iTRAQ-based proteomics analysis on insomnia rats treated with Mongolian medical warm acupuncture.

OBJECTIVE: To explore the proteomic changes in the hypothalamus of rats treated with Mongolian medical warm acupuncture for insomnia therapy based proteomics. METHOD: We used an iTRAQ-based quantitative proteomic approach to identify proteins that potential molecular mechanisms involved in the treatment of insomnia by Mongolian medical warm acupuncture. RESULT: In total, 7477 proteins were identified, of which 36 proteins showed increased levels and 45 proteins showed decreased levels in insomnia model group (M) compared with healthy control group (C), 72 proteins showed increased levels and 44 proteins showed decreased levels from the warm acupuncture treated insomnia group (W) compared with healthy controls (C), 28 proteins showed increased levels and 17 proteins showed decreased levels from the warm acupuncture-treated insomnia group (W) compared with insomnia model group (M). Compared with healthy control groups, warm acupuncture-treated insomnia group showed obvious recovered. Bioinformatics analysis indicated that up-regulation of neuroactive ligand-receptor interaction and oxytocin signaling was the most significantly elevated regulate process of Mongolian medical warm acupuncture treatment for insomnia. Proteins showed that increased/decreased expression in the warm acupuncture-treated insomnia group included Prolargin (PRELP), NMDA receptor synaptonuclear-signaling and neuronal migration factor (NSMF), Transmembrane protein 41B (TMEM41B) and Microtubule-associated protein 1B (MAP1B) to adjust insomnia. CONCLUSION: A combination of findings in the present study suggest that warm acupuncture treatment is efficacious in improving sleep by regulating the protein expression process in an experimental rat model and may be of potential benefit in treating insomnia patients with the added advantage with no adverse effects.

Effects of Er:YAG and Diode Laser Irradiation on Dental Pulp Cells and Tissues.

Vital pulp therapy (VPT) is to preserve the nerve and maintain healthy dental pulp tissue. Laser irradiation (LI) is beneficial for VPT. Understanding how LI affects dental pulp cells and tissues is necessary to elucidate the mechanism of reparative dentin and dentin regeneration. Here, we show how Er:YAG-LI and diode-LI modulated cell proliferation, apoptosis, gene expression, protease activation, and mineralization induction in dental pulp cells and tissues using cell culture, immunohistochemical, genetic, and protein analysis techniques. Both LIs promoted proliferation in porcine dental pulp-derived cell lines (PPU-7), although the cell growth rate between the LIs was different. In addition to proliferation, both LIs also caused apoptosis; however, the apoptotic index for Er:YAG-LI was higher than that for diode-LI. The mRNA level of odontoblastic gene markers-two dentin sialophosphoprotein splicing variants and matrix metalloprotease (MMP)20 were enhanced by diode-LI, whereas MMP2 was increased by Er:YAG-LI. Both LIs enhanced alkaline phosphatase activity, suggesting that they may help induce PPU-7 differentiation into odontoblast-like cells. In terms of mineralization induction, the LIs were not significantly different, although their cell reactivity was likely different. Both LIs activated four MMPs in porcine dental pulp tissues. We helped elucidate how reparative dentin is formed during laser treatments.

Humanizing Miniature Hearts through 4-Flow Cannulation Perfusion Decellularization and Recellularization.

Despite improvements in pre-clinical drug testing models, predictability of clinical outcomes continues to be inadequate and costly due to poor evidence of drug metabolism. Humanized miniature organs integrating decellularized rodent organs with tissue specific cells are translational models that can provide further physiological understanding and evidence. Here, we evaluated 4-Flow cannulated rat hearts as the fundamental humanized organ model for cardiovascular drug validation. Results show clearance of cellular components in all chambers in 4-Flow hearts with efficient perfusion into both coronary arteries and cardiac veins. Furthermore, material characterization depicts preserved organization and content of important matrix proteins such as collagens, laminin, and elastin. With access to the complete vascular network, different human cell types were delivered to show spatial distribution and integration into the matrix under perfusion for up to three weeks. The feature of 4-Flow cannulation is the preservation of whole heart conformity enabling ventricular pacing via the pulmonary vein as demonstrated by noninvasive monitoring with fluid pressure and ultrasound imaging. Consequently, 4-Flow hearts surmounting organ mimicry challenges with intact complexity in vasculature and mechanical compliance of the whole organ providing an ideal platform for improving pre-clinical drug validation in addition to understanding cardiovascular diseases.

