An in vitro anti-inflammatory effect of Thai propolis in human dental pulp cells

Abstract Objective To explore the potential for development of Thai propolis extract as a pulp capping agent to suppress pulpal inflammation from dental pulp infections. This study aimed to examine the anti-inflammatory effect of the propolis extract on the arachidonic acid pathway, activated by interleukin (IL)-1β, in cultured human dental pulp cells. Methodology Dental pulp cells, isolated from three freshly extracted third molars, were first characterized for their mesenchymal origin and treated with 10 ng/ml of IL-1β in the presence or absence of non-toxic concentrations of the extract from 0.08 to 1.25 mg/ml, as determined by the PrestoBlue cytotoxic assay. Total RNA was harvested and analyzed for mRNA expressions of 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2). Western blot hybridization was performed to investigate COX-2 protein expression. Culture supernatants were assayed for released prostaglandin E2 levels. Immunofluorescence was conducted to determine involvement of nuclear factor-kappaB (NF-kB) in the inhibitory effect of the extract. Results Stimulation of the pulp cells with IL-1β resulted in the activation of arachidonic acid metabolism via COX-2, but not 5-LOX. Incubation with various non-toxic concentrations of the propolis extract significantly inhibited upregulated COX-2 mRNA and protein expressions upon treatment with IL-1β (p<0.05), resulting in a significant decrease in elevated PGE2 levels (p<0.05). Nuclear translocation of the p50 and the p65 subunits of NF-kB upon treatment with IL-1β was also blocked by incubation with the extract. Conclusions Upregulated COX-2 expression and enhanced PGE2 synthesis upon treatment with IL-1β in human dental pulp cells were suppressed by incubation with non-toxic doses of Thai propolis extract via involvement of the NF-kB activation. This extract could be therapeutically used as a pulp capping material due to its anti-inflammatory properties.

Study of endoplasmic reticulum stress in human dental pulp cells induced by oxygen-glucose deprivation culture in vitro / 口腔疾病防治

Objective@#Oxygen-glucose deprivation (OGD) is used to mimic ischemia in vitro to observe whether endoplasmic reticulum (ER) stress is involved in human dental pulp cells (hDPCs) after OGD and to better understand the regulatory mechanism of hDPCs in ischemia.@*Methods@# hDPCs were cultured in glucose-free DMEM and hypoxia (volume fraction 2% O2) to establish an hDPCs OGD model in vitro, which mimics hDPCs ischemia in vitro. hDPCs were divided into a control group (normal culture) and an experimental group (OGD 0 h, 2 h, 4 h and 8 h groups). After pretreatment with OGD for 0, 2, 4 and 8 h, hDPC viability was measured by methylthiazol tetrazolium (MTT) assay. qRT-PCR was used to detect the mRNA expression of ER stress markers [splicing x-box binding protein1 (sXBP1), activating transcription Factor 4 (ATF4) and C/EBP homologous protein (chop)]. Western blot was used to detect the protein expression of ER stress markers [phosphorylated RNA-activated protein kinase-like ER-resident kinase (p-perk) and phosphorylated eukaryotic initiation factor-2α (p-eIF2α)]. @*Results@#Compared with OGD 0 h group, cell viability of hDPCs decreased when exposed to OGD treatment for 2 h, 4 h and 8 h. Compared with the control group, mRNA expressions of ER stress makers (sXBP1, ATF4 and chop) and the protein expressions of ER stress protein markers (p-perk andp-eIF2α) increased in OGD treatment cells after 4 h were higher in OGD cells. The differences were statistically significant (P<0.05).@*Conclusion@#The results indicate that ER stress response is involved in hDPCs in OGD treatment.

The effect of reduced glutathione on the toxicity of silver diamine fluoride in rat pulpal cells

Abstract Introduction Due to its ability to arrest untreated dental caries, silver diamine fluoride (SDF) has been advocated for indirect pulp capping procedures. However, the high concentrations of silver and fluoride in SDF raise concerns about its biocompatibility to pulpal tissues. Objectives This study aimed to investigate the effect of SDF on the viability, alkaline phosphatase (ALP) activity, and morphology of pulpal-like cells (RPC-C2A) and to evaluate the influence of reduced glutathione (GSH) on SDF-induced cytotoxicity and deposit formation on dentin. Methodology The cytotoxicity of diluted 38% SDF solutions (10-4 and 10-5), with or without the addition of 5 mM or 50 mM GSH, was evaluated at 6 and 24 hours. Cell viability was detected using WST-8 and the effect on ALP activity was performed using an ALP assay kit. Cell morphology was observed using a phase-contrast microscope. Scanning electron microscopy analysis was conducted to evaluate the effect of GSH incorporation or conditioning on SDF-induced deposit formation on dentin discs. Cytotoxicity data were analyzed by two-way analysis of variance (ANOVA) and Tukey post hoc tests (p<0.05). Results There were significant differences between the groups. The results demonstrated that all tested SDF dilutions caused a remarkable cytotoxic effect, while the addition of GSH prevented SDF-induced damage at 6-hour exposure time in the higher dilution of SDF. Dentin treated with plain SDF or GSH-incorporated SDF solution showed deposit formation with occluded dentinal tubules, unlike the other groups. Conclusion SDF severely disturbed the viability, mineralization-ability, and morphology of pulpal-like cells, while controlled concentrations of GSH had a short-term protective effect against SDF-induced damage. GSH showed an inhibitory effect on SDF-induced dentinal deposit formation. Further research is warranted to evaluate the effect of GSH on caries-arresting, anti-hypersensitivity, and antibacterial functions of SDF.

