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Changes in the proteoglycans of the intervertebral disc cartilaginous end-plate with ageing and degeneration Bishop, Paul Burton 1989

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CHANGES IN THE PROTEOGLYCANS OF THE INTERVERTEBRAL DISC CARTILAGINOUS END-PLATE WITH AGEING AND DEGENERATION By PAUL BURTON BISHOP B.Sc, The University of B r i t i s h Columbia, 1974 C , The Canadian Memorial Chiropractic College, 19 M.Sc, The University of B r i t i s h Columbia, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PATHOLOGY FACULTY OF MEDICINE We accept th is thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JULY 1989 ©. Paul Burton Bishop, 1989 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 P/rTH^06r,y The University of British Columbia Vancouver, Canada Date iZ.fifrf ABSTRACT This research examined the role of the cartilaginous end-plate (CEP) in ageing and degeneration of the human intervertebral disc (IVD). The matrix component affected primarily during degeneration of the IVD is proteoglycan (PG) (Pearce et a l . , 1987). The CEP, a hyaline cartilage found between the nucleus pulposus (NP) and the anulus fibrosus (AF) and the vertebral body, has been proposed as the source of the PG of the AF and NP. This study was undertaken to: 1) assess the similarity of CEP PG to PG from articular cartilage and IVD, (2) compare the CEP PG from healthy young discs with those from older degenerate discs ( 3 ) distinguish the changes in CEP PG due to ageing from those due to degeneration. The combined effects of ageing and degeneration were studied using end-plates from three healthy young spines and three post-mature spines; those to degeneration alone by comparison of two healthy with one degenerate disc in each of three spines. Altogether 86 CEP from 1 0 lumbar spines were examined. The CEP PG were prepared from 4 M guanidinium chloride tissue extracts by density gradient ultracentrifugation under associative conditions. PG were separated into high and low molecular weight (M) components by Sepharose C L - 2 B chromatography. The PG and the high M and low M fractions were analysed for hexose (hex) and hexuronate i i (hexA) as measures of keratan sulphate and chondroitin sulphate, respect ively. Also, the two fract ions were analysed by composite agarose-polyacrylamide gel electrophoresis. The CEP PG resembled the IVD PG more c lose ly than those of a r t i cu la r cart i lage PG: the f ract ion excluded from Sepharose CL-2B was low, the hex/hexA ra t i o was high, and f ive e lectrophoret ica l ly d i s t i n c t subspecies were seen. With degeneration, several properties of the CEP PG altered irrespective of age: the extractable .total proteoglycan f e l l , the ra t io hex/hexA rose and number of e lectrophoret ica l ly d i s t i nc t PG subspecies declined. With age, the sizes of the high M and low M fractions f e l l and the electrophoretic mobi l i t ies of the subspecies changed.^ These results suggested that degeneration involves both a conversion of aggregating to non-aggregating PG and the preferent ia l biosynthesis of a keratan sulphate-rich over a chondroitin sulphate-rich PG. i i i T A B L E OF CONTENTS PAGE A b s t r a c t i i T a b l e o f C o n t e n t s i v L i s t o f T a b l e s v i L i s t o f F i g u r e s v i i i L i s t o f A p p e n d i c e s x A b b r e v i a t i o n s x i A c k n o w l e d g e m e n t s x i i i 1 . 0 L i t e r a t u r e R e v i e w 1 1 . 1 I n t r o d u c t i o n 2 1 . 2 A n a t o m y o f t h e l u m b a r s p i n e a n d t h e h e a l t h y human i n t e r v e r t e b r a l d i s c 3 1 . 3 H i s t o l o g y o f t h e h e a l t h y i n t e r v e r t e b r a l d i s c 6 1 . 4 M e c h a n o c h e m i s t r y o f h e a l t h y c a r t i l a g e a n d i n t e r v e r t e b r a l d i s c 8 1 . 5 C a r t i l a g e a n d i n t e r v e r t e b r a l d i s c p r o t e o g l y c a n s 9 1 . 6 C h a n g e s i n c a r t i l a g e a n d i n t e r v e r t e b r a l d i s c w i t h a g e i n g a n d d e g e n e r a t i o n 14 1 . 7 B i o c h e m i c a l a n d f u n c t i o n a l p r o p e r t i e s . . o f t h e e n d - p l a t e a n d t h e p r e s e n t s t u d y 18 2 . 0 M e t h o d s 22 2 . 1 A n a l y t i c a l m e t h o d s 23 2 . 2 C o l l e c t i o n a n d g r a d i n g o f i n t e r v e r t e b r a l d i s c s 23 2 . 3 D i s s e c t i o n o f e n d - p l a t e s a n d d e t e r m i n a t i o n o f w a t e r c o n t e n t 24 2 . 4 P r e p a r a t i o n o f A l p r o t e o g l y c a n 25 2 . 5 G e l c h r o m a t o g r a p h y o f t h e A l p r o t e o g l y c a n 27 2 . 6 H i g h a n d l o w m o l e c u l a r w e i g h t c o m p o n e n t s 31 2 . 7 C o m p o s i t e a g a r o s e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s 32 2 . 8 S t a t i s t i c a l a n a l y s e s 33 i v 3.0 R e s u l t s 34 3.1.0 PART I C o m p a r i s o n o f t h e P r o t e o g l y c a n o f C a r t i l a g i n o u s E n d - P l a t e f r o m H e a l t h y I n t e r v e r t e b r a l D i s c s w i t h t h a t f r o m D e g e n e r a t e I n t e r v e r t e b r a l D i s c s 3.1.1 S e l e c t i o n o f s p e c i m e n s 35 3 . 1 . 2 D i s s e c t i o n o f t h e c a r t i l a g i n o u s e n d - p l a t e 46 3 . 1 . 3 Y i e l d a n d w a t e r c o n t e n t o f c a r t i l a g i n o u s e n d - p l a t e 53 3 . 1 . 4 C o m p o s i t i o n o f t h e A l f r a c t i o n s 56 3 . 1 . 5 G l y c o s a m i n o g l y c a n s o f t h e A l P r o t e o g l y c a n . . 58 3.1.6 S e p h a r o s e C L - 2 B g e l c h r o m a t o g r a p h y o f A l p r e p a r a t i o n s 61 3 . 1 . 7 C o m p o s i t i o n o f t h e A l p r o t e o g l y c a n p o p u l a t i o n s s e p a r a t e d b y S e p h a r o s e C L - 2 B . . . 69 3 . 1 . 8 C o m p o s i t e a g a r o s e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f A l p r o t e o g l y c a n s . . . . 72 3 . 2 . 0 PART I I C h a n g e s I n C a r t i l a g i n o u s E n d - P l a t e P r o t e o g l y c a n Due t o D e g e n e r a t i o n 3 . 2 . 1 S e l e c t i o n o f s p e c i m e n s 92 3 . 2 . 2 Y i e l d a n d w a t e r c o n t e n t o f c a r t i l a g i n o u s e n d - p l a t e 101 3 . 2 . 3 C o m p o s i t i o n o f e n d - p l a t e A l p r o t e o g l y c a n f r a c t i o n s 1 0 2 3 . 2 . 4 G l y c o s a m i n o g l y c a n s o f t h e A l P r o t e o g l y c a n . . 1 0 5 3 . 2 . 5 S e p h a r o s e C L - 2 B g e l c h r o m a t o g r a p h y o f A l p r o t e o g l y c a n 1 0 7 3 . 2 . 6 P r o p e r t i e s o f t h e p r o t e o g l y c a n p o p u l a t i o n s s e p a r a t e d b y S e p h a r o s e C L - 2 B . . . 1 1 6 3 . 2 . 7 C o m p o s i t e a g a r o s e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f A l p r o t e o g l y c a n s . . . . 1 1 9 4 . 0 D i s c u s s i o n 1 2 2 4.1 M o r p h o l o g i c a l c h a n g e s o f t h e e n d - p l a t e w i t h a g e i n g a n d d e g e n e r a t i o n 1 2 3 4 . 2 L i m i t a t i o n s o f c o l o r i m e t r i c a s s a y s 1 2 3 4 . 3 C h a r a c t e r i s t i c s o f t h e p r o t e o g l y c a n s o f e n d - p l a t e f r o m h e a l t h y d i s c s 1 2 4 4.4 C h a n g e s i n t h e e n d - p l a t e p r o t e o g l y c a n s w i t h a g e i n g a n d d e g e n e r a t i o n 1 2 8 4 . 5 C l i n i c a l s i g n i f i c a n c e . . . 1 3 5 R e f e r e n c e s 1 3 6 A p p e n d i x . 1 4 5 v LIST OF TABLES TABLE NO. TITLE PAGE 3.1.1 Donors of spines and grades of intervertebral discs • • • • 36 3.1.3 Comparisons of fresh weight, dry weight and water content of end-plate of healthy and degenerate discs 52 3.1.4 Composition of Al proteoglycan fractions from end-plates of healthy and degenerate discs 57 3.1.5 The effect of disc degeneration on the hexose and hexuronate contents of the Al proteoglycans 59 3.1.6 The modal partition coefficients of end-plate Al proteoglycans 68 3.1.7 Proportion of high M and composition of the high and low M fractions of Al proteoglycans taken from healthy and degenerate discs 70 3.1.8 Relative electrophoretic mobility of high and low M fractions of Al proteoglycans prepared from healthy and degenerate discs 88 3.2.1 Donors of Spines, grades and grade groups of intervertebral discs 94 3.2.2 Comparisons of fresh weight, dry weight and water content of end-plates of healthy and degenerate discs within three spines.... 103 3.2.3 Composition of Al proteoglycan fractions from end-plate of healthy and degenerate within three spines 104 3.2.4 The hexose and hexuronate contents of the Al proteoglycans of healthy and degenerate discs within the same spine 106 3.2.5 The modal partition coefficients of end-plate Al proteoglycans from healthy and degenerate discs within the same spine 115 vi TABLE NO. TITLE PAGE 3.2.6 Proportion of high M and composition of the high and low M fractions of Al proteoglycan taken from healthy and degenerate discs within the same spine 117 3.2.7 Relative electrophoretic mobility of high and low M proteoglycans prepared from healthy and degenerate discs of the same spine 120 4.3.1 A comparison of end-plate proteoglycans with those of articular cartilage, anulus fibrosus and nucleus pulposus 126 4.4.1 Comparison of the changes in the end-plate proteoglycan due to ageing and degeneration versus those due to degeneration alone 130 vi i LIST OF FIGURES FIGURE NO. TITLE PAGE 2.5.1 Calibration of Sepharose CL-2B column 29 3.1.1-1 Typical lumbar spine containing G r a d e I d i s c s 37 3.1.1-2 Lumbar spine containing Grade II discs 39 3.1.1- 3 Typical lumbar spine containing Grade IV/V discs 41 3.1.2- 1 The tissues of the intervertebral disc 44 3.1.2-2 Grade I intervertebral disc, cartilaginous end-plate and subchondral bone 47 ~i 3.1.2-3 Grade IV/V intervertebral disc, cartilaginous end-plate and subchondral bone 49 3.1.4-1 Density gradient centrifugation 54 3.1.6-1 Typical Sepharose CL-2B column chromatography profile of Al proteoglycan from end-plate of Grade I discs 62 3.1.6-2 Typical Sepharose CL-2B column chromatography profile of Al proteoglycan from end-plate of Grade IV/V discs 64 3.1.6-3 Typical Sepharose CL-2B column chromatography profile of Al proteoglycan from end-plate of Grade II discs 66 3.1.8-1 Composite agarose-polyacrlyamide gel electrophoresis of fractions of Al proteoglycan from end-plate of Grade I discs prepared by Sepharose CL-2B chromatography 73 vi i i FIGURE NO. TITLE PAGE 3.1.8-2 Composite agarose-polyacrylamide gel electrophoresis of fractions of Al proteoglycan from end-plate of Grade IV/V discs prepared by Sepharose CL-2B chromatography 75 3.1.8-3 Preparative composite agarose-polyacrylamide gel electrophoresis of the high M (Peak 1) fraction 78 3.1.8-4 Preparative composite agarose-polyacrylamide gel electrophoresis of the low M (Peak 2) fraction 80 3.1.8-5 CAPAGE analysis of high M (Peak 1) proteoglycan from end-plate of donor A,D and F 82 3.1.8-6 CAPAGE analysis of low M (Peak 2) proteoglycan from end-plate of donor A,D and F 84 —i 3.1.8-7 Densitometric scan of CAPAGE comparison of high M and low M proteoglycan fractions of end-plate from Grade 1,11 and IV/V discs 86 3.2.2-1 Lumbar spine of donor H 95 3.2.2-2 Lumbar spine of donor I 97 3.2.2-3 Lumbar spine of donor J 99 3.2.5-1 Sepharose CL-2B column chromatography of end-plate Al proteoglycan of L2/L3, L3/L4 and L5/S1 discs of donor H .... 108 3.2.5-2 Sepharose CL-2B column chromatography of end-plate Al proteoglycan of L2/L3, L4/L5 and L5/S1 discs of donor I .... 110 3.2.5-3 Sepharose CL-2B column chromatography of end-plate Al proteoglycan of L1/L2, L3/L4 and L4/L5 discs of donor J .... 112 ix LIST OF APPENDICES APPENDIX NO. TITLE PAGE 1 INTERVERTEBRAL DISC GRADING SCHEME 143 x LIST OF COMMONLY USED ABBREVIATIONS AF anulus fibrosus CAPAGE composite agarose-polyacrylamide gel electrophoresis CEP cartilaginous end-plate ChS chondroitin sulphate g gravity glcN N-acetylglucosamine galN N-acetylgalactosamine /g per gram GLM general linear model hex hexose hexA hexuronate _^  h hour(s) HA hyaluronate HABR hyaluronate binding region IVD intervertebral disc Kav partition coefficient between free f l u i d and available space KS keratan sulphate ml m i l l i l i t r e mm millimetre MRI magnetic resonance imaging ms milliseconds nm nanometre NP nucleus pulposus PAS periodic acid Schiff xi L I S T OF COMMONLY USED A B B R E V I A T I O N S ( C o n t ' d ) PG p r o t e o g l y c a n SAS s t a t i s t i c a l a n a l y s i s s y s t e m s SEM s c a n n i n g e l e c t r o n m i c r o s c o p y TE s p i n e c h o t i m e TR r e p e a t t i m e TZ t r a n s i t i o n z o n e UK U n i t e d K i n g d o m M. 1 m i c r o l i t r e Urn m i c r o m e t r e U m o l e m i c r o m o l e VO v o i d v o l u m e VT t o t a l v o l u m e wt w e i g h t w/v w e i g h t b y v o l u m e ACKNOWLEDGEMENTS I wish to recognize and thank the following people and/or organizations: Dr. R. H. Pearce, for his assistance, guidance and encouragement. Ms. J. M. Mathieson, Miss B.J. Grimmer and Dr. G.M. Bebault for their expert advice and assistance Dr.J.P. Thompson, Dr. I. Tsang, Dr. B. Ho, Dr. B. Flak, Dr. J. Sisler, Dr. J. Mayo, Dr. M. Adams, for their assistance in disc grading. Dr. P. Reid for his assistance and advice with histological staining techniques. Dr. D. Seccombe for his advice on experimental design. Arthritis Society for funding this research. Foundation for Chiropractic Education_and Research for a Research Fellowship. The Foundation for Chiropractic Education and Research, The Canadian Memorial Chiropractic College and the B.C. Chiropractic Association for other financial assistance. x i i i 1 . 0 LITERATURE REVIEW - l -1.1 Introduction Low back pain is a major c l i n i c a l and economic problem. It has reached epidemic proportions, with 80% of the human race experiencing low back pain at some time in their lives and as many as 60% of the normal population (UK) having experienced some degree of low back pain in the last year (Waddell, 1987). The cost of low back pain in the United States has over a recent four year period averaged in excess of $16 b i l l i o n (Frymoyer, 1988). Low back pain appears to be associated with disc degeneration, but many patients with radiographic evidence of disc degeneration are asymptomatic with no history of back pain (Nachemson, 1985). There is increasing evidence to suggest that the disc is just one biomechanical component of the spine and the loss of its functional capacity induces instability and degenerative changes in other components resulting in non-discogenic back pain (Roberts et a l . , 1989). The concept that a l l degenerative changes in the spine are initiated by disc degeneration has also been challenged (Kirkaldy-Willis et a l . , 1978). It is has been suggested that injury to other spinal tissues (e.g. facet joints) occurs f i r s t , thereby initiating degenerative changes that lead to additional biomechanical instability and eventual disc degeneration (Bishop, 1989). -2-1 . 2 Anatomy of the lumbar spine and the healthy human  intervertebral disc The human lumbar spine usually consists of five vertebrae (LI to L5) located between the thoracic spine and the sacrum. It contains five intervertebral discs identified by the adjacent vertebrae (L1/L2 to L5/S1). Along with the facet joints, the intervertebral discs are responsible for supporting the compressive loading to which the trunk is subjected. The intervertebral discs of the lumbar spine are wedge shaped, giving the spine an anteriorly convex or lordotic curve. The IVD consists of three distinct tissues: the gelatinous centre or nucleus pulposus (NP), the surrounding fibrous ring called the anulus fibrosus (AF) and the two cartilaginous end-plates (CEP) which separate the AF and NP from the vertebral bodies. The three disc tissues work in combination to evenly distribute stress applied to the vertebral column (Higuichi et a l . , 1982). The semi-fluid NP, enclosed by the lamellar structure of the AF, converts axial loads into tensile strain on the anular fibres and CEP (Lipson and Muir, 1981) . In the young spine, the NP occupies 35% (White and Panjabi, 1978) to 50% of the cross-sectional area of the disc and has a translucent appearance (Bijlsma, 1972). In the - 3 -lumbar spine the NP is located slightly posteriorly to the mid-line (Inoue, 1981). The NP in healthy, young discs is between 84-90% water (Naylor and Horton, 1955) with the remaining structure being comprised of polyanionic proteoglycan molecules entrapped in the tissue by an interlacing network of collagen. The AF in the lumbar disc is a concentrically ringed structure, made up of approximately 90 sheets of highly fibrous tissue (Panagiotacopulous and Pope, 1987). The AF fibres are arranged in a helicoid manner and in adjacent layers are oriented at 120 degrees to each other (White and Panjabi, 1978). It contains a densely packed array of collagenous fibres interspersed with proteoglycan in a lower ratio to collagen than in the NP (Galante, 1967). The central AF has fibres inserting directly into the CEP, while the peripheral AF has attachments that insert directly into the vertebral bodies (Sharpey's fibres). The AF is thicker and its individual layers better developed on the anterior aspect than on the posterior. The water content of .the AF in the healthy young IVD is approximately 78%. The cartilaginous end-plates of the healthy IVD are composed of hyaline cartilage that at skeletal maturity, contain fine collagenous f i b r i l s (Bernick and Caillet, 1982). Prior to skeletal maturity the CEP cover the entire vertebral body and disc. At approximately 12 years, ossification - 4 -centres appear in the outer regions of the end-plates (Zaoussis and James, 1958). With cessation of growth, the o s s i f i ca t i on centres fuse to form a bony r ing that surrounds the CEP (Coventry et a l . , 1945). During fe ta l development the blood supply of the IVD consists of a cap i l l a ry network that supplies the outer margins of the AF. These cap i l l a r i e s are most abundant in the posterolateral regions. The NP has no d i rect blood supply (Schmorl and Junghanns, 1971). By the fourth year of l i f e a l l of these blood vessels have been obl i terated. The blood supply to the CEP is poorly understood. Branches of the l a te ra l lumbar arter ies supply the vertebral bodies on a segmental basis. These vessels are in intimate contact with the end-plates, but do not supply them d i r e c t l y . Thus, the IVD is an avascular structure (the largest in the body) and from an ear ly age the AF, NP and CEP receive their supply of nutrients and dispose of their metabolic waste products exclus ively by d i f fus ion (Hoi1inshead, 1965). Early studies have shown that this process occurs v ia two routes: from the anular edge and through the end-plates (Brodin, 1955). Of the.se two pathways, passive d i f fus ion through the end-plates has been shown to be most important (Urban et a l . , 1977). The spinal motion segments surround the spinal cord and at each leve l contain many structures including ligaments and -5-jo int capsules, r i ch l y innervated by f ibres from the segmental meningeal nerves. In the healthy d i sc, branches of these f ibres (sinuvertebral nerves) terminate in the posterior surfaces of the AF (Pedersen et a l . , 1956). The nerve supplies of the NP and CEP are poorly understood. 