Experimental Study on Construction of Tissue Engineered Bone by Autologous Oxygen Release Nano-Bionic Scaffold Combined with Bone Morphogenetic Protein-2 Induced Bone Marrow Mesenchymal Stem Cells

2019 ◽  
Vol 9 (9) ◽  
pp. 1254-1260
Author(s):  
Fei Wang ◽  
Hongfang Wei ◽  
Chengdong Hu ◽  
Dongfeng Li ◽  
Xiwei Huo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Type Ii Collagen ◽  
Negative Control Group ◽  
Negative Control ◽  
Control Group ◽  
Type Ii ◽  
Oxygen Release ◽  
Fracture Group ◽  
Bionic Scaffold ◽  
Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) are used for bone tissue engineering. BMP-2 and autologous oxygen-releasing nano-bionic scaffolds promote bone differentiation of BMSCs. Our study intends to evaluate the role of autologous oxygen-releasing nano-bionic scaffolds combined with BMP-2-induced BMSCs in the construction of tissue engineered bone. Rat BMSCs were isolated and transfected with NC (negative control group) and BMP-2 (BMP-2 plasmid group), respectively. Healthy male SD rats were randomly and equally divided into fracture group, negative control group and the BMP-2 group which was implanted with autologous oxygen-releasing nano-bionic scaffolds to synthesize BMSCs and transfected with BMP-2 plasmids respectively followed by analysis of osteophytes growth, ALP activity, expression of BMP-2, type II collagen, Runx2 and OC by real time PCR, TGF-β1 secretion by ELISA and BMP-2 protein expression by western blot. BMSCs induced by autologous oxygen release nano-bionic scaffold combined with BMP-2 can significantly promote the increase of bone mineral density, increase the expression of Runx2 and OC, promote ALP activity, upregulate type II collagen, BMP-2 mRNA and protein, and TGF-β1 secretion compared to fracture group (P < 0.05). The BMSCs induced by autologous oxygen-releasing nanobionic scaffolds transfected with BMP-2 had a more significant effect on bone repair. Autologous oxygen-releasing nano-bionic scaffolds combined with BMP-2-induced BMSCs can promote bone healing by regulating BMP-2 and increasing osteogenesis at the bone defect.

scholarly journals Levels of matrix metalloproteinase-2 in committed differentiation of bone marrow mesenchymal stem cells induced by kartogenin

2019 ◽  
Vol 47 (7) ◽  
pp. 3261-3270
Author(s):  
Cheng Wang ◽  
Qiaohui Liu ◽  
Xiaoyuan Ma ◽  
Guofeng Dai
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Matrix Metalloproteinase ◽  
Type Ii Collagen ◽  
Matrix Metalloproteinase 2 ◽  
Type Ii ◽  
Proliferation And Differentiation ◽  
Marrow Mesenchymal Stem Cells

Objective To measure the inductive effect of kartogenin on matrix metalloproteinase-2 levels during the differentiation of human bone marrow mesenchymal stem cells (hMSCs) into chondrocytes in vitro. Methods In vitro cultured bone marrow hMSCs were grown to the logarithmic phase and then divided into three groups: control group (0 µM kartogenin), 1 µM kartogenin group and 10 µM kartogenin group. After 72 h of culture, cell proliferation and differentiation were observed microscopically. Matrix metalloproteinase-2 (MMP-2) in the cell supernatant and type II collagen levels in the cells were detected by enzyme linked immunosorbent assay and immunofluorescence staining, respectively. Results Kartogenin induced the proliferation and differentiation of hMSCs. With the increase of kartogenin concentration, the level of type II collagen was increased, while the level of MMP-2 decreased. Conclusion These findings indicate that kartogenin can induce hMSCs to differentiate into chondrocytes, and with the increase of kartogenin concentration, degeneration of the cartilage extracellular matrix may be inhibited.

