| Objective: 1.Prepare calcium sulfate bone cement incorporated with silk fibroin and Sema3A-loaded chitosan micropheres. 2. To search for physical and chemical properies of new bioactive materials which can be used as the substitute of bone repairing and drug carriers. 3. Evaluate the biocompatibility of the bone cement and osteogenesis effect.Methods: 1.Sema3 A was dissolved and mixed well with chitosan and then the solution was prepared with emulsification. SEM, particle size analysis and swelling rate determination were performed to study properties of the microspheres. The drug loading, encapsulation efficiency and drug release rate were determined by ELISA. 2. Aqueous solution 3%, 6% and 9%(w/v) of silk fibroin were prepared. 3. Different concentration of silk fibroin solution mixed with the plain calcium sulfate to prepare CSC/SF bone cement. The liquid-to-powder(L/P) ratio of composite cement was determined as 0.4 m L/g. The compressive strength of these bone cement was measured to determine the concentration of silk fibroin which can get the highest compressive strength. 4. Calcium sulfate powders incorporated with SLCM(0.5, 1, 5 wt.%) as the solid component were used to prepare the composite cement marked as CSC/SF/SLCM. 5. The initial and final setting time of different bone cement was measured on cement pastes. 6. Observe the collapsing antiimpact property of CSC applied 6% silk fibroin solution and 0.5% SLCM. 7. Analyze the mass loss of CSC, CSC/6SF and CSC/6SF/0.5SLCM at different time points. 8. The phase analysis of different composite calcium sulfate bone cement and calcium sulfate powders were performed by XRD. The typical chemical group of Sema3 A, SLCM, SF and CSC/SF/SLCM is analysed by FTIR. 9. SEM are used to observe the morphological of the bone cement before and after immersed in PBS for 8 weeks. 10. The extracts of the bone cements cultured with MC3T3-E1, The biocompatibility of bone cements in vitro was assessed by MTS. 11. The ostectomized cavities on the femur of Wistar rats were filled with CSC, CSC/SF and CSC/SF/SLCM, respectively. The bone tissues were stained with hematoxylin and eosin at 2, 4, 6, 8 weeks.Results: 1.The particle size distribution of the SLCM ranged from 5 to 18 μm and the average size was 13.72 μm. The SEM image of microspheres was shown that the particles were spherical shape with smooth surface. 2. The swelling rate was 77.02±5.57%. The size of SLCM became larger obviously after immersion in distilled water. The drug loading and the encapsulation efficiency rate of microspheres were 245.45±20.07 μg/g and 49.09±4.01%, respectively. 3. The burst rate of CSC/SF/0.5SLCM was 37.18±0.27% in the initial 1 d, followed by a slow and sustained drug release up to 28 d. After one week, the release speed of CSC/SF/0.5SLCM was 1.35±0.13% per week. 4. The compressive strength of CSC is 22.49±2.88 MPa. The compressive strength of the composite cement with 6% SF is 31.1±0.74 MPa. The composite cements with 0.5 wt% SLCM showed the highest compressive strength(39.17±1.96MPa) as compared with the other cements. 5. SF can prolong the initial and final setting times of bone cement. Interestingly, the setting time will be further prolonged with increasing the SLCM content. 6. The XRD spectrum of CSC, CSC/SF and CSC/SF/SLCM clearly indicated that they were composed of Ca SO4·2H2Ocraystals. Specially, the diffraction peaks at 2θ=11.6°、 20.7°、 29.1°、31.1°、33.4°, respectively, of Ca SO4·2H2O, at 2θ=14.7°、32.1°、25.7°、29.8°、42.2°of Ca SO4·0.5H2 O. The results indicated that the component of bone cement had almost changed from Ca SO4·0.5H2 O to Ca SO4·2H2O after incubated for 48 h and there was little Ca SO4·0.5H2 O in the composite bone cement. 7. The bands at 602,669 and 1116 cm-1 are assigned to the SO42- in the spectrum of CSC/6SF/0.5SLCM. The presence of absorption bands at 3548 and 3405 cm-1 was H2 O. This indicated that the CSC/6SF/0.5SLCM still contained H2 O after incubating for 72 h. The N-H stretching band appear at 2923 cm-1 which indicated the SF and SLCM existed. 8. The SEM showed the surface of these three kinds of bone cement was composed of needle-like crystals. There were numerous flakes and fine grains on the surface of CSC/SF and the surface was more compact with smaller voids. The microspheres in CSC/SF/SLCM were spherical in shape with smooth surface. The cements surfaces after immersion had a much larger voids than that before immersion. 9. Degradation assay: At the end of the 8th week, the weight loss was the lowest in CSC/6SF, followed by those in CSC/6SF/0.5SLCM and CSC. 10. The MTS assay indicated that these three type bone cements had no obvious effect on the cell viability contrasted with the negative control.11. SEM image of cells grown on CSC/6SF/0.5SLCM showed that MC3T3-E1 cells spread well with an intimate contact with the surface of the sample and kept elongated, spindle shaped morphology. 12. For CSC/SF/SLCM, extensive bone regeneration was observed characterized by osteocyte-populated woven bone that completely bridged the original defect area after 8 weeks.Conclusion: 1. Calcium sulfate powders incorporated with 0.5 wt.% SLCM as the solid component and 6% SF as the liquid component were showed the highest compressive strength. 2. Adding SF and SLCM into calcium sulfate improved the setting time of bone cement, which was expected to be applied to KP and VP.3.The vitro and vivo assay indicated that there were properties to promote proliferation, sustained release of drug and no toxicity. |
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