| Background:The long implantation time and the low osseointegration rates increase the risk of long-term stability. Approximately 6-12 weeks are required before clinical burden after implantation. It limits the clinical application of implant restoration. Research showed that the failure rate was highest on the early stage of healing. The more healing time increased the risk factors and raised the failure implantation rate. Therefore, the faster and better osseointegration is the pursuit goal and expectation of the clinician and the patients.The surface biological activity of titanium decreased with increasing storage time (time-dependent degradation).After the surface treatment of sand blasting and acid corrosion, the implant would change from relatively hydrophilic to hydrophobic when they were dried in the air.The factors influence the surface activity of implant including:surface morphology of molecular structure, elemental composition, surface free energy, dear hydrophobicity, chargestate.According to the research, the surface of the hydrophobic implant would reduce the protein adsorption, and at the same time the poor wettability affected the original condition and cellular responses of the material surface contacting with the organism’s blood components, leading to limited the implant’s biological activities even the osseointegratic effect at a long term. UV could restore the implant surface activity and improve the surface free energy and hydrophilia of aging implant after long-term storage. The restored activity promoted protein adsorption, osteoblast adhesion, proliferation and differentiation even synosteosis. All these results showed that the implant had a good biocompatibility. Our group developed a new implant with high surface energy and activity via processing titanium surface with UV. And we had proceeded a study on the physical and chemical analysis and cytology for it. The processed of titanium surface could promote the adhesion, proliferation, differentiation and mineralization of MG63 osteoblast. However, the excellent effect gradually disappeared after a long-term storage. The high surface energy and biological activity of titanium surface deal with other ways, gradually decreased with increasing storage time.Our group developed a new implant with high surface energy and activity via processing titanium surface with UV. And we had proceeded a study on the physical and chemical analysis and cytology for it. The processed of titanium surface could promote the adhesion, proliferation, differentiation and mineralization of MG63 osteoblast. However, the excellent effect gradually disappeared after a long-term storage. The high surface energy and biological activity of titanium surface deal with other ways, gradually decreased with increasing storage time.This suggested that the bioactivity of titanium surface had a certain timeliness. In addition, aging phenomenon of titanium surface was widespread and the reasons were still clear. Recently, a large number of studies have shown that, pure titanium surface in the air would adsorb inorganic ions and organic hydrocarbon compounds some impurities in a very short time, leading to surface pollution of implant and subsequent aging. Bioactivity of an aged and polluted pure titanium surface was lower than a new prepared one. The polluted pure titanium surface could change the surface chemical composition and surface energy, which affected adhesion, proliferation and differentiation of the early proteins and cells on the surface, and ultimately affected the building of osseointegration.Numerous studies indicate that the biological activity of the implant surface is closely related to the speed and strength bone tissue formed, There are two forms of graft healing the body and bone tissue after implant:Distance osteogenesis and Contact osteogenesis. Distance osteogenesis refers to new bone growth and bone wall from the original direction of the implant, and the final implant osseointegration produce surface attachment. And contact osteogenesis refers osteoblasts directly deposited on the surface in the implant, proliferation, and bone cells to form new bone original network. Studies have shown that contact osteogenic bone formation rate is 30% faster than the distance osteoblastic bone formation.Woven bone to bone contact surface of the explant help to increase the initial implant stability. Therefore, the establishment of contact osteogenesis mode, promote the formation of new bone tissue and shorten healing time after implant placement to reduce the risk of failure planting.Planting preparation process is complicated. There were a lot of cumbersome procedures from initial to final titanium materials has become a commodity, including machining, implant surface treatment, cleaning, disinfection, packaging and storage etc. It was inevitable for titanium to contact with the air in the process preparation.At present, storage in sealed vials at room temperature and atmospheric pressure is a conventional method which would cause the hydrocarbons and contaminants pollution in the air for a long time contact with the air. Whether in the conventional storage mode would continue to suffer from air pollution and continue to produce the aging? The research about this aspect has so far not reported. Therefore, based on these considerations, it is very necessary to isolate the implant from the air careful and avoid the changes of surface chemical structure caused by the air secondary population. Weather it is conducive to the formation of new early bone around implant after accepted the surface