In-vitro experiments on bio-functional calcium phosphate based coatings on titanium dental implant

Dental implants are a revolution in dentistry, but certain deficiencies still need to be addressed. One of the most severe threats to the success of dental implants is peri-implant infection. Existing coatings on titanium (Ti-6Al-4V) alloy surfaces rapidly lose antibacterial efficacy, reducing their ability to prevent peri-implant infectious disease. The objective of this paper was to investigate the dissolution capabilities and film properties of calcium phosphate (CaP) based layers on a titanium (Ti-6Al-4V) alloy surface produced with the radiofrequency magnetron sputtering method. These coatings have demonstrated good osseointegration capability due to their similarity to bone mineral matter. The bioactive coating materials are calcium phosphate, zinc chloride, and silver nitrate. Microstructural investigations of coated components were assessed using Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Energy Dispersive (EDX) analysis, and Atomic Force Microscopy (AFM). Bacterial adhesion to biomaterials is still a major issue when it comes to medical equipment. Antimicrobial peptides have generated interest due to the rise in antibiotic-resistant bacteria. The fact that there are concerns regarding the development of antibiotic resistance due to the development of traditional antibiotics to prevent illness reflects the complexity of the matter. The coated titanium samples were inoculated in nutrient broth and incubated at 37 °C for 24 h. The samples were tested against Staphylococcus aureus and Staphylococcus epidermidis for 72 h. A standard row containing between 1 * 105 CFU ml−1 and 1 * 107 CFU ml−1of fresh exponential culture was prepared. The plates were cultured at 37 °C and shaken (100 rpm) while the OD600 was monitored every 30 min. After 24 h, Staphylococcus aureus inhibition was higher than 70% in S4, and Staphylococcus epidermidis inhibition ranged from 60 to 70% in S4. Antimicrobial activity was found in the calcium phosphate coated samples (S4) against gram-positive Staphylococcus epidermidis and Staphylococcus aureus bacteria. The antimicrobial evaluation showed that titanium made with bioactive coating inhibited bacterial growth and biofilm formation.


