Ti substrates were obtained from Engineering Research Center for Biomaterials, Sichuan University. Glutaraldehyde, cetylpyridinium chloride, calcium acetate [Ca(CH₃COO)₂⋅H₂O], sodium silicate (Na₂SiO₃⋅9H₂O), and β-glycerophosphate disodium salt (C₃H₇Na₂O₆P⋅5H₂O) were purchased from Aladdin Industrial Co. (Shanghai, China). Alizarin red, CCK8, and Live-Dead Cell Staining Kit were provided by Sigma Chemical Co. (MO, United States). LIVE/DEAD^® BacLight^TM Bacterial Viability kit was received from Molecular Probes (CA, United States). Alkaline Phosphatase (ALP) Assay Kit, Bicinchoninic Acid Assay (BCA) Kit were obtained from Nanjing Jiancheng Biotechnology Institute (Nanjing, China). PrimeScript^® RT Reagent Kit and SYBR Premix ExTMTaq II were purchased from Takara Bio Inc. (Kyoto, Japan). Other chemicals were provided by Dingsheng Medical Instrument Reagent Co. (Wenzhou, China).

The HESP and MAO technologies were used to treat Ti for preparing target samples according to our previous study (Shen et al., 2020). Briefly, Ti substrates were first polished with gradient sandpapers, cleaned using detergent/alcohol and dried at 60°C. Then, the cleaned samples were treated for 100 s using a HESP device (Shot Peening Machine, Rösler, Germany) under 5 Mpa (pressure) and 0.1 mmA (strength). Glass beads with 0.25–0.3 mm in diameter were used as the shot. After that, HESP-pretreated specimens were further treated with different electrolytes (Tables 1, 2) for 5 min at 480 V using a MAO device developed by Xi’an Technological University. Electrolytes of the target specimens (named as MAO, Si-MAO, Si/Cu-MAO, respectively) evaluated in detail by following cells/bacteria tests.

The surface and cross-sectional morphologies of different coatings (MAO, Si-MAO, Si/Cu-MAO) were measured by scanning electron microscopy (SEM, Inspect-F, FEI, United States). The thickness and bonding strength of different coatings were characterized by an eddy current thickness gauge (ED200, Tianxing Research Institute, China) and scratch tester (Revetest Scratch Tester, CSM Instruments, Switzerland), respectively. The surface roughness (Ra), crystalline phase, chemical compositions, chemical states, and water contact angle were further tested by surface roughness meter (Perthometer M1, Mahr, Germany), X-ray diffraction (XRD, X’Pert Pro MPD, Philips, Dutch), energy dispersive spectrometry (EDS, Oxford Instruments, United Kingdom), X-ray photoelectron spectroscopy (XPS, Model PHI 5400, Perkin Elmer, United States), and contact angle measuring instrument (DSA30, Kruss, Germany), respectively.

The specimens were soaked in 6 mL of phosphate buffer saline (PBS) solution at 37°C. After 1, 4, 7, 10, and 14 day, we collected all the soaking solution and re-added another fresh PBS solution (6 mL). In addition, to determine the exact concentration of Si and Cu in the coatings, Si/Cu-MAO samples were soaked in 6 mL of hydrochloric acid (HCl, 3 mol/L) solution for 14 day under oscillatory conditions. The contents of Cu and Si in the PBS or HCl immersion solution were finally measured with an inductively coupled plasma emission spectrometer (ICAP-9000, Jarrell-Ash, United States).

The S. aureus (ATCC 6538) and S. mutans (ATCC 25175) were provided by the State Key Laboratory of Oral Diseases. After the determination of no other bacterial contamination by Gram stain, a single colony was selected to be inoculated on TSB/LB plates via the streak plate method and continued to culture for 24 h. The individual colonies on the plate were further transferred to liquid medium for culture 10 h before the following bacterial tests.

