INTRODUCTION: Heterotopic ossification is the formation of bone in the soft tissues of the body. It is an increasingly prevalent complication following certain fractures and major orthopedic surgeries. It leads to many complications, including swelling, redness, pain, and disability due to limited range of motion. Despite the prevalence of heterotopic ossification, the biological mechanisms responsible remain elusive. The current theory is that a stimulating event, such as musculoskeletal injury, releases a combination of circulating factors that promote heterotopic ossification, likely through the activation of the BMP-2 receptor is a primary pathway leading to heterotopic ossification. However, further research must confirm the underlying cellular and biochemical mechanisms.
OBJECTIVES: To compare the development of ectopic bone formation with and without muscle injury and determine the molecular effects the musculoskeletal injury has on BMP2-induced ectopic bone formation at the genetic level.
METHODS: Prx1/Ail2/Rag and Pax7/Ail2/Rag strains of mice were used in this study. Mice received two tamoxifen injections 48 hours apart, and surgery was performed following a one-month washout period. A gelatin sponge loaded with 0.1 µg of bone morphogenic protein 2 (BMP-2) was implanted in each hind limb against the femoral periosteum or intramuscularly, followed by muscle trauma to assigned animals by dropping a blunt mass. Mice were harvested on postoperative day (POD) 16, and radiographs, histological analysis, and quantitative polymerase chain reaction were performed on designated samples.
RESULTS: In the injury condition, mineralized bone tissue was clearly seen on radiograph images of subjects POD 16. Histological analysis on the periosteal implant subjects POD 16 confirmed no cartilage tissue or chondrocytes in the no injury group. However, cartilage and possible ectopic bone formation were seen in the injury samples. Prrx1 and Acta2 expression were significantly higher in the injury and no injury conditions than in the control. Sox9 was expressed significantly higher in the no injury condition compared to the control. Finally, Acan, Sox9, Runx2, and DMP1 were all expressed higher when the implant was placed on the periosteal surface rather than intramuscularly in the no injury condition.
CONCLUSIONS: The current study confirmed that gelatin sponge implants loaded with 0.1 µg of BMP2 and implanted on the periosteum and intramuscularly will induce ectopic bone growth. Muscle injury did affect the level of cartilage and ectopic bone formation. This demonstrates the additive effect musculoskeletal injury has on heterotopic ossification. The increased expression of Prrx1 confirms that Prrx1-expressing skeletal stem cells may contribute to ectopic bone formation. Higher Acta2 expression may be due to increased myofibroblasts following muscle injury or an unknown direct link between Acta2 and heterotopic ossification. Sox9 has been shown to activate the BMP signaling pathway known to promote heterotopic ossification. However, this link does not explain why there is only a significant increase in Sox9 expression in the no injury condition and no significant increase in BMP2 expression in any condition, nor Col10a1 and Acan. Further research is needed to confirm this study’s findings.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/48411 |
| Date | 15 March 2024 |
| Creators | Wilson, Melanie |
| Contributors | Bragdon, Beth C. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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