gms | German Medical Science

Objectives: Staphylococcus aureus is the major cause of osteomyelitis, particularly in implant-related bone infection. However, little is known on the influence of staphylococcal infection on the bone-forming osteoblasts after antimicrobial therapy. Thus, this study investigated whether S. aureus-infected osteoblasts still retain the ability to metabolise and produce mineralised extracellular matrix after antibiotic treatment.

Methods: Using the human osteoblasts-like Soas-2 cells, we established an in-vitro intracellular S. aureus-infection model. Following intracellular infection with high-grade S. aureus EDCC5055 strain, the osteoblasts were treated with rifampicin (RMP) and stimulated with osteogenic components up to 21-days.

Results and Conclusion: Immunofluorescence staining and transmission electron microscopic (TEM) imaging demonstrated the presence of intracellular bacteria within the infected osteoblasts. TEM images showed intact intracellular bacteria with dividing septa indicative of active replication. Also, resazurin assay showed an abnormally high metabolic activity of the infected osteoblasts, implying an increased intracellular bacteria proliferation. Treatment of the S. aureus-infected osteoblasts with a single dose of 8 µg/ml RMP revealed a significant reduction (p< 0.001) of the intracellular bacteria colony formation units as early as 2-hours post-infection. Also, single of dose of 8 µg/ml RMP sufficiently restored the metabolic activity of the infected osteoblasts akin to the uninfected groups (p >0.05). Alizarin red staining and quantification of the RMP-treated infected osteoblasts showed significantly lower amount of mineralised extracellular matrix after 7 days of osteogenesis (p<0.05). Interestingly, prolonged osteogenic stimulation with RMP-treatment up to 21 days substantially enhanced the extracellular matrix mineralisation level, comparable to the RMP-treated uninfected group. However, the untreated normal osteoblasts showed significantly more quantity of mineral deposits (p<0.001). TEM analysis of the RMP-treated infected osteoblasts at 21-days of osteogenesis revealed viable osteoblasts and newly differentiated osteocytes that are surrounded by densely distributed calcium crystal deposits within the extracellular matrix. Besides, residual colony of dead bacteria bodies and empty vacuoles of the fully degraded bacteria were embedded within the mineralised extracellular matrix. Gene expression of prominent bone formation markers, namely RUNX2, COLA1, BMP-2, SPARC, BGLAP, OPG and RANKL showed no significant difference between the infected and uninfected osteoblasts.

In conclusion, S. aureus infection can drastically impair osteoblasts metabolism and function. However, prompt antimicrobial therapy using optimal concentrations of potent antibiotics such as RMP can restore the metabolic activity of surviving osteoblasts. Furthermore, delay in early osteogenesis caused by bacterial infection can be significantly improved over time after successful treatment.