Coating of Sub-Micrometric Keratin Fibers on Titanium Substrates: A Successful Strategy for Stimulating Adhesion and Alignment of Fibroblasts and Reducing Bacterial Contamination

1Department of Applied Science and Technology, Politecnico di Torino, 10129 Torino, Italy; 2CNR-IPCB, Institute of Polymers, Composites and Biomaterials, 80125 Napoli, Italy; 3Department of Health Sciences, Università del Piemonte Orientale, 28100 Novara, Italy; 4Center for Translational Research on Autoimmune & Allergic Diseases—CAAD, 28100 Novara, Italy 5CNR-ISMAC, Institute for Macromolecular Studies, 13900 Biella, Italy; 6Department of Chemical, Materials and Industrial Production Engineering, University of Naples Federico II, Naples, Italy


Introduction
Effective soft tissue bonding and prevention of bacterial contamination are key issues in several implants such as trans-skin orthopedic and trans-mucosal dental implants [1,2]. Despite of a wide research on surface modification of titanium surfaces for bone contact applications, few research works focus on innovative surface treatments and coatings able to support soft tissue adhesion and limit bacterial contamination.
Fibroblasts, the main constituent of soft tissues, are generally rugo-phobic cells but they can align along specific directions (e.g., grooves) according to the well-known contact guidance phenomenon [3,4]. Moreover this kind of cell can be effective stimulated by biochemical factors such as keratin protein [5]. Following this rationale surfaces with oriented patterns and keratin enrichment can be suitable for the stimulation of fibroblast cells and the guidance of soft tissue adhesion in a healthy and effective way.
In addition, it is widely reported in literature that keratin has a high affinity for metal ions [6]. This ability is usually exploited in the water treatment field [7], but it can be also considered for the introduction of antibacterial properties in keratin-based materials.
Among different antibacterial metal ions, silver has been selected because it is well known for its broad spectrum antibacterial activity (based on a multiple mechanism of action), moreover it is associated with a low risk of resistance development, differently from antibiotics [8][9][10].
In the present research work, coatings of sub-micrometric keratin fibres were obtained onto commercially pure titanium substrates by electrospinning. Keratin was obtained from discarded wool following a green approach, which supports the employment of abundant local byproducts for the production of high added value coating intended for biomedical applications. Randomly oriented or aligned sub-micrometric fibres were deposited using stationary or rotating collectors respectively. Finally, deposited coatings were enriched with silver ions in order to confer also antibacterial functionalities.
Keratin was extracted from discarded wool by sulphitolysis with sodium metabisulphite and freeze dried, as previously described [12].
Randomly oriented keratin sub-micrometric fibres were deposited onto titanium substrates from keratin solutions 15% w/w in formic acid (reagent grade, 95%, Sigma-Aldrich, St. Louis, MO, USA) by means of a typical electrospinning setup composed by a plastic syringe, a highprecision syringe pump (KDS200, KD Scientific, Inc., Holliston, MA, USA) and a generator (SL50, Spellman High Voltage Electronics Corp., Hauppauge, NY, USA), which supplied a voltage of 25 kV. A stainless steel plate was used as collector and titanium disks substrates were attached to it for coating [12].
Varying the deposition conditions (time, temperature, humidity) different amounts of submicrometric fibers was obtained onto titanium substrates, with consequently different surface coverage degrees [12,13]. Occasionally some beads were also formed [12].
Keratin ability to bind metal ions [6] was exploited in order to confer antibacterial activity to coated surfaces, in this work. Titanium samples, coated with randomly oriented sub-micrometric keratin fibres, were soaked in silver nitrate aqueous solutions (0.01, 0.05 and 0.1 M) for 5 h at room temperature, as described in [15].
Surface topography of coated samples was investigated by means of field emission scanning electron microscopy equipped with energy dispersive spectroscopy (FESEM-EDS SUPRATM 40, Zeiss and Merlin Gemini Zeiss, Oerzen, Germany).
Cytocompatibility and cellular alignment were evaluated with human primary gingival fibroblasts (HGFs) from normal human gingiva, as representative cells for the possible final application of the proposed coatings and as previously reported [12]. Cells were seeded in a defined number (2 × 10 4 cells/specimen) directly onto samples' surface and cultivated up to 48 h after that cells morphology was evaluated by fluorescence imaging, after fixation with paraformaldehyde and staining with phalloidin and DAPI to visualize cytoskeleton f-actins filaments and nuclei, respectively.
Antibacterial activity was evaluated against Staphylococcus aureus (ATCC 25923). Coated samples were covered with 1 mL of LB medium containing 1 × 10 5 cells/ml and incubated for 90 min at 37 °C under agitation (120 rpm). Supernatants were then extracted and samples gently washed 3 times (with PBS) to remove non-adherent cells. Each specimen was rinsed with 1 ml of fresh LB medium and plate incubated (37 °C) up to 24 h for biofilm cultivation [12]. To assess the growth capacity of the bacterial strains in direct contact, bacterial viability was evaluated by colorimetric metabolic assay (XTT, Sigma-Aldrich). Moreover, the count of colony forming units (CFU) was also performed, as described in [15]; biofilm morphology was visually checked by SEM.