High glucose affects proliferation, reactive oxygen species and mineralization of human dental pulp cells

Abstract Diabetes is a group of metabolic disorders that can lead to damage and dysfunction of many organs including the dental pulp. Increased inflammatory response, reduction of dentin formation and impaired healing were reported in diabetic dental pulp. Hyperglycemia, which is a main characteristic of diabetes, was suggested to play a role in many diabetic complications. Therefore our aim was to investigate the effects of high glucose levels on proliferation, reactive oxygen species (ROS) production and odontogenic differentiation of human dental pulp cells (HDPCs). HDPCs were cultured under low glucose (5.5mM Glucose), high glucose (25 mM Glucose) and mannitol (iso-osmolar control) conditions. Cell proliferation was analyzed by MTT assay for 11 days. Glutathione and DCFH-DA assay were used to assess ROS and antioxidant levels after 24 h of glucose exposure. Odontogenic differentiation was evaluated and quantified by alizarin red staining on day 21. Expression of mineralization-associated genes, which were alkaline phosphatase, dentin sialophosphoprotein and osteonectin, was determined by RT-qPCR on day 14. The results showed that high glucose concentration decreased proliferation of HDPCs. Odontogenic differentiation, both by gene expression and mineral matrix deposit, was inhibited by high glucose condition. In addition, high DCF levels and low reduced glutathione levels were observed in high glucose condition. However, no differences were observed between mannitol and low glucose conditions. In conclusion, the results clearly showed the negative effect of high glucose condition on HDPCs proliferation and differentiation. Moreover, it also induced ROS production of HDPCs.

Resumo O diabetes abrange um grupo de distúrbios metabólicos que podem levar a danos e disfunções de muitos órgãos, incluindo a polpa dentária. Aumento da resposta inflamatória, redução da formação de dentina e comprometimento da cicatrização foram relatados na polpa dentária diabética. A hiperglicemia, que é uma característica determinante do diabetes, desempenha um papel importante em muitas complicações diabéticas. Portanto, nosso objetivo foi investigar os efeitos dos altos níveis de glicose na proliferação, produção de espécies reativas de oxigênio (ROS, em inglês) e diferenciação odontogênica das células da polpa dental humana (HDPCs, em inglês). As HDPCs foram cultivadas em condições de baixa glicose (glicose 5,5 mM), alta glicose (glicose 25 mM) e manitol (controle iso-osmolar). A proliferação celular foi analisada pelo ensaio MTT por 11 dias. Glutationa e DCFH-DA foram utilizados para avaliar os níveis de ROS e antioxidantes após 24 h de exposição à glicose. A diferenciação odontogênica foi avaliada e quantificada pela coloração com vermelho de alizarina no dia 21. A expressão de genes associados à mineralização, que eram fosfatase alcalina, sialofosfoproteína de dentina e osteonectina, foi determinada por RT-qPCR no dia 14. Os resultados mostraram que a alta concentração de glicose diminuiu a proliferação de HDPCs. A diferenciação odontogênica, tanto pela expressão gênica quanto pelo depósito da matriz mineral, foi inibida pela condição de alta glicose. Além disso, altos níveis de DCF e níveis reduzidos de glutationa foram observados na condição de alta glicose. No entanto, não foram observadas diferenças entre o manitol e as condições de baixa glicose. Em conclusão, os resultados mostraram claramente o efeito negativo da condição de alta glicose na proliferação e diferenciação de HDPCs. Além disso, essa condição também induziu a produção de ROS em HDPCs.

Biodentine Induce Proteínas Apoptóticas Caspasa 3 y PARP-1 en Células de la Pulpa Dental Humana / Biodentine Induces Apoptotic Proteins Caspase 3 and PARP - 1 on Human Dental Pulp Cells

