Study of optimal conditions for growth and osteogenic differentiation of dental pulp stem cells based on glucose and serum content.

Dental pulp stem cells (DPSCs) have shown promising characteristics in terms of their proliferation and osteogenic differentiation potential, which could be of greater benefit in regenerative dentistry. However, obstacles remain in the in vitro cultivation of DPSCs, which significantly affect their growth and differentiating ability. Therefore in this study, we demonstrated the growth and osteogenic differentiation of DPSCs in the presence of media containing different combinations of serum and glucose to get an optimized combination of both. DPSCs were cultured in media containing combinations of low glucose (LG), low serum (LS), high glucose (HG), and high serum (HS). The proliferation and osteogenic differentiation were assessed in DPSCs cultured with these different combinations of culture conditions. High glucose high serum condition significantly inhibited the proliferation of DPSCs and also affected their clonogenic potential, as evidenced by colony-forming units. Irrespective of the serum content, high glucose in the media also decreased the osteogenic potential of DPSCs confirmed by functional staining, and downregulation of osteogenesis-related genes. High glucose content in the culture media affects the growth and differentiation potential of the DPSCs. Hence, the culture conditions for the DPSCs should be reconsidered to utilize their maximum potential.

Adipogenic Stimulation and Pyrrolidine Dithiocarbamate Induced Osteogenic Inhibition of Dental Pulp Stem Cells Is Countered by Cordycepin.

BACKGROUND: dental pulp-derived stem cells are easy to access and collect and are an excellent source of stem cells for regenerative therapy. These cells can interact with many biomolecules and scaffolds and can pass on the instructive signals to the sites of regeneration where they are used. In this regard cordycepin, a potential biomolecule derived from medicinal mushrooms with a spectrum of bioactive properties such as antioxidant, anti-inflammatory, and anticancer has not yet been tested for its effect on human dental pulp stem cells. OBJECTIVE: the objective of the present study was to assess the in vitro adipogenic and osteogenic differentiation potential of human dental pulp stem cells with or without induction after administration of cordycepin. MATERIALS AND METHODS: human dental pulp stem cells DPSCs were isolated from a healthy permanent tooth extracted for orthodontic purposes after obtaining informed consent. Flow cytometry technique was used to assess the surface markers of these cells such as CD73, CD90, and CD105, CD34, CD45, and HLA-DR. Further, an MTT assay was performed on the cells after subjecting them to various concentrations of cordycepin. Following this, the adipogenic and osteogenic potential of the dental pulp stem cells was assessed with or without induction under the influence/absence of 5 µM of cordycepin. The results obtained were statistically analyzed and documented. RESULTS: it was found that the dental pulp stem cells showed strong positive expression for CD73, CD90, and CD105 and faint expression of CD34, CD45, and HLA-DR. MTT assay revealed that 5 µM was the optimum concentration of cordycepin for all the assays. Concerning adipogenesis experiments, there was a statistically significant lowering of all the 4 adipogenesis-related genes PPARγ, FABP4, LPL, and C/EBPα following cordycepin treatment in the presence of induction compared to the only induction group and untreated control cells (p < 0.05). In connection with osteogenesis, was found that there was a statistically significant increase in the expression of RUNX2, COL1A1, OSX and OCN genes along with the increase in alkaline phosphatase and alizarin red staining in the DPSC treated with cordycepin along with the presence of induction and simultaneous addition of PDTC compared to the control untreated cells and cells treated with induction and simultaneous addition of PDTC (p < 0.05). CONCLUSION: cordycepin can be exploited for its osteopromotive properties and can be used as a bioactive molecule alongside the administration of dental pulp stem cells in the area of regenerative biology and medicine.

Ultrasound Imaging versus Radiographs in Differentiating Periapical Lesions: A Systematic Review.

BACKGROUND: Ultrasonography is a non-invasive method of diagnosing periapical lesions while radiologic methods are more common. Periapical lesions due to endodontic infection are one of the most common causes of periapical radiolucency that need to be distinguished to help determine the course of treatment. This review aimed to examine the accuracy of ultrasound and compare it to radiographs in distinguishing these lesions in vivo. METHODS: This review process followed the PRISMA guidelines. A literature search of databases (PubMed, Scopus, Embase, and Web of Science) was conducted without any restrictions on time. Articles available in English were included. The selection was done according to the inclusion and exclusion criteria. The QUADAS-2 tool was used to assess the quality of the studies. RESULTS: The search provided a total of 87 articles, out of which, five were selected for the final review. In all the studies, ultrasound had higher accuracy in distinguishing periapical lesions. All the studies indicated a risk of bias, especially in patient selection. CONCLUSION: Within limitations, the study indicates that ultrasound is a better diagnostic tool to distinguish periapical lesions compared to radiographs but further studies with well-designed, rigorous protocols and low risk of bias are needed to provide stronger evidence.

