Determination and comparison rate of expression markers of osteoblast derived of Adipose derived stem cells markers in monolayer and pellet culture models

Abstract: Nowadays high accident rates, fractures leading to permanent bone disorders and the impossibility of bone transplant have made scientists to look for new methods of repairing injured bones. Considering the application of stem cells in bone tissue engineering, there exists the necessity to investigate various culture methods and suitable fields and scaffolds. Thus, we decided to induce adipose-derived stem cells into osteoblast cells in two systems of pellet culture and monolayer and compare osteogenic markers. Methods: Stem cells have been separated via mechanical and enzymatic methods and cultured in monolayer and pellet culture models with osteogenic medium. Then, RNA was separated from differentiated cells, complementary DNA (cDNA) was synthesized and amplified. Polymerase chain reaction (PCR) product was transferred to electrophoresis gel. The intensity of the bands was measured by Image-J software and analyzed by SPSS. Results: average osteopontin, osteocalcin and Runx2 genes in differentiated cells in the two culture systems showed a significant difference. The expression of osteocalcin, osteopontin and Runx2 gense in pellet system were more than monolayer systems in 21 days. Conclusion: This study indicated that pellet and monolayer culture systems are appropriate for bone engineering but osteocalcin, osteopontin and Runx2 genes expressions were different in the two culture system

An Effective Concentration of 5-Aza-CdR to Induce Cell Death and Apoptosis in Human Pancreatic Cancer Cell Line through Reactivating RASSF1A and Up-Regulation of Bax Genes

Background: Promoter hyper-methylation of tumor suppressor genes is a common event that occurs in cancer. As methylation is a reversible modification, agents capable of reversing an abnormal methylation status should help to combat cancer. 5-Aza-CdR is a DNA methyl-transferase inhibitor. The present study aimed to evaluate the effect of 5-Aza-CdR on the proliferation of human pancreatic cancer cell line (PANC-1) and the expression of RASSF1A and Bax genes. Methods: PANC-1 cells were cultured and treated with 5 and 10 µM/L of 5-Aza-CdR for 24, 48, 72, and 96 hours and the percentages of cell viability and apoptosis were measured by MTT and flow cytometry. RASSF1A gene promoter methylation was assessed by methyl-specific primer-PCR (MSP-PCR) and the expression of RASSF1A and Bax genes was measured using quantitative real-time PCR (qPCR). All quantitative data are presented as mean±SD (standard deviation). The one-way analysis of variance (ANOVA) with the LSD post hoc test was performed for statistical analysis using the SPSS software package, version 16.0. Results: 3-[4,5-dimethythiaziazol-2yl]-2,5-diphenyl tetrazoliumbr omide (MTT) assay revealed that 5-Aza-CdR significantly inhibit the growth and proliferation of PANC-1. The flow cytometry results showed over 40% and 70% of early and late apoptotic cells after treatment with 5 and 10 µm/L of 5-Aza-CdR, respectively. MSP-PCR data indicated that the treatment of cells with 10 µm/L 5-Aza-CdR resulted in partial demethylation of RASSF1A gene promoter. qPCR results showed significant re-expression of RASSF1A and up-regulation of Bax genes after 96 hours treatment of cells with 10 µm/L 5-Aza-CdR versus control cells (P<0.01). Conclusion: The result demonstrated that 5 and 10 µM of 5-Aza-CdR induce cell death and apoptosis by epigenetic reactivation of RASSF1A and up-regulation of Bax genes

An animal model study of osteochondral defect repair by human adipose stem cells and pomegranate fruit hydroalchoholic extract

Objective: Articular cartilage damages do not repair spontaneously. Tissue engineering is a promising approach to repair cartilage damage. Transforming growth factor-beta (TGF-β) members are the known induction factors in chondrogenic differentiation. However, hypertrophy of the chondrocytes resulting from mesenchymal stem cells (MSCs) induction by TGF-β is inevitable. Pomegranate fruit contains many ingredients which are useful in ensuring the health of organs. This study was designed to investigate the Pomegranate Fruit hydroalchoholic Extract (PFE) capability in human adipose derived stem cells (hASCs) differentiation into the chondrocytes on fibrin scaffold.Materials and Methods: Pomegranate fruit hydroalchoholic extract (PFE) was prepared. hASCs were isolated, expanded, labeled, and seeded on the fibrin scaffold. The constructs were divided into three groups including TGF-β3, PFE, and control. The constructs were induced for 14 days, then, the MTT assay, Real-Time Polymerase Chain Reaction (PCR), and histochemistry assessments were run, and finally, the constructs were transplanted into the knee defect of rats. The gross and histological assessments of the transplants were done after 8 weeks.Results: The viability rate, COL2A1, Aggrecan (ACAN) and COL10A1 genes expression levels, and histological criterion of the PFE samples were significantly higher than that of the control. The macroscopic grades and histological results of the PFE samples were close to that of the TGF-β3. The number of positive cells for COLІI protein were higher significantly in the PFE group than the control.Conclusion: PFE was effective in the chondrogenic induction of hASCs. Further studies are needed to find out the events of the chondrogenic induction using PFE

