Experimental Study On The Effect Of Multifunctional Biomimetic Mineralized Silk Fiber/Chitosan 3D Scaffolds On Bone Defect Repair

Part 1 Biomimetic mineralization and screening of silk fibroin fiber/chitosan 3D scaffolds Objective: To investigate the feasibility of using silk fibroin fiber/chitosan composite scaffolds as mineralization templates,and to study the effects of different timing of mineralization on the morphology,composition,physical and chemical properties of scaffolds and the behavior of MC3T3-E1 cells.Methods: The Antheraea Pernyi Silk Fibroin(AF)was prepared by chemical and physical combination method,and was blended with chitosan(CS)in the ratio of 3:4(W/W),and then the tussah silk fibroin fiber/chitosan(AF/CS)3D scaffold was prepared by freezedrying.After the scaffold was insoluble with Na OH aqueous solution,the scaffold was immersed in 5 times volume simulated body fluid(SBF)at a bath ratio of 1:1000 for bionic mineralization.Scanning microscope(SEM),Fourier transform infrared spectroscopy(FTIR),X-ray diffraction(XRD),mechanical study and hemolysis test were used to observe the structure,morphology,composition and toxicity analysis of the mineralized silk fibroin/chitosan scaffold(m AF/CS)at different time points(0,12 h,24h,48h).MC3T3-E1 cells were then inoculated on AF/CS scaffold and m AF/CS scaffold,and the adhesion genes were detected by CCK-8 assay,alive and dead staining,adhesion assay,alkaline phosphatase(ALP)staining,activity test,and Real-Time PCR(RT-PCR)to determine whether the mineralized AF/CS scaffold provides a bionic microenvironment that promotes proliferation,adhesion and differentiation,and to determine the optimal time of mineralization.Results: SEM results showed that AF/CS scaffold had good pore structure.With the extension of mineralization time,a relatively complete and dense mineralized layer was gradually formed on the surface of the scaffold.However,when the mineralization lasted for 48 h,a large amount of hydroxyapatite accumulated,resulting in the pore size of the scaffold becoming smaller or even closed.XRD and FTIR results showed that the crystal types of hydroxyapatite deposited on the surface of AF/CS template were almost the same after different mineralization time.The mechanical tests showed that the corresponding mechanical properties increased significantly with the extension of the mineralization time.The results of hemolysis test showed that the scaffolds had good blood compatibility before and after mineralization.In addition,cytotoxicity assay(CCK-8)and Calcein-AM/PI staining showed that the scaffolds have good cytocompatibility and promote proliferation after mineralization.But there appeared to be relatively more dead cells after 48 h of mineralization.The observation of cell morphology showed that biomimetic mineralization of scaffolds promoted the adhesion and growth of cells on the material surface.RT-PCR analysis also showed that the expression level of adhesion related genes in the m AF/CS group was significantly higher than that in the AF/CS group,and the difference was statistically significant(P<0.05).Compared with the unmineralized scaffolds,the alkaline phosphatase activity of the mineralized scaffolds was higher,especially the scaffolds that were mineralized for 24 hours.Conclusion: AF/CS scaffolds have good biological mineralization function as templates,and the mineralized AF/CS composite scaffolds have good cytocompatibility.Among them,the scaffolds mineralized for about 24 hours not only retain good pore structure,but also have the best biological evaluation effect,thus are suitable for bone repair scaffolds.Part 2 Preparation and in vitro study of biomimetic mineralized silk fibroin/chitosan 3D scaffolds loaded with salvianolic acid B and active strontium Objective: The functionalized biomimetic mineralized fibroin fiber/chitosan 3D scaffolds(m AF/CS-Sb@Sr)loaded with Salvianolic Acid B(Sb)and active strontium(Sr2+)were prepared by self-assembly of biomimetic mineralization,and their effects on osteogenesis/angiogenesis in vitro were studied.Methods: First,the m AF/CS 3D scaffold was prepared according to the steps in part 1,then Sb with concentration of 1mmol/L was dissolved in 5×SBF in 1: 9 Sr Cl2 and Ca Cl2 mole ratio,and incubated for 24 h to form hydroxyapatite coating containing Sr and/or Sb,then freeze-dried to obtain mineralized scaffold loaded with Sb and Sr(m AF/CS-Sr@Sb),mineralized scaffold loaded with Sb(m AF/CS-Sb)and mineralized scaffold loaded with Sr(m AF/CS-Sr).In addition,a simple m AF/CS branch was set up as the control group.The materials were characterized by SEM and FTIR,and the release curves of Sr2+ and Sb in vitro were measured.Human bone marrow mesenchymal stem cells(h MSCs)and human umbilical vein endothelial cells(HUVECs)were selected to study the cell models of osteogenesis and angiogenesis in vitro.For in vitro osteogenic ability evaluation,the scaffolds of each group were co-cultured with h MSCs cells,and the cytocompatibility was detected by CCK-8 and Calcein-AM/PI staining.Transwell assay was used to detect the effect of scaffolds on h MSCs migration.After 14 days of culture,alkaline phosphatase/alizarin red staining and quantitative analysis were performed.In addition,the relative expressions of Col Ⅰ,OPN,OCN and Runx2 were detected by RT-PCR and Western blot.To evaluate the ability of angiogenesis in vitro,the scaffolds of each group were co-cultured with HUVECs cells,then cell proliferation detection and tubulogenesis test were conducted,and the expressions