Human DPSCs Research Articles

Dysregulation of lncRNA TFAP2A-AS1 is involved in the pathogenesis of pulpitis by the regulation of microRNA-32-5p.

This study was designed to evaluate TFAP2A-AS1 expression in the dental pulp of teeth with or without pulpitis and to determine the function of TFAP2A-AS1 in pulp cells. GSE92681 was analyzed to filter out differentially expressed lncRNAs. Pulp samples from teeth with pulpitis and healthy teeth (control) were examined using real-time (RT) quantitative polymerase chain reaction (qPCR). Human dental pulp stem cells (hDPSCs) were cultured in a specific medium for osteogenic induction, or treated with lipopolysaccharide (LPS) to simulate inflammation. The viability and apoptosis of human DPSCs (hDPSCs) were determined by XTT assay and apoptosis detection kit. Inflammation was induced by LPS and assessed by measuring the expression and release of inflammatory cytokines after TFAP2A-AS1 knockdown. Osteogenic differentiation of hDPSCs was investigated by determining expression levels of osteogenic markers and alkaline phosphatase (ALP) activity after TFAP2A-AS1 overexpression. The downstream microRNA (miRNA) was predicted. Dual-luciferase reporter was used to confirm the binding between miR-32-5p and TFAP2A-AS1. The expression of TFAP2A-AS1 was evaluated in inflamed pulp using RT-qPCR. TFAP2A-AS1 had a discriminatory ability for healthy individuals and patients with pulpitis. The expression of TFAP2A-AS1 decreased upon the osteogenic differentiation of hDPSCs, and increased upon the LPS induction. TFAP2A-AS1 can reverse the osteogenic differentiation of hDPSCs, as evidenced by decreased levels of dentine sialophosphoprotein, dentin matrix protein-1, and ALP activity. TFAP2A-AS1 knockdown can promote cell proliferation of hDPSCs and relieve LPS-induced inflammation, as evidenced by decreased levels of TNF-α, IL-1β, and IL-6. miR-32-5p was identified as a downstream miRNA of TFAP2A-AS1. This study demonstrated the expression and potential function of TFAP2A-AS1 in the human dental pulp. TFAP2A-AS1 can inhibit odontogenic differentiation but promote inflammation in pulp cells.

Wnt/Ca2+ pathway inhibits neural differentiation of human dental pulp stem cells in vitro

Background/purposeDental pulp stem cells (DPSCs) have demonstrated significant potential for neuroregeneration. However, a full understanding of the specific mechanism underpinning the neural differentiation of DPSCs is still required. The Wnt signaling is crucial for the development of the embryonic neural system and the maintenance of adult neural homeostasis. This study aimed to investigate the role of the Wnt/Ca2+ pathway in the neural differentiation of human DPSCs (hDPSCs) and its modulation of the Wnt/β-catenin pathway. Materials and methodshDPSCs were cultured and divided into the control group and the neurogenic induction group (Neuro group). The mRNA and protein levels of neurogenic markers, Wnt/Ca2+, and Wnt/β-catenin pathway indicators were determined using Quantitative real-time PCR and Western blotting. After inhibition of the Wnt/Ca2+ pathway using a WNT5A short hairpin RNA (shRNA) plasmid and subsequent neurogenic induction, neurogenic markers and Wnt/β-catenin pathway indicators in the NC-sh-Neuro group and WNT5A-sh-Neuro group were determined using Quantitative real-time PCR and Western blotting. ResultsCompared with the control group, the expression of the Wnt/Ca2+ pathway indicators (WNT5A, Frizzled 2, calmodulin-dependent protein kinase IIa, and nuclear factor of active T cells 1) decreased in the Neuro group. Conversely, the expression of WNT3A, total β-catenin and active β-catenin in the Wnt/β-catenin pathway increased. Moreover, compared with the NC-sh-Neuro group, the WNT5A-sh-Neuro group exhibited a greater level of mature neural differentiation alongside elevated expression of the Wnt/β-catenin pathway indicators. ConclusionThe Wnt/Ca2+ pathway inhibited neural differentiation of hDPSCs and has a negative effect on the Wnt/β-catenin pathway in vitro.

