Enhanced antibacterial activity and superior biocompatibility of cobalt-deposited titanium discs for possible use in implant dentistry

Summary The clinical success of implants depends on rapid osseointegration, and new materials are being developed considering the increasing demand. Considering cobalt (Co) antibacterial characteristics, we developed Co-deposited titanium (Ti) using direct current (DC) sputtering and investigated it as a new material for implant dentistry. The material was characterized using atomic absorption spectroscopy, scanning electron microscopy-energy dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy. The material’s surface topography, roughness, surface wettability, and hardness were also analyzed. The Co thin film (Ti-Co15) showed excellent antibacterial effects against microbes implicated in peri-implantitis. Furthermore, Ti-Co15 was compatible and favored the attachment and spreading of MG-63 cells. The alkaline phosphatase and calcium mineralization activities of MG-63 cells cultured on Ti-Co15 remained unaltered compared to Ti. These data correlated well with the time-dependent expression of ALP, RUNX-2, and BMP-2 genes involved in osteogenesis. The results demonstrate that Co-deposited Ti could be a promising material in implant dentistry.


INTRODUCTION
Loss of teeth affects the facial appearance and poses serious issues affecting the individual's health, psychological, and behavioral aspects.To overcome these problems, advanced prosthetic dentistry offers treatment modalities such as dental implants, bridges, and dentures. 1According to the American Dental Association, around 5 million implants are surgically placed each year, with an increase of $9.1%. 2 In modern dentistry, Ti and its alloys are widely used for manufacturing dental implants, primarily due to their excellent mechanical properties, corrosion resistance, and biocompatibility. 3,4However, Ti implants lack antimicrobial activity, and the screw-like design favors bacterial colonization, 5 which occurs immediately after implant surgery.Research findings indicate that the adherence of salivary proteins to the implant surface facilitates the attachment of primary colonizers.The cell-to-cell attachment with secondary colonizers follows, forming a complex multi-species biofilm on implant surfaces. 6,7Biofilm formation (also called dental plaque, responsible for the inflammation of soft connective tissue) and subsequent peri-implantitis (the progressive loss of bone around the implant), causes implant failures in about 5-10% cases. 8,91][12] Recent studies propose surface modifications of the implant to improve its antimicrobial and osteointegration properties. 13,14The surface topography, chemical composition, and surface free energy of Ti play a pivotal role in initial osteoblast attachment, proliferation, and mineralization of the extracellular matrix during the osseointegration process. 15To improve host cell attachment and osseointegration, procedures such as sand-blasting, acid-etching, anodization, laser treatment, UV-photofunctionalization, and plasma treatment were explored, which cause implant surface modification at the micro-and nanoscale. 16,17][20] There is a race between bacteria and host cells for the attachment on the implant surface, and the antibacterial property of implant surfaces can certainly enhance early osseointegration, thus, ensuring the long-term survival of the implant. 213][24][25] Amongst the techniques that impart surface modification with metals, the 'sputtering' technique offers precise control over the process parameters, resulting in the formation of a uniform thin film.7][28] For the implant surfaces modified by metals, the antibacterial activity is governed by the metal ions released from the surface of the thin film, which cause physical damage to the membrane, inactivate cellular enzymes, and exert damaging effects on the genetic material, eventually leading to cell death. 29,30Due to the multi-level effects and broad-spectrum antibacterial activity of metals, researchers have focused on developing metallic surface coatings.
Co is an essential trace element and has been a constituent of implant alloys for over four decades. 313][34] Similar to our previous study, 22 we deposited a nanometer-thick layer of Co on Ti using DC sputtering and investigated it as a new material for implant dentistry.After fabrication, the physicochemical characterization was completed, and the antibacterial activity of the Co-deposited Ti was studied against periodontal pathogens.The in vitro biocompatibility of Co-deposited Ti was assessed using MG-63 cells.

