Effect of Enzymatic-based chemomechanical caries removal agent on proliferation and osteogenic differentiation of dental pulp stem cells.

To study the effect of glycyrrhizin (GA) on the viability and proliferation of dental pulp stem cells (DPSCs) compared with intracanal medicaments. Third molars of an adult donor were used to obtain the DPSCs. Flow cytometry was utilized to conduct phenotypic analysis for DPSCs. The methyl-thiazol tetrazolium (MTT) test was used to detect the cell viability. Cell proliferation assay was conducted at distinct time intervals: 3, 5, and 7 days. The flow cytometry analysis verified the positive expression of mesenchymal cell surface antigen molecules (CD73, CD90, and CD105) and the absence of hematological markers (CD14, CD34, and CD45) in the DPSCs. The cells that treated with concentrations more than 0.5 mg/mL of Ca(OH2) and triple antibiotic paste (TAP) gave significant decrease in viability in comparison to the untreated cells (p < 0.05). Also, the cells treated with concentrations 50 and 25 µM of GA showed no significant difference compared with the untreated cells (p > 0.05), while concentrations 12.5 and 6.25 µM expressed a significant increase in viability compared with the untreated cells (p < 0.05). At 7 days, cells treated with the three different concentrations of GA (12.5, 25, and 50 µM) demonstrated a significant increase in cell density compared with Ca(OH)2 and TAP-treated cells (p < 0.05). Based upon the potential of GA on DPSCs proliferation compared with Ca(OH)2 and TAP, It is conceivable to acknowledge that GA could be used as an intracanal medicaments for revascularization process of necrotic immature teeth. This study emphasizes the significance of assessing alternative root canal medicaments and their impact on the proliferation and viability of DPSCs. The results regarding GA, specifically its impact on the viability and growth of DPSCs, provide essential understanding for its potential application as an intracanal medicine. This study adds to the continuous endeavors in identifying safer and more efficient intracanal therapies, which are essential for improving patient outcomes in endodontic operations. How to cite this article: Alrashidi MA, Badawi MF, Elbeltagy MG, et al. The Effect of Glycyrrhizin on the Viability and Proliferation of Dental Pulp Stem Cells Compared to Intracanal Medicaments. J Contemp Dent Pract 2024;25(3):267-275.

Propolis is a natural substance produced by honeybees that has various biological properties including, anti-inflammatory, antioxidant and antimicrobial properties. Although previous studies have evaluated the antimicrobial effects of propolis in dentistry, its effects on dental pulp stem cell (DPSC) viability, migration, and differentiation are yet not well understood. The objective of this study was to investigate the effects of Chinese propolis on viability/proliferation, migration, differentiation and cytokine expression in DPSCs. Commercially available DPSCs (Lonza) were treated with aqueous extract of propolis (AEP) or ethanolic extract of propolis (EEP), and viability/proliferation was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays and quantification of nuclear staining. DPSC differentiation into mineralizing cells was evaluated with Alizarin red staining and cell migration was assessed using Boyden Chamber Transwell inserts. Cytokine expression was measured by RT-qPCR. AEP and EEP at 0.03 and 0.1 mg/mL did not affect DPSC viability/proliferation for up to 7-days treatment. Higher doses (0.33-33 mg/mL) induced a dose dependent decrease in DPSC viability/proliferation with a more prominent effect with EEP at 7 days. Neither AEP nor EEP induced DPSC differentiation into mineralizing cells, but both AEP and EEP (0.03-0.1 mg/ml) induced a dose dependent increase in DPSC migration. In addition, EEP prevents the upregulation of IL1b and IL6 but not IL8 and CCL2 in response to lipopolysaccharide stimulation. AEP has less potent anti-inflammatory effects and prevents only IL1b upregulation. This study provides new information about the biologic properties of ethanolic and aqueous extracts of propolis and shows that propolis, at doses that do not affect cell viability, induces DPSC migration and has anti-inflammatory properties. These data highlight the potential use of propolis as an alternative intra-canal medicament for regenerative endodontic procedures.

