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Öğe Assessment of spatially offset Raman spectroscopy to detect differences in bone matrix quality(Pergamon-Elsevier Science Ltd, 2023) Gautam, Rekha; Ahmed, Rafay; Haugen, Ezekiel; Unal, Mustafa; Fitzgerald, Sean; Uppuganti, SasidharSince spatially offset Raman spectroscopy (SORS) can acquire biochemical measurements of tissue quality through light scattering materials, we investigated the feasibility of this technique to acquire Raman bands related to the fracture resistance of bone. Designed to maximize signals at different offsets, a SORS probe was used to acquire spectra from cadaveric bone with and without skin-like tissue phantoms attenuating the light. Autoclaving the lateral side of femur mid-shafts from 5 female and 5 male donors at 100 degrees C and again at 120 degrees C reduced the yield stress of cortical beams subjected to three-point bending. It did not affect the volumetric bone mineral density or porosity. Without tissue phantoms, autoclaving affected more Raman characteristics of the organic matrix when determined by peak intensity ratios, but fewer matrix properties depended on the three offsets (5 mm, 6 mm, and 7 mm) when determined by band area ratios. The cut-off in the thickness of the tissue phantom layers was-4 mm for most properties, irrespective of offset. Matching trends when spectra were acquired without phantom layers between bone and the probe, & nu;1PO43 ? /Amide III and & nu;1PO43%(proline + OHproline) were higher and lower in the non-treated bone than in the autoclaved bone, respectively, when the thickness of tissue phantom layers was 4 mm. The layers, however, caused a loss of sensitivity to autoclaving related changes in & nu;3CO3/& nu;1PO43 ? and crystallinity. Without advanced post-processing of Raman spectra, SORS acquisition through turbid layers can detect changes in Raman properties of bone that accompany a loss in bone strength.Öğe Compositional assessment of bone by Raman spectroscopy(2021) Ünal, Mustafa; Ahmed, Rafay; Mahadevan-Jansen, Anita; Nyman, Jeffry S.Raman spectroscopy (RS) is used to analyze the physiochemical properties of bone because it is non-destructive and requires minimal sample preparation. With over two decades of research involving measurements of mineral-to-matrix ratio, type-B carbonate substitution, crystallinity, and other compositional characteristics of the bone matrix by RS, there are multiple methods to acquire Raman signals from bone, to process those signals, and to determine peak ratios including sub-peak ratios as well as the full-width at half maximum of the most prominent Raman peak, which is nu1 phosphate (nu 1PO4). Selecting which methods to use is not always clear. Herein, we describe the components of RS instruments and how they influence the quality of Raman spectra acquired from bone because signal-to-noise of the acquisition and the accompanying background fluorescence dictate the pre-processing of the Raman spectra. We also describe common methods and challenges in preparing acquired spectra for the determination of matrix properties of bone. This article also serves to provide guidance for the analysis of bone by RS with examples of how methods for pre-processing the Raman signals and for determining properties of bone composition affect RS sensitivity to potential differences between experimental groups. Attention is also given to deconvolution methods that are used to ascertain sub-peak ratios of the amide I band as a way to assess characteristics of collagen type I. We provide suggestions and recommendations on the application of RS to bone with the goal of improving reproducibility across studies and solidify RS as a valuable technique in the field of bone research.