Effects of mineral trioxide aggregate, BiodentineTM and calcium hydroxide on viability, proliferation, migration and differentiation of stem cells from human exfoliated deciduous teeth.

The aim of the study was to evaluate the effects of the capping materials mineral trioxide aggregate (MTA), calcium hydroxide (CH) and BiodentineTM (BD) on stem cells from human exfoliated deciduous teeth (SHED) in vitro. SHED were cultured for 1 - 7 days in medium conditioned by incubation with MTA, BD or CH (1 mg/mL), and tested for viability (MTT assay) and proliferation (SRB assay). Also, the migration of serum-starved SHED towards conditioned media was assayed in companion plates, with 8 µm-pore-sized membranes, for 24 h. Gene expression of dentin matrix protein-1 (DMP-1) was evaluated by reverse-transcription polymerase chain reaction. Regular culture medium with 10% FBS (without conditioning) and culture medium supplemented with 20% FBS were used as controls. MTA, CH and BD conditioned media maintained cell viability and allowed continuous SHED proliferation, with CH conditioned medium causing the highest positive effect on proliferation at the end of the treatment period (compared with BD and MTA) (p<0.05). In contrast, we observed increased SHED migration towards BD and MTA conditioned media (compared with CH) (p<0.05). A greater amount of DMP-1 gene was expressed in MTA group compared with the other groups from day 7 up to day 21. Our results show that the three capping materials are biocompatible, maintain viability and stimulate proliferation, migration and differentiation in a key dental stem cell population.

Effects of mineral trioxide aggregate, BiodentineTM and calcium hydroxide on viability, proliferation, migration and differentiation of stem cells from human exfoliated deciduous teeth

Abstract Objective: The aim of the study was to evaluate the effects of the capping materials mineral trioxide aggregate (MTA), calcium hydroxide (CH) and BiodentineTM (BD) on stem cells from human exfoliated deciduous teeth (SHED) in vitro. Material and Methods: SHED were cultured for 1 - 7 days in medium conditioned by incubation with MTA, BD or CH (1 mg/mL), and tested for viability (MTT assay) and proliferation (SRB assay). Also, the migration of serum-starved SHED towards conditioned media was assayed in companion plates, with 8 μm-pore-sized membranes, for 24 h. Gene expression of dentin matrix protein-1 (DMP-1) was evaluated by reverse-transcription polymerase chain reaction. Regular culture medium with 10% FBS (without conditioning) and culture medium supplemented with 20% FBS were used as controls. Results: MTA, CH and BD conditioned media maintained cell viability and allowed continuous SHED proliferation, with CH conditioned medium causing the highest positive effect on proliferation at the end of the treatment period (compared with BD and MTA) (p<0.05). In contrast, we observed increased SHED migration towards BD and MTA conditioned media (compared with CH) (p<0.05). A greater amount of DMP-1 gene was expressed in MTA group compared with the other groups from day 7 up to day 21. Conclusion: Our results show that the three capping materials are biocompatible, maintain viability and stimulate proliferation, migration and differentiation in a key dental stem cell population.

Expressão de DMP-1, DSPP, VEGF e FGF2 em células derivadas da polpa de dentes decíduos após o uso do laser de baixa intensidade / FGF2, VEGF, DSPP, and DMP-1 expression in cells of the pulp deciduous teeth with a low intensity laser

