牙周膜干细胞的研究进展

牙周膜干细胞可分化为成骨细胞或成牙骨质细胞、脂肪细胞和胶原形成细胞,是牙周组织工程中新的种子细胞。笔者就牙周膜干细胞的生物学特性、牙周膜干细胞的多向分化潜能、牙周膜干细胞的移植及其潜在的临床应用价值作一综述。     

Putative stem cells in regenerating human periodontium

Background and Objective:  Human postnatal stem cells have been identified in periodontal ligament, with the potential to regenerate the periodontium in vivo . However, it is unclear if periodontal ligament stem cells are present in regenerating periodontal tissues. The aim of this study was to identify and localize putative stem cells in block biopsies and explant cultures of regenerating human periodontal tissues.
Material and Methods:  Guided tissue regeneration was carried out on the molars of three human volunteers. After 6 wk, the teeth with the surrounding regenerating tissues and bone were surgically removed and processed for immunohistochemistry. The mesenchymal stem cell-associated markers STRO-1, CD146 and CD44 were used to identify putative stem cells. Cell cultures established from regenerating tissue explants were analysed by flow cytometry to assess the expression of these markers. Mineralization, calcium concentration and adipogenic potential of regenerating tissue cells were assessed and compared with periodontal ligament stem cells, bone marrow stromal stem cells and gingival fibroblasts.
Results:  STRO-1⁺, CD44⁺ and CD146⁺ cells were identified in the regenerating tissues. They were found mainly in the paravascular and extravascular regions. Flow cytometry revealed that cultured regenerating tissue cells expressed all three mesenchymal stem cell associated markers. The regenerating tissue cells were able to form mineral deposits and lipid-containing adipocytes. However, the level of mineralization in these cells was lower than that of periodontal ligament stem cells and bone marrow stromal stem cells.
Conclusion:  Cells with characteristics of putative mesenchymal stem cells were found in regenerating periodontal tissues, implying their involvement in periodontal regeneration.     

牙周膜维持结构和功能稳定的分子机制

牙周膜中存在有多向分化潜能的成体干细胞，能够分化形成成骨细胞和成牙骨质细胞，参与牙周组织骨改建、损伤修复及牙周再生，同时，牙周膜还具有抑制矿化、维持其结构与功能稳定的调节机制。本文就牙周膜细胞的分化能力和参与维持牙周膜结构与功能稳定的分子机制作简要综述。     

经典Wnt信号通路与牙周膜干细胞成骨分化

牙周炎是口腔最常见的疾病之一,累及牙周支持组织,随着疾病的进展将引起附着丧失、牙周袋形成、牙槽骨吸收,最终导致牙齿松动脱落。因被牙周炎破坏吸收的牙槽骨自愈能力十分有限,所以牙周炎的治疗目标是在彻底清除菌斑生物膜的基础上,争取获得较多的牙周组织再生。牙周膜干细胞作为最适宜进行牙周组织再生的细胞,被广泛研究。Wnt信号通路分为经典Wnt通路和非经典Wnt信号通路,为十分复杂而高度保守的通路传导途径。该通路与牙周膜干细胞成骨分化的关系十分密切,牙周膜干细胞的成骨分化又对牙周组织再生有重要意义。该文对经典Wnt信号通路与牙周膜干细胞成骨分化的研究概况作一综述。     

影响牙周膜干细胞成骨分化能力的因素

牙周膜干细胞(PDLSC)是维持牙周组织动态平衡和缺损修复的关键细胞,被认为是牙周组织工程的关键种子细胞之一.近年来的研究证明其有较强的的增殖和分化能力,然而在不同因素影响下其功能差异也较大.认识并研究这些影响因素不仅有助于深入了解和发掘PDLSC的生物学功能,而且对于今后基于PDLSC的牙周修复与再生治疗具有指导意义.该文就影响PDLSC成骨分化的多种因素作一叙述,展望其在牙周缺损修复治疗中的应用前景.     

