Octahedral Fe₃O₄ Nanozymes Penetrate and Remove Biofilms on Implants via Photomagnetic Response

Abstract

Dental implant papilla (DIP) is susceptible to bacterial adhesion and biofilm formation, and oral pathogenic biofilms can cause persistent oral infections. Enrichment of bacterial biofilms on implants can lead to soft tissue irritation and adjacent bone resorption, severely compromising dental health and potentially leading to periodontitis, implant loss and costly follow-up care. Nanozymes (NZs) are recently used in biofilm removal as they can induce the production of reactive oxygen species (ROS), which can kill bacteria. However, the short lifespan of ROS limits their diffusion distance, and affects their therapeutic efficacy. In this study, we prepared Fe₃O₄ nanoparticles (NZs) with different morphologies: flower-like (F-Fe₃O₄), hollow spherical (M-Fe₃O₄), octahedral (O-Fe₃O₄), and conventional nanoparticles (N-Fe₃O₄). The ferromagnetic properties of Fe₃O₄ NZs allow them to move and penetrate the biofilm under the action of a magnetic field. The saturation magnetic intensities of the four samples were as follows: F-Fe₃O₄ (23.1 emu g⁻¹), M-Fe₃O₄ (73.34 emu g⁻¹), O-Fe₃O₄ (96.06 emu g⁻¹), and N-Fe₃O₄ (52.15 emu g⁻¹). The synergistic combination of photothermal action and catalytic sterilization can effectively remove the biofilm. In addition, the prepared Fe₃O₄ nanozymes were able to maintain high biological activity on the implant surface with some osteogenic effect.

1. Introduction

In recent years, implant restoration has become an increasingly important approach for treating dental defects [1]. However, the failure rate of 5%–11% after 10–15 years of implant placement remains a significant concern [2,3]. The primary cause of implant failure is the accumulation of bacterial plaque and subsequent pathogenic infection, leading to peri-implantitis [4]. This condition results in biological complications and adversely affects the local immune microenvironment [5]. Dental implant papilla is characterized by irreversible inflammation of the dental implant papilla soft tissues and associated bone loss [6]. Treatment options for dental implant papilla typically include non-surgical, surgical, and combined approaches. Non-surgical treatments often involve chemical removal of biofilms using agents like chlorhexidine, hydrogen peroxide, saline-soaked cotton balls, and citric acid. Mechanical methods, such as air polishing systems, scrapers, and sonic descalers, are also employed to remove biofilms from implant surfaces, sometimes in conjunction with antibiotics [7,8]. Although antibiotics are highly effective in treating bacterial infections, the lack of proper antibiotic stewardship has significantly contributed to the emergence of antibiotic-resistant bacterial strains [9,10]. As a result, the need for alternative antimicrobial strategies has become a critical factor in addressing this issue. In this context, antibiotic-free approaches are emerging as a promising solution to the growing complexity of antimicrobial resistance [11,12]. At the same time, non-surgical therapies have not yielded the desired therapeutic outcomes in treating peri-implantitis [13,14], often leaving bacterial biofilms on implant surfaces that continue to pose a threat to the host’s oral health [15]. Mechanical debridement, while useful, can also damage the implant surface, compromising its biocompatibility [16,17].

In recent years, nanozymes have attracted great attention in the field of antibacterial [18,19]; nanozymes mainly play an antibacterial role by catalyzing the reaction to produce reactive oxygen species, which can destroy bacterial membranes, proteins and DNA, thus inhibiting or killing bacteria and not easy to induce bacterial resistance [20,21,22]. Nanozymes have broad-spectrum antimicrobial properties, low drug resistance, and good biocompatibility with minimal effect on host cells [23,24]. There have been reports of Fe₃O₄ nanoenzymes being used as antibacterial agents. Dextran-coated Fe₃O₄ nanoenzymes are effective antibacterial agents in the oral cavity, effectively inhibiting the occurrence of biofilm-related infections in the mouth without causing adverse effects on host tissues [25]. Additionally, research has developed a ZnO structure encapsulating Fe₃O₄, where the Fe₃O₄ core endows the nanoparticles with highly efficient photothermal properties, promoting the dispersion of dense biofilms and effectively killing bacteria within biofilms in vitro, thereby enhancing bactericidal efficacy. Strategies leveraging its photothermal effects are anticipated to advance the treatment of gingivitis and other intractable infectious diseases [26]. In oral medicine research, Fe₃O₄ particles are more commonly used than other iron oxides (such as Fe₂O₃, FeO) or iron-based mixed metal oxides (such as CoFe₂O₄, MnFe₂O₄, etc.), primarily due to their unique physicochemical properties, biocompatibility, and functional tunability, which better align with the specific requirements of the oral environment. Firstly, due to their unique magnetic responsiveness, Fe₃O₄ particles can assist in antibacterial activity through the magnetothermal effect (heat generation under an alternating magnetic field) or disrupt biofilm structures via magnetic mechanical forces (shear forces generated by the chain-like arrangement of particles under a static magnetic field). Secondly, it exhibits excellent biocompatibility. Iron ions released upon dissolution in the body can be cleared through iron metabolism pathways. However, excess metal ions (such as Mn²⁺, Co²⁺) in iron-based mixed oxides may interfere with normal cellular functions and even exhibit genotoxicity, limiting their application in sensitive tissues such as oral mucosa and dental pulp.

These advantages make antimicrobial nanomaterials a viable and effective strategy for biofilm removal [27,28]. However, the short lifetime of the free radicals produced by nanozymes results in a limited antimicrobial range of the materials, often making it difficult to penetrate deep into the biofilm.

