NL193264C - Method of manufacturing a blood-tight artificial vein. - Google Patents

Description

1 193264

Method of manufacturing a blood-tight artificial vein

The present invention relates to a method of manufacturing a blood-tight artificial vein, impregnating a flexible, porous, tubular artificial graft from the inside and coating it with a complex of collagen fibrils mixed with a plasticizer, and drying the collagen coating.

Such a method is known from GB 1,213,054. This document describes the application to a substrate with a porosity of 5000 ml / min.cm2 of a coating of collagen fibrils. After application, the collagen is both on the inner surface of the substrate and in its pores.

Replacing parts of human blood vessels with arteries is generally accepted today. Artificial veins can take different shapes and are made of very different materials. Among the well-accepted and successful arteries are those containing a biologically acceptable material and with an open space through which the blood can flow after the 15 arteries have been implanted. The artificial veins can be made from biologically acceptable fibers such as Dacron and Teflon, they can be woven or knitted, and also made from monofilament, multifilament or staple fiber yarn.

An important factor in choosing a substrate for arteries is the porosity of the wall of the tissue that makes up that vein. The porosity is important because it determines the tendency to bleed during and after implantation and also determines the ingrowth of tissues into its wall. It is desirable that the artery substrate be sufficiently blood-tight to prevent blood loss during implantation, but the structure should also be porous enough to allow fibroblasts and smooth muscle cells to grow inward so that the graft fuses with the surrounding tissue .

Blood-tight artificial veins with absorbable collagen reinforcement are known. They are described, for example, in DE 1,491,218. Here, a self-supporting collagen tube is introduced into a tubular substrate with a porosity greater than 2000 ml / min.cm2. Because the collagen tube is self-supporting and remains self-supporting even after joining with the substrate, a tube of a collagen mass with a textile substrate around it results. The collagen does not penetrate into the pores of the substrate.

US 3,425,418 describes a graft consisting of a textile substrate with a porosity greater than 10,000 ml / min.cm2 with two layers of collagen applied thereon. Due to the high porosity, the collagen naturally penetrates into the pores of the substrate.

The arteries described above are said to be suitable for many applications. However, it remains desirable to have a flexible tubular graft whose porosity is zero, yet which is sufficiently susceptible to ingrowth of surrounding tissue and which can also serve as a reservoir of slow-release drugs after implantation. This can be accomplished using a low porosity substrate with the collagen contained within the pores of the substrate.

The present invention now provides a method as described in the preamble, characterized in that the artificial graft has a porosity of 2000 to 3000 ml / min.cm2 (pure water, 120 mm mercury) and that the collagen fibrils are applied from an aqueous collagen slurry and massaged into the artificial graft and then dried and cross-linked, repeating the steps of applying an aqueous collagen slurry, massaging into the artificial graft and drying at least three times.

Preferably, the steps of applying an aqueous collagen slurry, massaging in the artificial graft, and drying are repeated at least five times.

It is furthermore advantageous if the collagen slurry contains 0.5 to 5.9 wt.% Collagen and 4 to 12 wt.% Plasticizer and the rest water. Even more preferably, the collagen slurry contains 1.5 to 4.0 wt% collagen and 6 to 10 wt% plasticizer and the balance water.

50 The collagen drug complex can be cross-linked by exposure to formaldehyde vapor.

Preferably, the source of the collagen is bovine skin that has undergone acid digestion leading to a high purity fibrillation dispersion.

Coating allows graft porosity to be reduced to less than 1% of coating porosity.

The porous tubular artificial grafts useful in this invention are preferably made by knitting or weaving Dacron multifilament yarns common to such products.

The incorporation of collagen slurry into the artificial graft can be performed by filling the artificial graft with a slurry of collagen fibrils and massaging by hand. After drying and cross-linking of the last layer, the grafts coated according to the invention have essentially zero porosity.

This invention is further elucidated on the basis of the accompanying drawings, of which figure 1 shows a collagen-coated artery according to the invention, partly in cross-section, figure 2 shows a branched artery of the type shown in figure 1, also partly in cross-section , and Figure 3 is a graph indicating reduced porosity with a series of collagen coatings according to the invention.

