Preparation Of Gelatin Grafted Agarose Thermosensitive Hydrogel And Its Application Of Skin Wound Repair With Loading Fibroblasts

During wound healing,fibroblasts play a prominent role by producing extracellular matrix,stimulating wound contraction,and inducing angiogenesis.However,it is difficult for direct fibroblasts transplantation to reconstruct lesion sites with large area or volume due to significant cell loss without carrier.To overcome these limitations,hydrogels had been widely used as cell carriers.By simply mixing the cells with the hydrogel precursor solution prior to crosslinking,the cells could be uniformly loaded in a three-dimensional(3D)hydrogel in which the loaded cells could survive,proliferate and promote skin repair.At present,seeking hydrogel for cells carrier is a key issue in biomaterial researches.Agarose hydrogel is non-immunogenic and thermosensitive.However,due to its low cell adhesion and its slow degradation,it was usually modified with biodegradable and bioactive materials.Having good bioactivity and degradation performance,gelatin was widely used in the field of biomaterials.However,it could not gel at body temperature,so it was usually crosslinked by toxic chemical crosslinking agents.Therefore,it is necessary to find a gelation method with better biocompatibility.This study intends to prepare gelatin grafted agarose hydrogel which can combine the advantages of both of agarose and gelatin.CDI(N,N-carbonyldiimidazole)were used to activate the hydroxyl groups on agarose for reacting with the amino groups on gelatin and three different grafting ratios of gelatin grafted-agarose thermosensitive hydrogel were obtained.The basic physicochemical properties and cytotoxicity,cell adhesion and degradability of them were evaluated.Fibroblasts were encapsulated in hydrogels,and their cell viability was measured by live/dead cell staining.Finally,fibroblasts-loaded hydrogels were used as a skin dressing to study its function in promoting skin repair.The results are as follows:By changing the concentrations of CDI and reacting conditions,three different grafting ratios(12.7%,40.3%,and 86.3%)of gelatin grafted-agarose were obtained and named as C1,C2,C3.The gelation time of degraded agarose,C1,C2 and C3 were 100 s,110s,120 S and 180 s and the gelation modulus were 850 Pa,1450 Pa,1130 Pa and 716 Pa respectively.The gelation temperature of the 4 hydrogels was all around 40 °C.Through cytotoxicity experiments and cell adhesion experiments,the hydrogels were proved having good cell compatibility and cell adhesion.The fibroblasts were loaded in the hydrogel.When cultured for 3 w,the cell viability in C3 was 93.2%,which indicated that the hydrogel provided a good environment for cells to grow in a 3D environment.Fibroblasts-loaded C3 hydrogels were used as skin dressings to study their ability to promote skin repair,and the fibroblasts-loaded C3 hydrogel has better repairing results than PBS or single C3 hydrogel.With good biocompatibility and degradability,the agarose grafted gelatin hydrogel can support fibroblasts to survive in the 3D environment and promote the healing of full-thickness defect of skin.These demonstrate that agarose-gelatin hydrogel has potential in the applications of cell carrier or tissue regeneration.