Safety and efficacy of PLGA(Ag-Fe₃O₄)-coated dental implants in inhibiting bacteria adherence and osteogenic inducement under a magnetic field

Abstract

Introduction

The placement of dental implants is performed in a contaminated surgical field in the oral cavity, which may lead to implant failure. Bacterial adhesion and proliferation (Streptococcus mutans, Porphyromonas gingivalis) often lead to implant infections. Although Ag nanoparticles hold great promise for a broad spectrum of antibacterial activities, their runoff from dental implants compromises their antibacterial efficacy and potentially impairs osteoblast proliferation. Thus, this aspect remains a primary challenge and should be controlled.

Materials and methods

In this study, PLGA(Ag-Fe₃O₄) was modified on the implanted tooth surface and was characterized by transmission electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The magnetic and antibacterial properties were also determined.

Results

Results showed that Ag successfully bonded with Fe₃O₄, and Ag-Fe₃O₄ not only exerted superparamagnetism but also exhibited antibacterial activity almost identical to silver nanoparticles (nano-Ag). The PLGA(Ag-Fe₃O₄) coating could significantly maintain the antibacterial activity and avoid bacterial adhesion to the implant. Compared with the blank control group, PLGA(Ag-Fe₃O₄) under magnetic field-coated samples had a significantly lower amount of colonized S. mutans (P<0.01). Osteoblast proliferation results showed that the coated samples did not exhibit cytotoxicity and could promote osteoblast proliferation as shown by MTT, alkaline phosphatase, and the nucleolar organizer region count.

Conclusion

We developed a novel Ag biologically compatible nanoparticle in this study without compromising the nano-Ag antibacterial activity, which provided continuous antibacterial action.

Keywords:

• Ag-Fe₃O₄
• PLGA
• dental implants
• antibacterial property

View correction statement:

Safety and Efficacy of PLGA(Ag-Fe3O4)-Coated Dental Implants in Inhibiting Bacteria Adherence and Osteogenic Inducement under a Magnetic Field [Corrigendum]

Introduction

Dental implants are embedded in the gum, which is a contaminated surgical field. The microbial composition may lead to periimplantitis. As a result of the host immune response to pathogenic bacteria, destruction of the tooth supporting tissues, in particular alveolar bone resorption,¹ may occur and then lead to the augmentation of the probing depth to form peri-implant pockets. These pockets provide an ideal environment for microorganism colonization, which causes inflammation and tissue destruction, and eventually lead to potential failure of the dental implant.² Bacterial adhesion ability is considered to be one of the principal factors in microbial reproduction, consequently forming and consolidating a biofilm on dental enamel, tooth root surfaces, and dental implants.^3,4 Periodontitis is one of the main causes of tooth loss in adults and is the primary reason for the need for dental implants in China.

Peri-implant pockets may yield up to 10⁷ different types of bacteria, which are in direct contact with the pocket epithelium. In oral inflammatory diseases, the bacteria Streptococcus mutans is considered as the principal microorganism associated with the beginning and development of inflammation due to its properties of metabolizing sucrose into lactic acid, synthesizing acids in a low pH environment, and producing extracellular and intracellular polysaccharides, which in turn promote further bacterial growth.^5,6 Bacterial adhesion on the surface of the teeth strongly improves bacteria–bacteria and bacteria–surface interconnection and consequently improves the adhesion and attachment of other microorganisms.⁷ Furthermore, bacteria and their products are able to alter osteoblast proliferation, thus resulting in implant failure.^8,9

Due to the complexity of the bacterial components, broad spectrum antibacterial action is important, and the usage of prophylactic antibiotics during implant placement remains indispensable. In the last few decades, researchers have focused their attention on silver nanoparticles (nano-Ag), which have shown broad spectrum antibacterial activity against various bacteria strains, including Streptococcus.¹⁰ Some mechanisms of this antibacterial action have been revealed, such as silver causing bacterial cell death by interfering with DNA replication, alteration of the bacterial peptides profile, increasing permeability, and interacting with thiol groups.¹¹ Despite such potential, widespread use of nano-Ag is limited, primarily because of unwanted side effects. Due to the runoff from the working point, dose escalation is usually adopted to prolong the action time of nano-Ag. In contrast to molecular agents, nano-Ag usually accumulates in the reticuloendothelial system (ie, liver and spleen), resulting in hepatic and renal injuries.¹² Additionally, a high nano-Ag concentration may not result in osteoblast proliferation, which may decrease the survival rate of dental implants.¹³ It is therefore important to enhance antibacterial activity and reduce bacterial adhesion ability without compromising biocompatibility.

