Green synthesis of nanohydroxyapatite with Elaeagnus angustifolia L. extract as a metronidazole nanocarrier for in vitro pulpitis model treatment.

The aim of this study is to introduce a dental capping agent for the treatment of pulp inflammation (pulpitis). Nanohydroxyapatite with Elaeagnus angustifolia L. extract (nHAEA) loaded with metronidazole (nHAEA@MTZ) was synthesized and evaluated using a lipopolysaccharide (LPS) in vitro model of pulpitis. nHAEA was synthesized through sol-gel method and analyzed using Scanning Electron Microscopy, Transmission Electron Microscopy, and Brunauer Emmett Teller. Inflammation in human dental pulp stem cells (HDPSCs) induced by LPS. A scratch test assessed cell migration, RT PCR measured cytokines levels, and Alizarin red staining quantified odontogenesis. The nHAEA nanorods were 17-23 nm wide and 93-146 nm length, with an average pore diameter of 27/312 nm, and a surface area of 210.89 m2/g. MTZ loading content with controlled release, suggesting suitability for therapeutic applications. nHAEA@MTZ did not affect the odontogenic abilities of HDPSCs more than nHAEA. However, it was observed that nHAEA@MTZ demonstrated a more pronounced anti-inflammatory effect. HDPSCs treated with nanoparticles exhibited improved migration compared to other groups. These findings demonstrated that nHAEA@MTZ could be an effective material for pulp capping and may be more effective than nHAEA in reducing inflammation and activating HDPSCs to enhance pulp repair after pulp damage.

Effect of HM-Exos on the migration and inflammatory response of LPS-exposed dental pulp stem cells.

AIM: The purpose of this study was to investigate the effects of human milk exosomes (HM-Exos) on the viability, migration, and inflammatory responses of lipopolysaccharide (LPS)-exposed human dental pulp stem cells (HDPSCs) in vitro. METHODS: HM-Exos were isolated, and dynamic light scattering (DLS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were used to analyze their physical properties (size and shape). To construct an in vitro inflammation model, HDPSCs were exposed to LPS. The MTT test and migration assay were used to investigate the effect of HM-Exos on cell proliferation and migration, and the quantitative polymerase chain reaction (qPCR) was used to assess the expression of inflammatory genes in HDPSCs. Data were analyzed using a one-way analysis of variance (ANOVA) with Tukey's post-test. RESULTS: DLS measurement revealed that HM-Exos were 116.8 ± 3.6 nm in diameter. The SEM and TEM images revealed spherical shapes with diameters of 97.2 ± 34.6 nm. According to the results of the cell viability assay, the nontoxic concentration of HM-Exos (200 µg/ml) was chosen for the subsequent investigations. The migration assay results showed that HM-Exos improved the potential of LPS-exposed HDPSCs to migrate. The qPCR results indicated that HM-Exos significantly reduced the expression of inflammatory cytokines such as TNF-α, IL-1ß, and IL-6 in HDPSCs after LPS stimulation. CONCLUSIONS: HM-Exos increased LPS-exposed HDPSCs migration and proliferation and reduced gene expression of inflammatory cytokines. They may be a viable candidate for pulpitis therapy.

Effects of hydroxyapatite nanorods prepared through Elaeagnus Angustifolia extract on modulating immunomodulatory/dentin-pulp regeneration genes in DPSCs.

Dental pulp stem cells (DPSCs) are a new type of mesenchymal stem cells (MSCs) found in the oral cavity with immunomodulation and tissue regeneration capacities. This study determined the impacts of nano-hydroxyapatite (nHA) prepared through Elaeagnus Angustifolia extract (EAE) to enhance the relative expression of immunomodulatory/dentin-pulp regeneration genes in DPSCs. To produce nHA and modified nHA via EAE (nHAEA), the sol-gel technique was used. The functional groups of nanoparticles (NPs), morphological, and optical features were determined using Fourier transform infrared (FTIR), X-ray diffraction (XRD), Scanning electron microscopy (SEM) together with energy-dispersive X-ray analysis (EDAX), and Transmission electron microscopy (TEM). The cell viability was then determined using the MTT method in the presence of various EAE, nHA, and nHAEA concentrations. Target gene expression was quantified using a real-time PCR procedure after treating DPSCs with an optimally non-toxic dose of EAE and NPs. The presence of the HA phase was reported with the XRD and FTIR results. According to the results of SEM and TEM, the rod-like NPs could be fabricated. nHAEAs were found to be characterized with low crystallite size, reduced diameter, lengthier, needle-like, and less agglomerated particles compared with nHA. The real-time PCR results demonstrated that nHAEA remarkably increased the expression of human leukocyte antigen-G5 (HLA-G5), vascular endothelial growth factor (VEGF), dentin sialophosphoprotein (DSPP), and interleukin6 (IL6) genes compared to the nHA group. These findings suggest that nHAEAs might have the potential application in the stemness capability of DPSCs for the treatment of inflamed/damaged pulp.

