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Lumican and fibromodulin in the periodontal ligament : a study in knockout mice Matheson, Stacey L. 2003

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Lumican and Fibromodulin in the Periodontal Ligament: A Study in Knockout Mice  By  S T A C E Y L. M A T H E S O N B S c , Dalhousie University, 1994 D D S , Dalhousie University, 2000 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E In T H E F A C U L T Y OF G R A D U A T E STUDIES (Department o f Oral Biological and Medical Sciences) W e accept this thesis as conforming to the required standard.  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A June 2003 © Stacey L . Matheson 2003  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or by his or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ABSTRACT Periodontal disease is one o f the most common diseases o f mankind and may present in otherwise healthy individuals or as part o f a medical condition or syndrome. The cause of periodontitis seems to be multifactorial involving both periodontal pathogens and host response. Periodontal disease involves breakdown o f collagen fibers o f the periodontal ligament, which manifests as increased probing depths and attachment loss around teeth. The integrity o f the periodontal ligament, then, seems important i n this disease process. Small leucine-rich proteoglycans have been located in the periodontal ligament. These are a family o f molecules i n the extracellular matrix that, among other things, play a role in collagen fibrillogenesis. This blinded, controlled study investigates the location o f lumican and fibromodulin, two class II small leucine-rich proteoglycans,  in oral  periodontal tissues and their role i n the collagen fibrillogenesis using adult mice that are singly or doubly deficient in these two small leucine-rich proteoglycans.  ii  <  T A B L E OF CONTENTS  ABSTRACT  ii  T A B L E OF C O N T E N T S  iii  LIST OF T A B L E S  vii  LIST OF FIGURES  xi  ACKNOWLEDGMENTS  xvi  C H A P T E R O N E - R E V I E W OF T H E L I T E R A T U R E  1  1.1 Introduction  1  1.2 Proteoglycans  2  1.3 Glycosaminoglycans  3  1.4 Small Leucine Rich Proteoglycans (SLRP)  5  1.4.1 Class I SLRP  6  1.4.2 Class II SLRP  7  1.4.3 Class III SLRP  7  1.5 Small Leucine Rich Proteoglycans of Oral Tissues 1.5.1 C l a s s I S L R P s  8 9  i. Decorin  9  ii. Biglycan  14  iii. PLAP-1  17  1.5.2 Class II SLRPs  17  i. Fibromodulin  17  ii. Lumican  19  iii. Osteoadherin / Osteomodulin  20  1.5.3 Localization of SLRP in Soft Oral Tissues  21  iii  i. Lining Mucosa  22  ii. Periodontal Ligament. .  26  iii. Pulp  30  1.5.4 Localization of SLRP in Hard Oral Tissues  31  i. Dentin / Predentin  32  ii. Cementum  35  iii. Bone  37  iv. Enamel  39  1.5.5 Localization of SLRP in Other Oral Tissues i. T M J disc  40 40  1.6 Collagen: A Basic Constituent of Oral Tissues  41  1.7 Collagen Synthesis  42  1.8 Collagen Fibrillogenesis  44  1.9 Collagen and Small Leucine Rich Proteoglycans  46  1.9.1 Interactions  46  1.9.2 The Binding Model  50  1.9.3 In Vitro Studies  51  1.9.4 Genetic Evidence  53  1.10 Implications of the Role of Small Leucine-Rich Proteoglycans in Dental Disease 56. 1.10.1 Proteoglycans and Glycosaminoglycans: Periodontal Disease Markers. 57 C H A P T E R T W O - A I M OF S T U D Y  62  CHAPTER THREE - MATERIALS A N D METHODS  63  3.1 Animals  63  3.2 Morphometric Analysis of Jaw Size  64  3.3 Analysis of Alveolar Bone Loss  64  3.4 Histomorphometric Analysis  64  iv  3.5 Immunohistochemical Analysis  67  3.6 Scanning Electron Microscopy  69  3.7 Statistical Analysis  69  CHAPTER FOUR - RESULTS  70  4.1 Immunohistochemical Analysis  70  4.2 Gross Morphometrical Analysis  71  4.3 Histomorphological Analysis  73  4.3.1 Teeth, Cementum, Alveolar Bone, Gingiva  73  4.3.2 Periodontal Ligament  74  4.3.2.1 Scanning Electron Microscopy of the Periodontal Ligament  78  4.3.2.2 Vasculature of the Periodontal Ligament  79  4.3.2.3 Tears in the Periodontal Ligament  80  C H A P T E R FIVE - DISCUSSION 5.1 Mouse Model  81 82  5.2 Fibromodulin and Lumican - Abundantly Expressed in Oral Tissues. . . . 82 5.3 Fibromodulin and Lumican Compensate for Each Other  84  5.4 No Gross Morphologic Changes in Jaws and Teeth in Knockout Mice . . .86 5.5 Most Oral Soft Tissues Showed no Gross Morphologic Changes  87  5.6 Collagen Fiber Bundles of the Knockout Periodontal Ligaments Have Altered Morphology  87  5.7 More Blood Vessels in Fibromodulin Knockout Periodontal Ligaments. . 97 5.8 More Disruptions in Fibromodulin Knockout Mouse Ligaments 5.9 Knockout Mice Express Mild Phenotypes in vivo C H A P T E R SIX - CONCLUSIONS A N D F U T U R E DIRECTIONS  99 100 102  6.1 Conclusions  102  6.2 Future Directions  103  REFERENCES  104  Appendix I  Tables  127  Appendix II  Figures  150  vi  LIST OF TABLES 1. The small leucine-rich proteoglycans (SLRPs). Class I S L R P s ( E M C 2 , Decorin, Biglycan, P L A P - 1 ) , Class II S L R P s (Osteoadherin, P R E L P - 1 , Keratocan, Lumican, Fibromodulin), Class III S L R P s (Mimecan, Opticin, Epiphycan) and others (yet unclassified: Chondroadherin, Nyctalopin) are shown. (DS = dermatan sulfate; P G = proteoglycan; K S = karatan sulfate; C S chondroitin sulfate; G A G = glycosaminoglycan; L R R = leucine-rich repeat). (Neame and K a y , 2000, Iozzo, 1998) 128 2. Glycosaminoglycans o f oral tissues (CS = chondroitin sulfate; D S = dermatan sulfate; H A = hyaluronic acid; H S = heparan sulfate; K S = keratan sulfate; P D L = periodontal ligament) (Bartold & Narayana, 1996) 129 3. Components o f dental soft tissues ( P D L = periodontal ligament, G A G s = glycosaminoglycans; K T = keratinized tissue) (Ten Cate, 1994) 130 4. Collagen composition i n dental and periodontal tissues. A b a b n e h e t a l , 1999)  (Ten Cate,  1994; 131  5. Components o f dental hard tissues ( H A = hydroxyapatite; P G s = proteoglycans; wt = weight; v o l = volume) (Ten Cate, 1994) 132 6. Types and distribution o f collagen. ( F A C I T = fibril-associated collagens with interrupted triple helices; P D L periodontal ligament) (Ten Cate, 1994; Bartold & Narayanan, 1996; Kadler et al, 1996; Kadler, 1995; Becker et al, 1991; Dublet et al, 1988; Butler et al, 1975; Huang et al, 1991; Sloan 1993; Karimbux et al, 1992) 133 7. The small leucine-rich proteoglycans ( S L R P ) that have been shown to bind fibrillar collagen thus far. References i n text 134 8. In vitro studies attesting the role o f small leucine-rich proteoglycans ( S L R P ) in collagen fibrillogenesis ( L U M = lumican; D C N = decorin; E C M = extracellular matrix; F M = fibromodulin; P G s = proteoglycans) 135 9. In vivo studies attesting to the role o f S L R P in collagen fibrillogenesis. For a more extensive review o f these studies, see Ameye & Young, 2002 ( D C N = decorin; K O = knockout; L U M = lumican; B G N = biglycan; F M = fibromodulin; P D L = periodontal ligament) 136 10. Sex (M=male; F=female) and age (months) o f all wild-type C D - I , fibromodulin knockout (FM-/-), lumican knockout ( L U M - / - ) and double knockout ( F M / L U M - / ) mice used in this study 137 11. Classification o f alveolar bone loss in mice as described by Wiebe et al, 2001 .138  Vll  12. List o f mice used for each staining and analyzed. Stains include: hematoxylin & eosin ( H & E ) , phosphotungstic acid hematoxylin ( P T A H ) , picrosirius red (PSR), anti-type I collagen antibody, anti-lumican ( a n t i - L U M ) antibody, anti-decorin (anti-DCN) antibody, anti-fibromodulin (anti-FM) antibody, anti-biglycan (antiB G N ) antibody. Stains were carried out on buccal-Ungual (Bu/Li) sections and mesio-distal ( M / D ) sections where indicated. Each slide contained 4 sections per slide. Refer to table 10 for age and gender o f mice 139 13.  Dilution table o f antibodies used, anti-decorin (anti-DCN), anti-biglycan (antiB G N ) , anti-lumican (anti-LUM), anti-fibromodulin (anti-FM) 140  14.  M i c e used for compensation study. Mesio-distal sections o f C D - I wild-type mice, fibromodulin knockout (FM-/-) mice, lumican knockout ( L U M - / - ) mice, and fibromodulin/lumican knockout ( F M / L U M - / - ) mice were chosen and divided into 4 groups as noted below. Each groups was stained separately. Each slide then received anti-lumican (anti-LUM), anti-decorin (anti-DCN), antifibromodulin (anti-FM) or anti-biglycan (anti-BGN) antibodies for an equal and standardized amount o f time. Sections from each groups were then compared and intensities o f immunostains were determined to be +++, ++, + or 0 stain 141  15.  Localization o f decorin, biglycan, lumican and fibromodulin i n dental tissues o f wild-type C D - I mice using non-standardized sections. Intensity o f immunostaining was noted on a relative basis as +++ (most intense), ++ (moderately intense) or + (least intense). - indicates no immunostaining detected. Intensity measures are relative to other tissues within the stain group and not relative to each other (CT=connective tissue) 142  16.  Maxillary j a w length measurements (millimeters), age (months) and sex (m = male; f = female) o f wild-type ( C D - I ) mice, fibromodulin knockout (FM-/-), lumican knockout ( L U M - / - ) , fibromodulin/lumican knockout mice. (NA=could not assess due to breakage) 143  17.  Jaw length comparisons o f pooled data. Pooled j a w lengths o f wild-type ( C D - I ) mice (n=5, mean age 6.6 months, male & female), fibromodulin knockout ( F M /-) (n=8, mean age 5.4 months, male & female), lumican knockout ( L U M - / - ) (n=6, mean age 5.5 months, male & female) and fibromodulin/lumican knockout (F/L-/-) (n=7, mean age 7.5 months, male & female) mice were compared using the student t statistic assuming equal variance. P values given for those comparisons that were statistically significant (*). N S = not significant 144  18.  Jaw length comparison o f different age groups (m = months) within the w i l d type ( C D - I ) , fibromodulin knockout (FM-/-), lumican knockout ( L U M - / - ) and fibromodulin/lumican knockout (F/L-/-) mouse groups. P value not shown for statistically insignificant comparisons. N S = not statistically significant 144  viii  19.  Jaw length comparisons o f similar ages (m = months) between different groups. Wild-type (CD-I), fibromodulin knockout, lumican knockout, fibromodulin/lumican knockout groups are compared. P value only shown for those comparisons that were statistically significant (*) using the student t-test assuming equal variance. N S = not statistically significant 144  20.  Jaw length comparisons o f males vs. females within the wild-type ( C D - I ) , fibromodulin knockout (FM-/-), lumican knockout (LUM-/-) and fibromodulin/lumican knockout (F/L-/-) groups. P value is given only for those comparisons that were statistically significant (*) using the student t-test assuming equal variance. N S = not statistically significant 145  21.  Jaw length comparisons o f males and females between the wild-type ( C D - I ) , fibromodulin knockout (FM-/-), lumican knockout (LUM-/-) and fibromodulin/lumican knockout (F/L-/-) groups. P value is given only for those comparisons that were statistically significant (*) using the student t-test assuming equal variance. N S = not statistically significant 145  22.  Jaw length comparisons o f male mice o f similar ages (m = months). Comparisons made between wild-type ( C D - I ) , fibromodulin knockout (FM-/-), lumican knockout ( L U M - / - ) and fibromodulin/lumican knockout (F/L-/-) groups. P value is given only for those comparisons that were statistically significant (*) using the student t-test assuming equal variance. N S = not statistically significant 145  23.  Jaw length comparisons o f female mice o f similar ages (m = months). Comparisons made between wild-type ( C D - I ) , fibromodulin knockout (FM-/-), lumican knockout ( L U M - / - ) and fibromodulin/lumican knockout (F/L-/-) groups. P value is given only for those comparisons that were statistically significant (*) using the student t-test assuming equal variance. N S = not statistically significant 146  24.  A . Table o f number o f blood vessels counted in the periodontal ligament o f C D 1 wild-type, fibromodulin knockout (FM-/-), lumican knockout ( L U M - / - ) and double knockout ( F M / L U M - / - ) mice. Table shows mice from which data was collected and blood vessels counted on buccal and lingual sides. B l o o d vessels were counted only i n the coronal two thirds o f the periodontal ligament (#=mouse number; Bu=buccal; Li=lingual) 147 B . Table o f number o f tears counted i n the periodontal ligament o f C D - I w i l d type, fibromodulin knockout (FM-/-), lumican knockout ( L U M - / - ) and double knockout ( F M / L U M - / - ) mice. Table shows mice from which data was collected and tears counted in the entire periodontal ligament (#=mouse number) 147  ix  25.  B l o o d vessels o f wild-type ( C D - I ) , fibromodulin knockout (FM-/-), lumican knockout ( L U M - / - ) , fibromodulin/lumican knockout (F/L-/-) mice are compared in total (pooled), as well as for buccal (Bu) and lingual ( L i ) periodontal ligament surfaces. P value is shown only for those comparisons that were statistically significant (*) with the student t-test assuming equal variance 148  26.  Tears o f the periodontal ligament are compared between wild-type ( C D - I ) , fibromodulin knockout (FM-/-), lumican knockout (LUM-/-), fibromodulin/lumican knockout (F/L-/-) mice are compared using pooled data. P value is shown only for those comparisons that were statistically significant (*) with the student t-test assuming equal variance 149  x  LIST OF FIGURES 1. Basic proteoglycan structure can be seen in the schematics o f decorin and biglycan small leucine-rich proteoglycans. Proteoglycans have a protein core with N - and C - terminal domains with glycosaminoglycans attached to it. In the case o f biglycan and decorin, the glycosaminoglycan chains can be dermatan sulfate (as in periodontal connective tissue) or chondroitin sulfate (CS), as depicted here, depending on tissue location. Larger proteoglycans have more complex structures 151 2. Structure o f small leucine-rich proteoglycan protein core protein (Neame and K a y , 2000) 152 3.  Structure o f similar small leucine-rich proteoglycans. Schematic demonstrated the similarities o f four small leucine-rich proteoglycans yet highlights the differences between class I members (decorin and biglycan) and class II members (fibromodulin and lumican) o f this family. Adapted from Iozzo, 1996 153  4.  Gingival fibers. Type I collagen fibers o f the gingiva have been classified according to their location and direction. There are four recognizable groups: the circular group, the dentogingival group, the dentoperiosteal group and the alveologingival group (Ten Cate, 1994) 154  5. Principle fibers o f the periodontal ligament. Type I collagen fiber bundles o f the periodontal ligament have been divided into 6 groups based on their location and orientation. They include the transeptal, alveolar crest, horizontal, oblique, apical and interradicular fibers. They are highly organized fibers running parallel to each other i n the ligament (Ten Cate, 1994) 155 6. Collagen biosynthesis demonstrating intracellular (hydroxylation, glycosylation, nucleation and propagation) and extracellular events (peptide cleavage, fibril formation and cross-linking) that lead to the formation o f a collagen molecule by fibroblasts. Specific amino acids on ribosomes form individual polypeptide chains that contain N and C terminal peptides. Some o f the lysine and proline residues are hydroxylated (vitamin C dependent enzyme driven processes) forming hydroxylisine and hydroxyproline. Sugar residues are added, a process known as glycosylation (an enzymatically driven process). Peptide chains then form a triple helix and are transported to the golgi apparatus where the procollagen molecules is completed and excreted from the cell. Next fibrillogenesis takes place where the N and C termini are eventually cleaved and 5 unit staggered microfibrils are formed (Kadler, 1995) 156 7. Triple helix o f collagen. Schematic representation o f collagen cased on a repeating triplet o f Glycine-proline-4-hydrohyproline. ( A ) shows a single collagen alpha chain, (B) shows three alpha chains folded into a triple helix with Glycine  XI  residues i n the center o f the molecule and (C) shows a cross-sectional view o f (B) looking down the axis o f the triple helix molecule (Kadler, 1995) 157 8. Collagen fibril structure. Collagen molecules aggregate to form a banded fibril. Negative staining techniques allow gaps to contain more stain and show up as dark areas i n electron microscope. Minerals in hard tissues accumulate in these gaps (Ten Cate, 1994) 158 9. M o d e l o f lateral fusion o f collagen molecules to form fibrils. Collagen molecules assemble into quarter-staggered arrays giving rise to fibril intermediates (65nm in mouse tendon). These fibril intermediates are stabilized by fibril-associated macromolecules, such as fibromodulin and lumican, to allow for fusion o f adjacent fibril intermediates i n the formation o f mature collagen fibrils. M o d e l based on study i n fibromodulin, lumican and double deficient mouse tendon ( E z u r a e t a l , 2000) 159 10. Contribution o f fibromodulin and lumican during collagen fibrillogenesis. In the model described i n figure 9, it is believed that lumican is expressed early i n ' development suggesting a role in the initial stages o f fibrillogenesis while fibromodulin expression increases during development suggesting a role in fibril maturation (Ezura et al, 2000) 160 11. The binding model o f decorin to type I collagen. Decorin, a class I small leucinerich proteoglycan, is thought to be horseshoe shaped and bind type I collagen fibrils on their concave side. Weber et al, 1996 estimate that each decorin molecule has enough space on the concave aspect o f the arc to accommodate one collagen molecule 161 12. Jaw length measurements. Photograph o f a defleshed, halved mouse maxilla showing where j a w length measurements were made using a Boley Gauge to the nearest tenth o f a millimeter. Measurements were made anteriorly at the osseous crest facial o f the incisor at the point o f incisor exit and posteriorly at the distal o f the third molar 162 13. Localization o f (A) fibromodulin, (B) lumican, (C) biglycan and (D) decorin in the dental and periodontal tissues o f wild-type mice. (A) Fibromodulin is expressed i n the connective tissues o f the periodontal ligament, gingiva and mucosa, pulp and predentin to similar degrees (++). (B) Lumican is expressed in the connective tissues o f the periodontal ligament (++) and gingival and mucosa (+++) with only weak expression evident i n pulp and predentin. (C) Biglycan is abundantly expressed i n the connective tissue o f the periodontal ligament and gingiva (+++) and to a lesser degree in that o f mucosa and predentin (++). (D) Decorin has a distribution pattern similar to that for lumican being abundantly expressed in the connective tissues o f the periodontal ligament (++) and even more i n gingiva and mucosal connective tissues (+++). Only weak decorin expression was seen i n pulp and predentin (+). (immunostain intensity: + = mild,  xn  ++ = moderate, and +++ = intense) (p=pulp; d=dentin; b=bone; c=cementum; e=epithelium; ct=connective tissue; pdl=periodontal ligament) 163 14. a. Relative expression o f small leucine-rich proteoglycans i n knockout mice. In order to determine i f expression o f other small leucine-rich proteoglycans was changed i n fibromodulin and lumican knockout mice, sections were immunostained with anti-lumican ( A , E , I , M ) , anti-fibromodulin ( B , F , J, N ) , antidecorin (C, G , K , O) and anti-biglycan (D, H , L , P) antibodies in C D - I wild-type ( A - D ) , lumican knockout (E-H), fibromodulin knockout (I-L) and double knockout ( M - P ) mice. More intense fibromodulin staining is seen in lumican knockout mice compared to wild-type mice (F vs. B ) and more intense lumican immunostaining is seen in fibromodulin knockout mice compared to wild-type mice (J vs. A ) . The intensities o f the other immunostainings are similar to that o f wild-type (b=bone; pdl=periodontal ligament; C T = connective tissue; e=epithelium; d=dentin; p=pulp) 164 b. Relative expression o f decorin and biglycan in knockout mice. First molar sections immunostained with anti-decorin ( A - D ) and anti-biglycan (E-H) antibodies o f wild-type ( A , E ) , lumican knockout (B,F), fibromodulin knockout (C,G) and double knockout (D,H) mice. N o marked differences i n immunostain intensity are noted between any o f the knockout mice and wild-type C D - I mice ((b=bone; pdl=periodontal ligament; C T = connective tissue; e=epithelium; d=dentin; p=pulp) 165 15. Teeth are fully erupted i n the knockout mice. Defleshed maxillary j a w halves o f wild-type C D - I ( A ) , fibromodulin (B), lumican (C) and double knockout (D) mice illustrating that all molars ( M l , M 2 and M 3 ) are fully erupted 166 16. Sections o f C D - I wild-type ( A ) , fibromodulin knockout (B), lumican knockout (C) and double knockout (D) mouse mandibular first molars stained with phosphotungstic acid hematoxylin. The first molars are fully erupted and have bone levels o f similar height. (Note: distal roots are missing due to sectioning angle). (b=bone; p=periodontal ligament; d=dentin, g=gingiva) 167 17. H & E stained sections o f the coronal third o f mandibular first molars o f C D - I wild-type ( A ) , fibromodulin knockout (B), lumican knockout (C) and double knockout (D) mice. Wild-type mouse periodontal ligament is full o f type I collagen fiber bundles (arrow) that are directed i n a 45 degree fashion from tooth to bone i n a relatively parallel course. Fiber bundles appear o f relatively even thickness and the spacing between the fiber bundles is evenly distributed (arrowhead) ( A ) . Fibromodulin knockout mouse periodontal ligament appears to have fibers o f variable diameters (arrows), with increased spacing between them (arrowheads) (B). Lumican knockout mice have fiber bundles o f variable diameters (arrows) and increased spacing between them (arrowheads) (C). Ligament i n the double knockout mice have a very disorganized arrangement o f collagen fiber bundles with variable fiber bundle diameters (arrows) and increased  xiii  spacing between them (arrowheads) (D). (p=periodontal ligament; d=dentin; b=bone) 168 18. Sections o f the coronal third o f mandibular first molars o f C D - I wild-type ( A ) ; fibromodulin knockout (B), lumican knockout (C) and double knockout (D) mice stained with phosphotungstic acid hematoxylin. Fiber bundles o f knockout mice are o f variable thickness (small arrows) and there is relatively more spacing between the fiber bundles (arrowheads) - features that are unlike those o f w i l d type mice. A n area o f external root resorption is noted i n fibromodulin knockout mouse root (large arrow) (B) (p=periodontal ligament; d=dentin; b=bone; c=cementum) 169 19. Sections o f mandibular first molar periodontal ligament microscope o f wild-type C D - I ( A ) , fibromodulin (B), lumican (C) and double knockout mice were stained with picrosirius red and analyzed under polarizing light. The periodontal space o f wild-type mice ( A ) are filled with collagen fiber bundles (small arrow) and the bundles have smooth outlines (large arrows). Fiber bundles o f fibromodulin knockout mice (B) are unorganized and appear to have dark stripes associated with them. Fiber bundles o f lumican knockout mice (C) have dark stripes associated with them. Fiber bundles o f double knockout mice (D) have altered outlines (large arrows) and vertical fibers are seen (small arrows) (c=cementum; p=periodontal ligament; b=bone) 170 20. Scanning electron micrographs o f C D - I wild-type ( A , E ) , fibromodulin knockout (B,F), lumican knockout ( C , G ) and double knockout mouse (D,H) first molar periodontal ligaments. A , E : Collagen fiber bundles in periodontal ligament o f normal mice were well organized and traversed the periodontal space without disruption ( A ) . The outlines o f the bundles were even and smooth (arrows) (E). B , F : Collagen fiber bundles o f fibromodulin knockout ligaments were disorganized (B) and had several interruptions along their lengths (F). The bundles were o f variable thickness and appeared slightly thinner than wild-type ligaments (B,F). A s well there was more inter fiber bundle spaces between them (F). Small branches are apparently protruding form the main fiber bundle (arrow) (F) C , G : Collagen fiber bundles o f lumican knockout mice were disorganized and thinner than the fibromodulin knockout and wild-type ligaments. M a n y small branches are seen coming off the main bundles (arrows) (G). D & H : Double knockout ligaments are very disorganized and their basic 45 degree orientation was essentially lost. The bundles appeared uneven and rough with small craterlike defects on the surface (arrowheads). Interfiber bundle spacing was greater then wild-type and small branches were seen coming off the main fiber bundles (arrows) 171 21. Examples o f tears and blood vessels that were counted in this study. B l o o d vessels were recognized as voids i n the ligament that had blood cells inside the lumen and endothelial cells lining the lumen. Tears were those voids with no cells associated with it and rough or stringy outline. Both phosphotungstic acid hematoxylin and  xiv  H & E stained sections were used for analysis (d=dentin; p=periodontal ligament; b=bone; v=vessels; t=tear) 172  XV  ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my thesis supervisor, D r . L a r i Hakkinen, for his guidance and support during the course o f this project. Thank you to Dr. Hannu Larjava for his support and advise throughout this project. Thank you to the members o f my committee, Drs. Edward Putnins and Doug Waterfield. Thank you to D r . D E Birk, Department o f Pathology, Anatomy and C e l l Biology, Jefferson Medical College, 1020 Locust Street, J A H 5 4 3 , Philadelphia, P A 19107, Dr. Chakravarti, John Hopkins University School o f Medicine, Baltimore, Maryland and Dr. Oldberg, University o f Lund, Sweden for providing the mice for this study. Thank you to Cristian Sperantia for his technical expertise throughout this project. Thank you to Yang Tonghua for her technical support. Thank you to a l l fellow graduate periodontics residents who shared the program with me for their support and encouragement. Thank you to Drs. Elaine Gordon, Edward Hannigan and Gary Foshay for their continued support and suggesting that I enter Periodontics. Most o f all, thank you to Jamie for his limitless support and understanding and Tiernan and Lauren who kept me laughing.  xvi  Chapter One - A Review of the Literature 1.1 Introduction. Small Leucine-Rich Proteoglycans Of Dental Tissues: Do They Play A Role In Dental Disease? Small leucine-rich proteoglycans have been localized i n several dental tissues. Deficiency or absence o f small leucine-rich proteoglycans may be implicated i n diseases associated with dental manifestations such as osteogenesis imperfecta, infantile progeroid syndrome, Ehlers-Danlos syndrome and Marfan syndrome (Beavan et al, 1993; Fedarko et al, 1992; Fushimi et al, 1989; Straub et al, 2002). It is suspected that these proteoglycans offer much in the way o f organization and strength to the periodontal ligament as well as influence the development and mineralization o f hard dental tissues, including enamel. It seems plausible that a deficiency or absence o f these seemingly inconspicuous molecules may allow for greater susceptibility to periodontal breakdown and perhaps altered mineralization or disease o f the hard dental tissues.  Periodontal disease, for example, is a very ubiquitous disease in our culture affecting one third to one half o f the American population (Albandar, 2002), with severe forms affecting relatively few (Brown & Loe, 1993). Periodontal disease has been linked to a number o f causes including virulent bacteria and host susceptibility. In some relatively rare cases, this increased susceptibility is attributed to a certain gene defect (Hart et al, 2000), as with Papillon Lefevre syndrome. However, in most cases, the underlying reason for rapidly progressing disease is unknown. Undoubtedly, type and virulence o f particular microbiota play an important role in these cases. However, recent advances in the understanding o f biological functions o f extracellular matrix molecules have provided novel information about their critical role in the development and maintenance o f  1  periodontal tissues. C e l l culture and in vivo studies using gene knockout animals (animals targeted for specific gene deletion) have suggested  that changes in the  expression o f extracellular matrix molecules may contribute to increased susceptibility to periodontal disease (discussed later).  In recent years, there have been very few reviews o f small leucine-rich proteoglycans o f oral tissues (Rahemtulla, 1984). M u c h o f the knowledge that has been gained thus far in this area has been based mainly on connective tissue research, particularly in cartilage (Embery, 2001). This paper reviews the small leucine-rich proteoglycans that have been localized i n dental tissues thus far and discusses how they may influence the biology and pathology o f these tissues. It should be noted that not all small leucine-rich proteoglycans have been investigated i n oral tissues and their exclusion in this review does not indicate their absence.  