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Directed collisions between epithelial cells and fibroblasts in vitro on micromachined substrata Damji, Amin 1992

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DIRECTED COLLISIONS BETWEEN EPITHELIAL CELLS ANDFIBROBLASTS IN VITRO ON MICROMACHINED SUBSTRATAbyAmin DamjiB.Sc., The University of British Columbia, 1985D.M.D., The University of British Columbia, 1992A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Oral Biology)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIADecember 1992© Amin Damji, 1992In 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.ORAL BIOLOGY, FACULTY OF DENTISTRYDepartment of ^The University of British ColumbiaVancouver, CanadaDate^December 22, 1992DE-6 (2/88)ABSTRACTDental implants contact several cell populations in vivoincluding fibroblasts (F) and epithelial (E) cells, but there is littleinformation on whether different implant materials or surfacetopographies alter cell interactions. In this study F and E cells werecultured separately or in combination on smooth or grooved titaniumsurfaces. Grooves 40 gm wide and 3 gm deep, separated by ridges40 gm wide were placed in silicon by micromachining, a techniqueoriginally developed for the fabrication of microelectroniccomponents, and these surfaces, or their epoxy replicas, were coatedwith titanium by vacuum deposition (99). E cells from porcineperiodontal ligament and F from human gingiva were cultured ongrooved as well as control flat surfaces as described previously(102), and cell interactions were observed using reflected lightdifferential interference contrast optics. Fibroblasts movedsignificantly faster on grooved surfaces, but the speed of E-celllocomotion was not significantly altered.^The grooves, however,guided the direction of locomotion for both cell types.^Whencultured on grooved surfaces in such a manner that the F and E cellscollided head-on, the F, but not the E cells, frequently demonstratedcontact inhibition of movement. However, after such collisions,significantly more F continued to invade the E cell sheet than wasobserved after F-E collisions on flat surfaces. A possibleexplanation of these observations is that the grooved surfaceproduces and maintains F-cell polarity so that the direction of celllocomotion is less readily altered by cell-cell interactions.TABLE OF CONTENTSPAGE ABSTRACT  ^iiLIST OF TABLES  ^viLIST OF FIGURES  ^viiACKNOWLEDGEMENTS  ^viiiCHAPTER 1: INTRODUCTIONA. The Social Behaviour of Cells in Culture^ 11. Contact Inhibition^ 12. Contact Inhibition in Epithelial Cells^53. Non-Reciprocal Contact Inhibition 64. The Mechanism of Contact Inhibition 75. The Significance of Contact Inhibition^8B. Cell Adhesion to Artificial Surfaces in vitro 91. Interference Reflection Light Microscopy (IRM)^10a. Focal Contacts  ^1 0b. Close Contacts  ^1 2c. Extracellular Matrix Contacts  ^1 22. Electron Microscopy^ 1 3a. Basement Membrane  ^1 3b. Hemidesmosomes  ^1 43. Molecules related to Cell Adhesion^1 4a. Extracellular Adhesion Molecules  ^1 5b. Membrane-bound Adhesion Molecules  ^1 5c. Cytoplasmic Adhesion Molecules  ^1 7ivC. Mechanisms of Cell Migration^ 1 7D. Quantification of Cell Motility & Persistence^20E. The Effect of Surface Topography on Cell Adhesionand Migration^ 23Contact Guidance^ 23a. Microexudate Hypothesis  ^24b. Microfilament Bundle Hypothesis  ^26c. Focal Contact Hypothesis  ^27d. Selective Adhesion Hypothesis  ^27e. Stochastic Model Hypothesis  ^28PROBLEM FORMULATION  ^30CHAPTER 2: MATERIALS AND METHODS A. Cell Culture^ 32B. Grooved Titanium-Coated Substrata^ 33a. Cleaning  ^34b. Oxidation  ^34c. Photolithography  ^34d. Oxide Patterning  ^35e. Final Etching  ^35C. Fabrication of Epoxy Substrata^ 36D. Preparation and Characterization of Surfaces^37E. Electron Microscopy^ 39F. Time-Lapse Studies 39G. Quantitation^ 40VCHAPTER 3: RESULTSA. Characteristics of Cell Motility on Flat and GroovedSurfaces Prior to Collisions^ 42a. Speed  ^42b. Direction  ^45c. Persistence  ^48B.^Fibroblast-Fibroblast (F-F) Collisions^ 52C. Epithelial-Epithelial (E-E) Collisions 54D. Fibroblast-Epithelial (F-E) Collisions^ 58CHAPTER 4: DISCUSSION^62FUTURE WORK^66CONCLUSIONS^69REFERENCES^70viLIST OF TABLESPageTable 1^Speed and Direction of Fibroblast LocomotionPrior to Contact^ 46Table 2^Speed and Direction of Epithelial Cell LocomotionPrior to Contact^ 47Table 3^Behavioural Responses Observed in F/F CellCollisions^ 55Table 4^Behavioural Responses Observed in F/E CellCollisions^ 56viiLIST OF FIGURESPageFig. 1^Schematic drawing of contact guidance on groovedsurfaces^ 25Fig. 2^Example of a micromachined surface with V-shapedgrooves^ 38Fig. 3^Path of a fibroblast on a grooved substratum^43Fig. 4^Path of a fibroblast on a smooth surface^44Fig. 5^Profile of square cumulative displacement offibroblasts moving on grooved (a) and smooth (b)surfaces prior to confrontation^ 50Fig. 6^Plot of mean square displacement of fibroblastsmoving on grooved and smooth surfaces prior toconfrontation^51Fig. 7^Sequence taken from a time-lapse cinefilm of twofibroblasts colliding on a grooved substratum^53Fig. 8^Sequence taken from a time-lapse cinefilm of E cellsheets colliding on a smooth surface^57Fig. 9^Sequence taken from a time-lapse cinefilm of afibroblast colliding with an E cell sheet on asmooth surface^ 59Fig.10^Electron micrographs of epithelial-fibroblastrelationship after collision on a grooved substratum 60viiiACKNOWLEDGEMENTSI am indeed grateful to my supervisor, Dr. D. M. Brunette, forhis guidance, advice and support throughout this project.I would also like to express my appreciation to Mrs. LesleyWeston for her technical instruction in cell culture technique,cinemicrography and electron microscopy. I also wish to thank Mr.Hiroshi Kato for preparing the micromachined wafers.All the people in the Department of Oral Biology and theFaculty of Dentistry provided a very friendly environment in whichto work. I would especially like to acknowledge Dr. Babak Chehroudi,Ms. Holly Maledy and Mrs. Diane Price for all their support andfriendship.And to my wife Reena, who believed in me, and to my parentsfor making the whole thing possible, I am especially indebted.The experiments in this thesis were supported by the MedicalResearch Council of Canada.1A. THE SOCIAL BEHAVIOUR OF CELLS IN CULTURECell locomotion in culture can be influenced by a number offactors, including the nature of the culture medium and of thesubstratum. The behaviour of individual cells can also be influencedby their neighbours, and hence a population of cells may be said toexhibit social behaviour (1).1. Contact Inhibition Normal cells cultured on a plane substratum usually arrangethemselves approximately into a layer one cell thick. This fact ledMichael Abercrombie and Joan Heaysman to perform a series ofexperiments (now regarded as classic) to investigate the corollaryof this observation that cells do not move over each other's surfaceto form a multilayered structure. They investigated the interactionsbetween fibroblasts migrating out from two explants that werecultured 1mm apart such that the outgrowths from them wouldcollide. They found that soon after colliding, the cells showed amarked change in their behaviour. Their speed of locomotiondecreased sharply, their direction of movement became random, andthe population density in the space between the explants becamealmost stable. The cells consequently became virtually stationaryand remained more or less as a monolayer on the substratum (2).They interpreted these observations as indicating that fibroblastsare somehow inhibited from moving over each other. The cellsshowed no tendency to reduce their speed of locomotion until afterthe two outgrowths had collided; thus they concluded that this2inhibition of movement was a result of contact between the cells,and thus named the phenomenon 'contact inhibition' (2).Abercrombie (3) has since defined the term as "the prohibition,when contact between cells occured, of continued movement such aswould carry one cell over the surface of another".One explanation of contact inhibition is that the surfaceactivity involved in fibroblast locomotion is paralysed in thoseparts of the cell's surface which are in contact with other cells;this would effectively stop any further movement in the directionthat led to the contact (2). This idea was supported by the observedinverse relationship between the velocity of fibroblasts and thenumber of other cells with which they were in contact. Thus, whileisolated fibroblasts lacking contacts with other cells had a meanvelocity of about 1.4 gm per minute, fibroblasts in contact with fiveother cells had a mean velocity of only about 0.7 gm per minute (1).The experiments of Abercrombie and Ambrose (4) confirmed thatlocomotory surface activity is indeed inhibited in regions of cellcontact. Their time-lapse films of colliding chick fibroblastsrevealed that when the leading lamella of a cell touched any part ofthe margin of another cell its ruffling activity stopped andmovement of the cell in that direction ceased. Subsequentinvestigations have shown that contact inbition is a complexprocess involving adhesion, paralysis and contraction (3).After two cells of the same type collide, the cells adhere toeach other in the region of contact. The ultrastructural studies ofHeaysman and Pegrum (5) have provided evidence of the formation ofan adhesion between cells in which cytoplasmic specializations,3similar to the adhesive plaques formed between cell and substratum,develop where the cells touch. After contact inhibition has occured,the cells subsequently separate as cytoplasmic processes are oftendrawn out between the cells in the region of their first contact. Theadhesion is accompanied by paralysis of the leading lamella; itsprotrusive activity and ruffling are reported to stop (3). In somecases, however, only a part of the lamella touches another cell; insuch cases paralysis is confined to that part of the lamella andruffling and protrusion continue unabated in adjacent regions of thelamella not involved in the contact (6). At about the same time asits locomotory activity is paralysed, the leading lamella contracts.This contraction is also localized but involves a considerableproportion of the lamella behind its leading edge and may be strongenough to separate the cells (3). While these events are happening, anew leading lamella is often being formed elsewhere on the cell andis normally responsible for changing the cell's direction ofmovement accordingly (3).The most reliable method of determining contact inhibition isto observe a series of collisions between cells and to then recordtheir outcome. Since contact inhibition should prevent one cell fromoverlapping another, the extent to which a population exists as amonolayer is often used as an assay for the phenomenon. As thisapproach is often tedious and time-consuming, quicker but moreindirect methods have been developed. Fixed and stained culturesare usually examined to determine the extent to which cells form amonolayer; the faintness and irregularity of the edges of the cellsmake it difficult to detect cytoplasmic overlaps, so overlaps4between nuclei are usually counted. The number of nuclear overlapsis expressed as a percentage of the number expected if the nucleihad been distributed at random, thus providing an overlap index forthe population of cells (2,7). An overlap index of 100 per centindicates that the cells are randomly arranged and any figure lessthan this implies that the cells are to some extent inhibited frommoving over each other; the more closely the index approaches zerothe more perfectly is the population organized as a monolayer.A low overlap index is often interpreted as evidence that thecells exhibit contact inhibition, and a high index as evidence for theopposite, but such inferences should be treated with caution sincefactors other than contact inhibition may lead to monolayering,while extensive overlapping of cells may not necessarily imply theabsence of contact inhibition. Armstrong and Lackie (8) mixed chickfibroblasts with rabbit polymorphonuclear leukocytes and found alow heterotypic overlap index. This finding could be interpreted asmeaning that collisions between these two different cell typesresult in contact inhibition. However, direct observation of thecells showed that such collisions did not result in the changescharacteristic of contact inhibition; protrusive activity and rufflingdid not stop and there was no contraction. The obvious conclusionwas that factors other than contact inhibition must have preventedthese cells from overlapping each other. Such monolayering withoutinhibition of locomotory activity has variously been termed 'type 2contact inhibition' (9), 'contact inhibition of the second kind' (10),and 'contact inhibition of overlapping' (11). Heaysman (12) howeverproposed that the term contact inhibition be retained for the contact5interaction leading to an inhibition of cellular locomotory activity,and