| BackgroundDentition defect and edentulous jaw are common diseases affecting the human population, however, traditional restorative treatments have many shortcomings and insufficient. Titanium implants are widely used in clinical practice by virtue of its excellent biocompatibility, corrosion resistance and high strength, etc. Although titanium material has good biocompatibility, for the biological inertness, since the TiO2 film is formed at the suface as soon as exposed in the air, which affects the combination between implant and bone tissue. Furthermore, the combination between implant and surrounding bone is related to the surface topography of implant because it is a kind of mechanical bond by growing new bone into the surface of the implants rather than chemical bonding.Lots of researchers developed variety of technologies in order to improve the ability to bind with the surrounding bone tissue as well as shorten the healing times. There are roughly three types of methods to modify titanium surfaces, including physical methods, chemical methods and electrochemical methods. To obtain good bioactivity for improving osseointegration, the surface properties of titanium were modified by two or more than two kinds of surface modification technologies in recent years. With the development of nanotechnology, nano-modified implant has become closer to the microenvironment in vivo because of the similar nano-scale structure of extracellular matrix protein. At present, uniform nanotubes can be formed by anodic oxidation, having a size effect and the quantum effect of nanostructures with a larger surface area. The structure is beneficial for the adhesion and deposition of protein. At the same time, it attracts widespread attention in terms of implant modification because of its potential ability of loading with drugs and bioactive materials.Icariin (C33H40O15, molecular weight 676.67) is an important component of traditional Chinese medicine, which is used for osteoporosis. Studies have shown that it can promote the proliferation of osteoblasts and the expression of bone-related genes, and can inhibit the activity of osteoclasts, thus facilitating the healing of bone tissue in patients with osteoporosis. What’s more, it is applied in bone tissue engineering gradually because of simple extraction process and stable chemical characteristics.Layer by layer self-assembly technique developed rapidly as a simple and versatile method of surface modification in the 1990s. Polyelectrolyte multilayers are alternately deposited by the use of contrast polyanion and polycation materials. Nowadays there are substantial researchers prepared self-assembled multilayers such as chitosan/hyaluronic acid, chitosan/DNA and chitosan/gelatin. Chitosan is a kind of biodegradable and non-toxic natural sugar, which has good biocompatibility and antibacterial property. Studies prepared bioactive scaffold using chitosan and gelatin, which exhibited good excellent properties of cell differentiation and adhesion. In addition, chitosan and gelatin have osteoinductive capacity and good prospects in bone tissue engineering. They can be used as coating materials for implant modification.In this study, TiO2 nanotubes were prepared and used to load with icariin. Sustained-release time of icariin was measured by HPLC and the effects on proliferation and activity of bone marrow mesenchymal stem cells were examined as well as the expression of bone-related genes. Chitosan/gelatin multilayers were prepared for further stimulating the microenvironment surrounding implants and exploring the effect on osteoblasts.Objection1. To prepare TiO2 nanotubes with different diameters by anodic oxidation.2. To explore the loading of icariin on TiO2 nanotubes and its sustained release.3. To investigate the effect of icariin activated TiO2 nanotubes on Mesenchymal stem cells.4. To construct the Chi/Gel multylayers by spin coated method.5. To expore the effect of Chi/Gel multylayers on the sustained-release of icariin.6. To explore the effect of implants coating by chitosan/gelatin multilayers on osteoblasts.The thesis is composed of the following two chapters:Chapter 1 The effect of TiO2 nanotubes loading with icariin on BMSCsMaterials and Methods1. Isolation, culture and identification of primary bone marrow mesenchymal stem cells1) Isolation and culture of mesenchymal stem cells BMSCs were derived from bone marrow of 2-week-old Sprague Dawley. The tibia and femur of SD rats were isolated under sterile conditions and the marrow cavity was flushed by complete medium. The marrow with complete medium was collected into tubes and centrifuged under 800 rpm for 3 min. Then the collected cells were resuspended in culture flasks and cultured in incubator.2) The measurement of growth curve MTT assay was used to measure the growth of cells. The second passage of BMSCs were seeded into 96-well culture plates with the density of 1×103/well. Absorbance value (OD) was measured after culturing for day 1,3,5,7 and 9. Then the growth curve of cells was draw with time as the abscissa and OD value as the vertical.3) The measurement of cell surface markers The third passage of cells were prepared and cell phenotype was analyzed by flow cytometric detection of CD 29, CD 44, CD 45, CD 34 and CD 90.4) The detection of multidirectional differentiation ability BMSCs were seeded at 5×104/well for osteogenic and adipogenic differentiation. Differentiation was induced by culturing cells in complete medium supplemented with osteogenesis induced factors for 21 days and concomitant induced factors for 14 days respectively. Then the cells were stained with Alizarin Red or Oil Red "O"and observed under inverted microscope.2. Preparation of TiO2 nanotubes and loading with icariin.1) Preparation of TiO2 nanotubes on titanium surface The TiO2 nanotubes (30 nm,80 nm diameters) with