C2K77 ELISA detects cleavage of type II collagen by cathepsin K in equine articular cartilage.

OBJECTIVES: Develop a species-specific ELISA for a neo-epitope generated by cathepsin K cleavage of equine type II collagen to: (1) measure cartilage type II collagen degradation by cathepsin K in vitro, (2) identify cytokines that upregulate cathepsin K expression and (3) compare cathepsin K with matrix metalloproteinase (MMP) collagenase activity in stimulated cartilage explants and freshly isolated normal and osteoarthritic (OA) articular cartilages. DESIGN: A new ELISA (C2K77) was developed and tested by measuring the activity of exogenous cathepsin K on equine articular cartilage explants. The ELISA was then employed to measure endogenous cathepsin K activity in cultured cartilage explants with or without stimulation by interleukin-1 beta (IL-1ß), tumour necrosis-alpha (TNF-α), oncostatin M (OSM) and lipopolysaccharide (LPS). Cathepsin K activity in cartilage explants (control and osteoarthritic-OA) and freshly harvested cartilage (control and OA) was compared to that of MMPs employing C2K77 and C1,2C immunoassays. RESULTS: The addition of Cathepsin K to normal cartilage caused a significant increase (P < 0.01) in the C2K77 epitope release. Whereas the content of C1,2C, that reflects MMP collagenase activity, was increased in media by the addition to cartilage explants of TNF-α and OSM (P < 0.0001) or IL-1ß and OSM (P = 0.002), no change was observed in C2K77 which also unchanged in OA cartilages compared to normal. CONCLUSIONS: The ELISA C2K77 measured the activity of cathepsin K in equine cartilage which was unchanged in OA cartilage. Cytokines that upregulate MMP collagenase activity had no effect on endogenous cathepsin K activity, suggesting a different activation mechanism that requires further study.

Early cathepsin K degradation of type II collagen in vitro and in vivo in articular cartilage.

OBJECTIVE: To characterize the initial events in the cleavage of type II collagen mediated by cathepsin K and demonstrate the presence of the resulting products in human and equine articular osteoarthritic cartilage. DESIGN: Equine type II collagen was digested with cathepsin K and the cleavage products characterized by mass spectrometry. Anti-neoepitope antibodies were raised against the most N-terminal cleavage products and used to investigate the progress of collagen cleavage, in vitro, and the presence of cathepsin K-derived products in equine and human osteoarthritic cartilage. RESULTS: Six cathepsin K cleavage sites distributed throughout the triple helical region were identified in equine type II collagen. Most of the cleavages occurred following a hydroxyproline residue. The most N-terminal site was within three residues of the previously identified site in bovine type II collagen. Western blotting using anti-neoepitope antibodies showed that the initial cleavages occurred at the N-terminal sites and this was followed by more extensive degradation resulting in products too small to be resolved by SDS gel electrophoresis. Immunohistochemical staining of cartilage sections from equine or human osteoarthritic joints showed staining in lesional areas which was not observed in non-arthritic sites. CONCLUSIONS: Cathepsin K cleaves triple helical collagen by erosion from the N-terminus and with subsequent progressive cleavages. The liberated fragments can be detected in osteoarthritic cartilage and may represent useful biomarkers for disease activity.

Changes in gene expression associated with matrix turnover, chondrocyte proliferation and hypertrophy in the bovine growth plate.

The aim of the study is to investigate the interrelationships between the expression of genes for structural extracellular matrix molecules, proteinases and their inhibitors in the bovine fetal growth plate. This was analyzed by RT-PCR in microsections of the proximal tibial growth plate of bovine fetuses in relationship to expression of genes associated with chondrocyte proliferation, apoptosis, and matrix vascularization. In the resting zone the genes for extracellular matrix molecule synthesis were expressed. Extracellular matrix degrading enzymes and their inhibitors were also expressed here. Onset of proliferation involved cyclic upregulation of cell division-associated activity and reduced expression of extracellular matrix molecules. Later in the proliferative zone we noted transient expression of proteinases and their inhibitors, extracellular matrix molecules, as well as activity associated with vascularization and apoptosis. With the onset of hypertrophy expression of proteinases and their inhibitors, extracellular matrix molecules, as well as activity associated with vascularization and apoptosis were significantly upregulated. Terminal differentiation was characterized by high expression of proteinases and their inhibitors, extracellular matrix molecules, as well as activity associated with apoptosis. This study reveals the complex interrelationships of gene expression in the physis that accompany matrix assembly, resorption, chondrocyte proliferation, hypertrophy, vascularization and cell death while principal zones of the growth plate are characterized by a distinct signature profile of gene expression.

