Bioprinting Human Skin Equivalents Using Reactive Jet Impingement

Abstract

Before most commercial products reach the market, their safety and efficiency must be evaluated. In vitro skin equivalents are extensively used to assess the toxicity and penetration of substances. However, their limited reproducibility and long manufacturing times have hindered their industrial implementation. The use of bioprinting systems has helped to automatise the production process, but the industrial scalability of these models as testing platforms is still limited. This research aimed to optimise the manufacturing of skin equivalents by incorporating the novel Reactive Jet Impingement (ReJI) bioprinting system. The combination ReJI process with the formulation of natural-based bioinks could help to improve the complexity of the models, recreating specific skin microenvironments and accelerating the skin formation process. The experimental results demonstrated the ability of ReJI to control cell distribution, ensuring the homogeneity of the models. In contrast to other bioprinting techniques, cell viability was not affected regardless of the printed cell density. By incrementing ten times the number of fibroblasts in the dermal models, it was possible to reduce their maturation time significantly. Meanwhile, the study of dermal bioinks allowed the identification of parameters that influence fibroblast behaviour. The selection of collagen sources and neutralisation method affected the physicochemical properties of the dermal models, along with the fibroblast proliferation and matrix remodelling. Similarly, the combination of fibrin with alginate and collagen enabled the customisation of the dermal layer. Finally, the incorporation of these biomaterials into the epidermal production also allowed an enhanced control over keratinocyte distribution and differentiation. This research demonstrates the suitability of ReJI for the high-throughput production of models and the possibility of decreasing the manufacturing times. Additionally, the selection of natural bioinks has shown to be an important factor in the recreation of different skin microenvironments and conditions, tailoring the cellular behaviour and functionality of the final models.