Effect of hylan on cartilage and chondrocyte cultures.

The protective role of hylan, a hyaluronan [hyaluronic acid (HA)] derivative, was studied in explanted bovine cartilage and isolated chondrocytes. Cartilage and chondrocytes were exposed to degradative enzymes (lysate from activated polymorphonuclear leukocytes), oxygen-derived free radicals (ODFR), conditioned media from mononuclear cells (MCCM), and interleukin-1 (IL-1), in the presence and absence of hylan. The effect of HA was also studied. In cartilage explants susceptibility to pertubation was evaluated in terms of 35S release and proteoglycan depletion and was compared to control cultures; high viscosity hylan was found to reduce 35S release in cartilage explants caused by degradative enzymes, ODFR, MCCM, and IL-1. The hylan effect was reversible and viscosity-dependent. In chondrocyte cultures, high viscosity hylan was effective in reducing cell injury caused by degradative enzymes and ODFR. The data suggest that the glycosaminoglycan hylan, as well as native HA, may mediate exposure to and/or response to stimuli associated with initiation of degenerative processes in cartilage tissues.     

Cartilage Wear Particles Induce an Inflammatory Response Similar to Cytokines in Human Fibroblast‐Like Synoviocytes

The synovium plays a key role in the development of osteoarthritis, as evidenced by pathological changes to the tissue observed in both early and late stages of the disease. One such change is the attachment of cartilage wear particles to the synovial intima. While this phenomenon has been well observed clinically, little is known of the biological effects that such particles have on resident cells in the synovium. The present work investigates the hypothesis that cartilage wear particles elicit a pro‐inflammatory response in diseased and healthy human fibroblast‐like synoviocytes, like that induced by key cytokines in osteoarthritis. Fibroblast‐like synoviocytes from 15 osteoarthritic human donors and a subset of three non‐osteoarthritic donors were exposed to cartilage wear particles, interleukin‐1α or tumor necrosis factor‐α for 6 days and analyzed for proliferation, matrix production, and release of pro‐inflammatory mediators and degradative enzymes. Wear particles significantly increased proliferation and release of nitric oxide, interleukin‐6 and ‐8, and matrix metalloproteinase‐9, ‐10, and ‐13 in osteoarthritic synoviocytes, mirroring the effects of both cytokines, with similar trends in non‐osteoarthritic cells. These results suggest that cartilage wear particles are a relevant physical factor in the osteoarthritic environment, perpetuating the pro‐inflammatory and pro‐degradative cascade by modulating synoviocyte behavior at early and late stages of the disease. Future work points to therapeutic strategies for slowing disease progression that target cell‐particle interactions. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1979–1987, 2019     

The synthetic processes of articular cartilage

The cells of cartilage are constantly remodeling the matrix in which they are suspended. The stimulus to initiate remodeling is probably the chondrocyte's response to physical and or chemical changes in the environment. Heat, pressure, friction, load, pH, and growth are examples of such factors, which, if altered, would have a dramatic effect on the cell's state of health. The mode of response by the chondrocyte is specific for a given stimulus. Elevated temperature, for example, switches on a set of genes, the heat shock genes, in chondrocytes. This results in the synthesis of a series of cellular protein that presumably in turn protects the cell from the injurious effects of heat. Load and pressure affect both the synthetic rate of matrix protein and the degradation rates of preexisting matrix. A number of low-molecular-weight proteins appear to be involved in anabolic and catabolic processes of cartilage. A protein recently isolated from synovium stimulates the synthesis of degradative enzymes in cartilage. This factor is probably involved in the remodeling process under normal physiologic conditions. More recently, it has been found in elevated levels under pathologic conditions such as in the synovial fluid of patients with rheumatoid and osteoarthritis. The mechanism by which this factor turns on the degradative pathway appears complex and is under investigation.     

Localization and expression of cartilage oligomeric matrix protein by human rheumatoid and osteoarthritic synovium and cartilage.

