The effects of keratin-coated titanium on osteoblast function and bone regeneration

Wool derived keratin, due to its demonstrated ability to promote bone formation, has been suggested as a potential bioactive material for implant surfaces. The aim of this study was to assess the effects of keratin-coated titanium on osteoblast function in vitro and bone healing in vivo. Keratin-coated titanium surfaces were fabricated via solvent casting and molecular grafting. The effect of these surfaces on the attachment, osteogenic gene, and osteogenic protein expression of MG-63 osteoblast-like cells were quantified in vitro. The effect of these keratin-modified surfaces on bone healing over three weeks using an intraosseous calvaria defect was assessed in rodents. Keratin coating did not affect MG-63 proliferation or viability, but enhanced osteopontin, osteocalcin and bone morphogenetic expression in vitro. Histological analysis of recovered calvaria specimens showed osseous defects covered with keratin-coated titanium had a higher percentage of new bone area two weeks after implantation compared to that in defects covered with titanium alone. The keratin-coated surfaces were biocompatible and stimulated osteogenic expression in adherent MG-63 osteoblasts. Furthermore, a pilot preclinical study in rodents suggested keratin may stimulate earlier intraosseous calvaria bone healing.


Introduction
Titanium implants are readily used in orthopaedics and craniofacial surgery and in particular, have found widespread usage in dentistry for the purpose of anchoring dental prostheses, providing a superior solution over conventional prostheses for the replacement of teeth lost as a result of dental caries, periodontal disease, trauma, or other tooth developmental abnormalities [1,2].These devices utilize the unique biocompatibility properties of titanium, which facilitates the apposition of bone directly onto the metal surface, a phenomena known as osseointegration [3].However, with increased clinical use and greater acceptance and popularity of implants, there are greater demands placed on implant systems from both clinicians and patients.In particular, there is demand for implant placement in sites where the quality of bone is less than ideal, such as those encountered in systemic conditions where the amount of mineralised tissue is reduced and/or bone healing is compromised [4].Studies have therefore focused on modifying the implant surface [5][6][7] to enhance the rate and or degree of bone deposition to ultimately improve integration with the surrounding bone tissue.
Keratin, a natural polymer extracted from wool, may be of use as a coating material on titanium implants as it is been shown to be both biocompatible, biodegradable and have osteogenic properties in vitro [8] and in vivo [9].Indeed, keratin scaffolds have been shown to support the adhesion, proliferation, and expression of various extracellular matrix proteins by fibroblasts [10,11] and osteoblasts [12] and have been tried in various biomedical applications such as wound healing, bone formation, nerve regeneration and tissue engineering [13].No study however has examined the osteogenic capacity of keratin when covalently coupled onto a titanium surface.
Once coated, the subsequent implant surface topography and chemistry are key factors influencing adherent cell behavior [14].In the initial phases of cell-biomaterial interactions, attachment determines subsequent spreading, proliferation, and differentiation [15].In this regard, the response of osteoblast cells to different micro-structured titanium surfaces has been well reported [16][17][18] where microrough implant surfaces have been shown to better support osteoblastic cell function compared to smooth surfaces [19].Thus, it is imperative to delineate how osteoblasts interact with keratin-coated titanium before any possible use in bone healing applications.
In this regard, our previous studies have described the preparation and physical properties of two keratin-coated titanium surfaces [20].This study aimed to first characterise the response of osteoblastic MG-63 cells to culture on these keratin-coated titanium surfaces in vitro, followed by a pilot in vivo preclinical study in rodents to assess their effect on bone healing of an intraosseous calvaria defect.
Briefly, to prepare the titanium for keratin coating, titanium discs were first treated with piranha solution (7:3 v/v of concentrated sulphuric acid (96%-98%, Sigma-Aldrich, USA) and hydrogen peroxide (30%, ChemSupply, Australia).The discs were then silanized using 3-aminopropyltriethoxysilane (99%, Sigma-Aldrich, USA) and crosslinked with glutaraldehyde (25%, Scharlau, Spain).Keratin was applied using two methods (1) solvent casting (Ti-KC), where a 1% keratin solution in sodium hydroxide was applied and air-dried for 24 h and (2) molecular grafting (Ti-KF), where discs were immersed in the same 1% keratin solution then incubated at 37 • C for 24 h, followed by washing and drying.All coated samples and untreated titanium (Ti) control samples were sterilized under UV light for 1 h prior to use.

