NL1008724C2 - Production of an artificial blood vessel comprising porous plastics tube coated with collagen material - Google Patents

Abstract

Production of an artificial blood vessel for implantation into a recipient comprises coating a porous plastics tube with a collagen material and crosslinking the collagen material with a compound capable of forming peptide bonds, the degree of crosslinking being such that 8-20 out of 27 free amino groups per 1000 amino acids are not crosslinked.

Description

NL 43433-Al / ho

Method for preparing a plastic blood vessel

The present invention relates to a method of preparing a plastic blood vessel for implantation in a receiver, the method comprising the steps of i) applying a collagen material to a porous tubular plastic support; and ii) cross-linking the collagen material.

Plastic blood vessels intended for implantation should be accepted as best as possible by the recipient's body. An important point here is that the plastic blood vessels are stable, i.e. the mechanical properties are maintained over time, and do not cause blood to clot, which could lead to blockage of the vessel or loose clots in the bloodstream. Collagen 15 is known to be a very thrombogenic material, but it is known in the art to crosslink a collagen with glutaraldehyde to yield collagen with reduced thrombogenicity.

The present invention aims to provide a method according to the preamble which gives a reduced thrombogenicity and an improved stability of the collagen material.

To this end, the method of the present invention is characterized in that the crosslinking of the collagen material takes place using a peptide bond promoter, the degree of crosslinking being chosen such that of the 27 free amino groups per thousand amino acids 8 to 20 amino groups are not modified in the peptide bond-comprising cross-linking.

In in vivo experiments, it has been found that thus prepared collagen materials exhibit improved stability.

In the present application, the term collagen material includes collagen, heat-treated collagen (gelatin) as well as fragments thereof obtained by chemical, physical or enzymatic cleavage. In particular, chemical cleavage includes partial hydrolysis, for example with acid.

Suitable collagen derived from bovine Achilles tendons has 27 free amino groups per 1000 amino acids. Although such bovine collagen can be used advantageously in the method of the present invention, collagen material from other sources can also be utilized. The number of free amino groups may differ slightly. As a result, the range defined in the characteristic does not change substantially. The free amino groups are the amino groups contained in the side chain of (hydroxyl) lysine.

Preferably, 12 to 16 amino groups are not involved in the peptide bond-comprising cross-linking.

In this range, it has been found that the inflammatory response upon implantation in vivo is significantly milder, given the lower neutrophil count of granulocytes, macrophages and giant cells (macrophages that phagocytose a collagen).

The peptide bonding agent is suitably a carbodiimide compound, preferably a water-soluble carbodiimide compounds and suitable N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide (EDC).

The collagen material can thus be cross-linked in a simple manner.

In addition to the carbodiimide, a succinimide compound is advantageously also present, such as suitably an optionally water-soluble N-hydroxysuccinimide compound.

The formation of peptide bonds can be promoted by the use of such an auxiliary compound.

According to a very favorable embodiment, the collagen material is provided with a blood-clotting inhibiting polysaccharide, such as heparin, heparan sulphate and dextran sulphate.

The chance of the formation of clots 35 is thus further reduced.

Advantageously, the blood coagulation inhibiting polysaccharide after the second step ii) is covalently linked to the amino groups not involved in a peptide bond after cross-linking, in particular by means of a peptide bonding agent 10087243.

Thus, suitable use can be made of the free amino groups remaining after the partial cross-linking of the collagen.

According to a preferred embodiment of the method according to the invention, the polysaccharide is reactivated, yielding a polysaccharide reactive with collagen material, before being contacted with the collagen material.

By carrying out the cross-linking in two steps and providing the polysaccharide, the properties of the plastic blood vessel can be better controlled compared to a one-step procedure.

