Browsing by Subject "Raman spectroscopy"
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Item Calcimimetics Alter Periosteal and Perilacunar Bone Matrix Composition and Material Properties in Early Chronic Kidney Disease(Wiley, 2022) Damrath, John G.; Moe, Sharon M.; Wallace, Joseph M.; Medicine, School of MedicineChronic kidney disease (CKD) affects 15% of Americans and greatly increases fracture risk due to elevated parathyroid hormone, cortical porosity, and reduced bone material quality. Calcimimetic drugs are used to lower parathyroid hormone (PTH) in CKD patients, but their impact on bone matrix properties remains unknown. We hypothesized that tissue-level bone quality is altered in early CKD and that calcimimetic treatment will prevent these alterations. To test this hypothesis, we treated Cy/+ rats, a model of spontaneous and progressive CKD-mineral and bone disorder (CKD-MBD), with KP-2326, a preclinical analogue of etelcalcetide, early in the CKD disease course. To measure tissue-level bone matrix composition and material properties, we performed colocalized Raman spectroscopy and nanoindentation on new periosteal bone and perilacunar bone using hydrated femur sections. We found that CKD and KP treatment lowered mineral type B carbonate substitution whereas KP treatment increased mineral crystallinity in new periosteal bone. Reduced elastic modulus was lower in CKD but was not different in KP-treated rats versus CTRL. In perilacunar bone, KP treatment lowered type B carbonate substitution, increased crystallinity, and increased mineral-to-matrix ratio in a spatially dependent manner. KP treatment also increased reduced elastic modulus and hardness in a spatially dependent manner. Taken together, these data suggest that KP treatment improves material properties on the tissue level through a combination of lowering carbonate substitution, increasing mineral crystallinity, and increasing relative mineralization of the bone early in CKD. As a result, the mechanical properties were improved, and in some regions, were the same as control animals. Therefore, calcimimetics may help prevent CKD-induced bone deterioration by improving bone quality in new periosteal bone and in bone tissue near osteocyte lacunae.Item Effects of Raloxifene and tibial loading on bone mass and mechanics in male and female mice(Taylor & Francis, 2022) Berman, Alycia G.; Damrath, John G.; Hatch, Jennifer; Pulliam, Alexis N.; Powell, Katherine M.; Hinton, Madicyn; Wallace, Joseph M.; Biomedical Engineering, School of Engineering and TechnologyRaloxifene (RAL) is a selective estrogen receptor modulator (SERM) that has previously been shown to cause acellular benefits to bone tissue. Due to these improvements, RAL was combined with targeted tibial loading to assess if RAL treatment during periods of active bone formation would allow for further mechanical enhancements. To do so, structural, mechanical, and microstructural effects were assessed in bone from C57BL/6 mice that were treated with RAL (0.5 mg/kg), tibial loading, or both for 6 weeks, beginning at 10 weeks of age. Ex vivo microcomputed tomography (CT) images indicated RAL and loading work together to improve bone mass and architecture, especially within the cancellous region of males. Increases in cancellous bone volume fraction were heavily driven by increases in trabecular thickness, though there were some effects on trabecular spacing and number. In the cortical regions, RAL and loading both increased cross-sectional area, cortical area, and cortical thickness. Whole-bone mechanical testing primarily indicated effects of loading. Further characterization through Raman spectroscopy and nanoindentation showed load-based changes in mineralization and micromechanics, while both loading and RAL caused changes in the secondary collagen structure. In contrast to males, in females, there were large load-based effects in the cancellous and cortical regions, resulting in increased whole-bone mechanical properties. RAL had less of an effect on cancellous and cortical architecture, though some effects were still present. In conclusion, RAL and loading work together to impact bone architecture and mechanical