On the Ion Implantation Synthesis of Ag-Embedded Over Sr-Substituted Hydroxyapatite on a Nano-Topography Patterned Ti for Application in Acetabular Fracture Sites

Introduction

Acetabular fracture is a frequently occurring disease in the field of orthopaedics. Pathological acetabular fractures, produced by osteoporotic bone, result from insufficient mechanical stress. The prevalence of these fractures among the elderly has lately shown a significant rise, mainly attributed to the combination of increased overall mortality and a relative decline in trauma cases among younger persons.¹ The chances of a positive clinical result are reduced in elderly people due to impaired physiologic reserve and reduced healing ability treatments. The advancement of bone substitutes, innovative imaging techniques, and fixation treatments might improve the future treatment of acetabular fractures.² According to the literature, the need for orthopaedic implants among patients has grown substantially in the last few years. Therefore, it is imperative that patients with orthopaedic disorders be treated and their quality of life as a whole be increased, depending on the development of an implant device.³ Subsequently, metallic biomaterials, such as titanium (Ti) and its alloys, are highly advantageous for fixing bone fractures and joint prostheses for their outstanding biocompatibility, increased tensile strength, enhanced corrosion resistance, wear resistance, and low allergy risk, and they have been extensively used as biomedical implant material.^4,5 Consequently, there is a persistent demand for enhanced healing response and accelerated bone growth in the context of acetabular fractures.⁶ To attain significant osseointegration between the implant surface and the bone, it is necessary to modify the surface of the implants.⁷ The application of an etching solution to the Ti-based material may result in the formation of an intricate and consistent arrangement of nano-sized pits on the surface, which can drastically improve the osteogenicity of the implant.⁸ The modified surface exhibits a notable resistance to corrosion, the capacity to adsorb proteins, the ability to stimulate the growth of bone cells, and ultimately ensures osseointegration between bone-to-implant contact. Additionally, it inhibits the formation of fibrous tissues to enhance osteoblast activity.⁹

Various techniques, such as plasma spray, electrochemical, sol-gel, sputtering, biomimetic, laser cladding, etc., are used to develop coatings on implant surfaces.^10–12 However, these techniques have certain limitations in various aspects. Therefore, there is a need to develop a novel coating that possesses exceptional capability to enhance osteogenicity and suppress bacterial activity without any adverse effects. Although plasma spray is a state-of-The-art technique, it generates thick coatings with various phases exhibiting mixed crystallinity and different dissolution rates of the coating composition.¹³

Furthermore, a bacterial infection might impact implant failure because a developed bacterial biofilm on the implant surface may render antibiotics ineffective. Hence, it is essential to develop surfaces that prevent the attachment of bacteria and enable the gradual release of antimicrobial agents that can effectively disrupt bacterial biofilms. To prioritize the development of such surfaces is essential to ensure the safety and effectiveness of implants.¹⁴

Consequently, the observed antibacterial properties of silver (Ag) have resulted in its widespread application in healthcare. Ag nanoparticles (AgNPs) are increasingly used in orthopaedic implants and treat implant-associated infections because of their ability to penetrate and destroy bacterial cells rapidly. It has been found from the literature that AgNPs can effectively prevent bacterial cells from further multiplying.¹⁵ Moreover, a study conducted by Zhang et al has shown that the incorporation of Sr and Ag to the titanium dioxide (TiO₂) coating has significant osteogenic and antibacterial properties.¹⁶ As stated earlier, HA is an essential component in promoting osseointegration at the interface of the bone and implant. However, the deposition of hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂, HA] onto titanium implant substrates via ion implantation has been suggested as a prospective strategy to enhance surface bioactivity and osteogenicity. As compared to plasma-sprayed HA coating, which may lead to issues such as the release of coating debris, infiltration of macrophages, encapsulation by fibrous tissues and possible implant failure,¹⁷ the apatite surface created by ion implantation can significantly enhance the early formation of bone into porous surfaces. This technique allows for precise control over the ion concentration, distribution, and depth of calcium and phosphorus deposited on the surface, resulting in a significant increase in adhesion strength.^18,19 The utilization of metal-incorporated calcium-phosphate (Ca-P) ceramics in biomedical applications, particularly in the field of bone regeneration, has generated significant interest.²⁰ So, the synthesis of HA by ion implantation provides outstanding mechanical properties simultaneously exhibiting higher osteogenicity. While conventional ceramic coatings are unsuitable for load-bearing applications or for application in extensive bone defects because of their weak mechanical properties, HA nanoparticles formed by ion implantation can overcome these drawbacks.^21,22

