9. Novel murine model of heterotopic ossification induced by spinal cord injury

Abstract

<h3>BACKGROUND CONTEXT</h3> Heterotopic ossification (HO) is a painful and debilitating condition causing abnormal bone formation in the soft tissues reported in up to 50% of polytrauma patients with spinal cord injury (SCI) and limb damage. In the absence of knowledge regarding the pathogenesis of HO, prophylactic treatment with NSAIDs, bisphosphonates, and radiation therapy do little to relieve the chronic pain and joint contractures resulting from HO or prevent its recurrence after surgical intervention to remove ectopic bone. A lack of clinically relevant animal models has been a major obstacle to understanding the pathogenesis of SCI-induced HO. <h3>PURPOSE</h3> The goal of this research is to characterize a clinically relevant mouse model of SCI-induced HO based on the hypothesis that simultaneous SCI and musculotendinous injury (MTI) can cause HO. <h3>METHODS</h3> Four-week-old C57BL/6J wild type mice were subjected to spinal cord transection between T10 and T11 using a pair of spring scissors via posterior open incision. The quadriceps were then crushed using a surgical clamp and a No. 15 surgical blade used to induce a 2mm MTI at the quadriceps insertion into patella in the LEFT hindlimb. The RIGHT hindlimb remained intact as internal procedural control. During the postoperative course, urine was expelled from the paraplegic using twice daily bladder massage. Soft food and water were placed in petri dishes on the bottom of the cages. Buprenorphine SR at 1mg/kg was administered subcutaneously on Day 0 and Day 4 to provide postoperative analgesia. All mice were euthanized at postoperative Day 21 when both hindlegs were dis-articulated at hip and ankle and fixed in formaldehyde overnight. High resolution micro-CT scans of the specimens were captured at the spatial resolution of 8µm. The volume of ectopic bone around the knee joint was quantified in a 3D reconstructed model. Undecalcified samples were then dehydrated, infiltrated, and embedded in low temperature MMA, and sectioned at 5µm. Consecutive sections were assessed by Von Kossa and Toluidine Blue staining to show ossification of peri-articular soft tissues, alkaline phosphatase (ALP) staining for osteoblasts, and tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts. Mann-Whitney U test was used for statistical analysis of ectopic bone volumes. <h3>RESULTS</h3> Of the seven mice euthanized at three weeks post SCI/MTI, two sustained patellar dislocation in the LEFT knee and were excluded from analyses. Quantitative microCT analysis of the remaining five revealed ectopic bone deposits in the peri-articular region of all LEFT knees but none in the RIGHT knees. The HO volume of the LEFT hindlimb was consistently higher than that of RIGHT hindlimb of each mouse (25th - 75th percentile 0.02 - 0.34 vs 0.00 - 0.00, P=0.09). Histological evaluation confirmed ectopic bone deposited in the peri-articular soft tissues of the LEFT knees while the musculotendinous tissue was intact on the RIGHT side. <h3>CONCLUSIONS</h3> This mouse model represents a novel, reproducible and clinically relevant preclinical model of SCI-induced HO that will facilitate investigation of pathogenic mechanisms and assessment of novel therapeutics for this condition. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.