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1.
Int. j. high dilution res ; 19(4): 25-34, 2020.
Article in English | LILACS, HomeoIndex | ID: biblio-1146572

ABSTRACT

BackgroundBreast cancer has been considered a public health problem and homeopathic treatments are becoming increasingly recommended due to its ways of action and absence of adverse effects. MCF-7 is an adenocarcinoma of human breast cell line useful as preclinicalmodel to screen therapeutic agents such as ultra-diluted Viscum album, an European plant which extract is commonly used in cancer therapy. AIMS MCF-7 and mesenchymal stem cells (MSC) were used to evaluate the in vitrocytotoxicity of homoeopathic Viscum album 1x10-3(VA3X). Methodscells were cultured for 24 hours in controlled environment (37.5oC and 5% CO2) in 96-well plates. After this time, VA3X was added to the culture medium in concentrations varying from 10 to 100 L/mL.A control group was maintained with culture medium only. Cells were cultivated for 48 hours in these conditions for evaluation of cell viability by MTT assay. ResultsHigher cytotoxicity was observed in MCF-7 when compared to MSC, as the lower concentration of VA3X was capable of inducing tumor cell death and not healthy cell death. The MTT assay results were that 42 L/mL of VA3X reduced MCF-7 cells viability to 50% and 62 L/mL reduced MSC cells to the same percentage, what means that tumor cells are more sensible to VA3X than heathy cells. ConclusionViscum albumpresented higher cytotoxic action on human breast cancer cell line culture than on mesenchymal stem cells. This medicine is extensively used against cancer, and the use of the homoeopathic form of it brings new possibilities as no or fewer adverse effects would be present.(AU)


Subject(s)
Humans , Breast Neoplasms/pathology , Adenocarcinoma/pathology , Homeopathic Therapeutics , Viscum album/toxicity , Mesenchymal Stem Cells/drug effects , MCF-7 Cells/drug effects , Breast Neoplasms/drug therapy , Adenocarcinoma/drug therapy , Cell Count , Cell Survival , Cell Culture Techniques
2.
J. appl. oral sci ; 27: e20180317, 2019. tab, graf
Article in English | LILACS, BBO | ID: biblio-984571

ABSTRACT

Abstract Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields. Objective Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved. Material and Methods ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA® software. Results RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone. Conclusions In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.


Subject(s)
Humans , Osteogenesis/drug effects , Tretinoin/pharmacology , Cell Differentiation/drug effects , Bone Morphogenetic Protein 2/drug effects , Mesenchymal Stem Cells/drug effects , Osteoblasts/drug effects , Osteogenesis/physiology , Reference Values , Time Factors , Osteocalcin/analysis , Osteocalcin/drug effects , Osteonectin/analysis , Osteonectin/drug effects , Cell Differentiation/physiology , Cells, Cultured , Blotting, Western , Reproducibility of Results , Analysis of Variance , Alkaline Phosphatase/analysis , Alkaline Phosphatase/adverse effects , Bone Morphogenetic Protein 2/metabolism , Mesenchymal Stem Cells/metabolism
3.
Braz. arch. biol. technol ; 62: e19180096, 2019. tab, graf
Article in English | LILACS | ID: biblio-1055376

ABSTRACT

Abstract Clinical research has shed the light on the relation between coagulation and inflammation. Coagulation cascade is activated in lung injury resulting in thrombotic and fibrotic lesions. Such a cascade is initiated by inflammation, then the two systems intense each other. New therapies that modulate coagulation and inflammation will be more successful than therapies targeting only one of them. Mesenchymal stem cells showed anti-inflammatory functions in animal models. The role of mesenchymal stem cells in methotrexate induced lung injury model was evaluated, but no studies scoped on the role of stem cells in coagulation associated with inflammation in such models. This study focuses on the therapeutic role of mesenchymal stem cells against the development of clotting in methotrexate induced lung injury rat model. Results showed that mesenchymal stem cells treatment for 4 weeks caused a decrease in lung activated coagulation factors; protease activated receptor-1, fibrinogen, plasminogen activator inhibitor-1 and platelet count with a decrease in inflammatory factors; tumor necrosis factor-α, interferon- γ, interleukin-8, monocyte chemoattractant protein-1 and total leukocyte count. Thus, mesenchymal stem cells have anti-inflammatory potency against clotting risk in methotrexate induced lung injury model. This opens the outlook for stem cells as a new therapy that moderates coagulation associated with inflammation.