Recently, the combination of noble metals and magnetic materials has attracted much attention,¹⁴ such as Ag-modified magnetic particles that achieved very high sensitivity for compounds dissolved in solutions using magnetic capture.¹⁵ Magnetic nanoparticles (Fe₃O₄) coupled with an active noble metal, such as silver (Ag-Fe₃O₄), result in a wide spectrum of desirable synergistic and complementary effects.¹⁶ Despite these promising effects, in the case of dental implants, the problem of biocompatibility is yet to be solved. Magnetite colloids are superparamagnetic and biocompatible,¹⁷ and nano-Ag is well known for its bactericide effect, potentially indicating a solution in regard to biosafety. Magnetic drug targeting employed nanoparticles as carriers, offering a promising treatment modality that avoids the side effects of conventional chemotherapy.¹⁴ The antibacterial activity of Ag-Fe₃O₄ against both Gram-positive and Gram-negative pathogens has been reported.¹⁸ In addition, paramagnetic implants can enhance the osteogenic response of pre-osteoblast cells.¹⁹

The healing of gums consists of an orderly series of events, such as osteoblast migration, proliferation, differentiation, and the formation of an extracellular matrix.²⁰ There is a considerable challenge to find an agent that improves both antibacterial activity and biocompatibility.²¹ Inspired by the unique properties of Ag-Fe₃O₄ nanoparticles, we demonstrated the feasibility of coating dental implants under an extracorporeal magnetic field with a reduced concentration of PLGA(Ag-Fe₃O₄) to improve biological compatibility without compromising antibacterial efficiency (Scheme 1). The permanent magnet was used to build the magnetic field as close to the PLGA(Ag-Fe₃O₄) as possible. PLGA(Ag-Fe₃O₄) was applied in vivo to the implanted tooth that contained a permanent magnet, allowing for Ag adhesion to the tooth surface without being removed by flushing water. Bacterial infection (such as by S. mutans) activates the host immune response to produce reactive oxygen species (ROS), leading to the destruction of the tooth supporting tissues (Scheme 1, left). In the implanted tooth coated with PLGA(Ag-Fe₃O₄), bacterial adhesion was weakened. Thus, the immune system did not produce ROS and the microenvironment around the implanted area stimulated osteoblast proliferation, thus improving the transplant success rate.

Materials and methods

Materials and animals

Silver nitrate (AgNO₃), ferric chloride (FeCl₃⋅6H₂O), ammonia water (NH₃⋅H₂O), and sodium hydroxide (NaOH) were purchased from Xilong Chemical Co, Ltd (Guangxi, China). PLGA (Resomer RG 502H) was purchased from Evonik, Essen, Germany. N-methyl pyrrolidone (NMP, 99%) was purchased from Acros Organics, Geel, Belgium. Trisodium citrate dehydrate (C₆H₅Na₃O₇⋅2H₂O) and ferrous chloride (FeCl₂⋅4H₂O) were purchased from Shanghai Richjoint Chemical Reagents Co, Ltd (Shanghai, China). Polyvinylpyrrolidone (PVP) was purchased from Shanghai Macklin Biochemical Co, Ltd (Shanghai, China). Agar was purchased from Sunshine Biotechnology Co, Ltd (Nanjing, China). All other reagents used were of analytical grade. Deionized water was used throughout the experiment.

Specific pathogen-free Sprague–Dawley (SD) rats of both genders (male, 200–215 g; female, 175–190 g) were obtained from the Experimental Animal Center of Nantong University, China. Five rats per cage were housed under controlled environmental conditions (22°C±3°C, 45%–70% relative humidity; 12 hour light/dark cycle). All animal experiments were approved by the Animal Care Committee of Nantong University (including guidelines for animal care and use, and guidelines for euthanasia of rats), which conducted the ethical review of the experiments.

Synthesis of PLGA-coated Ag-Fe₃O₄ nanoparticles

Ag-Fe₃O₄ nanoparticles were prepared by a hydrothermal technique.^22,23 In brief, FeCl₂⋅4H₂O (3.75 mmol) and PVP (20.00 mmol) were dissolved in 150 mL of deionized water. The mixed solution was stirred and heated to 90°C. NaOH solution (10 mL, 1 mol) was added to the mixture. After the mixed solution’s temperature reached 90°C, 20 mL of prepared AgNO₃ solution (2.5 mmol) and PVP solution (3.75 mmol) were added to the mixture. After stirring for 1 hour, the solution was cooled naturally and washed several times with deionized water and ethyl alcohol absolutely to remove all unreacted impurities.

PLGA (25% w/w, based on the total liquid formulation without drug) was dissolved in NMP at 25°C for 30 minutes under stirring in a glass vial. Ag-Fe₃O₄ was added and the mixture was vortexed for 3 minutes, followed by standing for 3 hours at 25°C.

