Differentiation Of Dental Pulp Stem Cells Research Articles

NOD2 is involved in regulating odontogenic differentiation of DPSCs suppressed by MDP through NF-κB/p65 signaling.

Dental pulp stem cells (DPSCs) are well known for their capable of both self-renewal and multilineage differentiation. Dental tissue diseases, include caries, are often accompanied by inflammatory microenvironment, and muramyl dipeptide (MDP) is involved in the inflammatory stimuli to influence the differentiation of DPSCs. Nucleotide-binding oligomerization domain 2 (NOD2), a member of the cytosolic Nod-like receptor (NLR) family, plays a key role in inflammatory homeostasis regulation, but the role of NOD2 in DPSCs differentiation under inflammatory is still unclear. In this study, we identified that MDP suppressed odontogenic differentiation of DPSCs via NOD2/ NF-κB/p65 signaling pathway. Alizarin red staining and ALP activity showed the odontogenic differentiation was suppressed by MDP in a concentration-dependent manner, and the expression of dentin differentiation marker protein dentin matrix protein 1 (DMP-1) and dentin Sialophosphoprotein (DSPP) also indicated the same results. The expression of NOD2 increased gradually with the concentration of MDP as well as the phosphorylation and nuclear translocation of p65, which meant NF-κB signaling pathway was activated. Further, the interference of NOD2 inhibited the phosphorylation and nuclear translocation of p65 and reversed the MDP-mediated decrease of odontoblast differentiation of DPSCs. Our study showed that MDP can inhibit the odontoblast differentiation of DPSCs in a concentration-dependent manner. The NF-κB signaling pathway was activated by increasing expression of NOD2. Interference of NOD2 reversed the negative ability odontoblast differentiation of DPSCs in the inflammatory environment. Our study might provide a theoretical basis for the clinical treatment for dentinogenesis of DPSCs.

The role of ZBTB16 in odontogenic differentiation of dental pulp stem cells

ObjectiveOdontogenic differentiation of dental pulp stem cells (DPSCs) is highly controlled by the activation of several transcription factors. The zinc finger and BTB domain-containing 16 (ZBTB16) gene encodes a BTB/POZ domain and zinc finger containing transcription factors and is involved in several biological processes, but little is known about its role in odontogenic differentiation. The main goal of the current study was to determine the role of ZBTB16 in odontogenic differentiation of DPSCs. DesignZBTB16, runt-related transcription factor 2 (RUNX2), and osterix (OSX) were silenced via small-hairpin RNA (shRNA) lentivirus. The odontoblastic differentiation of DPSCs was detected by alkaline phosphatase (ALP) staining, activity measurement, and alizarin red S staining in vitro. The gene and protein expression levels were assessed by RT-qPCR and western blotting. Further, an ectopic implantation experiment was performed to explore the role of ZBTB16 in mineralization regulation in vivo followed by histological examination. ResultsThe silencing of ZBTB16 attenuated ALP activity and mineralized nodules formation by DPSCs. In addition, knockdown of ZBTB16 impaired the expression of markers involved in odontogenic differentiation, including dentin sialophosphoprotein, dentin matrix acidic phosphoprotein 1, and collagen 1 in vitro and vivo. Silencing the OSX gene suppressed ZBTB16 expression and, in turn, OSX expression decreased after ZBTB16 knockdown. However, shRUNX2 did not suppress ZBTB16 expression and shZBTB16 did not affect RUNX2 expression. ConclusionsZBTB16 may play an important role in modulating the odontoblastic differentiation of DPSCs and act as a regulator of OSX in a possible feed-back cycle independent of RUNX2.

The role of complement C5a receptor in DPSC odontoblastic differentiation and in vivo reparative dentin formation

Therapeutic dentin regeneration remains difficult to achieve, and a majority of the attention has been given to anabolic strategies to promote dentinogenesis directly, whereas, the available literature is insufficient to understand the role of inflammation and inflammatory complement system on dentinogenesis. The aim of this study is to determine the role of complement C5a receptor (C5aR) in regulating dental pulp stem cells (DPSCs) differentiation and in vivo dentin regeneration. Human DPSCs were subjected to odontogenic differentiation in osteogenic media treated with the C5aR agonist and C5aR antagonist. In vivo dentin formation was evaluated using the dentin injury/pulp-capping model of the C5a-deficient and wild-type mice. In vitro results demonstrate that C5aR inhibition caused a substantial reduction in odontogenic DPSCs differentiation markers such as DMP-1 and DSPP, while the C5aR activation increased these key odontogenic genes compared to control. A reparative dentin formation using the C5a-deficient mice shows that dentin regeneration is significantly reduced in the C5a-deficient mice. These data suggest a positive role of C5aR in the odontogenic DPSCs differentiation and tertiary/reparative dentin formation. This study addresses a novel regulatory pathway and a therapeutic approach for improving the efficiency of dentin regeneration in affected teeth.

