The effect of vascular cell seeding on platelet deposition in an in vitro capillary perfusion model.

In view of the reported beneficial effects of cell seeding on the performance of synthetic vascular grafts, we determined the deposition of 111Indium-labelled human platelets in polyethylene capillary tubes covered with human endothelial cells (HEC) or smooth muscle cells (SMC) using an in vitro perfusion model. Platelet deposition decreased with increasing vascular cell coverage and was virtually absent when the number of adherent vascular cells of both types exceeded 50,000/cm2. Platelet deposition increased with increasing shear rate (300-900 s-1) only when surface coverage was less than 50,000 vascular cells/cm2. Deposition of Ca2+-ionophore A 23187-activated platelets in capillary tubes completely covered with SMC was significantly higher compared to capillaries covered with a similar number of HEC. When HEC-lined capillaries were treated with aspirin, only the deposition of activated platelets increased slightly, but significantly. This platelet reactivity was more evident when the endothelial lining was not confluent. These results demonstrate that, although seeding of HEC and SMC may both prevent the deposition of non-activated platelets to surfaces, non-aspirin-treated confluent HEC linings offer the best protection against deposition of activated platelets.

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.

Improved adhesion and proliferation of human endothelial cells on polyethylene precoated with monoclonal antibodies directed against cell membrane antigens and extracellular matrix proteins.

Endothelial cell seeding may improve the patency of synthetic vascular grafts provided that platelet reactivity of nonendothelialized sites is not increased. We have investigated if surface-adsorbed monoclonal antibodies directed against endothelial cell membrane proteins and against extracellular matrix proteins promote the adhesion and proliferation of cultured human endothelial cells, without causing platelet deposition at non-endothelialized sites. Adhesion of endothelial cells onto polyethylene coated with monoclonal antibodies directed against endothelial cell-specific membrane antigens, integrin receptors and glycoprotein CD31 was equal to or higher than adhesion onto fibronectin-coated polyethylene. Endothelial cells did not proliferate on these surface-adsorbed antibodies. However, pre-coating of polyethylene with mixtures of endothelial cell-specific monoclonal antibodies and monoclonal antibodies directed against fibronectin or von Willebrand factor, resulted in relatively high adhesion and optimal proliferation. Platelet reactivity of the polyethylene surface was found to significantly increase after adsorption of fibronectin, endothelial cell-specific monoclonal antibody or its Fc fragments. In contrast, adsorption of F(ab')2 fragments of endothelial cell-specific monoclonal antibody did not promote platelet deposition. Therefore, it is concluded that coating of vascular graft materials with mixtures of F(ab')2 fragments of monoclonal antibodies specifically directed against endothelial cells and against extracellular matrix proteins may be an effective way to both promote the growth of seeded endothelial cells and limit platelet-graft interaction.

Endothelialization of crosslinked albumin-heparin gels.

Crosslinked gels of albumin as well as heparinized albumin gels, potential sealants of prosthetic vascular grafts, were studied with regard to in vitro stability, binding of basic fibroblast growth factor (bFGF) and cellular interactions. A small percentage of the heparin present in these gels, was released during storage in SDS solution. During storage in cell culture medium at 37 degrees C, heparin release was 21-25 percent. Release of albumin did not occur. Human umbilical vein endothelial cells (HUVECs) rapidly adhered and subsequently spread on (heparinized) albumin gels, but proliferation was only observed if heparin was present in the gel. Binding of 125I-bFGF to heparinized albumin gel was 35 percent higher than to non-heparinized albumin gel. Growth of HUVECs occurred only on heparinized albumin gel loaded with bFGF and not on bFGF-loaded albumin gel. The number of platelets deposited under stationary conditions onto heparinized albumin gel was about twice the number found on nonheparinized albumin gel. Seeding of HUVECs on heparinized albumin gel, significantly reduced the number of platelets adhering to this surface. Moreover, no spreading of platelets was observed on substrates seeded with HUVECs. It can be concluded that crosslinked gels of albumin to which heparin is immobilized, are candidate sealants for prosthetic vascular grafts and suitable substrates for endothelial cell seeding.

Proteins involved in the Vroman effect during exposure of human blood plasma to glass and polyethylene.

The amounts of fibrinogen adsorbed to glass from various human blood plasmas have been measured as a function of time. The plasmas were 11 single donor plasmas, pooled plasma, a single donor high molecular weight kininogen (HMWK)-deficient plasma and HMWK-deficient plasma, which had been reconstituted with HMWK. For adsorption times between 1 min and 1 h more fibrinogen adsorbed from HMWK-deficient plasma compared with the amounts of fibrinogen which adsorbed from the other plasmas. This result supports the conclusion of several authors that HMWK is involved in the displacement of fibrinogen, initially adsorbed from normal human plasma to glass. Glass surfaces, pre-exposed to solutions of plasma and subsequently exposed to 1:1 diluted plasma, gives rise to a relatively high adsorption of HMWK which is independent of the plasma concentration of the precoating solution. The results indicate that HMWK from 1:1 diluted plasma is involved in the displacement of proteins from glass surfaces which had been pre-exposed to solutions with a low plasma concentration. Experiments with polyethylene as a substrate reveal that high density lipoprotein (HDL) from 1:1 diluted plasma is involved in the displacement of proteins from polyethylene surfaces which had been pre-exposed to solutions with a low plasma concentration. Moreover, evidence is presented that substantial amounts of albumin and fibrinogen, adsorbed from 1:1000 diluted plasma to glass and polyethylene, are displaced from the surfaces of these materials by proteins from 1:1 diluted plasma different from HMWK and HDL.

