Skip to main content
NCBI home page
Springer logoLink to Springer
. 2026 May 30;37(1):97. doi: 10.1007/s10856-026-07083-6

Antibacterial and osteogenic effect of EGCG/HAP/AgNPs composite coating on titanium surface

Jiayi Liao 1,2,3, Zhihua Li 1,2,3,✉
PMCID: PMC13429548  PMID: 42216990

Abstract

Titanium implants are widely used in dental applications; however, their clinical application is still limited by challenges such as bacterial infection and poor osseointegration. In this study, a composite coating consisting of epigallocatechin-3-gallate (EGCG), hydroxyapatite (HAP), and silver nanoparticles (AgNPs) was fabricated on the titanium surface to improve its antibacterial activity and osteogenic potential. The coating was prepared via alkali-thermal treatment, followed by EGCG adhesion and subsequent deposition of HAP and AgNPs. Surface characterization confirmed successful construction of the coating with improved hydrophilicity. The EGCG/HAP/AgNPs coating showed gradual changes in surface morphology and elemental composition after PBS immersion, suggesting partial coating degradation while retaining detectable residual Ag, Ca, and P on the titanium surface. The coating also exhibited effective antibacterial activity against Staphylococcus aureus and significantly promoted osteogenic differentiation of MC3T3-E1 cells, as evidenced by enhanced alkaline phosphatase activity and mineralized nodule formation. These results demonstrate that the EGCG/HAP/AgNPs composite coating possesses good antibacterial capacity, osteogenic activity, and biocompatibility, showing great potential for improving the clinical performance of titanium implants.

graphic file with name 10856_2026_7083_Figa_HTML.jpg

Introduction

Titanium and its alloys are widely used as biomedical materials because of their excellent biocompatibility and favorable mechanical properties. Studies have demonstrated that titanium and its alloys exhibit not only outstanding biocompatibility but also mechanical properties that closely resemble those of natural bone. including a bone-like elastic modulus and excellent fatigue resistance [1]. These unique material properties enable them highly suitable for a wide range of medical applications, particularly in dental implants.

However, because of their inherent biological inertness, titanium and its alloys exhibit limited intrinsic antibacterial activity, making bacterial colonization on implant surfaces a major clinical challenge [2]. Biofilm-related infections may subsequently develop into peri-implantitis, causing irreversible destruction of the surrounding soft tissues and bone [3]. This problem is particularly critical in the oral cavity, an open environment that harbors abundant bacterial pathogens, where bacteria biofilms are considered a key initiating factor for many oral diseases [4]. Bacterial adhesion and colonization on the surface of titanium implants can induce peri-implant bone resorption and eventually lead to implant failure [5]. Therefore, it is crucial to develop effective surface modification strategies to simultaneously enhance the antibacterial and osteogenic properties of titanium implants, thereby improving their stability and clinical performance [6].

Coating modification can significantly enhance the physicochemical and biological properties of titanium and titanium alloy surfaces, thereby endowing them with enhanced antibacterial and osteogenic activity [7]. After implantation, surface-modified implants can inhibit bacterial colonization while simultaneously promoting osteoregeneration, thereby reducing peri-implantitis-related bone resorption and enhancing long-term implant stability [8]. Hydroxyapatite/Ag nanoparticles (HAP/AgNPs) have been reported to exhibit favorable broad-spectrum antibacterial and bacteriostatic effects against various bacterial species [9, 10]. Moreover, compared with conventional titanium alloys, HAP/AgNPs-modified surfaces have shown superior performance in promoting cell spreading, proliferation, and adhesion, as well as in inducing osteogenic differentiation in human bone marrow stromal cells [11]. Mahmoud et al. reported that titanium dental implants functionalized with a HAP/AgNPs composite coating exhibited excellent antibacterial and osteogenic properties, resulting in improved in vivo stability [12, 13]. However, Nevertheless, the chemical inertness of titanium surfaces makes it difficult for HAP/AgNPs to directly establish effective interactions with the substrate.. At present, HAP/AgNPs composites are mainly immobilized on titanium surfaces through methods like electrodeposition [14] or plasma spraying [15]. However, these techniques are often associated with significant limitations, including procedural complexity, strict processing conditions, and poor clinical adaptability, which may ultimately hinder their broader translational application in dental implant therapy.

In recent years, natural polyphenolic compounds have attracted great interest from researchers because they can firmly adhere to the surface of most materials and serve as a platform for subsequent surface modification with functional molecules [16]. Among these compounds, epigallocatechin-3-gallate (EGCG), the main component of tea polyphenols in green tea, can be obtained in large quantities from green tea through conventional extraction processes. Owing to its abundant availability, low production cost, excellent biocompatibility, and multiple biofunctional properties, including antibacterial and osteogenic activities, EGCG has shown great potential for biomedical applications [17]. In addition, EGCG is rich in catechol and trihydroxyphenyl moieties, facilitating spontaneous polyphenolic coating formation on surface of materials [18]. Polyphenolic coatings can interact with other active organic molecules or metal ions, thereby functionalizing the material surface [19].

Therefore, this study aimed to prepare the EGCG/HAP/AgNPs coating on the titanium surface, characterize its physicochemical properties, and evaluate its biological properties, thereby providing an experimental basis for the application of the EGCG/HAP/AgNPs coating in the surface modification of titanium implants.