Synovium and cartilage from patients with osteoarthritis or rheumatoid arthritis were analyzed for expression of cartilage oligomeric matrix protein. Immunostaining of synovium with antiserum to cartilage oligomeric matrix protein demonstrated positive staining in both diseases. In osteoarthritis, there was positive staining within the synovial cells and immediately subjacent connective tissue, with less intense staining in the deeper connective tissue. In rheumatoid arthritis, there was less intense staining within the synovial cells and marked intense staining in the deeper connective tissue. In situ hybridization performed with an antisense digoxigenin-labeled riboprobe to human cartilage oligomeric matrix protein confirmed the presence of cartilage oligomeric matrix protein mRNA in the cells of the synovial lining in both types of synovium. Quantitative polymerase chain reaction with a cartilage oligomeric matrix protein MIMIC demonstrated increased cartilage oligomeric matrix protein mRNA in rheumatoid cartilage and synovium as compared with osteoarthritic cartilage and synovium, respectively; mRNA levels in rheumatoid synovium were similar to those from osteoarthritic chondrocytes. As a result of the high expression of cartilage oligomeric matrix protein from rheumatoid synovium, inflammatory synovium should be considered as a potential tissue source of cartilage oligomeric matrix protein in any investigation of biological markers of cartilage metabolism. The upregulated expression of cartilage oligomeric matrix protein in inflammatory tissues suggests its in vivo regulation by cytokines.     

Synovial factors and chondrocyte-mediated breakdown of cartilage: inhibition by hydrocortisone

Cartilage-synovium interactions were explored in a model culture system. Bovine nasal-cartilage discs were cocultured with minced rheumatoid synovium or synovium-conditioned media (SCM) in the presence or absence of hydrocortisone. Cartilage breakdown was assessed by the release of proteoglycan (PG) and hydroxyproline, and matrix biosynthesis by [35S]sulfate incorporation during pulse labeling. Chondrocyte-dependent breakdown in response to synovial factors (i.e., "catabolin" activity) was assessed by the difference in PG release between living and dead cartilages. Short-term contact with minced synovial membrane or exposure to its products released at a distance was sufficient to induce cartilage degradation in coculture; continued exposure was not required for breakdown to persist. Conditioned media from short-term synovial culture were similarly potent, and the induced breakdown was chondrocyte dependent. Matrix biosynthesis was inhibited in exposed cartilage but could be rapidly restored to normal on synovium removal despite the persistence of cartilage breakdown. Early hydrocortisone treatment suppressed the initiation of cartilage breakdown in cocultures and largely abolished the appearance of inductive factors in SCM. Later applications had little effect either in cocultures or in catabolin assays. We conclude that synovium-induced breakdown is an early event and that chondrocyte catabolic mechanisms once they have been activated are sufficient to maintain breakdown at a high level. Hydrocortisone, as well as limiting proteolysis, inhibits early tissue interactions at the level of synovial catabolin production or release.     

Studies on YKL-40 in knee joints of patients with rheumatoid arthritis and osteoarthritis. Involvement of YKL-40 in the joint pathology

OBJECTIVE: The presence of YKL-40 (human cartilage glycoprotein 39) in synovium, cartilage and synovial fluid (SF) from knee joints of patients with rheumatoid arthritis and osteoarthritis (OA) were related to histopathological changes in synovium and cartilage and to serum YKL-40 and other biochemical markers. METHODS: The localization of YKL-40 in synovium and cartilage was determined by immunohistochemistry. Synovial inflammation was estimated histologically and by magnetic resonance imaging (MRI). Biochemical markers of inflammation, neutrophil activation and cartilage metabolism were analysed. YKL-40 concentrations in serum and SF were determined by RIA and ELISA. RESULTS: In the synovium YKL-40 positive cells were found in lining and stromal cells (macrophages) and the number of YKL-40 positive cells was related to the degree of synovitis. In arthritic cartilage, YKL-40 was located to chondrocytes. YKL-40 levels in SF were higher in RA patients with moderate/severe or none/slight synovitis of the knee joint compared to OA patients with moderate/severe or none/slight synovitis. SF YKL-40 correlated with the synovial membrane and the joint effusion volumes determined by magnetic resonance imaging (MRI) and with other biochemical markers of intercellular matrix metabolism. SF YKL-40 was higher than serum YKL-40, and a relationship existed between the YKL-40 levels in SF and serum. Intraarticular glucocorticoid injection was followed by clinical remission and a decrease in serum YKL-40, which increased again at clinical relapse. CONCLUSIONS: YKL-40 in SF is derived from cells in the inflamed synovium, chondrocytes and SF neutrophils. Joint derived YKL-40 influences serum YKL-40. YKL-40 may be involved in the pathophysiology of the arthritic processes and reflect local disease activity.     

