人牙髓干细胞在聚乳酸-聚乙醇酸共聚物支架上粘附与增殖的研究

目的：研究成人牙髓干细胞（HDPSCs）在聚乳酸-聚乙醇酸共聚物（PLGA）支架上粘附与增殖的情况。方法：采用酶消化法分离、培养人牙髓干细胞,免疫组化法及体外诱导分化对细胞进行鉴定。将人牙髓干细胞与PLGA支架材料进行复合培养,扫描电镜（SEM）观察支架材料形态,细胞粘附、增殖及基质分泌情况;细胞计数检测其增殖力。结果：细胞接种2、5、10d,扫描电镜及细胞计数均显示HDPSCs与PLGA支架材料粘附紧密,生长状态良好,细胞明显增殖（P〈0.05）,有丰富的细胞外基质形成。结论：PLGA是一种适宜人牙髓干细胞粘附与增殖的支架材料。     

凝胶样支架材料对三维培养的人牙髓干细胞增殖的影响

目的　评价牙髓组织工程中不同浓度的两种可注射性凝胶样支架材料对牙髓干细胞增殖的影响。方法：配制不同浓度的I型胶原、肽段水凝胶（Puramatrix）支架材料,将牙髓干细胞分别接种于各组支架材料上;CCK-8方法检测细胞增殖情况;活死细胞染色观察细胞数量及形态。 结果　接种在各浓度支架材料上的牙髓干细胞均生长良好,其中1%的胶原支架和0.25%的水凝胶支架对牙髓干细胞的增殖有明显的促进作用;活死细胞染色观察牙髓干细胞在各浓度的两种支架材料中均生长良好,细胞数量随培养时间的延长呈递增趋势;细胞充分伸展,呈纺锤型。     

模拟微重力环境对人牙髓干细胞体外增殖能力的影响

目的：研究模拟微重力环境对人牙髓干细胞（human dental pulp stem cells,hDPSCs）在PLGA支架上体外增殖能力的影响。方法：将分离、鉴定后的人牙髓干细胞接种在PLGA支架上,采用MTT法检测普通环境和模拟微重力环境中培养24、48、72h的人牙髓干细胞在PLGA支架上的细胞活性。DAPI荧光染色和流式细胞术比较牙髓干细胞在两种环境培养3d的细胞数量以及细胞周期分布情况。结果：MTT显示模拟微重力组中各时间点的A值均高于普通环境培养组;DAPI荧光染色显示在模拟微重力下培养3d的人牙髓干细胞数量明显多于普通环境培养组;细胞周期分析结果表明模拟微重力组中S期细胞比例明显高于普通环境培养组（P〈0.05）。结论：接种在PLGA支架上的人牙髓干细胞在模拟微重力环境下较普通环境具有更高的体外增殖能力。     

Effects of Calcitonin Gene-related Peptide on Dental Pulp Stem Cell Viability,Proliferation, and Differentiation

IntroductionPulpitis is an inflammation of dental pulp caused by bacterial proliferation near or within pulpal tissues. In advanced stages, when the inflammation is associated with pulp necrosis, pulp preservation is dependent on dental pulp stem cells (DPSCs) that can differentiate into odontoblastlike cells and produce reparative dentin. In this study, we evaluated the influence of sensory neurons through calcitonin gene-related peptide (CGRP) on DPSC viability and proliferation and the ability of DPSCs to differentiate into mineralizing cells.MethodsCommercially available DPSCs were treated with varying doses of CGRP, and metabolic activity, viability, proliferation, and cell death were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays, trypan blue staining, 5-bromo-2'-deoxyuridine cell proliferation assay, and caspase-3 staining, respectively. DPSC differentiation was assessed with alizarin red staining and by quantifying messenger RNA expression of odontoblast makers.ResultsCGRP induced a dose-dependent decrease of DPSC metabolic activity that was prevented by the CGRP receptor antagonist CGRP 8-37. The decrease in the proportion of live cells induced by CGRP is associated with a decrease of cell proliferation but not with caspase-3–dependent apoptosis. Interestingly, dexamethasone-induced DPSC differentiation into mineralizing cells was neither inhibited nor enhanced by CGRP treatment.ConclusionsThe neuropeptide CGRP has an inhibitory effect on DPSC proliferation but does not enhance or inhibit the differentiation of DPSCs into mineralizing cells. This suggests that CGRP might negatively influence the ability of DPSCs to contribute to regenerative or tissue repair processes.     