Materials and methods

Sample preparation

The titanium specimens (Ø 1 cm, Xinbiao Metal Materials Co., Ltd. China) were sequentially polished using silicon carbide abrasive paper (#600, #800, #1000, #1200), and were ultrasonically cleaned sequentially in absolute ethanol and deionized water (30 min each), air-dried prior to use, and labeled as Group A (control group). Group A specimens were immersed in 5 mol/L NaOH (Maikelin Biochemical Technology Co., Ltd., China) solution at 60 °C for 12 h. Subsequently, the alkali-treated samples underwent ultrasonic cleaning in absolute ethanol followed by deionized water (30 min each), air-dried prior to use, and labeled as Group B. Group B specimens were immersed in 2 mg/mL EGCG (Maikelin Biochemical Technology Co., Ltd., China) solution (prepared in 10 mM Tris-HCl buffer, pH 8.5) for 24 h under light-protected conditions. Subsequently, the functionalized samples were sequentially ultrasonicated in absolute ethanol and deionized water (15 min each), air-dried and designated as Group C. AgNPs (50 nm, Sigma-Aldrich, Germany), HAP (100 nm, Maikelin Biochemical Technology Co., Ltd., China), and a mixture of AgNPs and HAP were separately added into deionized water, followed by ultrasonic dispersion for 30 min to prepare solutions with concentrations of 0.10 mg/mL AgNPs, 0.10 mg/mL HAP, and HAP/AgNPs solution containing 0.10 mg/mL HAP and 0.10 mg/mL AgNPs, respectively. Titanium specimens in group C were immersed into the AgNPs solution, HAP solution, and HAP/AgNPs solution, respectively, at 25 °C for 2 h. Subsequently, they were ultrasonically cleaned in absolute ethanol and deionized water for 10 min each, then dried for subsequent use. These samples were designated as D (AgNPs), E (HAP), and F (HAP/AgNPs), respectively.

Surface characterization

Surface wettability characterization was performed using a contact angle analyzer (OCA15EC, Dataphysics) by measuring the water contact angle (WCA) on titanium surfaces. Three equally spaced measurement locations were selected within the central area of each titanium samples surface. Using a micropipette, 10 μL of ultrapure water was precisely dispensed onto each location, and the morphological evolution of the droplet was recorded. Contact angle data were automatically calculated and acquired by the instrument’s integrated digital analysis system. Three titanium sheets were randomly selected per group (n = 3), and the arithmetic mean value of the measurements was determined as the representative value for each test condition.

Following vacuum drying treatment, all sample groups underwent surface conductive coating on the titanium sheets. An approximately 10 nm-thick gold/palladium (Au/Pd) alloy layer was deposited using a high-vacuum magnetron sputtering system under an argon atmosphere. SEM characterization was performed using a high-resolution field emission scanning electron microscope (Apreo 2S HiVac, Thermo Fisher Scientific) at accelerating voltages ranging from 3.0 to 5.0 kV. Surface morphological information was acquired using the secondary electron detector (ETD).

X-ray photoelectron spectroscopy (K-Alpha X system, Thermo Fisher Scientific) analysis was performed to characterize the chemical states of the functionalized surfaces. Measurements were conducted at a take-off angle of θ = 45°, involving both survey (wide) scans and high-resolution narrow scans. The acquired data enabled characterization of surface chemical states and semi-quantitative analysis of the coatings.

Evaluation of the stability of coating

Group F samples were immersed in PBS (1×, pH 7.4) with daily solution renewal. Coating stability was assessed by XPS and SEM at baseline (pre-immersion) and after 1, 7, 14, and 21 days of immersion.

Evaluation of coating biocompatibility

Cell Culture

Culture medium preparation:

Complete culture medium was prepared under sterile conditions. High-glucose Dulbecco’s Modified Eagle Medium (DMEM, Gibco, United States), supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL), was combined with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco, United States). Specifically, 45 mL of DMEM and 5 mL of FBS were mixed in a sterile 50 mL conical centrifuge tube. The complete medium was pre-warmed to 37 °C prior to use.

Cell thawing and initial seeding:

Cryopreserved MC3T3-E1 cells were rapidly retrieved from liquid nitrogen storage and immediately transferred to a 37 °C constant-temperature water bath for thawing. Concurrently, a sterile tissue culture flask containing complete medium was prepared. Upon complete dissolution of ice crystals within the cryovial, the cell suspension was aseptically transferred using a sterile Pasteur pipette into the prepared culture flask. The cells were gently resuspended by pipetting. The flask was then placed in a humidified incubator maintained at 37 °C with 5% CO₂. After 4–6 h of incubation to facilitate cell attachment, the medium containing cryoprotectant (DMSO) was aspirated and replaced with 3 mL of fresh, pre-warmed complete medium. Cell morphology and confluency were monitored daily thereafter under an inverted phase-contrast microscope, and the medium was refreshed every 24–48 h as required.

Routine medium changes:

For routine maintenance, spent culture medium was aspirated from the flask. The adherent cell monolayer was gently rinsed twice with 3 mL of sterile PBS (1×, pH 7.4) per wash to remove metabolic waste products and residual serum. After complete removal of the final PBS wash, 3 mL of fresh, pre-warmed complete medium was added to the flask. The flask was then returned to the 37 °C, 5% CO₂ incubator. This medium exchange procedure was performed every 24–48 h.

Cell subculturing (passaging):

Cells were subcultured upon reaching approximately 80–90% confluency, as assessed by phase-contrast microscopy. The spent medium was aspirated and the cell monolayer was washed twice with 3 mL of sterile PBS per wash. Following PBS removal, 1 mL of pre-warmed (37 °C) 0.25% (w/v) trypsin-EDTA solution was added to cover the cells. Incubation proceeded at 37 °C for 1–2 min to facilitate cell detachment. Enzymatic activity was then neutralized by adding 3 mL of complete medium containing FBS. The cell suspension was gently aspirated and dispensed several times using a pipette to achieve a single-cell suspension. The suspension was subsequently transferred to sterile conical tubes, centrifuged, and appropriate aliquots were seeded into new culture flasks. Fresh complete medium was added to achieve the desired final volume, and the flasks were gently swirled to ensure uniform cell distribution before being returned to the 37 °C, 5% CO₂ incubator for continued culture.

