N6 methyladenosine eraser FTO suppresses Staphylococcus aureus-induced ferroptosis of bone marrow mesenchymal stem cells to ameliorate osteomyelitis through regulating the MDM2/TLR4/SLC7A11 signaling pathway.

Osteomyelitis is a bone destructive inflammatory disease caused by infection. Ferroptosis is closely related to multiple inflammatory diseases, but the role of ferroptosis in Staphylococcus aureus (SA)-induced osteomyelitis remains unknown. In the present study, we found that SA treatment promoted the accumulation of iron, Fe2+ , lipid peroxide, and malondialdehyde, increased TFRC and reduced FTH1 and GPX4 to trigger ferroptosis in rat bone marrow mesenchymal stem cells (BMSCs). Interestingly, increased level of N6 methyl adenosine (m6A) modification along with decreased expression level of m6A eraser FTO were observed in SA-induced BMSCs, while upregulating FTO alleviated SA-triggered ferroptosis and protected cell viability in BMSCs. Mechanistically, MDM2 was identified as a target of FTO-mediated m6A demethylation, and FTO upregulation promoted MDM2 instability to downregulated TLR4 signal and elevate the expression of SLC7A11 and GPX4 in SA-induced BMSCs. Functional recovery experiments verified that overexpressing MDM2 or TLR4 reversed the inhibiting effect of FTO upregulation on ferroptosis in SA-treated BMSCs. Additionally, FTO upregulation restrained ferroptosis and pathological damage to bone tissue in SA-induced osteomyelitis model rats. Altogether, m6A eraser FTO alleviates SA-induced ferroptosis in osteomyelitis models partly through inhibiting the MDM2-TLR4 axis.

Management of infected bone defects of the femoral shaft by Masquelet technique: sequential internal fixation and nail with plate augmentation.

BACKGROUND: To evaluate the effectiveness of a sequential internal fixation strategy and intramedullary nailing with plate augmentation (IMN/PA) for bone reconstruction in the management of infected femoral shaft defects using the Masquelet technique. METHODS: We performed a retrospective descriptive cohort study of 21 patients (mean age, 36.4 years) with infected bone defects of the femoral shaft treated by the Masquelet technique with a minimum follow-up of 18 months after second stage. After aggressive debridement, temporary stabilisation (T1) was achieved by an antibiotic-loaded bone cement spacer and internal fixation with a bone cement-coated locking plate. At second stage (T2), the spacer and the locking plate were removed following re-debridement, and IMN/PA was used as definitive fixation together with bone grafting. We evaluated the following clinical outcomes: infection recurrence, bone union time, complications, and the affected limb's knee joint function. RESULTS: The median and quartiles of bone defect length was 7 (4.75-9.5) cm. Four patients required iterative debridement for infection recurrence after T1. The median of interval between T1 and T2 was 10 (9-19) weeks. At a median follow-up of 22 (20-27.5) months, none of the patients experienced recurrence of infection. Bone union was achieved at 7 (6-8.5) months in all patients, with one patient experiencing delayed union at the distal end of bone defect due to screws loosening. At the last follow-up, the median of flexion ROM of the knee joint was 120 (105-120.0)°. CONCLUSIONS: For infected femoral shaft bone defects treated by the Masquelet technique, sequential internal fixation and IMN/PA for the reconstruction can provide excellent mechanical stability, which is beneficial for early functional exercise and bone union, and does not increase the rate of infection recurrence.

DNMT1-mediated DNA methylation in toll-like receptor 4 (TLR4) inactivates NF-κB signal pathway-triggered pyroptotic cell death and cellular inflammation to ameliorate lipopolysaccharides (LPS)-induced osteomyelitis.