1.3 Histology of the healthy intervertebral disc The progressive d i f fe rent ia t ions that the intervertebral discs (IVDs) undergo after b i r th result in the three disc tissues (AF, NP and CEP) being distinguishable by the end of the f i r s t year. H i s to log i ca l l y , the IVD has been described as a "connective tissue organ", with each disc tissue having d i f ferent properties to f u l f i l i t s mechanical function (Urban and McMullin, 1988). The predominantly f i b r i l l a r structure of the AF is organized with the collagen f ibres t i g h t l y and regularly packed in an layered manner interspersed with proteoglycan (Buckwalter et a l . , 1976). Light and electron microscopic studies have demonstrated that the AF has a more dense f i b r i l l a r structure at i t s periphery than centra l ly near the NP. The t o t a l proteoglycan content of the AF increases from the periphery to the t rans i t ion zone (TZ) between the inner AF and NP (Spi lker, 1980). The collagen producing ce l l s located in the AF are biconvex in shape with elongated nuclei and are -6-distributed along the collagenous f i b r i l s . The resulting meshwork structure restricts the diffusion of large molecules by acting as a permeability barrier to control the transport of extracellular substances (Maroudas,1973). Unlike the lamellar organization of the collagenous network in the AF, the orientation of collagenous f i b r i l s in the NP is random with many large proteoglycan molecules scattered among and attached to the f i b r i l s (Inoue, 1 9 8 1 ) . Proteoglycan and water content are highest in the NP and lowest in the outer AF, while the reverse is true for collagen. The NP is vi r t u a l l y acellular with few elongated fibrocytes being evident (Pritzker, 1 9 7 7 ) . In the healthy IVD, the NP stains intensely with alcian blue, reflecting i t s high concentration of proteoglycans (Lipson & Muir, 1 9 8 1 ) . Histochemical studies have demonstrated that the glycosaminoglycans and collagen of the NP have properties similar to those of the CEP (Higuchi et a l . , 1 9 8 2 ; Herbert et a l . , 1 9 7 5 ; Inoue and Takeda, 1 9 7 5 ) . Studies conducted in mouse have shown only poorly developed Golgi complexes in the NP, unlikely to be capable of producing a proteoglycan rich matrix, while the CEP contains well-developed Golgi complexes (Higuchi et a l . , 1 9 8 0 ) . On this basis i t has been suggested that the CEP may produce the matrix substances of the NP. In the healthy IVD, the end-plates consist mainly of - 7 -hyaline cart i lage that stains pos i t i ve ly with a lc ian blue. Hematoxylin and eosin (H & E) staining of the CEP delineates a pink matrix from the blue nuclei of the many chondrocytes (Donisch and Trapp, 1971). Also present are a large number of fine collagenous f ibres that run throughout the hyaline cart i lage and glycosaminoglycan-rich matrix (Bernick and C a i l l e t , 1982). The CEP does not have a firm attachment to i t s adjacent vertebral body. Scanning electron microscopic (SEM) studies of the CEP shoved that there were no f i b r i l l a r connections between the CEP and the subchondral bone of the vertebral bodies, thus rendering the CEP vulnerable to horizontal shearing forces and possible injury (Inoue, 1981). 1 .4 Mechanochemistry of healthy cart i lage and intervertebral  disc In the mature non-degenerate state the intervertebral disc and end-plate cart i lage are avascular and have low ce l l u l a r densit ies. Therefore, i t is the properties of the extrace l lu lar matrix that are responsible for the physical properties of the tissue in vivo (Urban and Maroudas, 1980). The extracel lu lar matrices of cart i lage and disc consist ch ie f l y of collagenous f ibres embedded in a semi-f lu id gel of proteoglycan and water. At physiological pH the sulphate ester and glucuronic acid carboxyl groups hold a negative charge. This results in a molecule containing long chains of -8-polyanions that attract counterions in Donnan equil ibrium. The result ing osmotic pressure gradient produces the t i s sue ' s tendency to swell and imbibe water (Maroudas, 1973). From a functional point of view, the primary roles of the collagenous f ibre meshwork are to reta in the hydrated proteoglycan molecules and to l im i t the swell ing of the tissue (Eyre, 1979). The a b i l i t y of healthy cart i lage and intervertebral disc to absorb compressive loads and to redistr ibute mechanical forces e f fec t i ve l y , results from the f l u i d i t y and non-compressibility of the highly hydrated gel containing the proteoglycan molecules (Urban and Maroudas, 1981). 1.5 Carti lage and intervertebral disc proteoglycans The most studied proteoglycans are those isolated from bovine nasal cart i lage. Electron microscopic studies have shown that hyaline cart i lage proteoglycans have a bottle brush-l ike structure (Rosenberg et a l . , 1970). The proteoglycan molecules are made up of varying numbers, sizes and types of polyanionic sulphated polysaccharide chains (glycosaminoglycans) covalently attached to a shared protein core. The glycosaminoglycans are polysaccharides made up of disaccharide repeating units. Each unit consists of an amino sugar l inked by a g lycos id ic bond to a non-nitrogenous sugar. -9-The amino sugar or hexosamine may be either N-acetylglucosamine or N-acetylgalactosamine and the non-nitrogenous sugar either glucuronic acid and/or iduronic acid or galactose. Each disaccharide unit except hyaluronate may also contain one or more ester sulphate groups. (e.g. chondroitin sulphate contains a disaccharide of N-acetylgalactosamine and glucuronic acid and keratan sulphate a disaccharide of N-acetylglucosamine and galactose.) The most abundant glycosaminoglycans found in cart i lage are chondroitin sulphate (ChS), keratan sulphate (KS) and hyaluronate (HA) with dermatan sulphate and heparan sulphate found in some tissues. Chondroitin 4-sulphate and chondroitin 6-sulphate d i f f e r in the s i te of attachment of the ester sulphate group to the amino sugar residue. In cart i lage, chondroitin sulphate exists as a copolymer of C-4 sulphated and C-6 sulphated disaccharide units (Faltynek and S i lber t , 1978). Each proteoglycan monomer contains approximately 100 chondroitin sulphate chains each with a molecular weight of approximately 20,000 and 30-60 keratan sulphate chains having molecular weights averaging 5,000 (McDevitt, 1981). The ChS and KS are unevenly d istr ibuted along the protein core. Trypsin and chymotrypsin digestion of bovine tracheal and nasal cart i lage proteoglycans have shown that the KS-rich region of the protein core contains approximately 65% of the -10-to ta l KS and 10% of the ChS, while the ChS-rich region contains about 90% of the to ta l ChS and 20% of the to ta l KS (Heinegard and Axelsson, 1977). The core protein has a special ized section at the N-terminus that is capable of interact ing s p e c i f i c a l l y with the large chain, non-sulphated polysaccharide, hyaluronate (HA) (Hardingham and Muir, 1972). Proteoglycan molecules attach to chains of HA at intervals of approximately 30-40 disaccharide units through a non-covalent interact ion between their HA binding region (HABR) and a section of the HA chain at least 10 disaccharides long (Hascall and Heinegard, 1974). This interact ion is f a c i l i t a t e d and s tab i l i zed by a globular polypeptide termed a l ink protein (Hardingham, 1979). The l ink protein binds to both HA and the HABR and also protects the proteoglycan binding s i tes from proteo lyt ic digestion (Tengblad et a l . , 1984; Roughley et a l . , 1982). This gives r i se to extremely large aggregates having average molecular weights of approximately 250 m i l l i on (Inerot & Heinegard, 1982). A notable feature of the proteoglycan population in cart i lage and disc is i t s marked polydispers ity and heterogeneity. When proteoglycans are extracted from cart i lage, pur i f ied from other contaminating proteins and fractionated using gel chromatography, a broad range of molecular s izes is observed (Heinegard, 1972). This finding -11-re f lect s the differences in protein core length and the large var iat ion in the number and size of glycosaminoglycans attached to the protein core, as well as var iat ion in the number of proteoglycan monomeric subunits attached to the hyaluronic acid chain to form the aggregate. Several studies of the homology between cart i lage and disc proteoglycans have been undertaken (Stevens et a l . , 1979). The chief glycosaminglycans of the human disc are keratan sulphate (KS), chondroitin sulphate (ChS) and hyaluronate (Heinegard & Gardel l , 1967; Heinegard et a l . , 1979). In the mature disc,the rat ios of KS to ChS and of protein to ChS are higher than in bovine nasal cart i lage (Pearce and Grimmer, 1976). Art icu lar cart i lage (Kuettner and Kimura, 1985) and immature intervertebral disc (Buckwalter et a l . , 1985) have a high proportion of their proteoglycan in the aggregated form (approx. 85%). However, in the mature intervertebral d i sc, a much smaller proportion (30-40%) of the to ta l proteoglycans are present in the aggregate form (Ernes and Pearce, 1975). The mature disc proteoglycans appear either to lack or to have a non-functional HA binding region, since the addition of excess hyaluronate does not result in an increased proportion of aggregated molecules (McDevitt et a l . , 1981). A number of studies have compared the proteoglycan -12-populations of the three disc t issues. An electron microscopic comparison of the proteoglycans from AF,NP and CEP of infant disc (human) demonstrated that they share a basic common structure, which is in turn s imi lar to the structure of proteoglycan found in car t i l age. The electron micrographs ident i f ied NP and CEP aggregating and non-aggregating proteoglycans as having approximately the same s i ze. In addit ion, the NP appears to have two d i s t i nc t populations of aggregating proteoglycans of d i f ferent molecular s i ze, with the high molecular weight population resembling the aggregating PG seen in AF and CEP (Buckwalter et a l . , 1985). Further characterization of the disc proteoglycans has ident i f i ed addit ional differences. In human intervertebral d i sc, the AF contains a higher proportion of aggregating proteoglycans than the NP or CEP (Adams and Muir, 1976). Both the aggregating and non-aggregating proteoglycans of the intervertebral disc contain domains r i ch in KS (Lyons et a l . . 1981). The centre of the NP has a higher concentration of glycosaminoglycans than the other disc tissues (McDevitt, 1989) and also a KS concentration higher than that reported for any other connective tissue (Antonopoulous et a l . , 1969). Furthermore, when either disc AF, NP or a r t i cu la r ca r t i l age, proteoglycans are fractionated using composite agarose-polyacrylamide gel electrophoresis, multiple bands are observed suggesting that several d i s t i nc t sub-populations of -13-proteoglycans are present (McDevitt and Muir/1971; Roughley and Mason, 1976). Three distinct subspecies have been identified in cartilage (McDevitt & Muir, 1971; Heinegard et a l . , 1981). The NP aggregating fraction is made up of three distinct subspecies, while the non-aggregating proteoglycans from post-mature discs showed three electrophoretically distinct subpopulations, differing in KS and ChS content (Jahnke & McDevitt, 1988; DiFabio et a l . , 1987 ). Also, the AF proteoglycans have been shown to be comprised of at least two aggregating and three non-aggregating subspecies (DiFabio et a l . , 1987). The heterogeneity of the proteoglycans of the CEP, as assessed by electrophoresis, has not been investigated previously. 1.6 Changes in cartilage and intervertebral disc with ageing  and degeneration Ageing and degeneration of articular cartilage have been shown to be biochemically distinct processes (Inerot & Heinegard, 1982). While the total tissue content of glycosaminoglycans remains nearly constant with age, their relative proportions change markedly (Roughley & White, 1980). The relative proportions of keratan sulphate and hyaluronate increase at the expense of the ChS (Inerot & Heinegard, 1982). In addition, the ratio of chondroitin-6-sulphate to chondroitin-4-sulphate increases with age (Roughley & White, 1980 ) . -14-In degenerated a r t i c u l a r c a r t i l a g e , the proteoglycans are s m a l l e r ( I n e r o t and Heinegard, 1982). They a l s o c o n t a i n l e s s p r o t e i n and keratan sulphate and have decreased c h o n d r o i t i n - 6 - s u l p h a t e to c h o n d r o i t i n - 4 - s u l p h a t e r a t i o s (Inerot & Heinegard, 1982). Studies of o s t e o a r t h r i t i c a r t i c u l a r c a r t i l a g e have r e p o r t e d t h a t the r a t e of p r o t e o g l y c a n b i o s y n t h e s i s i s a c c e l e r a t e d 4 0 0 - f o l d . T h i s enhanced b i o s y n t h e s i s i s thought to r e p r e s e n t t i s s u e r e p a i r (Carney e t _ a l . , 1984 ). However, the r e p a i r process u s u a l l y f a i l s , p o s s i b l y s i n c e d e f e c t i v e PG, unable to form aggregates, i s the c h i e f b i o s y n t h e t i c product ( B r o c k l e h u r s t et a l . , 1984). I n t e r v e r t e b r a l d i s c degeneration i s d e f i n e d m o r p h o l o g i c a l l y , as c o n s i s t e n t b i o c h e m i c a l c r i t e r i a have yet to be e s t a b l i s h e d . A d e t a i l e d o b s e r v a t i o n a l study of degenerative changes i n 100 lumbar s p i n e s conducted by Vernon-Roberts has produced a s y s t e m a t i c d e s c r i p t i o n of i n t e r v e r t e b r a l d i s c degeneration (Vernon-Roberts, 1980). The f i r s t m o r p h o l o g i c a l l y i d e n t i f i a b l e event i n t h i s process i s l o s s of the g e l a t i n o u s c h a r a c t e r of the NP. I t becomes i n c r e a s i n g l y f i b r o u s i n appearance u n t i l i t can no longer be d i s t i n g u i s h e d from the AF. T h i s i s f o l l o w e d by the appearance of s p l i t s and c l e f t s i n the nucleus pulposus. These c l e f t s i n c r e a s e i n s i z e and number u n t i l they e v e n t u a l l y c o a l e s c e and extend i n t o the anulus f i b r o s u s . The p o s t e r i o r and p o s t e r o l a t e r a l r e g i o n s of the AF are favored l o c a t i o n s f o r -15-th is process to occur. This location is believed to be favored because of the AF being thinner on i t s posterior aspect as well as because the posterolateral regions having been weakened by the loss of blood vessels present in the fe ta l d isc. These changes are often accompanied by circumferential c l e f t s between the lamellae of the AF. As these tears enlarge, they predispose the disc to addit ional injury. Torsional s t ra in in part icu lar , w i l l further enlarge the gaps in the AF, possibly leading to disc herniation (K i rka ldy-Wi l l i s et a l . , 1978). Other c l e f t s can develop in the NP in the ax ia l d i rect ion within the d i sc. These can extend into the CEP to eventually produce extrusion of disc material into the trabecular bone of the vertebral body (Schmorl's nodes). The net effect of these changes is to reduce the height of the disc and thereby a l te r s i gn i f i can t l y the biomechanical equil ibrium that exists in the healthy motion segment. Histo log ica l studies of the disc have shown that with ageing, the matrix of the NP becomes less distinguishable from the matrix of the AF. After the age of 30, large chondroid ce l l s appear in increasing numbers in the NP (Pr i tzker, 1977). The fibrocytes in the AF produce an a lc ian blue staining matrix which separates the collagen f ibres surrounding each c e l l . This process (anular c e l l metaplasia) begins in the -16-t rans i t ion zone (TZ) between the AF and NP. At the same time, fraying and s p l i t t i n g of the innermost anular f ibres are seen and the large chondroid c e l l s in the NP begin to appear in the TZ. In more advanced stages of degeneration, large pools of a lc ian blue staining matrix appear between the anular f ib res . Recent studies suggest strongly that the depletion of proteoglycan from the entire lumbar spine predisposes the intervertebral disc to degeneration (Pearce e_t §_1., 1987). Many investigators have also linked degenerative changes in cart i lage with ageing to a slower turnover of matrix molecules (Poole, 1986). The most notable changes in the proteoglycans of cart i lage and disc with ageing and degeneration are reduction in the to ta l proteoglycan and water content with a concomitant re lat ive increase in collagen (Adams and Muir, 1986). In the intervertebral d i sc, these changes are most prominent in the NP. The proportion of aggregating proteoglycan in the disc decreases with ageing, while the proportion of non-aggregating proteoglycan increases. These changes are l i k e l y due to a decrease in the length of the protein core, since the ChS and KS chain lengths were unchanged (Pedr in i -Mi l le et a l . , 1980 ). Other investigators have reported a higher proportion of aggregated proteoglycan, a higher ra t io of glucosamine to galactosamine (KS to ChS) and a higher proportion of chondroitin 6-sulphate -17-re lat ive to chondroitin 4-sulphate in the NP of degenerate discs (Lyons et a l . , 1981). Studies have suggested that the intervertebral disc may be more capable of repair than a r t i cu l a r car t i lage. Animal model studies have shown that experimentally induced disc herniation stimulates a repair mechanism and that extensive enzymatic degradation of a lumbar disc in vivo results in the formation of a regenerated disc with PG properties v i r t u a l l y ident ica l to those of the o r i g ina l disc (Lipson & Muir, 1981; Bradford et a l . , 1983 ). 