We are developing new approaches to quantitative, label-free histological examination of tissues by infrared micro-spectroscopy. In this technique, an infrared spectrometer with a 2D detector array is attached to a microscope. It simultaneously measures infrared absorption spectra at 16,384 micron-size spots in a tissue section. Chemical composition, orientation and interactions of chemical groups are determined within each spot from unique spectral fingerprints of chemical compounds and plotted as 2D-images. To date, applications of this technique to research and diagnostics have been limited to dehydrated tissues because water strongly absorbs infrared light, resulting in optical interference artifacts. However, dehydration distorts biomolecular and tissue structure, smears out spectroscopic fingerprints, and degrades chemical and spectral resolution. To overcome this limitation, we designed and constructed an infrared chamber with thermo-mechanical stabilization, which allows keeping tissues in solution at desired temperature. By reducing the interference artifacts, we increased spectral reproducibility and chemical resolution by two orders of magnitude compared to commercial designs. Versatile solvent control and increased spectral accuracy of the new chamber allow qualitatively new experimental approaches. For example, with this technique we distinguish collagen from other proteins, resolve different glycosaminoglycans (GAG) and even quantify the extent of GAG sulfation in cartilage. Recently, we collected a spectral library of well-purified and characterized components of connective tissues, which we measured with significantly improved spectro-chemical resolution. We also developed a new approach to quantitative mapping of collagen orientation in tissue sections by polarized infrared hyperspectral imaging. In addition, we designed a new thermo-mechanically stabilized, flow-through chamber for high-definition Raman microspectroscopy, allowing a simultaneous additional characterization of bone specimens with polarized and fluorescence microscopies. We also adapted analytical autoradiographic imaging with micrometer spatial resolution, bridging it with high-definition microspectroscopic imaging to correlate tissue composition with biochemical processes. During the last year, we adapted in vivo dynamic labeling of bone formation surfaces with fluorescence dyes which allows to demarcate bone regions formed at given time points and to perform high-definition Raman microspectroscopy on the same samples. We further extended high-definition spectral library of model connective tissue compounds and continued developing computer analysis of the spectra, increasing chemical resolution and accuracy. We are currently utilizing this approach for characterization of collagen matrix organization in Osteogenesis Imperfecta, chondrodysplasias, bone tumors and other connective tissues pathologies. Specifically, we are focusing on studies of a knock-in mouse model of Diastrophic Dysplasia (DTD) caused by mutations in SLC26A2 sulfate/chloride antiporter. These mutations result in deficient sulfate uptake by chondrocytes, leading to undersulfation of proteoglycan GAG chains crucial for cartilage development and integrity. Like other inborn chondrodysplasias, DTD has delayed skeletal development, but exhibits an unusual progression. The undersulfation is normalized with age. Nonetheless, the articular cartilage degrades with age. To understand the mechanism of the progressive cartilage degradation, we collected 5-micron-resolution, quantitative images of distributions of major extra-cellular matrix components across femur head cartilage and growth plate in newborn DTD and wild type (WT) mice. We showed that in DTD mice, GAG sulfation was low compared to WT in the articular and proliferative zones but almost normal in the resting zone. In DTD mice, polarized infrared hyperspectral imaging revealed disruption of a dense layer of tangentially oriented collagen fibrils at the articular surface. The tangential collagen layer normally protects cartilage from frictional damage and synovial enzymes. Its disruption may cause articular proteoglycan depletion, a hallmark of early osteoarthritis, which we observed at birth and which further progresses with age despite the normalization of GAG sulfation. Collagen orientation in DTD mice was also disrupted throughout the femur head and growth plate. The disruption severity correlated with the extent of GAG undersulfation but not with densities of collagen, noncollagenous proteins and GAG chains, suggesting that GAG sulfation might be crucial for synthesis of the oriented matrix by cells. We used quantitative microradiography to study 35S-sulfate incorporation into cartilage explants, to show that variability of undersulfation across different cartilage regions in DTD was associated with faster chondroitin synthesis rate in the articular and proliferative zones. This observation explained the undersulfation normalization with age, when the cartilage growth slows down, and provided basis for developing a kinetic model for the regulation of GAG sulfation and new potential targets for DTD treatment. We are also focusing on bone disorders. We previously studied bones in the Brittle mouse model of Osteogenesis Imperfecta treated with normal stem cells labeled with green fluorescence protein. Using fluorescence and polarized-light observations, we distinguished matrix produced by non-fluorescent host and fluorescent donor cells and distinguished different types of bone material (woven, lamellar and fine-fibred) within femoral cortex. Using Raman microspectroscopy, we found that matrix mineralization heterogeneity near donor cells was lower within each material type, suggesting that better organization of matrix made by the donor cells may contribute to the amelioration of bone mechanical properties observed in the treated mice. We studied endocrine bone tumors caused by cyclic AMP signaling disruption in a mouse model with Prkar1a+/-/Prkaca+/- deletions in protein kinase A. Using polarized-light microscopy and Raman microspectroscopy, we found that tumor formation in adult mice causes periosteal deposition of immature cortical bone, in which collagen and mineral organizations are intermediate between those of woven and lamellar bone. These observations supported the hypothesis of the existence of periosteal adult stem cells capable of growing cortical bone. During the last year, we studied effects Prkar2a+/- and Prkar2b+/- deletions on new bone formation in tumors caused by Prkar1a+/- knock down in mouse using polarized-light microscopy, Raman microspectroscopy and dynamic bone labeling. We found unexpected partial compensation by such deletions of maturation of bone material at the levels of matrix mineralization and local collagen organization. Surprisingly, only Prkar2b+/-, but not Prkar2a+/-, was able to rescue global collagen organization, suggesting that regulatory subunit Prkar2b is involved in pathways responsible for global collagen organization in bone.
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