IN VITRO INITIATION OF BIOLOGICAL CALCIFICATION

生物钙化的体外引发

• 批准号: 2079234
• 负责人: ADELE L BOSKEY
• 金额: $ 17.19万
• 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/2079234
• 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).

在纵向骨生长过程中钙化软骨的形成是一种