Peter Ghosh

Increased Nerve and Blood Vessel Ingrowth Associated With Proteoglycan Depletion in an Ovine Anular Lesion Model of Experimental Disc Degeneration

Study Design. Nerves and blood vessel distribution in discs were localized immunohistochemically and correlated with the proteoglycan contents of normal and degenerate disc tissues. Objective. The aim of the present study was to systematically evaluate whether nerve and blood vessel ingrowth was associated with depletion of disc proteoglycans and degenerative changes in an established experimental model of disc degeneration. Summary of Background Data. Animal models of disc degeneration, allowing longitudinal study of pathogenic mechanisms, are limited. The ovine model enables systematic monitoring of blood vessel and nerve ingrowth during the development of disc degeneration after injury to the anulus fibrosus. Methods. Merino sheep received a controlled left anterolateral surgical defect in the outer anulus fibrosus of the L1–L2 and L3–L4 discs (lesion group); sham-operated controls received the retroperitoneal anterolateral approach only. Animals were killed 3, 6, 12, and 26 months postoperation, and the discs were collected for histology and compositional and morphologic analyses. Sagittal tissue sections were stained with toluidine blue and hematoxylin and eosin; Type IV collagen immunolocalization visualized blood vessel ingrowth, and nerves were immunolocalized using monoclonal antibodies to growth-associated protein (GAP-43), protein gene product 9.5, and glial fibrillary acidic protein. Results. Compositional and histologic results demonstrated early focal depletion 3–12 months postoperation of glycosaminoglycan associated with lesion development, increased blood vessel and nerve ingrowth, and infiltration of cells from the outer anulus fibrosus along the plane of the original defect. Blood vessel numbers in the outer to mid third of the anulus fibrosus were elevated in the lesion discs 3–6 months postoperation reaching a maximum at 12 months postoperation; nerves immunoreactive with protein gene product 9.5 (also maximal at 12 months postoperation) were often found associated (but not exclusively) with blood vessels, and some nerves were also reactive with GAP-43 and glial fibrillary acidic protein, but only at 12 months postoperation. Conclusions. Nerve and blood vessel ingrowth into the anulus fibrosis were strongly associated with proteoglycan depletion. The ovine anular lesion model of disc degeneration is a useful experimental model for the systematic evaluation of nerve and blood vessel development after anular injury.

Effects of transforming growth factor beta on proteoglycan synthesis by cell and explant cultures derived from the knee joint meniscus

Repair of meniscal tears depends in part upon the ability of the resident fibrochondrocytes to produce new extracellular matrix molecules including proteoglycans. Three culture systems have been used to investigate proteoglycan production by meniscal fibrochondrocytes from the inner, middle and outer zones of medial and lateral menisci of the sheep stifle joint. Cultures of meniscal explants, monolayered cells, and cells encapsulated in alginate beads were labeled with 35SO4H2 for 48 h in the absence and presence of transforming growth factor beta (TGF beta) and the proteoglycans were analysed by Sephacryl S-1000 chromatography. In general, the lateral meniscus produced more proteoglycan than the medial. Explants from the inner and middle zones produced predominantly aggrecan-like proteoglycan, together with a smaller proteoglycan population eluting with an average distribution coefficient of around 0.65. The outer meniscal zones synthesized less proteoglycan overall, the majority of which consisted of the smaller proteoglycans. These characteristic proteoglycan size profiles obtained with explant cultures also were preserved when cells isolated from the respective zones were cultured in alginate beads. Monolayer cell cultures, however, produced almost entirely small proteoglycans, regardless of their zone of origin. Chromatography of chondroitinase AC and ABC digested samples indicated that the small proteoglycan population comprised mostly dermatan sulphate-containing proteoglycans. In all meniscal zones and in all culture systems, TGF beta stimulated proteoglycan production by up to 100% and the proteoglycans were slightly larger. TGF beta also stimulated cell division in fibrochondrocyte monolayer cultures. Long term intermittent stimulation of alginate bead cultures with TGF beta resulted in large increases in proteoglycan synthesis, increased aggregation of large proteoglycan monomers, and an increase in the production of the larger of two small proteoglycans, putatively, biglycan.