Biocompatibility and mineralization activity of modified glass ionomer cement in human dental pulp stem cells

Background/purposeFortilin is a multi-functional protein involved in several cellular processes. It has been shown promising potential to be a bioactive molecule that can be incorporated in the dental materials. This study aimed to compare the biocompatibility and mineralization activities of modified glass ionomer cement (Bio-GIC) and Biodentine by direct and indirect method on human dental pulp stem cells (hDPSCs). Materials and methodsConventional glass ionomer cement (GIC), Bio-GIC (GIC supplemented with chitosan, tricalcium phosphate, and recombinant fortilin from Fenneropenaeus merguiensis), and Biodentine were examined in this study. Recombinant fortilin was purified and tested for its cytotoxicity by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay. Human DPSCs were treated with different material eluate for particular time intervals. At given time points, viability of hDPSCs was examined using MTT assay and calcium deposition was assessed by Alizarin red staining assay. Comparisons of the data among groups were analyzed by analysis of variance and Tukey's multiple comparisons. ResultsAll test materials demonstrated no cytotoxicity. In addition, Bio-GIC promoted cell proliferation at 72 h. For direct and indirect method, cells treated with Bio-GIC demonstrated significantly higher calcium deposition than other groups (P < 0.05). ConclusionBio-GIC and Biodentine are not cytotoxic to hDPSCs. Bio-GIC demonstrates enhanced calcium deposition comparable to Biodentine. Bio-GIC may be further developed as a bioactive material for dentin regeneration.

The Effect of the Dental Stem Cell Secretome on Tissue Regeneration: A Systematic Review

Secretome therapy is a promising approach in tissue regeneration because it can reproduce most of the advantages of cell-based therapies. This review aims to investigate the most prominent effect of using dental-derived secretome on tissue regeneration using a systematic review approach. A systematic electronic search was conducted via the PubMed, Scopus and Wiley online library databases for studies published in English up to October 2020. All the articles from the databases were screened, and the criteria for inclusion and exclusion were applied. Forty papers were included in the study, whereby there were 16 in vitro studies and 11 in vivo studies with different animal models. No clinical trial has been reported yet. The most studied DSCs were human SHEDs (12 studies), followed by human DPSCs (11) and human PDLSCs (5). The majority of the studies used secretome from human SHEDs and DPSCs. TGF-ß 1 is the most frequently detected protein in the secretome, which comes from six types of DSCs, followed by NGF and NT-3, which were found in five different types of DSC secretome. The compositions of the secretome were found to promote the regeneration of the tissues through their neurogenic, angiogenic, osteogenic and odontogenic effects, with the majority of studies reviewed reporting using them for nondental tissue regeneration. From this review, DSC-CM reported favorable tissue regeneration potential; however, many factors need to be explored in future research with regard to the application of secretome delivery, particularly DSC-CM, in the clinical setting.

Biological Activities of Glass Ionomer Cement Supplemented with Fortilin on Human Dental Pulp Stem Cells.

This study aimed to determine the most suitable recombinant fortilin and evaluate the biological activities of glass ionomer cement (GIC) incorporated with fortilin on human dental pulp stem cells (hDPSCs). Full-length and three fragments of Penaeus merguiensis fortilin were cloned and examined for their proliferative and cytoprotective effects on hDPSCs by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Human DPSCs were cultured with GIC supplemented with fortilin, tricalcium phosphate, or a combination of tricalcium phosphate and fortilin, designated as GIC + FL, GIC + TCP, and GIC + TCP + FL, respectively (n = 4 for each group). At given time points, hDPSCs were harvested and analyzed by MTT, quantitative reverse transcription polymerase chain reaction, alkaline phosphatase activity, and Alizarin Red assays. The full-length fortilin promoted cell proliferation and significantly increased cell survival. This protein was subsequently added into the GIC along with tricalcium phosphate to investigate the biological activities. All experimental groups showed reduced cell viability after treatment with modified GICs on days 1 and 3. The GIC + TCP + FL group significantly promoted odontoblastic differentiation at particular time points. In addition, alkaline phosphatase activity and calcium phosphate deposit were markedly increased in the GIC + TCP + FL group. Among all experimental groups, the GIC incorporated with fortilin and tricalcium phosphate demonstrated the best results on odontogenic differentiation and mineral deposition in hDPSCs.

Comparative evaluation of the effects of three hydraulic calcium silicate cements on odontoblastic differentiation of human dental pulp stem cells: an in vitro study.

