过表达β-catenin诱导人表皮干细胞向成釉质细胞分化

人表皮干细胞可作为上皮源性的成体干细胞可应用于人类牙齿再生,但是其诱导效率较低。该研究利用过表达手段上调Wnt/β-catenin信号通路核心因子β-catenin在人表皮干细胞的表达,以期提高诱导其向成釉质细胞分化的效率。分别构建β-catenin和β-catenin（S33Y）基因的慢病毒载体,转染293T细胞生产病毒液并感染人表皮干细胞,采用Western blot检测人表皮干细胞感染后β-catenin的蛋白表达水平;然后与具有诱导成牙潜能的小鼠牙胚间充质进行重组,移植裸鼠体内培养;嵌合体组织切片染色和免疫组化检测形成牙齿的效率（成牙率）和成釉质细胞分化的效率（成釉率）。结果显示,过表达β-catenin的人表皮干细胞的重组嵌合体的成釉率提高至100%。提示,过表达β-catenin可诱导人表皮干细胞向成釉质细胞分化。     

解离的小鼠牙胚上皮细胞在重聚发育过程仍维持牙齿发育基因的表达

为了解牙胚细胞解离重聚过程的细胞形态和分子机制,将小鼠帽状期牙胚解离细胞重聚,移植到小鼠肾囊膜下培养,组织切片,HE染色,观察再生牙齿的形态发生过程,并用原位杂交的方法进一步检测了与牙上皮发育密切相关的基因在再生牙上皮中的表达情况。结果发现,解离重聚的牙胚细胞在牙齿器官的再生过程重现了正常牙齿的形态发生过程;解离的牙上皮细胞在重聚和再生过程中保持Fgf8、Noggin和Shh等牙上皮发育基因表达。以上结果表明,即便是被解离形成分散状态的牙上皮细胞,在与牙胚间充质细胞重新聚合后,仍保持牙向分化的潜能。该结果为理解牙齿再生的机理提供新的实验数据,对利用干细胞进行牙齿再生的研究有重要的提示意义。     

CHIR-99021与FGF8协同诱导人表皮干细胞牙向分化

人表皮干细胞(human keratinocyte stem cells, hKSCs)可作为上皮源性的成体干细胞应用于牙齿再生,但是其诱导效率较低.本研究利用小分子化合物CHIR-99021提高hKSCs的Wnt/β-catenin信号活性,再与具有诱导成牙潜能的小鼠牙胚间充质重组,构建嵌合体,并移植裸鼠肾囊膜下培养20 d. 将嵌合体组织切片,并利用组织染色和免疫组化等方法鉴定牙齿结构. 结果显示,经FGF8诱导处理的hKSCs与小鼠牙胚间充质构成的嵌合体的成牙率为27.80%,其中成釉率仅为40.00%;经CHIR 99021诱导处理的hKSCs与小鼠牙胚间充质构成的嵌合体的成牙率仅为18.20%,其中成釉率高达100%;而CHIR 99021与FGF8协同作用,则进一步提高嵌合体成牙率至40.00%,其中成釉率也达75.00%. 进一步的研究发现,经CHIR-99021处理后,hKSCs的Wnt/β-catenin信号活性明显提高,同时FGF8的表达水平也显著上调. 以上结果表明,CHIR-99021可通过上调Wnt/β-catenin信号活性水平,同时促进FGF8表达,与FGF8协同,高效诱导hKSCs分化为具有分泌釉质功能的成釉质细胞. 研究结果对利用hKSCs作为上皮来源的成体细胞应用于人类牙齿再生的研究具有重要意义.     

过表达Msx1、Pax9和Bmp4对牙髓干细胞牙向分化的影响

在组织工程研究领域中,利用干细胞进行牙齿再生是一种途径。目前,研究认为牙齿的发育过程是上皮与间充质相互诱导的结果,利用干细胞进行再生牙齿时也需要有上皮源性和间充质源性干细胞的参与。牙髓干细胞是牙齿自体的干细胞,具有多向分化潜能,在牙齿再生中是一种理想的间充质源性干细胞。该研究通过慢病毒介导在牙髓干细胞中分别过表达人Msx1、Pax9和Bmp4基因,研究其对牙向分化的诱导潜能。过表达这三个基因均能显著提高牙髓干细胞碱性磷酸酶的水平,并且促使牙髓干细胞表达成牙本质细胞标志蛋白——牙本质涎磷蛋白、骨钙素、骨桥素和形成钙化组织。但在诱导牙向分化的能力上,三个基因有一定的区别。过表达Msx1基因对牙髓干细胞体外诱导牙向分化能力最为明显,其次是Bmp4基因,过表达Pax9在促进牙髓干细胞表达骨桥素和钙质形成上不是很显著。     

