Perfusion Culture Of Human Mensenchymal Stem Cells Seeded On PLGA Scaffold And Osteogenic Differention

Bone tissue engineering beckons a new frontier for clinical treatment to bone defects. hMSCs, as an ideal cell source, are widely used in the treatment of bony defects. PLGA scaffold has exhibited good biocompatibility in bone tissue engineering. Perfusion bioreactor can provided favorable conditions for the three-dimensional cultivation of bone tissue. The objective of this study was to determine the viability and growth of hMSCs in a three-dimensional porous PLGA scaffold in combination with perfusion bioreactor culture system. The scaffold has a porosity of 94.5%-98.0% and pore size of 280-450μm. hMSCs were seeded at a density of 2×10~6cells/ml, and the perfusion rate was 0.2ml/min. Firstly, three different cell density(1×10~6, 2×10~6, 4×10~6个/ml) were used in the seeding process, and a cell density of 2×10~6个/ml was found to be the more proper seeding density by the comparison of seeding efficiency. Comparison of cell status after 3-days culture under three different perfusion rates (0.2, 0.4, 0.8ml/min) generated the more proper perfusion rate (0.2ml/min). Cell viability by analyzing with MTT colorimetry increased with time, indicating a significant proliferation. The cell quantity based on DNA assay increased in a fast proliferation at first 6 days, and then in a slower proliferation. SEM images showed, at day 5, hMSCs has migrated into the scaffold holes, and a dense layer of extracellular matrix has been formed; and the entire surface of the scaffold was covered by a dense layer of extracellular matrix at day 11. FDA staining and H&E staining showed that the cells spreaded uniformly through the whole scaffold. ALP staining and ALP activity assay showed that the early differentiation began at 7 days after osteogenic differentiation, followed by a continuing differentiation. Moreover, ALP activity of osteogenic medium induced group is much higher than that of the control group.These results demonstrate the feasibility of culturing cell/PLGA in a flow perfusion bioreactor for bone tissue engineering applications.