Preparation of multilayer electrospun nanofibrous scaffolds containing soluble eggshell membrane as potential dermal substitute.

Electrospinning of natural and synthetic polymers has shown to be a great candidate for the fabrication of tissue engineering scaffolds due to their similarity to the nanofibrous structure of natural extracellular matrix (ECM). Moreover, the addition of ECM-like proteins could enhance the biocompatibility of these scaffolds. In this study, soluble eggshell protein (SEP) was first extracted and synthesized from the raw eggshell membrane. The characteristics and biocompatibility of the extracted SEP were evaluated using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) analysis and 3-(4,5- dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) (MTT) assay. For scaffolds fabrication, a three-layer nanofibrous composite structure was produced using the electrospinning technique. The outer layers composed of polyvinyl alcohol, chitosan, and extracted SEP while the middle layer composed of polyethylene oxide, gelatin, and zinc oxide nanoparticles (ZnO-NPs). For each layer, the electrospinning parameters were adjusted to form bead-free fibers. To improve fibers' stability against body fluids, the produced fibers were crosslinked using glutaraldehyde vapor. Several techniques such as scanning electron microscopy (SEM), energy dispersive X-ray, ATR-FTIR, swelling, tensile test, in vitro biodegradation, and MTT assay were implemented to evaluate the physical, chemical, and biological characterization of the fabricated fibers. The results showed that crosslinked fibers have adequate stability in water, suitable mechanical properties, and promising water uptake capacity. The MTT results also revealed that SEP and ZnO-NPs could increase scaffolds biocompatibility. Moreover, SEM photographs of cultured fibroblasts cells on the scaffolds showed that cells were well attached on the scaffolds and preserve their natural spindle shapes. Altogether, our findings demonstrated that the produced three-layer composite scaffolds are potential candidates for skin tissue engineering.