Odonto/Osteogenic Differentiation of Dental Pulp Stem Cells of Type 1 Diabetic Patients with Mineral Trioxide Aggregate/1α,25-Dihydroxyvitamin D3 Combination

IntroductionVitamin D3 plays an important role in the mineralization mechanism and is often deficient in diabetic patients. The objective of this study was to investigate the odonto/osteogenic differentiation potential of the combination of mineral trioxide aggregate (MTA)/1α,25-dihydroxyvitamin D3 (VD3) on dental pulp stem cells (DPSCs) of patients with type 1 diabetes mellitus (T1DM). MethodsDPSCs isolated from donors (control and T1DM) were cultured and characterized. Cell proliferation and wound healing assays were performed. DPSCs were exposed to 4 different media: growth medium (Dulbecco's modified Eagle's medium, 10% fetal bovine serum, antibiotic, and antimycotic), differentiation medium (DM) (growth medium plus ß-glycerophosphate and ascorbic acid), DM + MTA (DM plus 0.02 mg/mL MTA), and DM + MTA + VD3 (DM + MTA and 10 nmol/L vitamin D3). Odonto/osteogenic differentiation of DPSCs was evaluated by the alizarin red test, relative real-time polymerase chain reaction (dentin sialophosphoprotein, dentin matrix protein 1, collagen type 1 alpha 1, and osteocalcin), immunocytochemistry (antibone sialoprotein II, anti–dentin matrix protein 1, and anti–collagen type 1 alpha 1), and Western blot (dentin matrix protein 1 and osteocalcin) methods. ResultsThe proliferation rates of DPSCs isolated from controls were significantly higher than DPSCs isolated from T1DM in a time-dependent manner (P < .05). Alizarin red staining and the expression of odonto/osteogenic markers showed that odonto/osteogenic differentiation was more pronounced in controls (P < .05) compared with T1DM patients. Although DM + MTA caused the odonto/osteogenic differentiation in DPSCs derived from controls, DM + MTA + VD3 resulted in the odonto/osteogenic differentiation in DPSCs of T1DM patients (P < .05). ConclusionsOdonto/osteogenic differentiation was affected by both supplements used for differentiation and the systemic disease, diabetes mellitus. The differentiation potential of T1DM-derived DPSCs was clearly increased with the VD3 supplement, although it was not as efficient as in the controls. The VD3 supplement showed a positive effect on the differentiation of T1DM DPSCs compared with MTA alone.

Cell-derived Extracellular Matrix Proteins in Colloidal Microgel as a Self-Assembly Hydrogel for Regenerative Endodontics

IntroductionThis study investigated a colloidal microgel for angiogenic and odontogenic differentiation of cells in the presence of cell-derived extracellular matrix (ECM) proteins using a 3-dimensional culture model. MethodsViscoelastic properties of human dental pulp were determined to understand the native ECM environment. ECM proteins were extracted from dental pulp stem cell (DPSC) cultures, and MaxGel (Millipore Sigma, Burlington, MA) was used as a commercially available ECM protein. DPSCs were incubated in colloidal microgels in the presence of ECM proteins or gelatin methacryloyl (GelMA) as a bulk hydrogel (n = 9/group). The viability and odontogenic differentiation of DPSCs within hydrogels was determined using viability assays, mineralization staining, calcium and alkaline phosphatase assays, and quantitative polymerase chain reaction for odontogenic gene expression. Angiogenic properties of endothelial cells were determined using tubule formation assays and quantitative polymerase chain reaction to detect angiogenic gene expression. ResultsDental pulp had a higher elastic modulus than the viscous modulus, showing a solidlike response similar to hydrogels. DPSC-derived ECM showed higher collagen and GAG than MaxGel (P < .05). The viability of DPSCs was similar in colloidal microgels, whereas higher cell viability, calcium deposition, and alkaline phosphatase activity were observed in GelMA (P < .05). Colloidal microgels allowed tubule-like structures by endothelial cells, whereas no tubular formation was observed in GelMA. DPSC-derived ECM in colloidal microgel up-regulated odontogenic gene expression, whereas MaxGel up-regulated angiogenic gene expression (P < .05). ConclusionsColloidal microgels allowed cellular organization that can improve penetration and nutritional supply in a full-length root canal system. The bioactivity of cell-derived ECM proteins can be modified depending on the external stimulus.