Surface morphology and topology analysis
The surface morphology of the control (Ti) and Co-deposited Ti samples was studied using SEM, as depicted in Figures 1A-1D.The Ti surface showed parallel surface scratches, probably generated during the polishing process (Figure 1A).Deposition of Co on the Ti discs resulted in a change in surface morphology compared to the Ti (Figures 1B-1D).It was observed that the deposition of Co occurred in the form of a film, which contrasts with grainy islands reported in the case of Zn, Ag, and Cu following the sputtering protocol. 25,35,36The elemental analysis using EDS showed the presence of Co on all the deposited surfaces (Table S1).The atomic weight percent of Co increased with deposition time, indicating successful deposition.Figures 1E-1H represents the surface topography of the Ti and Co-deposited Ti discs observed using AFM.The Co-deposited surface showed a uniform thin film formed by coalesced Co nanograins.The 3D AFM images of the Ti and Co-deposited Ti disc are provided in the SI (Figure S1). Figure 1I shows a photograph of control and Co-deposited discs.From the images, the color of

Total metal content and ion release
The total Co content on the different sample surfaces was analyzed using AAS.After 5, 10, and 15 min of deposition, the Co content of the Ti surfaces was 3.47, 8.10, and 10.41 mg/mm 2 , respectively (Figure 2A).From the graph, it is evident that a time-dependent increase in the Co content was observed.These results are in good agreement with the results of the EDS analysis (Table S1). Figure 2B illustrates the release of Co from the Ti-Co 15 sample over 28 days.A biphasic ion release profile was observed in which an initial burst of Co around 24 h was followed by a sustained release up to 28 days.It is worth noting that the cumulative release was only 12.6%.The lower release of Co from the Ti-Co 15 sample indicates strong binding between the Co coating and the Ti surface.Such long-term release of Co in the peri-implant microenvironment could be a possible approach to preventing infection after surgery and the subsequent implant healing period.However, this would warrant in vivo studies.

Surface wettability
Physical adsorption of plasma proteins from the blood to the implant surface is one of the earliest biological events that govern osteoblast attachment and osseointegration success. 37Wettability and SFE of the implant surface have been shown to play a significant role in initial protein adsorption. 38As shown in Figure 2C (red bars), the control Ti disc exhibited a WCA of 87 , whereas Ti-Co 5 , Ti-Co 10 , and Ti-Co 15 showed WCAs of 56.4,49.5, and 44.8 , respectively.In the case of Ti-Co 5, the WCA is reduced by $30 , which is further reduced with time.The decrease in WCA can be attributed to the physicochemical properties of Co-deposited Ti discs, such as topography, SFE, and chemical composition.As per the Owens-Wendt geometric mean equation, the calculated SFE of Ti, Ti-Co 5 , Ti-Co 10 , and Ti-Co 15 were 37, 54.8, 60, and 63 mN/m, respectively (Figure 2C, blue bars).A study by Hao et al. (2014) showed that the intermediate wettability of 20-70 promoted the attachment, spreading, and osteogenic differentiation of hMCSCs. 39Authors found that this moderate wettability promoted the expression of avb 1 integrins in the MSCs, which could be the probable reason for improved osteogenesis.In our study, the better cell attachment, spreading, and expression of osteogenic genes on Co-deposited Ti discs could be due to 1) nanoscale surface topography, 2) improved surface wettability and SFE, and 3) the chemical properties of the surface.Overall, Co deposition on the Ti discs reduced the WCA and increased SFE compared to the control, which further proves the suitability of Co coating on dental implant materials.

Nanomechanical properties
Nanoindentation is a technique to assess the nanomechanical properties of thin coatings on biomaterials. 401][42][43] Figure 2D displays the load-displacement curves obtained from loading and unloading the Berkovich indenter onto the Ti-Co 15 surface.The Ti-Co 15 surface revealed an average hardness of 39.10 GPa and an average elastic modulus of 438.7 GPa (Table S2).The surface coatings applied to the biomaterials should possess high mechanical properties to ensure their stability during clinical use. 43The results showed the formation of a hard Co thin film on the Ti surface, which would resist mechanical damage during implantation.