of related angiogenesis differentiation genes VEGF and HIF-1ɑ were detected by RT-PCR.Results: SEM images showed that the m AF/CS,m AF/ CS-Sb,m AF/ CS-SR and m AF/CSSb@Sr scaffolds still maintained a good interoperable pore structure.FTIR and EDS spectra showed the successful incorporation of Sr2+ and Sb.Drug release results showed that Sr2+ had a more persistent release characteristic,while Sb had an initial burst release followed by sustained release for 10 days.For the evaluation of osteogenic ability in vitro,the survival and death test and CCK-8 test of h MSCs showed that the cells proliferated well on all scaffolds and showed high cell viability,and almost no dead cells were observed.The migration results at 12 h and 24 h showed that the number of migrating cells in m AF/CSSb@Sr group was significantly higher than that in m AF/CS,m AF/ CS-Sb and m AF/ CSSR groups.After standard osteogenic induction,alkaline phosphatase staining on all scaffolds was positive,of which the composite scaffolds in the m AF/CS-Sb@Sr group were more obvious than those in the other three groups.RT-PCR also showed that the coincorporation of Sr2+ and Sb in the scaffold significantly increased the expression of OPN,RUNX 2,OCN and ColⅠ genes.For the evaluation of in vitro angiogenesis ability,HUVECs cell proliferation assay showed that,compared with m AF/CS group and m AF/ CS-SR group,m AF/ CS-Sb and m AF/CS-Sb@Sr group had higher cell proliferation rate,and m AF/ CSSb group had the highest proliferation promoting effect(P<0.05).In the tube forming experiment,m AF/CS-Sb@Sr group showed the most prominent promoting effect on tube forming ability(P<0.05).In addition,RT-PCR analysis showed that the expression levels of angiogenesis related factors VEGF and HIF-1ɑ in m AF/CS-Sb@Sr group were significantly higher than those of the other three groups with statistical differences(P<0.05).Conclusion: 1.The functionalized biomimetic mineralized silk fibroin/chitosan 3D scaffold(mAF/CS-Sb@Sr)loaded with Sb and Sr2+ not only retained good interoperable pore structure and micro/nano morphology,but also sustainably released Sb and Sr2+,providing a good condition for bone repair.2.m AF /CS scaffolds loaded with Sb and Sr2+ have good cytocompatibility,and play a synergistic role in promoting angiogenesis and inducing osteogenic differentiation through the release of Sb and Sr2+.Part 3 In vivo study of biomimetic mineralized silk fibroin/chitosan 3D scaffolds loaded with salvianolic acid B and active strontium Objective: To evaluate the ability of functional biomimetic mineralized scaffolds(m AF/CS-Sb@Sr)loaded with Sb and Sr2+ to repair critical bone defects in the skull of rats.Methods: According to the different methods of skull defect treatment,50 male SD rats weighing 220-260 grams were randomly divided into five groups: group A: blank control group,group B: mineral fibroin fiber/chitosan scaffold group(m AF/CS group),group C: Sb loaded mineralized silk fibroin fiber/chitosan scaffold group(m AF/ CS-Sb group),group D: Sr2+ loaded mineralized silk fibroin fiber/chitosan scaffold group(m AF/ CS-Sr2+ group)and group E: Sb and Sr2+ loaded mineralized silk fibroin fiber/chitosan scaffold group(m AF/CSSb@Sr2+).The sterilized materials were implanted into the skull defects of SD rats.At 4 weeks and 8 weeks after surgery,5 rats in each group were randomly selected for micro-CT THREE-DIMENSIONAL reconstruction to observe the repair of bone defects,followed by histological analysis HE staining,Masson staining,and immunohistochemical ColⅠ evaluation to determine the formation of new bone.Results: Micro CT 3D reconstruction results showed that there was almost no new bone formation in the defect site of the control group in 4 weeks after implantation.In m AF/CS group,m AF/CS-Sb group,m AF/CS-Sr group and m AF/CS-Sb@Sr group,the newly formed bone grew from the defect edge to the gap of the scaffold.In particular,new bone growth was more pronounced in m AF/CS-Sb@Sr group(P<0.05).However,there was no significant difference in BV/TV and BMD between m AF/ CS-Sb and m AF/Cs-Sr groups(P>0.05).When the observation time was extended to 8 weeks after implantation,the new bone growth in all scaffold groups was significantly increased compared with that at 4 weeks,especially in the m AF/CS-Sb@Sr group,where the defect sites were mostly occupied by mineralized areas.Quantitative analysis showed that the BV/TV and BMD of m AF/CSSb@Sr group were higher than those of the other three groups(P<0.05)and that increased over time.In addition,HE staining,Masson staining and ColⅠ immunohistochemistry staining showed that the defect area in the control group was mainly composed of a large amount of fibrous connective tissue,and no obvious new bone formation was observed at 4 weeks after surgery.In contrast,a certain amount of new bone formation was observed in all scaffolds.At 8 weeks postoperatively,the number of new bone in the defect area of each scaffold group was significantly higher than that at 4 weeks,especially in the m AF/CSSb@Sr group,in which the defect area was significantly reduced,while in the control group,only a small amount of new bone tissue was observed.Conclusion: m AF/CS-Sb@Sr composite scaffolds not only simulate natural bone ECM through mineralization,providing an ideal microenvironment for osteogenesis,but also release Sr2+/Sb to synergistically promote angiogenesis and osteogenesis,showing a significantly stronger ability to regulate bone regeneration,thus it is an ideal bone tissue engineering repair material.