SE464009B - SYNTHETIC BLOOD SEED TRANSPLANT AND PROCEDURE FOR ITS PREPARATION - Google Patents

Description

464 009 2 growth of fibroblast and smooth muscle cells for adhesion of the graft at the host tissue. Synthetic blood vessels grafts of the type described in U.S. Patent 3,805,301 and 4,047,252, belonging to the holder of the present invention. elongated, flexible tubular bodies are produced. set by a yarn, such as Dacron. In the former patent the graft is a warp-knit tube, and in the latter issued the patent, it is a synthetic double velor plant, which is marketed under the Microvel brand. These types of grafts have sufficiently porous structures for to allow the growth of host tissue.

The general method of implantation includes a method of coagulation step, in which the graft is immersed in blood from the patient and may stand for a period of time sufficient for coagulation to take place. After pre-coagulation bleeding does not occur when the graft is implanted and tissue growth is not inhibited. Transplant infection is a very serious complication and occurs on average at two percent of transplant implants performed. It is associative row with a high risk of loss of a leg or arm and patient mortality is as high as 75% depending on the transplant the state of the state. Infection can usually be observed shortly thereafter surgery, but it can also take longer, which leading to more serious consequences.

An absorbable, collagen-reinforced graft is suggested in U.S. Patent 3,272,204, in which the collagen is obtained from the deep flexor muscle tendon of cattle. Another armed blood vessel prosthesis is described in U.S. Patent 3,479,670, which prosthesis includes a cylindrical tube of open mesh, wrapped with an outer helical casing of fused polypropylene monofibers filled with collagen fibrils to make the prosthesis impermeable to bacteria and fluids. The collages used the gene fibrils are the same as described in the US patent 3 272 204.

W ' ß 4e4äoo9 The synthetic blood vessel transplants proposed according to prior art are said to be suitable for many applications purpose. However, it is desirable to provide one flexible blood vessel graft that has no porosity (porosity zero), but which is susceptible to the growth of host tissue and serves as a reservoir for drug material such as can be slowly released from the surface of the graft after implantation. tion.

According to the invention there is provided a synthetic blood vessel graft, which includes a tubular, flexible porous graft substrate of a synthetic fiber with a porous 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 cross-linked collagen fibrils, which are complexed with one effective amount of a drug and mixed with a softener agents to make the graft blood-tight and flexible and to provide substantial release of medicinal products the part of the complex after implantation.

The drug material may include agents that promote cell division. antibacterial agents, antithrombogenic agents, fungal pesticides and antivirals and mixtures thereof to prevent graft infection by provide uninterrupted release of the drug moiety in the complex after implantation.

According to the invention there is also provided a synthetic blood vascular graft, which includes a tubular, flexible polyethylene terephthalate graft substrate with a porosity such as is less than about 3000 ml / min-cm ”(purified water at 120 mm Hg), the inner surface having crosslinked collagen fibrils, complexed with a drug and mixed with a softener both extending into the porous surface of the substrate structure and covers the outer surface, and wherein the collagen has 464 009 4 applied by treatment with a water slurry containing from 0.5 to 5.0% by weight of collagen fibrils and from 4 to l2% by weight of plasticizer.

The porous graft substrate may be a tubular blood vessel transplant made from a Dacron (registered trademark) material and it may be woven or knitwear. The source of collagen may be an aqueous fibril dispersion of collagen from cattle of high purity, which includes a plasticizer. The collagen is applied to the trans- the plant substrate by massage or kneading, so that covers the entire inner surface and penetrates the substrate to ensure impregnation of the porous structure of the substrate with the collagen fibril complex and covers its outer surfaces. Check- the gene can be cross-linked by exposure to formaldehyde vapor.

The collagen graft provides a flexible graft with good manageability.

