Early Characterization of Blast-related Heterotopic Ossification in a Rat Model

Abstract

Background Heterotopic ossification (HO) affects the majority of combat-related lower extremity wounds involving severe fracture and amputation. Defining the timing of early osteogenic-related genes may help identify candidate prophylactic agents and guide the timing of prophylactic therapy after blast and other combat-related extremity injuries. Questions/purposes Using a recently developed animal model of combat-related HO, we sought to determine (1) the timing of early chondrogenesis, cartilage formation, and radiographic ectopic bone development; and (2) the early cartilage and bone-related gene and protein patterns in traumatized soft tissue. Methods We used an established rat HO model consisting of blast exposure, controlled femur fracture, crush injury, and transfemoral amputation through the zone of injury. Postoperatively, rats were euthanized on Days 3 to 28. We assessed evidence of early ectopic bone formation by micro-CT and histology and performed proteomic and gene expression analysis. Results All rats showed radiographic evidence of HO within 28 days. Key chondrogenic (collagen type I alpha 1 [COL1a1], p = 0.016) and osteogenic-related genes (RuntThis work was supported by CDMRP (W81XWH-14-2-0010; PI-JAF) and BUMED (602115HP.3720.001.A1014; PI-JAF). Some of the authors are employees of the US Government. This work was prepared as part of their official duties. Title 17 U.S.C. §105 provides that ''Copyright protection under this title is not available for any work of the United States Government.'' Title 17 U.S.C. §101 defined a US Government work as a work prepared by a military service member or employees of the US Government as part of that person's official duties. The opinions or assertions contained in this paper are the private views of the authors and are not to be construed as reflecting the views, policy or positions of the Department of the Navy, Department of Defense nor the US Government. All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research 1 editors and board members are on file with the publication and can be viewed on request. Clinical Orthopaedics and Related Research 1 neither advocates nor endorses the use of any treatment, drug, or device. Readers are encouraged to always seek additional information, including FDA-approval status, of any drug or device prior to clinical use. Each author certifies that his or her institution approved the animal protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research. This work performed at the Naval Medical Research Center, Silver Spring, MD, USA. Conclusions We found that genes that regulate early chondrogenic and osteogenic signaling and bone development (COL1a1, RUNX-2, OCN, PHEX, and POU5F1) are induced early during the tissue reparative/healing phase in a rat model simulating a combat-related extremity injury. Clinical Relevance The ability to correlate molecular events with histologic and morphologic changes will assist re...

Targeted stimulation of retinoic acid receptor-γ mitigates the formation of heterotopic ossification in an established blast-related traumatic injury model

“…The etiology, pathophysiology and exact mechanism(s) orchestrating trauma-induced HO remaini poorly understood, although dysregulated immunological-neuro inflammation, production of local soluble osteogenic mediators, hypoxia and various risk factors have been identified as possible culprits [16, 19, 32]. Induction of chronic bacterial-induced inflammation and ostensibly, indolent infection of the residual limb and surrounding soft tissue are known clinical risk factors and are reported to be potentiators of ectopic bone formation [13].…”

“…After complete decalcification, tissues were embedded in paraffin wax, cut into serial 5µm thick longitudinal sections using a microtome and mounted onto glass slides for histology. Sections were used for immunohistochemistry or stained using hematoxylin-eosin (H&E) (Histoserv, Inc., Germantown, MD)[16]. …”

“…Reverse transcriptase polymerase chain reaction (RT-PCR) was used to convert 1 µg of RNA to cDNA. mRNA transcripts for 83 key genes involved in early chondrogenic, osteogenic and angiogenic signaling pathways (see Supplementary Table 1 for the complete list of genes and their function) were examined by real-time PCR using a custom low density microarray (Bio-Rad Laboratories, Hercules, CA) [16]. Relative gene transcript expression was calculated using the 2 −ΔΔCt method and normalized and compared to the gene expression levels of non-injured rat muscle tissue.…”

“…Heterotopic ossification, when triggered by traumatic injury or other major insults most often arises via endochondral ossification, one of the two essential processes by which the native skeleton forms and grows during embryogenesis and early postnatal life [16–19]. Skeletal elements including long bones, ribs and vertebrae are initially laid down as entirely cartilaginous structures in early embryogenesis.…”

“…Previously, we reported on the timing and kinetics of ectopic bone development in this trauma induced HO model. Using molecular and histological tools, we found that chondrogenesis became apparent approximately 3 days following injury and was subsequently followed by cartilage hypertrophy and endochondral bone formation within soft tissues surrounding the amputation site starting around post-injury day 10 [16]. Understanding the timing of these key developmental phases and events was quite important as it now provides a window for prophylactic administration of candidate therapies that could target mechanisms and pathways involved in endochondral ossification.…”