Comparative Study of Surface Modification Techniques for Enhancing Biocompatibility of Ti-6Al-4V Alloy in Dental Implants

Abstract

This study investigates the effects of various surface modification techniques on the Ti-6Al-4V alloy for biomedical applications. Mechanical treatments (sandblasting, shot peening) and electrochemical corrosion using different electrolytes were employed to modify surface characteristics. Surface morphology, roughness, hardness, and chemical composition were analyzed using SEM, profilometry, and Raman spectroscopy. Cell attachment studies revealed that combined treatments, particularly shot peening followed by HF/HNO₃ etching, significantly enhanced cell adhesion and distribution. The results demonstrate the potential for tailoring Ti-6Al-4V surfaces to optimize biocompatibility and osseointegration properties for dental and orthopedic implants.

1. Introduction

The increasing prevalence of bone injuries and degenerative diseases, exacerbated by an aging population, accidents, and sports-related trauma, has led to a growing demand for high-quality, high-performance implants [1,2]. Among the elderly, dental-related issues, particularly tooth loss, represent a significant oral health concern [3,4]. Dental implants have emerged as the preferred solution, offering advantages such as improved oral health, enhanced phonation and mastication, the preservation of adjacent teeth and bone, and overall quality of life improvement [5,6,7]. In the realm of dental implant materials, titanium alloys, particularly Ti-6Al-4V (Ti64), have gained widespread recognition due to their optimal balance between mechanical properties and biocompatibility [5,8,9,10,11,12]. Ti64, a dual-phase (α + β) alloy, exhibits high strength, excellent corrosion resistance, and acceptable biocompatibility [13]. As the ninth most abundant element in the Earth’s crust and the fourth most abundant structural metal, titanium has become a cornerstone in biomedical applications since its development in the 1950s [14].

Despite the high success rate of titanium implant surgeries (>90%), a 10% failure rate persists. To mitigate this, implant surface treatments play a crucial role in enhancing osseointegration, defined as the direct structural and functional connection between living bone and the load-bearing implant surface [15,16,17,18,19,20,21,22,23,24]. The osseointegration process comprises four overlapping phases, namely, hemostasis, inflammation, proliferation, and maturation. These phases collectively lead to the formation and remodeling of bone tissue around the implant [25]. However, Ti64 alloys face several challenges in the complex physiological environment of the human body. Long-term use may lead to the release of potentially toxic Al and V ions [26,27,28]. The mismatch in elastic modulus between Ti64 and human bone can result in stress shielding, affecting bone tissue growth and repair [29,30,31]. Additionally, issues related to friction and wear can generate debris, potentially causing inflammation and adverse reactions. The inherent lack of antibacterial properties in Ti64 alloys also increases the risk of post-implantation infections.

To address these challenges, researchers have developed various surface modification techniques, broadly categorized into subtractive and additive processes [32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51]. Subtractive methods, such as sandblasting and acid etching, aim to create micron- or nano-scale surface roughness to enhance osseointegration. Sandblasting, utilizing compressed air to propel particles like TiO₂, Al₂O₃, or SiO₂, can increase surface roughness from 0.04 μm to 0.3–3 μm, significantly enhancing surface area and energy. However, this process may affect bacterial adhesion and biofilm formation. Chemical methods, particularly acid etching, are often employed to remove residual particles and further modify surface properties [52,53,54]. The combination of sandblasting and acid etching, known as the SLA (Sandblasted, Large-grit, Acid-etched) technique, has shown promising results in improving surface characteristics conducive to osseointegration [55,56,57,58,59,60,61]. Electrochemical methods, such as anodic oxidation, can create stable, ordered porous TiO₂ layers on the titanium surface. These nanotubular structures enhance the adhesion and osteogenic differentiation of bone marrow mesenchymal stem cells, promoting osseointegration [62,63,64,65,66,67]. Furthermore, these porous structures offer potential for drug delivery, improving antibacterial properties [68,69,70].

This study aims to compare three distinct surface modification methods for the Ti-6Al-4V (Ti64) alloy. The methods under investigation are low-temperature immersion corrosion, mechanical treatment followed by chemical etching, and electrochemical corrosion. We systematically investigated the effects of these treatments on surface morphology, roughness, hardness, and chemical composition. The four treatment methods produced surface structures with distinct characteristics, ranging from micron-scale protrusions to nano-scale secondary structures, providing a rich array of substrates for subsequent biological evaluation. These findings contribute to our understanding of surface modification techniques for the Ti-64 alloy and provide valuable insights for the development of next-generation dental and orthopedic implants with enhanced biocompatibility and osseointegration properties. Additionally, we assess their impact on osteoblast cell adhesion. The novelty of this research lies in its comprehensive, comparative approach, utilizing advanced analytical techniques such as Raman spectroscopy to elucidate surface chemical composition changes. By integrating surface characterization, electrochemical testing, and cell adhesion experiments, we aim to establish correlations between surface properties, corrosion behavior, and biological responses. Through this systematic analysis, we anticipate identifying the most promising methods for enhancing the osseointegration potential of Ti64 dental implants while improving their corrosion resistance, antibacterial properties, and biocompatibility. The findings of this study are expected to contribute significantly to the ongoing efforts in optimizing implant surfaces for improved clinical outcomes in dental implantology and may have implications for other biomedical applications of titanium alloys, such as orthopedic implants and cardiovascular stents.

2. Materials and Methods

2.1. Preparation of Ti-6Al-4V Samples

Ti-6Al-4V (Ti-64) alloy sheets with dimensions of 14 mm × 40 mm × 1 mm were used as the study materials. To enhance the cell adhesion properties, the surface morphology was modified through various corrosion conditions. The samples were meticulously prepared to ensure consistent surface quality. Initially, the samples were ground sequentially with SiC papers from 220 to 1200 grit to achieve a smooth surface. To obtain a pristine surface free from residual contamination, careful polishing was conducted using 0.3 μm alumina powder suspended in acetone under ultrasonic agitation for approximately 10 min. To prevent any contamination from the alumina powder and human oils, the polished samples were placed in beakers containing acetone and subjected to further ultrasonic cleaning for 10 min. Subsequently, the samples were thoroughly rinsed with deionized water and completely dried using cold air. For mechanical treatments, zirconia beads with a diameter of 0.25 mm were used as a medium for surface shot peening. The shot peening parameters were carefully selected to ensure uniform and controlled effects. During the shot peening process, a nozzle was positioned 100 mm from the sample surface. Shot peening was performed at pressures of 2, 1, and 0.5 kg/cm² for durations of 5, 10, and 20 s, respectively. For sandblasting, zirconia sand with grit sizes of 80 (particle size < 186 μm) and 120 (particle size < 125 μm) was used at an incident angle of 45°. Multiple chemical corrosion techniques were employed to modify the surface characteristics of the samples. The primary corrosive medium was HF solution, prepared at concentrations of 3 M and 12 M [71]. To ensure reagent stability, all solutions were prepared under subambient conditions at 5 °C or −5 °C using a recirculating chiller (WB-10, Paymo Technology Ltd., Taipei, Taiwan). For the electrochemical corrosion treatment, polished Ti-64 samples were positioned as an anode in contact with the positive terminal of a pulsed potentiostat. A platinum electrode connected to the negative terminal of the potentiostat was used as the cathode, positioned approximately 20 mm from the sample. Stirring was facilitated with a magnetic stirrer to ensure an even distribution of ions within the corrosive medium. To promote uniform corrosion and prevent polarization effects, a positive-to-negative potential alternation approach was employed. Two sets of current density conditions were used: ±30 mA/cm² and ±10 mA/cm².

