3D Printed Silk Fibroin/Gelatin Hydrogel Scaffold And Its Application In Cartilage Regeneration

Once the cartilage been damaged,it’s hard to regenerate.Though there already have many treatments to repair cartilage damage in clinic,every treatment has their own disadvantage.The progress in study of cartilage tissue engineering open a new gate for cartilage repair.Scaffold materials for cartilage engineering should be biocompatible and have suitable mechanical performance and degradation rate.Among many methods of constructing porous scaffold,three-dimensional printing has the merits of higher productive and controllability.In this research,a silk fibroin/gelatin hydrogel is constructed by HRP enzymatically crosslink and we have systematically studied the factors that affects the gelling process induced by H₂O₂ permeation.After that by taking advantage of gelatin’s thermo-sensitivity,HRP enzymatically crosslink and conformation transition of silk fibroin,we successfully construct a 3d-printed hydrogel scaffold for cartilage regeneration.Firstly,three kinds of gelatin-tyramine with different degree of substitution was obtained and homogenous GT hydrogel with high solid content was made by soaking thermo-reversible gelatin hydrogel in H₂O₂ solution to induce enzymatically hydrogel.After adding silk fibroin and treat hydrogel with methanol solution,hydrogel’s mechanical performance can be significantly promoted.Among three kinds of SF/GT hydrogel,the mechanical performance of GT with the lowest substitution can be promoted most after treatment with methanol solution（compressive modulus increased from 0.4MPa to about1.5MPa after treatment with methanol solution）.By taking advantage of the thermo-sensitivity of gelatin and gelation process of soaking in hydrogen peroxide,the preparation of tubular hydrogels and the induction of mBMSCs seeded in the tubular hydrogel into endothelial cells in vitro were achieved.Then the sol-gel temperature and viscosity of three kinds of GT solution was studied.It was found that the higher the degree of substitution,the lower the sol-gel transition temperature.Under the same conditions,the viscosity of gelatin with low degree of substitution is larger,so LGT is finally selected as the printing material,and the appropriate printing temperature is set based on its gel-sol transition temperature to realize the 3D printing of GT and SF/GT porous hydrogel scaffolds.The physicochemical properties of the printed scaffolds showed that silk fibroin addition and conformational transformation significantly improved the mechanical properties of the scaffold and prolonged the degradation time of the scaffold in vitro.The growth and proliferation of seeded cell on the scaffolds were observed on every cell seeded scaffolds.Among all the scaffolds,the“SF₅GT₁₅/treatment with methanol”hydrogel has the highest compressive modulus（384±43kPa）,excellent compression fatigue property and longest degradation time in vitro which make it the most suitable scaffolds for cartilage regeneration and repair.Finally,we prepared hydrogel scaffolds with staggered holes by changing the spatial arrangement of filaments,which improved the efficiency of cell-seeding.Furthermore,by combining the hADSCs cell aggregates prepared in vitro,we compared the difference of Chondrogenic differentiation of hADSCs under two kinds of cell seeding methods:cell-aggregate seeding and cell suspension directly seeding.The results showed that stem upregulated the expression of relative chondrogenic gene and secreted cartilage matrix on the scaffolds under both cell seeding method.However,under cell aggregate seeding method,the stem cells expressed the more COL II and less COL I indicated that it tended to differentiate into hyaline cartilage,while under the cell suspension seeding method,the stem cells expressed the more COL I and less COL II which indicated it tended to differentiate into fibrous cartilage.Generally,the 3D printed silk fibroin/gelatin hydrogel scaffold constructed in this research has a broad application prospect in the field of cartilage tissue regeneration.