INGREDIENTS OF NON-ANTIGENIC ICL, DEGRADED, OF PER-ACETIC ACID Background of the Invention 1. Field of the Invention: This invention is in the field of implantable biological prostheses. The present invention is a biocompatible, elastic, non-antigenic tissue prosthesis, which can be designed in a variety of ways and used to repair, augment or replace mammalian tissues and organs. The prosthesis is gradually degraded and remodeled by host cells which replace the implanted prosthesis to restore structure and function and is useful for the repair and reconstruction of organs. 2. Brief Description of the Background of the Invention: Despite the increasing sophistication of medical technology, the repair and replacement of damaged tissues remain a serious, expensive and frequent problem in health care. Currently, implantable prostheses are made from a number of treated natural and synthetic materials. The ideal prosthetic material must be chemically inert, not carcinogenic, capable of withstanding mechanical stress, capable of being manufactured in the required form, and sterilizable, without still being physically modified by tissue fluids, or excite an external or inflammatory body reaction, inducing a state of allergy or hypersensitivity, or, in some cases, promote visceral adhesions (Jenkins SD et al., Surgery 9 (2): 392-398, 1983). For example, defects of the body wall that can not be closed with autogenous tissue due to trauma, necrosis or other causes, require repair, augmentation, or replacement with synthetic mesh. By reinforcing or repairing abdominal wall defects, various prosthetic materials have been used, including tantalum gauze, stainless steel mesh, DACRON®, ORLON®, FORTISAN®, nylon, woven polypropylene (MgARLEX®), expanded polytetrafluoroethylene microporous (GORE) -TEX®), silicone rubber reinforced with dacron (SILASTIC®), polyglactin 910 (VICRYL®), polyester (MERSILENE®), polyglycolic acid(DEXON®), processed sheep dermal collagen (PSDC®), degraded bovine pericardium (PERI-GOARD®), and preserved human dura (LYODURA®). Not a single prosthetic material has gained universal acceptance. The main advantages of metallic mesh are that they are inert, resistant to infection and can stimulate fibroplasia. Its main disadvantage is the fragmentation that occurs after the first year of implantation as well as the lack of malleability. Synthetic meshes have the advantage of being easily molded and, except for nylon, retain their tensile strength in the body. Its main disadvantage is the lack of unassability, susceptibility to infection, and its interference with wound healing. Synthetic absorbent meshes have the advantage of not being permanent at the implantation site, but often have the disadvantage of losing their mechanical strength, due to dissolution by the host, before adjusting the internal growth of the cell and the tissue. The most widely used material for replacement and for strengthening the abdominal wall during hernia repair is MARLEX®; however, several researchers have reported that with scar contraction, polypropylene mesh grafts become distorted and separate from surrounding normal tissue in a vortex of fibrous tissue. Others have reported moderate to severe adhesions when using llARLEX®. It is currently believed that GORE-TEX® is the most chemically inert polymer and has been found to cause minimal external body reaction when implanted. There is a greater problem with the use of polytetrafluoroethylene in a contaminated wound since it does not allow any macromolecular drainage, which limits the treatment of infections. Collagen first gained utility as a material for medical use because it was a natural biological prosthetic substitute that was supplied abundantly from various animal sources. The design goals for the original collagen prostheses were the same as for the synthetic polymer prostheses; The prosthesis must persist and act essentially as an inert material. With these objectives in mind, purification and degradation methods were developed to improve mechanical strength and decrease the rate of collagen degradation(Chvapil, M. et al., (1977) J. Bi omed. Mater. Res. 11:297-314; Kligman, A.M. et al., (1986) J. Dermatol. Surg. Oncol. 12 (4): 351-357; Roe, S.C. et al., (1990). Artif. Organs. 14: 443-448. oodroff, E.A. (1978). J. Bioeng. 2: 1-10). The degrading agents originally used include glutaraldehyde, formaldehyde, polyepoxides, diisocyanates (Borick P.M. et al., (1964) J. Pharm. Sci. 52: 1273-1275), and acyl azides. The processed dermal sheep collagen has been studied as an implant for a variety of applications. Before implantation, sheep dermal collagen is tanned with hexamethylene diisocyanate (van ache, PB et al., Biomaterials 12 (March): 215-223, 1991) or glutaraldehyde (Rudolphy, VJ et al., Ann Thorac Surg 52 : 821-825, 1991). Glutaraldehyde, probably the most widely used and studied degradation agent, was also used as a sterilizing agent. In general, these degrading agents generated collagen material that resembled more a synthetic material than a natural biological tissue, both mechanically and biologically. The native collagen of degradation reduces the antigenicity of the material (Chvapil, M. (1980) Reconstituted Collagen pp. 313-324 In: Viidik, A., Vuust, J. (Eds), Collagen Biology, Academic Press, London; Harjula, A. et al. (1980) Ann. Chir. Gynaecol. 69: 256-262.) By binding the antigenic epitopes, rendering them either inaccessible to phagocytosis or not recognizable by the immune system. However, data from studies using glutaraldehyde as the degradation agent are difficult to interpret since the glutaraldehyde treatment is also known to leave behind cytotoxic residues (Chvapil, M. (1980), supra, Cooke, A. and cois. (1983) Br. J. Exp. Path. 64: 172-176; Speer, DP et al.(1980) J. Biomed. Mater. Res. 14: 753-764; Wiebe, D. and cois. (1988) Surgery. 104: 26-33). Accordingly, it is possible that the reduced antigenicity, associated with the degradation of glutaraldehyde, is due to non-specific cytotoxicity rather than a specific effect on the antigenic determinants. Glutaraldehyde treatment is an acceptable way to increase the durability and reduce the antigenicity of collagen materials compared to those that do not degrade. However, glutaraldehyde degradation collagen materials significantly limit the body's ability to reshape the prosthesis (Roe, S.C. et al. (1990), supra). All of the above problems associated with traditional materials result, in part, from the body's inability to recognize any implant as "inert". Although of biological origin, the extensive chemical modification of collagen tends to make it "foreign". To improve the long-term performance of implanted collagen devices, it is important to retain many of the properties of natural collagen tissue. In this "tissue engineering" approach, the prosthesis is designed not as a permanent implant but as a scaffold or model for regeneration or remodeling. Tissue engineering design principles incorporate a requirement for isomorphic tissue replacement, where biodegradation of the implant matrix occurs at approximately the same functional rate of tissue replacement (Yannas, IV (1995) pp Regeneration Models). 1619-1635 In: Bronzino, JD (ed.), The Manual of Biomedical Engineering, CRC Press, Inc., Boca Raton, Florida). When such a prosthesis is implanted, it must immediately serve its mechanical requirement and / or biological function as a part of the body. The prosthesis must also support proper cellularization of the host through the internal growth of mesenchymal cells, and over time, through replacement of the isomorphic tissue, replacing it with host tissue, where the host tissue is a functional analogue of the original tissue . In order to do this, the implant must not produce a significant humoral immune response or be either cytotoxic or pyrogenic to promote healing and new tissue development. Prostheses or prosthetic material derived from explanted mammalian tissue have been extensively investigated for surgical repair or for the replacement of tissues and organs. The tissue is typically processed to remove cellular components that leave a natural tissue matrix. Further processing, such as degradation, disinfection or shaping, has also been investigated. US Pat. No. 3,562,820 to Braun discloses tubular, sheet and strip shapes of prostheses formed from submucosa bonded together by the use of a binder paste such as collagen fiber paste or by the use of an acid or alkaline medium US Patent No. 4,502,159 to Woodroof provides a tubular prosthesis formed from pericardial tissue in which tissue is cleaned of fats, fibers and foreign debris and then placed in phosphate-buffered saline. The pericardial tissue is then placed on a mandrel and the scar is then closed by suture and the tissue degraded afterwards. US Patent No. 4,703,108 to Silver provides a biodegradable matrix from solutions of soluble collagen or insoluble collagen dispersions which are cold-dried and then degraded to form a porous collagen matrix. US Patent No. 4,776,853 to Klement provides a process for preparing biological material for implantation which includes the extraction of cells using a hypertonic solution at an alkaline pH followed by a high-salt solution containing detergent; by subjecting the tissue to a protease-free enzyme solution and then to an anionic detergent solution. U.S. Patent No. 4,801,299 to Brendel discloses a method for processing complete structures derived from the body for implantation by treatment of body derived tissue with detergents to remove cellular structures, nucleic acids and lipids, in order to leave a matrix extracellular which