Dose-Dependent Effect of Cordycepin on Viability, Proliferation, Cell Cycle, and Migration in Dental Pulp Stem Cells.

OBJECTIVE: To examine the effect of Cordycepin on the viability, proliferation, and migratory properties of dental pulp-derived mesenchymal stem cells. MATERIALS AND METHODS: The pulp was derived from human premolar teeth extracted for orthodontic purposes after obtaining informed consent. The samples were transferred to the laboratory for processing. DPSCs were expanded and characterized using flow cytometry and differentiation to the bone, adipose, and cartilage cells was examined. MTT Assay was performed using various concentrations of Cordycepin. The growth curve was plotted for 13 days. Cell cycle analysis was performed by flow cytometry. Migratory ability was assessed by wound healing assay. ROS generation was detected by flow cytometry. Gene expression was quantified by RT-qPCR. Statistical analysis was performed. p < 0.05 was considered as significant and p < 0.01 was considered as highly significant (* p < 0.05, and ** p < 0.01). RESULTS: DPSCs expressed characteristic MSC-specific markers and trilineage differentiation. Cordycepin at lower concentrations did not affect the viability of DPSCs. The growth curve of cells showed a dose-dependent increase in cell numbers till the maximum dose. DPSCs treated with 2.5 µM Cordycepin was found to have a reduced G1 phase cell percentage. DPSCs treated with 2.5 µM and 5 µM Cordycepin showed a significant decrease in G2 phase cells. No significant difference was observed for S phase cells. Cordycepin treatment affected the migratory ability in DPSCs in a concentration-dependent manner. CONCLUSION: Cordycepin can be used at therapeutic doses to maintain stem cells.

Effect of Ascorbic Acid on Differentiation, Secretome and Stemness of Stem Cells from Human Exfoliated Deciduous Tooth (SHEDs).

Stem cells from human exfoliated deciduous teeth (SHEDs) are considered a type of mesenchymal stem cells (MSCs) because of their unique origin from the neural crest. SHEDs can self-renewal and multi-lineage differentiation with the ability to differentiate into odontoblasts, osteoblast, chondrocytes, neuronal cells, hepatocytes, adipocytes, etc. They are emerging as an ideal source of MSCs because of their easy availability and extraordinary cell number. Ascorbic acid, or vitamin C, has many cell-based applications, such as bone regeneration, osteoblastic differentiation, or extracellular matrix production. It also impacts stem cell plasticity and the ability to sustain pluripotent activity. In this study, we evaluate the effects of ascorbic acid on stemness, paracrine secretion, and differentiation into osteoblast, chondrocytes, and adipocytes. SHEDs displayed enhanced multifaceted activity, which may have applications in regenerative therapy.

Taurine Augments Telomerase Activity and Promotes Chondrogenesis in Dental Pulp Stem Cells.

BACKGROUND: Stem cell therapy has become an advanced and state-of-the-art procedure to regenerate lost tissues of the human body. Cartilage repair is a challenging task in which stem cells find potential application. One of the important biologic modifiers that can cause chondrogenic differentiation of stem cells is taurine. However, taurine has not been investigated for its effects on dental pulp derived stem cell (DPSC) chondrogenic differentiation. OBJECTIVE: The objective of the study was to investigate if taurine administration to DPSCs heralds chondrogenic differentiation as ascertained by expression of SOX9, COL2A1, ACAN, ELN, and COMP. The study also investigated if the differentiated cells synthesized glycosaminoglycans, a marker of cartilage formation. The study also aimed to assess proliferative activity of the cells after taurine administration by measuring the hTERT gene and protein expression. MATERIALS AND METHODS: DPSCs were obtained from a molecular biology laboratory and characterization of stem cell markers was done by flow cytometry. The cells were subjected to a MTT assay using various concentrations of taurine. Following this, hTERT gene and protein estimation was done in the control, telomerase inhibitor treated DPSC (TI-III), 10 µM taurine treated DPSC, and TI-III + 10 µM taurine treated DPSCs. A polymerase chain reaction was done to assess gene expression of SOX9, COL2A1, ACAN, ELN, and COMP genes and glycosaminoglycans were estimated in control cells, Induced DPSCs, induced and TI-III treated DPSCs, and 10 µM taurine treated DPSCs. RESULTS: DPSCs expressed CD73, CD90, and CD105 and did not express CD34, CD45, and HLA-DR, which demonstrated that they were mesenchymal stem cells. The MTT assay revealed that various concentrations of taurine did not affect the cell viability of DPSCs. A concentration of 10 µM of taurine was used for further assays. With regard to the hTERT gene and protein expression, the taurine treated cells expressed the highest levels that were statistically significant compared to the other groups. Taurine was also found to restore hTERT expression in telomerase inhibitor treated cells. With regard to chondrogenesis related genes, taurine administration significantly increased the expression of SOX9, COL2A1, ACAN, and ELN genes in DPSCs and caused a significant increase in glycosaminoglycan production by the cells. CONCLUSIONS: Taurine can be regarded a biologic modifier that can significantly augment chondrogenic differentiation of DPSCs and can find potential applications in regenerative medicine in the area of cartilage regeneration.