1.7 Biochemical and functional properties of the end-plate  and the present study This study was undertaken to determine whether or not the proteoglycans in the CEP are affected by ageing and/or degeneration of the intervertebral d isc. Since the disc is avascular, i t s nutr i t ion is largely dependent upon normally functioning end-plate (Ogata and Whiteside, 1981). The cap i l l a r y network that nourishes the disc is intimately associated with the end-plate and nutrients must f i r s t diffuse through the end-plate before reaching the AF and NP (Nachemson et a l . , 1970). The matrix composition of the end-plate and, in par t i cu la r , the proteoglycan content, plays a large part in determining the transport properties of nutrients (Maroudas, -18-1979). In addit ion, a normal end-plate PG composition acts as a barrier to prevent or slow the d i f fus ion of small proteoglycans of the AF and/or NP out of the disc (Bradford e_t a l . , 1983). The end-plate also plays a key role in disc biomechanics by enhancing i t s shock absorbing properties (Broberg, 1983). Loss of functional end-plate cart i lage reduces the a b i l i t y of the disc and the adjacent vertebral body to absorb impact and thereby a l te r the forces acting on the AF and NP (Coventry et a l . , 1945). Microscopic anomalies of the end-plate are thought to predispose the disc to the development of Schmorl's nodes (Roberts et a l . , 1989 ). Focal areas of end-plate are replaced by AF and NP tissue (Perey, 1957). With severe disc loading, the hydrostatic pressure of the NP causes the disc to expand into the subchondral bone leading to accelerated disc degeneration and vertebral compression fractures (Roberts et a l • , 1989; Eyring, 1969). Studies of changes in the end-plate with ageing and degeneration of the disc have demonstrated that the deeper cart i lage c e l l s become c a l c i f i e d , preventing the remaining CEP ce l l s from receiving nut r i t ion and leading to end-plate destruction. Also, the decrease in the supply of nutrients to the CEP impairs the maintenance of the AF and NP (Higuchi and Abe, 1987) and is thought to lead d i r e c t l y to degeneration of -19-the NP (Pr i tzker, 1977). Typical ly, the h i s to log ic features of CEP related to age occur in the th i rd decade. Ce l l death and focal loss of metachromasia are seen in the super f i c i a l layers with disorganization and clumping of the chondrocytes also apparent in the central region. In the early stages of degeneration, regenerative changes, such as increased metachromasia of t e r r i t o r i a l matrix may be seen. End-plate h i s to log ic change has been pos i t i ve ly correlated with disc space narrowing and degeneration of the nucleus pulposus (Aoki et a l . , 1987). Other studies have concluded that h i s to log ic changes associated with degeneration take place in the end-plate prior to changes in the AF or NP and have hypothesized that def ic iencies of matrix biosynthesis in the end-plate i n i t i a t e disc degeneration (Pr i tzker, 1977). Also, the CEP has been shown to have a much higher density of chondrocytes than the anulus fibrosus or nucleus pulposus (Buckwalter et a l . , 1985) and is therefore capable of contributing s i g n i f i c an t l y to the matrix biosynthesis in the disc AF and NP. L i t t l e was known previously of the biochemistry of the end-plate proteoglycans. If the end-plate is responsible for the biosynthesis of the matrix components of the AF and NP, then i t s proteoglycan composition should resemble that of the other disc t issues. However, the end-plate appears - 2 0 -h i s to l og i ca l l y s imilar to a r t i cu la r car t i lage, and therefore, as previously discussed, should have a proteoglycan composition that d i f f e r s markedly from that of the intervertebral disc. Recent studies have reported t o ta l end-plate proteoglycan contents intermediate between those of disc (AF and NP) and a r t i cu l a r cart i lage (Roberts et a l . , 1989). Other studies have demonstrated d i f f e r i n g proportions of aggregating proteoglycans in the end-plates of discs from postnatal and young adult donors (Donohue et a l . , 1988). The composition of the end-plate proteoglycans has also been shown to a l te r under the influence of abnormal mechanical stress. Ea r l i e r studies of CEP proteoglycans in discs from s c o l i o t i c spines had reported wide variat ions in concentration and composition of the aggregating and non-aggregating populations (Pedr in i -Mi l le et a l . , 1983). The spec i f i c aims of th is study are: 1) to characterize the proteoglycans of the carti laginous end-plate from healthy, young discs; 2) to compare the CEP PG from healthy discs of young donors with the CEP PG from degenerate discs of older donors and 3) to dist inguish between the changes in CEP PG due to ageing from those due to degeneration. -21-2 . 0 METHODS - 2 2 -2 . 0 METHODS 2.1 Analytical methods Unless otherwise stated, a l l chemicals used were either of analytical reagent grade or the best commercial grade available. Hexuronate was assayed using the carbazole-borosulphuric acid reaction (Bitter and Muir, 1961) with a sodium glucuronate monohydrate standard (Corn Products Refining Co., New York, N.Y., U.S.A.). Hexose was assayed using the anthrone reaction using a galactose standard (A Grade, Calbiochem-Behring, San Diego, CA, U.S.A.). 2.2 Collection and grading of intervertebral discs Lumbar spines free of the posterior elements were obtained within 48 h of death from the morgues of the Vancouver General and University Hospital-U.B.C site. Immediately after dissection magnetic resonance images (MRI) of the specimens were obtained in the mid-sagittal plane using the Picker Vista 2000 MR Proton Tomograph operating at 0.15T. Spin echo pulse sequences were used with a repeat time (TR) of 1916 ms or 2916 ms and an echo time (TE) of 40 ms. The spines were then wrapped in impermeable plastic and aluminium f o i l prior to freezing at -80°C. Each of the ten vertebral columns studied was cut, whilst frozen, in the mid-sagittal plane -23-using a band saw, rinsed in tepid water with brushing to remove bone fragments and photographed in colour. Typical ly, this procedure took one hour for each spine. The spines were stored at -80°C u n t i l needed. Discs were assessed on the basis of gross morphology and MRI using the grading scheme developed by Thompson et a l . (Appendix I ) . Morphological grades of I to V were assigned b l ind ly by Drs. P. Thompson (Orthopaedic Surgery), I. Tsang, M. Adams (Rheumatology), and R. Pearce (Pathology); MRI grades by D. L i , B. Flak, B. Ho (Diagnostic Radiology) and P. Thompson; a grade of I corresponded to a healthy, non-degenerate d i sc; grades II to V described increasingly severe degeneration. Each of the forty-three discs used in th is study was therefore graded independently by at least s ix d i f ferent individuals (including myself). Typica l ly , this procedure requiured 8-10 hours of preparation and interpretation for each spine. 2.3 Dissection of end-plates and determination of water  content At each level of the spine, the anulus fibrosus (AF) and nucleus pulposus (NP) were carefu l l y removed with a scalpel (Figures 3.1.2-2B and 3.1.2-3B; pp 55-57). The remaining remnants of the AF and NP were removed by scraping sideways -24-with the edge of a scalpel blade un t i l the perimeter of the carti laginous end-plate (CEP) was c lear ly v i s i b l e (Figures 3.1.2-2C and 3.1.2-3C). To avoid contamination of the CEP preparation with AF or NP, a rim of end-plate tissue was l e f t around the margins and the CEP removed from the in fer io r and superior aspects of the vertebral bodies u n t i l the subchondral bone was exposed (Figures 5d and 6d). This procedure was carried out on both the superior and in fer ior end-plates of each of the forty-three discs studied ( i .e . a t o t a l of eighty-s ix end-plates). Typica l ly , the dissection of each disc required a minimum of two hours. For the experiments conducted in Part I, the CEP of a l l discs from each lumbar spine were pooled, whilst in Part II, CEP adjacent to each disc were kept separately. A sample of each CEP dissection was f ixed in formalin (10%) for at least 7 days, embedded in para f f in , sectioned at 5 um and stained with haematoxylin and eosin (Cul l ing, 1974) (Figure 4D). The remaining tissue was weighed, sectioned at 20 um using a cryostat, then freeze-dried. Water content was estimated as the difference between fresh weight of the tissue following dissection and the dry weight after freeze-drying. 2.4 Preparation of A l proteoglycan Proteoglycan (PG) was extracted from the dry residue under d issociat ive conditions by suspension in 10 ml/g tissue -25-of 4M-guanidinium HCl containing 0.05M sodium acetate buffer, pH 5.8, and protease inhibitors, and shaking gently for 48 hours at 4°C (Hascall and Kimura, 1972). The protease inhibitors added immediately prior to the beginning of the extraction, were 6-aminocaproic acid (100 mM) for cathepsin D activity against proteoglycans, benzamidine-HCl (1 mM) for trypsin-like activity (Hascall & Kimura, 1972), disodium EDTA (10 mM) for metalloproteinases, phenylmethylsulphonyl fluoride in methanol (1 mM) for serine-dependent proteases and N-ethylmaleimide (10 mM) for th i o l proteases. The pH of 5.8 was above the optimum pH for acid proteases and below that for neutral proteases (Hascall and Kimura, 1972) and also close to optimal for extraction of proteoglycan (Hardingham & Muir, 1972). After extraction, the suspension was centrifuged at 15,000g for 60 min and the supernatant decanted. In some experiments, the pellet was then re-extracted using the same method. Hexuronate (HexA) was assayed to estimate the quantity of PG present. For the f i r s t two donor spines this procedure showed that 98% of the extractable hexuronate was present in the f i r s t extract. Therefore, a second extraction was not done in subsequent experiments. The extract was dialysed for 24 h at 4°C against 8 vol. 0.5 M sodium acetate, pH 5.8 containg protease inhibitors to reduce the guanidine-HCl concentration to 0.5 M (associative -26-conditions). Hyaluronate (Healon, Pharmacia, Uppsala, Sweden) equivalent in hexuronate to 2% of the proteoglycan hexuronate was added to assure maximum re-aggregation of the PG. The PG was prepared by cesium chloride density gradient centrifugation (Sajdera and Hascal l , 1970) using a start ing density of 1.42 g/ml at 100,000g at 10°C for 72 h. The tubes were cut into f ive sections of equal volume (Al to A5 from bottom to top) and the dens it ies, in g/ml, were determined by weighing a measured volume. Each f ract ion was dialyzed for 24 hours at 4°C against 0.05 M sodium acetate buffer, pH 5.8 and then for 24 hours at 4<>C against the column eluent described below. The y ie ld of PG, estimated as the sum of the molar amounts of hexuronate and hexose, was determined for a l l f ive f ract ions. The dialyzed Al f ract ion, containing the bulk of PG, was freeze-dried and stored at -80<>C u n t i l required (usually less than 7 days). 2.5 Gel chromatography of the Al proteoglycan A Sepharose CL-2B column (1.6 cm x 100 cm) was equi l ibrated with 8 volumes of 0.225 M sodium sulphate containing 0.033 M sodium phosphate buffer, pH 6.8 at 4°C. The flow rate was 2 ml/h and t h i r t y - f i v e 2.2 ml fractions were co l lected. The column eluent was monitored at 206 nm. Prior to gel f i l t r a t i o n of each A l PG sample, the column -27-was cal ibrated to determine the void volume (VO) and to ta l column volume (VT). The void volume (VO) was determined using a pa r t i a l l y pur i f ied proteoglycan extract (Al PG) from bovine nasal cart i lage (1.0 plmole HexA/ml) which contained a high proportion of aggregating PG having a molecular size greater than the exclusion l i m i t of Sepharose CL-2B (Heinegard et a l . , 1985). The to ta l volume (VT) of the column was determined using pyridoxal phosphate (6mM). A 1.0 ml sample was applied to the Sepharose CL-2B column and eluted with the column buffer described below. The absorbance at 280 nm of any fract ion with yellow color was recorded (Figure 2.5.1). Using th i s method and on the basis of t r i p l i c a t e runs, the void volume (± standard variation) was determined as 21.5 ml + 0.3 ml and the t o t a l volume was determined as 79.0 ml ± 0.5 ml (Figure 2.5.1). The column was cal ibrated prior to every appl icat ion of a CEP Al PG sample from each disc of the seven spines studied. Aliquots (1.0 ml) of Al PG were prepared from the freeze-dried CEP samples (1.0 jlmole HexA/ml of column buffer) and applied to the Sepharose CL-2B column previously equi l ibrated with column buffer (0.225 M sodium sulphate containing 0.033 M H3P04, pH 6.8) at 4<>C. The eluent was monitored at 206 nm, the flow rate of the column was 4 ml/hr and 2.2 ml fractions were collected (Figure 2.5.1). The mean values of the t r i p l i c a t e runs were plotted and -28-FIGURE 2.5.1 CALIBRATION OF SEPHAROSE CL-2B COLUMN A mixture of Al proteoglycan from bovine nasal septum (1.0 umoles hexA in a 1.0 ml volume) detected at 206 nm (VO) and pyridoxal phosphate (60 p.moles in a 1.0 ml volume) detected at 280nm (VT) was applied to the Sepharose CL-2B column. - 2 9 -F I G U R E 2 . 5 . 1 -30-the Kmode values (mean value of column elut ion volume corresponding to the point of highest absorbance at 206 nm) estimated for the aggregating (high M) and the non-aggregating (low M) PG populations. 2.6 High and low molecular weight components The Sepharose CL-2B column chromatography of the A l PG from CEP of a l l the samples studied showed two peaks of absorbance. The high and low molecular weight (high M and low M, respectively) PG components were separated by div id ing the eluate at the point of the minimum absorbance at 206 nm. In cases where the minimum absorbance was not obvious, l ines tangential to the nearest point of maximum rate of change of absorbance were constructed. The intersection of these l ines was taken as the div id ing point of the two peaks. The fractions contained within each of these peaks were then pooled, concentrated by pressure u l t r a f i l t r a t i o n (Amicon YM-10 membrane), freeze-dried and stored at -80°C. To determine the percentage of the to ta l Al PG contained in the high M f ract ion, the areas under the peak above and below the minimum were calculated by counting the squares on the recorded absorbance p lot . 2.7 Composite agarose-polyacrylamide gel electrophoresis Composite agarose-polyacrylamide gel electrophoresis -31-(CAPAGE) has been shown to be effect ive in resolving cart i lage proteoglycan into d i s t i nc t subspecies (McDevitt and Muir, 1971) . The CAPAGE analysis of the Sepharose CL-2B column chromatography fractions was done by pooling, across the elution p r o f i l e , 100 ul samples from groups of three fractions separated by one unsampled f ract ion. Each pool was concentrated and exchanged with d i s t i l l e d water using centr ifugal u l t r a f i l t r a t i o n (Centricon 30, Amicon Corp., Danvers, MA) to give 25 p i volumes containing 5-10 nmoles hexuronate. Each sample was mixed with an equal volume of 60% (w/v) sucrose/ 0.2% (w/v) Triton X-100/ 0.01% (w/v) bromphenol blue. Aliquots of 20 u.1 were examined by electrophoresis using the method of McDevitt & Muir (1971), modified to use ve r t i ca l gels 3mm thick on 140 mm x 160 mm frosted-glass plates (Heinegard et a l . , 1985). Each gel was pre-run for 1 h at a constant current of 50 mA and a maximum voltage of 250 V. The samples were then applied and run under the same conditions for up to 3 h. The CAPAGE analysis of high M fractions required a 2 h running time, while the analysis of the low M fractions required a running time of 3 h. After staining with tolu id ine blue and washing with 0.5M acetic acid (pH 2.5) and d i s t . water, the gels were dried in a i r at room temperature on GelBond (F.M.C. Corp.). - 3 2 -The preparative CAPAGE analysis carried out to assess the heterogeneity of the high M and low M PG species from CEP of Grade I discs used samples containing 1 plmole hexA in a volume of 250 p.1 d i s t i l l e d water. The electrophoresis was done under the conditions described above, except that a s ing le-wel l comb (preparative scale) was used when the gel was cast. The sample volume was applied to the gel with the stand on a level surface to d i s t r ibute the sample evenly across the wel l . The electrophoretic properties of the high and low M PG fractions of CEP from discs of d i f fe r ing grades were examined using the ana ly t i ca l CAPAGE method described previously. The gels were scanned at 525 nm with a recording absorptiometer (Autoscanner, Helena Laboratories). A chondroitin 4-sulphate standard prepared from bovine nasal septum cart i lage (Tengblad, unpublished results) was used as a reference for mobil ity; i t s average M, calculated from v i scos i ty measurements, was 22,400 (Wasteson, 1971). 