Tissue engineered periodontal products

Attainment of periodontal regeneration is a significant clinical goal in the management of advanced periodontal defects arising from periodontitis. Over the past 30 years numerous techniques and materials have been introduced and evaluated clinically and have included guided tissue regeneration, bone grafting materials, growth and other biological factors and gene therapy. With the exception of gene therapy, all have undergone evaluation in humans. All of the products have shown efficacy in promoting periodontal regeneration in animal models but the results in humans remain variable and equivocal concerning attaining complete biological regeneration of damaged periodontal structures. In the early 2000s, the concept of tissue engineering was proposed as a new paradigm for periodontal regeneration based on molecular and cell biology. At this time, tissue engineering was a new and emerging field. Now, 14 years later we revisit the concept of tissue engineering for the periodontium and assess how far we have come, where we are currently situated and what needs to be done in the future to make this concept a reality. In this review, we cover some of the precursor products, which led to our current position in periodontal tissue engineering. The basic concepts of tissue engineering with special emphasis on periodontal tissue engineering products is discussed including the use of mesenchymal stem cells in bioscaffolds and the emerging field of cell sheet technology. Finally, we look into the future to consider what CAD/CAM technology and nanotechnology will have to offer.     

Epigenetic modulation of dental pulp stem cells: implications for regenerative endodontics

Dental pulp stem cells (DPSCs) offer significant potential for use in regenerative endodontics, and therefore, identifying cellular regulators that control stem cell fate is critical to devising novel treatment strategies. Stem cell lineage commitment and differentiation are regulated by an intricate range of host and environmental factors of which epigenetic influence is considered vital. Epigenetic modification of DNA and DNA‐associated histone proteins has been demonstrated to control cell phenotype and regulate the renewal and pluripotency of stem cell populations. The activities of the nuclear enzymes, histone deacetylases, are increasingly being recognized as potential targets for pharmacologically inducing stem cell differentiation and dedifferentiation. Depending on cell maturity and niche in vitro, low concentration histone deacetylase inhibitor (HDACi) application can promote dedifferentiation of several post‐natal and mouse embryonic stem cell populations and conversely increase differentiation and accelerate mineralization in DPSC populations, whilst animal studies have shown an HDACi‐induced increase in stem cell marker expression during organ regeneration. Notably, both HDAC and DNA methyltransferase inhibitors have also been demonstrated to dramatically increase the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) for use in regenerative therapeutic procedures. As the regulation of cell fate will likely remain the subject of intense future research activity, this review aims to describe the current knowledge relating to stem cell epigenetic modification, focusing on the role of HDACi on alteration of DPSC phenotype, whilst presenting the potential for therapeutic application as part of regenerative endodontic regimens.     

Effect of leptin on differentiation of human dental stem cells

Oral Diseases (2011) 17 , 662–669 Objectives: Mesenchymal stem cells (MSCs) were identified in adult human periodontal ligament and dental pulp that are considered as potential stem cell sources for future clinical applications in dentistry. Leptin is known as an important regulator of mesenchymal differentiation. The objective of this study was to elucidate the role of leptin on proliferation and differentiation of dental MSCs. Materials and methods: Enhancement of cemento/odontoblastic differentiation of dental stem cells by leptin was confirmed by alizarin red S staining and alkaline phosphatase activity staining. In contrast, leptin reduced adipogenesis in both dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) confirmed by oil red O staining and RT‐PCR. The expression of adipogenic markers, lipoprotein lipase and proliferator‐activated receptor γ2 (PPARγ2), were suppressed in PDLSCs incubated on media supplemented with leptin for 2 weeks. Results: Leptin had a relatively stronger osteogenesis promoting effect and adipogenesis suppressing effect in PDLSCs than in DPSCs. Conclusions: Collectively, leptin had a relatively stronger promoting effect on cemento/odontoblastic differentiation and a suppressing effect on adipogenesis in PDLSCs than in DPSCs. This study has provided evidence that leptin acts as an important modulator of dental MSCs differentiation.     

Stem cell properties of human periodontal ligament cells

BACKGROUND AND OBJECTIVE: Stem cells have been used for regenerative therapies in various fields. The proportion of cells that possess stem cell properties in human periodontal ligament (PDL) cells is not yet well understood. In this study, we quantitatively characterized human PDL cells to clarify their stem cell properties, including self-renewal, multipotency, and stem cell marker expression. MATERIAL AND METHODS: PDL cells were obtained from extracted premolar or wisdom teeth, following which a proliferation assay for self-renewal, a differentiation assay for multipotency, immunostaining for STRO-1, and fluorescence-activated cell sorter (FACS) analysis for stem cell markers (including CD105, CD166, and STRO-1) were performed. RESULTS: Approximately 30% of 400 PDL cells were found to possess replicative potential and formed single-cell colonies, and 30% of these colonies displayed positive staining for STRO-1, 20% differentiated into adipocytes and 30% differentiated into osteoblasts. FACS analysis revealed that PDL cells, including cell populations, expressed the stem cell markers CD105, CD166, and STRO-1. CONCLUSION: The findings of this study indicated that PDL cells possess crucial stem cell properties, such as self-renewal and multipotency, and express the mesenchymal stem cell markers CD105, CD166, and STRO-1 on their cell surface, although there were some variations. Thus, PDL cells can be used for periodontal regenerative procedures.     

Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fibroblasts

Wada N, Wang B, Lin N‐H, Laslett AL, Gronthos S, Bartold PM. Induced pluripotent stem cell lines derived from human gingival and periodontal ligament fibroblasts. J Periodont Res 2011; 46: 438–447. © 2011 John Wiley & Sons A/S Background and Objective: Human induced pluripotent stem (iPS) cells, which have similar properties to human embryonic stem (hES) cells, have been generated from neonatal and adult human dermal fibroblasts by reprogramming. iPS cells have high pluripotency and differentiation potential, and may be a potential autologous stem cell source for future regenerative therapy. Material and Methods: iPS cell lines from human gingival fibroblasts and, for the first time, from periodontal ligament fibroblasts, were generated by reprogramming using a retroviral transduction cocktail of OCT3/4, SOX2, KLF4 and c‐MYC. iPS induction was investigated through expression of the embryonic stem cell markers SSEA4, OCT4, NANOG, GCTM‐2, TG30 and TRA‐1‐60. Following in vitro differentiation, the expression of genes for differentiation markers for ectoderm (SOX1, PAX6), mesoderm [RUNX1, T(Brachyury)] and endoderm (GATA4, AFP) was assessed by real‐time RT‐PCR. The ability to form teratomas following implantation into mouse testes was assessed by histology. Results: Human gingival fibroblast‐ and periodontal ligament fibroblast‐derived iPS cells showed similar characteristics to hES cells. Both sets of iPS cells displayed colony morphology comparable to that of hES cells and expressed the hES cell‐associated cell‐surface antigens, SSEA3, SSEA4, GCTM‐2, TG30 (CD9) and Tra‐1‐60, and the hES cell marker genes, OCT4, NANOG and GDF3. These iPS cells showed differentiation potential to form embryoid bodies in vitro and expressed genes for endoderm, ectoderm and mesoderm. Teratoma formation following implantation into mouse testes was observed. Conclusion: These results demonstrate that iPS cells can be successfully generated from adult human gingival and periodontal ligament fibroblasts.     

牙髓干细胞及其表面特异标志物

牙髓干细胞是位于牙髓组织中的一种成体干细胞,具有高度增殖与多向分化的能力。牙髓干细胞的出现,为牙髓病的治疗提供了新的治疗途径。下面就牙髓干细胞及其表面特异性标志物的研究进展作一综述。     

牙髓干细胞的生物学性能及临床应用研究进展

牙髓干细胞是一类具有自我更新、多向分化、高度增殖能力的外胚间充质干细胞,可为组织器官修复和多种疾病治疗提供细胞来源.本文通过总结相关文献,对牙髓干细胞的一般生物学特性进行论述,并探讨其成骨/成牙本质、成肌/成血管和神经再生的机制,以及其在糖尿病和眼部疾患的临床前应用.同时结合了国内外有关牙髓干细胞的最新临床应用进展,给...     

骨髓干细胞应用于牙槽骨修复的展望

干细胞是一种尚未充分分化,具有再生各种组织器官和人体潜在功能的细胞。骨髓间充质干细胞(MSC)是骨髓中的非造血干细胞,其在不同的诱导条件下能分化成成熟的间质细胞,如成骨细胞、成软骨细胞、脂肪细胞、肌腱细胞、网状细胞等。为了探讨骨髓间充质干细胞对骨修复的意义,本文对近年来国内外学者对于骨髓间充质干细胞应用于动物体内或体外成骨情况的研究现状作一综述。     

Tca-8113细胞系单细胞体外增殖的实验观察

目的:观察舌鳞癌Tca-8113细胞系的单细胞体外增殖能力,寻找肿瘤干细胞存在的依据,从而为鉴定其表面标记物奠定理论基础。方法:采用有限稀释法对舌鳞癌Tca-8113细胞进行单细胞体外培养,了解舌鳞癌细胞(Tca-8113)的分裂、增殖特点。结果:单细胞培养8d后,舌鳞癌Tca-8113细胞株中具有持续增殖能力的细胞占肿瘤细胞的10.85%,12d后,继续分裂增殖的细胞比例为5.23%,16d后,仅有5.09%的细胞继续增殖。结论:舌鳞癌Tca-8113细胞具有异质性,仅有少量细胞具有持续增殖能力,而这部分细胞是否就是舌鳞癌Tca-8113细胞的肿瘤干细胞。还有待进一步的实验研究证实。     