Ag nanoparticles were prepared as described by Ma et al.²⁴ In brief, AgNO₃ and Na₃ citrate solutions were filtered through a 0.22-μm microporous membrane and nano-Ag was prepared by adding Na₃ citrate solution to boiling AgNO₃ aqueous solution.

Characterization

The particle size and distribution of the present samples were studied by transmission electron microscopy (TEM). Measurements were performed using a JEM-200 CX microscope (JEOL, Tokyo, Japan) operated at an accelerating voltage of 80 kV.

X-ray powder diffraction (XRD) measurements were carried out in a D8-ADVANCE (Bruker AXS Inc, Madison, WI, USA) X-ray diffractometer using Cu-Kα radiation.

Fourier transform infrared (FTIR) spectra of the samples were obtained from a Paragon 1000 spectrometer (PerkinElmer Inc, Waltham, MA, USA). The signal resolution of the FTIR was 1 cm⁻¹ and a minimum of 16 scans was obtained and averaged within the range of 400–4,000 cm⁻¹.

The magnetic measurement was recorded at room temperature (300 K) by using a vibrating sample magnetometer (LKSM-7410).

To investigate the surface morphology of the planted tooth modified with PLGA(Ag-Fe₃O₄), scanning electron microscopy (SEM) images were obtained on a JSM-6700F microscope (JEOL).²⁵

Antibacterial experiment

To evaluate the antibacterial activity against Streptococcus, the effect of nano-Ag, Ag-Fe₃O₄, and PLGA(Ag-Fe₃O₄) on the bacterial growth kinetics media was quantitatively studied.²⁶ The inoculation of bacteria was carried out by growing strains in sheep blood agar plate at 37°C until a level of ~10⁹ CFU/mL of bacteria was reached. Then, 0.5 mL of 10⁹ CFU/mL bacterial suspension was added to 50 mL lysogeny broth liquid medium-contained teeth modified with nano-Ag, Ag-Fe₃O₄, or PLGA(Ag-Fe₃O₄) with 20 μg/mL Ag and incubated at 37°C. After incubation for 24 hours, the antibacterial efficacy was determined by measuring the OD at 540 nm.

To investigate the stability of antibacterial activity, nano-Ag, Ag-Fe₃O₄, or PLGA(Ag-Fe₃O₄) with 20 μg/mL Ag was incubated in PBS under magnetic stirring (600 rpm); the antibacterial activity was studied after the PBS was changed 2, 4, 6, 8, 10 times.

Bacterial adhesion plate colony count

Approximately 2 mL of bacterial suspension (1×10⁹ CFU/L) was co-cultured with teeth modified with nano-Ag, Ag-Fe₃O₄, or PLGA(Ag-Fe₃O₄) with 20 μg/mL Ag for 48 hours. All the samples were then taken out sequentially from the bacterial suspension and rinsed thrice with sterile PBS buffer for 30 seconds to remove non-adherent bacteria. The adherent bacteria were detached ultrasonically from the samples in 10 mL of the same PBS buffer for 5 minutes. After the solution was diluted to a certain factor, 50 mL of washing solution was uniformly coated on the sheep blood agar plate. After the solution was cultured anaerobically for 24 hours, the bacterial colonies were counted.

SEM observation of the surface adhesion of bacteria

After being co-cultured with bacterial suspension and washed, the samples were fixed with a volume fraction of 2.5% glutaraldehyde (Sigma-Aldrich Co, St Louis, MO, USA) for 24 hours at 4°C. The samples were removed and rinsed with sterile PBS buffer thrice, and then dehydrated with 30%, 50%, 70%, 80%, 90%, 100%, and 100% (v/v) graded ethanol, successively, with 15 minutes incubation at each concentration. The samples were dried by a vacuum dryer at a critical point of CO₂ and sprayed with gold coating before the SEM observation.

Stability behavior

In vitro stability behavior of Ag-Fe₃O₄ was tested as follows: Ag-Fe₃O₄ was added to 10 mL PBS (pH 7.4) at 37°C at the concentration of 50 μg/mL. After 2 days, the PBS was placed in a magnetic field, and the concentration in the supernatant was tested. The steps were repeated three times.

The stability of PLGA(Ag-Fe₃O₄) modified on the tooth surface was tested as which was dipped in 20 mL of PBS (pH 7.4) at 37°C with or without the magnetic field; the concentration of Ag in the supernatant was tested for 28 days.

Osteoblasts proliferation assay

Freshly isolated mouse osteoblasts were seeded into 96- well plates at a density of 1×10⁴ cells/well in the presence of nano-Ag, Ag-Fe₃O₄, PLGA(Ag-Fe₃O₄), and PLGA(Ag-Fe₃O₄) + M (static magnetic field) (Ag 20 μg/mL). The viability after culture was measured at 24 hours by MTT assay.

