Artificial Heart Polymer Surface Covalent Binding Of Heparin And Its Biocompatibility

in 10wt% TDI-benzene solution, containing 0.5wt% DBTDL as a catalyst, and then were mechanically rotated for 48h to activate the surfaces.1.1.2 Amplification of Reactive Moieties and Introduction of Spacer onto the SurfacesPolyfunctional polymers (PEP), including PVA and PEI, and spacer PEO were coupled to PU/PVC surfaces through condensation reactions between the -NCO group on PU/PVC surface and -OH (PVA, PEO), or -NH- (PEI) groups on the PFF.Modified PU-NCO/PVC-NCO surfaces were placed in 10wt% PEI/benzene solution (or 10wt% PVA-DMSO solution) , containing 0.5wt% DBTDL as a catalyst. The reaction proceeded for 24h at 60 ℃ (with mechanical mixing). Thus PEI or PVA was attached onto the surfaces. Said surfaces were modified with TDI again, followed by introduction of spacer PEO to obtain single-amplified surfaces. Said single-amplified surfaces were modified once again with polyfunctional polymers and the spacer, repeating the above mentioned processes so as to obtain double-amplified surfaces.1.1.3 Covalent Immobilization of Heparina. Covalent immobilization of heparin by ester bonds2g heparin was dissolved into 100ml formamide with its pH adjusted to about 1 by dehydrodrated sulfuric acid. Single-amplified or double-amplified PU/PVC surfaces were then placed into the above solution to react for 24 hours. Heparin molecules were then covalently attached onto the surfaces by esterification reaction between free -OH groups on PU/PVC surfaces and free -COOH on heparin.b. Covalent immobilization of heparin by urethane bondsLarge amount of -NH2 were introduced onto the single-amplified or double-amplified surfaces. Heparin molecules were then covalently immobilized onto above surfaces via urethane reactions between -NH2 groupson above surfaces and free -COOH on heparin. The process was as following: PEO was reacted with TDI, followed by reaction with diaminoethane to obtain NH2-PEO-NH2, which was then reacted with single-amplified or double-amplified surfaces to introduce -NH2 onto the surfaces.2g heparin was dissolved into 100ml formamide with its pH adjusted to about 1 by dehydrodrated sulfuric acid, followed by addition of diamino terminalized PEO and dripping of SOCI2, so as to obtain covalently heparinized surfaces via urethane bonds.c. Covalent immobilization of heparin by -NCO reaction Single-amplified or double-amplified surfaces were reacted with TDI, followed by addition of heparin/ formamide solution( 2g/100ml) with its pH adjusted to about 1 by dehydrodrated sulfuric acid, resulting in the covalently heparinized surfaces via condensation reactions between -NCO group and -COOH or -OH of heparin.1.2 FT-IR Spectroscopic AnalysisSurfaces obtained in varied processing stages were dried and then cut into pieces of 0.5cm2~1.0 cm . Prepared samples were then analyzed by AVATAR 360 FT-IR spectrum analyzer and the resulting spectra were compared with standard spectra to make sure that single-amplification or double-amplification processes succeeded, and that heparin molecules were covalently attached onto the surfaces.1.3 Quantificational Determination of Immobilized Surface Heparin Content by Colorimetric AnalysisImmobilized surface heparin content was quantificationally determined by toluidine blue colorimetric method. The absorbance of toluidine blue solution of varied surfaces was measured at 630 run and compared with standard curve representing standard concentrations of heparin, so as to indirectly obtain the surface heparin content.1.4 Statistic ApproachVariance analysis and t-test were carried out to statistically analyze the results of surface heparin content, which were denoted by mean ± standard deviation ("x±s) . PO.05 represented prominent deviation and P<0.01 represented non-prominent deviation. 2 Results2.1 AVATAR 360 FT-IR spectra of surfaces in varied processing stages showed that after surface activation, peaks at 2271 cm'1 could be observed, which was the characteristic absorbing peak of -NCO, indicating that -NCO group was attached onto the surfaces; after single-amplification and double-amplification, peaks centralized at 3400cm"1 ~ 3000cm"1 could be observed, which were the characteristic absorbing peaks of PVA> PEI and PEO, indicating the successful amplification of the surfaces; after heparinization, peaks at 3500cm" could be observed, which were the characteristic absorbing peak of heparin, indicating that heparin molecules were covalently immobilized onto the surfaces. AVATAR 360 FT-IR spectra of outcomes of esterification reaction showed peaks at 3040.26 cm'1, 1111.30 cm"1 and 1692.72 cm"1, which were characteristic absorbing peaks of ester, indicating that heparin molecules were covalently attached onto the surfaces by ester bonds; spectra of outcomes of urethane reaction showed peaks at 1690-1650 cm"'( y oo), 1650-1620 cm''( 8 NH), 3500-3050 cra"'( y Nh) and 1400 cm"'( y c-n and 8 NH), which were characteristic absorbing peaks of urethane, indicating that heparin molecules were covalently attached onto the surfaces by urethane bonds.2.2 The quantificational assay of the surfaces showed that compared to other methods, high surface heparin content had been achieved in heparinizedsurfaces by urethane reaction, ester reaction and -NCO reaction, with surface heparin content varying from 1.660ug-cm"2 to 18.869ng-cm"2. Without modification with polyfunctional polymers (PVA or PEI) and spacer PEO, surface heparin content was nearly 0. After the introduction of spacer PEO, small amount of heparin could be immobilized onto the surfaces. After the single-coupling of polyfunctional polymers (PVA or PEI) and spacer PEO, surface heparin content increased markedly (p<0.05), while after the double-coupling of polyfunctional polymers (PVA or PEI) and spacer PEO, .surface heparin content .increased, dramaticaly (p<0.05). Among the three methods, surface heparin content of heparinized surfaces by urethane bonds was the highest, while surface heparin content of heparinized surfaces by ester bonds and -NCO reaction were a bit lower.Part II Reaseach of Biocompatibility and Anticoagulation Activity of Heparinized SurfacesStability and anticoagulation activity of the above three heparinized surfaces were analyzed, so as to make sure that thus prepared surfaces could meet the requirements of long-term clinical application of artificial heart. Heparinized PU and PVC surfaces were assayed via in vitro and ex vivo experiments, and their biocompatibility evaluated, according to National Standard GB/T 16886.5-2000—Biocompatibility of clinical devices.1. Experimental1.1 Stability and Anticoagulation Activity of heparinized surfacesTo examine the stability of immobilized heparin, several heparinized surfaces were immersed in saline for varied times (Id, 3d, 5d, 7d, 15d, 2 Id, 28d) , and the amount of heparin released from the substrates was measuredaccording to toluidine blue method. Anticoagulation activity of heparinized surfaces after long time immersing was anlyzed by PT and APTT assay.1.2 Hemolysis test of heparinized surfacesHeparinized surfaces were immersed in saline by a proportion of lcm2:10ml, so as to obtain extracts. 72h later, 10ml extracts were diluted with 0.2ml fresh rabbit blood, centrifuged for 5min at lOOOr/min, then absorbance of extracts at 545nm was measured, taking distilled water as the positive reference and 0.9% NaCl as the negative reference. Hemolysis rate ( %) = Absorbance of sample - Absorbance of negative reference/Absorbance of positive reference - Absorbance of negative reference x 100%.1.3 Platelet Adhesion Test of heparinized surfaces10ml platelet blood extracted from human blood was diluted with calf blood serum to a concentration of 1.0 x 109/L, and then injected into the wells of cell cultivation board with heparinized materials immersed in it. The board was put into a CO2 incubator for 2h at 37°C, followed by rinsing, drying and surface gold painting, and was then scanned under S-520 SEM.1.4 Direct Contact GytotoxHy of Heparinized SurfacesHeparinized materials were cut into pieces of 0.7 cm*0.7 cm. Then the materials was cultivated with eugonic L-929 cells. By observing the status of L-929 cells, direct contact cytotoxity of heparinized material could be evaluated.1.5 MTT testAccording to the 12th part of National Standard—Biocompatibility of Clinical Devices, extracts of varied heparinized surfaces were prepared. L-929 cells were first inoculated into a 96-well board at a density of 104, each with a volume of 200ul. Then 80ul extracts with varying concentrations and controlliquid were added into each well. Day by day OD value was measured by BIO-RAD 550 ELISA machine. Cell growth curve was drew..1.6 Local Response After Muscle ImplantationAccording to the 6th part of National Standard—-Biocompatibility of Clinical Devices, heparinized materials were cut into pieces of 0.3 cmxl.O cm. Then heparinized materials were implanted into the vertebral column muscle of rabbits. After 1,4, 12 weeks, HE dyed specimen were made and local response after muscle implantation were evaluated with microscope.2 Results2.1 During the first 72h, small amount of heparin molecules could be observed to release from heparinized surfaces, while after that, little could be observed to release from the surfaces, and heparin releasing curve ran to a parallel line.2.2 PT and APTT time of heparinized surfaces increased dramatically. After 28d immersing in saline, anticoagulation activity of heparinized surfaces still remained well.2.3 Hemolysis rate of almost all heparinized surfaces were lower than 5%, except for the heparinized surfaces via -NCO reaction, which hemolysis rate exceeded 5%.2.4 Platelet adhesion test showed that without heparinization, large amount of round or elliptical platelets could be observed on PU and PVC surfaces, indicating a high platelet adhesion rate, while after covalent heparinization, net-like structure and branch-like structure were observed on the surfaces, and the amount of the blood platelet attached decreased markedly.2.5 The results of MTT test were in accordance with the results of direct contact cytotoxicity, indicating that no obvious cytotoxicity was observed except for the outcomes through-NCO reaction, namely, A5 > A6^ B5 > B6.2.6 The results of local response after muscle implantation showed that the inflammation response of heparinized PU and PVC surfaces via -NCO reaction was serious, with edema of striated muscle fibers, rupture of muscle fibers and large amount inflammation cells, while the inflammation responses of other heparinized surfaces were light, with only small amount inflammation cells.Results1. Heparinized PU and PVC surfaces were achieved by activation of PU and PVC surfaces, introduction of intermediate, such as polyfunctional polymers PEO and PEI and spacer PEO, so as to amplify reactive moieties onto the surfaces as well as covalent heparinization. AVATAR 360 FT-IR sprctroscopic analysis indicated the success of surface activation, single-amplification and double-amplification, and that heparin molecules were covalently attached onto the surfaces via ester bonds, urethane bonds and -NCO reaction.2 After single modification with polyfunctional polymers (PVA or PEI) and spacer PEO, surface heparin content increased markedly, while after double-coupling of polyfunctional polymers (PVA or PEI) and spacer PEO, surface heparin content increased even more dramaticaly. Among the three methods, surface heparin content of heparinized surfaces by urethane bonds was the highest, while the surface heparin content of heparinized surfaces by ester bonds and -NCO reaction were a bit lower. High surface heparin content...