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.

Inhibition of matrix metalloproteinases expression in human dental pulp cells by all-trans retinoic acid

All-trans retinoic acid（ATRA） inhibits matrix metalloproteinase（MMP）-2 and MMP-9 in synovial fibroblasts, skin fibroblasts,bronchoalveolar lavage cells and cancer cells, but activates MMP-9 in neuroblast and leukemia cells. Very little is known regarding whether ATRA can activate or inhibit MMPs in human dental pulp cells（HDPCs）. The purpose of this study was to determine the effects of ATRA on the production and secretion of MMP-2 and-9 in HDPCs. The productions and messenger RNA（mRNA） expressions of MMP-2 and-9 were accessed by gelatin zymography and real-time polymerase chain reaction（PCR）, respectively. ATRA was found to decrease MMP-2 level in a dose-dependent manner. Significant reduction in MMP-2 mRNA expression was also observed in HDPCs treated with 25 mmol？L21ATRA. However, HDPCs treated with ATRA had no effect on the pattern of MMP-9 produced or secreted in either cell extracts or conditioned medium fractions. Taken together, ATRA had an inhibitory effect on MMP-2 expression in HDPCs,which suggests that ATRA could be a candidate as a medicament which could control the inflammation of pulp tissue in vital pulp therapy and regenerative endodontics.

Self-assembly of differentiated dental pulp stem cells facilitates spheroid human dental organoid formation and prevascularization

BACKGROUND The self-assembly of solid organs from stem cells has the potential to greatly expand the applicability of regenerative medicine.Stem cells can self-organise into microsized organ units,partially modelling tissue function and regeneration.Dental pulp organoids have been used to recapitulate the processes of tooth development and related diseases.However,the lack of vasculature limits the utility of dental pulp organoids.AIM To improve survival and aid in recovery after stem cell transplantation,we demonstrated the three-dimensional(3D)self-assembly of adult stem cell-human dental pulp stem cells(hDPSCs)and endothelial cells(ECs)into a novel type of spheroid-shaped dental pulp organoid in vitro under hypoxia and conditioned medium(CM).METHODS During culture,primary hDPSCs were induced to differentiate into ECs by exposing them to a hypoxic environment and CM.The hypoxic pretreated hDPSCs were then mixed with ECs at specific ratios and conditioned in a 3D environment to produce prevascularized dental pulp organoids.The biological characteristics of the organoids were analysed,and the regulatory pathways associated with angiogenesis were studied.RESULTS The combination of these two agents resulted in prevascularized human dental pulp organoids(Vorganoids)that more closely resembled dental pulp tissue in terms of morphology and function.Single-cell RNA sequencing of dental pulp tissue and RNA sequencing of Vorganoids were integrated to analyse key regulatory pathways associated with angiogenesis.The biomarkers forkhead box protein O1 and fibroblast growth factor 2 were identified to be involved in the regulation of Vorganoids.CONCLUSION In this innovative study,we effectively established an in vitro model of Vorganoids and used it to elucidate new mechanisms of angiogenesis during regeneration,facilitating the development of clinical treatment strategies.

Genetic modification of miR-34a enhances efficacy of transplanted human dental pulp stem cells after ischemic stroke

Human dental pulp stem cells(hDPSCs) promote recovery after ischemic stro ke;however,the therapeutic efficacy is limited by the poor survival of transplanted cells.For in vitro expe riments in the present study,we used oxygen-glucose deprivation/reoxygenation in hDPSCs to mimic cell damage induced by ischemia/reperfusion.We found that miRNA-34a-5p(miR-34a) was elevated under oxygen-glucose deprivation/reoxygenation conditions in hDPSCs.Inhibition of miR-34a facilitated the prolife ration and antioxidant capacity and reduced the apoptosis of hDPSCs.Moreove r,dual-luciferase reporter gene assay showed WNT1and SIRT1 as the targets of miR-34a.In miR-34a knockdown cell lines,WNT1 suppression reduced cell prolife ration,and SIRT1 suppression decreased the antioxidant capacity.Togethe r,these results indicated that miR-34a regulates cell prolife ration and antioxidant stress via targeting WNT1 and SIRT1,respectively.For in vivo expe riments,we injected genetically modified hDPSCs(anti34a-hDPSCs) into the brains of mice.We found that anti34a-hDPSCs significantly inhibited apoptosis,reduced cerebral edema and cerebral infarct volume,and improved motor function in mice.This study provides new insights into the molecular mechanism of the cell prolife ration and antioxidant capacity of hDPSCs,and suggests a potential gene that can be targeted to improve the survival rate and efficacy of transplanted hDPSCs in brain after ischemic stroke.