ALP activity

For alkaline phosphatase (ALP) activity assessment, using 200 μL of radioimmunoprecipitation assay (RIPA) buffer, the total protein was extracted from osteoblasts cultured with Fe₃O₄ or PLGA(Ag-Fe₃O₄) at days 2, 4, and 6 of the differentiation period. For sedimentation of cell debris, the lysate was centrifuged at 15,000 rpm at 4°C for 15 minutes. After centrifugation, the supernatant was collected, and ALP activity was evaluated with an ALP assay kit (Bomei, Anhui, China). Finally, the enzyme activity (IU/L) was normalized against total protein (mg/dL).

Silver staining for nucleolar organizer region (AgNOR) staining

AgNOR histochemical staining was performed according to the literature.¹² In brief, after 4 days incubation, slides with osteoblasts were fixed in 95% ethanol for 50–60 minutes and then hydrated in distilled water. The silver staining was freshly prepared by dissolving 2% gelatin in 50% aqueous silver nitrate solution in a ratio of 1:2. Slides were incubated for 60 minutes at room temperature in the dark with this solution. After being stained, the slides were rinsed with distilled water, dehydrated with graded ethanol, cleared in xylene, and coverslipped. The software Image-pro plus 6.0 (Media Cybermetics, Rockville, MD, USA) was used for the analysis according to the manufacturer’s instructions.

Blood biochemistry index in vivo

Ten SD rats in each group were employed to evaluate the in vivo blood toxicology behavior. After the modification of nano-Ag, Ag-Fe₃O₄, and PLGA(Ag-Fe₃O₄) on the tooth (implanted with the permanent magnet) for 3 days, liver function markers, including ALP, alanine aminotransferase (ALT), aspartate aminotransferase (AST), the kidney function marker blood urea nitrogen (BUN), as well as creatinine (Cr) and globulin (GLB) were determined by an automated biochemical analyzer (Yoder, Nancy, France). The inflammatory cytokines (superoxide dismutase [SOD] and TNF-α) were analyzed by rat enzyme-linked immunosorbent assay kits.

Statistical analysis

The results were presented as mean ± standard deviation and analyzed using SPSS 13.0 (SPSS Inc, Chicago, IL, USA). The experimental data were examined for equal variance and normal distribution prior to statistical analysis. One-way analysis of variance was used for statistical analysis. Values were considered significantly different if P<0.05.

Results

Due to their reduced size, Fe₃O₄ nanoparticles behave as superparamagnets, ie, quasi-static magnetic measurements at room temperature that display the Langevin isotherm without hysteresis.²⁷ Ag nanoparticles are capable of killing bacteria by directly damaging the cytomembrane, and they are used in cancer therapy and medicine. However, nano-Ag runs off easily from the working point, thus working less effectively. Inspired by this, we demonstrated the safety and efficacy of a dental implant coating to inhibit bacterial proliferation and promote osteoblast proliferation by anchoring nano-Ag on the surface for a long duration.

PLGA(Ag-Fe₃O₄) nanocomposites preparation and characterization

In this study, Ag-Fe₃O₄ was prepared as follows: after the addition of NaOH to FeCl₂, Fe(OH)₂ was obtained. AgNO₃ also reacts with NaOH to form AgOH, and because AgOH is very unstable, it decomposed easily into Ag₂O. Then, the redox reaction between Ag₂O and Fe(OH)₂ produces Fe₃O₄ and Ag. Due to high surface energy, the Ag atoms aggregated rapidly, forming nanoparticles as a matrix of the nanocomposites. PVP adsorbed on the Ag nanoparticles surfaces prevented their further growth. In addition, some of the in situ generated Fe₃O₄ particles crystallized with Ag nanoparticles to form Ag-Fe₃O₄ nanocomposites resulting from their strong binding interaction, whereas others adhered to the surface of the nanocomposites. This outcome is in line with the results of previous studies.²² The reaction process is illustrated by Equations 1$3{\text{FeCl}}_2+6\text{NaOH}=3\text{Fe}{(\text{OH})}_2+6\text{NaCl}$(1) –3$3\text{Fe}{(\text{OH})}_2+{\text{Ag}}_2\mathrm{O}={\text{Fe}}_3{\mathrm{O}}_4+2\text{Ag}+3{\mathrm{H}}_2\mathrm{O}$(3) :

$3{\text{FeCl}}_2+6\text{NaOH}=3\text{Fe}{(\text{OH})}_2+6\text{NaCl}$(1)

$2{\text{AgNO}}_3+2\text{NaOH}=2{\text{Ag}}_2\mathrm{O}+2{\text{NaNO}}_3+{\mathrm{H}}_2\mathrm{O}$(2)

$3\text{Fe}{(\text{OH})}_2+{\text{Ag}}_2\mathrm{O}={\text{Fe}}_3{\mathrm{O}}_4+2\text{Ag}+3{\mathrm{H}}_2\mathrm{O}$(3)

