Development of Gallium Silicon Titanium Alloys for Dental Implants

Abstract

The aim of this study is to synthesize a new metallic aluminum and vanadium-free titanium alloy biomaterial for better osseointegration and implantation in the physiological system. The in vitro and in vivo methods were used to examine their biological compatibility, evaluated quantitatively and qualitatively. Results of Ga-Si-Ti alloy showed a higher ultimate tensile strength, yield strength and a higher percentage of elongation and more or less equal to Young’s modulus when compared with the Ti and Ti-Ga alloy. In vivo study, a PA view of whole-body radiography all groups exhibited a substantial difference in the linear bone density of newly formed bone. Ga-Si-Ti group showed the highest bone mineral density than Ti and Ti-Ga group in the micro CT ex vivo study. The study exhibited a significant difference between the groups and the proportion of cortical bone volume to trabecular bone volume BV/TV in percentage. This is related to the anti-resorptive action of gallium and osteoblastic property of silicon, in addition to the benefits of commercial pure-Ti alloy.

1. Introduction

1.1 Background

While the pride of humankind exists in a manner that helps different lives, having scientifically achieved the capability to replace the lost body parts along with their functions is considered to be the upfront of modern scientific civilization. The impact of the missing body parts and their dependence results in physical and psychosocial distress. The best assistance one can offer is to diminish, if not eliminate such an inability. In this direction, loss of teeth due to injury or any other pathology may lead to partial or complete edentulousness, that affects the psychosocial status and the functional mastication for which dental implants are preferred.

Study by the AAOMS reveals a 69% incidence of one tooth edentulous in the age group of 35–44 years [1]. Also, when people reach the age of 75 years, a minimum of one-fourth of the adult population would be completely edentulous. Rise, but the quality of life is not much improved. For instance, among the overall population, the percentage belonging to 65 years and above is on the rise. The total count of this category population in the year 2000 was 282 million and is expected to rise by 49%, rise to 420 million by 2050. Overall, the impact of an increase in population and a higher chance of that population being considered to be more than the age 65 leads to a considerable rise in the actual patient count, considering that 35 million people were older than age 65 in the year 2003. This number is required to increment by 87% by 2025. Hence, the increased need for dental implant treatments is due to the failures of a fixed prosthesis and the consequences, also the life longevity of the aging population and their dental needs.

Oral implantology has evolved and improved to be a centre of the art and technology of modern dentistry. The field demands its practitioners to have a distinguished knowledge of details in significant areas, including scientific updates, updating knowledge about different types of radiographs including regular radiographs, computed tomographic, Micro-CT, robust working knowledge about the anatomy, surgical procedures, prosthetic requirement and follow-up care. Every dentist who places or restores implants ought to be aware of the possible problems and should be able to know how to control them.

Oral implantology is a challenging field to master and the impact of failure may be a disastrous one. The dentist should know the material science and have awareness of how to prevent the failure of implants. Among various biomaterials, metallic biomaterials are used mainly and in various forms. The metals like Gold, Palladium and other noble metal alloys have the longest history of utilization. However, their high cost limits their applications. At present, the usable metallic materials in the medical science are Co-Cr-Mo, 316 L SS, Titanium-based alloys and miscellaneous alloys like amalgam and Gold. Titanium material resists corrosion, has strength to weight ratio, weight less and has good mechanical properties. Its biocompatibility, non-toxic, durable, easily available and cost-efficient makes Titanium, the choice of material for many uses in dental application

The mechanical strength and biocompatibility of Titanium make it the metal of choice for dental implants. Titanium with the mesoporous layer of Gallium and Silicon can act as a drug delivery system for enhancing osseointegration thereby expanding the clinical application of Ti alloy. Si is a potent anabolic element that promotes bone formation and Gallium inhibits bone osteolysis, review of literatures suggest verifying animal studies have shown that the synergistic impact of Ga and Si on promoting osseointegration can be applied to clinical trials.

The aim of this study is To Synthesize a new metallic Aluminum and Vanadium free Titanium alloy biomaterial for better osseointegration and implantation in the physiological system.

To Synthesize Gallium Silicon Titanium Alloy biomaterial.

To Characterize the mechanical and structural properties for use in dental implant application.

To Characterize the alloy for biocompatibility in the physiological system.

Advantage of gallium silicon titanium alloy implants to the population

• Gallium metal has the antiresorptive property and prevents the osteoclastic activity when implants placed in bone

• Silicon metal has the anabolic property and promotes the osteoblastic activity when implants placed in bone

• Titanium metal has high inert to the tissue and the metal density which is to replace missing hard tissue.

• Synergistic effect of Gallium silicon Titanium alloy would be having the inert tissue response with osteoclastic and promotes osteoblastic activity in bone promotes osseointegration of implant to bone in mankind with estrogen deficient females in old age and osteoporosis patient

2. Materials and methods

2.1 Synthesization of titanium, titanium gallium and gallium silicon titanium alloys

Code-263265-Gallium: 99.99% with molecular weight 69.72G/MOL, Code-343250-Silicon: 99.95% with molecular weight 28.09G/MOL and Code-GF96834493-1EA-Titanium: 99.6% were purchased from Aldrich and used as such with no further purification.

Titanium metal pieces are used as such.

Titanium Gallium alloy metal pieces were weighed as per the alloy composition described by [2] presented in Table 1.

Value	Titanium	Gallium	Total
Atomic %	98%	2%	100%
Weight Gm	19.423gm	0.577gm	20gm

Table 1.

Atomic and weight percentage of metals for Ti-Ga alloy.

Gallium Silicon Titanium alloy metal pieces are weighed and prepared as per the alloy composition described by [3] presented in Table 2.

Value	Gallium	Silicon	Titanium	Total
Atomic %	15%	5%	80%	100%
Weight gm	4.169 gm	O.577 gm	15.271gm	20gm

Table 2.

Atomic and weight percentage of metals for Ga-Si-Tialloy.

The arc melting technique was used for melting metals and making alloys using a vacuum arc furnace as shown in Figure 1. The heating is done by striking an arc between a tungsten electrode and the metal pieces Ti, Ga and Si, for alloy preparation were shown in Figure 2 that was placed in a water-cooled copper crucible. The melting was carried out in an evacuated chamber which was backfilled with inert Argon gas exactly before melting.

Figure 1.

Vacuum arc furnace.

Figure 2.

Metals used for alloy preparation.

The working principle of a laboratory arc melting unit is similar to the standard TIG welding unit. The heat required for melting the metal pieces in the crucible was generated by the electric arc between electrode and metal. Argon gas provides an inert atmosphere and prevents oxidation of the sample. Melting was repeated 3–4 times so that a homogeneous alloy is obtained. The typical process of flow of melting metals followed in this study is shown in Table 3.

