牙髓、牙周膜及脐带间充质干细胞三系分化能力的体外比较研究

目的:比较牙髓、牙周膜和脐带三种不同来源间充质干细胞的(Mesenchymal stem cells,MSCs)三系分化潜能.方法:培养分离人脐带间充质干细胞(Umbilical cord mesenchymal stem cells,UCMSCs)、牙髓干细胞(Dental pulp stem cells,DPSCs)和牙周膜干细胞(Periodontal ligament stem cells,PDLSCs);倒置显微镜观察细胞形态,体外诱导这3种MSCs向成骨、成脂和成软骨方向分化,通过茜素红染色、油红O染色、阿尔辛蓝染色分别鉴定细胞成骨、成脂和成软骨分化能力,进一步实时荧光定量PCR检测比较3种细胞成骨相关基因OCN和RUNX2及成脂相关基因PPAR-γ的表达水平.结果:这3种MSCs细胞形态略有不同,三者均可向成骨、成脂和成软骨方向分化,但其分化潜能有所差异,DPSCs成骨和成脂向分化能力强,UCMSCs则更适于成脂和成软骨向分化,而PDLSCs成软骨向分化较具优势.结论:DPSCs,PDLSCs和UCMSCs三系分化潜能不同,本研究为干细胞临床转化中选择理想细胞来源提供实验依据.     

人牙周膜干细胞的分离培养及体外诱导分化研究

目的:从成体人牙周组织中分离培养牙周膜干细胞,研究其生物学特性并进行诱导分化,为牙周组织工程提供可靠的种子细胞来源.方法:选取12～20岁的年轻患者因正畸拔除的健康牙齿,采用酶消化组织块培养法得到牙周膜细胞,待细胞达一定量后用有限元稀释法进行单细胞克隆,筛选牙周膜干细胞( PDLSCs).计算细胞克隆形成率(CFU- ...     

人牙周膜干细胞的初步鉴定及体外成骨诱导

目的：体外培养、鉴定人牙周膜干细胞（periodontal ligament stem cells,PDLSCs）并定向诱导分化为成骨细胞,探讨人PDLSCs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）,矿化诱导培养21d后检测钙结节形成情况、ALP活性,免疫细胞化学检测骨涎蛋白（BSP）、Ⅰ型胶原表达,RT—PCR检测ALP、BSP mRNA表达。结果：人PDLSCs体外诱导培养21d后可见钙结节形成,成骨细胞相关蛋白及mRNA均阳性表达。结论：人PDLSCs在体外诱导条件下具有成骨潜能。     

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。     

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...     

牙周膜干细胞成脂分化的研究进展

牙周膜干细胞( periodontal ligament stem cells,PDLSCs)被认为是牙周组织工程理想的种子细胞,具有多向分化潜能。间充质干细胞在成脂和成骨分化过程中具有相互制约的平衡关系,成脂分化的诱导因素通常是成骨分化的抑制因素,反之亦然。本文就近年来PDLSCs成脂分化的诱导、鉴定方法及相关转录因子的研究进展进行综述,分析PDLSCs成脂分化的机制,探讨通过抑制PDLSCs成脂分化促进其成骨分化的可行性,为治疗牙槽骨吸收等骨骼性疾病提供参考。     

成人牙周韧带干细胞分离培养及其定向分化的实验研究

干细胞(stem cell)是指具有高度增殖和自我更新能力,及多向分化潜能的细胞。在机体发育过程中,可存在处于不同分化等级的干细胞。我们对牙周韧带干细胞进行分离、培养,并进行脂肪化及矿化诱导,为进一步研究提供实验依据。1.资料和方法:选取年轻患者因正畸拔除的完好牙齿,刮取根中1/3牙周膜,采用组织块培养法得到牙周韧带细胞,待细胞达一定量后用有限稀释法进行单细胞克隆,筛选牙周韧带干细胞(periodontal ligament stem cells,PDLSCs),检测第三代PDLSCs波形蛋白和角蛋白表达,体外进行脂肪化及矿化诱导后对各克隆从碱性磷酸酶活性、矿化…     

