| Chapter 1 Research on the effection of domestic porous tantalum to thebiological behaviors and function of osteoblast in rabbitObjective:To investigate the biocompatibility and the effection of domestic porous tantalum scaffold in repairing of bone defect and promoting bone regeneration, the physical properties of domestic porous tantalum materials, proliferation of osteobla-sts, cell cycle, osteogenic secretion function were investigated, to provide the experi-mental basis for clinical application and experiments in vivo.Method:The morphological properties of the domestic porous tantalum were observed by the SEM. The osteoblasts were isolated from neonatal rabbits and identified. The extract fluid from porous tantalum was made according to the standards of ISO 10993-5/ISO 10993-12. Porous tantalum scaffold were autoclaved. The osteoblasts were divided into control (cells cultured in DMEM) group, porous tantalum extract group (cells cultured in extract) and porous tantalum scaffold and osteoblasts cocultured group. The proliferation of osteoblasts co-cultured with porous tantalum in vitro was detected by the CCK-8 assay. The osteoblasts were co-cultured with porous tantalum in vitro and the morphology, growth, proliferation, differentiation were observed by inverted phuse contrast microscope, scanning electron microscope and transmission electron microscope. The cell circle of osteoblasts was detected by flow cytometry. Elisa kit assay was applied to detected the amount of osteocalcin and collagen type I of three group cultured 3,5,7 days.Results:The observed result of canning electron microscope showed that the diameter of the micrograined texture in domestic porous tantalum is 20μm to 50μm, the pore sizes between micrograined texture ranged from 400μm to 600μm, which had a similar three-dimensional connected holes morphology to cancellous bone. The result of CCK-8 assay showed that the osteoblasts grew well and proliferated rapidly in extract of porous tantalum and no significant difference between three groups(P> 0.05). Inverted phase contrast microscope observed that osteoblasts and porous tantalum co-cultured, a large number of osteoblasts adhered to the porous tantalum scaffold edge. Scanning electron microscopic observed that osteoblasts well and adhered, spread extensively and proliferate rapidly on porous tantalum surface and internal pore, osteoblasts connected to each other, and secreted a large amount of extracellular matrix, which attached to flaky, gradually covered the material. Transmssion electron microscope observed the visible osteoblast ultrastructure size shape is normal, no differences in the ultrastructure of cells. Flow cytometry showed that there were normal diploid cells and similar cell cycle distribution in three groups. There were no significant difference in the cell cycle distribution between three groups (P>0.05). The result of quantitative determination of osteoblasts show that osteocalcin secreted in three groups, with incubation time, the secretion of osteocalcin increased gradually. In porous tantalum scaffold group osteocalcin secretion was higher than the other groups, there were significant difference beween three groups(P<0.05), there were no significant difference beween porous tantalum extract group and control group (P>0.05). The result of quantitative determination of collagen type I show that collagen type I secreted in three groups, at first with incubation time, the secretion of collagen type I increased gradually, the secretion was highest in 5d and then began to decline at the same time point. There was no statistically significant difference between the three groups (P=0.899); comparison of different time points in each group, the results showed there was a statistically significant difference in three groups at different time points (P<0.05); multiple comparisons showed there was no significant difference between 3d and 7d in porous tantalum group (P>0.05), there was statistical difference in the other group comparison (P<0.05).Conclusion:Domestic porous tantalum scaffolds have good biocompatibility, the secretion of osteocalcin increased after osteoblasts co-cultured with porous tantalum scaffold prompt that porous tantalum scaffold materials may promote osteoblast mineralization and osteogenesis.Chapter 2 Study on the effection of domestic porous tantalum to osteogenic factors and osteogenic transcription factor expressionObjective:To explore the domestic porous tantalum scaffold materials on the osteogenic effect mechanism through detecte the expression and changes of osteogenic facor of (osteocalcin, osteopontin, collagen type I, fibronectin) and osteogenic transcription factors (Runx-2, OSX, P38 