Coculture to vascularization transition in bioengineered skin grafts through VEGF-associated pathways tracked by exosomal biomarkers†

Inadequate vasculature poses a significant challenge in the clinical translation of tissue engineering constructs. Current strategies for vascularization typically recruit short-lived endothelial cells or induce mesenchymal stem cells (MSC) to differentiate into the endothelial lineage, often in combination with supporting pericytes or fibroblasts. However, endothelial-associated cocultures lack adaptive ability and form limited vasculature. In this study, we investigated the endothelial transdifferentiation of an MSC-fibroblast coculture loaded on a bioengineered graft and utilized the exosomes released by the coculture model as a biomarker to monitor the progress of vascularization inside the graft. To develop the pre-vascularized skin graft, dermal fibroblasts and MSC were seeded on a biocomposite chitosan/collagen/fibrinogen/D3 (CCF-D3) scaffold. The cocultured graft facilitated the differentiation of MSC to endothelial cells (MEnDoT). Additionally, it promoted vasculogenic sprouting through the VEGF–eNOS pathways, as evidenced by the expression of F-actin, VEGF-A, and downstream transcriptomic markers (CD31, CD34, eNOS, VEGF-A, VEGF-R2, PI3 K, and PLC-γ). Exosomes (∼130 nm diameter) were isolated from the coculture, and their spectral analysis revealed significant differences (p < 0.05) in the intensity ratio of nucleotides (952 cm⁻¹), polysaccharides (1071 cm⁻¹) and lipoproteins (1417 cm⁻¹), corresponding to vasculogenesis. The activation of the VEGF-associated pathway in the coculture model was validated using an inhibitor (dexamethasone), which was used to treat the coculture graft as a control. Thus, this study elucidated the vascularization of coculture constructs via the VEGF-associated pathway. It demonstrated the potential of exosome spectral fingerprints as promising biomarkers to monitor the vascularization progression inside the graft, paving the way for the development of standardized grafts for full-thickness skin tissue regeneration.