Prevascularization of electrospun PCL/PLA scaffolds using human adipose-derived stem and endothelial cells enhances vascular integration and host angiogenesis in vivo

Abstract

Background: Inadequate vascularization remains a major limitation in tissue engineering, often leading to graft failure due to limited oxygen and nutrient supply. Prevascularization, the formation of microvascular networks within scaffolds before implantation, aims to accelerate perfusion and improve graft integration. We developed bilayer electrospun poly(ε-caprolactone)/poly(l-lactide) (PCL/PLA) scaffolds prevascularized by co-culture of human adipose-derived mesenchymal stem cells (AD-MSCs) and human placental arterial endothelial cells (HPAECs). Methods: AD-MSCs were isolated from lipoaspirates and characterized by flow cytometry and functional assays. Bilayered PCL/PLA scaffolds were engineered with a wide-meshed layer for cell infiltration and a fine-meshed layer for mechanical stability. Scaffolds were seeded with AD-MSCs, HPAECs, or both (co-culture). Cell viability, adhesion, and apoptosis were analyzed histologically. Angiogenic and vasculogenic potential was evaluated in vitro and in vivo using the chick chorioallantoic membrane (CAM) assay. Results: AD-MSCs expressed characteristic markers, demonstrated adipogenic and osteogenic differentiation, and promoted angiogenesis in 2D co-culture. ELISA analyses indicated dynamic secretion of VEGF, HGF, and bFGF, reflecting both paracrine and contact-dependent AD-MSC–HPAEC interactions. On scaffolds, cells primarily adhered to the wide-meshed layer. Co-culture induced vessel-like structures within a multicellular stromal environment; monocultures did not support prevascularization. Five days post-implantation, prevascularized scaffolds exhibited human microvessels at the scaffold–CAM interface and in adjacent tissue, closely associated with AD-MSCs and containing chicken erythrocytes—indicating successful anastomosis and functional perfusion. Quantitative analysis showed a significant increase in vessel branching points in the host CAM tissue in response to AD-MSC-only (2.8-fold) and co-culture (3.5-fold) scaffolds versus acellular controls (p < 0.05). HPAEC-only scaffolds did not promote vascular outgrowth, likely due to poor cell survival. Conclusion: Scaffolds seeded with AD-MSCs enhanced host angiogenesis, while only co-cultures with HPAECs supported scaffold prevascularization and functional vascular integration in vivo. The stromal–endothelial combination enabled formation of perfused human microvessels and promoted host vascular remodeling. These findings underscore the translational potential of prevascularized scaffolds for improved graft integration.

1. Introduction

Bioengineered tissue constructs hold great promise for the treatment of complex injuries, chronic wounds, and congenital or acquired tissue defects. However, their clinical success critically depends on rapid and functional integration with the host vasculature. Inadequate vascularization remains a major challenge in regenerative medicine, particularly for metabolically active or large-scale tissues. Without early blood perfusion, implanted grafts are prone to hypoxia, nutrient deprivation, and impaired waste removal, leading to cell death and graft failure [1].

To promote vascular ingrowth, various strategies have been explored, including the optimization of scaffold porosity and surface topography [2, 3], the delivery of pro-angiogenic factors [4, 5], and cell-based approaches [6, 7]. However, endogenous angiogenesis, the sprouting of new vessels from the host vasculature, is inherently slow [8] and often insufficient to meet the metabolic demands of grafts in the critical early post-implantation phase. Prevascularization, defined as the formation of microvascular networks within a bioengineered tissue construct prior to implantation, has therefore emerged as a promising concept [1]. Its aim is to enable rapid anastomosis with host vessels upon implantation, thereby improving early perfusion, graft survival, and long-term tissue integration. Although no gold standard for prevascularization has been established to date, prefabricated microvascular networks represent a technically and biologically attractive strategy to overcome early ischemia.