MAPK) between three groups, to provide theoretical basis for the clinical application of domestic porous tantalum scaffold.Method:The osteoblasts were divided into control (cells cultured in DMEM) group, porous tantalum extract group (cells cultured in extract) and porous tantalum scaffold and osteoblasts cocultured group. The osteoblasts cultured 5d in three groups, immunocytochemical staining method was used to detect the expression of osteogenic factors Col-1, OC, FN, OPN and transcription factor Runx-2, OSX, p-P38 MAPK. Immunofluorescence staining detected the co-expression of OPN with Runx-2, OSX; P38 MAPK co-expression with Runx-2, OSX in osteoblasts cocultured with porous tantalum scaffold group. Western blotting and real-time fluorescence quantitative PCR method were used to detecte the protein and RNA expression of osteogenic factors Col-1, OC, FN, OPN and transcription factor Runx-2, OSX, p-P38 MAPK.Results:The result of immunocytochemical staining showed that the osteoblasts cultured 5d in three groups had the positive expression of OC, Col-1, FN, OPN, Runx-2, OSX and p-P38, cell cytoplasm had brown granules, the expression of all the factors in porous tantalum scaffold group were increased than other groups, especially the expression of OC, OPN, Runx-2, OSX were significantly higher than the other groups(P<0.05). The results of immunofluorescence staining show that in porous tantalum scaffold group OPN co-expression with Runx-2、OSX. The fluorescent dye labeled Runx-2, OSX was green, OPN was red in the cytoplasm, when they co-expression, the cytoplasm was yellow; and p-P38 was red in the cytoplasm which co-expression with Runx-2, OSX respectively, the cytoplasm was yellow. The western blot results show that in three groups the protein expression were consistented with immunocytochemical staining results, there were the expression of OC, Col-1, FN, OPN, Runx-2, OSX, p-P38 protein in three groups, and the expression of OC, OPN, Runx-2, OSX protein increased significantly in porous tantalum scaffold group (P<0.05). The results of real-time fluorescence quantitative PCR show there were the expression of OC, Col-1, FN, OPN, Runx-2, OSX, p-P38 mRNA in three groups, and the expression of OC, OPN, Runx-2, OSX mRNA increased significantly in porous tantalum scaffold group (P<0.05).Conclusion:Domestic porous tantalum scaffolds could effect the expression of osteogenic transcription factor Runx-2, OSX and osteogenic factor osteocalcin, osteopontin, collagen type I, fibronectin, promote osteoblast proliferation, growth and mineralization, promote bone formation, and domestic porous tantalum scaffolds is the ideal substitute material for bone transplantation.Chapter 3 Proteomics analysis of the osteoblast interaction of domestic porous tantalumObjective:To explore the mechanism of domestic porous tantalum scaffold materials which have three-dimensional structure influence the osteogenic related factor, and prove the preliminary work by relative isotopic labeling technique and absolute quantification (iTRAQ) technique to analysis the protein changes of MG63 cells respectively cultured in control (cells cultured in MEM) group, porous tantalum extract group (cells cultured in extract) and cell cocultured with porous tantalum scaffold group.Method:MG63 cells were divided into control (cells cultured in MEM, A) group, porous tantalum extract group (cells cultured in extract, B) and porous tantalum scaffold group (cells co-cultured with porous tantalum,C). Porous tantalum scaffold were autoclaved, the MG63 cell suspension were dropped on the surface of porous tantalum material in a humidified incubator containing 5% CO2 at 37℃, after 2h, turned the material to the other side and dropped the same concentration of cell suspension, stayed in a humidified incubator containing 5% CO2 at 37℃ for 2h, taken the material to a new hole, MEM culture solution was added to culture. Three groups of MG63 cells plates were placed in a humidified incubator containing 5% CO2 at 37℃ and the next day changed the liquid culture, continuous culture of 7d. The proliferation of MG63 cells co-cultured with porous tantalum in vitro was detected by the CCK-8 assay. The cells were co-cultured with porous tantalum in vitro and the growth, proliferation were observed by scanning electron microscope. Extracted of cellular proteins of three groups, Bradford method to quantitative protein, bidirectional fluorescence difference gel electrophoresis was used to examinate the protein extraction and quantitative differences between the three samples and made a preliminary analysis of protein samples. Using iTRAQ reagent labeled cell protein samples of A, B, C group, enzymatic peptides offline