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：利用啮齿动物牙周模型，确定调节 PPi/Pi 水平的因子（例如 ANK、NPP1、PHOSPHO1 和 TNAP）在牙根形成和牙骨质形成中的作用，并应用这些知识局部/系统地传递因子以再生牙周组织疾病。结果证明了 Pi/PPi 调节剂和功能性 SIBLING 家族基因/蛋白质在矿化过程中的重要性，强调需要微调生理化学/细胞分子因子以实现牙周组织形成/再生所需的稳态平衡。 1. ALPL 突变会导致低磷酸酯酶症 (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)，这种酶通过还原焦磷酸盐 (PPi)（一种矿化抑制剂）来促进矿化。通过敲入在人类亲属中发现的常染色体显性 ALPL 错义突变（导致碱基变化，A116T），创建了一种新型牙齿特异性 HPP (odonto-HPP) 小鼠模型。 Alpl+/A116T 小鼠的牙槽骨发生改变，包括射线可透性和吸收性病变、牙槽骨嵴上的类骨质积聚，以及通过 microCT 测量的几种骨特性的显着差异。尽管全身 ALP 与切牙牙骨质厚度显着相关，但脱细胞牙骨质的非显着变化似乎不会影响牙周附着或功能。 Alpl+/A116T 小鼠是第一个牙体低磷酸酯酶症模型，提供了有关牙槽发育和功能的见解，并为未来研究中测试潜在的牙科靶向治疗提供了新模型。福斯特等人 2016 2. Phospho1 KO 和 AlplxPhospho1 dKO：Phospho1 KO 小鼠的牙槽骨、外套牙本质和细胞牙骨质的矿化受到适度干扰，但脱细胞牙骨质和 PDL 似乎未受到干扰。与骨骼相反，Spp1（编码 OPN 的基因）的基因消融并没有改善 Phospho1 KO 牙槽组织的定性或定量缺陷。这些数据支持 PHOSPHO1 在牙槽骨、牙本质和细胞牙骨质的正确矿化中的作用，并且无细胞牙骨质形成可能不需要基质囊泡。茨威夫勒等人。 2016年 3. ANK、NPP1 和 TNAP 体内和体外：检查 Ank 和 Npp1 KO 动物揭示了与基因表达相关的补偿机制。此外，TNAP和NPP1在牙骨质矿化中的表达模式和功能表明，TNAP的早期表达创造了一个低PPi环境，允许AEFC启动，而NPP1的后期表达增加了Ppi，限制了AEFC并置。这些结果为我们的体内研究（将于 2016 年底开始）提供了理论依据，该研究使用啮齿类动物的牙周伤口愈合模型，通过慢病毒构建体传递控制 Pi/Ppi 的因子。 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 月重新评估成果和后续步骤。 项目 B：定义细胞外基质蛋白在牙周发生中的作用，重点关注 SIBLINGS 和胶原蛋白。 BSP：BSP 属于小整合素结合配体 N 连接糖蛋白 (SIBLING) 家族，这是一个酸性磷蛋白家族，在矿化组织中表达。我们报道 BSP KO 小鼠具有深刻的牙周表型，类似于 Alpl KO 小鼠。在此基础上，我们与 H. Goldberg 博士合作，检查了 KO 小鼠的肌腱/韧带功能，并报告说，根据该蛋白质已知的体外功能特性，BSP 可能是肌腱重新附着的有用治疗分子和骨韧带。马里诺维奇等人。 2016 OPN：根据 Ank-/- 小鼠 PDL 的蛋白质组学分析，我们将 OPN（一种 SIBLING）确定为感兴趣的因子。使用多种模型来确定 OPNs 与 PDL 区域的功能。我们的结论是，OPN 在发育过程中调节牙骨质矿化以及牙周结构和功能方面不具有非冗余或关键作用。然而，在伤口愈合模型中，OPN 功能的丧失可能会产生后果，目前多个小组正在对此进行探索。计划2016年9月投稿 正在进行中：为了进一步明确 BSP 对 Pi/PPi 调节相关基因的调节机制，使用 CRISPR CAS 技术生成了 Bsp KO 成牙骨质细胞 (OCCM30)。初步结果表明，BSP 在矿化过程中可能至少有两个作用，即调节局部 Pi/PPi 水平的调节剂和通过各种反馈环调节转录因子的表达。此外，我们正在开发 OPN x BSP dKO 以确定 OPN KO 是否会改变 BSP KO 表型。 胶原蛋白：从逻辑上讲，患有成骨不全（OI）（一种以低骨量、脆性和易碎骨以及骨折为特征的细胞外基质遗传性疾病）的个体将有明显的牙周缺陷。然而，迄今为止的报告重点关注一般骨病理学，在某些情况下，关注牙本质缺陷（牙本质发育不全（DI）），而对牙周组织的关注有限。作为脆骨联盟的一部分，我们与几名校外研究人员开展了一项多方合作项目，使我们能够从四种类型的成骨不全小鼠身上获取组织，并从成骨不全患者身上拔出牙齿。我们首次确定了由常染色体隐性遗传和常染色体显性 OI 引起的小鼠牙周变化。变化反映了发育缺陷，以及生命后期的功能改变和重塑。正在进行中：使用 CRISPR/Cas9 技术和 OCCM 细胞，我们将制备敲入 c.1546G &RT; T 细胞（模拟 Brtl+/- 小鼠）和 Crtap 完整基因 KO 以确定这些修饰对细胞功能的影响。 其他项目：IRF6、CL/P 和牙齿表现：Emily Chu DDS，博士，以完成她的博士学位。 （不会在 NIAMS/NIH 继续）。唇裂，无论有或没有腭受累（CLP），都与发育性牙齿异常的较高发生率相关。由于大多数 CLP 基因在整个口腔上皮细胞中表达，因此我们假设 CLP 基因在牙齿图案形成和釉质形成中发挥重要的功能作用。结果支持 IRF6 在牙齿数量、牙冠和牙根形态以及釉质形成中的作用，这可能是由于 Irf6 在上皮细胞类型的组织和极性中的功能作用。我们的数据强化了这样一种观念，即各种孤立的牙齿缺陷可以被视为受影响个体亲属的 CLP 谱系的一部分。楚等人。 2016年 此外，我们与 Lynda Bonewald 合作建立了一种新的永生化细胞系：鼠牙骨质细胞。赵等人。 2016年