MMP‐mediated collagen breakdown induced by activated protein C in equine cartilage is reduced by corticosteroids

The plasma serine protease activated protein C (APC) is synthesized by human chondrocytes at sites of pathological cartilage fibrillation. APC levels are increased in osteoarthritis (OA) synovial fluid, and in vitro APC has been shown to synergize with interleukin‐1β (IL‐1) to promote degradation from ovine cartilage. A model of equine cartilage degradation was established and used to explore corticosteroid activities. Intraarticular corticosteroids are a commonly prescribed treatment for joint disease, however their role in disease modification remains unclear. APC synergized with IL‐1 or tumor necrosis factor‐α (TNFα), promoting significant collagen degradation from equine cartilage explants within 4 days, but did not augment glycoaminoglycan (GAG) release. APC activated pro‐matrix metalloproteinases (MMP)‐2 but not pro‐MMP‐9, as assessed by gelatin zymography. APC did not directly activate pro‐MMP‐13. Dexamethasone, triamcinolone, and methylprednisolone acetate (MPA) were evaluated at concentrations between 10^{? 5}M and 10^?10M. High concentrations significantly increased GAG release from IL‐1+APC–treated explants. With the exception of MPA at 10^?10M, all concentrations of corticosteroids caused significant decreases in IL‐1+APC‐driven hydroxyproline loss. Treatment with corticosteroids suppressed expression of MMP‐1, ‐3, and ‐13 mRNA. The collagenolysis associated with IL‐1+APC synergy, and the inhibition of this effect by corticosteroids may involve gelatinase activation and downregulation of MMP expression, respectively. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:370–378, 2010     

Interaction of periosteal explants with articular chondrocytes alters expression profile of matrix metalloproteinases

Periosteal tissue is a source of growth factors and of osteochondral progenitor cells which makes it suitable for implantation in chondral defects as known in autologous chondrocyte implantation. The aim of this study was to determine the interaction between periosteal tissue and articular chondrocytes with respect to catabolic effectors such as matrix metalloproteinases (MMPs) and IL‐6. Human articular chondrocytes were cultured for up to 28 days as micromass pellets in coculture either with physical contact to periosteal explants or allowing paracrine interactions only. Expression, secretion, and activation of MMPs and IL‐6 were analyzed in chondrocytes, periosteum, and culture supernatants. Both coculture conditions influence gene expression levels of MMPs and IL‐6 in a time‐, culture‐, and tissue‐dependent manner. Coculturing of periosteum with chondrocytes promotes gene expression and secretion of IL‐6. In periosteum, physical contact inhibits MMP‐2 and MMP‐13 gene expression while paracrine coculture induces expression of IL‐6, MMP‐2, ‐7, and ‐13. Pro‐MMP‐2, ‐7, and ‐13 were detected in supernatants of all culture regimens whereas pro‐MMP‐9 was secreted from periosteum only. As a balanced amount of MMP activity is likely required to achieve sufficient integration of the regenerate tissue with the surrounding healthy cartilage, an exceeding expression of proteinases might result in degradation, hypertrophy or rejection of the graft. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1576–1585, 2010     

Glucosamine HCl reduces equine articular cartilage degradation in explant culture

Objective To determine whether glucosamine inhibits experimentally induced degradation of equine articular cartilage explants.Methods Articular cartilage was obtained from the antebrachio-carpal and middle joints of horses (2-8 years old) killed for reasons unrelated to lameness. Cartilage discs were harvested from the weight-bearing region of the articular surface and cultured. Media were exchanged daily and the recovered media stored at 4 degrees C. Explants were maintained in basal media 2 days prior to the start of four treatment days. On days 1-4 lipopolysaccharide (LPS, 10 microg/ml) or recombinant human interleukin-1 (rhIL-1, 50 ng/ml) were added to induce cartilage degradation. To test the potential protective effects of glucosamine, the compound was added in three concentrations (0.25, 2.5, or 25 mg/ml) and treatments were performed in triplicate. Controls included wells without LPS, rhIL-1beta, or glucosamine. Nitric oxide, proteoglycan and matrix metalloproteinases (MMP) released into conditioned media and tissue proteoglycan synthesis were measured as indicators of cartilage metabolism.Results Maximal nitric oxide production, proteoglycan release, and MMP activity were detected 1 day after the addition of LPS or rhIL-1beta to the media. The addition of 25 mg/ml of glucosamine prevented the increase in nitric oxide production, proteoglycan release and MMP activity induced by LPS or rhIL-1. Conclusions These data indicate that glucosamine can prevent experimentally induced cartilage degradation in vitro.     

