INTRODUCTION: Trauma to the musculoskeletal system can result in heterotopic ossification, a condition where aberrant bone tissue is synthesized and mineralized in the soft tissues of the body. Satellite cells expressing Pax7 are the predominant stem cell population found within adult skeletal muscle tissues. Once activated by trauma, satellite cells are primarily implicated in skeletal muscle regeneration by differentiating towards myocytes. Previous research in the lab has shown that no Pax7 derived cells were seen in either the fracture callus or periosteal ectopic bone induced by demineralized bone matrix (DBM). Questions however persist whether trauma can activate Pax7 cells to contribute to ectopic bone formation and whether muscle trauma will enhance the ability of DBM to induce ectopic bone in muscle.
OBJECTIVES: Identify how muscle trauma effects DBM-induced ectopic bone formation and characterize the contribution of the Pax7 satellite cell population in DBM-induced ectopic bone formation after muscle trauma.
METHODS: The tamoxifen inducible Pax7^tm1(cre/ER2)Gaka/J transgenic mice were crossed with B6.Cg-Gt(ROSA)26sor<tm14(CAG-tdTomato)Hze>/J to create Pax7/Ai14 reporter. These animals were subsequently crossed with B6,129S7-Rag1^tm1Mom/J mice. This created a transgenic reporter mouse that allows for the implantation of human DBM. Two tamoxifen doses (within 48 hours) were given to the animals approximately 31 days prior to surgery. Ectopic bone was induced by surgical implantation of DBM (50 mg) with 0.1 µg of bone morphogenic protein 2 (BMP-2) on the femoral periosteum or in the skeletal muscle tissue of the upper hind limb. Following implantation, mice received varying amounts of blunt force trauma to induce skeletal muscle trauma. Ectopic bone was then evaluated radiologically using plain film and micro-computed tomography, and histologically through fluorescence and brightfield microscopy. Micro-computed tomography allowed for the calculation of the ectopic bone volume, as well as the creation of 3D renderings of the ectopic bone. Fluorescence microscopy allowed for the visualization of recruited Pax7 positive satellite cells to the DBM-induced ectopic bone. Trichrome staining techniques allowed for the visualization and categorization of tissue types including skeletal muscle, un-mineralized and mineralized bone, and cartilage.
RESULTS: Muscle trauma did not significantly change the volume of ectopic bone that was induced by BMP-2 supplemented DBM that was implanted on either the periosteum or in skeletal muscle. However, the amount of BMP2 that was needed within DBM to induce ectopic bone within muscle was greatly decreased with muscle trauma. Intriguingly, muscle trauma resulted in the activation and recruitment of Pax7 positive cells to the DBM-induced ectopic bone in both periosteal and skeletal muscle implants.
CONCLUSIONS: Skeletal muscle trauma does not appear to impact the resulting bone volume of BMP2 supplemented DBM induced ectopic bone formation. However, the decreased dose of BMP-2 that was needed to induce ectopic bone formation within muscle suggests that trauma sensitized the stem cell populations that contribute to ectopic bone to BMP induction. The appearance of Pax7 within the newly formed ectopic bone with muscle trauma suggests that the muscle trauma effects the plasticity of Pax7 satellite enabling them to contribute to ectopic bone formation. Further research is needed to elucidate the molecular mechanism(s), which muscle trauma activates that favor ectopic bone formation and promotes the plasticity of Pax7 muscle satellite cells. These studies provide basis for the identification of novel therapeutic targets to treat heterotopic ossification.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/36582 |
| Date | 13 June 2019 |
| Creators | Moore, William |
| Contributors | Bragdon, Beth C., Gerstenfeld, Louis C. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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