| BackgroundIn the area of oral implantology, studying of titanium surface treatment is always a focus of scholars who aimed to improve the initial implant stability and osseointegration.The most commonly used surface treatment methods of titanium currently include several channels:chemical treatments, physical treatments and biological treatments. Wherein the chemical etching method of treatment is now recognized as a basic approach to reach clinical needs. Etching recipes varied, but the most widely used method is:49%H2SO4:19%HCl1:1by volume in the reaction for30minutes at60℃in water bath. In the traditional method, H2SO4, as a strong oxidant, oxidizes the surface of the Ti substrate into TiO2. The strong affinity of the chloride ions to the oxide film in HCl leads to the replacement of oxygen with chloride ions, producing the soluble product TiCl4-. That is, this combination performs faceting by first forming an oxide film and then corroding it, and the achieved surface topographies depend on the construction of the oxide film to a great extent. Therefore, sulfuric acid or hydrochloric acid, although with strong acidity, can’t perform etching for pure titanium alone, and the two acids need to work together. The tradiational etch combination can remove titanium oxide film and meet the clinical requirements of surface roughnes. However, due to both sulfuric acid and hydrochloric acid, which can occur completely ionized in aqueous solution, and a large number of free hydrogen ions exist, which can penetrate to itanium matrix, resulting in severe hydrogen embrittlement for titanium material, and reducing its mechanical properties. Meanwhile, concentrated sulfuric acid under heating could produce sulfur dioxide as a corrosive gas which induces contamination of the surface. Therefore, the tradiational etching solution does not poccess the ideal requirements in some areas, a new way to deal with the shortcoming is needed.In oral implant applications, due to the patient heavy bite occlusal force, tight occlusal clearance, lateral bite force, the proportion of crown and root structure is too large and wear of upper screw resulting in close contact with the base, in the slow and repeated stress on the superstructure, break induces the fracture of the screw, and the end of the screws were always located inside the base or in the implants. On one hand, when the implants made of titanium materials with a lower hardness, such as grade II, and osseointegration is good and the upper part of the implant is unreasonable which bears too much torque, may cause the implant fracture occurred. On another hand, the small diameter implants with narrow neck are likely to fracture near the weak point, when a large force was used to screw the implant into the bone, especially in the anterior mandiblewith high bone density.It is difficult to remove the broken implants and screws, which require the special equipments and tools, and the operation for a long time, the effect can not be expected, exerting patients with additional trauma and huge mental burden, is not conducive to maintaining a good doctor-patient relationship. Therefore, in the process of titanium etching, since a large number of free hydrogen ions exist, titanium sensitivity to hydrogen and severe long-term stress leads to fracture of implants, which is a kind of hydrogen embrittlement performance and should be avoided.Given the many adverse effects of hydrogen embrittlement caused to the clinical work, therefore, it is even more urgent to find a new etching method instead of the traditional etching methods should be proposed.About100years ago, researchers found that when the metal in the presence of hydrogen will reduce some of the mechanical properties of the metal, so that hydrogen embrittlement is known due to the mechanical properties of metal reducedsince the hydrogen permeation. Hydrogen embrittlement can reduce many properties of metallic material, such as elastic modulus, tensile strength, yield strength, fatigue strength and fracture toughness, etc.,can seriously affect the life span of the metal under the long-lasting metal stress. Damageto metal caused by hydrogen embrittlement is usually divided into six categories:a) hydrogen crisp (including delayed damage and delayed fracture) when the hydrogen in the metal reached a certain saturation concentration locally, can reduce the ductility and toughness of metals, in severe cases which can lead to delayed fracture occurs.b) hydrogen corrosion。Metal hydrogen underhigh temperature and high pressure will turn into methane since react with carbon. When the value of gas pressure reaches a certain critical level, the internal stress will lead to the generation of micro-cracks on the grain boundaries.c) hydride. Gap hydrides are formed when hydrogen into the transition element in the alloy latticed) microcracks. If an excess of hydrogen entered into the metal in the smelting process, these atoms can not diffuse and escape, and are retained to form hydrogen molecules in the material defects, which will occur the lattice distortion, and even the micro-cracks.e) hydrogen bubbling. Metal works in corrosive environments such as hydrogen sulfide, or for the hydrogen charging operation, when the hydrogen diffusion into the metal with largedefects