Examining the influence of muscle trauma on heterotopic ossification and stem cell recruitment
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Citation
Abstract
INTRODUCTION: Damage to the musculoskeletal system can cause heterotopic ossification, also known as ectopic bone formation, a condition where bone tissue develops in soft tissues. The cell marker Pax7 is used to identify satellite cells which are the stem cells used by the body when skeletal muscle is damaged. Damage to skeletal muscle activate satellite cells to begin the muscle regeneration process by differentiating into myocytes. Previous studies in the lab have shown that muscle injury creates an osteoinductive environment that enhances heterotopic ossification. However, the satellite cell’s role in heterotopic ossification still needs to be brought to light so the scientific community can better their understanding.
OBJECTIVE: Compare heterotopic ossification in mice that have received a muscle injury and mice that have not, evaluate Pax 7 satellite cell recruitment towards the site heterotopic ossification and identify key genes expressed in mice that have a muscle injury and mice that do not.
METHODS: Tamoxifen inducible B6.Cg-Pax7tm1(cre/ER2)Gaka/J transgenic mice were crossed
with B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J to create Pax7/Ai14 reporter.
These animals were then crossed with B6.129S7-Rag1tm1Mom/J mice. This created inducible reporter mice that were able to receive the absorbable gelatin sponge loaded with recombinant human bone morphogenic protein 2 (BMP-2). Two tamoxifen doses were given at 48 hours apart to the mice, followed by a washout period of 96 hours. The mice were then implanted with 0.2 cm3 of sterile absorbable gelatin sponge loaded with 0.3 micrograms of BMP-2 along the periosteal surface of the femur. Muscle injury was induced to the quadriceps using a blunt mass object in select mice following surgery. Mice were harvested at either post operative day 2, 8 or 16. Tissue samples were either prepared for histology or mRNA extraction. Samples prepared for histology allowed for analysis of ectopic bone formation and quantification of satellite cells. Samples prepared for mRNA extraction were further prepared for bulk RNA sequencing.
RESULTS: Muscle trauma did not significantly change the amount of ectopic bone induced by BMP-2 supplemented absorbable gelatin sponge that was implanted on the periosteal surface of the femur. Also, while we did see an increase in satellite cells recruited to the area of heterotopic ossification in injured mice it was not statistically significant. Interestingly, we observed osteoclasts as early as post operative day 2 in injured mice.
CONCLUSIONS: Skeletal muscle trauma does not appear to significantly impact the volume of ectopic bone in BMP-2 induced ectopic bone formation. However, the increase in Pax7 derived satellite cells in the tissues of injured mice may provide evidence for the satellite cell’s role in heterotopic ossification. Osteoclasts were also seen in injury mice as early as post operative day 2. This finding suggests that osteoclasts may have a role in ectopic bone formation in the initial stages of chondrogenesis. Further studies to make clear what osteoclast’s involvement in these stages are needed to better our understanding.