Ti-xCu (where x equals 0, 4 wt%, 6 wt%, or 8 wt%) bulk alloys were fabricated by mixing pure copper powders with contents at 0 wt%, 4 wt%, 6 wt%, or 8 wt% into commercial-grade pure titanium powder (99.99%). A planetary ball mill was employed to mix the powders at a rotational speed of 400 rpm to ensure thorough and uniform mixing of the powders. The shapes of the Ti and Cu powders are depicted in Figures 2A,B. The titanium powder predominantly exhibits a spherical form in Figure 2A, whereas the copper powder shows an irregular shape in Figure 2B. The pure titanium powder has a size of 15–45 µm in Figure 2C, while the pure copper powder has a particle size of less than 10 µm. Following a 1-h thorough mixing of the titanium and copper powders, Ti-xCu alloys with a relative density exceeding 99.5% were manufactured using the selective laser melting (SLM) technique. The resulting alloys were designated as Ti, Ti-4Cu, Ti-6Cu, and Ti-8Cu. SLM parameters with a scanning speed of 600 mm/s, 85 W of laser power, and 110 μm of space width were used. During the SLM process, a scanning pattern characterized by a zigzag trajectory with cross-hatching was employed. The orientation of the scanning strategy was consistently rotated by 90° when transitioning from one layer to the subsequent layer in Figure 2D.

Post-metallographic observations, samples were subjected to analysis with a scanning electron microscope (SEM, SU8010, Japan) coupled with an energy-dispersive spectrometer (EDS) to analyze their microstructure and elemental distribution across different phases. The alloy’s phase composition was identified via a D/MAX-2500PC X-ray diffractometer, equipped with a Cu-target Kα radiation source. The scan was conducted at a rate of 5° per minute over an angular range of 10°–80°. The acquired X-ray diffraction (XRD) data were subsequently processed using Jade 5.0 software to generate the corresponding XRD patterns.

The corrosion resistance of Ti-xCu was assessed using a conventional three-electrode workstation (Gamry REFERENCE 600+, USA). A saturated calomel electrode (SCE) functioned as the reference electrode (RE), a platinum electrode acted as the counter electrode (CE), and the sample, which had a polished surface area of 1 cm² post-encapsulation, served as the working electrode (WE). Electrochemical measurements were conducted in 0.9% NaCl at 37°C ± 1°C. After 1 h stabilization, the electrochemical impedance spectroscopy (EIS) was performed (1–2×10⁵ Hz), followed by potentiodynamic polarization (−0.5–1.5 V, 0.5 mV/s), with triplicate measurements for each sample.

In line with ISO 10271:2001, cylindrical samples measuring Φ10 mm × 10 mm (with a surface area exceeding 10 cm²) were placed in 0.9 wt% NaCl solution at an elution ratio of 1 cm²/mL and incubated at 37°C for 7 days, with triplicate samples for each group. Then, the immersed solution was analyzed for elemental concentration using an inductively coupled plasma atomic emission spectrometer (ICP-AES, Ultima 2).

The RAW264.7 murine macrophages and MC3T3-E1 murine osteoblasts (Cell Bank of Chinese Academy of Sciences) were cultured in DMEM and α-MEM (supplemented with 10% FBS, HyClone, USA), respectively, at 37°C/5% CO₂. Cells were passaged every 2–3 days. For experiments, macrophages (1 × 10⁵ cells/well) and osteoblasts (2 × 10⁴ cells/well) were seeded on Ti-xCu alloys in 24-well plates.

Ti-xCu alloys (n = 6) were placed into 24-well culture plates. Cells were then seeded onto the surfaces of these materials to evaluate their proliferative capacity at multiple time intervals using the CCK-8 assay. The CCK-8 reagent (Dojindo, Japan) was administered to each well following the protocol outlined in the CCK-8 kit manual. Following incubation of 1 day, 3 days, 5 days, and 7 days at 37°C, the optical density (OD) at a wavelength of 450 nm for each well was determined utilizing a spectrophotometric microplate reader using an iMark microplate reader (iMark, Bio-Rad Laboratories, USA).