For detecting the bacterial morphology, two bacteria (1 mL) were cultured on different specimens with a density of 1 × 10⁶ Colony-Forming Units (CFU)/mL. After 24 h, the bacteria attached to the samples were cleaned 3 times with PBS solution, fixed by 2.5% glutaraldehyde for 30 min at 4°C, and then dehydrated with gradient ethanol (30, 50, 75, 85, 95, and 100%). Finally, the bacterial samples were sprayed with gold and observed by SEM.

The bacteriostasis rate of S. aureus and S. mutans on different substrates were determined by the attachment film method (referring to GB/T21510-2008). Briefly, 20 μL of bacterial solution (1 × 10⁶ CFU/mL) was added to the surface of different specimens, covered with a cover glass, and cultured for 24 h at 37°C. Bacterial samples were then soaked in 20 mL of PBS solution for 5 min of vortex processing. After diluting 100 times, 0.1 mL of diluted bacteria solution was evenly coated on the agar plate and cultured for another 48 h. Finally, the colony was counted one by one and the bacteriostasis rate was statistically analyzed according to the following formula: Bacteriostasis rate (%) = (Bacterial number in MAO group - Bacterial number in experimental group) / Bacterial number in MAO group × 100%. The experimental group contained Si-MAO and Si/Cu-MAO in this study.

One milliliter of S. aureus or S. mutans (1 × 10⁶ CFU/mL) were seeded on different samples for 24 h. After cleaning 3 times with PBS solution, bacteria were stained using a LIVE/DEAD^® BacLight^TM Bacterial Viability kit. According to the kit instructions, 100 μL mixture solution of SYTO9 and PI were carefully added to the surface of different specimens and incubated for 15 min. Finally, the stained bacteria were observed using a confocal laser scanning microscope (CLSM, TCS SP2, LEICA, Germany). The final results were analyzed with Leica TCS SP2 Software and presented as 3D images.

MC3T3-E1 cells were cultured with low-sugar Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. When the fusion reached 80–90%, cells were digested by 0.25% trypsin and seeded on different substrates at a density of 1 × 10⁴ cells/cm². For morphology observation, cells cultured for 3 day were also fixed by 2.5% glutaraldehyde and dehydrated with gradient ethanol (30, 50, 75, 85, 95, and 100%). The dehydrated cells were further sprayed with gold and observed by SEM. In addition, for viability detection, Cell Counting Kit-8 (CCK8) assay was carried out. Briefly, cells at 1 × 10⁴ cells/cm² were cultured on different substrates for 4 and 7 day. After removing the culture medium, the mixture of CCK-8 solution (30 μL) and DMEM medium (270 μL) was added to each well and incubated for 2 h. 200 μL of the final solution was transferred to a 96-well plate and detected at 450 nm using an enzyme-labeled instrument (Multiskan Spectrum, Thermo Fisher Scientific Inc., United States).

The activity of ALP in MC3T3-E1 cells was determined by spectrophotometry. After culturing for 4 and 7 day on different samples, MC3T3-E1 cells were collected with 0.25% trypsin, creaked by repeated freeze-thaw treatment, and then detected using a ALP Assay Kit. Briefly, 30 μL of sample lysates, phenol standard solution, or double-distilled water were added to a 96-well plate, respectively. Fifty microliter of buffer and 50 μL of matrix solution were then added to each well in turn. After incubation for 15 min at 37°C, 150 μL of color developer was added to each well and detected at 520 nm using an enzyme-labeled instrument. Meanwhile, the concentration of total protein in each group was determined with a BCA Kit at 570 nm to standardize the final ALP activity.

After culturing for 7 and 14 day on MAO, Si-MAO, and Si/Cu-MAO substrates, MC3T3-E1 cells were fixed by 2.5% glutaraldehyde (30 min) and stained with commercial alizarin red staining solution (60 min). Then, the stained calcium nodule was dissolved using 10% cetylpyridinium chloride and measured at 540 nm.

