In Vitro Study Of Biocompatibility Of Silk Fibroin Materials

Silk fibroin(SF),extracted from Bombyx mori,is a naturally derived protein that can be processed into a wide range of material formats with tunable mechanical and degradation properties.It shows promise for numerous biomedical applications including drug delivery,tissue engineering,or implantable devices.In this thesis,we established in vitro cell culture models to study the biocompatibility of different SF biomaterials.SF films with different surface morphology,secondary structure and mechanical properties were prepared through addition of Glycerol(Gly)and polyethylene Glycol(PEG 400)to SF solution,and the mixture was dried at 4℃ or 37℃.The composite films prepared at 4℃ exhibited Silk I structure(mainly ɑ-helices)while those prepared at 37℃ were Silk II structure(mainly ?-sheets).At dry state,the elongation at break for the composite film dried at 4℃ and 37℃ reached 175.31±16.10% and 106.21±25.27%,respectively,43 and 26 times higher than the control sample of plain silk films prepared at the same condition.Human fibroblast HS.SK.865 and monocyte THP-1-derived macrophage were cultured on different SF films.The proliferation and morphology of fibroblast cells were determined,and the inflammatory markers including C-C chemokine receptor type 7(CCR7),interleukin-6(IL-6),tumor necrosis factor alpha(TNF-α)and interleukin-1β(IL-1β)were examined at 24 hrs and 48 hrs using real-time reverse transcription polymerase chain reaction(RT-PCR).After 5 days culture,the growth rate of fibroblast cultured on the 4℃ dried composite films was 6.1 times as high as that on the plain film,while the expression of CCR7 gene of the macrophage cells cultured on the 37℃ dried composite film was only 13.4% of that of the plain film after culturing for 24 hrs,indicating the soft,porous film that was prepared at the low temperature was more biocompatible than that prepared at the high temperature.SF spheres were prepared by mixing silk solution and polyethylene Glycol(PEG 10KDa),and further with ethanol.Spheres with different sizes(54.77±12.89 μm,5.44±1.16 μm,374.47±4.75 nm)were obtained with filtration,and further co-cultured with fibroblast HS.SK.865 and monocyte THP-1-derived macrophage.At low and medium dose,the small silk microspheres(S-SMSs)significantly inhibited proliferation of fibroblast cells while the larger silk microspheres(L-SMSs)and silk nanospheres(SNPs)did not.After cultured with macrophage for 24 hrs,the CCR7 gene expression for the S-SMS at medium concentration was 1.9 and 1.7 times as high as that of L-SMSs and TCP control,respectively.For SNPs,the IL-6 gene expression in the low concentration group was 61 and 51 times as high as that in the high concentration group and TCP group,whereas the IL-1β expression in the medium concentration group was 2.1 and 1.8 times as high as that in the high concentration and TCP group.Surprisingly low expression of IL-6 and IL-1β in the high concentration group might be due to the nanoparticle-induced cell apoptosis.Subsequent confocal microscopy study showed that in contrast to L-SMSs,S-SMSs and SNPs were endocytosed by the macrophage cells,which confirmed the results from cell proliferation and gene expression analysis.In conclusion,the cell culture model systems established in this study can be used to evaluate the biocompatibility and biosafety of silk biomaterials with different formats and properties,thus providing guidance for designing and developing biomaterial products in the future.