Laboratory of Oral Connective Tissue Biology

口腔结缔组织生物学实验室

• 批准号: 10006389
• 负责人: Martha Somerman
• 金额: $ 101.32万
• 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/10006389
• 关键词: Age; Alveolar; Animal Model; Biological Assay; Biology; Blood; Body Weight; Bone Marrow; Cartilage; Cell Maturation; Cell model; Cells; Cementoblast; Cementoclast; Cementogenesis; Clinical Trials; Collaborations; Collagen; Complement; Complex; Connective Tissue; Data; Defect; Dental; Dental Cementum; Dental Pulp; Dentin; Development; Epididymis; Equilibrium; Exhibits; Expression Profiling; Extracellular Matrix Proteins; Femur; Fostering; Gene Expression; Gene Family; Gene Proteins; Generations; Genes; Glucose; Histologic; Histology; Homeostasis; Image; In Vitro; Inflammatory; Insulin; Integrin Binding; Intramuscular; Kidney; Knockout Mice; Knowledge; Laboratories; Liver; Maps; Mediating; Microscopy; Minerals; Molecular; Morphology; Mus; Mutation; Natural regeneration; Operative Surgical Procedures; Oral; Orthodontic; Osteoclasts; Pancreas; Pattern; Periodontal Diseases; Periodontal Ligament; Periodontium; Phenotype; Plant Roots; Production; Property; Proteins; Proteomics; Publications; RGD (sequence); Reporting; Rodent Model; Role; Site; Stromal Cells; Subfamily lentivirinae; TNFSF11 gene; Technology; Time; Tissue Model; Tissues; Tooth Loss; Tooth Movement; Tooth root structure; Tooth structure; Triglycerides; Wolves; Wound Healing; alveolar bone; base; bone; cell type; craniofacial; cytokine; density; design; experimental study; extracellular; improved; in vitro Model; in vivo; in vivo Model; in vivo evaluation; lipid metabolism; macrophage; male; microCT; migration; mineralization; osteoclastogenesis; protein expression; regenerative; scaffold; second harmonic; transcriptome sequencing

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 Pi/PPi and SIBLING family genes/proteins during mineralization, highlighting the need for both physiochemical and cellular molecular factors to achieve homeostatic balance required for formation/regeneration of periodontal tissues. 1. Cell, Tissues and Animal Models of Periodontal Disease: a. Animal Models for regeneration of the periodontal apparatus: Previously, we reported factors controlling PPi/Pi levels have significant roles during formation of the periodontium. In a proof of principle experiment using Ibsp KO mice, exhibiting marked periodontal tissue destruction, TNAP lentivirus, intramuscular delivery, rescued the Ibsp KO phenotype, e.g., insertion of PDL into newly formed cementum and increased alveolar bone volume. In other experiments periodontal fenestration defects were created in ANK and ENPP1 KO mice and at 30 days post-surgery we noted increased cementum regeneration in tissues obtained from KO mice. Data to date suggest different effects of osteoclast like cells on wound healing between tissues analyzed from WT and KO mice and further between tooth root and surrounding bone. We are defining the osteoclast profile in a collaboration with Dr. Ozato s lab. These collective results highlight that factors modulating PPi/Pi levels at local periodontal sites promote cementogenesis. Thus, we initiated studies to delivery TNAP to local sites in Ibsp KO mice. Results are very promising (i.e., cementogenesis promoted) and now, with Dr. Nadine Samara, we are designing TNAP targeted to mineralized tissues for local delivery. We are using in vitro models to determine mechanisms by which TNAP activates mineralization and we continue to evaluate scaffold materials for improved local delivery. b. Ank, Enpp1, dKO and Ank, Alpl dKO mice and cells, in vitro: To determine whether effects of ANK and Enpp1 are additive/synergistic and if mechanistic differences exist in how they control cementogenesis, we generated Ank, Enpp1, dKO and Ank, Alpl dKO mice. Histological and microCT results indicate that acellular cementum phenotype exhibited by the Ank, Enpp1 dKO is a composite phenotype of the single KO. Ank, Alpl dKO mice exhibit improved cementogenesis compared to Alpl KOs, further supporting PPi as a key regulator in cementogenesis. Effects on cementum did not parallel effects on bone. Bone defects appeared more severe in Ank KO and dKO mice compared to Enpp1 KO mice, suggesting that ANK and ENPP1 have non-redundant roles in upregulating extracellular PPi. Additionally, although functional acellular cementum was formed in Ank, Alpl dKO mice, alveolar bone volume and mineral density were not improved compared to Alpl KO mice. Ongoing studies, using proteomics, qPCR