RESUMEN: Las patologías pulpares han sido un verdadero reto para la odontología principalmente por su tratamiento. Actualmente, existen numerosos biomateriales en el mercado que reportan tener propiedades inherentes en los tejidos dentarios. Sin embargo, diferentes estudios sobre múltiples líneas celulares expuestas a estos biomateriales demuestran resultados controversiales como biocompatiblidad y citotoxicidad celular. Biodentine, es un cemento endodóntico en base a silicatos cálcico de múltiples aplicaciones, que prestaría propiedades de biocompatibilidad como bioactividad celular, características que le permitirían incluso ser utilizado en contacto directo con la pulpa dental. El objetivo de este estudio es la evaluación in-vitro de Biodentine, sobre cultivos de células de la pulpa dental humana (CCPDH). Se prepararon discos de cemento de Biodentine™ de 2 x 6 mm, los que se expusieron a cultivos de células aisladas de la pulpa dental humana. Luego de 24, 48 y 72 horas de exposición, se realizaron ensayos de viabilidad celular utilizando el método colorimétrico MTT. También se realizaron ensayos de expresión proteica de dos proteínas involucradas en la vía de señalización de la apoptosis celular: Caspasa - 3 clivada y Poli (ADP-Ribosa) Polimerasa, PARP - 1. Existen diferencias estadísticamente significativas (p<0,05) en los ensayos de viabilidad celular entre las células expuestas a Biodentine y el grupo control, como también a medida que aumenta el tiempo de exposición (p<0,05). Por otra parte, también existen diferencias significativas (p<0,05) en la expresión de PARP- 1 en los grupos sometidos a Biodentine. Los resultados obtenidos en este estudio demuestran que Biodentine genera citotoxicidad celular en cultivos celulares de pulpa dental humana, por disminución de la viabilidad celular como por la expresión de proteínas apoptóticas. Es por esto que la utilización de este biomaterial debería ser estudiado y considerarse en cada caso clínico, especialmente como recubridor pulpar directo.

ABSTRACT: Oral pathologies have been a real challenge for dentistry, mainly due to its treatment. Currently, there are numerous biomaterials on the market that may present inherent properties in dental tissues. However, studies on multiple cell lines are based on biocompatible results such as biocompatibility and cellular cytotoxicity. Biodentine is endodontic cement based on calcium silicates of multiple applications, which would provide biocompatibility properties as cellular bioactivity, characteristics that will allow it to be used in direct contact with the dental pulp. The objective of this study is the in vitro evaluation of Biodentine, on cultures of cells of the human dental pulp (HDPC). Biodentine cement disks of 2 x 6 mm were prepared, and HDPC culture plates were introduced. After 24, 48 and 72 hours of exposure, cell viability tests were performed using the MTT colorimetric method. On the other hand, protein expression assays of two proteins involved in the signaling pathway of cell apoptosis Caspase-3 cleaved (cas-3 clv) and PARP-1 are carried out. There are statistically significant differences (p <0,05) in the cell viability tests between Biodentine and control group, as well as the exposure time increases (p <0,05). Otherwise, there are also significant differences (p <0,05) in the expression of PARP-1 in the groups, sometimes a Biodentine. The results in this study that Biodentine generates a cellular cytotoxicity in HDPC cultures, therefore, cell viability as the expression of apoptotic proteins. This is why the use of this biomaterial should be studied for each particular clinical case, especially as a direct pulp capping agent.

Effects of papain-based gel used for caries removal on macrophages and dental pulp cells

Abstract Papain-based gel is used for chemical-mechanical caries removal and present antimicrobial and anti-inflammatory activities. However, its effects on dental pulp cells and on macrophages remains largely unknown. Therefore, the aim of this study was to investigate whether the papain-based gel Papacárie Duo® acts as an immunomodulator in lipopolysaccharide (LPS)-activated macrophages and its effects on dental pulp cells . J774.1 macrophage and OD-21 dental pulp cells were stimulated with 0.5% and 5% of Papacárie Duo®, following pre-treatment or not with LPS. After 24 h, a lactate dehydrogenase assay was used to measure cytotoxicity, a tetrazolium-based colorimetric assay (MTT) was used to measure cell viability, and qRT-PCR was used to analyze relative gene expression of Ptgs2, Il10, Tnf, Mmp9, Runx2, Ibsp and Spp1. Papacárie Duo® was cytotoxic and reduced cell viability at 5% but not at 0.5% in both cultures. In macrophages, Papacárie Duo® increased the expression Il10 and LPS-induced Ptgs2, but it did not affect Tnf or Mmp9. In OD-21 cells, Papacárie Duo® inhibited Runx2 and Ibsp expression, but stimulated Spp1 expression. Papain-based gel presented a concentration dependent cytotoxicity, without affecting cell viability, for dental pulp cells and macrophages. Interestingly, the gel presented an inhibitory effect on pulp cell differentiation but modulated the activation of macrophages stimulated with LPS. We speculate that in dental pulp tissue, Papacárie Duo® would impair reparative dentinogenesis but could activate macrophages to perform their role in defense and inflammation.

