Proliferation Of hDPCs Research Articles

Synthesized Ceramide Induces Growth of Dermal Papilla Cells with Potential Contribution to Hair Growth.

BackgroundThe ceramide is known to play an important role in the formation of intracellular lipids, and play a crucial role as a barrier for skin and hair cuticle. Recent study has revealed that ceramide has potential effect on hair growth in a mouse model. However, the role of ceramide in human dermal papilla cells (hDPCs) known to play an important role in hair growth is not well understood yet.ObjectiveThe goal of this study was to investigate the effect of synthetic ceramides (oleyl and stearyl ceramides) on hair growth using hDPCs.MethodshDPCs were treated with synthesized ceramides. hDPCs viability was evaluated by MTT assay. The expression of hair growth related factors were investigated by western blot, real-time polymerase chain reaction and growth factor array. The expression of β-catenin was confirmed by immunofluorescence.ResultsTreatment with ceramides increased the expression of proteins affecting cell proliferation such as Bcl-2, BAX, phosphorylated-ERK and Cyclin D1. Also, ceramides treatment were increased the expression of several growth factors, including epidermal growth factor family, and promote the expression of Wnt/β-catenin and BMP2/4 signaling.ConclusionOur data suggest that synthetic ceramides stimulates hair growth by induction proliferation of hDPCs via modulation of Wnt/β-catenin and BMP2/4 signaling.

DLX3 Inhibits the Proliferation of Human Dental Pulp Cells Through Inactivation of Canonical Wnt/β-Catenin Signaling Pathway.

Homeodomain gene Distal-less-3 (Dlx3) plays an important role during tooth development. Our previous studies indicate that DLX3 inhibits proliferation of human dental pulp cells (hDPCs). However, the mechanism of DLX3 regulating proliferation of hDPCs and maintaining the quiescence of the cells remain unknown. Given the importance of canonical Wnt signaling in the proliferation of dental pulp cell and tooth development, we hypothesized that DLX3 inhibited proliferation of hDPCs through inactivation of canonical Wnt signaling. With overexpression or knock-down of DLX3 in primary hDPCs, we found DLX3 down regulated canonical Wnt signaling and its downstream target genes. And when the DLX3 overexpressed-cells were treated with lithium chloride, the proliferation inhibition by DLX3 was reversed. We also found that DLX3 enhanced the expression of DKK1 and the reduced proliferation of hDPCs by DLX3 was reversed with knock-down of DKK1. Furthermore, luciferase reporter assay and chromatin immunoprecipitation assay showed DLX3 was able to bind to Dkk1 promoter region from nucleotides (nt) -1656 to -1245, and stimulated Dkk1 promoter activity. Mutagenesis studies further revealed two DLX3 responsive elements in Dkk1 promoter. Taken together, our data indicate that DLX3 inhibits proliferation of hDPCs via inactivation of Wnt/β-catenin signaling pathway by directly binding to Dkk1 promoter and increasing its expression.

Novel effect of sildenafil on hair growth

BackgroundSildenafil, a phosphodiesterase 5 (PDE5) inhibitor, is known to increase the intracellular level of cyclic guanosine monophosphate (cGMP), which causes vasodilation. However, the effect of sildenafil on human hair follicles (hHFs) is unknown. ObjectiveThe purpose of this study was to determine the role of sildenafil in hair growth. MethodsWe investigated the expression of PDE5 in human dermal papilla cells (hDPCs) and hHFs. The effects of sildenafil on hDPC proliferation were evaluated using BrdU assays. The mRNA expression of growth factors and extracellular signal-regulated kinase (ERK) phosphorylation were investigated using real-time PCR and western blotting, respectively. Additionally, anagen induction and perifollicular vessel formation were evaluated using an in vivo mice model. ResultsWe confirmed high expression of PDE5 in hDPCs and hHFs. Sildenafil enhances proliferation of hDPCs and up-regulates the mRNA expression of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), which are responsible for hair growth. Additionally, sildenafil up-regulates the levels of phosphorylated ERK and accelerates anagen induction by stimulating perifollicular vessel formation after topical application in mice. ConclusionOur study demonstrates for the first time, the significant therapeutic potential of sildenafil on hair growth and its potential use in treatment of alopecia.

