NL8500240A - COLLAGEN COATED ARTISTS AND METHOD OF MANUFACTURE THEREOF. - Google Patents

Description

* 4 '· 1 1 - 1 -

Collagen-coated artery and method for the manufacture thereof.

This invention relates to artificial veins, and more particularly a tubular artificial graft having a series of layers of relaxed collagen fibrils that make the artificial vein blood-tight without having to undergo an uterine clotting.

\ Replacing parts of human blood vessels with arteries is generally accepted today. Artificial veins can take very different shapes and are made of very different materials.

10 Among the well accepted and successful arteries are those of biocompatible material containing an open space through which blood can flow after the artery has been implanted. The artificial veins can be made from biologically acceptable fibers such as Dacron and Teflon and can be woven or knitted or made from monofilament, multifilament or staple fiber yarn.

An important factor in choosing a particular artery substrate is the porosity of the wall of the tissue that makes up that vein. Porosity is important because it determines the tendency to bleed during and after implantation and also the growth of tissue into the wall of the artery. It is desirable that the artificial vein substrate be sufficiently blood-tight. it is possible to prevent blood loss during implantation, but the structure must also be sufficiently porous to allow fibroblasts and smooth muscle cells to grow inwards so that the graft grows on the tissue.

Artificial veins of the type described in U.S. Pat. Nos. 3,805,301 and 4,047,252 are elongated flexible tubular shapes made from a yarn such as 8500240

i V

-2--

Dacron. According to the former patent, the artificial vein is a run knitted tube and according to the second it is a double-velor artificial vein marketed under the name "Microvel". The structure of these arteries is porous enough to allow the surrounding tissue to grow inward.

The general method of implantation involves a coagulation in which the artery is immersed in the patient's blood and left long enough for blood to coagulate.

After this pre-clotting, no bleeding occurs when the artery is implanted and tissue growth is not impeded. However, it is desirable to make this pre-clotting unnecessary since it takes valuable time during surgery.

Blood-tight arteries with an enhancement of absorbable collagen have been proposed in U.S. Pat. No. 3,272,204. The type of collagen described was obtained from deep flexor tendon tendons of livestock. tendon-derived collagen is generally highly cross-linked and difficult to process with the enzymatic digestion proposed in the patent. Yet another reinforced artery is disclosed in U.S. Pat. No. 3,479,670, which discloses an open-mesh cylindrical tube surrounded by a screw-shaped reinforcement of fused polypropylene monofilament, which may have collagen fibrils as a filler to render the implant impermeable to bacteria and liquids. The collagen fibrils used are the same as described in patent 3,272,204.

The arteries previously described are said to be suitable for many applications. However, it remains desirable to have a flexible tubular graft that does not exhibit any porosity at all, yet is sufficiently susceptible to ingrowth of surrounding tissue and easier to process. 8500240-3 *.

Then what the existing technique provides. j

The invention provides a collagen 1 coated tubular artificial graft formed of a tubular porous structure of a biologically acceptable filamentous material with a cross-linked coating of at least three layers of collagen fibrils mixed with enough plasticizer to make the tubular graft blood-tight even without preliminary drafting. . The porous artificial vein can be a Dacron tubular graft and it can be knitted or woven.

Preferably, the source of the collagen is bovine skin that has undergone acid digestion leading to a high purity fibril dispersion. A purified aqueous collagen suspension with loom is massaged onto the artificial graft to cover the entire surface and create a smooth graft that feels good. After coating and drying at least three times, the collagen is cross-linked by exposing it to formaldehyde vapor. The porosity of the graft is thereby reduced to less than 1% of the porosity before coating.

Accordingly, it is an object of the present invention to provide an improved tubular artificial graft which is blood-tight, collagen-coated, and from which the collagen was obtained from bovine skin. Furthermore, the present invention contemplates an improved method for making such a collagen-coated tubular artificial graft, and for coating with collagen so that such a graft becomes blood-tight. Still other objects and advantages of the invention will be apparent in part from themselves and in part will be apparent from the description.