O objetivo do presente trabalho foi avaliar o efeito de diferentes densidades de energia do Laser de Baixa Intensidade na viabilidade e proliferação celular de fibroblastos derivados da polpa de dentes decíduos humanos e na expressão de RNAm para DMP- 1, DSPP, VEGF e FGF-2. Amostras de fibroblastos pulpares da polpa de dentes decíduos humanos foram obtidas de um Biorrepositório. Foram utilizadas células entre a 4ª e a 7ª passagem, irradiadas com Laser de Baixa Intensidade (InGaAlP) de acordo com os seguintes grupos experimentais: Grupo 1: 1,2 J/cm2 - 05 mW - 10s; Grupo 2: 2,5 J/cm2 - 05 mW - 20s; Grupo 3: 3,7 J/cm2 - 05 mW - 30s; Grupo 4: 5,0 J/cm2 - 05 mW - 40s; Grupo 5: 6,2 J/cm2 - 05 mW - 50s; Grupo 6: 2,5 J/cm2 - 10 mW - 10s; Grupo 7: 3,7 J/cm2 - 15 mW - 10s; Grupo 8: 5,0 J/cm2 - 20 mW - 10s; Grupo 9: 6,2 J/cm2 - 25 mW - 10s; Controle Negativo: DMEM 1% SFB ­ não irradiado; Controle Positivo: DMEM 10% SFB ­ não irradiado. As técnicas utilizadas para as análises de viabilidade e proliferação celular foram MTT e CV. A técnica utilizada para avaliação da expressão de RNAm para os alvos DMP-1, DSPP, VEGF e FGF-2 foi RT-PCR. Os resultados foram analisados pelo método ANOVA a dois critérios, seguido pelo teste de Tukey (p<0,05). Para o teste MTT, na comparação intragrupos observou-se que houve diferença estatisticamente significativa entre os períodos 6h, 12h e 24h, diminuindo a viabilidade com o passar do tempo, exceto para o Grupo 1. Na comparação intergrupos, o MTT mostrou menor viabilidade para o controle negativo em comparação com os outros grupos (p<0,05), exceto com grupo 5 (5mW/50 seg). Observou-se que os grupos com maiores potências (10mW, 15mW, 20mW e 25mW), menores tempos de aplicação (10 segundos) e densidades de energia entre 2,5 J/cm2 e 6,2 J/cm2, apresentaram estatisticamente maior viabilidade que o grupo com menor potência (5mW), maior tempo de aplicação (50 segundos) e densidade de energia de 6,2 J/cm2. Para o teste CV não houve diferença intragrupos, mas houve diferença intergrupos entre os controles positivo e negativo. Para a expressão de RNAm por RTPCR, os fatores de crescimento VEGF e FGF-2 foram expressos em grande quantidade no primeiro período experimental, enquanto que DMP-1 e DSPP não foram expressos de maneira significativa. De acordo com os resultados obtidos, frente as diferentes densidades de energia, sugere-se que a terapia a laser de baixa intensidade manteve os fibroblastos viáveis e aumentou a expressão de RNAm para VEGF e FGF-2.(AU)

This study aimed to evaluate the effect of different energy densities of Low Level Laser (LLL) on cell viability and proliferation of fibroblasts from the pulp of human primary teeth (DHPF) and on the RNAm expression of DMP-1, DSPP, VEGF and FGF-2. DHPF were obtained from a biorepository and used at passages 4th to 7th. The cells were irradiated with LLL (InGaAlP) according to the following experimental groups: Group 1: 1.2 J/cm2 - 05 mW - 10s; Group 2: 2.5 J/cm2 - 05 mW - 20s; Group 3: 3.7 J/cm2 - 05 mW - 30s; Group 4: 5.0 J/cm2 - 05 mW - 40s; Group 5: 6.2 J/cm2 - 05 mW - 50s; Group 6: 2.5 J/cm2 - 10 mW - 10s; Group 7: 3,7 J/cm2 - 15 mW - 10s; Group 8: 5.0 J/cm2 - 20 mW - 10s; Group 9: 6.2 J/cm2 - 25 mW - 10s; Negative Control: DMEM 1% SFB ­ not irradiated; Positive Control: DMEM 10% SFB ­ not irradiated. The techniques used to evaluate the cell viability/proliferation were MTT and Crystal Violet (CV) assays. RT-PCR was used to verify the RNAm expression of DMP-1, DSPP, VEGF, and FGF-2. Two-way ANOVA, followed by Tukey test (p<0.05) was used to analyze the results. In the intragroup comparison, MTT assay revealed statistically significant differences among the periods of 6h, 12h, and 24h, with viability reduction as time went by, except for Group 1. In the intergroup comparison, the MTT assay showed that the negative control had statistically lower viability than that of the other groups (p<0.05), except for Group 5 (5mW/50 s). The groups with higher powers (10mW, 15mW, 20mW, and 25mW), shortest application periods (10 s), and energy densities between 2.5 J/cm2 and 6.2 J/cm2 exhibited statistically higher viability than that of the group with small power (5mW), longer application period (50 s), and energy density of 6.2 J/cm2 . CV assay did not show intergroup statistically differences. In the intragroup comparison, CV assay revealed statistically significant differences between positive and negative controls (p<0.05). RT-PCR revealed increased RNAm expression of the growth factors VEGF and FGF-2 at the first experimental period, while DMP-1 and DSPP was not significant. Based on the results and different energy densities used, LLL maintained DHPF viability and increased the RNAm expression of VEGF and FGF-2.(AU)

Identification of extracellularly phosphorylated membrane proteins.

Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling.

Phototherapy up-regulates dentin matrix proteins expression and synthesis by stem cells from human-exfoliated deciduous teeth.

OBJECTIVES: The aim of this study was to evaluate the effects of infrared LED (850nm) irradiation on dentin matrix proteins expression and synthesis by cultured stem cells from human exfoliated deciduous teeth (SHED). METHODS: Near-exfoliation primary teeth were extracted (n=3), and SHED cultures were characterized by immunofluorescence using STRO-1, CD44, CD146, Nanog and OCT3/4 antibodies, before experimental protocol. The SHEDs were seeded (3×10(4) cells/cm(2)) with DMEM containing 10% FBS. After 24-h incubation, the culture medium was replaced by osteogenic differentiation medium, and the cells were irradiated with LED light at energy densities (EDs) of 0 (control), 2, or 4J/cm(2) (n=8). The irradiated SHEDs were then evaluated for alkaline phosphatase (ALP) activity, total protein (TP) production, and collagen synthesis (SIRCOL™ Assay), as well as ALP, collagen type I (Col I), dentin sialophosphoprotein (DSPP), and dentin matrix acidic phosphoprotein (DMP-1) gene expression (qPCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (α=0.05). RESULTS: Increased ALP activity and collagen synthesis, as well as gene expression of DSPP and ALP, were observed for both EDs compared with non-irradiated cells. The ED of 4J/cm(2) also increased gene expression of COL I and DMP-1. CONCLUSIONS: In conclusion, infrared LED irradiation was capable of biostimulating SHEDs by increasing the expression and synthesis of proteins related with mineralized tissue formation, with overall better results for the energy dose of 4J/cm(2). CLINICAL SIGNIFICANCE: Phototherapy is an additional approach for the clinical application of LED in Restorative Dentistry. Infrared LED irradiation of the cavity's floor could biostimulate subjacent pulp cells, improving local tissue healing.

Investigation of low-level laser therapy potentiality on proliferation and differentiation of human osteoblast-like cells in the absence/presence of osteogenic factors.

Several studies have shown that low-level laser irradiation (LLLI) has beneficial effects on bone regeneration. The objective of this study was to examine the in vitro effects of LLLI on proliferation and differentiation of a human osteoblast-like cell line (Saos-2 cell line). Cultured cells were exposed to different doses of LLLI with a semiconductor diode laser (659 nm; 10 mW power output). The effects of laser on proliferation were assessed daily up to seven days of culture in cells irradiated once or for three consecutive days with laser doses of 1 or 3 J/cm(2). The obtained results showed that laser stimulation enhances the proliferation potential of Saos-2 cells without changing their telomerase pattern or morphological characteristics. The effects on cell differentiation were assessed after three consecutive laser irradiation treatments in the presence or absence of osteo-inductive factors on day 14. Enhanced secretion of proteins specific for differentiation toward bone as well as calcium deposition and alkaline phosphatase activity were observed in irradiated cells cultured in a medium not supplemented with osteogenic factors. Taken together these findings indicate that laser treatment enhances the in vitro proliferation of Saos-2 cells, and also influences their osteogenic maturation, which suggest it is a helpful application for bone tissue regeneration.

Effect of nifedipine on the differentiation of human dental pulp cells cultured with mineral trioxide aggregate.