The study aimed to compare the response of human dental pulp stem cells (hDPSCs) towards three hydraulic calcium silicate cements (HCSCs) by measuring cytotoxicity and expression of dentinogenic genes. Dental pulps of five impacted mandibular third molars were extirpated as a source for hDPSCs. Next to culturing, hDPSCs were subjected to fluorescence-activated cell sorting after the third passage to validate stemness of the cells. Human DPSCs were exposed to diluted supernatants of OrthoMTA (OMTA), Biodentine (BD) and Calcium-Enriched Mixture (CEM) at concentrations 10, 25, 50 and 100% at the first, third and fifth day of culture. Then, cells were exposed to 10% concentrations supernatant of HCSCs to determine DSPP and DMP1 gene expression, using a quantitative polymerase-chain reaction. Data were analyzed using one-way and three-way ANOVA, followed by Tukey post hoc statistical tests. Optimal cell proliferation was observed in all groups, regardless of concentration and time-point. HCSC supernatants were non-cytotoxic to hDPSCs at all three time-points, except for 100% Biodentine on day five. On day seven, OMTA group significantly upregulated the expression of DSPP and DMP1 genes. On day 14, expression of DMP1 and DSPP genes were significantly higher in BD and OMTA groups, respectively. Biodentine significantly upregulated DMP1 gene expression over 14 days, whereas CEM was associated with only minimal expression of DSPP and DMP1 .

A novel mutation of SATB2 inhibits odontogenesis of human dental pulp stem cells through Wnt/\u03b2-catenin signaling pathway

BackgroundSATB2-associated syndrome (SAS) is a multisystem disorder caused by mutation of human SATB2 gene. Tooth agenesis is one of the most common phenotypes observed in SAS. Our study aimed at identifying novel variant of SATB2 in a patient with SAS, and to investigate the cellular and molecular mechanism of tooth agenesis caused by SATB2 mutation.MethodsWe applied whole exome sequencing (WES) to identify the novel mutation of SATB2 in a Chinese patient with SAS. Construction and overexpression of wild-type and the mutant vector was performed, followed by functional analysis including flow cytometry assay, fluorescent immunocytochemistry, western blot, quantitative real-time PCR and Alizarin Red S staining to investigate its impact on hDPSCs and the underlying mechanisms.ResultsAs a result, we identified a novel frameshift mutation of SATB2 (c. 376_378delinsTT) in a patient with SAS exhibiting tooth agenesis. Human DPSCs transfected with mutant SATB2 showed decreased cell proliferation and odontogenic differentiation capacity compared with hDPSCs transfected with wild-type SATB2 plasmid. Mechanistically, mutant SATB2 failed to translocate into nucleus and distributed in the cytoplasm, failing to activate Wnt/β-catenin signaling pathway, whereas the wild-type SATB2 translocated into the nucleus and upregulated the expression of active β-catenin. When we used Wnt inhibitor XAV939 to treat hDPSCs transfected with wild-type SATB2 plasmid, the increased odontogenic differentiation capacity was attenuated. Furthermore, we found that SATB2 mutation resulted in the upregulation of DKK1 and histone demethylase JHDM1D to inhibit Wnt/β-catenin signaling pathway.ConclusionWe identified a novel frameshift mutation of SATB2 (c.376_378delinsTT, p.Leu126SerfsX6) in a Chinese patient with SATB2-associated syndrome (SAS) exhibiting tooth agenesis. Mechanistically, SATB2 regulated osteo/odontogenesis of human dental pulp stem cells through Wnt/β-catenin signaling pathway by regulating DKK1 and histone demethylase JHDM1D.

Efficacy of Chitosan as Final Irrigating Solution on TGF-β1 Release from Root Canal Dentin and its Effect on Dental Pulp Stem Cells Response

Purpose: To study the effect of Chitosan final irrigating solution compared with EDTA using different irrigation techniques, on TGF-β1 release from root canal dentin, using enzyme-linked immunosorbent assay (ELISA), and to evaluate the effect of released TGF-β1 on Dental Pulp Stem Cells. Materials and Methods: Human DPSCs were isolated from impacted third molars. Forty eight human root segments were prepared to a standardized truncated cone-shaped canal with an open apex of 1 mm diameter, the volume of prepared segments were determined using CBCT. Samples were randomized into two experimental groups (n=24) regarding the final irrigating solution; Group I: 1.5% NaOCl followed by 17% EDTA, Group II: 1.5% NaOCl followed by 1% Chitosan, then subdivided into subgroups A and B (n = 12) according to the final irrigation technique: A: Needle Irrigation, B: XP-Endo Finisher agitation. The released TGF-β1 were quantified using ELISA at 4 hours, 1 day, and 3 days following irrigation. DPSCs response to1.5% NaOCl, 17% EDTA, 1% Chitosan, and different concentrations of released TGF- β1 was assessed using MTT assay.Results: No statistically significant difference in the quantity of TGF-β1 released by 17% EDTA and 1% Chitosan following needle irrigation or XP Endo Finisher agitation. 1% Chitosan significantly increased DPSCs viability compared to17% EDTA (P < 0.05). TGF-β1 showed significant expansion of viable DPSCs in a positive correlation to its concentration (P <0.001). Conclusions: 1% Chitosan effectively released TGF-β1 from root canal dentin as17% EDTA. The released TGF-β1 promoted DPSCs proliferation at picogram levels in a concentration dependent manner.

