MRM Insights: A Mouse Model for Studying Neurogenic Heterotopic Ossification in Spinal Cord Injury

Chan Gao

Ivy Sun

Every month, in the MRM Insights, a member of the MRM Network offers a unique perspective on the topics of stem cells and regenerative medicine. In this edition, Dr. Chan Gao, who serves as an Assistant Professor in the Departments of Medicine and Surgery, and Junior Scientist at the RI-MUHC, collaborates with Ivy Sun, an undergraduate research intern in his lab. Together, they present a mouse model for elucidating the mechanisms underlying spinal cord injury-associated neurogenic heterotopic ossification.

A Mouse Model for Elucidating the Mechanisms Underlying Spinal Cord Injury-associated Neurogenic Heterotopic Ossification

Heterotopic ossification (HO) is ectopic bone formation in soft tissues like muscle and tendon that causes pain, joint stiffness, and neurovascular compromise. Examples of injuries conducive to HO include those sustained in traffic accidents and falls, surgical replacement or reconstruction of joints, and repetitive overuse of muscle and tendon by elite athletes. HO is conceptualized as a form of aberrant tissue regeneration and repair characterized by prolonged inflammation. Available therapeutics for HO, including non-steroidal anti-inflammatory drugs (NSAIDs), bisphosphonate, and radiation therapy, are largely ineffective and raise safety concerns for long-term use.

Mouse model of SCI-associated NHO. Sensory neurons of dorsal root ganglion (DRG) from SCI+MTI mice exhibit distinct morphological characteristics when compared to those extracted from naïve Control mice. DRG neurons from Control mice (A) show normal neuronal bodies (blue arrows) and axonal connections (green arrows). DRG neurons from SCI+MTI mice (B) are characterized by abnormal cell bodies (orange arrows) and disrupted axonal connections (red arrows). Undecalcified bone stained with Von Kossa (upper) shows NHO formed in the damaged quadriceps with concomitant loss of orthotopic bone in SCI + MTI mice at postoperative 3 weeks (D) compared with naïve Control (C). Safranin O-stained adjacent sections (lower) show abnormal cartilage in the SCI+MTI knees. High magnification of ectopic bone (yellow square) in SCI+MTI knees shows organized columns of chondrocytes reminiscent of developing growth plate.

HO resulting from injuries to the central nervous system (CNS), i.e., brain and/or spinal cord, is called neurogenic HO (NHO). NHO was first recognized as a frequent and serious complication of traumatic spinal cord injury (SCI) in paraplegic veterans of World War I1. It is now diagnosed in more than 20% of individuals living with the consequences of severe SCI2 in which affects the muscles and tendons around big joints. Like orthotopic bone, ectopic bone is proposed to develop from progenitor cell condensation and osteogenic differentiation under the influence of local cytokines and growth factors11,12. Previous work using in vivo models has implicated endothelial cells, muscle fibro/adipogenic progenitors (FAPs), pericytes, and tenocyte precursors in HO3-7. Others suggest circulating osteogenic precursors (COP) in peripheral blood home to sites of bone remodeling and skeletal muscle injury to form ectopic bone8,9. This hypothesis is supported by clinical data documenting HO in specimens harvested from patients with predisposing conditions including CNS injuries10.

In the absence of a clinically relevant model to investigate pathogenetic mechanisms underlying SCI-associated NHO, we recently developed a mouse that mimics the clinical presentation of severe traumatic SCI with concomitant myotendinous injuries (SCI+MTI). Young adult mice undergo complete SCI by spinal cord transection at T9-10 followed by quadriceps muscle crush and tenotomy. Phenotypic characterization using histological analyses reveal ectopic bone and cartilage in the damaged quadriceps with concomitant loss of orthotopic bone in SCI+MTI mice euthanized at postoperative 3 weeks (Figure). Initial analysis of sensory neurons isolated from lumbar dorsal root ganglion (DRG) removed from below the SCI at postoperative 3 days reveal altered morphology. While this work associates sensory neurogenesis with osteogenesis in the context of SCI-associated NHO, the molecular pathways remain undefined. Further insight into those pathogenic mechanisms in our model will be obtained from unbiased proteomic profiling.

Proteomic analyses have been conducted on tissue specimens from patients with ossification of the posterior longitudinal ligament, thoracic ossification of ligamentum flavum and with traumatic HO. Statistically significant differences were demonstrated in pathways involved in inflammation and immunity, lipid metabolism, reactive oxygen species metabolism, angiogenesis, osteoblast differentiation of mesenchymal stem cells, and extracellular matrix mineralization17-19. Another pre-clinical study revealed dysregulation of TGFβ, MAPK, coagulation, and oxidative stress pathways in a rat model of skeletal muscle injury20. We will undertake a comprehensive proteomic analysis of DRG sensory neurons and quadriceps myotendinous tissue obtained from SCI+MTI mice. The approach will identify potential molecular targets for the development of novel diagnostic, prognostic and therapeutic applications.



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