Therapeutic effect of neurogrowth factor in the treatment of gingival pain and swelling in patients with dental pulp necrosis after root canal therapy

Objective: To explore the clinical efficacy of neurogrowth factor in the treatment of gingival pain and swelling in patients with dental pulp necrosis after root canal therapy and the effect on the serum inflammatory cytokines. Methods: A total of 156 patients with gingival pain and swelling after root canal therapy due to dental pulp necrosis were included in the study and randomized into the control group (n=78) and teh treatment group (n=78). The patients in the control group were given metronidazole tablets. On the above basis, the patients in the treatment group were given local injection of neurogrowth factors. 10-day treatment was regarded as one course, and the patients were continuously treated for 2 courses. The improvement of clinical symptoms before and after treatment in the two groups was evaluated. Gingival sulcus index and serum inflammatory cytokines before and after treatment in the two groups were detected and compared. Results: When compared with before treatment, the periodontal soft tissue swelling, tooth mobility, and periapical pain scores after treatment in the two groups were significantly reduced, and those in the treatment group were significantly lower than those in the control group. When compared with before treatment, the gingival sulcus bleeding index 1 and 2 courses after treatment in the two groups was significantly reduced, and that 2 courses after treatment was significantly lower than that after 1 course treatment. The gingival sulcus bleeding index 1 and 2 courses after treatment in the treatment group was significantly lower than that in the control group. When compared with before treatment, the serum IL-8 and IL-6 levels after treatment in the two groups, and TNF-α level after treatment in the treatment group were significantly reduced, and the above indicators in the treatment group were significantly lower than those in the control group. Conclusions: The neurogrowth factors in the treatment of gingival pain and swelling in patients with dental pulp necrosis after root canal therapy can effectively improve the clinical symptoms, and inhibit the inflammatory reaction, with a significant efficacy.

Conditioned medium from human dental pulp stem cells treats spinal cord injury by inhibiting microglial pyroptosis

Human dental pulp stem cell transplantation has been shown to be an effective therapeutic strategy for spinal cord injury.However,whether the human dental pulp stem cell secretome can contribute to functional recovery after spinal cord injury remains unclear.In the present study,we established a rat model of spinal cord injury based on impact injury from a dropped weight and then intraperitoneally injected the rats with conditioned medium from human dental pulp stem cells.We found that the conditioned medium effectively promoted the recovery of sensory and motor functions in rats with spinal cord injury,decreased expression of the microglial pyroptosis markers NLRP3,GSDMD,caspase-1,and interleukin-1β,promoted axonal and myelin regeneration,and inhibited the formation of glial scars.In addition,in a lipopolysaccharide-induced BV2 microglia model,conditioned medium from human dental pulp stem cells protected cells from pyroptosis by inhibiting the NLRP3/caspase-1/interleukin-1βpathway.These results indicate that conditioned medium from human dental pulp stem cells can reduce microglial pyroptosis by inhibiting the NLRP3/caspase-1/interleukin-1βpathway,thereby promoting the recovery of neurological function after spinal cord injury.Therefore,conditioned medium from human dental pulp stem cells may become an alternative therapy for spinal cord injury.

Dental pulp cell bank as a possible future source of individual hepatocytes

Mesenchymal stem cells(MSCs) as a source for regenerative medicine are now the subject of much clinical attention. There are high expectations due to their safety, low tumorigenic risk, and low ethical concerns. MSC therapy has been approved for acute graft-versus host diseases since 2015. Tooth-derived MSCs are known to have a great potential in their proliferation and differentiation capacities, even when compared with bone-marrow-derived MSCs. In particular, stem cells from human exfoliated deciduous teeth(SHEDs) are the best candidates for personal cell banking(dental pulp cell bank), because they can be obtained less invasively in the natural process of individual growth. SHEDs are known to differentiate into hepatocytes. There have been several studies showing the effectiveness of SHEDs on the treatment of liver failure in animal models. They may exert their effects either by repopulation of cells in injured liver or by paracrine mechanisms due to their immuneregulatory functions. Moreover, it may be possible to use each individuals' dental pulp cells as a future source of tailor-made differentiated hepatocytes in the context of a bioartificial liver or liver-on-a-chip to screen for drug toxicity.

