In Vivo Assessment of Printed Microvasculature in a Bilayer Skin Graft to Treat Full-Thickness Wounds

Abstract

Chronic wounds such as diabetic foot ulcers and venous leg ulcers are common problems in people suffering from type 2 diabetes. These can cause pain, and nerve damage, eventually leading to foot or leg amputation. These types of wounds are very difficult to treat and sometimes take months or even years to heal because of many possible complications during the process. Allogeneic skin grafting has been used to improve wound healing, but the majority of grafts do not survive several days after being implanted. We have been studying the behavior of fibroblasts and keratinocytes in engineered capillary-like endothelial networks. A dermo-epidermal graft has been implanted in an athymic nude mouse model to assess the integration with the host tissue as well as the wound healing process. To build these networks into a skin graft, a modified inkjet printer was used, which allowed the deposit of human microvascular endothelial cells. Neonatal human dermal fibroblast cells and neonatal human epidermal keratinocytes were manually mixed in the collagen matrix while endothelial cells printed. A full-thickness wound was created at the top of the back of athymic nude mice and the area was covered by the bilayered graft. Mice of the different groups were followed until completion of the specified experimental time line, at which time the animals were humanely euthanized and tissue samples were collected. Wound contraction improved by up to 10% when compared with the control groups. Histological analysis showed the neoskin having similar appearance to the normal skin. Both layers, dermis and epidermis, were present with thicknesses resembling normal skin. Immunohistochemistry analysis showed favorable results proving survival of the implanted cells, and confocal images showed the human cells' location in the samples that were collocated with the bilayer printed skin graft.

A comprehensive review on droplet-based bioprinting: Past, present and future

“…In one study Boland's group used it as a bioink constituent to investigate adhesion and proliferation of cells on collagen-coated cell repellant substrates [91]. Also, Boland's group fabricated a bilayer skin graft, which generated neoskin identical to native skin with microvessels [92]. In another study, fibrin-collagen bioink comprising one of the two cell types, AFSCs or mesenchymal stem cells (MSCs), was bioprinted into wound sites for treating surgical skin wounds [93].…”

“…It plays a significant role in wound healing and has been used to fabricate skin grafts [92,93]. Boland's group used fibrin to engineer micro-capillaries by bioprinting human dermal microvascular endothelial cells (HMVECs)-laden thrombin and Ca 2+ solution on a fibrinogen substrate [52].…”

Advances in bioprinted cell-laden hydrogels for skin tissue engineering

“…Bioprinting technologies are attractive to engineer a vascular tree within thick constructs by the precise layer by layer deposition of multiple cell types and ECMlike bioinks into prescribed spatial locations at high resolution [202]. Promising approaches to generate vascular networks have been reported using inkjet [34,209], laserassisted [203], and extrusion bioprinting [16,92,132]. In general, such approaches are based on four main strategies: (1) direct patterning vascular cells onto a receiving substrate [203], (2) continuous printing of polymeric bioink loaded with endothelial cells followed by polymer removal [96], (3) printing perfusable channels in a 3D construct for subsequent injection of a cell suspension into the empty channel [92], and (4) printing of multicellular spheroids [132].…”

“…To address the need for vascularization in skin substitutes, Yanez et al [209] used the inkjet technology to fabricate capillary-like endothelial networks into a dermoepidermal skin graft consisting of neonatal human dermal fibroblasts (NHDFs) and neonatal human epidermal keratinocytes (NHEKs) embedded in a fibrin-collagen matrix. HMVEC were mixed with thrombin and inkjet printed on top of a manually plated layer of collagen-NHDF cells containing fibrinogen.…”

3D Miniaturization of Human Organs for Drug Discovery

“…[102] Skin tissues were engineered recently. [103,104] A US-based research institute generated skin tissue in vitro with comparable biological and morphological characteristics to the native human skin. [105] The tissue consisted of alternating layers of collagen, fibroblasts, and keratinocytes.…”