Effects of high dose aspartame-based sweetener on the gut microbiota and bone strength in young and aged mice.

In a recent study examining the effects of manipulating the gut microbiome on bone, a control group of mice in which the microbiome was altered using a non-caloric, aspartame-based sweetener resulted in whole bone strength being 40% greater than expected from geometry alone, implicating enhanced bone tissue strength. However, the study was not designed to detect changes in bone in this control group and was limited to young male mice. Here we report a replication study examining how changes in the gut microbiome caused by aspartame-based sweetener influence bone. Male and female C57Bl/6 J mice were untreated or treated with a high dose of sweetener (10 g/L) in their drinking water from either 1 to 4 mo of age (young cohort; n = 80) or 1 to 22 mo of age (aged cohort; n = 52). Sweetener did not replicate the modifications to the gut microbiome observed in the initial study and did not result in an increase in bone tissue strength in either sex at either age. Aged male mice dosed with sweetener had larger bones (+17% femur section modulus, p<.001) and greater whole bone strength (+22%, p=.006) but the increased whole bone strength was explained by the associated increase in body mass (+9%, p<.001). No differences in body mass, whole bone strength, or femoral geometry were associated with sweetener dosing in males from the young cohort or females at either age. As we were unable to replicate the gut microbiota observed in the initial experiment, it remains unclear if changes in the gut microbiome can enhance bone tissue strength. Although prior work studying gut microbiome-induced changes in bone with oral antibiotics has been highly repeatable, the current study highlights the variability of nutritional manipulations of the gut microbiota in mice.

Microbiome-induced Increases and Decreases in Bone Matrix Strength can be Initiated After Skeletal Maturity.

Recent studies in mice have indicated that the gut microbiome can regulate bone tissue strength. However, prior work involved modifications to the gut microbiome in growing animals and it is unclear if the same changes in the microbiome, applied later in life, would change matrix strength. Here we changed the composition of the gut microbiome before and/or after skeletal maturity (16 weeks of age) using oral antibiotics (ampicillin + neomycin). Male and female mice (n = 143 total, n = 12-17/group/sex) were allocated into five study groups:1) Unaltered, 2) Continuous (dosing 4-24 weeks of age), 3) Delayed (dosing only 16-24 weeks of age), 4) Initial (dosing 4-16 weeks of age, suspended at 16 weeks), and 5) Reconstituted (dosing from 4-16 weeks following by fecal microbiota transplant from Unaltered donors). Animals were euthanized at 24 weeks of age. In males, bone matrix strength in the femur was 25-35% less than expected by geometry in mice from the Continuous (P=.001), Delayed (P=.005), and Initial (P=.040) groups as compared to Unaltered. Reconstitution of the gut microbiota led to a bone matrix strength similar to Unaltered animals (P=.929). In females, microbiome-induced changes in bone matrix strength followed the same trend as males but were not significantly different, demonstrating a sex-dependent response of bone matrix to the gut microbiota. Minor differences in chemical composition of bone matrix were observed with Raman spectroscopy. Our findings indicate that microbiome-induced impairment of bone matrix in males can be initiated and/or reversed after skeletal maturity. The portion of the femoral cortical bone formed after skeletal maturity (16 weeks) was small; suggesting that microbiome-induced changes in bone matrix occurred without osteoblast/osteoclast turnover through a yet unidentified mechanism. These findings provide evidence that the mechanical properties of bone matrix can be altered in the adult skeleton.

This study looked at how changes in the gut microbiome affect bone strength in adult mice. The gut microbiome of male and female mice was altered either before or after skeletal maturity. In male mice, those with altered microbiomes had weaker bones (a 25-35% reduction). Alterations to the gut microbiome after skeletal maturity had the same effect as lifelong changes, and restoration of an altered gut microbiome after skeletal maturity reversed the effect. Female mice showed a similar trend, but the changes were not statistically significant. The study concluded that changes in the gut microbiome can weaken bone strength in adult male mice in as short as two months, but this effect can be reversed by restoring the microbiome. These changes seem to occur without removal and replacement of bone tissue using the common bone remodeling processes, suggesting an unknown mechanism. This research provides new evidence that gut bacteria can affect bone strength suggesting the possibility that the microbiome can influence bone fragility.

Effects of long-term high dose aspartame on body mass, bone strength, femoral geometry, and microbiota composition in a young and aged cohort of male and female mice.

Background: Recent reassessment of the safety of aspartame has prompted increased evaluation of its effect on the health of a range of tissues. The gut microbiome is altered by oral aspartame. One prior study suggested that changes in the microbiome caused by aspartame could influence the strength of bone in young skeletally developing mice. Here we ask how aspartame influences bone in mice of different age and sex. Objective: The objective of this study was to determine the effect of aspartame on the bone strength and gut microbiota of young and aged mice. Methods: Male and female C57Bl/6J mice were untreated or treated with a high dose of aspartame in their drinking water from 1 month of age until 4 (young cohort; n = 80) or 22 months (aged cohort; n = 52). Results: In aged males, mice treated with aspartame had greater body mass, whole bone strength, and femoral geometry relative to untreated. Specifically, in aged males, aspartame led to 9% increase in body mass (p < 0.001), 22% increase in whole bone strength (p = 0.006), and 17% increase in section modulus (p < 0.001) relative to untreated mice. Aged males and females receiving aspartame had a different microbiota than untreated mice and a decreased abundance of Odoribacter. No differences in body mass, whole bone strength, or femoral geometry were associated with aspartame dosing in young males or young or aged females. Conclusions: Aspartame treated aged males had greater whole bone strength and the effect appeared to be explained by greater body mass. Aspartame treatment did not alter whole bone strength in young males or young or aged females despite the aspartame having a similar effect on the microbiota of both aged males and females.

Fragile X Messenger Ribonucleoprotein 1 (FMR1), a novel inhibitor of osteoblast/osteocyte differentiation, regulates bone formation, mass, and strength in young and aged male and female mice.

Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene mutations lead to fragile X syndrome, cognitive disorders, and, in some individuals, scoliosis and craniofacial abnormalities. Four-month-old (mo) male mice with deletion of the FMR1 gene exhibit a mild increase in cortical and cancellous femoral bone mass. However, consequences of absence of FMR1 in bone of young/aged male/female mice and the cellular basis of the skeletal phenotype remain unknown. We found that absence of FMR1 results in improved bone properties with higher bone mineral density in both sexes and in 2- and 9-mo mice. The cancellous bone mass is higher only in females, whereas, cortical bone mass is higher in 2- and 9-mo males, but higher in 2- and lower in 9-mo female FMR1-knockout mice. Furthermore, male bones show higher biomechanical properties at 2mo, and females at both ages. Absence of FMR1 increases osteoblast/mineralization/bone formation and osteocyte dendricity/gene expression in vivo/ex vivo/in vitro, without affecting osteoclasts in vivo/ex vivo. Thus, FMR1 is a novel osteoblast/osteocyte differentiation inhibitor, and its absence leads to age-, site- and sex-dependent higher bone mass/strength.