Effects of silicate-based composite material on the proliferation and mineralization behaviors of human dental pulp cells: An in vitro assessment.

The objective of this study was to investigate the effects of a silicate-based composite material on proliferation and mineralization of human dental pulp cells (hDPCs), which was compared with those of calcium hydroxide (Ca(OH)2, CH) and tricalcium silicate (Ca3SiO5, C3S). HDPCs were cultured with CH, C3S and tricalcium silicate/dicalcium silicate (Ca3SiO5/Ca2SiO4, C3S/C2S) composites extract. The CCK-8 assay showed that the composite material stimulated the proliferation of hDPCs. The odontogenic marker genes and DSPP protein expression were more significantly up-regulated by the C3S/C2S composite material compared with pure CH and C3S. HDPCs cultured with composite material extract exert stronger ALP activity and alizarin red S staining. C3S/C2S composite material was advantageous over pure C3S by showing enhanced ability to stimulate the proliferation and odontogenic differentiation of hDPCs, suggesting that the C3S/C2S composite materials possess desirable biocompatibility and bioactivity, and might be a new type of pulp-capping agent and dentin alternative materials.

Mineral Trioxide Aggregate Mixed with 5-Aminolevulinic Acid for the Photodynamic Antimicrobial Strategy in Hard Tissue Regeneration.

Aminolevulinic acid (ALA) based photodynamic antimicrobial strategy can provide good antimicrobial effects and be used for medical applications. The aim of this study was to apply this strategy to Mineral Trioxide Aggregate (MTA), which is commonly used as a filling material for root endings and by doing so, to increase the bactericidal capability of MTA, as well as to investigate its characterization, cytocompatibility, and odontogenic differentiation potential. MTA is known to be a derivative of calcium silicate (CS). In this study, MTA specimens with or without ALA and light treatment were prepared. Diametral tensile strength values (DTS), setting durations, X-ray diffraction (XRD) spectra, apatite-mineralization, and antimicrobial abilities of the MTA, were also analyzed. Human dental pulp cells (hDPCs) can proliferate into the newly formed matrix and differentiate into odontoblasts to reinforce and strengthen the root. Levels of hDPCs proliferation and its odontogenic capabilities when cultured on MTA with ALA and light treatment, and the percentages of cells existing in the various cell cycle stages, were further evaluated in this study. The results indicated that MTA added ALA with light treatment had greater antibacterial ability and cytocompatibility, compared to MTA alone. A higher percentage S phase of the cells cultured on MTA added ALA with light treatment was observed. Furthermore, hDPCs cultured on MTA added ALA with light treatment had the highest expression levels of the odontoblastic differentiation markers. ALA has great antimicrobial efficiency and is a potential material for future medical applications. ALA-based photodynamic antibacterial strategy applied in the MTA has great antibacterial ability, cytocompatibility, and odontoblastic differentiation potential, and can facilitate the development of root canal treatment.

Nature-derived lignan compound VB-1 exerts hair growth-promoting effects by augmenting Wnt/β-catenin signaling in human dermal papilla cells.

BackgroundVitexin is a kind of lignan compound which has been shown to possess a variety of pharmacological effects, such as anti-inflammatory, anti-oxidative and anti-cancer activities. However the effect of vitexin on hair regeneration has not been elaborated.MethodsThe proliferation of human dermal papilla cells (hDPCs) was examined by cell counting and continuous cell culture after vitexin compound 1 (VB-1) was treated. The expression of lef1, wnt5a, bmp2, bmp4, alpl and vcan was examined by RT-PCR. The expression of dkk1, tgf-β1, active-β-Catenin, and AXIN2 was examined by RT-PCR or immunoblotting. Hair shaft growth was measured in the absence or presence of VB-1.ResultsWe demonstrated that VB-1 significantly promotes the proliferation of hDPCs in a concentration-dependent manner within a certain concentration range. Among the hair growth-related genes investigated, dkk1 was clearly down-regulated in hDPCs treated with VB-1. The increased active β-Catenin and decreased AXIN2 protein levels suggest that VB-1 facilitates Wnt/β-catenin signaling in hDPCs in vitro. The expression of DP signature genes was also upregulated after VB-1 treatment. Our study further indicated that VB-1 promotes human hair follicle (HF) growth by HF organ culture assay.DiscussionVB-1 may exert hair growth-promoting effects via augmenting Wnt/β-catenin signaling in hDPCs.

