Transplantation of stem cells from human exfoliated deciduous teeth for bone regeneration in the dog mandibular defect

AIM: To investigate the effect of stem cells from human exfoliated deciduous teeth(SHED) transplanted for bone regeneration in the dog mandibular defect.METHODS: In this prospective comparative study, SHEDs had been isolated 5 years ago from human exfoliated deciduous teeth. The undifferentiated stem cells were seeded into mandibular bone through-andthrough defects of 4 dogs. Similar defects in control group were filled with cell-free collagen scaffold. After 12 wk, biopsies were taken and morphometric analysis was performed. The percentage of new bone formation and foreign body reaction were measured in each case. The data were subject to statistical analysis using the Mann-Whitney U and Kruskalwalis statistical tests. Differences at P < 0.05 was considered as significant level.RESULTS: There were no significant differences between control and SHED-seeded groups in connective tissue(P = 0.248), woven bone(P = 0.248) and compact bone(P = 0.082). There were not any side effects in transplanted SHED group such as teratoma or malignancy and abnormalities in this period.CONCLUSION: SHEDs which had been isolated and characterized 5 years ago and stored with cryopreservation banking were capable of proliferation and osteogenesis after 5 years, and no immune response was observed after three months of seeded SHEDs.

Stem cells from human exfoliated deciduous teeth ameliorate concanavalin A-induced autoimmune hepatitis by protecting hepatocytes from apoptosis

BACKGROUND Autoimmune hepatitis is a serious autoimmune liver disease that threatens human health worldwide,which emphasizes the urgent need to identify novel treatments.Stem cells from human exfoliated deciduous teeth(SHED),which are easy to obtain in a non-invasive manner,show pronounced proliferative and immunomodulatory capacities.AIM To investigate the protective effects of SHED on concanavalin A(ConA)-induced hepatitis in mice,and to elucidate the associated regulatory mechanisms.METHODS We used a ConA-induced acute hepatitis mouse model and an in vitro co-culture system to study the protective effects of SHED on ConA-induced autoimmune hepatitis,as well as the associated underlying mechanisms.RESULTS SHED infusion could prevent aberrant histopathological liver architecture caused by ConA-induced infiltration of CD3+,CD4+,tumor necrosis-alpha+,and interferon-gamma+inflammatory cells.Alanine aminotransferase and aspartate aminotransferase were significantly elevated in hepatitis mice.SHED infusion could therefore block ConA-induced alanine aminotransferase and aspartate aminotransferase elevations.Mechanistically,ConA upregulated tumor necrosisalpha and interferon-gamma expression,which was activated by the nuclear factor-kappa B pathway to induce hepatocyte apoptosis,resulting in acute liver injury.SHED administration protected hepatocytes from ConA-induced apoptosis.CONCLUSION SHED alleviates ConA-induced acute liver injury via inhibition of hepatocyte apoptosis mediated by the nuclear factor-kappa B pathway.Our findings could provide a potential treatment strategy for hepatitis.

Application of dental stem cells in three-dimensional tissue regeneration

Dental stem cells can differentiate into different types of cells.Dental pulp stem cells,stem cells from human exfoliated deciduous teeth,periodontal ligament stem cells,stem cells from apical papilla,and dental follicle progenitor cells are five different types of dental stem cells that have been identified during different stages of tooth development.The availability of dental stem cells from discarded or removed teeth makes them promising candidates for tissue engineering.In recent years,three-dimensional(3D)tissue scaffolds have been used to reconstruct and restore different anatomical defects.With rapid advances in 3D tissue engineering,dental stem cells have been used in the regeneration of 3D engineered tissue.This review presents an overview of different types of dental stem cells used in 3D tissue regeneration,which are currently the most common type of stem cells used to treat human tissue conditions.

