Para-nonylphenol impairs osteogenic differentiation of rat bone marrow mesenchymal stem cells by influencing the osteoblasts mineralization (original) (raw)
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Iranian journal of basic medical sciences, 2015
In previous investigations, we have shown para-nonylphenol (p-NP) caused significant reduction of proliferation and differentiation of rat bone marrow mesenchymal stem cells (MSCs) in vitro. In this study, we first treat the rats with p-NP, then carried out the biochemical and morphological studies on MSCs. Proliferation property of cells was evaluated with the help of MTT assay, trypan blue, population doubling number, and colony forming assay. Differentiation property was evaluated with quantitative alizarin red assay, measurement of alkaline phosphatase (ALP) activity as well as intracellular calcium content. In addition; morphological study, TUNEL test, activated caspase assay, and comet assay were performed to evaluate the mechanism of the cell death. The results showed significant reduction in the colony-forming-ability and population-doubling-number of extracted cells when compared to control ones. In addition, it was revealed that the p-NP treatment of rats caused significan...
2020
Objectives: Previously, it was found that the para-nonylphenol (p-NP) impairs the osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs); thus the aim of the present study was to evaluate the mechanism of the impairment. Methods: rBMSCs after 3 rd passage cultured in osteogenic media in the presence of 0, 0.5 and 2.5 µM p-NP for 5, 10, 15 and 20 days. The study investigated the viability of the cells using MTT assays. The mineralization was studied using Alizarin red quantification analysis. Using a flame-photometer, the electrolytes (sodium and potassium) were measured, and the level of calcium as well as ALT, AST, ALP and LDH was determined by commercial kits. The level of total-antioxidant, MDA and the activity of SOD and CAT were estimated with the help of a spectrophotometer. Gene expression was studied using rt-PCR. Results: The p-NP treatment of osteogenic differentiated MSCs showed intracellular electrolyte imbalance and variation of cellular metabolism. In addition, we observed oxidative stress due to the reduction of total antioxidant capacity and the imbalance of antioxidant enzymes activity. Investigating the genes involved in the osteogenic differentiation of MSCs to osteoblast showed that the 2.5 µM of p-NP reduced the expression of the ALP, SMAD, BMP and RUNX2 genes. Conclusion: The study concludes that this pollutant via influencing the genomics and metabolic imbalance, as well as oxidative induction, caused a reduction of mineralization and differentiation of MSCs. This environmental pollutant might cause osteoporosis, which necessitates raising public awareness, especially to those who live in the industrial area to prevent its drastic effect.
Revista eletrônica de Farmácia, 2012
Menopause is caused by the failure of ovarian hormones production and can produce unfavorable changes in lipid, glucose and insulin metabolism, in the coagulation/fibrinolysis system, as well as bone loss are some of its consequences. One of the current concerns is the relationship between the decrease in estrogen production and bone mass loss, which is considered a major risk factor for the development of osteoporosis in women. Osteoporosis is currently considered a worldwide public health problem and it is estimated that by 2030, more than one billion and 200 million women will be menopausal. One of the ways to treat menopause symptoms and decrease the chance of someone to develop osteoporosis is the use of the Hormone Replacement Therapy (HRT). However, this therapy has brought some risks to the health of some groups of women, especially those with a history of thromboembolic diseases and breast cancer in their families, and the use of phytoestrogens such as isoflavones is an alternative to the traditional treatment. Isoflavones are found mainly in soybeans (Glycine max), red clover (Trifolium pratense), Cimicifuga racemosa (of American origin) and rye. Objective: In this work we compare the ability of two phytoestrogens preparations (one is the soybean extract biotransformed by the fungus Aspergillus awamori (ESBF) produced by Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, and the other is Menoflavon ® 40 mg (Melbrosin International) composed