Subject(s)
Animals , Rats , Blood Coagulation , Methotrexate/administration & dosage , Lung Injury/chemically induced , Mesenchymal Stem Cells/drug effects , Inflammation/drug therapy , Models, Animal
4.
Braz. j. med. biol. res ; 51(2): e6520, 2018. tab, graf
Article in English | LILACS | ID: biblio-889032

ABSTRACT

Multiple growth factors can be administered to mimic the natural process of bone healing in bone tissue engineering. We investigated the effects of sequential release of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) from polylactide-poly (ethylene glycol)-polylactide (PELA) microcapsule-based scaffolds on bone regeneration. To improve the double emulsion/solvent evaporation technique, VEGF was encapsulated in PELA microcapsules, to which BMP-2 was attached. The scaffold (BMP-2/PELA/VEGF) was then fused to these microcapsules using the dichloromethane vapor method. The bioactivity of the released BMP-2 and VEGF was then quantified in rat mesenchymal stem cells (rMSCs). Immunoblotting analysis showed that BMP-2/PELA/VEG promoted the differentiation of rMSCs into osteoblasts via the MAPK and Wnt pathways. Osteoblast differentiation was assessed through alkaline phosphatase expression. When compared with simple BMP-2 plus VEGF group and pure PELA group, osteoblast differentiation in BMP-2/PELA/VEGF group significantly increased. An MTT assay indicated that BMP-2-loaded PELA scaffolds had no adverse effects on cell activity. BMP-2/PELA/VEG promoted the differentiation of rMSCs into osteoblast via the ERK1/2 and Wnt pathways. Our findings indicate that the sequential release of BMP-2 and VEGF from PELA microcapsule-based scaffolds is a promising approach for the treatment of bone defects.


Subject(s)
Animals , Rabbits , Rats , Polyesters/pharmacology , Polyethylene Glycols/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Vascular Endothelial Growth Factors/metabolism , Tissue Scaffolds , Bone Morphogenetic Protein 2/metabolism , Mesenchymal Stem Cells/cytology , Time Factors , Bone Regeneration , Signal Transduction/physiology , Cells, Cultured , Models, Animal , Cell Proliferation , beta Catenin/physiology , Nanoparticles , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Wnt Signaling Pathway/physiology
5.
J. appl. oral sci ; 25(5): 515-522, Sept.-Oct. 2017. graf
Article in English | LILACS, BBO | ID: biblio-893656

ABSTRACT

Abstract Hypersensitivity, local irritative and cytotoxic effects are known for the chemical components of Syzygium aromaticum and Cinnamomum zeylanicum contained in dental materials. However, there is no intimate data in dentistry using the whole extracts of these plants and introducing new ones. Salvia triloba is a well-known anti-inflammatory plant that correspondingly could be used in several dental traumas. Objectives: We aimed to show and compare the effect of S. aromaticum, C. zeylanicum, and S. triloba extracts on dental pulp stem cells (DPSCs) proliferation, differentiation, and immune responses. Material and Methods: Using xCELLigence, a real time monitoring system, we obtained a growth curve of DPSCs with different concentrations of the Extracts. A dose of 10 μg/mL was the most efficient concentration for vitality. Osteogenic differentiation and anti-inflammatory activities were determined by using an ELISA Kit to detect early and late markers of differentiation. Results: The level of osteonectin (ON, early osteogenic marker) decreased, which indicated that the osteogenic differentiation may be accelerated with addition of extracts. However, the level of osteocalcin (OCN, late osteogenic marker and sign of calcium granulation) differed among the extracts, in which S. aromaticum presented the highest value, followed by S. triloba and C. zeylanicum. Surprisingly, the determined calcium granules were reduced in S. aromaticum and S. triloba. In response to tumor necrosis factor alpha (TNF-α), S. triloba-treated DPSCs showed the most reduced level of IL-6 cytokine level. We suggest C. zeylanicum as a promising osteogenic inducer and S. triloba as a potent anti-inflammatory agent, which could be used safely in biocomposite or scaffold fabrications for dentistry. Conclusions: Because calcium granule formation and cell viability play a critical role in hard tissue formation, S. aromaticum in dentistry should be strictly controlled, and the mechanism leading to reduced calcium granule formation should be identified.


Subject(s)
Humans , Adolescent , Young Adult , Drugs, Chinese Herbal/pharmacology , Plant Extracts/pharmacology , Cinnamomum zeylanicum/chemistry , Syzygium/chemistry , Dental Pulp/cytology , Mesenchymal Stem Cells/drug effects , Anti-Inflammatory Agents/pharmacology , Osteogenesis/drug effects , Time Factors , Enzyme-Linked Immunosorbent Assay , Antigens, Differentiation/analysis , Osteocalcin/analysis , Osteonectin/analysis , Cell Differentiation/drug effects , Cells, Cultured , Calcium/analysis , Reproducibility of Results , Analysis of Variance , Cytokines/analysis , Dental Pulp/drug effects , Cell Proliferation/drug effects , Flow Cytometry
6.
Cell Journal [Yakhteh]. 2017; 19 (1): 146-158
in English | IMEMR | ID: emr-185801

ABSTRACT

Objective: We used sodium nitroprusside [SNP], a nitric oxide [NO] releasing molecule, to understand its effect on viability and proliferation of rat bone marrow mesenchymal stem cells [BM-MSCs]


