SPA inhibits hBMSC osteogenic differentiation and M1 macrophage polarization by suppressing SETD2 in acute suppurative osteomyelitis.

To clarify the impact of SETD2 on macrophage function in pediatric patients with acute suppurative osteomyelitis and to elucidate the precise underlying mechanism. To gain insights into the potential functions of SETD2, a comprehensive study was conducted utilizing a co-culture model of human bone mesenchymal stem cells (hBMSCs) and bone marrow-derived macrophages (THP-1). A range of techniques were employed, including quantitative polymerase chain reaction, western blotting, ELISA, alkaline phosphatase activity assays, alizarin red S staining, luciferase reporter gene assays, and chromatin immunoprecipitation, to unravel the intricate interactions and molecular mechanisms involving SETD2 in this system. It was observed that SETD2 expression was reduced in THP-1 cells stimulated by staphylococcal protein A (SPA). Furthermore, the downregulation of SETD2 resulted in elevated M1 macrophage polarization and glycolysis, effects that were mitigated by SPA stimulation. Notably, SPA-stimulated THP-1 cells exhibited an increase in HIF-1α expression, which exhibited an inverse correlation with SETD2 levels. Moreover, it was discovered that SETD2 functioned as a catalyst for H3K36me3 and bound to the HIF-1α gene, which, in turn, regulated HIF-1α expression. Furthermore, the suppression of HIF-1α abrogated the consequences of SETD2 downregulation on glycolysis and M1 macrophage polarization. Lastly, the study demonstrated that M1 macrophage polarization serves as a mediator for BMP4's inhibitory effect on osteogenic differentiation of hBMSCs. This research has uncovered a previously unknown role of SETD2 in macrophages during osteomyelitis, revealing its significance in the pathogenesis of this condition. These findings suggest SETD2 as a novel target for the treatment of osteomyelitis.

Nicotine promotes Staphylococcus aureus-induced osteomyelitis by activating the Nrf2/Slc7a11 signaling axis.

Although smoking is a significant risk factor for osteomyelitis, there is limited experimental evidence that nicotine, a key tobacco constituent, is associated with this condition, leaving its mechanistic implications uncharacterized. This study revealed that nicotine promotes Staphylococcus aureus-induced osteomyelitis by increasing Nrf2 and Slc7a11 expression in vivo and in vitro. Inhibition of Slc7a11 using Erastin augmented bacterial phagocytosis/killing capabilities and fortified antimicrobial responses in an osteomyelitis model. Moreover, untargeted metabolomic analysis demonstrated that Erastin mitigated the effects of nicotine on S. aureus-induced osteomyelitis by altering glutamate/glutathione metabolism. These findings suggest that nicotine aggravates S. aureus-induced osteomyelitis by activating the Nrf2/Slc7a11 signaling pathway and that Slc7a11 inhibition can counteract the detrimental health effects of nicotine.

Aberrant Expression of SLC7A11 Impairs the Antimicrobial Activities of Macrophages in Staphylococcus Aureus Osteomyelitis in Mice.

Staphylococcus aureus (S. aureus) persistence in macrophages, potentially a reservoir for recurrence of chronic osteomyelitis, contributes to resistance and failure in treatment. As the mechanisms underlying survival of S. aureus in macrophages remain largely unknown, there has been no treatment approved. Here, in a mouse model of S. aureus osteomyelitis, we identified significantly up-regulated expression of SLC7A11 in both transcriptomes and translatomes of CD11b+F4/80+ macrophages, and validated a predominant distribution of SLC7A11 in F4/80+ cells around the S. aureus abscess. Importantly, pharmacological inhibition or genetic knockout of SLC7A11 promoted the bactericidal function of macrophages, reduced bacterial burden in the bone and improved bone structure in mice with S. aureus osteomyelitis. Mechanistically, aberrantly expressed SLC7A11 down-regulated the level of intracellular ROS and reduced lipid peroxidation, contributing to the impaired bactericidal function of macrophages. Interestingly, blocking SLC7A11 further activated expression of PD-L1 via the ROS-NF-κB axis, and a combination therapy of targeting both SLC7A11 and PD-L1 significantly enhanced the efficacy of clearing S. aureus in vitro and in vivo. Our findings suggest that targeting both SLC7A11 and PD-L1 is a promising therapeutic approach to reprogram the bactericidal function of macrophages and promote bacterial clearance in S. aureus osteomyelitis.