treatment to avoid the secondary pollution? Whether it is more conducive to the implant bone integrated? These still need to carry out comparative study.Inert gas is colorless and tasteless monatomic gas at normal temperature and normal pressure. The outermost electrons electronic layer is filled "full", so they are very stable with little chemical reaction. Argon is one kind of inert gases with the un-active chemical properties which is commonly used as protection in industrial production. Argon is used as protective gas isolating from air pollution in metal welding to prevent the reaction between metal with oxygen under the high temperature. Previous research proved that plasma spraying to pure titanium under argon protection effectively isolates impurities in air pollution to maintain high surface energy of pure titanium, and also greatly reduce the adsorption the surface hydrocarbons to promote the cultivation of early bone formation around the body.The study of pure titanium implant surface treatment technology becomes a hot spot in the past few decades, including sandblast and acid etching, plasma coating titanium, hydroxyapatite coating, micro arc oxidation, laser processing, etc. The sandblast and acid etching has become the international most common way which is studied widely. No matter the roughness, surface morphology or osteoinductivity and biological compatibility, there are many research reports. Sandblasting the surface acid etching with mechanical properties and excellent biocompatibility properties has been generally acknowledged. The surface treatment method has become a more mature.This paper aims to study on whether the preparation of pure titanium implant prepared under argon protection can effectively reduce the contact with air to isolate from air pollution, so that the titanium implants can keep high surface energy and surface activity. It promotes early implant bone osseointegration around a body and the efficient establishment, and to improve the success rate of implants after2-4 week relatively dangerous period of higher risk.Objective:1. To study the effect on the physical chemical properties of pure titanium implant accepted the surface treatment to avoid the secondary pollution.2. To study the effect on the surface biological activity and early osteogenic of pure titanium implant accepted the surface treatment to avoid the secondary pollution.Materials and Methods:1. Preparation of titanium samplesCommercial fourth-grade pure titanium was cut into plates (1mm in thickness, 15mm in diameter) and titanium implants which are cylinder-shaped (3.5mm diameter 6mm effective length,0.75mm the depth of screw thread). Titanium plates were washed by 2 times of acetone, ethanol and deionized water, each for 15 minutes after polished with silicon carbide abrasive papers with 400#,800#,1000# and 1200. Furthermore, the 120μm diameters alumina particles were used to blast the samples perpendicularly under high pressure.18% hydrochloric acid and 49% sulfuric acid were further etched the samples at 60 ℃ degree for 30 minutes. Acetone, ethanol and the deionized water were used to wash the titanium plates and the implants successively two times under air protection and argon protection for 15 minutes respectively. A sealed vial was kept with the control samples. A sealed vial filled with argon was used to store the experimental samples after being sterilized by γ-ray of 25kGy for 12h. Then we thereby get the control samples and the argon protected samples(the experimental samples), these samples were used to detect or animal experiments two weeks later.2. Analysis of the sample surfaceWe observed the morphology of titanium surface by the scanning electron microscope (JSM-6330F, Japan Electronics Co, Ltd.). the size of contact angle of 1 ul water droplet on the titanium surface was measured by a surface contact angle measuring instrument (OCA15Pro, Dataphysics, Germany). The surface roughness was tested by the surface topography measurement. The chemical structure and compositions of titanium surface under argon protection in a high vacuum state was checked by the X-ray photoelectron spectroscopy (XPS) (ESCALAB 250; Thermo Fisher Scientific, US).3. Animals and Surgery1) The experimental animal and classifications of group. Four 15 months old healthy male beagle dogs (15 ± 0.2kg) were provided by Guangdong Medical Experimental Animal Center. The ethics protocols of animal experiments was approved by the Ethics Committee of Guangdong Provincial Stomatology Hospital. The dogs were reared on standard laboratory diet condition. According to the random principle, in 4 adult healthy male Beagle dogs were divided into 2 week group and 4 week group,2 dogs each group. The implants from two groups were implanted symmetrically on left and right of tibial, six implants were implanted in each side of the tibia. Each Beagle canine tibial implant 12 implants,48 implants were implanted in 4 Beagle dogs. There were 24 implants samples both in air exposure treatment group and argon treatment group.2) Implantation. General anesthesia was conducted by intramuscular injection of 0.2 mL/Sumianxin and 0.5 mL/kg Sodium pentobarbital. After the animals were fixed, they were in supine position, hair removal, disinfection and flapping towel in the surgery area. We cut skin along the surface of the tibial, and the subcutaneous tissue and periosteum to expose Tibial metaphyseala. Osteotomy in distal metaphyseal range of about 2cm-8cm was prepared at spacing of 1 cm, and it was in relatively uniform and comprised of the outer cortical bone marrow. It was