Laboratory of Oral Connective Tissue Biology

口腔结缔组织生物学实验室

• 批准号: 10244802
• 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/10244802
• 关键词: Age; Alveolar; Animal Model; Biological Assay; Biology; Blood; Body Weight; Bone Marrow; Cartilage; Cell model; Cells; Cementoclast; Cementogenesis; Clinical; Clinical Trials; Collaborations; Collagen; Complement; Complex; Connective Tissue; Contracts; Data; Defect; Dental; Dental Cementum; Dental Pulp; Dental crowns; Dentin; Development; Dose; Epididymis; Equilibrium; Euthanasia; Exhibits; Expression Profiling; Extracellular Matrix Proteins; Female; Femur; Fibroblast Growth Factor Receptors; Fostering; Future; Gene Expression; Gene Family; Gene Proteins; Generations; Genes; Germany; Glucose; Histologic; Histology; Homeostasis; Image; In Vitro; Inflammatory; Insulin; Integrin Binding; Intramuscular; Kidney; Knockout Mice; Knowledge; Laboratories; Liver; Manuscripts; Maps; Mediating; Microscopy; Minerals; Molecular; Morphogenesis; Morphology; Mus; Mutation; Natural regeneration; Operative Surgical Procedures; Oral; Orthodontic; Osteoclasts; Pancreas; Paper; Pattern; Periodontal Diseases; Periodontal Ligament; Periodontium; Phenotype; Plant Roots; Production; Property; Proteins; Proteomics; Publications; Publishing; RGD (sequence); Reporting; Rodent Model; Role; Site; Stromal Cells; Subfamily lentivirinae; TNFSF11 gene; Testing; Therapeutic; Time; Tissue Model; Tissues; Tooth Loss; Tooth Movement; Tooth root structure; Tooth structure; Triglycerides; United States National Institutes of Health; Wistar Rats; Wolves; alveolar bone; base; bone; craniofacial; cytokine; density; dental structure; design; disease phenotype; experimental study; extracellular; improved; in vivo; in vivo Model; in vivo evaluation; inhibitor/antagonist; lipid metabolism; male; microCT; mineralization; osteoclastogenesis; postnatal; protein expression; regenerative; second harmonic; transcriptome sequencing; wisdom tooth; wound healing

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: 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, TNAP lentivirus via intramuscular delivery, rescued the Ibsp KO periodontal disease phenotype, i.e., insertion of PDL into newly formed cementum and improved alveolar bone volume and mineral density. Thus, we initiated studies to deliver TNAP locally in Ibsp KO mice. Results were promising (i.e., cementogenesis promoted, manuscript to submit Oct.2020), leading to a collaborative studies with Drs. Samara and Shiloach at NIH, as well as Dr. Braddock to design TNAP targeted to mineralized tissues for local delivery. We have a contract with Thermofisher to design mineral targeted TNAP and anticipate being able to test this TNAP by Jan. 2021. Further, periodontal fenestration defects were created in ANK and ENPP1 KO mice. At 15 and 30 days post-surgery we noted increased cementum regeneration in tissues obtained from KO mice. Our data suggest different effects of osteoclast-like cells on wound healing between tissues analyzed from WT and KO mice and 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. 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 distinct mechanistic roles exist in cementogenesis, we generated Ank, Enpp1, dKO and Ank, Alpl dKO mice. Histological and microCT results indicate that acellular cementogenesis of Ank, Enpp1 dKOs was comparable to single KOs. Ank, Alpl dKO mice exhibit improved cementogenesis compared to Alpl KOs, further supporting PPi as a key regulator in cementogenesis. Bone defects appeared more severe in Ank KOs and dKO mice compared to Enpp1 KOs, suggesting that ANK and ENPP1 have non-redundant roles in upregulating extracellular PPi. Additionally, alveolar bone volume and mineral density were not improved compared to Alpl KO mice (publication, Bone 2020). Ongoing studies, using proteomics, qPCR microarray and RNA seq, 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 (manuscript to submit Dec.2020). To complement in vivo studies, 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: In continuing studies with Dr. Wolf investigating 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 microCT and histological analyses in tissues obtained from these orthodontically treated mice and data are being analyzed in Germany. 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 specific roles regulating mineralization of dentin and bone as well as in PDL and pulp, but not acellular cementogenesis. As a next step, we generated and are characterizing periodontal tissues obtained from Spp1(OPN) x Ibsp (BSP) dKO mice to determine if OPN loss 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. Histological analyses revealed defective periodontal complex and increased number of osteoclasts along the alveolar bone in dKO similar to Ibsp KO. Thickened alveolar crest was noted as a unique phenotype in dKO mice further suggesting complex interactions between OPN and BSP in maintaining periodontal homeostasis. Additionally, huge osteoclasts were 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. (manuscript to submit Feb. 2021) Ibsp-KAE: Toward defining BSP mechanistic functions in cementogenesis, we generated mice where the BSP RGD domain (integrin binding domain) was replaced by a non-functional KAE sequence (Ibsp-KAE). In collaboration with Dr. Ralston, results from histology and second harmonic generation microscopy 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. Micro CT analysis revealed a higher alveolar bone volume in older age Ibsp-KAE mice. In vitro data demonstrated comparable osteoclastogenesis potential between Ibsp-KAE, Ibsp KO, and 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, resulting in increased local osteoclast activity. 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. We anticipate submitting our manscript by Dec.2021. Another unanticipated finding was that Ibsp-KAE mice exhibit an increase in body weight over time vs WT and Ibsp KO mice. 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, we published a paper (Dev Dyn 2020), highlighting craniofacial and dental defects in tissues obtained from Crtap mice (inactivating mutations in the gene for cartilage-associated protein). Pan Fibroblast Growth Factor Receptor Inhibitor Infigratinib (BGJ398): In collaboration with QED Therapeutics, we are examining effects of BGJ398 on dentoalveolar development. Male and female Wistar rats were dosed daily (starting on 7 days postnatal) with vehicle, 0.1mg/kg BGJ398 (low-dose), or 1.0mg/kg BGJ398 (high-dose) until euthanasia on 37dpn. Third molars exhibited aberrant crown and root morphology in high-dose groups (100% females, 80% males), demonstrating sensitivity of early tooth morphogenesis to BGJ398. Clinically, the low-dose data implies a potential therapeutic treatment with minimal effect on dental structures. Future studies include analyses of genes/proteins associated with dentoalveolar development and potentially perturbed by BGJ398. Manuscript in progress to be submitted in December 2020.

