Long-term antibacterial activity of silver-containing hydroxyapatite coatings against Staphylococcus aureus in vitro and invivo.

BACKGROUND: The potential of silver-containing hydroxyapatite (Ag-HA) coatings to prevent orthopaedic implant-associated infection was explored previously; however, the resistance of Ag-HA coatings to late-onset orthopaedic infections is unknown. This study aimed to evaluate the long-term Ag+ elution and antibacterial properties of the Ag-HA coatings through in vitro and in vivo experiments. METHODS: Ag-HA-coated disc specimens were immersed in fetal bovine serum (FBS) for six months. Ag concentration was measured over time using inductively coupled plasma-mass spectrometry to evaluate Ag release. The hydroxyapatite (HA)- or Ag-HA-coated disc specimens were immersed in FBS for 3 months to elute Ag+ for in vitro experiments. Methicillin-resistant Staphylococcus aureus (MRSA) suspensions were inoculated onto each disc; after 48 h, the number of colonies and the biofilm volume were measured. HA- or Ag-HA-coated disc specimens were inserted under the skin of Sprague-Dawley rats for three months for in vivo experiments. In in vivo experiment 1, specimens were inoculated with MRSA and the number of colonies was counted after 48 h. In in vivo experiment 2, the specimens were inoculated with bioluminescent S. aureus Xen36 cells, and bioluminescence was measured using an in vivo imaging system. RESULTS: The Ag-HA-coated disc specimens continued to elute Ag+ after six months. The biofilm volume in the Ag-HA group was lower than in the HA group. In in vitro and in vivo experiment 1, the bacterial counts in the Ag-HA group were lower than those in the HA group. In in vivo experiment 2, the bioluminescence in the Ag-HA group was lower than that in the HA group on days 1-7 after inoculation. CONCLUSIONS: The Ag-HA-coated discs continued to elute Ag+ for a long period and exhibited antibacterial activity and inhibition of biofilm formation against S. aureus. The Ag-HA coatings have the potential to reduce late-onset orthopaedic implant-associated infections.

Time-dependent efficacy of combination of silver-containing hydroxyapatite coating and vancomycin on methicillin-resistant Staphylococcus aureus biofilm formation in vitro.

OBJECTIVE: We developed a silver-containing hydroxyapatite (Ag-HA) coating to prevent periprosthetic joint infection (PJI). Methicillin-resistant Staphylococcus aureus (MRSA) is the main PJI-causing bacteria. Previously, we had reported the combined effect of Ag-HA coating and vancomycin (VCM) on MRSA biofilm formation 24 h after MRSA inoculation. In this study, we investigated the time-dependent efficacy of Ag-HA coating and VCM on MRSA biofilm formation on Ti discs in vitro by three-dimensional confocal laser scanning microscopic analysis. RESULTS: For the Ti VCM and HA VCM groups, the total biofilm volumes per area at 96 h after MRSA inoculation were significantly larger than those at 48 h after MRSA inoculation, respectively (p < 0.001). In contrast, for the Ag-HA VCM group, the total biofilm volume per area at 96 h was significantly smaller than that at 48 h (p < 0.0001). Moreover, 96 h after MRSA inoculation, the total biofilm volume per area of the Ag-HA VCM groups was significantly smaller than those of the Ti VCM and HA VCM groups (p < 0.0001). Thus, the combination of Ag-HA and VCM might be useful for the prevention of MRSA-associated PJI.

Effect of bacterial media on the evaluation of the antibacterial activity of a biomaterial containing inorganic antibacterial reagents or antibiotics.

Several studies have been performed to assess the effectivesness of the antibacterial coating of a biomaterial to reduce surgical site infection. However, evaluations of these materials are inconsistent, and therefore it is difficult to compare their antibacterial performance. In this study, we evaluated the influence of different media such as nutrient broth (NB), Mueller-Hinton broth (MHB) and fetal bovine serum (FBS) on the antibacterial activity of AgNO3- or gentamicin-added bone cement using a modified ISO 22196 standard to devise a method to evaluate the antibacterial activity of biomaterials in vitro. The antibacterial activity results against Staphylococcus aureus and Escherichia coil were different in each medium. The antibacterial activity of AgNO3 in FBS was lower than the other media, whereas the antibacterial activity of gentamicin in FBS was higher than in the other media. It was assumed that the fluctuating antibacterial activity was influenced by serum components. The results showed that the ISO 22196 antibacterial evaluation method is suitable to evaluate antibacterial biomaterials after modifying the medium to FBS.

