Laboratory of Oral Connective Tissue Biology

口腔结缔组织生物学实验室

基本信息

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
项目类别：
财政年份：
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

项目摘要

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：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 和 SIBLING 家族基因/蛋白质在矿化过程中的重要性，强调了需要生理化学和细胞分子因子来实现牙周组织形成/再生所需的稳态平衡。 1. 牙周病的细胞、组织和动物模型：一个。牙周器再生的动物模型：之前，我们报道了控制 PPi/Pi 水平的因素在牙周膜形成过程中发挥重要作用。在使用 Ibsp KO 小鼠的原理证明实验中，表现出明显的牙周组织破坏，TNAP 慢病毒、肌内递送挽救了 Ibsp KO 表型，例如，将 PDL 插入新形成的牙骨质中并增加牙槽骨体积。在其他实验中，ANK 和 ENPP1 KO 小鼠产生牙周开窗缺陷，术后 30 天，我们注意到从 KO 小鼠获得的组织中牙骨质再生增加。迄今为止的数据表明，破骨细胞样细胞对 WT 和 KO 小鼠分析的组织之间以及牙根和周围骨骼之间的伤口愈合有不同的影响。我们正在与 Ozato 博士的实验室合作定义破骨细胞概况。这些集体结果强调了调节局部牙周部位 PPi/Pi 水平的因素促进牙骨质形成。因此，我们启动了将 TNAP 递送至 Ibsp KO 小鼠局部部位的研究。结果非常有希望（即促进牙骨质形成），现在，我们正在与 Nadine Samara 博士一起设计针对矿化组织的 TNAP，用于局部输送。我们正在使用体外模型来确定 TNAP 激活矿化的机制，并继续评估支架材料以改善局部输送。 b. Ank、Enpp1、dKO 和 Ank、Alpl dKO 小鼠和细胞，体外：为了确定 ANK 和 Enpp1 的作用是否是相加/协同的，以及它们控制牙骨质形成的机制是否存在差异，我们生成了 Ank、Enpp1、dKO 和 Ank， Alpl dKO 小鼠。组织学和显微CT结果表明Ank、Enpp1 dKO所表现出的脱细胞牙骨质表型是单个KO的复合表型。与 Alpl KO 相比，Ank、Alpl dKO 小鼠表现出改善的牙骨质形成，进一步支持 PPi 作为牙骨质形成的关键调节剂。对牙骨质的影响与对骨的影响并不平行。与 Enpp1 KO 小鼠相比，Ank KO 和 dKO 小鼠的骨缺陷显得更严重，这表明 ANK 和 ENPP1 在上调细胞外 PPi 方面具有非冗余作用。此外，虽然Ank、Alpl dKO小鼠中形成了功能性脱细胞牙骨质，但与Alpl KO小鼠相比，牙槽骨体积和矿物质密度没有改善。正在进行的研究使用蛋白质组学、qPCR 微阵列和 RNA seq 技术，重点是定义从 Ank 和 Enpp1 single 和 dKO 小鼠在牙根发育的各个阶段获得的 PDL 组织的蛋白质和基因表达谱。此外，如 1a 中所述，我们比较了 Ank 与 Enpp1 KO 小鼠的牙周伤口愈合能力。为了补充体内研究，Ank KO、Enpp1KO 和 dKO 成牙骨质细胞正在接受各种矿化条件，例如Pi 或 PPi。数据表明细胞类型之间破骨细胞因子/调节剂的基因表达存在差异。使用来自 Ank 和 Enpp1 单小鼠和 dKO 小鼠的骨髓基质细胞进行体外破骨细胞测定，以评估破骨细胞的分化和吸收与 ANK 和/或 ENPP1 损失的情况。 c.正畸牙齿移动：为了继续与 Wolf 博士一起研究，重点研究 Pi/PPi 在调节破骨细胞/破牙细胞分布中的作用，对 Ank-/- 小鼠进行正畸负荷。此外，为了表征牙周病受损对牙齿移动的影响，我们将 Ibsp-KAE（BSP RGD 被 KAE 取代）、Ibsp KO 和 WT 小鼠暴露于正畸负荷下。我们正在对这些经过正畸治疗的小鼠获得的组织进行显微 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，重点关注 SIBLINGS 和胶原蛋白。 BSPxOPN：2018 年（Foster 等人 Bone）我们得出结论，OPN 具有调节牙本质和骨矿化的非冗余作用，影响 PDL 和牙髓的组织特性，但不控制脱细胞牙骨质的并置。下一步，我们生成并表征从 Spp1(OPN) x Ibsp (BSP) dKO 小鼠获得的牙周组织，以确定 OPN 表达的丧失是否可以挽救 Ibsp KO 牙周表型。初步显微 CT 分析表明，与 WT 相比，dKO 小鼠的牙槽骨适当体积减少，但大于 Ibsp KO 小鼠，表明 OPN 和 BSP 在维持牙周稳态方面存在附加但复杂的相互作用。组织学分析显示，与 Ibsp KO 一样，dKO 中牙周复合体有缺陷，牙槽骨上的破骨细胞数量增加。增厚的牙槽嵴被认为是 dKO 小鼠的独特表型。一个有趣的发现是，在 Spp1 KO 小鼠股骨细胞中发现了巨大的破骨细胞，但在 Ibsp KO 或 dKO 细胞中却没有。与 Hanson Fong 博士合作，TEM 成像将用于进一步确定从这些 KO 小鼠体内和体外获得的破骨细胞的形态和功能。 Ibsp-KAE：作为定义 BSP 在牙骨质形成中功能机制的一步，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 博士合作的组织学和二次谐波发生显微镜结果表明，与 Ibsp KO 小鼠相比，Ibsp-KAE 小鼠表现出正常的牙骨质形成，但 PDL 紊乱，沿牙槽骨的破骨细胞显着增加，就像 Ibsp KO 小鼠一样。尽管破骨细胞增加，但显微 CT 分析显示，老年 Ibsp-KAE 小鼠的牙槽骨体积更高。检查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 获得的细胞的正常成熟与 WT 小鼠相当。基于这些结果，我们假设 Ibsp-KAE 小鼠中 PDL 插入减弱会触发炎症细胞因子的产生，进而促进 RANKL 和/或 RANK 的表达，导致局部破骨细胞活性增加。接下来，我们分析了 Ibsp KO 和 Ibsp-KAE 成牙骨质细胞的体外迁移特性。与 WT 细胞相比，Ibsp-KAE 细胞迁移速度较慢，且与 Ibsp KO 细胞相同，这提供了 RGD 区域丢失的证据，这是我们在体内注意到的介导 PDL 特性减弱的因素。另一个意想不到的发现是，与 WT 和 Ibsp KO 小鼠相比，Ibsp-KAE 小鼠的体重随着时间的推移而增加。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（此时仅使用雄性小鼠）是否表现出特定血液标志物的改变，包括胰岛素、葡萄糖和甘油三酯以及与脂肪代谢相关的特定组织，例如，肝脏、胰腺、肾脏和附睾。胶原蛋白：与 Nan Hatch 博士合作，于 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（软骨相关蛋白基因的失活突变）。