Development of a reconstructed human skin model for angiogenesis

Abstract

We have previously shown that reconstructed human skin engineered from autologous keratinocytes, fibroblasts, and sterilized donor allodermis stimulates angiogenesis within 5-7 days when placed on well-vascularized wound beds in nude mice. When this reconstructed skin was used clinically in more demanding wound beds, some grafts were lost, possibly due to delayed vascularization. As this reconstructed skin lacks any endothelial cells, our aim in this study was to develop an angiogenic reconstructed skin model in which to explore strategies to improve angiogenesis both in vitro and in vivo. We report that culture of small-vessel human dermal microvascular endothelial cells (HuDMECs) was achieved using magnetic beads coated with an antibody to platelet cell adhesion molecule as a means of purifying the culture. Keratinocytes, fibroblasts, and HuDMECs could be cultured from the same skin biopsy. Initial studies culturing HuDMECs and other sources of endothelial cells with the tissue-engineered skin showed that these cells were capable of slowly entering the dermis under standard culture conditions in vitro. In conclusion, this provides us with a model in which to explore strategies for improving angiogenesis in vitro and also establishes the culture methodologies for the production of reconstructed skin containing autologous keratinocytes, fibroblasts, and endothelial cells.

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.…”

Cell-Based Vascularization Strategies for Skin Tissue Engineering

“…Studies with HDMEC have shown that it may take up to 6 weeks to prepare therapeutic amounts for STE. 11 Our own experience with AB-BOEC also indicates that it may take several weeks to expand them to a clinically useful number. An important advantage of the latter compared to HDMEC is that the total amount of cells that can be generated is larger, so that excess cells may be cryopreserved for later use in the same patient.…”

“…27 Further refinement of the culture protocol has led to the development of serum-free culture techniques, making these cells safer for potential clinical use in STE. 28 However, despite optimization of techniques to obtain HDMEC, the limited yield requires time-consuming culture expansion (up to 6 weeks) and contamination with fibroblasts remains a challenging problem, 11,25 which currently impedes their use in a clinical setting. Although Nö r et al succeeded in creating functional vessels in a subcutaneous pocket when combining HDMEC with matrigel, Supp et al did not demonstrate functional connection to the host vasculature in a skin substitute.…”

“…Hence, when aiming to cultivate autologous cell-based skin substitutes, one must bear in mind that isolation and expansion of fibroblasts and keratinocytes also takes several weeks, so that all cell types must/can be grown simultaneously. 11 In some clinical cases, however, such as in chronic wounds, reconstruction can be delayed, which leaves sufficient time for isolation of autologous cells. Even in certain large burn cases, staged reconstruction is often required with multiple surgical interventions over a period of several months, such that these patients can still benefit from autologous cell approaches.…”

“…10 Hence, a major threat for clinical use of dermal substitutes is insufficient vascularization leading to loosening, infection or partial necrosis of the substitute. 11 Many approaches have been tested to vascularize skin substitutes. Several growth factor (GF) delivery techniques have been suggested to increase vascularization.…”