非牙源性干细胞在牙齿再生研究中的应用

近年来,干细胞治疗研究获取的成果为牙齿的修复和再生研究奠定了有力的理论基础.根据牙齿的发育特征,牙齿再生需要牙源性的上皮干细胞和牙源性的间充质干细胞,目前研究表明,牙源性的间充质干细胞可应用于牙齿再生,例如牙髓干细胞和牙周韧带干细胞.但是,人牙源性上皮干细胞仅存在于胚胎期,萌发后的牙齿并不存在牙上皮干细胞,因此学者们开始探索将非牙源性干细胞替代牙源性上皮细胞应用于牙齿再生研究.以下概述了胚胎干细胞、成体干细胞和诱导性多潜能性干细胞等非牙源性干细胞在牙齿再生中的研究进展.     

外胚间充质干细胞在牙组织工程的应用与基因调控

用干细胞构建组织工程化牙齿是近年来口腔医学领域的研究热点,外胚间充质干细胞是目前已知牙源性干细胞的共同前体细胞,细胞的生物学特性和成牙信号分子环境是牙齿发育与再生的核心与关键,并贯穿于牙齿形成的全过程,是研究牙组织工程最具潜力的种子细胞,明确外胚间充质干细胞成牙分化能力及相关表型特征和分化特性,对进一步深入认识牙齿发育与再生机理具有重要作用。     

脐带间充质干细胞牙向分化的可行性研究

再生医学近年来受到越来越多的重视。它开启了治疗由于老化,损伤及一些先天性缺陷所造成的缺损畸形的新途径。其临床应用已涉及到各种组织的修复,包括血液,皮肤,角膜,软骨和骨等。在口腔领域,目前治疗牙缺失主要依靠修复体,种植体和牙移植。然而这些方法都存在一定的缺陷。而通过再生医学的原理和方法实现牙再生治疗可以为机体提供有生命的,有功能的,相容性好的组织结构。种子细胞是牙再生的基础与关键。在牙再生研究中,牙髓间充质干细胞,牙乳头细胞,牙周膜间充质细胞,牙囊细胞及牙源性上皮细胞等牙源性干细胞常通过诱导分化为成釉细胞或成牙本质细胞来作为种子细胞应用,在临床上却难以获取,近来研究也有用骨髓间充质干细胞或脂肪间充质干细胞细胞等非牙源性干细胞者,但其牙向分化能力及分化调控机制还不明确。跻带间充质干细胞在新近的研究中较其它非牙源性干细胞表现出更大的优势,脐带间充质干细胞更原始、具有更高可塑性、更大扩增分化潜能。在此,本文就脐带间充质干细胞向牙细胞系分化的可能性做一论述,并对其可能实现的牙向分化给出可能的方法和策略,为牙再生种子细胞的选取提供新的思路。     

山羊角膜缘干细胞荧光蛋白(Venus)细胞株的建立及角膜上皮植片构建

角膜缘干细胞是角膜上皮更新与修复的来源,角膜上皮受损严重常会导致角膜盲。尽管近几年通过角膜缘干细胞移植术(LSCT)治愈角膜上皮受损的临床应用已被推广,但是对于角膜缘干细胞移植受损机体后的修复机理并不明确。为了实现角膜缘干细胞移植后的活体追踪,使用G418筛选标记有Venus荧光蛋白的角膜缘干细胞株(GLSC-V),并以其为种子细胞接种于去上皮羊膜上,体外培养21d构建成荧光角膜上皮植片。荧光倒置显微镜下观察GLSC-V的细胞质和细胞核均有绿色荧光表达,在体外培养荧光至少持续3个月。免疫荧光检测GLSC-V细胞P63、Integrinβ1均呈阳性表达,对GLSC-V细胞及未转染的GLSCs进行半定量RT-PCR检测显示,两组细胞皆未表达终末分化角膜上皮细胞基因k3、k12,GLSC-V中p63及pcna较未转染组细胞略上调,venus强表达。经HE染色观察构建的人工角膜组织由5～6层上皮细胞组成,组织中上表皮细胞个数少、体积大且呈扁平状;基底部细胞密集、体积小且成立方状。经免疫荧光检测仅组织基底部最基层细胞表达P63,上表皮细胞不表达。该人工角膜与正常角膜上皮组织结构特性相似,可用于移植,为研究角膜缘干细胞修复严重受损角膜上皮机理奠定基础。     

