Establishment And Osseointegration Studies Of Different Titanium Implant Surfaces

Part one: Establishment and osseointegration studies of a rough surface on titanium implantRoughened surfaces have been demonstrated to favor implant osseointegration. In vitro investigations have indicated roughened surfaces play an important role in osteoblastic attachment, differentiation, and matrix production, as well as their growth factor and cytokine production. In vivo studies have also demonstrated roughened surfaces significantly enhance bone-implant contact, new bone formation around implant and removal torque values compared with smooth surfaces. Clinic practices have shown high success rates after early loading. Even if in low-quality bone, such as the posterior maxilla, the success rate remained high.Therefore, an attention has always been focused on preparing roughened surfaces. The purpose of this study was to utilize the sandblasted and acid-etched treatments to establish a rough implant surface and analyze this surface by FSEM, EDX and XRD. Then, the effect of this surface on the bone-bonding ability was evaluated in vivo by removal torque tests and histomorphometric analysis. In part one of this study, the new surface was established and analyzed. The surface treatment was as follows: titanium plates were polished, sandblasted with green silicon carbide at the pressure of 4MPa and washed with acetone, 75% alcohol and distilled water in an ultrasonic cleaner, respectively, for 15 min. Subsequently, the specimens were chemically treated with a solution containing 0.11 mol/L HF and 0.09 mol/L HNO3 at room temperature for 10 min and dried in an oven at 50℃for 24h. Then, the specimens were treated with a solution containing 5.80 mol/L HC1 and 8.96 mol/L H₂SO₄ at 80℃for 30 min and dried in an oven at 50℃for 24h. Surface morphology of plates was observed with field-emission scanning electron microscopy (FSEM). The chemical composition of the surface was analyzed by an energy-dispersive electron probe X-ray microanalyzer(EDX). Surface crystal structure of both plates was analyzed by using an X-ray diffractometer (XRD) with a Cu anode.FSEM observation showed that the roughened surface was quite irregular. The multi-level pores appeared on the roughened surface. Microscopic evaluation demonstrated no residual particles from sandblasting for the roughened surface. The pits impacted by sandblast particles in a diameter of about 10-30um. Each pit embraced many micropits with a diameter of about 0.5～3um. EDX analysis showed that the chemical composition of rough surface was titanium. Five well-defined diffractions of titanium appeared on the XRD patterns. TiH₂ also appeared on the rough surface.In part two of this study, the bone-bonding ability of this rough implant was evaluated by mechanical tests, FSEM and EDX. Thread-shaped titanium implants with an external diameter of 3.0 mm and a length of 10 mm (n=100) were used in this study. The implants were separated into two groups: machined implants and roughened implants. 100 implants were inserted into bilateral femurs of 50 rabbits. Animals were euthanized at 2,4, 6, 8 and 12 weeks following the operation. Tissues were retrieved and prepared for removal torque tests (RTQ). The torqued implants were observed with FSEM and EDX. The RTQ values were statistically analyzed.The roughened implants showed greater RTQ values than did the machined implants at 2, 4, 6, 8, and 12 weeks (p<0.05). There were visible differences in bone apposition layers on both implant surfaces during all observation periods. Bone apposition layer on the roughened implant was more than that on the machined implant during all periods. During all observation periods, the content of Ca and P on the machined surface was always low. In contrast, the content of Ca and P on the rough surface was high after 2 weeks. After 4 weeks, the content somewhat decrease and did not change during 6 to 12 weeks.In part three of this study, the effect of the surface on peri-implant bone formation was evaluated by histomorphometric analysis. Thread-shaped titanium implants with an external diameter of 3.0 mm and a length of 8 mm (n=50) were used in this study. The implants were separated into two groups: machined implants and roughened implants. 50 implants were inserted into bilateral tibias of 25 rabbits. Starting the second day after the implantation, polychromatic fluorescence labeling was performed. The sequential administration of fluorescent dyes allows one to follow the direction and the topographic localization of new bone formation. Animals were euthanized at 2, 4, 6, 8 and 12 weeks. Tissues were retrieved and prepared for histomorphometric analysis. Bone to implant contact (BIC) and bone area in the threads were measured and statistically analyzed. Incandescent light and fluorescent microscopy evaluations demonstrated new bone formation on the roughened implant surface during the test period. In contrast, in the machined implant, new bone appeared to form on the old bone surfaces and gradually surrounded implant surfaces. During all periods, the roughened implant showed greater BIC and bone area in the threads compared to the machined implant. Significant differences were found among all parameters between two implants.These results showed this rough implant evidently improved the bone-bonding ability and shortened the healing period. This rough implant was favorable for early load. This rough treatment may be a choice for developing the domestic implant.Part two: Establishment and osseointegration studies of biomimetically and electrochemically deposited HA coatings on titanium implantPlasma-spraying is still the most popular technology commercially used for depositing HA coatings onto titanium-base implants. However, this process presents several drawbacks, including exposure of substrates to intense heat, residual thermal stresses in coatings, difficulty in evenly deposit HA coating on porous implants, alterations in HA structure due to the coating