Activin A suppresses interleukin-1-induced matrix metalloproteinase 3 secretion in human chondrosarcoma cells

The objective was to investigate the effect of activin A on matrix metalloproteinase 3 (MMP-3) production and to identify the role of activin A in chondroprotection. SW1353 cells, a human chondrosarcoma cell line, were stimulated with interleukin (IL) 1α and tumor necrosis factor (TNF) α, and the concentrations of activin A, follistatin, and MMP-3 secreted into the culture media were measured by enzyme-linked immunosorbent assay (ELISA). Activin A was added to cell cultures in the presence of IL-1α or TNFα to determine its effect on the production of MMP-3 and sulfated glycosaminoglycan (sGAG) (measured by Alcian blue assay). To study the mechanism responsible for the chondroprotective effects of activin A, the production of IL-1 receptor antagonist (IL-1ra) and tissue inhibitor for metalloproteinases 1 (TIMP-1) was examined by ELISA. Addition of IL-1α did not affect the production of activin A by cultured SW1353 cells. IL-1α and activin A inhibited the production of follistatin. Stimulation of SW1353 cells with activin A suppressed IL-1α-induced, but not TNFα-induced, MMP-3 expression. Activin A had no effect on the production of sGAG, IL-1ra, or TIMP-1, although it suppressed the induction of TIMP-1 and IL-1ra by IL-1α. This novel finding of MMP-3 inhibition by activin A suggests a new role of activin A in cartilage remodeling. Activin A may have therapeutic potential for preventing cartilage degradation.     

碱性成纤维细胞生长因子缓释微球对软骨细胞作用的实验研究

目的观察碱性成纤维细胞生长因子（bFGF）缓释微球对软骨细胞增殖和细胞外基质合成的影响。方法酶消化法从兔肩、膝关节获取软骨细胞,将bFGF微球加入软骨细胞培养液中,流式细胞仪观察细胞增殖情况。同时检测各组DNA总量及糖胺多糖总量。结果流式细胞仪检测显示培养2d后,bFGF组的G2/M＋S期百分数最高,4、8d后,bFGF微球组G2/M＋S期百分数最高。DNA总量对照组、bFGF组和bFGF微球组分别为（2.82±0.24）、（4.80±0.32）、（6.85±0.35）μg;糖胺多糖总量对照组、bFGF和bFGF微球组分别为（14.21±0.92）、（26.44±1.22）、（34.25±1.84）μg。结论bFGF促进了软骨细胞增殖,bFGF微球通过较长时间持续释放活性bFGF,明显促进了软骨细胞增殖与分化。     

rAAV2-hTGF-β1转染犬MSCs诱导分化为软骨细胞的研究

目的 探讨rAAV2-hTGF-β1体外转染犬MSCs定向分化为软骨细胞的可行性.方法 应用密度梯度离心法及贴壁筛选法分离培养犬MSCs,倒置显微镜及Giemsa染色观察细胞形态,流式细胞技术鉴定其表面标记物.取第三代MSCs,分别以感染复数(MOI)为1×105病毒基因数/细胞v.g./cell)、5×105v.g./cell的rAAV2-hTGF-β1进行转染.培养后定期取各组细胞进行相关检测.Western blot检测hTGF-β1的表达,ELISA法测定hTGF-β1的含量,RT-PCR检测Ⅱ型胶原、Aggrecan mRNA的表达,免疫细胞化学法检测Ⅱ型胶原的表达.结果 原代及传代培养的MSCs呈梭形外观,具有较强的增殖能力.细胞表面抗原CD29、CD44、CD105表达阳性,CD34、CD45表达阴性.rAAV2-hTGF-β1转染MSCs后,Western blot法检测到目的 蛋白hTGF-β1稳定表达:ELISA法检测转染组MSCs培养上清液中的hTGF-β1表达,且随时间的延长,各组hTGF-β1浓度逐渐增加,MOI 5×105组表达量明显高于MOI 1×105组(P<0.01);RT-PCR检测到各转染组Ⅱ型胶原、Aggrecan mRNA表达,免疫细胞化学法检测转染组细胞Ⅱ型胶原呈阳性表达,且高MOI值组表达强度明显高于低MOI值组.结论 rAAV2-hTGF-β1转染犬MSCs后可定向分化为软骨细胞,作为软骨组织工程的种子细胞是可行的.     