Effects of low intensity laser irradiation phototherapy on dental pulp constructs

AIM: To investigate low intensity laser irradiation phototherapy(LILIP) on the proliferation, mineralization and degradation of dental pulp constructs.METHODS: Stem cells from human exfoliated deciduous teeth(SHED) were grown to confluence and seeded on collagen scaffolds to create dental pulp constructs. LILIP was delivered to the dental pulp constructs using an 830 nm GaA IAs laser at an output power of 20 m W. The LILIP energy density was 0.4, 0.8, 1.2, and 2.4 J/cm2. After 8 d, the cell proliferation and degradation within the dental pulp constructs were measured using histologic criteria. After 28 d, the effect of LILIP on SHED mineralization was assessed by von Kossa staining.RESULTS: SHED proliferation within the dental pulp constructs varied after exposure to the 0.4, 0.8, 1.2,and 2.4 J/cm2 LILIP energy densities(P < 0.05). The maximum proliferation of SHED in nutrient deficient media was 218% after exposure to a 1.2 J/cm2 LILIP energy density. SHED grown in nutrient deficient media after exposure to a 0.4, 0.8, and 1.2 J/cm2 LILIP energy density, proliferated by 167-218% compared to the untreated(non-LILIP) control group(P < 0.05).SHED exposed to a 0.4, 0.8, and 1.2 J/cm2 LILIP energy density, and grown in optimal nutritional conditions and proliferated by 147%-164% compared to the untreated(non-LILIP) control group(P < 0.05). The exposure of SHED to the highest LILIP energy density(2.4 J/cm2) caused a reduction of the cell proliferation of up to 73% of the untreated(non-LILIP) control(P < 0.05). The amount of mineral produced by SHED increased over time up to 28 d(P < 0.05). The 0.8 and 1.2J/cm2 LILIP energy densities were the most effective at stimulating the increased the mineralization of the SHED from 150%-700% compared to untreated(nonLILIP) control over 28 d(P < 0.05). The degradation of dental pulp constructs was affected by LILIP(P <0.05). The dental pulp constructs grown in optimal nutritional conditions exposed to a 0.8 J/cm2 or 1.2 J/cm2 LILIP energy density had 13% to 16% more degradation than the untreated(non-LILIP) control groups(P < 0.05). The other LILIP energy densities caused a 1%degradation of dental pulp constructs in optimal nutritional conditions(P > 0.05).CONCLUSION: LILIP can enhance or reduce SHED proliferation, degradation and mineralization within dental pulp constructs. LILIP could promote the healing and regeneration of dental tissues.

Neural crest derived stem cells from dental pulp and tooth-associated stem cells for peripheral nerve regeneration

The peripheral nerve injuries,representing some of the most common types of traumatic lesions affecting the nervous system,are highly invalidating for the patients besides being a huge social burden.Although peripheral nervous system owns a higher regenerative capacity than does central nervous system,mostly depending on Schwann cells intervention in injury repair,several factors determine the extent of functional outcome after healing.Based on the injury type,different therapeutic approaches have been investigated so far.Nerve grafting and Schwann cell transplantation have represented the gold standard treatment for peripheral nerve injuries,however these approaches own limitations,such as scarce donor nerve availability and donor site morbidity.Cell based therapies might provide a suitable tool for peripheral nerve regeneration,in fact,the ability of different stem cell types to differentiate towards Schwann cells in combination with the use of different scaffolds have been widely investigated in animal models of peripheral nerve injuries in the last decade.Dental pulp is a promising cell source for regenerative medicine,because of the ease of isolation procedures,stem cell proliferation and multipotency abilities,which are due to the embryological origin from neural crest.In this article we review the literature concerning the application of tooth derived stem cell populations combined with different conduits to peripheral nerve injuries animal models,highlighting their regenerative contribution exerted through either glial differentiation and neuroprotective/neurotrophic effects on the host tissue.

