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. 2026 Mar 13;11(11):17860–17865. doi: 10.1021/acsomega.5c12419

Whiteness, Cytocompatibility, and Antibacterial Activity of Carbonate Apatite Granules Containing Zinc Oxide for Bone Regeneration

Zhibin Wang †,*, Masaya Shimabukuro ‡,*, Risa Miyake †, W M Ruvini Lasanthika Kumari Wijekoon †, Taiyo Yukawa †, Taishi Yokoi ‡, Eriko Marukawa †, Masakazu Kawashita ‡
PMCID: PMC13019271  PMID: 41908374

Abstract

Bone infections challenge orthopedic treatments, requiring artificial bone materials that combine antibacterial and osteogenic activities. Most inorganic antibacterial agents often compromise the aesthetic quality; it is necessary to impart antibacterial activities to artificial bone materials without compromising their aesthetic appearance. In this study, we focused on zinc oxide (ZnO) as an antibacterial agent, and ZnO-containing carbonate apatite (CAp) granules were fabricated from precursors composed of calcium carbonate (CaCO3) and ZnO via a pH-controlled dissolution–precipitation. The compositional conversion from CaCO3 to apatite in a sodium hydrogen phosphate (Na2HPO4) solution at pH 8.9 was terminated by the formation of zinc phosphate (Zn3(PO4)2·2H2O), whereas complete conversion was achieved in the solution at pH 7.0 without the formation of Zn3(PO4)2·2H2O. The whiteness values of ZnO-containing CAp granules prepared at pH 7.0 were comparable to those of pure CAp granules, indicating that ZnO incorporation did not affect the visual appearance. Furthermore, the ZnO-containing CAp granules exhibited significant antibacterial activity against methicillin-resistant Staphylococcus aureus while maintaining satisfactory cytocompatibility with osteoblast-like cells. These findings demonstrate that ZnO-containing CAp granules possess balanced aesthetic, antibacterial, and biological properties, suggesting their potential as promising artificial bone materials for orthopedic and implant applications where infection control and cytocompatibility are both required.

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Introduction

Bone infections are common complications in orthopedics, and effectively treating these infections while simultaneously promoting bone regeneration in infected bone defects remains a significant clinical challenge. To address this issue, imparting antibacterial activity to artificial bone materials has been considered an effective strategy for the treatment of infected bone defects. Calcium phosphate-based compounds, such as hydroxyapatite (HAp), β-tricalcium phosphate (β-TCP), and carbonate apatite (CAp), − have been widely employed as artificial bone materials. Among them, because natural bone is primarily composed of apatite integrated with type I collagen and contains carbonate substitutions that vary depending on age, species, and origin, , CAp has attracted considerable attention as a promising candidate material due to its compositional similarity to native bone. However, as these calcium phosphates cannot resist bacterial colonization in infected bone tissue, antibacterial activities have been conferred by incorporating antibacterial agents such as silver (Ag) or copper (Cu). − However, Ag- and Cu-based antibacterial agents often compromise the aesthetic appearance of artificial bones due to their inherent coloration. − Therefore, it is necessary to impart antibacterial activities to artificial bone materials without compromising their aesthetic appearance. Zinc oxide (ZnO) has attracted significant attention for its mild antibacterial activity, demonstrating effectiveness against a wide range of pathogenic bacteria. − Furthermore, ZnO is a white compound, which may provide antibacterial activity without compromising the aesthetic appearance of artificial bone materials.

One method for synthesizing carbonate apatite (CAp) is the compositional conversion of a precursor through a dissolution–precipitation reaction. − , In this process, the reaction system is supersaturated with respect to CAp, leading to its immediate precipitation upon dissolution of the precursor, CaCO3. In contrast, ZnO readily forms zinc phosphate upon dissolution during the dissolution–precipitation reaction. Because zinc phosphate is a sparingly soluble compound, − its precipitation during the conversion of CaCO3 to CAp is anticipated to terminate the reaction. Therefore, to prepare ZnO-containing CAp using this synthesis method, ZnO must be mixed with the CaCO3 precursor, while preventing the precipitation of zinc phosphate during compositional conversion from CaCO3 to CAp.

In the present study, we synthesized ZnO-containing CAp granules via a pH-controlled dissolution–precipitation method and systematically examined the influence of ZnO on aesthetic, antibacterial activity and cytocompatibility of the resulting samples. The aesthetic performance was evaluated by comparing the whiteness of the prepared samples with that of samples containing other antibacterial agents, while the antibacterial activity and cytocompatibility of ZnO-containing CAp was assessed in vitro against methicillin-resistant Staphylococcus aureus (MRSA) and MC3T3-E1 cells.