Surface chemical composition
XPS was employed to assess the surface chemical composition of the Ti and Co-deposited sample (Ti-Co 15 ).The XPS survey spectra of Ti showed prominent peaks at 285, 531, and 459 eV, which could be assigned to C 1s, O 1s, and Ti 2p, respectively, as shown in Figure 3A.The peak at 284.8 eV on the Ti surface could be due to carbon contamination.The Ti-Co 15 also showed C 1s and O 1s peaks at 285 and 531eV.The peaks at 781 and 796 eV were also seen and could be assigned to characteristic Co 2p 3/2 and Co 2p 1/2 doublet, confirming the Co deposition on the Ti disc.Due to the Co deposition, the characteristic Ti 2p peak at 458 eV was not detected in the Ti-Co 15 survey spectra.Figure 3B indicates the Co2p core-level spectra.The first pair of Co 2p 3/2 -2p 1/2 peaks at 780 and 795.1 eV could be assigned to the Co 2+ oxidation state.Similarly, the second pair of Co 2p 3/2 -2p 1/2 peaks at 781.5 and 797.1eV could be attributed to the Co 3+ oxidation state.Also, the corresponding O 1s spectrum showed peaks at 529.6 and 531.4 eV, corresponding to the Co-O bond (Figure 3C). 44The additional peak at 532 eV could be attributed to water adsorbed on the Ti-Co 15 surface (Figure 3C).The XPS data is in agreement with the previous research on Co 3 O 4 .Thus, the thin film contains Co 3 O 4 as a major chemical species.The detection of Co 3 O 4 in the coating might be due to the Co ions interacting with traces of oxygen in the chamber and further exposure to air under ambient conditions.

In vitro antibacterial assay
The antimicrobial activity of Co-deposited Ti discs was evaluated against the periodontal pathogens by the modified JIS Z 2801 method.The total viable count (TVC) analysis showed 100% antibacterial activity of Ti-Co 15 against Ss, Aa, Pg, Pg 93, and 53% against So (Figure 4).The antibacterial effects of Ti-Co 5 and Ti-Co 10 were lower (compared to Ti-Co 15 ) hinting at a concentration-dependent antibacterial activity.The antibacterial activity possibly correlates with the non-uniform nature of the Co thin films when the sputtering time was 5, 10 min.The antibacterial effect of the Co-deposited surface against each pathogen was statistically significant.6][47][48] A Cobased multifunctional wound dressing has shown potent concentration-dependent antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa. 33Mycosynthesized Co oxide nanoparticles using Aspergillus brasiliensis showed more or less similar antimicrobial potential against Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa and Escherichia coli) bacteria compared to ampicillin, streptomycin, gentamycin, and erythromycin. 49To the best of our knowledge, the results on the broad-spectrum antibacterial activity of Co on bacteria implicated in peri-implant infections are being reported for the first time.
A qualitative evaluation of live and dead cells on Ti and Co-deposited surfaces was performed using confocal microscopy.A reduction in live bacteria (green fluorescence) on the Co-deposited surfaces after 24 h of incubation was observed compared to the control (Figure 5).Ti-Co 15 surface showed a significant reduction in cell number compared to Ti, Ti-Co 5, and Ti-Co 10 .The antiadhesive activity data further supports the TVC assay results, hinting at contact killing of the bacteria due to Co released from the surface.
Figure 6 shows SEM images of periodontal pathogens grown on Ti and Co-deposited samples.It was observed that the Ti-Co 15 surface causes a significant reduction in bacterial cell population compared to the control.In addition to this, the Ti-Co 15 surface clearly showed a change in bacterial cell morphology and cell surface damage.Likewise, Ti-Co 5 and Ti-Co 10 samples showed only a slight reduction in bacterial cell number compared to Ti.The substantial bacterial cell reduction on the Ti-Co 15 surface could be attributed to the release of Co ions at high concentrations, rendering the surface unsuitable for bacterial cell attachment.Overall, the live-dead staining and SEM substantiate the TVC data.

In vitro biocompatibility Cell viability and proliferation assay
Osteoblast cell cultures have been widely used to understand bone-biomaterial interactions.Different osteoblast cell lines, such as MG-63, SaOs-2, MC3T3-E1, and primary human osteoblasts, have been employed to study the effect of implant surface modification on their bioactivities [50][51][52][53] Figure 7A shows the percentage cell viability of MG-63 cells on Ti and Co-deposited Ti samples after 24 h of culturing.A statistically significant difference in the cell viability was not observed between Ti and Co-deposited Ti samples indicating the excellent cytocompatibility of the Co-deposited surfaces.