1.2 Proteoglycans Proteoglycans are a group o f macromolecules that are found in the extracellular matrix and associated with cell membranes o f virtually all connective tissues in the body. For simplicity, the extracellular matrix proteoglycans can be divided into three groups (Iozzo, 1998). The hyalectans are a family o f proteoglycans that interact with hyaluronan and lectin and include aggrecan and versican. They form the gelatinous matrix i n which collagen fibrils are embedded. The basement membrane proteoglycans, such as perlecan, form the second group, and the small leucine-rich proteoglycans, the focus o f this paper, form the third group.  2  Structurally,  proteoglycans  contain  a  protein  core  that  has  O-  or  N - linked  oligosaccharides and covalently attached glycosaminoglycan side chains that are separate gene products (Fig. 1).  Functionally, proteoglycans act to regulate  cell growth  (Yamaguchi and Ruoslahti, 1988), cell adhesion (Lewandowska et al, 1987), collagen fibril formation (Vogel et al, 1984), extracellular matrix formation (Gallagher, 1989), maintain the transparency o f the cornea (Chakravarti et al, 1998), the tensile strength of skin and tendon (Danielson et al, 1997; Chakravarti et al, 1998), the viscoelasticity o f blood vessels, the compressive properties o f cartilage, the mineralized matrix o f bone (Iozzo, 2000), and bind, store and regulate the activity o f growth factors and cytokines (Yamaguchi et al, 1990). Some o f the functions o f proteoglycans are mediated by its protein core, while others by the glycosaminoglycan chain(s) that are attached to it. Other functions  seem to require  interactions with both the protein core and  attached  glyco samino glycans.  1.3 Glycosaminoglycans Glycosaminoglycans are linear hetero-polysaccharides o f repeating disaccharide units o f N-acetyl hexosamine and hexuronic acid. They are attached to the proteoglycan core proteins by trisaccharide galactose - galactose - xylose that recognize serine - glycine residues  and  various  amino  acids  i n the  core protein (Bourdon et  al,  1987).  Glycosaminglycans sometimes play an important role in determining the functional characteristics o f proteoglycans.  3  Seven species o f glycosaminoglycans have been identified: hyaluronan, dermatan sulfate, chondroitin 4-sulphate, chondroitin 6-sulphate, keratan sulphate, heparan sulphate, and heparin. Selective hexuronic acid residues within dermatan sulfate and heparan sulfate and selective sulphation o f the hexosamine residue within chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin and keratan sulfate allow for greater diversification, leading to the production o f glycosaminoglycan chains with specific properties. A n exception to this rule is hyaluronic acid, which is neither sulphated nor covalently bound to a protein core in connective tissue. It is found to aggregate with large proteoglycan aggregates like aggrecan and versican and with the cell surface proteoglycan, C D 4 4 (Embery, 2001).  The predominant glycosaminoglycans o f the periodontal tissues are hyaluronic acid, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, keratan sulfate and dermatan sulfate (Table 2). The number (from 1-100), size and type o f the glycosaminoglycan chains attached to the protein core can vary greatly depending on the cell/tissue origin. The glycosaminoglycan side chains mediate many functions o f proteoglycans. For example, glycosaminoglycans can potently bind and regulate the function o f growth factors, they mediate some o f the interactions o f proteoglycans with cells and the extracellular matrix and they are responsible in maintaining the appropriate conformation o f the proteoglycan molecule (Zimmermann and Ruoslahti, 1989; Sheikh et al, 1998; Toole, 1982; Comper and Laurent, 1978).  4  1.4 S m a l l L e u c i n e - R i c h Proteoglycans Small leucine-rich proteoglycans  comprise  a group  o f proteoglycans  (containing  chondroitin / dermatan sulfate or keratan sulfate glycosaminoglycan side chains) and glycoproteins (containing N-linked oligosaccharides) in the extracellular matrix of hard and soft tissues. They are, like larger proteoglycans, comprised o f a protein core and glycosaminoglycan side  chains.  The  distinguishing feature  o f small leucine-rich  proteoglycans, however, is the presence o f a central domain containing leucine-rich repeats i n the protein core (Fig. 2). This domain, which is responsible for most o f the functional activity o f these molecules, is flanked by smaller, less conserved, N-terminal and C-terminal cysteine clusters i n characteristic positions (Iozzo, 1997). The structure o f these molecules can be divided into four domains: domain I containing a signal peptide and a propeptide; domain II containing four evenly spaced cysteine residues and the glycosaminoglycan attachment site(s); domain III containing the leucine-rich repeats; domain I V containing two cysteine residues (Iozzo, 1998). Members o f the small leucinerich proteoglycan family differ in their numbers o f leucine-rich repeats, amino acid substitutions and glycosylation (Iozzo, 1997) (Fig. 3).  Small leucine-rich proteoglycans are involved in collagen fibrillogenesis, modulation o f growth factor activity, and regulation o f cell growth (Iozzo & Murdoch, 1996). They were first discovered as small proteoglycans o f bone and were subsequently found in the extracellular matrix o f most tissues. The relative abundance o f small leucine-rich proteoglycans in tissues varies during development and by location. This suggests a role in the differentiation and maintenance o f tissue structure (Johnson et al, 1997).  5  Knowledge i n the field o f small leucine-rich proteoglycans is expanding rapidly. In 1996, Iozzo noted in a review article the existence o f only 5 small leucine-rich proteoglycans. Today, just 7 years later, there are 15 distinct gene products i n this group (Table 1). It is only with the advent o f molecular biological and immunohistological techniques that they are being identified and their ultrastructural distribution and possible biological roles are beginning to be clarified (Embery et al, 2001). Small leucine-rich proteoglycans are divided into 3 classes based on their similarities in structure (Iozzo, 1997).  1.4.1 Class I Small Leucine-Rich Proteoglycans - This group o f molecules contains decorin, biglycan (Fisher et al, 1989) and asporin (Henry et al, 2001). Decorin and biglycan are the most homologous (-57% identical based on human amino acid sequences) o f all the small leucine-rich proteoglycans and are the only small leucine-rich proteoglycans to contain a pro-peptide (Krusius & Ruoslahti, 1986; Fisher et al, 1989), the function o f which remains unclear (Neame & K a y , 2000). Members of this group contain an N-terminal domain that is usually substituted with one (decorin) or two (biglycan) chondroitin sulfate/ dermatan sulfate side chains. They contain 10 leucine-rich repeats flanked by cysteine clusters. The cysteine residues have a unique spacing pattern and intervening amino acids, both o f which are maintained within each subfamily. For instance, the cysteine consensus sequence for class I small leucine-rich proteoglycans is CX3CXCX6C  and is different from that o f the other classes.  The class I members are  encoded by genes composed o f 8 exons with intron and exon junctions in highly conserved positions (Henry, 2001).  6  1.4.2 Class II Small Leucine-Rich Proteoglycans - This group contains fibromodulin (Oldberg et al, 1989), lumican (Blochberger et al, 1992), P R E L P (Bengtsson et al, 1995), keratocan (Corpuz et al, 1996) and osteoadherin (Sommarin et al, 1998). Members o f this group may exist as proteoglycans or glycoproteins (with no glycosaminoglycan chains) (Neame & K a y , 2000). They have a common gene structure o f 3 exons with cysteine spacing in the N-terminal region o f CX3CXCX9C.  The core proteins contain 10 leucine-  rich repeats (except P R E L P ) and can be substituted with N-linked keratan  sulfate  glycosaminoglycan side chains (Henry, 2001). A s a group, they have several amino acids in common and share a number o f conserved features (Corpuz et al, 1996). O n the amino acid level fibromodulin and lumican are 50% identical (Blochberger et al, 1992) while lumican, fibromodulin and keratocan are 35% identical (Corpuz et al, 1996). P R E L P is 36% similar to fibromodulin and 33% similar to lumican (Bengtsson et al, 1995), while osteoadherin is 42% similar to bovine keratocan, 38% similar to bovine fibromodulin, lumican and human P R E L P - 1 (Sommarin et al, 1998). Lumican and keratocan have a characteristic unlike other small leucine-rich proteoglycans in that outside o f the cornea, where  they  both  carry  keratan  sulfate,  they  are  poorly sulfated  or  unsulfated  glycoproteins (Corpuz et al, 1996).  1.4.3 Class III Small Leucine-Rich Proteoglycans - This group contains epiphycan/PGLb  (Shinomura  &  Kimata,  1992),  osteoglycin/mimecan  (Madisen et  al,  1990;  Funderburgh et al, 1997) and opticin (Reardon et al, 2000). They have a common gene structure composed o f 7-8 exons and only 6 leucine-rich repeats with cysteine spacing in the N-terminus region o f CXjCXCX^C. Osteoglycin/mimecan and epiphycan/PG-Lb can  7  be substituted with N-linked keratan sulfate glycosaminoglycan side chains and O-linked chondroitin sulfate / dermatan sulfate side chains respectively while opticin is substituted with O-linked sialylated oligosaccharides making it a glycoprotein rather than a proteoglycan (Henry, 2001).  Nyctalopin and chonroadherin are considered to be members o f the small leucine-rich proteoglycan family o f proteoglycans but have not been classified in any o f the above three classes. Although they have the leucine-rich repeat that shows some homology with the other members, their structure is quite different from the conventional small leucinerich proteoglycans perhaps resulting from early divergence during evolution o f this group of molecules (Henry et al, 2001).  Table 1 summarizes all members o f the small leucine-rich proteoglycan family known to date. A l l members are very similar in structure (Fig. 3), but are different at both the protein and gene levels (Neame and K a y , 2000).  1.5 S m a l l L e u c i n e - R i c h Proteoglycans O f O r a l Tissues Several small leucine-rich proteoglycans have been localized or associated with animal and human oral tissues. The expression pattern o f small leucine-rich proteoglycans seen during development o f body organs i n mice (Wilda et al, 2000), suggests that they play a role in development. Body organs develop through epitheliomesenchymal interactions. Teeth also form through the interaction o f these two entities such as when the epithelium invaginates into the mesenchyme o f the maxillary and mandibular processes (Ten Cate,  8  1994) and thus it might be imagined that small leucine-rich proteoglycans play a role in tooth development as well. It is not surprising, then, to find many small leucine-rich proteoglycans in oral and related tissues or associated with their development. The following discusses all the small leucine-rich proteoglycans that have thus far been localized in oral tissues. It should be noted, however, that not all small leucine-rich proteoglycans have been investigated in the oral cavity and omission o f some small leucine-rich proteoglycans here is not meant to suggest their lack o f existence in oral tissues.  1.5.1 Class I Small Leucine-Rich Proteoglycans In Oral Tissues Decorin Decorin ( A K A : D S - P G - I I ; P G II; P G - S 2 ; PG40) is the most studied member o f the small leucine-rich proteoglycan family. First detected i n cartilage and bone (Rosenberg et al, 1985; Fisher et al, 1983), it has since been found i n several body tissues such as articular cartilage, intervertebral discs (Neame & K a y , 2000), tendon, skin, gingiva (Fisher et al, 1989; Hakkinen et al, 1993), sclera (Coster and Fransson, 1981), mouse heart, kidney, small intestine, testes and liver (Henry, 2001). It was so named for its microscopic appearance on the collagen network, "decorating" the collagen fibers (Hocking, 1998).  The decorin gene is located on human chromosome 12q23 (Danielson et al, 1993) and distal end o f mouse chromosome 10 (Scholzen et al, 1994), the same site as fibromodulin and lumican (Chakravarti e& Magnuson, 1995) suggesting a possible overlap in function of these small leucine-rich proteoglycans. The mature protein is highly conserved across  9  species as shown i n comparison o f human, murine, bovine and avian forms (Weber et al, 1996).  Structurally, decorin is composed o f a 36.5 k D a core protein that contains 10 leucine-rich repeats with disulfide bond stabilized loops on either side. Decorin is secreted in proforms that are proteolytically modified in the extracellular matrix during maturation (Neame & K a y , 2000). The propeptide is short, consisting o f 14 amino acids and its functional role, i f any, remains unclear. Decorin usually carries one glycosaminoglycan chain (chondroitin sulfate or dermatan sulfate) which is found at the fourth amino acid position near the N-terminus, but it has been reported to have two glycosaminoglycan substitutions in chicken (Blaschke et al, 1996) or none at all as in the case o f human cartilage  (Johnstone  et  al,  1993).  Chondroitin  sulfate  is  the  predominating  glycosaminoglycan in the mineralized tissue (Fisher et al, 1989) while dermatan sulfate predominates in soft tissue (Bratt et al, 1992). Recently, a catabolically truncated form o f decorin has been identified in human skin. This truncated form in called decorunt and contains only 4 3 % o f the molecule - that containing the amino terminus (Carrino et al, 2003).  Based on the known functions o f decorin, it likely takes part in development and maintenance o f soft and hard tissues, mineralization o f hard tissues, wound healing and neovascularization. The best-documented function o f decorin is its ability to influence collagen organization and fibril formation through collagen binding (Vogel et al, 1984; Bidanset et al, 1992; Pogany et al, 1994; Schonherr et al, 1995). Targeted deletion o f  10  decorin i n animals leads to reduced tensile strength o f the skin due to the development o f abnormal collagen fibrils (Danielson et al, 1997). Additionally, decorin can bind growth factors (Yamaguchi et al, 1990; Yamaguchi & Ruoslahti, 1988), ions, including calcium (Embery et al, 1998) and zinc (Yang et al, 1999), epidermal growth factor receptor ( E G F R ) (Patel et al, 1998), and extracellular matrix molecules including fibronectin (Schmidt et al, 1991; Winnemoller et al, 1992), thrombospondin (Winnemoller et al, 1992) and complement component C l q (Krumdiek et al, 1992).  Aside from its very important role in collagen fibrillogenesis and organization, which w i l l be discussed in greater detail later, decorin has been referred to as the naturally occurring inhibitor o f transforming  growth factor -  beta (TGF-P)  (Ruoslahti &  Yamaguchi, 1991). TGF-(3 is a cytokine whose many functions include control o f cell proliferation,  differentiation,  adhesion  and deposition o f the  extracellular matrix,  processes that are very important in tissue growth and repair (Hocking et al, 1998). Decorin interacts with T G F - p (Yamaguchi et al, 1990) v i a its core protein (Hildebrand et al, 1994). When binding occurs, the activity o f TGF-P is modulated. This modulation may be through the blocking o f T G F - p receptors on cell surfaces (Hocking et al, 1998), the sequestration o f TGF-P in the extracellular matrix (Hildebrand et al, 1994) or by decreased production o f TGF-P through a negative feedback mechanism (Yamaguchi et al, 1990).  The role o f decorin as a TGF-P inhibitor is substantiated  by studies  in rats.  Administration o f decorin into rats with experimental glomerulonephritis (a fibrotic  11  disease characterized by increase extracellular matrix by mesangial cells due to an overproduction o f T G F - P as a result o f injury) results in a reduction o f pathological matrix accumulation (Border et al, 1992; Noble et al, 1992). Similar therapeutic findings were found using decorin gene therapy i n rats (Isaka et al, 1996).  Other studies have found that decorin only has an inhibitory effect on certain functions o f  TGF-P (Hausser et al, 1994) and that decorin-binding to TGF-P actually increases T G F P's ability to bind to receptors thereby increasing it function (Takeuchi et al, 1994). Hausser et al (1994) showed i n vitro that decorin added to osteosarcoma cells inactivated TGF-P's  function to up-regulate biglycan synthesis  but the  down regulation o f  proteoglycan 100 remained unaltered and did nothing to decrease T G F - p ' s function in monocyte cell proliferation. A s such, the exact role decorin has with regards to TGF-P binding remains unclear.  Decorin has also been shown to bind to the epidermal growth factor receptor ( E G F R ) with concurrent activation o f the mitogen-activated protein kinase-signaling pathway. Patel et al (1998) noted that activation o f this receptor results in elevation o f intracellular calcium ion concentration ([Ca++]) in single A431 squamous carcinoma cells. A s well, it has been shown that decorin may act as a natural inhibitor o f the E G F R signaling pathway (Csordas et al, 2000) resulting in growth suppression. However, these effects seem to be cell type specific. In periodontal fibroblasts, decorin suppresses cell growth by another mechanism (Hakkinen et al, 2000).  12  Decorin may also play a role in the mineralization process, the precise nature o f which remains unclear, although evidence suggests that its role may be that o f an inhibitor (Hoshi et al, 1999). It's glycosaminoglycan components have been shown to bind Ca++, interact with hydroxyapatite (Embery et al, 1998) and have been shown to be present in pre-mineralized tissues such as osteoid (Robey, 1996) and predentin (Takagi et al, 1990; Yoshiba et al, 1996). Immunolocalization studies o f mineralized tissues show that decorin concentration decreases around collagen fibers where calcification has started (Hoshi et al, 1999). A s well, decorin, like biglycan, decreases in proportion during the development  o f rabbit knee ligaments and has a precise location in these tissues  suggesting a developmental role. After injury, decorin, together with biglycan, must be reproduced i f complete healing is to occur (Kavanagh & Ashhurst, 2001).  Previous i n vitro and in vivo studies have shown that decorin plays a role in angiogenesis. Angiogenesis is the formation o f new capillaries from preexisting vessels  through  sprouting that occurs i n a variety o f physiological processes (embryonic development, ovarian follicle maturation and tissue repair) as well as pathological processes (diabetic retinopathy, tumor growth and metastasis, chronic inflammation) (Nelimarkka et al, 2001).  In vitro studies have shown that large vessels do not express decorin in  detectable amounts but when they begin to grow new capillaries, the endothelial cells synthesize decorin (Jarvelainen et al, 1992).  A s well, it has been shown that when  macrovasular endothelial cells are transduced to overexpress decorin in vitro, they form tubes in the collagen lattices while control cells do not (Schonnher et al, 1999). The presence o f decorin i n human atherosclerotic  13  plaques (Gutierrez et al, 1997) and  granulomatous  tissues (Schonherr et al, 1999) suggest an in vivo role as  well.  Inflammation may play a role in this process since decorin does not appear to be expressed by "resting endothelial cells" (Bosse et al, 1993) but is detectable when inflammation is present (Nelimarkka et al, 2001).  H o w decorin is involved in the  angiogenesis process can only be speculated at present. Whether it is decorin's ability to help organize and stabilize the extracellular matrix through it's binding o f collagen or its ability to interact with  TGF-P or E G F R or one o f its other many interactions remains to  be elucidated. Studies o f tumor growth, an angiogenic process, raise further questions regarding decorin's exact role in the angiogenesis process. Controlled animal studies show that decorin-expressing tumor xenografts  grow much slower than wild-type  counterparts and that this decreased growth may be due to a decreased expression o f vascular endothelial growth factor (Grant et al, 2002). Thus, although decorin seemingly has an important role i n angiogenesis and tumor growth suppression, its specific role remains unclear and may involve interaction o f other matrix constituents or be tissue or cell specific.  Biglycan Biglycan ( A K A : D S - P G - I ; P G - I ; P S - S I ) is a dermatan / chondroitin sulfate proteoglycan whose gene is located on the X chromosome (McBride et al, 1990). It is found in articular cartilage, intervertebral discs, skin, tendon, gingiva (Hakkinen et al, 1993), bone (Fisher et al, 1987), mouse heart, kidney, small intestine, testes, spleen, lung, liver (Wegrowski et al, 1995; Henry, 2001), collateral and cruciate ligaments and menisci o f rabbit knee joint, endothelial cells and smooth muscle cells (Stocker et al, 1991; M a r c u m & Thompson,  14  1991). Biglycan is found frequently associated with the cell surface and peri-cellular matrix (Neame & K a y , 2000) and often associated with specialized cells like skeletal myofibers, keratinocytes, and endothelial cells (Bianco et al, 1990).  Structurally, biglycan is secreted in pro-forms and then proteolytically modified in the extracellular matrix during maturation. The propeptide is short, consisting o f 21 amino acids and it is not clear whether it serves any functional role. Cleavage takes place prior to the aspartate residue ( . . . M M N - D E E . . . ) while the surrounding sites show considerable conservation suggesting that a common enzyme is involved in processing o f biglycan and decorin. The degree to which processing occurs is both age and tissue dependent with unprocessed pro-forms being particularly abundant in adult articular cartilage where they can represent 20% o f the molecules present (Neame & K a y , 2000). Biglycan possesses a domain structure similar to that o f decorin. It usually carries two glycosaminoglycan side chains (and so named biglycan (Fisher et al, 1989) attached to amino acid 5 and 11 within the protein core (Iozzo, 1997). A s with decorin, chondroitin sulfate-containing biglycan would appear i n mineralized tissue (Fisher et al, 1989) while the dermatan sulfate containing type occurs in soft tissue (Bratt et al, 1992). In addition, non-glycanated forms of biglycan have been found in adult cartilage (Johnstone et al, 1993).  Functionally, biglycan appears to play a role i n the mineralization process, although, like decorin, the precise nature o f its role remains to be determined. It has been suggested that the role o f decorin i n the mineralization process is one o f an inhibitor (Hoshi et al, 1999) while that o f biglycan is one o f a mineralization nucleator ( X u et al, 1998). It may do this  15  through its known ability to bind collagen (Pogany et al, 1994; Schonherr et al, 1995), or interact with soluble growth factors, like  TGF-P, to modulate their functional activity  (Iozzo & Murdoch, 1996) or hydroxyapatite (Bosky et al, 1997). Its glycosaminoglycan components have been shown to bind Ca++ and interact with hydroxyapatite (Embery et al 1998) and are present i n pre-mineralized tissues such as osteoid (Robey 1996) and predentin (Takagi et al 1990; Yoshiba et al 1996). Biglycan is also suspected o f being a repressor o f amelogenin expression and enamel formation (Goldberg et al, 2002). A l o n g with the above-mentioned interactions, bliglycan also binds to C q l and therefore may play a role in inflammation (Cox et al, 1970).  Like decorin, biglycan binds  TGF-P but with slightly different mechanisms. Through it's  binding, biglycan modulates the activity o f  TGF-P (Hildebrand et al, 1994). Although  how this modulation is carried out is unclear, it may be through sequestration o f the  TGF-  P in extracellular matrices (Hildebrand et al, 1994).  X u et al (1998) showed i n a study o f biglycan knockout mice that those deficient i n biglycan presented with an osteoporotic phenotype characterized by reduced growth rate and decreased bone mass (decreased amount and density o f trabecular bone and decreased  cortex  thickness  confirmed  with  high  resolution  radiographic  and  microradiographic techniques) that becomes more obvious with age. These findings were associated with a relative decrease i n osteoblastosteoclast ratio, attributing the reduction of bone mass to decreased formation o f bone. Previous studies have shown that biglycan levels may be related to stature in humans. Patients with Turners syndrome ( X O ) have  16  short stature and have decreased levels o f biglycan whereas patients with increased number o f sex chromosomes have increased limb length and increased levels o f biglycan (Vetter, 1993; Geerkens et al, 1995).  Periodontal Ligament Associated Protein-1 (PLAP-1) P L A P - 1 was discovered by Yamada et al (2001) i n freshly isolated human periodontal ligament cells using expression profiling techniques. P L A P - 1 m R N A expression was confirmed in vitro-maintained periodontal ligament cells. P L A P - 1 was classified as a new member o f the class I small leucine-rich proteoglycan family based on the alignment of its amino acid sequences. P L A P - 1 is unique from other members o f this group, however, in that it does not contain any glycosaminoglycan binding sites (making it not a real proteoglycan per se) and it has a characteristic stretch o f 16 aspartate acid residues at the N-terminal that may contribute to its function, which is presently unknown.  1.5.2 Class II Small Leucine-Rich Proteoglycnas Fibromodulin Fibromodulin, a keratan  sulfate proteoglycan, was first recognized as a 59-kDa  glycoprotein i n cartilage. It has since been located in cartilage, sclera, tendon (Plaas et al, 1990) and periodontal ligament (Watanabe et al, 1998) and is expressed by fibroblasts isolated from human periodontium (Hakkinen et al, 1996). Degradation products, although not found i n normal tissues, are apparent in arthritic cartilage, with a greater extent in rheumatoid arthritis joints than in osteoarthritis (Neame & K a y , 2000). The fibromodulin gene is located on human chromosome 12q (Sztrolovics et al, 1994).  17  Structurally, fibromodulin appears in different forms that seem to be age and location dependent.  The  leucine-rich  repeat  region  of  fibromodulin  has  potential  glycosaminoglycan binding sites allowing it to appear as a proteoglycan i n immature cartilage. However, fibromodulin may also appear as a glycoprotein, i n its unsulfated form, such as i n the case o f mature cartilage (Roughly et al, 1996). For this reason, fibromodulin has been described as a "part-time" proteoglycan. The molecule can contain up to 4 karatan sulfate chains (Plaas et al, 1990) and its structure bears much resemblance to lumican (Blochberger et al, 1992), another member o f this group. Fibromodulin form is location-dependent as well. In tracheal cartilage, for instance, the glycosaminoglycan chains  are  5-7  disaccharides  long whereas  in articular  cartilage,  they  are  8-9  disaccharides i n length (Lauder et al, 1996).  The function o f fibromodulin is suspect but revolves around its collagen-binding capacity implicating it i n collagen fibril formation (Blochberger et al 1992). Fibromodulin has been found to inhibit collagen fibrillogenesis (Hedbom & Heinegard, 1989) and has been shown to bind to collagen types I and II (Hedbom & Heinegard, 1993). Aside from its role i n collagen binding and collagen fibrillogenesis, fibromodulin appears to play a role in cell migration (Wilda et al, 2000) perhaps v i a its an association with fibronectin. A s well, fibromodulin has been shown to bind TGF-P via its protein core. In fact, this binding capacity appears to be stronger than the binding affinity between decorin or biglycan and T G F - p (Hidebrand et al, 1994).  18  Lumican Lumican is a keratan sulfate proteoglycan (Iozzo, 1997) whose gene, like that o f decorin, is found on the distal end o f mouse chromosome 10 (Chakravarti & Magnusson, 1995) and on human chromosome 12q21.3-22 (Chakravarti et al, 1995). Aside from bovine cornea, where it was first localized (Axelsson et al, 1978), lumican is located in corneal stroma o f chicken (Blochberger et al, 1992), heart valves (Funderburgh et al 1986), skin, skeletal muscle, intervertebral discs (Chakravarti & Magnuson, 1995), cartilage (Grover et al, 1995; Knudson & Knudson, 2001), cementum (Cheng et al 1996), predentin and dentin (Hall et al, 1997), bone (Raouf et al, 2002) but is scarce i n or absent i n brain, liver and spleen (Funderburgh et al, 1997).  Structurally, the amino acid terminals o f lumican may be sulfated (Oldberg et al, 1989) and the leucine-rich repeat region possesses potential glycosaminoglycan binding sites. The molecule is very similar in sequence homology in bovine, human, chick and mouse (Funderburgh et al, 1995; Grover et al, 1995; Funderburgh et al, 1993; Blochberger et al, 1992; Y i n g et al, 1997). The presence o f the sulfated form in the cornea implies that in this tissue it has specialized function while its role in other tissues seems elusive ( Y i n g et al, 1997). Lumican exists as a small leucine-rich proteoglycan in the cornea but may also appear as a glycoprotein in other tissues (Grover et al, 1995).  