Toll-like receptor 4 (TLR4) plays a critical role in various human diseases, and was associated with pyroptotic cell death and inflammatory responses. DNA methylation, which has stable and reversible properties, has been reported to alter the expression of target genes, including TLR4. However, the role of methylated TLR4 in osteomyelitis (OM) and the underlying molecular mechanisms remain unclear. RNA sequencing was used to identify differentially expressed genes and associated signaling pathways. RT-qPCR, Western blot, emzyme-linked immunosorbent assay (ELISA), cell counting kit-8 (CCK-8) and LDH assay kit were used to detect mRNA and protein expression of relevant genes, cell viability and the LDH activity, respectively. TLR4 methylation was detected by methylation-specific PCR (MSP) and verified by Chromatin immunoprecipitation (ChIP). Here, we found that DNA methyltransferase-1 (DNMT1)-mediated TLR4 demethylation significantly suppressed lipopolysaccharides (LPS)-induced pyroptosis and inflammatory response by inhibiting the TLR4/nuclear transcription factor-kappa B (NF-κB) axis. First, we confirmed TLR4 as the study target by mRNA transcriptome sequencing analysis, and TLR4 was observably high-expressed in both OM patients and LPS-treated osteoblastic MC3T3-E1. Then, we found that downregulation of DNMT1 blocked TLR4 promoter methylation modification, resulting in upregulation of TLR4. Simultaneously, functional experiments indicated that suppression of TLR4 or overexpression of DNMT1 promoted cell proliferation and inhibited cell pyroptosis and inflammation in LPS-induced MC3T3-E1, while upregulation of TLR4 restored the effects of DNMT1 silencing on OM progression. In addition, TLR4 elevated phosphorylation of IκB-α and NF-κB p65 in the NF-κB signal pathway, and inhibition of TLR4 or the NF-κB inhibitor PDTC reversed the influence of inhibition of DNMT1. In conclusion, our study demonstrated that DNMT1-mediated TLR4 DNA methylation alleviated LPS-induced OM by inhibiting the NF-κB signaling pathway.

Staphylococcus Aureus Induces Osteoclastogenesis via the NF-κB Signaling Pathway.

BACKGROUND Osteomyelitis is one of the refractory diseases encountered in orthopedics, while Staphylococcus aureus (S. aureus) is the most common causative organism in osteomyelitis. However, the precise mechanisms underlying the bone loss caused by S. aureus infection have not been well defined. Here, we investigated the effect of S. aureus on osteoclast differentiation and the probable molecular mechanism. MATERIAL AND METHODS RAW 264.7 cells were treated for 5 days with live S. aureus, inactivated S. aureus, and S. aureus filtrate. Then, the formation of osteoclast-like cells and resorption pits was observed, and the expression of osteoclast-specific genes (TRAP, MMP-9, cathepsin K, CTR and Atp6v0d2) was detected by real-time PCR. Moreover, key proteins in the signaling pathway associated with osteoclast differentiation were detected with Western blot. RESULTS The data showed that live S. aureus, inactivated S. aureus, and S. aureus filtrate induced osteoclast formation, promoted bone resorption, and increased the expression of osteoclast-specific genes in a dose-dependent manner in the absence RANKL. In addition, we found that the S. aureus-induced osteoclastogenesis was related to the degradation of IκB-a, phosphorylation of NF-κB p65, and increased expression of NFATc1. Thus, we used JSH-23 to inhibit NF-κB transcriptional activity. The effect of the S. aureus-induced osteoclastogenesis and the expression of osteoclast-specific genes and NFATc1 were inhibited, which indicated that the NF-κB signaling pathway plays a role in S. aureus-induced osteoclastogenesis. CONCLUSIONS This study demonstrated that S. aureus induces osteoclastogenesis through its cell wall compound and secretion of small soluble molecules, and the NF-κB signaling pathway plays a role in this process.

Clinical effects of early debridement, internal fixation, and Masquelet technique for childhood chronic haematogenous osteomyelitis of long bones.

BACKGROUND: Childhood chronic haematogenous osteomyelitis (CCHOM) is a severe condition in paediatric patients. The optimal timing of debridement and the subsequent method of bone reconstruction in CCHOM patients remain controversial. The purpose of this study was to assess the treatment efficacy of Masquelet technique with early debridement and internal fixation in CCHOM of long bones. METHODS: Between January 2016 and January 2021, a total of 21 patients (18 males, 3 females) with CCHOM of long bone were included. The mean age was 10.4 years (range, 2-18 years). All cases were treated by a two-stage surgical protocol of Masquelet technique. In the first stage, aggressive debridement, sequestrectomy, and inducing membrane by bone cement spacer were performed after definite diagnosis. In the second stage, cement spacer was removed, and autologous and allogeneic bone was grafted. Internal fixation was used for the first and/or second stage depending on stability requirements. The patients' clinical and imaging results were retrospectively analysed. RESULTS: The mean follow-up was 31.7 months (range, 21-61 months). None of the patients experienced recurrence of infection. Radiographic bone union time was 4.3 months (range, 2.5-11 months). Five cases underwent re-operation due to complications such as bone resorption or refracture. By the last follow-up visit, bones had healed and all of the patients had resumed daily living and sports activities. CONCLUSION: The Masquelet technique with early debridement and internal fixation is a viable surgical method for the management of large long bone defects of CCHOM patients.

