High-intensity exercise induces structural, compositional and metabolic changes in cuboidal bones--findings from an equine athlete model.

Fatigue fracture of cuboidal bones occurs in the human foot as well as the equine carpus. The racehorse provides a naturally-occurring model to study the effects of high-intensity exercise on the morphology and metabolism of cuboidal bones. We studied both the mineral and the collagenous matrix of the third (C(3)) and radial (C(r)) carpal bones of raced and non-raced Thoroughbred (TB) horses. We hypothesised that racehorses would show increases in the mineral component of these bones and post-translational modifications of the collagenous matrix alongside changes in markers of collagen remodelling and bone formation. C(3) and C(r) carpal bones were retrieved from raced TB horses (n=14) and non-raced TB horses (n=11). Standardised proximal-distal sections were taken from each bone and these were sliced transversely to study the proximal-distal differences in bone metabolism from the subchondral plate through to trabecular bone. Histomorphometry and bone mineral density measurements were performed in parallel with biochemical analyses including total collagen, collagen synthesis and cross-links, matrix metalloproteinases-2 and 9 and their inhibitors, calcium and phosphate, and bone alkaline phosphatase. The results of this study show that, while there is a net increase in bone formation in the racehorses, there is additionally an increase in bone collagen synthesis and remodelling, particularly within the trabecular regions of the bone. The increase in bone density would lead to greater stiffness, particularly in the cortical bone, and failure of this 'stiffer' cortical bone may result from its lack of support from the rapidly remodelling and structurally weakened underlying trabecular bone.

MT3-MMP (MMP-16) is downregulated by in vitro cytokine stimulation of cartilage, but unaltered in naturally occurring equine osteoarthritis and osteochondrosis.

Matrix degradation by metalloproteinases is considered a key feature in the loss of articular cartilage seen in many joint diseases. Membrane-type matrix metalloproteinase-3 (MT3-MMP) expression is elevated in human cartilage in end-stage osteoarthritis. We investigated whether MT3-MMP is similarly regulated in cartilage in two naturally occurring arthropathies in vivo and whether proinflammatory cytokines regulate its expression in vitro. MT3-MMP expression was evaluated in cartilage from horses with osteoarthritis and osteochondrosis and compared with age- and site-matched normal cartilage. MT3-MMP also was measured in normal cartilage stimulated with proinflammatory cytokines. MT3-MMP expression was not significantly altered in either osteoarthritis or osteochondrosis cartilage. However, gene expression was significantly downregulated by the addition of recombinant human interleukin-1beta, oncostatin M, or tumor necrosis factor-alpha to normal cartilage explants. The results suggest that MT3-MMP may not have a role in matrix destruction in equine cartilage diseases. Further work is required to characterize its regulation and function.

Use of biochemical markers of osteoarthritis to investigate the potential disease-modifying effect of tibial plateau levelling osteotomy.

OBJECTIVES: To evaluate the hypothesis that the concentration of the 1/20/5D4 epitope of keratan sulphate, cartilage oligomeric matrix protein and total sulphated glycosaminoglycans in synovial fluids from dogs with cranial cruciate ligament disease would be affected by tibial plateau levelling osteotomy. In addition, to evaluate the hypothesis that medial meniscal release or meniscal injury would alter the expression of these candidate biomarkers. METHODS: Forty-one dogs with naturally occurring cranial cruciate ligament disease were recruited prospectively. Synovial fluids were collected from the index joint before surgery and six weeks and six months postsurgery. Following tibial plateau levelling osteotomy, synovial fluids were assayed for 1/20/5D4 epitope of keratan sulphate and cartilage oligomeric matrix protein concentration using an inhibition ELISA and for sulphated glycosaminoglycans using a direct dye-binding assay. RESULTS: The sulphated glycosaminoglycans ratio did not change significantly during the study. Medial meniscal injury at entry was associated with lower concentrations of synovial fluid cartilage oligomeric matrix protein (P<0.05, unpaired t test). There was no association between medial meniscal release and the changes in marker concentrations, either from 0 to six weeks or 0 to six months. CLINICAL SIGNIFICANCE: Tibial plateau levelling osteotomy did not significantly alter the expression of the named candidate biomarkers. These findings reflect the limited nature of the arthrotomy or indicate that tibial plateau levelling osteotomy does not influence the progression of osteoarthritis (OA). From these studies, there is no evidence that tibial plateau levelling osteotomy affects cartilage metabolism.

The open reading frame of the Na(+)-dependent glutamate transporter GLAST-1 is expressed in bone and a splice variant of this molecule is expressed in bone and brain.

