Determining early markers of disease using Raman spectroscopy in a rat combat-trauma model of heterotopic ossification

Traumatic heterotopic ossification (HO) is the pathological formation of bone in soft tissue and is a debilitating sequela following acute trauma involving blast-related extremity musculoskeletal injuries, severe burns, spinal cord injury, and traumatic brain injury. Over 60% of combat related injuries and severe burns develop HO; often resulting in reduced mobility, chronic pain, ulceration, tissue entrapment, and reduced ambulation. Detection and prognosis is limited by current clinical imaging modalities (computed tomography, radiography, and ultrasound). This study identifies Raman spectral signatures corresponding to histological changes in a combat-trauma induced rat HO model at early time points prior to radiographic evidence of HO. HO was induced in Sprague-Dawley rats via blast over pressure injury, mid-femoral fracture, soft tissue crush injury, and limb amputation through the zone of injury. Rats were euthanized, and amputated limbs were formalin fixed and embedded in paraffin; 10 mu m sections were placed on gold slides, and paraffin was chemically removed. Tissues from sham-treated animals served as controls. Tissue maps consisting of Raman spectra were generated using a Raman microprobe system with an 80-90 mu m spot size and 785 nm excitation in regions exhibiting histological evidence of early HO development according to adjacent H&E sections. Factors were extracted from mapping data using Band-Target Entropy Minimization algorithms. Areas of early HO were highlighted by a Raman factor indicative of the presence of collagen. Identification of collagen as an early marker of HO prior to radiographic detection in a clinically relevant animal model serves to inform future clinical work.