Vitamin C-Incorporated Polycaprolactone Membranes Enhance Angiogenic–Osteogenic Coupling by Modulating Oxidative Stress In Vitro

Abstract

The study investigates the influence of vitamin C-incorporated polycaprolactone (PCL-Vit C) membrane in scavenging reactive oxygen species (ROS) and enhancing angiogenic–osteogenic coupling in vitro. Human umbilical vein endothelial cells and human fetal osteoblast cells were co-cultured on PCL membrane alone and PCL-Vit C membranes under osteogenic conditions to mimic the bone microenvironment. Angiogenic–osteogenic crosstalk was visualized by confocal microscopy, using PECAM1 and connexin-43 antibodies. Cell viability was assessed by XTT assay and ROS generation measured by flow cytometry. Alkaline phosphatase (ALP) and vascular endothelial growth factor (VEGF) release were determined calorimetrically and by ELISA (enzyme-linked immunosorbent assay), respectively. ALP, Col1, RUNX-2, OC, VEGF messenger RNA (mRNA) expressions were determined by quantitative polymerase chain reaction. Col1, OC, BMP-7, HIF-α protein levels were assessed by western blotting. Activation of P38, P44, and JNK signaling was also analyzed. PCL-Vit C membrane co-cultures showed abundant expression of gap junction protein, Cx43, and significantly increased viability compared to PCL alone. ROS production significantly declined in co-cultures grown on PCL-Vit C membranes, with elevated levels of VEGF and ALP. Expression of ALP, Col 1, RUNX2, OC, and VEGF mRNA was significantly higher in PCL-Vit C membranes. PCL-Vit C co-cultures further demonstrated significantly lower HIF-α levels, increased Col 1, OC, and BMP-7 levels, and enhanced activation of phospho-p38/P38, phospho-p44/44, and phospho-pJNK/JNK. PCL-Vit C membranes supported angiogenic–osteogenic coupling, accompanied by changes in oxidative stress, intercellular communication, and osteogenesis through activating key signaling pathways.