Mir-149 Promotes Survival and Differentiation of Bone Marrow Mesenchymal Stem Cells by Regulating Interleukin-6/Signal Transducer and Activator of Transcription 3 Signaling Pathway

2019 ◽  
Vol 9 (8) ◽  
pp. 1160-1166
Author(s):  
Guozhong Qin ◽  
Shaochuan Huo ◽  
Juehui Li ◽  
Yin Lian ◽  
Xiaoli Jin
Keyword(s):  
Bone Marrow ◽  
Signaling Pathway ◽  
Caspase 3 ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Alp Activity ◽  
Stat3 Signaling ◽  
Stat3 Signaling Pathway ◽  
Marrow Mesenchymal Stem Cells ◽  
Sirna Group

Bone marrow mesenchymal stem cells (BMSCs) can self-renew with multi-directional differentiation. Mir-149 is involved in various diseases, but whether Mir-149 regulates the survival and differentiation of BMSCs and related mechanisms remains unclear. BMSCs were isolated and randomly divided into Si-NC group, Mir-149 siRNA group, and Mir-149 siRNA + STAT3 inhibitor WP1066 group followed by analysis of the expression of Mir-149, RUNX2 and OPN mRNA by real time PCR, BMSCs proliferation by MTT assay, Caspase 3 activity, ALP activity, formation of type II collagen and IL-6 level by ELISA, as well as STAT3 signaling pathway expression by Western blot. Mir-149 expression was reduced in BMSCs of Mir-149 siRNA group, with promoted survival of BMSCs, decreased Caspase 3 activity, increased expression of RUNX2 and OPN, type II collagen formation, ALP activity, IL-6 secretion, as well as elevated pSTAT3 phosphorylation. The differences were statistically significant compared to Si-NC group (P < 0.05). Mir-149 siRNA + WP1066 inhibited pSTAT3 phosphorylation, reduced BMSCs survival, increased Caspase 3 activity, decreased RUNX2 and OPN expression, type II collagen production, ALP activity, as well as reduced IL-6 secretion. Compared with Mir-149 siRNA group, there were significant differences (P < 0.05). Down-regulation of Mir-149 in BMSCs can promote BMSCs survival and osteogenic differentiation by regulating IL-6/STAT3 signaling pathway.

scholarly journals Assessment the Genotoxic Potential of Fluoxetine and Amitriptyline at Maximum Therapeutic Doses for Four-Week Treatment in Experimental Male Rats

2021 ◽  
Vol 30 (1) ◽  
pp. 81-90
Author(s):  
Imad A. Al-Obaidi ◽  
Nada N. Al-Shawi
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Adult Male ◽  
Negative Control Group ◽  
Negative Control ◽  
Male Rats ◽  
Control Group ◽  
Group Iii ◽  
Group I ◽  
Hydrochloride Solution

Abstract At any moment, the continuous usage of medications can accompanied by DNA damage and the accumulation of such damages can cause serious consequences. Antidepressants are long-term used drugs and the incidence of their genotoxic impacts cannot be excluded. Therefore, this work was designed to investigate the possible genotoxic effects of the commonly used antidepressants (fluoxetine and amitriptyline) in adult male rats. Detection of DNA damage in individual cells was assessed by comet and micronucleus assays in three different cell populations i.e. liver, testis and bone marrow tissues of 24 swiss albino adult male rats. The animals were randomly allocated into three groups of 8 rats each: Group I - rats orally-administered distilled water via gavage tube for four weeks as a negative control. Group II - rats orally-treated with fluoxetine hydrochloride solution (7.2mg/kg/day) via gavage tube for four weeks. Group III - rats orally-treated with amitriptyline hydrochloride solution (27mg/kg/day) via gavage tube for four weeks. The results showed that both drugs (Group II and Group III) induced the same extent of DNA damage, as evidenced by a significantly higher DNA fragmentation in liver and testis tissues with increased frequencies of micronuclei formation in bone marrow tissues as compared with the negative control (Group I). These findings indicates that both Fluoxetine and Amitriptyline have genotoxic potentials and can induce the same extent of cytogenetic damage in rats. Special precautions and medical supervision should be taken in consideration with their uses.

