Study Of Growth And Cellular Properties Of Vascular Smooth Muscle Cells In Silk Fibroin Porous Scaffolds

The increasing morbidity of cardiovascular diseases in modern society has made it an urgent requirement to develop blood vessel substitutes especially those with internal diameters less than 6mm for replacement of diseased coronary and below-the-knee vessels.The current clinical treatment involving this disease is surgical replacement with autologous blood vessels or synthetic materials.However,small-caliber artificial blood vessels have not yet been clinically applied.The main reason is that thrombosis and neointimal thickening obstruct the artificial blood vessels.Therefore,the study of degradable porous artificial blood vessels inducing angiogenesis in situ has prospects for application,autogenous cell ingrowth can be achieved,and extracellular matrix is synthesized and deposited by these autologous cells to form biological blood vessels.In order to analyze the feasibility of silk fibroin in the application of artificial blood vessels,we have studied the synthesis of smooth muscle cells and extracellular matrix(elastin,collagen)in cultured regenerated silk fibroin scaffolds.In this paper,we used the freeze-drying method to prepare regenerated silk fibroin porous scaffold under the conditions of mass fraction of 4% silk fibroin solution and freezing temperature of-20 °C.The scaffold was well formed and its pore size is in the range of 60-200?m.Then we investigated the proliferation of smooth muscle cells on the material,the synthesis of extracellular matrix(elastin,collagen),and cell phenotype after seeding of smooth muscle cells,and the effect of transforming growth factor-?1(TGF-?1)on cell extracellular matrix synthesis and smooth muscle cell phenotype.First,we used DNA content analysis to study the proliferation of cells on porous scaffolds.The results showed that the number of vascular smooth muscle cells on silk fibroin membranes continued to increase from 1 to 3 weeks without the addition of TGF-?1 and the initial addition of TGF-?1.The number of cells at the fourth week of the material decreased,but the number of cells that continued to add TGF-?1 continued to increase.At the fourth week,the DNA content was 2.62 times that of the non-addition material and 1.28 times of the initial addition of the TGF-?1 material.Indicating that TGF-?1 promotes the proliferation of smooth muscle cells.Immunoenzymatic analysis showed that vascular smooth muscle cells had positive expression of elastin type I collagen and type III collagen on the porous silk fibroin scaffold,and the positive expression areas of the three proteins without TGF-?1 addition were almost the same over time.Without change,the positive expression of the three proteins no longer decreased after the continuous addition of TGF-?1,and the positive expression area of the material for continuous addition of TGF-?1 increased,at four weeks,the area percentage of elastin positive expression was 1.37 and 1.21 times higher than that of the other two types of collagen.Type I collagen was 1.38 and 1.11 times,and type III collagen was 1.47 and 1.18 times.but it became stable after 2 weeks.Real-time fluorescence quantitative PCR is used to study the effect of TGF-?1 on the expression of extracellular matrix protein m RNA on the porous silk fibroin scaffold.The results showed that the m RNA expression of elastin without TGF-?1 changed little,and the m RNA expression of type I collagen increased.The third week was 4.89 times of the first week,while the expression of type III collagen m RNA was decreased.The fourth week is only 0.38 times the first week.The m RNA expression of elastin and type III collagen in TGF-?1 added initially decreased after two weeks,but did not change significantly in the second 2 weeks,and the expression level of type I collagen m RNA increased significantly.The fourth week was 2.62 times to the first week.The m RNA expression levels of elastin,type I collagen,and type III collagen in TGF-?1 added continuously increased significantly in the first two weeks,which were 1.04,4.66,and 3.11 times of the first week,respectively,during the following period.becoming steady.To investigate the effect of TGF-?1 on the phenotype of smooth muscle cells and the expression of extracellular matrix protein m RNA,a real-time fluorescent quantitative PCR study of the contractile phenotypic factor and extracellular matrix protein of smooth muscle cells was performed after immobilizing the total RNA concentration of the material.The results showed that the smooth muscle cells on the silk fibroin porous scaffold transformed from a synthetic phenotype to a contractile phenotype after seeding,and the addition of TGF-?1 could initially enhance the ability of extracellular matrix protein synthesis of the cells and slow down the material.Smooth muscle cells convert from a synthetic phenotype to a contractile phenotype.