MG-63 proliferation and viability:
An 80 µl suspension of 10 000 MG-63 cells was seeded onto titanium surfaces in 24 well plates (Greiner CellStar, Sigma Aldrich, Australia) and incubated at 37 • C for 4 h.After this time the titanium surfaces were completely covered with culture medium.MG-63 proliferation on the keratin-coated titanium surfaces was subsequently assessed on days 1, 3, 7 and 14 (AlamarBlue™, Invitrogen, Thermofisher Scientific, USA).Uncoated titanium was used as a control surface.
MG-63 viability on days 1, 3 and 7 of culture on the keratin-coated titanium surfaces was also determined using a Live/Dead assay (Invitrogen, USA) and evaluated using custom, automated image analysis software (CellProfiler 4.2.1).Briefly, MG-63 culture images were captured on a Nikon Ti2 widefield inverted microscope using a Plan Apo λ 10× objective and 80 ms exposure.All live and dead cells were identified with a user-defined segmentation pipeline (supplementary S1) and percentage cell viability calculated.

MG-63 doubling time:
Counts of MG-63 cells adherent to keratin coated titanium were quantitated using a custom, automated image analysis pipeline generated with the open source CellProfiler (4.2.1) software.Briefly, MG-63 cells were cultured on the keratin-coated titanium samples (10 000 cells disc −1 ) and the cell nuclei stained (Hoechst 33 342; Sigma Aldrich 4533, Australia in live cell imaging solution; Invitrogen, Thermofisher Scientific).Live cell images were captured on days 1, 3 and 7 of culture and the nuclei identified from the blue (λex/em; 390/440) channel with a user-defined segmentation pipeline (supplementary S2).The percentage attachment and the cell population doubling time was subsequently calculated using online tools (www.doubling-time.com/compute_more.php).

MG-63 morphology and morphometry:
To qualitatively assess cell attachment, MG-63 cells (10 000 cells disc −1 ) cultured on keratin-coated titanium were fixed with 4% paraformaldehyde (PFA, Sigma Aldrich, USA) for 1 h at RT, dehydrated in ethanol and dried overnight in hexamethyldisilazane (Sigma Aldrich, USA).Dried samples were mounted onto stubs, sputter-coated with gold and examined by scanning electron microscopy.The morphology and degree of spreading of MG-63 cells were evaluated qualitatively using images captured on an Olympus FV3000 scanning confocal microscope after 1, 3, Morphometry of MG-63 cells cultured on keratin-coated titanium surfaces (10 000 cells disc −1 ) was assessed using an Olympus FV3000 scanning confocal microscope at days 1 and 3 of cell culture.Day 7 was excluded from morphometric analysis as the cells had reached confluence making it impossible to run the segmentation image analysis.The raw Olympus OIR image format files obtained were used for quantification.Morphometric analysis of representative images after days 1 and 3 of cell culture was carried out with CellProfiler software (4.0.1).Briefly, a traditional user-defined segmentation and thresholding pipeline was developed using classical image processing algorithms.All cell nuclei and cytoskeleton filaments were identified for each image (DAPI; 405 nm channel and Phalloidin; 568 nm channel respectively) (supplementary S3).Linear image adjustments were performed on images used in representative figures.250-300 cells (3 independent experiments each with two samples per subgroup) were used to evaluate cell morphology parameters such as cell area, perimeter, diameter, aspect ratio, and form factor.

