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.     

硅酸钙生物陶瓷对牙髓细胞体外增殖分化的影响

目的:应用硅酸钙(CaSiO3,CS)生物陶瓷作用于人牙髓细胞(human dental pulp cells,hDPCs),研究其对hDPCs增殖及向成牙本质细胞方向分化的影响。方法:从年轻健康患者(18²0岁)拔除的智齿或前磨牙牙髓组织中获取hDPCs进行培养。将质量浓度为0.2 g/mL的CS浸提液按1/2、1/4、1/8、1/16、1/32、1/64和1/128倍比稀释后作用于hDPCs。MTT实验检测不同质量浓度CS浸提液对hDPCs增殖的影响,进而筛选出最佳浓度。以最佳质量浓度(1/64倍比稀释)CS浸提液培养hDPCs 2、4 d后,Real-Time PCR检测以下hDPCs成牙本质相关基因的表达:牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)、牙本质基质蛋白-1(dentin matrix protein 1,DMP-1),Ⅰ型胶原(collagen typeⅠ,COL-Ⅰ)、骨钙蛋白(osteocalcin,OCN)、骨桥蛋白(osteopontin,OPN);培养7、14 d后碱性磷酸酶(alkaline phosphatase,ALP)染色及半定量检测ALP活性。结果:最佳质量浓度(1/64倍比稀释)CS浸提液能够促进hDPCs的增殖,对牙髓细胞DSPP、DMP-1、COL-1、OPN等成牙本质相关基因的表达有较为明显的促进作用。ALP染色及半定量分析显示该浓度的CS浸提液能够提高hDPCs分泌ALP的活性。结论:最佳质量浓度(1/64倍比稀释)CS浸提液能够明显促进hDPCs的增殖,提高成牙本质相关基因的表达,进而促进hDPCs向成牙本质细胞方向分化,为后期hDPCs结合CS支架材料进行牙本质再生的研究打下基础。     

The Effect of SIRT6 on the Odontoblastic Potential of Human Dental Pulp Cells

Introduction

The aim of this study was to investigate whether SIRT6 is expressed in human dental pulp as well as the effect of SIRT6 on proliferation and odontoblastic differentiation of human dental pulp cells (HDPCs).

Methods

Immunohistochemical and immunocytochemical assays were used to detect the expression of SIRT6 in human dental pulp tissue and HDPCs. To determine the effect of SIRT6 on odontoblast differentiation, HDPCs with loss (HDPCs SIRT6 knockdown) and gain (HDPCs SIRT6 overexpression) of SIRT6 function were developed, and their proliferation ability was examined. Odontogenic differentiation of HDPCs was determined by alkaline phosphatase (ALP) activity, ALP-positive cell staining, alizarin red staining, and von Kossa staining. Mineralization-related genes, including ALP, dentin sialophosphoprotein (DSPP), and dentin matrix acidic phosphoprotein 1, were determined by real-time quantitative polymerase chain reaction. Western blot analysis was performed to detect the expression of DSPP protein.

Results

SIRT6 was found in the dental pulp tissue and HDPCs. SIRT6 knockdown decreased ALP activity in HDPCs; calcium nodule formation ability; and the expression of mineralization-related genes such as ALP, DSPP, and DMP1, whereas these were increased with the overexpression of SIRT6.

Conclusions

SIRT6 is expressed in human dental pulp and participates in the odontoblast differentiation of HDPCs.     

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。     

胞内信号转导分子LIM矿化蛋白1在人牙髓细胞体外矿化过程中表达的实验研究

目的:研究胞内信号转导分子LIM矿化蛋白1在人牙髓细胞体外矿化过程中的表达。方法:利用组织块法体外培养人牙髓细胞,实验组培养液中加入矿化诱导剂,对照组不加矿化诱导剂。培养14d后,免疫细胞化学检测牙本质涎蛋白的表达;von kossa染色检测矿化结节的形成。培养期间,采用半定量RT-PCR技术监测LIM矿化蛋白1及碱性磷酸酶的表达,SPSS11.0软件分析结果。结果:培养14d,实验组牙本质涎蛋白免疫细胞化学呈阳性表达,von kossa染色阳性,矿化结节形成;对照组牙本质涎蛋白表达阴性,von kossa染色阴性。RT-PCR结果显示碱性磷酸酶及LIM矿化蛋白1在实验组和对照组各时段均表达。培养期间,实验组LIM矿化蛋白1的表达显著性高于对照组,其中培养第2天及9天时表达分别达到两个高峰,约为对照组的1.8倍及3.0倍。培养期间,碱性磷酸酶的表达显著性增高,其中培养14d时约为对照组的2.0倍。结论:首次发现LIM矿化蛋白1与人牙髓细胞的体外矿化过程相关,提示LIM矿化蛋白1可能在牙髓细胞的分化及矿化中发挥一定作用。     