TEM images of nano-Ag and Ag-Fe₃O₄ are shown in Figure 1A and B. The TEM images of the nano-Ag show its miniscule and regular shape. Ag-Fe₃O₄ morphology consisted of a single-crystal Ag core and polycrystallized Fe₃O₄ shell nanostructures that were amorphous (Figure 1B). Since Fe₃O₄ was bonded with nano-Ag, the size of the Ag-Fe₃O₄ particle was larger than either Fe₃O₄ or nano-Ag alone. Both Ag and Fe₃O₄ nanocrystallites formed composite nanoparticles instead of monocrystals, as demonstrated by the appearance of lattice fringes of the nanoparticles. The high-resolution TEM image (Figure 1C) shows that the nanoparticles comprised both Ag (~0.20 nm, which is consistent with Ag {200} planes of the fcc platinum structure) and Fe₃O₄ (~0.235 nm, which is consistent with Fe₃O₄ {400} planes of the fcc platinum structure) nanocrystallites, forming composite nanoparticles as demonstrated by lattice fringes of the nanoparticles. The PLGA can almost pack the Ag-Fe₃O₄ nanoparticles, and modify the surface of the implant (Figure 1D).

Figure 1 Characterization of PLGA(Ag-Fe₃O₄).

Notes: (A) TEM image of Ag nanoparticles. (B) TEM image of Ag-Fe₃O₄ nanoparticles. (C) HRTEM image. (D) SEM image of PLGA(Ag-Fe₃O₄) covered on the planted tooth. (E) XRD. (F) FTIR spectrum. (G) Room temperature magnetic hysteresis loops of Ag-Fe₃O₄. (H) Ag-Fe₃O₄ multiple release behavior response to the magnetic field. It meant that Ag was stably bonded with Fe₃O₄.

Abbreviations: PLGA, poly (D, L-lactic-co-glycolic acid); SEI, secondary electron image; TEM, transmission electron microscopy; M, static magnetic field; emu, electromagnetic unit; H, magnetic field strength; Oe, oersted; SEM, scanning electron microscopy; XRD, X-ray powder diffraction; FTIR, Fourier transform infrared; HRTEM, high-resolution transmission electron microscopy; WD, working distance (the distance from the objective lens to focus point).

The XRD patterns of Fe₃O₄ and Ag-Fe₃O₄ are shown in Figure 1E. The diffraction peaks located at 30°, 35.4°, 43°, 53.4°, 57°, and 62.6° correspond to the (220), (311), (400), (422), (511), and (440) planes in the Fe₃O₄ cubic lattice; these planes are associated with the spinel structure of magnetite phase (Fe₃O₄, reference JCPDS no. 82-1533). In addition, four peaks were found at 38°, 44°, 65°, and 77°, which represent Bragg reflections of Ag (111), (200), (220), and (311) reflection planes (JCPDS card no. 65-2871). These results are in good agreement with Fe₃O₄ XRD patterns reported in the literature.²⁸

To characterize the molecular nature of the material, FTIR spectra of the samples were observed (Figure 1F). The absorption bands at 3,342 and 1,646 cm⁻¹ belong to the vibrating peaks of a hydroxyl functional group on the surface of Fe₃O₄ and Ag-Fe₃O₄, indicating that the Ag-Fe₃O₄ composite particles have the same hydroxyl groups as Fe₃O₄. However, the characteristic absorption peak disappeared on the surface of Ag-Fe₃O₄ due to nano-Ag not being characterized by absorption in the infrared region.²⁹

To evaluate the magnetic response of Ag-Fe₃O₄ to an externally applied magnetic field, magnetization hysteresis curves were obtained by a superconducting quantum interference device (SQUID) magnetometer at 300 K by field cycling between −10 and 10 kOe, as shown in Figure 1G. The magnetic saturation value of Fe₃O₄ nanoparticles was 36.75 emu/g. As Ag connected with Fe₃O₄, a significant decrease in the saturation magnetization of Ag-Fe₃O₄ was observed; specifically, the magnetic saturation value was 23.36 emu/g. Although the composite sample exhibited weak ferromagnetism, there was neither remanence nor coercivity, indicating that the Ag-Fe₃O₄ sample obtained was superparamagnetic. The magnetic recyclability of Ag-Fe₃O₄ was tested in water by placing an external magnet near the glass bottle. The particles were attracted toward the magnet in a few seconds.

Furthermore, Ag-Fe₃O₄ nanoparticles showed long duration stability in vitro. When the Ag-Fe₃O₄ nanoparticles were placed in a magnetic field, the Ag concentration in the supernatant decreased quickly (Figure 1H). During the next 10 days, the phenomenon was repeated.

