成体干细胞及牙源性干细胞的研究进展

人体干细胞是在人体生长和发育过程中起主干作用的细胞,干细胞研究是目前生物医学研究的前沿领域,本文着重对成体干细胞和牙源性干细胞的研究现状及进展作一介绍。     

Epithelial stem cells in teeth

 Many tissues and organs maintain a process known as homeostasis, in which cells are replenished as they die as a result of apoptosis or injury. The continuously growing mouse incisors are an excellent model for studying the molecular mechanisms of cell homeostasis, renewal, and repair. We elucidated these mechanisms in mouse incisors by detecting adult stem cells and analyzing the stem cell lineage by bromodeoxyuridine (BrdU) labeling analysis. The stem cells divide slowly, giving rise to a daughter cell that remains in the cervical loop and a second daughter cell that enters the zone of rapidly dividing inner enamel epithelial cells (transit-amplifying cell population). During subsequent rounds of cell division, the latter cells move toward the incisal end and differentiate into ameloblasts that form the enamel matrix. Recent evidence from gene knockout mice suggests that fibroblast growth factor (Fgf10) plays an important role in the formation and maintenance of stem cells in the development of mouse incisors. The role of dental stem cells in odontogenic tumors is discussed. Received: January 22, 2002 / Accepted: April 16, 2002     

Dental pulp stem cells: State of the art and suggestions for a true translation of research into therapy

ObjectivesStem cells have the ability to rescue and/or repair injured tissue. In humans, it is possible to isolate different types of stem cells from the body. Among these, dental pulp stem cells (DPSCs) are relatively easily obtainable and exhibit high plasticity and multipotential capabilities. In particular they represent a gold standard for neural-crest-derived bone reconstruction in humans and can be used for the repair of body defects in low-risk autologous therapeutic strategies.SourcesAn electronic search was conducted on PubMed databases and supplemented with a manual study of relevant references.ResultsAll research described in this review highlight that DPSCs are mesenchymal stem cells that could be used in clinical applications. Unfortunately, very few clinical trials have been reported. Major obstacles imposed on researchers are hindering the translation of potentially effective therapies to the clinic. Both researchers and regulatory institutions need to develop a new approach to this problem, drawing up a new policy for good manufacturing practice (GMP) procedures. We strongly suggest that only general rules be standardized rather than everything. Importantly, this would not have an effect on the safety of patients, but may very well affect the results, which cannot be identical for all patients, due to physiological diversity in the biology of each patient. Alternatively, it would be important to study the role of specific molecules that recruit endogenous stem cells for tissue regeneration. In this way, the clinical use of stem cells could be successfully developed.ConclusionsDPSCs are mesenchymal stem cells that differentiate into different tissues, maintain their characteristics after cryopreservation, differentiate into bone-like tissues when loaded on scaffolds in animal models, and regenerate bone in human grafts. In summary, all data reported up to now should encourage the development of clinical procedures using DPSCs.     

Differences of isolated dental stem cells dependent on donor age and consequences for autologous tooth replacement

Objective

Autologous therapy via stem cell-based tissue regeneration is an aim to rebuild natural teeth. One option is the use of adult stem cells from the dental pulp (DPSCs), which have been shown to differentiate into several types of tissue in vitro and in vivo, especially into tooth-like structures. DPSCs are mainly isolated from the dental pulp of third molars routinely extracted for orthodontic reasons. Due to the extraction of third molars at various phases of life, DPSCs are isolated at different developmental stages of the tooth.

Design

The present study addressed the question whether DPSCs from patients of different ages were similar in their growth characteristics with respect to the stage of tooth development. Therefore DPSCs from third molars of 12–30 year-old patients were extracted, and growth characteristics, e.g. doubling time and maximal cell division potential were analysed. In addition, pulp and hard dental material weight were recorded.

Results

Irrespective of the age of patients almost all isolated cells reached 40–60 generations with no correlation between maximal cell division potential and patient age. Cells from patients <22 years showed a significantly faster doubling time than the cells from patients ≥22 years.

Conclusion

The age of patients at the time of stem cell isolation is not a crucial factor concerning maximal cell division potential, but does have an impact on the doubling time. However, differences in individuals regarding growth characteristics were more pronounced than age-dependent differences.     