microarray and RNA seq technologies are focused toward defining protein and gene expression profiles of PDL tissues obtained from Ank and Enpp1 single and dKO mice at various stages of tooth root development. Further, as mentioned in 1a, we are contrasting periodontal wound healing capabilities of Ank vs Enpp1 KO mice. To complement in vivo studies, Ank KO, Enpp1KO and dKO cementoblasts are being subjected to various mineralization conditions, e.g. Pi or PPi. and data suggest differences in gene expression of osteoclast factors/modulators between cell types. In vitro osteoclast assays using bone marrow stromal cells from Ank and Enpp1 single and dKO mice are being conducted to assess osteoclast differentiation and resorption with ANK and/or ENPP1 loss. c. Orthodontic tooth movement: To continued studies with Dr. Wolf, focused on investigate the role of Pi/ PPi in regulating osteoclast/odontoclast distribution, Ank-/- mice were subjected to orthodontic loading. Also, to characterize the effect of a compromised periodontia on tooth movement, we exposed Ibsp-KAE (BSP RGD replaced by KAE), Ibsp KO, and WT mice to orthodontic loading. We are performing micro CT and histological analyses in tissues obtained from these orthodontically treated mice. Project B: Defining the role of extracellular matrix proteins in periodontogenesis with a focus on SIBLINGS and collagen. BSPxOPN: In 2018 (Foster et al. Bone) we concluded that OPN has a non-redundant role regulating mineralization of dentin and bone, influences tissue properties of PDL and pulp, but does not control acellular cementum apposition. As a next step, we generated and are characterizing periodontal tissues obtained from Spp1(OPN) x Ibsp (BSP) dKO mice to determine if loss of OPN expression rescues the Ibsp KO periodontal phenotype. Preliminary micro CT analysis indicate decreased alveolar bone proper volume in dKO vs. WT, yet greater than that of Ibsp KO mice, suggesting additive, yet complex interactions between OPN and BSP in maintaining periodontal homeostasis. Histological analyses revealed defective periodontal complex and increased number of osteoclasts along the alveolar bone in dKO, like Ibsp KO. Thickened alveolar crest was noted as a unique phenotype in dKO mice. An intriguing finding was huge osteoclasts noted in cells from femurs of Spp1 KO mice but not Ibsp KO or dKO cells. In collaboration with Dr. Hanson Fong, TEM imaging will be used to define further the morphology and function of osteoclasts in vivo and in vitro, obtained from these KO mice. Ibsp-KAE: As a step in defining the mechanism of BSP function in cementogenesis, we generated mice where the BSP RGD domain (integrin binding domain) was replaced by a non-functional KAE sequence (Ibsp-KAE). Results from histology and second harmonic generation microscopy, a collaboration with Dr. Ralston, reveal that in contrast to Ibsp KO mice, Ibsp-KAE mice display normal cementogenesis, but disorganized PDL with significantly increased osteoclasts along the alveolar bone, like Ibsp KO mice. Despite increased osteoclasts, micro CT analysis revealed a higher alveolar bone volume in older age Ibsp-KAE mice. In vitro data examining osteoclastogenesis potential of femur macrophages, demonstrated normal maturation of cells obtained from Ibsp-KAE and Ibsp KO comparable to WT mice. Based on these results we hypothesize that the weakened PDL insertion in Ibsp-KAE mice triggers inflammatory cytokines production, which in turn promotes expression of RANKL and/or RANK, resulting in increased osteoclast activity locally. Next, we analyzed the migration properties of Ibsp KO and Ibsp-KAE cementoblasts in vitro. Ibsp-KAE cells migrated slower and at the same rate as Ibsp KO cells, when compared to WT cells providing evidence for loss of the RGD region as the factor mediating weakened PDL properties we noted in vivo. Another unanticipated finding was that Ibsp-KAE mice exhibit an increase in body weight over time vs WT and Ibsp KO mice. As a first step to understand this phenotype we have initiated studies to determine if these mice (using male mice only at this time) exhibit alterations in specific blood markers to include insulin, glucose and triglycerides and in specific tissues associated with fat metabolism, e.g., liver, pancreas, kidney and epididymis. Collagen: In collaboration with Dr. Nan Hatch, a publication was submitted June 2019, highlighting craniofacial and dental defects in tissues obtained from Crtap mice (inactivating mutations in the gene for cartilage-associated protein).

