Center for Biomedical Engineering, Brown University, Providence, RI, 02129, USA.
Center for Alternatives to Animals in Testing, Brown University, Providence, RI, 02129, USA.
Adv Sci (Weinh). 2022 Apr;9(10):e2103939. doi: 10.1002/advs.202103939. Epub 2022 Feb 1.
Dysregulation of extracellular matrix (ECM) synthesis, organization, and mechanics are hallmark features of diseases like fibrosis and cancer. However, most in vitro models fail to recapitulate the three-dimensional (3D) multi-scale hierarchical architecture of collagen-rich tissues and as a result, are unable to mirror native or disease phenotypes. Herein, using primary human fibroblasts seeded into custom fabricated 3D non-adhesive agarose molds, a novel strategy is proposed to direct the morphogenesis of engineered 3D ring-shaped tissue constructs with tensile and histological properties that recapitulate key features of fibrous connective tissue. To characterize the shift from monodispersed cells to a highly-aligned, collagen-rich matrix, a multi-modal approach integrating histology, multiphoton second-harmonic generation, and electron microscopy is employed. Structural changes in collagen synthesis and alignment are then mapped to functional differences in tissue mechanics and total collagen content. Due to the absence of an exogenously added scaffolding material, this model enables the direct quantification of cell-derived changes in 3D matrix synthesis, alignment, and mechanics in response to the addition or removal of relevant biomolecular perturbations. To illustrate this, the effects of nutrient composition, fetal bovine serum, rho-kinase inhibitor, and pro- and anti-fibrotic compounds on ECM synthesis, 3D collagen architecture, and mechanophenotype are quantified.
细胞外基质（ECM）合成、组织和力学的失调是纤维化和癌症等疾病的标志性特征。然而，大多数体外模型无法再现富含胶原蛋白组织的三维（3D）多尺度层次结构，因此无法反映天然或疾病表型。在此，通过将原代人成纤维细胞接种到定制的 3D 非粘附琼脂糖模具中，提出了一种新策略，以指导具有拉伸和组织学特性的工程 3D 环形组织构建体的形态发生，这些特性再现了纤维结缔组织的关键特征。为了表征从单分散细胞向高度排列的富含胶原蛋白基质的转变，采用了一种多模态方法，该方法整合了组织学、多光子二次谐波产生和电子显微镜。然后将胶原合成和排列的结构变化映射到组织力学和总胶原蛋白含量的功能差异上。由于没有添加外源性支架材料，该模型能够直接量化细胞衍生的 3D 基质合成、排列和力学变化，以响应相关生物分子扰动的添加或去除。为了说明这一点，定量研究了营养成分、胎牛血清、rho-kinase 抑制剂、促纤维化和抗纤维化化合物对 ECM 合成、3D 胶原结构和力学表型的影响。