is then sterilized before implantation. U.S. Patent No. 4,902,508 to Badylak discloses a three-layer tissue graft composition derived from a small intestine comprising the tunica submucosa, the muscularis mucosa and the stratum compactum of the tunica mucosa. The method for obtaining the tissue graft composition comprises the abrasion of the intestinal tissue followed by the treatment with an antibiotic solution. U.S. Patent No. 5,336,616 to Livesey discloses a method for processing biological tissues by treating the tissue to remove cells, treatment with a cryoprotective solution, freezing, rehydration, and finally, inoculation with cells to repopulate the tissue. A continuing goal of the researchers is to develop implantable prostheses that can be successfully used to replace or repair mammalian tissue, such as defects of the abdominal wall and vasculature. SUMMARY OF THE INVENTION The present invention overcomes the difficulties of currently available materials and provides a prosthetic device for use in the repair, augmentation, or replacement of damaged tissues and organs. This invention is directed to a prosthetic material which, when implanted in a mammalian host, overcomes controlled biodegradation accompanied by adequate cellular replacement in vivo, or formation of new tissue, such that the originally implanted prosthesis is remodeled and ultimately replaced. by cells and tissue derived from the host. The prosthesis of this invention, a material for tissue repair, comprises a non-antigenic collagen material derived from mammalian tissue. The collagen material is capable of stratifying and joining together to form sheets, tubes, or prostheses of complex, multi-layered shapes. The bonded collagen layers of the invention are structurally stable, manageable, semipermeable and suturable. Accordingly, an object of this invention is to provide a tissue repair fabric that does not exhibit many of the drawbacks associated with many of the grafts currently used clinically. Another object is to provide a prosthetic material that allows and facilitates the internal growth of the tissue and / or the regeneration of the organ at the implantation site, which is a sterile, non-pyrogenic and non-antigenic material derived from mammalian collagen tissue. Prostheses prepared from this material, when grafted onto a recipient host or patient, do not produce a significant humoral immune response. The prostheses formed from the material concomitantly overcome the controlled bioremodelation that occurs with an adequate cell replacement in vivo in such a way that the originally implanted prosthesis is remodeled by the living cells of the patient to form a regenerated organ or tissue. A further object of the present invention is to provide a simple, repeatable method for making a fabric repair fabric. Yet another object of this invention is to provide a method for the use of a novel multipurpose tissue repair fabric in autograft, allograft, and heterograft indications. Still a further object is to provide a novel tissue repair fabric that can be implanted using conventional surgical techniques. DETAILED DESCRIPTION OF THE INVENTION This invention is directed to designed tissue prostheses, which, when implanted in a mammalian host, can serve as a repair of functioning, augmentation or replacement, of body part or tissue structure, and will overcome the controlled biodegradation that occurs concomitantly with remodeling by host cells. The prosthesis of this invention, in its various modalities, therefore has double properties: First, it functions as a substitute body part, and second, while still functioning as a substitute body part, it functions as a remodeling model for the internal growth of the host cells. For this purpose, the prosthetic material of this invention, a tissue repair fabric, was developed comprising collagen tissue derived from a mammal that becomes non-antigenic and that is capable of binding itself or another. Although the prostheses will be illustrated through the construction of various devices and constructions, the invention is not limited thereto. It will be appreciated that the design of the device in its shape and thickness is selected depending on the final indication for construction. In the preferred embodiment, the collagen material from which the prostheses are formed, or the prosthesis itself, becomes sterile, non-pyrogenic, and non-antigenic. When the prosthesis is grafted onto a host or recipient patient it does not produce a significant humoral immune response. An acceptable level of response is one that does not demonstrate any significant increase in the antibody titer for collagen tissue proteins from baseline titer levels when the blood serum obtained from a recipient of a prosthesis is examined for antibodies. to proteins in collagen tissue extracts. In the preferred method, the tissue repair material or prosthesis itself becomes non-antigenic, while maintaining the ability for the prosthesis to concomitantly overcome the controlled bioremodelation that occurs with adequate cellular replacement in vivo. The method for preparing a non-antigenic prosthetic collagen material comprises disinfecting the material by a method to prevent microbial degradation of the material, preferably by using a solution comprising peracetic acid; and the degradation of the collagen material disinfected with a degradation agent, preferably l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Also in the preferred embodiment, the collagen tissues derived from the mammalian body are used to make said collagen material. Collagen tissue sources include, but are not limited to, intestine, thigh aponeurosis, pericardium, and dura mater. The most preferred material to be used is the tunica submucosa layer of the small intestine. The tunica submucosa is separated or divided into sheets preferably from the other layers of the small intestine. This layer is referred to hereinafter as the Intestinal Collagen Layer("ICL"). In addition, the collagen layers of the prosthetic device may be of the same collagen material, such as two or more capable of ICL, or of different collagen materials, such as one or more layers of ICL and one or more layers of thigh aponeurosis. . The submucosa, or the intestinal collagen layer (ICL), coming from a mammalian source, typically pigs, cows, or sheep, is mechanically cleaned by pressure extraction of the raw material between opposite rollers to remove the muscular layers (tunica muscularis) and the mucosa (mucous tunic). The tunica submucosa of the small intestine is harder and more rigid than the surrounding tissue, and the rollers will extract by pressure the softer components of the submucosa. In the examples that follow, the ICL was mechanically collected from the porcine small intestine using a Bitterling intestinal cleansing machine. Since the mechanically cleaned submucosa may have some hidden residue, visibly not apparent, that affects the consistency of the mechanical properties, the submucosa can be chemically cleaned to remove residues and other substances, other than collagen, for example, by soaking in regulatory solutions at 4 ° C, or by soaking with NaOH or trypsin, or other known cleaning techniques. Also, alternative means using detergents such as TRITON X-100 ™ (Rohm and Haas) or sodium dodecylsulfate (SDS) can be included in the chemical cleaning method; enzymes such as dispase, trypsin, or thermolysin; and / or chelating agents such as ethylenediaminetetraacetic acid (EDTA) or ethylenebis (oxyethylenitrile) tetraacetic acid (EGTA). After cleaning, the (ICL) should be decontaminated or disinfected, preferably with the use of dilute peracetic acid solutions as described in U.S. Patent No. 5,460,962, incorporated herein by reference. Decontamination or disinfection of the material is done to avoid degradation of the collagen matrix by bacteria or proteolytic enzymes. Other solutions and disinfectant systems for use with collagen are known in the art and can be used until, after the disinfecting treatment, there is no interference with the ability of the material to remodel. In a preferred embodiment, the prosthetic device of this invention has two or more overlapping collagen layers that are joined together. As used herein, "collagen bonded layers" means a composite of two or more layers of collagen material, the same or different, treated in such a way that the layers overlap each other and are held together sufficiently by auto- lamination. The joining of the collagen layers can be carried out in several different ways: by bonding or thermal welding, adhesives, chemical bonding, or sutures. In the preferred method, and in the examples that follow, the ICL is disinfected with a solution of peracetic acid at a concentration between about 0.01 and 0.3% v / v in water, preferably about 0.1%, at a neutralized pH of between approximately pH 6 and pH 8 and stored until used at approximately 4 ° C in phosphate buffered saline (PBS). The ICL is cut longitudinally and flattened on a flat, solid plate. One or more successive layers are then superimposed one on the other. A second solid flat plate is placed on top of the layers and the two plates are held together tightly. The entire apparatus, the clamped plates and the collagen layers are then heated for a time and under conditions sufficient to effect the union of the collagen layers together. The amount of heat applied must be high enough to allow the collagen to bind, but not so high as to cause the collagen to become irreversibly denatured. The time of heating and bonding will depend on the type of layer of collagen material used, the moisture