人牙髓干细胞体外分选分化的初步实验研究

目的体外培养人牙髓细胞,分选牙髓干细胞并诱导其分化。方法选择因正畸目的而拔除的健康完整双尖牙,酶消化法进行牙髓细胞培养,并进行来源鉴定。单抗Stro-1标记牙髓干细胞、免疫磁珠分选系统进行分选。矿化液定向诱导分选后的牙髓干细胞,比较诱导前后stro-1染色及改良Gomori钙钴法染色检测碱性磷酸酶（ALP）的改变。结果体外培养人牙髓细胞呈成纤维细胞样,抗波形丝蛋白染色阳性,抗角蛋白染色阴性。牙髓干细胞Stro-1检测阳性,牙髓细胞中干细胞阳性率约为10％。矿化诱导后细胞stro-1阴性、ALP阳性表达。结论采用免疫磁珠分选系统分离出人牙髓干细胞,初步验证干细胞分化潜能,为其后续生物学特性研究提供实验基础。     

Potential Roles of Bone Morphogenetic Protein 9 in the Odontogenic Differentiation of Dental Pulp Cells

IntroductionThe differentiation of dental pulp cells (DPCs) plays an important role in the repair of dental pulp injury. Bone morphogenetic protein 9 (BMP9) is one of the most effective BMPs to induce the differentiation of stem cells. However, the role of BMP9 in promoting the odontogenic differentiation of DPCs and dentinogenesis is worth knowing.MethodsFluorescence in situ hybridization and immunohistochemistry staining were performed to detect the BMP9 expression in human dental pulp. BMP9 was overexpressed in human DPCs (hDPCs), and the mineralization of hDPCs was tested by alkaline phosphatase staining and alizarin red staining. The expression of odontogenic differentiation-related genes was examined by quantitative real-time polymerase chain reaction and western blotting. The subcutaneous transplantation experiment was performed to test the odonto-induction ability of BMP9 in vivo. The rat direct pulp-capping experiment was performed to test the function of BMP9 in promoting dentin formation.ResultsBMP9 showed an increased expression in odontoblast layer at both the mRNA and protein levels. BMP9 enhanced the mineralization and induced the expression of odontogenic differentiation-related genes in hDPCs. More mineralized nodules, and increased expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP1) were detected in the beta-tricalcium phosphate scaffold/cells composites of BMP9 group compared with the control group. Meanwhile, there was thicker reparative dentin formation in the BMP9 group in the rat pulp exposure experiment.ConclusionsBMP9 participates in the process of DPC differentiation and promotes DPC mineralization and dentinogenesis. BMP9 might be a potential therapeutic target in the repair of dental pulp injury.     

The Role of Small Extracellular Vesicles Derived from Lipopolysaccharide-preconditioned Human Dental Pulp Stem Cells in Dental Pulp Regeneration

IntroductionRegenerative endodontics has created a desirable shift in the treatment paradigm despite current limitations of regenerative outcomes. Mesenchymal stem cells (MSCs) facilitate tissue regeneration and repair in a mild inflammatory environment. Small extracellular vesicles (sEVs) derived from MSCs play an imperative role in the paracrine modulation of regenerative responses modulated by MSCs. However, it remains unknown whether MSCs enhance dental pulp regeneration or whether this enhancement is mediated by sEVs in a mild inflammatory environment. The present study aimed to elucidate the effects of sEVs originated from lipopolysaccharide (LPS)-preconditioned human dental pulp stem cells (hDPSCs) on dental pulp regeneration.MethodsAll sEVs were isolated from hDPSCs cultured with or without LPS (ie, N-sEVs and L-sEVs, respectively). The effect of N-sEVs and L-sEVs on proliferation, migration, angiogenesis, and differentiation of rat bone marrow MSCs was identified in vitro. Moreover, N-sEVs or L-sEVs were implanted into rat pulpless root canal models, and the regenerated tissue in root canals was assessed via hematoxylin-eosin staining, Masson staining, and immunohistochemistry after 30 days of transplantation.ResultsBoth N-sEVs and L-sEVs could modulate BMSC proliferation, migration, angiogenesis, and differentiation. Both kinds of sEVs enhanced the structure of the regenerated tissue closer to that of a normal dental pulp in vivo. L-sEVs had a more significant effect than N-sEVs.ConclusionssEVs released by hDPSCs in a mild inflammatory microenvironment are capable of facilitating the regeneration of dental pulp through functional healing instead of scar healing, which has potential applications in regenerative endodontics.     

