Effect of angiopoietin 4 on odontogenic differentiation of dental pulp stem cells / 口腔疾病防治

Objective @# To investigate the effects of angiopoietin 4 (ANGPT4) on the odontogenic differentiation of human dental pulp stem cells. @* Methods @#This study has been reviewed and approved by the Ethics Committee, and informed consent has been obtained from patients. Human premolars were fixed, decalcified, dehydrated, embedded, and sectioned. Immunofluorescence staining was used to observe the expression and localization of ANGPT4. Human dental pulp stem cells (hDPSCs) were isolated and cultured in vitro. The growth state and morphology of hDPSCs were observed under an inverted phase contrast microscope. The expression of cell surface-related molecular markers was detected by flow cytometry. Alkaline phosphatase and alizarin red S staining were used to detect the odontogenic differentiation potential of hDPSCs. Oil-red O staining was used to detect the adipogenic differentiation potential of hDPSCs. RNA was extracted from hDPSCs at different time points after odontogenic induction, and RT-qPCR was used to analyze the mRNA expression of ANGPT4 and odontogenic-related genes during the odontogenic differentiation of hDPSCs in vitro. siRNA gene silencing technology was used to silence the expression of ANGPT4 in hDPSCs, and the silencing efficiency was detected by RT-qPCR and Western Blot. After silencing ANGPT4 in hDPSCs for 24 h, odontogenic induction was performed. Alkaline phosphatase and alizarin red S staining were performed on the 7th and 14th of induction to detect the odontogenic differentiation ability of hDPSCs after silencing ANGPT4@*Results @# Immunofluorescence staining of human premolars showed that ANGPT4 was expressed in odontoblasts and sub-odontoblastic cell-rich zone. hDPSCs were in good condition after 14 days of isolation and culture. Under the microscope, multiple cell colonies were observed, and the cell morphology was uniform and long spindle-shaped. The results of flow cytometry showed that hDPSCs expressed mesenchymal stem cell markers CD105 (90.42%) and CD90 (97.15%), but did not express hematopoietic cell markers CD45 (0.01%) and CD34 (0.08%). After odontogenic and adipogenic induction of hDPSCs, alkaline phosphatase staining, alizarin red S staining and oil red O staining were positive. The results of RT-qPCR after the odontogenic induction of hDPSCs showed that ANGPT4 was highly expressed on the 5th, 7th, 11th and 14th days of differentiation of hDPSCs (P<0.05), with the highest expression level on the 5th day. After hDPSCs were transfected with si-ANGPT4, the expression of ANGPT4 mRNA and protein was significantly down-regulated (P<0.05). The results of alkaline phosphatase staining showed that ALP staining intensity and area in the si-ANGPT4 group were significantly lower than those in the negative control. Alizarin red S staining showed that the formation of calcium nodules in the si-ANGPT4 group was significantly lower than that in the negative control.@* Conclusion@#ANGPT4 was expressed in odontoblasts and sub-odontoblastic cell-rich zone of human premolars. ANGPT4 may be a factor to promote the odontogenic differentiation of hDPSCs.

Potential role of dental pulp stem cells conditioned medium for odontoblastic differentiation

BACKGROUND: Functional bioengineered tooth regeneration using autologous or allogeneic alternative differentiated cells sources are thought to have a great potential in replacing conventional dentures. This study investigated the potential of dental pulp stem cells (DPSCs) conditioned medium for odontoblastic differentiation of Wharton's jelly mesenchymal stem cells (WJMSCs). The DPSCs derived from healthy adult permanent first molars were cultured at high confluence prior to conditioned medium collection. The WJMSCs were cultured in six different treatments, with varying ratios of culture media to DPSCs-conditioned medium. MTT assay was used to measure the rate of proliferation of WJMSCs, while immunocytochemistry staining was utilised to detect the expression of dental matrix protein 1 (DMP-1). The deposited calcium was detected and analysed via Alizarin-Red Staining (ARS). RESULTS: It was found that the proliferation of WJMSCs cultured under the mixture of complete medium and DPSCs conditioned medium showed significantly lower than the control; presumably the cells started to exit proliferative state prior differentiation. In 14 days of induction, the cells in all treatments showed osteoblastic-like morphology, calcium compound deposits were observed at day 7, 10 and 14 of differentiation suggested that DPSCs conditioned medium could lead to osteoblastic/odontoblastic differentiation. However, the DMP-1 protein can be seen only expressed minimally at day 14 of conditioned medium induction. CONCLUSIONS: In conclusion, DPSCs conditioned medium appeared as a potential odontoblastic induction approach for WJMSCs. To further investigate the stimulatory effects by DPSCs conditioned medium, specific signalling pathway need to be elucidated to enhance the differentiation efficiency.

