Osteogenic Gene Expression Correlates With Development of Heterotopic Ossification in War Wounds

Abstract

Background Heterotopic ossification (HO) is a frequent complication of modern wartime extremity injuries. The biological mechanisms responsible for the development of HO in traumatic wounds remain elusive. Question/purposes The aims of our study were to (1) characterize the expression profile of osteogenesis-related gene transcripts in traumatic war wounds in which HO developed; and (2) determine whether expression at the mRNA level correlated with functional protein expression and HO formation. Methods Biopsy specimens from 54 high-energy penetrating extremity wounds obtained at the initial and final surgical débridements were evaluated. The levels of selected osteogenic-related gene transcripts from RNA extracts were assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. As a result of its key role in osteogenesis, the concentration of BMP-2 in the effluent of 29 wounds also was determined. Conclusions Important differences exist in the osteogenic gene expression profile of wounds in which HO developed compared with wounds in which it did not develop. The upregulation of multiple osteogenesis-related gene transcripts indicates the presence of a proosteogenic environment necessary for ectopic bone formation in traumatic wounds.

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

“…Total RNA was isolated from skeletal muscle samples as previously described [9]. A custom-made low-density reverse transcription-polymerase chain reaction (RT-PCR) array consisting of 96 primer sets (including respective forward and reverse primers) for 83 rat-specific osteogenic, chondrogenic, adipogenic, and angiogenic genes as well as six housekeeping and seven quality control genes (SABiosciences, Gaithersburg, MD, USA) was used to assess gene expression (genes and their function listed in Supplemental Table 1 [Supplemental materials are available with the online version of CORR 1 .]).…”

“…A custom-made low-density reverse transcription-polymerase chain reaction (RT-PCR) array consisting of 96 primer sets (including respective forward and reverse primers) for 83 rat-specific osteogenic, chondrogenic, adipogenic, and angiogenic genes as well as six housekeeping and seven quality control genes (SABiosciences, Gaithersburg, MD, USA) was used to assess gene expression (genes and their function listed in Supplemental Table 1 [Supplemental materials are available with the online version of CORR 1 .]). Quantitative RT-PCR and dissociation curve analyses were performed as previously described [9]. Cycle threshold (Ct) measurements per samples were normalized using GAPDH.…”

“…In this report, we use our model to address (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 subsequent to calcium deposition, tissue mineralization, and ectopic bone formation. It is important to confirm the timing and upregulation of bone-related genes and proteins in our model because some observational clinical studies with soft tissue injury (without fracture or amputation) have shown elevated levels of such genes in a minority of cases [6,9,12].…”

Identifying the Cellular Mechanisms Leading to Heterotopic Ossification

“…However, it must be noted that these studies have not been validated in a civilian population and the authors' stress that the complex interrelationship between the inflammatory markers remains incompletely understood. The same group has also looked upstream at osteogenic gene expression as a predictor of HO in combat wounds [105]. They report a significant upregulation in transcriptional activity in key osteogenesis-related genes (ALPL, BMP-2, BMP-3, COL2A1, COLL10A1, COL11A1, COMP, CSF2, CSF3, MMP8, MMP9, SMAD1, VEGFA) in patients that developed HO compared to those who did not.…”

CORR Insights®: Do Inflammatory Markers Portend Heterotopic Ossification and Wound Failure in Combat Wounds?

“…The Regenerative Medicine Team from the Naval Medical Research Center is making progress in describing the massive, but poorly regulated immune response to blast injury. In previous work [1][2][3], the Regenerative Medicine Team identified numerous cytokines and chemokines produced in this response. Interestingly, some cytokines such as Il-6 are associated with acute inflammatory response, while others appear to be associated with osteogenesis and may contribute to the formation of heterotopic ossification (HO).…”