Lumican plays an important role in the maintenance o f corneal transparency, which is where it received its name (Blochberger et al, 1992). During mouse  development,  lumican appears at day 7 and subsequently appears in many organ systems with highest  19  concentration i n the heart and eyes (Oldberg et al, 1989). Initially i n the eye, the lumican is unsulfated. The appearance o f sulfated forms o f lumican in the eye is correlated with the development o f corneal transparency at day 15 (Funderburgh et al, 1986). Studies in mice that are missing lumican show that the mice lose their corneal transparency at 10 weeks indicating that lumican is essential for the maintenance but not initiation o f corneal transparency (Chakravarti et al, 1998). It is believed that lumican, like fibromodulin and decorin, acts v i a its collagen-binding capacity and its ability to control the collagen fibril diameters and interfibrillar spacing between them (Blochberger et al, 1992). In the cornea, the protein core o f lumican acts to limit collagen fibril diameter while its glycosaminoglycan side chains act i n the regular spacing o f fibrils and the acquisition o f corneal transparency  (Funderburgh et al, 1986; Cornuet et al, 1994; Scott, 1991).  Lumican also plays a role in corneal hydration due to its negatively charged carbohydrate components (Rawe et al, 1992; Bettelheim et al, 1975; Funderburgh et al, 1991).  In addition, lumican appears to play a role in the acquisition or maintenance o f skin tensile strength. Lumican knockout mice (homozygous mutation) show an 86% decrease in tensile strength o f skin, a finding that is correlated with irregular collagen fibril morphology and diameter, much the same as that seen i n decorin knockout mice (Chakravarti et al, 1998).  Osteoadherin/Osteomodulin Osteoadherin was first isolated from mineralized matrix o f bovine bone (Wendel et al, 1998). Called osteoadherin i n bovine tissue and osteomodulin in human and rat tissues, it  20  has actually been found to represent the same protein in different species (Buchaille et al, 2000). Since its discovery, osteoadherin has been found via in-situ hybridization studies in human and rat odontoblasts, i n the ameloblastic layer o f developing rat teeth and expressed by cells o f alveolar bone surrounding the teeth (Buchaille et al, 2000). This finding was consistent with the finding o f osteoadherin i n bovine bone through in vitro hybridization (Sommarin et al, 1998).  Although  structurally  similar  to  fibromodulin, lumican, keratocan,  and  PRELP,  osteoadherin is not closely related to them. It has a very acidic C-terminal peptide extension that clearly distinguishes it from the other mentioned proteoglycans above which may act to anchor the proteoglycan to the hydroxyapatite mineral in bone (Sommarin et al, 1998).  Although osteoadherin appears to be rather specific for bone, its function remains unclear. A s previously stated, it is an acidic small leucine-rich proteoglycan that binds to hydroxyapatite and may play a role in bone mineralization (Sommarin et al, 1998). A s well it appears to be involved in cell-matrix adhesion and binds to osteoblast integrin oc p ( W e n d e l e t a l , 1998). v  3  1.5.3 Localization Of Small Leucine-Rich Proteoglycans In Soft Oral Tissues Table 3 lists the components o f soft dental tissues. Inspection o f this table shows that each tissue contains cellular and acellular elements. The most predominant member o f the acellular elements is collagen. Various collagen types exist in the various dental  21  tissues (Table 4). The type o f collagen ultimately determines the nature o f the tissue. A l o n g with collagen, one finds various other components, notably proteoglycans.  O f the proteoglycans, a few small leucine-rich proteoglycans have been associated with oral tissues. Limited research exists in the field o f small leucine-rich proteoglycans in oral tissues, but with advances in laboratory techniques, one predicts that several more w i l l come to light. The following section discusses the small leucine-rich proteoglycans that have been found to be associated with soft dental tissues or their development to date.  L i n i n g M u c o s a and G i n g i v a The gingiva is the soft tissue that covers the alveolar process and surrounds the necks o f the teeth and hard palate while the lining mucosa is the soft tissue lining o f the remainder of the oral cavity (except the tongue). While both the gingiva and lining mucosa are comprised o f a top layer o f epithelium and an underlying layer o f connective tissue, they are unique oral tissues with different functions.  The gingiva can be divided into marginal, attached and interdental areas. The marginal gingiva is the unattached collar o f tissue around the teeth and has an outer (oral) surface (facing the oral cavity), an inner (sulcular) surface (facing the tooth) and a joining (junctional) surface attached to the tooth. The attached gingiva is continuous with the marginal gingiva and is bound tightly to the underlying alveolar bone. Gingiva is comprised o f a surface layer o f stratified squamous epithelium that tends to be keratinized in the oral extent and non-keratinized in the sulcular and junctional areas.  22  While many cell types are found i n the epithelium, the main cell type is the keratinocyte. Four cell layers (stratum) can be identified (basale, spinosum, granulosum, corneum) in keratinized epithelium, much the same as i n skin, while nonkeratinized epithelium presents with slightly different cell layers superficially (basale, spinosum, intermedium and superficiale) (Ten Cate, 1994).  Immediately under the epithelium is the basal lamina, sometimes referred to as the basement mambrane. The basal lamina consists o f two zones o f relatively equal thickness (45-50nm), the lamina lucida and the lamina densa. The lamina lucida is closest to the epithelium and appears as a structureless band in H&E-stained sections but there are slight condensations o f material present opposite the hemidesmosomes on the epithelial cell membrane called anchoring filaments as well as glycoproteins including bullous pemphigoid antigen. The lamina densa is between the lamina lucida and underlying connective tissue. The lamina densa consists o f type I V collagen in a chicken wire configuration, laminin, entactin, and proteoglycan rich in heparin sulfate. The lamina densa has anchoring fibrils inserting into it made o f type V I I collagen which extend into the underlying connective tissue through which type I and III collagen run on the connective tissue side, anchoring the epithelim to the connective tissue. Fibronectin and type V collagen are sometimes associated with the connective tissue surface o f the basal lamina. The primary role o f the basal lamina is to provide physical support to the epithelium. It also provides a surface for cell attachment and around endothelium, provides a filtration role (Ten Cate, 1994).  23  Underlying the epithelium is a layer o f connective tissue or lamina propria. The lamina propria can be divided into a more superficial papillary layer that is closest to the epithelium and a deeper reticular layer (so named for the netlike arrangement o f the collagen fibers here) that is contiguous with the periosteum o f the alveolar bone. The difference between these layers is poorly defined but is based on relative concentration and arrangement o f the collagen fibers. In the papillary layer the connective tissue fibers are thin and loosely arranged and many capillary loops are present. The reticular layer, on the other hand, has thick collagen fiber bundles that lie parallel to the surface plane. The lamina propria is comprised o f cells, blood vessels, neural elements, and fibers in an amorphous ground substance. The main cell type o f the lamina propria is the fibroblast while the main extracellular matrix component is collagen type I (Ten Cate, 1994).  Type I collagen fibers, which account for approximately 60% o f the gingival protein, although ultimately blended, can be divided into groups based on their location and orientation. They are named dentogingival, alveologingival, circular, periostogingival, transeptal group, transgingival, interpapillary, intercircular, and intergingival fibers (Fig. 4). These fiber groups act to attach the gingiva to the tooth and give rigidity and structure to the gingival margin to withstand forces o f mastication. Other important components o f the connective tissue layer include non-cellular elements such as: collagen type I V and V oxytalin  fibers,  eluanin  fibers,  elastin  fibers,  glycoproteins,  glycosaminoglycans,  proteoglycans, and cellular elements (8% by volume) like: fibroblasts (65% o f the cellular component) and blood cells including inflammatory cells. A more extensive  24  review on the histology o f the mucosa can be found i n Ten Cate (1994), Itoiz & Carranza (1996) and Holmstrup(1996).  Small leucine-rich proteoglycans were first noted in gingival tissues by Pearson & Pringle  (1986).  Their  work  in  bovine  gingiva  using  chromatography  and  immunochemistry techniques determined that there were small and large proteoglycans in gingival tissues. The small one was a dermatan sulfate proteoglycan that later was determined to be decorin (Rahemtulla, 1992).  Later, research localized decorin i n human gingival connective tissues. Hakkinen et al (1993) showed using immunohistochemical techniques to detect the protein core o f decorin that it was found throughout the human gingival connective tissue. Decorin staining was more intense i n the papillary regions o f the connective tissue than the reticular regions and it was found to be associated with collagen fiber bundles. This finding is similar to those found in human dermis (Bianco et al, 1990; Voss et al, 1986; Fleischmajer et al, 1991) and is supported by studies showing that papillary fibroblasts secrete more decorin than reticular fibroblasts in vitro (Schonherr et al, 1993) and in vivo (Schonherr et al, 1995). The increased intensity o f the decorin immunoreactivity in the subepithelial regions o f the connective tissue is not surprising i f one considers that decorin has been shown to bind to collagen types I-VI in vitro with the highest affinity for collagen type V I (Bidanset et al, 1992) which is highly concentrated in this subepithelial area (Chavrier et al, 1984; Becker et al, 1986; Rabanus et al, 1991). The localization o f decorin and its ability to regulate collagen fiber assembly (Vogel et al,  25  1984) i n other tissues, suggests that the decorin plays a similar role in gingival tissues. Decorin was also shown to stain intensely in the basement membrane zone, again similar to dermis (Voss et al, 1986) suggesting a role for decorin at this interface (Hakkinen et al, 1993).  Hakkinen et al (1993) showed in the same study that biglycan is weakly expressed in adult human gingival tissues showing some accumulation in the connective tissues under the epithelial rete ridges where it was localized as fine filaments on extracellular matrix fibers suggesting a role o f biglycan in collagen fibrillogenesis. In fact, biglycan can potently interact with type V I collagen that in turn interacts with type I collagen (Wiberg et al, 2002). Although biglycan is found i n developing epithelium o f human dermis (Bianco et al, 1990) and i n gastric epithlium (along with decorin) (Pohle et al, 2001), it was not (nor was decorin) localized in the epithelium o f adult human gingival epithelium (Hakkinen et al, 1993). The role o f biglycan i n human periodontal tissues is not clear at present.  Periodontal L i g a m e n t The periodontal ligament is that soft connective tissue found occupying the space between the cementum o f the tooth roots and the alveolar socket wall. While its most obvious role is to attach the teeth to bone, it has several other functions that vary from physical to formative to nutritional and sensory. Physical functions o f the periodontal ligament include transmission o f occlusal forces to the surrounding bone, protection to the blood vessels and nerves within it from masticatory forces, formative functions  26  include normal remodeling o f bone and cementum and regeneration, while it also plays roles in providing nutrition and lymphatic drainage to surrounding tissues and sensation in the way o f proprioception, pain and pressure (Carranza & Ubios, 1996).  The periodontal ligament is comprised o f cellular components, an extracellular matrix, blood vessels and nerves. The different cell types found i n the periodontal ligament include fibroblasts, epithelial cells, undifferentiated mesenchymal cells, endothelial cells, cells associated with the sensory system, bone associated cells and cementoblasts. The predominant cell type is the fibroblast, which i n rodent molars makes up approximately 35% o f the periodontal ligament volume (excluding blood vessels) (Beertsen, 1975) and in sheep incisors comprises approximately 20% (Berkovitz & Shore, 1995) o f the periodontal ligament volume. It is estimated that an adult human premolar would have a comparable cell density o f 25% i n the periodontal ligament (Beertsen et al, 1997). Fibroblasts are spindle-shaped and are oriented with their long axes parallel to the principle collagen fiber bundles. Their potential role includes the  synthesis  and  degradation o f collagen (Holmstrup, 1996) as well as the formation o f other extracellular matrix components.  The extracellular matrix o f the periodontal ligament is comprised o f collagen fibers, elastic fibers (oxytalan and eluanin) and ground substance (Ten Cate, 1994). Type I collagen, which forms  approximately  80% o f the  periodontal  ligament collagen  (Holmstrup, 1996) and type III collagen are the two most common types o f collagen found i n the periodontal ligament (Huang et al, 1991; Wang et al, 1980) in an  27  approximate 4:1 ratio (Butler et al, 1975) and they form banded fibrils. Collagen V is also found in minor amounts and is associated with collagen types I an III (Linsenmayer et al, 1983) found either buried within the fibril cores (Birk et al, 1988) or between the fibril bundle spaces (Becker et al, 1991). Several other minor collagens are found in the periodontal ligament including V I and X I I (Becker et al, 1991; Bronckers et al, 1986; Dublet et al, 1988; Karimbux et al, 1992; Sloan et al, 1993).  The collagen fibers o f the periodontal ligament, like those o f the gingiva, are arranged i n collagen fiber bundles and denoted the principle fiber groups (Fig. 5). These principle fibers occupy much o f the space between the tooth root and alveolar bone and are inserted into the tooth as Sharpes' fibers. The principle fibers are wavy in their course and as such are able to absorb applied forces. The fibers can be divided into groups including alveolar crestal, oblique, transeptal, horizontal, interradicular, and apical groups (Holmstrup, 1996).  Among the principle fiber groups one finds loose connective tissue in which cells, fibers (secondary, reticular, elastic), vessels and nerves are embedded. Other proteins o f the extracellular matrix include proteoglycans (Hakkinen et al, 1993; Embery et al, 1995) and glycoproteins (Zhang et al, 1993).  A m o n g the proteoglycans found in the periodontal ligament, are the small leucine-rich proteoglycans.  Few  proteoglycans  in  the  investigators periodontal  have  attempted  ligament.  28  Early  to  identify  research  small leucine-rich mainly  focused  on  glycosaminoglycan  components  of  connective  tissues  with  the  most  common  glycosaminoglycans found in periodontal ligament being hyaluronan, chondroitin 4sulfate, chondroitin 6-sulfate and dermatan sulfate (Munemoto et al, 1970). The small leucine-rich proteoglycans that have been isolated thus far in the periodontal ligament include decorin, biglycan, fibromodulin, lumican and P L A P - 1 , although not all have been investigated.  Decorin was first localized in the periodontal ligament by Pearson and Gibson (1982) and later by Pearson & Pringle (1986) who in animal studies noted that decorin o f the periodontal ligament is immunologically related to that o f skin, pulp and gingiva. Human and animal studies using immunolocalization techniques determined that decorin is associated with periodontal ligament collagen fibers both in the periodontal ligament proper (Hakkinen et al, 1993; Cheng et al, 1999) and the periodontal ligament fibers (Sharpey fibers) that insert into cementum (Ababneh et al, 1999). The function o f decorin in the periodontal ligament was investigated using a murine model where decorin knockout mice were compared to wild-type control mice. It was shown that mice deficient in decorin have increased numbers o f fibroblasts and altered collagen fibrils characterized by heterogenous shape and irregular profiles (Hakkinen et al, 2000). Thus it appears that decorin plays a role i n periodontal ligament collagen fibrillogenesis.  The other small leucine-rich proteoglycans mentioned above have been localized in the periodontal ligament but their functions have yet to be determined. Biglycan was localized i n human periodontal ligament using immunolocalization techniques (Hakkinen  29  et al, 1996; Abebneh et al, 1999) but was found to be less abundant than decorin in this tissue (Hakkinen et al, 1996). Fibromodulin was first localized in the periodontal ligament in animal studies using ion-exchange chromatography and electrophoresis as well as immunostaining (Watanabe et al, 1998) confirming previous in vitro reports that showed fibroblasts of the periodontium express fibromodulin (Hakkinen et al, 1996). As well, lumican was localized in the human periodontal ligament using standard indirect immunoperoxidase techniques and it was found to be associated with the collagen fibers inserting into cementum (Ababneh et al, 1999). Another small leucine-rich proteoglycan that appears to be unique to the periodontal ligament was isolated in human tissue using gene-profiling techniques. This small leucine-rich proteoglycan, named periodontal ligament associated protein -1 (PLAP-1) was found to be structurally similar to the class I small leucine-rich proteoglycans and appears to play a role in mineralization through its role in cytodifferentiation processes (Yamada et al, 2001).  Pulp The pulp is the soft connective tissue layer found in the center of the tooth which can be divided into four distinct zones at the histological level: the odontoblastic zone at the periphery, a cell free zone (the basal layer of Weil) under the odontoblastic zone, a cellrich zone containing mainly fibroblasts, and the pulp core that containing the major blood vessels and nerves of the pulp (Torneck, 1994). The cells of the pulp include odontoblasts,  fibroblasts,  undifferentiated  mesenchymal  cells,  as  well  as  immunocompetent cells, while collagen and ground substance are found extracellularly. The collagen component of the pulp is concentrated greatly in the apical third and is  30  comprised mainly o f types I and III in an approximate ratio o f 55:45. The ground substance o f the pulp is comprised o f glycosaminoglycans, glycoproteins,  water  (Torneck, 1994) and proteoglycans. A more extensive review on the histology o f the pulp can be found i n Torneck, 1994.  H a l l et al (1997) found using immunohistochemical techniques that lumican was weakly expressed throughout the pulp matrix and around odontoblasts in a fibrillar pattern. Later, Buchaille et al (2000) using in situ hybridization techniques, showed that osteoadherin (osteomodulin) m R N A is located in the pulp o f developing rat teeth, more highly concentrated i n the odontoblastic layer within it. Osteoadherin was also found in human odontoblasts lining the edge o f the pulp chamber i n partially developed human molars. It is believed from these studies that osteomodulin and lumican play a role in mineralization of hard dental tissues.  1.5.4 Localization Of Small Leucine-Rich Proteoglycans In Hard Oral Tissues The role o f proteoglycans in mineralized tissues has been underestimated in the past and it is only recently with the advent o f new laboratory techniques and the use o f genetically engineered mice that the importance o f these molecules in mineralized tissues is being elucidated. Perhaps the reason for the underestimation in the past is due to the apparent negligible quantity o f these molecules in the mineralized tissues (Jones and Leaver, 1972) and that in vitro studies showed that the amount o f glycosaminoglycans decreases as mineralization progresses (Baylink et al, 1972). In vitro, proteoglycans have been shown to have an inhibitory effect on hydoxyapatite formation and growth. However, since then  31  it has been shown that in solution, proteoglycans inhibit mineral nucleation and growth whereas on a surface, the same molecules promote mineral formation (Linde et al, 1989). Table 5 lists components o f dental hard tissues. A discussion o f small leucine-rich proteoglycans in these tissues follows.  Dentin / Predentin Dentin is the major mineralized tissue o f teeth and is closely related to bone in both chemical composition and mineralization process. There are several types o f dentin. The bulk o f dentin in the mature tooth is primary dentin. It outlines the pulp chamber and is characterized by multiple closely packed dentinal tubules  that traverse its  entire  thickness. Dentinal tubules contain the cytoplasmic processes o f the odontoblast cells that line the pulp space. Mantle dentin is the first dentin deposited by odontoblasts during tooth development and is found lining the outermost portion o f the primary dentin o f mature teeth. Secondary dentin, which is less organized than primary dentin, develops upon the completion o f root development. It is deposited at a slow rate by odontoblast cells lining the pulp chamber and results in a decreased pulp space. Tertiary dentin, also called reparative dentin, is produced in very localized areas in response to noxious stimuli. Its structure can be regular or irregular in nature depending on the nature and intensity o f the stimulus. Tertiary dentin is comprised o f collagen types I and III.  Mature dentin is made up o f 70% inorganic material, 20% organic material and 10% water by weight (45%, 33% & 22% by volume). Table 5 shows that the inorganic component is comprised o f hydroxyapatite while the organic portion is made up o f  32  collagen (mainly type I), glycoproteins, proteoglycans, phosphoproteins, and plasma proteins. Approximately 56% o f the mineralized phase is within the collagen. A more extensive review o f the histology o f dentin can be found in Torneck, 1994.  It has been suggested that there are two distinct groups o f proteoglycans i n dentin. The first one is i n predentin where glycosaminoglycans function to move collagen toward the mineralization front and where fibrils are packed and undergo mineralization. It is here that the small leucine-rich proteoglycans perhaps aid in fibrillogenesis, linking subunits and orienting collagen fibrils (Scott, 1996). The second group is in the dentin proper where glycosaminoglycans are secreted  distally by odontoblastic processes within  dentinal tubules at some distance from the dentin/predentin junction (Scott, 1996).  In the 1970's and 80's, several glycosaminoglycans have been found to be associated with dentin and predentin including heparan sulfate, dermatan sulfate, chondroitin 6sulfate, (Goldberg & Septier, 1986; Tenorio et al, 1990; Tagaki et al, 1990; Goldberg and Tagaki,  1993)  chondroitin 4-sulfate  (Embery,  1974;  Smalley & Embery,  1980;  Rahemtulla et al, 1984), hyaluronic acid (Clarke et al, 1965) and keratan sulfate (Linde, 1973). Only recently have studies been able to identify the actual proteoglycan to which these glycosaminoglycans probably belong.  Lumican was first discovered in human predentin and dentin by H a l l et al (1997) using immunohistochemical procedures. Lumican was located in predentin and, to a lesser extent, dentin. It was suggested that lumican might play a role in the mineralization  33  process o f dentin. Linde's finding o f keratan sulfate in dentin supports these findings ( L i n d e e t a l , 1973).  Embery et al (2001) found decorin and biglycan to be the most abundant small leucinerich proteoglycans in dentin with lumican and fibromodulin found in lesser amounts. In this study, immunohistological techniques and electron microscopy were used to show that antibodies recognizing chondroitin sulfate showed a decreasing gradient from pulp to mineralization front o f dentin with an opposite gradient occurring for keratan sulfate family o f small leucine-rich proteoglycans. Anti-decorin antibodies showed an increasing gradient toward the mineralization front. It has been suggested that these proteoglycans act to organize the collagen network to receive phosphoproteins and phospholipids as well as the possibility o f the spatially oriented glycosaminoglycan o f decorin and biglycan binding Ca++ and directing mineralization initiation.  Studies have been carried out on mice that have been genetically engineered to be deficient i n genes that code for one or two small leucine-rich proteoglycans. These studies suggest functional roles o f small leucine-rich proteoglycans. Studies comparing biglycan knockout and wild-type mice show that collagen fibrils i n predentin  are  decreased in the proximal third but increased in the central and distal third o f predentin in the knockout mice compared to w i l d type mice. This, together with the knowledge that small leucine-rich proteoglycans control collagen fibril diameter and assembly, confirms the role o f biglycan as a regulator o f extracellular matrix organization (Goldberg et al, 2002).  34  Cementum Cementum is a mineralized tissue that covers the roots o f teeth which facilitates attachment o f periodontal ligament collagen fibers to the tooth. Cementum can be divided into two types: acellular extrinsic fiber cementum  and cellular mixed  stratified  cementum. The latter is composed o f 3 intermingling types o f tissues: cellular intrinsic fiber  cementum,  acellular intrinsic fiber cementum  and  acellular extrinsic fiber  cementum. Cementum consists o f mineralized organic matrix, the primary constituent o f which is type I collagen with types III, I V and V I collagen present in minor amounts. A s well, a number o f non-collagenous proteins have been identified within cementum such as acidic glycoproteins, growth factors, attachment proteins and proteoglycans (Ababneh etal, 1999).  Early biochemical research by Bartold (1988) showed that the major glycosaminoglycan in human cementum was chondroitin sulfate while hyaluronic acid and dermatan sulfate were present i n small amounts. Later, immunohistochemical research localized the chondroitin sulfate proteoglycan to the pericellular environment and the extracellular matrix o f cementum (Bartold et al, 1990). A s well, Rahemtulla et al (1984) noted the presence o f two groups o f proteoglycans in hard tooth tissue (dentin and cementum) consisting i f chondroitin 4-sulfate and chondroitin 6-sulfate. N o mention was made in these studies regarding the core proteins o f the proteoglycans or the sulfation o f the molecules. These proteoglycans were later identified as decorin and biglycan (Cheng et al, 1999).  35  Cheng et al (1996) showed using immunohistological techniques that fibromodulin and lumican core proteins are localized i n cementum and precementum, on the walls o f lacunae and on cementocytes o f bovine teeth. Later, Cheng et al (1999) showed, using biochemical and immunochemical as well as immunostaining techniques, that biglycan core proteins are located in bovine cementoblasts and bovine precementum. Decorin was found to be mainly associated with bovine periodontal ligament collagen fibers and was also found i n the bovine cementum matrix. The differential tissue distributions o f the various small leucine-rich proteoglycans in cementum suggest that they play distinct roles in cementogenesis.  Cheng et al (1996, 1999) suggested that lumican, fibromodulin, biglycan and decorin play a role as inhibitors o f the mineralization o f cementum due to their specific location in the non-mineralized parts o f cementum. This suggestion is supported by studies that showed mineralization taking place in dentin upon removal o f the proteoglycans and those that show a decreasing proteoglycan content associated with mineralization and dentinogenesis (Tagaki et al, 1990; H a l l et al, 1997). However, other studies have shown that when immobilized on a solid surface, some proteoglycans act as promoters o f calcification (Lussi & Linde, 1993; Hunter & Weinert, 1996). In addition, it has been noted that the molecular weight o f biglycan in cementum is significantly less in cementum and this decreased molecular weight is due to smaller glycosaminoglycan side chains, the significance o f which remains to be determined (Cheng et al, 1999).  