2.8 S t a t i s t i c a l analyses Student's two-tai led t - tes t for non-paired data was used to examine the s ignif icance of differences between two groups; the NCSS programme was used for this procedure. Analysis of variance was used to examine for the presence of heterogeneity in data with nested or with more than two groups; s ignif icance was assessed using the variance rat io F; the SAS programme GLM (general l inear model) was used for this analysis. - 3 3 -3.0 RESULTS -34-3.1.0 PART I COMPARISON OF THE PROTEOGLYCAN OF CARTILAGINOUS END-PLATE FROM HEALTHY INTERVERTEBRAL DISCS WITH THAT FROM DEGENERATE INTERVERTEBRAL DISCS The i n i t i a l goals of this project were to compare the gross morphology, histological appearance and proteoglycan composition of cartilaginous end-plates of healthy, young discs with those of moderately to severely degenerate discs from older donors. 3.1.1 Selection of specimens As described in Table 3.1.1, the donors were either accident victims whose death did not involve trauma to the lumbar spine or others who had died acutely of a condition unrelated to the spine. The lumbar spines selected for investigation, with one exception, contained five intervertebral discs designated by the adjacent spinal segments (lumbar vertebrae (L) and sacrum (S)) (L1/L2, L2/L3, L3/L4, L4/L5 and L5/S1). Intervertebral disc (IVD) morphologic grades were assigned blindly by two rheumatologists and a chiropractor, on the basis of colour pictures of mid-sagittal sections (gross morphology) and the magnetic resonance imaging grades by three radiologists using - 3 5 -Table 3.1.1 Donors of spines and grades of intervertebral discs Donor Age (y) Cause of Death Disc Grade and Sex Gross MRI A 21F Brain injury I/I/I/I/I B 16M Skul l fracture I/I/I/I/I C 18F Brain Haemorrhage I/I/I/I/I D 33M Pulmonary embolus II/II/II/II/II E 64F Myocardial infarct IV/IV/IV/V/V F 9 5M Pulmonary embolus IV/IV/IV/IV/-G 62M Myocardial infarct V/IV/IV/IV/IV I/I/I/I/I I/I/I/I/I I/I/I/I/I II/II/II/II/II i v / i v / i v / i v / r IV/IV/IV/IV/-i v / m / i v / i v / r *Reading from l e f t to right are the grades assigned (I to V) to the discs L1/L2 to L5/S1. A dash s i gn i f i e s that the disc was not available for grading. Discs were graded on the basis of gross morphology (Gross) and magnetic resonance image (MRI). -36-FIGURE 3.1.1-1 TYPICAL LUMBAR SPINE CONTAINING GRADE I DISCS Color photograph ( l e f t ) and MRI (right) of mid-sagitta l section of the lumbar spine from a 16 year old male (Donor B). The L1/L2 to L5/S1 discs are shown and were each assigned a grade of I . - 3 7 -F I G U R E 3 . 1 . 1 - 1 FIGURE 3.1.1-2 LUMBAR SPINE CONTAINING GRADE II DISCS Col o r photograph ( l e f t ) and MRI ( r i g h t ) of m i d - s a g i t t a l s e c t i o n of the lumbar spine from a 33 year o l d male (Donor D). The L1/L2 to L5/S1 d i s c s are shown and were each a s s i g n e d a grade of I I . -39-F I G U R E 3 . 1 . 1 - 2 FIGURE 3.1.1-3 TYPICAL LUMBAR SPINE CONTAINING GRADE IV/V DISCS Color photograph ( l e f t ) and MRI (right) of mid-sagittal section of the lumbar spine from a 95 year old male (Donor F). The L1/L2 to L4/L5 discs are shown and were each assigned a grade of IV. Note that the L5/S1 disc was not obtained from th i s d issect ion, but was present in a l l of the other spines used. -41-FIGURE 3.1.1-3 MRI of mid-sagittal sections (See Figures 3.1.1-1, 3.1.1-2 and 3.1.1-3). The c r i t e r i a used to assign grades are described in Appendix I (Thompson, 1988). A grade of I corresponded to a healthy d i sc, showing no signs of degeneration; grades II to V described increasingly severe degeneration. Two pr inc ipa l groups of spines were used in this study. The f i r s t comprised three lumbar spines (Donors A,B and C)) that contained only grade I discs and the second contained grade IV or V discs (Donors E,F and G) . As l i s t ed in Table 3.1.1, the spines containing healthy discs were obtained from donors (two female and one male) between the ages of 16 and 21 years, while the spines containing degenerate discs were obtained from donors (two males and one female) between the ages of 62 and 95 years (Table 3.1.1). The lumbar spine of a 33 year old male (Donor D) containing f ive grade II discs was also studied. The gross morphology and MRI of typ ica l spines of the three groups (Donors B,D and F) are shown in Figures 3.1.1-1, 3.1.1-2 and 3.1.1-3. Table 3.1.1 l i s t s the grades assigned to each disc using gross morphology and MRI. The grading schemes, without exception, assigned the same grade to healthy (grade I) and mildly degenerate discs (grade I I ) , but often di f fered in their assessments of moderately (grade III) and severely degenerate (grades IV and V) d iscs. In each case of disagreement, the MRI grade was lower than the gross grade. - 4 3 -F I G U R E 3.1.2-1 T H E T I S S U E S O F T H E I N T E R V E R T E B R A L D I S C A s a g i t t a l section, stained with H . & E., of the Ll,2 disc of donor A, with the anterior aspect on the l e f t side: The three disc tissues l a b e l l e d as A, B and C, are shown at higher magnification below. A Nucleus pulposus (128x) B Anulus fibrosus (128x) C Cartilaginous end-plate (128x) D End-plate sample obtained from a dissec t i o n of the same spine(64x) -44-FIGURE 3.1.2-1 - 4 5 -3.1.2 Dissection of the carti laginous end-plate As shown in Figure 3.1.2-1, the cart i laginous end-plate (CEP) (Figure 3.1.2-1C) is readi ly distinguishable h i s to log i ca l l y from the other IVD tissues. However, both the nucleus pulposus (Figure 3.1.2-1 A) and the anulus fibrosus (Figure 3.1.2-1 B) are continuous with the CEP and can eas i ly contaminate any preparation of end-plate t i ssue. As previously described (see Methods), the dissect ion procedure involved removing the outer layer of the hyaline cart i lage to assure that the material used for later studies was not contaminated with other t i ssue. A tissue sample from a CEP preparation obtained using th is method is shown in Figure 3.1.2-1 D. The H and E sta in of the CEP preparation i l l u s t r a te s the usual appearance of hyaline cart i lage with sparse lacunae containing dark staining pyknotic nuclei interspersed within the homogeneous eosinophi l ic extrace l lu lar matrix. A sample of CEP tissue from each disc studied was examined in th is manner. A l l CEP dissections chosen for further study were shown by this method to be free of contaminating NP or AF. Figures 3.1.2-2 and 3.1.2-3 i l l u s t r a t e the differences between CEP dissections of healthy discs (grade I) and discs which have undergone degeneration (grade IV/V), respectively. As shown in Figure 3.1.2-2 A, the CEP in healthy discs is - 4 6 -FIGURE 3.1.2-2 GRADE I INTERVERTEBRAL DISC, CARTILAGINOUS END-PLATE AND SUBCHONDRAL BONE A Sag i t ta l section of a grade I disc B Horizontal section of the anulus fibrosus and nucleus pulposus of grade I disc C Cartilaginous end-plate of grade I disc (with anulus fibrosus and nucleus pulposus removed) D Subchondral bone of grade I disc (with end-plate removed) - 4 7 -FIGURE 3.1.2-2 FIGURE 3.1.2-3 GRADE IV/V INTERVERTEBRAL DISC, CARTILAGINOUS END-PLATE AND SUBCHONDRAL BONE A Sagitta l section of a grade IV disc B Anulus fibrous and nucleus pulposus of a grade IV disc C Cartilaginous end-plate of a grade IV disc (with the anulus fibrosus and nucleus pulposus removed) D Subchondral bone of a grade IV disc (with the end-plate removed) - 4 9 -FIGURE 3.1 . 2 - 3 - 5 0 -regular, approximately 3 mm thick, evenly contoured and separates the bone of the vertebrae above and below from the nucleus pulposus (NP) and the medial anulus fibrosus (AF). In degenerate discs, the CEP is disrupted and varies greatly in thickness, from less than 1 mm to the normal 3 mm (Figure 3.1.2-3 A). Also, in the healthy d isc, the NP and AF are c lea r l y demarcated; the NP is bulging and gelatinous, while the AF consists of uninterrupted concentric fibrous rings (Figure 3.1.2-2B). The degenerate disc shows an NP that is not c lea r l y demarcated from the AF; the NP and AF contain many c l e f t s that in some places have coalesced to form large f i ssures. Also the rings of the AF have been i n f i l t r a t e d by a mucinous substance (Figure 3.1.2-3B). When the NP and AF are removed to expose the underlying CEP, differences between grade I disc and grade IV/V disc CEP were evident in a l l specimens studied. Figure 3.1.2-2 C i l l u s t r a te s the typ ica l extent of the area covered by the end-plate in a healthy disc and demonstrates i t s even d i s t r ibut ion over the f u l l width of the NP and medial AF. In Figure 3.1.2-3 C (degenerate disc) the area covered by the CEP is markedly reduced with areas of c a l c i f i c a t i o n noted on the outer margins. The CEP also has many small d i scont inu i t ies and a generally inconsistent d i s t r ibut ion where i t remains intact. Figure 3.1.2-2 D shows the normal red, spongy character of the subchondral bone immediately beneath the CEP of a healthy d isc, in contrast -51-Table 3.1.3 Comparisons of fresh weight, dry weight and vater  content of end-plates of healthy and degenerate  d iscs Donor Mean Disc Fresh Dry Water Grade* Wt. Wt. Content Gross MRI (g) (g) (g/g fresh wt) A I I 4.20 0.95 0.772 B I I 6.40 1.62 0.747 C I I 1.23 0.32 0.739 D II II 4.42 1.31 0. 704 E IV IV 6.84 1.94 0.716 F IV IV 2.20 0.65 0 . 705 G IV IV 1. 41 0.50 0.644 *Denotes the average grades assigned to the f ive lumbar intervertebral discs Unpaired t - te s t s t a t i s t i c a l analysis Parameter Compar ison D.F. P Water Content Between groups 1 0. 058 Donors A7B and C were assigned to group 1; donors E,F and G to group 2 -52-with the c a l c i f i e d deposits (small white f lecks) in the subchondral bone of degenerate discs (Figure 3.1.2-3 D). 3.1.3 Yield and water content of cart i laginous end-plate The cart i laginous end-plates were dissected from each lumbar spine to produce a pool of ten end-plates for each of the seven lumbar spines (with the exception of donor F, which contained only eight end-plates). Table 3.1.3 l i s t s the weights of fresh tissue obtained from each spine. There was considerable var iat ion in the fresh weights of CEP pools obtained following dissect ion. This was for the most part due to differences in the physical size of the donors. For the purposes of standardization, a l l subsequent parameters are reported re la t i ve to one gram of dry t issue (except for water, for which wet weight was used). The mean water content (± standard deviation) of the CEP from spines containing grade I discs (0.753 g/g fresh wt t 0.019 g/g fresh wt) d i f fered from that of CEP from grade IV/V discs (0.688 g/g fresh wt ± 0.044 g/g fresh wt.). The probabi l i ty that the CEP from the group of three spines a l l containing grade I discs had the same water content as the CEP from the group of three spines containing only grade IV and V discs was 0.058 (by t - t e s t ) . The value for donor D overlapped those for the grade IV/V discs. Thus, despite the few specimens studied, the difference in water contents approached the conventional -53-FIGURE 3.1.4-1 DENSITY GRADIENT CENTRIFUGATION The densit ies of each f ract ion (sol id c i r c l e s ) obtained from the CsCl density centr ifugation are plotted for the Al (bottom) to A5 (top) f ract ions. The tota l proteoglycan, calculated as hexose plus hexuronate is plotted on the r ight ve r t i ca l ax is . -54-F I G U R E 3 . 1 . 4 - 1 Fraction -55-standard of s t a t i s t i c a l s i g n i f i c a n c e , i . e . , P<0.05. 3.1.4 Composition of the A l f r a c t i o n s The p r o t e o g l y c a n s were prepared from the CEP t i s s u e e x t r a c t u s i n g a s s o c i a t i v e CsCl d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . The e f f i c a c y of the technique i s dependent upon a c h i e v i n g a c o n t i n u o u s d e n s i t y g r a d i e n t from the top to the bottom of the c e n t r i f u g e tube ( F i g u r e 3.1 . 4-1). The r e s u l t i n g g r a d i e n t was d i v i d e d i n t o f i v e f r a c t i o n s o f equal volume. As i l l u s t r a t e d i n F i g u r e 3.1.4-1, the d e n s i t i e s of the f r a c t i o n s i n c r e a s e d s t e a d i l y from the top to the bottom of the tube. The d e n s i t i e s of the A l f r a c t i o n s ranged from 1.60g/ml to 1.68g/ml (Table 3 .1 . 4 ) . The t o t a l p r o t e o g l y c a n (PG) was estimated as the sum of the hexose (a measure o f k e r a t a n sulphate) and the hexuronate (a measure of the c h o n d r o i t i n s u l p h a t e ) . The hexose assay measures the amount of g a l a c t o s e , which i s p r e s e n t i n k e r a t a n s u l p h a t e and a l s o i n t h e r e l a t i v e l y few o l i g o s a c c h a r i d e s a t t a c h e d to the PG p r o t e i n c o r e . The hexuronate assay measures the amount of g l u c u r o n i c a c i d p r e s e n t i n c h o n d r o i t i n sulphate; none i s p r e s e n t i n keratan s u l p h a t e . The hexA assay i s a l s o o n l y an e s t i m a t e (of ChS), s i n c e o t h e r glycosaminoglycans c o n t a i n i n g hexA are p r e s e n t i n s m a l l amounts. As shown i n F i g u r e 3.1.4-1, between 76 and 85% of the e x t r a c t e d PG (hex + hexA) -56-Table 3.1.4 Composition of Al proteoglycan fractions from end-plates of healthy and degenerate discs Donor Density hex + hexA Fraction of ( g / m l ) ( H m o l e s / (hex + hexA) in g dry vt.) A l Component L % J A 1.68 153 76.1 B 1.60 152 78.8 C 1.64 129 81.0 D 1.62 155 79 .7 E 1.65 155 80.4 F 1.65 142 84 . 6 G 1.66 118 81.9 *The to ta l proteoglycan extracted vas separated into f ive fract ions (Al to A5) by density gradient centr i fugat ion. The Al fract ion had the highest density. Unpaired t - te s t s t a t i s t i c a l analysis Parameter Compar ison D. F. P Hex + HexA Betveen groups 1 0.661 Fraction (%) Betveen groups 1 0.122 Donors A,B and C vere assigned to group 1; donors E,F and G to group 2. -57-was p r e s e n t i n the A l f r a c t i o n . The d i s t r i b u t i o n of the t o t a l PG i n the d e n s i t y g r a d i e n t s e p a r a t i o n p r o f i l e was c o n s i s t e n t f o r CEP from both h e a l t h y and degenerate d i s c s (Grades I , I I and IV/V) (P=0.122 by t - t e s t ) . S ince the bulk of the PG occured i n the A l f r a c t i o n a l l a d d i t i o n a l c h a r a c t e r i z a t i o n s were c a r r i e d out e x c l u s i v e l y on t h i s f r a c t i o n . The t o t a l y i e l d of the p r o t e o g l y c a n e x t r a c t e d from the e n d - p l a t e t i s s u e d i d not d i f f e r between h e a l t h y and degenerate d i s c s (P=0.661 by t - t e s t ) (Table 3.1.4) and the percentage of the t o t a l PG p r e s e n t i n the A l f r a c t i o n d i d not change s i g n i f i c a n t l y with i n c r e a s i n g d i s c grade (P=0.122). Thus, d i f f e r e n c e s subsequently r e p o r t e d i n the c h a r a c t e r i z a t i o n of CEP A l PG f r a c t i o n s of h e a l t h y and degenerate d i s c s are not due t o d i f f e r i n g p r o p o r t i o n s of the p r o t e o g l y c a n i n the A l f r a c t i o n s a f t e r c e n t r i f u g a t i o n or to d i f f e r i n g c o n t e n t s of e x t r a c t a b l e p r o t e o g l y c a n s i n the t i s s u e . Furthermore, when these r e s u l t s are c o n s i d e r e d i n c o n j u n c t i o n w i t h the decrease i n water content observed w i t h i n c r e a s i n g d i s c grade, they suggest t h a t the o r i g i n a l PG may have been r e p l a c e d by another w i t h l e s s e r water b i n d i n g c a p a b i l i t y . 3.1.5 Glycosaminoglycans of the A l p r o t e o g l y c a n The A l p r o t e o g l y c a n s were analyzed f o r hexose and hexuronate t o e s t i m a t e the c o n t e n t s of k e r a t a n s u l p h a t e and -58-Table 3.1.5 The e f f e c t of d i s c degeneration on the hexose and  hexuronate contents of the A l p r o t e o g l y c a n s Donor hex hexA hex/ (p. moles/ (Umoles/ hexA g d r y vt) g dry wt) A 74.5 78.2 0.95 B 76.4 75.9 1.01 C 59 . 3 69 .8 0.85 D 80.6 74.1 1.10 E 116.4 38 .2 3.05 F 101.7 40.6 2.50 G 91.5 26.9 3. 