Resumo O gel à base de papaína é utilizando para remoção químico-mecânica do tecido cariado e apresenta propriedades antimicrobianas e anti-inflamatórias Entretanto, seu efeito sobre as células da polpa dentárias e macrófagos é desconhecido. Portanto, o objetivo deste estudo foi investigar o efeito de um gel de papaína (Papacárie Duo®) em células indiferenciadas da polpa dentária e a capacidade de induzir a ativação e síntese de mediadores inflamatórios por macrófagos estimulados com lipopolissacarídeo bacteriano (LPS). O gel de papaína foi diluído nas concentrações de 0,5 e 5%. Células indiferenciadas da polpa dentária OD-21 e macrófagos J774.1 foram mantidos em cultura com os diferentes estímulos por um período de estimulação de 24 h para realização do teste de citotoxicidade (Ensaio LDH) e para avaliação da viabilidade celular (Ensaio Colorimétrico MTT). A seguir foi realizada avaliação da expressão gênica relativa dos genes Ibsp, Runx2 e Spp1 em células OD-21; e dos genes Il10, Mmp9, Ptgs2 e Tnf em células J774.1, pelo método de transcrição reversa e reação em cadeia de polimerase em tempo real (qRT-PCR), após estimulação pelo período de 24 h. O extrato do gel diluído a 5% foi citotóxico às células da polpa dental, reduziu a viabilidade celular, inibiu a expressão de Runx2 e Ibsp e estimulou a expressão de Spp1. Em macrófagos, o extrato do gel foi citotóxico e reduziu a viabilidade celular na concentração de 5%. O LPS inibiu a viabilidade celular na presença ou não do extrato do gel, sem apresentar citotoxicidade. O extrato do gel induziu a expressão de Ptgs2 e Il10, sem alterar Tnf e Mmp9. O extrato do gel de papaína foi citotóxico, dependente da concentração, tanto em células da polpa dentária como em macrófagos, sem alterar a viabilidade celular. Interessantemente, apresentou efeito inibitório na diferenciação de células da polpa dentária e modulou a ativação de macrófagos estimulados com LPS. No tecido pulpar, o Papacárie Duo® poderia impedir a dentinogênese de reparação, porém ativar macrófagos para desempenhar seu papel na inflamação e defesa.

The role of APOD in the proliferation and migration of human dental pulp cells / 口腔疾病防治

Objective@#To explore the role of apoliprotein D (APOD) in the proliferation and migration of human dental pulp cells (DPCs) and to provide a basis for the use of APOD to promote pulp regeneration. @*Methods@#APOD expression in human dental pulp cells was inhibited by siRNA. The inhibition effect of APOD was confirmed by qPCR and Western blot. After APOD inhibition, colony formation experiments and CCK8 assays were employed to confirm the proliferation ability of dental pulp cells. Transwell assays were used to verify the cell migration ability after the inhibition of APOD expression.@*Results @# After inhibiting APOD expression, the colony formation rate in the si-apod group was reduced compared with the NC group, and the difference was statistically significant (t=7.624, P=0.002). The CCK8 experiment showed that the OD value in the si-apod group decreased at 3, 5 and 7 d compared with that in the NC group (P < 0.05). Transwell results showed that the number of cell divisions was 57.25 ± 4.03 in the si-apod group and 154.50 ± 8.39 in the NC group, and the difference was statistically significant (t=10.45, P < 0.001).@*Conclusion@# Inhibition of APOD expression in dental pulp cells inhibits their proliferation and migration ability.

Effect of Notch Signal and Autophage on MTA induced Proliferation of Human Dental Pulp Cells in Vitro / 现代生物医学进展

Objective:Mineral trioxide aggregate (MTA),a wildly used pulp capping material,could affect the proliferation and differentiation of dental pulp cells.The aim of this study is to study the roles of Notch signalling and autophagy in MTA induced human dental pulp cells (hDPCs) proliferation promotions.Methods:Healthy human third molars were collected and hDPCs were isolated by a combined digestion of collagenase Ⅰ and dispase Ⅱ.MTA extracts of different concentrations (0.5,1.0,2.0,5.0,10.0 mg/mL) were used to test the cytotoxicity by cells counting kit (CCK-8) assays and to select the optimum concentration for hDPCs survival..Expressions of Notch1,Hes1,LC3Ⅱ / Ⅰ and p62 in wild type and MTA treated hDPCs were detected by western blotting.Results:MTA extracts in a concentration of 1.0 mg/mL exerted most profoundly promotion effects on the proliferation of hDPCs among all concentrations tested.MTA of high concentration (10 mg/mL) was toxically to cells.Compared with that of wild type hDPCs,the expressions of Notch1 and Hes1(P＜0.05),or p62 and LC3Ⅱ/Ⅰ (P＜0.01) in MTA treated hDPCs were significantly increased.Much lower expression of Notch1 was detected in hDPCs when autophagy was induced by Earle's balanced salt solution (EBSS) starvation for 24 h.Conclusions:MTA could up-regulated hDPCs proliferation with highly relevant in stimulating Notch1-Hes1 signalling and inhibition of autophagy.The study is supposed to provide new insight in unrevealing the mechanisms of MTA mediated dental pulp cells proliferation.

