Effect of graphene oxide/ poly-L-lactic acid composite scaffold on the biological properties of human dental pulp stem cells.

BACKGROUND: Tissue engineering has attracted recent attention as a promising bone repair and reconstruction approach. Dental pulp stem cells (DPSCs) are pluripotent and can differentiate into bone cells with the correct environment and substrate. Therefore, suitable scaffold materials are essential for fabricating functional three-dimensional (3D) tissue and tissue regeneration. Composite scaffolds consisting of biodegradable polymers are very promising constructs. This study aims to verify the biological function of human DPSCs seeded onto composite scaffolds based on graphene oxide (GO) and poly-L-lactic acid (PLLA). METHODS: The surface morphology was observed under scanning electron microscopy (SEM). Chemical composition was evaluated with Fourier transform infrared (FTIR) spectroscopy. The biocompatibility of GO/PLLA scaffolds was assessed using phalloidin staining of cytoskeletal actin filaments, live/dead staining, and a CCK-8 assay. The effect of GO/PLLA scaffolds on cell osteogenic differentiation was detected through ALP staining, ALP activity assays, and alizarin red S staining, complemented by quantitative real-time PCR (qRT-PCR) analysis. RESULTS: Our data showed that GO and PLLA are successfully integrated and the GO/PLLA scaffolds exhibit favorable bioactivity and biocompatibility towards DPSCs. Additionally, it was observed that the 0.15% GO/PLLA scaffold group promoted DPSC proliferation and osteogenic differentiation by forming more calcium nodules, showing a higher intensity of ALP staining and ALP activity, and enhancing the expression levels of differentiation marker genes RUNX2 and COL1. CONCLUSIONS: These results demonstrate that the GO/PLLA scaffold is a feasible composite material suitable for cell culture and holds promising applications for oral bone tissue engineering.

Effects of Novel Nanoparticulate Bioceramic Endodontic Material on Human Dental Pulp Stem Cells In Vitro.

OBJECTIVES: This study aimed to investigate the in vitro effects of root canal filling and repair paste (nRoot BP) on human dental pulp stem cells (hDPSCs). METHODS: The effects of nRoot BP and iRoot BP Plus on the adhesion, proliferation, migration, and differentiation of hDPSCs were examined in vitro for 72 hours. The adhesion of cells was observed using immunofluorescence rhodamine ghost pen cyclic peptide staining and scanning electron microscopy (SEM). Cell density and changes in migration area were measured under a fluorescence inverted microscope. Fluorescent quantitative PCR was performed to detect genes related to odontogenesis and osteogenesis. RESULTS: Cells adhering to the surfaces of nRoot BP and iRoot BP Plus exhibited similar irregular polygonal morphologies, with cells extending irregular pseudopods to adhere to the materials. CCK-8 results indicated that the density of living cells for nRoot BP and iRoot BP Plus was lower than that of the blank control group at 3 and 5 days of culture. There was no significant difference in cell migration between the groups (P > .05). The migration ability of iRoot BP Plus and nRoot BP was similar to that of the control group. Both nRoot BP and iRoot BP Plus increased the expression of the RUNX2 gene, but there was no significant difference between the groups (P < .05). Furthermore, both nRoot BP and iRoot BP Plus downregulated the expression of the DSPP gene, with no significant difference between them (P > .05). CONCLUSIONS: nRoot BP exhibited a slight inhibition of hDPSC proliferation but did not affect the adhesion and migration of hDPSCs. The impact of nRoot BP on the osteogenic and odontogenic differentiation of hDPSCs was similar to that of iRoot BP Plus.

Decellularized human amniotic membrane scaffolds: influence on the biological behavior of dental pulp stem cells.