胎猪BMSC体外构建软骨的实验研究

目的比较胎猪骨髓间充质干细胞（Bone Marrow Mesenchymal Stem Cells,BMSCs）和成年猪BMSCs构建软骨能力的差异,寻找合适的同种异体组织工程软骨种子细胞来源。方法通过剖腹产手术获得胎龄为70 d的胎猪,胎猪骨髓液贴壁培养获得胎猪BMSCs;抽取成年猪骨髓液,经贴壁培养法获得成年猪BMSCs。两种细胞体外扩增培养后,观察第3代细胞形态,并进行成骨、成脂和成软骨诱导。分别取两种细胞以1×108 cells/mL的细胞终浓度,接种于聚乳酸包埋的聚羟基乙酸支架,体外诱导培养8周后取材。通过大体观察、糖胺聚糖（GAG）含量测定、总胶原含量测定、组织学,以及免疫组化等方法,对两种细胞构建的组织工程软骨的相关生物学特性进行比较。结果胎猪BMSCs比成年猪BMSCs具有更好的增殖和成骨、成脂和成软骨能力。胎猪BMSCs构建的软骨有良好的软骨外观,而且GAG含量和总胶原含量均高于成年猪BMSCs构建的软骨（P<0.01）。组织学和免疫组化显示,胎猪BMSCs构建的软骨组织结构致密,基质及Ⅱ型胶原显色程度均明显强于成年猪BMSCs构建的软骨。结论胎猪BMSCs是组织工程软骨较好的种子细胞来源。     

Tumor necrosis factor alpha can contribute to focal loss of cartilage in osteoarthritis

OBJECTIVE; To evaluate the potential for tumor necrosis factor alpha (TNFalpha)-induced focal loss of cartilage in osteoarthritic (OA) knee joints. DESIGN: Fresh cartilage from specified regions of OA joints was immunostained for TNF-receptor (R) bearing chondrocytes. Cartilage explants from the same regions were cultured with or without small amounts of TNFalpha and cumulative GAG release into supernatants measured. Concentrations of TNFalpha, p55 and p75 soluble (s) TNF-R in supernatants from cultured OA and non-arthritic (NA) synovium were measured by ELISA. RESULTS: TNF-R bearing chondrocytes were identified in OA cartilage; more specimens contained p55 TNF-R- than p75 TNF-R-bearing chondrocytes and differences in TNF-R distribution were apparent in cartilage from different regions of the same knees. TNFalpha at 5, 1, 0.5 and 0.25 ng/ml (but not 0.1 ng/ml) significantly increased glycosaminoglycans (GAG) release from cartilage explants in a dose-dependent manner. Variation in susceptibility to TNFalpha was observed in explants from different sites. TNFalpha and p75 sTNF-R, but not p55 sTNF-R, concentrations were significantly higher in OA, as compared with NA, supernatants. A significant correlation between TNFalpha and p75 sTNF-R measurements was apparent only in NA supernatants. CONCLUSIONS: Variations in chondrocyte TNF-R expression occur in OA cartilage in vivo. TNFalpha at concentrations produced by OA synovium in vitro, can degrade cartilage matrix. In most OA supernatants sTNF-R concentrations were insufficient to abrogate the effects of TNFalpha. Thus conditions exist in some OA knees for TNFalpha to contribute to focal loss of cartilage.     

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.     

一氧化氮在骨性关节炎发病机制中的作用

目的 研究一氧化氮与骨性关节炎发病的关系。方法 抽取骨性关节炎和类风湿性关节类患者的关节积液和血清,及关节镜下清理术中切除的骨性关节炎滑膜和退变软骨作为标本,用硝酸还原酶法测定其中的一氧化氮含量并进行比较。结果 骨性关节炎患者的软骨及滑膜中一氧化氮含量明显高于其血清及关节液,且软骨中一氧化氮含量明显高于滑膜。类风湿性关节炎患者关节液中一氧化氮浓度明显高于其血清及骨性关节炎的关节液。结论 骨性关节炎     