Cell counting kit-8

Three randomly selected titanium samples per experimental group per time point (n = 3) were completely dried and sterilized under ultraviolet (UV) light. Each sterilized disc was then placed into an individual well of a 48-well tissue culture plate. A 200 µL aliquot of MC3T3-E1 cell suspension, prepared at a density of 2 × 10⁵ cells/mL in complete culture medium, was carefully added to each well containing a titanium samples. The plate was transferred to a humidified cell culture incubator (bb150, Thermo Fisher Scientific) maintained at 37 °C with 5% CO₂. Cell viability/proliferation was assessed at designated time points (1, 4, and 7 days post-seeding). For each group, three independent discs were prepared for each time point (n = 3 per group per time point), and different wells were used for the measurements at days 1, 4, and 7. At each time point, the culture medium was not removed. Instead, 20 µL of CCK-8 reagent (Sigma-Aldrich, Germany) was added directly to each well. The plate was gently shaken to ensure mixing and returned to the incubator for an additional 4 h. Following the 4-h incubation with CCK-8, 100 µL of supernatant from each well was carefully transferred to a corresponding well of a 96-well plate. The absorbance of the transferred supernatant was measured at a wavelength of 450 nm using a microplate reader (Infinite 200 Pro, TECAN).

Osteogenesis test

MC3T3-E1 cells (5 × 103 ml–1) were seeded on the sample surfaces and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) for 24 h to allow attachment under standard culture conditions (37 °C, 5% CO₂, humidified atmosphere). Then the medium was replaced with osteogenic induction medium (DMEM supplemented with 10% FBS, 1% penicillin and streptomycin, 10 mM β-glycerophosphate, 50 μg/mL L-ascorbic acid, and 0.1 mM dexamethasone), which was changed every 3 days. Cells were cultured.

Alkaline phosphatase (ALP) staining and quantification

Following the 7-day co-culture period, ALP activity was assessed in parallel using two methods: (1) ALP Staining: Cellular ALP expression was visualized using a commercial Alkaline Phosphatase Staining Kit (Beyotime, China). Stained titanium samples were imaged using a stereo microscope (KL300 LED, LEICA) to document mineralization-associated ALP expression. (2) ALP Quantification: Total ALP activity was quantified using a commercial Alkaline Phosphatase Assay Kit (Beyotime, Chine). Absorbance was measured at 405 nm.

Alizarin red S (ARS) staining and quantification

Following the 7-day co-culture period, calcium nodules were stained using the Alizarin Red S Quantitative Staining Kit (Beyotime, China). Subsequently, the stained nodules were dissolved with 10% (w/v) cetylpyridinium chloride (CPC), and quantitative analysis was performed by measuring the absorbance at 562 nm.

Antimicrobial test

Culture media preparation

BHI broth was prepared by dissolving 19 g of Brain Heart Infusion (BHI) powder (Qingdao Haibo Biotechnology, China) in 500 mL of ultrapure water. The solution was transferred into a heat-resistant borosilicate glass bottle, sealed with aluminum foil, and wrapped in medical-grade non-woven sterilization packaging. Sterilization was performed in a vertical autoclave at 121 °C for 30 min. Sterilized BHI broth was stored at 4 °C and used within one week. For solid media, 38.5 g of BHI agar powder (Qingdao Haibo Biotechnology, China) was dissolved in 500 mL ultrapure water, transferred to a borosilicate bottle, and identically packaged and sterilized. Following autoclaving, the agar medium was cooled to 60 °C in a water bath, then aseptically poured into sterile Petri dishes within a laminar flow hood. Plates were air-dried at room temperature for 30 min to eliminate condensation, solidified completely, and stored inverted at 4 °C.

Bacterial revival and subculture

Glycerol stock cultures of Staphylococcus aureus (ATCC 25923) stored at –20 °C were thawed at room temperature. Using a sterile inoculation loop, bacterial suspension was transferred to 10 mL of sterile BHI broth in a culture tube. Inoculated broths were incubated at 37 °C for 16–18 h with constant agitation in an orbital shaker incubator (bb150, Thermofisher). Subsequently, fresh broth culture was streaked onto BHI agar plates using the quadrant streak method to obtain isolated colonies. Inoculated plates were inverted and incubated at 37 °C for 24 h. A single morphologically typical colony was aseptically transferred to fresh BHI broth using a sterile loop. After identical incubation, this subculture procedure was repeated once to ensure complete bacterial reactivation prior to experimental use.

Colony-forming unit (CFU) counting

The colony counting method was performed using Staphylococcus aureus as the model microorganism. Bacterial suspensions were adjusted to 108 CFU/mL in BHI broth. After UV sterilization, titanium samples were placed in a 48-well plate, inoculated with 20 μL of bacterial suspension, and incubated at 37 °C for 12 h. Subsequently, titanium samples were gently rinsed with PBS to remove non-adherent bacteria and transferred to 1.5-mL EP tubes containing fresh medium. Adherent bacteria were detached through three alternating cycles (3 min each) of ultrasonic washing and vigorous vortexing. The resulting suspensions were serially diluted in sterile PBS (pH 7.4) across five tenfold gradients (101–105). Aliquots (10 μL) from each dilution were spot-plated onto BHI agar plates, followed by 12-h incubation at 37 °C for colony enumeration. Triplicate samples were analyzed per group (n = 3).

Inhibition zone method

Staphylococcus aureus suspensions were adjusted to 105 CFU/mL. Using the spread plate technique, 100 μL of bacterial suspension was uniformly inoculated onto Agar-BHI medium plates. Titanium samples were aseptically placed on the agar surface, gently pressed to ensure full contact, and incubated at 37 °C for 24 h. The presence and diameter of inhibition zones surrounding the samples were observed to evaluate the antibacterial efficacy of coating-derived diffusible compounds.

Statistical analyses

All quantitative data are presented as mean ± standard deviation (mean ± SD). Statistical analyses were performed using IBM SPSS Statistics 23.0 (IBM Corp., Armonk, NY). Comparisons among multiple independent groups were assessed by one-way analysis of variance (One-Way ANOVA), followed by Tukey’s post hoc test. Statistical significance was defined as p < 0.05.