integrity, leading to greater improvements than either treatment individually.Item Exercise Alters Mineral and Matrix Composition in the Absence of Adding New Bone(2008-12) Kohn, David H.; Sahar, Nadder D.; Wallace, Joseph M.; Golcuk, Kurtulus; Morris, Michael D.The mechanical properties of bone are dictated by its amount, distribution and ‘quality’. The composition of the mineral and matrix phases is integral to defining ‘bone quality’. Exercise can potentially increase resistance to fracture, yet the effects of exercise on skeletal fragility, and how alterations in fragility are modulated by the amount, distribution and composition of bone, are unknown. In this investigation, the effects of exercise on the size, composition, mechanical properties and damage resistance of bones from mice of various ages, background strains and genetic makeup were assessed, as a means of testing the hypothesis that mechanical loading can improve skeletal fragility via compositional alterations. C57BL/6 mice (4-month-old males) ran on a treadmill for 21 days. Tibiae from exercised and control mice were analyzed for cross-sectional geometry, mechanical properties, microdamage and composition. Exercise significantly increased strength without increasing cross-sectional properties, suggesting that mechanical stimulation led to changes in the bone matrix, and these changes led to the improvements in mechanical properties. Consistent with this interpretation, the mineral/matrix ratio was significantly increased in exercised bones. The number of fatigue-induced microcracks was significantly lower in exercised bones, providing evidence that exercise modulates fatigue resistance. The ratio of nonreducible/reducible cross-links mirrored the damage data. Similar trends (exercise induced increases in mechanical properties without increases in cross-sectional properties, but with compositional changes) were also observed in 2-month-old biglycan-deficient and wild-type mice bred on a C57BL/6x129 genetic background.Item In Vivo Entombment of Bacteria and Fungi during Calcium Oxalate, Brushite, and Struvite Urolithiasis(Wolters Kluwer, 2020-12-23) Saw, Jessica J.; Sivaguru, Mayandi; Wilson, Elena M.; Dong, Yiran; Sanford, Robert A.; Fields, Chris J.; Cregger, Melissa A.; Merkel, Annette C.; Bruce, William J.; Weber, Joseph R.; Lieske, John C.; Krambeck, Amy E.; Rivera, Marcelino E.; Large, Timothy; Lange, Dirk; Bhattacharjee, Ananda S.; Romero, Michael F.; Chia, Nicholas; Fouke, Bruce W.; Urology, School of MedicineBackground: Human kidney stones form via repeated events of mineral precipitation, partial dissolution, and reprecipitation, which are directly analogous to similar processes in other natural and manmade environments, where resident microbiomes strongly influence biomineralization. High-resolution microscopy and high-fidelity metagenomic (microscopy-to-omics) analyses, applicable to all forms of biomineralization, have been applied to assemble definitive evidence of in vivo microbiome entombment during urolithiasis. Methods: Stone fragments were collected from a randomly chosen cohort of 20 patients using standard percutaneous nephrolithotomy (PCNL). Fourier transform infrared (FTIR) spectroscopy indicated that 18 of these patients were calcium oxalate (CaOx) stone formers, whereas one patient formed each formed brushite and struvite stones. This apportionment is consistent with global stone mineralogy distributions. Stone fragments from seven of these 20 patients (five CaOx, one brushite, and one struvite) were thin sectioned and analyzed using brightfield (BF), polarization (POL), confocal, super-resolution autofluorescence (SRAF), and Raman techniques. DNA from remaining fragments, grouped according to each of the 20 patients, were analyzed with amplicon sequencing of 16S rRNA gene sequences (V1-V3, V3-V5) and internal transcribed spacer (ITS1, ITS2) regions. Results: Bulk-entombed DNA was sequenced from stone fragments in 11 of the 18 patients who formed CaOx stones, and the patients who formed brushite and struvite stones. These analyses confirmed the presence of an entombed low-diversity community of bacteria and fungi, including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Aspergillus