Synoviocytes are more sensitive than cartilage to the effects of minocycline and doxycycline on IL‐1α and MMP‐13‐induced catabolic gene responses

The objective of this study was to determine the primary articular tissue target of doxycycline and minocycline. Synoviocytes—cartilage cocultures (n = 4) were treated with MMP‐13 (25 ng/mL medium) or IL‐1 (1.0 ng/mL medium) for 24 h. Doxycycline (4.3, 0.43, 0.043 µM) or minocycline (10, 1.0 or 0.1 µM) were then added and cultures were continued for 96 h. Cartilage and media were analyzed for GAG content. Quantitative PCR was used to measure cartilage MMP‐3, MMP‐13, aggrecan, COL2A1, ADAMTS‐4, and ADAMTS‐5 expression, and synoviocyte MMP‐3, MMP‐13, ADAMTS‐4, and ADMATS‐5 expression. Total and active MMP‐3, MMP‐13, and ADAMTS 4/5 enzymes were measured in culture medium. All concentrations of doxycycline and minocycline diminished GAG accumulation in the media. All concentrations of minocycline, but only the highest concentration of doxycycline decreased MMP‐3 and MMP‐13 expression in synoviocytes but not cartilage, and basal ADAMTS‐5 mRNA levels in both synoviocytes and cartilage. Only minocycline decreased active MMP‐13 protein in synoviocytes. In summary, the protective effects of tetracycline compounds are more pronounced in synoviocytes than cartilage, and following minocycline compared to doxycycline. Studies to determine the molecular mechanism of action of the tetracyclines in synoviocytes might lead to the design of targeted therapeutics for the treatment of OA or RA. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:522–528, 2010     

A critical role for collagen II in cartilage matrix degradation: Collagen II induces pro‐inflammatory cytokines and MMPs in primary human chondrocytes

We report a process that results in the acceleration of matrix degradation in human articular cartilage, a phenomenon commonly observed in osteoarthritis (OA). The study was conducted by (1) examining the potential of collagen II in modulating the gene expression profile of primary human chondrocytes (PHCs), and (2) investigating the involvement of pro‐inflammatory signaling cascades. We first tested the collagen II‐dependent induction of pro‐inflammatory cytokines and matrix metalloproteinases (MMPs) in PHCs. PHCs were incubated with or without monomeric (i.e., nonfibrillar) collagen II. Cells were then analyzed by RT‐PCR for the expression of MMP1, MMP3, MMP13, MMP14, and IL‐1β. ELISA was used to quantify IL‐6 and IL‐8 release. To examine the influence of collagen II signaling, specifically the role of MAPK p38, a p38‐inhibitor was added prior to collagen treatment. Changes in IκB concentration were monitored by immunoblot analysis to detect NFκB signaling. Results indicated that incubation of PHCs with collagen II did produce a dose‐dependent induction of MMP1, MMP3, MMP13, MMP14, as well as cytokines IL‐1β, IL‐6, and IL‐8. At the same time, inhibition of p38 and IκB degradation revealed that collagen II‐dependent gene induction also involves MAPK p38 and NFκB signaling. Thus, we provide evidence for a collagen II‐dependent feed‐forward mechanism whereby collagen II induces first MMPs and pro‐inflammatory cytokines and then release of collagen II fragments from mature collagen II fibers. This, in turn, induces more pro‐inflammatory cytokines and MMPs, and the process is repeated, which results in the acceleration and perpetuation of cartilage matrix degradation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:65–70, 2009     