Immunocytochemistry:
The expression of osteogenic proteins; OCN, OPN, ALP, RUNX2, COL-1, and BMP-2 was examined using immunofluorescence.Prior to the study, the primary antibody was titrated to determine the optimal concentration for staining (data not shown).Briefly, MG-63 cells cultured on keratin coated surfaces for 3 d were fixed with 4% PFA for 30 min.The cells were then blocked and permeabilized with PBS/0.3%Triton-X/3% BSA for 30 min.The fixed cells were incubated with the appropriate primary antibody: ALP (1:100, ab65834, Abcam, UK), OCN (1:500, ab93876, Abcam, UK), OPN (1:100, ab283656, Abcam, UK), BMP-2 (1:500, ab214821, Abcam, UK), COL-1 (1:500, ab138492, Abcam, UK), and RUNX2 (1:500, ab192256, Abcam, UK) at 4 • C for 24 h.(Research Resource Identifiers (RRID) of the primary antibodies are provided in supplementary S4.The cells were then incubated with a 1:500 mixture of secondary  were allocated for each healing time point (2 and 3 weeks) and intraosseous defects on either side of the cranium were created (figure 1).An intraosseous defect was selected over a critical defect to allow new bone formation from the cranium bone towards the implant as well as laterally from both sides of the defect to better simulate the clinical implant healing process.
Surgical procedures: All surgical procedures were performed under aseptic conditions to minimize potential for infection.General anaesthesia was administered with a Mediquip Isoflurane vaporizer to enable more precise control of the level of anaesthesia.Isoflurane (up to 5%) (ProVet, Australia) was used for induction followed by 1%-3% to maintain anaesthesia.The animals were kept on a heating pad during surgery and immediately postoperatively to maintain body temperature.The dorsal area of the cranium was then shaved and disinfected with 50 mg ml −1 Povidone-Iodine (Betadine, Mundipharma BV, Netherland).A sagittal incision was then made through the skin to expose the periosteum of the calvaria and cranial vertex.Following elevation of a full-thickness periosteal flap, two shallow bilateral circular defects 5 mm in diameter (figure 1(A)) were created using a trephine.After marking the defect area, an intraosseous defect ∼ 0.5 mm deep was created within the circular marked defect (figure 1(B)) using a round burr under copious isotonic saline irrigation to prevent heating of the bone and to remove bone debris.Calvaria defects were subsequently covered with one of the titanium discs (table 2 and figures 1(C), (D)).Keratin-coated discs were placed with the keratin facing the bone defect.The soft tissue was then closed (figure 1(E)) using resorbable coated sutures (Vicryl 5.0, Ethicon, Germany).
Postoperative analgesia (buprenorphine 0.01-0.05mg kg −1 ) was administered via intraperitoneal injection immediately after surgery.The animal's weight, overall condition, activity, food/water intake, and wound condition were noted for seven days postsurgery.After 2 and 3 weeks of healing, samples of the surgical defect with/without the titanium disc in situ were retrieved and fixed in 10% neutral buffered formalin (Sigma Aldrich, USA) for 24 h.The tissue specimens were subsequently embedded in methacrylate resin (Technovit 7200, Heraeus Medical, Macquarie Park, NSW) to facilitate the preparation of 20 µm undecalcified bone samples using a cutting/grinding system (EXAKT Advanced Technologies, Germany) for histological staining.
Histology: Sections were stained with 0.1% toluidine blue for 45 mins then scanned using an Olympus VS200 high speed brightfield and fluorescence histology slide scanner (Olympus, Tokyo, Japan) with 20x objective resulting in a resolution of 0.548 µm per pixel.QuPath software [22] was used to quantitate new bone area.Briefly, the target area i.e. new bone growth from the lateral margin of the defect towards the medullary line was manually outlined using the polygon annotation tool.Once outlined, QuPath automatically calculates the area within this described perimeter.

Statistical analysis:
For the in vitro studies, two-way or one-way ANOVA with Tukey post-hoc analysis was performed to calculate any significant difference in mean values between the samples.All experiments were performed in triplicate.A p-value of <0.05 was considered statistically significant.Due to technical limitations with the pilot animal study (see discussion), no statistical analysis of the results (new bone area) were performed.