Wnt/β-catenin and ERK pathway activation: A possible mechanism of photobiomodulation therapy with light-emitting diodes that regulate the proliferation of human outer root sheath cells.

Outer root sheath cells (ORSCs) play important roles in maintaining hair follicle structure and provide support for the bulge area. The hair growth promoting effects of photobiomodulation therapy (PBMT) have been reported, but the mechanisms for this in human ORCs (hORSCs) have rarely been studied. The aim of this study was to investigate the effect of various wavelengths of light-emitting diode (LED) irradiation on human ORSCs (hORSCs). LED irradiation effects on hORSC proliferation and migration were examined with MTT assay, BrdU incorporation assay and migration assays. hORSCs were irradiated using four LED wavelengths (415, 525, 660, and 830 nm) with different low energy levels. LED irradiation effects on the expression of molecules associated with the Wnt/β-catenin signaling and ERK pathway, hair stem cell markers, and various growth factors and cytokines in hORSCs were examined with real-time PCR and Western blot assay. The effect of the LED-irradiated hORSCs on cell proliferation of human dermal papilla cells (hDPCs) was examined with co-culture and MTT assay. PBMT with LED light variably promoted hORSC proliferation and suppressed cell apoptosis depending on energy level. LED irradiation induced Wnt5a, Axin2, and Lef1 mRNA expression and β-catenin protein expression in hORSCs. Phosphorylation of ERK, c-Jun, and p38 in hORSCs was observed after LED light irradiation, and ERK inhibitor treatment before irradiation reduced ERK and c-Jun phosphorylation. Red light-treated hORSCs showed substantial increase in IL-6, IL-8, TNF-a, IGF-1, TGF-β1, and VEGF mRNA. Light irradiation at 660 and 830 nm projected onto hORSCs accelerated in vitro migration. LED-irradiated hORSCs increased hDPCs proliferation when they were co-cultured. The conditioned medium from LED-irradiated hORSCs was sufficient to stimulate hDPCs proliferation. These results demonstrate that LED light irradiation induced hORSC proliferation and migration and inhibited apoptosis in vitro. The growth-promoting effects of LEDs on hORSCs appear to be associated with direct stimulation of the Wnt5a/β-catenin and ERK signaling pathway. Lasers Surg. Med. 49:940-947, 2017. © 2017 Wiley Periodicals, Inc.

MTA-induced Notch activation enhances the proliferation of human dental pulp cells by inhibiting autophagic flux.

To determine the roles of autophagy and Notch signalling in mineral trioxide aggregate (MTA)-mediated proliferation and differentiation of human dental pulp cells (hDPCs). hDPCs were separated from human healthy molar teeth using collagenase I/dispase II digestion and then cultured in α-MEM medium with 15% foetal bovine serum. hDPCs were seeded in 96-well plates, and cell counting kit assays were carried out to test their viability. Real-time quantitative polymerase chain reaction (qPCR) was used to evaluate ALP, Runx2, Notch1, Hes1 and Hey1 mRNA levels. Notch1, hes1, LC3 and p62 protein levels were quantified by Western blotting. Colocalization of LC3 and Notch1 was measured by immunofluorescence. Two-tailed Student's t-tests were used for statistical analysis. Autophagic flux was significantly (P<0.05) inhibited by MTA extracts, causing Notch degradation arrest. This resulted in the promotion of cell proliferation and inhibition of differentiation during the logarithmic phase of cell growth. MTA extract promoted the proliferation of hDPCs in part by activating Notch signalling through inhibition of autophagic flux during the early stage and, thus, might potentially induce rapid restoration of injured pulps.

Schwann cells secrete extracellular vesicles to promote and maintain the proliferation and multipotency of hDPCs.