Relationship between pre-radiographic cartilage damage following anterior cruciate ligament injury and biomarkers of cartilage turnover in clinical practice: a cross-sectional observational study.

OBJECTIVE: To determine whether differences in synovial fluid (SF) biomarkers of collagen and proteoglycan turnover are associated with pre-radiographic damage to articular cartilage and menisci following anterior cruciate ligament (ACL) injury and are of clinical value. METHOD: SF samples from ACL injured knees of 108 patients were obtained when damage to cartilages and menisci was evaluated arthroscopically. Concentrations of SF collagenase-generated cleavage neoepitope of type II collagen (C2C) were determined using ELISA and aggrecan-derived disaccharides of chondroitin-4-sulfate (Δdi-C4S), chondroitin-6-sulfate (Δdi-C6S), and keratan sulfate (KS), were measured in SF by High performance liquid chromatography (HPLC). RESULTS: Radiographic examination failed to detect any intra-articular degenerative changes. The number of high-grade cartilage lesions was positively associated with age, duration after injury and the level of C2C, and negatively with the level of KS. There was no association between the number of high-grade cartilage and meniscal lesions. Multivariable logistic regression revealed significant associations of increased C2C (adjusted Odds ratio (OR) of the upper quartile to remainder of 2.49, 95% Confidence interval (CI) = 0.85-7.27) and decreased KS (adjusted OR of the lower quartile to the remainder of 3.32, 95% CI = 1.19-9.24) with the presence of three or more high-grade cartilage lesions, independent of age and duration after injury. The combined impact of increased C2C and decreased KS was 22.8 (95% CI = 1.95-265.9), far exceeding the impact of each independent biomarker. CONCLUSION: Combinations of the C2C and KS as described here may offer greater ability to identify patients with early pre-radiographic high-grade cartilage damage compared to single clinical or biomarker parameters.

Application of biomarkers in the development of drugs intended for the treatment of osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is a chronic and slowly progressive disease for which biomarkers may be able to provide a more rapid indication of therapeutic responses to therapy than is currently available; this could accelerate and facilitate OA drug discovery and development programs. The goal of this document is to provide a summary and guide to the application of in vitro (biochemical and other soluble) biomarkers in the development of drugs for OA and to outline and stimulate a research agenda that will further this goal. METHODS: The Biomarkers Working Group representing experts in the field of OA biomarker research from both academia and industry developed this consensus document between 2007 and 2009 at the behest of the Osteoarthritis Research Society International Federal Drug Administration initiative (OARSI FDA initiative). RESULTS: This document summarizes definitions and classification systems for biomarkers, the current outcome measures used in OA clinical trials, applications and potential utility of biomarkers for development of OA therapeutics, the current state of qualification of OA-related biomarkers, pathways for biomarker qualification, critical needs to advance the use of biomarkers for drug development, recommendations regarding practices and clinical trials, and a research agenda to advance the science of OA-related biomarkers. CONCLUSIONS: Although many OA-related biomarkers are currently available they exist in various states of qualification and validation. The biomarkers that are likely to have the earliest beneficial impact on clinical trials fall into two general categories, those that will allow targeting of subjects most likely to either respond and/or progress (prognostic value) within a reasonable and manageable time frame for a clinical study (for instance within 1-2 years for an OA trial), and those that provide early feedback for preclinical decision-making and for trial organizers that a drug is having the desired biochemical effect. As in vitro biomarkers are increasingly investigated in the context of specific drug treatments, advances in the field can be expected that will lead to rapid expansion of the list of available biomarkers with increasing understanding of the molecular processes that they represent.

Serum cartilage oligomeric matrix protein (COMP) level is a marker of disease activity in relapsing polychondritis.