36  Human studies o f small leucine-rich proteoglycans i n cementum are few. Using a standard indirect immunoperoxidase technique on freshly extracted human permanent teeth, it was shown that decorin, lumican and biglycan are components o f the extracellular matrix o f cellular but not acellular cementum. They were found on cementocytes and at the borders and lumina o f lacunae and canaliculi surrounding the cells as well as associated with periodontal ligament fibers entering cellular and acellular cementum and by cementoblasts lining the root surface. Fibromodulin, on the other hand, could not be demonstrated in either human cementum type, unlike in bovine cementum, except associated with Sharpes' fibers that were anchored i n the cementum. Moreover, it was determined i n this controlled comparative study between healthy and periodontally infected teeth that neither age nor periodontal disease appeared to qualitatively influence the proteoglycan population o f cementum (Ababneh et al, 1999).  Bone  Holmstrup (1996) reviews alveolar bone microanatomy. Alveolar bone is that bone that surrounds and supports the teeth in the jaws. L i k e cementum, it contains blood vessels and nerves and is mineralized. Osteoblasts are the cells responsible for bone formation and they secrete osteoid, which later becomes mineralized. U p o n mineralization, osteoblasts become surrounded by mineralized tissue and become osteocytes. Small canals called canaliculi connect osteocytes and osteoblasts to each other. Osteoid is the precursor form o f mineralized bone and is comprised o f collagen (mainly type I with small amounts o f III and I V ) , glycoprotein and proteoglycans. Once secreted osteoid  37  becomes bone, nutrition is achieved through blood vessels that penetrate the bone through Haversian canals that are the center o f osteons in bone.  Mature bone matrix is dominated by mineral in the form o f hydroxyapatite (Table 5). The mineral crystals are aligned along the type I collagen fibrils, the predominant organic component in this tissue. The extracellular matrix o f bone also contains small chondroitin sulfate proteoglycans like decorin and biglycan as well as non-collagenous proteins (Sommarin e t a l , 1998).  The first small leucine-rich proteoglycans to be localized in bone were decorin and biglycan (Franzen and Heinegard, 1984; Fisher et al, 1989). Very little information is known about the proteoglycans o f alveolar bone as most research i n bone has been done on long bones from various species (Rahemtulla, 1992). A n i m a l studies in rabbit alveolar and basal bone have reported the presence o f chondroitin 4-sulfate and keratan sulfate in both bone types with a 1:1 ratio i n alveolar bone and 2:1 in basal bone (Waddington et al, 1988). In a later report, Waddington et al (1989) noted that chondroitin 4-sulfate made up 94% o f human alveolar bone with hyaluronic acid, dermatan sulfate and heparan sulfate present in minor amounts. These findings were later confirmed by Bartold et al (1990) using immunohistochemical techniques on rabbit, pig and human alveolar bone.  Osteoadherin has been localized as a minor leucine- and argenine-rich keratan sulfate proteoglycan found i n the mineralized matrix o f bone. It has been shown to bind osteoblasts v i a the  avP3 integrin i n a cation-dependent mechanism (Sommarin et al,  38  1998).  Buchaille et al (2000) also found osteoadherin in cells o f the alveolar bone  surrounding developing rat teeth.  The importance o f the small leucine-rich proteoglycans is highlighted in the recent studies o f biglycan knockout mice. X u et al (1998) developed a biglycan knockout mouse line to study biglycan's effect on development. M i c e deficient for biglycan gene were born normally but displayed a phenotype characterized by a reduced growth potential and reduced bone mass due to decreased bone formation that worsened with age (discussed above). Goldberg et al (2002) found in a study o f biglycan knockout mice that day-1 mandibles revealed the presence o f large unmerged nodules with large interglobular spaces in the bone between them, supporting the findings o f the osteoporosis phenotyope reported by X u et al (1998). Deletions i n two other connective tissue genes that are highly expressed in bone (decorin and osteonectin) failed to show similar phenotypes (Danielson et al, 1997; Gilmour et al, 1998) attesting biglycan's role in normal bone development. While the precise function o f small leucine-rich proteoglycans in bone remains to be elucidated, it is clear from these gene knockout studies that they indeed play a role in bone development and mineralization.  Enamel Enamel, the translucent hard tissue that covers the crowns o f teeth, has no collagen component and as such has a matrix completely different than bone and dentin. Enamel is composed o f unique proteins like amelogenin, ameloblastic enamelin and tuftelin (Ten Cate, 1994).  39  Buchaille et al (2000) were the first to show a small leucine-rich proteoglycan associated with enamel when they showed that osteroadherin is located in the ameloblastic layer i n developing rat teeth.  Goldberg et al (2002) i n studies comparing biglycan knockout mice and wild-type mice noted that biglycan knockout mice had enamel that was 3-5x thicker than wild-type mice. Instead o f the expected enamel rods, tubule-like structures were seen filled with stippled material and protracted Tomes' processes i n the outer enamel. It was suggested by the authors, after immunostaining for amelogenin, that the biglycan acts as a repressor o f amelogenin formation by ameloblasts and odontoblasts and this repression allows for normal enamel formation. Decorin and fibromodulin knockout mice did not develop the same abnormal enamel phenotype. Enamel formation in decorin knockout mice was repressed in the molars and aprismatic in the incisors while fibromodulin knockout mice appear to have normal enamel formation.  1.5.5 Localization Of Small Leucine-Rich Proteoglycans In Other Tissues Temporomandibular Joint (TMJ) Disc  The T M J disc is a fibrous connective tissue structure intervening between two bony components, the mandibular condyle and the glenoid fossa o f the temporal bone. It accommodates the biomechanical forces evoked by orofacial functions and facilitates movement o f the condyle-disc complex. Collagens and proteoglycans are prominent members o f the extracellular matrix in the T M J disc.  40  It has been shown previously that the distribution o f decorin and biglycan is different in the peripheral and central areas o f adult T M J discs in both bovine (Scott et al, 1989, 1995b) and rat (Mizoguchi et al, 1998) models. More recently, decorin and biglycan were analyzed using biochemical and immunolocalization techniques in extracellular matrix o f the T M J disc o f growing rats. It was shown that there are growth related-changes and regional differences i n the expression o f biglycan and decorin in the T M J disc o f growing rats. These changes may be a reflection o f changes in the biochemical environment caused by the development o f orofacial functions with decorin dramatically increasing with age and biglycan decreasing with age. It is interesting to note that during these changes i n proteoglycans, there is no real change reflected in glycosaminoglycan content (dermatin sulfate i n this case) in the T M J disc. A s well, decorin was shown to be more abundant in areas that undergo more tensile loading and biglycan more abundant in areas that undergo more compressive loading reflecting a difference in function o f these two proteoglycans (Kuwabara et al, 2002).  1.6 Collagen: A Basic Constituent Of Oral Tissues Collagen is a very important and ubiquitous structural component o f connective tissue extracellular matrices and provides the shape and form to many tissues and a surface to which many macromolecules, glycoproteins, polymers, inorganic ions and cells attach (Kadler, 1995).  There are more than 20 distinct types o f collagen in animal tissues (Kadler et al, 1996). The different collagens have different structures and can be generally classified into  41  fibrillar, non-fibrillar and F A C I T (fibril-associated collagens with interrupted triple helices) types. The non-fibrillar types may occur as net-like sheets underlying epithelial and endothelial cells or as fine filaments that anchor basement membranes to specialized structures in the skin. They may also occur as hexagonal lattices in cartilage and Descement's  membrane  providing a substrate for cell differentiation, proliferation,  migration and support (Kadler, 1995). However, fibrillar collagens (Types I, II, III, V , XI) make up the bulk o f all connective tissues in animals (Kadler, 1995; Kadler, 1996; Corsi et al, 2002) and are the focus o f the discussion on collagen herein. Table 6 lists the types o f collagen and their distribution i n dental and periodontal tissues.  The most abundant source o f fibrillar collagen is the dense connective tissue o f tendon, ligament, skin and bone. Here, fibrillar collagens are organized into fibrils, thicker fibers and  fiber  bundles.  Small  leucine-rich proteoglycans  play  a  role  in  collagen  fibrillogenesis. The rope-like fibers o f fibrillar collagen help maintain the integrity of these tissues (Kadler, 1995). Type I collagen is a fibrillar collagen and is the most abundant type found i n dental tissues (Ten Cate, 1994). Most research on collagen synthesis and fibrillogenesis is based on type I collagen.  1.7 Collagen Synthesis Collagen synthesis and fibrillogenesis is a highly regulated process (Fig. 6) consisting o f many steps and many players (Ten Cate, 1994; Kadler et al, 1995; Kadler et al, 1996). Collagen is comprised o f three polypeptide chains (alpha chains) that form a triple helix (Fig. 7). The alpha chains are constructed from repeating G l y - X - Y triplets, where X and  42  Y can be any amino acid but are frequently proline and hydroxyproline respectively. Glycine i n every third residue position o f each chain is a prerequisite for folding o f the three chains into the triple helix. The glycines are found facing the center o f the helix. The triple helix forms is a right-handed super helix that repeats every 30 residues. A more extensive review on the biosynthesis and fibrillogenesis o f collagen can be found in Kadler, 1995.  The synthesis o f individual collagen alpha chains is thought to occur as a two-step process: the first step occurring intracellularly and the second step occurring outside the cell (Fig. 6). Translation o f pre-procollagen molecules begins on free ribosomes, which later become associated with the endoplasmic reticulum in fibroblast cells. A m i n o acids on the ribosomes assemble to form the individual polypeptide chains. The chains first formed are substantially longer than those found in the final molecule because they contain amino acids at the N - and C - terminal ends that play important roles in the synthesis process and are later cleaved. Vitamin C-dependent enzymes act to hydroxylate the lysine and proline amino acids before helix formation begins. A s hydroxylation ensues, glucose residues are added to the molecules, a process known as glycosylation, another enzymatically-driven process. U p o n hydroxylation and glycosylation, the chains are aligned with the help o f disulfide bonds at the C-terminal ends and the triple helix formation begins. Once formed, the triple helix molecule is transported to the golgi apparatus, where it further remodels to form the procollagen molecule. Procollagen molecules are aligned i n a non-staggered configuration and are transported to the cell surface for excretion. The formation and excretion o f the procollagen molecule takes  43  approximately 35-60 minutes. Fibrillar collagens are formed as procollagens, which are excreted from the cell where it undergoes enzymatic processing at the N - and C - terminal ends to form collagen. The fragments then undergo a process known as fibrillogenesis, a self-assembly process.  1.8 Collagen Fibrillogenesis The process o f collagen fibrillogenesis is not fully understood (Kadler, 1995; Kadler 1996).  Since collagen fibrils  ultimately  determine the  architecture,  stability  mechanical attributes o f tissues, the details o f collagen fibrillogenesis are  and  important.  Determining how the process is regulated is important in understanding the assembly, function, pathology and healing o f connective tissues.  Once secreted, procollagen molecules are aligned extracellularly to form the typical banded collagen fibril (Fig. 8). Assembly o f fibrils is believed to be a self-assembly process driven by the amino acids that make up the molecule but is dependent on several types o f other molecules including other collagen types and proteoglycans, particularly the small leucine-rich proteoglycans (Kadler, 1995; Kadler, 1996; Ezura et al, 2000).  It is believed that there may be selective removal o f the C-terminal extension and partial removal o f the N-terminal extension o f the procollagen molecule that permits  the  alignment o f a five unit staggered microfibril. These microfibrils i n turn become aligned parallel to each other and staggered leaving a regular series o f gaps in the molecule. The size and shape o f the gaps is important since it is here that the minerals o f hard tissues,  44  such as bone and dentin and cementum, reside (Ten Cate, 1994). Once aligned, the remaining portion o f the N-terminus is lost and intermolecular bonding occurs that gives strength to the molecule. The bonding is comprised first o f hydrogen bonds with the development o f covalent bonds as the tissue matures (Ten Cate, 1994; Kadler, 1995).  Graham et al (2000) showed i n a study o f collagen fibril fusion i n tendon that fibroblasts synthesize transient early fibril intermediates  that fuse in an end-to-end fashion to  generate long fibrils. The intermediates were o f two types: unipolar fibrils, exhibiting a C - terminal and an N-terminal, and bipolar fibrils, exhibiting two N-terminals. End-toend fusion required the C-terminal o f the unipolar fibril intermediates. Small leucine-rich proteoglycans were noted to cover the long surfaces o f these fibrils but not the tips o f the fibrils, thus appearing to act as inhibitors o f lateral fusion. When small leucine-rich proteoglycans  were  absent,  the  fibril  intermediates  aggregated  laterally,  thus  demonstrating further that small leucine-rich proteoglycans promote end-to-end fusion o f collagen fibril intermediates and inhibit lateral fusion.  It has been proposed that collagen fibril growth in length and diameter occurs by accretion o f collagen fibril intermediates, the basic units in the growth o f fibrils (Ezura et al, 2000). In this model (Fig. 9), it is believed that fibril intermediates are stabilized through their interactions with fibril-associated macromolecules, such as the small leucine-rich  proteoglycans  lumican and  fibromodulin. The  fusion  o f the  fibril  intermediates generates a mature fibril in a multi-step manner. Progression through the growth process would occur through additive and like fusion. In this model, based on  45  studies o f mouse tendon i n lumican and fibromodulin and double knockout mice, it seems that lumican and fibromodulin have temporal functions associated with this process whereby  lumican functions  together with fibromodulin in the  early stages with  progressively less lumican and progressively more fibromodulin as the tissue matures (Fig. 10).  1.9.1 Collagen And Small Leucine-Rich Proteoglycans: Interactions The association o f proteoglycans with collagen has long been known. In the early 1960's, dermatan sulfate was isolated from collagen and it was determined that its interaction with the collagen fibrils increased the stability o f collagen and decreased its solubility (Toole et al, 1969). Since then, several small leucine-rich proteoglycans have been shown to bind collagen directly or be closely associated with collagen. It is through this interaction that they function i n collagen assembly and fibrillogenesis. Table 7 lists the small leucine-rich proteoglycans that have thus far been shown to associate with fibrillar collagens.  Other  molecules,  like  link  proteins  (Chandrasekhar  et  al,  1984),  glycosaminoglycans (Vogel et al, 1984; Vogel et al, 1987) and the N-terminus o f small leucine-rich proteoglycans (Vogel et al, 1987), were found to have little or no effect on collagen fibrillogenesis. It should be noted that not all small leucine-rich proteoglycans have been investigated at this point, and there are probably more small leucine-rich proteoglycan / collagen interactions that have yet to come to light.  In vitro studies have shown a direct interaction between decorin and fibrillar collagen (Vogel et al, 1984; Uldebjerg and Danielson, 1988). Later investigations determined that  46  decorin binds to the d and e bands o f collagen i n bovine skin (Scott, 1990) and adult and embryonic human skin (Fleischmajer et al, 1991). The interaction o f decorin with collagen is believed to be v i a binding o f the protein core o f the molecule (Vogel et al, 1984) at the 6  th  leucine-rich repeat (Kresse et al, 1997). Chemical processing o f the  molecule to remove the disulfide bonds o f decorin results in termination o f its effect on fibrillogenesis (Scott et al, 1996) while removal o f the N-terminus or glycosaminoglycans did not (Vogel et al, 1987). Noteably, the binding site o f decorin was found to be different than that o f lumican (Neame et al, 2000) and different than that o f fibromodulin (Hedbom & Heinegard, 1993).  Early investigations o f decorin / collagen interactions suggest that decorin may inhibit radial fibril growth by inhibiting cross-linking o f the molecules and may inhibit calcification o f mineralized tissues by occupying the gap regions where hydroxyapatite crystals are normally deposited (Scott and Orford, 1981). This inhibitory effect o f decorin on collagen fibrillogenesis was later demonstrated at the electron microscope level in studies o f bovine tendon and cartilage (Vogel et al, 1984; Vogel & Trotter, 1987). Later, it was shown that decorin might result in an increase in collagen fibril diameter (Kuc & Scott, 1997). Decorin's role in collagen fibrillogenesis is further  substantiated  by  investigations showing that a decrease in decorin and decorin m R N A occurs at the time when collagen fibril growth is occurring. Collagen fibril growth appears to be in a lengthwise fashion due to lateral fusion o f the molecules at a time when decorin content is decreasing (Birk et al, 1995).  47  Decorin has also been shown to interact with other fibrillar collagens, like type V . Its binding is specific and saturable and is thought to be v i a its protein core (Whinna et al, 1993) while both its core protein (Ehnis et al, 1997) and glycosaminoglycan chains (Font et al, 1993) can bind to collagen type X I V , a non-fibrillar collagen found associated with collagen type I.  Biglycan has been reported in the past to be unable to bind fibrillar collagens (Neame & K a y , 2000). However, recombinant radiolabeled biglycan produced in a eukaryotic system binds to microtiter wells coated with collagen types I, II, III, V and V I approximately as well as decorin (Cox et al, 1970). While immunohistological evidence has indicated that biglycan may interact with fibrillar collagens with much less affinity than decorin (Schonnher et al, 1995b), other methods o f binding cannot be ruled out. Binding o f biglycan to collagen may occur by way o f its glycosaminoglycan side chains (Midura et al, 1989) or v i a an ability to bind other collagens that are frequently found to be associated with collagen type I, such as collagen types V , V I and X I V (Hocking et al, 1998; Wiberg et al, 2002). Biglycan has been shown to bind other forms o f fibrillar collagens, such as collagen type V , which it binds in a specific and saturable way (Whinna et al, 1993). In this situation, it is speculated that biglycan binds with collagen via its protein core and glycosaminoglycan side chains, although the exact interaction has yet to be elucidated.  Fibromodulin binds with collagen I and II at a site other than the decorin binding site (Hedbom & Heinegard, 1993). Fibromodulin has been shown to compete with lumican  48  for binding sites on type I collagen, however (Svensson et al, 2000). It has been suggested  that fibromodulin affects  collagen fibrillogenesis. In vitro studies  on  developing chick metatarsal tendon revealed a 6-8 fold increase in fibromodulin m R N A levels from days 14-19 o f development when the metatarsal tendon is growing suggesting that fibromodulin regulates collagen fibril growth and matrix assembly (Nurminskaya & Birk, 1996). To determine what part o f the fibromodulin molecule binds to the collagen I fibril and what part o f the molecule is responsible for collagen fibrillogenesis inhibition, Font et al (1998) subjected fibromodulin to mild proteolysis to divide the molecule into its four domains. It was found that no single part o f the molecule acts to inhibit fibrillogenesis thereby suggesting that its action requires binding at several sites.  Lumican has been found to bind fibrillar collagen i n vitro (Rada et al, 1993). It competes for binding with fibromodulin (Svensson et al, 2000) at a site that is different than that o f decorin (Hedbom & Heinegard, 1993). L i k e the other small leucine-rich proteoglycans mentioned  above,  lumican is able to affect  collagen fibrillogenesis through  this  interaction. In the cornea for instance, it acts to maintain the transparency o f the cornea by enabling the maintenance o f thinner collagen fibers (Rada et al, 1993). Gene knockout studies (to be discussed i n greater detail later) show that its absence, like decorin, increases skin fragility (Chakravarti et al, 1998), albeit by a different mechanism (Neame et al, 2000).  Other small leucine-rich proteoglycans that have so far not been investigated in oral tissues are also being investigated for their role in fibrillogenesis o f collagen. Tasheva et  49  al (2002) i n an investigation o f mimecan knockout mouse skin and cornea showed using transmission electron microscopy that mimecan knockout mice have thicker collagen fibrils in the skin and cornea although these mice compared to wild-type mice displayed no evident pathological phenotype. N o changes were apparent in corneal transparency though the skin o f these animals was more fragile than in the wild-type mice. Further investigation is needed to determine i f there is any functional overlap with other small leucine-rich proteoglycans in these mice.  Mansson  et  al  (2001)  investigated  chondroadherin  in  vitro  using  recombinant  chondroadherin and collagen type II. It was found that chondroadherin bonds to collagen type II. The significance o f this interaction is not presently clear.  1.9.2 How Do Small Leucine-Rich Proteoglycans Interact With Collagen? The Binding Model With the knowledge that has been derived thus far, estimations have been made as to the structure o f the small leucine-rich proteoglycans. The current model o f decorin structure is based on the structure o f another protein that has a leucine-rich repeat region, the ribonuclease (RNAse) inhibitor (Kobe & Deisenhofer, 1993). Although the leucine-rich repeat o f this molecule allows it to be compared to decorin and perhaps other small leucine-rich proteoglycans, it is a very different molecule. For this reason, the structural model o f small leucine-rich proteoglycans should be treated with caution. It is believed that the leucine-rich repeat part o f the protein core allows the molecule to bend or fold into a horseshoe shape (Fig. 11) with the concave portion o f the molecule available for  50  interaction with collagen (Weber et al, 1996). Although the cys residues o f the small leucine-rich proteoglycans are closer than those o f the R N A s e inhibitor, it is believed that i f this bending occurs, there would be just enough space in the concave portion for a single tropocollagen triple helix to bind (diameter ~1.5nm) (Neame & K a y , 2000). It is speculated that the concave portion o f the "horseshoe" binds to collagen and the convex portion binds to a different collagen fibril. The glycosaminoglycan(s) are believed to extend away from the convex side o f the molecule to maintain interfibrillar space between the fibrils (Kobe & Deisenhofer, 1994; Reardon et al, 2000). The true structure w i l l not be fully appreciated, however, until the crystal structure is obtained (Neame & Kay, 2000).  Scott (1996) showed using rotary shadowing-electron microscopy that decorin, lumican, and fibromodulin are horseshoe-shaped.  It was suggested that decorin is a bidentate  ligand attached to two parallel neighboring collagen molecules i n the fibril, helping to stabilize fibrils and orient fibrillogenesis.  1.9.3 Small Leucine-Rich Proteoglycans In Collagen Fibrillogenesis: In Vitro Studies A s noted above, several in vitro studies have been done that attest to the role o f small leucine-rich proteoglycans in collagen fibrillogenesis. Other studies are discussed below (Table 8).  51  Recently, Neame et al (2000) in a study using recombinant decorin and lumican in collagen fibrillogenesis assay based on turbidity, found that decorin and lumican act independently on collagen fibril formation where lumican accelerates and decorin retards initial collagen fibril formation. It was determined that lumican and decorin do not compete for binding sites on collagen fibrils and that lumican and decorin act to increase collagen fibril stability to thermal denaturation. Lumican and decorin result in reduced overall turbidity suggesting a lower collagen fibril diameter. The presence o f both proteoglycans retarded fibril formation to a greater degree than either one alone - a synergistic effect.  Svensson et al (2000) in a study using recombinant lumican, fibromodulin and decorin proteoglycans  and a collagen fibril  formation / sedimentation  assay, found that  fibromodulin inhibits the binding o f lumican and vice versa. Fibromodulin and lumican do not affect the binding o f decorin to collagen or vice versa and fibromodulin binds to collagen with four times the affinity o f that o f lumican. Collagen was shown to have high and low affinity binding sites for fibromodulin and lumican (ie. each having two binding sites) with fibromodulin having the higher affinity.  Carlson et al (2002)  showed in culture that mutant lumican cells produced  an  unorganized extracellular matrix with altered fibril packing and structure compared to wild cell lines and showed that the cysteine-rich domain o f the lumican molecule to be an important factor in fibrillogenesis and stromal matrix assembly.  52  1.9.4  Small Leucine-Rich Proteoglycans In Collagen Fibrillogenesis: Genetic  Evidence Research i n recent years using genetically engineered animal models have essentially confirmed  that  small  leucine-rich proteoglycans  play  some  role  in  collagen  fibrillogenesis. Table 9 summarizes several o f these studies. The evidence is seen at the electron microscope level in most cases while in other cases severe phenotypes are observed.  Danielson et al (1997) in a study o f decorin knockout mice determined that they were viable but had fragile skin with reduced tensile strength. O n a microscopic level, it was noticed  that the  expressed  phenotype  was  accompanied  by  abnormal  collagen  morphology i n skin and tendon with coarser and irregular fibril profiles. The fibrils had increases and decreases i n mass along their length that may be the result o f altered lateral fusion o f the fibrils during fibrillogenesis due to the absence o f decorin.  X u et al (1998), in a study on biglycan knockout mice (discussed previously) found that mice with no biglycan expressed an osteoporotic phenotype characterized by reduced growth rate and decreased bone mass that became more obvious with age. H i g h resolution radiographic imaging showed that there was reduction i n the amount and density o f trabecular bone and reduced cortical thickness. It was determined that reduced bone mass was due to decreased bone formation rather than increased resorption.  53  Corsi et al (2002) studied the effect o f biglycan and decorin deficiency in single and double knockout mice. It was determined that biglycan knockout mice expressed a phenotype characterized by abnormal collagen fibrils o f bone, skin and tendon with thinning dermis occurring i n the skin without overt skin fragility. The decorin knockout mice showed similar skin phenotype but bone seemed to be unaffected. The findings in the double knockout mice were found to be additive i n dermis and synergistic i n bone manifested as severe skin fragility and marked osteopenia. Ultrastructural analysis o f collagen fibrils revealed a complete loss o f basic fibril geometry characterized by rough fibril profiles.  Biglycan and fibromodulin deficiencies were studied by Ameye et al (2002). M i c e deficient in biglycan and/or fibromodulin developed with gait impairment, ectopic tendon ossification and severe premature osteoarthritis. It was indicated that structurally weak tendons were responsible for the impairment, which in turn led to the ossification in the tendon as demonstrated by forced use o f the joints.  Fibromodulin deficiency was studied by Svensson et al (1999) who found, using transmission electron microscopy, that fibromodulin knockout mice have altered collagen fibril morphology i n tail tendon in that they were fewer i n number, had rough, irregular outlines i n cross-section. A s well, fibromodulin knockout tendon had on average more thin fibrils. These mice also showed a four-fold increase in lumican deposition suggesting a functional overlap o f these two small leucine-rich proteoglycans.  