Influence of 2-hydroxyethyl methacrylate (HEMA) exposure on angiogenic differentiation of dental pulp stem cells (DPSCs)

ObjectiveThe angiogenic differentiation of dental pulp stem cells (DPSCs) is important for tissue homeostasis and wound healing. In this study the influence of 2-hydroxyethyl methacrylate (HEMA) on angiogenic differentiation was investigated.MethodsTo evaluate HEMA effects on angiogenic differentiation, DPSCs were cultivated in angiogenic differentiation medium (ADM) in the presence or absence of non-toxic HEMA concentrations (0.1 mM and 0.5 mM). Subsequently, angiogenic differentiation was analyzed on the molecular level by qRT-PCR and protein profiler analyzes of angiogenic markers and flow cytometry of PECAM1. The influence of HEMA on angiogenic phenotypes was analyzed by cell migration and sprouting assays.ResultsTreatment with 0.5 mM HEMA during differentiation can lead to a slight reduction of angiogenic markers on mRNA level. HEMA also seems to slightly reduce the quantity of angiogenic cytokines (not significant). However, these HEMA concentrations have no detectable influence on cell migration, the abundance of PECAM1 and the formation of capillaries. Higher concentrations caused primary cytotoxic effects in angiogenic differentiation experiments conducted for longer periods than 72 h.SignificanceNon-cytotoxic HEMA concentrations seem to have a minor impact on the expression of angiogenic markers, essentially on the mRNA level, without affecting the angiogenic differentiation process itself on a detectable level.     

Quantification of angiogenic growth factors released by human dental cells after injury

OBJECTIVE: Angiogenesis is a key step in the dental pulp healing sequence which involves the dentine bridge formation. In a previous work, we showed that dental pulp cells secrete soluble factors which interact with endothelial cells and affect the process of angiogenesis. The objective of this work was to quantify the angiogenic growth factors released by mechanically injured human dental pulp cells and the effect of 2-hydroxyethyl methacrylate (HEMA) on this secretion. DESIGN: Pulp cells were prepared from immature third molars explants by the outgrowth method. Cell monolayers were either subjected to mechanical injuries or treated with increasing concentrations of HEMA. ELISA was used to quantify the secreted angiogenic growth factors in the culture media after different time periods of injury and after incubation with different concentrations of HEMA. RESULTS: Pulp cells secreted significant levels of PDGF-AB, VEGF and FGF-2. The concentration of these factors increased shortly (5h) after injury and returned to initial values after 1 day. HEMA treatment increased VEGF secretion but decreased that of FGF-2 in a dose-dependent manner while it did not affect PDGF-AB level. CONCLUSIONS: Dental pulp cells secrete angiogenic growth factors which play a pivotal role in angiogenesis which precedes the reparative dentine formation. PDGF-AB seems to play a major role because its level showed the highest increase in mechanically injured cells. The presence of HEMA affects both FGF-2 and VEGF levels and may partially explain the lack of dentine bridging after direct pulp capping with an adhesive system.     

Effect of IL-1ra on human dental pulp cells and pulpal inflammation

AIM: This study was conducted to investigate the effect of interleukin-1 receptor antagonist (IL-1ra) on the LPS-induced interleukin-1beta (IL-1beta) synthesis in human dental pulp cells and to assess the role of IL-1ra in pulpal inflammation. METHODS: IL-1beta from human dental pulp cells (HDP) was measured by sandwich ELISA; IL-1ra expression in pulpal tissue was detected by immunohistochemical staining. RESULTS: Stimulation of HDP with increasing concentrations of FnLPS resulted in dose-dependent IL-1beta production. The addition of IL-1ra reduced FnLPS-induced IL-1beta synthesis in human dental pulp cells. Significant inhibition of the FnLPS-induced IL-1beta synthesis was observed when IL-1ra was added before treating with FnLPS for 60 min. Large numbers of IL-1ra positive neutrophils, plasmacytes, endothelial cells and lymphocytes were observed in inflamed pulp tissue. CONCLUSIONS: IL-1ra could reduce LPS-stimulated IL-1beta synthesis, suggesting that IL-1ra may play a role in pulpitis.     

Effects of Notch ligand Delta1 on the proliferation and differentiation of human dental pulp stem cells in vitro

Objective

Notch signalling controls cell fate decisions in adult and embryonic tissues. The Notch ligand Delta1 is known to influence proliferation and differentiation of many kinds of tissue specific stem cells. In the present study, we investigated the role of Delta1 in the regulation of dental pulp stem cells (DPSCs) in vitro.

