Combined effects of bone morphogenetic protein-7 and mineral trioxide aggregate on the proliferation, migration, and differentiation of human dental pulp stem cells

Bioactive molecules present the potential to be used along with biomaterials in vital pulp therapy and regenerative endodontic treatment. Objective: The aim of this study was to assess the effects of the combined use of bone morphogenetic protein-7 (BMP-7) and mineral trioxide aggregate (MTA) on the proliferation, migration, and differentiation of human dental pulp stem cells (DPSCs). Methodology: For the proliferation analysis, DPSCs were incubated with a growth medium and treated with MTA and/or BMP-7 at different concentrations. For the following analyses, DPSCs were incubated with a differentiation medium and treated with MTA and/or BMP-7. Moreover, there were groups in which DPSCs were incubated with the growth medium (control), the differentiation medium, or DMEM/F12 containing fetal bovine serum, and not treated with MTA or BMP-7. Cell proliferation was analyzed using the WST-1 assay. The odontogenic/osteogenic differentiation was evaluated by immunocytochemistry, alkaline phosphatase (ALP) activity assay, alizarin red staining, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell migration was evaluated using a wound-healing assay. Data were analyzed using analysis of variance and Tukey test (p=0.05). Results: The use of BMP-7 with MTA presented no significant effect on cell proliferation in comparison with the treatment with MTA alone (p>0.05), but showed higher ALP activity, increased mineralization, and higher expression of DMP1 and DSPP when compared with other groups (p<0.05). Nestin expression was higher in the control group than in groups treated with MTA and/or BMP-7 (p<0.05). The cell migration rate increased after treatment with MTA when compared with other groups in all periods of time (p<0.05). At 72 hours, the wound area was smaller in groups treated with MTA and/or BMP-7 than in the control group (p<0.05). Conclusion: The use of BMP-7 with MTA increased odontogenic/osteogenic differentiation without adversely affecting proliferation and migration of DPSCs. The use of BMP-7 with MTA may improve treatment outcomes by increasing repair and regeneration capacity of DPSCs