The combination of silver-containing hydroxyapatite coating and vancomycin has a synergistic antibacterial effect on methicillin-resistant Staphylococcus aureus biofilm formation.

AIMS: Biofilm formation is intrinsic to prosthetic joint infection (PJI). In the current study, we evaluated the effects of silver-containing hydroxyapatite (Ag-HA) coating and vancomycin (VCM) on methicillin-resistant Staphylococcus aureus (MRSA) biofilm formation. METHODS: Pure titanium discs (Ti discs), Ti discs coated with HA (HA discs), and 3% Ag-HA discs developed using a thermal spraying were inoculated with MRSA suspensions containing a mean in vitro 4.3 (SD 0.8) x 106 or 43.0 (SD 8.4) x 105 colony-forming units (CFUs). Immediately after MRSA inoculation, sterile phosphate-buffered saline or VCM (20 µg/ml) was added, and the discs were incubated for 24 hours at 37°C. Viable cell counting, 3D confocal laser scanning microscopy with Airyscan, and scanning electron microscopy were then performed. HA discs and Ag HA discs were implanted subcutaneously in vivo in the dorsum of rats, and MRSA suspensions containing a mean in vivo 7.2 (SD 0.4) x 106 or 72.0 (SD 4.2) x 105 CFUs were inoculated on the discs. VCM was injected subcutaneously daily every 12 hours followed by viable cell counting. RESULTS: Biofilms that formed on HA discs were thicker and larger than those on Ti discs, whereas those on Ag-HA discs were thinner and smaller than those on Ti discs. Viable bacterial counts in vivo revealed that Ag-HA combined with VCM was the most effective treatment. CONCLUSION: Ag-HA with VCM has a potential synergistic effect in reducing MRSA biofilm formation and can thus be useful for preventing and treating PJI.Cite this article: Bone Joint Res. 2020;9(5):211-218.

Antibacterial Activity of Ag-Hydroxyapatite Coating Against Hematogenous Infection by Methicillin-Resistant Staphylococcus aureus in the Rat Femur.

Several antibacterial materials have been developed to prevent periprosthetic joint infection and thus prevent serious complications for patients and surgeons. However, no study has addressed the activity of antibacterial materials against hematogenous infection. The present study evaluated the antibacterial activity of a silver-containing hydroxyapatite-coated implant against methicillin-resistant Staphylococcus aureus (MRSA) hematogenous infection. Implants coated with hydroxyapatite and silver-hydroxyapatite were inserted into rats' right and left femurs, respectively, after which the animals were infected with S. aureus via a tail vessel. About 107 colony-forming units was the optimal bacterial number for the establishment of S. aureus hematogenous infection. Bacterial loads and C-reactive protein in the blood were measured to confirm bacteremia and inflammation. Fourteen days after the infection, bacterial loads were statistically lower in the femurs containing silver-hydroxyapatite-coated implants than in those with hydroxyapatite-coated implants (p = 0.022). Thus, silver-hydroxyapatite-coated implants might provide antibacterial activity against MRSA hematogenous infection in the postoperative period. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2655-2660, 2019.

Ability of zirconia double coated with titanium and hydroxyapatite to bond to bone under load-bearing conditions.

