Characterization of catabolin, the major product of pig synovial tissue that induces resorption of cartilage proteoglycan in vitro

1. Pig synovium in organ culture produces material which induces living cartilage to resorb its proteoglycan in vitro. 2. The bioassay for this material was to measure glycosaminoglycan released from explants of bovine nasal-septal cartilage cultured for 8 days. The performance of the assay was greatly improved by adding cortisol succinate (0.1μg/ml). This decreased the release of glycosaminoglycan from unstimulated cartilage without inhibiting its response to catabolic factors from the synovium. 3. By using this improved assay it was shown that 90% of the active materials in synovial culture medium were retained by dialysis membrane. 4. An active protein was partially purified from synovial culture medium by (NH₄)₂SO₄ precipitation, ion-exchange chromatography, gel filtration and preparative isoelectric focusing. 5. This protein, called catabolin, had mol.wt. 17000 and pI4.6. 6. Synovial culture medium concentrated in dialysis tubing was subjected to gel chromatography and found to contain one major active component, which was eluted at the same position as the partially purified catabolin. 7. The synovial culture medium was not inactivated by heating (70°C for 10min), nor were diluted preparations of partially purified catabolin, but concentrated crude preparations were thermolabile. 8. These results suggest that catabolin is the major substance produced by the synovial tissue in culture which induces resorption of proteoglycan of living cartilage in vitro. 9. Other cultured soft connective tissues produced catabolin-like activity. The example of sclera is shown, and production was inhibited by cortisol succinate (0.1μg/ml). 10. It is suggested that catabolin may be a general product of soft connective tissues in culture, and its physiological function may be to induce resorption of connective-tissue matrix after injury.     

Effects of copper and zinc on proteoglycan metabolism in articular cartilage

Co-Cultures of porcine articular cartilage and synovium or synovial conditioned medium were used as an in vitro model to mimic inflammatory events at the cartilage/synovial junction in degenerative joint disease. This model provides a useful tool to assess the anti-inflammatory and antiarthritic properties of pharmacological agents. In this study the effects of copper and zinc on (i) PG synthesis by cartilage and (ii) synovial-induced PG depletion have been investigated. Copper sulphate at a concentration of 0.01 mM did not stimulate PG synthesis significantly in cultured cartilage explants but completely abrogated the inhibitory effects of synovial tissue in co-culture experiments. This finding was supported by the histological demonstration of copper-dependent reversal of the PG depletion in cartilage exposed to synovial conditioned medium. Zinc sulphate at 0.01 mM had no effect on PG synthesis and was unable to protect cartilage against synovialinduced PG depletion. These results reveal possible mechanisms by which copper exerts its anti-inflammatory and anti-arthritic actions.     

Characterization of proteins from human synovium and mononuclear leucocytes that induce resorption of cartilage proteoglycan in vitro

Both human synovial tissue in culture and lectin-stimulated mononuclear leucocytes produced a protein that induced proteoglycan resorption in explants of bovine nasal cartilage and human articular cartilage. On gel filtration the protein had Mr 16000-20000 and on isoelectric focusing its pI was 5.2-5.3. The protein corresponded to catabolin, which has previously been identified as a product of cultured porcine synovial tissue and mononuclear leucocytes. The action of partially purified human catabolin was not inhibited by cortisol, although the activity of the leucocyte supernatants from which it had been isolated was inhibited. For this reason it is not possible to be sure that the active factor detected in the bioassay of the crude leucocyte culture supernatants is in fact catabolin.     

A tissue-culture model of cartilage breakdown in rheumatoid arthritis. Quantitative aspects of proteoglycan release

1. The destruction of articular cartilage in human rheumatoid and other arthritides is the result of diverse mechanical, inflammatory and local cellular factors. A tissue-culture model for studying cartilage-synovial interactions that may be involved in the final common pathway of joint destruction is described. 2. Matrix breakdown was studied in vitro by using bovine nasal-cartilage discs cultivated in contact with synovium. Synovia were obtained from human and animal sources. Human tissue came from patients with ;classical' rheumatoid arthritis, and animal tissue from rabbits with antigen-induced arthritis. 3. Cartilage discs increased their proteoglycan content 2-3-fold during 8 days in culture. Proteoglycan was also released into culture medium, approx. 70% arising from cartilage breakdown. 4. Synovial explants from human rheumatoid and rabbit antigen-induced arthritis produced equivalent stimulation of proteoglycan release. After an initial lag phase, the breakdown rate rose abruptly to a maximum, resulting in a 2-fold increase of proteoglycan accumulation in culture medium after 8-10 days. 5. High-molecular-weight products shed into culture media were characterized chromatographically and by differential enzymic digestion. Proteoglycan-chondroitin sulphate accounted for 90% of the released polyanion, and its partial degradation in the presence of synovial explants was consistent with limited proteolytic cleavage. 6. Rheumatoid synovium applied to dead cartilage increased the basal rate of proteoglycan release. Living cartilage was capable of more extensive autolysis, even in the absence of synovium. However, optimal proteoglycan release required the interaction of living synovium with live cartilage. These findings support the view that a significant component of cartilage breakdown may be chondrocyte-mediated.     