人牙髓干细胞的体外培养和鉴定

目的　研究第三恒磨牙来源的人牙髓干细胞的表型和生物学性状。方法　从成人健康阻生牙中获取牙髓,酶消化法分离获得牙髓干细胞,计算细胞克隆形成率(CFU-F);免疫组化、RT-PCR法检测细胞的表面分子表达; 流式细胞仪测定细胞周期;体外分化诱导实验检测细胞的多向分化能力。结果　分离获得的牙髓干细胞在体外具有一定的克隆形成能力,诱导条件下部分牙髓干细胞可向脂肪、肌细胞和成牙本质细胞方向分化,符合干细胞的特征。结论　成功的从人第三恒磨牙牙髓中分离得到牙髓干细胞。     

Rho激酶抑制剂Y-27632对人牙髓干细胞增殖能力的影响

目的: 初步研究Rho激酶抑制剂Y-27632对人牙髓干细胞(human dental pulp stem cells,hDPSCs)增殖能力的影响。方法: 采用体外贴壁式培养法培养人牙髓干细胞,应用Rho激酶抑制剂Y-27632,根据培养条件,分为普通培养液培养组(Con)及普通培养液+抑制剂Y-27632培养组(Con+Y),采用MTT法检测两种培养条件下人牙髓干细胞培养24、48、72、96 h细胞活性;采用DAPI染色法及流式细胞定量检测技术(FCM)比较两组在培养48 h的细胞数量及细胞周期分布情况。结果: MTT结果显示,实验组hDPSCs细胞增殖曲线较对照组明显上移,且增殖高峰期提前;培养48 h,DAPI染色结果显示,实验组细胞总数量明显多于对照组;FCM结果显示,对照组G₀/G₁期细胞比例为(66.8±6.84)%,S期细胞比例为(25.17±0.62)%,实验组G0/G1期细胞比例为(58.59±1.76)%,S期细胞比例为(31.34±1.16)%,实验组S期细胞比例明显高于对照组(P<0.05)。 结论: Rho激酶抑制剂Y-27632促进人牙髓干细胞DNA合成、细胞分裂及增殖。     

The Effect of Octenidine on Proliferation,Migration, and Osteogenic Differentiation of Human Dental Pulp and Apical Papilla Stem Cells

IntroductionThe research for alternative irrigating solutions is ongoing, since no “ideal” solution has yet been found. Octenidine dihydrochloride (OCT) has been indicated as an endodontic irrigant because it has adequate antimicrobial and biological properties. The present study aimed to assess the effects of OCT on proliferation, migration, and induction of the osteogenic phenotype of stem cells from human dental pulp and apical papilla.MethodsCells were collected from human third molars and exposed to different doses of OCT, chlorhexidine (CHX), sodium hypochlorite (NaOCl), and ethylenediaminetetraacetic acid (EDTA) to determine cell viability by alamarBlue assay; proliferation by bromodeoxyuridine incorporation; migration by the Transwell assay; alkaline phosphatase activity by thymolphthalein release; and production of mineralized nodules by alizarin red staining. The results were analyzed by 1- or 2-way analysis of variance and Tukey (α = .05).ResultsCHX promoted lower cell viability, followed by OCT, NaOCl, and EDTA, especially at intermediate doses (P < .05). Cells exposed to CHX had less proliferation than the other groups (P < .05). The Transwell assay revealed no differences among OCT, EDTA, and culture medium (control group) (P > .05). OCT and EDTA induced greater migration than CHX and NaOCl (P < .05). OCT and EDTA induced higher alkaline phosphatase activity than NaOCl and CHX (P < .05). No difference was detected among the groups using alizarin red staining (P > .05).ConclusionsOCT induced high migration, proliferation, and alkaline phosphatase activity of stem cells from human dental pulp and apical papilla, which could be advantageous for regenerative endodontic procedures.     