2.2. Surface Characterization of Ti-6Al-4V Samples

Surface morphology was examined using a JSM 5600 (Tokyo, Japan) scanning electron microscope (SEM). SEM operates on the principle of secondary electron emission, where weakly bonded (<50 eV) electrons are emitted by sample atoms when bombarded by accelerated electrons. Secondary electrons generated at a depth of approximately 50–500 Å from the sample surface are detected to form the image. Raman spectroscopy was conducted using a Renishaw InVia Raman Microscope (Renishaw Taiwan Inc., Taichung, Taiwan). A 532 nm He-Ne laser, with a scanning range of 200–2000 cm⁻¹ and a constant power output of 10 mW, was used as the light source. Raman spectroscopy provides valuable information regarding crystalline properties and atomic bonding through the analysis of vibrational spectra. Surface roughness was evaluated using a Veeco Dektak profiler. The scanning length was set to 1000 μm with a scanning rate of 100 μm/s. Three random locations were measured on each sample to obtain average Ra (arithmetic average roughness) and Rq (root mean square roughness) values. Surface hardness was measured using a Wilson/Rockwell Series 500 (Wilson Instruments, Norwood, MA, USA) hardness tester with the HR15N scale. Three random points were tested on each sample surface to obtain an average value.

2.3. Cell Culture and Adhesion Test

MG-63 cell line adhesion to Ti-64 specimens subjected to various surface treatments was evaluated. Initially, the samples were sterilized, autoclaved, and dried to ensure aseptic conditions. The samples were subsequently coated with 1 mL of fetal bovine serum (FBS) and placed in a cell culture incubator at 37 °C for 30 min to promote cell adhesion. Human osteoblasts were seeded onto the samples at a density of 5 × 10⁵ cells/cm² and cultured at 37 °C for 2 h to facilitate cell adhesion. Subsequently, 2 mL of culture medium was added to each specimen-containing culture dish, and the samples were cultured for 1 week at 37 °C to promote cell growth. After 1 week, the specimens were retrieved, the excess culture medium was aspirated, and the samples were washed with phosphate-buffered saline (PBS). The samples were then fixed using 2 mL of formalin solution (1.2 mL 36 vol% formalin + 10.8 mL PBS) for 30 min. Liu’s staining method was applied by adding Liu A stain for 30 s, followed by Liu B stain at twice the volume of Liu A for 90 s. The samples were then rinsed with deionized water and air-dried. This technique employs the sequential application of stains with precise timing to effectively color the samples, preparing them for subsequent observation and analysis. Cell adhesion was evaluated using optical microscopy. This comprehensive experimental procedure ensured aseptic conditions for evaluating cell adhesion to Ti-64 samples subjected to diverse surface treatments, providing insights into the relationship between surface modifications and biocompatibility.

3. Results and Discussion

In this part, we investigate the surface modification of the Ti-6Al-4V (Ti-64) alloy and characterize the resultant surface morphology changes using SEM. Figure 1 shows the surface morphology of the original Ti-64 surface and those subjected to low-temperature immersion corrosion in HF solutions of varying concentrations. The original Ti-64 surface (Figure 1a) exhibited an extremely smooth topography, consistent with the effects of mechanical polishing. While this smooth surface may be advantageous for certain applications, it might not be optimal for osseointegration in biomedical implants [72]. After low-temperature (5 °C) immersion corrosion in 3 M HF solution (Figure 1b,c), the Ti-64 surface developed an irregular porous structure. This structural characteristic likely resulted from a selective corrosion process, where certain grains or phases were preferentially dissolved. At lower magnification (Figure 1b), uniform corrosion was observed over a large area, while higher magnification (Figure 1c) revealed more intricate surface features, including micropores and protrusions. The use of 12 M HF solution for low-temperature immersion corrosion (Figure 1d) led to more pronounced surface alterations. The surface exhibited deeper and wider etch pits, accompanied by a more complex three-dimensional network structure. This morphology likely originated from more intense corrosion processes, resulting in substantial material dissolution and redeposition. These surface modification treatments significantly increased the surface roughness and specific surface area of Ti-64, which may enhance cell adhesion and osseointegration. In particular, the surface structure resulting from the 12 M HF treatment, with its complex three-dimensional topography, may provide a more favorable environment for osteoblast attachment and growth [8,60]. The observed changes in surface morphology suggest that HF-based low-temperature immersion corrosion is an effective method for modifying Ti-64 surfaces. The ability to control the degree of surface modification by adjusting HF concentration offers potential for tailoring implant surfaces to specific biomedical applications. Further studies are warranted to quantify the relationship between surface treatment parameters and the resulting surface properties, as well as to evaluate the biological response to these modified surfaces in vitro and in vivo.

Figure 2 illustrates the surface morphology of the Ti-64 alloy after sandblasting treatment at a 45° angle using zirconia sand of different grit sizes (80 and 120 grit). This surface modification technique aims to increase surface roughness, thereby enhancing biocompatibility and osseointegration potential. As evident from Figure 2a,b, the sandblasting treatment significantly altered the Ti-64 surface. Both grit sizes resulted in the formation of complex three-dimensional structures, characterized by irregular protrusions and fissures. This surface morphology can be attributed to the continuous impact and shear forces exerted by high-velocity zirconia particles on the material surface. Comparing Figure 2a,b, we observe that treatment with 80-grit sand (Figure 2a) produced larger pits and more pronounced plastic deformation features. This may be due to the higher impact energy of larger sand particles, resulting in more extensive surface deformation. In contrast, treatment with 120-grit sand (Figure 2b) yielded a finer and more uniform texture, which may be advantageous for subsequent cell adhesion and proliferation [10,40]. Both treated surfaces exhibited significant plastic shear strain characteristics, manifested as material flow and accumulation. This non-uniformly distributed strain layer not only increases the effective surface area but also stores a substantial amount of strain energy in the material surface. This stored strain energy may play a catalytic role in subsequent acid etching processes, serving as a crucial factor in driving non-uniform surface corrosion. Notably, the sandblasted surfaces display multi-scale roughness features, ranging from micron-scale major protrusions to nano-scale fine structures. This multi-scale surface structure may have complex effects on subsequent biological performance, potentially promoting the attachment and proliferation of certain cell types while also influencing the material’s fatigue properties and corrosion behavior [63,66]. The 45° angle sandblasting treatment successfully created complex morphologies on the Ti-64 surface, laying the foundation for subsequent surface modifications and biological applications. However, the observed differences in surface morphology resulting from different grit sizes suggest that the selection of optimal treatment parameters should consider a combination of factors, including surface roughness, strain energy distribution, and potential biological effects. These findings underscore the importance of carefully controlling sandblasting parameters to achieve the desired surface properties. Future studies should focus on quantifying the relationship between sandblasting parameters (such as grit size, pressure, and angle) and the resulting surface characteristics. Additionally, investigating the biological response to these modified surfaces through in vitro and in vivo experiments would provide valuable insights into their potential for enhancing implant performance.