PD-1/PD-L1 blockade is a potent adjuvant in treatment of Staphylococcus aureus osteomyelitis in mice.

There is no effective therapy for implant-associated Staphylococcus aureus osteomyelitis, a devastating complication after orthopedic surgery. An immune-suppressive profile with up-regulated programmed cell death 1/programmed death ligand 1 (PD-1/PD-L1) was identified based on our transcriptional data (GEO: GSE166522) from a mouse model of S. aureus osteomyelitis. PD-1/PD-L1 expression was up-regulated mainly in F4/80+ macrophages surrounding the abscess in S. aureus-infected bone. Mechanistically, PD-1/PD-L1 activated mitophagy to suppress production of mitochondrial reactive oxygen species (ROS), suppressing the bactericidal function of macrophages. Using neutralizing antibodies for PD-L1 or PD-1, or knockout of PD-L1 adjuvant to gentamicin markedly reduced mitophagy in bone marrow F4/80+ cells, enhanced bacterial clearance in bone tissue and implants, and reduced bone destruction in mice. PD-1/PD-L1 expression was also increased in the bone marrow from individuals with S. aureus osteomyelitis. These findings uncover a so far unknown function of PD-1/PD-L1-mediated mitophagy in suppressing the bactericidal function of bone marrow macrophages.

Long non-coding RNA KCNQ1OT1 overexpression promotes osteogenic differentiation of staphylococcus aureus-infected human bone mesenchymal stem cells by sponging microRNA miR-29b-3p.

Osteomyelitis (OM) is an orthopedic disease caused by bone infections in the bone cortex, bone marrow, periosteum, and surrounding soft tissues. Recent studies have implicated non-coding RNAs (ncRNAs) in the development of OM. However, little is known about the role of ncRNAs in the osteogenic differentiation during bone infection. In the present study, we investigated the role of KCNQ1OT1/miR-29b-3p axis in osteogenic differentiation in staphylococcus aureus (SpA)-infected human bone mesenchymal stem cells (hBMSCs). We first examined the expression of lncRNA KCNQ1OT1 and miR-29b-3p in the serum samples of OM patients and healthy controls. We also infected hBMSCs with different concentrations of SpA and studied the osteogenic differentiation after infection. Our results revealed that KCNQ1OT1 was downregulated while miR-29b-3p was upregulated in the serum samples of OM patients, as well as in SpA-infected hBMSCs. Overexpression of KCNQ1OT1 ameliorated the damage in hBMSCs caused by SpA infection. KCNQ1OT1 could support hBMSCs osteogenic differentiation by enhancing ALP activity, alizarin red S accumulation, expressions of osteogenic markers, and attenuating inflammatory responses after SpA infection. We further showed that miR-29b-3p was a downstream target of KCNQ1OT1, mediating the osteogenic differentiation of hBMSCs during SpA infection. Our data suggest that KCNQ1OT1 could ameliorate the SpA-induced suppression of osteogenic differentiation in hBMSCs by sponging miR-29b-3p. Modulating KCNQ1OT1 expression may serve as a strategy to ameliorate osteomyelitis.

Combination of kaempferol and azithromycin attenuates Staphylococcus aureus-induced osteomyelitis via anti-biofilm effects and by inhibiting the phosphorylation of ERK1/2 and SAPK.

