Objective: To determine if supplemental intraarticular α₂‐macroglobulin (α₂M) has a chondroprotective effect in a rat model of osteoarthritis (OA).Methods: Using Western blotting, mass spectrometry, enzyme‐linked immunosorbent assay (ELISA), and immunohistochemistry, α₂M was identified as a potential therapeutic agent through a comparison of α₂M concentrations in serum, synovial fluid (SF), and cartilage from normal subjects and patients with OA. In cultured chondrocytes, the effects of α₂M on interleukin‐1 (IL‐1)–induced cartilage catabolic enzymes were evaluated by Luminex assay and ELISA. In vivo effects on cartilage degeneration and matrix metalloproteinase 13 (MMP‐13) concentration were evaluated in male rats (n = 120) randomized to 1 of 4 treatments: 1) anterior cruciate ligament transection (ACLT) and saline injections, 2) ACLT and 1 IU/kg injections of α₂M, 3) ACLT and 2 IU/kg injections of α₂M, or 4) sham operation and saline injections. Rats were administered intraarticular injections for 6 weeks. The concentration of MMP‐13 in SF lavage fluid was measured using ELISA. OA‐related gene expression was quantified by real‐time quantitative polymerase chain reaction. The extent of OA progression was graded by histologic examination.Results: In both normal subjects and OA patients, α₂M levels were lower in SF as compared to serum, and in OA patients, MMP‐13 levels were higher in SF than in serum. In vitro, α₂M inhibited the induction of MMP‐13 by IL‐1 in a dose‐dependent manner in human chondrocytes. In the rat model of ACLT OA, supplemental intraarticular injection of α₂M reduced the concentration of MMP‐13 in SF, had a favorable effect on OA‐related gene expression, and attenuated OA progression.Conclusion: The plasma protease inhibitor α₂M is not present in sufficient concentrations to inactivate the high concentrations of catabolic factors found in OA SF. Our findings suggest that supplemental intraarticular α₂M provides chondral protection in posttraumatic OA.

Introduction: Previous observations implicate Indian hedgehog (Ihh) signaling in osteoarthritis (OA) development because it regulates chondrocyte hypertrophy and matrix metallopeptidase 13 (MMP-13) expression. However, there is no direct genetic evidence for the role of Ihh in OA, because mice with cartilage or other tissue-specific deletion of the Ihh gene die shortly after birth. We evaluated the role of Ihh in vivo via a Cre-loxP-mediated approach to circumvent the early death caused by Ihh deficiency. Methods: To evaluate the role of Ihh in OA development, Ihh was specifically deleted in murine cartilage using an Ihh conditional deletion construct (Col2a1-CreER ^T2 ; Ihh^fl/fl). The extent of cartilage degradation and OA progression after Ihh deletion was assessed by histological analysis, immunohistochemistry, real-time PCR and in vivo fluorescence molecular tomography (FMT) 2 months after OA was induced by partial medial meniscectomy. The effect of Ihh signaling on cartilage was compared between Ihh-deleted mice and their control littermates. Results: Only mild OA changes were observed in Ihh-deleted mice, while control mice displayed significantly more cartilage damage. Typical OA markers such as type X collagen and MMP-13 were decreased in Ihh-deleted mice. In vivo FMT demonstrated decreased cathepsins and MMP activity in knee joints of animals with deletion of Ihh. Conclusions: These findings support the protective role of Ihh deletion in surgically induced OA. Thus, our findings suggest the potential to develop new therapeutic strategies that can prevent and treat OA by inhibiting Ihh signaling in chondrocytes.

Purpose: To determine the importance of synovial fluid (SF) or subchondral bone marrow (BM) as nutrition sources in cartilage degeneration.Methods: Ninety‐five‐month‐old male rabbits were randomly divided into 5 groups according to sources of nutrition: SFBM‐both; BM‐only; SF‐only; None‐SFBM; and Free plug (unrestricted). Nutrition to 4‐mm‐diameter cylindrical osteochondral plugs created on the trochlea of the distal femurs was obstructed by Polyvinyl Chloride (PVC) cap. Cartilage changes were assessed after 4, 8, and 12 weeks by histology, immunohistochemistry, and real‐time PCR.Results: Cartilage in the BM‐only group suffered the greatest damage, followed by the None‐SFBM and SF‐only groups. Apoptosis was increased in the BM‐only and None‐SFBM groups compared with others. Cartilage was significantly thinner at all time points in the BM‐only and None‐SFBM groups when compared with SFBM‐both and Free plug, whereas in the SF‐only group, this difference occurred after 8 weeks. Compared with SFBM‐both and Free plug, expression of collagen II and aggrecan mRNAs in all groups was decreased but MMP‐3 increased, respectively.Conclusion: Our data indicate that SF‐derived nutrition is the dominant source of sustenance for adult cartilage structure and function. Cartilage damage is observed when the only nutrition source is the BM.

