Effect of Periodontal Ligament Stem Cells-Derived Conditioned Medium on Gene Expression and Differentiation of Tumor Necrosis Factor-α-Challenged Osteoblasts.

OBJECTIVES: Tumor necrosis factor-α (TNF-α) causes bone resorption in periodontitis. It induces the production of receptor activator of NF-κB ligand (RANKL) from osteoblasts, leading to the disturbance of bone homeostasis through RANKL, RANK, and osteoprotegerin (OPG) axis. This study aimed to explore the effect of periodontal ligament stem cells-derived conditioned medium (PDLSCs-CM) on gene expression related to bone homeostasis and the differentiation of TNF-α-challenged osteoblasts. MATERIALS AND METHODS: Human osteoblasts were cultured with 50 ng/mL of TNF-α and 0, 1, 10, and 100 µg/ mL of PDLSCs-CM. Osteoblasts cultured without TNF-α and PDLSCs-CM were served as control. Gene expression of RANKL, OPG, and interleukin-1ß (IL-1ß) was evaluated by reverse transcription quantitative polymerase chain reaction at 48 hours. The early-stage and late-stage differentiation of TNF-α-challenged osteoblasts without or with PDLSCs-CM was explored by alkaline phosphatase (ALP) activity and alizarin red staining, respectively, at day 1, 3, 6, 9, and 12. STATISTICAL ANALYSIS: Mann-Whitney U test was used to analyze the differences in gene expression of TNF-α-challenged osteoblasts at 24 and 48 hours, and Kruskal-Wallis test was used to analyze the effect of PDLSCs-CM on gene expression and ALP activity among all experimental groups using SPSS software version 21.0. Statistical significance was considered with p-value less than 0.05. RESULTS: Expression of RANKL, OPG and IL-1ß was significantly upregulated in TNF-α-challenged osteoblasts compared to the untreated control. The PDLSCs-CM at 1 and 10 µg/mL downregulated gene expression of TNF-α-challenged osteoblasts compared to the group without PDLSCs-CM, but the difference did not reach statistical significance. The ALP activity was decreased in TNF-α-challenged osteoblasts. The addition of PDLSCs-CM did not alter ALP activity of TNF-α-challenged osteoblasts. Alizarin red staining was comparable in the TNF-α-challenged osteoblasts cultured without or with PDLSCs-CM. CONCLUSIONS: The PDLSCs-CM did not alter gene expression involved in bone homeostasis and differentiation of TNF-α-challenged osteoblasts.

Stem cell-derived exosomes from human exfoliated deciduous teeth promote angiogenesis in hyperglycemic-induced human umbilical vein endothelial cells.

OBJECTIVE: To investigate the angiogenesis in human umbilical vein endothelial cells (HUVEC) under high glucose concentration, treated with exosomes derived from stem cells from human exfoliated deciduous teeth (SHED). METHODOLOGY: SHED-derived exosomes were isolated by differential centrifugation and were characterized by nanoparticle tracking analysis, transmission electron microscopy, and flow cytometric assays. We conducted in vitro experiments to examine the angiogenesis in HUVEC under high glucose concentration. Cell Counting Kit-8, migration assay, tube formation assay, quantitative real-time PCR, and immunostaining were performed to study the role of SHED-derived exosomes in cell proliferation, migration, and angiogenic activities. RESULTS: The characterization confirmed SHED-derived exosomes: size ranged from 60-150 nm with a mode of 134 nm, cup-shaped morphology, and stained positively for CD9, CD63, and CD81. SHED-exosome significantly enhanced the proliferation and migration of high glucose-treated HUVEC. A significant reduction was observed in tube formation and a weak CD31 staining compared to the untreated-hyperglycemic-induced group. Interestingly, exosome treatment improved tube formation qualitatively and demonstrated a significant increase in tube formation in the covered area, total branching points, total tube length, and total loop parameters. Moreover, SHED-exosome upregulates angiogenesis-related factors, including the GATA2 gene and CD31 protein. CONCLUSIONS: Our data suggest that the use of SHED-derived exosomes potentially increases angiogenesis in HUVEC under hyperglycemic conditions, which includes increased cell proliferation, migration, tubular structures formation, GATA2 gene, and CD31 protein expression. SHED-exosome usage may provide a new treatment strategy for periodontal patients with diabetes mellitus.

