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.

Leukemia/lymphoma-related factor (LRF) or osteoclast zinc finger protein (OCZF) overexpression promotes osteoclast survival by increasing Bcl-xl mRNA: A novel regulatory mechanism mediated by the RNA binding protein SAM68.

RANKL induces NFATc1, a key transcriptional factor to induce osteoclast-specific genes such as cathepsin K, whereas transcriptional control of osteoclast survival is not fully understood. Leukemia/lymphoma-related factor (LRF) in mouse and osteoclast zinc finger protein (OCZF) in rat are zinc finger and BTB domain-containing protein (zBTB) family of transcriptional regulators, and are critical regulators of hematopoiesis. We have previously shown that differentiation and survival were enhanced in osteoclasts from OCZF-Transgenic (Tg) mice. In the present study, we show a possible mechanism of osteoclast survival regulated by LRF/OCZF and the role of OCZF overexpression in pathological bone loss. In the in vitro cultures, LRF was highly colocalized with NFATc1 in cells of early stage in osteoclastogenesis, but only LRF expression persisted after differentiation into mature osteoclasts. LRF expression was further enhanced in resorbing osteoclasts formed on dentin slices. Osteoclast survival inhibitor such as alendronate, a bisphosphonate reduced LRF expression. Micro CT evaluation revealed that femurs of OCZF-Tg mice showed significantly lower bone volume compared to that of WT mice. Furthermore, OCZF overexpression markedly promoted bone loss in ovariectomy-induced osteolytic mouse model. The expression of anti-apoptotic Bcl-xl mRNA, which is formed by alternative splicing, was enhanced in the cultures in which osteoclasts are formed from OCZF-Tg mice. In contrast, the expression of pro-apoptotic Bcl-xs mRNA was lost in the culture derived from OCZF-Tg mice. We found that the expression levels of RNA binding splicing regulator, Src substrate associated in mitosis of 68 kDa (Sam68) protein were markedly decreased in OCZF-Tg mice-derived osteoclasts. In addition, shRNA-mediated knockdown of Sam68 expression increased the expression of Bcl-xl mRNA, suggesting that SAM68 regulates the expression of Bcl-xl. These results indicate that OCZF overexpression reduces protein levels of Sam68, thereby promotes osteoclast survival, and suggest that LRF/OCZF is a promising target for regulating pathological bone loss.

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.

2-Methacryloyloxyethyl Phosphorylcholine Polymer Coating Inhibits Bacterial Adhesion and Biofilm Formation on a Suture: An In Vitro and In Vivo Study.

Initial bacterial adhesion to medical devices and subsequent biofilm formation are known as the leading causes of surgical site infection (SSI). Therefore, inhibition of bacterial adhesion and biofilm formation on the surface of medical devices can reduce the risk of SSIs. In this study, a highly hydrophilic, antibiofouling surface was prepared by coating the bioabsorbable suture surface with poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate) (PMB). The PMB-coated and noncoated sutures exhibited similar mechanical strength and surface morphology. The effectiveness of the PMB coating on the suture to suppress adhesion and biofilm formation of methicillin-resistant Staphylococcus aureus and methicillin-susceptible Staphylococcus aureus was investigated both in vitro and in vivo. The bacterial adhesion test revealed that PMB coating significantly reduced the number of adherent bacteria, with no difference in the number of planktonic bacteria. Moreover, fluorescence microscopy and scanning electron microscopy observations of adherent bacteria on the suture surface after contact with bacterial suspension confirmed PMB coating-mediated inhibition of biofilm formation. Additionally, we found that the PMB-coated sutures exhibited significant antibiofouling effects in vivo. In conclusion, PMB-coated sutures demonstrated bacteriostatic effects associated with a highly hydrophilic, antibiofouling surface and inhibited bacterial adhesion and biofilm formation. Therefore, PMB-coated sutures could be a new alternative to reduce the risk of SSIs.

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.

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.

Prevention of bacterial adhesion and biofilm formation on a vitamin E-blended, cross-linked polyethylene surface with a poly(2-methacryloyloxyethyl phosphorylcholine) layer.