Osteomyelitis is bacterial infection of bone, commonly caused by Staphylococcus aureus. This work aims to study the potential of azithromycin and kaempferol against chronic osteomyelitis induced by azithromycin-resistant Staphylococcus aureus (ARSA). It was noticed that rats tolerated the treatments with no diarrhoea or weight loss; also, no deaths were observed in rats. The treatment by azithromycin alone failed to inhibit bacterial growth and also had no effect on the infection condition of bone, although the treatment decreased the levels of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α), but did not improve the oxidative stress levels. Kaempferol monotherapy slightly inhibited bacterial growth and bone infection; the treatment also inhibited the levels of IL-6 and (TNF-α). The treatment also improved the antioxidant status. However, the combined treatment of azithromycin and kaempferol significantly suppressed bacterial growth and bone infection and modulated oxidative stress. In vitro, the combined treatment inhibited the levels of IL-6 and TNF-α, and also suppressed the phosphorylation of ERK1/2 and stress-activated protein kinase (SAPK). The combined treatment also showed anti-biofilm activity in ARSA. The combination attenuates ARSA-induced osteomyelitis in rats compared with their treatments alone by reducing oxidative stress, inhibiting the phosphorylation of ERK1/2 and SAPK and inhibiting biofilm formation.

Staphylococcus aureus internalization impairs osteoblastic activity and early differentiation process.

Staphylococcus aureus is the most frequent aetiology of bone and joint infections (BJI) and can cause relapsing and chronic infections. One of the main factors involved in the chronicization of staphylococcal BJIs is the internalization of S. aureus into osteoblasts, the bone-forming cells. Previous studies have shown that S. aureus triggers an impairment of osteoblasts function that could contribute to bone loss. However, these studies focused mainly on the extracellular effects of S. aureus. Our study aimed at understanding the intracellular effects of S. aureus on the early osteoblast differentiation process. In our in vitro model of osteoblast lineage infection, we first observed that internalized S. aureus 8325-4 (a reference lab strain) significantly impacted RUNX2 and COL1A1 expression compared to its non-internalized counterpart 8325-4∆fnbAB (with deletion of fnbA and fnbB). Then, in a murine model of osteomyelitis, we reported no significant effect for S. aureus 8325-4 and 8325-4∆fnbAB on bone parameters at 7 days post-infection whereas S. aureus 8325-4 significantly decreased trabecular bone thickness at 14 days post-infection compared to 8325-4∆fnbAB. When challenged with two clinical isogenic strains isolated from initial and relapse phase of the same BJI, significant impairments of bone parameters were observed for both initial and relapse strain, without differences between the two strains. Finally, in our in vitro osteoblast infection model, both clinical strains impacted alkaline phosphatase activity whereas the expression of bone differentiation genes was significantly decreased only after infection with the relapse strain. Globally, we highlighted that S. aureus internalization into osteoblasts is responsible for an impairment of the early differentiation in vitro and that S. aureus impaired bone parameters in vivo in a strain-dependent manner.

Deficiency of Lipin2 Results in Enhanced NF-κB Signaling and Osteoclast Formation in RAW-D Murine Macrophages.

Lipin2 is a phosphatidate phosphatase that plays critical roles in fat homeostasis. Alterations in Lpin2, which encodes lipin2, cause the autoinflammatory bone disorder Majeed syndrome. Lipin2 limits lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. However, little is known about the precise molecular mechanisms underlying its anti-inflammatory function. In this study, we attempted to elucidate the molecular link between the loss of lipin2 function and autoinflammatory bone disorder. Using a Lpin2 knockout murine macrophage cell line, we showed that lipin2 deficiency enhances innate immune responses to LPS stimulation through excessive activation of the NF-κB signaling pathway, partly because of TAK1 signaling upregulation. Lipin2 depletion also enhanced RANKL-mediated osteoclastogenesis and osteoclastic resorption activity accompanied by NFATc1 dephosphorylation and increased nuclear accumulation. These results suggest that lipin2 suppresses the development of autoinflammatory bone disorder by fine-tuning proinflammatory responses and osteoclastogenesis in macrophages. Therefore, this study provides insights into the molecular pathogenesis of monogenic autoinflammatory bone disorders and presents a potential therapeutic intervention.

Local treatment of experimental mandibular osteomyelitis with an injectable biomimetic gentamicin hydrogel using a new rabbit model.