54  Ezura et al (2000) in a study o f fibromodulin, lumican and double knockout mice determined that all three groups o f mice exhibited altered collagen fibrils with the double knockout mice expressing a phenotype that was additive in nature - ie. worse than the single mutant phenotypes furthering the suggestion o f functional overlap between these two small leucine-rich proteoglycans. Three distinct abnormalities aware found i n collagen fibrils: an early presence o f fibril diameter heterogenicity in the double knockout mice, an abnormally large amount o f thin fibrils in later stages o f development, and irregular profiles o f the fibrils.  Chakravarti et al (1998) in a study on lumican knockout mice found that they too displayed skin fragility and laxity. In addition they expressed a phenotype characterized by corneal opacity. Ultrastructural analysis o f skin and cornea revealed altered collagen fibril morphology characterized by a significant proportion o f abnormally thick fibrils with altered interfibrillar spacing as well.  Jepsen et al (2002) in a study o f lumican, fibromodulin and double knockout mice determined that double knockout mice were small in body size, displayed a gait impairment, joint laxity and age-dependent osteoarthritis characterized by extreme tendon weakness. These findings were attributed to altered collagen fibril morphology and a disproportionate increase i n thin collagen fibrils.  55  1.10 Implications Of The Role Of Small Leucine-Rich Proteoglycans In Dental Disease Since most dental tissues have fibrillar collagen as their major non-cellular element (Tables 3, 4, 5), and since small leucine-rich proteoglycans play a role in collagen fibrillogenesis, one would suspect that small leucine-rich proteoglycans would be necessary for normal development and maintenance o f dental tissues.  Proteoglycans and collagen fibrils are the main constituents o f the connective tissues (Wilda et al, 2000). While there has been no direct link o f small leucine-rich proteoglycans i n the formation or progression o f periodontitis or caries, research indicates that there may be a role for these molecules in creating and maintaining the integrity of the dental soft tissues and development and mineralization o f dental hard tissues (Tables 8 and 9). If they do i n fact have a positive influence on the integrity o f these tissues, one must assume that a defect in small leucine-rich proteoglycans would increase the risk o f onset and progression o f dental disease such as dental caries and periodontitis.  Marked loss o f decorin and biglycan i n chronic inflamed periodontal tissues has been noted in a human study (Oksala et al, 1997) suggesting a role o f small leucine-rich proteoglycans i n maintaining the tissue integrity o f gingival tissues. A s well, alterations have been detected in small leucine-rich proteoglycan distribution during human gingival wound healing again suggesting a role i n organization and extracellular matrix integrity (Oksala et al, 1995). Moreover, a decrease or absence o f small leucine-rich proteoglycans has been found to be associated with some connective tissue diseases.  56  Decorin  deficiencies have been found associated with neonatal Marfan syndrome (Pulkkinen et al, 1990; Superti-Furga et al, 1992; Raghunath et al, 1993), infantile progeroid syndrome (Beavan et al, 1993), Ehlers-Danlos syndrome ( W u et al, 2001; Tajima et al, 1999; Quentin et al, 1990; Fushimi et al, 1989), and osteogenesis imperfecta (Dyne et al, 1996). Interestingly, these diseases have been shown to exibit dental manifestations including periodontitis (Straub et al, 2002; Pope et al, 1992; Reichert et al, 1999; Hartsfeld et al, 1990; M c K u s i c k , 1972). A s well, research i n gene knockout mice has shown that alterations i n decorin, biglycan, lumican and fibromodulin may produce changes that mimic diseases like Ehlers-Danlos syndrome (Danielson et al, 1997; Chakravarti et al, 1998; Corsi et al, 2000; Jepsen et al, 2002). The role o f small leucine-rich proteoglycans in maintaining tissue integrity has been inferred for years as evidenced by the use o f proteoglycans and glycosaminoglycan detection methods in periodontal disease.  1.10.1  Proteoglycans And Glycosaminoglycans As Periodontal Disease Markers  Several studies have examined the occurrence o f various extracellular matrix components in the gingival crevicular fluid in humans with various periodontal conditions including health. Most o f these studies evaluate the occurrence o f glycosaminoglycans and few are related to proteoglycans, especially small leucine-rich proteoglycans. It seems that the most valuable marker is chondroitin 4-sulfate, which is believed to originate i n alveolar bone (Oksala et al, 1993) although it may also originate in the soft tissue. The general trend is for glycosaminoglycan levels o f subjects with periodontal disease to be higher compared to those o f healthy individuals.  57  The first report o f glycosaminoglycan detection i n the gingival crevicular fluid was by Embery et al (1982) who used electrophoresis to find sulfated  glycosaminoglycans  present i n the gingival crevicular fluid o f chronic periodontitis subjects. Last et al (1985) investigated glycosaminoglycans i n the gingival crevicular fluid using electrophoresis as well. Gingival crevicular fluid was collected from subjects with chronic gingivitis, untreated  advanced  periodontitis,  early  periodontitis, juvenile  periodontitis,  teeth  undergoing active orthodontic movement, teeth in traumatic occlusion, and healing extraction sockets. Chondroitin 4-sulfate was found in all situations where degenerative changes were taking place i n the deeper periodontal tissues. Interestingly, these sulfated glycosaminoglycans could not be detected in areas o f recent periodontal treatment consisting o f periodontal surgery or daily subgingival irrigation with chlorhexidine.  Shibutani  et  al (1993)  developed  an  ELISA  test to  detect chondroitin  sulfate  glycosaminoglycans in the gingival crevicular fluid o f dogs with experimentally induced periodontitis. E L I S A values for chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate, although low, increased i n proportion with the severity o f inflammation present in these animals.  Giannobile  et  al  (1993)  investigated  the  quantities  of  chondroitin  sulfate  glycosaminoglycans i n the gingival crevicular fluid in health, gingivitis, and adult periodontitis maintenance and adult periodontitis non-maintenance  groups using a  safranin O binding assay. It was found that the amount o f glycosaminoglycans increased with  severity  and  control  of  periodontal  58  condition  such  that  the  levels  of  glycosaminoglycans were approximately 4 ng G A G / sample in health, 15 ng G A G / sample in gingivitis, 23 ng G A G / sample in maintained adult periodontitis and 120 ng G A G / sample in unmaintained adult periodontitis.  Smith et al (1995) examined the hyaluronan and chondroitin 4-sulfate i n the gingival crevicular fluid o f patients with chronic adult periodontitis at diseased and healthy sites before  and  after  treatment using electrophoresis.  Significantly  higher  levels o f  chondroitin 4-sulfate were detected at diseased sites prior to treatment correlating with probing depth and attachment levels. Sites that responded well to treatment (oral hygiene instruction and root planing) had lower levels o f chondroitin 4-sulfate than sites that did not respond well to treatment. Hyaluronan levels were less significantly associated with clinically successful treatment.  Hyaluronan being a rather ubiquitous component o f gingival crevicular fluid samples represents approximately 40% o f the total glycosaminoglycan content o f human gingiva. Its levels are believed to be constantly high due to the constant turnover o f the periodontal tissues and thus it is not a good disease marker (Embery et al, 1979). It tends to be absent i n certain disease situations such as acute necrotizing ulcerative gingivitis ( A N U G ) . Moreover, treatment o f A N U G has resulted in an increase o f hyaluronan to normal high levels (Last et al, 1987).  Heparan sulfate has been found i n gingival crevicular fluid o f orthodontic patients (Waddington et al, 1994).  59  Few studies have tried to detect parent proteoglycans in the gingival crevicular fluid. Waddington et al (1998) separated two proteoglycan species from gingival crevicular fluid. They were chondroitin sulfate-rich proteoglycans that reacted positively to antidecorin and anti-biglycan antibodies.  Oksala et al (1997) examined basement membrane heparan sulfate proteoglycan, C D 4 4 , syndecan (three heparan sulfate proteoglycans), decorin and biglycan i n chronically inflamed human periodontium using immunofluorescence microscopy o f tissue sections. It was found that basement membrane heparan sulfate proteoglycan was decreased in subepithelial and subendothelial basement membranes while C D 4 4 and syndecan were reduced i n epithelial cells but increased i n infiltrating lymphocytes. Decorin and biglycan levels were reduced i n the periodontal connective tissue o f chronically inflamed tissue with decorin localizing i n connective tissue along short rod-like structures. The results o f this study suggested that proteoglycan-dependent intercellular adhesion o f keratinocytes is decreased i n chronic inflammation while adhesion o f infiltrating lymphocytes is increased  i n this  state. Furthermore, the  disappearance  o f adhesion modulating  proteoglycans may regulate cell migration in inflamed periodontium.  These studies suggest that as periodontal tissues become inflamed, the proteoglycans (including the attached glycosaminoglycans) o f the tissues decrease allowing for the inflammatory process to enfold and begin the battle against periodontal pathogens. The lost proteoglycans (and glycosaminoglycans) are released into the gingival crevicular  60  fluid, which can be detected by various methods including E L I S A and electrophoresis. Chondroitin 4-sulfate seems to be a more sensitive marker for disease activity than chondroitin 6-sulfate, hyaluronan, or dermatan sulfate for determining active phases o f periodontal destruction at individual sites. Further research is required to determine the value o f this detection i n the clinical setting. The virtual absence o f soft tissue components i n the gingival crevicular fluid may be a reflection o f the very high turnover rate o f the periodontal tissues. Periodontal ligament has a turnover rate o f 15x that o f skin and 5x that o f alveolar bone (Sodek et al, 1977) with respect to its collagenous components. This high turnover rate may result in quick disappearance o f tissue breakdown products rather than their elution into the gingival crevicular fluid (Embery et al, 2000).  61  Chapter Two - Aim of the Study In the past, proteoglycans received little attention i n tissue research since they comprised a relatively small proportion o f tissue make-up. This small proportion, however, may not accurately reflect their importance. Over the last 30 years, methods o f study have improved to include the use o f immunohistochemical techniques  and genetically  engineered mice allowing new insights into the role and function o f these important molecules.  Proteoglycans are an important and integral part o f all periodontal tissues (cementum, periodontal ligament and bone especially). One must wonder, then, how these molecules influence the state o f these tissues. Could their absence (partial or complete), for instance, influence the initiation or progression o f periodontal disease or result in decreased regenerative ability o f the periodontium? Could alterations o f these molecules result in increased caries incidence? Only now, with the development and utilization o f newer laboratory techniques and the investigation into genetically engineered mice can we begin to understand the importance o f these molecules in the dental tissues.  Therefore this study w i l l analyze the gross anatomy o f dental and periodontal tissues as well as the structure and organization o f these tissues at the light and electron microscope levels in fibromodulin, lumican and double knockout adult mice compared to age and sex-matched C D - I wild-type mice. W e hypothesize that the absence o f lumican and / or fibromodulin w i l l result i n an altered morphology o f the periodontal ligament collagen fiber bundles.  62  Chapter Three - Materials and Methods 3.1 Animals 8 lumican ( L U M ) knockout mice, 8 fibromodulin ( F M ) knockout mice and 8 lumican / fibromodulin ( L U M / F M ) knockout mice aged 4.5 to 8 months (gifts from Dr. Birk, Jefferson Medical college, Philadelphia, P A , Dr. Chakravarti, John Hopkins University School o f Medicine, Baltimore, Maryland and Dr. Oldberg, University o f Lund, Sweden) and 10 age and sex-matched C D - I (wild-type) mice were used i n this study (Table 10). The generation and characterization o f the mice used in this study have been described in detail elsewhere (Svensson et al, 1999; Chakravarti et al, 1998; Jepsen et al, 2002).  Mouse heads were fixed i n 4% formalin i n neutral buffer for 3 weeks. The maxillae and mandibles were carefully dissected out, separated and divided in half at the midline. H a l f of each maxilla was defleshed in 2% potassium hydroxide for 2 weeks and examined under a dissecting microscope for gross morphological changes in jaws and teeth, presence or absence o f alveolar bone loss (see below), and to measure jaw sizes (see below). The mandibular samples were decalcified in 5% formic acid containing 0.9% sodium chloride for up to 7 weeks until no mineralized tissue was detected on radiographs. Both halves o f the mandible were embedded in paraffin using standardized procedures  (one half i n a mesio-distal orientation, the other i n a bucco-lingual  orientation) and sectioned at 6um. Standardized sections from the coronal and mid-third areas o f the tooth root were chosen from each mouse such that the widest diameter o f the pulp chamber was visible i n each section, the length and shape o f the roots and number o f  63  teeth per section (for mesio-distal sections) were the same to ensure that similar areas o f the teeth were being compared.  3.2 Morphometric Analysis of Jaw Size One half o f each defleshed maxilla was measured with a Boley Gauge. Only those jaws that remained intact after dissection were analyzed (Table 16). The tips o f the calipers were placed at the alveolar margin facial to the erupted incisor, where it emerges from the alveolus and at the distal o f the third molar (Fig. 12). The gauge was then read to 1/10 of a millimeter. Results were statistically analyzed with a student /-test to compare the single and double knockout mice to wild-type control mice and to compare the single knockout mice with the double knockout mice. A g e and sex were considered i n the statistical analysis.  3.3 Analysis of Alveolar Bone Loss One half o f all defleshed maxillae were analyzed under a dissecting microscope for bone loss using the classification system for bone loss i n mice described by Wiebe et al (2001). Bone loss i n this classification system was classified as follows: Grade I) horizontal component o f bone loss i n the furcation, Grade II) through & through furcations, and Grade III) through-and-through furcations with alveolar bone loss into the apical third o f the tooth root (Table 11).  3.4 Histomorphometric Analysis Paraffin sections from wild-type, fibromodulin, lumican, and double knockout mice were stained with phosphotungstic acid hematoxylin ( P T A H ) (Putchler et al, 1963) to study  64  collagen morphology and cells, hematoxylin and eosin ( H & E ) for general histology and picrosirius red (PSR) (see below) for analysis o f collagen organization and fiber morphology. Table 12 lists the mice used for each stain. Stained sections were mounted with Entellan mounting medium. T w o independent examiners (single blinded) analyzed the middle and coronal third areas o f the H & E and PTAH-stained mandibular first molar sections using a light microscope (Axiolab E by Zeiss) equipped with a 20x and 40x objective. Images were recorded with a N i k o n Coolpix 995 digital camera attached to a N i k o n Eclipse T S 100 microscope equipped with a 20x objective. The coronal and middle third areas o f the PSR-stained mandibular first molar sections were examined in the same manner using a polarizing light microscope (Carl Zeiss Jena Jenapol polarizing light microscope) equipped with a 20x objective. Angles on the polarizing light microscope were set so that at 0°, the sections were aligned with the long axis o f the periodontal ligament spaces. Images were recorded at 0°, 45° and 90° using a Canon E O S D60 digital camera with the following settings (aperture priority at A E , shutter speed o f 30, partial metering mode, 1.2 exposure compensation, ISO speed 100, manual focus).  Sections were analyzed under light and polarizing light microscopes and digital images o f sections were also examined using Photoshop 6.0 software for morphologic changes in the dental and periodontal tissues, presence or absence o f bone loss and cellular infiltrate indicative o f inflammation. Photoshop alterations only included cropping and sharpening of photos such that unsharp mask was set at 15%, 0 threshold level and 16 pixel radius. Photoshop 6.0 was used to take advantage o f the zoom feature.  65  P S R staining was carried out as follows. Deparaffinized section were immersed in picrosirius red (100ml saturated aqueous picric acid + . l g Gurr's Sirius red F 3 B ) for 1 hour and then immersed i n 1% acetic acid for 15 minutes (solution changed every 5 minutes) until solution was clear.  Parameters being examined were: gross morphology o f bone, dentin and cementum, the presence or absence o f external root resorption, the presence or absence o f bone loss (Wiebe et al, 2001), the presence or absence o f inflammatory cells i n H & E sections, gingival fiber morphology and orientation. The following parameters were analyzed in the periodontal ligament: relative amounts o f interfiber bundle space between collagen fiber bundles, relative thickness o f collagen fiber bundles (homogeneity), relative length of collagen fiber bundles (whether or not it was difficult to trace a bundle's path from tooth to bone), fiber bundle orientation, and fiber bundle outline (smooth and distinct or hazy and indistinct). Relative amount o f blood vessels were located i n the periodontal ligaments o f knockout mice and compared to those o f w i l d type mice. Non-standardized, representative sections o f 7 C D - I wild-type mice, 5 fibromodulin knockout mice, 5 lumican knockout mice and 6 double knockout mice were analyzed for differences in numbers o f blood vessels i n the coronal two-thirds o f the periodontal ligament. Blood vessels were recognized i n P T A H and H&E-stained sections as circular or rounded voids within the ligament lined with endothelial cells and containing recognizable blood cells within it. The blood vessels were counted on the buccal and lingual surfaces and were analyzed together and separately as such. Tears in the periodontal ligament collagen fiber bundles were a relatively frequent finding in some samples. Therefore, the number o f  66  breaks or disruptions were calculated from 6 CD-I wild-type mice (mice 1-6), 5 fibromodulin knockout mice (mice 1-5), 5 lumican knockout mice (mice 1-5) and 5 double knockout mice (mice 1-5) (Table 10) under light microscope at 20x objective and statistically comparedd.  3.5 I m m u n o h i s t o c h e m i c a l A n a l y s i s  For immunohistochemical localization of decorin, biglycan, lumican and fibromodulin deparaffinized sections of the mandibular first molars were incubated in a solution of 500uL 30% H202 + 50mL methanol for 30 minutes to block the endogenous peroxidase activity. Sections were rinsed and incubated with normal blocking serum (Vectastain; Vector Laboratories Inc., Burlingame, California) for 60 minutes at room temperature and then incubated with polyclonal antibodies against decorin (LF-113), biglycan (LF106) (gifts from Dr. Larry Fisher, National Institutes of Health, MIDR, Bethesda, Maryland), lumican (gifts from Svensson et al, 1999, Dept of Molecular Biology, University of Lund, Sweden), fibromodulin (gifts from Dr. Plaas et al, 1997, Shriner's Hospital for Crippled Children, Tampa Fl.) or type I collagen (Chemicon, Tamecula LA) (Table 13) at 4°Celsius for 16 hours.  After rinsing, sections were incubated with  biotinylated anti-rabbit antibody for 1 hour and then reacted with ABC avidin/peroxidase reagent (Vectastain Elite kit, Vector Laboratories Inc.). Sections were then rinsed and reacted with the Vector VIP substrate for peroxidase for 1-4 minutes. For localization experiments, the reaction was stopped when the immunostain was detected in the extracellular matrix under light microscope by dipping the sections in distilled water for  67  at least 2 minutes. B u / L i sections were used for localization o f S L R P in the dental tissues (Table 12).  To determine i f lack o f one or two proteoglycans was compensated by increased expression o f other members o f the small leucine-rich proteoglycan family within the gingiva, dental and periodontal tissues, a standardized staining procedure was used on mesio-distal sections so that intensities o f the various immunostains could be compared. Mesio-distal sections o f each mouse group (Table 14) were divided into 4 groups o f 4 (one mouse from each group) and the staining procedure was standardized as follows: one group was stained at a time and antibodies were applied to the groups o f sections in the same sequence such that antibodies ( a n t i - L U M , a n t i - D C N , anti-FM, anti-BGN) were applied to wild-type C D - I mouse sections, followed by fibromodulin knockout mouse sections, lumican knockout mouse sections and double knock-out mouse sections. Reactions were stopped when immunostaining was visually apparent under light microscope i n the extracellular matrix o f wild-type mouse sections (approximately 2.5 minutes). Reactions were stopped i n the same sequence as above to ensure equal exposure time to each group o f mouse sections.  A l l sections were mounted with Entellan mounting medium before being examined by two independent (single blinded) examiners using a light microscope (Axiolab E by Zeiss) with lOx, 20x and 40x objectives for localization o f proteoglycans within the gingiva, dental and periodontal tissues. The relative level o f proteoglycan expression was  68  determined using a scale o f 0-3 (0 = no stain; + = mild staining; ++ = moderate amount o f staining; +++ = extreme or intense staining).  3.6 Scanning Electron Microscopy Paraffin sections from the mid-root region o f the periodontal ligament o f mandibular first molars o f C D - I wild-type, fibromodulin, lumican, and double knockout mice were processed for scanning electron microscopy using standard procedures (Postek et al, 1980) and examined using a Cambridge 260 Stereoscan scanning electron microscope.  3.7 Statistical Analysis Jaw size, numbers o f blood vessels and tears in the periodontal ligament were compared using a student t-test assuming equal variance using the Microsoft Excel statistical package. Comparisons were made with pooled data and with age and sex-matched mice to control for these variables.  69  Chapter Four - Results 4.1 Immunohistochemical Analysis In order to compare the expression and localization o f fibromodulin and lumican to decorin and biglycan in periodontal and dental tissues o f wild-type C D - I mice, paraffin sections were stained with antibodies against decorin, biglycan, fibromodulin and lumican and analyzed under a light microscope using 5x, lOx, 20x and 40x objectives. A l l four proteoglycans localized abundantly in the mucosal, gingival and periodontal connective tissues (Table 15). Fibromodulin localized in the predentin, pulp, periodontal ligament and connective tissue o f the gingiva and mucosa at similar intensities (Fig. 13A).  Slight  immunoreactivity  was  detected  in  epithelial  basal  cells  while  no  immunoreactivity was noted in cementum or bone. Lumican was localized i n pulp connective tissue, periodontal ligament and connective tissue o f gingiva and mucosa in increasing intensity i n the order described (Fig. 13-B). N o lumican reactivity was observed i n bone, cementum, dentin or epithelium although a slight staining could be seen in the predentin layer. Biglycan immunoreactivity was located in pulp, predentin, periodontal ligament and gingival and mucosal connective tissue with the most intense staining occurring in the periodontal ligament. Moreover, gingival connective tissue appeared more intensely stained than mucosal connective tissue (Fig. 13-C). Decorin was located in pulp, predentin, periodontal ligament and gingival and mucosal connective tissue (Fig. 13-D). Most intense staining o f decorin appeared to be i n the connective tissue o f the gingiva and mucosa with most intense staining occurring i n the area immediate subjacent the epithelium.  70  The absence o f fibromodulin and lumican in the gene knockout mice was confirmed using anti-fibromodulin and anti-lumican antibodies. Fibromodulin, lumican and double knockout mice showed no corresponding proteoglycans in any o f the dental tissues (Fig. 14a - B , D , G , H ) although a slight cross-reactivity was noted in the basal epithelial layer in the fibromodulin knockout sections stained with anti-fibromodulin (Fig. 14a - G ) .  To determine  i f targeted deletion o f fibromodulin or lumican was associated with  compensation upregulation o f expression o f other members o f the small leucine-rich proteoglycan family, four groups o f mesio-distal sections (one per mouse group) o f fibromodulin, lumican, and double knockout and C D - I wild-type mice were stained with anti-lumican,  anti-fibromodulin,  anti-biglycan  or  anti-decorin  antibodies  using  a  standardized VIP-developing time. The findings showed that the relative staining for fibromodulin  was more intense in the periodontal ligament o f lumican knockout mice  than wild-type mice. Additionally, lumican reactivity was more intense in the connective tissue o f  fibromodulin  knockout mice than wild-type mice, including that in the  periodontal ligament (Fig. 14a - C , F). N o notable differences  in immunostaining  intensity were noted with anti-decorin or anti-biglycan immunostaining between  the  knockout mice and wild-type mice (Fig. 14b - A - H ) .  4.2 Gross M o r p h o l o g i c a l A n a l y s i s The gross morphology o f 8 fibromodulin knockout, 7 lumican knockout and 8 double knockout maxilla halves were analyzed and compared to those o f 10 age and sexmatched C D - I wild-type mice. A l l teeth, three molars and incisors, were present and  71  fully erupted i n all specimens (Fig. 15). Class I furcation defects (Wiebe et al, 2001) were detected i n all groups but there was no statistically significantly difference among the groups (data not shown). Only one specimen, fibromodulin knockout mouse #1, showed any appreciable amount o f bone loss, exhibiting class II furcation defects according to Wiebe e t a l (2001).  In order to analyze i f deletion o f fibromodulin, lumican or both proteoglycans affected j a w size, the length o f each maxilla was measured (Table 16). The mean j a w lengths o f the fibromodulin knockout (10.85 mm), lumican knockout (10.90 mm) and double knockout (10.79 mm) mice were smaller than those o f the wild-type mice (11.42 mm). The mean length o f the double knockout mouse jaws was also slightly smaller than that of either o f the single knockout mice as noted above. The differences between wild-type mice and fibromodulin knockouts as well as between wild-type and double knockouts were found to be statistically significant when data was pooled (Table 17). Other comparisons with pooled data were not found to be statistically significant.  In order to determine i f age o f the mice could explain the differences i n j a w size, the jaw size o f animals with different ages in each experimental group were compared (Tables 18, 19). The only statistically significant (P=.03) finding was found between the mean j a w lengths o f wild-type 8 month old mice (11.7mm) compared to 8 month old double knockout mice (10.85 mm). Generally, the findings showed that as age increased, jaw length increased and generally these findings were found to be statistically insignificant.  72  Gender analysis was also carried out to determine i f gender had an effect on jaw length. When males were compared to females within each group (Table 20), a statistically significant difference (P=.024) was found only within the fibromodulin group. Here, mean male jaw length (11.23 mm) was greater than mean female jaw length (10.62 mm). When males and females were compared between the groups (Table 21), the only statistically significant difference was found between the mean j a w lengths o f wild-type females (11.40 mm) compared to mean jaw lengths o f fibromodulin knockout females (10.62 mm) (P=.007). When age and gender were considered together (Tables 22, 23), a statistically significant result was seen between fibromodulin knockout 5.5 month old females (10.75 mm) and wild-type 6m female (11.4 mm) (P=.03). N o other statistically significant difference was found between any o f the groups.  