Bifidobacterium longum supplementation improves age-related delays in fracture repair

Abstract

Age-related delays in bone repair remains an important clinical issue that can prolong pain and suffering. It is now well established that inflammation increases with aging and that this exacerbated inflammatory response can influence skeletal regeneration. Recently, simple dietary supplementation with beneficial probiotic bacteria has been shown to influence fracture repair in young mice. However, the contribution of the gut microbiota to age-related impairments in fracture healing remains unknown. Here, we sought to determine whether supplementation with a single beneficial probiotic species, Bifidobacterium longum (B. longum), would promote fracture repair in aged (18-month-old) female mice. We found that B. longum supplementation accelerated bony callus formation which improved mechanical properties of the fractured limb. We attribute these pro-regenerative effects of B. longum to preservation of intestinal barrier, dampened systemic inflammation, and maintenance of the microbiota community structure. Moreover, B. longum attenuated many of the fracture-induced systemic pathologies. Our study provides evidence that targeting the gut microbiota using simple dietary approaches can improve fracture healing outcomes and minimize systemic pathologies in the context of aging.

Keywords: Bifidobacterium longum; aging; femur fracture; gut microbiome; probiotics.

FIGURE 1

Bifidobacterium longum supplementation attenuates post‐traumatic body mass loss and bone loss within the lumbar spine. (a) Study design depicting the random assignment of 18‐month‐old female mice to receive PBS (vehicle control) or B. longum (probiotic) for 2 weeks followed by creation of femoral fracture and assessments of healing and systemic effects. (b) Bifidobacterium longum supplementation blunted the decrease in body weight during the early post‐fracture period. Data are expressed as percentage change from pre‐fracture (day 0) weight. Two‐way ANOVA followed by Sidak's multiple comparisons testing, *p < 0.05 versus PBS (n = 10/group). Changes in B. longum lumbar spine (L1–L5) (c) bone mineral density (BMD), and (d) bone mineral content (BMC). Data are expressed as percentage change from pre‐fracture (day 0) values. Two‐way ANOVA followed by Sidak's multiple comparisons testing, *p < 0.05 versus PBS (n = 10/group). (e) Bifidobacterium longum supplementation increased the trabecular thickness (Tb.Th), bone surface density (BS/BV), and decreased structure model index (SMI) of the trabecular bone within the L3 vertebral body at day 35 post‐fracture. Student's t test, *p < 0.05 (n = 7‐8/group). Each data point represent an independent observation.

FIGURE 2

Supplementation with Bifidobacterium longum accelerates and enhances bone repair. (a) MicroCT analyses of fracture calluses at day 14 postfracture revealed a significant decrease in callus size (TV) and bone volume (BV), and an increase in callus bone volume fraction (BV/TV) in B. longum‐supplemented mice (n = 5–7/group). At day 21 postfracture, there was a significant increase in callus BV and BV/TV in mice supplemented with B. longum (n = 6–9/group). Student's t test, *p < 0.05. (b) Histomorphometric analyses showed a decreased callus size at day 14 postfracture in B. longum‐supplemented mice. Student's t test, *p < 0.05 (n = 4–5/group). Outliers were identified using the ROUT method in callus cartilage (excluded value 0.135%). (c) Bifidobacterium longum supplementation increased the maximum torque, strength, and energy at failure of fractured bones at day 35 postfracture. Student's t test, *p < 0.05 (n = 8–9/group). (d) Radiographs of two representative femora from each group at day 35 postfracture and radiographic scoring shows increased cortical bridging and remodeling in B. longum‐supplemented mice. Mann–Whitney U test, *p < 0.05 versus PBS (n = 7–9/group). Each data point represent an independent observation.

FIGURE 3

Bifidobacterium longum supplementation maintains the integrity of the intestinal barrier. (a) Bifidobacterium longum supplementation prevented increase in plasma FITC‐dextran concentrations after fracture. *p < 0.05 versus PBS (n = 6–7/group/timepoint). Values of the same color not sharing a common letter are significantly different, P < 0.05. (b) Serum endotoxin levels were significantly lower in B. longum‐supplemented mice at day 21 postfracture. *p < 0.05 versus PBS (n = 4–6/group/timepoint). Values of the same color not sharing a common letter are significantly different, p < 0.05. (c) Serum lipopolysaccharide binding protein (LBP) levels were significantly higher in B. longum‐supplemented mice at days 3, 14, and 21 postfracture. *p < 0.05 versus PBS. Values of the same color not sharing a common letter are significantly different, p < 0.05 (n = 4–5/group/timepoint). (d) Small intestine Aoah gene expression did not change in the B. longum‐supplemented mice but increased at days 14 and 21 postfracture in PBS‐treated mice. (e) Colon Aoah gene expression did not change in the B. longum‐supplemented mice but increased at day 7 postfracture in the PBS‐treated mice. Changes in (f) small intestine and (g) colon tight junction gene expression compared to baseline no fracture (day 0) shows a differential response to fracture in B. longum and PBS‐supplemented mice. Data are expressed as percent change from pre‐fracture (day 0) values. Two‐way ANOVA followed by Sidak's multiple comparison testing, values of the same color not sharing a common letter are significantly different, p < 0.05 (n = 4–5/group/timepoint).

FIGURE 4

Bifidobacterium longum supplementation influenced the fracture‐induced systemic inflammatory response. (a) Heat map of serum cytokines at day 3 (n = 4–5/group) and 7 (n = 5–6/group) postfracture. Serum IL‐10 and SDF1‐α increased in PBS control mice and VEGFa increased in B. longum‐supplemented mice at day 7 compared to day 3 postfracture. *p < 0.05. Each data point represent an independent observation. Outliers were identified using the ROUT method in IL‐10 (excluded value 126 pg/ml) and VEGFa (excluded values 40.3 and 74.7 pg/ml). (b) Fracture induced an increase in serum lipocalin‐2 levels in PBS control mice, which were significantly lower in B. longum‐supplemented mice. *p < 0.05 versus PBS (n = 4–5/group/timepoint). (c) Fracture induced a significant increase in small intestine lipocalin‐2 (Lcn2) gene expression in PBS control mice, but not in B. longum‐supplemented mice. Data were assessed using a two‐way ANOVA followed by Sidak's multiple comparison testing. Values of the same color not sharing a common letter are significantly different, p < 0.05 (n = 4–5/group/timepoint).

FIGURE 5

Bifidobacterium longum supplementation dampens fracture‐induced dysbiosis. (a) Principal coordinates ordination analyses (PCoA) of fecal beta diversity. (b) Fecal alpha diversity was not affected by fracture in B. longum‐supplemented mice. (c) Detailed relative abundance of bacterial taxa at the genera level within fecal samples prior to gavage (day −14), prior to fracture (day 0), and at days 3, 7, 14, and 21 postfracture. (d) Changes in the relative abundances of specific genera throughout fracture healing. (e) Bifidobacterium longum supplementation increased and maintained the abundance of Ralstonia mannitolilytica, Halobacterium hubeiense, Faecalibaculum rodentium, and Butyricimonas throughout fracture healing. Values of the same color not sharing a common letter are significantly different, p < 0.05. Data were assessed using a two‐way ANOVA followed by Sidak's multiple comparison testing. Each data point represent an independent observation (n = 4/group/timepoint).