1. Metal pieces are weighed as per the alloy composition	2. The copper mold is cleaned using Acetone	3. Metal pieces are placed at the centre of the mold
6. Switch ON the diffusion pump at 10^−3 mbar	5. Ensure that there is no leakage or open gap	4. Chamber is evacuated using a rotary pump
7. Let vacuum reach 10^−5 mbar inside the chamber	8. Switch ON chiller for water flow to mold & electrode	9. Switch ON the power supply
12. Strike arc again & melt the pieces to form a button	11. Melt Ti getter to consume O2 in chamber (if any)	10. Strike the arc using the electrode and mold
13. Flip ingot and rebuild vacuum to desired level	14. Repeat the process for 2–3 times to obtain an ingot	15. Perform suction casting if required

Table 3.

Process of a vacuum arc melting.

Triplets in each sample of Titanium, Titanium Gallium, Gallium Silicon Titanium alloys were made into a sample size of 1 mm length x 5 mm diameter (Figure 3). Samples were polished with sandpaper no: 60, 220, 240, 320, 400, 600, 1200 and 2400, cleaned with alcohol and then bathed with distilled water as described by [4]. Samples were divided into three groups, viz. Ti, Ti-Ga and Ga-Si-Ti groups for experimentation.

Figure 3.

Sample size used for in-vitro study.

2.2 Characterization of the mechanical and structural properties of Ti, Ti-Ga and Ga-Si-Ti alloys

The microstructural phases and visualization of the microstructure of the alloys and the mechanical and structural properties of the experimental alloys were studied.

2.2.1 Determination of microstructural phases

The microstructural phases of the alloys Ti, Ti-Ga and Ga-Si-Ti were assessed by X-ray diffraction (XRD). The X-ray diffraction was done in Bragg-Brentano θ-20 Geometry method using X-Ray Diffractometer (Smart Lab, 9 kW- Rikagu, Japan instrument) as per [5].

2.2.2 Visualization of microstructure of the alloys

The samples were mirror polished with colloidal silica for 10 minutes before etching. Eventually etching was done with Kroll’s reagent (Sigma Aldrich) for 4–5 seconds, reagent consist of 5% Nitric acid, 10% Hydrofluoricacid and 85% water and visualized with an inverted metallurgical microscope. De winter victory model no 4100, Germany as per the procedure of [2].

2.2.3 Scanning electron microscopic study

Triplet samples from Ti, Ti-Ga and Ga-Si-Ti alloys groups were dried at room temperature and fixed in glutaraldehyde 2%. The sample was immersed with alcohols (50%, 75%, 95% and 100%) for dehydration. The sample was placed over absorbent paper for 48 hours and analyzed with SEM (Scanning Electron Microscope) and EDX (Energy Dispersive X-ray Analysis)as per the procedure used by [6, 7].

2.2.4 Tensile strength test for the alloys

The tensile strength of the alloys was measured by using ASTM E8 as per the following specification. The samples were cut by an Electric Discharge Machine (EDM), rinsed with acetone and degreased with NaOH and distilled water and then measured with a Vernier caliper. The sample was held at the tensile fixture and pulled at the velocity of the speed of 1 mm per minute and the results were measured as described by [8].

2.2.5 Compression test for the alloys

Compression testing was done on the samples as per ASTM E9 specification and the results were recorded. The model was held in the compression fixture as sample edges were flattened and the flatness was checked with the dye gauge and the results were recorded as per [9].

2.2.6 Microhardness test for the alloys

In the VHN test, the applied force was smooth without causing any effect and held in contact for a duration of 10–15 seconds. ASTM 384 VHN was tested at 200gF/1.96133 N using cylindrical specimens of 11 mm in height of the sample. 6 readings were taken for each specimen with ‘The Wilson VH1150’ digital micro-hardness tester machine and the values were recorded as described by [8].

2.3 Characterization of the alloy for biological compatibility in the physiological system

1 mm long x 5 mm width in size Ti, Ti-Ga, Ga-Si-Ti alloys samples were prepared for in-vitro studies (Figure 3). The samples were polished with sandpaper and were cleaned with alcohol followed by distilled water. The samples were sterilized through steam autoclave 121$°C$ for a duration of 30 minutes 15 pounds per square inch of pressure.

2.3.1 Study of the bioactivity of the Ti, Ti-Ga and Ga-Si-Ti alloys using simulated body fluid in-vitro study

Simulated body fluid is prepared according to [9]. The pH of 1.0 SBF/liter fluid was adjusted to pH 7.4 using 1NHCL, (i.e.) 1NHCl ----- > 8.3 ml of HCL in 91.7 ml H₂O and Six-well plates were used for the samples of all the three groups. The triplet sample from each group was soaked in the prepared simulated body fluid (Figure 4). Each plate of 10 ml of SBF fluid was treated with albumin and kept at room temperature and the fluid was changed every day after 24 hours for 21 days. Using SEMand EDX spectrometry, the sample surfaces were assessed qualitatively.

Figure 4.

Triplet sample in simulated body fluid In 6 well plate.

2.3.2 Evaluation of cytotoxicity of the Ti, Ti-Ga and Ga-Si-Ti alloys using SaoS-2 cell line in-vitro study

SaoS-2 cell line purchased from NCCS PUNE, INDIA was cultured with Mc Coy’s 5A medium in 10% FBS (Gibco, In-vitrogen Bio-Services India, Bangalore, India) and 1% penicillin-streptomycin (HIMEDIA) and were maintained in this at 37°C with 5% CO_2. The cell line was used for assessing cell interaction and alloys with regard to cell adhesion, viability and proliferation. Their transformation of SaoS-2 cells into osteocytes and calcium deposition at the surface of the alloy was measured through qualitative and quantitative evaluation methods.

Before seeding of cells, the samples were treated in a culture medium for 24 hours. Then the Cells were separated using a standard protocol of [10] for cell density and incubated at 37°C with 5% CO₂ in an 8x12 well plate. The cell line and alloys of three combinations in triplicate were incubated as per the procedure of [11] which is shown in Figure 5.

Figure 5.

Triplet sample in SaoS-2 cells in 8x12 well plates.

2.3.3 Alamar blue assay

The vitality and cytotoxic character of the samples were determined using the cell viability reagent alamar blue. The pH McCoy’s 5A medium was adjusted to the pH of 7.0 to 7.4 and incubated at 37°C in sealed plates to prevent evaporation. Since the Alamar blue is photosensitive, so the incubation was done under darkness as described by [12].

2.3.3.1 Cell adhesion study

After 24 hours of cell seeding, adhesion was assessed, by adding 10% Alamar blue dye to the cell medium. After 4 hours, the dyed media was aspirated and the same was measured for their adhesion to alloys at 570 nm and 600 nm as per the procedure adopted by [13].