Laser Photobiomodulation 808nm: Effects on Gene Expression in Inflammatory and Osteogenic Biomarkers in Human Dental Pulp Stem Cells.

The tissue engineering of dental oral tissue is tackling significant advances and the use of stem cells promises to boost the therapeutical approaches of regenerative dentistry. Despite advances in this field, the literature is still scarce regarding the modulatory effect of laser photobiomodulation (PBM) on genes related to inflammation and osteogenesis in Postnatal Human Dental Pulp Stem cells (DPSCs). This study pointedly investigated the effect of PBM treatment in proliferation, growth and differentiation factors, mineralization, and extracellular matrix remodeling genes in DPSCs. Freshly extracted human third molars were used as a source for DPSCs isolation. The isolated DPSCs were stimulated to an inflammatory state, using a lipopolysaccharide (LPS) model, and then subjected or not to laser PBM. Each experiment was statistically evaluated according to the sample distribution. A total of 85 genes related to inflammation and osteogenesis were evaluated regarding their expression by RT-PCR. Laser PBM therapy has shown to modulate several genes expression in DPSCs. PBM suppressed the expression of inflammatory gene TNF and RANKL and downregulated the gene expression for VDR and proteolytic enzymes cathepsin K, MMP-8 and MMP-9. Modulation of gene expression for proteinase-activated receptors (PARs) following PBM varied among different PARs. As expected, PBM blocked the odontoblastic differentiation of DPSCs when subjected to LPS model. Conversely, PBM has preserved the odontogenic potential of DPSCs by increasing the expression of TWIST-1/RUNEX-2/ALP signaling axis. PBM therapy notably played a role in the DPSCs genes expression that mediate inflammation process and tissue mineralization. The present data opens a new perspective for PBM therapy in mineralized dental tissue physiology.

Hypoxia-inducible factor 1\u03b1 induces osteo/odontoblast differentiation of human dental pulp stem cells via Wnt/\u03b2-catenin transcriptional cofactor BCL9

Accelerated dental pulp mineralization is a common complication in avulsed/luxated teeth, although the mechanisms underlying this remain unclear. We hypothesized that hypoxia due to vascular severance may induce osteo/odontoblast differentiation of dental pulp stem cells (DPSCs). This study examined the role of B-cell CLL/lymphoma 9 (BCL9), which is downstream of hypoxia-inducible factor 1α (HIF1α) and a Wnt/β-catenin transcriptional cofactor, in the osteo/odontoblastic differentiation of human DPSCs (hDPSCs) under hypoxic conditions. hDPSCs were isolated from extracted healthy wisdom teeth. Hypoxic conditions and HIF1α overexpression induced significant upregulation of mRNAs for osteo/odontoblast markers (RUNX2, ALP, OC), BCL9, and Wnt/β-catenin signaling target genes (AXIN2, TCF1) in hDPSCs. Overexpression and suppression of BCL9 in hDPSCs up- and downregulated, respectively, the mRNAs for AXIN2, TCF1, and the osteo/odontoblast markers. Hypoxic-cultured mouse pulp tissue explants showed the promotion of HIF1α, BCL9, and β-catenin expression and BCL9-β-catenin co-localization. In addition, BCL9 formed a complex with β-catenin in hDPSCs in vitro. This study demonstrated that hypoxia/HIF1α-induced osteo/odontoblast differentiation of hDPSCs was partially dependent on Wnt/β-catenin signaling, where BCL9 acted as a key mediator between HIF1α and Wnt/β-catenin signaling. These findings may reveal part of the mechanisms of dental pulp mineralization after traumatic dental injury.