Results

As shown in Fig. 1, the WCA of the untreated titanium surface in the control group (Group A) was 101.3 ± 3.2°, indicating a highly hydrophobic surface. After alkali–thermal treatment (Group B), surface hydrophilicity was markedly improved, with the WCA decreasing to 63.9 ± 2.8°. Following EGCG modification (Group C), the WCA further decreased to 23.7 ± 3.1°, indicating a substantial increase in hydrophilicity. In contrast, subsequent incorporation of AgNPs or HAP resulted in a slight increase in WCA. The EGCG/AgNPs coating (Group D) exhibited a WCA of 30.4 ± 2.3° and the EGCG/HAP coating (Group E) showed a slightly lower WCA of 28.7 ± 0.8°. The EGCG/HAP/AgNPs composite coating (Group F) presented a WCA of 30.4 ± 2.5°.

Fig. 1.

Fig. 1

Open in a new tab

Water contact angle (WCA) measurements of titanium surfaces in each experimental group, indicating changes in surface hydrophilicity after different treatments. A Group A (control group); B Group B (alkali-thermal treatment); C Group C (EGCG); D Group D (EGCG/AgNPs); E Group E (EGCG/HAP); F Group F (EGCG/HAP/AgNPs)

The surface of Group A (control group) appeared relatively smooth. After Alkali-thermal treatment (Group B), the titanium surface exhibited a porous network-like structure. In Group D (EGCG/AgNPs), clusters of AgNPs with approximately from 50 to 100 nm in size were observed on the EGCG-coated surface. Group E (EGCG/HAP) showed dense, rod-like HAP aggregates covering the EGCG coating, with particles sizes around 100–500 nm. In Group F (EGCG/HAP/AgNPs) larger micron-sized clusters can be saw on the surface (Fig. 2).

Fig. 2.

Fig. 2

Open in a new tab

Scanning electron microscope (SEM) images showing surface morphologies of titanium samples in each group. A Group A (control group); B Group B (alkali-thermal treatment); C Group C (EGCG); D Group D (EGCG/AgNPs); E Group E (EGCG/HAP); F Group F (EGCG/HAP/AgNPs)

XPS analysis of Group B samples revealed a Na 1 s peak at binding energies of 1070–1075 eV, which is characteristic of Na₂TiO₃ formation, confirming the successful alkali–thermal treatment of the titanium surface. Following EGCG modification (Group C), the C 1 s peak intensity increased, while the Na 1 s signal was markedly decreased. In Groups D and F, a distinct Ag signal was detected, confirming the successful incorporation of AgNPs onto the titanium surface. Meanwhile, Groups E and F exhibited characteristic Ca and P peaks, indicating the presence of hydroxyapatite. Notably, Group F simultaneously showed Ag, Ca, and P signals, demonstrating the successful construction of the EGCG/HAP/AgNPs composite coating (Fig. 3 and Table 1).

Fig. 3.

Fig. 3

Open in a new tab

X-ray photoelectron spectroscopy (XPS) analysis illustrating the elemental composition of titanium surfaces across the experimental groups

Table 1.

The elemental composition of the surfaces of each group of titanium samples

Atomic (%) C O Ca P Ti Ag
A 40.74 42.72 0.53 0.21 15.74 0.06
B 24.15 50.07 0.16 0.00 25.61 0.00
C 43.46 43.23 0.25 0.00 13.06 0.00
D 30.18 46.04 0.35 0.39 19.16 3.89
E 29.16 46.43 4.21 3.39 16.74 0.07
F 30.98 43.85 4.13 3.01 14.37 3.67
Open in a new tab

This study investigated the stability of the coating during immersion in PBS. As shown in the SEM images (Fig. 4), before PBS immersion, the coating surface exhibited clusters of varying sizes. XPS results (Table 2) indicated that the surface atomic concentrations of Ag, Ca, and P were 3.67%, 4.13%, and 3.01%, respectively. After one day of immersion, the contents of HAP and AgNPs decreased significantly. SEM images (Fig. 4) showed substantial loss of small-sized clusters, predominantly attributed to AgNPs. According to XPS (Fig. 5), the Ag content decreased from 3.67% to 1.32% after one day of immersion. During the subsequent immersion period, the coating degradation rate slowed. By day twenty-one, residual coating material was still detectable on the titanium surface. XPS analysis further showed that the surface concentrations of Ca and P decreased to 2.49% and 1.63%, respectively, while the Ag content declined to 0.68%. These results indicate that the EGCG/HAP/AgNPs coating underwent gradual surface changes during PBS immersion, while residual Ag, Ca, and P were still detectable after 21 days. However, because no quantitative analysis of Ag⁺ or Ca²⁺ in the immersion solution was performed, these XPS results should be interpreted as evidence of coating stability and surface compositional changes rather than direct evidence of ion release.

Fig. 4.

Fig. 4

Open in a new tab

The surface morphologies of each group of titanium sheets after being immersed in PBS solution for 1, 7, 14 and 21 days. a Control group, b 1 day, c 7 days, d 14 days, e 21 days

Table 2.

The elemental composition of the titanium sheets in each group after being immersed in PBS solution for 1, 7, 14 and 21 days

Atomic (%) C O Ca P Ti Ag
a 30.98 43.85 4.13 3.01 14.37 3.67
b 28.49 47.66 3.06 2.30 17.15 1.32
c 32.34 45.24 2.78 1.95 16.14 1.55
d 28.46 47.92 3.27 2.31 17.25 0.79
e 39.00 43.12 2.49 1.63 13.07 0.68
Open in a new tab

a. control group, b. immersed for 1 day, c. immersed for 7 days, d. immersed for 14 days, e. immersed for 21 days.

Fig. 5.