Activation of Transient Receptor Potential Ankyrin 1 and Vanilloid 1 Channels Promotes Odontogenic Differentiation of Human Dental Pulp Cells

IntroductionTransient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) are thermosensitive channels that play an important role in thermal sensation or tooth pain by regulating intracellular Ca²⁺ concentration that is essential for pulp tissue repair. The aim of this study was to evaluate the role of TRPA1 and TRPV1 channels in the odontogenic differentiation of human dental pulp cells (HDPCs).MethodsHDPCs were isolated from healthy human intact third molars and cultured in odontogenic differentiation medium. Gene and protein expression levels of TRPA1 and TRPV1 channels during the odontogenic differentiation of HDPCs were evaluated by real-time quantitative polymerase chain reaction and Western blot analysis. HDPCs were then treated with channel agonists or antagonists, and the expression levels of odontogenic markers dentin sialophosphoprotein (DSPP) and osteopontin (OPN) were examined. Alkaline phosphatase activity and alizarin red staining were also conducted to detect mineralization levels.ResultsConsistent with the mineralization degree and DSPP and OPN expression, messenger RNA and protein expression of TRPA1 and TRPV1 channels was up-regulated during the odontogenic differentiation of HDPCs. The application of TRPA1 or TRPV1 agonists increased the mineralized nodules of alizarin red staining and alkaline phosphatase activity and up-regulated the messenger RNA and protein expression of DSPP and OPN, respectively, with the highest values reached on the seventh day (P < .05). On the contrary, the mineralization level and DSPP and OPN expression could be suppressed by using the antagonists of these 2 channels.ConclusionsTRPA1 and TRPV1 channels not only showed up-regulated expression along with the odontogenic differentiation of HDPCs but also could affect the odontogenic differentiation by regulating intracellular Ca²⁺ concentration.     

Enhancing Effects of Immobilized Chondroitin Sulfate on Odontogenic Differentiation of Dental Pulp Stem Cells and Reparative Dentin Formation

IntroductionChondroitin sulfate (CS) is a major proteoglycan involved in the mineralization of the organic matrix of dentin. In this study, the roles of CS immobilized in cross-linked collagen I (Col I) hydrogels on odontogenic differentiation of dental pulp stem cells (DPSCs) and reparative dentin formation were investigated.MethodsDifferent concentrations of CS were incorporated into the genipin–cross-linked Col I hydrogels (CS-0.05, CS-0.1, and CS-0.2, respectively). The influences of CS on the proliferation and odontogenic differentiation of DPSCs were investigated. Finally, the effect of the functionalized hydrogel on the formation of reparative dentin was analyzed in a rat pulp capping model in vivo.ResultsCS improved the proliferation of DPSCs seeded on the hydrogels (P < .05). CS also enhanced the mineralization activities and increased the expression levels of the odontogenic-related proteins of DPSCs on days 7 and 14 (P < .05). In vivo, CS-0.1 hydrogel induced reparative dentin formation with higher quality compared with mineral trioxide aggregate.ConclusionsCS immobilized in Col I hydrogels could induce odontogenic differentiation of DPSCs in vitro and promote homogeneous mineralized barrier formation in vivo. CS–Col I hydrogel has the potential for reparative dentin formation of high quality in direct pulp capping.     

内毒素对重组人成骨蛋白-1诱导人牙髓细胞活性的影响

目的：研究具核梭杆菌的内毒素（ＬＰＳ）对重组人成骨蛋白－１（ｒｈＯＰ－１）诱导牙髓细胞增殖、分化的影响。方法：采用噻唑盐比色测定（ＭＴＴ）、酶动力学方法和放射免疫技术。结果：在较低浓度ＬＰＳ作用时,可协同增加ｒｈＯＰ－１对牙髓细胞增殖和碱性磷酸酶（ＡＬＰａｓｅ）活性,但对骨钙素（ＢＧＰ）无明显影响,而高浓度ＬＰＳ则起抑制作用。结论：提示适度的ＬＰＳ在牙髓对龋病的防御反应过程中起着一定调节作用。     

Effect of Verapamil,a Calcium Channel Blocker,on the Odontogenic Activity of Human Dental Pulp Cells Cultured with Silicate-based Materials

Introduction

This study examines how calcium silicate cement extracts influence the behavior of human dental pulp cells (hDPCs) through calcium channels and active mitogen-activated protein kinase pathways, in particular extracellular signal-related kinase (ERK).

Methods

HDPCs are treated with various silicon concentrations both with and without verapamil, after which the cells’ viability and odontogenic differentiation markers are determined by using PrestoBlue assay and Western blot, respectively.

Results

The silicon promoted cell proliferation and inhibited calcium channel blockers. It was also found that silicon increased ERK and p38 activity in a dose-dependent manner. Furthermore, it raised the expression and secretion of alkaline phosphatase, osteocalcin, dentin sialophosphoprotein, and dentin matrix protein-1. In addition, statistically significant differences (P < .05) have been found in the secretion of osteocalcin in ERK inhibitor + verapamil between the silicon concentrations; these varations are dose-dependent and indicate that ERK signaling is involved in the silicon-induced odontogenic differentiation of hDPCs.