and exist as non-metallic inclusions may cause hydrogen blistering.f) rheologydegradation. After the hydrogen atoms enter into the metal, the metal can lead to a decrease in toughness of the alloy as high temperature creep resistance decreases.Therefore, involving a new etching recipe, while reaching clinical requirements of proper surface roughness and surface morphology as much as possible, to reduce the infiltration of titanium hydrogen, becomes very necessary.There was many a studies about the different kinds of organic acid etching of titanium researches, including oxalic acid, hydrofluoric acid, phosphoric acid, hydrochloric acid, nitric acid, etc..But by theoretical analysis, hydrochloric acid and oxalic acid, in theory, can not reducehydrogen ions; nitric acid has a strong passivative effect, can not reach the requirement of surface roughness.Hydrofluoric acid, phosphoric acid are not strong acid, in theory, can reduce the amount of hydrogen ions permeating into materials. However, although the hydrofluoric acid is a weak acid, a low concentration of hydrofluoric acid can react violently with the titanium, and it is difficult to control with highly toxic, corrosive ability, easy to cause heal ulcers to the operators. Phosphoric acid with moderate strength acidity and low toxicity can significantly promote the formation of hydroxyapatite, and is conducive to bone formation around the implant, and do not have oxidation, be absence of the oxidation. It is prone to cause the first stage ionization, but difficult to initiate the secondor thethird ionization. This phenomenon indicates that in the phosphoric acid solution, the concentration of free hydrogen ions is significantly lower than those of sulfuric acid, and therefore may reduce the number of free hydrogen ions into the interior part of the titanium material. However, pure phosphoric acid at room temperature can not etch the titanium. Therefore, there are many scholars who worked on physicochemical and biological characteristics of the phosphoric acid and phosphoric acid reaction with the titanium, in which the reaction occurs within the two approaches:including hydrothermal (180-800℃) and electrochemical treatments, which include anodic oxidation, anodic corrosion and cathodic reduction. However,some of these studies failed to estimate the adverse impact of the hydrothermal treatment, which could aggravate the permeation of hydrogen into Ti substrates. Other studies lead to a considerably limited faceting effect that cannot satisfy the roughness requirement. Through our investigation, we discovered that Ti could be considerably facetted by a combination of inorganic acid and acid fluoride. Therefore, a large of pre-preliminary experiments found that adding the acid fluoride to an inorganic acid, the corrosive effects may occur on the titanium at room temperature. A further concentration of the sodium phosphate had been screened, using6%,8%,10%,12%,16%,18%,25%,30%,35%,40%,46%,50%,55%and62%phosphoric acid respectively and0.1mol/1NaF acid etching of titanium, found that35%,46%and62%phosphoric acid(the concentration of3.57mol/1,4.62mol/1,6.32mol/1) and sodium fluoride in the process of etching the titanium, the corrosion reaction occurs with proper reaction speed, and the surface roughness reach the clinical requirements of titanium when reaction processes for30minutes, and there are differences between the three groups.Furthermore, by varying the concentration of sodium fluoride, the surface topography of the titanium shows stable changes. In summary, through theoretical studies and the results of the pre-experiment, to eliminate the negative factors on heat treatment and electrochemical treatment, our group expect the new etch recipe, compared to conventional etching method, could significantly reduces the amount of hydrogen permeability material, achieve the desired surface roughness clinical, and be more conducive to the adsorption of extracellular matrix proteins and osteoblasts. We primarily focus on the performance of this combination with respect to the surface roughness, the hydrogen content permeating into the Ti substrate, calcium phosphates deposition ability, zeta potential, adhesive ability with FN, VN and MG-63. This study investigates the surface topography, roughness, surface chemistry, hydrogen content, hydrogen distribution and growth ability in simulated body fluid (SBF) of titanium samples treated using various methods, and adhesive ability with FN, VN and MG-63are performed and detected by western blot and immunohistochemisitry. We hope that this study will produce a novel surface with a morphology, composition and hydrogen content that is appropriate for dental implantation.CHAPTER ONE Phosphoric acid and sodium fluoride:a novel etching combination on hydrogen embrittlement of titaniumObjective:We investigate whether a novel and inexpensive etching method, H3PO4+NaF, on titanium, could obtain both a lower hydrogen content and superior calcium phosphates deposition performance, while achieving proper surface roughness, in comparison with the traditional etching method.Method:(1):Pretreatment of samples:Ti plate of grade IV has been processed, then were treated with different concentrations of H3PO4+NaF at ambient temperature without auxiliary implementations, as group A, B and C, when the concentrations of phosphoric acid were3.57mol/1,4.62mol/1and6.32mol/1respectively. Samples were treated by the traditional method as sulfuric acid and hydrochloric acid at60℃for30min(group T).The samples were then maintained in simulated body fluid with pH7.4for10days and20days at37℃.