process. Biomimetic deposition and electrochemical deposition are two of the most promising new processes. Both processes have the potential to be used to coat porous titanium implants. Moreover, these processes can produce nano-hydroxyapatite (nano-HA) on the metallic substrate surface. This part of the study used the biomimetic and electrochemical depositions to coat the rough surface with nano-HA. Two surface coatings were analyzed by FSEM, XRD and FTIR. Then, effects of the coatings on the bone-bonding ability were evaluated in vivo by removal torque tests and histomorphometric analysis. This study provides guidelines for their medical applications by comparing the effects of two coatings.In part four of this study, the biomimetic and electrochemical coatings were analyzed. The biomimetic deposited CaP (BDCaP) coating was produced previously mentioned by Barrere. Roughened titanium plates were first soaked into supersaturated Ca-P solution (SBF-A) with high concentrations of salts and inhibitors of crystal growth to favor heterogeneous nucleation. The samples were soaked into SBF-A for 24 h at 37℃. Subsequently to pretreatment in SBF-A, the samples were immersed into a second solution (SBF-B) for 48 h at 37℃in order to grow the biomimetic coating. The electrochemically deposited HA (EDHA) coating was performed previously mentioned by Ye. Roughened plates were used as the working electrode (cathode) for the deposition of HA, while a platinum (Pt) plate as the counter electrode. The electrolytes were prepared by dissolving reagent-grade Ca(NO₃)₂ and NH₄H₂PO₄ (or NaH₂PO₄) with Ca/P ratio being 1.67 in deionized water and their PH values were adjusted by ammonia. To improve to the conductivity of the electrolytes, 0.1M NaO₃ was added. The deposition process was conducted with a DC power source operated at 3.0V and in electrolytes with 6x10^-4M Ca²⁺ concentration at 85℃. Surface analysis of two coatings was performed by FSEM, XRD and FTIR.FSEM observation showed that many sharp crystal flakes by biomimetic depostion were deposited on the rough plate surface. The sharp crystal flake was approximately 2-5μm in length and 100 nm in thickness. These rose-like shapes were found in some areas. Homogeneous coatings of electrochemical processing were deposited on the plate surfaces. The coating was characterized by rod-like crystals with a hexagonal cross section and diameters of about 70-80 nm. The rod-like ones were oriented and dense. XRD pattern confirmed that the BDCaP was substantially composed of crystalline HA and OCP. XRD pattern of the EDHA showed that the coating exhibited typical apatite peaks at 20 of 25.9°and 31-33°. The peaks of the coating match well with the standard HA patterns. FTIR spectra of the BDCaP also confirmed that the coating consists of HA and OCP crystals. FTIR spectra of the EDHA showed that the coating consists of pure HA crystals.In part five of this study, the bone-bonding ability of the BDCaP and EDHA implants was evaluated by mechanical tests, FSEM and EDX. The animal experiment and test methods were similar to which in part two of this study.The EDHA implants showed significantly greater RTQ values than did the roughened and BDCaP implants at 2, 4, 8, and 12 weeks. At 6 weeks, there were no significant differences among three implants. No differences were found between the BDCaP and roughened implants. FSEM observation of the torqued implants showed that bone apposition appeared on implant surfaces. At high magnification, the BDCaP coating disappeared while a thick tissue formed on the EDHA implant surfaces. After 6 weeks, the EDHA coating was only seen in some areas. During 4-12 weeks, the attached bone tissue on the EDHA implant surface was more than that on the BDCaP implant surface. The Ca and P contents on the BDCaP implant surfaces were low after 2-4 weeks, then continuously decreased during 6-8 weeks and hoisted after 12 weeks. In contrast, the Ca and P contents on the EDHA implant surfaces hoisted during 2-4 weeks, then decreased after 6 weeks and then hoisted after 8-12 weeks. During the periods, the Ca and P peaks of the EDHA implant were always higher than that of the BDCaP implant.In part six of this study, effects of the BDCaP and EDHA coatings on peri-implant bone formation was evaluated by histomorphometric analysis. The animal experiment and test methods were similar to which in part three of this study.After 2 weeks, the woven bone was formed on the BDCaP and EDHA implant surfaces. After 4 weeks, new bone on the EDHA implant surfaces became mature while new bone on the BDCaP implant surface was mainly woven. The new bone on the BDCaP implant surface became mature after 6 weeks. During 4-8 weeks, the EDHA implant significantly increased the BIC and bone area while the BDCaP failed to increase the BIC and bone area during all periods.These results show the EDHA process has a better ability of improving implant osseointegration compared to the BDCaP process. The electrochemical deposition is a promising process for clinical practice. Conclusion1. A roughened method was established by using sandblasting and double acid-etching treatments, which formed irregular and porous surface with levels of nanometer to micro.2. Compared to the machined surface, the roughened surface evidently improved new bone formation around implant and the bonding strength between implant and bone.3. Biomimetic deposition adopted in this paper can form nano-HA on the roughened surface, which failed to improve new bone formation around implant and the bonding strength between implant and bone.4. Electrochemical deposition adopted in this paper can also form nano-HA on the roughened surface, which was demonstrated to improve new bone formation around implant and the bonding strength between implant and bone.5. Compared to biomimetic deposition, electrochemical deposition is favorable for implant osseointegration.6. The roughened method and electrochemical deposition of HA are favorable for clinically developing surfaces of implants.