Evaluation of dentinogenesis inducer biomaterials: an in vivo study

Abstract When exposure of the pulp to external environment occurs, reparative dentinogenesis can be induced by direct pulp capping to maintain pulp tissue vitality and function. These clinical situations require the use of materials that induce dentin repair and, subsequently, formation of a mineralized tissue. Objective: This work aims to assess the effect of tricalcium silicate cements and mineral trioxide aggregate cements, including repairing dentin formation and inflammatory reactions over time after pulp exposure in Wistar rats. Methodology: These two biomaterials were compared with positive control groups (open cavity with pulp tissue exposure) and negative control groups (no intervention). The evaluations were performed in three stages; three, seven and twenty-one days, and consisted of an imaging (nuclear medicine) and histological evaluation (H&E staining, immunohistochemistry and Alizarin Red S). Results: The therapeutic effect of these biomaterials was confirmed. Nuclear medicine evaluation demonstrated that the uptake of 99mTc-Hydroxymethylene diphosphonate (HMDP) showed no significant differences between the different experimental groups and the control, revealing the non-occurrence of differences in the phosphocalcium metabolism. The histological study demonstrated that in mineral trioxide aggregate therapies, the presence of moderate inflammatory infiltration was found after three days, decreasing during follow-ups. The formation of mineralized tissue was only verified at 21 days of follow-up. The tricalcium silicate therapies demonstrated the presence of a slight inflammatory infiltration on the third day, increasing throughout the follow-up. The formation of mineralized tissue was observed in the seventh follow-up day, increasing over time. Conclusions: The mineral trioxide aggregate (WhiteProRoot®MTA) and tricalcium silicate (Biodentine™) present slight and reversible inflammatory signs in the pulp tissue, with the formation of mineralized tissue. However, the exacerbated induction of mineralized tissue formation with the tricalcium silicate biomaterial may lead to the formation of pulp calcifications

Comparison of odontoblast differentiation capacity between stem cells from human exfoliated deciduous teeth and dental pulp stem cells / 中南大学学报(医学版)

OBJECTIVES@#To explore the difference in odontoblast differentiation capacity between stem cells from human exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSCs), and to examine the expression level of ephrinB1 in odontoblast differentiation of these stem cells.@*METHODS@#The stems cells were divided into a SHED group and a DPSCs group. After odontoblast differentiation induction, the above 2 groups were also randomly divided into a 3 d group and a 7 d group, respectively.The calcium deposition was detected by alkaline phosphatase (ALP) staining and alizarin red staining.The mRNA and protein expressions of ephrinB1, dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) were detected by real-time PCR and Western blotting.@*RESULTS@#ALP staining and alizarin red staining showed that there was stronger mineralization capacity in the SHED group than that in the DPSCs group. The relative mRNA and protein expressions of DMP-1, DSPP, and ephrinB1 in the SHED group were higher than those in the DPSCs group except for the protein expression of DMP-1 in the SHED 3 d group (all <0.05).@*CONCLUSIONS@#SHED has stronger odontoblast differentiation capacity than DPSCs. In addition, ephrinB1 may be involved in the processes of odontoblast differentiation in the SHED and DPSCs.

Effect of grape seed extract (GSE) on functional activity and mineralization of OD-21 and MDPC-23 cell lines

Abstract Recent studies on functional tissue regeneration have focused on substances that favor cell proliferation and differentiation, including the bioactive phenolic compounds present in grape seed extract (GSE). The aim of this investigation was to evaluate the stimulatory potential of GSE in the functional activity of undifferentiated pulp cells and odontoblast-like cells. OD-21 and MDPC-23 cell lines were cultivated in odontogenic medium until subconfluence, seeded in 24-well culture plates in a concentration of 2x104/well and divided into: 1) OD-21 without GSE; 2) OD-21+10 µg/mL of GSE; 3) MDPC-23 without GSE; 4) MDPC-23+10 µg/mL of GSE. Cell proliferation, in situ detection of alkaline phosphatase (ALP) and total protein content were assessed after 3, 7 and 10 days, and mineralization was evaluated after 14 days. The data were analyzed by ANOVA statistical tests set at a 5% level of significance. Results revealed that cell proliferation increased after 10 days, and protein content, after 7 days of culture in MDPC-23 cells. In situ ALP staining intensity was higher in undifferentiated pulp cells and odontoblast-like cells after 7 and 10 days, respectively. A discrete increase in MDPC-23 mineralization after GSE treatment was observed despite OD-21 cells presenting a decrease in mineralized nodule deposits. Data suggest that GSE favors functional activity of differentiated cells more broadly than undifferentiated cells (OD-21). More studies with different concentrations of GSE must be conducted to confirm its benefits to cells regarding dentin regeneration.

