MECHANISM FOR BONE SIALOPROTEIN TO NUCLEATE BIOAPATITE

骨唾液酸蛋白使生物磷灰石成核的机制

• 批准号: 6375089
• 负责人: Ronald Joseph Midura
• 金额: $ 17.95万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6375089
• 关键词: SDS polyacrylamide gel electrophoresis; X ray crystallography; apatites; bone sialoprotein; confocal scanning microscopy; density gradient ultracentrifugation; electron microscopy; extracellular matrix proteins; glycoproteins; high performance liquid chromatography; laboratory rat; normal ossification; northern blottings; nucleic acid hybridization; osteoblasts; periosteums; protein biosynthesis; protein purification; protein structure function; sedimentation; southern blotting; tissue /cell culture; vesicle /vacuole

Mineralization of bone is of critical importance to its numerous functions and can be perturbed in local or systemic musculoskeletal disorders. Our understanding of skeletal development, fracture healing, and bone remodeling will be enhanced by insights into the anabolic processes of bone matrix formation and biomineralization. There is considerable interest in the identification of biological molecules which nucleate bone mineral (bioaptite). This proposal hypothesizes that newly synthesized bone sialoprotein (BSP) nucleate bioapatite when associated with membrane- delimited vesicles. This hypothesis will be tested using natural periosteum and three osteoblastic culture models. We have observed spherical (sub)micron-sized apatite-containing particles ("bioapatite vesicles") in postnatal periosteum of rat tibia, as well as in primary and cell line osteoblastic cultures. They contain BSP and are strikingly similar in appearance to mineralized structures observed in osteoid ("crystal ghost aggregates"). Four aims are proposed addressing the structure/function relationship of BSP and apatite in "bioapatite vehicles". (1) Characterize the structure of bioapatite vehicles isolated from postnatal periosteum. (2) Spatially locate the major components of purified bioapatite vehicles in postnatal periosteum in situ. (3) Identify and structurally characterize any bioapatite vehicle counterparts from osteoblastic cultures. (4) Over-express BSP from an inducible gene construct transfected into osteoblastic cells. Aims 1 and 2 will provide a precise compositional database to compare the structure of bioapatite vehicles to that of matrix vesicles and crystal ghost aggregates. Aim 3 will identify relevant culture models to assess mechanisms of formation and determine essential molecular components. Aim 4 will directly test whether over-expression of BSP enhance apatite nucleation and whether it is a critical structural component for bioapatite vehicle integrity. This proposed research will yield new insight into apatite formation by osteoblasts because it will analyze a unique nucleation structure and ascertain the function of BSP in these entities. Ultimately, e believe that characterization of these novel biomineralization structures will lead to a better understanding of the mechanisms for bine mineralization and perhaps lead to new treatments to improve mineralization disorders of bone.

骨矿化对其众多功能至关重要，在局部或全身性肌肉骨骼疾病中可能受到干扰。我们对骨骼发育、骨折愈合和骨重塑的理解将通过深入了解骨基质形成和生物矿化的合成代谢过程而得到加强。鉴定成核骨矿物（生物磷灰石）的生物分子引起了相当大的兴趣。本研究假设新合成的骨涎蛋白（BSP）与膜分隔囊泡相关时形成生物磷灰石核。这一假设将使用天然骨膜和三种成骨细胞培养模型进行检验。我们在大鼠胫骨出生后骨膜以及原代和细胞系成骨细胞培养中观察到球形（亚）微米大小的含磷灰石颗粒（“生物磷灰石囊泡”）。它们含有BSP，在外观上与在类骨中观察到的矿化结构（“晶体幽灵聚集体”）惊人地相似。在“生物磷灰石载体”中，提出了解决BSP和磷灰石结构/功能关系的四个目标。(1)表征从出生后骨膜分离的生物磷灰石载体的结构。(2)原位定位纯化生物磷灰石载体在出生后骨膜中的主要成分。(3)从成骨细胞培养中鉴定和结构表征任何生物磷灰石载体。(4)通过诱导型基因构建体转染成骨细胞过表达BSP。目的1和2将提供一个精确的成分数据库，以比较生物磷灰石载体的结构与基质囊泡和晶体鬼聚体的结构。目标3将确定相关的培养模型，以评估形成机制并确定基本的分子成分。Aim 4将直接测试BSP的过表达是否会增强磷灰石成核，以及它是否是生物磷灰石载体完整性的关键结构成分。这项研究将对成骨细胞形成磷灰石产生新的见解，因为它将分析一种独特的成核结构，并确定BSP在这些实体中的功能。最终，我们相信这些新的生物矿化结构的特征将导致更好地理解骨矿化机制，并可能导致新的治疗方法来改善骨矿化障碍。