| Owing to the emergence of stent thrombosis (ST), in-stent restenosis (ISR) and other clinical complications after the stent implantation, the modification of cardiovascular stent surface have gradually changed from single-molecule, single-functional designs to multi-molecule, multi-functional ones. Based on the theories that endothelial cell layer has good property of natural anti-coagulation and endothelialization on material’s surface is absolutely necessary for the cardiovascular stent, the surface modification pursuing both the anti-coagulation function and promoting endothelialization micro-environment becomes a new hotspot. However, there still exist some problems that need to resolve, for example, the design of the micro-environment including the selection of different functional biomolecules, the restriction and equilibrium between two functional biomolecules. In this paper, heparin, acting as anticoagulant, was used as the target molecule to construct a bifunctional micro-environment of anticoagulation/promoting endothelialization on the Ti-based material surface, combining with the promoting endothelialization molecule type IV collagen. Series of characterization tests, optimization as well as in vitro and in vivo animal tests were carried out to evaluate the properties of the multi-functional layer.Firstly, negatively charged surface is produced with hydroxyl reactive groups on the Ti-based materials after alkaline activation, then large number of amino groups are introduced to the surface by assembling a polylysine media layer or silanizing with APTES. Then the biological modification layer, type IV collagen/heparin biological modification layer, was obtained based on the former layer through coating two different molecules, type IV collagen and heparin. Some quantitative and qualitative characterizations, such as FTIR, SEM, AFM, the water contact angle test, the immunofluorescence staining and quartz crystal microbalance with dissipation (QCM-D) were carried out, it was indicated that heparin and type IV collagen can be fixed on the Ti-surface through layer-by-layer electrostatic self-assembly. The experimental parameters results of process optimization indicate that the optimum parameter are assembling a polylysine media layer, the heparin concentration is5mg/mL, the concentration of type IV collagen is25μg/mL, the optimal number of double layer is15, and the best reaction time of heparin and type IV collagen is6minutes and25minutes respectively. The in vitro static results show that the bionic layer has anticoagulant properties and good cytocompatibility, on which ECs and EPCs could be promoted to adhere, grow, proliferate and migrate. The in vitro dynamic blood tests show that the bionic layer has a poor performance of hemocompatibility, on which thrombosis can easily happen, indicating there exist the competition and balance effects between heparin and type Ⅳ collagen with two different functions in different environment. The in vivo animal experiment results manifest that the bionic surface can encourage new endometrial growth which has more endothelialization but thicker endothelial neointimal, this maybe related to that IV collagen belongs to non-selective biomolecule and could promote the migration and proliferation of both smooth muscle cells and other cells.Moreover, the interaction and the competition-equilibrium mechanisms of heparin and type IV collagen were also studied. It was found that during the competition of anticoagulant and procoagulant, heparin prevent thrombosis mainly via endogenous coagulation pathway, and reduce platelet binding through the charge repulsion effect. In addition, the heparin also can inhibit the expression of GPⅡ b/Ⅲa on the surface of the prophase platelets. While the type Ⅳ collagen play a role on promoting clotting factor XII self-activating then resulting in starting extrinsic coagulation pathways, and binding with the platelet receptors VWF-A3, α2β1and Glycoprotein Ⅵ through collagen sequences PGQOGVMGF, GFOGER and (GPO)x leading to facilitating platelet adhesion and activated, thereby causing the degeneration of fibrinogen to fibrin. In the competition of inhibiting and promoting endothelialization, fragment3(CB3(Ⅳ)) on type IV collagen, constituted of amino acid residues of peptide chain, show the advantage that could promote the cell proliferation by binding with the integrate receptors α1β1and α2β1on the endothelial cell surface. On the contrary, heparin represents a role of inhibiting the proliferation of endothelial cells through the ODC gene expression mediated by the select protein kinase C (PKC) which could be prevented by glycosaminoglycan on the heparin. In addition, compared to type IV collagen with the positive charges, the heparin with the negative charges possesses opposite effect on the endothelial cells which bear negative charges on the surface. Ultimately, heparin and type IV collagen could achieve a relative equilibrium condition with the competitions during the extrinsic coagulation pathway, platelet adhesion and endothelial cell adhesion and proliferation.Finally, due to the type IV collagen in this work also promotes the smooth muscle cells adhesion and growth, GREDVY polypeptide which has the endothelial cells selectivity adhesion property was chosen to construct the GREDVY polypeptide/heparin bionic layer. FTIR, AFM, the water contact angle test, and the immunofluorescence staining etc. were also used to characterize the GREDVY polypeptide/heparin bionic layer. It was shown that heparin and GREDVY polypeptide can be jointly fixed by electrostatic binding on Ti. The in vitro static experiments, including the platelet adhesion experiments, APTT test, cell culture experiments, competitive binding experiments of endothelial cells and platelets, were used for hemocompatibility and cytocompatibility contrastive evaluation, and the evaluation of competitive adhesion and growth experiments of endothelial cells and smooth muscle cells on Ti and GREDVY polypeptide/heparin bionic layer. The results showed that GREDVY polypeptide/heparin bionic layer not only represents a good hemocompatibility and promoting endothelial cells adhesion, but also inhibits smooth muscle cells adhesion and proliferation obviously.In short, it is feasible to construct a dual-function or multifunction surface using the biological molecules with different functions. The cross-distribution of the two biomolecules on the outer layer may provide the realization of the dual-function or multifunction. But the implementation of the multi-function is not a simple sum of several functions, there is a competition and balance. The competition and balanced relationship will be adjusted with the changes of external biological environments, and a new balance can be achieved after a new round of competition. Progress in this work provides an important reference for surface microenvironment building on the artificial materials contact the blood, including the selection and design of biological molecules, which opens up new ideas for the construction of multi-function micro-environment on the cardiovascular scaffold surface. |
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