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THE FIBRILLAR ORGANI ZATION OF COLLAGEN IN CONNECTIVE TISSUE
A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biophysics at
Massey University
ALAN SMITHSON CRAIG
1984
DEDICATION
Thi s thes is is ded icated to my wife - Wendy and our ch ildren
Michael , Kim , Kirsten and Hadley .
ABsrRAcr
Al though cer tain aspects of connective t issue structure have been stud i ed in considerable deta i l, comparatively l i ttle e ffort has been devoted to studying one of the l argest structural uni ts present in mos t tissues the col lagen fibr i l . In this thes i s electron microscope observations have been made on the transverse dlinens ions o f f ibr i ls from t i ssues as d iverse as cornea, sk in and tendon . Collagen f ibr i l d iameter d istr ibutions have been measured for such tissues from a w ide range of animals predominantly mammals, .but also f i sh , amphibians, repti les and b i rds - at varying stages o f development . These data have allowed the growth o f col lagen fibr i ls to be s tud ied quant i tat ively and their si ze d istr ibutions to be related to the i r mechanical a ttr ibutes . Di seased tissues o r tissues conta ining anomalous fibr i l diameter d is t r ibut ions have also been stud ied and , where poss ible, the data have been related to the a ltered mechan i cal propert ies of the tissue and to its mode of growth and development . I n a coord i na ted study wi th other research workers , the content of the ind iv idual glycosarninoglycans in a ti ssue have been shown to be related to the mass-average d iameters of the col lagen f ibr ils in those t i ssues . These results prov ide a bas i s for urrlerstand ing the feedback mechani sm by which f ibr i l s i ze d i str ibutions may be mod i f i ed in l i ne with chang ing mechanica l needs and ind icate the furrlarnenta l steps in
the growth and development of f ibr il s .
In add i tion to these stud ies, two other speci fic problems were addressed . In the f irst, the ultrastructure of a spec ialized
connective tissue the cornea was stud ied in deta i l . By ma intain ing precise exper imental protocols and measurement procedures i t was shown, contrary to the prev ious 'data of others, tha t the
collagen f ibr i l s i n mammals , b i rds , repti les , amph ibians and car t i lag inous fish were simi lar to one another but · signif icantly di fferent to the corneal stromal f ibr ils of the bony fish. Further stud ies , which indicated that the fibr i ls were constant in d i a�eter across the width of the stroma , clar i f ied prev ious results which had ind icated a g radual change in d iameter with varying depth in ti1e stroma . An age-related study of fibr i l diameters in the cornea was also undertaken . The second problem investigated was the degree o f shr inkage i ntroduced dur ing the preparative procedures for electron microscopy . I n col laborative stud ies with others , X-ray and electron microscope observations were made on the same tissue in hydrated and dehydrated states respectively. Analyses of these data indicated that sign i f icant lateral shr inkage does i ndeed occur in f ibr ils from foetal or imnature tissues as well as in mature tissues conta ining only small d iameter f ibr ils . Throughout the thesis possible sources of artefact introduced by . the technique of electron microscopy have been considered and the data interpreted conservatively.
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ACKNOWLEDGEMENTS
Many people have helped me in a variety of ways to get this thesis into its present form.
In my earlier years at DSIR Keith Williamson introduced me to, and gave me sound guidance in, the principles and techniques of electron microscopy. Subsequently my Director, Ray Bailey, gave me
the encouragement and provided the impetus for me to embark on this present course of study.
Throughout this thesis the experimental results obtained by electron microscopy have, where possible, been related to biochemical and X-ray diffraction data kindly made available to me by Barbara Brodsky, Eric Eikenberry, 11ichael Flint, Gerry Gillard and Isabel Williams. Gary Thomas and Bob Fletcher supplied me with histogram plotting and population deconvolution progra� and together provided me with oft-needed statistical advice. Doug Hopcroft has been responsible for the excellent maintenance of the electron microscopes and Ray Bennett printed the micrographs. I was assisted by m�1y friends in typing the manuscript but it was June Tipoki who bore the lion's share of this chore.
Finally, and of greatest importance to me, my research colleague and supervisor, David Parry, not only instigated this project but displayed an un-ending enthusiasm for it. I am indebted to his necessary and continual encouragement throughout my writing-up.
To all of these people, and to many others whom I have personally acknowledged, I sincerely thank you for your support.