Influence of the Arg-Gly-Asp-Ser sequence on the biological effects of bioactive glass on human dental pulp cells / 北京大学学报(医学版)

Objective:Positive effects of bioactive glass (BG) on proliferation,mineralization,and differentiation of human dental pulp cells (hDPCs) was already verified in various former studies.The Arg-Gly-Asp-Ser sequence (RGDS) was confirmed of affecting cell adhesion.Before further investigation,the objective of this study is to investigate whether RGDS can affect the effects of BG on the adhesion,proliferation and mineralization of hDPCs.Methods: hDPCs were harvested from third molars of 18-25-year-old individuals after informed consent.Enzyme digestion technique was used.The 4th to 6th ge-neration of hDPCs were used for all experiments.The cells of the experimental groups were cultured in Dulbecco minimum essential medium (DMEM) containing ionic dissolution products of BG and RGDS of seve-ral concentrations (12.5 mg/L,25.0 mg/L,50.0 mg/L,100.0 mg/L,200.0 mg/L).DMEM containing ionic dissolution products of BG without RGDS was used for cell culture as control group.Cell adhesion was tested 4 h after cell seeding by MTT assay.Cell proliferation was examined at 1,3,5,7,and 9 d after cell seeding by MTT assay.Cell mineralization was investigated on days 14 and 28 by alizarin red staining.After being stained and dried,mineralized nodules were dissolved by cetylpyridinium chloride (CPC) for semi-quantitative test.Results were statistically analyzed by one way ANOVA,SPSS (version 19.0) and P<0.05 was considered to be significant.Results: Cell adhesion in BG group showed no difference from that in DMEM group.Compared with BG group,hDPCs in BG+RGDS groups suggested weaker cell adhesion.When the concentration of RGDS increased,the adhered cell number decreased.hDPCs cultured with BG and RGDS showed lower proliferation activity in the early stage,while no significant difference was observed after 3 d.BG group promoted the mineralization of hDPCs compared with positive control group,negative control group and RGDS group.No significant difference was observed between BG+RGDS group and BG group or between RGDS group and positive control group.Conclusion: BG promotes proliferation and mineralization without affecting cell adhesion of hDPCs.Unbounded RGDS inhibits cell adhesion,but has no influence on the positive effects of BG on the proliferation and mineralization of hDPCs.

Induction of Prostaglandin E₂ by Porphyromonas gingivalis in Human Dental Pulp Cells

Cyclooxygenase-2 (COX-2)-mediated prostaglandin E₂ (PGE₂) plays a key role in development and progression of inflammatory responses and Porphyromonas gingivalis is a common endodontic pathogen. In this study, we investigated induction of COX-2 and PGE₂ by P. gingivalis in human dental pulp cells (HDPCs). P. gingivalis increased expression of COX-2, but not that of COX-1. Increased levels of PGE₂ were released from P. gingivalis-infected HDPCs and this PGE₂ increase was blocked by celecoxib, a selective COX-2 inhibitor. P. gingivalis activated all three types of mitogen-activated protein kinases (MAPKs). P. gingivalis-induced activation of nuclear factor-κB (NF-κB) was demonstrated by the results of phosphorylation of NF-κ B p65 and degradation of inhibitor of κB-α (IκB-α). Pharmacological inhibition of each of the three types of MAPKs and NF-κB substantially attenuated P. gingivalis induced PGE2 production. These results suggest that P. gingivalis should promote endodontic inflammation by stimulating dental pulp cells to produce PGE₂.

Melatonin Rescues Human Dental Pulp Cells from Premature Senescence Induced by H₂O₂

Although anti-aging activities of melatonin, a hormone secreted by the pineal gland, have been reported in senescence-accelerated mouse models and several types of cells, its impact and mechanism on the senescence of human dental pulp cells (HDPCs) remains unknown. In this study, we examined the impact of melatonin on cellular premature senescence of HDPCs. Here, we found that melatonin markedly inhibited senescent characteristics of HDPCs after exposure to hydrogen peroxide (H₂O₂), including the increase in senescence-associated β-galactosidase (SA-β-gal)-positive HDPCs and the upregulation of p21 protein, an indicator for senescence. In addition, as melatonin attenuated H₂O₂-stimulated phosphorylation of c-Jun N-terminal kinase (JNK), while selective inhibition of JNK activity with SP600125 significantly attenuated H₂O₂-induced increase in SA-beta-gal activity. Results reveal that melatonin antagonizes premature senescence of HDPCs via JNK pathway. Thus, melatonin may have therapeutic potential to prevent stress-induced premature senescence, possibly correlated with development of dental pulp diseases, and to maintain oral health across the life span.