Comparison of internal and external fixation after debridement in the Masquelet technique for Cierny-Mader type IV tibial post-traumatic osteomyelitis.

OBJECTIVE: To compare the effect of internal fixation vs. external fixation after debridement in stage I of the Masquelet technique for Cierny-Mader (C-M) type Ⅳ chronic post-traumatic tibial osteomyelitis. METHODS: This retrospective observational study included patients with tibial osteomyelitis who underwent staged treatment with the Masquelet technique between January 2016 and June 2020 at the 920 Hospital of Joint Logistic Support Force of the PLA. The patients were grouped according to the fixation they received after stage I. Infection recurrence, time to radiological bone healing and full weight-bearing, self-rating anxiety scale (SAS) score, Hospital for Special Surgery (HSS) Knee Score, and American Orthopedic Foot and Ankle Society (AOFAS) Ankle-Hindfoot Score were compared. RESULTS: Sixty-three patients were included (50 males and 13 females). There were 40 and 23 patients with internal and external fixation, respectively. There were no significant differences between the two groups regarding the preoperative and intraoperative data (all P>0.05). After stage I operation, the infection control rates were 85.0% and 82.6% in the internal and external fixation groups (P=0.803), and these rates were 92.5% and 95.7% after stage II (P=0.621). There were no differences in the SAS scores (P=0.278), time to radiological union (P=0.795), time to full weight-bearing (P=0.725), AOFAS scores (P=0.302), HSS scores (P=0.085), and complication rates (P=0.593). There were 27 times complications in 19 patients, with an incidence of 42.9%, without significant differences between groups. CONCLUSION: There were no differences between the two fixation methods after debridement in stage I of the Masquelet technique for C-M type Ⅳ chronic post-traumatic tibia osteomyelitis.

Evaluation of the mechanical properties and clinical application of nickel-titanium shape memory alloy scaphoid arc nail.

To investigate the mechanical and biomechanical properties of nickel-titanium (Ni-Ti) shape memory alloy scaphoid arc nail (NT-SAN) fixator as well as study the surgical method of treating carpal scaphoid fractures and evaluate its clinical efficacy. (1) Static and dynamic bending tests with embedded axial bending fixture were conducted to study the mechanical properties. (2) To evaluate biomechanical strength and fatigue, 32 scaphoid samples were classified into four groups to perform the fixation rigidity test: intramedullary Kirschner fixation (group A), Kirschner straddle nail fixation (group B), screw nail fixation (group C), and NT-SAN fixation (group D). Next, 24 scaphoid waist fracture models were classified to conduct fatigue experiments as follows: Kirschner straddle nail fixation (group E), screw nail fixation (group F), and NT-SAN fixation (group G). (3) The Krimmer score chart was used for clinical evaluations. (1) NT-SAN showed excellent mechanical performance and a long lifespan. (2) NT-SAN was fixated with a strong intensity and an anti-fatigue outcome. (3) Ninety-eight interviewed patients were satisfied with the therapeutic effects of the arc nail (satisfaction rate: 95.92%). The designed strength and hardness of NT-SAN corresponded with the anatomical characteristics of the scaphoid, and the designed mechanical properties met the biomechanical requirements of a scaphoid fracture. The fatigue strength can meet the requirements of bone healing after the scaphoid fracture. Clinical trials on NT-SAN scaphoid fracture treatment have shown that the surgery is simple and the clinical results are satisfactory. The therapeutic level of NT-SAN is III; thus, it is worth promoting.

Muscle pedicle bone flap transplantation for treating femoral neck fracture in adults: a systematic review.

