Regulation of human mesenchymal stem cells differentiation into chondrocytes in extracellular matrix-based hydrogel scaffolds
Regulation of human mesenchymal stem cells differentiation into chondrocytes in extracellular matrix-based hydrogel scaffolds
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DOI：
10.1016/j.colsurfb.2013.10.001
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发表时间：
2014-02-01
影响因子：
5.8
通讯作者：
Dai, Jianwu
中科院分区：
文献类型：
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作者：
Du, Mingchun;Liang, Hui;Dai, Jianwu
为了诱导人骨髓间充质干细胞(HMSCs)在三维微环境中分化为软骨细胞，我们利用软骨细胞外基质(ECM)成分硫酸软骨素(CS)和胶原(Col)制备了多孔水凝胶支架。浊度和粘度实验表明，当pH=2-3时，可以通过pH触发共沉淀形成水凝胶。酶联免疫吸附试验(ELISA)证实水凝胶支架可以像设想的那样可控地释放生长因子。释放转化生长因子-β(TGF-β)刺激hMSCs向软骨细胞分化，然后释放胶原结合域-碱性成纤维细胞生长因子(CBD-bFGF)促进分化并保持软骨细胞表型。在体外细胞培养实验中，比较细胞在不同微环境中的分化过程：二维培养为对照，三维培养为无生长因子(CC)的支架材料，含CBD-bFGF的样品(CC-C)，含转化生长因子-β的样品(CC-T)，含CBD-bFGF/TGF-β的样品(CC-CT)。实时定量聚合酶链式反应(RT-PCR)显示CD44和CD105的hMSC标记基因减少，而II型胶原和聚集素的标记基因增加，尤其是CC-CT标本。免疫组化结果进一步证实，CC-CT标本中CD44的hMSC标志蛋白消失，而II型胶原的软骨细胞标志蛋白出现。这些结果表明，含有生长因子的ECM水凝胶支架可以为hMSCs向软骨细胞分化提供合适的3D细胞生态位，并且分化过程可以受到可控释放的生长因子的调控。(C)2013爱思唯尔B.V.保留所有权利。
To induce human mesenchymal stem cells (hMSCs) to differentiate into chondrocytes in three-dimensional (3D) microenvironments, we developed porous hydrogel scaffolds using the cartilage extracellular matrix (ECM) components of chondroitin sulfate (CS) and collagen (COL). The turbidity and viscosity experiments indicated hydrogel could form through pH-triggered co-precipitation when pH = 2-3. Enzyme-linked immunosorbent assay (ELISA) confirmed the hydrogel scaffolds could controllably release growth factors as envisaged. Transforming growth factor-beta (TGF-beta) was released to stimulate hMSCs differentiation into chondrocytes; and then collagen binding domain-basic fibroblast growth factor (CBD-bFGF) was released to improve the differentiation and preserve the chondrocyte phenotype. In in vitro cell culture experiments, the differentiation processes were compared in different microenvironments: 2D culture in culture plate as control, 3D culture in the fabricated scaffolds without growth factors (CC), the samples with CBD-bFGF (CC-C), the samples with TGF-beta (CC-T), the samples with CBD-bFGF/TGF-beta (CC-CT). Real-time polymerase chain reaction (RT-PCR) revealed the hMSC marker genes of CD44 and CD105 decreased; at the same time the chondrocyte marker genes of collagen type II and aggrecan increased, especially in the CC-CT sample. lmmunostaining results further confirmed the hMSC marker protein of CD 44 disappeared and the chondrocyte marker protein of collagen type II emerged over time in the CC-CT sample. These results imply the ECM-based hydrogel scaffolds with growth factors can supply suitable 3D cell niches for hMSCs differentiation into chondrocytes and the differentiation process can be regulated by the controllably released growth factors. (C) 2013 Elsevier B.V. All rights reserved.