KDM6B epigenetically regulates odontogenic differentiation of dental mesenchymal stem cells

Mesenchymal stem cells （MSCs） have been identified and isolated from dental tissues, including stem cells from apical papilla, which demonstrated the ability to differentiate into dentin-forming odontoblasts. The histone demethylase KDM6B （also known as JMJD3） was shown to play a key role in promoting osteogenic commitment by removing epigenetic marks H3K27me3 from the promoters of osteogenic genes. Whether KDM6B is involved in odontogenic differentiation of dental MSCs, however, is not known. Here, we explored the role of KDM6B in dental MSC fate determination into the odontogenic lineage. Using shRNA-expressing lentivirus, we performed KDM6B knockdown in dental MSCs and observed that KDM6B depletion leads to a significant reduction in alkaline phosphate （ALP） activity and in formation of mineralized nodules assessed by Alizarin Red staining. Additionally, mRNA expression of odontogenic marker gene SP7 （osterix, OSX）, as well as extracellular matrix genes BGLAP （osteoclacin, OCN） and SPP1 （osteopontin, OPN）, was suppressed by KDM6B depletion. When KDM6B was overexpressed in KDM6B-knockdown MSCs, odontogenic differentiation was restored, further confirming the facilitating role of KDM6B in odontogenic commitment. Mechanistically, KDM6B was recruited to bone morphogenic protein 2 （BMP2） promoters and the subsequent removal of silencing H3K27me3 marks led to the activation of this odontogenic master transcription gene. Taken together, our results demonstrated the critical role of a histone demethylase in the epigenetic regulation of odontogenic differentiation of dental MSCs. KDM6B may present as a potential therapeutic target in the regeneration of tooth structures and the repair of craniofacial defects.

TET1 knockdown inhibits the odontogenic differentiation potential of human dental pulp cells

Human dental pulp cells （hDPCs） possess the capacity to differentiate into odontoblast-like cells and generate reparative dentin in response to exogenous stimuli or injury. Ten-eleven translocation 1 （TET1） is a novel DNA methyldioxygenase that plays an important role in the promotion of DNA demethylation and transcriptional regulation in several cell lines. However, the role of TET1 in the biological functions of hDPCs is unknown. To investigate the effect of TET1 on the proliferation and odontogenic differentiation potential of hDPCs, a recombinant shRNA lentiviral vector was used to knock down TET1 expression in hDPCs. Following TET1 knockdown, TET1 was significantly downregulated at both the mRNA and protein levels. Proliferation of the hDPCs was suppressed in the TET1 knockdown groups. Alkaline phosphatase activity, the formation of mineralized nodules, and the expression levels of DSPP and DMP1 were all reduced in the TETl-knockdown hDPCs undergoing odontogenic differentiation. Based on these results, we concluded that TET1 knockdown can prevent the proliferation and odontogenic differentiation of hDPCs, which suggests that TET1 may play an important role in dental pulp repair and regeneration.

Treated dentin matrix induces odontogenic differentiation of dental pulp stem cells via regulation of Wnt/β-catenin signaling

Treated dentin matrix(TDM)is an ideal scaffold material containing multiple extracellular matrix factors.The canonical Wnt signaling pathway is necessary for tooth regeneration.Thus,this study investigated whether the TDM can promote the odontogenic differentiation of human dental pulp stem cells(hDPSCs)and determined the potential role of Wnt/β-catenin signaling in this process.Different concentrations of TDM promoted the dental differentiation of the hDPSCs and meanwhile,the expression of GSK3βwas decreased.Of note,the expression of the Wnt/β-catenin pathway-related genes changed significantly in the context of TDM induction,as per RNA sequencing(RNA seq)data.In addition,the experiment showed that new dentin was visible in rat mandible cultured with TDM,and the thickness was significantly thicker than that of the control group.In addition,immunohistochemical staining showed lower GSK3βexpression in new dentin.Consistently,the GSK3βknockdown hDPSCs performed enhanced odotogenesis compared with the control groups.However,GSK3βoverexpressing could decrease odotogenesis of TDM-induced hDPSCs.These results were confirmed in immunodeficient mice and Wistar rats.These suggest that TDM promotes odontogenic differentiation of hDPSCs by directly targeting GSK3βand activating the canonical Wnt/β-catenin signaling pathway and provide a theoretical basis for tooth regeneration engineering.

An amelogenin-based peptide hydrogel promoted the odontogenic differentiation of human dental pulp cells

Amelogenin can induce odontogenic differentiation of human dental pulp cells(HDPCs),which has great potential and advantages in dentine-pulp complex regeneration.However,the unstability of amelogenin limits its further application.This study constructed amelogenin self-assembling peptide hydrogels(L-gel or D-gel)by heating-cooling technique,investigated the effects of these hydrogels on the odontogenic differentiation of HDPCs and explored the underneath mechanism.The critical aggregation concentration,conformation,morphology,mechanical property and biological stability of the hydrogels were characterized,respectively.The effects of the hydrogels on the odontogenic differentiation of HDPCs were evaluated via alkaline phosphatase activity measurement,quantitative reverse transcription polymerase chain reaction,western blot,Alizarin red staining and scanning electron microscope.The mechanism was explored via signaling pathway experiments.Results showed that both the L-gel and D-gel stimulated the odontogenic differentiation of HDPCs on both Day 7 and Day 14,while the D-gel showed the highest enhancement effects.Meanwhile,the D-gel promoted calcium accumulation and mineralized matrix deposition on Day 21.The D-gel activated MAPK-ERK1/2 pathways in HDPCs and induced the odontogenic differentiation via ERK1/2 and transforming growth factor/smad pathways.Overall,our study demonstrated that the amelogenin peptide hydrogel stimulated the odontogenic differentiation and enhanced mineralization,which held big potential in the dentine-pulp complex regeneration.