Tendon synovial cells secrete fibronectin in vivo and in vitro

The chemistry and cell biology of the tendon have been largely overlooked due to the emphasis on collagen, the principle structural component of the tendon. The tendon must not only transmit the force of muscle contraction to bone to effect movement, but it must also glide simultaneously over extratendonous tissues. Fibronectin is classified as a cell attachment molecule that induces cell spreading and adhesion to substratum. The external surface of intact avian flexor tendon stained positively with antibody to cellular fibronectin. However, if the surface synovial cells were first removed with collagenase, no positive reaction with antifibronectin antibody was detected. Analysis of immunologically stained frozen sections of tendon also revealed fibronectin at the tendon synovium, but little was associated with cells internal in tendon. The staining pattern with isolated, cultured synovial cells and fibroblasts from the tendon interior substantiated the histological observations. Analysis of polyacrylamide gel profiles of 35S-methionine-labeled proteins synthesized by synovial cells and internal fibroblasts indicated that fibronectin was synthesized principally by synovial cells. Fibronectin at the tendon surface may play a role in cell attachment to prevent cell removal by the friction of gliding. Alternatively, fibronectin, with its binding sites for hyaluronic acid and collagen, may act as a complex for boundary lubrication.     

Cell migration after synovium graft interposition at tendon repair site

Background

We have recently reported that interpositional synovium grafts from tendon sheath have a potential to accelerate tendon healing when implanted at the repair site. The purpose of this study was to investigate the effect of orientation of the synovium after synovium graft transplantation, by comparing the ability of cells from the visceral and parietal surfaces to migrate into the tendon in a canine tissue culture model.

Methods

The synovium graft was placed within a complete tendon laceration, with either the visceral or parietal surface facing the proximal end of the lacerated tendon. The number of migrating cells was quantified by a cell migration assay. Qualitative immunohistochemistry and confocal laser microscopy were also used at day 10.

Results

Many labeled synovial cells were observed within the tendon to which the visceral surface of the synovium graft was facing. Migrated cells were also observed on the parietal side, but there were fewer cells compared to visceral surface cells. Migrating cells all expressed α-smooth muscle actin.

Conclusion

We found that graft orientation affected cell migration. Whether this finding has clinical significance awaits in vivo study.     

Use of 1.5% glycine as a nonconductive fluid medium for arthroscopic electrosurgery

Sixty-five surgical cases were studied to determine whether 1.5% glycine, used as an irrigation solution during electrosurgery to the knee, had any histological effect on the synovial cells or the articular cartilage. Histologic specimens of the articular cartilage were stained with Safranin-O to assess proteoglycans-enhanced chrondrocyte function, and the synovium was stained with pentachrome. The results indicated that there were no adverse effects, either short term or long term, when viewing both the synovium and the articular cartilage histopathologically after glycine irrigation subsequent to the electrothermal procedure.     

Production of catabolin by synovium from an experimental model of osteoarthritis

Catabolic factors from synovium, or catabolins, may be important in the pathogenesis of osteoarthritis. The synovial production of catabolin in osteoarthritis was studied using the experimental model of osteoarthritis induced by section of the anterior cruciate ligament. At 1, 4, and 32 weeks after operation, the synovium was harvested, cultured, and the synovial conditioned medium assayed for catabolic activity. The synovial conditioned medium from the operated joints was significantly more capable of inducing cartilage destruction (p less than 0.05) than was the synovial conditioned medium from the unoperated knees. However, when the results were expressed relative to the weight of synovium, the differences were not significant. These results suggest that the increased production of catabolic synovial factors in early experimental osteoarthritis is probably a result of the synovial hypertrophy.     

A biphasic tumor of the mediastinum with features of synovial sarcoma. A report of four cases

Four cases of a biphasic mediastinal tumor histologically identical to synovial sarcoma of soft tissue were observed. The tumors presented as solitary mediastinal masses. Although the tumors were frequently adherent to adjacent pleura or pericardium, none appeared to be arising from a mesothelial surface. All cases were composed of an intimate admixture of keratin-positive epithelial cells and vimentin-positive spindle cells with areas of transition, hyalinization, and calcification. Follow-up was available on three patients, who died of their disease 10 months, 14 months, and 4 years after diagnosis, respectively. Although synovial sarcoma usually occurs in deep soft tissues near joints in the extremities, it has been reported in locations removed from synovial, tendon sheath, and bursal structures. This report adds a previously unrecognized location for synovial sarcoma, the mediastinum. Occurrence in this location further supports an origin from pluripotential mesenchyme as opposed to synovium. The differential diagnosis includes mesothelioma, thymoma, germ cell tumors, malignant peripheral nerve sheath tumor with glandular differentiation, and metastatic carcinoma.     

