ECM coating modification generated by optimized decellularization process improves functional behavior of BMSCs
ECM coating modification generated by optimized decellularization process improves functional behavior of BMSCs
复制标题
通过优化的脱细胞工艺产生的 ECM 涂层修饰可改善 BMSC 的功能行为
DOI：
10.1016/j.msec.2019.110039
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发表时间：
2019-12-01
影响因子：
7.9
通讯作者：
Zhao, Jiyuan
中科院分区：
文献类型：
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作者：
Li, Mei;Zhang, Tingxia;Zhao, Jiyuan
骨间充质干细胞（BMSCs）已广泛应用于组织工程和再生医学领域。然而，BMSCs数量较少以及体外扩增后干细胞特性的丧失限制了BMSCs的临床应用。在本研究中，培养成骨细胞以沉积细胞外基质（ECM），然后去除细胞（脱细胞）以产生ECM涂层基质。优化脱细胞过程，最大限度地去除细胞和细胞成分，并保留整合的 ECM，这被证明有利于 BMSC 体外扩增。脱细胞后，TFFF-ECM（经triton X-100（T）和3次冻融循环（FFF）脱细胞）中仅检测到不到2％的残留DNA和细胞蛋白，远低于传统脱细胞方法（triton X-100（T）和NH4OH（N））产生的TN-ECM。同时，采用TFFF法脱细胞后，ECM成分和结构保存得最好。检测到更多的 ECM 蛋白，结构蛋白（纤连蛋白和胶原蛋白）在 TFFF-ECM 中表现为经典网络纤维。在功能上，各种脱细胞ECM（dECM）均被证明能够促进BMSCs增殖和成骨分化能力，从而维持BMSCs的干性。重要的是，在早期阶段，TFFF-ECM上培养的细胞比其他类型的dECM上培养的细胞生长得更快，并且在体外培养时，TFFF-ECM有利于保持高表达OCT4和NANOG的BMSCs的干性。蛋白质组学分析表明ECM中的蛋白质在多种生物活性和信号通路中发挥作用，有助于BMSC的干性维持。因此，本研究优化的温和脱细胞工艺增强了 dECM 对 BMSC 体外培养的有效性，并可能进一步应用于基于 BMSC 的组织修复。
Bone mesenchymal stem cells (BMSCs) have been widely applied in tissue engineering and regenerative medicine. However, small number of BMSCs and loss of stem cell characteristics after expansion in vitro limited clinical use of BMSCs. In the present study, osteoblasts were cultured to lay down extracellular matrix (ECM) and then the cells were removed (decellularization) to generate ECM coating substrates. The decellularization process was optimized to maximally remove cells and cellular components, along with integrated ECM retained which was demonstrated to be beneficial for BMSCs expansion in vitro. After decellularization, only less than 2% of residual DNA and cellular proteins were detected in TFFF-ECM (decellularized by triton X-100 (T) and three freeze/thaw cycles (FFF)), which was much less than that in TN-ECM generated by traditional decellularization method (triton X-100 (T) and NH4OH (N)). Meanwhile, ECM components and structure were preserved best after decellularization by TFFF method. More ECM proteins were detected, and structure proteins (fibronectin and collagen) exhibited as classic network fibers in TFFF-ECM. Functionally, all kinds of decellularized ECM (dECM) were demonstrated to promote BMSCs proliferation and osteogenic differentiation capacity, thus maintain the stemness of BMSCs. Importantly, cells cultured on TFFF-ECM grew faster than the cells on other kinds of dECM at early stage and TFFF-ECM was beneficial to preserve stemness of BMSCs with high expression of OCT4 and NANOG when cultured in vitro. Proteomic analysis showed the proteins in ECM functioned in multiple biological activities and signaling pathways, which contributed to stemness maintenance of BMSC. Thus, the mild decellularization process optimized in this study enhanced the effectiveness of dECM for BMSCs culture in vitro and maybe further applied to BMSCs based tissue repair.