Gene expression profiling of chondrocytes from a porcine impact injury model

OBJECTIVE: To identify differentially expressed genes between axially impacted and control articular cartilage taken from porcine patellae maintained in organ culture for 14 days. METHODS: Porcine patellae were impacted perpendicular to the articular surface to create an impact injury. Intact patellae (control and impacted) were maintained in culture for 14 days. Total RNA was then extracted from the articular cartilage beneath the impaction and used to prepare two Serial Analysis of Gene Expression (SAGE) libraries. Approximately 42,500 SAGE long tags were sequenced from the libraries. The expression of select genes was confirmed by quantitative real-time PCR analysis. RESULTS: Thirty-nine SAGE tags were significantly differentially expressed in the impacted and control libraries, representing 30 different annotated pig genes. These genes represented gene products associated with matrix molecules, iron and phosphate transport, protein biosynthesis, skeletal development, cell proliferation, lipid metabolism and the inflammatory response. Twenty-three of the 30 genes were down-regulated in the impacted library and five were up-regulated in the impacted library. Quantitative real-time PCR follow-up of four genes supported the results found with SAGE. CONCLUSION: We have identified 30 putative genes differentially expressed in a porcine impact injury model and validated these findings for four of these genes using real-time PCR. Results using this impact injury model have contributed further evidence that damaged chondrocytes may de-differentiate into fibroblast-like cells and proliferate in an attempt to repair themselves. Additional work is underway to study these genes in further detail at earlier time points to provide a more complete story about the fate of chondrocytes in articular cartilage following an injury.     

Chondroitin sulphate impedes the migration of a sub-population of articular cartilage chondrocytes

OBJECTIVE: To determine whether chondroitin sulphate (CS) impedes the migration of primary articular chondrocytes. DESIGN: Articular chondrocytes were isolated from young and skeletally mature bovine animals. Boyden chambers were used to quantify chondrocyte migration on aggrecan in the presence and absence of CS chains. A novel in vitro model of cell migration into articular cartilage explants was designed to visualise and quantify the migration of labelled chondrocytes into cartilage matrix which had been treated with chondroitinase ABC to remove CS chains present. RESULTS: A consistent trend of increased migration with both age groups of a sub-population of chondrocytes was demonstrated on aggrecan in the absence of CS. These data were supported by results from the in vitro model of chondrocyte migration which demonstrated increasing numbers of a chondrocyte sub-population from both age groups of cartilage migrating into the chondroitinase ABC digested cartilage explants with time in culture. Minimal migration of these chondrocytes was demonstrated into phosphate buffered saline (PBS) treated control explants. CONCLUSIONS: We confirm that a sub-population of chondrocytes isolated from both young and skeletally mature articular cartilages have the ability to migrate. We also demonstrate that CS chains inhibit the migration of these articular chondrocytes and that their removal by chondroitinase ABC digestion enhances the migration of these chondrocytes. Such findings may provide a clinical application for improving cell-based cartilage repair strategies by enhancing integration between endogenous and repair tissue.     