Expressions of cell polarity related proteins CDC42and PAR3 during tooth germ development in mice / 吉林大学学报(医学版)

Objective:To study the expressions of cell polarity related proteins CDC42 and PAR3 during tooth germ development in the mice,and to discuss their possible roles during tooth development in the mice.Methods:The whole heads were obtained from the mouse embryo on the days 13.5,14.5,16.5 and 18.5 (E13.5,E14.5,E16.5 and E18.5) and the mice on the postnatal days 1 (PN1) and 5 (PN5).The tissues were fixed in paraformaldehyde,decalcified,dehydrated,embedded in paraffin,and sectioned.The histology of tooth germ was observed by HE staining.The expressions of CDC42 and PAR3 during tooth germ development were detected by immunohistochemistry staining.Results:The HE staining results showed that E13.5,E14.5,E16.5 and E18.5were the bud stage,the cap stage,the early and the late bell stage of tooth germ development,respectively;the tooth germ of PN1 mice showed the matured odontoblasts and ameloblasts;the tooth germ of PN5 mice showed the completed tooth crown development.The immunohistochemistry staining results showed that CDC42 expressed in the tooth germ of the mice at E13.5,E14.5 and E16.5;the CDC42 expression at E 18.5 was reduced compared with E13.5,E14.5 and E16.5;CDC42 mainly expressed in the odontoblasts and ameloblasts of the tooth germ of the mice at PN1 and PN5;PAR3 weakly expressed in the tooth germ of the mice at E13.5 and E14.5,and it was increased at E16.5 and E18.5.At PN1 and PN5,the expressions of PAR3 were decreased compared with E18.5.Conclusion:CDC42 and PAR3 partieipat in the mouse tooth development;during the early stage of tooth germ development,they may be involved in the proliferation and migration of mouse dental germ;during the late stage,CD42 and PAR3 may be involved in the differentiation of the odontoblasts and the ameloblasts,especially in the establishment and maintenance of cell polarity.

cAMP-responsive-element-binding protein promotes the differentiation of human stem cells from the apical papilla via inhibition of the TGF-β1 pathway / 口腔疾病防治

Objective @#This study aimed to investigate the effect of cAMP-responsive-element-binding protein (CREB) overexpression on the differentiation of human stem cells from the apical papilla (hSCAPs), stimulated by transforming growth factor- beta (TGF-β1). @*Methods@#Cells were isolated from human immature third molars via enzymatic digestion. Four experimental groups were set up: ①a control group, receiving normal mineralization inducer (α-MEM, 10% FBS, 10 mmol/L β-sodium glycerophosphate, 50 μg/mL vitamin C, 10 nmol/L dexamethasone); ② a TGF-β1 group, receiving normal mineralization inducer and 5 μg/mL TGF-β1; ③ a TGF-β1+LV-empty group, receiving normal mineralization inducer and the transfected empty virus vector with 5 μg/mL TGF-β1; and ④ a TGF-β1+ov-CREB group, receiving normal mineralization inducer and the transfected CREB-overexpressing viral vector, with 5 μg/mL TGF-β1. The transfected cells were cultured in odontogenic medium in the presence or absence of TGF-β1 for 2 weeks. Alizarin red staining was used to detect mineralized nodules, and the mRNA expression of the mineralization genes runt-related transcription factor 2 (RUNX2), dentin sialophosphoprotein (DSPP) and alkaline phosphatase (ALP) was measured by qPCR. @*Results @#Compared with the control group (1.12 ± 0.11), TGF-β1 inhibited the deposition of calcium minerals (0.67 ± 0.12) (P < 0.05) via hSCAPs and inhibited the mRNA expression of RUNX2 (0.60 ± 0.03), DSPP (0.43 ± 0.12) and ALP (0.69 ± 0.05) (P < 0.05). In contrast, overexpression of CREB attenuated the effect of TGF-β1 on hSCAPs, resulting in the development of a high number of mineralized nodules (1.27 ± 0.10) (P < 0.01) and increased RNA levels of RUNX2 (1.33 ± 0.07), DSPP (1.32 ± 0.11) and ALP (1.26 ± 0.03) (P<0.05) compared with those in the TGF-β1 group. @*Conclusion@#Overexpressed CREB promotes odontogenic differentiation of hSCAPs by interfering with TGF-β1.

Expressions of cell polarity related proteins CDC42 and PAR3 during tooth germ development in mice / 吉林大学学报(医学版)

