MECHANISM FOR BONE SIALOPROTEIN TO NUCLEATE BIOAPATITE

骨骼的矿化对其众多功能至关重要，并可在局部或全身肌肉骨骼疾病中受到干扰。通过对骨基质形成和生物矿化的合成代谢过程的洞察，我们对骨骼发育、骨折愈合和骨重塑的理解将得到加强。在鉴定成核骨矿物(黑磷灰石)的生物分子方面有相当大的兴趣。这一建议假设，新合成的骨涎蛋白(BSP)在与膜分隔的囊泡结合时形成生物磷灰石。这一假设将使用天然骨膜和三种成骨细胞培养模型进行验证。我们在出生后的大鼠胫骨骨膜以及原代和成骨细胞培养中观察到了球形(亚)微米级的含有磷灰石的颗粒(“生物磷灰石囊泡”)。它们含有BSP，在外观上与类骨质中观察到的矿化结构(“晶体幽灵集合体”)惊人地相似。提出了解决生物磷灰石载体中BSP和磷灰石结构/功能关系的四个目标。(1)对从出生后骨膜分离的生物磷灰石载体的结构进行表征。(2)对纯化后的生物磷灰石载体在出生后骨膜中的主要成分进行空间定位。(3)从成骨细胞培养物中鉴定和鉴定任何生物磷灰石载体的结构特征。(4)将诱导型基因载体转入成骨细胞，获得高表达的BSP。AIMS 1和2将提供一个精确的成分数据库，将生物磷灰石载体的结构与基质小泡和晶体幽灵聚集体的结构进行比较。目标3将确定相关的培养模式，以评估形成机制和确定必要的分子成分。目的4将直接测试BSP的过度表达是否促进磷灰石成核，以及它是否是生物磷灰石载体完整性的关键结构成分。这项拟议的研究将为成骨细胞形成磷灰石提供新的见解，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，对这些新的生物矿化结构的表征将有助于更好地理解骨骼矿化的机制，并可能导致改善骨骼矿化障碍的新疗法。

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.