牙根发育启动相关信号通路的研究进展

在牙齿发育过程中,当牙冠发育完成后,上皮根鞘的形成启动了牙根发育,然而关于牙根发育启动的分子调控机制目前尚未明了。该文就近年来关于牙根发育启动的信号通路,主要包括Shh信号通路、TGF-β信号通路、Nfic家族、成纤维细胞生长因子家族以及Notch信号通路等的研究进展作一综述。     

发育期根端复合体是牙根牙周发育的生长调控中心

首次强调了哺乳动物牙根发育过程中根端组织的完整性,并首次提出了发育期根端复合体（DAC）的概念,这个概念的提出是基于牙根与牙周组织发育的同步性、二者之间结构及功能上的整体性以及发育期牙根根端组织各种细胞成分间密切的相关性。文中详细论述了DAC的组织学、细胞学特性及其形成牙根和牙周组织的能力,证明了DAC是成体环境中具有“胚胎性”特征的组织,作为牙根和牙周组织共同发育的生长和调控中心持续存在于牙根发育的整个过程中。DAC包含形成牙根和牙周组织所必需的所有的前体细胞和成牙的微环境,在体内、外均表现出极强的发育能力以及再生牙根和牙周组织的能力,有望成为牙根和牙周组织工程优秀的种子细胞来源。     

Damage of the Hertwig's epithelial root sheath: effect upon root growth after autotransplantation of teeth in monkeys

Abstract The effect of damage to the Hertwig's epithelial root sheath (HERS) during autotransplantation was examined in 12 green vervet monkeys. During the transplantation of permanent incisor tooth germs, either one-half or the entire root sheath was amputated or a contusion injury made to the root sheath. Transplanted incisors served as controls, where attempts were made not to injure the HERS. After an observation period of 9 months, a histologic examination of the transplanted teeth was performed. This examination showed variations in root growth among the various experimental groups. Thus, arrested root formation with ingrowth of bone from the base of the socket was the normal finding in the groups where either the entire HERS was amputated or contused. The group with partial amputation of HERS showed formation of a new but diminutive root. The control group showed teeth with normal root formation. The development of pulp necrosis usually led to complete or almost complete arrest of root formation irrespective of group. The present study indicated that the HERS can function normally subsequent to trauma elicited by surgical separation of the follicle and the post-transplantation disturbances in nutrition. Furthermore, a regenerative potential appeared to exist in this structure, even if just a fraction of it was left intact.     

上皮根鞘细胞表面超微结构及牙根发育中上皮根鞘增殖力的变化

目的:观察体外培养的上皮根鞘细胞超微结构及牙根发育中上皮根鞘增殖力的变化。方法:体外培养大鼠上皮根鞘细胞,扫描电镜观察。制备出生后5 d、7~8 d和15 d的SD大鼠下颌磨牙石蜡切片,增殖细胞核抗原,检测免疫染色。结果:上皮根鞘细胞表面有许多微绒毛,细胞间连接紧密。牙根发育中上皮根鞘增殖力有序变化。结论:上皮根鞘细胞具有典型的上皮细胞表面结构特性。牙根发育中上皮根鞘有序断裂与其增殖力变化有关。     

大鼠Hertwig''''s上皮根鞘细胞的分离培养

目的:建立大鼠Hertwig's上皮根鞘细胞分离、培养和纯化的方法.方法:分离出生后7d SD大鼠上下颌第一、二磨牙牙胚,切取牙冠颈部组织,酶消化法原代培养,利用差速传代纯化上皮细胞.用角蛋白14和波形丝蛋白染色确定细胞来源.结果:原代培养细胞为上皮和间充质混合细胞,经2～3次差速传代后可获得纯化的上皮根鞘细胞.免疫组化染色角蛋白14阳性,波形丝蛋白阴性.结论:利用酶消化法及差速传代法培养并获得纯化的上皮根鞘细胞.     

骨形态发生蛋白信号通路在牙根发育中的作用

骨形态发生蛋白（BMP）家族是调节细胞生命活动的重要因子,几乎参与了所有组织的发育。BMP介导的信号通路在牙发育过程中发挥十分重要的作用,而牙根发育是牙发育的一部分,是上皮和间充质相互作用的复杂过程。上皮和牙胚间充质中的BMP信号通路在牙根发育中的作用也有所不同,本文综述了BMP信号通路在牙根发育中作用的研究进展。     

