Fabriation And Characterization Of CS/Gel/n HAp/CNTs Composite Scaffold

The appearance of bone tissue engineering has provided a new direction for the repair of bone tissue defects,and preparation of tissue engineering scaffolds is the key to construct tissue engineering bone,which has attracted wide attention in recent years.The material performance of bone tissue engineering scaffold determines the final characteristics of the composite scaffold.Therefore,it is crucial to choose suitable materials for the bone tissue engineering scaffold.Chitosan(CS)is a natural polymer material with non-toxic,stable,antibacterial,biodegradable and good biocompatibility.Gelatin(Gel)is a biopolymer produced by collagen hydrolysis.It has low immunogenicity,non-toxic,absorbable properties and can also promote cell adhesion.Hydroxyapatite(nHAp)is one of the main components of natural bone.It has good biocompatibility and can induce the occurrence of new bone and has good bone conductivity.Multi-walled carbon nanotubes(MWCNTs)has excellent mechanical property,good thermal conductivity and electrical conductivity.Therefore,in order to prepare non-toxic,degradable,suitable for mechanical strength and good biocompatible scaffolds,chitosan,gelatin,hydroxyapatite and multi walled carbon nanotubes were compounded to prepare composite scaffolds to make their advantages complementary.In this study,CS/Gel/nHAp/MWCNTs composite scaffolds containing different MWCNTs ratios were prepared by freeze-drying,and the water absorption,porosity,mechanical properties and so on of the composite scaffolds were characterized.Then,cell-scaffold composite was constructed,and the biological properties of the composite were characterized.In this study,CS/Gel/nHAp scaffolds,CS/Gel/nHAp/0.3%MWCNTs scaffolds,CS/Gel/nHAp/0.6%MWCNTs scaffolds and CS/Gel/nHAp/0.9%MWCNTs scaffolds were prepared by freeze-drying.The composite scaffolds have rich and interconnected porous structure with large holes and small holes.The diameter of large holes is between 120μm-330μm,and the diameter of small holes is between 20μm-70μm,which can provide sufficient space for cell proliferation and growth,ensure the delivery of nutrients and metabolic waste,and provide a balanced growth environment.By infrared spectrum and EDS analysis of the composite scaffolds,it was found that the scaffolds were physical blends by freeze-drying,and no chemical reactions occurred,and CS,Gel,nHAp and MWCNTs had been successfully incorporated into the scaffolds.With the increase of MWCNTs content in the scaffolds,the porosity and water absorption of the composite scaffolds first increase and then decrease,and the contact angle first decrease and then increase,and the degradation rate of the scaffolds are slowed down.After adding MWCNTs,the elastic modulus of the composite scaffolds increase significantly,and with the increase of MWCNTs content,the elastic modulus of composite scaffolds increase gradually,and the elastic modulus of the CS/Gel/nHAp/0.9%MWCNTs scaffold is the largest,which is about 3.4 times that of the elastic modulus of the CS/Gel/nHAp scaffold.The MC3T3-E1 cells cultured in this study have good morphology and good proliferation ability in vitro.After the alkaline phosphatase staining,a large amount of bluish purple substance appeared in the cell matrix.Alizarin red staining showed a large number of red calcium nodules around the cells,indicating the MC3T3-E1 have good osteogenic differentiation ability in vitro.The cell scaffold composite was observed by scanning electron microscope,and it was found that the cells had good walls on each composite scaffold and the synapses were connected between the cells,but the number of cells on the CS/Gel/nHAp/0.6%MWCNTs scaffold was more than that of the other groups.Compared with other scaffolds,the cells were more evenly distributed and maintained better cell viability on the CS/Gel/nHAp/0.6%MWCNTs scaffold by fluorescence staining.CCK detection revealed that the cell viability of CS/Gel/nHAp/0.3%MWCNTs scaffolds and CS/Gel/nHAp/0.6%MWCNTs scaffolds was higher than that of CS/Gel/nHAp scaffolds after 5 days of culture.ALP staining showed that the addition of MWCNTs could promote the secretion of ALP.The results showed that the scaffold with proper MWCNTs had good physical and chemical properties and biological properties,and could support the cell adhesion,proliferation and differentiation in vitro.