In vitroreconstruction of a human capillary‐like network in a tissue‐engineered skin equivalent

Abstract

For patients with extensive burns, wound coverage with an autologous in vitro reconstructed skin made of both dermis and epidermis should be the best alternative to split-thickness graft. Unfortunately, various obstacles have delayed the widespread use of composite skin substitutes. Insufficient vascularization has been proposed as the most likely reason for their unreliable survival. Our purpose was to develop a vascular-like network inside tissue-engineered skin in order to improve graft vascularization. To reach this aim, we fabricated a collagen biopolymer in which three human cell types keratinocytes, dermal fibroblasts, and umbilical vein endothelial cells were cocultured. We demonstrated that the endothelialized skin equivalent (ESE) promoted spontaneous formation of capillary-like structures in a highly differentiated extracellular matrix. Immunohistochemical analysis and transmission electron microscopy of the ESE showed characteristics associated with the microvasculature in vivo (von Willebrand factor, Weibel-Palade bodies, basement membrane material, and intercellular junctions). We have developed the first endothelialized human tissue-engineered skin in which a network of capillary-like tubes is formed. The transplantation of this ESE on human should accelerate graft revascularization by inosculation of its preexisting capillary-like network with the patient's own blood vessels, as it is observed with autografts. In addition, the ESE turns out to be a promising in vitro angiogenesis model.

Engineered vascularized bone grafts

“…Their differentiation state prior to coimplantation with ECs, however, is an important parameter that supports the network, because the population that remained in an undifferentiated proliferated state formed stronger, more mature networks. The hMSCs fulfill at least two roles in supporting the vasculogenesis process, synthesis of trophic factors (22,23) and contributing the mural component (9,19,21,24,25), neither of which is well understood at present. Both in vitro and in vivo, the tubular networks formed by monocultures of ECs such as HUVECs are not stable.…”

“…Both in vitro and in vivo, the tubular networks formed by monocultures of ECs such as HUVECs are not stable. Inclusion of mesenchymal-derived cells that can serve as pericytes such as the 10T1/2 cell line, embryonic and dermal fibroblasts, or hMSCs generates networks that are more robust and can anastomose to form functional vessels in vivo (5,7,9,11,28). Furthermore, it is suggested that 10T1/2 cells differentiate into SMCs or pericytes through heterotypic interactions with ECs (10,11,13).…”

Rapid Anastomosis of Endothelial Progenitor Cell–Derived Vessels with Host Vasculature Is Promoted by a High Density of Cotransplanted Fibroblasts

“…23 The 1:5 ratio of fibroblasts to ECs used in our previous study was based on previous reports in the literature. 6,12 However, the recent work by Melero-Martin et al…”

“…The cells should be clinically available, easy to purify and expand in vitro, and, ideally, autologous, thus avoiding host immune responses. Generation of functional vessels in vivo has been reported using mature ECs such as human umbilical vein ECs (HUVECs) 11,12 and human dermal microvascular ECs. 13 However, either these cells are unavailable for adults, or they require an invasive procedure to harvest.…”

The Effects of Constant Flow Bioreactor Cultivation and Keratinocyte Seeding Densities on Prevascularized Organotypic Skin Grafts Based on a Fibrin Scaffold

“…Fibroblasts play an important role in organizing the clot, which includes the secretion of extracellular matrix and the promotion of angiogenesis. 14,15 To our knowledge, bi-layered prevascularized skin grafts so far have been only engineered based on the so-called ''self-assembly approach,'' [16][17][18] collagenbased scaffolds, 19,20 de-cellularized donor dermis, 21 or use of a de-cellularized porcine jejunal segment as suggested by Groeber et al 22 All of these methods are characterized by specific shortcomings: The self-assembly approach takes approximately 6 weeks to form a prevascularized neo-dermis, which is a significantly longer timeframe than our employed method. Collagen-based scaffolds, de-cellularized donor dermis, or xenogenous templates cannot offer a complete autologous approach; drawbacks such as transmission of infectious diseases, allergic reactions, or intricate experimental setup have to be taken into account as a possible future issue regarding human implementation.…”