Leyh Michaela, Seitz Andreas, Dürselen Lutz, Springorum Hans-Robert, Angele Peter, Ignatius Anita, Grifka Joachim, Grässel Susanne
Stem Cell Res Ther. 2014 Jun 10;5(3):77. doi: 10.1186/scrt466.
In the present study, we established a novel in vitro coculture model to evaluate the influence of osteoarthritis (OA) cartilage explants on the composition of newly produced matrix and chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs) and the phenotype of OA chondrocytes. In addition, we included a "tri-culture" model, whereby a mixture of BMSCs and chondrocytes was cultured on the surface of OA cartilage explants.
Gene expression analysis, protein and glycosaminoglycan (GAG) assays, dot-blot, immunofluorescence, and biomechanical tests were used to characterize the properties of newly generated extracellular matrix (ECM) from chondrocytes and chondrogenically differentiated BMSCs and a mix thereof. We compared articular cartilage explant cocultures with BMSCs, chondrocytes, and mixed cultures (chondrocytes and BMSCs 1:1) embedded in fibrin gels with fibrin gel-embedded cells cultured without cartilage explants (monocultures).
In general, co- and tri-cultured cell regimens exhibited reduced mRNA and protein levels of collagens I, II, III, and X in comparison with monocultures, whereas no changes in GAG synthesis were observed. All co- and tri-culture regimens tended to exhibit lower Young's and equilibrium modulus compared with monocultures. In contrast, aggregate modulus and hydraulic permeability seemed to be higher in co- and tri-cultures. Supernatants of cocultures contained significant higher levels of interleukin-1 beta (IL-1β), IL-6, and IL-8. Stimulation of monocultures with IL-1β and IL-6 reduced collagen gene expression in BMSCs and mixed cultures in general but was often upregulated in chondrocytes at late culture time points. IL-8 stimulation affected BMSCs only.
Our results suggest an inhibitory effect of OA cartilage on the production of collagens. This indicates a distinct modulatory influence that affects the collagen composition of the de novo-produced ECM from co- and tri-cultured cells and leads to impaired mechanical and biochemical properties of the matrix because of an altered fibrillar network. We suggest that soluble factors, including IL-1β and IL-6, released from OA cartilage partly mediate these effects. Thus, neighbored OA cartilage provides inhibitory signals with respect to BMSCs' chondrogenic differentiation and matrix composition, which need to be accounted for in future cell-based OA treatment strategies.
在本研究中，我们建立了一种新型的体外共培养模型，以评估骨关节炎（OA）软骨外植体对新生成基质组成、人骨髓间充质干细胞（BMSCs）软骨形成分化以及OA软骨细胞表型的影响。此外，我们还纳入了一种“三培养”模型，即将BMSCs和软骨细胞的混合物培养在OA软骨外植体表面。
采用基因表达分析、蛋白质和糖胺聚糖（GAG）检测、斑点印迹、免疫荧光和生物力学测试，以表征软骨细胞、软骨形成分化的BMSCs及其混合物新生成的细胞外基质（ECM）的特性。我们将关节软骨外植体与BMSCs、软骨细胞以及混合培养物（软骨细胞和BMSCs 1:1）包埋于纤维蛋白凝胶中的共培养物，与未与软骨外植体共培养的纤维蛋白凝胶包埋细胞（单培养物）进行了比较。
总体而言，与单培养物相比，共培养和三培养细胞方案中I、II、III和X型胶原蛋白的mRNA和蛋白质水平降低，而GAG合成未见变化。与单培养物相比，所有共培养和三培养方案的杨氏模量和平衡模量往往较低。相反，共培养和三培养中的聚集模量和水力渗透率似乎较高。共培养物的上清液中白细胞介素-1β（IL-1β）、IL-6和IL-8水平显著更高。用IL-1β和IL-6刺激单培养物通常会降低BMSCs和混合培养物中的胶原蛋白基因表达，但在培养后期软骨细胞中往往会上调。IL-8刺激仅影响BMSCs。
我们的结果表明OA软骨对胶原蛋白的产生具有抑制作用。这表明存在一种独特的调节影响，影响共培养和三培养细胞新生成的ECM的胶原蛋白组成，并由于纤维网络改变导致基质的机械和生化特性受损。我们认为，OA软骨释放的包括IL-1β和IL-6在内的可溶性因子部分介导了这些作用。因此，相邻的OA软骨对BMSCs的软骨形成分化和基质组成提供抑制信号，这在未来基于细胞的OA治疗策略中需要加以考虑。