In order to promote that cells forming part of the wall of a natural blood vessel, in particular endothelial cells, cover the plastic blood vessel as quickly as possible, and thereby further reduce the chance of clotting, the method according to the invention advantageously turns into a growth factor especially a polysaccharide-binding growth factor, such as bFGF, aFGF or VEGF.

bFGF (basic Fibroblast Growth Factor), aFGF and VEGF are a growth factor that promotes the proliferation of (seeded) endothelial cells. aFGF is acidic Fibroblast Growth 25 Factor and VEGF Vascular Endothelial Growth Factor. By using a growth factor binding the polysaccharide, a release of the growth factor can be easily achieved where this growth factor is required. The polysaccharide is suitably selected from the group consisting of heparin, heparan sulfate, chondroitin sulfate, hyaluronic acid and dextran sulfate.

According to a further favorable embodiment, the plastic blood vessel is inoculated in a last step with endothelial cells compatible with the body of the recipient, such as autologous endothelial cells.

The present invention will now be elucidated by means of the following exemplary embodiments.

EXAMPLE I

1. Preparation of collagen.

1008724 1 '4

Type I insoluble collagen from bovine Achilles tendon (Sigma, St. Louis, Missouri, USA, C 99879, portion 23H7065) was allowed to swell overnight in 0.52 M acetic acid (1 g collagen / 50 ml) at 4 ° C . The mixture was dispersed for 4 minutes along with 50 g of crushed ice in a Philips mixer and then homogenized at 4 ° C for 30 minutes using an Ultra-Turrax T25 (IKA Labortechnik, Staufen, Germany). The resulting slurry was filtered through a number of filters (Cellector 10 screen, Bellco, Feltham, UK), with a pore size decreasing from 140 μηι to 10 μτη.

2. Coating plastic blood vessel with collagen.

The filtered suspension obtained above was deaerated at a pressure of 0.06 mbar. The collagen was introduced into the lumen of plastic blood vessels (Double velor Dacron or Cooley knitted Dacron, Meadox Medicals, Oakland, NJ, USA) using a syringe and under slightly elevated pressure, the collagen suspension was passed through the porous wall of the plastic blood vessel squeezed.

Instead of Dacron, the plastic blood vessel can also be made from a plastic selected from the group consisting of i) polyurethane; ii) PTFE; iii) polyethylene tephthalate; and iv) polylactic acid, wherein the plastic is optionally subjected to a treatment known in the art to render it hydrophilic before being covered with collagen.

Excess collagen was removed from the lumen of the blood vessel and the plastic blood vessel thus treated was allowed to dry at room temperature. After repeating this procedure 5 times, the collagen-coated blood vessel thus was impermeable to water as determined according to ISO-DIS 7198-1 (Technical Committee Iso-Tc, Cardiovascular implants-Tubular vascular prostheses. Part 1: Synthetic vascular prostheses, Iso-Dis 7198-1, biz. 1 - 52, 1990). SEM analysis 35 showed complete coverage of the surface of the plastic blood vessel with collagen.

3. Cross-linking of collagen applied to a plastic blood vessel.

The collagen was cross-linked using N-1 1008724 (3-dimethylaminopropyl) -'-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS). The dried collagen-coated plastic blood vessels were washed in a 0.05 M 2-morpholinoethanesulfonic acid buffer (MES buffer, pH 5.40) for 1 hour. Then, the plastic blood vessels were immersed in a solution of EDC and NHS in MES buffer, and cross-linking was performed with gentle shaking. Per gram of collagen, 1.731 g of EDC and 0.415 g of NHS in 215 ml of MES buffer were used. After 4 hours, cross-linking was stopped by washing with a 0.1 M Na 2 HPO 4 solution for 2 hours.

These treatments resulted in both hydrolysis of activated carboxylic acid groups and hydrolysis of residual amounts of EDC. Then it was washed 4 times for 30 minutes with deionized water.