Materials and Methods

Sample Preparation

ZnO powder (Fujifilm Wako Pure Chemical, Osaka, Japan) was mixed with CaCO3 powder (Sakai Chemical Industry Co., Ltd., Osaka, Japan) at ZnO contents of 0, and 10 wt % relative to CaCO3. The ZnO used in this study was crystals less than 1 μm in size (Figure S1). The mixtures were placed in a stainless-steel mold and uniaxially pressed at 150 MPa using an oil pressure machine. The pressed samples were then heated to 350 °C at a rate of 5 °C·min–1 and maintained at this temperature for 24 h. After heating, the samples were crushed and sieved to obtain CaCO3 granules of 600–1000 μm in size. The granules were immersed in a 1 mol·L–1 sodium hydrogen phosphate (Na2HPO4; Fujifilm Wako Pure Chemical Industries, Osaka, Japan) solution at pH of 8.9, or in the same solution adjusted to pH 7.0 using H3PO4. This immersion process was conducted at 80 °C for 7 days. After immersion, the resulting granules were washed by immersion in distilled water at 80 °C for 30 min. The CAp granules prepared from CaCO3 containing 0 and 10 wt % ZnO were designated as CA0Z and CA10Z, respectively.

Sample Characterization

The crystalline phases of samples were characterized using X-ray diffraction (XRD; MiniFlex600; Rigaku Corp., Tokyo, Japan) with Cu Kα radiation. Diffraction patterns were recorded over a 2θ range of 10 to 60° with a step size of 0.02°. Fourier-transform infrared (FT-IR) spectroscopy (FT/IR-6200; JASCO, Tokyo, Japan) was used to analyze the chemical structures of the samples. The ZnO content was calculated based on the Zn concentration measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES; ICPE-9800; Shimadzu Corp., Kyoto, Japan). For the ZnO content analysis, the samples were completely dissolved in nitric acid solution, and the Zn concentration was quantified via ICP-AES. The ZnO content was then calculated using the measured Zn concentration, solution volume, molecular weight ratio of Zn to ZnO, and the weight of the dissolved sample. The microstructures of the samples were observed by field-emission scanning electron microscope with energy-dispersive X-ray spectrometry (FE-SEM/EDS; JSM-7900F; JEOL, Tokyo, Japan). The overall appearance structures of the samples were observed using an S9D stereomicroscope (Leica Microsystems, Wetzlar, Germany) equipped with a Digital Sight 1000 camera (Nikon, Tokyo, Japan). The whiteness value of each sample was measured using a whiteness meter (AWM-216; Guangdong Amittari Instruments Co. Ltd., Guangdong, China) (n = 3), which was calibrated with a standard white board (whiteness value = 77.2). For comparison, CAp powders were mixed with 10 wt % Ag (99.9%, Alfa Aesar, Haverhill, MA) or Cu (99%, Alfa Aesar) powders, and their whiteness was measured. Unlike the main samples, which were fabricated as ZnO-containing CAp granules to assess whiteness, antibacterial activity, and cytocompatibility, these mixtures were not granulated but were prepared merely as powder mixtures of CAp with Ag or Cu nanoparticles for whiteness comparison. The amount of Zn ion released from the samples was evaluated using ICP-AES. The samples were immersed in a physiological saline (Otsuka Pharmaceutical, Tokyo, Japan) at 37 °C for 24 h. After immersion, the Zn ion concentration in the supernatant was measured using ICP-AES (n = 3).

Cytocompatibility Evaluation

Before evaluations, all samples were subjected to dry-heat sterilization at 170 °C for 3 h. All sterilized samples were immersed in the medium overnight to prevent the burst release of Zn ions. As described in our previous study, , MC3T3-E1 cells (Riken BioResource Center, Ibaraki, Japan) were maintained in a cell culture medium, which was an α modification of Eagle’s minimum essential medium (MEMα; Fujifilm Wako Pure Chemical, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; HyCloneTM, Cytiva, Logan, UT), 100 U·mL–1 penicillin, 100 μg·mL–1 streptomycin, and 0.25 μg·mL–1 amphotericin B (Fujifilm Wako). The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Extracts were obtained by incubating the samples at a ratio of 0.1 g per 1 mL of medium for 24 h. Cytotoxicity tests were performed using the sample extracts. MC3T3-E1 cells were cultured in the regular culture medium at an approximate initial concentration of 10,000 cells·well–1 for 24 h. The regular culture medium was then replaced with the prepared material extracts. After 24 h of incubation, cell viability was assessed using the CCK-8 assay (n = 5).