Previously we have reported expression of an mRNA with homology to the Na(+)-dependent glutamate transporter, GLAST-1, in bone. Here we demonstrate that the complete open reading frame of GLAST-1 mRNA and corresponding 69 kDa protein are expressed in rat bone in vivo. We have also discovered a novel splice variant (GLAST-1a), lacking exon 3, expressed in rat bone and brain. A 55 kDa protein detected by anti-GLAST antibody in rat cerebellum corresponds to the molecular weight of unglycosylated GLAST-1a. This has led us to propose that GLAST-1a has an opposite orientation in the cell membrane to GLAST-1.

Elevated levels of proteolytic enzymes in the aging human vitreous.

PURPOSE: To identify whether aging of human vitreous is accompanied by an elevation in degradative enzymes within the tissue. METHODS: Human vitreous samples from donors aged 10 to 88 years were placed in two groups based on donor age of less than or more than 50 years. Homogenized samples were analyzed by gelatin substrate zymography for matrix metalloproteinases (MMP). Serine proteinases were detected by casein substrate zymography, and a specific antibody was used to confirm the identity of, and to quantify, the serine proteinase, plasmin. RESULTS: Progelatinase A (ProMMP-2) was present in all the vitreous samples but did not show an age-related increase. MMP-2 was also present at low levels. Progelatinase B (ProMMP-9) was found in approximately 80% of samples analyzed, but neither its presence nor level of activity was age dependent. Of the serine proteinase activities detected, an enzyme of approximately 80 kDa was identified by Western blot analysis as plasmin(ogen). Quantitative analysis revealed a significant increase in plasmin(ogen) with age. CONCLUSIONS: This study shows there is an age-related increase in potential degradative activity in human vitreous that may be responsible for degenerative changes such as vitreous liquefaction. The data suggest that increased levels of these enzymes precede, or are indicative of, underlying ocular disease in some individuals.

Evaluation of cartilage, synovium and adipose tissue as cellular sources for osteochondral repair.

Osteochondral lesions are a major cause of pain and disability in several species including dogs, horses and human beings. The objective of this study was to assess three potential sources of canine cells for their osteochondral regenerative potential. Cartilage, synovium and adipose tissue cells were grown in pellet culture in chondrogenic or osteogenic media. Cartilage-derived pellets displayed the best chondrogenic differentiation as indicated by significantly higher COL2A1 and SOX9 mRNA expression, greater glycosaminoglycan content, and higher retention of Safranin-O stain compared to the synovium and adipose-derived cells. Following application of the osteogenic media, all three cell sources exhibited small areas of positive alizarin red staining. Poor intracellular alkaline phosphatase activity was found in all three cell types when stimulated although osteocalcin and RUNX2 expression were significantly increased. Cells isolated and cultured from canine articular cartilage retained their specific chondrocytic phenotype. Furthermore, canine adipocytes and synovial cells did not undergo chondrogenic differentiation and did not exhibit evidence of multipotency. Although osteogenic differentiation was initiated at a genomic level, phenotypic osteoblastic differentiation was not observed. The findings of this study suggest that cells isolated from canine adipose tissue and synovium are sub-optimal substitutes for chondrocytes when engineering articular cartilage in vitro.

Variations in cell morphology in the canine cruciate ligament complex.

Cell morphology may reflect the mechanical environment of tissues and influence tissue physiology and response to injury. Normal cruciate ligaments (CLs) from disease-free stifle joints were harvested from dog breeds with a high (Labrador retriever) and low (Greyhound) risk of cranial cruciate ligament (CCL) rupture. Antibodies against the cytoskeletal components vimentin and alpha tubulin were used to analyse cell morphology; nuclei were stained with 4',6-diamidino-2-phenylindole, and images were collected using conventional and confocal microscopy. Both cranial and caudal CLs contained cells of heterogenous morphologies. Cells were arranged between collagen bundles and frequently had cytoplasmic processes. Some of these processes were long (type A cells), others were shorter, thicker and more branched (type B cells), and some had no processes (type C cells). Processes were frequently shown to contact other cells, extending longitudinally and transversely through the CLs. Cells with longer processes had fusiform nuclei, and those with no processes had rounded nuclei and were more frequent in the mid-substance of both CLs. Cells with long processes were more commonly noted in the CLs of the Greyhound. As contact between cells may facilitate direct communication, variances in cell morphology between breeds at a differing risk of CCL rupture may reflect differences in CL physiology.

Chondrocytes harvested from osteochondritis dissecans cartilage are able to undergo limited in vitro chondrogenesis despite having perturbations of cell phenotype in vivo.

