Evaluation of dental pulp stem cells response to flowable nano-hybrid dental composites: A comparative analysis.

BACKGROUND: Flowable resin composites (FRC) are tooth-colored restorative materials that contain a lower filler particle content, and lower viscosity than their bulk counterparts, making them useful for specific clinical applications. Yet, their chemical makeup may impact the cellular population of the tooth pulp. This in-vitro study assessed the cytocompatibility and odontogenic differentiation capacity of dental pulp stem cells (DPSCs) in response to two recent FRC material extracts. METHODS: Extracts of the FRC Aura easyflow (AEF) and Polofil NHT Flow (PNF) were applied to DPSCs isolated from extracted human teeth. Cell viability of DPSCs was assessed using MTT assay on days 1, 3 and 7. Cell migration was assessed using the wound healing assay. DPSCs' capacity for osteo/odontogenic differentiation was assessed by measuring the degree of mineralization by Alizarin Red S staining, alkaline phosphatase enzyme (ALP) activity, and monitoring the expression of osteoprotegerin (OPG), RUNX Family Transcription Factor 2 (RUNX2), and the odontogenic marker dentin sialophosphoprotein (DSPP) by RT-PCR. Monomer release from the FRC was also assessed by High-performance liquid chromatography analysis (HPLC). RESULTS: DPSCs exposed to PNF extracts showed significantly higher cell viability, faster wound closure, and superior odontogenic differentiation. This was apparent through Alizarin Red staining of calcified nodules, elevated alkaline phosphatase activity, and increased expression of osteo/odontogenic markers. Moreover, HPLC analysis revealed a higher release of TEDGMA, UDMA, and BISGMA from AEF. CONCLUSIONS: PNF showed better cytocompatibility and enhancement of odontogenic differentiation than AEF.

Photobiomodulation and low-intensity pulsed ultrasound synergistically enhance dental mesenchymal stem cells viability, migration and differentiation: an invitro study.

Novel methods and technologies that improve mesenchymal stem cells (MSCs) proliferation and differentiation properties are required to increase their clinical efficacy. Photobiomodulation (PBM) and low-intensity pulsed ultrasound (LIPUS) are two strategies that can be used to enhance the regenerative properties of dental MSCs. This study evaluated the cytocompatibility and osteo/odontogenic differentiation of dental pulp, periodontal ligament, and gingival MSCs after stimulation by either PBM or LIPUS and their combined effect. MTT assay, cell migration assay, osteo/odontogenic differentiation by AR staining and ALP activity, and expression of osteo/odontogenic markers (OPG, OC, RUNX2, DSPP, DMP1) by RT-qPCR were evaluated. Statistical analysis was performed using ANOVA, followed by Tukey's post hoc test, with a p-value of less than 0.05 considered significant. The results showed that combined stimulation by PBM and LIPUS resulted in significantly the highest viability of MSCs, the fastest migration, the most dense AR staining, the most increased ALP activity, and the most elevated levels of osteogenic and odontogenic markers. The synergetic stimulation of PBM and LIPUS can be utilized in cell-based regenerative approaches to promote the properties of dental MSCs.

The Effect of Clopidogrel and Ticagrelor on Human Adipose Mesenchymal Stem Cell Osteogenic Differentiation Potential: In Vitro Comparative Study.

Ticagrelor (TICA) and clopidogrel (CLP) are extensively used antiplatelet drugs that act by antagonizing the P2Y12 receptors that are found on platelets in addition to bone cells. Aim. The purpose of this study was to investigate the effect of clopidogrel and ticagrelor on stem cells osteogenic differentiation in vitro. Methods. Human adipose-derived mesenchymal stem cells (hAd-MSCs) were divided into (1) control group, (2) osteogenic group (osteo group), (3) clopidogrel group (CLP group), and (4) ticagrelor group (TICA group). The osteogenic differentiation potential was determined by mineralization nodule formation using Alizarin Red S staining, measuring ALP enzyme activity by alkaline phosphatase assay. Quantitative determination for osteogenic markers included osteocalcin (OC); runt-related transcription factor 2 (RUNX2) performed using western blot; osteoprotegerin (OPG) using enzyme-linked immunosorbent assay (ELISA) and inflammatory markers; and tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) measured using real-time polymerase chain reaction quantitative (RT-PCR) and ELISA. Results. In comparison to all study groups, the TICA group showed significant increase in the mineralized extracellular matrix, ALP enzyme activity, and bone markers expression as RUNX2 (P < 0.0001), OC, and OPG (P < 0.05). The expression of IL-6 and TNF-α was determined by RT-qPCR and ELISA techniques. TICA and CLP significantly decreased both markers compared to the control group. The TICA group showed statistically significant lower levels of both markers (P < 0.0001) than the CLP and control groups via the ELISA technique. Conclusion. TICA may possess a positive effect on hAd-MSCs osteogenic differentiation compared to CLP.

