Daniel Burkhardt

Matrix metalloproteinase 13–deficient mice are resistant to osteoarthritic cartilage erosion but not chondrocyte hypertrophy or osteophyte development

OBJECTIVE To investigate the role of matrix metalloproteinase 13 (MMP-13; collagenase 3) in osteoarthritis (OA). METHODS OA was surgically induced in the knees of MMP-13-knockout mice and wild-type mice, and mice were compared. Histologic scoring of femoral and tibial cartilage aggrecan loss (0-3 scale), erosion (0-7 scale), and chondrocyte hypertrophy (0-1 scale), as well as osteophyte size (0-3 scale) and maturity (0-3 scale) was performed. Serial sections were stained for type X collagen and the MMP-generated aggrecan neoepitope DIPEN. RESULTS Following surgery, aggrecan loss and cartilage erosion were more severe in the tibia than femur (P<0.01) and tibial cartilage erosion increased with time (P<0.05) in wild-type mice. Cartilaginous osteophytes were present at 4 weeks and underwent ossification, with size and maturity increasing by 8 weeks (P<0.01). There was no difference between genotypes in aggrecan loss or cartilage erosion at 4 weeks. There was less tibial cartilage erosion in knockout mice than in wild-type mice at 8 weeks (P<0.02). Cartilaginous osteophytes were larger in knockout mice at 4 weeks (P<0.01), but by 8 weeks osteophyte maturity and size were no different from those in wild-type mice. Articular chondrocyte hypertrophy with positive type X collagen and DIPEN staining occurred in both wild-type and knockout mouse joints. CONCLUSION Our findings indicate that structural cartilage damage in a mouse model of OA is dependent on MMP-13 activity. Chondrocyte hypertrophy is not regulated by MMP-13 activity in this model and does not in itself lead to cartilage erosion. MMP-13 deficiency can inhibit cartilage erosion in the presence of aggrecan depletion, supporting the potential for therapeutic intervention in established OA with MMP-13 inhibitors.

The proteoglycans of the canine intervertebral disc

The high-buoyant-density proteoglycans of the nucleus pulposus and annulus fibrosus of the beagle intervertebral disc have been isolated by CsCl density gradient ultracentrifugation. The sulphated proteoglycans were labelled in vivo with 35SO4, 24 h and 60 days prior to killing. The hydrodynamic size and aggregation of the 24 h, 60 day and resident (from hexuronic acid and hexosamine analysis) proteoglycan subunit populations were determined by Sepharose CL-2B chromatography in the presence or absence of excess hyaluronic acid. The hydrodynamic size of the keratan sulphate-proteoglycan core protein complexes were also determined by Sepharose CL-2B chromatography after chondroitinase ABC digestion of proteoglycans. When initially synthesised (24 h) or after 60 days, the percentage aggregation and hydrodynamic size of the proteoglycans derived from the annulus fibrosus were larger than those present in the nucleus pulposus. Hexosamine, hexuronic and protein determination of the high-buoyant-density fractions showed that the proteoglycans of the nucleus pulposus were richer in chondroitin sulphate than those in the annulus. However there was no difference in Mr of the chondroitin sulphate and keratan sulphate attached to the proteoglycans of the two disc regions, nor were differences detected by HPLC between the proportions of chondroitin 4-sulphate and chondroitin 6-sulphate present in these high-density fractions. In contrast, the low-buoyant-density (1.54 greater than p greater than 1.45) proteoglycan fractions and tissue residues remaining after 4 M GuHCl extraction were found to contain dermatan sulphate, suggesting the presence of a third proteoglycan species possibly associated with the collagen of the fibrocartilagenous matrix.

Spinal biomechanics and aging are major determinants of the proteoglycan metabolism of intervertebral disc cells.

Study Design. The proteoglycan metabolism of ovine disc nucleus pulposus and anulus fibrosus cells was investigated in relation to age, spinal level, and intrinsic spinal biomechanical properties. Objective. To evaluate the hypothesis that with aging loss of proteoglycans from the lumbosacral disc exceeds that from upper lumbar discs because of its proximity to a rigid segment, the sacrum. Summary of Background Data. The proteoglycan and associated water of the disc decreases with aging. Methods. Proteoglycans were extracted directly from the disc tissues using 4 M GuHCl and examined by composite agarose polyacrylamide gel electrophoresis. Disc cells were cultured in alginate beads, and their metabolic activity was assessed by 3H-thymidine incorporation into DNA and by bioreduction of a cell proliferation dye. Newly synthesized proteoglycans were radiolabeled with 35S, and their molecular weight distributions and ability to aggregate with hyaluronan were determined by Sephacryl S1000 gel chromatography. Resident proteoglycans extracted from disc tissues with 4 M GuHCl were similarly evaluated. A group of adult animals also were studied biomechanically to evaluate the range of spinal motion (L4/L5 to L7/S1). Results. In contrast to the neonatal proteoglycan samples, the biosynthesis of proteoglycans by nucleus pulposus cells of adult discs increased progressively toward the sacrum. This correlated with increased metabolic activity. Analysis of the resident proteoglycans by composite agarose polyacrylamide gel electrophoresis indicated that although the aggrecan-1 population was present almost exclusively in the neonatal group, it was the aggrecan-2 population that predominated in the adult discs, and it became progressively more heterogeneous with aging and proximity of the disc to the sacrum. Conclusions. The proteoglycans of the lumbosacral disc of adult animals turned over faster than proteoglycans of adjacent lumbar discs. The reduced proteoglycan content and ability to aggregate, particularly in the nucleus pulposus of lumbosacral discs, indicated that proteoglycan catabolism exceeded the rate of biosynthesis. These events in the lumbosacral disc are thought to be determined mechanically by its proximity to the sacrum.