Materials and Methods: This experimental study evaluated the viability and morphology of MSCs in the presence of SNP [100 to 2000 micro M] at 1, 5, and 15 hours. We chose the 100, 1000, and 2000 micro M concentrations of SNP for one hour exposure for further analyses. Cell proliferation was investigated by the colony forming assay and population doubling number [PDN]. Na[+], K[+], and Ca2[+] levels as well as activities of lactate dehydrogenase [LDH], alkaline phosphatase [ALP], aspartate transaminase [AST], and alanine transaminase [ALT] were measured


Results: The viability of MSCs dose-dependently reduced from 750 micro M at one hour and 250 micro M at 5 and 15 hours. The 100 micro M caused no change in viability, however we observed a reduction in the cytoplasmic area at 5 and 15 hours. This change was not observed at one hour. The one hour treatment with 100 micro M of SNP reduced the mean colony numbers but not the diameter when the cells were incubated for 7 and 14 days. In addition, one hour treatment with 100 micro M of SNP significantly reduced ALT, AST, and ALP activities whereas the activity of LDH increased when incubated for 24 hours. The same treatment caused an increase in Ca2[+] and reduction in Na+ content. The 1000 and 2000 micro M concentrations reduced all the factors except Ca2[+] and LDH which increased


Conclusion: The high dose of SNP, even for a short time, was toxic. The low dose was safe with respect to viability and proliferation, especially over a short time. However elevated LDH activity might increase anaerobic metabolism


Subject(s)
Animals, Laboratory , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Cell Survival , Cell Proliferation/drug effects , Lactate Dehydrogenases , Anaerobiosis
7.
Braz. oral res. (Online) ; 31: e17, 2017. tab, graf
Article in English | LILACS | ID: biblio-839523

ABSTRACT

Abstract Periodontitis develops as a result of a continuous interaction between host cells and subgingival pathogenic bacteria. The periodontium has a limited capacity for regeneration, probably due to changes in periodontal ligament stem cells (PDLSCs) phenotype. The aim of this study was to evaluate the effects of lipopolysaccharides from Porphyromonas gingivalis (PgLPS) on mesenchymal phenotype and osteoblast/cementoblast (O/C) potential of PDLSCs. PDLSCs were assessed for Toll-like receptor 2 (TLR2) expression by immunostaining technique. After, cells were exposed to PgLPS, and the following assays were carried out: (i) cell metabolic activity using MTS; (ii) gene expression for IL-1β, TNF-α and OCT-4 by real-time polymerase chain reaction (RT-qPCR); (iii) flow cytometry for STRO-1 and CD105, and (iv) osteogenic differentiation. PDLSCs were positive for TLR2. PgLPS promoted cell proliferation, produced IL-1β and TNF-α, and did not affect the expression of stem cell markers, STRO-1, CD105 and OCT-4. Under osteogenic condition, PDLSCs exposed to PgLPS showed a similar potential to differentiate toward osteoblast/cementoblast phenotype compared to control group as revealed by mineralized matrix deposition and levels of transcripts for RUNX2, ALP and OCN. These results provide evidence that PgLPS induces pro-inflammatory cytokines, but does not change the mesenchymal phenotype and osteoblast/cementoblast differentiation potential of PDLSCs.


Subject(s)
Humans , Osteogenesis/drug effects , Periodontal Ligament/cytology , Lipopolysaccharides/toxicity , Porphyromonas gingivalis , Mesenchymal Stem Cells/drug effects , Time Factors , Gene Expression , Osteocalcin/analysis , Cell Differentiation/drug effects , Cell Survival/drug effects , Cells, Cultured , Tumor Necrosis Factor-alpha/analysis , Statistics, Nonparametric , Cell Proliferation/drug effects , Alkaline Phosphatase/analysis , Octamer Transcription Factor-3/analysis , Toll-Like Receptors/analysis , Core Binding Factor Alpha 1 Subunit/analysis , Interleukin-1beta/analysis , Mesenchymal Stem Cells/metabolism , Real-Time Polymerase Chain Reaction , Flow Cytometry
8.
Braz. j. med. biol. res ; 49(6): e5273, 2016. tab, graf
Article in English | LILACS | ID: biblio-951687

ABSTRACT

The present study tested the hypotheses that i) transforming growth factor beta 1 (TGF-β1) enhances differentiation of rat bone marrow mesenchymal stem cells (MSCs) towards the cardiomyogenic phenotype and ii) intramyocardial implantation of the TGF-β1-treated MSCs improves cardiac function in heart failure rats. MSCs were treated with different concentrations of TGF-β1 for 72 h, and then morphological characteristics, surface antigens and mRNA expression of several transcription factors were assessed. Intramyocardial implantation of these TGF-β1-treated MSCs to infarcted heart was also investigated. MSCs were initially spindle-shaped with irregular processes. On day 28 after TGF-β1 treatment, MSCs showed fusiform shape, orientating parallel with one another, and were connected with adjoining cells forming myotube-like structures. Immunofluorescence revealed the expression of cardiomyocyte-specific proteins, α-sarcomeric actin and troponin T, in these cells. The mRNA expression of GATA4 and Nkx2.5 genes was slightly increased on day 7, enhanced on day 14 and decreased on day 28 while α-MHC gene was not expressed on day 7, but expressed slightly on day 14 and enhanced on day 28. Transmission electron microscopy showed that the induced cells had myofilaments, z line-like substances, desmosomes, and gap junctions, in contrast with control cells. Furthermore, intramyocardial implantation of TGF-β1-treated MSCs to infarcted heart reduced scar area and increased the number of muscle cells. This structure regeneration was concomitant with the improvement of cardiac function, evidenced by decreased left ventricular end-diastolic pressure, increased left ventricular systolic pressure and increased maximal positive pressure development rate. Taken together, these results indicate that intramyocardial implantation of differentiated MSCs enhanced by TGF-β1 improved cardiac function in heart failure rats.


