Effect of hyaluronic acid-gelatin double-network hydrogel on osteogenic differentiation of rat bone marrow mesenchymal stem cells / 上海交通大学学报（医学版）

Objective • To construct a natural polymers-based hydrogels with enhanced mechanical property through double network (DN) strategy under the premise of retaining the good biocompatibility of natural polymers, and then evaluate the beneficial effect of hydrogels on the osteogenic differentiation of rat bone mesenchymal stem cells (rBMSCs). Methods • Methacrylated hyaluronic acid (HAMA) and methacrylated natural gelatin (GelMA) were synthesized through reaction of HA and Gel with methacrylic anhydride (MA), respectively. Thereafter, HA-Gel DN hydrogels were fabricated through two-step photo-crosslinking. The basic physicochemical properties of hydrogels were evaluated by scanning electron microscopy, swelling, compression and degradation analysis. Hydrogels were applied as substrate materials for rBMSCs culture in vitro. Cell viability, attachment, spreading and proliferation were assessed by CCK-8 analysis and fluorescent staining analysis. The osteogenic differentiation of rBMSCs was determined by quantitative PCR and Western blotting analysis. Results • In comparison to HA hydrogels and Gel hydrogels, HA-Gel DN hydrogels showed more suitable physicochemical properties, such as more suitable water absorption and water retention [(12.6± 0.7) fold, (10.3± 0.4) fold], stronger mechanical property [(43.7± 5.6) kPa] and slower degradation rate [(82.3±3.9)% for 12 weeks] for osteogenic differentiation of rBMSCs. Experiment in vitro revealed that HA-Gel DN hydrogels had good biocompatibility, quantitative PCR revealed that it could promote the expression of osteogenic genes including Runx2, BSP, OPN, OCN, OSX and ALP. Western blotting revealed that the HA-Gel DN hydrogels also increased the levels of osteogenic proteins (OPN, OSX and BSP). Conclusion • HAGel DN hydrogels have good biocompatibility and promote the osteogenic differentiation of rBMSCs, which provide a new experimental basis for DN hydrogels becoming the potential material for bone defects repair.

The effects of phenytoin on the differentiation of rat bone marrow mesenchymal stem cells into vascular endothelial cells / 实用口腔医学杂志

Objective:To investigate the effects of PHT on the differentiation of rat bone marrow mesenchymal stem cells(BMSCs) into vascular endothelial cells(VECs).Methods:rBMSCs were cultured in indirect coculture system with VECs and independent culture system in the presence of phenytoin at 0,20 and 40 μg/ml respectively.After 1 4 d culture the mRNA expression levels of the intercellular adhesion molecule-1 (ICAM-1 ),vascular cell adhesion molecules-1 (VCAM-1 ),vascular endothelial growth factor re-ceptor -2(KDR)and Runt-related transcription factor 2 (RUNX2)were detected by real-time PCR.Results:The expression levels of ICAM-1 ,KDR and RUNX2 were upregulated in rBMSCs by PHT treatment.The expression levels of ICAM-1 ,KDR and RUNX2 in the co-culture groups were higher than those in the independent culture groups of rBMSCs treated by PHT at the same concentra-tion.The expression level of VCAM-1 in co-culture groups was lower than that in the independent culture groups.Conclusion:PHT may promote rBMSCs differentiation into rVECs in the co-culture system.

Baicalin induces osteogenic differentiation of rat bone marrow derived mesenchymal stem cells via Wnt/β-catenin signaling pathway / 中国药理学通报

