MECHANISM FOR BONE SIALOPROTEIN TO NUCLEATE BIOAPATITE

骨的矿化对其许多功能至关重要，并且可在局部或全身性肌肉骨骼疾病中受到干扰。通过深入了解骨基质形成和生物矿化的合成代谢过程，我们对骨骼发育、骨折愈合和骨重建的理解将得到加强。有相当大的兴趣，在确定的生物分子，成核骨矿物质（bioaptite）。这个提议假设新合成的骨唾液蛋白（BSP）与膜界定的囊泡结合时使生物磷灰石成核。将使用自然骨膜和三种成骨细胞培养模型来检验这一假设。我们已经观察到球形（亚）微米大小的磷灰石颗粒（“生物磷灰石囊泡”）在出生后的大鼠胫骨骨膜，以及在原代和细胞系成骨细胞培养。它们含有BSP，在外观上与类骨质中观察到的矿化结构（“晶体幽灵聚集体”）惊人相似。提出了四个目标，解决BSP和磷灰石的结构/功能关系的“生物磷灰石车辆”。(1)表征从出生后骨膜分离的生物磷灰石载体的结构。(2)在出生后骨膜原位定位纯化生物磷灰石载体的主要成分。(3)识别并从结构上表征任何来自成骨细胞培养物的生物磷灰石载体对应物。(4)从转染成骨细胞的诱导基因构建体中过度表达BSP。目的1和2将提供一个精确的组成数据库，以比较生物磷灰石载体的结构与基质囊泡和晶体鬼聚集体的结构。目标3将确定相关的文化模式，以评估形成机制，并确定必要的分子组成。目标4将直接测试BSP的过度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，因为它将分析独特的成核结构，并确定BSP在这些实体中的功能。最后，我们相信，这些新的生物矿化结构的表征将导致更好地理解骨矿化的机制，并可能导致新的治疗方法，以改善骨矿化障碍。

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.