Laboratory of Oral Connective Tissue Biology

口腔结缔组织生物学实验室

• 批准号: 9359803
• 负责人: Martha Somerman
• 金额: $ 99.6万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9359803
• 关键词: Ablation; Affect; Alkaline Phosphatase; Amelogenesis; Animals; Attention; Biology; CRISPR/Cas technology; Cell Line; Cell physiology; Cells; Cementoblast; Cementogenesis; Cementum Formation; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Collagen; Complex; Connective Tissue; Data; Defect; Dental; Dental Cementum; Dental crowns; Dentin; Dentinogenesis Imperfecta; Development; Diphosphates; Disease; Doctor of Philosophy; Environment; Enzymes; Epithelial Cells; Equilibrium; Extracellular Matrix; Extracellular Matrix Proteins; Extramural Activities; Family; Feedback; Fracture; Future; Gene Expression; Gene Expression Regulation; Gene Proteins; Genes; Genetic; Glycoproteins; Human; Hypophosphatasia; In Vitro; Incidence; Incisor; Individual; Integrin Binding; Knock-in; Knockout Mice; Knowledge; Laboratories; Lesion; Life; Ligaments; Ligands; Link; Lip structure; Logic; Manuscripts; Maps; Measures; Missense Mutation; Modeling; Modification; Molecular; Morphology; Mus; Mutation; National Institute of Arthritis and Musculoskeletal and Skin Diseases; Natural regeneration; Oral; Osteogenesis Imperfecta; Osteoid; Outcome; Pathology; Patients; Pattern; Periodontal Diseases; Periodontal Ligament; Periodontium; Phenotype; Phosphoproteins; Plant Roots; Play; Process; Property; Proteins; Proteomics; Reporting; Research Personnel; Rodent; Rodent Model; Role; Skeleton; Staging; Structure; Subfamily lentivirinae; T-Lymphocyte; Technology; Tendon structure; Testing; Therapeutic; Thick; Time; Tissues; Tooth Abnormalities; Tooth Loss; Tooth structure; United States National Institutes of Health; Vesicle; Wound Healing; alveolar bone; base; bone; bone mass; cell type; immortalized cell; in vivo; in vivo Model; inhibitor/antagonist; insight; interest; kindred; microCT; mineralization; mouse model; novel; oral cavity epithelium; regenerative; skeletal; targeted treatment; transcription factor

Project A: Establish the role of factors regulating PPi/Pi levels, e.g., ANK, NPP1, PHOSPHO1, and TNAP, in root formation and cementogenesis and apply this knowledge to deliver factors locally/systemically to regenerate periodontal tissues, using rodent models of periodontal disease. Results demonstrate the importance of regulators of Pi/PPi and of functional SIBLING family genes/proteins during mineralization, highlighting the need for fine tuning physiochemical/cellular molecular factors toward achieving a homeostatic balance required for formation/regeneration of periodontal tissues. 1. Mutations in ALPL result in hypophosphatasia (HPP), a disease causing defective skeletal mineralization. ALPL encodes tissue nonspecific alkaline phosphatase (TNAP), an enzyme that promotes mineralization by reducing pyrophosphate (PPi), a mineralization inhibitor. A novel mouse model for tooth-specific HPP (odonto-HPP) was created by knocking in an autosomal dominant ALPL missense mutation (causing a base change, A116T), which was identified in a human kindred. Alpl+/A116T mice featured alterations in the alveolar bone, including radiolucencies and resorptive lesions, osteoid accumulation on the alveolar bone crest, and significant differences in several bone properties measured by microCT. Non-significant changes in acellular cementum did not appear to affect periodontal attachment or function, though systemic ALP correlated significantly with incisor cementum thickness. The Alpl+/A116T mouse is the first model of odontohypophosphatasia, providing insights on dentoalveolar development and function and providing a new model for testing potential dental-targeted