Effects Of Fluoride-ion Implantation On The Biocompatibility And Antibacterial Activity Of Titanium

ObjectiveImplanted prosthodontic denture, as a kind of medical appliances, became the major way to recover the defect and absence of dentition. The surface character and morphology of the implanted denture determined the post-implantation surviving as well as tissue cells' adhesion, proliferation and differentiation on the surfaces. Titanium and its alloy took a major part in the prosthodontic field because of their excellent biocompatibilities, mechanical characteristics, corrosion resistances and processablilities. However, titanium could not be biologically integrate with the bone tissue, and meanwhile titanium did not acquire bactericidal capabilities then shortly after the implantation, the dental plaques could be identified around the implanted dentures. To obtain a better bone integration, many researchers applied the surface activation processing to titanium using surface modifying techniques which rendered the material with biocompatibilities in a certain extent and sound results had been reported. But reports on the modified bactericidal properties were still few.A lot of statistic data showed, in clinic, the dental plaque deposits on the cervical part of the implanted dentures, by damaging the soft tissue barriers and the osteo-titanium integrating surfaces via bacterial surface materials, toxins and metabolites, caused a certain portion of the total loosing and shedding of the implanted dentures due to the peripheral inflammation and eventually the loss of the osteo-titanium integrating surfaces and failure of the denture implantation. Porphyromonas gingivalis was considered the maily bacterium causing the peri-implantitis. Hence, To prevent the occurrence of peri-implantitis, it is extremely necessary to inhibit the initial adhesion and proliferation of Porphyromonas gingivalis.The effects of implantation materials on osteoblast activities were multiplex. On cell level, they could influence the morphology, attachment, migration and proliferation. On molecule level, they could influence the expression of the bone-related genes. On evaluation of the bioactivities of implanted materials, an important index was to observe if this material could promote massive cell adhesion and normal morphologies forming in early stage of cell seeding, the morphological changes were biological signals would intrigued a series of cell behaviors. The attachments of the cells were relative to the pathway of the cell signals, whose conductions were based on the formation of vinculin. The integrins were conbined with the matrix protein and vinculin, then the compounds were formed and influenced the morphology of F-actin. Further more, the cell morphology and secreted function were changed. Collagen-1 was considered the most important composition of the fiberation collagen, and the expressions of mRNA and protein were regard as the symbol of bone formation and rebuilding. Hence, the judgement of biological potency to the implantation materials were according to the expressions of mRNA and protein.At the time that bioceramics coating techniques were still facing many obstacles, the ion implantantion techniques which could modify the surface physical properties and chemical constitutions and enhance the biocompatibilities of the titanium became the focusing of current studies. Plasma immersion ion implantation has brought the intensified notices around the world and it began to be applied in biomedical field. As a necessary microelement for organic activities, fluoride took a significant part in the developing and maintaining of the physiological structures of the bones. At the same time, fluoride had been proved acquiring a good property of anti-bacterial capabilities. Currently, researchers begin to carry out studies on the inhibiting effects of fluoride to gram negative bacteria to achieve the goal of preventing periodontitis and peri-implantitis or peri-implantoclasia. In our study, we applied the plasma immersion ion implantation technique to implant fluoride ion into the smooth surfaces of pure titanium. The aim of our present study was to evaluate the physical structure and chemical composition of the modified surfaces as well as assess the effect on the biocompatibility and antibacterial activites, which were pivotal in evaluating the viability of a material used for implanting into human body.Methods1. Fluoride ion implantation and microscopical analysisThe test specimens in the form of discs 10mm in diameter and 1mm thick were ground down and polished one side as a mirror finish and then they were sequentially ultrasonicated in acetone, absolute alcohol and deionized water, air-dried and stored in a desiccator. Prior to plasma immersion ion implantation processing, the samples were sputtered by argon