G.H.M. Engbers

Immobilization of heparin to EDC/NHS-crosslinked collagen. Characterization and in vitro evaluation.

In the present study, heparin immobilization to a non-cytotoxic crosslinked collagen substrate for endothelial cell seeding was investigated. Crosslinking of collagen using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) resulted in a material containing 14 free primary amino groups per 1000 amino acid residues (E/N14C). At a fixed molar ratio NHS:EDC of 0.6, the amount of heparin covalently immobilized to E/N14C increased with increasing molar ratios of EDC to heparin carboxylic acid groups (Hep-COOH), to a maximum of approximately 5-5.5 wt% at a ratio of 2. Upon incubation in cell culture medium of endothelial cells, 4 to 7% of the immobilized heparin was released during 11 days. Immobilization of increasing amounts of heparin to E/N14C progressively reduced activation of contact activation proteases. Optimal anticoagulant activity, as measured by thrombin inhibition, was obtained after heparin immobilization using a ratio of EDC to Hep-COOH of 0.2-0.4 (14-20 mg heparin immobilized per gram of collagen). Platelets deposited to (heparinized) E/N14C showed only minor spreading and aggregation, although heparin immobilization slightly increased the number of adherent platelets. The results of this study suggest that heparin immobilization to EDC/NHS-crosslinked collagen may improve the in vivo blood compatibility of this material.

Improved endothelialization of vascular grafts by local release of growth factor from heparinized collagen matrices

Endothelial cell seeding, a promising method to improve the performance of small-diameter vascular grafts, requires a suitable substrate, e.g. crosslinked collagen. In addition, the growth of seeded endothelial cells can be improved by local release of a heparin-binding protein, basic fibroblast growth factor (bFGF). In this study, the influence of immobilization of heparin to collagen, crosslinked using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS), on the binding and release of bFGF was determined. Heparin was immobilized also using EDC and NHS. Furthermore, the effects of the release of bFGF from (heparinized) EDC/NHS-crosslinked collagen on the proliferation of seeded endothelial cells was studied in vitro. Immobilization of increasing amounts of heparin to EDC/NHS-crosslinked collagen (containing 14 free epsilon-amino groups per 1000 amino acid residues, E/N14C) resulted in binding of increasing amounts of bFGF to the material. Maximal bFGF binding was observed for E/N14C containing 20-30 mg heparin immobilized per gram of collagen which was obtained using a molar ratio of EDC to heparin-carboxylic acid groups of 0.4 for heparin immobilization (E/N14C-H(0.4)). Up to concentrations of 320 ng bFGF/ml, 10% of the added bFGF bound to E/N14C, while binding of bFGF to E/N14C-H(0.4) was 22%. The initial release rate of bFGF bound to E/N14C was much higher compared to bFGF bound to E/N14C-H(0.4): respectively, 30 vs. 2% in the first 6 h. After 10 days, the bFGF release from E/N14C and E/N14C-H(0.4) amounted to 83 vs. 42%, respectively. Binding of increasing amounts of bFGF resulted in increased growth of human umbilical vein endothelial cells (HUVECs) seeded on both E/N14C and E/N14C-H(0.4). Nevertheless, after 6 and 10 days of proliferation cell numbers on E/N14C-H(0.4) where higher than cell numbers on E/N14C, irrespective of the bFGF concentration used for loading of the matrix. It is concluded that heparinized, EDC/NHS-crosslinked collagen is a good synthetic vascular graft coating for in vivo endothelial cell seeding.

Cross-linking and characterisation of gelatin matrices for biomedical applications.