niger. Bacterial cells approximately 1 μm in diameter were also optically observed to be entombed and well preserved in amorphous hydroxyapatite spherules and fans of needle-like crystals of brushite and struvite. Conclusions: These results indicate a microbiome is entombed during in vivo CaOx stone formation. Similar processes are implied for brushite and struvite stones. This evidence lays the groundwork for future in vitro and in vivo experimentation to determine how the microbiome may actively and/or passively influence kidney stone biomineralization.Item Instrumental and Chemometric Analysis of Automotive Clear Coat Paints by Micro Laser Raman and UV Microspectrophotometry(2012-07-19) Mendlein, Alexandra Nicole; Siegel, Jay A.; Goodpaster, John V. (John Vincent); Li, LeiAutomotive paints have used an ultraviolet (UV) absorbing clear coat system for nearly thirty years. These clear coats have become of forensic interest when comparing paint transfers and paint samples from suspect vehicles. Clear coat samples and their ultraviolet absorbers are not typically examined or characterized using Raman spectroscopy or microspectrophotometry (MSP), however some past research has been done using MSP. Chemometric methods are also not typically used for this characterization. In this study, Raman and MSP spectra were collected from the clear coats of 245 American and Australian automobiles. Chemometric analysis was subsequently performed on the measurements. Sample preparation was simple and involved peeling the clear coat layer and placing the peel on a foil-covered microscope slide for Raman or a quartz slide with no cover slip for MSP. Agglomerative hierarchical clustering suggested three classes of spectra, and principal component analysis confirmed this. Factor loadings for the Raman data illustrated that much of the variance between spectra came from specific regions (400 – 465 cm-1, 600 – 660 cm-1, 820 – 885 cm-1, 950 – 1050 cm-1, 1740 – 1780 cm-1, and 1865 – 1900 cm-1). For MSP, the regions of highest variance were between 230 – 270 nm and 290 – 370 nm. Discriminant analysis showed that the three classes were well-differentiated with a cross-validation accuracy of 92.92% for Raman and 91.98% for MSP. Analysis of variance attributed differentiability of the classes to the regions between 400 – 430 cm-1, 615 – 640 cm-1, 825 – 880 cm-1, 1760 – 1780 cm-1, and 1860 – 1900 cm-1 for Raman spectroscopy. For MSP, these regions were between 240 – 285 nm and 300 – 370 nm. External validation results were poor due to excessively noisy spectra, with a prediction accuracy of 51.72% for Raman and 50.00% for MSP. No correlation was found between the make, model, and year of the vehicles using either method of analysis.Item Multi-scale Analysis of Bone Chemistry, Morphology and Mechanics in the oim Model of Osteogenesis Imperfecta(2014-08) Bart, Zachary R.; Hammond, Max A.; Wallace, Joseph M.Osteogenesis imperfecta is a congenital disease commonly characterized by brittle bones and caused by mutations in the genes encoding Type I collagen, the single most abundant protein produced by the body. The oim model has a natural collagen mutation, converting its heterotrimeric structure (two α1 and one α2 chains) into α1 homotrimers. This mutation in collagen may impact formation of the mineral, creating a brittle bone phenotype in animals. Femurs from male wild type (WT) and homozygous (oim/oim) mice, all at 12 weeks of age, were assessed using assays at multiple length scales with minimal sample processing to ensure a near-physiological state. Atomic force microscopy (AFM) demonstrated detectable differences in the organization of collagen at the nanoscale that may partially contribute to alterations in material and structural behavior obtained through mechanical testing and reference point indentation (RPI). Changes in geometric and chemical structure obtained from µ-Computed Tomography and Raman spectroscopy indicate a smaller bone with reduced trabecular architecture and altered chemical composition. Decreased tissue material properties in oim/oim mice are likely driven by changes in collagen fibril structure, decreasing