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IntroductionSoft tissue calcification, including both dystrophic calcification and heterotopic ossification, may occur following injury. These lesions have variable fates as they are either resorbed or persist. Persistent soft tissue calcification may result in chronic inflammation and/or loss of function of that soft tissue. The molecular mechanisms that result in the development and maturation of calcifications are uncertain. As a result, directed therapies that prevent or resorb soft tissue calcifications remain largely unsuccessful. Animal models of post-traumatic soft tissue calcification that allow for cost-effective, serial analysis of an individual animal over time are necessary to derive and test novel therapies. We have determined that a cardiotoxin-induced injury of the muscles in the posterior compartment of the lower extremity represents a useful model in which soft tissue calcification develops remote from adjacent bones, thereby allowing for serial analysis by plain radiography. The purpose of the study was to design and validate a method for quantifying soft tissue calcifications in mice longitudinally using plain radiographic techniques and an ordinal scoring system.MethodsMuscle injury was induced by injecting cardiotoxin into the posterior compartment of the lower extremity in mice susceptible to developing soft tissue calcification. Seven days following injury, radiographs were obtained under anesthesia. Multiple researchers applied methods designed to standardize post-image processing of digital radiographs (N = 4) and quantify soft tissue calcification (N = 6) in these images using an ordinal scoring system. Inter- and intra-observer agreement for both post-image processing and the scoring system used was assessed using weighted kappa statistics. Soft tissue calcification quantifications by the ordinal scale were compared to mineral volume measurements (threshold 450.7mgHA/cm3) determined by μCT. Finally, sample-size calculations necessary to discriminate between a 25%, 50%, 75%, and 100% difference in STiCSS score 7 days following burn/CTX induced muscle injury were determined.ResultsPrecision analysis demonstrated substantial to good agreement for both post-image processing (κ = 0.73 to 0.90) and scoring (κ = 0.88 to 0.93), with low inter- and intra-observer variability. Additionally, there was a strong correlation in quantification of soft tissue calcification between the ordinal system and by mineral volume quantification by μCT (Spearman r = 0.83 to 0.89). The ordinal scoring system reliably quantified soft tissue calcification in a burn/CTX-induced soft tissue calcification model compared to non-injured controls (Mann-Whitney rank test: P = 0.0002, ***). Sample size calculations revealed that 6 mice per group would be required to detect a 50% difference in STiCSS score with a power of 0.8. Finally, the STiCSS was demonstrated to reliably quantify soft tissue calcification [dystrophic calcification and heterotopic ossification] by radiographic analysis, independent of the histopathologi...

Heterotopic ossification (HO), or the pathologic formation of bone within soft tissues, is a significant complication following severe injuries as it impairs joint motion and function leading to loss of the ability to perform activities of daily living and pain. While soft tissue injury is a prerequisite of developing HO, the exact molecular pathology leading to trauma-induced HO remains unknown. Through prior investigations aimed at identifying the causative factors of HO, it has been suggested that additional predisposing factors that favor ossification within the injured soft tissues environment are required. Considering that chondrocytes and osteoblasts initiate physiologic bone formation by depositing nanohydroxyapatite crystal into their extracellular environment, we investigated the hypothesis that deposition of nanohydroxyapatite within damaged skeletal muscle is likewise sufficient to predispose skeletal muscle to HO. Using a murine model genetically predisposed to nanohydroxyapatite deposition (ABCC6-deficient mice), we observed that following a focal muscle injury, nanohydroxyapatite was robustly deposited in a gene-dependent manner, yet resolved via macrophage-mediated regression over 28 days post injury. However, if macrophage-mediated regression was inhibited, we observed persistent nanohydroxyapatite that was sufficient to drive the formation of HO in 4/5 mice examined. Together, these results revealed a new paradigm by suggesting the persistent nanohydroxyapatite, referred to clinically as dystrophic calcification, and HO may be stages of a pathologic continuum, and not discrete events. As such, if confirmed clinically, these findings support the use of early therapeutic interventions aimed at preventing nanohydroxyapatite as a strategy to evade HO formation. Electronic supplementary material The online version of this article (10.1007/s00223-018-0502-5) contains supplementary material, which is available to authorized users.

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Chronic diseases in growing children, such as autoimmune disorders, obesity, and cancer, are hallmarked by musculoskeletal growth disturbances and osteoporosis. Many of the skeletal changes in these children are thought to be secondary to chronic inflammation. Recent studies have likewise suggested that changes in coagulation and fibrinolysis may contribute to musculoskeletal growth disturbances. In prior work, we demonstrated that mice deficient in plasminogen, the principal protease of degrading and clearing fibrin matrices, suffer from inflammation-driven systemic osteoporosis and that elimination of fibrinogen resulted in normalization of IL-6 levels and complete rescue of the skeletal phenotype. Given the intimate link between coagulation, fibrinolysis, and inflammation, here we determined if persistent fibrin deposition, elevated IL-6, or both contribute to early skeletal aging and physeal disruption in chronic inflammatory conditions. Skeletal growth as well as bone quality, physeal development, and vascularity were analyzed in C57BL6/J mice with plasminogen deficiency with and without deficiencies of either fibrinogen or IL-6. Elimination of fibrinogen, but not IL-6, rescued the skeletal phenotype and growth disturbances in this model of chronic disease. Furthermore, the skeletal phenotypes directly correlated with both systemic and local vascular changes in the skeletal environment. In conclusion, these results suggest that fibrinolysis through plasmin is essential for skeletal growth and maintenance, and is multifactorial by limiting inflammation and preserving vasculature.