SE464058B - SYNTHETIC BLOOD CEREAL TRANSPLANT AND PROCEDURES AND MEDICINES IN THE FORM OF COLLAGEN FIBRILLY SLAMMING FOR ITS PREPARATION - Google Patents

Description

Is sufficiently blood tight to prevent blood loss during implantation, but the structure must be porous enough to allow fibroblast and smooth muscle cell ingrowth to bind the graft to the host tissue. Synthetic blood vessel grafts of the type described in U.S. Patents 3,805,301 and 4,047,252, to the assignee of the present invention, are elongate, flexible tubular bodies made of a yarn, for example Dacron. In the former patent the graft is a warp-knit tube, and in the later granted patent it is a synthetic double velor graft, which is marketed under the trademark Microvel. These types of grafts have a sufficiently porous structure to allow the growth of host tissue. The general implantation procedure involves, as a step, pre-coagulation, in which the graft is immersed in the patient's blood and allowed to stand for a sufficient period of time for coagulation to take place. After pre-coagulation, bleeding does not occur when the graft is implanted and tissue growth is not prevented. However, it is desirable to avoid pre-coagulation, as it requires valuable time during surgical treatment.

Blood-tight absorbable collagen-reinforced grafts have been proposed in U.S. Patent 3,272,204. The type of collagen described is obtained from the deep flexor muscle tendon of cattle. Late-obtained collagen is generally highly cross-linked and difficult to treat by the enzyme digestion treatment described in the patent. An additional reinforced blood vessel prosthesis is described in U.S. Patent 3,479,670, which discloses an open mesh cylindrical tube wrapped in an outer helical sheath of fused polypropylene monofibers, which may be filled with collagen fibrils to render the prosthesis impermeable to bacteria and fluids. . The collagen fibrils used are the same as described in patent 3,272,204. 3,464,058 The synthetic blood vessel grafts proposed in the prior art are said to be suitable for many applications. However, the desire remains to be able to provide a flexible blood vessel graft that exhibits virtually no porosity (porosity zero), while remaining sufficiently susceptible to host tissue ingrowth and being easier to process than in the prior art.

According to the invention there is provided a synthetic blood vessel graft comprising a tubular, flexible porous substrate made of a synthetic fiber having a porosity of less than about 3000 ml / min-cm * (purified water at 120 mm Hg), the graft substrate on the inner surface and across the porous substrate to the outer surface have crosslinked collagen fibrils mixed with a plasticizer to make the graft blood tight and flexible and the collagen fibrils being added by applying an aqueous slurry of collagen fibrils which have been massaged or kneaded. through the substrate and crosslinked after application. The porous graft substrate may be a tubular blood vessel graft made from a Dacron (registered trademark) material and may be woven or knitted. The source of collagen may be a dispersion of fibrils in water, which may be derived from high purity bovine skin collagen and which includes a plasticizer. The collagen is applied to the graft substrate by massage or kneading so that it covers the entire inner surface and penetrates into the substrate to ensure intimate mixing of the collagen fibril complex into the porous structure of the substrate and extends over its outer surface. The collagen is preferably crosslinked by exposure to formaldehyde vapor.

The collagen graft is blood-tight, flexible with good handling and is resorbed by the body after implantation.

The invention further provides a synthetic blood vessel graft comprising a tubular, flexible polyethylene terephthalate graft substrate having a porosity of less than about 3000 ml / min-cm * (purified water at 120 mm Hg), the inner surface being treated with at least five applications of crosslinked collagen fibrils mixed with a plasticizer extending into the porous structure of the substrate and covering the outer surface, each application being with a water slurry containing from 0.5 to 5.0 weight percent collagen fibrils and from 4 to 12 weight percent plasticizer. .

The collagen source is preferably derived from bovine skin (ox skin), which has been treated by acid digestion to give a fibrill dispersion with a high degree of purity. An aqueous purified collagen slurry containing a plasticizer is applied to the synthetic blood vessel graft by massage or kneading, so that it covers the entire surface and provides a flexible graft with good handling. After preferably at least three cycles of treatment and drying, the collagen is crosslinked by exposure to formaldehyde vapor. The porosity of the graft can thereby be reduced to less than 1% of the porosity of the graft before treatment.