4. Determination of the number of free e-amino groups per 1000 amino acid residues.

The number of free primary amino groups remaining in the product obtained in section 3 was determined using 2,4,6-trinitrobenzenesulfonic acid (TNBS, Fluka, 20 Buchs, Switzerland) according to a method slightly adapted by Wang et al. (Ref. 1). , 2, 3). Reaction of TNBS with primary amino groups leads to the formation of trinitrophenyl-lysine residues. Samples of approximately 4 mg (cross-linked) collagen were incubated with 1.0 ml 4 w / v% NaHCO 3 solution at room temperature for 1 hour. Then 1.0 ml of a 0.5 w / v% TNBS aqueous solution was added and the mixture was heated at 40 ° C for 2 hours. Then the collagen samples were hydrolyzed by incubation in 3.0 ml 6 MHCl at 60 ° C for 90 minutes. The absorbance was measured after addition of 5 ml of water and cooling to room temperature at 345 nm. A molar absorption coefficient of 14,600 l / mol cm was used to calculate the number of unreacted amino groups (ref. 2).

5. Heparin immobilization.

35 The preparation below uses a sodium salt of heparin (Bufa Chemie, Castricum, The Netherlands). This heparin from porcine mucosa had the following properties: MW = 12,500, activity: 195 IU / mg, number of carboxylic acid groups 18.75 mol / mol heparin.

1008724 6

The product obtained at point 3 was equilibrated for at least 30 minutes in 0.05 M MES buffer (pH 5.60). EDC and NHS were added to a 2 w / v% solution of Na-heparin in MES buffer in a molar ratio of heparin-5-COOH: EDC: NHS of 1: 0.4: 0.28. After pre-activation for 10 minutes, the collagen coated and cross-linked blood vessel was added. For the immobilization reaction, a molar ratio of heparin to free collagen e-amino groups of 2: 1 was used. After 2 hours, the reaction was stopped by replacing the reaction mixture with a 0.1 M Na 2 HPO 4 solution and washing for 2 hours. This treatment resulted in hydrolysis of both activated carboxylic acid groups and residual EDC. Non-chemically bound adsorbed heparin was removed by washing with a 4 M NaCl solution (4 times 24 hours). After washing with water (3 times 24 hours), the plastic blood vessel was ready.

6. BFGF binding.

In a favorable embodiment, the blood vessel obtained at point 5 may be provided with a growth factor 20 which promotes the proliferation of seeded (in vitro) or naturally provided (in vivo) endothelial cells. Suitable is this growth factor bFGF (basic Fibroblast Growth Factor), in particular human bFGF. Suitable is recombinant human bFGF from Gibco, Paisley, UK). The plastic blood vessel obtained at point 5 is incubated in phosphate buffered saline (PBS) for at least 30 minutes. Subsequently, the materials were incubated in a solution of bFGF in PBS (0-600 ng bFGF / ml PBS) further containing 1 mg / ml BSA (A 7030, Sigma, St. Louis, MO, USA). After incubating at room temperature for 90 minutes with gentle shaking, the materials were washed twice with PBS for 5 minutes.

EXAMPLE II

Using the experiment described below it has been shown that the growth of endothelial cells on collagen substrates cross-linked according to the invention is better than on an un-cross-linked collagen or a fibronectin-coated substrate. The greater presence of endothelial cells in vivo reduces the chance of unwanted clots forming and blocking the blood vessel.

Endothelial cells were isolated from an umbilical cord (Human Umbilical Vein Endothelial Cells; HUVECs) and cultured as described by Wachem of P.B. et 5 al. (Ref. 4). After harvesting, the cells were grown to the third pass in polystyrene tissue culture bottles (TCPS) (Costar, Cambridge, MA, (USA), which culture bottles were pre-coated with a solution of partially purified human fibronectin (Fnc, 2 mg / ml; gift from the Central Laboratory of the 10 Netherlands, Red Cross Blood Transfusion Service (CLB, Amsterdam, Netherlands)) in culture medium without serum.

The culture medium consisted of a mixture of equal volumes of RPMI 1640 and MI99, supplemented with 100 U / ml penicillin (Pen), 100 µg / ml streptomycin (Strep), 2.5 μg / ml Fungizone, 2 mM L-glutamine (all from Gibco, Paisley, UK) and 20% human serum. The cultivation was done at 37 ° C and under a humidified atmosphere of 5% CO2 and 95% air.

HUVECs were detached from the culture bottles by incubation with trypsin (Gibco, 0.05% with 0.02% EDTA in PBS). Trypsin was inactivated by addition of culture medium.

