Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan.
Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan.
J Biosci Bioeng. 2021 Jun;131(6):686-695. doi: 10.1016/j.jbiosc.2021.02.005. Epub 2021 Mar 26.
Angiogenesis is a pressing issue in tissue engineering associated with restoration of blood supply to ischemic tissues and promotion of rapid vascularization of tissue-engineered grafts. Fibroblast growth factor-2 (FGF-2) plays a vital role in processes such as angiogenesis and is an attractive candidate for tissue engineering. While skeletal muscle tissue engineering is established, the role of FGF-2 in endothelial function to promote angiogenesis after transplantation is unclear. Here, a culture system comprising a five-layered sheet of human skeletal muscle cells co-incubated on green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) mimicking in vivo angiogenesis was used to investigate the role of FGF-2 in vascularization of engineered tissues. The basal level of FGF-2 in cultured media of skeletal muscle cell sheets was undetectable. Therefore, cell sheets co-incubated with GFP-HUVECs were exogenously treated with 10 ng/mL FGF-2, and endothelial network formation was evaluated. After prolonged culture, the endothelial network length and connectivity increased following treatment with FGF-2 as compared with control treatment. The numbers of medium and long endothelial networks significantly increased inside the sheet longer than 0.2 and 0.4 cm, respectively, after FGF-2 treatment. Time-lapse microscopy monitoring dynamic endothelial behavior revealed that FGF-2-mediated maintenance of endothelial connection and retardation of endothelial network disconnection after 72 h. The present study suggests the precise role of FGF-2 in maintaining endothelial connection and the extent of the endothelial network in skeletal muscle cell sheets. This understanding can be applied to design in vitro pre-vascularized tissue and graft integration prospects.
血管生成是组织工程中一个紧迫的问题，它与恢复缺血组织的血液供应和促进组织工程移植物的快速血管化有关。成纤维细胞生长因子 2（FGF-2）在血管生成等过程中起着至关重要的作用，是组织工程的一个有吸引力的候选物。虽然骨骼肌组织工程已经建立，但 FGF-2 在移植后促进血管生成的内皮功能中的作用尚不清楚。在这里，使用了一种包含五层共培养的人骨骼肌细胞的培养系统，这些细胞与表达绿色荧光蛋白的人脐静脉内皮细胞（GFP-HUVEC）共培养，模拟体内血管生成，以研究 FGF-2 在工程组织血管化中的作用。骨骼肌细胞片培养介质中 FGF-2 的基础水平无法检测到。因此，将 GFP-HUVEC 共培养的细胞片用 10ng/mL 的 FGF-2 进行外源性处理，并评估内皮网络的形成。经过长时间培养后，与对照处理相比，FGF-2 处理后内皮网络长度和连通性增加。与对照处理相比，在 FGF-2 处理后，长度超过 0.2 和 0.4cm 的细胞片中，中长内皮网络的数量显著增加。延时显微镜监测动态内皮行为表明，FGF-2 介导的维持内皮连接和延缓内皮网络断开发生在 72 小时后。本研究表明 FGF-2 在维持内皮连接和骨骼肌细胞片中内皮网络的程度方面的精确作用。这种理解可以应用于设计体外预血管化组织和移植物整合的前景。