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.

PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells(HDDPCs) and HDDPC-derived iPS cells

The ability of human deciduous tooth dental pulp cells（HDDPCs） to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a Piggy Bac（PB）-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing td Tomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.

Gelatin-biofermentative unsulfated glycosaminoglycans semi-interpenetrating hydrogels via microbial-transglutaminase crosslinking enhance osteogenic potential of dental pulp stem cells

Gelatin hydrogels by microbial-transglutaminase crosslinking are being increasingly exploited for tissue engineering,and proved high potential in bone regeneration.This study aimed to evaluate,for the first time,the combination of enzymatically crosslinked gelatin with hyaluronan and the newly developed biotechnological chondroitin in enhancing osteogenic potential.Gelatin enzymatic crosslinking was carried out in the presence of hyaluronan or of a hyaluronan–chondroitin mixture,obtaining semi-interpenetrating gels.The latter proved lower swelling extent and improved stiffness compared to the gelatin matrix alone,whilst maintaining high stability.The heteropolysaccharides were retained for 30 days in the hydrogels,thus influencing cell response over this period.To evaluate the effect of hydrogel composition on bone regeneration,materials were seeded with human dental pulp stem cells and osteogenic differentiation was assessed.The expression of osteocalcin(OC)and osteopontin(OPN),both at gene and protein level,was evaluated at 7,15 and 30 days of culture.Scanning electron microscopy(SEM)and two-photon microscope observations were performed to assess bone-like extracellular matrix(ECM)deposition and to observe the cell penetration depth.In the presence of the heteropolysaccharides,OC and OPN expression was upregulated and a higher degree of calcified matrix formation was observed.Combination with hyaluronan and chondroitin improved both the biophysical properties and the biological response of enzymatically crosslinked gelatin,fastening bone deposition.

Magnetic bioassembly platforms towards the generation of extracellular vesicles from human salivary gland functional organoids for epithelial repair

Salivary glands(SG)are exocrine organs with secretory units commonly injured by radiotherapy.Bio-engineered organoids and extracellular vesicles(EV)are currently under investigation as potential strategies for SG repair.Herein,three-dimensional(3D)cultures of SG functional organoids(SGo)and human dental pulp stem cells(hDPSC)were generated by magnetic 3D bioassembly(M3DB)platforms.Fibroblast growth factor 10(FGF10)was used to enrich the SGo in secretory epithelial units.After 11 culture days via M3DB,SGo displayed SG-specific acinar epithelial units with functional properties upon neurostimulation.To consistently develop 3D hDPSC in vitro,3 culture days were sufficient to maintain hDPSC undifferentiated genotype and phenotype for EV generation.EV isolation was performed via sequential centrifugation of the conditioned media of hDPSC and SGo cultures.EV were characterized by nanoparticle tracking analysis,electron microscopy and immunoblotting.EV were in the exosome range for hDPSC(diameter:88.03±15.60 nm)and for SGo(123.15±63.06 nm).Upon ex vivo administration,exosomes derived from SGo significantly stimulated epithelial growth(up to 60%),mitosis,epithelial progenitors and neuronal growth in injured SG;however,such biological effects were less distinctive with the ones derived from hDPSC.Next,these exosome biological effects were investigated by proteomic arrays.Mass spectrometry profiling of SGo exosomes predicted that cellular growth,development and signaling was due to known and undocumented molecular targets downstream of FGF10.Semaphorins were identified as one of the novel targets requiring further investigations.Thus,M3DB platforms can generate exosomes with potential to ameliorate SG epithelial damage.