직접 마우스 외상 / 이소성 골화의 모델을 굽기

Summary

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

Abstract

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

Introduction

Heterotopic ossification (HO) is the formation of ectopic bone in which osteo-potent cells are aberrantly induced to form endochondral bone outside of the skeleton. While the details of the HO formation pathway are still largely unknown, an accepted paradigm includes three key factors: an inflammatory inciting incident, a permissive niche, and mesenchymal stem cells capable of forming bone.^1-3 HO is a common comorbidity complicating over 60% of major burn injuries, 65% of combat-related injuries, and 10% of invasive orthopaedic surgery cases.^4,5 However, it is often difficult to predict where HO will form because it can occur at sites of local injury or at distant locations that may be otherwise uninjured. This variability in location makes it difficult to intervene prophylactically to prevent reactive bone formation in a locally targeted manner. There are also congenital forms of HO such as fibrodysplasia ossificans progressiva (FOP) in which patients are prone to the development of robust HO in response to minor trauma or inflammatory insult. Powerful animal models using transgenic mice have reproduced this phenotype and provided insight to the molecular pathways that may also be important in trauma induced HO.^6-9 Translational research into the pathogenesis of non-congenital HO has used a wide variety of constructs ranging from injury alone to the implantation of exogenous osteo-inductive materials and/or cells.^10-13

In our prior work we have validated a simple and reliable model of HO formation in mice which does not require the administration of any exogenous material.^14-17 This model created two key conditions to initiate HO: local trauma and global inflammation. This was achieved through the use of an Achilles tenotomy (local trauma) combined with a distant burn injury (global inflammation). Mice received both treatments concurrently and were found to develop a robust amount of HO that could be analyzed by histologic, radiologic, and molecular means. Interestingly, concurrent burn injury significantly increased the amount of HO that formed and accelerated its developmental time-course.^14-16 HO developed at predictable sites around the calcaneus, ankle joint, and tibia/fibula of the limb that received the tenotomy. The reliability of HO development at a known location allowed for focused examination of molecular and histologic features in the early stages of ectopic ossification.^14,17 To date, 100% of mice (over 50 animals) with a tenotomy and concurrent burn injury have developed HO. Additionally, longitudinal 2D and 3D imaging and spectroscopic analysis were conducted to examine the growth pattern and biochemical make-up of HO.^15,16

Protocol

윤리 문 : 실험 동물 연구를위한 연구소의 여덟 번째 판 (ILAR, 2011)과 같다 : 모든 동물의 절차는 실험 동물의 사용 및 관리에 대한 가이드에 제공된 지침에 정의 된대로 좋은 동물 관행에 엄격한에 따라 수행 하였다 미시간 대학의 기관 동물 관리 및 사용위원회 (PRO0001553)에 의해 승인했다. 1. 마우스 외과 적 치료 참고 : 사용 8-10주 된 C57BL가 / 6 마​​우스. …

Representative Results

이 본 연구에서, 프로토콜은 외상의 이전에 발행 된 마우스 모델에 대한 설명 / 호. 14 ~ 17이 부상을 구울 부분 두께의 아킬레스 건 절단술 및 글로벌 염증 모욕과 동시에 지역화 근골격계 손상의 생성을 포함 유도 구울 수 있습니다. 이 시리얼 이미징 따라 할 수있는 건 절단술 사이트에서 반응 뼈의 안정적인 형성을 초래한다. 현재까지 모두 아킬레스 건 절단술을 유지하고 부상을 구울 수…

Discussion

Heterotopic ossification represents a major functional impairment faced by patients that sustain trauma, burns, and invasive musculoskeletal procedures. The most at-risk population are soldiers in modern conflicts with major blast injuries from mechanisms such as improvised explosive devices (IED).¹⁸ Improved body armor and forward positioned medical units allows for improved survival of major extremity injury. After initial stabilization and repair of their extremity injury, these patients are at high risk…

Materials

C57BL/6 mice	Jackson Laboratory	664	8-10 weeks old
Isoflurane – Fluriso	VET one, Boise, ID	V1 501017
Buprenorphine – Buprenex	Reckitt Benckiser Healthcare	NDC 12496-0757-1	0.3 mg/ml solution
Betadine	Owens and Minor, Mechanicsville, VA	2047PVP202
5-0 Vicryl sutures	Ethicon, Summerville, NJ	J493
Tegaderm Film, 6cm x 7cm	3M	1624W	Cut in half to properly cover burn site
µCT – GE eXplore Locus SP	GE Healthcare Pre-Clinical Imaging, London, ON, Canada
Microview 2.2 Advanced Bone Analysis Application	GE Healthcare Pre-Clinical Imaging, London, ON, Canada