Somatic stem cells for regenerative dentistry

Complex human tissues harbour stem cells and/or precursor cells, which are responsible for tissue development or repair. Recently, dental tissues such as periodontal ligament (PDL), dental papilla or dental follicle have been identified as easily accessible sources of undifferentiated cells. The dental stem cell biology might provide meaningful insights into the development of dental tissues and cellular differentiation processes. Dental stem cells could also be feasible tools for dental tissue engineering. Constructing complex structures like a periodontium, which provides the functional connection between a tooth or an implant and the surrounding jaw, could effectively improve modern dentistry. Dental precursor cells are attractive for novel approaches to treat diseases like periodontitis, dental caries or to improve dental pulp healing and the regeneration of craniofacial bone and teeth. These cells are easily accessible and, in contrast to bone-marrow-derived mesenchymal stem cells, are more closely related to dental tissues. This review gives a short overview of stem cells of dental origin.     

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组织工程学是应用细胞生物学和工程学原理,研究和开发用于修复和改善人体组织器官损伤后的功能和形态的一门科学,如何获取增殖能力强,又具有成骨潜能的种子细胞对于骨组织工程的构建是至关重要的,寻找能够满足要求和易于操作的种子细胞是组织工程中的关键环节。本文对组织工程骨的种子细胞培养及其应用做一综述。     

The use of adult stem cells in rebuilding the human face

BACKGROUND: Stem cells have been isolated from a variety of embryonic and postnatal (adult) tissues, including bone marrow. Bone marrow stromal cells (BMSCs), which are non-blood-forming cells in marrow, contain a subset of skeletal stem cells (SSCs) that are able to regenerate all types of skeletal tissue: bone, cartilage, blood-supportive stromal cells and marrow fat cells. METHODS: Bone marrow suspensions are placed into culture for analysis of their biological character and for expansion of their number. The resulting populations of cells are used in a variety of assays to establish the existence of an adult SSC, and the ability of BMSC populations to regenerate hard tissues in the craniofacial region, in conjunction with appropriate scaffolds. RESULTS: Single-cell analysis established the existence of a true adult SSC in bone marrow. Populations of ex vivo expanded BMSCs (a subset of which are SSCs) are able to regenerate a bone/marrow organ. In conjunction with appropriate scaffolds, these cells can be used to regenerate bone in a variety of applications. CONCLUSIONS: BMSCs have the potential to re-create tissues of the craniofacial region to restore normal structure and function in reconstructing the hard tissues of a face. Ex vivo expanded BMSCs with scaffolds have been used in a limited number of patients to date, but likely will be used more extensively in the near future.     

Oral biosciences: The annual review 2022

BackgroundThe Journal of Oral Biosciences is devoted to advancing and disseminating fundamental knowledge concerning every aspect of oral biosciences.HighlightThis review features review articles in the fields of “Bone Cell Biology,” “Tooth Development & Regeneration,” “Tooth Bleaching,” “Adipokines,” “Milk Thistle,” “Epithelial–Mesenchymal Transition,” “Periodontitis,” “Diagnosis,” “Salivary Glands,” “Tooth Root,” “Exosome,” “New Perspectives of Tooth Identification,” “Dental Pulp,” and “Saliva” in addition to the review articles by the winner of the “Lion Dental Research Award” (“Plastic changes in nociceptive pathways contributing to persistent orofacial pain”) presented by the Japanese Association for Oral Biology.ConclusionThe review articles in the Journal of Oral Biosciences have inspired its readers to broaden their knowledge about various aspects of oral biosciences. The current editorial review introduces these exciting review articles.     

