Division of Orthopaedic Surgery, McGill University, Montreal, Canada.
Tissue Eng Part A. 2010 Nov;16(11):3449-55. doi: 10.1089/ten.TEA.2010.0091. Epub 2010 Aug 18.
A major drawback of current cartilage and intervertebral disc tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients express type X collagen (COL10), a marker of late-stage chondrocyte hypertrophy (associated with endochondral ossification). Parathyroid hormone (PTH) regulates endochondral ossification by inhibiting chondrocyte differentiation toward hypertrophy. In this study, we investigated the effect of PTH on expression of COL10 in MSCs from OA patients and analyzed the potential mechanisms related to its effect. MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA. Expanded cells were then incubated for 0-48 h without (control) or with 100 nM PTH (1-34). Protein expression and phosphorylation were measured by Western blot. Results showed that PTH (1-34) inhibited expression of COL10 in MSCs from OA patients in a time-dependent manner. In parallel, PTH (1-34) stimulated expression of COL2, a marker of chondrogenic differentiation. Results also showed that PTH (1-34) inhibited in a sustained manner the phosphorylation of p38 and AKT protein kinase signaling pathways. Interestingly, the modulation of COL2 and COL10 gene expression was significant as rapidly as after 1 h in the presence of PTH (1-34); changes in the phosphorylation of p38 and AKT were significant only after 6 h. This suggests that while p38 and AKT protein kinase signaling pathways may not be required to initiate the regulation of expression of COL2 and COL10 by PTH (1-34), these pathways may modulate later events necessary for preventing precocious MSC hypertrophy.
当前软骨和椎间盘组织工程的一个主要缺点是,来自骨关节炎(OA)患者的间充质干细胞(MSCs)表达 X 型胶原(COL10),这是晚期软骨细胞肥大(与软骨内骨化有关)的标志物。甲状旁腺激素(PTH)通过抑制软骨细胞向肥大分化来调节软骨内骨化。在这项研究中,我们研究了 PTH 对 OA 患者 MSCs 中 COL10 表达的影响,并分析了与其作用相关的潜在机制。从接受全髋关节置换术的 OA 供体骨髓腔内抽吸物中获得 MSCs。然后将扩增的细胞在无(对照)或有 100 nM PTH(1-34)的情况下孵育 0-48 小时。通过 Western blot 测量蛋白质表达和磷酸化。结果表明,PTH(1-34)以时间依赖性方式抑制 OA 患者 MSCs 中 COL10 的表达。同时,PTH(1-34)刺激 COL2 的表达,COL2 是软骨分化的标志物。结果还表明,PTH(1-34)以持续的方式抑制 p38 和 AKT 蛋白激酶信号通路的磷酸化。有趣的是,在存在 PTH(1-34)的情况下,COL2 和 COL10 基因表达的调节在 1 小时内就非常显著;p38 和 AKT 的磷酸化变化仅在 6 小时后才显著。这表明,虽然 p38 和 AKT 蛋白激酶信号通路可能不是启动 PTH(1-34)调节 COL2 和 COL10 表达所必需的,但这些通路可能调节随后发生的、对防止 MSC 过早肥大所必需的事件。