As a preclinical study, we evaluated the ability of hydroxyapatite and titanium on zirconia (HTOZ) to bond to bone under load-bearing conditions in animal experiments. HTOZ, HA, and Ti on Co-Cr alloy (HTOC) and Ti on Co-Cr alloy (TOC) were implanted into the weight-bearing portion of the femoral condyles of nine beagle dogs. Femurs were extracted 4, 12, and 52 weeks after implantation and examined mechanically by pullout testing, and histologically by toluidine blue staining, SEM, and calculation of the affinity index. The interfacial shear strengths (mean+/-SD) of the HTOZ, HTOC, and TOC groups were 4.42+/-0.453, 3.90+/-0.903, and 4.08+/-0.790 MPa at 4 weeks; 6.82+/-2.64, 6.00+/-1.88, and 6.63+/-1.63 MPa at 12 weeks; and 13.98+/-1.94, 11.95+/-1.51, and 10.78+/-0.83 MPa at 52 weeks. There were no significant differences in the interfacial shear strengths between the three groups at any time. Affinity indices (mean+/-SD) obtained from SEM images of the HTOZ, HTOC, and TOC groups were 49.6+/-6.52%, 43.3+/-10.43%, and 23.7+/-3.95% at 4 weeks; 55.0+/-6.72%, 51.5+/-3.07%, and 28.6+/-4.09% at 12 weeks; and 59.1+/-6.73%, 63.0+/-6.40%, and 34.3+/-6.72% at 52 weeks. HA-coated implants (HTOZ, HTOC) had significantly higher affinity indices than non-HA-coated implants (TOC) at all times. HTOZ has the ability to bond to bone equivalent to HTOC and TOC. HTOZ is an excellent material for components of cementless joint prostheses.

Silver-Containing Hydroxyapatite Coating Reduces Biofilm Formation by Methicillin-Resistant Staphylococcus aureus In Vitro and In Vivo.

Biofilm-producing bacteria are the principal causes of infections associated with orthopaedic implants. We previously reported that silver-containing hydroxyapatite (Ag-HA) coatings exhibit high antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). In the present study, we evaluated the effects of Ag-HA coating of implant surfaces on biofilm formation. Titanium disks (14-mm diameter, 1-mm thickness), one surface of which was coated with HA or 0.5%-3.0% Ag-HA with a thermal spraying technique, were used. In vitro, the disks were inoculated with an MRSA suspension containing 4 × 105 CFU and incubated for 1-2 weeks. In vivo, MRSA-inoculated HA and 3% Ag-HA disks (8.8-10.0 × 108 CFU) were implanted subcutaneously on the back of rats for 1-7 days. All disks were subsequently stained with a biofilm dye and observed under a fluorescence microscope, and biofilm coverage rates (BCRs) were calculated. The BCRs on the Ag-HA coating were significantly lower than those on the HA coating at all time points in vitro (p < 0.05). Similar results were observed in vivo (p < 0.001) without argyria. Ag-HA coating reduced biofilm formation by MRSA in vitro and in vivo; therefore, Ag-HA coating might be effective for reducing implant-associated infections.

Silver oxide-containing hydroxyapatite coating supports osteoblast function and enhances implant anchorage strength in rat femur.

Antibacterial silver with hydroxyapatite (Ag-HA) is a promising coating material for imparting antibacterial properties to implants. We previously reported that 3% (w/w) silver with HA (3% Ag-HA) has both antibacterial activity and osteoconductivity. In this study, we investigated the effects of Ag-HA on the in vitro osteoblast function and the in vivo anchorage strength and osteoconductivity of implants. Production of the osteoblast marker alkaline phosphatase, but not cytotoxicity, was observed in cells of the osteoblast cell line MC3T3-E1 cultured on the 3% Ag-HA-coated surface. These results were similar to those observed with silver-free HA coating. In contrast, a significant high level of cytotoxicity was observed when the cells were cultured on a 50% Ag-HA-coated surface. The anchorage strength of implants inserted into the femur of Sprague-Dawley (SD) rats was enhanced by coating the implants with 3% Ag-HA. On the 3% Ag-HA-coated surface, both metaphyseal and diaphyseal areas were largely covered with new bone and had adequate osteoconductivity. These results suggest that 3% Ag-HA, like conventional HA, promotes osteogenesis by supporting osteoblast viability and function and thereby contributes to sufficient anchorage strength of implants. Application of 3% Ag-HA, which combines the osteoconductivity of HA and the antibacterial activity of silver, to prosthetic joints will help prevent postoperative infections.

Acute and subacute toxicity in vivo of thermal-sprayed silver containing hydroxyapatite coating in rat tibia.