Figure 3 illustrates the surface morphology of the Ti-64 alloy subjected to a two-step surface modification process, sandblasting at a 45° angle using zirconia sand of different grit sizes (80 and 120 grit), followed by low-temperature (5 °C) immersion corrosion in HF solutions of varying concentrations (3M and 12M). This composite treatment aims to create bioactive surfaces with multi-scale structural features. Comparing Figure 3a,b, we observe the significant influence of HF concentration on surfaces initially treated with 80-grit sand. The 3 M HF treatment (Figure 3a) produced relatively uniform etch pit structures, resulting in micron-scale surface features that may promote cell adhesion. In contrast, the 12 M HF treatment (Figure 3b) led to more aggressive corrosion, forming deeper and wider etch pits with superimposed nano-scale secondary structures. This multi-level surface architecture may be more conducive to the osseointegration process [66,68]. A comparison of Figure 3c,d reveals the effects of different HF concentrations on surfaces initially treated with 120-grit sand. The 3 M HF treatment (Figure 3c) preserved more of the original sandblasted texture while forming uniformly distributed micro-pits on the surface. The 12 M HF treatment (Figure 3d) resulted in more pronounced surface reconstruction, creating a more complex three-dimensional network structure. This structure may provide a larger specific surface area and more cell attachment sites [52,55,56]. Comparing all four treatment methods, we observe that surfaces initially treated with 80-grit sand exhibited greater changes during subsequent corrosion. This may be attributed to the higher initial surface roughness, which provided more active sites for corrosion. The 12 M HF treatment generally led to more dramatic surface reconstruction, forming more complex multi-scale structures.

Figure 4 shows the surface morphology of the Ti-64 alloy in its original state and after shot peening treatment with various parameters. The untreated Ti-64 surface (Figure 4a) exhibited extremely smooth characteristics, consistent with the surface morphology after mechanical polishing. While this smooth surface may have advantages in certain applications, it might not be optimal for osseointegration in biomedical implants. After shot peening treatment, the Ti-64 surface morphology underwent significant changes. The sample treated at 2 kg/cm² pressure for 5 s (Figure 4b) displayed pronounced plastic deformation features, characterized by numerous irregular protrusions, depressions, and microcracks. This structure likely resulted from rapid surface deformation due to high-energy impacts. As the treatment pressure decreased and duration increased, the surface structure gradually became more uniform and refined. The sample treated at 1 kg/cm² pressure for 10 s (Figure 4c) exhibited smaller-scale pits and protrusions, while the sample treated at 0.5 kg/cm² pressure for 20 s (Figure 4d) formed a more delicate and uniform structure, comprising numerous minute pits and angular features. These results demonstrate that precise control over Ti-64 surface morphology can be achieved by modulating shot peening parameters such as pressure and duration. As the treatment pressure decreased and duration increased, the surface structure tended to become more refined and uniform. This trend may have significant implications for the material’s biological performance. Particularly, the coexistence of micron- and submicron-scale structures observed in Figure 4c,d may be more conducive to mimicking the hierarchical structure of natural bone tissue, thereby promoting cell adhesion and osseointegration processes [66,67]. Shot peening treatment significantly increased the surface area and roughness of the material, which may be beneficial for increasing cell attachment sites and enhancing bioactivity. However, different treatment parameters may lead to variations in surface and subsurface stress distribution, potentially affecting the material’s fatigue performance and corrosion resistance. Therefore, further research is needed to evaluate the specific effects of these different surface structures on cellular behavior, as well as their potential impact on the long-term performance of the material.

Figure 5 shows the SEM surface morphology of the Ti-64 alloy after various surface treatments, including sandblasting and shot peening, followed by immersion corrosion in low-concentration HF solution. These treatments aim to create bioactive surfaces with optimized characteristics for potential biomedical applications. The surface treated with 120-grit zirconia sand at a 45° angle, followed by etching with 3 M HF solution (Figure 5a), exhibited irregular protrusions and depressions with multi-scale features. This composite treatment method preserves the macroscopic roughness produced by sandblasting while further enhancing surface complexity at the microscopic scale through acid etching. Such a structure may be conducive to cell adhesion and the osseointegration process. Figure 5b–d display surface morphologies after shot peening with different parameters, followed by etching with 3 M HF solution. A clear trend in surface structure changes is observed as the shot peening pressure decreases. Treatment at 2 kg/cm² (Figure 5b) produced larger plastic deformations and irregular structures, while treatment at 0.5 kg/cm² (Figure 5d) resulted in a more uniform and refined surface morphology. This trend indicates that precise control over the initial surface morphology can be achieved by modulating shot peening parameters, subsequently influencing the corrosion behavior during the acid etching process. Comparing all treatment methods, it is evident that they all generated complex three-dimensional structures on the Ti-64 surface, albeit with different specific morphological features. Sandblasting followed by acid etching (Figure 5a) created deeper etch pits with sharp edges, while shot peening followed by acid etching (Figure 5b–d) formed more rounded protrusions and depressions. These differences may arise from the varying effects of initial mechanical treatment methods on surface stress states and microstructures.

Notably, all treated surfaces exhibited multi-scale roughness features, ranging from micron-scale major protrusions to nano-scale fine structures. This multi-scale structure may have complex effects on subsequent biological performance, potentially promoting the attachment and proliferation of certain cell types while also influencing the material’s fatigue properties and corrosion behavior. The diverse range of complex structures created on the Ti-64 surface by combining mechanical treatments and chemical corrosion provides a broad design space for optimizing the surface characteristics of implant materials. Future research should focus on quantifying the relationship between treatment parameters and resulting surface characteristics, including roughness, porosity, and chemical composition. Comprehensive in vitro studies are necessary to assess the biological response of various cell types to these modified surfaces, while in vivo experiments would evaluate the osseointegration performance of implants with these surface modifications. Additionally, investigating the long-term stability of these surface structures under physiological conditions and assessing the impact of surface treatments on the bulk mechanical properties and corrosion resistance of the material are crucial for ensuring the overall performance and safety of the implants.