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：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小鼠的原理实验证明中，通过肌肉内递送TNAP慢病毒，营救了IBSP KO牙周疾病表型，即将PDL插入新形成的胶质性并改善肺泡骨体积和矿物质密度。因此，我们启动了研究以在IBSP KO小鼠中局部提供TNAP。结果是有希望的（即促进了水泥生成，手稿提交10月2020年），从而与DRS进行了协作研究。 NIH的Samara和Shiloach以及Braddock博士设计了针对矿化组织的TNAP，用于局部分娩。我们与Thermofisher有一份合同，以设计针对矿物质的TNAP，并预计能够在2021年1月之前测试该TNAP。此外，在ANK和ENPP1 KO小鼠中创建了牙周室外缺陷。手术后15天和30天，我们注意到从KO小鼠获得的组织中骨髓再生的增加。我们的数据表明，破骨细胞样细胞对从WT和KO小鼠分析的组织以及牙齿根和周围骨之间的组织之间的伤口愈合的不同影响。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。 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，如果在水泥生成中存在独特的机械作用，我们会产生ANK，ENPP1，DKO和ANK，ANK，ANK，Alpl DKO小鼠。组织学和Mi​​croCT的结果表明，ANK的细胞胶结发生，ENPP1 DKO与单个KOS相当。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。与ENPP1 KOS相比，ANK KOS和DKO小鼠中的骨缺损表现更严重，这表明ANK和ENPP1在上调细胞外PPI中具有非冗余的作用。 另外，与Alpl KO小鼠相比，牙槽骨体积和矿物质密度没有提高（出版物，骨骼2020）。正在进行的研究，使用蛋白质组学，QPCR微阵列和RNA SEQ，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（手稿提交12月2020年）。为了补充体内研究，正在进行使用来自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，以评估用ANK和/或ENPP1损失评估破骨细胞分化和吸收。 c。正畸牙齿运动：在与沃尔夫博士进行的继续研究中，研究了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观和组织学分析，并且正在德国分析数据。 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以及PDL和PDL和PULP的矿化作用的特定作用，而不是细胞的胶结胶质发生。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中的肺泡骨适当体积降低，但大于IBSP KO小鼠。组织学分析表明，牙周复合物缺陷和沿肺泡骨的破骨细胞数量增加，类似于IBSP KO。在DKO小鼠中，增厚的牙槽冠被认为是一种独特的表型，进一步表明OPN和BSP之间在维持牙周稳态时之间的相互作用。此外，在SPP1 KO小鼠股骨的细胞中发现了巨大的破骨细胞，但没有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。 （手稿提交2021年2月） IBSP-KAE：朝着定义胶结生成中的BSP机械功能，我们生成了小鼠，其中BSP RGD结构域（整合素结合结构域）被非功能KAE序列（IBSP-KAE）取代。与Ralston博士合作，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小鼠表现出正常的胶结生成，但PDL散布，沿肺泡骨（如IBSP KO小鼠）沿肺泡骨的破骨细胞显着增加。微型CT分析显示，年龄较大的IBSP-KAE小鼠的肺泡骨体积较高。体外数据表现出IBSP-KAE，IBSP KO和WT小鼠之间可比的破骨细胞生成潜力。基于这些结果，我们假设IBSP-KAE小鼠的PDL插入弱触发了炎症细胞因子的产生，这反过来促进了RANKL的表达，从而增加了局部破骨细胞活性。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。我们预计将在12月2021年12月之前提交我们的手稿。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。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，胰腺，肾脏，肾脏和附子症。 胶原蛋白：与Nan Hatch博士合作，我们发表了一篇论文（Dev Dyn 2020），强调了从CRTAP小鼠获得的组织中的颅面和牙齿缺陷（软骨相关蛋白的基因中的突变失活）。 PAN成纤维细胞生长因子受体抑制剂Impigratinib（BGJ398）：与QED Therapeutics合作，我们正在研究BGJ398对齿状肺泡发育的影响。每天用车辆服用雄性和雌性Wistar大鼠（从产后7天开始），0.1mg/kg BGJ398（低剂量）或1.0mg/kg BGJ398（高剂量）直至37dpn上的安乐死。第三磨牙在高剂量组（100％女性，80％男性）中表现出异常的牙冠和根形态，表现出早期牙齿形态发生对BGJ398的敏感性。在临床上，低剂量数据意味着潜在的治疗治疗，对牙齿结构影响最小。未来的研究包括对与牙道肺泡发育相关的基因/蛋白质的分析，并可能受到BGJ398的干扰。手稿进行，将于2020年12月提交。