■慢性骨髓炎(骨感染)的治疗仍然是一个临床挑战;特别是,它需要增强抗生素药物的递送来治疗细胞内金黄色葡萄球菌(S.金黄色葡萄球菌),防止感染复发和抵抗。以前的研究发现,用于治疗肌肉骨骼疾病的非侵入性冲击波可以改变细胞通透性,然而,目前尚不清楚冲击波是否会改变慢性骨髓炎的细胞膜通透性。此外,目前尚不清楚这种通透性的变化是否会促进抗生素进入成骨细胞发挥抗菌作用。
■在我们的研究中,采用台盼蓝染色确定对成骨细胞模型无明显损伤的冲击波参数;采用BODIPY®FL万古霉素检测冲击波对成骨细胞模型细胞膜通透性的影响;采用高效液相色谱-质谱(HLPC-MS)检测冲击波对抗生素进入成骨细胞模型的影响;采用平板菌落计数法检测金黄色葡萄球菌对成骨细胞模型中抗生素的清除作用。为了探索机制,用平板菌落计数法检测不同脉冲冲击波对金黄色葡萄球菌的影响,此外,免疫荧光法检测成骨细胞P2X7受体,检测细胞外ATP水平。此外,观察P2X7受体拮抗剂KN-62或A740003对休克辅助抗生素细胞内抗菌活性的影响。然后,我们使用金黄色葡萄球菌建立慢性胫骨骨髓炎大鼠模型,并研究冲击波辅助抗生素治疗大鼠慢性骨髓炎的有效性和安全性。
■在0.21mJ/mm2下施加多达400个冲击波脉冲,对细胞内金黄色葡萄球菌感染的成骨细胞模型的活力没有显着影响。令人惊讶的是,这种冲击波治疗显著增强了BODIPY®FL万古霉素对成骨细胞模型细胞的递送.此外,冲击波疗法增加了亲水性抗生素(万古霉素和头孢呋辛钠)的递送,但不是亲脂性抗生素(利福平和左氧氟沙星),提高了细胞内抗菌作用。之后,我们发现冲击波治疗增加了ATP(P2X7受体激活剂)的细胞外浓度,而KN-62或A740003,P2X7受体抑制剂,细胞内抗菌活性降低。然后,我们发现0.1mL1×1011CFU/mLATCC25923金黄色葡萄球菌适用于大鼠慢性骨髓炎的建模。此外,冲击波辅助万古霉素治疗与最强的抗菌和成骨作用在测试的治疗中被证实在体内通过影像学检查,微生物培养,和组织病理学,具有良好的安全性。
■我们的结果表明,冲击波可以通过以P2X7受体依赖性方式改变细胞膜通透性来促进抗生素进入成骨细胞进行抗菌。此外,考虑到大鼠骨髓炎模型的抗菌和成骨效率以及高度的安全性,因此,冲击波辅助万古霉素治疗可能是慢性骨髓炎的一种可能的辅助治疗.
UNASSIGNED: Treatment of chronic osteomyelitis (bone infection) remains a clinical challenge; in particular, it requires enhanced delivery of antibiotic drugs for the treatment of intracellular Staphylococcus aureus (S. aureus), which prevents infection recurrence and resistance. Previous studies have found that noninvasive shock waves used to treat musculoskeletal diseases can alter cell permeability, however, it is unclear whether shock waves alter cell membrane permeability in chronic osteomyelitis. Furthermore, it remains unknown whether such changes in permeability promote the entry of antibiotics into osteoblasts to exert antibacterial effects.
UNASSIGNED: In our study, trypan blue staining was used to determine the shock wave parameters that had no obvious damage to the osteoblast model; the effect of shocks waves on the cell membrane permeability of osteoblast model was detected by BODIPY®FL vancomycin; high performance liquid chromatography-mass spectrometry (HLPC-MS) was used to detect the effect of shock wave on the entry of antibiotics into the osteoblast model; plate colony counting method was used to detect the clearance effect of shock wave assisted antibiotics on S. aureus in the osteoblast model. To explore the mechanism, the effect of different pulses of shock waves on S. aureus was examined by plate colony counting method, besides, P2X7 receptor in osteoblast was detected by immunofluorescence and the extracellular ATP levels was detected. Furthermore, the effect of P2X7 receptor antagonists KN-62 or A740003 on the intracellular antibacterial activity of shock-assisted antibiotics was observed. Then, we used S. aureus to establish a rat model of chronic tibial osteomyelitis and investigated the efficacy and safety of shock-wave assisted antibiotics in the treatment of chronic osteomyelitis in rats.
UNASSIGNED: The viability of the osteoblast models of intracellular S. aureus infection was not significantly affected by the application of up to 400 shock wave pulses at 0.21 mJ/mm2. Surprisingly, the delivery of BODIPY®FL vancomycin to osteoblast model cells was markedly enhanced by this shock wave treatment. Furthermore, the shock wave therapy increased the delivery of hydrophilic antibiotics (vancomycin and cefuroxime sodium), but not lipophilic antibiotics (rifampicin and levofloxacin), which improved the intracellular antibacterial effect. Afterwards, we discovered that shock wave treatment increased the extracellular concentration of ATP (the P2X7 receptor activator), while KN-62 or A740003, a P2X7 receptor inhibitor, decreased intracellular antibacterial activity. We then found that 0.1 mL of 1 × 1011 CFU/mL ATCC25923 S. aureus was suitable for modeling chronic osteomyelitis in rats. Besides, the shock wave-assisted vancomycin treatment with the strongest antibacterial and osteogenic effects among the tested treatments was confirmed in vivo by imaging examination, microbiological cultures, and histopathology, with favorable safety.
UNASSIGNED: Our results suggest that shock waves can promote the entry of antibiotics into osteoblasts for antibacteria by changing the cell membrane permeability in a P2X7 receptor-dependent manner. Besides, considering antibacterial and osteogenic efficiency and a high degree of safety in rat osteomyelitis model, shock wave-assisted vancomycin treatment may thus represent a possible adjuvant therapy for chronic osteomyelitis.