Exogenous FGF-2 prolongs endothelial connection in multilayered human skeletal muscle cell sheet

Abstract

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 invivo 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 10ng/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 0.4cm, 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 72h. 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 invitro pre-vascularized tissue and graft integration prospects.