Probiotics: Can it modulate fracture healing?

Abstract

Objective: Fractures remain a huge burden and their management adversely affects individuals' function and productivity during the lengthy healing period. Gut microbiota exerts a systemic influence on diverse aspects of host physiology, including bone. The primary objective of this study was to evaluate if oral probiotic treatment before or after a fracture in a mouse model could increase cytokines and biomarkers essential for bone healing with subsequent improvement in the biomechanical properties of the healed callus.

Methods: Femoral osteotomy and intramedullary pinning were performed on C57BL/6 mice. Group 1 received either control PBS or probiotic via oral gavage for 5 weeks before fracture (pre-fracture). Group 2 received equivalent treatments for 4 weeks only after fracture (post-fracture). Fracture calluses were harvested on day 3 and 7 for RT-qPCR to quantify osteogenic-related inflammatory cytokines and bone biomarkers. Fractured femurs were evaluated day 28 post-osteotomy via microstructural analysis (μCT) and biomechanical testing (torsion).

Results: Mice treated with probiotics pre-fracture (group 1) showed significantly increased gene expression on day 3 of cytokines TGF-β, IL-6 and IL-17F and a corresponding increase in gene expression on day 7 for Col1 and Runx2. Significant improvement was also seen in bone volume fraction, bone mineral density, tissue mineral density, maximum yield torque, stiffness and strain energy. Mice treated with probiotics post-fracture (group 2), demonstrated no changes in cytokine or bone marker gene expression with no significant changes on microstructural analysis. However, significant increases were seen in twist angle at failure and strain energy, with a corresponding reduction in torsional stiffness.

Conclusion: Our results suggest that oral probiotic administration, before or after a fracture, may sufficiently alter the gut flora microenvironment leading to improved bone healing biomechanical properties. The use of probiotics may provide a cost-effective and low-risk adjunctive therapy to improve fracture healing.

Fig 1. Proposed experimental fracture model to determine the effects of probiotic treatment pre-fracture (Group 1) vs. probiotic treatment post-fracture (Group 2).

Fig 2. Group 1 probiotic treatment pre-fracture.

A: Cytokine gene expression at day 3 after fracture using quantitative RT-PCR demonstrated increase in pro-inflammatory cytokines IL-6 and IL-17F (*P<0.05) with no difference in the expression of the anti-inflammatory cytokine TGF-β. No differences were seen at day 7. B: Bone marker gene expression (Col1, Col2 –Type 1, Type 2 Collagen) expression was only increased at day 7 after fracture for Col1 with no differences seen on day 3. Results showed VSL#3 promoted expression of Col1 at day 7. *P<0.05. C: Gene expression of transcription factor RUNX2 was elevated at day 3 and day 7 after fracture. *P<0.05. Means are presented ±SEMs.

Fig 3. Group 1 stereologic analysis of fractured limb callus at day 28 after fracture—μCT analysis confirmed significantly higher bone mineral density and tissue mineral density in the probiotic treatment pre-fracture group compared to control with increase in bone volume fraction.

*P<0.05.

Fig 4. Group 1 biomechanical testing of fractured limb callus at day 28 after fracture–Mechanical assessment of samples showed significantly higher maximum yield torque, strain energy and torsional stiffness in the probiotic treatment pre-fracture group *P<0.05.

Fig 5. Group 2 probiotic treatment post-fracture–No significant differences were detected in the gene expression of pro-inflammatory cytokines or mature bone markers in the fracture callus on day 3 and 7 compared to untreated controls P>0.05.

Means are presented ±SEMs.

Fig 6. Group 2 stereologic analysis of fractured limb callus at day 28 after fracture—μCT analysis confirmed no differences in bone volume, total volume, bone mineral density and tissue mineral density in the probiotic treatment post-fracture group compared to control.

P>0.05.

Fig 7. Group 2 biomechanical testing of fractured limb callus at day 28 after fracture–Mechanical assessment of samples showed a higher twist angle and strain energy in the probiotic treatment post-fracture group with a decrease in torsional stiffness *P<0.05.