Enhanced adipogenic differentiation and reduced collagen synthesis induced by human periodontal ligament stem cells might underlie the negative effect of recombinant human bone morphogenetic protein-2 on periodontal regeneration

Song D‐S, Park J‐C, Jung I‐H, Choi S‐H, Cho K‐S, Kim C‐K, Kim C‐S. Enhanced adipogenic differentiation and reduced collagen synthesis induced by human periodontal ligament stem cells might underlie the negative effect of recombinant human bone morphogenetic protein‐2 on periodontal regeneration. J Periodont Res 2011; 46: 193–203. © 2010 John Wiley & Sons A/S Background and Objective: Recombinant human bone morphogenetic protein‐2 (rhBMP‐2) is a potent inducer for the regeneration of mineralized tissue, but has a limited effect on the regeneration of cementum and periodontal ligament (PDL). The aim of the present study was to determine the effects of rhBMP‐2 on the in vitro and in vivo biologic activity of well‐characterized human PDL stem cells (hPDLSCs) and to elucidate the underlying mechanism of minimal periodontal regeneration by rhBMP‐2. Material and Methods: hPDLSCs were isolated and cultured, and then transplanted into an ectopic subcutaneous mouse model using a carrier treated either with or without rhBMP‐2. Comprehensive histologic, histometric and immunohistochemical analyses were performed after an 8‐wk healing period. The effects of rhBMP‐2 on the adipogenic and osteogenic/cementogenic differentiation of hPDLSCs were also evaluated. The effect of rhBMP‐2 on both soluble and insoluble collagen synthesis was analyzed, and the expression of mRNA and protein for collagen types I, II, III and V was assessed. Results: In the present study, rhBMP‐2 promoted both adipogenic and osteogenic/cementogenic differentiation of hPDLSCs in vitro, and the in vivo potential of hPDLSCs to form mineralized cementum and organized PDL tissue was down‐regulated following treatment with rhBMP‐2. Collagen synthesis, which plays a crucial role in the regeneration of cementum and the periodontal attachment, was significantly reduced, with associated modification of the relevant mRNA and protein expression profiles. Conclusion: In summary, the findings of the present study suggest that enhanced adipogenic differentiation and inhibition of collagen synthesis by hPDLSCs appear to be partly responsible for the minimal effect of rhBMP‐2 on cementum and PDL tissue regeneration by hPDLSCs.     

The dynamic healing profile of human periodontal ligament stem cells: histological and immunohistochemical analysis using an ectopic transplantation model

Kim Y‐T, Park J‐C, Choi S‐H, Cho K‐S, Im G‐I, Kim B‐S, Kim C‐S. The dynamic healing profile of human periodontal ligament stem cells: histological and immunohistochemical analysis using an ectopic transplantation model. J Periodont Res 2012; 47: 514–524. © 2012 John Wiley & Sons A/S Background and Objective: Human periodontal ligament stem cells (hPDLSCs) have been reported to play the pivotal role in periodontal regeneration. However, the dynamic cellular healing process initiated by hPDLSCs still remains to be elucidated. In the present study, the sequence of regeneration by hPDLSCs was assessed using histological and immunohistochemical observation in an ectopic transplantation model, which is a well‐standardized assessment tool that excludes the innate healing factors from the animals. Material and Methods: Human periodontal ligament stem cells that were isolated and characterized from teeth (n = 12) extracted for the purpose of orthodontic treatment were transplanted with carriers into ectopic subcutaneous pouches in immunocompromised mice (n = 20). Animals were killed after several different healing periods: 3 d (n = 4), 1 (n = 4), 2 (n = 4), 4 (n = 4) and 8 wk (n = 4). Histological analysis for regenerated tissues formed by hPDLSCs was conducted using hematoxylin and eosin, Masson’s trichrome and picrosirius red staining. In addition, immunohistochemical staining was performed to observe the sequential expression of osteogenic/cementogenic and periodontal ligament tissue‐specific markers associated with periodontal regeneration. Results: The whole healing process by transplanted hPDLSCs could be broadly divided into four distinctive phases. In the first phase, proliferated hPDLSCs migrated evenly all over the carrier, and collagenous tissues appeared in the form of amorphous collagen matrices. In the second phase, collagen fibers were well arranged among the carriers, and cementoid‐like tissues were observed. In the third phase, the formation of mature collagen fibers, resembling Sharpey’s fibers, was associated with active mineralization of cementum‐like tissues, and in the fourth phase, the maturation of cementum‐like tissues was observed on carrier surfaces. Various osteogenic/cementogenic markers related to the regeneration processes were expressed in a well‐orchestrated time order. Interestingly, well‐organized cementum‐like and periodontal ligament fiber‐like tissues and cells with early and late osteogenic/cementogenic markers were frequently observed in the secluded area of carrier surfaces. We termed this area the cell‐rich zone. Conclusion: The results from this study clearly demonstrated the sequential histological changes during periodontal tissue regeneration by hPDLSCs. Understanding of this process would potentially enable us to develop better cell‐based treatment techniques.     