In vitro responses of human pulp cells and 3T3 mouse fibroblasts to six contemporary dental restoratives

In vitro responses of human primary pulp cells (HPCs) and 3T3 mouse fibroblasts to six contempo-rary commercial dental restoratives were evaluated using the WST-1 assay. The results show that Fuji II is not cytotoxic to both cells. Fuji II LC is not cyto-toxic to HPCs but cytotoxic to 3T3 cells, indicating that 3T3 cells are more vulnerable to 2-hydroxyethyl methacrylate (HEMA) than HPCs. Vitremer is very cytotoxic probably due to having diphenyliodonium chloride and HEMA in it. Z100 is very cytotoxic probably due to having triethylene glycol dimethacry-late (TEGDMA) in it. P60 is cytotoxic but less cyto-toxic than Z100 probably due to no TEGDMA in it. Durelon is the most cytotoxic among the six materials studied probably due to the high cytotoxicity of zinc ions. Additionally, the cytotoxcity of the tested mate-rials was found to be dose-dependent.

Human dental pulp stem cells: Applications in future regenerative medicine

Stem cells are pluripotent cells, having a property of differentiating into various types of cells of human body. Several studies have developed mesenchymal stem cells(MSCs) from various human tissues,peripheral blood and body fluids. These cells are then characterized by cellular and molecular markers to understand their specific phenotypes. Dental pulp stem cells(DPSCs) are having a MSCs phenotype and they are differentiated into neuron, cardiomyocytes, chondrocytes, osteoblasts, liver cells and β cells of islet of pancreas. Thus, DPSCs have shown great potentiality to use in regenerative medicine for treatment of various human diseases including dental related problems. These cells can also be developed into induced pluripotent stem cells by incorporation of pluripotency markers and use for regenerative therapies of various diseases. The DPSCs are derived from various dental tissues such as human exfoliated deciduous teeth, apical papilla, periodontal ligament and dental follicle tissue. This review will overview the information about isolation, cellular and molecular characterization and differentiation of DPSCs into various types of human cells and thus these cells have important applications in regenerative therapies for various diseases. This review will be most useful for postgraduate dental students as well as scientists working in the field of oral pathology and oral medicine.

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation

BACKGROUND Validation of the reference gene(RG)stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction(RT-qPCR)data normalisation.Commonly,in an unreliable way,several studies use genes involved in essential cellular functions[glyceraldehyde-3-phosphate dehydro-genase(GAPDH),18S rRNA,andβ-actin]without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes.Furthermore,such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recom-mend two or more genes.It impacts the credibility of these studies and causes dis-tortions in the gene expression findings.For tissue engineering,the accuracy of gene expression drives the best experimental or therapeutical approaches.We cultivated DPSCs under two conditions:Undifferentiated and osteogenic dif-ferentiation,both for 35 d.We evaluated the gene expression of 10 candidates for RGs[ribosomal protein,large,P0(RPLP0),TATA-binding protein(TBP),GAPDH,actin beta(ACTB),tubulin(TUB),aminolevulinic acid synthase 1(ALAS1),tyro-sine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,zeta(YWHAZ),eukaryotic translational elongation factor 1 alpha(EF1a),succinate dehydrogenase complex,subunit A,flavoprotein(SDHA),and beta-2-micro-globulin(B2M)]every 7 d(1,7,14,21,28,and 35 d)by RT-qPCR.The data were analysed by the four main algorithms,ΔCt method,geNorm,NormFinder,and BestKeeper and ranked by the RefFinder method.We subdivided the samples into eight subgroups.RESULTS All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm.The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs.Either theΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes.However,geNorm analysis showed RPLP0/EF1αin the first place.These algorithms’two least stable RGs were B2M/GAPDH.For BestKeeper,ALAS1 was ranked as the most stable RG,and SDHA as the least stable RG.The pair RPLP0/TBP was detected in most subgroups as the most stable RGs,following the RefFinfer ranking.CONCLUSION For the first time,we show that RPLP0/TBP are the most stable RGs,whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

