MXPA99001763A - Graft prosthesis, materials and methods - Google Patents

Abstract

A graft prosthesis (11), materials and method for implanting, transplanting, replacing, or repairing a part of a patient. The graft prosthesis includes a purified, collagen-based matrix structure removed from a submucosa tissue source. The submucosa tissue source is purified by disinfection and removal steps to deactivate and remove contaminants, thereby making the purified structure biocompatible and suitable for grafting on and/or in a patient.

Description

PROSTHESIS FOR GRAFTING; MATERIALS AND METHOD TO IMPLANT, TRANSPLANT, REPLACE OR REPAIR ONA PART OF A PATIENT DESCRIPTION REFERENCE TO RELATED REQUESTS This application claims priority over the US Patent Applications Nos. Of Series 60 / 024,542 and 60 / 024,693, filed August 23, 1996 and September 6, 1996, respectively, each of which is hereby incorporated by reference in its entirety. TECHNICAL FIELD The present invention relates in general to a medical structure and, in particular, to grafts for grafts and to materials and methods therefor. BACKGROUND OF THE INVENTION I Tissue implants have been manufactured in purified form and derived from collagen-based materials and have been described in the scientific literature. Cohesive films of high tensile strength have been manufactured using collagen molecules or collagen-based materials. However, aldehydes have generally been used to crosslink the collagen molecules to produce films having a high tensile strength. With these REF. 29471 types of materials, the aldehydes can be removed from the film, for example by hydrolysis. Because such residues are cytotoxic, the films are poor tissue implants. Other techniques have been developed to produce collagen-based tissue implants that avoid the problems associated with collagen molecules cross-linked with aldehydes. One such technique is illustrated in US Pat. No. 5,141,747, in which the collagen molecules are crosslinked or coupled to their amino epsilon lysine groups, followed by denaturation of the coupled and preferably modified collagen molecules. The described use of such collagen material is for repair of the eardrum membrane. While such membranes are described as exhibiting good physical properties and which can be sterilized by subsequent processes, they are not capable of remodeling or generating cell growth or, in general, promoting new growth and healing of damaged or diseased tissue structures. In general, researchers of surgical techniques have worked for many years to develop new techniques and materials to be used in implants and grafts, to replace or repair damaged or diseased tissue structures, for example, blood vessels, muscles, ligaments, tendons and Similar. Currently, it is not uncommon, for example, for an orthopedic surgeon to harvest an autologous or allogeneic label tendon to be used as a replacement for a fragment of the cruciate ligament of the patella. Surgical methods of such techniques are known. In addition, it has been common for surgeons to use implantable prostheses formed of plastic, metallic and / or ceramic structures to reconstruct or replace physiological structures. However, despite its wide use, prostheses implanted by surgery present numerous risks for the patient. Researchers have also attempted to develop satisfactory polymeric or plastic materials to serve as functional tissue structures and / or other connective tissues, for example, those involved in hernias and joint injuries by dislocation. It has been found that it is difficult to provide a durable plastic material that is suitable for long-term connective tissue replacement ... The tissues surrounding the plastic material can become infected and the difficulties in treating such infections often lead to failure of the implant or prosthesis. As mentioned above, various collagen-based materials have also been used for the aforementioned tissue replacements; however, these materials still do not exhibit the tensile strength requirement and also presented problems with infections and other immunogenic responses, encapsulation, or presented other problems when they might have a load of antibiotics, growth factors and the like. For example, U.S. Patent No. 4,956,178 describes a submucosa collagen matrix that is obtained from the mammalian intestinal tract.; however, it was described that the collagen matrix is loaded with antibiotics. In a related patent, U.S. Patent No. 5,372,821, it is disclosed that a submucosa collagen matrix can be sterilized by conventional techniques, for example, tanning with aldehyde, propylene oxide, gamma radiation and peracetic acid. Non-specific processing steps are described except that the submucosa layer first detaches from the surrounding tissue prior to the sterilization treatment. Therefore, there is a need to obtain better purified forms of collagen-based matrices from tissue sources. Also, there is a need to provide a process whereby the ease of removal of such matrices from tissue sources is improved to obtain such improved purified products. The present invention addresses these needs. BRIEF DESCRIPTION OF THE INVENTION According to a preferred embodiment of the present invention there is provided a graft prosthesis that includes a matrix structure based on purified collagen, coming from a tissue source of submucosa, wherein the purified structure has a level of contaminants which makes the purified structure biocompatible. Another preferred embodiment of the present invention provides a graft prosthesis that includes a matrix structure based on purified collagen, from a tissue source of submucosa, wherein the purified structure has an endotoxin level of less than 12 endotoxin units per gram. Another preferred embodiment of the present invention provides a graft prosthesis that includes a matrix structure based on purified collagen, coming from a tissue source of submucosa, wherein the purified structure has a content level of nucleic acids of less than 2 micrograms per milligram . Another preferred embodiment of the present invention provides graft prostheses that include a matrix structure based on purified collagen, originating from a tissue source of submucosa, wherein the purified structure has a virus level of less than 500 plaque forming units per gram. The present invention also provides a graft prosthesis that includes a matrix structure based on purified collagen, coming from a tissue source of submucosa, wherein the purified structure has a level of processing agents of less than 100,000 parts per million per kilogram. Another embodiment of the present invention relates to a method for obtaining a collagen-based matrix from a tissue source of submucosa. The method includes treating the tissue source of submucosa with a disinfecting agent to obtain a tissue source of disinfected submucosa and removing the collagen-based matrix from the tissue source of disinfected submucosa. Another preferred embodiment of the present invention provides a method for obtaining a collagen-based matrix from a tissue source of submucosa, which includes obtaining a tissue source of submucosa that has been treated with a disinfecting agent and removing the collagen-based matrix from the tissue source of submucosa.