content and the thickness of the material, and the heat applied. A typical range of heat is from about 50 ° C to about 75 ° C, more typically from 60 ° C to 65 ° C and more typically 62 ° C. A typical range of times will be from about 7 minutes to about 24 hours, typically about one hour. The degree of heat and the amount of time that heat is applied can be easily deduced through routine experimentation by varying the heat and time parameters. The joining step can be carried out in a conventional oven, although other heat appliances or applications can be used, including, but not limited to, water baths, laser energy, or electric heat conduction. Immediately after heating and bonding, the apparatus is cooled, in an air or water bath, to a range of between room temperature to 20 ° C and 1 ° C. The rapid cooling, called tempering, will immediately or almost immediately stop the heating action. To carry out this step, the apparatus must be cooled, typically in a water bath, with a temperature preferably between about 1 ° C to about 10 ° C, more preferably about 4 ° C. Although cooling temperatures below 1 ° C can be used, care must be taken not to freeze the collagen layers, which can cause structural damage. In addition, temperatures above 10 ° C can be used in tempering, but if the tempering temperature is too high, then the heating can not be stopped in time to sufficiently fix the collagen layers in their current configuration. The prosthetic material or multilayer construction is preferably degraded. The degradation of the attached prosthetic device provides strength and some durability to the device to improve the handling properties. The degradation agents should be selected in order to produce a biocompatible material capable of being remodeled by the host cells. The various types of degrading agents are known in the art and can be used such as ribose and other sugars, oxidative agents and dehydrothermal methods(DHT). A preferred degradation agent is the carbodiimide hydrochloride of l-ethyl-3- (3-dimethylaminopropyl) (EDC). The degrading solution containing EDC and water may also contain acetone. In a preferred embodiment, sulfo-N-hydroxysuccinimide is added to the degradation agent (Staros, J.V., Biochem 21, 3950-3955, 1982). In a preferred embodiment, a method comprising the disinfection with peracetic acid and its subsequent degradation with EDC of the ICL material is carried out to reduce the antigenicity of the material. The immunoreactive proteins present in ICL are not sterilized, not degraded, either they are reduced or removed, or their epitopes have been modified in such a way that they no longer produce a significant humoral immune response. However, graft implants of this material show an initial, transient, inflammatory response as a result of a wound healing response. As used herein, the term "non-antigenic" means that it does not produce a significant humoral immune response in a host or patient in which a prosthesis is implanted. An acceptable level of response is one that does not demonstrate a significant increase in the antibody titer for collagen tissue proteins from baseline titer levels when blood serum obtained from a recipient of a prosthesis is examined for antibodies to proteins in collagen tissue extracts. For a patient or host previously not sensitized to collagen tissue proteins, the preferred serum antibody titer is 1:40 or less. The prostheses of the preferred embodiment are also preferably non-pyrogenic. When a prosthesis that is pyrogenic is grafted onto a host or recipient patient, it will provoke a feverish reaction in the patient, thus affecting the ability of the prosthesis to remodel. The pyrogens are examined by intravenous injection of a solution containing a sample of material in three test rabbits. A temperature sensing probe is placed in the rectum of rabbits to monitor changes in temperature. If there is an increase in temperature in any rabbit above 0.5 ° C, then the test for that sample is continued in five more rabbits. If no more than three of the eight rabbits show individual temperature increases of 0.5 ° C or more and the sum of the eight individual maximum temperature increments does not exceed 3.3 ° C, the material under examination meets the requirements for the absence of pyrogens. . (Pyrogen Test (151), pp. 1718-1719.In: The United States Pharmacopeia (USP) 23 The United States Pharmacopeial Convention, Inc., Rockville, MD). The tissue repair fabric of this invention, which functions as a substitute body part, can be flat, tubular, or complex in geometry. The shape of the fabric repair fabric will be decided by its intended use. In this way, when the joining layers of the prosthesis of this invention are formed, the mold or plate can be adapted to accommodate the desired shape. The tissue repair fabric can be implanted to repair, augment or replace damaged or diseased organs, such as abdominal wall defects, pericardium, hernias and various other organs and structures including, but not limited to, bone, periosteum, perichondrium , intervertebral disc, articular cartilage, dermis, epidermis, intestine, ligaments and tendons. In addition, the tissue repair fabric can be used as a vascular or intra-cardiac plaster, or as a heart replacement valve. For example, flat sheets can be used to support the prolapsed or hypermobile organs by using the leaf as a sling for the organs. This sling can support organs such as a bladder or a uterus. Tubular grafts can be used, for example, to replace cross sections of tubular organs such as vasculature, esophagus, trachea, intestine and fallopian tubes. These organs have a basic tubular shape with an external surface and a luminal surface. In addition, planar sheets and tubular structures can be formed together to form a complex structure in order to replace or augment the cardiac or venous valves. In addition to functioning as a substitute body part or support, the second function of the prosthesis is that of a bioremodelation model or scaffold. "Bioremodelation" is used herein to refer to the production of structural collagen, vascularization, and epithelialization by internal growth of host cells at a functional rate approximately equal to the biodegradation rate of the implanted prosthesis by cells and host enzymes . The tissue repair fabric retains the characteristics of the prosthesis originally implanted while remodeling the host throughout, or substantially all, the host tissue, and as such, is functional as an analogue of the tissue that it repairs or replaces. The mechanical properties include mechanical integrity such that the fabric repair fabric resists slipping during bioremodelation, and is additionally manageable and suturable. The term "manageable" means good handling properties. The term "suturable" means that the mechanical properties of the layer include the suture retention that allows needles and suture materials to pass through the prosthesis material at the time of suturing the prosthesis in sections of native tissue, a process known as anastomosis. During suturing, such prostheses should not tear as a result of the tensile forces applied to them by suture, nor should they tear when the suture is knotted. The sutureability of the tissue repair fabric, i.e., the ability of the prosthesis to resist tearing while suturing, refers to the intrinsic mechanical strength of the prosthesis material, the thickness of the graft, the tension applied to the suture, and the speed at which the knot is closed. As used herein, the term "non-slip" means that the biomechanical properties of the prosthesis impart durability so that the prosthesis does not stretch, distend, or expand beyond normal limits after implantation. As described below, the total tension of the implanted prosthesis of this invention is within acceptable limits. The prosthesis of this invention acquires a tensile strength as a function of cellular bioremodelation after implantation by replacement of structural collagen by host cells at a rate greater than the loss of mechanical strength of the implanted materials due to biodegradation and The remodeling. The fabric repair fabric of the present invention is "semi-permeable", even when degraded. The semi-permeability allows the internal growth of the host cells for remodeling or for the placement of the collagen layer. The "non-porous" quality of the prosthesis prevents the passage of fluids that try to retain them by implanting the prosthesis. Conversely, pores may be formed in the prosthesis if the quality is required for an application of the prosthesis. The mechanical integrity of the prosthesis of this invention is also found in its ability to fold or bend, as well as the ability to cut or reduce the prosthesis obtaining a clean edge without delaminating or abrading the edges of the construction. Additionally, in another embodiment of the invention, sheared or cut collagen fibers can be included between the collagen layers by adding bulk to the construct and providing a mechanism for a differential rate of remodeling by the host cells. The properties of the construction that incorporate the fibers can be altered by variations in the length and diameter of the fibers; variations in the proportion of the fibers used, and fully or partially degraded fibers. The length of the fibers can vary from 0.1 cm to 5.0 cm. In another embodiment of the invention, collagen filaments, such as those set forth in U.S. Patent No. 5,378,469 and incorporated herein by reference, may be incorporated into the multilayer fabric repair fabric for reinforcement or for different speeds functional remodeling. For example, a propeller or "twister" of a braid of 20 to 200 denier of collagen filament can be applied to the surface of the fabric repair fabric. The size of the diameter of the helix or the collagen filament braid may vary from 50 to 500 microns, preferably from 100 to 200 microns. In this way, the properties of the fabric repair fabric layer can be varied by the geometry of the filament used for reinforcement. The functionality of the design will depend on the geometry of the braid or twine. Additionally, collagen filament constructions such as a felted woolen or woven fabric, or woven, woven, three-dimensional woven fabric can be incorporated between the layers or on the construction surface. Some modalities may also include a collagen gel between the layers alone or with a drug, growth factor or antibiotic to function as a delivery system. Additionally, a collagen gel could be incorporated with a filament or filament construction between the layers. As will be appreciated by those skilled in the art, many of the embodiments that incorporate the gel, filament or collagen filament construction will also affect physical properties such as elasticity, radial strength, kink resistance, suture retention, and flexibility. The physical properties of the filament or the filament construction can also vary as the filaments are degraded. In some embodiments, additional collagen layers may be added to the outer or inner surfaces of the collagen layers attached to create a smooth flow surface for final application as described in PCT International Publication No. WO 95 / 22301, the contents of which are incorporated herein by reference. This layer of soft collagen also promotes cellular attachment of the host which facilitates internal growth and bioremodelation. As described in PCT International Publication No. WO 95/22301, this soft collagen layer can be made of fibrillar collagen extracted with acid or non-fibrillar, which is predominantly type I collagen, but can also include other types of collagen. Collagen The collagen used can be derived from any number of mammalian sources, typically skin or bovine, porcine, or sheep tendons. The collagen has preferably been processed by acid extraction to result in a high purity fibril or gel dispersion. The collagen can be extracted by acid from the collagen source by the use of a weak acid, such as acetic, citric or formic acid. Once extracted into the solution, the collagen can be precipitated by salt using NaCl and recovered using standard techniques such as centrifugation or filtration. The details of the collagen extracted by acid from the bovine tendon are described, for example, in US 5,106,949, incorporated herein by reference. The collagen dispersions or gels for use in the present invention are generally at a concentration of about 1 to 10 mg / mL, preferably, about 2 to 6 mg / mL, and more preferably at about 3 to 5 mg / mL and a pH of about 2 to 4. A preferred solvent for collagen is diluting acetic acid, for example, from about 0.05 to 0.1%. Other conventional solvents for collagen may be used since such solvents are compatible. Once the prosthetic device has been produced, can be dried, packaged and sterilized with gamma irradiation, typically 2.5 Mrad, and stored. Terminal sterilization employing chemical solutions such as peracetic acid solutions as described in U.S. Patent No. 5,460,962, incorporated herein, may also be used. In the examples that follow, the ICL is cut longitudinally and flattened outward on a glass plate, although any firm, non-insulated, inert mold can be used. In addition, the mold can be of any shape: flat, round, or complex. In a complex or round mold, the lower and upper mold pieces will be constructed in an appropriate manner in order to form the complete prosthesis in the desired shape. Once so constructed, the prosthesis will maintain its shape. Thus, for example, if the prosthesis is configured in a round shape, it can be used as a heart valve leaflet replacement. The multi-layer fabric repair fabric can be tubularized by various alternative means or combinations thereof. The multilayer fabric repair fabric can be formed into a tube in either the normal or inverted position. The tube can be processed mechanically by suturing, using interrupted sutures with suitable suture material and it is advantageous since it allows the tube to be cut and shaped by the surgeon at the time of implantation without unraveling. Other processes for suturing the submucosa may include adhesive bonding, such as the use of fibrin-based gums or industrial-type adhesives such as polyurethane, vinyl acetate or polyepoxy. Preferably, thermal bonding techniques including laser welding or thermal welding of the scar, followed by annealing, can also be used to seal the sides of the tube thus formed. Other mechanical means are possible, such as using tubular rivets of special shape or staples. With these tubing techniques, the ends of the sides can either be bumped or overlapped. If the sides overlap, the cut suture once the tube is formed. In addition, these tubing techniques are typically made on a mandrel to determine the desired diameter. The structural tube thus formed can be maintained on the mandrel or another bolt suitable for further processing. In order to control the functional rates of biodegradation and consequently the rate of decrease of prosthesis strength during bioremodelation, the prosthesis is preferably degraded, using a suitable degradation agent, such as l-ethyl-3-3- carbodiimide hydrochloride. dimethylaminopropyl) (EDC). The degradation of the prosthesis also helps to avoid luminal slipping, by keeping the diameter of the tube uniform, and by increasing the resistance of the burst. The bond strength of a multi-layer suture or prosthesis is increased when thermal bonding or dehydration methods are used. It is believed that the degradation of the intestinal collagen layer also improves the suture retention strength by improving the resistance to slit propagation. The collagen can be deposited on the internal or external surface of the ICL as described in Example 5 of US Pat. No. 5,256,418, incorporated herein by reference. In summary, when the fabric repair fabric is for tubing, the multilayer fabric is accommodated at one end by Luer adjustments and the collagen dispersion fills the tube. This step can also be carried out as described in the above-mentioned patent application using a hydrostatic pressure head. The inner layer of collagen can also be deposited by flowing the collagen at both ends of the tube simultaneously. The tube is then placed in a bath of polyethylene glycol 20% (PEG) in saline regulated with isotonic phosphate (PBS), of neutral pH. The osmotic gradient between the internal collagen solution and the external solution of PEG in combination causes simultaneous concentration and placement of the collagen along the inner diameter of the internal structural layer wall. The tube is then removed from the PEG bath and a glass rod with a desired diameter of the prosthesis inner diameter is inserted into the collagen solution oralternatively, one end of the prosthesis is closed and air pressure is applied internally to keep the inner diameter of the tube open. The prosthesis is then allowed to dry and subsequently rehydrated in PBS. The collagen coating thus formed, in the form of a dense fibrillar collagen, fills slight irregularities in the intestinal structural layer, thus resulting in a prosthesis with both a smooth flow surface and a uniform thickness. The procedure also facilitates the attachment of the collagen gel to the intestinal collagen layer. A collagen layer of varying thickness and density can be produced by changing the deposition conditions that can be determined by changes in the routine parameter. The same procedures can be used to apply the collagen to the outer surface of the ICL to create a three-layer prosthesis. The construction of the prosthesis is thrombogenic in replacement of small diameter blood vessel. It can only be used in vascular applications in high flow vessels (large diameter). Therefore, the prosthesis should become non-thrombogenic if it should be useful for repair or replacement of small diameter blood vessels. Heparin can be applied to the prosthesis by a variety of well-known techniques. For illustration, heparin can be applied to the prosthesis in the following three ways. First, the benzalkonium heparin solution (BA-Hep) can be applied to the prosthesis by immersing the prosthesis in the solution and then drying it in the air. This procedure treats collagen with an ionically bound BA-Hep complex. Second, EDC can be used to activate heparin, then heparin is covalently attached to the collagen fiber. Third, EDC can be used to activate the collagen, then protamine is covalently bound to the collagen and then heparin is ionically bound to the protamine. Many other coating, joining and clamping methods that are well known can also be used. Tissue repair fabric treatment can be carried out with drugs in addition to or in substitution of heparin. Drugs may include, for example, growth factors to promote vascularization and epithelialization, such as macrophage-derived growth factor (MDGF), platelet-derived growth factor (PDGF), endothelial cell-derived growth factor (ECDGF). ), antibiotics to attack any potential infection of the surgical implant; or nerve growth factors incorporated in the internal collagen layer when the prosthesis is used as a conduit for nerve regeneration. In addition to or in substitution of drugs, matrix components such as proteoglycans or glycoproteins or glycosaminoglycans can be included within the construct.
