Studies On The Biological Activity Of Micro-and Nano-hydroxyapatite Enhanced Sodium Alginate/chitosan Bone Scaffold

Purpose:According to the previous experimental methods,micro-hydroxyapatite/nanohydroxyapatite/sodium alginate/chitosan plastic microspheric scaffolds containing VEGF and vancomycin were prepared under sterile environment.Then,they co-cultured with BMSCs and further explored their effects on the proliferation,osteogenesis and adhesion of BMSCs,which laid a preliminary foundation for the clinical application of the new scaffolds.Methods:1.The primary BMSCs were isolated and cultured from the bilateral femur and tibia of SD rats using the whole bone marrow adherence screening method,and the cells were successively passaged to the third generation for osteogenic induction,adipogenic induction and subsequent experiments.2.According to the previous experimental method,sodium alginate/ chitosan multilayer sustained-release microspheres with VEGF were prepared in a sterile environment,and they were added to the mixed matrix composed of vancomycin,micro-hydroxyapatite,nano-hydroxyapatite,sodium alginate and chitosan.The micro-hydroxyapatite/nano-hydroxyapatite/sodium alginate/chitosan plastic microspheric scaffold loaded with VEGF and vancomycin was fabricated after lyophilization.3.The effect of micro-hydroxyapatite/nano-hydroxyapatite/sodium alginate/chitosan scaffold on the proliferation activity of BMSCs was detected by MTT assay.The effects of scaffolds on the osteogenic activity of BMSCs were comprehensively evaluated by the calcium release and alizarin red staining,ALP staining,ALP activity assay and Real-time PCR that detected the transcription of ALP,BMP2,OPN,and Runx2 osteogenesis-related genes in BMSCs after osteogenic induction.Otherwise,BMSCs were inoculated with scaffolds.The stem cells were treated with DAPI,and the cell adhesion in the co-culture system was observed with a laser confocal microscope to determine the effect of scaffolds on the adhesion of BMSCs.Results:1.The long spindle-shaped BMSCs were successfully isolated and cultured from the bilateral femur and tibia of SD rats.The calcium nodules were observed after osteogenic induction and lipid droplets were observed after adipogenic induction.2.The micro-hydroxyapatite/nano-hydroxyapatite/sodium alginate/chitosan plastic microspheric scaffolds with VEGF and vancomycin were successfully prepared under sterile environment and they had good morphology.3.In the MTT cell proliferation experiment,the blank group,the sodium alginate/ chitosan scaffold group and the micro-hydroxyapatite/nano-hydroxyapatite/sodium alginate/chitosan scaffold group did not show statistical differences(P> 0.05).4.In the osteogenic activity-related test,compared with the sodium alginate/chitosan scaffold,the calcium ion release amount of microhydroxyapatite/nano-hydroxyapatite/sodium alginate/chitosan scaffold in phosphate buffer was not statistically differences.(P>0.05).Not only the alizarin red staining was significantly deepened,which meaned extracellular calcium salt deposition was significantly increased,but also the ALP staining was deeper.Besides,ALP activity expression and ALP,BMP2,OPN and Runx2 osteogenesis-related gene transcription expression levels were also significantly improved(P <0.05).5.In the cell adhesion experiment,blue round and round-like nuclei were three-dimensionally distributed in two kinds of scaffolds observed by the laser confocal microscope.However,the number of attached cells increased significantly in micro-hydroxyapatite/nano-hydroxyapatite/sodium alginate/chitosan.Conclusions:1.The spindle-shaped BMSCs were successfully extracted from rats and identified by osteogenic induction and adipogenic induction,which were consistent with the characteristics of stem cells.2.The micro-hydroxyapatite/nano-hydroxyapatite/sodium alginate/chitosan scaffolds loaded with VEGF and vancomycin were successfully prepared.3.The micro-hydroxyapatite and nano-hydroxyapatite did not interfere with the effect of sodium alginate / chitosan scaffold on cell proliferation activity,but they could significantly improve its osteogenic activity and adhesion to cells.