Antibacterial Activity Evaluation

Before evaluations, all samples were subjected to dry-heat sterilization at 170 °C for 3 h. All sterilized samples were immersed in the medium overnight to prevent the burst release of Zn ions. For antibacterial activity evaluation, MRSA was cultured in Tryptic Soy Broth medium (Sparks, MD) at 37 °C for 24 h. The optical density of the bacterial suspensions was measured at 600 nm (OD600) using a CO8000 Biowave cell density meter (WPA Ltd., Cambridge, U.K.). The suspensions were then diluted to achieve a concentration of 3.2 × 107 CFU·mL –1. A total of 50 μL of bacterial suspension was added to 0.05 g of each sample, followed by incubation at 37 °C for 24 h (n = 3). The BacTiter-Glo Microbial Cell Viability Assay was employed to evaluate the antibacterial efficacy of each sample.

Statistical Analysis

Statistical analyses were performed using the Kaleida Graph software (version 4.1.1; Synergy Software, Eden Prairie, MN). Student’s t test was performed for two-group comparisons. Statistical significance was set at p < 0.05 in this study. All experiments were conducted with independently prepared scaffold samples, and the reported n values refer to biological replicates.

Results and Discussion

Before immersion in the Na2HPO4 solution, the crystalline phase of the CA0Z precursor consisted of a single phase of CaCO3 (Figure A). Moreover, the XRD patterns of the CA10Z precursor were consistent with both CaCO3 and ZnO (Figure A). After immersion in the Na2HPO4 solution at pH 8.9 for 7 days, diffraction peaks attributed to peaks attributed to apatite were observed from CA0Z and CA10Z, and the peaks attributed to CaCO3, ZnO, and zinc phosphate hydrate (Zn3(PO4)2·2H2O) were observed only from CA10Z (Figure B). In contrast, after immersion in the Na2HPO4 solution at pH 7.0 for 7 days, the peaks attributed to apatite were observed from CA0Z and CA10Z, whereas the peaks attributed to ZnO were detected only in CA10Z (Figure C). Thus, the CA10Z prepared at pH7.0 consisted of two phases: apatite and ZnO. Furthermore, CaCO3, the precursor of apatite, and zinc phosphate formed at pH 8.9, was not detected in the XRD pattern of CA10Z prepared at pH 7.0. These findings indicate that the compositional conversion from CaCO3 to apatite was inhibited in Na2HPO4 solution at pH 8.9 due to by the formation of Zn3(PO4)2·2H2O from ZnO in the sample. Therefore, the formation of crystalline Zn3(PO4)2·2H2O was suppressed at pH 7.0, whereas alkaline phosphate species promote its precipitation at pH 8.9. In general, the phosphate speciation and pH influenced the resulting zinc phosphate phase. , ZnO becomes unstable in alkaline phosphate environments, where deprotonated phosphate species promote the formation of zinc phosphate. Furthermore, the ZnO partially dissolves even at pH 7.0, and the released Zn ions may react with phosphate ions at pH 7.0. However, interfacial studies have shown that the transformation pathways of ZnO are highly pH dependent and kinetically controlled. Consequently, while the formation of Zn3(PO4)2·2H2O is favored at pH 8.9, such processes can be kinetically hindered at pH 7.0 within compositional transformation from CaCO3 to apatite. In other words, the chemical conversion from CaCO3 to apatite was accomplished by 7 days of immersion in the Na2HPO4 solution at pH 7.0, even in the presence of ZnO within the CaCO3 granules. Therefore, subsequent evaluations were conducted on samples immersed in a Na2HPO4 solution at pH 7.0.

1.

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X-ray diffraction patterns of the samples before immersion (A), after immersion in Na2HPO4 solution at pH 8.9 (B), and pH 7.0 (C).

The FTIR spectra of the CA0Z and CA10Z showed characteristic absorption bands that correspond to CAp (Figure A), which includes phosphate (located at ∼1190–976, 961, and 660–520 cm–1) and carbonate (located at ∼1650–1300 and 873 cm–1) groups. Furthermore, no absorption bands attributed to hydroxyl groups (631 cm–1) were detected in CA0Z and CA10Z (Figure A). These results confirmed that these samples were composed of apatite, wherein the OH– and PO4 3– sites were completely or partially substituted by CO3 2–. Therefore, the samples were mainly composed of AB-type CAp prepared from CaCO3 through a dissolution–precipitation reaction at pH 7.0 condition. The ZnO contents in the CA0Z and CA10Z were 0 and 7.4 ± 0.2 wt %, respectively (Figure B). In other words, the CA10Z was composed of CAp and approximately 7 wt % of ZnO.