4.3 H i s t o m o r p h o l o g i c a l Analysis A total o f 110 sections including the first molar, gingiva and periodontal ligament at the coronal and middle thirds o f the root surface o f the first molar were stained with phosphotungstic acid hematoxylin ( P T A H ) , hematoxylin and eosin ( H & E ) and picrosirius red (PSR) and analyzed under light microscope for the presence o f inflammatory cellular infiltrate in the connective tissues o f the gingiva and relative alveolar bone loss as well as morphological characteristics o f teeth, root cementum, gingiva, periodontal ligament and bone.  73  4.3.1 Teeth, Cementum, Alveolar Bone And Gingiva Morphological and histological analysis o f sections stained with H & E and P T A H show that the teeth o f fibromodulin, lumican and double knockout mice developed normally and that there were no gross morphological differences i n the dental and periodontal tissues o f the wild-type mice compared to those o f the knockout mice excluding enamel, which, due to processing, could not be compared. The periodontal tissues appeared healthy and there was no evidence o f inflammatory cellular infiltrate in the connective tissues o f any o f the groups (Fig. 16).  Amounts o f cementum differed from section to section in all animals and there was abundant cementum formed at the apical third o f the roots i n all groups. This finding appeared to be i n keeping with the location o f the section o f the tooth and age o f the specimen (data not shown). Bone lining the socket was found to be jagged in some areas and smooth in others, which appeared to be a random finding in all groups. N o conclusive changes were noted in the pulp tissue, although some samples appeared to have an increased number o f cells (data not shown). N o gross morphological differences were noted i n the gingival tissues o f the different groups i n that the amounts o f connective tissue, number o f cells, cellular organization, and thickness o f epithelial layers seemed relatively proportional (Fig. 16).  4.3.2 Periodontal Ligament In all animal groups, periodontal ligament fiber bundles on the buccal surfaces were different than the lingual surfaces with more unorganized fiber bundles found on the  74  lingual surface. A s well, periodontal ligament fiber bundles were randomly organized in the apical third o f all root surfaces in all four groups. This random organization increased in an apical direction i n all groups and therefore, only the middle and coronal thirds o f buccal surfaces were used to compare collagen organization and morphology in the periodontal ligament.  Sections stained with H & E (Fig. 17A), P T A H (Fig. 18A) and P S R combined with polarizing light microscope (data not shown) showed that the coronal third portion o f the periodontal ligament o f wild-type mice appeared to be filled with well-organized collagen fiber bundles with little space between them. The space between the fiber bundles appeared to be evenly distributed and homogenous i n nature. The fiber bundles were homogenous in thickness relative to each other and for the entire length o f the fiber from tooth to bone and were oriented, in a parallel fashion, at 45° to the root surface. Moreover, the fiber bundles could easily be traced from tooth to bone surface and appeared to have well defined, smooth edges and had dark bands associated with them as seen in the mid-third periodontal ligament area o f PSR-stained sections analyzed under a polarizing light microscope (Fig. 19A).  Compared to wild-type mice, the periodontal ligaments o f fibromodulin knockout mice stained with H & E (Fig. 17B), P T A H (Fig 18B) and P S R combined with polarizing light microscope (data not shown) displayed collagen fiber bundles in the coronal third area that were not homogenous i n thickness along the length o f the fiber bundles or among the different fiber bundles. There appeared to be a relative increase in the number o f thick  75  fiber bundles in the ligament relative to all other groups, giving the ligament fiber bundles a clumpy or curly appearance. The fiber bundles had a less well-defined outline and often appeared fuzzy. Although the basic orientation o f the periodontal collagen ligament fiber bundles was apparent, running at 45° from tooth to bone surface, it was difficult to consistently trace the fiber bundles from tooth to bone surface. A s well, the spaces between the fiber bundles were not evenly distributed throughout the ligament with narrow and wide spaces scattered throughout. Overall, there appeared to be more spacing between the collagen fiber bundles than that found in wild-type mice, although this was not quantified. PSR-stained sections analyzed under polarizing light microscope shows that fiber bundles o f the mid-third area o f the periodontal ligament appear to have hazier outlines and relatively more dark bands associated with them compared to those o f wild-type mice (Fig. 19B).  Lumican knockout mice expressed a periodontal ligament phenotype different from that of wild-type mice and fibromodulin knockout mice as seen in coronal third sections stained with H & E (Fig. 17C), P T A H (Fig, 18C) and P S R (data not shown). Collagen fiber bundles could usually be traced from tooth to bone surface but not in all cases. There appeared to be an increase in space between the fiber bundles compared to those o f the wild-type mice and fibromodulin knockout mice. A s well, the fiber bundles often appeared to be non-homogenous i n thickness along the length o f the fiber bundles or among fiber bundles within the ligament (although the fiber bundles were more homogenous in this regard than those o f fibromodulin knockout mice). Unlike the fibromodulin knockout mice, there appeared to be a preponderance o f thin fiber bundles  76  throughout the ligament giving the periodontal ligament fiber bundles a appearance. L i k e the periodontal ligaments o f  fibromodulin  stringy  knockout mice, lumican  knockout mice displayed mid-third area collagen fiber bundles with hazy outlines and more dark bands associated with them than those found i n wild-type mice (Fig. 19C).  The double knockout mice expressed a periodontal ligament phenotype that contained some o f the attributes o f both single knockout mice described above as seen in coronal third sections stained with H & E (Fig. 17D), P T A H (Fig. 18D) and P S R (Fig. 19D). There was usually increased space between collagen fiber bundles o f the periodontal ligament compared to that found i n either o f the single knockout or wild-type mice. The collagen fiber bundles were non-homogenous in thickness along their lengths and among the different fiber bundles within the ligament with thick and thin fiber bundles readily apparent. A s well, it was difficult to trace the fiber bundles from tooth to bone surface and the basic orientation o f 4 5 ° from tooth to bone was altered. The combination o f all the above findings gave the double knockout mouse periodontal ligament an overall random appearance, one that was notably worse than either o f the single knockout mice. Mid-third PSR-stained sections o f the periodontal ligament showed that the dark bands associated with the fiber bundles in the other groups were not readily apparent in these ligaments (Fig. 19D).  The  above morphological findings were confirmed with analysis o f mid-third root  sections stained with H & E , P T A H and P S R (data not shown) as well as sections immunostained for type I collagen (data not shown). Analysis o f sections stained with  77  antibodies against type I collagen showed a diffuse staining in the periodontal ligament o f the wild-type mice and more discreet staining i n the periodontal ligament o f gene knockout mice. Different fiber bundle widths seemed to be apparent in the single and double knockout mice and fiber bundles appeared to be wavier than those o f the w i l d mice.  4.3.2.1 Scanning Electron Microscopy Of Periodontal Ligament Scanning electron microscopy was performed  to analyze the morphology o f the  periodontal ligament fiber bundles i n more detail. The periodontal ligament fiber bundles of wild-type C D - I mice (Fig. 2 0 - A , E ) appear well organized with proper angulation traversing the space between tooth and bone. The fiber bundles appear to have smooth outlines and surface texture and the spacing between the bundles appears relatively even throughout the ligament. A t 2.62kX magnification, the fiber bundles o f wild-type mouse had a sheet-like appearance. B y contrast, the fibromodulin knockout mice (Fig. 20-B, F) have periodontal ligament fiber bundles that appear unorganized, clumpy, o f varying thickness and have many disruptions. A s well, they appear to have a rough surface texture. The relative amount o f spacing between the collagen fiber bundles is greater than in wild-type mice. The lumican knockout mouse periodontal ligament fiber bundles are different that that o f wild-type and fibromodulin knockout mice (Fig 20-C, G). They are thinner than either wild-type and fibromodulin knockout mice, with a stringy appearance and have increased amounts o f inter-fiber bundle spacing. A s well, a roughened fiber bundle outline is apparent. The double knockout mouse ligament fiber bundles (Fig. 20D , H) are o f varying thickness and have altered surface texture compared to the other  78  groups. They have visible "crater-like" defects on the surface o f the fiber bundles and more inter fiber bundle spacing than wild-type, like the other knockout groups. Another notable finding is that all three knockout groups display very thin filamentous structures branching out from the main fiber bundles o f the periodontal ligament (Fig. 20-E-H). These thin branches were not apparent in the wild-type mouse ligament fiber bundles (Fig. 20 E).  4.3.2.2 V a s c u l a t u r e O f T h e P e r i o d o n t a l L i g a m e n t Aside from the differences in collagen fiber bundle morphology, more blood vessels were located i n the periodontal ligaments o f knockout mice, compared to those o f wild-type mice (Tables 24, 25; F i g 21). The number o f blood vessels was higher on the buccal side of the periodontal ligament than the lingual side i n all groups, except in the fibromodulin knockout mice where a more even distribution was found (discussed further in a later section). Relatively few, scattered small blood vessels were noted i n the periodontal ligament o f the wild-type mice. Overall, the mean number o f blood vessels was higher in the fibromodulin knockout mice than any o f the other groups. A s well, the mean number of blood vessels was higher i n the periodontal ligaments o f double knockout mice than wild-type or lumican knockout mice. However, when total numbers and means o f blood vessels were compared among the different groups, no statistically significant differences were noted.  N o statistical significance was noted when comparing the blood vessels o f the buccal aspects o f the periodontal ligament. When lingual periodontal ligaments were compared,  79  however, fibromodulin knockout mice showed statistically significantly more blood vessels than wild-type mice, lumican knockout mice and double knockout mice (mean number o f 2.2, 0, .25 and 1.2 respectively). Other blood vessel comparisons in the lingual periodontal ligament yielded statistically insignificant results (Table 25).  4.3.2.3 Tears I n T h e P e r i o d o n t a l L i g a m e n t Histological stained sections o f periodontal ligaments showed that in many samples there were breaks or tears i n the periodontal ligament collagen fiber bundles. Tears were identified as irregularly shaped voids within the ligament or between the ligament and bone or between the ligament and tooth characterized by absence o f stain that contained no cells and often exhibited visibly torn tissue fragments within the void (Fig. 21). Tears were analyzed as pooled data only since some tears were quite large and wrapped around the apex o f the tooth and thus appeared on both the buccal and lingual surfaces.  Virtually no tears were detected in the periodontal ligaments o f the wild-type mice. Fibromodulin knockout mice exhibited a higher mean number o f tears i n the periodontal ligament than the other three groups. However, no statistically significant differences were noted when all groups were compared (Table 26).  80  Chapter Five - Discussion  Periodontal ligament collagen fiber bundles mediate the attachment o f the tooth to the alveolar bone. Periodontal disease involves breakdown o f this collagen fiber attachment. Certain inherited conditions, such as Ehlers-Danlos syndrome, involving altered collagen fibrillogenesis are associated with severe periodontal disease at an early age (Pope et al, 1992; Reichert et al, 1999; Hartsfeld et al, 1990; M c K u s i c k , 1972) suggesting that altered collagen fibrils may predispose to periodontal disease. The mechanisms that regulate collagen fiber organization in the periodontal ligament are poorly understood (Kadler, 1995). Fibromodulin and lumican belong to a family o f small leucine-rich proteoglycans that associate with collagen fibrils (Scott, 1986; Pringle & Dodd, 1990; Hedlund et al, 1994; B i r k et al, 1995), can modulate collagen fibril formation and inhibit lateral growth of fibrils in vitro (Vogel et al, 1984; Hedbom & Heinegard, 1993; Rada et al, 1993) and in vivo (Svensson et al, 1999; Ezura et al, 2000). They are expressed i n a number o f collagenous connective tissues and play a significant role i n defining tissue integrity through their abilities to interact with other extracellular matrix molecules and potentially regulate collagen fibrillogenesis (Chakravarti et al, 1998; Svensson et al, 1999; Ezura et al, 2000; Ameye et al, 2002; Jepsen, 2002). A i m s o f the present immunohistochemical and scanning electron microscope study were to determine:  1) i f fibromodulin and  lumican are expressed in the dental and periodontal tissues o f normal (wild-type) mice and how their  distribution compares  to that o f the  class  I small leucine-rich  proteoglycans, decorin and biglycan, 2) how the distribution o f these four proteoglycans changes i n mice that are missing lumican, fibromodulin or both o f the genes that code for these proteoglycans, 3) i f lumican and fibromodulin effect  81  collagen fiber bundle  fiber bundle morphology and organization in the dental tissues. This study shows for the first time, the major roles o f fibromodulin and lumican i n regulation o f periodontal ligament structure.  5.1 The Mouse Model In order to study the function o f fibromodulin and lumican, the periodontal tissues o f adult fibromodulin, lumican and double knockout mice were analyzed in this study. Although the anatomical nature o f mouse dentition is different from that o f humans, the structure and organization o f the periodontal tissues is similar. A s a consequence, the essential features o f periodontitis are held in common with humans (Page & Schroeder, 1982). Since the continuously erupting incisor teeth o f mice are structurally and functionally different than human teeth (Page & Schroeder, 1982), only the molar teeth were included i n this analysis. M i c e have 3 molars in each quadrant. U p o n reaching occlusion, which occurs at about 1 month o f age, the molars continue to erupt i n a buccoocclusal direction. During continued eruption, cementum is deposited apically and cusp tips wear away to compensate (Page & Schroeder, 1982).  5.2 Fibromodulin And Lumican Are Abundantly Expressed In Oral Tissues Fibromodulin, lumican, decorin and biglycan were all localized in the periodontal ligament o f wild-type mice i n the present study. This supports findings o f previous studies (Hakkinen et al, 2000; Cheng et al, 1999; Ababneh et al, 1999; Hakkinen et al, 1996; Hakkinen et al, 1993; Watanabe et al, 1998). This study localizes for the first time in the healthy adult C D - I wild-type mouse, fibromodulin and lumican to the gingival  82  connective tissue, and fibromodulin, decorin and biglycan to pulp. Fibromodulin was localized in the predentin, pulp, periodontal ligament and connective tissue o f the gingiva and mucosa at similar intensities (++) and to the basal cells o f the epithelium to a lesser degree (+) while no immunoreactivity was detected i n cementum or bone. Lumican was localized in the connective tissue o f the pulp (+), periodontal ligament (++), gingiva (+++)  and mucosa (+++)  in increasing intensity in the order described, a similar  distribution to that o f decorin. N o lumican reactivity was observed in bone, cementum, dentin or epithelium, although a slight staining could be seen in the predentin layer. Biglycan immunoreactivity was located in the connective tissue o f pulp (+), predentin (+), periodontal ligament (+++), gingiva (+++) and mucosa (++). Moreover, gingival connective tissue appeared more intensely stained than mucosal connective tissue. Decorin showed a similar distribution pattern to lumican with localization occurring in pulp (+), predentin (+), periodontal ligament (++) and gingival (+++) and mucosal (+++) connective tissues. The most intense staining o f decorin appeared i n the connective tissue of the gingiva and mucosa with most intense staining occurring in the area immediate subjacent the epithelium. Thus, decorin and lumican seem to be more strongly expressed in gingival and mucosal connective tissue as compared with periodontal ligament while biglycan is most abundantly expressed i n periodontal ligament and fibromodulin shows relatively even expression level in various periodontal connective tissues.  This study did not localize biglycan in alveolar bone, although it is abundantly expressed in bone (Bianco et al, 1990). Furthermore, we did not localize the other small leucine-rich proteoglycans to the other dental hard tissues, although decorin, biglycan, fibromodulin  83  and lumican were expressed i n the predentin to varying degrees. Previous studies have demonstrated the existence o f these proteoglycans in mineralized tissues (Hall et al, 1997; Embery et al, 2001; Goldberg et al, 2002; Cheng et al, 1996; Cheng et al, 1999; Ababneh et al, 1999; Bartold et al, 1990). The lack o f immunospecificity for these proteoglycans in the oral hard tissues in our study may be due to their loss during the demineralization treatment or the fixation, dehydration and embedding steps during processing (Goldberg et al, 2002). Although decorin and biglycan have been detected in epithelial cells o f the gastric tissue using immunohistological techniques (Pohle et al, 2001) and biglycan has been detected in epithelium o f developing human dermal tissue (Bianco et al, 1990), no decorin or biglycan was found i n oral mucosal epithelium supporting findings o f previous reports on dental tissues (Hakkinen et al, 1993; Hakkinen et al, 2000).  Slight fibromodulin immunoreactivity was noted i n the basal epithelial  layers o f fibromodulin knockout mice. Since these mice are lacking the fibromodulin genes necessary to synthesize fibromodulin, it is believed that the staining may be the result o f cross-reactivity with other proteins i n this tissue. The distribution o f the small leucine-rich proteoglycans in different dental tissues would suggest that they have unique functional roles i n the tissues where they accumulate. Several o f the small leucine-rich proteoglycans show an overlap i n distribution, suggesting a possible overlap in function in these tissues.  (  5.3 Fibromodulin And Lumican Compensate For Each Other To determine i f any compensation upregulation was occurring among the small leucinerich proteoglycans in the knockout mice, mesiodistal sections o f fibromodulin, lumican,  84  double knockout and wild-type mice were stained with anti-lumican, anti-fibromodulin, anti-biglycan and anti-decorin antibodies using standardized procedures. It was found that fibromodulin was more intensely stained i n the periodontal ligament o f lumican knockout mice than wild-type mice and lumican was more intensely stained in the periodontal ligament o f fibromodulin knockout mice than wild-type mice. These findings may suggest a compensation mechanism between lumican and fibromodulin in single knockout mice.  Compensation between lumican and fibromodulin is an expected finding since both o f these small leucine-rich proteoglycans share the same binding site on type I collagen with fibromodulin having a higher affinity (Svensson et al, 2000). Compensatory changes in expression between  different  small leucine-rich proteoglycans  have  already  been  observed using protein analysis (Svensson et al, 1999; Svennson et al, 2000; Ezura et al, 2000; Jepsen et al, 2002) or suggested based on severity o f double knockout phenotypes compared to single knockout phenotypes (Ameye et al, 2002; Corsi et al, 2002). For lumican and fibromodulin, a compensation mechanism was noted previously. Jepsen et al (2002) and Svensson et al (1999) noted a 2-fold and 4-fold increase in fibromodulin respectively using lumican protein analysis in fibromodulin knockout mouse flexordigotorum longus tendon o f the limb and tail tendon respectively. Jepsen et al (2002) reported, however, that lumican knockout mice expressed less fibromodulin than w i l d type mice, a contradiction to this report. One reason for this might be that different small leucine-rich proteoglycans, including fibromodulin, may play different roles i n different tissues  since  Jepsen's  study  was  on  mouse  85  tendon.  In this  study,  we  used  immunohistochemical stains to analyze the abundance o f proteoglycans. This method is only semi-quantitative, and should be confirmed by biochemical analysis of, these proteoglycans in the periodontal ligament. However, results o f the staining intensities observed correlate with the severity o f phenotypes observed in the knockout mice.  5.4 No Gross Morphologic Changes In Jaws And Teeth In Knockout Mice The jaws o f mice deficient i n fibromodulin, lumican or both were intact and all teeth were present. A difference was noted in lengths o f maxillary mouse jaws among the groups. The mean mouse j a w length o f the single and double knockout mice were smaller than the that o f the wild-type mice. The jaws o f the double knockout mice, on average, were smaller than either o f the single knockout varieties. Although these findings were not found to be statistically significant in this report, they do support previous findings that mice deficient i n lumican, fibromodulin or both have smaller bodies than wild-type mice with double knockout mice being the smallest (Jepsen et al, 2002). Perhaps the reason for the lack o f significance i n this study is due to small sample size used. A s well, a more accurate measure o f j a w length may be reflected i n mandibular jaw length measurements rather than maxillary j a w length as reported here. It should be noted, however, that a previous report noted that fibromodulin knockout mice developed with no gross anatomical defects and grew to normal size (Svensson et al, 1999) while lumican knockout mice were significantly smaller (70-80% body weight) than wild-type littermates at birth (Chakravarti et al, 1998).  86  Fibromodulin,  lumican and  double  knockout  mice  showed  no  gross  structural  abnormalities i n hard dental tissues, including bone, cementum and dentin (excluding enamel), compared to those o f wild-type mice using the investigative  techniques  employed in this study. A l l teeth o f the knockout and wild-type mice were present and fully erupted and there was no evidence o f bone loss or inflammation that would indicate periodontitis i n any o f the mice studied. Periodontitis does not usually occur naturally in mice (Page & Schroeder, 1982), although some studies have reported it i n some wild strains (Sheppe et al, 1965; Wiebe et al, 2001). To this extent, it is not surprising that no alveolar bone loss indicative o f periodontal disease was found in any o f the knockout mice. Bone loss was found associated with one fibromodulin knockout mouse as determined by the presence o f class II furcation defects (Wiebe et al, 2001), which may be a result o f hair impaction (Page & Schroeder, 1982). N o bone loss was detected i n any of the other mice.  5.5 Most Oral Soft Tissues Showed No Gross Morphologic Changes N o gross structural morphologic changes were noted among the dental soft tissues, including pulp, mucosal and gingival epithelium, mucosal and gingival connective tissues in any o f the knockout mice compared to wild-type mice.  5.6 Collagen Fiber Bundles Of The Knockout Periodontal Ligaments Have Altered Morphology Some general trends were noted i n the periodontal ligament o f all mouse  groups.  Collagen fiber bundles were more disorganized i n the apical third o f the tooth root, which  87  might be due to the continuing migration o f these teeth and the constant, quick deposition of cementum at the apex (Page & Schroeder, 1982). For this reason, we used the coronal and middle third o f the root to analyze collagen fiber bundle morphology and organization i n the periodontal ligament. A s well, collagen fiber bundles on the buccal surface o f the root were more organized than those on the lingual surface o f the root as seen i n H & E , P T A H , P S R and immunostained bucco-lingual sections, which might be explained by the fact that the teeth erupt i n an occluso-buccal direction (Page & Schroeder, 1982).  Wild-type mice displayed collagen fiber bundles i n a sheet-like arrangement with smooth outlines, o f relatively even thickness that traversed the space between tooth and bone such that fibers could almost be traced across the periodontal ligament space. A s well, the spaces between the fiber bundles were evenly distributed. These findings support previous reports i n the literature. Fiber bundles cross the entire width o f the periodontal space but branch en route and anastomose with adjacent fiber bundles to form a complex three dimensional network (Sloan, 1978b; Sloan 1979b) and as a result, it may be impossible to trace an intact network o f bundles across the periodontal space (Sloan & Carter, 1995). Collagen fibrils in the rat periodontal ligament (Berkovitz et al, 1981) and of the mouse tendon (Ezura et al, 2000) have been reported to have a unimodal distribution with collagen fibrils being small and o f essentially equal diameter. However, these reports are based on studies o f collagen fibrils  (diameter ~50nm) using a  transmission electron microscope, whereas this study analyzed collagen fiber bundles (diameter ~10um) with a scanning electron microscope. The sheet-like arrangement o f  88  collagen fiber bundles has been reported previously in scanning electron microscope study o f continuously erupting rat incisors (Carter & Sloan, 1994). Molars were analyzed in this study confirming a three-dimensional model o f the periodontal ligament (Sloan, 1978a). In this model, the bundles closest to bone and cementum are round with the alveolar bundles being larger than the latter. The middle zone o f fiber bundles in this model are arranged into thin sheets that form a series o f flattened compartments running along the axis o f the tooth.  Fibromodulin, lumican, and double knockout mice displayed periodontal ligaments with abnormal morphology and organization o f the collagen fiber bundles with a relatively increased amount o f space between them. Although the basic fiber bundle orientation was apparent in many samples o f all four groups o f mice, the periodontal ligaments o f the knockout mice were characterized by disorganized fiber bundles o f varying widths as seen under light and scanning electron microscopy. A l l three knockout ligaments had relatively thinner bundles than wild-type. Fibromodulin knockout fiber bundles appeared clumpy, lumican knockout bundles appeared stringy and double knockout ligaments contained fiber bundles with both o f these characteristics. Moreover, scanning electron micrographs o f the periodontal ligaments revealed the presence o f several small branches protruding from the main collagen fiber bundles present i n all knockout groups.  Graham et al (2000) showed i n a study o f collagen fibril fusion i n tendon that fibroblasts synthesize transient, early fibril intermediates that fuse in an end-to-end fashion to generate long fibrils. Small leucine-rich proteoglycans were noted to cover the long  89  surfaces of these fibrils but not the tips of the fibrils, thus appearing to act as inhibitors of lateral  fusion.  intermediates  When  small  aggregated  leucine-rich  laterally,  thus  proteoglycans  were  absent,  demonstrating  that  small  the  fibril  leucine-rich  proteoglycans promote end-to-end fusion o f collagen fibril intermediates and inhibit lateral fusion. Ezura et al (2000) proposed that collagen fibril intermediates are stabilized through their interactions with fibril-associated macromolecules,  such as the small  leucine-rich proteoglycans lumican and fibromodulin while fusion o f fibril intermediates occurred in a multi-step manner to generate a mature fibril. In this model, based on studies o f mouse tendon in lumican and fibromodulin and double knockout mice, it seems that lumican and fibromodulin have temporal functions associated with this process whereby  lumican functions  together with fibromodulin in the  early  stages with  progressively less lumican and progressively more fibromodulin acting as the tissue matures.  