Autologous synovial fluid enhances migration of mesenchymal stem cells from synovium of osteoarthritis patients in tissue culture system

Synovial fluid from osteoarthritic knee contains mesenchymal stem cells (MSCs). One of the possible reservoirs of MSCs in synovial fluid is synovial tissue, and synovial fluid may induce mobilization of MSCs into synovial fluid in osteoarthritis patients. Here, we investigated whether synovial fluid expanded synovial MSCs in a tissue culture system. Human synovium and synovial fluid were obtained from osteoarthritis patients during total knee arthroplasties. In the tissue culture system, autologous synovial fluid expanded synovial cells statistically higher than αMEM + FBS, and the addition of TGFβ3 to αMEM + FBS increased expansion to a similar level in all 11 donors. The addition of decorin or anti‐TGFβ neutralizing antibody to synovial fluid partially inhibited synovial cell expansion. In cell culture assay, synovial fluid proliferated synovial cells fewer than αMEM + FBS. The expanded synovial cells in synovial fluid retained multipotentiality and showed surface markers similar to those of MSCs. We demonstrated that autologous synovial fluid enhanced expansion of MSCs in tissue culture of synovium from osteoarthritis patients by promoting cell migration. This effect was partially affected by TGFβ. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1413–1418, 2008     

Induction of chondrogenesis and superficial zone protein accumulation in synovial side population cells by BMP‐7 and TGF‐β1

Synovial cells are known to contain a sub‐population of cells with multipotent differentiating capacity including chondrocytes. However, the stem/progenitor cells in synovial cells have not been well characterized. Stem/progenitor cells can exclude Hoechst 33342 dye, and the cell fraction with this property is called “side population (SP).” SP cells are present in many adult tissues. The aim of this investigation was to identify, isolate, and characterize SP cells from bovine synovium. Hoechst dye efflux and fluorescence activated cell sorting showed that synovial cells contained 0.60% SP cells. In the presence of verapamil, an inhibitor of ABC transporters critical for the dye efflux property, the SP cell fraction was not observed, indicating the critical role of ABC transporters. Isoforms of ABC transporters (ABCG2 and ABCB1 mRNA) were highly expressed in SP cells derived from the synovial cells by real‐time RT‐PCR analysis. Bone morphogenetic protein‐7 (BMP‐7) induced type II collagen mRNA expression characteristic of chondrogenesis in articular cartilage with both SP and non‐SP cells. In addition, expression of SZP mRNA, a marker of the surface layer of articular cartilage, was significantly up‐regulated by BMP‐7, and the protein accumulation of SZP was stimulated by both BMP‐7 and TGF‐β1. Thus, synovial cells contain ABC transporter‐dependent SP cells. These findings demonstrate that side population cells of synovium differentiate toward an articular chondrocyte phenotype of the surface layer and have direct implications for tissue engineering and regeneration of articular cartilage. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:485–492, 2008     

Expression of cartilage-related genes in bovine synovial tissue.

The synovium contains mesenchymal stem cells with chondrogenic potential. Although synovial and articular cartilage tissue develop from a common pool of mesenchymal cells, little is known about their genetic commonalities. In the present study, the mRNA levels for several cartilage-related proteins, namely, cartilage oligomeric matrix protein (COMP), Sox9, aggrecan, and collagen types I, II, IX, X, and XI, were measured using the real-time polymerase chain reaction. Our data reveal the synovium of calf metacarpal joints to physiologically express not only type I collagen but also COMP, Sox9, aggrecan, and collagen types X and XI. The mRNA levels for the latter five proteins lie between 2% and 15% of those in articular cartilage. We speculate that these genes are being expressed by chondroprogenitor cells, whose presence in the synovium reflects a common ontogenetic phase in the fetal development of this tissue and of articular cartilage.