40 Unpaired t - t e s t s t a t i s t i c a l a n a l y s i s Parameter Comparison D.F. P Hex Between groups 1 0.021 HexA Between groups 1 0.001 Hex/HexA Between groups 1 0.002 Donors A,B and C were assig n e d to group 1; donors E,F and G to group 2. -59-c h o n d r o i t i n s u l p h a t e , r e s p e c t i v e l y . Both the hex and hexA components of the CEP A l PG d i f f e r e d s i g n i f i c a n t l y between h e a l t h y and degenerate d i s c s (P=0.021 and 0.001 by t - t e s t , r e s p e c t i v e l y ) . The mean hexose content of the CEP A l PG (± standard d e v i a t i o n ) i n c r e a s e d from 70.1 ±9.4 )imoles/g d r y wt i n h e a l t h y d i s c s to 103.2 ±12.5 ^ moles/g d r y wt i n degenerate d i s c s . The hexose i n the A l f r a c t i o n of CEP from grade II d i s c s was int e r m e d i a t e between h e a l t h y and degenerate d i s c s (80.6 }imoles/g dry wt) (Table 3.1.5). The mean hexA content decreased with i n c r e a s i n g d i s c grade. The CEP A l PG from grade I d i s c s had a hexA c o n c e n t r a t i o n (± standard d e v i a t i o n ) of 74.6 ± 4.3 nmoles/g d r y wt , while the grade IV/V d i s c hexA c o n c e n t r a t i o n had decreased to 35.2 Hmoles/g d r y wt (±7.3). The hexA c o n c e n t r a t i o n i n A l f r a c t i o n s of CEP from grade II d i s c s was c l o s e to that of h e a l t h y d i s c s , namely 74.1 Jimoles/g dry wt. (Table 3.1.5). Thus, the c h o n d r o i t i n sulphate (ChS) content of the CEP A l PG decreased with i n c r e a s i n g d i s c grade, w h i l s t the keratan sulphate (KS) content i n c r e a s e d . Only the l a t t e r e f f e c t was apparent a t the e a r l y stage of d e g e n e r a t i o n . The decrease i n hexA content and i n c r e a s e i n hexose c o n c e n t r a t i o n r e s u l t e d i n marked d i f f e r e n c e s i n the r a t i o hex/hexA. The r a t i o , which r e f l e c t s the r e l a t i v e c o n c e n t r a t i o n s of t i s s u e KS and ChS i n the p r o t e o g l y c a n , was appr o x i m a t e l y 1:1 i n grade I and II and 3:1 i n grade IV/V d i s c s . T h i s suggests that the CEP of h e a l t h y d i s c s c o n t a i n e d -60-approximately equal amounts of KS and ChS w h i l s t i n the CEP of the degenerate d i s c s , there was approximately three times more KS than ChS (Table 3 . 1 . 5 ) . 3.1.6 Sepharose CL-2B g e l chromatography of the  A l p r e p a r a t i o n s The A l PG from each end-plate p r e p a r a t i o n was f r a c t i o n a t e d i n t o a high M component, thought to represent the aggregating PG, and a low M component comprised of non-aggregating PG molecules. A l i q u o t s of A l PG (1 Umole hexA i n a 1 ml volume) were a p p l i e d to the column and t r i p l i c a t e runs were completed f o r each donor pool of 8-10 end-plates to assess v a r i a b i l i t y w i t h i n samples. P r e v i o u s s t u d i e s have demonstrated t h a t aggregating p r o t e o g l y c a n i s excluded from the most porous p a r t i t i o n i n g g e l commercially a v a i l a b l e (Sepharose CL-2B), while non-aggregating PG i s able to penetrate the g e l ( H a s c a l l & Heinegard, 1974). F i g u r e 3.1.6-2 i l l u s t r a t e s the Sepharose CL-2B column chromatography e l u t i o n p r o f i l e o btained from an a l i q u o t (1.0 Umole hexA i n a 1.0 ml. volume) of CEP A l PG from grade I d i s c s (Donor B). A la r g e peak (Peak 1) of absorbance at 206 nm was d e t e c t e d near the v o i d volume and another broad peak (Peak 2) near the middle of the e l u t i o n p r o f i l e . Three r e p l i c a t e runs produced a s i m i l a r r e s u l t with the mean Kmode (± standard d e v i a t i o n ) of the high molecular weight (high M) -61-FIGURE 3.1.6-1 TYPICAL SEPHAROSE CL-2B COLUMN CHROMATOGRAPHY PROFILE OF A l PROTEOGLYCAN FROM END-PLATE OF  GRADE I DISCS An a l i q u o t of A l PG (1.0 jimole hexA i n a 1.0 ml volume) from CEP of grade I d i s c s (Donor B) was a p p l i e d to the column and e l u t e d . The Kmode of each peak was recorded as the p a r t i t i o n c o e f f i c i e n t of the s p e c i e s of highest absorbance. -62-FIGURE 3.1.6-1 Fraction number -63-FIGURE 3.1.6-2 SEPHAROSE CL-2B CHROMATOGRAPHIC PROFILE OF A l PROTEOGLYCAN FROM END-PLATE OF GRADE II DISCS An a l i q u o t of A l PG (1.0 Jimole hexA i n a 1.0 ml volume) from CEP of grade II d i s c s (Donor D) was a p p l i e d to the column and e l u t e d . The Kmode of each peak was recorded as the p a r t i t i o n c o e f f i c i e n t of the s p e c i e s of hig h e s t absorbance. -64-FIGURE 3.1.6-2 FIGURE 3.1.6-3 TYPICAL SEPHAROSE CL-2B COLUMN CHROMATOGRAPHIC PROFILE OF A l PROTEOGLYCAN FROM END-PLATE OF  GRADE IV/V DISCS An a l i q u o t of A l PG (1.0 u.mole hexA i n a 1.0 ml volume) from CEP of grade IV/V d i s c s (Donor G) was a p p l i e d to the column and e l u t e d . The Kmode of each peak was recor d e d as the p a r t i t i o n c o e f f i c i e n t of the sp e c i e s of h i g h e s t absorbance. -66-FIGURE 3.1.6-3 Table 3.1.6 The modal p a r t i t i o n c o e f f i c i e n t s of end-plate A l proteoglycan Donor Di s c Kmode  Grade* High M Low M A I 0.05, B I 0.02, C I 0.01, D II 0.12, E IV 0.22, F IV 0.20, G IV 0 . 28, Measurement e r r o r 0 .05, 0 .09 0 .55, 0.53, 0. 55 0 .06, 0 .06 0 .48, 0 .48, 0. 48 0 .06, 0 .02 0 .52, 0.50, 0. 50 0 .15, 0 .15 0 .82, 0.80, 0 . 80 0 .26, 0 .26 0 . 88, 0.80, 0 . 82 0 .26, 0 . 26 0 .78, 0.72, 0 . 76 0 . 26, 0 . 26 0 .74, 0 . 76, 0 . 74 0 .024 0 .021 A l i q u o t s of A l PG were a p p l i e d to a c a l i b r a t e d Sepharose CL-2B column. The absorbance of the el u e n t was monitored a t 206 nm. The Kmode of each peak was recorded as the e l u t i o n volume of the s p e c i e s of h i g h e s t absorbance a t 206 nm. *The grades l i s t e d are the average of a l l d i s c s i n each s p i n e . Nested a n a l y s i s of v a r i a n c e Kmode Comparison D.F. P by F - t e s t High M Group vs donor 2/4 0.0002 Donor vs e r r o r 4/14 0.3331 Low M Group vs donor 2/4 0.0001 Donor vs e r r o r 4/14 0.0003 Donors A,B and C were a s s i g n e d to group 1; donor D to group 2 and donors E,F and G to group 3. -68-peak determined as 0.047 ± 0.025 (see Table 3.1.6). The Kmode of each peak i s the p a r t i t i o n c o e f f i c i e n t of the species of highest absorbance. This procedure yielded s i m i l a r bimodal elution p r o f i l e s f o r a l i q u o t s of equal volume and concentration from CEP of grade II and grade IV/V discs (Figures 3.1.6-3 and 3.1.6-4). The Kmode values obtained from rep l i c a t e elutions showed good r e p r o d u c i b i l i t y with a root mean square measurement error close to 0.02 (Table 3.1.6). Since the column was c a l i b r a t e d prior to each run, the mean Kmode values could be compared for differences i n e l u t i o n volume. The r e s u l t s l i s t e d i n Table 3.1.6 show that the Kmode values d i f f e r e d s i g n i f i c a n t l y between the healthy and degenerate discs for both the high and low M peaks. The measurement error for the high M peak was i n s u f f i c i e n t l y small to distinguish between donors (P=0.33), while c l e a r differences between donors were apparent for the low M peak (P=0.0003). 3.1.7 Composition of the Al proteoglycan populations  separated by Sepharose CL-2B Studies of a r t i c u l a r c a r t i l a g e have shown that degeneration i s associated with greatly enhanced biosynthesis of proteoglycan and have suggested that the newly synthesized proteoglycan may have a defective c a p a b i l i t y of forming aggregates. The following experiment compared the c a p a b i l i t y -69-Table 3.1.7 P r o p o r t i o n of high M and composition of the high M and low M f r a c t i o n s of A l p r o t e o g l y c a n s taken from h e a l t h y and degenerate d i s c s Donor P r o p o r t i o n Hexose/Hexuronate of High M High M Low M (%) A 14 0.90 1.00 B 9 0.86 1.10 C 16 0.82 1.14 D 15 0.91 0.91 E 21 2. 46 3.30 F 39 2.89 2.67 G 68 3.32 2.78 One-way a n a l y s i s of v a r i a n c e Parameter Comparison D.F P r o p o r t i o n of High M High M hex/hexA Low M hex/hexA Between groups Between groups Between groups 0.099 0.001 0.001 Donors A,B and C were assigned to group 1; donor D to group 2 and donors E,F and G to group 3. -70-of d i s c end-plate p r o t e o g l y c a n , from h e a l t h y and degenerate s p i n e s , to form aggregates. The r e s u l t s l i s t e d i n Table 3.1.7 show t h a t the average percentage (± standard d e v i a t i o n ) of t o t a l p r o t e o g l y c a n (hex plus hexA) excluded from Sepharose CL-2B, i n c r e a s e d from 13% ± 4% f o r the h e a l t h y d i s c CEP to 43% ± 24% of the t o t a l PG i n the degenerate d i s c CEP . The p r o b a b i l i t y t h a t the p r o p o r t i o n of a g g r e g a t i n g PG was the same f o r both groups was 0.099 by a n a l y s i s of v a r i a n c e . In grade II d i s c CEP (Donor D), the f r a c t i o n of t o t a l PG co n t a i n e d i n Peak 1 was 15% (Table 3.1.7), c l o s e to the value f o r group I. Thus, d e s p i t e the lar g e d i f f e r e n c e s i n the mean values, the h i g h d i s p e r s i o n of data d i d not permit the c o n c l u s i o n of any s i g n i f i c a n t d i f f e r e n c e between the groups. Studies of a r t i c u l a r c a r t i l a g e have a l s o r e p o r t e d changes i n the c o n c e n t r a t i o n s of keratan s u l p h a t e and c h o n d r o i t i n s ulphate a s s o c i a t e d with d e g e n e r a t i o n and ageing. The r e l a t i v e p r o p o r t i o n s of keratan sulphate and c h o n d r o i t i n sulphate i n CEP A l PG were determined by hexose and hexuronate assays, r e s p e c t i v e l y . The r a t i o of hex/hexA i n both the high M and low M f r a c t i o n s d i f f e r e d s i g n i f i c a n t l y i n h e a l t h y and degenerate d i s c s (P=0.001). For the high M f r a c t i o n , the r a t i o (± standard d e v i a t i o n ) increased from a mean of 0.86 ±0.04 f o r h e a l t h y d i s c CEP to a mean of 2.89 ± 0.043 i n degenerate d i s c CEP. In the low M f r a c t i o n the hex/hexA -71-(standard d e v i a t i o n ) i n c r e a s e d from 1.08 ± 0.07 to 2.92 ± 0.34. As was the case f o r the u n f r a c t i o n a t e d samples, the hex/hexA values f o r grade II d i s c s resembled c l o s e l y those of grade I d i s c s . 3.1.8 Composite agarose-polyacrylamide g e l e l e c t r o p h o r e s i s of  A l P r o t e o g l y c a n s The p r o t e o g l y c a n p o p u l a t i o n s of d i s c AF and NP and of bovine n a s a l c a r t i l a g e are known to be heterogeneous and p o l y d i s p e r s e (McDevitt and Muir, 1971; Jahnke and McDevitt, 1988; D i F a b i o et a l . . 1986). In the present study, the h e t e r o g e n e i t y of the CEP A l PG was assessed u s i n g composite agarose-polyacrylamide g e l e l e c t r o p h o r e s i s (CAPAGE). The i n i t i a l CAPAGE a n a l y s i s examined samples f r a c t i o n a t e d by Sepharose CL-2B chromatography, s i n c e other s t u d i e s have r e p o r t e d poor e l e c t r o p h o r e t i c r e s o l u t i o n u s i n g u n f r a c t i o n a t e d p r o t e o g l y c a n (DiFabio et a l . , 1986; Jahnke and McDevitt, 1988). The CAPAGE an a l y s e s of the f r a c t i o n a t e d A l PG from CEP of grade I and grade IV/V d i s c s are i l l u s t r a t e d i n F i g u r e s 3.1.8-1 and F i g u r e 3.1.8-2, r e s p e c t i v e l y . These r e s u l t s show a g e n e r a l i z e d i n c r e a s e i n e l e c t r o p h o r e t i c m o b i l i t y of the PG molecules with d e c r e a s i n g molecular s i z e . F i g u r e s 3.1.8-1 and 3.1.8-2 a l s o i l l u s t r a t e t h a t when examined by CAPAGE the CEP PG r e p r e s e n t a mixture of d i s c r e t e subspecies r a t h e r than a s i n g l e p o l y d i s p e r s e -72-FIGURE 3.1.8-1 COMPOSITE AGAROSE-POLYACRYLAMIDE GEL ELECTROPHORESIS OF FRACTIONS OF A l  PROTEOGLYCAN FROM END-PLATE OF GRADE I DISCS  PREPARED BY SEPHAROSE CL-2B CHROMATOGRAPHY CAPAGE a n a l y s i s of f r a c t i o n s from the Sepharose CL-2B chromatography of A l PG from CEP of grade I d i s c s (Donor C). A l i q u o t s (100 p:l) of three s u c c e s s i v e f r a c t i o n s were pooled to reduce the number of samples to e i g h t . Each lane c o n t a i n e d 5-10 nmoles hexA i n a volume of 20 U l . Lane A shows the e l e c t r o p h o r e t i c m o b i l i t y of the h i g h e s t molecular s i z e f r a c t i o n ( e l u t e d a t VO) r e l a t i v e to a c h o n d r o i t i n sulphate standard (lanes I and J ) , with lanes B to H demonstrating the m o b i l i t i e s of PG f r a c t i o n s of d e c r e a s i n g molecular s i z e ( i n c r e a s i n g VE). -73-FIGURE 3.1.8-1 -74-FIGURE 3.1.8-2 COMPOSITE AGAROSE-POLYACRYLAMIDE GEL ELECTROPHORESIS OF FRACTIONS OF A l  PROTEOGLYCAN FROM END-PLATE OF GRADE IV/V  DISCS PREPARED BY SEPHAROSE CL-2B  CHROMATOGRAPHY CAPAGE a n a l y s i s of f r a c t i o n s from the Sepharose CL-2B chromatography of the A l PG from CEP of grade IV/V d i s c s (Donor E ) . A l i q u o t s (100 u l ) of three s u c c e s s i v e f r a c t i o n s were pooled to reduce the number of samples to e i g h t . Each lane c o n t a i n e d 5-10 nmoles hexA i n a volume of 20 u l . Lane B shows the e l e c t r o p h o r e t i c m o b i l i t y of the h i g h e s t molecular s i z e f r a c t i o n ( e l u t e d at VO) r e l a t i v e to a c h o n d r o i t i n sulphate standard ( l a n e s A and J ) , with lanes C to I demonstrating the m o b i l i t i e s of PG f r a c t i o n s of d e c r e a s i n g molecular s i z e ( i n c r e a s i n g VE). -75-- 7 6 -p o p u l a t i o n . A comparison of the CAPAGE a n a l y s i s of grade I d i s c CEP PG with an e q u i v a l e n t amount of grade IV/V d i s c CEP PG, showed with degeneration, a l o s s of the high M PG p o p u l a t i o n s t a i n e d with t o l u i d i n e blue and of low M PG near the t o t a l volume of the column, with an apparent i n c r e a s e i n the amount of the PG p o p u l a t i o n of i n t e r m e d i a t e M. The l o s s of s t a i n e d p r o t e o g l y c a n m a t e r i a l on a CAPAGE g e l r e f l e c t s a l o s s of a n i o n i c glycosaminoglycan s i d e chains such as KS and/or ChS and not n e c e s s a r i l y a l o s s of p r o t e i n s i n c e t o l u i d i n e blue i s an a n i o n i c dye which binds c h i e f l y to the e s t e r s u lphate groups of the glycosaminoglycan and not to the p r o t e i n core ( K i t a h a r a , 1979). The aggregable and non-aggregable p r o t e o g l y c a n s of AF and NP have been shown to c o n t a i n e l e c t r o p h o r e t i c a l l y d i s t i n c t s u b p o p u l a t i o n s (Jahnke and McDevitt, 1988; D i F a b i o et a l . . 1986). To i n v e s t i g a t e t h i s p o s s i b i l i t y i n the present study, A l PG f r a c t i o n s e l u t e d as a high M and as a low M peak from the Sepharose CL-2B column were pooled s e p a r a t e l y , c o n c e n t r a t e d and analyzed u s i n g p r e p a r a t i v e s c a l e CAPAGE. Fi g u r e 3.1.8-3 i l l u s t r a t e s the r e s u l t obtained from peak 1 (high M) of CEP A l PG of grade I d i s c s . T h i s a n a l y s i s demonstrates that the p o p u l a t i o n of PG molecules w i t h i n Peak 1 was comprised of at l e a s t three d i s t i n c t subspecies (Bands I, II and I I I ) . F i g u r e 3.1.8-4 i l l u s t r a t e s t h a t the low M PG p o p u l a t i o n (Peak 2) i s made up of a t l e a s t two -77-FIGURE 3.1.8-3 PREPARATIVE COMPOSITE AGAROSE-PPLYACRYLAMIDE GEL ELECTROPHORESIS OF THE HIGH MOLECULAR  WEIGHT (PEAK 1) FRACTION The f r a c t i o n s c o n t a i n e d w i t h i n Peak 1 (high M) of three Sepharose CL-2B column chromatography s e p a r a t i o n s were conce n t r a t e d to a f i n a l volume of 600 p.1. A l i q u o t s c o n t a i n i n g 1.0 Jimole hexA i n a volume of 250 )il were a p p l i e d to the p r e p a r a t i v e g e l (CAPAGE) f o r a n a l y s i s (see Method). -78-FIGURE 3.1.8-3 Band I Band II Band III - 7 9 -FIGURE 3.1.8-4 PREPARATIVE COMPOSITE AGAROSE-POLYACRYLAMIDE GEL ELECTROPHORESIS OF THE LOW MOLECULAR  WEIGHT (PEAK 2) FRACTION The f r a c t i o n s c o n t a i n e d w i t h i n Peak 2 (low M) of three Sepharose CL-2B column chromatography s e p a r a t i o n s were concentrated to a f i n a l volume of 600 U l . A l i q u o t s c o n t a i n i n g 1.0 p:mole of hexA i n a volume of 250 u l were a p p l i e d to the p r e p a r a t i v e g e l (CAPAGE) f o r a n a l y s i s (see Methods). -80-FIGURE 3.1.8-4 Band IV Band V -81-FIGURE 3.1.8-5 CAPAGE ANALYSIS OF HIGH M (PEAK 1) PROTEOGLYCAN FROM END-PLATE OF DONOR  A,D AND F Samples of the high M PG f r a c t i o n containing 5-10 nmoles hexA in a 20 ul volume were applied to each lane. Lanes A (donor A) and E (donor B) contained PG from grade I d i s c s ; lane B contained PG from grade IV/V discs (donor F) and lane F from grade II discs (donor D). Lanes C and D contained a standard of ChS (see Methods). -82-FIGURE 3 .1.8 c -83-FIGURE 3.1.8-6 CAPAGE ANALYSIS OF PEAK 2 (LOW M) PROTEOGLYCAN FROM END-PLATE OF DONORS A,D AND F Samples of the low M PG f r a c t i o n c o n t a i n i n g 5-10 nmoles hexA i n a 20 u l volume were a p p l i e d t o each l a n e . Lane A c o n t a i n e d PG from grade I d i s c s (donor A); lane B from grade I I d i s c s (donor D) and lane C from grade IV/V d i s c s (donor F ) . Lane D contained a s t a n d a r d o f ChS (see Methods). -84-FIGURE 3.1.8-6 A B C D -85-FIGURE 3.1.8-7 DENSITOMETRIC SCANS OF CAPAGE COMPARISON OF HIGH M AND LOW M PROTEOGLYCAN FRACTIONS OF  END-PLATE FROM GRADE I,II AND IV/V DISCS Donor A contained o n l y grade I d i s c s ; donor D o n l y grade II d i s c s and donor F onl y grade IV/V d i s c s . A l l scans show m o b i l i t i e s r e l a t i v e to the same ChS standard. -86-FIGURE 3.1.8-7 > a? CC Donor F - Peak 1 Mobility relative to ChS -87-Table 3.1.8 R e l a t i v e e l e c t r o p h o r e t i c m o b i l i t y of high and low M p r o t e o g l y c a n s prepared from h e a l t h y and degenerate d i s c s D i s c Mobi l i t y * Grade High M Low M I 0.10 0.55 0 . 