Impact of Sintering Duration on the Mechanical and Bioactive Properties of Pure Ti, Ti-Al Alloy, and Ti-Al-HAp Composite for Biomedical Applications

Orthopedics, a clinical medicine subdivision, focuses on diagnosing and treating illnesses and injuries of the musculoskeletal system, including extremities and the spine. It has evolved significantly due to advancements in biomaterials science [1]. Metals such as cobalt and titanium-based alloys are favored in orthopedic applications due to their high strength, fracture resistance, and suitable biocompatibility [2, 3]. Commercial pure titanium (CP-Ti) and its alloys are widely used in orthopedic implants in the human body, including dental implants, crowns, bridges, joint replacements, bone fixation plates, and more [4] for their mechanical stability and inertness to biological reactions [5]. CP-Ti and its alloys exhibit excellent biocompatibility, high corrosion resistance to body fluids, and desirable mechanical properties such as a high strength-to-weight ratio [6].

For non-load-bearing applications, titanium and its alloys are recommended for their excellent ability to integrate well with bone tissue without causing significant adverse effects [7]. To meet implants' demanding mechanical and biological requirements, titanium alloys are processed using various techniques, such as powder metallurgy (P/M), casting, cold working, machining, hot working, and additive manufacturing [8]. The two main techniques for manufacturing Ti alloy implants are powder metallurgy and casting [9]. The P/M method, in particular, is favored due to its cost-effectiveness and ability to produce near-net-shape parts with excellent surface quality [10]. This technique has been instrumental in fabricating titanium alloys with optimized properties for biomedical applications [11]. The success of the P/M process (including powder mixing, compacting powder, and the sintering process [12]) depends on thorough characterization and control of the metal powder, including the distribution of particle size, structure, sintered material characteristics, and behaviors during compression and sintering stages [10].

Despite the advantages of titanium and its alloys, challenges remain in optimizing their properties for biomedical applications. The processing conditions, such as sintering temperature and time, as well as the incorporation of other elements like aluminum and hydroxyapatite, significantly influence the material’s final properties, including mechanical strength, micro-hardness, and biocompatibility [13]. The sintering temperature directly depends on the melting temperature of the base element (Ti), which is approximately 1678℃ [14]. The sintering temperature optimum range is usually limited between 60% to 80% of the melting temperature of the base element, therefore, in this case, the sintering temperature is 1,006℃ to 1,342℃. The high sintering temperature led to a closure of the pores and reduced their total number, causing them to be of a spheroidal shape in morphology [15]. Longer sintering times improve densification but at diminishing rates due to the gradual loss of the driving force for sintering. Extended sintering time offers diminishing benefits after the material reaches a near-equilibrium state [16]. By optimizing these parameters—temperature and time, the sintering process can be tailored to produce materials with specific properties required for biomedical and industrial applications [17].

One challenge in using titanium alloys for biomedical applications is their limited biocompatibility, primarily due to their bioinert nature. The hydroxyapatite (HAp) with the chemical formula of Ca₁₀(PO₄)₆(OH)₂ is commonly incorporated into titanium alloys to address this limitation and is the greatest hopeful bio-ceramic material because of its exclusive biocompatibility properties. The presence of Calcium-phosphate ceramic (bioactive oxide) in this material leads to improved bioactivity. The HAp is an outstanding bioactive agent transporter for the growth of tissue due to its composition similar to that of natural bone [18]. The HAp incorporation provides the calcium (Ca) and phosphorus (P) distribution, which could aid better bone osseointegration than implants made of pure metal alloys. Combining Ti alloys with HAp ceramic creates a unique bio-material with excellent biological and mechanical properties [19]. Moreover, adding aluminum (Al) to titanium further enhances its mechanical properties, such as tensile strength, hardness, and fatigue resistance, making it more suitable for high-stress applications like joint replacements and bone fixation. The addition of aluminum also helps to improve the material’s resistance to wear and corrosion, which are critical factors in the longevity and reliability of implants. This makes titanium-aluminum alloys highly desirable for use in the medical field, especially for load-bearing implants [20].