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Fourier transform infrared (FTIR) spectra (A) and zinc oxide (ZnO) content (B) in the CA0Z and CA10Z. The bar graph presents the data as mean ± standard deviation (SD). “*” indicate statistically significant differences between the indicated groups (p < 0.05).

SEM images revealed that CA0Z and CA10Z retained their granular macro-architecture (Figure ). This indicates that the dissolution–precipitation reaction at pH 7.0 induced the compositional conversion from CaCO3 to CAp without macro-structural alterations, regardless of the presence or absence of ZnO in the CaCO3 precursors (Figure S2). Furthermore, differences in the microarchitecture and composition were observed between the samples (Figure ). High-magnification and EDS mapping images revealed that CA0Z exhibited plate-like crystals composed mainly of Ca, and this microarchitecture was similar to that of CAp reported previously. In contrast, CA10Z exhibited hexagonal crystals, in which both Ca and Zn were detected. Thus, the presence of ZnO in the CaCO3 precursor resulted in CAp granules that retained their macrostructure but displayed microstructural and compositional alterations.

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Scanning electron microscopy (SEM) images, Ca and Zn elements mapping images obtained from the surfaces of CA0Z and CA10Z.

Stereoscopic microscopy images revealed that the CA0Z and CA10Z were white granular samples (Figure A). Furthermore, the whiteness values of CA0Z and CA10Z were at a comparable level (Figure B), indicating that the presence of ZnO in the CaCO3 precursor did not affect the whiteness of resultant CAp granules. When 10 wt % of Ag or Cu nanoparticles were mixed with CAp powder, the whiteness values were 46.8 ± 0.2 and 49.2 ± 0.3, respectively. Therefore, it is expected that the presence of Ag and Cu-based antibacterial agents in the CaCO3 precursors affect the whiteness of resultant CAp. Previously, we reported the CAp containing Ag3PO4 granules, for achieving the osteogenic and antibacterial activities. The color of Ag3PO4-containing CAp gradually became yellower with increasing Ag3PO4 content (Figure S3). Furthermore, it has been reported that the color of Ag-doped HAp changes with heat treatment, becoming yellow at 400 °C and turning gray up to 1000 °C. In addition to Ag-doped HAp, the slight single doping of iron, manganese, and Cu change the color of hydroxyapatite from white into yellow-orange, cyan, and red-violet, respectively. Thus, while antibacterial agents such as Ag and Cu are effective, their incorporation into calcium phosphate materials often results in undesirable color changes. Therefore, evaluating the whiteness of sample is essential to confirm that antibacterial functionality can be introduced without compromising the aesthetic requirements for clinical bone substitute applications.

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Stereoscopic microscopy images (A) and the whiteness values (B) of CA0Z and CA10Z. The bar graph presents the data as mean ± SD.

To determine the Zn ion release behavior of the samples, CA0Z and CA10Z were immersed in physiological saline for 24 h. The amount of Zn ions released from CA0Z and CA10Z were 0 and 12.0 ± 0.9 μmol·L–1, respectively (Figure A). Therefore, CA10Z potentially releases a slight amount of Zn ions in vivo. Furthermore, the viabilities of MC3T3-E1 cells incubated with CA0Z and CA10Z extracts were 100 ± 4.1, and 77.8 ± 6.0% (Figure B), whereas the viabilities of MRSA in CA0Z and CA10Z groups were 100 ± 16, and 28.1 ± 1.2% (Figure C). In general, Zn ions contribute to the protein function disruption, membrane damage, oxidative stress, and disruption of nutrient uptake. − Furthermore, the direct contact of ZnO with bacterial cell walls leads to the disruption of cell wall. , Therefore, it is considered that CA10Z exhibited the antibacterial activity due to the release of Zn ions and the contact effect between the surface ZnO and MRSA.

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Amount of Zn ions released from the samples to the physiological saline during 24 h of immersion (A). Viabilities of MC3T3-E1 cells (B) and methicillin-resistant Staphylococcus aureus (MRSA) (C) after 24 h of incubation with CA0Z and CA10Z. The bar graph presents the data as mean ± SD “*” indicate statistically significant differences between the indicated groups (p < 0.05).