Methods

DPSCs were isolated from impacted third molars. Expression of human Notch1, 2 and Delta1 in DPSCs were detected by immunochemistry. Delta1 overexpressed DPSCs were constructed by a retroviral method. Delta1 transduced DPSCs proliferation changes were examined by means of colony-forming assay, BrdU incorporation assay and cell cycle analysis. Delta1 transduced DPSCs were cultured in differentiation-inductive medium. The nodule formation and DSPP expression were evaluated.

Results

It was shown that the Notch receptors and Delta1 ligand were expressed throughout the proliferation and differentiation process of cultured dental pulp stem cells. Furthermore, it was found in our study that Delta1 could significantly enhance the proliferation of DPSCs and permit DPSCs differentiating into odontoblast-like cells in differentiation-inductive environments.

Conclusions

Our findings verified that Notch-Delta1 signalling was expressed in human DPSCs in vitro and appeared to play pivotal role in DPSCs proliferation enhancement and differentiation regulation, thereby consistent with the hypothesis that the Notch pathway controls stem cell fate during pulp regeneration.     

五倍子水提取物对内毒素损伤人牙髓细胞超微结构的影响

目的：观察五倍子水提取物对内毒素损伤人牙髓细胞超微结构的影响。方法：采用组织块法体外培养人牙髓细胞，以5μg／mL五倍子水提取物和100μg／mL内毒素分别或共同作用于第5代牙髓细胞，并设空白对照组，在透射电镜下观察细胞超微结构的改变。结果：100μg／mL内毒素对牙髓细胞超微结构有不同程度的损伤，加入5μg／mL五倍子水提取物后，牙髓细胞超微结构损伤明显减轻，细胞器结构基本恢复正常。结论：低浓度五倍子水提取物能够抑制内毒素对人牙髓细胞超微结构的损伤作用。     

人牙髓干细胞在钛金属表面向成骨细胞样细胞分化的实验研究

目的：探讨矿化液诱导条件下人牙髓干细胞( human dental pulp stem cells,HDPSCs)在钛金属表面向成骨细胞样细胞分化的潜力。方法将HDPSCs接种于钛金属板表面,于第3、7d时观察其增殖状态。将HDPSCs接种于钛金属板表面进行矿化液诱导,于诱导的第0、3、5、7、14、21、28d时,采用RT-PCR检测其牙本质涎磷蛋白( DSPP)、碱性磷酸酶( ALP)、骨涎蛋白( BSP)和骨钙素( OCN)的基因表达。 Western印迹分析、免疫荧光检测BSP及OCN蛋白表达。对照组为矿化液诱导培养皿中的HDPSCs,检测指标、方法基本相同,免疫细胞化学染色检测OCN蛋白表达。结果钛金属板上 HDPSCs增殖迅速,表现出良好的生物相容性。两组细胞均自诱导第3天ALPmRNA呈高表达、DSPP始终不表达。培养皿组BSP、OCNmRNA于第5天开始表达,并随时间延长而表达增高;钛金属组BSP、OCNmRNA表达于第14天出现第一峰值,随后有所下降,28天再次升高。两组BSP蛋白表达趋势与其RT-PCR结果一致,第5天 OCN染色阳性。结论钛金属表面 HDPSCs 经矿化液诱导向成骨细胞样细胞分化。     

牙本质粘结剂对人牙髓细胞毒性的体外研究

目的：评价全酸蚀牙本质粘结剂和自酸蚀牙本质粘结剂对人牙髓细胞的毒性作用.方法：用人牙髓细胞为实验细胞,采用MTT比色分析法,对4 种牙本质粘结剂（Prime ＆ Bond NT,Single Bond,Xeno Ⅲ,iBond）进行体外细胞毒性研究.结果：不同浓度的粘结剂稀释液均可使人牙髓细胞的形态有所改变.4 种牙本质粘结剂的细胞毒性有显著性差异,且作用时间和浓度的改变对其细胞毒性有影响.全酸蚀粘结剂比自酸蚀粘结剂的细胞毒性强.结论：4 种牙本质粘结剂在体外对人牙髓细胞均有一定程度的细胞毒性,其中Single Bond的毒性较强,临床使用粘结剂时应合理选择粘结剂和掌握固化时间.     

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。     

Biological characteristics of dental pulp stem cells and their potential use in regenerative medicine

BackgroundRegenerative medicine has emerged as a multidisciplinary field with the promising potential of renewing tissues and organs. The main types of adult stem cells used in clinical trials are hematopoietic and mesenchymal stem cells (MSCs). Stem cells are defined as self-renewing clonogenic progenitor cells that can generate one or more types of specialized cells.HighlightMSCs form adipose, cartilage, and bone tissue. Their protective and regenerative effects, such as mitogenic, anti-apoptotic, anti-inflammatory, and angiogenic effects, are mediated through paracrine and endocrine mechanisms. Dental pulp is a valuable source of stem cells because the collection of dental pulp for stem cell isolation is non-invasive, in contrast to conventional sources, such as bone marrow and adipose tissue. Teeth are an excellent source of dental pulp stem cells (DPSCs) for therapeutic procedures and they can be easily obtained after tooth extraction or the shedding of deciduous teeth. Thus, there is increased interest in optimizing and establishing standard procedures for obtaining DPSCs; preserving well-defined DPSC cultures for specific applications; and increasing the efficiency, reproducibility, and safety of the clinical use of DPSCs.ConclusionThis review comprehensively describes the biological characteristics and origins of DPSCs, their identification and harvesting, key aspects related to their characterization, their multilineage differentiation potential, current clinical applications, and their potential use in regenerative medicine for future dental and medical applications.     