Subject(s)
Animals , Male , Bone Marrow Transplantation/methods , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/drug effects , Heart Failure/surgery , Time Factors , RNA, Messenger/analysis , Cell Differentiation , Polymerase Chain Reaction , Reproducibility of Results , Treatment Outcome , Myosin Heavy Chains/analysis , Myocytes, Cardiac/drug effects , Microscopy, Electron, Transmission , GATA4 Transcription Factor/analysis , Homeobox Protein Nkx-2.5/analysis
9.
Biol. Res ; 48: 1-7, 2015. graf
Article in English | LILACS | ID: biblio-950809

ABSTRACT

BACKGROUND: Low survival rate of transplanted cells compromises the efficacy of cell therapy. Hexokinase II (HKII) is known to have anti-apoptotic activity through its interaction with mitochondria. The objective was to identify miRNAs targeting HKII and investigate whether miRNA-mediated modulation of HKII could improve the survival of mesenchymal stem cells (MSCs) exposed to H2O2. The expression of HKII in MSCs exposed to H2O2 was evaluated, and HKII-targeting miRNA was screened based on miRNA-target prediction databases. The effect of H2O2 on the expression of the selected HKII-targeting miRNA was examined and the effect of modulation of the selected HKII-targeting miRNA using anti-miRNA on H2O2-induced apoptosis of MSC was evaluated. RESULTS: H2O2 (600 µM) induced cell death of MSCs and decreased mitochondrial HKII expression. We have identified miR-181a as a HKII-targeting miRNA and H2O2 increased the expression of miR-181a in MSCs. Delivery of anti-miR-181a, which neutralizes endogenous miR-181a, significantly attenuated H2O2-induced decrease of HKII expression and disruption of mitochondrial membrane potential, improving the survival of MSCs exposed to H2O2. CONCLUSIONS: These findings suggest that H2O2-induced up-regulation of miR-181a contributes to the cell death of MSCs by down-regulating HKII. Neutralizing miR-181a can be an effective way to prime MSCs for transplantation into ischemic tissues.


Subject(s)
Humans , Apoptosis , MicroRNAs/metabolism , Mesenchymal Stem Cells/pathology , Glioma/pathology , Hexokinase/metabolism , Hydrogen Peroxide/toxicity , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Cell Differentiation , Cell Movement , Cell Survival , Reactive Oxygen Species , Semaphorins/genetics , Semaphorins/metabolism , MicroRNAs/antagonists & inhibitors , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/enzymology , Real-Time Polymerase Chain Reaction , Glioma/metabolism , Hydrogen Peroxide/administration & dosage , Mitochondria/enzymology , Neoplasm Invasiveness
10.
Biol. Res ; 48: 1-8, 2015. ilus, graf, tab
Article in English | LILACS | ID: lil-734619

ABSTRACT

BACKGROUND: Mesenchymal stem cells (MSCs) are considered the best candidate in stem cells therapy due to their multipotent differentiation ability, low expression of co-stimulatory molecules (CD80, CD86, CD34 and HLA-II) and immunosuppression effects on in vivo immune responses. MSCs were now widely used in clinical trials but received no encourage results. The major problem was the fate of engrafted MSCs in vivo could not be defined. Some studies indicated that MSCs could induce immune response and result in the damage and rejection of MSCs. As toll like receptors (TLRs) are important in inducing of immune responses, in this study we study the role of TLR7 in mediating the immune status of MSCs isolated from umbilical cord. RESULTS: Our results indicated that TLR7 agonist Imiquimod could increase the proliferation of PBMC isolated from healthy human volunteers and release of lactate dehydrogenase (LDH) in supernatant from PBMC-UCMSCs co-culture system. Flow cytometry and quantitative PCR also confirmed the regulated expression of surface co-stimulatory molecules and pro-inflammatory genes (IL-6, IL-8, IL-12, TGF-β and TNF-α). And the down-regulation expression of stem cell markers also confirmed the loss of stemness of UCMSCs. We also found that the osteo-differentiation ability of UCMSCs was enhanced in the presence of Imiquimod. CONCLUSION: To our knowledge, this is the first report that activation of TLR7 pathway increases the immunogenicity of UCMSCs. Extensive researches have now been conducted to study whether the change of immune status will be help in tumor rejection based on the tumor-tropism of MSCs.