therapies in future studies. Foster et al 2016 2. Phospho1 KO and AlplxPhospho1 dKO: Phospho1 KO mice featured modest disturbances in mineralization of alveolar bone, mantle dentin, and cellular cementum, yet acellular cementum and PDL appeared undisturbed. In contrast to the skeleton, genetic ablation of Spp1 (gene encoding OPN) did not ameliorate qualitative or quantitative defects in Phospho1 KO dentoalveolar tissues. These data support a role for PHOSPHO1 in proper mineralization of alveolar bone, dentin, and cellular cementum and that matrix vesicles may not be required for acellular cementum formation. Zweifler et al. 2016 3. ANK, NPP1 and TNAP in vivo and vitro: Examining Ank and Npp1 KO animals revealed a compensatory mechanism related to gene expression. Also, the expression patterns and functions of TNAP and NPP1 in cementum mineralization suggest that the early expression of TNAP creates a low PPi environment allowing for AEFC initiation, while later expression of NPP1 increases Ppi, restricting AEFC apposition. These results provide the rationale for our in vivo studies (to begin late 2016) using periodontal wound healing models in rodents to deliver factors controlling Pi/Ppi, using lentivirus constructs. 4. In an effort to define specific functions for ANK vs ENPP1, we are developing a double KO (dKO). We will reevaluate outcomes and next steps in Oct. 2016. Project B: Defining the role of extracellular matrix proteins in periodontogenesis with a focus on SIBLINGS and collagen. BSP: BSP belongs to the Small Integrin Binding Ligand N-linked Glycoprotein (SIBLING) family, a family of acidic phosphoproteins, expressed in mineralized tissues. We reported that BSP KO mice have a profound periodontal phenotype, similar to Alpl KO mice. Building on this, in collaboration with Dr. H. Goldberg, we examined tendon/ligament function in KO mice and reported that, based on the known in vitro functional properties of the protein, BSP may be a useful therapeutic molecule in the reattachment of tendons and ligaments to bone. Marinovich et al. 2016 OPN: We identified OPN, a SIBLING, as a factor of interest based on proteomic analysis of PDL from Ank-/- mice. Several models were used to determine OPNs function with the PDL region. We conclude that OPN does not have a non-redundant or critical role in regulating cementum mineralization and periodontal structure and function during development. Yet, in wound healing models loss of OPN function may have consequences, which is being explored by several groups at this time. Plan to submit manuscript Sept.2016 Ongoing: To further define the mechanistic aspects of BSPs regulation of genes associated with Pi/PPi modulation, Bsp KO cementoblasts (OCCM30), using CRISPR CAS technology, were generated. Preliminary results suggest that BSP may have at least two roles in the mineralization process, i.e., regulating modulators of local Pi/PPi levels and regulating expression of transcription factors through various feedback loops. In addition, we are developing an OPN x BSP dKO to determine if OPN KO would alter the BSP KO phenotype. Collagen: Logic would suggest that individuals with osteogenesis imperfect (OI), a heritable disorder of the extracellular matrix characterized by low bone mass, brittle and fragile bones, and bone fractures, would have marked periodontal defects. Yet, reports to date focus on general bone pathology and, in some cases, dentin defects (dentinogenesis imperfecta (DI)), with limited attention to the periodontium. Undertaking a multi-collaborative project, with several extramural investigators as part of the Brittle Bone Consortium, has enabled us to obtain tissues from four types of OI mice as well as extracted teeth from OI patients. For