of Trifolium pratense isoflavones) to stimulate osteogenesis in vitro, using osteoblasts cultures derived from mesenchymal stem cells. Methods: Evaluation of cell viability by the MTT method and formation of mineralized matrix. Results: a) In the viability assays, ESBF was 106.3% viable at a concentration of 0.5 μg/mL, 93.1% viable at a concentration of 1 μg/mL and 92.4% viable at a concentration of 4 μg/mL. For these concentrations of 0.5 μg/mL, 1 μg/mL and 4 μg/mL ESBF, the main soybean metabolites (daidzein (D) and genistein (G)) were determined by HPLC and we found 1.181 nM D and 0.922 nM G; 2.361 nM D and 1.845 nM G; and 9.445 nM D and 7.379 nM G, respectively. When using Menoflavon ® for the viability assays, we found 97.8%, 85.4% and 86.5% of live cells at a concentration of 0.5 μg/mL, 1 μg/mL and 4 μg/mL, respectively, corresponding to 7.5 ng/mL G and 28.75 ng/mL D; 15 ng/mL G and 57.5 ng/mL D; 60 ng/mL G and 230 ng/mL D, respectively. b) The formation of mineralized matrix on day 14 of culture by ESBF was 105.7%, 114.8% and 101.7%, and at day 21 of culture, it was 105.2%, 79.9% and 79.5%, at a concentration of 0.5 μg/mL, 1 μg/mL and 4 μg/mL, respectively. When using Menoflavon ® , the formation of mineralized matrix on day 14 of culture was 104.1%, 83.6% and 106.4%, and at day 21 of culture, it was 98.6%, 87.8% and 83.5%, at a concentration of 0.5 μg/mL, 1 μg/mL and 4 μg/mL, respectively. D and G values were the same as presented above (item a). Conclusion: With the results obtained, it is not possible to conclude which isoflavones preparation out of the two is the best to induce bone mineralization.
The mechanism of para-nonylphenol (PNP) reducing the proliferation and differentiation of bone marrow mesenchymal stem cells (MSCs) is not known. The present study was designed to investigate the mechanism. Methods: MSCs were extracted under sterile condition from Wistar rat and cultured in DMEM, containing 15 % FBS and penicillin/streptomycin until the 3 rd passage, then cells were treated with 0, 0.5 and 2.5 µM of PNP for 5, 10, 15 and 20 days. We studied the viability, proliferation, cell cycle and morphology of the cells. In addition, the concentration of total protein, sodium, potassium and calcium and the activity of metabolic enzymes (ALT, AST and LDH) were determined. Also, induction of oxidative stress was estimated by determining the total antioxidant and MDA levels in addition to the activity of SOD and CAT. Results: The concentrations of PNP caused a significant increase in metabolic enzymes activity and reduced the total protein dose dependently from day 5 to day 20. But only the higher PNP concentration reduced the sodium level and increased the calcium concentration during the treatment period. In addition, we observed a significant decrease in the total antioxidant level and of SOD and CAT activities whereas a significant increase in MDA was seen. Also, PNP stopped the cell cycle at "S" and "G2/M" phases. Conclusion: Para-NP, used in many industries, was able to reduce the viability and proliferation of the MSCs via metabolic and electrolyte imbalance and by induction of oxidative stress and cell cycle disruption.
P-Nonylphenol (p-NP) as a non ionic surfactant is used in many industries such as pesticides, cosmetics, detergents and so on. Our previous study showed that the p-NP causes reduction in viability of the rats Bone Marrow Mesenchymal Stem cells (MSCs). The aim of the present study was to investigate the mechanism of the cell death due to p-NP exposure. The cells, after 3 rd passage were treated with 100µM p-NP for a period of 36 hrs, then using TUNEL, caspase and comet assay as well as fluorescent dye like Hochest, acridine orange and monodansylcadaverin (MDC) staining the mechanism of cell death was studied. The results of this study showed that the p-NP treated cells were TUNEL positive and also, activated caspase-3 enzyme in their cytoplasm was visualized. Hochest staining showed chromatin condensation and nuclei breakage, at which the nuclear breakage was further confirmed by comet assay. In addition, staining the cells with acridine orange revealed that the cytoplasm of the treated cells contained numerous vesicles. Furthermore, staining of the cells with MDC showed the presence of the double layer autophagic vacuoles. In conclusion, the induced cell death due to p-NP toxicity was determined to be caspase dependent apoptosis as well as autophagy.