Stem cells and the dental pulp: potential roles in dentine regeneration and repair

The dentine-pulp complex displays exquisite regenerative potential in response to injury. The postnatal dental pulp contains a variety of potential progenitor/stem cells, which may participate in dental regeneration. A population of multipotent mesenchymal progenitor cells known as dental pulp stem cells with high proliferative potential for self-renewal has been described and may be important to the regenerative capacity of the tissue. The nature of the progenitor/stem cell populations in the pulp is of importance in understanding their potentialities and development of isolation or recruitment strategies, and allowing exploitation of their use in regeneration and tissue engineering. Various strategies will be required to ensure not only effective isolation of these cells, but also controlled signalling of their differentiation and regulation of secretory behaviour. Characterization of these cells and determination of their potentialities in terms of specificity of regenerative response will form the foundation for development of new clinical treatment modalities, whether involving directed recruitment of the cells and seeding of stem cells at sites of injury for regeneration or use of the stem cells with appropriate scaffolds for tissue engineering solutions. Such approaches will provide an innovative and novel biologically based new generation of clinical treatments for dental disease.     

舌鳞癌Tca8113细胞株干细胞相关差异表达基因的筛选及分析

目的应用抑制消减杂交（SSH）技术筛选舌鳞癌干细胞相关差异表达基因。方法以舌鳞癌Tca8113细胞株中的醛脱氢酶（ALDH）高表达（ALDHbr）细胞与低表达（ALDHlow）细胞互为实验组和对照组,进行正反向消减杂交后建立差异表达基因文库,随机挑选差异基因克隆进行测序及生物信息学分析。结果经Blast比对分析,得到62条差异表达基因;功能聚类发现多数与细胞周期调节、细胞分化等生物行为相关;信号通路分析显示有11条基因参与的12条信号通路发生显著改变。结论应用SSH技术成功筛选出了与舌鳞癌干细胞相关的一些基因,进一步研究这些基因对探索舌鳞癌干细胞特异性表面标志物具有重要意义。     

诱导多能干细胞研究现状及展望

Takahashi和Yamanaka通过瞬时高表达外源性的转录因子,首次得到了诱导多能干细胞,这种具有多潜能细胞特性细胞的诞生,预示着一种新的干细胞研究方法的到来。本文就诱导多能干细胞的发展历程及最新进展作一综述。     

牙髓干细胞分化的研究进展

牙髓干细胞(DPSC)是一种具有高度增生、自我更新能力和多相分化潜能的成体干细胞,在一定条件下可向特定的细胞类型分化,在牙髓修复和牙齿再生中发挥着重要的作用。本文主要就DPSC成骨向分化、成牙本质向分化的研究进展作一综述。     

Tissue engineering of teeth using adult stem cells

Tooth development, a process which occurs in the developing embryo, involves the reciprocal and sequential signalling between epithelial and mesenchymal tissue of the developing first branchial arch. The oral epithelium produces the first inductive signals for odontogenesis at around E10.0, which trigger off a cascade of events that result in the formation of a tooth. We have engineered a tooth in vitro by harnessing the basic principles of odontogenesis and the inductive capability of the oral epithelium of the developing embryo. We replaced the mesenchymal portion of the developing mandibular primordium with aggregates of stem cells from embryos as well as stem cells taken from adult mice. The cell aggregates were covered with embryonic epithelium from E10.0 mouse embryos to form recombinant explants. In vitro culture of these recombinant explants resulted in the induction of early tooth marker genes in the cell aggregates, indicating that the cells were able to respond to the odontogenic signals produced by the oral epithelium. In vivo culture of explants resulted in the induction of Dspp within the cell aggregates indicating that tooth tissue was present. Three recombinant explants, where the cell aggregates consisted of adult bone marrow cells, produced teeth. To determine whether the oral cavity would be able to sustain the growth of an implanted tooth germ, E14.5 molar rudiments were implanted into the diastema region of the maxilla of adult mice. The resulting teeth appeared to be normal in size and were connected to the underlying bone. These experiments are an indication that it is possible to induce odontogenesis and engineer a tooth using adult cells of non-dental origin. They also indicate that developing tooth germs could be successfully implanted into the gingiva of patients.     