Introduction
An implant is an artificial device to replace a damaged or missed biological operation in a living body. The implants comprise biomedical materials like titanium, silicone, and apatite; they are selected based on their functionalities [1]. Different types of implant devices like pacemakers, concealer implants, and dental implants are used based on their biological operation [2]. The dental implant is the most used device for replacing a missing or decayed tooth root. Yet, those dental implants can replace the tooth root, but their biocompatibility is minimum [3]. If the implant is not osseointegrated, it will fail, resulting in alveolar bone resorption and implant loss. Osseointegration failure can be caused by various reasons, including inadequate bone quality and volume, poor dental hygiene, periodontitis, smoking, and systemic ill health [4]. Furthermore, osseointegration is influenced by implant features such as shape, surface roughness, and substance. Hence, to enhance biocompatibility, the dental implant material is coated with carbon or the same dental implant material [5].
The proper selection of an implant material is usually a difficult task because biocompatibility must be ensured as well as the material's endurance and manufacturability. Several approaches have been used to improve metal implant biocompatibility and osteogenic potential, ranging from surface modifications involving inorganic mineral coatings to implant surface coatings to modify peri-implant tissue reactivity and extend implant life [6]. A coating material protects the body's organs from metal ions generated by metallic implant materials [7]. The effectiveness of an implant material is determined not only by its physicochemical properties but also by its surface. Recently the osteoconductive coatings materials on the implant surface, such as calcium phosphate (CaP), tri-calcium phosphate (Ca 3 (PO 4 ) 2 ), zinc chloride (ZnCl 2 ), and silver nitrate (AgNO 3 ), ammonium dihydrogen phosphate ((NH 4 )H 2 PO 4 ), calcium chloride (CaCl 2 ) protein, peptide, and cellularmatrix coating and hydroxyapatite (HA) have yielded promising results in terms of osteoblast cell adhesion and cell function [8]. Furthermore, when choosing a coating material, the capacity to promote implant bone bonding is a significant issue to be considered. Consequently, the surface layer must be non-porous, dense, and adhere well to the substrate of the implant material. [9]. One disadvantage of using metals as biomaterials is that they are typically synthetic materials without biological function. Surface modification is required to add bio function to metals because bio function cannot be added during manufacturing processes such as melting, casting, forging, and heat treatment. Surface modification could improve corrosion resistance, biocompatibility, and biofunction of the surface layer. Many techniques for surface modification of metals are being researched for these purposes, and some are being commercialized. Thermal sputtering, dip coating, electrophoretic deposition, magnetron sputtering, colloidal solution deposition, plasma spraying, pulsed laser deposition, and sol-gel methods can all be used to coat metal surfaces with bioactive compounds to improve bone healing and metal implant integration [10]. With these coatings, the healing period ranges from one to six months and the implant coating should possess both properties of a faster healing process and prevent the implant from microorganisms for a longer lifetime [11].
Inorganic coatings such as calcium phosphates or hydroxyapatite have promoted and accelerated bone formation in implant surroundings. Organic or biologically active molecules functionalized on implant surfaces promote cell adhesion. Nano-hydroxyapatite is used as a single coating or in a composite coating with collagen, bioglass, or titanium dioxide to mimic the bio-environment of native bones. [12]. Using a plasma-sprayed hydroxyapatite coating on the implant surface is a typical way to improve biocompatibility and stimulate the rapid formation of bone tissue [13]. In the absence of this coating, adhesion, delamination, a reduction in coatings, and cohesive failure with porosity can occur. However, advances in coating techniques have rekindled interest in Radiofrequency (RF) magnetron sputtering. It is used to sinter a target of a pure compound of phosphates to achieve a suitable substrate with a coating of chemical compositions. This method produces quite a tiny amount (films at the nanoscale), which is detrimental for dental implants since thin layers disintegrate quickly. [14]. High energy electrons strike gas molecules or atoms to form positive ions, which are then used to bombard the hydroxyapatite target, depositing the target material onto the implant substrate material after ionization and an excitation-relaxation reaction. The ability of the sputtering coating to interact with living bone tissue and form strong bonds with it is its most intriguing property. Because of its uniform coating layer on metal surfaces, the sputtering coating is today's most efficient and widely used method on metallic implants. [15].
The HA or other coating materials like bioactive glass or polymers require additional materials for antibacterial growth on the dental implant. A uniform, highly adherent coating only a few microns thick was produced by using RF magnetron sputtering to coat a titanium alloy implant with a bioactive material in the earlier days [16]. The thickness of the coating will decide whether a direct implantation method succeeds or fails. The thickness of coatings can be measured using SEM by examining cross-sections of the coating and measuring the distance between the substrate and the top of the coating. Coating thicknesses ranging from 0.05 to 200 μm can be achieved using different implantation procedures [12]. Therefore, this paper proposes an RF sputtering coating, an experimental and anti-bacterial analysis of different combinations of coating material with tricalcium phosphate (Ca 3 (PO 4 ) 2 ), zinc chloride (ZnCl 2 ), and silver nitrate (AgNO 3 ) on titanium dental implant surface to achieve both the osseointegration and anti-bacterial properties. This study looked at the film properties of HA coatings made using RF sputtering to improve osseointegration and stability in alveolar bones. In figure 1 depicts a thorough examination of the proposed coating material and sample preparation processes, as well as detailed experimental data.

Materials
The substrate material was used to make the substrate specimens Ti-6Al-4V; the chemical composition was (in wt.%): (Ti − 90.05%, Al 5.45%-6.95%, V 3.75%-4.75%, Fe − 0.28%, O − 0.18%, H − 0.016%, C − 0.07%, N − 0.03%, the rest − 0.38%). Two different specimens' shapes (plates/discs) were purchased from metal M s −1 Nation alloys suppliers. As substrates, metallic plates measuring 75 mm in length, 25 mm in width, and 0.5 mm in thickness were employed. For the microbiological test, another round-shaped sample with a diameter of 7 mm and a thickness of 0.5 mm was used. To remove debris and contaminants, all-round discs and rectangular metallic plates were polished with 400 grit silicon carbide sheets, then ultrasonically treated for 10 min at room temperature (25 ± 1°C) per component in ethanol/acetone (1:10, substrate to solvent ratio). After the solvent treatments, all discs/plates were soaked with distilled water.