Mandibular osteomyelitis (OM) is a challenging disease. Our objective was to assess a new OM model in rabbits induced by arsenic trioxide and to assess the efficacy of local treatment of OM using injectable gentamicin-collagen hydrogels (GNT-COLL). OM was induced unilaterally by controlled confinement of arsenic trioxide paste to the root canal of lower incisors of rabbits, while OM progression was characterized for 16 weeks. On the other hand, two injectable COLL hydrogels functionalized with GNT were prepared and characterized for physicochemical properties; a simple GNT-COLL and a nanohydroxyapatite (nHA)- loaded hydrogel (GNT-COLL/nHA). The two hydrogels were evaluated to treat OM model, while a multidose intramuscular GNT solution served as positive control. Outcomes were assessed by standard methods at 4 and 12 weeks post-surgery. The clinical, radiographical, and histopathological findings provided evidence for the validity of the arsenic-induced OM. The results demonstrated that a single intra-lesional injection of the two hydrogels was more suppressive to OM compared to multidose systemic GNT. The composite GNT-COLL/nHA hydrogel proved to induce early preservation of alveolar bone (ridge) length and higher amount of bone area\total area at 4 weeks (40.53% ± 2.34) followed by GNT-COLL (32.21% ± 0.72). On the other hand, the positive control group revealed the least ridge length and bone area\total area (26.22% ± 1.32) at 4 weeks. Both hydrogels successfully arrested OM with no signs of recurrence for up to 12 weeks. Therefore, results support the greater advantages of the composite hydrogel as an osteogenic/antibiotic delivery system in OM treatment.

The multi-kinase inhibitor dasatinib suppresses autoinflammation and increases bone density in a mouse model for chronic recurrent multifocal osteomyelitis.

Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disease that presents with bone destruction and pain. Although genetic studies have identified signalling pathways involving CRMO, molecularly targeted drugs remain unavailable. We used an animal model of CRMO as an in vivo screening system for candidate therapeutic agents. A gain-of-function mutation in Fgr, a member of Src family kinases (SFKs), causes peripheral paw inflammation and reduced bone mineral density (BMD) in Ali18 mice. The SFK inhibitor dasatinib was selected for administration to Ali18 mice daily for 2 weeks. Local inflammation and BMD were assessed by clinical scoring and computed tomography, respectively. Pilot studies in a small number of animals showed that dasatinib administration effectively suppressed the early phase of autoinflammation in Ali18 mice. Serial oral gavage of dasatinib to a group of Ali18 mice confirmed significant suppression of paw swelling with no side effects. Histological analysis revealed that abnormal proliferative bone marrow cells and inflammatory infiltration into the skin in the affected area were clearly reduced in the animals with dasatinib administration. Further, trabecular BMD in Ali18 long bones was restored to levels similar to that found in wild type mice. Our results indicate that autoinflammation and related-bone phenotypes were completely suppressed by the dasatinib kinase inhibitor in CRMO model animals. Thus, it is strongly suggested that dasatinib can be used for clinical treatments of CRMO with the combination of molecular diagnosis of the FGR locus. SIGNIFICANCE OF THE STUDY: Autoinflammation and related-bone phenotypes were effectively suppressed by the kinase inhibitor dasatinib in CRMO model animals. In combination with molecular analysis of the FGR locus, dasatinib is a strong candidate for the clinical treatments of CRMO. We propose that the animal model employed in this study can be used to screen this and other potential drugs for CRMO.

Tumor necrosis factor-α promotes Staphylococcus aureus-induced osteomyelitis through downregulating endothelial nitric oxide synthase.