Strontium (Sr) has undergone extensive research and is well recognized for its osteogenic properties, whereas the amount of Sr substituted in the Ca site of HA impacts the shape, structure of apatite, and crystallinity of Ca-P ceramics.^23,24 These characteristics, in turn, affect the solubility of the coating, the in vitro behaviour of osteoblasts and osteoclasts and the in vivo bone remodelling processes. Due to ongoing dissolution and precipitation, the SrHA surface exhibits reactivity and biodegradability, forming apatite resembling osseous tissue on its surface. SrHA stimulates the formation of new bone by bone marrow stem cells (BMSCs), minimizes the time required for bone healing, and improves the integration of implants with the surrounding bone tissue.²⁵

The present investigation focuses on the synthesis of Sr substituted HA on a rotating cpTi implant using Ca²⁺, P²⁺ and Sr²⁺ ions produced from a low energy ion beam linear accelerator and subsequently, Ag²⁺ was flash coated (low-dose implantation) after the formation of SrHA onto the nano-topography patterned cpTi surface. Furthermore, various analytical techniques were employed to assess the mechanical and biological properties of the specimens. The specimens were also modified using two different surface modification techniques, such as cathodic deposition (CD) and Low Temperature High Speed Collison (LTHSC) methods, and their properties were compared. Hence, this study provides invaluable insights into the surface structure design of biomedical implants that promote successful osteogenesis, making it an essential contribution to the biomedical fields.

Materials and Methods

Pretreatment of Ti Sheet

Commercially pure titanium (cpTi, grade 4) surface was polished in a single direction longitudinally to form nano patterns using 600–1200 SiC grit papers. cpTi substrates were etched in a mixture of 1 part HF, 4 parts HNO₃ and 5 parts water. Nano-topographical cpTi surfaces were further processed by boiling in a 10 M NaH₂ solution at 60°C for 24 h. Then, the substrates were washed with deionized water and dried overnight in a dark atmosphere at room temperature. The cpTi specimens were then sintered at 600°C at a linearly increasing temperature of 5°C/min for 1h to obtain the nano-topographical surface on Ti. Then, the substrate underwent ultrasonic cleaning using deionized water, acetone, and toluene. After drying in air, the Ti substrates were kept in a desiccator until they were coated with Ag-SrHA by ion implantation, CD and LTHSC techniques.²⁶

Fabrication of Rotating Specimen Holder for Ion Implantation Technique

The Ti substrate was rolled into a cylindrical shape and mounted on a rotating specimen holder driven by an electric motor. The idea behind the use of a cylindrical sample holder is to (i) synthesize multiple specimens in a single loading (cut into pieces after implantation), (ii) reduce specimen loading time- and (iii) enhance the line-of-sight area under 3D rotation of the specimen. Subsequently, Ca²⁺, P²⁺, and Sr²⁺ ions at different energies were implanted on its surface. Before cell culture, both the control and ion-implanted Ti were sterilized using ultraviolet radiation and kept in a desiccator at room temperature.

Ag Flash-Coated Sr-HA Deposition on Ti Surface by Ion Implantation (SrHA-Ag/Ti (Ion-Implanted))

Using a low-energy linear accelerator (50 keV Ion implanter, Source: Multi-cathode source of negative ions by cesium sputtering ion source), implantation of Ca²⁺, P²⁺ and Sr²⁺ ions was carried out. CaO was used as the source cathode material for the Ca²⁺ ions, while GaP was utilised as the source cathode material for the PO₄^3- ions and SrH₂ was used as the source cathode material for obtaining Sr²⁺ ions. A rotating cylindrical cpTi (purity 99.2%), with dimensions of Ф 9.6 mm × height 15 mm affixed on the sample holder was implanted with ⁴⁰Ca, ³¹P, and ⁸⁸Sr beams. A uniformly implanted area of 10 × 30 mm² was taken as the active implantation area. To synthesize SrHA, ion energies of 100 KeV for Ca²⁺ and, 75 KeV for P²⁺, 180 KeV for Sr²⁺ were used with a beam current density of 100 nA/cm², 200 nA/cm² and 230 nA/cm² for Ca²⁺, P²⁺ and Sr^2+, respectively. The purpose of choosing different ion energies was to implant the ions (Ca²⁺, P²⁺ and Sr²⁺) at the same implantation depth so that subsequent physical conditions would bring them together to interact. The vacuum of the implantation chamber was kept at 4 × 10⁻⁵ Pa during P²⁺ implantation. The Ca²⁺ implantation was carried out by backfilling oxygen at a pressure of 1.7 × 10⁻³ Pa in the implantation chamber to obtain CaO on the Ti surface. At the end of Ca²⁺ ion implantation, the maximum concentration of Ca²⁺ and the dose of Ca²⁺ (which is the number of implanted Ca²⁺ atoms/cm²) was enhanced in the Ti substrate with a fluence of 2×10¹⁶ Ca²⁺ ions/cm². In contrast, the fluence for P²⁺ was 1.32 × 10¹⁶ P²⁺ ions/cm² and for Sr²⁺ it was 0.2 × 10¹⁶ Sr²⁺ ions/cm²). Because of the O₂ partial pressure, TiO₂ and CaO have been formed onto the Ti substrate during implantation, resulting in the sputtering yield dropping below one (the number of sputtered ions is less than the implanted ions). Similar was the case for P²⁺ ions that formed PO_4, and Sr²⁺ ions formed SrO in the process of O₂ backfilling. Subsequent heating of the implanted Ti substrate at 80°C for 3h, Ca and Sr react with O₂ and H₂O, forming Ca(OH)₂ and Sr(OH)₂. In this condition, the surface contains CaO, SrO, Ca (OH)₂ and Sr (OH)₂ as the major components implanted in the TiO₂ matrix. In the present phase, these finely dispersed amorphous phases are in a ready-to-react form for the synthesis of SrHA in subsequent annealing. After annealing this implanted Ti substrate at 600°C for 5 min in a fast-annealing furnace, a crystalline phase of SrHA was produced. Annealing the Ti substrate had three beneficial effects: the first one was the formation of SrHA, the second one was conversion from an amorphous phase to a crystalline phase, and the third advantage was to overcome the implantation damage. Since amorphous SrHA has detrimental effects in a Ti implant surrounded by body tissues, the present crystalline form of SrHA is beneficial for bioimplant applications.^26,27