In the construction of artificial hip joint replacements, the surface and substrate of a cross-linked polyethylene (CLPE) liner are designed to achieve high wear resistance and prevent infection by bacteria. In this study, we fabricated a highly hydrophilic and antibiofouling poly(2-methacryloyloxyethyl phosphorylcholine [MPC]) (PMPC)-graft layer on the vitamin E-blended CLPE (HD-CLPE(VE)) surface. The 100-nm-thick, smooth, and electrically neutral PMPC layer was successfully fabricated on the HD-CLPE(VE) surface using photoinduced graft polymerization. The PMPC-grafted HD-CLPE(VE) was found to prevent bacterial adherence and biofilm formation on the surface because of the formation of a highly hydrophilic polyzwitterionic layer on the surface of HD-CLPE(VE), which can serve as an extremely efficient antibiofouling layer. The number of bacterial adhered on the PMPC-grafted HD-CLPE(VE) surface was reduced by 100-fold or more by PMPC grafting, regardless of the biofilm-production characteristics of the strains. In contrast, vitamin E blending did not affect bacterial adhesion. Moreover, the number of planktonic bacteria did not differ significantly, regardless of PMPC grafting and vitamin E blending. In conclusion, the PMPC-grafted HD-CLPE(VE) provided bacteriostatic effects associated with smooth, highly hydrophilic surfaces with a neutral electrostatic charge owing to the zwitterionic structure of the MPC unit. Thus, this modification may prove useful for the production of artificial hip joint replacement materials. STATEMENT OF SIGNIFICANCE: Our preliminary in vitro findings suggest that improved bacteriostatic performance of the HD-CLPE(VE) surface in orthopedic implants is possible via PMPC grafting. The results also indicate that surface modifications affect the anti-infection properties of the orthopedic implants and demonstrate that the application of a PMPC-grafted HD-CLPE(VE) surface may be a promising approach to extend the longevity and clinical outcomes of total hip arthroplasty. Further research is needed to evaluate the resistance to infection of PMPC-grafted HD-CLPE(VE) in terms of the varieties of biofilm formation tests including fluid flow conditions and animal experiments, which may offer useful clues to the possible performance of these materials in vivo.

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.

The human apoptosis inhibitor NAIP induces pyroptosis in macrophages infected with Legionella pneumophila.

Human nucleotide oligomerization domain-like receptor family apoptosis inhibitory protein (NAIP) prevents apoptosis by inhibiting caspase-3, -7, and -9. Four functional Naip exist in the murine genome, each of which is equally similar to human NAIP. Among them, Naip5 induces pyroptosis by promoting caspase-1 activation in response to Legionella pneumophila infection in macrophages. However, the contribution of human NAIP to this response is unclear. To investigate the role of human NAIP in macrophage survival, we stably expressed human NAIP in RAW264.7 macrophages. Human NAIP inhibited camptothecin-induced apoptosis in macrophages; however, it promoted cytotoxicity in L. pneumophila-infected cells. This cytotoxicity was associated with caspase-1. In addition, human NAIP restricted the intracellular growth of L. pneumophila. L. pneumophila flagellin was required for cytotoxicity, caspase-1 activation, and restriction of intracellular bacterial growth. Expression of murine Naip5 produced comparable results. These data indicate that human NAIP regulates the host response to L. pneumophila infection in a manner similar to that of murine Naip5 and that human NAIP and murine Naip5 regulate cell survival by inhibiting apoptosis or by promoting pyroptosis in response to specific cellular signals.

The transcription factor FBI-1/OCZF/LRF is expressed in osteoclasts and regulates RANKL-induced osteoclast formation in vitro and in vivo.

OBJECTIVE: Since transcription factors expressed in osteoclasts are possible targets for regulation of bone destruction in bone disorders, we investigated the expression of the transcription factor FBI-1/OCZF/LRF (in humans, factor that binds to inducer of short transcripts of human immunodeficiency virus type 1; in rats, osteoclast-derived zinc finger; in mice, leukemia/lymphoma-related factor) in patients with rheumatoid arthritis (RA), and assessed its role in osteoclastogenesis in vivo. METHODS: Expression of FBI-1/OCZF was investigated in subchondral osteoclasts in human RA and in rat adjuvant-induced arthritis (AIA) using immunostaining and in situ hybridization, respectively. Transgenic mice overexpressing OCZF (OCZF-Tg) under the control of the cathepsin K promoter were generated, and bone mineral density and bone histomorphometric features were determined by peripheral quantitative computed tomography, calcein double-labeling, and specific staining for osteoclasts and osteoblasts. LRF/OCZF expression and the consequence of LRF inhibition were assessed in vitro with RANKL-induced osteoclast differentiation. RESULTS: FBI-1/OCZF was detected in the nuclei of osteoclasts in rat AIA and human RA. RANKL increased the levels of LRF messenger RNA and nuclear-localized LRF protein in primary macrophages. In OCZF-Tg mice, bone volume was significantly decreased, the number of osteoclasts, but not osteoblasts, was increased in long bones, and osteoclast survival was promoted. Conversely, inhibition of LRF expression suppressed the formation of osteoclasts from macrophages in vitro. CONCLUSION: FBI-1/OCZF/LRF regulates osteoclast formation and apoptosis in vivo, and may become a useful marker and target in treating disorders leading to reduced bone density, including chronic arthritis.