Dental pulp stem cells: Novel cell-based and cell-free therapy for peripheral nerve repair

The regeneration of peripheral nerves comprises complicated steps involving a set of cellular and molecular events in distal nerve stumps with axonal sprouting and remyelination. Stem cell isolation and expansion for peripheral nerve repair(PNR) can be achieved using a wide diversity of prenatal and adult tissues, such as bone marrow or brain tissues. The ability to obtain stem cells for cell-based therapy(CBT) is limited due to donor site morbidity and the invasive nature of the harvesting process. Dental pulp stem cells(DPSCs) can be relatively and simply isolated from the dental pulps of permanent teeth, extracted for surgical or orthodontic reasons. DPSCs are of neural crest origin with an outstanding ability to differentiate into multiple cell lineages. They have better potential to differentiate into neural and glial cells than other stem cell sources through the expression and secretion of certain markers and a range of neurotropic factors;thus, they should be considered a good choice for PNR using CBT. In addition,these cells have paracrine effects through the secretion of neurotrophic growth factors and extracellular vesicles, which can enhance axonal growth and remyelination by decreasing the number of dying cells and activating local inhabitant stem cell populations, thereby revitalizing dormant or blocked cells,modulating the immune system and regulating inflammatory responses. The use of DPSC-derived secretomes holds great promise for controllable and manageable therapy for peripheral nerve injury. In this review, up-to-date information about the neurotrophic and neurogenic properties of DPSCs and their secretomes is provided.

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.

Therapeutic potential of dental pulp stem cell transplantation in a rat model of Alzheimer’s disease

Dental pulp stem cells are dental pulp-derived mesenchymal stem cells that originate from the neural crest.They exhibit greater potential for the treatment of nervous system diseases than other types of stem cells because of their neurogenic differentiation capability and their ability to secrete multiple neurotrophic factors.Few studies have reported Alzheimer’s disease treatment using dental pulp stem cells.Rat models of Alzheimer’s disease were established by injecting amyloid-β1–42 into the hippocampus.Fourteen days later,5×106 dental pulp stem cells were injected into the hippocampus.Immunohistochemistry and western blot assays showed that dental pulp stem cell transplantation increased the expression of neuron-related doublecortin,NeuN,and neurofilament 200 in the hippocampus,while the expression of amyloid-βwas decreased.Moreover,cognitive and behavioral abilities were improved.These findings indicate that dental pulp stem cell transplantation in rats can improve cognitive function by regulating the secretion of neuron-related proteins,which indicates a potential therapeutic effect for Alzheimer’s disease.This study was approved by the Animal Ethics Committee of Harbin Medical University,China(approval No.KY2017-132)on February 21,2017.

Adrenaline in pulp capping treatment of reversible pulpitis

BACKGROUND The role of epinephrine in the treatment of pulp capping in patients with reversible pulpitis is not clear.AIM To explore the role of epinephrine in the treatment of pulp capping in patients with reversible pulpitis.METHODS A total of 100 patients with reversible pulpitis who were treated in Anhui Jieshou People's Hospital from January 2020 to December 2021 were included in the study.They were categorized into an observation group(n=50;treatment with adrenaline)and a control group(n=50;treatment with zinc oxide eugenol paste).The 24-h postoperative pain,regression time of gingival congestion and redness,clinical efficacy,and incidence of adverse reactions were compared between the groups.Patients were further categorized into the ineffective and effective treatment groups based on clinical efficacy.Logistic multiple regression analysis explored factors affecting the efficacy of pulp capping treatment.RESULTS A significant difference in 24-h postoperative pain was observed between the groups(P<0.05),with a higher proportion of grade I pain noted in the observation group than in the control group(P<0.01).The regression time of gingival congestion and swelling was lower in the observation group(2.61±1.44 d and 2.73±1.36 d,respectively)than in the control group(3.85±1.47 d and 4.28±1.61 d,respectively)(P<0.05).The 2-wk postoperative total effective rate was lower in the control group(80.00%)than in the observation group(94.00%)(P<0.05).The incidence of adverse reactions was not significantly different between the control(14.00%)and observation(12.00%)groups(P>0.05).The proportion of adrenaline usage was lower(P<0.05)and that of anaerobic digestion by Streptococcus and Fusobacterium nucleatum was higher in the ineffective treatment group than in the effective treatment group(P<0.05).Logistic multiple regression analysis revealed adrenaline as a protective factor(P<0.05)and anaerobic digestion by Streptococcus and F.nucleatum as risk factors for pulp capping in reversible pulpitis(P<0.05).CONCLUSION Adrenaline demonstrated therapeutic efficacy in pulp capping treatment for reversible pulpitis,reducing pain and improving clinical symptoms safely.It is a protective factor for pulp capping,whereas Streptococcus and F.nucleatum are risk factors.Targeted measures can be implemented to improve clinical efficacy.