Ag²⁺ ions of energy 100 KeV were implanted on the as-formed SrHA ion-implanted Ti surface with a concentration as low as 1.1 × 10¹⁵ ions/cm² that can act as an antibacterial agent. The Ag²⁺ ions were implanted at a depth of 35 nm from the surface of Ti. The whole process of implantation of Ca²⁺, P²⁺, Sr²⁺, and Ag²⁺ ions was carried out over a span of 196 hours, excluding the post-implantation treatments.

Depth Profile Analysis

The maximum depth profile was measured at 70 nm, 69 nm and 75 nm for Ca²⁺, P²⁺, and Sr²⁺ ion implantation, respectively, whereas the maximum range approached 30 nm, 32 nm, and 30 nm for Ca²⁺, P²⁺, and Sr²⁺ ion implantation as depicted in Figures 1–3 respectively. The depth profile calculations were carried out by SRIM software.²⁸ Under modified vacuum conditions, the depth profiles of the implanted ions were drastically changed.

Figure 1 (a). Depth distribution of Ca ions inside the Ti and (b) range of ions inside Ti.

Figure 2 (a). Depth distribution of P ions inside Ti and (b) range of ions inside Ti.

Figure 3 (a). Depth distribution of Sr ions inside Ti and (b) range of ions inside Ti.

Preparation of [SrHA-Ag/Ti (LTHSC)) and (SrHA-Ag/Ti (CD)]

The LTHSC technique was used to coat the cpTi surface with Ag-SrHA powder, which had been prepared through a wet chemical process. A straight cylindrical nozzle was used to discharge nitrogen gas, released from a cylinder operating at 10⁻³ torr, known as the carrier gas. The SrHA powder was introduced into a powder hopper, which was then carried by a nitrogen carrier gas. The gap between the nozzle and the substrate was maintained 1 cm apart. Allowing the Ag-SrHA powder to collide with the cpTi surface via the spray nozzle resulted in the formation of Ag-SrHA coating layers.²⁹ Similarly, Ag-SrHA deposition on cpTi was carried out using electrochemical deposition as described in the literature.^30,31

The present investigation focuses on the synthesis of Ag-incorporated SrHA on a Ti substrate, their adhesion strength and biological behaviour. So, in comparative analysis, only the biological studies pertaining to CD and LTHSC specimens have been carried out.

Physical Characterization

Scanning Electron Microscope (SEM)

Using a scanning electron microscope (SEM, JSM-6700FM, JEOL, Japan), the morphological characteristics of the cpTi surface and SrHA-Ag/Ti (ion-implanted) have been examined.⁴

X-Ray Photoelectron Spectroscopy (XPS)

The elemental profile of ion-implanted Ti surface was determined by employing XPS (MICROLAB-350, Britain) with A1-Kα (1486.6 eV) as the excitation source.³²

Fourier Transform Infrared Spectroscopy (FTIR)

Bruker FTIR Spectrophotometer (Alpha model) was used to conduct Fourier Transform Infrared Spectroscopy (FTIR) on the as-formed specimens to determine the functional groups of the coating composition. An infrared (IR) beam is reflected from the titanium surface at various (almost close to glancing) angles of incidence as part of the measuring procedure. Two different IR beams were used to record the spectrum: one polarised perpendicular and the other polarised parallel to the surface of the mounted specimen. On the basis of the ratio of S to N (signal-to-noise ratio), multiple averaged computer scanning was performed for the specimen during the data-acquiring process. At a resolution of 4 cm⁻¹, the spectra have been obtained in the spectrum of 4000–400 cm⁻¹; all generated spectra have been computed with double-digit accuracy.^33,34