In this paper, we prepared magnetic Fe₃O₄ nanozymes with photothermal activity, which have the potential to address the existing challenge. Under the influence of a magnetic field, Fe₃O₄ nanozymes can experience mechanical vibrations that disrupt the biofilm and facilitate penetration into its interior [29], thereby ensuring the effective lethal range of the nanozymes. Furthermore, these nanozymes exhibit beneficial photothermal properties, which, when exposed to infrared radiation, can enhance their bactericidal efficacy even further [26,30]. The biofilm-removing properties of the prepared nanozymes are closely related to the magnetic, photothermal properties, and enzymatic activities, and their morphologies play a key role in influencing these properties. Therefore, Fe₃O₄ nanozymes with different morphologies are constructed, including flower-like (F-Fe₃O₄), hollow spherical (M-Fe₃O₄), octahedral (O-Fe₃O₄) and normal nanoparticles (N-Fe₃O₄), the relationship between the biofilm clearance performance and the morphology of the Fe₃O₄ nanozymes are systematically investigated, which may provide guidance for the designing of antibacterial agent for dental implant. We hope that through systematic research, we will not only be able to explore the potential applications of nanomaterials, comprehensively assess their safety and efficacy, and ensure that they meet strict regulatory standards, but also provide the theoretical and experimental basis for their market launch.

2. Materials and Methods

2.1. Materials

Glycerin (Aladdin), iron (III) nitrate nonahydrate (Fe(NO₃)·9H₂O, Macklin, AR, Shanghai, China), sodium hydroxide (NaOH, Aladdin, AR, Shanghai, China), ethylene glycol (Aladdin, AR, Shanghai, China), isopropyl Alcohol (Aladdin, AR, Shanghai, China), ethanol (Macklin, AR, Shanghai, China), iron (III) chloride hexahydrate (FeCl₃·6H₂O, Aladdin, AR, Shanghai, China), iron sulfate heptahydrate (FeSO₄·7H₂O, Macklin, AR, Shanghai, China), sodium acetate (NaAc, Sigma-Aldrich, AR, St. Louis, MI, United States), hydrazinium hydrate solution (Aladdin, Shanghai, China), sodium dodecylbenzenesulfonate (Aladdin, Shanghai, China), propidium iodide (PI, Beyotime Biotechnology, Shanghai, China), 4 midino-2-phenylindole (DAPI, Beyotime Biotechnology, Shanghai, China).

2.2. Preparation of Nanozymes

To prepare F-Fe₃O₄, 105 mL of isopropanol was added to a beaker containing 0.97 g of Fe (NO₃)₃·6H₂O and 15 mL of glycerol. The mixture was stirred thoroughly. Next, 1 mL of deionized water was added dropwise, and the stirring was continued for 10 min to ensure complete mixing of the solution. The resulting mixture was transferred to a stainless steel autoclave and heated at 190 °C for 12 h. After the reaction, the autoclave was allowed to cool naturally to room temperature. The precipitate was then separated by centrifugation (10,000 rpm, 5 min), washed three times with ethanol, and dried overnight in an oven (DZF-1, Tianjin Jinli Instrument Equipment Technology Co., Ltd., Tianjin, China) at 70 °C. Finally, the obtained powder was annealed in a nitrogen stream at 350 °C for 3 h, then dried again in an oven at 70 °C.

To prepare M-Fe₃O₄, 2.7 g of ferric chloride hexahydrate (FeCl₃·6H₂O) was dissolved in 80 mL of ethylene glycol, followed by the addition of 0.8 g of sodium dodecylbenzene sulfonate as a surfactant. Sodium acetate (0.7 mol/L) was then added and fully dissolved into the solution. The resulting mixture was transferred to a polytetrafluoroethylene (PTFE)-lined reactor. The reaction was carried out in an electrically heated oven at 180 °C for 12 h. After the reaction, the product was centrifuged (10,000 rpm, 5 min) and washed with deionized water and anhydrous ethanol. Finally, the washed product was dried in a vacuum drying oven.

To prepare O-Fe₃O₄, 0.2 g of FeSO₄·7H₂O and 4 g of NaOH were dissolved in a minimal amount of water using sonication. Then, 20 mL of ethylene glycol was added to the FeSO₄·7H₂O solution and stirred to form a light green solution. Next, a NaOH solution was added, followed by the dropwise addition of hydrazine hydrate (N₂H₄·H₂O). The solution was stirred for 30 min to ensure homogeneity. The mixture was transferred to a PTFE-lined stainless steel reactor and heated at 200 °C for 48 h. After the reaction was completed, the product was allowed to cool naturally to room temperature. The product was then washed repeatedly with deionized water and ethanol, followed by vacuum drying.

To prepare N-Fe₃O₄, 0.264 g of FeCl₃·6H₂O and 0.274 g of FeSO₄·7H₂O were dissolved in 10 mL of deionized water and heated at 50 °C to facilitate dissolution. Meanwhile, 0.36 g of NaOH was dissolved in 60 mL of deionized water in a separate container. The NaOH solution was then added dropwise to the FeCl₃ solution, with continuous stirring for 15 h. The resulting suspension was centrifuged (10,000 rpm, 5 min) and washed three times with ethanol and deionized water. Finally, the washed product was dried in a vacuum drying oven at 60 °C.

2.3. Characterization Techniques

Scanning electron microscopy (SEM, S-4800, Hitachi, Tokyo, Japan) was used to observe the morphology and surface features. Transmission electron microscopy (TEM, F200X G2, Thermo Fisher Scientific, Waltham, MA, USA) was used to observe the deeper structure of the samples and to obtain information on the size of the corresponding nanozymes. The elemental composition of the samples was determined by energy dispersive spectroscopy (EDS, F200X G2, Thermo Fisher Scientific, Waltham, MA, USA) analysis. In addition, X-ray diffraction (XRD, D8 Discover, Rigaku, Tokyo, Japan) was used to provide further information on the chemical composition and crystal structure of the samples.

