High-definition infrared micro-spectroscopic imaging of biomaterials

生物材料的高清红外显微光谱成像

• 批准号: 7968726
• 负责人: Edward Mertz
• 金额: $ 7.9万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968726
• 关键词: Aftercare; Age; Biochemical; Biocompatible Materials; Birth; Cartilage; Cell Culture Techniques; Cells; Chemicals; Chloride Ion; Chlorides; Chondrocytes; Collagen; Collagen Fibril; Collagen Type II; Comb animal structure; Computer Analysis; Connective Tissue; Cytoskeleton; Degenerative polyarthritis; Dehydration; Diagnostics Research; Dysplasia; Enzymes; Epiphysial cartilage; Exhibits; Fingerprint; Fluorescence; Glycosaminoglycans; Goals; Growth; Heterogeneity; High Pressure Liquid Chromatography; Image; Inorganic Sulfates; Kinetics; Knock-in Mouse; Label; Libraries; Life; Light; Maps; Measures; Mechanics; Microscope; Modeling; Monitor; Morphologic artifacts; Mus; Mutation; Newborn Infant; Optics; Osteogenesis Imperfecta; Pathology; Property; Proteins; Proteoglycan; Regulation; Reproducibility; Resolution; Rest; Severities; Skeletal Development; Solutions; Solvents; Specimen; Spectrum Analysis; Spottings; Stem cells; Structure; Surface; Techniques; Temperature; Tissues; Unspecified or Sulfate Ion Sulfates; Water; Wild Type Mouse; absorption; aggrecan; antiporter; articular cartilage; base; biglycan; bone; cartilage development; chemical fingerprinting; chemical group; chondrodysplasia; decorin; density; design; design and construction; detector; femur head; hyaluronate; improved; interest; mineralization; mouse model; novel strategies; polarized light; polysulfated glycosaminoglycan; spectroscopic imaging; sulfation; uptake

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.

我们正在开发利用红外显微光谱学对组织进行定量的、无标记的组织学检查的新方法。在这项技术中，一个带有2D探测器阵列的红外光谱仪被连接到显微镜上。它同时测量组织切片中16,000微米大小的斑点的红外吸收光谱。根据化合物独特的光谱指纹确定每个斑点内化学基团的化学组成、取向和相互作用，并绘制成2D图像。到目前为止，这项技术在研究和诊断中的应用仅限于脱水组织。水强烈吸收红外光，造成光学干涉伪影。然而，脱水会扭曲生物分子结构和组织结构，涂抹光谱指纹，并降低化学和光谱分辨率。 为了克服这一局限，我们设计并建造了一种具有热机械稳定性的红外室，允许组织在期望的温度下保持在溶液中。通过减少干扰伪影，与商业设计相比，我们将光谱重复性和化学分辨率提高了两个数量级。多才多艺的溶剂控制和新色谱室提高的光谱精确度使新的实验方法成为可能。例如，使用这项技术，我们将胶原与其他蛋白质区分开来，分解不同的糖胺多聚糖(GAG)，甚至量化软骨中GAG硫酸盐化的程度。最近，我们通过结合模型软骨素和透明质酸的红外和高效液相色谱表征，提高了不同类型硫酸基氨基己酸链的光谱化学拆分能力。我们还开发了一种新的方法，通过偏振红外高光谱成像来定量绘制软骨切片上的胶原定位图。 在过去的一年里，我们将模型化合物的高清晰度光谱库扩展到聚集素、双聚糖、核心蛋白和II型胶原。我们还开发了一种新的计算机光谱分析，提高了化学分辨率和准确性。我们目前正在利用这种方法来表征成骨不全、软骨发育不良和其他结缔组织病理中的胶原基质组织。 具体地说，我们专注于SLC26A2硫酸盐/氯逆向转运蛋白突变引起的遗传性发育不良(DTD)敲入小鼠模型的研究。这些突变导致软骨细胞对硫酸盐的摄取不足，导致对软骨发育和完整性至关重要的蛋白多糖Gag链的硫酸盐化不足。像其他先天性软骨发育不良一样，DTD延缓了骨骼发育，但表现出不同寻常的进展。未充分的硫酸盐化作用随着年龄的增长而正常化。尽管如此，关节软骨会随着年龄的增长而退化。为了了解软骨降解的机制，我们收集了新生DTD和野生型(WT)小鼠股骨头软骨和生长板上主要细胞外基质成分分布的5微米分辨率的定量图像。在DTD小鼠中，与WT相比，关节和增殖区的GAG硫酸盐化程度较低，但在静息区几乎正常。DTD中的低硫酸盐化似乎与关节和增殖区较快的生长速度有关，这与当软骨生长随年龄减慢时的低硫酸盐化正常化一致。在DTD小鼠中，偏振红外高光谱成像显示关节表面有一层密集的切向取向的胶原纤维破裂。切向胶原层的存在通常是为了保护软骨免受摩擦损伤和滑膜酶的伤害。它的破坏可能会导致关节蛋白多糖耗竭，这是我们在出生时观察到的早期骨关节炎的一个特征，尽管GAG硫酸盐正常化，但随着年龄的增长，这种情况会进一步发展。DTD小鼠的整个股骨头和生长板的胶原蛋白取向也被破坏。破坏的严重程度与GAG硫化不足的程度有关，但与胶原、非胶原蛋白和GAG链的密度无关，提示GAG硫化可能是细胞合成定向基质的关键。基于这些观察，我们提出了一个调节GAG硫化的动力学模型，并提出了治疗DTD的新的潜在靶点。 在过去的一年里，我们将红外技术的进步应用到拉曼显微镜中，并开发了一种新的热机械稳定的流通式小室，它可以同时对骨骼标本进行三重表征。该小室允许记录骨骼的高清晰度拉曼光谱，特别是在用绿色荧光蛋白标记的正常干细胞治疗后，脆性成骨不全小鼠模型的骨骼。通过荧光和偏振光观察，我们区分了非荧光宿主细胞和荧光供体细胞产生的基质，并区分了股骨皮质内不同类型的骨材料(编织、板层和细纤维)。我们发现，在每种材料类型中，供体细胞附近基质矿化的异质性都有所降低，这表明更好地组织供体细胞基质可能有助于改善接受治疗的小鼠的骨力学性能。