Influence of Lithium- and Zinc-Containing Bioactive Glasses on Pulpal Regeneration.

OBJECTIVE: To evaluate the potential of modified bioactive glasses containing lithium and zinc as pulp capping materials by investigating the odontogenic differentiation and mineralization response in the tooth culture model. MATERIALS AND METHODS: Lithium- and zinc-containing bioactive glasses (45S5.1Li, 45S5.5Li, 45S5.1Zn, 45S5.5Zn, 45S5.1Zn sol-gel, and 45S5.5Zn sol-gel), fibrinogen-thrombin, and biodentine were prepared to assess Axin2 gene expression at 0, 30 minutes, 1 hour, 12 hours, and 1 day and DSPP gene expression at 0, 3, 7, and 14 days in stem cells from human exfoliated deciduous teeth (SHEDs) using qRT-PCR. The experimental bioactive glasses incorporated with fibrinogen-thrombin and biodentine were placed on the pulpal tissue in the tooth culture model. Histology and immunohistochemistry were analyzed at 2 weeks and 4 weeks. RESULTS: Axin2 gene expression for all experimental groups was significantly higher than the control at 12 hours. The DSPP gene expression for all experimental groups was significantly higher than the control at 14 days. The presence of mineralization foci was significantly higher at 4 weeks for the modified bioactive glasses 45S5.5Zn, 45S5.1Zn sol-gel, and 45S5.5Zn sol-gel as well as Biodentine compared with the fibrinogen-thrombin control. CONCLUSION: Lithium- and zinc-containing bioactive glasses increased Axin2 and DSPP gene expression in SHEDs and can potentially enhance pulp mineralization and regeneration. Zinc-containing bioactive glasses are a promising candidate to be used as pulp capping materials.

Evaluation of the Efficacy of Human Dental Pulp Stem Cell Transplantation in Sprague-Dawley Rats with Sensorial Neural Hearing Loss.

OBJECTIVES: The purpose of the present study was to evaluate the efficacy of spiral ganglion neuron (SGN) regeneration after dental pulp stem cell (DPSC) transplantation in a rat sensorineural hearing loss (HL) model. MATERIALS AND METHODS: Sham or experimental HL was induced in adult Sprague-Dawley rats by cochlear round window surgery. An HL rat model was established with a single 10 mM ouabain intratympanic injection. After 7 days, the rats received DPSCs, stem cells from human exfoliated deciduous teeth (SHED), or culture medium in the sutural area to establish four groups: sham, HL-DPSC, HL-SHED, and HL-medium. Histological analyses were performed at 4, 7, and 10 weeks after transplantation, and the number of SGNs, specific SGN protein expression, and the function of SGNs were evaluated. STATISTICAL ANALYSIS: Data were statistically by MS Excel and SPSS v.15.0. Intergroup level of significance was determined via a one-way analysis of variance and Duncan's multiple range test with 95% confidence intervals. RESULTS: New SGN formation was observed in the HL-DPSC and HL-SHED rat groups. The number of SGNs was significantly higher in the HL-DPSC and HL-SHED groups than in the HL-medium group over 4 to 10-week survival period. HL-DPSC rats exhibited higher SGN density compared with that in HL-SHED group, which was statistically significant at week 10. The regenerated SGNs expressed cochlear wiring regulator GATA-binding-protein 3. Moreover, the SGNs from the HL-DPSC group also exhibited a higher expression of synaptic vesicle protein and regulated action potential-dependent neurotransmitter release compared with SGNs from the HL-SHED group. CONCLUSIONS: Our findings suggest that DPSCs and SHED repair and regenerate SGNs in rat HL model. Dental pulp stem cells represent a promising treatment strategy for restoring damage to the sensory circuits associated with deafness.

