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.     

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。     

牙齿发育的分子机制

在牙齿发育过程中,已知BMPs和FGFs信号通路在上皮和间充质之间的序的相互作用中起着重要的作用。BMP4是启动牙形态发生的一个关键的信号分子。转录因子Msx基因主要起信号介导和传递的作用。已发现鸟类仍具有牙齿发育的潜能。鸟类牙齿发育停止在牙原基阶段的可能原因是Bmp4不在预定的牙胚组织中表达,导致发育信号的传递受阻。     

体外培养的人牙胚上皮细胞的成釉分化

人表皮干细胞可以作为牙齿再生中上皮源性的种子细胞,但是其成釉分化的效率低下. 本研究分离培养了人牙胚上皮细胞,利用E13.5的小鼠牙间充质与其重组,构建重组牙胚,对其成釉分化的潜能和机制进行研究. 研究结果发现,体外培养的P1代人牙胚上皮细胞成釉率高达50%. 随着传代次数的增加,成釉率明显下降. 通过对牙上皮发育分化相关基因的表达检测和分析表明,重组牙胚成牙分化能力和成釉潜能的下降与牙上皮发育相关基因的表达状态密切相关. 特别是FGF8表达水平的下调以及PITX2不同亚型在人牙胚细胞中表达量的不均衡,可能是导致人牙胚细胞成釉潜能下降并丧失的主要原因. 本研究结果为理解牙齿再生过程中上皮源性的种子细胞的成釉机制提供了新的实验数据,对进一步提高表皮干细胞在牙齿再生过程中的成釉率有指导意义.     

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。     

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。     

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。     

成釉质细胞分化机制的研究进展

牙齿的发育是由牙上皮和神经脊来源的间充质之间的连续的相互诱导产生的.上皮和间充质之间连续的相互诱导,使得上皮细胞分化为具有分泌牙釉质功能的成釉质细胞,而间充质细胞分化为具有分泌牙本质功能的成牙本质细胞.成釉质细胞的正常分化对于形成正常的牙釉质是至关重要的,本文主要对细胞信号分子、细胞黏附分子、釉质特异性基因和转录因子等在成釉质细胞分化过程中的作用做一概述.     

鸟类仍保留牙齿发生定位的分子机制

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等认为在进化进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组织仍保留与哺乳动物早期牙齿发生相类似的信号通路,能表达相关的基因并产生相应的信号分子,鸟类牙齿发育停滞在牙原基时期的可能原因是Bmp4不在预定牙上皮组织中表达,导致发育信号的传递受阻,因此,鸟类胚胎的牙原基组织是一个很好的实验模型,用于研究上皮与间充质组织之间的相互作用,导致器官发育的分子机制。在本研究中,我们又进一步对鸡胚发育中与牙原基定位的有关分子信号通路进行了研究,研究证实了,与小白鼠的发育相似,在鸡胚发育中,在任何可见的牙齿发生了形态变化出现之前,Pax9作为预定牙间充质的标记基因（Fig.1),利用体外器官培养,组织重组和原位杂交等方法,证实了Pax9在下颚间充质中的定位表达是由其上方上皮中的两种信号分子所决定（Fig.2),其中FGF8诱导Pax9的表达（Fig.3).而BMP4则抑制该表达（Fig.4)。通过这两种信号之间的诘抗作用决定了间充质中Pax9的表达部位,亦即牙原基的发生部位,因此,与小白鼠相似,在鸟类中牙原基的发生部位是由两类具有诘抗作用的信号分子所决定的,本研究结果进一步证实了在鸟类胚胎发育过程中仍保留与牙齿发育有关的早期信号通路。     

Reconstitution of Telomerase Activity Extends the Proliferative Life-span and Maintains the Neurogenetic Potential of Mesenchymal Stem Cells Derived from Human Fetal Muscle

目的 :通过重建端粒酶活性延长胎儿肌肉源间充质干细胞寿命 ,并对其成神经潜能进行研究 ,为组织工程神经修复提供种子细胞。方法 :将人端粒酶催化亚基 (hTERT)基因通过脂质体转染法导入胎儿肌肉源间充质干细胞 ,RT PCR检测hTERTmRNA的表达 ,TRAP PCR检测细胞端粒酶活性。用bFGF诱导已重建端粒酶活性的肌肉源间充质干细胞向神经细胞分化 ,免疫荧光及免疫印迹法检测分化情况。结果 :转染hTERT的胎儿肌肉源间充质干细胞能稳定表达端粒酶活性。转染后传 75代的细胞经bFGF诱导仍维持着自我更新及向神经细胞分化的潜能 ,且无恶性转化倾向。结论 :重建端粒酶活性可延长胎儿肌肉源间充质干细胞寿命并维持自我更新及成神经潜能 ,为建立组织工程标准细胞系提供了新的实验手段     