2.4. In Vitro Photothermal Performance Testing

Four samples with different morphologies were irradiated with an 808 nm laser (MDL-III-808-2.5W, Changchun New Industry Optoelectronic Tech. Co., Ltd., Changchun, China) at power densities of 1 W cm⁻², 1.5 W cm⁻², and 2 W cm⁻². The temperature changes were monitored using a FLIR-1910582 thermal camera. The effect of sample concentration on temperature was investigated by preparing samples at concentrations of 0, 0.25, 0.5, 1, and 1.5 mg mL⁻¹ and irradiating them with a 1 Wcm⁻² 808 nm laser for 5 min.

To assess photothermal stability, the samples were subjected to five switching cycles of 808 nm laser irradiation (1 W cm⁻²). Photothermal conversion efficiency was measured by exposing the samples to the 808 nm laser for 5 min, followed by a 5 min cooling period after the laser was turned off. The temperature data were recorded at 1 min intervals during the cooling phase.

2.5. HRP-Mimicking Activity

The peroxidase-like activity of Fe₃O₄ samples was evaluated using catalytic oxidation of 3,3′,5′,5′-tetramethylbenzidine (TMB). When TMB is oxidized, it forms a blue charge transfer complex (oxTMB) with an absorption peak at 652 nm. Enzyme-linked immunosorbent assay was used to test samples of different forms (DNM-9602, Beijing Perlong New Technology Co. Ltd., Beijing, China), and the change in TMB absorption intensity was monitored by UV-Vis spectroscopy at 10 min intervals.

TMB was dissolved in alcohol to a concentration of 200 µg mL⁻¹, and the nanomaterials were suspended in HAc/NaAc buffer (0.1 M, pH 6.5) at 1 mg mL⁻¹. The TMB solution and sample suspension were mixed at a 1:1 ratio, and peroxidase-like activity was quantified by recording changes in absorption. The relationship between activity and pH was tested using a 50 mM H₂O₂ concentration.

2.6. Biocompatibility Testing

We first tested for cytocompatibility using the cck-8 assay, MC3T3-E1 cells were seeded in 96-well plates at 5000 cells/well. Sterilized samples were dispersed in fresh medium at concentrations of 0.125, 0.25, and 0.5 mg mL⁻¹ and incubated for 24 h. After replacing the medium with the sample suspension, cells were cultured for 24 or 48 h. The CCK-8 working solution was added and the culture was continued for 1–4 h. Absorbance at 450 nm was measured to assess cell viability.

Cell morphology was then observed using live–dead cell staining. For live–dead staining, cells cultured with samples at 0.5 mg mL⁻¹ concentration were stained with Calcein-AM and propidium iodide (PI) at a 1000-fold dilution. After 30 min of incubation at 37 °C, cells were observed under a fluorescence microscope (EVOS M7000, Thermo Fisher Scientific, Massachusetts).

2.7. Osteogenic Performance Testing

We first tested for cytotoxicity. Soak titanium plates (5 mm in diameter) that have been sanded with sandpaper in hydrogen peroxide (H₂O₂) solutions of different concentrations (3%, 1%, and 0.5%). Seed 10,000 rBMSCs onto the titanium plates, wait 4 h for the cells to adhere, then add 1 mL of complete culture medium. After culturing the rBMSCs for one day, calculate the cell survival rate using the CCK-8 assay.

Then we did an alkaline phosphatase (ALP) stain. ALP activity was analyzed qualitatively on H₂O₂-treated titanium sheets using the BCIP/NBT chromogenic kit. rBMSCs cells were cultured for 7 days, and ALP staining was performed. The stained samples were observed under an inverted microscope.

After 7 days of culture, cells were lysed with RIPA buffer, and the total ALP activity was measured using p-nitrophenyl phosphate. Protein content was quantified using a BCA kit. The ALP activity per unit of protein was calculated to quantify the ALP activity.

We detected the expression of osteogenesis genes by RT-PCR (LightCycler@96, Roche, Basel, Switzerland). rBMSCs were cultured on H₂O₂-treated titanium sheets for 7 days. RNA was extracted, and cDNA was synthesized. RT-PCR was performed using specific primers, and gene expression was analyzed using the 2^−∆∆Ct method, with GAPDH as the internal control. The primer sequences used in this part of the experiment are shown in Table S1.

2.8. In Vitro Antimicrobial Performance Test

We first performed biofilm cultures of Streptococcus mutans. Streptococcus mutans were cultured overnight, transferred to BHI medium, and activated for 3 h until the bacterial concentration reached 1.0 OD at 600 nm. A 10 × 10 mm titanium plate was used as a model for dental implant infection. S. mutans (ATCC25175, Beijing Bio-Tech Co., Ltd., Beijing, China) and artificial saliva were introduced onto the titanium plate, and biofilm formation was allowed in an anaerobic incubator at 37 °C for 30 min. The plates were then incubated for up to 3 days.

Biofilms were formed on Petri dishes and treated with the samples in NaAc buffer (pH = 4.5) at a concentration of 2 mg mL⁻¹. H₂O₂ (0.5%) was added, and the samples were exposed to a magnetic field for biofilm removal. After 30 min, biofilm removal was confirmed using confocal fluorescence microscopy. The samples were evaluated for biofilm removal and bacterial killing using CCK-8 and crystal violet assays.