Proliferation experiments were performed on cross-linked collagen films with varying degrees of cross-linking. Non-cross-linked collagen matrix and fibronectin-coated TCPS served as controls. Collagen films were incubated overnight in a solution of penicillin (200 U / ml) and streptomycin (200 μg / ml) in PBS, at 37 ° C. The films were placed in wells of a 12-well tissue culture plate and secured with sterilized Viton O-rings (Eriks, Alkmaar, Netherlands). The seeding density was 10,000 cells 30 cm 2. The culture medium was replaced three times a week and instead of 20% human serum contained 5% human serum, 5 U / ml heparin (Bufa Chemie, Castricum, The Netherlands) and 0.3 ng / ml recombinant human bFGF (Gibco).

After incubation for 3, 7 or 10 days, the number of HUVECs 35 on the different films was determined using a Burker chamber. HUVECs were detached from the collagen films by incubation with 400 μl solution of collagenase (Sigma C 6885, type 2, 1 mg / ml) and bovine serum albumin (BSA, Sigma, A-6003, 5 mg / ml) in PBS, after removal of the culture medium.

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The results shown in Table 1 below clearly show that cross-linking of collagen using EDC and NHS had a positive impact on the growth of the seeded HUVECs. This was even better than the growth on fibronectin coated TCPS, which is the standard substrate for endothelial cell culture.

_TABLE 1_

Number of free cell density on day1 Number / cm3 NHj groups 10 per 1000 amino acids__3_ 7__10_ 10 6526 (879) 3 26261 (2927) 46278 (2814) 14 10417 (1035) 25571 (2432) 45243 (2740) 18 8503 (1193) 30779 ( 2735) 51455 (2806) 22_ 8879 (904) 22245 (1190) 29367 (3706) 15 _27__14495 (1234) __ 21461 (628) __ 21649 (1489) fibronectin__18473 (497) __ 26920 (954) __ 35140 (2456) 1 On day 0 with 10,000 cells / cm2 (n = 4).

20 2 Standard deviation

EXAMPLE III

This experiment used cross-linked collagen according to example I point 3, which after cross-linking had 25 free e-amino groups per 1,000 amino acid residues. Heparin was immobilized on these collagen films using a molar ratio of EDC: heparin carboxylic acid groups of 0.4 and at a pH of 5.60. The films were placed in wells of a 12-well tissue culture plate and secured with sterilized Viton O-rings (Eriks, Alkmaar, Netherlands). Collagen films were incubated overnight in a solution of penicillin (200 U / ml) and streptomycin (200 μg / ml) in PBS, at 37 ° C. After thorough washing with sterile PBS, the collagen films were incubated with 0.44 ml bFGF solution in PBS containing 1 mg / ml BSA for 90 minutes at room temperature. After the incubation, the films were again washed with PBS (2 times 5 minutes), and HUVECs obtained as in Example II were seeded at a density of 10,000 cells / cm2.

40 Two collagen films not treated with bFGF solution served as controls. HUVECs were grown using the culture medium 1008724 9 described in Example II supplemented with 5% human serum. The culture medium of one of the two collagen films used as control was supplemented with heparin (5 U / ml) and bFGF (0.3 ng / ml). This control is referred to as a positive control, while the collagen film 5 grown in the same medium as bFGF-treated collagen films is referred to as a negative control. The culture medium was changed 3 times a week.

TABLE 2 10 Cell Density at Day1 Count / cm 'ng / ml bFGF during | incubation 3 7 10 5 18,417 (1,984) 2 29,147 (3,151) 35,893 (4,352) 11 26,230 (2,812) 41,384 (4,949) 35,395 (1,821) 15 19 19,243 (2,645) 60,658 (6,237) 64,047 (4,425) 37 30,246 (1,627) 60,554 (2,697) 66,139 (1,228) | 204_33,258 (1,716) 73,292 (5,993) 69,025 (950) negative control 10,950 (1,516) 13,931 (1,130) 13,680 (3,194) positive control 13,962 (1,273) 22,025 (4,675) 28,771 (2,255) 20 1 On day 0, sowing 10,000 cells / cm2 (n = 4).