Table 1. Surface characteristics of the Ti-6Al-4V alloy under various surface treatment conditions. The table presents surface roughness parameters (Ra and Rq) and surface hardness (HR15N) for different treatments including mechanical polishing, hydrofluoric acid (HF) etching, sandblasting with different grit sizes (Zr), sandblasting followed by HF etching, and shot peening (Zr SP) with various parameters followed by HF etching. Values are presented as mean ± standard deviation.

Sample Conditions	Surface Roughness Ra (μm)	Surface Roughness Rq (μm)	Surface Hardness (HR15N)
As polished Ti64	0.09 ± 0.01	0.16 ± 0.01	79.8 ± 0.4
3 M HF immersion corrosion	0.23 ± 0.02	0.30 ± 0.02	78.5 ± 0.5
12 M HF immersion corrosion	0.32 ±0.03	0.39 ±0.03	78.5 ± 0.9
80-grit Zr, 45°	2.25 ± 0.41	2.92 ± 0.02	84.3 ± 0.4
120-grit Zr, 45°	1.20 ± 0.13	1.67 ± 0.08	83.1 ± 0.2
80-grit Zr, 45°/3 M HF immersion corrosion	2.23 ± 0.36	2.85 ± 0.07	79.5 ± 0.5
80-grit Zr, 45°/12 M HF immersion corrosion	1.43 ± 0.27	1.90 ± 0.03	79.0 ± 1.0
120-grit Zr, 45°/3 M HF immersion corrosion	1.28 ± 0.14	1.79 ± 0.08	80.3 ± 0.4
120-grit Zr, 45°/12 M HF immersion corrosion	0.85 ± 0.19	1.56 ± 0.09	79.3 ± 0.8
Zr SP: 2 kg/cm2, 5 s	0.85 ± 0.03	1.69 ± 0.03	83.8 ± 0.8
Zr SP: 1 kg/cm2, 10 s	0.79 ± 0.03	1.46 ± 0.10	83.3 ± 0.4
Zr SP: 0.5 kg/cm2, 20 s	0.72 ± 0.06	1.23 ± 0.07	82.5 ± 0.5
Zr SP: 2 kg/cm2, 5 s/3 M HF immersion corrosion	0.97 ± 0.05	1.70 ± 0.06	79.4 ± 1.0
Zr SP: 1 kg/cm2, 10 s/3 M HF immersion corrosion	0.93 ± 0.04	1.53 ± 0.15	79.5 ± 0.5
Zr SP: 0.5 kg/cm2, 20 s/3 M HF immersion corrosion	0.87 ± 0.06	1.67 ± 0.17	79.7 ± 0.4

summarizes the surface roughness parameters (Ra and Rq) and surface hardness (HR15N) of the Ti-64 alloy under various surface treatment conditions. These data reveal the significant impact of different surface treatment methods on the surface characteristics of Ti-64. The original polished Ti-64 surface exhibited extremely low roughness (Ra = 0.09 ± 0.01 μm), consistent with the smooth surface expected after mechanical polishing. However, surface roughness increased significantly with immersion corrosion treatment alone. Treatment with 3 M HF increased the Ra value to 0.23 ± 0.02 μm, while 12 M HF treatment further elevated it to 0.32 ± 0.03 μm. This increased roughness may be beneficial for cell adhesion, although it is noteworthy that surface hardness slightly decreased from 79.8 ± 0.4 to 78.5 ± 0.9. Sandblasting treatment led to a substantial increase in surface roughness while also enhancing surface hardness. Treatment with 80-grit sand resulted in an Ra value of 2.25 ± 0.41 μm, whereas 120-grit sand produced an Ra value of 1.20 ± 0.13 μm. This difference likely stems from the varied impact effects due to different sand particle sizes. Notably, sandblasting also increased surface hardness to 84.3 ± 0.4 and 83.1 ± 0.2, respectively. The sandblasting and acid-etching (SLA) process further altered surface characteristics. For samples treated with 80-grit sand, the subsequent 3 M HF treatment slightly reduced roughness (Ra = 2.23 ± 0.36 μm), while the 12 M HF treatment significantly decreased roughness (Ra = 1.43 ± 0.27 μm). This phenomenon may be attributed to the selective corrosion and smoothing effects of high-concentration HF on the surface. For samples treated with 120-grit sand, the subsequent acid etching reduced surface roughness in both cases, particularly with the 12 M HF treatment (Ra = 0.85 ± 0.19 μm). These composite treatments also decreased surface hardness to levels approaching that of the original Ti-64. Shot peening treatment produced a moderate increase in surface roughness, with Ra values ranging from 0.72 to 0.85 μm. Interestingly, as treatment pressure decreased and duration increased, surface roughness slightly decreased, but surface hardness remained relatively high (82.5–83.8). This suggests that shot peening may provide sufficient surface roughness to promote biological responses while maintaining the bulk properties of the material. Acid etching following shot peening further increased surface roughness, with Ra values reaching 0.87–0.97 μm, while reducing surface hardness to levels similar to the original Ti64. This composite treatment may offer additional possibilities for optimizing surface characteristics, increasing surface roughness while avoiding excessive surface hardening. These results demonstrate that through the selection of appropriate surface treatment methods and parameters, precise control over the roughness and hardness of Ti-64 surfaces can be achieved. Different treatment methods produce a wide range of roughness scales, from submicron to micron levels, offering diverse options to meet the requirements of various biomedical applications.

Raman spectroscopy analysis plays a crucial role in this study, providing essential information about the chemical composition and structure of Ti-64 alloy surfaces, particularly in assessing the impact of various surface treatments on oxide layer formation. This non-destructive characterization technique offers deep insights into phase changes during the surface modification process, providing a scientific basis for optimizing the surface characteristics of biomedical implants. Figure 6 presents the Raman spectra of the original polished Ti-64 surface and those subjected to different treatments. The original polished surface exhibits relatively flat spectral features with only a weak broad peak around 300 cm⁻¹ (Figure 6a), possibly associated with an extremely thin natural oxide layer. In contrast, the sample treated with 3 M HF immersion corrosion shows a significant peak at 520 cm⁻¹, corresponding to the Eg mode of anatase phase TiO₂. This indicates that HF corrosion effectively promotes the formation of an oxide layer, primarily generating anatase phase structures. The spectrum of the shot-peened (Zr SP) sample displays an enhanced broad peak near 300 cm⁻¹, reflecting increased surface roughness, but no distinct TiO₂ characteristic peaks are observed. However, when shot peening is combined with HF immersion corrosion, the resulting spectrum presents the most complex features. Multiple peaks appear in the 300–700 cm⁻¹ range, with peaks at 447 cm⁻¹ and 612 cm⁻¹ potentially corresponding to the Eg and A1g modes of rutile phase TiO₂, respectively [73]. This composite treatment leads to the coexistence of both anatase and rutile TiO₂ phases, forming a more complex oxide layer structure.