30 0.65 0.45 II 0.47 0.65 0.78 IV 0.48 0.65 0.75 0.92 *The m o b i l i t y of each band was measured r e l a t i v e to the same ChS s t a n d a r d . -88-e l e c t r o p h o r e t i c a l l y d i s t i n c t subspecies (Bands IV and V). Since the CAPAGE experimental c o n d i t i o n s r e q u i r e d to achieve the optimum s e p a r a t i o n of the high and low M PG subspecies d i f f e r e d , the m o b i l i t i e s of the f i v e bands c o u l d not be compared on a s i n g l e g e l . However, the m o b i l i t i e s of the three high M and two low M bands were thought to d i f f e r s i n c e the g e l chromatographic p a r t i t i o n c o e f f i c i e n t s of t h e i r parent p r o t e o g l y c a n p o p u l a t i o n s ( i . e . the high and low M f r a c t i o n s ) d i f f e r e d and s i n c e e a r l i e r experiments had demonstrated a n o n - s p e c i f i c d i f f e r e n c e i n t h e i r e l e c t r o p h o r e t i c p r o p e r t i e s (F i g u r e s 3.1.8-1 and 3.1.8-2). The e l e c t r o p h o r e t i c a l l y d i s t i n c t PG s u b s p e c i e s of the high and low M f r a c t i o n s were then i d e n t i f i e d f o r CEP from d i s c s at v a r i o u s stages of degeneration. F i g u r e s 3.1.8-5, 3.1.8-6 and 3.1.8-7 i l l u s t r a t e the e f f e c t of i n c r e a s i n g d i s c grade on the d i s t r i b u t i o n of these s u b s p e c i e s . Since d i f f e r e n t experimental c o n d i t i o n s are r e q u i r e d to o b t a i n the optimum s e p a r a t i o n of high and low M s u b s p e c i e s , these comparisons were made on separate g e l s . In a d d i t i o n , equal amounts of PG (as determined by hexA assay) from each CEP p r e p a r a t i o n were a p p l i e d to the g e l s to assess the r e l a t i v e c o n c e n t r a t i o n s of each subspecies (using d e n s i o m e t r i c s c a n s ) . F i g u r e 3.1.8-5 compares the high M f r a c t i o n (Peak 1) from grade I d i s c s (Donor A) i n Lane A with Peak 1 from grade II d i s c s (Donor D) i n Lane B. A r e f e r e n c e s t a n d a r d of -89-c h o n d r o i t i n sulphate (see Methods) was a p p l i e d to Lane C. The d e n s i t o m e t r i c scans of these g e l s are shown i n F i g u r e 3.1.8-7 (Donor A, Peak 1 and Donor D, Peak 1) with the e l e c t r o p h o r e t i c m o b i l i t i e s of the peaks shown, a l l r e l a t i v e to the same c h o n d r o i t i n sulphate (ChS) standard (RChS). The PG p o p u l a t i o n i n Peak 1 from grade I d i s c CEP showed three d i s t i n c t s ubspecies (RChS=0.10 / RChS=0.30 and RChS=0.45) (Table 3.1.8), while the Peak 1 PG from grade II d i s c CEP showed one broad band, having an e l e c t r o p h o r e t i c m o b i l i t y (RChS=0.47) s i m i l a r to the subspecies of h i g h e s t m o b i l i t y present i n Peak 1 of grade I d i s c s (Table 3.1.8). The p r o f i l e of the band suggested t h a t i t was l i k e l y comprised of two bands with o v e r l a p p i n g e l e c t r o p h o r e t i c m o b i l i t i e s . In F i g u r e 3.1.8-5 the e l e c t r o p h o r e t i c p r o p e r t i e s of Peak 1 CEP PG from grade I d i s c s (Lane E) were compared with those from grade IV/V d i s c s (Lane F ) . Lane D contained the ChS standard. The d e n s i t o m e t r i c scan of the g e l shown i n F i g u r e 3.1.8-5 (Lanes E and F) show that the PG c o n t a i n e d i n Peak 1 from CEP of grade IV/V d i s c s (Donor F) i s comprised of a s i n g l e s p e c i e s with an RChS=0.48 (Figure 3.1.8-7; Table 3.1.8). F i g u r e 3.1.8-6 i l l u s t r a t e s the e l e c t r o p h o r e t i c p r o p e r t i e s of the PG p o p u l a t i o n s c o n t a i n e d i n Peak 2 of CEP from grade I (Lane A), grade II (Lane B) and grade IV/V (Lane C) d i s c s . Lane D contained the ChS standard. As shown i n F i g u r e 3.1.8-7 the PG p o p u l a t i o n i n Peak 2 of CEP from grade I d i s c s (Donor A) was comprised of two d i s t i n c t -90-subspecies with RChS=0.55 and 0.65 (Table 3.1.8). In grade II d i s c CEP (Donor D), Peak 2 c o n t a i n s two subsp e c i e s with RChS=0.65 and 0.78 (Table 3.1.8). In grade IV/V d i s c CEP (Donor F ) , the Peak 2 PG c o n s i s t e d of three d i s t i n c t s u bspecies having RChS=0.65 / 0.75 and 0.92 (Table 3.1.8). The d e n s i t o m e t r i c scan a l s o showed evidence of t r a c e l e v e l s of high M PG s u b s p e c i e s , which may have r e s u l t e d from contamination d u r i n g g e l l o a d i n g . -91-3.2.0 PART I I CHANGES IN CARTILAGINOUS END-PLATE PROTEOGLYCAN DUE TO DEGENERATION 3.2.1 S e l e c t i o n of specimens In the p r e v i o u s experiments, the e f f e c t of age c o u l d not be d i s t i n g u i s h e d from the e f f e c t of de g e n e r a t i o n because these two c h a r a c t e r i s t i c s tended t o change t o g e t h e r i n the specimens used. A n a l y s i s of a s u f f i c i e n t number of specimens t o t e s t which of the v a r i a b l e s was important was c o n s i d e r e d i m p r a c t i c a l . Rather, the comparison of d i s c s o f d i f f e r e n t grades w i t h i n the same s p i n e was used t o i s o l a t e the e f f e c t of degeneration. The donors used i n t h i s i n v e s t i g a t i o n were s e l e c t e d by the same i n c l u s i o n / e x c l u s i o n c r i t e r i a (e.g. no evide n c e of di s e a s e known t o a f f e c t the spine) used i n the p r e v i o u s experiments. A l s o , the grades were a s s i g n e d as d e s c r i b e d before (Appendix I ) . D i s c s of grades I and I I were f u r t h e r assigned to grade group 1 and d i s c s of grades I I I and IV t o grade group 2. The CEP from t h r e e d i s c s of each lumbar s p i n e were used, two of s i m i l a r grade and one of c l e a r l y d i f f e r i n g -92-grade (Table 3.2.1). The c o n t r a s t between the degenerate and h e a l t h y d i s c s was compared with the c o n t r a s t between the two h e a l t h y d i s c s . The s p i n e s best s u i t e d f o r these experiments would c o n t a i n i n a d d i t i o n to two or more h e a l t h y d i s c s , a t l e a s t one d i s c having undergone marked degenerative change (grade IV or V) . Of the three s p i n e s s e l e c t e d f o r t h i s study, o n l y one (Donor H), contained a grade IV d i s c . The remaining donor spines (Donors I and J ) , both contained h e a l t h y d i s c s and one d i s c t h a t had undergone moderate degenerative change (grade I I I ) . The lack of i d e a l donors was thought to be due to the u n l i k e l y s e t of c l i n i c a l circumstances t h a t would give r i s e to t h i s c o n f i g u r a t i o n (e.g. a h i s t o r y of trauma focussed at the l e v e l of a s i n g l e s p i n a l segment i n the presence of otherwise normal d i s c s ) and to the p r e v i o u s l y r e p o r t e d f i n d i n g t h at a l l d i s c s w i t h i n a lumbar spine are depleted of p r o t e o g l y c a n p r i o r to the m o r p h o l o g i c a l appearance of d e g e n e r a t i v e changes and are t h e r e f o r e more l i k e l y to degenerate s i m u l t a n e o u s l y (Pearce et a l . , 1987). As shown i n Table 3.2.1, the two g r a d i n g systems d i f f e r e d i n t h e i r assessment of s e v e r a l d i s c s , with the MRI based c l a s s i f i c a t i o n c o n s i s t e n t l y r e p o r t i n g a higher grade. However, there were no i n s t a n c e s of these d i f f e r e n c e s r e s u l t i n g i n d i s p a r i t i e s i n the grade group assignment. D i f f e r e n t lumbar d i s c s were s e l e c t e d f o r study f o r the -93-Table 3.2.1 Donors of s p i n e s , qrades and qrade qroups of i n t e r v e r t e b r a l d i s c s Donor Age (y) Cause of Death Disc Grade Grade Group & Sex Gross MRI H 27M C e r v i c a l f r a c t u r e L2/L3 I II 1 . L3/L4 I I 1 L5/S1 III IV 2 I 26F C e r v i c a l f r a c t u r e L2/L3 I I 1 L4/L5 II II 1 L5/S1 I I I I I I 2 J 32M Myoca r d i a l i n f a r c t L1/L2 I II 1 L3/L4 I II 1 L4/L5 I I I I I I 2 D i s c s were graded on the b a s i s of gross morphology and magnetic resonance imaging (MRI). D i s c s of grade I and II were as s i g n e d to grade group 1; d i s c s of grades III and IV to grade group 2. -94-FIGURE 3.2.2-1 LUMBAR SPINE OF DONOR H s e c U o n ^ ? ^ ( l e f t ) and MRI ( r i g h t ) of m i d - s a g i t t a l K ? K ? L S ^ ^ - a r o i l male) -95-FIGURE 3.2.2-1 - 9 6 -FIGURE 3.2.2-2 LUMBAR SPINE OF DONOR I Colour photograph ( l e f t ) and MRI ( r i g h t ) of m i d - s a g i t t a l s e c t i o n of lumbar spine of donor I (26 year o l d female). The L1/L2 through L5/S1 d i s c s are shown. - 9 7 -FIGURE 3.2.2-2 FIGURE 3.2.2-3 LUMBAR SPINE OF DONOR J Colour photograph ( l e f t ) and MRI ( r i g h t ) of m i d - s a g i t t a l s e c t i o n of lumbar spine of donor J (32 year o l d male). The L1/L2 through L5/S1 d i s c s are shown. - 9 9 -d i f f e r e n t donors (Figures 3.2.2-1, 3.2.2-2 and 3.2.2-3). In each donor the most degenerate disc occurred at the most caudal l e v e l , i n keeping with previously reported patterns of human lumbar disc degeneration (Miller et a l . , 1988). The CEP A l PG of discs selected for study i n part 2 of th i s investigation were characterized in the same manner as those of part 1. Thus, changes i n the s p e c i f i c properties of the CEP proteoglycans at t r i b u t e d to ageing and degeneration (part I) could be d i r e c t l y compared with changes i n the same properties of the CEP observed between discs of d i f f e r i n g grade within the same spine. A two-way analysis of variance was used to assess the significance of the experimental r e s u l t s . In addi t i o n to the estimate of the s i g n i f i c a n c e of the differences between discs of d i f f e r e n t grades, t h i s method of analysis allowed f o r comparisons to be made both between donors and to assess by interaction the consistency between donors of any e f f e c t s observed. 3.2.2 Yie l d and water content of ca r t i l a g i n o u s end-plate The superior and i n f e r i o r CEP from each of the three discs selected for study from each lumbar spine were dissected. The two end-plates from each disc were pooled and each of the three p a i r s analyzed separately. The weights of fresh tissue obtained from each disc varied widely between -101-s p i n e s as would be expected from donors of d i f f e r i n g age, sex and p h y s i c a l s t a t u r e (Table 3.2.2). A l l comparisons between d i s c s , with the e x c e p t i o n of water content, were made r e l a t i v e to the dry weights l i s t e d i n Table 3.2.2 . The mean water contents of the CEP from the group 1 d i s c s was c o n s i s t e n t l y higher than t h a t of the group 2 d i s c s f o r a l l donors s t u d i e d (P=0.018 by F - t e s t with P f o r interaction=0.211) (Table 3.2.2b). These r e s u l t s are i n agreement with those obtained i n p a r t I of t h i s study (Table 3.1.3) where the end-plates from four or f i v e lumbar d i s c s were pooled from an e n t i r e lumbar s p i n e . However, the r e s u l t s from i n d i v i d u a l d i s c end-plates were s u b s t a n t i a l l y more s i g n i f i c a n t (P=0.018 versus P=0.058). 3.2.3 Composition of end-plate A l p r o t e o g l y c a n f r a c t i o n s The d e n s i t i e s of the A l f r a c t i o n s of the C s C l g r a d i e n t s ranged from 1.60 g/ml to 1.66 g/ml (Table 3.2.3). As was found i n p a r t I, 75 to 82% of the t o t a l e x t r a c t e d PG (hex + hexA) was present i n the A l f r a c t i o n (Table 3.2.2) and the r e c o v e r y of PG i n the A l f r a c t i o n was c o n s i s t e n t between donors and d i s c grades. Thus, any d i f f e r e n c e s i n the p r o p e r t i e s of the CEP A l PG between d i s c s and donors c o u l d not be a t t r i b u t e d to d i f f e r e n c e s i n buoyant d e n s i t i e s or p r o t e o g l y c a n r e c o v e r y i n the A l f r a c t i o n . The donors d i f f e r e d i n the t o t a l PG per gram d r y t i s s u e -102-Table 3.2.2 Comparisons of f r e s h we i g h t , d r y weight and water conten-of e n d - p l a t e s of h e a l t h y and degenerate d i s c s w i t h i n t h r e e s p i n e s Donor Dis c Grade Grade Fresh Dry Water Gross MRI Group Wt. Wt. Content (g) (g) (g/g f r e s h wt) H L2/L3 I II 1 0.682 0.171 0.749 L3/L4 I I 1 0.702 0.184 0.738 Mean f o r grade group 1 0.744 L5/S1 I I I IV 2 0.488 0.167 0.658 I L2/L3 I I 1 0.931 0. 201 0.784 L4/L5 II II 1 0.973 0.262 0.730 Mean f o r grade group 1 0.757 L5/S1 III I I I 2 0.955 0. 345 0.639 J L1/L2 I II 1 0.480 0.140 0.708 L3/L4 I II 1 0.330 0.100 0 .697 Mean f o r grade group 1 0.703 L4/L5 II I I I I 2 0.340 0.110 0.676 V a r i a t i o n w i t h i n grade group 0.022 b) Two-way a n a l y s i s of v a r i a n c e of water c o n t e n t Comparison D.F. P by F - t e s t Donor vs e r r o r 2/3 0.469 Grade group vs e r r o r 1/3 0.018 I n t e r a c t i o n vs e r r o r 2/3 0.211 71Q 3-Table 3.2.3 Composition of A l p r o t e o g l y c a n f r a c t i o n s from end-plate  of h e a l t h y and degenerate d i s c s w i t h i n t h r e e s p i n e s Donor D i s c D e n s i t y Grade Hex + HexA P r o p o r t i o n of Group (hex + hexA) i n (g/ml) (umoles/ A l F r a c t i o n g f r e s h wt) (_%) H L2/L3 L3/L4 Mean f o r 1.64 1.60 grade 1 1 group 1 141 135 138 78.2 80.1 L5/S1 1.62 2 96 77.6 I L2/L3 L4/L5 Mean f o r 1.66 1.62 grade 1 1 group 1 168 172 170 81.8 78.2 L5/S1 1.62 2 160 78. 4 J L1/L2 L3/L4 Mean f o r 1.65 1.66 grade 1 1 group 1 121 116 119 77.4 75.9 L4/L5 1.62 2 99 75.6 V a r i a t i o n w i t h i n grade group 4 Two-way a n a l y s i s of v a r i a n c e of hex + HexA Compar i s o n D.F. P by F - t e s t Donor vs e r r o r 2/3 0.0008 Grade group vs e r r o r 1/3 0.0028 I n t e r a c t i o n vs e r r o r 2/3 0.0342 -104-weight as measured by the sum of hex and hexA (P=0.0008) (Table 3.2.3). An i n c r e a s e i n d i s c grade r e s u l t e d i n a decrease i n t o t a l CEP A l PG (P <0.003), p a r t i c u l a r l y e v i d e n t i n two of the three donors s t u d i e d (Table 3.2.3c). Thus, d e s p i t e d i f f e r e n c e s between donors, d i s c s of grade group 2 had l e s s p r o t e o g l y c a n then those of group 1. 3.2.4 Glycosaminoglycans of the A l p r o t e o g l y c a n The A l p r o t e o g l y c a n s were analysed f o r hexose and hexuronate to estimate the c o n t e n t s of k e r a t a n s u l p h a t e and c h o n d r o i t i n s u l p h a t e , r e s p e c t i v e l y . The hexose r e s u l t s l i s t e d i n Tables 3.2.4 showed t h a t f o r two of the t h r e e donors s t u d i e d , the KS content i n c r e a s e d and t h a t when a l l three donors were c o n s i d e r e d , the i n c r e a s e with d i s c grade approached s t a t i s t i c a l s i g n i f i c a n c e (P=0.074 by F - t e s t ) . The hexuronate (hexA) c o n c e n t r a t i o n s of the CEP A l PG showed a marked and s i g n i f i c a n t decrease of ChS with i n c r e a s i n g grade group (P=0.0015 by F - t e s t ) t h a t was c o n s i s t e n t amongst a l l donors s t u d i e d (P f o r i n t e r a c t i o n s . 1 0 0 ) (Table 3.2.4). The changes observed i n the CEP A l KS and ChS c o n c e n t r a t i o n s with i n c r e a s i n g grade group suggest t h a t d e g e n e r a t i o n may be a s s o c i a t e d w i t h s e l e c t i v e glycosaminoglycan b i o s y n t h e s i s and/or d e g r a d a t i o n . S t u d i e s of a r t i c u l a r c a r t i l a g e A l PG have r e p o r t e d Table 3.2.4 The hexose and hexuronate c o n t e n t s of the A l p r o t e o g l y c a n s of healthy and degenerate d i s c s w i t h i n the same s p i n e Donor D i s c Grade Hex HexA Hex/ Group (umoles/g (umoles/g HexA dry wt) d r y wt) H L2/L3 1 L3/L4 1 Mean f o r grade group L5/S1 2 I L2/L3 1 L4/L5 1 Mean f o r grade group L5/S1 2 J L1/L2 1 L3/L4 1 Mean f o r grade group L4/L5 2 V a r i a t i o n w i t h i n grade group 70.5 64.9 67.7 70.5 70.4 70.5 1.00 0.93 0.91 71.1 25.4 2.80 78.2 90.6 84.4 89.8 81.2 85.5 0.87 1.11 0.99 112.1 47.7 2.35 71.8 72.3 72.1 49.9 43.5 46.7 1.44 1.66 1.55 72.8 26.0 2.80 3.9 4.4 0.14 Two-way a n a l y s i s of v a r i a n c e Parameter Comparison D.F. P by F-Hex Donor vs e r r o r Grade group vs I n t e r a c t i o n vs e r r o r e r r o r 2/3 1/3 2/3 0.022 0.074 0.117 HexA Donor vs e r r o r Grade group vs I n t e r a c t i o n vs e r r o r e r r o r 2/3 1/3 2/3 0.006 0.002 0.100 Hex/HexA Donor vs e r r o r Grade group vs I n t e r a c t i o n vs e r r o r e r r o r 2/3 1/3 2/3 0.039 0.001 0.170 -106-marked i n c r e a s e s i n the KS/ChS with d e g e n e r a t i o n (Poole e_t a l . , 1984). For the p r e s e n t study, the changes i n KS and ChS with i n c r e a s i n g grade group r e s u l t e d i n changes i n the KS/ChS r a t i o (hex/hexA) from between 0.9S and 1.55 i n h e a l t h y d i s c s to between 2.35 and 2.80 i n degenerate d i s c s (P=0.001). These changes were c o n s i s t e n t amongst a l l donors s t u d i e d (P f o r i n t e r a c t i o n s . 1 7 ) and c l o s e l y resemble the changes observed i n p a r t I where the KS/ChS i n c r e a s e d from 0.94 + 0.08 to 2.98 ± 0.45 with an i n c r e a s e i n grade group. 