Contact Angle

Measurement of the contact angle of the as-formed specimens with simulated body fluid (SBF) at pH 7.2 and Dulbecco’s modified eagle’s medium (DMEM) (cell culture media) was carried out using the contact angle setup (VCA optima, USA). The angle of contact of the stationary liquid droplets with the specimen substrates was quantified.³⁵

Adhesion Strength

The adhesion strength of the coatings on the specimens was assessed using a universal material testing machine (CMT4204, Sans Material Test Instruments Co., Ltd.) at a crosshead speed of 2 mm/min. Maximum load was measured when the coating was peeled out of the substrate. The adhesion strength (σ) was determined using the following formula:

,

where “a” is the contact area of the coating with the stainless steel substrate, and F is the highest load when the coatings were peeled.³²

Cell Culture

MC3T3-E1 pre-osteoblast cells (NCCS, Pune) were cultured in DMEM (GIBCO) medium supplemented with 10% fetal bovine serum (GIBCO), 100 mg mL⁻¹ of streptomycin, and 100 units mL⁻¹ of penicillin. The mixture was then incubated in a humidified atmosphere with 5% CO₂ at a temperature of 37°C. The cultured media was replaced with fresh media to eliminate non-adherent cells on every alternative day for two weeks.³⁶

Live/Dead Osteoblast Cell Analyses

The viability of MC3T3-E1 pre-osteoblast cells was determined using a Live and Dead viability/Cytotoxicity Kit (Molecular Probes, USA). Then the specimens were incubated for 7 days in standard culture conditions and were washed 3 to 4 times and treated with 2.5% glutaraldehyde (Solarbio Company, Beijing, China) solution for 2h at 4°C. The specimens were then transferred to a new 24-well plate. After rinsing with PBS twice, they were left to stain at room temperature in a dark environment. Again, the specimens were added with 1 mL of a prepared solution (consisting of 2 µM calcein AM and 4 µM EthD-1) and incubated for 10 min in the dark. The fluorescence microscope (Olympus, Tokyo, Japan) was used to analyze the Live/Dead osteoblast cells qualitatively.³⁷

Osteoblast Cell Viability Test by MTT Assay

The cell viability was assessed by culturing them on specimens for 1, 7, and 14 days. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to quantify the functionality and viability of mitochondria in the cells. The tetrazolium salt MTT undergoes conversion by mitochondrial succinic dehydrogenase of live cells, resulting in the formation of a dark-blue insoluble compound known as formazan. A solution of MTT (5 mg/mL, Sigma Aldrich) was added to each well, and the cells were incubated at 37° C for 4 h. After the removal of growth media, dimethyl sulfoxide (DMSO) was put into each well to facilitate the dissolution of cells. Cell viability was determined by quantifying the optical density of the coloured substance in the solution using a spectrophotometer (Microplate Reader EL 308; Bio-Teck Instrument, Winooski, VT) which was configured to measure at a wavelength of 450 nm. The blank reference was extracted from wells without cellular structures.^29,38

Gene Expression Using Quantitative Real-Time Polymerase Chain Reaction (qRTPCR)

The osteogenic gene expression of MC3T3-E1 pre-osteoblast cells was assessed using a real-time polymerase chain reaction (qRT-PCR; Bio-Rad Laboratories) to quantify the mRNA expression of marker gene levels such as Runt-related transcription factor 2 (RUNX2), type-1 collagen (COL1), and osteocalcin (OCN). To extract RNA, cells were seeded at a density of 4 × 10⁴ cells/well for 1, 7, and 14 days, then lysed with TRIzol (Invitrogen). Using a superscript II first-strand cDNA synthesis kit (ThermoFisher), 1 mg of RNA was reverse-transcribed into complementary DNA (cDNA).³⁹ The forward and reverse primer sequence has been depicted in Table 1.

Table 1 Forward and Reverse Primer Sequences

Bacterial Viability

The antibacterial properties of composites were tested using S. aureus, the most prevalent gram +ve bacteria that causes osteomyelitis. Bacteria viability was evaluated using the MTT test in vitro. The bacteria were cultured in a Mueller-Hinton Broth (MHB) medium. After that, the as-formed specimens were incorporated into the centre of a 24-well plate and put in a CO₂ incubator at 37°C with 1 mL of 1 × 10⁶ CFU/mL bacterial suspension in each well. The bacteria culture was carried out for time periods of 10 h, 20 h and 30 h, and the adherent bacteria were evaluated for bacteria viability test by adding 150 μL bacteria suspension with 150 μL MTT solution (5 mg/mL) and incubated at 37°C for 6 h. The experiment was conducted three times for each test. The bacterial viability was assessed by measuring the optical density (OD) of the substrate solution.⁴⁰

Statistical Analysis

The experiments were performed (n = 3 for contact angle and adhesion strength and n = 5 for other biological studies), and the outcomes were expressed as mean ± standard deviation for the synthesized coatings. The statistical analyses were conducted using one-way Analysis of Variance (ANOVA) with Tukey’s multiple comparison tests (Prism, version 5.0). A significance level of p < 0.05 was chosen to determine the statistical significance of the observed differences between the groups.