Blocking Stat3 to Regulate the Balance of Th17/Treg in Cgvhd Therapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5420-5420
Author(s):  
Jianyu Weng ◽  
Xin Huang ◽  
Xiaomei Chen ◽  
Qiong Luo ◽  
Jianhua Su ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Negative Control Group ◽  
Treg Cells ◽  
Negative Control ◽  
Control Group ◽  
Technology Planning ◽  
Planning Project ◽  
Target Organs ◽  
Significant Difference

Abstract Background: Our preliminary research found that STAT3, IL-17A, and IL-21 expressed in cGVHD patients. So, we provide the blocking STAT3 signal to the induction of Treg cells differentiation, and to provide experimental basis on new targets of cGVHD immunotherapy. Methods: 1. Mice spleen CD4+ CD62L+ naiveT cells were separated by immune magnetic bead and then activated for 72 h. After 96 h infection with STAT3-shRNA and negative control lentivirus, the Th subgroup proportion were measured by flow cytometry. Th related cytokines levels test by Luminex. Real time quantitative PCR was to detect STAT3 and Th subgroup related transcription factor mRNA levels. CD117+ mouse bone marrow stem progenitor cells were sorted by flow cytometry, and transfected by STAT3-shRNA. Inhibition of STAT3 gene in mRNA level was measured at 96 h. Cell proliferation activity was test with CCK8 kit, and cell apoptosis rate determined by flow cytometry. Differentiation of CD117+ cells was induced by 2.2% of methyl cellulose and different cytokines. 2. BALB/c female mice, after the linear accelerator 700cGy of whole body irradiation, accepted miHA mismatched male B10. D2 mice bone marrow cells and spleen cells (8 x 106, 1:1). Randomly assigned 6 mice of cGVHD clinical score of 0.6 or above to each group. After STAT3-shRNA or negative control lentivirus treatment, the observe end point was 58th day after transplantation. The clinical and pathologic scores compared. Th17 and Treg cells measured by flow cytometry. Th related cytokines measured by Luminex. Purpose genes in blood and protein expression levels in target organs were found by Q-PCR and western blot test, respectively. Results: 1. The Th17 / Treg ratio of shRNA group was significantly decreased than that in the NC group (P < 0.05). Except for the Foxp3 gene, other purpose genes, including T-bet, Gata3, RORγt, TGFβ, Notch1, and Jagged2 mRNA levelsin interference group were cut. GM-CSF, IFN-gamma, beta, IL- 3, IL-2, IL-4, IL-6, TNF alpha, IL-17, IL-22a, IL-27, and IL-9 factor expression levels were significant difference between shRNA and negative control group (P < 0.05). There was no significant difference of cell proliferation activity, early apoptosis rate, and differentiation ability in STAT3-shRNA treated CD117+ bone marrow, compared with negative control group and blank control group (P > 0.05). 2. After 50th day, shRNA treatment group appear hair recovery, energy recovery, weight gain, shortness of breath better, mean of cGVHD score decreased. At the 58th day, clinical scores of cGVHD between shRNA treatment group and the negative control group overall mean difference was statistically significant (P < 0.05). cGVHD pathological score of lungs in shRNA treat group reduced (P < 0.05). STAT3mRNA levels in peripheral blood, phosphorylated STAT3, and STAT3 expression level of lung declined than control groups. The proportion of Th17 / Treg cells of spleen was significant reduced in shRNA group, compared with negative control group (P < 0.05). Conclusion: 1. STAT3 knocking down in naïve CD4 + Th cells induced the increased Treg cells, and the decreased Th17 cells. IL-2 confirmed to promote the growth of Treg cells. It speculated that blocking STAT3 might bring Th9 cells differentiation. STAT3 blocking in CD117+ stem progenitor cells have no significant effect on the proliferation, apoptosis and differentiation, validation the safety of STAT3-shRNA. 2. STAT3-shRNA treatment cGVHD mice in vivo achieved curative effect. The main target organs was the lung, which might be closely related to the fall in the proportion of Th17 /Treg. STAT3 may be used as a new target for immunotherapy of cGVHD. Acknowledgment The project was sponsored by grants from National Natural Science Foundation of China (No. 30972790; No.81270648; No.81370665; No.81300446), Provincial Natural Science Foundation of Guangdong (No. S2012010009560), Provincial Science and Technology Planning Project of Guangdong (No.2013B021800186; No.2013B021800201), and Science and Technology Planning Project of Guangzhou (No. 201400000003-4, 201400000003-1). Disclosures No relevant conflicts of interest to declare.

