Characterisation of elastin from chicken skin and its potential as functional biomaterial in tissue engineering

Abstract

Chicken skin, a by-product of poultry processing, contains essential elastin, a high-value material with huge potential to be used in diverse applications, including regenerative medicine. Despite its promising features, elastin is not widely used due to its insolubility. Therefore, this study was performed to characterise elastin isolated from chicken skin and explore the fabrication of a three-dimensional (3D) hybrid collagen/elastin (Col/Elas) bioscaffold for skin tissue engineering (TE) application. The study was divided into two phases. The first phase was carried out to isolate and characterise elastin from chicken skin and to verify its quality compared to commercial-grade elastin. The second phase aimed to fabricate and characterise the 3D hybrid Col/Elas bioscaffold for skin TE application. In the first phase, elastin was extracted from chicken skin using a non-enzymatic approach with sodium chloride (NaCl), sodium hydroxide (NaOH), and oxalic acid treatment before freeze-drying. The results revealed that high-quality elastin powder was successfully isolated with approximately 4 ± 0.09% yield and a molecular weight of 19 kDa. The extracted elastin powder contained a high amount of glycine and proline and a low amount of hydroxyproline, methionine, and histidine. According to the Fastin elastin assay, elastin accounts for 77% of the total protein content. Both extracted and commercial elastin exhibited characteristic amides A, B, I, and II peaks at high denaturation temperatures of 322.9 °C and 331.2 °C, respectively, proving that the two samples are equivalent and have a high degree of thermal stability. From the microscopic analysis, the extracted elastin appeared to have uniform structures, which are in line with the X-ray Diffraction (XRD) observation, confirming that it was primarily amorphous with a low presence of crystallinity. The antioxidant and antihypertensive (ACE inhibition) activities of elastin were assessed and demonstrated comparable properties with commercial-grade elastin. The toxicological test also suggested that the extracted elastin was non-toxic and could be safely incorporated at concentrations lower than 0.5 mg/mL. The second phase of this work focused on the potential utilisation of the extracted elastin as a bioscaffold for skin TE application. The 3D hybrid Col/Elas bioscaffold that combined the ovine tendon collagen type-I (Col) and the extracted elastin was fabricated by freeze-drying and then crosslinked with 0.1% (w/v) genipin (GNP). The results demonstrated a uniform interconnected porous structure with pore sizes ranging from 127 ± 21.8 μm to 245 ± 35.3 μm, good porosity (> 70%), and high water uptake capacity (> 1200%). Compared to the control (Col) bioscaffold, the biodegradation rate of the fabricated Col/Elas bioscaffold was lower (0.10 mg/h). The Energy Dispersive X-ray (EDX) analysis detected 59.06 ± 1.36% to 70.66 ± 2.89% carbon, 6.02 ± 0.20% to 7.09 ± 0.69% nitrogen, and 23.79 ± 0.65% to 32.93 ± 0.98% oxygen in the bioscaffold. Besides, the Fourier Transform Infrared (FTIR) analysis revealed that Col and elastin remained in the scaffold and exhibited similar functional amides. The combined elastin and Col also recorded a positive effect through the increased Young's modulus values and improved attachment and proliferation of primary Human Dermal Fibroblasts (HDF) and Human Epidermal Keratinocytes (HEK). In-vitro biocompatibility analysis revealed no toxic effects, indicating that the bioscaffold is safe to be used. In conclusion, the encouraging findings in this study imply that extracted elastin from chicken skin could be used and integrated as a bioscaffold for skin TE application.