Schwann cells (SCs) are the principal glial cells in peripheral nerve system, involved in neuropathies with great regenerative potential. Dental pulp cells have been reported to maintain neurogenic potential. In contrast, the regulatory role of SCs on human dental pulp cells (hDPCs) development remains undefined. SC secretion and SC-derived extracellular vesicles (EVs) were collected and used to treat hDPCs; and proliferation and multiple differentiation of hDPCs were detected after EVs treatments. Finally, we analysed the proteomes of SC-EVs and SCs through mass spectrum. In this study, we found SC secretion showed a predominantly regulatory role on the development of hDPCs. Further, we identified EVs from SC secretion with similar function as SC secretion in regulating hDPCs proliferation and multipotency. And expression of transcription factor Oct4 was upregulated after treatment of both SC secretion and EVs, as well as Sox2 and Nanog. We detected abundant enrichment of Oct4 in EVs, which might be responsible for the upregulation of stem cell-related genes in hDPCs. Through proteome and western blot analysis, we found enriched TGFβs in EVs, indicating that accelerated hDPCs proliferation may be mediated by activated TGFβ-Samd and TGFβ-MAPK signalling. In summary, our study sheds light on critical regulatory ability of SC-derived EVs on hDPCs proliferation and multipotency, suggesting great implications for seeding cells used in tissue engineering.

The effects of Biodentine/polycaprolactone three-dimensional-scaffold with odontogenesis properties on human dental pulp cells.

To determine the feasibility of using three-dimensional printed Biodentine/polycaprolactone composite scaffolds for orthopaedic and dental applications. The physicochemical properties and the odontogenic differentiation of human dental pulp cells (hDPCs) were investigated. Biodentine was well-suspended in ethanol and dropped slowly into molten polycaprolactone with vigorous stirring. The Biodentine/polycaprolactone composite scaffolds were then fabricated into controlled macropore sizes and structures using an extrusion-based three-dimensional (3D) printer. The mechanical properties, bioactivity, and the proliferation and odontogenic differentiation of human dental pulp cells (hDPCs) cultured on the scaffolds were evaluated. Biodentine/polycaprolactone scaffolds had uniform macropores 550μm in size with established interconnections and a compressive strength of 6.5 MPa. In addition, the composite scaffolds exhibited a good apatite-forming ability and were capable of supporting the proliferation and differentiation of hDPCs. The composite scaffolds fabricated by an extrusion-based 3D printing technique had similar characteristics to Biodentine cement, including bioactivity and the ability to promote the differentiation of hDPCs. These results indicate that the composite scaffold would be a candidate for dental and bone regeneration.

The Characteristics of Mineral Trioxide Aggregate/Polycaprolactone 3-dimensional Scaffold with Osteogenesis Properties for Tissue Regeneration

IntroductionThe aim of this study was to investigate whether the mineral trioxide aggregate/polycaprolactone (MTA/PCL) hybrid 3-dimensional (3D) scaffold supplies a suitable microenvironment for the osteogenic differentiation of human dental pulp cells (hDPCs) and to further consider the effect of the MTA/PCL composite on the biological performance of hybrid scaffolds. MethodsMTA was suspended in absolute alcohol and dropped slowly into PCL that was generated with the printable MTA-matrix. Then, the MTA/PCL composite was prepared into highly uniform scaffolds with controlled macropore sizes and structure using a 3D printing technique. Mechanical properties and the apatite precipitation of the scaffolds were evaluated as well as the cell response to the scaffolds by culturing hDPCs. ResultsThe results showed that the MTA/PCL 3D scaffold had uniform, 450-μm, high-porosity (70%) macropores and a compressive strength of 4.5 MPa. In addition, the MTA/PCL scaffold could effectively promote the adhesion, proliferation, and differentiation of hDPCs. ConclusionsThe 3D-printed MTA/PCL scaffolds not only exhibited excellent physical and chemical properties but also enhanced osteogenesis differentiation. All of the results support the premise that this MTA/PCL porous scaffold would be a useful biomaterial for application in bone tissue engineering.