preseparation, LC-MS/MS mass spectrometry analysis, after strong cation ion exchange chromatography, reversed-phase liquid chromatography separation, using ABI-5600 mass spectrometry analysis and analysised the mass spectral data, detected and identificated the proteins, screened the differential proteins. Screened the different proteins which participated in the regulation of osteoblast. Statistics the differences of protein quantitative information, analyze the biological information of data. Analysis the molecular function, biological process, the participation of the subcellular localization of the different protein, looking for the biological function which significantly associated with different proteins, completed the analysis of interactions between proteins, to explore the different protein involved in the major metabolic pathways and signal transduction pathways between three groups.Results:MG63 cells were cultured in three different environments, the cells were in good condition, the morphology of the cells were polygonal simplification or long fusiform; ALP staining show positive results. The result of CCK-8 assay showed that the osteoblasts grew well and proliferated rapidly in extract of porous tantalum and no significant difference between three groups(P>0.05). There was a significant difference in different time (F=2302.523, P<0.001). The interaction between group and time (F=4.661, P<0.001). Results of individual effects show that there was no statistical significance between the three groups in the rest of the time except 7d (P>0.05). Scanning electron microscope observated that MG63 cells grew and adhesived in the material surface and pore, during the early days, cells arranged sparse;with incubation time the cells were connected to each other, gradually covered the surface of the material, and extends into the pores of the material, later the cells secreted large amounts of matrix, covered most of the surface.The result of protein quantitative analysis show that the bands of three samples were clear, uniform distribution and no obvious high abundance in line with the test standard. There were differences between three samples by fluorescence two-dimensional difference gel electrophoresis, achieve to iTRAQ test standard. The experimental results of iTRAQ according to the original data of the p-value, select the P is less than or equal to 0.05 to filter, the difference is greater than or equal to 1.5 or multiple fold difference is less than or equal to 0.666, the results were analyzed, There were 56 different protein between control group and porous tantalum extract group, there were 150 different protein between porous tantalum extract group and porous tantalum scaffold group. The data of different protein was map to DAVID database, between porous tantalum extract group and porous tantalum scaffold group, there were 6 protein can not identify,50 protein were identified include 35 up-regulated proteins,15 down-regulated proteins, analysis the biological process, molecular functionand and cellular components of different proteins, screened 6 up-regulated proteins,2 down-regulated proteins which related to experiment. Between porous tantalum scaffold and control group, there were 26 protein can not identify,124 protein were identified include 104 up-regulated proteins,20 down-regulated proteins, analysis the biological process, molecular functionand and cellular components of different proteins, screened 12 up-regulated proteins,1 down-regulated proteins which related to experiment. Kinesin-1, actin, keratin 10 is consistent with the differential expression in porous tantalum extract and porous tantalum scaffold group; in porous tantalum scaffold group cytoskeleton associated protein, gelsolin, catalase, phospholipase A2, integrin beta-1, calcium binding protein A4, extracellular matrix protein-1 differentially up regulated.Conclusion:Screened the different protein of control group, porous tantalum extract group and porous tantalum scaffold group by iTRAQ to reveal the interaction mechanism in porous tantalum material and cell. Preliminary work suggests the three-dimensional structure of porous tantalum could promote cell proliferation, adhesion, cell matrix secretion. By proteomic technology analysis found that most of different proteins in porous tantalum scaffold group mainly involved in cell adhesion, movement, secretion which illustrate the three-dimensional structure, high porosity and micropore structure of porous tantalum could promote cell adhesion, secretion, and provide a theoreticalbasis and new direction for the further research of domestic porous tantalum materials. |
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