Subject(s)
Humans , Adjuvants, Immunologic/pharmacology , Aminoquinolines/pharmacology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/immunology , /agonists , Antigens, CD/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Flow Cytometry , Gene Expression Regulation/drug effects , /analysis , /analysis , /analysis , L-Lactate Dehydrogenase/drug effects , L-Lactate Dehydrogenase , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Membrane Proteins/drug effects , Osteogenesis/drug effects , Real-Time Polymerase Chain Reaction , Transforming Growth Factor beta/analysis , Tumor Necrosis Factor-alpha/analysis
11.
Scientific Journal of Kurdistan University of Medical Sciences. 2014; 19 (4): 79-90
in Persian | IMEMR | ID: emr-153688

ABSTRACT

Environmental contaminants such as bisphenol A, in addition to the environmental problems can affect human health. Epigallocatechingallat has been found to have antioxidant and cytoprotective properties in cultured cells but its effect on toxicity induced by bisphenol A has not yet been determined in mesenchymal stem cells. The aim of this investigation was to study the protective role of epigallocatechingallat in rat bone marrow mesenchymal stem cells following treatment with bisphenol A[an oxidative stress inducer]. Material and In this experimental study, rat bone marrow mesenchymal stem cells were extracted using flashing-out method and cultured in DMEM containing 15% FBS and 100U/ml Pen/Strep. At the end of the third passage, cells were divided into 4 groups: control, bisphenol A, bisphenol A + epigallocatechingallat and epigallocatechingallat. The groups were treated for 12, 24, 36, and 48 hrs. After treatment with bisphenol A and epigallocatechingallat, viability, morphology, rate of DNA damage and calcium content of the cells were evaluated. Data were analyzed by one way ANOVA. p<0.05 was considered significant. Bisphenol A caused a significant reduction in the viability and calcium content of the cells. In addition, morphological changes such as nuclear breakage and chromatin condensation, as well as cytoplasm shrinkage, were observed in the group treated with bisphenol A. We found alterations in these parameters in the group of bisphenol A+ epigallocatechingallat which were similar to those observed in the control ones. Epigallocatechingallat can produce a protective role against the toxic effects of bisphenol A in the mesenchymal stem cells


Subject(s)
Animals, Laboratory , Mesenchymal Stem Cells/drug effects , Bone Marrow , Benzhydryl Compounds , Phenols , Rats , Protective Agents , Cell Survival
12.
Article in English | WPRIM | ID: wpr-223878

ABSTRACT

BACKGROUND: Demineralized bone matrix (DBM) is used for bone healing due to its osteoinductivity, but it requires a carrier for clinical application. Here, we report the effects on the osteoinductivity of DBM by use of a poloxamer 407-based hydrogel as the carrier, compared to sterile water. METHODS: DBM-W and DBM-H represent 27 wt% of DBM with sterile water and DBM with a poloxamer 407-based hydrogel, respectively. Both of the compositions were applied to human mesenchymal stem cell (MSC) cultures, and monitored for alkaline phosphatase (ALP) staining and ALP activity. Six 10-week-old athymic nude rats were used for abdominal muscle grafting with either DBM-W or DBM-H, and were tested by plane radiography, microfocus X-ray computed tomography (CT), and decalcified histology to evaluate ectopic bone formation. RESULTS: The DBM-W group showed stronger ALP staining at 7, 14, and 21 days of treatment, and significantly higher ALP activity at 7 and 14 days of treatment, compared to the DBM-H group. Plane radiography could not confirm the radio-opaque lesions in the rat ectopic bone formulation model. However, ectopic bone formation was observed in both groups by micro-CT. Compared to the DBM-H group, the DBM-W group showed higher bone volume, percent bone volume and trabecular number, and the difference in percent bone volume was statistically significant. Decalcified histology found bony tissue with lamellation in both groups. CONCLUSIONS: Our results suggest that poloxamer 407-based hydrogel has efficacy as a DBM carrier since it shows ectopic bone formation, but its effects on the quality and quantity of osteoblastic differentiation in rat abdominal ectopic bone and MSC are considered negative.