Examples of grafts according to the invention will be described below with reference to the accompanying drawing, in which: Fig. 1 is a partial cross-section of a collagen-treated synthetic blood vessel graft according to the invention; Fig. 2 is a partial cross-section of a branched, tubular graft of the type shown in Fig. 1; and Fig. 3 is a graph showing the reduction of porosity after a series of collagen treatments according to the invention. 5,464,058 In the following description, all percentages refer to percentages by weight of the total composition, unless otherwise indicated.

A synthetic blood vessel graft 10 constructed and arranged in accordance with the invention is shown in Figure 1. The graft 10 comprises a tubular substrate member 12 made of a biocompatible filamentary or fibrous synthetic material, preferably a polyethylene terephthalate such as Dacron. The substrate 12 is a porous warp knitted fabric or fabric of Dacron having an inner and outer velor surface of the type described in U.S. Patent 4,047,252. The tubular member 12 is made of Dacron, but any biologically compatible wire or fibrous material may be used for the substrate, provided that it can be processed or formed into a porous structure, which allows tissue to grow and maintains an open lumen for blood flow.

The tubular member 12 has a collagen coating on the inner surface which is shown as 16. The collagen coating 16 is formed by at least three treatments with an aqueous collagen fibril and plasticizer dispersion which has been crosslinked by exposure to formaldehyde vapor. Figure 2 shows a branched collagen-treated graft 20. The graft 20 comprises a main tube portion 22 and two branches 24. The main tube portion 22 and the branched portions 24 are constituted by a knitted Dacron substrate 26 with an inner surface treated with collagen 28 by at least three applications of collagen fibrils.

Substrates for porous blood vessel grafts suitable for use in the invention are preferably made from Dacron multifibre yarns by knitting or weaving methods commonly used in the manufacture of these products. In general, the porosity of the Dacron substrate varies from 2000 to 3000 mt / min.- cm * (purified water at 120 mm Hg). Crosslinked collagen is applied by repeatedly filling a tubular substrate with a collagen and plasticizer slurry and manual massage or kneading as well as removing the excess, after which the deposited dispersion is allowed to dry. After the final application, the collagen is crosslinked by exposure to formaldehyde vapor, air dried and finally vacuum dried to remove excess moisture and excess formaldehyde. The treated grafts according to the invention have virtually no porosity.

The following examples are set forth to illustrate the process for producing purified collagen from bovine skin and treated grafts of the invention. The examples are intended to illustrate the invention without limiting it.

Example 1 Fresh calfskins were mechanically skinned from young calves, fetuses or stillborn and washed in a rotating vessel with cold running water until the water was observed to be free of surface dirt, blood and / or tissues. The subcutis was mechanically cleansed to remove contaminating tissue, such as fat and blood vessels. The skins were then cut lengthwise into strips about 12 cm wide and inserted into a wooden or plastic vessel of the type commonly used in the leather industry.

The skins were freed from hair using a rinsing solution of 1 M Ca (OH) 2 for 25 hours. Alternatively, the skins can be freed from hair by mechanical means or by a combination of chemical and mechanical means. After hair removal, the skins were cut into small pieces, approximately 25.4 x 25.4 mm, and washed in cold water.

After washing, 120 kg of the calfskins were introduced into a vessel with 260 l of water, 2 l of NaOH (50%) and 0.4 l of H 2 O 2 (35%). The components were slowly mixed for 12 to 15 hours at 4 ° C and washed with an excess of tap water for 40 minutes to give partially cleansed skins. The partially cleansed skins were treated in a solution of 260 l of water, 1.2 l of NaOH (50%) and 1.4 kg of CaO for 5 minutes with slow mixing. This treatment was continued twice a day for 25 days. After this treatment, the solution was decanted and discarded, and the skins were washed with an excess of tap water for 90 minutes with constant stirring.

The skins were acidified by treatment with 14 kg of HCl (35%) and 70 l of water while the skins were subjected to vigorous stirring. The acid was allowed to penetrate the skins for about 6 hours. After acidification, the skins were washed in an excess of tap water for about 4 hours or until a pH of 5.0 was reached. The pH of the skins was readjusted to 3.3-3.4 using acetic acid with 0.5% preservative. The cleansed skin was then passed through a meat grinder and extruded under pressure through a series of filter screens with a constantly decreasing mesh size. The end product was a white homogeneous smooth paste of pure collagen derived from bovine skin.