Thus, CA10Z exhibited the antibacterial activity against MRSA, owing to presence of ZnO in CAp granules. When the cell viability is less than 70%, the material is considered cytotoxic in accordance with the stipulations of the International Organization for Standardization (ISO) standard 10993–5. Therefore, the cytotoxicity test revealed that CA10Z showed acceptable cytocompatibility for osteoblast-like cells. Furthermore, the Zn ions were released from CA10Z into the physiological saline, suggesting that the released Zn ions play a crucial role in antibacterial activity and cytocompatibility. In other words, these results indicate that the Zn ions released from CA10Z contribute to exhibiting antibacterial activity against MRSA while maintaining acceptable cytocompatibility with osteoblast-like cells. According to previous report, the minimum inhibitory concentration (MIC) of Zn ions was determined to be 0.1 μmol·L–1, and cell proliferation remained above 80% at concentrations below 100 μmol·L–1. Our results are consistent with this suitable range of Zn ions, the amount of Zn ions released from CA10Z was approximately 12 μmol·L–1, and was within the suitable range for achieving both antibacterial activity and cytocompatibility. Therefore, CA10Z exhibited significant antibacterial activity against MRSA while maintaining acceptable biocompatibility, indicating that the antibacterial activity of CA10Z was mainly attributed to the released Zn ions. The release of Zn ions from CA10Z interferes with essential bacterial enzymatic and metabolic processes, leading to growth inhibition. Furthermore, the surface of CA10Z showed different crystal morphology compared with CA0Z (Figure ), indicating that ZnO crystals presented on the surface in CA10Z. These crystals potentially enhance physical contact between ZnO and bacteria, which can disturb the bacterial membrane and weaken cell integrity. As a limitation, the long-term biocompatibility and osteogenic performance of CA10Z remain to be fully clarified. Although CA10Z exhibited acceptable cytocompatibility according to ISO 10993–5, further in vitro and in vivo osteogenesis evaluations are required to comprehensively assess its biological performance. Although the antibacterial activity was evaluated using Gram-positive bacteria in this study, further evaluation using Gram-negative bacteria such as Escherichia coli is necessary to further understand the antibacterial mechanism. Furthermore, the efficacy of CA10Z on infection prevention must be revealed by the animal infection models.

This study demonstrated that ZnO-containing CAp granules had superior advantages on whiteness, antibacterial activity, and cytocompatibility, highlighting its promise for orthopedic or implant applications where infection control alongside cell viability is critical. Our findings have significant implications for the design of artificial bone materials using ZnO, potentially leading to innovative medical devices for treatment and prevention of bone infection.

Conclusion

In this study, ZnO-containing CAp granules were fabricated from precursors composed of CaCO3 and ZnO via a pH-controlled dissolution–precipitation. The compositional conversion from CaCO3 to apatite in the Na2HPO4 solution at pH 8.9 was terminated by the formation of Zn3(PO4)2·2H2O. In contrast, this compositional conversion was accomplished by the Na2HPO4 solution at pH 7.0 without the formation of Zn3(PO4)2·2H2O. The whiteness values of ZnO-containing CAp granules prepared at pH 7.0 conditions were comparable to those of pure CAp granules. Furthermore, the ZnO-containing CAp granules exhibited significant antibacterial activity against MRSA while maintaining acceptable cytocompatibility with osteoblast-like cells. These findings highlight the potential of the ZnO-containing CAp granules as an artificial bone material for orthopedic or implant applications, combining antibacterial activity, preserved aesthetics, and cytocompatibility.

Supplementary Material

ao5c12419_si_001.pdf (207.5KB, pdf)

Acknowledgments

This study was partially supported by the Young Innovative Medical Scientist Unit (Institute of Science Tokyo) and the Project on Design & Engineering by Joint Inverse Innovation for Materials Architecture (DEJI2MA) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c12419.

  • SEM image of ZnO used in this study; SEM images of CA0Z and CA10Z precursors; and a photograph of silver phosphate containing CAp granules (PDF)

Z.W.: Investigation, data analysis, data curation, writingoriginal draft. M.S.: Investigation, data analysis, data curation, visualization, writingoriginal draft, review and editing, conceptualization, funding acquisition, supervision. R.M.: Investigation. W.M.R.L.K.W.: Investigation. T.Y.: Investigation. T.Y.: Visualization. E.M.: Review & editing. M.K.: Review & editing.

The authors declare no competing financial interest.

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