Omidi et al. [21] utilized spark plasma sintering (SPS) to fabricate titanium-hydroxyapatite (HAp) composites with varying HAp content (0%-40%) in multiple layers. They observed an increase in grain size and an initial rise in Vickers microhardness, which decreased at higher HAp content. The maximum compressive strength was achieved with five-layer samples, emphasizing the need to optimize HAp content. However, sintering conditions and the impact of alloying elements like aluminum were not explored, which is a focus of the current study. Kobayashi et al. [22] investigated the biocompatibility of Ti-HAp composites fabricated by compressive shear, using MC3T3-E1 mouse osteoblasts. They found that HAp remained stable at 800℃ but decomposed slowly at 1100℃, with higher HAp content reducing cell proliferation. This study provided insights into biocompatibility but did not address the influence of sintering temperature on mechanical properties or the combination of titanium with other elements, a topic covered in the present research [22]. Farrahnoor and Zuhailawati [23] studied Ti-Nb-HAp composites through powder metallurgy, finding that XRD analysis revealed phases like TiO₂ and Ti2P as HAp content increased. Maximum bioactivity occurred at 15 wt.% HAp, though sintering conditions were not considered. The current research aims to investigate both the mechanical and biological properties of Ti-based composites under varying sintering conditions.

Shanmuganantha et al. [24] fabricated Ti-HAp and Ti-CaSiO₃ composites using hot press compression and vacuum sintering, showing good biocompatibility with human osteoprogenitor cells. While they emphasized bioactivity and titanium strength, the study did not examine the effect of alloying elements like aluminum on mechanical properties, which this research addresses [24]. Arivazhagan et al. [25] developed Ti-6Al-4V implants enhanced with HAp, significantly improving biomechanical and biological performance. While they demonstrated HAp’s potential for osseointegration, the study did not explore sintering conditions or alloying elements, which will be investigated in the present work [25]. Sadlik et al. [26] fabricated porous Ti/HAp composites with varying HAp content using powder metallurgy. They observed that introducing HAp decreased microhardness but increased biological activity, though the effects of sintering conditions or alloying elements were not addressed. These aspects will be further explored in this study. Yaşar and Ekmekci [27] studied the effects of micro- and nano-HAp powder sizes on surface topography and biocompatibility of Ti-6Al-4V alloy using Electrical Discharge Machining (EDM). They found that micro-HAp reduced surface roughness and improved wettability, while nano-HAp had minimal effects. However, this study did not investigate bulk mechanical properties, such as hardness and compressive strength, which will be examined in the current research [27]. While studies have examined the interaction of titanium with hydroxyapatite or aluminum separately (e.g., Omidi et al. [21]; Yaşar and Ekmekci [27]), no comprehensive research has investigated the combined effect of all three elements in a single sintering process.

Our study contributes a new perspective by examining the impact of this three-component mixture and provides quantitative evidence of its influence on the mechanical and microstructural properties of the sintered material.

Although previous studies have explored the biocompatibility and mechanical properties of Ti-HAp composites, there is a gap in understanding the combined effects of aluminum and HAp in Ti-based composites, particularly with varying Ti particle sizes. The impact of sintering time on mechanical and biological properties has also not been thoroughly investigated. This study aims to fill these gaps by preparing Ti-based materials (CP-Ti, Ti-6%Al alloys, and Ti-6%Al-2%HAp composites) using powder metallurgy, focusing on optimal sintering times and assessing density, compressive strength, micro-hardness, and biological properties for biomedical applications.

3.1 The powder characterization

3.1.1 Chemical composition characterization

Elemental analyses of the powders (Ti1, Ti2, Al, and HAp) were performed by X-ray fluorescence (XRF) analysis using the SPECTRO Analytic Instrument model (XEPOS) at 100 V.

3.1.2 Particle size characterization

The particle sizes of each powder (Ti1, Ti2, Al, and HAp) were measured using a DLS Bookhaven 90Plus Particle- USA Size Analyzer following ASTM B822-20 [32].

3.2 Sample characterization

3.2.1 The density and relative porosity measurements

The apparent density, relative density, and relative porosity of each sample produced after sintering at various sintering times were calculated using the Archimedes method with a density measurement according to ASTM ISO3369:1975 standard procedures. Each sample's dry weight (W_d) was measured. Subsequently, these samples were immersed (saturated) in water for 24 hours, followed by measuring their weight (w_n), and were calculated according to the subsequent rule in Eq. (1) [33]. Three readings were taken for each sample, and the average apparent density was calculated. Additionally, the values were compared with the standard deviation, and the error bar was determined. The theoretical density (ρ_theoretical) for each specific composition is 4.5 g/cm³ for 100% Ti, 4.392 g/cm³ for 94% Ti-6Al, and 4.3648 g/cm³ for 92% Ti-6% Al-2% HAp. Each sample's relative density (ρ_relative) was calculated according to Eq. (2) [34]. Finally, the relative porosity of each sample was determined according to Eq. (3) [35].