Effects of Epigallocatechin Gallate, an Antibacterial Cross-linking Agent, on Proliferation and Differentiation of Human Dental Pulp Cells Cultured in Collagen Scaffolds

IntroductionThis study aimed to evaluate the efficacy of epigallocatechin gallate (EGCG), an antibacterial cross-linking agent, on the proliferation and differentiation of human dental pulp cells (hDPCs) cultured in hydrogel collagen scaffolds. MethodsThe odontogenic differentiation induced by EGCG was evaluated by alkaline phosphatase (ALP) activity and odontogenic-related gene expression using real-time polymerase chain reaction. The antibacterial effect of EGCG was investigated by a disc diffusion assay in comparison with glutaraldehyde. Proliferation was analyzed by cell number counting under both optical and confocal laser scanning microscopes. To assess the mechanical properties of collagen treated with EGCG, the setting time, surface roughness, and compressive strength were measured. ResultsEGCG itself did not up-regulate the odontogenic-related markers (P > .05) although ALP activity was slightly increased. The proliferation and differentiation of hDPCs cultured in collagen increased significantly in the presence of EGCG (P < .05). The antibacterial activity of EGCG was similar to that of glutaraldehyde. The setting time of collagen was significantly shortened when it was treated with EGCG (P < .05). The surface roughness and compressive strength of the cross-linked collagen were higher than those of collagen without EGCG (P < .05). ConclusionsOur results showed that EGCG, the antibacterial cross-linking agent, promoted the proliferation and differentiation of hDPCs cultured in collagen scaffolds. Furthermore, the enhanced mechanical properties of collagen scaffolds induced by EGCG may play important roles in cell behavior. Consequently, the application of EGCG to collagen scaffolds might be beneficial for regenerative endodontic therapy.

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 TET1-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.

Behaviour of human dental pulp cells cultured in a collagen hydrogel scaffold cross-linked with cinnamaldehyde.

To investigate the effects of the cross-linking agent cinnamaldehyde (CA) on differentiation of human dental pulp cells (hDPCs) cultured in a collagen hydrogel, which may be useful as a scaffold for regenerative endodontic therapy. The odontogenic potential of hDPCs exposed to CA was examined using alkaline phosphatase (ALP) activity, Alizarin red S staining and real-time polymerase chain reaction for odontogenic gene expression. The morphological features of hDPCs cultured in CA-treated collagen were evaluated by scanning electron microscopy. Determination of cell numbers for evaluating proliferation was assessed by optical and fluorescence microscopy. To assess the mechanical properties of collagen treated with CA, setting time, compressive strength and surface roughness were measured. Statistical analysis was performed using Student's t-test compared with control (P=0.05). CA per se did not increase ALP activity, calcium nodule formation and expression of odontogenic-related markers (P>0.05). On the contrary, the proliferation and odontogenic differentiation of hDPCs cultured in a collagen scaffold was promoted in the presence of CA (P<0.05). The setting time was significantly shortened, and the compressive strength and surface roughness were increased by treatment with CA (P<0.05). Cross-linking of collagen scaffolds by CA had beneficial effects with respect to attachment, proliferation and differentiation of hDPCs. Consequently, the application of cross-linking agents such as CA may represent a new strategy for dentine-pulp complex regeneration.

Enhancement of Human Hair Growth Using Ecklonia cava Polyphenols.

