| Background:To repair of the tissue and organ defects, the application of biological materials is becoming more and more extensive. This kind of material used to repair or replace the damaged tissues and organs to restore its function without any adverse effects on the human body. Biocompatibility is an important factor for material in the clinical application. Williams David proposed that a good biomaterial can achieve the desired objectives, but also does not cause any other local or systemic reaction. In plastic surgery, biomaterials are often used as filling materials, the filling material mainly divided into two kinds, the natural materials and artificial biological materials. Biomaterials can depart into including metal materials, inorganic non metallic materials and polymer materials.At present, silicon rubber, high density polyethylene and polytetrafluoroethylene etc are commonly used in the plastic surgery, these materials have its own advantages and disadvantages. Silicone rubber as a traditional plastic material, its first application is as the nose prosthesis. Because of its excellent physical and chemical properties, silicone rubber is extensively used in clinical. However its biocompatibility is poor, so improve the biocompatibility is necessary. Using modern theory to modificat the silicone rubber surface will greatly improve its clinical application value. The surface modification of silicone rubber can improve cell compatibility. The principle of surface modification is to change the adsorption behavior of protein. The affinity and hydrophobicity of the material directly affect the surface protein adsorption type and quantity. One method of the surface modification is to change the material’s hydrophilic / hydrophobic property.Hydroxyapatite has an excellent biocompatibility, non-toxic, non irritating, no rejection, no aging, non allergic, non carcinogenic. At the same time, hydroxyapatite has a good bone conductivity, HA in the binding between the bone, and bone tissue can achieve the ideal biological combination. However hydroxyapatite is brittlen,so it is difficult to carve the shape.This paper combined with the advantages of the two materials; avoid the disadvantages of two kinds of material. We coated hydroxyapatite on the surface of silicone rubber in previous study, and found that the hydroxyapatite coated SR material has a good biomechanical properties and compatibility. Based on previous study, we further studied the biocompatibility of HA/SR, detected the biological safety of the materials, and studied the molecular mechanism of biocompatibility of HA/SR.Research method:This part is divided into four parts.Part I: the biocompatibility and safety assessment of HA/SR1 the biocompatibility of materials testingOn the basis of the previous studies, we satined the cells by actin tracker to evaluate cell adhesion, used CCK8 to assess the cell proliferation, used SEM to observe the cell shap. The molecular detection of zyxin, talin and vinculin were detected by immunofluorescence.2. The biological safety evaluation of materialsThrough the material hemolysis rate, body weight, liver and kidney function, organ surface observation, implantation experiment and HE staining, Masson staining, immunohistochemical staining(CD4,CD68, TNF-a, a-SMA, elastin,), we studied the biology safety of HA/SR.Part 2: Study on the adsorption of OPN on the surface of materials:1, the surface protein adsorption of the wound fluid in rats:We collected the wound fluid in rats, soaked SR, HA-1/SR, HA-2/SR with the wound fluid, using scanning electron microscope to study the surface of the material, analyzed surface water contact angle, and using WB certification the OPN protein.2, the expression and secretion of OPN in the surface of materialsSR, HA-1/SR and HA-2/SR three kinds of material surface in vitro cultured human dermal fibroblasts, collected and cultured supernatant, extracted the total protein, WB experiments to verify the secretion and expression of OPN surface.Part 3: To analysis of the important role of OPN in HA/SR cells attached to the surface of the material.1. The effect of OPN on cell adhesion in surface of materialsHuman dermal fibroblasts were cultured on the surface of SR, HA-1/SR, and HA-2/SR. In each group of materials we added exogenous recombinant human OPN protein and OPN antibody, observed the adhesion of cells in each case.2. The role of OPN in cell migrationTrans well was used to test the effect of OPN on cell migration by using the exogenous OPN protein as a cell chemotaxis. Combined with the cell adhesion of OPN protein on the cell adhesion and migration, we reveald the importance of OPN on the hydroxyapatite coated silicone rubber surface.Part 4: Study on the molecular mechanism of HA/SR cell compatibility1, RNA interference technology to reduce the expression of MMP9, we