离心管培养软骨作为半月板移植替代物的可能性研究

目的比较培养关节软骨和半月板软骨细胞的生物学特性,探讨培养软骨作为半月板移植替代物的可能性. 方法分别自3周龄大耳白兔关节软骨和半月板分离软骨细胞,行单层传代培养和离心管培养.将离心管培养形成的软骨和6周龄兔半月板行组织学和透射电镜观察,比较关节软骨细胞和半月板纤维软骨细胞的生长曲线.流式细胞术检测第2、4代关节软骨细胞和半月板纤维软骨细胞周期. 结果第4代关节软骨细胞呈去分化,似成纤维细胞.离心管关节软骨细胞培养能形成软骨,半月板纤维软骨细胞不能形成软骨.培养软骨和半月板的组织学及超微结构差异显著,培养软骨中软骨细胞5%呈现凋亡.第2、4代关节软骨细胞中亚二倍体细胞比例明显多于第2、4代半月板纤维软骨细胞(P<0.05). 结论半月板来源的软骨细胞经离心管培养不能形成软骨组织,关节软骨细胞离心培养形成软骨不能作为半月板的移植物,关节软骨和半月板软骨组织存在明显的区别.     

Gene expression and proliferation analysis in young, aged, and osteoarthritic sheep chondrocytes effect of growth factor treatment.

Cartilage is a support tissue with a poor capacity to self-repair. Its cells, chondrocytes, are responsible for synthesizing and renewing the matrix that surrounds them in a constant turnover mechanism. Autologous chondrocyte implantation (ACI) is one of the techniques that promises to be an alternative to common strategies for chondral lesions. To apply this technique, a large amount of cells must be obtained. In our work, we studied the state of cells from different cartilage (young, aged, and osteoarthritic sheep) cultured in monolayer by analyzing their proliferation rate using bromodeoxyuridine and their gene expression profile by RT-PCR. A decrease was found in expression of type II collagen and aggrecan in aged, osteoarthritic, and passaged chondrocytes. Treatment of cells with growth factors aFGF, IGF-I, TGF-beta, and OP-1 improved the proliferation rate in all cells studied and stimulated gene expression of type II collagen, aggrecan, and TGF-beta. Osteoarthritic cells showed a poor response according to matrix gene expression, while young cells responded properly, and aged chondrocytes showed a moderate response. These results suggest that the state of cartilage may affect the behavior of cultured chondrocytes.     

Smooth muscle actin expression by human articular chondrocytes and their contraction of a collagen-glycosaminoglycan matrix in vitro.

Recent studies have demonstrated that human articular chondrocytes can express the gene for a contractile muscle actin, alpha-smooth muscle actin (SMA), in situ. One objective of this work was to evaluate the SMA-content of isolated human articular chondrocytes using Western blot analysis and to correlate the amount of SMA in the cells with passage number and the number of days in culture. A second objective was to determine if articular cartilage-derived cells expressing the gene for SMA in vitro also continue to express type II collagen. A final aim of the current study was to determine if SMA-containing cartilage-derived cells were capable of contracting a collagen glycosaminoglycan analog of extracellular matrix in vitro. Articular chondrocytes were isolated from 13 patients undergoing total joint arthroplasty. Cells were serially passaged through passage 7. Samples were allocated for Western blot analysis of SMA. Cells in monolayer culture were also stained immunohistochemically for SMA and type II collagen. Cells from passage 3 and 7 were seeded into a porous type I collagen-glycosaminoglycan matrix and the diameter of the scaffolds measured every other day for 21 days. Immunohistochemistry of the articular cartilage samples revealed SMA in the articular chondrocytes in situ with a greater percentage of cells staining positive in the superficial half (60 +/- 1.2%; mean +/- SEM) of the cartilage than in the basal half (28 +/- 1.3%). There was an increasing amount of SMA in the cells in monolayer culture with passage number and a meaningful correlation of the SMA content with the days in culture (linear regression analysis; R2 = 0.72). Double staining for SMA and type II collagen showed that type II collagen-expressing cells in monolayer could also express SMA. SMA-containing cells were found to contract the collagen glycosaminoglycan matrix, with the cells containing more SMA (passage 7 cells) displaying more matrix contraction than those with a lesser amount of SMA (passage 3 cells). The results indicate that control of the expression of SMA may be important when employing articular chondrocytes, expanded in monolayer culture, for implantation alone or in a cell-seeded matrix for cartilage repair procedures.     

