New Method for Raman Investigation of the Orientation of Collagen Fibrils and Crystallites in the Haversian System of Bone

Abstract

Knowledge of the organization of the components of bone is of primary importance in understanding how this tissue responds to stresses and provides a starting point for the design and development of biomaterials. Bone structure has been the subject of numerous studies. The mineralized fiber arrangement in cortical bone is either a twisted or orthogonal plywood structure. Both mineral models coexist in compact bone. Raman polarized spectroscopy offers definite advantages in the study of biological samples, enabling the simultaneous analysis of mineral and organic components and the determination of molecular orientation through the polarization properties of the Raman scattering. In this study, we used the Raman polarization approach to simultaneously investigate the orientation of collagen fibrils and apatite crystals in human cortical bone. Raman bands ratios were monitored as a function of sample orientation. Specific ratios were chosen--such as nu(3) PO(4)/nu(1) PO(4), amide III (1271 cm(-1))/amide III (1243 cm(-1)), and amide I/amide III (1243 cm(-1))--due to their sensitivity to apatite-crystal and collagen-fibril orientation. Based on this original approach, spatial changes were monitored as a function of distance from the Haversian canal. The results revealed simultaneous tilting in intra-lamellar collagen-fibril and mineral crystal orientations. These results are consistent with a twisted plywood organization in the Haversian bone structure at the lamellar level. But at molecular level, the co-alignment of the collagen fibrils and the apatite crystal is observed in the innermost lamellae and becomes gradually less ordered as the distance from the Haversian canal increases. This work highlights the interest of Raman spectroscopy for the multiscale investigation of bone structure.

Polarization in Raman spectroscopy helps explain bone brittleness in genetic mouse models

“…Notable differences in RS intensity between orthogonal bone orientations [ Fig. 3(e)] occur mostly at locations previously reported by the Penel group 8,26 to be sensitive to polarization: ν3 phosphate (1045 cm −1 ), amide III δðNHÞ band (1273 cm −1 ), 44 and amide I (1668 cm −1 ). The most striking differences occur at peak shoulders and would, therefore, be overlooked by many standard analyses.…”

“…However, traditional peak ratios only report a small portion of the known spectral changes that occur with polarization as previously characterized by others. 8,18,[25][26][27] Moreover, limiting analysis to established peak ratios may overlook crucial aspects of complex toughness phenotypes if they manifest as subtle peak widening or shifting.…”

Bone Material Properties in Osteogenesis Imperfecta

“…(15) Although their long axes align with that of the adjacent collagen fibril, the planes of stacks around and between fibrils may not be similar; there is some evidence that they vary significantly across local areas. (54) In addition to the highly ordered mineral platelet arrays/stacks, there is a disorganized hydrogen-phosphate phase, with both the ordered and disordered mineral elements being strongly associated with both citrate and water. (1) A recent article suggests that citrate acts to maintain the ordered platelet parallel arrays and also holds nonplatelet hydrogen-phosphate in a relatively immobile, highly hydrated phase between the platelets, thus maintaining a degree of disorder and preventing crystals increasing in size or thickness (which would result in increased bone fragility).…”

Relationship between the v2PO4/amide III ratio assessed by Raman spectroscopy and the calcium content measured by quantitative backscattered electron microscopy in healthy human osteonal bone

“…We chose the v 2 PO 4 and the amide III bands as it was previously shown that they have minimal susceptibility to tissue organization/orientation effects. [18][19][20][21][22] The Ca content (Ca mass fraction) was derived from BE signal intensities (gray levels in qBEI image) that reflect the local electron density of the bone material. For this purpose, the gray levels were standardized against reference materials (carbon and aluminum) with known electron densities (atomic numbers).…”