| Objective:To characterize the pore size,porosity,water swelling rate,mechanical properties,biocompatibility and degradability of silk fibroin-collagen scaffolds prepared by 3D printing,and compare the difference between 3D printing and scaffold prepared by freeze-drying method.Observe whether 3D printing scaffolds and ADSCs can successfully construct tissue engineering fat in vivo.Methods:1.Silk fibroin-collagen scaffolds were prepared by 3D printing and freeze-drying(FD).The silk fibroin concentration was 3.9%,the collagen concentration was 1.95%,and the mixing ratio was 2:1.2.Generally observed the appearance of the 3D printing scaffold(experimental group)and FD scaffold(control group)prepared by silk fibroin-collagen were.The porosity,water swelling rate,pore size and mechanical properties of the two groups of scaffolds were measured respectively;isolate and extract the adipose-derived mesenchymal stem cells(ADSCs).The third generation ADSCs were inoculated on the two kinds of scaffold material separately and cultured in vitro,and using the CCK-8 reagent detect the OD values of cells at 450 nm wavelength,and the toxicity of the scaffold to the cells and the biocompatibility with the cells,and after 14 days of cultivation,the cell-scaffold complex was fixed and sampled.The adhesion and extension of the cells on the scaffold materials and the secretion of the matrix were observed by scanning electron microscopy.3.The two groups of ADSCs-scaffold complexes were cultured and implanted into rat subcutaneous.The changes of scaffolds were observed at 2w,4w and 6w after implantation,understanding the degradation of the two groups of scaffolds.The implant materials were taken for HE staining,Masson staining,and immunohistochemical staining to understand the inflammatory response,degree offibrosis and adipogenesis.Results: 1.General observation: 3D scaffold is generally white,grid-like,internal structure is regular,thickness is uniform,surface pores are regularly distributed,pore size is basically the same,soft under wet conditions,certain elasticity;scanning electron microscopy shows that the cells in the 3D scaffold can fully adhere and stretch,protrude from the pseudopod,and secrete a large amount of extracellular matrix.The FD scaffold is generally white and has a dense sheet-like structure.It is relatively regular and has different thickness.The surface is slightly concave and convex.See the bubble structure.The inside of the scaffold has different sizes of holes,disordered arrangement and poor connectivity.It is tough and not easy to deform.Scanning electron microscopy shows that the cells in the FD scaffold can fully adhere and stretch,protrude from the pseudopod,and secrete a large amount of extracellular matrix.2.The data were analyzed by SPSS 21.0 statistical software,which accorded with normal distribution and homogeneity of variance.The difference between the two groups was tested by independent sample t test,and P<0.05 was used as the criterion for judging the difference significance.The aperture of the 3D printing scaffold is(226.06±28.40)μm,and the aperture of the FD scaffold is(186.91±33.93)μm.There was no significant difference in the pore size between the two groups(P=0.619);the porosity of the 3D printing scaffold is(94.01±6.08)%,the porosity of the FD scaffold is(80.37 ± 2.72)%,there was a significant difference in porosity between the two groups(P=0.024);the water swelling ratio of the 3D printingscaffold is(301.63±30.03)%,the water swelling rate of FD scaffold is(208.33±45.37)%.There was a significant difference in the rate of water swelling between the two groups(P=0.041).The elastic modulus of 3D printing scaffold is(34.66±13.74)kPa.the elastic modulus of the FD scaffold is(32.87 ± 12.43)kPa,and there was no significant difference in the elastic modulus between the two groups(P=0.836).3.The CCK-8 reagent was used to detect the OD value at the wavelength of 450 nm after co-culture of ADSCs-scaffolds.The results showed that the OD value increased gradually with the increase of the culture days,and the OD value of the 3D group was slightly higher than that of the FD group.The number of cells on the 3D scaffold was higher than that of the FD group,but there was no significant difference in cell compatibility between the two groups(P=0.061).4.Scanning electron microscopic observation of ADSCs-scaffold after co-culture: See the cells in both groups of scaffold can fully adhere to stretch,extend the pseudopod,and secrete a large numberof extracellular matrix.5.Observing the scaffold at 2w,4w,6w at every time points:both groups of scaffold were covered by a layer of envelope which the 3D group was thicker than the FD group,and the new blood vessels formed on the surface of the envelope,see the thin layer of fat under the envelope,the scaffold gradually becomes smaller as time goes by.Histological observation showed that both the 3D group and the FD group had inflammatory cell infiltration,multinucleated giant cells formed,see damaged scaffold materials,there were fat cells under the fibrous membrane,and the fibers proliferated greatly.The inflammatory reaction is gradually reduced over time,and the 3D group inflammatory reaction and the degree of fibrosis was lighter than that of the FD group;Immunohistochemical staining:vacuolar adipocytes with specific brown-black color were observed in both groups.Conclusions: 1.Through research,it is found that the 3D printing scaffold has suitable pore size,porosity,water swelling ratio and spatial structure superior to FD scaffold,which is conducive to cell adhesion and proliferation,and has good biocompatibility with cells.2.The 3D printing scaffold is degradable in vivo and has good degradation performance.3.The 3D printing scaffold has a mild inflammation and fibrosis in the body,and can form adipose tissue in the body,which is suitable for the research of adipose tissue engineering. |
PDF Full Text Request