Antibiotic Perturbation of Gut Microbiota Alters Osseointegration Around an Intramedullary Nail in Mice With or Without Osteomyelitis

Abstract

Treatment of osteomyelitis requires prolonged antibiotic therapy which significantly alters the gut microbiota. While the influences on bone mass and microstructure have been extensively studied, it is poorly understood what impact the changes in gut microbiota may have on the host response to osseointegration around an intramedullary nail implanted. Here, we explored the influence of gut microbiota on the bone osseointegration process around an implant under two conditions: implantation of an intramedullary nail in the bone marrow cavity and chronic osteomyelitis(CO) induced by Staphylococcus aureus infection. Body weight, hepatorenal functions, serum levels of proinflammatory cytokines were monitored. The composition of gut microbiota was assessed via 16S rRNA sequencing, and the bone condition was analyzed via micro-computed tomography and hematoxylin and eosin staining, safranin O-fast green and Goldner's trichrome. Osteoblastogenesis and osteoclastogenesis were assessed by detecting tartrate-resistant acid phosphatase and osterix expression. We found that perturbation of gut microbiota led to delayed osseointegration and increased levels of proinflammatory cytokines in the serum (p<0.05), lower bone mass (p<0.05), deficient endochondral ossification and bone formation, reduced osteoblastogenesis (p<0.05) and enhanced osteoclastogenesis (p<0.001). Survival rates (p=0.002) and bacterial loads (p=0.0363) in bone differed significantly between the CO and antibiotic-treated CO mice, but cytokines levels, bone mineral density, and bone formation did not differ, likely because of the severely damaged bone structure. In summary, antibiotic treatment perturbed the gut microbiota and significantly interfered with the bone osseointegration around the nail by increasing proinflammatory cytokine levels in circulation, inhibiting osteoblastogenesis, enhancing osteoclastogenesis, and thus leading to higher pathogen colonization as well as higher mortality postinfection. This report of ours is the first to clarify how antibiotic-induced alterations in the gut microbiota may affect bone osseointegration, helping us understand the role of gut microbiota disorders in osteoblastogenesis and osteoclastogenesis following implant insertion with or without infection. Funding Statement: This study was supported by National Natural Science Foundation of China [grant no. 81802182], Guangdong Medical Science Research Foundation [grant no. B2019040], Science and technology program of Guangdong Province [grant no. 2016B090913004], the Innovation Leader Team Program of Guangzhou [grant no.201809010014] and Natural Science Foundation of Guangdong Province [grant no. 2019A1515012115]. Declaration of Interests: All authors declare no competing interests. Ethics Approval Statement: All animal experiments of ours were approved by Nanfang Hospital Animal Ethics Committee and conducted in accordance with all relevant ethical principles and guidelines set by the Animal Welfare Act and the NIH Guide for Care and Use of Laboratory Animals.