2型糖尿病伴牙周炎患者牙周膜干细胞骨向分化研究

目的:比较牙周炎和2型糖尿病伴牙周炎患者牙周膜干细胞成骨分化能力。方法:体外组织块法和有限稀释法克隆化培养牙周炎患者牙周膜干细胞(P-PDLSCs组)和2型糖尿病伴牙周炎患者牙周膜干细胞(D-PDLSCs组),计算克隆形成率,免疫荧光检测细胞表型分子CD146、STRO-1进行干细胞鉴定,矿化诱导后茜素红染色观察矿化结节形成,实时定量聚合酶链反应(real time PCR)检测成骨相关基因表达。结果:P-PDLSCs组和D-PDLSCs组细胞的克隆形成率分别为(25.6±2.7)%和(17.9±1.7)%(P﹤0.05);P-PDLSCs组CD146和STRO-1表达明显高于D-PDLSCs组;成骨诱导21 d后茜素红染色,2组均出现不同程度的矿化结节,P-PDLSCs组的矿化能力较D-PDLSCs组强;成骨诱导1周后D-PDLSCs组碱性磷酸酶(alkaline phosphatase,ALP)、Runx-2和Ⅰ型胶原蛋白(type-Ⅰcollagen,Col-Ⅰ)的mRNA表达均明显低于P-PDLSCs组(P<0.05)。结论:糖尿病伴牙周炎患者牙周膜干细胞成骨分化能力低于单纯牙周炎患者牙周膜干细胞。     

人牙周膜干细胞的分离鉴定及骨形态发生蛋白-2对其趋化效应的研究

目的研究人牙周膜干细胞（PDLSCs）对骨形态发生蛋白-2（BMP-2）的趋化反应。方法通过有限稀释法分离、培养人PDLSCs,利用免疫荧光染色检测人PDLSCs波形丝蛋白及干细胞表面标志物STRO-1的表达,检测人PDLSCs多向分化能力,通过克隆形成实验和5-溴-2-脱氧尿嘧啶核苷（BrdU）共培养的方法检测其干细胞特性。利用24孔的Transwell细胞培养室来检测人PDLSCs对BMP-2的趋化反应,光镜下计迁移至滤膜下侧面的不同视野的细胞数。结果人PDLSCs抗波形丝蛋白染色阳性,表达干细胞表面标志物STRO-1,体外诱导培养的人PDLSCs能够向成骨细胞和成脂细胞分化,具有较高的自我更新能力,并在体外呈克隆状生长。在100、200 ng·mL-1 BMP-2实验组,Transwell细胞培养室中迁移的细胞数目显著多于空白对照组（P<0.01）。结论BMP-2对人PDLSCs有趋化效应。     

炎症微环境下芦丁对牙周膜干细胞成骨分化能力的影响

目的 研究芦丁（rutin）对炎症微环境下牙周膜干细胞成骨分化能力的影响。方法 采用有限稀释法分离纯化获得牙周膜干细胞,采用流式细胞术鉴定牙周膜干细胞。以脂多糖（lipopolysaccharide, LPS）刺激牙周膜干细胞,建立体外炎症模型。实验分为4组,第1组使用α-MEM培养基培养牙周膜干细胞,第2组使用含有脂多糖的α-MEM培养基培养牙周膜干细胞,第3组在含有脂多糖的α-MEM培养基加入芦丁培养牙周膜干细胞,第4组使用含有芦丁的α-MEM培养基培养牙周膜干细胞。通过碱性磷酸酶染色、碱性磷酸酶活性测试、茜素红染色、RT-PCR以及蛋白免疫印迹等方法检测成骨分化能力的改变。采用SPSS 17.0软件包对数据进行统计分析。结果 CCK-8和碱性磷酸酶活性测试结果显示,10 μmol/L芦丁对炎症状态下牙周干细胞增殖和成骨分化作用最明显。碱性磷酸酶染色和茜素红染色结果显示,10 μmol/L芦丁可以改善炎症微环境下牙周膜干细胞的成骨分化能力。RT-PCR、蛋白免疫印迹结果显示,芦丁可以增强炎症状态下COL1、ALP、RUNX2等成骨基因和成骨蛋白的表达。结论 芦丁可以增强炎症微环境下牙周膜干细胞的成骨分化能力。     

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.     

