| ObjectiveDue to trauma,infection,cancer,osteomyelitis surgery debridement,and bone and joint defects caused by a variety of congenital diseases,bone and joint replacement is required in clinical.There are many materials used for bone implantation,including macromolecules,inorganic ceramics and metal materials,but the implantation effect is not ideal.As more and more bone implant materials were discoveried,there is an increasing number of studies on bone and joint replacement therapy.Titanium oxide nanotubes(TiO2),modified by conventional nanomechanics of tissue engineering materials using nano technology,has many advantages of unique biological properties,better biocompatibility,and facilitates bone integration much faster than traditional materials in the field of bone implantation and joint replacement.The application of this research has become the focus of attention.In this study,titanium dioxide modified with different diameter TiO2 nanotubes and osteoblast MG-63 were cultured in vitro to observe the effect of adhesion,proliferation and differentiation on osteoblasts,and to explore the possible mechanism,which provides theoretical basis for the development of nanomaterials in clinical application.MethodsThe bone implant materials andmetal material,were divided into four groups.three of which were treated with 50nm,100nm,CDW(Coaxial double wall,CDW)pattern.While the control group was titanium without any treatment.MG-63 cells were respectively implanted on the surface of nanotubes with 50 nm.100 nm,CDW pattern and the titanium of the control group.1.SEM(Search engine marketing,SEM)method was used to detect MG-63 cell growth status and cell morphology in the four respectively;2.MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MT T)method was used to detect MG-63 cell proliferation in the four respectively;3.ALP(Alkaline phosphatase,ALP)test-kit was used to detect activity changes of MG-63 cell’s alkaline phosphatase in the four respectively;4.Immunofluorescence method was used to assess the expression of DPT in MG-63 cell in the four respectively;5.Western blot method was used to determine the protein expression of DPT,P-FAK,FAK,ERK,F-actin and OPN in MG-63 cell in the four respectively.Results1.SEM observation showed that the osteoblasts on the surface of the four groups presented different attachment morphology and quantity after 3 days of composite culture with osteoblasts.The quantity of osteoblasts on the surface of the 50nm group and the CDW group were more than those in the control group and the 100nm group The osteoblasts on the 100nm group were flattened,stretching a larger range of cells out of more pseudopodia.2.MTT analysis indicated that the MG-63 activity which co-cultured in three kinds of nanotubes were increased faster compared with that of control group.MG-63 cells’activity was increased significantly on the day of 3,5 and 7,with a time dependence.3.ALP test proved that the expression of ALP were increased in the nanotube co-cultured group with 50nm,100nm,CDW compared with the titanium co-cultured group.4.Immunofluorescence showed that the expression of DPT were increased in the nanotube co-cultured group with 50nm,100nm,CDW compared with the titanium co-cultured group.5.Western blot results showed that The expression levels of DPT,P-FAK,FAK,ERK,F-actin and OPN were increased in the nanotube co-cultured group with 50nm,100nm,CDW compared with the titanium co-cultured group.ConclusionCo-cultured nanotubes with 50nm,100nm,CDW pattern can promote the growth,spread,proliferation and differentiation of osteoblasts much more than which with titanium.Also it has good biocompatibility.Which mechanism may be related with the increasing of the expression of ALP and the activation of signaling pathway DPT/FAK/ERK. |