Chitosan-based bioactive hydrogels for osteogenic differentiation of dental pulp stem cells

Platelet-rich plasma (PRP) is an emerging therapeutic application because PRP contains various growth factors that have beneficial effects on tissue regeneration and engineering. Mesenchymal stem cells and PRP derived from peripheral blood have been well studied. In this study, we investigated the effects of PRP derived from human umbilical cord blood (UCB-PRP) on proliferation, alkaline phosphatase (ALP) activity, and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHEDs), dental pulp stem cells (DPSCs), and periodontal ligament stem cells (PDLSCs). Three types of dental stem cells were primarily isolated and characterized by flow cytometric analysis. Dental stem cells were exposed to various concentrations of UCB-PRP, which resulted in the proliferation of dental stem cells. Treatment with 2% UCB-PRP resulted in the highest level of proliferation. The ALP activity of DPSCs and PDLSCs increased following treatment with UCB-PRP in a dose-dependent manner up to a concentration of 2%. ALP activity decreased with higher concentration of UCB-PRP. The effects of UCB-PRP on calcium deposition were similar to those on proliferation and ALP activity. Treatment with 2% UCB-PRP resulted in the highest calcium depositions in DPSCs and PDLSCs; however, treatment with 1% UCB-PRP resulted in the highest calcium deposition in SHEDs. The concentrations of platelet-derived growth factor-AB and transforming growth factor-β1 in UCB-PRP were investigated and found to be comparable to the amounts in peripheral blood. Overall, UCB-PRP had beneficial effects on the proliferation and osteogenic differentiation of dental stem cells. Determination of the optimal concentration of UCB-PRP requires further investigation for clinical applications.

The objective of this study is to compare the effects of the two calcium phosphate composite scaffolds on the attachment, proliferation, and osteogenic differentiation of rabbit dental pulp stem cells (DPSCs). One nano-hydroxyapatite/collagen/poly (l-lactide) (nHAC/PLA), imitating the composition and the micro-structure characteristics of the natural bone, was made by Beijing Allgens Medical Science & Technology Co., Ltd. (China). The other beta-tricalcium phosphate (β-TCP), being fully interoperability globular pore structure, was provided by Shanghai Bio-lu Biomaterials Co, Ltd. (China). We compared the absorption water rate and the protein adsorption rate of two scaffolds and the characterization of DPSCs cultured on the culture plate and both scaffolds under osteogenic differentiation media (ODM) treatment. The constructs were then implanted subcutaneously into the back of severely combined immunodeficient (SCID) mice for 8 and 12 weeks to compare their bone formation capacity. The results showed that the ODM-treated DPSCs expressed osteocalcin (OCN), bone sialoprotein (BSP), type I collagen (COLI) and osteopontin (OPN) by immunofluorescence staining. Positive alkaline phosphatase (ALP) staining, calcium deposition and calcium nodules were also observed on the ODM-treated DPSCs. The absorption water rate and protein adsorption rate of nHAC/PLA was significantly higher than β-TCP. The initial attachment of DPSCs seeded onto nHAC/PLA was significantly higher than that onto β-TCP; and the proliferation rate of the cells was also significantly higher than that of β-TCP on 1, 3, and 7 days of cell culture. The ALP activity, calcium/phosphorus content and mineral formation of DPSCs + β-TCP were significantly higher than DPSCs + nHAC/LA. When implanted into the back of SCID mice, nHAC/PLA alone had no new bone formation, newly formed mature bone and osteoid were only observed in β-TCP alone, DPSCs + nHAC/PLA and DPSCs + β-TCP, and this three groups displayed increased bone formation over the 12-week period. The percentage of total bone formation area had no difference between DPSCs + β-TCP and DPSCs + nHAC/PLA at each time point, but the percentage of mature bone formation area of DPSCs + β-TCP was significantly higher than that of DPSCs + nHAC/PLA. Our results demonstrated that the DPSCs on nHAC/PLA had a better proliferation, and that the DPSCs on β-TCP had a more mineralization in vitro, much more newly formed mature bones in vivo were presented in DPSCs + β-TCP group. These findings have provided a further knowledge that scaffold architecture has different influence on the attachment, proliferation and differentiation of cells. This study may provide insight into the clinical periodontal bone tissue repair with DPSCs + β-TCP construct.