Effect of EDTA on Attachment and Differentiation of Dental Pulp Stem Cells

Introduction

In regenerative endodontics, it is believed that EDTA induces odontoblast differentiation by releasing growth factors from the dentin matrix. The aim of this study was to evaluate the effect of EDTA on the attachment and differentiation of dental pulp stem cells (DPSCs). We also investigated whether the behavioral changes of DPSCs could be caused by biochemical components released from EDTA-treated dentin.

Methods

Cells were obtained from human third molars, and the stem-like nature of the cells was investigated by flow cytometric analysis. DPSCs were seeded on EDTA-treated and untreated dentin slices. After 3 days of culture, cell attachment was evaluated by cell density, fibronectin 1 gene expression level using quantitative real-time polymerase chain reaction, and scanning electron microscopy. After 21 days of culture, the expression of differentiation genes was investigated by quantitative real-time polymerase chain reaction, and calcification was observed using alizarin red S staining. To investigate the EDTA-induced growth factor release, DPSCs were cultured with or without direct contact with the EDTA-treated dentin surface.

Results

After 3 days of culture, both the cell density and fibronectin expression level were significantly higher in the EDTA-treated dentin group. After 3 weeks, the DPSCs on the EDTA-treated dentin surfaces showed higher expression levels of dentin sialophosphoprotein and dentin matrix protein 1, whereas the DPSCs cultured without direct contact with the EDTA-treated dentin surfaces did not exhibit these findings.

Conclusions

Our results showed that EDTA induced cell attachment and odontoblastic/osteoblastic differentiation, which was observed only in the group in which the DPSCs were placed in direct contact with the EDTA-treated dentin surfaces. These findings suggest that EDTA is beneficial for achieving successful outcomes in regenerative endodontics.     

神经生长因子对人牙髓细胞增殖与分化作用的研究

目的 研究神经生长因子对体外培养的人牙髓细胞增殖和分化作用的影响。方法 组织块法行人牙髓细胞的原代培养后，采用四唑盐比色法和酶动力学方法，测定不同浓度（1、10、100U／mL）的神经生长因子对体外培养的第5-8代人牙髓细胞增殖及碱性磷酸酶活性的作用。结果 与对照组相比，10U／mL的神经生长因子可显著促进人牙髓细胞的增殖（P〈0．05）；100U／mL的神经生长因子可显著促进碱性磷酸酶的活性（P〈0．01）。结论 不同浓度的神经生长因子可显著促进人牙髓细胞的增殖与分化。     

Evaluation of the Biodistribution of Human Dental Pulp Stem Cells Transplanted into Mice

Introduction

Several studies have attempted to use human dental pulp stem cells (hDPSCs) for pulp-dentin complex regeneration in vitro. However, the safety of such applications should be first evaluated in vivo before their use in clinical trials. The purpose of this study was to investigate the in vivo fate of intrapulpally transplanted hDPSCs.

模拟微重力环境对人牙髓干细胞体内增殖能力的影响

目的：研究模拟微重力环境对聚乳酸-羟基乙酸（PLGA）支架上人牙髓干细胞（hDPSCs）体内增殖能力的影响。方法：分离、培养hDPSCs并进行鉴定。以PLGA作为支架材料培养人牙髓干细胞,将hDPSCs-PLGA复合物分别在模拟微重力环境和普通重力环境下培养72h,然后移植到裸鼠背部皮下。植入4周后取出组织块,分别进行HE染色、Masson染色、Ki67和I型胶原免疫组化检测。结果：模拟微重力环境下细胞密度、胶原生成量、I型胶原表达量和Ki67阳性细胞数量明显高于普通环境（P〈0.01）。结论：模拟微重力环境培养的hDPSCs的体内增殖能力高于普通重力组。     

Ionic Extraction of a Novel Nano-sized Bioactive Glass Enhances Differentiation and Mineralization of Human Dental Pulp Cells

Introduction

This study aimed to investigate the effects of a novel nano-sized 58S bioactive glass (nano-58S BG) on the odontogenic differentiation and mineralization of human dental pulp cells (hDPCs) in vitro.