Cell attachment and spreading behavior
The attachment and spreading of osteoblasts on the Ti surface is the first key event that profoundly influences cell proliferation and differentiation. 54As evidenced by the cell morphology, the spreading behavior of MG-63 cells on the Co-deposited Ti surface was much different from that of those grown on the Ti surface after 24 h.As seen in Figure 7C, the MG-63 cells grown on the control surface appear spindleshaped, flat polygonal in shape, with a few round cells.Morphologically, the cells grown on Ti-Co 5 and Ti-Co 10 surfaces were more or less similar to those on Ti (Figures 7D and 7E).Contrary to this, the cells grown on the Ti-Co 15 surface appeared large, flat, and polygonal with excellent spreading, almost covering the entire deposited surface (Figure 7F).The differences in cell attachment and spreading behavior on the Ti-Co 15 surface can be attributed to improved wettability and nanoscale roughness. 55Moreover, the enhanced cell attachment and spreading of osteoblast on the Ti-Co 15 surface could speed up the contact osteogenesis process (i.e., de novo bone formation on the implant surface), ensuring the early and long-term survival of the implant.

Monitoring the alkaline phosphatase activity
ALP is an important enzyme involved in bone formation and is a marker of early osteogenic differentiation. 56The data on the ALP activity of MG-63 osteoblasts cultured on Ti and Co-deposited Ti surfaces for 7 and 14 days are depicted in Figure 8A.On day 7, the difference in the ALP activity between the control and Co-deposited Ti was not statistically significant.However, on day 14, significantly higher ALP activity (p < 0.05) was observed on Ti-Co 15 compared to Ti, Ti-Co 5 , and Ti-Co 10 .Recently, Co-doped tricalcium phosphate scaffolds have been shown to promote proliferation with a substantial increase in the ALP activity of human bone marrow stromal cells (BMSCs). 57In a similar study, Co incorporated hydroxyapatite (HA) scaffold (Co-HA, 1.25% Co content) supported more ALP activity of MG-63 cells compared to the HA scaffold. 58Thus, the results in the present study are in agreement with earlier reports.

Assessment of calcium deposition
The mineralization efficiency of MG-63 cells on Ti and Co-deposited Ti surfaces was assessed by performing an ARS assay.After 14 days, no significant difference in the mineralization efficiency of the MG-63 cells was observed between Ti and Co-deposited Ti surfaces, as shown in Figures 8D-8G and B. At 21 days, red-colored calcium-rich deposits were observed on all the surfaces (Figures 8H-8K).Interestingly, the distribution of calcium-rich deposits was observed over the entire Ti-Co 15 surface compared to others (Figures 8K and 8B).As

Monitoring the expression of osteogenesis-related genes
Co is an essential trace element that the human body requires and is an integral part of vitamin B12. 594][65] The effect of Co deposition on the expression of ALP, Col1a, BMP-2, and RUNX-2 (osteogenesis-related) genes in MG-63 osteoblasts was assessed by qPCR (Figure 9).ALP is responsible for the formation of hard tissue; it hydrolyzes pyrophosphate and provides inorganic phosphate to facilitate ECM mineralization.BMP-2 recruits undifferentiated mesenchymal stem cells and differentiation into osteoblast in early osteogenesis.Col1a is an inherent part of the extracellular bone matrix.RUNX-2 is an important transcription factor that controls downstream osteogenic gene expression. 66We did not observe a statistically significant difference in the expression of the ALP gene between Ti and Co-deposited Ti surfaces after 14 days.However, on day 21, a significantly higher ALP gene expression was observed on all Co-deposited Ti surfaces compared to control (p < 0.01 for Ti-Co 5 and Ti-Co 10 , p < 0.001 for Ti-Co 15 ) (Figure 9A).On day 14, for Ti-Co 10 and Ti-Co 15 surfaces, the relative gene expression of Col1a was significantly low (Ti-Co 10 p < 0.05, and Ti-Co 15 p < 0.01).Whereas the expression of Col1a was restored on day 21 with no statistically significant differences between native Ti and Co-deposited surfaces (Figure 9B).Also, the relative gene expression of BMP-2 was similar on the Co-deposited and non-deposited surfaces after 14 and 21 days.However, on day 21, the Ti-Co 10 surface showed significantly higher BMP-2 expression (p < 0.01) than Ti (Figure 9C).In the case of RUNX-2, a significantly higher gene expression was observed on the Ti-Co 15 sample (p < 0.001) at the 14 th and 21 st day compared to the Ti (Figure 9D).