BACKGROUND: Infections of Staphylococcus aureus (S. aureus) often result in osteomyelitis, which is the acute or chronic infections of the bone marrow or bones. TNF-α is long recognized as a key factor contributing to the pathogenesis of osteomyelitis, but little is known about the underlying molecular mechanism. METHODS: Expression levels of TNF-α, and several candidate genes, including endothelial nitric oxide synthase (eNOS), known to be downregulated by TNF-α were analysed in MC3T3-E1 cells with S. aureus infection and osteomyelitis patient blood. MicroRNA(miR)-129-5p was predicted and experimentally verified to target eNOS. Alizarin red sulfate (ARS) and alkaline phosphatase (ALP) staining assays were conducted on MC3T3-E1 cells with S. aureus infection to assess the role of TNF-α/miR-129-5p/eNOS on mineralization defect. RESULTS: TNF-α and miR-129-5p were upregulated while eNOS was downregulated in MC3T3-E1 cells with S. aureus infection and osteomyelitis patients, showing inversely correlated expression profiles. MiR-129-5p directly binds to the 3'-UTR of eNOS mRNA to suppress eNOS expression in MC3T3-E1 cells. TNF-α blocker inhibited miR-129-5p and elevated eNOS expression, likely contributing to rescued mineralization defect in S. aureus-infected MC3T3-E1 cells. During S. aureus infection, upregulated TNF-α increases endogenous miR-129-5p expression, which in turn inhibits eNOS, contributing to osteomyelitis. CONCLUSION: Our study thereby proposes a novel signalling cascade involving TNF-α/miR-129-5p/eNOS in the pathogenesis of osteomyelitis, which may also serve as therapeutic targets.

A case of bone destruction caused by chronic non-bacterial osteomyelitis (CNO) successfully repaired with a tumour necrosis factor-α (TNF-α) inhibitor, adalimumab.

Chronic Non-bacterial Osteomyelitis (CNO) is an autoinflammatory bone disorder that causes non-bacterial and non-neoplastic osteomyelitis. CNO appeared to the long bone, clavicle, pelvis, and spine on children commonly. This time, we report a case with osteomyelitis of the mandible for the adult-onset. A 25-year-old woman presented pustulosis palmaris/pustular psoriasis after the extraction of the lower right tooth 1 year before hospitalisation. She felt pain and swelling of the right jaw and an antibiotic, NSAIDs, and glucocorticoids were ineffective. The cortical osteotomy of right mandibular bone was carried out 2 months before hospitalisation, but the symptom was not improved and she was admitted to our hospital. For pustular psoriasis with CNO, we treated her with adalimumab and the pain and swelling in her right jaw disappeared immediately. One and two years after the treatment, osteolytic and sclerotic bone lesion and osteomyelitis were improved in both Computed Tomography (CT) and Magnetic Resonance Imaging (MRI). An anti-TNF-α antibody may be an effective therapy for CNO resistant to conventional treatment.

Receptor Activator of Nuclear Factor kappa-B Ligand is Not Regulated During Chronic Osteomyelitis in Pigs.

Bone loss is a major complication of osteomyelitis and from numerous in-vitro studies, it has been concluded that the bone lysis is caused by elevated expression of the receptor activator of nuclear factor κB ligand (RANKL), leading to increased osteoclast activity. However, we failed to find any relationship between bone loss and osseous RANKL expression in a porcine model of acute and chronic implant-associated osteomyelitis (IAO) due to Staphylococcus aureus or in chronic osteomyelitis lesions in slaughter pigs. Surprisingly, we found that the expression of RANKL was reduced during chronic bone infections. This is in line with the few studies conducted on human samples. A significant bone loss was observed in IAO lesions and in lesions from slaughter pigs, but with no indication of osteoclast involvement using histochemistry, immunohistochemistry for RANKL, receptor activator of nuclear factor kappa-B, osteoprotegerin and cathepsin K, and high-throughput quantitative real-time polymerase chain reaction on bone tissue from osteomyelitic lesions. A strong inflammatory response was seen in the infected animals and, therefore, we propose proteolytic enzymes induced by inflammation to be a major component of the bone loss. Furthermore, we found a significant upregulation of the IL26 gene in infected animals, which can inhibit RANKL-induced osteoclastogenesis, but has no homologue in mice. This finding emphasises that neither murine models nor in-vitro studies can mirror human disease development completely. The present study emphasises that the interactions between microorganisms, the immune system and bone cells in osteomyelitis are too complex to be accurately represented by an in-vitro model.

[Comparative assessment of mineral metabolism in shoulders and crura in patients with osteomyelitis.] / Сравнительная оценка минерального обмена у пациентов с остеомиелитом плеча и голени.