Preferential adsorption of high density lipoprotein from blood plasma onto biomaterial surfaces.

In the present study a two step enzyme immuno assay (EIA) was used for the investigation of the adsorption of proteins and lipoproteins from solutions and from blood plasma onto polymer surfaces. It was found that only a small adsorption of the major blood proteins occurred from plasma. Evidence is presented that the reason for this adsorption behaviour is a preferential adsorption of high density lipoprotein (HDL).

Adhesion of endothelial cells and adsorption of serum proteins on gas plasma-treated polytetrafluoroethylene.

From in vitro experiments it is known that human endothelial cells show poor adhesion to hydrophobic polymers. The hydrophobicity of vascular prostheses manufactured from Teflon or Dacron may be the reason why endothelialization of these grafts does not occur after implantation in humans. We modified films of polytetrafluoroethylene (Teflon) by nitrogen plasma and oxygen plasma treatments to make the surfaces more hydrophilic. Depending on the plasma exposure time, modified polytetrafluoroethylene surfaces showed water-contact angles of 15-58 degrees, versus 96 degrees for unmodified polytetrafluoroethylene. ESCA measurements revealed incorporation of both nitrogen- and oxygen-containing groups into the polytetrafluoroethylene surfaces, dependent on the plasma composition and exposure time. The thickness of the modified surface layer was approximately 1 nm. The adhesion of cultured human endothelial cells from 20% human serum-containing culture medium to modified polytetrafluoroethylene surfaces with contact angles of 20-45 degrees led to the formation of a monolayer of cells, which was similar to the one formed on tissue culture polystyrene, the reference surface. This was not the case when endothelial cells were seeded upon unmodified polytetrafluoroethylene. Surface-modified expanded polytetrafluoroethylene prosthesis material (GORE TEX soft tissue) also showed adhesion of endothelial cells comparable to cell adhesion to the reference surface. The amounts of serum proteins, including fibronectin, adsorbed from serum-containing medium to modified polytetrafluoroethylene surfaces were larger than those adsorbed to unmodified polytetrafluoroethylene. Moreover, the modified surfaces probably allow the exchange of adsorbed serum proteins with cellular fibronectin.

Platelet deposition in a capillary perfusion model: quantitative and morphological aspects.

The capillary perfusion model according to Cazenave and co-workers was characterized by investigating the effects of protein precoating, perfusion time and shear rate on platelet deposition using 111Indium labelling of human platelets and scanning electron microscopy (SEM). Compared with uncoated polyethylene, platelet deposition was increased after precoating with purified human von Willebrand factor, fibrinogen or fibronectin, and decreased by preadsorbed immunoglobulin G, albumin or whole plasma. Platelet aggregates were observed on immunoglobulin G-coated polyethylene, whereas all other surfaces showed single adherent platelets. Complete platelet spreading was only observed after precoating with fibronectin. The quantitative data concerning platelet deposition were evaluated by using the convective-diffusion theory. Our results indicate the applicability of this perfusion model for the in vitro testing of biomaterials.

In vitro leucocyte adhesion to modified polyurethane surfaces. I. Effect of ionizable functional groups.

To study the effect of ionizable functional groups on the adhesion of leucocytes to surfaces, both poly(ethyleneimine) and poly(acrylic acid) were immobilized on polyurethane films, resulting in the introduction of amine and carboxylic acid groups, respectively. This was confirmed by contact angle measurements and XPS analysis. In vitro adhesion of granulocytes and lymphocytes on untreated and modified surfaces was compared. The number of adherent cells on modified surfaces as a function of time was significantly higher than on untreated surfaces. This effect was most pronounced for the adhesion of lymphocytes to surfaces modified with amine groups. In this case, the number of adherent cells after 1 h of exposure was three times higher than on untreated surfaces. A moderate enhancement of leucocyte adhesion was observed in the case of surfaces modified with carboxylic acid groups. There is evidence that these groups were not ionized under the experimental conditions used. The modification procedures described may be used to improve polyurethane filters for the removal of leucocytes from blood.

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.

Deposition of endothelial fibronectin on polymeric surfaces.

