Guangdong Police College, Guangzhou 510440, Guangdong, China.
College of Chemistry and Materials Science, Jinan University, 601 West Huangpu Avenue, Guangzhou 510632, Guangdong, China.
ACS Appl Mater Interfaces. 2024 Oct 23;16(42):56801-56814. doi: 10.1021/acsami.4c12847. Epub 2024 Oct 10.
Oriented fibers in bone lamellae are recognized for their contribution to the anisotropic mechanical performance of the cortical bone. While increasing evidence highlights that such oriented fibers also exhibit osteogenic induction to preosteoblasts, little is known about the effect of the overlay angle between lamellae on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). In this study, bone lamellae-like fibrous matrices composed of aligned core-shell [core: polycaprolactone (PCL)/type I collagen (Col I) + shell: Col I] nanofibers were seeded with human BMSCs (hBMSCs) and then laid over on each other layer-by-layer (L--L) at selected angles (0 or 45°) to form three-dimensional (3D) constructs. Upon culture for 7 and 14 days, osteogenic differentiation of hBMSCs and mineralization within the lamellae assembly (LA) were characterized by real-time PCR, Western blot, immunofluorescent staining for osteogenic markers, and alizarin red staining for calcium deposition. Compared to those of random nanofibers (LA-RF) or aligned fibers with the overlay angle of 45° (LA-AF-45), the LA of aligned fibers at a 0° overlay angle (LA-AF-0) exhibited a noticeably higher osteogenic differentiation of hBMSCs, i.e., elevated gene expression of OPN, OCN, and RUNX2 and protein levels of ALP and RUNX2, while promoting mineral deposition as indicated by alizarin red staining and mechanical testing. Further analyses of hBMSCs within LA-AF-0 revealed an increase in both total and phosphorylated integrin β1, which subsequently increased total focal adhesion kinase (FAK), phosphorylated FAK (p-FAK), and phosphorylated extracellular signal kinase ERK1/2 (p-ERK1/2). Inhibition of integrin β1 and ERK1/2 activity effectively reduced the LA-AF-0-induced upregulation of p-FAK and osteogenic markers (OPN, OCN, and RUNX2), confirming the involvement of integrin β1-FAK-ERK1/2 signaling. Altogether, the overlay angle of aligned core-shell nanofiber membranes regulates the osteogenic differentiation of hBMSCs via integrin β1-FAK-ERK1/2 signaling, unveiling the effects of anisotropic fibers on bone tissue formation.
骨板中的定向纤维因其对皮质骨各向异性力学性能的贡献而被认可。虽然越来越多的证据表明，这种定向纤维也对成骨前体细胞具有成骨诱导作用，但对于骨板之间的覆盖角对骨髓间充质干细胞（BMSCs）成骨分化的影响知之甚少。在这项研究中，由定向核壳[核：聚己内酯（PCL）/I 型胶原蛋白（Col I）+壳：Col I]纳米纤维组成的类似骨板的纤维基质被接种了人骨髓间充质干细胞（hBMSCs），然后以选定的角度（0 或 45°）分层（L--L）铺设在一起，形成三维（3D）结构。培养 7 天和 14 天后，通过实时 PCR、Western blot、成骨标志物的免疫荧光染色和茜素红染色检测钙沉积，对 hBMSCs 的成骨分化和骨板组装（LA）内的矿化进行了表征。与随机纳米纤维（LA-RF）或 45°覆盖角的定向纤维（LA-AF-45）相比，0°覆盖角的定向纤维的 LA（LA-AF-0）明显促进了 hBMSCs 的成骨分化，即 OPN、OCN 和 RUNX2 的基因表达和 ALP 和 RUNX2 的蛋白水平升高，同时通过茜素红染色和力学测试促进了矿化沉积。对 LA-AF-0 内的 hBMSCs 的进一步分析表明，整合素β1 的总蛋白和磷酸化蛋白均增加，随后总粘着斑激酶（FAK）、磷酸化 FAK（p-FAK）和磷酸化细胞外信号调节激酶 ERK1/2（p-ERK1/2）增加。整合素β1 和 ERK1/2 活性的抑制有效地降低了 LA-AF-0 诱导的 p-FAK 和成骨标志物（OPN、OCN 和 RUNX2）的上调，证实了整合素β1-FAK-ERK1/2 信号通路的参与。总之，定向核壳纳米纤维膜的覆盖角通过整合素β1-FAK-ERK1/2 信号通路调节 hBMSCs 的成骨分化，揭示了各向异性纤维对骨组织形成的影响。