Hybrid and Composite Scaffolds Based on Extracellular Matrices for Cartilage Tissue Engineering

Cartilage consists of chondrocytes and a special extracellular matrix (ECM) having unique biochemical, biophysical, and biomechanical properties that play a critical role in the proliferation and differentiation of cells inherent to cartilage functions. Cartilage tissue engineering (CTE) requires recreating these microenvironmental physicochemical conditions to lead to chondrocyte differentiation from stem cells. ECM-derived hybrid scaffolds based on chondroitin sulfate, hyaluronic acid, collagen, and cartilage ECM analogs provide environments conducive to stem cell proliferation. In this review, we describe hybrid scaffolds based on these four cartilage ECM derivatives; we also categorize these scaffolds based on the methods used for their preparation. The use of hybrid scaffolds is increasing in CTE to address the complexity of cartilage tissue. Thus, a comprehensive review on the topic should be a useful guide for future research. Scaffolds fabricated from extracellular matrix (ECM) derivatives are composed of conducive structures for cell attachment, proliferation, and differentiation, but generally do not have proper mechanical properties and load-bearing capacity. In contrast, scaffolds based on synthetic biomaterials demonstrate appropriate mechanical strength, but the absence of desirable biological properties is one of their main disadvantages. To integrate mechanical strength and biological cues, these ECM derivatives can be conjugated with synthetic biomaterials. Hence, hybrid scaffolds comprising both advantages of synthetic polymers and ECM derivatives can be considered a robust vehicle for tissue engineering applications. © Copyright 2019, Mary Ann Liebert, Inc., publishers 2019

The effect of stem cells and vascular endothelial growth factor on cancer angiogenesis

The formation of new vessels from pre-existing vessels is known as angiogenesis. The process is controlled by stimuli and inhibitors. Angiogenesis starts as a result of the unbalance of these factors, where balance has a tendency toward the stimulus. One of the most important factors promoting angiogenesis is the vascular endothelial growth factor (VEGF). In addition to being involved in vascular regeneration in normal tissues, VEGF also takes part in tumor tissue angiogenesis. These factors affect endothelial cells (ECs) directly as well as differentiate tumor cells from endothelial cells and play an active role in tumor tissue angiogenesis. Angiogenesis partakes in the growth and proliferation of tumor tissue. Because anti-angiogenic treatment is favorable in existing cancer therapies, the potential benefits should be considered. One of these new therapies is cell therapy using mesenchymal stem cells (MSCs). Research on MSCs remains controversial because much of the earlier research on MSCs has shown their effectiveness, but more recent research has identified harmful effects of these cells. This article reviews the role of stem cells and their secretions in the angiogenesis of tumor tissues

Comparison of aggrecan gene expression in chondrogenesis of adipose-derived stem cells in pellet and micromass culture systems

Background: Nowadays, Human adipocyte-derived stem cells (hADSCs) has been widely used in tissue engineering because of its unique features such as extraction from more sources, more easily and non-invasive extraction methods. In order to increase cell-cell interactions, similar to embryonic pre-cartilage condensation, the use of three dimensional (3D) high-density cell culture systems such as Pellet and Micromass that simulates optimal condensation in chondrogenesis in vivo is necessary. Also, these culture systems provide the proper diffusion of nutrients. Aggrecan is a proteoglycan and one of the important components of extracellular matrix of cartilage tissue that plays an important role in the organization of the extracellular matrix. The high concentrations of aggrecan produces the osmotic properties that is necessary to normal tissue function of cartilage. In current study, Aggrecan gene expression was investigated during chondrogenesis of hADSCs in two Pellet and Micromass culture systems. Methods: This experimental study was done in Department of Anatomical Sciences Department of Faculty Medical in Isfahan University of Medical Sciences, Iran, from April 2013 to January 2015. First, the abdominal adipose tissue was obtained from three patients after obtaining written consent during their liposuction surgeries. ADSCs were extracted by mechanical and enzymatic methods and were cultured in monolayer culture. Then, in order to induction of chondrogenic differentiation, 5&times;105 cells of third passage (P3) were transferred to three-dimensional culture systems Pellet and Micromass containing chondrogenic mediums in experimental groups of 7 and 14 days. The evaluation of aggrecan gene expression was performed by real-time PCR technique. Results: Gene expression analysis revealed that aggrecan was significantly increased in micromass culture at day 14 compared to Pellet culture at days 14 and 7 (P&le;0.01). Also, aggrecan was significantly increased in Micromass culture at day 7 compared to Pellet culture at day 7 (P&le;0.05). Conclusion: Due to higher expression of aggrecan gene in Micromass culture compared to Pellet culture, this system may be more efficient than Pellet culture in synthesis of aggrecan in chondrogenic differentiation of ADSCs