Keratin-coated titanium
Details of the surface characteristics of the titanium surfaces coated with wool keratin using APTES as the silane agent and glutaraldehyde as the cross-linking agent have been previously reported [20].Briefly, the solvent cast titanium surface (Ti-KC) exhibited a thick continuous keratin coating while the molecular grafted titanium surface (Ti-KF) had a thinner, non-uniform keratin coating over the titanium surface.Both coatings similarly increased surface roughness (Ra ∼ 0.45 µm) compared to untreated titanium (Ra ∼ 0.3 µm), with the molecular-grafted surface (Ti-KF) also demonstrating higher wettability (contact angle ∼ 47 • ) c.f. Ti-KC (contact angle ∼ 73 • ).
Both coated titanium surfaces were chemically stable over 28 d, with the solvent cast titanium surface (Ti-KC) exhibiting good adhesion and tensile bond strength of the coated keratin [20].

Osteoblast function in vitro MG-63 proliferation, Viability and Doubling time:
MG-63 proliferation on the molecular grafted keratin-coated surface (Ti-KF) was almost identical to that seen on uncoated titanium (figure 2(A)).While MG-63 proliferation was initially significantly (p < 0.05) lower at days 3 and 7 on the solvent cast keratin-coated titanium surface (Ti-KC), by day 14, MG-63 proliferation levels on all surfaces were essentially the same (figure 3(A)).No significant difference in cell viability (>85%, figure 2(B)) over 7 d was seen between the surfaces tested.Immunofluorescence micrographs of stained MG-63 cells cultured on the different titanium surfaces (figure 2(C)) and total cell numbers (figure 2(D)), reflected that seen for cell proliferation.Similarly, no significant surface effects on MG-63 doubling time were noted i.e. untreated titanium (39.27 ± 9.2 h), solvent cast titanium (31.03 ± 6 h), and molecular grafted titanium (33.6 ± 6.4 h).

MG-63 Morphology and Attachment:
To determine any effects of the keratin coatings on cell attachment and morphology, confocal microscopy (figure 3) and SEM (figure 4) analyses were performed.After 1 d in culture, fibroblast shaped cells with filopodialike cytoplasmic extensions were seen on Ti-KF and untreated titanium surfaces whereas cells on the Ti-KC surface were rounder in shape.After 3 d of culture, cells on the Ti-KC surface now exhibited a spindle-shaped morphology like those on Ti-KF.No further obvious visual differences in cell morphology were observed in cells on untreated titanium and both keratin-coated titanium surfaces after 3 d.
SEM micrographs similarly showed that MG-63 cells were well-attached displaying flattened, elongated spindle shapes on the untreated titanium and molecular grafted titanium surface (Ti-KF) after day 1 in culture.Again, MG-63 cells on the solvent cast titanium surface (Ti-KC) were more sparse presenting as round or polygonal shaped cells.By day 3 MG-63 cells on the Ti-KC surface were displaying a morphology similar to that seen with culture on Ti-KF.After 7 d culture no further differences could be determined as, cells started to be organized into multilayers on all surfaces.
The qualitative morphological observations (figures 3 and 4) were subsequently confirmed by automated single cell image analysis (supplementary S3) using cell; area, perimeter, diameter, form factor and aspect ratio (figure 5  factor (figure 5(E)) describes cell shape (circular or elongated), and aspect ratio (figure 5(F)) quantifies cell elongation.On day one of culture on the solvent cast titanium surface (Ti-KC), the MG-63 cells had a significantly more rounded morphology with smaller cytoplasmic area, diameter, perimeter, aspect ratio and larger form factor, compared to the cells on the untreated titanium and molecular grafted titanium (Ti-KF) surfaces.This morphometric data thus suggests restricted MG-63 spreading with fewer cellular protrusions on the solvent cast titanium surface.Conversely, a large cytoplasmic area, diameter, perimeter and aspect ratio with less form factor, was observed in cells on the molecular grafted titanium surface (Ti-KF) suggesting that the osteoblastic cells were more spread out, similar to cells cultured on untreated titanium.After 3 d of culture, no further morphometric differences in cell appearance were noted among the surfaces.Both the qualitative and quantitative analyses of osteoblast morphological changes suggest that initial cell attachment was not as good on the Ti-KC surface compared to the Ti-KF surface at the initial time point (day 1).Data from day 7 was not included as it was not possible to analyse individual cell morphology due to confluency by this time.