2.3.3.2 Eosin staining of the alloys

The staining of the samples was carried out for three alloy groups. The samples cleaned with PBS were dipped in 10% formalin for duration of ten minutes. Followed by eosin staining for one minute, washed with water, absolute ethanol was used for drying the samples for three minutes and observed in bright field microscopy to observe the surface of the alloys with cell adhesion.

2.3.3.3 Viability and proliferation study

Viability and proliferation were measured at 48 hours and 96 hours after cell seeding. 10% alamar blue dye included in the medium whenever medium was changed. Six hours after adding the dye the medium containing alamar blue dye was aspirated and measured at 570 nm and 600 nm in a spectrometer.

The proliferation rate was estimated by comparing the percentage decrease in matching wells after 96 hours to 48 hours using the formula below.

Proliferation rate, $P=\frac{R96-R48}{R48}\times 100$ R96 and R48 are percentage reductions of viability tests performed at 96 and 48 hours, respectively.

2.3.3.4 Study of the samples using SaoS-2 cell line for mineralization in-vitro study

Mineralization of SaoS-2 Cells with the surface of the alloy for cell seeding density was done according to the technique followed by [14]. The cells have been cultured for 3 weeks and the medium was changed every three days. The mineralization procedure was carried out as per the procedure of [15]. The calcium deposits in the cell culture were measured using alizarin red dye. We utilized 1 milliliter of a 40 mM solution that had a pH of 4.2 and left it at room temperature for 10 minutes. In order to remove the dye, the wells were washed with phosphate buffer saline and distilled water five times before being examined under a stereomicroscope to obtain the images.

2.4 In-vivo study for evaluation of Ti, Ti-Ga and Ga-Si-Ti alloy as biomaterial implants

Study Design: An implant designed to promote bone growth may require at least two months and upto six months for bone regeneration and to study the localized tissue reaction. With the current crossover research study, the goal is to evaluate the newly synthesized Ga-Si-Ti implant for its biocompatibility in comparison with Ti and Ti-Ga implants. This removes the anatomical variation and avoids bias in evaluating osseointegration. The test was carried out to assess for reaction around the implants as per ISO-Standard No: 10993–6 and 10,993–11.

2.4.1 Implants

Non-threaded cylindrical implants were fabricated according to the surgical anatomy and the diameter of the femur bone of the rat. The implants were surgically placed on the right side femur of the rats. Three types of implants viz. (i) Ti (ii) Ti-Ga and (iii) Ga-Si-Ti in size of 1.5 m diameter and 3 mm length are shown in Figure 6. SEM images of Ti, Ti-Ga and Ga-Si-Ti implants are shown in Figures 7–9.

Figure 6.

Sample size used for in-vivo study.

Figure 7.

SEM images of Ti.

Figure 8.

SEM images of Ti-Ga.

Figure 9.

SEM images of Ga-Si-Ti.

2.4.2 Characteristic details of the experimental groups

The study was conducted on male Wistar rats (Rattus /Norvegicus) of about 8 weeks of age with the body weight of 200–250 gm. In total, 18 rats were studied in this research. The experimental rats randomly split into three groups in which the Ti, Ti-Ga and Ga-Si-Ti metal alloys were implanted on the right femur bone and the rats were kept under observation for normal health.

The IAEC of Sathyabama Institute of Science and Technology issued the ethical clearance with the IAEC Number: SU/CLATR/IAEC/XV/152/2020. The experimental animals were housed in the Sathyabama Institute of Science and Technology animal experimental laboratory in Chennai, Tamil Nadu, India. The rats were fed a standard maintenance rat diet and water ad libitum.

Experiments were performed according to CPCSEA guidelines and conforming to NIH guidelines for the animal.

2.4.3 Surgical procedures

Surgical procedures (Figure 10) were carried out under general anesthesia, as per the procedure adopted by [16] with Ketamine (40-90 mg) and Xylazine 5-10 mg (Rompun) by the intraperitoneal path. The surgical site was tonsured and disinfected with povidone-iodine solution & an injection of Lidocaine with Adrenaline (1:80000) was infiltrated at the site of incision to prevent bleeding.

Figure 10.

Animal surgery and implant placement.

A stab incision was made along the imaginary line between the hip joint and along the lateral aspect of the thigh. The medial aspect of the femur bone was exposed on the right side. With a 1.5 mm surgical drill, a 2 mm x 3 mm defect was created in the medial aspect of the femur and under irrigation with normal saline.

The implants were placed in the defect and secured with a 3–0 silk suture around the femur bone. Then the muscles were secured followed by a skin suture (Figure 10). The skin wound was protected by Healex Spray contains benzocaine 0.36%w/w and cetrimide 6.5%w/w (Shreya life sciences Pvt. limited) and Nebasulf powder contains Bacitracin (250.0 IU), sulfacetamide (60.0 mg) and Neomycin (5.0 mg) (Abbott healthcare PVT LTD). Gentamycin 20 mg/kg/day for five days and Buprenorphine 0.04 mg/kg/day for 3 days) were given intra-peritoneal as post-operative antibiotic and analgesic respectively. Hydration of animals was maintained with 5 ml of dextrose saline on the day of surgery.

2.4.4 Radiographic analysis

Radiographic evaluation was carried to assess the position of implant with the surrounding bone and to conduct a study to evaluate the new bone formed along the implants.

2.4.4.1 Digital radiography

Two types of digital radiography were done in the present study.

2.4.4.1.1 Posterior anterior view of the whole body

Posterior Anterior view of the whole body [17] using Sirona orthophos Xg radiographic machine, a direct digital radiograph was taken for comparing the bone density of the femur with implant and normal femur bone of the same animal at a different time interval, i.e. 14th, 28th, 42nd and 56th day. Once the standard images were produced and the post-implantation were aligned by selecting reference points on the implant images as per [18] following the selection of a region of interest on the images, the follow-up images were subtracted from the baseline image and negative images used as digital subtraction of radiographic images for evaluating the bone density changes. The quantitative assesment of newly formed bone and the mineralization around implants was analyzed using Sidexis Xg software for calculating the density of the bone using grayscale. Qualitative assessment was done using the images which showed decreased grayscale inferring mineral bone loss and increased grayscale inferring bone mineral gain.

2.4.4.1.2 Paralleling technique

The second method of digital radiography used paralleling technique to the film (phosphor plates) and implanted femur bone of rat by using x-mind x-ray unit setting with 60kVp, 4 Ma, with an exposure time of 0.4 Seconds to produce the highest quality of digital image with most negligible radiation. Soredex Digora Optime processing unit was used in which best contrast at lower exposure digital image can be achieved. The photon detector used in the system is a phosphor plate with the following characteristics with a dimension of 31 mm x 41 mm and the image pixel of 1034x1368 according to ISO 10993-1and ISO10993-5 non-toxic & non-irritative to biological systems, so the hygienic bag made of latex-free food-grade polyethylene was used over phosphorylates while taking a radiograph. Linear measurements of bone densitometric values were obtained using Gray values [19].