Effect of Notch signaling and autophagy on MTA induced differentiation of human dental pulp cells in vitro / 口腔疾病防治

Objective@# The aim of this study is to investigate the roles of Notch signaling and autophagy on mineral trioxide aggregate (MTA) induced differentiation of human dental pulp cells (hDPCs). @*Methods @#Third molars from healthy human were collected and hDPCs were isolated by a combined digestion of collagenase Ⅰ and dispaseⅡ. Real time PCR were used to test the mRNA expression levels of alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and dentin sialophoprotein (DSPP) in MTA treated hDPCs in different time (24 h, 3 d and 7 d). The mineralization nodules formed by hDPCs with or without MTA treatment were detected by Von Kossa staining. Expressions of Notch1, Jagged1, Hes1, LC3Ⅱ/LC3 Ⅰand p62 in wild type and MTA treated hDPCs were detected by western blotting.@*Results@#MTA extracted in a concentration of 0.1 mg/mL could promote the differentiation of hDPCs. Compared with that of wild type hDPCs, the expressions of Notch1, Hes1, or Jagged1 and p62 (P<0.01) in MTA treated hDPCs were significantly increased. MTA treatment showed inhibition effects on autophagy flux similar to Bafilomycin A1, a specific inhibitor of fusion between autophagosomes and lysosomes. @*Conclusion@#MTA could promote hDPCs differentiation with highly relevant in stimulating Notch1-Jagged1-Hes1 signaling and inhibition of autophagy flux.

The effects of erythropoietin on the proliferation and osteogenic differentiation of human dental pulp cells / 实用口腔医学杂志

Objective:To evaluate the effects of erythropoietin (EPO)on the proliferation and osteogenic differentiation of human dental pulp cells(hDPCs)in vitro.Methods:Isolated hDPCs were cultured and identified.The cells were treated by EPO and the proliferation of the cells was determined by CCK-8 assay.After incubation with EPO at 20 U /ml in osteogenic induction medium for 7 and 1 4 days,the mineralization of hDPCs was observed by alkaline phosphatase (ALP)activity assay and alizarin red staining.Real-time PCR was utilized to detect the expression of odontogenesis-related genes.Results:EPO enhanced the proliferation of hDPCs in a time-and dose-dependent manner.After treatment with EPO,ALP activity and the minerialized nodes of the cells increased(P <0.05);the expression levels of odontogenesis-related genes DSPP,OCN,OSTERIX and RUNX2 were upregulated(P <0.05).Con-clusion:EPO can promote proliferation and differentiation of human dental pulp cells.

Effect of hypoxia on angiogenesis-related proteins in human dental pulp cells

Dental pulp is a highly vascularized tissue with high regenerative potential. Revascularization of severed vasculature in the tooth is required for pulp healing during avulsed tooth treatment. In this study, the relative expression of angiogenesis-related proteins was determined in human dental pulp cells using a human angiogenesis proteome profiler array. The proteome profiler array detected differentially expressed angiogenesis-related factors under conditions of hypoxia, which enhances the angiogenic potential of dental pulp cells. We confirmed that hypoxia regulates the mRNA expression of angiogenesis-related factors, including CXCL16 in dental pulp cells. Furthermore, conditioned media of hypoxic pulp cells induced tube-like structures of vascular endothelial cells, which were reduced by the neutralization of CXCL16 function. In conclusion, our data show that angiogenesis-related factors are differentially expressed by hypoxia in dental pulp cells and suggest that CXCL16 may involve in the revascularization of hypoxic dental pulp.

Dose-responses of Stem Cells from Human Exfoliated Teeth to Infrared LED Irradiation

Despite several reports regarding tissue regeneration, including pulp repair induced by different light sources, only limited data have been reported concerning the effects of light-emitting diodes (LED) on stem cells from human exfoliated deciduous teeth (SHEDs). The aim of this study was to evaluate the effects of different energy densities of infrared LED on the cell viability, number of cells and mineralized tissue production by SHEDs. SHEDs were obtained from near-exfoliation primary teeth (n=3), seeded in plain DMEM (104 cells/cm2), and irradiated by a LED prototype (LEDTable 850 nm, 40 mW/cm2) delivering 0 (control), 2, 4, 8, 15 or 30 J/cm2 (n=9). Cell viability (MTT assay), cell proliferation (trypan blue assay), and mineralized nodule (MN) formation (alizarin red stain) were assessed 12 and 72 h post-irradiation. Data were subjected to Kruskal-Wallis and Mann-Whitney tests (α=0.05). Cells irradiated with 2 or 4 J/cm2 exhibited higher metabolism at 72 h, and all energy densities provided increase in cell proliferation after 12 h. Regarding MN formation, the best results were observed at 72 h after SHED irradiation with 8 and 15 J/cm2. It was concluded that the cell viability, cell number and MN formation by pulp cells are enhanced after exposure to infrared LED irradiation. Overall, the greatest SHED biostimulation was obtained with 4 and 8 J/cm2.