Prenylflavonoids isolated from Macaranga tanarius stimulate odontoblast differentiation of human dental pulp stem cells and tooth root formation via the mitogen-activated protein kinase and protein kinase B pathways.

To identify odontogenesis-promoting compounds and examine the molecular mechanism underlying enhanced odontoblast differentiation and tooth formation. Five different nymphaeols, nymphaeol B (NB), isonymphaeol B (INB), nymphaeol A (NA), 3'-geranyl-naringenin (GN) and nymphaeol C (NC) were isolated from the fruit of Macaranga tanarius. The cytotoxic effect of nymphaeols on human DPSCs was observed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effect of nymphaeols on odontoblast differentiation was analysed with Alizarin Red S staining and odontoblast marker expression was assessed using real-time polymerase chain reaction and Western blot analysis. The molecular mechanism was investigated with Western blot analysis. In order to examine the effect of INB on dentine formation in the developing tooth germ, INB-soaked beads were placed under the tooth bud explants in the collagen gel; thereafter, the tooth bud explant-bead complexes were implanted into the sub-renal capsules for 3weeks. Tooth root formation was analysed using micro-computed tomography and histological analysis. Data are presented as mean±standard error (SEM) values of three independent experiments, and results are compared using a two-tailed Student's t-test. The data were considered to have statistical significance when the P-value was less than 0.05. Three of the compounds, NB, INB, and GN, did not exert a cytotoxic effect on human DPSCs. However, INB was most effective in promoting the deposition of calcium minerals in vitro (P<0.001) and induced the expression of odontogenic marker genes (P<0.05). Moreover, this compound strongly induced the phosphorylation of mitogen-activated protein (MAP) kinases and protein kinase B (AKT) (P<0.05). The inhibition of p38 MAP, c-Jun N-terminal kinase (JNK), and AKT substantially suppressed the INB-induced odontoblast differentiation (P<0.001). In addition, isonymphaeol B significantly induced the formation of dentine and elongation of the tooth root in vivo (P<0.05). Prenylflavonoids, including INB, exerted stimulatory effects on odontoblast differentiation and tooth root and dentine formation via the MAP kinase and AKT signalling pathways. These results suggest that nymphaeols could stimulate the repair processes for dentine defects or injuries.

Transplantation of human dental pulp stem cells ameliorates diabetic polyneuropathy in streptozotocin-induced diabetic nude mice: the role of angiogenic and neurotrophic factors

BackgroundDental pulp stem cells (DPSCs) have high proliferation and multi-differentiation capabilities that maintain their functionality after cryopreservation. In our previous study, we demonstrated that cryopreserved rat DPSCs improved diabetic polyneuropathy and that the efficacy of cryopreserved rat DPSCs was equivalent to that of freshly isolated rat DPSCs. The present study was conducted to evaluate whether transplantation of cryopreserved human DPSCs (hDPSCs) is also effective for the treatment of diabetic polyneuropathy.MethodshDPSCs were isolated from human impacted third molars being extracted for orthodontic reasons. Eight weeks after the induction of diabetes in nude mice, hDPSCs (1 × 105/limb) were unilaterally transplanted into the hindlimb skeletal muscle, and vehicle (saline) was injected into the opposite side as a control. The effects of hDPSCs were analyzed at 4 weeks after transplantation.ResultshDPSC transplantation significantly ameliorated reduced sensory perception thresholds, delayed nerve conduction velocity, and decreased the blood flow to the sciatic nerve in diabetic mice 4 weeks post-transplantation. Cultured hDPSCs secreted the vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) proteins. A subset of the transplanted hDPSCs was localized around the muscle bundles and expressed the human VEGF and NGF genes at the transplanted site. The capillary/muscle bundle ratio was significantly increased on the hDPSC-transplanted side of the gastrocnemius muscles in diabetic mice. Neutralizing antibodies against VEGF and NGF negated the effects of hDPSC transplantation on the nerve conduction velocity in diabetic mice, suggesting that VEGF and NGF may play roles in the effects of hDPSC transplantation on diabetic polyneuropathy.ConclusionsThese results suggest that stem cell transplantation with hDPSCs may be efficacious in treating diabetic polyneuropathy via the angiogenic and neurotrophic mechanisms of hDPSC-secreted factors.

