.2645. FUTURE DIRECTIONSIt is known that there are considerable changes in human jaw movementsassociated with the different dentitions that appear during postnatal growth. It is possiblethat these variations in masticatory envelopes are due to a change in relative musclefibre growth, by the utilization of different contraction strategies, or a combination ofboth. Whereas it seems that shifts in the human masseter fibres’ angulation occur duringgrowth, detailed data on such alterations are not available. It would be therefore usefulto determine the angulations of masseter fibres in humans at different ages, andcorrelate these with functional jaw movements.Alternatively, it is possible that these changes are due to muscle contractionpatterns which simply become more refined with age. This assumption would beconsistent with the view that ontogenetic changes in oral behaviour are caused by thedevelopment of a different motor pattern (presumably on a suitable anatomicalsubstrate), and not by a major change in morphology other than size. Whether motor-unit (MU) territories are also restricted to small muscle portions, and whetherdifferential contraction of the masseter muscle also occurs in young humans still remainsto be confirmed. If it is assumed that in young humans MU territories are restricted totendinous boundaries and that the contraction pattern is homogeneous, the stimulus foradoption of complex regional motor contraction patterns might be the result of learning.That is, after experimenting with various contraction patterns, the growing individualwith a more complex dentition might select a group of muscle fibres which work mostefficiently for the given task.265Given the variability in length and type of fibre terminations in the adultmuscle, and their implication in muscle force production, a more detailed evaluation ofthe distribution of different types of fibre endings within the muscle and within the MUwould be useful. Since it is not easy, at present, to study detailed MU anatomy inhumans, an animal model would be indicated, which would follow the methods describedby Ounjian et a! (1991) and Eldred et a! (1993). Briefly, if it is assumed that musclefibres belonging to a MU taper towards their ends forming an elliptic shape, the sum offibre areas measured on a single section through the approximate midlength of the MU,would not represent the true physiological cross-sectional area (CSA) of the unit. Thesuggested, more adequate method consist of measuring the CSA of a single MU, definedas the sum of the maximal areas to be found anywhere along the length of each of theMU fibres. The presence of MU territories of distinct shapes would infer that productionof muscle tension varies within the muscle.The use of MR imaging facilities enables an individualized assessment ofmuscle cross-sectional size, of internal muscle structure, and of craniofacial architecturein living subjects, which could be used for longitudinal or cross-sectional studies ofcraniofacial development or jaw biomechanics. With the development of softwaredesigned to reconstruct three-dimensional shapes directly from grey density valuesobtained from three-dimensional imaging techniques, the development of individualizedreconstructions of human anatomy will be extremely simplified.The ability to combine MR images with jaw movement data, measured withsix degrees of freedom, is an advanced technology, useful in the study of masseter266attachment movement. This method could also be used to study masseter attachmentdisplacement combined with electromyographic recording of different muscle regions,and in many other studies of attachment displacement, in which mandibular motion issubjected to different situations, such as by using bite splints of different designs, orchewing on different food consistencies. This type of experiments could prove to beuseful in the evaluation of masticatory dysfunctions.Muscle