人胚胎干细胞向神经上皮祖细胞的诱导分化

人胚胎干细胞具有自我更新和多向分化潜能,是研究早期胚胎发育和细胞替代治疗的重要细胞来源.采用一种与小鼠成纤维细胞共培养的方法进行人胚胎干细胞的神经诱导,可产生高纯度的神经上皮祖细胞,其神经上皮特异性基因的表达有一定的时空性;诱导生成的神经上皮祖细胞具有增殖潜能并可分化为神经元和星型胶质细胞,是潜在的神经干细胞.人胚胎干细胞来源的神经上皮祖细胞为研究神经发育和神经诱导提供了新材料,也为神经系统疾病的细胞替代治疗提供了新的细胞来源.     

鸟类仍保留牙齿发生定位的分子机制(英文)

众所周知现代鸟类不长牙齿,而其侏罗纪和白垩纪的祖先则长有牙齿。然而,在发育中鸡胚口腔中却残留着牙齿发生的原基,在形态上与哺乳动物臼牙牙原基极为相似。现代鸟类的胚胎组织是否具有牙齿发育的潜能,目前已有不少研究者对这一问题进行了探讨。Kollar和Fisher 等人将鸡胚胎下颌靠近口腔面的上皮与小鼠的牙间充质进行组织重组实验,并植入小鼠眼球中作intraocular grafting培养。他们的实验结果表明重组后的组织块可以发育形成牙齿的结构,包括形成成釉细胞(ameloblast),并能分泌釉质。Kollar等认为在进化过程中鸟类牙齿的消失并非由于口腔上皮中有关釉质合成的遗传信息的丢失,而是牙齿发育过程中的组织之间所必须的相互作用(次级诱导)受阻而造成的。Lemus和Fuenzalida等人的实验结果进一步证实了这一结论。他们用鹌鹑胚胎躯体的上皮组织与蜥蚁或兔子的牙间充质重组后,用鸡胚绒毛膜法进行培养,得到了发育很好的牙齿结构。发现鹌鹑的上皮细胞也可以分化形成釉质细胞,并分泌牙釉质。Cummings 将鹌鹑胚胎的牙上皮组织与小鼠胚胎的牙间充质组织重组后也得到类似的结果。根据小白鼠牙齿发育中已知的调控分子信号通路,我们曾对鸟类不长牙齿的分子机制进行了研究。我们的研究发现鸟类牙胚组织仍保留     

Induction of human keratinocytes into enamel-secreting ameloblasts

Mammalian tooth development relies heavily on the reciprocal and sequential interactions between cranial neural crest-derived mesenchymal cells and stomadial epithelium. During mouse tooth development, odontogenic potential, that is, the capability to direct an adjacent tissue to form a tooth, resides in dental epithelium initially, and shifts subsequently to dental mesenchyme. Recent studies have shown that mouse embryonic dental epithelium possessing odontogenic potential is able to induce the formation of a bioengineered tooth crown when confronted with postnatal mesenchymal stem cells of various sources. Despite many attempts, however, postnatal stem cells have not been used successfully as the epithelial component in the generation of a bioengineered tooth. We show here that epithelial sheets of cultured human keratinocytes, when recombined with mouse embryonic dental mesenchyme, are able to support tooth formation. Most significantly, human keratinocytes, recombined with mouse embryonic dental mesenchyme in the presence of exogenous FGF8, are induced to express the dental epithelial marker PITX2 and differentiate into enamel-secreting ameloblasts that develop a human-mouse chimeric whole tooth crown. We conclude that in the presence of appropriate odontogenic signals, human keratinocytes can be induced to become odontogenic competent; and that these are capable of participating in tooth crown morphogenesis and differentiating into ameloblasts. Our studies identify human keratinocytes as a potential cell source for in vitro generation of bioengineered teeth that may be used in replacement therapy.     

Constitutively active PTH/PTHrP receptor in odontoblasts alters odontoblast and ameloblast function and maturation