ρ_{a$=\frac{wd}{wd-wn}*\check{D}$}                   (1)

ρ_{relative$=\frac{\rho a}{\rho theoretical}$}                   (2)

Relative Porosity=$\left\{ 1-\left( \frac{\rho relative}{~\rho theortical} \right) \right\}*100$            (3)

where,

ρ_a = the apparent density (g/cm³),

w_d = the dry sample weight (g),

w_n = the immersed test sample weight (g), and

$\check{D}$ = the water density (g/cm³).

3.2.2 Compression tests

The compression strengths of all samples were determined following the ASTM E9 standard using a universal testing machine (Laryee Technology Co. LTD) [36].

3.2.3 Microhardness tests

The microhardness of all samples was measured using a microhardness testing apparatus (LAYREE, Netherlands) following ASTM E18-07. Vickers hardness testing was performed by applying a pyramidal-shaped diamond indenter to the sample surface under a load of 5 N for 15 seconds [37].

3.3 Extra Inspections

The best-performing sample from the tests above was exposed to extra inspections such as XRD, SEM, EDS, AAS, and MTT assay. 

3.3.1 X-ray diffraction inspection

The microstructural phases of the best-performing sample in this study were analyzed using X-ray diffraction (XRD). The measurements were performed with a Shimadzu X-Ray Diffractometer (model XRD-6000), which employs a Cu X-ray tube and operates within a 2θ range of -6° to 163°, with an angular resolution of 0.002°.

3.3.2 Morphology inspections

The surface morphologies of the best-performing sample were analyzed using scanning electron microscopy (SEM), while the detailed elemental composition and distribution were determined through energy dispersive X-ray spectroscopy (EDS), both performed using a Thermo Scientific Axia model Scanning Electron Microscope (SEM) made in the Netherlands.

3.3.3 Atomic Absorption Spectroscopy (AAS) characterization

Atomic Absorption Spectroscopy (AAS) was employed to quantify the ions released at three specific time intervals (3, 7, and 14 days) from the best-performing sample in this study. The measurements were performed in Ringer’s solution, a fluid formulated to mimic the ionic composition of human body fluids, ensuring physiological relevance in the analysis (the composition of Ringer’s solution is shown in Table 3). The sample was immersed in a 100 mL glass beaker containing Ringer’s solution, incubated in a water bath at 37℃, and aliquots of the solution were extracted using a syringe at 3, 7, and 14 days. Ion release was monitored throughout the 14-day immersion period. The extracted solutions were analyzed using a novAA 400P atomic absorption spectrometer (Germany). Figure 2 illustrates the procedure of this test [38].

Table 3. Components of Ringer’s solution

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Figure 2. The incubation during the AAS testing process

3.3.4 MTT assay characterization

The MTT assay, a tetrazolium-based method, was employed to evaluate cell viability (%) and cytotoxicity (%) using the Promega GloMax Discover GM3000. MG63 human bone fibroblast cells (10³ cells/well) were seeded in 100 μL of DMEM, supplemented with Ti-6%Al-2%HAp powder (0.1 ng/mL) in its nanoscale form (nHAp). The cells were incubated at 37℃ for 24, 48, and 72 hours, with careful monitoring of incubation conditions to maintain optimal growth, including a consistent 5% CO₂ level to preserve pH balance in the medium. Following the incubation periods, 10 μL of MTT reagent was added to each well and incubated for an additional 4 hours. This reagent is reduced by viable cells to form purple formazan crystals. After incubation, formazan crystals were solubilized by adding 100 μL of DMSO and allowing the reaction to complete at room temperature for 24 hours. The absorbance was then measured at 550 nm using a spectrophotometer to quantify the formazan produced as shown in Figure 3. Control wells (untreated cells) showed approximately 100% viability, and the cell viability of the treated samples was calculated using the following formula [39]: 

Cell viability (%) = (Mean absorbance of treated cells / Mean absorbance of control cells)*100%.                    (4)

In this study, the response of MG63 cells to Ti-6%Al-2%HAp powder was also assessed through the MTT assay. The results of the mitochondrial activity assay demonstrated that the tested sample had no toxic effects on the MG63 cells, with cell viability rates remaining above 70% after incubation for 1, 2, and 3 days. These findings support the biocompatibility of Ti-Al-HAp, confirming its potential as a viable biomaterial for biomedical applications, including implants.