OBJECTIVES: Relapsing polychondritis (RP) is a rare and severe disease which may lead to destruction of elastic cartilages. Until now, no reliable biomarker of disease activity in RP has been available. This study was designed to measure serum levels of cartilage biomarkers during both active and inactive phases of the disease. METHODS: Serum levels of cartilage oligomeric matrix protein (COMP), chondroitin sulfate 846 epitope (CS846) of proteoglycan aggrecan and collagen type II collagenase cleavage neoepitope (C2C) were measured retrospectively in 21 subjects with RP. The Wilcoxon matched-pairs signed-rank test was used for statistical comparisons of biomarker levels in active and inactive phases of RP. RESULTS: Only the serum level of COMP was significantly increased during disease flares. Steroids did not alter the serum cartilage-related biomarker levels. However, during the active phase, C2C levels were significantly higher in steroid treated patients compared with non-steroid treated patients. CONCLUSIONS: This study suggests that serum COMP level may be useful for monitoring disease activity of RP. Further prospective studies are required to confirm this result.

Evidence to suggest that cathepsin K degrades articular cartilage in naturally occurring equine osteoarthritis.

OBJECTIVE: The mechanisms leading to degeneration of articular cartilage in osteoarthritis (OA) are complex and not yet fully understood. Cathepsin K (CK) is a cysteine protease which can also cleave the triple helix of type II collagen. This exposes a neoepitope that can now be identified by specific antibodies. The aim of this study was to obtain evidence suggesting a role for CK in naturally occurring equine OA in both lesional and peri-lesional regions. METHODS: Articular cartilages (n=12 horses; 5 healthy, 7 OA) were harvested from animals postmortem. A gross macroscopic examination, histologic (Safranin O-Fast Green and Picrosirius red staining) and immunohistochemical evaluation were performed. Samples were divided into normal appearing cartilage, peri-lesional and lesional cartilage. Cartilage degradation in the samples was graded histologically and immunohistochemically. CK and possible CK cleavage were detected immunohistochemically with specific anti-protein and anti-neoepitope antibodies, respectively. A comparison of CK neoepitope (C2K) production with the collagenase-generated neoepitope produced by matrix metalloproteinases (MMP)-1, 8 and 13 (C2C) was also assessed immunohistochemically. RESULTS: CK and CK cleavage were significantly more abundant in OA cartilage (both peri-lesional and lesional) when compared to remote cartilage within the sample joint or cartilage from healthy joints. The immunohistochemical pattern observed for CK degradation (C2K) was similar to that of collagenase degradation (C2C). Macroscopic cartilage changes and histologic findings were significantly correlated with immunohistochemistry results. CONCLUSION: The data generated suggests that CK may be involved in cartilage collagen degradation in naturally occurring osteoarthritis.

Serum concentrations of type II collagen biomarkers (C2C, C1, 2C and CPII) suggest different pathophysiologies in patients with hip osteoarthritis.

BACKGROUND: Cartilage destruction in osteoarthritis (OA) involves excessive degradation and increased synthesis of cartilage matrix macromolecules including type II collagen and proteoglycans. Cartilage biomarkers exist for the measurement of cartilage matrix turnover and may reveal differences in patients with OA. OBJECTIVE: To determine whether there are detectable differences in and relationships between biomarkers of type II collagen (CII) degradation (C2C, C1, 2C) and synthesis (CP II) in patients with only hip OA (OHOA) and those suffering from multiple sites OA (MSOA). PATIENTS AND METHODS: Fifty-six patients classified as MSOA or OHOA. Minimum hip joint space width (Min JSW) measured by computer from standard radiographs. Serum measurement of CII synthesis C-propeptide (CPII) and cleavage of type II (C2C) and types I and II (C1, 2C) collagens. Aggrecan metabolism was assessed by serum CS 846 assay. Step to step logistic regression to determine the effect of the quantitative data on the assignment to each subgroup. RESULTS: Twenty-four subjects were classified with MSOA. Among the 32 OHAO patients, 15 had bilateral hip OA and 17 had unilateral hip OA. The latter were classified with "Isolated hip OA" (IHOA). CPII levels were significantly lower in patients with MSOA than in those with OHOA (99.9+/-50.3ng/mL versus 141.9+/-81.2ng/mL, p=0.04. OR= 0.18 for CPII >120 ng/mL, p<0.005). C2C levels were also lower in MSOA (9.7+/-2.3ng/mL) versus OHOA (11.4+/-3.2ng/mL, p=0.03. OR= 0.26 for C2C >10 ng/mL, p=0.02). There was an inverse correlation between min JSW and C2C only in patients with IHOA (r=0.50, p= 0.02). CONCLUSION: Hip OA, in patients with MSOA, might be related to alteration in CII metabolism which may result in a deficient type II collagen repair process. The significant relationship between C2C and JSW in IHOA suggests that this marker is of value in assessing cartilage degradation patients with involvement of a single joint.

