Preparation Of Silk Fibroin Macroporous Hydrogels By Cryo-photocrosslinking

Biocompatible hydrogel materials with a macroporous structure have broad application prospects in the fields of tissue engineering,selective cell capture,and cell culture.Existing materials are often difficult to meet the functional requirements of macroporous structure(about 100 μm),mechanical strength,biocompatibility,activity regulation,etc.,especially the balance between macroporous structure and mechanical strength.In response to the above problems,this study developed a through-hole structure construction method based on cryo-photocrosslinking based on silk fibroin,a commonly used natural biological material in the field of tissue engineering.Its innovation is that compared with the conventional ice gel system,the photo-oxidative cross-linking of silk fibroin is carried out under freezing conditions,so that the silk fibroin’s tyrosine can be used without introducing any exogenous pore-forming agent and cross-linking agent.The acid group realizes cross-linking,and obtains a pure protein macroporous material system.The material can be made into a monolithic column structure and can maintain a stable structure under a flow system,which can be used for cell capture and in situ culture.The specific research contents are as follows:(1)Construction and characterization of silk fibroin macroporous gels.Using ruthenium bipyridine and ammonium persulfate as catalysts,the photo-oxidative crosslinking of frozen silk fibroin solution was successfully achieved under the excitation of visible light.The effects of key process parameters such as freezing stability,silk fibroin concentration,catalyst dosage,and illumination time on the pore characteristics and gel strength of macroporous ice gel were optimized.The results show that gels with clear pore structure can be formed in the range of silk fibroin concentration of 50-200 mg/m L and freezing temperature of-20～-10 ℃,and the pore size range is about 70～100 μm.The gel properties were characterized by mechanical tester,thermogravimetric analyzer and rheometer.The results show that the pores of the gel have good penetration.Under the condition of the linear flow rate of the solution of 11 cm/min,the flow resistance is less than 5Pa;the maximum swelling rate is about 1900%.It can be recovered after recovery and remains stable for a long time in PBS solution;the thermal decomposition temperature is about 180 ℃,and the thermal stability of silk fibroin material is effectively improved by chemical cross-linking and physical cross-linking.(2)The macroporous silk fibroin gel material was functionalized and used for cell capture and in situ culture.The feasibility of one-pot doping of functional proteins to introduce biologically active functional units was verified.The results showed that the red fluorescent protein as a tracer molecule could be uniformly distributed on the pore wall of the gel and could retain its structural activity.Furthermore,a macroporous silk fibroin monolith material with EGFR-specific binding activity was prepared by mixing anti-epidermal growth factor receptor(EGFR)nanobodies with silk fibroin.Studies have shown that the material can effectively capture human epidermal cancer cells A431 highly expressing EGFR;under immobilization conditions,the relative activity of the cells in the gel is close to 90%,and the cells can continue to grow and spread in the gel.In this study,a technical method for the preparation of silk fibroin hydrogels with a perforated macroporous structure by cryo-photocrosslinking was established.This technology has the characteristics of simple and rapid reaction,no introduction of exogenous molecules,and easy integration of biologically active units.The ability of a monolithic column to work under flow conditions.This material system is expected to provide potential solutions for researches such as circulating tumor cell capture,in situ culture,and in vitro tumor modeling.