Promotion of the Differentiation of Dental Pulp Stem Cells into Oligodendrocytes by Knockdown of Heat Shock Protein 27

Stem cell-based therapy has been evaluated in many different clinical trials for various diseases. This capability was applied in various neurodegenerative diseases, such as multiple sclerosis, which is characterized by demyelination, axonal injury, and neuronal loss. Dental pulp stem cells (DPSCs) are mesenchymal stem cells from the oral cavity that have been studied with potential application for the regeneration of different tissues. Heat shock protein 27 (HSP27) regulates neurogenesis in the process of neural differentiation of placenta multipotent stem cells. Here, we hypothesize that HSP27 expression is also critical for the neural differentiation of DPSCs. An evaluation of the possible role of HSP27 in the differentiation of DPSCs was performed using gene knockdown and neural immunofluorescent staining. We found that HSP27 played a role in the differentiation of DPSCs and that knockdown of HSP27 in DPSCs rendered cells to oligodendrocyte progenitors; i.e., small hairpin specific for HSP27 DPSCs exhibited NG2-positive immunoreactivity and gave rise to oligodendrocytes or type-2 astrocytes. This neural differentiation of DPSCs may have clinical significance in the treatment of patients with neurodegenerative diseases. In conclusion, our data provide an example of the oligodendrocyte differentiation of a DPSC model, which may be applied in human regenerative medicine.

LIM Mineralization Protein-1 Enhances the Committed Differentiation of Dental Pulp Stem Cells through the ERK1/2 and p38 MAPK Pathways and BMP Signaling.

Tissue regeneration is the preferred treatment for dentin and bone tissue defects. Dental pulp stem cells (DPSCs) have been extensively studied for their use in tissue regeneration, including the regeneration of dentin and bone tissue. LIM mineralization protein-1 (LMP-1) is an intracellular non-secretory protein that plays a positive regulatory role in the mineralization process. In this study, an LMP-1-induced DPSCs model was used to explore the effect of LMP-1 on the proliferation and odonto/osteogenic differentiation of DPSCs, as well as the underlying mechanisms. As indicated by the cell counting kit-8 assay, the results showed that LMP-1 did not affect the proliferation of DPSCs. Overexpression of LMP-1 significantly promoted the committed differentiation of DPSCs and vice versa, as shown by alkaline phosphatase activity assay, alizarin red staining, western blot assay, quantitative real-time polymerase chain reaction assay, and in vivo mineralized tissue formation assay. Furthermore, inhibiting the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) pathways using specific pathway inhibitors showed that the ERK1/2 and p38 MAPK pathways attenuated the differentiation of DPSCs. Besides, the expression of BMP signaling pathway components were also determined, which suggested that LMP-1 could activate BMP-2/Smad1/5 signaling pathway. Our results not only indicated the underlying mechanism of LMP-1 treated DPSCs but also provided valuable insight into therapeutic strategies in regenerative medicine.

Biological reactions of dental pulp stem cells cultured in presence of new xenograft bone substitutes from different sources: An in vitro study.

This study aimed to compare the biological reactions of dental pulp stem cells (DPSCs) cultured on new xenograft bone substitutes derived from camel and bovine bones. DPSCs were cultured and placed on different xenograft materials including Bone Plus (bovine), Camel Bone, and demineralized bovine bone matrix. The viability and proliferation of cells were evaluated by the methyl thiazolyl tetrazolium assay after 24, 48, and 72 h. The alkaline phosphatase (ALP) test and Alizarin red staining were performed at 7 and 21 days to assess the osteoblastic differentiation of cells. Osteocalcin (OCN) gene expression was evaluated qualitatively at 3-, 7- and 14-days using polymerase chain reaction (PCR). Semi-quantitative PCR was also performed using ImageJ software. Data were analyzed by ANOVA and Tukey's honestly significant difference test. The cell proliferation rate was significantly different among the three xenograft bone substitutes at 24-, 48- and 72 h (P < 0.05). The ALP activity of DPSCs in all three xenograft bone substitute groups was greater than that in the control group (P < 0.05). Alizarin red staining showed no significant difference in the formation of calcified nodules among the groups. Qualitative and semi-quantitative PCR displayed that the expression of OCN gene in the Camel Bone and Bone Plus groups was higher than that in the demineralized bovine bone matrix group. The Camel Bone xenograft caused a high proliferation rate and optimal osteogenic differentiation of DPSCs qualitatively and semi-quantitatively in vitro. Further studies are required on this xenograft bone substitute.