Differences in biomarkers of type II collagen in atrophic and hypertrophic osteoarthritis of the hip: implications for the differing pathobiologies.

BACKGROUND: Cartilage destruction in osteoarthritis (OA) involves the excessive degradation and increased synthesis of cartilage matrix macromolecules including type II collagen (CII) and proteoglycans. The lack of osteophytes (atrophic form of OA) has been shown to be a disease severity factor in hip OA. Since osteophyte formation involves endochondral ossification and a cartilage intermediate, atrophic OA may also exhibit differences in cartilage turnover compared to hypertrophic OA. Cartilage serum biomarkers may offer an opportunity to identify such differences in patients. AIM: To determine whether serum levels of cartilage biomarkers can distinguish between the presence and absence of osteophyte formation in patients with atrophic and hypertrophic hip OA. PATIENTS AND METHODS: Fifty-six patients (mean age/standard deviation (SD): 62/11; mean body mass index (BMI)/SD: 27/11) with symptomatic hip OA (American College of Rheumatology criteria; mean Lequesne index/SD: 8.3/4) were classified as having an atrophic or hypertrophic form of OA, according to the absence or presence, respectively, of any osteophyte on a standard radiograph of the pelvis. Minimum joint space width (minJSW) and angles of dysplasia [centre-edge (CE) and head-neck-shaft (HNS)] were determined by computerized measurements. The following serum markers were used which are commercial kits from Ibex Diagnostics (Montreal, QC): proteoglycan aggrecans turnover: CS 846; CII synthesis: C-propeptide (CPII), cleavage by collagenase of type II (C2C) and type I and II (C1,2C) collagens. STATISTICS: Patients with atrophic and hypertrophic OA were compared for each variable and step to step logistic regression was used to determine the effect of variables on the belonging to each group. Correlations were examined using linear regression or Spearman test. RESULTS: CPII serum levels were significantly lower in the atrophic OA patients (77.3 vs 117.4 ng/mL). There were no significant differences between groups for C2C, C1,2C and CS 846 . CPII and C2C concentrations were highly correlated in hypertrophic OA (P=0.002) but not in atrophic OA (P=0.8). CONCLUSION: Atrophic hip OA is characterized by reduced synthetic activity involving type II collagen synthesis. This could account in part for the absence of osteophyte formation. The highly significant correlation between CPII and C2C in hypertrophic but not in atrophic OA suggests that the physiological coupling between CII formation and degradation may be lost in atrophic OA. These differences may therefore help explain the absence of osteophyte in atrophic OA and its association with more rapid disease progression.

Defects in articular cartilage metabolism and early arthritis in fibroblast growth factor receptor 3 deficient mice.

Fibroblast growth factor (FGF) receptor 3 has been identified as a key regulator of endochondral bone development and of post-natal bone metabolism through its action on growth plate chondrocytes and osteoblasts, respectively. It has also been shown to promote chondrogenesis and cartilage production by cultured pre-chondrogenic cells in response to FGF18. In the current studies, we show that the absence of signaling through Fgfr3 in the joints of Fgfr3(-/-) mice leads to premature cartilage degeneration and early arthritis. Degenerative changes in cartilage matrix included excessive proteolysis of aggrecan core protein and type II collagen, as measured by neo-epitope immunoreactivity. These changes were accompanied by increased expression of metalloproteinase MMP13, type X collagen, cellular hypertrophy and loss of proteoglycan at the articular surface. Using a novel micro-mechanical indentation protocol, it was shown that articular cartilage in the humeral head of 4-month-old Fgfr3(-/-) mice was less resistant to compressive force and less stiff than that of littermate controls. These results identify Fgfr3 signaling as a potential target for intervention in degenerative disorders of cartilage metabolism.