Examples of the grafts according to the invention will be described below with reference to the accompanying drawing, at which: Fig. 1 is a partial cross-section of a collagen-treated device. synthetic blood vessel transplant according to the invention ningen; Fig. 2 is a partial cross-section of a branched, tubular grafts of the type shown in Fig. 1; Fig. 3 is a diagram illustrating uninterrupted release of tetracycline from a collagen slurry in rabbits; Fig. 4 is a diagram illustrating uninterrupted release of tetracycline at different collagen gel concentrations; and Fig. 5 is a diagram illustrating uninterrupted release of 5,464,009 tetracycline in a colloid gel at different concentrations and dosages.

In the following description, all percentages are by weight. percent of the total composition, unless otherwise stated specifically stated.

A synthetic vascular graft 10 constructed and arranged in accordance with the invention is shown in Fig. 1.

The graft 10 comprises a tubular substrate portion 12 which is made of a biologically compatible wire or fiber synthetic material, preferably polyethylene terephthalate, such as Dacron. The substrate 12 is a porous warp knitted fabric. material or fabric 14 of Dacron with an inner and outer velor surface of the type described in U.S. Patent 4,047,252 the tubular part 12 is made by Dacron, but which biologically compatible wire or fibrous material such as preferably can be used for the substrate, provided that it can manufactured with a porous structure, which allows tissue growth and maintains an open lumen for blood flow.

The inner surface of the tubular member 12 is treated with glue. gene, shown as 16, which also extends into the pores of the substrate. The collagen component 16 is formed by a series of at least three applications of collagen fibrils. Figure 2 shows a branch, collagen-treated graft 20. The graft 20 includes a tubular main body 22 and two branches 24. The tubular the main part 22 and the branched parts 24 are made of one knitted Dacron substrate 26. The inner surface of the substrate 26 is treated. through at least three applications of collagen 28 in fibril form.

Porous substrates for blood vessel transplants, which are suitable for used according to the invention, are preferably prepared from Dacron multifibre yarn by knitting or weaving which were widely used in the manufacture of these products dukter. In general, the porosity of the Dacron substrate varies from 464 009 e 2000 to 3000 ml / min-cm * (purified water at 120 mm Hg). The the cross-linked collagen is applied to the inner surface of the graft by repeatedly filling a tubular substrate with a slurry of collagen fibrils and plasticizers and manual massage or kneading and removal of the excess, after which the deposited dispersion is allowed to dry. After the final During the application, the collagen is crosslinked by exposure to formaldehyde steam, air dried and then vacuum dried to remove excess moisture and excess formaldehyde.

The treated grafts according to the invention have as good as no porosity.

The following examples are given to illustrate the method of position of purified collagen of bovine skin and treated grafts according to the invention. The examples are given to illustrate the invention and are not intended to be limiting the same.

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 found to be free from surface dirt, blood and / or tissues. The subcutis is cleansed was made mechanically to remove contaminating tissue, such as fat and blood vessels. Then cut the skins in the longitudinal direction to strips with a width of about 12 cm and inserted into one wooden or plastic vessels of the type commonly used within the leather industry.

The skins were depilated using a rinsing solution of 1 M Ca (OH) 2 for 25 hours. Alternatively, the skins can be depilated by mechanical means or by a combination of chemical and mechanical means. After depilation, the skins are cut into small pieces pieces with a size of about 25.4 mm x 25.4 mm and washable in cold water. 7 464 009 After washing, 120 kg of the calfskins were introduced into a vessel with 260 liters of water, 2 liters of NaOH (50%) and 0.4 liters of H2O2 (35%). The components were mixed slowly for 12 to 15 minutes hours at 4 ° C and washed with an excess of water for 30 minutes to give partially cleansed skins. The partially cleansed skins were treated in a solution of 260 liters water, 1.2 liters of NaOH (50%) and 1.4 kg of CaO for 5 minutes with slow mixing. This treatment was continued for two times daily for 25 days. After this treatment decant- the solution was discarded and the skins were washed with an excess of tap water 90 minutes under constant stirring.

The skins were acidified by treatment with 14 kg of HCl (35%) and 70 liters 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 had been reached.

The pH of the skins was readjusted to 3.3 to 3.4 using acetic acid with 0.5% preservative. The purified hu- it was then passed through a meat grinder and extruded under pressure through a series of filter sieves with constant decrease mesh size. The end product was a white, homogeneous smooth paste pure collagen derived from bovine skin.

