Basic Research On Titania Nanotubes For Osteogenesis And Antibacterial Applications

Titanium(Ti) implants are widely used in the field of orthopedics and dental implants because of their high biocompatibility and good mechanical properties. However, implant-associated infections remain as one of the most serious postoperative complications. There are two main reasons for the high incidence of infection: Ti bioactivity is not ideal and thus may lead to the formation of a fibrous capsule around the implant, which can prevent the contact between host immunity sentinel cells and bacterial molecules; Bacteria can easily adhere to the implant and form a biofilm, which provides the bacteria with high resistance to host defenses and antimicrobial therapies. Hence, implant coatings with enhanced osteogenic activity and antibacterial property are necessary for preventing infections. Titania nanotubes(TNTs) with relatively close elasticity modulus compared to bone tissue, high surface-to-volume ratio, and hollow structures have been demonstrated to have high bioactivity and to be superior platforms for local drug delivery applications. TNTs loaded with antibiotics may demonstrate desired bioactivity and antibacterial property, and thus can be used to prevent implant-associated infections.Objective:TNTs with three different diameters were prepared at three different voltages. The response of bone marrow mesenchymal stem cells(BMSCs) and the adhesion of Staphylococcus aureus(S. aureus) on the three TNTs were systematically investigated simultaneously to choose the most favorable TNTs for implants.Octenidine dihydrochloride(OCT)/poly(lactic-co-glycolic acid)(PLGA) was infiltrated into TNTs using a simple solvent-casting technique. The diameter of TNTs decreased to the chose favorable diameter after OCT/PLGA loading by controlling the experimental conditions. The response of BMSCs and the adhesion of S.aureus on the synthesized drug carrier were systematically investigated simultaneously. The synthesized drug carrierwith enhanced excellent biocompatibility and relatively long term antibacterial ability were expected.Methods:Part11. TNTs with various tube diameters were fabricated through anodization on a Ti sheet using a mixture of 0.50 wt% NH4 F + 10 vol% H2 O in glycerol at 10,30, and 60 V, respectively, and designated as NT10,NT30, and NT60, respectively.The surface topography were observed with Field emission scanning electron microscope(FE-SEM).The crystal structures were analyzed using X-ray diffractomer(XRD). Contact angle measurements were performed by a contact angle measuring system.2. The bioactivity of TNTs was evaluated by in vitro BMSCs culture. FE-SEM was used to show the cell morphology. DAPI staining of the nucleus followed by cell counting was used to evaluate cell adhesion. CCK-8 method was used to determine cell activity. Intracellular alkaline phosphatase(ALP) activity was measured by commercial kit. Sirius red staining was used to determine collagen secretion. Alizarin red staining was used to display extracellular matrix mineralization. Real-time quantitative polymerase chain reaction(q RT-PCR) was used to monitor the gene expressions.3. The antibacterial property of TNTs was evaluated by in vitro S.aureus culture. The live bacteria were quantitated by the plate-counting method.Part21. NT60 were synthesized through anodization as above-mentioned. OCT/PLGA was infiltrated into NT60 using a simple solvent-casting technique. The surface topography, the tube length and the infiltration of PLGA were observed with FE-SEM. Contact angle measurements were performed by a contact angle measuring system.2. The bioactivity of OCT/PLGA-NT60 was evaluated by in vitro BMSCs culture.Cell morphology, cell activity, intracellular ALP activity, collagen secretion, extracellular matrix mineralization and gene expressions were monitored as above-mentioned.3. In vitro OCT release from OCT/PLGA-NT60 was investigated.OCT-NT60 which was fabricated usinga vacuum-assistedphysical adsorption method was also investigated and used as a control. The antibacterial property of OCT/PLGA-NT60 was evaluated by in vitro S.aureus culture. The bacterial adhesion was observed with FE-SEM, the inhibition zone was detectedby the disk-diffusion method and the antibacterial rate was calculated by the plate-counting method.Results:Part11. TNTs with three different diameters were fabricated on a Ti sheet through anodization. The average diameter of NT10, NT30, and NT60 were about 30, 100 and 200 nm respectively. XRD results indicated that all three types of TNTs had anatase peaks after annealing at 450℃. The contact angles on TNTs were smaller than the control Ti, and they decreased as the diameter increased.2. NT10 could promote the adhesion and early proliferation of BMSCs, but there was no obvious difference in the later proliferation with time;Furthermore, BMSCs on NT10 appeared to be round without noticeable extentions, and thus could not promote ALP activity, collagen secretion, extracellular matrix mineralization and osteogenesis-related gene expressions. The initial cell adhesion and cell proliferation on NT30 were slightly inhibited, but the inhibitory effect was neither serious nor long, the cell growth on NT30 caught up with time; Meanwhile, NT30 could promote cell spreading, and thus promote ALP activity, collagen secretion, extracellular matrix mineralization and osteogenesis-related gene expressions. NT60 demonstrated the best ability to promote cell spreading and osteogenic differentiation; however, it clearly impaired cell adhesion and proliferation and this trend became more evidient with time.3. The colony forming units on the TNTs were markedly decreased compared with the control Ti, and this thrend beame more evidient as the tube diameter increased.Part21. OCT/PLGA was infiltrated into NT60 using a simple solvent-casting technique without completely filling the nanotubes, and the diameter of TNTs decreased to the chosen favorable diameter of 100 nm after loading. The contact angle on OCT/ PLGA-NT60 was bigger than NT60, but still smaller than the control Ti.2. OCT/PLGA-NT60 could promote initial cell adhesion. The early cell proliferation on OCT/PLGA-NT60 was slightly inhibited, but the cell growth caught up with time. Meanwhile, OCT/PLGA-NT60 could promote cell spreading, and thus showed good osteogenesis-inducing ability.3.OCT/PLGA-NT60 enabled slow and sustained OCT release, and thus exhibited good long-term antibacterial ability.Conclusions:TNTs with three different diameters were fabricated using weakly acidic electrolyte including NH4 F and glycerol through electrochemical anodization. The average diameter of NT10, NT30, and NT60 were about 30, 100 and 200 nm respectively. NT30 with a diameter of 100 nm could improved BMSCs osteogenic differentiation to the utmost without blocking cell proliferation and simultaneously decreased S. aureus adhesion, and thus should be the optimal choice for the bone part of the implant.OCT/PLGA was infiltrated into NT60 using a simple solvent-casting technique, and the diameter of NT60 decreased to the chosen favorable diameter of 100 nm after loading.OCT/PLGA-NT60 could supported BMSCs proliferation and demonstarated good osteogenesis-inducing ability. Also, OCT/PLGA-NT60 enabled sustained OCT release and exhibited good long-term antibacterial ability.Thus, OCT/PLGA-NT60 are highly attractive for the bone part of the implant because of their high bioactivity and long-term antibacterial efficacy.