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: 10008741
• 负责人: Edward Mertz
• 金额: $ 21.05万
• 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/10008741
• 关键词: Adult; Age; Amish; Amyloid fibers; Anterior; Antineoplastic Agents; Atomic Force Microscopy; Autophagocytosis; Biochemical; Biochemical Process; Biocompatible Materials; Birth; Bone Matrix; Bone neoplasms; Bovine Cartilage; Carbon Nanotubes; Cartilage; Cartilage Matrix; Cell Culture Techniques; Cells; Chemicals; Chlorides; Chondrocytes; Chondroitin; Collagen; Collagen Fibril; Communities; Connective Tissue; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Defect; Degenerative polyarthritis; Dehydration; Deposition; Diagnostic; Dyes; Dysplasia; Endocrine; Exhibits; Extracellular Matrix; FKBP10 gene; Femur; Fibroblasts; Financial compensation; Fingerprint; Fluorescence; Fluorescence Microscopy; Fluorescence Polarization; Glycosaminoglycans; Goals; Green Fluorescent Proteins; HIV; Heterogeneity; Histologic; Human; Image; Immature Bone; Knock-in Mouse; Knock-out; Label; Libraries; Light; Magnetic Resonance Imaging; Mature Bone; Measures; Mechanics; Methods; Microradiography; Microscope; Monitor; Morphologic artifacts; Mus; Mutation; National Institute of Arthritis and Musculoskeletal and Skin Diseases; National Institute of Biomedical Imaging and Bioengineering; National Institute of Neurological Disorders and Stroke; Newborn Infant; Optics; Osteoblasts; Osteogenesis; Osteogenesis Imperfecta; Pathologic; Pathology; Penetration; Peptides; Placenta; Polarization Microscopy; Protein-Restricted Diet; Proteins; Proteoglycan; Reproducibility; Research; Research Personnel; Resolution; Rest; Sampling; Scientist; Severity of illness; Signal Transduction; Skeletal Development; Solvents; Specimen; Spectrum Analysis; Spottings; Stem cell transplant; Structure; Surface; Syndrome; Techniques; Technology; Temperature; Time; Tissues; Unspecified or Sulfate Ion Sulfates; Water; absorption; antiporter; articular cartilage; bone; bone fragility; bone quality; bone strength; cancer cell; cartilage development; celecoxib; chemical fingerprinting; chemical group; cortical bone; design; design and construction; detector; disease phenotype; femur head; improved; in vivo; interest; mechanical properties; mineralization; mouse model; neurotoxicity; novel strategies; null mutation; overexpression; polysulfated glycosaminoglycan; prevent; quantitative imaging; receptor; spectroscopic imaging; tumor; uptake

We are developing new approaches to quantitative, label-free histological examination of tissues by infrared (IR) micro-spectroscopy. In this technique, an infrared spectrometer with a 2D detector array is attached to a microscope. It simultaneously measures IR 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 IR 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 IR 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. 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 IR hyperspectral imaging. To correlate tissue composition with biochemical processes, we bridged this high-definition (HD) microspectroscopic imaging with analytical autoradiographic imaging with micrometer spatial resolution. We also designed a new thermo-mechanically stabilized, flow-through chamber for HD Raman microspectroscopy, allowing a simultaneous additional characterization of bone specimens with Raman, polarized and fluorescence microscopies. 