To give the grafts sufficient flexibility in dry conditioner, plasticizers are added to the collagen slurry before application. Suitable plasticizers include glycerol or biologically compatible materials containing several OH groups, e.g. sorbitol, or others biologically acceptable plasticizers. In a collagen slurry containing from 0.5 to 5.0% by weight of collagen, there is a soft in an amount of from 4 to 12% by weight.

Among the most important properties obtained during treatment of a synthetic blood vessel transplant with collagen fibrils 464 ÛÛ9 s according to the invention is reduction of the porosity of the porous the substrate to about zero. Twenty randomly selected untreated Synthetic blood vessel transplants by Microvel Dacron have one average purity for purified water of 1796 ml / min-cm * at 120 mm Hg with a standard deviation of 130. After several collagen applications, the porosity is reduced to zero. Following examples illustrate the method of treating graft the substrate.

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

The collagen slurry includes 8% glycerol, 17% ethanol and the rest consists of water and it has a viscosity of 30,000 cP. The syringe is placed at one end of a Meadox Medicol Microvel "Dacron graft with a diameter of 8 mm and a length of about 12 cm. The slurry is injected into the lumen of The microvel graft and massaged or kneaded manually to cover the entire inner surface with the collagen slurry and ensure penetration into the pores of the Dacron material, so that the material becomes completely impregnated thereby. Possibly excess collagen slurry is removed through any of the open the 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 through exposure to formaldehyde vapor for 5 minutes. The cross-linked the graft is then air dried for 15 minutes and vacuum dried then 24 hours to remove moisture and any excess shot of formaldehyde.

Example 3 The blood density of a collagen-treated blood vessel plants prepared according to Example 2 were tested in the following manner.

A Microvel graft measuring 8 mm x 12 cm was attached WE 9 464 009 at a blood reservoir at a pressure of 120 mm Hg due to the height of the reservoir. Heparin-stabilized blood was brought to pass through the graft and blood collected through the graft was determined and expressed in ml per minute and cmz. 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 zero, since the value is within the margin of error in the investigation.

To compare this result with the blood loss for untreated Microvel graft repeated the experiment with use of an untreated graft. The average the porosity was 36 ml / min-cm ”.

Example 4 The porosity of a collagen-treated fabric graft is reduced to less than 1 percent of an untreated graft after three coatings. A standard test used to measure The water porosity of a graft is as follows. An aqueous pillars corresponding to a pressure of 120 mm Hg may pass through a 0.5 cm * orifice with a sample of graft over the mouth for a minute. The amount of water collected was measured.

Number of milliliters of water collected per minute per cm * area was calculated. Several readings were made for each sample. The porosity is indicated as follows: porosity = ml / min / cm 2 The water porosity of a Microvel graft fabric was approximately 1900 ml / min / cm 2. The porosity after treatment was as follows: 464 009 10 Number of treatments Porosity 0 1900 266 146 14 Gk fl nb-Luhål-J U "I In all cases, it was collagen that was used for impregnation of the fabric a softened slurry originating from bovine skin prepared in the same manner as the composition of Example 2.

On the basis of these results, it is appropriate to apply collagen slurry through at least three or four treatments with fibrils, and preferably four or five treatments with drying between each application and crosslinking to fix the collagen at the substrate.

According to the invention, the collagen is modified so that it contains taking a drug or anticoagulant, such as heparin, to prevent infection and inhibit coagulation ring along the inner surface of the prosthesis. As stated is the collagen complexed with one or more drugs, such as antibacterial antifungal agents, antimicrobial agents or fungicides agents, to prevent transplantation infection. Common anti- bacterial agents that can be used include oxacillin, gentamicin, tetracycline, cephalosporin and the like, which may complexed with the collagen fibrils prior to application to the the plant substrate.

In addition to reduced porosity show with collagen treated blood vessel grafts according to the invention reduced thrombus genicity compared to untreated grafts.