Over Kanserlerinde Bmp-7 ve Bmp-2 Nin İnvazyon ve Metastazdaki Etkisi

Ovarian cancer is clinically important, because they are diagnosed late and have metastasis when they are diagnosed. Epithelial-mesenchymal transition (EMT) plays role in the pathogenesis of ovarian cancer. Bone morphogenetic proteins (BMP), transcription factors and integrins are effective in the EMT process. Our hypothesis in this study is that there is an inverse correlation between BMP 2 and BMP 7 immunoreactivity in ovarian cancers. The immunoreactivities of BMP-2, BMP-7, ZEB2, ITG α5 and ITG β1 were determined by indirect immunoperoxidase in high grade serous ovarian cancer with and without lymph node metastasis and both indirect immunoperoxidase and immunofluorescence method was used for determing the immunreactivity of these molecules in SKOV-3 cell line. Data was evaluated by H-score analysis and the difference between the groups was statistically determined. The immunoreactivity of BMP-2 and ZEB2 was found significantly stronger in both ovary and lymph node samples with lymph node metastasis (p <0.000, p <0.000). BMP-7 immunereactivity was observed stronger in ovarian tissues without lymph node metastasis. The immunoreactivity of ITG α5 immunoreactivity was stronger in ovarian tissues without lymph node metastasis (p <0.001). The tumor cells showed stronger ITG β1 immunoreactivity in lymph node samples with lymph node metastasis and ovary samples without lymph node metastasis (p <0.001). In vivo and in vitro results of our study showing strong positive immunreativity of BMP-2 and weak immunoreactivity of BMP-7 in tumor cells confirmed our hypothesis and showed BMP-2 and BMP-7 has inverse correlation. Changes in the expression of BMPs affect progression in ovarian cancer. Increase in the level of BMP-7 expression may play a role in protective mechanisms from ovarian cancers.Çalışmamız Hacettepe Üniversitesi Bilimsel Araştırmalar Birimi (TSA-2017-13046) tarafından desteklenmiştir.ONAY SAYFASI iii YAYIMLAMA VE FİKRİ MÜLKİYET HAKLARI BEYANI iv ETİK BEYAN v TEŞEKKÜR vi ÖZET vii ABSTRACT Vİİİ İÇİNDEKİLER ix SİMGELER ve KISALTMALAR xii ŞEKİLLER xiv TABLOLAR xvii 1. GİRİŞ 1 2. GENEL BİLGİLER 3 2.1 Over kanserleri 3 2.2 Epitel-mezenkimal geçiş (EMT) 4 2.3 Kemik morfojen protein (BMP) 6 2.3.1 BMP’ler ve kanser 8 2.4 Çinko parmak E-box bağlayıcı homeobox 2 (ZEB2) 9 2.5 Metastaz 10 2.6 İntegrinler 12 3. GEREÇ ve YÖNTEM 16 3.1 Gereç 16 3.1.1 Hasta gruplarının belirlenmesi 16 3.2. Histokimya ve İmmunohistokimya Yöntemi 16 3.2.1 Doku Kesitlerinin Alınması 16 3.2.2. Kullanılan Malzemelerin Hazırlanması 16 3.2.3. Histokimya Yönteminin Uygulanması 21 3.2.4 İmmunohistokimya Yönteminin Uygulanması 22 3.3 Hücre Kültürü 25 3.3.1 Over kanseri hücre hattı kültürü 25 3.3.2. SKOV-3 Over Kanseri Hücrelerinde İmmunositokimya Yönteminin Uygulanması 25 3.4 Kesitlerin Görüntülenmesi 29 3.5 İmmunohistokimya ve İmmunositokimya İşaretlenmesinin Değerlendirilmesi 29 3.6 İstatistiksel analiz 29 4. BULGULAR 31 4.1 Hasta verilerinin değerlendirilmesi 31 4.1.1 Hastaların demografik özellikleri 31 4.2 Seröz over karsınom vakalarında over dokusu ve lenf nodunun histopatalojik değerlendirilmesi 31 4.3 Hasta Gruplarında Tümör Hücreleri ve Tümör Stromasında İmmunohistokimyasal İşaretlenmelerinin Değerlendirilmesi 34 4.3.1 BMP-2 ve BMP-7 immunreaktivitesi 34 4.3.2 ZEB2 immunreaktivitesi 35 4.3.3 ITG α5 Ve ITG β1 İmmunreaktivitesi 36 4.4 BMP-2, BMP-7, ZEB2, ITG α5 ve ITG β1 immunreaktivitelerinin gruplar arası karşılaştırılması 36 4.4.1 BMP-2 immunreaktivitesinin gruplar arası karşılaştırılması 37 4.4.2 BMP-7 immunreaktivitesinin gruplar arası karşılaştırılması 39 4.4.3 ZEB2 immunreaktivitesinin gruplar arası karşılaştırılması 41 4.4.4 ITG α5 İmmunreaktivitesinin Gruplar Arası Karşılaştırılması 43 4.4.5 ITG β1 İmmunreaktivitesinin Gruplar Arası Karşılaştırılması 45 4.5 BMP-2 Ve BMP-7 İmmunreaktivitelerinin Karşılaştırılması 47 4.6 SKOV-3 Hücre Hattı Kültürü 48 4.7 SKOV-3 Over Adenokarsinoma Hücre Hattına ait Hücrelerde İmmünperoksidaz ve İmmünfloresan İşaretlemelerin Değerlendirilmesi 49 4.8 SKOV-3 over adenokarsinoma hücre hattı hücrelerinde ITG α5, ITG beta1’in çift immünofloresan işaretlemesi 55 4.9 SKOV-3 Over Adenokarsinoma Hücre Hattına Ait Hücrelerde İmmünperoksidaz ve İmmünfloresan İşaretlemelerin H-skor ve İstatistiksel Analiz Değerlendirilmesi 56 5. TARTIŞMA 58 6. SONUÇLAR 63 7. KAYNAKLAR 66 8. EKLER EK-1. Tez Çalışması İle İlgili Etik Kurul İzni 9. ÖZGEÇMİŞOver kanserleri geç dönem tanı koyulmaları ve tanı aldıklarında metastaz yapmış olmaları nedeniyle klinikte önem taşımaktadır. Epitel-mezenkim geçişi (EMT) over kanserlerinin patogenezinde rol oynamaktadır. Kemik morfojen proteinleri (BMP), transkripsiyon faktörleri ve integrinler EMT sürecinde etkilidir. Bu çalışmada hipotezimiz, over kanserlerinde BMP-2 ve BMP-7 immünreaktiviteleri arasında ters korelasyon olduğudur. Çalışmamızda, lenf nodu tutulumu olan ve olmayan yüksek dereceli seröz over kanserinde BMP-2, BMP-7, ZEB2, ITG α5, ITG β1 immünreaktiviteleri indirekt immünperoksidaz ile, SKOV-3 hücre hattında indirekt immünperoksidaz ve immünofloresan yöntemi ile işaretlendikten sonra H-skor analiz ile değerlendirildi. Gruplar arası fark istatistiksel olarak belirlendi. BMP-2 ve ZEB2 immünreaktivitesi lenf nodu tutulumu olan hem over hem lenf nodu örneklerinde anlamlı olarak daha güçlü bulundu (p<0,000, p<0,000). BMP-7 ile lenf nodu tutulumu olmayan over dokusu örneklerinde daha güçlü immünreaktivite gözlendi. ITG α5 immünreaktivitesi lenf nodu tutulumu olmayan over dokusu örneklerinde daha güçlü saptandı (p<0,001). ITG β1 immunreaktivitesi lenf nodu tutulumu olmayan over dokusu ve lenf nodu tutulumu olan lenf nodu örneklerinde anlamlı olarak daha güçlü bulundu (p<0,001). Çalışmamızın in vivo ve in vitro sonuçları tümör hücrelerinde BMP-2 ile güçlü immunreaktivite, BMP-7 ile zayıf immünreaktivite gözlenmesi hipotezimizi doğrulamaktadır. Over kanserinde BMP-2 ve BMP-7 ifadeleri ters korelasyon göstermektedir. BMP’lerdeki değişiklikler over kanserinde progresyonu etkilemektedir. BMP-7 artışı koruyucu mekanizmalarda rol oynayabilir