The tissue repair fabric can be laser drilled to create micron-sized pores through the completed prosthesis to aid internal cell growth by using excimer lasers (eg at wavelengths KrF or ArF). The pore size may vary from 10 to 500 microns, but is preferably from about 15 to 50 microns and the spacing may vary, but approximately 500 microns are preferred to the center. The tissue repair fabric can be laser drilled at any time during the process to make the prosthesis, but it is preferably done before decontamination or sterilization. Gaps or spaces can also be formed by the phase inversion method. At the moment of stratifying the ICL, crystalline particles are distributed between the layers, which are insoluble in the liquid heat source to be bound, but must be soluble in the tempering bath or the degrading solution. If laser or dry heat is used to join the layers, then any soluble crystalline solid can be used since it is soluble in the tempering bath or the degrading solution. When the crystalline solid solubilizes and diffuses, a space that has occupied the solid remains. The size of the particles may vary from 10 to 100 microns, but is preferably from about 15 to 50 microns and the separation may vary between the particles when distributed between the layers. The number and size of the gaps formed will also affect the physical properties(ie, elasticity, resistance to twisting, suture retention, manageability). The following examples are provided to better elucidate the practice of the present invention and should not be construed in any way to limit the scope of the present invention. Those skilled in the art will recognize that various modifications may be made to the methods described herein while not departing from the spirit and scope of the present invention. EXAMPLES Example 1: Collection and Processing of the Porcine Bowel Intestinal Collagen Coating The small intestine of a pig was harvested and mechanically separated, using a Bitterling Gutter Cleaning Machine (Nottingham, UK) which forcibly removed the fat, the muscle and mucosal layers of the submucosa tunic using a combination of mechanical action and rinsing using hot water. The mechanical action can be described as a series of rollers that compress and separate the successive layers of the submucosal tunic when the intact intestine passes between them.
The tunica submucosa of the small intestine is harder and more rigid than the surrounding tissue, and the rollers remove the softer components of the submucosa under pressure. The result of cleaning the machine was such that only the submucosa layer of the intestine remained. Finally, the submucosa was decontaminated with 0.3% peracetic acid for 18 hours at 4 ° C and then rinsed in phosphate-buffered saline. The product that remained was a layer of intestinal collagen (ICL). Example 2: Various Welding Temperatures and EDC Concentrations of the ICL The effects of the welding temperature (followed by tempering), the welding time, the concentration of 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide (EDC), the concentration of acetone and the degradation time, after welding to a welding resistance were examined for the application of two-layer ICL tube. The ICL was derived from porcine as described in Example 1. The strength qualities were measured using a suture retention test and a final tensile strength test (UTS). The ICL was inverted and tensioned on a pair of mandrels which were inserted into an ICL support structure. The mandrels were made of stainless steel tube with an external diameter of 4.75 mm. The ICL and the mandrels were then placed in a dehydration chamber set at a relative humidity of 20% at 4 ° C for approximately 60 minutes. After dehydration, the ICL was removed from the camera and the mandrels. The lymphatic end areas were removed and the ICL was manually clamped around the mandrel twice to form a "non-welded" double layer construction. The clamped ICL was returned to the dehydration chamber and allowed to dry for another 90 minutes still at a relative humidity of 20% at about 50% +/- 10% humidity. To determine the amount of moisture present in a sample construction, a CEM ™ oven was used. A THERMOCENTER ™ furnace was installed for the temperature treatment designed for constructions to be welded. The temperatures examined for welding varied from 55 ° to 70 ° C. Once the constructions were placed in the oven, the oven was allowed to equilibrate before starting the synchronization. The constructions were allowed to remain in the chamber for the time required for that condition. The welding times varied from 7 to 30 minutes. As soon as the time was complete the constructions were removed from the chamber and placed in a 4 ° C water bath for approximately 2 to 5 minutes. The welded constructions were returned to the dehydration chamber for approximately 30 minutes until dehydrated to approximately 20% +/- 10%. After dehydration, the constructs were inserted into a vessel containing EDC in either deionized water or deionized water and acetone at the appropriate concentrations for the conditions examined. The EDC concentrations examined were 50, 100, and 200 mM. The acetone concentrations examined were 0, 50 and 90% in water. The duration time for degradation was determined by the conditions examined. The degradation times were 6, 12 and 24 hours. After degradation, the construction was removed from the solution and rinsed with phosphate buffered saline at physiological pH (PBS) three times at room temperature. The welded and degraded construction was then removed from the mandrel and stored in PBS until examination. In addition to the thirty constructions that were prepared, two other double layer constructions were prepared by welding at 62 ° C for 15 minutes and degradation in 100 mM EDC in 100% H20 for 18 hours. The suture retention test was used to determine the ability of a construction to maintain a suture. A piece of construction was secured in a CHATTILION ™ force measuring device and a hold of 1-2 mm was taken with a 6-0 SURGILENE ™ suture, pushed through a wall of the construction and secured. The device is then pushed into the suture to determine the force required to tear the construction material. The average suture breaks between 400-500 g of force; surgeons tend to be 150 g force. The welding / material strength test was carried out to determine the UTS of a construction. Sample rings of 5 mm lengths were cut from each tube and each was examined for its final tensile strength test (UTS) using an MTS ™ mechanical examination system. Three sample rings were cut from each tube for three test thrusts made for each construction for a total of 90 thrusts. A ring was placed in the MTS ™ fasteners and pushed at a speed of 0.02 kg force / sec until the weld slipped or broke, or until the material broke (instead of welding). Example 3: Various Welding Temperatures of the ICL The effect of the welding temperature and the tempering after welding on the welding resistance was examined for the application of the ICL's two-layer pipe.
A 10-foot long ICL sample was cut along its length and prepared as in the procedure outlined in Example 2. Six 6 mm diameter tubes ranging from 15-20 cm in length were prepared for each temperature condition. The tubes were subjected to a temperature condition while they were moistened for 3.5 hours. The temperature conditions were: ambient temperature (20 ° C), 55 ° C, 62 ° C, and 62 ° C, followed immediately by bathing in a 4 ° C bath for one minute. All tubes were then degraded in EDC. The six tubes were placed together in 300 mL of 100 mM EDC overnight at room temperature. The tubes were then rinsed with phosphate-buffered saline of physiological resistance after degradation. Sample rings of 5 mm lengths were cut from each tube and each was examined for its final tensile strength test (UTS) using an MTS ™. Five sample rings were taken from each tube for 5 test thrusts in each of the 6 tubes per condition for a total of 30 thrusts. The solder strength was less consistent for tubes joined by dehydration at room temperature compared to the other temperature treatments when examined using the UTS test. One of the six welded tubes at room temperature had UTS measurements comparable to those of the other treatments. For tubes welded at other temperatures with or without tempering, there was no difference in welding resistance. After the UTS examination, it was determined that the rupture of the material was not a separation of the weld but a failure of the material in all cases. Example 4: The Antigenicity of the Degradated Intestinal Collagen Layer Fresh samples of the porcine submucosa intestinal layer were obtained after the cleaning step as described in Example 1. The samples were then left untreated and stored in water, they were immersed in saline solution regulated with physiological resistance phosphate, treated with 0.1% peracetic acid, or treated with 0.1% peracetic acid and then degraded with l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride ( EDC). The samples were then extracted with a solution