To give the grafts sufficient flexibility in the dry state, a biologically acceptable plasticizer is added, such as a biologically compatible material containing several OH groups, e.g. glycerol or sorbitol, to an aqueous collagen slurry before application. A collagen slurry containing from 0.5 to 5.0% by weight of collagen contains the plasticizer with from 4 and 12% by weight.

From 10 to 25% ethanol may be present to accelerate evaporation of the water.

The most significant property obtained in treating a synthetic blood vessel graft with collagen and plasticizer according to the invention is the reduction of the porosity of the porous substrate to about zero. For comparison, twenty randomly selected untreated synthetic Meadox Microvel blood vessel grafts had an average water porosity of 1796 ml / min-cm * at 120 mm Hg and a standard deviation of 130. After treatment according to the invention, the porosity is reduced to zero. . The following examples illustrate the method of treating the graft substrate according to the invention.

Example 2 A 50 cm 3 syringe is filled with a water slurry of 2% purified bovine skin collagen prepared according to Example 1.

The collagen slurry contains 8% glycerol, 17% ethanol and the rest water and has a viscosity of 30,000 CP. The syringe is placed at * one end of a "Meadox Medical Microvel" Dacron graft with a diameter of 8 mm and a length of approximately 12 cm. The slurry is injected into the lumen of the Microvel graft and massaged by hand over the entire inner surface.

Any excess collagen slurry is removed through one of the open ends. The graft is allowed to dry for about 1/2 hour at room temperature. The treatment and drying steps are repeated three more times.

After the fourth application, the collagen is crosslinked by being exposed to formaldehyde vapor for 5 minutes. The crosslinked graft is then air dried for 15 minutes and then vacuum dried for 24 hours to remove moisture and any excess formaldehyde.

Example 3 The blood density of a collagen-treated blood vessel graft prepared according to Example 2 was tested in the following manner. A 8 mm x 12 cm Microvel graft was attached to a blood vessel at a pressure of 120 mm Hg due to the height of the reservoir. Heparin-stabilized blood was passed through the graft. Blood collected through the graft was determined and expressed in ml per min-cm ”. The porosity in five experiments was determined to be 0.04, 0.0, 0.0, 0.04 and 0.03.

This represents an average porosity of 0.022 ml / min-cm * which was considered to be zero, since the value is within the margin of error of the test.

To compare this result with the blood loss for untreated Microvel, the experiment was repeated using an untreated graft tissue. The average porosity was 36 ml / min-cm 2.

Example 4 The porosity of a collagen-treated tissue graft is reduced to less than 1% after three applications, as shown below. A standardized water porosity test used to measure the water porosity of a graft is as follows. A water column corresponding to a pressure of 120 mm Hg may pass through a 0.5 cm * orifice with a sample of the graft over the orifice for 1 minute. The amount of water collected was measured. The amount of water in milliliters collected per minute per cm ”area was calculated. Several measurements were made for each sample. The porosity is stated as follows: porosity = ml / min / cm '.

The water porosity of a Microvel graft material was approximately 1900 ml / min / cm 2. The porosity after treatment was as follows: Number of treatments Porosity The collagen was in all cases a softened slurry obtained from bovine skin prepared in accordance with the composition 464 D58. described in Example 2. These results are shown in the diagram in Figure 3. On the basis of these results, it is convenient to apply the collagen slurry by at least three treatments with fibrils, and preferably four or five treatments, with drying between each application and cross-section. binding after the last treatment to fix the collagen to the substrate.

In addition to reduced porosity, collagen-treated blood vessel grafts of the invention exhibit reduced thrombogenicity compared to untreated grafts. The following examples show significantly less thrombogenicity of collagen-impregnated blood vessel grafts compared to control samples.

Example 5 Antithrombogenicity was evaluated in vitro by the method of Imai and Nose (J. Biomed, Mater Res. 6, 165, 1972). In accordance with this method, a volume of 0.25 ml of ACD blood (citric acid stabilized) was mixed with 25 μl of 0.1 M CaCl 2 and applied to the inner surface of a collagen-treated Microvel graft prepared according to Example 2. large volume was applied to an untreated Microvel graft as a control sample. The same geometry of the blood stain was observed after 5, 10 and 15 minutes. The coagulation reaction was stopped by adding 5 ml of distilled water to the samples.

