Early detection of burn induced heterotopic ossification using transcutaneous Raman spectroscopy

Abstract

Introduction Heterotopic ossification (HO), or the abnormal formation of bone in soft tissue, occurs in over 60% of major burn injuries and blast traumas. A significant need exists to improve the current diagnostic modalities for HO which are inadequate to diagnose and intervene on HO at early time-points. Raman spectroscopy has been used in previous studies to report on changes in bone composition during bone development but has not yet been applied to burn induced HO. In this study, we validate transcutaneous, in-vivo Raman spectroscopy as a methodology for early diagnosis of HO in mice following a burn injury. Methods An Achilles tenotomy model was used to study HO formation. Following tenotomy, mice were divided into burn and sham groups with exposure of 30% surface area on the dorsum to 60° water or 30° water for 18 seconds respectively. In-vivo, transcutaneous Raman spectroscopy was performed at early time points (5 days, 2 and 3 weeks) and a late time point (3 months) on both the tenotomized and non-injured leg. These same samples were then dissected down to the bone and ex-vivo Raman measurements were performed on the excised tissue. Bone formation was verified with Micro CT and histology at corresponding time-points. Results Our Raman probe allowed non-invasive, transcutaneous evaluation of heterotopic bone formation. Raman data showed significantly increased bone mineral signaling in the tenotomy compared to control leg at 5 days post injury, with the difference increasing over time whereas Micro CT did not demonstrate heterotopic bone until three weeks. Ex-vivo Raman measurements showed significant differences in the amount of HO in the burn compared to sham groups and also showed differences in the spectra of new, ectopic bone compared to pre-existing cortical bone. Conclusions Burn injury increases the likelihood of developing HO when combined with traumatic injury. In our in-vivo mouse model, Raman spectroscopy allowed for detection of HO formation as early as 5 days post injury. Changes in bone mineral and matrix composition of the new bone were also evidenced in the Raman spectra which could facilitate early identification of HO and allow more timely therapy decisions for HO patients.

Identifying the Cellular Mechanisms Leading to Heterotopic Ossification

“…In vivo Raman spectroscopy has been employed to detect bone mineral deposition in a rodent HO model as soon as 5 days post injury [106]. In a similar manner, the same group have developed a near infra-red optical imaging system that can demonstrate HO formation 5 days after injury [107].…”

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

“…First, clinically relevant animal models have only recently been developed [21,30]. Second, clinical diagnostic methods are unable to accurately predict the site of HO development [5,20]. Third, analysis of human tissues obtained early in the débridement process has not been performed.…”

mTOR inhibition and BMP signaling act synergistically to reduce muscle fibrosis and improve myofiber regeneration

“…In vivo development of HO was assessed with microCT scans 20 days after injury (GE Healthcare Biosciences, using 80 kVp, 80 mA, and 1,100 ms exposure). Images were reconstructed and HO volume formation was analyzed using a calibrated imaging protocol as previously described (39).…”