TABLE OF COOTENTS
ABSTRAcr
N::.KNOWLEDGEMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF F IGU RES
CHAPTER 1: THE COLLAGENOUS COMPONENT OF CONNECTIVE TI SSU ES
1.1 INTRODUCTION
1.2 THE STRUCTURAL HIERARCHY OF COLLAGEN
1.2.1 The collagen molecule
1.2.2 The collagen fibr i l
1 . 3 THE NATURE OF COLLAGENOUS T ISSUES
1.3.1 The cel lular components
1.3.2 The f ibrous components
1.4 SCOPE AND AIMS OF THIS THESI S CHAPTER 2: MATERIALS AND METHODS
2.1 INSTRUMENTAL METHOD
2.2 COLLECTION OF SPECIMENS
2. 3 PREPARATIVE PROCEDURES
2.4 SECTION ING AND STAINING TECHN IQUES
2.5 PHOTOMICROGRAPHY AND MENSURATION METHODS
2. 6 ANALYTICAL METHOOS ·
CHAPTER 3: THE CORNEA
3 . l I NTRODUCriON
3.2 RESULTS AND D ISCUSS ION
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Page
111 V
v i
X
x i i i
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2 8
ll
14
1 8
21 30 33 34 35 37
39 47
CHAPTER 4: EXPERIMENTAL OBSERVATIONS ON THE GROWTH AND DEVELOPMENT OF COLLAGEN F I BRILS
4 . 1 I NTRODUCTION
4 . 2 TENDONS AND LIGAMENTS
4 . 2 . 1 Av ian Me ta tarsal Tendon
4 . 2 . 2 Mamma l ian Tendons and Ligamen ts
4 . 3 SKINS
4 . 4 OTHER TISSU ES
CHAPTER 5: COLLAGEN FIBRIL ASSEMBLY D ISORDERS
5 . 1 INTRODUCTION
5 . 2 F IBRILLAR MALFORMAT IONS
5 . 2 . 1 Hered i tab le Disorders
5 . 2 . 2 Natura l ly Occurring Mal forma tions
5 . 3 CHANGING FIBRIL DIAMETER D ISTRIBUTI ONS
5 . 3 . 1 I nduced Disorders
5 . 3 . 2 Acqui red Disorders
5 . 4 CONCLUS I ONS
CHAPTER 6: INTERPRETATION AND SIGN I FICANCE OF COLLAGEN FIBRIL DIAMETER DISTRI BUTION DATA
6 . 1 CONSI DERAT IONS OF THE LIMITATIONS IMPOSED BY ELECTRON MICROSCOPY
6 . 2 ANALYSIS OF THE DIAMETER DISTRI BUTIONS THAT ARE SK�RP AND UNIMODAL
6 . 3 ANALYSI S OF DIAMETER DISTRIBUTIONS THAT ARE HETEROGENEOUS
6 . 4 ANALYSIS OF BROAD DISTRIBUTIONS OF COLLAGEN FIBRIL
Page
61
63 70 84
89
9 3
9 6
102
105 116 120
123
128
1 3 9
DIAMETER 141
6 . 5 CORRELATION BETWEEN ELECTRON M ICROSCOPE AND X-RAY DATA 143
CHAPTER 7 : THE GRCMTH AND DEVELOPMENT OF CONNECTIVE TISSUES AND THE RELATIONSH I P BETWEEN COLLAGEN FIBRIL DIAMETER DISTRIBUTIONS AND MECHANICAL PROPERT IES
7 . 1 INTRODUCT ION
7 . 2 CORRELATIONS BETWEEN COLLAGEN F IBRIL DIAMETER DISTRIBUTIONS AND T ISSUE ATTRIBUTES
7. 2 . 1 Foetal Development
7. 2 . 2 Modes of Col lagen Fibr i l Development for Al tr icious and Precoci ous Animals
7. 2 . 3 Post-natal Development
7 . 2 . 4 Form of the Col lagen Fibr i l Diameter D i s tr ibut ion at Matur i ty
7 . 2 . 5 Form of b�e Collagen Fibr i l Diameter Distr ibut ion
Page
149
152
155 157
159
at Senescence 164
7 . 2 . 6 Correlat ion Between Mass-average D iameter and
Ultimate Tensi le Streng th 164
7 . 2 . 7 Form o f the Diameter Distribut ion and the Mechanical Propert ies of the T i ssue
CHAPTER 8: THE RELATIONSH I P BETWEEN GLYCOSAMINOGLYCAN
COMPOS IT ION AND COLLAGEN FIBRIL DIAMETERS: A POSSIBLE MECHANISM FOR F IBRI LLOG ENESIS
8 . 1 INTRODUCT ION
8 . 2 DO GLYCOSAMINOGLYCANS MEDI ATE CONTROL?
