| Objectives:To study the effects of different doses of BTX-A on the capsule formation of silicone prosthesis and the biological behavior of fibroblasts.Methods:1.Material preparation(1)For the preparation of silica gel,the computerized numerical control(CNC)engraving equipment was used to process a 10.0 cm×10.0 cm×0.1 cm mold.The polydimethylsiloxane(PDMS)prepolymer was solidified on the mold and reinforced in a constant temperature drying oven at 60-75 ℃ for 1 h to prepare smooth PDMS.(2)Cutting: The smooth PDMS was cut into circular sheets with the sizes of 96,24 and 6 well plates for cell experiments.Square PDMS silicone sheets with a size of 1.0cm×1.0 cm were cut for animal experiments.(3)Disinfection: The cut PDMS silicone sheet was sterilized overnight by immersing in 75% ethanol,and then soaked in sterile PBS for 2 hours before use.2.The effect of different doses of BTX-A on the capsule formation of silicone prosthesis(1)Operation implementation: A total of 10 Sprague-Dawley rats were selected,and four longitudinal surgical incisions of about 1 cm in length were made at two points on each side of the median line on the back of each rat,and the subcutaneous fascia tissue was bluntly separated into the surgical incision.A square piece of 1.0 cm × 1.0 cm PDMS silicone sheet was subcutaneously implanted in each of the four incisions.After suture with No.4 silk suture,a 1 m L empty needle was used to inject into the normal skin beside the surgical incision,and 0.1 ml normal saline was injected around the implant material and set as the control group.The rest were injected with the same volume of BTX-A(Lanzhou Institute of Biological Products)normal saline solution with different concentrations,and the doses were 5.0,2.5 and 1.0 U,respectively.After 3 months,the PDMS silicone sheet and surrounding capsule tissue were removed surgically.(2)Imaging observation: Before removing the encapsulated tissue,the thickness of fibrous tissue around the PDMS silicone sheet prosthesis in vivo was detected by high-resolution small animals ultrasound imaging system(HR-US).(3)Histological measurement: HE staining and Masson staining were performed to observe the thickness of capsule tissue and the arrangement of collagen fibers.(4)Immunohistochemical assessment: the expression of collagen type Ⅰ(COL-Ⅰ),collagen type Ⅲ(COL-Ⅲ),α-smooth muscle actin(α-SMA)and proliferating cell nuclear antigen(PCNA)was observed by immunohistochemical staining,and the corresponding H-score was calculated for semi-quantitative analysis and comparison.3.Effects of different doses of BTX-A on the biological behavior of fibroblasts(1)Cell proliferation activity: Cells were seeded on the surface of the material,and BTX-A complete medium containing BTX-A 0,2.0,5.0,and 10.0 U/ m L complete medium was added,respectively.The cell activity at 48 hours of culture was detected by CCK-8method.(2)Cell migration ability: The cells of each group cultured for 48 hours after inoculation were taken,and the migration of cells on the surface of silica gel was observed by the live cell imaging system workstation.(3)Cytoskeleton: the fibroblasts in each group were stained by immunofluorescence after 48 hours of culture,and the cytoskeleton organization was observed by laser confocal microscope.(4)Proliferating cell nuclear antigen(PCNA)and α-smooth muscle actin(α-SMA)were detected by Western blot after 72 hours of culture.Results:1.Imaging examination in animal experiments showed that with the increase of BTX-A dose,the thickness of fibrous tissue around the prosthesis in rats decreased gradually.The difference between 5.0 U BTX-A group and control group was statistically significant(P < 0.05).2.Histological observation showed that the thickness of capsule in each experimental group decreased with the increase of BTX-A dose,and the difference was statistically significant compared with the control group(P < 0.05).The imaging measurement results were correlated with the histological measurement results(P < 0.05).3.The results of immunohistochemistry showed that there was no significant difference in the proportion of collagen component structure in each group,but the H-score of α-SMA decreased with the increase of BTX-A dose,and the difference between 2.5 U and 5.0 U groups was statistically significant(P < 0.05).4.CCK-8 test results showed that there was no significant difference in cell activity among each group after 48 hours of culture(P > 0.05).5.The recording results of live cell workstation showed that with the increase of concentration,the speed of cell migration increased significantly,and the results were statistically significant(P < 0.05).6.Cytoskeleton staining showed that the cells in 5.0 and 10.0 U/ml groups showed shorter longitudinal axis and smaller area than the control group.7.The results of Western blot showed that the expression of α-SMA and PCNA decreased in the treatment of BTX-A,but the difference was not statistically significant.Conclusion:1.BTX-A can inhibit the expression of α-SMA in the capsule tissue and reduce the thickness of the capsule at 3 months after implant,but the specific dose needs to be further explored.2.BTX-A can promote the migration of normal skin-derived fibroblasts on the surface of the material and the change of cell morphology,which can help to evenly cover the capsule around the prosthesis in the early stage.At the same time,α-SMA has a downward trend,although there is no statistical difference,it may still play a role in the prevention of capsule.3.BTX-A may reduce collagen deposition by affecting the biological behavior of fibroblasts,but the specific mechanism needs to be further studied. |
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