Abstract
Tissue engineering plays an important role in the production of skin equivalents for the therapy of chronic and especially burn wounds. Actually, there exists no (cellularized) skin equivalent which might be able to satisfactorily mimic native skin. Here, we utilized a laser-assisted bioprinting (LaBP) technique to create a fully cellularized skin substitute. The unique feature of LaBP is the possibility to position different cell types in an exact three-dimensional (3D) spatial pattern. For the creation of the skin substitutes, we positioned fibroblasts and keratinocytes on top of a stabilizing matrix (Matriderm®). These skin constructs were subsequently tested in vivo, employing the dorsal skin fold chamber in nude mice. The transplants were placed into full-thickness skin wounds and were fully connected to the surrounding tissue when explanted after 11 days. The printed keratinocytes formed a multi-layered epidermis with beginning differentiation and stratum corneum. Proliferation of the keratinocytes was mainly detected in the suprabasal layers. In vitro controls, which were cultivated at the air-liquid-interface, also exhibited proliferative cells, but they were rather located in the whole epidermis. E-cadherin as a hint for adherens junctions and therefore tissue formation could be found in the epidermis in vivo as well as in vitro. In both conditions, the printed fibroblasts partly stayed on top of the underlying Matriderm® where they produced collagen, while part of them migrated into the Matriderm®. In the mice, some blood vessels could be found to grow from the wound bed and the wound edges in direction of the printed cells. In conclusion, we could show the successful 3D printing of a cell construct via LaBP and the subsequent tissue formation in vivo. These findings represent the prerequisite for the creation of a complex tissue like skin, consisting of different cell types in an intricate 3D pattern.
Highlights
Major burn injuries often prove difficult in therapy due to their complexity, the high risk of infection, the large area which might be affected and the potential destruction of deeper skin layers including the dermis
Operations and Macroscopic Evaluation To evaluate skin constructs in vivo generated via laser-assisted bioprinting (LaBP), we printed 20 layers of a keratinocyte cell line (HaCaT) on top of 20 layers of a fibroblast cell line (NIH3T3) by an established laserassisted bioprinting procedure
The skin constructs were placed into fullthickness skin wounds in the dorsal skin fold chamber preparation in mice in such a way that the constructs and the surrounding skin laid in close contact to each other
Summary
Major burn injuries often prove difficult in therapy due to their complexity, the high risk of infection, the large area which might be affected and the potential destruction of deeper skin layers including the dermis. The availability of autologous splitthickness skin grafts and keratinocytes for wound coverage is limited, especially in case of large burned areas. Several skin substitutes like IntegraH and MatridermH are already employed in the clinical application, being complemented by the use of autologous split-thickness skin grafts [1,2,3]. While IntegraH serves to prepare the wound bed in preparation for transplantation with autologous split-thickness skin three weeks later, MatridermH is used in a single step procedure and must be covered immediately. Full success in burn wound regeneration has not been reached yet, neither under functional nor under aesthetic aspects. Neither hair follicles nor sebaceous and perspiratory glands can be regenerated
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