| Background:Due to a bone-forming mechanism at the TiO2interface called "osseointegration", titanium is widely used as material for implants in reconstructive and restorative surgeries for osteoporotic fractures and edentulous jaws. For decades, dental implant has been widely used in restoration of missing tooth, and get some very good clinical results.Nevertheless, there are also many problems unsolved. The implant area bone-titanium contact percentage remains at50～65%, which is far below the ideal100%. In addition, the long healing time after implant surgery limit the clinical application of titanium implant. Much effort is needed to get faster and stronger integration between implant surface and host bone tissue.The osseointegration of dental implants is dependent on their surface bioactivity, which are determining factors for rapid and intimate bone-implant contact. The concept of bioactivity means the ability of direct connection between the endogenous material surface and the host bone tissues with bone-bonding, and without intervening connective tissue, no matter it is rough surface or not.Normally, titanium was regarded as a bioinert material because of the TiO2membrane outside the surface. In order to get better bioactivity, treatment of titanium surface has been a primary focus of implant science and technology. Various modification techniques, such as sandblasted and acid-etched(SLA), micro-arc oxidizing(MAO) and HA coating have been used to increase the bioactivity of titanium surface.In recent years, the effects ultraviolet (UV) irradiation on titanium surface had earned consideration. In our previous study, we had reported ultraviolet (UV) irradiation can be used to decrease hydrocarbon contamination and obtain better bioactivity of titanium surface, which will increased the protein absorption and cell attachment, proliferation, differentiation and final osteointegration on it. But further study proved that these effects degrade over time. On other type of titanium surface, it has been also reported that the biological activity of old titanium surface was significantly lower than a new one.At present, the reasons for the decreasing of physicochemical properties and bioactivity of the titanium surface is not yet clear. Some papers had reported that titanium surface exposed to the air will adsorb hydrocarbons from air and these contaminations may change the surface composition and surface energy which affected the protein absorption and cell attachment, proliferation, differentiation and final osteointegration.In clinical applications, it is impossible to get a new titanium implant immediately after processing. All the titanium implants go through different periods of aging process. Therefore, the preservation of the implant surface properties is important, as it relates to the actual performance of implant used in clinics.Objective:1. To investigate the time-dependent change of physicochemical properties and bioactivity of acid-etched titanium surface with reference to protein absorption and cell culture in vitro.2. To investigate the effects of storage methods on time-related changes of titanium surface physicochemical properties, bioactivity and the potential mechanism.Methods:1. Specimen preparationCommercially pure titanium discs (TA2, Baoji, Shanxi Province, China),15mm in diameter and1.5mm in thickness, were prepared, and treated with a mixture of18%HCl and49%H2SO4to get acid-etched surface, as experiment specimen.Experiment1:The experiment discs were divided into4groups. Group1:new titanium discs (control), the discs were used immediately after processing; Groups2～4:titanium discs placed in sealed containers and stored in a dark room for3days,2weeks,4weeks respectively.Experiment2:The experiment discs were divided into5groups. Group1:new titanium discs (control); Group2:traditional preservation method, titanium discs placed in sealed containers and stored in a dark room; Group3-5:titanium discs were submerged in ddH2O/NaCl solution (0.15mol/L)/CaCl2(0.15mol/L) solution immediately after processing. Group2-5were stored for3days,2weeks,4weeks before tested2. Surface characterizationThe surface morphology of the discs was examined with scanning electron microscopy (SEM)(S-3700N, Hitachi, JAPAN) and Optical Profiler (NT1100, Veeco, USA). X-ray photoelectron spectroscopy (XPS)(ESCALAB250, Thermo-VG Scientific, US) was used for determination of surface composition and elemental oxidation state of the titanium surfaces. The wettability of the discs was determined by means of an automatic contact angle measuring device (OCA15, Dataphysics, German).3. Protein adsorption assayBovine serum albumin was used as model protein. The protein concentration was quantified by means