Influence of Curcumin Nanocrystals on the Early Osteogenic Differentiation and Proliferation of Dental Pulp Stem Cells

Regenerative therapies are the developed method for regrowing, repairing, or exchanging damaged or diseased cells, tissues, or organs. Bone injury regeneration is a complicated procedure. For this reason, stimulation of the osteogenic differentiation impact on mesenchymal stem cells (MSCs) is a critical method. The goal of the current study was to test the biocompatibility and induction of early osteogenic differentiation by curcumin nanocrystals in human dental pulp stem cells (DPSCs) which are MSCs derived from dental pulp. In this study, curcumin nanocrystals were produced through the spray‐drying technique and then were characterized using conventional methods. Then, cytotoxicity and proliferative properties of curcumin nanocrystals were evaluated in different concentrations at 48 hours, 72 hours, and 7 days which were measured by the MTT test, and a special kit was used to determine the activity of alkaline phosphatase (ALP). The results of this study displayed that the prepared nanocurcumin with the mean particle size of 128 nm, spherical morphology, and the negative surface charge did not show a toxic effect on dental pulp cells in most concentrations for 48 and 72 hours and was toxic only at a concentration of 25 μM. Also, comparing the toxicity of different doses at 7 days showed that at concentrations of 2.5, 5, 10, and 25 μM, cell growth was significantly inhibited. ALP activity was increased in two weeks compared to one week and also compared to that of the control group significantly (p &lt; 0.05). Considering the positive possessions of nanocurcumin on the osteogenic differentiation of DPSCs and its low toxicity, this substance can be considered in the planning of regenerative protocols.

YBX1 Promotes the Inclusion of RUNX2 Alternative Exon 5 in Dental Pulp Stem Cells.

Background and ObjectivesRUNX2 plays an essential role during the odontoblast differentiation of dental pulp stem cells (DPSCs). RUNX2 Exon 5 is an alternative exon and essential for RUNX2 transcriptional activity. This study aimed to investigate the regulatory mechanisms of RUNX2 exon 5 alternative splicing in human DPSCs.Methods and ResultsThe regulatory motifs of RUNX2 exon 5 were analyzed using the online SpliceAid program. The alternative splicing of RUNX2 exon 5 in DPSCs during mineralization-induced differentiation was analyzed by RT-PCR. To explore the effect of splicing factor YBX1 on exon 5 alternative splicing, gaining or losing function of YBX1 was performed by transfection of YBX1 overexpression plasmid or anti-YBX1 siRNA in DPSCs. Human RUNX2 exon 5 is evolutionarily conserved and alternatively spliced in DPSCs. There are three potential YBX1 binding motifs in RUNX2 exon 5. The inclusion of RUNX2 exon 5 and YBX1 expression level increased significantly during mineralization-induced differentiation in DPSCs. Overexpression of YBX1 significantly increased the inclusion of RUNX2 exon 5 in DPSCs. In contrast, silence of YBX1 significantly reduced the inclusion of exon 5 and the corresponding RUNX2 protein expression level. Knockdown of YBX1 reduced the expression of alkaline phosphatase (ALP) and osteocalcin (OC) and the mineralization ability of DPSCs, while overexpression of YBX1 increased the expression of ALP and OC and the mineralization ability of DPSCs.ConclusionsHuman RUNX2 exon 5 is conserved evolutionarily and alternatively spliced in DPSCs. Splicing factor YBX1 promotes the inclusion of RUNX2 exon 5 and improves the mineralization ability of DPSCs.

Melatonin-Medicated Neural JNK3 Up-Regulation Promotes Ameloblastic Mineralization.