Mechanical force-induced midpalatal suture remodeling in mice

Mechanical stress is an important epigenetic factor for regulating skeletal remodeling, and application of force can lead to remodeling of both bone and cartilage. Chondrocytes, osteoblasts and osteoclasts all participate and interact with each other in this remodeling process. To study cellular responses to mechanical stimuli in a system that can be genetically manipulated, we used mouse midpalatal suture expansion in vivo. Six-week-old male C57BL/6 mice were subjected to palatal suture expansion by opening loops with an initial force of 0.56 N for the periods of 1, 3, 5, 7, 14 or 28 days. Periosteal cells in expanding sutures showed increased proliferation, with Ki67-positive cells representing 1.8 ± 0.1% to 4.5 ± 0.4% of total suture cells in control groups and 12.0 ± 2.6% to 19.9 ± 1.2% in experimental/expansion groups (p < 0.05). Starting at day 1, cells expressing alkaline phosphatase and type I collagen were seen. New cartilage and bone formation was observed at the oral edges of the palatal bones at day 7; at the nasal edges only bone formation without cartilage appeared to occur. An increase in osteoclast numbers suggested increased bone remodeling, ranging from 60 to 160% throughout the experimental period. Decreased Saffranin O staining after day 3 suggested decreased proteoglycan content in the secondary cartilage. Micro-CT showed a significant increase in maxillary width at days 14 and 28 (from 2334 ± 4 μm to 2485 ± 3 μm at day 14 and from 2383 ± 5 μm to 2574 ± 7 μm at day 28, p < 0.001). The suture width was increased at days 14 and 28, except in the oral third region at day 28 (from 48 ± 5 μm to 36 ± 4 μm, p < 0.05). Bone volume/total volume was significantly reduced at days 14 and 28 (50.2 ± 0.7% vs. 68.0 ± 3.7% and 56.5 ± 1.0% vs. 60.9 ± 1.3%, respectively, p < 0.05), indicative of increased bone marrow space. These findings demonstrate that expansion forces across the midpalatal suture promote bone resorption through activation of osteoclasts and bone and cartilage formation via increased proliferation and differentiation of periosteal cells. Mouse midpalatal suture expansion would be useful in further studies of the ability of mineralized tissues to respond to mechanical stimulation.     

离心管内组织工程软骨培养的研究

目的　探讨离心管内 5种材料与软骨细胞培养形成组织工程软骨。方法　分别将脱脂脱蛋白骨、羟基磷灰石、糖蛋白微孔膜、胶原海绵和明胶海绵置于离心管底 ,按 3× 10 6个细胞 /管加入 3周龄兔关节软骨细胞 ,离心 ( 2 0 0r/min) 5min后连续培养 2周。进行组织学检查。结果　 5种支架材料与软骨细胞复合培养均能形成软骨样组织。软骨细胞保持卵圆形和多角形 ,细胞外基质形成。 5种组织工程软骨具有不同的组织学结构 ,细胞及其细胞外基质形态差异明显。结论　支架材料与软骨细胞具有良好的生物相容性 ,软骨细胞于离心管内培养可形成组织工程软骨。     

益气化瘀方对体外培养的大鼠颈椎间盘软骨细胞超微结构的影响

研究益气化瘀方 (主药 :黄芪、丹参、川芎等 )对体外培养的大鼠颈椎间盘软骨细胞超微结构的影响。 8～ 10周龄SD大鼠 70只 ,随机均分为生理盐水对照组 ,芬必得组 ,中药低、中、高剂量组 ,每组 14只。定时、定量胃饲相应药物 ,4天后分别处死大鼠取血清 ,相同条件下制备益气化瘀方含药血清和对照血清 ,取体外培养的大鼠颈椎间盘软骨细胞进行研究 ,采用透射电镜及流式细胞仪检测软骨细胞超微结构及细胞凋亡率。结果显示 ,中药组能促进细胞DNA合成(S期 ) ,与对照组相比 ,差异有非常显著的意义 (P <0 0 1)。表明益气化瘀方具有促进大鼠颈椎间盘软骨细胞生长、增殖及促进细胞DNA合成的作用。     

24,25-(OH)2D3 Regulation of Matrix Vesicle Protein Kinase C Occurs Both During Biosynthesis and in the Extracellular Matrix