Areas of specific sites visualized through digital images were selected for densitometric analysis. In this study, linear, cortical (CBD), trabecular (TBD) bone densities and Newly Formed Bone Density (NFBD) were measured for qualitative evaluation by serial radiograph. Quantitative assessments were done by one measurement in each area per image along with bone density around implants and the point density was measured using Gray values.

2.4.4.2 Computed tomography study

Computed Tomography was done using the Somato go-now scanner in which the beam is collimated using the material ultra-fast ceramic collimation and the CT detectors of the scan utilizing a CT X-ray source of 80–120 Kv with strong filtration to produce a monochromatic beam. The width of the collimator is 0.6 mm and the true resolution of the machine is 1 mm.

In the present study, 300–450 window levels and 1300–1500 window widths were used. The data reconstruction was done using the software, to calculate the distance, pixels and intensity for image analysis. Thirty two images were recorded per rotation of 16.0 x 0.7 collimation. The spiral accusation with pitch factor is 0.8 ratios. All the specimens were scanned with the same parameters and data were collected by a computer. The image was reconstructed to evaluate the linear attenuation, co-efficient of each voxel in a slice assigned with CT number [16] or Hounsfield number to each voxel. The mean Hounsfield Units were calculated and statistically analyzed. CT scan was obtained on the 28th and 56th days to evaluate the osseointegration in terms of the newly developed bone. After the implantation, there were disparities in the density of the trabecular and cortical bones. Computed Tomographic scans have been assessed in two and three-dimension formatted images for studying the bone mineralization in response to implant material placed in critical defect created for evaluating the biocompatibility of the implant with the bone.

2.4.5 PolyFluoroChrome study (PFCs)

Experimental rats of all the three groups were injected with PFC according to the procedure described by [20]. Post-operatively the animals were administered with intra-peritoneal injected with Alizarin Red S (30 mg/kg body wt) on the 7th and 14th day, Tetracycline (12 mg/kg body wt) on 21st and 28th day and finally Calcein (4 mg/kg body wt) on 35th and 42nd day respectively.

2.4.6 Sacrifice of the experimental animals

The animals were sacrificed on the 56th day and femur bones with implants were retrieved and stored in 70% ethanol at 4°C for histopathological study and ex-vivo Micro-CT.

Euthanasia: All experimental rats were euthanized by giving a 5x dose of Ketamine\Xylazine (500/50 mg/kg) intraperitoneally. The femur bones with implants were retrieved and subjected to histopathological study. The carcass of the experimental animals was disposed of as per the Biological Waste management program of the Institution through M/s GJ Multiclave (India) Pvt. Ltd., Tambaram Sanatorium, Chennai - 600,047.

2.4.7 MICRO-CT study

Micro-CT scan of rat femur with explant was done according to [16] .The scanner used was a SCANCO Medical CT 40. The following values were used in an easily configurable measuring methodology: 12 μm spatial resolution, 70 kVp beam energy, 114 A beam intensity, 300 ms integration time and 1024 × 1024 image matrix. Explants from rats were isolated, washed with PBS and stored in ethanol in an Eppendorf tube. The density of bone mineral was calculated in g.cm⁻³. BMD refers to combined density of defined volume with bone and soft tissue. The study’s calibration of bone mineral density was done by 2 mm–4 mm phantom rod pairs inside a tube of water matching the animal’s bone diameter in the ex-vivo study. After scanning the phantom rods, a bone sample with three different implant materials ROI was observed for BMD for calibration.

2.5 Histomorphometric analysis of bone remodeling using Polyfluorochrome (PFC) dyes in-vivo and ex-vivo

Specimen Preparation for Histology and Histomorphometric analysis was done as per the procedure adopted by [21].

Reagents: 10% NBF, Alcohol (70%, 80%, 96% and 100%), CH₃COCH₃ (Sigma Aldrich, Bangalore), MMA (Sigma Aldrich, Bangalore), C₁₄H₁₀O₄ (Sigma Aldrich, Bangalore), 5% NaOH, KMnO4 (Merck, Maharashtra) Methylene Blue (Thermo Fischer, Mumbai), Distilled water, Acid Fuschin (Sigma Aldrich, Bangalore) Saturated Picric Acid (Sigma Aldrich, Bangalore), Cyanoacrylate glue (Alteco chemicals, Japan), Stevenel’s blue and Van Gieson’s Picrofuchsin stain.

Specimens were fixed in 10% NBF, dehydrated in increasing ethanol concentrations (70–100%), cleared using acetone alcohol mixture and embedded in MMA. Resin blocks of specimens were prepared after polymerization in MMA under vacuum conditions for 7–10 days at room temperature. The PMMA block was split into wide sections (70-100 m) using a saw microtome (ACCUTOME 100, Struers, Denmark). It was adhered to a glass slide and polished using a grinder polisher (ECOMET 3000, Buehler, Germany). The sections were stained with Stevenel’s blue and Vangieson’s picro fuchsin and examined in a trinocular transmitted light microscope (Nikon Ni-E). Photomicrographs were taken with a camera (Nikon DS Ri1) attached to the trinocular microscope (Nikon Eclipse) for histomorphometry and examined in a fluorescence microscope.

The samples were observed in violet, blue and green illumination in Leica fluorescent microscope attached to a computer with Leica system software installed. Images were acquired in 10x magnification. For every sample, different illuminations were used for the same focused field to obtain the standard images. Thus, the obtained images were superimposed in the order of green illumination followed by a violet after blue, which corresponds to the excitation of dyes in the same order as administered to the rats. This was accomplished by using GIMP version 2 software. The dimensions inside individual images were measured using NIH-Image J software according to techniques described by [22].

2.5.1 Histomorphometry

Bone Implant Contact (BIC), Rate of Bone Apposition (RBA) and Osteoblast proximity were the parameters evaluated and all the measurements were made with NIH-Image J. The marking techniques were done according to [23]. The total perimeter of implant areas of implant-bone contact was marked and BIC was estimated as contacting area percentage to the whole implant perimeter as per the formula adopted by [24]. Kulak and Dempster, [25] calculated MAR by dividing the mean distance between fluorescence labels by the time interval between them.

2.6 Statistical analysis

Quantitative data obtained from in-vitro, cytotoxic test done with alamar blue assay to evaluate the adhesion, viability and proliferation with SaoS-2 cell line, in which two-tailed test, which is used in null hypothesis testing and statistical significance bone calcium mineralization and in-vivo radiographic analysis of the bone were statistically analyzed utilizing the SPSS software (version 21.0; IBM corporation, Armonk, NY, USA). Significance level was fixed at 5% (α = 0.05).