The chondroprotective drugs, arteparon and sodium pentosan polysulphate, increase collagenase activity and inhibit stromelysin activity in vitro

The effects of the chondroprotective drugs, sodium pentosan polysulphate (SP54) and Arteparon (glycosaminoglycan polysulphate), on the in vitro activities of the purified matrix metalloproteinases interstitial collagenase (matrix metalloproteinase 1, MMP1) and stromelysin (MMP3) were examined. Both drugs produced concentration-dependent enhancement of the degradation of type I collagen fibrils by purified human fibroblast collagenase and rat tumour collagenase. Rat collagenase activity was increased by drug concentrations above 0.5 microgram/mL, whereas human collagenase activity was only increased by higher drug concentrations, above 5 micrograms/mL. The concentration dependence of the increase in rat collagenase activity was similar for both drugs, with a maximal 3-fold increase at 50 micrograms/mL. In contrast, human collagenase activity was increased to a greater extent by SP 54 compared to Arteparon, with maximal increases at 5000 micrograms/mL of 6-fold and 2-4-fold, respectively. Both drugs produced concentration-dependent inhibition of the proteoglycan-degrading activity of both human fibroblast stromelysin and rat tumour stromelysin. Rat and human stromelysin activities were inhibited at drug concentrations above 0.005 microgram/mL, with a similar concentration dependence for both drugs. Fifty percent inhibition of rat stromelysin was produced by concentrations of each drug in the 0.5-5 microgram/mL range. The pattern of inhibition of human stromelysin was similar, except that drug concentrations in the 500-5000 micrograms/mL range produced 50% inhibition. The possible modes of action for these drug effects and their possible pharmacological significance are discussed.

Inhibition of synovial fluid lysosomal glycosidases by anti-arthritic gold preparations.

The ability of currently available anti-arthritic gold preparations to inhibit lysosomal glycosidases from rheumatoid synovial fluid and normal human serum was studied in vitro.It was shown that these preparations differ markedly in their ability to inhibit the enzymes. Gold thioglucose (Solganal) did not inhibit β-glucuronidase (β-GLUC), β-N-acetylglucosaminidase (β-NAG) or hyaluronidase (HASE). Chloro(triethylphosphine)gold (SK&F 36914) was a potent inhibitor of β-NAG only. Sodium aurothiomalate (Myochrysine and sodium 3-aurothio-2-propanol-1-sulphonate (Allyochrisine) were inhibitors of all three enzymes, notably β-GLUC.Kinetic analysis of inhibition by aurothiomalate demonstrated apparent competitive inhibition with β-GLUC, but non-competitive inhibition with HASE and β-NAG β-GLUC was also strongly inhibited by silver and copper thiomalates.The concentrations of these drugs required for effective inhibition of lysosomal glycosidases probably exceed those attained in serum and therefore preclude this action extracellularly. It is suggested that durg sequestration and retention within phagocytic cells facilitates inhibition of glycosaminoglycan catabolism that mediates cleavage of glucuronidic linkages of hyaluronic acid and chondroitin sulphates.The hypothesis that gold dompounds act in vivo by attenuating the activity of lysosomal enzymes is discussed in relation to these and previous findings.

Changes in gait after bilateral meniscectomy in sheep: effect of two hyaluronan preparations

BackgroundThis study examined the effect of bilateral meniscectomy on ground reaction forces (GRFs) in sheep, and the therapeutic effect of two hyaluronan (HA) preparations.MethodsEighteen sheep were subjected to bilateral lateral meniscectomy and were treated from 16 to 20 weeks postoperatively with intraarticular Hyalgan (Fidia Farmaceutici), HYADD4-G (a novel amide derivative; Fidia Farmaceutici), or saline placebo (n = 6 per group). GRFs were assessed at baseline and 6, 12, 16, 22, and 26 weeks postoperatively. Rheological parameters and HA content of synovial fluid samples were assessed using micro-Fourier rheometry.ResultsMeniscectomy significantly reduced GRF and abolished the normal two-peak vector. GRF deficits were partially ameliorated by both HA preparations: Hyalgan increased peak vertical forces at 6 weeks post-treatment (week 22), while HYADD4-G increased vertical impulse post-treatment. Both HA treatments, but not saline placebo, restored a twopeak composite force vector at 6 weeks post-treatment. Neither HA preparation significantly modulated osteoarthritis (OA) severity, or synovial fluid parameters.ConclusionsThis study showed that GRF responses to bilateral meniscectomy in sheep mimic available data for human meniscectomy and OA patients. However, this time course suggests that gait deficits are temporally unrelated to observed cartilage or synovial fluid changes. The bilateral ovine meniscectomy model demonstrates modest but quantifiable changes in GRF that mimic human OA and are amenable to modification by known OA therapies such as HA.