The present invention also relates to a composition that includes a structure containing collagen from a tissue source, initially containing the structure and other tissue, wherein the structure containing collagen has a level of endotoxins no greater than 12 units of endotoxins per gram. The present invention also provides a matrix containing purified collagen obtained from a tissue source of a mammal, wherein the matrix includes mammalian submucosa tissue and which is obtained by a process that includes disinfecting the mammalian tissue source and then removing the structure of the disinfected mammalian tissue source obtained. In preferred aspects, the present invention provides purified forms of submucosa tissue collagen matrices derived from the alimentary, respiratory, urinary or genital tract of animals, wherein the matrices have a bioburden level substantially zero and / or which are essentially free from pyrogens. A matrix of preferred collagen can be implanted into a human or animal patient without causing a cytotoxic response, infection, rejection of the implant or any other deleterious effect in the majority of patients. While a preferred implantable collagen matrix according to some aspects of the present invention comprises mainly the submucosa fabric, the collagen matrix in this case may also comprise partial layers of lamellar muscular mucosa, muscular mucosa, lamina propria, an extract layer compact and / or other tissue materials, depending on the source from which it is derived. Further, in accordance with the present invention, there is provided a delaminated submucosa fabric collagen matrix which is derived from the alimentary, respiratory, urinary or genital tract of animals or humans, wherein the collagen matrix of purified submucosa is produced by the delamination or detachment of a dismfected submucosa tissue source to obtain the collagen matrix of delaminated submucosa tissue. An advantageous matrix can be obtained, for example, by a process comprising treating a source of unprocessed, non-delaminated submucosa fabric from the alimentary, respiratory, urinary or genital tract of animals, with a disinfecting agent, which is followed by the delamination or detachment of the collagen matrix of the submucosa tissue from the tissues that surround it. The preferred collagen matrix has a bioburden substantially zero and can be implanted into a human or animal patient without causing any cytotoxic response, infection, rejection of the implant or any other harmful effect in the majority of patients. Still in accordance with the present invention, there is provided a method for obtaining a collagen matrix of pure delaminated submucosa fabric, in a substantially sterile state, comprising delaminating a tissue source of dismfected submucosa fabric to obtain the collagen matrix of submucosa tissue delaminated. A preferred method comprises treating a source of non-delaminated submucosa fabric from the alimentary, respiratory, urinary or genital tract of animals or humans, with a disinfecting agent, which is followed by delamination or detachment of the submucosa fabric from the other tissues. attached to said submucosal fabric. Still in accordance with the present invention, there is provided a highly pure submucosa fabric collagen matrix derived from the alimentary, respiratory, urinary or genital tract of animals, which has a substantially zero bioburden and wherein the submucosa fabric collagen matrix is substantially it does not contain surface residues, that is, it does not substantially include muscle tissue, mucous layers, lipids or cellular debris. The preferred collagen matrix can be implanted into a human or animal patient without causing any cytotoxic response, infection, rejection of the implant or any other harmful effect on the patient. Still in accordance with the present invention, there is provided a highly pure submucosa fabric which is derived from the alimentary, respiratory, urinary or genital tract of animals and wherein the submucosa fabric is delaminated or peeled off in a substantially sterile condition, comprising growth factors and it is produced by rinsing the delaminated submucosa fabric source with a solvent, for example water, followed by a treatment with a disinfecting agent, preferably a peracid, at a pH of about 1.5 to about 10, followed by delamination or detachment of the fabric. submucosa of the tissues that surround it. The peracid is buffered at pH levels greater than 7. Desirably, the collagen matrices produced in this manner have a highly substantial content of one or more growth factors. Still in accordance with the present invention, there is provided a tissue graft composition that includes a collagen matrix of submucosa fabric that is essentially free of pyrogens. More preferably, said compositions will include a submucosa fabric collagen matrix having a pyrogen content of about 1 endotoxin unit per gram (EU / g) or less. Still in accordance with the present invention, a highly pure submucosa fabric as described above will demonstrate angiogenesis in vivo upon implantation in a human or animal patient. The present invention relates to implantable tissue constructions, to a process for producing such implantable tissue constructions and to their use to promote the regrowth and healing of diseased or damaged tissue structures.

More particularly, the present invention relates to purified forms of a collagen matrix of submucosa fabric suitable for use as an implantable tissue, and to methods of producing such purified forms of this implantable collagen-based tissue. These and other aspects of the present invention will be apparent to those skilled in the art upon review of the following description. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 provides a perspective view of a prosthesis structure for tubular graft in accordance with the present invention. DETAILED DESCRIPTION OF THE INVENTION For the purposes of promoting an understanding of the principles of the present invention, reference will now be made to certain preferred embodiments thereof and specific language will be used to describe them. However, it should be understood that it is not intended to limit the scope of the invention in such forms as alterations, further modifications, and applications of the principles thereof as described herein, which ordinarily can occur to those skilled in the art. to which the invention relates. In the descriptions herein, a number of terms are used. In order to provide a clear and consistent understanding of the description and the claims, the following definitions are provided. Bioburden - refers to the number of live microorganisms, expressed in colony forming units (CFU), that are on and / or in a given amount of the material. Examples of microorganisms include bacteria, fungi and their spores. Disinfection - refers to a reduction in the bioburden of a material. . Sterile - refers to a condition in which a material has a bioburden such that the probability of having a live microorganism (CFU) on and / or in a given section of the material, is one in a million or less. Pyrogen - refers to a substance that produces a febrile response after being introduced into a host. Endotoxin - refers to a particular pyrogen that is part of the cell wall of a gram-negative bacterium. Endotoxins are continuously released from bacteria and contaminate the materials. Purification - refers to the treatment of a material to remove one or more contaminants that are in the material, for example contaminants with which the material occurs in nature and / or microorganisms or components thereof that are in the material. Illustratively, the contaminants may be those that are known to cause toxicity, infectivity, pyrogenicity, potential irritation, reactivity, hemolytic activity, carcinogenicity and / or immunogenicity. Biocompatibility - refers to the ability of a material to pass the biocompatibility tests established in the Standard of the International Standards Organization (ISO) No.10993 and / or of the Pharmacopoeia of the United States (US Pharmacopeia, USP) 23 and / or in the Memorandum of the Blue Book of the Food and Drug Administration of the United States (Food and Drug Administration, No. G95-1, entitled "Use of International Standard ISO-10993, Biological Evaluation of Medical Devices Part-1: Evaluation and Testing 'Typically, these tests evaluate the toxicity, infectivity, pyrogenicity, potential irritation, reactivity, hemolytic activity, carcinogenicity and / or immunogenicity of a material.A structure or biocompatible material when introduced into most of patients will not cause any adverse reaction or response.In addition, it is contemplated that biocompatibility may be affected by other contaminants such prions, surfactants, oligonucleotides and other agents or contaminants that affect biocompatibility. Pollutant - refers to an unwanted substance on, attached to, or within a material. This includes, but is not limited to: bioburden, endotoxins, processing agents such as antimicrobial agents, blood, blood components, viruses, DNA, RNA, spores, fragments of unwanted tissue layers, cell debris and mucosa. Submucosal fabric - refers to a layer of connective tissue containing collagen, present under the mucosa in most parts of the alimentary, respiratory, urinary and genital tracts of animals. As described above, the present invention generally provides prostheses for grafts and materials that include a matrix structure based on purified collagen, and methods for obtaining and using same. Prostheses for advantageous grafts of the present invention are obtained from a tissue source of submucosa, for example including animal tissues such as human tissues or from other mammals, for example porcine, bovine or ovine tissues. The submucosa fabric, like many animal tissues, is generally aseptic in its natural state, as long as the human being or the animal does not suffer from infection or disease. This is particularly the case since the submucosal fabric is an inner layer within the alimentary, respiratory, urinary and genital tract of animals. Accordingly, it is generally not exposed to bacteria or other cellular debris such as the epithelium of the intestinal tract. A feature of the present invention is the discovery that by disinfecting the tissue source of the submucosa fabric prior to