For the in-vitro model, mineralization occurs at different rates in each study group when different materials are used. So, to understand mineralization, calcium nodules deposited were evaluated for the 4th, 7th, 8th, 11th, 14th and 21st day. To compare the mean values between variables recorded at a different time points, repeated measures ANOVA was applied to find the group means.

For the in-vivo model, linear bone density of PA view of the whole body was compared between all the three groups of Ti, Ti-Ga and Ga-Si-Ti implanted bone with adjacent side normal bone. Medial cortical, lateral cortical, trabecular bone and new bone formation were recorded on 14th, 28th, 42nd and 56th day on both the implanted side as well as adjacent normal bone. Non-parametric The implanted bone was compared to normal bone using the Mann-Whitney U test (similar to the post hoc Tukey test) and the implanted bone was compared between groups using the Kruskal-Wallis test.

Digital subtraction linear bone density of the PA view of the body was compared between the three implanted bone groups of Ti, Ti-Ga and Ga-Si-Ti. On the 14th and 28th days, the first indications of newly formed bone was documented using the digital subtraction approach. To examine the changes within each group, the Wilcoxon signed-rank test was used. For intergroup comparison, the Kruskal-Wallis test was used.

For the in-vivo model, to evaluate the point density in digital radiography using paralleling technique, the non-parametric statistical test of the Kruskal-Wallis test was applied.

Mean difference intergroup comparison concerning Hounsfield Units of the CT scan and mean/density in micro-CT, both were analyzed using the non-parametric Kruskal Wallis test and one-way Anova test respectively.

Histomorphometry analysis of bone was evaluated through BIC; it is described as the amount of contacting area to the total perimeter of implant in percentage, osteoblast proximity which is the distance between the first osteoblast and the implant and finally, the Rate of Bone Apposition (RBA). The BIC (Bone Implant Contact), RBA and Osteoblast proximity were analyzed using Kruskal Wallis Test.

3. Results

In the microstructure evaluation of Ti, Ti-Ga and Ga-Si-Ti exhibited ‘α’ phase.

In the mechanical study, the Ga-Si-Ti alloy showed a higher Ultimate tensile strength (780 MPa), Yield strength (595 MPa) and a higher percentage of elongation (18.76%) and more or less equal to Young’s modulus (1.16x105 MPa) and Compressive at fracture (1696 MPa) when compared with the Ti and Ti-Ga alloy.

In the Simulated Body Fluid study, among the three samples, the Ga-Si-Ti alloy showed more precipitation on its surface and a higher Ca/P ratio (1.8%) in the precipitation.

In-vitro evaluation of cytotoxicity of the Ti, Ti-Ga and Ga-Si-Ti alloys revealed the ability of SaoS-2 cells to adhere to the surfaces of all three alloys, which indicated the recognition of the experimental alloys by the cells as surfaces to grow on. No statistical differences observed in the adhesion, viability and proliferation properties between the groups. In mineralization in-vitro study, Alizarin dye exposed the Ca and Phosphate mineralization on day 7th, 14th and 21st day of the existence of spherulites on the surfaces of all three group alloys was investigated and confirmed. Quantitative analysis of mineralization revealed that the Ga-Si-Ti alloy group showed more mean number (212 $\pm$ 2.28) of calcified nodules than cpTi (130 $\pm$ 4.36) and Ti-Ga (137 $\pm$ 2.65) group alloys.

In the in-vivo study, a PA view of whole-body radiography all groups exhibited a substantial difference in the linear bone density of newly formed bone. However, compared with Ti and Ti-Ga groups, the Ga-Si-Ti group had the highest mean bone density of newly formed bone.

The CT study revealed an increase in bone density in terms of higher Hounsfield Units for trabecular bone with Ga-Si-Ti group (802.92 ± 226) on day 56 when compared to Ti (673 ± 73.37) and Ti-Ga (708.43 ± 166.54) groups and higher Hounsfield Units of new bone density in Ga-Si-Ti, followed by Ti-Ga group and then Cp-Ti group.

Ga-Si-Ti group showed the highest bone mineral density (459.9792 mg/cm3) than Ti and Ti-Ga group in the micro CT ex-vivo study. In addition, the study exhibited a significant difference between the groups and the Ga-Si-Ti group implants in the proportion of cortical bone volume to trabecular bone volume BV/TV in percentage showed a clinical significance with the highest BV/TV bone volume of 25.07%.

In the present study, Poly Fluorochrome tracers (Alizarin red, Tetracycline and Calcein) clearly showed the remodeling pattern. When compared to other implants, Ga-Si-Ti alloy implants promoted cell proliferation and differentiation and improved the BIC. Ga-Si-Ti alloy implants exhibited ∼98% BIC and much closer osteoblasts presence to the surface of the implants.

4. Discussion

Titanium is a “miracle metal” in dentistry and its alloys have become the preferred metals for preparing bone biomaterials. The most popular implant materials are Ti, Ti-6Al-4 V, Ti-6Al-7Nb and Ti-13Nb-13Zr. Since Cp-Ti is relatively pliable, V and Al are hazardous and Nb and Zr are brittle, there is considerable interest in the development of superior Ti alloys. Considering the vast potential of Gallium and Silicon, a ternary Ti alloy containing 15% Gallium and 5% Silicon was synthesized in order to enhance this scenario and expand the clinical application of Titanium. In addition, there were few investigations conducted on the structural and mechanical properties of Ga-Si-Ti alloys and there is currently no information on the in vitro and in vivo biocompatibility of Ga-Si-Ti alloy as a biomaterial. This has been the motivation for the present study.

4.1 Microstructural properties

Microstructure analysis is useful to interpret an alloy’s mechanical and corrosion resistance properties.

X-ray diffractometry was used to evaluate the structural features of Ti, Ti-Ga and Ga-Si-Ti alloys. The results revealed a single $\alpha$-Ti phase with a partially stable HCP crystal structure. In Ti-Ga and Ga-Si-Ti alloys, the $\alpha$ - peak could be observed. Similar outcomes were reported for Cp-Ti alloy [26]; Ti-Ga alloy [2] and Ga-Si-Ti alloy [3, 26]. According to [2] Ga is a stabilizing element that, when dissolved in Ti, elevates the eutectic transformation temperature from α (hcp) to β (bcc). Ti-Ga alloys, on the other hand, have a short solidification interval and a solubility of up to 20% in the α -Ti phase. Despite the presence of β-stabilizing Silicon in the Ga-Si-Ti alloy, the Ga-Si-Ti alloy exhibited α-phase because the Si content was only 5%. Consequently, all three alloys exhibited α-phase, which was validated by XRD. This result concurs with the microstructural tests conducted by [27] on the Ti-15Nb-5Si alloy. SEM images of Ti, Ti-Ga and Ga-Si-Ti alloys indicated irregular grain boundaries in the present investigation. These uneven grain boundaries were attributed by [2] to the non-equilibrium cooling process during casting.