Target preparation
The Ti-6Al-4V (Grade 5) plates/discs were coated with bioactive powders. The magnetron sputtering targets (50 mm diameter × 5 mm thickness) was sintered from bioactive powder (99.95 percent purity, particle size > 30 nm, density 2.13 g cm −3 ). In figure 2 represents first, the coating powder is converted into target form by mixing the concentrations that are available as T1 with 100% tricalcium phosphate (Ca 3 (PO 4 ) 2 ), target T2 with 75% tricalcium phosphate (Ca 3 (PO 4 ) 2 ) + 25% zinc chloride (ZnCl 2 ), T3 as 50% tricalcium phosphate (Ca 3 (PO 4 ) 2 ) + 25% zinc chloride (ZnCl 2 ) + 25% silver nitrate (AgNO 3 ). Initially, a 7 mm diameter and 0.5 mm thick Ti-6Al-4V discs are taken, and it is cleaned with acetone for the microbiological test. • For making the target, the circular sputtering die is cleaned with acetone and graphite to remove the dust particles. The sample powders are put into the die and applied to a 300 kN load for 60 s [17]. Then they are removed carefully, and the green target is formed. The 20 g target has been prepared, and it should be sintered at around 1600°C to harden and kept at a temperature of around 600°C for 1 h while the furnace cooled [18].

Sputtering process
The in-vitro examination was carried out by coating the titanium Ti-6Al-4V round disc (6 discs per sample) with the target samples T1, T2, and T3 it was subjected to a sputtering process. RF sputtering (Hind High Vacuum, Karnataka) is a plasma-based deposition process that develops a magnetized confined plasma on a target material's surface.
Positively charged energetic ions in the plasma interact with the negatively charged target material, ejecting or 'splattering' atoms and depositing particles on the substrate [19]. Different parameters, such as power, operating pressure, and substrate-to-target distance, were employed using magnetron sputtering equipment in figure 3. The spray chamber pressure was maintained at the base pressure is less than or equal to 1.55 × 10 -5 Torr, and the operating pressure is 1.5 × 10 -3 Torr. Then, argon gas was injected into the instrument as an aerosol at a rate of 160 cm 3 min −1 .
The surface was prepared using magnetron sputtering at a power of 160 W. During sputtering, the power given to the targets was increased by 10 W over 3 min and then retained at 160 W till a sputtering was completed [20]. The target is loaded in a concerned holder. The substrate, i.e., Ti-6Al-4V disc, is loaded on the topmost part of the machine. A high vacuum is created after 30 min, and plasma is generated using RF sputtering. To achieve sputtering 200-240 mA range of power is used. Thus, the target ions get deposited on the substrate. The coating is carried out for an hour. Further denoted as coated samples S1-titanium plate/disc coated with Ca 3 (PO 4 ) 2 , sample S2 -Ti plate/disc coated with Ca 3 (PO 4 ) 2 + ZnCl 2 , Sample S3 -Ti plate/disc coated with Ca 3 (PO 4 ) 2 + ZnCl 2 + AgNO 3. The coating thickness and microstructural were examined using a Scanning Electron Microscope (SEM), and Energy-Dispersive x-ray (EDX) analysis was used to investigate element distribution.  Atomic Force Microscope (AFM) was employed to investigate three-dimensional roughness characteristics (nm).

