Identification of multipotent stem cells from adult dog periodontal ligament

Periodontal diseases, which are characterized by destruction of the connective tissues responsible for restraining the teeth within the jaw, are the main cause of tooth loss. Periodontal regeneration mediated by human periodontal ligament stem cells (hPDLSCs) may offer an alternative strategy for the treatment of periodontal disease. Dogs are a widely used large-animal model for the study of periodontal-disease progression, tissue regeneration, and dental implants, but little attention has been paid to the identification of the cells involved in this species. This study aimed to characterize stem cells isolated from canine periodontal ligament (cPDLSCs). The cPDLSCs, like hPDLSCs, showed clonogenic capability and expressed the mesenchymal stem cell markers STRO-1, CD146, and CD105, but not CD34. After induction of osteogenesis, cPDLSCs showed calcium accumulation in vitro. Moreover, cPDLSCs also showed both adipogenic and chondrogenic potential. Compared with cell-free controls, more cementum/periodontal ligament-like structures were observed in CB-17/SCID mice into which cPDLSCs had been transplanted. These results suggest that cPDLSCs are clonogenic, highly proliferative, and have multidifferentiation potential, and that they could be used as a new cellular therapeutic approach to facilitate successful and more predictable regeneration of periodontal tissue using a canine model of periodontal disease.     

聚己内酯静电纺丝支架与人牙周膜干细胞的生物相容性

目的：观察荧光标记的人牙周膜干细胞在聚己内酯静电纺丝支架上的生长情况,评价聚己内酯静电纺丝支架与人牙周膜干细胞的生物相容性.方法：采用有限稀释法,体外分离培养人牙周膜干细胞.用荧光染料对牙周膜干细胞进行标记,检测细胞增殖情况,评价荧光标记对细胞生长特性的影响.制备聚己内酯静电纺丝支架,与牙周膜干细胞直接接触共培养7d,激光共聚焦显微镜观察细胞在支架材料上的黏附和增殖情况,扫描电镜观察细胞在支架材料上的生长状态.结果：成功分离培养人牙周膜干细胞;荧光染料标记后细胞发红色荧光,标记对细胞形态和生长特性无显著影响.激光共聚焦显微镜观察,可见牙周膜干细胞能够在聚己内酯静电纺丝支架上黏附增殖,局部细胞生长融合.扫描电镜观察,可见牙周膜干细胞在聚己内酯静电纺丝支架上黏附牢固,可进入支架内部复层生长.结论：荧光标记的牙周膜干细胞在聚己内酯静电纺丝支架上生长良好,聚己内酯静电纺丝支架与牙周膜干细胞具有良好的生物相容性,有望作为牙周组织工程的载体材料.     

牙周膜干细胞的研究进展

牙周组织工程是牙周病治疗研究的热点,牙周膜干细胞是其关键种子细胞之一。本文就牙周组织工程的相关研究和牙周膜干细胞的来源、分离与培养、细胞表型、生物学特性、功能影响因素和分子调控进行综述,并对其面临的挑战和前景作一讨论。     

改良法分离培养人牙周膜干细胞

目的 改良法体外分离培养人牙周膜干细胞(PDLSC),并进行鉴定.方法采用酶消化组织块法获得人牙周膜细胞,通过有限稀释法克隆化培养、分离得到PDLSC,用含10%FBS的α-MEM培养液培养并传代:测定克隆形成率:免疫组织化学检测角蛋白及波形蛋白表达:流式细胞术分析细胞周期及表面标志物STRO-1、CD146的表达;并...     