Effect of pulp capping materials on odontogenic differentiation of human dental pulp stem cells: An in vitro study.

OBJECTIVES: Migration and differentiation of human dental pulp stem cells (hDPSCs) is a vital and key factor in the success of reparative dentin formation for maintenance of pulp vitality. Pulp capping materials are used to stimulate DPSCs to induce new dentin formation. Thus, the aim of the present study was to compare the response of DPSCs to four commercially available pulp capping materials: a bioactive bioceramic (Material 1), a nonresinous ready-to-use bioceramic cement (Material 2), a bioactive composite (Material 3), and a biocompatible, dual-cured, resin-modified calcium silicate (Material 4). MATERIALS AND METHODS: hDPSCs were isolated and cultured from freshly extracted teeth and were then characterized by flow cytometry and multilineage differentiation. Discs prepared from pulp capping materials were tested with hDPSCs and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, cell migration assay and odontogenic differentiation assay was performed. Expression of osteogenic markers (osteopontin, RUNX family transcription factor 2, osteocalcin) and the odontogenic marker (dentin sialophosphoprotein) was detected using reverse transcription-polymerase chain reaction. RESULTS: Materials 1, 2, and 3 generated more cell viability than Material 4. Furthermore, Material 4 showed the least wound exposure percentage, while Material 3 showed the highest percentage. Enhanced mineralization was found in hDSCPs cultured with Material 3, followed by Material 1, and then Material 2, while Material 4 revealed the least calcified mineralization. CONCLUSIONS: The results of this study were inconclusive regards contemporary bioceramic materials designed for vital pulp therapy as they have different effects on hDPSC. Further testing for cytotoxicity using live-dead staining, animal experiments, clinical trials, and independent analyses of these biomaterials is necessary for clinicians to make an informed decision for their use.

Enhancing the anticancer potential of metformin: fabrication of efficient nanospanlastics, in vitro cytotoxic studies on HEP-2 cells and reactome enhanced pathway analysis.

Metformin (MET), an oral antidiabetic drug, was reported to possess promising anticancer effects. We hypothesized that MET encapsulation in unique nanospanlastics would enhance its anticancer potential against HEP-2 cells. Our results showed the successful fabrication of Nano-MET spanlastics (d = 232.10 ± 0.20 nm; PDI = 0.25 ± 0.11; zeta potential = (-) 44.50 ± 0.96; drug content = 99.90 ± 0.11 and entrapment efficiency = 88.01 ± 2.50%). MTT assay revealed the enhanced Nano-MET cytotoxicity over MET with a calculated IC50 of 50 µg/mL and > 500 µg/mL, respectively. Annexin V/PI apoptosis assay showed that Nano-MET significantly decreased the percentage of live cells from 95.49 to 93.70 compared to MET and increased the percentage of cells arrested in the G0/G1 phase by 8.38%. Moreover, Nano-MET downregulated BCL-2 and upregulated BAX protein levels by 1.57 and 1.88 folds, respectively. RT-qPCR revealed that Nano-MET caused a significant 13.75, 4.15, and 2.23-fold increase in caspase-3, -8, and - 9 levels as well as a 100 and 43.47-fold decrease in cyclin D1 and mTOR levels, respectively. The proliferation marker Ki67 immunofluorescent staining revealed a 3-fold decrease in positive cells in Nano-MET compared to the control. Utilizing the combined Pathway-Enrichment Analysis (PEA) and Reactome analysis indicated high enrichment of certain pathways including nucleotides metabolism, Nudix-type hydrolase enzymes, carbon dioxide hydration, hemostasis, and the innate immune system. In summary, our results confirm MET cytotoxicity enhancement by its encapsulation in nanospanlastics. We also highlight, using PEA, that MET can modulate multiple pathways implicated in carcinogenesis.

Enhanced human periodontal ligament stem cell viability and osteogenic differentiation on two implant materials: An experimental in vitro study.

Background: Surface roughness of dental implants impacts the survival of adult periodontal stem cells and rate of differentiation. This research was conducted to test how human periodontal ligament stem cells behaved on yttria stabilized tetragonal zirconia polycrystals and polyetheretherketone (PEEK) discs with different surface topographies. Methods: Discs roughening was prepared by sandblasting. Stem cells were cultivated on zirconia discs with a polished surface, PEEK discs with a polished surface, sandblasted zirconia discs and sandblasted PEEK discs. Cells viability was assessed after 24, 48, 72 hours. Scanning electron microscopy was used to examine the adherence and attachment of cells. Osteoblastic differentiation capacity was studied by checking the mineralization clusters development through alizarin red S staining and alkaline phosphatase assay. ANOVA and the Tukey post hoc test were used for the statistical analysis. Results: Polished PEEK discs showed lower cell viability, whereas roughened sandblasted zirconia and PEEK discs showed the highest proliferation rates and cell viability percent. The osteogenic differentiation was enhanced for rough surfaces in comparison to polished surfaces. Sandblasted zirconia and PEEK discs showed a markedly increased mineralized nodule development and ALP enzyme activity compared to the polished surface and control. Conclusions: Micro- topographies creation on the PEEK implant surface enhances stem cell attachment, viability, and osteogenic differentiation.