Aim To investigate the role of Wnt/β-cate-nin signaling pathway on the baicalin-induced osteo-genic differentiation in rat bone marrow derived mesen-chymal stem cells ( rBMSC ) . Methods rBMSC was isolated and cultured by adherence screening method. Alkaline phosphatase ( ALP) amount, CFU-FALP and mineralized nodules were compared between each ba-icalin group and vehicle control group at different time points. Real time q-PCR was employed to evaluate the mRNA level of Wnt signaling-related marker ( Wnt10a, GSK-3β,β-catenin and LEF1) after baica-lin treatment. Protein expression of β-catenin and Runx2 was measured by Western blot. Results Ba-icalin significantly increased ALP activities from day 3 to day 7 . The formation of CFU-FALP and mineralized nodules remarkably increased after rBMSC was treated with1, 10, 50 μmol · L-1 baicalin. mRNA levels of Wnt10a, β-catenin, GSK-3β, LEF1and osteocalcin were enhanced significantly in baicalin-treated group compared to control group. Protein expression of β-catenin and Runx2 was also elevated. Conclusion Baicalin ( 0. 1 to 50 μmol · L-1 ) promotes the osteo-genic differentiation and maturation of rBMSC, in which Wnt/β-catenin signaling pathway might be in-volved.

Development of cell model of polymer/liquid crystal and effect of their elasticity on adhesion of rBM-MSCs / 中国病理生理杂志

[ ABSTRACT] AIM:To develop the cell model of polymer/liquid crystal and to study the effect of their elasticity on the adhesion of rat bone marrow mesenchymal stem cells (rBM-MSCs).METHODS: Using the method of solvent e-vaporation induced phase separation, the cell model of polymer/liquid crystal was constructed.The surface morphology and phase separation structure were determined by polarized optical microscopy ( POM) , scanning electron microscopy ( SEM) and small angle X-ray scattering ( SAXS ) .rBM-MSCs were separated and expanded by adherent culture.The surface markers of rBM-MSCs were detected by flow cytometry.The cells were induced to osteogenic differentiation and adipogenic differentiation for 2 weeks.After 3 passages, the cells were divided into 4 groups, including total PU control group, 10%membrane group, 30%membrane group and 50%membrane group.The cells were then incubated with rhodamine phalloi-din for cytoskeleton staining and were observed under the confocal laser scanning microscope after cultured for 24 h.RE-SULTS:The cell model of polymer/liquid crystal was constructed successfully using the method of solvent evaporation in-duced phase separation.Flow cytometry results showed that the rBM-MSCs positively expressed CD29, CD44 and CD90, and negatively expressed CD34 and CD45.After stained with alizarin red S and oil red O, the calcium nodule and lipid droplets in rBM-MSCs were observed obviously.The cytoskeleton staining result indicated that the area in total PU control group, 10%membrane group and 30%membrane group were greater, and the actin microfilaments were also clearer than that in 50%membrane group.CONCLUSION:The cell model with suitable content of liquid crystal made a contribution to the rBM-MSCs’ adhesion, but too much liquid crystal inhibits cell adhesion.

Adipogenesis of Sprague Dawely rats mesenchymal stem cells: a morphological, immunophenotyping and gene expression follow-up study / 대한해부학회지

Mesenchymal stem cells (MSCs) offer significant promise as a multipotent source for cell-based therapies and could form the basis for the differentiation and cultivation of tissue grafts to replace damaged tissue. However, no gene expression follow up analysis has been undertaken to characterize the in vitro adipogenic differentiated MSCs. The main goal of this study was to focus on MSCs and to analyze their differentiation capacity. To achieve this aim, bone marrow MSCs from sprague dawely rats were isolated, expanded in monolayer culture and characterized with respect to their cluster of differentiation (CD) and ability for adipogenic differentiation capacity. The expression of CD44, CD45, CD29, CD34, and CD90 on bone marrow derived MSCs was characterized using flow cytometry. Adipogenesis was determined by staining with oil-red O and reverse transcription polymerase chain reaction assessments of lipoprotein lipase, leptin, adiponectin and adipocyte genes at different time intervals, after 4, 7, 14, and 21 days. Our results revealed that the pattern of CD marker expression was highly positive significant with CD29, CD44, and CD90 when compared with CD34 and CD45. MSCs showed proliferative potential and were capable of adipogenic differentiation characterized by reddish brown-droplets following staining with oil-red O and expression of molecular bands of genes. These results demonstrate, at the morphological, immunophenotyping and gene expression levels, the multipotency of MSCs and thus highlight their potential therapeutic value for cell-based tissue engineering.

