de Windt Tommy S, Saris Daniel B F, Slaper-Cortenbach Ineke C M, van Rijen Mattie H P, Gawlitta Debby, Creemers Laura B, de Weger Roel A, Dhert Wouter J A, Vonk Lucienne A
1 Department of Orthopaedics, University Medical Center Utrecht , Utrecht, The Netherlands .
2 MIRA Institute for Biotechnology and Technical Medicine, University Twente , Enschede, The Netherlands .
Tissue Eng Part A. 2015 Oct;21(19-20):2536-47. doi: 10.1089/ten.TEA.2014.0673. Epub 2015 Aug 12.
Using a combination of articular chondrocytes (ACs) and mesenchymal stromal cells (MSCs) has shown to be a viable option for a single-stage cell-based treatment of focal cartilage defects. However, there is still considerable debate whether MSCs differentiate or have a chondroinductive role through trophic factors. In addition, it remains unclear whether direct cell-cell contact is necessary for chondrogenesis. Therefore, the aim of this study was to investigate whether direct or indirect cell-cell contact between ACs and MSCs is essential for increased cartilage production in different cellular environments and elucidate the mechanisms behind these cellular interactions. Human ACs and MSCs were cultured in a 10:90 ratio in alginate beads, fibrin scaffolds, and pellets. Cells were mixed in direct cocultures, separated by a Transwell filter (indirect cocultures), or cultured with conditioned medium. Short tandem repeat analysis revealed that the percentages of ACs increased during culture, while those of MSCs decreased, with the biggest change in fibrin glue scaffolds. For alginate, where the lack of cell-cell contact could be confirmed by histological analysis, no difference was found in matrix production between direct and indirect cocultures. For fibrin scaffolds and pellet cultures, an increased glycosaminoglycan production and type II collagen deposition were found in direct cocultures compared with indirect cocultures and conditioned medium. Positive connexin 43 staining and transfer of cytosolic calcein indicated communication through gap junctions in direct cocultures. Taken together, these results suggest that MSCs stimulate cartilage formation when placed in close proximity to chondrocytes and that direct cell-cell contact and communication through gap junctions are essential in this chondroinductive interplay.
联合使用关节软骨细胞（ACs）和间充质基质细胞（MSCs）已被证明是一种可行的单阶段基于细胞治疗局灶性软骨缺损的方法。然而，关于MSCs是通过营养因子分化还是具有软骨诱导作用仍存在相当大的争议。此外，软骨形成是否需要直接的细胞间接触仍不清楚。因此，本研究的目的是探讨在不同细胞环境中，ACs与MSCs之间的直接或间接细胞间接触对于增加软骨生成是否至关重要，并阐明这些细胞相互作用背后的机制。将人ACs和MSCs以10:90的比例培养在藻酸盐珠、纤维蛋白支架和微球中。细胞在直接共培养中混合、通过Transwell滤器分离（间接共培养）或用条件培养基培养。短串联重复分析显示，培养过程中ACs的百分比增加，而MSCs的百分比下降，在纤维蛋白胶支架中变化最大。对于藻酸盐，组织学分析可证实缺乏细胞间接触，直接和间接共培养之间的基质产生没有差异。对于纤维蛋白支架和微球培养，与间接共培养和条件培养基相比，直接共培养中糖胺聚糖产生增加，II型胶原沉积增加。连接蛋白43阳性染色和胞质钙黄绿素转移表明直接共培养中通过缝隙连接进行通讯。综上所述，这些结果表明，当MSCs与软骨细胞紧密相邻放置时会刺激软骨形成，并且直接的细胞间接触和通过缝隙连接的通讯在这种软骨诱导相互作用中至关重要。