Fig. 5

Open in a new tab

The elemental composition of the titanium sheets in each group after being immersed in PBS solution for 1, 7, 14 and 21 days. a Control group, b 1 day, c 7 days, d 14 days, e 21 days

This study evaluated the biocompatibility of the coating. As shown in Fig. 6, no significant difference in MC3T3 cell proliferation was detected between the EGCG coating group (Group C) and the control group (Group A), indicating good biocompatibility of the EGCG surface coating. In contrast, the EGCG/AgNPs coating (Group D) resulted in a significant reduction in MC3T3 cell proliferation compared with the control group, indicating evident cytotoxicity. Notably, the EGCG/HAP/AgNPs coating (Group F) showed significantly improved cell proliferation relative to Group D, with no significant difference observed compared with the control group.

Fig. 6.

Fig. 6

Open in a new tab

Cell viability of MC3T3-E1 cells cultured on titanium surfaces in different groups, evaluated using the Cell Counting Kit-8 (CCK-8) assay. *p < 0.05, **p < 0.01, ***p < 0.001

In this study, the ALP activity of osteoblasts on surfaces on titanium in group B showed a slight increase compared to the control group. Following EGCG incubation, the expression of ALP on the coated surface was further elevated. In contrast, the EGCG/AgNPs group (Group D) exhibited lower ALP levels than the control. Notably, Group E (EGCG/HAP) demonstrated significantly higher ALP activity compared to the control, while Group F (EGCG/HAP/AgNPs) also showed markedly increased ALP expression, albeit to a lesser extent than Group E. These results indicate that the EGCG/HAP/AgNPs coating effectively promotes ALP expression (Fig. 7).

Fig. 7.

Fig. 7

Open in a new tab

Alkaline phosphatase (ALP) staining and quantitative analysis of MC3T3-E1 cells cultured on titanium samples, assessing early osteogenic differentiation. *p  <  0.05, **p  < 0.01, ***p  <  0.001. A Group A (control group); B Group B (alkali-thermal treatment); C Group C (EGCG); D Group D (EGCG/AgNPs); E Group E (EGCG/HAP); F Group F (EGCG/HAP/AgNPs)

ARS staining and quantitative results (Fig. 8) showed that compared with the control group, the EGCG/AgNPs group (Group D) exhibited a reduced level of calcium nodule formation. In contrast, both the EGCG/HAP group (Group E) and the EGCG/HAP/AgNPs group (Group F) showed significantly higher calcium deposition than the control group, indicating that this composite coating markedly enhances the mineralizing function of osteoblasts.

Fig. 8.

Fig. 8

Open in a new tab

Alizarin Red S (ARS) staining and quantitative analysis of calcium nodule formation in MC3T3-E1 cells cultured on titanium samples for 21 days. *p  <  0.05, **p  <  0.01, ***p  <  0.001. A Group A (control group); B Group B (alkali-thermal treatment); C Group C (EGCG); D Group D (EGCG/AgNPs); E Group E (EGCG/HAP); F Group F (EGCG/HAP/AgNPs)

EGCG/HAP/AgNPs group (Fig. 9) showed a significant reduction in bacterial colonies compared to the control group. Moreover, no significant difference was observed in colony counts between the EGCG/HAP/AgNPs group (Group F) and the EGCG/AgNPs group, indicating that the incorporation of HAP did not compromise the coating’s antibacterial performance. The inhibition zone results confirmed that the EGCG/HAP/AgNPs coating could be released into the surrounding environment, thereby effectively inhibiting the growth and proliferation of adjacent bacteria [20]. Furthermore, EGCG itself contributes to antibacterial activity. Bacterial colony counting revealed that the Staphylococcus aureus count in the EGCG-coated group (Group C) was significantly lower than that in the control group, demonstrating that EGCG possesses inherent antibacterial properties. However, no clear inhibition zone was observed in the EGCG coating group (Group C) or the control group in the inhibition zone assay (Fig. 10).

Fig. 9.

Fig. 9

Open in a new tab

Antibacterial activity of titanium samples against Staphylococcus aureus evaluated by colony-forming unit (CFU) assay. a Representative images of bacterial colony growth after treatment with serially diluted bacterial suspensions recovered from the tested titanium samples (Groups A–F). Serial dilutions (10⁰–10⁵) are indicated on the left. b Quantitative analysis of viable bacterial counts (CFU/ml) recovered from each group (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 10.

Fig. 10

Open in a new tab

Inhibition zone assay demonstrating antibacterial effects of different coatings on titanium surfaces against Staphylococcus aureus. A Group A (control group); B Group B (alkali-thermal treatment); C Group C (EGCG); D Group D (EGCG/AgNPs); E Group E (EGCG/HAP); F Group F (EGCG/HAP/AgNPs)

Discussion

Alkali–thermal treatment is a common and effective strategy for titanium surface modification. This treatment leads to the formation of a nanoscale porous structure on the titanium surface (Fig. 2), which increases the specific surface area. As a result, the modified surface exhibits enhanced surface reactivity, provides abundant active sites, and facilitates subsequent interactions between the titanium substrate and functional molecules [21]. Following EGCG incubation (Group C), the hydrophilicity of the titanium surface was further improved, as evidenced by a marked reduction in the water contact angle to approximately 23.7 ± 3.1°. This pronounced decrease in WCA suggests successful surface coverage by EGCG molecules, which are rich in hydrophilic phenolic hydroxyl groups. Moreover, the distinct changes in surface wettability observed after loading with EGCG, AgNPs, HAP, or their combination indirectly confirm the successful construction of the corresponding coatings. Overall, the EGCG/HAP/AgNPs composite coating maintained relatively high surface hydrophilicity, which is favorable for subsequent biological interactions.

Following alkali-thermal treatment, a Na₂TiO₃ layer forms on the titanium surface [22]. This layer is thermodynamically unstable in aqueous environments, where dynamic ion exchange between Na⁺ and H₃O⁺ occurs, resulting in the gradual transformation into an amorphous, Ti–OH–rich hydrated titania gel layer [21]. After incubation in EGCG solution, the intensity of the Na 1 s signal decreased, which can be attributed partly to the hydrolysis of Na₂TiO₃ and partly to the coverage by EGCG molecules. The changes in the surface elemental composition of the titanium samples from each group, as revealed by XPS analysis, provide evidence supporting the successful construction of the respective coatings.