Cross-linking of gelatin A and B with N,N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was optimised by varying the NHS/ EDC molar ratio at constant EDC concentration. Native and cross-linked gelatin gels were characterised using the degree of swelling, the number of free amine groups, the phase transition temperature, and titration of the carboxylic acid residues. The cross-linking reaction was most efficient at a NHS to EDC molar ratio of 0.2. At higher NHS/EDC molar ratios, the reaction of EDC with NHS becomes more pronounced, thereby reducing the effective amount of EDC for cross-linking. Swelling measurements of cross-linked gelatin gels gave deviating results when no NHS was used, which was explained by heterogeneous localisation of cross-links in the gelatin gel. The incorporation of undesired compounds into the gelatin gels during the cross-linking reaction was not observed. At optimal NHS to EDC molar ratio, gelatin A and B were cross-linked using increasing EDC/COOHgelatin molar ratios. A range of samples varying from very low cross-link density to very high cross-link density (at high EDC/COOHgelatin) was obtained. Stability of the gels is enhanced with increasing cross-link density, but a minimal cross-link density is required to obtain gelatin gels which are stable at 40°C.

Binding and release of basic fibroblast growth factor from heparinized collagen matrices

Endothelial cell seeding is a promising method to improve the performance of small-diameter vascular grafts. Growth of endothelial cells seeded on the luminal surface of synthetic vascular grafts, coated with a matrix suitable for cell seeding (e.g. collagen), can be accelerated by local, sustained release of basic fibroblast growth factor (bFGF). In this study two potential matrices for in vivo endothelial cell seeding were studied with respect to bFGF binding and release: collagen crosslinked using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), as well as heparinized EDC/NHS-crosslinked collagen. bFGF binding was determined after incubation of circular samples (10 mm diameter) with 0.25 ml bFGF solution for 90 min. Immobilization of increasing amounts of heparin, also using EDC and NHS, to crosslinked collagen containing 14 free primary amino groups per 1000 amino acid residues (E/N14C) resulted in binding of increasing amounts of bFGF. A plateau in bFGF binding was observed for heparinized E/N14C containing approximately 2.0-3.0 wt% of immobilized heparin which was obtained using a molar ratio of EDC to heparin-carboxylic acid groups of 0.4 during heparin immobilization (E/N14C-H(0.4)). At concentrations up to 840 ng bFGF/ml, 10% of the added bFGF bound to E/N14C, while binding of bFGF to E/N14C-H(0.4) amounted to 22%. Both E/N14C and E/N14C-H(0.4) pre-loaded with bFGF showed sustained bFGF release. A burst release of 30% in endothelial cell culture medium (CM) was observed for E/N14C during the first 6 h, compared to 2% release from E/N14C-H(0.4). After 28 days, the bFGF release from E/N14C and E/N14C-H(0.4) in CM amounted to 100 and 65%, respectively. Combined results of binding and release of bFGF indicate that compared to E/N14C, E/N14C-H(0.4) is the substrate of choice for bFGF pre-loading and subsequent endothelial cell seeding.

In vivo biocompatibility of carbodiimide-crosslinked collagen matrices: Effects of crosslink density, heparin immobilization, and bFGF loading

Collagen matrices, crosslinked using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (E) and N-hydroxysuccinimide (N), were previously developed as a substrate for endothelial cell seeding of small-diameter vascular grafts. In the present study, the biocompatibility of various EN-crosslinked collagen matrices was evaluated following subcutaneous implantation in rats for periods up to 10 weeks. The effects of the crosslink density, referred to as the number of free primary amino groups per 1,000 amino acid residues (EN10, EN14, EN18, or EN22), the amount of heparin immobilized to EN14, and the effect of preloading heparinized EN14 with basic fibroblast growth factor (bFGF) on the induced tissue reaction were studied. EN-crosslinked collagen was biocompatible at both early and late time intervals, and matrices with high crosslink densities (i.e., EN14, EN10) especially demonstrated a significantly decreased antigenic response when compared to noncrosslinked collagen. Furthermore, increased crosslinking resulted in a decreased degradation rate. Immobilization of heparin onto EN14 resulted in a similar to EN14 (thus without heparin) or somewhat reduced tissue reaction, but fibrin formation and vascularization were increased with increasing quantities of immobilized heparin. Matrices preloaded with bFGF also demonstrated good biocompatibility, especially in combination with higher amounts of immobilized heparin. The latter matrices [EN14 with high heparin and bFGF, thus EN14-H (0.4)F and EN14-H(1.0)F] demonstrated significantly increased vascularization for periods up to 3 weeks. Neither heparin immobilization nor bFGF preloading induced an increased antigenic response. It is concluded that the results of this study justify further evaluation of bFGF preloaded, heparin immobilized EN14 collagen, as a matrix for endothelial cell seeding in experimental animals.