Dentin regeneration based on tooth tissue engineering: A review.

Missing or damaged teeth due to caries, genetic disorders, oral cancer, or infection may contribute to physical and mental impairment that reduces the quality of life. Despite major progress in dental tissue repair and those replacing missing teeth with prostheses, clinical treatments are not yet entirely satisfactory, as they do not regenerate tissues with natural teeth features. Therefore, much of the focus has centered on tissue engineering (TE) based on dental stem/progenitor cells to create bioengineered dental tissues. Many in vitro and in vivo studies have shown the use of cells in regenerating sections of a tooth or a whole tooth. Tooth tissue engineering (TTE), as a promising method for dental tissue regeneration, can form durable biological substitutes for soft and mineralized dental tissues. The cell-based TE approach, which directly seeds cells and bioactive components onto the biodegradable scaffolds, is currently the most potential method. Three essential components of this strategy are cells, scaffolds, and growth factors (GFs). This study investigates dentin regeneration after an injury such as caries using TE and stem/progenitor cell-based strategies. We begin by discussing about the biological structure of a dentin and dentinogenesis. The engineering of teeth requires knowledge of the processes that underlie the growth of an organ or tissue. Then, the three fundamental requirements for dentin regeneration, namely cell sources, GFs, and scaffolds are covered in the current study, which may ultimately lead to new insights in this field.

Effect of PCL/nHAEA nanocomposite to osteo/odontogenic differentiation of dental pulp stem cells.

PURPOSE: The green synthesis of nanoparticles has recently opened up a new route in material production. The aim of this study was to evaluate the effect of nanohydroxyapatite (nHA) synthesized from Elaeagnus angustifolia (EA) extract in polycaprolactone (PCL) nanofibers (PCL/nHAEA) to odontogenic differentiation of dental pulp stem cells (DPSCs) and their potential applications for dentin tissue engineering. METHODS: Green synthesis of nHA via EA extract (nHAEA) was done by the sol-gel technique. Then electrospun nanocomposites containing of PCL blended with nHA (P/nHA) and nHAEA (P/nHAEA) were fabricated, and the characterization was evaluated via X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and the contact angle. The morphology of nanofibers and the cell adhesion capacity of DPSCs on nanofibers were evaluated using SEM. Cytocompatibility was assessed by MTT. Osteo/odontogenic differentiation ability of the nanocomposites were assessed using alkaline phosphatase (ALP) activity, alizarin red S (ARS) staining, and quantitative real-time polymerase chain reaction (qPCR) technique. RESULTS: Viability and adhesion capacity of DPSCs were higher on P/nHAEA nanofibers than PCL and P/nHA nanofibers. ARS assay, ALP activity, and qPCR analysis findings confirmed that the nHAEA blended nanofibrous scaffolds substantially increased osteo/odontogenic differentiation of DPSCs. CONCLUSION: PCL/nHAEA nanocomposites had a noticeable effect on the odontogenic differentiation of DPSCs and may help to improve cell-based dentin regeneration therapies in the future.

Expression of immunomodulatory and tissue regenerative biomarkers in human dental pulp derived-mesenchymal stem cells treated with curcumin: an in vitro study.

BACKGROUND: Human Dental pulp derived-mesenchymal stem cells (hDP-MSCs) have the capability of selfrenewal, multipotency, as well as immunosuppressive properties. They are ideal candidates for regenerating damaged dental tissue and treating inflammation-related diseases. However, methods (such as genetic variation) to improve the immunomodulatory and regenerative efficiency of MSCs in different diseases still need to be developed. Curcumin (CUR) is known for its broad applications in regenerative medicine and the treatment of inflammatory disorders via its anti-inflammatory and anti-oxidant effects. This study was conducted to investigate the effect and underlying mechanisms of CUR on the immunomodulatory and regenerative function of hDP-MSCs and whether treating these cells with CUR can improve therapeutic efficacy. METHODS AND RESULTS: hDP-MSCs were isolated from dental pulp and then treated with CUR. Cell viability rate was observed in hDP-MSCs after treatment of CUR by MTT assay. Real-time quantitative (RT-PCR) was applied to estimate the expression of immunomodulatory and regenerative genes after treatment of CUR. The RT-PCR results showed that VEGF-A and STAT3 markers were up-regulated while HLA-G5 and VCAM-1 markers were down-regulated by CUR (20 µM) treatment in hDP-MSCs (P < 0.001). Besides, this research indicated that there were no significant changes in the expressions of RelA and DSPP after 48 h (P = 0.33, P = 1). CONCLUSION: Our findings demonstrate that CUR can enhance the immunomodulatory and regenerative effects of hDP-MSCs and improve their therapeutic efficacy. These findings can give an understanding of the mechanism for improving restorative and immunomodulatory activity in hDP-MSCs by curcumin.