OBJECTIVE: The objective of this study was to assess the characterization of human acellular amniotic membrane (HAAM) using various decellularization methods and their impact on the proliferation and differentiation of human dental pulp stem cells (DPSCs). The goal was to identify scaffold materials that are better suited for pulp regeneration. METHODS: Six different decellularization methods were used to generate the amniotic membranes. The characteristics of these scaffolds were examined through hematoxylin and eosin (H&E) staining, scanning electron microscopy (SEM), and immunohistofluorescence staining (IHF). The DPSCs were isolated, cultured, and their capacity for multidirectional differentiation was verified. The third generation (P3) DPSCs, were then combined with HAAM to form the decellularized amniotic scaffold-dental pulp stem cell complex (HAAM-DPSCs complex). Subsequently, the osteogenic capacity of the HAAM-DPSCs complex was evaluated using CCK8 assay, live-dead cell staining, alizarin red and alkaline phosphatase staining, and real-time quantitative PCR (RT-PCR). RESULTS: Out of the assessed decellularization methods, the freeze-thaw + DNase method and the use of ionic detergent (CHAPS) showed minimal changes in structure after decellularization, making it the most effective method. The HAAM-DPSCs complexes produced using this method demonstrated enhanced biological properties, as indicated by CCK8, alizarin red, alkaline phosphatase staining, and RT-PCR. CONCLUSION: The HAAM prepared using the freeze-thaw + DNase method and CHAPS methods exhibited improved surface characteristics and significantly enhanced the proliferation and differentiation capacity of DPSCs when applied to them. The findings, therefore demonstrate the capacity for enhanced pulp regeneration therapy.

Preparation of PDA-GO/CS composite scaffold and its effects on the biological properties of human dental pulp stem cells.

Reduced graphene oxide (rGO) is an graphene oxide (GO) derivative of graphene, which has a large specific surface area and exhibited satisfactory physicochemical characteristics. In this experiment, GO was reduced by PDA to generate PDA-GO complex, and then PDA-GO was combined with Chitosan (CS) to synthesize PDA-GO/CS composite scaffold. PDA-GO was added to CS to improve the degradation rate of CS, and it was hoped that PDA-GO/CS composite scaffolds could be used in bone tissue engineering. Physicochemical and antimicrobial properties of the different composite scaffolds were examined to find the optimal mass fraction. Besides, we examined the scaffold's biocompatibility by Phalloidin staining and Live and Dead fluorescent staining.Finally, we applied ALP staining, RT-qPCR, and Alizarin red S staining to detect the effect of PDA-GO/CS on the osteogenic differentiation of human dental pulp stem cells (hDPSCs). The results showed that PDA-GO composite was successfully prepared and PDA-GO/CS composite scaffold was synthesized by combining PDA-GO with CS. Among them, 0.3%PDA-GO/CS scaffolds improves the antibacterial activity and hydrophilicity of CS, while reducing the degradation rate. In vitro, PDA-GO/CS has superior biocompatibility and enhances the early proliferation, migration and osteogenic differentiation of hDPSCs. In conclusion, PDA-GO/CS is a new scaffold materialsuitable for cell culture and has promising application prospect as scaffold for bone tissue engineering.

Functionalized self-assembled peptide RAD/Dentonin hydrogel scaffold promotes dental pulp regeneration.

RADA16-I is an ion-complementary self-assembled peptide with a regular folded secondary conformation and can be assembled into an ordered nanostructure. Dentonin is an extracellular matrix phosphate glycoprotein functional peptide motif-containing RGD and SGDG motifs. In this experiment, we propose to combine RAD and Dentonin to form a functionalized self-assembled peptide RAD/Dentonin hydrogel scaffold. Furthermore, we expect that the RAD with the addition of functional motif Dentonin can promote pulp regeneration. The study analyzed the physicochemical properties of RAD/Dentonin through circular dichroism, morphology scanning, and rheology. Besides, we examined the scaffold's biocompatibility by immunofluorescent staining, CCK-8 method, Live/Dead fluorescent staining, and 3D reconstruction. Finally, we applied ALP activity assay, RT-qPCR, and Alizarin red S staining to detect the effect of RAD/Dentonin on the odontogenic differentiation of human dental pulp stem cells (hDPSCs). The results showed that RAD/Dentonin spontaneously assembles into a hydrogel with aß-sheet-based nanofiber network structure.In vitro, RAD/Dentonin has superior biocompatibility and enhances adhesive proliferation, migration, odontogenic differentiation, and mineralization deposition of hDPSCs. In conclusion, the novel self-assembled peptide RAD/Dentonin is a new scaffold material suitable for cell culture and has promising applications as a scaffold for endodontic tissue engineering.