BACKGROUND: This systematic review was conducted to gather available evidence on the effectiveness of muscle pedicle bone flap transplantation in adult patients with femoral neck fractures. METHODS: Databases such as PubMed, EMBASE, IEEE, Web of Science, and Cochrane library were searched from their dates of inception until March 2021. Two reviewers independently selected the interventional studies on the assessment of the effectiveness of muscle pedicle bone flap transplantation for femoral neck fractures; data extraction and assessment of the methodological quality as per the Institute of Health Economics quality appraisal checklist were also performed by the reviewers. The effectiveness and complication outcomes were assessed by calculating the average rates. RESULTS: Overall, 20 studies with 1022 patients were included in this review. Notably, the methodologic quality of the included studies was typically poor. The average effective rates were as follows: good, 73.4%; fair, 15.4%; and poor, 10.9%. Moreover, the average nonunion rate, average avascular necrosis rate, average collapse rate, and the overall reoperation rate were 9.0%, 6.7%, 4.7%, and 7.3%, respectively. CONCLUSIONS: This systematic review of heterogeneous studies with varying number of patients and varying surgical techniques indicated that muscle pedicle bone flap transplantation provides promising results with low rates of avascular necrosis and nonunion. Nevertheless, further controlled studies are required to ascertain the effectiveness of muscle pedicle bone flap transplantation in treating femoral neck fracture.

[Nitinol memory alloy two foot fixator with autologous cancellous bone grafting for old scaphoid fracture and nonunion].

OBJECTIVE: To summarize the effectiveness of nitinol memory alloy two foot fixator with autologous cancellous bone grafting in treating old scaphoid fracture and nonunion. METHODS: Between January 2013 and January 2017, 11 patients of old scaphoid fracture and nonunion were treated with nitinol memory alloy two foot fixator and autologous cancellous bone grafting. All patients were male with an average age of 26.1 years (range, 18-42 years). The fractures were caused by sport in 3 cases, falling in 7 cases, and a crashing object in 1 case. The interval between injury and operation was 6-18 months (mean, 8.9 months). Postoperative outcome measures included operation time, fracture healing time, grip strength, range of motion (ROM) of flexion, extension, ulnar deviation, and radial deviation, Mayo score, visual analogue scale (VAS) score, and the Disabilities of the Arm, Shoulder, and Hand (DASH) score. RESULTS: The operation time was 35-63 minutes (mean, 48 minutes). All incisions had primary healing with no infection and loosening or breakage of internal fixator. All patients were followed up 12-30 months (mean, 20.7 months). X-ray films showed that fracture healing was achieved in all patients with an average time of 15 weeks (range, 12-25 weeks). All internal fixators were removed after 10-12 months of operation (mean, 11.2 months). At last follow-up, the grip strength, ROMs of flexion, ulnar deviation, and radial deviation were superior to those before operation ( P<0.05), no significant difference was found in ROM of extension between pre- and post-operation ( t=0.229, P=0.824). There were significant differences in above indexes between affected and normal sides ( P<0.05). At last follow-up, the Mayo, VAS, DASH scores were also significantly superior to those before operation ( P<0.05). CONCLUSION: For the old scaphoid fracture and nonunion, Ni-Ti arched shape-memory alloy fixator and autologous cancellous bone grafting can obtain good effectiveness, which is an effective treatment.

[Effect of staphylococcal lipoteichoic acid on differentiation of RAW264.7 cells into osteoclasts].

Objective: To investigate the effect of staphylococcal lipoteichoic acid (LTA-sa) on RAW264.7 cells differentiation into osteoclasts. Methods: RAW264.7 cells were cultured with LTA-sa of 100 ng/mL (group A), LTA-sa of 200 ng/mL (group B), LTA-sa of 400 ng/mL (group C), receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) of 100 ng/mL as positive control (group D), and equal volume of PBS as blank control (group E) respectively for 5 days. And then, tartrate resistant acid phosphatase staining (TRAP) was used to detect the formation of osteoclast-like cells, Image-Pro Plus 6.0 software to measure the areas of bone resorption pits in Corning Osteo Assay Surface (COAS) wells, and MTT assay to observe the proliferation activity of RAW264.7 cells in group A, B, C, and E. Results: After cultured for 5 days, the formation of osteoclast-like cells and bone resorption pits were observed in all groups. The number of osteoclast-like cells and the area of bone resorption pits in groups A, B, C, and D were more than those in group E. And with the increased concentration of LTA-sa, the indexes in groups A, B, and C increased gradually, but were lower than those in group D, and differences were significant between groups ( P<0.05). At 5 days after culture, there was no significant difference in absorbance value among the experimental groups (groups A, B, C, and E) ( P>0.05). Conclusion: LTA-sa has promoting effect on RAW264.7 cells differentiation into osteoclasts.