Increased synthesis of matrix metalloproteinases by aortic smooth muscle cells is implicated in the etiopathogenesis of abdominal aortic aneurysms.

PURPOSE The objective of this study was to identify the metalloproteinases elaborated by medial smooth muscle cells (SMCs) isolated from abdominal aortic aneurysm (AAA) and control arterial tissues and to ascertain if the levels produced by AAA SMCs were elevated. METHODS SMC monolayers cultured from the outgrowth cells of tunica media explants were established, and their identity was determined by fluorescent microscopy by using a fluorescein isothiocyanate conjugated anti-SMC alpha-actin antibody. Matrix metalloproteinases (MMPs) produced by SMC monolayers in serum-free culture were examined by gelatin zymography and Western blotting with monoclonal antibodies to MMP-2, 3, and 9. RESULTS Serum-free media from AAA SMCs contained metal-dependent elastolytic activity that cleaved the synthetic substrate succinyl trialanyl 4-nitroanilide (pH optima 7.2) and also 14C-insoluble elastin. The level of proteolytic activity found in these cultures was significantly greater than from control SMC media. Zymography established that AAA SMC media samples contained metal-dependent gelatinases of 50 to 64 and 92 kDa, which were identified respectively as MMP-2 and 9 by Western blotting by using monoclonal antibodies to these proteases. CONCLUSION Medial SMCs isolated from AAA tissue produce significantly higher levels of MMP-9 and 2 than SMCs from control arterial tissues. These proteinases have the capacity to degrade elastin and a range of extracellular matrix proteins. From these data, we suggest SMCs may be involved in the abnormal degradation of the aortic wall in AAA through the excessive metalloproteinase activity produced by SMCs.

The pathobiology of osteoarthritis and the rationale for the use of pentosan polysulfate for its treatment

OBJECTIVES Structure-modifying osteoarthritis (OA) drugs (SMOADs) may be defined as agents that reverse, retard, or stabilize the underlying pathology of OA, thereby providing symptomatic relief in the long-term. The objective of this review was to evaluate the literature on sodium pentosan polysulfate (NaPPS) and calcium pentosan polysulfate (CaPPS), with respect to the pathobiology of OA to ascertain whether these agents should be classified as SMOADs. METHODS Published studies on NaPPS and CaPPS were selected on the basis of their relevance to the known pathobiology of OA, which also was reviewed. RESULTS Both NaPPS and CaPPS exhibit a wide range of pharmacological activities. Of significance was the ability of these agents to support chondrocyte anabolic activities and attenuate catabolic events responsible for loss of components of the cartilage extracellular matrix in OA joints. Although some of the anti-catabolic activities may be mediated through direct enzyme inhibition, NaPPS and CaPPS also have been shown to enter chondrocytes and bind to promoter proteins and alter gene expression of matrix metalloproteinases and possibly other mediators. In rat models of arthritis, NaPPS and CaPPS reduced joint swelling and inflammatory mediator levels in pouch fluids. Moreover, synoviocyte biosynthesis of high-molecular-weight hyaluronan, which is diminished in OA, was normalized when these cells were incubated with NaPPS and CaPPS or after intraarticular injection of NaPPS into arthritic joints. In rabbit, canine, and ovine models of OA, NaPPS and CaPPS preserved cartilage integrity, proteoglycan synthesis, and reduced matrix metalloproteinase activity. NaPPS and CaPPS stimulated the release of tissue plasminogen activator (t-PA), superoxide dismutase, and lipases from vascular endothelium while concomitantly decreasing plasma levels of the endogenous plasminogen activator inhibitor PAI-1. The net thrombolytic and lipolytic effects exhibited by NaPPS and CaPPS may serve to improve blood flow through subchondral capillaries of OA joints and improve bone cell nutrition. In geriatric OA dogs, NaPPS and CaPPS reduced symptoms, as well as normalized their thrombolytic status, threshold for platelet activation, and plasma triglyceride levels. These hematologic parameters were shown to be abnormal in OA animals before drug treatment. Similar outcomes were observed in OA patients when CaPPS or NaPPS were given orally or parenterally in both open and double-blind trials. CONCLUSIONS The data presented in this review support the contention that NaPPS and CaPPS should be classified as SMOADs. However, additional long-term clinical studies employing methods of assessing joint structural changes will be needed to confirm this view.