乏氧微环境下机械应力对人牙周膜干细胞成骨分化的影响

人牙周膜干细胞是牙周组织中具有自我更新和多向分化潜能的一类成体干细胞,在特定诱导条件下可以成骨分化。牙周膜处于一个相对乏氧的环境,人牙周膜干细胞在牙周膜中承载着多种机械应力。探讨乏氧环境下机械应力对人牙周膜干细胞成骨分化的影响更接近于体内环境,更有助于获得真实的牙周组织改建的实验室资料。该文对乏氧微环境下机械应力对人牙周膜干细胞成骨分化的影响做一系统性回顾。     

Stimulation of Periodontal Ligament Stem Cells by Dentin Matrix Protein 1 Activates Mitogen‐Activated Protein Kinase and Osteoblast Differentiation

Background: Periodontitis can ultimately result in tooth loss. Many natural and synthetic materials have been tried to achieve periodontal regeneration, but the results remain variable and unpredictable. We hypothesized that exogenous treatment with dentin matrix protein 1 (DMP1) activates specific genes and results in phenotypic and functional changes in human periodontal ligament stem cells (hPDLSCs). Methods: hPDLSCs were isolated from extracted teeth and cultured in the presence or absence of DMP1. Quantitative polymerase chain reactions were performed to analyze the expression of several genes involved in periodontal regeneration. hPDLSCs were also processed for immunocytochemical and Western blot analysis using phosphorylated extracellular signal‐regulated kinase (pERK) and ERK antibodies. Alkaline phosphatase and von Kossa staining were performed to characterize the differentiation of hPDLSCs into osteoblasts. Field emission scanning electron microscopic analysis of the treated and control cell cultures were also performed. Results: Treatment with DMP1 resulted in the upregulation of genes, such as matrix metalloproteinase‐2, alkaline phosphatase, and transforming growth factor β1. Activation of ERK mitogen‐activated protein kinase signaling pathway and translocation of pERK from the cytoplasm to the nucleus was observed. Overall, DMP1‐treated cells showed increased expression of alkaline phosphatase, increased matrix, and mineralized nodule formation when compared with untreated controls. Conclusion: DMP1 can orchestrate a coordinated expression of genes and phenotypic changes in hPDLSCs by activation of the ERK signaling pathway, which may provide a valuable strategy for tissue engineering approaches in periodontal regeneration.     