Dental pulp cell bank as a possible future source of individual hepatocytes

Mesenchymal stem cells(MSCs) as a source for regenerative medicine are now the subject of much clinical attention. There are high expectations due to their safety, low tumorigenic risk, and low ethical concerns. MSC therapy has been approved for acute graft-versus host diseases since 2015. Tooth-derived MSCs are known to have a great potential in their proliferation and differentiation capacities, even when compared with bone-marrow-derived MSCs. In particular, stem cells from human exfoliated deciduous teeth(SHEDs) are the best candidates for personal cell banking(dental pulp cell bank), because they can be obtained less invasively in the natural process of individual growth. SHEDs are known to differentiate into hepatocytes. There have been several studies showing the effectiveness of SHEDs on the treatment of liver failure in animal models. They may exert their effects either by repopulation of cells in injured liver or by paracrine mechanisms due to their immuneregulatory functions. Moreover, it may be possible to use each individuals' dental pulp cells as a future source of tailor-made differentiated hepatocytes in the context of a bioartificial liver or liver-on-a-chip to screen for drug toxicity.

Effects of low intensity laser irradiation phototherapy on dental pulp constructs

AIM: To investigate low intensity laser irradiation phototherapy(LILIP) on the proliferation, mineralization and degradation of dental pulp constructs.METHODS: Stem cells from human exfoliated deciduous teeth(SHED) were grown to confluence and seeded on collagen scaffolds to create dental pulp constructs. LILIP was delivered to the dental pulp constructs using an 830 nm GaA IAs laser at an output power of 20 m W. The LILIP energy density was 0.4, 0.8, 1.2, and 2.4 J/cm2. After 8 d, the cell proliferation and degradation within the dental pulp constructs were measured using histologic criteria. After 28 d, the effect of LILIP on SHED mineralization was assessed by von Kossa staining.RESULTS: SHED proliferation within the dental pulp constructs varied after exposure to the 0.4, 0.8, 1.2,and 2.4 J/cm2 LILIP energy densities(P < 0.05). The maximum proliferation of SHED in nutrient deficient media was 218% after exposure to a 1.2 J/cm2 LILIP energy density. SHED grown in nutrient deficient media after exposure to a 0.4, 0.8, and 1.2 J/cm2 LILIP energy density, proliferated by 167-218% compared to the untreated(non-LILIP) control group(P < 0.05).SHED exposed to a 0.4, 0.8, and 1.2 J/cm2 LILIP energy density, and grown in optimal nutritional conditions and proliferated by 147%-164% compared to the untreated(non-LILIP) control group(P < 0.05). The exposure of SHED to the highest LILIP energy density(2.4 J/cm2) caused a reduction of the cell proliferation of up to 73% of the untreated(non-LILIP) control(P < 0.05). The amount of mineral produced by SHED increased over time up to 28 d(P < 0.05). The 0.8 and 1.2J/cm2 LILIP energy densities were the most effective at stimulating the increased the mineralization of the SHED from 150%-700% compared to untreated(nonLILIP) control over 28 d(P < 0.05). The degradation of dental pulp constructs was affected by LILIP(P <0.05). The dental pulp constructs grown in optimal nutritional conditions exposed to a 0.8 J/cm2 or 1.2 J/cm2 LILIP energy density had 13% to 16% more degradation than the untreated(non-LILIP) control groups(P < 0.05). The other LILIP energy densities caused a 1%degradation of dental pulp constructs in optimal nutritional conditions(P > 0.05).CONCLUSION: LILIP can enhance or reduce SHED proliferation, degradation and mineralization within dental pulp constructs. LILIP could promote the healing and regeneration of dental tissues.