that the monolayering of cells without such inhibition should betermed 'substratum-dependent inhibition of locomotion'.Since its original discovery in cultures of embryonic chick-heart fibroblasts, contact inhibition has been shown to occur invitro between normal fibroblasts derived from a wide variety ofembryonic and adult tissues. It is known to occur not only whenfibroblasts of the same type collide (i.e. homotypic contactinhibition) but also when fibroblasts derived from different tissues,or even from different animals, collide (i.e. heterotypic contactinhibition). Furthermore 'self-contact inhibition' in cultures ofchick-heart fibroblasts has also been described (13). This occurswhen two lamellae from the same fibroblast make contact with eachother; both lamellae are paralysed and contract as if they belongedto two different cells.2. Contact Inhibition in Epithelial CellsAlthough less intensively studied than fibroblasts, there isevidence that epithelial cells in vitro also display contactinhibition. Unlike fibroblasts, epithelial cells may move over aplane substratum either as single isolated cells or as coherentsheets (14). Dissociated epithelial cells from a number of differentsources are strongly monolayered in culture, and there is someevidence that collisions between them lead to the formation of anadhesion and local paralysis of the locomotory machinery, but noretraction phase is observed (15).6The evidence for the occurence of contact inhibition within andbetween sheets of epithelial cells is well known. That leadinglamellae are usually present only at the edges of sheets or islandsof epithelial cells has been known since the earliest days of tissueculture (16). Similarly, the formation of leading lamellae by thenewly exposed edges of cells in the margins of spontaneouslyoccuring or deliberately created gaps in epithelial sheets has beendescribed by several workers (17,18). It is also well known that theformation of a leading lamella is inhibited in areas of contactbetween sheets of homologous epithelial sheets colliding in culture(19, 20). In all, these observations are consonant with the beliefthat contact inhibition occurs between individual epithelial cells invitro.3.  Non-Reciprocal Contact Inhibition Collisions between epithelial cells and fibroblasts have notbeen studied extensively, but in a number of cases fibroblasts havebeen reported to display contact inhibition after colliding with anepithelial sheet (15). Parkinson and Edwards (21) found thatfibroblasts from the chick choroid show contact inhibition aftercolliding with sheets of pigmented retina epithelial cells but thatthere is no reciprocal response from the epithelial cells; the leadinglamellae at the edge of the epithelial sheet are quite unaffected bycontact with the fibroblasts and the sheet continues its forwardmovement without interuption. This is a clear example of 'non-reciprocal contact inhibition', a phenomenon first described incollisions in culture between normal chick fibroblasts and mouse7MCIM sarcoma cells (22).^The extent to which non-reciprocalcontact inhibition occurs between different cell types is unknownbecause only a small number of cell types have been examined.4. The Mechanism of Contact Inhibition There have been many attempts to explain the characteristictriad of adhesion, paralysis and contraction which typifies contactinhibition. One possible explanation was that leading lamellae maybe unable to bend sufficiently to surmount another cell, or, if bentsufficiently, may be unable to maintain locomotory activity (3). Thefindings of Vesely and Weiss (9) do not support this hypothesis.They demonstrated that if colliding mouse fibroblasts (which areknown to display homotypic contact inhibition) are fixed withgluteraldehyde, other living cells of the same type move freely overtheir surfaces without any inhibition. Another hypothesis presentedby Abercrombie (3) was that the inhibition of locomotory activityresults from the action of molecules diffusing through gap junctionsformed between colliding cells. However, this signal hypothesis hasbeen unsupported by Heaysman and Turin (23), who observed thatfibroblasts exhibited contact inhibition when they collided withdead fibroblasts which had been fixed by membrane stabilizingagents and presumably could not transmit signals. Another tentativeexplanation emphasized the adhesivity of the membrane. In thisview, which is based on differential adhesion, cells are less likelyto establish an efficient attachment to the surface of other cellsthan to the substratum. In support of this hypothesis, fibroblastscultured on nonadhesive substrata have been shown to clump8together instead of following the principle of contact inhibition ofmovement according to which cells would disperse on thesubstratum (24). Another similar hypothesis (5) interprets contactinhibition of movement as a consequence of mechanical failure ofthe attachment site between two cells. In an attachment site, twofocal contacts often form with microfilament bundles insertingfrom both cells. Two bundles of microfilaments may exert morestress on the adhesion plaque than one. This possibly results infailure of the attachment and the subsequent diversion of cellstowards the substratum.5. The Significance of Contact Inhibition Contact inhibition influences the social behaviour of cells inculture in several ways. In cultures whose population density is notuniform contact inhibition causes cells to move away from a densely-populated area towards a sparsely-populated one (25). Celllocomotion occurs preferentially in this way because those cellswhich move towards the sparsely-populated area will be lessinhibited by contact with other cells. This effect has been clearlyseen in the centrifugal migration of cells from an explant of tissuein vitro (26). Vasiliev et al. (27) observed similar orientedmovements when a dense and essentially immobile monolayer ofcells was 'wounded' by scraping away some cells in order to create acell-free area on the substratum. The cells at the edge of thewound, now no longer in contact with neighbouring cells on all sides,started to move towards the cell-free space.^This directedmovement persisted until the space was repopulated.^Such9observations suggest that, to some extent, the initiation, directionand cessation of cell locomotion in culture can be explained on thebasis of contact inhibition, and it is thought that, in somesituations, the same is true in vivo.B. CELL ADHESION TO ARTIFICIAL SURFACES in vitroMuch of our knowledge regarding cell/substratum adhesionoriginates from studies of cells in culture, particularly fibroblastsand epithelial cells. Using in vitro techniques, great insight hasbeen achieved into how cells interact with their environment at themicroscopic and molecular level. The process of cell adhesion invitro differs in serum-free and serum-containing medium; however,the events happening in the presence of serum are thought likely toresemble the behaviour of cells in vivo. In the presence of serum,the adhesion of cells to a foreign object is a multi-step process.The initial step is the adsorption of the medium and adhesionmolecules to the surface. The chemical and physical nature of asubstrate surface can influence the quality and quantity of theadsorbed adhesion molecules (28,29). The second step in celladhesion is the actual initial contact of the cell with thesubstratum, a process that may involve electrostatic forces (30).As both the surface of the cell and currently-used tissue-culturesubstrata are negatively charged, they would be expected to repeleach other and thus inhibit contact or adhesion. Weiss (31),however, suggests that the charges on the surfaces of cells are1 0distributed as patches of greater-and lesser-than-average chargedensity. Weiss noted that the areas with lesser-than-averagecharge density could approach the substratum closely enough tofacilitate adhesion. The third and fourth steps of attachment andspreading are complementary and involve reorganization of thecytoskeleton and direct interaction of the cell membrane with thesubstratum.1.  Interference Reflection Light Microscopy (IRM) Cell adhesion to a substratum has been studied at the lightmicroscope, electron microscope and molecular levels. The principlelight microscopic technique used to study the cell-substratuminterface is interference reflection microscopy (IRM). IRM enablesan observer to determine how closely various areas of a cell adhereto optically clear substrata (32). Using IRM, Izzard and Lochner (33)classified the contact between cells and their substratum into twomain types: focal contacts and close contacts. A third type of cell-substratum contact, the extracellular matrix (ECM) contact, has beenreported by Taylor (43).a. Focal ContactsFocal contacts appear in IRM as black areas 0.25-10 gm wideand 2-10 gm long. They represent regions where the distance of thecell membrane from the substratum is 10-15 nm. Focal contactswere first observed in the electron microscope by Abercrombie et al.(34), who called them "adhesion plaques". They speculated that11stress fibres associated with the focal contacts generate tension topull the whole cell forward. In agreement with Abercrombie,Heaysman and Pergum (35) reported that fibroblasts can move onlyover a substratum to which they form focal contacts, and it has alsobeen reported that epithelial cells, which migrate in the form of asheet, develop focal contacts primarily at the periphery of the sheetat the leading edge (36). These findings led to the hypothesis thatfocal contacts were also involved in the migration of epithelialcells, since it is believed that the motile power of an epithelialsheet is located at its free edge close to the ruffling membrane (36).It also should be noted that some degree of adhesion is generallyrequired for most cell types to migrate, but if adhesion becomesexcessive it may be inhibitory. In this connection, Singer (37)reported a cell-adhesion molecule, fibronectin, at some focalcontacts of fibroblasts. He suggested that perhaps two kinds offunctionally distinct focal contacts exist: (i) fibronectin-negativefocal contacts that might be relatively unstable adhesion sites,characteristic of moving cells, (ii) fibronectin-associated focalcontacts that might be characteristic of stationary fibroblasts.Despite growing evidence of the role of focal contacts in celllocomotion, several studies indicate that focal contacts are moreprominent in cells displaying little or no motility, and that highlymotile cells can migrate without forming apparent focal contacts(38,39). Thus focal contacts may not be an absolute requirement forthe locomotion of some cell types. It is possible that the variationin the role of focal contacts may reflect the properties of differenttypes of cells.12b. Close ContactsClose contacts also mediate cell-substratum attachment andare characterized as broad areas, approximately 30 nm from thesubstratum, that appear grey in the IRM. Close contacts are largerand more diffuse than focal contacts, and their biochemicalcomposition has been more difficult to define (40). It has beensuggested that they may be involved in generating traction forforward protusion of the lamellipodium (41). Close contacts are theonly form of attachment observed in some highly mobile cells,suggesting that close contacts alone may be adequate for rapid cellmigration (38,39).c. Extracellular Matrix ContactsA third class of cell-substratum attachment, the extracellularmatrix contacts (ECMC) appear white in the IRM, indicatingapproximately 100 nm or more separation from the substratum.When viewed in the electron microscope, the space between the celland the substratum contains strands of extracellular matrix. Intheir investigation of ECMC, Singer et al. (42) reported closetransmembrane association of fibronectin-containing fibres andactin filaments, as well as an accumulation of heparan sulfateproteoglycans. They have termed the ECMC "fibronexus". Theyspeculated further that these sites could function during cellmigration along tracts of fibronectin-containing extracellularmatrix in vivo. ECMC have been reported in fibroblasts and epithelial13cells attaching to various substrata such as epoxy, titanium and gold(43,44).2.  