(2):Observation of surface morphology:The surface morphologies were examined using a scanning electron microscope (SEM, HitachiS-3700N), The Scaleplate feature of the software provided with the instrument was used to measure the diameters of the round pits;10measurements were performed for each pit, and the values were averaged.(3):Detection of surface roughness:The surface roughnesses were then determined through white-light interferometry(Taylor Hobson CII).The surface roughness, and the values of Ssk and Sku were obtained using Talymap surface analysis software.(4):Detection of surfacecompositions and contents:XRD patterns of sub-groups Al, B1, B10, and Cl and of group T were obtained by X-ray diffractometry (Bruker, D8-advance). X-ray photoelectron spectroscopy (Kratos, Axis Ultra DLD) was used to quantify the P and F contents of Al, B1and B10. The obtained data were processed by Casa XPS.(5):Detection of hydrogen contents:Inert gas fusion thermal conductivity analysis was used to determine the hydrogen contents in the Ti samples (Leco, TCH600) according to ASTM E1447-09. The samples were put into the graphite crucible. The air remaining in the graphite crucible was degassed under40A under a helium atmosphere. The hydrogen in the sample was released in the form of H2and converted into water by rare earth.(6):Detection of hydrogen distribution:The hydrogen distributions in the transverse sections of sub-group B1and group T were detected by time-of-flight secondary ion mass spectrometry (ToF-SIMS, Model2100Trift, Physical Electronics, USA) according to ASTM E1504-06. Before the measurement, the samples were prepared by cross-section polishing (JEOL, SM-09010) according to ASTM E1078-09to prevent contamination and to ensure smoothness. The H mapping results were obtained using Wincadence software.(7):Detection of elastic modulus:Universal material testing machine (Instron5565) were involved to examine the elastic modulus of samples treated by different etching methods. Specifically, the bending test was limited within the elastic deformation range (0-800N) at ambient temperature.(8):Hydroxyapatite deposition on Ti plate:Simulated body fiulde were prepared according the reference. The HA deposition abilities of different etching combinations were evaluated by immersing the samples, as B1, B4, B10and T, into SBF for20days. Scanning electron microscopy (SEM, Hitachi S-3700N) and X-ray photoelectron spectroscopy (Kratos, Axis Ultra DLD) was used to observe the morphology and to quantify the Ca and P contents of B1,B4,B10and T.Results:The surface roughnesses of groups A, B, and C are in the range of0.47±0.03μm to0.87±0.05μm. The surface topography switches from small round profile to deep V-shape groove dramatically as increasing the concentration of NaF. P and F attach onto the surface synchronously.The hydrogen contents of new groups are in the range of30.9±3.8ppm to86.9±6.0ppm, and that of group T is287±16.5ppm. The distribution of hydrogen atoms were mainly inside of the grains of titanium. Calcium salt coverage areas are larger than that of traditional etching group T (F=4.507, P=0.010), the calcium phosphorus ratio of the subgroup B1, B4are closer to those of hydroxyapatite, with non-selective deposition site. The surface roughness of group T was0.59±0.04μm; hydrogen content of287±16.5ppm, show the significant differences (former:F=10.349, P<0.001; latter:F=221.945, P<0.001);hydrogen distribution concentrate on the boundaries between the crystal lattice material, and only calcium salt deposited in the etch pits.Conclusion:Meeting withthe roughness of clinical needs, the novel etching combination could significantly reducing the hydrogen content as well. The hydrogen is mainly located inside the titanium grains, which could reduce the probability of intergranular fracture caused by hydrogen embrittlement theoretically. By changing the concentration ratio of the components,it could obtain different characteristics of the surface topography. The synchronous attachments of phosphorous and fluorine onto the surface and regularity of its surface morphology transformation may affect the deposition of calcium phosphates. CHAPTER TWO Influence of titanium treated by phosphoric acid and sodium fluoride on the early adsorption of protein and osteoblastsObjective:To study the nature of the surface charge of the samples treated by phosphoric acid and sodium fluoride, and the early adsorption capacity to fibronectin and vitronectin as well as to human osteosarcoma cells MG-63.Methods:(1):Pretreatment of samples.Ti plate of grade IV has been processed according to experimental conditions and instrumentation requirements, then were treated with different concentrations of H3PO4+NaF at ambient temperature without auxiliary implementations, and treated by the traditional method as sulfuric acid and hydrochloric acid at60℃for30min.We randomly selected subgroup B1, B4and B10with conventional acid group T for comparison.