Radiographic joint damage in rheumatoid arthritis is associated with differences in cartilage turnover and can be predicted by serum biomarkers: an evaluation from 1 to 4 years after diagnosis.

INTRODUCTION: The objective of this study was to determine whether serum biomarkers for degradation and synthesis of the extracellular matrix of cartilage are associated with, and can predict, radiographic damage in patients with rheumatoid arthritis (RA). METHODS: Clinical and radiographic data of 87 RA patients were recorded 1 year after disease onset and then annually up to four years. Serum concentrations of four cartilage biomarkers were determined at these time points: a neoepitope formed by collagenase cleavage of type II collagen (C2C), a neoepitope formed by collagenase cleavage of type II collagen as well as type I collagen (C1,2C), a carboxy propeptide of type II procollagen formed during synthesis (CPII), and a cartilage proteoglycan aggrecan turnover epitope (CS846-epitope). Biomarker concentrations between patients with rapid radiographic progression (>7.3 Sharp/van der Heijde units per year) and those with slow radiographic progression (<2.3 units per year) were compared. In addition, we evaluated the long-term and short-term predictive value of each biomarker for progression of radiographic damage. RESULTS: Patients with rapid radiographic progression had higher C2C, higher C1,2C, and higher CS846-epitope levels than slow progressors. CPII levels showed no differences. Most importantly, the long-term radiographic progression for C2C, for C1,2C, and for CS846-epitope can be predicted by the biomarker value at year 1 after disease onset. C2C was also a predictor for joint space narrowing and annual radiographic damage during the subsequent year. CONCLUSION: This study shows that the concentration of serum biomarkers of cartilage collagen breakdown and proteoglycan turnover, but not of collagen synthesis, are related to joint destruction in RA. The use of these biomarkers may be of value when studying progression of joint damage in patients with RA.

Early post-traumatic osteoarthritis-like changes in human articular cartilage following rupture of the anterior cruciate ligament.

OBJECTIVE: Injury to the anterior cruciate ligament (ACL) frequently leads to post-traumatic osteoarthritis (OA). In this study we determined whether early degenerative changes characteristic of idiopathic OA are induced in articular cartilage following ACL injury. METHODS: A small sample of femoral articular cartilage was removed at surgery, as part of ACL reconstruction, from a total of 50 patients with ACL injuries. Of these, 28 underwent surgery less than 1 year post-injury. Control cartilages were obtained from the same site from 21 persons at autopsy. All cartilages were examined for molecular changes. The content of type II collagen, its cleavage by collagenases and its denaturation were determined by immunoassay. The total content of glycosaminoglycan (GAG), which is principally aggrecan, was measured colorimetrically. Data were expressed per unit DNA (GAG and collagen content) or as a percentage of total collagen cleaved or denatured. Other cartilages from the same site (8 controls, 12 less than 1 year and 8 more than 1 year post-injury) were frozen sectioned and examined histologically to determine by Mankin grading cartilage degeneration. RESULTS: Histological analyses revealed that control subjects exhibited staining for proteoglycan, which was reduced in some patients following ACL rupture. Degeneration of the articular surface was sometimes observed 1 year after ACL rupture. Although the Mankin grade increased with time after rupture these changes were not significant. Immunoassays, however, revealed an increase in GAG content within 1 year which was maintained after 1 year although no longer significant. No changes in total type II collagen content were observed during the period of study. However, there were significant increases in the denaturation and cleavage of type II collagen less than and more than 1 year post-ACL rupture. Total type II collagen content was directly correlated with GAG content in all three groups, with the significance being weakest at more than 1 year. After 1 year an inverse correlation was observed between total type II collagen content and collagen cleavage as well as denaturation. CONCLUSIONS: These observations reveal that joint instability resulting from ACL injury rapidly results in degenerative changes characteristic of those seen in idiopathic OA at arthroplasty and in experimental OA following ACL surgery. These changes may contribute to the development of post-traumatic OA that is commonly observed following ACL injury. The observations support and extend conclusions from other studies on human and animal articular cartilage and synovial fluids post-ACL injury that have revealed a rapid onset of damage to type II collagen and an initial increase in proteoglycan content characteristic of experimental OA post-ACL injury. This study provides direct evidence for the rapid development of degenerative changes characteristic of OA following ACL injury.