Robust expression of SIRT6 inhibits pulpitis via activation of the TRPV1 channel.

Invasion of dentinal tubules and pulp tissue by pathogenic bacteria may cause infection leading to pulpitis. Sirtuin 6 (SIRT6) is a NAD-dependent protein deacetylase encoded by the SIRT6 gene. The effect of SIRT6 on lipopolysaccharide (LPS)-induced pulpitis and its mechanism of action were discussed in this study. Dental pulp cells (DPCs) were extracted from human teeth and injected with LPS to induce inflammation. The cells injected with LPS showed substantially decreased expression of SIRT6. The overexpression of SIRT6, induced by plasmid-transfection of DPCs with SIRT6 overexpressing vector, led to a marked decrease in proinflammatory cytokines (IL-6, IL-1ß, and TNF-α) and deactivation of NF kappa B pathway. Additionally, dentin matrix protein-1 (DMP1), a promoter of inflammation in dental pulp tissues, was downregulated. Further investigation revealed that SIRT6 promotes ubiquitination of the transient receptor potential vanilloid 1 (TRPV1) channel, leading to its degradation and deactivation. The role of TRPV1 in the anti-inflammatory effects of SIRT6 was determined through incubation of SIRT6-expressing dental pulp stem cells (DPSCs) with capsaicin. This incubation counteracted the effect of SIRT6 on cytokines and DMP1. The injection of lentivirus-SIRT6 attenuated LPS-induced pulpitis in vivo by suppressing TRPV1 activity. Thus, SIRT6 inhibits the TRPV1 channel during LPS-induced inflammation of dental pulp. SIGNIFICANCE OF THE STUDY: This study discussed the effect of sirtuin 6 (SIRT6) on lipopolysaccharide (LPS)-induced pulpitis as well as its mechanism of action and found that SIRT6 may be a negative regulator of pulpitis. Additionally, low expression of SIRT6 and high expression of transient receptor potential vanilloid 1 (TRPV1) in LPS-treated human dental pulp cells are closely associated with proinflammatory cytokines, dentin matrix protein 1 expression, and activation of the NF-κB pathway, which indicated that TRPV1 may be a biomarker for pulpitis and the SIRT6-TRPV1-CGRP axis maybe a clinical target due to their role regulating inflammation and neuropathic pain.

Downregulation of miR-224-5p Promotes Migration and Proliferation in Human Dental Pulp Stem Cells.

INTRODUCTION: Pulp regeneration, as a treatment for pulp necrosis, has significant advantages over root canal therapy for the preservation of living pulp. To date, research on pulp regeneration has mainly focused on the transplantation of pulp stem cells into the root canal, but there is still a lack of research on the migration of pulp cells into the root canal via cell homing. Stem cells from the apical tooth papilla (SCAP) are recognized as multidirectional stem cells, but these cells are difficult to obtain. MicroRNAs are small noncoding RNAs that play crucial roles in regulating normal and pathologic functions. We hypothesized that some types of microRNAs might improve the migration and proliferation function of dental pulp stem cells (DPSCs), which are easily obtained in clinical practice, and as a result, DPSCs might replace SCAP and provide valuable information for regenerative endodontics. METHODS: Magnetic activated cell sorting of DPSCs and SCAP was performed. Next-generation sequencing was performed to examine DPSCs and SCAP miRNAs expression and to identify the most significant differentially expressed miRNA. CCK-8 and transwell assays were used to determine the impact of this miRNA on DPSCs proliferation and migration. RESULTS: The most significant differentially expressed miRNA between DPSCs and SCAP was miR-224-5p. Downregulating miR-224-5p promoted DPSCs proliferation and migration; the opposite results were observed when miR-224-5p was upregulated. CONCLUSION: MiR-224-5p promotes proliferation and migration in DPSCs, a finding that is of great significance for further exploring the role of dental pulp stem cells in regenerative endodontics.