In vitro and in vivo evaluation of gelatin-chondroitin sulphate hydrogels for controlled release of antibacterial proteins.

Chemically cross-linked gelatin-chondroitin sulphate (ChS) hydrogels, impregnated in Dacron, were evaluated as drug delivery systems for antibacterial proteins. The gelatin-chondroitin sulphate gels, plain or impregnated in Dacron, were cross-linked with a water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The release of lysozyme and recombinant thrombocidin (rTC-1), an antibacterial protein derived from human blood platelets, from the gelatin-ChS gels in Dacron in phosphate-buffered saline at 37 degrees C was determined, and compared to the release from gelatin gels in Dacron and plain gelatin-ChS gels. The incorporation of chondroitin sulphate into gelatin gels, caused a marked increase in lysozyme loading capacity, and a slower release rate. The relative release profiles for rTC-1 and lysozyme were equal for cross-linked gelatin as well as for cross-linked gelatin-ChS gels. Furthermore, rTC-1 showed no loss of antibacterial activity after 1 week of release. The lysozyme concentration profiles in the samples and in the surrounding medium as a function of time were calculated using mathematical solutions for Ficks second law of diffusion for a semi-infinite composite medium, which is a schematic representation of a slab in a surrounding medium. The biocompatibility and degradation of the Dacron matrices impregnated with gelatin-ChS gels was studied after implantation in subcutaneous pockets in rats. Chemically cross-linked gelatin-Ch5 gels showed a mild tissue reaction, and almost complete degradation within 18 weeks of implantation.

Endothelial cell seeding of (heparinized) collagen matrices: effects of bFGF pre-loading on proliferation (after low density seeding) and pro-coagulant factors

Endothelial cell seeding to improve the performance of small-diameter vascular grafts requires a suitable substrate, such as crosslinked collagen. In addition to providing a suitable substrate for adhesion and growth of endothelial cells, proliferation of seeded endothelial cells can be enhanced by local, sustained release of basic fibroblast growth factor (bFGF, a heparin-binding growth factor for endothelial cells). We have previously shown that collagen crosslinked using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) supports adhesion and proliferation of human umbilical vein endothelial cells (HUVECs). In the present study, HUVECs were seeded on (heparinized) EDC/NHS-crosslinked collagen, pre-loaded with bFGF. Proliferation of HUVECs on (heparinized) crosslinked collagen increased with increasing amounts of pre-loaded bFGF. The minimal cell-seeding density required for proliferation proved to be very low after pre-loading the substrates with bFGF, and was 4-fold lower for heparinized crosslinked collagen compared to crosslinked collagen (250 versus 1000 cells/cm(2)). Pro-coagulant properties (von Willebrand factor secretion and tissue factor expression) of HUVECs seeded on (heparinized) crosslinked collagen, with or without pre-loading of bFGF, were comparable to those of HUVECs on TCPS. It is concluded that heparinized, EDC/NHS-crosslinked collagen pre-loaded with bFGF is a candidate matrix for in vivo endothelial cell seeding of synthetic vascular graft materials.

In vivo compatibility and degradation of crosslinked gelatin gels incorporated in knitted Dacron