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Figure 3. Block diagram of MTT assay

5.1 Particle size analysis

Figures 4-7 show the examination chart revealed effective diameters of the starting powders, 484.9 nm for Ti1 powder, 174.6 nm for Ti2 powder, 112.5 nm for Al powder, and 72.5 nm for HAp powder, respectively, with minor size variations. Using a combination of coarse and fine particles in sintered materials can significantly impact both compressibility and microhardness. The fine particles fill the voids between the coarse particles, leading to a denser structure that reduces compressibility. This results in a material that is less prone to deformation under pressure.

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Figure 4. Size distribution of Ti1 powder

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Figure 5. Size distribution of Ti2 powder

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Figure 6. Size distribution of Al powder

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Figure 7. Size distribution of HAp powder

Additionally, the mixture of particle sizes enhances the bonding between particles during sintering, improving the material’s overall hardness. The fine particles contribute to a more uniform distribution of stress, which increases the microhardness by enhancing resistance to scratching and wear. However, an imbalance in the ratio of coarse to fine particles can lead to variability in hardness due to weaker regions in the material [40]. Particle size plays a crucial role in the sintering process. A reduction in particle size enhances the sintering process. Smaller particles possess a larger pore-to-solid interfacial area, which generates a stronger driving force for sintering. This leads to increased diffusion mechanisms. Greater surface area accelerates surface diffusion, finer grain sizes facilitate grain boundary diffusion, and larger interparticle contact areas promote volume diffusion. Table 4 shows the powder properties of each material used in this work.

Table 4. Starting powder properties

Component	Particle Size (nm)	Purity (%)
Titanium1 (Ti1)	484	98.81
Titanium2 (Ti2)	174	98.76
Aluminum (Al)	112	98.32
Hydroxyapatite (HAp)	72	95.43

5.2 X-ray fluorescence (XRF) analysis

Figures 8-11 show the chemical analysis results for (Ti1, Ti2, Al, and HAp) powders, respectively. XRF analysis reveals the purity of each powder, along with the elements that appeared as impurities and their respective percentages. This analysis also showed that Ti1 powder, Ti2 powder, Al powder, and Hap powder concentrations reached 98.81%, 98.76%, 98.32%, and 95.43%, respectively. This figure shows that the powders used to produce these alloys are pure. The XRF analysis of as-received powders confirms the existence of the essential elements.

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Figure 8. XRF analysis of Ti1 powder

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Figure 9. XRF analysis of Ti2 powder

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Figure 10. XRF analysis of Al powder

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Figure 11. XRF analysis of HAp powder

5.3 Density and porosity results

Figures 12-14 present the density measurements for the various samples, showing the apparent density (g/cm³), relative density (%), and relative porosity (%) as functions of sintering times (60, 90, and 120 minutes). The data indicate that both apparent and relative densities increase, while relative porosity decreases, with extended sintering times. Specifically, increasing the sintering time from 60 to 120 minutes resulted in significant increases in both apparent and relative densities, accompanied by a decrease in relative porosity. The highest density values for all compositions (group C: T_C, TA_C, and TAH_C) were recorded at a sintering time of 120 minutes. These results highlight the crucial role of prolonged sintering in reducing porosity and enhancing densification.

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Figure 12. Apparent density – sintering time chart

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Figure 13. Relative density – sintering time chart

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Figure 14. Relative porosity - sintering time chart

Among all the samples, TAH_C—sintered at 1,300℃ for 120 minutes—exhibited the highest relative density (93.31%) and the lowest relative porosity (0.7862%), with an apparent density of 4.067 g/cm³. Longer sintering times facilitate pore closure, reducing pore number and promoting spheroidal pore structure formation [41]. Furthermore, the incorporation of aluminum (Al = 2.7 g/cm³) and hydroxyapatite (HAp = 3.14 g/cm³) contributes to enhanced densification, bringing the measured densities closer to the theoretical density of titanium (4.5 g/cm³). Notably, aluminum aids in the formation of the α-phase in titanium alloys, which is less dense than the β-phase [42].