549-P: Transplantation of Human Dental Pulp Stem Cells Ameliorates Diabetic Polyneuropathy via Increasing Angiogenic and Neurotrophic Gene Expression in the Transplanted Muscles

Transplantation of cryopreserved DPSCs ameliorated diabetic neuropathy equal to freshly isolated DPSCs. Human DPSCs (hDPSCs) can be isolated from teeth extracted at a young age and can be cryopreserved until use. The aim of this study is to clarify the therapeutic mechanism of hDPSCs transplantation on diabetic polyneuropathy. We collected human impacted third molars from adult at Aichi-Gakuin University hospital. Written informed consent was obtained from each donor. Identification of hDPSCs was analyzed by FACS and differentiation capabilities. Diabetes was induced by injection of STZ in 6 week-old BALB/cAJcl-nu/nu mice. Eight weeks after STZ injection, hDPSCs were transplanted into unilateral hindlimb skeletal muscles of normal and diabetic mice. Four weeks after transplantation, sciatic blood flow (SNBF), sciatic motor/sensory nerve conduction velocity (MNCV/SNCV) and current perception threshold (CPT) were assessed. To elucidate the therapeutic effects of hDPSCs, diabetic mice were treated with neutralizing antibody soon after transplantation. Immunohistological and gene expression analysis of hindlimb skeletal muscles were also performed at the end of the experiments. Diabetic mice showed significant reductions in SNBF, MNCV and SNCV and increase in CPTs in the control side compared with normal mice. Transplantation of hDPSCs significantly ameliorated the impaired SNBF, MNCV, SNCV and CPTs in the hDPSCs-injected side. The transplanted hDPSCs were located around the muscle bundles and expressed the human VEGF and NGF genes. Capillary/muscle bundle ratio was also increased in the hDPSCs-injected side. Furthremore, the effects of hDPSCs transplantation were cancelled by neutralizing antibodies of VEGF and NGF. These results suggest that cell therapy using hDPSCs may be useful for treatment of diabetic neuropathy via the angiogenic and neurotrophic factor by hDPSCs secretion. Disclosure M. Hata: None. M. Omi: None. N. Nakamura: None. M. Miyabe: None. M. Ito: None. E. Makino: None. S. Kanada: None. T. Ono: None. Y. Imanishi: None. T. Himeno: None. J. Nakamura: Research Support; Self; Astellas Pharma Inc., Boehlinger Ingelheim Japan Co., Ltd., Daiichi Sankyo, Eli Lilly Japan K.K., Japan Tobacco Inc., Kaken Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K.K., Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Otsuka Pharmaceutical Co., Ltd., Pfizer Japan Inc., Sanofi K.K., Sanwa Kagaku Kenkyusho, Shionogi &amp; Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker’s Bureau; Self; Abbott Japan Co., Ltd., ARKRAY, Astellas Pharma Inc., AstraZeneca K.K., Boehlinger Ingelheim Japan Co., Ltd.,, Daiichi Sankyo, Eli Lilly Japan K.K., Fukuda Denshi, Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K.K., Mylan, Novartis Pharma K.K., Novo Nordisk Pharma Ltd, Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Terumo Medical Corporation. H. Kamiya: Speaker’s Bureau; Self; Astellas Pharma Inc., AstraZeneca K.K., Boehringer Ingelheim K.K., Daiichi Sankyo, Eli Lilly Japan K.K., Fukuda Denshi, Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K.K., Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. S. Ozawa: None. J. Takebe: None. T. Matsubara: None. K. Naruse: None.

Regenerative potential of human dental pulp stem cells in the treatment of stress urinary incontinence: In vitro and in vivo study.

