Critical-sized (8 mm) bilateral segmental defects were created in the femora of 12-week-old male RNU rats (Taconic Biosciences Inc., Hudson, NY) under isoflurane anesthesia (61). Limbs were stabilized by custom internal fixation plates that allow controlled transfer of ambulatory loads in vivo (43) and secured to the femur by four bicortical miniature screws (J.I. Morris Co., Southbridge, MA). Animals were given subcutaneous injections of buprenorphine (0.04 mg/kg) every 8 hours for the first 48 hours postoperatively and 0.013 mg/kg every 8 hours for the following 24 hours, with or without carprofen (4 to 5 mg/kg) every 24 hours for 72 hours. In addition, 5 ml of 0.9% NaCl was administered subcutaneously to aid in recovery. All procedures were performed in strict accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and the policies of the Institutional Animal Care and Use Committee at Case Western Reserve University (protocol no. 2015-0081) and the University of Notre Dame (protocol no. 14-05-1778).

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