Example 5 A homogeneous slurry of collagen from bovine skin that 11 464 009 prepared according to Example 1 was prepared, which contained 1% collagen from bovine skin, 8% glycerol, 17% ethanol and the rest water. Ceclor, a cephalosporin antibiotic from Eli Lilly and Company, which inhibits the growth of Staphylococcus aureus and Escherichia coli, were incorporated into the slurry in a concentration of 20 mg per ml. Collagen slurry included Ceclor was kneaded like a coating on a double velor fabric of Dacron on both sides with 1/2 hour drying between the treatments, so that the fabric was impregnated with the slurry.

The treatment added 3.1 mg of collagen per cm 2.

As a comparative sample, double velor fabric was impregnated by Dacron with the same collagen slurry, but without the Ceclor antibiotic.

The comparative sample had a coating of 4.1 mg collagen per cmz.

Both pieces of treated and impregnated fabric were dipped 1 minute in 4% formaldehyde-10% glycerol solution and vacuum dried for 64 hours and sterilized using gamma- radiation.

The antimicrobial activity of the collagen-treated laid blood vessels transplanted with Dacron fabric impregnated with Ceclor was determined by agar diffusion analysis. l cm ”large cloth patches were placed on inoculated agar surfaces, resulting in growth inhibitory zones, indicating that the antibiotic was active against §¿ aureus (34 mm inhibition zone) and §¿ coli (29 mm inhibition zone). For comparison, use untreated, collagen-treated blood vessel transplant tissue did not show any antimicrobial effect. The results are given in the following Tables I and II. 464 009 12 TABLE I FABRIC TREATED WITH COLLAGEN AND ANTIBIOTICS Plate l Plate 2 Plate 3 šå f » §¿ aureus 36 mm 31 mm 35 mm 34 mm Height 33 mm 28 mm 27 mm 29 mm TABLE-II FABRIC TREATED WITH COLLAGEN Plate l Plate 2 Plate 3 x3 §¿ aureus 0 O 0 §¿ coli 0 0 0 Example 6 A collagen slurry prepared according to Example 1 and contained in containing 1.3% collagen protein (determined by its hydroxy- proline content) was mixed in a ratio of 1: 3 with water (W) to form a 3.3% by weight homogeneous collagen gel (G). The pH of the collagen gel was adjusted to 3.8 and 20 mg of tetracyc- flax (TC) was added per milliliter of gel. Immediately before injection In two rabbits, collagen gel-tetracycline compound was mixed. plexet with glutaraldehyde (0.3 ml 3% glutaraldehyde per ml gel) and was injected into the subcutaneous tissue with needles no. 18. For comparison injected two rabbits with a similar dose of tetracycline and water, 20 mg TC / ml water / kg body weight.

To study the rate at which tetracycline is released from the injected site, blood was collected at different times. points from the rabbit's ear vein. The level of TC in the blood was measured according to Wilson et al. described procedure (Clin. Chem.

Acta., 36; 260, l972). The results of the TC analysis of the blood from all four rabbits collected within 2 hours 13 464 009 7 days after injection are shown in Figure 3.

Figure 3 shows that after injection of TC into water, the drug the product reaches its maximum in serum within two hours as shown with curve A. After ll hours, TC can no longer be detected. Then tetracycline was administered in a collagen gel, which was cross-linked the one with glutaraldehyde (10 G. + 30 W), the TC content remained in serum stable for about 6 days as shown by curve B. Administration Thus, the release of TC into collagen gel prolonged the effective release of the administration of the drug 25 times compared to injection in only an aqueous medium.

Example 7 The test described in Example 6 was repeated using of collagen gel at two different concentrations for the final the injection. In addition, the tetracycline content was 30 mg oxytetra- cyclin (OTC) / ml gel / kg body weight at a dose of 1 ml / kg body weight or 50% more tetracycline per dose than in Example 6.

The results shown in Figure 4 show that the concentration of collagen in the gel affects the rate at which OTC released from the collagen matrix. The denser the collagen gel is, the slower the release of the drug. In it In this example, the kinetics of OTC release were studied during a total of 124 hours after injection of the tested complex in the subcutaneous tissue of a total of six rabbits.