This work aimed to investigate the effect of Sophora flavescens polysaccharide on proliferation and osteogenic differentiation of dental pulp stem cells. The dental pulp stem cells were isolated from rat dental crown tissue and the surface biomarkers were identified by flow cytometry. The osteogenic differentiation of dental pulp stem cells was assessed by alizarin red staining and alkaline phosphatase staining. The dental pulp stem cells were treated with different doses of Sophora flavescens polysaccharide for different time points and cell viability was assessed by cell counting kit 8. Cell migration was detected by Transwell and wound healing assay. The expression of collagen I, osteocalcin I, runt-related transcription factor 2 and beta-catenin were measured by quantitative real time polymerase chain reaction assay and Western blotting assay. The cluster of differentiation 90 was highly expressed and cluster of differentiation 45 expressions was relatively low on surface of isolated dental pulp stem cells. Sophora flavescens polysaccharide suppressed the proliferation of dental pulp stem cells. The treatment with low dose Sophora flavescens polysaccharide (0.2 mg/ml) promoted the migration and osteogenic differentiation of dental pulp stem cells, but higher concentration of Sophora flavescens polysaccharide (1 and 10 mg/ml) suppressed the migration and osteogenic differentiation of dental pulp stem cells. Treatment with low concentration of Sophora flavescens polysaccharide up-regulated the expression of osteogenic biomarkers. Low dose Sophora flavescens polysaccharide can promote the migration and osteogenic differentiation of dental pulp stem cells and up-regulated the expression of osteogenic genes.

Platelet-rich fibrin (PRF) modified nano-hydroxyapatite/chitosan/gelatin/alginate scaffolds increase adhesion and viability of human dental pulp stem cells (DPSC) and osteoblasts derived from DPSC

Injectable thermo-sensitive hydrogels have a potential application in bone tissue engineering for their sensitivities and minimal invasive properties. Human dental pulp stem cells have been considered a promising tool for tissue reconstruction. The objective of this study was to investigate the proliferation and osteogenic differentiation of dental pulp stem cells in injectable thermo-sensitive chitosan/β-glycerophosphate/hydroxyapatite hydrogel invitro. The chitosan /β-glycerophosphate hydrogel and chitosan/β-glycerophosphate/hydroxyapatite hydrogel were prepared using the sol-gel method. The injectability of chitosan /β-glycerophosphate hydrogel and chitosan/β-glycerophosphate/hydroxyapatite hydrogel was measured using a commercial disposable syringe. Scanning electron microscopy was used to observe the inner structure of hydrogels. Then dental pulp stem cells were seeded in chitosan /β-glycerophosphate hydrogel and chitosan/β-glycerophosphate/hydroxyapatite hydrogel, respectively. The growth of dental pulp stem cells was periodically observed under an inverted microscope. The proliferation of dental pulp stem cells was detected by using an Alamar Blue kit, while cell apoptosis was determined by using a Live/Dead Viability/Cytotoxicity kit. The osteogenic differentiations of dental pulp stem cells in chitosan /β-glycerophosphate hydrogel and chitosan/β-glycerophosphate/hydroxyapatite hydrogel were evaluated by alkaline phosphatase activity assay and mRNA expression of osteogenesis gene for 21 days in osteogenic medium. The results indicated that there was no significant difference between chitosan /β-glycerophosphate hydrogel and chitosan/β-glycerophosphate/hydroxyapatite hydrogel in injectability. Cells within the chitosan/β-glycerophosphate/hydroxyapatite hydrogel displayed a typical adherent cell morphology and rapid proliferation with high cellular viability after 14 days of culture. Dental pulp stem cells seeded in chitosan/β-glycerophosphate/hydroxyapatite hydrogels had a higher alkaline phosphatase activity and better up-regulation of gene expression levels of Runx-2, Collagen I, alkaline phosphatase and osteocalcin than in chitosan /β-glycerophosphate hydrogels after osteogenic differentiation. These results demonstrated that the chitosan/β-glycerophosphate/hydroxyapatite hydrogel had excellent cellular compatibility and the superiority in promoting dental pulp stem cells osteogenic differentiation invitro, showing that the combination of dental pulp stem cells and chitosan/β-glycerophosphate/hydroxyapatite hydrogel has the potential to be used for bone tissue engineering.