BackgroundEcklonia cava is a brown alga that contains various compounds, including carotenoids, fucoidans, and phlorotannins. E. cava polyphenols (ECPs) are known to increase fibroblast survival. The human dermal papilla cell (hDPC) has the properties of mesenchymal-origin fibroblasts.ObjectiveThis study aims to investigate the effect of ECPs on human hair growth promotion in vitro and ex vivo.MethodsMTT assays were conducted to examine the effect of ECPs on hDPC proliferation. Hair growth was measured using ex-vivo hair follicle cultures. Real-time polymerase chain reaction was performed to evaluate the mRNA expression of various growth factors in ECP-treated hDPCs.ResultsTreatment with 10 µg/ml purified polyphenols from E. cava (PPE) enhanced the proliferation of hDPCs 30.3% more than in the negative control (p<0.001). Furthermore, 0.1 µg/ml PPE extended the human hair shaft 30.8% longer than the negative control over 9 days (p<0.05). Insulin-like growth factor-1 (IGF-1) mRNA expression increased 3.2-fold in hDPCs following treatment with 6 µg/ml PPE (p<0.05). Vascular endothelial growth factor (VEGF) mRNA expression was also increased 2.0-fold by 3 µg/ml PPE (p<0.05). Treatment with 10 µg/ml PPE reduced oxidative stress in hDPCs (p<0.05).ConclusionThese results suggest that PPE could enhance human hair growth. This can be explained by hDPC proliferation coupled with increases in growth factors such as IGF-1 and VEGF. Reducing oxidative stress is also thought to help increase hDPCs. These favorable results suggest that PPE is a promising therapeutic candidate for hair loss.

A promising scaffold with excellent cytocompatibility and pro-angiogenesis action for dental tissue engineering: Strontium-doped calcium polyphosphate.

Angiogenesis is of great importance in dental tissue engineering. Therefore, it is quite necessary for dental tissue engineering to develop a new scaffold with bioactivity to induce angiogenesis. This study was performed to determine the ability of 1% strontium-doped calcium polyphosphate (SCPP) to induce angiogenesis by detecting the levels of VEGF and bFGF from cultured human dental pulp cells (hDPCs). Results of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) tests indicated the growth of hDPCs on 1%SCPP scaffolds was more superior than that on calcium polyphosphate (CPP) and hydroxyapatite (HA) scaffolds. The detection of MTT indicated 1%SCPP could accelerate proliferation of hDPCs. Further analyzed with ELISA demonstrated the protein levels of VEGF, bFGF from cultured hDPCs on 1%SCPP scaffolds were significantly higher than that on CPP and HA scaffolds. These findings above suggested 1%SCPP could be a promising scaffold for dental tissue engineering to induce angiogenesis.

Bee Venom Promotes Hair Growth in Association with Inhibiting 5α-Reductase Expression.

Alopecia is an important issue that can occur in people of all ages. Recent studies show that bee venom can be used to treat certain diseases including rheumatoid arthritis, neuralgia, and multiple sclerosis. In this study, we investigated the preventive effect of bee venom on alopecia, which was measured by applying bee venom (0.001, 0.005, 0.01%) or minoxidil (2%) as a positive control to the dorsal skin of female C57BL/6 mice for 19 d. Growth factors responsible for hair growth were analyzed by quantitative real-time PCR and Western blot analysis using mice skins and human dermal papilla cells (hDPCs). Bee venom promoted hair growth and inhibited transition from the anagen to catagen phase. In both anagen phase mice and dexamethasone-induced catagen phase mice, hair growth was increased dose dependently compared with controls. Bee venom inhibited the expression of SRD5A2, which encodes a type II 5α-reductase that plays a major role in the conversion of testosterone into dihydrotestosterone. Moreover, bee venom stimulated proliferation of hDPCs and several growth factors (insulin-like growth factor 1 receptor (IGF-1R), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)2 and 7) in bee venom-treated hDPCs dose dependently compared with the control group. In conclusion, bee venom is a potentially potent 5α-reductase inhibitor and hair growth promoter.

Hair growth-promotion effects of different alternating current parameter settings are mediated by the activation of Wnt/β-catenin and MAPK pathway.