2.9. In Vivo Experiments and Histological Analysis

The animal experiments were approved by the Biomedical Ethics Committee. Twenty-four SD rats (220–250 g) were acclimated for 7 days before surgery. We took 48 purchased titanium rods (diameter 5 mm) and sterilized them with ultraviolet light for later use. We selected 12 smooth titanium rods for direct use. The remaining 36 titanium rods were reserved for subsequent use after the formation of S. mutans biofilm on their surfaces. Among these, 12 contaminated titanium rods were selected for direct use; 12 contaminated titanium rods were treated with a mixture of 0.5% H₂O₂ and 500 μg/mL O-Fe₃O₄ for 15 min, and 12 contaminated titanium rods were treated with 3% H₂O₂ for 15 min. During the treatment process, the solution was simultaneously exposed to 808 nm near-infrared light at a power of 1.5 W cm⁻² for 5 min, and a magnetic field was applied to drive sample movement. The four groups were labeled as Ti, Ti@S.mutans, Ti@S.mutans + O-Fe₃O₄, and Ti@S.mutans + 3% H₂O₂, respectively. After anesthesia and surgical preparation were administered, titanium rods were implanted into the femoral trochanteric fossae of the rats. Seven days after implantation, digital X-ray photography (XPLORER 1600, Beijing Zhongtuo Yiting Technology Co., Ltd., Beijing, China) was used to take X-ray images of the implantation site in rats to confirm whether the samples had fallen out. One week after surgery, some of the rats were euthanized. The implants were then removed and placed in BHI culture medium at 37 °C for 24 h. After this time, the OD₆₀₀ value of the BHI culture medium was measured. Fresh blood samples were collected for a complete blood count analysis, and the rats’ major organs were subjected to H&E staining. Six weeks postoperatively, the rats were euthanized and their femurs with implants were removed. The femur samples were then subjected to H&E and Masson staining.

Subsequently, in vivo antimicrobial experiments were conducted using two types of samples: titanium plates (diameter 5 mm) and titanium plates infected with S. mutans (T@S.mutans). The experimental mice were divided into four groups: the Control group, which received titanium implants; the Inflammation group (T@S.mutans), which received T@S.mutans implants without further treatment; the Inflammation + O-Fe₃O₄ group (T@S.mutans + O-Fe₃O₄), which received T@S.mutans implants followed by injection with 20 µL of O-Fe₃O₄ + 0.5% H₂O₂ mixed solution using a magnetic field to disrupt the biofilm, and then irradiated with an 808 nm laser for 5 min; and the Inflammation + H₂O₂ group (T@S.mutans +3% H₂O₂. One day after implanting T@S.mutans, we injected them with 20 µL of 3% H₂O₂. We implanted the treated titanium plates into the backs of the mice. One day after treatment, we euthanized some of the mice, removed the titanium plates, and performed plate coating. Seven days after treatment, we euthanized the mice, remove the treated skin tissue, and performed H&E staining.

2.10. Statistical Analysis of Data

All experimental data were repeated three times, with three parallel samples in each group. The results are expressed as mean ± standard deviation (Mean ± SD).

3. Results

3.1. Characterization of Nanozyme

The crystal structures of the synthesized samples were analyzed using X-ray diffraction (XRD). As shown in Figure 1i, all the diffraction peaks of F-Fe₃O₄ correspond to the Fe₃O₄ phase with a face-centered cubic structure. The diffraction peaks of the M-Fe₃O₄ sample at 2θ values of 30.5°, 35.4°, 43.2°, 53.6°, 57.1°, and 62.7° correspond to the (220), (311), (400), (422), (511), and (440) crystal planes of cubic Fe₃O₄, respectively. Similarly, the diffraction peaks of N-Fe₃O₄ at 2θ values of 30.5°, 35.4°, 43.2°, 53.6°, 57.1°, and 62.7° also match the (220), (311), (400), (422), (511), and (440) crystal planes of cubic Fe₃O₄, confirming their cubic crystal structure.

As shown in Figure 1a, F-Fe₃O₄ exhibits an internally hollow shell structure with a shell thickness of approximately 150 nm. The magnified transmission electron microscopy (TEM) image in Figure 1e reveals that the nanosheets are composed of interconnected nanozymes, giving the material a highly porous texture. The inner hollow shell of M-Fe₃O₄, shown in Figure 1b, has a thickness of about 70 nm and consists of many smaller nanozymes. The TEM image of O-Fe₃O₄ in Figure 1c clearly shows the presence of square and rhombic structures, confirming its octahedral morphology. In contrast, the TEM image of N-Fe₃O₄ demonstrates a tendency to agglomerate (Figure 1d), likely due to the combined effects of its small size and magnetic properties. SEM and TEM analyses confirm the successful synthesis of Fe₃O₄ nanozymes with four distinct morphologies. The specific particle size distribution of each group of samples is shown in Figure S1. Hysteresis line analysis of four groups of Fe₃O₄ samples was carried out at room temperature by an integrated physical property measurement system, as shown in Figure S2. The prepared Fe₃O₄ samples had different magnetic properties, among which the O-Fe₃O₄ samples had the highest saturation magnetization intensity, while the F-Fe₃O₄ samples had the lowest saturation magnetization intensity. The reason for the different magnetic properties of the samples may have been due to the different micromorphology of the prepared samples. Because the spherical shape has lower surface energy and good particle dispersion, it also exhibited weaker magnetic anisotropy and therefore a nearly isotropic magnetic response in an external magnetic field. On the other hand, octahedral iron tetroxide has a highly symmetrical geometry and also exhibits strong magnetic anisotropy. In general, the octahedral-shaped particles had some magnetization directionality and could be better aligned in the external magnetic field compared to the spherical and cubic shaped ferric tetroxide [31,32].

