A zinc bar (Zn ≥ 99.99%) was cut into disk specimens with dimensions of Φ10 × 2 mm. These samples were ground with sandpapers to 2000#, ultrasonically cleaned with deionized water, respectively, and then dried in a drying cabinet. Whereafter, the samples were exposed to an acidic calcium phosphate electrolyte consisting of 0.12 M NaH₂PO₄, 0.20 M CaCl₂, and 0.10 M Na₂EDTA·2H₂O with the pH of 3.50 for 6 h at 100°C in autoclaves. Then, the obtained Ca-P-coated samples were washed 3 times using deionized water and dried. The Li-OCP particles were fabricated by the following method: 0.009 mol NH₄PO₃ and 0.03 mol CH₄N₂O were mixed in 300 ml of distilled water for 30 min at room temperature (named A solution), while 0.01188 mol calcium acetate and 0.00012 mol of lithium chloride were mixed in 300 ml of distilled water for 30 min at room temperature (named B solution). Subsequently, solution B was added to solution A and stirred for 5 min (solution C). Solution C was heated to 90°C in a water bath with stirring for 2 h. The Li-OCP particles were obtained after the reaction. The PLA powders and Li-OCP particles were mixed and PLA was dissolved in chloroform solvent to fabricate the PLA/Li-OCP solution. Then, the Ca-P-coated samples were dipped in PLA/Li-OCP solution and pulled out rapidly to produce the PLA/Li-OCP coating on the Ca-P-coated samples (named PLA/Li-OCP-coated sample). The weight ratio of the PLA powders and Li-OCP particles was 400:1.

A scanning electron microscope was used to observe the morphologies of coated zinc. The chemical compositions and phase of the Ca-P coatings were analyzed by energy spectroscopy (EDS) and x-ray diffraction, with the radiation source being Cu-K α with a scanning speed of 5°. The chemical valence state of coatings was characterized by x-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, Thermo Fisher) equipped with a monochromatic Al-K α (1,486.60 eV). The C1s peak located at the binding energy of 284.80 eV was used for the charge correction. The wettability of coatings was analyzed by a contact angle meter.

The corrosion rate of the coated samples was studied via immersion test in simulated body fluid (SBF) solution at 37 ± 0.50°C for 21 days. The volume of solution to the immersed area of the surface was 1.25 ml/cm². To imitate a dynamic circulation environment in the human body, the SBF solution was refreshed every 3 days. At each time point, the pH value was recorded by a pH meter. After 21 days of immersion, the immersed coated zinc was washed by deionized water and dried in air. Scanning electron microscopy was used to observe the morphology.

The corrosion resistance of coated zinc was measured via potential polarization curve and electrochemical impedance spectroscopy (EIS) using a Princeton Model 273A electrochemical work station in SBF solution. In the electrochemical test, the pure zinc and coated zinc were used as the working electrode, platinum sheet was used as the auxiliary electrode, and saturated mercuric electrode was used as the parameter electrode. In the dynamic potential polarization test, an applied potential ranged from −0.50 V to +1.50 V relative to OCP with a scanning velocity of 1 mV/s. The frequency ranging from 105 Hz to 10–2 Hz was used applying 10 mV perturbation in the EIS test.

Pre-osteoblast cell lines MC3T3-E1 were used to analyze the cytotoxicity. The MC3T3-E1 were cultured with studied samples in Dulbecco’s modified Eagle’s medium (DMEM) containing 100 U/ml penicillin, 10% fetal bovine serum (FBS), and 100 μg/mg streptomycin in an incubator with humid 5% CO₂. The cytotoxicity was measured using MTT according to operating instructions. The studied coated zinc and uncoated zinc were soaked in DMEM to extract the immersion at 37°C for 3 days. The cells with a density of 5× 10⁴/well were seeded in a 96-well plate for 1, 3, and 5 days, respectively. The medium was refreshed every day. According to the manufacturer’s protocol, the cell viability in terms of optical density (OD) values was obtained at a wavelength of 490 nm in a microplate reader. Meanwhile, 5×10⁴ cells/ml were seeded on samples in 24-well plates and cultured for 3 days to assess the cell morphology attached to studied samples. After that, the cells attached to the samples were rinsed with PBS solution, and then fixed on the surface of samples using 4% paraformaldehyde at 25°C for 25 min and subsequently rinsed with PBS. Thereafter, the cell microfilament was stained by 1.0% (v/v) FITC-phalloidin dye for 30 min, and then nuclei were stained by 1 mg/ml DAPI for 10 min at 37°C. The cell morphology was analyzed by a fluorescence microscope.