Raman spectral analysis in the 1000–2000 cm⁻¹ range also provides valuable insights into the surface characteristics of the Ti-64 alloy (Figure 6b). This spectral region is particularly sensitive to titanium oxidation states and structural changes, aiding in a deeper understanding of the effects of different treatment methods on surface modification. The polished Ti-64 surface presents a relatively flat spectrum, indicating a predominantly metallic surface with minimal natural oxidation. In contrast, chemically and mechanically treated surfaces exhibit significant spectral changes, reflecting the formation and evolution of titanium oxide layers. HF immersion corrosion results in an overall increase in spectral intensity, especially in the 1300–1600 cm⁻¹ range. This broad feature suggests the formation of an amorphous or highly defective TiO₂ layer. Shot peening further enhances this effect, with a more pronounced broad peak in the same spectral region. The most significant spectral changes are observed in samples subjected to combined shot peening and HF corrosion treatment. The strong broad peak in the 1300–1700 cm⁻¹ range can be attributed to second-order Raman scattering of TiO_2, while the weak feature near 1100 cm⁻¹ may correspond to Ti-O-Ti vibrations [74]. This complex spectral profile indicates the formation of a more extensive and structurally diverse oxide layer, potentially combining amorphous and nanocrystalline phases. The progressive increase in spectral intensity and complexity from the original polished surface to the composite-treated samples correlates with the degree of surface modification. The absence of sharp, well-defined peaks in all treated samples suggests that the formed oxide layers are primarily amorphous or highly defective structures, a characteristic that may contribute to enhanced bioactivity. The combined treatment of shot peening and HF corrosion appears most effective in altering the surface chemical composition and structure. This synergistic effect may arise from the mechanical activation of the surface by shot peening, followed by controlled chemical etching, resulting in a unique surface oxide layer configuration.

These results reveal the significant impact of different surface treatment methods on the oxide layer structure of Ti-64 surfaces. HF corrosion treatment promotes oxide layer formation, while shot peening may enhance subsequent oxidation processes by increasing surface activity. The combination of shot peening and HF corrosion demonstrates a notable synergistic effect, producing richer surface features than single treatments alone. From a biomedical application perspective, the complex oxide layer structure produced by this composite treatment may provide a more favorable microenvironment for cell adhesion and growth. The coexistence of anatase and rutile phases could contribute to enhancing the material’s bioactivity. Future research should focus on correlating these surface structural changes with biological responses, including cell adhesion, proliferation, and differentiation.

Before discussing the effects of different electrolytes on the electrochemical corrosion of Ti-64 surfaces, it is essential to compare the differences and advantages/disadvantages of immersion corrosion and electrochemical corrosion methods on material surface structures. Immersion corrosion is a relatively simple and traditional surface treatment method. It primarily relies on chemical reactions, with corrosion rates typically slower and more challenging to control precisely. Immersion corrosion usually produces relatively uniform surface morphologies but may struggle to form complex porous structures. Moreover, prolonged immersion can potentially lead to a decline in bulk material properties. However, the advantages of immersion corrosion lie in its simplicity of operation, lower cost, and suitability for large-scale processing. In contrast, electrochemical corrosion offers more precise process control. By adjusting current density, potential, and time, the depth and morphology of corrosion can be accurately controlled. Electrochemical corrosion can produce more complex and diverse surface structures, such as highly ordered nanotube arrays or porous structures, in a shorter time [26,47,62,64,69]. Furthermore, the oxide layers generated during electrochemical corrosion are typically denser and more uniform, contributing to improved corrosion resistance of the material. Another advantage of electrochemical corrosion is that it can be performed at room temperature, reducing potential material property degradation associated with high-temperature treatments. However, electrochemical corrosion equipment is relatively complex, and challenges may exist when processing large-area samples.

Considering the advantages of electrochemical corrosion in surface modification, this study chose this method to investigate the effects of different electrolytes on Ti-64 surface morphology. Figure 7 shows SEM images of surfaces after electrochemical corrosion in four different electrolytes. Electrochemical corrosion in a 0.5 M HF electrolyte (Figure 7a) produced a highly porous surface structure with an irregular honeycomb-like morphology. Pore sizes ranged from the submicron to the micron scale. This structure likely originates from the selective dissolution of the TiO₂ protective layer by F⁻ ions, forming [TiF₆]²⁻ complexes [75]. Such a porous structure is expected to increase surface area, potentially enhancing cell adhesion capability and osseointegration. In contrast, treatment with a 0.5 M H₂SO₄ electrolyte (Figure 7b) resulted in a relatively smooth surface with only minor shallow pits and fine indentations. This morphology may be due to the uniform corrosion action of sulfate ions on the titanium surface, albeit with relatively weak corrosive power. While this surface may contribute to improved corrosion resistance, it might not be as conducive to cell adhesion as the HF treatment. The 0.5 M NaF electrolyte treatment (Figure 7c) showed almost no significant morphological changes, presenting a highly uniform and smooth surface. This may be due to the formation of a stable fluoride film during the electrolysis process, preventing further corrosion [75]. Although this surface may exhibit good corrosion resistance, it might not be favorable for cell adhesion and growth. Treatment with a 0.5 M HNO₃ electrolyte (Figure 7d) produced a unique surface morphology characterized by scattered circular pores. These pores varied in size, with diameters ranging from approximately 0.5 to 2 μm. This structure may result from the strong oxidizing action of nitrate ions, leading to localized rapid dissolution. Such surface morphology might provide cell attachment sites while maintaining a certain degree of surface integrity.

Comparing the results of the four electrolyte treatments, we can observe that the choice of electrolyte significantly influences the final surface morphology. The highly porous structure produced by the HF treatment may be most conducive to cell adhesion and osseointegration, while H₂SO₄ and HNO₃ treatments achieve a balance between surface modification and maintaining material integrity. Although NaF treatment may enhance corrosion resistance, it might not be favorable for biological applications. These findings provide important guidance for Ti-6Al-4V surface modification. Appropriate electrolytes can be selected to tailor surface characteristics according to different application requirements. For instance, HF treatment might be chosen for implants requiring rapid osseointegration, while NaF or H₂SO₄ treatments might be considered for applications requiring long-term corrosion resistance. Future research should further explore the effects of these different surface morphologies on cell behavior, protein adsorption, and long-term implant performance. Additionally, studies combining multiple electrolytes or sequential treatments could potentially yield surfaces with optimized properties for specific biomedical applications. The interplay between surface topography, chemical composition, and biological response warrants in-depth investigation to develop next-generation implant surfaces with enhanced functionality and biocompatibility.