Gelatin gels were applied to porous Dacron meshes with the aim of using these gels for local drug delivery. In this article, the biocompatibility and degradation of gelatin gels with different crosslink densities applied in Dacron were studied in vivo by subcutaneous implantation in rats. Dacron discs were treated with carbon dioxide gas plasma to improve hydrophilicity, and subsequently impregnated with gelatin type B. The gelatin samples were crosslinked to different extents using various amounts of water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). After 6 h, 2, 5, and 10 days, and 3, 6, and 10 weeks of postimplantation, the tissue reactions and biodegradation were studied by light microscopy. The early reaction of macrophages and polymorphonuclear cells to crosslinked gelatin was similar to or milder than Dacron. Giant cell formation was predominantly aimed at Dacron fibers and was markedly reduced in the presence of a crosslinked gelatin coating. At week 10 of implantation, the crosslinked gelatin gels were still present in the Dacron matrix. The gelatin degradation was less for samples with the highest crosslink density. The gelatin gel with the lowest crosslink density showed clear cellular ingrowth, starting after 6 weeks of implantation. The intermediate and high crosslinked gelatin gels showed little or no ingrowth. In these gels, giant cells were involved in the phagocytosis of gelatin parts at week 10. Application of carbodiimide crosslinked gelatin gels in Dacron is suitable for medical applications because of the good biocompatibility of the gels and the possibility of adapting the degradation rate of gelatin to a specific application.

Proliferation of endothelial cells on surface-immobilized albumin-heparin conjugate loaded with basic fibroblast growth factor

Seeding of endothelial cells (ECs) on the luminal surface of small-diameter vascular grafts is a promising method to avoid occlusion of these prostheses. Immobilization of basic fibroblast growth factor (bFGF) to substrates used to coat or fill porous prostheses may enhance the formation of a confluent monolayer of ECs. Human umbilical vein endothelial cells (HUVECs) were grown on bFGF-loaded albumin-heparin conjugate bound to CO2 gas-plasma-treated polystyrene. In the order of 2-3 ng/cm2 bFGF had to be immobilized to form a confluent monolayer of HUVECs. The most prominent effect of surface-immobilized bFGF was stimulation of the proliferation shortly after seeding, resulting within 3 days in confluent cell monolayers with high density. In contrast, in cultures with 0.3 ng/mL bFGF in the medium instead of bFGF bound to the surface, it took almost a week before the cell layers reached confluency. Binding of bFGF to heparin and the biological activity of bFGF towards ECs were not influenced by the (radio-)labeling of bFGF with iodine. However, only a minor part of the bFGF used in this study displayed heparin affinity. Furthermore, degradation and multimerization of labeled bFGF in time occurred when the growth factor was stored at 20 degrees -37 degrees C. This limits the use of labeled bFGF to short-term (hours) experiments. In conclusion, bFGF loading of vascular graft surfaces through complexation of bFGF with a heparin-containing matrix probably will lead to more rapid formation of a confluent monolayer of ECs on graft surfaces upon seeding of the cells.

Controlled delivery of antibacterial proteins from biodegradable matrices

Prosthetic valve endocarditis is an infrequent, but serious complication of cardiac valve replacement. The infection is caused by the adherence of bacteria to the prosthetic valve or to tissue at the site of implantation. Recently it was shown that antibacterial peptides from blood platelets are involved in clearance and killing of bacteria adhering to vegetations induced in a model for prosthetic valve endocarditis using rabbits. The application of these antibacterial proteins in a release system, incorporated in the Dacron sewing ring of the prosthetic heart valve would diminish the incidence of endocarditis. In this study a release system for small cationic proteins based on cross-linked gelatin was developed and characterised. Furthermore, the system was evaluated with respect to the uptake and in vitro release of lysozyme, a small cationic protein that was chosen as a model protein for small cationic antibacterial proteins. Variation of gelatin type (A and B), and cross-link density resulted in differences in swelling, thermal behaviour, and number of charged groups. Lysozyme uptake was proportional to swelling, but was governed by the number of anionic groups. The latter was also observed for the release profiles: when the amount of free carboxylic acids is higher (gelatin B compared to gelatin A), the lysozyme release lasts for a longer time period. The release into solidified agarose medium, as a model for heart muscle tissue, was measured. After 50 h, 40-100% of the lysozyme was released, which is in accordance with the aimed release period of 24-48 h. The adsorption experiments in vitro suggest an influence of the electrostatic interactions between lysozyme and gelatin. This hypothesis was validated with a mathematical model which takes both diffusion and adsorption interactions into account.