Detection of aggrecanase- and MMP-generated catabolic neoepitopes in the rat iodoacetate model of cartilage degeneration.

OBJECTIVE: To characterize the time course of aggrecan and type II collagen degradation in the rat iodoacetate model of cartilage degeneration in relationship to the temporal sequence that has been described in human osteoarthritis (OA). DESIGN: Rats were injected intra-articularly in one knee joint with iodoacetate and damage to the tibial plateau was assessed from digitized images captured using an image analyzer. The articular cartilage from the tibial plateau was harvested, extracted and glycosaminoglycan (GAG) content was measured using the dimethylmethylene blue (DMMB) assay. Cartilage aggrecan neoepitopes were detected in cartilage extracts by Western blotting using antibodies recognizing the aggrecanase-generated C-terminal neoepitope NITEGE (BC-13) and the MMP-generated C-terminal neoepitope DIPEN (BC-4). A type II collagen collagenase-generated neoepitope was detected in cartilage extracts by ELISA using the Col2-3/4Cshort antibody; denatured collagen was detected using the Col2-3/4m antibody. RESULTS: Degenerative joint changes and proteoglycan (GAG) loss progressed with time after iodoacetate injection. Western blotting of cartilage extracts of iodoacetate treated rats demonstrated an increase in both aggrecanase- and MMP-generated epitopes with the NITEGE aggrecanase neoepitope being significantly elevated on days 7, 14 and 21 while DIPEN the MMP neoepitope was significantly elevated on days 7 and 14. The type II collagen neoepitope recognized by Col2-3/4Cshort was significantly increased in cartilage extracts of rats at days 14 and 21 after iodoacetate injection. CONCLUSION: The proteoglycan fragments extracted from the knee cartilage of rats after the intra-articular injection of iodoacetate appeared to result from cleavage at both aggrecanase and MMP sites. Cleavage of type II collagen by collagenase was also detected after iodoacetate injection and occurred subsequent to the initiation of aggrecan loss. These observations serve to demonstrate similarities in the mechanisms of cartilage degeneration induced by iodoacetate to those seen in articular cartilage in OA.

Distinct phases of coordinated early and late gene expression in growth plate chondrocytes in relationship to cell proliferation, matrix assembly, remodeling, and cell differentiation.

Although much has been learned about growth plate development and chondrocyte gene expression during cellular maturation and matrix remodeling in the mouse, there has been a limited study of the interrelationships of gene expression between proteinases, growth factors, and other regulatory molecules in the mouse and in other species. Here we use RT-PCR of sequential transverse sections to examine the expression profiles of genes involved in chondrocyte growth, differentiation, matrix assembly, remodeling, and mineralization in the bovine proximal tibial growth plate. Specifically, we studied the expression of genes encoding COL2A1 and COL10A1, the latter a marker of cellular hypertrophy, the matrix metalloproteinases (MMP), MMP-13 and MMP-9, as well as the transcriptional factors, Sox9 and Cbfa1, the growth factors basic fibroblast growth factor (bFGF), parathyroid hormone-related peptide (PTHrP), transforming growth factor (TGF)beta1, and beta2, Indian hedgehog (Ihh), and the matrix protein osteocalcin. These were analyzed in relationship to cell division defined by cyclin B2 expression. Two peaks of gene expression activity were observed. One was transient, limited, and located immediately before and at the onset of cyclin B2 expression in the early proliferative zone. The other was generally much more pronounced and was located in the early hypertrophic zone. The upregulation of expression of COL2A1, its transcriptional activator Sox9, osteocalcin, MMP-13, and TGFbeta2 was observed immediately before and at the onset of cyclin B2 expression and also in the hypertrophic zones. The upregulation of COL10A1, Cbfa1, MMP-9, TGFbeta-1, and Ihh gene expression was associated exclusively with the terminal differentiation of chondrocytes at the time of mineral formation in the extracellular matrix. In contrast, bFGF and PTHrP expression was observed in association with the onset of cyclin B2 expression and hypertrophy. This initial cluster of gene expression associated predominantly with matrix assembly and onset of cell proliferation is therefore characterized by expression of regulatory molecules distinct from those involved at hypertrophy. Together these results identify separate phases of coordinated gene expression associated with the development of the physis in endochondral bone formation.