3.2. Photothermal Performance Testing of Nanozymes

To evaluate the photothermal properties of the materials, we leveraged their strong absorption in the near-infrared (NIR) region, testing both irradiated and non-irradiated samples at different concentrations. As shown in Figure S3a, the sample solutions with identical concentrations were heated to 65 °C, 63.2 °C, 48.2 °C, and 56 °C, respectively, under 5 min of NIR light irradiation. In contrast, the temperature change in the control (deionized water) was negligible. The photothermal conversion efficiencies for F-Fe₃O₄, M-Fe₃O₄, O-Fe₃O₄, and N-Fe₃O₄ were 65.56%, 54.5%, 17.28%, and 36.72%, respectively (Figure S3b). To visualize the temperature change in the sample solution with laser irradiation, the solution was irradiated with the laser for 5 min and temperature infrared photographs were taken at 1 min intervals (Figure 2a–d). The variation in photothermal performance among the four samples is primarily attributed to differences in their specific surface areas, as a larger surface area facilitates more efficient heat transfer.

Moreover, the temperature increase was found to be concentration-dependent. We prepared nanozyme suspensions at different concentrations, irradiated them with NIR light (1 W cm⁻²) for 5 min, and recorded the temperature changes. As shown in Figure 2e–h, the temperature rise in the samples increased in parallel with the nanozyme concentration. We also examined how the heating rate of the samples varied with laser power. When irradiated with 808 nm lasers of different intensities (1 W cm⁻², 1.5 W cm⁻², and 2 W cm⁻²), the heating rate of each group accelerated as the laser power increased (Figure 2i–l).

In addition to high photothermal conversion efficiency, an ideal photothermal material must also exhibit good photothermal stability to ensure long-term performance. To test the stability of the Fe₃O₄ samples, we subjected each sample to five thermal cycles. The photothermal cycling stability curves, shown in Figure S4, demonstrated consistent heating and cooling trends across all cycles, with the highest plateau temperature at 5 min remaining nearly constant. These results confirm that the Fe₃O₄ samples possessed excellent photothermal stability, making them suitable for long-term applications. To demonstrate the physical properties of the samples prepared in this paper, Table S2 compares their photothermal performance and saturation magnetization with those of Fe₃O₄ materials reported in other studies. Table S2 shows the photothermal conversion efficiency and saturation magnetic intensity of engineered Fe₃O₄ reported in this study and other studies. It can be seen that the Fe₃O₄ with different microstructures prepared in this study exhibit significant differences in photothermal performance and magnetic properties. The highest photothermal conversion efficiency was achieved by F-Fe₃O₄, reaching 65.56%, while most of the other literature reported values around 55%. The highest saturation magnetic strength was 96.06 emu g⁻¹ for O-Fe₃O₄, which is higher than the highest value of approximately 80 emu g⁻¹ reported in the comparison literature.

Additionally, we evaluated the enzyme catalytic performance of the Fe₃O₄ samples. Fe₃O₄ nanoenzymes had peroxidase activity and could react with H₂O₂ to generate hydroxyl radicals (·OH) and hydroxide ions (OH⁻). The generation of ·OH is a strong oxidizing agent that can oxidize a variety of organic substrates, thereby exhibiting peroxidase activity. The peroxidase-like activities of all groups of Fe₃O₄ samples increased with decreasing pH, as shown in Figure 3a–d. The peroxidase-like activity of Fe₃O₄ nanozymes was correlated with the concentration of ferric ions in solution, and the samples had better peroxidase-like activities in acidic conditions, mainly because acidic conditions are more favorable for the release of ferric ions from Fe₃O₄. The catalytic activity of each group of Fe₃O₄ nanozymes was then evaluated as a function of time. As shown in Figure 3e–h, the catalytic experiments demonstrated that the absorption peak at 653 nm of the TMB solution increased over time, indicating a gradual increase in the production of hydroxyl radicals (·OH). This trend suggests that the Fe₃O₄ nanozymes effectively catalyzed the production of ·OH from H₂O₂ in the solution, with the catalytic activity continuing until the substrate was consumed. These results confirm that Fe₃O₄ acts as an efficient peroxidase-like enzyme, capable of effectively catalyzing the generation of highly reactive and toxic ·OH radicals from H₂O₂.

3.3. In Vitro Biocompatibility Analysis

The biocompatibility of the four sample groups was assessed using MC3T3-E1 cells, and the results are presented in Figure 4. Among the samples, O-Fe₃O₄ exhibited the highest biocompatibility, as shown in Figure 4a, with cell viability remaining above 90% even at a concentration of 500 μg mL⁻¹. In comparison, the other three samples maintained cell viability of over 80% at a concentration of 125 μg mL⁻¹.

Furthermore, after 24 h of co-incubation with MC3T3-E1 cells, live–dead cell staining and actin staining were performed. As shown in Figure 4b, there were very few dead cells in each sample group, and the cells appeared to be in good condition. Actin staining revealed a reticular distribution of actin filaments within the cells, with clear cell borders and intact cytoskeletons, further confirming that the cells were in a healthy growth state. These findings suggest that the samples had low cytotoxicity and were biocompatible. In addition, to explore the reasons for the differences in biocompatibility, PBS was used to soak each sample for one day before measuring the Fe concentration in the solution. As shown in Table S3, the greater the release of Fe ions, the poorer the biocompatibility. The release of Fe ions leads to iron death in cells.