Chronic osteomyelitis in long tubular bones is generally aggravated by metabolic imbalance in patients' organisms affecting mineral metabolism. It is critically important that the extent of this imbalance should be determined prior to surgical intervention to choose optimal methodology, proper monitoring of recovery and adequate prognostication of final results. With this in mind a comparative assessment has been carried out to shed light on the intensity of mineral metabolism in the blood serum of patients suffering from osteomyelitis in their shoulders and crura. Dynamics of how indices of electrolytes and activity of acidic and alkaline phosphatases vary have been studied in blood serum samples in 24 patients, 18 patients suffering from osteomyelitis in crus bones and 6 - in shoulders. In addition, correlations between total calcium, inorganic phosphate and phosphatase index have been computed. The mineral metabolism indices which were exhibited by the patients having shoulder osteomyelitis and by the patients with crus osteomyelitis are statistically meaningful differences prior to surgery and 2-3 days after surgery. 21 days after surgical intervention the activity of osseous remodeling enzymes, content of total calcium and phosphates as well as the correlations with the phosphatase index show no significant differences. These specific features of mineral metabolism turn out to level out in the process of treating chronic osteomyelitis in upper and lower limbs with transosseous compression-distraction osteosynthes. It has been the first study aimed at discovering comparative characteristics of mineral metabolism in patients with shoulder osteomyelitis and the ones with crus osteomyelitis at different times following surgical intervention. The study looks into correlations between the electrolytes and the mineralization index and shows specific traits exhibited by patients who suffering from osteomyelitis in shoulder and crus went through reparative regeneration at different periods of treatment.

Hydrogen sulfide promotes lipopolysaccharide-induced apoptosis of osteoblasts by inhibiting the AKT/NF-κB signaling pathway.

Apoptosis of osteoblasts plays a crucial role in osteomyelitis. Hydrogen sulfide (H2S) levels are increased in the pathophysiological processes of osteomyelitis. However, the effect of H2S on the apoptosis of osteoblasts remains unclear. To investigate the specific role of H2S in osteoblast apoptosis, MC3T3-E1 and hFOB cells were treated with NaHS or Na2S, a donor of H2S, and lipopolysaccharide (LPS), during osteomyelitis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, flow cytometry analysis, western blotting, immunofluorescence, polymerase chain reaction, and Alizarin red staining were performed to examine the effects of H2S on osteoblast cell apoptosis, cell osteogenic differentiation, and AKT kinase (AKT)/nuclear factor kappa B (NF-κB) signaling. Hydrogen sulfide increased cell apoptosis, and inhibited the proliferation and osteogenic differentiation of osteoblast cells impaired by LPS. H2S increased apoptosis through upregulation of the FAS ligand (FASL) signaling pathway. H2S-induced apoptosis was alleviated using a FAS/FASL signaling pathway inhibitor. Treatment with NaHS also increased cell apoptosis by downregulating AKT/NF-κB signaling. In addition, treatment with an AKT signaling pathway activator decreased apoptosis and reversed the inhibitory effects of H2S on osteogenic differentiation. Hydrogen sulfide promotes LPS-induced apoptosis of osteoblast cells by inhibiting AKT/NF-κB signaling.

Two-staged time-dependent materials for the prevention of implant-related infections.

Infection is a main cause of implant failure. Early implant-related infections often occur in the first 4 weeks post-operation. Inhibiting bacterial adhesion and biofilm formation at the early stage and promoting subsequent implant osseointegration are important for implant success. Our previous studies demonstrated that dimethylaminododecyl methacrylate (DMADDM) provided dental materials with antibacterial effects. In the present study, DMADDM and hydroxyapatite (HA) are loaded on to the titanium (Ti) surface via poly dopamine (PDA) self-polymerization. This local DMADDM-delivery Ti is referred as Ti-PHD. Here we report the two-staged capability of Ti-PHD: (1) in the first stage, releasing DMADDM during the high-infection-risk initial period post-implantation for 4 weeks; (2) then in the second stage, enhancing osteogenesis and promoting osseointegration. Ti-PHD has a porous surface with higher average roughness and greater hydrophilicity than pure Ti. Its biocompatibility is verified in vitro and in vivo. During the first 4 weeks of release, both DMADDM remaining on Ti surface and DMADDM released into the soaking medium greatly reduced the adherence and growth of pathogens. This is further confirmed by the prevention of bone destruction in a rat osteomyelitis model. After releasing DMADDM for 4 weeks, Ti-PHD promotes osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and new bone formation around the implants in vivo. This article represents the first report on the two-staged, time-dependent antibacterial and osteogenesis effects of Ti-PHD, demonstrating its potential for clinical applications to inhibit implant-associated infections. STATEMENT OF SIGNIFICANCE: The present study develops a two-staged time-dependent system for local dimethylaminododecyl methacrylate (DMADDM) delivery via Ti implant (referred to as Ti-PHD). DMADDM and hydroxyapatite (HA) are loaded on to the Ti surface with poly dopamine (PDA). Ti-PHD can release DMADDM during the high-risk period of infection in the first stage, and then promote osseointegration and new bone formation in the second stage. This bioactive and therapeutic Ti is promising to inhibit infections and enhance implant success.