Plasma membranes and matrix vesicles isolated from rat costochondral resting zone chondrocyte cultures contain predominantly protein kinase C alpha (PKCα) and PKCζ, respectively, and the level of PKC specific activity in these membrane fractions is regulated by 24,25-(OH)₂D₃ [14]. In the present study, we examined whether the effect of 24,25-(OH)₂D₃ on membrane PKC is via genomic mechanisms during biogenesis and through a nongenomic mechanism after the matrix vesicles are resident in the matrix. There was a dose-dependent decrease in matrix vesicle PKC specific activity and a significant increase in plasma membrane enzyme activity in cultures treated for 90 minutes with 10⁻⁹–10⁻⁷ M 24,25-(OH)₂D₃. However, at 12 hours, matrix vesicle PKC was stimulated, but no effect was seen in the plasma membranes, suggesting that the effect seen at 90 minutes was due to a direct action of the hormone on PKC activity in the membrane, and that the effect seen at 12 hours was due to new matrix vesicle production with altered PKC content. Neither actinomycin D nor cycloheximide inhibited matrix vesicle PKC at 30, 60, or 90 minutes, but by 12 hours, these inhibitors blocked the effect of the hormone. 24,25-(OH)₂D₃-dependent plasma membrane PKC was sensitive to both actinomycin D and cycloheximide at early time points, but by 12 hours, no effect of the inhibitors was seen. Monensin did not alter basal plasma membrane PKC activity or the 24,25-(OH)₂D₃-dependent increase, suggesting that this increase was due to translocation of cytosolic PKC rather than new membrane synthesis. Monensin did not affect matrix vesicle PKC at early time points, but it decreased 24,25-(OH)₂D₃-dependent enzyme activity at later times, indicating that new matrix vesicle production was blocked. At least part of the effect of 24,25-(OH)₂D₃ on PKC involved phospholipase A₂ (PA₂). Quinacrine (a PA₂ inhibitor) alone had no effect on matrix vesicle PKC, but in cultures treated for 12 hours with quinacrine and 24,25-(OH)₂D₃, a synergistic increase in matrix vesicle PKC was observed. Quinacrine caused a time-dependent decrease in matrix vesicle PKC and a dose- and time-dependent increase in plasma membrane PKC when incubated directly with the membranes, supporting the hypothesis that PA₂ plays a role in the nongenomic regulation of PKC by 24,25-(OH)₂D₃. Experiments using anti-isoform specific antibodies showed that 24,25-(OH)₂D₃ modulated the distribution of PKCα, β, and ζ between the plasma membrane and matrix vesicle compartments via translocation and new PKC synthesis. Thus, the data support the hypothesis that 24,25-(OH)₂D₃ regulates matrix vesicles through two pathways: a genomic one at the stage of biosynthesis and packaging, and a second nongenomic mechanism acting directly upon matrix vesicles in the matrix. These data also indicate that matrix vesicle regulation consists of complex events with several different points of regulation. Received: 11 October 1996 / Accepted: 25 April 1997     

三种软骨细胞分离培养方法对细胞骨架的影响比较

目的 比较分离培养人类软骨细胞的3种方法，探讨各种方法的特点及对细胞骨架的影响。方法 分别采用酶消化法、组织贴块法、酶消化结合组织贴块法分离软骨细胞，测量每种方法分离的软骨细胞瓶底汇合形成单层时间，进行比较，以甲苯氨蓝染色、Ⅱ型胶原免疫组织化学评价细胞的生物特性，并用磁驱动原子力显微镜（MAC mode AFM）对二代细胞表面形态及细胞骨架特性进行检测。结果 MAC mode AFM检测显示，酶消化结合组织贴块法分离软骨细胞表面形态、细胞骨架分布更接近生理状态，甲苯氨蓝、Ⅱ型胶原免疫组织化学染色显示强阳性，进行方差分析及q检验，3种方法分离的软骨细胞瓶底汇合形成单层时间差异有统计学意义（P〈0．05）。结论 酶消化结合组织贴块法细胞分离效率高、生长速度快、细胞骨架特性保持更好、细胞纯度高，在细胞骨架研究中可优先选用。