Microbiological test
The antimicrobial property of the suggested coated disc was tested using the most common infectious agents responsible for tooth damage namely S. aureus and S. epidermidis [21]. In this current study, S. aureus (ATCC 29 213) and S. epidermidis (MTCC 4350) were procured from American Type Culture Collection and Microbial Type Culture Collection centers respectively, and maintained in Nutrient broth containing 5 g sodium chloride, 5 g peptone, 3 g beef extract in 1 l of ddH 2 O at 37°C. The fresh exponential culture was used to prepare a standard row ranging from 1 * 10 5 CFU ml −1 and 1 * 10 7 CFU ml −1 . Every 30 min, the OD 600 was measured as the plates were cultured while being shaken (100 rpm) at 37°C. To conduct further analysis, it was determined how long it took to reach a 0.1 threshold, and the calibration was used to calculate the initial bacterial concentration [22]. All the chemicals used for nutrient broth preparation were obtained from HiMedia, Mumbai. The initial pH of the Nutrient broth was adjusted to 7.2, and both cultures were grown till they reach the log phase [23]. The solutions were treated with coated titanium discs to evaluate the suggested coating powder's antibacterial behavior.
The absorbance of both strains of cells was measured at 620 nm after they were grown on 12 nm well polystyrene microtiter plates. A 1.5107 cell ml −1 cell suspension was injected in nutritional broth, and the plate was heated at 37°C for 72 min to allow the cells to attach to the surface [24]. Washing the wells with sterile 1X PBS eliminated the non-adherence cells. Then, 500 μl of 0.02 percent crystal violet stain was applied for biofilm formation measurement. Finally, the crystal violet suspension was added to other 96 well plates after being dissolved in 150 μl of 33% acetic acid. This process is to verify the incubation of bacteria to the plates, and this layer is called biofilm [25].
The term biofilm is the extra layer formed on the disc after incubation of the pathogen for three days. The subsequent procedure is used to examine this layer using an SEM microscope. The reacted discs are immersed in 25% glutaraldehyde overnight at 4°C. The specimens are rinsed thrice with 100 μl of 0.1% osmium tetroxide for 30 min at 4°C [24].
The discs are subjected to a dehydration process for different ethanol concentrations (50% −100%) for five minutes [24 31]. At room temperature, the discs are dried, and they can view under SEM to analyze the bacterial formation. Figures 5(a), and (b) represent the SEM images of the biofilm formatted on the discs, and it means the identification of biofilm formation by reacting the discs with 500 μl of 0.02% crystal violet stain [26]. The adhesion of bacteria to the device surface is a critical stage in the pathophysiology of implant-related illnesses. Methods of creating and manipulating material qualities to decrease bacterial formation and hence control microbial activities are additional appealing techniques than standard methods that release or inject antibiotics or biocides (e.g. catheter lock solutions) [27]. The creation of functional surfaces through topographical modifications (e.g. surface texturing) is a promising technique to reduce initial microbial attachment and the frequency of persistent infections on implant surfaces [28]. Surface modification with micro-or nanoscale features reduces surface contact area and affects surface energy, and this strategy has been demonstrated to be effective in limiting bacterial adherence and biofilm formation. It indicates the number of bacteria accumulated on both coated and uncoated discs for 72 h with bacterial pathogens, and it was also washed thrice with phosphate solution [29].

Results and discussion
3.1. Coating thickness and interface microstructure 3.1.1. Scanning electron microscope SEM, FTIR, AFM, and an EDX were used to examine the coated plates' structural integrity. EDX is used to explore the chemical element distribution in the coated disc. The SEM image of the surface shows that the titanium substrate coats the bioactive materials well, traps them on the titanium surface layer, and binds (there is no space) them to the titanium substrate.
However, the research found that dip coating with bioactive compounds resulted in poor substrate adhesion and the absence of some mechanical properties, in addition to the problem that other coating processes typically face. [30]. The coating is smooth, dense, and hard, with no inherent problems (voids and cracks), and has excellent adherence to the titanium substrate, as seen by SEM pictures in figure 6. The difficulty of metallic implants mixing harmoniously with the surrounding bone is a common drawback. The incompatibility of metallic implants with natural bone is due to challenges in the chemical composition of the two materials.
It is used to increase the stability and osseointegration of dental implants by coating them with a bioactive material such as Ca 3 (PO 4 ) 2 , which has a chemical content similar to bone. The surface treatment has been demonstrated to improve osseointegration and control metal-based implant limitations, including releasing toxic ions, wear, and corrosion [31]. Surface modification is the optimal solution in implant dentistry. A smooth and crack-free coating has great potential for biological connections between synthetic alternatives and living tissue. This finding revealed that bioactive material was the coating's primary ingredient phase. Furthermore, the coating material's chemical composition did not modify during sputtering.
The layer thickness of three titanium coated samples was determined using SEM cross-sectional images in this work. The average thickness of the 3-coating samples is 205.8 nm shown in figure 7. Even though a thick layer allows for more time for osseointegration, earlier research has found that the film cracks at a thickness of 185 to 290 nm. The appropriate layer thickness for osseointegration is between 190 to 245 nm [32].
According to EDX analysis of the substrate's cross-section, the hydroxyapatite particles were integrated with the substrate with no gaps in the interface. Ca, P, Al, V, Zn, and Ag, the primary components of the coated substrate, were found in the deep microstructure (not the interface line) of a typical cross-section of the coating and titanium interfaces shown in figure 8. It shows the presence of elements in the titanium disc. These elements also represent the bonding information and strength between the particles. In these elements, the coated disc has higher vanadium, and calcium components, and it produces a stronger bonding and thick coating over the disc. When EDX mapping of the substrate's cross-section is employed with an implant surface, the biocompatibility of titanium devices increases, which may aid in osseointegration [33]. The elemental composition of the titanium disc in weight % is presented in table 1.