The overexpression of SIRT1 inhibited osteoarthritic gene expression changes induced by interleukin‐1β in human chondrocytes

In this study, we examined the effects of overexpression of SIRT1 on IL‐1β‐induced gene expression changes in human chondrocytes to explore a protective role of SIRT1 in human chondrocytes. SIRT1 was overexpressed in human chondrocytes by expression plasmid under stimulation with IL‐1β. SIRT1 was also inhibited by siRNA under stimulation with IL‐1β. Gene expression changes were examined by real‐time PCR. The interaction of SIRT1 and p65 (NF‐κB) were examined by Western blotting. SIRT1, MMP‐13, and ADAMTS‐5 expressions in human cartilage were examined by immunohistochemistry. IL‐1β stimulation significantly up‐regulated MMP‐1, 2, 9, and 13 and ADAMTS‐5. Overexpression of SIRT1 significantly inhibited the up‐regulation of those genes caused by IL‐1β while the inhibition of SIRT1 further increased them. In addition, the overexpression of SIRT1 markedly reduced the IL‐1β‐induced acetylation of p65. SIRT1 expression was clearly detected in the non‐OA cartilage while MMP‐13 and ADAMTS‐5 were undetectable. In contrast, in the OA cartilage, SIRT1 expression was decreased while MMP‐13 and ADAMTS‐5 were increased. Our observations suggested that SIRT1 can play a protective role by suppressing IL‐1β‐induced expressions of cartilage‐degrading enzymes partially through the modulation of the NF‐κB pathway. SIRT1 overexpression might be a new therapeutic approach for OA. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 531–537, 2013     

Effective knock down of matrix metalloproteinase‐13 by an intra‐articular injection of small interfering RNA (siRNA) in a murine surgically‐induced osteoarthritis model

This study investigated the effect of MMP‐13 gene knock down on cartilage degradation by injecting small interfering RNA (siRNA) into knee joints in a mouse model of osteoarthritis (OA). OA was induced in male C57BL/6 mice by destabilization of medial meniscus (DMM) surgery. Change of Mmp13 expression over time was determined by qPCR analysis from 3 days to 6 weeks after surgery. Mmp13 and control chemically modified siRNA were injected into the knee joint 1 week after surgery and expression levels were assessed in synovium by qPCR 48 h later. Cartilage degradation was histologically assessed 8 weeks after DMM surgery according to OARSI recommendations. Mmp13 expression levels were elevated 1 week after surgery and peaked at 77 fold at 2 weeks compared to expression at 3 days. A 55% decrease of Mmp13 levels in cartilage was observed 48 h after injection of Mmp13 siRNA (p = 0.05). Significant reduction in the histological score at 8 weeks after surgery was observed in the Mmp13 siRNA‐treated group compared to the control siRNA group (p < 0.001). Intra‐articular injection of Mmp13 siRNA at the early phase of OA development resulted in effective knock down of Mmp13 expression and delay in cartilage degradation in vivo. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1175–1180, 2014.     