To reduce the incidence of implant-associated infection, we previously developed a novel coating technology using hydroxyapatite (HA) containing silver (Ag). This study examined in vivo acute and subacute toxicity associated with the Ag-HA coating in rat tibiae. Ten-week-old rats received implantation of HA-, 2% Ag-HA-, or 50% Ag-HA-coated titanium rods. Concentrations of silver in serum, brain, liver, kidneys, and spleen were measured in the acute phase (2-4 days after treatment) and subacute phase (4-12 weeks after treatment). Biochemical and histological examinations of those organs were also performed. Mean serum silver concentration peaked in the acute phase and then gradually decreased. Mean silver concentrations in all examined organs from the 2% Ag-HA coating groups showed no significant differences compared with the HA coating group. No significant differences in mean levels of glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, lactate dehydrogenase, creatinine, or blood urea nitrogen were seen between the three groups and controls. Histological examinations of all organs revealed no abnormal pathologic findings. No acute or subacute toxicity was seen in vivo for 2% Ag-HA coating or HA coating. Ag-HA coatings on implants may represent biologically safe antibacterial biomaterials and may be of value for reducing surgical-site infections related to implantation.

Silver oxide-containing hydroxyapatite coating has in vivo antibacterial activity in the rat tibia.

Bacterial infection is a serious postoperative complication of joint replacement. To prevent infections related to implantation, we have developed a novel antibacterial coating with Ag-containing hydroxyapatite (Ag-HA). In the present study, we examined the antibacterial activity of Ag-HA implant coatings in the medullary cavity of rat tibiae. Forty 10-week-old rats received implantation of Ag-HA- or HA-coated titanium rods, then were inoculated with ∼1.0 × 10(2) colony-forming units of methicillin-resistant Staphylococcus aureus. Bacterial counts were calculated for rats euthanized at 24, 48, and 72 h postoperatively. Serum levels of Ag (in the Ag-HA group only) were calculated for rats euthanized at 24, 48, 72 h and 4 weeks. Radiographic evaluations of bone infection were also performed at 4 weeks. Tibiae from both groups showing infection were evaluated histologically. Significant differences in bacterial counts were seen at 24, 48, and 72 h. Mean concentrations of Ag in serum peaked about 48 h after implantation, then gradually decreased. Mean radiographic scores for infection were significantly lower with Ag-HA implants than with HA implants. Histological examination showed better results for abscesses, bone resorption, and destruction of cortical bone around Ag-HA-coated implants. These results indicate that Ag-HA coatings may help prevent surgical-site infections associated with joint replacement.

In vivo antibacterial and silver-releasing properties of novel thermal sprayed silver-containing hydroxyapatite coating.

One of the serious postoperative complications associated with joint replacement is bacterial infection. In addressing this problem, we have previously described the development of a novel thermal spraying technology combining silver (Ag) showing antibacterial activity with hydroxyapatite (HA) displaying good biocompatibility and osteoconductivity, and reported the in vitro properties. This study evaluated serum Ag ion concentrations and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) using a subcutaneous rat model. HA loaded with 3 wt % of silver oxide (Ag-HA) and plain HA were sprayed on the surface of titanium disks. Ag-HA- or HA-coated samples were implanted into the back subcutaneous pockets of male Sprague-Dawley rats. Mean serum Ag ion concentration in the Ag-HA group increased to more than 50 ppb by 48 h after implantation, then decreased gradually to baseline levels. Mean (+/- standard error of the mean) number of viable MRSA on HA coating was (1.5 +/- 0.5) x 10(5), which is significantly more than the (1.1 +/- 0.4) x 10(4) on Ag-HA coating (p < 0.001). Ag-HA coating offers good abilities to release Ag ions and kill MRSA in vivo.

Antibacterial properties of nanostructured silver titanate thin films formed on a titanium plate.