INTRODUCTION: Mineral trioxide aggregate (MTA) can induce differentiation of the dental pulp cells into odontoblast-like cells and generate a dentin-like mineral structure. The mechanisms underlying MTA-induced odontoblastic differentiation in human dental pulp cells (HDPCs) are not completely understood. The purpose of this study was to evaluate the effect of nifedipine as calcium channel blocker on MTA-induced odontoblastic differentiation in HDPCs. METHODS: HDPCs extracted from maxillary supernumerary incisors and third molars were directly cultured on MTA with or without nifedipine in the culture medium. Cell growth and expression of odontoblastic differentiation markers were determined by using methyl-thiazol-diphenyl-tetrazolium assay and reverse transcription-polymerase chain reaction analysis, respectively. Phosphorylation of mitogen-activated protein kinase was measured by Western blotting, and calcium deposition was assessed by using alizarin red S staining. RESULTS: MTA at a concentration of 1 mg/mL significantly up-regulated the expression of dentin sialophosphoprotein and dentin matrix protein-1 and enhanced mineralized nodule formation. However, nifedipine attenuated the MTA-induced odontoblastic differentiation in HDPCs. In addition, MTA-induced mineralization was blocked by inhibition of extracellular signal-regulated kinase (ERK), p38, and Jun N-terminal kinase (JNK) by using U0126, SB203580, and SP600125, respectively. Furthermore, phosphorylation of ERK and JNK in response to MTA was inhibited when the medium was supplemented with nifedipine. CONCLUSIONS: This study showed that calcium ions released from MTA play an important role in odontoblastic differentiation of HDPCs via modulation of ERK and JNK activation.

A high-throughput cell-based Gaussia luciferase reporter assay for identifying modulators of fibulin-3 secretion.

An R345W mutation in fibulin-3 causes its inefficient secretion, increased intracellular steady-state levels, and the macular dystrophy, Malattia Leventinese (ML), a disease similar to age-related macular degeneration. It is unknown whether R345W causes ML through increased intracellular levels, by the secretion of a potentially aggregation-prone protein, or both. To identify small molecules that alter the secretion of fibulin-3, we developed ARPE19 retinal cell lines that inducibly express wild-type (WT) or R345W fibulin-3 fused to an enhanced Gaussia luciferase (eGLuc2). Screening of the Library of Pharmacologically Active Compounds demonstrated that these cell lines and the GLuc assay are suitable for high-throughput chemical screening. Two estrogen-related compounds enhanced fibulin-3 secretion, whereas a diverse series of small molecules reduced fibulin-3 secretion. A counterscreen identified compounds that did not substantially alter the secretion of unfused eGLuc2, demonstrating at least partial selectivity for fibulin-3. A secondary assay using untagged fibulin-3 confirmed that the top three inhibitory compounds reduced R345W fibulin-3 secretion. Interestingly, in untagged fibulin-3 studies, one compound, phorbol 12-myristate 13-acetate, reduced R345W fibulin-3 secretion while minimally enhancing WT fibulin-3 secretion, the desired activity and selectivity we sought for ML. The identified compounds could serve as tools for probing the etiology of fibulin-3-related diseases.

In vitro evaluation of the odontogenic potential of mouse undifferentiated pulp cells.

The aim of this study was to evaluate the odontogenic potential of undifferentiated pulp cells (OD-21 cell line) through chemical stimuli in vitro. Cells were divided into uninduced cells (OD-21), induced cells (OD-21 cultured in supplemented medium/OD-21+OM) and odontoblast-like cells (MDPC-23 cell line). After 3, 7, 10 and 14 days of culture, it was evaluated: proliferation and cell viability, alkaline phosphatase activity, total protein content, mineralization, immunolocalization of dentin matrix acidic phosphoprotein 1 (DMP1), alkaline phosphatase (ALP) and osteopontin (OPN) and quantification of genes ALP, OSTERIX (Osx), DMP1 and runt-related transcription factor 2 (RUNX2) through real-time polymerase chain reaction (PCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests (p<0.05). There was a decrease in cell proliferation in OD-21 + OM, whereas cell viability was similar in all groups, except at 7 days. The amount of total protein was higher in group OD-21 + OM in all periods; the same occurred with ALP activity after 10 days when compared with OD-21, with no significant differences from the MDPC-23 group. Mineralization was higher in OD-21+OM when compared with the negative control. Immunolocalization demonstrated that DMP1 and ALP were highly expressed in MDPC-23 cells and OD-21 + OM cells, whereas OPN was high in all groups. Real-time PCR revealed that DMP1 and ALP expression was higher in MDPC-23 cell cultures, whereas RUNX2 was lower for these cells and higher for OD-21 negative control. Osx expression was lower for OD-21 + OM. These results suggest that OD-21 undifferentiated pulp cells have odontogenic potential and could be used in dental tissue engineering.