Osteoprotective Effect of Cimiracemate in Glucocorticoid-Induced Osteoporosis by Osteoprotegerin/Receptor Activator of Nuclear Factor κ B/Receptor Activator of Nuclear Factor Kappa-Β Ligand Signaling

Pharmacology ◽  
2019 ◽  
Vol 103 (3-4) ◽  
pp. 163-172
Author(s):  
Zheng Ding ◽  
Huifeng Shi ◽  
Wei Yang
Keyword(s):  
Bone Marrow ◽  
Negative Control Group ◽  
Negative Control ◽  
Control Group ◽  
Nuclear Factor ◽  
Methylprednisolone Acetate ◽  
Histopathological Changes ◽  
Mediators Of Inflammation ◽  
Receptor Activator ◽  
Nuclear Factor Κ B

Objectives: Present investigation determines the protective effect of cimiracemate A against glucocorticoid-induced osteoporosis rat. Methods: Osteoporosis was induced by injecting methylprednisolone acetate (21 mg/kg) for the period of 6 weeks, and the rats were treated with cimiracemate A 5 and 10 mg/kg, p.o. 60 min after the administration of methylprednisolone acetate (21 mg/kg) for the duration of 6 weeks. Effect of cimiracemate A was observed by estimating the microarchitecture of bone and histopathological changes by micro-CT scan and light microscope. Moreover, lipid profile, mediators of inflammation, and parameters that affect bone formation were determined in the serum and western blot assay, and reverse transcription polymerase chain reaction was done for the estimation of protein expression in the bone tissues. Moreover, cytotoxic effect of cimiracemate A on bone marrow macrophages and bone marrow stromal cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: Result of the investigation suggests that treatment with cimiracemate A ameliorates the microarchitecture of bone and histopathological changes in the glucocorticoid-induced osteoporosis rat. Level of lipid and mediators of inflammation was significantly reduced in the serum of cimiracemate A-treated rats than the negative control group. However, the activity of tartrate-resistant acid phosphatase and the level of collagen type I fragments in the serum were found to be reduced, and osteocalcin level was enhanced in cimiracemate A-treated rats than the negative control group. Moreover, treatment with cimiracemate A attenuates the expression of receptor activator of nuclear factor kappa-Β ligand (RANKL), receptor activator of nuclear factor κ B (RANK), and osteoprotegerin (OPG) protein in glucocorticoid-induced osteoporosis rats. Conclusion: In conclusion, our study suggests that cimiracemate A protects the glucocorticoid-induced osteoporosis by regulating the RANKL/RANK/OPG signaling pathway.

scholarly journals Study of the probable genotoxic effects of Zolone (Phosalone) exposure in mice bone marrow derived cells

2021 ◽  
Vol 43 (1) ◽  
Author(s):  
Zohre Khodabandeh ◽  
Mahmoud Etebari ◽  
Mehdi Aliomrani
Keyword(s):  
Bone Marrow ◽  
Organophosphorus Pesticide ◽  
Negative Control Group ◽  
Negative Control ◽  
Control Group ◽  
Genotoxic Effects ◽  
Red Bone Marrow ◽  
Significant Difference ◽  
Bone Marrow Derived Cells ◽  
Dose Dependent