牙髓干细胞的研究进展

牙髓干细胞是来源于牙髓组织中的一种成体干细胞,该种细胞具有高度增殖、自我更新的能力和多向分化潜能。牙髓干细胞的研究对牙髓再生、牙体修复等牙组织工程将产生重要的意义。该文就牙髓干细胞的研究现状作一综述,并对其应用前景进行探讨。     

Identification of novel epithelial stem cell-like cells in human deciduous dental pulp

It is well known that interactions between epithelial components and mesenchymal components are essential for tooth development. Therefore, it has been postulated that both types of stem cells might be involved in the regeneration of dental hard tissues. Recently, mesenchymal dental pulp stem cells that have odontogenic potential were identified from human dental pulp. However, the existence of epithelial cells has never been reported in human dental pulp. In the present study, we isolated and characterized epithelial cell-like cells from human deciduous dental pulp. They had characteristic epithelial morphology and expressed epithelial markers. Moreover, they expressed epithelial stem cell-related genes such as ABCG2, Bmi-1, ΔNp63, and p75. Taken together, our findings suggest that epithelial stem cell-like cells might exist in human deciduous dental pulp and might play a role as an epithelial component for the repair or regeneration of teeth.     

Stem cells of dental pulp

Any clinician dreams to obtain the regeneration of the destroyed organ for his patient. In the human being, the regeneration of complex structures is not possible, except the liver and the bone marrow, which can be regenerated because of the presence of adult stem cells in these tissues. The stem cells have two principal properties: they ensure their self-renewal and they have the ability to differentiate into several cellular types. Using specific markers allowing the identification of the stem cells in bone marrow, stem cells were observed in dental pulp tissues. Although the origin, the identification, and the localization of these stem cells of dental pulp remain under consideration, the optimism in research on stem cells permits to believe that the knowledge on dental stem cells will lead to their use in therapeutics.     

Bone morphogenetic proteins in dentin regeneration for potential use in endodontic therapy

The human dentition is indispensable for nutrition and physiology. The teeth have evolved for mastication of food. Caries is a common dental problem in which the dentin matrix is damaged. When the caries is deep and the dental pulp is exposed, the pulp has to be removed in many cases, resulting ultimately in loss of the tooth. Therefore, the regeneration of dentin-pulp complex is the long-term goal of operative dentistry and endodontics. The key elements of dentin regeneration are stem cells, morphogens such as bone morphogenetic proteins (BMPs) and a scaffold of extracellular matrix. The dental pulp has stem/progenitor cells that have the potential to differentiate into dentin-forming odontoblasts in response to BMPs. Pulpal wound healing consists of stem/progenitor cells release from dental pulp niche after noxious stimuli such as caries, migration to the injured site, proliferation and differentiation into odontoblasts. There are two main strategies for pulp therapy to regenerate dentin: (1) in vivo method of enhancing the natural healing potential of pulp tissue by application of BMP proteins or BMP genes, (2) ex vivo method of isolation of stem/progenitor cells, differentiation with BMP proteins or BMP genes and transplantation to the tooth. This review summarizes recent advances in application of BMPs for dentin regeneration and possible use in endodotic therapy.     

Application of dental stem cells in three-dimensional tissue regeneration

Dental stem cells can differentiate into different types of cells. Dental pulp stem cells, stem cells from human exfoliated deciduous teeth, periodontal ligament stem cells, stem cells from apical papilla, and dental follicle progenitor cells are five different types of dental stem cells that have been identified during different stages of tooth development. The availability of dental stem cells from discarded or removed teeth makes them promising candidates for tissue engineering. In recent years, three-dimensional (3D) tissue scaffolds have been used to reconstruct and restore different anatomical defects. With rapid advances in 3D tissue engineering, dental stem cells have been used in the regeneration of 3D engineered tissue. This review presents an overview of different types of dental stem cells used in 3D tissue regeneration, which are currently the most common type of stem cells used to treat human tissue conditions.     

IGFBP5 promotes angiogenic and neurogenic differentiation potential of dental pulp stem cells