Results

Scanning Electron Microscopy Study

The morphological attributes and surface nano topography of (a) cpTi, as well as (b) SrHA-Ag/Ti (ion-implanted) were illustrated in SEM micrographs (Figure 4). Both the cpTi and SrHA-Ag/Ti (ion-implanted) surface exhibited almost similar surface nano topography with visible coating in the later surface.

Figure 4 Morphological attributes and surface nano topography of (a) cpTi, as well as (b) SrHA-Ag/Ti (ion-implanted).

X-Ray Photoelectron Spectroscopy Study

With the aid of XPS, the elemental profile of the surface was estimated, and elements, such as Ca, P, Sr, Ti and O were detected (Figure 5).

Figure 5 The elemental composition of ion-implanted Ti spectrum using XPS.

Fourier Transform Infra-Red Spectroscopy Study

FTIR spectrum showed the formation of SrHA with several unique absorption peaks as shown in Figure 6. The PO₄³⁻ (544 cm⁻¹, 614 cm⁻¹, and 1033 cm⁻¹) and OH⁻ (1654 cm⁻¹ and 3491 cm⁻¹) functional groups are ascribed to the presence of SrHA.

Figure 6 FTIR spectrum of as-fabricated specimen.

Contact Angle

The contact angles of the synthesized specimens were quantified using two separate measuring fluids, DMEM and SBF. The results were then compared to cpTi, as depicted in Figure 7. The ion-implanted SrHA-Ti demonstrated a reduced angle of 35° and 40° in SBF and DMEM. The contact angles for the cathodically deposited SrHA-Ti and LTHSC deposited SrHA-Ti implants were 43°, 41° for SBF and 45°, 49° for DMEM, respectively.

Figure 7 Contact angle of as-fabricated specimen. *p < 0.05, denotes a significant difference.

Adhesion Strength

The adhesion strength of the ion-implanted specimen was 43 ± 2.3 MPa, while that of the specimen with cathodic deposition had an adhesion strength of 6.3 ± 0.31 MPa, and the LTHSC deposited specimen showed a strength of 19 ± 0.92 MPa. The adhesion strength of the ion-implanted specimen was found to be significantly higher than the other two specimens (Figure 8).

Figure 8 Adhesion strength of as-fabricated coatings. *p < 0.05, denotes a significant difference.

Cell Proliferation

The MTT assay was used to evaluate cell proliferation on the specimens at 1, 7, and 14 days of incubation (Figure 9). The as-formed specimens had significantly higher cell proliferation than cpTi at all points of the incubation period. The cell proliferation on the SrHA-Ag/Ti (ion-implanted) surface was considerably higher than that of SrHA-Ag/Ti (CD) and SrHA-Ag/Ti (LTHSC). However, there was no significant difference in cell viability between SrHA-Ag/Ti (CD) and SrHA-Ag/Ti (LTHSC) specimens.

Figure 9 MTT assay using osteoblast cells. *p < 0.05, denotes a significant difference.

Bacterial Viability

The antibacterial activity was assessed by examining the viability of S. aureus (Figure 10). Antibacterial efficacy was measured after incubating the Ag-incorporated SrHA specimens at 37°C for 24h. On Ag-incorporated SrHA, the viability of S. aureus was reduced by around 40–60%. The presence of Ag in the Sr-HA ion-implanted surface resulted in a decrease in S. aureus by about 60%, indicating a similar pattern of 40% and 30% reduction, respectively, in specimens synthesized by cathodic and LTHSC deposition.

Figure 10 Bacteria viability on as-fabricated specimens. *p < 0.05, denotes a significant difference.

Live/Dead Cell Fluorescence Analysis

The qualitative Live/Dead cell analyses of the as-formed coatings were visualized using a fluorescence microscope to understand the activities of osteoblast survival status (Figure 11). It was found that a number of MC3T3-E1 cells had grown on all specimens as the culture time increased, suggesting that the SrHA surfaces induce a conducive environment for cell adherence, differentiation and proliferation.

Figure 11 Fluorescence micrographs of osteoblast cells after 7 days of culture.