Parathyroid hormone (PTH)-related protein (PTH-rP) is an important autocrine/paracrine attenuator of programmed cell differentiation whose expression is restricted to the epithelial layer in tooth development. The PTH/PTHrP receptor (PPR) mRNA in contrast is detected in the dental papilla, suggesting that PTHrP and the PPR may modulate epithelial-mesenchymal interactions. To explore the possible interactions, we studied the previously described transgenic mice in which a constitutively active PPR is targeted to osteoblastic cells. These transgenic mice have a vivid postnatal bone and tooth phenotype, with normal tooth eruption but abnormal, widened crowns. Transgene mRNA expression was first detected at birth in the dental papilla and, at 1 week postnatally, in odontoblasts. There was no transgene expression in ameloblasts or in other epithelial structures. Prenatally, transgenic molars and incisors revealed no remarkable change. By the age of 1 week, the dental papilla was widened, with disorganization of the odontoblastic layer and decreased dentin matrix. In addition, the number of cusps was abnormally increased, the ameloblastic layer disorganized, and enamel matrix decreased. Odontoblastic and, surprisingly, ameloblastic cytodifferentiation was impaired, as shown by in situ hybridization and electron microscopy. Interestingly, ameloblastic expression of Sonic Hedgehog, a major determinant of ameloblastic cytodifferentiation, was dramatically altered in the transgenic molars. These data suggest that odontoblastic activation of the PPR may play an important role in terminal odontoblastic and, indirectly, ameloblastic cytodifferentiation, and describe a useful model to study how this novel action of the PPR may modulate mesenchymal/epithelial interactions at later stages of tooth morphogenesis and development.     

Gene deployment for tooth replacement in the rainbow trout (Oncorhynchus mykiss): a developmental model for evolution of the osteichthyan dentition

Repeated tooth initiation occurs often in nonmammalian vertebrates (polyphyodontism), recurrently linked with tooth shedding and in a definite order of succession. Regulation of this process has not been genetically defined and it is unclear if the mechanisms for constant generation of replacement teeth (secondary dentition) are similar to those used to generate the primary dentition. We have therefore examined the expression pattern of a sub-set of genes, implicated in tooth initiation in mouse, in relation to replacement tooth production in an osteichthyan fish (Oncorhynchus mykiss). Two epithelial genes pitx2, shh and one mesenchymal bmp4 were analyzed at selected stages of development for O. mykiss. pitx2 expression is upregulated in the basal outer dental epithelium (ODE) of the predecessor tooth and before cell enlargement, on the postero-lingual side only. This coincides with the site for replacement tooth production identifying a region responsible for further tooth generation. This corresponds with the expression of pitx2 at focal spots in the basal oral epithelium during initial (first generation) tooth formation but is now sub-epithelial in position and associated with the dental epithelium of each predecessor tooth. Co-incidental expression of bmp4 and aggregation of the mesenchymal cells identifies the epithelial-mesenchymal interactions and marks initiation of the dental papilla. These together suggest a role in tooth site regulation by pitx2 together with bmp4. Conversely, the expression of shh is confined to the inner dental epithelium during the initiation of the first teeth and is lacking from the ODE in the predecessor teeth, at sites identified as those for replacement tooth initiation. Importantly, these genes expressed during replacement tooth initiation can be used as markers for the sites of "set-aside cells," the committed odontogenic cells both epithelial and mesenchymal, which together can give rise to further generations of teeth. This information may show how initial pattern formation is translated into secondary tooth replacement patterns, as a general mechanism for patterning the vertebrate dentition. Replacement of the marginal sets of teeth serves as a basis for discussion of the evolutionary significance, as these dentate bones (dentary, premaxilla, maxilla) form the restricted arcades of oral teeth in many crown-group gnathostomes, including members of the tetrapod stem group.     

Newly established cell lines from mouse oral epithelium regenerate teeth when combined with dental mesenchyme

The present study attempted to examine whether clonal cell lines of the oral epithelium can differentiate into ameloblasts and regenerate tooth when combined with dental germ mesenchyme. Clonal cell lines with a distinct morphology were established from the oral epithelium of p53-deficient fetal mice at embryonic day 18 (E18). The strain of mouse is shown to be a useful source for establishing clonal and immortalized cell lines from various tissues and at various stages of development. Tooth morphogenesis is almost completed and the oral epithelium is segregated from the dental epithelium at E18. In RT-PCR analysis of cell lines, mucosal epithelial markers (cytokeratin 14) were detected, but ameloblast markers such as amelogenin and ameloblastin were not detected when cells were cultured on plastic dish. They formed stratified epithelia and expressed a specific differentiation marker (CK13) in the upper layer when cultured on feeder layer or on collagen gel for 1–3 wk, demonstrating that they are of oral mucosa origin. Next, bioengineered tooth germs were prepared with cell lines and fetal molar mesenchymal tissues and implanted under kidney capsule for 2–3 wk. Five among six cell lines regenerated calcified structures as seen in natural tooth. Our results indicate that some oral epithelial cells at E18 possess the capability to differentiate into ameloblasts. Furthermore, cell lines established in the present study are useful models to study processes in tooth organogenesis and tooth regeneration.     