Prediction of adolescent happiness based on spiritual lifestyle with the mediation of mental health

Background and Aim: Happiness is one of the constructs proposed in positive psychology, which is related to many concepts in psychology. The aim of the present study was to predict the happiness of teenagers based on spiritual lifestyle with the mediation of mental health.  Methods:The research design was a correlational type in which happiness was the dependent variable, spirituality was the independent variable, and mental health was the mediating variable. The statistical population of students were studying in high schools in Tehran, and 300 people were selected by cluster sampling. The tools included Hall and Edwards' spirituality questionnaire (1996), Schneider's life expectancy questionnaire (2002) and revised Oxford happiness scale (2001). The data were analyzed by path analysis.  Results: The results show that spirituality and mental health are predictors of happiness and mental health has a relative mediating role in predicting happiness. Mental health is also predicted by spirituality. Conclusion: In the end, it was concluded that strategic hope completely mediates between the level of satisfaction and lack of negative feelings with spiritual lifestyle, and functional hope mediates partially between perception management and despair

Investigation of Cytocidal Activity of Bacillus Thuringiensis Parasporal Toxin on CCRF-CEM Cell Line

Background & Objective: Parasporin is a parasporal protein of Bacillus thuringiensis and exhibits special cytocidal activity against human cancer cells. Similar to other insecticidal Bacillus thuringiensis crystal toxins, parasporin shows target specificity and damages the cellular membrane. In this study, different strains of Bacillus thuringiensis isolated from various regions of Iran and their cytocidal activity against CCRF-CEM cell line and human erythrocyte were investigated.   Materials & Methods: Fifty soil samples were collected from different Iranian provinces, and characterization was performed based on protein crystal morphology by phase-contrast microscope and variations of Cry protein toxin using SDS-PAGE. After parasporin was processed with proteinase K, the active form was produced and protein activity on the cell line was evaluated. Results: Parasporal inclusion proteins showed different cytotoxicity against acute lymphoblastic leukemia cells (ALL), but not against normal lymphocyte. Isolated parasporin demonstrated no hemolytic activity against human erythrocyte. It appears that these proteins have the ability to differentiate between normal lymphocytes and leukemia cells and have specific receptors on specific cancer cell lines. Conclusion: Our results provide evidence that the parasporin-producing organism is a common member in Bacillus thuringiensis populations occurring in the natural environments of Iran

Use of Sine Shaped High-Frequency Rhythmic Visual Stimuli Patterns for SSVEP Response Analysis and Fatigue Rate Evaluation in Normal Subjects