In vitro evaluation of the odontogenic potential of mouse undifferentiated pulp cells

The aim of this study was to evaluate the odontogenic potential of undifferentiated pulp cells (OD-21 cell line) through chemical stimuli in vitro. Cells were divided into uninduced cells (OD-21), induced cells (OD-21 cultured in supplemented medium/OD-21+OM) and odontoblast-like cells (MDPC-23 cell line). After 3, 7, 10 and 14 days of culture, it was evaluated: proliferation and cell viability, alkaline phosphatase activity, total protein content, mineralization, immunolocalization of dentin matrix acidic phosphoprotein 1 (DMP1), alkaline phosphatase (ALP) and osteopontin (OPN) and quantification of genes ALP, OSTERIX (Osx), DMP1 and runt-related transcription factor 2 (RUNX2) through real-time polymerase chain reaction (PCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests (p<0.05). There was a decrease in cell proliferation in OD-21 + OM, whereas cell viability was similar in all groups, except at 7 days. The amount of total protein was higher in group OD-21 + OM in all periods; the same occurred with ALP activity after 10 days when compared with OD-21, with no significant differences from the MDPC-23 group. Mineralization was higher in OD-21+OM when compared with the negative control. Immunolocalization demonstrated that DMP1 and ALP were highly expressed in MDPC-23 cells and OD-21 + OM cells, whereas OPN was high in all groups. Real-time PCR revealed that DMP1 and ALP expression was higher in MDPC-23 cell cultures, whereas RUNX2 was lower for these cells and higher for OD-21 negative control. Osx expression was lower for OD-21 + OM. These results suggest that OD-21 undifferentiated pulp cells have odontogenic potential and could be used in dental tissue engineering.

O objetivo foi avaliar o potencial odontogênico de células indiferenciadas da polpa (OD-21) por meio de indução química in vitro. As células foram divididas em grupos: controle (OD-21), induzido (OD-21 em meio suplementado/OD-21 + OM), e células odontoblastóides (MDPC-23). Após 3, 7, 10 e 14 dias, avaliou-se proliferação e viabilidade celular, proteína total e fosfatase alcalina (ALP), mineralização, imunolocalização da proteína da matriz dentinária 1 (DMP1), ALP e osteopontina (OPN), assim como a expressão dos genes ALP, OSTERIX (Osx), DMP1 e fator de transcrição RUNX2 por PCR em tempo real. Os dados foram avaliados pelo teste de Kruskal-Wallis seguido pelo teste de Mann-Whitney U (p<0.05). Houve diminuição na proliferação celular em OD-21 + OM, com viabilidade celular similar em todos os grupos, exceto aos sete dias. O conteúdo de proteína total foi maior no grupo OD-21 + OM em todos os períodos; o mesmo ocorreu com a atividade de ALP quando comparada com o grupo OD-21, além de apresentar resultados similares ao grupo MDPC-23. A mineralização foi maior em OD-21 + OM quando comparada com o controle negativo. A imunolocalização demonstrou expressão de DMP1 e ALP em MDPC-23 e OD-21 + OM, enquanto que todos os grupos foram positivos para OPN. A expressão gênica de DMP1 e ALP foi maior nas culturas de MDPC-23, enquanto que a de RUNX2 foi menor para estas células e maior no controle negativo. A expressão de OSTERIX foi menor em OD-21 + OM quando comparada aos outros grupos. Sugere-se que as células indiferenciadas da polpa da linhagem OD-21 apresentam potencial odontogênico e poderiam ser usadas para a engenharia tecidual.

Expression of Notch signalling-related genes in normal and differentiating rat dental pulp cells.

Notch signalling is of fundamental importance to various processes during embryonic development and in adults. The possible role of Hey1, an important Notch signalling component, in odontoblast differentiation was evaluated in this study. Primary cultured dental pulp cells, derived from upper incisors of 5-week-old Wistar rats, were placed in alpha-modification of Eagle's minimal essential medium supplemented with 10% Fetal Bovine Serum (FBS), and ascorbic acid (AA) and beta-glycerophosphate (beta-GP), with or without dexamethasone, and cultured on dishes coated with collagen type IA for 7 days. Conventional and real-time Polymerase Chain Reaction (PCR) was performed to determine the expression of Notch-related genes and dentin sialophosphoprotein as a marker of odontoblast differentiation. Dentin sialophosphoprotein and Hey1 expression was significantly increased and decreased in the presence of AA + beta-GP compared with controls, respectively. These findings suggest that Hey1 may be a negative regulator in odontoblast differentiation.

Wnt5a promotes differentiation of human dental papilla cells.

