T.K.F. Taylor

The knee joint meniscus. A fibrocartilage of some distinction.

It is now well established that the meniscus performs a number of roles that are important to the efficient performance of the knee joint. Of particular importance is the recognition of its load-bearing function and its stabilization of the joint during flexion-extension. The compression of the wedge-shaped meniscus during loading is translated into circumferential stresses, and the collagen fiber distribution and orientation is well adapted for this mechanical role. Proteoglycans of the meniscus have structural characteristics in common with those of articular cartilage but do not appear to influence the tensile properties of menisci. With aging and degeneration, compositional changes take place within the meniscus that reduce its ability to transmit tensional stresses, and this contributes to failure. Total meniscectomy is not a benign procedure. Partial excision has less deleterious effects on the joint. Surgical repair of meniscus lesions has now emerged as a procedure of some significance and laboratory research suggests that a solution to this problem may be within reach. Experimental studies in dogs also indicate that chondroprotective agents such as semisynthetic sulfated polysaccharides may protect articular cartilage after meniscectomy.

A comparative analysis of the differential spatial and temporal distributions of the large (aggrecan, versican) and small (decorin, biglycan, fibromodulin) proteoglycans of the intervertebral disc.

This study provides a comparative analysis of the temporal and spatial distribution of 5 intervertebral disc (IVD) proteoglycans (PGs) in sheep. The main PGs in the 2 and 10 y old sheep groups were polydisperse chondroitin sulphate and keratan sulphate substituted species. Their proportions did not differ markedly either with spinal level or disc zone. In contrast, the fetal discs contained 2 slow migrating (by composite agarose polyacrylamide gel electrophoresis, CAPAGE), relatively monodisperse chondroitin sulphate‐rich aggrecan species which were also identified by monoclonal antibody 7‐D‐4 to an atypical chondroitin sulphate isomer presentation previously found in chick limb bud, and shark cartilage. The main small PG detectable in the fetal discs was biglycan, whereas decorin predominated in the 2 and 10 y old IVD samples; its levels were highest in the outer annulus fibrosus (AF). Versican was most abundant in the AF of the fetal sheep group; it was significantly less abundant in the 2 and 10 y old groups. Furthermore, versican was immunolocalised between adjacent layers of annular lamellae suggesting that it may have some role in the provision of the viscoelastic properties to this tissue. Versican was also diffusely distributed throughout the nucleus pulposus of fetal IVDs, and its levels were significantly lower in adult IVD specimens. This is the first study to identify versican in ovine IVD tissue sections and confirmed an earlier study which demonstrated that ovine IVD cells synthesised versican in culture (Melrose et al. 2000). The variable distribution of the PGs identified in this study provides further evidence of differences in phenotypic expression of IVD cell populations during growth and development and further demonstrates the complexity of the PGs in this heterogeneous but intricately organised connective tissue.

REGULATION OF GELATINASE-A (MMP-2) PRODUCTION BY OVINE INTERVERTEBRAL DISC NUCLEUS PULPOSUS CELLS GROWN IN ALGINATE BEAD CULTURE BY TRANSFORMING GROWTH FACTOR-β1AND INSULIN LIKE GROWTH FACTOR-I

The aim of this study was to gain information relevant to disc repair processes. Limited degradation of the collagen matrix by matrix metalloproteases (MMPs) may facilitate the loosening of cell‐cell and cell‐matrix interactions within the injured intervertebral disc (IVD) to favour the penetration of blood vessels and migration of fibroblasts into the defect to promote repair processes. Gelatinase A (MMP‐2) has a particularly important role to play in angiogenesis, in the present study we investigated the in vitro regulation of MMP‐2 by Transforming Growth Factor‐β1 (TGF‐β1) and Insulin‐like Growth Factor‐1 (βIGF‐I) in cells from the nucleus pulposus (NP) of the ovine IVD. Ovine NP cells were grown in alginate bead cultures in complete medium (10% foetal calf serum) for 7 days, established in serum‐free conditions for 24h, then stimulated with TGF‐β1 (0.1 or 10ng/ml) or IGF‐I (2 or 50ng/ml) ±Concanavalin A (20μg/ml) for an additional 48h. Conditioned medium was examined for matrix metalloproteases using gelatin zymography, Tissue Inhibitor of Metalloproteinase 2 (TIMP‐2) and Membrane Type 1 Matrix Metalloproteinase (MT1‐MMP) were immunolocalised in beads. Pro (72kDa) and active (59kDa) MMP‐2 were the major gelatinolytic MMPs detected in control cultures, the TGF‐β1 and IGF‐I treatments significantly decreased levels of the active MMP‐2, inclusion of Concanavalin A resulted in a complete reversal of this trend with IGF‐I, and to a lesser extent with TGF‐β1. Cell surface levels of TIMP‐2 and MT1‐MMP were decreased by the TGF‐β1 treatment while IGF‐I only appeared to decrease TIMP‐2 expression. The findings of this study provide some insight as to why dense avascular connective tissues such as the intervertebral disc have such a poor healing potential.