Electron MicroscopyThe advent of electron microscopy has led to the observationof two structures associated with the attachment of epithelial cellsto various substrata, namely the basement membrane andhemidesmosomes.a. Basement MembraneThe basement membrane (BM) is a three-layer, complexmixture of mainly non-fibrillar collagen, glycoproteins andproteoglycans. It is always found between epithelium andconnective tissue and is frequently reported at the epithelial-substratum interface (45-49). The BM is usually organized intothree layers: (i) the "lamina lucida" appears as a 10-50 nm-thickpale layer associated with the epithelial cell membrane; (ii) the"lamina densa" is a 20-300 nm-thick electron-dense layer closer tothe substratum; and (iii) the "lamina fibroreticularis" or "sublaminadensa" is a poorly-defined layer with fine anchoring fibrils andcollagen fibers, that probably originates from the connective tissue.In addition to mediating adhesion of epithelial cells, numerous otherfunctions have been attributed to the BM, including a role as aselective molecular barrier, and participation in tissuemorphogenesis and cell differentiation or regeneration (50).14b. HemidesmosomesHemidesmosomes (HD) have been reported regularly at theepithelial cell-substratum interface (45,48). They appear as denseplaques, in diameter and 20 nm from the substratum (36).The hemidesmosome was named for its morphological similarity toone half of the "desmosomes" that attach epithelial cells together(51). Recent characterization of the molecular components of thejunctions show that they are in fact quite unrelated implying thatstructural similarity is fortuitous (46). HD, like desmosomes,consist of an electron-dense plaque at the cytoplasmic side of theplasma membrane, into which intermediate filaments are attached.On the extracellular side of HD, fine filaments, possibly composed ofcollagen type VII (52), cross the lamina lucida and attach to thelamina densa and may extend into the sublamina densa (53). HD havebeen identified at the interface of epithelial cells cultured onseveral implant surfaces such as epoxy, carbon, ceramics andtitanium (45,48).3. Molecules Related to Cell Adhesion Cell attachment to artificial surfaces can also be examinedfrom a molecular perspective. The cell membrane does not attach toa substrate directly, but rather attachment is mediated byextracellular, membrane-bound and cytoplasmic adhesion molecules.15a. Extracellular Adhesion MoleculesGlycoproteins are probably the most important extracellularadhesion molecules. Fibronectin, a high molecular weight (440,000D) glycoprotein, is perhaps the most comprehensively studiedglycoprotein. It has the ability to bind to both the cell membraneand other extracellular components such as collagen andproteoglycans. Adhesion glycoproteins in other tissues andstructures include: laminin in the basement membrane, osteonectinin mineralized or soft connective tissue, chondronectin in cartilageand tenascin in embryonic tissue (50,54).Another family of extracellular adhesion molecules that canassociate with the cell membrane are proteoglycans. Structurally,proteoglycans consist of a protein core in which serine-glycinesequences serve as attachment sites for one or moreglycoseaminoglycan (GAG) chains (50). These chains function bymeans of their high-affinity binding sites to collagen, fibronectin,growth factors and cell membrane.Structural proteins such as the collagens and elastin(s), whichare fibrous components of the extracellualr matrix, also participatein the cell- adhesion process. Collagens can bind to the cellmembrane through two mechanisms, either through their arginine-glycine-arspartic acid sequence or indirectly by means of otherattachment molecules such as proteoglycans or glycoproteins (50).b. Membrane-bound Adhesion MoleculesMembrane-bound adhesion molecules or cell surface receptorsare essential for mediating the attachment of the extracellular16matrix to intracellular components. The most important group ofthese receptors is a family of related molecules termed integrins(55). Morphologically, integrins are composed of a largeextracellular domain, a small transmembrane domain, and anintracellular tail (56,50). Other cell-surface molecules are theproteoglycans syndycan and CD44 that contain heparan andchondroitin sulfate chains (57). The extracellular domains of thesemolecules bind to fibronectin, laminin, tenascin and collagen. Aswith integrins, the intracellular domains have high affinity to theactin component; however, the mechanisms of such interactions arenot clear (50).Several cell types exhibit large hyaluronan-dependentpericellular coats. The structure of these coats depends on theinteraction of pericellular hyaluronan (HA) with a HA-bindingprotein (HABP); antibody to HABP has been shown to block coatformation. Such HA-dependent coats may be important ininfluencing cell interaction and behaviour as HA has been shown toact as a direct mediator of both homotypic and heterotypic cell-celladhesion (118).The expression of fibronectin and integrins was recentlystudied in cultured periodontal ligament epithelial cells (PLE) byUitto et. al. (119). Their results indicate that PLE cells synthesizeand utilize fibronectin-containing material for their attachment,spreading and migration; and that cell-substratum and cell-cellcontacts are at least partly mediated through integrin-type cell-surface receptors.1 7c. Cytoplasmic Adhesion MoleculesMuch of our knowledge of the cytoplasmic domain ofattachment molecules comes from observations of the cytoplasmiccomponents of focal contacts in fibroblasts, and hemidesmosomes inepithelial cells. It appears that at focal contacts the actinfilaments attach to integrins indirectly via a series of cytoplasmicproteins including vinculin, talin, alpha actinin and an actin-specificcapping protein (58).In summary, it appears that the adhesion of the cell to theextracellular matrix and subsequently to the substratum is acomplex phenomenon involving the cooperation of at least threegroups of adhesion molecules.C. MECHANISMS OF CELL MIGRATIONThe study of cell migration has been accomplished mainly invitro. The mechanisms of cell migration have been studied mostextensively in fibroblasts, phagocytes and epithelial cells. Althoughthere is no agreement on the mechanism of cell locomotion, itappears that cell locomotion involves various stages includingadhesive interactions, force generation, protrusion of cellularprocesses and relocation.One of the earliest speculations on the mechanism of cellmovement made in the 18th century suggested that a transition fromsolid to liquid status was required to provide the motile force18necessary to drive cells forward (59). Although the majorcytoplasmic protein actin has the ability to form sol-to-geltransitions, this theory was overshadowed by the discovery ofmyosin. Myosin, the major protein of muscle cells, interacts withactin to produce the sliding movements of the myofibrils thatresults in muscle contraction. Since the intermittent distributionof myosin on the actin-containing microfilament shows similaritiesto that in muscle cells, it was proposed that a mechanism similar tothat of force generation in muscle cells may be operative in non-muscle cells (56). Abercrombie et al. (60,61) proposed a model inwhich continuous contractions of the stress fibres associated withfocal contacts of the leading lamellae exerted a force on thesubstratum strong enough to pull the cell forward. Although stressfibres have been shown to shorten as the cell moves (62), thistheory does not account for cells that do not possess focal contactsand yet are capable of locomotion (63). Huxley (64) and Small (65)elaborated on a theory that proposed that the forward movement of acell results from a forward flow of the cytoplasmic constituents,that in turn are propelled by the sliding movement of the membrane-bound actin filaments and myosin molecules. A broader theoryproposed by Dunn (62) suggests that cells move by the coordinationof force generation by the cytoplasmic actin/myosin meshworks,which are distributed at the leading lamellae and throughout thecell.The actin/myosin model of cell movement soon becamecriticized as new findings revealed that non-muscle cells possess avariety of myosin-like molecules and that many of these molecules19differ in structure and function from myosin of the muscle cells (65-67). Proponents of these new findings boldly ignore the role ofcytoskeletal proteins and emphasize the role of direct incorporationof molecules into the cell membrane, and subsequent recyclingduring endo/exocytosis, as a potential mechanism of locomotion(68). Heath and Holifield (71) have recently criticized this theory,stating that cell motility is not driven by bulk membrane flow.Recently the old notion of sol-to-gel transformation of actin hasbeen revived, with a new interpretation that this mechanismgenerates osmotic forces to drive the cell forward (69).Epithelial cells, however, migrate in a sheet or unit in whichcells within the sheet form long lasting desmosomal attachments(18.) Dipasquale (70) demonstrated that in vitro cells located at themargin of the sheet display marked surface activity includingruffling, and suggested that the marginal cells may provide themotive power for the migration of an epithelial sheet. In their viewepithelial cell locomotion resembles a train in which the marginalcells act like the locomotive and the non marginal cells are draggedpassively behind like boxcars. This theory may have in vivo validityin the case of epithelial cells migrating to cover a wound in thecornea (36).The leap-frog or tracked-vehicle model, proposed by Krawczyk(72) and Gibbins (73), suggests that cells several microns behind theleading edge of a migrating epithelium proliferate. These cells thenmove up, forward and over the basal cells and then move down toattach to the substratum. In this way new cells are layered down inan advancing front. Evidence for this theory is more indirect than20the train theory as it rests on observations of histological sectionstaken at long intervals that need to be interpreted to infer thedynamic events of cell locomotion (72,74).Much still remains to be learnt about the mechanismsunderlying cell migration and factors regulating such mechanisms.It seems probable that not one single mechanism but rather an arrayof mechanisms may be involved in cell migration, and equallypossible that different mechanisms may dominate in a particularpopulation of cells.D. QUANTIFICATION OF CELL MOTILITY & PERSISTENCEThe ability of cells to locomote actively is important in manybiological processes. Embryogenesis, the immune response, andwound repair are examples in which individual cell motility occurswithin a multicellular organism. Many cell types are able tomaintain their locomotive properties in tissue culture. Cell motilityin vitro has been studied for many years and from these studiesdetailed qualitative description of cell morphology as well asquantitative information on cell motility has been presented for avariety of cell types and a variety of experimental conditions(24,34,56).The dynamic morphology of migrating cells, particularlyfibroblasts, is highly complex. Recent work has attempted toquantify both cell morphology and motility and thereby elucidate thecellular and molecular machinery that drives and controls these21phenomena. To date, analysis of cell morphology and themeasurement of cell motility have been studied with automated andinteractive video-enhanced imaging systems as well as with 16 mmtime-lapse films.Many researchers are interested in finding a technique whichwould be applicable to cinephotomicrographic analysis of cellmovement and which would allow them to determine throughstatistical analysis whether cells were moving randomly or whetherthey were under the influence of any external or internal forces.On a flat substrata a cell in a simple random walk turns atrandom intervals and the the probability of turning through a largeangle is the same as the probability of turning through a very smallangle. If the steps become very short, either because the frequencyof turning increases or because the speed is very low, then theoverall displacement is very small. It is clear that if the cell is toachieve a significant displacement in a realistic time then thefrequency of turning must be restricted or the speed must beinfinitely high. An alternative to restricting the frequency ofturning is to restrict the angle through which turns are made, thusyielding a considerably greater net displacement. It is possible thatgrooved substrata impose this very restriction on the cells. As themaximum permitted angle of turn decreases, the path of a cellbecomes straighter and the displacement increases to a limit set bythe speed of the cell.Since cells have an upper limit to the speed at which they canmove, and yet make significant net displacements in uniformenvironments, there must be some constraint upon turning behaviour22which has its origin within the cell. From observation it is knownthat cells tend to be polarized to a certain extent. Recognizing thatthe active protrusions of the leading lamellae of a fibroblast arerestricted to a particular area of the cytoplasm, it follows that theturning behaviour is likely to be restricted by the necessity ofrearranging the motile machinery in order to make a turn. Hence theturning behaviour would be limited by internal constraints and thatthe path of a moving cell would show the phenomenom ofpersistence.An objective way of demonstrating this persistence is toexamine the displacement of cells as a function of time. Thismethod was used by Gail and Boone (107) who investigated theeffect of 'persistence' on the pure random walk model, and foundtheoretically and confirmed experimentally that the motility of apersisting cell could indeed be characterized by an augmenteddiffusion constant. They showed that measurements of the meansquare distance traveled as a function of time provided a convenientmeasure of cellular motility. It is intuitively clear that a cellwhich tends to persist in its direction of motion will undergo alarger displacement in a given time than would the same cellwithout directional persistence. The authors confirmedtheoretically that the mean square displacement of a persisting cellalways exceeds that expected of a similar cell without persistence.23E. THE EFFECT OF SURFACE TOPOGRAPHY ON CELL ADHESIONAND MIGRATIONA major factor affecting cell adhesion as well as cellmigration is the macroscopic and microscopic geometry of thesubstrate surface. Surface topography might influence cells in avariety of ways, including confining diffusion channels in and out ofcells, limiting access to nutrients and escape of waste products, andinducing stress and strain on the cell membrane (75). Commonly allcell types, excluding those that grow in suspension, live in a milieuwith some type of topography. This topography could be provided byother cells, extracellular matrix, other organisms or by artificialmaterials.