Hepatocyte growth factor enhances the ability of dental pulp stem cells to ameliorate atherosclerosis in apolipoprotein E-knockout mice

BACKGROUND Atherosclerosis(AS),a chronic inflammatory disease of blood vessels,is a major contributor to cardiovascular disease.Dental pulp stem cells(DPSCs)are capable of exerting immunomodulatory and anti-inflammatory effects by secreting cytokines and exosomes and are widely used to treat autoimmune and inflam-mation-related diseases.Hepatocyte growth factor(HGF)is a pleiotropic cytokine that plays a key role in many inflammatory and autoimmune diseases.AIM To modify DPSCs with HGF(DPSC-HGF)and evaluate the therapeutic effect of DPSC-HGF on AS using an apolipoprotein E-knockout(ApoE-/-)mouse model and an in vitro cellular model.METHODS ApoE-/-mice were fed with a high-fat diet(HFD)for 12 wk and injected with DPSC-HGF or Ad-Null modified DPSCs(DPSC-Null)through tail vein at weeks 4,7,and 11,respectively,and the therapeutic efficacy and mechanisms were analyzed by histopathology,flow cytometry,lipid and glucose measurements,real-time reverse transcription polymerase chain reaction(RT-PCR),and enzyme-linked immunosorbent assay at the different time points of the experiment.An in vitro inflammatory cell model was established by using RAW264.7 cells and human aortic endothelial cells(HAOECs),and indirect co-cultured with supernatant of DPSC-Null(DPSC-Null-CM)or DPSC-HGF-CM,and the effect and mechanisms were analyzed by flow cytometry,RT-PCR and western blot.Nuclear factor-κB(NF-κB)activators and inhibitors were also used to validate the related signaling pathways.RESULTS DPSC-Null and DPSC-HGF treatments decreased the area of atherosclerotic plaques and reduced the expression of inflammatory factors,and the percentage of macrophages in the aorta,and DPSC-HGF treatment had more pronounced effects.DPSCs treatment had no effect on serum lipoprotein levels.The FACS results showed that DPSCs treatment reduced the percentages of monocytes,neutrophils,and M1 macrophages in the peripheral blood and spleen.DPSC-Null-CM and DPSC-HGF-CM reduced adhesion molecule expression in tumor necrosis factor-αstimulated HAOECs and regulated M1 polarization and inflammatory factor expression in lipopolysaccharide-induced RAW264.7 cells by inhibiting the NF-κB signaling pathway.CONCLUSION This study suggested that DPSC-HGF could more effectively ameliorate AS in ApoE-/-mice on a HFD,and could be of greater value in stem cell-based treatments for AS.