2 Standard deviation

Table 2 shows that bFGF promotes growth of HUVECs 25 with bFGF concentrations above about 19 ng / ml resulting in maximum growth.

J 008724 10

REFERENCES

1. Gilbert, D.L. et al. "Macromolecular release from collagen monolithic devices". J. Biomed. Mater. Res. 1221-1239 (1990).

2. Wang, C.L., T. Miyata, et al. (1978). "Collagen induced platelet aggregation and release. I. Effects of the side-chain modifications and role of argynyl residues". Bio-chim. Biophys. Acta 544: 555-567.

10 3. Olde Damink, L.H.H., P.J. Dijkstra, et al. (1995). "Glutaraldehyde as a crosslink agent for collagen-based biomaterial". J. Mat. Sci .: Mat Med. £: 460-472.

4. Wachem of P.B. et al. (Thromb. Haemos., £ 6 (2), pp. 189-192 (1986).

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Claims (19)

1. A method of preparing a plastic blood vessel for implantation in a receiver, the method comprising the steps of i) applying a collagen material to a porous tubular plastic support; and ii) cross-linking the collagen material, characterized in that the cross-linking of the collagen material takes place using a peptide bond promoter, the degree of cross-linking being chosen such that of the 27 free amino groups per thousand amino acids 8 to 20 amino groups are not modified in the peptide bond-comprising cross-linking. 2. A method according to claim 1, characterized in that 12 to 16 amino groups are not involved in the cross-linking comprising a peptide bond. A method according to claim 1 or 2, characterized in that the peptide bond promoting agent formation is a carbodiimide compound. 4. A method according to claim 3, characterized in that the carbodiimide compound is a water-soluble carbodiimide compound. Process according to claim 4, characterized in that the water-soluble carbodiimide compound is N- (3-dimethyl-aminopropyl) -N'-ethylcarbodiimide (EDC). The method according to any one of claims 3 to 5, further comprising a succinimide compound. Process according to claim 6, characterized in that an optionally water-soluble N-hydroxysuccinimide compound is used as the succinimide compound. A method according to any one of the preceding claims, characterized in that the collagen material is provided with an anticoagulant polysaccharide. 9. A method according to claim 8, characterized in that when the blood coagulation inhibiting polysaccharide is used a polysaccharide selected from the group consisting of heparin, heparan sulfate and dextran sulfate. 1008724 Method according to claim 8 or 9, characterized in that the blood coagulation inhibiting polysaccharide is covalently coupled after the second step ii) to the amino groups not involved in a peptide bond after cross-linking. Method according to claim 10, characterized in that the polysaccharide is coupled to collagen by means of a peptide bonding agent. 12. A method according to claim 11, characterized in that the polysaccharide is preactivated with the peptide bonding agent before being contacted with collagen. 13. A method according to any one of the preceding claims, characterized in that after cross-linking the plastic blood vessel is provided with a growth factor which promotes the growth of endothelial cells. A method according to claim 13, characterized in that a growth factor binding a polysaccharide is used as the growth factor. 15. A method according to claim 14, characterized in that the polysaccharide is selected from the group consisting of heparin, heparan sulfate, chondroitin sulfate, hyaluronic acid and dextran sulfate. 16. A method according to claim 14 or 15, characterized in that the polysaccharide-binding growth factor is bFGF, aFGF or VEGF. A method according to any one of the preceding claims, characterized in that the plastic blood vessel is manufactured from a plastic selected from the group consisting of i) polyurethane; ii) PTFE; iii) Dacron; iv) polyethylene terephthalate; v) polylactic acid, optionally subjecting the plastic to a treatment to render it hydrophilic before coating with collagen. 18. A method according to any one of the preceding claims, characterized in that the blood vessel is boneed with compatible endothelial cells. A method according to claim 18, characterized in that the compatible endothelial cells are autologous endothelial cells. 1008724