Olvera Dinorath, Sathy Binulal N, Kelly Daniel J
Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland.
Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin, Dublin 2, Ireland.
ACS Biomater Sci Eng. 2020 Sep 14;6(9):5145-5161. doi: 10.1021/acsbiomaterials.0c00337. Epub 2020 Aug 12.
The bone-ligament interface transitions from a highly organized type I collagen rich matrix to a nonmineralized fibrocartilage region and finally to a mineralized fibrocartilage region that interfaces with the bone. Therefore, engineering the bone-ligament interface requires a biomaterial substrate capable of maintaining or directing the spatially defined differentiation of multiple cell phenotypes. To date the appropriate combination of biophysical and biochemical factors that can be used to engineer such a biomaterial substrate remain unknown. Here we show that microfiber scaffolds functionalized with tissue-specific extracellular matrix (ECM) components can direct the differentiation of MSCs toward the phenotypes seen at the bone-ligament interface. Ligament-ECM (L-ECM) promoted the expression of the ligament-marker gene tenomodulin () and higher levels of type I and III collagen expression compared to functionalization with commercially available type I collagen. Functionalization of microfiber scaffolds with cartilage-ECM (C-ECM) promoted chondrogenesis of MSCs, as evidenced by adoption of a round cell morphology and increased SRY-box 9 () expression in the absence of exogenous growth factors. Next, we fabricated a multiphasic scaffold by controlling the spatial presentation of L-ECM and C-ECM along the length of a single electrospun microfiber construct, with the distal region of the C-ECM coated fibers additionally functionalized with an apatite layer (using simulated body fluid) to promote endochondral ossification. These ECM functionalized scaffolds promoted spatially defined differentiation of MSCs, with higher expression of observed in the region functionalized with L-ECM, and higher expression of type X collagen and osteopontin (markers of endochondral ossification) observed at the end of the scaffold functionalized with C-ECM and the apatite coating. Our results demonstrate the utility of tissue-specific ECM derived components as a cue for directing MSC differentiation when engineering complex multiphasic interfaces such as the bone-ligament enthesis.
骨 - 韧带界面从富含高度有序的I型胶原蛋白的基质过渡到非矿化的纤维软骨区域，最后过渡到与骨相接的矿化纤维软骨区域。因此，构建骨 - 韧带界面需要一种能够维持或引导多种细胞表型在空间上特定分化的生物材料基质。迄今为止，可用于构建这种生物材料基质的生物物理和生化因素的适当组合仍然未知。在这里，我们表明用组织特异性细胞外基质（ECM）成分功能化的微纤维支架可以引导间充质干细胞（MSC）向在骨 - 韧带界面所见的表型分化。与用市售I型胶原蛋白功能化相比，韧带ECM（L - ECM）促进了韧带标记基因肌腱调节蛋白（）的表达以及I型和III型胶原蛋白表达水平的提高。用软骨ECM（C - ECM）对微纤维支架进行功能化促进了MSC的软骨形成，这在没有外源性生长因子的情况下通过呈现圆形细胞形态和增加SRY盒9（）表达得到证明。接下来，我们通过控制L - ECM和C - ECM沿单个电纺微纤维构建体长度的空间呈现来制造一种多相支架，C - ECM包被纤维的远端区域还用磷灰石层（使用模拟体液）进行功能化以促进软骨内骨化。这些ECM功能化支架促进了MSC在空间上的特定分化，在用L - ECM功能化的区域中观察到更高的表达，在用C - ECM和磷灰石涂层功能化的支架末端观察到X型胶原蛋白和骨桥蛋白（软骨内骨化标记物）的更高表达。我们的结果证明了组织特异性ECM衍生成分在构建复杂的多相界面（如骨 - 韧带附着点）时作为引导MSC分化线索的效用。
