Functions of extracellular matrix proteins in dental and skeletal mineralization

项目摘要/摘要 牙周复合体，包括牙骨质、牙周膜和牙槽骨，是牙齿的关键。 依恋和功能。牙周病是地球上最常见的疾病之一，会导致牙周 破坏和牙齿脱落，并影响健康和生活质量。然而，牙周再生是可能的， 目前的治疗方法是不可预测的，很少是真正的再生，而且许多缺乏生物学基础。这条路 对再生的影响尚不清楚，部分原因是成牙骨质细胞的起源和分化 牙骨质)以及牙骨质形成中的调控过程仍然知之甚少。骨涎蛋白(IBSP) 基因；BSP蛋白)是一种与矿化组织相关的多功能细胞外基质(ECM)蛋白， 骨骼形成和骨骼重塑。IBSP基因敲除(IBSP-/-)小鼠功能缺失 牙骨质、牙周膜脱离、缺陷性牙槽骨和细胞性牙骨质矿化，以及牙槽骨 吸收和牙齿脱落。BSP功能的潜在机制尚不清楚，尽管BSP 三个功能结构域，包括一个胶原结合结构域，多聚谷氨酸(Polye)基序，促进 矿化，以及启动细胞信号的精氨酸-甘氨酸-天冬氨酸(RGD)整合素结合域。 我们认为BSP是研究牙骨质形成和牙槽骨愈合的唯一候选因子。 因为它是有选择地表达的，是正常功能所必需的，并且是通过非冗余机制运行的(S) 有别于其他增长因素。根据我们的初步数据，我们的假设是成牙骨质细胞 表达BSP的外胚间充质细胞与成骨细胞不同；BSP通过RGD结构域向骨细胞发出信号 通过胶原结合域将矿物质沉积到胶原纤维上；以及BSP 促进牙槽骨愈合。这些假说将通过三个具体的目标来检验：(1)定义起源和 条件消融IBSP的成牙骨质细胞转录组的对比研究 并利用IBSP-TOPAZ小鼠内源性黄色荧光蛋白的表达进行体外纯化 对成牙骨质细胞进行荧光激活细胞分选，并进行转录分析；(2)分析 BSP在骨骼和牙齿中发挥作用的机制是通过确定BSP与胶原和 成牙骨质细胞BSP、RGD和胶原结合域失活的体外和遗传学分析 (3)评价BSP在磨牙槽骨修复中的作用。 小鼠的愈合模型和人类受试者的截骨愈合模型以映射BSP的表达，以及 确定当BSP被消融或特定功能域被灭活时，小鼠的愈合结果。

PROJECT SUMMARY/ABSTRACT The periodontal complex, including cementum, periodontal ligament (PDL), and alveolar bone, is critical for tooth attachment and function. Periodontal diseases are among the most prevalent on earth, causing periodontal destruction and tooth loss, and affecting health and quality of life. Periodontal regeneration is possible, however, current therapies are unpredictable, few are truly regenerative, and many lack a biologic foundation. The path to regeneration remains unclear, in part, because origins and differentiation of cementoblasts (cells that produce cementum), and regulatory processes in cementogenesis, remain poorly understood. Bone sialoprotein (Ibsp gene; BSP protein) is a multifunctional extracellular matrix (ECM) protein associated with mineralized tissues, skeletal formation, and bone remodeling. Ibsp knockout (Ibsp-/-) mice feature absence of functional acellular cementum, PDL detachment, defective alveolar bone and cellular cementum mineralization, and alveolar bone resorption and tooth loss. The underlying mechanisms of BSP function remain unknown, although BSP harbors three functional domains, including a collagen-binding domain, polyglutamic acid (polyE) motifs that promote mineralization, and an arginine-glycine-aspartic acid (RGD) integrin-binding domain that initiates cell signaling. We propose that BSP is a unique candidate factor for studying cementum formation and alveolar bone healing because it is selectively expressed, essential for proper function, and operates by non-redundant mechanism(s) distinct from other growth factors. Based on our preliminary data, our hypotheses are that cementoblasts are BSP-expressing ectomesenchymal cells distinct from osteoblasts; BSP signals bone cells via the RGD domain in bone remodeling and directs mineral deposition onto collagen fibrils via the collagen-binding domain; and BSP promotes alveolar bone healing. These hypotheses will be tested by 3 specific aims: (1) To define the origin and transcriptome of cementoblasts using conditional ablation of Ibsp from ectomesenchymal vs. epithelial cell populations, and use endogenous yellow fluorescent protein expression in Ibsp-topaz mice to ex vivo purify cementoblasts by fluorescence-activated cell sorting (FACS) and perform transcriptomic analysis; (2) To analyze the mechanism by which BSP functions in bones and teeth by defining the binding site of BSP on collagen and analyzing inactivation of BSP RGD and collagen-binding domains in cementoblasts in vitro and genetically engineered mouse lines in vivo; and (3) To evaluate the role of BSP in alveolar bone repair using a molar socket healing model in mice and an osteotomy healing model in human subjects to map BSP expression, and determine healing outcomes in mice when BSP is ablated or specific functional domains have been inactivated.