mechanics studies have commonly suggested that in a pennatestructure, muscle fibre contraction promote parallel sliding of the central aponeurosisrelative to the attachment tendons. Since MU territories were found to be mostlyconfined within tendinous boundaries, it is possible that differential contraction couldapproximate the central aponeurosis to one of the attachment tendons, changing in thatway the orientation of the resultant muscle-force vector. While it is theoretically possibleto investigate this supposition with MR imaging of the muscle when the jaw is kept atdifferent relationships relative to the maxilla, the required long imaging sessions makesit impractical. In recent years, ultrasound imaging has been increasingly used in clinicaland experimental investigations in living subjects. Its non-invasiveness, availability andease of usage makes it a useful and more practical tool to investigate internalaponeuroses movement during natural function.It is possible that high threshold MUs in the human masseter have morerobust firing properties than the low threshold units studied in this thesis. If this is so,data collection of the functional behaviour of MUs, with known locations relative tointernal muscle structures, would provide additional valuable data for the development267of a model of the internal mechanics of the masseter. Ultimately, such informationwould add to our understanding of motor control patterns in the orofacial region.The stereotaxic technique developed in this thesis could be utilized in a varietyof functional studies. The size and location of high threshold MU territories could bestudied in the masseter, to confirm whether they are also restricted to tendinousboundaries, or if they extend over a large volume passing across tendons. If the latterassumption proves correct, it would mean that only low threshold units are capable ofdifferential contraction. In addition, motor control studies, in which it is important tolocate the electromyographic recording sites relative to internal muscle structure, couldbe performed.Finally, the relationship between form and function in the masticatory systemcould be further explored to include other muscles and bone. With the same techniquesutilized in this thesis, anatomical variations of other muscles and bones could beexplored between individuals of similar or of different age groups. The displacement ofdifferent jaw muscles’ attachment sites, and the motion of the mandible and condylecould be also analyzed. 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The newidentification of m. temporalis superficialis, m. maxillo-mandibularis and zygomaticomandibularis in the human anatomy. [Jap] Acta anat nippon (Tokyo) 37:260-267.Young SW (1984) Nuclear magnetic resonance imaging. In Basic Principles. RavenPress, New York, pp 11.297Xiguang L, Zhang Y, Chen Y, Chen YX (1986) Applied anatomy of the massetericnerve. Zhonghua Kouqiangke Zozhi 21:226-227.Zajac FE (1989) Muscle and tendon: properties, models, scaling, and application tobiomechariics and motor control. Crit Rev Biomed Eng 17(4):359-411.Zey A (1939) Funktion des Kauapparatus und Schadelgestaltung bei denWiederkäuern. Med Inaug Diss, Frankfurt am Main.2987. APPENDIXThe orthogonal displacements of four tendinous insertion points, when thejaw is moved from tooth intercuspation to different functional positions, are presentedseparately for each living subject. The explanation of the methods, the anatomicallocation of the insertion points, and the discussion of the results are presented in section3.2 of this thesis.299Table XVI Displacements of four insertion points of subject I at different jawpositions. Displacements are expressed as orthogonal distances (in mm) calculated fromthe insertion’s coordinate point at maximum intercuspation.Insertion PointsTask 1 2 3 4x 0.40 0.59 0.53 0.59Working Side y -2.05 -4.10 -3.76 -4.39z -4.14 -6.59 -2.94 -4.23x 