Introduction: Melatonin, an endogenous neurohormone, modulates the biological circadian rhythms of vertebrates. It functions have been reported in previous stomatological studies as anti-inflammation, antioxidant, osseointegration of dental implants and stimulation to dental pulp stem cells differentiation, but its role in ameloblastic differentiation and mineralization has been rarely studied. Objective: To reveal the effects of melatonin on the mineralization of ameloblast lineage cells (ALCs), and to identify the change in gene expression and the potential mechanism based on ribonucleic acid sequencing (RNA-seq) analysis. Method: ALCs were induced in melatonin-conditioned medium. After 7-days culture, Western blot, real-time PCR, alkaline phosphatase (ALP) activity test, RNA-seq were accordingly used to detect the change in molecular level. After 1-month odontogenic induction in melatonin medium, Alizarin Red-S (ARS) staining showed the changes of mineral nodules. Differentially expressed genes (DEGs), enrichment of functions and signaling pathways analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) database were performed. The JNK3 antagonist (JNK3 inhibitor IX, SR3576) and β-arrestin1 (Arrb1) overexpression were applied to confirm the fluctuation of melatonin-medicated JNK3 and Arrb1 expression. Results: In this study, we found out melatonin contributed to the ameloblastic mineralization, from which we can observed the elevated expression of enamel matrix protein, and increased ALP activity and mineralized nodules formation. RNA-seq analysis showed the up-regulation of neural JNK3 and down-regulation of Arrb1 in ALCs. Meanwhile, phosphorylated JNK3 deficiency (phosphorylated JNK3 inhibitor---SR3576 added to culture medium) led to mineralization delay, and Arrb1 overexpression proved Arrb1 takes bridge between melatonin receptors (MTNR) and JNK3 in MAPK signaling pathway.

In vitro evaluation of injectable Tideglusib-loaded hyaluronic acid hydrogels incorporated with Rg1-loaded chitosan microspheres for vital pulp regeneration

Injectable systems receive attention in endodontics due to the complicated and irregular anatomical structure of root canals. Here, injectable Tideglusib (Td)-loaded hyaluronic acid hydrogels (HAH) incorporated with Rg1-loaded chitosan microspheres (CSM) were developed for vital pulp regeneration, providing release of Td and Rg1 to trigger odontoblastic differentiation of human dental pulp stem cells (DPSC) by Td and vascularization of pulp by Rg1. The optimal concentrations were determined as 90 nM and 50 μg/mL for Td and Rg1, and loaded in HA and CSM in HAH, respectively. Odontogenic (COL1A1, ALP, OCN, Axin-2, DSPP, and DMP1) and angiogenic (VEGFA, VEGFR2, and eNOS) differentiation of DPSC cultured in the presence of hydrogels was shown at gene expression level. Our results suggest that our injectable hydrogel formulation has potential to improve strategies for vital pulp regeneration. In vivo evaluations are needed to test the feasibility and potential of these hydrogels for vital pulp regeneration.

MiR-140-3p enhanced the osteo/odontogenic differentiation of DPSCs via inhibiting KMT5B under hypoxia condition

Human dental pulp stem cells (DPSCs) have emerged as an important source of stem cells in the tissue engineering, and hypoxia will change various innate characteristics of DPSCs and then affect dental tissue regeneration. Nevertheless, little is known about the complicated molecular mechanisms. In this study, we aimed to investigate the influence and mechanism of miR-140-3p on DPSCs under hypoxia condition. Hypoxia was induced in DPSCs by Cobalt chloride (CoCl2) treatment. The osteo/dentinogenic differentiation capacity of DPSCs was assessed by alkaline phosphatase (ALP) activity, Alizarin Red S staining and main osteo/dentinogenic markers. A luciferase reporter gene assay was performed to verify the downstream target gene of miR-140-3p. This research exhibited that miR-140-3p promoted osteo/dentinogenic differentiation of DPSCs under normoxia environment. Furthermore, miR-140-3p rescued the CoCl2-induced decreased osteo/odontogenic differentiation potentials in DPSCs. Besides, we investigated that miR-140-3p directly targeted lysine methyltransferase 5B (KMT5B). Surprisingly, we found inhibition of KMT5B obviously enhanced osteo/dentinogenic differentiation of DPSCs both under normoxia and hypoxia conditions. In conclusion, our study revealed the role and mechanism of miR-140-3p for regulating osteo/dentinogenic differentiation of DPSCs under hypoxia, and discovered that miR-140-3p and KMT5B might be important targets for DPSC-mediated tooth or bone tissue regeneration.

