Open fractures of the leg are a reconstructive challenge. Early restoration of the soft tissue envelope has dramatically improved the outcome of these fractures with regard to deep infection and function. However the ideal tissue for covering open fractures remains controversial. Muscle is said to be superior, but the mechanism is unclear. A higher blood flow was thought to be the key factor although this has not been established. Angiogenesis is vital for fracture healing, and Vascular Endothelial Growth Factor (VEGF), has been shown to be pivotal. We have developed a novel murine open tibial fracture model to compare the vascularity of muscle and fasciocutaneous tissue and investigate their role in angiogenesis during fracture healing. An open tibial fracture, stripped of periosteum, was created in female C57Bl/10 mice, under Home Office Approval. Skeletal stabilization was achieved with a 0.38mm diameter intramedullary pin. Animals were divided into experimental groups which allowed exclusive comparison of the soft tissues. A piece of sterile, inert material (Polytetrafluoroethylene, PTFE), was inserted at the fracture site to exclude either muscle posteriorly (Fasciocutaneous group) or skin and fascia anteriorly (Muscle group). A control group was devised consisting of fracture plus stripping but no PTFE, the Skeletal Injury only group. Animals were harvested at days 3, 5, 7, 9, 14, 21 and 28 days post-fracture and healing was assessed histologically. Bridging of the fracture site, by callus containing woven bone was quantified by histomorphometry. Endpoint assessment was also achieved by peripheral Quantitative Computed Tomography (pQCT) and mechanical testing. Immunohistochemistry was performed on specimens, to estimate vascularity using an antibody to factor VIII related antigen, which selectively demonstrates vascular endothelium. Vascular densities were determined within the muscle and fasciocutaneous tissues adjacent to the fracture sites, by manual counting of immunostained vessels at high magnification (x500) using an eyepiece graticule. VEGF levels were measured by ELISA in tissue specimens and the level of the VEGF antagonist soluble VEGF Receptor 1 (sFlt-1) was also assayed to determine relative drive for new blood vessel formation. All statistical analyses were performed by 1 Way and 2 Way ANOVA. Fracture healing was more advanced beneath muscle. There was earlier bridging of the fracture site by callus, with 50% more bone content and a stronger union beneath muscle compared to fasciocutaneous tissue (p<0.05). However, significantly greater vascular densities per unit area were observed in fasciocutaneous tissue compared to muscle (p<0.0001) at all time points during fracture healing. The relative excess of VEGF over sFlt-1 receptor, defined as the “angiogenic drive”, was statistically significant during the time course of fracture healing (p=0.0001). We have studied the exclusive effects of muscle and fasciocutaneous tissue on the healing of open tibial fractures stripped of periosteum. We have demonstrated faster bridging beneath muscle compared to skin and fascia alone, comparable to our fracture control. However, there were significantly higher vascular densities in fasciocutaneous tissue compared to muscle. Temporal distribution of factors known to be pivotal for the angiogenic process have been established within soft tissues, demonstrating that the coverage of fractures by soft tissues plays an important role in establishing the biological milieu and creating the microenvironment for fractures to heal. Our results contradict the widely held view that muscle provides superior coverage of open fractures because of a higher capillary density. The data suggest that vascularity is not the only factor influencing the choice of soft tissues. Extrapolating our data to clinical practice, muscle should be the tissue of choice for covering high-energy open distal tibial shaft fractures.
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