1.61 2.32 1.88 2.15Balancing Side y -3.94 -5.73 -5.43 -5.97z -1.92 -4.19 -0.89 -2.07x 0.32 0.39 0.42 0.43Open y -2.98 -5.08 -4.73 -5.37z -3.40 -5.87 -2.16 -3.47300Table XVII Displacements of four insertion points of subject II at different jawpositions. Displacements are expressed as orthogonal distances (in mm) calculated fromthe insertion’s coordinate point at maximum intercuspation.Insertion PointsTask 1 2 3 4x -0.81 -1.09 -0.66 -0.97Working Side y -3.90 -5.07 -4.18 -5.28z -6.16 -6.02 -3.39 -3.89x 1.45 1.42 1.36 1.36Balancing Side y -4.17 -5.52 -4.56 -5.82z -5.18 -5.06 -1.89 -2.52x 1.03 0.75 1.16 0.84Open y -5.03 -6.31 -5.35 -6.56z -5.79 -5.65 -2.74 -3.30301Table XVffl Displacements of four insertion points of subject ifi at different jawpositions. Displacements are expressed as orthogonal distances (in mm) calculated fromthe insertion’s coordinate point at maximum intercuspation.Insertion PointsTask 1 2 3 4x -1.16 -1.38 -0.83 -1.14Working Side y -0.75 -2.42 -2.54 -3.82z -4.28 -5.18 0.81 -1.41x 1.99 2.56 1.68 2.35Balancing Side y -3.73 -5.16 -5.45 -6.43z -1.05 -2.13 3.86 1.41x -0.03 -0.28 0.46 0.08Open y -2.92 -4.63 -4.74 -6.07z -2.25 -3.18 2.92 0.65302Table XIX Displacements of four insertion points of subject IV at different jawpositions. Displacements are expressed as orthogonal distances (in mm) calculated fromthe insertion’s coordinate point at maximum intercuspation.Insertion PointsTask 1 2 3 4x -1.61 -1.87 -1.33 -1.16Working Side y -0.80 -1.53 -1.36 -2.10z -4.59 -5.24 -3.56 -2.87x 1.11 1.50 1.11 1.26Balancing Side y -5.51 -6.10 -6.05 -6.77z -0.13 -0.91 0.88 1.57x 0.33 0.54 0.34 0.43Open y -5.02 -5.74 -5.66 -6.51z -2.33 -3.16 -1.16 -0.37303BIOGRAPHICAL INFORMATIONNAME:M6nica De Lorenzo TonndorfMAILING ADDRESS:1983 Gainsborough DriveAtlanta, Ga 30341USAPtACE ND DATOF BIRIH:Rio de Janeiro, Ri, BrazilJuly 08, 1963EDUCATION (Colleges and Universities attended, dates, and degrees):Federal University of Rio de Janeiro (UFRJ), Brazil1982 - 1986 Doctor of Dental Surgery (DDS)POSITIONS HELD:1983 - 1984 Teaching Assistant in Histology and Embryology - (UFRJ)1984 - 1987 Teaching Assistant in Temporomandibular disorders (Clinics) - (UFRJ)1985 - 1986 Teaching Assistant in Fixed Prosthodontics (Clinics) - (UFRJ)1986 - 1987 Assistant Instructor in Prosthodontics (Clinics) - (UFRJ)1987 - 1992 Teaching Assistant in Dental Occlusion and Temporomandibular Disorders(UBC)1988 - 1992 Teaching Assistant in Fixed Prosthodontics (pre-cinical course) - (UBC)PUBLICATIONS (if necessary, use a second sheet):See attached page.AWARDS:1987 - 1988 Rotary International Scholarship1988 - 1989 Government of Canada Award (WUSC)1988 Dr. George S. Beagrie Award1989 P.E.O. Chapter Sisterhood Award1989 Medical Research Council of Brazil (CNPq) Fellowship - declined1989- 1991 Brazilian Ministry of Education Fellowship (CAPES)c?tJ presentedto the Special Collections Division, University Library.DE•5PUBUCATIONSTonndorf M.L., Sasaki, K. and Hannam, A.G. Single-wire recording of regional activityin the human masseter muscle. Brain Res. Bull. 23:155-159, 1989.Tonndorf M.L. and Hannam, A.G. Effects of bite side on human masseter motor unitbehaviour. J. Dent. Res. 70:553, 1991.Tonndorf M.L. and Hannam, A.G. Masseter muscle attachment sites at different jawpositions. J. Dent. Res. 71:117, 1992.Tonndorf M.L. and Hannam, A.G. Motor unit territory in the human masseter muscle.J. Dent. Res. 72:371, 1993.Tonndorf, M.L., Connell D.G. and Harmam, A.G. Stereotactic location of EMG needleelectrode scans relative to tendons in the human masseter muscle. J. Neurosci. Methods(in press), 1993.Tormdorf, M.L. and Hannam, A.G. Motor unit territory relative to tendon in the humanmasseter muscle. Muscle & Nerve (in press), 1993.Hagiwara, M., Hannam, A.G., Korioth, T.W.P. and Tormdorf, M.L. Three-dimensionalmotion of the human mandibular condyle. J. Dent. Res. 72:267, 1993.


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