^Curtis (75) in a recent review, divides the surfacetopographies of materials into two categories:^those producedinadvertantly during the tooling of a prosthesis and those produceddeliberately to achieve certain desired end results.Contact Guidance Although first observed by Harrison (76), contact guidance wasnamed and substantially studied by Weiss (77,78), who definedcontact guidance as the tendency of cells to be guided by the shapeof the substratum to which they are attached (figure 1). He observedthat cells preferentially oriented and migrated along parallel linesengraved in glass or the oriented bundles of collagen naturallypresent in fish scales. Later studies indicated that not only groovedsurfaces oriented and directed cell migration but substrata madefrom uniform glass cylinders also demonstrated the same ability (79-2483). Contact guidance has been observed in diverse cell populationsincluding fibroblasts, neutrophils and epithelium (79-82). Recentstudies using more controlled microfabrication techniques haveshown that fibroblasts respond hierarchically to grooves ofdifferent dimensions, with large grooves dominating the effects ofsmaller grooves (79). Epithelial cells on grooves 3-60 gm-deep,with 30-220 gm-spacing, appear to be affected by the repeatspacing of the grooves more than by their depth, and morepronounced alignment has been noticed on densely-spaced groovedsurfaces than on surfaces with widely spaced grooves (80).Recently Clark et al. (97) reported that on shallow grooves (0.2-1.9gm), repeat spacings between 4-24 gm have little effect onfibroblasts' and epithelial cell's orientation, and groove depthdetermines the extent of cell alignment. Several mechanisms havebeen hypothesized to explain contact guidance.a. Microexudate hypothesisThis theory, proposed by Weiss (78), suggests that colloidalexudates secreted from the cells first become oriented along thelong axis of the grooves. Subsequently, the leading edge of a cellwould track on these molecules and thus orient the locomotion ofthe cell. Trinkaus (84) believes strongly that this theory is the besthypothesis yet available for explaining orientation in the movementsof most cells in vivo. In Trinkaus' view, Weiss's hypothesis could bethe readiest explanation for the directed migration of pigment cellsalong blood vessels, the movement of nerve axons along bloodvessels, and the movement of neural crest cells along the side of the25(e)^..^......^.....\";_f(g)^............^RidgeGroof......,^e ,Figure 1. Schematic drawing of contact guidance on groovedsurface26neural tube. This theory, however, has not been endorsed by in vitroexperimental tests. Curtis and Varde (85) showed orientation onuniform glass cylinders of cells that would not be expected toproduce any microexudate orientation. Yet another flaw is that cellsorient with the grooves in serum-free medium faster than the timenormally required for protein synthesis and secretion (78).b. Microfilament bundle hypothesisThis theory, originally suggested by Curtis and Varde (85), wassupported by Dunn and Heath (86) who observed the behaviour offibroblasts on simple patterned substrata with sharp changes ininclination. Their theory was based on the relative rigidity as wellas contractile ability of stress fibres inserting into the focalcontacts. Dunn and Heath proposed that stress fibres of fibroblastssense the geometry of a substratum, and are unable to bend oversharp changes of inclination in a substratum. Thus fibroblasts couldnot locomote efficiently in a direction that involves crossing such asharp inclination. A criticism of this theory arises fromobservations that indicate not all cells that move develop focalcontacts and stress fibres, and yet such cells exhibit contactguidance (87,88). In addition, Brunette's (80) observation that cellscan be oriented on grooves of small dimension, which are less likelyto interfere with stress fibres, does not support this theory.2 7c. Focal contact hypothesisOhara and Buck (89) suggested that the mechanical stiffness offocal contacts determines cell orientation and thus contactguidance. There is a tendency for focal contacts to form and align inthe direction of the grooves and ridges, simply because thesubstratum available for focal contact formation would beunrestricted. This would result in alignment and polarization of thecytoskeleton, and ultimately the entire cell itself. This theory,however, does not explain the behaviour of some cell types, such asleukocytes, or fungal hyphae, which are contact guided and yet do notseem to have focal contacts (75). This theory was later modified byCurtis (75), who described contact guidance as the ability of cells toreact to discontinuities by forming attachment sites to them. Incontrast to Ohara and Buck, Curtis made no presumption about thestructure of these adhesion sites; however, he showed that theattachment sites frequently associated with actin condensation maybe disturbed as the size of the discontinuities becomes comparableto or greater than a cell's dimensions. Curtis speculated that thisinterference with the adhesion sites would be enough to exert aneffect on cell orientation.d. Selective adhesionIt has also been suggested that contact guidance occursbecause cells may show preferential adhesion to a specific area orto a particular shape on the substratum. For example, on groovedsubstrata, cells may adhere selectively to the walls of the groove,the ridges, or the edges where the ridges and groove walls intersect.28Selective adhesion as a means of orienting cells has been shown inseveral experiments in which channels were cut or scratched in anonadhesive surface coating to expose the more adhesive underlyingsurface. These experiments used glass substrata coated withchrome (90), gold (91), or brain phospholipids (92). Polystyrenesubstrata were also used, in which case substrata were coated withfine lines of silicon monoxide (93) or selectively sulphonated (94).These experiments indicate that adhesive heterogeniety of thesubstratum can alone induce orientation in spreading cells andsubsequently influence their directions of translocation.Nevertheless, this theory appears unsatisfactory in the case ofcontact guidance of cells cultured on uniform glass cylinders, wheredifferential adhesion could not play a role (95).e. Stochastic modelThis recently suggested model (96), proposes that cells reactprobabilistically to the topographical features of a surface. Theprobability of a cell making a successful protrusion and adhesion ina given direction may be reduced by specific features of a surfacetopography, and similarly protrusions and adhesion made in otherdirections might be favored. Thus cell shape and direction oflocomotion would be determined probabilistically by the surface. Aslight deviation in any step involved in cell locomotion has thechance of being sufficiently magnified to induce cell orientation.The probabilistic nature of cell response to a topographical featurewas also addressed by Clark et al. (97), who suggested that specifictopographical features could reduce the probability of a cell making29a successful protrusion and/or contact in a given direction. Thisreduction could be due to a number of factors; however, Clark et al.favored Dunn and Heath's (86) proposal on relative inflexibility ofmicrofilament bundles as the most economical explanation. Thus itappears that no single hypothesis can sufficiently explain contactguidance, and it is possible that more than one mechanism isoperative30PROBLEM FORMULATIONDental implants, percutaneous devices, and natural teethencounter unique problems occasioned by their anatomical location,for they must penetrate a stratified epithelium and extend into amicrobe-rich environment. The survival of both teeth and dentalimplants is threatened by the migration of epithelium down the rootof the implant or tooth. In the oral cavity, this apical migrationleads to the formation of pockets in which food debris and bacteriacan be trapped, and ultimately to the epithelium walling off thetooth or implant, which is exfoliated.In attempts to defeat this problem and promote tissueintegration, manufacturers of implants have produced devices thatvary markedly in their surface topography. For the most part thesevariations in surface design represent empirical attempts tooptimize performances that are based more on biomechanical andbiomaterial considerations than on the application of principles ofcell behaviour. Such empiricism has been justified because ourknowledge of how surfaces affect cell behaviour is inadequate.The subject matter of this thesis addresses this very problem.The experiments in this study have been specifically designed toinvestigate a number of key issues: how do surface topographicfeatures such as grooves affect the social interaction of differentcell populations? How do grooves affect cell orientation andmigration in relation to speed, direction and persistence?Many of the reported observations of collisions betweenfibroblasts and epithelial cells to date are generally made form31experiments employing smooth substrata.^It may be possible,however, to obtain different results by modifying the shape of thesurface to which the cells are attached.A specific problem in studying collisions between cells is thatit is difficult to control the specific regions of the cells that comeinto contact. In typical cell-confrontation experiments the angle atwhich the cells approach each other is not controlled, so that insome collisions leading lamellae (LL) confront each other whereas inother collisions a LL encounters the side of another cell. Recently,micromachining, a process developed for microelectronicsfabrication, has been introduced as a means of preparing groovedsubstrata that can control the direction of cell locomotion (98-101).This study examines collisions of fibroblasts and epitheliumunder conditions where the orientation of collisions has beencontrolled by growing cells on grooved titanium-coated surfacesprepared by micromachining. The results of these directed collisionexperiments have implications for the design of dental implantsurfaces so that they can be rationally based on the appliedprinciples of cell locomotory behaviour. The titanium-coatedsubstrata represent an in vitro model of a dental implant surface;thus it is of great importance to determine what is the effect ofsuch a surface on fibroblast-epithelial cell social interaction. Thisinvestigation is also the first of its kind in that it compares thebasic parameters of cell locomotion and behaviour on smooth versusgrooved titanium-coated surfaces.32MATERIALS AND METHODSCELL CULTURE Epithelial cells, derived from porcine epithelial rests ofMalassez (a group of cells found in the periodontal ligament), andhuman gingival fibroblasts were isolated and cultured as describedby Brunette et al. (102). This technique results in the growth ofboth epithelial cells and fibroblasts. The two cell types could beseparated by the tendency of fibroblasts to be less resistant todetachment by trypsin (103). In brief, the cells were cultured inalpha Minimal Essential Medium (MEM)1 supplemented with 15% fetalbovine serum2 and antibiotics (penicillin G3 100 pg/ml, gentamycin50 p.g/ml, fungizone4 3 jig/m1) at 37°C in a humidified atmosphereof 95% air 5% CO2. Epithelial cells were removed from the growthsurface and suspended using a trypsin solution6 (0.25% trypsin, 0.1%glucose, citrate-saline buffer, pH 7.8) and seeded onto the substrataat a cell-population density of 2 x 104 cells/cm2. In theconfrontation experiments 3 pl droplets containing 1 x 106 cells/ml(F) or 5 x 106 cells/ml (E) were placed 2 mm apart on