Background: Recent EEG-SSVEP signal based BCI studies have used high frequency square pulse visual stimuli to reduce subjective fatigue. However, the effect of total harmonic distortion (THD) has not been considered. Compared to CRT and LCD monitors, LED screen displays high-frequency wave with better refresh rate. In this study, we present high frequency sine wave simple and rhythmic patterns with low THD rate by LED to analyze SSVEP responses and evaluate subjective fatigue in normal subjects.Materials and Methods: We used patterns of 3-sequence high-frequency sine waves (25, 30, and 35 Hz) to design our visual stimuli. Nine stimuli patterns, 3 simple (repetition of each of above 3 frequencies e.g., P25-25-25) and 6 rhythmic (all of the frequencies in 6 different sequences e.g., P25-30-35) were chosen. A hardware setup with low THD rate (&lt;0.1%) was designed to present these patterns on LED. Twenty two normal subjects (aged 23–30 (25 ± 2.1) yrs) were enrolled. Visual analog scale (VAS) was used for subjective fatigue evaluation after presentation of each stimulus pattern. PSD, CCA, and LASSO methods were employed to analyze SSVEP responses. The data including SSVEP features and fatigue rate for different visual stimuli patterns were statistically evaluated.Results: All 9 visual stimuli patterns elicited SSVEP responses. Overall, obtained accuracy rates were 88.35% for PSD and &gt; 90% for CCA and LASSO (for TWs &gt; 1 s). High frequency rhythmic patterns group with low THD rate showed higher accuracy rate (99.24%) than simple patterns group (98.48%). Repeated measure ANOVA showed significant difference between rhythmic pattern features (P &lt; 0.0005). Overall, there was no significant difference between the VAS of rhythmic [3.85 ± 2.13] compared to the simple patterns group [3.96 ± 2.21], (P = 0.63). Rhythmic group had lower within group VAS variation (min = P25-30-35 [2.90 ± 2.45], max = P35-25-30 [4.81 ± 2.65]) as well as least individual pattern VAS (P25-30-35).Discussion and Conclusion: Overall, rhythmic and simple pattern groups had higher and similar accuracy rates. Rhythmic stimuli patterns showed insignificantly lower fatigue rate than simple patterns. We conclude that both rhythmic and simple visual high frequency sine wave stimuli require further research for human subject SSVEP-BCI studies

Procaine and Saline Have Similar Effects on Articular Cartilage and Synovium in Rat Knee

PubMedWoSScopu

Photobiomodulation with polychromatic light (600-1200 nm) improves fat graft survival by increasing adipocyte viability, neovascularization, and reducing inflammation in a rat model

Objectives Unpredictability with the final volume and viability of the graft are the major concerns in fat grafting. An experimental study was conducted to increase graft retention using photobiomodulation (PBM) with polychromatic light in near-infrared region (600-1200 nm) by utilizing its stimulatory effects on angiogenesis, neovascularization, adipocyte viability, and anti-inflammatory properties. Methods A total of 24 rats were divided into four groups (n = 6) according to the applied polychromatic light protocol to the recipient site (none, before fat transfer, after fat transfer, and combined). In all groups, inguinal fat pad was excised, measured for volume and weight, and transferred to the dorsum of the rat. At the end of the experiment, fat grafts were harvested from the recipient site for volume and weight measurements, histological, and immunohistochemical evaluation. Results Intergroup comparison revealed that fat graft retention regarding weight and volume, was significantly superior in Group IV (p = 0.049 and p = 0.043, respectively), which polychromatic light was applied both before and after transfer of the graft. Hematoxylin-eosin and Masson's trichrome stained sections showed absence of necrosis, fibrosis, inflammation, cyst formation, and increased vascularization of both inner and outer zones of the grafts in Group IV. Also, immunohistochemical staining scores for perilipin (indicator for adipocyte viability), CD31 and VEGF (indicators for angiogenesis and neovascularization) were significantly higher (p < 0.001). Ki67 scores were significantly lower in this group because of anti-inflammatory environment (p < 0.001). Conclusions Application of PBM to the recipient site before and after fat transfer improved outcomes in rats at 56 day after fat grafting by means of volume retention, increased neovascularization and adipocyte viability and reduced necrosis, fibrosis and inflammation

Prediction of individual differences in risky behaviour in young adults via variations in local brain structure

In recent years the problem of how inter-individual differences play a role in risk-taking behaviour has become a much debated issue. We investigated this problem based on the well-known balloon analogue risk task (BART) in which participants inflate a virtual balloon opting for a higher score in the face of a riskier chance of the balloon explosion. In this study, based on a structural Voxel Based Morphometry (VBM) technique we demonstrate a significant positive correlation between BART score and size of the grey matter volume in the anterior insula in riskier subjects. Although the anterior insula is among the candidate brain areas that were involved in the risk taking behaviour in fMRI studies, here based on our structural data it is the sole area with a significant structural variation among different subjects. Also a seemingly conflicting finding is discussed where the anterior insula is shown to be more active in risk aversive subjects

Image_4_Use of Sine Shaped High-Frequency Rhythmic Visual Stimuli Patterns for SSVEP Response Analysis and Fatigue Rate Evaluation in Normal Subjects.PDF