AIM: To investigate the role of Wnt5a in the process of differentiation of human dental papilla cells (HDPCs). METHODOLOGY: Recombinant adenovirus encoding full-length Wnt5a cDNA was constructed to investigate the biological role of Wnt5a on the differentiation of HDPCs. The effect of Wnt5a on HDPCs differentiation was determined by ALP activity assay, ALP staining and mineral induction assay. Mineralization-related gene expressions were assessed by RT-PCR. RESULTS: Immunostaining revealed Wnt5a expression in the odontoblast layer and dental papilla tissue. Over-expression of Wnt5a by transfecting HDPCs with an Wnt5a-carrying construct increased ALPase activity and the formation of mineralized nodules of HDPCs. RT-PCR analysis showed that the expressions of mineralization-related genes, such as bone sialoprotein, collagen type I, osteonectin, osteopontin (OCN), dentine matrix protein-1 were up-regulated by Wnt5a. CONCLUSIONS: Wnt5a promoted differentiation of HDPCs.

Hydrogen gas reduced acute hyperglycemia-enhanced hemorrhagic transformation in a focal ischemia rat model.

Hyperglycemia is one of the major factors for hemorrhagic transformation after ischemic stroke. In this study, we tested the effect of hydrogen gas on hemorrhagic transformation in a rat focal cerebral ischemia model. Sprague-Dawley rats (n=72) were divided into the following groups: sham; sham treated with hydrogen gas (H(2)); Middle Cerebral Artery Occlusion (MCAO); and MCAO treated with H(2) (MCAO+H(2)). All rats received an injection of 50% dextrose (6 ml/kg i.p.) and underwent MCAO 15 min later. Following a 90 min ischemic period, hydrogen was inhaled for 2 h during reperfusion. We measured the level of blood glucose at 0 h, 0.5 h, 4 h, and 6 h after dextrose injection. Infarct and hemorrhagic volumes, neurologic score, oxidative stress (evaluated by measuring the level of 8 Hydroxyguanosine (8OHG), 4-Hydroxy-2-Nonenal (HNE) and nitrotyrosine), and matrix metalloproteinase (MMP)-2/MMP-9 activity were measured at 24 h after ischemia. We found that hydrogen inhalation for 2 h reduced infarct and hemorrhagic volumes and improved neurological functions. This effect of hydrogen was accompanied by a reduction of the expression of 8OHG, HNE, and nitrotyrosine and the activity of MMP-9. Furthermore, a reduction of the blood glucose level from 500+/-32.51 to 366+/-68.22 mg/dl at 4 h after dextrose injection was observed in hydrogen treated animals. However, the treatment had no significant effect on the expression of ZO-1, occludin, collagen IV or aquaporin4 (AQP4). In conclusion, hydrogen gas reduced brain infarction, hemorrhagic transformation, and improved neurological function in rats. The potential mechanisms of decreased oxidative stress and glucose levels after hydrogen treatment warrant further investigation.

Nerve growth factor promotes differentiation of odontoblast-like cells.

Contemporary strategies in tooth repair markedly rely on the newest findings on the cellular and biological components of dental development. Among several identified bioactive molecules, neurotrophins were recently proposed to affect tooth germ cell proliferation, differentiation, and cell-extracellular matrix interactions. The present study attempted to explore the effect of nerve growth factor (NGF) on a spontaneously immortalized dental papilla mesenchymal cell line. NGF induced differentiation of odontoblast-lineage cells with subsequent biomineralization in vitro. Here we showed that normalized transcript levels of tissue-specific markers such as DSPP and DMP1 were elevated significantly, indicating cell differentiation and maturation processes. We performed innovative gene expression analysis of TM14, a matricellular protein and novel member of the fibulin family. TM14 expression followed a pattern similar to that of DMP1, which suggests its important role in cell-matrix and intercellular interactions during dentin calcification. Alkaline phosphatase enzyme assay confirmed the extracellular matrix calcifications in NGF-supplemented groups. Thus, NGF was characterized as a potent promoter of mineralization during dentin formation. For the first time, we included TM14 in odontoblast genotype analysis and proved that NGF also promotes in vitro odontoblast differentiation. Collectively, these results highlight the importance of NGF during tooth morphogenesis, as well as urge the elaboration of complex epithelial-mesenchymal tissue cultures, where further elucidation of the signaling factor network could be completed.