Methods

Extractions were prepared by incubating nano-58S BG, 45S5 BG, or 58S BG particulates in Dulbecco modified Eagle medium at 1% w/v for 24 hours and were filtrated through 0.22-μm filters. The supernatants were used as BG extractions. The hDPCs were cultured in nano-58S BG, 45S5 BG, and 58S BG extractions. The proliferation of hDPCs was evaluated using the methylthiazol tetrazolium assay. Odontogenic differentiation was evaluated based on the real-time polymerase chain reaction of differentiation- and mineralization-related genes, namely, alkaline phosphatase (ALP), collagen type I, dentin sialophosphoprotein (DSPP), and dentin matrix protein 1. The gene expressions were verified using ALP activity assessment, immunocytochemistry staining of osteocalcin and DSPP, and mineralization assay using alizarin red S stain.

Results

All BG extractions up-regulated the expression of odontogenic genes, and the most significant enhancement was in the nano-58S BG group. All BG extractions, especially nano-58S, increased ALP activity, osteocalcin and DSPP protein production, and mineralized nodules formation.

Conclusions

Compared with regular BG, the novel nano-58S BG can induce the differentiation and mineralization of hDPCs more efficiently and might be a better potential candidate for dentin-pulp complex regeneration.     

Cell Wall Mannan of Candida Attenuates Osteogenic Differentiation by Human Dental Pulp Cells

IntroductionCandida spp. has recently been introduced to interact with conventional carious bacteria, leading to dental caries progression and virulence ability. Evidence regarding the influence of Candida spp. on human dental pulp cell response remains unknown. This study aimed to investigate the effects of Candida albicans mannans on cytotoxicity, cell proliferation, osteogenic differentiation, and inflammatory-related gene expression in human dental pulp cells (hDPCs).MethodshDPCs were treated with cell wall mannans isolated from C. albicans, Candida krusei, Candida glabrata, Candida tropocalis, Candida parapsilosis, and Candida dubliniensis. Cell viability was performed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Osteogenic differentiation– and inflammatory-related gene expression were determined using a real-time polymerase chain reaction. Mineralization was examined using alizarin red S staining.ResultsThe treatment of mannans isolated from C. albicans, C. krusei, C. glabrata, C. tropocalis, C. parapsilosis, and C. dubliniensis at concentrations ranging from 10–100 μg/mL did not affect cytotoxicity or cell proliferation. Mannans isolated from C. albicans, C. glabrata, and C. tropocalis significantly attenuated mineralization. However, cell wall mannans isolated from C. krusei, C. parapsilosis, and C. dubliniensis did not significantly influence mineral deposition in hDPCs. C. albicans cell wall mannans significantly attenuated osteogenic differentiation–related gene expression (RUNX2, ALP, and ENPP1). Interestingly, IL12 messenger RNA expression was significantly upregulated when treated with C. albicans cell wall mannans. The addition of recombinant IL12 significantly decreased mineralization in hDPCs.ConclusionsC. albicans cell wall mannans attenuated osteogenic differentiation in hDPCs and up-regulated inflammatory-related gene IL12 expression.     