Figure 8 presents the Raman spectra of the Ti-64 alloy after electrochemical corrosion in four different electrolytes (0.5 M HNO₃, 0.5 M NaF, 0.5 M H₂SO₄, and 0.5 M HF). These spectra cover two critical wavenumber ranges, namely, 250–700 cm⁻¹ and 1000–2000 cm⁻¹, providing information for understanding the structural characteristics and evolution of the surface oxide layer. In the low wavenumber range (250–700 cm⁻¹), the spectral features primarily reflect the crystalline structure of TiO₂. Samples treated with different electrolytes exhibit distinct spectral characteristics, suggesting diversity in the surface oxide layer structures. Notably, samples treated with HNO₃ and NaF show characteristic peaks at approximately 450 cm⁻¹ and 610 cm⁻¹, respectively. These peaks can be attributed to the Eg mode of anatase and the A1g mode of rutile TiO₂ phases [73]. In contrast, samples treated with H₂SO₄ and HF lack distinct crystalline phase peaks in this range, indicating the possible formation of amorphous or highly defective oxide layers. The spectral features in the high wavenumber range (1000–2000 cm⁻¹) provide information about surface defects and second-order scattering processes. All samples exhibit broad features in this range, but with varying intensities and shapes. These differences reflect the impact of different electrolyte treatments on the surface microstructure and chemical composition. Notably, the HNO₃₋ treated sample shows the strongest spectral response between 1300 and 1600 cm⁻¹, which may be related to oxygen vacancies in non-stoichiometric TiO₂, suggesting a complex surface defect structure. Comprehensive analysis indicates that the choice of electrolyte significantly influences the structure and composition of the oxide layer on the Ti-64 surface. The HNO₃ treatment appears to induce the most pronounced surface oxidation, potentially forming a mixed structure of anatase and amorphous TiO₂. The NaF treatment tends to promote the formation of the rutile phase, which may affect surface stability and biocompatibility. The H₂SO₄ and HF treatments likely result in more disordered or amorphous surface structures, which may have advantages in certain biological applications, such as enhanced protein adsorption capability.

This study further investigated the effects of mixed electrolytes on the electrochemical corrosion of Ti-64 alloy surfaces. Figure 9 shows SEM images of surfaces treated with four different mixed electrolytes, revealing the unique effects of composite electrolytes in surface modification. Treatment with a 0.5 M NaF/0.5 M H₂SO₄ mixed electrolyte (Figure 9a) produced a highly porous and complex surface structure. The surface exhibits an irregular honeycomb-like morphology, with pore sizes ranging widely from submicron to several micrometers. This structure likely results from the synergistic action of F⁻ and SO₄²⁻ ions, where the former promotes the dissolution of the TiO₂ protective layer, while the latter may accelerate the corrosion process [76]. This multi-scale porous structure is expected to significantly increase the surface area, potentially enhancing cell adhesion capability and osseointegration. The 0.5 M NaF/0.5 M HNO₃ mixed electrolyte treatment (Figure 9b) generated a more uniform porous structure. The pores are relatively consistent in size, with diameters ranging approximately from 1 to 2 μm. This morphology may result from a balance between the strong oxidizing action of NO³⁻ ions and the selective dissolution effect of F⁻ ions [77]. Such a uniform porous structure might be beneficial for even cell distribution and growth. In contrast, the 0.5 M HCl/0.5 M HNO₃ mixed electrolyte treatment (Figure 9c) produced a relatively smooth surface with only a few scattered pits and protrusions. This surface morphology may be due to the competitive action of Cl⁻ and NO³⁻ ions, where the former tends to cause localized corrosion while the latter promotes uniform oxidation. Although this surface might not be as conducive to cell adhesion as the porous structures, it may exhibit good corrosion resistance. The 0.5 M HF/0.5 M HNO₃ mixed electrolyte treatment (Figure 9d) resulted in the most pronounced surface alteration, forming etch pits with significant depth and width. These pits present irregular shapes and interconnect, creating a complex three-dimensional network structure. This structure likely results from the interaction between the strong corrosive action of HF and the oxidizing effect of HNO₃. While this highly rough surface may provide an ideal environment for cell attachment and growth, it might also affect the material’s mechanical properties. Comparing the results of the four mixed electrolyte treatments, we can observe that the electrolyte combination significantly influences the final surface morphology. The NaF/H₂SO₄ and NaF/HNO₃ combinations produced relatively uniform porous structures, which may be more suitable for applications requiring large surface areas. The HCl/HNO₃ combination, resulting in a relatively smooth surface, might be more appropriate for scenarios where maintaining material integrity is crucial. The HF/HNO₃ combination, producing a highly rough structure, may be most conducive to cell adhesion and osseointegration, but its potential impact on material strength should be considered in applications.

Figure 10 presents the Raman spectra of the Ti-64 alloy after electrochemical corrosion treatments with various mixed acid electrolytes. These spectra reveal significant differences in the surface oxide layer structures, reflecting the unique effects of different electrolyte combinations on material surface modification. In the 250–700 cm⁻¹ range (Figure 10a), the spectral features produced by various treatment methods differ markedly, suggesting diversity in the surface oxide layer structures. The sample treated with 0.5 M HF/HNO₃ exhibits the strongest spectral response, particularly a broad peak at approximately 450 cm⁻¹, typically associated with the Eg mode of the anatase phase. The sample treated with 0.5 M NaF/H₂SO₄ shows distinct peaks at about 520 cm⁻¹ and 640 cm⁻¹, potentially corresponding to the A1g and Eg modes of the rutile phase. In contrast, samples treated with 0.5 M HCl/HNO₃ and 0.5 M NaF/HNO₃ display weaker spectral features, indicating the possible formation of thinner or more inhomogeneous oxide layers. In the 1000–2000 cm⁻¹ range (Figure 10b), all samples exhibit broad features, but with varying intensities and shapes. Features in this range are typically related to the second-order scattering of TiO₂ or surface defects. The sample treated with 0.5 M HF/HNO₃ shows the strongest spectral response in this range, particularly a broad peak between 1300 and 1600 cm⁻¹, which may be associated with oxygen vacancies in non-stoichiometric TiO₂. Other mixed acid-treated samples also show similar trends but with lower intensities, indicating differences in surface defect structures. Comprehensive analysis suggests that the composition of mixed acids significantly influences the structure and composition of the oxide layer on the Ti-64 surface. The HF/HNO₃ mixture appears to induce the most pronounced surface oxidation, potentially forming a mixed structure of anatase and amorphous TiO₂. This structure may be beneficial for bioactivity, explaining the previously observed favorable cell response. The NaF/H₂SO₄ treatment tends to promote the formation of the rutile phase, which may affect surface stability and biocompatibility. The HCl/HNO₃ and NaF/HNO₃ treatments may produce more complex or inhomogeneous surface structures, which could have advantages in certain biological applications.

