Synthesis And Characterization Of Functional Coatings On The Titanium Surface

Objective:Titanium and titanium alloys have been widely used in oral, craniofacial and joints as well as other areas owing to its good biocompatibilty. However, the implants are more prone to failure when the patients suffering from trauma, infection and other systemic diseases. In order to meet the needs of the patient with bone disorders or bone physiological defects, more and more scientists paid a lot of attentions to the functional implants through surface modification techniques. Our group has successfully prepared chitosan/gelatin(CS/G) coatings on the titanium surface via electrophoretic deposition(EPD). CS/G implants may promote the new bone formation around the implants and will not interrupt the osteointegration. CS/G coatings herein will act as substrate on the functional implants. Many different kinds of bioactivitors and metal ions can be co-deposited on the surface via EPD. So CS/G substrate can be used as a smart coating on implant surface.Strontium represents a new promising candidate to prevent and treat osteoporosis. Based on the results of Part one, we explored further to prepare CS/G coatings loaded with strontium via EPD. The physico-chemical properties of Sr/CS/G coatings were respectively observed. The lifespan of such implants modified with Sr/CS/G coatings might be prolonged.PART ONE-Observation of the physico-chemical properties and bone response in vivo of implants modified with CS/G coatingsExp.l Preparation of chitosan/gelatin coatingsMaterials and methods:The titanium rods were processed to be disks($1.5cm, depth lmm). The titanium disks were sand-blasted and acid-etched in sequence. The chiosan/gelatin were prepared according to this concentration:chitosan0.6%, gelatin1.4%. SA disks act as cathod and platinum acts as anode. In certain electric field, the CS/G coatings could be formulated on the SA disk surface.Results:A uniform porous hydrogel appeared on the SA disk surface. The dried coatings was thin film and there was a strong bond strength between the film and disk. Exp.2The physico-chemical properties of CS/G coatingsMaterials and methods:The thickness of CS/G coatings were measured with digital micrometer. The structure of the coatings immersed in PBS were observed with fluorescence microscope owing to the autofluorescene of chitosan. The dried coatings could absorb water in the presence of PBS. The weight of absorbed water is measured and analyzed as swelling ratio. In addition, lysozyme is one of the enzymes which can degrade chitosan. In this research, the weight decrease of CS/G coatings were measured after the CS/G disk degraded in the lysozyme-PBS solution for a certain while.Results:All scaffold-like coatings displayed highly porous structure with excellent interconnectivity and the pore size ranged from O50μm to O350um. The wall became gradually thinner and thinner. The swelling ratio shot up within lOmin. After that, the weight of the absorbed PBS declined gradually and then remained stable at around1000%from50to120min. The weight of the coatings plummeted over the first14hours. Subsequently, the weight of the coatings descended gradually and decreased significantly after28days (p=0.044). The decreased weight was due to not only chitosan degradation but also gelatin dissolution.EXP.3The growth of MC3T3-E1cells on the CS/G coatingsMaterials and methods:MC3T3-E1cells were seeded on the CS/G disks and SA disks. Cultured for2days, the cells were fixed and stained (Rh and DAPI) to observe the morphology. The proliferation and ALP activity (alkaline phosphatase) were respectively analyzed at different time point.Results:The F-actins of cells which are the cytoskeletal structure and affect cell migration displayed red fluorescence. Through observing the morphology of F-actin, the lanky filopodia appeared clearly. Meanwhile, the images showed that CS/G coatings could sustain MC3T3-E1cell attachment and migration. For both groups, the number of MC3T3-E1cells at7days was significantly higher than that at1and3days. However, no remarkable effects of CS/G coatings on cell proliferation were found compared with SA disks at1,3and7days. ALP activity is expressed as ALP divided by DNA. For CS/G implant group, ALP activity decreased from7to day28 days after cell seeding. However, no significant difference in ALP activity between CS/G and SA groups was detected at7,14and18days.EXP.4The in vivo bone response to CS/G implantsMaterials and methods:The implants were designed and processed in sequence. Thirty-two implants were placed into the distal femurs of rabbits. They were injected with fluorescent dyes at different time. They were sacrificed at2,4,8and12weeks postoperatively. BIC and BV/TV were analyzed with Micro-CT and histomorphologic sections. The dyes were observed with CLSM.Resluts:The dynamic new bone formation around CS/G implants was observed with CLSM. The coatings showed three-dimensional macroporous structure in vivo. This scaffold was biocompatible and could bridge the osteoblast attachment, migration, and secretion. At the end of experiment, the CS/G coatings were completely degraded. The Micro-CT based bone morphologic and morphometric analyses revealed a sharp contrast about osteogenic behavior(BV/TV) at8and12weeks from the gap level of the implants. There is no significant difference about BIC between CS/G implants and SA implants. There was no difference of the parameters of BV/TV and BIC from the upper of the implants between the two groups.PART-II The physico-chemical poroperties of Sr/CS/G coatingsEXP.5The physico-chemical poroperties of Sr/CS/G coatingsMaterials and methods:The detailed protocals were same with EXP.l. lg S1CI2were added into the CS/G solutions. Then the Sr/CS/G coatings were made via EPD as CS/G coatings. Surface morphology of the coatings was observed by scanning electron microscope. The element analysis of the coatings was observed with EDS. X-ray diffraction pattern (XRD) of the coatings was measured using an X-ray Diffractometer. The CS/G solution and Sr/CS/G solution were respectively observed with TEM. MC3T3-E1cells were seeded on the Sr/CS/G coatings and their morphology was observed by SEM.Results:A lot of particles existed on the surface of Sr/CS/G coatings with a diameter of1-4μm and the particles were made up of smaller microspheres and these microspheres were packed with a layer of organic materials. The results of EDS indicated that Sr/CS/G coatings included C、O、Cl Sr elements. The atom percentages of Cl and Sr in the non-particle areas were7.25%and3.5%and the atom percentages of Cl and Sr in the particle areas were2.52%and8.77%.. According to the analysis of matter phase of XRD patterns of, it is judged that the diffraction peaks of SrCl26H2Oand SrCO3existed in the XRD patterns of Sr/CS/G coatings. The results of Sr2+release from the Sr/CS/G coatings into PBS showed Sr2+could be continually released within1months. The images of TEM indicated that nanoparticles were formed in the Sr/CS/G solution with a diameter of200-400nm. The nanoparticles appeared like core-shell structure. In addition, MC3T3-E1cells could grow and spread more widely on the Sr/CS/G coatings observed with SEM.Conclusions:1. CS/G coatings could be prepared on the titanium surface via EPD.2. Through observing the physical-chemical properties CS/G coatings, it was found that CS/G coatings fabricated via EPD were degradable, good water absorption and had the ability to form a three-dimensional porous scaffolds.3. CS/G coatings could sustain the attachment and migration of MC3T3-E1cells.4. The dried CS/G coating could recover its three-dimentional hydrogel structure in the presence of water. It can be completely degraded in vivo and promote the new bone formation.5. Sr/CS/G coating could be successfully fabricated via EPD through adding the strontium chloride into the CS/G solution.