Human dental pulp stem/stromal cells in clinical practice

Dental pulp stem/stromal cells(DPSCs)are fibroblast-like,neural crest-derived,and multipotent cells that can differentiate into several lineages.They are relatively easy to isolate from healthy and inflamed pulps,with little ethical concerns and can be successfully cryopreserved and thawed.The therapeutic effects of DPSCs derived from animal or human sources have been extensively studied through in-vitro and in-vivo animal experiments and the findings indicated that DPSCs are effective not only for dental diseases but also for systemic diseases.Understanding that translational research is a critical step through which the fundamental scientific discoveries could be translated into applicable diagnostics and therapeutics that directly benefit humans,several clinical studies were carried out to generate evidence for the efficacy and safety of autogenous or allogeneic human DPSCs(hDPSCs)as a treatment modality for use in cell-based therapy,regenerative medicine/dentistry and tissue engineering.In clinical medicine,hDPSCs were effective for treating acute ischemic stroke and human exfoliated deciduous teeth-conditioned medium(SHED-CM)repaired vascular damage of the corpus cavernous,which is the main cause of erectile dysfunction.Whereas in clinical dentistry,autologous SHED was able to rege-nerate necrotic dental pulp after implantation into injured teeth,and micrografts enriched with autologous hDPSCs and collagen sponge were considered a treatment option for human intrabony defects.In contrast,hDPSCs did not add a significant regenerative effect when they were used for the treatment of post-extraction sockets.Large-scale clinical studies across diverse populations are still lacking to provide robust evidence on the safety and efficacy of hDPSCs as a new treatment option for various human diseases including dental-related problems.

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.

In-Vitro Cellular Responses of Human Dental Primary Cells to Dental Filling Restoratives

In vitro cytotoxicity of six contemporary commercial dental filling restoratives on human dental primary cells, pulp cells (HPCs) and human gingival fibroblasts (HGFs), were tested using WST-1 assay. Continuous 3T3 mouse fibroblast cell lines were used for comparison. The results show that conventional glass-ionomer cement (GIC) Fuji II is not cytotoxic to all the cells. Resin-modified GIC (RMGIC) Fuji II LC is not cytotoxic to both HPCs and HGFs but cytotoxic to 3T3 cells. RMGIC Vitremer and resin composite Z100 are very cytotoxic to all the cells. Resin composite P60 is cytotoxic but much less cytotoxic than Z100. Polycarboxylate cement Durelon is the most cytotoxic among the six tested materials. It was found that continuous 3T3 cell lines were more vulnerable to leachable cytotoxic components than primary HPCs and HGFs. It was also found that the cytotoxcity of the tested materials was dose-dependent.

Dental pulp stem cells stimulate neuronal differentiation of PC12 cells

Dental pulp stem cells(DPSCs) secrete neurotrophic factors which may play an important therapeutic role in neural development, maintenance and repair. To test this hypothesis, DPSCs-conditioned medium(DPSCs-CM) was collected from 72 hours serum-free DPSCs cultures. The impact of DPSCs-derived factors on PC12 survival, growth, migration and differentiation was investigated. PC12 cells were treated with nerve growth factor(NGF), DPSCs-CM or co-cultured with DPSCs using Transwell inserts for 8 days. The number of surviving cells with neurite outgrowths and the length of neurites were measured by image analysis. Immunocytochemical staining was used to evaluate the expression of neuronal markers NeuN, microtubule associated protein 2(MAP-2) and cytoskeletal marker βIII-tubulin. Gene expression levels of axonal growth-associated protein 43 and synaptic protein Synapsin-I, NeuN, MAP-2 and βIII-tubulin were analysed by quantitative polymerase chain reaction(qRT-PCR). DPSCs-CM was analysed for the neurotrophic factors(NGF, brain-derived neurotrophic factor [BDNF], neurotrophin-3, and glial cell-derived neurotrophic factor [GDNF]) by specific ELISAs. Specific neutralizing antibodies against the detected neurotrophic factors were used to study their exact role on PC12 neuronal survival and neurite outgrowth extension. DPSCs-CM significantly promoted cell survival and induced the neurite outgrowth confirmed by NeuN, MAP-2 and βIII-tubulin immunostaining. Furthermore, DPSCsCM was significantly more effective in stimulating PC12 neurite outgrowths than live DPSCs/PC12 co-cultures over the time studied. The morphology of induced PC12 cells in DPSCs-CM was similar to NGF positive controls;however, DPSCs-CM stimulation of cell survival was significantly higher than what was seen in NGF-treated cultures. The number of surviving PC12 cells treated with DPSCs-CM was markedly reduced by the addition of anti-GDNF, whilst PC12 neurite outgrowth was significantly attenuated by anti-NGF, anti-GDNF and anti-BDNF antibodies. These findings demonstrated that DPSCs were able to promote PC12 survival and differentiation. DPSCs-derived NGF, BDNF and GDNF were involved in the stimulatory action on neurite outgrowth, whereas GDNF also had a significant role in promoting PC12 survival. DPSCs-derived factors may be harnessed as a cell-free therapy for peripheral nerve repair. All experiments were conducted on dead animals that were not sacrificed for the purpose of the study. All the methods were carried out in accordance with Birmingham University guidelines and regulations and the ethical approval is not needed.