MG-63 osteogenic gene expression:
Broadly, a ⩾ 2fold increase in osteogenic gene expression following culture on the keratin-treated titanium surfaces (compared to untreated titanium), was observed at some point over the 7 d of observation.The precise temporal nature of this response however was variable e.g., OCN expression was highest at days 1 & 3 whereas OPN and BMP-2 expression was highest at day 7 (figures 6(A)-(F)).Comparing the gene expression response between culture on Ti-KC or Ti-KF at each observation point, these were also largely similar except for OCN, where fold changes were significantly higher in MG-63 cells on the Ti-KF surface (4.6 ± 3.1 & 6.9 ± 5.2) c.f Ti-KC (0.3 ± 0.015 & 1.01 ± 0.32) at the earliest time-points i.e. days 1 & 3 respectively (figure 6 A).The highest fold changes increase (∼10fold) in expression levels were seen with BMP-2 from days 3-7 (figure 6(F)) by cells on both keratin-coated titanium surfaces.

MG-63 osteogenic protein expression:
Using immunocytochemistry (figure 8(A)), the expression of osteogenic proteins by MG-63 cells cultured on the test surfaces was only assessed on day 3 when the level of cell confluency was amenable to single cell immunofluorescence microscopy.In general, quantification of these results (figure 8(B)) showed significantly higher expression of OCN, OPN and COL-1 was noted on the keratin-coated titanium surfaces compared to untreated titanium whereas ALP and RUNX2 expression was low on all surfaces.Interestingly, significantly lower BMP-2 expression was seen on the keratin-coated titanium surfaces at this early time-point.

In vivo bone healing:
The study protocol was successful with no animals dying or showing any signs of inflammation during the post-surgery study period.Histological observations of bone healing at 2 and 3 weeks showed some new bone tissue present in all samples (figure 9).Pre-existing cortical bone and newly formed bone were separated by a cement line, with the cortical bone being predominantly composed of lamellar and compact tissue with osteocytes in the lacunae.The new bone exhibited a woven structure, with new bone apposition observed mainly at the peripheral region of the defect covered by the titanium discs.In the defect site of all implanted samples, new osteoid matrix was apparent.
Unfortunately, while some of the implanted discs were lost from the defect site during sample processing/grinding (figure 9 control Ti disc), the area of new bone could still be quantitated using the slide scanner software (supplementary S5).At 2 weeks in both the blank and control, new bone area increased     further with 3 weeks of healing i.e. 27.7 ± 9.8 mm 2 to 86.2 ± 36.5 mm 2 and 51.2 ± 7.8 mm 2 to 118.1 ± 42.65 mm 2 respectively (figures 10(A) and (B)).In the keratin-coated titanium treatment groups, both surface treatments facilitated higher percentages of new bone area at 2 weeks compared to the control samples (191 ± 130.3 mm 2 & 125.3 ± 57.4 mm 2 ) for Ti-KC and Ti-KF respectively (figure 10(A)).After 3 weeks however, the mean new bone area was slightly lower (145 ± 106 mm 2 & 96.9 ± 36.3 mm 2 ) for Ti-KC and Ti-KF respectively (figure 10(B)).

