牙胚细胞条件培养液诱导大鼠胚胎面突外胚间充质细胞向成牙本质样细胞分化

目的：探讨牙胚细胞条件培养液（TGC—CM）在诱导面突外胚间充质细胞向成牙本质样细胞分化中的作用。方法：取妊娠12．5d SD大鼠胎鼠第4代下颌突外胚间充质细胞,在牙胚细胞条件培养液（TGC—CM）的诱导下,通过形态学观察、免疫组化、RT—PCR等方法,探索牙胚细胞条件培养液诱导面突外胚间充质细胞向成牙本质样细胞分化的可能。结果：面突外胚间充质细胞在诱导培养基中生长良好。培养7d,在诱导组中细胞出现核极化,有很长的突起,细胞平行排列。抗牙本质涎蛋白（DSP）呈阳性反应。RT—PCR显示mRNA水平表达成牙本质细胞特异的牙本质涎磷蛋白（DSPP）和牙本质基质蛋白-1（DMP-1）。结论：面突外胚间充质细胞在含有多种细胞因子的TGC—CM的作用下能分化为成牙本质样细胞,能为研究牙齿的分化和发育提供良好的模型和实验依据。     

永生化成牙本质细胞表达牙本质基质蛋白的实验研究

目的　探讨永生化人成牙本质细胞样细胞系hTERT-hOd-l表达牙本质基质蛋白的情况。方法　矿化液培养hTERT-hOd-l细胞5周,检测骨钙素(OC)分泌量和碱性磷酸酶(ALP)活性。采用免疫组织化学、RT-PCR和原位杂交方法检测Ⅰ型胶原、骨涎蛋白(BSP)、牙本质基质蛋白1 (DMP1)以及成牙本质细胞标志物牙本质涎磷蛋白(DSPP) 和牙本质涎蛋白(DSP)在细胞中的表达。结果　在矿化液诱导下,hTERT-hOd-l细胞ALP活性和OC分泌量升高。 hTERT-hOd-l细胞在mRNA水平上表达BSP、DMP1和DSPP,在蛋白质水平上表达DSP和Ⅰ型胶原。结论　hTERT- hOd-l细胞在体外表达牙本质基质蛋白,具有矿化的潜能。     

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.     

Leptin Induces Odontogenic Differentiation and Angiogenesis in Human Dental Pulp Cells via Activation of the Mitogen-activated Protein Kinase Signaling Pathway

Introduction

Up-regulation of odontogenic differentiation, dentin formation, and angiogenesis in dental pulp are key factors in vital pulp therapy. The aim of this study was to investigate whether leptin could promote odontogenic differentiation and angiogenesis in human dental pulp cells (hDPCs). In addition, the involvement of the intracellular signaling pathway in these effects was determined.

脂多糖诱导牙髓细胞DNA双链断裂的研究

目的:探讨低剂量条件下脂多糖诱导牙髓细胞DNA损伤方法,为研究脂多糖诱导牙髓细胞DNA双链断裂及DNA修复提供实验模型。方法:10 μg/L脂多糖连续刺激牙髓细胞1、3、6次后,采取MTT及TUNEL分别检测其对牙髓细胞增殖及凋亡的作用;采用q-PCR及免疫印迹检测γ-H2A.X的mRNA及蛋白水平的表达;使用免疫荧光及免疫组化法观测γ-H2A.X在体内、外牙髓细胞的表达。结果:与对照组相比,10 μg/L脂多糖连续刺激对牙髓细胞增殖及凋亡均无显著性差异;脂多糖连续刺激6次后细胞免疫荧光显示在细胞核检测到γ-H2A.X的阳性表达,且蛋白及mRNA水平的表达较对照组有显著性增加(P<0.05);免疫组化结果表明在脂多糖诱导4、6、8 d后,γ-H2A.X在大鼠牙髓组织较对照组表达水平显著升高(P<0.05)。结论:低剂量脂多糖连续刺激可诱导牙髓细胞产生DNA双链断裂。     

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.     

Stem Cell Regulatory Gene Expression in Human Adult Dental Pulp and Periodontal Ligament Cells Undergoing Odontogenic/Osteogenic Differentiation

IntroductionDuring development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes.MethodsIn this study, we investigated the differential expression of 84 stem cell–related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation.ResultsOur results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor β (TGF-β)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration.ConclusionsThis study has generated an overview of stem cell–related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-β/BMP, and cadherin signaling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration.     