Enzymatic activity and distribution of phospholipase A2 in human cartilage

Extracellular phospholipase A2 (PLA2) with proinflammatory activity has recently been discovered in synovial fluids in inflammatory arthritides. In the search for the sources of synovial fluid PLA2, human synovium and articular cartilage were found to contain large quantities of the enzyme. In rheumatoid arthritis (RA), PLA2 activity in synovium, superficial and deep layers of articular cartilage was 20 +/- 14 (SEM), 168 +/- 62 and 533 +/- 176 nmol/min/mg protein respectively. Corresponding values in osteoarthritis (OA) were 49 +/- 11, 569 +/- 109 and 1709 +/- 243 nmol/min/mg protein, all significantly higher (p less than .01) than in RA. Nasal septal cartilage contained much less PLA2, 19 +/- 5.6. PLA2 in human articular and nasal cartilage has sn-2 specificity, a neutral pH optimum and absolute calcium dependence. High PLA2 concentration in articular cartilage may imply that, at least in part, cartilage is the source of PLA2 in the joint space. Since RA cartilage and synovium have less PLA2 activity than the corresponding OA tissues, additional sources of PLA2 in RA synovial fluids are implicated.     

Stimulation of the synthesis of collagenase activator protein in cartilage by a factor present in synovial-conditioned medium

We have purified a low molecular weight protein from medium conditioned by calf synovium with physical and biological properties similar to the leukocyte cytokine interleukin 1 (IL-1). The factor is active in stimulating the synthesis (three- to fivefold) of collagenase activator protein (CAP) by the surface (1-2 mm) of articular cartilage while CAP synthesis in the deeper zones of articular cartilage is not affected. Recombinant mouse IL-1 and commercially available purified human IL-1 are also capable of stimulating cartilage to synthesize and secrete CAP. The synthesis of other proteins, including collagenase, appeared to be unaffected by either the synovial factors or the human and mouse IL-1.     

Effects of short term ether and pentobarbital anesthesia on bone and cartilage metabolism.

The effects of stress due to brief (4--5 min) ether and pentobarbital anesthesia vs. decapitation on assays of seven enzymes in homogenates of synovium, articular and epiphyseal cartilage, and metaphyseal and cortical bone were compared. Etherization caused twofold changes in synovial and articular cartilage G-6-PDH, LDH, CPK, glutamic DH, and ICDH based on tissue dry weight and DNA content. Pentobarbital anesthesia produced only slightly lower activities, per gram DNA, of LDH, acid phosphatase, and glutamic-DH in cortical bone. Epiphyseal cartilage metabolism was unaffected by either mode of anesthesia. No difference could be detected between levels of enzyme activities of the several tissues taken from rats that had been decapitated or anesthetized with pentobarbital. The changes in enzyme activities suggested that pentobarbital was non-stressful and appropriate to metabolic studies in the skeleton.     

The effects of proteolytic enzymes on the mechanical properties of adult human articular cartilage.