上皮根鞘结构对牙根发育的影响

目的: 研究上皮根鞘的结构对牙根发育的影响。方法: 选择出生后(postnatal, PN)8 d的SD大鼠, 切取第一磨牙牙胚的颈部组织, 用胶原酶和dispase酶消化, 1组直接离心形成细胞团, 另1组加用胰酶消化后, 离心形成细胞团。将细胞团体外培养4 h后, 种植到母鼠肾被膜下, 4周后取材观察。结果: 胶原酶和dispase酶消化可将上皮根鞘解离成碎片, 加用胰酶消化后, 上皮根鞘被解离为单个细胞。含有上皮根鞘碎片的细胞团种植后形成牙根样结构, 而含有上皮根鞘离散细胞的细胞团则形成管状牙本质和骨样牙本质块。结论: 上皮根鞘的结构对其引导牙根发育具有重要作用。     

建期上皮剩余的形态学和细胞活性研究

目的追踪观察上皮剩余在牙齿萌出和建!过程中的形态分布和细胞活性改变。方法细胞角蛋白14（ CK14）标记小鼠上皮剩余细胞，光镜观察其分布和细胞形态，透射电镜观察上皮剩余细胞的超微结构。PV免疫组化两步法检测CK14和增殖细胞核抗原（ PCNA）在上皮剩余细胞中的表达。结果自牙齿萌出前到建!后期上皮剩余的外形和分布改变明显，建!后期上皮细胞簇更为规则，分布由牙根表面的较广泛分布向根分叉和牙颈等部位局限，与成熟期上皮剩余相似。建!阶段，上皮剩余沿牙根表面呈现类网格样布局，细胞数量较萌出前期明显增加，细胞增殖活性检测显示此时上皮剩余特别是根分叉水平细胞增殖活跃。建!后期上皮剩余细胞数量开始减少，透射电镜结果显示簇内出现细胞凋亡征象。结论上皮剩余不是仅仅作为无功能的剩余物，其可能是上皮根鞘功能的延伸或持续，在建!过程中发挥积极作用，从而完成牙周组织的发育和维持牙周内环境的稳定。     

Hertwig's epithelial root sheath, enamel matrix proteins, and initiation of cementogenesis in porcine teeth

OBJECTIVES: The aim of this study was to analyze the association between Hertwig's epithelial root sheath (HERS) cells, enamel matrix proteins (EMPs), and cementogenesis. MATERIAL AND METHODS: Porcine teeth were examined at the beginning of root formation by light and transmission electron microscopy. Colloidal gold immunocytochemistry was used to analyze the protein expression of amelogenin and ameloblastin. RESULTS: Before and during disintegration of HERS, its cells displayed the cytologic features of protein synthesis and secretion. While some cells assumed an ameloblast-like phenotype, others extended their territory away from the root surface. A collagenous matrix filled the widening intercellular spaces, and tonofilaments and desmosomes were still present in cells featuring the morphologic characteristics of cementoblasts. Labeling for amelogenin was observed but ameloblastin was not immunodetected. Labeling was associated with organic matrix deposits that were sporadically and randomly distributed both along the root surface and away from it among the dissipated epithelial cells. CONCLUSIONS: These findings suggest that HERS' cells occasionally assume a lingering ameloblastic activity at the beginning of root formation in the pig. While the results do not support the hypothesis of a causal relationship between EMPs and cementogenesis, they lend support to the concept of an epithelial origin of cementoblasts.     

Effects and distribution of the enamel matrix derivative Emdogain® in the periodontal tissues of rat molars transplanted to the abdominal wall

Abstract – The enamel matrix derivative Emdogain^® (EMD) has been found to promote regeneration of lost periodontal tissues. We have studied the effects and distribution of EMD in the periodontal tissues of maxillary rat molars transplanted to a subcutaneous position in the abdominal wall. The molars were transplanted with or without EMD either immediately after extraction or after drying for 30 min. After 2 days, 1, 2 or 4 weeks the rats were killed and the teeth were examined by means of light microscopy and immunohistochemistry with anti-amelogenin antibodies. Teeth transplanted immediately after extraction showed formation of alveolar bone separated from the dental roots by a periodontal space, regardless of the use of EMD. Among the teeth that were transplanted with EMD after drying for 30 min, new alveolar bone was formed in five out of eight teeth after 2 and 4 weeks. None of the teeth that were dried for 30 min and transplanted without EMD showed alveolar bone formation. Only one tooth transplanted with EMD showed root resorption after drying, while resorption was noted in all teeth transplanted without EMD. All teeth that were transplanted with EMD and none of the teeth that were transplanted without EMD showed an immunohistochemical reaction for amelogenin. After 2 days, amelogenin was precipitated on all surfaces exposed at the transplantation procedure. Later, the immunoreactive material was redistributed to cells at the root surface, where it was still demonstrable after 4 weeks. In conclusion, EMD is accumulated in cells at the root surface and promotes regeneration of the periodontal tissues of the transplanted teeth. It also seems to promote healing of root resorption.     