Stem cell-derived exosomes from human exfoliated deciduous teeth promote angiogenesis in hyperglycemic-induced human umbilical vein endothelial cells

Abstract Objective To investigate the angiogenesis in human umbilical vein endothelial cells (HUVEC) under high glucose concentration, treated with exosomes derived from stem cells from human exfoliated deciduous teeth (SHED). Methodology SHED-derived exosomes were isolated by differential centrifugation and were characterized by nanoparticle tracking analysis, transmission electron microscopy, and flow cytometric assays. We conducted in vitro experiments to examine the angiogenesis in HUVEC under high glucose concentration. Cell Counting Kit-8, migration assay, tube formation assay, quantitative real-time PCR, and immunostaining were performed to study the role of SHED-derived exosomes in cell proliferation, migration, and angiogenic activities. Results The characterization confirmed SHED-derived exosomes: size ranged from 60-150 nm with a mode of 134 nm, cup-shaped morphology, and stained positively for CD9, CD63, and CD81. SHED-exosome significantly enhanced the proliferation and migration of high glucose-treated HUVEC. A significant reduction was observed in tube formation and a weak CD31 staining compared to the untreated-hyperglycemic-induced group. Interestingly, exosome treatment improved tube formation qualitatively and demonstrated a significant increase in tube formation in the covered area, total branching points, total tube length, and total loop parameters. Moreover, SHED-exosome upregulates angiogenesis-related factors, including the GATA2 gene and CD31 protein. Conclusions Our data suggest that the use of SHED-derived exosomes potentially increases angiogenesis in HUVEC under hyperglycemic conditions, which includes increased cell proliferation, migration, tubular structures formation, GATA2 gene, and CD31 protein expression. SHED-exosome usage may provide a new treatment strategy for periodontal patients with diabetes mellitus.

Optimal culture conditions for neurosphere formation and neuronal differentiation from human dental pulp stem cells.

OBJECTIVES: Human dental pulp stem cells (DPSCs) have been used to regenerate damaged nervous tissues. However, the methods of committing DPSCs into neural stem/progenitor cells (NSPCs) or neurospheres are highly diverse, resulting in many neuronal differentiation outcomes. This study aims to validate an optimal protocol for inducing DPSCs into neurospheres and neurons. METHODOLOGY: After isolation and characterization of mesenchymal stem cell identity, DPSCs were cultured in a NSPC induction medium and culture vessels. The durations of the culture, dissociation methods, and passage numbers of DPSCs were varied. RESULTS: Neurosphere formation requires a special surface that inhibits cell attachment. Five-days was the most appropriate duration for generating proliferative neurospheres and they strongly expressed Nestin, an NSPC marker. Neurosphere reformation after being dissociated by the Accutase enzyme was significantly higher than other methods. Passage number of DPSCs did not affect neurosphere formation, but did influence neuronal differentiation. We found that the cells expressing a neuronal marker, ß-tubulin III, and exhibiting neuronal morphology were significantly higher in the early passage of the DPSCs. CONCLUSION: These results suggest a guideline to obtain a high efficiency of neurospheres and neuronal differentiation from DPSCs for further study and neurodegeneration therapeutics.

Odontogenic gene expression profile of human dental pulp-derived cells under high glucose influence: a microarray analysis.

Hyperglycemia, a major characteristic of diabetes, is considered to play a vital role in diabetic complications. High glucose levels have been found to inhibit the mineralization of dental pulp cells. However, gene expression associated with this phenomenon has not yet been reported. This is important for future dental therapeutic application. OBJECTIVE: Our study aimed to investigate the effect of high glucose levels on mineralization of human dental pulp-derived cells (hDPCs) and identify the genes involved. METHODOLOGY: hDPCs were cultured in mineralizing medium containing 25 or 5.5 mM D-glucose. On days 1 and 14, RNA was extracted and expression microarray performed. Then, differentially expressed genes (DEGs) were selected for further validation using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. Cells were fixed and stained with alizarin red on day 21 to detect the formation of mineralized nodules, which was further quantified by acetic acid extraction. RESULTS: Comparisons between high-glucose and low-glucose conditions showed that on day 1, there were 72 significantly up-regulated and 75 down-regulated genes in the high-glucose condition. Moreover, 115 significantly up- and 292 down-regulated genes were identified in the high-glucose condition on day 14. DEGs were enriched in different GO terms and pathways, such as biological and cellular processes, metabolic pathways, cytokine-cytokine receptor interaction and AGE-RAGE signaling pathways. RT-qPCR results confirmed the significant expression of pyruvate dehydrogenase kinase 3 (PDK3), cyclin-dependent kinase 8 (CDK8), activating transcription factor 3 (ATF3), fibulin-7 (Fbln-7), hyaluronan synthase 1 (HAS1), interleukin 4 receptor (IL-4R) and apolipoprotein C1 (ApoC1). CONCLUSIONS: The high-glucose condition significantly inhibited the mineralization of hDPCs. DEGs were identified, and interestingly, HAS1 and Fbln-7 genes may be involved in the glucose inhibitory effect on hDPC mineralization.