The EDAX analysis and mapping validated the distribution of Ga in the Ti-Ga alloy and the distribution of Ga and Si in the Ti-Si alloy. Vizureanu et al., [28] performed a similar study of Ti-Mo-Si alloys to validate the presence of Si and Mo in Ti-Mo-Si alloys.

4.2 Mechanical properties

Some of the properties like compression strength, tensile strength, modulus and elongation are important mechanical properties to be assessed in newly synthesized metals for use as biomaterials. In this study, the above properties of Ti, Ti-Ga and Ga-Si-Ti were done to assess the suitability of Ga-Si-Ti alloy as an implant, particularly as a dental implant.

The ultimate compression strength of Ti, Ti-Ga and Ga-Si-Ti alloys was found to be similar, viz. 1656 MPa, 1690 MPa and 1696 MPa respectively. The results obtained in this study are comparable to the compression strength recorded by [29] for Commercial pure-Titanium (1074 MPa) and Ti-64 alloy (1661.6 MPa).

Regarding the selection of an alloy for usage as a biomaterial, the tensile strength and modulus of the alloy are to be evaluated for assessing its fatigue strength and ductility. In the present experiment, the Ti, Ti-Ga and Ga-Si-Ti alloys showed 579 MPa, 612 MPa and 780 MPa as ultimate tensile strength and 1.10x10⁵ MPa, 1.13x10⁵ MPa and 1.16x10⁵ MPa as Young’s modulus, respectively. The percentage of elongation was observed as 17.9%, 17.6% and 18.76% for the Ti, Ti-Ga and Ga-Si-Ti alloy respectively. The findings of this study are in conformity with the findings of other investigations, which found that alloys made of Cp-Ti, Ti-Ga and Ga-Si-Ti have comparable levels of strength, modulus and elongation [2, 3, 29].

The observations made using Ti-Ga alloy are comparable to those made by [2, 30]. The present study revealed a higher ultimate tensile strength and percentage of elongation and more or less equal Young’s modulus to Ga-Si-Ti alloy when compared to those mechanical properties shown by Ti and Ti-Ga alloys.

Comparing the tensile strength, yield strength, percentage of elongation and compressive at fracture of Ga-Si-Ti alloy with those of Cp-Ti and Ti-Ga alloy reveals that Ga-Si-Ti alloy has superior mechanical properties.

Similarly, the Vicker’s hardness values observed in the present study for Ti, Ti-Ga and Ga-Si-Ti were 177.5 ± 4.18, 271.6 ± 16.49and 240.3 ± 9.89. The hardness values for Ti and Ti-Ga alloys found in this study are similar to those found by [2]. The Ti-15NbxSi alloy was given a hardness value of 313 ± 7 by [27]. This is a slightly higher than the hardness of the Ga-Si-Ti alloy from this study, where they used two $\beta$-stabilizing alloys. According to [31], the addition of Tantalum and Zirconium to Titanium improved the wear resistance of the alloy and the addition of Zirconium boosted the micro-hardness of the Titanium zirconium composite. In comparison, Choi et al., [5] correlated the increased value of hardness of Ti alloy with the melting procedure used for the alloy preparation. The mechanical properties observed in the current investigation after the addition of 2% Ga in Ti-Ga and 15% Ga in Ga-Si-Ti alloy have increased the strength and micro-hardness monotonically. This observation is related to solid solution strengthening of Ga as observed in [2] study on the evaluation of mechanical properties of Ti-Ga binary alloy. Antonova et al., [3] demonstrated that Ga can be employed effectively to improve the mechanical properties of Ti alloys, particularly Ti-Si alloys. A perusal of the literature revealed that there is a paucity of information on the effect of the addition of Ga and the combined effect of Ga and Si on Ti alloy and the present study contributed to a better understanding of the role of Ga and Si in the composition of Ti alloy. Both the addition of 2% Ga to Cp-Ti and the addition of 15% Ga and 5% Si to Cp-Ti alloy increased the mechanical properties of Ti alloy, according to the results of this study.

However, the Young’s modulus reported in this research for the Ti, Ti-Ga and Ga-Si-Ti alloys is somewhat more than that of human bone. The modulus value obtained for the Ga-Si-Ti alloy of this study is more or less similar to the best known and comm only used Ti and Ti-6Al-4 V alloy and it was considered as close to the modulus of human bone. Thus the results obtained in the study revealed that the elements Gallium and Silicon can be added to the Titanium alloy for improving its mechanical properties. Hence, the results obtained as a dental implant biomaterial, a novel Ga-Si-Ti alloy with improved structural and mechanical capabilities as compared to the widely used Ti and Ti-based alloys was proposed in this study.

4.3 Simulated body fluid

In the present study, the bioactivity of the Ti, Ti-Ga and Ga-Si-Ti alloy the samples were tested by immersing them for 21 days in Simulated Body Fluid (SBF). Immersion of the biomaterials in simulated body fluid (SBF) is an extensively used method to investigate the bioactivity of Ti and its alloys biomaterial. Kokubo, [32] opined that the bioactivity of the biomaterial can be predicted based on the formation of apatite on the surface of biomaterials when immersed in SBF and by the analysis of the apatite so formed. In the present investigation, SBF was prepared as per the procedure of [33] which is comparable to human plasma. After the immersion in SBF for 21 days, the visualization of Ti, Ti-Ga and Ga-Si-Ti alloy samples using SEM revealed increased precipitation on their surfaces. However, based on the SEM images no conclusion can be drawn on the bioactivity of the alloys since there were no differences observed between the samples in the apatite formation activity.

The element of analysis of such formed precipitate on the Ti, Ti-Ga and Ga-Si-Ti alloy samples revealed the presence of a maximum percentage of Titanium and the presence of Calcium, Phosphorus, Sodium and Oxygen along with Gallium in Ti-Ga and Gallium and Silicon in Ga-Si-Ti alloy sample immersion study. When the Ca/P ratio was calculated, it was higher in the Ga-Si-Ti alloy sample than in the Ti alloy sample. and Ti-Ga alloy samples after their immersion in SBF for 3 weeks duration. Stenport et al., [34] studied the Ca/P ratio for biomaterials immersed in SBF for 2 weeks and suggested increase in immersion duration for proper assessment of the bioactivity of the biomaterials. The bioactivity seen in terms of precipitation for the Ti-based alloys samples in this study is consistent with the findings of [35] where they demonstrated that the Titanium implants have become bioactive once they get oxidized in a calcium hydroxide solution.

