Construction And Evaluation Of Bone Marrow Mesenchymal Stem Cells Combined With Thermosensitive Hydrogel Three-dimensional Micro-vironment In Vitro Culture System

Objective:The construction of three-dimensional microenvironment composites were made with BMSCs as seed cells and thermosensitive hydrogel scaffold as carrier. To evaluate the biocompatibility of bone marrow mesenchymal stem cells (BMSCs) and thermosensitive hydrogel three-dimensional. To explore the biological characteristics of cell proliferation and differentiation of bone marrow mesenchymal stem cells in the three-dimensional microenvironment..o provide experimental evidence and the experimental basis of which carrying out related to tissue engineering and applied basic researchMethod:1.Optimize the three-dimensional microenvironment scaffolds materials:Preparation of Thermosensitive hydrogel by Chitosan, Glycerol2-phosphate Disodium Salt Hydrate as raw material, and Hydroxyethyl-cellulose Medium Viscosity as crosslinker. Chitosan, Glycerol2-phosphate Disodium Salt Hydrate, Hydroxyethyl-cellulose Medium Viscosity were mixed according to the different volume ratio, divided into six experimental groups, evaluated the time of gelation and rigidity.2. Establish a stable bone marrow mesenchymal stem cells resources:Separation of the BMSCs from the bone marrow of new born SD rats, culturing MSCs with low Glucose Dulbecco’s modified Eagle’s medium containing10%Fetal bovine serum, cells reached70%of the bottom area of the flasks, the passaged culturing to the fourth generation, subsequent generation of BMSCs passage time stabilized at four or five days. Generations of cells were confirmed as maintain multi-lineage differentiation potential and low differentiated state.3.Choose the third passaged MSCs weel growing as seed cells and thermosensitive hydrogel as scaffold, culturing with low Glucose Dulbecco’s modified Eagle’s medium containing10%Fetal bovine serum2weeks, Add to cardiac tissue lasate culture with hive days, Construct three-dimensional microenvironment culture system. The cells’growth were observed under inverted phase contrast microscope, hematoxylin and eosin staining, A0staining and scanning electron microscopy, evaluating of the interaction between the cells, and thermosensitive hydrogel in three-dimensional microenvironment culture system.RusuIt:Bone marrow mesenchymal stem cells seeded in the hydrogel aater24h the cells remained spherical, slow growth within48hours,72h spherical cells began to increase, attached to the surface thermosensitive hydrogel. Continuous culture for two weeks, take the cell-scaffold hematoxylin and eosin staining, a large number of round cells grow along the stent sur[ace, some cells can cross between the bracket. Under the scanning electron microscope, showing a large number of cells to maintain round, heterogeneity, the ability to divide, adhesion on the scaffolds, part of the cell surface visible in exocytosis secretion membrane particles.2.Myocardium lysate induced five days, by hematoxylin and eosin staining showed cell fusiform and package the scaffold material, by scanning electron microscope, it can be observed in cells from circular to fusiform cells between the traction.Conclusion:Optimization can be constructed with good aperture rate of thermosensitive hydrogel. Bone marrow mesenchymal stem cells with hydrogel scaffold has good biocompatibility, can maintain a low differentiated state and active proliferation. Well in simulating the in vivo tissue structure in a specific three-dimensional micro-environment culturing system. And can promote the directed differentiation of bone marrow mesenchymal stem cells.