EGCG, rich in phenolic hydroxyl groups, not only adheres to the titanium surface efficiently but also acts as a versatile platform for anchoring AgNPs, HAP, and HAP/AgNPs composites onto the titanium substrate. In Group F (EGCG/HAP/AgNPs), AgNPs were adsorbed onto the HAP surface and further agglomerated into larger micron-sized clusters. Studies have shown that porous HAP clusters can adsorb AgNPs to form HAP/AgNPs composite materials. Ag⁺ can replace Ca²⁺ ions within the HAP structure, leading to an interaction between the two components [23, 24].

Numerous studies have reported the cytotoxicity of AgNPs. For instance, AgNPs have been shown to induce cytotoxicity in macrophages [25] and human T cells [26]. Decreased cell viability and proliferative capacity have also been observed in neurons, liver cells, skin cells, and alveolar epithelial cells following treatment with AgNPs [27–31]. Furthermore, Kim et al. reported subacute oral cytotoxic effects and tissue accumulation after 90 days of AgNPs treatment in rats [32]. The cytotoxicity of AgNPs is generally associated with factors such as their size, shape, concentration, and the rate of Ag⁺ release [33, 34]. When combined with HAP, the EGCG/HAP/AgNPs coating (Group F) exhibited significantly reduced cytotoxicity compared to Group D (Fig. 6), which may be attributed to the following two factors. First, the altered Ag⁺ release kinetics: the cytotoxicity of AgNPs is often enhanced by a faster Ag⁺ release rate, which can lead to rapid intracellular accumulation of Ag⁺ and subsequent cell damage [34]. The high specific surface area and porous structure of HAP allow it to adsorb AgNPs, thereby delayed the release of Ag⁺ [24, 35]. Second, the increased particle size: the cytotoxicity of AgNPs is known to be size-dependent [35]. After adsorption onto the HAP surface, AgNPs undergo further aggregation forming larger clusters (Fig. 2). This significant increase in particle size likely contributed to the markedly reduced cytotoxicity, to the extent that no significant difference was observed compared to the control group (Fig. 6).

Osteogenic performance is a key indicator for evaluating the potential application of biomaterials in bone tissue engineering, with alkaline phosphatase (ALP) being widely recognized as an early marker of osteoblastic differentiation [36]. During osteogenic differentiation, the expression of ALP precedes that of late-stage markers such as osteocalcin and type I collagen, making it widely used as an early indicator of osteogenic differentiation [37]. The enzymatic activity of ALP plays a critical role in extracellular matrix mineralization by hydrolyzing pyrophosphate to release phosphate groups, thereby promoting the deposition of hydroxyapatite crystals. Additionally, ALP is involved in regulating intracellular phosphate metabolism, supplying necessary substrates for mineralization [38].

The formation of calcium nodules is a marker of osteoblast maturation [39]. Alizarin Red S (ARS), a calcium-chelating agent, specifically binds to calcium salts forming an orange-red complex, thereby labeling mineralized nodules. In the later stages of osteoblast differentiation, calcium and phosphate deposits form mineralized structures within the extracellular matrix. Under acidic conditions, ARS binds to calcium ions to produce an insoluble red precipitate, with staining intensity positively correlated with the degree of mineralization. This method allows indirect assessment of the mineralization capacity of osteoblasts through quantitative or semi-quantitative analysis of the stained area and color intensity [40].

The antibacterial efficacy of the EGCG/HAP/AgNPs coating primarily relies on the broad-spectrum antibacterial properties of AgNPs. Released Ag⁺ ions can interact with thiol groups in bacterial membrane proteins, disrupting the integrity of the cell membrane [41]. Additionally, AgNPs can inhibit the activity of antioxidant enzymes within bacteria, causing ROS-mediated DNA damage and exerting antibacterial effects [42]. AgNPs also inhibit biofilm formation, further contributing to their antimicrobial activity [43].

Furthermore, EGCG contributes to antibacterial activity. Bacterial colony counting revealed that the Staphylococcus aureus count in the EGCG-coated group (Group C) was significantly lower than that in the control group, demonstrating that EGCG possesses inherent antibacterial properties. EGCG can disrupt bacterial membrane integrity and increase membrane permeability. Its polyphenolic structure can also chelate metal ions, interfere with bacterial metabolism, and inhibit the synthesis of key bacterial enzymes [44], thereby enhancing the synergistic antibacterial effect of the coating. However, no clear inhibition zone was observed in the EGCG coating group (Group C) or the control group in the inhibition zone assay (Fig. 10), suggesting that the antibacterial effect of EGCG may depend on direct contact with bacteria.

This study has several limitations. First, although XPS analysis after PBS immersion showed changes in the surface elemental composition of the coating, the release of Ca²⁺ and Ag⁺ ions was not directly quantified. Therefore, the ion-release behavior and release kinetics of the EGCG/HAP/AgNPs coating remain to be further investigated using quantitative methods. Second, the antibacterial evaluation was limited to Staphylococcus aureus, however, the oral microbiota is highly complex and contains many anaerobic bacteria, such as Porphyromonas gingivalis, which are closely associated with peri-implant infections. Future studies should include additional oral pathogens and multispecies biofilm models to more comprehensively evaluate the antibacterial performance of the coating. Third, the biocompatibility and osteogenic potential of the coating were assessed only using MC3T3-E1 cells. Further studies involving other relevant cell types are needed to better evaluate the biological response to the coating. Finally, no animal experiments were performed in the present study. Therefore, the long-term coating stability, osseointegration capacity, and antibacterial efficacy of the EGCG/HAP/AgNPs coating under in vivo conditions remain to be validated.