of 0.5 M NaCl / 0.1 M tartaric acid for about 18 hours. Two sodium dodecylsulfate-polyacrylamide gels of 12% Tris-glycine (Novex Precast Gels cat # EC6009) were then passed and then transferred after about 18 hours to 0.45 μ of nitrocellulose paper. ICL tartaric acid extracts, either treated or untreated, were run against a standard control band containing: 10 μl of Pre-stained Kaleidoscope Standards (Bio-Rad cat # 161-0324): 2 μl sw weight standards low-molecular-weight bioassay SDS-PAGE (Bio-Rad cat # 161-0306): 6 μl charge regulator; 10 μl of control standard were loaded to each band. The gel was soiled for approximately 2 hours with 1% dry fat-free milk (Carnation) in phosphate-buffered saline. The gel was then rinsed three times with saline regulated with borate / Tween with 200 μl of rinse per band. The primary antibody in 200 μl of Rb serum and borate-buffered saline (100 mM boric acid: 25 mM sodium borate: 150 mM NaCl) / Tween was added to each band at various titer ranges (1: 40, 1: 160, 1: 640 and 1: 2560). The gel was then incubated at room temperature for one hour on an oscillating platform (Bélico Biotechnology) with the speed set to 10. The gel was then rinsed three times with saline buffered with borate / Tween. The secondary antibody, sheep anti-rabbit Ig-Ap (Southern Biotechnology Associates Inc. Cat # 4010-04) at a dilution of 1: 1000 was added to the bands at 200 μl per band and the gel was incubated for one hour at room temperature. environment on an oscillating platform. The nitrocellulose membrane was then immersed in an AP color development solution while it was incubated at room temperature on an oscillating platform until the color development was completed. The development was stopped by rinsing the membrane in deionized water for ten minutes on an oscillating platform while changing the water once during the ten minutes. The membrane was then air dried. The results obtained from the gel analysis suggest that the antigenicity of the ICL derived from porcine treated with peracetic acid and EDC has a greatly reduced antigenicity compared to the other treatments. Example 5: Six-Layer Tissue Repair Fabric as an Abdominal Wall Plaster The six layers of porcine intestinal collagen were placed on top of each other on a glass plate. A second glass plate was then placed on top of the intestinal collagen layers and tightly attached to the first plate. The apparatus was placed in a conventional type oven at 62 ° C for one hour. Immediately after warming, the apparatus was placed in a water bath at 4 ° C for ten minutes. The apparatus was disassembled, the intestinal collagen layers were removed, and treated with 100 mM l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in 50% acetone for four hours at 25 ° C. . The material was bulked and sterilized by gamma irradiation (2.5 Mrad). The tissue repair fabric was sutured in a 3 cm x 5 cm defect in the midline of New Zealand White rabbits (4 kg) using a continuous 2-0 prolene suture. The animals were sacrificed at four weeks, ten weeks, and 16 weeks, and were examined completely, mechanically, and histologically. The entire examination showed minimal inflammation and swelling. The graft was covered with a layer of bright tissue that appeared to be continuous with the parietal peritoneum. The small blood vessels could be observed proceeding circumferentially from the periphery to the center of the plaster. Mechanically the graft was stable without observed reherniation. The histological examination revealed relatively few inflammatory cells and those that were observed were basically close to the margin of the graft (due to the presence of proleny suture material). The peritoneal surface was smooth and completely covered by the mesothelium. Example 6: Two-Layer Tissue Repair Fabric as a Pericardial Repair Plaster Two layers of porcine intestinal collagen were placed on top of each other on a glass plate. A second glass plate was then placed on top of the intestinal collagen layers and tightly attached to the first plate. The apparatus was placed in a conventional type oven at 62 ° C for one hour. Immediately after heating the apparatus was placed in a water bath at 4 ° C for 10 minutes. The apparatus was disassembled, the intestinal collagen layers were removed, and treated with 10 mM l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in 50% acetone for 4 hours at 25 ° C. . The material was bulked and sterilized by gamma irradiation (2.5 Mrad). A 3 x 3 cm portion of New Zealand white rabbit pericardium was cut and replaced with a piece of tissue repair fabric of the same size(anastomized with interrupted sutures of 7-0 proleno).
The animals were sacrificed at four weeks and at 180 days, they were examined completely, mechanically and histologically. The entire examination showed minimal inflammation and swelling. The small blood vessels could be observed proceeding circumferentially from the periphery to the center of the graft. Mechanically the graft was stable without adhesion to either the sternum or pericardial tissue. The histological examination revealed relatively few inflammatory cells and those that were observed were basically close to the margin of the graft (due to the presence of proleny suture material). Example 7: Hernia Repair Device A prototype device for hernia repair using ICL was developed by having a hollow internal region. When the device is completed it has a round conformation attached to the periphery and an expanded internal region that is inchaded by the inclusion of saline regulated with phosphate of physiological resistance. The inner region can optionally accommodate a coil of wire to add rigidity or another substance for the structural support or supply of the substance. To assemble ICL multilayer sheets, lengths of 15 cm of the ICL of lymphatic ends were reduced and cut below the side with the ends to form a sheet. The leaves were hit dry with Texwipes. On a clean glass plate (6"x 8"), the sheets were stratified with the mucous side down. In this case, two two-layer and two four-layer plasters were constructed by stratifying either two or four layers of ICL onto the glass plates. A second glass plate (6"x 8") was placed on top of the last ICL layer and the plates were held together and then placed in a hydrated oven at 62 ° C for one hour. The constructions were then quenched in deionized water at 4 ° C for about 10 minutes. The glass plates were then removed from the bath and a plate was removed from each poultice. The ICL layers now attached were then smoothed to remove any wrinkles or bubbles. The glass plate was then replaced after the ICL layers and returned to the hydrated oven for 30-60 minutes until dried. The plasters were removed from the furnace and partially rehydrated by spraying with phosphate-buffered saline of physiological resistance. For the construction of a double layer construction, a double-layer plaster was removed from the glass plates and placed on the double layer plaster even on the other glass plate. An annular plate was placed(outside = 8, 75 cm; anterior - 6 cni) on the second plaster. Approximately 10 cc of saline regulated with phosphate of physiological resistance was injected through a measuring needle between the two double layer plasters. A second plate was then placed on top of the annular plate and clamped together. For construction of a four-layer construction, the same steps were followed except that two four-ply plasters were used instead of two two-ply plasters. The constructions were placed in a hydrated oven at 62 ° C for one hour. The constructions were then quenched in deionized water at 4 ° C for approximately fifteen minutes. The constructs were then degraded in 200 mL of 100 mM EDC in 50% acetone for about 18 hours and then rinsed with deionized water. The constructions were then reduced to the shape with a razor blade to the size of the outer edge of the annular plate. Example 8: Invertebrate Disc Replacement ICL, dense fibrillar collagen and hyaluronic acid were configured to approximate exactly the structure and anatomical composition of an invertebrate disc. Floppy disks were prepared Dense fibrinous collagen containing hyaluronic acid. 9 mg of sodium salt of hyaluronic acid derived from a bovine trachea (Sigma) was dissolved in 3 mL of 0.5 N acetic acid. 15 mL of 5 mg / mL of collagen (Antek) were added and mixed. The mixture was centrifuged to remove air bubbles. Three trans-perforations (Costar) in a plate of 6 perforations (Costar) were added 5mL of the solution. To the area outside the trans-perforation N600 PEG was added to cover the lower part of the membranes. The plate was maintained at 4 ° C on an orbital shaker table at low speed for approximately 22 hours with an exchange of PEG solution after 5.5 hours. The PEG solution was removed and the trans-perforations were dehydrated at 4 ° C / 20% Rh overnight. To join ICL multilayer sheets, lengths of 15 cm of ICL were reduced from the lymphatic ends and the sides were cut with the ends to form a leaf. The leaves were hit dry with Texwipes. On a clean glass plate the