The expression of ALP, collagen I (Col I), and osteocalcin (OCN) genes was measured by real-time fluorescence quantitative PCR (RT-qPCR). After culturing for 7 day, the total RNA of MC3T3-E1 cells was extracted through the Trizol method. The mRNA was then reverse transcribed into complementary deoxyribonucleic acid (cDNA) and determined using a PrimeScript^® RT Reagent Kit and SYBR Premix ExTMTaq II, respectively. MAO group was considered the control group in this study. The expression of target genes was standardized with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. The primers were shown in Table 3.

MC3T3-E1 cells (1 × 10⁴ cells/cm²) and S. mutans (5 × 10⁵ cells/cm²) were co-cultured on different specimens for 24 h. For SEM observation, the adherent cells were cleaned 3 times with PBS solution, fixed for 40 min with 2.5% glutaraldehyde, and then dehydrated with gradient ethanol (30, 50, 75, 85, 95, and 100%). The treated samples were sprayed with gold and observed by SEM. Next, to directly observe the living and dead MC3T3-E1 cells on different substrates, the adherent cells were stained with a commercial Live-Dead Cell Staining Kit for another 20 min. The stained cells were visualized using fluorescence microscope (IX71, OLYMPUS, Japan). In addition, the ALP activity and osteogenic genes (ALP and OCN) expression of the co-cultured MC3T3-E1 cells were further determined (refer to 2.8 and 2.10).

All experiments were independently repeated 3 times. The data was represented by means ± standard deviation. SPSS20.0 packages were used for statistical analysis in this study. The one-way analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) tests were used to determine the differences between different groups. ^∗p < 0.05 (confidence level: 95%) and ^∗∗p < 0.01 (confidence level: 99%) indicated significant differences between groups.

In order to obtain the best Si/Cu-doped porous structures, the initial concentrations of Na₂SiO₃⋅9H₂O and Cu(CH₃COO)₂⋅2H₂O in the electrolyte were investigated. We selected a concentration range of 0–0.08 moL/L for Na₂SiO₃⋅9H₂O to be used in this study, because higher concentration (>0.08 moL/L) would cause the electrolyte to be cloudy and affect the integrity of target coatings. From Figure 1A, it was found that the ability of the obtained samples to promote osteoblast proliferation increased gradually with the increase of initial SiO₃^2– (Si1 < Si2 < Si3). Therefore, Si3 (also named as Si-MAO) were selected to further prepare the target Si/Cu-doped materials. After adding different concentrations of Cu²⁺ to Si3, the biological activity of target samples was Cu²⁺ concentration-dependent (increasing at first and then decreasing, Figure 1B), which was consistent with previous studies (Cao et al., 2012; Li K. et al., 2019). Since the main reason for adding Cu²⁺ was to endow titanium with excellent antibacterial properties, Si/Cu2 (also named as Si/Cu-MAO) with similar bioactivity to Si-MAO was selected for follow-up studies.

The key variables that play a role in affecting the biocompatibility of implant materials include surface morphology, roughness, chemical composition/state, crystalline structure, wettability, and coating stability (Chen et al., 2019; Abaricia et al., 2020). Hence, the in-depth surface characterization of MAO, Si-MAO, and Si/Cu-MAO were conducted. The SEM images (Figure 2A) showed no significant difference in the surface morphology of MAO, Si-MAO, and Si/Cu-MAO, which presented some porous structures (pore size: ∼4 μm). These pores were considered to be the channels for micro-arc discharges during MAO treatment. No obvious cracks were observed on these porous coatings, which was related to the pretreatment of HESP. In our previous study (Shen et al., 2020), we have demonstrated that the formation of coating cracks can be significantly inhibited by the residual compressive stress generated by the HESP treatment. In addition, the cross-sectional SEM images (Figure 2A) and eddy current thickness gauge result (Figure 2B) showed that that the porous coatings of MAO, Si-MAO, and Si/Cu-MAO (about 8.4 μm) were tightly bound to titanium substrates.

EDS results (Table 4) showed that the contents of Ca, P, Ti, and O in MAO samples were about 43.8 ± 0.4, 41.7 ± 0.4, 7.5 ± 0.3, and 7.1 ± 0.2 wt%, respectively. Furthermore, Si was also successfully detected in Si-MAO and Si/Cu-MAO groups (2.4 ± 0.2 and 2.3 ± 0.5 wt%), while Cu was only observed in the latter. The content of Cu (1.4 ± 0.1 wt%) in the Si/Cu-MAO coating was consistent with the range reported in the previous study (Yao et al., 2014).