Parathyroid Hormone Secretion and Receptor Expression Determine the Age-Related Degree of Osteogenic Differentiation in Dental Pulp Stem Cells.

OBJECTIVE: To demonstrate the levels of parathyroid hormone secretion and genetic expressions of parathyroid hormone (PTH) and PTH1 receptor (PTH1R) genes in the dental pulp stem cells (DPSCs) from different age groups before and after induction of osteogenic differentiation. In addition, we also wanted to check their correlation with the degree of osteogenic differentiation. METHODS: Human primary DPSCs from three age groups (milk tooth (SHEDs), 7-12 years old; young DPSCs (yDPSCs), 20-40 years old; old DPSCs (oDPSCs), 60+ years old) were characterized for mesenchymal stem cell (MSC) markers. DPSCs were subjected to osteogenic differentiation and functional staining. Gene expression levels were analyzed by qRT-PCR. Surface receptor analysis was done by flow cytometry. Comparative protein levels were evaluated by ELISA. RESULTS: All SHEDs, yDPSCs, and oDPSCs were found to be expressing mesenchymal stem cell markers. SHEDs showed more mineralization than yDPSCs and oDPSCs after osteogenic induction. SHEDs exhibited higher expression of PTH and PTH1R before and after osteogenic induction, and after osteogenic induction, SHEDs showed more expression for RUNX2, ALPL, and OCN. Higher levels of PTH were observed in SHEDs and yDPSCs, and the number of PTH1R positive cells was relatively lower in yDPSCs and oDPSCs than in SHEDs. After osteogenic induction, SHEDs were superior in the secretion of OPG, and the secretions of ALPL and PTH and the number of PTH1R positive cells were relatively low in the oDPSCs. CONCLUSIONS: The therapeutic quality of dental pulp stem cells is largely based on their ability to retain their stemness characteristics. This study emphasizes the criterion of aging, which affects the secretion of PTH by these cells, which in turn attenuates their osteogenic potential.

Comparative evaluation of push-out bond strength of ProRoot MTA, bioaggregate and biodentine.

OBJECTIVE: To evaluate the push-out bond strength of Biodentine (BD) in comparison with two available calcium silicate based materials, bioaggregate (BA) and ProRoot MTA (WMTA). MATERIALS AND METHODS: One hundred and twenty-three Root dentin slices of freshly extracted single Rooted human teeth were randomly divided into three groups (n = 41) according to the used test material: WMTA, BA, BD. After canal space preparation, the filling materials were placed inside the lumen of the slices. After 72 hours, the maximum force applied to materials at the time of dislodgement was recorded and slices were then examined under a stereomicroscope at ×40 magnification to determine the nature of bond failure. Analysis of variance (ANOVA) test was used to compare means of push-out bond strength. Post-hoc test was then accomplished for multiple comparisons. Chi-square test was used to determine if there is significant association between the type of material and type of failure. RESULTS: The mean push-out bond strength ± standard deviation in MPa values of WMTA, BA and BD were 23.26 ± 5.49, 9.57 ± 3.45, 21.86 ± 6.9, respectively. There was no significant difference between the means of WMTA and BD (p = 0.566), but the mean of BA was significantly lower than those of WMTA and BD (p = 0.000). Under stereomicroscope, WMTA and BA showed a majority of mixed type of failure than cohesive failure, while BD showed the opposite. No adhesive failure was observed in any specimen. CONCLUSION: The findings of the present study imply that the force needed for BD displacement is similar to WMTA and significantly higher than the force required to displace BA.

A comparative study of the debridement efficacy and apical extrusion of dynamic and passive root canal irrigation systems.

