Fibrinolysis is essential for fracture repair and prevention of heterotopic ossification
Masato Yuasa, Nicholas A. Mignemi, Jeffry S. Nyman, Craig L. Duvall, Herbert S. Schwartz, Atsushi Okawa, Toshitaka Yoshii, Gourab Bhattacharjee, Chenguang Zhao, Jesse E. Bible, William T. Obremskey, Matthew J. Flick, Jay L. Degen, Joey V. Barnett, Justin M.M. Cates, Jonathan G. Schoenecker
Masato Yuasa, Nicholas A. Mignemi, Jeffry S. Nyman, Craig L. Duvall, Herbert S. Schwartz, Atsushi Okawa, Toshitaka Yoshii, Gourab Bhattacharjee, Chenguang Zhao, Jesse E. Bible, William T. Obremskey, Matthew J. Flick, Jay L. Degen, Joey V. Barnett, Justin M.M. Cates, Jonathan G. Schoenecker
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Research Article Bone biology Hematology Hepatology Nephrology Pulmonology

Fibrinolysis is essential for fracture repair and prevention of heterotopic ossification

Abstract

Bone formation during fracture repair inevitably initiates within or around extravascular deposits of a fibrin-rich matrix. In addition to a central role in hemostasis, fibrin is thought to enhance bone repair by supporting inflammatory and mesenchymal progenitor egress into the zone of injury. However, given that a failure of efficient fibrin clearance can impede normal wound repair, the precise contribution of fibrin to bone fracture repair, whether supportive or detrimental, is unknown. Here, we employed mice with genetically and pharmacologically imposed deficits in the fibrin precursor fibrinogen and fibrin-degrading plasminogen to explore the hypothesis that fibrin is vital to the initiation of fracture repair, but impaired fibrin clearance results in derangements in bone fracture repair. In contrast to our hypothesis, fibrin was entirely dispensable for long-bone fracture repair, as healing fractures in fibrinogen-deficient mice were indistinguishable from those in control animals. However, failure to clear fibrin from the fracture site in plasminogen-deficient mice severely impaired fracture vascularization, precluded bone union, and resulted in robust heterotopic ossification. Pharmacological fibrinogen depletion in plasminogen-deficient animals restored a normal pattern of fracture repair and substantially limited heterotopic ossification. Fibrin is therefore not essential for fracture repair, but inefficient fibrinolysis decreases endochondral angiogenesis and ossification, thereby inhibiting fracture repair.

Authors

Masato Yuasa, Nicholas A. Mignemi, Jeffry S. Nyman, Craig L. Duvall, Herbert S. Schwartz, Atsushi Okawa, Toshitaka Yoshii, Gourab Bhattacharjee, Chenguang Zhao, Jesse E. Bible, William T. Obremskey, Matthew J. Flick, Jay L. Degen, Joey V. Barnett, Justin M.M. Cates, Jonathan G. Schoenecker

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Figure 8

Fibrinogen knockdown rescues hard-tissue callus union.

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Fibrinogen knockdown rescues hard-tissue callus union. Representative 3D...
Representative 3D and 2D μCT images, safranin O–stained sections, and immunofluorescence microscopy for fibrin in Plg–/– and Plg–/– Fbglo mice. 3D μCT images demonstrate heterotopic ossification (yellow arrowheads) and poorly macroscopically remodeled hard-tissue fracture callus in Plg–/– mice for 42 DPF formation. 2D μCT of the fracture sites in Plg–/– Fbglo mice shows cortical bridging by hard-tissue callus (yellow sharps), whereas this is not shown in Plg–/– mice (orange arrowheads). Histological sections confirmed united fractures with the presence of reactive woven bone in Plg–/– Fbglo mice in contrast to the disorganized callus with residual soft-tissue callus in Plg–/– mice (purple arrowheads). Immunofluorescence for fibrin confirms markedly less fibrin in the fracture site in Plg–/– Fbglo mice (red stain, fibrin, with DAPI blue counterstain). White dotted line denotes border of the femur and fracture callus. Scale bars: 1 mm. n ≥ 5 for each genotype.