A new strategy for bone regeneration: electrospun wool keratin scaffolds with gold nanoparticles as antimicrobial biomaterial

1 Introduction Recovery and valorisation of biopolymers obtained from renewable and cheap sources may not have a trivial impact on biomedical applications. In the regenerative medicine field, the repair and reconstruction of bone defects is considered one of the major challenges. To overcome these issues, the application of bone tissue engineering practices based on the biopolymer exploitation is easily accessible and represents a viable alternative to the conventional use of bone grafts to induce new functional bone regeneration. In this research work, keratin derived from wool fibres was used as the main substrate for producing scaffolds applicable in this sector. Keratin is a cysteine-rich protein that can be extracted from different sources, including low–cost, renewable and abundant biosources such as raw wool (1). Wool keratin is biocompatible, biodegradable, bioresorbable, non-immunogenic, does not induce inflammatory responses, and improves cellular adhesion. Also, nanofibrous keratin scaffolds may accelerate cell viability, proliferation and osteogenesis, suggesting that keratin may represent a promising polymer in bone tissue engineering. To better mimic the composition and behaviour of bone tissue, the novelty proposed in this work concerns the application of the electrospinning technique to obtain nanofibrous membranes with a high surface-to-volume ratio. Furthermore, gold nanoparticles (GNPs) were added to enhance antibacterial properties (2). Biocompatibility was evaluated by performing cell viability tests on human osteoblast-like SaOs-2 cells. Additionally, the anti-adhesive properties of electrospun keratin scaffolds with/without gold nanoparticles were evaluated against bacteria in order to study the effect of gold nanoparticles against bacterial adhesion. 2 Experimental 2.1 Materials and Methods 2.1.1 Preparation of electrospun keratin-scaffolds Extracted keratin powder was dissolved in formic acid and kept overnight under mild stirring at 20°C. Concentrated GNP dispersion was added to the keratin solution before electrospinning. After the process, the electrospun keratin nanofibers were collected on aluminium or polypropylene non-woven and thermally treated at 180°C for 4h in an oven to make them water-insoluble. 2.1.2 Cell viability test Human osteoblast-like SaOs-2 cells were cultured on two different materials (Ker and Ker 0.1% GNPs) for 1, 3 and 7 days. These cells are used to study proliferation and differentiation because they grow rapidly in vitro and have osteoinductive properties. Cell viability has been determined by CCK8 assay on cells attached to both substrates and to TCP (tissue culture plate) control, represented by cells grown in the well. 2.1.3 Early process of bacterial adhesion on keratin-based membranes The anti-adhesive properties of electrospun keratin scaffolds against bacteria have been explored using two model strains: Staphylococcus aureus ATCC 25923 as Gram-positive bacterium and Escherichia coli ATCC 25922 as Gram-negative bacterium. Microorganisms were seeded on samples and incubated at 37°C for 3h. After the incubation time, bacteria that remained adherent to the specimens were diluted and plated on agar plates. After incubation 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 results of cell viability assay (data not shown) demonstrated that SaOs-2 cells were capable to adhere and grow up to 7 days on both Ker and Ker 0.1% GNPs samples, as supported by SEM images acquired after 7 days of cell growth (Figure 1). The obtained quantitative data demonstrate that cell viability is higher on Ker 0.1% GNPs than Ker. Hence, GNPs stimulate and promote SaOs-2 cells growth on Ker-based membranes. After 7 days of incubation on samples, cells show a decrement in growth, probably due to the initial differentiation phase. Regarding the early process of bacterial adhesion (after 3h), Ker 0.1 % GNPs samples show a reduced bacterial adhesion compared to the pure Ker. The anti-adhesive effect of Ker 0.1% GNPs was different against the Gram-negative E. coli and the Gram-positive S. aureus. In particular, the sample with GNPs was able to reduce of ca. 20% the adhesion of E. coli. Interestingly, it reduced S. aureus of ca. 90% after 3h (Figure 2). 4 Conclusions The presence of gold nanoparticles in keratin-based scaffolds not only promotes cell growth on the surface of the scaffolds but also reduces the early process of bacterial adhesion (after 3h) of bacteria, in particular of Gram-positive S. aureus. The anti-adhesive effect seems significant for Gram-positive bacteria. In conclusion, the nanofibrous keratin-based membranes based on the preliminary biocompatibility studies, may represent a promising biomaterial for bone tissue engineering applications. Further studies will be performed to better evaluate their potential and their antibacterial properties.