牙源性干细胞治疗帕金森病的研究进展

帕金森病(PD)是第二常见的神经退行性疾病,主要是由于中脑黑质(SN)多巴胺能神经元的丧失而导致运动功能障碍。牙源性干细胞(DSCs)来自颅神经嵴,易于采集,具有很强的增殖和分化能力,能够促进神经修复和再生。DSCs可以分化成新的多巴胺神经元并分泌大量神经营养因子(NTFs)改善神经功能,同时还可以通过免疫调节抑制神经炎性反应,DSCs可以有效减轻PD大鼠的运动功能障碍,并在再生医学领域中扮演着重要的角色。

miR-26b对人脱落乳牙牙髓干细胞及人脐带间充质干细胞增殖、迁移和成骨分化的影响

背景:microRNA-26b(miR-26b)对干细胞的多种功能具有重要的调节作用,但其对人脱落乳牙牙髓干细胞和人脐带间充质干细胞生物学特性的影响尚不清楚。目的:探讨miR-26b对人脱落乳牙牙髓干细胞和人脐带间充质干细胞增殖、迁移和成骨分化的影响。方法:培养及鉴定人脱落乳牙牙髓干细胞和人脐带间充质干细胞;用miR-26 mimics(实验组)和miRNAs mimics对照(对照组)转染2种细胞,构建过表达模型用于后续实验。CCK-8法检测过表达miR-26b细胞的增殖能力;Transwell和划痕实验分析过表达miR-26b细胞的迁移能力;RT-qPCR法检测过表达miR-26b细胞成骨诱导后成骨标志物的表达。结果与结论:①转染miR-26b模拟物可提高2种细胞中miR-26b表达量,促进人脱落乳牙牙髓干细胞增殖,对人脐带间充质干细胞的扩增无明显影响;②相较于对照组,2种细胞过表达miR-26b后迁移能力增强;③miR-26b在2种细胞成骨分化过程中表达水平降低;④相较于对照组,2种细胞过表达miR-26b后,成骨相关基因骨钙素、骨桥素、碱性磷酸酶、Ⅰ型胶原蛋白mRNA水平均下调。结果表明,过表达miR-26b能促进人脱落乳牙牙髓干细胞增殖、迁移,抑制其成骨分化;也促进人脐带间充质干细胞迁移,抑制其成骨分化,但对其增殖无明显影响。

高糖对乳牙牙髓干细胞生长及成骨分化能力的影响

目的探讨高糖对乳牙牙髓干细胞(SHED)生长及成骨分化能力的影响。方法从河南大学赛思口腔医院口腔颌面外科收集拔除的6~8岁无牙体牙髓牙周疾病的健康儿童的滞留乳牙,体外培养获得SHED,实验分为5.5 mmol·L^(-1)低糖组及25 mmol·L^(-1)高糖组。用细胞计数CCK-8试剂盒(CCK-8)检测两组细胞的增殖速度。分别用含两种不同糖浓度的成骨矿化液诱导21 d后,行茜素红染色实验,实时荧光定量PCR(QPCR)技术检测骨向分化相关基因碱性磷酸酶(ALP)、Runt相关转录因子2(RUNX2)和骨钙素(OCN)、骨桥蛋白(OPN)表达的差异。结果CCK-8检测结果显示,培养2~6 d时高糖组的OD值小于低糖组(P<0.05);成骨诱导后,高糖组的钙化结节数量少于低糖组,且高糖组的RUNX-2、ALP、OCN和OPN的相对表达量低于低糖组(P<0.05)。结论高糖抑制SHED的生长及成骨分化能力。

