Mechano-chemistry of human femoral diaphysis revealed by correlative Brillouin–Raman microspectroscopy

Abstract

Brillouin–Raman microspectroscopy is presented as an innovative label-free all-optical investigation approachable to characterize the chemical composition and the mechanical properties of human tissues at micrometric resolution. Brillouin maps unveil mechanical heterogeneities in a human femoral diaphysis, showing a ubiquitous co-existence of hard and soft components, even in the most compact sections. The novel correlative analysis of Brillouin and Raman maps shows that the relative intensity of Brillouin peaks is a good proxy for the fraction of mineralized fibers and that the stiffness (longitudinal elastic modulus) of the hard component is linearly dependent on the hydroxyapatite concentration. For the soft component, a gradient of composition is found, ranging from an abundance of proteins in the more compact, external, bone to abundance of lipids, carotenoids, and heme groups approaching the trabecular, inner, part of the diaphysis. This work unveils the strong potential of correlative mechano-chemical characterization of human tissues at a micrometric resolution for both fundamental and translational research.

Applications of Vibrational Spectroscopy for Analysis of Connective Tissues

“…Recently, Cardinali et al [ 154 ] described an innovative application of Raman spectral imaging by using correlative Brillouin–Raman microspectroscopy to evaluate the spatial correlation of bone tissue mechanical and compositional properties at the microscale. Briefly, Brillouin spectroscopy is a nondestructive light-scattering technique which can assess the elastic modulus of a tissue, allowing identifying and mapping the distribution of hard and soft components within bone.…”

“…The Brillouin image ( b ) showing the distribution of hard components has a strong spatial correlation with the Raman image ( c ) of the distribution of tissue mineralization (R = 0.78). Image source: Cardinali et al [ 154 ].…”

Brillouin and Raman Micro-Spectroscopy: A Tool for Micro-Mechanical and Structural Characterization of Cortical and Trabecular Bone Tissues

“…The sample is mounted on an xyz translation stage for mapping and imaging. More details of the instrumentation can be found in [16,26].…”

“…The addition of a correlated Raman analysis allows us to assign these two components to the presence in the same scattering volume of an ordered phase of collagen bundles and a not-ordered phase of poorly oriented and mineralized collagen fibrils, non-collagenous proteins, and water. Whereas the first one is responsible for bone and cartilage tissue resistance to the tensile and compressive stresses, the latter is fundamental to producing a more accessible environment for metabolite diffusion and cellular growth and survival [25,26].…”

Non-contact elastography methods in mechanobiology: a point of view

“…c Average mechanical properties, collected in 2D maps, with 3 μm steps, over the 4 main regions of the diaphysal ring of a human femur. d Distribution of point averaged longitudinal elastic moduli (adapted from Cardinali et al 2020) the advantage of the technique for non-contact deep 3D mechanical characterization. Indeed, as no local contact is needed, either for detecting the signal nor for inducing the stress field, this technique is fully suited to operate in experimental conditions mimicking the in vivo situation, where physical access to the cells or to tissues cannot be assured.…”