Identifying the role of muscle trauma on heterotrophic ossification
Murphy, Jr., Paul Gilbert
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INTRODUCTION: Musculoskeletal trauma can lead to the abnormal accumulation of ectopic bone tissue, a process otherwise known as heterotrophic ossification. Many stem cell populations have been implicated in the osteogenic response initiated by injured musculoskeletal tissue. Pax7 expressing muscle stem cells, called satellite cells, have been identified as the major contributor to skeletal muscle repair. Additionally, cells positive for Pax7 are involved in the formation of ectopic bone that is induced by DBM implantation in the presence of trauma. In an attempt to better understand the mechanism of ectopic bone formation following injury, the presence of myogenic, osteogenic, chondrogenic, and adipogenic gene markers were explored within and in proximity to the implanted DBM. OBJECTIVES: To establish the contribution of post-natal muscle stem cells and associated relevant molecular mechanisms involved in the DBM induced ectopic bone formation following muscle trauma. METHODS: Tamoxifen inducible Pax7tm1(cre/ER2)Gaka/J mice were crossed with B6.Cg-Gt(ROSA)26sor<tm14(CAG-tdTomato)Hze>/J. Animals were back crossed with B6,129S7-Rag1tm1Mom/J mice, creating a Pax7/Ai14/Rag reporter enabling ectopic bone to be induced with human DBM. Mice were dosed with two injections of tamoxifen, 48 hours apart, then allowed a 30-day washout period. Animals received an implant of 50 mg of human DBM supplemented with 0.1 µg of bone morphogenetic protein 2 (BMP-2) and were placed on the femoral periosteum or intramuscularly on the upper hind limb, in order to induce ectopic bone formation. Muscle trauma was introduced to each limb after DBM surgery by a blunt impact trauma. The DBM implant, surrounding muscle and the femur were harvested either two, eight, or sixteen days following surgery. Tissue samples were radiographed, after which either mRNA or histological analysis was carried out. Gene expression was analyzed by real time qPCR using mRNA extracted from implant or muscle surrounding the implant. Tissue samples destined for histology were frozen, sectioned and then fixed on slides for processing. Fast Green/Safranin-O staining allowed for visualization of the cartilage and bone tissue at the site of the ectopic bone. Immunofluorescence was used to observe the presence of Pax7 positive, osteogenic and chondrogenic cells adjacent to or interacting with the DBM implant. RESULTS: Pax7 and Prx1 expressions within periosteal and intramuscular implants did not differ significantly in terms of time post-surgery or implant location. Certain chondrogenic (ColX) and osteogenic (Sp7 and DMP1) followed previously established patterns in expression following periosteal implantation surgery. Finally, the expression of Pax7 and Myf5 closely mirror one another in the muscle tissue which surrounded the periosteal implant. CONCLUSIONS: The combination of DBM implantation surgery and muscle contusion yields a large and varied genetic and physiological response. Many of the genes involved in the formation of ectopic bone following muscle injury were characterized. The data reaffirms previously studied patterns of expression following periosteal DBM administration, and suggests that this pattern is delayed for those implants located within the muscle.