Relation between cell density and the secretion of von Willebrand factor and prostacyclin by human umbilical vein endothelial cells.

In this study, the relation between the density of human umbilical vein endothelial cells (HUVECs) cultured on TCPS and (crosslinked) collagen, and the secretion of von Willebrand factor (vWF) and prostacyclin (PGI2) was determined. Collagen was crosslinked using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in combination with N-hydroxy-succinimide (NHS), resulting in a matrix containing 14 free primary amino groups per 1000 amino acid residues after crosslinking (E/N14C). HUVECs were seeded on E/N14C, non-crosslinked collagen (N-Coll) and fibronectin-coated TCPS at densities ranging from 2500 to 50,000 cells/cm2. After 1 day of culture, both basal and A23187-stimulated secretion of vWF (expressed per 1,000,000 cells) was considerably increased at low cell densities (i.e. below 5000 cells/cm2) on all substrates. Secretion of PGI2 gradually increased with decreasing cell densities below 10,000 cells/cm2. After 10 days of proliferation, cell numbers on all substrates exceeded 50,000 cells/cm2, irrespective of the seeding density. Concomitantly, the initial higher secretion of PGI2 and vWF at the lowest seeding densities was decreased after longer times of culture, to values comparable to those obtained for higher seeding densities.

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.

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.

Endothelial cell seeding on crosslinked collagen: effects of crosslinking on endothelial cell proliferation and functional parameters.

Endothelial cell seeding, a promising method to improve the performance of small-diameter vascular grafts, requires a suitable substrate, such as crosslinked collagen. Commonly used crosslinking agents such as glutaraldehyde and formaldehyde cause, however, cytotoxic reactions and thereby hamper endothelialization of currently available collagen-coated vascular graft materials. The aim of this study was to investigate the effects of an alternative method for crosslinking of collagen, using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS), on various cellular functions of human umbilical vein endothelial cells (HUVECs) in vitro. Compared to non-crosslinked type I collagen, proliferation of seeded endothelial cells was significantly increased on EDC/NHS-crosslinked collagen. Furthermore, higher cell numbers were found with increasing crosslink densities. Neither the morphology of the cells nor the secretion of prostacyclin (PGI2), von Willebrand factor (vWF), tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) was affected by the crosslink density of the collagen substrate. Therefore, EDC/NHS-crosslinked collagen is candidate substrate for in vivo application such as endothelial cell seeding of collagen-coated vascular grafts.

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.

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.

TGF-beta and bFGF affect the differentiation of proliferating porcine fibroblasts into myofibroblasts in vitro.

Fibroblasts and myofibroblasts are involved in the foreign body reaction to biomaterials, especially in capsule formation. However, contraction or detachment of the capsule can lead to complications. Biocompatibility of biomaterials may be improved by the application of proteins regulating the differentiation or activation of (myo)fibroblasts. Myofibroblasts, differentiating from fibroblasts can be identified by the expression of alpha-smooth muscle actin (alpha-SM actin). We investigated the influence of proliferation and quiescence on the differentiation of porcine dermal cells and whether transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (bFGF) are involved in the differentiation of proliferating cells. Porcine cells were used because pigs increasingly function as in vivo models while little is known of the characteristics of their cells. Serum-free cultured, quiescent fibroblasts differentiated into myofibroblasts, while proliferating fibroblasts cultured in the presence of serum containing TGF-beta, formed alpha-SM actin-negative cell clusters. After reaching confluency, these clusters started to expressing alpha-SM actin. Moreover, these proliferating cells produced TGF-beta from day 4 onwards while bFGF did not. Differentiation into myofibroblasts was inhibited by bFGF and to an even greater extent by antibodies to TGF-beta. Further, two theories concerning the role of the myofibroblast in tissue contraction in view of two biomaterial application will be discussed.