3.4. Effect of Hydrogen Peroxide (H₂O₂) on Titanium Implants and Biocompatibility

In clinical settings, peri-implantitis is typically treated with mechanical biofilm removal followed by secondary sterilization using 3% medical-grade hydrogen peroxide (H₂O₂). However, high concentrations of H₂O₂ can corrode titanium implants and adversely affect the growth and differentiation of peri-implant cells, ultimately slowing wound healing [33]. To investigate the effects of H₂O₂ on titanium implants, Ti disks were treated with various concentrations of H₂O₂, and the biocompatibility of the treated implants was assessed. As shown in Figure S5, high concentrations of H₂O₂ caused significant damage to cellular biocompatibility, with cell viability dropping below 50% after treatment with 3% H₂O₂. In contrast, lower concentrations of H₂O₂ did not have a significant impact on the cytocompatibility of the Ti disks. Notably, the treatment with a 0.5% H₂O₂ solution mixed with O-Fe₃O₄ did not adversely affect the cytocompatibility of the Ti disks. ALP staining of cells cultured on titanium wafers treated with different H₂O₂ concentrations revealed that titanium wafers from the control group and those treated with the 0.5% H₂O₂ + O-Fe₃O₄ mixed solution exhibited stronger ALP expression on their surfaces (Figure 5a). This suggests that these treatments caused minimal corrosion to the titanium surface, and the ability of rBMSCs to differentiate into osteoblasts was not hindered. In contrast, titanium wafers treated with 3% H₂O₂ showed reduced ALP expression, indicating that prolonged exposure to 3% H₂O₂ corroded the titanium surface and significantly inhibited the osteogenic differentiation of rBMSCs.

The quantitative analysis of ALP activity (Figure 5c) confirmed the visual findings from ALP staining. The ALP activity of cells on titanium wafers treated with 0.5% H₂O₂ and 0.5% H₂O₂ mixed with O-Fe₃O₄ was not significantly different from that of the control titanium group. However, a significant reduction in ALP activity was observed on titanium wafers treated with 3% H₂O₂, further supporting the detrimental effect of high-concentration H₂O₂ on osteoblast differentiation.

The expression of key genes related to osteogenesis, including ALP, BMP-2, Col-1, Runx-2, and OPN, on the surface of rBMSCs cultured on titanium wafers treated with different H₂O₂ solutions was assessed by RT-PCR analysis (Figure 5b). The expression of ALP, a critical early marker of osteogenesis, decreased gradually with increasing H₂O₂ concentration. This downregulation of ALP suggests that high concentrations of H₂O₂ hinder the osteogenic differentiation of rBMSCs. Similarly, the expression of Col-1, an essential component of the bone matrix, was notably suppressed by 3% H₂O₂. These findings indicate that prolonged exposure to 3% H₂O₂ negatively affects the expression of osteogenic genes and the differentiation of rBMSCs on titanium surfaces. In contrast, the growth and differentiation of cells on titanium implants treated with 0.5% H₂O₂ remained largely unaffected, highlighting the lower cytotoxicity and better osteogenic potential of the lower concentration of H₂O₂.

The results indicate that while 3% medical H₂O₂ significantly impairs the osteogenic differentiation of cells on titanium implants, lower concentrations (such as 0.5%) have a less pronounced effect. Additionally, the use of O-Fe₃O₄ in combination with 0.5% H₂O₂ mitigates the negative effects on cell viability and osteogenic differentiation, suggesting a potential therapeutic approach for improving peri-implant tissue healing and preventing implant corrosion.

3.5. In Vitro Analysis of Antimicrobial Properties

As demonstrated in previous biocompatibility studies, high concentrations of H₂O₂ can impair wound healing. However, lower concentrations of H₂O₂ can be converted into hydroxyl radicals (·OH), which are highly reactive oxygen species (ROS) with stronger oxidizing capabilities than H₂O₂ itself. This increased reactivity makes ·OH more effective at causing oxidative damage to bacteria [34].

In the antimicrobial tests, as shown in Figure 6, no significant antibacterial effect was observed in the control group following 808 nm laser irradiation and H₂O₂ treatment. This indicates that neither the 808 nm laser irradiation nor 0.5% H₂O₂ alone, or in combination, inhibited the growth of S. aureus. However, the antibacterial effect of F-Fe₃O₄ was the most pronounced under 808 nm laser irradiation. The addition of 0.5% H₂O₂ to the F-Fe₃O₄ sample group further enhanced the antibacterial effect, likely due to the generation of ·OH from the peroxide-like catalytic activity of the Fe₃O₄ nanozymes. The combination of NIR laser irradiation and 0.5% H₂O₂ demonstrated a significantly stronger antibacterial effect compared to photothermal therapy alone or treatment with 0.5% H₂O₂ alone. This suggests that the synergistic generation of ·OH and localized heating leads to more aggressive bacterial killing.

In Figure 6b, the antimicrobial efficacy against S. mutans was also evaluated. No notable antibacterial effect was seen in the control group after 808 nm laser irradiation and 0.3% H₂O₂ treatment, indicating that neither the laser nor H₂O₂ alone significantly inhibited the growth of S. mutans. However, under 808 nm laser irradiation, the photothermal properties of the Fe₃O₄ samples inhibited the growth of S. mutans, with the effect corresponding to their photothermal performance. When 0.3% H₂O₂ was added, all four Fe₃O₄ sample groups catalyzed the production of ·OH from H₂O₂, leading to a significant inhibition of S. mutans growth. The combined effect of photothermal heating and ·OH generation resulted in substantial antibacterial activity, confirming that the synergy between heating and reactive oxygen species enhances bacterial killing efficiency.

To evaluate the biofilm removal capacity of the Fe₃O₄ samples, we first established a biofilm infection model on smooth titanium (Ti) sheets using S. mutans. The process of biofilm formation on the Ti sheet was observed using SEM, and the microstructure of the biofilm at various infection time points is shown in Figure S6. After 4 h, the S. mutans showed a low activity globular distribution on the surface of the Ti sheet, with only a small portion of the bacteria aggregated into a cluster. When the infection time was extended to 8 h, the S. mutans started to form chain-like growth and cross-linked to form clumps of aggregated colonies. After 12 h, the cross-linking between S. mutans increased, the distribution of long chains of bacteria on the surface decreased, and the clumps of aggregated colonies gradually fused to form a larger colony. After 1 day of infection, the cross-linking between S. mutans was more intense, showing overlapping and fusion, forming a dense web-like structure. After 2 and 3 days of infection, the biofilm still maintained a dense reticular structure similar to that at 1 day. Therefore, after 1 day of infection, S. mutans successfully covered the entire surface of the titanium sheet and formed a dense biofilm, and the biofilm infection model was successfully established. Therefore, the smooth Ti sheet infected with S. mutans for 1 day was selected for biofilm removal experiments in the subsequent experiments.

