Evaluating Surface Properties And The Cell Compatibility Of Carbon Ion Implanted Silicone Rubber

Background and Objectives:Nowadays, surgery is the main method to reconstitute and repair body surfaceorganism and organ malformation which brought by inborn or external injury. Implant ofsoft tissue filling material is the fundamental therapeutics method, which also consistentwith modern medical science tendency. Compare to the other materials，silicone rubber hasgood physical chemistry performance, physiology inertia, bearing corrosion and facility toelaboration, is still the most usual implant material in clinic use. But it also has intensivehydrophobic surface, and the materials hydrophobicity will induce shortage ofbiocompatibility, around the material will form fibrous connective tissue and encapsulationwill occurrence. With the development of encapsulation,material will disfiguration and shift.Therefore, suitable epi-reshaping using in silicone rubber will improve cell compatibilityand elevate the clinic therapeutic efficacy of the soft tissue implanting polymer materials,and is benefit to study of surface characteristic and cell biology proceeding. According tothe literature, the silicone rubber surface modification methods include the graftcopolymerization, the biomimetic coating method and so on, but there exists someproblems which limited the effect of the surface modification of silicone rubber. The graftcopolymerization method is by a chemical reaction on the basis of activation of the polymerintroducing a specific chemical group, the entire process is not only cumbersome steps, butthe unstable chemical group also restricts the modified effect. And the biomimetic coatingmethod is a method by spraying, sputtering or the like, on the basis of maintaining thecharacteristics of the material itself, covered with a layer of biologically active substance inthe material surface. This modified method also has more cumbersome steps, and theoperating conditions is difficult to control, restrictions its popularization and application.Based on previous work, we would like using ion injection to sharping the surface ofsilicone rubber (SR), take the anthropometric compose element carbon (C) to treat SR, acquired carbon-silicone rubber (C-SR). Ion implantation techniques previously used forthe metal and alloy material modification, it can select the type of ion source injectionmodified and micro-graphic structure is formed in the surface of the modified materials, sothat the microscopic structure of the surface of the material has been changed, but it doesnot transform the material’ properties, for the surface modification of silicone rubberhaving a good feasibility. In our research, the SEM(Scanning electron microscope),AFM(Atomic Force Microscope), FTIR(Fourier transform infrared spectrometer), XPS(Fourier transform infrared spectrometer), XRD(X-ray diffraction) and the water contactangle were used to analyze the surface properties of modified materials. And CellProliferation Experiment，Immunofluorescence and Western Blot technology were used toanalyze the biocompatibility of modified materials. We hope that can be prepared a linewith clinical need, and fewer side effects, a new soft tissue filler material.Methods:Part I SR preparation and C ion injection and observed their microscopic morphology1. At room temperature (Temp20℃, humidity50%), add agitated uniformity A, Bcomposition of liquid silicone rubber into metal plate tooting and let the liquid siliconerubber thiolaction at room temperature. After solidification completed, choosing the surfacesmooth and integrity SR to do the next experiment. The SR chaffs were depart as threegroups, each group received10keV,1*1015ion/cm2,3*1015ion/cm2,1*1016ion/cm2dosage ion injection. Through three kinds of dosage, we gained three kind of C-SR, and wename them as C-SR1, C-SR2, C-SR3.2. Through SEM and AFM to observe and compare the different of their microscopicmorphology.Part II Texting Surface properties of SR and C-SRUsing FTIR(Fourier transform infrared spectrometer), XPS(Fourier transform infraredspectrometer), XRD(X-ray diffraction) and the water contact angle to analyze and evaluatethe surface properties of modified materials. For statistical analyze, we used SPSS20.0（P=0.05）.Part III Evaluating the cell compatibility of Carbon ion implanted silicone rubber1. Cell preparation: the human dermal fibroblasts were cultivated on the surface ofmodified materials in vitro. 2. Experimental groups:1) Normal group: The cells were seeded onto the cell culture plates without materials.2) SR group: The cells were seeded onto the surface of SR;3) C-SR1group: The cells were seeded onto the surface of C-SR1;4) C-SR2group: The cells were seeded onto the surface of C-SR2;5) C-SR3group: The cells were seeded onto the surface of C-SR3.3. Using the confocal laser scanning microscopy to observe the framework andattachment of cell, and the CCK-8assay was used to detect the bioactivity of thefibroblasts.4. Using the Immunofluorescence,Real-time PCR and Western blot to measure theexpression of the protein of OPN, Vinculin, Zyxin and Talin.Part IV Research the mechanism of the improved cytocompatibility of materials1. Prior to joining exogenous OPN monoclonal antibodies and recombinant OPNprotein exogenous circumstances, cytoskeletal proteins (actin) expression are staining byimmunofluorescence, while comparing the state of cell growth and cell adhesion of eachgroup of materials.2. Using Western Blot technique to detect OPN expression in the culture medium ofeach group of materials.3. Using Trans-well chemotaxis assay to detect whether the