Intra‐articular resveratrol injection prevents osteoarthritis progression in a mouse model by activating SIRT1 and thereby silencing HIF‐2α

We investigated the feasibility of the intra‐articular injection of resveratrol for preventing the progression of existing cartilage degeneration in a mouse model of osteoarthritis (OA). The effects of resveratrol on the expression of silent information regulator 2 type 1 (SIRT1), hypoxia‐inducible factor‐2α (HIF‐2α) and catabolic factors in OA cartilage was explored. OA was induced in the mouse knee via destabilization of the medial meniscus (DMM). Resveratrol was injected weekly into the operated knee beginning 4 weeks after surgery. The OA phenotype was evaluated via histological and immunohistochemical analyses at 8 weeks after DMM. Western blot analysis was performed to identify whether resveratrol modulated the interleukin (IL)‐1β‐induced expression of HIF‐2α in human chondrocytes. Histologically, resveratrol treatment preserved the structural homeostasis of the articular cartilage and the subchondral bone. Following resveratrol injection, the expression of collagen type II was retained, but the expression of inducible nitric oxide synthase and matrix metalloproteinase‐13 was reduced in OA cartilage. Moreover, the administration of resveratrol significantly induced the activation of SIRT1 and the inhibition of HIF‐2α expression in mouse OA cartilage and in IL‐1β‐treated human chondrocytes. These findings indicate that the intra‐articular injection of resveratrol significantly prevents the destruction of OA cartilage by activating SIRT1 and thereby suppressing the expression of HIF‐2α and catabolic factors. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1061–1070, 2015.     

Gait and behavior in an IL1β‐mediated model of rat knee arthritis and effects of an IL1 antagonist

Interleukin‐1 beta (IL1β) is a proinflammatory cytokine that mediates arthritic pathologies. Our objectives were to evaluate pain and limb dysfunction resulting from IL1β over‐expression in the rat knee and to investigate the ability of local IL1 receptor antagonist (IL1Ra) delivery to reverse‐associated pathology. IL1β over‐expression was induced in the right knees of 30 Wistar rats via intra‐articular injection of rat fibroblasts retrovirally infected with human IL1β cDNA. A subset of animals received a 30 µl intra‐articular injection of saline or human IL1Ra on day 1 after cell delivery (0.65 µg/µl hIL1Ra, n = 7 per group). Joint swelling, gait, and sensitivity were investigated over 1 week. On day 8, animals were sacrificed and joints were collected for histological evaluation. Joint inflammation and elevated levels of endogenous IL1β were observed in knees receiving IL1β‐infected fibroblasts. Asymmetric gaits favoring the affected limb and heightened mechanical sensitivity (allodynia) reflected a unilateral pathology. Histopathology revealed cartilage loss on the femoral groove and condyle of affected joints. Intra‐articular IL1Ra injection failed to restore gait and sensitivity to preoperative levels and did not reduce cartilage degeneration observed in histopathology. Joint swelling and degeneration subsequent to IL1β over‐expression is associated limb hypersensitivity and gait compensation. Intra‐articular IL1Ra delivery did not result in marked improvement for this model; this may be driven by rapid clearance of administered IL1Ra from the joint space. These results motivate work to further investigate the behavioral consequences of monoarticular arthritis and sustained release drug delivery strategies for the joint space. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:694–703, 2011     

Comparison of gene expression patterns in articular cartilage and dedifferentiated articular chondrocytes

During monolayer culture, articular chondrocytes dedifferentiate into fibroblast‐like cells. The mechanisms underlying this process are poorly understood. We sought to further characterize dedifferentiation by identifying an extended panel of genes that distinguish articular cartilage from dedifferentiated chondrocytes. Thirty‐nine candidate marker‐genes were identified from previous studies on articular‐cartilage gene‐expression. Real‐time PCR was used to evaluate the mRNA levels for these candidates in calf articular cartilage and dedifferentiated articular chondrocytes. Twenty‐two of the candidate marker genes exhibited at least a two‐fold difference in gene expression in the two cell types. Twelve of these genes had at least a ten‐fold difference in gene expression. Tenascin C (TNC), type I collagen (COL1A1), and hypoxia‐inducible factor 1 alpha (HIF1α) showed the highest relative expression levels in dedifferentiated chonodrocytes. Type II collagen (COL2A1), type XI collagen (COL11A2), and superficial zone protein (SZP) showed the highest relative expression levels in articular cartilage. In contrast to previous findings, fibromodulin mRNA, and protein levels were higher in dedifferentiated chondrocytes. Compared to smaller subsets of markers, this panel of 12 highly differentially expressed genes may more precisely distinguish articular cartilage from dedifferentiated chondrocytes. Since many of the genes up‐regulated in dedifferentiated chondrocytes are also expressed during cartilage development, dedifferentiated chondrocytes may possess features of cartilage precursor cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:234–245, 2012     

