DENTIN BIOMINERALIZATION

描述（申请人提供）：生物矿化是脊椎动物和无脊椎动物中最广泛和最重要的过程之一。我们一直致力于研究骨和牙本质的矿化，其中磷灰石晶体特别生长在I型胶原基质中。虽然这两种组织矿化的整体机制可能是相似的，但我们采用了牙本质系统，因为它代表了比骨骼相对简单的系统。基本的工作假设是，某些酸性的、磷酸化的、非胶原的细胞外基质蛋白大分子（NCP）首先通过与胶原纤维表面的相互作用定位，然后指导晶体沉积的成核和相对于纤维轴的某些特定结晶习惯方向的取向。假设NCP与生长晶体的进一步相互作用可以在习惯、形状和大小方面特异性地调节晶体的生长。虽然牙本质中存在许多NCP，但我们的研究重点是牙本质的主要磷酸化蛋白：磷酸化蛋白（Phosphophoryn） [PP]。PP本质上是非常酸性的，其特点是丝氨酸残基含量非常高，磷酸化水平约为85%。

DESCRIPTION (provided by applicant): Biomineralization is one of the most widespread and important processes in both vertebrates and invertebrates. We have directed our efforts to study the mineralization of bone and dentin in which apatite crystals are specifically grown within type I collagen matrices. Although it is likely that the overall mechanisms involved in mineralization of the two tissues are similar, we have adopted the dentin system since it represents a relatively simpler system than bone. The basic working hypothesis has been that certain acidic, phosphorylated, non-collagenous extracellular matrix protein macromolecules (NCP) are first localized by interaction with the collagen I fibril surfaces, which then direct the nucleation of crystal deposits and orientation of certain specific crystallographic habit directions relative to the fibril axes. Further interactions of the NCP with the growing crystals are postulated to specifically regulate crystal growth in terms of habit, shape and size. Although there are many NCP present in dentin, we have focused our efforts on the primary phosphoprotein of dentin: Phosphophoryn ([PP]). PP is very acidic in nature and characterized by a very high content of serine residues, at about an 85 percent level of phosphorylation. In this proposal we plan to introduce the gene encoding for PP to over-express it in fibroblast, osteoblast and odontoblast cells, respectively. This will enable us to establish an in vivo system to study biomineralization. We will characterize the mineralization of the crystals in these three separate cells to ascertain the nucleation and crystal growth directions. This approach will allow us to address many important questions: Is the post-translationnal modification of PP similar in fibroblast in contrast to osteoblast and/or odontoblast cells? Will the mineralized crystal have the same crystallographic properties when the nucleating protein, such as PP, is expressed in a fibroblast without the rest of the NCP, which is usually expressed in an osteoblast or odontoblast? We will also study the effect of phosphorylation on the Mineralization process in vitro by expressing the recombinant PP and studying its in vitro phosphorylation on the mineral. These studies should yield new information related to the specific role of phosphoproteins in biomineralization. This knowledge will be extremely valuable in determining the mechanisms involved in the biomineralization process particularly since the studies are conducted in an in vivo model in comparison to the traditional approaches of in vitro studies of biomineralization. This data will also provide basic knowledge for designing treatment of ectopic mineralization specifically in diseases as such kidney stones or failing heart valve prosthesis.