Department of Health Sciences and Technology, ETH Zürich, CH-8093 Zürich, Switzerland.
ZHAW School of Life Sciences and Facility Management, Institute for Chemistry and Biotechnology, Center for Cell Biology and Tissue Engineering, CH-8820 Wädenswil, Switzerland.
Biomater Sci. 2019 Nov 1;7(11):4519-4535. doi: 10.1039/c9bm00868c. Epub 2019 Aug 22.
Macromolecular crowding is used by tissue engineers to accelerate extracellular matrix assembly in vitro, however, most mechanistic studies focus on the impact of crowding on collagen fiber assembly and largely ignore the highly abundant provisional matrix protein fibronectin. We show that the accelerated collagen I assembly as induced by the neutral crowding molecule Ficoll is regulated by cell access to fibronectin. Ficoll treatment leads to significant increases in the amount of surface adherent fibronectin, which can readily be harvested by cells to speed up fibrillogenesis. FRET studies reveal that Ficoll crowding also upregulates the total amount of fibronectin fibers in a low-tension state through upregulating fibronectin assembly. Since un-stretched fibronectin fibers have more collagen binding sites to nucleate the onset of collagen fibrillogenesis, our data suggest that the Ficoll-induced upregulation of low-tension fibronectin fibers contributes to enhanced collagen assembly in crowded conditions. In contrast, chemical cross-linking of fibronectin to the glass substrate prior to cell seeding prevents early force mediated fibronectin harvesting from the substrate and suppresses upregulation of collagen I assembly in the presence of Ficoll, even though the crowded environment is known to drive enzymatic cleavage of procollagen and collagen fiber formation. To show that our findings can be exploited for tissue engineering applications, we demonstrate that the addition of supplemental fibronectin in the form of an adsorbed coating markedly improves the speed of tissue formation under crowding conditions.
大分子拥挤被组织工程师用于加速细胞外基质在体外的组装，然而，大多数机制研究都集中在拥挤对胶原蛋白纤维组装的影响上，而很大程度上忽略了丰富的临时基质蛋白纤维连接蛋白。我们表明，中性拥挤分子 ficoll 诱导的胶原蛋白 I 组装的加速受细胞对纤维连接蛋白的获取的调节。ficoll 处理会导致表面附着的纤维连接蛋白的量显著增加，细胞可以很容易地收获这些纤维连接蛋白来加速纤维原纤维的形成。FRET 研究表明，ficoll 拥挤还通过上调纤维连接蛋白组装来上调低张力状态下纤维连接蛋白纤维的总量。由于未拉伸的纤维连接蛋白纤维具有更多的胶原蛋白结合位点来引发胶原蛋白纤维原纤维的形成，我们的数据表明，ficoll 诱导的低张力纤维连接蛋白纤维的上调有助于增强拥挤条件下的胶原蛋白组装。相比之下，在细胞接种之前，用化学交联剂将纤维连接蛋白交联到玻璃基质上，可以防止早期力介导的纤维连接蛋白从基质上收获，并抑制在 ficoll 存在下胶原蛋白 I 组装的上调，尽管已知拥挤环境会驱动前胶原蛋白的酶切和胶原蛋白纤维的形成。为了表明我们的发现可以被用于组织工程应用，我们证明了以吸附涂层形式添加补充纤维连接蛋白可以显著提高在拥挤条件下组织形成的速度。
