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.

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.

[Anatomical double bundle reconstruction of medial patellofemoral ligament with allograft tendon in the treatment of patellar dislocations].

OBJECTIVE: To investigate the clinical therapeutic results of allograft tendon for anatomical reconstruction of medial patellofemoral ligament (MPFL) for the treatment of patellar dislocations. METHODS: From September 2008 to June 2013, 16 patients with patellar dislocation underwent MPFL reconstructions. There were 2 males and 14 females, aged 11 to 27 years old (16 years old on average). Patellar dislocations occurred in 11 left and 5 right knees. The disease course ranged from 3 to 10 years. The frequency of dislocation ranged from 9 to 33 times (19 times on average). Affected knee joints showed patellar instability; the range of action for patella obviously increased. The X-ray films showed patellar dislocation. The preoperative Q angle was (36 ± 9)°, and the congruence angle was (63 ± 18)°. Reconstruction was performed via allograft tendon. Allograft tendon was fixed through the superomedial pole of the patella, and the other end was fixed at the natural MPFL insertion site near the medial femoral condyle with an interference screw in a bone tunnel. All the patients were evaluated postoperatively; Kujala patellofemoral scores, objective knee function, complications, and reoperations were assessed. RESULTS: Primary healing was achieved in all cases. No infection or necrosis and absorption of grafts was observed. All the patients were followed up for an average of 16.4 months (ranged, 10 to 24 months) postoperatively. At the latest follow-up, all the patients had no pain, swelling and patellar instability; neither patella redislocation nor fracture occurred. The X-ray films showed good position of tunnel 6 months after operation, and the congruence angle was (5 ± 9)°, showing statistically significant difference when compared with preoperation (P < 0.05). The postoperative Q angle was (17 ± 8)°, the Kujala knee function score improved significantly from 45.20 ± 9.20 to 89.30 ± 6.40 at the latest follow-up, showing statistically significant difference (P < 0.05). CONCLUSION: MPFL reconstruction improves clinical symptoms. Anatomical MPFL reconstruction is effective for patellar dislocation, and it offers good recovery of the premorbid patella mechanics. The interference screw provides firm fixation. Allograft can avoid the graft harvest site morbidity, but it increases the cost of the surgery.