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

Abstract

Organotypic full-thickness skin grafts (OTSG) are already an important technology for treating various skin conditions and are well established for skin research and development. These obvious benefits are often impaired by the need of laborious production, their noncomplete autologous composition, and, most importantly, their lack of included vasculature. Therefore, our study focused on combining a prevascularized dermal layer with an epidermis to cultivate full-thickness skin grafts incorporating capillary-like networks. It has been shown that prevascularization accelerates ingrowth of tissue-engineered grafts, and it is a prerequisite to circumvent diffusion limits due to graft thickness. To obtain such a graft, we chose a dermal layer incorporating human umbilical vein endothelial cells (HuVEC) amid human dermal fibroblasts within a fibrin-based scaffold, seeded apically with human foreskin keratinocytes (hfKC). Our research investigated the used concept's feasibility, as well as the effect of hfKC addition on the development of a well-connected capillary-like network after approximately 21 days. In addition, we evaluated the utilization of a custom-made constant flow bioreactor for simplified cultivation of these grafts, therefore possibly easing graft production and presumably increasing their cost effectiveness. Skin grafts were assessed by conventional two-dimensional histology. In addition, software-assisted three-dimensional evaluation of the capillary-like structure networks was performed by twophoton laser scanning microscopy (TPLSM) and subsequent image processing was done with ImagePro Ò Analyzer 7.0 software, thereby evaluating its platform technology power in the field of prevascularized skin grafts. All samples showed a capillary-like structure network, but we could report a significant reduction of its total length after 14 days of tri-culture with 5 · 10 5 /cm 2 seeded hfKC, possibly indicating nutritional deficiencies for this particular high cell density experimental setup. Lower concentrations of hfKC did not affect the formation of the capillary-like structures significantly. The developed bioreactor simplified cultivation of prevascularized OTSG. However, a flow-dependent reduction of capillary-like structures in 1 and 5 mL/min flow conditions occurred. We conclude that our technique for creating prevascularized OTSG is feasible. In addition, TPLSM is well suited for analyzing the prevascularization process. We hypothesize that the handling benefits of our bioreactor can be preserved by using considerably lower flow rates while not impairing the forming of capillary-like structure networks.

A first vascularized skin equivalent for as an alternative to animal experimentation

“…To date, perfusion of vessel-like structures in pre-vascularized models has only been achieved upon grafting to an animal model (Gibot et al, 2010;Schechner et al, 2003;Tremblay et al, 2005). The current model can be regarded as an improvement on a previously published pre-vascularized skin model, which only enabled medium flow underneath the tissue and left out the perfusion of vessels in the construct (Helmedag et al, 2015). In this previous model the fluidic compartment was separated from the tissue by a porous membrane, limiting mass transport.…”

Macrophages significantly enhance wound healing in a vascularized skin model

“…The stiffness and viscosity of fibrin glues could inhibit cell migration and differentiation, slowing the formation of capillary‐like structures and thus increasing the risk of transplant necrosis . We therefore chose a final fibrin concentration of 5 mg/mL as previously recommended …”

“…28 We therefore chose a final fibrin concentration of 5 mg/mL as previously recommended. 21 To optimize the cultivation conditions for all four cell types in our model, we mixed equal quantities of the EGM-2, KGM, and RPMI-1640 GlutaMAX media (for the support of endothelial cells, keratinocytes and macrophages, respectively) resulting in the development of a functional epidermal multilayer. The macrophage-specific RPMI-1640 medium appeared to confer a proangiogenic effect and thus support the development of the epidermis.…”

Methodologies in creating skin substitutes

“…Another method to improve cell penetrance includes the unique method of UV cross-linking scaffold solutions with live cells[212], however this requires specific materials for the scaffold as previously mentioned altering the mechanical properties of the skin substitute. Finally, although quite expensive, there is support for the use of bioreactors for different dynamic seeding techniques including rotational seeding for skin substitutes[213]. The use of bioreactors allows for uniform distribution of cells throughout hydrogels and similar seeding densities between different hydrogels decreasing variation in tissue engineering and allowing for mass manufacturing of bioengineered tissues.…”