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??Objective    To study the influence of exosome secreted by the human bone marrow mesenchymal stem cells ??hBMMSCs?? on proliferation and differentiation of periodontal ligament stem cells ??hPDLSCs????and to provide experimental basis and evidence of the mechanism of hBMMSCs in promoting periodontal tissue regeneration. Methods    hPDLSCs were cultured by enzyme digestion method and hBMMSCs were cultured by centrifugation method?? and the immunophenotype was identified by flow cytometry. Supernatant of hBMMSCs in P3 was collected?? exosome was extracted by kit and observed by SEM??and CD63 level was tested by WB. The experiment group was treated by exosome of hBMMSCs and control group was treated by DD water. hPDLSCs proliferation was tested by MTT and colony unite forming was tested in 7 days. After osteogenetic induction?? ALP and Alizarin Red staining were performed and quantitative analysis was carried. Meanwhile osteogenesis-related gene expression was tested by qPCR. Results    hPDLSCs positively expressed CD29?? CD44?? CD90?? CD146 and Stro-1?? hBMMSCs positively expressed CD29?? CD44?? CD90 and CD106??both negatively expressed CD14?? CD34 and CD45. Exosome secreted by hBMMSCs were small ball-shaped under SEM and expressed CD63 strongly by WB. The proliferation and colony unite forming of experimental group was significantly higher than control group ??P < 0.05??. The quantitative expression of ALP and alizarin red staining after osteogenetic induction were significantly stronger than the control group ??P < 0.05??. The osteogenetic gene expression??including Runx2?? OCN?? ALP?? BSP and Col - ?? was significantly higher than that of control group by qPCR ??P < 0.05??. Conclusion    The exosome secreted by hBMMSCs can promote??proliferation and osteogenesis of hPDLSCs in vitro.     

牙周膜干细胞的研究进展

牙周病是一种由菌斑微生物引起的慢性感染性疾病,可引起牙周支持组织的破坏和丧失,最终导致牙齿松动脱落。牙周病治疗的最终目标是修复和重建受损的牙周支持组织。从牙周膜中分离获取的间充质干细胞具有成体干细胞的特性及多重分化潜能,可以分化为骨组织和牙周支持组织等多种类型的组织,这对牙周组织修复再生和牙周组织工程具有重大意义,因而备受关注。本文就牙周膜干细胞、牙周膜干细胞的生物学特性、牙周膜干细胞的影响因素及其调控机制等研究进展作一综述。     

人乳牙牙周膜干细胞的生物学特性研究

目的:体外分离纯化人乳牙牙周膜组织来源的干细胞,研究其生物学特性、表面标记物及多向分化能力。方法:用酶消化法和有限稀释法对正常人乳牙牙周膜组织进行原代培养,并通过免疫细胞化学方法和流式细胞仪对其来源进行鉴定,进而从增殖能力、克隆形成能力和多向分化能力等方面对乳牙牙周膜干细胞的生物学特性进行研究。结果:分离培养获得的人乳牙牙周膜干细胞在形态上与恒牙牙周膜干细胞相似,均为长梭形成纤维细胞样;免疫细胞化学和流式细胞仪分子表型鉴定显示乳牙牙周膜干细胞阳性表达间充质来源的表面标志STRO-1,CD146,CD29和CD90,造血系来源的标志物CD34为阴性;细胞周期,MTT和克隆形成率的检测结果显示,人乳牙牙周膜干细胞的增殖能力强于恒牙牙周膜干细胞;人乳牙牙周膜干细胞在体外诱导条件下可以向骨,脂肪细胞方向分化;同时RT-PCR检测发现,矿化诱导后细胞成骨相关基因(ALP,OCN,Col I)的表达上调,成脂诱导后细胞成脂相关基因(PPAR-γ,C/EBPα)的表达上调。结论:人乳牙牙周膜中确实存在间充质来源的干细胞,并且具有较强的增殖能力和多向分化能力,为牙周组织工程种子细胞提供了新的可能来源。     