Objective: To study the expressions of cell polarity related proteins CDC42 and PAR3 during tooth germ development in the mice, and to discuss their possible roles during tooth development in the mice. Methods: The whole heads were obtained from the mouse embryo on the days 13. 5, 14. 5, 16. 5 and 18. 5 (E13. 5, E14. 5, E16. 5 and E18. 5) and the mice on the postnatal days 1 (PN1) and 5 (PN5). The tissues were fixed in paraformaldehyde, decalcified, dehydrated, embedded in paraffin, and sectioned. The histology of tooth germ was observed by HE staining. The expressions of CDC42 and PAR3 during tooth germ development were detected by immunohistochemistry staining. Results: The HE staining results showed that E13. 5, E14. 5, E16. 5 and E18. 5 were the bud stage, the cap stage, the early and the late bell stage of tooth germ development, respectively; the tooth germ of PN1 mice showed the matured odontoblasts and ameloblasts; the tooth germ of PN5 mice showed the completed tooth crown development. The immunohistochemistry staining results showed that CDC42 expressed in the tooth germ of the mice at E13. 5, E14. 5 and E16. 5; the CDC42 expression at E 18. 5 was reduced compared with E13. 5, E14. 5 and E16. 5; CDC42 mainly expressed in the odontoblasts and ameloblasts of the tooth germ of the mice at PN1 and PN5; PAR3 weakly expressed in the tooth germ of the mice at E13. 5 and E14. 5, and it was increased at E16. 5 and E18. 5. At PN1 and PN5, the expressions of PAR3 were decreased compared with E18. 5. Conclusion: CDC42 and PAR3 participat in the mouse tooth development; during the early stage of tooth germ development, they may be involved in the proliferation and migration of mouse dental germ; during the late stage, CD42 and PAR3 may be involved in the differentiation of the odontoblasts and the ameloblasts, especially in the establishment and maintenance of cell polarity.

Optimisation of polymerase chain reaction conditions for detection of mineralization markers in isolated odontoblasts from human teeth

The present study aimed to determine the best polymerase chain reaction (PCR) conditions for amplification of odontoblast markers; alkaline phosphatase (ALP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and osteopontin (OPN). Informed consent was obtained from the individuals prior to tooth extraction. RNA was extracted from odontoblasts obtained from extracted teeth using innuPREP RNA Mini kit (Analytik Jena, Germany). Five selected target factors in enhancing PCR: primer concentration, extension time, number of cycles, annealing time, and annealing temperature were manipulated to yield the correct size of amplicons. One step reverse transcriptase PCR reactions were performed using MyTaq One-Step RT-PCR kit (Bioline, USA) with a C1000 Thermal Cycler (Bio-Rad, USA) in a 25 µL reaction, keeping the amount of 2 ng/µL RNA, 0.25 µL reverse transcriptase, 0.5 µL RiboSafe Rnase inhibitor and 1X MyTaq One-Step Mix, constant. The optimal conditions were determined to be 400nM of primers for DMP1 and DSPP, 200 nM for ALP and OPN; 30 seconds of extension time and 35 PCR cycles for all genes; 10 seconds of annealing time for ALP, DMP1 and DSPP, 7 seconds for OPN. The annealing temperature were 56.4°C for ALP, 58.6°C for DMP1, 52.7°C for DSPP, and 56.3°C for OPN, respectively. The optimized PCR protocols produced the correct size of odontoblast markers.

Recombinant human transforming growth factor β1 promotes dental pulp stem cells proliferation and mineralization / 北京大学学报(医学版)

Objective: To explore suitable concentration of recombinant human transforming growth factor β1 (rhTGF-β1) usage and study the effect of rhTGF-β1 on differentiation of dental pulp stem cells (DPSCs).Methods: DPSCs were isolated from the undiseased third molars of people aged 18-25 years and cultured according to instructions in vitro.Different concentrations (1 , 6 , 10 μg/L) of rhTGF-β1 were added to the culture medium to examine DPSCs proliferation by CCK-8 (cell counting kit-8) assay.The suitable concentration was then selected.For differentiation, the DPSCs were incubated for 7 or 14 days with rhTGF-β1 supplemented with osteo/odontoblastic induction medium containing 10 nmol/L dexamethasone, 10 mmol/L b-glycerophosphate, 50 g/L ascorbate phosphate, 10 nmol/L 1,25-dihydroxyvitamin D3 and 10% fetal bovine serum.The cells were then washed 3 times with phosphate-buffered saline and sonicated with 1%Triton X-100 for 30 minutes on ice.Cellular alkaline phosphatase (ALP) activity was assayed with p-nitrophenyl phosphate as the substrate.The enzyme activity was expressed as p-nitrophenyl produced per milligram of protein [bicinchoninic acid (BCA) protein assay kit].To examine mineral nodule formation, the cultured cells were fixed in 4% paraformaldehyde and washed in water, and the mineralization of the extracellular matrix was assayed by 1% alizarin red S staining and elution of staining was examined as optical density (D) under microplate reader.The mean difference was considered significant at 0.05 and 95% confidence interval.Results: The DPSCs had ty-pical fibroblast morphology and could form mineral nodules after being cultured with osteo/odontoblstic induction medium for 14 days.6 μg/L rhTGF-β1 significantly promoted the DPSCs proliferation on the 3rd and 5th days.After the incubation of osteo/odontoblastic induction medium, the DPSCs with the 6 μg/L rhTGF-β1 increased ALP activities compared with the control;D values in the 6 μg/L rhTGF-β1 group was 0.31±0.03, while the control group was 0.02±0.01(P<0.05).The total protein content in the 6 μg/L rhTGF-β1 group was (2 775.46±83.54) mg/L, and the control group was (1 432.20±110.83) mg/L (P<0.05).To eliminate the cells proliferation influence, relative ALP activities, which was defined as the total ALP divided by the total protein content, the 6μg/L rhTGF-β1 group was 6 times higher than the control group.Alizarin red S staining showed increased mineral nodule formation in the rhTGF-β1 group.The elution of staining under microplate reader also showed more optical density in the 6 μg/L rhTGF-β1-treated cells (0.83±0.02) than that in the control groups (0.55±0.05, P<0.05).Conclusion: 6 μg/L rhTGF-β1 could significantly promote DPSCs proliferation and odontoblastic differentiation in vitro.