OPN and MMP9canpromote cell migration, and through gelatin zymography is used to test MMP9activity levelof intracellular situation of each group materials.4. Based on the successfully constructed of MMP9-RNAi model, by adding exogenousrecombinant OPN or not, and using laser scanning confocal microscopy to obsearve theexpression of MMP9and OPN, and the changes of cell morphology, proliferation andadhesion.Results:1. We have prepared SR and used multifunction ion implanter successfully preparedC-SR modified materials in a preset conditions by three different ion implantationconditions.2. We observed that the microscopic morphology has no significant difference betweenSR and C-SR by SEM. But from the AFM micrographs, SR exhibits a smoother and more homogeneous surface compared with C-SR1, C-SR2and C-SR3. The surfaces of C-SR arecomposed of larger irregular peaks and deeper valleys than the surface of SR.3. FTIR results showed that the C-SR have no significant change with SR, suggestingthat the C-SR did not change the organic functional constitute of SR. The XRD patterns ofC-SR and SR exhibit characteristic peaks consistent with crystalline materials. It is evidentthat C ion injection into the SR matrix changes the surface microstructure. And no distinctXRD patterns among the three kinds of C-SR have been observed, possibly because thedifference among C-SR1, C-SR2and C-SR3is too small to be detected by our method. Toestimate the composition and chemical states of C-SR and SR, XPS analysis was employed.The injection also changes the surface charge distribution, silicone oxygen rate andchemical-element distribution due to C ion incorporation, which may facilitate adhesionand proliferation of fibroblasts. With the improvement of C ion injection dosage, Si contenton the surface of SR decreased, the possible reason is that C replaced Si and precede ionbond. The distinct XRD patterns have been observed among the three C-SR, possiblybecause the difference of dosage. Water contact angle was hire to detect the hydrophilicityof the SR materials, the figure of data showed that C ion injection can decrease the watercontact angle, and with the improvement of C ion injection dosage, water contact angledecreased much more, this indicate that C ion injection can change the hydrophilicity of SR,and may change the protein adsorption and profit the adhesion and proliferation offibroblasts.4. Through cystoskeleton specific staining, the images show that on the surface ofC-SR, cells growth much better than which on the surface of SR, to stretch out filopodium,microfilament spread much more regularity, cell morphous is much spreading.5. Using CCK-8to study the cell proliferation status, the result shows that comparingto SR, cells on C-SR surface have better viability. The data have significant difference,P<0.05.6. Western-blot and immunofluorescence reveal that cells on the suface of C-SRexpressed OPN/Vinculin/Zyxin/Talin much more than those on the surface of SR, inaddition, with the C ion dosage improvement，the protein level of talin increased(P<0.05).7. Real-time PCR reveal that cells on the suface of C-SR expressed OPN/Vinculinmuch more than those on the surface of SR, in addition, with the C ion dosage improvement，the protein level of talin increased(P<0.05).8. Studies on the role of OPN, we added exogenous OPN monoclonal antibodies, eachgroup of materials can be observed cytoskeleton protein levels are decreased, and the cellmorphology, adhesion conditions are affected, while in adding exogenous recombinantOPN protein after reversed this phenomenon.9. Using Western Blot to detect the OPN’s extracellular secretion, it as a bridge proteinrole in regulating the expression of related proteases, thereby promoting cell adhesion,proliferation and migration.10. Using Trans-well chemotaxis assay to evaluate the migration ability of OPN andMMP9, OPN and MMP9were observed with the promotion of the role of cell migration;further zymography experiments obtained in C-SR material MMP9activity wassignificantly higher than the SR.11. MMP9-RNAi model was successfully constructed, and based on this, we foundthat the expression of OPN and MMP9were decreased without adding exogenousrecombinant OPN protein and like with the cell adhesion, growth state of proliferation,andwhile it can be restoration by adding exogenous recombinant OPN protein. Comprehensiveindicates exogenous OPN can increase MMP9expression, and they are together toparticipate in the the mechanism of the improved cytocompatibility of materials.Conclusions:1. The production method of C-SR is simple, the process is easy and has betterfeasibility.2. The surface physical and chemical properties of C-SR is stable, C-SR maintain theexcellent characteristics while also improving the hydrophobic properties of the SR. So,C-SR has a better organization of affinity compare to SR.3. The growth state, proliferative capacity, cell adhesion ability of human dermalfibroblast cultured on the C-SR are better than SR. And C-SR has a better biocompatibilitythan SR, it is more suitable for long-term clinical implant into repairing the defect bodyparts.4. From the study of the mechanism of C-SR improved cell compatibility, we believethat the change of physical and chemical properties of the material surface will impact thematerial surface microenvironment, thereby selectively enhances the adsorption of OPN and other proteins, and OPN will bind with its specificity relevant receptor to mediatesignal transduction, and up-regulation the expression of MMP9, causing the correspondingcellular response, thereby increasing the cytocompatibility of the material.