Research Centre for Experimental Orthopedics, Orthopaedic University Hospital Heidelberg, Heidelberg, Germany.
Department of Orthopedics, Trauma Surgery and Spinal Cord Injury, Orthopaedic University Hospital Heidelberg, Heidelberg, Germany.
J Cell Physiol. 2018 Nov;233(11):8962-8970. doi: 10.1002/jcp.26832. Epub 2018 Jun 1.
Early loss of up to 50% of cells is common for in vitro chondrogenesis of mesenchymal stromal cells (MSC) in pellet culture, reducing the efficacy and the tissue yield for cartilage engineering. Enhanced proliferation could compensate for this unwanted effect, but relevant signaling pathways remain largely unknown. The aim of this study was to identify the contribution of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), insulin-like growth factor (IGF), and hedgehog (HH) signaling toward cell proliferation during chondrogenesis and investigate whether a further mitogenic stimulation is possible and promising. Human MSC were subjected to chondrogenesis in the presence or absence of pathway inhibitors or activators up to Day 14 or from Days 14 to 28, before proliferation, DNA and proteoglycan content were quantified. [3H]-thymidine incorporation revealed arrest of proliferation on Day 3, after which cell division was reinitiated. Although BMP signaling was essential for proliferation throughout chondrogenesis, IGF signaling was relevant only up to Day 14. In contrast, FGF and HH signaling drove proliferation only from Day 14 onward. Early BMP4, IGF-1, or FGF18 treatment neither prevented early cell loss nor allowed further mitogenic stimulation. However, application of the HH-agonist purmorphamine from Day 14 increased proliferation 1.44-fold (p < 0.05) and late BMP4-application enhanced the DNA and proteoglycan content, with significant effects on tissue yield. Conclusively, a differential and phase-dependent contribution of the four pathways toward proliferation was uncovered and BMP4 treatment was promising to enhance tissue yield. Culture forms less prone to size limitations by nutrient/oxygen gradients and a focus on early apoptosis prevention may be considered as the next steps to further enhance chondrocyte formation from MSC.
体外微团培养的间充质基质细胞（MSC）软骨形成中，早期细胞丢失高达 50%是常见的，这降低了软骨工程的疗效和组织产量。增强增殖可以补偿这种不良影响，但相关信号通路在很大程度上仍然未知。本研究的目的是确定骨形态发生蛋白（BMP）、成纤维细胞生长因子（FGF）、胰岛素样生长因子（IGF）和 hedgehog（HH）信号在软骨形成过程中对细胞增殖的贡献，并研究是否可以进一步进行有丝分裂刺激，以及这种刺激是否有前景。将人 MSC 置于存在或不存在信号通路抑制剂或激活剂的情况下进行软骨形成，直至第 14 天或第 14 天至第 28 天，然后定量增殖、DNA 和蛋白聚糖含量。[3H]-胸苷掺入表明增殖在第 3 天停止，之后细胞分裂重新开始。尽管 BMP 信号在整个软骨形成过程中对于增殖是必需的，但 IGF 信号仅在第 14 天之前才是相关的。相比之下，FGF 和 HH 信号仅从第 14 天开始驱动增殖。早期 BMP4、IGF-1 或 FGF18 处理既不能防止早期细胞丢失，也不能允许进一步有丝分裂刺激。然而，从第 14 天开始应用 HH-激动剂 purmorphamine 可使增殖增加 1.44 倍（p<0.05），并且晚期 BMP4 处理可增强 DNA 和蛋白聚糖含量，对组织产量有显著影响。总之，揭示了四个途径对增殖的不同和阶段依赖性贡献，并且 BMP4 处理有望提高组织产量。培养形式较少受到营养/氧气梯度的限制，并且关注早期细胞凋亡的预防可能被认为是进一步增强 MSC 形成软骨细胞的下一步。