A Histological Study on Venom Apparatus of Montivipera xanthina (Gray, 1849) (Serpentes, Viperidae)

ABSTRACT This is the first report describing morphological and histological structure of venom apparatus in Montivipera xanthina. The venom gland apparatus of M. xanthina was composed of four distinct parts, main venom gland, primary duct, accessory gland and secondary duct. The main venom gland was composed of acini. The accessory gland was formed by long and ramified tubules. Nine fangs were detected in one half of the upper jaw each at different stage of development. Only one tooth was functional and the eight remaining fangs form a replacement series. The venom apparatus of viperid snakes are the most effective venom-delivery system among vertebrates and have been the focus of scientific interests for many years. Despite this interest, there are few studies including venom-delivery system of venomous snakes. The results presented here may be common among venomous snakes and model of the viperid design of the venom apparatus.

Transforming Growth Factor -beta 1 (TGF-beta 1) immunoreactivity in heterotopic grafts of adult dental apical papilla / Inmunoreactividad del Factor Transformador del Crecimiento-beta 1 (TGF-beta 1) en la papila apical dental adulta heterotópicamente trasplantada

To analyze the expression of transforming growth factor-beta 1 inheterotopic grafts of adult dental apical papilla. Methodology: The apical papilla of adult Wistar rats was grafted in the ear of the same donor rats. 1, 3, 7 and14 days after grafting, rats were perfused and the tissue containing the graft was processed for histological conventional technique and for immunohistochemical detection of transforming growth factor-beta 1. Results: Heterotopically grafted apical papilla developed osteoid dentine. In an early post-grafting stage, odontoblast-like cells organized themselves in palisade and synthesized dentine. However, newly formed dentine possessed the structural appearance of reactive osteoid dentine, which was systematically destroyed by the activity of osteoclaste-like cells. Transforming Growth Factor-beta 1 was observed in mesenchymal cells, extracellular matrix of the graft and surrounding host tissue, while odontoblast-like cells were systematically devoid of immunoreactivity. Conclusion: The different expression of transforming growth factor-beta 1 between normal tissue and grafted tissue development suggests that in heterotopic graft conditions the inflammatory mediation of the transforming growth factor-beta 1 prevails against its morphogenetic role...

Analizar la expresión del factor transformador del crecimiento-beta1 en trasplantes heterotópicos de papila dental del incisivo de la rata adulta. Metodología: La papila apical del incisivo de 12 ratas Wistar adultas fue trasplantada en la oreja de las mismas ratas donantes, y perfundidas 1, 3, 7 y 14 días postrasplante. El tejido fue procesado para histología convencionaly para la detección inmunohistoquímica del factor transformador del crecimiento-beta1. Resultados: La papila apical trasplantada desarrolló osteodentina. En fases tempranas postrasplante se observaron células parecidas a los odontoblastos que se organizaron en empalizada y segregaron dentina que se depositó sobre su superficie apical o secretora. Esta dentina evolucionó a osteodentina caracterizada por perder su estructura tubular e incluir a las células odontoblásticas en lagunas de su matriz. Finalmente, la osteodentina presentó procesos líticos mediados por células de tipo osteoclasto. Durante todo el proceso la expresión del factor transformador del crecimiento-beta1 se restringió a las células mesenquimales, a la matriz del trasplante y a las zonas circundantes del huésped, estando ausente en los odontoblastos, a diferencia de lo que sucede durante la odontogénesis normal. Conclusión: La diferente localización de la expresión del Factor Transformador de crecimiento beta1 entre el tejido hospedero y el trasplantado sugieren que en condiciones de trasplante heterotópico de papila dental la mediación inflamatoria del Factor Transformador de crecimiento beta1 prevalece sobre su papel morfogenético...

