Long Term Probiotic Use Increases Bone Density in Female Mice

Probiotic bacteria are micro‐organisms that when taken in adequate amounts have beneficial effects on the host. A previously published study from our laboratory demonstrated that short term (4 week) treatment of male but not female mice, with Lactobacillus reuteri (ATCC 6475) lead to increased bone density. In the present study we investigate the long term (8 weeks) effects of L. reuteri treatment in female mice. Female Balb/c mice were treated by gavaging with L . reuteri (1x10 9 cfu/ml) or vehicle control (MRS broth) three times per week. In addition, L. reuteri (1.5x10 8 cfu/ml) or vehicle was added to the drinking water for the duration of the study. At 4 and 8 weeks femoral trabecular bone parameters were measured by µCT. Bone marrow (BM) cellular phenotype was analyzed by flow cytometry and RANKL, OPG and TGFβ gene expression evaluated by qPCR. Similar to our earlier studies in fertile female mice, we did not observe any effect of LR on bone parameters at 4 week treatment. Interestingly however, at 8 weeks a significant 30.3% increase in femoral trabecular bone volume fraction was identified in L. reuteri treated mice. Examination of BM cellular phenotype and gene expression demonstrated that L. reuteri increased numbers of CD4 + T cells and significantly reduced RANKL expression at 4 weeks but not at 8 weeks. OPG and TGFβ expression were found to be elevated at 4 weeks, with TGFβ expression significantly increased at 8 weeks in the L. reuteri cohort. Together our data indicates that long term L. reuteri treatment increases femoral trabecular bone in female mice via the regulation of immune cells and expression of key genes involved in osteoclastogenesis