The effect of LED photobiomodulation on the proliferation and osteoblastic differentiation of periodontal ligament stem cells: in vitro.

BACKGROUND: The aim of this study was to investigate the influence of three different light-emitting diode (LED) wavelengths on the proliferation and osteoblastic differentiation of periodontal ligament stem cells (PDLSCs) in vitro. METHODS: PDLSCs seeded on 96- and 24-well plates, for proliferation and osteoblastic differentiation, respectively, were irradiated daily by LED light with peak emission wavelengths of 630, 680, and 830 nm at constant energy densities of 3.5 J/cm2. Cultures were grown for 8 days for the proliferation assay, 10 days for the alkaline phosphatase (ALP) assay, and 28 days for Alizarin red staining. Mitochondrial activity, ALP enzyme level, and the ability to form calcium phosphate deposits were measured and compared across cultures. RESULTS: Results obtained from statistical analysis of the experimental data indicated that the rate of proliferation (P < 0.05) in 830-nm irradiated cultures were significantly higher than the control samples at day 6 and 8; whereas, for the 630- and 680-nm groups, test results showed lower proliferation rates at day 8. For osteoblastic differentiation, significantly greater mineralization than the control samples was detected in the red-light groups (630 and 680 nm) during the late differentiation period (P < 0.001), which was supported by a higher ALP activity of the 630- and 680-nm groups in the early stage (P < 0.01). CONCLUSION: The results of this study demonstrate that the PDLSCs responded differently to specific LED wavelengths. For enhancing cellular proliferation, 830-nm LED irradiation was more effective. On the other hand, the wavelengths of 630 and 680 nm were better for stimulating osteoblastic differentiation.

Effect of surface chemistry-modified polycaprolactone scaffolds on osteogenic differentiation of stem cells from human exfoliated deciduous teeth.

The aim of this study was to investigate the effect of surface modification of a polycaprolactone scaffold on promoting osteogenic differentiation of stem cells from human exfoliated deciduous teeth. Four different polycaprolactone scaffold were evaluated: untreated; coated with hyaluronic acid; coated with gelatin; and coated with hyaluronic acid and then with gelatin. The resulting scaffolds were characterized using scanning electron microscopy and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). Human stem cells were cultured on the modified scaffolds placed in osteogenic differentiation medium. During culture, the osteogenic potential of the stem cells was examined by evaluating alkaline phosphatase activity and staining intensity, expression of osteoblastic-specific genes, and matrix mineralization. Scanning electron microscopy and ATR-FTIR confirmed productive biomacromolecular surface treatment of the polycaprolactone scaffold. All scaffolds permitted differentiation of stem cells into osteoblastic cells, but the gelatin-coated polycaprolactone scaffold facilitated osteogenesis of a larger number of stem cells than the untreated and the hyaluronic acid-coated scaffolds. We demonstrate that gelatin is an appropriate macromolecule for modifying the surface of an electrospun polycaprolactone fibre scaffold that is used subsequently in bone tissue engineering applications.

Optimization of poly (lactic-co-glycolic acid)-bioactive glass composite scaffold for bone tissue engineering using stem cells from human exfoliated deciduous teeth.

