| Objective:The changes of early biological behaviors of osteoblasts such as adhesion,proliferation,differentiation,spreading and morphology were observed through the combined stimulations of materials surface chemistries and vitamin D3 on the osteoblasts,exploring the possible mechanism of combined stimulation on the biological behavior of osteoblasts.Methods:Using blank tissue culture slides as a control,the surface of hydroxyl,amino and methyl groups were constructed by silicon-alkyl self-assembly technology.The surface of the materials were characterized by water contact angle and X-ray photoelectron spectroscopy?XPS?.Resuscitating MC3T3-E1 to investigate the effects of the simple chemical stimulation on the osteoblast early adhesion,proliferation,differentiation,and cell morphology as a control group.10-7mol/L,10-8mol/L,10-9mol/L vitamin D3 medium were prepared by dissolving vitamin D3 in absolute ethyl alcohol,investigating the differences of the early response behavior of osteoblasts under the condition of different concentrations of vitamin D3 and materials surface chemistries combined stimulations,at different time points after the addition of vitamin D3,blood cell count board,CCK-8,ALP,BCA detection kit,alizarin red dye and immunofluorescence staining were used to detect the early cell adhesion,proliferation and differentiation,cell focal adhesions and cytoskeleton structure under different concentrations of vitamin D3 and materials surface chemistries combined stimulations.Results:Water contact angles show that the hydrophilicity of the surface of each group of materials change obviously.The hydrophilicity of the hydroxyl surfaces are enhanced,while the surface of methyl groups are highly hydrophobic,and the surface of amino groups have suitable hydrophilicity,the existence of each element on the surface of the materials were detected by XPS Si spectrum and C spectrum,the results of the two tests prove that the surface of the tissue cultured slides with different chemical groups?-OH,-NH2,-CH3?were successfully prepared.In the simple chemical stimulation group,it was found that the surface of the chemical groups can influence the biological behavior of osteoblasts,the surfaces of NH2 promote cell adhesion,proliferation and differentiation,and increase cell activity,the surfaces of OH and CH3 are inhibited,the rule is NH2>Glass>OH?CH3.The main reason for this result is the different surface hydrophilicity and surface charge of each group of materials.It was found between the materials surface chemistries-vitamin D3 combined stimulation groups and the control groups that medium and high concentrations of vitamin D3(10-8mol/L?10-7mol/L)could promote the early differentiation of osteoblasts and the formation of focal adhesions and cytoskeleton actin filaments?F-actin?structures,and inhibit the early proliferation and adhesion of osteoblasts on the surface of each group of materials;low concentrations of vitamin D3(10-9mol/L)can promote the early proliferation of osteoblasts and inhibit the early differentiation of osteoblasts and the formation of focal adhesions and cytoskeleton actin filaments?F-actin?structures on the surface of each group of materials.Conclusion:The combination of vitamin D3 and materials surface chemistries can induce the early responses of osteoblasts on each material surfaces,but the response degrees are different.The mechanism may be the differences in cell focal adhesion and cytoskeleton caused by materials surface chemistries-vitamin D3 combined stimulation on material surfaces,which it leads to a series of differences in early cell behaviors subsequently,therefore,it is necessary to study the relationship between the early behavior of osteoblasts and the combined stimulation of vitamin D3 and materials surface chemistries,it can provide a strong theoretical basis for the treatment of osteoporosis and the construction of tissue engineering bone model in vitro. |
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