Antibacterial activity of various surfaces against methicillin-resistant Staphylococcus aureus (MRSA) was studied. Sodium titanate thin film with a porous network structure and sodium titanate nanotube thin film were formed on titanium surfaces through the reaction of titanium plates with NaOH solutions. Through a silver ion-exchange treatment, Na(+) ions in sodium titanate were exchanged with Ag(+) ions in silver acetate solution, along with the loading of silver nanoparticles on the titanate surfaces. Results of silver ion elution tests of the thin films in fetal bovine serum solution indicate that the release period and the number of silver ions released from the silver titanate thin films can be controlled by altering the crystal structure, nanostructure, and thickness of the titanate phase. The silver ion-exchanged titanate thin films showed high antibacterial activity against MRSA. It was also revealed that although the crystal structure of titanate itself has no large antibacterial effect, higher antibacterial activity mainly arises from the silver ions held in the interlayer spacing of the titanate. The obtained results should aid the development of more convenient and inexpensive antibacterial implants.

Apatite-forming ability of titanium compound nanotube thin films formed on a titanium metal plate in a simulated body fluid.

We compared the apatite-forming ability of a sodium titanate nanotube thin film, an anatase-type titanium dioxide nanotube thin film, and a silver nanoparticle/silver titanate nanotube nanocomposite thin film, in simulated body fluid. The ability of the silver nanoparticle/silver titanate nanotube nanocomposite thin film is slightly higher than that of the anatase-type titanium dioxide nanotube thin film and significantly higher than that of the sodium titanate nanotube thin film. The high ability of the silver nanoparticle/silver titanate nanotube nanocomposite thin film is a newly observed phenomenon, which is probably due to the crystal structure of silver titanate--specifically, to the surface atomic arrangement, the large amount of Ti-OH formed on the nanotube surface, or both. The anatase-type titanium dioxide nanotube thin film and the silver nanoparticle/silver titanate nanotube nanocomposite thin film may have bright prospects for future use in implant materials such as artificial joints. The silver nanoparticle/silver titanate nanotube nanocomposite thin film is particularly promising for its antibacterial properties.

Development of novel thermal sprayed antibacterial coating and evaluation of release properties of silver ions.

Several studies have addressed the use of antibacterial coating to reduce implant-associated infections. In this study, novel silver (Ag)-containing calcium-phosphate (CP) coating technology based on the thermal spraying method was developed. The coating's physical and chemical properties, in vitro antibacterial activity, hydroxyapatite (HA)-forming ability, and release of Ag ions were evaluated. An amorphous structure of the coating was confirmed by X-ray diffraction, and Ag residue in the coating was determined by elementary analysis. The coating showed strong antibacterial activity to methicillin-resistant Staphylococcus aureus in fetal bovine serum (FBS) along with HA-forming ability in simulated body fluid. Therefore, it is expected that the coating would confer antibacterial and bone bonding abilities to the implant surface. Time course release testing of Ag ions from the coating on immersion in FBS showed pronounced Ag release for up to 24 h after immersion, with consistent strong antibacterial activity at the early postoperative stage. In repeated testing, the amount of released Ag ions was about 6500 parts per billion (ppb, microg/L) for the first release test, after which it gradually decreased. However, retention of significant release of Ag ions after a sixth repeat implies that Ag release from the coating is slow in FBS.

Plate, wire, mesh, microsphere, and microtube composed of sodium titanate nanotubes on a titanium metal template.

Sodium titanate nanotube/titanium metal composites were synthesized by hydrothermal treatment of titanium metals with various morphologies such as plate, wire, mesh, microsphere, and microtube at 160 degrees C in aqueous NaOH solution and by the subsequent fixation treatment by calcination at 300 degrees C. The surface of the composite was covered with sodium titanate nanotubes with a diameter of approximately 7 nm, and the core part of the composite was titanium metal phase. The raw titanium metal acts as a template or a morphology-directing agent of micrometer size or more to arrange the nanotubes as well as a titanium source for the formation of nanotubes. The concentration of titanium species increases in the reaction solution as the dissolution of titanium metal is accelerated by the reaction between titanium and OH-. Furthermore, with an increase in concentration of titanium species in the reaction solution, the titanium species are re-precipitated as sodium titanate nanotubes onto the titanium metal. Titanium metal with a large surface area and volume can form sodium titanate nanotubes on the surface of the titanium metal, though titanium metal with a small volume and surface area tends to dissolve with the hydrothermal treatment. Even in the synthesis using titanium metal with a small volume and surface area, sodium titanate nanotubes are formed and cover the surface of the titanium metal by adding another titanium metal as a source of titanium species in the reaction solution.