Gene Expression Studies Using qRT-PCR

The levels of gene expression associated with osteogenesis on the as-formed specimens were assessed using qRT-PCR (Figure 12). The number of viable osteoblast cells on all the as-formed specimens on days 7 and 14 was significantly high (p > 0.05). As seen in Figure 12a, on day 1, gene expression of RUNX2 of SrHA-Ag/Ti (ion-implanted) was significantly higher than the control cpTi and the coating deposited by CD and LTHSC techniques. However, on day 7, no noticeable difference was observed among the as-formed specimens along with the control cpTi. On day 14, SrHA-Ag/Ti (ion-implanted) specimen exhibited significantly higher RUNX2 gene expression than others. Further, on day 14, gene expressions of OCN (Figure 12b) and COL1 (Figure 12c) were significantly high on day 14, and there was a significant increase in the expression of the OCN gene on SrHA-Ag/Ti (ion-implanted) that leads to improved osseointegration.

Figure 12 Analysis of osteogenic relative gene expressions of (a) RUNX2, (b) COL1, and (c) OCN. *p < 0.05, denotes a significant difference.

Discussion

Prior studies have investigated the efficacy of ion implantation as a method to enhance the osseointegration of Ti implants. However, surface treatments have mainly focused on modifying surface properties, including surface chemistry, roughness, and energy. Therefore, it is essential to optimize the biological and mechanical properties of a metallic implant to obtain an ideal bioimplant surface.⁴¹ Although plasma spray is a state-of-The-art technique for surface modification of Ti implants, it has several disadvantages that include various phases of calcium phosphate in the coating, giving rise to surface delamination and changes in the crystal structure of the matrix, subsequently releasing the coated nanoparticles that may cause inflammatory reactions.⁴² To overcome these drawbacks of plasma spray, the conventional ion implantation technique has been utilized in the present study, which is supposed to unalter the nano-topography patterned on the Ti surface without changing its surface chemistry.^13,43 The primary drawback of conventional ion implantation is that it is a line-of-sight process limited to the implantation of two-dimensional surfaces. However, with the use of a rotating sample holder, this problem has been largely solved in the present investigation.⁴⁴ Ion implantation technique has not been found to affect the texture of the surface. Nano-topographies may directly correspond to the size of protein and membrane receptors, which could enhance osteoblast differentiation and tissue regeneration.⁴⁵ On the other hand, considering the importance of nano-topographical features, the synergistic effects of dual-length scale materials show promising results in improving osseointegration.⁴⁶

In the present investigation, once the coating was formed on the patterned surfaces using cathodic and LTHSC techniques, the nano-topographic patterns were concealed by the Ag-SrHA coating formed on them.⁴⁷ On the other hand, in the ion implantation technique, the coating formed was a gradient layer conglomerated with the composition of the patterns as well as underneath the cpTi surface.^48–50 Thus, the efficacy of nano-topography patterned surfaces in interacting with the osteoblasts is retained. These treatments bring out changes in surface topography by a sputtering process while leaving the surface chemistry unaltered.⁵¹ On the other hand, the gradient layer accounts for the high adhesion strength of the coating, giving rise to a coating with long-term stability. This study has investigated the impact of nano-topography patterned surfaces on osteoblast and bacteria cells after impregnating these surfaces with osteogenic and antibacterial agents using ion implantation technique, and the biological studies on this surface have been compared with Ag-SrHA coated specimens synthesized by cathodic deposition³⁰ and LTHSC technique.²⁹

It was evident from the XPS spectrum that SrHA-Ag coatings have been formed on the nano-topography patterned cpTi surfaces. While in both CD and LTHSC techniques, Ag²⁺ and Sr²⁺ have substituted the Ca site of HA inside the apatite lattice, Ag²⁺ has been flash-coated on ion-implanted SrHA coating. Since Sr and Ca have similar chemical and physical properties, substituting Sr at the Ca site in HA is an elementary procedure. Subsequently, the FTIR result validated the presence of PO₄³⁻ and OH⁻ band peaks, as depicted in Figure 6. The peaks associated with the PO₄³⁻ group resulted from the symmetrical P–O stretching, while the peaks assigned to the OH⁻ group were generated due to the stretching modes of O–H bands.^52,53 Apart from the PO₄³⁻ and OH⁻ groups, no impurities or additives were detected, indicating that the HA phase has been formed. Moreover, depth analysis using SRIM software^9,54 demonstrated that several ranges of gradient layers were formed on the cpTi surface by the deposition of Ca²⁺, Sr²⁺, and P²⁺. Even though the dosis of the ions were calculated to obtain (Ca+Sr)/P ratio of 1.67, from proton-induced X-Ray emission (PIXE)⁵⁵ spectroscopic analysis, the (Ca+Sr)/P ratio was found to be 1.615, which is almost close to the ideal Ca/P ratio (1.67) of HA.