组织工程骨干细胞的研究进展

随着再生医学的迅速发展,组织工程骨为骨缺损的修复提供了一个新方法,因而成为研究的热点,而干细胞作为组织工程成骨过程中非常重要的环节,同样受到了极大的关注。干细胞的特点是具有能够在特定条件下分化为不同类型细胞的能力。本文就能够分化为成骨细胞的干细胞的分类及各自特点的相关研究进行综述。     

体外观察三种支架材料对人乳牙牙髓干细胞生物学行为的影响

目的：观察乳牙牙髓干细胞（SHEDs）在3种羟基磷灰石（HA）复合支架材料上黏附、增殖、分化情况。方法：利用正常替换的乳牙分离培养 SHEDs,免疫组织化学法鉴定细胞来源,体外诱导实验观察细胞分化能力。将 SHEDs 分别接种在羟基磷灰石／β-磷酸三钙（HA／TCP）,羟基磷灰石／胶原（HA／COL）和羟基磷灰石／聚乙丙交酯（HA／PLGA）支架材料上复合培养,于4、6、8、10 h 4个时间点检测各细胞黏附率,MTT 比色法检测 SHEDs 增殖情况;碱性磷酸酶（ALP）活性检测、能谱分析钙含量;Von Kossa 染色和灰度扫描细胞矿化能力。结果：SHEDs 在 HA／COL 上的黏附少于 HA／PLGA 和 HA／TCP（P ＜0．05）;HA／PLGA对 SHEDs 矿化诱导能力最强,其次是 HA／TCP,HA／COL 最弱。结论：SHEDs 在 HA／PLGA 上的黏附率、增殖率及矿化能力优于HA／TCP 和 HA／COL。     

牙周膜干细胞用于犬牙槽嵴组织工程化增高的实验研究

目的：探讨牙周膜干细胞复合生物支架材料增高重建牙槽嵴效果,为缺牙后牙槽嵴重度萎缩的种植义齿修复奠定基础.方法：分离培养牙周膜干细胞（PDLSCs）,将其分别与骨组织工程支架材料羟基磷灰石-磷酸三钙（HA/TCP）及附着龈组织工程材料脱细胞真皮基质（ADM）复合诱导后,植入犬双尖牙缺失致牙槽嵴缺损部位,PDLSCs-HA/TCP植入骨缺损区,PDLSCs-ADM植入粘骨膜缺损区.植入32周,大体测量植入前后增高牙槽嵴高度;X-线片显示牙槽嵴高度与密度变化;组织学观察边界区新生牙槽骨结构,未植入做对照.结果：细胞-材料复合体植入后牙槽嵴高度显著增高,由移植前4.15＆#177;0.20,增加到移植后9.30＆#177;0.15（P＜0.05）.X-线显示邻牙标记部位冠方有类骨密度硬组织形成,牙槽嵴明显增高.组织学显示：与对照组比较,细胞材料移植组可见缺损区有新生骨样组织形成,可见骨陷窝和骨细胞样结构.结论：PDLSCs分别与骨及粘骨膜组织工程支架材料HA/TCP、ADM复合后体内移植,可显著增高牙槽嵴高度,是牙槽嵴增高重建、实现无牙合种植修复的有效途径.     

Pulp tissue from primary teeth: new source of stem cells

SHED (stem cells from human exfoliated deciduous teeth) represent a population of postnatal stem cells capable of extensive proliferation and multipotential differentiation. Primary teeth may be an ideal source of postnatal stem cells to regenerate tooth structures and bone, and possibly to treat neural tissue injury or degenerative diseases. SHED are highly proliferative cells derived from an accessible tissue source, and therefore hold potential for providing enough cells for clinical applications. In this review, we describe the current knowledge about dental pulp stem cells and discuss tissue engineering approaches that use SHED to replace irreversibly inflamed or necrotic pulps with a healthy and functionally competent tissue that is capable of forming new dentin.     

骨髓基质干细胞接种于聚乙醇酸支架中异位成骨的观察

目的　将骨髓基质干细胞接种于聚乙醇酸支架中 ,进行异位骨再造的研究。方法　分离、培养、扩增兔骨髓基质干细胞 ,细胞长满后用基因重组人骨形成蛋白 - 2诱导 3天 ;收集细胞 ,按 5× 10 7/ml的浓度接种于聚乙醇酸支架中 ,体外继续培养 5天后 ,植入裸鼠背部皮下组织中 .于植入术后 2月取材 ,通过大体标本、组织学检查观察新骨的形成情况。结果　细胞接种后 1天 ,即可贴附于材料表面 ,扫描电镜观察 ,细胞在材料表面伸展良好。植入后 2个月 ,在裸鼠背部可形成明显的块状组织 ,质地坚硬 ;取材后见形成的组织为暗红色 ,表面光滑 ;组织学检查见新形成的组织由骨细胞和骨小梁构成。结论　聚乙醇酸负载骨髓基质干细胞 ,可以在异位形成骨组织。     

