Department of Orthopaedic Surgery and Traumatology, University of Turku and Turku University Hospital, Turku, Finland.
Tissue Eng Part C Methods. 2012 Sep;18(9):658-66. doi: 10.1089/ten.TEC.2011.0675. Epub 2012 May 14.
The development of in vitro culturing techniques for osteoblastic differentiation of human mesenchymal stem cells (hMSC) is important for cell biology research and the development of tissue-engineering applications. Dexamethasone (Dex) is a commonly used supplement, but the optimal use of Dex treatment is still unclear. By adjusting the timing of Dex supplementation, the negative effects of long-term Dex treatment could be overcome. Transient Dex treatment could contribute toward minimizing broad donor variation, which is a major challenge. We compared the two most widely used Dex concentrations of 10 and 100 nM as transient or continuous treatment and studied inter- and intraindividual variations in osteoblastic differentiation of hMSC. Characterized bone marrow-derived hMSC from 17 female donors of different age groups were used. During osteoblastic induction, the cells were treated with 10 or 100 nM Dex either transiently for different time periods or continuously. Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity and staining for ALP, von Kossa, collagen type I, and osteocalcin. Cell proliferation, cell viability, and apoptosis were also monitored. The strongest osteoblastic differentiation was observed when 100 nM Dex was present for the first week. In terms of inter- and intraindividual coefficients of variations, transient treatment with 100 nM Dex was superior to the other culture conditions and showed the lowest variations in all age groups. This study demonstrates that the temporary presence of 100 nM Dex during the first week of induction culture promotes hMSC osteoblastic differentiation and reduces inter- and intraindividual variations. With this protocol, we can reproducibly produce functional osteoblasts in vitro from the hMSC of different donor populations.
体外培养技术对于人骨髓间充质干细胞（hMSC）成骨分化的发展对于细胞生物学研究和组织工程应用的发展非常重要。地塞米松（Dex）是一种常用的补充剂，但 Dex 治疗的最佳使用方法仍不清楚。通过调整 Dex 补充的时间，可以克服长期 Dex 治疗的负面影响。短暂的 Dex 处理可以有助于最小化广泛的供体变异，这是一个主要挑战。我们比较了两种最广泛使用的 Dex 浓度 10 和 100 nM 作为短暂或连续处理，并研究了 hMSC 成骨分化的个体间和个体内差异。使用来自不同年龄组的 17 名女性供体的特征性骨髓来源的 hMSC。在成骨诱导期间，用 10 或 100 nM Dex 处理细胞，短暂处理不同时间段或连续处理。通过测量碱性磷酸酶（ALP）活性和 ALP、von Kossa、胶原 I 和骨钙蛋白染色来评估分化。还监测细胞增殖、细胞活力和细胞凋亡。当存在 100 nM Dex 的第一周时，观察到最强的成骨分化。就个体间和个体内变异系数而言，短暂处理 100 nM Dex 优于其他培养条件，并且在所有年龄组中显示出最低的变异。这项研究表明，在诱导培养的第一周短暂存在 100 nM Dex 可促进 hMSC 成骨分化并减少个体间和个体内的差异。使用该方案，我们可以从不同供体群体的 hMSC 体外重复性地产生功能性成骨细胞。