Our objective was to characterize the variation in gene expression for key genes associated with chondrogenic phenotype of osteochondrosis (OC)-affected and normal chondrocytes, and to identify whether OC chondrocytes can redifferentiate and regain a phenotype similar to normal chondrocytes if appropriate chondrogenic signals are given. Equine articular cartilage removed at surgery to treat clinically significant OC lesions was collected (n = 10), and the gene expression evaluated and compared to aged-matched normal samples (n = 10). Cartilage was harvested from normal (n = 4) and OC (n = 3) joints from horses at necropsy. Chondrogenic pellet cultures were established following monolayer proliferation. After 14 days in culture, the pellets were assessed by histochemical and pellet weight analysis, assay of glycosaminoglycan (GAG) content, and gene expression. Chondrocytes from OC cartilage expressed significantly more Coll-I, -II, -III, and -X than chondrocytes from normal cartilage (all p < 0.0001). Furthermore, OC chondrocytes expressed significantly more MMP-13, ADAMTS-4 (both p < 0.0001), and TIMP-1 (p < 0.001) and significantly less TIMP-2 and TIMP-3. Pellets created from OC chondrocytes contained significantly less GAG (p = 0.0069) and expressed significantly less Sox9 and significantly more superficial zone protein (SZP) (p = 0.0105) than pellets created from normal cartilage. The results suggest that chondrocytes from OC cartilage at the time of surgical treatment have perturbations in phenotype compared to cells from normal cartilage. Despite these differences, following monolayer expansion and pellet culture under chondrogenic conditions, chondrocytes derived from OC cartilage retain some ability to undergo chondrogenic differentiation and synthesize an appropriate cartilage-like matrix. However, this chondrogenic differentiation potential is inferior to that seen in aged-matched normal chondrocytes.

Type II collagen deposition in cruciate ligament precedes osteoarthritis in the guinea pig knee.

OBJECTIVE: To examine the collagens in cruciate ligaments of young Dunkin-Hartley guinea pigs, to determine whether a change in specific collagen types is an early feature of the spontaneous osteoarthritis (OA), which consistently develops in the medial compartment of the knee in this strain. DESIGN: Collagen types I, II, III, IX, and XI were detected by immunofluorescence microscopy in the anterior and posterior cruciate ligaments of animals at 3, 4-5 and 12 weeks of age. Type II collagen in PCL was further analysed by confocal microscopy or biochemical assay after cyanogen bromide digestion, SDS-PAGE and immunoblotting. Interfibrillar proteoglycans were visualized by transmission electron microscopy. RESULTS: Collagen types I and III formed the bulk of fibrous mid-ligament tissue in all animals. Typical cartilage collagens, types II, IX and XI, were identified by immunolabeling where ligaments attached to tibial bone. Type II collagen, normally restricted to the fibrocartilage attachment sites, was also found at separate foci in anterior fiber bundles of the posterior cruciate ligament in 12-week-old animals. Biochemical data confirmed these observations which, together with electron microscopy showing large atypical proteoglycan structures, suggested the deposition of fibrocartilage within the fibrous mid-ligament. CONCLUSIONS: Cruciate ligaments, especially posterior cruciate ligament in Dunkin-Hartley guinea pigs synthesize cartilage-like matrix in mid-ligament prior to the appearance of classical signs of OA.

Characterization of type XI collagen-glycosaminoglycan interactions.

Using competitive binding experiments, it was found that native type XI collagen binds heparin, heparan sulfate, and dermatan sulfate. However, interactions were not evident with hyaluronic acid, keratan sulfate, or chondroitin sulfate chains over the concentration range studied. Chondrocyte-matrix interactions were investigated using cell attachment to solid phase type XI collagen. Pretreatment of chondrocytes with either heparin or heparinase significantly reduced attachment to type XI collagen. Incubation of denatured and cyanogen bromide-cleaved type XI collagen with radiolabeled heparin identified sites of interaction on the alpha1(XI) and alpha2(XI) chains. NH(2)-terminal sequence data confirmed that the predominant heparin-binding peptide contained the sequence GKPGPRGQRGPTGPRGSRGAR from the alpha1(XI) chain. Using rotary shadowing electron microscopy of native type XI collagen molecules and heparin-bovine serum albumin conjugate, an additional binding site was identified at one end of the triple helical region of the collagen molecule. This coincides with consensus heparin binding motifs present at the amino-terminal ends of both the alpha1(XI) and the alpha2(XI) chains. The contribution of glycosaminoglycan-type XI collagen interactions to cartilage matrix stabilization is discussed.