مقالات پذیرفته شده در سومین کنگره بین المللی زیست پزشکی
Evaluation of cardiomyocyte s interaction with bacterial cellulose scaffold
Evaluation of cardiomyocyte s interaction with bacterial cellulose scaffold
Mohaddeseh Salehghamari,1Zeinab Neshati,2,*Mansour Mashreghi,3
1. Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran 2. Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran 3. Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran
Introduction: Myocardial infarction (MI) is one of heart diseases that every year puts many lives at risk. In MI, due to vessel closure and lack of blood supply to cardiomyocytes, fibrosis is developed. Therefore, fibrotic tissue must be replaced with intact cardiomyocytes. Tissue engineering is suggested as a solution for induction of cardiac regeneration. Bacterial cellulose (BC) due to mechanical properties, interconnected pores, inexpensive production and biocompatibility is useful as a scaffold in tissue engineering.
Methods: Bacterial cellulose (BC) was produced by Gluconacetobacter xylinus (PTCC 1734) in Hestrin and Schramm (HS) medium (2% (w/v) D-glucose, 0.5% (w/v) pepton, 0.5% (w/v) yeast extract, 2.7% (w/v) Na2HPO4 (Disodium hydrogen phosphate), 1.15% (w/v) citric acid) and conditions of static culture for 7 days at 28oC and pH=6. HS medium was sterilized in 121oC for 20 min. BC membrane was removed from the top of the HS medium, and then was placed in NaOH 1% (w/v) at 80oC and then distilled water each for 1 h. BC was neutralized with deionized water. Characterization of BC was done by SEM (scanning electron microscope), FTIR (Fourier-transform infrared spectroscopy), XRD (X-ray powder diffraction) and AFM (Atomic force microscope) and compared with collagen as a standard scaffold in tissue engineering. SEM images were taken for investigation of the pores and their diameters. FTIR was used to display chemical functional groups. XRD represents crystallinity of scaffold. AFM is used to measure nanofibrils diameter.
Cardiomyocytes were isolated from 2-day- old Wistar neonatal rats. Briefly, these cells were isolated using mechanical and enzymatic methods. Fibroblasts were seperated from cardiomyocytes through attachment ability to culture plates. At the last stage, cardiomyocytes were passed through the cell strainer for removal of undigested tissues. Cardiomyocytes were counted and seeded in 48 well plate covered with gelatin solution, bacterial cellulose membrane or collagen. After overnight incubation, cells were treated with mitomycin C. SEM images were taken at 1, 3, 5 and 7 days after mitomycin C treatment. Cell proliferation assay was done at day 7. Briefly, medium of cells in 48 well plates was removed, 200 l of 10% MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (0.5 mg/ml in PBS) was added to each well and cells were incubated for 4 h in dark condition. Then, medium was removed and 200 l DMSO was added to each well. Absorbance of samples was read with Elisa reader at 545 nm. Absorbance of cells cultured on BC was read 30 minutes after DMSO addition. HDF cells (human dermal fibroblasts) were seeded on BC as control.
Results: In SEM pictures diameter of BC pores was around 90 nm. FTIR displayed chemical functional groups. XRD showed peaks in 2θ=14o, 16o and 22o. BC nanofibers diameter was around 100-200 nm according to AFM analysis. FTIR and XRD results for collagen were consistent with other articles. Phase contrast and SEM images showed that cardiomyocytes could adhere to gelatin and BC but no cells were attached on collagen. However, cell density was decreased on BC from 3 day to 7 day, but cells were showing contractile activity. MTT assay results showed that cell viability for cardiomyocytes cultured on gelatin was more than BC, as these cells were not able to attach on BC for a long time. HDF cells could adhere to the surface of culture dish and BC for 10 days without decreasing in cell density, and results of MTT assay for these cells was the same for both culture dish surface (no scaffold) and BC and around 100% at day 10 of culture.
Conclusion: Cardiomyocytes could attach on BC, but its combination with gelatin makes it a better scaffold for cardiomyocytes, probably. Cardiomyocytes are differentiated cells, thus they did not proliferate and their viability based on MTT assay was not as well as fibroblasts.