Effect of rapamycin on human periodontal ligament stem cells that have been exposed to sodium hypochlorite.

AIMS: The present study investigated the effect of rapamycin on the viability and osteogenic differentiation potential of human periodontal ligament stem cells (hPDLSCs) in the presence of sodium hypochlorite (NaOCl). MAIN METHODS: After determining the minimum inhibitory concentration of NaOCl and optimum concentration of rapamycin, the viability of hPDLSCs was evaluated using the MTT assay subsequent to their exposure to NaOCl, rapamycin, or a combination of both. Osteogenic differentiation was evaluated by the cell mineralization assay performed by alizarin red S staining, alkaline phosphatase activity, and monitoring the expression of osteogenic genes markers Runt-related transcription factor 2, osteocalcin, and osteoprotegerin, using real-time quantitative polymerase chain reaction (RT-qPCR). The expression of autophagy-related genes PI3K, Akt, and mTOR, was also analyzed with RT-qPCR. KEY FINDINGS: Stem cells treated with rapamycin showed the highest percentage of viable cells in the presence of NaOCl. The same trend was observed for all osteogenic differentiation assays. The hPDLSCs treated with rapamycin demonstrated the highest calcium nodule deposition, alkaline phosphatase activity, and the expression of osteogenic gene markers. These effects were not adversely affected by the presence of NaOCl. Rapamycin significantly inhibited mTOR gene expression, while there were no differences in the gene expression of PI3K and Akt. SIGNIFICANCE: Rapamycin counteracts the cytotoxic effect of NaOCl by enhancing the viability and osteogenic differentiation potential of hPDLSCs. Rapamycin appears to accomplish these processes via autophagy activation, by inhibiting mTOR gene expression. The incorporation of rapamycin in regenerative endodontic therapy may encourage a higher success rate.

Effect of Different Sealers on the Cytocompatibility and Osteogenic Potential of Human Periodontal Ligament Stem Cells: An In Vitro Study.

BACKGROUND: There is tendency for unavoidable sealer extrusion in some clinical cases. This might adversely affect host stem cells and affect healing. This study aimed to investigate the effect of different sealers on the cytocompatibility and osteogenic potential of human periodontal ligament stem cells (hPDLSCs). METHODS: The cytotoxic effect of the extracted elutes of VDW.1Seal (VDW.1), Endosequence BC Sealer HiFlow (ES), GuttaFlow-2 (GF), and ADSeal (AD-S) on the hPDLSCs was determined using the MTT assay. Cell proliferation and migration were assessed by the scratch wound healing assay. Osteogenic differentiation potential was assessed. Measurement of pH values and calcium ions release was performed. RESULTS: GF had a significantly higher percentage of viable cells. The cell migration assay showed that GF demonstrated the lowest open wound area percentage. GF and AD-S showed the highest calcium nodule deposition. GF demonstrated higher ALP activity than ES. Expression of RUNX2 and OC genes was similar for all sealers, while OPG gene expression was significantly higher for VDW.1 and GF. ES and AD-S displayed the highest pH values on day 1. Calcium ion release of ES and VDW.1 was significantly the highest. CONCLUSIONS: GuttaFlow-2 and VDW.1Seal sealers have favorable behavior toward host stem cells.

Effect of local application of platelet-rich fibrin scaffold loaded with simvastatin on peri-implant bone changes.

Aim: The purpose of this study was to compare the effects of autologous platelet-rich fibrin (PRF) alone and PRF loaded with SIM on peri-implant bone changes and implant stability in patients undergoing implant rehabilitation. Settings and Design: This was a nonrandomized controlled split-mouth study. Materials and Methods: The study included 8 males between the ages of 45 and 60 years. Each patient received two implants, one on each side of the arch. One side was treated with PRF alone and the other side with PRF loaded with SIM at the time of osteotomy. A cone-beam computed tomography was used to evaluate bone changes around the insertion of implant sites at 3, 6, and 12 months postoperatively. The secondary outcome included measuring implant stability using Osstell device at baseline and 3 months postinsertion. To compare groups at different time periods, data were examined using a two-way analysis of variance. Statistical Analysis Used: The results were compared between the groups using a two-way analysis of variance, followed by a post hoc Bonferroni test. To examine total bone changes and stability comparisons between the two groups at the end of the trial, an unpaired t-test was utilized. Results: The mean crestal bone-level changes in the SIM/PRF group were significantly lower than the PRF group, with a mean shift of 0.9788 ± 0.04853 versus 1.356 ± 0.0434, respectively (P < 0.0001). There was no significant difference between the two groups in implant stability. Conclusion: Peri-implant application of SIM/PRF resulted in less bone changes than PRF alone, which may prove to be beneficial for the long-term success of implants. SIM showed promising results in limiting peri-implant bone resorption providing new clinical application for SIM in dental implant rehabilitation.

