Characterizing heterotopic ossification development following muscle injury
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Abstract
INTRODUCTION: Musculoskeletal trauma is a well-known cause of heterotopic ossification, also known as ectopic ossification, a pathological process in which bone forms within soft tissues. Previous studies in the lab have shown that muscle trauma increases the amount of bone formed in demineralized bone matrix-induced animal models. Moreover, recent studies have shown that ectopic ossification's underlying mechanism follows a specific gene expression progression similar to endochondral ossification. Because of the inherent complexity of the biological composition of DBM, the need to use immune deficient animals with human DBM, and the experimental objective to clarify the role of BMP2 in the inductive process on musculoskeletal trauma on ectopic bone formation a simpler and better defined model of inducing ectopic bone formation was examined. For the studies reported here, defined concentrations of BMP2 were added to gelatin sponges in combination with a defined muscle injury to induce ectopic bone formation on bone formation.
OBJECTIVE: Characterize the effects muscle trauma on ectopic bone formation using absorbable gelatin sponge/BMP2 – induction model for ectopic bone formation. Bone volume and structure were assessed by MicroCT. The quantitative expression of specific mRNAs was used to examine: the induction and recruitment of stem cells: skeletal cell development and differentiation; and BMP signaling.
METHODS: Tamoxifen inducible B6.Cg-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 an inducible reporter mouse capable of receiving the absorbable gelatin sponge loaded with recombinant human bone morphogenic protein 2 (BMP-2). Between 8-10 weeks of age, mice received two tamoxifen injections within 48 hours apart, followed by a washout period of 96 hours. Animals were implanted with 0.2 cm^3 of sterile absorbable gelatin sponged loaded with 0.3 µg of BMP-2 along the periosteal surface of the femur bilaterally. Muscle injury was induced in select animals following surgery. Animals were harvested at either postoperative day (POD) two, eight, or sixteen. Tissue samples were either prepared for plain film radiographs and micro-computed tomography (µCT) or mRNA extraction. Samples selected for imaging were radiographed using plain film and then proceeded for µCT. Micro-computed tomography allowed for analysis of the ectopic bone and the creation of 3D renderings. Implant samples selected for mRNA extraction were further prepared to analyze gene expression using real time quantitative polymerase chain reaction (qPCR).
RESULTS: Muscle trauma did not significantly change the characteristics of ectopic bone formed in the BMP-2 supplemented absorbable gelatin sponge implant. However, a batch effect had been observed between samples that received two different batches of BMP-2, which differed significantly in tissue volume and trabecular morphology. Gene expression showed a progressive pattern of expression. Sox2 expression peaked early in POD 2 samples. In comparison, Prx1 and α-SMA expression peaked later on at POD 8 and 16. Early markers of chondrogenesis, Sox9, and Acan, peaked at POD 8, while late makers of chondrogenesis, Col10a1, and osteogenesis markers, RUNX2, Sp7, and DMP-1, peaked at POD16. Expression of various members of the BMP gene family showed that BMPs were endogenously induced over the course of ectopic bone formation and suggests that BMP signaling is central to progressive development of ectopic bone formation.
CONCLUSION: Skeletal muscle injury does not appear to significantly impact the formation of ectopic bone in gelatin sponge implants supplemented with 0.3 µg of BMP-2. However, batch 2 samples showed some distinction between injured and noninjured samples, but the sample size was too small to make any firm conclusion. Further studies with the same protocol would help create a more definitive conclusion. RNA analysis reaffirmed a previous study's findings showing a progression of gene expression similar to endochondral ossification. However, this study produced evidence suggesting a delay in either osteogenesis or in the apoptosis and resorption of the hypertrophic cartilage within this model. Further studies could introduce later harvest times at 21 or 31 days postoperative, allowing a better understanding of when osteogenesis peaks during ectopic ossification.