| Part I:Preparation and characterization of fibroin/hydroxyapatite three-phasic osteochondral scaffoldsObjective:To construct silk fibroin/hydroxyapatite three-phase osteochondral scaffold with platelet-derived growth factor(PDGF)sustained release and characterize its physical and chemical properties.Methods:Different concentrations of silk fibroin solution were prepared,and different concentrations of silk fibroin scaffolds were prepared by freeze-drying and methanol immersion.According to the anatomical characteristics of cartilage,the appropriate concentration of silk fibroin solution was selected to prepare cartilage phase and interface layer respectively.Hydroxyapatite scaffolds were prepared by high temperature sintering as bone scaffolds.Hydroxyapatite scaffold and silk fibroin scaffold were joined into three-phase osteochondral bionic scaffold by repeated freeze-thaw method.Platelet-derived growth factor(PDGF)was loaded on the scaffold after the modification of scaffold by polydopamine(PDA).The scaffolds were divided into 4 groups:blank group(HSF group),PDGF loaded group without modification by PDA(HSP-1 group),PDA modified group(HSP-2 group),and PDGF loaded group with modification by PDA(HSPP group).The pore size distribution of the scaffold was analyzed by SEM and NanoMesurer software,the porosity of scaffolds was evaluated by liquid displacement method,the chemical groups and bonds of scaffolds were analyzed by FTIR,the hydrophilicity was analyzed by water absorption,and the mechanical properties were tested by biomechanical testing machine.Finally,the release of PDGF was detected by ELISA at 1d,2d,3d,4d,5d,6d,7d,14d,21d and 28d,and the drug release capability was evaluated.Results:After identification and analysis,5%of SF solution was used to fabricate cartilage layer of scaffolds and 20%of SF solution was used to fabricate interface layer of scaffolds.The pore diameters of the scaffolds were 110.13±29.38?m and 96.53±33.72 ?m respectively.Scanning by SEM showed that the scaffold was spongy in shape and honeycomb in surface and section.The porosity of the scaffolds were 42.5±6.6%,44.9±8.1%,42.3 ± 4.3%,41.7±5.4%respectively,with no significant difference(P>0.05).FTIR was used to analyze the scaffolds,and it was found that the peaks mainly appeared at 1650 cm-1,1516 cm-1 and 1232 cm-1,which were characteristic peaks of SF and HA respectively and PDGF had no obvious effect on wave crest.There was no significant difference in water absorption between groups(P>0.05).Biomechanical compression modulus of the scaffolds of each group were shown around 8 kPa with no significant difference between the groups(the P>0.05).In HSP-1 group,because PDGF was physically adsorbed on the scaffold surface,it burst released 52.5%on the first day and 67.74%on the third day.Then the release curve became stable,and the cumulative release was nearly 73.41%on the last 28 days.In HSPP group,PDGF was released only 12.53%on the first day,then gradually released until 52.07%on the seventh day,and continued to release within the next three weeks,with a cumulative release of 71.74%on the twenty-eighth day.Conclusion:The silk fibroin/hydroxyapatite three-phase osteochondral bionic scaffold has good pore size,porosity,hydrophilicity and mechanical properties,and can release PDGF slowly and continuously after PDA modification.Part II:Effects of silk fibroin/hydroxyapatite three-phase osteochondral bionic scaffold on migration,proliferation and chondrogenesis of SMSCs in vitroObjective:To investigate the effect of silk fibroin/hydroxyapatite three-phase osteochondral bionic scaffold on migration,adhesion,proliferation and chondrogenesis of rabbit SMSCs in vitro.Methods:SMSCs were obtained by enzymatic digestion and purified by limited dilution.The phenotype of primary SMSCs was identified by flow cytometry and multilineage differentiation ability of SMSCs.The P3 SMSCs were used to detect the biocompatibility of the scaffolds.Cells were planted on the scaffolds.On the 1 st and 7th day,the adherence and growth of cells on the scaffolds were observed by SEM.The proliferation rate of SMSCs was measured by CCK-8 method on 1st,3rd,5th and 7th day.The ability of drug-loaded scaffolds to promote cell migration was detected by transwell co-culture method.The morphological