Caracterización estereológica de odontoblastos en pulpas dentarias humanas sanas y con pulpitis reversible / Stereological characterization of odontoblasts in normal healthy and reversible pulpitis in human dental pulps

La reacción y reparación dentinaria depende del número de odontoblastos. Los métodos para obtener estimaciones fiables sobre la cantidad de odontoblastos en la pulpa dental han sido subjetivos y sesgados, sobre todo al evaluar los cambios cuantitativos y la potencial capacidad reparativa en presencia de caries. El objetivo de este trabajo fue estimar y comparar cuantitativamente el número, densidad y volumen de odontoblastos en dientes sanos y con diagnóstico de pulpitis reversible producto de caries a través de herramientas estereológicas. Se utilizaron dientes premolares humanos obtenidos de exodoncias, divididos en un grupo sano y otro cariado. Fueron fijados y descalcificados con ácido nítrico al 5%. Siguiendo el protocolo del orientator se obtuvieron 5 secciones de 5 mm teñidas por H-E de cada diente. Se aplicó el recuento estereológico de los odontoblastos con el test multipropósito M42. Se estimaron las densidades de número (Nv), volumen (Vv) y superficie (Sv), y calcularon las Medias (±DE) por diente, y Medias (±EE) por grupo. Las diferencias entre grupos se analizaron mediante la prueba T, con un valor p 0,05 de significación estadística. En dientes sanos, la Media (±EE) para Nv de odontoblastos fue 0,409x105/mm3 (±0,018x105/mm3), para Vv 19,714% (±1,43%) y para Sv 21,016 mm2/mm3 (±1,425 mm2/mm3). En dientes cariados, la Nv fue 0,521x105 /mm3 (±0,023x105/mm3), la Vv 24,686% (±1,625%) y la Sv 23,203 mm2/mm3 (±1,364 mm2/mm3). Al comparar las Nv, los odontoblastos del grupo con caries aumentaron significativamente (p=0,0062), al igual que la Vv (p=0,0197). Existe un aumento del número de odontoblastos en los dientes con pulpitis reversible, lo que condicionaría su capacidad de respuesta. La metodología empleada puede ser aplicable para determinar el comportamiento pulpar y cuantificar variables de respuesta odontoblástica en tratamientos restauradores atraumáticos de manera imparcial y reproducible.

Dentinal reaction and repair depends on factors like the amount of odontoblasts. Methods for obtaining reliable estimates of the number of odontoblasts in the dental pulp have been subjective and biased, especially when assessing the potential quantitative changes and reparative capacity in the presence of cavities. The aim of this study was to estimate and quantitatively compare, through stereological tools, the number, density and volume of odontoblasts in healthy teeth, diagnosed with reversible pulpitis due to caries. We used human premolars obtained from extractions, divided into groups of healthy teeth and teeth with caries, fixed and decalcified in 5% nitric acid. Following the orientator protocol, five 5-µm-thick sections stained by HE were obtained from each tooth. Stereological counting for odontoblast with M42 multipurpose test was applied. Numerical density (Nv), volume density (Vv) and surface density (Sv) were estimated, and the mean (± SD) for each tooth, and Mean (± SE) per group were calculated. Differences between groups were analyzed by t test, with p 0.05 for statistical significance. In the healthy teeth group, the mean (± SE) for Nv was 0.409x105/mm3 (±0.018x105/mm3), Vv 19.714% (±1.43%) and to Sv 21.016 mm2/mm3 (±1.425 mm2/mm3) odontoblast cells. In the caries teeth group, the Nv was 0.521x105 /mm3 (±0.023x105/mm3), Vv 24.686% (±1.625%) and Sv 23.203 mm2/mm3 (±1.364 mm2/mm3). When comparing Nv, an increased in odontoblasts significantly (p = 0.0062), as well as Vv (p = 0.0197) in caries teeth group. There is an increased number of odontoblasts in teeth with reversible pulpitis, which would condition its responsiveness. The methodology can be applied to determine pulp behavior, and quantify variables of odontoblastic response in atraumatic restorative treatments in an impartial and reproducible form.

Differentiation of odontoblast-like cells from human exfoliated deciduous teeth-derived stem cells by transforming growth factor β3 in combination with heparin in vitro / 实用医学杂志

Objective To investigate the ability of human exfoliated deciduous teeth-derived stem cells (SHED) to differentiate into odontoblast-like cells. Methods SHEDs were isolated by enzyme digestion method. The 3nd passage SHEDs were incubated with 25 ng/mL recombinant human TGF-β3 , or with TGF-β3 in combination with heparin. The DSPP expression was detected by Q-PCR and Western-blotting assay. Alizarin red staining, immunhistochemistry assay and alkaline phosphatase(AKP) activity assay were performed, respectively. Result The AKP activity was enhance by TGF-β3 in combination with heparin. Alizarin red staining was positive in TGF-β3-heparin groups, with the increase of DSPP expression at both mRNA and protein level. Conclusion TGF-β3 in combination with heparin can enhance the differentiation of human exfoliated deciduous teeth-derived stem cells into odontoblast-like cells.