Virulence of methicillin-resistant Staphylococcus aureus modulated by the YycFG two-component pathway in a rat model of osteomyelitis.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) strains present an urgent medical problem in osteomyelitis cases. Our previous study indicated that the YycFG two-component regulatory pathway is associated with the bacterial biofilm organization of MRSA strains. The aim of this study was to investigate the regulatory roles of ASyycG in the bacterial biofilm formation and the pathogenicity of MRSA strains using an antisense RNA strategy. METHODS: An ASyycG-overexpressing MRSA clinical isolate was constructed. The bacterial growth was monitored, and the biofilm biomass on bone specimens was examined using scanning electron microscopy and confocal laser scanning microscopy. Furthermore, quantitative RT-PCR (QRT-PCR) analysis was used to measure the expression of yycF/G/H and icaA/D in the MRSA and ASyycG strains. The expression of the YycG protein was quantified by Western blot assays. We validated the role of ASyycG in the invasive ability and pathogenicity of the strains in vivo using histology and peptide nucleic acid fluorescent in situ hybridization. RESULTS: The results showed that overexpression of ASyycG lead to a reduction in biofilm formation and exopolysaccharide (EPS) synthesis compared to the control MRSA strains. The ASyycG strains exhibited decreased expression of the yycF/G/H and icaA/D genes. Furthermore, Western blot data showed that the production of the YycG protein was inhibited in the ASyycG strains. In addition, we demonstrated that ASyycG suppressed the invasive ability and pathogenicity of the strain in vivo using an SPF (specific pathogen free) rat model. CONCLUSION: In summary, the overexpression of ASyycG leads to a reduction in biofilm formation and bacterial pathogenicity in vivo, which provides a potential target for the management of MRSA-induced osteomyelitis.

Staphylococcus aureus Infects Osteoclasts and Replicates Intracellularly.

Osteomyelitis (OM), or inflammation of bone tissue, occurs most frequently as a result of bacterial infection and severely perturbs bone structure. OM is predominantly caused by Staphylococcus aureus, and even with proper treatment, OM has a high rate of recurrence and chronicity. While S. aureus has been shown to infect osteoblasts, it remains unclear whether osteoclasts (OCs) are also a target of intracellular infection. Here, we demonstrate the ability of S. aureus to intracellularly infect and divide within OCs. OCs were differentiated from bone marrow macrophages (BMMs) by exposure to receptor activator of nuclear factor kappa-B ligand (RANKL). By utilizing an intracellular survival assay and flow cytometry, we found that at 18 h postinfection the intracellular burden of S. aureus increased dramatically in cells with at least 2 days of RANKL exposure, while the bacterial burden decreased in BMMs. To further explore the signals downstream of RANKL, we manipulated factors controlling OC differentiation, NFATc1 and alternative NF-κB, and found that intracellular bacterial growth correlates with NFATc1 levels in RANKL-treated cells. Confocal and time-lapse microscopy in mature OCs showed a range of intracellular infection that correlated inversely with S. aureus-phagolysosome colocalization. The propensity of OCs to become infected, paired with their diminished bactericidal capacity compared to BMMs, could promote OM progression by allowing S. aureus to evade initial immune regulation and proliferate at the periphery of lesions where OCs are most abundant.IMPORTANCE The inflammation of bone tissue is called osteomyelitis, and most cases are caused by an infection with the bacterium Staphylococcus aureus To date, the bone-building cells, osteoblasts, have been implicated in the progression of these infections, but not much is known about how the bone-resorbing cells, osteoclasts, participate. In this study, we show that S. aureus can infect osteoclasts and proliferate inside these cells, whereas bone-residing macrophages, immune cells related to osteoclasts, destroy the bacteria. These findings elucidate a unique role for osteoclasts to harbor bacteria during infection, providing a possible mechanism by which bacteria could evade destruction by the immune system.