Effect of the antineoplastic agent busulfan on rat molar root development

Objective

The antineoplastic bifunctional-alkylating agent busulfan (Bu) induces developmental anomalies. We examined histopathological changes in the molar roots of rats that received Bu at different stages of root formation.

Design

At different developmental stages, i.e., on postnatal days (P) 13, 15, and 19, rats were administered 7.5 mg/kg of Bu dissolved in dimethyl sulfoxide (DMSO) and then killed on P 30. After micro-computed tomography analysis, the maxillary first molars underwent immunohistochemical analysis for cytokeratin 14 (CK14), nestin, and dentin sialoprotein (Dsp). This was followed by histomorphometric analysis.

Results

The rats receiving Bu at an early stage (i.e., P 13 and P 15) showed osteodentin formation and complete destruction of the Hertwig's epithelial root sheath (HERS). Cells around osteodentin showed nestin and Dsp immunoreactivity. The root lengths in rats treated with Bu at P 13 (1228.44 ± 62.17 μm) and P 15 (1536.08 ± 109.71 μm) were lower than that in the control rats (1674.10 ± 40 μm). A narrowed apical foramen and an increased amount of osteodentin were also present, depending on the rat's age at the time of treatment (P < 0.05).

Conclusion

Busulfan treatment in juvenile rats resulted in abnormal root development, depending on the stage at which Bu was administered. This abnormal development may result from the destruction of the HERS. The administration of Bu caused a shortage of HERS cells, which are required for normal root development. This disturbs root formation, resulting in osteodentin formation and a narrowed apex foramen.     

Reactions of periodontal ligament epithelial cell clusters and OX6-immunopositive cells to experimental tooth movement and periodontitis

Tadokoro O, Kawahara I, Vandevska‐Radunovic V. Reactions of periodontal ligament epithelial cell clusters and OX6‐immunopositive cells to experimental tooth movement and periodontitis. J Periodont Res 2011; 46: 584–591.©2011 John Wiley & Sons A/S Background and Objective: The aim of this study was to investigate reactions of periodontal ligament epithelial cell clusters and major histocompatibility complex class II (OX6)‐immunopositive cells to simultaneously induced tooth movement and periodontitis employing Waldo’s method. Material and Methods: Elastic gums were inserted between the right upper first and second molars of rats. Animals were killed by intracardiac perfusion on days 1, 3, 7 and 14 after the experimental procedures, and maxillary molars were decalcified and processed for OCT compound. Cytokeratin and OX6 antibodies to detect epithelial and immunocompetent cells were used for double‐fluorescence immunohistochemistry. Immunostained sections of rat upper molar regions were examined with a fluorescence microscope. Results: Large periodontal ligament epithelial cell clusters appeared and became contiguous with each other, and OX6‐immunopositive cells surrounded the clusters over time in the periodontal ligament near the gum insertion site. In the periodontal ligament distant from the gum insertion site, epithelial cell clusters and OX6‐immunopositive cells were scattered. After 14 d, thickened epithelium and elongated rete pegs were found close to large epithelial cell clusters in the periodontal ligament near the gum insertion site. Conclusion: These findings suggest proliferation and/or aggregation of periodontal ligament epithelial cells, and interaction between OX6‐immunopositive cells and the periodontal ligament epithelial cells, in response to tooth movement and periodontal inflammation. This method may be a useful experimental model to elucidate the relationship between rete pegs and periodontal ligament epithelial cell clusters in inflammatory conditions.     

Hypothermic insult to the periodontium: a model for the study of aseptic tooth resorption

Abstract – The aim of the current investigation was to define an animal model for the study of hard tissue resorption by examining the responses of the periodontal ligament (PDL) to both single and multiple episodes of hypothermic injury to the crowns of rat teeth. A group of 12 male rats weighing 200–250 g were anesthetized, and pellets of dry ice (CO₂) were applied once to the crowns of the right first maxillary molars for continuous periods of 10 or 20 min. Animals were sacrificed at 2, 7, 14 and 28 days and tissues were processed for routine histological examination. A second group of eight animals and a third group of 12 animals were subjected to three applications of dry ice over a period of 1 week and sacrificed at 2 and 14 days respectively after the final application. In addition to thermal insult, the periodontium of teeth from a fourth group of six rats was subjected to mechanical trauma. Examination of the sections from the group undergoing a single freezing episode revealed that, by 1 week, shallow resorption lacunae had appeared on the root surface. These became more extensive after 14 days. At the same time hyaline degeneration was evident in the PDL. Within this group, teeth subjected to the longer 20‐min application times generally showed more extensive injuries. By 28 days, evidence of repair was observed with reparative cementum beginning to line the resorption lacunae in the root dentin. Sections from animals subjected to multiple episodes of thermal trauma and those subjected to additional mechanical insult showed more extensive external root resorption than those from single‐injury animals. It was concluded that low temperature stimuli applied to the crowns of rat molars were capable of eliciting a sterile degenerative response in the PDL which, in turn, resulted in external root resorption. Furthermore, the degree of this tissue injury was commensurate with the duration and number of exposures to the trauma. The results also indicated that progression of the resorptive process required periodic exposure to the injury, in the absence of which repair to the damaged root occurred.     