CXCL10 is upregulated in synovium and cartilage following articular fracture

The objective of this study was to investigate the expression of the chemokine CXCL10 and its role in joint tissues following articular fracture. We hypothesized that CXCL10 is upregulated following articular fracture and contributes to cartilage degradation associated with post‐traumatic arthritis (PTA). To evaluate CXCL10 expression following articular fracture, gene expression was quantified in synovial tissue from knee joints of C57BL/6 mice that develop PTA following articular fracture, and MRL/MpJ mice that are protected from PTA. CXCL10 protein expression was assessed in human cartilage in normal, osteoarthritic (OA), and post‐traumatic tissue using immunohistochemistry. The effects of exogenous CXCL10, alone and in combination with IL‐1, on porcine cartilage explants were assessed by quantifying the release of catabolic mediators. Synovial tissue gene expression of CXCL10 was upregulated by joint trauma, peaking one day in C57BL/6 mice (25‐fold) versus 3 days post‐fracture in MRL/MpJ mice (15‐fold). CXCL10 protein in articular cartilage was most highly expressed following trauma compared with normal and OA tissue. In a dose dependent manner, exogenous CXCL10 significantly reduced total matrix metalloproteinase (MMP) and aggrecanase activity of culture media from cartilage explants. CXCL10 also trended toward a reduction in IL‐1α‐stimulated total MMP activity (p = 0.09) and S‐GAG (p = 0.09), but not NO release. In conclusion, CXCL10 was upregulated in synovium and chondrocytes following trauma. However, exogenous CXCL10 did not induce a catabolic response in cartilage. CXCL10 may play a role in modulating the chondrocyte response to inflammatory stimuli associated with joint injury and the progression of PTA. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1220–1227, 2018.

     

MT1‐MMP and Type II Collagen Specify Skeletal Stem Cells and Their Bone and Cartilage Progeny

Skeletal formation is dependent on timely recruitment of skeletal stem cells and their ensuing synthesis and remodeling of the major fibrillar collagens, type I collagen and type II collagen, in bone and cartilage tissues during development and postnatal growth. Loss of the major collagenolytic activity associated with the membrane‐type 1 matrix metalloproteinase (MT1‐MMP) results in disrupted skeletal development and growth in both cartilage and bone, where MT1‐MMP is required for pericellular collagen dissolution. We show here that reconstitution of MT1‐MMP activity in the type II collagen‐expressing cells of the skeleton rescues not only diminished chondrocyte proliferation, but surprisingly, also results in amelioration of the severe skeletal dysplasia associated with MT1‐MMP deficiency through enhanced bone formation. Consistent with this increased bone formation, type II collagen was identified in bone cells and skeletal stem/progenitor cells of wildtype mice. Moreover, bone marrow stromal cells isolated from mice expressing MT1‐MMP under the control of the type II collagen promoter in an MT1‐MMP‐deficient background showed enhanced bone formation in vitro and in vivo compared with cells derived from nontransgenic MT1‐MMP‐deficient littermates. These observations show that type II collagen is not stringently confined to the chondrocyte but is expressed in skeletal stem/progenitor cells (able to regenerate bone, cartilage, myelosupportive stroma, marrow adipocytes) and in the chondrogenic and osteogenic lineage progeny where collagenolytic activity is a requisite for proper cell and tissue function.     

基质金属蛋白酶-13在骨性关节炎发病中的活性调控研究

目的 研究一氧化氮(NO)是否通过膜型基质金属蛋白酶-1(MT1-MMP)间接激活基质金属蛋白酶-13酶原(pro-MMP-13).方法 购买并传代人软骨肉瘤细胞(SW1353),用NO供体S-亚硝基-N-乙酰基青霉胺(SNAP),SNAP+NO清除荆氧合血红蛋白(OxyHb)和SNAP+组织金属蛋白酶抑制物-2(TI...     

The orphan G-protein coupled receptor RDC1: evidence for a role in chondrocyte hypertrophy and articular cartilage matrix turnover

OBJECTIVE: RDC1 is a class A orphan G-protein coupled receptor of unknown function. The purpose of this study was to identify compound RDC1 agonists and use these as tools to determine the effect of RDC1 activation in human chondrocytes and cartilage explant tissue. METHODS: Computational chemistry was employed to build a homology model of the RDC1 receptor. A virtual screen of in-house compounds was then performed and positive hits screened for their ability to invoke a Ca2+ response in a recombinant RDC1 HEK293 cell line, as measured by FLIPR. The effect of RDC1 activation on human chondrocytes and cartilage explant gene expression was determined by quantitative real-time polymerase chain reaction (PCR), and these effects validated as being mediated by RDC1 using siRNA antisense. RESULTS: Tissue expression profiling demonstrated that RDC1 expression was predominant in cartilage tissue. Treatment of human primary chondrocytes with RDC1 agonist induced a Ca2+ response, suggesting the receptor is active in this tissue type. Treatment for 24h with RDC1 agonist led to altered expression of a number of genes associated with chondrocyte hypertrophy and increased matrix degradation in human primary chondrocytes, and elevated total matrix metalloproteinase (MMP) activity in cartilage explant. Transfection with RDC1 siRNA caused a >90% reduction in human primary chondrocyte RDC1 expression and significantly reduced the impact of RDC1 agonist on the previously identified RDC1-regulated genes. CONCLUSIONS: RDC1 activation in human chondrocytes and cartilage explant leads to changes in gene expression and activity associated with chondrocyte hypertrophy, angiogenesis and increased matrix degradation, suggesting signalling via the RDC1 receptor may play an important role in the early development of osteoarthritis (OA).     

