Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Republic of Korea; Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University Graduate School, Republic of Korea.
Department of Biochemistry, Medical College, Inha University, Republic of Korea.
Acta Biomater. 2014 Jun;10(6):2750-61. doi: 10.1016/j.actbio.2014.01.021. Epub 2014 Jan 24.
Control over the interface of biomaterials that favors the initial adhesion and subsequent differentiation of stem cells is one of the key strategies in bone tissue engineering. Here we engineer the interface of biopolymer electrospun fiber matrices with a fusion protein of fibronectin 9-10 domain (FNIII9-10) and osteocalcin (OCN), aiming to stimulate mesenchymal stem cell (MSC) functions, including initial adhesion, growth and osteogenic differentiation. In particular, a specific tethering of FNIII9-10-OCN protein was facilitated by the hydroxyapatite (HA) mineralization of the biopolymer surface through a molecular recognition of OCN to the HA crystal lattice. The FNIII9-10-OCN anchorage to the HA-mineralized fiber was observed to be highly specific and tightly bound to preserve stability over a long period. Initial cell adhesion levels, as well as the spreading shape and process, of MSCs within 24h were strikingly different between the fibers linked with and without fusion protein. Significant up-regulations in the mRNA expression of adhesion signaling molecules occurred with the fusion protein link, as analyzed by the reverse transcriptase polymerase chain reaction. The expression of a series of osteogenic-related genes at later stages, over 2-3weeks, was significantly improved in the fusion protein-tailored fiber, and the osteogenic protein levels were highly stimulated, as confirmed by immunofluorescence imaging and fluorescence-activated cell sorting analyses. In vivo study in a rat calvarium model confirmed a higher quantity of new bone formation in the fiber linked with fusion protein, and a further increase was noticed when the MSCs were tissue-engineered with the fusion protein-linked fiber. Collectively, these results indicate that FN-OCN fusion protein links via HA mineralization is a facile tool to generate a biointerface with cell-attractive and osteogenic potential, and that the engineered fibrous matrix is a potential bone regenerative scaffold.
控制有利于干细胞初始黏附和随后分化的生物材料界面是骨组织工程的关键策略之一。在这里，我们通过纤维连接蛋白 9-10 结构域（FNIII9-10）和骨钙素（OCN）融合蛋白对生物聚合物电纺纤维基质的界面进行了工程改造，旨在刺激间充质干细胞（MSC）的功能，包括初始黏附、生长和成骨分化。特别是，通过 OCN 对 HA 晶格的分子识别，促进了 FNIII9-10-OCN 蛋白在生物聚合物表面的羟基磷灰石（HA）矿化的特定固定。观察到 FNIII9-10-OCN 锚定到 HA 矿化纤维上具有高度特异性和紧密结合，可在很长一段时间内保持稳定性。在 24 小时内，MSC 的初始细胞黏附水平以及细胞的扩展形状和过程在与融合蛋白相连和不相连的纤维之间有明显差异。通过逆转录聚合酶链反应分析，发现融合蛋白连接时粘附信号分子的 mRNA 表达显著上调。在融合蛋白定制纤维中，在稍后阶段（2-3 周），一系列成骨相关基因的表达显著改善，并且通过免疫荧光成像和荧光激活细胞分选分析证实了成骨蛋白水平的高度刺激。在大鼠颅骨模型的体内研究中证实，在与融合蛋白相连的纤维中形成了更多的新骨，并且当将 MSC 与融合蛋白相连的纤维组织工程化时，发现了进一步的增加。总的来说，这些结果表明，通过 HA 矿化的 FN-OCN 融合蛋白连接是一种生成具有细胞吸引力和成骨潜力的生物界面的简便工具，并且工程化纤维基质是一种有潜力的骨再生支架。