The present invention is further elucidated with reference to the accompanying drawings, of which figure 1 shows a collagen-coated artery vein according to the invention, partly in cross-section, 8500240-4 * 5 figure 2 a branched artery of the figure 1 depicts a cut type, also partially in section, and FIG. 3 is a graph showing the reduced porosity with a series of collagen coatings according to the invention.

An artificial vein 10 is shown in Figure 1. Artificial vein 10 has a tubular substrate 12 of biologically acceptable filamentary plastic, preferably of polyethylene terephthalate such as Dacron. Substrate 12 is a porous, run knitted Dacron fabric with an inner and an 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 wire-shaped material, provided that it is possible to make a porous structure of that material which allows tissue to grow inwards 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 branches 24 are of a Dacron knitting 26 with on the inside a covering 28 made up of at least three layers of collagen fibrils.

The porous tube substrates to be used in this invention are preferably made by knitting or weaving from Dacron multifilament yarns which are commonly used for this type of product. In general, the porosity of the Dacron substrate ranges from 2000 to 3000 ml / min.cm2 (pure water at 120 mm of mercury). The inner crosslinking of cross-linked collagen is applied by filling the tube substrate with a suspension of 8500-240% collagen with plasticizer and massaging by hands, 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 5 and is first air dried and then vacuum dried to remove excess moisture and free formaldehyde. The grafts coated according to the invention have essentially zero porosity.

The following non-limiting Examples 10 illustrate the preparation of pure collagen from bovine skin and the production of coated arteries according to the invention.

Example I

Fresh calf skins were cut mechanically and poured into a rotating vessel with cold running water. washed until they are 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 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) for 25 minutes (Also, the skins can be depilated mechanically or by a combination of chemical and mechanical treatment). After the treatment, the skins were cut into pieces about 2 cm x 2 cm and washed with cold water.

After washing, 120 kg of bovine skin were placed in a vessel with 260 liters of water, 2 liters of 50% NaOH 30 and 0.4 liters of 35% Oj. 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 now treated with a solution of 1.2 liters of 50% NaOH and 1.4 kg of CaO in 260 liters of water for 5 minutes, with slow stirring. This treatment was carried out 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 liters of water while stirring vigorously. 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 10 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.

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 main 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 zero. For comparison, the porosity of randomly chosen uncoated arteries from Meadox Microvel had an average water porosity of 1796 ml / min.cm? at 120 mm 30 of mercury, with a standard deviation of 130. After coating according to the invention, the porosity is 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 I. The collagen- 8500240

* ψ I

7 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 added by one of the! open ends lined. The artificial vein was allowed to dry at room temperature for about 10 hours. Drying coating 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 artificial vein was air dried for 15 minutes and then under vacuum for 24 hours to remove moisture and residual formaldehyde.

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. 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.crf, which can 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.crf.

Example IV

That the porosity of a graft-35 tissue with three collagen coatings is reduced to about 1% was demonstrated as follows with a standard porosity test. Water was passed under a pressure of 120 mm of mercury through a tissue-closed opening of 1 cm 2 for one minute. The amount of water collected was measured and the permeability calculated per minute per cm 2. Multiple determinations were made on each sample. The porosity of a micro-sheet graft tissue was about 1900 ml / min.cm2. After coating, it was found:

Number of coatings Porosity 10 0 1900 1 266 2 146 3 14 4 5 15 5 2 6 0

In all cases, the collagen coating was obtained from bovine skin, as described in Example II. These results are also shown in Figure 3. On this basis, 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 attach the whole to the 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

The thrombogenity was determined in vitro by the method of Imai and Nose (J. Biomed. Mater. Res. J, (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 35 micro-collagen coated tissue according to Example II. As a blank, the same amount was placed on an uncoated 8500240 * * - 9 - 4. fabric microvel. 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:

Impregnated the collagen Just!

Water the tissue with blood quickly slowly

Thrombus formation in; 5 min. 0 ++ i 10 min. + +++ 15 min. ++ ++++ 15 A comparison of 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 enter the knitted Dacron's fabric.