XPS results (Figure 3A) further proved that Si and Cu were successfully doped in the Si/Cu-MAO coatings. By comparing with the NIST X-ray Photoelectron Spectroscopy Database, the fitting peak of Si2p near 102.5 eV was determined to be silicate radical (SiO₃^2–). The characteristic peaks of CuSiO₃/Cu₃(PO₄)₂ [Cu2p1/2 (955.0 eV) and Cu2p3/2 (934.7 eV)] and CuO [Cu2p1/2 (952.8 eV), Cu2p3/2 (933.0 eV), Cu2p3/2 sat (944.0 eV) and Cu2p3/2 sat (940.5 eV)] were also observed in the fitting curves of Cu2p, indicating that Cu existed in a bivalent form (Cu²⁺).

XRD results (Figure 3B) showed that the three materials have similar patterns except for the difference of peak strength. Only the characteristic peaks of anatase (JCPDS 21-1272) and titanium (JCPDS 05-0682) were observed. This indicates that there was no change in the crystalline structure of MAO coating after the addition of Cu and Si. Furthermore, there was no significant differences observed in the surface roughness (∼2.2 μm, Figure 2C), critical loads (∼14 N, Figure 2D), and water contact angle (∼54°, Figure 3C) among the three materials.

To detect the release behavior of Cu²⁺ and SiO₃^2–, Si/Cu-MAO substrates were soaked in PBS/HCl solution for 1, 4, 7, 10, and/or 14 day. Figure 3D showed that the total contents of Cu and Si in the Si/Cu-MAO coatings were about 5.8 and 13.5 μg/cm², respectively. Comparing the amount at different time, Cu²⁺ and SiO₃^2– released on day 1 was the largest (reaching 0.51 ± 0.03 and 1.3 ± 0.10 μg). With the extension of the soaking time, the amount of Cu²⁺ and SiO₃^2– gradually decreased: 0.42 ± 0.03 and 0.96 ± 0.09 μg (4 day), 0.32 ± 0.03 and 0.79 ± 0.04 μg (7 day), 0.30 ± 0.02 and 0.75 ± 0.03 μg (10 day), and 0.27 ± 0.03 (14 day) and 0.66 ± 0.04 μg, respectively. Further statistics showed that the release of Si and Cu accounted for about 33.2 and 31.3% of the total added ions within 14 day. The long-term slow-release property of the Cu²⁺ and SiO₃^2– can endow Si/Cu-MAO specimens with longer antibacterial and osteogenic induction functions.

S. aureus is a common pathogen of implantable infection and has strong pathogenicity. Recent studies have confirmed that S. aureus is also a common cause of peri-implantitis (Ulu et al., 2018; Karthikeyan et al., 2019). Moreover, S. mutans in the oral cavity is also a common bacterium in inducing peri-implantitis, and can significantly increase the pathogenicity of other bacterial infections (Geremias et al., 2017; Lemos et al., 2019). Therefore, we have selected both S. aureus and S. aureus to be used for antibacterial studies of our target materials in this study. No significant difference in antibacterial activity was observed between MAO and Si-MAO groups (Figure 3A), which indicated no contribution of Si to antibacterial property. Compared to MAO and Si-MAO, Si/Cu-MAO samples showed a better bacteriostatic effect on S. aureus and S. mutans, and the antibacterial rate was about 99.1 ± 2.3% and 98.7 ± 3.5%, respectively. This was further confirmed by the Live/Dead staining results as shown in Figure 5. The staining images (Figure 5A) demonstrated the two types of bacteria on the surface of MAO and Si-MAO were stained green (living bacteria), while almost all bacteria on Si/Cu-MAO were stained red (dead bacteria). The dead/total rate (Figure 5B) of bacteria on Si/Cu-MAO substrates were 96.4 ± 3.2% (S. aureus) and 94.1 ± 3.9% (S. mutans), respectively. These results suggested that Si/Cu-MAO had an excellent broad-spectrum germicidal efficacy against the common oral bacteria.