the first time, we identify periodontal changes in mice resulting from autosomal recessive and autosomal dominant OI. Changes reflect developmental defects, as well as altered function and remodeling during later stages in life. Ongoing: Using CRISPR/Cas9 technologies and OCCM cells we will prepare Knock-in c.1546G &RT; T cells (mimic Brtl+/- mouse) and Crtap complete gene KO to define the effects of these modifications on cell function. Other projects: IRF6, CL/P and dental manifestations: Emily Chu DDS, PhD for fulfillment of her PhD. (Will not be continued at NIAMS/NIH). Clefting of the lip, with or without palatal involvement (CLP), is associated with a higher incidence of developmental tooth abnormalities. Because most CLP genes are expressed throughout the oral epithelium, we hypothesized that CLP genes play an important functional role in tooth patterning and amelogenesis. Results support a role for IRF6 in tooth number, crown and root morphology and amelogenesis that is likely due to a functional role of Irf6 in organization and polarity of epithelial cell types. Our data reinforce the notion that various isolated tooth defects could be considered part of the CLP spectrum in relatives of an affected individual. Chu et al. 2016 In addition, we have established a new immortalized cell line: Murine Cementocytes in collaboration with Lynda Bonewald. Zhao et al. 2016

项目A：确定调节PPI/PI水平的因素的作用，例如ANK，NPP1，Phospho1和TNAP，在根形成和胶合发生中，并将这些知识应用于局部/系统地在局部/系统地提供牙周组织，使用牙周疾病模型再生牙周组织。结果证明了矿化过程中PI/PPI和功能性同胞家族基因/蛋白质的调节剂的重要性，强调需要微调生理化学/细胞分子因子对实现牙周组织形成/再生所需的稳态平衡。 1。ALPL的突变导致下局部（HPP），这是一种导致骨骼矿化缺陷的疾病。 ALPL编码组织非特异性碱性磷酸酶（TNAP），一种酶，该酶通过还原焦磷酸（PPI）（一种矿化抑制剂）来促进矿化。通过敲击常染色体显性alpl误义突变（导致基本变化，A116T）来创建一种用于牙齿特异性HPP（odonto-HPP）的新型小鼠模型，该模型已在人类的亲属中鉴定出来。 Alpl+/A116T小鼠在牙槽骨骨中发生了改变，包括放射性损伤和吸收性病变，骨质骨骨上的骨质骨会积聚，以及通过Microct测得的几种骨特性的显着差异。尽管系统性ALP与切牙正质厚度显着相关，但细胞胶质的不显着变化似乎并没有影响牙周附着或功能。 Alpl+/A116T小鼠是Odontohypophophatasia的第一个模型，它提供了有关牙道肺泡发育和功能的见解，并为未来研究中测试潜在的牙科靶向疗法提供了新的模型。 Foster等人2016 2。Phosho1KO和Alplxphospho1 DKO：Phospho1 KO小鼠在肺泡骨，地幔牙本质和细胞胶质的矿化方面具有适度的干扰，但伴有细胞胶质和PDL出现。与骨骼相反，SPP1的遗传消融（编码OPN）不能改善磷酸化牙齿牙道肺泡组织中的定性或定量缺陷。这些数据支持磷酸1在肺泡骨，牙本质和细胞胶质的适当矿化中的作用，并且可能不需要基质囊泡来形成细胞胶状囊泡。 Zweifler等。 2016 3。ANK，NPP1和TNAP在体内和体外：检查ANK和NPP1 KO动物显示了与基因表达相关的补偿机制。同样，TNAP和NPP1在质矿化中的表达模式和功能表明，TNAP的早期表达产生了较低的PPI环境，允许AEFC启动，而后来的NPP1表达会增加PPI，从而限制了AEFC的应用。这些结果为我们的体内研究（开始于2016年底）使用啮齿动物中的牙周伤口愈合模型为我们的体内研究提供了理由，以使用慢病毒构建体传递控制PI/PPI的因素。 4。为了定义ANK与ENPP1的特定功能，我们正在开发双KO（DKO）。我们将在2016年10月重新评估结果和下一步。 项目B：定义细胞外基质蛋白在牙周发生中的作用，重点是兄弟姐妹和胶原蛋白。 BSP：BSP属于在矿化组织中表达的酸性磷蛋白家族的小型整合素结合配体N-连接糖蛋白（同胞）家族。我们报告说，BSP KO小鼠具有强烈的牙周表型，类似于Alpl KO小鼠。在此基础上，我们与H. Goldberg博士合作研究了KO小鼠中的肌腱/韧带功能，并报告说，基于蛋白质的已知体外功能性能，BSP可能是肌腱和肌韧带的肌腱和韧带的重新分子中有用的治疗分子。 Marinovich等。 2016年OPN：我们确定了基于ANK - / - 小鼠PDL的蛋白质组学分析的兄弟姐妹OPN，这是一种感兴趣的因素。使用几种模型来确定PDL区域的OPN函数。我们得出的结论是，OPN在调节发育过程中调节牙骨质矿化以及牙周结构和功能中没有非冗余或关键作用。但是，在伤口愈合模型中，OPN功能的损失可能会产生后果，目前正在探索几个小组。计划提交手稿2016年9月 正在进行：为进一步定义了与PI/PPI调制相关的基因的机械方面，生成了使用CRISPR CAS技术的BSP KO COMENTOBLASTS（OCCM30）。初步结果表明，BSP在矿化过程中至少具有两个角色，即调节局部PI/PPI水平的调节剂，并通过各种反馈回路调节转录因子的表达。此外，我们正在开发一个OPN X BSP DKO，以确定OPN KO是否会改变BSP KO表型。 胶原蛋白：逻辑表明，成骨的个体不完美（OI），这是一种可遗传的细胞外基质疾病，其特征是低骨质量，脆性和脆弱的骨骼以及骨折的疾病，其牙周骨折的特征是明显的牙周缺陷。然而，迄今为止的报告集中在一般的骨骼病理学上，在某些情况下，牙本质缺陷（牙本质发生不完美（DI）），对牙周的关注有限。进行多个授权的项目，其中几个是校外研究者作为脆性骨骼财团的一部分，使我们能够从四种类型的OI小鼠中获取组织，并从OI患者那里提取牙齿。我们第一次确定了由常染色体隐性和常染色体显性OI导致的小鼠的牙周变化。变化反映了发育缺陷，以及在生命后期阶段的功能和重塑改变。正在进行：使用CRISPR/CAS9技术和OCCM单元格，我们将准备C.1546G＆RT; T细胞（模拟BRTL +/-小鼠）和CRTAP完成基因KO，以定义这些修饰对细胞功能的影响。 其他项目：IRF6，CL/P和牙齿表现形式：Emily Chu DDS，博士学位，用于履行其博士学位。 （不会在Niams/NIH继续进行）。唇裂，有或没有palat骨受累（CLP）的嘴唇与发育异常的发生率更高有关。由于大多数CLP基因在整个口服上皮中都表达，因此我们假设CLP基因在牙齿构图和没有变性中起着重要的功能作用。结果支持IRF6在牙齿数，牙冠和根部形态和休闲发生中的作用，这可能是由于IRF6在组织和上皮细胞类型的极性中的功能作用所致。我们的数据加强了这样一种观念，即在受影响个体的亲属中，各种孤立的牙齿缺陷可以被视为CLP谱系的一部分。 Chu等。 2016 此外，我们已经建立了一种新的永生细胞系：与Lynda Bonewald合作的鼠胶质细胞。 Zhao等。 2016