On the other hand, surface wettability is one of the most important attributes that affect cell adhesion, protein adsorption, blood coagulation, platelet adhesion/activation, and tissue growth for biomedical applications.⁵⁶ It is an important factor in the evaluation of hydrophilic characteristics and, therefore, the attachment and proliferation of osteoblast cells. It has been found that surface roughness may impact hydrophilicity, which is a crucial factor for the nucleation and cell development events that lead to osseointegration.^57,58 The key findings of this study indicate that the surface of the SrHA-Ag/Ti (ion-implanted) specimen is mostly hydrophilic in nature as compared to the other two modified surfaces, such as SrHA-Ag/Ti (CD) and SrHA-Ag/Ti (LTHSC). The decreased contact angle of the SrHA-Ag/Ti (ion-implanted) may be attributed to the effect of nano-topography patterned surface that remains intact even after ion implantation. Moreover, the drawback associated with acetabular displacement is the interfacial bonding strength between the substrate and the implant.^22,59 As per ASTM standard F1147, the minimum tensile strength of a Ca-P coating must be 34.5 MPa, and it has been achieved only by the ion implantation technique.⁶⁰ Upon analyzing the adhesion strength, it was observed that the specimen obtained through ion implantation exhibited the maximum bonding strength when compared to the specimens obtained using CD and LTHSC techniques. Moreover, in the ion implantation technique, the implanted ions form a gradient layer inside the Ti matrix. Hence, surface delamination problems do not arise. The presence of nano-topography patterns in the SrHA-Ag/Ti (ion-implanted) surface enhances the interface adhesive force between the cpTi surface with osteoblast cells. Although all three investigations assessed the antibacterial properties of Ag²⁺ ions and osteogenicity through the formation of osseous tissues by MC3T3-E1 cells cultured on SrHA-Ag/Ti surface, ion implantation techniques exhibited superior properties.

For developing novel coatings having both antibacterial and pro-osteogenic characteristics, bi-functionalization of Ti implants has been adopted. The deposition of osteogenic agents on Ti implants favours surface osteogenic properties.⁶¹ Several in vitro investigations have shown that Sr promotes bone formation by activating the calcium-sensing receptor and inhibiting bone resorption by uplifting osteoprotegerin (OPG) and reducing receptor activators of nuclear factor kappa β ligand (RANKL).^40,62 OPG, a protein generated by osteoblasts, inhibits RANKL-induced osteoclastogenesis by acting as a RANKL decoy receptor. The OPG/RANKL ratio consequently plays a key role in regulating bone resorption and osteoclastogenesis. The calcium-mimicking structure of Sr stimulates the CaSR/PI3K/Akt signal pathway, preventing GSK3 production. This promotes the expression, nuclear localization, and transcriptional activity of β-catenin. Additionally, Sr may enhance the expression of RUNX2, ALP, OCN, and COL1 via upregulating Wnt5a pathway.⁶³ The antibacterial properties, along with osteogenicity, are important parameters for an implant to be used for acetabular fracture. Ag is a broad-spectrum antibacterial and antifungal agent that can even inhibit methicillin-resistant S. aureus strains.⁶⁴ It has been reported that AgNPs can readily act against bacterial cells more effectively than their micron-sized counterparts. Moreover, the size and shape of the AgNPs play a major role in bactericidal action. Morones et al⁶⁵ reported that AgNPs of 1–10 nm diameter exhibited optimum bactericidal action.⁶⁶ From recent studies, it has been observed that the nano-Ag released from the surface of the implant penetrates the bacterial cell walls, which leads to membrane damage, and subsequently interacts with sulfur and phosphorus-containing biological components, including DNA, RNA, proteins, and enzymes by resulting in the production of reactive oxygen species (ROS). Increased ROS induces an apoptosis-like response, DNA damage, and lipid peroxidation, ultimately resulting in the death of the bacteria.⁶⁷ However, its application in the biomaterials field was compromised because of its cytotoxicity resulting from excess concentrations of Ag. Additionally, it should be considered that higher concentrations of Ag might have cytotoxic effects on mammalian cells. A maximum concentration of 0.7% of Ag has been found to be non-toxic for biomedical implants.⁶⁸ Hence, in the present investigation, we have taken the concentration of Ag to be 0.5%. According to the bacterial viability results, SrHA-Ag/Ti (CD) showed reduced bacterial cell viability as compared to SrHA-Ag/Ti (ion-implanted) and SrHA-Ag/Ti (LTHSC) surfaces. CD and LTHSC specimens resulting in lower bacterial viability may be ascribed to the presence of Ag in HA lattice structure, which leads to better antibacterial activity. On the other hand, ion-implanted specimens involve flash coating Ag on its surface resulting in comparatively lower antibacterial activities. This may be due to less available Ag ions because of their attachment to the Ti lattice. Moreover, since the Ag ions were implanted in the near-surface regions of Ti, there would be a higher loss of these ions by sputtering, giving rise to less available Ag. However, the release of Ag persisted spontaneously from the specimens prepared by other techniques.