Bacterial biofilms are inherently resistant to treatments due to their complex multilayered structure, consisting of bacteria and EPS. The EPS matrix helps trap antibiotics and biocides, impeding their ability to penetrate the biofilm and kill the bacteria. Biofilms also enhance bacterial resistance to antimicrobial agents and physical disruption.

In our study, we utilized the unique properties of Fe₃O₄ nanozymes, including magnetic responsiveness, photothermal activity, and peroxidase-like catalytic activity to remove biofilms. Using these properties, mechanical, thermal, and chemical disruption of biofilms can be performed.

We first tested the mechanical removal of biofilm using the magnetic properties of the Fe₃O₄ samples. The samples were subjected to a magnetic field for 30 min, and the detached bacteria were washed off. The remaining biofilm was stained with crystal violet, and the amount of remaining biofilm was quantified by measuring the optical density at 600 nm (OD₆₀₀). Figure 7a shows that O-Fe₃O₄ exhibited the most effective mechanical biofilm removal compared to the other samples. The application of 0.5% H₂O₂ and 808 nm laser irradiation (for 5 min) while applying the magnetic field further enhanced the biofilm removal efficiency, showing a significant improvement compared to magnetic field alone.

Next, the live bacterial count in the biofilm was assessed using the CCK-8 assay. Figure 7b illustrates that O-Fe₃O₄ showed the best biofilm removal capacity, consistent with the crystal violet results. Further comparison of the four Fe₃O₄ samples under combined treatments of H₂O₂, 808 nm laser irradiation, and magnetic field demonstrated the following ranking of biofilm removal effectiveness: O-Fe₃O₄ > M- Fe₃O₄ > F-Fe₃O₄> N-Fe₃O₄ (Figure 7c). Notably, the O-Fe₃O₄ group showed the greatest biofilm removal ability, while the F-Fe₃O₄ group showed the weakest effect, with minimal damage to the biofilm.

To further confirm the bacterial damage caused by the treatment, biofilms were subjected to live–dead cell staining and analyzed using laser confocal microscopy (AXR, Nikon), as shown in Figure 7d. In the magnetic-field-only group, a significant portion of S. mutans remained intact within the biofilm. However, in the groups treated with 0.5% H₂O₂ and 808 nm laser irradiation, the bacterial population was notably reduced, with fewer live cells detected. The F-Fe₃O₄ group exhibited the least effective biofilm disruption, and a layer of bacterial biofilm remained visible under the microscope. The above results indicate that O-Fe₃O₄ nanozymes have the most effective biofilm removal performance, which may be due to the different micromorphology of the prepared Fe₃O₄ nanozymes, which directly affects their magnetic, photothermal, and biological properties. Although F-Fe₃O₄ showed the best performance in pure antimicrobial experiments, O-Fe₃O₄, which had better magnetic properties, killed the biofilm the most when confronted with thicker biofilms. Under the influence of a magnetic field, magnetic Fe₃O₄ can navigate between biofilms, disrupting their dense structure and promoting material penetration. Thus, differences in magnetic properties lead to variations in biofilm removal capability. In this study, F-Fe₃O₄ did not exhibit the most prominent magnetic properties, resulting in suboptimal performance in biofilm removal.

3.6. In Vivo Biological Analysis of Fe₃O₄ Nanozymes

In vivo experiments were conducted to evaluate the biological safety and efficacy of Fe₃O₄ nanozymes for biofilm removal and infection prevention in an animal model. Titanium rods were implanted into rats, and several assays were performed to assess the biocompatibility, infection control, and bone tissue regeneration.

The long-term behavior of Fe₃O₄ nanozymes after oral implantation is closely related to its physicochemical properties (such as size, surface modification, and crystal structure) and the biological environment (such as local blood flow, enzyme activity, and immune response).

First, due to their small size, nanoparticles may become mechanically trapped within the pore structure of the implant site (such as microporous surfaces of dental implants) or physically adsorb to collagen and glycosaminoglycans in the extracellular matrix (ECM), forming stable deposits.

Second, positively charged particles (such as amino-modified Fe₃O₄) are prone to electrostatic adsorption with negatively charged cell membranes (phospholipid bilayers) or ECM components (heparan sulfate), increasing the likelihood of retention.

Under the influence of an external magnetic field (such as orthodontic magnets, implant-integrated magnetic components), Fe₃O₄ nanozymes can be directed to accumulate and fixate at target sites, resisting the erosion caused by saliva flow or chewing movements, thereby achieving long-term retention. For example, magnetically controlled Fe₃O₄ nanozymes can be anchored in periapical tissues during root canal treatment via an external magnetic field, thereby prolonging the duration of antimicrobial action.

In clinical practice, for peri-implantitis, after mechanically scraping off the biofilm, 3% medical-grade hydrogen peroxide (H₂O₂) is typically used as an adjunctive antimicrobial agent. As the concentration of the H₂O₂ solution increases, its antimicrobial efficacy also increases. The 3% H₂O₂ concentration exhibits greater antimicrobial activity, enabling a more effective comparison of the antimicrobial performance of different material groups in vivo.