An artificial vein 10 according to the invention is shown in figure 1. Artificial vein 10 has a tubular substrate 12 of biologically acceptable filamentary plastic, preferably of a polyethylene erephthalate such as Dacron. Substrate 12 is a porous, run knitted Dacron fabric with an inner and outer surface of velor as described in U.S. Pat. No. 4,047,252. However, this tube 12 may also be made of any other biologically acceptable filamentary material provided that it is possible to make a porous structure of that material which allows tissue to grow in and leaves room for blood flow.

The tubular part 12 has a coating of collagen (16) on the inner surface. The collagen coating 16 is made up of at least three layers of an aqueous dispersion of collagen fibrils with plasticizer which is cross-linked by exposure to formaldehyde vapor.

Figure 2 shows a branched collagen-coated artery 20. The graft 20 has a main tube 22 with two branches 24. The main tube 22 and the branches 24 are of a Dacron knitting 26 with an inner lining 28 made up of at least three layers of collagen fibrils.

The porous tube substrates to be used in this invention are preferably knitted or weaved from Dacron multifilament yarns which are commonly used for this type of product. In general, the porosity of the Dacron substrate varies between 2000 and 3000 ml / min.cm2 (pure water, 120 mm mercury). The inner coating of cross-linked collagen is applied by filling a tube-substrate with a suspension of collagen with plasticizer and massaging by hand, removing the excess and allowing the deposited dispersion to dry. After the final application, the collagen coating is cross-linked by exposure to formaldehyde vapor and air-dried and then under vacuum to remove excess moisture and free formaidehyd. The grafts coated according to the invention have essentially zero porosity.

The following non-limiting examples illustrate the preparation of pure collagen from bovine skin and the production of coated artificial veins according to the invention.

Example I

Fresh calf skins were cut mechanically and washed in a rotating drum with cold running water 40 until they were free from floor dirt, blood and tissues. The subcutaneous tissue was mechanically cleared of tissue, including adipose tissue and blood vessels. The skins were then longitudinally cut to about 12 cm wide strips which were placed in a wooden or plastic tub, as is customary in the leather industry. The skins were depilated by keeping them in contact with 1 M Ca (OH) 2 for 25 minutes. (The skins can also be depilated mechanically or by a combination of chemical and mechanical treatment.) After treatment, the skins were cut into pieces of about 2½ cm x 2½ cm and washed with cold water.

After washing, 120 kg of bovine skin was placed in a vessel with 260 liters of water, 2 liters of 50% NaOH and 0.4 liters of 35% H 2 O 2. The contents were stirred slowly at 4 ° C for 12 to 15 hours and then rinsed with plenty of tap water for 30 minutes to give partially purified skins. These partially purified skins were treated for 50 minutes with a solution of 1.2 liters of 50% NaOH and 1.4 kg of CaO in 260 liters of water for 50 minutes, with slow stirring. This treatment was performed twice a day for 25 days. The solution was decanted and discarded each time and finally the skins were washed with plenty of tap water for 90 minutes with continuous stirring.

The skins were acidified by treating them with a mixture of 14 kg of 35% HCl and 70 55 liters of water with vigorous stirring. The acid was allowed to soak into the skins for about 6 hours. After acidification, the skins were washed with plenty of tap water for about 4 hours until a pH of 5.0 was reached. The pH of the skins was adjusted to 3.3-3.4 with acetic acid containing 0.5% preservative. The purified skin then passed through a meat grinder and was then extruded through a series of filter sieves of ever smaller mesh size. The final product was a white, homogeneous, smooth paste of pure bovine skin collagen.

In order to give the grafts in the dry state sufficient flexibility, a biologically acceptable plasticizer is added to the aqueous collagen suspension before application. A collagen suspension containing between 0.5 and 5% by weight of collagen contains between 4 and 12% by weight of plasticizer. Between 10 and 25% ethanol can be present to accelerate the evaporation of the water.

The major change that occurs when coating a tubular artificial graft with collagen and plasticizer according to the invention is that the porosity of the porous substrate drops to about 10 degrees. For comparison, the porosity of randomly chosen uncoated Meadox Microvel arteries had an average water porosity of 1796 ml / min.cm2 at 120 mm of mercury, with a standard deviation of 130. After coating according to the invention, the porosity was reduced to zero . The following example illustrates this.