its delamination or detachment, the aseptic state of the submucosa fabric can be preserved or substantially preserved, particularly if the delamination process occurs. sterile In particular, it has been found that disinfecting the submucosa tissue source, followed by a removal of a purified matrix that includes the submucosa fabric, for example by delaminating the submucosa fabric of the muscular tunic and the mucosal tunic, minimizes the exposure of the fabric. submucosa to bacteria and other contaminants. In turn, this makes it possible to minimize the exposure of the isolated submucosa fabric matrix to disinfectants or sterilizing agents if desired, thereby substantially preserving the inert biochemistry of the submucosa fabric and many of the beneficial effects of the submucosa fabric. . An implantable collagen matrix of submucosal fabric according to the present invention, as indicated above, can be obtained from the alimentary, respiratory, urinary or genital tract of animals. Preferably, the tissues of the submucosa fabric, which are based on collagen and therefore are predominantly collagen, are derived from the mammalian alimentary tract and preferably from the intestinal tract of pigs. A more preferred source of whole small intestine is harvested from mature adult pigs weighing more than about 450 pounds (204 kilograms). The intestines taken from healthy non-diseased animals will contain blood vessels and a supply of blood within the intestinal tract, as well as several microbes such as E. coli contained within the lumen of the intestine. Therefore, disinfecting the entire bowel prior to delamination of the submucosa fabric substantially removes these contaminants and provides a preferred implantable submucosal tissue, which is substantially free of blood and blood components as well as of any other microorganisms, pyrogens or others. pathogens that may be present. Indeed, it is believed that this method substantially retains the inert aseptic state of the submucosa fabric, although it should be understood that the present invention is not intended to be limited by any theory. It is also desirable that the collagen matrix according to the present invention be substantially free of any antibiotic, antiviral agent or any type of antimicrobial agent that could affect the inert biochemistry of the matrix and its effectiveness when implanted. In the past, a method for the treatment of such tissue material was to rinse the delaminated tissue in saline and immerse it in an antimicrobial agent, for example, as described in US Pat. No. 4,956,178. While such techniques may optionally be practiced with the isolated submucosa of the present invention, the preferred processes according to the present invention avoid the use of antimicrobial agents and the like, which not only could affect the biochemistry of the collagen matrix, but also which can also be unnecessarily introduced into the patient's tissues. As described above, it has been discovered that a highly pure form of a collagen matrix of implantable submucosa fabric can be obtained by first disinfecting a source of submucosa fabric before removing a purified collagen matrix including the layer of the submucosa fabric, for example, by delaminating or detaching the submucosa fabric source, certain processing advantages have also been discovered as well as better properties of the resulting submucosa fabric layer obtained by this process, including a greater ease of removing adhering tissues from the layer submucosa and a characteristic profile low in contaminants The processes of the present invention desirably include first rinsing the source of submucosa fabric one or more times with a solvent, suitably with water.The step of rinsing is followed by a treatment with an agent The disinfectant, desirably, is an oxidizing agent Preferred disinfecting agents are peroxy compounds, preferably peroxy organic compounds and more preferably perished. Such disinfecting agents are desirably used in a liquid medium, preferably a solution, with a pH of about 1.5 to about 10, preferably a pH of about 2 to about 6 and more preferably a pH of about 2 to about 4. In the In accordance with the methods of the present invention, the disinfecting agent will generally be used under conditions and for a period which provides for the recovery of the matrix characteristics of purified submucosa as described in the present invention, preferably exhibiting a bioburden essentially of 0 and / or essentially pyrogen-free. In this regard, the desirable processes of the present invention include immersing the tissue source (e.g., immersing or bathing in a liquid medium containing the disinfecting agent for a period of at least about 5 minutes, typically in the range of about 5 minutes. minutes at about 40 hours and more typically in the range from about 0.5 hours to about 5 hours.A preferred peroxy disinfecting agent is hydrogen peroxide.The concentration of hydrogen peroxide can vary from about 0.05 to 30% by volume. the concentration of hydrogen peroxide is about 1 to 10% by volume and more preferably about 2 to 5% by volume The solution may or may not be buffered at a pH of about 5 to 9. Preferably, the pH is about 6 to 7.5.These concentrations can be diluted in water or in an aqueous solution of approximately about 30% in volume of alcohol. The most preferred alcohol is ethanol. The temperature of the solution can vary from about 15 to 50 ° C. Preferably, the temperature of the solution is about 20 to 40 ° C. More preferably, the temperature of the solution is from about 32 to 37 ° C. The exposure time can vary from approximately 10 to 400 minutes. Preferably, the exposure time is from about 120 to 240 minutes. More preferably, the exposure time is from 180 to 210 minutes. A preferred organic peroxide disinfecting agent is perpropanoic acid. The concentration of the perpropanoic acid can vary from about 0.1 to 10% by volume. Preferably, the concentration of the perpropanoic acid is from about 0.1 to 1.0% by volume and more preferably from about 0.2 to 0.5% by volume. These concentrations of perpropanoic acid can be diluted in water or in an aqueous solution of about 2 to about 30% by volume of alcohol. Preferably, the alcohol is ethanol. The submucosa tissue tissue source can be exposed to the organic peroxide solution for periods of about 15 minutes to about 40 hours and more typically in the range of about 0.5 to about 8 hours. Other peroxy disinfectants are suitable for use, as described in "Peroxygen Compounds", S. Block, in Disinfection, Sterilization and Preservation, S. Block, Editor, 4th Edition, Philadelphia, Lea &; Febiger, pp. 167-181, 1991; and "Disinfection with peroxygens", M.G.C. Baldry and J.A.L. Fraser, in Industri al Bi ocídes, K. 1 ^. 10 Payne, Editor, New York, John Wiley and Sons, pp. 91-116, 1988. Another oxidizing disinfecting agent is (1,6-di (4-chlorophenyldiguanido) hexane) chlorhexidine in its digluconate form. The digluconate concentration of Chlorhexidine may vary from about 0.1 to 15% by weight. Preferably, the digluconate concentration of ^^ Chlorhexadine is from about 0.1 to 2% by weight and more preferably from about 0.2 to 5% by weight. The solution may or may not be buffered at a pH of about 5 to 8. Preferably, the pH is about 5.5 to 7. These concentrations may be diluted in water or in an aqueous solution of about 2 to about 20% by volume of alcohol. Preferably, the alcohol is ethanol at a concentration of about 5 to 10%. The temperature of the solution can vary from about 15 to 30 ° C. The exposure time can vary from approximately to 400 minutes. Preferably, the exposure time is approximately 30 to 60 minutes. Other chlorinated agents are described in "Chlorhexidine", G. W. Denton, in Disinfection, Sterilization and Preservation, S. Block, Editor, 4th Edition, Philadelphia, Lea & Febiger, pp. 274-289, 1991. In preferred preparative processes, a peracid or other disinfecting agent can be dissolved in an aqueous solution of dilute alcohol, preferably the alcohol will have from one to about 6 carbon atoms and the alcohol generally constitutes about 1. 30% in volume of the solution. Preferably, the alcohols for use in the present invention are selected from the group consisting of ethanol, propanols and butanols. Ethanol is the most preferred alcohol for these purposes. When a peracid is used in disinfection, it is preferably selected from the group consisting of peracetic acid, perpropanoic acid or perbenzoic acid. Peracetic acid is the most preferred disinfectant agent. The peracetic acid is preferably diluted in about 2 to about 10% by volume of an alcohol solution. The concentration of peracetic acid may vary, for example, from about 1.0 volume to F about 0.5 volume%. Preferably, the concentration of peracetic acid is about 0.1 to about 0.3% by volume. Hydrogen peroxide can also be used as a disinfecting agent. Alternatively, or in addition, the tissue source of submucosa tissue, for example from the small intestine, can be disinfected using disinfectants such as glutaraldehyde, formalin and the like, which are also known for their ability to introduce substantial cross-linking. to collagen matrices, in contrast to the action of other disinfectants such as perishes, which can be used to disinfect without introducing such crosslinks. Additionally, the submucosa fabric source can be treated with radiation, for example gamma radiation, for disinfection purposes. Variations in the disinfection process 20 may also include the following: 1. The intestine is treated with 0.2% peracetic acid, 5% ethanolic solution, in a ratio of 10: 1 of solution with respect to the ratio of intestine by weight . The solution has a pH of 2.6. The solution and the intestine are mixed vigorously for two hours. . The intestine is treated with 1% peracetic acid, 25% ethanolic solution, at a ratio of 5: 1 of the solution with respect to the ratio of intestine by weight. The solution has a pH of 2. The solution and the intestine are mixed vigorously for one hour. . The intestine is treated with 1% peracetic acid, 15% ethanol, and 10% hydrogen peroxide solution, at a ratio of 5: 1 of the solution with respect to the ratio of intestine by weight. The solution and the intestine are mixed vigorously for one hour. 4. The whole small intestine is rinsed four times with highly purified water for 15 minutes.