the grooved orsmooth substrata. After 2 hours the unattached cells were removed1Terry Fox Media Lab, Vancouver, B.C.2Bocknek, Toronto, Ont.3Sigma, St. Louis, MO, USA4Gibco, Grand Island, NY, USA6Worthington TPCK33by washing with medium and the cell populations allowed to growand migrate towards each other.GROOVED TITANIUM-COATED SUBSTRATAGrooved culture substrata were prepared by a technique calledmicromachining. This technology has been utilized in a number ofapplications as it produces grooves, slots or pits of precisedimensions in silicon or gallium-arsenide wafers. The particularmicromachining technique employed in this study was adopted fromthat developed in the Department of Electronical Engineering of theUniversity of British Columbia by Camporese et al. (104) for thefabrication of high quality photomasks for solar cells.Micromachining allows excellent control over the production ofgrooved or pitted surfaces because different aspects of the groovesand pits are controlled by different steps involved in themicromachining process.The silicon wafers used in this study were the n-type (100)silicon wafers.6 These wafers were 5 centimeters in diameter, 200-350 gm in thickness, and had polished front surfaces and bright-etched rear surfaces. The micromachining process7 involves severalstages:svirginia Semiconductor Inc., Fredericksburg, Va., USA7Micromachined wafers were prepared by Hiroshi Kato, Dept. ofElectrical Engineering, U.B.C.34a. CleaningA vigorous cleaning criterion was used to obtain highly cleansilicon wafers. The method involved the following steps: (i) tenminutes in a solution of 300 ml H20, 60 ml 30% H202, 60 ml 30%NH4OH at 750-850C, (ii) thirty seconds in 10% HF, and (iii) tenminutes in a solution of 300 ml H20, 60 ml HCL, 60 ml H202. Waferswere rinsed for at least ten minutes in distilled water, after eachstep of cleaning. Finally wafers were immersed in isopropyl alcoholfor four minutes and blow-dried in filtered nitrogen.b. OxidationThe objective of this stage was to grow a layer of silicondioxide on the front and rear surfaces of the silicon wafer. This wasachieved by using wet oxygen at 1150°C for two hours in a furnace.8This procedure produced oxides 0.6 gm thick on each side of thesilicon wafer.c. PhotolithographyThis stage begins with the production of a computer-generatedmaster pattern of the grooved surface to produce photomasks.Currently, photomasks are produced by a computerized optical-pattern generator and the pattern is then etched in chromium-gold.The next step of this phase involved coating the front surface8Two inch tube furnaces (No.7), Fairchild Semiconductor Corp., USA.35(polished side) of the silicon wafer with negative photoresist.0Wafers were then exposed through the photomask with a 320 nmwave-length UV light, developed and baked at 160°C. It should benoted that the alignment of the photomask on the silicon wafer playsa significant role in obtaining precise groove dimensions dictated byanisotropic etching. Slight misalignment of the photomask maycause a less predictable etching.d. Oxide patterningBuffered HF was used to remove the unprotected oxide layersresulting from the UV light exposure. Then the remaining developedphotoresist was removed in microstrip (NMP) solvent.10 Thisprocedure produced a silicon wafer whose front surface waspatterned with an oxide layer. For example, wafers used for groovedsurfaces had strips of silicon oxide.e. Final etchingThe wafers were preferentially etched in 19% potassiumhydroxide solution at 80°C. The potassium hydroxide etches awaythe silicon, leaving the oxide layer intact. The speed of etchingvaries according to the crystalline arrangement of the siliconwafers. The etch rate is typically 1.4 gm/minute, which willdecrease 300 times if silicon wafer with crystal orientation "111"is used.0Microposit S-1400 Seried (Shipley), Newton, Massachusetts, USA.loMicrostrip 2001, Olin Hunt Specialty Inc., West Paterson, NJ, USA36The shape of the grooves or pits is dictated by the crystalorientation of the silicon wafer. For example silicon wafer 110produces vertical- walled grooved surfaces and silicon wafer 100produces V-shaped grooves. The depth of grooves can be controlledby the time of etching; and the desired repeat spacing, the "pitch"(comprising one groove and one ridge between the grooves) can beincorporated in the design of the master pattern. Figure 2 shows anexample of a micromachined surface with V-shaped grooves. Thegrooves used in this study were 40 gm wide at the top of the groove,3 gm deep and were separated by ridges 40 gm in width.FABRICATION OF EPDXY SUBSTRATAAlthough micromachining uses silicon in the production oftopographies with desired properties, the topography can betransferred to other materials that are more suitable for biologicalexperimentation. Vinyl-silicone base impression materials11 wereused to take the impression of the micromachined silicon wafers andimpressions were used to cast replicas of the originalmicromachined surfaces in Epotek12, an epoxy resin that is claimedto be biocompatible. The epoxy replicas were baked for 3 days at60°C prior to being used for in vitro experimentation. These epoxysubstrates were used for confrontation experiments that wereinitially recorded on video tape and subsequently they were fixedand sectioned for electron microscopy. For enhanced optics, allExaflex (G-C) Dental Industrial Corporation, Tokyo, Japan.12Epotek 302-3 (Epoxy Technology), Billerca, Mass., USA37other experiments were set-up on titanium-coated silicon wafersand the resulting confrontations were recorded on 16mm cinefilm.PREPARATION AND CHARACTERIZATION OF SURFACESThe silicon wafers and the epoxy replicas were then coatedwith approximately 50 nm of titanium. The initial set of surfacesused were "sputter" coated. This technique, however ,sometimesresulted in the introduction of tiny 'bubbles' in the titanium layerthat were evident when viewed under enhanced optics. Theremaining set of silicon wafers and epoxy replicas were thereforecoated with titanium by vacuum deposition,13 which generallyyielded a clean, bubble-free surface coating. Both surfaces werecleaned using a detergent14 specifically formulated for tissueculture with ultrasonication, rinsed twenty times with deionizedsterile filtered water, and then incubated for two hours with serum-free tissue culture medium in which the antibiotics were raised toten times the concentration used in normal growth media. After afuther five rinses with sterile deionized water, the titanium-coatedsilicon wafers and the titanium-coated epoxy substrata were storedin individual sterile petri dishes. Titanium substrata prepared inthis way have been characterized by X-ray photo emissionspectroscopy and found to have a surface layer of TiO2, whichprovides an excellent substratum for cell attachment (105,106).13Randex 3140 Sputtering system, 815 San Antonio Rd, Palo Alto, CA147X Cleaning Solution, Flow Laboratories, Mclean, VA 22102, USA38Figure 2. Example of a micromachined surface with V-shapedgrooves.39ELECTRON MICROSCOPYSections were taken of the titanium-coated epoxy substratesin the zone where F-E cell confrontation had occured using a SorvallMT-215 microtome. One gm-thick sections were cut in anorientation such that the grooves were cut in cross-section. Thesesections were then stained with Toluidine Blue and examined underthe light microscope-16 to confirm that the section had been obtainedfrom the cell confrontation zone. For transmission electronmicroscopy (TEM), fifty-to-sixty nanometer sections of the epoxywere cut with a diamond knife. The sections were then stained withalcoholic uranyl acetate and aqueous lead citrate, and viewed underan electron microscope) 7TIME-LAPSE STUDIES Grooved or control flat substrata were mounted on glass slidesand placed in a Pentz chamber18 which was in turn placed in a stageincubator and viewed with reflected light differential interferencecontrast optics. Timing and exposure of the 16 mm cinefilm19 wascontrolled by an cinemicrographic system or recorded on video tape15Sorval MT-2, Porter Blum Ultra-MicrotomeisZeiss Photomicroscope, Oberkoken, W. Germany17phillips 30018Bachofer, Reutlingen, W. GermanyisKodak 2415, Opti-Quip, Highland Mills, N.Y.40using an image processing system20 and a time lapse videorecorder.ziIn the time-lapse studies, the two cell populations wereplaced on the stage incubator and filmed only after it wasestablished that collisions were likely to occur within 24 hours. Nodifference in cell motility was noted in the cells between the early(0-4 hrs) and late (20+ hrs) periods of the experiment.QUANTITATION The time lapse films were backprojected using a LW dataanalyzer which allows for frame-by-frame projection onto a screenand the position of the cell's centroid marked onto an acetate sheet.For fibroblasts the centroid was defined as that point where thelong axis of the cell and a line at right angles to the long axis at thecell's widest point intersect. For epithelial cells the centroid of thenucleus was used because cell boundaries were sometimesindistinct. The path of each cell thus consisted of an ordered set ofpoints representing the sequential position of the cell's centroid.Although one frame of film was exposed every minute, the positionof the cell was determined only every 20 minutes (i.e. every 20thframe) because this time allowed for sufficient movement that thepoints were clearly separated from one another and the distancecould be measured accurately. The change in position that wasmeasured every 20 minutes will be called a step in this study. In20Hamamatsu Mark 221Panasonic 805041addition the angle made by the line connecting the two points and, inthe case of grooved surfaces the direction of the grooves, wasdetermined. For smooth surfaces the direction of each step wasmeasured relative to a line whose direction was selected randomlyusing a random number table. For pairs of colliding cells, the timeof onset of collision was noted to the nearest frame and theprogress of the cells before and after contact noted. In addition thenumber of cells which exhibited contact paralysis and retractionafter contact as well as those that continued in their originaldirection of motion, were counted. Data were entered into theUniversity of British Columbia's main frame computer (IBM) forstatistical analysis using the SPSS statistical package.Statistical analysis of data in Tables 1 and 2 was performedusing a Mann-Whitney U-test. A Bayesian approach of statisticalanalysis (120), similar to that used for growth curves, was used toanalyse the persistence data (Figs. 5 & 6). The Students' t-test wasused to determine the existence of a significant difference betweenthe coefficients for cells cultured on grooved and smooth surfaces.The data for behavioural responses observed in cell collisionexperiments presented in Tables 3 and 4 were arranged into 2 X 2contingency tables and analyzed using the chi-square statistic.42RESULTSCHARACTERISTICS OF CELL MOTILITY ON SMOOTH AND GROOVEDSURFACES PRIOR TO COLLISIONS A. SpeedExamples of the paths of fibroblasts on grooved and smoothsurfaces are illustrated in figures 3 and 4 respectively. The datafor each cell were obtained by measuring each step in its path whileit was in the field of the microscope. A total of 125 fibroblast cellswere examined, yielding in excess of 1700 step measurements. Onaverage each path for fibroblasts on the grooved surface comprised10 steps whereas 20 steps were measured for the slower-movingcells on the smooth surface. The mean values for the speed andangle of the steps were calculated for each cell, and these data werecombined to give the information in Tables 1 and 2, in which cellsare the unit of statistical analysis. Both epithelial and fibroblastcells were classified on the basis of whether they were moving on asmooth surface or, if on the grooved substrata, within the grooves oron the ridges. It should be noted that the cells observed in thisstudy were plated at a high cell-population density, and migratedfrom the initial spot of concentrated cells to confront the other cellpopulation. The fields chosen, however, for observation weresufficiently far from the location of the droplet that fewinteractions with fibroblasts travelling in the same direction werenoted. Thus, the speed and angle of their steps were not influencedby interactions with other cells prior to the confrontation. At the43G^1^2^ 9^ 14R 1 2 33^4^56^7^a^9••■■•_,....................—..—.....e....„.....................1.:50 gmFigure 3. Path of a fibroblast on a grooved substratumG=groove, R=ridge.1450 gm i^i44Figure 4. Path of a fibroblast on a smooth surface4 5time of confrontation the cells were not in contact with any cellother than the one being confronted.A striking observation was that fibroblasts moved at roughlytwice the speed on grooved surfaces than they did on flat ones(Table 1). The frequency distribution of speed was markedly skewedto the right and approximated an exponential distribution rather thana Gaussian distribution.