1、Development of thrombus-resistant and cell compatible crimped polyethylene terephthalate cardiovascular grafts using surface co-immobilized heparin and collagen運用將肝素和膠原在運用將肝素和膠原在PET織狀移植織狀移植物表面共固化的技術，以提高其抗物表面共固化的技術，以提高其抗血栓性能血栓性能以及細胞相容性以及細胞相容性AbstractShort-term patency of polyethylene terephthalate (P

2、ET) cardiovascular grafts is determined mainly by the inherent thrombogenicity and improper endothelialization following grafts implantation.The aim of the present study was to immobilize heparin to develop thrombus resistant grafts. Additionally, collagen was co-immobilized to enhance the host cell

3、 compatibility.woven and knitted forms of crimped PET grafts of graftsSurface modifyproduce functional carboxyl groupscovalent immobilization of heparin or co-immobilization of heparin/collagenthe end-point connectCharacterized byFTIRXPSBlood coagulation testplatelet deposition testhost cell adhesio

4、n and growth testFig. 1. SEM micrographs of unmodified woven (A) and knitted (B) forms of crimped PET grafts. The multifilament PET threads in woven grafts are fabricated in an over-and-under pattern,while PET threads in knitted grafts are looped.Methods1.Surface modification of crimped PET grafts B

5、riefly, slices (2 2 cm2) from woven and knitted forms of crimped PET grafts were cleaned in 20ml of 50% ethanol solution for 15min in an ultrasonic water bath (Sonorex RK 100H, Bandelin,Germany). Subsequently, the grafts were rinsed several times with distilled water under stirring and then oven dri

6、ed at 55 C for 2 h. The cleaned grafts were dipped in 20ml of 1% NaOH solution and incubated in a boiling water bath for 1 h. The modified grafts were rinsed with distilled water and oven dried at 55 C for 2 h. The obtained grafts are abbreviated as PET-COOH.PET20ml of 50% ethanol solution，15min ult

7、rasonic water bath distilled waterstirringoven dried 55 C, 2hcleaned grafts 20ml of 1% NaOH solution boiling water bath ，1hPET-COOH2.Grafting of ethylenediamine onto PET-COOH Crimped PET-COOH were immersed in 15 ml of MES buffer (0.1 M, pH 5.5) and hydrated for about 2 h. Carboxyl group on the surfa

8、ce was activated by adding 0.1 M of EDC and 5 mM of sulfo-NHS and incubated under gentle stirring at 100 rpm/min (IKA C-MHGHS7, Germany) at room temperature for 3 h. A solution of 50mM of ethylenediamine was slowly added to the activated PET-COOH. The conjugation between one terminal amino group of

9、ethylenediamine and one activated carboxyl group on the surface of PET-COOH was completed overnight at room temperature while stirring. Afterwards, grafts were rinsed with distilled water under sonication for 5 min and dried under dust free-airflow. The obtained grafts are abbreviated as PET-NH2.3.I

10、mmobilization of heparin onto PET-NH2 Briefly, PET-NH2 (2 2 cm2)were immersed in 10 ml sodium phosphate buffer (0.1 M, pH 6.8) containing 150 l of 50% glutaraldehyde and incubated overnight at room temperature under gentle stirring at 100 rpm/min. Afterwards, grafts were rinsed with distilled water

11、under sonication for 5 min and dipped into 10 ml of heparin solution (1 mg/ml) in sodium carbonate buffer (0.5 M, pH 9.5) at 4 C. The excess aldehydes and the formed Schiff bases were reduced by adding 500 l Tris buffer (0.2 M,pH 8.5) and incubated at 4 C for 1 h. Finally, the grafts were washed wit

12、h sodium phosphate buffer and rinsed with distilled water under sonication for 5 min and dried at room temperature under dust free airflow. The obtained grafts are abbreviated as PET-Hep.4.Co-immobilization of heparin and collagen onto PET-NH2 Collagen co-immobilization was optimized using several r

13、atios of heparin/collagen at 50:50, 55:45, 60:40, 65:35, 70:30, 75:25 w/w. The immobilization of heparin and collagen was performed onto PET-NH2as previously described. Only the optimized heparin/collagen ratio at 70:30 w/w (0.7 mg/ml and 0.3 mg/ml, respectively) was considered in this work. The obt

14、ained grafts were abbreviated as PET-Hep-Col and subjected to all biological experimentsCollagenHeparinResults1. FTIR surface characterization of functionalized PET-COOH2.XPS surface analysis of PET-NH2, PET-Hep and PET-Hep-Col3.In-vitro biocompatibility of PET-Hep and PET-Hep-Col grafts3.1Platelet

15、deposition The platelet deposition on PET-Hep and PET-Hep-Col was assessed using appropriate continuous flow condition and the number of adhered platelets was calculated per unit surface area of the studied graft. The platelet deposition was significantly reduced on PET-Hep and PET-Hep-Col. The diff

16、erence between woven and knitted forms was almost insignificant P 0.05. Specifically, the platelets deposited on woven and knitted PET-Hep and PET-Hep-Col exhibited fewest pseudopodia and aggregation compared to unmodified PET. From the statistical point of view, the number of deposited platelets on

17、 both woven and knitted PET-Hep decreased by 3 and 2.8 fold compared to unmodified PET at P = 0.001. Lower platelets were deposited on PET-Hep-Col showed only 1.7 and 1.8 fold decrease in number per unite surface compared to unmodified PET at P = 0.013.2Blood coagulation testFig. 7. SEM micrographs

18、of blood clot formed on woven PET grafts. Clot formed on unmodified PET (A), PET-Hep (B), and PET-Hep-Col (C) is shown. The magnified micrographs of unmodified PET (A1) showed well-formed enormous clots accompanied by intensive fibrin fibers, while PET-Hep (B1) and PET-Hep-Col (C1) illustrated clot

19、resistance surface.3.3L929 cells adhesion and growth on PET-Hep and PET-Hep-Col graftsConclusion Immobilization of heparin on crimped PET grafts showed potential inhibition of platelet deposition and blood clot formation. The approach to immobilize heparin by the end-point provided the needful flexibilityfor optim