Expression of a stable articular cartilage phenotype without evidence of hypertrophy by adult human articular chondrocytes in vitro

Chondrocytes that were isolated from adult human articular cartilage changed phenotype during monolayer tissue culture, as characterized by a fibroblastic morphology and cellular proliferation. Increased proliferation was accompanied by downregulation of the cartilage-specific extracellular matrix proteoglycan, aggrecan, by cessation of type-II collagen expression, and by upregulation of type-I collagen and versican. This phenomenon observed in monolayer was reversible after the transfer of cells to a suspension culture system. The transfer of chondrocytes to suspension culture in alginate beads resulted in the rapid upregulation of aggrecan and type-II collagen and the downregulation of expression of versican and type-I collagen. Type-X collagen and osteopontin, markers of chondrocyte hypertrophy and commitment to endochondral ossification, were not expressed by adult articular chondrocytes cultured in alginate, even after 5 months. In contrast, type-X collagen was expressed within 2 weeks in a population of cells derived from a fetal growth plate. The inability of adult articular chondrocytes to express markers of chondrocyte hypertrophy has underscored the fundamental distinction between the differentiation pathways that lead to articular cartilage or to bone. Adult articular chondrocytes expressed only hyaline articular cartilage markers without evidence of hypertrophy.     

兔关节软骨细胞聚集培养的生物学性状观察

目的 观察聚集培养软骨细胞生物学性状的变化,为软骨细胞移植建立合适的体外培养方法。方法2001年11月至2004年6月酶消化法分离成年兔关节软骨细胞,分别低密度单层培养和高密度聚集培养,组化及免疫组化法观察细胞表型变化。结果低密度培养时,前3代细胞增殖迅速,但很快去分化,3代以后增殖缓慢,细胞表型大部丢失;聚集培养时,软骨细胞去分化速度减缓;传3代后细胞聚集培养,细胞表型部分恢复。结论聚集培养利于维持软骨细胞表型,原代细胞聚集培养或传代培养后聚集培养是较好的获取大量优良软骨细胞的培养方式。     

Endogenous versus exogenous growth factor regulation of articular chondrocytes

Anabolic growth factors that regulate the function of articular chondrocytes are candidates for articular cartilage repair. Such factors may be delivered by pharmacotherapy in the form of exogenous proteins, or by gene therapy as endogenous proteins. It is unknown whether delivery method influences growth factor effectiveness in regulating articular chondrocyte reparative functions. We treated adult bovine articular chondrocytes with exogenous recombinant insulin‐like growth factor‐I (IGF‐I) and transforming growth factor‐beta1 (TGF‐β1), or with the genes encoding these growth factors for endogenous production. Treatment effects were measured as change in chondrocyte DNA content, glycosaminoglycan production, and aggrecan gene expression. We found that IGF‐I stimulated chondrocyte biosynthesis similarly when delivered by either exogenous or endogenous means. In contrast, exogenous TGF‐β1 stimulated these reparative functions, while endogenous TGF‐β1 had little effect. Endogenous TGF‐β1 became more bioactive following activation of the transgene protein product. These data indicate that effective mechanisms of growth factor delivery for articular cartilage repair may differ for different growth factors. In the case of IGF‐I, gene therapy or protein therapy appear to be viable options. In contrast, TGF‐β1 gene therapy may be constrained by a limited ability of chondrocytes to convert latent complexes to an active form. Published 2013 by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 32:54–60, 2014.     

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     

Effects of intermittent hydrostatic pressure and BMP‐2 on osteoarthritic human chondrocyte metabolism in vitro

Purpose

This study examined effects of intermittent hydrostatic pressure (IHP) and a chondrogenic growth factor, bone morphogenetic protein‐2 (BMP‐2), on anabolic, catabolic, and other metabolic markers in human osteoarthritic (OA) chondrocytes in vitro.

Methods

Articular chondrocytes, isolated from femoral OA cartilage and maintained in high‐density monolayer culture, were examined for effects of BMP‐2 and IHP on gene expression of matrix‐associated proteins (aggrecan, type II collagen, and SOX9) and catabolic matrix metalloproteinases (MMP‐2 and MMP‐3) and culture medium levels of the metabolic markers MMP‐2, nitric oxide (NO), and glycosaminoglycan (GAG). The results were analyzed using a mixed linear regression model to investigate the effects of load and growth factor concentration.

Results

IHP and BMP‐2 modulated OA chondrocyte metabolism in accordance with growth factor concentration independently, without evidence of synergism or antagonism. Each type of stimulus acted independently on anabolic matrix gene expression. Type II collagen and SOX9 gene expression were stimulated by both IHP and BMP‐2 whereas aggrecan was increased only by BMP‐2. IHP exhibited a trend to decrease MMP‐2 gene expression as a catabolic marker whereas BMP‐2 did not. NO production was increased by addition of BMP‐2 and IHP exhibited a trend for increased levels. GAG production was increased by BMP‐2.