regular nanostructure were prepared by anodic oxidation on titanium substrates.2) Loading of icariin on TiO2 nanotubes Ethanol/DMSO was used to dissolve icariin as an organic solvent. The treated specimens were soaked in different concentrations of the above liquid and ultrasound for 5 min. Then nanotubes activated by icariin were obtained after air drying.3) The characterization of TiO2 nanotubes Field emission scanning electron microscope (FE-SEM) was used to observe the surface topography before and after treatment by different voltage. The surface hydrophilicity of different groups was evaluated by measuring water contact angle.4) The detection of sustained-release time of icariin Different specimens loading with icariin were placed into PBS liquid. And 1 mL of PBS solution was cellected and measured at predetermined time points using HPLC, the data was used to calculate the sustained release.3. The effect of TiO2 nanotubes loading with icariin on BMSCs1) Observation of the surface morphology of BMSC s BMSCs were seeded onto specimens at a density of 1×104/well. The cells were fixed using paraformaldehyde (4%) after 24 hours. Then TRITC Phalladin was added as well as DAPI. The morphology of the cells was observed using inverted microscope.2) Cell migration assay The prepared specimens were put into 24-well plate, BMSCs were added into each well at a density of 1×105mL. Sterilized tips of 20 ul were used to scratch after incubation for 36 hours. The potential of migration was estimated under fluorescence microscopy after 24 hours.3) Adhesion and proliferation of BMSCs BMSCs were seeded on the surface of different groups and cultured. Then CCK-8 kit was used to detect the adhesion and proliferation at selected time points.4) The expression of osteoblast-related genes The expression of bone-related genes such as OC, COL-I, Runx2, OPN and BSP were measured by qRT-PCR assay. Furthermore, the activity of alkaline phosphatase in each group was detected.5) Statistical analysis SPSS 20.0 software was used for statistical analysis. All data are expressed as mean ± standard deviation. Comparisons between groups were tested by one-way analysis of variance (ANOVA), least significant difference (LSD) post hoc tests when the variance was regular, otherwise Dunnett’s T3 test was used. All differences were considered significant when P<0.05.Results1. Bone marrow mesenchymal stem cells of femur and tibia from SD rats were obtained by adherent culture of whole bone marrow. The expression of surface markers and multidirectional differentiation indicated that the cells had potentials for differentiation ability and the characteristic of mesenchymal stem cells.2. The walls of the nanotubes were smooth and the diameter was uniform after electrolyzing by ethylene glycol. The diameters of the nanotubes were 30 nm,50 nm and 80 nm as voltage changed for 10v,20v and 30v.3. The contact angle of nanotubes reduced significantly compared with titanium. The contact angle of 80 nm diameter was smaller than the diameter of 30 nm, while icariin showed little effect on the contact angle of titanium surface.4. The experimental results showed that icariin can be loaded on nanotubes by the physical adsorption. Icariin can be sustained-release for up to 3 days, the burst release phynomenon happended in the first day, then the release rate decreased.5. Inverted fluorescence microscope was used to observe the morphology of the cytoskeleton on different titanium surfaces, showing that less pseudopodias on the surface of titanium. More and longer lamellipodia can be observed on the surface of TiO2 nanotubes treated with 10 voltage, while cells expressed more pseudopodias on the nanotubes with 30 voltage. Icariin had no significant effect on the surface morphology of BMSCs.6. BMSCs migrated slightly better on the surface of nanotubes with 30 nm diameter. The migration of BMSCs in NTio+ICAo.os group expressed much better than other groups, suggesting that icariin can help BMSCs to migrate.7. The numbers of BMSCs attached to the surface of titanium were detected using CCK-8 kit at predesigned time points. The results showed that titanium group and specimens treated by 10 voltage can be beneficial to the growth of BMSCs than other groups, and the difference had statistically significant at 2 hours. Icariin failed to promote the adhesion on the surface significantly. In addition, there was no significant difference among the groups at first day. The cell proliferation in titanium and NTio groups expressed better than in NT30 group at third day, while the groups treated with 30 voltage can promote the proliferation of BMSCs when culturing for 7 days. Icariin failed to promote the proliferation of BMSCs.8. The expression of osteogenesis related genes increased significantly in experiment groups, especially for OC, BSP and OPN. The experiment groups loading with icariin can enhance the expression of genes to some extent and the difference was statistically significant.9. While there were no significant difference among the groups for the ALP activity except for the groups of NT10+ICA0.05 and NT30+ICA0.05 at day 7.Chapter 2 The effect of TiO2 nanotubes modified by Chi/Gel multilayers on osteoblastsMaterials and Methods1. Isolation, culture and identification of primary osteoblasts Osteoblasts of SD rats were obtained by trypsin and collagenase digestion, then the cells were purified by differential adhesion and the morphology was observed under microscope. The osteoblasts were identified by alkaline phosphatase staining, immunohistochemical staining of collagen I and alizarin red staining.2. The preparation and characterization of TiO2 nanotubes modified by Chi/Gel multilayers1) Chitosan and gelatin solution were prepared with the concentration of 10 mg/mL. Spin coating method was used to prepare the multilayers. The rotational speed was 4000 