Zein Protein/Hydroxyapatite Composite Scaffold In Vitro Study Of Bone Tissue Engineering

Objective: Seven kinds of zein protein/hydroxyapatite composite scaffolds arecompared by vitro experiments in order to search suitable proportion ofhydroxyapatite in this composite scaffolds for human bone marrow mesenchymalstem cell growth，and make sure of using ultrasonic agitation in the compositescaffolds production process is beneficial to the human bone marrow mesenchymalstem cell adhesion and growth.Methods: First, the pure zein protein solution and composite solution of the pure zeinprotein solution and different proportions hydroxyapatite (respectively5%,10%,15%)were prepared, and the latter is divided into two parts. One part was stired by ultrasonicagitation, and the other was stired by the common method of mixing. Then7groups ofmaterials are prepared through the electrostatic spinning. Second the healthy peoplewere selected（Age amount10to40years old）,and their bone marrow blood wascollected.The primary generation human bone marrow mesenchymal stem cells wereacquired by the whole blood culture, and continuous cultured to the fifthgeneration.Then the cell were reserved after identified by flow cytometry andpluripotential differentiation.Third the specified number of the fifth generation of humanbone marrow mesenchymal stem cells were planted into7groups of materials,then theHoechst staining was done on the first day and third day,the cytoactive of each groupwere evaluated under the laser confocal microscope.At the same time, the shape ofhuman bone marrow mesenchymal stem cells were observed by electron microscope;At last the specified number of the fifth generation human bone marrow mesenchymalstem cells were planted on the7groups of materials, and the proliferation of humanbone marrow mesenchymal stem cell were evaluated in different materials by theCCK-8experiment in order to analyze which proportion of hydroxyapatite in thiscomposite scaffolds is more suitable for human bone marrow mesenchymal stem cellgrowth by statistics. Results:1、The adhesion and growth of human bone marrow mesenchymal stemcells on7groups of materials were seen at the first day after Hoechst staining bysanning confocal laser microscopy, but the number of cells was small.There was nosignificant differences among the groups.At the third day the human bone marrowmesenchymal stem cells on the7groups of materials increased significantly with goodactivity after Hoechst staining observed by confocal laser microscopy.There was nosignificant differences between each group of human bone marrow mesenchymal stemcell number by naked eyes.2、The round sharp human bone marrow mesenchymal stem cells were found on allthe four groups of materials which contained10%,15%hydroxyapatite by the electronmicroscope on the third day,most of which were in composite solution stired byultrasonic agitation. The large number of human bone marrow mesenchymal stemcells which looked like stellate, spindle, multiple foot shape could be seen on theremaining three groups of material.Cells juncted into flake.Cell synapses and filamentsof material were winding.3、The results of CCK-8experiment:The human bone marrow mesenchymal stem cellson the two groups of materials which contained5%hydroxyapatite grew faster thanpure zein protein group(P<0.05). Furthermore,the cells on the materials whichcontained5%hydroxyapatite without ultrasonic agitation grew faster than the oneswith ultrasonic agitation (P<0.05). In addition,the cell on the materials containing thesame proportion of hydroxyapatite without ultrasonic agitation grew faster than theones on the materials with ultrasonic agitation (P<0.05).Conclusion：1、Observed by the laser confocal microscope and electron microscope,the human bone marrow mesenchymal stem cells can adhere and grow on the zeinprotein composite scaffolds containing different proportions of hydroxyapatite.2、The zein protein composite scaffolds containing5%hydroxyapatite withoutultrasonic agitation is the most suitable for the human bone marrow mesenchymal stemcell adhesion and growth.3、In the process of this kind of material production using ultrasonic agitation is disadvantageous to human bone marrow mesenchymal stem cells adhesion andgrowth.