Involvement of miR-146a-5p/neurogenic locus notch homolog protein 1 in the proliferation and differentiation of STRO-1+ human dental pulp stem cells.

Dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAPs) are oral mesenchymal stem cells capable of self-renewal and have a potential for multilineage differentiation. Increasing evidence shows that microRNAs (miRNAs) play important roles in stem cell biology. Here, we focused on exploring miR-146a-5p and its relationship to the undifferentiated status of STRO-1+ SCAPs and STRO-1+ DPSCs, as well as its role during STRO-1+ DPSC differentiation and proliferation. Our data indicated that baseline miR-146a-5p expression is significantly lower in STRO-1+ SCAPs than in STRO-1+ DPSCs and increased in the latter during osteogenic induction. Moreover, we identified miR-146a-5p as a key miRNA that promotes osteo/odontogenic differentiation of STRO-1+ DPSCs and attenuates cell proliferation. Additionally, it was observed that STRO-1+ DPSC mineralization results in the downregulation of notch receptor 1 (NOTCH1) and hes family bHLH transcription factor 1 (HES1). Interference with neurogenic locus notch homolog protein 1 (Notch 1) signaling was verified to enhance differentiation and suppress STRO-1+ DPSC proliferation. It was further observed that miR-146a-5p directly targets the 3'-untranslated region (3'-UTR) of NOTCH1 and inhibits expression of both NOTCH1 and HES1mRNAs and Notch 1 and transcription factor HES-1 (HES-1) proteins in STRO-1+ DPSCs. We conclude that miR-146a-5p exerts its regulatory effect on STRO-1+ DPSC differentiation and proliferation partially by suppressing Notch signaling.

The efficacy of platelet-rich fibrin as a scaffold in regenerative endodontic treatment: a retrospective controlled cohort study.

BACKGROUND: Blood Clot (BC) or platelet concentrates have been used as scaffold in regenerative endodontic treatment (RET). The aim of this retrospective study was to compare the performance of platelet-rich fibrin (PRF) with BC in inducing root development and periapical lesion healing after tooth revascularization. METHODS: Five patients receiving RET using PRF as a scaffold were matched 1:1 to a previous cohort of 5 patients who underwent tooth revascularization by provoking periapical bleeding. Clinical signs and symptoms were examined at follow-ups. Periapical lesion healing and root development were monitored radiographically. The resolution of clinical signs and symptoms as well as periapical radiolucency was observed in all patients (100%). RESULTS: Root elongation, dentinal wall thickening and apex closure were found in most cases (80% in both groups). There was no significant difference between the groups in terms of clinical sign resolution, root development and periapical healing. CONCLUSIONS: Within the limits of this study, PRF achieved comparable outcomes to BC in terms of clinical sign and symptom resolution, periapical lesion healing and continued root development in RET.

Concurrent preoperative chemotherapy and three-dimensional conformal radiotherapy followed by surgery for oral squamous cell carcinoma: a retrospective analysis of 104 cases.

