The Construction Of A SDF-1α And TGF-β1 Loaded Porous Silk Fibroin-gelatin Sponge Scaffolds And The Preliminary Study In Cartilage Repairment

Objective: In this study,we prepared a silk fibroin(SF)/gelatin sponge copolymer porous scaffold loaded with SDF-1α andTGF-β1 by lyophilization.The physical and chemical properties and chondrogenic differentiation ability of rat bone marrow mesenchymal stem cells(rBMSCs)on the scaffold were studied which laid a foundation for in situ induced cartilage repair experiment in vivo and provides a basis for new therapy of cartilage defect.Method:(1)The constitution and groups of prepared scaffolds were listed below(G0:gelatin sponge;GS:G0+2wt.% SF;(2)GST: G0+2wt.% SF+300ng/ml TGF-β1;GSS: G0+2wt.% SF+150ng/ml SDF-1α;GSTS: G0+2wt.% SF+150ng/ml SDF-1α+300ng/ml TGF-β1),the morphology of the scaffold was observed by scanning electron microscopy(SEM).The structure of the scaffold was determined by Fourier transform infrared spectroscopy(FTIR).The water absorption,degradation properties cumulative and release rate of active factors of the scaffold were detected by immersing the scaffold in phosphate buffered saline(PBS).(2)Effects of scaffolds on rBMSCs: 1.Cytocompatibility: 3-5 generations of rBMSCs were planted on scaffolds.After 1 d of culture,live-dead staining was used to observe cell viability;after 1,3,and 5 d,cck-8 kits were used to measure the proliferation of cells.2.Cell migration performance: The chemotaxis ability of the scaffold to cells was examined using a monolayer cell scratch model.3.The expression of genes and proteins involved in cartilage differentiation was detected using qPCR and WB techniques.Results :(1)SEM photographs show that the GS composite scaffold has a porous structure with a pore size of about 127±15 μm.The FTIR results show that the main molecular groups of each scaffold are protein amide bonds and hydrogen-oxygen bonds,and no other molecular groups appear;The water absorption performance of GS was good,but the water absorption of the G0 stent was lower than that of the G0.The degradation rate of GS was significantly lower than that of the G0;the concentration of SDF-1α and TGF-β1 released by GS was significantly lower than that of G0,suggesting that the active factor was able to be released for a longer period of time.(2)Live-dead staining results showed that the cytocompatibility of each group was good.The results of CCK8 indicated that rBMSCs could proliferate well on the scaffold;the single-layer cell scratch model suggested that the scaffolds loaded with two factors show the best chemotaxis effect;qPCR showed that the load factor-bearing scaffold group had the ability to promote the expression of cartilage-related genes;WB showed that the Sox9 protein expressed by rBMSCs on the two groups loaded with TGF-β1 was significantly higher than that of the GS group,indicating that the composite scaffold has good chondrogenesis property.Conclusion: The silk fibroin/gelatin sponge porous composite scaffold with pore diameter suitable for cartilage growth was successfully prepared.The GS composite scaffold has good water absorption performance,degradation performance and sustained release property of active factors.In vitro research showed that rBMSCs proliferate well in scaffolds.Load factor GS scaffolds have the ability to recruit rBMSCs and induce chondrogenic differentiation of rBMSCs.The SDF-1α and TGF-β1 silk fibroin-gelatin sponge composite porous scaffolds constructed in this study accorded with the requirements of in situ induced regeneration of cartilage tissue engineering materials,and laid a good foundation for further in vivo cartilage repair experiments.