Striking differences were observed between the two grafts tested, and the following semi-quantitative parameters were established: 11 464 058 Collagen impregnated Untreated The rate of soaking of the graft matrix with blood fast slow Blood clot formation within 5 min. 0 ++ 10 min. + +++ 15 min. ++ ++++ A comparison of the clot formation on the inner surface of the collagen-impregnated Microvel graft and the control Microvel graft gave the following. In the collagen-impregnated graft, no fibrin-coagulum formation occurred within 5 minutes. At 15 minutes, the coagulation on the collagen-impregnated graft was much less than in the corresponding untreated grafts.

The surface of the Microvel graft in contact with the drop of blood almost behaved like hydrophobic. It took between 10 and 15 seconds for the blood to penetrate the tissue of the knitted Dacron graft. This differs from the collagen-impregnated graft, in which blood penetrated the graft matrix uniformly and rapidly.

After 5 minutes, no blood clot residue could be detected on the collagen-treated graft. At the same time, there was a thin but clear blood clot on the surface of the untreated control Microvel graft. After 10 and 15 minutes, the total volume of blood clots present on the inner surface of the graft was smaller in the collagen-treated graft than in the control samples. 464 058 12 Example 6 The thrombogenicity of collagen impregnated Microvel grafts was tested in vitro (dogs) as follows. A femoral artery venous (AV) shunt was installed in deeply anesthetized Greyhounds. A prosthesis material with a length of 5 cm was adapted at both ends with conical plastic tubes for better handling.

This allowed easy insertion of a sample graft segment into the arterial shunt. After insertion, a venous clamp was slowly removed and the arterial end was then slowly opened.

The blood flow was allowed to circulate through the implant for 10 minutes or 30 minutes. Then both ends of the shunt were reclosed with clamps and the inserted prosthesis was removed. The excess blood was drained and the weight determined. The presence of blood clots adhering to the surface of the graft was observed macroscopically. The grafts were then washed (three times) in excess distilled water and weighed again.

A standard 6 mm diameter Dacron-Microve1 graft was used as a control. This graft was pre-coagulated before insertion into the shunt. This test thus provided both visual and objective gravimetric information on the thrombogenicity of the tested surface. The weight of blood seeping through the graft wall was also determined to determine the difference between the samples examined.

No bleeding was observed with collagen-impregnated grafts.

After insertion of a pre-coagulated control graft into the AV shunt, an average of 30 ml of blood / 5 cm graft length was lost during the first 5 minutes. For the next 5 minutes, only 3-5 ml of blood was lost. With one of the control grafts, which was tested for 30 minutes, a minimum bleeding of 1 ml / min / 5 cm continued through the pre-coagulated graft throughout the test. 13 464 058 The collagen-impregnated grafts implanted for 10 or 30 minutes showed the same pattern of resistance to thrombosis observed macroscopically. A thin even layer of shiny protein material covers the collagen layer. After washing repeatedly in distilled water, a continuous film of proteins (fibrin) can be seen in most prostheses. A typical blood clot was not observed in the test prostheses.

Of the five pre-coagulated untreated Dacron graft implants tested, three showed distinct multiple thrombosis. These were located across the direction of blood flow and covered or exceeded 1/3 to 1/2 of the circumference. In the remaining two prostheses, a similar skin of protein layer covered the inner surface. The outer surfaces of each control graft contained large blood clots due to continuous bleeding through the wall.

Based on these observations, the thrombogenicity of collagen-impregnated Dacron vascular grafts was significantly lower than that of pre-coagulated control grafts. This may be due to either decreased blood clot formation due to the collagen or that blood clots formed in the control grafts due to the need for pre-coagulation. Because blood coagulation is a reaction involved in excessive cellular reaction with the fibrotic replacement material, it is beneficial to reduce blood clot formation in the matrix of Dacron grafts, leading to less risk of embolism.