Cellular fibronectin is deposited on tissue culture polystyrene during the adhesion and spreading of cultured human endothelial cells (HEC). Following the seeding of HEC upon this polymer, larger amounts of fibronectin are deposited as both cell density and incubation time increase. Our results indicate that the ability to deposit cellular fibronectin onto a polymeric surface is a condition for the spreading and proliferation of HEC.

Adhesion and spreading of cultured endothelial cells on modified and unmodified poly (ethylene terephthalate): a morphological study.

The in vitro adhesion and spreading of human endothelial cells (HEC) on hydrophobic poly(ethylene terephthalate) (PETP) and moderately wettable tissue culture poly(ethylene terephthalate) (TCPETP) were studied with light microscopy and electron microscopy. Numbers of HEC adhering on TCPETP were always higher than those found on PETP. When cells were seeded in the presence of serum, extensive cell spreading on both PETP and TCPETP was observed after the first 30 min. Thereafter, spread cells appeared to withdraw from the PETP surface, resulting in irregularly shaped cells. Complete cell spreading occurred on TCPETP. Complete cell spreading also occurred on PETP and TCPETP when HEC had first been seeded from phosphate buffer solution and serum was supplied after 30 min. Furthermore, HEC spread on both PETP and TCPETP when the surfaces were precoated with protein(s), which promotes cell adhesion. However, when plasma was used for the coating, spread cells did not proliferate in a monolayer pattern. This study shows that TCPETP is, in general, a better surface for adhesion and proliferation of HEC than is PETP, suggesting that vascular prostheses with a TCPETP-like surface will perform better in vivo than prostheses made of PETP.

Adhesion of cultured human endothelial cells onto methacrylate polymers with varying surface wettability and charge.

The adhesion of human endothelial cells (HEC) onto a series of well-characterized methacrylate polymer surfaces with varying wettabilities and surface charges was studied either in serum-containing (CMS) or in serum-free (CM) culture medium. HEC adhesion in CMS onto (co)polymers of hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA) was found to be optimal on the moderately wettable copolymer (mol ratio 25 HEMA/75 MMA). Positively-charged copolymers of HEMA or MMA with trimethylaminoethyl methacrylate-HCl salt (TMAEMA-Cl), both with mol ratios of 85/15 and a negatively-charged copolymer of MMA with methacrylic acid (MAA), mol ratio 85/15, showed high numbers of adhering HEC. In CM, HEC adhered onto the three charged copolymers mentioned above, but neither onto the copolymer of HEMA and MAA (mol ratio 85/15) nor onto the HEMA/MMA co- and homopolymers. Complete cell spreading in CM was only observed on the positively-charged copolymers.

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.

Interaction of cultured human endothelial cells with polymeric surfaces of different wettabilities.

The in vitro interaction of human endothelial cells (HEC) and polymers with different wettabilities in culture medium containing serum was investigated. Optimal adhesion of HEC generally occurred onto moderately wettable polymers. Within a series of cellulose type of polymers the cell adhesion increased with increasing contact angle of the polymer surfaces. Proliferation of HEC occurred when adhesion was followed by progressive flattening of the cells. Our results suggest that moderately wettable polymers exhibit a serum and/or cellular protein adsorption pattern that is favourable for growth of HEC.

Adherence and proliferation of endothelial cells on surface-immobilized albumin-heparin conjugate.

Small-diameter vascular grafts rapidly fail after implantation, due to occlusion caused by thrombosis. This problem cannot be overcome using medication. A promising improvement of graft patency is the seeding of endothelial cells (EC) on the luminal surface of the vascular graft. Conjugates of albumin and heparin, which were developed to obtain nonthrombogenic coatings, could form an ideal coating for vascular grafts. Besides presenting anticoagulant function, heparin will bind proteins with cell adhesive properties, thus facilitating adherence of EC to the graft surface. EC were able to grow to confluency on CO(2) gas plasma-treated polystyrene (PS-CO(2)) coated with albumin-heparin conjugate. CO(2) gas plasma treatment resulted in the introduction of functional groups at the surface (e.g., hydroxyl, aldehyde, carboxylic acid, and epoxide groups). Addition of albumin-heparin conjugate to the functionalized surface in an aqueous solution with pH 8.2 yielded a stable monolayer of covalently bound conjugate. The number of cells adhering and proliferating on this surface was comparable to the number of cells on fibronectin-coated PS-CO(2). However, the structure and size of EC proliferating on surface-immobilized albumin-heparin was more irregular. Long-term adherence might be improved by adding fibronectin to the albumin-heparin surface, either as a mixture with albumin-heparin or in a separate incubation step.

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.

Surface modification of copolyether-urethane catheters with poly(ethylene oxide).

Pellethane 2363 80A catheters were modified with poly(ethylene oxide) in order to improve their blood compatibility. Contact angle measurements showed that Pellethane 2363 80A surfaces had increased wettability after this modification. The results of in vitro blood compatibility tests showed that surface modification with poly(ethylene oxide) resulted in a five-fold reduction of platelet deposition. Activation of coagulation was not affected.