Effects of phytosomal curcumin treatment on modulation of immunomodulatory and pulp regeneration genes in dental pulp mesenchymal stem cells.

Dental pulp stem cells (DPSCs) are a new population of mesenchymal stem cells (MSCs) located in the oral cavity with potential capacities for tissue regeneration and immunomodulation. The purpose from this study was to determine effects of curcumin nanoparticle into phytosomal formulation (PC) on the relative expression of DSPP, VEGF-A, HLA-G5, VCAM1, RelA and STAT3 genes which are among the most important factors influencing processes of immunomodulatory and tissue regenerative by DPSCs. After isolation and culture of DPSCs, these cells were characterized according to predetermined criteria including flow cytometric analysis for detection of the most important cell surface markers and also evaluation of multilineage differentiation potential. Then, the MTT method was employed to check the cell viability in treatment with different concentrations of PC. Following DPSCs' treatment with an optimal-non-toxic dose of this nanoparticle, quantification of expression of target genes was performed using real-time PCR procedure. According to results of immunophenotyping analysis and cell differentiation experiments, the isolated cells were confirmed as MSCs as more than 99% of them expressed specific mesenchymal markers while only about 0.5% of them were positive for hematopoietic marker. The real-time PCR results indicated that PC significantly reduced the expression of RelA, STAT3, VCAM1 and HLA-G5 genes up to many times over while optimally enhanced the expression of DSPP and VEGF-A genes, although this enhance was statistically significant only for VEGF-A (all P < 0.001). The study suggests that PC affects the stemness capabilities of DPSCs and it may facilitate the development of MSCs-based therapeutics in regenerative dentistry.

Dental pulp stem cells for reconstructing bone defects: A systematic review and meta-analysis.

Background: Bone reconstruction with appropriate quality and quantity for dental implant replacement in the alveolar ridge is a challenge in dentistry. As dental pulp stem cells (DPSCs) could be a new perspective in bone regeneration in the future, this study investigated the bone regeneration process by DPSCs. Methods: Electronic searches for articles in the PubMed, EMBASE, and Scopus databases were completed until 21 April 2022. The most important inclusion criteria for selecting in vivo studies reporting quantitative data based on new bone volume and new bone area. The quality assessment was performed based on Cochrane's checklist. Results: After the title, abstract, and full-text screening of 762 studies, 23 studies were included. A meta-analysis of 70 studies that reported bone regeneration based on new bone area showed a statistically significant favorable influence on bone tissue regeneration compared to the control groups (P<0.00001, standardized mean difference [SMD]=2.40, 95% CI: 1.55‒3.26; I2=83%). Also, the meta-analysis of 14 studies that reported new bone regeneration based on bone volume showed a statistically significant favorable influence on bone tissue regeneration compared to the control groups (P=0.0003, SMD=1.85, 95% CI: 0.85‒2.85; I2=84%). Conclusion: This systematic review indicated that DPSCs in tissue regeneration therapy significantly affected bone tissue complex regeneration. However, more and less diverse preclinical studies will enable more powerful meta-analyses in the future.

Immunomodulatory effects of phytosomal curcumin on related-micro RNAs, CD200 expression and inflammatory pathways in dental pulp stem cells.

Human dental pulp stem cells (hDPSCs) have significant potential of immunomodulatory for therapeutic and regenerative biomedical applications compared to other mesenchymal stem cells (MSCs). Nowadays, alteration of gene expression is an important way to improve the performance of MSCs in the clinic. MicroRNAs (miRs) and CD200 are known to modulate the immune system in MSCs. Curcumin is famous for its anti-inflammatory impacts. Phytosomal curcumin (PC) is a nanoparticle synthesized from curcumin that removes the drawbacks of curcumin. The purpose of this research was to assess the effects of PC on the expression of the CD200 and four key miRNAs in immune system. PC (30 µM) treatment of hDPSCs could ameliorate their immunoregulatory property, presented by reduced expressions of miR-21, miR-155 and miR-126, as well as enhanced expressions of miR-23 and CD200. The PC was also able to reduce PI3K\AKT1\NF-κB expressions that were target genes for these miRs and involved in inflammatory pathways. Moreover, PC was more effective than curcumin in improving the immune modulation of hDPSCs. Evidence in this study suggested that PC mediates immunoregulatory activities in hDPSC via miRs and CD200 to regulate PI3K\AKT1\NF-κB signalling pathways, which may provide a theoretical basis for PC in the treatment of many diseases. SIGNIFICANCE OF THE STUDY: Autoimmune diseases or tooth caries are partly attributed to global health problems and their common drug treatments have several side effects. The goal of this study is dentin regeneration and autoimmune diseases treatment via stem cell-based approaches with phytosomal curcumin (PC), for the first time. Because dental pulp stem cells have unique advantages (including higher immunomodulatory capacity) over other mesenchymal stem cells, we considered them the best option for treating these diseases. Using PC, we try to increase the immunomodulatory properties of these cells.