Effects of different forms of chitosan on intercellular junctions of mouse fibroblasts in vitro

Chitosan is a linear polysaccharide that has many biomedical applications. We compared the effects of chitosan, in both solution and membranous form, on intercellular adhesion of Swiss 3T3 mouse fibroblasts. Cells were grown as spheroidal cell cultures. Some control cell spheroids were cultured without chitosan and two experimental groups were cultured with chitosan. Chitosan in solution was used for one experimental group and chitosan in membranous form was used for the other. For each group, intercellular adhesion was investigated on days 5 and 10 of culture. Transmission electron microscopy revealed well-defined cellular projections that were more prominent in cells exposed to either membranous or solution forms of chitosan than to the chitosan-free control. Immunocytochemical staining of ICAM-1 and e-cadherin was used to determine the development of intercellular junctions. Compared to the weakly stained control, strong reactions were observed in both chitosan exposed groups at both 5 and 10 days. Cells were treated with 5-bromo-2-deoxyuridine (BrdU) and incubated with anti-BrdU primary antibody to assess proliferation. Both the solution and membranous forms of chitosan increased proliferation at both 5 and 10 days. Cellular viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT). The MTT assay indicated high cell viability; maximum viability was obtained with the solution form of chitosan at day 5. Chitosan exposure increased the number of intercellular junctions and showed a significant proliferative effect on 3T3 mouse fibroblasts

Differences between solution and membrane forms of chitosan on the in vitro activity of fibroblasts

Background: Chitosan, a linear polysaccharide, has been recently used in biomedical applications. In vitro studies have demonstrated its effect on cellular growth and its stimulatory action on cellular layer formation. Aims: The present study aims to compare the proliferative effects of chitosan in two forms, membranous and solution forms, on Swiss 3T3 mouse embryonic fibroblasts. Study Design: In vitro study. Methods: Three experimental groups were formed: cells were cultured in a normal medium without chitosan (Control Group); cells were cultured either in a medium containing 2.0% chitosan in membranous form (Membrane Group) or chitosan solution at a concentration of 2.0% (Solution Group).Two different methods were used in the experiments: cells cultured on the medium containing chitosan in solution or membranous forms (method 1); and chitosan solution or membranous forms were added into the medium containing previously cultured cells (method 2). Results: Scanning electron microscopic investigations of the experimental groups revealed cells with well-defined cellular projections, intact cellular membranes and tight intercellular junctions. They were especially prominent in the membrane group of method 1 and in the membrane and solution groups of method 2. Mouse monoclonal anti-collagen 1 primary antibody was used to indicate collagen synthesis. Prominent collagen synthesis was detected in the membrane groups on the 10th day of culture for both methods. Bromodeoxyuridine (BrdU) and MTT assays were performed in order to assess cellular proliferation and viability, respectively. BrdU labelling tests indicated a higher proliferation index in the membrane group of method 1 on the 5th and 10th days. For the second method, the membranous form on the 10th day and solution form on the 5th day were the most effective groups in terms of cellular proliferation. MTT results reflected a high cellular viability in method 1 on the 5th day of treatment with the membranous form, whereas cellular viability was highest in the solution form of method 2 on the 5th day. Conclusion: The membranous form of chitosan induced a significant proliferative effect and increased the ratio of cell-to-cell junctions of Swiss 3T3 mouse embryonic fibroblasts. Conveniently, the solution form also resulted in enhanced cell proliferation and viability compared to the control group. As the solution form is easy to prepare and apply to cells compared to the membrane form, the application of Chitosan directly to media appears to be a convenient alternative for tissue engineering approaches

Notch signaling pathway in cumulus cells can be a novel marker to identify poor and normal responder IVF patients

PURPOSE: To identify expression of Notch signaling proteins and its ligands in human cumulus cells which were obtained by follicle aspiration and to compare the differences of this protein expression between the normal and poor responder patients. METHODS: 47 patients who applied to the assisted reproductive treatments with various infertility problems were included to the study. Controlled ovarian hyperstimulation was performed by using GnRH agonist and gonadotropins. Serum hormon levels were measured by using Chemilluminescent Microparticle Immunoassay method for each patient. After ultrasonographic ovarian follicle screening, oocytes were retrievaled. Cumulus cells obtained from the follicles were cultured for 72 h and immunuhistochemistry were performed for Notch1, Notch2, Notch3, Notch4, Jagged1 and Jagged2 proteins. Histological score (HSCORE) were applied to all of the samples. The association between Notch and its ligands protein expressions and the oocyte-embryo quality and fertilization rates were investigated. RESULTS: Significant differences were observed between the mean values of age, AMH and FSH in the 2 groups, respectively (p < 0.05). However, the mean female infertility duration and total gonadotropin dose did not differ significantly between normal and poor responder groups. All the patients cumulus cells expressed Notch1, Notch2, Notch3, Notch4, Jagged1 and Jagged2. There was a significant difference (p < 0.05) only for Notch2 between the 2 groups and a positive correlation between Notch2 and Notch3 (r = 547, p = 0.00) expressions were noted. Furthermore, no correlations were observed between the following: Notch1, Notch2, Notch3, Notch4, Jagged1, and Jagged2 expression; mature oocyte number; fertilization rates, and embryo quality percentage in both of the groups. CONCLUSION: Notch signalling proteins can be an indicator for understanding the ovarian response in ovulation induction