Effects of heme oxygenase‐1 on bacterial antigen‐induced articular chondrocyte catabolism in vitro

This study tested the hypothesis that heme oxygenase‐1 (HO‐1) expression counteracts bacterial antigen‐induced catabolic metabolism in human articular chondrocytes. HO‐1 expression was induced in chondrocytes by the iron‐containing porphoryin, hemin. Anti‐catabolic and anti‐apoptotic effects of HO‐1 expression were evaluated following bacterial antigen (lipopolysaccharides, LPS) activation of chondrocytes by quantification of cytokine and cartilage matrix protein expression. Effects of HO‐1 over‐expression on chondrocyte matrix metabolism were evaluated using plasmid‐driven protein synthesis. Hemin increased HO‐1 expression and LPS increased interleukin‐1beta and interleukin‐6 gene and protein expression in chondrocytes. Hemin‐induced HO‐1 decreased LPS‐induced interleukin‐1beta and interleukin‐6 gene and protein expression. Increased HO‐1 expression partially reversed LPS‐suppression of aggrecan and type II collagen gene expression and suppressed LPS‐induced gene expression of IL‐6, inducible nitric oxide synthase (iNOS), matrix metalloproteinases (MMPs), and IL‐1beta. HO‐1 induction was inversely correlated with LPS‐induced chondrocyte apoptosis. HO‐1 over‐expression in chondrocytes decreased matrix protein gene expression. With LPS activation, increased HO‐1 expression decreased chondrocyte catabolism, partially reversed LPS‐dependent inhibition of cartilage matrix protein expression and protected against apoptosis. Without LPS, hemin‐induced HO‐1 and plasmid‐based over‐expression of HO‐1 inhibited cartilage matrix gene expression. The results suggest that elevated HO‐1 expression in chondrocytes is protective of cartilage in inflamed joints but may otherwise suppress matrix turn over. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1943–1949, 2013     

The involvement of beta1 integrin in the modulation by collagen of chondrocyte-response to transforming growth factor-beta1.

The physiologic response of chondrocytes to maintenance of the matrix and response to injury likely involves signaling from multiple sources including soluble cytokines, mechanical stimulation, and signaling from the extracellular matrix. The signaling from the extracellular matrix may serve to effect cell differentiation and to modulate the response to cytokines. We have previously reported that type II collagen modulates the response of bovine articular chondrocytes to TGF-beta1. The molecular nature of the signaling mechanism has not been elucidated but presumably involves a similar mechanism by which the cell attaches to the surrounding matrix. An alginate bead culture system is utilized to which exogenous type II collagen is added. The inclusion of type II collagen results in an alteration of integrin expression with a down regulation of alpha2. The response of the chondrocyte to TGF-beta1 can be modulated by the inclusion of exogenous type II collagen. The modulation of DNA and proteoglycan synthesis was blocked by the treatment of anti-beta1 integrin antibody (4B4) or by cyclic RGD containing peptides. These events occur at concentrations that block cell adhesion to type II collagen. Linear RGD containing peptides and anti-anchorin antibodies had no effect on the modulation by type II collagen. These results suggest that type II collagen binding by chondrocytes at least in part occurs through the beta1 integrin. This binding results in modulation of the cell response to TGF-beta1. This modulation may serve to provide physiologic specificity to the cytokine-signaling cascade. An understanding of the regulatory milieu of the chondrocyte may permit the stimulation of an intrinsic repair of articular cartilage in the future. A near term application of this understanding can be made to tissue engineering attempts at articular cartilage repair.     