The effects of the lysosomal proteinase cathepsin D on the mechanical properties of adult human articular cartilage were examined in detail in 7 joints within the age range 21 to 72 years. The results of a preliminary study on the effects of the lysosomal proteinase cathepsin B1 and clostridial collagenase on the mechanical properties of cartilage are also presented. Cartilage which had been incubated with either cathepsin D or cathepsin B1 showed increased deformation in uniaxial compression perpendicular to the articular surface. The enzyme-treated cartilage also showed decreased tensile stiffness at low values of stress. This effect was more pronounced in specimens from the deeper zone of cartilage than in specimens from the superficial zone. It was also more pronounced in specimens which were aligned perpendicular to the predominant alignment of the collagen fibres in the superficial zone than in specimens which were parallel to the collagen fibres. At higher stresses the tensile stiffness of the treated cartilage was not significantly different from that of the untreated tissue. The tensile fracture stress of the cartilage was also not significantly reduced by the action of cathepsin D. In contrast to the effects observed with the cathepsins, the preliminary results obtained by incubating cartilage for 24 h with clostridial collagenase showed that both the tensile stiffness and the fracture stress were considerably lower than the corresponding values for the untreated tissue. Biochemical analysis of the incubation media, and the specimens, revealed that a large proportion of the proteoglycans was released from the cartilage by each of the three enzymes. The proportion of the total collagen which was released from the cartilage was different for each enzyme: cathepsin D released between 0 and 1.5 per cent, cathepsin B1 released between 2.3 and 4.3 per cent and collagenase released between 5.3 and 27.8 per cent of the collagen after 24 h.     

Doublecortin is expressed in articular chondrocytes

Articular cartilage and cartilage in the embryonic cartilaginous anlagen and growth plates are both hyaline cartilages. In this study, we found that doublecortin (DCX) was expressed in articular chondrocytes but not in chondrocytes from the cartilaginous anlagen or growth plates. DCX was expressed by the cells in the chondrogenous layers but not intermediate layer of joint interzone. Furthermore, the synovium and cruciate ligaments were DCX-negative. DCX-positive chondrocytes were very rare in tissue engineered cartilage derived from in vitro pellet culture of rat chondrosarcoma, ATDC5, and C3H10T1/2 cells. However, the new hyaline cartilage formed in rabbit knee defect contained mostly DCX-positive chondrocytes. Our results demonstrate that DCX can be used as a marker to distinguish articular chondrocytes from other chondrocytes and to evaluate the quality of tissue engineered or regenerated cartilage in terms of their "articular" or "non-articular" nature.     

Identification of link proteins in canine synovial cell cultures and canine articular cartilage

Link proteins are glycoproteins in cartilage that are involved in the stabilization of aggregates of proteoglycans and hyaluronic acid. We have identified link proteins in synovial cell cultures form normal canine synovium using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunofluorescence, and immunolocation with specific antibodies by electrophoretic transfer. We have also found evidence for the synthesis of link proteins in these cultures by fluorography of radiolabeled synovial cell extracts. We have identified a 70,000 mol-wt protein in canine synovial cell culture extracts that has antigenic cross-reactivity with the 48,000-mol-wt link protein. Three link proteins were identified in normal canine articular cartilage. These results indicate that link proteins are more widely distributed in connective tissues than previously recognized and may have biological functions other than aggregate stabilization.     

Topographic localization of a 116,000-dalton protein in cartilage

A disulfide-bonded greater than 400,000-dalton (greater than 400-kD) protein with 116-kD subunits in hyaline cartilage from several species has recently been described. It constitutes 2-4% of the total noncollagenous protein in 4 M guanidinium chloride extracts of normal articular cartilage and accounts for most of the total noncollagen, nonproteoglycan protein synthesized in short-term organ cultures of canine articular cartilage. In the present study, immunofluorescence techniques were used to examine the topographic distribution of the 116-kD subunit protein in normal cartilage. In specimens of normal adult articular cartilage from several species, the protein was located throughout the matrix. More intense staining was observed at the articular surface than in the remainder of the uncalcified cartilage. In contrast, in fetal cartilage, the protein was uniformly distributed throughout the matrix without a marked increase in surface staining. Normal canine menisci and annulus fibrosus also demonstrated moderate fluorescence after incubation with the antiserum to the 116-kD subunit protein. Normal canine nucleus pulposus, synovium, aorta, and monolayer cultures of canine synovial cells exhibited only weak immunofluorescence after incubation with the antiserum. Therefore, the 116-kD subunit protein appears to be a ubiquitous matrix protein in cartilage.     

Identification of catabolin,a protein from synovium which induces degradation of cartilage in organ culture

Explants of porcine synovium produce a factor which causes degradation of the matrix of live cartilage in organ culture. Cartilage degradation was measured as release of glycosaminoglycan from explants of bovine nasal septum. Fractionation of synovial culture medium showed the factor to be a protein of 20,000 mol.wt. and iso-electric point pH = 4.6. The factor has been named catabolin.     

