IN VITRO INITIATION OF BIOLOGICAL CALCIFICATION

生物钙化的体外引发

• 批准号: 3158276
• 负责人: ADELE L BOSKEY
• 金额: $ 15.73万
• 依托单位: HOSPITAL FOR SPECIAL SURGERY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3158276
• 关键词: X ray crystallography; basement membrane; calcification; cartilage development; cell differentiation; chick embryo; electron microscopy; extracellular matrix proteins; histochemistry /cytochemistry; immunochemistry; in situ hybridization; membrane proteins; messenger RNA; normal ossification; osteoarthritis; phosphates; proteoglycan; rickets; tissue /cell culture

The formation of calcified cartilage during longitudinal bone growth is a dynamic process which involves cellular action, cell-matrix interactions, and physicochemical phenomena. The process leading to biological calcification has not yet been fully explained. The long range goal of this study is to establish which factors govern the initial deposition of mineral and which regulate the development of that mineral. This investigation will use the differentiating chick limb-bud mesenchymal cell system which, depending on culture conditions, consistently forms either organized (normal) or randomly (dystrophic) mineralized cartilage matrices resembling those in tissues. A combination of physical chemistry and cell biology techniques will be used to evaluate the roles of inorganic and organic phosphate, matrix proteins, and vascular invasion, in cartilage calcification. Analyses will be based on quantitative techniques for characterizing the mineral, the organic matrix, and their interactions. X-ray diffraction, electron microscopy, and Fourier transform infrared microscopy will provide data on the nature of the mineral, its crystal size and perfection, its organization in the matrix, and its relative abundance. Immunocytochemistry and histochemistry, combined with in situ hybridization and analyses of cell mRNAs provide data on the composition of the mineralizing matrix and the cells expressing that matrix. It is postulated that phosphate (P) has a function in the regulation of calcification beyond its physicochemical role. To define this function the following hypotheses will be tested: a) Removal of P from matrix proteins will prevent chondrocyte-mediated calcification, b) P exerts its effect on the matrix at the post-translational rather than the genomic level, c) Mineralization is dependent on kinase-mediated phosphorylation of matrix proteins, d) Mineralization is affected by phosphorylation of cellular, as opposed to matrix, proteins, and e) P ions exert their effects by regulating mitochondrial and matrix calcium levels, or, by regulating the activities of enzymes involved in control of initiation of calcification and crystal growth. It is further suggested that in vivo mineralization could be regulated by modification, of those factors which cause initial mineral deposition. To determine why mineral forms at discrete sites in the matrix we will evaluate the hypotheses that the site of initial mineralization is determined by: a) The presence of specific matrix proteins, b) The activity, shape, turnover and maturation of the cell, and c) The presence of degradative activities. The reason cartilage calcification starts in the proximity of blood vessels in vivo will be examined in the culture system by testing hypotheses that a) Vascular invasion alters pH facilitating initial mineral deposition, b) The presence of basement membrane proteins promotes mineralization, and c) Endothelial cells provide factors which accelerate mineralization. The understanding of the cartilage calcification mechanism afforded by this study will aid the development of new therapies for diseases of impaired calcification (eg. growth disorders, rickets) and of accelerated or dystrophic calcification (eg. osteoarthritis).

骨纵向生长过程中钙化软骨的形成是 涉及细胞行为、细胞-基质相互作用的动态过程， 和物理化学现象。 导致生物的过程 钙化尚未完全解释。 长期目标是 这项研究的目的是确定哪些因素控制着初始沉积 矿物质并调节该矿物质的发育。 这 调查将使用分化鸡肢芽间充质 根据培养条件，一致形成的细胞系统 有组织（正常）或随机（营养不良）矿化软骨 类似于组织中的基质。 身体素质的结合 将使用化学和细胞生物学技术来评估其作用 无机和有机磷酸盐、基质蛋白和血管 侵袭、软骨钙化。 分析将基于 用于表征矿物、有机物的定量技术 矩阵及其相互作用。 X射线衍射、电子显微镜、 傅里叶变换红外显微镜将提供有关自然的数据 矿物的性质、晶体大小和完美程度、其组织结构 基质及其相对丰度。 免疫细胞化学和 组织化学，结合原位杂交和细胞分析 mRNA 提供有关矿化基质和 表达该基质的细胞。 据推测，磷酸盐 (P) 具有 调节钙化的功能超出其物理化学性质 角色。 为了定义该函数，将测试以下假设： a) 从基质蛋白中去除 P 将防止软骨细胞介导的 钙化，b) P 对基质发挥作用 翻译后水平而非基因组水平，c) 矿化是 依赖于激酶介导的基质蛋白磷酸化，d) 矿化受细胞磷酸化的影响，而不是 基质、蛋白质和 e) P 离子通过调节发挥作用 线粒体和基质钙水平，或者通过调节活性 参与控制钙化和晶体起始的酶 生长。 进一步表明体内矿化可能是 通过修改调节，导致初始矿物质的那些因素 沉积。 确定矿物在不同地点形成的原因 矩阵我们将评估初始位置的假设 矿化由以下因素决定： a) 特定基质的存在 蛋白质，b) 细胞的活性、形状、周转和成熟， c) 存在降解活动。 软骨的原因 体内血管附近开始钙化 通过测试以下假设在培养系统中进行检查：a) 血管 入侵改变 pH 值，促进初始矿物质沉积，b) 基底膜蛋白的存在促进矿化，c) 内皮细胞提供加速矿化的因子。 这 了解由此提供的软骨钙化机制 研究将有助于开发针对受损疾病的新疗法 钙化（例如生长障碍、佝偻病）和加速或 营养不良性钙化（例如骨关节炎）。