Department for Trauma Surgery, Regensburg University Medical Center, Regensburg, Germany.
Cells Tissues Organs. 2010;192(3):158-66. doi: 10.1159/000313399. Epub 2010 Apr 20.
Induction of chondrogenesis in mesenchymal stem cells (MSCs) with TGF-beta leads to a hypertrophic phenotype. The hypertrophic maturation of the chondrocytes is dependent on the timed removal of TGF-beta and sensitive to hypertrophy-promoting agents in vitro. In this study, we have investigated whether TGF-beta3, which has been shown to be more prochondrogenic compared to TGF-beta1, similarly enhances terminal differentiation in an in vitro hypertrophy model of chondrogenically differentiating MSCs. In addition, we tested the impact of the time of chondrogenic conditioning on the enhancement of hypertrophy. MSCs were chondrogenically differentiated in pellet culture in medium containing TGF-beta1 or TGF-beta3. After 2 or 4 weeks, chondrogenic medium was switched to hypertrophy-inducing medium for 2 weeks. Aggregates were analyzed histologically and biochemically on days 14, 28 and 42. The switch to hypertrophy medium after 14 days induced hypertrophic cell morphology and significant increase in alkaline phosphatase activity compared to the chondrogenesis only control using both TGF-beta1 and TGF-beta3. After 28 days predifferentiation, differences between hypertrophic and control groups diminished compared to 14 days predifferentiation. In conclusion, chondrogenic conditioning with both TGF-beta isoforms similarly induced hypertrophy in our experiment and allowed the enhancement of the hypertrophic chondrocyte phenotype by hypertrophic medium. Enhancement of hypertrophy was seen more clearly after the shorter chondrogenic conditioning. Therefore, to utilize this experimental model as a tool to study hypertrophy in MSC chondrogenesis, a predifferentiation period of 14 days is recommended.
转化生长因子-β（TGF-β）诱导间充质干细胞（MSCs）向软骨细胞分化会导致细胞出现肥大表型。软骨细胞的肥大成熟依赖于 TGF-β的适时去除，并且在体外对促进肥大的试剂敏感。在这项研究中，我们研究了与 TGF-β1 相比，具有更强软骨形成能力的 TGF-β3 是否同样会增强体外软骨形成的 MSC 肥大分化模型中的终末分化。此外，我们还测试了软骨形成条件作用时间对促进肥大的影响。MSCs 在含 TGF-β1 或 TGF-β3 的培养基中以微球体培养的方式进行软骨分化。2 或 4 周后，将软骨形成培养基切换为肥大诱导培养基培养 2 周。在第 14、28 和 42 天对聚集物进行组织学和生化分析。与仅用 TGF-β1 和 TGF-β3 的软骨形成对照相比，在第 14 天切换到肥大培养基会诱导出肥大细胞形态，并显著增加碱性磷酸酶活性。在预分化 28 天后，与预分化 14 天后相比，肥大组与对照组之间的差异减小。总之，在我们的实验中，两种 TGF-β 同工型的软骨形成条件作用均会相似地诱导肥大，并且允许通过肥大培养基增强肥大的软骨细胞表型。在较短的软骨形成条件作用后，更明显地增强了肥大作用。因此，为了将此实验模型用作研究 MSC 软骨形成中肥大的工具，建议将预分化时间设为 14 天。