ICAM-1 expression on chondrocytes in rheumatoid arthritis: induction by synovial cytokines

The intercellular adhesion molecule-1 (ICAM-1) was found by immunostaining chondrocytes in cartilage from three patients with rheumatoid arthritis. Expression of ICAM-1 was restricted to chondrocytes in areas of erodedcartilage adjacent to the invading synovial tissue. Toluidine blue staining of these areas demonstrated severe depletion of the cartilage extracellular matrix. In areas of undamaged cartilage there was no ICAM-1 expression. Since ICAM-1 is not constitutively expressed on normal human articular cartilage, but could be induced in vitro by exogenous IL-1alpha, TNFalpha and IFNgamma or by co-culturing cartilage with inflammatory rheumatoid synovium, we conclude that the induction of ICAM-1 on rheumatoid chondrocytes results from the synergistic action of a variety of cytokines produced by the inflammatory cells of the invading pannus.     

MicroRNA-146a is linked to pain-related pathophysiology of osteoarthritis

Because miR-146a is linked to osteoarthritis (OA) and cartilage degeneration is associated with pain, we have characterized the functional role of miR-146a in the regulation of human articular cartilage homeostasis and pain-related factors. Expression of miRNA 146a was analyzed in human articular cartilage and synovium, as well as in dorsal root ganglia (DRG) and spinal cord from a rat model for OA-related pain assessment. The functional effects of miR-146a on human chondrocytic, synovial, and microglia cells were studied in cells transfected with miR-146a. Using real-time PCR, we assessed the expression of chondrocyte metabolism-related genes in chondrocytes, genes for inflammatory factors in synovial cells, as well as pain-related proteins and ion channels in microglial cells. Previous studies showed that miR-146a is significantly upregulated in human peripheral knee OA joint tissues. Transfection of synthetic miR-146a significantly suppresses extracellular matrix-associated proteins (e.g., Aggrecan, MMP-13, ADAMTS-5, collagen II) in human knee joint chondrocytes and regulates inflammatory cytokines in synovial cells from human knee joints. In contrast, miR-146a is expressed at reduced levels in DRGs and dorsal horn of the spinal cords isolated from rats experiencing OA-induced pain. Exogenous supplementation of synthetic miR-146a significantly modulates inflammatory cytokines and pain-related molecules (e.g., TNFα, COX-2, iNOS, IL-6, IL8, RANTS and ion channel, TRPV1) in human glial cells. Our findings suggest that miR-146a controls knee joint homeostasis and OA-associated algesia by balancing inflammatory responses in cartilage and synovium with pain-related factors in glial cells. Hence, miR-146a may be useful for the treatment of both cartilage regeneration and pain symptoms caused by OA.     

Toward regeneration of articular cartilage

Articular cartilage is classified as permanent hyaline cartilage and has significant differences in structure, extracelluar matrix components, gene expression profile, and mechanical property from transient hyaline cartilage found in the epiphyseal growth plate. In the process of synovial joint development, articular cartilage originates from the interzone, developing at the edge of the cartilaginous anlagen, and establishes zonal structure over time and supports smooth movement of the synovial joint through life. The cascade actions of key regulators, such as Wnts, GDF5, Erg, and PTHLH, coordinate sequential steps of articular cartilage formation. Articular chondrocytes are restrictedly controlled not to differentiate into a hypertrophic stage by autocrine and paracrine factors and extracellular matrix microenvironment, but retain potential to undergo hypertrophy. The basal calcified zone of articular cartilage is connected with subchondral bone, but not invaded by blood vessels nor replaced by bone, which is highly contrasted with the growth plate. Articular cartilage has limited regenerative capacity, but likely possesses and potentially uses intrinsic stem cell source in the superficial layer, Ranvier's groove, the intra‐articular tissues such as synovium and fat pad, and marrow below the subchondral bone. Considering the biological views on articular cartilage, several important points are raised for regeneration of articular cartilage. We should evaluate the nature of regenerated cartilage as permanent hyaline cartilage and not just hyaline cartilage. We should study how a hypertrophic phenotype of transplanted cells can be lastingly suppressed in regenerating tissue. Furthermore, we should develop the methods and reagents to activate recruitment of intrinsic stem/progenitor cells into the damaged site. Birth Defects Research (Part C) 99:192–202, 2013 . © 2013 Wiley Periodicals, Inc .     