plasma for 10 minutes. Fluoride-ion implantation was carried out at State Key Laboratory of Advanced Welding Production Technology of Harbin Institute of Technology.The chemical composition of the modified surfaces was characterized by X-ray Photoelectron Spectroscopy and the physical structure was characterized by scanning electron microscope. The fluoride ion-implanted titanium were immersed in the pure water for 37℃for 72h, then the concentrations of fluorine ions released from the immersed water were detected through the method of electrochemistry.2. The effect of fluoride ion-implanted titanium on antibacterial activityPorphyromonas gingivalis were cultured on the modified titanium and thenumbers of the bacterial were counted via colony-forming units.After Porphyromonas gingivalis culture for 48h, the samples were harvested as washing with phosphate-buffered saline then fixing with 2.5% glutaraldehyde in 0.1M cacodylic acid buffer overnight. After fixation, the samples were sequentially dehydrated in ethanol for 10min each, immersed in isoamyl acetate for 1.5min, a critical point drying fluid and sputter-coated with a thin layer of Au-Pd. Morphologies of the bacterials were observed and captured by SEM. 3. The effect of fluoride ion-implanted titanium on the biocompatibilityMG-63 cells were cultured in 96-well cell culture plates at a density of 5,000cells/ml. After the adherence of the cells, theα-MEM medium was replaced by the leaching liquor of the fluoride ion-implanted titanium and non-implanted titanium. The effects on the generation of MG-63 were examined by MTT.After MG-63 culture for 48h, the samples were harvested as washing with phosphate-buffered saline then fixing with 2.5% glutaraldehyde in 0.1M cacodylic acid buffer overnight. After fixation, the samples were sequentially dehydrated in ethanol for 10min each, immersed in isoamyl acetate for 1.5min, a critical point drying fluid and sputter-coated with a thin layer of Au-Pd. Morphologies of the osteoblasts were observed and captured by SEM.Cell counting was carried out using acridine orange stain method. At the appropriate time interval of incubation (6,24,48h), samples were fixed in 95% ethanol and stained in 4×10^-4mg/ml acridine orange for 1min. After rinsed with PBS, the samples were examined under fluorescent microscope, and the number of attached cells was counted in a 2mm² randomly selected areas.Cell cycle was analyzed by simple staining flow cytometric analysis. The MG-63 cells were harvested at the same point of culture for 48h as described in previous protocol by treating with 0.25% trypsin-EDTA and incubated with 70% alcohol at 4℃in dark for 24 hours. After fixation, cells were stained at 4℃in dark for 30 minutes and then analyzed on a flow cytometer collecting 10,000 events .The staining buffer recipe was: 0.05‰PI, 0.02‰RNase, 0.01M Triton-X-100, 0.1% Sodium Citrate.In order to investigate the formation of focal adhesion plaque, MG-63 were seeded onto the surfaces of the modified discs in 24-well cell culture plates at a density of 20,000 cells/ml and quantified by morphometric analysis using an immunofluorescence microscope after cell cultured for 6h, 24h, 48h respectively.In order to investigate the formation and expression of collagen-I, MG-63 were seeded onto the surfaces of the modified discs in 24-well cell culture plates at a density of 20,000 cells/ml and quantified by the analysis using an immunofluorescence microscope, reverse transcription polymerase chain reaction and Western Blotting after cell cultured for 6h, 24h, 48h respectively.Results1. Chemical composition and Physical structure of the modified surfacesThe XPS survey spectra in the binding energy range of 0-1350 eV indicated that the main effect of fluoride-ion implantation on the chemical states was the appearances of Titanium, Oxygen,fluorine and Carbon signals. The fitting results clearly proved that titanium dioxide (TiCh) existed on the surface layers of the non-implanted samples, while titanium dioxide (TiO₂) and titanium trifluoride (TiF₃) existed on the surface layers of the fluoride-implanted samples.There were a great deal of homogeneously sporadic distributed granular masses on fluoride ion-implanted surfaces, which appeared impacting into the basal material or depositing on its surface. Moreover, with the increasing time of fluoride ion implantation, the number of the granular masses deposited on the modified surfaces increased largely accompanied with the appearance of interstice among the homogeneously distributed granular masses. The concentration of fluorine ions released from fluoride ion-implanted titanium was less than 0.10mg/L of the detection limit.2. The effects of fluoride ion-implanted titanium on the antibacterial activityThe colony-forming units of Porphyromonas gingivalis on fluoride ion-implanted titanium was