sheets were stratified with the mucosal side below a thickness of 5 layers and a second glass plate was placed on top of the fifth layer. Five five-ply plasters were constructed. The plates with the ICL in the medium were held together and placed in a hydrated oven at 62 ° C for one hour. The constructions were then annealed in RODI water at 4 ° C for about ten minutes after they were removed from the quench bath and stored at 4 ° C until the disk assembly. To another glass plate was placed a large plaster. A slightly smaller plaster was placed on the first plaster aligned with one edge of the larger plaster. A poultice was cut in half and a hole was cut in the middle of approximately the size of each of the DFC diskettes. With the central holes aligned, the two halves were placed on the second plaster aligned at the same edge. Three rehydrated DFC / HA disks were placed inside the central hole. Another slightly smaller plaster was placed on the two halves containing the DFC disks and one larger plaster was placed on the smaller plaster both aligned at the same edge. A second glass plate was placed on top of the construction. The resulting shape was that of a flange, with the thickest side being the aligned edges that lean to the opposite side. The device thus formed was placed in a hydrated oven at 62 ° C for one hour and then warmed in RODI water at 4 ° C for about ten minutes. The device was then degraded in lOOmM EDC (Sigma) in 90% acetone (Baxter) for approximately five hours and then rinsed with three exchanges of phosphate buffered saline. The edges of the device were then reduced with a razor blade. Example 9: The Formation of a Vascular Graft Construction The proximal jejunum of a pig was collected and processed with an Intestine Cleansing Machine (Bitterlin, Nottingham, United Kingdom) and then decontaminated with a peracetic acid solution as described in Example 1. ICL treated with peracetic acid (PA-ICL) was cut in a longitudinally open manner and the lymphatic end areas were removed to form an ICL sheet. The ICL sheets were wrapped around 6.0 mm diameter stainless steel mandrels to form double layer constructions. The constructions (in the mandrels) were then placed in a THERMOCENTER ™ balanced oven chamber set at 62 ° C for about 1 hour. The constructions were removed from the chamber and placed in a water bath at 4 ° C for about 2 to 5 minutes. Constructs were chemically degraded in 50 L of 100 mM l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) in a 50/50 water / acetone solution for 18 hours to form vascular graft constructions (PA / EDC -ICL) degraded EDC, treated with peracetic acid. The constructions were removed from the mandrels and rinsed with water to remove the residual EDC solution. After removal of the mandrels, a layer (approximately 200 μm thick) of type I collagen extracted from the bovine tendon was placed on the luminal surface of the constructions according to the method described in U.S. Patent No. 5,256,418, incorporated in the present. The polycarbonate burrs (Luer lock fits that are conically shaped at one end) were sealably fixed to either end of the constructions and the constructions were placed horizontally in a placement crack. A 50 mL container of 2.5 mg / mL of collagen extracted with acid, prepared by the method described in US Pat. No. 5, 106,949, incorporated herein, was fastened through the burrs. The collagen was allowed to fill the inner diameter of the ICL tube and then placed in a shaking bath of 20% MW 8000 polyethylene glycol (Sigma Chemicals Co.) for 16 hours at 4 ° C. The apparatus was dismantled and a 4 mm diameter glass rod was placed in the ICL tube filled with collagen to fix the luminal diameter. The prosthesis was allowed to dry. The lumped DFC layer was covered with benzalkonium chloride heparin (HBAC) by immersing the grafts three times in a solution of 800 U / mL HBAC and allowed to dry. Finally, the graft received a final chemical sterilization treatment in 0.1% v / v peachy acid. The graft was stored in a dry state until the implant procedure. Example 10: Implant Studies in Animal Models Twenty-five mongrel dogs weighing 15-25 kg were allowed to fast overnight and then anesthetized with intravenous thiopental (30 mg / kg), intubated and maintained with halothane and nitrous oxide. Cefazolin (1000 mg) was administered intravenously preoperatively as well as postoperatively. Each dog received either aortic diversion grafts or a femoral interposition graft. For aortic diversion grafts, a midline abdominal incision was made and the aorta was exposed from the renal arteries to the bifurcation, followed by administration of intravenous heparin (100 U / kg). The grafts (6 mm x 8 cm) were placed between the distant infra-renal aorta (end-to-side anastomosis) and the aorta closest to the bifurcation(end-to-side anastomosis). The aorta was ligated away from the proximal anastomosis. The incisions were closed and the dogs were kept on aspirin for 30 days after surgery. For the femoral interposition grafts, the animals were opened bilaterally, the femoral arteries were exposed and a length of 5 cm was cut. The grafts (4mm x 5cm) were anastomized in an end-to-end fashion towards the femoral artery. On the contralateral side, a control graft was placed. The incisions were closed and the animals were kept on aspirin for 30 days after surgery. The post-operative follow-up varied from 30 days to 360 days.
Blood was collected from the pre-implant, and four and eight weeks after implantation. The animals were sacrificed at various time points (30 days, 60 days, 90 days, 180 days, and 360 days). White New Zealand rabbits weighing 3.5-4.5 kg were allowed to fast overnight, and then anesthetized with acepromazine (20 mg) and ketamine (40 mg), intubated and maintained with ketamine (50 mg / mL). , they were injected intravenously as needed. Penicillin (60,000 U) was administered intraoperatively preoperatively. A midline abdominal incision was made and the aorta was exposed from the renal arteries to the bifurcation, followed by administration of intravenous heparin (100 U / kg). A length of 3 cm of aorta was cut, and the grafts (2.5 mm x 3 cm) were anastomized in an end-to-end fashion towards the aorta. The incisions were closed and the animals were kept without anticoagulant therapy after surgery. The post-operative follow-up varied from 30 days to 360 days. The animals were sacrificed at various time points (30 days, 60 days, 90 days, 180 days and 360 days). The implants together with the adjacent vascular tissues obtained from the sacrificed animals were fixed for transmission electron microscopy (TEM) analysis for 4 hr in a solution of 2.0% parafolmaldehyde, 2.5% glutaraldehyde in 0.1 M sodium cacodylate pH of 7.4. The samples were then post-fixed at 0% to 1.0% (in 0.1 M sodium cacodylate) and stained in group with 2.0% uranyl acetate (aqueous). After secondary fixation all specimens were dehydrated in a series of graduated ethanol and propylene oxide and placed in Epox 812 (Ernest F. Fulla, Rochester, NY USA). The ultrathin sections (~ 700 nm) were stained with uranyl acetate and lead citrate. The sections were examined in a JEOL Instruments JEM100S at 80 kV. For the scanning electron microscope (SEM), the samples were fixed for 18 hours in a Karnovsky solution at medium resistance and rinsed 5 times in Sorensen's phosphate buffer before post-fixation in 1.0% Os04 for 1 hour . The samples were then rinsed twice in Sorensen's phosphate buffer and three times in double distilled water. Dehydration was carried out through a series of ethanol (50%, 70%, 90% and 100%), followed by critical point drying. The samples were assembled and covered with 60/40 gold / palladium. ICL graft explants from dogs and rabbits were examined histologically to evaluate the internal cellular growth of the host. Masson's trichromatic staining of a 60-day explant showed significant host infiltration. The darker blue dye showed the ICL collagen while the matrix surrounding the myofibroblasts, stained with lighter blue, showed an abundance of host collagen. The high energy amplification of the section showed numerous intermixed cells within the ICL. The inflammatory response observed at 30 days was resolved and the cellular response was predominantly myofibroblastic. The surface of the remodeled graft was aligned by endothelial cells as demonstrated by SEM and the dyeing of Factor VIII. At 360 days, a mature 'neo-artery' had formed. The neo-adventitia was composed of fascicles of host collagen populated by cells similar to fibroblasts. The cells and matrices of the remodeled construct appeared quite mature and similar to the tissue. Example 11: Generation of Anti-ICL Antibodies Fresh samples of porcine submucosa intestinal layer were obtained after the cleaning step as described in Example 1 but were not treated with peracetic acid. The samples were left untreated(NC-ICL), were treated with 0.1% peracetic acid (PA-ICL), or treated with 0.1% peracetic acid and then degraded with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride ( PA / EDC-ICL).