The Role of Interleukin 6 in Osteogenic and Neurogenic Differentiation Potentials of Dental Pulp Stem Cells

IntroductionStudies have shown that there is a significantly higher concentration of interleukin 6 (IL-6) in inflamed pulp tissues compared with healthy pulp tissues. The aims of this study were to investigate the baseline differences between mesenchymal stem cells (MSCs) isolated from healthy human dental pulp stem cells (H-DPSCs) and inflamed dental pulp stem cells (I-DPSCs) and their correlation to IL-6 and to determine whether IL-6 can affect the differentiation potentials of these cells.MethodsMSCs isolated from healthy and inflamed pulp tissues were cultured and characterized in vitro. The levels of secreted IL-6 in the culture supernatants from H-DPSCs and I-DPSCs were measured by enzyme-linked immunosorbent assay. IL-6 and neutralizing IL-6 were added to H-DPSCs and I-DPSCs, respectively. Immunofluorescence staining, alizarin red staining, and Western blotting were performed to assess the differentiation potentials of H-DPSCs and I-DPSCs. The independent unpaired 2-tailed Student's t-test was performed after quantification analysis.ResultsH-DPSCs and I-DPSCs showed a similar expression of MSC-associated markers including CD44, CD73, CD90, and CD105, whereas H-DPSCs showed a lower level of IL-6, lower osteogenic differentiation potentials, and higher neurogenic differentiation potentials compared with I-DPSCs. The addition of IL-6 to H-DPSCs increased osteogenic potentials and decreased neurogenic potentials, whereas the neutralization of IL-6 for I-DPSCs led to decreased osteogenic potentials and increased neurogenic potentials.ConclusionsThe findings of this study indicated IL-6 has the capacity to enhance osteogenesis while hindering neurogenesis of DPSCs.     

Growth and Differentiation Factor-5 Regulates the Growth and Differentiation of Human Dental Pulp Cells

Introduction

Growth and differentiation factor-5 (GDF-5) is a multifunctional protein that regulates the development and repair in many tissues. The purpose of this study was to investigate whether GDF-5 may influence the proliferation, differentiation, and collagen turnover of human dental pulp cells.

Methods

Human dental pulp cells were treated with different concentrations of GDF-5 (0–500 ng/mL). Morphology of pulp cells was observed under a microscope. Cell proliferation was evaluated by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Immunofluorescent assay was used to observe the percentages of cell mitosis. Collagen content was measured by Sircol collagen assay. Tissue inhibitor of metalloproteinase-1 level in the culture medium was measured with enzyme-linked immunosorbent assay and Western blotting. Cell differentiation was evaluated by alkaline phosphatase (ALP) staining and ALP enzyme activity assay.

Results

After exposure of dental pulp cells to various concentrations of GDF-5, cell number was up-regulated significantly in dose-dependent manner. GDF-5 also stimulated mitosis of dental pulp cells as indicated by an increased percentage of binucleated cells from 28% to 35%–45%. GDF-5 did not affect the collagen content and tissue inhibitor of metalloproteinase-1 level of pulp cells. GDF-5 decreased the ALP activity of pulp cells as analyzed by ALP staining and enzyme activity assay, with 14%–44% of inhibition.

Conclusions

GDF-5 revealed mitogenic and proliferative activity to dental pulp cells. GDF-5 showed inhibitory effect on ALP activity but little effect on the collagen turnover. These events are crucial in specific stages of dental pulp repair and regeneration. GDF-5 may be potentially used for tissue engineering of pulp-dentin complex.     

Satb2过表达对牙髓干细胞增殖和迁移能力的影响

目的:通过慢病毒介导Satb2(special AT-rich binding protein 2,Satb2)感染人牙髓干细胞(human Dental pulp stem cells,hDPSCs),观察Satb2过表达对人牙髓干细胞迁移和增殖能力的影响。方法:构建Satb2过表达慢病毒感染人牙髓干细胞,通过筛选得到稳定过表达Satb2细胞克隆。CCK8实验检测过表达Satb2对人牙髓干细胞的增殖能力的影响。划痕实验和Transwell细胞迁移实验观察细胞迁移能力的变化。免疫荧光染色观察细胞骨架微管的变化。结果:过表达Satb2的人牙髓干细胞相比对照组增殖能力增强(P＜0.05),微管更粗大,细胞的迁移能力增强。结论:过表达Satb2能使人牙髓干细胞的增殖和迁移能力增强。