PLGA(Ag-Fe₃O₄) antibacterial activity in vitro

To investigate the antibacterial activity of PLGA(Ag-Fe₃O₄) in the mouth, S. mutans was chosen as the model bacterium. Bacterial growth kinetics were observed after administration of nano-Ag, Ag-Fe₃O₄, and PLGA(Ag-Fe₃O₄) at the Ag concentration of 20 μg/mL, and the results were determined by measuring the OD at 540 nm. PLGA(Ag-Fe₃O₄) nanoparticles were able to reduce S. mutans growth in a similar manner as nano-Ag. The antibacterial action of Ag-Fe₃O₄ was also similar to that of nano-Ag (Figure 2A).

Figure 2 (A) Antibacterial results. PLGA(Ag-Fe₃O₄) nanoparticles were able to reduce Streptococcus mutans growth as nano-Ag, Ag-Fe₃O₄. **P<0.01. (B) Antibacterial activity test after washing. PLGA(Ag-Fe₃O₄) under magnetic field can be anchored on the tooth surface and exhibited a long-term antibacterial effect. (C) Results of S. mutans adhered to the samples surface. (D) SEM image of S. mutans adhered to the surface of different samples.

Abbreviations: Abs, absorbance; PLGA, poly (D, L-lactic-co-glycolic acid); SEM, scanning electron microscopy; M, static magnetic field; SEI, secondary electron image; WD, working distance (the distance from the objective lens to focus point).

Anchoring the antibiotic material on the tooth surface is the key factor of the efficacy of PLGA(Ag-Fe₃O₄). Figure 2B shows that nano-Ag could not be adsorbed on the tooth surface after washing in the simulation test, while Ag-Fe₃O₄ improved the inhibition rate of S. mutans to 31% after being washed 10 times under the magnetic field. The results of viable bacteria adhering to the different experimental material surfaces are shown in Figure 2C. This indicated that Ag-Fe₃O₄ (Ag ion connects with Fe₃O₄) not only exhibited magnetically recyclable properties but also showed simultaneous antibacterial activity. After modification with PLGA, the inhibition rate increased to 60%, suggesting that PLGA(Ag-Fe₃O₄) remained stable and exhibited bacteriostatic abilities.

SEM observation of the surface adhering bacteria

SEM results of the adherence of S. mutans on the samples’ surface are shown in Figure 2D. A large number of S. mutans colonies adhered on the surface of the tooth covered with nano-Ag, as most of the nano-Ag was washed away. In contrast, the surface of PLGA(Ag-Fe₃O₄)-coated samples under the magnetic field showed very few S. mutans colonies.

Osteogenic proliferation test results

When the PLGA(Ag-Fe₃O₄) was modified on the tooth surface, the Ag nanoparticles were stably bonded with Fe₃O₄, and fixed on the tooth for >20 days in vitro (Figure 3A). Measurement of ALP activity was conducted to evaluate the ability of the PLGA(Ag-Fe₃O₄) scaffold to induce osteogenic differentiation (Figure 3B). An analogous increasing trend in ALP activity was observed among the groups. The activity of the enzyme increased as the duration of induction increased. In this regard, osteogenic cells cultured on with Fe₃O₄ and PLGA(Ag-Fe₃O₄) presented higher ALP activity at days 4 (P<0.05) and 6 (P<0.01), compared with PBS-treated groups.

Figure 3 (A) Storage stability test result of PLGA(Ag-Fe₃O₄) nanoparticles. (B) ALP activity of osteoblasts in PBS, Fe₃O₄, or PLGA(Ag-Fe₃O₄) nanoparticles after 2, 4, and 6 days of differentiation culture; *P<0.05. **P<0.01. (C) Viability of osteoblasts incubated with different concentrations of samples for 24 hours. n=6; **P<0.01. (D) AgNOR staining in nucleoli of osteoblasts cultured with different nanoparticles. Original magnification 1,000×.

Abbreviations: M, static magnetic field; PLGA, poly (D, L-lactic-co-glycolic acid); ALP, alkaline phosphatase; AgNOR, silver staining for nucleolar organizer region.

In Figure 3C, the L₉₂₉ cell line co-cultured with PLGA(Ag-Fe₃O₄) + M at different concentrations showed a high proliferation rate throughout the entire culture incubation time. Cell viability was significantly higher using 100 mg/L PLGA(Ag-Fe₃O₄) + M compared to the other groups (P<0.01).

After being stained by a silver impregnation method, the borders of nucleolar organizer regions and the nucleus were clearly visible. The nuclei were stained pale yellow, whereas AgNOR dots or aggregates produced dark staining. As illustrated in Figure 3D, keratinocytes co-cultured with Ag nanoparticles were smaller than the other groups, and fewer AgNOR dots or aggregates were observed. Most of the nuclei in the Ag-Fe₃O₄ group contained one or two AgNORs, while a large number of AgNOR proteins were observed in the PLGA(Ag-Fe₃O₄) and PLGA(Ag-Fe₃O₄) + M groups.