The composite samples consist of particles of various sizes: Ti1 (484 nm), Ti2 (174 nm), Al (112 nm), and HAp (72 nm). The moderate porosity observed, which is typical of titanium-based implants, facilitates tissue adhesion while maintaining sufficient mechanical strength. the porosity level also hurts (negative effect) the biomaterial implant's mechanical properties. The porosity presence can reduce the mechanical strength of the biomaterial implant.  In the structure of a material, the pores can perform as stress concentrators, which leads to fatigue strength reduction [43].

This balance is crucial for minimizing adverse biological reactions and promoting healing [44]. Additionally, the relative density of sample TAH_C (93.31%) aligns with typical values reported for titanium-based implants, which range from 85% to 95% [45], while the density of Ti-6Al-4V alloy typically ranges between 4.43 to 4.48 g/cm³ [46].

Hoseinzadeh et al. [47] investigate how varying sintering times affect the microstructure, phase, density, and mechanical and tribological properties of Cf/Si₃N₄ composites. Findings indicate that optimal sintering time enhances densification.

5.4 Compression test results

Figure 15 shows each sample's compressive strength (MPa) as a function of sintering times. The data indicate that the compressive strength of the samples increases with longer sintering times. Specifically, the compressive strength continues to rise with sintering times of 60, 90, and 120 minutes, reaching its peak at 120 minutes for group C. The maximum compressive strengths of group C (T_C=511 MPa, TA_C=492 MPa, and TAH_C=476 MPa) were achieved under the highest sintering conditions (1,300℃ for 120 minutes), which also led to a reduction in porosity, as previously discussed.

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Figure 15. Compressive strength - sintering time chart

The varying particle sizes (Ti1=484 nm, Ti2=174 nm, Al=112 nm, HAp=72 nm) contributed to improved toughness by enhancing phase homogeneity and particle interactions, resulting in increased material strength and resistance to deformation. However, the addition of aluminum (Al) and hydroxyapatite (HAp) to titanium (Ti) led to a decrease in compressive strength, as expected. While pure Ti exhibits high strength, the incorporation of Al alters its microstructure, and HAp, although beneficial for biocompatibility, is brittle and does not significantly contribute to compression strength [48]. The compressive strength of pure Ti typically ranges from 130 to 170 MPa, with some reports indicating values between 240 and 550 MPa depending on factors such as purity and processing conditions. In comparison, the compressive strength of human trabecular bone is between 2 and 80 MPa [49] while the compressive strength of Ti-6Al-4V alloy typically ranges between 1074 to 1661.6 MPa [50]. D. Kumar, E. Zhang, and F. Nguyen focus on the effect of sintering duration on the mechanical strength and bioactivity of Ti-6Al-4V/HAp composites. Results show that an optimal sintering time balances mechanical properties with bioactivity [51].

5.5 Micro-hardness test results

Figure 16 illustrates the micro-hardness test results as a function of sintering times. The measurements were conducted using a 4.9 N load with a dwell time of 15 seconds per sample. The results indicate a gradual increase in micro-hardness (HV) with increasing sintering time (60, 90, and 120 minutes). The micro-hardness continued to increase with longer sintering times, reaching its peak at 120 minutes for all compositions in group C (T_C = 379 HV, TA_C = 351 HV, and TAH_C = 298 HV). The observed increase in hardness is attributed to the enhanced densification at longer sintering times (1,300℃ and 120 minutes), which reduces porosity and strengthens the material. Additionally, the variation in particle size during fabrication improved phase homogeneity and particle interactions, leading to increased resistance to deformation. Ti (Ti1 and Ti2), Al, and HAp particles contributed to this effect by minimizing crack propagation and enhancing material toughness. Furthermore, the addition of Al and HAp to Ti resulted in lower hardness [26, 52]. The Vickers hardness of Ti-6Al-4V typically ranges from 330 to 400 HV [53].

The standard deviation and standard error for the obtained results as shown in Tables 5-7.

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Figure 16. Microhardness -sintering time chart

Table 5. Statistical results of apparent density

Group	Sample	Std. Deviation	Std. Error
A	TA	0.0265	±0.57
	TAA	0.0165	±0.52
	TAHA	0.0392	±0.51
B	TB	0.00896	±0.53
	TAB	0.0117	±0.48
	TAHB	0.00711	±0.51
C	TC	0.02057	±0.38
	TAC	0.01143	±0.31
	TAHC	0.00941	±0.3

Table 6.  Statistical results of microhardness

Group	Sample	Std. deviation	Std. error
A	TA	0.54	±1.18
	TAA	1.47	±0.85
	TAHA	1.87	±1.08
B	TB	2.54	±1.47
	TAB	1.85	±1.07
	TAHB	1.34	±0.77
C	TC	0.57	±1.48
	TAC	1.07	±0.62
	TAHC	1.02	±0.59

Table 7. Statistical results of compression.