easy for observation and histological comparison. Gentamicin was injected intramuscularly into the animals at 80mg for 5days to prevent from the infection.3) Fluorescent double labeling method. Calcein was injected at 5mg/ml on 3,4 day prior to sacrifice,Tetracycline which was bone fluorescent marker was injected at 25mg/ml on 13,14 day prior to sacrifice.4) Sample processing. The animals were sacrificed 2 and 4 weeks later by a lethal overdose of anesthetic and removed the tibial segments containing the implantation. The bones were fixed by 10% formalin, and were dehydrated with graded alcohol, and finally embedded with resin.5) The experimental test samples. We cut specimens into 150μm thick slices by hard tissue slicer (Leica SP1600, Leica Company, Germany) in the locations of far, near and middle. Then they were polished to about 30μm slices and observe by the fluorescence microscope (Olympus BX41, Olympus Co, Japan). After stained by toluidine, the slices were observe by an optical microscope. Finally quantitative analysis of implant-bone contact (BIC) was analyzed by the image analysis software (Image-pro Express 6.0, Media Cybernetics Inc, USA) and BA (Bone area). Every implantation was measured three times, and the average value was recorded for statistical analysis.4. Statistical AnalysisAll measurement data are expressed as mean ± standard deviation. Statistical data were completed by SPSS 13.0 statistical software. T-test was used for two independent samples. P<0.05 was considered statistically significant.Results:1. The titanium morphology and surface hydrophilicityNo difference of the morphology of titanium surface between the experimental group and the control group(P>0.05). They both were rough containing nano-pores with uniform surface.The immediately prepared titanium plates either prepared under air protection or argon protection had better hydrophilicity which were essentially zero. After 3 days and 2 weeks, the titanium surface hydrophilicity decreased of both groups. Compared with the samples exposed in the air, the titanium surface hydrophilicity exposed in argon had small decrease, (p<0.01).2. The surfaces chemical structure and compositionTwo groups of titanium plates had the same chemical elements containing Ti, C, O, Al, N and other elements. Wherein, compared with the control group, elements in the experimental group were significantly different(C43.6% vs.24.3%, Ti 29.5% vs.17.8% and O 32.5% vs.38.3%. These results indicated that argon protection can effectively isolate the carbon-containing gas and the other organic adsorbents from the air, but the argon gas would not adsorbed to the titanium surface which causes pollution. Based on the chemical structure and composition, compared with the control group, C element binding energies decreased at 284.8 eV and 286.4 eV. The 284.8 eV energy binding peak came from CC and CH, the main hydrocarbons in the air,286.4 eV binding energy peak came from CO which were mainly from carbohydrate in the air, but the 286.4 eV mainly from the adsorption of organic compounds in the air. That indicated that the titanium plates in the air would absorb pollutants from the air as the time goes on because of the contact with the air, while argon protection can effective isolate physical adsorption from pollutants in the air to avoid contamination.3. Implantation and bone integration measurementThe implantation was implanted at 2cm of distal tibial metaphyseal locations of Beagles. The spacing was 1cm. Four proximal implants were made into hard tissue section to calculate BIC. Two distal implants were used to assess Torsion output moment. Early anatomical experiments proved that approximately 2cm-9cm region of distal tibial metaphyseal was comprised of outer cortical bone with uniform and the central canal. There were two kinds of fluorescence in hard tissue, yellow and green. They represented the colors of tetracycline and calcein, respectively. Yellow was slightly weaker than green because the injection of tetracycline needed longer time for metabolism and green can cover yellow. Further, only little bone was formed in the marrow cavity in the sections in both two groups, therefore cortical bone area was used for BIC calculation between two groups. In the hard tissue sections stained blue with toluidine, newborn bone tissue was stained light blue in bone ring, osteoblasts and osteoid ingredients can also existed. Compared with the control group, the bone ring had better osteogenesis in the experimental group with a large number of new bones and thick trabecular. The bone implant contact BIC were 48.39 ± 5.83% and 71.51 ± 7.61% of the control group and the experimental group, respectively. BIC in the experimental group was significantly higher than that the control group (p<0.01), Torsion output torque value in the experimental group was higher than the control group (p<0.05).Conclusion:1. Surface inevitably implant treated by conventional preparation way would be polluted by organic matter impurities form the air which reduced surface bioactivity.e.2. Conventional surface modification of titanium implant isolated air population would not impact on the implant surface topography.3. Argon is expected to be used as gas protection the process of implantation preparation as a stable and inert gas.4. Conventional surface modification of titanium implant that exposures in the air for a short period of time would be contaminated, and the main reason is the increase of hydrocarbons. There may be some other factors also needed further study. |
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