3.2.5 Sepharose CL-2B g e l chromatography of A l  p r o t e o g l y c a n The CEP A l PG was f r a c t i o n a t e d i n t o h i g h and low M components by g e l chromatography. F i g u r e s 3.2.5-1, 3.2.5-2 and 3.2.5-3 i l l u s t r a t e the Sepharose CL-2B e l u t i o n p r o f i l e s of CEP A l PG from the d i s c s of donors s t u d i e d (Donors H, I and J) . In donor H, the prominence of the h i g h and low M peaks of the degenerate d i s c d i f f e r e d markedly from those of the degenerate d i s c s i n donors I and J . While a l o s s of t o t a l PG with i n c r e a s i n g d i s c grade was c o n s i s t e n t l y observed i n the three donor spines (Table 3.2.3), i n donor H an a d d i t i o n a l l o s s of a g g r e g a t i n g PG, with an a s s o c i a t e d concomitant g a i n of non-aggregating PG, had occurred (Table 3.2.6). T h i s may i n p a r t r e f l e c t the l a r g e r d i f f e r e n c e i n d i s c grades compared i n -107-FIGURE 3.2.5-1 SEPHAROSE CL-2B COLUMN CHROMATOGRAPHY OF END-PLATE A l PROTEOGLYCAN OF L2/L3, L3/L4 AND  L5/S1 DISCS OF DONOR H A l i q u o t s of A l PG (1.0 )imole hexA i n a 1.0 ml volume) from CEP of the L2/L3, L3/L4 and L5/S1 d i s c s of donor H were a p p l i e d t o the column. F r a c t i o n s were e l u t e d under the c o n d i t i o n s d e s c r i b e d i n the Methods. The Kmode of each peak was recorded as the p a r t i t i o n c o e f f i c i e n t of the s p e c i e s of h i g h e s t absorbance. -108-FIGURE 3.2.5-1 -109-FIGURE 3.2.5-2 SEPHAROSE CL-2B COLUMN CHROMATOGRAPHY OF END-PLATE A l PROTEOGLYCAN OF L2/L3, L4/L5 AND L5/S1 DISCS OF DONOR I A l i q u o t s of A l PG (1.0 u.mole hexA i n a 1.0 ml volume ) from CEP of the L2/L3, L4/L5 and L5/S1 d i s c s of donor I were a p p l i e d to the column. F r a c t i o n s were e l u t e d under the c o n d i t i o n s d e s c r i b e d i n the Methods. The Kmode of each peak was recorded as the p a r t i t i o n c o e f f i c i e n t of the s p e c i e s of h i g h e s t absorbance. -110-FIGURE 3.2.5-2 -111-FIGURE 3.2.5-3 SEPHAROSE CL-2B COLUMN CHROMATOGRAPHY OF END-PLATE A l PROTEOGLYCAN OF L1/L2, L3/L4 AND  L4/L5 DISCS OF DONOR J A l i q u o t s of A l PG (1.0 Hmole hexA i n a 1.0 ml volume) from CEP of the L1/L2, L3/L4 and L4/L5 d i s c s of donor J were a p p l i e d t o the column. F r a c t i o n s were e l u t e d under the c o n d i t i o n s d e s c r i b e d i n the Methods. The Kmode of each peak was r e c o r d e d as the p a r t i t i o n c o e f f i c i e n t of the s p e c i e s of h i g h e s t absorbance. -112-FIGURE 3.2.5-3 -113-Donor H. In donors I and J , where the d i f f e r e n c e between d i s c grades was s m a l l e r , the low M PG f r a c t i o n s were d e p l e t e d i n the degenerate d i s c s , w h i l e the prominence of the hig h M peaks remained c o n s t a n t . The r e s u l t s l i s t e d i n Table 3.2.5 show t h a t Kmode v a l u e s f o r the low M (assumed t o be non-aggregating) PG f r a c t i o n were s i g n i f i c a n t l y d i f f e r e n t f o r healthy (grade group 1) and degenerate (grade group 2) d i s c s (P=0.05 by F - t e s t ) . Changes i n the Kmode v a l u e s a s s o c i a t e d with i n c r e a s i n g d i s c grade f o r the a g g r e g a t i n g PG f r a c t i o n , were l e s s profound (P=0.08 by F - t e s t ) . The changes i n Kmode i n both PG p o p u l a t i o n s were not c o n s i s t e n t amongst a l l donors. The l a r g e s t v a r i a t i o n s i n Kmode (high and low M f r a c t i o n s ) were p r e s e n t i n Donor H, where the d i f f e r e n c e i n d i s c grade was the g r e a t e s t (Table 3.2.5) . These r e s u l t s d i f f e r from those o b t a i n e d i n p a r t I of t h i s study where the combined e f f e c t s of a g e - r e l a t e d change and d i s c d e g e n e r a t i o n were e v a l u a t e d . Those r e s u l t s i d e n t i f i e d more pronounced d i f f e r e n c e s i n the Kmode of both the a g g r e g a t i n g and non-aggregating PG f r a c t i o n s w i t h i n c r e a s i n g d i s c grade f o r a l l donors s t u d i e d . In the f i r s t s e r i e s , the d i f f e r e n c e s i n grades were c o n s i d e r a b l y g r e a t e r . -114-Table 3.2.5 The modal p a r t i t i o n c o e f f i c i e n t s of end-plate A l pr o t e o g l y c a n s from h e a l t h y and degenerate d i s c s w i t h i n the same spine Donor Grade Kmode and Group High M Low M D i s c  Donor H L2/L3 1 0.04, 0.02, 0.04 0.44, 0.42, 0.48 L3/L4 1 0.04, 0.06, 0.04 0.48, 0.54, 0.50 Mean of grade group 1 0.04 0.48 L5/S1 2 0.15, 0.09, 0.12 0.74, 0.71, 0.70 Donor I L2/L3 1 0.02, 0.02, 0.04 0.46, 0.44, 0.52 L4/L5 1 0.04, 0.06, 0.06 0.48, 0.48. 0.52 Mean of grade group 1 0.04 0.48 L5/S1 2 0.02, 0.06, 0.03 0.58, 0.58, 0.60 Donor J L1/L2 1 0.05, 0.05, 0.05 0.52, 0.50, 0.52 L3/L4 1 0.02, 0.06, 0.06 0.50, 0.52, 0.50 Mean of grade group 1 0.05 0.51 L4/L5 2 0.07, 0.02, 0.02 0.54, 0.56, 0.52 P a r t i a l l y nested two-way a n a l y s i s of v a r i a n c e Kmode Comparison D.F P by F - t e s t High M Donor vs d i s c 2/3 0.18 Grade group vs d i s c 3/3 0.08 Low M Donor vs d i s c 2/3 0.48 Grade group vs e r r o r 3/3 0.05 -115-3.2.6 P r o p e r t i e s of the p r o t e o g l y c a n p o p u l a t i o n s s e p a r a t e d by  Sepharose CL-2B Changes i n the percentage of t o t a l PG p r e s e n t i n the aggregate form i n the CEP of d i s c s having undergone de g e n e r a t i o n and ageing may r e f l e c t enhanced p r o t e o g l y c a n b i o s y n t h e s i s of a g g r e g a t i n g PG or s e l e c t i v e d e g r a d a t i o n of non-aggregating p r o t e o g l y c a n s . The r e s u l t s l i s t e d i n Table 3.2.6 show t h a t the p r o p o r t i o n of the t o t a l PG i n the high M (aggregating) f r a c t i o n d i f f e r e d between h e a l t h y and degenerate d i s c s , but i n a manner i n c o n s i s t e n t amongst donors. Donor H showed a marked l o s s of a g g r e g a t i n g PG with i n c r e a s i n g d i s c grade group; Donor J showed an i n c r e a s e , while the change i n p r o p o r t i o n of t o t a l PG i n the a g g r e g a t i n g form i n Donor I was i n c o n c l u s i v e (Table 3.2.6). In view of the marked changes the hex/hexA of the t o t a l CEP A l PG underwent wi t h i n c r e a s i n g d i s c grade, hex/hexA r a t i o s were measured f o r both the high and low M components, assumed to be a g g r e g a t i n g and non-aggregating, r e s p e c t i v e l y . S i g n i f i c a n t i n c r e a s e s i n the hex/hexA, s u g g e s t i n g i n c r e a s e d p r o p o r t i o n s of KS r e l a t i v e to ChS, were i d e n t i f i e d i n the high M f r a c t i o n (P=0.0027) c o n s i s t e n t l y amongst a l l donors (P f o r i n t e r a c t i o n s . 8 4 ) (Table 3.2.6). The low M f r a c t i o n (non-aggregating) shoved a s i m i l a r r e s u l t w i t h the KS/ChS i n c r e a s i n g from 0.86 to 1.53 i n h e a l t h y d i s c s (grade group 1) -116-Table 3.2.6 P r o p o r t i o n of hig h M and c o m p o s i t i o n s of the h i g h and  low M f r a c t i o n s of A l p r o t e o g l y c a n taken from h e a l t h y  and degenerate d i s c s w i t h i n the same s p i n e s Donor Grade P r o p o r t i o n of Hex/HexA and Group High M High M Low M D i s c {%} Donor H L2/L3 1 32 0.97 0.89 L3/L4 1 39 1.17 0.82 Mean f o r grade group 1 36 1.07 0.86 L5/S1 2 9 2.71 3.11 Donor I L2/L3 1 22 0.80 0.88 L4/L5 1 30 0.95 1.09 Mean f o r grade group 1 26 0.88 0.99 L5/S1 2 38 2.79 2.23 Donor J L1/L2 1 29 0.90 1.46 L3/L4 1 26 1.51 1.59 Mean f o r grade group 1 28 1.21 1.53 L4/L5 2 41 2.94 3.17 V a r i a t i o n w i t h i n grade group 5 0.27 0.10 -117-Two-way a n a l y s i s of v a r i a n c e Parameter Comparison D.F, P by F - t e s t P r o p o r t i o n of High M Donor vs e r r o r Grade group vs e r r o r I n t e r a c t i o n vs e r r o r 2 /3 1/3 2 /3 0 .456 0 .923 0 .023 hex/hexA High M Donor vs e r r o r Grade group vs e r r o r I n t e r a c t i o n vs e r r o r 2 /3 1/3 2 /3 0 .538 0 .030 0 .844 hex/hexA Low M Donor vs e r r o r Grade group vs I n t e r a c t i o n vs 2 /3 0 .010 e r r o r 1/3 0 .0002 e r r o r 2 /3 0 .026 -118-to betveen 2.23 to 3.17 i n degenerate d i s c s (grade group 2)(Table 3.2.6). Thus, vhen these r e s u l t s are c o n s i d e r e d w i t h the l o s s of t o t a l PG v i t h i n c r e a s i n g grade group, the C h S - r i c h PG appear to be l o s t more e x t e n s i v e l y than the K S - r i c h PG. In a d d i t i o n , a d i s p r o p o r t i o n a t e b i o s y n t h e s i s of K S - r i c h PG may be t a k i n g plac e r e s u l t i n g i n h i g h e r l e v e l s of KS and a l s o c o n t r i b u t i n g to the i n c r e a s e i n KS/ChS observed v i t h i n c r e a s i n g d i s c grade group. 3.2.7 Composite a g a r o s e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of  A l P r o t e o g l y c a n s In p a r t 1 of t h i s study, f i v e e l e c t r o p h o r e t i c a l l y d i s t i n c t PG s u b s p e c i e s were i d e n t i f i e d i n the CEP of h e a l t h y d i s c s . The d i s t r i b u t i o n of the subsp e c i e s was shovn to a l t e r vhen the d i s c undervent degeneration and a g e i n g . The experiments c a r r i e d out i n p a r t two of t h i s s t u d y i n v e s t i g a t e d the e f f e c t of i n c r e a s i n g d i s c grade (degeneration) a l o n e , on the e l e c t r o p h o r e t i c h e t e r o g e n e i t y of the CEP A l PG. The r e s u l t s of the CAPAGE a n a l y s i s of the hig h and low M components of the CEP A l PG are summarized i n Table 3.2.7. For each d i s c grade s t u d i e d , the g e l s v i t h the best r e s o l u t i o n of bands vere scanned. In g e n e r a l , the p a t t e r n s seen f o r d i s c s of the same grade vere s i m i l a r , but the r e s o l u t i o n i n s e v e r a l g e l s was poor. -119-Table 3.2.7 R e l a t i v e e l e c t r o p h o r e t i c m o b i l i t y of h i g h and low M p r o t e o g l y c a n s prepared from h e a l t h y and degenerate d i s c s of the same spine D i s c Grade Number M o b i l i t y *  MRI Group of D i s c s High M Low M Grade S t u d i e d * *  II I I I IV 1 2 2 0.08 0.25 0.44 0.61 0.66 0. 46 0 0 60 71 0. 49 0 0. 60 75 0.50 0 0 0 62 70 86 *The m o b i l i t y of each band was measured r e l a t i v e to the same ChS s t a n d a r d . **For each d i s c grade, o n l y the g e l s showing the best r e s o l u t i o n of bands were scanned. -120-The e l e c t r o p h o r e t i c p r o p e r t i e s of the CEP A l PG v a r i e d w i t h i n grade groups as w e l l as between grade groups (Table 3.2.7). The r e s u l t s l i s t e d i n Table 3.2.7 confirmed the p r e v i o u s f i n d i n g t h a t the h i g h M f r a c t i o n ( a g g r e g a t i n g PG) of grade I ( h e a l t h y ) d i s c CEP PG c o n t a i n e d t h r e e e l e c t r o p h o r e t i c a l l y d i s t i n c t s ubspecies and the low M f r a c t i o n (non-aggregating PG) two s u b s p e c i e s . In grade II and I I I d i s c s , the two s u b s p e c i e s of the high M f r a c t i o n with the lowest e l e c t r o p h o r e t i c m o b i l i t y were not p r e s e n t and one of the s u b s p e c i e s of the low M f r a c t i o n had i n c r e a s e d r e l a t i v e to the grade I d i s c s . The most degenerate d i s c (grade IV) was comprised of a high M f r a c t i o n with o n l y one e l e c t r o p h o r e t i c band and a low M f r a c t i o n t h a t c o ntained an a d d i t i o n a l h i g h m o b i l i t y band not seen i n any of the other d i s c grades. Thus, i n both the study i n v e s t i g a t i n g the combined e f f e c t s of d e g e n e r a t i o n and ageing and t h i s study i n v e s t i g a t i n g d e g e n e r a t i o n alone, s i m i l a r changes i n the d i s t r i b u t i o n of e l e c t r o p h o r e t i c PG s u b s p e c i e s were observed. -121-4.0 DISCUSSION 4.1 M o r p h o l o g i c a l changes of the end-plate w i t h ageing and  d e g e n e r a t i o n In the l i m i t e d number of specimens s t u d i e d , the gross anatomy of the e n d - p l a t e and/or subchondral bone appeared to change with d i s c grade. Healthy d i s c s showed a r e g u l a r and evenly contoured CEP extending over the width of the NP and inner t w o - t h i r d s of the AF. In the advanced s t a g e s of degeneration (Grade IV/V d i s c s ) the CEP was markedly thinned and i r r e g u l a r and, i n some i n s t a n c e s , d i s r u p t e d , a l l o w i n g increased areas of the AF to come i n c o n t a c t w i t h the subchondral bone. Gross d i f f e r e n c e s were e v i d e n t i n the subchondral bone a f t e r removal of the CEP between h e a l t h y and degenerate d i s c s . The s u r f a c e beneath the CEP i n degenerate d i s c s o f t e n showed evidence of s c l e r o t i c change and/or c a l c i u m d e p o s i t i o n which c o u l d i n h i b i t the flow of n u t r i e n t s to the NP, while the subchondral bone of h e a l t h y d i s c s was f r e e of s c l e r o s i s and c a l c i u m d e p o s i t s , c o n f i r m i n g p r i o r o b s e r v a t i o n s (F i g u r e s 3.1.1-2 and 3.1.1-3). 4.2 L i m i t a t i o n s of c o l o r i m e t r i c assays The hexuronate and hexose assays were used to estimate the l e v e l s of c h o n d r o i t i n sulphate and k e r a t a n s u l p h a t e r e s p e c t i v e l y . Assays f o r glucosamine and galactosamine have been used by other i n v e s t i g a t o r s f o r the same purpose (Adams and Muir, 1976). The l a t t e r method i s more cumbersome -123-s i n c e i t r e q u i r e s q u a n t i t a t i v e h y d r o l y s i s of the g lycosaminoglycans. T h i s i s d i f f i c u l t to a c h i e v e without d e s t r u c t i o n . A l s o the two hexosamines have u s u a l l y been separated by ion exchange chromatography. T h e r e f o r e , the simpler d i r e c t c o l o r i m e t r i c assays were used. C o n d i t i o n s of the hexose assay method used i n t h i s study were chosen t o a v o i d i n t e r f e r e n c e from pentoses, h e x u r o n i c a c i d s and 2-deoxypentoses ( S c o t t & M e l v i l l e , 1953). N e v e r t h e l e s s , t h i s measure of hexose i n c l u d e d the g a l a c t o s e and mannose pr e s e n t i n the o l i g o s a c c h a r i d e s a t t a c h e d to the p r o t e o g l y c a n p r o t e i n core and any hexose p r e s e n t i n the r e g i o n of the g l y c o s a m i n o g l y c a n 1 s attachment to the p r o t e i n c o r e . Since the c o n t r i b u t i o n s from both of these s o u r c e s was s m a l l , the measure of k e r a t a n s u l p h a t e by t h i s method was c l o s e to the true v a l u e . S i m i l a r i l y , the c a r b a z o l e r e a c t i o n , used to assay the hexuronate, i s a measure of c h o n d r o i t i n s u l p h a t e but w i l l r e a c t a l s o w i t h the g l u c u r o n i c a c i d of h y a l u r o n a t e , r e p r e s e n t i n g about 2% of the hexuronate of the d i s c (Hardingham and Adams, 1976). Hexoses and pentoses a l s o produce the same c o l o r as hexuronate with t h i s r e a c t i o n , but with an e x t i n c t i o n c o e f f i c i e n t of about 10% of t h a t with hexuronate. 4.3 C h a r a c t e r i z a t i o n of the proteoglycans of e n d - p l a t e from  h e a l t h y d i s c s -124-The p r o t e o g l y c a n s of a r t i c u l a r c a r t i l a g e and i n t e r v e r t e b r a l d i s c have many common p r o p e r t i e s . However, t h e i r two most s i g n i f i c a n t d i f f e r e n c e s are the p r o p o r t i o n of t o t a l PG p r e s e n t as aggregate and the r a t i o of k e r a t a n s u l p h a t e t o c h o n d r o i t i n s u l p h a t e (McDevitt, 1988). As d e s c r i b e d i n Table 4.2.1, the end-plate resembles the i n t e r v e r t e b r a l d i s c i n both these r e s p e c t s . The most c o n v i n c i n g evidence of t h i s i s the r e l a t i v e l y low p r o p o r t i o n of a g g r e g a t i n g PG found i n the end-plate (24%). The comparable s m a l l amount of aggregate PG found i n the AF and NP has been a t t r i b u t e d to the l a c k of f u n c t i o n a l h y a l u r o n a t e b i n d i n g r e g i o n s (HABR), s i n c e the a d d i t i o n of exogenous hy a l u r o n a t e d i d not r e s u l t i n i n c r e a s e d aggregate f o r m a t i o n (Hardingham & Adams, 1976; McDevitt et a l . , 1981). The absence of f u n c t i o n a l HABRs c o u l d a r i s e v i a two p o s s i b l e mechanisms. T h i s r e g i o n of the newly s y n t h e s i z e d PG c o u l d be degraded by p r o t e a s e s a f t e r b i o s y n t h e s i s (Roughley e t a l . , 1982) or two or more d i f f e r e n t PG molecules c o u l d be s y n t h e s i z e d d i r e c t l y . In a d d i t i o n , the HABR might be p r e s e n t , but i n a form i n c a p a b l e of r e a c t i n g with h y a l u r o n a t e to form aggregates. The b i o s y n t h e s i s by the end-plate of PG molecules l a c k i n g an HABR would p r o v i d e a d d i t i o n a l support to the h y p o t h e s i s t h a t the s i t e of AF and NP p r o t e o g l y c a n b i o s y n t h e s i s i s the e n d - p l a t e chondrocytes. The f i n d i n g s of t h i s s t u d y suggest t h a t a d d i t i o n a l experiments should be -125-Table 4.3.1 A comparison of end-plate p r o t e o g l y c a n s v i t h those of a r t i c u l a r c a r t i l a g e , anulus f i b r o s u s and nucleus pulposus The r e f e r e n c e used f o r each parameter quoted appears i n parentheses. T i s s u e Water P r o p o r t i o n hex/ CAPAGE Content of High M hexA Subspecies LM L%) L%J A r t i c u l a r 60-80 58-85 0.6-0.9 2-3 C a r t i l a g e (a) (b) (c,d) (e) Anulus 72-78 >30-40; 53 0.4-1.5 5 F i b r o s u s ( f , g ) ( h , i , j ) (k) (11,0,1) Nucleus 84-90 <30-40; 53 0.8-1.8 4-6 Pulposus ( f , g ) (m, j ) (k) (n,o,1) End- P l a t e * 74 24 1.2 5 *Mean v a l u e s from 9 donors References; a) I n e r o t & Heinegard, 1982; b) Roughley & Mort, 1986; c) Heinegard et a l . . 