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 HD Raman microspectroscopy on the same samples. We use these techniques to characterize collagen matrix organization in osteo- and chondro-dysplasias, bone tumors and other connective tissues pathologies. Specifically, we studied 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. DTD has delayed skeletal development and exhibits an unusual progression: The undersulfation is normalized with age, but the articular cartilage degrades with age. Our quantitative imaging of extra-cellular matrix components in femur head cartilage in newborn mice showed that in DTD mice, GAG sulfation was low compared to wild type (WT) in the articular and prehypertrophic zones but almost normal in the resting zone. In DTD mice, polarized IR hyperspectral imaging revealed disruption of a dense layer of tangentially oriented collagen fibrils at the articular surface. Disruption of this protective layer 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. Using quantitative microradiography of 35S-sulfate incorporation into cartilage explants, we showed that variability of undersulfation across different cartilage regions in DTD was associated with faster chondroitin synthesis rate in the articular and prehypertrophic zones. This observation explained the undersulfation normalization with age. We assisted NIBIB scientists using our technology to demonstrate penetration of functionalized carbon nanotubes inside cancer cells that overexpress hylauronate receptors, validating this approach to intracellular delivery of anticancer agents. We investigated effects of stem cell transplantation on bone quality in the Brittle mouse model of Osteogenesis Imperfecta (OI). Using fluorescence and polarized-light microscopy, we distinguished matrix produced by host cells and green-fluorescent-protein-labeled donor cells within different types of bone (woven and lamellar) in 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 increased bone strength in the treated mice. We studied endocrine bone tumors caused by cyclic AMP signaling disruption in mice with different combinations of deletions of subunits in protein kinase A. Using polarized-light microscopy, Raman microspectroscopy, and dynamic bone labeling, we found that tumor formation in adult Prkar1a+/-Prkaca+/- mice causes periosteal deposition of immature cortical bone. We found partial compensation of Prkar1a+/- deletion effects on local maturation of bone material, matrix mineralization and collagen organization by additional Prkar2a+/- or Prkar2b+/- deletions. We showed that Celecoxib treatment of R1a+/-Ca+/- mice improved organization and mineralization of cortical bones covering the tumors. We developed non-destructive quantification of collagen content in cell cultures using HD Raman microspectroscopy. We applied this method to human fibroblast cultures from recessive osteogenesis imperfecta caused by FKBP10-null mutations. We found reduced deposition of collagen in the extracellular matrix despite synthesis of normal collagen quantities by these cells and by ppib-/- mouse osteoblasts. However, the deposition did not correlate with the disease severity and phenotype for other FKBP10 mutations causing Kuskokwim syndrome, suggesting complexity of FKBP10 functions. We showed that collagen R780 substitutions causing OI result in reduced matrix deposition. We used a G610C mouse model of OI occurring in Old Order Amish community . We showed that unexpectedly high brittleness of G610C mouse femurs is partly due to abolished anterior-posterior drift preventing replacement of woven bone laid at young age by lamellar bone. Furthermore, mature lamellar bone, whose hypermineralization contributes to bone fragility in human OI, was