Laboratory of Oral Connective Tissue Biology

口腔结缔组织生物学实验室

• 批准号: 9155481
• 负责人: Martha Somerman
• 金额: $ 107.1万
• 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/9155481
• 关键词: Age; Alkaline Phosphatase; Animals; Apical; Biological Assay; Biology; Body Weight; Bone remodeling; Cells; Cementoblast; Cementogenesis; Cementum Formation; Clinical Trials; Collagen; Complex; Congenital Abnormality; Connective Tissue; Data; Defect; Dental; Dental Cementum; Dental Enamel; Dental Pulp Calcification; Dentin; Dentinogenesis Imperfecta; Deposition; Development; Diet; Disease; Environment; Enzymes; Equilibrium; Exhibits; Extracellular Matrix Proteins; Family; Fiber; Functional disorder; Gene Expression; Gene Proteins; Genes; Goals; Growth; Health; Homeostasis; Hormones; Human; Hypophosphatasia; In Situ Hybridization; In Vitro; Incisor; Individual; Intestines; Knock-in Mouse; Knockout Mice; Knowledge; Laboratories; Length; Lesion; Life; Logic; Maintenance; Malnutrition; Malocclusion; Maps; Measures; Metabolism; Minerals; Missense Mutation; Modeling; Molecular; Morphology; Mus; Natural regeneration; Null Lymphocytes; Oral; Oral health; Osteocytes; Osteogenesis Imperfecta; Osteoid; Pathology; Pattern; Periodontal Diseases; Periodontal Ligament; Periodontium; Phenotype; Plant Roots; Play; Property; Proteins; Reporting; Research; Rodent; Rodent Model; Role; Serum; Site; Soft Diet; Staging; Staining method; Stains; Surface; Testing; Time; Tissues; Tooth Exfoliation; Tooth Loss; Tooth structure; Transcript; Vesicle; Width; Wild Type Mouse; Wound Healing; alveolar bone; base; bone; calcium phosphate; craniofacial; craniofacial complex; dentin matrix protein 1; design; human subject; improved; in vitro Assay; in vivo; in vivo Model; insight; kindred; long bone; microCT; mineralization; mouse model; novel; programs; protein expression; regenerative; repaired; skeletal; transcriptome sequencing

A. Hypothesis: Factors regulating PPi/Pi levels, e.g., ANK, NPP1, PHOSPHO1, and TNAP, are involved in root formation and cementogenesis. The findings to date demonstrate the importance of regulators of Pi/PPi and of functional SIBLING family genes/proteins during mineralization, and highlight the importance of fine tuning physiochemical and cellular molecular factors toward achieving a homeostatic balance required for formation and for regeneration of periodontal tissues. 1. Teeth obtained from Alpl KO mice, beyond the known cementum phenotype (no acellular cementum, altered PDL and exfoliation of teeth due to lack of PDL attachment to the root surface in humans and mice) exhibit a marked dentin and enamel phenotype also seen in humans. Further, these defects were corrected by TNAP enzyme replacement in mice and, in clinical trials, in humans. 2. ChimAP, a novel treatment for hypophosphatasia (HPP), intestinal-like chimeric alkaline phosphatase (ChimAP), improved skeletal mineralization in Alpl-/- mice. 3. 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 no detectable changes in long bone properties, but exhibited 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. 4. Phospho1 KO and AlplxPhospho1 dKO: Teeth obtained from AlplxPhospho1 dKO mice exhibit a more profound dentin phenotype than those comparable tissues obtained from KO of either Alpl or Phospho1, demonstrating the important function of PHOSPHO1 in controlling Pi levels in matrix vesicles (MVs), synergistically with TNAP. 5. ANK, NPP1 and TNAP in vivo and vitro: Examining Ank and Npp1 KO animals, demonstrated a compensatory mechanism related to gene expression. Cementum analysis of tissues obtained from either Npp1 or Ank KO mice (i.e., decreased PPi locally) exhibited