Example II

A 50 ml syringe was filled with an aqueous suspension of 2% purified bovine skin collagen, prepared as described in Example 1. The collagen suspension contained 8% glycerol, 17% ethanol with the balance water, and had a viscosity of 30,000 cps. The syringe was placed in one end of a Dacron artificial vein "Meadox Medical Microvel" of 12 mm length and 8 mm diameter. The suspension was injected into this artery and it was massaged by hand so that the entire inner surface was covered with the collagen suspension. A little excess collagen suspension was drained through one of the open ends. The artery was allowed to dry at room temperature for about one Vz. Drying was repeated three times.

After the fourth coating was applied, the collagen coating was cross-linked by exposing it to formaldehyde vapor for 5 minutes. The cross-linked artery was air dried for 15 minutes and then under vacuum for 24 hours to remove moisture and formaldehyde residues.

Example III

The blood density of the collagen-coated arteries obtained according to Example II was determined as follows. A 8 mm x 12 cm micro sheet graft was stretched over the outlet of a blood reservoir; the hydrostatic height put a pressure of 120 mm mercury on this sheet. Heparin-stabilized blood was passed through that sheet. The blood passed through was collected and the permeability in ml per min.cm2 was calculated. Five tests found porosities of 0.04, 0.0, 0.0, 0.04 and 0.03. This means an average porosity of 0.022 ml / min.cm2, which may be equated with zero, since it was within the experimental error of this study.

For comparison, that transmittance measurement was also performed on an uncoated microfibre piece; the mean porosity was now 36 ml / min.cm.

Example IV

That the porosity of a graft fabric with three collagen coatings is reduced to about 1% was demonstrated with a standard porosity test as follows. Water was passed under a pressure of 120 mm of mercury through a tissue-closed opening of a V 2 cm 2 for one minute. The amount of water collected was measured and the permeability per minute per cm2 was calculated. Multiple determinations were made on each sample. The porosity of a microvel 45 graft tissue was about 1900 ml / min.cm2. After coating, it was found:

Number of coatings Porosity 0 1900 50 1 266 2 146 3 14 4 5 5 2 55 6 0 193 264 4

In all these cases, the collagen coating was obtained from bovine skin, as described in Example II. These results are also shown in Figure 3. On the basis of this, it is preferable to apply a collagen coating of at least three layers of fibrils, more preferably four or five layers, with intermediate drying after each application, and finally cross-linking to form the whole. substrate.

In addition to being much less porous, the collagen-coated vascular grafts of the invention are also much less thrombogenic than the untreated tissues. This is evident from the following examples.

Example V

Thrombogenicity was determined in vitro by the method of Imai and Nose (J. Biomed. Mater, Res. 6, (1972) 165). According to this method, 0.25 ml of citric acid stabilized blood was mixed with 25 µl of 0.1 M CaCl 2 and placed on the inner surface of a microveline coated fabric according to Example II. As a blank, the same amount was placed on an uncoated Microvel fabric. After 5, 10 and 15 minutes the same shape of blood stain was always seen. The solidification was stopped by adding 5 ml of distilled water to the samples. Notable differences were seen between the two tissues tested, and the following semi-quantitative assessment was expressed below:

With collagen Impregnated 20 -

Watering the tissue with blood quickly slowly

Thrombus formation in 5 min. 0 ++ 10 min. + +++ 25 15 min. ++ ++++

A comparison of the thrombus formation on the inner surface of a collagen coated fabric Microvel and the blank fabric Microvel was as follows. In the collagen impregnated tissue, there was no blood clot within 5 minutes. After 15 minutes, the clot on the collagen coated fabric was much less than on the corresponding untreated fabric.

The surface of the Microvel tissue that contacted the drop of blood was almost hydrophobic. It took about 10 to 15 seconds for the blood to penetrate knitted Dacron's tissue. This contrasts with the collagen-impregnated fabric that was quickly and evenly soaked through the blood. After 5 minutes, no residual clot was found on the collagen-coated tissue. At the same time, a thin but irreversible clot was present on the surface of the ordinary blank. After 10 and 15 minutes, the total volume of clotting on the inner surface in the collagen-coated tissue was less than in the blank.

Based on these in vitro observations without blood flow, the collagen-coated 40 micro-knitted Dacron fabric quickly soaked with blood without any clotting occurring within 5 minutes. The blank then shows clotting. Later, after 10 and 15 minutes, the amount of clotting on the collagen-impregnated fabric was less than with the ordinary blank fabric.

