Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina, USA.
Tissue Eng Part A. 2013 Jan;19(1-2):299-306. doi: 10.1089/ten.TEA.2012.0015. Epub 2012 Sep 26.
This study investigates the effects of cyclic hydrostatic pressure (CHP) on chondrogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3-D) agarose constructs maintained in a complete growth medium without soluble chondrogenic inducing factors. hASCs were seeded in 2% agarose hydrogels and exposed to 7.5 MPa CHP for 4 h per day at a frequency of 1 Hz for up to 21 days. On days 0, 7, 14, and 21, the expression levels of collagen II, Sox9, aggrecan, and cartilage oligomeric matrix protein (COMP) were examined by real-time reverse transcriptase-polymerase chain reaction analysis. Gene expression analysis found collagen II mRNA expression in only the CHP-loaded construct at day 14 and at no other time during the study. CHP-loaded hASCs exhibited upregulated mRNA expression of Sox9, aggrecan, and COMP at day 7 relative to unloaded controls, suggesting that CHP initiated chondrogenic differentiation of hASCs in a manner similar to human bone marrow-derived mesenchymal stem cells (hMSC). By day 14, however, loaded hASC constructs exhibited significantly lower mRNA expression of the chondrogenic markers than unloaded controls. Additionally, by day 21, the samples exhibited little measurable mRNA expression at all, suggesting a decreased viability. Histological analysis validated the lack of mRNA expression at day 21 for both the loaded and unloaded control samples with a visible decrease in the cell number and change in morphology. A comparative study with hASCs and hMSCs further examined long-term cell viability in 3-D agarose constructs of both cell types. Decreased cell metabolic activity was observed throughout the 21-day experimental period in both the CHP-loaded and control constructs of both hMSCs and hASCs, suggesting a decrease in cell metabolic activity, alluding to a decrease in cell viability. This suggests that a 2% agarose hydrogel may not optimally support hASC or hMSC viability in a complete growth medium in the absence of soluble chondrogenic inducing factors over long culture durations. This is the first study to examine the ability of mechanical stimuli alone, in the absence of chondrogenic factors transforming growth factor beta (TGF-β)3, TGF-β1 and/or bone morphogenetic protein 6 (BMP6) to induce hASC chondrogenic differentiation. The findings of this study suggest that CHP initiates hASC chondrogenic differentiation, even in the absence of soluble chondrogenic inductive factors, confirming the importance of considering both mechanical stimuli and appropriate 3-D culture for cartilage tissue engineering using hASCs.
本研究旨在探讨循环静压（CHP）对三维（3-D）琼脂糖构建体中无可溶性软骨诱导因子的人脂肪来源干细胞（hASC）软骨分化的影响。将 hASC 接种于 2%琼脂糖水凝胶中，并在 1 Hz 频率下每天施加 7.5 MPa CHP4 小时，持续 21 天。在第 0、7、14 和 21 天，通过实时逆转录聚合酶链反应分析检测 II 型胶原、Sox9、聚集蛋白聚糖和软骨寡聚基质蛋白（COMP）的表达水平。基因表达分析发现，只有在第 14 天加载 CHP 的构建体中检测到 II 型胶原 mRNA 的表达，而在研究期间的其他任何时间都没有检测到。与未加载对照相比，加载 CHP 的 hASC 在第 7 天表现出 Sox9、聚集蛋白聚糖和 COMP 的 mRNA 表达上调，表明 CHP 以类似于人骨髓间充质干细胞（hMSC）的方式启动 hASC 的软骨分化。然而，到第 14 天，加载 hASC 构建体的软骨标志物 mRNA 表达显著低于未加载对照。此外，到第 21 天，所有样本的 mRNA 表达几乎都无法测量，表明细胞活力下降。组织学分析验证了加载和未加载对照样本在第 21 天都缺乏 mRNA 表达，细胞数量明显减少，形态发生变化。与 hASC 和 hMSC 的比较研究进一步检测了这两种细胞类型在 3-D 琼脂糖构建体中的长期细胞活力。在整个 21 天的实验期间，无论是加载 CHP 的还是未加载对照的 hMSC 和 hASC 构建体，细胞代谢活性都明显下降，表明细胞代谢活性下降，暗示细胞活力下降。这表明，在缺乏可溶性软骨诱导因子的情况下，在完整生长培养基中，2%琼脂糖水凝胶可能无法长时间支持 hASC 或 hMSC 的活力。这是第一项研究，单独检查机械刺激的能力，在没有转化生长因子β（TGF-β）3、TGF-β1 和/或骨形态发生蛋白 6（BMP6）等软骨形成因子的情况下，诱导 hASC 软骨形成分化。本研究结果表明，CHP 可启动 hASC 软骨形成分化，即使在缺乏可溶性软骨诱导因子的情况下也是如此，这证实了在使用 hASC 进行软骨组织工程时，不仅要考虑机械刺激，还要考虑适当的 3-D 培养。