OBJECTIVES: The objectives of this study were to assess the clinical effects of an integrated program consisting of concurrent preoperative combined paclitaxel and nedaplatin chemotherapy and three-dimensional conformal radiotherapy followed by surgery intended to cure oral squamous cell carcinoma and to determine whether this integrated program is feasible and effective with respect to the treatment of oral squamous cell carcinoma. METHODS: A total of 104 biopsy-confirmed patients who presented with oral squamous cell carcinoma for the first time were included in this study. Concurrent preoperative combined paclitaxel and nedaplatin chemotherapy and three-dimensional conformal radiotherapy were administered to these patients. The most common treatment regimen consisted of infusions of paclitaxel (135-175 mg/m2/day), infusions of nedaplatin (150 mg; 80-100 mg/m2/day), and irradiation at doses ranging from 1.5 Gy twice daily to 30-40 Gy over 3-4 weeks. The clinical variables evaluated herein included the local recurrence rate, distant metastasis rate, postoperative survival rate, and degree of mouth opening restriction. RESULTS: The median follow-up time for surviving patients was 60 months, and the median time to progression for all patients was 57.69 months (95% confidence interval, 56.09 to 59.29 months, and the 3-year disease-free survival probability was 97.11%). The effectiveness rate of the integrated program was 98.08%, and the surgery resection rate was 100%. Only a few postoperative adverse reactions were observed. The local recurrence and distant metastasis rates were 1.92% (2 patients) and 2.88% (3 patients), respectively. The titanium rejection and infection reaction rate that led to restriction of mouth opening was only 2.88% (3 patients). Finally, the 5-year post-surgery survival rate was 91.35% (95 patients). CONCLUSION: Preoperative combined paclitaxel and nedaplatin chemotherapy and three-dimensional conformal radiotherapy have significant clinical effects leading to positive anti-tumor results in patients with oral squamous cell carcinoma. These treatments also increase the likelihood that patients will undergo successful surgical treatment. The integrated program described herein can increase long-term survival and surgery resection rates and is associated with only a limited number of adverse reactions.

Transfection of truncated bone morphogenetic protein receptor-II into oral squamous cell carcinoma cell line Tca8113 and inhibitory effect on proliferation and inductive effect on apoptosis.

Bone morphogenetic proteins (BMPs), one of the crucial regulators in embryonic development and bone formation, have been implicated in epithelium-derived tumors. Previous results showed the involvement of overexpression of BMP 2, 4, 5 in the carcinogenesis of oral epithelia. The ability of BMP receptor-II mutant to modify the malignant phenotype of oral squamous cell carcinoma cell line Tca8113 by blocking the BMP signal transduction pathway has been proposed. In this study, a negative truncated mutant of the BMP receptor-II (tBMPR-II) was transfected into Tca8113 cells. The effects were evaluated though RT-PCR, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, BrdU staining, cell cyclin assay, TdT-mediated dUTP nick end labeling (TUNEL) staining, and cell cycle protein detection. Overexpression of tBMPR-II gene transfection truncates the expression of BMPR-II mRNA expression, but not BMP 2, 4, 5. tBMPR-II resulted in a remarkable inhibition of cell proliferation and viability compared with control Tca8113. The inhibitory effects were partly attributed to the induction of apoptosis and cell cycle arrest in G(0) /G(1) accompanied by downregulation of the intracellular cell cycle proteins of cyclin D1 and cyclin-dependent kinases 4, as well as the upregulation of p27 and p57. Loss of BMP signals correlates tightly with suppression of cell proliferation, induction of apoptosis, and benign transformation of Tca8113 cells phenotype.

PHEX neutralizing agent inhibits dentin formation in mouse tooth germ.

The mutation of phosphate-regulating gene with homologies to endopeptidases on the X-chromosome (PHEX) can lead to human X-linked hypophosphatemic rickets which displays hypo-mineralization in bone and dentin. To study its possible roles in teeth, PHEX antibody was injected into pregnant mice on E15 to explore its roles on the formation of enamel and dentin. Mallory trichrome staining results showed that arrangements of ameloblasts and odontoblasts were irregular after PHEX antibody treatment. Differentiation of odontoblasts and the formation of dentin were inhibited. Spatiotemporal distribution of PHEX protein was observed in various stages of tooth germ. Immunohistochemical results showed positive PHEX signals appeared in the inner enamel epithelium on E16 and became stronger on E18. Ameloblasts and odontoblasts showed much higher PHEX expression on P1 and P3. Expression of PHEX in odontoblasts decreased accordingly. However, enamel formation was only slightly affected. The findings proved that a decrease in PHEX expression could suppress dentin formation.