Biochemical detection results

The teeth were treated with the same dose of nano-Ag and PLGA(Ag-Fe₃O₄) + M (the magnetic field was generated by the permanent magnet in the implanted tooth). Liver function markers, including ALP, ALT, and AST, as well as Cr, GLB, and the kidney function marker BUN were also normal during the test procedure (Figure 4A), suggesting no clear hepatic and renal disorders in rats after PLGA(Ag-Fe₃O₄) + M treatment for 3 days. Alternatively, after the nano-Ag treatment, ALP, ALT, and Cr levels were significantly higher than those of the control group (P<0.05).

Figure 4 (A) Parameters of serum biochemistry profiles after administration of PLGA(Ag-Fe₃O₄) for 3 days. (B) The concentration of Ag in blood after treatment with nano-Ag and Ag-Fe₃O₄. Error bars represent standard error (n=5). (C) The degree of inflammation factors, including SOD and TNF-α in different samples-treated group. *P<0.05, **P<0.01.

Abbreviations: ALP, alkaline phosphatase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; ALT, alanine aminotransferase; Cr, creatinine; GLB, globulin; PLGA, poly (D, L-lactic-co-glycolic acid); SOD, superoxide dismutase; TNF-α, tumor necrosis factor-α.

Scheme 1 Schematic diagram of PLGA(Ag-Fe₃O₄)-coated on dental implants.

Notes: Bacterial infection (such as the one by Streptococcus mutans) activates host immune response to produce ROS, leading to the destruction of the tooth supporting tissues (left). In the planted tooth (center) covered with PLGA(Ag-Fe₃O₄), bacterial adhesion ability weakened (top right). Thus, the immune system did not produce ROS and the microenvironment around the planted area stimulated osteoblast proliferation and improved the transplant success rate (bottom right).

Abbreviations: PLGA, poly (D, L-lactic-co-glycolic acid); ROS, reactive oxygen species; N, magnetic north pole; S, magnetic south pole.

To probe into the causes for those differences, the change of the concentration of silver in the blood was detected. The silver ion passed into the bloodstream and reached the systemic circulation, causing some dose–response toxic side effects. Figure 4B shows that the Ag in the Ag-Fe₃O₄ + M and PLGA(Ag-Fe₃O₄) + M treated groups mainly accumulated in the blood. The silver concentration in the blood did not greatly increase in the PLGA(Ag-Fe₃O₄) + M-treated group; rather, it remained at a relatively low level.

TNF-α and SOD levels were also evaluated after the rats were treated with nano-Ag, Ag-Fe₃O₄, and PLGA(Ag-Fe₃O₄) + M (Figure 4C). Rats treated with PLGA(Ag-Fe₃O₄) + M for 3 days showed less inflammation as indicated by TNF-α and SOD levels that were significantly lower than those in the nano-Ag and Ag-Fe₃O₄ groups. These data suggest that PLGA(Ag-Fe₃O₄) + M is harmless in vivo.

Discussion

It is both a challenge and contradiction to improve biological compatibility without compromising antibacterial activity in a dental implant. Silver, as an excellent antibacterial agent, causes toxicity, including DNA and chromosome damage, due to its accumulation. In spite of its broad spectrum antibacterial activity against various strains of bacteria, widespread use of nano-Ag is still limited. At high concentrations, nanomaterials may compromise cell proliferation since they can easily enter into the cytoplasm and cross the nuclear membrane, implying that the particles can reach the nucleus.³⁰ Early research reported on the green synthesis of Ag nanoparticles using different biological sources such as plant extracts,^31,32 bacteria,³³ fungi,³⁴ and enzymes.³⁵ Nevertheless, the present study is the first to reduce the concentration of nano-Ag on the implanted tooth to improve biosafety without a reduction in antibacterial activity.

A wide spectrum of desirable synergistic and complementary effects can be obtained when the superparamagnetism nanoparticles (Fe₃O₄) are coupled with silver.^23,36 However, the biosafety and efficacy of adherence inhibition involve problems that have yet been solved. In an early study, Prucek et al³⁷ considered the combination of magnetite nanoparticles and nano silver for use in disinfection and biomedical applications, such as the targeted transport of drugs, as well as for its removal by an external magnetic field. Amarjargal et al²³ reported an enhanced magnetic moment and strong catalytic and antibacterial activities of the Ag-Fe₃O₄ nanocomposite, demonstrating its potential application in water treatment and biomedical applications, with the ability to be subsequently removed by means of an external magnetic field.