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。结果表明，矿化过程中PI/PPI和兄弟姐妹家族基因/蛋白质的重要​​性，强调了对实现牙周组织形成/再生所需的生理化学和细胞分子因子的需求。 1。牙周疾病的细胞，组织和动物模型： 一个。牙周设备再生的动物模型：以前，我们报道了控制PPI/PI水平的因素在牙周形成过程中具有重要作用。在使用IBSP KO小鼠的原理实验的证明中，表现出明显的牙周组织破坏，TNAP慢病毒，肌肉内递送，拯救了IBSP KO表型，例如，将PDL插入新形成的骨质骨骼中，并增加了肺泡骨体积。在其他实验中，在ANK和ENPP1 KO小鼠中产生了牙周室外缺陷，在手术后30天，我们注意到从KO小鼠获得的组织中骨髓再生的增加。迄今为止的数据表明，破骨细胞之类的细胞对来自WT和KO小鼠分析的组织之间的伤口愈合以及牙齿根和周围骨之间的进一步影响。我们正在与Ozato S Lab的合作中定义破骨细胞的轮廓。这些集体结果强调了调节局部牙周部位调节PPI/PI水平的因素促进胶结发生。 因此，我们启动了研究将TNAP传递到IBSP KO小鼠的本地位点。结果非常有前途（即促进了胶结发生），现在，对于Nadine Samara博士，我们正在设计针对矿化组织的TNAP，以供局部递送。我们正在使用体外模型来确定TNAP激活矿化的机制，并继续评估脚手架材料以改善局部输送。 b。在体外：ANK，ENPP1，DKO和ANK，Alpl DKO小鼠和细胞：确定ANK和ENPP1的影响是否是加性/协同作用，并且是否存在机械差异，是否存在机械差异，我们会产生ANK，ENPP1，DKO，DKO和ANK，Alpl DKO小鼠。组织学和Mi​​croCT的结果表明，ANK表现出的细胞胶质表型，ENPP1 DKO是单个KO的复合表型。 与Alpl Kos相比，ANK，Alpl DKO小鼠表现出改善的胶结发生，进一步支持PPI作为胶结生成中的关键调节剂。 对骨骼的影响对骨骼没有平行影响。 与ENPP1 KO小鼠相比，ANK KO和DKO小鼠的骨缺损似乎更为严重，这表明ANK和ENPP1在上调细胞外PPI中具有非冗余作用。 此外，尽管与Alpl KO小鼠相比，尽管在ANK中形成了功能性细胞牙骨质，但ALPL DKO小鼠，肺泡骨体积和矿物质密度却没有提高。正在进行的研究，使用蛋白质组学，QPCR微阵列和RNA SEQ技术专注于在牙齿根发育的各个阶段从ANK和ENPP1单一和DKO小鼠获得的PDL组织的蛋白质和基因表达谱。此外，如1A所述，我们正在与ANK与ENPP1 KO小鼠的牙周伤口愈合能力进行对比。为了补充体内研究，ANK KO，ENPP1KO和DKO胶质细胞受到各种矿化条件，例如PI或PPI。 数据表明细胞类型之间破骨细胞因子/调节剂的基因表达差异。 使用来自ANK和ENPP1单一和DKO小鼠的骨髓基质细胞的体外破骨细胞测定，以评估破骨细胞分化和通过ANK和/或ENPP1损失的吸收。 c。正畸牙齿运动：为了与沃尔夫博士进行持续研究，重点是研究PI/PPI在调节破骨细胞/odontoclast分布中的作用，ANK - / - 小鼠接受了正畸载荷。 