Preparation And Properties Of Enzymatic Crosslinking Injectable Hydrogel

Objective:Jawbone defect is a common medical disease in human beings at present.How to treat bone defect and accelerate regeneration of bone tissue is a medical problem we are facing.At present,due to limited source,donor injury and immune rejection,the common use of autologous transplantation and allotransplantation is limited.Bone tissue engineering has become a new alternative method for bone transplantation by creating an active artificial osteogenic microenvironment in the bone defect area,whose core element is biological scaffold material.Hydrogels is a hot research topic in tissue engineering and regenerative medicine.Hydrogels have high water content similar to extracellular matrix and excellent biomimetic properties,which are ideal three-dimensional cell culture and cell delivery systems.Cells wrapped in the hydrogel can be easily injected into the target site,thereby avoiding additional open surgery and benefiting patients with irregular,deep or closed defects.Hydrogels made from silk fibroin have attracted wide attention due to their natural safety,good biocompatibility,controllable degradation and ability to combine with other materials.They have broad application prospects in the field of biomedical science.However,the traditional silk fibroin hydrogel network has simple structure,simple function and poor adaptability to complex application environment.It greatly limits its application and development.In this study,an injectable SF/GO composite hydrogel was prepared using silk fibroin protein and GO as raw materials by enzymatic cross-linking.Multiple groups of gel materials were formed by changing GO content.The differences in physical and chemical properties,in vitro cytocompatibility and osteogenesis of composite gels with different GO concentrations were studied.By changing the ratio of SF/GO in the composite gel,the properties of the composite gel was optimized,and the feasibility of the composite gel as scaffold material for bone tissue repair was discussed.Methods:In this study,SF/GO composite hydrogels were prepared by enzyme catalyzed cross-linking method,and the effect of GO addition ratio on the physical and chemical properties of the composite gel was studied.Scanning electron microscopy（SEM）,X-ray diffraction（XRD）,Fourier transform infrared spectrometer（FT-IR）,X-ray photoelectron spectroscopy（XPS）,universal material testing machine and drainage method were used to investigate the morphology of the scaffold.The secondary structure,chemical states of element and mechanical properties of porosity were characterized and compared.The effect of GO content on the properties of the composite gels was studied.In addition to bone marrow mesenchymal stem cells（BMSCs）between the parcel in the gel,with a laser scanning confocal microscope（LSCM）and CCK-8 method to detect the parcel in the SF/GO hydrogel of BMSCs in the process of gelation,cell survival and proliferation of in vitro cell compatibility evaluation material.Finally,BMSCs were inoculated into the gel for osteogenic differentiation for 7,14 and 21 days.The osteogenic ability of the materials was evaluated by Alkaline phosphatase（ALP）staining and activity determination,alizarin red S（ARS）staining and Ca²⁺semi-quantitative analysis.Results:1.After adding HRP/H₂O₂crosslinking agent,SF/GO mixture quickly formed a jelly like gel.With the increase of GO mass fraction,the formation time of composite gel was prolonged（SF group 14.25±4.5,SF/0.05%GO group 17.75±2.2,SF/0.1%GO group 18.5±2.3,SF/0.2%GO group 24±3.6,SF/0.4%GO group 24±4.2）.Between SEM results showed that SF,and GO not appear obvious phase separation phenomenon,has the good compatibility,between each group of gel material are porous structure of interpenetration,hole size and shape of varies with the percentage of the GO add,the higher the content of the GO,the smaller aperture of compound gel（90.49+/-0.74 SF group,92.36+/-0.57 SF/0.05%GO group,SF/0.1%GO group 92.64+/-0.81,93.13+/-0.22 SF/0.2%GO group,SF/0.4%GO group93.24±0.89）.The results of XRD and FT-IR showed that with the increase of GO content,the irregular curled structure in the composite gel decreased gradually,and the content ofβ-sheet structure increased gradually.XPS results showed that compared with SF hydrogel,the C/O ratio in SF/GO composite gel increased,while the N/O ratio almost remained unchanged.Compression mechanics test results show that the addition of GO significantly improves the compressive strength and failure strain of hydrogel.2.Cell experiments in vitro showed that the cells coated in the gel were uniformly and three-dimensional distributed in the colloidal scaffold and maintained good cell morphology.With the extension of culture time,the cells in the gel proliferated a lot,compared with the other groups,the SF/0.05%GO group had the best cell growth in the hydrogel.Osteogenetic differentiation in vitro cultivation experiment results show that the parcel in the cells with the gel osteogenesis induction medium for 7 days and 14 days,four groups of cells are visible to certain osteogenesis,alkaline phosphatase is on the rise,alizarin red staining results similar to alkaline phosphatase staining results,and compared with other groups of materials,SF/0.05%GO group expression of ALP activity and calcium salt deposition is better than other groups.（P<0.05）.Conclusion:In conclusion,we successfully prepared SF/GO coated composite gel using two materials,SF and GO,by means of enzyme cross-linking.With the addition of GO,the pore size of the scaffold becomes more regular,the porosity changes less,and the mechanical properties are improved to a certain extent.In addition,the compound gel with low GO concentration showed better cell growth,proliferation and osteogenic differentiation than the SF hydrogel without or with high GO concentration.Therefore,the injectable BMSCs hydrogel is a good biomedical material and has potential application prospects in the field of bone tissue engineering.