Results and Discussion
FESEM observations of randomly oriented and aligned sub-micrometric keratin fibres are reported in Figure 1. Depending on the process parameters, different coating density can be obtained. Different degrees of substrate coverage can be of interest in order to combine, for example, substrate topographical properties with biochemical stimuli of keratin fibres, as reported by the authors in [12,13].
Moreover, mats of randomly oriented fibers or aligned fibers can be obtained by changing the type of collector (stationary or rotating collector, respectively). The second typology of fibres can be of interest, again, with the final aim to combine biochemical signals from keratin with topographical stimulation of substrate topography and fibres direction. In fact, fibroblast cells are highly sensitive to contact guidance [3,4] as well as to keratin biochemistry [5].
Biological tests confirm this hypothesis. HGF show to align along the fibres direction in presence of a defined direction of keratin deposition (Figure 2), thus confirming keratin fibers ability to drive fibroblasts adhesion and spread as also previously demonstrated by the Authors [12,13].  (Figure 3i) underline that they are constituted mainly by silver. Looking at the high density keratin samples (Figure 3d-f), it can be noted that the titanium substrate is hindered by the high density keratin layer, so it is almost impossible to analyze separately fibres and beads as well as to observe eventual precipitates on the titanium substrates. EDS analysis on the sample area (Figure 3l) evidences the presence of titanium, keratin constituents and silver.
It seems that in the case of high keratin density coating, almost all silver ions have been loaded onto keratin fibres, as previously reported by the authors [15]. On the other hand, low keratin density coating maintains a portion of titanium surface free from coating and in this case, silver has been loaded on the surface in two forms: into keratin fibres/beads and in the form of submicrometric precipitates on the metallic substrate. Since keratin is able to attract and retain metallic ions and titanium is able to reduce silver ions to metallic silver (due to its negative redox standard potential) [6,7,16] it can be supposed that ionic silver has been introduced into keratin while metallic silver has been precipitated on the metallic substrate.
Antibacterial tests revealed the ability of all Ag-doped coated samples to reduce bacterial adhesion and biofilm formation (Figure 4). In fact, S. aureus viability was significantly reduced by comparing Ag-doped specimens and keratin control ones (Figure 4a, p < 0.05, indicated by §) as well as the number of viable colonies was significantly lowered (Figure 4b, p < 0.05, indicated by §). Finally, SEM images confirmed that bacteria in contact with Ag ions grafted onto specimens' surface were dead as they presented typical membrane damages due to Ag ions accumulation (Figure 4c).  This observation, in addition to what previously discussed about topography, underlines that the possibility to tailor keratin coverage of titanium surfaces give the possibility to select the different stimuli to be administered to cells (topographical or biochemical), but also to select, different antibacterial strategies, (such as silver ions or nanoparticles) and silver loading ability.
All the Ag-doped coatings maintain complete biocompatibility for HGF cells, as reported by the authors in [15].
The rationale and main findings of the research is summarized in Figure 5. Sub-micrometric keratin fibres coating have been deposited onto titanium substrates by electrospinning with random or oriented disposition. The so-produced coatings resulted perfectly biocompatible and able to support HGF adhesion and proliferation, as well as alignment in case of sub-micrometric keratin fibres deposited with a preferential direction. Silver enrichment of modified surfaces depends on the coating density and, in all cases, allows the production of biocompatible coatings able to reduce bacterial adhesion. In conclusion the here described coatings can match the main requirements of titanium surfaces facing soft tissues, such as transmucosal dental implants, which main problems are poor gum sealing and bacterial contamination.

Conclusions
Keratin obtained by discarded wool by a green approach was successfully used for the preparation of high added value coatings intended for biomedical applications. Sub-micrometric keratin nanofibers were obtained with random orientation on plane Ti-disks by means of electrospinning deposition with stationary collector while oriented fibres were produced by means of the application of a rotating collector. The ability of keratin to bind metal ions was exploited for fibres enrichment with antibacterial silver ions.

Conflicts of Interest:
The authors declare no conflict of interest.