The early treatment and rapid closure of acute or chronic wounds is essential for normal healing and prevention of hypertrophic scarring. The use of split thickness autografts is often limited by the availability of a suitable area of healthy donor skin to harvest. Cellular and non-cellular biological skin-equivalents are commonly used as an alternative treatment option for these patients, however these treatments usually involve multiple surgical procedures and associated with high costs of production and repeated wound treatment. Here we describe a novel design and a proof-of-concept validation of a mobile skin bioprinting system that provides rapid on-site management of extensive wounds. Integrated imaging technology facilitated the precise delivery of either autologous or allogeneic dermal fibroblasts and epidermal keratinocytes directly into an injured area, replicating the layered skin structure. Excisional wounds bioprinted with layered autologous dermal fibroblasts and epidermal keratinocytes in a hydrogel carrier showed rapid wound closure, reduced contraction and accelerated re-epithelialization. These regenerated tissues had a dermal structure and composition similar to healthy skin, with extensive collagen deposition arranged in large, organized fibers, extensive mature vascular formation and proliferating keratinocytes.
INTRODUCTION AND OBJECTIVES: Many reconstructive procedures require skin grafts for closure. Currently, the clinical standard for wound closure is the use of autologous skin grafts or flap. However, this method is often limited by the donor skin availability and is not applicable to extensive wound coverage. Cell-based approaches have become a promising modality for patients requiring skin wound repair as part of reconstruction. We previously have shown that keratinocytes and dermal fibroblasts, delivered to the wound with a bioprinter, are able to rapidly repair skin wounds in animal models. In this study, we aim to bioprint engineered skin that would facilitate wound healing as well as exhibit functionality, such as pigmentation and hair induction/formation. METHODS: Human keratinocytes, melanocytes, dermal fibroblasts, and follicle dermal papilla cells were cultured and expanded for bioprinting. Human fibroblasts and papilla cells were suspended in a printable hydrogel majorly composed of fibrin. These cells were printed first in order to create the dermal layer. Subsequently, human keratinocytes and melanocytes were suspended in the same hydrogel as the dermal layer and were printed to create the epidermal layer. The two layered constructs with a dimension of 1cmx1cm were bioprinted to mimic the thickness of normal rodent skin, followed by cross-linking with thrombin. The bilayered skin constructs were cultured for 5 days and then implanted subcutaneously onto nude mice.RESULTS: Most of the bioprinted cells were viable, maintained their phenotypic characterization, and remained in their spatial orientation within the skin construct. After a week of implantation, the bioprinted skin constructs showed revascularization and maintained their structural integrity. A gel-only group (used as control) did not maintain its structure and was not retrievable after one week. Histological analyses show the presence of bilayered anatomical skin configuration. Masson's Trichrome staining confirmed the presence of extracellular matrix (ECM) in the cell-containing constructs.CONCLUSIONS: We show that four different types of skin cells can be delivered in layers using a bioprinter to generate a skin construct. The bioprinted skin constructs are capable of forming and maintaining their skin-like structure even after 1 week of implantation. This initial study suggests that skin bioprinting may become a method to generate clinically demanding skin tissues for patients who require additional skin for coverage during reconstructive procedures.
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