TNF-α通过ERK1/2-Runx2信号通路调控SHED成骨分化能力的实验研究

目的:探讨肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)对人脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth,SHED)骨分化能力的影响,分析ERK1/2-Runx2信号通路在该调控过程中的变化。方法:从6~8岁健康儿童正常乳恒牙替换即将脱落的乳切牙中分离和培养SHED,取第三代细胞,分为对照组(成骨诱导剂培养)、观察组(成骨诱导剂和TNF-α共培养)和激动剂组(成骨诱导剂、TNF-α和ERK通路激动剂共培养)。采用茜素红染色评价成骨分化功能,采用Western印迹检测SHED细胞中Osterix、OPN、ERK1/2、pERK1/2和Runx2的蛋白表达水平,应用qRT-PCR检测Osterix、OPN、ERK1/2、pERK1/2和Runx2 mRNA的表达。采用SPSS 26.0软件包对数据进行统计学分析。结果:3组细胞成骨分化能力比较结果显示,3组细胞中均可见红棕色矿化结节。3组组间相比,对照组矿化结节最多,激动剂组次之,观察组最少。与对照组相比,观察组和激动剂组的Osterix、OPN蛋白和mRNA表达水平显著下降,而激动剂组Osterix、OPN蛋白和mRNA表达水平显著高于观察组;3组细胞的ERK1/2蛋白和mRNA表达水平无显著差异,而观察组和激动剂组pERK1/2和Runx2的蛋白和mRNA表达水平显著高于对照组,激动剂组的蛋白及mRNA表达水平显著高于观察组。结论:TNF-α对SHED成骨分化具有抑制作用,该作用可能与抑制ERK1/2-Runx2信号通路有关。

Comparison of Osteogenesis Between Two Kinds of Stem Cells from Goat Combined Calcium Phosphate Cement in Tissue Engineering

To explore the possible mechanism of osteogenesis for deciduous teeth stem cells (DTSCs) in vivo/ vitro, stem cells from goat deciduous teeth (SGDs) were firstly isolated, induced and transplanted into immunocompromised mice. The SGDs's mineralization pattern and osteogenesis were compared with bone marrow messenchymal stem cells (BMMSCs) from goats. SGDs have similar osteogenic differentiation pattern in vitro and bone-like tissue formation mechanism in vivo to BMMSCs; moreover SGDs have stronger alkaline phosphatase (ALP) gene expression and osteopontin (OPN) gene expression levels than BMMSCs; also SGDs can form more bone-like tissues than BMMSCs when cell-scaffold compounds are transplanted into immunocompromised mice. This pre-clinical study in a large-animal model confirms that DTSCs may be an appropriate source of stem cells in repairing bone defects with tissue engineering.

乳牙牙髓干细胞分化前后的外泌体miRNA筛选研究

目的 分析乳牙牙髓干细胞分化前后的外泌体中miRNA差异表达谱。方法 原代提取乳牙牙髓干细胞,在矿化诱导培养基中进行分化。超速离心法提纯外泌体并进行鉴定;利用miRNA基因芯片检测分化前后的外泌体中miRNA的表达谱,验证差异表达的miRNA,预测其靶基因,进行GO分析和KEGG通路的功能分析。结果 在未分化和分化的乳牙牙髓干细胞的外泌体中共发现215个差异表达的miRNA,最相关的KEGG通路为FoxO、Ras和MAPK等信号通路。GO分析包括多细胞生物的发育和结构形态发生的调控。结论 乳牙牙髓干细胞分化前后的外泌体miRNA可能会参加调控成牙本质向分化过程,为牙髓修复提供了理论依据。

骨碎补总黄酮对乳牙牙髓干细胞增殖及成骨向分化的影响

目的初步探索不同浓度骨碎补总黄酮(total flavone of rhizoma drynariae)对乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth)增殖及成骨分化能力的影响。方法2020年3月—2021年8月黑龙江省医院检验科初步筛选出适宜浓度的RDTF,三代分离、培养SHEDs,通过流式细胞术及茜素红染色分别对细胞表面标志物和成骨分化潜能进行鉴定。实验分为4组(0时段为对照组,24、48、72 h组),每组设置3个复孔,采用不同浓度RDTF处理SHEDs后钙钴法染色,Western blot法检测其成骨相关蛋白表达情况。结果结果显示,所获取的细胞为具有成骨分化能力的SHEDs。与对照组相比,24、48、72 h内各浓度组均对SHEDs增殖无明显影响,差异无统计学意义(P>0.05),12.5、25 mg/L RDTF组的Runt相关转录因子2(Runx2)及骨钙素(OCN)的蛋白表达均显著上调,其中25 mg/L RDTF组对Runx2表达的促进效果最明显(249.681±14.653),差异有统计学意义(F=102.7,P<0.001)。结论RDTF对SHEDs的增殖无显著影响,可促进SHEDs成骨分化。

