Immobilization Of Laminin,Heparin And SDF-1? On Ti Surface And Investigation Of The Anticoagulation/Endothelialization Promoting(Inducing)Multi-Biofunctional Layer

The thrombosis and restenosis caused by implantation of vascular stent have been become a hot point,which were considered associated with the injury of vascular intimal after stent implantation.The integrated endothelial layer was seen as the natural barrier of vascular that maintaining the balance of intravascular microenviroment and sustain the vascular patency.Therefore,to realize the rapid re-endothelialization of injured vascular is the key to solve the problem of thrombosis and restenosis.In this article,laminin(Ln),heparin(Hep)and Stromal cell-derived factor-la(SDF-la)was utilized to construct anticoagulation/promoting(inducing)endothelialiation multi-biofunction layer.Firstly,the negatively charged hydroxyl was introduced onto the Ti surface by alkali activation,then the surface was bonded by positively charged PLL based on electrostatically binding to introduce amino group,the c-terminus of Ln and Hep was activated by EDC/NHS/MES crosslink system and subsequently binding to the amino surface.Finally,the SDF-1? was added to the surface.The FTIR and XPS results indicated the the alkali activation was successful and PLL and Hep were binding onto the activated surface.The Hep release assay showed that the Hep released amount increased with the Ln concentration and there was a burst release of Hep in all test groups within 1 day.The SDF-la release result disclosed that the SDF-1? slowly release in the PBS,the total amount released during 28 days was about 5.26±1.09 ng.The AFM result showed that the roughness of alkali activated surface significantly improved.Water contanct angle result indicated that compare with Ti,the hydrophilia of co-immobilized surface enhanced and correlated with laminin concentration ratio;The Ln binding density and AT III binding ability indicated the Ln density and AT III binding amount was increasing with the increasing of laminin concentration ratio.The hemocompatibility results indicated that the amount and activation degree of platelets adhered on the Ln/Hep co-immobilized surfaces were significantly decreased compare to Ti,the L(50)H and L(100)H showed larger platelet adherent and activation while they dereased when the Ln concentration reached 150 ?g/ml,the adherent platelet number became larger when SDF-1? were added to the L(200)H surface.The Ln/Hep modified surfaces prolonged the APTT and presented remarkable effect when Ln concentration reached 150 ?g/ml,the L(200)H-SDF shortened the APTT compared with L(200)H.The amount and activation of adsorbed fibrinogen was increased among Ln/Hep co-immobilized surfaces,but seems no significant changes with the increasing of Ln concentration,in terms of fibrinogen activation ratio,the Ln/Hep samples were comparable with Ti.The cytocompatibility results indicated that the growth and proliferative activity of ECs and EPCs on the L(200)H was better than Ti surface,the cell number on the L(200)H-SDF surface increased compared with Ti surface,and the EPCs apoptosis have been alleviated while co-immobilized with Ln/Hep.After introduced the SDF-1?,the growth and proliferation of ECs were significantly improved while didn't show significant difference on the growth and proliferation of EPCs compare with L(200)H.The migration of ECs and the chemotaxis of EPCs were significantly promoted on the L(200)-SDF surface than on Ti surface.The growth and proliferation of SMCs were inhibited by Ln/Hep co-immobilized surfaces,among the modified samples,L(50)H,L(150)H and L(200)H showed the most obvious inhibitory effect while L(200)-SDF didn't show significant difference with L(200)H.The research of mechanism indicated that the the characteristics and properties of bio-functional layer on the biomaterial surface has significant effect on the surface anticoagulation/promoting(inducing)endothelialization.Except for the surface roughness,hydrophilia and charge,the molecule species,molecular activity,molecular loading quantity and interactions between molecules will sifnificantly influence cells and blood.Large number of negative charged-OH was introduced to the surface and the roughness increased obviously,the adhesion of platelets and cells were inhibited.After bonding PLL to the surface,the strong negative surface became weaker while the introduction of-NH3 caused highly cytotoxicity.The coimmobilized samples with different Ln/Hep concentration ratio resulted in different biocompatibility effect due to the bonding amount of Ln and Hep,as well as Hep activity.In conclusion,this paper constructed a favourable anticoagulation/promoting(inducing)endothelialization surface through bonding of Hep,Ln and SDF-1?,which provides effective reference for subsequent modification of cardiovascular device.