Discussion
This study showed that both solvent cast and molecular grafted keratin-coated titanium surfaces did not adversely affect the long term (14 d) proliferation, attachment, or viability of MG-63 cells in vitro.This agrees with previous studies which have also demonstrated the ability of keratin to support high density cell growth over even longer periods (23-43 d) in culture [23].In the short-term however (days 1-7), lower cell proliferation on the solvent cast titanium surface (Ti-KC) surface was observed although levels subsequently increased to those seen on molecular grafted titanium surface (Ti-KF) by 14 d.
Cell proliferation on biomaterial surfaces is significantly influenced by the surface's properties, such as topography (roughness) and chemistry (hydrophilicity) [24].Despite considerable evaluation of implant surfaces however, there are still no accepted guidelines for analysis of roughness measurements [25].The roughness average 'Ra' , used in this study, is defined as the average of surface heights and depths across the biomaterial surface, and gives a good representation of height variation.'Micro-scale' surface features (0.5-1.5 µm) have been suggested to influence tissue-level interactions and improve mechanical interlocking while nanoscale features (1-100 nm) activate biological responses that involve elements such as proteins and cells, which influences osteoblastic behavior [26][27][28].Although the link between different height, spatial, or functional roughness parameters with specific biological responses is yet to be fully delineated, rough surfaces are generally accepted to enhance osteoblastic proliferation and subsequent osteogenesis [29][30][31].
It is unlikely however that the difference in initial proliferation of MG-63 cells observed on the Ti-KC surface in this study could be attributed to surface roughness, as both of the keratin-coated surfaces (i.e.Ti-KC and Ti-KF) exhibited a similar Ra value (0.43 µm and 0.46 µm respectively c.f 0.28 µm for untreated titanium) [20].This suggests potential for other surface properties to play important roles in cell-biomaterial interactions influencing cellular responses.In this regard, the surface energy, or wettability, of a material has garnered significant research interest in recent years.Hydrophilic surfaces lead to a more favourable conformational protein arrangements which can enhance cellular attachment [32].Studies have also shown that even relatively mildly hydrophilic biomaterials with contact angles from 40 • to 90 • can impact cell adhesion and proliferation [33], suggesting that the lower proliferation of MG-63 cells on Ti-KC may be influenced by its higher contact angle (72.8 • ) when compared to Ti-KF (contact angle = 47 • ) [20].Interestingly, another in vivo study also suggested that any differences observed between the hydrophilic and hydrophobic discs occurred in the early stages of wound healing, such as the first eight days suggesting surface energy is only influential on initial cellular interactions [34].
Clearly research needs to continue to evaluate which surface property or combination of properties will lead to the desired cellular response appropriate for its application.Furthermore, given our experimental protocol, it was not possible to differentiate any effect(s) of keratin alone, independent of any induced physiochemical effects i.e., topography and/or wettability.Further studies using keratin coatings with varying degrees of surface roughness and/or wettability would be required to address this question.
The excellent cell viability on both keratin-coated titanium surfaces is also in agreement with previous research that showed high viability with the use of keratin composite materials [35,36].Studies using hydroxyapatite-keratin nanocomposite fibres have shown that it was the presence of keratin in these hybrid surfaces that promoted favourable conditions for cell attachment [35], this also suggests that the lower proliferation on the Ti-KC surface was more likely not related to cytotoxicity, but rather to lower levels of cell attachment.No significant difference in doubling time were seen among the surfaces examined (Ti-KC, 31.03±6 h and Ti-KF, 33.6 ± 6.4 h), which were similar to that seen (∼29 h) with culture on keratin sponges [23].While studies have shown 1.3-1.9times higher doubling times of MG-63 cells cultured on different substrates [37,38], the results of this study indicate that the growth rate of MG-63 cells on all surfaces was similar despite any poor initial cell attachment.
Although substrate topography is known to influence cellular function on implant surfaces [15], where a reduction in cell spreading and cytoplasmic extensions often leads to hindered cell attachment [39], the exact nature of this effect is still unclear.Our qualitative and quantitative analyses of MG-63 attachment and morphology showed that cells on the Ti-KC surface were initially more round in shape with less cellular protrusions than cells on the Ti-KF surface.This finding is similar to other studies which also reported a more circular cell morphology on flat surfaces rather than microgrooved or nanopatterned surfaces [21].In contrast however, other studies using MG-63 cells have shown round osteoblast cells with few focal adhesion on rough surfaces compared to smooth surfaces [40][41][42].Saos-2 osteoblast cells were similarly shown to appear as circular cells with small actin filaments after 1 d in vitro cultured on a hybrid keratin-hydroxyappatite-PLLA membrane [35].Nonetheless, quantitative morphological analysis (figure 5) of the MG-63 cells showed that a similar degree of cell spreading was ultimately achieved regardless of the underlying substrate.