Cycle‐dependent matrix remodeling gene expression response in fatigue‐loaded rat patellar tendons

Expression profiling of selected matrix remodeling genes was conducted to evaluate differences in molecular response to low‐cycle (100) and high‐cycle (7,200) sub‐failure‐fatigue loading of patellar tendons. Using our previously developed in vivo patellar tendon model, tendons were loaded for 100 or 7,200 cycles and expression of selected metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and collagens were quantified by real‐time RT‐PCR at 1‐ and 7‐day post‐loading. Expression profiles were also obtained from lacerated tendons as an acute injury model. The high‐cycle group showed upregulation of TIMP‐1, ‐2, Col3a1, and Col5a1, and downregulation TIMP‐4 at both time points, upregulation of MMP‐2 at 7‐day post‐loading and downregulation of MMP‐13 and ‐14 at 1‐day post‐loading, suggesting overall repair/remodeling. In contrast, the low‐cycle loaded group showed upregulation of MMP‐2, ‐3, ‐13, and Col12a1 at both time points, upregulation of TIMP‐1, ‐2, ‐3, Col3a1, and integrin β1 and downregulation of integrin α11 at 1‐day post‐loading and upregulation of Col1a1 at 7‐day post‐loading, consistent with a hypertrophic (adaptive) pattern. Lacerated tendons showed a typical acute wound response with upregulation of all examined remodeling genes. Differences found in tendon response to high‐ and low‐cycle loading are suggestive of the underlying mechanisms associated with a healthy or damaging response. Published by Wiley Periodicals, Inc. J Orthop Res 28:1380–1386, 2010     

In Vivo Dual Fluorescence Imaging to Detect Joint Destruction

Diagnosis of cartilage damage in early stages of arthritis is vital to impede the progression of disease. In this regard, considerable progress has been made in near‐infrared fluorescence (NIRF) optical imaging technique. Arthritis can develop due to various mechanisms but one of the main contributors is the production of matrix metalloproteinases (MMPs), enzymes that can degrade components of the extracellular matrix. Especially, MMP‐1 and MMP‐13 have main roles in rheumatoid arthritis and osteoarthritis because they enhance collagen degradation in the process of arthritis. We present here a novel NIRF imaging strategy that can be used to determine the activity of MMPs and cartilage damage simultaneously by detection of exposed type II collagen in cartilage tissue. In this study, retro‐orbital injection of mixed fluorescent dyes, MMPSense 750 FAST (MMP750) dye and Alexa Fluor 680 conjugated monoclonal mouse antibody immune‐reactive to type II collagen, was administered in the arthritic mice. Both dyes were detected with different intensity according to degree of joint destruction in the animal. Thus, our dual fluorescence imaging method can be used to detect cartilage damage as well as MMP activity simultaneously in early stage arthritis.     