Previous studies had demonstrated that copper substances/ions could harm the nucleic acid, protein, and lipid of bacteria by changing bacterial membrane potential or inducing excessive active oxygen production (Hong et al., 2012; Tambosi et al., 2018; Zhang et al., 2020). The antibacterial mechanism of Si/Cu-MAO to S. aureus and S. mutans might be also as follows: firstly, when bacteria attached to Si/Cu-MAO, the doped copper directly destroyed the membrane potential and induced the death of bacteria (contact bacteriostasis; Zhang et al., 2020); secondly, the released Cu²⁺ could bind to the sulfhydryl and amino groups in the bacterial membrane/protein, destroy the energy metabolism and respiratory system of the bacteria, resulting in bacterial death (release bacteriostasis; Tambosi et al., 2018). Therefore, the synergistic effect of contact and release modes endowed Si/Cu-MAO with broad-spectrum antibacterial effect.

The attachment of S. aureus and S. mutans to different substrates were observed through SEM, as shown in Figure 4B. A large number of S. aureus and S. mutans were observed on the surfaces of MAO and Si-MAO, indicating that the addition of SiO₃^2– had no obvious antibacterial effects. Compared with MAO and Si-MAO groups, there was a significant reduction of both bacteria on the surfaces of Si/Cu-MAO samples indicated with red arrows (dead bacteria). This further showed that Si/Cu-MAO could inhibit the early adhesion and proliferation of S. aureus and S. mutans, which was confirmed by the above results of bacteriostasis rate (Figures 4A, 5).

To determine the effects of additive SiO₃^2– and Cu²⁺ on cell morphology and viability, SEM observation and CCK-8 measurement were carried out. From SEM results (Figure 6A), it was found that all MC3T3-E1 cells adhered tightly to the surfaces of different materials after 3 day of culture. Compared with the MAO group, the cells on Si-MAO and Si/Cu-MAO groups produced more filamentous pseudopods (red arrows), which extended into the pore structures. The result (Figure 6B) of cell viability also showed that Si-MAO and Si/Cu-MAO significantly (p < 0.05 or 0.01) improved the proliferation of MC3T3-E1 cells compared with MAO group after culturing for 4 and 7 day. However, no significant difference of cell viability was observed between Si-MAO and Si/Cu-MAO groups.

In this study, to obtain the best bacteriostatic effect, we chose the highest concentration of Cu²⁺ that MC3T3-E1 cells could tolerate as the parameter of sample preparation (Figure 1B). Thus, the properties of Si-MAO and Si/Cu-MAO in promoting pseudopodia formation and cell proliferation were mainly caused by Si. It had been proved that Si had great potential to cause cell microenvironment alkalization and activate the Ca²⁺ channels to increase the Ca²⁺ influx, thus promoting the early cell adhesion and pseudopods formation (Liu et al., 2013; Wu et al., 2014). In addition, Si could also combine with O₂ to form silicate with three-dimensional network structures, then adsorb free proteins and interact with cell integrin, and finally promote the adhesion, proliferation and differentiation of osteoblasts (Henstock et al., 2015).

As a non-specific phosphomonoesterase, ALP had been proved to greatly hydrolyze inorganic pyrophosphate and increase the formation of hydroxyapatite (Siller and Whyte, 2018; Yu et al., 2018). Thus, ALP activity was frequently selected as a typical early marker of osteoblast differentiation. In addition to ALP, the mineralization level of osteoblasts was also evaluated as a late indicator of osteogenic differentiation in previous researches (Yu et al., 2018; Su et al., 2020). After both 4 and 7 day of culture, the ALP activity of MC3T3-E1 cells in Si-MAO and Si/Cu-MAO groups was significantly (p < 0.05 or 0.01) higher than that in MAO group (Figure 7A). The mineralization results (Figure 7B) also confirmed that Si-MAO and Si/Cu-MAO substrates were more effective in promoting osteoblast differentiation. In addition, to further verify the osteoinductive potential of different samples at the molecular level, the expression of ALP, collagen I (Col I) and osteocalcin (OCN) gene was measured after 7 day. The expression trends (Figure 7C) of the three genes were similar to ALP activity and mineralization level: compared with MAO, both Si-MAO and Si/Cu-MAO significantly (p < 0.05) enhanced the gene expression of ALP, Col I, and OCN.

