Bone mineralization and collagen formation observed with principal component analysis of Raman scattering from healing calvarial defects

摘要

Bone healing is a complex process involving molecular changes. Bone matrix consists of collagen proteins that serve asthe framework and minerals, calcium and phosphate, are deposited into the matrix accordingly. Raman spectroscopy is apromising technique to study bone mineral and matrix environments simultaneously. We studied the bone compositionusing 785 nm excitation during healing of subcritical calvarial defects without disrupting the fracture. Calvarial defects(in vivo) were created using 1 mm burr drill on the parietal bones of Sprague-Dawley rats (n=8). After 7 days, subjectswere sacrificed and an additional defect (control) was created. Principal component analysis was utilized for the analysisof Raman spectra and helped in classifying normal and healing bone. Principal component 1 (PC1) shows that the majorvariation between in vivo and control defects and normal bone surface is at 958 cm~(-1) (ʋ1 phosphate band). PC2 shows amajor variation at 1448 cm~(-1) (CH_2 deformation). PC2 score distinguishes in vivo defects from normal surface and controldefects. The decrease in crystallinity and mineral to matrix ratio at the healing site as revealed by Raman confirms thenew bone formation. Scanning electron and optical microscopy show the formation of newly generated matrix by meansof bony bridges of collagens. The surface roughness increases by 23% from control to in vivo defects, as revealed byoptical profiler. Overall, the new collagen formation shows the scaffolding of the bone is growing during healing.