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 clotting 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 micro-knitted Dacron fabric quickly soaked with blood without any clotting occurring within 5 minutes. The blank 8500240 * - 10 - does show 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

The thrombogenicity 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 10 synthetic resin 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, "Dacron Microvel", was used as blank. This graft had pre-clotted before insertion. Testing showed, both by eye and by objective weight, 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. The veins impregnated with collagen did not trickle blood at all.

When an anticoagulant comparator 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 comparative grafts tested for 30 minutes, bleeding of at least 1 ml / min per 5 cm continued throughout the test.

8500240

'IO

«- 11 -

The collagen-impregnated arteries 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 coagulation is not seen in such an artery.

Of the five tested Dacron implants 10 that had undergone pre-coagulation, three had clearly multiple coagulations. 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 as a result of continued bleeding through the wall.

Based on these observations, the throrogeneity of Dacron's collagen-impregnated grafts is significantly less than comparative coagulation grafts. This may be due to less clotting by the collagen coating as well as clotting on the comparison grafts due to pre-clotting. 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 leading 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 coagulation, but that is not all. Conventional porous grafts, although necessary for long term application, necessitated the surgeon to clot the graft with the patient's blood to avoid excessive blood loss during implantation. As a rule this pre-solidification 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 thromboinity 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.

8500240

Claims (17)

1. Tubular artificial graft consisting of a smoothly porous tubular substrate with at least the inner surface a cross-linked coating of at least three layers of collagen fibrils mixed with enough plasticizer to make the graft blood-tight and smooth. Artificial graft according to claim 1/10, the porous substrate of which is polyethylene terephthalate. Artificial graft according to claim 2, of which the porous substrate is knitted. Artificial graft according to claim 2, of which the porous substrate is woven. An artificial graft according to any one of the preceding claims, wherein both inner and outer surface are of velor. Artificial graft according to any one of the preceding claims, the fibrils of which are cross-linked by exposing them to formaldehyde vapor. An artificial graft according to any one of the preceding claims, the plasticizer of which is a biologically acceptable polyhydric material. Artificial graft according to claim 7, of which the plasticizer is sorbitol. An artificial graft according to claim 7, the plasticizer of which is glycerol. An artificial graft according to any one of the preceding claims, the coating of which was applied from an aqueous suspension with 0.5 to 5.0 wt% collagen fibrils and 4 to 12 wt% plasticizer. An artificial graft according to any one of the preceding claims, the collagen fibrils of which were obtained from bovine skin. Artificial graft according to any one of the preceding claims, of which both inner and outer surfaces are coated 850024Q * * 4ft -Ht. A method of making a blood-dense artery, comprising: providing a tubular, flexible and porous substrate, placing an aqueous suspension of collagen fibrils with plasticizer on at least the inner surface of the substrate, massaging the substrate to ensure intimate distribution of the 10 collagen fibrils in the porous structure of the substrate, drying the collagen, placing a second layer of collagen-plasticizer suspension on the first layer of the substrate and drying Placing a third layer of collagen-with-plasticizer slurry on the substrate and drying, cross-linking the collagen coating by exposing it to formaldehyde vapor, and drying under vacuum to remove residual formaldehyde. 20 14. Suspension of collagen fibrils to be used in making a blood-tight artery containing 0.5 to 5.0 wt% collagen fibrils and 4.0 to 12.0 wt% biologically acceptable plasticizer and the remainder is water . A suspension according to claim 14, the collagen fibrils of which were obtained by acid digestion of bovine skin. Suspension according to claim 14 or 15, the plasticizer of which is sorbitol and / or glycerol. 17. A tubular artificial graft consisting of a tubular, flexible and porous substrate of polyethylene terephthalate, with at least five layers of cross-linked collagen fibrils mixed with plasticizer on the inner surface thereof, ......... .... ........................................ 8500240 5 w Λ - / Which layers were formed from an aqueous suspension containing 1.5 to 4.0 wt% collagen fibrils and 6 to 10 wt% plasticizer. 85 00 240