After culturing the MC3T3-E1 cell line on the surfaces of various material groups, the samples were carefully transferred to a new 24-well plate using sterile forceps and washed three times with PBS. A staining solution containing calcein-AM and propidium iodide (PI) (Beyotime, China) was then added, followed by incubation at 37°C for 15 min. Subsequently, the samples were rinsed three times with PBS and imaged under a fluorescence microscope. Red fluorescence indicated dead cells, whereas green fluorescence represented live cells. Cell viability was calculated as the percentage of live cells relative to the total cell count.

To evaluate cell adhesion and spreading across various samples, rhodamine phalloidin and DAPI staining were employed to delineate the cytoskeleton and nuclei of the cells, respectively. Ti-xCu alloys were placed in 24-well plates, and cells were seeded onto these surfaces. The samples were fixed with 4% paraformaldehyde for 20 min and then incubated in the dark at 37°C for 30 min. Subsequently, the samples were subjected to DAPI staining for 5 min followed by three PBS washes. The cytoskeleton and nuclei of MC3T3-E1 cells were examined using a fluorescence microscope (Olympus, Japan), with images captured for blue emissions (under 400–440 nm excitation) and red emissions (under 510–550 nm excitation).

According to international standard ISO 10993-12 (Zhang et al., 2016), the immersed samples were stored in air at 37°C and 5% CO₂ with a specific surface area-to-volume ratio of 1.25 cm²/mL for 72 h to prepare the extracts. MC3T3-E1 cells were inoculated in 24-well plates.

RAW264.7 macrophages (1 × 10⁵ cells/well, n = 4) were cultured on Ti-xCu for 6 h/72h, MC3T3-E1 osteoblasts (n = 4) were cultured on Ti-xCu substrates in osteogenic induction medium for 7 days. Total RNA was extracted and reverse-transcribed using a PrimeScript RT kit (Takara, Japan). Quantitative PCR was performed on a LightCycler480 system (Roche) with SYBR Green mix, using NCBI-derived primers (Table 1; synthesized by Shanghai Shenggong Biotech). Gene expression was normalized to GAPDH via the 2^−ΔΔCT method.

Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) suspensions adjusted to a McFarland standard of 0.25 were co-cultured with Ti-xCu alloys, respectively. After 24-h incubation, 100-μL aliquots were taken from each culture and evenly spread onto nutrient agar plates. These plates were then incubated at 37°C in a humidity-controlled chamber with a humidity of 90% for 24 h. Once the incubation was complete, the number of bacterial colonies on each plate was counted. The antibacterial efficacy was evaluated using the following formula: Antibacterial rate = ( 1 − Experimental   group   colony   count   Control   group   colony   count ) × 100 %

Twelve clean-grade, 6-week-old male ICR mice weighing 20–30 g were used in the study. The mice were obtained from Shanghai Slack Laboratory Animal Co., Ltd. (Production License No. SCXK 2012-0002). This animal experiment was approved by the Experimental Animal Ethics Committee of Fujian Medical University (Approval No. IIACUC FJMU 2023-0184). Following a 7-day acclimation period with ad libitum access to food and water, mice underwent air pouch establishment. Dorsal fur was shaved, and 4 mL sterile air was injected subcutaneously on days 0 and 4 to induce pouch formation. Successful pouch development was confirmed on day 5 by the absence of inflammatory signs. On day 7, animals were anesthetized (1% sodium pentobarbital, 1 mL/100 g body weight), and implants were aseptically inserted into the pouches with subsequent wound closure. After 7 days post-implantation, major organs were harvested, fixed in 4% paraformaldehyde, and embedded in paraffin. Tissue sections (10 μm) were prepared for hematoxylin and eosin (H&E) staining and histological examination.

All displayed data were represented as mean ± standard deviation (SD). Statistically significant differences were analyzed by one-way analysis of variance (ANOVA) and Dunnett’s t-tests. P < 0.05 was considered significant. *P < 0.05, **P < 0.01, ***P < 0.001.

