Study Of Osteogenesis On The Surfaces Of Biomaterials Modified By PPy-CS And FK506

Part. IEffect of Osseointegration at the Bone-implant Interface with the Superficial Modification by PPy-CSObjective:The aim of this study was to discuss the in vivo biocompatibility and osseointegration of implants modified by PPy-CS in rabbits.Methods:Seventeen female New Zealand white rabbits weighed about 3.20 ± 0.20 kg were selected in this study. The bioabsorbable polymer composite screws coated by PPy-CS were called "coated screws". While the uncoated bioabsorbable polymer composite screws and commercially available stainless steel cortical screws were used as reference implants. All the screws were implanted into the rabbit’s femur (1 screw) and tibia (2 screws), three for test specimens and three for controls. Twelve rabbits were observed for 12 and 26 weeks, while other five rabbits were used for the twist test at 12 weeks. Systemic effects were evaluated from food and water consumption, body weight, body temperature, clinical signs, blood samples, internal organs weights and histological examination. Local effects were studied from bone tissue and surrounding soft tissue histology. New bone formation was evaluated by the micro-computed tomography (p.-CT), hard tissue histology, and tetracycline labelling, twist tests as well.Results:The rabbits grew well after the implant operation. All the food and water consumption, body weight, body temperature, clinical signs, results of blood samples test, internal organs weights and histological examination, local surrounding soft tissue histology were analysed and there were no significant difference between the two groups(P>0.05), which showed there were no systemic side effects in coated group. There were significant differences between the two groups (P<0.05) from the perspective of new bone formation detected by μ-CT, hard tissue histology, tetracycline labelling at 12 and 26 week time period. And twist tests also showed significant difference between the two groups (P<0.05).Conclusions:PPy-CS coated bioabsorbable polymer composite screws have performed well in vivo biocompatibility and osseointegration in rabbits in the process of 12 and 26 weeks’ observation.Part. ⅡThe Osteogenesis of FK506 Modified on the Surface of Porous Scaffolds in vitroObjective:The present study aims at investigating the in vitro osteogenesis effect of FK506 modified on the surface of porous scaffolds.Methods:Nano-sized β-TCP (nβ-TCP) was prepared with a wet grinding method from micro-sized β-TCP (mβ-TCP) and polylactic acid (PLA) was put into the nβ-TCP to obtain the weight 30 percent of β-TCP in the mixed solution. Then FK506 with different weight were selected and put into the prior solution, all the three ingredients were mixed uniformly and this composite was poured into the polytetrafluoroethylene mould. With freeze-drying, different types of porous scaffolds modified by FK506 were completed and all these scaffolds were made into the same size of 5mm×5mm×3mm. FK506 sustained release curve was tested by ELISA Kit in the time point of 0.33、0.66、1、2、6、12、24、48、96、144、336、720 hours for the several types of FK506 modified porous scaffolds. After bone marrow mesenchymal stem cells (BMSCs) from the volunteer’s ilium were labeled with DiI, BMSCs 1×106 cells/30μL were evenly seeded drop by drop into the different type of porous scaffolds. The composites of cell-scaffold were cultured in vitro for one week and then were observed under SEM. At the time point of 1 week and 2 week, the OCN was tested through the ELISA Kit from the cultural solution and ALP value was obtained after the composites of cell-scaffolds were grinded and under splitting decomposition.Results:Five types of FK506 modified porous scaffolds with different weight percent (0%、2%、4%、6%、8%) were obtained. The releasing quantity of FK506 from the 5 types of porous scaffolds was gained through the FK506 ELISA Kit, and the FK506 sustained releasing curve was obtained. The BMSCs grew well in the several porous scaffolds under the observation of SEM. After the composites of cell-scaffold were cultured in vitro for one week while the several types of porous scaffolds were grinded under splitting decomposition aiming at detecting ALP value. The average ALP value of 6wt% group showed higher than that of other 4 groups; however, there was no significant difference among the 5 groups after statistical analysis. At the second week, all the ALP value of 5 groups rose and the average ALP value of 6% group was highest among the 5 groups (P<0.05). The OCN value of 6 wt% group showed higher than that of other 4 groups, and there was a significant difference in 6 wt% group among the 5 groups after statistical analysis (P<0.05) at first week time point. While at second week time point, the average OCN value of 6% and 8% groups showed a significant difference with that of other 3 groups individually (P<0.05). However, the OCN value did not show statistically significant difference between 6% and 8% groups (P>0.05).Conclusions:BMSCs could grow well in the FK506 modified porous N-PLA/β-TCP scaffolds,6wt% appeared perfect in the drug sustained releasing curve. Through the test of ALP and OCN in vitro,6wt% FK506 modified porous N-PLA/β-TCP scaffolds might show better osteogenesis in vitro.Part. ⅢThe in vivo Biocompatibility and Osteogenesis of FK506 Modified on the Surface of Porous Scaffolds in RabbitsObjective:The purpose of this study was to investigate the in vivo biocompatibility and osteogenesis of FK506 modified on the surface of porous scaffolds in rabbits and a more effective kind of porous scaffolds in enhancing new bone formation would be determined in combination with former in vitro experiment.Methods:5 kinds (wt%) of FK506 modified porous scaffolds were completed and all the test blocks were made into the same size of 5mmx5mmx3mm, which were prepared under radiosterilization. After bone marrow mesenchymal stem cells (BMSCs) from the volunteer’s ilium were labeled with Dil and Brdu, BMSCs 1×106 cells/30μL were evenly seeded into the five kind porous scaffolds. The composites of cell-scaffold were cultured in vitro for two weeks and then were implanted into the subcutaneous tissue on 8 rabbits’backs. Pure porous scaffolds without any cells were implanted into another 4 ribbits as the control. After the operation, food and water consumption, body weight, body temperature, clinical signs, the histology of local soft tissue around the blocks were observed. New woven bone formation, on the one hand, was evaluated by the blocks’ fast frozen-sectioned histology of OCN immunofluorescence、ALP immunohistochemistry; on the other hand, was evaluated by the routine paraffin embedding histology of HE staining、OCN and Col I immunohistochemistry.Results:All the composites of cell-scaffold and pure porous scaffolds were implanted into the subcutaneous tissue on rabbits’back successfully. There were not any abnormal clinic signs、food and water consumption、changes of weight etc in rabbits’ system toxic reaction. Local abnormal effects such as redness、swelling、heat or abnormal cysts around the implants were not found postoperatively. After euthanasia at 4 weeks and 8 weeks postoperative, there was not any obvious inflammatory reaction in the implants surrounding tissue through gross and microscopic observation. And Dil-labeled BMSCs survived in the blocks under the observation of fluorescent microscope. Brdu-labeled BMSCs in the blocks slices appeared antigen-antibody reaction, which also further proved BMSCs could survive in these five kinds of porous scaffolds. At 4 week time, all the blocks from 5 groups were observed under microscopy after routine HE staining and immunohistochemistry. The new woven bone、ALP、OCN and Col I in 6% group was obviously more than that in other 4 groups (P<0.05). At 8 week time, the new woven bone in 6% and 8% groups were more than that in other 3 groups (P<0.05), while there was no statistical difference between 6% and 8% groups. The ALP and Col I in 6% groups were more than other 4 groups (P<0.05). While OCN in 6% group showed no statistical difference with that in other 4 groups (P>0.05), though the average expression of OCN at 8 week was more than that at 4 week.Conclusions:N-PLA/β-TCP porous scaffolds modified by FK506 in the combination with BMSCs showed good biocompatibility in rabbits and the in vivo osteogenesis property in 6wt% group demonstrated better than that in other groups.