Fibronectin‐α4β1 Interactions in Hepatic Cold Ischemia and Reperfusion Injury: Regulation of MMP‐9 and MT1‐MMP via the p38 MAPK Pathway

Liver ischemia‐reperfusion injury (IRI) remains a challenging problem in clinical settings. The expression of fibronectin (FN) by endothelial cells is a prominent feature of the hepatic response to injury. Here we investigate the effects of the connecting segment‐1 (CS‐1) peptide therapy, which blocks FN‐α4β1 integrin leukocyte interactions, in a well‐established model of 24‐h cold liver IRI. CS‐1 peptides significantly inhibited leukocyte recruitment and local release of proinflammatory mediators (COX‐2, iNOS and TNF‐α), ameliorating liver IRI and improving recipient survival rate. CS1 therapy inhibited the phosphorylation of p38 MAPK, a kinase linked to inflammatory processes. Moreover, in addition to downregulating the expression of matrix metalloproteinase‐9 (MMP‐9) in hepatic IRI, CS‐1 peptide therapy depressed the expression of membrane type 1‐matrix metalloproteinase (MT1‐MMP/MMP‐14) by macrophages, a membrane‐tethered MMP important for focal matrix proteolysis. Inhibition of p38 MAPK activity, with its pharmacological antagonist SB203580, downregulated MMP‐9 and MT1‐MMP/MMP‐14 expressions by FN‐stimulated macrophages, suggesting that p38 MAPK kinase pathway controls FN‐mediated inductions of MMP‐9 and MT1‐MMP/MMP‐14. Hence, this study provides new insights on the role of FN in liver injury, which can potentially be applied to the development of new pharmacological strategies for the successful protection against hepatic IRI.     

Altered proteolytic activity and expression of MMPs and aggrecanases and their inhibitors in Kashin–Beck disease

Kashin–Beck disease (KBD) is a chronic, deforming endemic osteoarticular disease with altered metabolism of the cartilage matrix. Matrix metalloproteinases (MMPs), aggrecanases (ATAMTSs), and their inhibitors (TIMPs) play important roles in cartilage formation and matrix degradation. This study investigated these proteases and inhibitors in young KBD cartilage. The percentages of chondrocytes staining for MMP‐1/‐13 and MMP‐generated DIPEN neoepitope, aggrecanase‐generated ITEGE neoepitope in aggrecan in KBD patients were significantly higher than in controls. However, TIMP‐1 was significantly less numerous than in controls in the superficial and middle zones of KBD samples, the percentage of chondrocytes staining for the TIMP‐2 was significantly higher than in controls. Staining for MMP‐1/‐13 and, TIMP‐1/‐2 in KBD patients was prominent in the superficial zone and the middle zone of articular cartilage. Staining for ITEGE and DIPEN neoepitopes in KBD samples was prominent in the superficial zone and the middle zone of articular cartilage. The strongest staining for the MMP and aggrecanase‐generated neoepitopes was adjacent to areas of chondronecrosis. These results indicated that KBD cartilage destruction depends on collagen‐ and aggrecan‐degrading proteases such as collagenases (MMP‐1/‐13), as well as aggrecanases. Increased TIMP‐2 level adjacent to necrotic areas suggest that attempted repair mechanism are also activated. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:47–55, 2015.     

Cartilage degradation independent of MMP/aggrecanases

OBJECTIVE: To identify and characterize a cartilage degradation mechanism that is independent of the proteolytic cleavages by matrix metalloproteinases (MMPs) and aggrecanases. METHODS: The sensitivity of glycosaminoglycan (GAG) release and collagen release to an MMP/aggrecanase inhibitor, AG3340, was compared using a bovine nasal cartilage explant culture. The release of matrix proteins and hyaluronan (HA) from the culture was analyzed by immunoblotting and radioimmunoassay, respectively. Induction of HA-degrading activity by retinoic acid was examined using the cartilage explant culture and a primary culture of chondrocytes. Degradation of the matrix components of cartilage was also characterized in vivo using an acute arthritis model induced by an intra-articular injection of interleukin 1alpha (IL-1alpha). RESULTS: AG3340 did not effectively inhibit GAG release at a concentration of more than 10muM, while 10nM of the inhibitor completely suppressed collagen degradation. Retinoic acid induced the release of the aggrecan G1 domain, link protein and HA into the culture medium, and the release of these molecules was not completely inhibited by 10muM of AG3340. The molecules were released as ternary complexes. Retinoic acid induced HA degradation in the explant culture and hyaluronidase activity in the primary culture of chondrocytes. The release of the G1 domain of aggrecan and link protein into the synovial fluid was also observed in the IL-1alpha-induced acute arthritis model. CONCLUSION: A novel mechanism by chondrocyte-derived hyaluronidase(s) is involved in the release of the matrix components from cartilage, and the hyaluronidase(s) and MMPs/aggrecanases act in a coordinated manner in cartilage degradation.     

