B .1;Ì,lrl,i.i .1. ¿;.i ,l r¡ c',litL'r(:i:i t; i.ri 'uit:i rl coit; i,.l' ¡i¡;,r s, trJ),cit rì epos:i tcrÌ :ì l: tltr,-: li.¡::i vcrs,i t¡r l-,;i.!;i'¡. .¡'¡ . ì;r,ri r:¡; ;1"¡lL:'i.1.¡i;-l-ç -[' o r" -l (j,'rÌ] at.tar ìr ], (] i;o c c¡ l¡yi.rr lr: " ì.)lte: -io-15 [J.i.gr,.0,1. ri:rc: l,,lts t'. OXYTALAN FIBRES IN MARSUPIAL PERIODONTAL LIGAMENTS A proJect report submítted in partial- fulfllnent for the degree of Master of Dental Surgery. by I^IAYNE JOHN SA}.,IPSON, B.D.S., I{ONS. B.SC. (DENT). Department of Dental llealÈh University of Adelaíde Adelaide, South AusÈralia. t97 4. l- TABLE OF CONTENTS Page Number LIST OF TABLES. vi. LIST OF FIGURES. vií. LIST OF DIAGRAMS. x. BAR GRAPHS. xii. SI'MMARY. xiii. SIGNED STATB{EI{I. xv. ACKNO!üLEDGN4EI.TIS. xvi. INTRODUCTION. I ATMS OF THE INVESTIGATION. 3 CHAPTER 1 - LITERATURE REVIEI,I: OXYTAIAN 4 1.1. Discovery. 4 t.2. Distríbutl-on' 5 (a) l{an. 5 (b) Aninals. I 1.3. Functional Consideratlons. 11 L.4. Non-pathologic Changes . 13 1.5. Pathologic Changes. L4 1 .6. EIecÈronmlcroscoPY. 15 L.7 . Formatíon and DeveloPment. 18 1 .8. HístochemistrY, 19 (a) OemonstraÈlon of oxytalan. 19 (b) Mechanisms of stalning 2T (i) aldehyde fuchsln 2l (1i) orcein 23 (tií) resorcin fuchsl-n 24 (iv) other dyes. 25 (c) Reliabílity of hístochemLcal proeedures. 25 1l- Table of Contents (continued) Page Number CHAPTER 2 - THE FIBROUS CONNECTIVE TISSUES 29 2.I. ExPlanatorY Notes- 29 2.2. CoLLagen. 31 2.3. Reticulin. 36 2.4. A' Comparíson BeËween Collagen and Retícu1-in, 3B (a) SimilariËies. 38 (b) Differences. 39 2.5. ElasËic Fibres. 40 2.6. A' Comparison Between Collagen and Elastic Fibres' 44 (a) SÍmilaríti-es. 44 (b) Differences. 46 2.7. OxYtalan. 47 2.8. Cornparison Between Elastic and oxytalan Fibres, 47 ^ (a) Similaríties. 47 (b) Differences. 49 CHAPTER 3 - CRITERIA FOR CALLING A FIBRE OXYTALAN 51 3.1. Pre-elastic Fibres. 51 3.2. OxYtalan Fíbres. 51 CHAPTER 4 . }'IATERIALS AND METIIODS 55 4.1. Materials. 55 4.2. Methods' 55 (a) AnatomY. 56 (b) Histologic PreParation 56 (i) fixatíon 56 (ii) decalcification 56 (iií) nettralízatíon 59 (iv) processing 59 (v) paraffin enbeddíng 59 (vi) sectioning. 60 (c) staining. 60 (d) EnzYme digestions. 62 (e) Investigation of dye properËies 63 (i) dye solutions 63 (ii) pll analysis 64 (iii) specÈroPhotometry. 64 l- aa Table of Contents (contínued) Page Number 4.2. (f) MicroscoPY 65 (i) light microscoPe 65 (ií) Phase contrast 65 (iii) dark field 65 (iv) ultra violet mícroscoPY' 66 (g) l'lethylation and saponification tests for carboxYl grouPs. 66 (h) PhotograPhY. 67 4.3. Three-Djmensional Reconstruction, 67 (a) Technique. 67 (b) Calculations. 70 77 CHAPTER 5 - FINDINGS 5.1. Gross AnatomY' 77 (a) Mandible. 77 (b) DenËítion 83 (i) wombat 83 (ii) wa1-labY B4 (iii) Possum 84 (iv) marsuPial míce. 87 5.2.HistologyoftheMarsupialMandibularDenËition' 87 (a) DenËal hisÈologic observaÈions 87 (i) wombat 87 (íi) wallabY 90 (ííi) Possum 93 (iv) marsuPial mice. 94 (b) Distributlon of Èhe connective tissues 96 (í) nerve fibres 97 (ií) collagen 97 (iii) retículin 97 (ív) mucoPolYsaccharides 97 (v) elastic fibres 100 (vi) oxYtalan fibres. LO2 (c) Regional distribution of oxytalan fibres 106 (i) wombaÈ 106 (ii) wallabY 113 1V. Table of Contents (continued) Page Number 5.2. (c) (íii) possum r23 (iv) marsupial mice. L26 (d) Summary of the arrangement of oxytalan fibres r29 (i) cemental attachment 133 (ii) periodontal ligament 133 (iii) supra-alveolar regíon 135 (iv) apex 135 (v) pulp 13s (vi) blood vessels. 