人脱落乳牙牙髓干细胞治疗非口腔疾病的研究进展

人脱落乳牙牙髓干细胞(SHEDs)作为一类具有较强增殖能力且能够多向分化的牙源性干细胞,易于获取且符合伦理要求,在干细胞治疗中具有很大潜力。目前关于SHEDs在非口腔疾病中的治疗作用缺乏阐述,本文就SHEDs在神经系统、消化系统、心血管系统、泌尿系统、免疫系统、内分泌系统及呼吸系统疾病中的最新治疗进展做一综述,以期为SHEDs在非口腔疾病中的临床应用提供参考。

基于RNA测序的SHED体外连续扩增后差异表达基因的研究

目的:利用RNA测序(RNA sequencing,RNA-seq)技术研究人脱落乳牙干细胞(stem cells derived from human exfoliated deciduous teeth,SHED)体外长时期扩增至20代(P20)后基因表达的差异,初步探讨其体外扩增衰老相关的信号通路。方法:从健康儿童脱落的乳牙中分离牙髓干细胞,在常规条件下将其扩增培养至20代,利用RNA-seq筛选出差异表达的基因,并对其进行相关生物学信息分析,寻找SHED体外连续扩增衰老相关的信号通路。结果:RNA-seq结果显示,SHED第4代(P4)和P20代差异表达的基因共有1884个,其中上调表达的基因有575个,下调表达基因1309个,这些差异表达的基因分布在生物过程(biological progress,BP)、细胞组成(cellular component,CC)和分子功能(molecular function,MF)等生物学过程中。早、晚期代次的SHED差异基因及相关蛋白之间的作用主要富集在剪接体、核糖体、细胞周期、p53通路相关的信号通路上。结论:本研究揭示了SHED体外连续扩增培养至第20代后基因表达谱的改变,为后续进一步研究其细胞体外衰老机制指明了方向。

白藜芦醇促进人乳牙牙髓干细胞的增殖和成骨分化

背景:白藜芦醇是一种天然非黄酮类多酚化合物。研究表明,白藜芦醇具有抗氧化、抗炎、抗衰老和抗肿瘤等多种生物活性,并且能促进骨髓间充质干细胞的成骨分化,但其是否能调节乳牙牙髓干细胞的成骨分化尚不清楚。目的:研究白藜芦醇对人乳牙牙髓干细胞增殖和成骨分化能力的影响。方法:组织块法分离培养人乳牙牙髓干细胞,不同浓度的白藜芦醇处理成骨诱导的人乳牙牙髓干细胞,CCK-8检测细胞活性,茜素红染色检测矿化结节数量,RT-qPCR检测成骨相关基因的mRNA表达及Western blot检测Runt相关转录因子2的蛋白水平。结果与结论:①CCK-8检测结果显示,处理第3天时,较高浓度(20,40,80,100μmol/L)白藜芦醇显著抑制细胞增殖,因此,后续实验中选用1,5,10μmol/L白藜芦醇处理乳牙牙髓干细胞,检测其对成骨分化的作用;②与成骨诱导对照组相比,10μmol/L白藜芦醇组乳牙牙髓干细胞的矿化结节数量显著增多;③与成骨诱导对照组相比,10μmol/L白藜芦醇组乳牙牙髓干细胞的碱性磷酸酶、Runt相关转录因子2和骨钙素的mRNA表达量升高;④与成骨诱导对照组相比,白藜芦醇组乳牙牙髓干细胞的Runt相关转录因子2蛋白水平呈浓度依赖性增加;⑤结果表明,适宜浓度的白藜芦醇可提高人乳牙牙髓干细胞的增殖和成骨分化能力。