Afterwards, the intestine is subjected to an electron radiation beam of 1.5 MRAD. 5. The whole small intestine is rinsed four times with highly purified water for 15 minutes. As it moves along a conveyor belt, the intestine is subjected to high intensity pulse light, which disinfects the intestine. After the treatment described above, the layer of submucosa fabric delaminates or detaches from its source, for example from the whole intestine, from the uterus of the cow and the like. It has been found that by following this web-after-disinfection procedure, it is easier to separate the submucosal fabric layer from the tissues attached to it, for example at least the muscle tunic tissue, as compared to the layer detachment. of submucosal fabric before disinfection. Furthermore, it has been found that the resulting submucosa fabric layer, in its most preferred form, exhibits superior histology, since it has less tissue and debris adhered to its surface, as compared to a layer of submucosa fabric obtained by first delaminating the layer of submucosa. Submucosal fabric from its source and then disinfecting it. In addition, a more uniform submucosa fabric fabric can be obtained by this process, and a submucosa fabric having the same or similar physical and biochemical properties can be obtained more consistently in each separate processing. Importantly, a highly purified, substantially sterile submucosa fabric is obtained by this process. The web treatment of the submucosal fabric source is preferably carried out using a disinfected or sterile coated machine, to produce a submucosa fabric that is substantially sterile and has been minimally processed. A suitable deck machine is the Stridhs Universal Machine Model 3-U-400 from Hog Casing, commercially available from AB Stridhs Maskiner, Gotoborg, Sweden. Therefore, the bioburden levels measured are minimum or substantially zero. Of course, other means may be employed to delaminate the submucosa fabric source, without departing from the spirit of the present invention, including for example hand delamination. It has also been found that the most preferred processes in accordance with the present invention will not only eliminate or significantly reduce the contaminants contained in the collagen matrix of submucosa fabric, but also produce a fabric that substantially does not exhibit degradation of its physical and physical properties. mechanical, for example, differential porosity (ie, when one side of the submucosa layer has a greater porosity than the other side) and good strength, for example resistance to breakage. Likewise, it has been discovered that the most preferred processes do not affect the differential porosity of the submucosa tissue collagen matrix, which finally affects the efficiency level of this tissue implant. For example, the tissue is not necessarily treated with a crosslinking agent or a material that alters the porosity or inherent native structure of the collagen matrix. In addition, when hydrogen peroxide is used, the matrix as a whole has a higher porosity as well as a higher oxygen content. This helps ensure the absence of contaminants, for example endotoxins, pyrogens and the like. Likewise, in an advantageous form, the collagen-based matrices of the present invention (for example including the submucosa tissue), demonstrate the ability to induce active angiogenesis, i.e., an internal growth of the blood vessels within the tissue matrix. . In this regard, these preferred matrices of the present invention will contain beneficial components with which matrices naturally occur, including for example one or more glycosaminoglycans, glycoproteins, proteoglycans and / or growth factors (eg, Growth Transformer Factor-a). , and / or Fibroblast Growth Factor 2 (basic)). Preferred collagen-based matrices of the present invention, preferably matrices containing submucosa, are also characterized by their low levels of contaminants, which are set forth in Table 1 below, which shows each level of contaminant taken individually or in any combination with any or all of the other levels of pollutants described. The abbreviations in Table 1 are as follows: CFU / g = colony forming units per gram; PFU / g = plaque forming units per gram; μg / g = micrograms per milligram; ppm / kg = parts per million per kilogram.

TABLE 1 The even more preferred collagen-based matrices of the present invention contain an endotoxin level of less than 1 EU / g and preferably less than 0.5 EU / g. Collagen-based matrices according to the present invention can be processed in any number of ways to obtain collagenous matrices useful both in vitro and in vivo. For example, the submucosa can be configured in some way to obtain tissue grafts useful in vascular applications, for example as is generally described in U.S. Patent Nos. 2,127,903 and 4,902,508. 7th hour with reference to Figure 1, for use in vascular grafts, a generally tubular graft prosthesis structure 11 is formed with or including the collagen-based matrix 12, whose diameter "D" approximates that of the recipient blood vessel. In one embodiment, this can be completed by manipulating a tubular segment or sheet of the submucosa fabric to define a cylinder having a diameter "D" approximately equal to that of the recipient blood vessel, and suturing, joining or in some way securing the longitudinal seam 13 to form a properly dimensioned tubular vascular graft having a lumen 14 for the passage of blood. In illustrative preparative procedures, the graft is formed on a sterile mandrel or rod having an outer diameter approximately equal to that of the vessel to be grafted.