Takadama et al., [36] concluded that the evaluation of the production of apatite on the surface of the biomaterials submerged in SBF, as well as the elemental characterization of apatite by EDX spectrometry, were both qualitatively and quantitatively effective in predicting the biomaterial’s in vivo bone bioactivity [37]. Using the SBF model, they observed a variation in the development of calcium phosphates on Titanium implants with different types of surfaces. Qiu et al., [2], reported that the incorporation of Ga into pure Ti enhanced resistance to corrosion in both regular artificial saliva and saliva with fluoride. The bioactivity property observed with Ti, Ti-Ga and Ga-Si-Ti in the current study also agrees with the previous studies of [38, 39]. In these studies, they correlated the bioactivity characteristics of Cp-Ti and Ti-based alloys with their good corrosion resistance and biocompatibility properties.

Tadashi Kokubo and Hiroaki Takadama, [33] also opined that the SBF method of study could be of use in the development of new bioactive materials and also for screening and forecasting their biocompatibility property in animal testing. In a review by [40], it was concluded that the elements like Oxygen, Nitrogen, Calcium, Phosphorus and Sodium implanted into Titanium alloys improved the mechanical properties, bioactivity and the cyto-compatibility of Titanium and its alloys. The increased precipitation and the elemental analysis of the apatite in the present study were indicative of the Ti, Ti-Ga and Ga-Si-Ti alloys bioactivity. Moreover, among the three alloy samples, the Ga-Si-Ti alloy sample found to be superior in bioactivity in SBF, since it showed both the increased precipitation on its surface and a higher Ca/P ratio in the precipitate. Hence based on this SBF model in-vitro study, it is suggested that the Ti, Ti-Ga and Ga-Si-Ti alloys are suitable for in vivo study in experimental animals.

4.4 Cytotoxicity of Ti, Ti-Ga and Ga-Si-Ti alloys

Newly synthesized biomaterials should undergo rigorous study to determine their biocompatibility before their use in human body. In-vitro cytotoxicity test is used as one of the screening tests for evaluating the biocompatibility of biomaterials and it is often conducted using cell line such as SaoS-2, L929 and MG63. Prideaux et al., [15] demonstrated the usefulness of SaoS-2 cells in their in-vitro cytotoxicity study for determining the biocompatibility of biomaterials. Przekora, [41] reviewed the in-vitro cytotoxicity assay using SaoS-2 cell line in in-vitro model and concluded that SaoS-2 cell line was more suitable to evaluate the biocompatibility of biomaterials.

In the present study the properties such as adhesion, the viability and proliferation rate of SaoS-2 cells on the surfaces of Ti, Ti-Ga and Ga-Si-Ti alloy samples were studied. The adhesion of SaoS-2 cells on the surfaces of the alloys clearly indicated that the biocompatibility of these alloys using in-vitro cell line models. The SEM images of the study revealed that the cells were well attached over the surfaces of all the three sample of the alloys.

The Alamar Blue viability assay and the proliferation assay in this study suggested that the Ti, Ti-Ga and Ga-Si-Ti alloy samples can support and promote the cell growth on their surfaces. Even though there were no difference observed in the adhesion and proliferation of SaoS-2 among the three samples a slight decrease in the viability assay was observed with Ga-Si-Ti alloy sample and this could be related to the alkaline phosphatase activity of SaoS-2 cell line as reported by [42]. It is demonstrated in the present study that all the SaoS-2 cells which were viable proliferated throughout the cell culture experiment. This indicate the all the three alloys are not cytotoxic, among the three Ti showed more proliferation than Ga-Si-Ti and Ti-Ga.This observation of the present study is in agreement with [42].

This finding concurred with the observation of [43] where a decreased SaoS-2 cells viability in the presence of SiO₂. Many studies confirmed that the alloy Ti is non-toxic due to their unique ability that its surfaces can form TiO₂ has a stable passive film layer, making it superior in terms of biocompatibility. In a review by [44] mentioned that the Ti based biomaterials such as Ti and Ti 64 alloys exhibited good cell adhesion in the in-vitro studies and they opined that the Ti and its alloys are more promising biomaterials. Chandler et al., [45] evaluated the cytotoxicity of Gallium and reported that Ga has no significant cytotoxicity. The present in-vitro cytotoxicity study also revealed that the metals Gallium and Silicon were also non-toxic since the study revealed that the samples Ti-Ga and Ga-Si-Ti showed adhesion and proliferation similar to Ti alloy sample.

These observations were in agreement with the in-vitro cytotoxicity studies of [2] for Ti-Ga alloy and [28] for Ti-Mo-Si alloy. Cochis et al., [46] also confirmed that addition of the 2% and 20% Gallium to Titanium alloys containing 3% Silicon were nontoxic in their cytotoxicity evaluation study. Even though review of literatures revealed that the Ti, Ti based alloys biomaterials containing Gallium and Silicon are biocompatible, there is no in vitro studies to demonstrate the biocompatibility aspect of 15% Ga and 5% Silicon in Titanium alloy for comparison of Ga-Si-Ti alloy of the present study.

4.5 Mineralization

Mineralization is a process by which cells deposit inorganic calcium and phosphate on the surface of biomaterials and this mechanism of mineralization in the in-vitro research is focused on the formation of bone nodules and around the implants [47]. In the present study the Alizarin Red dye exposed the Calcium Phosphate mineralization of SaoS-2 cells at 4th, 7th, 14th and 21st days of culture with Ti, Ti-Ga and Ga-Si-Ti alloy samples in the cells. The SEM EDX examination indicated the presence of spherulites on the surfaces of each of the three alloys and the quantitative analysis with Leica stereomicroscope showed an enhanced number of calcified nodules on the Ga-Si-Ti alloy (212 ± 2.8) than Ti (130 ± 4.36) and Ti-Ga (137 ± 2.65) alloys on 21st day of observation and during the different days of study period. The statistical analysis showed a significant difference between Ti and Ti-Ga as well as between Ti and Ga-Si-Ti in calcified nodule formation. A considerable difference was also detected between Ti-Ga and Ga-Si-Ti alloys. In this study, it is clearly observed that the Ga-Si-Ti alloys has a maximum calcified nodules at the end of 21st day of experiment. Hence it is inferred from the present study that the presence of Silicon in Ga-Si-Ti alloy has enhanced the proliferation and Calcium, Phosphate nodule formation. He et al., [43] obtained similar results in the presence of Silica nanoparticles with SaoS-2 cells. Martinez-Ibanez et al., [48] also confirmed the increase in proliferation and mineralization with Si coated Ti alloy in their in-vitro study using mesenchymal stem cells.

The current findings with Ga-Si-Ti also concurred with the work of [49] where they observed a decreased viability and significantly enhanced proliferation and mineralization nodule formation in the presence of silicate ions. Hence based on the observations of this in-vitro study on the adhesion, viability proliferation and Ga-Si-Ti alloy with enhanced mineralization characteristics is in-vitro biocompatible and a potential alloy for the manufacture of dental biomaterials.