Fourier transform infrared spectroscopy
FTIR analysis uses absorption of radiant energy in transmitting and vibrating mode for functional group determination. Its primary function is to identify organic substances such as water, sulphate, nitrite, phosphate, carbonate, nitrate, silicones, ammonium ions, silicon dioxide, and other inorganic substances that can be detected with this technique. In figure 9 displays composite FTIR spectra as well as the peak of absorption for different composites. This method is mainly employed to detect intermetallic compounds such as impurities, uncured monomers, and oxides. [34]. An infrared beam containing frequencies ranging from 500 Hz to 1000 Hz is sent to the sample 500 cm −1 to 4000 cm −1 .
The samples will absorb different frequencies depending on the functional group or connection between the atoms. Figure 9 indicates that IR light absorbs very little, showing that Cu, P, V, and Ag are pure after the sputtering procedure. This demonstrates that bio-coating has been achieved. The peak at 513 cm −1 shows the vibrational frequency of Ag-O ionic bonding groups [34]. Symmetric and asymmetric stretching vibrations of [PO4] 3− groups appear at 1060 and 1030-1096 cm −1 , respectively [28]. Stretching of (CO 3 ) 2− occurs between 1400-1500 cm −1 . Metal oxides and metal OH bonds (based on the alloy type) occur at 1000−450 cm −1 on the alloy surface. The presence of carbonate groups was confirmed by the bands at 1420-1490 cm −1 and 875 cm −1 [31]. The additional bands at 1430 cm −1 , 1562 cm −1 , and 1660 cm −1 can be explained by combining the -OH, (CO 3 ) 2− , and -COOH groups. Due to the creation of C-O bond vibrations during the casting process, a spectrum can be seen between 1500 and 2000 cm −1 . This bond helps form a good coating on the implant and protects from bacterial inhibition [34].

Atomic force microscope
This work used AFM to investigate three-dimensional roughness characteristics (nm). The AFM image of the specimen exposed to plasma for 10 min was also obtained. A Nanoscope V (Veeco, USA) made the AFM recordings. Table 2 shows the changes in surface roughness, the mean values of the parameters (estimated from 3 to 5 images), and the standard deviations. Figure 10 and the data in table 2 clearly illustrate that after depositing the titanium substrate, the surfaces become smoother. However, sample S1's surface had some scratches and imperfections. Samples S2 and S3 have substantially smoother surfaces. While the average Ra parameter of sample S1 is 70.392 nm, it is only 47.832 nm in sample S3.
Similarly, the Rpv average of the peak-to-valley distance is 378.296 nm and 338.546 nm, respectively. The titanium with bioactive material coating had a significantly higher surface roughness than uncoated titanium substrates.

