Raman spectral classification of mineral- and collagen-bound water's associations to elastic and post-yield mechanical properties of cortical bone

Abstract

Water that is bound to bone’s matrix is implied as a predictor of fracture resistance; however, bound water is an elusive variable to be measured nondestructively. To date, the only nondestructive method used for studying bone hydration status is magnetic resonance variants (NMR or MRI). For the first time, bone hydration status was studied by short-wave infrared (SWIR) Raman spectroscopy to investigate associations of mineral-bound and collagen-bound water compartments with mechanical properties. Thirty cortical bone samples were used for quantitative Raman-based water analysis, gravimetric analysis, porosity measurement, and biomechanical testing. A sequential dehydration protocol was developed to replace unbound (heat drying) and bound (ethanol treatment) water in bone. Raman spectra were collected serially to track the OH-stretch band during dehydration. Four previously identified peaks were investigated: I3220/I2949, I3325/I2949 and I3453/I2949 reflect status of organic-matrix related water (mostly collagen-related water) compartments and collagen portion of bone while I3584/I2949 reflects status of mineral-related water compartments and mineral portion of bone. These spectroscopic biomarkers were correlated with elastic and post-yield mechanical properties of bone. Collagen-water related biomarkers (I3220/I2949 and I3325/I2949) correlated significantly and positively with toughness (R2=0.81 and R2=0.79; p<0.001) and post-yield toughness (R2=0.65 and R2=0.73; p<0.001). Mineral-water related biomarker correlated significantly and negatively with elastic modulus (R2=0.78; p<0.001) and positively with strength (R2=0.46; p < 0.001). While MR-based techniques have been useful in measuring unbound and bound water, this is the first study which probed bound-water compartments differentially for collagen and mineral-bound water. For the first time, we showed an evidence for contributions of different bound-water compartments to mechanical properties of wet bone and the reported correlations of Raman-based water measurements to mechanical properties underline the necessity for enabling approaches to assess these new biomarkers noninvasively in vivo to improve the current diagnosis of those who may be at risk of bone fracture due to aging and diseases.

Bone quality changes associated with aging and disease: a review

“…[5][6][7] Bone turnover is generally measured by histomorphometry, 8 although it can be approximated from urinary turnover-markers. 9 Macroscopic cortical porosity has been measured by microcomputed tomography (microCT) 10 MRI; 11 however, newer methods exist on the basis of Raman spectroscopy 12,13 and short-wave infrared Raman spectroscopy 14 that have also been applied to measure water content. Damage has been assessed by basic Fuschin staining 15 and more recently by synchrotron radiation transmission X-ray microscopy with an X-ray negative stain.…”

Non-destructive NIR spectral imaging assessment of bone water: Comparison to MRI measurements

“…It is clear that a limiting factor in the investigation of water in bone is the lack of an appropriate laboratory tool for such studies. Although gravimetric analyses can be performed [50, 52], such studies leave the tissues of interest dehydrated, and not suitable for most further analyses. We envision NIRSI being useful for assessment of water content at high spatial resolution in animal models of disease and for evaluation of the effect of therapeutics on bone quality.…”

“…More importantly, these techniques cannot map bound and free water in cortical bone at spatial resolutions beyond imaging resolution, which is on the order of 0.5mm for whole bone cross section studies[45]. Recently, a custom built short wave Raman spectrometer was utilized to investigate water in bone at high spatial resolution, but such analyses would be challenging using standard Raman spectrometers [42, 52]. …”

Novel Raman Spectroscopic Biomarkers Indicate That Postyield Damage Denatures Bone's Collagen

“…The applied load and actuator displacement were recorded to construct load‐displacement curves. These curves were then converted to stress‐strain curves to calculate postyield mechanical properties including toughness, PYT, and maximum flexural (ultimate) strength (σ max ) . Toughness was reported as work to fracture (ie, the area under the stress‐strain curve) and PYT was reported as the area under the postyield region of the stress‐strain curve.…”

“…Toughness was reported as work to fracture (ie, the area under the stress‐strain curve) and PYT was reported as the area under the postyield region of the stress‐strain curve. Applying the flexure formula to the maximum force provided maximum flexural (ultimate) strength …”