^Accordingly the comparison betweensurfaces was performed using a Mann-Whitney U-test.^Nosignificant difference was found between the speed of fibroblasts inthe grooves and those on the ridges. However, cells on groovedsurfaces were significantly faster (p<.001) than cells on a smoothsurface. In contrast epithelial cells exhibited no significantdifferences in speed on grooved and smooth surfaces (Table 2).B. DirectionFor both fibroblasts (Table 1) and epithelium (Table 2), thedirection of cell migration was controlled significantly on thegrooved substrata. The average angle made by the steps offibroblasts to the grooves was 4.7°÷4.10 for cells on grooves and9.1°+6.80 for cells on the ridges, whereas the average angle made bysteps of fibroblasts on the smooth surface to a randomly selectedreference line was 53.9°+15.60. Similar results were observed withepithelial cells. In summary the grooved substratum directed thelocomotion of both fibroblasts and epithelium, but influenced thespeed of only the fibroblasts, not the epithelium.46TABLE 1SPEED AND DIRECTION OF FIBROBLASTLOCOMOTION PRIOR TO CONTACTAVG.SPEEDS.D. AVG.ANGLES.D.Grooves 24.1 11.1 4.7 4.1 55Ridges 21.7 10.6 9.1 6.8 23Smooth 11.3 13.6 53.9 15.6 464 7TABLE 2SPEED AND DIRECTION OF EPITHELIALLOCOMOTION PRIOR TO CONTACTS.D. nAVG.SPEEDS.D. AVG.ANGLEGrooves 9.3 5.0 13.8 4.5 10Ridges 9.0 4.1 13.9 5.9 1 3Smooth 8.4 2.8 41.7 27.0 1148C. PersistenceAlthough the locomotion of fibroblasts in a flat culture dishhas been described as random, Gail and Boone (107) observed thatfibroblasts had a tendency to persist in their direction of movement.Furthermore they demonstrated theoretically and confirmedexperimentally that the motility of a persisting cell could becharacterized by an augmented diffusion constant of a random-walkmodel. The mean square displacement as a function of time isdirectly proportional to the diffusion constant of the random-walkmodel. A comparitive profile of the square cumulative displacementof fibroblasts moving on grooved versus smooth surfaces is shown inFig. 5. The mean square displacement of 51 fibroblasts on groovedsubstrata and 33 fibroblasts on smooth substrata as a function oftime is shown in Fig. 6. Only cells that were observed for at least160 minutes were included in the analysis. It is clearly evident thatcells on the grooved substrata showed a larger displacement at anygiven time, and thus a stronger directional persistence than cells ona smooth control substrate. Because the same cells are beingobserved, the values at each time point plotted in Fig. 6 aredependent on the previous time point. This follows because thevalue of the cumulative displacement for any cell at any time isinfluenced by the location of the cell at the previous point in time.Because the values at each time point are thus not independent, atraditional statistical approach is not appropriate. Therefore thevalue of the augmented diffusion constant for each cell wasdetermined independently. The value of the mean augmenteddiffusion constant for a population of fibroblasts moving on smooth49and grooved substrata was then estimated by averaging theindividual augmented diffusion constants obtained from the curverelating square cumulative displacement with time (see Fig. 5). Thismethod of analysis, which can be considered as a Bayesian approach,is warranted in such situations in which data on the change of somemeasurement for individual members of a group are collected over aperiod of time, and when the analysis does not allow the usualassumptions of independence to be made. The augmented diffusionconstant for cells on the grooved substratum was 71.2±7.2 (S.E.)nearly eight fold higher than the constant for cells on the smoothsurface (9.3±2.5). This difference was statistically significant(p<.01).50Fig a: Profile of Sq. Cum. Displacement -- Groove Surface8820^40^60^80^100^120^140^160Time (in Minutes)Fig b: Profile of Sq. Cum. Displacement -- Smooth Surface20^40^60^80^100^120^140^160Time (in Minutes)Figure 5. Profile of square cumulative displacement of fibroblastsmoving on (a) grooved (51 cells) and (b) smooth (33 cells) surfacesprior to confrontation140 160I^1^r^I^I^I^1^120 40 60 80 100^120051Trims (In Woes)Figure 6. Plot of mean square displacement of fibroblasts movingon grooved and smooth surfaces prior to confrontation.52FIBROBLAST-FIBROBLAST (F-F) COLLISIONSIn these experiments fibroblasts were plated to form smallcolonies separated by the length of the grooved substratum so thatthe cells which collided were, as a general rule, both movingtowards each other and the interaction observed wasleading-lamella (LL) with LL. Fibroblasts commonly exhibited one oftwo types of behaviour. On contact some cells demonstratedparalysis which was then followed by retraction so that the cellmoved in the opposite direction to that from which it came. Theresponse has been described as Cl of locomotion type 1 (CI-1) (83).In the second predominant form of behaviour both cells paused uponcontact of their LL after which one of the pair deflected laterally tothe opposite wall of the groove (Fig. 7). Both cells continuedlocomotion in the same direction as prior to their encounter albeitat a slower pace than that observed before contact. When the tworound cell bodies of the fibroblasts subsequently became alignedparallel to each other there was a tendency for the cells to pausebriefly. Then with a discernible increase of speed, each cell movedaway in its respective direction to reach a cell-free area ,andtypically returned to a more central position within the groove. Thisresponse will be referred to as deflection. F-F confrontations on aflat surface occasionally resulted in a deflection but this cellbehaviour was more commonly observed on grooved substrata.53Figure 7.^Sequence taken from a time-lapse cinefilm of twofibroblasts colliding on a grooved substratum.^The deflectionmanuever is illustrated.^(a) time 0, fibroblasts initially on left andright indicated by white and black arrows respectively, (b) 60 min.,the cells collide, (c) 120 min. (d) 135 min. fibroblast (white arrow)crosses to wall of groove, and then continues in its originaldirection (e) 180 min. (f) 285 min.54Table 3 demonstrates that on the grooved substrata the mostcommon behaviour was deflection whereas on the smooth substrataCI-1 was by far the most common response. The proportions of cellsdemonstrating the two responses were statistically differentbetween the grooved and smooth substrata when analyzed by meansof a 2 x 2 contingency table (p < .0001).EPITHELIAL-EPITHELIAL (E-E) COLLISIONS Unlike fibroblast cells, which move as individuals, E cellsmoved as a sheet. On both grooved and flat surfaces, sheets of Ecells adhered to each other upon contact (Fig. 8). There were noinstances observed of E cells crawling over or under one another. Onoccasion, however, after the sheets made contact an E cell from onesheet would migrate along the seam formed between the two sheets.55TABLE 3BEHAVIOURAL RESPONSES OBSERVED IN FIBROBLAST / FIBROBASTCELL COLLISIONSDeflection^CI-1GroovedSmooth^24^53^23P<0.000156TABLE 4BEHAVIOURAL RESPONSES OBSERVED IN FIBROBLAST / EPITHELIALCELL COLLISIONSAdvance^CI-1Grooved^53^23Smooth^106^20P<.0257Figure 8. Sequence taken from a time-lapse cinefilm of E cellsheets colliding on a smooth surface. Arrows indicate the leadinglamellae of the cells. (a) time 0, (b) 28 min., (c) 60 min., cellscollide, (d) 77 min., (e) 86 min., (f) 159 min.58FIBROBLAST-EPITHELIAL (F-E) COLLISIONS In these experiments separate colonies of epithelial cellsand fibroblasts initially spaced 2 mm apart came into contactthrough the growth and migration of the cell populations. On thegrooved substratum the collisions were head-on, i.e. F-LL to E-LL.On the smooth smooth surface the type of collision varied so thatF-LL to E-side and E-LL to F-side as well as E-LL to F-LL wereobserved. On both smooth and grooved substrata the effects ofcollision on the cell populations were nonreciprocal; the motion ofthe E cells was not affected in a discernible manner. In allexperiments the E-cell sheet eventually covered the microscopicfield. Fibroblasts, however, demonstrated either CI-1 or invaded theE-cell sheet. Fig. 9, which is a series of frames abstracted from acinefilm, illustrates the predominant response that occurred whenfibroblasts collided with an E-cell sheet on a smooth substratum.EM sections of confronting cell populations demonstrated that Ecells, identified by their tonofilaments and desmosomes, were mostfrequently located superior to fibroblasts (Fig. 10). In somelocations the fibroblasts and E cells formed an elaborate multilayerin which E cells were sandwiched between fibroblasts as has beenobserved previously with E cells derived from the cell rests ofMalassez (108). Fibroblasts responded differently from E cells onthe two substrata. As contact guidance is not unidirectional, manyof the observed fibroblast cells retreated prior to collision. Ongrooved substrata a total of 53 cells invaded the E-cell sheet and 23demonstrated CI-1 whereas on the smooth surfaces 106 invaded thesheet and only 20 cells demonstrated CI-1 (Table 4).5 9Figure 9. Sequence taken from a time-lapse cinefilm of a fibroblast colliding with anE-cell sheet on a smooth surface, black arrow indicates the leading lamellae of the &cellsheet, the white arrow the fibroblast. (a) time 0, fibroblast contacts sheet, (b) 89min., fibroblast lamellae widens, (c) 307 min., (d) 310 min., fibroblast lamellacontracts, (e) 329 min., fibroblast reapproaches E-cell sheet, (f) 351 min., fibroblastlamella underlaps E cell sheet, (g) 368 min., fibroblast lamella is completely under Esheet, but fibroblast cell body remains outside the sheet, (h) 392 min., fibroblastcontinues to advance under the E-cell sheet, (i) entire fibroblast is under E-cell sheet,(j) 427 min., (I) 492 min., fibroblast continues advancing under E-cell sheet.60Figure 10.^Electron^micrographs^of^epithelial-fibroblastrelationship after collision on a grooved substratum.^E cellsidentified by tonofilaments (T) and desmosomes (D) are locatedabove fibroblasts (F) which contact titanium (11) -coated epoxysubstratum.61The two substrata differed significantly in the proportion of cellsexhibiting CI-1 and invasive behaviour when the data were analyzedusing a 2 x 2 contingency table (p < .02).62DISCUSSIONThis study employed titanium-coated grooved substrata tocontrol the direction of cell locomotion so that directconfrontations of leading lamellae could be observed A titaniumcoating was used to ensure strong adhesion of cells to thesubstratum and to model the conditions existing on titaniumimplants. Heaysman (109) has noted that substratum adhesivenesscan have a significant effect in modifying Cl of cell movement. Inher view many instances of apparent failure of contact inhibitionmay be due to a change in cell-substratum adhesion such that lessactual close contact occurs. As cells adhere to titanium morestrongly than to glass or plastic (106), such failures of adhesionmay be minimized in this study, relative to other investigationsperformed on more commonly used substrata such as glass ortissue-culture plastic. The grooves on the substrata influenced cellbehaviour in several ways affecting the speed, direction andpersistence of movement.The locomotory behaviour of cells on grooved titaniumsurfaces exhibited some properties that could be predicted fromprevious studies on other substrata. Fibroblasts as well as theircytoskeletal filaments (99) including microfilaments andmicrotubules (109) were aligned with the grooves and it was notsurprising that fibroblasts moved in the direction of the grooves.Moreover, the observation that fibroblasts move at roughly doublethe speed on the grooved substrata than they do on a smooth surfaceagrees in general with the observations of Wood (83) who found that63the leading mesenchyme cells migrating from an explant of finmoved at a faster rate on grooved quartz than on a flat surface. Thelimits of speed at which a cell can crawl are thought to be set bythe assembly constants for the reorganization of its machinery, therate of forward protrusion and the decay characteristics ofposterior attachments (110). The increased speed observed ongrooved substrata probably results from the polarization offibroblasts observed on these substrata. There is some evidencethat the bipolar organization of a fibroblast cell depends upon theintegrity of the microtubular system (111), and others in this areaof research have found that microtubules, as noted above, have beenobserved to align with grooves in the substrata (112). Suchconsistently polarized cells would be expected to move more rapidlybecause the organization of their cytoskeleton and organelles wouldnot have to be modified, as would occur when cells change theirdirection of locomotion.In contrast E cells did not increase their speed on groovedsurfaces. The explanation for this difference between fibroblastsand E cells may originate