Location of putative stem cells in human periodontal ligament

BACKGROUND AND OBJECTIVE: The origin of cells in the mature periodontium, and the location of their progenitors, are still unknown. It is also unknown whether inflammation influences the number and distribution of these cells within the periodontium. Molecules such as STRO-1, CD146 and CD44 have been identified on a variety of mesenchymal stem cells. The aim of this study was to identify and localize putative stem cells in diseased and healthy human periodontal ligament using cell-surface markers for mesenchymal stem cells. MATERIAL AND METHODS: Healthy and periodontitis-affected teeth were collected, fixed in 10% neutral-buffered formalin, decalcified and embedded in paraffin in preparation for immunohistochemistry. Antibodies against STRO-1, CD146 and CD44 were used to identify putative stem cells in the periodontal ligament. RESULTS: Putative stem cells were identified in both healthy and diseased periodontal ligament. They were mainly located in the paravascular region and small clusters of cells were also found in the extravascular region. Wider distributions of these cells were detected in sections of diseased ligament. CONCLUSION: Within the periodontal ligament of both healthy and diseased teeth, cells have been identified consistent with their identification as putative stem cells. The presence of an inflammatory reaction associated with periodontitis may enhance the number of these cells.     

人牙周膜干细胞的分离培养及初步鉴定

目的 体处分离培养人类牙周膜干细胞并对其生物学性状进行初步鉴定.方法 采用有限稀释法进行牙周膜干细胞克隆筛选,获得单细胞克隆来源细胞,检测其克隆形成率,并采用免疫组织化学染色、碱性磷酸酶染色、免疫荧光染色等方法鉴定牙周膜干细胞的组织来源及生物学特性.结果 有限稀释法获得的牙周膜干细胞克隆株呈簇状生长,排列紧密,细胞呈圆形或椭圆形,细胞较小,排列形成紧密的团块状.免疫组化染色显示细胞波形蛋白阳性,角蛋白阴性;碱性磷酸酶染色阳性;早期间充质干细胞的标志物STRO-1荧光染色显示细胞发出明亮的红色荧光,表达阳性.结论 牙周膜中存在具有高增殖能力的干细胞,克隆化分离培养的人牙周膜干细胞具有强克隆形成能力,具有间充质干细胞表型和生物学特性.     

牙周膜干细胞膜片的构建及其生物学特性的研究

目的:探讨牙周膜干细胞膜片的构建及其生物学特性.方法:采用胶原酶消化人牙周膜组织,获得牙周膜干细胞(Periodontal ligament stem cells,PDLSCs),经体外鉴定、扩增后构建PDLSCs细胞膜片,并通过倒置显微镜、HE染色、扫描电镜(SEM)对PDLSCs细胞膜片形态学进行检测.此外,细胞膜...     

人牙周膜干细胞体外诱导分化为神经元样细胞的实验研究

目的体外定向诱导人牙周膜干细胞分化为神经元样细胞,探讨人牙周膜干细胞的多向分化潜能。方法体外分离、培养人牙周膜细胞,待细胞达一定量后用有限稀释法进行克隆化培养,筛选鉴定牙周膜干细胞（PDLSC）,用含10 μg/L碱性成纤维细胞生长因子（bFGF）的预诱导培养液诱导培养24 h,然后换用含5 mmol/L β-巯基乙醇（β-ME）的诱导培养液诱导培养6 h,光镜下观察诱导细胞形态学的改变,免疫荧光、RT-PCR等方法检测鼠抗人神经元特异性烯醇化酶（NSE）、神经丝蛋白（NF）和胶质纤维酸性蛋白（GFAP）的表达。同时以未诱导细胞为对照。结果体外诱导培养6 h细胞即发生形态学改变,可看到典型的神经元样细胞;免疫荧光、RT-PCR检测诱导细胞表达神经元细胞特异性标志NSE、NF,未诱导细胞无表达。结论人牙周膜干细胞在体外可以诱导分化为神经元样细胞,具有多向分化的潜能。     

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。     

几种胚胎干细胞标志在牙周膜细胞中的表达及意义

目的检测部分胚胎干细胞标志在牙周膜细胞中的表达。方法通过酶解组织块法分离牙周膜细胞,利用多向分化实验和流式细胞术分析表面标志对获得细胞进行鉴定。利用免疫荧光法和RT-PCR法检测牙周膜细胞中Oct4、Nanog、Sox2、SSEA-4、TRA-1-60、TRA-1-81这些胚胎干细胞标志的表达。结果牙周膜细胞为成纤维细胞样,可分化为成骨细胞和成软骨细胞,可均一表达间充质细胞标志CD44、CD90、CD105,异质性表达间充质标志CD146、Stro-1。细胞免疫荧光实验显示牙周膜细胞表达部分胚胎干细胞标志:Oct4、Nanog、SSEA-4、TRA-1-60阳性,Sox2、TRA-1-81阴性。半定量RTPCR结果显示P3和P8代牙周膜细胞表达Oct4和Nanog,表达量差异不大,Sox2则为阴性。结论牙周膜细胞除了表达常用间充质干细胞标志外,也可表达部分胚胎干细胞标志,这对牙周膜细胞的鉴定和进一步分选纯化有一定应用价值。     