另外，为了表征牙周牙齿受损对牙齿移动的影响，我们暴露了IBSP-KAE（BSP RGD被KAE代替），IBSP KO和WT小鼠对正畸负载。我们正在从这些正畸治疗的小鼠获得的组织中进行微CT和组织学分析。 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。 BSPXOPN：在2018年（Foster等人），我们得出的结论是，OPN具有调节牙本质和骨骼矿物质的非冗余作用，影响了PDL和PULP的组织特性，但不能控制链细胞的胶质胶质。下一步，我们生成并正在表征从SPP1（OPN）X IBSP（BSP）DKO小鼠获得的牙周组织，以确定OPN表达的损失是否挽救了IBSP KO牙周表型。初步的微CT分析表明，DKO与WT的肺泡骨适当体积降低，但大于IBSP KO小鼠的肺泡骨骼适当的体积，这表明OPN和BSP之间的添加剂却复杂的相互作用在维持牙周稳态方面。组织学分析表明，牙周复合物缺陷和沿着DKO中肺泡骨的破骨细胞数量增加，例如IBSP KO。在DKO小鼠中，增厚的牙槽冠被认为是独特的表型。一个有趣的发现是SPP1 KO小鼠股骨的细胞中指出的巨大破骨细胞，而不是IBSP KO或DKO细胞。与Hanson Fong博士合作，TEM成像将用于进一步定义从这些KO小鼠中获得的体内和体外破骨细胞的形态和功能。 IBSP-KAE：作为定义BSP功能机理的一步，我们生成了小鼠，其中BSP RGD结构域（整联蛋白结合结构域）被非功能性KAE序列（IBSP-KAE）取代。与Ralston博士的合作，组织学和第二次谐波生成显微镜的结果表明，与IBSP KO小鼠相比，IBSP-KAE小鼠显示出正常的胶结发生，但PDL杂乱无章，沿肺泡骨（如IBSP KO小鼠）沿肺泡骨（如IBSP KO小鼠）造成了明显增加的骨质成体。尽管破骨细胞增加了，但微CT分析显示，年龄较大的IBSP-KAE小鼠的肺泡骨体积较高。研究股骨巨噬细胞破骨细胞生成潜力的体外数据表明，从IBSP-KAE和IBSP KO获得的细胞正常成熟与WT小鼠相当。基于这些结果，我们假设IBSP-KAE小鼠中的PDL插入弱触发了炎症细胞因子的产生，这反过来促进了RANKL和/或等级的表达，从而在局部增加了骨细胞活性。接下来，我们在体外分析了IBSP KO和IBSP-KAE胶质细胞的迁移特性。 与WT细胞相比，IBSP-KAE细胞的迁移速度较慢，并且与IBSP KO细胞相同，而WT细胞提供了RGD区域丢失的证据，因为它是介导的弱PDL特性的因子，我们在体内指出。 另一个意外的发现是，随着时间的流逝，IBSP-KAE小鼠体重增加了，与WT和IBSP KO小鼠相比会增加。作为了解这种表型的第一步，我们已经开始研究，以确定这些小鼠（仅此时仅使用雄性小鼠）是否在特定的血液标记物中显示出改变，包括胰岛素，葡萄糖和甘油三酸酯以及与脂肪代谢相关的特定组织，例如肝脏，肝脏，胰腺，肾脏，肾脏和子女。 胶原蛋白：与Nan Hatch博士合作，2019年6月提交了一份出版物，强调了从CRTAP小鼠获得的组织中的颅面和牙齿缺陷（与软骨相关蛋白的基因失活突变）。