Apesar de diversos estudos envolvendo regeneração tecidual, incluindo o reparo pulpar induzido por diferentes fontes de luz, dados limitados têm sido reportados a respeito dos efeitos da irradiação com diodos emissores de luz (LED) sobre células-tronco de dentes decíduos esfoliados (SHEDs). O objetivo do presente estudo foi avaliar os efeitos de diferentes doses de energia (DE) do LED infravermelho sobre a viabilidade celular, número de células viáveis e produção de nódulos mineralizados (NM) por SHEDs. As células foram obtidas a partir de dentes decíduos próximos ao período de esfoliação (n=3), semeadas em DMEM completo (10⁴ células/cm2) e irradiadas utilizando um protótipo de LED (LEDTable 850 nm, 40 mW/cm²⁾ com as doses de 0 (controle), 2, 4, 8, 15 ou 30 J/cm² (n=9). A viabilidade celular (MTT), o número de células viáveis (trypan blue assay) e a formação de NM (alizarin red stain) foram realizados 12 e 72 h após a irradiação. Os dados foram avaliados utilizando os testes Kruskal-Wallis e Mann-Whitney (α=0,05). As células irradiadas com 2 ou 4 J/cm² exibiram uma maior viabilidade em 72 h, e todas as DE aumentaram o número de células viáveis após 12 h. Para a formação de NM, os melhores resultados foram observados 72 h após a irradição das SHEDs, com as doses de 8 e 15 J/cm². Concluiu-se que a viabilidade celular, o número de células e a formação de NM por células pulpares são aumentados após exposição ao LED infravermelho. De um modo geral, a melhor bioestimulação celular (SHEDs) foi obtida com 4 e 8 J/cm².

The effects of cadherin-11 transfection on the migration and differentiation of rat dental pulp cells / 实用口腔医学杂志

Objective:To investigate the migration and odontogenesis of rat dental pulp cells transfected with adenovirus vector enco-ding cadherin-11.Methods:The dental pulp cells were cultured and transfected with pDC316-mCMV-EGFP-Cadherin-11.Cad-11 protein expression of the cells was examined by immunohistochemistry staining.The odontogenic differentiation of the cells was studied by alizarin red staining and ALP staining.The adhesion and migration of the cells were tested.Results:Transfection of pDC316-mC-MV-EGFP-Cadherin11 promoted Cad-11 protein expression and ALP activity,increased calcified nodule formation(P <0.05),adhe-sion and migration of rat dental pulp cells(P <0.05).Conclusion:Cadherin-11 may promote the odontoblast differentiation and mi-gration of rat dental pulp cells.

The effects of 2-hydroxyethyl methacrylate on the proliferation and migration of human dental pulp cells / 实用口腔医学杂志

Objective:To assess the effects of HEMA on the proliferation and migration of human dental pulp cells(hDPCs).Meth-ods:hDPCs were obtained using tissue explant culture technique in vitro,and then cells of the 3rd -5th passages were treated by differ-ent concentrations of HEMA for 24,48 and 72 h respectively.Cell proliferation was examined by MTT assay.Cell migration was ob-served by Transwell method.Results:The proliferation ability of hDPCs decreased when exposed to HEMA in both time and concentra-tion dependent manner(vs control,P <0.05).Cell proliferation at 24 h expossure was statistically higher than that at 48 h and 72 h(P<0.05).The migration of hDPCs was significantly reduced in HEMA groups at different concentrations(vs control,P <0.05).Con-clusion:HEMA inhibits the proliferation and migration of human dental pulp cells in vitro.

Electron Beam Irradiation of Resin Luting Agents - a Cytotoxic Evaluation on Dental Pulp Cells.

Aim: The aim of this study was to evaluate the cytotoxicity of three resin luting agents Rely X luting cement, Rely X luting 2 cement and Clearfil SA luting cement before and after electron beam irradiation. Materials and Methods: Growth and maintenance of cell cultures of human pulp cells was done in Dulbecco’s modified Eagle’s Medium (DMEM). The test samples were divided into two Categories: Irradiated Category and Non-radiated Category. Samples in Irradiated category were exposed to electron beam radiation at 200Gy. Three subgroups of radiated category and non radiated category were made. All the samples were subjected to MTT assay and spectrophotometric analysis and their cytotoxicity was assessed. Statistical analysis was done using t test. Results: Evaluation of Rely X luting Cement showed that radiated samples of powder, liquid and set cements showed decreased cell viability than non radiated samples. In Case of Rely X luting 2 cement, radiated samples showed increased cell viability for Paste A and Paste B samples. But in set material, irradiated samples showed decreased cell viability as compared to non radiated samples. For Clearfil SA luting Cement, Paste B showed increased cell viability for radiated samples. Paste A and Set cement of radiated samples showed decreased cell viability than non radiated samples. Conclusion: In the present study , the increased cytotoxicity of irradiated samples may be due to increase in the release of unbound monomers which may be due to chain breakage after irradiation and a reduction in the cytotoxicity which may be due to the cross linking of unbound monomers during irradiation.