Hemolysis assay
Blood compatibility is one of the essential criteria that biomaterials must satisfy before clinical application.Blood incompatibility of biomaterial may lead to severe complications such as hemolysis, coagulation, and immune rejection, ultimately resulting in implant failure. 67The hemocompatibility of Co-deposited Ti was studied using a hemolysis assay.Figure 8C displays the results of the incubation of blood with Ti and Co-deposited Ti discs for 1 h.Ti and all Co-deposited surfaces revealed <5% hemolysis compared to Triton X-100 (positive control, complete hemolysis) (Figure S2).These data suggested that Co-deposited surfaces possess excellent hemocompatibility.

DISCUSSION
Ti is widely used for the fabrication of dental implants, but it lacks effective bioactivity and antibacterial activity, which delays the osseointegration process.Ti alloys possess a higher elastic modulus compared to natural the bone which is responsible for the stress shielding effect, but these were shown to release cytotoxic aluminum and vanadium ions. 68These issues were resolved by the design of b-type Ti alloys, containing higher amount of b phase stabilizers such as Mo, Zr, and Ta.Ti-15Mo, Ti-24Nb-4Zr-8Sn, and Ti-35Nb-2Ta-3Zr, are some examples of the latter. 69The porous b-type Ti alloys can be prepared using powder metallurgy, additive manufacturing (3D printing), and FAST-forge. 69Though these alloys have excellent physico-chemical properties they lack effective bioactivity.Thus, continued research to improve the antibacterial properties and osseointegration potential of the Ti implant will essentially promote the use of implants as a long-lasting treatment for edentulous individuals.In the past various techniques were used to modify the Ti surface and coating with Ag, Cu, Zn, Sr, Ga, Sn, and Au conferred antibacterial activity. 70Amongst physical techniques, sputtering, has been widely used for the deposition of metals on different surfaces owing to ease of use, achieving the purity of coating, and uniform film deposition. 22Few investigations have reported the broad-spectrum antibacterial activity of Co. [45][46][47][48] Recently, Co-Cr alloy was shown to maintain the tribological properties and corrosion behavior in acidic artificial saliva-lubricated conditions, which implies the possible use of such alloy in dental implant construction. 71In the time-course experiment, an increase in Co content with deposition time was observed, suggesting the success of sputtering in the surface modification.It has been shown that osteoblast cells respond to the topographic features of the extracellular matrix such as pits, pores, and fibers with dimensions ranging between 5 and 200 nm. 72In addition, the AFM images of Co-deposited Ti surfaces, (especially Ti-Co 15 ), show distinct Co nanograins (30-70 nm diameter) which might be a parameter contributing to improved cell attachment.Also, it has been reported that nanoscale surface topography promotes protein adsorption and conformation which in turn influences cell attachment. 73Additionally, Co deposition improved the hydrophilicity and SFE of the native Ti surface.Taken together, earlier findings corroborate the results of the present study on Codeposited Ti using DC sputtering as a dental implant material.
Peri-implantitis is the most common cause of implant failures, with bacterial infection being the root cause.1][12] In this study, we assessed the antibacterial potential of Co-deposited Ti specifically against periodontal pathogens belonging to yellow (S. sanguinis, S. oralis), purple (A.actinomycetemcomitans), and red (P.gingivalis, P. gingivalis 93 (Indian strain)) complexes.It was noteworthy that Co-deposited Ti, especially Ti-Co 15 , showed excellent antibacterial activity against periodontal pathogens.Although metals possess remarkable antibacterial activity with meagre chances of developing resistance in bacteria, the minute concentrations may be cytotoxic to the host cells.Keeping this in mind, we investigated the effect of Co coating on the viability, attachment, spreading, and proliferation potential of the MG-63 osteoblasts.The results showed that Co coating on Ti did not have any cytotoxic effect on MG-63 osteoblasts; the Ti-Co 15 surface supported more cell attachment and spreading, one of the most critical steps during implant healing. 54In addition, the Ti-Co 15 surface exhibited more ALP activity and mineralization efficiency, reaffirming the possibility of Co coating for the surface modification of Ti to enhance its osseointegration.The qPCR assay revealed that, at 14 days an overall increase in ALP gene expression on the Co-deposited Ti (especially on Ti-Co 15 ) compared to control Ti continued up to 21 days.A study by Kargozar et al. showed a similar trend of increase in ALP gene expression when SaOS-2 cells (human osteosarcoma cell line) were treated with Co-substituted bioglass. 74Based on these results, it may be concluded that Co ions have a positive effect on the ALP gene expression.In the case of Col1a gene expression, previous studies have shown that higher concentrations of Co ions (0.25-1 mM) had a negative impact on the expression of the Col1a gene in MG-63 cells. 75,76In our investigation we found a reduction in the Col1a gene expression on the Co-deposited Ti at 14 days in a dose-dependent manner i.e., more reduction on Ti-Co 15 followed by Ti-Co 10 , and Ti-Co 5 which could be attributed to higher Co release during the initial period of incubation.However, it is important to note that the Col1a gene expression normalized at 21 days probably due to the depletion of Co ions from the surface during incubation.Moreover, MG-63 cells cultured on the Ti-Co 15 surface showed higher expression of BMP-2 and RUNX-2 genes, thus proving the safety of Co coating for in vivo use.Surface modification of dental implants using metal nanoparticles provides the advantages of antibacterial activity and improved osseointegration.However, the difficulties in the degradation and excretion of these metal nanoparticles might pose serious health disadvantages such as cytotoxicity, bioaccumulation, and systemic toxicity. 77Our findings suggest that although Co-deposited Ti surfaces do not have any cytotoxic effect on the MG-63 osteoblasts, the in vivo compatibility studies are warranted to evaluate the excretion, bioaccumulation, and systemic health consequences of Co coating.It is worth noting that the sputter deposition of Co on biomaterial surfaces may prevent biomaterial-associated infections, ensuring successful bio-integration.