To investigate cell differentiation, certain gene markers were investigated such as COL1, which primarily contributes to the extracellular matrix (ECM) of bone; RUNX2, which is expressed during osteoblast formation; and OCN, which is a late indicator of osteogenic differentiation.⁶⁸ The current investigation provided strong evidence that all of the markers for osteogenic gene expression were significantly higher in the SrHA-Ag/Ti (ion-implantation) specimen than in the other two modified surfaces. Braux et al discovered that the presence of Sr²⁺ ions stimulates the growth of human primary bone cells. Furthermore, the osteogenic gene expression in the presence of Ag ions indicates that the cytotoxic effect of the Ag ions did not affect the osteoblast cells.⁶⁹ Additionally, the MTT assay study confirmed that there is an increase in cell viability on all three substrates (SrHA-Ag/Ti (CD), SrHA-Ag/Ti (LTHSC), and SrHA-Ag/Ti (ion-implanted)), exhibiting their cytocompatibility nature. In a similar study, Lee et al²⁹ found that there was no significant difference in cell proliferation between the control (Ti) and the HA-coated Ti surfaces via the LTHSC technique. Additionally, a study by Lafzi et al,⁷⁰ measured osteogenic gene expression in MC3T3-E1 cells on nano-HA-coated Ti substrates. Their results showed better expression on day 7 as compared to day 14, suggesting better gene expressions. Another study demonstrated that SrHA-Ag coatings, along with silicon co-doping, exhibited superior biological characteristics as compared to HA and HA-Ag coatings while maintaining similar anti-bacterial effects on HA-Ag coating.⁷¹ The live/dead fluorescence staining on all the specimens exhibited their cytocompatible nature, and Ag is shown to have the least effect on the growth of osteoblast cells. Simultaneously, it can be observed that there is the highest number of live cells in the SrHA-Ag/Ti (ion-implanted) specimen. Therefore, the surfaces that were ion-implanted with SrHA-Ag/Ti were found to be suitable for cell adhesion and proliferation. Geng et al discovered that Sr doping to the HA coating layer on Ti implants can mitigate the cytotoxicity of Ag incorporated into the coating. By stimulating the osteoblasts and inhibiting the osteoclasts, the later is also known to disrupt the process of osteogenesis when incorporated. Nevertheless, the replacement of Sr in Ag/SrHA nanoparticles may effectively mitigate the adverse consequences of Ag and amplify the biological efficacy of HA. Therefore, the synthesized Ag/SrHA nanoparticles have the potential to be used in biomedical implants, specifically in orthopaedic and dental applications, due to their exceptional ability to promote bone growth and prevent bacterial infections.^72,73 The study demonstrated that the nano-scale pattern of the Ti surface has strong mechanical interlocking capability with surrounding bone, which may enhance the stability of the implant as compared to smooth surfaces. Moreover, the presence of nano/micron-patterned surface roughness enhances the process of protein adsorption, promotes the attachment of osteoblasts, helps in the development of osteoblasts into bone cells, and facilitates the integration of implants with surrounding bone tissues in living organisms.

Conclusions

In the present study, Ca²⁺, P²⁺, and Sr²⁺ were implanted into a nano-topography patterned Ti surface to synthesize Sr-HA/Ti; Ag was subsequently implanted on the as-formed specimen as a flash antibacterial coating. The surface topography of the ion-implanted surface retained its initial structure. The SrHA-Ag coating exhibited optimal osteoblast function while concurrently minimizing bacterial adherence. The inclusion of Ag in the SrHA coating showed a more effective ability to prevent infection caused by S. aureus bacteria. The presence of Sr had a beneficial effect on the surface of SrHA-Ag, leading to a considerable improvement in the attachment, proliferation, and differentiation of MC3T3-E1 cells. The adhesion strength of the ion-implanted coating was superior to the other two coatings and in accordance with ASTM standard protocol. Due to its enhanced adhesion, cytocompatibility, and strong antibacterial capabilities, SrHA-Ag/Ti (ion-implanted) may exhibit great promise for treating infectious bone defects and promoting the integration of bone to implant contacts in acetabular fossa defect sites. The findings of this research demonstrated that SrHA-Ag coating using the ion implantation method exhibits considerable promise as a viable substitute for the state-of-The-art plasma spray technique. The use of ion implantation to synthesize strontium-substituted hydroxyapatite is intriguing. Sr incorporation into HA can enhance bone regeneration and reduce osteoporosis risk. Combining SrHA with Ag²⁺ ions for antibacterial properties is novel. Few studies have explored this dual functionality. The synthesized specimens may be used as implant materials in critical body parts such as acetabular fracture sites, neurocranium or viscerocranium sites, involving high-cost implant applications. Further research may be conducted on the large-scale production of cost-effective SrHA-Ag coatings that can be used as a common implant material.