Diagnosing Healing Calvarial Defects through Spectroscopic Methods


Student thesis: Doctoral Thesis

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Award date15 Apr 2019


Bone fracture healing is a complex process involving cellular and biochemical changes to regenerate the damaged bone and rebuild the biomechanical properties and original geometry. Bone matrix consists of collagen proteins that serve as the framework, and bone minerals, that are mostly calcium and phosphate, are deposited into the matrix accordingly. Bone, in its natural balance of components, provide extraordinary toughness and stiffness. However, bone fractures and diseases like osteoporosis, osteogenesis imperfecta, osteoarthritis etc. cause brittleness, fragility, loss of bone mass and severe pain. Through examination of bone tissue from healing and normal surface and control groups using rat models, this dissertation demonstrates the ability of spectroscopic methods including Raman spectroscopy, X-Ray fluorescence spectroscopy, electron microscopy, optical microscopy and optical profiling, to advance the knowledge of bone compositional changes during natural healing.

Rat calvarial defect is a non-load bearing, versatile and reproducible orthotropic model that allows studying bone regeneration. This helps with evaluation of biomaterials and bone tissue engineering approaches. A bone healing model is proposed that involves subcritical calvarial defects, which heal spontaneously, and optical methods to study the healing bone without mechanical perturbation. For this purpose, shallow defects (200 - 250 µm in depth) were made on the parietal bones of rats. Consequently, a heterogeneous material known as callus is formed to stabilize and eventually close the defect. The proposed method allowed studying the bone regeneration without removal of the callus. This is a favorable approach for accurate assessment of compositional changes.

Raman spectroscopy is a promising technique to study bone mineral and matrix environments simultaneously. Raman bone parameters mineral to matrix ratio, carbonate to phosphate ratio, crystallinity and collagen alignment etc. are directly related to bone health. First, 1mm defects were created on the skulls (in vivo) of Sprague-Dawley rats. After 7 and 14 days of healing, the subjects were sacrificed, and additional defects were similarly created (control). Raman spectroscopy (785nm) was performed at the two time points and defect types. Spectra were quantified by mineral/matrix ratio, carbonate/phosphate ratio and crystallinity. Results revealed that mineral/matrix of in vivo defects was lower than that of controls by ~34% after 7 days and ~21% after 14 days. Carbonate/phosphate was 8% and 5% higher while crystallinity was 7% and 3% lower, respectively. Optical profiling showed that surface roughness increases 1.2% from controls to in vivo after 7 days, then decreased 13% after 14 days. Overall, the results showed maturation of mineral crystals during healing and agreed with microscopic assessment.

Second, principal component analysis (PCA) followed by linear discriminant analysis (LDA) were applied to the Raman spectra to help identify the biochemical content contained within. Raman spectra were obtained using 785nm excitation as before. Principal component 1 (PC1) of PCA shows that the major variation between in vivo and control defects and normal bone surface is at 958 cm-11 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. Optical profiling shows the increase in surface roughness in in vivo defects compared to controls. 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.

Both major components like calcium and phosphorus, and trace elements like iron, zinc, strontium etc., play important roles in bones health. Iron is vital for oxygen transport and known to play important role in collagen synthesis. Zinc is known for bone growth and development. Third, major and trace components during healing of subcritical calvarial defects were investigated. Energy dispersive X-ray fluorescence (EDXRF) was used to examine the defect types without mechanically perturbing the healed bone. Fundamental parameters method (FP) was used for quantitative analysis. Compared to control defects, iron has been found to increase locally at healing site by ~6.7-fold after 7 days that drops by more than 50% after 14 days. Similarly, zinc and potassium concentrations are increased in in vivo defects whereas calcium and phosphorus found decreased. Raman results show the large variation at collagen peaks after 7 days, which significantly reduced after 14 days. New collagen formation is also observed with scanning electron microscopy, optical microscopy, and surface profiling. Iron in particular, and also likely zinc, serve as catalyst for collagen formation during bone healing.

Lastly, this thesis concludes and describes the potential of future work. New directions with some preliminary results are proposed based on our methodology and assessment that could further improve our knowledge regarding fracture healing process.

    Research areas

  • Raman spectroscopy, calvarial defects, Bone healing, Tissue characterization