Ovine endometrial cell culture on nanofiber scaffold: an approach for uterine tissue engineering

Background: In this study, a novel polycaprolactone/ gelatin/ polydimethylsiloxane (PCL/G/PDMS) scaffold with improved mechanical properties and controllable porous structure was prepared through electro spinning method. Good functional uterine endometrial cells are vital for fertilized egg implantation, repair, regeneration and normal activity of the uterus. This study introduced endometrial cell primary culture in vitro and cell culture on PCL/G/PDMS scaffold as the initial step for uterine tissue engineering.Objective: Produced scaffold can be used in any case to repair the endometrial damage, in order to provide a good environment for implantation of the embryo and to reduce the infertility in the future. Using tissue engineering to create and reconstruct the uterine endometrium is one of the best reliable option for this purpose.Materials and Methods: PCL, G and PDMS were purchased from Sigma-Aldrich® and trifluoroacetic acid (C2HF3O2) from Merck® companies. A PCL/G/PDMS copolymer was synthesized with volume ratios of difference (To fabricate scaffolds with optimum the diameter of nanofibers, porosity, hydrophilicity, biodegradability, biocompatibility, stress and strain of scaffolds, the effect of PCL/G/PDMS mixture ratio influencing the scaffolds characteristics were investigated via RSM). Fibers were electrospun onto an aluminum foil. The designed scaffolds were evaluated by a scanning electron microscope (SEM). Endometrial tissue samples from the uterus were collected in a sterile condition from five sheep after slaughtering in a slaughterhouse. Endometrial cells were isolated from endometrial samples. Isolated cells were cultured in cell culture medium. After sterilization under UV light for 20 min, to enumerate the cells, the population doubling time (PDT) was determined and 10^4 cells were cultured into 24-well culture plates containing scaffolds. The cells were incubated in a humidified standard incubator at 37oC with 5% CO2. Cells were harvested at days 3, 7, and 10 for growth and evaluated using H&E staining and MTT test. By triplicate assay the activity and survival of the cells on nanofibers were calculated as a percentage of samples to controls. In this study, the random scaffold was designed for the uterine endometrium layer and parallel scaffold for the uterine myometrium layer. Also, the diameter of nanofibers, porosity, hydrophilicity, biodegradability, biocompatibility, stress and strain of scaffolds have been studied.Results: The SEM results showed that diameter of the fiber is at the nanoscale. The PCL/G/PDMS scaffolds significantly promoted endometrial cell attachment and proliferation comparing to PCL scaffold alone. Endometrial cell viability Was acceptable by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay. The H & E results also showed that the cells developed and replicated well on the scaffold. endometrial cells have grown on the parallel scaffold in line with nano fibers and on the randomistic scaffold have grown in all directions. This indicates that the cells are compatible with the Nano-fiber scaffolds produced. All quantitative and qualitative parameters indicate that the production scaffold is suitable for tissue engineering of the uterus.Conclusion: These findings could be used in tissue engineering as the first step for uterine tissue engineering. Also, PCL/G/PDMS scaffold can be candidates for other regenerative cell therapy and tissue engineering.

کلیدواژه ها:

Tissue engineering ، Nanofiber ، Elastin ، Ovine endometrial Cells