<p>Background: Recent EEG-SSVEP signal based BCI studies have used high frequency square pulse visual stimuli to reduce subjective fatigue. However, the effect of total harmonic distortion (THD) has not been considered. Compared to CRT and LCD monitors, LED screen displays high-frequency wave with better refresh rate. In this study, we present high frequency sine wave simple and rhythmic patterns with low THD rate by LED to analyze SSVEP responses and evaluate subjective fatigue in normal subjects.</p><p>Materials and Methods: We used patterns of 3-sequence high-frequency sine waves (25, 30, and 35 Hz) to design our visual stimuli. Nine stimuli patterns, 3 simple (repetition of each of above 3 frequencies e.g., P25-25-25) and 6 rhythmic (all of the frequencies in 6 different sequences e.g., P25-30-35) were chosen. A hardware setup with low THD rate (<0.1%) was designed to present these patterns on LED. Twenty two normal subjects (aged 23–30 (25 ± 2.1) yrs) were enrolled. Visual analog scale (VAS) was used for subjective fatigue evaluation after presentation of each stimulus pattern. PSD, CCA, and LASSO methods were employed to analyze SSVEP responses. The data including SSVEP features and fatigue rate for different visual stimuli patterns were statistically evaluated.</p><p>Results: All 9 visual stimuli patterns elicited SSVEP responses. Overall, obtained accuracy rates were 88.35% for PSD and > 90% for CCA and LASSO (for TWs > 1 s). High frequency rhythmic patterns group with low THD rate showed higher accuracy rate (99.24%) than simple patterns group (98.48%). Repeated measure ANOVA showed significant difference between rhythmic pattern features (P < 0.0005). Overall, there was no significant difference between the VAS of rhythmic [3.85 ± 2.13] compared to the simple patterns group [3.96 ± 2.21], (P = 0.63). Rhythmic group had lower within group VAS variation (min = P25-30-35 [2.90 ± 2.45], max = P35-25-30 [4.81 ± 2.65]) as well as least individual pattern VAS (P25-30-35).</p><p>Discussion and Conclusion: Overall, rhythmic and simple pattern groups had higher and similar accuracy rates. Rhythmic stimuli patterns showed insignificantly lower fatigue rate than simple patterns. We conclude that both rhythmic and simple visual high frequency sine wave stimuli require further research for human subject SSVEP-BCI studies.</p

Table_5_Use of Sine Shaped High-Frequency Rhythmic Visual Stimuli Patterns for SSVEP Response Analysis and Fatigue Rate Evaluation in Normal Subjects.DOCX

<p>Background: Recent EEG-SSVEP signal based BCI studies have used high frequency square pulse visual stimuli to reduce subjective fatigue. However, the effect of total harmonic distortion (THD) has not been considered. Compared to CRT and LCD monitors, LED screen displays high-frequency wave with better refresh rate. In this study, we present high frequency sine wave simple and rhythmic patterns with low THD rate by LED to analyze SSVEP responses and evaluate subjective fatigue in normal subjects.</p><p>Materials and Methods: We used patterns of 3-sequence high-frequency sine waves (25, 30, and 35 Hz) to design our visual stimuli. Nine stimuli patterns, 3 simple (repetition of each of above 3 frequencies e.g., P25-25-25) and 6 rhythmic (all of the frequencies in 6 different sequences e.g., P25-30-35) were chosen. A hardware setup with low THD rate (<0.1%) was designed to present these patterns on LED. Twenty two normal subjects (aged 23–30 (25 ± 2.1) yrs) were enrolled. Visual analog scale (VAS) was used for subjective fatigue evaluation after presentation of each stimulus pattern. PSD, CCA, and LASSO methods were employed to analyze SSVEP responses. The data including SSVEP features and fatigue rate for different visual stimuli patterns were statistically evaluated.</p><p>Results: All 9 visual stimuli patterns elicited SSVEP responses. Overall, obtained accuracy rates were 88.35% for PSD and > 90% for CCA and LASSO (for TWs > 1 s). High frequency rhythmic patterns group with low THD rate showed higher accuracy rate (99.24%) than simple patterns group (98.48%). Repeated measure ANOVA showed significant difference between rhythmic pattern features (P < 0.0005). Overall, there was no significant difference between the VAS of rhythmic [3.85 ± 2.13] compared to the simple patterns group [3.96 ± 2.21], (P = 0.63). Rhythmic group had lower within group VAS variation (min = P25-30-35 [2.90 ± 2.45], max = P35-25-30 [4.81 ± 2.65]) as well as least individual pattern VAS (P25-30-35).</p><p>Discussion and Conclusion: Overall, rhythmic and simple pattern groups had higher and similar accuracy rates. Rhythmic stimuli patterns showed insignificantly lower fatigue rate than simple patterns. We conclude that both rhythmic and simple visual high frequency sine wave stimuli require further research for human subject SSVEP-BCI studies.</p