肿瘤干细胞与化疗耐药

化疗是晚期肿瘤患者综合序列治疗的重要组成部分,但肿瘤细胞的耐药性常导致临床化疗失败。肿瘤干细胞能驱动肿瘤生成,如果化疗药物不能有效靶向作用于肿瘤干细胞,即使肿瘤缩小,残存的肿瘤干细胞也会迅速驱动肿瘤的复发。本文主要总结近年来关于肿瘤干细胞参与化疗耐药可能分子机制的研究成果,和靶向使肿瘤干细胞对治疗敏感性增加的新方法。     

复层牙周膜细胞膜片组织再生的实验性研究

目的:制备复层牙周膜干细胞膜片(MUCPs)与单层牙周膜干细胞膜片(MCPs),并评价其生物学活性和组织再生能力。方法:将构建的2种细胞膜片通过免疫组化和扫描电镜等方法观察其生物学活性。再将2种膜片分别与2种支架材料陶瓷牛骨(CBB)与预处理牙本质片(TDM)复合构建不同的生物性牙根,植入裸鼠皮下,观察组织再生情况。结果:体外结果显示MUCPs高表达Col-I、COL-III、Fibronectin、Laminin等细胞外基质的结构蛋白。体内结果显示在复层细胞膜片组可以观察到再生出更明显的类似于牙周复合组织的矿化沉积和牙周膜样纤维。结论:MUCPs具有更为良好的生物学活性和组织再生能力,将为临床牙周组织缺损的治疗提供一种新的治疗方法。     

人牙髓干细胞的体外分离、培养及鉴定

目的 :体外分离、培养人牙髓干细胞 ,从不同角度对其进行鉴定。方法 :采用胶原酶和Dispase酶联合消化法培养人牙髓干细胞 ,有限稀释法分离纯化 ,将克隆形成的细胞通过透射电镜观察其超微结构、流式细胞仪细胞周期分析及抗波形丝蛋白 (Vimentin)、CD44、骨粘素 (ON)、牙本质涎蛋白 (DSP)免疫组化染色方法进行鉴定。结果 :通过有限稀释法获得了人牙髓干细胞克隆 ,透射电镜下观察这种克隆形成细胞具有干细胞典型的超微结构特点 ,大多数细胞处于G0 /G1期 ,为细胞周期的静止期 ,并具有牙源性未分化间充质细胞的表型特点。结论 :克隆化培养是人牙髓干细胞较为有效的分离纯化方法 ,该方法分离的人牙髓干细胞具有干细胞的结构、细胞周期及表型特点。     

人乳牙牙髓干细胞在干细胞治疗中的应用

乳牙牙髓干细胞（SHED）是牙源性干细胞的一种,属外胚间充质干细胞。作为一种理想的干细胞来源,SHED在干细胞治疗中有良好的应用前景。本文阐述了SHED的生物学特征及其在干细胞治疗中的优势,探讨了SHED在组织再生和修复中发挥的多向分化潜能、细胞分泌功能和免疫调节功能等方面的功能作用。此外,本文还介绍了SHED在各系统、器官疾病治疗中的临床应用,重点阐述了用SHED进行干细胞移植在牙髓—牙本质再生、颌骨再生、神经系统疾病治疗和免疫系统疾病治疗方面的研究进展。     

毛囊bulge干细胞培养方法的比较研究

目的通过对限定性角质形成细胞无血清培养基（DK-SFM）和3T3滋养层培养毛囊bulge干细胞的比较,寻求既能方便获得大量毛囊bulge干细胞,又能减少其分化的理想培养条件。方法采用DK-SFM和3T3滋养层分别培 养毛囊bulge干细胞,通过倒置显微镜观察毛囊bulge干细胞的形态,免疫荧光染色检测毛囊bulge干细胞的特异性标 记细胞角蛋白19（CK19）和分化相关抗体群34（CD34）的表达鉴定毛囊bulge干细胞。通过比较2种方法培养的毛囊bulge干细胞的克隆形成率和CD34阳性率的差异,分别比较毛囊bulge干细胞的增殖能力和干细胞的数量,综合评估 2种培养方法的优劣。结果倒置显微镜观察2种培养方法所得毛囊bulge干细胞均为铺路石状。免疫荧光染色检测 2种培养方法培养的毛囊bulge干细胞CK19和CD34均呈阳性。DK-SFM和3T3滋养层培养的毛囊bulge干细胞克隆形成 率分别为69.4%和62.2%。流式细胞仪检测DK-SFM培养的毛囊bulge干细胞中CD34阳性率为72.3%;3T3滋养层培养 的毛囊bulge干细胞中CD34阳性率为34.7％。结论DK-SFM和3T3滋养层培养作为毛囊bulge干细胞的2种培养方法, 均可以获得未分化的毛囊bulge干细胞。用3T3滋养层培养毛囊bulge干细胞的方法比较复杂,且所获的细胞混杂有 3T3细胞,但用此种方法可获得大量的毛囊bulge干细胞。用DK-SFM培养毛囊bulge干细胞方法简单,但细胞不易贴 壁,且数量较少,但能有效分离出较纯的毛囊bulge干细胞,保持较好的细胞活性。