rpm for 40 s each layer. Total coating layers referred to as T/(Chi/Gel)5Chi and T/ICA/(Chi/Gel)5Chi.2) The surface hydrophilicity of samples was measured by contact angle analyzer. X-ray photoelectron spectroscopy was used to analysis the surface elements on each group. Furthermore, The surface roughness and topography were evaluated using Atomic Force Microscopy (AFM). The amount of icariin released from T+ICA group and T+ICA+LBL group were examined by high performance liquid chromatograph (HPLC).3. The effect of TiO2 nanotubes modified by Chi/Gel multilayers on osteoblasts1) The observation of cytoskeleton Osteoblasts were seeded onto specimens at a density of 2×104/well. Cells were stained and the morphology was observed using inverted microscope.2) Cell migration assay Osteoblasts were added on each specimen at a density of 2×104/mL, then proliferation was measured by CCK-8 Kits at day 1,3,5 and 7.3) The expression of bone-related genes The expression of bone-related genes such as COL-I, OPN, OPG and RANKL were measured by qRT-PCR assay.4) The activity of alkaline phosphatase The activity of ALP was measured after being cultured for 7 days and 14 days according to instruction.5) Bone-related protein was measured by Western Blot Cells were seeded on titanium surfaces in each group and the intracellular proteins were extracted after 10 days. The expression levels of OPN and COL-I protein were detected by Western Blot.6) Statistical analysis SPSS 20.0 software was used for statistical analysis. All data are expressed as mean ± standard deviation. Comparisons between groups were tested by one-way analysis of variance (ANOVA), least significant difference (LSD) post hoc tests when the variance was regular, otherwise Dunnett’s T3 test was used.. All differences were considered significant when P<0.05.Results1. Culture and identification of primary osteoblasts The osteoblasts were observed by enzymatic digestion and purified by differential adherence method. The expression of alkaline phosphatase and collagen type I were positive. In addition, bone nodules can be observed after visible mineralization, which revealed the character of osteoblasts.2. Characterization of titanium modified by Chi/Gel multilayers1) The results of AFM revealed that the roughness of titanium was smallest. The roughness increased significantly after modification by nanotubes. The roughness of titanium loading with icariin did not affect the topography significantly. The roughness of titanium coated by Chi/Gel multilayer film was reduced.2) The measurements of electron spectroscopy and contact angle showed that the Chi/Gel multilayer film covered titanium surface completely.3. The effect of TiO2 nanotubes modified by Chi/Gel multilayers on osteoblasts1) The observation of cytoskeleton The Observation showed that osteoblasts spread well with irregular morphology and more pseudopods. While the cells became smaller on the surface of nanotubes, the expansion of cells was not sufficient. In addition, icariin failed to change the cell morphology significantly. Cells spread well with a large number of filopodias and lamellipodias on the Chi/Gel multilayer film, and T+ICA+LBL group spread best compared with T+LBL group.2) The proliferation of osteoblasts The number of cells was least on TiO2 nanotubes compared with other groups at the first day, there was not statistically significant. The proliferation of cells was higher in T+ICA+LBL group at day 3,5 and 7, followed by T+LBL group. The proliferation of T+ICA group was higher than that of NT group at each time point, which indicated that icariin can improve the proliferation of osteoblasts.3) The expression of bone-related genes There were some differences about the expression of bone-related genes among these groups after being cultured for 7 days and 14 days. The expression levels of COL-Ⅰ was higher in T+ICA group than T+LBL group after 7 days. While There was no significant difference about the expression level of OPN in each group. The expression of OPG was significantly higher in T+ICA group than in T+LBL group, while it was lower in NT group and T+LBL group than that of titanium group significantly. After incubation for 14 days, the expression of COL-Ⅰ and OPN in experiment groups was significantly higher than pure titanium group, while there was not statistically significant about the expression of OPG and RANKL among groups.4) The detection of Alkaline Phosphatase The expression level of ALP in T+ICA+LBL group and T+ICA group was significantly higher than those of pure titanium (P<0.05) after being cultured for 14 days.5) The expression of COL-Ⅰ and OPN The expression of OPN in each experimental group was significantly higher than those of pure titanium group except NT group. In addition, the expression of COL-Ⅰ in T+ICA+LBL group and T+ICA group was significantly higher than in other groups and the difference was statistically significant.Conclusion1. BMSCs were isolated and cultured through adherent culture of whole bone marrow successfully.2. TiO2 nanotube structures loading with icariin can be prepared successfully by anodic oxidation method combined with physical adsorption. Icariin can release from the TiO2 nanotubes for 3 days.3. The experiments in vitro showed that icariin can promote the spreading and proliferation of BMSCs on the surface of nanotubes, and larger diameter of nanotubes can increase the expression of bone-related genes.4. Primary osteoblasts can be observed by enzyme digestion and purified by differential attachment method.5. Chi/Gel multilayers can be constructed by spin coating method, and the structure can prolong the release time to 5 days.6. Chi/Gel multilayer coating and icariin can improve the adhesion, proliferation and the expression of bone-related protein of primary osteoblasts. |
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