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.

Variation in proteoglycan metabolism by articular chondrocytes in different joint regions is determined by post-natal mechanical loading.

In this study we investigated the hypothesis that cartilage from defined regions of ovine stifle joints, which were subjected to differing mechanical stresses, contained phenotypically distinct chondrocyte populations. Chondrocyte phenotypes were identified by the relative biosynthesis of the proteoglycans (PGs) aggrecan, biglycan and decorin. Articular cartilage (AC) from adult and neonatal ovine stifle joints were examined. Cells were cultured as both full-depth AC explants and in alginate beads after their isolation from the AC matrix. When chondrocytes from the various topographical regions of adult ovine knee joints were cultured as explants they demonstrated a consistent difference with regard to the metabolism of aggrecan and decorin. Significantly, this topographically-dependent phenotypic expression of PGs was preserved when the chondrocytes were cultured in alginate beads. In adult joints, chondrocytes from the central region of the tibial plateau not covered by the meniscus, which is subjected to high mechanical loads in-vivo, synthesized less aggrecan but more decorin than cells from regions covered by the meniscus. When chondrocytes from identical AC regions of neonatal ovine joints were cultured as explants, no topographical difference in aggrecan nor decorin metabolism could be detected. The results of this study, in association with the existing literature, lead us to propose that post-natal mechanical loading of AC could select for chondrocyte clones or induce a lasting modulation of chondrocyte phenotypic expression in different joint regions. Such cellular changes could result in the synthesis of PG populations that confer properties to AC most suited to resist the variable mechanical stresses in the different joint regions. This study serves to emphasize the importance of using cartilage from identical joint areas when examining PG metabolism by chondrocytes. Further investigation into the relationship between mechanical loading, regional chondrocyte phenotype selection and the response of these cells to anabolic and catabolic factors may provide important insights into the focal nature of AC degeneration in osteoarthritis.

An injectable hydrogel incorporating mesenchymal precursor cells and pentosan polysulphate for intervertebral disc regeneration

Intervertebral disc (IVD) degeneration is one of the leading causes of lower back pain and a major health problem worldwide. Current surgical treatments include excision or immobilisation, with neither approach resulting in the repair of the degenerative disc. As such, a tissue engineering-based approach in which stem cells, coupled with an advanced delivery system, could overcome this deficiency and lead to a therapy that encourages functional fibrocartilage generation in the IVD. In this study, we have developed an injectable hydrogel system based on enzymatically-crosslinked polyethylene glycol and hyaluronic acid. We examined the effects of adding pentosan polysulphate (PPS), a synthetic glycosaminoglycan-like factor that has previously been shown (in vitro and in vivo) to this gel system in order to induce chondrogenesis in mesenchymal precursor cells (MPCs) when added as a soluble factor, even in the absence of additional growth factors such as TGF-β. We show that both the gelation rate and mechanical strength of the resulting hydrogels can be tuned in order to optimise the conditions required to produce gels with the desired combination of properties for an IVD scaffold. Human immunoselected STRO-1+ MPCs were then incorporated into the hydrogels. They were shown to retain good viability after both the initial formation of the gel and for longer-term culture periods in vitro. Furthermore, MPC/hydrogel composites formed cartilage-like tissue which was significantly enhanced by the incorporation of PPS into the hydrogels, particularly with respect to the deposition of type-II-collagen. Finally, using a wild-type rat subcutaneous implantation model, we examined the extent of any immune reaction and confirmed that this matrix is well tolerated by the host. Together these data provide evidence that such a system has significant potential as both a delivery vehicle for MPCs and as a matrix for fibrocartilage tissue engineering applications.

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.