<i>Akkermansia Muciniphila</i> Promotes Bone Fracture Healing by Enhancing Preosteoclast-Associated Type H Vessel Formation

Abstract

Background: Promoting bone fracture repair is of great clinical significance due to the increasing traumatic incidence and extensive medical burden. Owing to the critical role of adequate blood supply plays in fracture healing, improving revascularization of fractured area is one of the major measures in fracture treatment. Moderate local inflammation is considered to trigger angiogenesis, whereas systemic inflammation exerts reverse effect on vessels formation. Previous studies showed replenishment with Akkermansia muciniphila (A. muc), a mucin-degrading gut probiotic, ameliorate systemic inflammation by tightening intestinal barrier and thereby to protect against diabetes, obesity, atherosclerosis, etc. The purpose of this study is to determine whether supplementation of this inflammation-modulating microbe can promote fractured bone healing and to investigate underlying mechanism. Methods: Femoral fracture operation and intramedullary fixation were performed in 8-week-old female C57BL/6 mice. Fractured mice were intragastrically administrated with A. muc or equivalent vehicle for 1, 2, 4 or 6 weeks. Micro computed tomography (µCT) scanning and terminal biomechanical test were conducted to evaluate the fracture healing quality. Histological, immunohistochemical and immunofluorescent staining were carried out to assess composition of callus, osteogenic, osteoclastic and angiogenic activities, as well as local inflammatory responses. Intestinal permeability was tested by detecting colonic mRNA levels of gap junction protein. Circulatory lipopolysaccharide (LPS) levels of different groups were also tested. In addition, the abundances of A. muc were measured at various time points (1, 2, 4 and 6 weeks post fracture). Finding: A. muc was successfully cultured in vitro and intragastric gavage of this probiotic successfully colonized in gut of mice. Increased abundance of A. muc led to accelerated and strengthened bone-fracture healing, evidenced by µCT analyses and three-point bending test, as well as more chondroid matrix in callus at 2 weeks post fracture (WPF) and promoted osteogenesis at 6 WPF compared to vehicle-treated mice. Tartrate-resistant acid phosphatase (TRAP) staining revealed no significant difference in the number of osteoclast between A. muc group and control group at 6 WPF. Notably, more preosteoclasts were found in fracture area of A. muc-treated mice at 2 WPF. At 2 WPF, more TRAP+ cells positive for platelet-derived growth factor-BB (PDGF-BB) were detected on surface of woven bone of mice administrated with A. muc, coinciding with increased formation of type H vessels, which highly express the endothelial markers CD31 and Endomucin (CD31hiEmcnhi). Compared to vehicle group, A. muc-treated mice showed higher mRNA levels of the gap junction proteins and lower gene expression of inflammatory cytokines in intestines, consistent with lower systemic LPS. At the meantime, immunohistochemical analyses revealed less pro-inflammatory factors positive staining areas in fractured bone of A. muc-treated mice, compared to vehicle-treated mice. Interpretation: At early stage of fracture healing process, A. muc treatment reduces intestinal permeability and alleviates local inflammatory responses, which possibly induce high levels of PDGF-BB secreted by preosteoclasts and increase type H vessel formation in callus, thereby to promote bone fracture healing. Our study proposes that A. muc treatment has potential as a promising strategy for bone fracture healing. Funding Statement: This work was supported by the National Natural Science Foundation of China (Grant No. 81670807, 81871822, 81600699), the Excellent Young Scientist Award of National Natural Science Foundation of China (Grant No. 81522012), the Thousand Youth Talents Plan of China (Grant No. D1119003), the Hunan Youth Talent Project (Grant No. 2016RS3021), the Innovation Driven Project of Central South University, China (Grant No. 2016CX028, 2019CX014), and the Fundamental Research Funds for the Central Universities of Central South University, China (Grant No. 2017zzts211). Declaration of Interests: The authors declare: None. Ethics Approval Statement: Animal care and experimental procedures were approved by the Ethical Review Board at Xiangya Hospital of Central South University.