BackgroundTo study the odontogenic potential of dental pulp stem cells (DPSCs) after induction with three different bioactive materials: activa bioactive (base/liner) (AB), TheraCal LC (TC), and mineral trioxide aggregate (MTA), when combined with two different types of scaffolds.MethodsDPSCs were isolated from freshly extracted premolars of young orthodontic patients, cultured, expanded to passage 4 (P), and characterized by flow cytometric analysis. DPSCs were seeded onto two scaffolds in contact with different materials (AB, TC, and MTA). The first scaffold contained polycaprolactone-nano-chitosan and synthetic hydroxyapatite (PCL-NC-HA), whereas the second scaffold contained polycaprolactone-nano-chitosan and synthetic Mg-substituted hydroxyapatite (PCL-NC-Mg-HA). DPSC viability and proliferation were evaluated at various time points. To assess odontoblastic differentiation, gene expression analysis of dentin sialophosphoprotein (DSPP) by quantitative real-time polymerase chain reaction (qRT-PCR) and morphological changes in cells were performed using inverted microscope phase contrast images and scanning electron microscopy. The fold-change in DSPP between subgroups was compared using a one-way ANOVA. Tukey's test was used to compare the fold-change in DSPP between the two subgroups in multiple comparisons, and P was set at p < 0.05.ResultsDSPP expression was significantly higher in the PCL-NC-Mg-HA group than in the PCL-NC-HA group, and scanning electron microscopy revealed a strong attachment of odontoblast-like cells to the scaffold that had a stronger odontogenic differentiation effect on DPSCs than the scaffold that did not contain magnesium. MTA has a significantly higher odontogenic differentiation effect on cultured DPSCs than AB or TC does. The combination of scaffolds and bioactive materials improves DPSCs induction in odontoblast-like cells.ConclusionsThe PCL-NC-Mg-HA scaffold showed better odontogenic differentiation effects on cultured DPSCs. Compared to AB and TC, MTA is the most effective bioactive material for inducing the odontogenic differentiation of cultured DPSCs.

This study aims to evaluate the impact of asiaticoside (AC) on the viability and proliferation of dental pulp stem cells (DPSCs), considering the known negative effects of routinely used intracanal medicaments. This evaluation will be compared with the outcomes from using traditional intracanal medicaments, specifically triple antibiotic paste (TAP) and calcium hydroxide [Ca(OH)2]. The DPSCs were obtained from the third molars of an adult donor. The application of flow cytometry was employed to do a phenotypic analysis on DPSCs using CD90, CD73, CD105, CD34, CD14, and CD45 antibodies. The methylthiazol tetrazolium (MTT) assay was employed to assess cellular viability. The cells were treated with different concentrations of TAP and Ca(OH)2 (5, 2.5, 1, 0.5, and 0.25 mg/mL), along with AC (100, 50, 25, 12.5, and 6.25 µM). A cell proliferation rate was performed at 3, 5, and 7 days. The characterization of DPSCs was conducted by flow cytometry analysis, which verified the presence of mesenchymal cell surface antigen molecules (CD105, CD73, and CD90) and demonstrated the absence of hematopoietic markers (CD34, CD45, and CD14). Cells treated with concentrations over 0.5 mg/mL of TAP and Ca(OH)2 showed a notable reduction in cell viability in comparison to the untreated cells (p < 0.05). Additionally, the cells treated with different concentrations of AC 12.5, 6.25, 25, and 50 µM did not differ significantly from the untreated cells (p > 0.05). Nevertheless, cells treated with concentrations of 100 µM showed a significant reduction in viability compared to the untreated cells (p < 0.05). After a period of 7 days, it was noted that cells exposed to three different concentrations of AC (50, 25, and 12.5 µM) had a notable rise in cell density in comparison to TAP and Ca(OH)2 (p < 0.05). Furthermore, cells that were exposed to a concentration of 12.5 µM exhibited the highest cell density. The cellular viability of the AC-treated cells was superior to that of the TAP and Ca(OH)2-treated cells. Moreover, the AC with a concentration of 12.5 µM had the highest degree of proliferation. This study underscores the importance of evaluating alternative root canal medicaments and their effects on DPSCs' growth and vitality. The findings on AC, particularly its influence on the survival and proliferation of DPSCs, offer valuable insights for its probable use as an intracanal medication. This research contributes to the ongoing efforts to identify safer and more effective intracanal treatments, which are crucial for enhancing patient outcomes in endodontic procedures. How to cite this article: Alazemi MJ, Badawi MF, Elbeltagy MG, et al. Examining the Effects of Asiaticoside on Dental Pulp Stem Cell Viability and Proliferation: A Promising Approach to Root Canal Treatment. J Contemp Dent Pract 2024;25(2):118-127.