Changes in subchondral bone in cartilage resurfacing--an experimental study in sheep using different types of osteochondral grafts.

OBJECTIVE: This article addresses the subchondral bone integrity in cartilage resurfacing by comparing fresh, untreated auto-, xeno-, and photooxidized osteochondral allo- and xenografts. Photooxidation was expected to improve mechanical stability of the osteochondral grafts through an improved linkage of the collagen fibers within the bone matrix. DESIGN: Untreated auto- and xenografts and with photooxidation pretreated allo- and xenografts were surgically implanted in femoral condyles of sheep (n=40). After 2, 6, 12 and 18 months results were evaluated histologically using non-decalcified bone embedded in acrylic resin. Qualitative evaluation was performed with emphasis on bone matrix, biomechanical stability of graft anchorage, formation of cystic lesions, and bone resorption and formation. Quantitative evaluation of the total subchondral bone area was conducted histomorphometrically. Statistical analysis (factorial ANOVA test) was used to compare differences between groups with respect to the percentage of bone matrix and fibrous tissue per section. RESULTS: Subchondral bone resorption was fastest in untreated, fresh autografts, followed by photooxidized allografts, untreated, fresh xenografts and last pretreated photooxidized xenografts. Cystic lesions were seen in all types of grafts, but were most pronounced at 6 months in autografts and least in photooxidized grafts. Cyst-like lesions had subsided substantially in the untreated auto- and photooxidized xenografts, if no graft dislocation occurred during the healing period. Mononuclear cell infiltration and an increase in the presence of multinuclear cells were observed at 2 months, mostly in untreated autografts, followed by photooxidized allo- and untreated xenografts. They were much higher in numbers compared to photooxidized grafts, at least in the early specimens at 2 months. Graft stability was linked to the rate of bone resorption. CONCLUSION: Substantial resorption of the subchondral bone, involving the development of cyst-like lesions, lead to dislocation and finally to cartilage matrix degradation of the grafts. The process of photooxidation decreased the speed of bone resorption in osteochondral grafts and, thus, improved graft stability and cartilage survival. These results suggest that the remodeling of the subchondral bone of the host and the graft within the first 6 months is an important factor in graft stability and overall results of cartilage resurfacing.

Presence and mechanism of knee articular cartilage degeneration after meniscal reconstruction in dogs.

OBJECTIVE: Partial meniscectomy is the golden standard for treating a bucket-handle tear in the meniscus of the knee, but it inevitably leads to articular cartilage degeneration. Surgical creation of an access channel between the lesion and the vascularized synovial lining is intended to induce ingrowth of repair tissue and thus avoid degeneration of articular cartilage. DESIGN: The presence and mechanism of cartilage degeneration were evaluated in 24 canine menisci after a longitudinal lesion and access channel had been created in the avascular part of the meniscus. In 12 menisci the channel was implanted with a porous polymer scaffold, while the remaining 12 were left empty. Evaluation was performed using routine histology and antibodies directed against denatured type II collagen (Col2-3/4M). RESULTS: Articular degeneration was apparent in the polymer implant group and the empty channel group. This consisted of fibrillation, loss of chondrocytes and decreased proteoglycan content. Areas of fibrillated cartilage always showed positive labeling with the collagen degradation antibody Col2-3/4M. Collagen degradation was also visible in non-fibrillated areas. The upper zone of the cartilage showed swelling especially in the implant group, with empty cell lacunae and moderate levels of Col2-3/4M antibody labeling. DISCUSSION: This reconstruction technique cannot be considered superior to partial meniscectomy. We propose that degradation of the collagen type II network is a result of cartilage fibrillation and vice versa.

Elevation of a collagenase generated type II collagen neoepitope and proteoglycan epitopes in synovial fluid following induction of joint instability in the dog.

