The Effects Of Different Stiffness Of Silicone Rubber On Proliferation And Collagen Synthesis Of Human Dermal Fibroblasts

Background:The silicone Rubber(SR),such as silicone nose implants,Silicone breast implants and expander,which has become the most commonly used implant material in plastic surgery due to its stable physical and chemical properties including invariabilit y and non-degradation in vivo.However,on account of the low compatibility between the surface of silicone rubber implant and human tissues,a capsular fibrous capsule which composed of a large amount of collagen and a few of fibroblasts will eventually form to package the silicon implant.The capsule may gradually thicken as time going,and contracture may come up under some unknown factors.Eventually,the contracture capsule may lead to the deformation and displacement of the silicon prosthesis,and further lead to local tissue stiffness,deformity and pain,which can seriously affect the prognosis of surgery and bring bad experience to patients.Indeed,the formation and contracture of capsule is an outcome of wound healing after silicon prosthesis implantation.It has been demonstrated that this process is closely related to the fibroblasts.Studies have shown that the physicochemical properties of biomaterials such as surface stiffness,morphology,hydrophilic or hydrophobic properties,electric charges,chemical groups,etc.have an impact on cell adhesion,proliferation,differentiation and other biological behaviors.Objective:To explore the relationship between the physicochemical properties of silicone rubber implant surface and the biological behavior of fibroblasts.To determine the etiology of complications related to silicone rubber implant and invent new silicone rubber materials with better structural properties and improve the clinical treatment effect of capsule.The objective of this study is to explore the effects of the stiffness of silicone rubber on the proliferation and collagen synthesis of Human Dermal Fibroblasts(HDFs),and to provide ideas for the development of better silicone rubber materials.Methods:1 Preparation of SR with different stiffness and detection of surface propertiesSilicon rubber production : A-type and B-type silicone gel were adequately mixed in a ratio of 1:9,2:8,…,9:1.And then,We poured the silicon gel mixture into a custom grooved mold with a size of 100 mm * 100 mm * 1 mm.After the silicone rubber solidifies at room temperature,we selected the silicone rubber sample which has smooth surface and no bubbles.Surface stiffness detection: We measured the stiffness of each silicon rubber sample with the Shore Type A Hardness Tester(note: the group A:B=1:9 did not achieve complete curing at room temperature,and was abandoned).Detection of tensile strength and elongation at break: The microcomputer universal material testing machine was used for tensile test to detect the tensile strength and elongation at break of SR in each sample.Surface topography measurement: Atomic Force Microscopy(AFM)was used to detect the surface micromorphology of silicon rubber samples with the highest and lowest stiffness.Detection of surface chemical groups and element composition: The chemical functional groups and constituent elements on the surface of the two groups with the highest and lowest stiffness were detected by Fourier infrared spectrometer(FTIR)and X-ray electron spectroscopy(XPS)respectively.Surface hydrophilic and hydrophobic detection: The surface water contact angle of the above two groups of silicone rubber samples was measured by the Water Contact Angle Tester.2 Effects of different stiffness SR on fibroblasts proliferation and collagen synthesisFibroblasts acquisition: The primary human dermal fibroblasts were extracted from the foreskin of healthy boy(obtain the informed consent of the patients and the ethical review),and the fourth generation of cells were taken after passage for subsequent experiments.Observe the adhesion of cells on the surface of silicone rubber samples: HDFs was inoculated on the surface of silicone rubber samples with different stiffness,and the cell adherent growth and cell morphology were respectively observed under an inverted microscope at 24 hours,48 hours and 72 hours.Cell proliferation assay: CCK-8(Cell Counting kit-8)was used to detect HDFs Cell proliferation on silicone rubber samples with different stiffness.Detection of HDFs collagen synthesis: We selected two groups of silicon rubber samples with the highest stiffness(Stiff group,A:B=9:1)and the lowest stiffness(Soft group,A:B=2: 8)to experiment.