Abstract Background and aim Approximately, 2 million tonnes of pesticides are utilized annually worldwide. Phosalone (Pln), an organophosphorus pesticide, acts as an insecticide and acaricide to control pests of crops such as nuts, citrus fruits, pomegranates, stone fruits, grapes, potatoes, and artichokes. The purpose of this study was to evaluate the possible genotoxic effects following exposure to Pln in the cells derived from mouse red bone marrow. Materials and methods Sixty mice were divided into 6 groups including cyclophosphamide (40 mg/kg, IP) and Pln (6, 12, 20, and 40 mg/kg) exposure by gavage. After 1 and 5 days of exposure, animals were euthanized and the genotoxicity assays were done on bone marrow extracted cells. Results Comet assay shows a time and dose-dependent toxicity which further DNA degradation is observed after 5-day exposure (p < 0.05). Also, Pln significantly increased the MnPCE/PCE ratio after 12 and 20 mg/kg administration while no significant difference was reported between the doses of 6 and 40 mg/kg BW with the negative control group. Conclusion Our results suggested a serious concern about its potential effects on biological life and related disease inductions. However further studies need to confirm the exact mechanism of Pln genotoxicity and the cause of diverse response of its activity at 40 mg/kg. This study also showed that increasing the dose of Pln reduces the MnNCE/Total cells ratio, which may indicate the possibility of bone marrow suppression. All of the above results emphasize the need to seriously limit the use of this compound as an agricultural pesticide.

Ex Vivo Induction of Megakaryocytic Differentiation of Umbilical Cord Blood CD34+ Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 318-318
Author(s):  
Xin Guan ◽  
Meng Qin ◽  
Bin Shen ◽  
Yu Zhang ◽  
Wenhong Jiang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cord Blood ◽  
Ex Vivo ◽  
Negative Control Group ◽  
Negative Control ◽  
Control Group ◽  
Mouse Bone Marrow ◽  
Post Transplantation ◽  
Cd34 Cells

Abstract Background and Objectives: Due to platelet shortage, megakaryocytes have been regarded as an effective substitute to the alleviation of frequent thrombocytopenia after stem cell transplantation. However, ex vivo expansion of megakaryoblasts and their subsequent differentiation into mature megakaryocytes for clinical applications remains a challenge. Here, we describe the development of a two-stage culture system for producing megakaryocytes from cord blood CD34+ cells. Design and Methods: Firstly, we expanded CD34+ hematopoietic progenitor/stem cells for 6 days in a serum-free culture system (IMDM basal medium with the addition of biotin, putrescine, insulin, human serum albumin, selenium, and some other nutrients) supplemented with stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (TPO), interleukin 3 (IL-3), low density lipoprotein (LDL), StemRegenin 1 (SR1), and DMSO. CD34+ cells expansion was monitored by flow cytometric analysis of cell surface markers coupled with cell counting. Subsequently, these expanded cells were induced toward the megakaryocytic lineage for additional 7 days in the same serum-free medium as above supplemented with SCF, TPO, IL-3, IL-6, IL-11, granulocyte-macrophage colony-stimulating factor (GM-CSF), and LDL. Megakaryocytes were detected by flow cytometry using antibodies against specific cell surface markers including CD41a and CD42b. Differentiated megakaryocytes were also confirmed by morphological criteria such as cell size and DNA polyploidy. To functionally evaluate induced megakaryocytes, these cells were transplanted into sublethally irradiated NOD/SCID mice. Viability and cell being of these mice were monitored after injection. Mice of the negative control group (n=3) were injected with saline. In the experimental group (n=6), each mouse was injected with 1.0×107 cells from the second stage of culture. Results: After the first stage culture, proliferation folds of total cells and CD34+ cells were 85.65±7.03 and 62.91±4.36, respectively. The calculated yield from each CD34+ cell was between 1.0×104 to 1.5×104 CD41+ megakaryocytes with a purity of CD41+ and CD42+ cells reaching 93.7%±2.8% and 80.3%±5.8%, respectively. Differentiated cells were morphologically discernible as they were much larger than starting CD34+ cells with apparent lobular nuclei and numerous α-granules. In addition, about 32.67%±7.43% of induced megakaryocytes exhibited 4N or larger DNA content (4N 17.6%±4.12%; 8N 10.93%±2.48%; >8N 3.23%±1.34%). In mouse studies, samples collected from the negative control group contained no cells positive for human CD41a and CD42b markers. In the experimental group, human platelets were detected in mouse peripheral blood 3 days post-transplantation. At day 14 post-transplantation, the percentage of platelets derived from injected human megakaryocytes reached 13.6%±6.2%. Human megakaryocytes were also detected in mouse bone marrow 7 days post-transplantation, peaking at day 14 (~2.38% of total bone marrow megakaryocytes). Conclusions: We have established a stem cell expansion and differentiation platform that can be adapted to large-scale production of mature megakaryocytes from umbilical cord blood cells. Significantly, induced megakaryocytes are capable of engrafting in mouse bone marrow and producing platelets after transplantation into irradiated NOD/SCID mice. Therefore, our experimental platform is capable of producing a sufficient number of functional megakaryocytes for various clinical applications in the future. Disclosures Qin: Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment. Ren:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment.