Sclerosing osteomyelitis of Garré: A confusing clinical diagnosis.

We describe a rare case of sclerosing osteomyelitis of Garré in a 63-year-old woman with uncontrolled right thigh pain. She had suffered from lower back pain and radiating pain on the right lower leg for a year and 4 months and so had spine surgery 8 months ago. But the right thigh pain persisted, and the levels of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) remained abnormal. Right femur radiographs showed cortical thickening on the proximal femur. Magnetic resonance images showed T2 hypersignal intensity lesions in the proximal femur. Under suspicion of osteoid osteoma or sclerosing osteomyelitis, surgery was performed with biopsy, bone curettage, and drilling. The culture was negative, and the biopsy showed chronic osteomyelitis. Despite surgery, the levels of CRP and ESR still remained abnormal. After using venous antibiotics, the pain subsided and CRP and ESR levels turned to normal. Followed by 6 weeks of oral antibiotics, pain was relieved after 1-year follow-up.

Osteoclastic expression of higher-level regulators NFATc1 and BCL6 in medication-related osteonecrosis of the jaw secondary to bisphosphonate therapy: a comparison with osteoradionecrosis and osteomyelitis.

BACKGROUND: With an increasing indication spectrum of antiresorptive drugs, the medication-related osteonecrosis of the jaw secondary to bisphosphonate therapy [MRONJ (BP)] is continuously gaining clinical relevance. Impaired osteoclast function, accompanied by altered cell morphology and expression of osteoclastic effector proteins, contributes to the pathogenesis of MRONJ (BP). However, the underlying regulatory mechanisms at a transcriptional level are unaddressed so far. These mechanisms are crucial to the development of disease-characteristic osteoclastic anomalies, that contribute to the pathogenesis of MRONJ (BP). NFATc1 is considered a master upstream osteoclastic activator, whereas BCL6 acts as osteoclastic suppressor. The present study aimed to elucidate the NFATc1 and BCL6 mediated osteoclastic regulation and activity in MRONJ (BP) compared to osteoradionecrosis (ORN) and osteomyelitis (OM) and normal jaw bone. METHODS: Formalin-fixed jaw bone specimens from 70 patients [MRONJ (BP) n = 30; OM: n = 15, ORN: n = 15, control: n = 10] were analyzed retrospectively for osteoclast expression of NFATc1 and BCL6. The specimens were processed for H&E staining and immunohistochemistry. The histological sections were digitalized and analyzed by virtual microscopy. RESULTS: Osteoclastic expression of NFATc1 and BCL6 was significantly higher in MRONJ (BP) specimens compared to OM and control specimens. NFATc1 and BCL6 labeling indices revealed no significant differences between MRONJ (BP) and ORN. The ratio of nuclear BCL6+ osteoclasts to cytoplasmic BCL6+ osteoclasts revealed significantly higher values for MRONJ (BP) specimens compared to OM and controls. CONCLUSION: This study displays that osteoclasts in MRONJ (BP) tissues feature increased expression of the higher-level regulators, paradoxically of both NFATc1 and BCL6. These observations can help to explain the genesis of morphologically altered and resorptive inactive osteoclasts in MRONJ (BP) tissues by outlining the transcriptional regulation of the pathomechanically relevant osteoclastic effector proteins. Furthermore, they strengthen the etiological delineation of MRONJ (BP) from OM and extend the osteoclast profiles of MRONJ (BP), OM and ORN and thus could lead to a better histopathological differentiation that can improve treatment decision and motivate new therapeutic concepts.