(2):Zeta potential sample surface test:Use a solid surface zeta potential analyzer for analysis with Helmholtz-Smoluchowski model.Two identical samples of each experimental group were put into the adjustable gap in the sample cell, face-to-face, spacing0.1mm-0.2mm. KCl electrolyte was used to(at a concentration of1×10-6mol/1) calibrate curve and test sample surface flatness.Then detect the charge and its numberon the sample surface in acidic and basic environments, and the surface potential of the sample curves plotted at different pH conditions.Data obtained were analyzed by the Attract software.(3):Sample surface detection of fibronectin and vitronectin adsorption capacity: SD rats, male,8-10weeks old,180-220g,16.Only one sample was implanted in one femoral bone marrow cavitiesof each leg.Sample was allowed to maintaine for30minutes.Truncated femur samples were washed with deionized water sufficiently, aiming to wash off the loosely attached proterins. Then samples were washed by SDS solution (50mM/L Tris,2%SDS,5%β-mercaptoethanol), vibrated for15min, centrifugedfor15minutes with1000r/min, the supernatant was moved, and stroed in ultra-low temperature refrigerator.Western blot was used for the semi-quantitative detection of the target protein. Firstly,we determined the total protein concentration with the BCA kit,-20℃to save for backup.Wash the gel with SDS-PAGE running buffer and load samples into gel.Run gel at100V for20min through the stacking part of the gel andturn the volts up to140V for40min after the proteins have gone through the stack and are migrating through the resolvinggel. And the membrane was immersed in blocking solution overnight at4℃. The target protein antibodies (1μg/ml, according to the requirements of dilution) were added,4℃closed overnight. Then the film was transferred to the target protein secondary antibody(1:5000), and developed by chemiluminescence.(4):Early adsorption ability of samples surfacesto human osteosarcoma cells MG-63:Subgroup B1, B4and B10with conventional acid group T were randomly selected for comparison.SD rats, male,8-10weeks old,180-220g,32.Only one sample was implanted in one femoral bone marrow cavitiesof each leg.Sample was allowed to maintaine for30minutes.Truncated femur samples were washed with deionized water sufficiently.Four treatment groups B1, B4, B10and T, have four parallel samples respectively. Samples obtained were placed in96-well plates, and MG-63cells with80%contact rate were rinsed, digestion, centrifugation, resuspended in cell count, and then were seeded as a density of1×105/cm2onto the titanium sheet surface. Samples were placed in37℃, the volume fraction in the conventional5%CO2incubator incubation culture, were detected cell adhesion capacity, as well as changes in cell morphology at6h,12h,18h and24h. Cells were fixed using3%-4%paraformaldehyde at room temperature for30minutes; then0.1%Triton X-100was added96-500μL/hole,allowed to stand for3minutes,to increase cell permeability. F-actin green fluorescent phalloidin working fluid was added100μl/well in the dark conditionat room temperature, stained for30minutes in the dark.Quantitative software Tanon colon of cell number was used to calculate cell number in per field of view under the microscope, and for each sample to obtain the number of5fields of view, the data was performed one-way ANOVA statistical analysis.Results:Zeta potential of all test sample surfaces are negative, showing significant difference(F=70.719,P<0.001), those of subgroups B10and B1are the lowest and the highest absolute value, respectively, and that of B4is similar to that of group T. Subgroup B10poccessed the highest adsorption capacities of fibronectin and vitronectins, showing significant difference with other groups(former:F=116.692, P<0.001; latter:F=13.937, P=0.002). While, the capacities of B1are the lowest, B4and group T on fibronectin adsorption capacity are similar, but the group T on vitronectin adsorption capacity is better than that of B4, and is similar to that of B10. The results of MG-63cells’number on each group are consistent with those of fibronectin adsorption ability of each group(F=701.542, P<0.001),.Maybe the amount of phosphate and fluorine attached onto H3PO4group are benefit for the MG-63cellsspread speed, and their mature are faster than that of other groups.Samples treated by traditional etching method as group T is contaminated by sulfur, whose cell spreading and maturation are slow.Conclusion:The high surface roughness and surface morphology with sharp edges and cusps induce the sample surfaceto poccess a low absolute value of zeta potential. Therefore, the surface adsorption of proteins and cells in surrounding are the most feasible, such as the subgroup B10.The sample surface with the round pits obtain high absolute value of zeta potential, less prone to guide the adsorption with surrounding substance, such as the subgroup B1. due to a secondary pore structure of the surface, group T may haveaffecting the adsorption of proteins and cells. Samples treated by new etch recipe with attached phosphate element and fluorine element may be beneficial to accelerate the speed of cell mature. The residual sulfur of traditional etching formulations not possessesthis effect. |
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