Photo-crosslinked mono-component type II collagen hydrogel as a matrix to induce chondrogenic differentiation of bone marrow mesenchymal stem cells

2017 ◽  
Vol 5 (44) ◽  
pp. 8707-8718 ◽  
Author(s):  
Ke Yang ◽  
Jing Sun ◽  
Dan Wei ◽  
Lu Yuan ◽  
Jirong Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Triple Helix ◽  
Chondrogenic Differentiation ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Collagen Hydrogel ◽  
Component Type ◽  
Marrow Mesenchymal Stem Cells ◽  
P Type

Type II collagen methacrylamide with a triple helix was developed for 3D construction of a cartilaginous ECM-like microenvironment to induce chondrogenic differentiation of BMSCs.

scholarly journals Cell therapy as a new approach on hepatic fibrosis of murine model of Schistosoma mansoni infection

2019 ◽  
Author(s):  
N. ALsulami Muslimah
Keyword(s):  
Hepatic Fibrosis ◽  
Negative Control Group ◽  
Treated Group ◽  
Positive Control ◽  
Negative Control ◽  
Control Group ◽  
Liver Histopathology ◽  
Marrow Mesenchymal Stem Cells ◽  
And Function

AbstractSchistosomiasis is an acute and chronic disease caused by blood flukes (trematode worms) of the genus Schistosoma. Schistosomiasis is disease that are prevalent in or unique to tropical and subtropical regions. Previous studies have shown that the role of bone marrow mesenchymal stem cells (BMSCs) therapy in improvement of hepatic fibrosis. Therefore, the current study was designed to assess the therapeutic role of BMSCs in murine schistosomiasis mansoni. BMSCs derived male mice were intraperitoneal injected into female mice that received S. mansoni cercariae through subcutaneous route. Mice were divided into four groups: negative control group (noninfected non treated); positive control group (infected non treated); BMSCs treated group; and untreated group. Liver histopathology and immunohistochemically were evaluated. BMSC intraperitoneal injection resulted in a significant reduction in liver collagen, granuloma size, and significant increase in OV-6 expression in the Schistosomiasis treated mice group. There was overall improvement of the pathological changes of the liver. The findings support that BMSCs has a regenerative potential in the histopathology and function of the liver tissue by decreasing liver fibrosis.

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