CLINICAL RELEVANCE: Measurement of markers of cartilage pathology in synovial fluid may provide clinical rheumatologists and osteoarthritis (OA) researchers important information for early diagnosis of OA as well as a method for monitoring disease progression and response to treatment. This study demonstrates the value of this approach in an established model of OA (cranial cruciate ligament rupture) at a point distant enough from the original surgical manipulation so as to have little to no effect on the marker concentrations. OBJECTIVE: The objective of this study was to determine whether measurement of markers of cartilage collagen cleavage and proteoglycan turnover in synovial fluid from a canine model could be used to detect cartilage changes following the onset of joint instability during the development of OA. DESIGN: A model of joint instability that develops OA was created in 18 mature dogs using monopolar radiofrequency energy (MRFE). MRFE was arthroscopically applied to one cranial cruciate ligament (CCL) while the contralateral CCL was sham treated. The treated CCLs ruptured approximately 8 weeks (55 +/- 1.6 days) after MRFE treatment. Synovial fluid was collected at time zero prior to MRFE treatment, 4 weeks after MRFE treatment, and at 4, 8, and 16 weeks after CCL rupture. Synovial fluid concentrations of the neoepitope COL2-3/4C long (type II collagen cleavage by collagenase) and epitopes 3B3(-) (proteoglycan aggrecan sulfation) and 846 (associated with aggrecan synthesis) were analyzed. RESULTS: Compared to sham treated joints, the synovial fluid concentrations of COL2-3/4C long and 3B3(-) were significantly increased 2.2 fold and 2.9 fold, respectively, in joints with MRFE treated CCLs following CCL rupture. Concentrations of the 846 epitope in synovial fluid showed a trend toward an increase, which was not significant, after CCL rupture. CONCLUSIONS: Concentrations of the collagenase-cleaved type II collagen neoepitope and 3B3(-) epitope in synovial fluid were significantly increased by 4 weeks and remained elevated for at least 16 weeks after CCL rupture. This suggests that in dogs the COL2-3/4C long neoepitope and 3B3(-) epitope are sensitive markers for changes in joint cartilage turnover in joints that are developing OA.

Excessive degradation of type II collagen in articular cartilage in equine osteochondrosis.

Articular osteochondrosis (OCD) occurs in both man and animals. The etiology remains to be determined. Studies of OCD lesions in animals may provide clues as to its pathogenesis. The aim of our study was to determine whether there was evidence for increased degradation namely proteoglycan (PG) release and type II collagen cleavage in articular cartilage harvested from OCD lesions. We examined ex vivo explants at post-mortem from equine OCD lesions and macroscopically normal site and age matched cartilage. These were cultured over a 10 day period in serum-free medium. Type II collagen cleavage was measured in articular cartilage and media using an Elisa assay to detect the COL2-3/4C(short) epitope, which is generated on cleavage of the triple helix of type II collagen by collagenases. PG release was measured by a dye-binding assay. Cumulative release of PG and COL2-3/4C(short) and their contents in cartilage at the end of the culture period were determined. In OCD lesions there was a significant increase in type II collagen cleavage by collagenase but no evidence for increase of PG degradation. These findings point to a selective increase in type II collagen cleavage by collagenases, in OCD lesions of the kind observed in osteoarthritis. Further work is needed to determine whether changes represent primary or secondary events in the pathogenesis of OCD.

Detection and analysis of cartilage degeneration by spatially resolved streaming potentials.

Cartilage molecular changes in osteoarthritis are most commonly related to the degradation and loss of proteoglycan and collagen fibrils of the extracellular matrix, which directly influence tissue stiffness and compression-generated streaming potentials. In this study, we evaluated the potential of a new technique, spatially resolved mapping of streaming potentials, to non-destructively indicate cartilage health or degeneration. Matched pairs of bovine cartilage/bone explant disks were cultured for 11 days in a serum free medium with and without interleukin-lalpha (IL-1alpha). The electromechanical properties (static stiffness, dynamic stiffness and streaming potentials) of cartilage disks were measured during unconfined compression using a mechanical tester coupled with a linear array of eight 50 microm diameter platinum-iridium microelectrodes. After 11 days of culture, the proteoglycan content of IL-1alpha treated disks was significantly reduced and the denatured and cleaved collagen content was increased compared to control disks. These biochemical alterations were concomitant with the reductions in the amplitudes of the static stiffness, the dynamic stiffness and the streaming potential profile as well as changes in the shape of the streaming potential profile. We found that spatial mapping of streaming potentials presents several advantages for the development of a clinical instrument to evaluate the degeneration of articular cartilage.