Development of a novel PCR method to comprehensively analyze salivary bacterial flora and its application to patients with odontogenic infections.

OBJECTIVE: The objective of this study was to develop a novel polymerase chain reaction (PCR) method to comprehensively analyze salivary bacterial flora. STUDY DESIGN: The bacterial flora in the saliva of 10 healthy persons and 11 patients with odontogenic infections were examined using a DNA extraction method with a high level of cell destruction efficiency and a novel universal primer set to amplify approximately 580 bp of the 16S rDNA sequence. RESULTS: Streptococcus (54.5%), Neisseria (14.7%), Actinomyces (8.4%), Gemella (4.1%), Granulicatella (3.8%), and Prevotella (1.4%) were dominant in a total of 1655 clones examined from the saliva of the healthy subjects. The dominant genera differed among the patients with odontogenic infections (a total of 823 clones) and were entirely different from those of the healthy subjects. CONCLUSION: This novel comprehensive salivary bacterial flora analysis method may be a useful supportive method to identify causative agents of odontogenic infections.

Inhibition of histone deacetylase suppresses osteoclastogenesis and bone destruction by inducing IFN-beta production.

Osteoclasts are bone-resorptive multinucleated cells that are differentiated from hemopoietic cell lineages of monocyte/macrophages in the presence of receptor activator of NF-kappaB ligand (RANKL) and M-CSF. Downstream signaling molecules of the receptor of RANKL, RANK, modulate the differentiation and the activation of osteoclasts. We recently found that histone deacetylase inhibitors (HDIs), known as anticancer agents, selectively suppressed osteoclastogenesis in vitro. However, the molecular mechanism underlying inhibitory action of HDIs in osteoclastogenesis and the effect of HDIs on pathological bone destruction are still not remained to be elucidated. In this study, we show that a depsipeptide, FR901228, inhibited osteoclast differentiation by not only suppressing RANKL-induced nuclear translocation of NFATc1 but also increasing the mRNA level of IFN-beta, an inhibitor of osteoclastogenesis. The inhibition of osteoclast formation by FR901228 was abrogated by the addition of IFN-beta-neutralizing Ab. In addition, treatment of adjuvant-induced arthritis in rats revealed that FR901228 inhibited not only disease development in a prophylactic model but also bone destruction in a therapeutic model. Furthermore, immunostaining of the joints of therapeutically treated rats revealed significant production of IFN-beta in synovial cells. Taken together, these data suggest that a HDI inhibits osteoclastogenesis and bone destruction by a novel action to induce the expression of osteoclast inhibitory protein, IFN-beta.

Two histone deacetylase inhibitors, trichostatin A and sodium butyrate, suppress differentiation into osteoclasts but not into macrophages.

Histone deacetylase (HDAC) inhibitors are emerging as a new class of anticancer therapeutic agents and have been demonstrated to induce differentiation in some myeloid leukemia cell lines. In this study, we show that HDAC inhibitors have a novel action on osteoclast differentiation. The effect of 2 HDAC inhibitors, trichostatin A (TSA) and sodium butyrate (NaB), on osteoclastogenesis was investigated using rat and mouse bone marrow cultures and a murine macrophage cell line RAW264. Both TSA and NaB inhibited the formation of preosteoclast-like cells (POCs) and multinucleated osteoclast-like cells (MNCs) in rat bone marrow culture. By reverse transcription-polymerase chain reaction analysis, TSA reduced osteoclast-specific mRNA expression of cathepsin K and calcitonin receptor (CTR). In contrast, TSA and NaB did not affect the formation of bone marrow macrophages (BMMs) induced by macrophage colony-stimulating factor as examined by nonspecific esterase staining. Fluorescence-activated cell sorting analysis showed that TSA did not affect the surface expression of macrophage markers for CD11b and F4/80 of BMMs. TSA and NaB also inhibited osteoclast formation and osteoclast-specific mRNA expression in RAW264 cells stimulated with receptor activator of nuclear factor-kappa B (NF-kappa B) ligand (RANKL). Transient transfection assay revealed that TSA and NaB dose dependently reduced the sRANKL-stimulated or tumor necrosis factor alpha (TNF-alpha)-stimulated transactivation of NF-kappa B-dependent reporter genes. The treatment of RAW264 cells with TSA and NaB inhibited TNF-alpha-induced nuclear translocation of NF-kappa B and sRANKL-induced activation of p38 mitogen-activated protein kinase (MAPK) signals. These data suggest that both TSA and NaB exert their inhibitory effects by modulating osteoclast-specific signals and that HDAC activity regulates the process of osteoclastogenesis.