Bone formation includes both the expression of genes responsible for osteoblast differentiation and mineralisation and the subsequent synthesis/secretion of effector proteins [43].While the morphological results of this study suggest that coating titanium with keratin by either method produces a bioactive surface facilitating osteoblast adhesion and proliferation, the effect(s) of these keratin-coated titanium surfaces on osteoblast genotype and phenotype have not been delineated.Of these genes, ALP expression is an early marker of osteoblast differentiation; OPN an indicator of osteogenic differentiation; OCN is expressed in the later stages of osteoblast differentiation and is associated with mineralisation activity; COL-1 is a marker of extracellular bone matrix synthesis; BMP-2 is potent inducer factor for osteoblast differentiation while RUNX2 is a transcription factor and early indicator of immature osteoblast differentiation and an important regulator of ALP, OCN, COL-1, and OPN [14,21,[44][45][46].
Studies have shown that keratin scaffolds can indeed improve the expression of osteoblast differentiation markers [47,48] suggesting keratin may be a suitable biomaterial for tissue engineering.Furthermore a UV-crosslinked keratinchitosan composite was shown to upregulate osteogenic gene expression thereby promoting osteogenic differentiation [49].In the present study, distinct gene expression profiles were observed over the 7 day study, and the expression of OCN, OPN, RUNX2 and COL-1 all increased between days 1 and 3 whereas BMP-2 expression increased primarily at days 7. BMP-2 is known to induce the expression of RUNX2 which in turn promotes the expression of COL-1 and OPN which are involved in bone matrix formation and mineralisation [50].In terms of any coatedsurface effects, only OCN expression was significantly increased on the Ti-KF surface whereas OPN, ALP, RUNX2, COL-1, and BMP-2 gene expression were all similar on either keratin coating across all time points.These findings show osteogenic gene expression on both keratin-coated surfaces was generally higher compared to that on uncoated titanium which may prove to be beneficial in bone regeneration applications.However, apart from OCN expression, there was no significant difference in osteogenic differentiation between either modified surface.
A key consideration for any coating on titanium is the nature of its attachment to, and subsequent stability on, the titanium surface to ensure the coating will not delaminate during implantation [51].Our previous research has shown pre-treatment of the titanium surface with 3-aminopropyltriethoxysilane and the crosslinker glutaraldehyde afforded covalent binding of keratin onto titanium by both solvent casting and molecular grafting techniques.Degradation of the keratin coating from the titanium surfaces in vitro, showed an initial (24 h) release of keratin that may be conducive for osteoblast attachment and proliferation [20].No further increase in the rate of keratin degradation over the following 4-6 weeks however was observed, and in this regard, the coating was deemed to be relatively 'stable' [20].In the present study, keratin released from the titanium surfaces in vivo was not examined however and is a limitation of the study.Further preclinical in vivo studies are essential to demonstrate the feasibility and or suitability of potential coating polymers for subsequent clinical use.Keratin's biocompatibility and slow degradation rate however suggests significant potential for in vivo use [8,10].
In our pre-clinical pilot study, healing periods of 2 or 3 weeks were selected to examine bone formation in a novel intraosseous calvaria defect.An intraosseous defect model has been used in other studies to assess bone regeneration and osseointegration around implants [52].These authors generated three-walled defects in sheep femurs and showed that early osseointegration was enhanced using anodized discs compared to machined discs [52].In another ovine model, a porous keratin-hydroxyapatite composite was shown to promote greater new bone formation [53,54] while degradation of keratin-HA from a coated titanium implant was comparable to the rate of bone regeneration in the defect site [53].Further in vivo investigations have shown that the keratin-HA composites exhibit remarkable biocompatibility and osseointegration, with complete integration into bone occurring within 12 weeks [54].Accelerated bone repair in a rodent, critical-size calvaria defect model using a keratin-HA-rhBMP-2 hybrid scaffold has also been shown [55].These studies all suggest keratin has significant potential as a biomaterial able to assist in bone healing.
Histological and morphological analysis of calvaria sections in the present study showed the formation of both new osteoid material, and bone fill area from the lateral margins of the defects, with all treatment options.After 2 weeks healing however, treatment with keratin-coated titanium samples resulted in significantly higher percentages of new bone fill area.Variation in the results due to the limited number of sections able to be obtained per sample and the inability to obtain sections from identical regions of the defect site where the samples were implanted, meant no statistical analyses were performed due to these limitations.Regardless, the individual results suggest there was a clear difference in the bone fill area between the control and keratin-coated titanium samples at 2 weeks, suggesting keratin may have a stimulatory effect on bone formation in the early stages of healing.
While we acknowledge that the defect model used in this study cannot reproduce implant osseointegration and hence is a limitation of the study, another study in sheep using the femoral condyle as the surgical site, also demonstrated a novel keratin hydrogel increased osseointegration around implants earlier when compared to controls [56].Moreover, after 16 weeks, the %BIC was still almost twice as great in test implants as controls.In the present study however, by 3 weeks of healing, the levels of new bone formation in animals treated with keratin coated samples were by then similar to those seen in the control group.Overall, the results from this pilot in vivo study suggest that keratin applied as a polymer coating onto titanium may promote the early formation of new bone in osseous defects.To validate these findings, further studies utilising a larger sample size with more healing time points (early and late) are recommended.Similarly, comparison with bone formation using the standard 'critical size' calvaria defect model would also be useful.

