A Biocompatibility Study Of Silk Fibroin And Polycaprolactone Microfiber Scaffolds

Objective:The comparative analysis of the rat costal chondrocytes proliferation between SF and PCL scaffolds.To explore their feasibility as scaffold materials,so as to provide experimental basis for promoting cartilage repair by tissue engineering technology in the future.Methods:The selected rat was 4 weeks old SD rat,the rat chest was dissected with microsurgical instruments,the costal cartilage was isolated and the soft tissue was carefully removed,the rat was obtained costal chondrocytes and chondrocytes were proliferated and differentiated to the third generations.The preparation of the SF and PCL scaffolds of the same quality(3mg)and(10mg),two kinds of scaffolds were divided into two groups-SF groups and PCL groups,then the rat costal chondrocytes were cultured on the two groups of the scaffolds.MTS cell proliferation assay and phalloidin staining were evaluated the cell proliferation,attachment and distribution at 1,4 and 7 day.At day 7,the morphology,microstructure and cell attachment of the scaffolds in the two groups were observed under scanning electron microscope(SEM).At day 14,quantifying the mRNA relative expression levels on the two groups of the scaffolds,and expression levels of chondrocyte-related genes such as SOX 9,Col Ⅱ,Col Ⅰ,Col X and ALP.At day 14,the cells in the two groups of scaffolds were stained with type 2 collagen by DAB colorimetry to observe the secretion of extracellular matrix(ECM).At day 14,the GAGs/dsDNA content of cells in the two groups of scaffolds was determined by DMMB testing reagent.Result:1.MTS cell proliferation assay:The average ± standard deviation of the absorbance values at 490nm at 1,4 and 7 day on the two groups of scaffolds were as follows:SF(0.143± 0.043)and PCL(0.384±0.007),SF(0.765±0.167)and PCL(1.013 ±0.039),SF(0.871 ±0.096)and PCL(0.834±0.031).The absorbance of the SF groups and the PCL groups increased at 1,4 and 7 day,and the SF groups increased more obviously at day 7.However,at day 7 the absorbance value of the PCL groups decreased compared with day 4.The stents of the two groups were statistically compared according to the time and days,and the results were statistically significant(*p<0.05;**p<0.01;**p<0.001).2.Phalloidin staining results:With the development of time,chondrocytes proliferated in large numbers on both groups of scaffolds under the fluorescence microscope at 7 day.However,chondrocytes on the SF scaffolds showed a morphological distribution of aggregation and growth,while chondrocytes on the PCL scaffolds showed a scattered distribution.3.DAB staining results:At 14 day type 2 collagen content on the SF scaffolds was significantly higher than that on the PCL scaffolds.4.SEM observation results:Average diameter of SF and PCL fiber was 13.47μm and 14.94.The proliferation of chondrocytes were along the scaffold fibers in the two groups,and the scaffold fibers were wrapped.The proliferating cells and the ECM filled the pores between the scaffold fibers,and chondrocytes tended to aggregate and grow into clusters inside the SF scaffolds.5.RT-PCR test results:The quantifying the mRNA relative expression of chondrocyte-related genes was analyzed at day 14.At day 14,the expression of SOX-9,Col Ⅱ and Col Ⅰ on the SF groups were higher than the PCL groups.The Col X and ALP-relatively late stage markers of chondrogenesis-on the PCL groups were expressed slightly higher than the silk groups at day 14.6.GAGs/dsDNA test results:At day 14,the content of GAGs/dsDNA in the two groups of cells was determined by DMMB testing reagent,and the results showed that the cells on the SF scaffolds could secret more GAGs,which was statistically significant(**p<0.01).Conclusion:1.The proliferation rate of chondrocytes on the SF scaffolds was higher than the PCL scaffolds.It also maintained the gene phenotype of chondrocytes.It secreted more type 2 collagen and GAGs,but the cells on the PCL scaffolds showed dedifferentiation.2.The SF scaffold has excellent biocompatibility and simulates an ECM environment that is more suitable for cell survival,which enables cells to adhere to the scaffold quickly and firmly.3.The SF scaffold is a carrier of chondrocyte proliferation in the future,provides a feasible scheme for clinical diagnosis and treatment of patients who are difficult to recover joint function due to cartilage injury.