Engineered-Skin of Single Dermal Layer Containing Printed Hybrid Gelatin-Polyvinyl Alcohol Bioink via 3D-Bioprinting: In Vitro Assessment under Submerged vs. Air-Lifting Models

Abstract

Three-dimensional (3D) in vitro skin models are frequently employed in cosmetic and pharmaceutical research to minimize the demand for animal testing. Hence, three-dimensional (3D) bioprinting was introduced to fabricate layer-by-layer bioink made up of cells and improve the ability to develop a rapid manufacturing process, while maintaining bio-mechanical scaffolds and microstructural properties. Briefly, gelatin-polyvinyl alcohol (GPVA) was mixed with 1.5 × 106 and 3.0 × 106 human dermal fibroblast (HDF) cell density, together with 0.1% genipin (GNP), as a crosslinking agent, using 3D-bioprinting. Then, it was cultured under submerged and air-lifting conditions. The gross appearance of the hydrogel’s surface and cross-section were captured and evaluated. The biocompatibility testing of HDFs and cell–bioink interaction towards the GPVA was analyzed by using live/dead assay, cell migration activity, cell proliferation assay, cell morphology (SEM) and protein expression via immunocytochemistry. The crosslinked hydrogels significantly demonstrated optimum average pore size (100–199 μm). The GPVA crosslinked with GNP (GPVA_GNP) hydrogels with 3.0 × 106 HDFs was proven to be outstanding, compared to the other hydrogels, in biocompatibility testing to promote cellular interaction. Moreover, GPVA–GNP hydrogels, encapsulated with 3.0 × 106 HDFs under submerged cultivation, had a better outcome than air-lifting with an excellent surface cell viability rate of 96 ± 0.02%, demonstrated by 91.3 ± 4.1% positively expressed Ki67 marker at day 14 that represented active proliferative cells, an average of 503.3 ± 15.2 μm for migration distance, and maintained the HDFs’ phenotypic profiles with the presence of collagen type I expression. It also presented with an absence of alpha-smooth muscle actin positive staining. In conclusion, 3.0 × 106 of hybrid GPVA hydrogel crosslinked with GNP, produced by submerged cultivation, was proven to have the excellent biocompatibility properties required to be a potential bioinks for the rapid manufacturing of 3D in vitro of a single dermal layer for future use in cosmetic, pharmaceutic and toxicologic applications.