Dental stem cells for dental pulp regeneration have become a new strategy for pulpitis treatment. Angiogenesis and neurogenesis play a vital role in the pulp-dentin complex regeneration, and appropriate growth factors will promote the process of angiogenesis and neurogenesis. Insulin-like growth factor-binding protein 5 (IGFBP5) is involved in the regulation of tooth growth and development. A previous study showed that IGFBP5 enhanced osteo/odontogenic differentiation of dental stem cells. Our research intends to reveal the function of IGFBP5 in the angiogenic and neurogenic differentiation of human dental stem cells. Human dental pulp stem cells (DPSCs) were used in the present study. Lentiviral IGFBP5 shRNA was used to silence the IGFBP5. Retroviruses expressing Wild-type IGFBP5 were used to over-express IGFBP5. Angiogenic and neurogenic differentiation were carried out by in vitro study. Real-time RT-PCR and western blot results showed that over-expression of IGFBP5 upregulated the expressions of angiogenic markers, including VEGF, PDGFA and ANG-1, and neurogenic markers, including NCAM, TH, Nestin, βIII-tubulin, and TH, in DPSCs. Moreover, microscope observation confirmed that over-expression of IGFBP5 enhanced neurosphere formation in DPSCs in size and amount. Immunofluorescence staining results showed that over-expression of IGFBP5 also prompted the percentage of Nestin and βIII-tubulin positive neurospheres in DPSCs. While depletion of IGFBP5 downregulated the expressions of VEGF, PDGFA, ANG-1, NCAM, TH, Nestin, βIII-tubulin, and TH, it decreased the neurosphere formation and percentage of Nestin and βIII-tubulin positive neurospheres in DPSCs. In conclusion, our results revealed that IGFBP5 promoted angiogenic and neurogenic differentiation potential of DPSCs in vitro and provided the possible potential target for enhancing directed differentiation of dental stem cells and dental pulp-dentin functional regeneration.     

Hard tissue regeneration capacity of apical pulp derived cells (APDCs) from human tooth with immature apex

Recent studies indicate that dental pulp is a new source of adult stem cells. The human tooth with an immature apex is a developing organ, and the apical pulp of this tooth may contain a variety of progenitor/stem cells, which participate in root formation. We investigated the hard tissue regeneration potential of apical pulp derived cells (APDCs) from human tooth with an immature apex. APDCs cultured with a mineralization-promoting medium showed alkaline phosphatase activity in porous hydroxyapatite (HA) scaffolds. The composites of APDCs and HA were implanted subcutaneously in immunocompromised rats and harvested at 12 weeks after implantation. In histological analysis, the APDCs/HA composites exhibited bone- and dentine-like mineralized tissues in the pore areas of HA. This study suggests that the human tooth with an immature apex is an effective source of cells for hard tissue regeneration.     

Establishment of in vitro culture system for evaluating dentin–pulp complex regeneration with special reference to the differentiation capacity of BrdU label-retaining dental pulp cells

We have proposed the new hypothesis that dental pulp stem cells play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with progenitors. This study aimed to establish an in vitro culture system for evaluating dentin–pulp complex regeneration with special reference to the differentiation capacity of slow-cycling long-term label-retaining cells (LRCs). Three intraperitoneal injections of BrdU were given to pregnant ICR mice to map LRCs in the mature tissues of born animals. The upper bilateral first molars of 3-week-old mice were extracted and divided into two pieces and cultured for 0, 1, 3, 5 and 7 days using the Trowel’s method. We succeeded in establishing an in vitro culture system for evaluating dentin–pulp complex regeneration, where most odontoblasts were occasionally degenerated and lost nestin immunoreactivity because of the separation of cell bodies from cellular processes in the dentin matrix by the beginning of in vitro culture. Numerous dense LRCs mainly resided in the center of the dental pulp associating with blood vessels throughout the experimental periods. On postoperative days 1–3, the periphery of the pulp tissue including the odontoblast layer showed degenerative features. By Day 7, nestin-positive odontoblast-like cells were arranged along the pulp–dentin border and dense LRCs were committed in the odontoblast-like cells. These results suggest that dense LRCs in the center of the dental pulp associating with blood vessels were supposed to be dental pulp stem/progenitor cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells.     

Isolation of pluripotent stem cells from human third molar dental pulp

Potent stem/progenitor cells have been isolated from normal human dental pulps, termed dental pulp stem cells (DPSCs). However, no study has described the presence of stem cell populations in human dental pulp from the third molar with embryonic phenotypes. The dental pulp tissue was cultured in media with the presence of LIF, EGF, and PDGF. In the present study, we describe a new population of pluripotent stem cells that were isolated from dental pulp (DPPSC). These cells are SSEA-4(+), Oct4(+), Nanog(+), FLK-1(+), HNF3beta(+), Nestin(+), Sox2(+), Lin28(+), c-Myc(+), CD13(+), CD105(+), CD3(-), CD45(-), CD90(low), CD29(+), CD73(low), STRO-1(low) and CD146(-). We have investigated by SEM analysis and q-RT-PCR the capacity of DPPSCs to 3D differentiate in vitro using the Cell Carrier 3D glass scaffold into tissues that have similar characteristics to embryonic mesoderm and endoderm layers. These data would support the use of these cells, which are derived from an easily accessible source and can be used in future regeneration protocols for many tissue types that differentiate from the three embryonic layers.