While these models offer information on the formation of collagen fibrils, they do not explain how suprafibrillar structures, like the collagen fiber bundles of the periodontal ligament, are formed. The collagen binding model of Weber et al (1996) and rotary shadowing-electron  microscopy  (Scott,  1996)  suggest  that  small  leucine-rich  proteoglycans are horseshoe shaped which, based on decorin structure, would have just enough space i n the concave portion for a single tropocollagen triple helix to bind (Weber et al, 1996). I f this is so, then what role do small leucine-rich proteoglycans play i n the formation o f collagen fiber bundles of the periodontal ligament?  90  Different tissues have different suprafibrillar architectures (Giraud-Guille, 1996). For example, cornea, bone osteons, tendon and dermis have individual collagen fibril arrangements. Corneal fibrils are arranged in multiple layers or parallel lamellae, bone osteons have concentric lamellae, tendon fibers exhibit a crimp in their structure and dermis has a complex three-dimensional weave. These different suprafibrillar structures are characterized by distances on the scale o f several micrometers, compared to the 0.3 um length o f a single collagen molecule (Hulmes, 2002).  The periodontal ligament is comprised o f parallel collagen fiber bundles (principle fibers) that extend across the periodontal space in a complex wavy course (Sloan, 1978b). They are named according to their location and direction. A "bundling mechanism" was described by Yamamoto & Wakita (1991) and is based on observations o f the developing rat molar. This mechanism proposes that loose fibril bundles develop into tight fibril bundles as root formation takes place and involves the formation o f compartments within the tissue by cellular processes. However, how the exact mechanism involved i n the bundling o f fibers remains unclear, but seems to most certainly involve the small leucinerich proteoglycans, fibromodulin, lumican (as seen in this study) and decorin (Hakkinen et al, 2000). It is possible that the constituent collagen fibrils arrange themselves parallel to each other forming in essence, one large collagen fiber, in which case the so-called "collagen fiber bundle" would be more appropriately termed a "collagen fibril  bundle"  (Fig. 23). Alternatively, the fibrils may be arranged in smaller subunits (fibers) that come together to form bundles o f collagen fibers (Fig. 24). The term "collagen fiber bundle" is frequently associated with the periodontal ligament in the literature and would certainly  91  imply the latter. This model is supported by the scanning electron microscope findings in this study using a micrometer scale since collagen fibrils  have a reported  diameter  measured i n nanometers.  Scanning electron microscopy o f fibromodulin, lumican and double knockout mouse periodontal ligaments revealed the presence o f very thin filamentous structures that appeared to be branching from the larger collagen fiber bundle. These branching structures were not present i n wild-type mice. The filamentous structures seen may represent single collagen fibers that have broken away or failed to fuse with the rest o f the collagen fiber bundle and is suggestive o f altered lateral fusion o f the fibers within the fiber bundle. It is clear that small leucine-rich proteoglycans play a role in the organization o f the collagen fiber bundles o f the periodontal ligament but seems likely that the mechanism by which they do so is different than that suggested at the molecular / fibril level.  Fibromodulin knockout mice in this study displayed periodontal ligament fiber bundles that were thinner than those o f the wild-type but thicker than lumican knockout mice. Those o f lumican knockout mice seemed  relatively thinner  as seen under light  microscope in coronal and mid-root sections stained with H & E , P T A H , under polarizing light microscope with P S R stained sections and in scanning electron micrographs. Jepsen et al (2002) and Ezura et al (2000) reported in studies o f mouse limb tendon that fibromodulin knockout mice had a higher number o f thinner fibrils and lumican knockout mice had a higher number o f thick fibrils compared to wild-type mice. Moreover, it  92  appears that lumican and fibromodulin have differential expression during mouse tendon development  with lumican being expressed  early and acting predominantly  and  fibromodulin expression increasing with age assuming a predominant role in collagen fibrillogenesis (Ezura et al, 2000). From this, it seems that lumican is required for early fibril development while fibromodulin in required for maturation o f collagen fibrils. The different findings may be due to the different levels o f analyzation since these studies looked at individual collagen fibrils using a 50 nm scale and this study looked at fiber bundles using a 10 urn scale. In the present study, we analyzed at histological and scanning  electron  microscope  levels, the  organization  and  morphologies  of  the  periodontal ligament fiber bundles that are formed by collagen fibers that have joined together. W e did not analyze the diameter o f the individual collagen fibers. It is possible that the lack o f proteoglycans may effect differently the formation o f the collagen fibrils and how these fibers j o i n to form highly organized fiber bundles typical o f the periodontal ligament.  The rapid turnover o f the periodontal ligament may result in phenotypes that differ from that i n other tissues. It might also be possible that the constant renewal experienced in the periodontal ligament may alter the expression o f lumican and fibromodulin i n this tissue and the phenotypes expressed. The turnover rate o f the periodontal ligament is very high and has been estimated to be 15x that o f skin and 5x that o f alveolar bone with respects to its collagenous components  (Sodek et al, 1977). Turnover and remodeling in the  periodontal ligament involve rapid synthesis and breakdown o f matrix components most notably the meshwork o f type I collagen fiber bundles that stretches out  93  between  cementum and bone (Beersten et al, 1997). One might expect this turnover rate to be quite high i n the mouse relative to other animal models especially humans considering their shorter life span and that their molars are continuously erupting (Page & Schroeder, 1982).  The findings o f collagen fiber bundle morphology and organization were  further  substantiated with analysis o f collagen fibers labeled with anti-type I collagen antibody. The periodontal ligament o f the wild-type mice were found to have a diffuse staining pattern compared to those o f knockout mice which demonstrated a more discreet staining o f the fibers. This finding might suggest homogenous fiber bundle diameters i n the periodontal ligament o f the wild-type mice. The increased inter-bundle spacing and heterogenous nature o f the fiber bundles o f the knockout periodontal ligaments may make the staining appear more discreet. Hakkinen et al (2000) noted that decorin knockout mice, which were found to have altered collagen fibril diameters by transmission electron microscopy, had more diffuse staining o f the periodontal ligaments than wild-type mice. The reason for this different collagen fiber organization at the light microscope level may be the effect o f different small leucine-rich proteoglycans being investigated.  Picrosirius red stain and polarizing light microscopy was used i n this study to investigate periodontal ligament collagen fiber bundle morphology. Picrosirius red is a strong anionic dye that stains collagen i n such a way that the long axes o f the elongated dye molecules are aligned with the collagen molecules resulting in an enhanced birefringency and is thus thought to be somewhat specific to collagen (Junqueira et al, 1979), although  94  Sirius red has been reported to bind to other basic amino acids v i a electrostatic forces (Nielsen et al, 1998). The enhanced birefringency o f collagen can be appreciated with a polarizing light microscope.  Previous studies using this technique have reported that fibers o f varying thickness show different colors under the polarizing light microscope. Some report that thick collagen fibers (1.6 - 2.4 urn) appear as yellow / green in color while thinner collagen fibers (<.8 um) show a red / orange polarization color (Hirshberg et al, 1999). Other work determined that thin fibrils show a yellow / green polarization color and thick fibers show a red / orange polarization color (Dayan et al, 1989). The different collagen types being analyzed as well as perhaps the different tissues being examined can explain the discrepancy i n these reports. The different colors have been attributed not only to varying collagen fiber thickness, but also to the tightness o f the pack between collagen fibers as well as their alignment. Tightly packed, well-organized collagen fibers tend to have a polarization color o f red / orange when stained with picrosirius red (Dayan et al, 1989). The different staining colors were not detected in this study presumably because the fibers were all within the "thin range" and all stained red / orange. Collagen fiber diameters o f mammalian periodontal ligament are relatively small with mean diameters in the order o f 45-55 n m with a unimodal distribution (Berkovitz et al, 1981; Luder et al, 1988). In other connective tissues, like tendon, fibril diameters may reach 250 nm. There is evidence that the diameter o f collagen fibers increase with age and the small diameter of the periodontal ligament fibers could be the result o f high turnover rate o f this structure (Berkovitz et al, 1981) or a lack o f mature collagen fibrils. Crocodile have been  95  reported to have large diameter fibril up to 250 nm, where collagen fiber turnover is slow (Berkovitz and Sloan, 1979). There are no significant differences i n fibril diameter in continuously growing incisors (higher turnover rate) and non-continuously growing rat molars (lower turnover rate) (Berkovitz and M o x h a m , 1989). A t the same time there is no increase in human fibril diameter in the maturing human periodontal ligament (Luder et al, 1988). There is no reported diameter for mouse periodontal ligament fiber bundles, although one would presume that it was smaller than those reported above given the smaller dimensions o f the mouse dentition and the presumed rapid turnover o f the fiber bundles around the continuously erupting teeth (Page & Schroeder, 1982).  Analysis o f sections stained with picrosirius red revealed the presence o f dark bands that appeared to be associated with the collagen fiber bundles o f wild-type, fibromodulin and lumican knockout mouse periodontal ligaments. The banding is consistent with previous reports o f crimping o f collagenous tissues (Diamant et al, 1972; Keller & Gathercole, 1976). The "crimp" is a term that is used to describe the quantifiable periodicity o f structure o f various collagenous tissues. In polarized light, the crimp can be recognized as a regular banding o f dark lines across a collagenous bundle. Polarized light analysis of the periodontal ligament fiber bundles has demonstrated this crimping arrangement previously (Diamant et al, 1972). The increased "banding" seen in the single knockout mice and relative absence i n the double knockout mice further suggests alterations i n the collagen fiber bundles o f the periodontal ligament o f these mice.  96  r  5.7 More Blood Vessels In Fibromodulin Knockout Periodontal Ligament Using H & E and PTAH-stained sections, blood vessels appeared to be more numerous on the buccal side o f the periodontal ligament than the lingual side in the wild-type, lumican knockout and double knockout groups while a more even distribution was found in the fibromodulin knockout group. Having a higher number o f blood vessels on one surface than the other might be explained by the direction o f eruption o f the molar teeth. One would expect a higher number o f blood vessels i n mouse molars to be on the lingual / tension side o f the periodontal ligament since the molars erupt in a buccal direction (Rygh & Brudvik, 1995) although the forces o f eruption and migration cannot be compared to horizontal orthodontic forces. The significance o f the more even distribution in the fibromodulin knockout mice is unclear from this study but may be due to altered eruption o f the teeth due to alterations in the collagen fiber bundles o f the periodontal ligament. Double knockout mouse periodontal ligaments had more blood vessels than the lumican knockout mice and wild-type mouse periodontal ligaments. Fibromodulin knockout mice, however, displayed more blood vessels in the periodontal ligaments that any o f the other groups. These results were only found to be statistically significant when lingual surfaces were compared.  A case report o f a type VIII Ehlers-Danlos syndrome patient receiving orthodontic therapy noted that the patient experienced severe apical root resorption and alveolar bone loss during the orthodontic therapy. The author suggested that these findings might be attributed to altered collagen fibers in the periodontal ligament making them weaker and unable to withstand routine orthodontic forces on the tension side o f the periodontal  97  ligament. He suggested  that this decreased  structural  integrity resulted  i n over-  compression on the opposite side o f the tooth which then led to increased hyalinization and circular disorders that were enhanced by vascular fragility leading to resorption o f cementum and dentin (Karrer et al, 2000).  Previous i n vitro and in vivo studies suggest that at least one small leucine-rich proteoglycan, decorin, is involved in angiogenesis (Jarvelainen et al, 1992; Schonnher et al, 1999; Gutierrez et al, 1997; Nelimarkka et al, 2001). In vitro studies suggest that decorin promotes blood vessel growth since: 1. large vessels express decorin when they begin to grow new capillaries (Jarvelainen et al, 1992) and 2. macrovascular endothelial cells form tubes i n collagen lattices when they are transduced to overexpress decorin in vitro, while control cells do not (Schonnher et al, 1999). While the exact mechanism by which decorin regulates blood vessel growth is unclear, it may be due to its ability to bind collagen or interact with T G F - p or E G F R . The findings o f the present study suggest that other small leucine-rich proteoglycans, such as fibromodulin, may also play a role i n this process. The fact that fibromodulin and double knockout mouse periodontal ligaments had increased amounts o f blood vessels suggest that presence o f fibromodulin would down-regulate blood vessel growth.  H & E and PTAH-stained sections were analyzed and effort was made to count only those structures that contained a lumen, lined with endothelial cells and what appeared to be blood cells inside. In some sections the blood cells appeared to be stained o f equal intensity to pulp cells (presumably blood cells) and darker than epithelial cells, but in  98  other sections both blood cells in the periodontal ligament and pulp stained with equal intensity as the epithelium raising questions to their true identity. To confirm these findings, further research should be employed using immunohistochemical techniques using antibodies against endothelial cells (antibodies for CD31 or von Willebrands Factor) to accurately identify blood vessels. One possibility is that with collection of the blood vessel data, what appears to be or what was counted as one blood vessel might actually be one blood vessel that was cut several times during sectioning. This would be dependent on the angulation of the cut or the curvature of the vessel. In any case, it is clear that the fibromodulin was different than the other mouse groups and further investigation is warranted to determine what these differences are.  5 . 8 More Disruptions In Fibromodulin Knockout Mouse Periodontal Ligaments In histological sections, disruptions or breaks in the periodontal ligament appeared more frequently in the of fibromodulin knockout mice than the other mouse groups (not statistically significant). It is possible that the breaks are a result of sectioning procedures and represent artifacts in the periodontal ligament. Interestingly, however, the wild-type mice had very few breaks compared to any of the knockout mice. It is possible that the knockout mouse ligaments are lacking the structural integrity to resist these breaks during the processing of the samples. Small leucine-rich proteoglycans play a role in collagen fibrillogenesis and they also have been noted to provide the tendon with maintenance of hydration thereby increasing tissue strength to tensile stresses (Cribb & Scott, 1995). The existence of thinner fibrils due to decreased lateral fusion (Ezura et al, 2002) or reduced cross-linking of collagen molecules (Kadler, 1995) might also reduce the structural  99  integrity of this tissue. Other studies have shown that mice lacking lumican and decorin have increased skin fragility (Danielson et al, 1997; Chakravarti et al, 1998; Corsi et al, 2002) and those lacking lumican, fibromodulin or both (Jepsen et al, 2002) and biglycan, fibromodulin or both (Ameye et al, 2002) have dramatically less tendon stiffness. Certainly, the seemingly increased susceptibility of the periodontal ligament to tearing raises questions about the structural integrity of the periodontal ligament to periodontal breakdown and further analysis is required to determine the exact role of lumican and fibromodulin in providing strength to the periodontal ligament.  5 . 9 Fibromodulin Knockout, Lumican Knockout And Double Knockout Mice Express Mild Oral Phenotypes In Vivo Mice deficient in a small leucine-rich proteoglycans exhibit microscopic and macroscopic phenotypes. Biglycan knockout mice show macroscopic phenotypes in that they exhibit decreased growth rate characterized by decreased bone mass (Xu et al, 1998). They also exhibit a microscopic phenotype characterized by thin skin but no macroscopic phenotype is seen in the skin of these mice (Corsi et al, 2002). Decorin knockout mice exhibit macroscopic changes in skin characterized by increased skin fragility associated with thinning of the skin (Danielson et al, 1997). They also exhibit microscopic changes in bone characterized by decreased average fibril diameter and size range compared to wild-type mice but no macroscopic phenotype as a result of this (Corsi et al, 2002). These findings might be explained by the relative abundance of biglycan in bone relative to decorin and decorin in skin relative to biglycan while ultrastructural changes noted may imply similar roles in these tissues. Similarly, lumican deficiency results in a  100  macroscopic  phenotype  characterized  by  decreased  transparency  o f the  cornea  (Chakravarti et al, 1998), a tissue where it is abundant. Macroscopic effects o f missing small leucine-rich proteoglycans seem to be more severe than microscopic ones that can only be observed at the microscopic level.  The apparent morphological changes in the periodontal ligament collagen fiber bundles seen in this study expressed by lumican, fibromodulin and double knockout mice appeared not to be associated with clear functional defects. This may be due to the existence o f other small leucine-rich proteoglycans compensating for those that are missing thereby  partially masking the  effect  o f the  missing small  leucine-rich  proteoglycans. It has been suggested that the small leucine-rich proteoglycans may substitute or compensate for each other i n these defect states (discussed above). Our findings also suggested that fibromodulin and lumican compensated for each other when one o f the proteoglycans was missing. However, the most likely explanation is that mice do not naturally get inflammatory periodontal disease and therefore, structurally abnormal periodontal ligament cannot contribute to increased susceptibility to the disease. Perhaps i f the animals are induced to develop periodontal disease, the functional significance o f the lack o f certain proteoglycans could be better answered.  101  Chapter Six - Conclusions And Future Directions 6.1 Conclusions 1.  Fibromodulin, lumican, decorin and biglycan are abundantly expressed in the connective tissues o f the periodontal ligament, gingiva and pulp.  2. The absence o f fibromodulin, lumican or both results in morphological alterations o f the collagen fiber bundles i n the periodontal ligament characterized by altered surface morphology, relative increase in inter-bundle spacing, increased random organization o f collagen fiber bundles as seen under light, polarizing and scanning electron microscopy. 3. A more severe phenotype is seen in mice lacking both fibromodulin and lumican with regard to collagen fiber bundle morphology than single knockout or w i l d type mice suggesting a synergistic effect. 4. Fibromodulin and lumican appear to compensate for each other i n the periodontal ligament o f mice. 5. The absence o f fibromodulin and / or lumican may result i n increased number o f blood vessels i n the periodontal ligament. 6. The absence o f fibromodulin and / or lumican may result in decreased integrity o f the periodontal ligament because there appeared to be an increased number of tears i n the ligaments o f these mice after sectioning. 7. This study suggests that collagen fibers bind together to form collagen fiber bundles and fibromodulin and lumican play a role in this process.  102  6.2 F u t u r e Directions Although significant progress has been made in the membership, structure and function of small leucine-rich proteoglycans, much work remains to be done. A s more members o f this family are targeted for deletion in transgenic mice, and as we understand more about the molecular structure o f the small leucine-rich proteoglycans we may start to approach an understanding o f their role in oral tissues.  Future research with regards to fibromodulin and lumican can be done to determine i f the alterations noted in this study would decrease the integrity o f the periodontal tissues to the extent that it would lead to an increased susceptibility to periodontal destruction o f the collagen fiber attachment o f the teeth. This can be done by inducing periodontitis in transgenic mice. A s well, further research is required to determine the nature and extent of the role o f fibromodulin and lumican on the periodontal ligament vasculature. D o these small leucine-rich proteoglycans regulate angiogenesis in the ligament and i f they do what are the  repercussions  o f this? 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(DS = dermatan sulfate; P G = proteoglycan; K S = karatan sulfate; C S = chondroitin sulfate; G A G = glycosaminoglycan; L R R = leucine-rich repeat) (Neame and Kay, 2000; Iozzo, 1998). Other names  GAG (#)  Chromosome  Exons  Cys Spacing  LRR  EMC2  Human 9q21.3  8  CX CXCX6C  10  Asporin  8  CXjCXCXdC  10  8  CX^CXCXsC  10  8  CX3CXCX6C  10  8  CXjCXCXoC  10  Decorin (36kDa)  DS-PG-II; PG II; PGS2; PG40  DS/CS * (1**)  Human 9q229q21.3 Human 12q21.3-23 Mouse 10  Biglycan (38kDa)  DS-PG-I; PG-I; PG-S1  DS/CS (1-2)  Human Xq28 Mouse X  PLAP-1  3  (periodontal  ligament associated protein)  Osteoadherin  Osteomodulin  PRELP-1 (proline  KS (2-3)  9q22-9q21.3  3  CX CXCX C  10  KS (2-3)  Human lq32 Mouse I  3  CX,CXCX C  10  argenine-rich end leucine-rich repeat protein)  3  9  9  Keratocan  KSPG 37A protein  KS (3-5)  Human 12q Mouse 10  3  CX CXCX,C  10  Lumican (38kDa)  KSPG 37B  KS (3-4)  Human 12q21.3q22 Mouse 10 Human lq32 Mouse 10  3  CX CXCX,C  10  3  CX3CXCX9C  10  Human 9q229q21.3  7-8  CX CXCX C  6  Human lq32 Mouse I  7-8  CX CXCX,,C  6  Human 12q21.3 Mouse 10  7-8  CX CXCX«C  6-7  Fibromodulin (42kDa) Mimecan  KS (2-4)  Osteoglycin, KSPG25, OIF (osteoinductive factor)  KS (2-3)  Opticin Epiphycan (36kDa)  PG-Lb, DSPG3  DS/CS (2-3)  Chondroadherin Nyctalopin * adult chick cornea contains keratan sulfate. ** avian decorin contains two glycosaminoglycan side chains.  128  3  3  2  6  2  2  Table 2. Glycosaminoglycans (GAG) of oral tissues (CS = chondroitin sulfate; DS = dermatan sulfate; HA = hyaluronic acid; HS = heparan sulfate; KS = keratan sulfate; PDL = periodontal ligament) (refs in text). Dental Tissue G A G Bone CS, DS, HA, HS, KS Dentin CS, HA, HS, KS Cementum CS, DS, HA, KS, HS Gingiva DS, HA, CS, HS, KS PDL DS, CS, HA, HS, KS Pulp DS, CS, HA, KS  129  Table 3. Components o f dental soft tissues ( P D L = periodontal ligament, G A G s = glycosaminoglycans; K T = keratinized tissue) (Ten Cate, 1994). Cellular PDL  Osteoblasts, osteoclasts, fibroblasts (35% in mouse molar), epithelial cell rests of malassez, macrophages, cementoblasts, undifferentiated mesynchymal cells  Connective Tissue (Lamina propria)  Fibroblast, histiocyte, macrophage, monocyte, mast cell, PMNs, lymphocyte, plasma cell, endothelial cell, inflammatory cells (lymphocyte, plasma cells)  Gingival Epithelium (KT) Basement Membranes  Keratinocytes, nonkeratinocytes (melanocytes, Langerhans' cells, Merkel's cells, lymphocytes)  Pulp  Odontoblasts, fibroblasts, undifferentiated mesenchymal cells, macrophages, other immunocompetent cells  130  Acellular Collagen fiber bundles (51% in mouse molar), collagen types I, III, V , VI, V l l , XII, ground substance (glycoproteins, GAGs, PGs, glycolipids), vessels, nerves Collagen I, III, IV, VII, V (in inflamed tissue), ground substance (PGs, glycoproteins), elastic fibers, fibronectin, vessels, nerves Hyaluronic acid  Collagen IV and VII, laminins, proteoglycans (perlecan), entactin, bullous pemphigoid antigen, other glycoproteins Collagen I & III in a ratio of 55:45, ground substance (GAGs, glycoproteins, water), vessels, nerves  Table 4. Collagen composition in dental and periodontal tissues (Ten Cate, 1994; Ababneh et al, 1999).  Tissue  Collagen type  Bone  I III IV V  Location  References  Bone matrix, Sharpey's fibers Sharpey's fibers, endosteal spaces Basement membranes o f blood vessels and nerves Bone matrix  Takitaetal, 1987  Cementum  VI X XI I  PDL  III IV V VI I  Principle and secondary fibers  III  Principle and secondary fibers  IV V  Basement membranes o f blood vessels and nerves Collagen fibers  VI  Fine fibers  Sharpey's fibers, fibrillar cementum Sharpey's fibers and cementum  I  IV  Lamina propria. M a i n component o f all layers. Lamina propria. M a i n l y i n upper layers under epithelium and within blood vessel walls. Basement membranes  V  Collagen fibers, blood vessels  VI  Microfibrils  III  Dentin  I  Pulp  V I III  Romanos et al, 1991; Becker et al, 1986 Bronckers et al, 1986; Becker et al, 1986 Becker et al, 1986  Becker etal, 1991  Wang etal, 1980  Sharpey's fibers  XII Gingiva  Wang etal, 1980  Dentin matrix Course cross banded fibrils Fine branched filaments associate with plasma membranes and fibroblasts  131  Takita et al, 1987; Becker et al, 1991; Butler et al, 1975; Huang et al, 1991 Wang et al, 1980; Takita et al, 1987; Becker etal, 1991; Butler et al, 1975; Huang etal, 1991; Wang et al, 1980 Romanos et al, 1991  Romanos et al, 1991; Becker et al, 1991 Becker et al, 1991; Romanos et al, 1991; Sloan etal, 1993 Dublet et al, 1988; Karimbux et al, 1992 Chavrier et al, 1981; 1984; 1994; 1999; Takitaetal, 1987 Wang et al, 1980; Chavrier et al, 1981; 1984; 1994; 1999; Takita et al, 1987; Wang etal, 1980 Chavrier et al, 1981; 1984; 1994; 1999; Becker etal, 1986 Romanos et al, 1991; Rabanus et al, 1991; Becker et al, 1986; Schuppan et al, 1986 Romanos et al, 1991; Rabanus et al, 1991; Becker etal, 1986 Takita et al, 1987; Magloire et al, 1983 Bronckers et al, 1986 Magloire et al, 1982 Martinez et al, 2000; Magloire et al, 1982; Wang etal, 1980  T a b l e 4. Collagen composition in dental and periodontal tissues (Ten Cate, 1994- Ababneh et  al, 1999).  Tissue  Collagen type  Location  References  Bone  I  Bone matrix, Sharpey's fibers Sharpey's fibers, endosteal spaces Basement membranes o f blood vessels and nerves Bone matrix  Takita et al, 1987  111  IV V  Cementum  VI X XI I  PDL  III IV V VI I  Principle and secondary fibers  III  Principle and secondary fibers  IV V  Basement membranes o f blood vessels and nerves Collagen fibers  VI  Fine fibers  Sharpey's fibers, fibrillar cementum  I III  IV V VI Dentin  I  Pulp  V I III  Romanes etal, 1991; Becker etal, 1986 Bronckers et al, 1986; Becker et al, 1986 Becker et al, 1986  Becker etal, 1991  Wang etal, 1980  Sharpey's fibers and cementum Sharpey's fibers  XII Gingiva  Wang etal, 1980  Lamina propria. M a i n component o f all layers. Lamina propria. M a i n l y i n upper layers under epithelium and within blood vessel walls. Basement membranes Collagen fibers, blood vessels Microfibrils Dentin matrix Course cross banded fibrils Fine branched filaments associate with plasma membranes and fibroblasts  131  Takita et al, 1987; Becker et al. 1991; Butler etal, 1975; Huang et al, 1991 Wang etal, 1980; Takita et al, 1987; Becker etal, 1991; Butler et al, 1975; Huang et al, 1991; Wang et al, 1980 Romanosetal, 1991  Romanosetal, 1991; Becker et al, 1991 Becker et al, 1991; Romanos et al, 1991; Sloan etal, 1993 Dublet et al, 1988; Karimbux et al, 1992 Chavrier et al, 1981; 1984; 1994; 1999; Takita etal, 1987 Wang et al, 1980; Chavrier et al, 1981; 1984; 1994; 1999; Takita et al, 1987; Wang et al, 1980 Chavrier et al, 1981; 1984; 1994; 1999; Becker etal, 1986 Romanos et al, 1991; Rabanus et al, 1991; Becker et al, 1986; Schuppan et al, 1986 Romanos et al, 1991; Rabanus et al, 1991; Becker etal, 1986 Takita et al, 1987; Magloire et al, 1983 Bronckers et al, 1986 Magloire et al, 1982 Martinez et al, 2000; Magloire et al, 1982; Wang etal, 1980  Table 5 . Components o f dental hard tissues ( H A = hydroxyapatite; P G s = proteoglycans; wt = weight; v o l = volume) (Ten Cate, 1994).  