F-doped TiO2 microporous coating on titanium with enhanced antibacterial and osteogenic activities

To enhance bacterial resistance and osteogenesis of titanium (Ti) -based implants, TiO2/calcium-phosphate coatings (TiCP) doped with various amounts of fluorine (F) (designated as TiCP-F1, TiCP-F6, and TiCP-F9) were prepared on Ti by micro-arc oxidation. The F doped TiCP coatings possess a microporous structure (pore size of 3–4 μm in average diameter) which is evenly covered by nano-grains of 30–60 nm in size. Successful F incorporation into TiCP was determined by X-ray photoelectron spectroscopy, and it shows weak influence on the microstructure, phase compositions, surface roughness and wettability of TiCP. All the coatings bonded firmly to the Ti substrates and showed enduring high adhesion strength in biological circumstances. The bacterial resistance and osteogenesis of the coatings were evaluated by implanting testing materials in vitro and in an infected rabbit model caused by bacteria. Both the in vitro and in vivo results indicated that TiCP and TiCP-F1 were of much higher osteogenic activity compared with Ti but lacking of bacterial resistance, whereas TiCP with high F addition (TiCP-F6 and TiCP-F9) exhibited both dramatically improved bacterial resistance and osteogenesis. In summary, TiCP-F6 possessed the best antibacterial and osteogenic activities, especially exhibited excellent osseointegration efficacy in the infected rabbit model.


Antibacterial activity evaluation
The bacteria were grown overnight in Luria-Bertani (LB) medium containing 10 g L -1 peptone, 5 g L -1 NaCl, and 5 g L -1 beef extract. The pH was adjusted to be between 7.0 and 7.2 using 1 M NaOH solution. S. aureus and E. coli were then re-suspended in phosphate buffered saline (PBS) at a concentration of 10 7 and 10 6 cells/ml (calibrated in terms of colony forming units (CFU) using the spread plate method), respectively. One ml of the bacterial suspension discussed above was added to each sample and incubated for 24 h at 37 o C. Then the bacteria on the samples were dissociated, collected and inoculated into a standard agar culture medium. After incubation at 37 o C for another 24 h, the live bacteria were counted in accordance with the National Standard of China GB/T 4789.2 protocol. The antibacterial ratio was calculated using the formula, A.R.= (A-B)/A×100%, where A.R. means the antibacterial ratio, A is the average number of bacteria on the control specimen, the flat Ti (CFU per specimen), and B is the average number of bacteria on the testing specimen (CFU per specimen).
In the FESEM examination, 1 ml of the bacterial suspension discussed above was inoculated to each Ti sample and incubated for 24 h at 37 o C. The samples were fixed, dehydrated in a series of ethanol solutions for 30 min each followed by the final dehydration conducted in absolute ethanol twice, dried in the hexamethyldisilizane ethanol solution series, and finally observed with FESEM.

MSC harvest and culture
The animal experiments were conducted according to the ISO 10993-2:1992 animal welfare requirements and approved by the Institutional Animal Care and Use Committee (IACUC) of Xi'an Jiaotong University. Briefly, bone marrow was aspirated from the femora and tibias, from which the mononucleated cells were isolated via density gradient centrifugation. The cells obtained were plated in cell culture flasks containing 20 ml of a-MEM containing 10% FBS and 1% antibiotics, and cultured at 37 o C in a humidified atmosphere of 5% CO2 and 95% air.
Non-adherent cells were removed and the adherent cells were collected for further expanding.

In vivo osteogenic and antibacterial activities
The animal experiments were conducted according to the ISO 10993-2:1992 animal welfare requirements and approved by the Institutional Animal Care and Use Committee (IACUC) of Xi'an Jiaotong University. Twenty-four adult New Zealand male rabbits 3 months in age weighing 2-3 kg were used. S. aureus was chosen to create osteomyelitis. After intraperitoneal injection of 4% chloral hydrate (0.9 ml/100 g body weight) and sterilization with povidone iodine, 4 holes of 1 cm distance from each other were sequentially drilled using a disinfected hand-operated drill (2 mm in diameter) on the left femur of rabbit. Four Ti or the coated Kirschner wires were classificatorily implanted into the drilled holes of each femur, as shown in Schematic 1A. Subsequently, 20 μl of the PBS-diluted suspension of S. aureus with a density of infected model. The Ti Kirschner wire together with 20 μl PBS injected into the medullary cavity (denoted as Ti+PBS) was set as a control. After bacterial inoculation, the fascia and skin were sutured. Following surgery, the rabbits were housed in the separated cages and allowed to eat and drink ad libitum up to weeks 8 when they were sacrificed by intraperitoneal injection of overdose pentobarbitone sodium.
Immediately thereafter killing the rabbits, the femurs containing implants were The biomechanical pull-out test was used to assess the strength of bone-implant integration. The femurs containing implants (n=3 for each group) were harvested after 8 weeks of implantation in the infected rabbit model , which were partially embedded in PMMA with the implants' top being horizontal. The testing machine (Shimadzu, AGS-10kNG, Japan) was used to pull the implant vertically out at a cross-head speed of 1 mm/min. The load-displacement curve was recorded and the maximum pull-out force was then calculated.