a marked increase in protein expression for two SIBLINGS, OPN and DMP1, and further, cementoblasts exposed to Pi in vitro also exhibited an increase in transcripts for these two genes. We noted cementoblasts in vitro expressed Alpl gene and TNAP protein early, whereas induction of Enpp1 gene/protein expression required mineralization conditions, similar to in vivo findings. These patterns were confirmed in human teeth where rich TNAP expression in the PDL is contrasted by specific localization of NPP1 to root-lining cementoblasts. These studies clarify the expression patterns and functions of TNAP and NPP1 in cementum mineralization, suggesting 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 highlighted the importance of Pi/PPi modulation for cementogenesis and provide the rationale for our ongoing in vivo studies using periodontal wound healing models in rodents to deliver factors controlling Pi/PPi, both locally at the defect site as well as systemically. B. Hypothesis: Hormones involved in regulating mineral metabolism e.g., PTH, 1,25D, and FGF23 modulate the activity of DMP-1, a key extracellular matrix protein belong to the SIBLING family. Murine cementoblasts (OCCM-30), osteocyte-like cells (MLO-Y4, and MLO-A5) known to express Dmp1 were used to analyze effects of PTH and 1,25D in regulating DMP1 as well as other factors involved in mineral homeostasis, by employing RNA-seq. Collectively, the results suggest that both PTH and 1,25D share complementary effects toward maintaining mineral homeostasis by partly regulating similar genes/proteins associated with calcium and phosphate metabolism, while also exerting distinct roles on factors modulating mineral metabolism. Regulating genes/proteins mutually governed by PTH and 1,25D may be a viable approach for designing new therapies for preserving mineralized tissue health. C. Hypothesis: Extracellular matrix proteins, especially those within the SIBLING family, play a vital role in development and maintenance of the periodontium. We have focused on determining the mechanism for lack of cementum formation in Bsp null mice using in vivo assays, (TEM/SEM; IHC, in situ hybridization and special stains for collagen and mineral deposition), in vitro assays, (using Bsp null cell functional analysis and RNA-Seq analysis from freshly isolated PDL tissues and OCCM cells with BSP KO). While healthy wild-type (WT) mice exhibit a strong integration of Sharpeys fibers at the periodontal ligament (PDL)-root surface, Bsp-/- mice feature a lack of cementum ultrastructure, loss of Sharpeys fibers integration, and detachment of PDL from the root surface. In a closely related project in the laboratory of our collaborator, Dr. Harvey Goldberg, the effect of soft diet on incisor malocclusion and periodontal destruction in Bsp-/- mice was used to better understand the role of BSP in wider periodontal function. Bsp-/- mice in the soft diet group featured normal body weight, long-bone length, and serum ALP activity, suggesting that tooth dysfunction and malnutrition contribute to growth and skeletal defects reported in Bsp-/- mice. Bsp-/- incisors erupted at a slower rate, which likely leads to the observed thickened dentin and enhanced mineralization of dentin and enamel towards the apical end. We propose that the decrease in eruption rate is due to lack of acellular cementum and associated defective periodontal attachment, noted in both soft and hard diet groups. These data demonstrate the importance of BSP in maintaining proper periodontal function and alveolar bone remodeling, and point to dental dysfunction as a causative factor of skeletal defects observed