For example, the rod is inserted into the lumen of a segment of submucosal fabric that retains its native tubular shape. The redundant tissue is then collected and the desired diameter of the lumen is achieved by suturing along the length of the graft (for example, using two continuous suture lines or a single interrupted suture line) or using some other recognized tissue securing technique. in this field. Alternatively, a sheet of the submucosa fabric of the present invention is wrapped around the rod to form an overlapped seam, which may be sutured, glued or secured in some other manner, to obtain the tubular graft construction. In preferred forms, the internal luminal surface of the graft may be formed on the mucosal side of the submucosal tissue. The submucosa fabric of the present invention possesses highly desirable mechanical properties for tissue graft materials in vascular applications, including a low porosity index, a high yield and a high burst resistance. Those skilled in the art will note that the preferred tissue graft material will have sufficiently low porosity to prevent surgical hemorrhage and yet have sufficiently high porosity to allow the extension of a newly growing vasa vasorum through the graft material, to nourish the neo-intima layer and the luminal surface. The submucosa fabric fabric of the present invention can also be processed to provide fluidized compositions, for example using techniques such as those described in US Patent No. 5,275,826. In this regard, solutions or suspensions of the submucosa fabric can be prepared by grinding and / or digestion of the submucosa fabric with a protease (e.g., trypsin or pepsin), for a period sufficient to solubilize the tissue and form a substantially homogeneous solution. . The submucosa raw material, desirably, is ground by tearing, cutting, milling, liquefying or the like. Crushing the submucosa in a frozen or freeze-dried state is advantageous, although good results can also be obtained by subjecting a suspension of pieces of the submucosa fabric to a treatment with a high speed blender and removing the water, if necessary, by centrifugation. and decanting the remains. The crushed submucosa fabric can be dried, for example by lyophilization, to form a powder. Then, if desired, the powder can be hydrated; that is, it can be combined with water or a regulatory saline solution and optionally other pharmaceutically acceptable excipients, to form a fluid tissue graft composition, for example having a viscosity of about 2 to about 300,000 cps at 25 EC. The higher viscosity graft compositions may have a gel or paste consistency. The fluidized submucosal fabric of the present invention finds its use as an injectable heteroin for tissues, for example, soft or bone tissues, that need repair or augmentation, more typically to correct tissue defects induced by trauma or disease. The fluidized submucosa compositions of the present invention are also advantageously used as fillers for implant constructions comprising, for example, one or more submucosal fabric sheets formed in sealed (sutured) patches for use in cosmetic or trauma treatment procedures. In an illustrative preparation, the submucosa fabric prepared in accordance with the present invention is reduced to small pieces (eg, cut) which are emptied into a flat bottom stainless steel container. Liquid nitrogen is introduced into the container to freeze the specimens, which are then crushed in a frozen state, to obtain a thick submucosa cloth powder. Such processing can be carried out, for example, with a manual screw press with a brass ingot placed on the frozen specimens. The ingot serves as an interphase between the specimens and the spindle of the press. Liquid nitrogen can be added periodically to specimens of submucosa cloth, to keep them frozen. Other methods can be used to crush the submucosa fabric specimens, to produce a submucosa fabric powder useful in accordance with the present invention. For example, submucosa fabric specimens can be lyophilized and then ground using a hand-held screw press or other milling media. Alternatively, the submucosa fabric can be processed in a high cut blender to produce, after removing the water and drying, a submucosa fabric powder. The submucosa fabric powder can be further comminuted using a precooled mortar and a pistil, to produce a product consisting of finer particles. Once again, liquid nitrogen is used as necessary to keep the frozen solid particles during final grinding. The powder can be easily hydrated using, for example, buffer saline to produce a fluidized tissue graft material of the present invention, at the desired viscosity. To prepare another preferred fluidized material, a submucosa fabric powder can be sifted through a wire screen, collected and subjected to proteolytic digestion to form a substantially homogeneous solution. For example, the powder can be digested with 1 mg / ml pepsin (Sigma Chemical Co., St. Louis MO.) And 0.1 M acetic acid, adjusting to pH 2.5 with HCl, for a period of 48 hours at room temperature . After this treatment, the reaction medium can be neutralized with sodium hydroxide to inactivate the peptic activity. Subsequently, the solubilized submucosa can be concentrated by saline precipitation from the solution and separated for further purification and / or lyophilized to form an intestinal submucosa solubilized with protease, in powder form. The fluidized submucosa fabric compositions of the present invention have wide application in tissue replacements, tissue augmentations and / or repairs. The fluidized submucosal compositions can be used to induce new growth of natural connective tissue or bone in an area that has an existing defect. By injecting an effective amount of a fluidized submucosa composition at the site of a tissue defect or wound that needs healing, you can take advantage of the biotropic properties of the submucosa fabric. It is also possible to form large surface area constructions by combining two or more segments of submucosa fabric in accordance with the present invention, for example using techniques such as those described in US Patent No. 2,127,903 and / or International Publication No. WO 96 / 32146, dated October 17, 1996, Published International Application No. PCT / US96 / 04271, filed April 5, 1996. Thus, a plurality of submucosa fabric strips may be fused, for example by compression of overlapping areas of the strips under dehydrating conditions, to form a planar construction having a surface area greater than that of any other flat surface of the individual strips used to form said construction. The submucosa fabric according to the present invention can also be used to prepare tissue graft constructions useful in orthopedic soft tissue applications, for example in the repair of tendons or ligaments, employing techniques that have been applied in this field to others. materials for grafting of natural or synthetic origin. For example, repair techniques such as those generally described in US Patent Nos. 2,127,903 and 5,281,422 can be carried out using the submucosa fabric in accordance with the present invention. For tendon and ligament replacement applications, a segment of the submucosal fabric can be preconditioned by longitudinal stretching to an elongated length. For example, a segment of sub-mask fabric may be conditioned by a prolonged application of a load on the longitudinal axis of the segment (eg, suspending a segment weight) for a period sufficient to allow an extension of from about 10 to about 20% of the tissue segment. The graft material can also be preconditioned by stretching it in the lateral dimension. Then, the submucosal fabric segment can be configured, alone or in combination with other segments, in a variety of ways to serve as a replacement for the ligament or tendon, or as a substitute or as a patch for a torn or damaged ligament or tendon. For applications such as connective tissue graft, the segment is desirably configured to have a one layer or multiple layer configuration, wherein at least the opposite end portions and / or the opposite side portions are shaped to have multiple layers of the graft material, to obtain a reinforcement to be attached to physiological structures such as bones, tendons, ligaments, cartilages and muscles. In a ligament replacement application, the opposite ends will be attached to a first and a second bone, respectively, wherein the bones are typically articulated as in the case of the knee joint. In a tendon replacement application, a first end of the graft construction will be attached to a bone and a second end will be attached to a muscle. As indicated above, in connective tissue applications, it will be advantageous to form, manipulate or shape the end portions of the graft construction to be joined, for example, to a The bone structure, in such a way as to reduce the possibility of the graft tearing at the junction point. For these purposes, the submucosa fabric graft material can be partially folded or inverted to provide multiple layers for better grip, for example, with nails or staples. Alternatively, a segment of submucosal fabric can be folded back or ^^ 10 on itself to join the end portions, to obtain a first connective portion that is to be attached, for example, to a first bone and a bend in the intermediate portion to provide a second connective portion to be attached to a second bone, articulated with respect to the first bone. For example, one of the end portions of the submucosa tissue graft can be adapted to be pulled ^ through a tunnel, for example, in the femur and attached to it, while the other end portion can be adapt to be pulled through a tunnel in the tibia and join it to the same, to obtain a substitute for the natural cruciate ligament, where the segment that is being adapted is placed under tension between the tunnels to obtain a ligament fusion , that is, a function of tension and position that is provided by a normal ligament. Because the grafts used in orthopedic applications are typically placed under tension in their surgical facility, it is preferable to combine two or more tissue segments to obtain a multi-layer graft construction. Another object of the present invention, therefore, is to provide grafts in which two or more submucosa segments are arranged so that their end portions are together and join the end portions and / or side portions adapted to be joined. to a bone, tendon, ligament or other physiological structure. One method for providing a double segment can be to pull a tubular segment internally within another segment, to obtain a double-walled tube, whose joined ends can be attached, for example, to a bone, tendon or ligament. These double segments will provide better resistance to tensile and tensile stress. In other forms, multiple segments of submucosa fabric or strips can be arranged in a braided configuration, for example, in a diamond braid or braid configuration as a curtain cord, or in a mesh configuration, including multiple interlocking handles with handles neighboring, which can be very useful in the repair of ligaments or tendons.