Fabrication of Co-deposited Ti
Circular polished discs of Grade 5 titanium alloy (Ti 6 Al 4 V), 10 mm diameter and 2 mm thickness (Bhagyashali metal, Mumbai, India), were used in the study.The discs were cleaned to remove the surface contaminants from the cutting and machining.Firstly, the discs were washed with a warm detergent solution, followed by five washes with water, and dried.The discs were then treated with acetone, isopropanol, and ethanol for 15 min each in the ultrasonic water bath with three distilled water washes in between and dried.The Co thin film was deposited on the Ti discs using a DC sputtering machine (Hind HiVac, Bangalore, India).Co disc (99.99% pure, diameter 3", thickness 2 mm) was used as a target with a fixed target to substrate distance of 3 cm.The deposition was carried out in an argon (UHP grade) plasma atmosphere (0.1 mbar working pressure and 100 mA current) for varying time intervals (5, 10, and 15 min).Co-deposited samples were abbreviated as Ti-Co 5 , Ti-Co 10 , and Ti-Co 15, respectively.The undeposited Ti discs were used as control.

Surface morphology and topology analysis
The surface morphology and elemental composition of the samples were determined using scanning electron microscopy (SEM) (MA15 EVOâ; Carl Zeiss, Baden-Wurttemberg, Germany) coupled with energy dispersive X-ray spectroscopy (EDS) (Oxford Instruments, Abingdon, United Kingdom).Before analysis, the samples were sputter deposited with a thin gold film, and images were captured at 20kV accelerating voltage.
The samples' surface topography and average surface roughness were measured using atomic force microscopy (AFM) (Innovaâ, Bruker, Massachusetts, USA).Antimony doped silicon tip (resonance frequency 300 kHz and nominal elastic constant 40 N/m) was used for scanning the surface in tapping mode.Gwyddion (version 2.59), a freeware, was used to visualize the AFM images and determine the average roughness (Ra).
The sputter deposition rate of Co and coating thickness of the Co-deposited Ti was assessed using the surface profilometry technique.The experimental details and results obtained are presented in the supplemental information (SI) section (Figures S3 and S4).

Total metal content and release study
Quantitative estimation of the total Co content of the deposited samples was performed using atomic absorption spectrophotometer (AAS) (ContrAA 800D, Analytikjena, Jena, Germany).Initially, all the samples were immersed in 1 mL of concentrated nitric acid (HNO 3 ) for 12 h to dissolve the Co coating completely.The resulting solution was diluted up to 10 mL using double distilled water in a standard flask and used directly for estimation. 22he release of Co from the Ti-Co 15 sample was monitored in 10 mM phosphate-buffered saline (PBS, pH 7.2) at different time intervals using inductively coupled plasma-atomic emission spectrophotometry (ICP-AES) (ARCOS, SPECTRO analytical instruments GmbH, Germany) available at Indian Institute of Technology, Mumbai, India.The sample was immersed in 1 mL of PBS and incubated at 37 C.At pre-determined time intervals (0, 1, 3, 7, 14, 21, and 28 days), the solution was withdrawn and replaced with the same quantity of fresh PBS.The resulting solution was digested in concentrated HNO 3 for 12 h and further diluted to 5 mL in a standard flask using double distilled water and used for the estimation. 22

