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水平的因素，例如ANK，NPP1，Phosho1和TNAP，参与了根形成和胶结发生。 迄今为止的发现表明，矿化过程中PI/PPI的调节剂以及功能性同级家族基因/蛋白质的重要​​性，并强调了微调生理学和细胞分子因子对实现稳态平衡的重要性，并强调了牙周组织的形成和再生组织再生所需的稳态平衡。 1。从Alpl Ko小鼠获得的牙齿，除了已知的牙骨质表型（没有骨质骨质，PDL改变，PDL改变和牙齿的去角质，由于人类和小鼠的根表面缺乏PDL附着）表现出明显的牙本质和搪瓷表型。此外，这些缺陷通过小鼠的TNAP酶替代以及在人类中的临床试验中纠正。 2. Chimap，一种新型的次磷酸（HPP），肠状嵌合碱性磷酸酶（Chimap）的治疗方法，改善了Alpl - / - 小鼠的骨骼矿化。 3。一种用于牙齿特异性HPP（Odonto-HPP）的新型小鼠模型是通过敲打常染色体显性alpl误差突变（导致基本变化的A116T）来创建的，该突变是在人类幼虫中鉴定出来的。 ALPL+/A116T小鼠没有长骨特性的可检测变化，但在牙槽骨骨上显示出改变，包括放射性牙齿和解抗性病变，瘤骨在牙槽骨骨波峰上积累，并且在微观上测量的几个骨特性上存在显着差异。 4。磷酸1 KO和Alplxphospho1 DKO：从Alplxphospho1 DKO小鼠获得的牙齿比从Alpl或Phospho1的KO获得的可比组织表现出更深刻的牙本质表型，这表明了磷酸1的重要功能在phospho1中的重要功能（Mvs）（Mvs）与Synaps synapsiptime tnn nnergistimestime tnnn.gistriestime。 5。ANK，NPP1和TNAP体内和体外：检查ANK和NPP1 KO动物，证明了与基因表达相关的补偿机制。对从NPP1或ANK KO小鼠获得的组织（即PPI降低）获得的骨质分析表现出明显的蛋白质表达增加，对于两个兄弟姐妹，OPN和DMP1，其他两个基因的胶质细胞也显示出对Pi in velro的胶质细胞的增加。我们注意到早期的体外胶质成素细胞表达ALPL基因和TNAP蛋白，而诱导ENPP1基因/蛋白质表达需要矿化条件，类似于体内发现。这些模式在人的牙齿中得到了证实，其中在PDL中丰富的TNAP表达与NPP1的特异性定位对根胶质细胞相反。这些研究阐明了TNAP和NPP1在质矿化中的表达模式和功能，这表明TNAP的早期表达产生了较低的PPI环境，允许AEFC启动，而后来的NPP1表达增加了PPI，限制了AEFC的aefc appososition。这些结果强调了PI/PPI调节对胶结发生的重要性，并为我们正在进行的体内研究提供了使用啮齿动物中牙周伤口愈合模型的基本原理，以提供控制PI/PPI的因素，这些因素都在缺陷部位以及系统地。 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的活性是一个关键的细胞外基质蛋白属于同胞家族。 鼠胶质细胞（OCCM-30），已知表达DMP1的已知的骨细胞样细胞（MLO-Y4和MLO-A5）用于分析PTH和1,25D在调节DMP1以及其他涉及矿物稳态中涉及的因素中的效应，该因素通过使用RNA-Seq。总体而言，结果表明，PTH和1,25D的共同互补作用都通过部分调节与钙和磷酸盐代谢相关的相似基因/蛋白质来维持矿物质稳态，同时也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相互控制的基因/蛋白质可能是设计用于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，尤其是兄弟姐妹家族中的蛋白质，在牙周的发育和维持中起着至关重要的作用。我们的重点是确定使用体内测定（TEM/SEM； IHC，原位杂交和用于胶原蛋白和矿物质沉积的特殊染色）的BSP NULL小鼠中缺乏牙骨质形成的机制，在体外测定（使用BSP Null细胞功能分析以及来自新鲜隔离PDL Tissue and scp ko的RNA-NULL细胞功能分析，并将虽然健康的野生型（WT）小鼠在牙周韧带（PDL） - 根表面表现出强大的夏普纤维整合，但BSP - / - 小鼠缺乏胶质性超微结构，尖锐纤维纤维的损失，并从根表面脱离PDL。在我们的合作者Harvey Goldberg博士实验室中的一个密切相关的项目中，软饮食对BSP - / - 小鼠中切牙不良和牙周销毁的影响，用于更好地了解BSP在更广泛的牙周功能中的作用。软饮食组中的BSP - / - 小鼠具有正常体重，长骨长度和血清ALP活性，这表明牙齿功能障碍和营养不良有助于BSP - / - 小鼠报告的生长和骨骼缺陷。 BSP - / - 切牙以较慢的速率爆发，这可能导致观察到的牙本质增厚，并增强了牙本质和搪瓷朝根部的矿化。我们认为，散发速率的降低是由于缺乏细胞牙骨质和相关的牙周附着，在软饮食组中都指出。这些数据表明，BSP在维持适当的牙周功能和肺泡骨重塑方面的重要性，并将牙齿功能障碍指向BSP - / - 小鼠中观察到的骨骼缺陷的病因。 D.确定与胶原蛋白功能对牙周组织相关的疾病的影响。逻辑表明，成骨的个体不完美（O.I.）将标有牙周缺陷。然而，迄今为止的报告集中在一般骨骼病理学上，在某些情况下，牙本质缺陷（Dentinogenogeny Imperfecta（D.I.））以及迄今为止研究牙周/水泥缺陷的报告受到限制。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，异位浆钙化和肺泡骨缺损，而Gly610Cys +/-小鼠在年轻人时代的形态相对正常。 AR模型表现出重大的牙周变化。 CRTAP - / - 和PPIB - / - 小鼠都具有平行表型，包括增加的细胞胶质，以及降低的细胞胶质，牙本质宽度和根长度。我们第一次确定了由AR和AD OI导致的小鼠的牙周变化。变化反映了发育缺陷，以及在生命后期阶段的功能和重塑改变。这些发现将提供对OI人类受试者口腔牙科健康的见解。