Adipogenesis of Sprague Dawely rats mesenchymal stem cells: a morphological, immunophenotyping and gene expression follow-up study / 대한해부학회지

Mesenchymal stem cells (MSCs) offer significant promise as a multipotent source for cell-based therapies and could form the basis for the differentiation and cultivation of tissue grafts to replace damaged tissue. However, no gene expression follow up analysis has been undertaken to characterize the in vitro adipogenic differentiated MSCs. The main goal of this study was to focus on MSCs and to analyze their differentiation capacity. To achieve this aim, bone marrow MSCs from sprague dawely rats were isolated, expanded in monolayer culture and characterized with respect to their cluster of differentiation (CD) and ability for adipogenic differentiation capacity. The expression of CD44, CD45, CD29, CD34, and CD90 on bone marrow derived MSCs was characterized using flow cytometry. Adipogenesis was determined by staining with oil-red O and reverse transcription polymerase chain reaction assessments of lipoprotein lipase, leptin, adiponectin and adipocyte genes at different time intervals, after 4, 7, 14, and 21 days. Our results revealed that the pattern of CD marker expression was highly positive significant with CD29, CD44, and CD90 when compared with CD34 and CD45. MSCs showed proliferative potential and were capable of adipogenic differentiation characterized by reddish brown-droplets following staining with oil-red O and expression of molecular bands of genes. These results demonstrate, at the morphological, immunophenotyping and gene expression levels, the multipotency of MSCs and thus highlight their potential therapeutic value for cell-based tissue engineering.

A comparison of vitamin A and leucovorin for the prevention of methotrexate-induced micronuclei production in rat bone marrow

INTRODUCTION: Methotrexate, a folate antagonist, is a mainstay treatment for childhood acute lymphoblastic leukemia. It is also widely used in a low dose formulation to treat patients with rheumatoid arthritis. In rats, methotrexate is known to induce micronuclei formation, leading to genetic damage, while vitamin A is known to protect against such methotrexate-induced genetic damage. Leucovorin (folinic acid) is generally administered with methotrexate to decrease methotrexate-induced toxicity. OBJECTIVES: We aimed to determine whether vitamin A and leucovorin differed in their capacity to prevent formation of methotrexate-induced micronuclei in rat bone marrow erythrocytes. The present study also aimed to evaluate the effect of combined treatment with vitamin A and leucovorin on the formation of methotrexate-induced micronuclei. METHODS: Male and female Wistar rats (n=8) were injected with 20 mg/kg methotrexate (single i.p. dose). The control group received an equal volume of distilled water. The third and fourth groups of rats received vitamin A (5000 IU daily dose for 4 successive days) and leucovorin (0.5 mg/kg i.p. dose for 4 successive days), respectively. The fifth and sixth groups of rats received a combination of vitamin A and a single dose of methotrexate and a combination of leucovorin and methotrexate, respectively. The last group of rats received a combination of leucovorin, vitamin A and single dose of methotrexate. Samples were collected at 24 hours after the last dose of the treatment into 5 percent bovine albumin. Smears were obtained and stained with May-Grunwald and Giemsa. One thousand polychromatic erythrocytes were counted per animal for the presence of micronuclei and the percentage of polychromatic erythrocyte was determined. RESULTS: Comparison of methotrexate-treated rats with the control group showed a significant increase in the percentage of cells with micronuclei and a significant decrease polychromatic...

Problems in osteogenic differentiation of rat bone marrow stromal cells

The affect of body position on 32 biochemical indexes / 中华检验医学杂志

Objective To investigate the affect of body positions on biochemical indexes. Methods By autogenous contrast and cross matched survey, 107 volunteers divided into 3 season patches of winter, spring and summer, blood samples were drawn from the same part in both standing and lying positions。From19 persons, blood samples were collected respectively after standing and sitting for 15 min, lying for 15 min and 30 min and then sitting for another 15 min。 The blood samples were analyzed for 32 biochemical indexes on analyzer。Results 25 biochemical indexes in sitting position were significantly different from those in lying position (P0。05)。Conclusions Changing body position can result in obvious physiological variation of 28 biochemical indexes, particularly of those related to protein. Such result may lead to abnormality in some marginal values. It suggests body position should not be neglected in analyzing laboratory data.