骨髓间充质干细胞来源的外泌体促进牙周再生的体外研究

目的    研究人骨髓间充质干细胞（human bone marrow mesenchymal stem cells, hBMMSCs）分泌的外泌体（exosome）对人牙周膜干细胞（human periodontal ligament stem cells, hPDLSCs）增殖和分化功能的影响,为进一步明确hBMMSCs促进牙周组织再生的机制提供实验基础和证据。方法    分别用酶消化法和离心法培养hPDLSCs和hBMMSCs,并用流式细胞术检测其间充质表面标志物。收集培养的第3代hBMMSCs上清液,利用试剂盒提取exosome,电镜下观察其结构,Western Bolt法检测其CD63表达水平。用hBMMSCs分泌的exosome处理hPDLSCs设为实验组,四甲基偶氮唑盐（MTT）法检测hPDLSCs的增殖情况,克隆形成率检测多克隆形成能力,成骨诱导后碱性磷酸酶（ALP）染色和茜素红染色并定量,同时实时荧光定量PCR（qPCR）检测成骨相关基因表达。结果    流式细胞术检测,hPDLSCs阳性表达CD29、CD44、CD90、CD146和Stro-1,hBMMSCs阳性表达CD29、CD44、CD90和 CD106,两者均阴性表达CD14、CD34和CD45。hBMMSCs分泌的exosome电镜下呈小球状,Western Bolt法检测其强表达CD63。实验组hPDLSCs经MTT法检测其增殖速率和克隆形成能力显著高于对照组（P < 0.05）。实验组hPDLSCs成骨诱导后ALP染色、茜素红染色定量表达显著强于对照组（P < 0.05）。qPCR检测成骨基因Runx2、OCN、ALP、BSP和Col-Ⅰ的表达,实验组显著高于对照组（P < 0.05）。结论    hBMMSCs分泌的exosome可促进牙周膜干细胞的体外增殖和成骨分化能力。     

Biologic interaction of three-dimensional periodontal fibroblast spheroids with collagen-based and synthetic membranes

Background: Cell‐based therapy using autologous cells has been suggested as a potential approach for periodontal tissue regeneration. Spheroid systems are a form of three‐dimensional cell culture that promotes cell matrix interaction, which could recapitulate the aspect of cell homeostasis in vivo. The aim of this study is to assess the interaction of periodontal fibroblast spheroids with synthetic and collagen‐based membranes that have been used in guided tissue regeneration. Methods: Commercially available normal human periodontal ligament fibroblasts were grown in spheroid forms using a liquid overlay technique and then transplanted onto a collagen‐based and a polyglycolic acid–based membrane. The biologic interaction of the spheroids with the membranes was assessed using basic histology, Alamar blue tissue viability assay, scanning electron microscopy, and immunohistochemical analysis. Results: Periodontal fibroblast spheroids adhered to both membranes, and the cells were able to proliferate and migrate from the spheroids both horizontally and vertically into the membrane scaffolds. Immunohistochemical analysis showed expression of collagen type I, periostin, and Runx2 by the periodontal fibroblasts. Conclusion: Periodontal fibroblast spheroids were able to grow three‐dimensionally on the biologic membranes and may have the potential to be used together with guided tissue regeneration approaches as an adjunct for periodontal regeneration.     

人脐带Wharton's Jelly来源间充质干细胞与人牙周膜干细胞成骨分化能力的对比研究

目的:比较人脐带Wharton's Jelly来源间充质干细胞(human umbilical cord Wharton's Jelly-derived mesechymal stem ceils,hUCWJMSCs)与人牙周膜干细胞(periodontal mesenchymal stem cells,hPDLSCs)成骨分化能力.方法:体外培养hUC-WJMSCs和hPDLSCs.MTT法检测细胞增殖情况;成骨诱导后测定细胞的ALP活性,茜素红染色检测细胞矿化能力,Real-timePCR分析OPN和Runx2基因的表达.结果:hUCWJMSCs增殖能力高于hPDLSCs;经矿化诱导后hPDLSCs ALP表达、矿化结节形成高于hUCWJMSCs(P＜0.05);Runx2在hPDLSCs中表达高于hUCWJMSCs(P ＜0.05);而hUCWJMSCs中OPN表达高于hPDLSCs(P＜0.05).结论:hUCWJMSCs、hPDLSCs均具有成骨分化能力,hPDLSCs成骨分化能力较强.     