The submucosa fabric of the present invention can also be used to provide an orthopedic graft to be used as a connective tissue to hold together pieces of fractured bones in an appropriate orientation in the body, wherein the tissue segment is formed to serve as a wrapping for the fracture around segments of fractured bones and that remains attached to the bone. In still other orthopedic applications, the submucosa fabric of the present invention can be used to repair bone tissue, for example using the general techniques described in US Patent No. 5,641,518. Thus, a submucosal fabric in the form of powder can be implanted in a damaged or diseased bone region to be repaired. The submucosa fabric powder can be used alone or in combination with one or more additional bioactive agents, such as physiologically compatible minerals, growth factors, antibiotics, chemotherapeutic agents, antigens, antibodies, enzymes and hormones. Preferably, the powder-shaped implant.e will be compressed to a predetermined three-dimensional shape, which will be implanted in the bone region and will substantially retain its shape during the replacement of the graft by endogenous tissues. The submucosa fabric according to the present invention can also be used as a cell growth substrate, illustratively in sheet form, paste or gel, in combination with nutrients that support the growth of cells, for example eukaryotic cells such as endothelial cells, fibroblast cells, fetal skin, osteosarcoma and adenocarcinoma cells (see for example, International Publication No. WO 96/24661 dated August 15, 1996, Published International Application No. PCT / US96 / 01842 filed February 9, 1996. In preferred forms, the submucosa fabric substrate composition will support the proliferation and / or differentiation of mammalian cells, including cells of human beings The submucosa web of the present invention can also serve as a collagenous matrix in compositions for producing transformed cells (see for example International Publication WO 96/25179 filed August 22, 1996, International Application Published No. PCT / US96 / 02136 filed on February 16, 1996; and International Publication No. WO 95/22611 dated August 24, 1995, Published International Application No. PCT / US95 / 02251 filed on February 21, 1995. Such compositions for cellular transformation will generally include purified submucosal fabric in accordance with the present invention, for example in the form of paste or fluidized, in combination with a recombinant vector (for example a plasmid) containing a nucleic acid sequence with which the target cells in vitro or in vivo will be genetically transformed. Target cells for transformation may include, for example, bone progenitor cells. The submucosa fabric of the present invention can also be used in repairs of body walls, including for example the repair of abdominal wall defects such as hernias, using techniques analogous to those described in Ann. Plast. Surg. , 1995, 35: 3740380; and J. Surg. Res. , 1996, 60: 107-114. In such applications, the preferred tissue submucosal tissue grafts of the present invention promote favorable organization, vascularity and consistency in the remodeled tissue. In dermatological applications, the submucosa fabric of the present invention can be used in the repair of partial or full thickness wounds and in the dermal augmentation using the general grafting techniques that are known in this field and in the scientific literature (see for example Armáis of Plástic Surgery 1995, 35; 381-388). In addition, in the burn treatment area, it is known that they provide a dermal substitute upon which cultured epidermal grafts (preferably cultured epidermal autografts or AECs) are transplanted. Such cultured grafts typically involve keratocyte and / or fibroblast transplants over the dermal substitute. In accordance with the present invention, the purified submucosa fabric can be used as a dermal substitute, for example, in sheet form and the AEC can be transplanted onto the submucosa fabric. In one embodiment of this aspect of the present invention, keratocytes can be transplanted, for example by seeding or transferring a sheet of keratocytes, on the mucosal side of the submucosa tissue. Fibroblasts can also be transplanted on the mucosal side and / or on the opposite (abluminal) side of the submucosa tissue. The submucosa fabric according to the present invention can also be used in tissue grafts in urogenital applications. For example, the submucosa fabric can be used in the repair of the urinary bladder to provide a platform for the regeneration of the bladder, using techniques corresponding to those described in US Pat. No. 5,645,860; Urology, 1995, 46: 396-400; and J. Urology, 1996, 155: 2098. In fluidized form, the submucosal fabric of the present invention can also be used in an endoscopic injection procedure to correct vesicoureteral reflux. In such applications, submucosal injection can be performed, for example, in the area under the ureteral orifice of a patient, to induce smooth muscle growth and the formation of collagen at the site of injection. In other areas, the tissue graft constructions formed with the fabric, submucosa of the present invention, can be used in neurological applications, for example in techniques that require a dural substitute to repair defects caused by trauma, tumor resection or surgical procedures. . In order to promote a better understanding of the present invention and its features and advantages, the following specific examples are provided. It should be understood that these specific examples are illustrative and not limiting of the present invention. EXAMPLE 1 Thirty feet of whole intestine from a mature adult pig is rinsed with water. This material is then treated with a 0.2% by volume peracetic acid solution in an aqueous solution of 5% by volume ethanol, for a period of two hours with agitation. The layer of submucosal tissue is delaminated from the entire intestine in a disinfected machine. The delaminated submucosa fabric is rinsed four (4) times with sterile water and impurities or contaminants such as endotoxins, microorganisms and pyrogens are tested. It was found that the resulting fabric had a bioburden level essentially zero. The layer of submucosal tissue was easily and consistently separated from the entire intestine and was found to have minimal tissue remnants on its surface. EXAMPLE 2 A 10-foot section of porcine complete intestine was washed with water. After rinsing, this section of the intestinal collagen source of submucosa fabric was treated for approximately two and a half hours with a solution of 0.2% by volume acetic acid in an aqueous solution of 5% by volume ethanol with stirring. After treatment with peracetic acid, the submucosal fabric layer delaminated from the entire intestine. The resulting submucosal fabric was rinsed four (4) times with sterile water. It was found that bioburden was essentially zero. EXAMPLE 3 A small section of the intestinal collagen material of submucosal fabric was implanted subcutaneously in a rat. In a period of 72 hours, significant angiogenesis was observed. EXAMPLE 4 Two sections of small intestine were processed by different methods. The first section was rinsed with tap water, disinfected for two hours in an aqueous solution of 5% by volume ethanol comprising 0.2% by volume of peracetic acid, of pH approximately 2.6, delaminated to the submucosa fabric, rinsed with purified water, it was