Conclusion

In this study, EGCG/HAP/AgNPs composite coating was successfully constructed on the titanium surface through alkali-thermal treatment. The results demonstrated that this coating possesses good antibacterial capacity, osteogenic activity, and biocompatibility, showing great potential for improving the clinical performance of titanium implants.

Acknowledgements

We thank for Prof. Dr. Zhihua Li for his kindly instruction. We would like to express our gratitude to Professor Junchao Wei in the field of polymer materials from Nanchang University for his guidance on this research.

Author contributions

JL: software, writing—original draft preparation, visualization. ZL: conceptualization, methodology, data curation, investigation, supervision, writing, reviewing and editing.

Data availability

The data and materials used in the current study are available from the corresponding author upon reasonable request.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Consent for publication

Consent forms for publication was obtained from all subjects involved in the study.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Song C, Liu L, Deng Z, Lei H, Yuan F, Yang Y, et al. Research progress on the design and performance of porous titanium alloy bone implants. J Mater Res Technol. 2023;23:2626–41. [Google Scholar]
  • 2.Spriano S, Yamaguchi S, Baino F, Ferraris S. A critical review of multifunctional titanium surfaces: New frontiers for improving osseointegration and host response, avoiding bacteria contamination. Acta Biomater. 2018;79:1–22. [DOI] [PubMed] [Google Scholar]
  • 3.Darby I. Risk factors for periodontitis & peri-implantitis. Periodontology 2000. 2022;90:9–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Gendron R, Grenier D, Maheu-Robert L-F. The oral cavity as a reservoir of bacterial pathogens for focal infections. Microbes Infect. 2000;2:897–906. [DOI] [PubMed] [Google Scholar]
  • 5.Schwarz F, Derks J, Monje A, Wang HL. Peri-implantitis. J Clin Periodontol. 2018;45:S246–S266. [DOI] [PubMed] [Google Scholar]
  • 6.Rautray TR, Narayanan R, Kwon TY, Kim KH. Surface modification of titanium and titanium alloys by ion implantation. J Biomed Mater Res Part B: Appl Biomater. 2010;93:581–91. [DOI] [PubMed] [Google Scholar]
  • 7.Civantos A, Martinez-Campos E, Ramos V, Elvira C, Gallardo A, Abarrategi A. Titanium coatings and surface modifications: toward clinically useful bioactive implants. ACS Biomater Sci Eng. 2017;3:1245–61. [DOI] [PubMed] [Google Scholar]
  • 8.Xue T, Attarilar S, Liu S, Liu J, Song X, Li L, et al. Surface modification techniques of titanium and its alloys to functionally optimize their biomedical properties: thematic review. Front Bioeng Biotechnol. 2020;8:603072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Andrade FAC, de Oliveira Vercik LC, Monteiro FJ, da Silva Rigo EC. Preparation, characterization and antibacterial properties of silver nanoparticles–hydroxyapatite composites by a simple and eco-friendly method. Ceram Int. 2016;42:2271–80. [Google Scholar]
  • 10.Silva-Holguín PN, Reyes-López SY. Synthesis of hydroxyapatite-Ag composite as antimicrobial agent. Dose-Response. 2020;18:1559325820951342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Tian B, Chen W, Dong Y, Marymont JV, Lei Y, Ke Q, et al. Silver nanoparticle-loaded hydroxyapatite coating: structure, antibacterial properties, and capacity for osteogenic induction in vitro. RSC Adv. 2016;6:8549–62. [Google Scholar]
  • 12.Abo-Elmahasen MMF, Dena ASA, Zhran M, Albohy SA. Do silver/hydroxyapatite and zinc oxide nano-coatings improve inflammation around titanium orthodontic mini-screws? In vitro study. Int Orthod. 2023;21:100711. [DOI] [PubMed] [Google Scholar]
  • 13.Aboelmahasen MMF, Othman SS, Dena ASA, Zhran M, Ma M, El-Destawy MT, et al. Histomorphometric and CBCT comparison of osseointegration around orthodontic titanium miniscrews coated with different nanoparticles: An in-vivo animal study. Int Orthod. 2024;22:100823. [DOI] [PubMed] [Google Scholar]
  • 14.Mokabber T, Cao H, Norouzi N, Van Rijn P, Pei Y. Antimicrobial electrodeposited silver-containing calcium phosphate coatings. ACS Appl Mater interfaces. 2020;12:5531–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Ignatev M, Rybak T, Colonges G, Scharff W, Marke S. Plasma sprayed hydroxyapatite coatings modified with silver nanoparticles. Acta Met Slov. 2013;19:20–29. [Google Scholar]
  • 16.Chen Y, Liu Q. Oxidant-induced plant phenol surface chemistry for multifunctional coatings: Mechanism and potential applications. J Membr Sci. 2019;570-571:176–83. [Google Scholar]
  • 17.Mereles D, Hunstein W. Epigallocatechin-3-gallate (EGCG) for clinical trials: more pitfalls than promises? Int J Mol Sci. 2011;12:5592–603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Barrett DG, Sileika TS, Messersmith PB. Molecular diversity in phenolic and polyphenolic precursors of tannin-inspired nanocoatings. Chem Commun. 2014;50:7265–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Zhang X, Li Z, Yang P, Duan G, Liu X, Gu Z, et al. Polyphenol scaffolds in tissue engineering. Mater Horiz. 2021;8:145–67. [DOI] [PubMed] [Google Scholar]
  • 20.Nakayama M, Shimatani K, Ozawa T, Shigemune N, Tomiyama D, Yui K, et al. Mechanism for the antibacterial action of epigallocatechin gallate (EGCg) on Bacillus subtilis. Biosci, Biotechnol, Biochem. 2015;79:845–54. [DOI] [PubMed] [Google Scholar]