ObjectivesTo evaluate the regenerative potential of human dental pulp stem cells (hDPSCs) in an animal model of stress urinary incontinence (SUI). SUI, an involuntary leakage of urine, is due to physical stress involving an increase in bladder pressure and a damage of external urethral sphincter affecting muscles and nerves. Conventional therapies can only relieve the symptoms. Human DPSCs are characterized by peculiar stemness and immunomodulatory properties and might provide an alternative tool for SUI therapy.Materials and methodsIn vitro phase: hDPSCs were induced towards the myogenic commitment following a 24 hours pre‐conditioning with 5‐aza‐2′‐deoxycytidine (5‐Aza), then differentiation was evaluated. In vivo phase: pudendal nerve was transected in female rats to induce stress urinary incontinence; then, pre‐differentiated hDPSCs were injected in the striated urethral sphincter. Four weeks later, urethral sphincter regeneration was assayed through histological, functional and immunohistochemical analyses.ResultsHuman DPSCs were able to commit towards myogenic lineage in vitro and, four weeks after cell injection, hDPSCs engrafted in the external urethral sphincter whose thickness was almost recovered, committed towards myogenic lineage in vivo, promoted vascularization and an appreciable recovery of the continence. Moreover, hDPSCs were detected within the nerve, suggesting their participation in repair of transected nerve.ConclusionsThese promising data and further investigations on immunomodulatory abilities of hDPSCs would allow to make them a potential tool for alternative therapies of SUI.

HEMA Effects on Autophagy Mechanism in Human Dental Pulp Stem Cells

Autophagy is a complex mechanism that permits the degradation of cellular components in order to enhance cell homeostasis, recycling the damaged, dysfunctional, or unnecessary components. In restorative dentistry practice, free resin monomers of 2-hydroxyethyl methacrylate (HEMA) can be released. The aim of this study was to investigate the effect of HEMA on proliferation and autophagy in human dental pulp stem cells (hDPSCs). Human DPSCs were treated with different concentrations of HEMA (3 and 5 mmol L−1). To evaluate the proliferation rate, MTT and trypan blue assays were used. Autophagic markers such as microtubule-associated protein 1 light chain 3 (LC3-I/II) and ubiquitin-binding protein (p62) were analyzed through immunofluorescence observations. Beclin1, LC3-I/II, and p62 were evaluated by means of Western blotting detection. Considering that activity of extracellular signal–regulated kinase (ERK) and its phosphorylated form (pERK) mediates several cellular processes, such as apoptosis, autophagy, and senescence, the involvement of ERK/pERK signaling was also evaluated. Obtained results showed a decreased cell proliferation associated with morphological changes in HEMA-treated cells. The Western blot results showed that the expression levels of Beclin1, LC3-I/II, and ERK were significantly elevated in HEMA-treated cells and in cells co-treated with rapamycin, an autophagic promoter. The expression levels of p62 were significantly reduced compared to the untreated samples. Protein levels to the autophagic process, observed at confocal microscopy confirmed the data obtained from the Western blot. The up-regulation of ERK and pERK levels, associated with nuclear translocation, revealed that ERK pathway signaling could act as a promoter of autophagy in dental pulp stem cells treated with HEMA.

The Effect of Melanocyte Stimulating Hormone and Hydroxyapatite on Osteogenesis in Pulp Stem Cells of Human Teeth Transferred into Polyester Scaffolds

Presently, tissue engineering is employed in the restoration and repair of tissue defects. Degradable scaffolds, stem cells and stimulating factors are employed in this method. In this study, the effect of melanocyte-stimulating hormone (MSH) and/or hydroxyapatite (HA) on proliferation, osteoblast differentiation, and mineralization of human dental pulp stem cells (hDPSCs) seeded on PLLA-PCL nanofibrous scaffolds was evaluated. For this aim, PLLA-PCL-HA nanofibrous scaffolds were fabricated using electrospinning method. FE-SEM images exhibited that all nanofibers had bead-free morphologies with average diameters ranging from 150–205 nm. Human DPSCs seeded into PLLA-PCL nanofibers were treated with MSH. Cell viability, proliferation, morphology, osteogenic potential, and the expression of tissue-specific genes were assessed by means of MTT assay, FE-SEM, alizarin red S staining, and RT-PCR analysis. hDPSCs exhibited improved adhesion and proliferation capacity on the PLLA-PCL-HA nanofibers treated with MSH compared to other groups (p<0.05). Additionally, PLLA-PCL-HA nanofibers treated with MSH exhibited significantly higher mineralization and alkaline phosphatase activity than other groups. RT-PCR results confirmed that PLLA-PCL-HA nanofibers enriched with MSH could significantly unregulated the gene expression of BMP2, osteocalcin, RUNX2 and DSPP that correlated to osteogenic differentiation (p<0.05). Based on results, incorporation of HA nanoparticles in PLLA-PCL nanofibers and addition of MSH in media exhibited synergistic effects on the adhesion, proliferation, and osteogenesis differentiation of hDPSCs, and therefore assumed to be a favorable scaffold for bone tissue engineering applications.

