Functions of extracellular matrix proteins in dental and skeletal mineralization

项目总结/摘要 牙周复合体包括牙骨质、牙周膜和牙槽骨，是牙齿健康的重要组成部分 附件和功能。牙周病是地球上最普遍的疾病之一， 破坏和牙齿脱落，影响健康和生活质量。然而，牙周膜再生是可能的， 目前的疗法是不可预测的，很少有真正的再生，许多缺乏生物学基础。路径 部分原因是成牙骨质细胞（产生牙骨质的细胞）的起源和分化， 牙骨质）和牙骨质形成中的调节过程仍然知之甚少。骨唾液酸蛋白 BSP蛋白）是与矿化组织相关的多功能细胞外基质（ECM）蛋白， 骨骼形成和骨重塑。Ibsp基因敲除（Ibsp-/-）小鼠的特征是缺乏功能性非细胞 牙骨质、牙周膜脱离、牙槽骨缺损和细胞牙骨质矿化以及牙槽骨 吸收和牙齿脱落。BSP功能的潜在机制仍不清楚，尽管BSP具有 三个功能结构域，包括胶原结合结构域、促进胶原结合的聚谷氨酸（polyE）基序， 在一些实施方案中，所述多肽包含一个或多个整合素结合结构域，所述整合素结合结构域包含矿化结构域和启动细胞信号传导的精氨酸-甘氨酸-天冬氨酸（RGD）整合素结合结构域。 我们认为BSP是研究牙骨质形成和牙槽骨愈合的一个独特的候选因子 因为它是选择性表达的，对正常功能至关重要，并通过非冗余机制运行 与其他生长因子不同。根据我们的初步数据，我们的假设是，成牙骨质细胞是 与成骨细胞不同的表达BSP的外胚间充质细胞; BSP通过RGD结构域向骨细胞发出信号 在骨重建中，通过胶原结合结构域引导矿物质沉积到胶原纤维上; 促进牙槽骨愈合。这些假设将通过3个具体目标进行检验：（1）确定起源， 外胚间充质细胞与上皮细胞中Ibsp条件性消融对成牙骨质细胞转录组影响 群体，并使用内源性黄色荧光蛋白表达的Ibsp-topaz小鼠离体纯化 通过荧光激活细胞分选术（FACS）检测成牙骨质细胞并进行转录组学分析;（2）分析 BSP在骨和牙齿中的作用机制，通过确定BSP在胶原蛋白上的结合位点， 体外和遗传分析成牙骨质细胞中BSP RGD和胶原结合结构域的失活 研究BSP在磨牙牙槽骨修复中的作用 小鼠中的愈合模型和人类受试者中的截骨愈合模型以绘制BSP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.