  • 21.Li P, De Groot K. Calcium phosphate formation within sol-gel prepared titania in vitro and in vivo. J Biomed Mater Res. 1993;27:1495–1500. [DOI] [PubMed] [Google Scholar]
  • 22.Sedó J, Saiz-Poseu J, Busqué F, Ruiz-Molina D. Catechol-based biomimetic functional materials. Adv Mater. 2013;25:653–701. [DOI] [PubMed] [Google Scholar]
  • 23.Bee S-L, Bustami Y, Ul-Hamid A, Lim K, Abdul Hamid Z. Synthesis of silver nanoparticle-decorated hydroxyapatite nanocomposite with combined bioactivity and antibacterial properties. J Mater Sci: Mater Med. 2021;32:106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Zhang B, Yao R, Li L, Wang Y, Luo R, Yang L, et al. Green tea polyphenol induced Mg2+-rich multilayer conversion coating: toward enhanced corrosion resistance and promoted in situ endothelialization of AZ31 for potential cardiovascular applications. ACS Appl Mater interfaces. 2019;11:41165–77. [DOI] [PubMed] [Google Scholar]
  • 25.Eom H-J, Choi J. p38 MAPK activation, DNA damage, cell cycle arrest and apoptosis as mechanisms of toxicity of silver nanoparticles in Jurkat T cells. Environ Sci Technol. 2010;44:8337–42. [DOI] [PubMed] [Google Scholar]
  • 26.Yang Z, Liu Z, Allaker R, Reip P, Oxford J, Ahmad Z, et al. A review of nanoparticle functionality and toxicity on the central nervous system. J R Soc interface. 2010;7:S411–S422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Hussain S, Hess K, Gearhart J, Geiss K, Schlager J. In vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol Vitr. 2005;19:975–83. [DOI] [PubMed] [Google Scholar]
  • 28.Samberg ME, Oldenburg SJ, Monteiro-Riviere NA. Evaluation of silver nanoparticle toxicity in skin in vivo and keratinocytes in vitro. Environ health Perspect. 2010;118:407–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Foldbjerg R, Olesen P, Hougaard M, Dang DA, Hoffmann HJ, Autrup H. PVP-coated silver nanoparticles and silver ions induce reactive oxygen species, apoptosis and necrosis in THP-1 monocytes. Toxicol Lett. 2009;190:156–62. [DOI] [PubMed] [Google Scholar]
  • 30.Asharani P, Hande MP, Valiyaveettil S. Anti-proliferative activity of silver nanoparticles. BMC cell Biol. 2009;10:65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Kim YS, Song MY, Park JD, Song KS, Ryu HR, Chung YH, et al. Subchronic oral toxicity of silver nanoparticles. Part fibre Toxicol. 2010;7:20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Pv A. Cytotoxicity and genotoxicity of silver nanoparticles in human cells. AcS Nano. 2009;2:279–90. [DOI] [PubMed] [Google Scholar]
  • 33.Vazquez-Muñoz R, Borrego B, Juárez-Moreno K, García-García M, Morales JDM, Bogdanchikova N, et al. Toxicity of silver nanoparticles in biological systems: does the complexity of biological systems matter? Toxicol Lett. 2017;276:11–20. [DOI] [PubMed] [Google Scholar]
  • 34.Kittler S, Greulich C, Diendorf J, Koller M, Epple M. Toxicity of silver nanoparticles increases during storage because of slow dissolution under release of silver ions. Chem Mater. 2010;22:4548–54. [Google Scholar]
  • 35.Kim TH, Kim M, Park HS, Shin US, Gong MS, Kim HW. Size-dependent cellular toxicity of silver nanoparticles. J Biomed Mater Res Part A. 2012;100:1033–43. [DOI] [PubMed] [Google Scholar]
  • 36.Liu W, Zhang L, Xuan K, Hu C, Liu S, Liao L, et al. Alpl prevents bone ageing sensitivity by specifically regulating senescence and differentiation in mesenchymal stem cells. Bone Res. 2018;6:27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Gaharwar AK, Singh I, Khademhosseini A. Engineered biomaterials for in situ tissue regeneration. Nat Rev Mater. 2020;5:686–705. [Google Scholar]
  • 38.Golub EE, Boesze-Battaglia K. The role of alkaline phosphatase in mineralization. Curr Opin Orthop. 2007;18:444–8. [Google Scholar]
  • 39.Gregory CA, Grady Gunn W, Peister A, Prockop DJ. An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem. 2004;329:77–84. [DOI] [PubMed] [Google Scholar]
  • 40.Vester H, Holzer N, Neumaier M, Lilianna S, Nüssler AK, Seeliger C. Green Tea Extract (GTE) improves differentiation in human osteoblasts during oxidative stress. J Inflamm. 2014;11:15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Wei Y, Chen M, Li M, Wang D, Cai K, Luo Z, et al. Aptamer/hydroxyapatite-functionalized titanium substrate promotes implant osseointegration via recruiting mesenchymal stem cells. ACS Appl Mater Interfaces. 2022;14:42915–30. [DOI] [PubMed] [Google Scholar]
  • 42.Qayyum S, Oves M, Khan AU. Obliteration of bacterial growth and biofilm through ROS generation by facilely synthesized green silver nanoparticles. PloS one. 2017;12:e0181363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Adewunmi Y, Namjilsuren S, Walker WD, Amato DN, Amato DV, Mavrodi OV, et al. Antimicrobial activity of, and cellular pathways targeted by, p-anisaldehyde and epigallocatechin gallate in the opportunistic human pathogen Pseudomonas aeruginosa. Appl Environ Microbiol. 2020;86:e02482–19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Steinmann J, Buer J, Pietschmann T, Steinmann E. Anti-infective properties of epigallocatechin-3-gallate (EGCG), a component of green tea. Br J Pharm. 2013;168:1059–1073. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data and materials used in the current study are available from the corresponding author upon reasonable request.

Articles from Journal of Materials Science. Materials in Medicine are provided here courtesy of Springer

RESOURCES