studied the relationship between OPN and MMP. By means of mass spectrometry and bioinformatics, OPN and MMP9 were used to clarify the specific molecular mechanism to regulate the cytoskeleton rearrangement on the HA/SR surface.2, Study in vitro cultured human dermal fibroblasts on HA-1/SR, HA-2/SR, SR extracted the total protein, SDS-PAGE separation, after the gel imaging system to collect pictures, analysis and cut the difference between the point of protease analysis, EMBL, NCBI and other database analysis of the possible molecular mechanism, used WB to vertification the result.Reserch resultsThe main results are as follows:Biological compatibility test of materials(1) Throug CCK8, cell skeleton staining, SEM, we can see HA/SR have a good compatibility.(2) The cell adhesion molecular as vinculin, talin, zyxin and other detection of cell adhesion molecules on the surface of the material is further studied. It is revealed that hydroxyapatite coated silicone rubber can improve the expression of the surface cell adhesion molecules.(3) The biocompatibility of HA/SR was further confirmed by in vivo implantation, HE stain found that HA/SR has a good biocompatibility.2, the toxicological properties of materials and biological safety evaluationUsing Masson HE and ICH of, CD68, a-SMA,, TNF-a, CD4, elastin, we studied the toxic property of HA/SR. The experimental results showed that the HA/SR are safe enough.3, OPN role as a bridge protein(1) By eye observing, scanning electron microscopy analysis and water contact angle experiment on the surface of the material, we found that the surface water contact angle decreased after wound fluid in rats soaking, which may be related to the surface of the material.(2) OPN protein expression and secretion improved on the surface of HA/SR, and the adsorption capacity of OPN protein on the surface of HA/SR was bigger than that of SR, which may be related to the compatibility of HA/SR.(3) The exocrine OPN protein and intracellular OPN protein improved on the surface of the HA/SR. Secretion OPN protein and intracellular OPN protein expression are higher than that of cells on the surface of SR further indicated that OPN in material surface adsorption to HA/SR material is related to cell compatibility.(4) By the addition of exogenous OPN and OPN antibody blocking experiments, we can see that the addition of exogenous OPN can enhance the adhesion of the material surface cells, while OPN antibody blocking weakened the cell adhesion, and the results show that the compatibility of HA/SR surface cells may be related to OPN.(5) Through the trans well experiment, OPN as exogenous protein added to the cells chemotaxis chamber outer chamber can significantly improve the fiber cells of the wear ability of films revealed the OPN may at the material cell interface, improve the ability of cell migration. Through the analysis of the surface protein layer and the blocking of OPN antibody, WB, Transwell and other experimental techniques were used to define the important role of OPN in the material/cell interface, OPN protein is important in the cell adhesion and migration of hydroxyapatite coated silicone rubber.4, the molecular mechanism of HA/SR cell compatibility(1) The activity of MMP9 on the surface of HA/SR was significantly increased, which indicated that the activity of MMP9 was enhanced with the increase of OPN activity in the SR surface coating, which indicates that MMP9 may have some correlation with OPN.(2) After MP9 interference the number of adherent cells on the surface of the material reduced, cell morphology also showed the disordered state, cytoskeletal collapse, fixed state, but the morphology and the change of the quantity of adhesion reduction can be reversed by OPN, suggesting that MMP9 is OPN downstream molecules involved in cell adhesion, improve the compatibility of material surface.(3) Mass spectrometry analysis results by NCBI and EMBL database, find out the main differences protein through literature search and analysis we propose EF1? mediated γ-actin may contribute to cytoskeletal rearrangement, thus affecting the surface of cell compatibility, and the conjecture is verified by EF1? interference caused cell adhesion to diminish the experimental.Conclusion:1. HA/SR has good biocompatibility.2, HA/SR is safe and non-toxic, accord with the biological materials national medical biological evaluation of standard, has good biological safety.3, The adsorption of OPN on the surface of HA/SR has an important effect on cell adhesion and migration, and it is a bridge protein in the process of improving the biocompatibility of HA/SR cells.4, OPN may enhance the cell’s description of the HA/SR by mediate MMP9, and the regulation of γ-actin on the regulation of EF1? plays an important role in the enhancement of HA/SR surface cell compatibility. |
PDF Full Text Request