The aforementioned results of pseudopodia formation, cell proliferation, ALP activity, mineralization and gene expression showed that Si/Cu-MAO was slightly better than Si-MAO, but there was no significant difference between them. This indicated that the osteogenic effects of Si-MAO and Si/Cu-MAO was mainly attributed to Si rather than Cu. Sun et al. had reported that the Si-doped hydroxyapatite could promote the proliferation and differentiation of osteoblasts via up-regulating MAPK and Wnt signaling pathways (Sun et al., 2018). Dong et al. also claimed that SiO₃^2– could promote the Col I and OCN synthesis of MSCs by activating the BMP-2/Smad1/5/RUNX2 signaling pathway (Dong et al., 2016). Although Cu²⁺ also had superior biocompatibility, they were very dose-dependent (Lin et al., 2016; Li K. et al., 2019). High concentrations of Cu²⁺ would lead to significant cytotoxicity through the oxidative stress pathway (Cao et al., 2012; Li K. et al., 2019). The release of Cu²⁺ from Si/Cu-MAO was about 0.51 μg (∼8 μM) at the first day, which had been close to its safe concentration (∼10 μM) for MC3T3-E1 cells (Li K. et al., 2019). Therefore, compared with Si-MAO, Si/Cu-MAO did not significantly promote the spreading, proliferation and osteogenic differentiation of MC3T3-E1 cells, nor did it show obvious cytotoxicity in this study.

Studies have shown that when bacteria are present, they competitively adhere to the implant surface with the host cells (Subbiahdoss et al., 2011). By forming a dense bacterial membrane or secreting a large amount of toxin, these adherent bacteria induce apoptosis of repair cells and ultimately prevent the formation of new bone (Hoffman et al., 2005; Subbiahdoss et al., 2011; Shen et al., 2019b). The ideal implantation material is expected to inhibit bacterial infection without affecting the normal physiological function of osteoblasts. Therefore, in this study, to further investigate the protective effects of different materials on surface cells in the presence of bacteria, MC3T3-E1 cells and S. mutans were further co-cultured for 24 h. The SEM images (Figure 8A) showed that MC3T3-E1 cells on the surfaces of MAO and Si-MAO were round, and many bacteria adhered to the cell surface (cell location images). Many bacteria were also observed in the empty spaces around the cells (bare location images). Compared with the above two groups, the number of bacteria in the cell and bare parts of the Si/Cu-MAO group decreased significantly. The cell spreading of MC3T3-E1 cells (red arrows) on the surface of Si/Cu-MAO group was almost unaffected by bacteria. Live/Dead staining images (Figure 8A) showed that there were a large number of dead cells (red color) on MAO and Si-MAO, and the remaining living cells (green color) also shriveled into a round shape. However, only a small number of cells died on Si/Cu-MAO substrates, indicating that it could ensure the survival of MC3T3-E1 cells effectively in the presence of S. mutans. Meanwhile, we found that the round living cells on MAO and Si-MAO appeared to be larger than those in Si/Cu-MAO group, most likely due to the presence of a large number of living bacteria (also appeared green) on the cell surface. Figures 8B,C further showed that only the co-cultured MC3T3-E1 cells on Si/Cu-MAO substrates had significant ALP activity and genes (ALP and OCN) expression. The other two groups showed almost no enzyme activity and gene expression. These results further proved that Si/Cu-MAO substrates had good antibacterial activity and could maintain the normal biological behavior of surface osteoblasts under infection conditions.