Wettability study
The wettability and surface free energy (SFE) of the Ti and Co-deposited samples were assessed using contact angle (CA) measurement.The CA measurements were carried out on a drop shape analyzer (DSA25S, Kruss Scientific, Hamburg, Germany) instrument in static mode using double distilled water and di-iodomethane as a wetting medium (2 mL) at room temperature (RT).The representative contact angle is the mean of three independent measurements.The SFE was calculated using Owens-Wendt geometric mean equation. 78 Where q is the measured contact angle, g L is the surface tension of the liquid, g D L and g P L are dispersive and polar components of the liquids.The SFE ½g S = g D S + g P S was determined by measuring the CA of water and di-iodomethane.

Nanomechanical properties
The nanomechanical properties (hardness and Young's modulus) of the Ti-Co 15 surface were evaluated by nanoindentation test using a G200 Nano indenter (Agilent, California, United States).A diamond Berkovich indenter was employed based on a continuous stiffness-measuring technology.A maximum load of 5 mN was applied on the surfaces during the test.A total of 10 indentations in randomly selected areas were fixed using 4% paraformaldehyde (PFA) for 15 min and permeabilized in 0.1 % Triton X-100 for 5 min.Cells were stained with rhodaminephalloidin (actin stain, 30 min) and Hoechst (nuclear stain, 10 min) dyes and observed under CLSM.Samples were washed twice with PBS after each step. 79nitoring alkaline phosphatase (ALP) activity Intracellular ALP activity of MG-63 cells cultured on Ti and Co-deposited Ti samples were assessed spectrophotometrically by determining the enzymatic conversion of p-nitrophenol phosphate (pNPP) to yellow-coloured p-nitrophenol (pNP) as described by Wang et al. (2016). 80nitially, 2 x 10 4 cells were cultured for 7 and 14 days on the Ti, and Co-deposited Ti surfaces.After incubation, samples were washed with PBS, and cells were lysed using RIPA buffer for 1 h at 4 C. Lysates were centrifuged at 12000 rpm for 30 min at 4 C, and the supernatant was used for the assay.50 mL of lysate was added to 100 mL of pNPP solution (10 mM Tris buffer, pH 9, and supplemented with 1 mM MgCl 2 ) and incubated at 37 C for 30 min.The absorbance of the coloured product was recorded at 405 nm, and the amount of p-nitrophenol formed was estimated using a standard curve.The enzyme activity was normalized to total protein content measured using BCA assay.

Assessment of calcium deposition
The effect of Co deposition on the mineralization efficiency of MG-63 cells was assessed using alizarin red S (ARS) dye.Cells (2 x 10 4 ) were seeded on the Ti and Co-deposited Ti samples and allowed to proliferate in a-MEM until monolayer formation (6 days).The normal growth medium was replaced with osteogenic medium (a-MEM supplemented with 50 mg/mL ascorbic acid, 10 mM b-glycerophosphate, and 100 nM dexamethasone) in which the cells were grown for 14 and 21 days.Before staining, the monolayer was washed with PBS and then fixed with 4% PFA for 15 min at RT, followed by washing with PBS to remove excess PFA.ARS dye (40 mM, pH 4.2, 500 mL) was added to each sample and incubated for 20 min.Subsequently, the unbound dye was removed, and the stained monolayer was washed five times with distilled water and visualized under a stereomicroscope (Leica M205A, Wetzlar, Germany).For quantitation, stained monolayers were stored at -20 C. The dye was extracted in 500 mL of 10% (w/v) cetylpyridinium chloride solution for 1 h, and then absorbance of the coloured solution was recorded at 405 nm. 81,82nitoring the expression of osteogenesis-related genes The relative expression of osteogenesis-related genes viz, Alkaline phosphatase (ALP), Collagen 1 (Col1a), Bone morphogenetic protein 2 (BMP-2), and Runt-related transcription factor 2 (RUNX-2) were studied by the real-time qPCR.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the reference gene.After 14 and 21 days of incubation, the total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA).Subsequently, the complementary DNA (cDNA) was synthesized from 1 mg of total RNA using ReadyScriptâ cDNA synthesis kit (Sigma-Aldrich, St. Louis, United States).The qPCR was performed using LightCyclerâ 480 SYBR Green I Master mix on the LightCyclerâ480 platform (Roche Diagnostics, Switzerland).The qPCR cycles consist of an initial denaturation step at 94 C for 5 min followed by 40 amplification cycles consisting of 94, 58, and 72 C for 10, 30, and 30 seconds, respectively.The relative gene expression was studied using the comparative 2 -DDCT method according to Livak and Schmittgen (2001). 83Dissociation curves were generated for each reaction to ensure specific amplification.Threshold values (C T ) generated from the software tool (Roche LC 2.0) were employed to quantify relative gene expression.The sequence of primers used in the study is listed in Table S3.