Example VI

45 The thrombogenity of collagen-impregnated fabrics Microvel was determined in vitro as follows. In greyhounds, an artery / vein shunt was applied under deep anesthesia. A 5 cm long tube was provided with synthetic cones at both ends for better handling. This made the material easy to place in the artery. After insertion, a vein clamp was slowly removed and then slowly at the end of the artery. The blood was allowed to flow through this implant for 10 minutes or 30 minutes. Then both ends of the shunt were again clamped and the implant was removed. The blood was drained and then weighed. The presence of clots on the graft surface was observed macroscopically. The graft was then washed three times with plenty of distilled water and reweighed.

A standard artificial vein with a diameter of 6 mm served as a blank, "Dacron Microvel". This graft 55 had pre-solidified prior to insertion. Testing showed both the eye and the objective weighing that the tested surface was thrombogenic. How much blood trickled through the graft wall was also determined to record the difference between the samples tested.

Claims (3)

5 193264 No blood dripped through the veins impregnated with collagen. When an image-graded comparison graft was placed in the shunt, an average of 30 ml of blood per 5 cm was lost in the first 5 minutes, but only 3-5 ml of blood in the next 5 minutes. In one of the comparison grafts tested for 30 minutes, bleeding was at least 1 ml / min. per 5 cm throughout the test. The artificial veins impregnated with collagen for 10 to 30 minutes showed the same resistance to thrombus formation as observed macroscopically. A thin, smooth layer of proteinaceous material then covers the collagen layer. After repeated washing with distilled water, most arteries show a continuous layer of protein (fibrin). A typical clotting is not seen in such an artery. 10 Of the five tested Dacron implants that had undergone a presentation, three clearly showed multiple clots. These were perpendicular to the direction of blood flow, covering 1/3 to 1/2 of the circumference. In the other two arteries, a similar proteinaceous layer covered the inner surface. The outer surfaces of all comparison grafts carried large clots due to the continuous bleeding through the wall. Based on these observations, the thrombogenity of Dacron collagen-impregnated grafts is clearly less than comparison grafts that have undergone visualization. This may be due to less clotting by the collagen coating as well as clotting on the comparison grafts due to pre-clotting. Since blood clotting is a phenomenon that plays a role in the excessive cell response in fiber replacement, it is advantageous to form blood clots with a matrix to counteract the Dacron fibers, which leads to less chance of embolism. By applying at least three layers of plasticizer collagen fibrils to a porous substrate according to the invention, highly desirable improvements are achieved when the graft is introduced into a human by a surgeon to replace a blood vessel. The expected benefits include the elimination of the need for representation, but that's not all. Conventional porous grafts, while necessary for long-term application, necessitated the surgeon to clot the graft with the patient's blood to avoid excessive blood loss upon implantation. As a rule, this cluttering is a time-consuming activity that requires some skill and experience. Therefore, a primary object of coating with collagen was to eliminate the need for this pre-clotting. In addition, the porous substrates for artificial veins form an ideal matrix for tissue growth. In addition, the markedly lower thrombogenicity of collagen-impregnated arteries leads to the risk of embolism. Coating an artificial vein with a suspension of collagen with plasticizer according to the invention also provides a tubular graft that remains flexible and easy to handle. It is thus seen that the aforementioned objectives are well achieved. 35 A method of manufacturing a blood-tight artificial vein, impregnating from the inside a flexible, porous, tubular artificial graft and coating it with a complex of collagen fibrils mixed with a plasticizer, and drying the collagen coating, characterized in that artificial graft has a porosity of 2000 to 3000 ml / min.cm2 (pure water, 120 mm of mercury) and that the collagen fibrils are applied from an aqueous collagen slurry and massaged into the artificial graft and then dried and cross-linked, with the steps of applying a 45 aqueous collagen slurry, massage into the artificial graft and repeat drying at least three times. The method according to claim 1, characterized in that the collagen slurry contains 0.5 to 5.0 wt.% Collagen and 4 to 12 wt.% Plasticizer and the remainder water. A method according to any one of the preceding claims, characterized in that the collagen is cross-linked by exposure to formaldehyde vapor. Hereby 2 sheets drawing