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。我们致力于研究骨和牙本质的矿化，其中磷灰石晶体特定地生长在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，因为它代表了一种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、非胶原性的细胞外基质蛋白大分子(NCP)首先通过与I型胶原纤维表面的相互作用来定位，然后指导晶体沉积的成核和某些特定的晶体习性方向相对于纤维轴的取向。NCP与生长的晶体进一步的相互作用被假设为在习性、形状和大小方面专门调节晶体的生长。虽然牙本质中存在许多NCP，但我们主要研究牙本质的主要磷蛋白：磷蛋白(Phophoryn，[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，大约有85%的磷酸化水平。 在这个方案中，我们计划引入PP编码基因，分别在成纤维细胞、成骨细胞和成牙本质细胞中过表达PP。这将使我们能够建立一个体内系统来研究生物矿化作用。我们将对这三个独立细胞中的晶体的矿化进行表征，以确定成核和晶体生长方向。这种方法将使我们能够解决许多重要的问题：与成骨细胞和/或成牙本质细胞相比，成纤维细胞中PP的翻译后修饰类似吗？当成核蛋白，如PP，在成纤维细胞中表达，而不表达NCP的其余部分时，矿化晶体是否具有相同的结晶学特性？我们还将通过表达重组PP并研究其在矿物上的体外磷酸化来研究磷酸化对体外矿化过程的影响。 这些研究应该会产生与磷蛋白在生物矿化中的具体作用相关的新信息。这一知识在确定生物矿化过程中涉及的机制方面将非常有价值，特别是因为这些研究是在体内模型中进行的，而不是传统的生物矿化体外研究方法。这些数据还将为设计异位矿化治疗提供基础知识，特别是在肾结石或失败的心脏瓣膜假体等疾病中。

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.