Preparation And Biological Evaluations Of Bio-functionalized Collagen Coatings

Bio-functionalized coating is the promising candidate to regulate the interactions between metallic implant and cells/tissues by activating the implant surface,then promote the osseointegration.However,it is still a challenge to realize the rational design of bio-function and controlled preparation of coatings availably.This work focuses on the design of coatings according to the bio-functions that are required in the osseointegration process,which is relied on the key factors affecting cell growth.Collagen,the major extracellular matrix,was taken as the primary deposition component and was deposited onto the metallic implant surface to form the coatings with bio-functions of protein sustained release,high cell affinity and magnetically assisted mechanical stimulation by different electrochemical deposition methods.In addition,the characterizations and in vitro cell assays were performed to evaluate the bio-functions of coatings.Moreover,the regulation mechanisms of coatings on cells were discussed,and the research achievements mainly contain the following four aspects:1.Controllable electrodeposition of collagen coatingsIn situ constant potential electrodeposition method.Here the bovine serum albumin(BSA)was successfully incorporated into the collagen coating to form the BSA/collagen composite coating.The mineralization degree and BSA content(17-173 ng/cm2)could be regulated by the BSA amount in the electrolyte,mixing sequences of the electrolyte and deposition potentials.Alternating potential electrodeposition method.In this work,a collagen coating with multi-layers(1-4 layers)consists of weakly mineralized layer and highly mineralized layer alternatively was prepared via alternating potential electrdeposition method by programmatically setting the positive and negative potentials(1-7 segments).Compared to constant potential electrodeposition method,it is an effective way to reduce the mineralization degree and improve the adhesion strength of the coating(1.25-fold).A magnetic collagen coating,which was incorporated with different distributed iron oxide nanoparticles(inner/outer located or homogeneously located),was also deposited onto the substrate by alternating potential electrodeposition method with controllable saturation magnetization(Ms=0-7.303 emu/g).Magnetically assisted electrodeposition method.The collagen fibrils were magnetized by IOPs and orientationally deposited onto the substrate under magnetic field.The collagen fiber diameter(300 nm-2 ?m)and orientation degree could be regulated by mass ratios of IOPs and collagen,also the magnetic field intensity during deposition and drying process.2.Collagen coating with protein sustained releaseThe release behavior of bioactive molecular is affected by their locations,and prolong the release path can inhibit the burst release.As a model protein,BSA was inner located into the collagen coating by in situ constant potential electrodeposition method and the release behavior of BSA,as well as the biological effect was evaluated.The BSA release behavior exhibited a two-stage release:an initial slow stage and a subsequent fast stage.Compared to the collagen coating,the BSA/collagen coating well supported the cell proliferation on it.The cellular proliferation is positive correlation with the BSA loading,which indicates the BSA released from the coating can synergize collagen to maintain the cell viability.Therefore,loading BSA into the coatings could enhance cellular adhesion and maintain the cell viability on the coatings,which may be beneficial to the further bone integration.3.Collagen coating with high cell affinityHigher binding sites and ordered structures on the surface are benefical to the cell affinity of the coating.The low mineralized collagen coating was prepared by alternating potential electrodeposition method and compared to the highly mineralized surface,cells on the coating with lower mineralization degree presented larger spreading area and more pseudopodia because more binding sites were exposed for recognition,which supported cell adhesion and proliferation and enhanced the cell affinity(1.6-fold).In the case of low mineralization,the highly oriented collagen could further increase the affinity of the cells to the surface of the collagen coating,exhibiting better cell adhesion and proliferation results.This was because the cells arranged in the orientation of the coating surface due to the contact-guided effect,which resulted in the better skeleton development,thicker actin filaments,and better spread state,demonstrating the higher cell affinity(1.7-fold).4.Collagen coating with magnetically mechanical stimulationThe swollen magnetic collagen coating deforms under the stimulation of an external magnetic field and mechanically stimulated the cells on it.The random and aligned magnetized collagen coating were prepared by alternating potential electrodeposition method and magnetically assisted electrodeposition method,respectively.When the magnetic field acted on the random magnetized collagen coating,the cells showed the highest ALP activity,indicating that the cells could sense and respond to the mechanical stimuli produced by the coating deformation.The mechanical stimuli were related to the content of IOPs in the coating.When the ratios of magnetic particles and collagen in the electrolyte was 0.67,the coating showed the best ability to induce osteogenic differentiation.Placing the IOPs on the outer layer of the coating could further enhance the mechanical stimulation and significantly promote the osteogenic differentiation of the cells.When magnetic fields with specific directions are applied to the aligned magnetized collagen coating,the cells sensed mechanical stimuli in different directions.When the direction of the magnetic field coincided with the orientation of the collagen(0°),the cells exhibited the highest degree of alignment along the collagen fibers,with the greatest osteogenic differentiation.While the direction of the magnetic field was perpendicular to the orientation of the collagen,the osteogenic differentiation was impaired to some extent.