8 . 2 . 1 Previous Concepts 8 . 2 . 2 Observati ons 8 . 2 . 3 Hypothes i s
8 . 3 CONCLUSIONS CHAPTER 9: SUMMA RY
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165
l70
172 176
184 190 195
Page APPENDICES
APPENDIX l: GENETICALLY DISTINCT COLLAGEN TYPES AND THEIR
DISTRIBUT ION IN THE BODY T ISSUES 200
APPENDIX 2: DIMENS IONS. OF THE PARAMETERS OF PROPOSED
COLLAGEN SUB-FIBRILLAR ASSEMBL IES 201
APPENDIX 3: SOUECE OF CORNEAS FOR COMPARATI VE STUDY AND STATE OF PRESERVATION PRIOR TO PREPARATION FOR ELECTRON
MICROSCOPY 202
APPENDIX 4: PROCESSED MATERIAL SUPPLIED BY OTHER RESFAECH WORKERS
APPENDIX 5: BACTERIAL COLLAGENASE TREATED FLEXOR TENDONS FROM HORSE . EXPERIMENTAL PROTOCOL , CLINICAL AND POST-MORTEM OBSERVATIONS .
REFERENCES
203
203a 204
LIST OF TABLES
Page Table l . l Components of connective t i ssues in matu re animals . 16 Table 3 . 1 Mean d iameters of populations of collagen fibr i l s
f rom the corneal stromal lamellae o f the adul t vertebrates stud ied .
Table 3 . 2 Mean collagen f ibr i l d iameters o f the corneal stroma as cited in var ious works .
Table 3 . 3 Mean d iameters o f populations of collagen fibr ils f rom corneal stromal lamel lae in developing frog ,
48
52
rat , guinea pig , man and some neonatal mammal s . 55 Table 3 . 4 Var iat ion of mean col lagen fibr i l d iameter with depth
below the anter i or surface of corneal stroma .
Table 3 . 5 Collagen f ibr i ls of the vertebrate corneal stroma hav ing d iameters wh ich are not s imple multi?les of -s nm .
Table 4 . 1 Mean and mass-average d iameters o f collagen f ibr ils in chick metatarsal tendons .
Table 4 . 2 Resolution of multimodal d i str ibuti ons of col lagen f ibr i ls in chick metatarsal tendons .
Table 4 . 3 Modal centre- to-centre and mean sur face separations of collagen f ibr i l s in foetal metatarsal tendons .
Table 4 . 4 Precocious-altr icious class ification o f neonate placental mammals .
Table 4 . 5 Mean and mass-average d iameters o f col lagen f ibr i l d iameter d istr ibutions o f tendons and l igaments in
the developing sheep .
Table 4 . 6 Resolut ion of sub-populat ions o f collagen f ibr i l d iameters in sheep tendons a nd l igaments ; The ir
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59
6 1
66
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73
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compa r i son with the sharp unimodal d istr ibut ion
.
observed . 79
Table 4 . 7 Mean and mass-average d iameters of col lagen fibr i l d iameter d i str ibuti ons o f tendons from neonate to
adult gu i nea pigs . 80
Table 4 . 8 Mean and mass-average d iamete rs o f col lagen f ibr i l d iameter d istr ibutions o f flexor tendons i n foetal
to ma ture ra ts . 85
Table 4 . 9 Resolut i on o f bimodal d istr ibut ions of collagen f ibr i l d iameters in rat tendons and the ir compar ison
to the sharp unimodal d istr ibutions observed . 86
·rable 4 . 10 Mean and mass-average d iameters of col lagen f ibr i l
d iameter d istr ibut ions i n sk i n . 90 Table 4 . 11 Mean and mass-ave rage d iameters of co l lagen f ibr i l
d iameter d istr ibutions recorded from some
m isce l la neous ti ssues . 9 2
Table 5 . 1 Col lagen fibr i l diameter d istr ibut ion data from horse superficial d ig ital f lexor tendon after
treatment with bacter i a l col lagenase . 1 1 3 Table 5 . 2 Resolut i on o f sub-popula ti ons o f col lagen fibr ils i n
horse super fic i a l d ig ital flexor tendon treated with
bacter ial col lagenase . 114
Table 6 . 1 Diameters of col lagen fibr i l s as determi ned by
electron microscope and X- ray d i ffraction stud ies . 145 Table 7 . 1 Form of the col lagen fibr i l d iameter d istr ibution
i n foetal to senescent t issues . 153
Table 7 . 2 Mean and mass-average d i ameters of col lagen fibr ils
i n tendons , ligaments and sk i ns in perinatal animal s . 154 Table 7.3 Bir th-mass of animals expressed as percentages of
adult-mass .
