Early Characterization of Blast-related Heterotopic Ossification in a Rat Model

Background: Heterotopic ossification (HO) affects the majority of combat-related lower extremity wounds involving severe fracture and amputation. Defining the timing of early osteogenic-related genes may help identify candidate prophylactic agents and guide the timing of prophylactic therapy after blast and other combat-related extremity injuries. Questions/purposes: Using a recently developed animal model of combat-related HO, we sought to determine (1) the timing of early chondrogenesis, cartilage formation, and radiographic ectopic bone development; and (2) the early cartilage and bone-related gene and protein patterns in traumatized soft tissue. Methods: We used an established rat HO model consisting of blast exposure, controlled femur fracture, crush injury, and transfemoral amputation through the zone of injury. Postoperatively, rats were euthanized on Days 3 to 28. We assessed evidence of early ectopic bone formation by micro-CT and histology and performed proteomic and gene expression analysis. Results: All rats showed radiographic evidence of HO within 28 days. Key chondrogenic (collagen type I alpha 1 [COL1α1], p = 0.016) and osteogenic-related genes (Runt-related transcription factor 2 [RUNX-2], p = 0.029; osteoclacin [OCN], p = 0.032; phosphate-regulating neutral endopeptidase, X-linked [PHEX], p = 0.0290, and POU domain class 5 transcription factor [POU5F], p = 0.016) and proteins (Noggin [NOG], p = 0.04, OCN, p = 0.02, RUNX- 2, p = 0.04, and substance P-1 [SP-1], p = 0.01) in the injured soft tissue, normalized to the contralateral limb and/or sham-treated naïve rats, increased on Days 3 to 14 postinjury. By 14 days, foci of hypertrophic chondrocytes, hyaline cartilage, and woven bone were present in the soft tissue surrounding the amputation site. Conclusions: We found that genes that regulate early chondrogenic and osteogenic signaling and bone development (COL1α1, RUNX-2, OCN, PHEX, and POU5F1) are induced early during the tissue reparative/healing phase in a rat model simulating a combat-related extremity injury. Clinical Relevance: The ability to correlate molecular events with histologic and morphologic changes will assist researchers and clinicians to understand HO and hence formulate therapeutic interventions.