Increased truncated TrkB receptor expression and decreased BDNF/TrkB signaling in the frontal cortex of reeler mouse model of schizophrenia.

Heterozygous reeler mouse has been used as an animal model for schizophrenia based on several neuropathological and behavioral abnormalities homologous to schizophrenia. Since some of these abnormalities are primarily associated with altered BDNF signaling we investigated BDNF signaling in the frontal cortex of reeler mice in order to shed some light on the neuropathology and treatment of schizophrenia. BDNF, TrkB receptor isoforms (full-length and truncated), reelin, GAD67, GAD65, p75NTR, and NRH-2 levels were measured in the frontal cortex samples from reeler (B6C3Fe a/a-Reln rl/+) and wild-type (WT) mice. BDNF protein levels were significantly higher in reeler compared to WT. The protein levels of full-length TrkB were not altered in reeler mice, but both mRNA and protein levels of truncated TrkB were significantly higher. Protein analysis showed that TrkB activity, as indicated by the levels of tyrosine-phosphorylated TrkB, was lower in reeler mice. We did not find any significant change in the levels of p75NTR and NRH-2, regulatory proteins of TrkB signaling, in the reeler mice. Furthermore, we found significant reduction in reelin and GAD67 expressions, but not GAD65 expression in reeler compared to WT mice. In summary, molecular processes associated with defective BDNF signaling in reeler mice provide new therapeutic targets for neuroprotective pharmacotherapy for schizophrenia.

A Comparison of 3 tumor markers (MIA, TA90IC, S100B) in stage III melanoma patients.

PURPOSE: There is no consensus regarding the optimal tumor markers for melanoma. We compared 3 tumor markers, TA90-immune complex (TA90IC), melanoma-inhibiting activity (MIA) protein, and S100B protein in Stage III melanoma patients undergoing adjuvant vaccine immunotherapy. EXPERIMENTAL DESIGN: The serum of 75 patients representing 3 prognostic cohorts was assayed for the tumor markers prior to initiating immunotherapy and at 6 follow-up time points. Upper limits of normal for TA90IC, MIA and S100B were set at OD 0.41, 8.5 ng/ml, and 2.5 microg/l, respectively. RESULTS: At least 1 marker became elevated prior to 41 (80 percent) of 51 recurrences. TA90IC was the earliest elevated marker in 29 (57 percent), MIA in 11 (22 percent), and S100B in 4 (8 percent). Multivariate regression analysis revealed that TA90IC was an independent predictor of survival when elevation occurred between 2 weeks and 3 months, whereas MIA was an independent predictor at 4-6 months. In the poor prognostic cohort, mean values for MIA and S100B increased progressively, whereas TA90IC exhibited a parabolic curve. CONCLUSION: In this patient population, TA90IC and MIA were complementary; elevation of the immune complex preceded elevation of the tumor antigen in patients who developed recurrence. Additional studies in populations not receiving vaccine will further clarify the clinical utility of these assays.

Immunohistochemical and electron microscopic study of the meridian-like system on the surface of internal organs of rats.

Threadlike structures on the surfaces of mammalian internal organs were recently observed, and they can be the anatomical basis of the meridian-like network of internal organs as detected by the Bi-Digital O-Ring test imaging method. Morphological and histological studies were performed to reveal their novel features by using confocal and electron microscopy, and there is evidence that reveals that they have a ductal structure in which some liquid flows. They form a part of the network of acupuncture meridians that is thought to be a circulatory system distributed throughout an animal body. In order to confirm further the circulatory function it was necessary to provide evidences for the existence of endothelial cells at the inner boundaries of the microchannels that form the threadlike ducts. In this work we performed immunohistochemical studies for the identification of endothelial cells. In addition, we identified the extracellular matrices between the microchannels in the threadlike structure by using antifibronectin, elastin, laminin, and collagen type IV. Since immunohistochemical data are not sufficient for definite proof we supplemented the analysis with ultra structural information by using transmission electron microscopy (TEM). Yet TEM images show only ultrathin sections (50 nm) of a specimen, we needed to take 3-dimentional images to provide a visual demonstration of the microchannels in the threadlike ducts. We conducted high voltage electron microscopic studies for this purpose and obtained corroborating tomograms that exhibit the microchannels. In conclusion, the meridian-like tissues are ducts with microchannels, which implies the circulatory function of the acupuncture meridian network.