Table 7 . 4 Max imum mass-average d iameter of col lagen f ibr i l s in adul t connective t i ssues .
Table 8 . 1 Glycosaminog lycan content and mass-average co llagen f ibr i l d iameter in sk in as a function of age .
Table 8 . 2 Glycosaminog lycan content and mass-average co llagen f ibr i l d iameter in tendon as a functi on o f age .
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169
177
179
LIST OF FIGURES
Page
Figure 1 . 1 Space- f i l l ing model o f the col lagen molecule . 3 Figure 1 . 2 Schematic representat ion of the procollagen molecule . 5 Figure 1 . 3 Schematic representation of collagen synthes is and
f ibr il logenesi s .
Figure 1 . 4 Electron micrograph o f negatively sta i ned col lagen fibr i l s with a d iagram showing that the D-per iod can be accounted for by a regular stagger ing of col lagen molecules .
Figure 1 . 5 Electron micrograph o f transverse sect ions o f the collagen fibr i ls in foetal rat tai l-tendon .
Fi gure 1 . 6 Electron microg raph o f transverse sections o f the collagen fibr i l s in adult rat ta i l- tendon .
Fi gure l . 7 Electron micrographs o f transverse sect ions o f elastic f ibres at varying stages of development .
Figure 2 . 1 Cross-sectional drawing o f a transmiss ion electron microscope column.
Figure 2 . 2 S impl i f ied ray d iag ram of a transmiss i on electron microscope .
Figure 2 . 3 Ray diag ram i llustrat ing the depth of f ield 1n an electromagnetic lens .
Figure 3 . 1 Electron micrographs o f transverse sections through (a) the total thickness o f the corneal stromata o f the snake a nd (b) a portion o f the corneal stromata o f the magpie .
Figure 3 . 2 Electron microg raphs o f transverse and long i tud inal sections o f sutural f ibres in the corneal stroma ta o f the dogfish .
6
10
12
1 3
1 7
24
25
29
4 1
42
Figure 3 . 3 Electron micrographs o f transverse sections qf the collagen fibrils from the corneal stroma of ( a) bony
fish (gold fish) and ( b) cartilaginous fish ( stingray) • 50 Figure 3 . 4 Electron micrographs of transverse sections of the
collagen fibrils from the corneal stroma of ( a ) cartilaginous fish (dog fish) , ( b ) amphibian
( salamander ) , (c) reptile ( snake) , (d) bird (magpie) and (e) mammal ( rabbit) .
Figure 4 . 1 Low magnification electron micrograph o f the cellular elements and collagen fibrils in an 18 day foetal
51
chick metatarsal tendon . 67
Figure 4 . 2 Electron micrographs showing (a) the sharp unimodal distribution o f collagen fibril diameters in 14 day
foetal chick metatarsal tendon and (b) a collagen fibril diameter distribution which may be resolved into several distinct populations in 18 day foetal chick metatarsal tendon .
Figure 4 . 3 Electron micrographs of bacterially-contaminated 15 day chick metatarsal tendon showing collagen fibrils falling into "close-arrays".
Figure 4 . 4 Frequency and mass distributions o f collagen fibril diameters in sheep flexor tendons .
Figure 4 . 5 Frequency and mass distributions o f collagen fibril diameters. in sheep extensor tendons .
Figure 4 . 6 Frequency and mass distributions of collagen fibril diameters in sheep suspensory ligaments .
Figure 4 . 7 Frequency and mass distributions of collagen fibril diameters in guinea pig flexor tendons .