Seven days post-implantation, digital X-ray imaging was used to assess the positioning of the titanium rods and confirm that none of the implants had been dislodged. As shown in Figure 8a, the X-ray images indicated that all rods remained in place without any signs of displacement. Following the imaging, the titanium rods were removed for bacterial culture analysis. Figure 8b shows that infection was observed only in the Ti@S.mutans group, where S. mutans infection had occurred. No infection was observed in the other experimental groups, indicating that Fe₃O₄-based treatments, especially when combined with H₂O₂, were effective in preventing bacterial colonization at the implant sites.

H&E staining of sections of heart, liver, spleen, lung, and kidney from each group of rats implanted with titanium rods, as shown in Figure 8c, revealed no toxic reaction in any of the groups. The heart sections showed normal myocardial structure without inflammatory cell infiltration or other signs of abnormality. This indicates that the implanted titanium rods did not cause significant damage to the cardiac tissue. The liver sections showed normal hepatocyte arrangement and hepatic lobular structure with no areas of necrosis or inflammatory reaction. The splenic sections showed normal structure of splenic corpuscles and red and white medulla, and no abnormal proliferation or inflammatory reaction was observed. The lung sections showed normal alveolar structures and bronchioles, and no inflammation, fibrosis, or other abnormal changes were observed in the lungs. The kidney sections showed normal tubular and glomerular structures, and no tubular dilatation, necrosis, or inflammation was observed.

In order to observe the bone tissue around the titanium rods, H&E staining and Masson staining were performed on the bone tissue around the titanium rods, as shown in Figure 9a, and the H&E results showed that there was significant inflammation in the Ti+ S. mutans group compared to the other groups.

The bone tissue appeared blue in Masson’s stain, while the collagen fibers appeared red. As shown in Figure 9b, it can be seen that the Ti@S.mutans group had low bone tissue content, indicating that the presence of biofilm was not conducive to the formation of bone tissue around the titanium rods, and several other groups observed that the bone tissue around the titanium rods was tightly arranged, and the collagen fibers organically combined with the bone matrix to form a structurally complete and tight bone tissue network. In conclusion, implantation of titanium rods treated with O-Fe₃O₄ and 3% H₂O₂ does not cause bone tissue infection, and O-Fe₃O₄ can effectively remove biofilm.

H&E staining of the mice’s internal organs did not reveal the presence of O-Fe₃O₄ nanozymes or any other abnormalities. This suggests that after subcutaneous injection, the O-Fe₃O₄ nanozymes did not accumulate in the internal organs and did not cause any noticeable damage. Furthermore, it indicates that the injected nanozymes did not provoke any adverse or foreign body reactions, nor did they have any significant effects on the internal organs of the mice.

In vitro biofilm removal experiments demonstrated that O-Fe₃O₄ effectively eliminates biofilm. To further verify its biofilm removal effect in vivo, titanium plates infected with Streptococcus mutans biofilm were first implanted under the skin of mice. Then, 20 μL of an O-Fe₃O₄ + 0.5% H₂O₂ mixed solution was injected at the implantation site. Biofilm removal and sterilization were performed using a magnetic field and an 808 nm laser. One day after treatment, the titanium plates were removed, and the bacteria on the plates were shaken into PBS solution and plated. The results, shown in Figure S7, indicated that the T@S.mutans +O-Fe₃O₄ group had the fewest bacterial colonies. This suggests that the in vivo biofilm removal effect of O-Fe₃O₄ was superior to that of the T@S.mutans +3% H₂O₂ group, confirming that O-Fe₃O₄ can also effectively remove biofilm in vivo.

Orbital blood was collected from the mice, and routine blood tests were conducted to assess whether the implanted samples induced inflammation. Routine blood tests, which include white blood cell (WBC) count and absolute neutrophil count (Neu #), are key indicators of the body’s inflammatory response. As shown in Figure S8, the blood parameters of all groups remained within normal ranges, and WBC counts were stable without abnormal increases. This suggests that the implanted samples and sterilization treatments did not trigger systemic inflammation.

To further investigate whether implantation caused localized inflammation in the surrounding skin, H&E staining was performed on the skin at the site of the titanium plate. Inflammatory cells, which stain dark purple or blue, were examined. The results, shown in Figure S9, revealed that the T@S.mutans group exhibited significantly more inflammatory cells compared to the other groups, likely due to the presence of the biofilm. In contrast, the T@S.mutans +O-Fe₃O₄ group showed no significant inflammation compared to the control group, indicating that the biofilm was effectively removed without causing an inflammatory reaction in the surrounding skin tissue.

4. Conclusions

Four types of Fe₃O₄ nanozymes with varying morphologies were synthesized using hydrothermal and co-precipitation methods. Among these, the octahedral O-Fe₃O₄ nanozymes exhibited the highest biofilm removal capabilities, attributed to their superior magnetic properties. Under the influence of a magnetic field, these nanozymes can penetrate deeply into the biofilm, ensuring sufficient interaction between the bacteria and the nanozymes. Consequently, the bacteria can be effectively eliminated by the O-Fe₃O₄ nanozymes due to their photothermal and enzyme-like activities. Additionally, these nanozymes preserve the bioactivity of the implant surface and possess osteogenic properties. Overall, these nanozymes provide a simplified, non-antibiotic approach to the clinical treatment of peri-implantitis, offering an effective alternative to antibiotics and contributing to the reduction in bacterial resistance. Although nanoenzymes have high-efficiency biofilm removal capabilities, due to the complexity and variability of the oral cavity, nanoenzymes that rely on physical and chemical conditions to take effect may have certain limitations. In the future, low-toxicity or non-toxic materials can be screened for the preparation of nanoenzymes, and safer surface modification strategies can be developed to ensure safety in oral treatment.