Evaluate the growth and adhesion of osteoblast cells on nanocomposite scaffold of hydroxyapatite/titania coated with poly hydroxybutyrate

Background: The generation of bioartificial bone tissues may help to overcome the problems related to donor site morbidity and size limitations. Materials and Methods: In this paper, hydroxyapatite (HA) powder was made out of bovine bone by thermal analysis at 900°C and first, and then, porous HA (50 weight percentage) was produced by polyurethane sponge replication method. In order to improve the scaffold mechanical properties, they have been coated with poly hydroxybutyrate. In terms of phase studies, morphology, and specifying agent groups, the specific characterization devices such as X-ray diffraction and Fourier transform infrared, were employed. To compare the behavior of cellular scaffolds, they were divided into four groups of scaffolds. The osteoblast cells were cultured. To perform phase studies, analysis of Methylthiazole tetrazolium (MTT) and Trypan blue were carried out for the viability and attachment on the surface of the scaffold, and the specification of Scanning electron microscopy was employed for the morphology of the cells. Results: The results of MTT analysis performed on four groups of scaffolds have shown that Titanium oxide (Tio2 ) had no effect on cell growth alone and HA was the main factor of growth and cell osteoblast adhesion on the scaffold. Moreover, the results showed that the use of coating with poly-3-hydroxybutyrate saved the factors and placed the osteoblasts within the pore. Since the main part of bone consists of HA, the TiO2 accelerates the formation of apatite crystals at the scaffold surface which is the evidence for bone tissue regeneration. Conclusions: It is likely that the relation between HA and TiO2 leads to an increase in osteoblast adhesion and growth of cells on the scaffold surface

Transcriptomic comparison of osteopontin, osteocalcin and core binding factor 1 genes between human adipose derived differentiated osteoblasts and native osteoblasts

Background: There are significant limitations in repair of irrecoverable bone defects. Stem-cell therapy is a promising approach for the construction of bone tissue. Mesenchymal stem cells (MSCs) have been introduced as basic tools for bone tissue generation. Through MSCs, adipose-derived stem cells (ADSCs) are more interesting. Since the similarity of native osteoblasts and differentiated osteoblasts from ADSCs in terms of gene expression pattern is unknown, this study was designed to compare gene expression patterns of some genes involved in osteogenesis between human native osteoblasts and adipose-derived differentiated osteoblasts. Materials and Methods: Realtime qRT-PCR was used for studying the gene expression of osteocalcin, osteopontin, and core binding factor alpha 1 (Cbfa1) in human native osteoblasts and adipose derived osteogenic osteoblasts at days 7, 14, 21, and 28 of differentiation. Results: This study demonstrated that native osteoblasts and differentiated osteoblasts, cultured in common osteogenic medium, have significant differences in gene expression levels for osteocalcin and osteopontin. Compared to native osteoblasts, these genes are expressed lower in all four groups of differentiated osteoblastic cells. We also found, there is a progressive increase in cbfa1 expression over the differentiation period of ADSCs from day 7 to day 28. Conclusions: Our findings help for better assessment of adipose-derived differentiated cells as a source for cell-based therapy

Effect of low frequency ultrasound waves on the morphology and viability of cultured human gingival fibroblasts