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     

Protective effect of atorvastatin in cultured osteoarthritic chondrocytes

The aim of our study was to evaluate the in vitro effect of an HMG‐CoA reductase inhibitor, atorvastatin, on the expression of significant anabolic and catabolic genes in human osteoarthritic chondrocytes and to explore the metabolic pathways involved in this process. Human articular osteoarthritic chondrocytes were cultured in the presence and absence of atorvastatin (10 and 50 µmol/L) for 24 h. Metalloproteinase 13 (MMP‐13), collagen type II (COL2A1), and aggrecan (AGC) mRNA expression levels were evaluated by real‐time PCR, and protein expression levels by Western blot analysis. IL‐1β levels in culture medium was analyzed with ELISA. The effect of the treatment with the mevalonate isoprenoid derivatives farnesol and geranylgeraniol, or the cholesterol precursor squalene, was evaluated in the atorvastatin osteoarthritic chondrocyte cultures. Incubation of osteoarthritic chondrocyte cultures with atorvastatin produced a significant dose‐dependent reduction in IL‐1β production. Atorvastatin supplementation in cultures produced a decrease in MMP‐13 mRNA and protein expression levels, which was reversed by the addition of farnesol. Regarding AGC and COL2A1 mRNA expression, a significant increase was observed only in chondrocytes cultures treated with 50 µmol/L atorvastatin. Our findings suggest that atorvastatin may have potential chondroprotective effects mostly by reducing cartilage degradation. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:110–115, 2010     

Connective tissue growth factor mRNA expression pattern in cartilages is associated with their type I collagen expression

Connective tissue growth factor (CTGF) has been identified as a secretory protein encoded by an immediate early gene and is a member of the CCN family. In vitro CTGF directly regulates the proliferation and differentiation of chondrocytes; however, a previous study showed that it was localized only in the hypertrophic chondrocytes in the costal cartilages of E 18 mouse embryos. We described the expression of CTGF mRNA and protein in chondrocytes of different types of cartilages, including femoral growth plate cartilage, costal cartilage, femoral articular cartilage, mandibular condylar cartilage, and cartilage formed during the healing of mandibular ramus fractures revealed by in situ hybridization and immunohistochemistry. To characterize the CTGF-expressing cells, we also analyzed the distribution of the type I, type II, and type X collagen mRNA expression. Among these different types of cartilages we found distinct patterns of CTGF mRNA and protein expression. Growth plate cartilage and the costal cartilage showed localization of CTGF mRNA and protein in the hypertrophic chondrocytes that expressed type X collagen mRNA with less expression in proliferating chondrocytes that expressed type II collagen mRNA, whereas it was also expressed in the proliferating chondrocytes that expressed type I collagen mRNA in the condylar cartilage, the articular cartilage, and the cartilage appearing during fracture healing. In contrast, the growth plate cartilages or the costal cartilages were negative for type I collagen and showed sparse expression of CTGF mRNA in the proliferating chondrocytes. We found for the first time that CTGF mRNA could be differentially expressed in five different types of cartilage associated with those expressing type I collagen. Moreover, the spatial distribution of CTGF mRNA in the cartilages with type I collagen mRNA suggested its roles in the early differentiation, as well as in the proliferation and the terminal differentiation, of those cartilages.     

Autologous solution protects bovine cartilage explants from IL‐1α‐ and TNFα‐induced cartilage degradation

Osteoarthritis (OA) is characterized by deterioration of articular cartilage driven by an imbalance of pro‐ and anti‐inflammatory cytokines. To address the cartilage deterioration observed in OA, an autologous protein solution (APS) has been developed which has been shown to inhibit the production of destructive proteases and inflammatory cytokines from chondrocytes and monocytes, respectively. The purpose of this study was to determine the chondroprotective effect of APS on IL‐1α‐ or TNFα‐challenged bovine articular cartilage explants. Cartilage explants were cultured in the presence or absence of recombinant inflammatory cytokines, IL‐1α and TNFα. Explants under equivalent inflammatory conditions were pretreated with recombinant antagonists IL‐1ra, sTNF‐RI, or APS to measure their inhibition of matrix degradation. Explants were further evaluated with Safranin‐O, Masson's Trichrome, and Hematoxylin and Eosin histological staining. APS was more effective than recombinant antagonists in preventing cartilage matrix degradation and inhibited any measurable IL‐1α‐induced collagen release over a 21‐day culture period. APS treatment reduced the degree of Safranin‐O staining loss when cartilage explants were cultured with IL‐1α or TNFα. Micrographs of APS treated cartilage explants showed an increase in observed cellularity and apparent cell division. APS may have the potential to prevent cartilage loss associated with early OA. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1929–1935, 2013     

