| Upon the implantation of an implant, the initial event is protein adsorption on the implant. The state of the adsorbed protein is essentially important for the success of the implant. It has been widely proved that the surface properties of biomaterials regulate the amount, type and conformation of the adsorbed proteins. Our group recently reported that the adsorption force of fibronectin(FN) on material surfaces is controlled by surface chemistry as well. Unfortunately, the mechanism by which the surface properties regulate protein adsorption remains elusive. Clarification of the mechanism may provide valuable guidance for the design and modification of biomaterials or biomaterials surface.Molecular dynamics simulation(MD) is a powerful tool to provide microscale information on a macromolecule, including the location and speed of the composing atoms, the inter-atom interaction force, and various energies relating to the macromolecule. The combination of MD with computer graphics technology may promise a visual and molecular-level understanding of a protein. This study aims to clarify the mechanism by which the surface hydrophilicity/hydrophobicity regulates the adsorption of FN by using MD technology together with experiments. To this end, a series of self-assembled monolayers(SAMs) with various hydrophilicity was constructed by using Au-SH self-assembly method, and the amount, bioactivity and organization of FN on SAMs were detected. For MD simulation, FNIII10 was selected, and Material Studio(MS), Visual Molecular Dynamics(VMD) and NAnoscale Molecular Dynamics(NAMD) were employed to predict the effects of hydrophilicity on the adsorption sites, organization, RGD flexibility and interaction energy of FNIII10. Finally, the results from experiments and MD simulation were combined to propose the mechanism by which surface hydrophilicity regulate FN adsorption. The main works and conclusions are listed as follows:(1) Preparation of SAMs with various hydrophilicity: Gold-alkyl thiol self-assembly technology was employed to produce SAMs with various hydrophilicity, including OH-SAMs(100%-OH), 90%-OH-SAMs(-OH :-CH3 = 90 : 10), 65%-OH-SAMs(-OH :-CH3 = 65 : 35) and CH3-SAMs(100%-CH3). Their static water contact angles are 24.1±2.0o, 45.2±2.0o, 76.4±2.0o, and 105.0±2.0o, respectively. The results from X-photoelectron spectroscopy(XPS) verified the successful preparation of the SAMs as well.(2) Experimental studies: Various SAMs were incubated in FN solution of 20μg/mL at 37 oC for 2h, and the effects of surface hydrophilicity on FN adsorption were investigated.①The results from Micro BCA detection indicate that FN density on SAMs increases with increasing surface hydrophobicity, following a pattern of OH-SAMs ≈ 90%-OH-SAMs < 65%-OH-SAMs < CH3-SAMs.②The results form ELISA show that RGD activity in FN decreases with increasing surface hydrophobicity, following OH-SAMs ≈ 90%-OH-SAMs > 65%-OH-SAMs > CH3-SAMs.③AFM observations indicate that the organization of FN on SAMs tends to become more compact with increasing surface hydrophobicity. FN molecules on OH-SAMs aggregate loosely with some irregular agglomerates, while on CH3-SAMs FN molecules are the most compact.(3) The pdb files for OH-SAM and CH3-SAM were built by using MS. Thereafter, the psf files for various SAMs and FN-III10 were built by using VMD. The equilibrium simulations of FN-III10 and various SAMs were performed by using NAMD at the NVT system for 1ns and 500 ps, respectively.(4) After the equilibrium simulations, FN-III10 and SAMs were put together for further equilibrium simulations at NVT system for 1ns, providing information on RMSD(Root Mean Square Deviation) values, adsorption sites and interaction energy.① On hydrophilic OH-SAMs, hydrogen bonding is the primary interactions between FN-III10 and SAMs. With the increase of-CH3 content on SAMs, the interaction force is switching to hydrophobic interactions.② The simulated RMSD values demonstrate that the flexibility of RGD decreases with increasing hydrophobicity of SAMs. Correspondingly, the bioactivity of RGD decreases as well, following a pattern of OH-SAMs > 90%-OH-SAMs > 65%-OH-SAMs > CH3-SAMs, which is consistent with the results from ELISA.③ On OH-SAMs, FNIII10 molecules present with their major axis parallel to the SAMs. With the increase of hydrophobicity, the major axis of FNIII10 gradually rotates and finally the minor axis of FNIII10 becomes parallel to the surface on CH3-SAMs. Accordingly, FNIII10 molecules organize more compact on hydrophobic SAMs than on hydrophilic SAMs, leading to more adsorbed proteins on hydrophobic SAMs. The simulated pattern on the amount and organization of the adsorbed proteins(OH-SAMs < 90%-OH-SAMs < 65%-OH-SAMs < CH3-SAMs) is consistent with the results from Micro BCA and AFM.④ The interaction energy was shown to increase with the increasing of surface hydrophobicity, following a pattern of OH-SAMs < 90%-OH-SAMs < 65%-OH-SAMs < CH3-SAMs.(5) According to the experimental data and MD results, a mechanism for the regulation of surface hdyrophilicity to FN adsorption was proposed: Strong surface hydrophobicity promises more hydrophobic interaction sites between FN and surfaces and thus higher interaction energy, leading to restricted flexibility of FN skeletons and thus RGD sequences; the restricted RGD sequence is hard to alter its conformation to adapt cell adhesion, demonstrating reduced bioactivity. On the other hand, strong hydrophobicity promotes the arrangement of FN molecules with their minor axis parallel to the surface, leading to more compact aggregation and thus higher FN density on the surface. |
