Fabrication And Properties Of Fish-Sourced Collagen Fibrillar Gel Cross-Linked By Chlorogenic Acid And Procyanidin

Collagen is an important biomaterial in tissue engineering and regenerative medicine for good biocompatibility.However,the application of collagen from terrestrial mammals,such as pig and cow,is limited by the outbreak of zoonotic diseases like mad cow disease and foot-and-mouth disease,as well as religious barries.As a result,marine collagen has attracted attention due to similar amino acid composition and properties,as well as the extensive sources.Tilapia(Oreochromis mossambicus)is one of the important farmed fish in the world.A large number of by-products will be produced during processing.There is abundant collagen in fish skin,which can self-assemble into collagen fibrillar hydrogel(CFG).However,pure collagen-based material shows poor mechanical properties,low thermal stability,and rapid degradation,which make it difficult to meet requirements of medical materials.EDC/NHS is a cross-linking agent for collagen,but the degree of cross-linking on collagen fibrils is low.Although genipin shows better cross-linking,it is expensive.Polyphenols are used to modify CFG because of interaction with proteins in various ways and high safety.In this study,chlorogenic acid(CGA)and procyanidins(PC)was applied to modify CFG for good antioxidant and bacteriostatic activity.In order to discuss the cross-linking comprehesnsively,laccase was used to catalyze them into oxidized chlorogenic acid(LC)and oxidized procyanidin(LP),respectively,and the structures and properties of cross-linked hydrogels were investigatd.The results are as follows:(1)In order to meet medical requirements for collagen from fish skin,tilapia skin was firstly pretreated with Na OH solution to remove pigment on the surface,and the self-assembly was maintained.The optimal pretreatment conditions were as follows: the concentration of Na OH solution was 1 M,temperature was 30℃,and solid-liquid ratio was 1:50.The collagen could self-assemble into collagen fibrillar gel(CFG),which was cross-linked with CGA and PC,respectively.The optimum concentrations were separately 6 and 8 m M,and the corresponding times were 8 and12 h at 28°C.Attenuated total reflectance-Fourier transform infrared spectrophotometer(ATR-FTIR)and X-ray photoelectron spectroscopy(XPS)showed that there were hydrogen bonds between phenolic hydroxyl groups of polyphenols and carbonyl groups of hydrogels,well consistent with the results of molecular docking simulation.X-ray diffraction(XRD),scanning electron microscopy(SEM),and porosity results showed that the fibrillar network of CFG-PC was denser,which led to a significant improvement in water absorption,water retention,mechanical properties,and enzymatic resistance,while a decrease in thermal stability.Unfortunately,CGA had no significant effect on the structure and properties of CFG.Noticeably,both CGA and PC imparted good antioxidant and antibacterial properties to the hydrogel.The above results suggested that PC cross-linking not only improved the mechanical properties and enzymatic resistance evidenalty,but also endowed the gels with antioxidant and antibacterial properties.(2)To improve the modification effect,CGA was firstly catalyzed by laccase into LC.The optimum catalytic conditions were p H 4.5,reaction temperature 40°C,laccase dosage 0.4 U/mg and reaction time 12 h.LC with different concentrations were used to cross-link CFG,and the optimum cross-linking concentration was 3mg/m L.ATR-FTIR and XPS showed that the quinone groups of LC reacted with the amino groups of collagen fibers by Schiff base and Michael addition.As a result,LC densified the structure of collagen fiber network,and furthermore,the water absorption and retention properties,mechanical properties,thermal stability and enzymatic resistance were significantly improved,and it was better than CGA.In addition,thehydrogels cross-linked by LC showed good antioxidant properties and blood compatibility.The above results indicated that LC can effectively improve the performance of CFG.(3)To further enhance the performance of hydrogels,PC was catalyzed by laccase,and the optimum conditions were p H 5.0,reaction temperature 40°C,laccase dosage 0.12 U/mg,and reaction time 8 h.For CFG,optimum cross-linking concentration of LP was 4 mg/m L.ATR-FTIR and XPS showed that there was covalent effect between LP and collagen fibrils,and XRD and SEM displayed that LP made the collagen fibril network denser.The hydrogels cross-linked by LP had better water absorption and retention properties,enzymatic resistance,antioxidant properties,and blood compatibility,and it should be noted that the mechanical properties were superior to PC-modified.Additionally,thermal stability of hydrogels was firstly increased and then decreased,and the denaturation temperature reached the highest at the LP concentration of 2 mg/m L.The above results indicated that LP can further improve the mechanical properties and thermal stability of CFG.In summary,polyphenols and the derivatives can effectively improve the properties of aquatic collagen fibrillar gels.The related results not only provide foundations for the development and application of fish-sourced collagen biomaterials,but also provide references for exploiting other aquatic collagen-based biomaterials.