Low‐intensity pulsed ultrasound (LIPUS) increases the articular cartilage type II collagen in a rat osteoarthritis model

In this study, the effect of low‐intensity pulsed ultrasound (LIPUS) on cartilage was evaluated in a rat osteoarthritis (OA) model using serum biomarkers such as CTX‐II (type II collagen degradation) and CPII (type II collagen synthesis) as well as histological criteria (Mankin score and immunohistochemical type II collagen staining). OA was surgically induced in the knee joint of rats by anterior cruciate/medial collateral ligament transection and medial meniscus resection (ACLT + MMx). Animals were divided into three groups: sham‐operated group (Sham), ACLT + MMx group without LIPUS (?LIPUS), and ACLT + MMx group with LIPUS (+LIPUS; 30 mW/cm², 20 min/day for 28 days). CTX‐II levels were elevated in both ?LIPUS and +LIPUS groups compared to that in the Sham group after the operation, but there was no significant difference between +LIPUS and ?LIPUS groups, suggesting that LIPUS does not affect the degradation of type II collagen in this model. In contrast, CPII was significantly increased in +LIPUS group compared to ?LIPUS and Sham. Moreover, histological damage on the cartilage (Mankin score) was ameliorated by LIPUS, and type II collagen was immunohistochemically increased by LIPUS in the cartilage of an OA model. Of interest, mRNA expression of type II collagen was enhanced by LIPUS in chondrocytes. Together these observations suggest that LIPUS is likely to increase the type II collagen synthesis in articular cartilage, possibly via the activation of chondrocytes and induction of type II collagen mRNA expression, thereby exhibiting chondroprotective action in a rat OA model. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:361–369, 2010     

Type II collagen modulates the composition of extracellular matrix synthesized by articular chondrocytes.

The articular cartilage extracellular matrix (ECM) interfaces with chondrocytes and influences many biological processes important to cartilage homeostasis and repair. The alginate bead culture system can be viewed as a model of cartilage repair in which the chondrocyte attempts to recreate the pericellular matrix while maintaining a differentiated phenotype. The purpose of this study was to evaluate the alteration in epitopes of proteoglycan and tenascin synthesized by chondrocytes in the presence of exogenous extracellular type II collagen. We evaluated the effects on four biomarkers associated with the creation of the denovo matrix using ELISA and immunohistochemistry: keratan sulfate epitope (5D4), 3B3(-) neoepitope of chondroitin-6- sulfate, 3B3(+) chondroitinase-generated epitope of chondroitin-6-sulfate, and tenascin-C expression. TGF-beta1 stimulated the production of 3B3(+), 5D4, and tenascin-C in a dose-dependent manner and decreased 3B3(-) levels. Following the addition of exogenous type II collagen, 3B3(-) increased and tenascin-C decreased but did not change the direction of TGF-beta1 effects. In contrast, 5D4 expression decreased in the presence of collagen II as TGF-beta1 increased to 10 ng/ml. Interestingly, the amount of 3B3(+) epitope was not affected by the incorporation of type II collagen. Immunohistochemistry found there was no significant difference in distribution of these biomarkers in the presence and absence of extracellular type II collagen incorporation. These results elucidate the subtle biochemical differences in ECM synthesized by chondrocytes in the presence of type II collagen and further characterize the role played by ECM in the TGF-beta1 regulation of the articular cartilage physiology.     