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。

Wnt信号通路在调控牙髓干细胞多向分化及炎症损伤修复中的作用

人牙髓干细胞(hDPSC)和乳牙牙髓干细胞(SHED)均属于间充质干细胞,具有多向分化潜能。Wnt信号通路包括经典Wnt/β-catenin信号通路和非经典Wnt信号通路,参与牙齿的生长发育过程,能促进牙损伤的修复。在炎症状态下,激活的Wnt信号通路可加快牙损伤修复,参与hDPSC的增殖与成牙本质向分化过程。在非炎症状态下,Wnt信号通路参与调控hDPSC和SHED成骨、成牙本质方向分化,抑制其成脂向分化。

人乳牙牙髓干细胞的体外培养观察

目的:体外分离、培养人乳牙牙髓干细胞。方法:采用酶消化法将人乳牙牙髓分离培养,以获得人乳牙牙髓干细胞。有限稀释法分离纯化,测定细胞克隆形成率,细胞计数法测生长曲线,细胞爬片行HE染色、碱性磷酸酶染色、抗波形蛋白(vimentin)免疫组化染色。结果:通过有限稀释法获得了克隆形成率为11～24个/103,细胞群体倍增时间为39.84h,HE染色细胞形态为梭形、细胞体小、胞核大的干细胞。且碱性磷酸酶染色阳性,抗波形蛋白(vimentin)免疫组化染色阳性。结论:人乳牙牙髓中可分离培养出牙髓干细胞。

人乳牙牙髓干细胞体外成骨特性的实验研究

目的研究人乳牙牙髓干细胞体外成骨特性。方法从健康儿童滞留乳牙牙髓组织中分离培养乳牙牙髓干细胞(SHED)后,经流式细胞仪分选出两组细胞。A组:CD34+/CD117+双阳性表达的细胞,B组:剩余的混杂细胞。研究两组细胞各自成骨特性:运用流式细胞仪分选标记干细胞,以免疫细胞化学技术,荧光定量PCR技术检测成骨细胞标记物,并做矿化染色。结果分离培养的细胞呈成纤维细胞样生长,经分选的A、B两组细胞在第30天时免疫细胞化学检测发现均有成骨细胞的标记物RUNX-2、OC、BSP表达,在培养第40天时运用荧光定量PCR检测分析发现A、B两组细胞表达RUNX-2、OC、BSPmRNA基因水平的差异有显著性意义:A组高于B组;在第50天A、B两组细胞运用VonKossa's染色法和茜素红染色法检测,结果显示A组细胞呈阳性表达,B组细胞呈阴性表达。结论纯化后的CD34+/CD117+双阳性表达的SHED具有体外自行成骨特性。

山羊乳牙牙髓细胞分离培养与矿化诱导的体外研究

目的:分离培养山羊乳牙牙髓细胞,研究其矿化诱导前、后生物学特性的改变。方法:采用改良酶解组织块法培养山羊乳牙牙髓细胞,应用免疫组化方法(SAB法)对第2代细胞进行来源检测,经矿化诱导培养的第4代细胞与常规培养细胞做对照,进行相关生物学特性检测,包括细胞增殖能力、矿化能力、细胞形态改变、蛋白OCN表达、相关成骨基因(ALP、COL-I、OCN、OPN)表达水平。结果:改良酶解组织块法可较快地培养出山羊乳牙牙髓细胞,细胞爬出时间为培养的第3～4天。第2代细胞抗波丝蛋白阳性,抗角蛋白阴性,证明其间充质来源。MTT法测定显示,未诱导细胞的增殖能力明显高于矿化诱导后的细胞。与未诱导细胞相比较,矿化诱导的细胞ALP染色、钙结节茜素红染色均呈强阳性,免疫组化OCN阳性表达。诱导14d后,定时定量PCR检测证实成骨基因OCN、ALP显著上调。结论:本实验采用改良酶解组织块法成功培养出山羊乳牙牙髓细胞;连续矿化诱导培养14d后,山羊乳牙牙髓细胞分泌矿化基质,具有向成骨细胞分化和形成骨组织的潜能。