The mRNA expression of osteogenic differentiation-related genes including RUNX2, BMP, and OCN was detected by quantitative PCR (qPCR) analysis. The cells MC3T3-E1 with a density of 5 × 10⁴/well were cultured with studied samples in DMEM in a 24-well plate for 3 and 5 days. According to operating instructions, the mRNA expression of osteogenic genes including RUNX2, BMP, and OCN was analyzed by qPCR each time.

The one-way ANOVA was used for statistical analysis. There is a significance between the studied groups when p < 0.05.

Figures 1A–F show the morphology of pure zinc, the Ca-P-coated samples, and PLA/Li-OCP-coated samples. As for the pure zinc without coating, scratches could be found on the pure zinc after being ground with sandpapers to 2000# (Figure 1A,B). With Ca-P coating treatment, at low magnification (Figure 1C), the coatings on pure zinc formed at different temperatures seem compact and dense without evident defects, indicating that the Ca-P coatings could be successfully formed on the pure zinc substrate. At high magnification (Figure 1D), numerous good crystals could be detected in all coatings, but the morphology of those coatings was different. In the case of the PLA/Li-OCP-coated samples, the morphology of Ca-P coating was covered by the PLA/Li-OCP coating. Moreover, the nano Li-OCP particles cannot be well observed in the PLA coating. The EDS results obtained from zones Ⅰ and Ⅱ are listed in Table 1, which suggested that the ratio of Ca/P for the Ca-P coating was close to 1, and the C element appeared in the PLA/Li-OCP coating, which belonged to PLA. However, the Li element cannot be detected by EDS because Li would emit an x-ray signal that exists at a relatively low intensity of less than 55 eV. This is because the conventional EDS detectors absorb 100% of x-rays of approximately 55 eV (Lin et al., 2021).

Figure 2A shows the results of the contact angle of studied samples. Hydrophilicity is one of the indicators to assess the ability of cell adhesion on the implant in the initial stage. As can be seen, the value of contact angle of pure zinc was 121.54 ± 0.81°, and the values of contact angle for the Ca-P- and PLA/Li-OCP-coated samples were 61.12° ± 1.43 and 80.68° ± 1.05°, respectively, indicating that there was no significant difference in the water contact angle among the coatings obtained by different temperatures. Thus, the presence of coatings on the zinc could decrease the water contact angle, suggesting that the coating could improve the hydrophilicity of the surface compared to pure zinc. Figure 2B shows the XRD of the samples with and without coatings. The main phase of the Ca-P coatings was dicalcium phosphate anhydrous CaHPO₄, whose ratio of Ca/P was consistent with the EDS results in Table 1. When the zinc was exposed to the electrolyte, heterogeneous nucleation of CaH₂EDTA began to occur on the substrate. Meanwhile, upon processing the hydrothermal treatment, the increasing temperature promoted the precipitation of the CaH₂EDTA crystal on the zinc surface. However, the further increasing temperature resulted in the dissolution of CaH₂EDTA crystals, releasing calcium ions. Then, the calcium ions react with dihydrogen phosphate ions to form CaEDTA 2 − + H + ⇌ CaHEDTA − (3–1) CaHEDTA − + H + ⇌ CaH 2 EDTA ↓ (3–2) CaH 2 EDTA ⇌ Ca 2 + + H 2 EDTA 2 − (3–3) Ca 2 + + HPO 4 2 − ⇌ CaHPO 4 ↓ (3–4)