Hepatocyte growth factor stimulates root growth during the development of mouse molar teeth

Sakuraba H, Fujiwara N, Sasaki‐Oikawa A, Sakano M, Tabata Y, Otsu K, Ishizeki K, Harada H. Hepatocyte growth factor stimulates root growth during the development of mouse molar teeth. J Periodont Res 2012; 47: 81–88. © 2011 John Wiley & Sons A/S Background and Objective: It is well known that tooth root formation is initiated by the development of Hertwig’s epithelial root sheath (HERS). However, relatively little is known about the regulatory mechanisms involved in root development. As hepatocyte growth factor (HGF) is one of the mediators of epithelial–mesenchymal interactions in rodent tooth, the objective of this study was to examine the effects of HGF on the root development of mouse molars. Material and Methods: The HERS of mouse molars and HERS01a, a cell line originated from HERS, were used in this study. For detection of HGF receptors in vivo and in vitro, we used immunochemical procedures. Root development was assessed by implanting molar tooth germs along with HGF‐soaked beads into kidney capsules, by counting cell numbers in HERS01a cell cultures and by performing a 5′‐bromo‐2′‐deoxyuridine (BrdU) assay in an organ‐culture system. Results: HGF receptors were expressed in the enamel epithelium of molar germs as well as in HERS cells. HGF stimulated root development in the transplanted tooth germs, the proliferation of HERS01a cells in culture and HERS elongation in the organ‐culture system. Examination using BrdU revealed that cell proliferation in HERS was increased by treatment with HGF, especially that in the outer layer of HERS. This effect was down‐regulated when antibody against HGF receptor was present in the culture medium. Conclusion: Our results raise the possibility that HGF signaling controls root formation via the development of HERS. This study is the first to show that HGF is one of the stimulators of root development.     

Enamel matrix protein adsorption to root surfaces in the presence or absence of human blood

Background: The clinical use of an enamel matrix derivative (EMD) has been shown to promote formation of new cementum, periodontal ligament (PDL), and bone and to significantly enhance the clinical outcomes after regenerative periodontal surgery. It is currently unknown to what extent the bleeding during periodontal surgery may compete with EMD adsorption to root surfaces. The aim of this study is to evaluate the effect of blood interactions on EMD adsorption to root surfaces mimicking various clinical settings and to test their ability to influence human PDL cell attachment and proliferation. Methods: Teeth extracted for orthodontic reasons were subjected to ex vivo scaling and root planing and treated with 24% EDTA, EMD, and/or human blood in six clinically related settings to determine the ability of EMD to adsorb to root surfaces. Surfaces were analyzed for protein adsorption via scanning electron microscopy and immunohistochemical staining with an anti‐EMD antibody. Primary human PDL cells were seeded on root surfaces and quantified for cell attachment and cell proliferation. Results: Plasma proteins from blood samples altered the ability of EMD to adsorb to root surfaces on human teeth. Samples coated with EMD lacking blood demonstrated a consistent even layer of EMD adsorption to the root surface. In vitro experiments with PDL cells demonstrated improved cell attachment and proliferation in all samples coated with EMD (irrespective of EDTA) when compared to samples containing human blood. Conclusion: Based on these findings, it is advised to minimize blood interactions during periodontal surgeries to allow better adsorption of EMD to root surfaces.     

牙本质牙髓复合体的形成研究(一)牙本质牙髓复合体的新概念及其在牙齿发育中的形成研究

牙本质及牙本质牙髓复合体的形成研究一直是口腔医学研究的重大课题。近年来,生命前沿科学领域的飞速发展和重大进展,也掀起了牙本质牙髓形成研究的热潮。本文阐述了对牙本质牙髓复合体概念的新认识,系统回顾了牙齿发育过程中、牙齿发育完成后,牙本质牙髓复合体体内形成研究、体外形成研究的新进展,及这些研究对临床治疗的指导意义和应对策略,分析了面临的挑战。这对我们把握该领域的研究现状和进一步深入研究奠定了必要的基础。