Bone quality assessment of osteogenic cell cultures by Raman microscopy

The use of autologous stem/progenitor cells represents a promising approach to the repair of craniofacial bone defects. The calvarium is recognized as a viable source of stem/progenitor cells that can be transplanted in vitro to form bone. However, it is unclear if bone formed in cell culture is similar in quality to that found in native bone. In this study, the quality of bone mineral formed in osteogenic cell cultures were compared against calvarial bone from postnatal mice. Given the spectroscopic resemblance that exists between cell and collagen spectra, the feasibility of extracting information on cell activity and bone matrix quality were also examined. Stem/progenitor cells isolated from fetal mouse calvaria were cultured onto fused‐quartz slides under osteogenic differentiation conditions for 28 days. At specific time intervals, slides were removed and analyzed by Raman microscopy and mineral staining techniques. We show that bone formed in culture at Day 28 resembled calvarial bone from 1‐day‐old postnatal mice with comparable mineralization, mineral crystallinity, and collagen crosslinks ratios. In contrast, bone formed at Day 28 contained a lower degree of ordered collagen fibrils compared with 1‐day‐old postnatal bone. Taken together, bone formed in osteogenic cell culture exhibited progressive matrix maturation and mineralization but could not fully replicate the high degree of collagen fibril order found in native bone.In this Raman spectroscopic study, we examined the quality of bone formed in vitro by fetal mouse calvarial stem/progenitor cells under osteogenic differentiation conditions. We characterized bone mineral and matrix cell culture components and detected the presence of lipid and glycosaminoglycan‐like components. Bone formed in vitro at Day 28 was similar to 1‐day‐old postnatal mouse calvarial bone in terms of mineralization, mineral crystallinity, and collagen crosslink ratios, but differed in the degree of collagen fibril order.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/148347/1/jrs5521_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148347/2/jrs5521.pd

Is photobleaching necessary for Raman imaging of bone tissue using a green laser?

AbstractRaman microspectroscopy is widely used for musculoskeletal tissues studies. But the fluorescence background obscures prominent Raman bands of mineral and matrix components of bone tissue. A 532-nm laser irradiation has been used efficiently to remove the fluorescence background from Raman spectra of cortical bone. Photochemical bleaching reduces over 80% of the fluorescence background after 2 h and is found to be nondestructive within 40 min. The use of electron multiplying couple charge detector (EMCCD) enables to acquire Raman spectra of bone tissues within 1–5 s range and to obtain Raman images less than in 10 min

External Bone Size Is a Key Determinant of Strength‐Decline Trajectories of Aging Male Radii

Given prior work showing associations between remodeling and external bone size, we tested the hypothesis that wide bones would show a greater negative correlation between whole‐bone strength and age compared with narrow bones. Cadaveric male radii (n = 37 pairs, 18 to 89 years old) were evaluated biomechanically, and samples were sorted into narrow and wide subgroups using height‐adjusted robustness (total area/bone length). Strength was 54% greater (p < 0.0001) in wide compared with narrow radii for young adults (<40 years old). However, the greater strength of young‐adult wide radii was not observed for older wide radii, as the wide (R2 = 0.565, p = 0.001), but not narrow (R2 = 0.0004, p = 0.944) subgroup showed a significant negative correlation between strength and age. Significant positive correlations between age and robustness (R2 = 0.269, p = 0.048), cortical area (Ct.Ar; R2 = 0.356, p = 0.019), and the mineral/matrix ratio (MMR; R2 = 0.293, p = 0.037) were observed for narrow, but not wide radii (robustness: R2 = 0.015, p = 0.217; Ct.Ar: R2 = 0.095, p = 0.245; MMR: R2 = 0.086, p = 0.271). Porosity increased with age for the narrow (R2 = 0.556, p = 0.001) and wide (R2 = 0.321, p = 0.022) subgroups. The wide subgroup (p < 0.0001) showed a significantly greater elevation of a new measure called the Cortical Pore Score, which quantifies the cumulative effect of pore size and location, indicating that porosity had a more deleterious effect on strength for wide compared with narrow radii. Thus, the divergent strength–age regressions implied that narrow radii maintained a low strength with aging by increasing external size and mineral content to mechanically offset increases in porosity. In contrast, the significant negative strength–age correlation for wide radii implied that the deleterious effect of greater porosity further from the centroid was not offset by changes in outer bone size or mineral content. Thus, the low strength of elderly male radii arose through different biomechanical mechanisms. Consideration of different strength–age regressions (trajectories) may inform clinical decisions on how best to treat individuals to reduce fracture risk. © 2019 American Society for Bone and Mineral Research.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149566/1/jbmr3661_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149566/2/jbmr3661.pd

Realising the potential of infrared microscopy in combinatorial chemistry

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Realising the potential of infrared microscopy in combinatorial chemistry

In combinatorial chemistry, infrared microscopy has been synonymous with aiding the optimisation of solid-phase reaction protocols, as well as providing invaluable information regarding the diffusion characteristics of a wide range of polymeric supports.  In this project, infrared microscopy is used to interrogate the functional group distribution of chloromethylated polystyrene (CPS) beads after coupling with 4-cyanophenol, an IR-tagging agent.  This characterisation approach provided evidence for homogeneous site distributions, as well as providing a unique insight into the chemical reactivity and accessibility of reagents in highly cross-linked CPS beads.  A IR-microscope fitted with a linear pixel-array detector was also used to create spatially resolved multi-layered IR-images of a large collection of polymer beads supporting carbonyl and nitrile monomers.  Strategies for obtaining high quality spectra from both imaging and mapping IR-microscopes without compromising on sample area, analysis time, or spatial resolution are also discussed. The last decade has also seen considerable interest in developing miniaturised IR-flow cells for tandem on-line separation and IR-identification of synthetic compounds.  In this project, micro-litre volume flow cells were fabricated without the need for expensive microfabrication techniques (under patent review by the University of Southampton, Sept 2003).  The flow cell was hyphenated with an IR-microscope and HPLC-pump and was used to record the IR-spectra of a series of 3-alkylamino-iidazo[1,2-α]pyridines and pyrazines using stopped flow-injection techniques. The multi-component synthesis of 3-alkylamino-imidazo[1,2-α]pyrimidines also gave rise to the unexpected synthesis of the 2-alkylamino-imidazo[1,2-α]pyrimidines.  The structures of both derivatives were confirmed by X-ray crystallography and a mechanistic rationale for their formation was proposed.</p