CaHPO₄, as a kind of biological CaP ceramics, shows excellent biocompatibility to the bone, and has been used as a coating to improve the proliferation and maturation of osteoblastic cells (Chow, 1999). As for the PLA/Li-OCP-coated samples, the main phase of the coating was Ca₈H₂(PO₄)₆∙5H₂O. The total spectrum of the Ca-P coating in Figure 2C shows that the peaks of O1s, Zn2p, Ca2p, and P2p were detected, while Li 1s was found in PLA/Li-OCP coating except for O1s, Zn2p, Ca2p, and P2p in Figure 2D; this indicated that the Li was doped into the OCP particles.

The potentiodynamic polarization curves are shown in Figure 3, and the corrosion potential (E _corr ) and the corrosion current density (I _corr ) are listed in Table 2. Compared with the pure zinc, there was no significant difference in the E _corr between the pure zinc and Ca-P-coated samples, but the E _corr for the PLA/Li-OCP sample shifted to positive. The E _corr value of the bare zinc was −1.12 ± 0.03 V, and the E _corr value for the Ca-P-treated sample and PLA/Li-OCP samples was −1.18 ± 0.12 V and −1.03 ± 0.07 V, respectively. In thermodynamics, the positive shift of corrosion potential indicated that the corrosion tendency of pure zinc was inhibited by the presence of coatings on the substrate. Meanwhile, I _corr in the coated specimens was decreased compared with the bare zinc, in which the PLA/Li-OCP sample exhibited the lowest corrosion current density. This indicated that the PLA/Li-OCP coatings could effectively act as a barrier and block the infiltration of electrolytes into the substrate. With regard to the coated samples, the I _corr of the Ca-P-coated sample was reduced from 8.26 × 10^–5 A/cm² of pure zinc to 1.32 × 10^–5 A/cm². When treated with PLA/Li-OCP on the Ca-P-coated sample, the I _corr was further reduced to 6.56 × 10^–6 A/cm². As mentioned above, the PLA/Li-OCP-coated samples had the best corrosion resistance.

The EIS was used to further study the corrosion resistance of the coated samples. Figure 4A shows the Nyquist curves of studied specimens without coatings in SBF solution. In all cases, two semicircle-like curves were found in the Nyquist curves. In high frequency, the capacitive loop generally corresponds to the charge transfer resistance of corrosion products. Herein, the capacitive loop was assigned to the Ca-P coatings, while this capacitive loop in high frequency was attributed to the corrosion product for the bare zinc (Jamesh et al., 2015). The capacitive loop represented the adsorption process during corrosion. In the lower-frequency region, as a rule, the diameter of the semicircle-like curves can be represented by the polarization resistance of the coatings. In Figure 4A, the PLA/Li-OCP sample exhibited the biggest diameter of the semicircle-like curves, whereas pure zinc showed the smallest diameter of the semicircle-like curves. The Bode-impendence plots in Figure 4B show that a remarkable increase of impedance value was obtained in the coated samples compared with the bare zinc. The impedance for pure zinc, Ca-P, and PLA/Li-OCP-coated samples was ∼242.13, ∼336.65, and ∼1200 Ω cm², respectively. As expected, the PLA/Li-OCP samples exhibited the greatest impedance at the lowest-frequency zone, indicating that the PLA/Li-OCP samples had the best corrosion resistance. Moreover, all the samples revealed an inductive character at the lower frequencies, a characteristic of pitting corrosion (Wu et al., 2017). The bode-phase plots in Figure 4C demonstrate that there was a significant increase in the phase angle in the medium-frequency region as the treatment temperature increased. The phase angle was reduced from −30.25° of pure zinc to −55° of the PLA/Li-OCP sample. Besides, two time constants can be found in the coated samples. Normally, in the high frequencies, the time constant is assigned to double-layer capacitance and the corresponding charge transfer resistance, while one is related to the film resistance of the corrosion product in low frequency (Wojciechowski et al., 2016). The equivalent electrical circuit was applied to fit the EIS spectra (Figures 4D,E). R_s means the solution resistance. CPE1 and R_ct are the constant phase element (CPE) and corrosion product resistance, while the constant phase element and the inner layer resistance were represented by CPE2 and R_f, respectively (Jamesh et al., 2015). Moreover, the capacitance to remit the “scatter effect” at the interface between electrode and solution was replaced by CPE with a symbol of Q. In general, higher values of R_ct and R_f mean a greater corrosion resistance of the sample. A lower CPE1 value represents a lower corrosion area on the surface, while a higher CPE2 means the relatively thin and incompact film on the metal. The corresponding results fitted from the circuit elements are listed in Table 3, which was a good fit confirmed by chi-square (χ ²) values of about 10^–3. The values of R_ct of the coated zinc for the Ca-P- and PLA/Li-OCP-coated samples were 275.34 and 275.34 Ω cm², respectively, which were greater than 276.82 Ω cm² of pure zinc; the values of R_f of the coated zinc for the Ca-P- and PLA/Li-OCP-coated samples were 25.34 and 156.47 × 10³ Ω cm², respectively, indicating that the corrosion resistance of the inner layer was increased with temperature; besides, lower CPE1 and CPE2 values were also found in the coated zinc, indicating a near-capacitive behavior of the zinc modified by coatings. It is observed that the R_ct resistance of the coating was significantly larger than the R_f resistance of the outer layer, suggesting that the compact coating can favor more effective protection on the substrate from corrosion.