1979; d) Stevens et a l . , 1979; e) Heinegard e t a l . , 1981; f ) Naylor e t a l . , 1955; g) Urban & Maroudas, 1981; h) Ernes and Pearce, 1975; i ) Hardingham & Adams, 1976; j ) Donohue e t a l . , 1988; k) Pearce R, p e r s o n a l communication; 1) D i F a b i o et a l . , 1986; m) Adams & Muir, 1976; n) Jahnke & McDevitt, 1988; o) Stanescu e t a l . , 1980; -126-d i r e c t e d t o i n v e s t i g a t i n g t h i s p o s s i b i l t y . The water content of the end-plate PG n e i t h e r s t r o n g l y supports or r u l e s out a resemblance to d i s c p r o t e o g l y c a n s (Table 4.2.1). While the water content of d i s c t i s s u e s i s g e n e r a l l y h i g h e r than t h a t of a r t i c u l a r c a r t i l a g e , both t i s s u e s f a l l i n t o the same broad range, g i v i n g l i m i t e d value to comparisons. The CAPAGE a n a l y s i s of the CEP PG i n t h i s study, confirmed the f i n d i n g s i n other t i s s u e s t h a t s e v e r a l d i s t i n c t s u b s p e c i e s o c c u r . The broad bands r e f l e c t the p o l y d i s p e r s e nature of the molecules under study and the l a r g e molecular weights of the p r o t e o g l y c a n s preclude the use of monodisperse molecular weight standards as e l e c t r o p h o r e t i c g e l markers (Wasteson, 1971). The pro t e o g l y c a n s of the CEP appear to have e l e c t r o p h o r e t i c p r o p e r t i e s that resemble the PG p o p u l a t i o n s of the i n t e r v e r t e b r a l d i s c more c l o s e l y than those of a r t i c u l a r c a r t i l a g e (Table 4.3.1). The a g g r e g a t i n g PG f r a c t i o n of end-plate from h e a l t h y d i s c s have the same number of subspecies found i n h e a l t h y d i s c NP and the non-aggregating PG f r a c t i o n of CEP resemble the non-aggregating PG p o p u l a t i o n i n mature d i s c AF and NP. These f i n d i n g s are c o n s i s t e n t with the hyp o t h e s i s t h a t the PG found i n the d i s c has i t s o r i g i n i n the CEP and t h a t , i n a d d i t i o n to mediating the flow of n u t r i e n t s to the NP, the end-plate p l a y s an a c t i v e r o l e i n the s y n t h e s i s of the p r o t e o g l y c a n s of the other d i s c t i s s u e s . However, i f -127-d e g r a d a t i v e enzymes are abundant i n the d i s c and d e g r a d a t i o n products are r e t a i n e d i n the d i s c , these f a c t o r s c o u l d a f f e c t the p r o t e o g l y c a n s of the NP, AF and CEP even though t h e i r o r i g i n s were d i f f e r e n t . 4.4 Changes i n the end-plate p r o t e o g l y c a n w i t h ageing and  d e g e n e r a t i o n In d i s c AF, NP and a r t i c u l a r c a r t i l a g e , a g e i n g and d e g e n e r a t i o n are thought to be d i s t i n c t b i o c h e m i c a l processes, producing d i f f e r e n t changes i n t i s s u e p r o t e o g l y c a n composition ( I n e r o t & Heinegard, 1982; Lyons et a l . , 1981; Pearce e_t a l . , 1987). The changes i n the end-plate p r o t e o g l y c a n s o c c u r r i n g with ageing and degeneration which were i n v e s t i g a t e d i n t h i s study are l i s t e d i n Table 4.3.1. In the f i r s t p a r t of t h i s study, the PG of end-plates from grade I and grade IV/V d i s c s of d i f f e r e n t s p i n e s were compared, while i n p a r t two, the PG from e n d - p l a t e s from d i s c s of more s i m i l a r grade (grade II versus grade IV, grade II versus grade I I I and grade I versus grade I I I ) from w i t h i n the same spine were compared (see Methods f o r a complete d e s c r i p t i o n of the s e l e c t i o n of specimens used f o r s t u d y ) . The v a l i d i t y of the comparisons of the p r o t e o g l y c a n s i n the two p a r t s of t h i s s t u d y may have been l i m i t e d by the l e s s e r d i f f e r e n c e s i n grade i n P a r t I I . The water content of the AF and NP i n d i s c changes with -128-age (Urban & M c M u l l i n , 1985). The main l o s s of water takes p l a c e i n the NP ( P a n a g i o t a c o p u l o s et a l . , 1987), r e d u c i n g i t s l o a d - b e a r i n g c a p a b i l i t y and i n c r e a s i n g the v i s c o e l a s t i c demands made upon the AF (Panagiotacopulos et a l . , 1979). S t u d i e s comparing water c o n t e n t of h e a l t h y d i s c s w i t h degenerate d i s c s , r e p o r t a 10% l o s s i n the NP and inner AF (Lyons et a l . , 1981). In mature a r t i c u l a r c a r t i l a g e , the water content decreases from 10-17% due to ageing (Maroudas, 1973), while i n o s t e o a r t h r i t i c a r t i c u l a r c a r t i l a g e , the water content i s 10% l e s s than i n normal c a r t i l a g e (Maroudas e t a l . , 1986) The decrease i n water c o n t e n t seen i n the e n d - p l a t e with ageing and d e g e n e r a t i o n of the d i s c was of b o r d e r l i n e s i g n i f i c a n c e . I t was l i k e l y due c h i e f l y to the e f f e c t of d i s c d e g e n e r a t i o n alone, s i n c e equal l o s s e s of water were seen i n end-plate i n both p a r t s of t h i s study (Table 4.3.1). Since the a b i l i t y of e n d - p l a t e to imbibe water i s due to the p r o t e o g l y c a n , a decrease i n water content i s o f t e n seen i n c o n j u n c t i o n with l o s s of p r o t e o g l y c a n (McDevitt, 1988). In these experiments the t o t a l t i s s u e PG d i d not change c o n s i s t e n t l y with a g e i n g or d e g e n e r a t i o n amongst a l l the donors s t u d i e d . The l e s s e r decrease i n end-plate water content observed i n t h i s s t u d y may be p a r t i a l l y due to the replacement of one type of p r o t e o g l y c a n , having a h i g h charge d e n s i t y and t h e r e f o r e a h i g h water b i n d i n g c a p a c i t y , by a PG -129-Table 4.4.1 Comparison of the changes i n the en d - p l a t e proteoglycans  due to ageing and d e g e n e r a t i o n versus those due to  de g e n e r a t i o n alone Parameter Changes due t o Age ing/Degeneration (Part I)  Changes due t o Degeneration (P a r t I I ) Water Content decreased by 10% decreased by 10% Hex + HexA no change s l i g h t decrease i n some donors A l PG hex/hexA Kmode High M Kmode Low M P r o p o r t i o n of High M hex/hexA High M hex/hexA Low M CAPAGE High M CAPAGE Low M in c r e a s e d by approx. 3x moderate i n c r e a s e marked i n c r e a s e no change i n c r e a s e d by approx. 3x in c r e a s e d by approx. 3x l o s s of subsp e c i e s I and II g r e a t l y i n c r e a s e d m o b i l i t y of s p e c i e s IV & V and appearance of another s u b s p e c i e s i n c r e a s e d by approx. 3x m i l d i n c r e a s e m i l d i n c r e a s e no change i n c r e a s e d by approx. 3x i n c r e a s e d by approx. 3x l o s s of su b s p e c i e s I and II m i l d l y i n c r e a s e d m o b i l i t y of s p e c i e s V and appearance of another s u b s p e c i e s -130-with a lower charge d e n s i t y . T h i s e f f e c t c o u l d be achieved by r e p l a c i n g a C h S - r i c h p r o t e o g l y c a n , having two n e g a t i v e charges per r e p e a t i n g u n i t , (one e s t e r sulphate and one c a r b o x y l ) with a K S - r i c h p r o t e o g l y c a n t h a t has o n l y one n e g a t i v e charge per r e p e a t i n g u n i t (one e s t e r s u l p h a t e ) . Ageing and d e g e n e r a t i o n a f f e c t the m o l e c u l a r s i z e of end-plate p r o t e o g l y c a n s (Table 4.3.1). P r e v i o u s s t u d i e s of end-plate p r o t e o g l y c a n s have i d e n t i f i e d two f r a c t i o n s , w e l l - r e s o l v e d by chromatography and thought to r e p r e s e n t a g g r e g a t i n g and non-aggregating PG p o p u l a t i o n s ( P e d r i n i ejt a l . , 1983). Comparisons of end-plate PG from ten day-old and s i x month-old i n f a n t s have shown t h a t the m o l e c u l a r s i z e of the non-aggregating e n d - p l a t e PG f r a c t i o n d e c r e a s e s (Buckwalter e t a l . . 1985). In a r t i c u l a r c a r t i l a g e , an accumulation of low M p r o t e o g l y c a n s r e s u l t i n g from ageing have a l s o been r e p o r t e d ( B a y l i s s et a l . , 1985). In the present i n v e s t i g a t i o n , a geing and d e g e n e r a t i o n were found to have d i f f e r e n t e f f e c t s on the molecular s i z e of the end-plate p r o t e o g l y c a n s (Table 4.3.1). Both the a g g r e g a t i n g (high M) and non-aggregating (low M) PG p o p u l a t i o n s were reduced i n molecular s i z e to a g r e a t e r degree as a r e s u l t of ageing than as a r e s u l t of d e g e n e r a t i o n (Table 4.3.1). While the experimental r e s u l t s appear to suggest t h a t t h i s e f f e c t was g r e a t e r on the non-aggregating p r o t e o g l y c a n s , s u b s t a n t i a l changes i n the m o l e c u l a r s i z e d i s t r i b u t i o n of the a g g r e g a t i n g -131-p r o t e o g l y c a n may have o c c u r r e d , but to an e x t e n t i n s u f f i c i e n t to a l l o w the molecules to penetrate the g e l . A number of i n v e s t i g a t i o n s have ass e s s e d the e f f e c t s of d e g e n e r a t i o n on the c o m p o s i t i o n of d i s c p r o t e o g l y c a n s . Comparisons of p r o t e o g l y c a n s from mature d i s c (AF and NP i n a l l cases) with those from immature d i s c and from h e a l t h y d i s c with d i s c from s c o l i o t i c s p i n e s have demonstrated changes i n the estimated r e l a t i v e p r o p o r t i o n s of the glycosaminoglycans (ChS and KS). P r e v i o u s s t u d i e s of human i n f a n t i n t e r v e r t e b r a l d i s c (immature) have r e p o r t e d the glcN/galN i n AF and NP as 0.24:1 (Buckwalter e t a l . , 1985) as compared w i t h a 1:1 r a t i o i n mature d i s c (Pearce, p e r s o n a l communication). S t u d i e s comparing the t i s s u e s of d i s c s from s c o l i o t i c s p i n e s with those from normal s p i n e s r e p o r t e d the glcN/galN i n CEP as 0.9:1 i n normal s p i n e s and 0.8:1 i n s c o l i o t i c s p i n e s ( P e d r i n i - M i l l e et a l . , 1983) E a r l i e r i n v e s t i g a t i o n s of the changes i n p r o t e o g l y c a n content of a r t i c u l a r c a r t i l a g e with ageing, have r e p o r t e d an i n c r e a s e i n the g l c N / g a l N with i n c r e a s i n g age, r e s u l t i n g from a r e l a t i v e decrease i n the ChS c o n c e n t r a t i o n coupled with an increase i n KS c o n c e n t r a t i o n ( B a y l i s s et a l . , 1978; B a y l i s s , 1985; Maroudas et a l . , 1986). The r e l a t i v e p r o p o r t i o n s of end-plate C h S - r i c h PG decreased and the K S - r i c h PG increased with d i s c d e g e n e r a t i o n , t h a t i s , with i n c r e a s i n g d i s c grade. The r a t i o of KS/ChS, r e f l e c t e d by the r a t i o of hex/hexA, i n c r e a s e d from 1:1 i n CEP -132-of h e a l t h y d i s c s to 3:1 i n CEP of degenerate d i s c s . T h i s i n c r e a s e was found to take p l a c e i n both the excluded (or high M) and i n c l u d e d (or low M) PG f r a c t i o n s . The i n c r e a s e d r a t i o was due to both a moderate decrease i n the ChS c o n c e n t r a t i o n and a marked i n c r e a s e i n the KS c o n c e n t r a t i o n , s u g g e s t i n g t h a t the C h S - r i c h PG was p r e f e r e n t i a l l y degraded e n z y m a t i c a l l y and/or the b i o s y n t h e s i s of PG r i c h i n KS was s t i m u l a t e d as has been observed i n o s t e o a r t h r i t i c c a r t i l a g e (McDevitt et a l . , 1981; Maroudas et a l . , 1986). S t u d i e s of AF and NP i n degenerate d i s c s have suggested t h a t i n c r e a s e d p r o p o r t i o n s of a g g r e g a t i n g p r o t e o g l y c a n s , h i g h i n K S - r i c h molecules, are a s s o c i a t e d with a r e p a i r process (McDevitt, 1988). The f i n d i n g s of t h i s s t u d y suggest t h a t a s i m i l a r process may occur i n the e n d - p l a t e . Degeneration a l s o appears to change the r e l a t i v e p r o p o r t i o n s of end-plate p r o t e o g l y c a n s u b s p e c i e s , while ageing changes the molecular s i z e of the s u b s p e c i e s (Table 4.3.1). The l o s s of the two slowest moving bands on the e l e c t r o p h o r e t i c g e l suggests t h a t the two l a r g e s t a g g r e g a t i n g PG s u b s p e c i e s have been degraded, p o s s i b l y to form a d d i t i o n a l amounts of the t h i r d a g g r e g a t i n g s u b s p e c i e s . S i m i l a r l y , the two non-aggregating s u b s p e c i e s may have undergone enzymatic d e g r a d a t i o n to produce the t h i r d s u b s p e c i e s seen with d e g e n e r a t i o n . The apparent decrease i n molecular s i z e of the two non-aggregating s u b s p e c i e s , r e f l e c t e d by t h e i r i n c r e a s e d -133-m o b i l i t i e s on the CAPAGE g e l , was most pronounced i n end-plate PG from d i s c s t h a t had undergone ageing and o n l y m i l d l y e v i d e n t i n e n d - p l a t e from degenerate d i s c s . T h i s i s c o n s i s t e n t w i t h the e a r l i e r f i n d i n g t h a t decreases i n the molecular s i z e of the non-aggregating PG f r a c t i o n , as determined by g e l chromatography, are due c h i e f l y to ageing and not d e g e n e r a t i o n . The p r o p e r t i e s of end-plate p r o t e o g l y c a n s i d e n t i f i e d i n t h i s study are not l i k e l y to have been a f f e c t e d by the protease a c t i v i t y a s s o c i a t e d with post-mortem changes, s i n c e p r e v i o u s s t u d i e s have shown t h a t e x t r a c t s of post-mortem i n t e r v e r t e b r a l d i s c do not have any d e g r a d a t i v e e f f e c t on p r o t e o g l y c a n s (Stevens et a l . , 1979). The f i n d i n g s of t h i s s t u d y are s i g n i f i c a n t i n t h a t they p r o v i d e a d d i t i o n a l s t r o n g support f o r the hypothesis t h a t d i s c d e g e n e r a t i o n begins i n the e n d - p l a t e and t h a t the i n i t i a t i o n of d e g e n e r a t i o n i n v o l v e s changes i n the composition of the end-plate p r o t e o g l y c a n . The c h i e f l i m i t a t i o n s of t h i s s t u d y are r e l a t e d to the s m a l l number of donors s t u d i e d and the presence of u n c o n t r o l l e d v a r i a b l e s . Groups of t h r e e donor spines were used to a s s e s s the e f f e c t s of ageing and/or degeneration on end-plate p r o t e o g l y c a n c o m p o s i t i o n . I r r e s p e c t i v e of the s m a l l number of donors used, the d i f f e r e n c e s between groups were shown to be s t a t i s t i c a l l y s i g n i f i c a n t . The main v a r i a b l e s not -13 4-c o n t r o l l e d i n t h i s study are the e f f e c t s of d i f f e r e n t p a t i e n t / d o n o r c l i n i c a l h i s t o r i e s (e.g. s e d e n t a r y versus a c t i v e l i f e s t y l e ) and i n p a r t I I , the l e v e l of lumbar spine d i s c s used f o r the purposes of comparison. These v a r i a b l e s are i n h e r e n t to a l l post-mortem i n v e s t i g a t i o n s of the spine and of i n d i v i d u a l lumbar d i s c s and cannot be c o n t r o l l e d i n any p r a c t i c a l manner. 4 . 5 C l i n i c a l s i g n i f i c a n c e These f i n d i n g s j u s t i f y the r e c e n t l y i n t e n s i f i e d i n t e r e s t i n the r o l e of the CEP i n d i s c d e g e n e r a t i o n (Aoki et a l . , 1987; Roberts et a l . , 1989). C l i n i c a l s t u d i e s have focussed on i n j u r y t o the AF as primary i n d i s c d e g e n e r a t i o n and have regarded the d i s c as a whole as an i n e r t s t r u c t u r e incapable of r e g e n e r a t i o n . 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A scheme for the assessment of ageing and degeneration from the gross morphology of the human intervertebral disc, Rased on Vernon-Roberts (1980) GRADE NUCLEUS ANULUS END-PLATE VERTEBRAL BODY Gel-like, bulging, bluish-white Discrete lamellae, white Hyaline, uniform thickness Margins rounded II Fibrous tissue extending from anulus Chondroid or mucinous material between lamellae Irregular thickness of cartilage Margins pointed III Consolidation of fibrous tissue Extensive chondroid or mucinous material; loss of anular-nuclear demarcation Focal defects in cartilage Early osteophytes or osteophytes at margins IV Horizontal clefts parallel to end-plate Focal disruptions Fibrocartilaginous tissue extends from subchondral bone; irregularity and focal sclerosis of sub-chondral bone Osteophytes <2mm V Clefts extend though nucleus and anulus Diffuse sclerosis Osteophytes >2mm From Thompson, Pearce and Schechter (in preparation) -147-Table 2. A scheme for the assessment of ageing and degeneration using magnetic resonance images of human lumbar intervertebral disc. T2-weighted spin-echo images with T K 2000msec and T E 80msec are used. GRADE NUCLEUS ANULUS END-PLATE VERTEBRAL BODY I Homogeneous, bright; demarcation distinct II Horizontal dark band extends across anulus centrally III Signal intensity diminished; gray tone with dark and bright stippling IV Proportion of gray signal reduced; larger bright and dark regions Gross loss of disc height; bright and dark signals dominant Homogeneous, dark gray Areas of increased signal intensity Indistinguishable from nucleus Single dark line Increase in central concavity Line less distinct Indistinguishable from Focal defects in line nucleus; some bright and dark signals contiguous with nucleus Signals contiguous with nucleus Defects and areas of thickening Margin rounded Tapering of margins Small dark projections from margins Projections < 2mm with same intensity as bone marrow Projections > 2mm with same intensity as bone marrow From Thompson, Ho, Pearce and Schechter (in preparation) -148-

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