化脓链球菌脂磷壁酸对人牙周膜干细胞分化能力的影响

目的 研究脂磷壁酸(LTA)对牙周膜干细胞(PDLSCs) 分化能力的影响,从而探讨G+细菌毒素LTA对人牙周膜干细胞再生牙周组织的影响.方法 用0.1、1和10μg/ml的LTA作用于PDLSCs,采用MTT方法及流式细胞术检测细胞的增殖及凋亡情况,使用碱性磷酸酶染色和茜素红染色对细胞的成骨分化能力进行比较;应用油红O染色鉴定成脂分化能力,实时定量PCR检测成骨成脂相关基因的变化,研究LTA在PDLSCs分化中的作用.结果 1μg/ml及10μg/ml LTA显著降低PDLSCs增殖率,细胞凋亡增加.ALP染色及活性结果表明,早期成骨能力各组之间没有显著差异;茜素红染色结果及钙离子浓度定量测定显示,随LTA浓度升高,PDLSCs晚期成骨能力降低,并具有LTA浓度依赖性.成骨相关基因Runx2表达表现相同变化趋势.油红O染色结果表明,随着LTA浓度的增加,PDLSCs的成脂分化能力升高,成脂相关基因PPARγ表达升高,具有LTA浓度依赖性.结论 LTA降低PDLSCs成骨分化能力,同时促进细胞成脂分化能力,表明LTA抑制PDLSCs介导的牙周组织再生.     

A Synthetic Oligopeptide Derived From Enamel Matrix Derivative Promotes the Differentiation of Human Periodontal Ligament Stem Cells Into Osteoblast‐Like Cells With Increased Mineralization

Background: In a previous study, the authors obtained a synthetic peptide (SP) for useful periodontal tissue regeneration. Periodontal ligament stem cells (PDLSCs) have multiple potentiality to contribute to tissue regeneration. The aim of this experiment is to investigate the effect of SP on human PDLSCs. Methods: Periodontal ligament cells were obtained from healthy adult human third molars and used to isolate single PDLSC‐derived colonies. The mesenchymal stem cell nature of the PDLSCs was confirmed by immunohistochemical evaluation of STRO‐1 expression. Proliferation and osteoblastic differentiation were investigated by culturing PDLSCs in normal or osteogenic medium with and without SP (100 ng/mL). Osteoblast differentiation was assessed by measuring alkaline phosphatase (ALP) activity, osteocalcin production, mRNA expression of osteonectin, mineralization, and calcium deposition. Results: Isolated PDLSCs were immunohistochemically positive for vimentin and STRO‐1 and negative for cytokeratin. A greater number of calcified nodules were observed in osteogenic medium culture with SP than without. In the early and later stages of PDLSC culture with SP, osteonectin production and osteocalcin production were increased. SP in culture with osteogenic medium significantly enhanced proliferation of PDLSCs, as well as ALP activity, expression of osteonectin, osteocalcin production, formation of calcified nodules, and mineralization. Conclusions: SP enhances the formation of calcified nodules and osteocalcin production in the culture of PDLSCs into osteoblast‐like cells and is a useful material for periodontal tissue regeneration.     

High‐power,red‐light‐emitting diode irradiation enhances proliferation,osteogenic differentiation,and mineralization of human periodontal ligament stem cells via ERK signaling pathway

1 Background

Light‐emitting diode (LED) is attracting attention as a new light source for phototherapy. However, its effects on periodontal tissue regeneration remain unknown. The aim of this study was to examine the effects of high‐power, red LED irradiation on human periodontal ligament stem cells (PDLSCs), which play an important role in periodontal tissue regeneration.

2 Methods

PDLSCs were derived from adult human third molars. The light source was red LED (peak wavelength: 650 nm). Energy densities ranging from 0 to 10 J/cm² were tested to determine the optimal dose. PDLSC proliferation was measured using two parameters: live cell protease and ATP levels. After the cells were induced to differentiate, the effect of LED irradiation on osteogenic differentiation and mineralization was examined, with particular focus on the extracellular signal‐regulated kinase (ERK)1/2 signaling pathway using an ERK inhibitor (PD98059).