OBJECTIVE: The aim of this study was to develop a composite scaffold with the optimal poly(lactic-co-glycolic acid) (PLGA) and bioactive glass proportions to provide an environment for bone tissue regeneration and repair. DESIGN: PLGA-bioactive glass composite scaffolds were prepared using a salt-leaching technique with different percentages of bioactive glass (0%, 10 %, and 15 % [w/w]) with PLGA. The resulting scaffolds were characterized using scanning electron microscopy and energy dispersive X-ray spectroscopy (SEM-EDS), and water contact angle, dynamic mechanical, and pH analysis. The scaffold biocompatibility was investigated using stem cells from human exfoliated deciduous teeth (SHED) and rat experiments. RESULTS: SEM-EDS confirmed the successful fabrication of three-dimensional PLGA-bioactive glass scaffolds. The results showed that 10 % bioactive glass with PLGA exhibited favorable properties including increased pore size, hydrophilicity, and mechanical properties. The growth medium pH was increased for scaffolds containing bioactive glass. All scaffolds were biocompatible, and 10 % bioactive glass composite scaffolding showed better attachment, growth, and proliferation of SHED compared to the other scaffolds. Moreover, it enhanced osteogenic differentiation of SHED in vitro and in vivo. CONCLUSIONS: Salt-leaching-derived PLGA-bioactive glass composite scaffolds were successfully established. PLGA with 10 % bioactive glass had adequate physical properties and bioactivity, and it could be considered as a composite for bone tissue engineering applications.

Optimal culture conditions for neurosphere formation and neuronal differentiation from human dental pulp stem cells

Abstract Objectives Human dental pulp stem cells (DPSCs) have been used to regenerate damaged nervous tissues. However, the methods of committing DPSCs into neural stem/progenitor cells (NSPCs) or neurospheres are highly diverse, resulting in many neuronal differentiation outcomes. This study aims to validate an optimal protocol for inducing DPSCs into neurospheres and neurons. Methodology After isolation and characterization of mesenchymal stem cell identity, DPSCs were cultured in a NSPC induction medium and culture vessels. The durations of the culture, dissociation methods, and passage numbers of DPSCs were varied. Results Neurosphere formation requires a special surface that inhibits cell attachment. Five-days was the most appropriate duration for generating proliferative neurospheres and they strongly expressed Nestin, an NSPC marker. Neurosphere reformation after being dissociated by the Accutase enzyme was significantly higher than other methods. Passage number of DPSCs did not affect neurosphere formation, but did influence neuronal differentiation. We found that the cells expressing a neuronal marker, β-tubulin III, and exhibiting neuronal morphology were significantly higher in the early passage of the DPSCs. Conclusion These results suggest a guideline to obtain a high efficiency of neurospheres and neuronal differentiation from DPSCs for further study and neurodegeneration therapeutics.

Odontogenic gene expression profile of human dental pulp-derived cells under high glucose influence: a microarray analysis

Abstract Hyperglycemia, a major characteristic of diabetes, is considered to play a vital role in diabetic complications. High glucose levels have been found to inhibit the mineralization of dental pulp cells. However, gene expression associated with this phenomenon has not yet been reported. This is important for future dental therapeutic application. Objective Our study aimed to investigate the effect of high glucose levels on mineralization of human dental pulp-derived cells (hDPCs) and identify the genes involved. Methodology hDPCs were cultured in mineralizing medium containing 25 or 5.5 mM D-glucose. On days 1 and 14, RNA was extracted and expression microarray performed. Then, differentially expressed genes (DEGs) were selected for further validation using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. Cells were fixed and stained with alizarin red on day 21 to detect the formation of mineralized nodules, which was further quantified by acetic acid extraction. Results Comparisons between high-glucose and low-glucose conditions showed that on day 1, there were 72 significantly up-regulated and 75 down-regulated genes in the high-glucose condition. Moreover, 115 significantly up- and 292 down-regulated genes were identified in the high-glucose condition on day 14. DEGs were enriched in different GO terms and pathways, such as biological and cellular processes, metabolic pathways, cytokine-cytokine receptor interaction and AGE-RAGE signaling pathways. RT-qPCR results confirmed the significant expression of pyruvate dehydrogenase kinase 3 (PDK3), cyclin-dependent kinase 8 (CDK8), activating transcription factor 3 (ATF3), fibulin-7 (Fbln-7), hyaluronan synthase 1 (HAS1), interleukin 4 receptor (IL-4R) and apolipoprotein C1 (ApoC1). Conclusions The high-glucose condition significantly inhibited the mineralization of hDPCs. DEGs were identified, and interestingly, HAS1 and Fbln-7 genes may be involved in the glucose inhibitory effect on hDPC mineralization.