with the nature of E-cell locomotion, inwhich cells behave as a connected population rather than as acollection of isolated individuals. The changes in cell polarity andcytoskeletal reorganization of an E-cell sheet are thought to beinfrequent on either smooth or grooved surfaces and thus thegrooves did not appreciably alter E-cell speed.Gail and Boone (107) demonstrated that the persistence ofcell motion is proportional to the square of the displacement. Ongrooved substrata the displacement of fibroblasts increased64markedly over that found on smooth substrata because the anglebetween the steps was small and aligned with the grooves, and theirspeed was roughly double that of cells on a smooth surface. Thus,not surprisingly, the persistence of motion of fibroblasts ongrooved substrata was greater, in fact roughly eight fold greater,than on a smooth surace. Dunn and Brown (117) have recentlydeveloped a rather sophisticated mathematical approach to analyzepersistence. In the present thesis however, a more simple andtraditional method of approach was utilized based on the model byGail and Boone (107) whose method is sufficiently powerful todemonstrate a difference in fibroblast cell persistence on groovedversus smooth surfaces. In future studies it may be profitable toutilize the Dunn and Brown method to analyze cell persistencebecause this method attacks the problem at the level of theemergent properties of cell motility and then seeks a molecularexplanation rather than trying to predict the emergent propertiesfrom a study of the molecular interactions.Some of the results of the collision experiments could bepredicted from other studies of cell confrontation. For example CI-1occurs more frequently in LL-LL E-F cell collisions than LL to sidecollisions (113). In the E-F confrontations reported here, CI-1 wasmore prevalent in the head- on collisions of LL that occurred ongrooved surfaces than in those occuring on the smooth substratum,on which more LL-side collisions occurred. Like other workers(21,24), we have found that F-E confrontations were nonreciprocal;the E cells did not demonstrate any discernible CI-1 on contact withfibroblasts. Moreover, as is characteristic of E cells in culture, E-E65cell collisions normally resulted in union of the two E-cell sheets(24).A novel finding in our study, however, came in the F-Fconfrontations, where the most frequent result of head-on collisionsof fibroblasts on grooved substrata was not CI-1, which featuresretraction (109), but rather an unusual interaction, termed here adeflection, in which the two cells, after a brief pause, made alateral shift and continued in their original direction of locomotion.Such behaviour was rare on flat surfaces. In our view the mostlikely explanation of this result is that the substratum-imposedalignment of cytoskeletal filaments and cell polarity inhibited thecells from markedly changing direction and thus modified thedirectional outcome of the cell collisions.The behaviour of the deflected cells was similar to thatreported by Elsdale (114) and Erickson (115) who observed thatcontacting cells often slide along one another, aligned and looselyassociated. They suggested that this response stems from paralysisof the parts of the lamellipodia that touch each other, with theremaining active regions then determining the motion. They alsofound that the alignment response only occurs when the anglebetween the contacting cells is small.An important but poorly understood feature of cell socialbehaviour that has been implicated in the mechanism of contactguidance is the tendency of cells to adhere to each other along theirmargins (106). This tendency was noted in the deflection maneuversas the cells paused when the cell bodies were aligned. At this timethere would be a maximum opportunity for cell margins to adhere to66one another, but this tendency was often not sufficient to stop thecells from continuing in their original direction of motion.Edelstein-Keshet and Ermentrout (116) have also examined thecontact response between fibroblast cells and found that cell-cellinteractions can lead to self-organization and mediatemorphogenetic pattern formation. Their mathematical modelsuggests that fibroblasts reorient and align with each other uponcontact and show that such contact responses account for theformation of multicellular patterns called parallel arrays.It is widely recognized that cell populations do not exhibiteither complete Cl or a complete failure of Cl, but that thefrequency with which Cl occurs varies both with differentexperimental procedures and with different cell types (109). In thisstudy the novel experimental conditions included a titanium-coatedgrooved substratum that led to the observation of some unexpectedcell social behaviour. Grooves are only one of the topographies thatcan be produced by micromachining, and additional insights into cellbehaviour might be obtained by employing other precisely specifiedsurface configurations.FUTURE WORKThe studies in this thesis were mainly concerned with thebehavioural responses observed in directed collisions betweenepithelial and fibroblast cells on micromachined substrata. Theresults of these experiments revealed some unexpected cell socialbehaviour. The molecular mechanisms underlying such behaviour,however, have not been well elucidated. Future research should67therefore be directed towards this area. A few suggestions relatingto experimental design are presented below.The increased speed and persistence of motion exhibited byfibroblasts on grooved substrata is thought to result from the stablepolarization of the cytoskeleton of fibroblasts observed on thesesubstrata. This hypothesis should be tested by performingexperiments that investigate the arrangement of cytoskeletalelements on grooved versus smooth substrata and how they changewith time.The most frequent result observed in head-on collisonsbetween two fibroblasts on grooved substrata was deflection. Inthis response the cells, after a brief pause on contact, make alateral shift and slide by one another in polarized fashion. Suchbehaviour is thought to occur as a result of substratum-imposedalignment of cytoskeletal filaments. It would be of great interestto examine the status of cytoskeletal elements before, during andafter such a response so as to determine whether filamentalignment is a possible cause or effect of the deflection response.A powerful technique that would be useful to test the abovehypotheses is optical sectioning by means of confocal microscopy.Confocal microscopy enables sections of a cell to be obtained alongthe optic axis of the microscope. In this way, the shape, orientationand precise location of structures such as actin filaments or focalcontacts can be determined. By combining immunofluorescenttechniques with confocal microscopy, the sequence and spatialdistribution of cytoskeletal elements could be determined before,during and after cell collision.68In this study, the cell confrontation experiments wereperformed on grooved as well as smooth substrata. In each of theexperiments, however, the confronting cell populations (F-E, F-F, E-E) were both either directed (i.e. on a grooved surface) or non-directed (i.e. on a smooth surface). Additional insight into cellsocial behaviour might be obtained if experiments are conducted inwhich one cell population is directed and made to confront a secondcell population that is non-directed, and visa versa. Alternatively,as grooves are only one of the topographies that can be produced bymicromachining, one might consider employing other preciselyspecified surface configurations.By understanding the mechanisms underlying the phenomena ofcell locomotion and behaviour it should be possible to improve thedesign of percutaneous devices and dental implants by placingsurfaces with precisely defined topographies on these artificialdevices.69CONCLUSIONSSpecific conclusions from the experiments in this thesis canbe summarized as follows:1. Grooved surfaces increase the speed of fibroblast locomotionand tightly control the direction of locomotion.2. Grooved surfaces control the direction of locomotion but donot alter epithelial cell speed.3. Fibroblasts on grooved substrata showed a stronger directionalpersistence, roughly eight fold greater, than cells on a smoothcontrol substratum.4. The most frequent result observed in head-on collisionsbetween fibroblasts on grooved substrata was not contactinhibition type 1, but rather deflection.5. Contact inhibition of movement of fibroblasts by epithelialcells occurs more frequently in leading lamella (LL) to LLcollision than in LL to side collision.6. Contact inhibition is not reciprocal between the fibroblastsand epithelial cells used in this study. Epithelial cells did notexhibit contact inhibition on colliding with fibroblasts, butsome fibroblasts were contact-inhibited by epithelium.70REFERENCES1. M. Abercrombie and J.E.M. Heaysman, "Observations on thesocial behaviour of cells in tissue culture. I. Speed ofmovement of chick heart fibroblasts in relation to theirmutual contacts", Exp. Cell Res., vol 5, p.11, 19532. M. Abercrombie and J.E.M. Heaysman, "Observations on thesocial behaviour of cells in tissue culture. II. 'Monolayering' offibroblasts", Exp. Cell Res., vol 6, p.293, 19543. M. Abercrombie,"Contact inbihition in tissue culture", In Vitro,vol. 6, p.128, 19704. M. Abercrombie and E.J. Ambrose, "Interference microscopestudies of cell contacts in tissue culture", Exp. Cell Res., vol.15, p.332, 19585. J.E.M. Heaysman and S.M. Pegrum, "Early contacts betweenfibroblasts: an ultrastructural study", Exp. Cell Res., vol. 78,p.71, 1973a6. J.P. Trinkaus, T. Betchaku and L.S. Krulikowsky, "Localinhibition of ruffling during contact inhibition of cellmovement", Exp. Cell Res., vol. 64, p.291, 19717. M. Abercrombie, D.M. Lamont and E.M. Stephenson, "Themonolayering in tissue culture of fibroblasts from differentsources", Proc. Roy. Soc. B., vol. 170,p.349, 19688. P.B. Armstrong and J.M. Lackie, "Studies on intercellularinvasion In Vitro using rabbit peritoneal neutrophilgranulocytes (PMNs) I. Role of contact inhibition oflocomotion", J. Cell Biol., vol. 65, p.439, 1975719. P. Vesely and A. Weiss, "Cell locomotion and contact inhibitionof normal and neoplastic rat cells", Int. J. Cancer, vol. 11, p.64,197310. A. Harris, "Contact inhibition of cell locomotion", in R.P. Cox(ed.), Cell Communication (John Wiley and Sons, New York), pp.147-85, 197411. D.R. Garrod and M.S. Steinberg, "Cell locomotion within acontact inhibited monolayer of chick embryonic liverparenchyma cells", J. Cell Sc., vol. 18, p.405, 1975.12. J.E.M. Heaysman, "Contact inhibition of locomotion:reappraisal", Mt. Rev. CytoL, vol. 55, p.49, 1978.13. T. Ebendal and J.P. Heath, "Self-contact inhibition of movementin cultured chick heart fibroblasts", Exp. Cell Res., vol. 110,p.469,^1977.14. C.A. Middleton, "The control of epithelial cell locomotion intissue culture", in Locomotion of Tissue Cells (Ciba FoundationSymposium 14, Elsevier, Amsterdam), pp.251-70, 1973.15. C.A. Middleton, "Cell contacts and the locomotion of epithelialcells", in R. Bellairs, A. Curtis and G. Dunn (eds.), CellBehaviour (Cambridge University Press), pp.159-82, 1982.16. S.J. Holmes, "The behaviour of the epidermis of Amphibianswhen cultivated outside the body", J. exp. Zoo!., vol 17, p.281,1914.17. K.M. Wilbur and R. Chambers, "Cell movements in the healing ofmicrowounds In Vitro", J. exp. Zoo!., vol. 91, p.287, 1941.18. R.B. Vaughan and J.P. Trinkaus, "Movements of epithelial cellsheets In Vitro", J. Cell Sc., vol. 1, p.407, 1966.7219. M. Abercrombie and C.A. Middleton, "Epithelial-mesenchymalinteractions affecting locomotion of cells in culture", in R.Fleischmajer and R.E. Billingham (eds.), Epithelial MesechymalInteractions (Williams and Wilkins, Baltimore), pp. 56-63,196820. B.A. Flaxman and B.K. Nelson, "Ultrastructural studies of theearly junctional zone formed by keratinocytes showing contactinhibition of movement In Vitro", J. Invest. Dermatol., vol. 63,p.326, 1974.21. E.R. Parkinson and J.G. Edwards, "Non-Reciprocal ContactInhibition of Locomotion of Chick Embryonic ChoroidFibroblasts by^Pigmented Retina Epithelial Cells", J. CellSc., 33:103-120, 1978.22. J.E.M. Heaysman, "Non-reciprocal contact inhibition",Experientia, vol. 26, p.1344, 1970.23. J.E.M. Heaysman and L. Turin,"Interactions between living andzinc-fixed cells in culture", Exp Cell Res, 101, 419-422, 1976.24. C.A. Middleton and J.A. Sharp,"The social behaviour of cells inculture", in Cell Locomotion , Croom Helm, London & Canberra,137, 1984.25. M. Abercrombie, "The crawling movement of metazoan cells",Proc. Roy. Soc. 8, vol. 207, p.129, 198026. M. Abercrombie and J.E.M. Heaysman, "The directionalmovement of fibroblasts emigrating from cultured explants",Ann. Med, exp. Biol. Fenn., vol. 44, p.161, 1966.27. J.M. Vasiliev, I.M. Gelfand, L.V. Domnina and R.I. Rapaoport,"Wound healing processes in cell cultures", Exp. Cell Res., vol.54, p.83, 1969.7328. R.L. Williams and D.F. Williams,"The spacial resolution ofprotein adsorption on surfaces of heterogeneous metallicbiomaterials", J Biomed Mater Res , 23, 339-350, 1989.29. D. Smith,"Discussion Surface factors", J Oral Imp. SpecialIssue, 2 (XIV), 417-422, 1988.30. C.A. King, J.E. M. Heaysman and T.M. Preston, "Experimentalevidence for the role of long range forces in fibroblasts-substrate interaction", Exp. Cell Res. , 119,406, 1979.31. L. Weiss,"Cell adhesion", Int Dent J, 28(1): 7-17,1975.32. A.S.G. Curtis, "The mechanism of adhesion of cells to glass", JCell Biol., 20,199, 1964.33. C.S. Izzard and L.R. Lochner, "Cell-to-substrate contacts inliving fibroblasts: an interference reflexion study with anevaluation of the technique", J Cell Sci, 21,129, 1976.34. M. Abercrombie, J. Heaysman and S.M. Pegrum, "The locomotionof fibroblasts in culture. IV. Electron microscopy of theleading lamellae", Exp Cell Res, 67, 359 ,1971.35. J .E. M. Heaysman and S.M. Pegrum, "Early cell contacts inculture", Cell Behaviour , R. Bellaires, A. Curtis and G. Dunn,Eds., Cambridge University Press, London, 49, 1982.36. D.M. Brunette, "Interactions of epithelial cells with foreignsurfaces" CRC Rev Biocompatibility, 1,4:323-369, 1986.37. 