divided into two samples and it was quickly frozen. The second section was rinsed with running water, delaminated to the submucosa fabric, rinsed with purified water, placed in a 10% neomycin sulfate solution for 20 minutes (as described in US Patent No. 4,902,508). , rinsed with purified water, divided into two samples and quickly frozen. The four samples prepared above were tested for bioburden and endotoxin level. The first two samples have biocharges of less than 0.1 CFU / g and endotoxin levels of less than 0.1 EU / g. The second two samples had respective bioburdens of 1.7 and 2.7 CFU / g and respective endotoxin levels of 23.9 and 15.7 EU / g. EXAMPLE 5 Three sections of small intestine were processed by different methods. The first one was rinsed with running water, disinfected for 2 hours in an aqueous solution of 5% by volume ethanol comprising 0.2% by volume of peracetic acid, of pH approximately 2.6, delaminated to the submucosa fabric, rinsed with purified water and it froze quickly. The second was rinsed with running water, delaminated to the submucosa fabric, rinsed with purified water, disinfected in accordance with the methods of Example I of US Patent No. 5,460,962 (treatment for 40 hours in a 0.1% aqueous solution). in volume of peracetic acid, regulated at pH 7.2) and quickly frozen. The third was rinsed with running water, delaminated to the submucosa fabric, rinsed with purified water, disinfected in accordance with the methods of Example 2 of US Patent No. 5,460,962 (treatment in a 0.1% peracetic acid solution in volume with high salt content, buffered to pH 7.2) and quickly frozen. All three samples were tested for endotoxins. The endotoxin levels were < 0.14 EU / g for the first sample, > 24 EU / g for the second sample and > 28 EU / g for the third sample. EXAMPLE 6 Two sections of porcine small intestine were infected with 7 x 10 plaque forming units (PFU) of virus. Both were exposed to 0.18% peracetic acid, 4.8% ethanol aqueous solution in a ratio of nine to one by weight of the solution with respect to the material. A first sample was immersed in this solution for 5 minutes; the second sample was submerged for 2 hours. The material processed for 5 minutes exhibited 400 PFU per gram of material. The material processed for 2 hours exhibited zero PFU per gram of material. EXAMPLE 7 A purified submucosa fabric, prepared as described herein, was tested for its content of nucleic acids. Four samples of material weighing 5 mg each were subjected to DNA / RNA extraction in the manner detailed in the DNA / RNA isolation kit of Amersham Lifescience Inc., Arlington Heights, Illinois. Quantification of nucleic acids was performed by spectrophotometric determination of the optical densities of the solution at 260 and 280 nm. The average content of nucleic acids was 1.9 ± 0.2 μm per milligram of material. A small intestine submucosa, prepared as described in US Patent No. 4,902,508, it was tested to determine its content of nucleic acids. Four samples of material weighing 5 mg each were subjected to DNA / RNA extraction as detailed in the Amersham DNA / RNA isolation kit.

The quantification of nucleic acids was performed by spectrophotometric determination of the optical densities of the solution at 260 and 280 nm. The average content of nucleic acids was 2.4 ± 0.2 μm per milligram of material. EXAMPLE 8 Sections of submucosa fabric prepared in accordance with the methods described herein, were sent to an independent testing laboratory (NamSA, Inc., Northwood, Ohio) to test biocompatibility as described in the ISO 10993 Standard. Samples were tested for Acute Systemic Toxicity according to USP (United States Pharmacopoeia, USP), Acute Intracutaneous Toxicity according to USP, Cytotoxicity, LAL Endotoxin, Material Mediated Pyrogenicity, Hemolysis by Direct Contact and Primary Skin Irritation. The samples passed all the tests, indicating that the material is biocompatible. It will be noted that variations of the above-described methods are intended to be within the scope of the present invention. For example, the tissue source of the submucosa tissue, eg stomach, whole intestine, cow uterus and the like, can be partially delaminated, treated with a disinfecting or sterilizing agent followed by delamination of the submucosa fabric. Illustratively, adhered mesenteric layers and / or serosa layers of the whole intestine can be advantageously removed prior to treatment with the disinfecting agent, followed by delamination of the remaining submucosal tissue. These steps can - or not to be followed by additional disinfection steps, for example enzymatic purification and / or removal of the nucleic acids. Alternatively, the submucosa fabric source can be minimally treated with a disinfecting agent or the like, then delaminated the submucosa fabric of the muscle tunic and the mucosal tunic, followed by a complete disinfection treatment to obtain the desired levels of pollutants All these variations and modifications are contemplated as part of the process described herein and within the scope of the present invention. Furthermore, it will be noted that the publications 15 cited herein are indicative of the techniques that experts in the field have and, therefore, each publication is incorporated in its entirety as a reference. It is noted that in relation to this date, the best method known by the applicant to bring the said invention to practice is that which is clear from the present description of the invention.

Claims (64)

1. CLAIMS Having described the invention as an antecedent, what is contained in the following is claimed as property. A graft prosthesis characterized in that it comprises: a matrix structure based on purified collagen from a tissue source of submucosa, wherein the modified structure has a level of contaminants such that it makes it biocompatible.
2. 2. The graft prosthesis according to claim 1, characterized in that the purified structure has an endotoxin level of less than 12 endotoxin units per gram.
3. 3. The graft prosthesis according to claim 2, characterized in that the level of endotoxins is less than 10 units of endotoxin per gram.
4. 4. The prosthesis for grafting according to claim 3, characterized in that the level of endotoxmas is less than 5 units of endotoxin per gram.
5. 5. The graft prosthesis according to claim 4, characterized in that the level of endotoxins is less than 1 unit of endotoxin per gram.
6. 6. The graft prosthesis according to claim 1, characterized in that the purified structure has a bioburden level of less than 2 colony forming units per gram.
7. 7. The graft prosthesis according to claim 6, characterized in that the level of bioburden is less than 1 colony forming units per gram.
8. 8. The graft prosthesis according to claim 7, characterized in that the level of bioburden is less than 0.5 colony forming units per gram.
9. 9. The graft prosthesis according to claim 1, characterized in that the purified structure has a nucleic acid content of less than 10 micrograms per milligram.
10. 10. The graft prosthesis according to claim 9, characterized in that the content of nucleic acids is less than 2 micrograms per milligram.
11. The graft prosthesis according to claim 1, characterized in that the purified structure has a virus level of less than 500 plaque forming units per gram.
12. 12. The prosthesis for grafting according to claim 11, characterized in that the level of virus is less than 100 plaque forming units per gram.