Electrical stimulation is being used in variable skin therapeutic conditions. There have been clinical studies demonstrating the positive effect of electrical stimuli on hair regrowth. However, the underlying exact mechanism and optimal parameter settings are not clarified yet. To investigate the effects of different parameter settings of electrical stimuli on hair growth by examining changes in human dermal papilla cells (hDPCs) in vitro and by observing molecular changes in animal tissue. In vitro, cultured hDPCs were electrically stimulated with different parameter settings at alternating current (AC). Cell proliferation was measured by MTT assay. The Ki67 expression was measured by immunofluorescence. Hair growth-related gene expressions were measured by RT-PCR. In animal model, different parameter settings of AC were applied to the shaved dorsal skin of rabbit for 8 weeks. Expression of hair-related genes in the skin of rabbit was examined by RT-PCR. At low voltage power (3.5 V) and low frequency (1 or 2 MHz) with AC, in vitro proliferation of hDPCs was successfully induced. A significant increase in Wnt/β-catenin, Ki67, p-ERK and p-AKT expressions was observed under the aforementioned settings. In animal model, hair regrowth was observed in the entire stimulated areas under individual conditions. Expression of hair-related genes in the skin significantly increased on the 6th week of treatment. There are optimal conditions for electrical stimulated hair growth, and they might be different in the cells, animals and human tissues. Electrical stimuli induce mechanisms such as the activation of Wnt/β-catenin and MAPK pathway in hair follicles.

Short-term effects of calcium ions on the apoptosis and onset of mineralization of human dental pulp cells in vitro and in vivo.

Calcium ions (Ca2+) are a major constituent of most pulp-capping materials and have an important role in the mineralization of human dental pulp cells (hDPCs). A previous study by our group has shown that increased levels of Ca2+ can promote hDPC-mediated mineralization in long-term cultures (21 days). However, the initiation of mineralization occurs in the early stage of osteogenic inductive culture, and the effects of Ca2+ on the mineralization of hDPCs in short-term cultures (five days) have not been studied in detail. Furthermore, the underlying mechanism by which Ca2+ stimulates the mineralization of hDPCs has remained controversial. A strong correlation between mineralization and cell apoptosis and/or death has been identified. Thus, the present study hypothesized that Ca2+ may promote the onset of hDPC-mediated mineralization through inducing their apoptosis and/or death. To verify this hypothesis, Ca2+ was added to the growth culture medium and osteogenic culture medium at various concentrations. Alizarin Red S staining and reverse transcription-polymerase chain reaction analysis were used to evaluate the onset of mineralization. Furthermore, the cell counting kit-8 and fluorescein isothiocyanate-Annexin V/propidium iodide double-staining method were adopted to detect the proliferation and apoptosis of hDPCs in the growth culture medium. An animal experiment and scanning electron microscopic observation of ceramic graft implants were applied to measure the mineralization in vivo. The results showed that 5.4 and 9.0 mM Ca2+ accelerated the onset of mineralized matrix nodule formation, promoted osteopontin mRNA expression and induced marked cell apoptosis and necrosis, but had no obvious effect on cell proliferation. These findings indicated a positive association between cell apoptosis and/or death and the timing of formation as well as the quantity of extracellular mineralization induced by Ca2+ in short-term cultured hDPCs.

Evaluation of a Bioceramic as a Pulp Capping Agent In Vitro and In Vivo

IntroductionThis study aims to investigate the effects of the bioceramic iRoot BP Plus (Innovative Bioceramix Inc, Vancouver, Canada) as a pulp capping agent in vitro and in vivo. MethodsIn vitro, human dental pulp cells (hDPCs) were seeded into plates with the prepared iRoot BP Plus or mineral trioxide aggregate (MTA) packed in the bottom of different wells. The proliferation of hDPCs was determined using the 3,(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Meanwhile, 2 animal models of direct pulp capping and pulpotomy were applied in Wistar rats in vivo. The exposed pulps were capped with iRoot BP Plus or MTA. After 1 and 4 weeks, maxillary segments were obtained and prepared for histologic analysis. ResultshDPCs grew very well even in the place contacted with MTA or iRoot BP plus in vitro. MTA and iRoot BP Plus both enhanced the proliferation of hDPCs (P < .05). In vivo, results revealed that few inflammatory cells were present in the pulpal area corresponding to the pulp exposure. A slight layer of newly generated matrix was also observed next to MTA and iRoot BP Plus after 1 week. A complete reparative dentin bridge with polarizing odontoblastlike cells was detected in all specimens in the iRoot BP Plus group after 4 weeks. ConclusionsiRoot BP Plus exhibited good biocompatibility to pulp tissue and induced the proliferation of dental pulp cells and the formation of reparative dentin bridge. iRoot BP plus may be used as a pulp capping material for vital pulp therapy