降钙素对兔膝骨关节炎发病过程中细胞因子及关节软骨细胞凋亡的试验研究

目的探讨降钙素(CT)对兔膝骨关节炎发病过程中细胞因子及关节软骨细胞凋亡情况的影响。方法将36只健康大耳白兔随机分为空白对照组(A组)、模型组(B组)和CT治疗组(C组),B、C两组采用改良Hulth法复制膝骨关节炎模型,A组除切开皮肤和关节腔外,不做其他处理,从术后第6周开始C组每天皮下注射CT 5u/kg,A、B组给与等量生理盐水皮下注射,连续注射20 d后处死动物。观察膝骨关节炎(KOA)发病过程中关节软骨病理组织学的变化,关节液中细胞因子IL-1、TNF-α、NO的变化。关节软骨Ⅱ型胶原及基质金属蛋白酶-1(MMP-1)的变化,以及CT对上述指标的影响。结果与A组相比,B组JP和Mankin’s评分明显升高,关节软骨损伤明显加重,关节液中IL-1、TNF-α、NO含量明显升高(P<0.05),关节软骨Ⅱ型胶原含量明显降低,MMP-1活性和软骨细胞凋亡百分数明显升高(P<0.05);与B组相比,C组关节液中IL-1、TNF-α、NO含量明显降低(P<0.05),关节软骨损伤减轻、Ⅱ型胶原含量明显升高,MMP-1活性和软骨细胞凋亡百分数明显降低(P<0.05)。结论 CT对KOA的治疗作用与细胞因子及细胞外基质合成与分解代谢失衡的恢复有关。     

Intra‐articular autologous uncultured adipose‐derived stromal cell transplantation inhibited the progression of cartilage degeneration

The role of uncultured adipose‐derived stromal cells for osteoarthritis treatment remains unclear despite sporadic reports supporting their use in clinical settings. This study aimed to evaluate the therapeutic effects of autologous uncultured adipose‐derived stromal cell transplantation in a rabbit osteoarthritis model. Uncultured adipose‐derived stromal cells isolated from rabbits were administered via intra‐articular injection into the knees after osteoarthritis onset. Animals were sacrificed at 8 and 12 weeks after osteoarthritis onset to compare the macroscopic, histological, and immunohistochemical characteristics between the uncultured adipose‐derived stromal cell and control groups. Co‐culture assay was also performed. The chondrocytes isolated from the model were co‐cultured with adipose‐derived stromal cells. The cell viability of chondrocytes and expression of chondrocyte‐specific genes in the co‐culture (uncultured adipose‐derived stromal cell) group were compared with the mono‐culture (control; chondrocytes only) group. In macroscopic and histological analyses, the uncultured adipose‐derived stromal cell group showed less damage to the cartilage surface than the control group at 8 and 12 weeks after osteoarthritis onset. In immunohistochemical and co‐culture assay, the uncultured adipose‐derived stromal cell group showed higher expression of collagen type II and SRY box‐9 and lower expression of matrix metalloproteinase‐13 than the control group. The cell viability of chondrocytes in the uncultured adipose‐derived stromal cell group was higher than that in the control group. Intra‐articular autologous uncultured adipose‐derived stromal cell transplantation inhibited the progression of cartilage degeneration in a rabbit osteoarthritis model by regulating chondrocyte viability and secreting chondrocyte‐protecting cytokines or growth factors, which promote anabolic factors and inhibit catabolic factors. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1376–1386, 2019.     

Tissue neogenesis and STRO‐1 expression in immature and mature articular cartilage

This study determined the potential for neotissue formation and the role of STRO‐1+ cells in immature versus mature articular cartilage. Cartilage explants from immature and mature bovine knee joints were cultured for up to 12 weeks and stained with safranin‐O, for type II collagen and STRO‐1. Bovine chondrocyte pellet cultures and murine knee joints at the age of 2 weeks and 3 months, and surgically injured cartilage, were analyzed for changes in STRO‐1 expression patterns. Results show that immature explants contained more STRO‐1+ cells than mature explants. After 8 weeks in culture, immature explants showed STRO‐1+ cell proliferation and newly formed tissue, which contained glycosaminoglycan and type II collagen. Mature cartilage explants showed only minimal cell expansion and neotissue formation. Pellet cultures with chondrocytes from immature cartilage showed increased glycosaminoglycan synthesis and STRO‐1+ staining, as compared to pellets with mature chondrocytes. The frequency of STRO‐1+ cells in murine knee joints significantly declined with joint maturation. Following surgical injury, immature explants had higher potential for tissue repair than mature explants. In conclusion, these findings suggest that the high percentage of STRO‐1+ cells in immature cartilage changes with joint maturation. STRO‐1+ cells have the potential to form new cartilage spontaneously and after tissue injury. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:96–102, 2010     