Effect of Fluoride-Treated Enamel on Indirect Cytotoxicity of a 16% Carbamide Peroxide Bleaching Gel to Pulp Cells

The aim of this study was to evaluate the possibility of fluoride solutions applied to enamel to protect pulp cells against the trans-enamel and transdentinal cytotoxicity of a 16% carbamide peroxide (CP) bleaching gel. The CP gel was applied to enamel/dentin discs adapted to aicial pulp chambers (8 h/day) during 1, 7 or 14 days, followed by fluoride (0.05% or 0.2%) application for 1 min. The extracts (culture medium in contact with dentin) were applied to MDPC-23 cells for 1 h, and cell metabolism (MTT assay), alkaline phosphatase (ALP) activity and cell membrane damage (flow cytometry) were analyzed. Knoop microhardness of enamel was also evaluated. Data were analyzed statistically by ANOVA and Kruskal-Wallis tests (α=0.05). For the MTT assay and ALP activity, significant reductions between the control and the bleached groups were observed (p<0.05). No statistically significant difference occurred among bleached groups (p>0.05), regardless of fluoride application or treatment days. Flow cytometry analysis demonstrated 30% of cell membrane damage in all bleached groups. After 14 days of treatment, the fluoride-treated enamel presented significantly higher microhardness values than the bleached-only group (p<0.05). It was concluded that, regardless of the increase in enamel hardness due to the application of fluoride solutions, the treated enamel surface did not prevent the toxic effects caused by the 16% CP gel to odontoblast-like cells.

Resumo O objetivo do presente estudo foi avaliar o possível efeito protetor de soluções fluoretadas aplicadas sobre o esmalte dentário frente à citotoxicidade trans-amelodentinária de um gel clareador com 16% de peróxido de carbamida (PC). O gel de PC foi aplicado sobre discos de esmalte/dentina adaptados a câmaras pulpares aiciais (8 h/dia) durante períodos de 1, 7 ou 14 dias, seguido de aplicação de soluções fluoretadas (0,05% ou 0,2%) durante 1 min. Os extratos (meio de cultura em contato com a dentina) foram aplicados sobre células MDPC-23 durante 1 h, seguido de análise do metabolismo celular (teste do MTT), atividade de fosfatase alcalina (ALP) e danos à membrana celular (citometria de fluxo). A microdureza Knoop do esmalte dental foi avaliada. Os dados foram analisados pelos testes de ANOVA e Kruskal-Wallis. Para o teste do MTT e atividade de ALP, redução significante entre os grupos controle e clareados foram observados (p<0,05). Nenhuma diferença entre os grupos clareados foi observada (p>0,05), independente da aplicação das soluções fluoretadas ou tempo de tratamento. A análise por citometria de fluxo demonstrou lesão à membrana celular em torno de 30% para todos os grupos clareados. Após 14 dias de tratamento, os espécimes clareados e fluoretados apresentaram aumento significante na microdureza do esmalte (p<0,05). Pôde-se concluir que apesar do aumento na dureza do esmalte decorrente da aplicação das soluções fluoretadas, este tratamento não preveniu os efeitos tóxicos causados pelo gel com 16% de PC sobre as células odontoblastóides. .

Efeito protetor da vitamina E (α-Tocoferol) contra a atividade citotóxica do peróxido de hidrogênio / Protective effect of vitamin E (α-tocopherol) against the cytotoxicity of hydrogen peroxide

O clareamento dental de consultório, realizado com géis que apresentam elevada concentração de peróxido de hidrogênio (PH), pode causar danos intensos para as células da polpa dentária. Dentro deste contexto, a administração de agentes antioxidantes previamente ao clareamento dental, tem sido considerada uma terapia promissora, pois pode eliminar ou pelo menos minimizar os efeitos adversos deste procedimento clínico estético. Assim, o objetivo do presente estudo foi avaliar o possível efeito protetor da Vitamina E (alfa-Tocoferol / α-T) contra à ação citotóxica do PH sobre células odontoblastóides (MDPC-23). Para isto, células foram semeadas em placas de 96 wells durante 72 horas e então submetidas a diferentes tempos de pré-tratamento (1, 4, 8 e 24 horas) com variadas concentrações de α-T (1, 3, 5 e 10 mM). Após os períodos de pré-tratamento, as células foram expostas ou não ao PH (0,018%) durante 30 minutos. Nos grupos controle positivo e negativo, as células foram somente expostas a uma solução de PH (0,018%) ou meio de cultura, respectivamente. O metabolismo celular foi avaliado pelo teste de MTT. A absorbância foi transformada em porcentagem e analisada pelo Teste de Kruskal-Wallis, complementado por Mann-Whitney (α=0,05%). Todas as concentrações de αT e tempos de pré-tratamento propostos neste estudo apresentaram proteção das células contra os efeitos citotóxicos do PH. Porém, os melhores resultados foram obtidos com as concentrações de 1 e 3 mM (126% e 97%, respectivamente) por 24 horas em relação a 41% de metabolismo celular do grupo controle positivo (p<0,05). Portanto, as células pré-tratadas com α-T por 24 horas, em concentrações baixas (1 e 3 mM), apresentaram os melhores resultados de viabilidade celular, ou seja, maior efeito protetor frente à agressão direta do PH

