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 16,000 micron-size spots in a tissue section. Chemical composition, orientation and interactions of chemical groups within each spot are determined 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. Water strongly absorbs infrared light, causing 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 improved spectro-chemical resolution of different types of sulfated GAG chains by combing infrared and HPLC characterization of model chondroitins and hyaluronate. We also developed a new approach to quantitative mapping of collagen orientation across cartilage sections by polarized infrared hyperspectral imaging. During the last year, we extended high-definition spectral library of model compounds to aggrecan, biglycan, decorin and type II collagen. We also developed a new 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 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 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. In DTD mice, GAG sulfation was low compared to WT in the articular and proliferative zones but almost normal in the resting zone. The undersulfation in DTD appeared to be associated with faster growth rate in the articular and proliferative zones, consistent with the undersulfation normalization when the cartilage growth slows down with age. 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 is normally present to protect 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 the growth plate. The severity of the disruption 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. Based on these observations, we proposed a kinetic model for the regulation of GAG sulfation and new potential targets for DTD treatment. During the last year, we carried over infrared advances into Raman microspectrsocopy and developed a new thermo-mechanically stabilized, flow-through chamber which allowed a simultaneous triple characterization of bone specimens. The chamber allowed recording high-definition Raman spectra of bones in the Brittle mouse model of Osteogenesis Imperfecta, in particular, after treatment 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. We found that heterogeneity of matrix mineralization near donor cells was decreased within each material type, suggesting that better organization of donor cells matrix may contribute to amelioration of bone mechanical properties observed in the treated mice.
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