As depicted in Figure 3, the XRD patterns for the Ti-xCu (x = 0, 4 wt%, 6 wt%, 8 wt%) alloys exhibit a high similarity among the four groups. Predominantly, the pure Ti- and Cu-bearing Ti alloys consist of the α phase. In Ti-xCu alloys, the intensity of the diffraction peak for the Ti₂Cu phase increases with the addition of copper. When the copper content reaches 8 wt%, the diffraction peak for the Ti₂Cu phase becomes the strongest. Additionally, XRD patterns demonstrate that the diffraction peak around 40° shifts to higher angles as the copper content increases. This is because with the increase of copper content, the diffraction peak near 40° in the XRD pattern (Figure 3) shifts to the right. The observed phenomenon primarily results from lattice distortion induced by copper incorporation into the titanium matrix. Given copper’s smaller atomic radius compared to titanium, its dissolution in the α-Ti lattice causes lattice contraction, consequently reducing interplanar spacing (d-spacing). According to Bragg’s law (nλ = 2*d* sinθ), a reduced d-value increases the diffraction angle (θ), shifting the peak toward higher angles. At higher Cu content (≥4 wt%), Ti₂Cu precipitation further distorts the lattice. The tetragonal Ti₂Cu structure, differing from hexagonal α-Ti, additionally influences peak positions.

Figure 4 displays the SEM images along with their respective EDS spectra for the microstructures of Ti and the Ti-4Cu, Ti-6Cu, and Ti-8Cu alloys. In Figure 4A, the Ti microstructure is characterized by small α-phase grains that are consistently dispersed within the matrix. Figures 4B–D show that after alloying the Cu into Ti, the resulting microstructure of the alloy is dominated by lamellar Ti₂Cu (marked as bright regions in the enlarged image) and an α-Ti matrix (marked as dark regions in the enlarged image). A significant observation is the progressive enlargement of the Ti₂Cu area and the concurrent increase in grain size with increased Cu content in the matrix. Notably, at 8 wt%Cu content, the amount of Ti₂Cu surpasses that of the matrix. In this study, the laser power of 85 W provides sufficient energy density for complete powder melting without excessive penetration, ensuring molten pool stability. These parameters maintain a cooling rate (approximately 10⁵–10⁶ K/s), which suppresses macroscopic segregation of Cu elements and promotes the uniform distribution of nanoscale Ti2Cu precipitates. However, at 8 wt% Cu, the molten pool stability decreases owing to Cu’s high latent heat and altered surface tension, resulting in heterogeneous solidification and a broader Ti₂Cu size distribution. This phenomenon is consistent with the solute enrichment mechanism revealed in the study by Zhang et al. (2019). The study shows that high Cu content intensifies constitutional supercooling at the solidification front, thereby affecting the nucleation and growth of precipitates.

Figure 5A depicts the potentiodynamic polarization curves of Ti-xCu alloys in a 0.9 wt% NaCl solution at 37°C, respectively. The corresponding corrosion parameters are listed in Table 2. The curves for the four groups are notably consistent, each showing a sequence of activation and passivity, indicative of a similar electrochemical corrosion behavior. The transition from the cathodic to anodic potential signifies the alloy’s corrosion potential (E_corr), after which the curve enters the passive phase, in which the current density increases linearly with the increase in corrosion potential. Alloying Cu into the pure Ti leads to a general upward shift in the curves compared to pure Ti, which corresponds to a decrease in the I_corr. The I_corr values for Ti and the Ti-4Cu, Ti-6Cu, and Ti-8Cu alloys are 3.76 × 10⁻⁸, 1.93 × 10⁻⁸, 1.37 × 10⁻⁸, and 2.84 × 10⁻⁸ A cm², respectively. A decreasing I_corr is found with higher Cu content. As the Cu reaches 8 wt%, the I_corr increases to 2.84 × 10⁻⁸ A cm². Despite this increase, the Ti-6Cu alloy still exhibits a lower corrosion current than the Ti alloy.