Odontogenic capability: bone marrow stromal stem cells versus dental pulp stem cells

Background information. Although adult bone‐marrow‐derived cell populations have been used to make teeth when recombined with embryonic oral epithelium, the differences between dental and non‐dental stem‐cell‐mediated odontogenesis remain an open question. Results. STRO‐1⁺ (stromal precursor cell marker) DPSCs (dental pulp stem cells) and BMSSCs (bone marrow stromal stem cells) were isolated from rat dental pulp and bone marrow respectively by magnetic‐activated cell‐sorting techniques. Their odontogenic capacity was compared under the same inductive microenvironment produced by ABCs (apical bud cells) from 2‐day‐old rat incisors. Co‐cultured DPSCs/ABCs in vitro showed more active odontogenic differentiation ability than mixed BMSSCs/ABCs, as indicated by the accelerated matrix mineralization, up‐regulated alkaline phosphatase activity, cell‐cycle modification, and the expression of tooth‐specific proteins and genes. After cultured for 14 days in the renal capsules of rat hosts, recombined DPSC/ABC pellets formed typical tooth‐shaped tissues with balanced amelogenesis and dentinogenesis, whereas BMSSC/ABC recombinants developed into atypical dentin—pulp complexes without enamel formation. DPSC and BMSSC pellets in vivo produced osteodentin‐like structures and fibrous connective tissues respectively. Conclusions. DPSCs presented more striking odontogenic capability than BMSSCs under the induction of postnatal ABCs. This report provides critical insights into the selection of candidate cells for tooth regeneration between dental and non‐dental stem cell populations.     

Localization and quantitation of 125I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages

We have shown earlier that epidermal growth factor (EGF) inhibits morphogenesis and cell differentiation in mouse embryonic teeth in organ culture. This inhibition depends on the stage of tooth development so that only teeth at early developmental stages respond to EGF (A-M. Partanen, P. Ekblom, and I. Thesleff (1985) Dev. Biol. 111, 84-94). We have now studied the quantity and pattern of EGF binding in teeth at various stages of development by incubating the dissected tooth germs with 125I-labeled EGF. Although the quantity of 125I-EGF binding per microgram DNA stays at the same level, localization of 125I-EGF binding by autoradiography reveals that the distribution of binding sites changes dramatically. In bud stage the epithelial tooth bud that is intruding into the underlying mesenchyme has binding sites for EGF, but the condensation of dental mesenchymal cells around the bud does not bind EGF. At the cap stage of development the dental mesenchyme binds EGF, but the dental epithelium shows no binding. This indicates that the dental mesenchyme is the primary target tissue for the inhibitory effect of EGF on tooth morphogenesis during early cap stage. During advanced morphogenesis the binding sites of EGF disappear also from the dental papilla mesenchyme, but the dental follicle which consists of condensed mesenchymal cells surrounding the tooth germ, binds EGF abundantly. We have also studied EGF binding during the development of other embryonic organs, kidney, salivary gland, lung, and skin, which are all formed by mesenchymal and epithelial components. The patterns of EGF binding in various tissues suggest that EGF may have a role in the organogenesis of epitheliomesenchymal organs as a stimulator of epithelial proliferation during initial epithelial bud formation and branching morphogenesis. The results of this study indicate that EGF stimulates or maintains proliferation of undifferentiated cells during embryonic development and that the expression of EGF receptors in different organs is not related to the age of the embryo, but is specific to the developmental stage of each organ.     

Notch2 protein distribution in human teeth under normal and pathological conditions

Notch signaling is essential for the appropriate differentiation of many cell types during development and, furthermore, is implicated in a variety of human diseases. Previous studies have shown that although the Notch1, -2, and -3 receptors are expressed in developing and injured rodent teeth, Notch2 expression was predominant after a lesion. To pursue the role of the Notch pathway in tooth development and disease, we have analyzed the expression of the Notch2 protein in embryonic and adult wounded human teeth. During the earlier stages of tooth development, the Notch2 protein was expressed in the epithelium, but was absent from proliferating cells of the inner enamel epithelium. At more advanced stages, Notch2 was expressed in the enamel-producing ameloblasts, while it was absent in mesenchyme-derived odontoblasts that synthesize the dentin matrix. Although Notch2 was not expressed in the pulp of adult intact teeth, it was reexpressed during dentin repair processes in odontoblasts and subodontoblastic cells. Transforming growth factor beta-1, which stimulates odontoblast differentiation and hard tissue formation after dental injury, downregulated Notch2 expression in cultured human dental slices, in vitro. These observations are consistent with the notion that Notch signaling is an important element in dental physiological and pathogenic conditions.