Subject(s)
Animals , Bone Matrix/physiology , Cell Culture Techniques , Decalcification Technique , Excipients/pharmacology , Hydrogels/pharmacology , Male , Mesenchymal Stem Cells/drug effects , Osteogenesis/drug effects , Poloxamer/pharmacology , Rats , Rats, Nude
13.
Acta cir. bras ; 28(5): 353-360, May 2013. ilus
Article in English | LILACS | ID: lil-674155

ABSTRACT

PURPOSE: To evaluate in vitro ability the of three different biomaterials - purified hydroxyapatite, demineralized bone matrix and castor oil-based polyurethane - as biocompatible 3D scaffolds for canine bone marrow mesenchymal stem cell (MSC) intending bone tissue engineering. METHODS: MSCs were isolated from canine bone marrow, characterized and cultivated for seven days with the biomaterials. Cell proliferation and adhesion to the biomaterial surface were evaluated by scanning electron microscopy while differentiation into osteogenic lineage was evaluated by Alizarin Red staining and Sp7/Osterix surface antibody marker. RESULTS: The biomaterials allowed cellular growth, attachment and proliferation. Osteogenic differentiation occurred in the presence of hydroxyapatite, and matrix deposition commenced in the presence of the castor oil-based polyurethane. CONCLUSION: All the tested biomaterials may be used as mesenchymal stem cell scaffolds in cell-based orthopedic reconstructive therapy.


Subject(s)
Animals , Dogs , Bone Matrix , Bone Substitutes/therapeutic use , Castor Oil/therapeutic use , Durapatite/therapeutic use , Mesenchymal Stem Cells/drug effects , Polyurethanes/therapeutic use , Tissue Scaffolds , Bone Regeneration/drug effects , Cell Adhesion , Cells, Cultured , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Materials Testing , Microscopy, Electron, Scanning , Mesenchymal Stem Cells/cytology , Osteogenesis/drug effects , Surface Properties , Tissue Engineering
14.
Braz. j. med. biol. res ; 46(5): 405-416, maio 2013. graf
Article in English | LILACS | ID: lil-675676

ABSTRACT

Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2′-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.


Subject(s)
Adult , Humans , Middle Aged , Adipocytes/drug effects , Cell Differentiation/drug effects , Deoxycytidine/pharmacology , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Mesenchymal Stem Cells/drug effects , Adipocytes/cytology , Blotting, Western , Cells, Cultured , Cell Proliferation/drug effects , DNA Methylation , Epigenomics , Fluorescent Antibody Technique , Polymerase Chain Reaction/methods , Up-Regulation
15.
Arq. bras. endocrinol. metab ; 57(2): 98-111, Mar. 2013. ilus, tab
Article in Portuguese | LILACS | ID: lil-668746

ABSTRACT

OBJETIVO: Avaliar se a triiodotironina (T3) aumenta a diferenciação osteogênica das células-tronco mesenquimais do tecido adiposo (CTM-TA) de ratas adultas ovariectomizadas e com osteoporose e compará-lo ao de ratas adultas e jovens sem osteoporose. MATERIAIS E MÉTODOS: CTM-TA foram cultivadas em meio osteogênico e distribuídas em sete grupos: 1) CTM-TA de ratas jovens sem osteoporose; 2) CTM-TA de ratas adultas sem osteoporose; 3) CTM-TA de ratas adultas com osteoporose e 4, 5, 6 e 7) CTM-TA de ratas adultas com osteoporose tratadas com T3 (0,01 nM, 1 nM, 100 nM e 1.000 nM). AVALIARAM-SE: atividade da fosfatase alcalina, conversão do dimetiltiazol (MTT), porcentagem de nódulos de mineralização, celularidade e quantificação de transcriptos gênicos para colágeno I, osteocalcina, osteopontina e Bmp-2. RESULTADOS: Independente da dose, T3 reduziu a conversão do MTT, a atividade da fosfatase, a porcentagem de células e a expressão de colágeno I em pelo menos uma das doses e dos períodos estudados (p < 0,05). Mas o tratamento com T3 não alterou o número de nódulos de mineralização e a expressão de osteopontina e Bmp-2 em culturas de CTM-TA de ratas adultas com osteoporose (p > 0,05). CONCLUSÃO: T3 apresenta efeitos negativos sobre alguns fatores envolvidos na diferenciação osteogênica de CTM-TA, sem, no entanto, reduzir a formação de nódulos de mineralização e a expressão de proteínas ósseas.


OBJECTIVE: To examine if triiodothyronine (T3) increases osteogenic differentiation adipose tissue derived stem cells (ASCs) from ovariectomized adult rats with osteoporosis compared with young rats and adult rats without osteoporosis. MATERIALS AND METHODS: The ASCs were cultured in osteogenic medium and distributed into seven groups: 1) ASCs of young rats without osteoporosis; 2) ASCs of adult rats without osteoporosis; 3) ASCs of adult rats with osteoporosis and 4, 5, 6 and 7) ASCs of adult rats with osteoporosis treated with T3 (0.01 nM, 1 nM, 100 nM and 1,000 nM). We analyzed alkaline phosphatase activity, dimethylthiazol (MTT) conversion, percentage of mineralized nodules, cellularity and quantification of gene transcripts for collagen I, osteocalcin, osteopontin and Bmp-2. RESULTS: Regardless of the dose, T3 reduced the MTT conversion, alkaline phosphatase activity, percentage of cells and the expression of collagen I in at least one of the doses and periods studied (p < 0.05). But, the treatment with T3 does not modify the number of mineralized nodules and the expression of osteopontin and Bmp-2 in culture of ASCs from adult rats with osteoporosis (p > 0.05). CONCLUSION: T3 has a negative effect on some factors involved in osteogenic differentiation of ASCs from adult rats with osteoporosis, without; however, reduce the formation of mineralized nodules and the expression of bone proteins.


