S. Agarwal1, J. Peterson1, O. Eboda1, S. Loder1, C. Brownley1, A. Donneys1, D. Fine1, K. Stettnichs1, K. Ranganathan1, S. Wang1, S. Buchman1, P. Cederna1, B. Levi1 1University Of Michigan,Surgery,Ann Arbor, MI, USA
Introduction: Clinical and laboratory experience has shown that diabetes contributes to delayed wound and bony healing. However, the relationship between diabetes and the generation of heterotopic ossification (HO) following trauma has not been previously studied. By understanding how diabetes potentiates ectopic bone formation, we may be able to identify treatment strategies which target similar local or systemic factors to prevent HO in patients following trauma, large surface-area burns, and surgical procedures.
Methods: Male leptin-deficient (ob/ob) or wild type mice (C57BL/6 background) underwent 30% total body surface area burn injury with left hind limb Achille’s tenotomy. At 7 weeks, mice were injected with Microfil contrast and hind limbs were imaged with micro-CT to quantify aggregate vessel volume. Vessel volumes were normalized to hind limb mass to account for differences in limb size between diabetic and wild type mice. A second set of mice underwent micro-CT every 2 weeks up to 9 weeks to quantify HO volume (Hounsfield unit threshold 1000) after adjusting images to remove normal tibia and fibula bone.
Results: HO volume in diabetic mice was significantly lower than in wild type mice at 9 weeks following burn and tenotomy injury (3.99 mm3 v. 6.20 mm3, p<0.01). Diabetic mice exhibited a decreasing trend in HO volume from week 5 to week 9 based on micro-CT (r=-0.52, p =0.15), suggesting that HO had actually resorbed in these mice. The mean vessel volume in the hind limbs of diabetic mice 7 weeks after burn and tenotomy was significantly greater than the mean vessel volume in wild type mice (20.6 mm3 v. 4.7 mm3, p <0.05). When adjusting for limb mass, we found that diabetic legs continued to have more vessel volume (14.1 mm^3/g v. 7.2 mm3/g). Further examination of vessel architecture showed that the diabetic hind limbs had small vessel growth with disorganized character, while the wild type hind limbs had fewer, but larger vessels.
Conclusion: Here we use Microfil with micro-CT imaging to compare vessel formation between diabetic (leptin-deficient) and wild-type mice in a model of ectopic bone formation. Our findings demonstrate that despite robust small vessel formation at the site of hind limb trauma, diabetic mice produce less ectopic bone than wild type mice. We also found that diabetic mice actually exhibit resorption of ectopic, suggesting that these mice may be unable to sustain bone growth. This may be related to the disorganized pattern of vessel growth characteristic of the wounds in diabetic mice, as demonstrate by Microfil. Our findings may elucidate additional targets, such as the local vascular milieu, to prevent initial ectopic bone growth or recurrence following surgical excision.