The pH values of SBF soaked with studied samples with and without coatings for 21 days are presented in Figure 5A. When the zinc and its alloys are exposed to the electrolyte, the chemical reaction listed as the following would have occurred (Ma et al., 2015; Hernandez-Escobar et al., 2019): Anodic reaction : Zn →  Zn 2 + + 2 e - (3–5) Cathodic reaction : H 2 O + 1 / 2 O 2 → 2 OH - (3–6) Total reaction : Zn 2 + +   2 OH - Zn → ( OH ) 2 (3–7)

Proceeding with the anode reaction, the degradation of zinc and its alloys begins to occur. Most of the OH⁻in the cathode reaction would react with Zn²⁺ ions to form zinc hydroxide on the surface of pure zinc, while the rest of the OH⁻would cause an increase of pH value in the surrounding environment, which will promote the deposition of insoluble calcium and phosphorus salt on the zinc surface. The SBF solution immersed with the bare zinc exhibited the highest pH value approaching 7.89 on the first day, which sharply increased for 9 days, and then reduced after 12 days of immersion. With the deposition of the complex corrosion products on zinc surface, a relatively dense protective film dominantly consisting of zinc hydroxide would result in the decrease of corrosion rate. With the extension of the immersion time, the thicker corrosion layer further reduced the corrosion rate of zinc. At the same time, a degradation behavior occurred, resulting in the damage of the corrosion product layer. Then, the electrolyte infiltrated into the substrate to proceed with the degradation. This phenomenon could be reflected by the fluctuant pH curve, suggesting an alternative in pit corrosion, generation, and degradation of corrosion products on zinc. In the initial immersion time, the pH values of the SBF solutions immersed with CaP-coated samples significantly decreased, indicating that all the coatings could improve the degradation rate of pure zinc. Noticeably, it can be seen that there was a different degradation behavior among the coated samples. As for the Ca-P-coated samples, the pH value began to increase when the samples were immersed for 1 day. This suggested that some electrolyte permeated into the substrate. In contrast, the PLA/Li-OCP samples showed the lowest pH value concerning the other groups, indicating that more compact and stable coatings were deposited on pure zinc. After 12 days, the pH was gradually reduced and approached a lower level for the coated zinc. Moreover, a more fluctuant profile of the pH values in the bare zinc was observed with respect to the zinc with Ca-P coatings. With regard to coated samples, such a phenomenon was not evident in the coated groups, especially for the PLA/Li-OCP group.