Variations of the proteoglycans of the canine intervertebral disc with ageing.

A group of young (2.0 +/- 0.6 years) (group 1) and old (9.7 +/- 1.5 years) (group 2) beagle dogs were given Na2 35SO4 (1.0 mCi/kg) intravenously 60 days prior to being killed to radiolabel their proteoglycans. Lumbar discs were removed and dissected into nucleus pulposus and annulus fibrosus. Proteoglycans were extracted at 4 degrees C from these tissues with buffered 4.0 M Gdn-HCl containing proteinase inhibitors, and purified by CsCl density gradient ultracentrifugation. The average hydrodynamic size and ability of the purified proteoglycans to aggregate in the presence of excess hyaluronic acid was determined by Sepharose CL-2B chromatography. The galactosamine/glucosamine ratios of these proteoglycans as well as their non-aggregating fractions were also ascertained. The proteoglycan content of discs of old animals was significantly less than in the young. The proportion of 35S-labelled, or non-labelled proteoglycans which could aggregate in the presence of hyaluronic acid was also much lower in the preparations isolated from the older discs. In contrast, the average hydrodynamic size of the non-aggregating proteoglycans isolated from the annuli fibrosi of group 2 animals were larger than the corresponding population of group 1 animals. Aminosugar analysis of these same proteoglycan fractions from older animals afforded galactosamine/glucosamine ratios (mean 1.81 +/- 0.14) which were less than the younger age group (mean 2.63 +/- 0.40). These data suggest that with ageing and degeneration the proteoglycans of the beagle disc undergo increased degradation with the accumulation in the annulus fibrosus of a population which is of larger average hydrodynamic size and richer in keratan sulphate than proteoglycans present in younger tissues.

The collagenous and non-collagenous protein of the canine intervertebral disc and their variation with age, spinal level and breed.

The total nitrogen, collagen and non-collagenous protein (NCP) content of the nucleus pulposus (NP), transitional zone (TZ) and annulus fibrosus (AF) of the beagle (a chondrodystrophoid breed) and greyhound (a non-chondrodystrophoid breed) intervertebral discs have been determined over the age range birth to 124 months. Discs were sampled at three spinal levels - lumbosacral, lumbar and thoracic regions. The beagle NP attains an average NP collagen content of 25% dry weight before 11 months of age, whereas the greyhound NP content is less than 5% collagen for most of its life. The NCP of the young greyhound disc is greater than that of the beagle of corresponding age. Plots of the ratio of collagen/NCP for discal regions at various ages highlight the large differences between the two breeds of dog. It is suggested that these differences in protein distribution are related to the elevated incidence of intervertebral disc prolapse in the beagle.

A Comparative Chemical and Histochemical Study of the Chondrodystrophoid and Nonchondrodystrophoid Canine Intervertebral Disc

The chemical composition of the intervertebral disc of 9-month-old chondrodystrophoid and nonchondrodystrophoid dogs was studied for collagen, noncollagenous protein and glycosaminoglycan. Content of these substances differed significantly between breeds. The differences were most marked in the nucleus pulposus; the noncollagenous protein content of the nonchondrodystrophoid breed was higher than in that of the chondrodystrophoid dogs. The total nitrogen value of the nonchondrodystrophoid nuclei pulposi was less than that of the corresponding chondrodystrophoid discs mainly because of the high collagen content of the latter discs. Histochemically, it was found that the nuclei pulposi of the nonchondrodystrophoid breed contains larger amounts of glycosaminoglycan than in the discs of the chondrodystrophoid breeds.