13. 13. The graft prosthesis according to claim 12, characterized in that the virus level is less than 1 plaque forming unit per gram.
14. The graft prosthesis according to claim 1, characterized in that the purified structure has a level of processing agents of less than 100,000 parts per million per kilogram.
15. 15. The graft prosthesis according to claim 14, characterized in that the level of processing agents is less than 1,000 parts per million per kilogram.
16. 16. The graft prosthesis according to claim 15, characterized in that the level of processing agents is less than 100 parts per million per kilogram.
17. 17. The graft prosthesis according to claim 1, characterized in that the purified structure has a fungal level of less than 2 colony forming units per gram.
18. 18. The prosthesis for grafting according to claim 17, characterized in that the level of fungi is less than 1 colony forming units per gram ..
19. 19. The graft prosthesis according to claim 18, characterized in that the level of fungi is less than 0.5 colony forming units per gram.
20. 20. The graft prosthesis according to claim 1, characterized in that the purified structure comprises a tissue source of delaminated submucosa.
21. 21. The graft prosthesis according to claim 1, characterized in that the purified structure comprises a tissue source of disinfected and delaminated submucosa.
22. 22. The graft prosthesis according to claim 1, characterized in that the purified structure comprises a tissue source of dismfected submucosa and then delaminated.
23. 23. A graft prosthesis characterized in that it comprises: a matrix structure based on purified collagen from a tissue source of submucosa, wherein the purified structure has an endotoxin level of less than 12 units of endotoxin per gram.
24. 24. A graft prosthesis characterized in that it comprises: a matrix structure based on purified collagen from a tissue source of submucosa, wherein the purified structure has a nucleic acid content of less than 2 micrograms per milligram.
25. 25. A graft prosthesis characterized in that it comprises: a matrix structure based on purified collagen from a tissue source of submucosa, wherein the purified structure has a virus level less than 500 plaque forming units per gram.
26. 26. A graft prosthesis characterized in that it comprises: a matrix structure based on purified collagen from a tissue source of submucosa, wherein the purified structure has a level of processing agents of less than 100,000 parts per million per kilogram.
27. 27. A method for obtaining a collagen-based matrix from a tissue source of submucosa, characterized in that it comprises: treating the tissue source of submucosa with an I disinfecting agent to obtain a tissue source of disinfected submucosa; and removing the collagen-based matrix from the tissue source of disinfected submucosa.
28. 28. The method according to claim 27, characterized in that the tissue source of submucosa comes from the alimentary tract of a mammal.
29. 29. The method according to claim 28, characterized in that the mammal is a pig.
30. 30. The method according to claim 29, characterized in that the tissue source 5 of submucosa comes from the small intestine of a pig.
31. 31. The method according to claim 27, characterized in that the disinfecting agent is an oxidizing agent.
32. 32. The method according to claim 10, characterized in that the agent • Disinfectant is a peroxy compound.
33. 33. The method according to claim 32, characterized in that the disinfecting agent is an organic peroxy compound.
34. 34. The method according to claim 33, characterized in that the disinfecting agent is a peracid.
35. 35. The method according to claim 34, characterized in that the peracid is 20 selects from the group consisting of peracetic acid, perpropanoic acid and perbenzoic acid.
36. 36. The method according to claim 35, characterized in that the peracid is peracetic acid. 25
37. 37. The method of compliance with 15 claim 40, characterized in that the medium is a claim 34, characterized in that the treatment includes treating the tissue source of submucosa with a medium containing an alcohol and the peracid.
38. 38. The method according to claim 37, characterized in that the alcohol has 1 to about 6 carbon atoms.
39. 39. The method according to claim 38, characterized in that the alcohol is selected from the group consisting of ethanol, propanols and butanols.
40. 40. The method according to claim 39, characterized in that the alcohol is ethanol.
41. 41. The method according to claim 40, characterized in that the medium is an aqueous solution of ethanol containing from about 0.1 to about 0.3% by volume of peracetic acid.
42. 42. The method according to claim 34, characterized in that the treatment includes treating the tissue source of submucosa with a medium containing the peracid and having a pH of from about 2 to about 6.
43. 43. The method according to claim 42, characterized in that the medium has a pH of from about 2 to about 4.
44. 44. The method according to claim 43, characterized in that the peracid is peracetic acid and the medium contains from about 0.1 to about 0.3% by volume of peracetic acid.
45. 45. A method for obtaining a collagen-based matrix from a tissue source of submucosa, characterized in that it comprises: obtaining a tissue source of submucosa which has been treated with a disinfecting agent; and removing the collagen-based matrix from said submucosa tissue source.
46. 46. The method according to claim 45, characterized in that the tissue source of submucosa comes from a small intestine.
47. 47. The method according to claim 46, characterized in that the disinfection includes treating the tissue source of submucosa with an oxidizing agent.
48. 48. The method according to claim 47, characterized in that the treatment includes contacting the tissue source of submucosa with an aqueous medium containing the oxidizing agent.
49. 49. The method according to claim 47, characterized in that the treatment includes contacting the tissue source of submucosa with an aqueous medium containing a peroxy compound.
50. 50. The method according to claim 49, characterized in that the peroxy compound is a peracid.
51. 51. The method according to claim 50, characterized in that the peracid is peracetic acid.
52. 52. The method according to claim 51, characterized in that the medium comprises an alcohol.
53. 53. The method according to claim 52, characterized in that the alcohol is ethanol.
54. 54. The method according to claim 51, characterized in that the small intestine comes from a pig.
55. 55. A composition characterized in that it comprises: a structure containing collagen removed from a tissue source that initially contained that structure and other tissues, wherein the structure containing collagen has a level of endotoxins no greater than 12 units of endotoxin per gram.
56. 56. The composition according to claim 55, characterized in that the collagen-containing layer is the submucosa and the tissue source is the small intestine.
57. 57. The composition according to claim 56, characterized in that the tissue source is small pig intestine.
58. 58. The composition according to claim 55, characterized in that the level of endotoxins is less than 10 endotoxin units per gram.
59. 59. The composition according to claim 58, characterized in that the level of endotoxins is less than 5 units of endotoxin per gram.
60. 60. The composition according to claim 50, characterized in that the endotoxin level is less than 1 endotoxin unit per gram.
61. 61. The composition according to claim 60, characterized by the level of endotoxins is less than 0.5 units of endotoxin per gram.
62. 62. A matrix containing purified collagen obtained from a mammalian tissue source, characterized in that the matrix comprises mammalian submucosa tissue and residual contaminants from the mammalian tissue source, wherein the structure is obtained by a process comprising disinfecting the tissue of the mammalian tissue. mammal and then remove the disinfected mammalian tissue structure.
63. 63. The composition according to claim 62, characterized in that the disinfection includes contacting the mammalian tissue source with an aqueous solution containing a peracid.
64. 64. The composition according to claim 63, characterized in that the peracid is peracetic acid.