2010
Bone marrow contains cell type termed mesenchymal stem cells (MSC), first recognized in bone marrow by a German pathologist, Julius Cohnheim in 1867. That MSCs have potential to differentiate in vitro in to the various cells lines as osteoblast, chondroblast, myoblast and adipoblast cells lines. Aims of our study were to show in vivo capacity of bone marrow MSC to produce bone in surgically created non critical size mandible defects New Zeland Rabbits, and then in second part of study to isolate in vitro MSC from bone marrow, as potential cell transplantation model in bone regeneration. In vivo study showed new bone detected on 3D CT reconstruction day 30, on all 3 animals non critical size defects, treated with bone marrow MSC exposed to the human Bone Morphogenetic Protein 7 (rhBMP-7). Average values of bone mineral density (BMD), was 530 mg/cm3, on MSC treated animals, and 553 mg/cm3 on control group of 3 animals where non critical size defects were treated with iliac crest autol...
International Journal of Hematology-Oncology and Stem Cell Research
Background: In vitro impact of dihydrotestosterone (DHT) and 17-estradiol (E2) in osteogenic differentiation of castrated rat bone marrow mesenchymal stem cells (rBMMSC) still need to be clarified. Materials and Methods: The viability, proliferation and density of cultured rBMMSC isolated from sham operated (Sham) and castrated (Cast) male rats were evaluated. rBMMSC were cultured with osteogenic differentiating medium (ODM) in the presence of DHT (5,10 nM) and E2 (10,100 nM). Osteogenesis was evaluated by alizarin red staining and measurement of calcium deposition and bone alkaline phosphatase (BALP) activity. Results: Population doubling (PD) of rBMMSC isolated from Cast rats was significantly lower (P<0.05) compared to that isolated from Sham rats. rBMMSC from Cast rats showed low scattered calcified nodule after culturing in ODM and did not cause a significant increase in calcium deposition and B-ALP activity compared to rBMMSCs from Sham rats. Exposure of rBMMSC isolated fr...
Nitric Oxide-biology and Chemistry, 2008
Human adult bone marrow contains hematopoietic lineage cells as well as a heterogeneous population of cells making up the marrow stroma . Stromal cells include adipocytes, reticular cells, endothelial cells, and fibroblastic cells. They are in direct contact with hematopoietic cells and support the differentiation of Background: It has been frequently reported that culture-expanded mesenchymal stem cells from bone marrow of healthy donors can be induced to differentiate to osteocytic lineage. This study examined the potential for osteogenic differentiation of mesenchymal stem cells obtained from two patients with myeloproliferative disorders. Methods: Mesenchymal stem cells were derived from bone marrow aspirates obtained in an outpatient clinic from two patients, one with polycythemia vera and the other with essential thrombocythemia. Nucleated bone marrow cells were directly cultured in flasks. Adherent fibroblastic cells in monolayers were isolated by removing nonadherent cells during medium changes. Osteogenic differentiation was induced in expanded adherent cells for 2 weeks in osteogenic medium containing 100 nmol/ L dexamethasone, 10 mmol/L β-glycerophosphate, and 0.05 mmol/L L-ascorbic acid-2-phosphate. Osteogenic differentiation was evaluated by alkaline phosphatase staining and determination of calcium in deposited minerals on culture plates. The expression of osteopontin mRNA was determined by reverse transcription-polymerase chain reaction. Results: After induction in osteogenic medium, the expression of alkaline phosphatase in mesenchymal stem cells became more intense. Induced alkaline phosphatase-positive cells assumed an irregular shape with multiple spiculate projections, while uninduced alkaline phosphatase-positive cells had a flattened polygonal shape or were elongated. Calcium deposition on the plates of induced cells was 0.39 ± 0.03 µmol/well in cells from the patient with polycythemia vera and 0.54 ± 0.03 µmol/ well in cells from the patient with essential thrombocythemia, but was not detectable in uninduced cells from either patient. Induction by osteogenic medium markedly increased the expression of osteopontin mRNA in stem cells derived from both patients. Conclusions: In this study, mesenchymal stem cells obtained from aspiration of bone marrow in patients with myeloproliferative disorders were expanded by culture. After osteogenic induction, these cells were shown to be able to differentiate into osteocytic lineage in vitro.