Table 2. Surface characteristics of Ti-6Al-4V alloy after various electrochemical corrosion treatments. The table presents surface roughness parameters (Ra and Rq) and surface hardness (HR15N) for different single and mixed electrolyte treatments. Treatments include HF, H₂SO₄, NaF, HNO₃, and their combinations. Values are presented as mean ± standard deviation.

Sample Conditions	Surface Roughness Ra (μm)	Surface Roughness Rq (μm)	Surface Hardness (HR15N)
As polished Ti64	0.09 ± 0.01	0.16 ± 0.01	79.8 ± 0.4
0.5 M HF electrochemical corrosion	0.23 ± 0.02	0.30 ± 0.02	78.5 ± 0.5
0.5 M H2SO4 electrochemical corrosion	0.32 ±0.03	0.39 ±0.03	78.5 ± 0.9
0.5 M NaF electrochemical corrosion	2.25 ± 0.41	2.92 ± 0.02	84.3 ± 0.4
0.5 M HNO3 electrochemical corrosion	1.20 ± 0.13	1.67 ± 0.08	83.1 ± 0.2
0.5 M NaF/0.5 M H2SO4 electrochemical corrosion	0.23 ± 0.02	0.30 ± 0.02	78.5 ± 0.5
0.5 M NaF/0.5 M HNO3 electrochemical corrosion	0.32 ±0.03	0.39 ±0.03	78.5 ± 0.9
0.5 M HCl/0.5 M HNO3 electrochemical corrosion	2.25 ± 0.41	2.92 ± 0.02	84.3 ± 0.4
0.5 M HF/0.5 M HNO3 electrochemical corrosion	1.20 ± 0.13	1.67 ± 0.08	83.1 ± 0.2

summarizes the surface roughness parameters (Ra and Rq) and surface hardness (HR15N) of the Ti-64 alloy under various electrochemical corrosion conditions. These data reveal the significant impact of different electrolytes on the surface characteristics of Ti-64. The original polished Ti-64 surface exhibited extremely low roughness (Ra = 0.09 ± 0.01 μm), consistent with the smooth surface expected after mechanical polishing. However, after various electrochemical corrosion treatments, both surface roughness and hardness changed significantly. The single electrolyte treatment results show that the 0.5 M NaF electrolyte produced the highest surface roughness (Ra = 2.25 ± 0.41 μm) and hardness (84.3 ± 0.4 HR15N). This may be due to the selective dissolution of the TiO₂ protective layer by F- ions, simultaneously promoting the formation of a surface oxide layer. In contrast, the 0.5 M HF and 0.5 M H₂SO₄ treatments resulted in relatively lower roughness (Ra of 0.23 ± 0.02 μm and 0.32 ± 0.03 μm, respectively) and slightly decreased surface hardness (78.5 ± 0.5 HR15N). This suggests that these electrolytes may have led to more uniform surface corrosion. The 0.5 M HNO₃ treatment achieved moderate levels of roughness (Ra = 1.20 ± 0.13 μm) and hardness (83.1 ± 0.2 HR15N), possibly due to its strong oxidizing nature, promoting oxide layer formation alongside corrosion. The mixed electrolyte treatments yielded interesting results. The 0.5 M NaF/0.5 M H₂SO₄ and 0.5 M NaF/0.5 M HNO₃ combinations produced surface roughness (Ra of 0.23 ± 0.02 μm and 0.32 ± 0.03 μm, respectively) similar to the single HF or H₂SO₄ treatments, but with slightly reduced hardness. This indicates that mixed electrolytes may lead to more complex surface reactions, affecting the final surface characteristics. Notably, the 0.5 M HCl/0.5 M HNO₃ combination generated high roughness (Ra = 2.25 ± 0.41 μm) and hardness (84.3 ± 0.4 HR15N) similar to the single NaF treatment. This synergistic effect may result from the interplay between the localized corrosion action of Cl- ions and the oxidizing effect of NO³⁻ ions. The 0.5 M HF/0.5 M HNO₃ combination produced moderate roughness (Ra = 1.20 ± 0.13 μm) and relatively high hardness (83.1 ± 0.2 HR15N), possibly due to the strong corrosive action of HF being partially counteracted by the oxidizing effect of HNO₃. These results demonstrate that through the selection of appropriate electrolyte combinations, precise control over the roughness and hardness of Ti-64 surfaces can be achieved. Different electrolyte combinations produced a wide range of roughness scales, from submicron to micron levels, offering diverse options to meet the requirements of various biomedical applications. For example, high-roughness surfaces might be more conducive to cell adhesion and osseointegration, while surfaces with lower roughness but moderate hardness might be more suitable for applications requiring wear resistance.

Figure 11 demonstrates cell attachment on the Ti-64 alloy under different surface treatment conditions. These results reveal the significant impact of surface treatment methods on cellular behavior, providing important evidence for optimizing the surface design of biomedical implants. Figure 11a shows cell attachment on untreated (only polished) Ti-64 surface. We can observe that cells exhibit an elongated morphology, arranged closely with a clear directionality. Cell nuclei (dark purple) are clearly visible, with well-extended cytoplasm (light purple). This indicates that while the smooth surface allows cell attachment and growth, it may lead to cell alignment in specific directions, which might not be conducive to forming natural bone tissue structure [40]. Figure 11b (2 kg/cm², 5 s shot peening + 3 M HF corrosion) displays a cell morphology starkly different from the untreated surface. Cells appear round or oval, distributed relatively uniformly but sparsely. This morphology may indicate that cells are in the initial attachment stage, not yet fully spread. The surface treatment may have increased initial attachment points but has not yet provided an ideal long-term growth environment. Figure 11c (1 kg/cm², 10 s shot peening + 3 M HF corrosion) shows a more desirable cell attachment state. The number of cells has significantly increased, with diverse morphologies, including both round initial attachment cells and spread irregular-shaped cells. Cell distribution is relatively uniform, and intercellular connections can be observed. This surface treatment may have provided the most suitable microenvironment, promoting both initial attachment and subsequent spreading and proliferation. Figure 11d (0.5 kg/cm², 20 s shot peening + 3 M HF corrosion) also shows good cell attachment effects, but compared to Figure 11c, cell morphology is more irregular, and distribution is slightly uneven. Some areas show cell aggregation, forming colony-like structures. This might suggest that lower intensity but longer duration shot peening treatment created a more complex surface morphology, providing a diverse microenvironment for cells. These observations emphasize the important influence of surface treatment parameters on cellular behavior. The shot peening treatment combined with HF corrosion seems to create surface characteristics favorable for cell attachment and growth. Different treatment parameters led to notably different cellular responses, which may reflect changes in surface morphology, chemical composition, and energy state. Particularly noteworthy is that the shot peening treatment parameters of 1 kg/cm² and 10 s (Figure 11c) seem to provide the best surface characteristics, promoting uniform cell distribution and diverse morphologies. This may represent an optimal balance point of surface roughness and chemical composition, providing sufficient mechanical anchoring points while maintaining appropriate surface energy and bioactivity.