Through at least three treatments with a collagen fibril and plasticizer slurry of a synthetic porous blood vessel graft substrate according to the invention, specific desirable improvements are obtained when the graft is surgically inserted into a human patient as a blood vessel replacement. The expected benefits include, but are not limited to, eliminating the need for precoagulation. Conventional porous grafts, although proven necessary for long-term openness, have made it necessary for the surgeon to pre-coagulate the graft with the patient's blood to prevent excessive blood loss at the time of implantation. The pre-coagulation step is usually a time-consuming step, requiring some experience and knowledge. It has therefore been a primary purpose of the collagen treatment to completely eliminate the need for pre-coagulation of synthetic grafts.

The porous synthetic blood vessel grafts provide an ideal matrix for tissue ingrowth while eliminating the need for precoagulation. In addition, the significantly lower thrombogenicity of collagen-impregnated synthetic blood vessel grafts reduces the risk of embolism. Treatment of a synthetic blood vessel graft with a collagen and plasticizer slurry through a series of treatments according to the invention also provides a blood vessel graft that remains flexible and with good manageability.

Claims (20)

15 464 058 PATENT CLAIMS 1. l. Synthetic blood vessel graft, characterized in that it comprises a tubular flexible porous substrate, made of a synthetic fiber having a porosity of less than about 3,000 ml / min-cm * (purified water at 120 mm Hg) , wherein the graft substrate on the inner surface and across the porous substrate to the outer surface has crosslinked collagen fibrils mixed with a plasticizer to make the graft blood tight and flexible, and wherein the collagen fibrils have been supplied by applying an aqueous slurry of collagen fibrils having kneaded through the substrate and crosslinked after application. A blood vessel graft according to claim 1, characterized in that the porous substrate is polyethylene terephthalate. Blood vessel graft according to claim 2, characterized in that the porous substrate is knitted. A blood vessel graft according to claim 1, characterized in that the porous substrate is woven. 5. A blood vessel graft according to claim 1, characterized in that the inner and outer surfaces of the substrate have a velor surface. Blood vessel grafts according to claim 1, characterized in that the collagen fibrils have been cross-linked by exposure to formaldehyde moss. Blood vessel graft according to claim 1, characterized in that the plasticizer is a biologically compatible polyvalent material. Blood vessel grafts according to claim 7, characterized in that the plasticizer is sorbitol. Blood vessel transplant according to claim 7, characterized in that the plasticizer is glycerol. 10. l0. Blood vessel grafts according to claim 1, characterized in that the collagen coating has been prepared from a deposited water slurry of from 0.5 to 5.0% by weight of collagen fibrils and from 4 to 12% by weight of plasticizer, based on the total weight of the slurry. 11. ll. Blood vessel grafts according to claim 1, characterized in that the collagen fibrils are derived from bovine skin. 12. l2. A method of making a collagen-containing synthetic blood vessel graft, characterized in that it comprises: providing a porous tubular flexible synthetic graft substrate; applying a water slurry of collagen fibrils to the cavity of the tubular substrate; kneading the slurry into the substrate to ensure intimate mixing between the collagen fibril complex and the porous structure of the substrate; drying of the collagen; crosslinking of the collagen and the following vacuum drying. 13. l3. Synthetic blood vessel graft, characterized in that it comprises a tubular flexible graft substrate of polyethylene terephthalate having a porosity of less than about 3,000 ml / min-cm * (purified water at 120 mm Hg), the inner surface being treated with at least five applications of cross-linked collagen fibrils mixed with a plasticizer extending into the porous structure of the substrate and covering the outer surface, each application being a water slurry containing from 0.5 to 5.0% by weight of collagen fibrils and from 4 to 12% by weight of plasticizer. 17 464 1058 A blood vessel graft according to claim 13, characterized in that the plasticizer is selected from sorbitol and glycerol. A blood vessel graft according to claim 1, characterized in that the slurry of collagen is applied at least three times and dried between the applications. 16. A method according to claim 12, characterized in that the crosslinking takes place by means of formaldehyde vapor. 17. A method according to claim 12, characterized in that the slurry is applied at least three times and dried between the applications. 18. Collagen fibril slurry for the manufacture of a blood-tight, synthetic blood vessel graft, characterized in that it comprises about 0.5 to 5.0% of collagen fibrils and 4.0 to 12.0% of a biologically compatible plasticizer, the remainder being water. A slurry according to claim 18, characterized in that the collagen fibrils have been obtained by acid digestion of bovine skin. A slurry according to claim 18, characterized in that the plasticizer is selected from sorbitol and glycerol.