المخلص: أهداف البحث: كان الهدف من هذه الدراسة هو التحقيق في تأثير سعة الاهتزاز للموجات الميكانيكية بالموجات فوق الصوتية (27 كيلو هرتز) على قدرة الخلايا اللثوية البشرية على الحياة وتشكلها التي تم زرعها على مادة حيوية. طرق البحث: تم زرع الخلايا اللثوية البشرية على أطباق زرع الأنسجة وسطح سبائك التيتانيوم ''تي آي 6 ايه 14 في'' في مجموعتين تشمل ثلاثة أيام وسبعة أيام من زرع الخلايا. تعرضت الخلايا لثلاث سعات اهتزاز لمدة 20 دقيقة يوميا. تم استخدام صور مجهر المسح الإلكتروني لتحديد شكل الخلايا. النتائج: لأول مرة، عند مستوى معين من سعة الاهتزاز المقارن بشدة 260 مللي وات / سم2، تم تحديد كيفية فقدان الخلايا الملتصقة اتصالها. تكاثرت الخلايا اللثوية البشرية التي تلقت مستوى معين من سعة الاهتزاز المقارن بشدة 50 مللي وات / سم2 بشكل كبير، في حين كانت السعات الأعلى لها آثارا سلبية. الاستنتاجات: أظهر مسح صور المجهر الإلكتروني للأرومات الليفية اللثوية البشرية على أقراص التيتانيوم عند مستوى معين من سعة الاهتزاز مقارنة بكثافة 50 ميغاواط / سم2 بتشكل سداسي ملحوظ، والتي كانت تسمى نمط قرص العسل في هذا البحث التجريبي، وفي اليوم السادس لوحظ أن الخلايا اللثوية البشرية تكاثرت على أطباق زرع الأنسجة بمعدل أعلى وخلايا جديدة متصلة بشكل موحد على طبقة من الخلايا. يشير هذا إلى تأثير الأنسجة الخلوية كركيزة لنمو الخلايا الليفية اللثوية البشرية الجديدة تحت الموجات فوق الصوتية منخفضة الكثافة. Abstract: Objectives: The aim of this study was to investigate the effect of the vibration amplitude of mechanical ultrasound waves (27 kHz) on the viability and morphology of human gingival fibroblasts (hGFs) when cultured on a biomaterial substrate. Method: hGFs were seeded on tissue culture plates (TCPs) and an Ti6Al4V titanium alloy surface in two groups for three days and seven days of cell culture. The cells were subjected to three vibration amplitudes for 20 min each day. Scanning electron microscope (SEM) images were used to characterize cell morphology. Results: Experiments showed that hGF cells became detached from their plates at a vibration amplitude comparable to an intensity of 260 mW/cm2. In addition, hGfs that received a vibrational amplitude comparable to an intensity of 50 mW/cm2 underwent significant proliferation proliferated significantly; however, cells receiving higher amplitudes suffered from adverse effects. Conclusions: SEM images of hGFs on titanium disks at vibration amplitude comparable to an intensity 50 mW/cm2 showed a remarkable hexagonal architecture, which we refer to as a honeycomb pattern. On day 6 the observed hGFs on TCPs, proliferated at a higher rate and new cells attached uniformly on the existing layer of cells. These data indicate the effect of cellular tissue as a substrate on the growth of new hGFs under low-intensity ultrasound

The effect of CTB on P53 protein acetylation and consequence apoptosis on MCF-7 and MRC-5 cell lines

Background: P300 is a member of the mammalian histone acetyl transferase (HAT) family, an enzyme that acetylates histones and several non-histone proteins including P53 (the most important tumor suppressor gene) during stress, which plays an important role in the apoptosis of tumor cells. Hereby, this study describes the potency of CTB (Cholera Toxin B subunit) as a P300 activator to induce apoptosis in a breast cancer cell line (MCF-7) and a lung fibroblast cell line (MRC-5) as a non-tumorigenic control sample. Materials and Methods: MCF-7 and MRC-5 were cultured in RPMI-1640 and treated with or without CTB at a concentration of 85.43 μmol/L, based on half-maximal inhibitory concentration (IC50) index at different times (24, 48 and 72 h). The percentage of apoptotic cells were measured by flow cytometry. Real-time quantitative RT-PCR was performed to estimate the mRNA expression of P300 in MCF-7 and MRC-5 with CTB at different times. ELISA and Bradford protein techniques were used to detect levels of total and acetylated P53 protein generated in MCF-7 and MRC-5. Results: Our findings indicated that CTB could effectively induce apoptosis in MCF-7 significantly higher than MRC-5. We showed that expression of P300 was up-regulated by increasing time of CTB treatment in MCF-7 but not in MRC-5 and the acetylated and total P53 protein levels were increased more in MCF-7 cells than MRC-5. Conclusion: CTB could induce acetylation of P53 protein through increasing expression of P300 and consequently induce the significant cell death in MCF-7 but it could be well tolerated in MRC-5. Therefore, CTB could be used as an anti-cancer agent