Bone  Inorganic Components 67% HA  Organic 33%  Components 25% collagen (I, V, VI, X, XI), 5% noncollagenous material (osteocalcin, sialoprotein (also known as osteopontin), phosphoprotein, osteonectin, bone specific protein), osteoblasts, osteocytes, osteoclasts  Cementum  50%  90%  Collagen (I, III, IV, VI), PGs, glycoproteins, phosphoproteins, cementoblasts, cementocytes  Dentin  70% (wt) 45% (vol)  20% (wt) 33% (vol)  Enamel  96%  Collagen (I, V), glycoproteins, PGs, phosphoproteins, plasma proteins, odontoblasts TRAP peptide sequence (Tyrosine rich enamel amelogenin protein) interlaced between the crystals  HA  "*  HA & minerals  4%  132  Table 6 . Types and distribution o f collagen. ( F A C I T = fibril-associated collagens with interrupted triple helices; P D L periodontal ligament) (Ten Cate, 1994; Bartold & Narayanan 1996; Kadler et al, 1996; Kadler, 1995; Becker et al, 1991; Dublet et al, 1988; Butler et al 1975- Huang et al, 1991; Sloan 1993; Karimbux et al, 1992; Rabanus et al, 1991). ' Types of collagen Fibrillar I  II III  V  XI FACIT  Basement Membrane Collagens Short Chain Collagens Domains MACIT's Multiplexins Others  Location Skin, bone, gingiva, PDL, cementum, dentin, pulp, tendon, ligament, cornea, inter-vertebral disc, kidney, muscle, salivary gland, cartilage, menisci, artery, granulation tissue, intracellular fibrils, calvaria, most other connective tissues Cartilage, vitreous humor, notochord, intervertebral disc, ear, developing bone, cornea, placenta, menisci Widespread distribution particularly where type I collagen is found. Embryonic connective tissue, pulp, PDL, skin, gingiva, aorta, bone, cartilage, tooth, ligament, muscle, eye, tendon, salivary gland, dentin, cementum, placenta, lung, kidney, menisci, liver, artery, Schwann cells, granulation tissue Widespread distribution particularly where type I collagen is found. Basement membranes, blood vessels, ligament, skin, dentin, gingiva, PDL, most soft tissues, corneal stroma, bone, spleen, placenta, muscle, ear, vitreous humor, teeth, menisci, oral mucosa, mammary gland, cartilage, perichondrium, lung, granulation tissue, kidney, artery, uterus Typically found associated with type II collagen predominantly in cartilage, heart, skeletal muscle, calvaria, skin, brain Cartilage, vitreous and developing cornea Soft tissue, including PDL All tissues  IX XII XIV XVI XIX IV  Basement membranes  VIII  Descemet's membrane, fetal heart  X XIII  Cartilage, pericellular matrix Fetal epidermis, intestinal mucosa  XVII XV XVIII VI VII  Soft tissues including the PDL, skin, cornea, tendon, gingiva as well as cementum (minor amounts) Anchoring fibrils, calvaria, tendon, PDL, cartilage, cervix  133  Table 7. The small leucine-rich proteoglycans ( S L R P ) that have been shown to bind fibrillar collagen thus far (Schonnher et al, 1995; Hedbom & Heinegard, 1989; Hansson et al, 2001; Neame et al, 2000; Font et al, 1998; Rada et al, 1993; Vogel et al, 1987;Bidanset et al, 1992; Whinna et al 1993; Hedbom & Heinegard, 1993).  SLRP  Collagen  Decorin Biglycan Fibromodulin Chondroadherin Lumican  I, II, III, V i,v  I, II, XI II I  134  Table 8 .  In vitro studies attesting the role of small leucine-rich proteoglycans ( S L R P ) i n  collagen fibrillogenesis ( L U M = lumican; D C N = decorin; E C M = extracellular matrix; F M = fibromodulin; P G s = proteoglycans). Study Carlson et al, 2002  Study design - SLRP studied Cultured mutant L U M cells  Neame et al, 2000  Recombinant D C N and L U M P G in collagen fibrillogenesis assay based on turbidity.  Svensson et al, 2000  Recombinant L U M , F M and D C N PGs and a collagen fibril formation/sedimentation assay  Findings Mutant L U M cells produced an unorganized E C M with altered fibril packing and structure compared to wild type cells The cys domain of the L U M molecule is an important factor in collagen fibrillogenesis D C N and L U M act independently on collagen fibril formation L U M accelerates initial collagen fibril formation D C N retards initial collagen fibril formation L U M and D C N do not compete for binding sites on collagen fibrils L U M & D C N act to increase collagen fibril stability to thermal denaturation L U M & D C N result in reduced overall turbidity suggesting a lower collagen fibril diameter. The presence of both PGs retarded fibril formation to a greater degree than either one alone - a synergistic effect. F M inhibits the binding of L U M and vice versa. F M and L U M do not affect the binding of D C N to collagen or vice versa. F M binds to collagen with 4x the affinity of that of L U M . High and low affinity binding sites for F M and L U M (ie. each have two binding sites) with higher affinity for them being had by F M .  135  Table 9. In vivo studies attesting to the role of SLRP in collagen fibrillogenesis. For a more extensive review of these studies, see Ameye & Young, 2002 (DCN = decorin; KO = knockout; L U M = lumican; BGN = biglycan; FM =fibromodulin;PDL = periodontal ligament). Study  Danielson et al, 1997 Chakravarti et al, 1998  Xu et al, 1998  Svensson et al, 1999 Ezura et a I, 2000  Hakkinen et al, 2000  Corsi et al, 2002  Ameye et al, 2002  SLRP studied DCN KO  mouse skin LUM KO mouse tail, skin, eyes  BGN KO mouse bone FM KO mouse tendon LUM KO. FM KO, and double KO mouse leg tendon DCN KO mouse PDL BGN and DCN and double KO mice skin and bone  BGN and FM and double KO mice tendon  2002  LUM and FM and double KO mice tendon  Goldberg et  BGN KO dentin, enamel  Jepsen et al,  al, 2002  Findings  DCN KO mice have fragile skin with reduced tensile strength. Electron microscope showed irregular outline and size variability of collagen fibrils. Suspect uncontrolled lateral fusion of collagen fibrils. LUM is strongly expressed during development of the mouse LUM does not affect expression of DCN Loosely arranged fibril orientation in the homozygous mutants LUM KO mice had skin that was more compliant and weaker than normal Corneal transparency was reduced in LUM KO mice. Fibril morphology was uniformly thin in wild and single LUM KO (LUM -/+) LUM -/- mouse corneas showed three key differences: 1. thicker fibrils present in addition to normal diameter fibrils. 2. many irregular shaped fibrils 3. increased interfibrillar spacing. Similar findings were seen in mouse tail cross sections and back skin. BGN KO mice exhibit an osteoporotic phenotype characterized by reduced growth rate and decreased bone mass that becomes more obvious with age. Altered morphologic phenotype in mouse tail tendon characterized by fewer and abnormal collagen fibrils. Fibers were disorganized and had irregular and rough outlines and more thin fibers were evident than normal. Premature presence of collagen fibril heterogeneity in 4d double KO tendon compared to wild type tendon. Abnormally large number of small diameter collagen fibrils in later stages of development especially at 3m. Collagen fibrils had irregular profiles Abnormal morphology and organization of collagen fibrils associated with increased numbers of fibroblasts. In skin, BGN KO mice exhibited collagen fibrils that were irregular in profile, had a broader size range and were less tightly packed. Skin in these mice was thinner but not fragile. DCN KO mice have thin and fragile skin. Double KO mice show macroscopic and microscopic skin changes that are worse than either single KO mice (additive effect). In bone, BGN KO mice exhibit a larger average fibril diameter and an osteopenic phenotype while DCN KO mice show smaller average fibril diameter but no macroscopic phenotype. Double KO mice bone exhibits abnormally shapedfibrilsand an osteopenic phenotype worse than either single KO mice - a synergistic effect. Both single KO and double KO mutants showed joint specific and age specific ectopic ossification in tendons but were larger and more numerous in double KO mice. Double KO mice also show gate impairment and severe osteoarthritis that occurs at a much earlier age than either single KO mice. This osteoarthritic change was found to be due to structurally weak tendons. Double KO mice were also smaller than normal and single KO mice. LUM KO mice showed slightly smaller body weight and size, slight decrease in FM expression, increased average fibril size, and no tendon stiffness. FM KO mice showed slightly smaller body weight and size, decreased tendon stiffness, increased LUM expression, decrease in average fibril size. Double KO mice displayed considerably smaller body size and weight, severe gate abnormality, joint laxity, osteoarthritis, bowed legs and marked tendon stiffness decrease with increased average number of thinfibersubpopulations. BGN KO mice displayed altered enamel formation that was 3-5x thicker than normal, decreased collagen fibril diameter in proximal third of predentin, increased collagen fibril diameter in central and distal thirds of predentin.  136  Table 1 0 . Sex (M=male; F=female) and age (months) o f all wild-type C D - I , fibromodulin knockout ( F M - / - ) , lumican knockout ( L U M - / - ) and double knockout ( F M / L U M - / - ) mice used i n this study.  U Wildtype  Sex  Age (m)  # FM-I-  Sex  Mouse  Age (m)  U LVM-I-  5.5  5.5  1 2 3 4  5.5  5  5.5  6  5  7  Sex  Mouse  Age (m)  # FM/LUM-/-  4.4 4.5  1 2  6  3  6  4  6  5  6  6  6  7  Sex  Mouse  Age (m)  Mouse  1 2 3 4 5 6 7  8 9  10  M M M F F F F F M M  5  1  5  2  5  3  6  4  6  5  6  6  8  7  8  8  F F F F F M M M  5.5 5.5  M M M M F F F  8  5  8 8  137  F F F F F M M M  8 8 8 8 8 8 8  4.5  T a b l e 11. Classification o f alveolar bone loss in mice as described by Wiebe et al, 2001. Grade I II  III  Description Exposure of the furcation and horizontal bone loss extending into the furcation. Exposure of the furcation with a through-andthrough defect from the buccal to the oral surface of the tooth. Through-and-through furcation defect with horizontal bone loss extending into the apical third of the root.  138  Table 12. List o f mice used for each staining and analyzed. Stains include: hematoxylin & eosin ( H & E ) , phosphotungstic acid hematoxylin ( P T A H ) , picrosirius red (PSR), antitype I collagen antibody, anti-lumican ( a n t i - L U M ) antibody, anti-decorin (anti-DCN) antibody, anti-fibromodulin (anti-FM) antibody, anti-biglycan (anti-BGN) antibody. Stains were carried out on buccal-lingual (Bu/Li) sections and mesio-distal ( M / D ) sections where indicated. Each slide contained 4 sections per slide. Refer to table 10 for age and gender o f mice. Stain H&E (Bu/Li) H&E (M/D) PTAH (Bu/Li) PTAH (M/D) PSR (Bu/Li) Anti-type I collagen (Bu/Li) Anti-LUM/Anti-DCN (Bu/Li) Anti-LUM/Anti-DCN (M/D) Anti-FM/ Anti-BGN (Bu/Li) Anti-FM/Anti-BGN (M/D)  CD-1#  FM-/-#  LUM-/-#  FM/LUM-/-#  1,2,3,4,5,6 1,2,3,6 1,2,4,5,6 1,2,3,6 1,3,4,5,6 1,2,3,4,5,6 1,2,3,4,5,6 1,2,3,6 1,2,3,4,5,6 1,2,3,6  1,2,4,5 2,4,5 1,2,3,4,5 2,3,4,5 1,2,4,5 1,2,3,4,5 1,2,3,4,5 2,3,4,5 1,2,3,4,5 2,3,4,5  1,3,4,5 1,2,3,4,5 1,2,3,4,5 1,2,3,4,5 1,2,3,4,5 1,2,3,4,5 1,2,3,4,5 1,2,3,4 1,2,3,4,5 1,2,3,4  1,2,3,4,5 1,3,4,5 1,2,3,4,5,8 1,2,3,4,5 1,2,3,4,5 1,2,3,4,5,8 1,2,3,4,5,8 1,2,3,4 1,2,3,4,5,8 1,2,3,4  139  T a b l e 13. Dilution table of antibodies used, anti-decorin (anti-DCN), anti-biglycan (antiB G N ) , anti-lumican (anti-LUM), anti-fibromodulin (anti-FM). Antibody Anit-mouse D C N Anti-mouse B G N Anti-human F M Anti-mouse L U M Anti-mouse collagen type I  Source Rabbit Rabbit Rabbit Rabbit Rabbit  Dilution 1/500 1/500 1/1000 1/1000 1/500  140  T a b l e 14. M i c e used for compensation study. Mesio-distal sections o f C D - I wild-type mice, fibromodulin-deficient (FM-/-) mice, lumican-deficient ( L U M - / - ) mice, and fibromodulin & lumican-deficient ( F M / L U M - / - ) mice were chosen and divided into 4 groups as noted below. Each groups was stained separately. Each slide then received antilumican (anti-LUM), anti-decorin (anti-DCN), anti-fibromodulin (anti-FM) or antibiglycan (anti-BGN) antibodies for an equal and standardized amount o f time. Sections from each groups were then compared and intensities o f immunostains were determined to be +++, ++, + or 0 stain.  anti-LUM & anti-DCN antibody Group CD-I FM-/- LUM-/- FM/LUM-/A 1 2 1 1 B 2 3 2 2 C 3 4 3 3 D 6 5 4 4  141  anti-FM & anti-BGN antibody CD-I FM-/- LUM-/- FM/LUM-/1 2 1 1 2 3 2 2 3 4 3 3 6 5 4 4  Table 15. Localization o f decorin, biglycan, lumican and fibromodulin i n dental tissues o f wild-type C D - I mice using non-standardized sections. Intensity o f immunostaining was noted on a relative basis as +++ (most intense), ++ (moderately intense) or + (least intense). - indicates no immunostaining detected. Intensity measures are relative to other tissues within the stain group and not relative to each other (CT=connective tissue). Tissue Periodontal ligament Gingival C T Mucosal C T Gingival epithelium Mucosal epithelium Pulp Bone Cementum Dentin/predentin (p)  Decorin  Biglycan Fibromodulin Lumican  ++ +++ +++ + -  +++ +++ ++ + -  ++ ++ ++ + + ++ -  ++ +++ +++ + -  -KP)  ++(P)  +++(p)  •KP)  142  fa fa fa  fa  11.0 10.8 10.2 | 11.3 10.8 NA 11.0 10.4  Sex Age  ro <* in  ss m  S fa  r - 00  fa fa  10.1 11.8 11.0 11.0  NA  in  10.2 11.3  LUM -/-  Length  5s2  in 00 00 oo oo 00 00 00 "St  FM/LUM-/-  Length Age  Sex  O  in  CN ro  X  4>  fa-  ss  fa  in in in in <n in in in >n in in in m ro  ! | | |  d  m  10.5 10.4 10.9 11.0 11.4 11.3 11.0  Age  fa fa fa  Length  UO  j_ CN ro  Sex  fa  et H  oo  «n SO  Length Age  m  m  10.9  NA NA NA  11.6 11.2  NA  CD-I  fa fa fa fa fa  _  CN ro  m  r- oo  m  11.9  11.5  NA  vo 00 00 oo 00  o  Table 17. Jaw length comparisons of pooled data. Pooled jaw lengths of wild-type (CD-I) mice (n=5, mean age 6.6 months, male & female), fibromodulin knockout (FM-/-) (n=8, mean age 5.4 months, male & female), lumican knockout (LUM-/-) (n=6, mean age 5.5 months, male & female) and fibromodulin/lumican knockout (F/L-/-) (n=7, mean age 7.5 months, male & female) mice were compared using the student t statistic assuming equal variance. P values given for those comparisons that were statistically significant (*). NS = not significant.  Groups Compared P value Significance CD-I vs F M - / CD-1 vs. L U M - / CD-1 vs. F/L-/F M - / - vs. L U M - / F M - / - vs. F/L-/L U M - / - vs. F/L-/-  P=.03 >.05 P=.02 >.05 >.05 >.05  * NS * NS NS NS  Table 18. Jaw length comparison of different age groups (m = months) within the wild-type (CD1), fibromodulin knockout (FM-/-), lumican knockout (LUM-/-) and fibromodulin/lumican knockout (F/L-/-) mouse groups. P value not shown for statistically insignificant comparisons. NS = not statistically significant.  Group  Ages compared P value Significance  CD-I  5m vs. 6m 5m vs. 8m 6m vs. 8m 5m vs. 5.5m 4.5m vs. 6m 4.5m vs. 8m  FM-/LUM-/F/L-/-  >.05 >.05 >.05 >.05 >.05 >.05  NS NS NS NS NS NS  Table 19. Jaw length comparisons of similar ages (m = months) between different groups. Wildtype (CD-I), fibromodulin knockout, lumican knockout, fibromodulin/lumican knockout groups are compared. P value only shown for those comparisons that were statistically significant (*) using the student t-test assuming equal variance. N S = not statistically significant. Groups compared  P value Significance  CD-I 5m vs. F M - / - 5m CD-I 5m vs. L U M - / - 4.5m CD-I 5m vs. F/L-/- 4.5m F M - / - 5m vs. F/L-/-4.5m L U M - / - 4.5m vs. F/L-/- 4.5m F M - / - 5m vs. L U M - / - 4.5m CD-I 6m vs. F M - / - 5.5m CD-I 6m vs. L U M - / - 6m CD-I 6m vs. F/L-/-4.5m CD-I 6m vs. F/L-/- 8m F M - / - 5.5m vs. L U M - / - 6m F M - / - 5.5m vs. F/L-/- 4.5m L U M - / - 6m vs. F/L-/- 4.5m L U M - / - 6m vs. F/L-/- 8m CD-I 8m vs. F/L-/-8m  >.05 >.05 >.05 >.05 >.05 >.05 >.05 >.05 >.05 >.05 >.05 >.05 >.05 >.05 .03  NS NS NS NS NS NS NS NS NS NS NS NS NS NS  *  144  Table 20. Jaw length comparisons of males vs. females within the wild-type (CD-I), fibromodulin knockout (FM-/-), lumican knockout (LUM-/-) and fibromodulin/lumican knockout (F/L-/-) groups. P value is given only for those comparisons that were statistically significant (*) using the student t-test assuming equal variance. N S = not statistically significant. Groups compared CD-I male vs. female F M - / - male vs. female L U M - / - male vs. female F/L-/- male vs. female  P value >.05 .02 >.05 >.05  Significance NS  * NS NS  Table 21. Jaw length comparisons of males and females between the wild-type (CD-I), fibromodulin knockout (FM-/-), lumican knockout (LUM-/-) and fibromodulin/lumican knockout (F/L-/-) groups. P value is given only for those comparisons that were statistically significant (*) using the student t-test assuming equal variance. N S = not statistically significant. Groups compared CD-I male vs. F M - / - male CD-I male vs. L U M - / - male CD-I male vs. F/L-/- male F M - / - male vs. L U M - / - male F M - / - male vs. F/L-/- male L U M - / - male vs. F/L-/- male CD-I female vs. F M - / - female CD-I female vs. L U M - / - female CD-I female vs. F/L-/- female FM-/-female vs. L U M - / - female F M - / - female vs. F/L-/- female L U M - / - female vs. F/L-/- female  P value >.05 >.05 >.05 >.05 >.05 >.05 .007 >.05 >.05 >.05 >.05 >.05  Significance NS NS NS NS NS NS He  NS NS NS NS NS  Table 22. Jaw length comparisons of male mice of similar ages (m = months). Comparisons made between wild-type (CD-I), fibromodulin knockout (FM-/-), lumican knockout (LUM-/-) and fibromodulin/lumican knockout (F/L-/-) groups. P value is given only for those comparisons that were statistically significant (*) using the student t-test assuming equal variance. NS = not statistically significant. Groups compared CD-I 5m male vs. F M - / - 5m male CD-I 5m male vs. L U M - / - 4.5m male CD-I 5m male vs. L U M - / - 6m male CD-I 5m male vs. F/L-/- 4.5m male F M - / - 5m male vs. L U M - / - 4.5m male F M - / - 5m male vs. L U M - / - 6m male F M - / - 5m male vs. F/L-/- 4.5m male L U M - / - 4.5m male vs. F/L-/- 4.5m male CD-I 8m male vs. F/L-/- 8m male  P value >.05 >.05 ?  ? >.05 >.05 >.05 >.05 9  145  Significance NS NS NS NS NS NS NS NS NS  Table 23. Jaw length comparisons of female mice of similar ages (m = months). Comparisons made between wild-type (CD-I), fibromodulin knockout (FM-/-), lumican knockout (LUM-/-) and fibromodulin/lumican knockout (F/L-/-) groups. P value is given only for those comparisons that were statistically significant (*) using the student t-test assuming equal variance. N S = not statistically significant. Groups compared CD-I 6m female vs. F M - / - 5.5m female CD-1 6m female vs. L U M - / - 6m female CD-1 6m female vs.F/L-/- 8m female F M - / - 5.5m female vs. L U M - / - 6m female F M - / - 5.5m female vs. F/L-/- 8m female L U M - / - 6m female vs. F/L-/- 8m female CD-I 8m female vs. F/L-/- 8m female  P value .03 >.05 >.05 >.05 >.05 >.05 >.05  146  Significance * NS NS NS NS NS NS  T a b l e 24a. Table o f number o f blood vessels counted i n the periodontal ligament o f C D 1 wild-type, fibromodulin knockout (FM-/-), lumican knockout ( L U M - / - ) and double knockout ( F M / L U M - / - ) mice. Table shows mice from which data was collected and blood vessels counted on buccal and lingual sides. B l o o d vessels were counted only in the coronal two thirds o f the periodontal ligament (#=mouse number; Bu=buccal; Li=lingual).  Wild-type # Bu Li 1 4 0 2 3 0 3 2 0 4 2 0 5 1 0 Means  2.4  F M - / -  # 1 2 3 4  5  0  Bu  L U M - / -  2 4 4 1 0  Li 1 2 3 2 3  # Bu 1 3 2 0 3 1 4 2  2.2  2.2  1.5  Li 0 0 0 1  .25  FM/LUM-/# Bu Li 1 2 1 2 3 1 3 3 2 4 2 2 5 2 0 8 1 1 2.2 1.2  Table 24b. Table o f number o f tears counted in the periodontal ligament o f C D - I wildtype, fibromodulin knockout (FM-/-), lumican knockout ( L U M - / - ) and double knockout ( F M / L U M - / - ) mice. Table shows mice from which data was collected and tears counted in the entire periodontal ligament (#=mouse number).  Means  W i d-type Tears # 1 0 1 0 2 2 3 0 4 2 1 5 6 2 1  FM-/Tears 1 4 2 0 3 5 2 4  1 2 3 4  5  5  #  2  #  2.6  LUM-/Tears 1 3 0 2 1  1.4  147  FM[/LUM-/Tears 1 0 2 1 3 1 4 4 5 2 8 0  #  1.3  Table 25. Blood vessels of wild-type (CD-I), fibromodulin knockout (FM-/-), lumican knockout (LUM-/-), fibromodulin/lumican knockout (F/L-/-) mice are compared in total (pooled), as well as for buccal (Bu) and lingual (Li) periodontal ligament surfaces. P value is shown only for those comparisons that were statistically significant (*) with the student t-test assuming equal variance.  Groups compared Pooled P value Bu P value Li P value CD-I vs. F M - / CD-1 vs. L U M - / CD-1 vs. F/L-/F M - / - vs. L U M - / F M - / - vs. F/L-/L U M - / - vs. F/L-/-  >.05 >.05 >.05 >.05 >.05 >.05  >.05 >.05 >.05 >.05 >.05 >.05  .0004* >.05 .007* .005* >.05 >.05  148  Table 26. Tears of the periodontal ligament are compared between wild-type (CD-I), fibromodulin knockout (FM-/-), lumican knockout (LUM-/-), fibromodulin/lumican knockout (F/L-/-) mice are compared using pooled data. P value is shown only for those comparisons that were statistically significant (*) with the student t-test assuming equal variance.  Groups compared P value Significance CD-I vs. F M - / -  CD-1 vs. LUM-/CD-1 vs. F/L-/F M - / - vs. L U M - / F M - / - vs. F/L-/L U M - / - vs. F/L-/-  >.05 >.05 >.05 >.05 >.05 >.05  NS NS NS NS NS NS  149  Appendix II - Figures  150  F i g u r e 1. Basic proteoglycan structure can be seen in the schematics o f decorin and biglycan small leucine-rich proteoglycans. Proteoglycans have a protein core with N - and C - terminal domains with glycosaminoglycans attached to it. In the case o f biglycan and decorin, the glycosaminoglycan chains are either chondroitin sulfate (CS), as depicted here, or dermatan sulfate, as i n the periodontal connective tissues, depending its tissue distribution. Larger proteoglycans have much more^complex structures.  BIGLYCAN (CS-PGI)  DECORIN (CS-PGII)  151  Basic structure of the small leucine-rich proteoglycan protein core (Neame & Kay, 2000). F i g u r e 2:  Cysteine cluster N-terminal domain  LRRs  UJ  C-terminal  domain (osteoadherin) Disulfide bonds  152  Figure 3. A schematic o f the molecular organization o f some representative small leucine-rich proteoglycans. Similarities can be seen among decorin and biglycan (two class I family members) and fibromodulin and lumican (two class II family members). The core proteins do not represent their actual lengths nor do the sites o f glycosylation and leucine-rich repeats represent their actual location.  153  F i g u r e 4. Gingival fibers. Type I collagen fibers o f the gingiva have been classified according to their location and direction. There are four recognizable groups: the circular group, the dentogingival group, the dentoperiosteal group and the alveologingival group (Ten Cate, 1994).  154  Figure 5. Principle fibers o f the periodontal ligament. Type I collagen fiber bundles o f the periodontal ligament have been divided into 6 groups based on their location and orientation. They include the transeptal, alveolar crest, horizontal, oblique, apical and interradicular fibers. They are highly organized fibers running parallel to each other in the ligament (Ten Cate, 1994).  155  Figure 6 . Collagen biosynthesis demonstrating intracellular (hydroxylation, glycosylation, nucleation and propagation) and extracellular events (peptide cleavage, fibril formation and cross-linking) that lead to the formation o f a collagen molecule by fibroblasts. Specific amino acids on ribosomes form individual polypeptide chains that contain N and C terminal peptides. Some o f the lysine and proline residues are hydroxylated (vitamin C dependent enzyme driven processes) forming hydroxylisine and hydroxyproline. Sugar residues are added, a process known as glycosylation (an enzymatically driven process). Peptide chains then form a triple helix and are transported to the golgi apparatus where the procollagen molecules is completed and excreted from the cell. Next fibrillogenesis takes place where the N and C termini are eventually cleaved and 5 unit staggered microfibrils are formed (Kadler, 1995)  155  F i g u r e 7. Triple helix o f collagen. Schematic representation o f collagen cased on a repeating triplet o f Glycine-proline-4-hydrohyproline. ( A ) shows a single collagen alpha chain, (B) shows three alpha chains folded into a triple helix with Glycine residues in the center o f the molecule and (C) shows a cross-sectional view o f (B) looking down the axis of the triple helix molecule (Kadler, 1995).  157  Figure 8 . Collagen fibril structure. Collagen molecules aggregate to form a banded fibril. Negative staining techniques allow gaps to contain more stain and show up as dark areas in electron microscope. Minerals i n hard tissues accumulate in these gaps (Ten Cate 1994).  A p p r o x i m a t e l y 1/4 s t a g g e r *-  300nm  '  158  Figure 9. Model of lateral fusion of collagen molecules to form fibrils. Collagen molecules assemble into quarter-staggered arrays giving rise to fibril intermediates (65 nm in mouse tendon). These fibril intermediates are stabilized by fibril-associated macromolecules, such as fibromodulin and lumican, to allow for fusion of adjacent fibril intermediates in the formation of mature collagen fibrils. Model based on study in fibromodulin, lumican and double knockout mouse tendon (Ezura et al, 2000).  Molecular Assembly  Progressional Growth by Fusion  » m>  w  » l  65 nm  w  I  Intermediates (Subunits) i  92nm\  g  2  n m  112nm  I* c  ) 0 nnmV m\ 130 30 nm 130  130nm  \  v A  W 184 nm  151  260-400 nm  10. Contribution of fibromodulin and lumican during collagen fibrillogenesis. In the lateral fusion model described in figure 9, it is believed that lumican is expressed early in mouse tendon development suggesting a role in the initial stages of collagen fibrillogenesis. Fibromodulin expression increases during development suggesting a role in tendon fibril maturation (Ezura et al, 2000). Figure  IfcO  F i g u r e 11: The binding model. Decorin, a class I small leucine-rich proteoglycan, is thought to be horseshoe-shaped and bind type I collagen fibrils on their concave side. Weber et al, 1996 estimate that each decorin molecule has enough space on the concave aspect of the arc to accommodate one collagen molecule.  161  Figure 12: Jaw length measurements. Photograph of a defleshed, halved mouse maxilla showing where jaw length measurements were made using a Boley Gauge to the nearest tenth of a milliimeter. Measurements were made anteriorly at the osseous crest facial of the incisor at the point of incisor exit and posteriorly at the distal of the third molar (M1 = 1st molar; M2 = 2nd molar; M3 = 3rd molar).  l<o2_  F i g u r e 13. L o c a l i z a t i o n of fibromodulin (A), l u m i c a n (B), b i g l y c a n ( C ) a n d decorin (D) in dental a n d periodontal t i s s u e s (p=pulp; d=dentin; b=bone; c = c e m e n t u m ; e=epithelium; ct=connective tissue; pdl=periodontal ligament) ( i m m u n o s t a i n intensity: mild = +, m o d e r a t e = ++, intense = +++).  I4.3  IC4  1<O5  Figure 15: Defleshed maxillary jaw halves of wild-type (A),fibromodulin knockout (B), lumican knockout (D), and double knockout mice (C). All teeth are fully erupted (M1 = 1st molar, M2 = 2nd molar, M3 = 3rd molar).  Figure 16. S e c t i o n s of  CD-1 wild-type (A), fibromodulin knockout (B), l u m i c a n knockout (C) a n d d o u b l e knockout (D) m o u s e mandibular first m o l a r s stained with p h o s p h o t u n g s t i c a c i d h e m a t o x y l i n . T h e first m o l a r s are fully erupted a n d h a v e b o n e levels of similar height. Distal roots are m i s s i n g d u e to s e c t i o n i n g angle. (b=bone; p=periodontal ligament; d=dentin; g=gingiva).  I (.7  F i g u r e 17. H & E s t a i n e d s e c t i o n s of the coronal third of m a n d i b u l a r first molars of CD-1 wild-type (A), fibromodulin knockout (B), l u m i c a n k n o c k o u t (C) a n d d o u b l e knockout (D) m i c e . T h e periodontal ligament of wild-type m i c e a r e filled with type I c o l l a g e n fiber b u n d l e s that are of relatively e v e n t h i c k n e s s a n d properly oriented (arrow). T h e s p a c i n g b e t w e e n the b u n d l e s is e v e n l y distributed, (arrowhead). L i g a m e n t s of the knockout m i c e h a v e fiber b u n d l e s of u n e v e n t h i c k n e s s (arrows) with u n e v e n s p a c i n g b e t w e e n t h e m (arrowheads). T h e d o u b l e k n o c k o u t m o u s e ligaments h a v e the m o s t r a n d o m a r r a n g e m e n t of c o l l a g e n fiber b u n d l e s .  lb?  Figure 18. First m a n d i b u l a r molar sections stained with p h o s p h o t u n g s t i c acid hematoxylin of C D - 1 wild-type (A), fibromodulin k n o c k o u t (B), l u m i c a n knockout (C) a n d d o u b l e knockout (D) mice. S m a l l a r r o w s point to fiber b u n d l e s of the k n o c k o u t m i c e that are of variable t h i c k n e s s a n d a r r o w h e a d s point to a r e a s of i n c r e a s e d inter fiber bundle s p a c i n g - features unlike wild-type. L a r g e arrow points to a r e a of external root resorption (B). (d=dentin; p=periodontal ligament; b=bone; c = c e m e n t u m ) .  Figure 19. 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