Subject(s)
Animals , Female , Rats , Adipose Tissue/cytology , Mesenchymal Stem Cells/drug effects , Osteoporosis , Osteogenesis/drug effects , Triiodothyronine/pharmacology , Age Factors , Alkaline Phosphatase/metabolism , Cell Differentiation , Mesenchymal Stem Cells/enzymology , Mesenchymal Stem Cells/physiology , Ovariectomy , Osteogenesis/physiology , Osteoporosis/pathology , Rats, Wistar , Real-Time Polymerase Chain Reaction
16.
Arq. bras. endocrinol. metab ; 57(1): 62-70, fev. 2013. graf, tab
Article in Portuguese | LILACS | ID: lil-665764

ABSTRACT

OBJETIVO: Avaliar se a adição de T3 aumenta o potencial osteogênico das células-tronco mesenquimais da medula óssea (CTM-MO) de ratas adultas normais comparado ao de ratas jovens. MATERIAIS E MÉTODOS: CTM-MO foram cultivadas em meio osteogênico e separadas em seis grupos: 1) CTM-MO de ratas jovens; 2) CTM-MO de ratas adultas; 3, 4, 5 e 6) CTM-MO de ratas adultas com T3 nas concentrações de 0,01; 1; 100 e 1000 nM, respectivamente. Foram avaliados: atividade da fosfatase alcalina, conversão do dimetiltiazol (MTT) e síntese de colágeno aos sete, 14 e 21 dias e celularidade e número de nódulos de mineralização aos 21 dias de diferenciação. RESULTADOS: T3 reduziu significativamente a conversão do MTT, a atividade da fosfatase alcalina, a síntese de colágeno e a formação dos nódulos de mineralização em pelo menos uma das doses e dos períodos estudados (p < 0,05). Os valores foram menores quando comparados aos das CTM-MO de ratas jovens e adultas sem T3 (p < 0,05). CONCLUSÃO: T3 apresenta efeitos negativos sobre os fatores envolvidos na diferenciação osteogênica das CTM-MO de ratas adultas.


OBJECTIVE: To examine if triiodothyronine (T3) increases osteogenic differentiation in bone marrow mesenchymal stem cells (BMMSCs) of adult rats compared with young rats. MATERIALS AND METHODS: BMMSCs were cultured in osteogenic medium and distributed into six groups: 1) BMMSCs of young rats; 2) BMMSCs of adult rats; 3, 4, 5 and 6) BMMSCs of adult rats with T3 (0.01, 1, 100 to 1000 nM). We analyzed alkaline phosphatase activity, dimethylthiazol (MTT) conversion, and collagen synthesis at 7, 14, and 21 days, and percentage of cells per field and number of mineralized nodules at 21 days of differentiation. RESULTS: T3 reduced MTT conversion, alkaline phosphatase activity, collagen synthesis, and the synthesis of mineralizalized nodules in at least one of the doses and periods studied (p < 0.05). Values were lower when compared with young and adult rats BMMSCs (p < 0.05) without T3. CONCLUSION: T3 has a negative effect on the factors involved in osteogenic differentiation of BMMSC from adult rats.


Subject(s)
Animals , Female , Rats , Bone Marrow Cells/drug effects , Cell Differentiation/drug effects , Mesenchymal Stem Cells/drug effects , Osteogenesis/drug effects , Triiodothyronine/pharmacology , Analysis of Variance , Alkaline Phosphatase/metabolism , Bone Marrow Cells/cytology , Cells, Cultured , Calcification, Physiologic/drug effects , Collagen/metabolism , Models, Animal , Mesenchymal Stem Cells/cytology , Phenotype , Rats, Wistar , Tetrazolium Salts/metabolism , Thiazoles/metabolism
17.
Braz. j. med. biol. res ; 46(1): 39-51, 11/jan. 2013. tab, graf
Article in English | LILACS | ID: lil-665801

ABSTRACT

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.


Subject(s)
Animals , Male , Mice , Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Hematopoietic Stem Cells/drug effects , Mesenchymal Stem Cells/drug effects , Piperazines/pharmacology , Pyrimidines/pharmacology , Cell Proliferation , Cell Survival , Cells, Cultured , Colony-Forming Units Assay , Fibroblasts , Hematopoietic Stem Cells/cytology , Immunohistochemistry , Mesenchymal Stem Cells/cytology
18.
Journal of Dental Medicine-Tehran University of Medical Sciences. 2013; 26 (1): 55-63
in English, Persian | IMEMR | ID: emr-143239