hypermineralized in G610C mice as well. We developed and used an HDIR method to show that cultured G610C osteoblasts deposited less dense collagen matrix, explaining hyperminerlaization and brittleness of OI bone material. Our treatment with low-protein diet activating autophagy, rescued the hypermineralization defect, pointing at autophagy as OI treatment target. We have shown that osteoblast-specific knockout of Autophagy protein 5 (Atg5) in G610C mice reduces bone matrix hypermineralization and are presently characterizing effects of Atg5 overexpression. We developed force microscopy to analyze and measure mechanical properties of mouse cartilage matrix to study effect of matrix structure and composition on its mechanical properties. We assisted NIAMS researchers in micro-spectroscopic characterization of the structure in poorly ordered peptides from HIV-Rev1 protein. Currently, we are quantifying with HDIR composition and structure of bovine cartilage to assist developing quantitative in vivo MRI imaging of cartilage composition. We assisted NINDS researchers in characterization of the structure of HIV-Tat peptide forming amyloid fibers and causing neurotoxicity.

我们正在开发通过红外 (IR) 显微光谱对组织进行定量、无标记组织学检查的新方法。在这项技术中，带有二维探测器阵列的红外光谱仪连接到显微镜上。它同时测量组织切片中 16,384 微米大小点的红外吸收光谱。化学基团的化学成分、方向和相互作用是根据化合物的独特光谱指纹确定每个点内的，并绘制为二维图像。迄今为止，该技术在研究和诊断中的应用仅限于脱水组织，因为水强烈吸收红外光，导致光学干涉伪影。然而，脱水会扭曲生物分子和组织结构，抹掉光谱指纹，并降低化学和光谱分辨率。 为了克服这一限制，我们设计并建造了具有热机械稳定性的红外室，可以将组织保持在所需温度的溶液中。通过减少干扰伪影，与商业设计相比，我们将光谱再现性和化学分辨率提高了两个数量级。新腔室的多功能溶剂控制和更高的光谱精度允许采用新的实验方法。例如，通过这项技术，我们可以将胶原蛋白与其他蛋白质区分开来，解析不同的糖胺聚糖 (GAG)，甚至量化软骨中 GAG 硫酸化的程度。我们收集了经过充分纯化和表征的结缔组织成分的光谱库，并以显着提高的光谱化学分辨率进行测量。我们还开发了一种通过偏振红外高光谱成像定量绘制组织切片中胶原蛋白方向的新方法。为了将组织成分与生化过程联系起来，我们将这种高清（HD）显微光谱成像与具有微米空间分辨率的分析放射自显影成像结合起来。 我们还设计了一种用于高清拉曼显微光谱的新型热机械稳定流通室，允许使用拉曼、偏振和荧光显微镜同时对骨样本进行额外表征。我们采用荧光染料对骨形成表面进行体内动态标记，从而可以划分在给定时间点形成的骨区域，并对相同样品进行高清拉曼显微光谱分析。 我们使用这些技术来表征骨和软骨发育不良、骨肿瘤和其他结缔组织病理中的胶原基质组织。具体来说，我们研究了由 SLC26A2 硫酸盐/氯化物逆向转运蛋白突变引起的骨性发育不良 (DTD) 敲入小鼠模型。这些突变导致软骨细胞对硫酸盐的吸收不足，从而导致对软骨发育和完整性至关重要的蛋白聚糖 GAG 链硫酸化不足。 DTD 会延迟骨骼发育并表现出不寻常的进展：硫酸盐不足随着年龄的增长而正常化，但关节软骨会随着年龄的增长而退化。我们对新生小鼠股骨头软骨细胞外基质成分的定量成像表明，在 DTD 小鼠中，与野生型 (WT) 相比，关节区和肥大前期区的 GAG 硫酸化水平较低，但在休息区几乎正常。在 DTD 小鼠中，偏振红外高光谱成像显示关节表面切向定向胶原纤维致密层的破坏。这种保护层的破坏可能会导致关节蛋白多糖耗竭，这是早期骨关节炎的一个标志，我们在出生时就观察到了这种情况，尽管 GAG 硫酸化已正常化，但随着年龄的增长，这种情况会进一步恶化。通过对软骨外植体中掺入的 35S-硫酸盐进行定量显微摄影，我们发现 DTD 中不同软骨区域的硫酸不足的变异性与关节和肥大前期区域更快的软骨素合成速率相关。这一观察结果解释了硫酸盐不足随着年龄的增长而正常化。 我们协助 NIBIB 科学家使用我们的技术证明功能化碳纳米管在过度表达透明质酸受体的癌细胞内的渗透，验证了这种抗癌药物的细胞内递送方法。 我们研究了干细胞移植对成骨不全（OI）脆性小鼠模型骨质量的影响。使用荧光和偏光显微镜，我们区分了股骨皮质中不同类型的骨（编织骨和层状骨）内宿主细胞和绿色荧光蛋白标记的供体细胞产生的基质。使用拉曼显微光谱，我们发现每种材料类型中供体细胞附近的基质矿化异质性较低，这表明供体细胞产生的更好的基质组织可能有助于增加治疗小鼠的骨强度。 我们研究了蛋白激酶 A 亚基缺失不同组合的小鼠中因环 AMP 信号传导破坏引起的内分泌骨肿瘤。使用偏光显微镜、拉曼显微光谱和动态骨标记，我们发现成年 Prkar1a+/-Prkaca+/- 小鼠的肿瘤形成会导致未成熟皮质骨的骨膜沉积。我们发现额外的 Prkar2a+/- 或 Prkar2b+/- 缺失可以部分补偿 Prkar1a+/- 缺失对骨材料局部成熟、基质矿化和胶原蛋白组织的影响。我们发现塞来昔布治疗 R1a+/-Ca+/- 小鼠可改善覆盖肿瘤的皮质骨的组织和矿化。 我们使用高清拉曼显微光谱法对细胞培养物中的胶原蛋白含量进行无损定量。我们将此方法应用于由 FKBP10 缺失突变引起的隐性成骨不全症的人成纤维细胞培养物。我们发现，尽管这些细胞和 ppib-/- 小鼠成骨细胞合成了正常数量的胶原蛋白，但细胞外基质中胶原蛋白的沉积却减少了。然而，沉积物与引起 Kuskokwim 综合征的其他 FKBP10 突变的疾病严重程度和表型无关，表明 FKBP10 功能的复杂性。我们发现，导致 OI 的胶原蛋白 R780 替代会导致基质沉积减少。 我们使用了发生在旧秩序阿米什社区的成骨不全症 G610C 小鼠模型。我们发现，G610C 小鼠股骨出人意料的高脆性部分是由于前后漂移的取消，阻止了年轻时铺设的编织骨被板层骨取代。此外，成熟的板层骨的过度矿化会导致人类成骨不全症患者的骨脆性，而在 G610C 小鼠中，成熟的板层骨也过度矿化。我们开发并使用 HDIR 方法来显示培养的 G610C 成骨细胞沉积密度较低的胶原基质，解释了 OI 骨材料的过度矿化和脆性。我们用低蛋白饮食激活自噬的治疗挽救了过度矿化缺陷，将自噬作为 OI 的治疗目标。我们已经证明，G610C 小鼠中成骨细胞特异性敲除自噬蛋白 5 (Atg5) 可减少骨基质过度矿化，目前正在表征 Atg5 过表达的影响。 我们开发了力显微镜来分析和测量小鼠软骨基质的力学性能，以研究基质结构和成分对其力学性能的影响。 我们协助 NIAMS 研究人员对 HIV-Rev1 蛋白中排列不良的肽的结构进行显微光谱表征。目前，我们正在对牛软骨的 HDIR 成分和结构进行量化，以协助开发软骨成分的定量体内 MRI 成像。 我们协助 NINDS 研究人员表征形成淀粉样纤维并引起神经毒性的 HIV-Tat 肽的结构。