α-SMA ELISA kit and Collagen Ⅰ ELISA kit were respectively used to quantificat the expression of α-SMA and Collagen Ⅰof HDFs.3 Initial study on the mechanism of SR stiffness affecting fibroblast proliferation and collagen synthesisExperimental groups and treatment methods: normal group : Soft+PBS,Stiff+PBS;3-MA group : Soft+ 3-MA(5mm),Stiff+ 3-MA(5mm);Rapamycin group : Soft+ rapamycin(100nM),Stiff+ rapamycin(100nM).Detection of autophagy marker protein: The HDFs were inoculated onto the silicone rubber material.After pre-incubation for 24 hours,the autophagy inhibitor and the inducer were added.Four hours after the treatment,the autophagy marker protein Beclin 1,LC3 II and p62 were detected by Western Blot.And then,Compare and analyze the Western Blot results.Effect of autophagy on HDFs proliferation: The cell proliferation of the above groups was detected by CCK-8 kit,and the expression of cytoskeletal protein(F-actin)in HDFs was observed by laser confocal microscopy.Effect of autophagy on collagen synthesis of HDFs: The α-SMA ELISA kit and Collagen I ELISA kit were used to detect the expression of α-SMA and Collagen I in HDFs in 3-MA group and rapamycin group.Results:1.Eight groups of silicon rubber film materials with different hardness were prepared by simple method,and the hardness of each group was quantitatively determined.It was found that the hardness of silicon rubber gradually decreased with the increase of b-type liquid silica gel proportion,and the hardness of all materials was between 3.0~13.0 HA.2.The tensile test results showed that the elongation at break of the material increased with the increase of SR hardness,but the tensile strength of SR was lower when the hardness was too high or too low.3.The surface morphology of silicone rubber in Stiff group and Soft group was observed by AFM and results confirmed that there was no significant difference between the two groups.The surface morphology was not a factor in the difference of cell biological behavior in this experiment.4.The results of FTIR and XPS showed no significant difference,indicating that there was no significant difference in the surface chemical functional groups of SR between the two groups,and the surface chemical properties were not the factor causing the difference in cell biological behavior.5.There was no significant difference in the water contact Angle of SR surface between the two groups,indicating that the change of hardness did not cause a change of hydrophilic and hydrophobic properties of SR surface.6.By observing the adhesion of HDFs on SR surfaces with different hardness in each group,the cells were able to adhere to the wall and grow well.The morphology of the cells was in the form of normal fibroblasts with sufficient spread,which indicating that different proportions of type A and type B silica gel had no obvious cytotoxicity.7.The results of proliferation experiments showed that the proliferation ability of HDFs increased with the increase of hardness,which in Stiff group was significantly better than that of Soft group.It indicates that the hardness of SR has a significant effect on the proliferation of HDFs-high hardness SR promotes the proliferation of HDFs.8.The expression ofα-SMA and Collagen I in HDFs by ELISA showed that the expression of α-SMA and Collagen I in the Soft group was significantly higher than that in the Stiff group,indicating that SR hardness can significantly affect the collagen synthesis of HDFs and affect the fibroblasts transformation.9.Autophagy-related marker protein detection and intervention in autophagy have shown that inhibition of autophagy can inhibit HDFs proliferation and enhance the expression of α-SMA and Collagen I,and induction of autophagy can promote HDFs proliferation and decrease the expression levels ofα-SMA and Collagen I.Conclusions:1.The different hardness silicone rubber materials prepared by physical mixing of type A and type B silica gels have no significant difference in surface physical properties except for hardness.It can be basically determined that the hardness of silicone rubber is the main factor affecting the conclusion of the study.2.Silicone rubber hardness is an important factor affecting the proliferation of HDFs and their transformation into myofibroblasts and collagen synthesis.3.Autophagy may be one of the ways in which the hardness of silicone rubber affects the proliferation of HDFs and its transformation into myofibroblasts and collagen synthesis.However,how SR hardness mediates autophagy remains to be further studied.