of a microplate reader at562nm. The amount of protein adsorbed by the specimens was determined from the difference in protein concentration between the later and initial protein solution.4. Osteoblastic cell cultureMG63osteoblast-like cells were used in these experiments. The cells were seeded onto the tested discs in24-well plates at a density of2.4×104cells/cm2. Initial attachment of cells was evaluated by measuring the amount of cells attached to titanium substrates after30min,1h,2h and4h of incubation. The adherent cells were fixed with4%paraformaldehyde and then stained with the fluorescent dye Hoechst (nucleolus, Blue color; Sigma). Cell adhesion was evaluated by counting the number of stained nuclei on each sheet.After4h of incubation, specimens stained with fluorescent dye rhodamine phalloidin. Fluorescence microscope was used to examine cell morphology and cytoskeletal arrangement of the osteoblasts seeded onto titanium surfaces.The proliferation of cells was quantified in terms of cell density at1and3culture days using MTS based colorimetry. The amount of formazan product was measured using a microplate reader at490nm.After7days of culture, the alkaline phosphatase (ALP) activities of the samples were determined by a colorimetric assay using an ALP reagent SIGMA FAST p-Nitrophenyl phosphate (Sigma-Aldrich, Missouri, USA) containing p-nitrophenyl phosphate (p-NPP) as the substrate. The absorbance of p-nitrophenol formed was measured at405nm.The mineralization capability of cultured osteoblasts was examined by Alizarin red staining. After incubation for14days.The cultures were stained with40mM Alizarin Red in distilled water for10min at room temperature. Subsequently, the cells were washed five times with ddH2O and images were acquired. For quantitative analysis, the stained cultures were dissolved in500μL10%Cetylpyridinium chloride in10mM sodium phosphate. Color intensity was measured by a microplate reader at620nm absorbance.5. Statistical analysisAll the experiments described above were performed in triplicate. A one-way ANOVA was used to assess the statistical significance of the results between groups. All statistical analyses were performed using SPSS13.0software. P<0.05was considered statistically significant and P<0.01was considered to be highly statistically significant.Result:1. Scanning electron microscope (SEM) analysis and quantitative topographical analysis demonstrated no morphological differences among all surfaces as a result of storage method. The acid etched surface exhibited a porous microstructure with spherical pores. The micropore diameter was approximately1～3μm. Quantitative measurements of surface roughness showed no differences in any of the calculated roughness parameters among different groups, indicating that all the storage methods would not change the surface topography.2. X-ray photoelectron spectroscopy (XPS) indicated the chemical composition varied with surface types. In tradition method, the atomic percentage of carbon continued to increase with storage time, from26.0%to48.2%, indicating more adsorption of CO2and other organic molecules from the atmosphere. At the same time, the composition of Ti and O decreased with storage time. The contents of Ti decreased from19.7%to14.1%, while O decreased from53.4%to36.9%. When stored in solution, the concentration of C on the titanium surface only increased slightly as the storage time increased, indicating storing the titanium discs in solution can protect titanium surface from the hydrocarbon contamination in atmosphere significantly.3. Contact angle measurement indicated that the surface of new titanium surface exhibited superhydrophilic property with a contact angle of0.0°. But in tradition method, the surface property changed from being hydrophilic to hydrophobic as the storage time increased, and the contact angle changed from0.0°to113°. In contrast, the titanium discs stored in solution maintained the superhydrophilic (contact angle<5°) even after4weeks, but the area of2mL ddH2O spread over the titanium surface decreased with time. We hypothesis this phenomenon was caused by hydrocarbons contamination in the storage solution. This study showed that the rate of surface energy loss can be reduced by storing implant in solution, but cannot be eliminated.4. The protein adsorption rates on titanium discs stored in various protocols with different storage times are presented as a percentage relative to the total amount incubated for1h. At different storage time point, protein adsorption rate of CaCl2group was highest. At3days and4weeks storage time, protein adsorption rate of CaCl2group even higher than new surface. While the surface stored in tradition method showed the lowest protein absorption