A single blunt impact on cartilage promotes fibronectin fragmentation and upregulates cartilage degrading stromelysin‐1/matrix metalloproteinase‐3 in a bovine ex vivo model

Post‐traumatic osteoarthritis (PTOA) is characterized by progressive cartilage degeneration in injured joints. Since fibronectin‐fragments (Fn‐fs) degrade cartilage mainly through up‐regulating matrix metalloproteinases (MMPs) and pro‐inflammatory cytokines, we hypothesized that Fn‐fs play a key role in PTOA by promoting chondrolysis in and around injured cartilage. To test this hypothesis, we profiled the catabolic events focusing on fibronectin fragmentation and proteinase expression in bovine osteochondral explants following a single blunt impact on cartilage with a drop tower device which created partial‐thickness tissue damage. Injured and control explants were cultured for up to 14 days. The presence of Fn‐fs, MMPs (‐1, ‐3, ‐13), ADAMTS‐5 in culture media and in cartilage was determined with immunoblotting. The daily proteoglycan (PG) depletion of cartilage matrix was assessed with DMMB assay. The effect of explant‐conditioned media on chondrocytes was also examined with immunoblotting. Impacted cartilage released significantly higher amount of native Fn, three chondrolytic Fn‐fs and PG than non‐impacted controls did. Those increases coincided with up‐regulation of MMP‐3 both in culture media and in impacted cartilage. These findings support our hypothesis that PTOA may be propelled by Fn‐fs which act as catabolic mediators through up‐regulating cartilage‐damaging proteinases. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:811–818, 2014.     

Estrogen modulates iodoacetate‐induced gene expression in bovine cartilage explants

Estrogen loss may be involved in onset or progression of osteoarthritis. Estrogen receptors are present in chondrocytes, thus estrogen may exert effects directly on cartilage. However, studies on direct estrogen effects on cartilage are limited. We investigated, in an in vitro cartilage explant model, whether estrogen prevents damage or stimulates repair after damage induced by addition of iodoacetate (IA), as an experimental model for osteoarthritis. We used healthy bovine cartilage explants. Prevention experiment: Explants precultured with/without estradiol (E) for 3 days were cultured with IA for 4 h on day 0, and subsequently cultured as in preculture: with/without E. Explants were harvested at day 2 for gene expression analysis. Repair experiment: At day 0, explants were cultured with IA for 4 h on day 0, and subsequently cultured without E or with E. Explants were harvested at days 2, 10, and 14 for gene expression analysis. IA transiently downregulated most genes tested, whereas vascular endothelial growth factor (VEGF) was upregulated on day 2. On day 14, transforming growth factor β (TGFB)1 and TGFB3 were upregulated, and matrix metalloproteinase (MMP)13 and VEGF downregulated. Estradiol affected gene expression of aggrecan (AGC)1, MMP2, MMP14, tissue inhibitor of metalloproteinase (TIMP)2, TGFB2, and TGFB3. Prevention experiment: Estradiol did not significantly affect IA‐induced changes in gene expression (no significant interaction). Repair experiment: Estradiol affected IA‐induced changes in expression of collagen (COL)2, MMP2, MMP3, MMP13, MMP14, TIMP2, TGFB2, TGFB3, and VEGF. Estradiol affects expression of anabolic and catabolic genes in bovine cartilage explants and modulates the effects of IA. These effects of estradiol may be beneficial for cartilage maintenance and repair. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:607–615, 2010