Contents lists available at ScienceDirect Bone journal homepage: www.elsevier.com/locate/bone Case Report A novel rat model of heterotopic ossification after polytrauma with traumatic brain injury Rhys D. Brady a,b, ⁎ , Michael Z. Zhao a , Ker R. Wong a , Pablo M. Casilla-Espinosa a,b , Glenn R. Yamakawa a , Ryan C. Wortman a , Mujun Sun a , Brian L. Grills c , Richelle Mychasiuk a , Terence J. O'Brien a,b , Denes V. Agoston d , Peter V.S. Lee e , Stuart J. McDonald a , Dale L. Robinson e , Sandy R. Shultz a,b a Department of Neuroscience, Central Clinical School, Monash University, Melbourne, VIC, Australia b Department of Medicine, Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC, Australia c Department of Physiology, Anatomy, and Microbiology, La Trobe University, Bundoora, VIC, Australia d Department of Anatomy, Physiology, and Genetics, Uniformed Services University, Bethesda, MD, USA e Department of Biomedical Engineering, The University of Melbourne, Parkville, VIC, Australia ARTICLE INFO Keywords: Ectopic bone Fracture Musculoskeletal injury Femur Computed tomography Brady model ABSTRACT Neurological heterotopic ossification (NHO) is characterized by abnormal bone growth in soft tissue and joints in response to injury to the central nervous system. The ectopic bone frequently causes pain, restricts mobility, and decreases the quality of life for those affected. NHO commonly develops in severe traumatic brain injury (TBI) patients, particularly in the presence of concomitant musculoskeletal injuries (i.e. polytrauma). There are cur- rently no animal models that accurately mimic these combinations of injuries, which has limited our under- standing of NHO pathobiology, as well as the development of biomarkers and treatments, in TBI patients. In order to address this shortcoming, here we present a novel rat model that combines TBI, femoral fracture, and muscle crush injury. Young adult male Sprague Dawley rats were randomly assigned into three different injury groups: triple sham-injury, peripheral injury only (i.e., sham-TBI + fracture + muscle injury) or triple injury (i.e., TBI + fracture + muscle injury). Evidence of ectopic bone in the injured hind-limb, as confirmed by micro- computed tomography (μCT), was found at 6-weeks post-injury in 70% of triple injury rats, 20% of peripheral injury rats, and 0% of the sham-injured controls. Furthermore, the triple injury rats had higher ectopic bone severity scores than the sham-injured group. This novel model will provide a platform for future studies to identify underlying mechanisms, biomarkers, and develop evidence based pharmacological treatments to combat this debilitating long-term complication of TBI and polytrauma. 1. Introduction Neurological heterotopic ossification (NHO) is characterized by the formation of bone in soft tissue and around joints following an insult to the central nervous system (CNS) [1]. Traumatic brain injury (TBI) is one of the most common forms of CNS insult and is frequently induced by motor vehicle collisions, warzone injuries, slips and falls, and as- saults [2]. Due to the high impact nature of these events, TBI often occurs in the presence of a concomitant peripheral injury (e.g., fracture, muscle crush) [3]. Studies suggest that NHO is common in these polytrauma patients [4–7], with a recent study reporting that up to ~70% of trauma cases featuring TBI combined with severe injuries to the extremities resulting in NHO [4–8]. The formation of NHO typically occurs within 1 to 3 months of these combined injuries, with acute symptoms including warmth, edema, erythema, and muscle loss [3]. Early NHO diagnosis is therefore difficult as these symptoms are asso- ciated with other inflammatory conditions which commonly occur in patients with TBI and polytrauma [9–15]. As the formation of ectopic bone progresses, it can cause severe chronic pain, nerve entrapment, and reduced range of motion, which are associated with difficulty in sitting, standing, and walking [3]. Current intervention occurs after the manifestation of NHO and is limited to surgical resection, which is invasive, can result in recurrence, and frequently leaves patients with functional deficits [13,16,17]. There are neither prophylactic clinical treatments, nor predictive bio- markers for NHO after TBI, though the identification of these would https://doi.org/10.1016/j.bone.2020.115263 Received 17 December 2019; Received in revised form 1 February 2020; Accepted 3 February 2020 ⁎ Corresponding author at: Department of Neuroscience, Monash University, 6th Floor, The Alfred Centre, 99 Commercial Road, Melbourne, VIC 3004, Australia. E-mail address: rhys.brady@monash.edu (R.D. Brady). Bone 133 (2020) 115263 Available online 04 February 2020 8756-3282/ © 2020 Published by Elsevier Inc. T