Polyetheretherketone and titanium surface treatments to modify roughness and wettability – Improvement of bioactivity and antibacterial properties

• PEEK and titanium both sustain cell adhesion, proliferation and differentiation. • Air-plasma treatment increases PEEK and titanium surface wettability. • Surface properties affect cell behavior depending on material nature. • Micropatterned titanium surface induce dental pulp stem cell differentiation. Among the materials available for implant production, titanium is the most used while polyetheretherketone (PEEK) is emerging thanks to its stability and to the mechanical properties similar to the ones of the bone tissue. Material surface properties like roughness and wettability play a paramount role in cell adhesion, cell proliferation, osteointegration and implant stability. Moreover, the bacterial adhesion to the biomaterial and the biofilm formation depend on surface smoothness and hydrophobicity. In this work, two different treatments, sandblasting and air plasma, were used to increase respectively roughness and wettability of two materials: titanium and PEEK. Their effects were analyzed with profilometry and contact angle measurements. The biological properties of the material surfaces were also investigated in terms of cell adhesion and proliferation of NIH-3T3 cells, MG63 cells and human Dental Pulp Stem Cells. Moreover, the ability of Staphylococcus aureus to adhere and form a viable biofilm on the samples was evaluated. The biological properties of both treatments and both materials were compared with samples of Synthegra® titanium, which underwent laser ablation to obtain a porous micropatterning, characterized by a smooth surface to discourage bacterial adhesion. All cell types used were able to adhere and proliferate on samples of the tested materials. Cell adhesion was higher on sandblasted PEEK samples for both MG63 and NIH-3T3 cell lines, on the contrary, the highest proliferation rate was observed on sandblasted titanium and was only slightly dependent on wettability; hDPSCs were able to proliferate similarly on sandblasted samples of both tested materials. The highest osteoblast differentiation was observed on laser micropatterned titanium samples, but similar effects, even if limited, were also observed on both sandblasted materials and air plasma treated titanium. The lowest bacterial adhesion and biofilm formation was observed on micropatterned titanium samples whereas, the highest biofilm formation was detected on sandblasted PEEK samples, and in particular on samples not treated with air-plasma, which displayed the highest hydrophobicity. The results of this work showed that all the tested materials were able to sustain osteoblast adhesion and promote cell proliferation; moreover, this work highlights the feasible PEEK treatments which allow to obtain surface properties similar to those of titanium. The results here reported, clearly show that cell behavior depends on a complex combination of surface properties like wettability and roughness and material nature, and while a rough surface is optimal for cell adhesion, a smooth and less hydrophilic surface is the best choice to limit bacterial adhesion and biofilm formation.

The effect of JD005 compound on odontogenic differentiation of human dental pulp stem cells

Preservation of natural teeth and their structure is critical for chewing, swallowing, and better speech. Dentin and dental tissues are crucial in maintaining natural teeth and their structure, but treatment of dental tissue and dentin fracture is chal-lenging for dentistry. Hence, scientific understanding needs to be translated to discover novel dental treatments. Dental pulp stem cells (DPSCs) are mesenchymal stem cells (MSCs) isolated from the third molar pulp. These cells have similar characteristics to MSCs with a unique property where DPSCs can differentiate into dentin forming odontoblast-like cells. Here, we made an effort to evaluate the potential substrate that induces odontoblastic differentiation of DPSCs. In this study, JD005, a modified A2A antagonist from ZM-241385, was determined to enhance OD. Specifically, JD005 treatment signifi-cantly enhanced the OD markers like alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), OPN, dentin sialophosphoprotein (DSPP), and dentin matrix protein 1 (DMP1). Additionally, the DPSCs treated with JD005 showed enhanced SMAD phosphorylation associated with differentiation. These observations distinctively suggest that JD005 po-tentially contributes to dentin regeneration via odontoblast differentiation.

Epigenetic regulation of dental pulp stem cells and its potential in regenerative endodontics.