Passive loss of proteoglycan from articular cartilage explants

The addition of proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 0.25 mM benzamidine hydrochloride, 6.25 mM EDTA, 12.5 mM 6-aminohexanoic acid and 2 mM iodoacetic acid) to explant cultures of adult bovine articular cartilage inhibits proteoglycan synthesis as well as the loss of the macromolecule from the tissue. Those proteoglycans lost to the medium of explant cultures treated with proteinase inhibitors were either aggregates or monomers with functional hyaluronic acid-binding regions, whereas proteoglycans lost from metabolically active tissue also included a population of monomers that were unable to aggregate with hyaluronate. Analysis of the core protein from proteoglycans lost into the medium of inhibitor-treated cultures showed the same size distribution as the core proteins of proteoglycans present in the extracellular matrix of metabolically active cultures. The core proteins of proteoglycans appearing in the medium of metabolically active cultures showed that proteolytic cleavage of these macromolecules occurred as a result of their loss from the tissue. Explant cultures of articular cartilage maintained in medium with proteinase inhibitors were used to investigate the passive loss of proteoglycan from the tissue. The rate of passive loss of proteoglycan from the tissue was dependent on surface area, but no difference in the proportion of proteoglycan aggregate to monomer appearing in the medium was observed. Furthermore, proteoglycans were lost at the same rate from the articular and cut surfaces of cartilage. Proteoglycan aggregates and monomer were lost from articular cartilage over a period of time, which indicates that proteoglycans are free to move through the extracellular matrix of cartilage. The movement of proteoglycans out of the tissue was shown to be temperature dependent, but was different from the change of the viscosity of water with temperature, which indicates that the loss of proteoglycan was not solely due to diffusion. The activation energy for the loss of proteoglycans from articular cartilage was found to be similar to the binding energies for electrostatic and hydrogen bonds.     

The effect of retinoic acid on proteoglycan biosynthesis in bovine articular cartilage cultures

The addition of retinoic acid to adult bovine articular cartilage cultures produces a concentration-dependent decrease in both proteoglycan synthesis and the proteoglycan content of the tissue. Total protein synthesis was not affected by the presence of retinoic acid, indicating that the inhibition of proteoglycan synthesis was not due to cytotoxicity. The proteoglycans synthesized in the presence of retinoic acid were similar in hydrodynamic size, ability to form aggregates with hyaluronate, and glycosaminoglycan composition to those of control cultures. However, the presence of larger glycosaminoglycan chains suggests that the core protein was substituted with fewer but longer glycosaminoglycan chains. In cultures maintained with retinoic acid, a decreased ratio of the large proteoglycan was synthesized relative to the small proteoglycan compared to that measured in control cultures. In cultures maintained with retinoic acid for 1 day and then switched to medium with 20% (v/v) fetal calf serum, the rate of proteoglycan synthesis and hexuronate contents increased within 5 days to levels near those of control cultures. Within 2 days of switching to medium with 20% (v/v) fetal calf serum, the relative proportions of the proteoglycan species were similar to those produced in cultures maintained in medium with 20% (v/v) fetal calf serum throughout. The rate of proteoglycan synthesis by bovine articular cartilage cultures exhibited an exponential decay following exposure to retinoic acid, with estimated half-lives of 11.5 and 5.3 h for tissue previously maintained in medium alone or containing 20% (v/v) fetal calf serum, respectively. The addition of 1 mM benzyl beta-D-xyloside only partially reversed the retinoic acid-mediated inhibition of proteoglycan synthesis. This indicates that the inhibition of proteoglycan synthesis by retinoic acid was due to both a decreased availability of xylosylated core protein and a decreased capacity of the chondrocytes to synthesize chondroitin sulfate chains.     

Soluble recombinant neprilysin induces aggrecanase-mediated cleavage of aggrecan in cartilage explant cultures.

Neprilysin (neutral endopeptidase, enkephalinase, CALLA, CD10, NEP) is a regulatory Zn metallopeptidase expressed in the brush border membranes of the kidney and has been found in porcine chondrocytes and rat articular cartilage as well as other cell types and tissues. Although its function in cartilage is not currently known, previous observations of high levels of NEP enzymatic activity in the synovial fluid of arthritic patients and on the chondrocyte membranes of human osteoarthritic cartilage have led to the hypothesis that NEP is involved in the inflammation or degradation pathways in articular cartilage. Our study localized endogenous NEP to the membranes of mature bovine articular chondrocytes in a tissue explant model and demonstrated that the addition of soluble recombinant NEP (sNEP) to the culture medium of bovine cartilage explants leads to the degradation of aggrecan through the action of aggrecanase. A 6-day exposure to sNEP was necessary to initiate the degradation, suggesting that the chondrocytes were responding in a delayed manner to an altered composition of regulatory peptides. This NEP-induced degradation was completely inhibited by the NEP inhibitors thiorphan and phosphoramidon. These results suggest that NEP is present as a transmembrane enzyme on articular chondrocytes where it can cleave regulatory peptides and lead to the induction of aggrecanase.     

Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction.

Rheumatoid arthritis is a chronic inflammatory joint disease, leading to cartilage and bone destruction. In this study, we investigated the effects of local IL-4 application, introduced by a recombinant human type 5 adenovirus vector, in the knee joint of mice with collagen-induced arthritis. One intraarticular injection with an IL-4-expressing virus caused overexpression of IL-4 in the mouse knee joint. Enhanced onset and aggravation of the synovial inflammation were found in the IL-4 group. However, despite ongoing inflammation, histologic analysis showed impressive prevention of chondrocyte death and cartilage erosion. In line with this, chondrocyte proteoglycan synthesis was enhanced in the articular cartilage. This was quantified with ex vivo 35S-sulfate incorporation in patellar cartilage and confirmed by autoradiography on whole knee joint sections. Reduction of cartilage erosion was further substantiated by lack of expression of the stromelysin-dependent cartilage proteoglycan breakdown neoepitope VDIPEN in the Ad5E1 mIL-4-treated knee joint. Reduced metalloproteinase activity was also supported by markedly diminished mRNA expression of stromelysin-3 in the synovial tissue. Histologic analysis revealed marked reduction of polymorphonuclear cells in the synovial joint space in the IL-4-treated joints. This was confirmed by immunolocalization studies on knee joint sections using NIMP-R14 staining and diminished mRNA expression of macrophage-inflammatory protein-2 in the synovium tissue. mRNA levels of TNF-alpha and IL-1beta were suppressed as well, and IL-1beta and nitric oxide production by arthritic synovial tissue were strongly reduced. Our data show an impressive cartilage-protective effect of local IL-4 and underline the feasibility of local gene therapy with this cytokine in arthritis.     

Hyaluronan injection in murine osteoarthritis prevents TGFbeta 1-induced synovial neovascularization and fibrosis and maintains articular cartilage integrity by a CD44-dependent mechanism

Introduction

The mechanism by which intra-articular injection of hyaluronan (HA) ameliorates joint pathology is unknown. Animal studies have shown that HA can reduce synovial activation, periarticular fibrosis and cartilage erosion; however, its specific effects on the different cell types involved remain unclear. We have used the TTR (TGFbeta1 injection and Treadmill Running) model of murine osteoarthritis (OA), which exhibits many OA-like changes, including synovial activation, to examine in vivo tissue-specific effects of intra-articular HA.

Methods

The kinetics of clearance of fluorotagged HA from joints was examined with whole-body imaging. Naïve and treated knee joints were examined macroscopically for cartilage erosion, meniscal damage and fibrosis. Quantitative histopathology was done with Safranin O for cartilage and with Hematoxylin & Eosin for synovium. Gene expression in joint tissues for Acan, Col1a1, Col2a1, Col3a1, Col5a1, Col10a1, Adamts5 and Mmp13 was done by quantitative PCR. The abundance and distribution of aggrecan, collagen types I, II, III, V and X, ADAMTS5 and MMP13 were examined by immunohistochemistry.

Results

Injected HA showed a half-life of less than 2 h in the murine knee joint. At the tissue level, HA protected against neovascularization and fibrosis of the meniscus/synovium and maintained articular cartilage integrity in wild-type but not in Cd44 knockout mice. HA injection enhanced the expression of chondrogenic genes and proteins and blocked that of fibrogenic/degradative genes and proteins in cartilage/subchondral bone, whereas it blocked activation of both groups in meniscus/synovium. In all locations it reduced the expression/protein for Mmp13 and blocked Adamts5 expression but not its protein abundance in the synovial lining.

Conclusions

The injection of HA, 24 h after TGFbeta1 injection, inhibited the cascade of OA-like joint changes seen after treadmill use in the TTR model of OA. In terms of mechanism, tissue protection by HA injection was abrogated by Cd44 ablation, suggesting that interaction of the injected HA with CD44 is central to its protective effects on joint tissue remodeling and degeneration in OA progression.