High Glucose Affects Proliferation, Reactive Oxygen Species and Mineralization of Human Dental Pulp Cells.

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.

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.

Neuronal differentiation of dental pulp stem cells from human permanent and deciduous teeth following coculture with rat auditory brainstem slices.

Sensorineural hearing loss is a common disability found worldwide which is associated with a degeneration of spiral ganglion neurons (SGN). It is a challenge to restore SGN due to the permanent degeneration and viability of SGN is requisite for patients to receive an advantage from hearing aid devices. Human dental pulp stem cells (DPSC) and stem cells from human exfoliated deciduous teeth (SHED) are self-renewing stem cells that originate from the neural crest during development. These stem cells have a high potential for neuronal differentiation. This is primarily due to their multilineage differentiation potential and their relative ease of access. Previously, we have shown the ability of these stem cell types to differentiate into spiral ganglion neuron-like cells. In this study, we induced the cells into neural precursor cells (NPC) and cocultured with auditory brainstem slice (ABS) encompassing cochlear nucleus by the Stoppini method. We also investigated their ability to differentiate after 2 weeks and 4 weeks in coculture. Neuronal differentiation of DPSC-NPC and SHED-NPC was higher expression of specific markers to SGN, TrkB, and Gata3, compared to monoculture. The cells also highly expressed synaptic vesicle protein (SV2A) and exhibited intracellular calcium oscillations. Our findings demonstrated the possibility of using DPSCs and SHEDs as an autologous stem cell-based therapy for sensorineural hearing loss patients.

Effect of Bioceramic Materials on Proliferation and Odontoblast Differentiation of Human Stem Cells from the Apical Papilla.

INTRODUCTION: In regenerative endodontic treatment (RET), practitioners favor the placement of bioceramics as sealing materials over blood clots. It is important to understand the interaction between sealing material and cells in the root canal. The purpose of this study was to compare the effectiveness of various bioceramic materials (ProRoot MTA [Dentsply, Tulsa, OK], Biodentine [Septodont, Saint-Maur-des-Fossés, France], and RetroMTA [BioMTA, Seoul, Korea]) as sealing materials in RET for the proliferation and differentiation of stem cells from the apical papilla (SCAPs). METHODS: SCAPs were seeded at 20,000 cells/well and cultured with soluble agents of testing materials through a transwell culture plate. The proliferation of SCAPs was investigated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on days 1, 3, 7, and 14 of testing. Alizarin red staining and quantitative real-time polymerase chain reaction were used for SCAP differentiation at different time points (1, 7, 14, and 21 days). The odontoblast genes expressed are dentin matrix acidic phosphoprotein 1, dentin sialophosphoprotein, osteocalcin, and matrix extracellular phosphoglycoprotein, which were used in this study. The SCAPs were cultured in odonto/osteogenic induction medium and also contacted soluble agents from the testing materials. RESULTS: All 3 tested biomaterials induced SCAP proliferation. The Biodentine, ProRootMTA, and RetroMTA groups showed significant SCAP proliferation on days 7 and 14 compared with the control. In regard to odontoblastic differentiation, only Biodentine showed positive alizarin red staining. The highest expressions of dentin matrix acidic phosphoprotein 1, dentin sialophosphoprotein, and matrix extracellular phosphoglycoprotein were found on day 21 in the Biodentine group. The expression of osteocalcin was found to be significant on day 7. CONCLUSIONS: Biodentine, ProRootMTA, and RetroMTA can induce SCAP proliferation. Biodentine induced significant SCAP differentiation among the 3 materials.

Optimal Xeno-free Culture Condition for Clinical Grade Stem Cells from Human Exfoliated Deciduous Teeth.