space available for mineral nucleation and growth, as supported by a reduction in mineral crystallinity. Multi-scale analyses of this nature offer much in assessing how molecular changes compound to create a degraded, brittle bone phenotype.Item Multi-scale analysis of morphology, mechanics, and composition of collagen in murine osteogenesis imperfecta(2013-11-06) Bart, Zachary Ryan; Wallace, Joseph; Na, Sungsoo; Yokota, Hiroki, 1955-; Schild, John H.Osteogenesis imperfecta is a rare congenital disease commonly characterized by brittle bones caused by mutations in the genes encoding Type I collagen, the single most abundant protein produced by the body. The murine model (oim) exists as a natural mutation of this protein, converting its heterotrimeric structure of two Col1a1 molecules and a single Col1a2 molecule into homotrimers composed of only the former. This defect impacts bone mechanical integrity, greatly weakening their structure. Femurs from male wild type (WT), heterozygous (oim/+), and homozygous (oim/oim) mice, all at 12 weeks of age, were assessed using assays at multiple length scales with minimal sample processing to ensure a near-physiological state. Atomic force microscopy (AFM) demonstrated detectable differences in the organization of collagen at the nanometer scale that may partially attribute to alterations in material and structural behavior obtained through mechanical testing and reference point indentation (RPI). Changes in geometric and chemical structure through the use of µ-Computed Tomography and Raman spectroscopy respectively indicate a smaller, brittle phenotype caused by oim. Changes within the periodic D-spacing of collagen point towards a reduced mineral nucleation site, supported by reduced mineral crystallinity, resulting in altered material and structural behavior in oim/oim mice. Multi-scale analyses of this nature offer much in assessing how molecular changes can compound to create a degraded, brittle phenotype.Item Quantification of minerals associated to enamel caries process by raman spectroscopy(2020) Sungkapreecha, Siras; Masatoshi, Ando; Lippert, Frank; Hara, AndersonBackground: Stimulated Raman spectroscopy (SRS) is a nondestructive tool for biochemical characterization of tissues. The aims were: 1) To evaluate the ability of SRS and Spontaneous Raman spectroscopy (SpRS) to differentiate among sound, demineralized and remineralized bovine enamel by phosphate and carbonate ratio (P/C-Ratio); and 2) To determine the correlation between the outcomes of transverse microradiography (TMR: Integrated mineral loss (ΔZ) and lesion depth) and P/C-Ratio. Material and Methods: Thirty, 5×5×2-mm ground and polished bovine enamel blocks were prepared. The surface was divided into 3 equal areas. Each area was chemically demineralized (demin) by Carbopol demineralized solution for 0 (Sound-Demin), 24 (24h-Demin), and 48h (48h-Demin), respectively. Then, specimens were sectioned for TMR analysis, and the remaining one part of each specimen was remineralized (remin) for 15days using a pH-cycling model (Sound-Remin, 24hD-Remin=24h-Demin and remineralization, 48hD-Remin = 48h-Demin and remineralization). Demin and remin groups were scanned to obtain P/C-Ratio by SpRS and SRS. SRS was further scanned from 0 (surface) up to 100 µm into the dentine at 10-µm intervals. Remineralized specimens were sectioned for TMR analysis. Wilcoxon signed-rank tests were used to compare between TMR and SpRS/SRS. Spearman correlation coefficients were used to correlate among TMR, SpRS, and SRS. A 5-percent significance level was used for each test. Results: As demin time increased, both ΔZ and lesion depth were increased. After remineralization, both values were decreased. There were significant differences between demine and remin groups and between demin times. For SpRS, Sound-Demin had significantly larger P/C-Ratio than 24h-Demin and 48h-Demin (p ≤ 0.001). The 24h-Demin had significantly larger values than 48h-Demin (p = 0.048). Sound-Remin had larger P/C-Ratio than 24hD-Remin (p = 0.316) and 48hD-Remin (p = 0.015). 24hD-Remin was larger than 48hD-Remin (p = 0.269). 24hD-Remin had significantly larger P/C-Ratio than 24h-Demin (p ≤ 0.001). 