Staphylococcus aureus Protein A induces osteoclastogenesis via the NF­κB signaling pathway.

Staphylococcus aureus (S. aureus) is the most common organism causing osteomyelitis, and Staphylococcus aureus protein A (SpA) is an important virulence factor anchored in its cell wall. However, the precise mechanisms underlying the bone loss caused by SpA have not been well understood. The present study aimed to investigate the effect of SpA on osteoclast differentiation, and the probable mechanism was investigated. Raw264.7 cells were treated with SpA in the absence or presence of receptor­activated (NF)­κB ligand for 5 days, and morphological and biochemical assays were used to assess osteoclastogenesis and explore the underlying mechanisms. Data demonstrated that SpA induced osteoclast differentiation and promoted bone resorption in a dose­dependent manner in the absence or presence of RANKL. In addition, the expression of osteoclast­specific genes, such as the tartrate resistant acid phosphatase, matrix metalloproteinase­9, cathepsin K, calcitonin receptors and d2 isoform of the vacuolar ATPase Vo domain, were enhanced by SpA. Furthermore, the SpA­induced osteoclast differentiation was associated with the degradation of inhibitor of κB­α, phosphorylation of NF­κB p65 and increased expression of nuclear factor of activated T­cells. However, by treatment with JSH­23, an NF­κB inhibitor, the formation of osteoclast­like cells and resorption pits was significantly reduced, and the expression of osteoclast­specific genes was also inhibited. Collectively, in the present study SpA induced osteoclast differentiation, promoted bone resorption, and the NF­κB signaling pathway was involved in this process.

Absorbable scaphoid screw development: a comparative study on biomechanics.

BACKGROUND: The scaphoid is critical for maintaining the stability and movement of the wrist joints. This study aimed to develop a new internal fixator absorbable scaphoid screw (ASS) for fixation of the scaphoid waist after fracture and to test the biomechanical characteristics of ASS. MATERIALS AND METHODS: An ASS was prepared using polylactic acids and designed based on scaphoid measurements and anatomic features. Twenty fractured scaphoid waist specimens were randomly divided into experimental and control groups (n=10/group). Reduction and internal fixation of the scaphoid were achieved with either Kirschner wires (K-wires) or ASS. A moving target simulator was used to test palmar flexion and dorsal extension, with the range of testing (waist movement) set from 5° of palmar flexion to 25° of dorsal extension. Flexion and extension were repeated 2,000 times for each specimen. Fracture gap displacements were measured with a computerized tomography scanning. Scaphoid tensile and bending strengths were measured by using a hydraulic pressure biomechanical system. RESULTS: Prior to biomechanical fatigue testing, fracture gap displacements were 0.16±0.02 mm and 0.22±0.02 mm in the ASS and K-wire groups, respectively. After fatigue testing, fracture gap displacements in the ASS and the K-wire groups were 0.21±0.03 mm and 1.52±0.07 mm, respectively. The tensile strengths for the ASS and K-wire groups were 0.95±0.02 MPa and 0.63±0.02 MPa, respectively. CONCLUSION: Fixation using an ASS provided sufficient mechanical support for the scaphoid after fracture.

[STUDY ON MOLECULAR MECHANISM OF OSTEOCLAST DIFFERENTIATION INDUCED BY STAPHYLOCOCCAL PEPTIDOGLYCAN].