The in-office tooth bleaching using gels with high concentrations of hydrogen peroxide (HP) may cause irreversible damage to pulp tissue. Therefore, the administration of antioxidazing agents previously to the tooth bleaching procedures has been considered as a promising therapy, mainly due the capacity of these agents to prevent, or at last reduce, the negative side-effects caused by toxic products used in this esthetic treatment. Therefore, the aim of the present in vitro study was to evaluate the protective activity of vitamin E (alfa-Tocoferol / α-T) against the toxic effects of HP applied to cultured odontoblast-like cells (MDPC-23). Cells were seeded in wells of 96-well plates for 72 hs and then pre-treated with different concentrations of α-T (1, 3, 5, and 10 mM) for variable periods (1, 4, 8, and 24 hs). Following, the cells were exposed to a HP solution (0,018%) for 30 min. In positive and negative control groups, the cells were exposed only to HP solution (0,018%) or culture medium, respectively. The cell metabolism was assessed by MTT assay and the absorbance numeric data, expressed as percentage values, were subjected to the statistical analysis of Kruskal-Wallis complemented by Mann-Whitney test (α=0.05%). All α-T concentrations and pre-treatments evaluated in this study showed cell protection against the cytotoxic effects of HP. However, considering the MDPC23 cells in the negative control group as presenting 100% metabolism, it was observed that the most relevant data occurred when the cells were pre-treated with α-T at 1 and 3 mM (126% and 97% of cell metabolism, respectively) for 24hs compared to the positive control group (41%) (p<0.05). Based upon the methodology used in the present investigation, it can be concluded that low concentrations of α-T (1 and 3 mM) applied for 24 hs to the cultured odontoblast-like MDPC-23 cells provide the best protective effects against the cytotoxicity caused by hydrogen peroxide

Smad4 Mediated TGF-beta/BMP Signaling in Tooth Formation Using Smad4 Conditional Knockout Mouse

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.

A Trial of Screening of Genes Involved in Odontoblasts Differentiation from Human Dental Pulp Stem Cells

This study investigated the genes involved in the differentiation of odontoblasts derived from human dental pulp stem cells (hDPSCs). hDPSCs isolated from human tooth pulp were validated by fluorescence activated cell sorting (FACS). After odontogenic induction, hDPSCs were analyzed investigated by Alizaline red-S staining, ALP assay, ALP staining and RT-PCR. Differential display-polymerase chain reaction (DD-PCR) was performed to screen differentially expressed genes involved in the differentiation of hDPSCs. By FACS analysis, the stem cell markers CD24 and CD44 were found to be highly expressed in hDPSCs. When hDPSCs were treated with agents such as beta-glycerophosphate (beta-GP) and ascorbic acid (AA), nodule formation was exhibited within six weeks. The ALP activity of hDPSCs was found to elevate over time, with a detectable up-regulation at 14 days after odontogenic induction. RT-PCR analysis revealed that dentin sialophosphoprotein (DSPP) and osteocalcin (OC) expression had increased in a time-dependent manner in the induction culture. Through the use of DD-PCR, several genes were differentially detected following the odontogenic induction. These results suggest that these genes may possibly be linked to a variety of cellular process during odontogenesis. Furthermore, the characterization of these regulated genes during odontogenic induction will likely provide valuable new insights into the functions of odontoblasts.

MicroRNA Analysis during Cultured Odontoblast Differentiation

MicroRNAs (miRNAs, miRs) are about 21-25 nucleotides in length and regulate mRNA translation by base pairing to partially complementary sites, predominantly in the 3'-untranslated region (3'-UTR) of the target mRNA. In this study, the expression profile of miRNAs was compared and analyzed for the establishment of miRNA-related odontoblast differentiation using MDPC-23 cells derived from mouse dental papilla cells. To determine the expression profile of miRNAs during the differentiation of MDPC-23 cells, we employed miRNA microarray analysis, quantitative real-time PCR (qRT-PCR) and Alizaline red-S staining. In the miRNA microarray analysis, 11 miRNAs were found to be up- or down-regulated more than 3-fold between day 0 (control) and day 5 of MDPC-23 cell differentiation among the 1,769 miRNAs examined. In qRT-PCR analysis, the expression levels of two of these molecules, miR-194 and miR-126, were increased and decreased in the control MDPC-23 cells compared with the MDPC-23 cells at day 5 of differentiation, respectively. Importantly, the overexpression of miR-194 significantly accelerated mineralization compared with the control cultures during the differentiation of MDPC-23 cells. These results suggest that the miR-194 augments MDPC-23 cell differentiation, and potently accelerates the mineralization process. Moreover, these in vitro results show that different miRNAs are deregulated during the differentiation of MDPC-23 cells, suggesting the involvement of these genes in the differentiation and mineralization of odontoblasts.