Collagen formation observed from healing calvarial defects with principal component analysis of Raman scattering

Bone healing is a complex process involving molecular changes. Bone matrix consists of collagen proteins that serve as the framework and minerals, calcium and phosphate, are deposited into the matrix accordingly. Raman spectroscopy is a promising technique to study bone mineral and matrix environments simultaneously. We studied the bone composition using 785 nm excitation during healing of subcritical calvarial defects without disrupting the fracture. Calvarial defects (in vivo) were created using a 1 mm burr drill on the parietal bones of Sprague-Dawley rats (n = 12). After 7 days, subjects were sacrificed and an additional defect (control) was created. Principal component analysis was utilized for the analysis of Raman spectra and helped in classifying normal and healing bone. Principal component 1 (PC1) shows that the major variation between in vivo and control defects and normal bone surface is at 958 cm-1 (ν1 phosphate band). PC2 shows a major variation at 1448 cm-1 (CH2 deformation). PC2 score distinguishes in vivo defects from normal surface and control defects. The decrease in crystallinity and mineral to matrix ratio at the healing site as revealed by Raman confirms the new bone formation. Scanning electron and optical microscopy show the formation of newly generated matrix by means of bony bridges of collagens. The surface roughness increases by 23% from control to in vivo defects, as revealed by optical profiler. Histology shows the decreased depth of in vivo defects and new blood vessels formation. Overall, the new collagen formation shows the scaffolding of the bone is growing during healing.