Regenerative endodontics (RE) therapy means physiologically replacing damaged pulp tissue and regaining functional dentin–pulp complex. Current clinical RE procedures recruit endogenous stem cells from the apical papilla, periodontal tissue, bone marrow and peripheral blood, with or without application of scaffolds and growth factors in the root canal space, resulting in cementum-like and bone-like tissue formation. Without the involvement of dental pulp stem cells (DPSCs), it is unlikely that functional pulp regeneration can be achieved, even though acceptable repair can be acquired. DPSCs, due to their specific odontogenic potential, high proliferation, neurovascular property, and easy accessibility, are considered as the most eligible cell source for dentin–pulp regeneration. The regenerative potential of DPSCs has been demonstrated by recent clinical progress. DPSC transplantation following pulpectomy has successfully reconstructed neurovascularized pulp that simulates the physiological structure of natural pulp. The self-renewal, proliferation, and odontogenic differentiation of DPSCs are under the control of a cascade of transcription factors. Over recent decades, epigenetic modulations implicating histone modifications, DNA methylation, and noncoding (nc)RNAs have manifested as a new layer of gene regulation. These modulations exhibit a profound effect on the cellular activities of DPSCs. In this review, we offer an overview about epigenetic regulation of the fate of DPSCs; in particular, on the proliferation, odontogenic differentiation, angiogenesis, and neurogenesis. We emphasize recent discoveries of epigenetic molecules that can alter DPSC status and promote pulp regeneration through manipulation over epigenetic profiles.

Overexpression of long noncoding RNA MCM3AP-AS1 promotes osteogenic differentiation of dental pulp stem cells via miR-143-3p/IGFBP5 axis.

MCM3AP-AS1 regulates the cartilage repair in osteoarthritis, but how it regulates osteogenic differentiation of dental pulp stem cells (DPSCs) remains to be determined. DPSCs were isolated and induced for osteogenic differentiation. MCM3AP-AS1 expression was increased along with the osteogenic differentiation of DPSCs, whose expression was positive correlated with those of OCN, alkaline phosphatase (ALP) and RUNX2. On contrary, miR-143-3p expression was decreased along with the osteogenic differentiation and was negatively correlated with those of OCN, ALP and RUNX2. Dual-luciferase reporter gene assay showed that miR-143-3p can be negatively regulated by MCM3AP-AS1 and can regulate IGFBP5. MCM3AP-AS1 overexpression increased the expression levels of osteogenesis-specific genes, ALP activity and mineralized nodules during DPSC osteogenic differentiation, while IGFBP5 knockdown or miR-143-3p overexpression counteracted the effect of MCM3AP-AS1 overexpression in DPSCs. Therefore, this study demonstrated the role of MCM3AP-AS1/miR-143-3p/IGFBP5 axis in regulating DPSC osteogenic differentiation.

MiR-6807-5p Inhibited the Odontogenic Differentiation of Human Dental Pulp Stem Cells Through Directly Targeting METTL7A.

Background: Tooth tissue regeneration mediated by mesenchymal stem cells (MSCs) has become the most ideal treatment. Although the known regulatory mechanism and some achievements have been discovered, directional differentiation cannot effectively induce regeneration of tooth tissue. In this study, we intended to explore the function and mechanism of miR-6807-5p and its target gene METTL7A in odontogenic differentiation.Methods: In this study, human dental pulp stem cells (DPSCs) were used. Alkaline phosphatase (ALP), Alizarin red staining (ARS), and calcium ion quantification were used to detect the odontogenic differentiation of miR-6807-5p and METTL7A. Real-time RT-PCR, western blot, dual-luciferase reporter assay, and pull-down assay with biotinylated miRNA were used to confirm that METTL7A was the downstream gene of miR-6807-5p. Protein mass spectrometry and co-immunoprecipitation (Co-IP) were used to detect that SNRNP200 was the co-binding protein of METTL7A.Results: After mineralized induction, the odontogenic differentiation was enhanced in the miR-6807-5p-knockdown group and weakened in the miR-6807-5p-overexpressed group compared with the control group. METTL7A was the downstream target of miR-6807-5p. After mineralized induction, the odontogenic differentiation was weakened in the METTL7A-knockdown group and enhanced in the METTL7A-overexpressed group compared with the control group. SNRNP200 was the co-binding protein of METTL7A. The knockdown of SNRNP200 inhibited the odontogenic differentiation of DPSCs.Conclusion: This study verified that miR-6807-5p inhibited the odontogenic differentiation of DPSCs. The binding site of miR-6807-5p was the 3′UTR region of METTL7A, which was silenced by miR-6807-5p. METTL7A promoted the odontogenic differentiation of DPSCs. SNRNP200, a co-binding protein of METTL7A, promoted the odontogenic differentiation of DPSCs.