OBJECTIVE: To investigate the molecular mechanism of osteoclast differentiation induced by Staphylococcal peptidoglycan (PGN-sa). METHODS: Raw264.7 cells were stimulated with PGN-sa and with PGN-sa+SC75741[a potent inhibitor of nuclear factor κB (NF-κB) activation] in a concentration of 200 ng/mL. The protein expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) was tested at 0, 1, 2, and 3 days; the proteins related to osteoclast differentiation of extracellular regulated protein kinases (ERK), p38, c-Jun N-terminal kinase (JNK), NF-κB, inhibitor of NF-κB (IκB-α), Akt, and the phosphorylation forms of p38, ERK, JNK, Akt, NF-κB were measured at 0, 5, 10, 20, 40, and 60 minutes by Western blot. In addition, Raw264.7 cells were stimulated with PGN-sa in the concentrations of 100 ng/mL (group A), 200 ng/mL (group B), 400 ng/mL (group C), and with PBS (group D) for 1, 2, and 3 days; the expression levels of tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), and IL-6 were detected by ELISA. RESULTS: The results of Western blot showed that the expression of NFATc1 increased gradually with time, showing significant difference between different time points (P<0.05). However, after SC75741 was added, the expression of NFATc1 was inhibited at 2 and 3 days, showing significant difference when compared with no addition of SC75741 (P<0.001). After stimulation of PGN-sa, the expression of IkB-α decreased significantly at 5 and 10 minutes when compared with those at the other time points (P<0.001), and returned to normal at 20 minutes. Meanwhile, the expression of p-NF-κB increased significantly at 5 and 10 minutes when compared with those at the other time points (P<0.001), and returned to normal at 20 minutes; and the expression of p-NF-κB at 5 minutes was significantly higher than that at 10 minutes (P<0.001). After the addition of SC75741, there was no change in the expressions of IκB-α and p-NF-κB, showing no significant difference between different time points P>0.05). Moreover, the expressions of ERK, p38, JNK, NF-κB, Akt, p-p38, p-ERK, p-JNK, and p-Akt showed no significant change between different time points P>0.05). ELISA results showed that there were no expressions of TNF-α and IL-1α in groups A-D at different time points. The expression of IL-6 had an increasing trend with time prolonged in each group, showing significant differences between different time points (P<0.05). Moreover, at 1 day after culture, the expression of IL-6 showed no significant difference among groups P>0.05). At 2 and 3 days after culture, the expression of IL-6 in groups A-C showed an increasing trend and was significantly higher than that in group D, showing significant difference among groups (P<0.05). CONCLUSIONS: PGN-sa can promote osteoclast differentiation through NF-κB signaling pathway, and IL-6 may play a role in this process.

[EFFECT OF STAPHYLO CO CCAL PEPTID O GLYCAN ON OSTEO CL AST DIFFERENTIATION].

OBJECTIVE: To investigate the effect of Staphylococcal peptidoglycan (PGN-sa) on raw264.7 cells differentiating into osteoclasts. METHODS: There were 5 groups in the experiment: 100 ng/mL PGN-sa group, 200 ng/mL PGN-sa group, 400 ng/mL PGN-sa group, positive control group [100 ng/mL receptor activator of nuclear factor κB ligand (RANKL)], and blank control group (PBS). Raw264.7 cells were cultured with different concentrations of PGN-sa, RANKL, or PBS for 5 days, and then tartrate resistant acid phosphatase (TRAP) staining was used to detect the formation of osteoclast-like cells; Image-Pro Plus 6.0 software was used to detect the bone resorption areas of osteoclast-like cells; and MTT assay was used to observe the proliferation activity of raw264.7 cells. RESULTS: TRAP staining showed that PGN-sa and RANKL can induce raw264.7 cells to differentiate into osteoclast-like cells; different concentrations of PGNsa groups had more osteoclast-like cells formation than blank control group (P<0.05), and the number of osteoclast-like cells significantly increased with the increase of PGN-sa concentrations (P<0.05). Bone resorption cavity experiment showed that bone resorption cavities were obvious in different concentrations of PGN-sa groups and in positive control group, and the area of bone absorption cavities was increased with the increasing PGN-sa concentrations, showing significant difference between groups (P<0.05). MTT assay showed that no significant difference was found in the absorbance (A) value between different concentrations of PGN-sa groups and blank control group, and between different concentrations of PGN-sa groups (P>0.05). CONCLUSIONS: PGN-sa can promote raw264.7 cells to differentiate into osteoclasts with bone resorption activity.