Öğe Effect of ribose incubation on physical, chemical, and mechanical properties of human cortical bone(Elsevier Ltd., 2023) Ünal, Mustafa; Uppuganti, Sasidhar; Dapaah, Daniel Y.; Ahmed, Rafay; Pennings, Jacquelyn S.; Willett, Thomas L.Raman spectroscopy (RS) is sensitive to the accumulation of advanced glycation end-products (AGEs), and it measures matrix-sensitive properties that correlate with the fracture toughness of human cortical bone. However, it is unclear whether sugar-mediated accumulation of AGEs affects the fracture toughness of human cortical bone in a manner that is consistent with the negative correlations between amide I sub-peak ratios and fracture toughness. Upon machining 64 single-edge notched beam (SENB) specimens from cadaveric femurs (8 male and 7 female donors between 46 years and 61 years of age), pairs of SENB specimens were incubated in 15 mL of phosphate buffered saline with or without 0.1 M ribose for 4 weeks at 37 °C. After acquiring 10 Raman spectra per bone specimen (n = 32 per incubation group), paired SENB specimens were loaded in three-point bending at a quasi-static or a high loading rate approximating 10−4 s−1 or 10−2 s−1, respectively (n = 16 per incubation group per loading rate). While 2 amide I sub-peak ratios, I1670/I1640 and I1670/I1610, decreased by 3–5% with a 100% increase in AGE content, as confirmed by fluorescence measurements, the ribose incubation to accumulate AGEs in bone did not affect linear elastic (KIc) nor non-linear elastic (KJc) measurements of bone's ability to resist crack growth. Moreover, AGE accumulation did not affect the change in these properties when the loading rate changed. Increasing the loading rate increased KIc but decreased KJc. Ribose incubation did not affect mineral-related RS properties such as mineral-to-matrix ratios, Type B carbonate substitutions, and crystallinity. It did however increase the thermal stability of demineralized bone (differential scanning calorimetry), without affecting the network connectivity of the organic matrix (i.e., maximum slope during a hydrothermal isometric tension test of demineralized bone). In conclusion, RS is sensitive to AGE accumulation via the amide I band (plus the hydroxyproline-to-proline ratio), but the increase in AGE content due to ribose incubation was not sufficient to affect the fracture toughness of human cortical bone.Öğe Sensitivity of the amide I band to matrix manipulation in bone: a Raman micro-spectroscopy and spatially offset Raman spectroscopy study(Royal Soc Chemistry, 2023) Ahmed, Rafay; Unal, Mustafa; Gautam, Rekha; Uppuganti, Sasidhar; Derasari, Shrey; Mahadevan-Jansen, AnitaThe fracture resistance of bone arises from the hierarchical arrangement of minerals, collagen fibrils (i.e., cross-linked triple helices of & alpha;1 and & alpha;2 collagen I chains), non-collagenous proteins, and water. Raman spectroscopy (RS) is not only sensitive to the relative fractions of these constituents, but also to the secondary structure of bone proteins. To assess the ability of RS to detect differences in the protein structure, we quantified the effect of sequentially autoclaving (AC) human cortical bone at 100 & DEG;C (& SIM;34.47 kPa) and then at 120 & DEG;C (& SIM;117.21 kPa) on the amide I band using a commercial Raman micro-spectroscopy (& mu;RS) instrument and custom spatially offset RS (SORS) instrument in which rings of collection fiber optics are offset from the central excitation fiber optics within a hand-held, cylindrical probe. Being clinically viable, measurements by SORS involved collecting Raman spectra of cadaveric femur mid-shafts (5 male & 5 female donors) through layers of a tissue mimic. Otherwise, & mu;RS and SORS measurements were acquired directly from each bone. AC-related changes in the helical status of collagen I were assessed using amide I sub-peak ratios (intensity, I, at & SIM;1670 cm(-1) relative to intensities at & SIM;1610 cm(-1) and & SIM;1640 cm(-1)). The autoclaving manipulation significantly decreased the selected amide I sub-peak ratios as well as shifted peaks at & SIM;1605 cm(-1) (& mu;RS), & SIM;1636 cm(-1) (SORS) and & SIM;1667 cm(-1) in both & mu;RS and SORS. Compared to & mu;RS, SORS detected more significant differences in the amide I sub-peak ratios when the fiber optic probe was directly applied to bone. SORS also detected AC-related decreases in I-1670/I-1610 and I-1670/I-1640 when spectra were acquired through layers of the tissue mimic with a thickness & LE;2 mm by the 7 mm offset ring, but not with the 5 mm or 6 mm offset ring. Overall, the SORS instrument was more sensitive than the conventional & mu;RS instrument to pressure- and temperature-related changes in the organic matrix that affect the fracture resistance of bone, but SORS analysis of the amide I band is limited to an overlying thickness layer of 2 mm.
