Pilot study for detection of early changes in tissue associated with heterotopic ossification: moving toward clinical use of Raman spectroscopy

Abstract

Over 60% of combat-wounded patients develop heterotopic ossification (HO). Nearly 33% of them require surgical excision for symptomatic lesions, a procedure that is both fraught with complications and can delay or regress functional rehabilitation. Relative medical contraindications limit widespread use of conventional means of primary prophylaxis, such as nonspecific nonsteroidal anti-inflammatory medications and radiotherapy. Better methods for risk stratification are needed to both mitigate the risk of current means of primary prophylaxis as well as to evaluate novel preventive strategies currently in development. We asked whether Raman spectral changes, measured ex vivo, could be associated with histologic evidence of the earliest signs of HO formation and substance P (SP) expression in tissue biopsies from the wounds of combat casualties. In this pilot study, we compared normal muscle tissue, injured muscle tissue, very early HO lesions ( < 16 d post-injury), early HO lesions ( > 16 d post-injury) and mature HO lesions. The Raman spectra of these tissues demonstrate clear differences in the Amide I and III spectral regions of HO lesions compared to normal tissue, denoted by changes in the Amide I band center (p < 0.01) and the 1340/1270 cm⁻¹ (p < 0.05) band area and band height ratios. SP expression in the HO lesions appears to peak between 16 and 30 d post-injury, as determined by SP immunohistochemistry of corresponding tissue sections, potentially indicating optimal timing for administration of therapeutics. Raman spectroscopy may therefore prove a useful, non-invasive and early diagnostic modality to detect HO formation before it becomes evident either clinically or radiographically.

• Heterotopic ossification
• muscle
• Raman spectroscopy
• substance P
• war wounds

Declaration of interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper. The views expressed in this manuscript are those of the authors and do not reflect the official policy of the Department of the Army, Department of the Navy, the Department of Defense or the United States Government. We are military service members (or employee of the US Government). This work was prepared as part of our official duties. Title 17 U.S.C. 105 provides the “Copyright protection under this title is not available for any work of the United States Government”. Title 17 U.S.C. 101 defines a US Government work as a work prepared by a military service member or employee of the US Government as part of that person's official duties. Drs. Nicole Crane and Katherine Cilwa are employees of the Henry M. Jackson Foundation for the Advancement of Military Medicine.

We certify that all individuals who qualify as authors have been listed; each has participated in the conception and design of this work, the analysis of data (when applicable), the writing of the document and the approval of the submission of this version; that the document represents valid work; that if we used information derived from another source, we obtained all necessary approvals to use it and made appropriate acknowledgements in the document; and that each takes public responsibility for it.

This effort was supported (in part) by the US Navy Bureau of Medicine and Surgery under the Medical Development Program and Office of Naval Research work unit number (602115HP.3720.001.A1015), USAMRMC Military Medical Research and Development award OR090136, as well as the Orthopedic Trauma Research Program grant # OTRP W81XWH-07-1-0222. This study was approved by the Walter Reed Military Medical Center Institutional Review Board in compliance with all Federal regulations governing the protection of human subjects. The WRNMMC IRB approved protocol numbers are 352334 and 374863, and the protocol titles are “The Use of the Vacuum Assisted Wound Closure Device in Treating Extremity Wounds” and “The Orthopedic Discarded Tissue Protocol”. The multidisciplinary care of these patients would not have been possible without the dedicated efforts of everyone at the Walter Reed National Military Medical Center. Both civilian and military personnel have rendered skilled and compassionate care for these casualties. All of our efforts are dedicated to those who have been placed in harm's way for the good of our nation.

Supplementary material available online

Supplementary Figure 1.