Introduction: To investigate if decreased histone deacetylase 4 (HDAC4) is associated with human osteoarthritis (OA) cartilage degeneration by releasing HDAC4 inhibition of runt-related transcription factor-2 (Runx2) resulting in increase of OA cartilage degeneration-related genes. Methods: The mRNA and protein levels of HDAC4, Runx2, matrix metalloproteinase (MMP)-13, Indian hedgehog (Ihh) and type X collagen were detected by performing real-time PCR (RT-PCR), western blotting and immunohistochemistry on specimens from human OA and normal cartilage. To further explore the mechanism of regulation of Runx2 and OA-related genes by HDAC4, changes in these OA-related genes were further quantified by RT-PCR after overexpression of HDAC4 and knockdown of HDAC4 by siRNA. Runx2 and MMP-13 promoter activities were measured by dual luciferase assays. Results: The levels of HDAC4 in the cartilage from OA patients and healthy 40- to 60-year-old donors were decreased to 31% and 65% compared with specimens from 20- to 40-year-old healthy donors, respectively (P <0.05). Decreased HDAC4 was associated with increased Runx2 and other OA-related genes in human OA cartilage, specifically: MMP-13, Ihh and type X collagen. Exogenous HDAC4 decreased the mRNA levels of Runx2, MMP1, MMP3, MMP-13, type X collagen, Ihh, ADAMTS-4 and -5, and increased the mRNA of type II collagen. In addition, the data also shows that overexpression of HDAC4 not only decreased the expression of interleukin (IL)-1β, Cox2 and iNos and increased the expression of aggrecan, but also partially blocked the effect of IL-1β on expression of catabolic events in human OA chondrocytes. HDAC4 also inhibited Runx2 promoter activity and MMP13 promotor activity in a dose-dependent manner. In contrast, inhibition of HDAC4 by TSA drug had an opposite effect. Conclusions: Our study is the first to demonstrate that decreased HDAC4 contributes, at least in part, to the pathogenesis of OA cartilage degeneration. Thus, HDAC4 may have chondroprotective properties by inhibiting Runx2 and OA-related genes.

Background: Chondrosarcoma is a disease that does not respond to conventional cytotoxic chemotherapy and expression of MMP1 is a marker for a poor prognosis. The mechanism of increased MMP1 expression in chondrosarcoma is not completely known. Our goal is to identify molecular pathways that could serve as therapeutic targets. Chondrosarcoma become hypoxic as they grow, are capable of eliciting an angiogenic response, and typically metastasize to the lungs. The present study determined the effect of hypoxia and specifically HIF-1a on expression of CXCR4 and MMP1 and their role in chondrosarcoma cell invasion.Results: CXCR4 and its ligand, SDF1, are upregulated in primary chondrosarcoma tumors compared to normal articular cartilage, and CXCR4 was upregulated in chondrosarcoma cell line JJ compared to normal chondrocytes. Hypoxia and specifically HIF-1a increased CXCR4 and MMP1 expression in JJ cell line and chondrosarcoma invasionin vitro. The hypoxia mediated increase in MMP1 expression and chondrosarcoma invasion could be inhibited by siRNA directed at HIF-1a or CXCR4, the CXCR4 inhibitor AMD3100, as well as with ERK inhibitor U0126 and ERK siRNA.Conclusions: Chondrosarcoma cell invasion is increased by hypoxia induced expression of CXCR4 and MMP1 and is mediated by HIF-1a and ERK. Both invasion and MMP1 can be inhibited with CXCR4 blockade, suggesting that CXCR4/SDF1 signaling may be a therapeutic target for chondrosarcoma.