Correlation between numbers of cells in human dental pulp and age: Implications for age estimation

ObjectiveThe aim of this study was to investigate correlations between dental pulp cell count of odontoblasts, subodontoblasts and fibroblasts and age, within different age groups. Formulation of regression equations using the dental pulp cell count for predicting age was attempted.DesignEighty-one extracted teeth were grouped into two age groups (6–25 years, 26–80 years). The teeth were demineralized and histological sections were prepared for cell count. Regression equations were generated from regression analysis of cell count and tested for age estimation.ResultsThe number of dental pulp cells were found to increase until around the third decade of life and following this, the odontoblasts and subodontoblasts cell numbers began to decline while the fibroblasts seemed to remain almost stationary. The Pearson correlation test revealed a significant positive correlation between the cell number for all type of cells and age in the 6–25 years group (r = +0.791 for odontoblasts, r = +0.600 for subodontoblasts and r = +0.680 for fibroblasts). In the 26–80 years age group, a significant negative correlation of the odontoblasts (r = −0.777) and subodontoblasts (r = −0.715) with age was observed but for fibroblasts, the correlation value was negligible (r = −0.165). Regression equations generated using odontoblasts and subodontoblasts cell number were applicable for age estimation. The standard error of estimates (SEEs) were around ± 5 years for 6–25 years and ± 8 years for 26–80 years age groups. The mean values of the estimated and chronological ages were not significantly different.ConclusionsA significant correlation between the cell count of odontoblasts and subodontoblasts with age was demonstrated. Regression equations using odontoblasts and subodontoblasts cell number can be used to predict age with some limitations.     

五倍子水提取物对内毒素诱导人牙髓细胞分泌IL-6的影响

目的:观察不同浓度五倍子水提取物对内毒素诱导人牙髓细胞分泌IL-6的影响.方法:采用组织块法体外培养人牙髓细胞,以含不同浓度五倍子水提取物(1.25、2.5、5.10、20 μg/mL)、25μg/mL内毒素和20mL/L新生牛血清的DMEM培养基作用于第5代人牙髓细胞,用放射免疫法测定IL-6含量,采用单因素方差分析(完全随机设计)和t检验对实验数据进行统计学分析.结果:低浓度(1.25、2.5、5、10、20μg/mL)五倍子水提取物能明显抑制25μg/mL内毒素诱导人牙髓细胞分泌IL-6,这种抑制作用在一定范围内呈浓度依赖性.结论:低浓度五倍子水提取物具有抑制内毒素诱导人牙髓细胞分泌IL一6的作用.     

EphrinB2 signaling enhances osteogenic/odontogenic differentiation of human dental pulp stem cells

ObjectiveTo investigate the role of the EphrinB2 signaling pathway in the osteogenesis/odontogenesis of human dental pulp stem cells (DPSCs).DesignThe endogenous expression levels of EphrinB2 and its cognate receptors EphB2 and EphB4 in DPSCs were analyzed by qRT-PCR and Western blotting after 7, 14 and 21 days of osteogenic/odontogenic induction culture. Additionally, the phosphorylation of EphrinB2, EphB4 and ERK1/2 proteins at early time-points following osteogenic induction, were also investigated by Western blots. Subsequently, we investigated whether supplementation of recombinant EphrinB2-Fc within the induction milieu can enhance the osteogenic/odontogenic differentiation of DPSCs.ResultsEndogenous gene and protein expression levels of EphrinB2, EphB2 and EphB4 were upregulated in induced versus non-induced DPSCs, over 21 days of osteogenic/odontogenic induction. Western blots showed increase in phosphorylated EphrinB2, EphB4 and ERK1/2 proteins at early time-points following osteogenic induction. Preliminary investigation of a concentration range (0, 0.5, 1 and 2 μg/ml) of recombinant EphrinB2-Fc within osteogenic induction media, showed that 0.5 μg/ml was optimal for enhancing the osteogenic/odontogenic differentiation of DPSCs over a culture duration of 14 days. Subsequently, more comprehensive qRT-PCR analysis with 0.5 μg/ml EphrinB2-Fc revealed significant upregulation of several key osteogenic marker genes in treated versus untreated DPSCs after 21 days of osteogenic/odontogenic induction. By 7 days of osteogenic induction, DPSCs treated with 0.5 μg/ml EphrinB2-Fc exhibited significantly more calcium mineralization (Alizarin red S staining) and alkaline phosphatase activity than the untreated control.ConclusionsEphrinB2 signaling plays a key role in the osteogenic/odontogenic differentiation of DPSCs.     