BACKGROUND AND OBJECTIVES: Stem cells from human exfoliated deciduous teeth (SHED) are a promising clinical resource for various tissue defects, including lumbar spondylosis, neural compression, and cleft palate. Use of media containing animal-derived serum carries potential risk of infectious diseases and unwanted immunogenicity. To increase the potential utility of SHED for clinical application, SHED was adapted to xeno-free conditions. METHODS: Define xeno-free culture media were compared with the conventional serum containing media in the culture of SHED. Cultured SHED in different media were further characterized through proliferative capacities, cellular phenotype, and differentiation potential. RESULTS: Selected xeno-free media were capable of supporting the growth of SHED. MSCGM-CD Bulletkit medium greatly increased the number and proliferate capacity of colony-forming unit-fibroblast than SHED cultured in other media. In addition, the characteristic surface markers expression and multipotent differentiation potential of SHED in the MSCGM-CD Bulletkit medium were comparable to those observed with serum-containing medium. CONCLUSIONS: The xeno-free medium described herein has the potential to be further used for the safe expansion and to determine efficient way to produce clinical grade dental stem cells for therapeutic applications.

Differentiation of stem cells from human deciduous and permanent teeth into spiral ganglion neuron-like cells.

OBJECTIVE: Stem cells from pulp tissue are a promising cell-based therapy for neurodegenerative patients based on their origin in the neural crest. The aim of this study was to differentiate and evaluate the ability of human dental pulp stem cells from permanent teeth (DPSC) and stem cells from human exfoliated deciduous teeth (SHED) to differentiate into spiral ganglion neurons. DESIGN: After isolation and characterization of mesenchymal stem cell properties, DPSC and SHED were treated with the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and glial cell-derived neurotrophic factor (GDNF). The differentiation was identified by immunostaining and qRT-PCR analysis of neuronal markers and measuring intracellular calcium activity. RESULTS: After 2 weeks of induction, morphological changes were observed in both DPSC and SHED. The differentiated cells expressed neuron-specific class III beta-tubulin, GATA binding protein 3 (GATA3) and tropomyosin receptor kinase B, protein markers of spiral ganglion neurons. These cells also showed upregulation of the genes encoding these proteins, namely GATA3 and neurotrophic receptor tyrosine kinase 2. Intracellular calcium dynamics that reflect neurotransmitter release were observed in differentiated DPSC and SHED. CONCLUSION: These results demonstrate that dental pulp stem cells from permanent and deciduous teeth can differentiate into spiral ganglion neuron-like cells.

High glucose promotes the osteogenic differentiation capability of human periodontal ligament fibroblasts.

Periodontal ligament fibroblasts (PDLFs) are important cells, which are involved in maintaining tooth integrity. Diabetes has been found to be associated with periodontal disease in a bidirectional manner. The aim of the present study was to investigate the stemness properties of human PDLFs (HPDLFs) in high glucose conditions. HPDLFs were analyzed for their osteogenic differentiation capacity by inducing the cells with osteogenic medium in various glucose concentrations. The gene expression was then examined using reverse transcription­quantitative polymerase chain reaction analysis, and examinations of alkaline phosphatase activity and nodule formation were performed. The results of the gene expression analysis revealed that high glucose media induced the expression of NANOG, octamer-binding transcription factor 4, (sex determining region Y)­box 2, cluster of differentiation 166 (CD166), PERIOSTIN and ß­CATENIN following culture of the cells for 3 days. Alkaline phosphatase activity increased following 14 days in the high glucose condition. In addition, higher numbers of calcified nodules were formed on day 28 in the group cultured with high glucose. The results showed that high glucose induced bone formation by elevating the expression of stem cell markers, particularly CD166, and this induction may be regulated through ß-CATENIN.

Using Oral and Colon Cancer Cells for Studying the Anticancer Properties of Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are of importance in defense mechanism of many organisms and are potential candidate for treatment of infections in animals and humans. AMPs exhibit a wide range of immunomodulatory activities related to innate immunity, wound healing, and inflammation. AMPs also serve as drug delivery vectors, antitumor agents, and mitogenic agents. Here, we describe the investigation of anticancer and cytotoxic activities of antimicrobial peptides by colorimetric MTT assay using smooth muscle, dental pulp stem cell, human colon cancer cell line (SW620), and human oral squamous carcinoma cell line (HSC4).