135 5.3. Enzpre Reactions, r42 (a) Oxytalan. L42 (b) Elastic fibres. 145 (c) Collagen. r45 (d) Other Ëissues. r45 5 .4. InvestigaËion of Dye ProPerties, r46 (a) Dyes. 146 (b) pH analysis. 153 (c) Spectrophotometric analysis. r54 (d) MethylaËíon and saponification tests for carboxyl groups. r64 5.5. Microscopy, 164 (a) Phase conÈrast. 164 (b) Dark field. 165 (c) Fluorescence. 165 5.6. Three-dimensional reconstruction. t66 CHAPTER 6 - DISCUSSION 168 6.1. Anímals 168 6"2" Oxytalan Fibres, 170 (a) Distributíon. L70 (b) Attachments. 176 (c) Three-dimensional reconstruction. 177 (d) Eruptíng teeËh. 178 (e) Functional consíderatioris. 178 (f) Relationship to elasÈic fibres 181 (i) similarities TB2 v Table of Contents (continued) Page Number 6.2 (f) (íi) differences. 183 (g) Relationship to other fibres. l84 (h) staining reactions 184 (i) oxidation 186 (ií) reactive groups 186 (iíi) dyes. 190 (i) Spectrophotometry. t99 (j ) Fluorescent studies. 200 (k) Enzymes. 200 CHAPTER 7 - CONCLUSIONS 203 CHAPTER 8 - APPENDICES 2t0 8.1. Appendix I. 2t0 Lísts of elastic fibre dyes. I "2. Appendíx II. 213 The animals studied. 8.3 . Appendix III. 2TB Histologic preparation of the specimens. 8. 4. Appendíx IV. 220 Conventional st.ainíng techniques. 8.5. Appendix V. 229 Code used to systematise the recording of histologic sections for the presence of oxytalan fibres. 8.6 Appendix VI. 230 Enzyme digesËions. 8.7 Appendix VII. 232 Additional oxytalan-positive dye solutions. 8.8 Appendix VIII. 240 Spectrophotometric analysís of representatíve staining solutions for the dyes used ín this study. 8.9 Appendix IX. 244 Methylation and saponificatíon tests for carboxyl groups. CHAPTER 9 BIBLIOGRAPIIY 245 vt. LIST OF TABLES Page Number Table 1. Staining reactions of oxytalan and elastic fibres frorn Fullmer and Lillie 1958. 53 Table 2. Effect of enzymatic hydrolysís on staining reactions of formalín fixed oxytalan and elastic fibres vríth aldehyde fuchsin from Fullmer and Lí1lie 1958. s4 Table 3. composite findings of the staÍning reactions of the marsupials studied and the results of Fullrner and Lillie (1958). 104 Table 4. Iron-orceín reactions (Roman et al. 1967; Lillie et al. 1968) . 104 Table 5. OxyÈalan and elastic fibre distríbution in the wombat (aldehyde fuchsin sËaining) . 111 Table 6. Distribution of oxytalan fibres in the wallaby (aldehyde fuchsin staining technique). 119 Table 7. DistríbuÈion of oxytalan fibres in the possum (aldehyde fuchsin staining technique) . r24 Tabte 8. Distribution of oxytalan fibres in the marsupial rnice (aldehyde fuchsin staínÍng technique). 130 Table 9. Effects of enzyne digestions as determined by staíning with aldehYde fuchsin. r43 Table I0. List of dyes investigated. L47 Table 11. List of dyes found to reacÈ positively with oxytalan-líke structures. t49 Table 12. Oxytalan posítive dyes, pH values, and manipulaÈions of some dye solutions. 