Polymeric vs hydroxyapatite-based scaffolds on dental pulp stem cell proliferation and differentiation

AIM: To evaluate adhesion, proliferation and differentiation of human dental pulp stem cells(h DPSCs) on four commercially available scaffold biomaterials. METHODS: hD PSCs were isolated from human dental pulp tissues of extracted wisdom teeth and established in stem cell growth medium. h DPSCs at passage 3-5 were seeded on four commercially available scaffold biomaterials, SureO ss(Allograft), Cerabone(Xenograft), PLLA(Synthetic), and OSTEON Ⅱ Collagen(Composite), for 7 and 14 d in osteogenic medium. Cell adhesion and morphology to the scaffolds were evaluated by scanning electron microscopy(SEM). Cell proliferation and differentiation into osteogenic lineage were evaluated using DNA counting and alkaline phosphatase(ALP) activity assay, respectively. RESULTS: All scaffold biomaterials except Sure Oss(Allograft) supported h DPSC adhesion, proliferation and differentiation. hD PSCs seeded on PLLA(Synthetic) scaffold showed the highest cell proliferation and attachment as indicated with both SEM and DNA counting assay. Evaluating the osteogenic differentiation capability of hD PSCs on different scaffold biomaterials with ALP activity assay showed high level of ALP activity on cells cultured on PLLA(Synthetic) and OSTEON ⅡCollagen(Composite) scaffolds. SEM micrographs also showed that in the presence of Cerabone(Xenograft) and OSTEON Ⅱ Collagen(Composite) scaffolds, the h DPSCs demonstrated the fibroblastic phenotype with several cytoplasmic extension, while the cells on PLLA scaffold showed the osteoblastic-like morphology, round-like shape. CONCLUSION: PLLA scaffold supports adhesion, proliferation and osteogenic differentiation of hD PSCs. Hence, it may be useful in combination with hD PSCs for cell-based reconstructive therapy.

Therapeutic effects of dental pulp stem cells on vascular dementia in rat models

Dental pulp stem cells are a type of adult stem cells with strong proliferative ability and multi-differentiation potential. There are no studies on treatment of vascular dementia with dental pulp stem cells. In the present study, rat models of vascular dementia were established by two-vessel occlusion, and 30 days later, rats were injected with 2 × 10^(7) dental pulp stem cells via the tail vein. At 70 days after vascular dementia induction, dental pulp stem cells had migrated to the brain tissue of rat vascular dementia models and differentiated into neuronlike cells. At the same time, doublecortin, neurofilament 200, and Neu N m RNA and protein expression levels in the brain tissue were increased, and glial fibrillary acidic protein m RNA and protein expression levels were decreased. Behavioral testing also revealed that dental pulp stem cell transplantation improved the cognitive function of rat vascular dementia models. These findings suggest that dental pulp stem cell transplantation is effective in treating vascular dementia possibly through a paracrine mechanism. The study was approved by the Animal Ethics Committee of Harbin Medical University(approval No. KY2017-132) in 2017.