Ⅱ型胶原海绵修复兔膝关节软骨缺损的组织学观察

目的:观察Ⅱ型胶原海绵修复兔膝关节软骨缺损的组织学变化。方法:将28只成年雄性新西兰兔制作膝关节缺损模型并随机分成2组,A组在缺损局部植入Ⅱ型胶原海绵,B组不植入以作对照。手术后2、4、6、8、10、12周分别作HE、Safranin O染色及S-100蛋白检测。结果:(1)HE染色显示A组2周后即出现新生软骨细胞,12周后新生软骨组织完全填满缺损区并与周围组织整合,而B组直至术后第12周仍由纤维组织所填充;(2)S-100蛋白免疫组化检测结果显示A组新生软骨细胞具有正常兔软骨细胞的表型;(3)Safranin O染色结果显示A组新生软骨细胞具有分泌软骨基质的功能。结论:Ⅱ型胶原具有较强的诱导软骨细胞生长的能力,其诱导生长的新生软骨具有正常透明软骨的表型和功能。     

Interaktion zwischen humanen Chondrozyten und extrazellulärer Matrix in vitro

BACKGROUND: Autologous chondrocyte transplantation (ACT) has had reasonable success for repairing small articular cartilage defects. A limiting factor for ACT is, however, the in vitro cultivation of chondrocytes because it leads to dedifferentiation. Therefore, the goal of this work was to optimize the monolayer culture of chondrocytes in vitro. MATERIAL AND METHOD: Human articular chondrocytes were plated on either collagen type II or untreated surfaces. The cells were evaluated morphologically and with immunoblotting. RESULTS: On collagen type II surfaces, a stable chondrogenic phenotype, expression of beta1-integrin, and a significant activation of phosphorylated intracellular proteins and the adaptor protein Shc could be observed up to day 20 in culture. Treatment with beta1 integrin antibody led to a loss of cell adhesion (82%). The results indicate that on collagen type II, beta1-integrin receptors are activated. Through the activation of Shc, these stimulate the Ras-MAPK pathway, which stabilizes the chondrogenic phenotype. CONCLUSION: Our results provide a practical and low-cost solution for improved long-term chondrocyte cultivation, thus providing a new perspective for using ACT on larger or arthrotic cartilage defects.     

The proteoglycan synthesis repertoire of rabbit chondrocytes maintained in type II collagen gels

Repair of experimental articular cartilage lesions employing cultured rabbit articular chondrocytes requires a detailed knowledge of the phenotypic stability of these cells. A suitable matrix vehicle for use in chondrocyte transplantation is a much sought-after component of any transplantation paradigm. We studied the proteoglycan synthesis repertoire of young immature rabbit articular chondrocytes maintained in chick type II collagen gels or collagen gels supplemented with recombinant human transforming growth factor-beta 1 (rhTGF beta 1). Maintenance of chondrocytes in type II collagen gels increased the percentage 35SO4-labeled proteoglycans reaching equilibrium in the A1D1 or D1 fraction of CsCl density gradient when compared to chondrocytes maintained in polystyrene microwell cultures. Although rhTGF beta 1 supplementation increased the percentage of A1D1/D1 proteoglycan by chondrocytes grown on polystyrene, rhTGF beta 1 did not augment this percentage increase in A1D1/D1 when added to collagen II gels. Rabbit chondrocytes synthesized two core proteins derived from the high-density aggregatable proteoglycans. LI and LII have apparent molecular sizes of 480 kDa and 390 kDa, respectively. Both core protein forms were found in the medium fraction, but the predominant core protein form associated with the cell fraction was LI. Maintenance of chondrocytes in collagen II gels increased synthesis of both core proteins. In addition to the large core proteins, three other core proteins with properties on SDS PAGE characteristic of the small dermatan sulfate proteoglycans, biglycan and decorin, were identified. Synthesis of these core proteins was stimulated by maintenance in collagen gels. Furthermore, they were preferentially retained in the gel matrix. Chondrocytes maintained on glass or in type II collagen gels stained with monoclonal antibodies specific for chondroitin-6-sulfate, chondroitin-4-sulfate and keratan sulfate. However, while chondrocytes grown on glass slides failed to stain with monoclonal antibody 3B3 in the absence of chondroitinase ABC digestion, chondrocytes grown in collagen II gels stained intensely in the absence of enzyme pretreatment. These results were confirmed by Western blots.