Three Dimensional Bioprinting of a Vascularized and Perfusable Skin Graft Using Human Keratinocytes, Fibroblasts, Pericytes, and Endothelial Cells

Abstract

Multilayered skin substitutes comprising allogeneic cells have been tested for the treatment of nonhealing cutaneous ulcers. However, such nonnative skin grafts fail to permanently engraft because they lack dermal vascular networks important for integration with the host tissue. In this study, we describe the fabrication of an implantable multilayered vascularized bioengineered skin graft using 3D bioprinting. The graft is formed using one bioink containing human foreskin dermal fibroblasts (FBs), human endothelial cells (ECs) derived from cord blood human endothelial colony-forming cells (HECFCs), and human placental pericytes (PCs) suspended in rat tail type I collagen to form a dermis followed by printing with a second bioink containing human foreskin keratinocytes (KCs) to form an epidermis. In vitro, KCs replicate and mature to form a multilayered barrier, while the ECs and PCs self-assemble into interconnected microvascular networks. The PCs in the dermal bioink associate with EC-lined vascular structures and appear to improve KC maturation. When these 3D printed grafts are implanted on the dorsum of immunodeficient mice, the human EC-lined structures inosculate with mouse microvessels arising from the wound bed and become perfused within 4 weeks after implantation. The presence of PCs in the printed dermis enhances the invasion of the graft by host microvessels and the formation of an epidermal rete.

Skin Wound Healing Process and New Emerging Technologies for Skin Wound Care and Regeneration

“…In a study conducted by Baltazar and colleagues, it was shown that 3D bioprinting succeeded in fabricating vascularized human skin in vitro from human cells, with morphological and biological similarity to in vivo human skin. An implantable skin graft, including a perfusable microvascular system, was obtained by 3D printing; the prototype was made by combining a bioink with foreskin dermal fibroblasts (FBs) and human endothelial cells (ECs), with a second bioink including foreskin keratinocytes (KCs) to form the dermis and epidermis, respectively [ 121 ]. Won and colleagues conducted another innovative study on decellularized ECM derived from a pig dermis; the authors obtained a printable bio-ink that was mixed with human dermal fibroblasts (HDFs) to produce a construct loaded with human cells.…”

Monitoring calcium-induced epidermal differentiation in vitro using multiphoton microscopy

“…It should be noted that the in vitro skin culture protocols can be further refined to produce cultures fully resembling native human tissue. 57,58 It is plausible that high cell density and the real-time control of Ca 2þ gradient are, among others, important parameters needed for the development of improved in vitro tissue culturing protocols. For this purpose, MPM provides a feasible approach, potentially enabling real-time tissue culture optimization.…”

A Concise Review on Tissue Engineered Artificial Skin Grafts for Chronic Wound Treatment: Can We Reconstruct Functional Skin Tissue In Vitro?

“…Nevertheless, constructs containing both types of the cells allow keratinocyte-fibroblast crosstalk through the release of GFs and cytokines, which is very important for a fast healing process and promotion of re-epithelialization [49,50]. To produce functional dermo-epidermal constructs, researchers frequently use various advanced techniques like 3D bioprinting [56,57] or electrospinning [58].…”