ABSTRACT

The exfoliated human deciduous tooth contains multipotent stem cells [Stem Cell from Human Exfoliated Deciduous tooth [SHED]] that identified to be a population of highly proliferative and clonogenic. These cells are capable of differentiating into a variety of cell types including osteoblast/osteocyte, adiopcyte, chondrocyte and neural cell. The aim of this study was to evaluate the differentiation of SHED to osteoblast in standard osteogenic medium and comparing the results with medium which supplemented with glucosamine in form of chitosan. Dental pulp cells were isolated from freshly extracted primary teeth, digested with 4 mg/ml collogenase/dispase, and grown in Dulbecco's modified Eagle's medium with 10 percent fetal bovine serum. The clonogenic potential of cells was performed after 3 weeks of culture. Flowcytometric analysis, performed at day 21 of culture to identify surface markers of mesenchymal stem cells. The cells from 3rd passage were used for osteogenic differentiation in routine osteoinductive medium. Chitosan [10 microg/ml] was added to the culture medium of case group. Alizarin Red Staining and Alkaline Phosphatase [ALP] activity were done to evaluate osteogenic differentiation in the developing adherent layer on the third passage. The results were analyzed using T-test. For the analysis of normal distribution of data, non-parametric Kolmogrov-Smirnov test was used. The colonogenic efficiency was more than 80%. Flowcytometric analysis showed that the expression of mesenchymal stem cell marker CD90, CD 105 and CD146 were positive in SHED, while hematopoietic cell marker CD34, CD45 and endothelial cell marker CD31 were negative. Quantitative analysis of Alizarin Red Staining demonstrated that: mineralized nodule formation was higher in the group supplemented with glucosamine [chitosan]. Results from Alkaline Phosphatase activity test, on day 21, demonstrated a significantly higher ALP activity in the group supplemented chitosan [P<0.001]. Stem cells isolated and cultured from exfoliated deciduous teeth pulp can be differentiated to osteoblast. Addition of chitosan can be beneficial to promote osteogenic differentiation of these cells


Subject(s)
Osteogenesis , Mesenchymal Stem Cells/drug effects , Tooth, Deciduous , Tooth Exfoliation , Osteoblasts , Glucosamine
19.
Journal of Medicinal Plants. 2013; 12 (46): 50-59
in Persian | IMEMR | ID: emr-140338

ABSTRACT

Osteoporosis is one of the most prevalent disease in current century .Estrogen deficiency is the basic cause of osteoporosis in menopaused women. Hormone Replacement Therapy [HRT] can increase the risk of breast and ovary cancers. Medicinal plant are natural source of secondary metabolite and can a reliable source for treating osteoporosis. Ferula gummosa [Galbanum] has been used in traditional medicine since ancient time. This study is focused on determining the effect of Galbanum root ethanolic extract on osteogenesis progress in Human Mesenchymal Stem Cells [hMSCs]. The Bone Marrow hMSCs were seeded at 12 well plates and treated with different amount of Galbanum extracts [0.5 to 100 micro g/ml]. Extract cytotoxicity were measured using MTT method .Effect of extract on osteogenesis was evaluated in time interval 7 and 14 days using_ Alkaline Phosphatase_ enzyme activity method. The data analysis revealed a significant increase in cell proliferation in range of [0.5 to 5] micro g/ml after 24, 48 and 72 hour of treatment with galbanum extract. Analysis of result revealed a significant increase in alkaline phosphatase [ALP] enzyme activity in the range of 1 to 10 micro g/ml compared with control group. Ferula gumossa has been used in Iranian folk medicine for many years. Our in vitro study showed that Frula gummosa extract has osteoprotective effect


Subject(s)
Humans , Plant Extracts , Ethanol , Osteogenesis/drug effects , Mesenchymal Stem Cells/drug effects , Alkaline Phosphatase
20.
Article in English | WPRIM | ID: wpr-219416

ABSTRACT

The use of mesenchymal stem cells (MSCs) has emerged as a potential new treatment for myocardial infarction. However, the poor viability of MSCs after transplantation critically limits the efficacy of this new strategy. The expression of microRNA-210 (miR-210) is induced by hypoxia and is important for cell survival under hypoxic conditions. Hypoxia increases the levels of hypoxia inducible factor-1 (HIF-1) protein and miR-210 in human MSCs (hMSCs). miR-210 positively regulates HIF-1alpha activity. Furthermore, miR-210 expression is also induced by hypoxia through the regulation of HIF-1alpha. To investigate the effect of miR-210 on hMSC survival under hypoxic conditions, survival rates along with signaling related to cell survival were evaluated in hMSCs over-expressing miR-210 or ones that lacked HIF-1alpha expression. Elevated miR-210 expression increased survival rates along with Akt and ERK activity in hMSCs with hypoxia. These data demonstrated that a positive feedback loop involving miR-210 and HIF-1alpha was important for MSC survival under hypoxic conditions.


Subject(s)
Cell Survival , Cobalt , Gene Expression Regulation/physiology , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Mesenchymal Stem Cells/drug effects , MicroRNAs/metabolism , Oxygen/pharmacology , Oxygen Consumption , RNA, Small Interfering/metabolism
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