Alcoholism: Clinical & Experimental Research, 2004
Background: Alcohol-induced osteoporosis is characterized by a considerable suppression of osteogenesis. The objective of this investigation was to determine the effect of alcohol on gene expression, protein synthesis, and mineralization in human bone marrow-derived mesenchymal stem cells induced toward osteogenic differentiation in vitro. Methods: Human bone marrow-derived mesenchymal stem cells induced toward osteogenesis were cultured in the presence or absence of 50 mM alcohol. Stem cells were characterized by using SH2 antibody to the cell-surface antigen CD105/endoglin, and their proliferation in the presence of alcohol was quantified. The expression of genes for early, middle, and late markers of the osteogenic lineage was quantified by Northern analysis, and bone matrix protein synthesis was assayed. The effect of alcohol on cell-mediated matrix mineralization in terminally differentiated cultures was determined by von Kossa staining. Results: Fluorescence-activated cell sorting analysis of human mesenchymal stem cells separated with a Percoll gradient proved 99% homogeneity by using SH2 antibody to the surface antigen CD105. Dosedependent inhibition of proliferation of these stem cells occurred at concentrations greater than 50 mM alcohol. Gene expression of osteoblast-specific factor 2/core binding factor a1 (Osf2/Cbfa1), type I collagen, alkaline phosphatase, and osteocalcin (early, middle, and late markers for osteogenesis, respectively) was analyzed with and without osteogenic induction and treatment with 50 mM alcohol. After induction, Osf2/Cbfa1 levels were unresponsive to alcohol. To determine the effect of alcohol on human mesenchymal stem cell progression along the osteogenic pathway, messenger RNA (mRNA) levels for type I collagen, alkaline phosphatase, and osteocalcin were examined after osteogenic induction. After osteogenic induction, alcohol down-regulated the gene expression of type I collagen and significantly reduced its synthesis. Alcohol did not alter mRNA expression of alkaline phosphatase, a midstage marker for osteogenesis, but significantly decreased its activity compared with osteogenic induction alone. After induction, osteocalcin remained unchanged by alcohol at both the mRNA and protein levels. Histochemistry revealed decreased alkaline phosphatase staining and fewer alkaline phosphatase-positive cells in alcohol-treated human mesenchymal stem cell cultures. von Kossa staining revealed a reduction in the number of mineralizing nodules in stem cell cultures after alcohol treatment. Conclusions: Collectively, the data suggest that alcohol alters osteogenic differentiation in human bone marrow-derived mesenchymal stem cell cultures during lineage progression and provide further insight into alcohol-induced reduced bone formation.
Toxicology in Vitro, 2006
The purpose of this study was to investigate the in vitro effects of resveratrol (RSVL) and cyclosporin A (CsA) on proliferation and osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cell (BMSC) cultures. Application of RSVL (10 À8 -10 À6 mol l À1 ) resulted in a dose-dependent increase in [ 3 H]-thymidine incorporation, alkaline phosphatase (ALP) activity and calcium deposition of BMSCs cultures, which was accompanied with the increase of NO production and cGMP content. Concurrent treatment with the estrogen receptor antagonist ICI182,780 (10 À7 mol l À1 ) or the NO synthase inhibitor, N x -nitro-L-arginine methyl ester (6 · 10 À3 mol l À1 ) abolished the RSVL (10 À6 mol l À1 )-induced increase in NO production and cGMP content and eliminated the RSVL-induced increase in proliferation and osteoblastic differentiation of BMSCs. In contrast, CsA (10 À6 -10 À5 mol l À1 ) dosedependently decreased [ 3 H]-thymidine incorporation, ALP activity and calcium deposition of BMSCs cultures, which was accompanied with the reduction of NO production in the conditioned media. Concurrent treatment with RSVL (10 À6 mol l À1 ) significantly reversed the CsA (3 · 10 À6 mol l À1 )-mediated decrease in NO production and restored the proliferation and differentiation potential of BMSCs. Our data suggest that (1) the NO/cGMP pathway may play an important role in both RSVL-induced and CsA-inhibited proliferation and osteoblastic differentiation of mouse BMSCs, and (2) RSVL may act through an ER/NO/cGMP pathway to reverse the inhibitory effect of CsA on BMSC cultures. Taken together, the data suggest that RSVL may prevent osteoporosis induced by CsA.