Plasmin Prevents Dystrophic Calcification After Muscle Injury

Abstract

Extensive or persistent calcium phosphate deposition within soft tissues after severe traumatic injury or major orthopedic surgery can result in pain and loss of joint function. The pathophysiology of soft tissue calcification, including dystrophic calcification and heterotopic ossification (HO), is poorly understood; consequently, current treatments are suboptimal. Here, we show that plasmin protease activity prevents dystrophic calcification within injured skeletal muscle independent of its canonical fibrinolytic function. After muscle injury, dystrophic calcifications either can be resorbed during the process of tissue healing, persist, or become organized into mature bone (HO). Without sufficient plasmin activity, dystrophic calcifications persist after muscle injury and are sufficient to induce HO. Downregulating the primary inhibitor of plasmin (α2-antiplasmin) or treating with pyrophosphate analogues prevents dystrophic calcification and subsequent HO in vivo. Because plasmin also supports bone homeostasis and fracture repair, increasing plasmin activity represents the first pharmacologic strategy to prevent soft tissue calcification without adversely affecting systemic bone physiology or concurrent muscle and bone regeneration. © 2016 American Society for Bone and Mineral Research.

Trauma-Induced Nanohydroxyapatite Deposition in Skeletal Muscle is Sufficient to Drive Heterotopic Ossification

“…Scale bar represents 500 nm. Image was obtained from a WT C57BL/6J following CTX injury at 3 days post injury when macrophage infiltration to damaged tissue is greatest [ 36 ]. c Longitudinal STiCSS analysis of Abcc6 +/− treated with either control (PBS) or clodronate-filled liposomes beginning at the time of injury.…”

“…Following adequate anesthesia with isoflurane, focal muscle injury was induced via intramuscular injection of 40 µL of 10 nM cardiotoxin (Accurate Chemical and Scientific Corp, Westbury, NY) into the posterior compartment of the lower leg using a lateral approach with a 28.5 g, 0.5 mL, insulin syringe as previously described [ 36 , 37 ]. Both the right and left posterior compartment muscles of the lower extremity were injured and analyzed by radiography for the presence of skeletal muscle calcification.…”

“…µCT images of the injured hind limbs were acquired following sacrifice at 55 kVp, 145 µA, 200 ms integration, 500 projections per 180° rotation, with a 20 µm isotropic voxel size (µCT40, Scanco Medical AG, Bassersdorf, Switzerland). After reconstruction, a volume of interest comprising the region of soft tissue calcification within the posterior compartment of the lower extremity was selected as previously described [ 36 ]. Mineralized tissue within the volume of interest was segmented from soft tissue using a threshold of 220/1000 (or 450.7 mgHA/cm 3 ), a Gaussian noise filter of 0.2, and support of 1.…”

“…Slides were then dehydrated and cleared in xylene before mounting with Permount. Histological quantification of skeletal muscle damage was assessed by light microscopy at × 200 magnification (Axio imager a1, ZEISS; Oberkochen, Germany) as previously described [ 36 ]. At least 3 mice were analyzed per group, with > 2 sections per mouse and > 4 images per section (i.e., minimum of 24 images per group).…”

“…Calculation for sample size was based upon previously published investigations [ 36 , 37 ]. Previously, we determined that 3 mice per groups were necessary to detect a 100% change in soft tissue calcification quantified by radiographic analysis.…”