changes of cytoskeleton and nucleus were observed by FITC-Phalloidin and DAPI fluorescence staining during cartilage differentiation of SMSCs when cells were co-cultured with scaffolds using Transwell plate.Assessment of the ability of SMSCs to differentiate into cartilage by pellet culture.In addition,the ability of scaffolds to promote differentiation was evaluated by detecting the changes of cartilage marker proteins in supernatants of each group during co-culture.Results:The purified SMSCs were identified by flow cytometry.The expression of CD90 and CD 105 were 99.90%and 97.90%respectively,and CD34 was 0.00%,and SMSCs can differentiate into bone,fat and cartilage after induction.SMSCs adhered well to the scaffolds in each group and had the strongest proliferation ability on the scaffolds in the SPP group.The scaffolds in SPP group had a significant effect on promoting cell migration,compared with SF group,SP-1 group and SP-2 group,there was a significant difference(P<0.05).After 7 days of scaffold-induced chondrogenic differentiation of SMSCs,nuclear and skeleton staining showed that the number of cells in SPP group increased significantly,and the activity of cells was better.Skeleton staining showed that the skeleton protein was more oriented and accorded with the normal chondrocyte expression.After pellets cultured,toluidine blue and alcian blue staining in the experimental group indicated the production of a large number of extracellular matrix of chondrocytes.PDGF released from SPP group could promote the synthesis of Col-II and GAG in SMSCs without increasing the synthesis of Col-I.Conclusion:The biocompatibility of silk fibroin/hydroxyapatite three-phase osteochondral bionic scaffold is excellent,which can promote the adhesion and proliferation of SMSCs on the scaffold,promote the migration of SMSCs in vitro,and promote the differentiation of SMSCs into cartilage.Part III:Application of silk fibroin/hydroxyapatite three-phase osteochondral bionic scaffold in repairing rabbit osteochondral injury modelObjective:To investigate the feasibility of silk fibroin/hydroxyapatite three-phase osteochondral bionic scaffold in promoting the migration of SMSCs from synovium around knee joint to repair osteochondral injury in rabbits.Methods:According to preoperative design,50 sexually mature New Zealand white rabbits were randomly divided into 5 groups:Blank control group,HSF group,HSP-1 group,HSP-2 group and HSPP group.A rabbit model of critical osteochondral defect of knee joint was constructed.The corresponding scaffolds were implanted in groups.MRI images were performed 12 weeks and 24 weeks after operation to evaluate the cartilage repair.At 12W and 24W after operation,the specimens were taken for gross histological score,histological staining and immunohistochemical staining to observe cartilage regeneration and scaffold degradation.Result:Compared with HSF group,HSP-1 group and HSP-2 group,the cartilage of HSPP group was repaired satisfactorily.The morphological score of new cartilage in HSPP group was significantly higher than that in other control group(P<0.05).MRI imaging scores of each group showed that cartilage regeneration could be achieved in HSPP group at an early stage,and the process of cartilage repair was significantly better than that of other groups(P<0.05).Histological analysis of new tissues in each group showed that the morphology of new osteochondral tissue in HSPP group was better than that in other groups,and the positive rate of cartilage specific staining was significantly higher than that in other groups,especially 24 weeks after operation,which was close to normal osteochondral tissue.Immunohistochemical results showed that the regenerated osteochondral tissue contained a large amount of collagen type ?,and we found that the degradation of scaffolds in HSPP group was also higher,which was related to the inflammation caused by cell adhesion and proliferation,which could accelerate the degradation of scaffolds.Conclusion:Silk fibroin/hydroxyapatite three-phase osteochondral biomimetic scaffold can recruit SMSCs from the synovium around the injured knee joint in rabbits,and differentiate into cartilage under PDGF induction,so as to achieve regeneration and repair of the injured knee joint. |
PDF Full Text Request