significantly less than that on non-implanted titanium (P<0.01). Further more, with the extending of the fluoride ion implanting time, the colony-forming units decreased apparently.The number of Porphyromonas gingivalis adhered to the surface of non-implanted titanium was more and the morphous was regular. On the other hand, Porphyromonas gingivalis adhered to the surface of fluoride-implanted titanium appeared to be sligh abnormity and grew together as chains of cells. Further more, P. g appeared to be widely damaged with irregular morphous and considerable pieces of disaggregation of the bacterium left with the extending of the fluoride ion implantation time.3. The effects of fluoride ion-implanted titanium on the biocompatibilityCompared to non-implanted titanium, the generation of MG-63 cultured on fluoride ion-implanted titanium was conspicuous. Moreover, the number of the cells increased largely with the extending of fluoride ion implantation time (P<0.01). But there was no significant difference between F-Ti-7h and F-Ti-5h (P>0.05).SEM analysis revealed that fluoried ion-implanted titanium surfaces did influence the morphologies of MG-63 cells in vitro. Cells grew on the discs showed relatively well spreading and flattening, as well with an increased cell-to-substrate contact ratio compared to those grew on the non-implanted titanium discs which were mainly round-shaped. Moreover, with the increasing of the implanting time of fluoride ion, the density of attached cells enhanced and they secreted a considerable amount of extracellular matrix substances which wrapped the granula on the sample surfaces.Time-dependent increases were observed in numbers of attached and proliferated cells on all samples. At 6h after cell seeding, no significant differences among five substrates were identified (p>0.05). But after 24h, the cells attached on F-Ti increased more rapidly than those on cp-Ti (p<0.05). This trend became more obvious along with the extended time of fluoride ion implantation, while there was no significant difference in cell number between F-Ti-5h and F-Ti-7h (P>0.05).At 48h after cell seeding, no significant differences between F-Ti and cp-Ti were observed in the percentages of cells in G₀/G₁ and G₂/M, while the percentages of cells in S phase on fluoride ion-implanted titanium were shown much higher than those on non-implanted titanium. No apoptotic peaks were observed before the G₁ peak.Time-dependent increases were observed in the formation of focal adhesion plaque on the two samples of fluoride ion-implanted titanium and non-implanted titanium. At 6h after cell seeding, the immunofluorescent intensity of focal adhesion plaque on fluoride ion-implanted titanium was more than that on non-implanted titanium (p<0.05). But after 24h, no significant differences between the two samples (p>0.05).Time-dependent increases were observed in the formation of collagen-I on the two samples. At 6h and 24h after cell seeding, the immunofluorescent intensity of collagen-I on fluoride ion-implanted titanium was significantly more than that on non-implanted titanium (p<0.01). While after 48h, the difference between fluoride ion-implanted titanium cp-titanium was significant (p<0.05).Time-dependent increases were observed in the expression of collagen-I mRNA and protein on the two samples. The relative mRNA and protein levels of fluoride ion-implanted titanium were higher than those on non-implanted titanium (p<0.05).Conclusion1. The chemical elements of fluoride ion-implanted titanium were Titanium, Oxygen, Fluorine and Carbon, and they existed on the surface layers of the fluoride ion-implanted samples as titanium dioxide and titanium trifluoride.2. The titanium surfaces subjected to the implantation of fluoride ions presented the morphologies of homogeneous distributing and sporadic granulate masses.3. The concentration of fluoride ion released from the fluoride ion-implanted titanium was less than 0.10mg/L of the detection limit.4. Fluoride ion-implanted titanium had the effects on inhibiting the generation of Porphyromonas gingivalis.5. Fluoride ion-implanted titanium had the effects on the attachment and morphology of Porphyromonas gingivalis.6. The modified layer of fluoride ion-implanted titanium had no toxicity to osteoblasts.7. In comparison with the non-implanted titanium, the cells growing on the fluoride ion-implanted titanium showed the better morphologies, attachment and proliferation. 8. The immunofluorescent intensity of focal adhesion plaque on fluoride ion-implanted titanium was higher than that on non-implanted titanium.9. The immumofluorescent intensity of collagen-I on fluoride ion-implanted titanium was higher than that on non-implanted titanium.10. The relative mRNA and protein level of collagen-I on the fluoride ion-implanted titanium were higher than that on non-implanted titanium.