Peptide Chitosan/Dextran Core/Shell Vascularized 3D Constructs for Wound Healing

Abstract

Three-dimensional (3D) bioprinting has emerged to create novel cell-based therapies for regenerative medicine applications. Vascularized networks within engineered constructs are required, and toward this end, we report a promising strategy using core-shell (c/s) extrusion 3D-bioprinting technology that employs biomimetic biomaterials to construct regenerative, prevascularized scaffolds for wound care. A custom-designed cell-responsive bioink consisting of a 13% (w/v) cell-laden gelatin methacryloyl (GelMA) shell surrounding a peptide-functionalized, succinylated chitosan (C)/dextran aldehyde (D) cell-laden core was successfully bioprinted resulting in organized microdesigns exhibiting excellent cell viability and subsequent vessel formation. Our templating strategy takes advantage of GelMA's intrinsic thermoreversible properties of low degree of acryloyl functionalization used in combination with a lightly, chemically cross-linked peptide-CD core to serve as temporal structural supports that stabilize during extrusion onto a cooled platform. Mechanical integrity was further strengthened layer-by-layer via GelMA UV photo-cross-linking. We report the first example of GelMA used in combination with a peptide-CD bioink to c/s 3D-bioprint regenerative, prevascularized constructs for wound care. Particular cell adhesion and proteolytic peptide-CD functionalized pair combinations, P15/MMP-2 and P15/cRGD, were found to significantly increase growth of human bone-marrow-derived mesenchymal stems cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs). The constructs delivered two cell types: hBMSCs in the shell bioink and HUVECs within the core bioink. Cord-like, natural microvascularization was shown with endothelial cell marker expression as confirmed by immunofluorescence (IF) staining exhibiting tubelike structures. In addition, in vitro skin wound healing activity of the construct showed a ∼twofold rate of wound closure. Overall, c/s 3D-bioprinted, peptide-CD/GelMA constructs provided the appropriate microenvironment for in vitro stem and endothelial cell viability, delivery, and differentiation. We foresee these custom constructs as representing a fundamental step toward engineering larger scale regenerative, prevascularized tissues.

Keywords: 3D bioprinting; chitosan; coaxial; gelatin methacryloyl; stem cell; vascularization.