牙周膜干细胞向脂肪细胞方向定向诱导分化实验

目的探讨人牙周膜干细胞（PDLSCs）向脂肪细胞定向分化潜能,分析其分化过程中形态与功能变化。方法免疫磁珠法分离人PDLSCs,用成脂诱导液连续诱导21 d,通过倒置显微镜、透射电镜、流式细胞仪、RTPCR、Western blot及免疫荧光方法观察诱导细胞形态、结构变化,分析其表面脂肪细胞特异性标志---低密度脂蛋白（LPL）和过氧化物酶体增殖物受体γ（PPAR-γ）的基因及蛋白表达时相及表达量,油红O染色检测脂滴分泌情况。结果诱导21 d,诱导细胞呈脂肪细胞样圆形细胞,超微结构观察可见胞浆中大量脂肪细胞特征性脂滴。流式分析结果显示,有96.54%的PDLSCs向脂肪细胞分化。诱导细胞表达脂肪细胞特异性表面标志LPL mRNA和PPAR-γmRNA及PPAR-γ蛋白,且随诱导时间延长PPAR-γ蛋白表达量增加。诱导细胞产生油红O阳性染色的脂滴。结论人PDLSCs在适当条件下可定向分化为脂肪细胞样细胞,并具有脂肪细胞形态、结构和功能特点,具有可塑性。     

免疫磁珠法分离纯化人牙周膜干细胞

目的：建立免疫磁珠法体外分离纯化人牙周膜干细胞的方法。方法：采用STRO-1作为磁珠分离细胞的表面标志,利用间接法免疫磁珠分离细胞,描绘其生长曲线及免疫组化染色检测抗波形丝蛋白（Vimentin）、骨粘连素（ON）、骨桥蛋白（BSP）的表达。结果：通过免疫磁珠法获得了人牙周膜STRO-1^＋细胞,观察其生长曲线表明其为慢周期性;该细胞抗波形丝蛋白（Vimentin）、骨粘连素（ON）、骨桥蛋白（BSP）弱阳性表达。结论：免疫磁珠法是有效分离纯化牙周膜干细胞的方法,该方法分离的人牙周膜STRO-1^＋细胞具有干细胞的超微结构、细胞周期及表型特点。     

人CD146＋牙周膜干细胞的分选及其干细胞特性的鉴定

目的：通过酶消化法及流式细胞分选技术分选CD146＋牙周膜干细胞（Periodontal ligament cells,PDLCs）,探讨其干细胞特性。方法：采用流式细胞技术从人牙周膜细胞中分选CD146＋PDLCs,并对其进行免疫组化染色（角蛋白、波形丝蛋白及Stro-1）,成骨、成脂诱导分化及克隆形成实验鉴定其干细胞特性。结果：CD146＋PDLCs表达波形丝蛋白及Stro-1,具有成骨及成脂分化能力,体外培养能形成细胞克隆。结论：以CD146为分选指标的流式细胞分选技术可作为牙周膜干细胞筛选的方法。     

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。     

Periodontal ligament stem cells: an update and perspectives

Chronic periodontitis is a serious infectious and inflammatory oral disease of humans worldwide. Conventional treatment modalities are effective for controlling periodontal disease. However, the regeneration of damaged periodontal tissues remains a major challenge in clinical practice due to the complex structure of the periodontium. Stem cell‐based regenerative approaches combined with the usage of emerging biomaterials are entering a new era in periodontal regeneration. The present review updates the current knowledge of periodontal ligament stem cell‐based approaches for periodontal regeneration, and elaborates on the potentials for clinical application.