Figure 5B presents the Nyquist diagrams of Ti and the Ti-4Cu, Ti-6Cu, and Ti-8Cu alloys. All curves exhibit a semicircular shape, suggesting single-layer film corrosion. The capacitive arc reflects charge transfer resistance, which is a measure of the electrode reaction’s velocity and a determinant of the passive film’s corrosion resistance. Comparing the radii of the capacitive arcs in the Nyquist diagrams reveals the stability and corrosion resistance of the Ti-xCu alloys. That is, a larger arc radius corresponds to higher impedance and stronger corrosion resistance. As observed in Figure 5B, the capacitive arc radius does not change significantly with increasing Cu content, suggesting that the addition of Cu does not adversely affect the stability of the passive film. Figures 5C,D present the Bode diagrams for the Ti-xCu alloys. In the high-frequency range (1–100 kHz), the phase angle of all alloys decreases as frequency increases, while the impedance value |Z| remains largely unchanged, indicating that impedance at high frequencies is linked to the solution resistance. In the low-frequency range (<100 Hz), as frequency decreases, the phase angle of all alloys approaches 90°. A phase angle near 90° typically signifies a stable, dense passive film. At 0.01 Hz, Ti-xCu alloys exhibit phase angles similar to pure Ti, suggesting Cu addition minimally affects passive film durability or corrosion resistance. Additionally, the Bode plot’s slope of ∼ −1 (0.1–1,000 Hz) indicates capacitive behavior consistent with a single-layer passive film, further confirming the formation of a dense protective layer.

The Bode and Nyquist diagrams indicate that a single-layer film forms on the surface. An equivalent electrical circuit (EEC) featuring a single constant-phase element (CPE) is used to model this corrosion, as inserted in Figure 5D. The CPE, denoted by Q, describes the deviation of the double-layer parameters at the electrode–solution interface from capacitance C and is independent of frequency. It is related to the surface roughness, composition, porous electrodes, adsorption reactions, and other material conditions. The admittance and impedance of the CPE are given by Z CPE = 1 Y 0 j ω − n where Y₀ is the CPE’s magnitude, j is the imaginary unit, and n is an exponent less than 1 for the CPE. A decreasing n value closer to 1 indicates fewer pores in the passive layer, signifying a denser layer. In the equivalent circuit, R_s and R_p represent the resistance of the electrolyte and the passive layer, respectively, while Q signifies the capacitance of the passive film. Table 3 lists the electrochemical parameters of the Ti-xCu alloys derived from the equivalent circuit model. The polarization resistance R, of Ti is 1.94 × 10⁶ Ω cm which increases with Cu content to 2.34 x 10⁶ Ω cm for Ti-4 Cu. A higher polarization resistance indicates better corrosion resistance. However, R_p slightly decreases when the copper content reaches 6 wt% and 8 wt%. Additionally, the n values for Ti-xCu are very close, suggesting a dense passive layer and good corrosion resistance for Cu-bearing Ti alloys.

Figures 6A,B illustrate the release pattern of Ti and Cu ions in a 0.9 wt% NaCl solution, respectively. The profiles clearly show that Ti and Cu ions increase with higher concentrations of Cu content. To assess the antibacterial performance of Ti-xCu alloys, these samples were cultured with E. coli and S. aureus for 24 h, respectively, as shown in Figure 6C. The data from Figure C demonstrate that pure Ti has a high density of bacterial colonies, while Ti-4Cu, Ti-6Cu, and Ti-8Cu show a marked decrease in colony count. This suggests that Ti-4Cu, Ti-6Cu, and Ti-8Cu possess superior antibacterial properties. Meanwhile, the antibacterial rates for these alloys are above 99%.

Figure 7A shows MC3T3 cell adhesion on Ti-xCu alloys after 3-day co-culture, with blue (nuclei) and red (actin filaments) fluorescence staining. Cells exhibit intact cytoskeletons with polygonal-to-spindle morphologies and clearly visible nuclei, proteins, and pseudopodia, indicating healthy osteoblast growth and high activity. Figure 7B shows live/dead staining of osteoblasts on Ti-xCu alloy surfaces. The viability of osteoblasts exceeded 90% on all Ti-xCu groups (Figure 7C), with no statistically significant differences observed among groups. The CCK-8 assay demonstrated significantly increased osteoblast proliferation in Cu-containing groups after 5 days of co-culture (Figure 7D). At day 7, the Ti-6Cu group exhibited superior proliferation compared to other groups. These results indicate that Ti-xCu alloys exert no significant inhibitory or cytotoxic effects on osteoblasts, with the Ti-6Cu alloy showing optimal cytocompatibility.

