Manufacture, Modifications, Atibacterial And Osteointegration Activities Researches Of The Titania Nanotube Coatings

BACKGROUNDTitanium and titanium alloys are widely used in implant because of their excellent physical and biological properties.Case reports of implant failure for loose and infection are not seldom, so surface modify of implant is always the research hotpot in material field. Surface of implants can impact the Osteointegration between material and bone and a lot of methods were used to change the implant surface morphology. Bone is a nano composite structure made up of hydroxyapatite, collagen and other biological molecules, so bone cells may be more compliant to nanoscale surface compare with macro and micro level surface. The stuy of titania nanotubes (TiO2-NT) is a hotpot of material field.The dimension of TiO2-NT can be adjusted by the anodic oxidation voltage. It’s controversial for osteogenesis abilities of mesenchymal stem cells (MSCs) and osteoblasts (OB) influenced by the dimension of TiO2-NT. Osteointegration involved in both bone formation and bone resorption, but few researches were carried out to explain the relationships between nanotebe dimension and the formation or activities of osteoclasts(OC).The loose and infection of implants are the most serius complications considered by doctors. The implants which can promote the integration of bone and prevent the occurrence of infection will greatly accelrate the development of internal fixation technique. Zinc (Zn) which is very important for osteoblasts activities, alkaline phosphatase (ALP) activity and collagen synthesis is an important trace element in the formation of bone. The antibacterial ability of Zn were observed in the material containing Zn such as zinc ceramic and zinc sulfate used widely in stomatology. So TiO2-NT loaded with Zn may improve osteointegration and antibacterial abilities for implants.The old patients received implant surgeries become more and more with the aging of population and the improvement of living standard. The loose and sinking of implant induced by osteoposis are serious problems considered by the doctors and patients. Strontium (Sr) is the unique clinical drug to enhance bone formation and inhibit bone absorption now, but oral strontium may induce systemic adverse reaction such as nausea, diarrhea and headache. TiO2-NT loaded with Sr may improve osteointegration for the osteoprosis patients.OBJECTIVEDifferent demesions of TiO2-NT are produced to study the relationship between osteogenesis activities, formation and activities of osteoclasts and nanotubes dimesion. Zn is loaded into TiO2-NT to get an osteogenisis and antibacterial implant coating. TiO2-NT are modified by Sr and then to study the formation and activities of osteoclasts in vitro and osteointegration in osteoporosis rat models.METHODSTiO2-NT were produced by anodic oxidation. Field emission scanning electron microscopy was used to observe the dimension, integrity and uniformity of nanotubes.Deferent dimensions of TiO2-NT were produced at10,40and60V voltage. In vitro experiments, Bone marrow mononuclear cells (BMMs) were cultured on sample surface then the formation of osteoclasts, TRAP activity and staining, osteoclasts related gene expressing on different dimesion samples were researched. Mesenchymal stem cells (MSCs) were seeded on samples to observe the mineralization of extracellular matrix.Zn was loaded into TiO2-NT by hydrothermal reaction in zinc acetate and different processing times to get various the quantities of Zn loaded. In vitro experiments, protein absorption assay, cell adhesion numbers counted by DAPI staining, cytotoxity detected by LDH kit, cell proliferation observed by CCK-8, cell shape observed by SEM, ALP activities detected by ALP kit, osteoblasts related genes expression detected by realtime-PCR, alizarin red staining and antibacterial activities in culture medium and culture dish were carry out. In vivo experiments, implants were inserted into the tibias of rats and micro-CT was used to detect bone parameters and biomechanical testing was carried out to assess the maximum pull output force.Sr was loaded into TiO2-NT by hydrothermal method in Sr(OH)2solution and different processing times to get the various quantities of Sr loaded. In vitro experiments, psteoclasts formed on samples and around samples were observed by cytoskeleton fluorescence staining, DAPI and TRAP staining. Osteoclast activities were evaluated by TRAP activity and bone absorption assay. Osteoporosis rat models were established and implants were inserted into upper tibias of rats. Micro-CT and bone slices were used to assess bone formation and parameters around the implants.RESULTSTiO2-NT materials, having nanotube diameters of30,80, and120nm, were produced separately by anodization at10,40, and60V, respectively. The introduction of TiO2-NT to titanium substrates significantly reduced the formation and activities of osteoclasts on samples. With the enlargement of the nanotubes diameter, the osteoclasts number, tartrate-resistant acid phosphatase staining and activity, and related gene expressions of osteoclasts were further reduced. Osteogenic ability was enhanced by increasing the nanotube diameters.We produced coatings containing TiO2-NT incorporated with zinc (NT-Zn) on Ti substrates by anodization and hydrothermal treatment. The content of incorporated zinc was adjusted by duration of hydrothermal treatment. NT-Zn implants not only show better bone formation ability in vitro and in vivo to enhance osteointegration between bone and implants, but also can effectively inhibit growth of bacteria to prevent occurrence of infection. The cytotoxicity of local high concentration of zinc on NT-Zn3h specimen in vitro can be solved by dilution effect in vivo.NT-Sr coatings on Ti substrates were produced by anodization and hydrothermal treatment. Sr was distributed to the outer wall and inner wall of nanotubes. The slow release curve of Sr was described from immersing in PBS. The osteoclast formation and activities on samples and around samples were significantly inhibited. Osteoprosis rat models were successfully established by bilateral ovaries removing and the implants were inserted into upper of rat tibias. The trabecular bone, bone amount and osteointegration around NT-Sr implants were remarkablely inhanced.CONCLUTIONTiO2-NT coating can inhibit the formation and activities of osteoclasts. Moreover, TiO2-NT can control the formation and activities of osteoclasts by altering their nanoscale dimension, without dependency or side effects. The osteogenic abilities of MSCs were enhanced as the nanotubes diameter increased. Overall, the larger diameter nanotubes implant, such as NT60, was better able to inhibit bone absorption and enhanced bone formation to prevent implants loose and failure, especially for osteoporosis patients.Zn was added into TiO2-NT on Ti implants to form NT-Zn by a simple method involving zinc acetate immersion. NT-Zn implants not only show better bone formation ability in vitro and in vivo to enhance osteointegration between bone and implants, but also can effectively inhibit growth of bacteria to prevent occurrence of infection. The cytotoxicity of local high concentration of zinc on NT-Zn3h speciman in vitro can be solved by dilution effect in vivo. NT-Zn structure implants which were osteogenic and antibacterial may be used as a novel type of implant materials on the promise of further human experiment.Sr was added into TiO2-NT on Ti implants to form NT-Sr by hydrothermal treatment and can slowly release. NT-Sr can inhibit osteoclasts formation and activities in vitro and and got more bone amount, enhance bone parameters, better osteointegration in vivo. The activities caued by NT-Sr was proportional to the amount of Sr loaded. NT-Sr coating produced by a simple and economical method can be an ideal material for osteoprosis patients.