Role of Ku70 in the apoptosis of inflamed dental pulp stem cells.

The study aimed to investigate the effects of DNA repair proteins on cell apoptosis in human DPSCs during inflammation. Lipopolysaccharide (LPS) was used to stimulate inflammation in dental pulp in vivo and in vitro. We identified the activation of DSB response and DNA repair proteins in inflamed pulp tissue and in LPS-treated human DPSCs. Then we transfected the cells with Ku70 (a key protein involved in NHEJ) siRNA and detected the expression changes of γ-H2A.X, DNA repair proteins and cell apoptosis. Immunohistochemical staining showed that at 4 and 6days of pulpitis the expression of Ku70 and γ-H2A.X significantly increased. The levels of γ-H2A.X, Ku70, Xrcc4, and Rad51 increased considerably in the LPS-treated DPSCs. Furthermore, decreased expression of Ku70 could increase the number of γ-H2A.X foci, apoptotic cells and reduce cell viability in DPSCs. The results indicate that NHEJ pathway was the main mechanism involved in DNA damage response induced by repeated LPS stimulation in DPSCs. Meanwhile, the findings suggested that Ku70 serves importantly in the apoptosis of DPSCs in the inflammatory environment.

Neuro-fuzzy method for predicting the viability of stem cells treated at different time-concentration conditions.

Dental stem cells isolated for human dental pulp are an excellent source for regenerative medicine and dentistry. Simulation of clinical scenario is one of the crucial challenges for evaluation of the efficacy of DPSCs in various regenerative therapies. In this study we evaluated the viability of DPSCs after treatment with artificial bacterial lipopolysaccharides (LPS) as the main component responsible for inducing inflammatory response in majority of the inflammatory conditions in clinical scenario. Although a number of studies have previously treated stem cells with LPS from bacteria, however the accuracy level of the outcome was not established. Here we have analyzed the outcome using adaptive neuro-fuzzy inferences system (ANFIS) to predict the viability of human DPSCs after treatment with bacterial LPS.

Human DPSCs fabricate vascularized woven bone tissue: a new tool in bone tissue engineering

Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells that have been successfully used in human bone tissue engineering. To establish whether these cells can lead to a bone tissue ready to be grafted, we checked DPSCs for their osteogenic and angiogenic differentiation capabilities with the specific aim of obtaining a new tool for bone transplantation. Therefore, hDPSCs were specifically selected from the stromal–vascular dental pulp fraction, using appropriate markers, and cultured. Growth curves, expression of bone-related markers, calcification and angiogenesis as well as an in vivo transplantation assay were performed. We found that hDPSCs proliferate, differentiate into osteoblasts and express high levels of angiogenic genes, such as vascular endothelial growth factor and platelet-derived growth factor A. Human DPSCs, after 40 days of culture, give rise to a 3D structure resembling a woven fibrous bone. These woven bone (WB) samples were analysed using classic histology and synchrotron-based, X-ray phase-contrast microtomography and holotomography. WB showed histological and attractive physical qualities of bone with few areas of mineralization and neovessels. Such WB, when transplanted into rats, was remodelled into vascularized bone tissue. Taken together, our data lead to the assumption that WB samples, fabricated by DPSCs, constitute a noteworthy tool and do not need the use of scaffolds, and therefore they are ready for customized regeneration.

Dental Pulp Tissue Regeneration Using Dental Pulp Stem Cells Isolated and Expanded in Human Serum