3 Results

LED irradiation at 8 J/cm² led to a significant increase in PDLSC proliferation and enhanced Runx2 and Osterix mRNA expression, Alkaline phosphatase activity, procollagen type I C‐peptide and osteocalcin production, calcium deposition, and alizarin red S staining. In addition, LED induced the activation of ERK1/2, and the effects of LED on PDLSC proliferation, differentiation, and mineralization could be suppressed by treatment with PD98059.

4 Conclusions

The results of this study show that 650‐nm high‐power, red, LED irradiation increases PDLSCs proliferation, and osteogenic differentiation and mineralization, mediated by ERK1/2 activation. These findings suggest that LED may be a useful tool for periodontal tissue regeneration.     

Cell Responses to Conditioned Media Produced by Patient‐Matched Stem Cells Derived From Healthy and Inflamed Periodontal Ligament Tissues

Background: Periodontal ligament stem cells (PDLSCs) derived from clinically compromised teeth with periodontitis are considered a readily accessible cell source, but their impaired stem cell functionalities, as observed in various in vitro and in vivo models, necessitate further investigation of these inflamed cells before their translation into therapeutic applications. In this study, the effects of conditioned media (CM) produced by stem cells derived from human healthy periodontal ligament tissues (H‐PDLSCs) or inflamed periodontal ligament tissues (I‐PDLSCs), referred to as H‐CM and I‐CM, respectively, on the biologic properties of H‐PDLSCs and I‐PDLSCs from the same donor are compared to explore the extent to which inflamed cells can be rescued by their extrinsic environment (i.e., by H‐CM). Methods: H‐CM and I‐CM were prepared from in vitro cell cultures, and the cellular responses of H‐PDLSCs and I‐PDLSCs to patient‐matched H‐CM and I‐CM were investigated in terms of colony‐forming ability, cell proliferation, and adipogenic/osteogenic differentiation. Results: In H‐CM and I‐CM, H‐PDLSCs and I‐PDLSCs exhibited similar adipogenic potential. However, when incubated in I‐CM, both cell types demonstrated an increased capacity to proliferate but a decreased capacity to differentiate into osteoblasts. Significantly, the impaired osteogenic differentiation of I‐PDLSCs was partially rescued by incubation in H‐CM under osteo‐inducing conditions. Conclusion: The CM of patient‐matched H‐PDLSCs and I‐PDLSCs differed, and the impaired osteogenic differentiation of inflamed stem cells had the potential to be rescued, at least partially, for therapeutic use via changing the cell culture microenvironment in vitro.     

糖基化终末产物对人牙周膜干细胞骨向分化能力影响的研究

目的:探讨糖基化终末产物(AGEs-HSA)对人牙周膜干细胞(HPDLSC)骨向分化能力的影响。方法:体外组织块法和有限稀释法克隆化培养牙周膜干细胞,流式细胞仪检测牙周膜细胞表型分子CD44、CD146、stor-1、CD90,对其进行干细胞鉴定,将培养出的牙周膜干细胞与不同浓度的AGE-HSA共同培养,并矿化诱导后茜素红染色观察钙结节形成情况、碱性磷酸酶染色观察ALP活性、实时定量聚合酶链反应(real timePCR)检测成骨基因表达情况。结果:流式细胞仪细胞表型分析CD44、CD146、stor-1、CD90表达呈阳性。成骨诱导21 d后茜素红染色,对照组和实验组均出现不同程度的矿化结节,定量分析显示1、10、100、200μg/mLAGEs组矿化能力均比对照组低,差异均有统计学意义(P<0.05);不同浓度的实验组之间矿化能力均有统计学差异(P<0.05),且随着AGEs浓度的升高,矿化能力逐渐降低。成骨诱导7 d ALP染色比较发现各实验组均比对照组减弱。成骨诱导1周后RT-PCR检测各实验组的成骨基因ALP、Runx-2、Col-1、Runx-2 mRNA表达水平均较对照组低,差异有统计学意义(P<0.05);不同浓度的实验组之间各成骨基因的表达也有统计学差异(P<0.05)。结论:AGEs能抑制HPDLSC的骨向分化,并在一定范围内呈浓度依赖性。