White New Zealand rabbits were immunized with 0.5 mg of any of the three types of ICL samples (NC-ICL, PA-ICL, or PA / EDC-ICL) to generate anti-serum. Initially, rabbits were injected subcutaneously with 0.5 mL of untreated ICL homogenized in complete Freund's adjuvant (1: 1, 1 mg / mL). The substitute rabbits received 0.5 mL of phosphate-buffered saline in Freund's complete adjuvant. Rabbits were boosted every 3 to 4 months with 0.5 mL of the appropriate form of ICL in incomplete Freund's adjuvant (0.25 mg / mL). The serum was collected 10-14 days after each boost. Example 12: Generation of ICL Extracts and Characterization of Potentially Antigenic Proteins Associated with Native Collagen Proteins were extracted from NC-ICL, PA-ICL, or PA / EDC-ICL using tartaric acid (Bellon, G. et al., ( 1988) Anal Biochem 175: 263-273) or TRITON X-100 (Rohm and Haas). The NC-ICL, PA-ICL or PA / EDC-ICL, powdered, (10% w / v) was mixed with either TRITON X-100 tartaric acid (Rohm and Haas) (0.1 M tartaric acid, 0.5 M NaCl) extraction buffer (TEB, 1% TRITON X-100 in 20 mM Tris-HCL) (pH 7.2), 2 mM EGTA, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 25 mg / mL of each of aprotinin, leupeptin, and pepstatin (Sigma, San Luis, MO)). The mixtures were incubated overnight at 4 ° C. The extracts were filtered through gauze to remove the residues, dialyzed against PBS and concentrated using Centriprep-30 (Amicon, Danvers, MA). The extracts were stored at -80 ° C until they were used. Tartaric acid and TEB extracts were separated in 10% polyacrylamide gels by SDS-PAGE according to Laemmli (Laemmli, United Kingdom (1970) Na ture 221: 680-685). The gels either stained with silver (Bio-Rad, Hercules, CA) or were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL). The multiple protein bands were visualized in the NC-ICL extracts by silver staining. In contrast, only two bands were visible in the PA-ICL extracts and no protein bands were observed in the bands containing PA / EDC-ICL. These results suggest that the treatment of peracetic acid and EDC, in combination, leads to a decrease in proteins without extractable collagen in ICL. The immunoblot was transferred overnight using a Trans-Stain Cell (Bio-Rad) in Tris-Glycine from 20% methanol transfer buffer. Nitrocellulose membranes containing proteins transferred with ICL were blocked with Blotto regulator (1% fat-free dry milk in borate-buffered saline with 0.1% Tween-20 (BBS / Tween)) for 1 hour at room temperature. The nitrocellulose membranes were transferred to a multi-screen apparatus containing 12 individual bands. The membranes were rinsed three times with BBS / Tween. The positive control or test serum (100 μL / band) was added to the membrane and ran at room temperature for 1 hour. Each band was rinsed three times with BBS / Tween. Secondary antibodies: sheep anti-rabbit Ig labeALPH or sheep anti-sheep Ig labeALPH (Southern Biotechnology) were added to the appropriate bands (100 μL / band) and strepatabidin-AP (100 μL) was added to one of the bands that contains the molecular weight standards of Kaleidoscope (Bio-Rad). An alkaline phosphatase conjugate substrate kit (Bio-Rad) was used to visualize the immunoblots. Rabbit anti-NC-ICL serum, generated by repeated immunization with NC-ICL, was used to potentially detect immunoreactive proteins. The sera of the immunized rabbits recognized the antigens with molecular weights in the range of <; 30, 40-70, and > 100 kDa in the tartaric acid extract. This same serum was examined in immunoblots of TEB extracts from NC-ICL. The immunoreactive proteins were detected with ranges of molecular weights similar to those detected in the tartaric acid extract, with additional reactivity detected in the range of 70-100 kDa. The results indicated that NC-ICL contains multiple proteins that are immunoreactive and these proteins can be extracted by tartaric acid or TEB. The greater number of immunoreactive proteins present in the TEB extract correlated with the increase in proteins extracted using TEB compared to tartaric acid. Example 13: Effect of PA or EDC Treatment of ICL on the Antigenicity of Type I Collagen in ICL The serum of rabbits immunized with NC-ICL, PA-ICL, or PA / EDC-ICL (serum prepared as described in Example 11) or type I collagen extracted with acid (Organogenesis, Canton, MA) was examined for antibodies specific to type I collagen by ELISA. ELISA plates (Immulon II, NUNC, Bridgeport, NJ) were covered with 200 mL / 1 mg / mL perforation of type I collagen extracted with 0.05 M acid carbonate buffer (pH 9.6) overnight at 4 ° C. Plates were rinsed twice with PBS / Tween-20 (0.1%). Serum samples from animals or rabbit anti-collagen type I antibody (Southern Biotechnology, Birmingham, AL) were added to perforations (100 mL / well) and incubated for 1 hour at room temperature. Plates were rinsed three times with PBS / Tween. Secondary antibodies: sheep anti-rabbit Ig labeled ALPH or sheep anti-sheep Ig labeled ALPH (Southern Biotechnology) were added to the appropriate perforations and incubated at room temperature for 1 hour. Plates were rinsed three times with PBS / Tween. Substrate (1 mg / mL) of P-nitrophenyl phosphate (PNPP) was added to each perforation (100 mL / perforation). The absorbance was read at 405 nm on a SpectraMax microplate reader (Molecular Devices, Sunnydale, CA). Anti-collagen type I antibodies could not be detected in serum from rabbits immunized with any form of ICL, at a serum dilution of 1:40. In contrast, rabbits immunized with purified type I collagen had an antibody titer of 1: 2560. These data suggest that degradation is not necessary to reduce the antigenicity to type I of collagen, since rabbits immunized with NC-ICL did not generate type I anti-collagen antibodies. These data therefore suggest that the immunodominant proteins in NC-ICL are proteins without collagen. Also, the effect of PA and EDC on the reduction of the antigenicity of the ICL is directed towards proteins without collagen.
Example 14: Effects of Disinfection and Degradation on the Antigenicity of the ICL The effect of the treatment of PA and EDC on the antigenicity of the ICL was determined by using an anti-NC-ICL antiserum to examine the immunoreactive proteins present in tartaric acid or ects fromTEB of ICL treated with PA or PA / EDC. The PA-ICL tartaric acid ects and the PA / EDC-ICL TEB ects were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes for immunoblot analysis, as described in Example 12 The specific antiserum of NC-ICL was used to examine the immunoreactive proteins in each ect. Even when PA-ICL and PA / EDC-ICL immunoblots were overexposed, no reactivity could be detected in the bands containing anti-NC-ICL antibodies thus suggesting that the immunoreactive proteins detected in the NC-ICL are either missing or their epitopes have been modified in such a way that they are no longer recognized by the anti-NC-ICL anti-serum. To address this latter result, serum from rabbits immunized with either PA-ICL or PA / EDC-ICL was also examined. No antibody binding was detected in any of the above bands. These data indicate that although the rabbits were immunized with modified ICL, they did not generate antibodies that could recognize proteins ected with modified ICL. These results suggest that the proteins removed or modified during the disinfection and degradation process are the same proteins responsible for the antigenicity of NC-ICL. The antibody response of the rabbits immunized with PA-ICL or PA / EDC-ICL was analyzed by immunostaining, as described in example 12. This approach was taken to ensure that the lack of reactivity of the anti-NC-ICL serum with PA / EDC-ICL was due to the absence of proteins in the ICL and not due to an inability to ect proteins that could be accessible to the immune system in vivo since the degradation of the collagen materials with EDC could reduce the amount and quality of the protein ected from the ICL. Anti-ICL antiserum was generated using PA-ICL or PA / EDC-ICL to immunize rabbits. Serum from these rabbits was examined for protein-specific antibodies in either tartaric acid or TEB protein ects of NC-ICL. The anti-PA-ICL recognized the 207, 170, and 38-24 kDa proteins recognized by the anti-NC-ICL, but lost reactivity with the lower molecular weight proteins. No bands were detected by anti-PA / EDC-ICL serum from a rabbit. Serum from another anti-PA / EDC-ICL rabbit reacted with the 24-38 kDa proteins. These data suggest that both PA-ICL and PA / EDC-ICL are less antigenic than NC-ICL. The antigenic epitopes of ICL either are removed during the process of disinfection and degradation or are modified to reduce their antigenicity. In any case, disinfection and degradation resulted in a material whose antigenicity was significantly reduced. Example 15: Determination of Humoral Immune Response in Graft Vessels The humoral immune response of the dogs to the ICL graft components was examined to determine whether the ICL should retain its antigenicity in order to stimulate cell internal growth in the graft. Pre-implant blood samples were collected four and eight weeks after the implantation of the fifteen dogs that received PA / EDC-ICL vascular grafts. The serum from each blood sample was examined for the antibodies to both the tartaric acid and the TEB ects of NC-ICL. Even at a serum dilution of 1:40, none of the dogs examined had antibodies that reacted with ICL proteins. These same serum samples were examined for the presence of anti-collagen type I antibodies by ELISA. All serum samples were negative for antibodies to type I collagen at a serum dilution of 1:40. Masson's trichromatic staining of the explant paraffin sections from these dogs showed infiltration of the host cells. These results demonstrated that PA / EDC-ICL does not produce an antibody response when the host is actively remodeling the material. Although the above invention has been described in some detail by way of illustration and example for the purpose of clarity and understanding, it will be obvious to one of experience in the art that certain changes and modifications may be made within the scope of the appended claims.