48hD-Remin had significantly larger P/C-Ratio than 48h-Demin (p ≤ 0.001). For SRS, at surface (0 µm), for demin group, Sound-Demin had significantly larger P/C-Ratio than 24h-Demin (p = 0.020) and 48h-Demin (p = 0.032). 24h-Demin had larger value than 48h-Demin; but no significant difference (p = 0.117). Among remin groups, Sound-Remin was not statistical significance different for 24hD-Remin (p = 0.172) and 48hD-Remin (p = 0.134). However, 24hD-Remin was smaller; but not statistical significance different from 48hD-Remin (p = 0.688). At deeper levels (10 µm to 100µm), it was found that 1) After demineralization, Sound-Demin had significantly larger P/C-Ratio than 24h-Demin and 48h-Demin at 0 µm to 20 µm, and 80 µm to 100µm; Sound-Demin had significantly larger P/C-Ratio than 48h-Demin; and no statistical significance differences were found among Sound-Demin and 24h-Demin, 24h-Demin and 48h-Demin. 2) After remineralization, no statistical significance differences were found among Sound-Remin, 24hD-Remin, and 48hD-Remin. 3) Sound-Demin had significantly larger P/C-Ratio than Sound-Remin at 0 µm ,10 µm, 20 µm; and no statistical significance differences were found at levels deeper than 30 µm. 4) No statistical significance differences were found between 24h-Demin and 24hD-Remin from 0 µm to 70µm; and 24hD-Remin had significantly larger P/C-Ratio than 24h-Demin from 80 µm to 100 µm. 5) No statistical significance differences were found between 48h-Demin and 48hD-Remin. For correlation, moderate correlation was found between SpRS demineralized/remineralized groups and ΔZ, and between SpRS demineralized groups and lesion depth. Conclusion: SpRS and SRS have the potential to quantify demineralization through calculation of the phosphate and carbonate ratio. In addition, SpRS can detect the change of remineralization. A nondestructive caries detection approach using SpRS and SRS would be beneficial in clinical practice.Item Removing or truncating connexin 43 in murine osteocytes alters cortical geometry, nanoscale morphology, and tissue mechanics in the tibia(Elsevier, 2016-07) Hammond, Max A.; Berman, Alycia G.; Pacheco-Costa, Rafael; Davis, Hannah M.; Plotkin, Lilian I.; Wallace, Joseph M.; Biomedical Engineering, School of Engineering and TechnologyGap junctions are formed from ubiquitously expressed proteins called connexins that allow the transfer of small signaling molecules between adjacent cells. Gap junctions are especially important for signaling between osteocytes and other bone cell types. The most abundant type of connexin in bone is connexin 43 (Cx43). The C-terminal domain of Cx43 is thought to be an important modulator of gap junction function but the role that this domain plays in regulating tissue-level mechanics is largely unknown. We hypothesized that the lack of the C-terminal domain of Cx43 would cause morphological and compositional changes as well as differences in how bone responds to reference point indentation (RPI) and fracture toughness testing. The effects of the C-terminal domain of Cx43 in osteocytes and other cell types were assessed in a murine model (C57BL/6 background). Mice with endogenous Cx43 in their osteocytes removed via a Cre-loxP system were crossed with knock-in mice which expressed Cx43 that lacked the C-terminal domain in all cell types due to the insertion of a truncated allele to produce the four groups used in the study. The main effect of removing the C-terminal domain from osteocytic Cx43 increased cortical mineral crystallinity (p=0.036) and decreased fracture toughness (p=0.017). The main effect of the presence of the C-terminal domain in other cell types increased trabecular thickness (p<0.001), cortical thickness (p=0.008), and average RPI unloading slope (p=0.004). Collagen morphology was altered when either osteocytes lacked Cx43 (p=0.008) or some truncated Cx43 was expressed in all cell types (p<0.001) compared to controls but not when only the truncated form of Cx43 was expressed in osteocytes (p=0.641). In conclusion, the presence of the C-terminal domain of Cx43 in osteocytes and other cell types is important to maintain normal structure and mechanical integrity of bone.