IntroductionDental pulp–derived stem cells (DPSCs) have the potential to regenerate dentin and dental pulp tissue because of their differentiation capacity and angiogenic properties. However, for regenerative approaches to gain regulatory and clinical acceptance, protocols are needed to determine more feasible ways to cultivate DPSCs, namely, without the use of xenogeneic-derived components (animal sera) and exogenous growth factors. MethodsIn this study, human DPSCs were isolated from third molars and expanded in standard culture conditions containing fetal bovine serum (DPSCs-FBS) or conditions containing human serum (DPSCs-HS). After cell characterization and evaluation of their angiogenic secretome, DPSCs were seeded in tooth slice/scaffolds and implanted subcutaneously into immunodeficient mice. After 30 days, tooth slices were retrieved and evaluated for dental pulp tissue regeneration. Immunohistochemistry and confocal microscopy were used to quantify blood vessel formation and evaluate predentin and dentin formation. ResultsAfter culture, DPSCs-HS produced concentrations of angiogenic growth factors equivalent to DPSCs-FBS. Additionally, in DPSCs-HS, several angiogenic factors were produced in at least 1-fold higher concentrations than in DPSCs-FBS. In vivo, it was determined that DPSCs-HS produced a robust angiogenic response and regeneration of dentin equivalent to DPSCs-FBS. ConclusionsThese findings show that DPSCs can be isolated and expanded to clinical scale numbers in media devoid of animal serum or exogenous growth factors and still maintain their pulp regenerative properties. The implications of these findings are significant for further development of clinical protocols using DPSCs in cell therapies.

Osteogenic/Odontogenic Bioengineering with co-Administration of Simvastatin and Hydroxyapatite on Poly Caprolactone Based Nanofibrous Scaffold.

Purpose: Statin is an effective factor for promoting osteogenesis. The aim of the present study was to evaluate the effect of simvastatin (SIM) and/or HA addition on changes in osteogenesis levels by human DPSCs transferred onto three-dimensional (3D) nanofibrous Poly (ε-caprolactone) (PCL)/Poly lactic acide (PLLA) polymeric scaffolds. Methods: For this purpose, a 3D nanofibrous composite scaffold of PCL/PLLA/HA was prepared by electrospinning method. SIM was added to scaffolds during DPSCs culturing step. Cell proliferation and osteogenic activity levels were assessed by using MTT assay and Alizarin Red assay methods. In addition, the expression of genes responsible for osteogenesis, including BMP2, Osteocalcin, DSPP and RUNX2, were determined before and 2 weeks after incorporation of SIM. Results: The MTT assay showed that PCL/PLLA/HA scaffolds seeded with DPSCs has significant (p<0.05) more proliferative effect than PCL/PLLA or DMEM cultured cells, additionally SIM administration improved this result over the PCL/PLLA/HA scaffolds without SIM treatment. SEM imaging revealed improved adhesion and probably osteogenic differentiation of DPSCs on PCL/PLLA/HA nanofibers treated with SIM, moreover the alizarin red assay ensured significant (p<0.05) higher mineralization of this group. Finally, real time PCR confirmed the positive regulation (P<0.05) of the expression of osteo/odontogenesis markers BMP2, Osteocalcin, DSPP and RUNX2 genes in PLLA-PCL-HA (0.1)-SIM group. Conclusion: As a result, addition of simvastatin with incorporation of hydroxyapatite in PCL-PLLA scaffolds might increase the expression of osteogenesis markers in the DPSCs, with a possible increase in cell differentiation and bone formation.

Isolation, Characterization, and Differentiation of Dental Pulp Stem Cells in Ferrets

IntroductionThe ferret canine tooth has been introduced as a suitable model for studying dental pulp regeneration. The aim of this study was to isolate and characterize ferret dental pulp stem cells (fDPSCs) and their differentiation potential. MethodsDental pulp stem cells were isolated from freshly extracted ferret canine teeth. The cells were examined for the expression of stem cell markers STRO-1, CD90, CD105, and CD146. The osteo/odontogenic and adipogenic differentiation potential of fDPSCs was evaluated. Osteogenic and odontogenic marker genes were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) on days 1, 4, and 8 after osteo/odontogenic induction of fDPSCs including dentin sialophosphoprotein (DSPP), dentin matrix protein-1, osteopontin, and alkaline phosphatase. Human dental pulp cells were used as the control. The results were analyzed using 3-way analysis of variance. ResultsfDPSCs were positive for STRO1, CD90, and CD105 and negative for CD146 markers with immunohistochemistry. fDPSCs showed strong osteogenic and weak adipogenic potential. The overall expression of DSPP was not significantly different between fDPSCs and human dental pulp cells. The expression of DSPP in osteo/odontogenic media was significantly higher in fDPSCs on day 4 (P < .01). The overall expression of dentin matrix protein-1, osteopontin, and alkaline phosphatase was significantly higher in fDPSCs (P = .0005). ConclusionsfDPSCs were positive for several markers of dental pulp stem cells resembling human DPSCs and appeared to show a stronger potential to differentiate to osteoblastic rather than odontoblastic lineage.