Multidifferentiation potential of dental-derived stem cells

Tooth-related diseases and tooth loss are widespread and are a major public health issue.The loss of teeth can affect chewing,speech,appearance and even psychology.Therefore,the science of tooth regeneration has emerged,and attention has focused on tooth regeneration based on the principles of tooth development and stem cells combined with tissue engineering technology.As undifferentiated stem cells in normal tooth tissues,dental mesenchymal stem cells(DMSCs),which are a desirable source of autologous stem cells,play a significant role in tooth regeneration.Researchers hope to reconstruct the complete tooth tissues with normal functions and vascularization by utilizing the odontogenic differentiation potential of DMSCs.Moreover,DMSCs also have the ability to differentiate towards cells of other tissue types due to their multipotency.This review focuses on the multipotential capacity of DMSCs to differentiate into various tissues,such as bone,cartilage,tendon,vessels,neural tissues,muscle-like tissues,hepatic-like tissues,eye tissues and glands and the influence of various regulatory factors,such as non-coding RNAs,signaling pathways,inflammation,aging and exosomes,on the odontogenic/osteogenic differentiation of DMSCs in tooth regeneration.The application of DMSCs in regenerative medicine and tissue engineering will be improved if the differentiation characteristics of DMSCs can be fully utilized,and the factors that regulate their differentiation can be well controlled.

Single CD271 marker isolates mesenchymal stem cells from human dental pulp

Mesenchymal stem cells （MSCs） are a promising tool in regenerative medicine due to their capacity to differentiate into multiple lineages. In addition to MSCs isolated from bone marrow （BMSCs）, adult MSCs are isolated from craniofacial tissues including dental pulp tissues （DPs） using various stem cell surface markers. However, there has been a lack of consensus on a set of surface makers that are reproducibly effective at isolating putative multipotent dental mesenchymal stem cel^s （~M^Cs）. II1 ~his stucly, we used clif^et（~nt combinations of surface markers （CD51/CD140a, CD271, and STRO-1/CD146） to isolate homogeneous populations of DMSCs from heterogeneous dental pulp cells （DPCs） obtained from DP and compared their capacity to undergo multilineage differentiation. Fluorescence-activated cell sorting revealed that 27.3% of DPCs were CD51＋/CD140a＋, 10.6% were CD271＋, and 0.3% were STRO-1＋/CD146＋. Under odontogenic conditions, all three subsets of isolated DMSCs exhibited differentiation capacity into odontogenic lineages. Among these isolated subsets of DMSCs, CD271＋ DMSCs demonstrated the greatest odontogenic potential. While all three combinations of surface markers in this study successfully isolated DMSCs from DPCs, the single CD271 marker presents the most effective stem cell surface marker for identification of DMSCs with high odontogenic potential. Isolated CD271＋ DMSCs could potentially be utilized for future clinical applications in dentistry and regenerative medicine.

Wireless electrical stimulation generated by piezoelectric nanomaterial promotes the dental pulp regeneration via regulating mitochondrial Ca^(2+)/PKA signaling pathway

The induction of dental pulp stem cells(DPSCs)into odontogenic differentiation is a cutting-edge method of dental pulp regeneration treatment.However,it remains a challenge to develop biomaterials and ther-apies that can induce odontogenic differentiation.Here,we propose a wireless electrical stimulation strat-egy to induce DPSCs odontogenic differentiation via K_(0.5)Na_(0.5)NbO_(3)piezoelectric nanoparticles(KNN)and polarized KNN(PKNN),which can be endocytosed by DPSCs.In vitro,several odontogenic differentiation indexes were also increased in DPSCs treated with KNN and PKNN,and the increase was more obvious in the PKNN group.Intracellular wireless electrical field promoted mitochondrial calcium concentration via mitochondrial calcium unidirectional transporter(MCU),increased the production of adenosine triphos-phate(ATP),and induced odontogenic differentiation through the activation of the cAMP-PKA signaling pathway.In vivo,dental pulp-like tissue was induced by electrical stimulation wirelessly with KNN and PKNN,which was more clinically friendly compared with the wired device,and the induction was more obvious in the PKNN group consistent with in vitro experiments.In conclusion,this work demonstrates the potential of PKNN as an effective stimulus that can induce odontogenic differentiation of DPSCs and be applied to dental pulp regeneration treatment.

Effects of different aperture-sized type I collagen/silk fibroin scaffolds on the proliferation and differentiation of human dental pulp cells

This study aimed at evaluate the effects of different aperture-sized type I collagen/silk fibroin(CSF)scaffolds on the proliferation and differentiation of human dental pulp cells(HDPCs).The CSF scaffolds were designed with 3D mapping software Solidworks.Three different aperture-sized scaffolds(CSF1-CSF3)were prepared by low-temperature deposition 3D printing technology.The morphology was observed by scanning electron microscope(SEM)and optical coherence tomography.The porosity,hydrophilicity and mechanical capacity of the scaffold were detected,respectively.HDPCs(third passage,1105 cells)were seeded into each scaffold and investigated by SEM,CCK-8,alkaline phosphatase(ALP)activity and HE staining.The CSF scaffolds had porous structures with macropores and micropores.The macropore size of CSF1 to CSF3 was 421627 lm,579636 lm and 707643 lm,respectively.The porosity was 69.862.2%,80.162.8%and 86.563.3%,respectively.All these scaffolds enhanced the adhesion and proliferation of HDPCs.The ALP activity in the CSF1 group was higher than that in the CSF3 groups(P<0.01).HE staining showed HDPCs grew in multilayer within the scaffolds.CSF scaffolds significantly improved the adhesion and ALP activity of HDPCs.CSF scaffolds were promising candidates in dentine-pulp complex regeneration.