炎症微环境对牙周组织再生的影响

慢性牙周炎是一种以牙周组织破坏为特征的慢性炎症性疾病。牙周病的最终治疗目的是牙周组织再生。而牙周炎时形成的局部炎症微环境,不仅造成牙周组织的破坏,而且会影响牙周组织再生过程,比如影响牙周膜干细胞的增殖分化以及生长因子的作用等。本文就炎症微环境对牙周组织再生的影响作一综述。     

组织工程细胞片在牙周组织再生中的作用

牙周组织工程系指将在体外以理想的种子细胞和细胞外基质构建的具有生命活性的复合体植入牙周缺损部位,对牙周缺损部位进行结构功能性重建。组织工程细胞膜片构建主要有温敏培养皿、刮取法、胶原凝胶法、磁力组织工程法、表面粗糙颗粒单分子层法和聚合电解质等。牙周组织工程细胞片主要有牙周膜干细胞和骨髓间充质干细胞片,分别将其与不同的载体叠加并植入动物体内,可发现细胞外基质和纤维黏附,牙骨质样组织和牙周组织再生。将人牙周膜成纤维细胞和人脐静脉内皮细胞、人真皮成纤维细胞和人脐静脉内皮细胞共培养,所形成的细胞片可生成类似原生血管的管状结构。目前的组织工程细胞片的生物力学性能较差,与传统支架结合仍然存在着材料降解引起的组织纤维化和免疫排斥等问题,组织工程细胞片在牙周组织工程应用中具有较大应用前景;但是,要实现牙周组织的完全再生,其技术有待进一步的研究。本文就细胞片的构建、不同的牙周组织工程细胞片、细胞片工程面临的问题等研究进展作一综述。     

Periodontal regeneration following implantation of cementum and periodontal ligament-derived cells

Nuñez J, Sanz‐Blasco S, Vignoletti F, Muñoz F, Arzate H, Villalobos C, Nuñez L, Caffesse RG, Sanz M. Periodontal regeneration following implantation of cementum and periodontal ligament‐derived cells. J Periodont Res 2012; 47: 33–44. © 2011 John Wiley & Sons A/S Background and Objective: The periodontal regeneration of bone defects is often unsatisfactory and could be largely improved by cell therapy. Therefore, the purpose of this study was to evaluate the regenerative potential of implanting canine cementum‐derived cells (CDCs) and canine periodontal ligament‐derived cells (PDLDCs) in experimentally created periodontal intrabony defects in beagle dogs. Material and Methods: Cells were obtained from premolars extracted from four beagle dogs. Three‐wall intrabony periodontal defects, 3 mm wide and 4 mm deep, were surgically created in their second and fourth premolars and plaque was allowed to accumulate. Once the defects were surgically debrided, periodontal regeneration was attempted by random implantation of collagen sponges embedded with 750,000 CDCs, 750,000 PDLDCs or culture medium. After 3 mo of healing, specimens were obtained and periodontal regenerative outcomes were assessed histologically and histometrically. Results: The histological analysis showed that a minimal amount of new cementum was formed in the control group (1.56 ± 0.39 mm), whereas in both test groups, significantly higher amounts of new cementum were formed (3.98 ± 0.59 mm in the CDC group and 4.07 ± 0.97 mm in the PDLDC group). The test groups also demonstrated a larger dimension of new connective tissue, resulting in a significantly more coronal level of histological attachment. Conclusion: This proof‐of‐principle study suggests that cellular therapy, in combination with a collagen sponge, promoted periodontal regeneration in experimental intrabony periodontal defects.     

Stem cells and periodontal regeneration

Periodontitis is an inflammatory disease which manifests clinically as loss of supporting periodontal tissues including periodontal ligament and alveolar bone. For decades periodontists have sought ways to repair the damage which occurs during periodontitis. This has included the use of a range of surgical procedures, the use of a variety of grafting materials and growth factors, and the use of barrier membranes. To date periodontal regeneration is considered to be biologically possible but clinically unpredictable. Recently, reports have begun to emerge demonstrating that populations of adult stem cells reside in the periodontal ligament of humans and other animals. This opens the way for new cell-based therapies for periodontal regeneration. For this to become a reality a thorough understanding of adult human stem cells is needed. This review provides an overview of adult human stem cells and their potential use in periodontal regeneration.