Figure 4 . 8 Frequency and mass distributions o f collagen fibril
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68
71
76
77
78
81
diameters in guinea pig extensor tendons. 8 2 Figure 4.9 Frequency and mass distributions of collagen fibril
diameters in guinea pig diaphragmatic tendons. 83
Figure 4 . 10 Frequency and mass distributions of collagen. fibril
...
diameters in rat forelimb flexor tendons. 87 Figure 4 . 11 Frequency and mass distributions of collagen fibril
diameters in rat hindlimb flexor tendons. 88 Figure 5 . 1 Electron micrographs of transverse sections of the
collagen fibrils in lamb skin; (a) and (c) are from an animal suffering from dermatosparaxis and (b) is
from a control. 97
Figure 5 . 2 Electron micrograph of transverse sections of the
collagen fibrils in dermatosparactic lamb tendon. 97 Figure 5. 3 Electron micrographs of transverse sections of the
collagen fibrils in (a) nor�al greyhound dermis (b)
dysplastic greyhound-dermis and (c) papillary layer
of dysplastic greyhound-dermis. lOO
Figure 5 . 4 Electron micrographs of sections through the dysplastic greyhound-dermis showing (a) abnormal lysosomal activity in a dermal fibrocyte and details of (b) lamellar, (c) fibrinoid, and (d)
"electron-dense" lysosomal inclusions. 101 Figure 5 . 5 Electron micrographs of transverse sections of
collagen fibrils from (a) unstretched and (b) stretched Cuvierian tubules of the sea cucumber
Holothuria forskali. 104
Figure 5 . 6 Frequency and mass distributions of collagen fibril diameters in normal rat skin and in healing
11longitudinal11 and 11transverse11 wounds. 107
Figure 5 . 7 Frequency and mass d i str ibutions o f col lagen f ibr i l d iameters taken from the r i ght ( contralateral) superf ic ial digital f lexor tendons o f horses whose left super ficial flexor tendon had been treated with bacter i al col lagenase .
Figure 5 . 8 Frequency and mass d istr ibuti ons of collagen f ibr i l d iameters from the superf ic ial d ig i tal f lexor tendons of hor se s at var ious times after treatment with
bacter i al col lagenase .
Figure 5 . 9 Electron m icrographs of transverse sections of the collagen f ibr ils in (a) normal hor se super f ic ial d i g i ta l flexor tendon , (b) 24 hours after bacter ial collagenase treatment and (c) 4 weeks after bacter ial col lagenase treatment .
Figure 5 .10 Frequency and mass d i str ibutions o f col lagen fibril d iameter s in Dupuytren ' s co ntractu re and nodules , and
110
111
112
in normal pabnar fasc i a . 118
Figure 6.1 Electron m icrographs of ( a) overfocus and (b) underfocus Fresnel fringes in a holey plastic
support f i bn.. 129
Figure 6.2 ( a ) H i stogram showing all data obta i ned from sharp unimodal col lagen fibr i l d iameter d i str ibutions hav ing means in the range 14 - 44 nm. (b) Graph
showing relationship between observed collagen fibr i l
d iameters a nd hypothetical sub-fibri llar uni ts . 131 Figure 6 . 3 Diagram showing the proj ected d imens i ons of a
cyl indr ical f ibr i l lying obl iquely in a thin section . 1 34 Figure 6 . 4 Bar d iagram showing the spread o f recorded diameters
from sharp unimodal d i str ibuti ons and the d i f ferences (xvi)
in measurement made by two observers . 137 Figure 6 . 5 Electron microraph of transver se secti ons of col lagen
f ibr i ls in the developing lamprey sk in . Fibr i l s are of uniform d iameter and appear to have electron trans lucent "cores " .
Figure 6 . 6 Frequency d i str ibutions of collagen f ibr i l d ia�eters in 18 day foetal chick metatar sal tendons as measured by three independent observers .
Figure 7 . 1 Electron microg raphs showing transverse sections of the col lagen f ibr i l s from the sk ins of lamprey , r at and trout .
Fi gure 7 . 2 Frequency and mass d i str ibutions of collagen f ibr i l
140
142
160
d iameters in sk ins from lamprey, rat and trout . 161 Figure 7 . 3 Electron micrographs show ing the lamellar arrangement
of the col lagen fibr i l s in lamprey sk in . 163
Figure 7.4 Graphs of ( a ) tens i le strength and mass-average col lagen fibr i l d iameter versus age for rat sk in and
(b) tens i le streng th and mass-average col lagen f ibr il diameter versus age for rat- ta i l tendon .
Figure 8 . 1 Graphs of t i ssue percentage contents of hyaluronic ac id , chond r o i t i n sulphate and dermatan sulpha te versus col lagen f ibr i l mass-average d iameter .
166
183