Conclusions
Covalently bound keratin on titanium did not negatively impact on MG-63 cell attachment, morphology, or subsequent proliferation but increased osteogenic gene and protein expression in adherent MG-63 cells suggesting this surface may be useful in boneimplant applications.A preclinical pilot study subsequently showed both keratin-coated titanium surfaces enabled earlier bone healing after 2 weeks healing compared to untreated titanium.These findings suggest that a keratin-coated titanium surface has the potential to accelerate bone healing around implants.

Figure 1 .
Figure 1.Clinical photographs of the surgical procedures.(A) Circular marks (2 × 5 mm diameter) were created on the calvaria.(B) Intraosseous defects (∼0.5 mm deep) created within the marked area.(C) Group 1: defect on left side was uncovered (blank), defect on right side was covered with an untreated titanium disc (Ti).(D) Group 2: defects were covered with a solvent cast keratin-coated titanium disc (left side) and molecular grafted keratin-coated titanium disc (right side).(E) The periosteum and skin were closed, and animals were allowed to heal for 2 or 3 weeks.(Scale bar = 5 mm).The study followed institutional guidelines for humane animal treatment and complied with relevant legislation from the National Health and Medical Research Council, Australia.

Figure 7 .
Figure 7. Osteogenic function of MG-63 osteoblast cells following culture on untreated titanium (Ti), solvent cast keratin-coated (Ti-KC), and molecular grafted keratin-coated titanium (Ti-KF) surfaces.(A) ALP enzymatic activity over 21 d of culture.The dashed line shows the lowest concentration of standard used in the assay.(B) MG-63 mineralisation determined using alizarin red staining over 21 d of culture.

Figure 9 .
Figure 9. Toluidine blue stained ground sections after 2 weeks of healing showing (A) the defect either not covered (blank) or covered with untreated Ti (control lost during processing) or (B) keratin-coated titanium (Ti-KC and Ti-KF).Newly formed bone is visible from the lateral bone margins into the defect.Scale bar = 100 µm.'NB'-newly formed bone, 'OB'-old bone and 'O'-new osteoid.

Table 1 .
Primer sequences of osteogenic genes used for RT-qPCR.
antibody (donkey anti-rabbit IgG-Alexa Fluor TM 647, Invitrogen, Thermofisher Scientific) and Hoechst 33 342 (10 µM, Sigma Aldrich, 4533, Australia) for 1 h in the dark.Fluorescence images were acquired with a Ti2 Nikon widefield inverted microscope (10x objective) and semi-quantitatively analyzed using the Glencoe Omero software (Seattle, WA).2.3.In vivo studiesRat calvaria defect model: Ethical approval for this study was obtained from the Griffith University Animal Ethics Committee (GU Ref No: DOH/03/20/AEC) and all experiments were performed in accordance with the 'ARRIVE' guidelines (https://arriveguidelines.org/).Twelve adult athymic female rats (CBH RNU, ARC, Western Australia) approximately 10-12 weeks of age weighing 180 ± 20 g were maintained with both food and water ad libitum.Three animals per surgical group (table 2)