Fabrication and characterization of bilayered tissue scaffolds incorporating bioactive agents for skin tissue engineering applications

Aktürk, Ömer
In this study, it was aimed to fabricate tissue scaffolds from different biological polymers (collagen, silk fibroin and sericin) for skin tissue engineering applications. For this purpose, bilayered scaffolds composed of epidermal (collagen/sericin films) and dermal (collagen sponges, collagen matrices or silk fibroin matrices) layers were produced with different biomaterial fabrication methods. Casting and solvent evaporation (film), lyophilization/freeze-drying (sponge) and dry/wet electro-spinning (micro/nanofibrous matrices) methods were used to obtain the scaffolds. Different sizes (10-55nm) of gold nanoparticles (AuNPs) with spherical shapes were synthesized and one size group (37 nm) was incorporated into dermal layers. Collagen based scaffolds were cross-linked with glutaraldehyde and silk fibroin based ones were treated with ethanol to increase the aqueous stability. The suitability of these scaffolds to skin tissue engineering applications was evaluated by means of physicochemical characterization, in vitro biocompatibility, antibacterial assessment, intra-dermal irritation, skin sensitization, genotoxicity and in vivo rat skin wound healing tests. The resistance of collagen based scaffolds to hydrolytic and enzymatic degradation was enhanced significantly after cross-linking, on the contrary collagen nanofibrous scaffolds had tendency to degrade quickly. Especially silk fibroin based scaffolds had the lowest degradation levels. Based on water swelling, silk fibroin based scaffolds and collagen sponge groups were similar (10-30 g/g) due to their similar porosities (70-80%) and pore size distributions (10-200 µm). However, water swelling of collagen nanofibrous scaffolds was lower (3 g/g) in comparison due to their low porosity (10%) and small pore sizes (0-8 µm). The water vapor transmission rates of bilayered scaffolds were statistically similar (about 1300-1400 g/m2/day) and they were confirmed to have good oxygen permeability, which was attributed to collagen/sericin film. Tensile strength of collagen based scaffolds enhanced significantly after cross-linking. AuNPs incorporation into collagen based scaffolds did not have a significant effect on tensile strength values. Yet, AuNPs incorporation significantly increased tensile strength of silk fibroin based scaffolds. Also, tensile strength decreased dramatically when the testing was done in wet conditions. On the other hand, elongation at break of all the wet scaffolds were improved significantly reaching to even above 100% values. Elastic modulus of all the scaffolds increased significantly after cross-linking indicating increased hardness, on the contrary elastic modulus decreased significantly in wet conditions as they became more extensible and softer in aqueous environment. The cytotoxic effect of AuNPs on keratinocyte and fibroblasts were size and dose dependent. None of the scaffolds (epidermal and dermal layers) had a cytotoxic effect (cell viability > 85%) on L929 fibroblasts. Cell seeding tests showed that, fibroblasts and keratinocytes attached and gained their natural morphology (spread or polygonal) in 1 day and proliferated on the scaffolds during 3 days incubation period according to SEM analysis. Antibacterial assessment tests based on the agar disc diffusion method provided strong evidence that AuNPs fabricated in this study had an antibacterial potential against S. Aureus. Also, the OD measurement and SEM analysis showed that S. Epidermidis could not attach onto scaffolds containing AuNPs compared to control without AuNPs. In vivo tests results showed that all the groups had significantly better wound contraction than untreated controls on post-operative 14th day. Early stage re-epithelization started to form in collagen based and silk fibroin based scaffold groups, with low inflammation, and high fibrosis and granulation tissue formation compared to MatridermTM. Also, the groups containing honey or AuNPs started to recover their mechanical properties much quicker than MatridermTM according to biomechanical tests. All these results suggested that all the groups, especially the groups containing honey or AuNPS have equal or slightly better in vivo healing effect than MatridermTM. According to animal toxicology evaluation and genotoxicity tests on BLCS-AuX, there were no adverse skin reactions (irritation or sensitization) or genotoxicity. As a conclusion, it could be suggested that all of these bilayered skin substitutes, especially the groups containing bioactive agents (honey or AuNPs) could be very valuable skin substitute materials for use in the management of skin wounds.