Effects Of Three-Dimensional Microenvironment Constructed By Microfluidic Chip Technology On Proliferation And Differentiation Of BMSCs

Objective: This study used microfluidic chip technology to prepare alginic acid microgel spheres containing bone marrow-derived mesenchymal stem cells to simulate the microenvironment of the extracellular matrix.The correlation between stem cells and the extracellular environment and the research of new bone repair materials provide the basis.The application prospect of stem cell tissue engineering in biomedicine is prospected.Methods: 1.Prepare alginic acid microgel spheres containing bone marrow mesenchymal stem cells using microfluidic chip technology,observe the growth of mesenchymal stem cells in the microgel spheres with an optical microscope,and observe the microgel spheres with an inverted fluorescence microscope.The status of the inner nucleus;2.Contrast cell-loaded microgel spheres with two-dimensional planar cultured cells,and use living and dead staining and CCK-8 reagent to detect the survival and proliferation of mesenchymal stem cells in microgel spheres;3.Use light microscope and scanning electron microscope to observe the mineralization of microgel balls after osteogenesis induction,and use Fourier transform infrared spectrum analysis and energy scattering x-ray spectrum analysis to detect the mineralization components of microgel balls;4.Compare the cell-loaded microgel spheres with two-dimensional planar cultured cells and test the bone-forming ability of the cell-loaded microgel spheres by alkaline phosphatase activity;5.Using the tibial bone marrow cavity ablation model,the cell-loaded microgel spheres were implanted into the rat tibial bone marrow cavity,with gel only and cells only as a control group.Tibial specimens were embedded in paraffin, sectioned,and hematoxylin-Eosin staining validates the ability of bone repair in vivo.Results: 1.Under the light microscope,the diameter of the cells increased with the increase of the culture time,and reached a peak of 36.379 ?m on the 7th day.Under the fluorescence microscope,the cells in the microgel spheres were seen as multinucleated cell clusters.2.On the first day after making microgel spheres,the cell survival rate and cell viability of the cell-containing microgel group were lower than those of the uncoated gel group,and the differences between the two groups were statistically significant(P <0.05);At the following time points(days 3,5,7,and 11),there was no significant difference in cell survival rate and cell viability between the two groups(P>0.05).The cell survival rate of the two groups could reach more than 98%.Rising rapidly in the first 3 days,then slowing down,reaching a peak metabolic rate on the 7th day;3.Under the light microscope,the microgel spheres were found to diffuse mineral deposits from the cells to the surface of the microgel spheres after 7 days of osteogenesis induction,and the microgel spheres after 21 days of osteogenesis induction was seen under a scanning electron microscope.The surface of the microgel spheresl is rough,and a large number of calcified crystals can be seen.Infrared spectrum analysis shows that compared to the osteogenic induction culture,the surface of the microgel showed a large amount of phosphate deposition and type?collagen after 21 days of induction.Energy scattering x-ray spectrum analysis showed Before the osteogenic induction culture,the content of Ca and P in the microgel spheres increased significantly after 21 days of induction;4.The alkaline phosphatase activity of the cell-loaded microgel ball group increased faster than the unencapsulated control group,and reached its peak earlier;5.The results of animal experiments showed that a large number of new bone trabeculae appeared in the medullary cavity of the cell-containing microgel ball group,which extended from the cortical edge and matched the decrease of adipocytes in the bone marrow.Conclusion: The preparation of alginic acid microgel spheres containing bone marrow mesenchymal stem cells by using microfluidic chip technology can not only ensure the survival and growth and proliferation of cells in the microgel spheres,but also promote osteogenic differentiation,mineralization and deposition of cells.