Establishing a technique for isolation and characterization of human periodontal ligament derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are extensively used in tissue regenerative procedures. One source of MSCs is the periodontal ligament (PDL) of teeth. Isolation of MSCs from extracted teeth is reasonably simple, being less invasive and presenting fewer ethical concerns than does the harvesting of MSC’s from other sites. The objectives of this study were to isolate and characterize the PDL stem cells (PDLSC) from healthy adults’ extracted teeth and then to characterize them by comparing them with bone-marrow derived MSCs (BMMSC).MethodsThe PDL tissue was scraped from the roots of freshly extracted teeth to enzymatically digest using collagenase. The cells were sub-cultured. Flow-cytometric analysis for the MSC surface-markers CD105, CD73, CD166, CD90, CD34, CD45 and HLA-DR was performed. To confirm the phenotype, total RNA was extracted to synthesize cDNA and which was then subjected to RT-PCR. The gene-expression for Oct4A, Sox2, NANOG and GAPDH was determined by gel-electrophoresis. To assess their multilineage potential, cells were cultured with osteogenic, chondrogenic and adipogenic medium and then stained by Alizarin-red, Alcian-blue and Oil-Red-O respectively. MSCs from the bone-marrow were processed similarly to serve as controls.ResultsThe cells isolated from extracted teeth expanded successfully. On flow-cytometric analysis, the cells were positive for CD73, CD90, CD105, CD166 and negative for CD34, CD45 and HLA-DR. The PDLSCs expressed Oct4A, Sox2, and NANOG mRNA with GAPDH expression. Cells cultured in the osteogenic, chondrogenic and adipogenic media stained positive for Alizarin-red, Alcian-blue and Oil- Red-O respectively. The surface marker expression and the trilineage differentiation characteristics were comparable to those of the BMMSCs.ConclusionsThe periodontal ligament tissue of extracted teeth is a potential source of therapeutically useful MSCs. Harvesting them is not invasive and are a promising source of MSC as the PDLSCs showed characteristics similar to those of the highly regarded MSC’s derived from bone-marrow.     

成人骨髓基质干细胞体外培养与鉴定

目的：本实验旨在建立一套体外分离培养人骨髓基质干细胞（human bone marrow stem cell HMSCs)的方法，观察HMSCs，体外培养并传代，观察原代及传代细胞的细胞形态及生长规律，第4代细胞用矿化液连续培养30天，在倒置显微镜下观察矿化结节形成情况。结果：体外利用骨髓基质细胞培养液成功培养了HMSCs,并连续传代5－7代，利用第4代细胞用矿化液连续培养30天，矿化结节形成，在细胞间有钙盐沉积。结论：HMSCs可以在体外培养成功，并可定向诱导为成骨细胞，有希望作为极佳的种子细胞应用于临床。     

颌突外胚间充质干细胞参与根尖缺损修复作用的研究

目的:探讨颌突外胚间充质干细胞参与根尖缺损修复的作用。方法:将外胚间充质干细胞复合锻烧骨后移植入大鼠根尖缺损处,以大鼠成纤维细胞复合煅烧骨、空白煅烧骨作为对照,1月后取材,经HE染色鉴定。结果:实验组修复的效果明显好于对照组,形成牙髓牙骨质样物。结论:提示移植的外胚间充质干细胞向该局部组织细胞分化,并参与根尖组织的修复。     

Post exposure prophylaxis to occupational injuries for general dentist

The generation of dental structures and/or entire teeth in the laboratory depends upon the manipulation of stem cells and requires a synergy of all cellular and molecular events that finally lead to the formation of tooth-specific hard tissues, dentin and enamel. This review focuses on the different sources of stem cells that have been used for making teeth in vitro and their relative efficiency. Embryonic, post-natal and adult stem cells were assessed and proved to possess an enormous regenerative potential, but their application in dental practice is still limited due to various parameters that are not yet under control such as the high risk of rejection, cell behaviour, long tooth eruption period, appropriate crown morphology and suitable colour. Nevertheless, the development of biological approaches for dental reconstruction using stem cells is promising and remains one of the greatest challenges in the dental field.