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 6

Plasmin is not required for soft-tissue callus formation, but is essential for vascularization of fracture callus.

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Plasmin is not required for soft-tissue callus formation, but is essenti...
(A) Safranin O– and MSB-stained sections and immunofluorescence for VEGF-A and fibrin in the fracture callus at 14 DPF. Safranin O staining reveals the presence of the soft-tissue callus, while MSB-stained sections demonstrate that, while reduced in quantity compared with WT mice, the newly formed bone is histologically indistinguishable from newly formed bone in WT mice. VEGF immunofluorescence staining reveals VEGF expression within the hypertrophic chondrocytes (avascular soft-tissue callus, yellow asterisks) of WT and Plg–/– mice. Fibrin immunofluorescence reveals excessive fibrin deposition (white arrowheads) at the fracture site and in the hard-tissue callus of Plg–/– mice at 14 DPF compared with WT mice. Scale bars: 1 mm (top row); 100 μm (second, third, and fourth rows). (B) Representative 3D μCT reconstructions of angiogram contrast–perfused femurs show reduced revascularization of the fracture callus in Plg–/– mice at 14 DPF (color scale bar indicates vessel size range: black = 0.00 mm, red = 0.18 mm). Immunohistochemistry for CD31-positive vessels (red arrowheads) in the fracture site (yellow arrows). The interface of avascular soft-tissue callus and vascularized hard-tissue callus is marked by a black dashed line. The fracture gap is defined as the region between hard-tissue calluses (black double arrows). Scale bars: 1 mm (top row); 100 μm (middle and bottom rows). (C) Statistical analysis (Student’s t test) comparing number (top) and total area (bottom) of CD31-positive blood vessels in the callus. Four sections in each sample were averaged to constitute a single replicate (1 n). ***P < 0.01. Error bars represent SD. n ≥ 4 for each genotype.