1.1. Singer, "Fibronectin-Cytoskleton Relationships", Biology ofECM: A series Fibronectin , D.F. Mosher (ed.), Academic PressInc. 1989.38. J.R. Couchman and D.A. Rees,"Actomyosin organisation foradhesion, spreading, growth and movement in chickfibroblasts", Cell Biol Int Rev, 3,431 ,1979.7439. J. Kolega, M.S. Shure, W.T. Chen and N.D. Young, "Rapid cellulartranslocation is related to close contacts formed betweenvarious cultured cells and their substrata", J Cell Sc., 54,23-34, 1982.40. J.V. Small,"Microfilament-based motility in non-muscle cells",Cur Opin Cell Biol , 1,75-79, 1989.41. G. Rinnerthaler, B. Geiger and J.V. Small,"Contact formationduring fibroblast locomotion: involvement of membrane rufflesand microtubules", J Cell Biol , 106, 747-760, 1988.42. 1.1. Singer, "The fibronexus: a transmembrane association offibronectin-containing fibers and bundles of 5 nmmicrofilaments in hamster and human fibroblasts", Cell16,675-685, 1979.43. A.C. Taylor, "Adhesion of cells to surfaces", in Adhesion inBiological System , R.D. Manly, Ed., Academic Press, new York,51, 1970.44. J.A. Jansen, "Epithelial cell adhesion to dental implantmaterials", thesis, catholic university, Nijmegan, Netherlands,1984.45. T.R.L. Gould, D.M. Brunette and L. Westbury, "The attachmentmechanism of epithelial cells to titanium in vitro ", J DentRes, 16:611-616, 1981.46. P.K. Legan, J.E. Collins and D.R. Garrod, "The molecular biologyof desmosomes and hemidesmosomes: what's in a name?",BioEssays, 14(6): 385-393, 199247. P.D. Yurchenco and J.C. Schittny, "Molecular architecture ofbasement membranes", FASEB Journal, 4: 1577-1590, 19907548. J.A. Jansen, J.R. de Wijn, J.M.L. Wolters-Lutgerhorst and P.J. vanMullem,"Adhesion of guinea-pig epidermal cells to oral implantmaterials", Proceeding of Int. Cong. Tiss. Integration , (May1985), D.van Steenberghe, T. Albrektsson, P.I. Branemark, etal.(eds), Excerpta Medica, 1986.49. B.A. Flaxman, M.A. Lutzner and E.J. Van Scott,"Ultrastructure ofcell attachment to substratum in vitro ", J Cell Biol , 36, 406-410, 1968.50. V-J. Uitto and H. Larjava,"Extracellular matrix molecules andtheir receptors. An overview with special emphasis onperiodontal tissue", Critical Rev Oral Biol Oral Med , 2: 323-354, 199151. D.E. Kelly,"Fine structures of desmosomes, hemidesmosomes,and an epidermal globular layer in developing newt epidermis",J Cell Biol , 28,51-73, 1966.52. L.Y. Sakai, D.R. Keene, N.P. Morris and R.E. Burgeson,"Type VIIcollagen is a major structural component of anchoring fibrils",J Cell Biol , 103, 1577-1586, 1986.53. J. Ellison and D.R. Gerrod, "Anchoring filaments of theamphibian epidermal-dermal junction traverse the basallamina entirely from the plasma membrane ofhemidesmosomes to dermis", J Cell Sci , 72, 163-172, 1984.54. H.K. Kleinman, M.L. McGarvey, M.L. Hassell et. al., "The role oflaminin in basement membranes and in the growth, adhesionand differentiation of cells", in The role of ExtracellularMatrix in Development, Trelstad, R.L., Alan R. Liss, New York,123, 1984.55. S.M. Albelda and C.A. Buck,"Integrins and other cell adhesionmolecules", FASEB J, 4,2868, 1990.7656. B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts and J.D.Watson, "Molecular Biology of The Cell" Garland Publ., 2nd Ed.,1989.57. M. Jalkanen, S. jalkanen and M. Bernfield,"Cell surfaceproteoglycan mediated binding of extracellular effectormolecules", in Biology of Extracellular Matrix Series , ed. byR.P. Mecham, Academic Press, New York, in Press.58. B. Geiger,"Cytoskeleton-associated cell contacts", Curr OpinCell Biol , 1, 103-109, 1989.59. T.P. Stossel,"How cells crawl", Amer Sci, 78, 408, 1990.60. M Abercrombie, G.A. Dunn and J.P. Heath,"Locomotion andcontraction in non-muscle cells", Contractile Systems in Non-muscle Tissue, Elsevier, North-Holland Biomedical Press, 3-11, 1976.61. M. Abercrombie,"The crawling movement of metazoan cells",Proc Roy Soc B, 207,129, 1980.62. G.A. Dunn,"Mechanisms of fibroblast locomotion", in A.S.G.Curtis and J.D. Pitts (eds), Cell Adhesion and Motility,Cambridge University Press, 409, 1980.63. J.A. Heath and G.A. Dunn,"Cell-to-substratum contacts of chickfibroblasts and their relation to microfilament system", J CellSci, 29,197, 1978.64. H.E. Huxley,"Muscular contraction and cell motility", Nature,243, 445, 1973.65. J.V. Small, G. Isenberg and J.E. Celis,"Polarity of actin at theleading edge of cultured cells", Nature, 272, 638, 1978.66. E.D. Korn and J.A. Hammer,"Myosins of nonmuscle cells", AnnualRev Biophys Biophyc Chem, 17,23,1988.7767. E.D. Korn and J.A. Hammer,"Myosin I", Curr Opin Cell Biol, 2, 57-61, 1990.68. M.S. Bretscher,"How animal cells move", Scientific Amer,72-90,1989 .69. G.F. Oster and A.S. Perelson,"The physics of cell motility", JCell Sci, Suppl 8, 35, 1987.70. A. DiPasquale,"Locomotory activity of epithelial cells inculture", Exp Cell Res, 94,191, 1975.71. J.P. Heath and B. F. Holifield, Cell motility and thecytoskeleton, 18: 245-257, 199172. W.S. Krawczyk,"A pattern of epidermal cell migration duringwound healing", J Cell Biol, 49, 247, 1971.73. J.R. Gibbins,"Epithelial migration in organ culture. Amorphological and time lapse cinematographic analysis ofmigrating stratified squamous epithelium", Pathology, 10, 207,1978.74. J.J. Scuibba,"Regeneration of the basal lamina complex duringepithelial wound healing", J Periodontal Res, 12,204, 1977.75. A.S.G. Curtis and P. Clark,"The effects of topographic andmechanical properties of materials on cell behaviour", CRC inBiocompatibility, 5(4): 343-362, 1990.76. R.G. Harrison,"The reaction of embryonic cells to solidstructures", J Exp Zoo!, 17, 521, 1914.77. P. Weiss,"Nerve patterns: the mechanics of nerve growth",Growth, Suppl. 5,163, 1941.78. P. Weiss,"Cell contact", Int Rev Cytol, 7, 391, 1958.7879. D.M. Brunette, "Fibroblasts on micromachined substrata orienthierarchically to grooves of different dimensions", Exp CellRes, 164,11-26,1986b.80. D.M. Brunette, "Spreading and orientation of epithelial cells ongrooved substrata" , Exp Cell Res, 167,203-217,1986c.81. G.A. Dunn and A.F. Brown, "Alignment of fibroblasts on groovedsurfaces described by a simple geometric transformation", JCell Sc!, 83,313-340,1986.82. P.C. Wilkinson, J.M. Shields and W.S. Haston,"Contact guidanceof^human neutrophil leukocytes", Exp Cell Res, 140, 55-62, 1982.83. A. Wood, "Contact guidance on microfabricated substrata: theresponse of teleost fin mesenchyme cells to repeatingtopographical patterns", J Cell Sci, 90,667-681,1988.84. J.P. Trinkaus,"Cells Into Organs, The forces that shape theembryo", Prentice-Hall Inc., USA, second ed., 1984.85. A.S.G. Curtis and M. Varde,"Control of cell behaviour:Topological factors", J Nat! Cancer lnst, 33,15-26, 1964.86. G.A. Dunn and J.P. Heath," A new hypothesis of contact guidancein tissue cells", Exp Cell Res, 101,1-14, 1976.87. P. Bongrand, C. Capo and R. Depieds,"Physics of cell adhesion",Prog Surface Sci, 12,217-286, 1982.88. P.C. Wilkinson and J.M. Lackie,"The influence of contactguidance on chemotaxis of human neutrophil leukocytes", ExpCell Res, 145, 255-264, 1983.89. P.T. Ohara and R.C. Buck,"Contact guidance in vitro", Exp CellRes, 121,235-249, 1979.7990. M.D. Rosenberg,"Cell guidance by alterations in monomolecularfilms", Science, 139, 411-12, 1963.91. G. Albrecht-Buehler,"The angular distribution of directionalchanges of guided 313 cells", J Cell Biol, 80, 53-60, 1979.92. 0. Yu. lvanova and L.B. Margolis,"The use of phospholipid filmfor shaping cell cultures", Nature, 242, 200-1, 1973.93. A. Cooper, H.R. Munden and G.L. Brown,"The growth of mouseneuroblastoma cells in controlled orientations on thin films ofsilicon monoxide", Exp Cell Res, 103, 435-9, 1976.94. A.K. Harris,"The behavior of cultured cells on substrata ofvariable adhesiveness" Exp Cell Res, 77, 285-297,1973.95. G.A. Dunn, "Contact guidance of cultured tissue cells: a surveyof potentially relevant properties of the substratum", in CellBehaviour, R. Bellairs, A.S.G. Curtis and G. Dunn, Eds.,Cambridge University Press, London,247,1982.96. D.M. Brunette, "The effects of implant surface topography onthe behaviour of cells" , Int J Oral Maxillofac Implants, 3,231-246,1988.97. P. Clark, P. Connolly, A.S. G. Curtis, J.A.T. Dow and C.D.W.Wilkinson,"Topographical control of cell behaviour: II. multiplegrooved substrata", Development, 108, 635-644, 1990.98. D.M. Brunette, G.S. Kenner, and T.R.L. Gould, "GroovedTitanium Surfaces Orient Growth and Migration of Cells fromHuman Gingival Explants", J. Dent. Res., 62:1045-1048, 1983.99. D.M. Brunette, "Fibroblasts on Micromachined Substrata OrientHierarchically to Grooves of Different Dimensions", Exp. CellRes., 164:11-26, 1986.80100. D.M. Brunette, "Spreading and orientation of epithelial cells ongrooved substrata", Exp. Cell Res., 167:203-217, 1986.101. P. Clark, V.P. Connoll, A.S.G. Curtis, J.A.T. Dow and C.D.Wilkinson "Topographical control of cell behaviour onmultiple grooved surfaces", Development,108:635-644, 1990.102. D.M. Brunette, A.H. Melcher and H.K. Moe "Culture and Origin ofEpithelium-like and Fibroblast-like Cells from PorcinePeriodontal Ligament Explants and Cell Suspensions", ArchsOral Biol., 21:393-400, 1976103. R.B. Owens,"Glandular epithelial cells from mice: A method forselective cultivation", J Natn Cancer lnst, 52, 1375-1378,1974.104. D.S. Camporese, T.P. Lester and DI. Pulfrey "Development ofFine Line Silicon Shadow Masks for the Deposition of Solar CellGrids", Proc. 15th IEEE Photovoltaic Specialists Conference.,527-529,1981.105. R.D. Fletcher, G. Schneider, M.N. Labant and J.N. Albertson, Jr.,"An In Vitro Adhesion Technique for Measuring Cell Adhesionto Rigid Materials", J. Dent. Res. 58:1750-1751, 1979.106. D.M. Brunette, "The Effect of Surface Topography on CellMigration and Adhesion", in Surface Characterization ofBiomaterials, Ed. B.D. Ratner, Elsevier Science Publishers,203-217, 1988.107. M.H. Gail and C.W. Boone, "The locomotion of mouse fibroblastsin tissue culture", Biophysical Journal, 10:980-993, 1970.108. D.M. Brunette, R.J. Kanoza, Y. Marmary, J. Chan and A.H.Me!cher "Interaction between fibroblast-like andepithelial-like cells in cultures derived from monkeyperiodontal ligament", J. Cell. Sci., 27:127-140, 1977.81109. J.E.M. Heaysman, "Contact Inhibition of Locomotion: AReappraisal", Int. Review of Cytology, 55:49-66, 1978.110. J.M. Lackie, "Cell Movement and Cell Behaviour", Allen &Unwin Ltd. 175-254, 1986.111. U. Euteneuer and M. Schliwa, "Persistant directional motilityof cells and cytoplasmic fragments in the absence ofmicrotubules", Nature (Lond.), 310:58-61, 1984.112. C. Oakely, unpublised observations, UBC113. J.G. Edwards and E.K. Parkinson, "Locomotory InteractionsBetween Epithelial Cells and Fibroblasts In Vitro", in CellBehaviour, Ed. by R. Bellairs, A. Corks and G. Dunn, Cambridge,349-357, 1982.114. T. Elsdale, "The generation and maintenance of parallel arraysin cultures of diploid fibroblasts", in Biology of Fibroblasts byE. Kulonen and J. Pikkarainen, Academic Press, N.Y., 1973115. C.A. Erickson, "Analysis of the formation of parallel arrays byBHK cells in vitro", Exp Cell Res, 115:303-315, 1978116. L. E. Keshet and G.B. Ermentrout, "Contact response of cellscan mediate morphogenetic pattern formation",Differentiation, 45:147-159, 1990117. G.A.Dunn and A.F. Brown, "A unified approach to analysing cellmotility", J. Cell Sci, 8, 81-102, 1987118. E.D. Hay, "Cell Biology of Extracellular Matrix", 2nd Ed., PlenumPublishing Corp., 305-341, 1991119. V.J. Uitto, H. Larjava, J. Peltonen and D.M. Brunette, "Expressionof fibronectin and integrins in cultured periodontal ligamentepithelial cells", J. Dent. Res. 71(5): 1203-1211, 199282120. T. Fearn, "A Bayesian approach to growth curves", Biometrika ,62(1): 89-100, 1975


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