4.6 In-vivo radiographic study

To evaluate the position of the implants in the implant bed, regular radiographs were taken. Qualitatively by using radiographic images. The decreased grayscale is indicating bone mineral loss, while an increase in grayscale indicates bone mineral gain. Macroscopic variations in bone mineral density for a localized change can be visualized in normal radiography if it is more than 12%. Hence the digital radiograph was done to evaluate the bone density changes that can be detected with follow-up radiograph by using digital radiograph even if there is 1–5% (Matteson et al. 1996).

Khojastepour et al., [50] and Han et al., [51] assessed the implant position and new bone formation qualitatively using digital radiographs.

In the present study, the bone mineral density around the implant was evaluated using the Posterior-Anterior view of whole-body radiography and Paralleling radiography qualitatively and quantitatively. The quantitative evaluation was done by using density calculation software.

In the posterior-anterior view of whole-body radiographic study on bone density of medial cortical, lateral cortical, Trabecular bone and new bone formation along the implant were calculated for the implanted bones with adjacent side normal bone on the 14th, 28th, 42nd and 56th day after implantation in Ti, Ti-Ga and Ga-Si-Ti groups and compared.

The PA view of the whole-body radiographic study on the bone changes around the implants showed a substantial alteration in bone mineral density and the analysis revealed the highest bone mineral density in the Ga-Si-Ti group when compared to Ti-Ga and Ti group. The changes were markedly noted along with the trabecular bone and newly formed bone densities between Ti and Ti-Ga on 28th and 42nd day and Ti and Ga-Si-Ti on 28th, 42nd and 56th day, whereas during the investigation, there were no significant differences in bone density between Ti-Ga and Ga-Si-Ti, indicating that there is little change in the newly formed bone between the two groups.

When compared with implanted and adjacent side non implanted bone, no change was observed in the medial cortical and lateral cortical bone mineral density, since there was no variation in the nutrition and management of experimental rats.

Whereas with trabecular bone mineral density assessment, the Ti groups showed decreased density on the 14th, 28th and 42nd days. This observation is correlated with inflammation changes, whereas the radio-opaque appearance clinically on the 56th day proved that there is healing and osteocyte formation. This is in agreement with the observation of [52] in which the whole body of rat radiographic evaluation was done.

However, the radio-opaque region was observed with Ti-Ga and Ga-Si-Ti groups on the 28th day itself. This difference between Ti-Ga and Ga-Si-Ti group alloys is attributed to the presence of Ga elements in Ti-Ga group alloy and Ga and Si elements in Ga-Si-Ti group alloy and their role in osseointegration. This is in agreement with the observation of [53].

The assessment of newly formed bone in Ti, Ti-Ga and Ga-Si-Ti group implants revealed no mineralization changes on the 14th day and it was observed clinically as radiolucent legion. Whereas the changes in newly formed bone in the Ti group were found to be significantly increased on the 28th, 42nd and 56th day. However, the density observed on the 56th day was near approximate to the cortical bone of non-implanted bone on the adjacent side, whereas this observation was observed on the 42nd day in the Ti-Ga group and the 28th day within the Ga-Si-Ti group respectively. This is similar to the observation in the in-vitro study on Si plays in bone anabolic regenerative approaches by [54].

The bone mineral density study in the present experiment is also similar to the study conducted by [50] in mice and based on this study it is suggested that the PA view radiograph technique can also be used for the evaluation of the bone mineral density of new biomaterials.

4.7 CT study

The bone mineral density of cortical, trabecular and newly formed bone around the implants was measured in the Hounsfield unit in this CT investigation. On the 28th day, there was no significant difference between groups in cortical, trabecular, or newly formed bone. On the 56th day, the Ti-Ga and Ga-Si-Ti implants had a high bone mineral density in the cortical, trabecular and newly formed bone, whereas the Ti-Ga and Ga-Si-Ti implants had a high bone mineral density in the cortical, trabecular and newly formed bone. It is evident from the study that Ti-Ga and Ga-Si-Ti showed increased bone mineral density, which infers that there was good osseointegration in Ga-Si-Ti and Ti-Ga group implants than Ti implants. This is in agreement with the observation of [55].

In the present CT Study, the study provided in-situ details of mineralization of cortical, trabecular and newly formed bone and the osseointegration of the implants. Hence it is inferred that the increased mineralization in trabecular bone observed in the study is indicative of promotion of osseointegration by the presence of metal elements Gallium and Silicon in Ga-Si-Ti alloy implants. This is in agreement with the observation of [56].

4.8 Micro-CT study

However, the observation on trabecular volume (BV/TV), bone mineral density, trabecular thickness, mean trabecular number, mean trabecular separation revealed a better osseointegration in Ga-Si-Ti implant and it was also can be correlated with histo-morphometric study. The observation on increase in trabecular bone volume around the Ga-Si-Ti and other implants is in agreement with micro-CT study of [57] Further in the present study the BV/TV is 25% and it corresponds with increase in trabecular bone which is indicative of better osseointegration and it is good in all the three groups.

4.9 Histology and Histomorphometric study

Histological analysis revealed the new bone formation between the implant surface and the existing bone. Good osteoblastic activity and mononuclear cells are observed in Ti-Ga alloy implants. New bone formation around implants with Chondrogenic foci, with lacunae and chondrocyte were seen. Intra-peritoneal injections of Alizarin, Tetracycline and Calcein made it possible to evaluate the kinetics of bone growth and bone remodeling histologically around the implants. A higher mineral apposition was detected at 4 weeks was greater than that found at 8th weeks in all three groups. Bone Implant Contact (BIC), Mineral Apposition Rate (MAR) and Osteoblast proximity were the parameters evaluated. Ga-Si-Ti alloy implants have exhibited maximum BIC ∼ 98%. Ga has antiresorptive effects on bone and bone fragments, preventing osteoclast resorption.

5. Conclusion

It is evident from the present investigation that the addition of Gallium and Silicon elements with Cp-Ti alloy has improved the mechanical characteristics of Cp-Ti alloy. Studies conducted in vitro demonstrated that all three alloys are bioactive and non-cytotoxic. In the animal experiment, the enhanced osseointegration property of Ga-Si-Ti alloy implants is related to the synergistic anti-resorptive action of Gallium and osteoblastic property of Silicon, in addition to the benefits of Cp-Ti alloy.

This research concludes by stating that the combination of elements Gallium and Silicon to Titanium has not only enhanced the mechanical and physical properties of Cp-Ti alloy, but also the biological qualities of Cp-Ti alloy and that the newly synthesized Ga-Si-Ti alloy implant may be a promising biomaterial in the dental field.

Conflict of interest

The authors declare no conflict of interest.