rate, and decreased with storage time significantly. The surface property was stable when the discs were stored in solution, protein absorption rate did not significantly changed with storage time. In addition, an intriguing phenomenon is that the surface of titanium discs submerged in ddH2O/NaCl solution (0.15mol/L)/CaCl2(0.15mol/L) solution showed different protein absorption.5. In this experiment, on all surfaces, cell attachment increased with incubation time. The new surface showed very good cell attachment results. At30min incubation time, the adherent cell number on new surface was about40%of the total cell numbers seeded on the discs, and after4hours incubation, almost all the cells seeded on the disc had attached to the surface.When the titanium discs were stored in tradition method, the adherent cell numbers on titanium surface decreased with the storage time. The cell number on the surface of4-week old was only about5%of that of new surface after30min incubation. Even after4hours incubation, only about50%of total cells attached to the surface of4-week old titanium surface storage in tradition method. At each incubation time adopted in this study, all the surface stored in solution showed better cell attachment than tradition method.CaCl2group was the best one, showed no statistical difference in the cell adhesion with new surface, and the tradition method was worse.6. After4h of incubation, Fluorescence microscope was used to examine cell morphology and cytoskeletal arrangement of the osteoblasts seeded onto titanium surfaces. The cells seeded on the new titanium surface were noticeably larger and spread better, exhibiting a flattened morphology that was elongated omnidirectionally with numerous lamellipodia. When the titanium disc were stored in tradition method, on3-day old surface, we also found some cells had spread into a triangle and flat shape, but most of them maintained a round shape. The cells seeded on the surface of2-week old and4-week old showed a rather round morphology with sparse filopodial extensions, but no significant morphological difference was observed between them. On the4-week old surface stored in ddH2O/NaCl solution (0.15mol/L)/CaCl2(0.15mol/L) solution, the cells were noticeably larger and spreaded better on these surfaces, showed no significant morphological difference with new surface. 7. When the titanium discs were stored in tradition method, at both incubation time points, the cell proliferation decreased with the storage time. After1day incubation, on the4-week old surface stored in ddH2O/NaCl solution (0.15mol/L)/CaCl2(0.15mol/L) solution, the cell density were noticeably larger than the4-week old surface stored in tradition method. Even after3day incubation, this trend still existed. In addition, CaCl2group showed significantly higher proliferative activity than ddHaO group and NaCl group, but showed no significant morphological difference with new surface.8. After seven days of culture, the ALP activities of the samples were determined by a colorimetric assay using an ALP reagent SIGMA FASTp-nitrophenyl phosphate (p-NNP)(Sigma-Aldrich, Missouri,USA) containing p-NPP as the substrate. The ALP activity of osteoblasts on different titanium surfaces are not obviously affected by the storage method, there was no difference among the tested titanium discs.After incubation for14days, the mineralization capability of cultured osteoblasts was examined by alizarin red staining. Matrix mineralization is dramatically affected by the storage methods. New surface and CaCl2group induce highest mineralization and no obvious difference in matrix mineralization can be found between these two groups. But the titanium surface stored in tradition method induced lower mineralization than other surface.Conclusion:1. Hydrocarbons contamination on titanium surface significantly affect the bioactivity of acid-etched surfaces, including protein adsorption and osteoblast cell response.2. Traditional methods of storing an implant in a sealed container cannot protect the surface from hydrocarbons contamination.3. The present study has revealed that the storage method significantly affected titanium surface bioactivity. Traditional storage method shows the worse effect on maintaining titanium surface bioactivity, which degrade with storage time significantly. Storing the titanium discs in solution can protect the surface from the hydrocarbon contamination of surrounding environment and enhance surface energy. 4. The charge of ions in the storage solution may play an important role in affecting the bioactivity evaluation of titanium surface. Bivalence cation plays a positive role in helping the interactivity between protein/cell and titanium surface. |
PDF Full Text Request