BACKGROUND: Root canal irrigation carries a risk of extrusion of irrigant into the periapical tissues which can be associated with pain, swelling, and tissue damage. Studies have shown less extrusion with sonic or apical negative pressure devices compared with syringe and side-port needle or passive ultrasonic irrigation with continuous irrigant flow. This study aimed to evaluate the effectiveness of the EndoVac irrigation system, regarding 1) debris removal and 2) the control of apically extruded irrigating solution. METHODS: Fifty extracted human single-rooted teeth were used in this study. The teeth were then randomly divided into three experimental groups according to the type of irrigation used and one control group. In group 1, irrigation was performed using the EndoVac irrigation system. In group 2, irrigation was performed using a 30-gauge, tip-vented irrigation needle. In group 3, irrigation was performed using a 30-gauge, side-vented irrigation needle. The control group received instrumentation with no irrigation to serve as a control for cleaning efficiency. Root canal instrumentation was performed using the Profile NiTi rotary system with a crown-down technique. All of the experimental teeth were irrigated with the same amount of 5.25% sodium hypochlorite. The amount of extruded irrigating solution was then measured by subtracting the post-instrumentation weight from the pre-instrumentation weight using an electronic balance. The cleanliness of debris removal was evaluated using scanning electron microscopy. RESULTS: EndoVac irrigation had the least amount of extrusion followed by the side-vented and tip-vented method. The difference between the groups was statistically significant (P <0.01). As for the cleaning results, the debris collection in the EndoVac and tip-vented groups was the least in the apical third. In the control and the side-vented groups, the debris was the greatest in the apical third, but this difference was not significant among the three experimental groups. CONCLUSIONS: The EndoVac irrigation system extruded significantly less irrigant solution than either needle irrigation system. Debris collection was the least in the apical third for the EndoVac irrigation system. No significant difference was found in the cleaning efficiency among the three irrigation systems.

An in vitro evaluation of the cytotoxicity of varying concentrations of sodium hypochlorite on human mesenchymal stem cells.

AIM: To evaluate and compare the cytotoxicity of various concentrations of sodium hypochlorite on immortalized human bone marrow mesenchymal stem cells (MSCs). MATERIALS AND METHODS: The 5.25 percent sodium hypochlo-rite (NaOCl) at concentrations of 0.5, 0.1, 0.025, 0.0125, and 0.005 mg/ml were used to assess the cytotoxic effect on MSCs. Immortalized human bone marrow mesenchymal stem cells (hTERT-MSCs) were exposed to NaOCl at 5 different concentrations. Cell viability was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and alamarBlue assays. The cell morphology changes were assessed with scanning electron microscopy (SEM) after exposure to 2, 4, and 24 hour incubation. The ethidium bromide/acridine orange (EB/ AO) fuorescent stain was applied to the cells in the 8-chamber slides after they were incubated with the testing agents for 2 and 4 hours to detect live and dead cells. The observations were quantitatively and qualitatively analyzed. RESULTS: The cell viability study using MTT assay and AB assay showed significant reduction with varying concentration at 2 and 4 hours incubation period. The cell viability decreased with the higher percentage of NaOCl. The exposure time also revealed an inverse relation to the cell viability. The SEM analysis showed reduction in the number of cells and morphological alterations with 0.5 mg/ml at 2 and 4 hours compared to 0.025 mg/ml NaOCl. Destruction of the cells with structural alterations and lysis was evident under fuorescence microscope when the cells were exposed to 0.5 mg/ml NaOCl. CONCLUSION: Within the limitations of this in vitro study it can be concluded that NaOCl is toxic to the human bone marrow MSCs. The cell lysis was evident with higher concentration of sodium hypochlorite. From the observations, it can be concluded that a lower concentration of NaOCl may be used as endodontic irrigant due to its cytotoxic properties. Further studies are mandatory to evolve a consensus on the optimal concentration of sodium hypochlorite to be used as endodontic irrigant.

An in vitro comparative study of the adaptation and sealing ability of two carrier-based root canal obturators.

The study was done to assess the sealing ability and adaptation of RealSeal 1, and to compare it with Thermafil. 65 single-rooted extracted teeth were selected and root canal treatment was performed. Root canals were obturated with RealSeal 1 or Thermafil. A double chamber bacterial leakage model using E. faecalis was developed to assess the sealing ability. Samples were monitored daily for 60 days. After the bacterial leakage test, samples were embedded in resin and sectioned horizontally at 2 and 4 mm from the apical foramen. Specimens were examined under scanning electron microscope and digitally photographed. AutoCAD software was used to measure the gap between the canal surface and obturation material. Results were statistically analyzed using nonparametric Kaplan-Meier survival analysis for the bacterial leakage and t-test to compare the means of gap in RealSeal 1 and Thermafil at 2 and 4 mm. There was no significant difference between the RealSeal 1 and Thermafil with respect to leakage over time. At 2 mm and 4 mm, RealSeal 1 had significantly more gaps than Thermafil. From the observations it can be concluded that RealSeal 1 and Thermafil have comparable performance in terms of adaptation and sealing ability.