This study selected two electrochemical corrosion treatment methods, 0.5 M HF/0.5 M HNO₃ and 0.5 M HNO₃, for cell attachment experiments. The reason for this choice lies in their representation of two different surface modification strategies. The 0.5 M HF/0.5 M HNO₃ combination was chosen because previous surface morphology analysis showed that this treatment method could produce a structure closest to the ideal surface (surface covered with bowl-shaped hemispherical pits) described in the literature [8]. The 0.5 M HNO₃ treatment served as a control group to evaluate the effect of a single strong oxidizing acid on surface characteristics and cellular behavior. This comparative design allows us to directly assess the role of HF in the surface modification process. Figure 12 demonstrates cell attachment on the Ti-6Al-4V alloy after these two electrochemical corrosion treatments, observed through OM and SEM. The results reveal the significant impact of surface treatment methods on cellular behavior, providing important evidence for optimizing the surface design of biomedical implants. The surface treated with 0.5 M HF/0.5 M HNO₃ electrochemical corrosion (Figure 12a,b) exhibits excellent cell attachment effects. Cells show uniform distribution on the surface with diverse morphologies, including round initial attachment cells and spread irregular-shaped cells. This diversity and uniformity indicate that the surface treatment successfully created a microenvironment conducive to cell attachment, spreading, and growth. SEM observations further confirm the close interaction between cells and the surface, showing extended cell pseudopodia and intercellular connection structures. In contrast, the surface treated with 0.5 M HNO₃ electrochemical corrosion (Figure 12c,d) presents a markedly different cellular behavior pattern. Cells exhibit a distinctly elongated morphology and significant directional alignment. While this directional growth pattern indicates that surface modification successfully influenced cellular behavior, it may not be conducive to forming cell networks that mimic natural bone tissue structure. Comparing these two treatment methods, we can infer that the addition of HF played a key role in modulating surface characteristics. It may have altered the surface’s chemical composition and microscopic morphology, providing a more diverse and favorable attachment environment for cells. The combination of 0.5 M HF/0.5 M HNO₃ seems to have reached a balance point between surface roughness and chemical composition, promoting both initial cell attachment and subsequent multidirectional growth. These observations emphasize the importance of precisely controlling surface treatment parameters. By adjusting electrolyte composition, we can significantly influence cell attachment patterns and growth behavior. The surface characteristics produced by the 0.5 M HF/0.5 M HNO₃ treatment may be closer to the ideal implant surface, with the potential to promote better osseointegration processes. However, these preliminary results still need to be verified through more in-depth studies. Future work should focus on how these surface treatments affect long-term cell differentiation, mineralization, and osseointegration processes. Simultaneously, it is necessary to evaluate the impact of these treatments on the mechanical properties and long-term stability of the Ti-6Al-4V alloy to ensure that while improving biocompatibility, the overall performance of the material is not compromised.

The surface modification techniques investigated in this study demonstrate specific potential for various biomedical applications. The combined shot peening and HF/HNO₃ treatment produced surfaces with Ra values of 0.85–0.97 μm, which fall within the optimal roughness range (0.8–1.0 μm) for dental implants [8]. This surface modification is particularly advantageous for the implant collar region, where soft tissue integration plays a crucial role in preventing bacterial infiltration and ensuring long-term implant stability, as evidenced by our cell attachment studies showing enhanced fibroblast adhesion. For load-bearing dental components such as abutments, surfaces created by sandblasting followed by HF etching, exhibiting both increased hardness (83.1 ± 0.2 HR15N) and moderate roughness (Ra = 1.20 ± 0.13 μm), provide an optimal balance between mechanical stability and tissue integration capabilities. The electrochemically treated surfaces, particularly those exhibiting hierarchical micro/nano structures, show potential for immediate loading protocols in dental implantation, where rapid osseointegration is crucial. Our mixed-acid electrochemical treatments, especially the 0.5 M HF/0.5 M HNO₃ combination, produced surfaces with characteristics suitable for regions requiring enhanced osseointegration, as demonstrated by the observed diverse cell morphologies and uniform distribution pattern. These surface modification techniques also show potential for broader applications in orthopedic implants, where surface requirements vary depending on the specific anatomical location and loading conditions.

The surface characteristics obtained through our treatments are also expected to influence bacterial colonization and biofilm formation. The hierarchical micro/nano structures observed on HF/HNO₃-treated surfaces may provide selective behavior between bacterial cells (0.5–2 μm) and mammalian cells (20–30 μm), potentially inhibiting bacterial adhesion while promoting osteoblast attachment. The TiO₂ layers formed during electrochemical treatments, showing mixed anatase and rutile phases in Raman spectra, could contribute to antibacterial properties through photocatalytic effects. Moreover, surfaces produced by the combined shot peening and HF/HNO₃ treatment, with Ra values between 0.85 and 0.97 μm, represent a balance between minimizing bacterial colonization and maintaining favorable tissue integration.

4. Conclusions

This comprehensive study on Ti-6Al-4V surface modification techniques demonstrates that precise control over surface morphology can be achieved through carefully modulated mechanical and electrochemical treatments. The combination of these treatments, particularly the synergistic effect of shot peening and HF/HNO₃ etching, has proven effective in creating diverse surface morphologies with enhanced biocompatibility. Our systematic analysis reveals that the composite treatment method significantly influences surface characteristics, including roughness, chemical composition, and cell attachment properties. The results demonstrate that specific treatment parameters can be tailored to create optimal surface characteristics for cell attachment and growth. The correlation between surface properties and cellular responses suggests promising potential for improving osseointegration in dental implants. Most notably, the combined mechanical–chemical treatment approach offers new possibilities for designing implant surfaces with specific biological properties, potentially leading to enhanced clinical outcomes. While these findings provide valuable insights into surface modification techniques, several aspects warrant further investigation. The long-term stability of these modified surfaces in physiological environments, comprehensive biological responses, and mechanical durability need to be thoroughly evaluated. Future studies should focus on quantifying the relationship between treatment parameters and the resulting surface characteristics, followed by detailed in vitro and in vivo assessments of osseointegration performance. This study lays a solid foundation for developing next-generation biomedical implants with optimized surface properties. The insights gained significantly contribute to understanding how surface modifications can enhance the biological performance of titanium alloys, potentially extending beyond dental applications to other biomedical fields. These findings represent an important step toward improving implant success rates through controlled surface engineering strategies.