Conclusions

The current study shows that silicon ions released from calcium silicate substrates play a key role in odontoblastic differentiation of hDPCs through calcium channels and modulate ERK activation.     

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.     

Lysophosphatidic Acid Rescues Human Dental Pulp Cells from Ischemia-induced Apoptosis

Introduction

Dental pulp is particularly susceptible to ischemic conditions (hypoxia and serum deprived) because it is commonly exposed to trauma, inflammation, chronic caries injury, and pulpitis. We investigated the apoptotic response of human dental pulp cells (HDPCs) to varying levels of oxygen and serum to mimic different degrees of ischemia, tested whether lysophosphatidic acid (LPA) could reverse ischemia-induced apoptosis, and investigated the possible mechanisms of LPA.

Methods

HDPCs were cultured under conditions mimicking serum deprivation and ischemia for 2 days with or without LPA at 25 μg/mL. Flow cytometry and JC-1 fluorescence were used to detect any apoptotic change. Western blotting was used to measure the expression of the apoptosis regulators B-cell lymphoma 2 (Bcl-2) and Bax, focal adhesion kinase (FAK), Src, extracellular signal-regulated kinase (ERK), and Akt.

Results

Flow cytometry and JC-1 immunofluorescence showed that ischemia could induce apoptosis of HDPCs in 2 days and treatment with LPA could reduce cell death significantly. To clarify the molecular mechanisms, Western blot results showed up-regulation of both proapoptotic Bax and antiapoptotic Bcl-2 during apoptosis. LPA functioned as an antiapoptotic cytokine by activation of the phosphorylation of FAK and ERK. No statistically significant difference was found in the activation levels of p-Src or p-Akt.

Conclusions

A self-defense mechanism functioned during cell apoptosis. LPA could effectively rescue HDPCs from ischemia-induced apoptosis via regulation of Bax and Bcl-2 and the activation of phosphorylated FAK and phosphorylated ERK. LPA is a potent candidate for biological therapy of chronic pulpal inflammatory diseases.     

人牙髓干细胞体外分选分化的初步实验研究

目的 体外培养人牙髓细胞,分选牙髓干细胞并诱导其分化.方法 选择因正畸目的 而拔除的健康完整双尖牙,酶消化法进行牙髓细胞培养,并进行来源鉴定.单抗Stro-1标记牙髓干细胞、免疫磁珠分选系统进行分选.矿化液定向诱导分选后的牙髓十细胞,比较诱导前后Stro-1染色及改良Gomori钙钴法染色检测碱性磷酸酶(ALP)的改变.结果 体外培养人牙髓细胞呈成纤维细胞样,抗波形丝蛋白染色阳性,抗角蛋白染色阴性.牙髓干细胞Stro-1检测阳性,牙髓细胞中干细胞附性率约为10%.矿化诱导后细胞Stro-1阴性、ALP阳性表达.结论 采用免疫磁珠分选系统分离出人牙髓干细胞,初步验证干细胞分化潜能,为其后续生物学特性研究提供实验基础.     

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.

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     

Mesenchymal Stem Cells Derived from Human Inflamed Dental Pulp Exhibit Impaired Immunomodulatory Capacity In Vitro

IntroductionDental pulp stem cells (DPSC) are very attractive in regenerative medicine. In this study, we focused on the characterization of the functional properties of mesenchymal stem cells derived from DPSCs. Currently, it is unknown whether inflammatory conditions present in an inflamed dental pulp tissue could alter the immunomodulatory properties of DPSCs. This study aimed to evaluate the immunomodulatory capacity in vitro of DPSCs derived from healthy and inflamed dental pulp.MethodsDPSCs from 10 healthy and inflamed dental pulps (irreversible pulpitis) were characterized according to the minimal criteria of the International Society for Cell Therapy, proliferation, differential potential, and colony-forming units. Furthermore, the immunomodulatory capacity of DPSCs was tested on the proliferation of T lymphocytes by flow cytometry and the in vitro enzyme activity of indoleamine 2, 3-dioxygenase.ResultsThere were no significant differences in the DPSC characteristics and properties such as immunophenotype, tridifferentiation, colony-forming units, and proliferation of the DPSCs derived from normal and inflamed pulp tissue. Furthermore, there were significant differences in the immunomodulatory capacity of DPSCs obtained from human healthy dental pulp and with the diagnosis of irreversible pulpitis.ConclusionsOur results showed that DPSCs isolated from inflamed dental pulp showed typical characteristics of MSCs and diminished immunosuppressive capacity in vitro in comparison with MSCs derived from healthy dental pulp. Further investigation in vivo is needed to clarify the mechanism of this diminished immunosuppressive capacity.