Conclusions

This study confirmed the hypothesis that human OA chondrocytes respond to a specific type of mechanical load, IHP, through enhanced articular cartilage macromolecule gene expression and that IHP, in combination with a chondrogenic growth factor BMP‐2, additively enhanced matrix gene expression without interactive effects. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:361–368, 2011     

Inhibition of Chondrocyte Terminal Differentiation and Matrix Calcification by Soluble Factors Released by Articular Chondrocytes

Chondrocytes do not undergo terminal differentiation in normal articular cartilage, whereas growth plate chondrocytes synthesize ALPase and induce matrix calcification terminally. Articular chondrocytes in osteoarthritic joints have been reported to express the terminal differentiation phenotypes, suggesting that terminal differentiation of articular chondrocytes is inhibited in normal joints. In the present study, we investigated the underlying inhibitory mechanism of the terminal differentiation in articular cartilage using a culture on type II collagen-coated dishes or a novel culture model on Millipore filters. ALPase activity increased from day 7 to day 8 in growth plate chondrocyte cultures on the collagen-coated dishes, but not in articular chondrocyte cultures. The ALPase expression of growth plate chondrocytes on the collagen-coated dish was completely inhibited when the same number of articular chondrocytes was mixed in the growth plate chondrocyte cultures. When articular chondrocytes or growth plate chondrocytes were maintained on Millipore filters held in 16-mm dishes, they started to synthesize ALPase. The ALPase expression of the chondrocytes on Millipore filters was inhibited by the presence of articular chondrocytes maintained on the bottom collagen-coated substratum in the same dishes. These results indicate that factors that diffused into the medium through the Millipore filters are involved in the inhibition of terminal differentiation. Since the conditioned medium from articular chondrocyte cultures did not affect the ALPase expression, it is considered that the soluble factors, which are continuously released from articular chondrocytes, are responsible for the inhibition of terminal differentiation. Received: 23 April 1998 / Accepted: 12 March 1999     

Influence of the growth factors PDGF‐BB,TGF‐β1 and bFGF on the replicative aging of human articular chondrocytes during in vitro expansion

Decreasing replicative potential and dedifferentiation of articular chondrocytes during expansion in cell culture are essential limitations for tissue engineering and cell therapy approaches. Telomeres and telomerase play a key role in cell development, aging, and tumorigenesis. There is evidence that growth factors are involved in regulating telomerase activity. Therefore, the objective was to evaluate the effect of selected growth factors on telomere biology of serially passaged chondrocytes. Human articular chondrocytes were isolated from cartilage of three patients undergoing total knee arthroplasty. The chondrocytes were cultured in monolayer with the growth factors PDGF‐BB, TGF‐β1, and bFGF. Telomere length was measured by telomere restriction fragment length assay, and telomerase activity was determined by quantifying the gene expression of its catalytic subunit hTERT by rtPCR. Chondrocytes cultured with PDGF‐BB and TGF‐β1 showed a significantly higher proliferation rate than control cells. None of the growth factor cultures revealed an accelerated rate of telomere shortening. Telomerase was not expressed in significant amounts in any of the chondrocyte cultures. Growth factor treatment of chondrocyte cell cultures for cell therapy purposes can be regarded as safe in terms of telomere biology. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:354–360, 2010     

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     

Cartilage-derived morphogenetic protein-1 and -2 are endogenously expressed in healthy and osteoarthritic human articular chondrocytes and stimulate matrix synthesis

OBJECTIVE: We investigated whether chondrocytes derived from osteoarthritic cartilage may lose their responsiveness to cartilage-derived morphogenetic protein-1, -2 (CDMP-1, -2) and osteogenic protein-1 (OP-1) compared with healthy cells, thus leading to an impaired maintenance of matrix integrity. DESIGN: Chondrocytes were isolated from articular cartilage from patients with and without osteoarthritic lesions. Cells were grown as monolayer cultures for 7 days in a chemically defined serum-free basal medium (BM) in the presence of recombinant CDMP-1, -2, and OP-1. Glycosaminoglycan synthesis was measured by [35S]Sulfate incorporation into newly synthesized macromolecules. Cell proliferation was investigated by [3H]Thymidine incorporation. The endogenous gene expression of CDMPs/OP-1 and their respective type I and type II receptors was examined using RT-PCR. The presence of CDMP proteins in tissue and cultured cells was detected by Western immunoblots. RESULTS: mRNAs coding for CDMPs and their respective receptors are endogenously expressed not only in healthy, but also in osteoarthritic cartilage. CDMP proteins are present in both normal and osteoarthritic articular cartilage and cultured chondrocytes. CDMP-1, CDMP-2 and OP-1 markedly increased glycosaminoglycan synthesis in both healthy (P< 0.01) and osteoarthritic (P< 0.05) human articular chondrocytes. A comparison of the glycosaminoglycan biosynthetic activity between healthy and osteoarthritic samples revealed no detectable difference, neither in stimulated nor in unstimulated cultures. [(3)H]Thymidine incorporation showed that CDMPs/OP-1 did not affect cell proliferation in vitro. CONCLUSION: CDMPs and OP-1 exert their anabolic effects on both healthy and osteoarthritic chondrocytes indicating no loss in responsiveness to these growth factors in OA. The endogenous expression of CDMPs/OP-1 and their receptors suggest an important role in cartilage homeostasis.     

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     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.