Monocyte monolayer assay in pre-transfusion testing: A magic key in transfusing patients with recurrent bad cross-match due to alloimmunization.

BACKGROUND: The monocyte monolayer assay (MMA) is an in-vitro assay that can predict the outcome of blood transfusion of antigen positive units when serologically compatible blood is not available. MATERIALS AND METHODS: Fifty-four patients testing positive by the antibody screening test using gel agglutination were further examined by the alloantibody identification panel to determine alloantibody specificity. After determining and categorizing the antibodies, patients' samples were examined using the MMA to determine the clinical significance of the detected alloantibodies. We also tested 2 seeding methods (24-well cell culture plates versus 8-well chamber-slides) and 3 visualization/staining techniques (unstained phase contrast, Leishman and Giemsa staining). RESULTS: 35 out of the 54 cases (64.8%) had a monocyte index of >5% which is predictive of occurrence of hemolytic reaction after transfusion; 23 cases with antibodies known to be clinically significant [anti-C, anti-E, anti-c, anti-K, anti-Fy(a), anti Fy(b), anti-JK(b)], 2 with Anti-M specificity, 7 cases with autoantibodies and 3 cases with multiple antibodies. On the other hand, 19 out of the 54 (35.2%) cases included in the study showed a monocyte index of <5% which is predictive of absence of hemolytic reaction after transfusion. The 8-well chamber-slides were better than the 24-well culture plates, as the latter showed a lot of un-phagocytosed RBCs in the background. Also, Leishman staining was better than Giemsa staining with better and clearer differentiation between the RBCs, monocytes and phagocytic vacuoles. CONCLUSION: MMA can be used as a surrogate cross-match test for the selection of blood units in cases where antigen-negative blood units are not available.

The sole and combined effect of simvastatin and platelet rich fibrin as a filling material in induced bone defect in tibia of albino rats.

Statins like simvastatin (SIM) have demonstrated to have pleiotropic actions other than their conventional use as antilipidemic drugs. Also, nowadays natural scaffolds like platelets rich fibrin (PRF) showed promising results on bone regeneration. Aim This study compare the regenerative power of SIM and PRF added locally each as a sole filling material on induced bone defect and evaluate the combined effect using PRF loaded with SIM. MATERIALS AND METHODS: A critical size bone defect was induced in 48 male albino rats of average weight 150-200 g and were divided into 4 groups according to the filling material. Control, PRF, SIM, and SIM/PRF group. Each group was subdivided according to the sacrificing period into two subgroups (one and two-months postoperatively). Tibial specimens were evaluated histologically using masson trichrome (MT) special stain to detect areas of new bone formation, immunohistochemically using anti- BMP2 and anti-VEGF, serum levels of Osteoprotegerin (OPG), RANKL, osteocalcin and alkaline phosphatase enzyme (ALP) were measured one and two months postoperatively using ELISA, Finally bone mineral density (BMD) at the bone defect area was analyzed using digital X-ray one and two-months postoperatively. RESULTS: The percentage of newly formed bone increased significantly in the three groups vs the control group with the highest significant increase (p < 0.001) in the SIM/PRF group one month postoperatively. Also, SIM/PRF group was the only group which showed significant bone maturation two-months postoperatively compared to the other groups. Immunohistochemical analysis showed significant increase in positively stained BMP-2 and VEGF expression (p < 0.001) in the three groups vs the control group with the highest significant increase (p < 0.001) in the SIM/PRF group. Serum bone anabolic markers increased significantly in the SIM and SIM/PRF groups. In contrast, RANKL serum level decreased significantly in the SIM and SIM/PRF group one month postoperatively with no significant decrease in the PRF group vs the control group. Digital X-ray results revealed the highest BMD percent change was found in the SIM/PRF group and showed complete bone healing two-months postoperatively.

WITHDRAWN: Response to letter to the editor: The sole and combined effect of simvastatin and platelet rich fibrin as a filling material in induced bone defect in tibia of albino rats.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, https://doi.org/10.1016/j.bone.2019.01.007. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.