Study Of Cardiac Muscle Tissue Engineering On PLGA-Based Scaffold

This study investigates construction of cardiac myocyte cell- three dimentional biodegradable polymer foams of PLGA scaffold complex in a bioreactor so as to unveil the possibility of generating 3-D cardiac muscle tissue under the environment that mimics microgravity and supply a 3-dimensional model for physiological and pharmacological studies in vitro.In the first part of the study, we tested and evaluated the biocompatibility of PLGA foams.The tests of cytotoxicity and intramuscular implantation were carried out to biological effects of polymer foams of PLGA. The primary results showed that ploymer foams of PLGA had good biocompatibility in vitro, which were no significant toxicity to body. These results exhibit that the novel porous PLGA foams may be suitable for use as a biodegradable scaffold for regeneration of cardiac tissue.In the second part of the study, l~2day old neonatal rat cardiac muscle cells were isolated by sequential digestion and pre-plating methods , and the acquired cells were cultured in DMEM containing 10% fetal bovine serum. The cell type was identified by H-E and anti-sarcomeric a -actin stains.Cardiac cell beating and metabolic indexes, including specific consumption rate of glucose,specific production rate of lactate,lactate transform rate were primarily confirmed. The isolated cardiac myocytes adhered to the bottom of the flasks 24 hours after plating and began to contract spontaneously and by day 3~4 formed synchronously contracting networks.In the third part of the study, the PLGA foams were shaped into the form of a sheet which diameter was about 5mm. Then cardiac myocyte were seeded onto the PLGA foams with a cellular density of 2x107 cells/ml. The cell-polymer constructs were transferred into rotary cell culture system (RCCS) and incubated for 7 days. Cells cultured in 75mL flasks served as control. Morphological changes of the cells were detected by light microscope and metabolic rate was recorded.Ultrastructure of the cells growing in porous polymer was observed by transmission electron microscopy. Content of total DNA and protein in the newly-formed tissue were analyzed. HE and anti-sarcomeric a -actin stains wereperformed in comparison with native cardiac muscle. At the same time,the isolated cardiac myocytes were seeded into Cytodex-3 microcarrier in RCCS and cultured for 7 days.Morphological changes of the cells on microcarrier were observed by light microscope and scan electron microscopy.Metabolic rate of the cells was measured. When incubated for 7 days in RCCS, cell-polymer constructs formed a continuous outer tissue layer containing cells aligned with each other. The cell population in the interior of the construct was less in density than the outer part. Transmission electron microscopy demonstrated subcellular elements characteristic of cardiac myocytes in the outermost layer of constructs. Positive stains of anti-sarcomeric a -actin suggested presence of cell population of differentiated cardiac myocytes in these constructs. Construct biomass was not significantly different from that in neonatal rat ventricle and approximately 40% of that in adult rat ventricles. Metabolic activity of cells of constructs cultured in RCCS was higher than that in flasks. Cells adhered into cytodex-3 microcarrier 12 hours after seeding and formed cells groups . Metabolic activity of the cells on microcarrier was vigorous.The above results demonstrated that dissociated cardiac myocyte cultured on 3-dimensional scaffolds in RCCS under favorable conditions can form engineered constructs with structural and functional features resembling those of native cardiac tissue.