Figures 6A–G display the morphology of the studied samples immersed in SBF for 21 days, and the corresponding EDS patterns are shown in Figure 6G. It can be seen that lots of pits were displayed in pure zinc, indicating that pure zinc suffered from pitting corrosion, as seen in Figure 6A,D; moreover, the EDS patterns obtained from point a and b in Figure 6D suggested that the corrosion products consisted of Ca and P. In the case of the Ca-P-coated sample in Figure 6B, a crack could be observed on the coating, as marked by the ellipse, and the EDS patterns from point c in Figure 6E indicated that this region should be assigned to the corrosion products. This is because the Zn element appeared in this region compared with point b. The presence of Zn demonstrated that the occurrence of pit corrosion resulted in the released zinc ions from the substrate reacting with calcium phosphate to form corrosion products, suggesting that the protection of Ca-P coating locally became invalid, resulting in the electrolyte permeating into the substrate. Concerning the PLA/Li-OCP coating (Figure 6C), there was still a compact and dense PLA coating without obvious defects on the substrate. This suggested that the PLA coating could provide ample protection to prevent the electrolyte from infiltrating the substrate. This is further confirmed by the EDS patterns in point e in Figure 6F, which showed that no Zn element was found in the coating, and a small amount of Ca and P was found, indicating sufficient protection from the coating although there was degradation of the PLA coatings.

The cell viability after cells were seeded on studied samples with and without coatings for 1, 3, and 5 days is shown in Figure 7. It can be seen that there was almost no cell activity when the cells were exposed to the bare zinc during culture time, which suggested severe cytotoxicity of the material. This could be attributed to the high zinc ion concentration and pH value that induced cell apoptosis. In contrast, such a case was improved in that cell activity could be detected when the cells were co-cultured with the coated samples. As for the Ca-P-treated group, the maximum cell viability was found on the first day and then the value of viability slightly decreased with time. The cell viability for the PLA/Li-OCP group was higher than that of the Ca-P-treated samples at any time point, and the cell viability gradually increased after 3 and 5 days of culture. This revealed that the PLA/Li-OCP sample showed the greatest cytocompatibility with the ability for cell proliferation due to the degradable rate being considerably improved by the coating obtained at this temperature. Figure 8 shows photographs of MC3T3-E1 cells exposed on the studied samples for 3 days. In Figure 8, alive cells failed to adhere to the pure zinc surface, showing lytic morphology, a characteristic of dead cells. This case was slightly changed in the Ca-P-coated samples. In contrast, these conditions were changed by the presence of PLA/Li-OCP coatings. It can be seen that the cells attached to the PLA/Li-OCP coatings show a healthy spreading morphology, indicating that the PLA/Li-OCP coatings could favor the attachment and proliferation of cells. This observation coincided with the results of cell activity.

To further evaluate the effect of coating on cell proliferation, RT-PCR was applied to measure the related osteogenic genes at the mRNA level including runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and bone morphogenetic protein (BMP). Figures 9A–C show the expression levels of RUNX2, BMP, and OCN after cells were cultured with samples for 3 and 5 days. As expected, the expression of all related osteogenic genes in the bare zinc was much low during culture time, whereas fabricating the Ca-P coatings on the zinc could slightly enhance the expression of related osteogenic genes. After 3 days of culture, the expression of RUNX2 and BMP on Ca-P-treated samples was higher than that on the pure zinc, and the PLA/Li-OCP samples present the greatest gene expression among the other groups. After 5 days of culture, the expression of the osteogenic genes in PLA/Li-OCP-treated samples was higher than that of the Ca-P-treated samples and pure zinc. As mentioned above, the PLA/Li-OCP coating could improve the osteogenic activity and osteogenesis of pure zinc.