人牙髓干细胞培养及其细胞表型的分析研究

目的 :分离培养来源于人体牙髓组织的干细胞并对其细胞表型进行分析。方法 :采用胶原酶消化法获得人牙髓组织的单细胞悬液 ,14d后挑取细胞克隆传代培养 ,随机选取 2株细胞克隆扩大培养 ,以骨髓间充质干细胞为参照 ,利用免疫组化方法通过与外胚间充质干细胞的比较对其表型进行分析。结果 :人的牙髓干细胞呈集落状生长 ,有细胞克隆形成 ,细胞表达多种细胞表面标志。结论 :人牙髓组织中存在有呈克隆样生长的成体干细胞 ,细胞表型呈现多样性。     

人乳牙牙髓干细胞和成人牙髓干细胞研究进展

近年来,成体干细胞不断地从不同的组织中被分离出来,该类细胞具有多向分化潜能、较强的增殖能力和持久的自我更新能力,具备充当组织工程种子细胞的天然优势。2000年和2003年,研究者先后从成人牙髓组织和人乳牙牙髓组织中分离出具有干细胞特征的细胞,这两种细胞的发现对牙组织工程将产生重要的意义。现就这两种成体干细胞的研究进展做一综述,并展望其应用前景。     

人牙髓细胞在离体牙髓腔内向成牙本质细胞样细胞分化的研究

目的观察人牙髓细胞在离体牙髓腔内向成牙本质细胞样细胞分化的潜能。方法将原代培养的人牙髓细胞接种至处理过的离体牙髓腔内,2周后固定、脱钙、包埋、切片、染色,显微镜下观察其生长及分化情况。结果牙髓细胞在牙本质表面生长良好,部分细胞伸出胞质突伸入牙本质小管中,表现出成牙本质细胞样细胞的形态。结论牙本质可以诱导牙髓细胞向成牙本质细胞样细胞分化,可为组织工程化牙髓的研制提供实验依据。     

体外培养的冠髓和根髓细胞中碱性磷酸酶的活性

目的：比较根部和冠部牙髓培养细胞的碱性磷酸酶活性，探讨牙髓干细胞在牙髓中的定位。方法：用组织块法分别培养根髓、冠髓细胞，用Gomori碱性磷酸酶染色法对根、冠髓培养细胞在加入条件孵育液(20％DMEM 10nmol／L地塞米松 10mmol/L β-甘油酸钠 50mg／L L-抗坏血酸)后的第5、10、15天的碱性磷酸酶活性进行测定，从牙髓干细胞的功能角度，探讨其在牙髓中的定位。结果：根髓、冠髓细胞均出现碱性磷酸酶染色阳性，染色强度在第5天无差别，在第10、15天根髓染色强度大于冠髓。结论：碱性磷酸酶的活性在根髓中大于冠髓。牙髓干细胞可能存在于全部牙髓之中．其密度在根髓中大于冠髓。     

高糖对人牙髓细胞增殖及氧化应激的影响

目的 探讨高糖对人牙髓细胞(HDPCs)增殖和氧化应激的影响,以探索高糖环境对牙髓的影响机制.方法 分离培养牙髓细胞,鉴定组织来源,实验分为4组:低糖组(葡萄糖5.5 mmol/L)、正常组(葡萄糖25 mmol/L)、高糖组(葡萄糖50mmol/L)、高渗组(与50 mmol/L组渗透压相等,并与5.5 mmol/L...     

体外培养的人牙髓细胞表型分析

目的：分离培养来源于人体的牙髓细胞并对其细胞表型进行分析。方法：采用组织块法培养人牙髓细胞,根据牙髓的细胞成分选择特异性抗体,利用免疫组化技术对其细胞表型进行分析。结果：Ⅲ型胶原在体外培养的牙髓细胞中广泛表达,ON和OPN表达也较广泛,阳性细胞呈星形,伸出多个胞浆突起互相连接;α-平滑肌肌动蛋白和CD34表达细胞为集落状,前者呈细长的梭形,后者呈圆形;Ⅰ型胶原、BSP和OC仅在少量细胞中表达,且细胞形态不规则;DSP、NF、CD14及CD45均为阴性。结论：体外培养的人牙髓细胞表型呈现多样性,表达平滑肌、内皮细胞及骨的标志物。