In Figure 4, curve A shows that OTC in water reaches its maximum in serum shortly after injection and can not be detected after 18 or 20 hours. Curve B shows the OTC concentration in serum for OTC complexed with a collagen matrix at a weight ratio between gel complex and water of 1:20 and curve C at 3:20. The release of OTC is faster for the less concentrated the gel according to curve B.

Example 8 Collagen gel containing 3% collagen, measured as dry matter, 464 G09 14 was mixed with tetracycline to two concentrations, which contained (A) 50 mg TC / ml and (B) 100 mg TC / ml gel. After mixture with 0.3 ml 3% glutaraldehyde (Gl) per ml gel (G) Complex A was injected at a dose of 2 ml / kg body weight and complex B was injected at a dose of 1 ml / kg. Plasma level con- concentrations of TC in mg / ml are shown in curves A and B in Figure 5.

Complex A was also injected at a dose of 1 ml / kg and is shown through curve C in Figure 5. The actual plasma levels of tetra- cyclin during the period up to 5 days after injection is shown in figure 5. the values in Figure 5 show that both the actual concentration of tetracycline and the surface geometry of the implant the level of play of the drug release from the gel and the level of tetracycline in plasma.

Claims (16)

15 464 009 PATENT CLAIMS Synthetic blood vessel graft, characterized in that it comprises a tubular flexible porous graft substrate of a synthetic fiber having a porosity of less than about 3,000 ml / min-cm * (purified water at 120 mm Hg), graft substrate on the inner surface and across the porous substrate to the outer surface have crosslinked collagen fibrils, which are complexed with an effective amount of a drug and mixed with a plasticizer to make the graft blood tight and flexible and to provide continuous release of the drug moiety in the complex after implantation. Blood vessel transplant according to claim 1, characterized in that the drug component of the complex is a pharmaceutical agent selected from the group consisting of antimicrobial agents, antibacterial agents, antifungal agents, antithrombogenic agents, cell division promoters and mixtures thereof. . Blood vessel grafts according to claim 1, characterized in that the collagen fibril drug complex is delivered in at least three applications, which consist of the supply of water slurries of collagen fibrils which have been dried between the applications 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 4, characterized in that the porous substrate is knitted. Blood vessel graft according to claim 4, characterized in that the porous substrate is Woven. 464 009 16 Blood vessel transplant according to claim 4, characterized in that the inner and outer surface of the substrate has a velor surface. Blood vessel grafts according to claim 1, characterized in that the complex of collagen fibrils and drugs has been crosslinked by exposure to formaldehyde vapor. Blood vessel transplant according to claim 1, characterized in that the plasticizer is a biologically compatible polyvalent material. Blood vessel transplant according to claim 9, characterized in that the plasticizer is sorbitol. ll. Blood vessel grafts according to claim 9, characterized in that the plasticizer is glycerol. A process for the manufacture of a collagen-containing synthetic blood vessel graft for drug delivery, characterized in that it comprises: providing a porous tubular flexible synthetic graft substrate; applying a water slurry of a complex of collagen fibrils, complexed with a drug and mixed with a plasticizer, to at least the inner surface of the 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. A method according to claim 12, characterized in that the steps of applying a water slurry of collagen fibrils to the substrate, kneading and drying are repeated at least three times. f! 01 17 464 009 l4. Synthetic blood vessel graft, characterized in that it comprises a tubular flexible graft substrate of polyethylene terephthalate of a synthetic fiber with a porosity of less than about 3,000 ml / min-cm "(purified water at 120 mm Hg), the inner surface has at least five applications of cross-linked collagen fibrils, complexed with a drug and 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. 15. A method according to claim 12, characterized in that the crosslinking takes place by means of formaldehyde vapor. Blood vessel grafts according to claim 1, characterized in that the water slurry contains from 0.5 to 5.0% by weight of collagen fibrils and from 4 to 12% by weight of plasticizer.