150 Table 13. Dyes found to stain elastic fi-bres by Horobin and James (1970). 210 Table 14. A list of dyes whích have been successful in demonstrating elastic fibres (from Pearse' 1968) 212 Table 15. Code used to systematíse the recording of histologíc sections for the presence of oxytalan fibres. 229 Table 16. SpectrophoËometric analysis of representative staining solutíons for the dyes used ín thís study. 240 v11 LIST OF FIGURES Page Number Figure 1. Radiographic testing of specÍmen decalcificaÈion. 57 Fígure 2. Anatomy of the wombaË skull and mandibl-e. 78 Figure 3. Anatomy of the wallaby mandible. 79 Figure 4. Anatomy of the possum nandible. 80 Figure 5. Anatomy of the skull and mandíble of A. Flavípes and S. crassicaudaÈa. 81 Figure 6. Nerve flbre in Èhe possum periodontal ligarnent. 98 Figure 7. Nen¡e fibre formatlons in Ëhe gingiva of A. flavipes. 98 Fígure B. Reticulin adjacent to the wallaby gingival epitheliun. 99 Fígure 9. Positive periodlc acid-Schiff reaction for oxytalan- líke structures in the wallaby perlodontal 1ígament. 99 Figure 10. Elastíc fibres 1n the i-nferior dental artery of the wombat. 101 Figure 11. Iron-orceín reactíon for elastic fíbres in the wa1-laby gíngíval corÍum. 101 Flgure 12. Non-oxidÍzed control section of wombat perlodontal IÍgament. 103 Fígure 13. Oxidized section adjacent to that shown in Figure 12. 103 Figure 14. Non-oxidlzed secËion in the apical thírd region of the wombat periodontal ligament. 105 Fígure 15. Oxidized secÈion adjacent Èo that shown in Fígure 14. 105 Figure 16. Branching oxytal-an fíbres embedded ln the cementum of a wombat molar. 107 Figure 17. Apíco-occlusally alígned oxytalan fíbres in the wombat perlodontal ligament. 107 Fígure 18. Oxytalan flbres linkíng blood vessels in the wombat períodontal f-igament. 110 Ftgure 19. Oxytalan ln the immature pulp of a contlnuously eruptfng wombat molar. 110 Figure 20. Gradatíons ín oxytalan fibre sizes in Ëhe wallaby períodontal ligament. 115 v111. List of Fieures (conËinued) Page Number Figure 21. Network of fine, branching oxytalan fibres adjacent the cementum of a wallaby molar. 115 figure 22. Relationship of an oxytalan fibre and a periodontal blood vessel in the wallaby. 116 Figure 23. OxyÈalan fíbres accompanying a blood vessel into alveolar bone. 116 Figure 24. Oxytalan fibre Èracts. TL7 Figure 25. Oxytalan fíbre sizes and appearance of elastic recoil in the wallaby períodontal ligament. TT7 Figure 26. Fine, branching oxytalan network passing beneath Èhe apex of a wallaby molar. L22 Fígure 27. OxyÈalan fíbres and fibre tracts in the periodontal ligarnent of an erupting wallaby molar. t22 Fígure 28. ûxytalan fíbres in the pulp Èissue and around Èhe apex of the possum incisor. r27 Figure 29. Oxytalan fÍbre cascade ínto the periodontal ligamenË of A. FlaviPes. I27 Figure 30. Relationshi-p between oxytalan fíbres and perlodontal blood vessels ín S. crassícaudat,a. I28 Figure 31. Oxytalan fíbres embedded in Èhe cementum and emerging into the wallaby periodontal lígament. 134 Figure 32. Ilne rreshwork of oxytalan fibres located near the cemental surface of a wall-aby molar. 134 Figure 33. Oxytalan fibre tracts (wallaby molar). L36 Figure 34. Oxytalan fit¡re Èracts (wallaby incisor). 136 Figure 35.