Effects of Calcitonin Gene-related Peptide on Dental Pulp Stem Cell Viability, Proliferation, and Differentiation

Background: Shock wave-enhanced emission photoacoustic streaming (SWEEPS) is a novel irrigation activation method based on photoacoustic streaming. The aim of this study was to look into the impact of SWEEPS on the attachment and survival of dental pulp stem cells (DPSCs). Materials and Methods: In this in vitro study, 34 standardized root segments were randomly allocated into two groups: SWEEPS and the conventional conditioning group. After the irrigation human DPSCs were seeded on the internal walls of these samples, and the attachment and survival of 30 of them were assessed on different days. The remaining two samples were observed using a scanning electron microscope (SEM). Independent sample t-test, Mann–Whitney U-test, one-way ANOVA, Kruskal–Wallis, and two-way ANOVA were used for data analysis with the level of significance = 0.05. Results: The viability of DPSCs was significantly greater in the SWEEPS group in comparison with the conventional conditioning group (P = 0.029). Both groups have shown a significant increase in the viability of DPSCs over time (P = 0.0001, P = 0.003). SEM results have shown a smear layer-free surface with firmly attached DPSCs in the SWEEPS group. Conclusion: The results of this study indicated that active irrigation using SWEEPS could provide a superior surface in terms of viability and attachment of DPSCs compared to the conventional conditioning method.

The role of NMT induction on odontogenic proliferation and differentiation of dental pulp stem cells

Characterization of silver diamine fluoride cytotoxicity using microfluidic tooth-on-a-chip and gingival equivalents

Dental pulp stem cell is a new type of mesenchymal stem cell that has a potential for tissue regeneration. Gelatin sponges are often used for hemostasis in dental surgery. In this study, we aimed to evaluate the dental pulp stem cells' proliferation and osteogenic differentiation in different layer-by-layer-modified gelatin sponge scaffolds including the G, G + P (gelatin sponge+ poly-l-lysine modification), G + M (gelatin sponge + mineralization modification), and G + M + P (gelatin sponge + mineralization modification + poly-l-lysine modification) groups in vitro and assessed them in vivo. The results showed that dental pulp stem cells had a great potential for osteogenic differentiation. In vitro, the G + M + P group not only enhanced the adhesion and proliferation of dental pulp stem cells but also facilitated their osteogenic differentiation. However, alkaline phosphatase activity was prohibited after modification. In vivo, both dental pulp stem cells and cells from nude mice grew well on the scaffold, and G + M and G + M + P groups could promote the mineralization deposit formation and the expression of osteocalcin in osteogenic differentiation of dental pulp stem cells. In conclusion, the combination of dental pulp stem cells and G + M + P scaffold has a great potential for bone tissue engineering.