Hemolysis assay
The hemocompatibility of the Co-deposited Ti samples was determined as per the procedure described by Patil et al. (2022). 84Briefly, freshly drawn sheep blood was processed by centrifugation at 500 x g to concentrate the RBCs.The RBC pellet was washed with normal saline until a clear supernatant appeared.Then concentrated RBCs were diluted with saline (1:9).500 mL of diluted RBC suspension was added over the Ti and Co-deposited Ti samples (in triplicate) and incubated at 37 C for 1 h.After incubation, the RBC suspension was collected in a fresh microcentrifuge tube and centrifuged at 500 x g for 5 min.Following this, Drabkin's reagent was added, and the cyanmethemoglobin formed was estimated by recording the absorbance at 540 nm using a multi-well plate reader (SynergyHT, Biotek, USA).Saline and Triton X-100 (0.1%, 50mL) were used as negative and positive controls, respectively.Percentage hemolysis was calculated using the following formula.

QUANTIFICATION AND STATISTICAL ANALYSIS
All the experiments were carried out in triplicates (n=3).All the data values are represented as meanGSD.Data analysis was performed using GraphPad Prism 5 software.One-and two-way ANOVA was applied to the data set, followed by Bonferroni post hoc test to estimate the statistical significance between the control and test groups.

Figure 2 .
Figure 2. Physicochemical characteristics of Co deposited Ti discs Quantitative estimation of total Co content deposited on Ti discs (A).Evaluation of Co ion release from Ti-Co 15 surface over 28 days (B).Assessment of water contact angle and surface free energy of the Ti and Co-deposited Ti discs (C).Force-displacement curve of Ti-Co 15 surface using nanoindentation technique (D).*** and &&& indicate p < 0.001 when compared with Ti.

Figure
Figure 7B indicates the results of the cell proliferation assay.All the Co-deposited Ti surfaces supported the proliferation of MG-63 cells at day 3 compared to the Ti surface.However, the cell proliferation data on day 5 showed a statistically insignificant difference.A higher absorbance, indicating cell proliferation, was observed on day 7 on the Ti-Co 15 surface compared to the Ti, Ti-Co 5 , and Ti-Co 10 surfaces.Thus, it is interesting to note that Co deposition on Ti surfaces favored the proliferation of MG-63 cells.The proliferation data supports the applicability of Co for dental implant surface modification.

Figure 5 .
Figure 5. Live-dead staining of periodontal pathogens grown on Ti and Co-deposited Ti discs Scale bar represents 50 mm.

Figure 6 .
Figure 6.Scanning electron microscopy images of periodontal pathogens on Ti and Co-deposited Ti discs Scale bar represents 10 mm.

Figure 7 .
Figure 7. Viability, proliferation and visualization of MG-63 cells cultured on Co-deposited Ti discs Percentage cell viability of MG-63 cells cultured on Ti and Co-deposited Ti discs (A).Cell proliferation ability of MG-63 cells cultured on Ti and Co-deposited Ti discs (B).** indicates p < 0.01, and ***p < 0.001 when compared with Ti.Confocal microscopic images of the cytoskeleton and nuclear staining of MG-63 cells cultured on Ti (C), Ti-Co 5 (D), Ti-Co 10 (E), and Ti-Co 15 (F) for 24 h.Scale bar represents 100 mm.