Nitric Oxide-Induced Apoptosis of Human Dental Pulp Cells Is Mediated by the Mitochondria-Dependent Pathway

Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. NO is produced by nitric oxide synthase (NOS), and NOS is abundantly expressed in the human dental pulp cells (HDPCs). NO produced by NOS can be cytotoxic at higher concentrations to HDPCs. However, the mechanism by which this cytotoxic pathway is activated in cells exposed to NO is not known. The purpose of this study was to elucidate the NO-induced cytotoxic mechanism in HDPCs. Sodium nitroprusside (SNP), a NO donor, reduced the viability of HDPCs in a dose- and time-dependent manner. We investigated the in vitro effects of nitric oxide on apoptosis of cultured HDPCs. Cells showed typical apoptotic morphology after exposure to SNP. Besides, the number of Annexin V positive cells was increased among the SNP-treated HDPCs. SNP enhanced the production of reactive oxygen species (ROS), and N-acetylcysteine (NAC) ameliorated the decrement of cell viability induced by SNP. However, a soluble guanylate cyclase inhibitor (ODQ) did not inhibited the decrement of cell viability induced by SNP. SNP increased cytochrome c release from the mitochondria to the cytosol and the ratio of Bax/Bcl-2 expression levels. Moreover, SNP-treated HDPCs elevated activities of caspase-3 and caspase-9. While pretreatment with inhibitors of caspase (z-VAD-fmk, z-DEVD-fmk) reversed the NO-induced apoptosis of HDPCs. From these results, it can be suggested that NO induces apoptosis of HDPCs through the mitochondria-dependent pathway mediated by ROS and Bcl-2 family, but not by the cyclic GMP pathway.

In vitro evaluation of the odontogenic potential of mouse undifferentiated pulp cells

The aim of this study was to evaluate the odontogenic potential of undifferentiated pulp cells (OD-21 cell line) through chemical stimuli in vitro. Cells were divided into uninduced cells (OD-21), induced cells (OD-21 cultured in supplemented medium/OD-21+OM) and odontoblast-like cells (MDPC-23 cell line). After 3, 7, 10 and 14 days of culture, it was evaluated: proliferation and cell viability, alkaline phosphatase activity, total protein content, mineralization, immunolocalization of dentin matrix acidic phosphoprotein 1 (DMP1), alkaline phosphatase (ALP) and osteopontin (OPN) and quantification of genes ALP, OSTERIX (Osx), DMP1 and runt-related transcription factor 2 (RUNX2) through real-time polymerase chain reaction (PCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests (p<0.05). There was a decrease in cell proliferation in OD-21 + OM, whereas cell viability was similar in all groups, except at 7 days. The amount of total protein was higher in group OD-21 + OM in all periods; the same occurred with ALP activity after 10 days when compared with OD-21, with no significant differences from the MDPC-23 group. Mineralization was higher in OD-21+OM when compared with the negative control. Immunolocalization demonstrated that DMP1 and ALP were highly expressed in MDPC-23 cells and OD-21 + OM cells, whereas OPN was high in all groups. Real-time PCR revealed that DMP1 and ALP expression was higher in MDPC-23 cell cultures, whereas RUNX2 was lower for these cells and higher for OD-21 negative control. Osx expression was lower for OD-21 + OM. These results suggest that OD-21 undifferentiated pulp cells have odontogenic potential and could be used in dental tissue engineering.

O objetivo foi avaliar o potencial odontogênico de células indiferenciadas da polpa (OD-21) por meio de indução química in vitro. As células foram divididas em grupos: controle (OD-21), induzido (OD-21 em meio suplementado/OD-21 + OM), e células odontoblastóides (MDPC-23). Após 3, 7, 10 e 14 dias, avaliou-se proliferação e viabilidade celular, proteína total e fosfatase alcalina (ALP), mineralização, imunolocalização da proteína da matriz dentinária 1 (DMP1), ALP e osteopontina (OPN), assim como a expressão dos genes ALP, OSTERIX (Osx), DMP1 e fator de transcrição RUNX2 por PCR em tempo real. Os dados foram avaliados pelo teste de Kruskal-Wallis seguido pelo teste de Mann-Whitney U (p<0.05). Houve diminuição na proliferação celular em OD-21 + OM, com viabilidade celular similar em todos os grupos, exceto aos sete dias. O conteúdo de proteína total foi maior no grupo OD-21 + OM em todos os períodos; o mesmo ocorreu com a atividade de ALP quando comparada com o grupo OD-21, além de apresentar resultados similares ao grupo MDPC-23. A mineralização foi maior em OD-21 + OM quando comparada com o controle negativo. A imunolocalização demonstrou expressão de DMP1 e ALP em MDPC-23 e OD-21 + OM, enquanto que todos os grupos foram positivos para OPN. A expressão gênica de DMP1 e ALP foi maior nas culturas de MDPC-23, enquanto que a de RUNX2 foi menor para estas células e maior no controle negativo. A expressão de OSTERIX foi menor em OD-21 + OM quando comparada aos outros grupos. Sugere-se que as células indiferenciadas da polpa da linhagem OD-21 apresentam potencial odontogênico e poderiam ser usadas para a engenharia tecidual.