Group	Sample	Std. deviation	Std. error
A	TA	3.86	±4.58
	TAA	4.55	±3.22
	TAHA	6.68	±8.19
B	TB	6.16	±4.36
	TAB	6.21	±4.32
	TAHB	3.73	±2.65
C	TC	5.29	±4.25
	TAC	2.95	±2.08
	TAHC	3.74	±2.65

5.6 X-ray diffraction (XRD) analyzing results

Figure 17 illustrates the XRD patterns obtained from the best-performing sample, TAH_C. The multiple peaks observed indicate the presence of various compounds resulting from the decomposition of hydrocarbons and their interaction with Ti and Al. The XRD analysis of TAH_C identified the following compounds: TiP₂O₇, Ti, Al₂Ti, CaTiO₃, TiO₂, CaTi₄P₆O₂₄, and Na₂Ti₃O₇, along with their respective standard cards. In the context of biomaterials, analyzing the presence of compounds such as TiP₂O₇, TiO₂, Al₂Ti, Na₂Ti₃O₇, CaTi₄P₆O₂₄, and CaTiO₃ formed after sintering Ti with Al and hydroxyapatite (HAp) at high sintering time (120 minutes at 1,300℃) provides valuable insights into their potential effects on biocompatibility, mechanical properties, and stability. For example, CaTiO₃ has attracted interest for its bioactivity, especially in bone repair applications. It demonstrates compatibility with bone tissue and can promote apatite formation on its surface when exposed to bodily fluids, which is an indicator of bioactivity. Additionally, it has good chemical stability and hardness, which could contribute to mechanical integrity in load-bearing implants. The combination of Ca and Ti in CaTiO₃ can be beneficial in orthopedic implants and bone scaffolds, as calcium is crucial for bone cell signaling and formation, while Titanium Pyrophosphate (TiP₂O₇) has structural stability and wear resistance, but limited research on its specific interactions with biological systems exists. However, phosphate-based compounds promote osteoconductivity, potentially benefiting bone integration in implants or bone grafts. The presence of these ceramic compounds in sample TAH_C leads to no need for coating or additional processes that make the product more biocompatible [54-61].

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Figure 17. XRD patterns analysis results of the sample TAH_C

5.7 Analysis results of energy dispersive X-ray spectroscopy (EDS)

Figure 18 illustrates the EDS analysis results of the best-performing sample TAH_C. This test was conducted to examine the effect of adding 2% HAp and 6% Al with Ti on the microstructure of the sample and their impact on other properties of Ti elements produced by P/M technology. The chemical surface analysis revealed significant peaks for titanium, aluminum, calcium, phosphorus, and oxygen, with a small amount of silicon from the raw materials. EDS examination showed that the sample TAH_C had high purity.

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Figure 18. EDS analysis of the sample TAH_C

5.8 SEM analysis

Figure 19 shows the SEM examination conducted on the best-performing sample TAH_C. Observation is shown in Figure 13, displaying the sample mentioned above at 50 µm. The grains and pores in this figure exhibit varying sizes and dimensions, with particle (grain) sizes ranging from approximately 16 µm to 40 µm in TAH_C.  SEM analysis revealed greater homogeneity in particle size distribution within the sample TAH_C, contributing to its higher sintering time (120 minutes at 1,300℃). The Gwyddion application was used to determine the Roughness Average (Ra) in nanometers (nm) for the sample TAH_C. Based on the SEM image of the sample, the surface roughness average of this sample (3D image) was obtained. The Roughness Average R_a (nm) of the sample TAH_C is 137nm. This result was obtained because of the present nanoscale particle size in HAp (72nm) and Al (112nm), as well as being affected by higher sintering time [62]. The nanostructure material caused the implant surface to have a nano-roughness surface, with a nano-roughness surface, (less than 1 μm). The surface with this nano-roughness improves the adsorption of a protein and the cellular responses, leading to more and quicker, reliable osseointegration [63].