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.     

Biocompatibility of New Pulp-capping Materials NeoMTA Plus,MTA Repair HP,and Biodentine on Human Dental Pulp Stem Cells

Introduction

The aim of the present study was to evaluate the in vitro cytotoxicity of MTA Repair HP, NeoMTA Plus, and Biodentine, new bioactive materials used for dental pulp capping, on human dental pulp stem cells (hDPSCs).

Hydrogen Peroxide Induces Apoptosis in Human Dental Pulp Cells via Caspase-9 Dependent Pathway

Introduction

Reactive oxygen species are a group of metabolic intermediates produced during oxidative metabolism in eukaryotic cells. They include superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (·OH), and ¹O₂. Of these intermediates, H₂O₂ is the most stable. Dental pulp cells can be invaded by tooth bleaching, laser radiation, and dental materials. This can influence the intracellular level of reactive oxygen species. Apoptosis, which is the best-known form of programmed cell death, is pivotal to tissue development and regeneration. Little information is available regarding the relationship between H₂O₂ and apoptosis of human dental pulp cells (hDPCs). The purpose of this study was to investigate whether H₂O₂ can induce apoptosis in hDPCs and its signaling way.

Methods

HDPCs were obtained by using a modified tissue explant technique in vitro and cultured at 37°C, 20% O₂ (5% CO₂, 95% air) in Dulbecco modified Eagle medium. Cell viability was investigated by methyl-thiazol-tetrazolium assay. Cell apoptosis was detected by using the annexin V–fluorescein isothiocyanate/propidium iodide apoptosis assay and flow cytometry. Expression of activated caspase-3, cleaved caspase-9, and β-actin was analyzed by using Western blot.

Results

Cell viability of hDPCs decreased more in treated groups than in the control group from days 1 to 7. The relative number of apoptotic cells and the expression of activated caspase-3 and cleaved caspase-9 were much higher in groups exposed to 20 and 50 μmol/L H₂O₂.

Conclusions

These results imply that low concentrations of H₂O₂ are cytotoxic to hDPCs and induce apoptosis in hDPCs in a caspase-9–dependent way.     

Minced Pulp as Source of Pulpal Mesenchymal Stem Cells with Odontogenic Differentiation Capacity

Introduction

Pulp tissue regeneration is becoming a reality after discovery of mesenchymal stem cells (MSCs) residing in the pulp tissues through various clinical innovations, although MSC transplantation into the pulp space has met with challenges of in vitro cell expansion and cultures. As a way to circumvent the regulatory and technical complexities of in vitro MSC culture, we investigated the use of minced pulp tissues as a source of pulpal MSCs for tissue regeneration.

牙髓干细胞在组织再生及细胞治疗中的研究进展

<正>在人体发育过程中,组织器官高度分化,行使不同功能,组成复杂的人体结构。一部分组织器官仍保留着一定再生功能。如上皮组织、肝组织等,而大部分组织器官只具有部分再生能力。如骨、软骨、神经组织。这些组织器官在创伤、感染、肿瘤等疾病破坏后再生能力较弱,给医学带来极大的挑战。近年来,组织再生及细胞治疗的发展为解决上述问题展现出美好的前景。干细胞用于组织再生及细胞治     

破骨细胞分化因子诱导小鼠骨髓细胞形成破骨细胞的实验研究

目的:探讨破骨细胞分化因子(ODF)和巨噬细胞集落刺激因子(M-CSF)联合应用进行体外诱导小鼠骨髓细胞形成破骨细胞(OC)的能力。方法:收集5～6周小鼠骨髓细胞,在含M-CSF(10 ng/ml)的α-MEM全培养液中培养24h,然后将悬浮生长的细胞接种到24孔培养板,并加入不同浓度的ODF和M-CSF。,通过观察抗酒石酸盐酸性磷酸酶(TRAP)染色的阳性细胞和能否在骨磨片上形成吸收陷窝来鉴定OC形成情况。结果:在只加入ODF或M-CSF一种细胞因子时,未见有TRAP阳性或CTR阳性细胞形成,同时加入ODF和M-CSF,可形成典型的OC。TRAP阳性多核细胞的数目和培养液中钙离子浓度的增加与ODF的浓度呈正相关。结论:小鼠骨髓来源的单核细胞在ODF和M-CSF共同作用下可形成具有骨吸收功能的OC,为体外研究OC的分化发育和功能调节提供了一种新的方法。