The polyester PLGA, which has been approved by Food and Drug Administration for many applications, is biocompatible and biodegradable by hydrolysis of its ester linkages under physiological conditions and is excreted from the body as carbon dioxide and water via the Krebs cycle.³⁸ In this study, we developed a coated PLGA(Ag-Fe₃O₄)-implanted tooth obtained under an extracorporeal magnetic field that used a modest concentration of this nanoparticle. TEM, XRD, and FTIR results showed that Ag was connected to the surface of the Fe₃O₄ particle. Ag-Fe₃O₄ nanoparticles were well dispersed and did not agglomerate easily. Results for several analyses of broad spectrum antibacterial activity of the nano-Ag revealed that various bacterial strains died upon contact with the silver iron. The antibacterial effect of Ag-Fe₃O₄ could thus be attributed to the silver ions. In addition, Ag-Fe₃O₄ showed good magnetic properties through its magnetic saturation value that was less than that of Fe₃O₄. Ag-Fe₃O₄ with antibacterial activity and good magnetic properties might be used in biomedicine. In this study, since a permanent magnet was implanted in the implant tooth, few Ag-Fe₃O₄ nanoparticles were washed away in the magnetic field.

Controlling bacterial burden is currently one of the most important problems in the implanted gum preparation.³⁹ In clinical practice, antibacterial agents are used in high dose to prevent infection, leading to low biosafety. Thus, “antibacterial” and “biosafety” seem to represent an irreconcilable contradiction. Bacterial infections such as those due to S. mutans lead to the host immune response to produce ROS, consequently leading to the destruction of the tooth supporting tissues. One of the purposes of this study was to control inflammation and promote osteoblast proliferation.

PLGA(Ag-Fe₃O₄) was adopted to improve biological compatibility without compromising bacteriostatic efficiency. When the nano-Ag is firmly bonded with Fe₃O₄, it can significantly increase the concentration of the drug in the treatment site and hence decrease the drug dosage. As Ag-Fe₃O₄ was applied to the implanted tooth, the use of a magnetic field anchored the Ag to the tooth surface, making it resistant to flushing water. When the implanted tooth was covered with PLGA(Ag-Fe₃O₄), the bacterial adhesion ability weakened. Thus, the immune system did not produce ROS, and the microenvironment of the implanted area stimulated osteoblast proliferation, which increased the success rate of the dental implant.

According to our results, nano-Ag was stably anchored onto the surface of the tooth in the PLGA(Ag-Fe₃O₄) + M- treated group. The antibacterial effect of PLGA(Ag-Fe₃O₄) + M was significantly higher than that of the nano-Ag group after washing six times. Further, less S. mutans adhesion was observed on the tooth surface, indicating that anchoring the silver on the tooth surface is an effective method to prevent bacterial infections.

Acute and chronic nano-Ag exposure causes remarkable damages in various organs, such as the liver and kidney.⁴⁰ Since liver injury occurs due to the cumulative nano-Ag effect, and a dose–response relationship exists, effective protection from such damage can be obtained by reducing the concentration used. Despite using the same Ag concentration, the Ag level on the tooth surface in PLGA(Ag-Fe₃O₄) + M was significantly higher than that of the nano-Ag. Alternatively, the Ag level in the blood and liver was higher with nano-Ag than with PLGA(Ag-Fe₃O₄) + M. Further, in this study, the concentration of silver iron in the liver and kidney of the PLGA(Ag-Fe₃O₄) + M group was significantly lower than the concentration in the nano-Ag group. The serum biochemical parameter results showed that PLGA(Ag-Fe₃O₄) + M resulted in no damage to the liver. SOD and TNF-α results showed that nano-Ag caused remarkably more liver and kidney damage than PLGA(Ag-Fe₃O₄) + M. Hence, the present results showed that PLGA(Ag-Fe₃O₄) + M exhibited magnetic properties without losing antimicrobial properties, leading to minimal side effects.

Conclusion

This work analyzed the ability of nano-Ag to improve biological compatibility without compromising antibacterial activity. PLGA(Ag-Fe₃O₄) + M showed the best combination in terms of magnetic properties, antibacterial ability, and osteoblast-promoted proliferation and thus might represent another important and promising application. Therefore, this work provides experimental evidence for further clinical research to improve antibacterial action, reducing the unwanted side effects and improving antibacterial activity.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (nos. 51472115 and 81570952), the Scientific Research Foundation of Graduate School of Nanjing University (no. 2016CL13), the Jiangsu Province Natural Science Foundation of China (nos. BK 20170143, BK20161114), and the Medical Science and Technology Development Foundation, Nanjing Department of Health (nos. ZKX16054, ZKX13050). Major Project supported by Medical Science and Technology Development Foundation, Nanjing Department of Health (no. ZDX16008).

Disclosure

The authors report no conflicts of interest in this work.