in Bsp-/- mice. D. To determine the effect of disorders associated with collagen function on periodontal tissues. Logic would suggest that individuals with osteogenesis imperfect (O.I.) would have marked periodontal defects. Yet, reports to date focus on general bone pathology and in some cases dentin defects (dentinogenesis imperfecta (D.I.)), and reports to date investigating periodontal/cementum defects are limited. Our studies show, compared to controls, AD Brtl+/- mice featured thinner dentin, ectopic pulp calcification and alveolar bone defects, while Gly610Cys+/- mice had relatively normal morphology at young ages. AR models exhibited significant periodontal changes. Both Crtap-/- and Ppib-/- mice featured parallel phenotypes including increased acellular cementum, and reduced cellular cementum, dentin width and root length. For the first time, we identify periodontal changes in mice resulting from AR and AD OI. Changes reflect developmental defects, as well as altered function and remodeling during later stages in life. These findings will provide insights into oral-dental health of human subjects with OI.

A. 假设：调节 PPi/Pi 水平的因子，例如 ANK、NPP1、PHOSPHO1 和 TNAP，参与牙根形成和牙骨质形成。 迄今为止的研究结果证明了 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，并强调了微调理化和细胞分子因素对于实现牙周组织形成和再生所需的稳态平衡的重要性。 1. 从 Alpl KO 小鼠获得的牙齿，除了已知的牙骨质表型（没有无细胞牙骨质、改变的 PDL 以及由于人类和小鼠中缺乏 PDL 附着到牙根表面而导致牙齿脱落），表现出明显的牙本质和牙釉质表型，在人类。此外，这些缺陷通过小鼠中的 TNAP 酶替代得到纠正，并且在临床试验中，在人类中也得到了纠正。 2. ChimAP，一种治疗低磷酸酯酶症（HPP）的新型疗法，肠样嵌合碱性磷酸酶（ChimAP）可改善 Alpl-/- 小鼠的骨骼矿化。 3. 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 小鼠的长骨特性没有可检测到的变化，但牙槽骨表现出变化，包括射线可透性和再吸收性病变、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。 4. Phospho1 KO 和 AlplxPhospho1 dKO：从 AlplxPhospho1 dKO 小鼠获得的牙齿比从 Alpl 或 Phospho1 KO 获得的类似组织表现出更深刻的牙本质表型，证明 PHOSPHO1 在控制基质囊泡 (MV) 中 Pi 水平方面的重要功能，与 TNAP 具有协同作用。 5. ANK、NPP1 和 TNAP 体内和体外：检查 Ank 和 Npp1 KO 动物，证明与基因表达相关的补偿机制。对从 Npp1 或 Ank KO 小鼠（即局部 PPi 减少）获得的组织进行的牙骨质分析显示，两个 SIBLINGS、OPN 和 DMP1 的蛋白表达显着增加，此外，体外暴露于 Pi 的成牙骨质细胞也显示出以下转录物的增加：这两个基因。我们注意到体外成牙骨质细胞早期表达 Alpl 基因和 TNAP 蛋白，而 Enpp1 基因/蛋白表达的诱导需要矿化条件，与体内发现相似。这些模式在人类牙齿中得到了证实，其中 PDL 中丰富的 TNAP 表达与 NPP1 特定定位于根衬成牙骨质细胞形成对比。这些研究阐明了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的后期表达则增加PPi，限制AEFC并置。这些结果强调了 Pi/PPi 调节对牙骨质形成的重要性，并为我们正在进行的体内研究提供了理论基础，该研究使用啮齿类动物的牙周伤口愈合模型来传递控制 Pi/PPi 的因子，无论是在缺损部位的局部还是全身。 B. 假设：参与调节矿物质代谢的激素，例如 PTH、1,25D 和 FGF23 调节 DMP-1 的活性，DMP-1 是属于 SIBLING 家族的关键细胞外基质蛋白。 使用已知表达 Dmp1 的鼠成牙骨质细胞 (OCCM-30)、骨细胞样细胞 (MLO-Y4 和 MLO-A5) 来分析 PTH 和 1,25D 在调节 DMP1 以及涉及矿物质稳态的其他因素中的作用，通过使用 RNA 测序。总的来说，结果表明，PTH 和 1,25D 通过部分调节与钙和磷酸盐代谢相关的相似基因/蛋白质，在维持矿物质稳态方面具有互补作用，同时还对调节矿物质代谢的因素发挥不同的作用。调节由 PTH 和 1,25D 共同控制的基因/蛋白质可能是设计保护矿化组织健康的新疗法的可行方法。 C. 假设：细胞外基质蛋白，尤其是 SIBLING 家族中的蛋白，在牙周组织的发育和维护中发挥着至关重要的作用。我们专注于使用体内测定（TEM/SEM；IHC、原位杂交和胶原蛋白和矿物质沉积的特殊染色）、体外测定（使用 Bsp 缺失细胞）确定 Bsp 缺失小鼠牙骨质形成缺乏的机制。对新鲜分离的 PDL 组织和 OCCM 细胞进行功能分析和 RNA-Seq 分析（BSP KO）。健康野生型 (WT) 小鼠在牙周膜 (PDL) 根表面表现出 Sharpeys 纤维的强烈整合，而 Bsp-/- 小鼠则缺乏牙骨质超微结构、Sharpeys 纤维整合丧失以及 PDL 从牙周膜上脱落。根面。在我们的合作者 Harvey Goldberg 博士实验室的一个密切相关的项目中，利用软饮食对 Bsp-/- 小鼠门牙咬合不正和牙周破坏的影响来更好地了解 BSP 在更广泛的牙周功能中的作用。软饮食组的 Bsp-/- 小鼠体重、长骨长度和血清 ALP 活性正常，表明牙齿功能障碍和营养不良导致 Bsp-/- 小鼠的生长和骨骼缺陷。 Bsp-/- 切牙萌出速度较慢，这可能导致观察到的牙本质增厚以及牙本质和牙釉质朝向根尖端的矿化增强。我们认为，萌出率下降是由于缺乏无细胞牙骨质和相关的牙周附着缺陷，软饮食组和硬饮食组均注意到这一点。这些数据证明了 BSP 在维持适当的牙周功能和牙槽骨重塑方面的重要性，并指出牙齿功能障碍是 Bsp-/- 小鼠中观察到的骨骼缺陷的致病因素。 D. 确定与胶原蛋白功能相关的疾病对牙周组织的影响。从逻辑上讲，成骨不全（O.I.）的个体会有明显的牙周缺陷。然而，迄今为止的报告侧重于一般骨病理学和某些情况下的牙本质缺陷（牙本质发育不全（D.I.）），迄今为止调查牙周/牙骨质缺陷的报告有限。我们的研究表明，与对照组相比，AD Brtl+/- 小鼠具有较薄的牙本质、异位牙髓钙化和牙槽骨缺陷，而 Gly610Cys+/- 小鼠在年轻时具有相对正常的形态。 AR 模型表现出显着的牙周变化。 Crtap-/- 和 Ppib-/- 小鼠均具有平行的表型，包括无细胞牙骨质增加、细胞牙骨质减少、牙本质宽度和牙根长度减少。我们首次确定了 AR 和 AD 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。这些发现将为了解成骨不全症人类受试者的口腔健康提供见解。