Figure 8A shows the alkaline phosphatase (ALP) staining results of osteoblasts cultured in Ti-xCu alloy extracts for 7 days. In the 6Cu and 8Cu groups, the samples displayed progressively larger clusters of purple calcium nodules, with the Ti-6Cu group exhibiting the most intense staining (P < 0.05 vs. other groups, Figure 8C), indicating higher ALP expression levels. Figure 8B presents Alizarin Red S (ARS) staining of osteoblasts cultured in Ti-xCu alloy extracts for 14 days. The Ti-6Cu and Ti-8Cu groups demonstrated significantly higher ARS staining intensity than other groups (P < 0.05), suggesting enhanced extracellular matrix mineralization (Figure 8D). To further ascertain the impact of Cu in titanium alloys on osteogenic differentiation, RT-PCR was utilized to evaluate the expression of osteogenic genes in cells cultured with Ti-xCu alloys, the expression of the alkaline phosphatase (Alp) gene in Figure 8E indicates that on Ti-4, 6, 8 Cu groups had higher expression, and Ti-6Cu had the highest performance (P < 0.05), indicating enhanced early osteogenic activity. Collagen type 1 (Col-1) expression was increased in the Ti-4Cu and Ti-6Cu groups, indicating enhanced collagen synthesis, osteoblast differentiation, and bone matrix mineralization. These findings suggest that optimal levels of Cu content in pure Ti, especially in the Ti-6Cu, can effectively promote osteogenic differentiation, making it a potential candidate material for bone tissue engineering.

Figure 9A depicts the morphology of RAW264.7 on Ti-xCu alloy after 3 days of co-culturing to assess the production of inflammatory and anti-inflammatory cytokines following macrophage culture with Ti, Ti-4Cu, Ti-6Cu, and Ti-8Cu for 6 h and 3 days. The findings revealed a greater number of star-shaped macrophages on the surfaces of the copper-enriched titanium alloys, Ti-6Cu and Ti-8Cu, as opposed to those on Ti-4Cu. Figures 9B–F depict the levels of inflammatory cytokines (Il-1β, Il-6, and Tnf-α) and anti-inflammatory cytokines (Il-1 and, Arg-1) in macrophages cultured on Ti-xCu alloys at 6 h and 3 days. Initially, at the 6-h mark, there is an upregulation of inflammatory cytokines Il-1β and Tnf-α, particularly in Ti-6Cu, indicating an early inflammatory response. After 3 days of culture, the expression of Tnf-α decreases. Regarding the anti-inflammatory cytokines Il-10 and Arg-1, the titanium alloys containing copper generally tend to increase the expression levels of these cytokines at all observed time points. However, it is important to note that there is an exception for Ti-8Cu, compared to Ti-6Cu, where a decrease in IL-10 expression is observed. This shift suggests that Cu-bearing Ti alloys, especially those with higher Cu content, may initially trigger inflammation but then promote a transition toward an anti-inflammatory and tissue-regenerative state in macrophages. It is known that the elongation of macrophages can boost the M2 subtype’s capacity to produce anti-inflammatory cytokines and shield cells against stimulation by interferon-gamma and lipopolysaccharide. Consequently, the copper-containing titanium alloys, Ti-6Cu and Ti-8Cu, may have the ability to stimulate the polarization of macrophage RAW264.7 cells, offering a potential advantage over the Ti-4Cu alloy.

The in vivo biocompatibility of Cu-bearing Ti alloys was tested through animal trials. After a 7-day implantation period in mice, the biosafety of the heart, liver, spleen, lungs, and kidneys was assessed using H&E staining, as shown in Figure 10. Histological analysis revealed preserved cellular architecture across all examined tissues. Cardiomyocytes exhibited regular fiber alignment without structural damage, interstitial congestion, or inflammatory infiltration. Hepatic tissue maintained normal lobular organization with intact sinusoids, devoid of congestion or hemorrhage. Splenic lymphocytes displayed consistent density and clear red–white pulp demarcation. Pulmonary histology showed thin alveolar septa without inflammatory exudates or hemorrhage. Renal tissues demonstrated normal tubular epithelium and glomeruli, absent degenerative changes or interstitial inflammation. The outcomes show no significant histopathological changes in the tissue sections analysis, which suggests that the Ti-4Cu, Ti-6Cu, and Ti-8Cu alloys have excellent in vivo compatibility and release ions at safe levels that do not cause organ toxicity.