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Figure 19. SEM image of sample TAH_C

5.9 Atomic Absorption Spectroscopy (AAS) analyzing results

This study measured the ion release from the best-performing sample TAH_C in Ringer’s solution. After 14 days in Ringer’s solution, the maximum Titanium ion release was 0.912 ppm from TAH_C, as shown in Figure 20. This result is consistent with previous studies, which have reported the ion release of titanium in biological solutions, where titanium ions are typically released in low concentrations due to corrosion or surface degradation [64]. After 14 days in Ringer’s solution, the Aluminum ion release from the sample TAH_C was 0 ppm, as illustrated in Figure 14. This finding suggests minimal or no release of aluminum, which is consistent with the inert behavior of aluminum in the titanium-based alloys under the experimental conditions [65].

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Figure 20. Atomic Absorption Spectroscopy analysis of sample TAH_C

Titanium, known for its non-toxicity, is naturally removed by the body and bonds well with bone. The titanium ion release is usually minimal due to its high biocompatibility, and the body can effectively handle and eliminate any excess titanium ions [66]. It has been shown that when titanium ions are released, they do not accumulate to toxic levels and are excreted without causing harm [67]. Furthermore, the ion release stabilizes after a period due to adsorption processes, with various responses occurring on the implant surface when interacting with bone [68]. Adsorption of metal ions is maintained by cell generation at the implant’s surface, preventing further ion release and ensuring the material’s long-term stability and biocompatibility [69].

5.10 MTT assay: Analyzing results

Formazan crystals, when dissolved, show absorbance at 500-600 nanometers, with a darker color indicating more viable cells. Materials like Titanium (Ti), Aluminum (Al), and Hydroxyapatite (HAp) used in bone fixation are evaluated for their impact on the human body. Titanium is known for its corrosion resistance, durability, and biocompatibility, forming a protective titanium oxide layer that makes it non-toxic [70]. Aluminum enhances the mechanical properties but poses toxicity risks when accumulated in the body, though proper coating minimizes this risk [71]. Hydroxyapatite, a natural bone component, is highly biocompatible and aids bone healing by enhancing the interaction between the implant and the surrounding bone tissue [72]. Ti-Al-HAp materials degrade slowly, releasing minimal ions, typically remaining within safe limits unless under excessive corrosion, making them safe with low toxicity when adhering to medical standards [73].

The best cell viability recorded was 86.7% for cells on a powder mix after three days. Hydroxyapatite aids in bone integration, offering excellent biological and mechanical properties. The MTT assay showed a 13.3% cytotoxicity rate after three days, indicating moderate biocompatibility, which is considered acceptable in initial evaluations, as shown in Figure 21. Titanium’s interaction with tissues is minimal due to its inertness, while aluminum, linked to Alzheimer’s disease over extended periods, dissolves more quickly in powder form, releasing trivalent aluminum ions [74].  Aluminum itself is a controversial material that is controversial for human exposure. The accumulation of aluminum in the body can be toxic, as increased levels have been associated with health risks such as neurotoxicity and kidney disease. In TiAlHap, aluminum is incorporated with titanium and hydroxyapatite to form an integrated material with enhanced mechanical and chemical properties. Toxicity concerns revolve around the potential release of aluminum ions into the body due to corrosion or degradation. However, when these materials are properly manufactured and coated, the risk of aluminum leakage is low, reducing the potential health hazards. Hydroxyapatite is a natural component of human bones and teeth, making it a highly biocompatible material. It does not cause significant immune reactions or toxicity in the body [75].

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Figure 21. The toxicity of cell MG63 – time chart

Powder metallurgy technology at 1,300℃ transforms aluminum into a crystalline state, eliminating its toxicity long-term and addressing titanium’s inertness. Adding hydroxyapatite improves the interaction between bone and fixation plates, enhancing osteointegration, reducing toxicity, and aiding in healing damaged bones [76]. The cytotoxicity of the tested material was evaluated using the MTT assay, following the ISO 10993-5 guidelines for in vitro cytotoxicity testing. This standard provides a framework for assessing the biological effects of medical device materials on cultured mammalian cells. In this study, MG63 osteoblast-like cells were used to determine the viability and metabolic activity in response to the material extracts [77].

My supervisors: Prof. Dr. Niveen Jamal Abdulkader and Assistant. Prof. Dr. Laith Wadah, Prof. Dr. Kadum Kamel, all materials engineering department/ UOT staff, my family, and Mrs. Maysaloon Fakher (Iraqi writer).