Intervertebral Disc Reconstitution After Chemonucleolysis With Chymopapain is Dependent on Dosage: An Experimental Study in Beagle Dogs

Study Design. The current report describes a study in beagles in which the effects of intradiscal injection of three doses of chymopapain were evaluated with respect to the reduction of disc width and reconstitution of the nucleus pulposus. Objectives. To establish an intradiscal dose of chymopapain that would achieve optimal reduction in disc height followed by maximum reconstitution of the nucleus pulposus. Summary of Background Data. Earlier reports of the efficacy of high and low doses of chymopapain for chemonucleolysis have provided conflicting data, and a scientific basis for an appropriate dose is lacking. Methods. Four mature, female beagles were subjected to chemonucleolysis using three doses of chymopapain as Chymodiactin (31, 63 and 125 picokatals/disc) injected into the L2‐L3, L1‐L2, and L3‐L4 discs. Disc widths were monitored radiographically over 32 weeks. Proteoglycans were radiolabeled by intravenous injection with Na235SO4 (1 mCi/kg) 24 hours before sacrifice, and their specific activities (disintegrations per minute/mg proteoglycan), hydrodynamic size, and ability to aggregate determined. Results. Sixty‐three picokatals of Chymodiactin produced optimal disc reconstitution after chemonucleolysis. A reduction in disc height of approximately 35% was evident within 1 month and this slowly returned to approximately 90% of the preinjection value after 32 weeks. The nucleus pulposus contained approximately 75% of the proteoglycan content of control tissues, and most of these formed aggregates with hyaluronan. Disc collagen levels remained relatively unaffected by treatment. Conclusions. This study demonstrates that an effective reduction in disc width compatible with later reconstitution of the nucleus pulposus can be achieved experimentally with an appropriate dose of chymopapain. These data clearly indicate that an optimal dose of chymopapain for chemonucleolysis in humans needs to be established.

Differential Expression of Proteoglycan Epitopes and Growth Characteristics of Intervertebral Disc Cells Grown in Alginate Bead Culture

This study assessed the growth characteristics and proteoglycan (PG) production in vitro of cells isolated from various zones; annulus fibrosus (AF), the transitional zone (TZ, an opalescent region situated between the AF and nucleus pulposus, NP) and the NP of the ovine intervertebral disc. The isolated cells were subsequently cultured in calcium alginate microspheres. Cellular metabolic activity was assessed over the 10 days of culture by the bioreduction of a tetrazolium dye substrate to a coloured formazan chromophore which could be measured colorimetrically at 490 nm. These data were correlated with the DNA content of beads measured using the fluorescent dye Hoechst 33258. Viable and non-viable cells in alginate beads were also assessed histochemically using the fluorescent dyes 5-chloromethylfluorescein diacetate and ethidium homodimer-1. PGs synthesised in culture were examined immunohistologically using monoclonal antibodies to defined glycosaminoglycan side chain PG epitopes. The results obtained clearly showed that AF and TZ cells differed from NP cells in the measured indices of cellular metabolism and in the extent of matrix deposition. In contrast to cells from the NP the cells from the AF and TZ were more metabolically active and programmed more to cellular proliferation than to matrix production.

Variation of Collagenous and Non-Collagenous Proteins of Human Knee Joint Menisci with Age and Degeneration

The chemical composition has been determined in inner, middle and peripheral zones of 50 human knee joint menisci of ages 0–86 years. Of these, 12 showed areas of degeneration which were analysed sepa

Elevated synthesis of biglycan and decorin in an ovine annular lesion model of experimental disc degeneration.

The aim of this study was to extend our earlier observations on the changes that occur in the proteoglycans (PGs) of discs subjected to experimental injury to the annulus fibrosus (AF). We employed the alginate bead culture method to examine the metabolism of the dermatan sulphate (DS) containing PGs by cells derived from different regions of ovine discs that had been subjected to experimental annular injury. This was compared with the metabolism of the DS-PGs by cells isolated from equivalent regions of normal sham-operated discs. Six months after induction of the annular lesion, AF cells isolated from the lesion produced significantly higher levels of decorin and biglycan in alginate bead culture than did cells from equivalent zones of the controls. Decorin and biglycan were identified in culture media samples by immunoblotting, using specific antibodies (6-B-6, LF-96), and also by positive identification of their de-glycosylated core proteins. The core protein of the DS-PGs has been shown to inhibit type I/II collagen fibrillogenesis, to negatively regulate the action of transforming growth factor-β (TGF-β) and to diminish cellular proliferation in vitro; events which may be detrimental to tissue repair. The findings are therefore consistent with our previous observation the annular lesions in the avascular inner annulus have no capacity to heal.