Effects Of Graphene Oxide On Osteogenic Activity Of Calcium Phosphate Cements In Vitro

Calcium phosphate cements(CPCs)are bioactive bone cements which hydration products are similar to human bone tissue,and the hydration process have the characteristics of plasticity and low heat.However,due to the factors such as stomata and solid/liquid phase,the mechanical properties of the hydration products are weak and the application is limited.Graphene oxide(GO)has two-dimensional layered structure of carbon nano-materials,and its excellent mechanical properties are often remarkablely used as a reinforcing and toughening additives to combine with polymer or ceramic materials.In addition,GO has a mixed structure equipped with various functional groups containing oxygen to promote its dispersion and hydrophilic.The characteristics above are conducive for GO to improve the mechanical properties of cementitious materials.GO has been added to the CPCs in the early stage.The compressive strength of GO/CPCs composite materials has been effectively enhanced,and the surface morphology of the materials has been modified.However,there is little study on the biocompatibility and osteo-induction of the GO/CPCs composites.In this study,we use ethanol as milling medium,and different qualities of GO are added into ?-Ca3(PO4)2-Ca HPO4-Ca CO3 series powder of the classic CPCs to make GO/CPCs mix cement solid powders:GO-0(GO 0wt%)?GO-1(GO 0.06wt%)?GO-2(GO 0.24wt%)?GO-3(GO 0.96wt%)by the ball milling method.With sodium phosphate buffer as liquid phase,the cement paste is modulated and puts into the mold according to a certain solid-liquid ratio.The hydration reaction is under the condition of 37 ?,humidity 100% for a week.After a week,the GO/CPCs are removed from the mould and polished to 0.1±0.01 g.Then MC3T3-E1 cells are cultured with the materials in basal media or osteogenic media(24-well cell culture plate)and repeat three times.After culturing for 1,3 and 7 days,the cell morphology on the surface of materials is observed by Scanning electron microscopy(SEM),the viability of the cells is evaluated by MTT testing,the osteo-induction of cells is investigated by alkaline phosphatase(ALP)assay and the gene expression level of osteocalcin(OCN),osteopontin(OPN)or type I collagen(COLL)derived from MC3T3-E1 cells.The proliferation and differentiation of MC3T3-E1 cells influenced by GO/CPCs in vitro is explored.The results are as follows:In basal media: MC3T3-E1 cell get along well with GO/CPCs and have produced cytoplasmic synapses which the diameter increased with the amount of GO and culturing time.The cell viability and ALP activity of cells increase with the increase of GO content.The relative expression of OCN,OPN and COLL are higher in GO-1 cells than in GO-0 cells,and the OCN m RNA content rises 58-fold in GO-1 cells.The results above reveal that GO/CPCs are non-toxic to MC3T3-E1 cell and can promote the cell vitality with the increase of the GO content.GO can promote the expression level of OCN and regulate the expression temporally of osteoblast gene OCN,OPN and COLL which indicates that GO has bone-inducing effect toward MC3T3-E1 cell.In osteogenic media: The diameter of cytoplasmic synapses,the cell viability and ALP activity of MC3T3-E1 cells decrease gradually with the increase of GO content.Real-time PCR assay result reveals: GO/CPCs materials promote the genes above expression are lower than that in GO-0 cells.The above results show that the role of GO and bone inducible factors are antagonistic to MC3T3-E1 cell proliferation and differentiation.