Biofilm formation
A qualitative micromorphological investigation of the specimens S1, S2, S3, S4 with Staphylococcus aureus and Staphylococcus epidermidis was performed using scanning electron microscopy. Figures 11(a) and (b) show the standard reference point of SEM images of the specific biomaterial samples. The microscopic images showed that more bacteria accumulated on an uncoated alloy than titanium disc coated with Ca 3 (PO 4 ) 2 + ZnCl 2 + AgNO 3 surfaces. The surface characteristics of the biomaterial are one of the factors that influence biofilm formation. The capacity of a clinical Staphylococcus aureus strain and a Staphylococcus epidermidis strain to produce biofilms was investigated using three distinct biomaterial combinations. Bacteria generated on all biomaterial specimens S1, S2, S3, and S4 tested. The S1 surface was more densely inhabited after 72 h than S2, S3, and S4 surfaces. The bacterial formations on the surface of S4 were discovered to be more distributed and horizontally spread than others on the other biomaterials studied.
Bacterial adhesion was influenced by surface roughness, electrostatic forces, and the chemical composition of the biomaterials utilized. As a result, studying biofilms developed on common dental biomaterials is critical for long-term effectiveness in treating implant-associated illnesses [27]. The number of organisms on biomaterial discs changed depending on the materials utilized. Peri-implant infections are mostly caused by Staphylococcus aureus and Staphylococcus epidermidis, which can lead to implant failure. Figure 11 shows the % of biofilm inhibition in the various samples. A median biofilm inhibition of 89 (70; 100) % for Staphylococcus aureus and 85 (60; 110) % for Staphylococcus epidermidis was obtained using the concentration of 500 μl of 0.02% crystal violet stain. Based on these results, the coated sample S3 surface using a concentration of 500 μl of  0.02% crystal violet stain was considered as the optimal process, and to produce a significant anti-biofilm effect on sample S3 (25%) was significantly lower than on other samples S1, S2, and S4 (>50%).

Bacterial adhesion
From the quantitative perspective, an examination of the data presented in figure 12 shows that bacterial adhesion increased over time for both the coated and uncoated discs. Depending on the substance utilized, the number of microorganisms adhering to biomaterial discs varied [35]. However, at all-time points, there is a considerable difference in the quantity of bacterial adhesion between the two strains examined, with Staphylococcus aureus bacterial adhesion (3.6 nm) and Staphylococcus epidermidis bacterial adhesion (1.5 nm) on the S3 surface. Surface treatment with nanoscale or nanoscale structures minimizes surface interfacial bonding and influences contact area, and this method is shown to be beneficial in restricting bacterial adhesion and biofilm development. These in vitro tests indicate that S3-coated surfaces have a lot of potential for usage in dental implanted devices. This helps to prolong the implant material's lifetime [36].

4. Conclusion
In this article's findings, the FTIR spectroscopy of all the coated plate/disc exhibits the same morphology across the entire infrared wavelength apart from a limited region, establishing bonding between active and passive elements. The spectrum describes the uniform coating of sample S3 because it is uniform. SEM, EDX, and AFM confirmed that bioactive material with different morphology and a uniform coating was formed on the Titanium surface. Bacterial adhesion on three commercial surfaces was tested, with comparable results (Staphylococcus aureus) or better (Staphylococcus epidermidis) than those obtained with laboratory-prepared surfaces, exemplifying the biosurfactant's ability to prevent the colonization of Dental implants, regardless of surface morphology. In vitro, antibacterial evaluation demonstrated a significantly lower amount of Staphylococcus aureus bacterial adhesion  (3.6 nm) and Staphylococcus epidermidis bacterial adhesion (1.5 nm) on the S3 surface. In conclusion, titanium implant coating with 50% Ca 3 (PO 4 ) 2 + 25% ZnCl 2 + 25% AgNO 3 proved to be a potential technique for extending the lifetime of dental implants while restricting staphylococcal adherence and minimizing biofilm formation on titanium surfaces. All the results demonstrate that perhaps the proposed materials are effective when applied in medicine as implants with compatible surfaces and that they are chemically like natural bioactive materials. The author(s) would like to thank Mepco Schlenk Engineering College, Sivakasi, and Madurai Kamaraj University, Madurai, for providing excellent facilities and constant support extended to carry out the research work.

Data availability statement
All data supporting this study's findings are included within the article (and any supplementary files).

Declaration of conflicting interests
The author(s) declared no potential conflicts of interest for the research, authorship, and /or publication of this article.