Shock wave assisted intracellular delivery of antibiotics against bone infection with Staphylococcus aureus via P2X7 receptors

Abstract

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Keywords

• Chronic osteomyelitis
• Shock wave
• Cell membrane permeability
• Osteoblasts
• P2X7 receptors