Potier Esther, Ito Keita
1 Orthopaedic Biomechanics, Department of Biomedical Engineering, Eindhoven University of Technology , Eindhoven, The Netherlands .
Tissue Eng Part A. 2014 Dec;20(23-24):3241-51. doi: 10.1089/ten.TEA.2013.0703.
Bone marrow stromal cells (BMSCs) have shown promising potential to stop intervertebral disc degeneration in several animal models. In order to restore a healthy state, though, this potential should be further stimulated. Notochordal cells (NCs), influential in disc development, have been shown to stimulate BMSC differentiation, but it is unclear how this effect will translate in an environment where resident disc cells (nucleus pulposus cells [NPCs]) could also influence BMSCs. The goal of this study was, therefore, to evaluate the effects of NCs on BMSCs when cocultured with NPCs, in a simplified 3D in vitro system. Bovine BMSCs and NPCs were mixed (Mix) and seeded into alginate beads. Using culture inserts, the Mix was then cocultured with porcine NCs (alginate beads) and compared to coculture with empty beads or porcine skin fibroblasts (SFs, alginate beads). NPCs alone were also cocultured with NCs, and BMSCs alone cultured under chondrogenic conditions. The effects of coculture conditions on cell viability, matrix production (proteoglycan and collagen), and gene expression of disc markers (aggrecan, type II collagen, and SOX9) were assessed after 4 weeks of culture. The NC phenotype and gene expression profile were also analyzed. Coculture with NCs did not significantly influence cell viability, proteoglycan production, or disc marker gene expression of the Mix. When compared to NPCs, the Mix produced the same amount of proteoglycan and displayed a higher expression of disc marker, indicating a stimulation of the BMSCs (and/or NPCs) in the Mix. Additionally, during the 4 weeks of culture, the NC phenotype changed drastically (morphology, gene expression profile). These results show that NCs might not be as stimulatory for BMSCs in an NPC-rich environment, as believed from individual cultures. This absence of effects could be explained by a mild stimulation provided by (de)differentiating NCs and the costimulation of BMSCs and NPCs by each other.
骨髓基质细胞（BMSCs）在多种动物模型中已显示出阻止椎间盘退变的潜在前景。然而，为了恢复健康状态，这种潜力还应进一步激发。脊索细胞（NCs）在椎间盘发育中具有重要作用，已被证明能刺激BMSC分化，但尚不清楚在椎间盘驻留细胞（髓核细胞[NPCs]）也能影响BMSCs的环境中，这种作用将如何转化。因此，本研究的目的是在简化的三维体外系统中，评估NCs与NPCs共培养时对BMSCs的影响。将牛BMSCs和NPCs混合（Mix）并接种到海藻酸钠珠中。然后使用培养插入物，将Mix与猪NCs（海藻酸钠珠）共培养，并与空珠或猪皮肤成纤维细胞（SFs，海藻酸钠珠）共培养进行比较。单独的NPCs也与NCs共培养，单独的BMSCs在软骨形成条件下培养。培养4周后，评估共培养条件对细胞活力、基质产生（蛋白聚糖和胶原蛋白）以及椎间盘标志物（聚集蛋白聚糖、II型胶原蛋白和SOX9）基因表达的影响。还分析了NC的表型和基因表达谱。与NCs共培养对Mix的细胞活力、蛋白聚糖产生或椎间盘标志物基因表达没有显著影响。与NPCs相比，Mix产生的蛋白聚糖量相同，并且显示出更高的椎间盘标志物表达，表明Mix中的BMSCs（和/或NPCs）受到了刺激。此外，在4周的培养过程中，NC的表型发生了显著变化（形态、基因表达谱）。这些结果表明，在富含NPCs的环境中，NCs对BMSCs的刺激作用可能不如单独培养时所认为的那样。这种无效应可能是由于（去）分化的NCs提供的轻度刺激以及BMSCs和NPCs之间的相互共刺激所导致的。
