A new scenario for bone regeneration: development of electrospun keratin-based scaffolds added with gold nanoparticles

1. Introduction One of the main constituents of tissue engineering relates to scaffolds, whose function is to promote cellular adhesion, proliferation and differentiation until the formation of new tissue. From this perspective, it is fundamental to discover renewable sources for biomedical applications such as supporting membranes. Wool keratin has drawn significant attention because of its numerous positive properties, such as being biodegradable, biocompatible, non-immunogenic and improving cellular adhesion (1). In the regenerative medicine field, the repair of bone tissue represents an important challenge due to its complex anatomical structure as well as possessing strong regeneration capability. The present manuscript proposes an innovative strategy based on bone tissue engineering: the production of electrospun wool keratin scaffolds as alternative biomaterials. To improve the antibacterial properties, gold nanoparticles (GNPs) were added to the starting solution (2). The electrospinning technique was applied to obtain nanofibrous membranes with a high surface-to-volume ratio that may better mimic the behaviour of bone tissue (3). Biocompatibility was evaluated by performing cellular viability tests on human osteoblast-like SaOs-2 cells: the results showed that GNPs favour cell growth on the surface of keratin-based scaffolds. Additionally, the anti-adhesive properties of the produced scaffolds were assessed to investigate the effect of GNPs. These reduced the early process of bacterial adhesion of the Staphylococcus aureus strain. 2. Materials and Methods 2.1 Preparation of electrospun keratin-scaffolds Keratin powder extracted from wool was dissolved in formic acid and kept under mild stirring overnight. Concentrated GNPs dispersion was added to the keratin-based solution before electrospinning. At the end of the process, the nanofibers were collected and thermally treated at 180 °C for 4h. 2.2 Cellular viability test Human osteoblast-like SaOs-2 cells were cultured for 1, 3 and 7 days on two different materials: pure keratin nanofibres and GNPs-keratin nanofibres. These cells possess osteoinductive properties, so they are used to study proliferation and differentiation stages. Cellular viability has been determined by CCK8 assay on cells attached to both materials and to tissue culture plate (TCP) control, represented by cells grown in the well. 2.3 Early process of bacterial adhesion on keratin-based scaffolds The anti-adhesive properties of electrospun keratin scaffolds have been explored using the bacterial strain Staphylococcus aureus ATCC 25923. Microorganisms were seeded on samples and incubated at 37 °C for 3h. After the incubation time, the survival bacteria were diluted and plated on agar plates and subsequently incubated at 37 °C for 24h. The colonies formed from surviving adherent cells were counted and expressed as CFUs (colony-forming units)/mL. 3. Results and Discussion The outcomes of the cell viability assay demonstrated that SaOs-2 cells were capable of adhering and growing up to 7 days on both pure keratin and GNPs-keratin nanofibres. The obtained quantitative data evidence that cell viability is higher on GNPs-keratin than keratin. Hence, GNPs stimulate and promote SaOs-2 cell growth on keratin-based membranes. As regards the early process of bacterial adhesion, GNPs-keratin samples show a reduced bacterial adhesion than the pure keratin. 4. Conclusions The addition of gold nanoparticles in keratin-based scaffolds not only favours cell growth on the scaffold's surface but also reduces the early process of adhesion of the S. aureus bacterium. Therefore, the electrospun keratin scaffolds may represent a promising biomaterial for bone tissue engineering applications, even if further studies will be performed to better understand their efficacy. References (1) Sanchez Ramirez DO. et al., 2022, 10.3390/jfb14010005 (2) Abdalla SSI. et al., 2020, doi: 10.2174/1567201817666191227094334 (3) Sarrami P. et al., 2022, 10.1016/j.ijbiomac.2022.09.117