MXPA99010251A - Chemical cleaning of biological material - Google Patents

Abstract

The invention is directed to collagenous tissues which have been treated to remove non-collagenous components such as cells, cellular debris, and other extracellular matrix components, such as proteoglycans and glycosaminoglycans, normally found in native tissues. Treatment of the tissue with alkali, chelating agents, acids and salts removes non-collagenous components from the collagenous tissue matrix while controlling the amount of swelling and dissolution so that the resultant collagen matrix retains its structural organization, integrity and bioremodelable properties. The process circumvents the need to use detergents and enzymes which detrimentally affect the cell compatibility, strength and bioremodelability of the collagen matrix. The collagenous tissue matrix is used for implantation, repair, or use in a mammalian host.

Description

CHEMICAL CLEANING OF BIOLOGICAL MATERIAL BACKGROUND OF THE INVENTION 1. Field of the Invention: This invention is in the field of tissue engineering. The invention is directed to collagenous tissues that have been treated to remove non-collagenous components such as cells, cell debris and other extracellular matrix components, such as proteoglycans and glycosaminoglycans, normally found in natural tissues. The treatment of tissue with alkali, chelating agents, acids and salts, removes the non-collagenous components of the collagenous tissue matrix while controlling the amount of volume increase and dissolution so that the resulting collagen matrix retains its structural organization, integrity and bioremovable properties. The process avoids the need to use detergents and enzymes that affect in a harmful way the cellular compatibility, the resistance and biore pradability of the collagen matrix. The collagenous tissue matrix is used for implantation, repair or use in a mammalian host. 2. Brief Description of the Background of the Invention: The field of tissue engineering combines the methods of engineering with the principles of the biological sciences to understand the structural and functional relationships in tissues of normal and pathological mammals. The purpose of tissue engineering is the development and ultimate application of biological substitutes to restore, maintain or improve tissue functions. (Skalak, R. and Fox, C.F., "Tissue Engineering" ("Ingeni ería de Tej i dos"), Alan R. Liss Inc. N.Y. (1988)). Collagen is the main structural protein in the body and constitutes approximately one third of the total body proteins. It comprises most of the organic matter of the skin, tendons, bones and teeth and occurs as fibrous inclusions in most other structures of the body. Some of the properties of collagen are its high resistance to stress; its ability to exchange ions, due in part to the binding of electrolytes, metabolites and drugs; its low antigenicity, due to the masking of the determinants of antigenic potential by the helical structure, and its low - extensibility, semipermeability and solubility. In addition, collagen is a natural substance for cell adhesion. These and other properties make collagen an appropriate material for the engineering and production of tissues of implantable biological substitutes and bioremovable prostheses. Because collagen is a major component of these biological substitutes, a method is needed to obtain sufficient amounts of collagen that is consistent in quality. There is presently a need for an improved method for the removal of non-collagenous components such as cells, cell debris and other extracellular matrix components, such as proteoglycans and glycosaminoglycans, normally found in natural tissues to produce a matrix of substantially pure natural collagen. Some of these non-collagenous structures that are present in natural tissues are believed to be antigenic and will produce a chronic inflammatory response when implanted in a host. However, there is a variety of methods in the art for cleaning the collagenous tissue which has resulted in collagenous compositions with different characteristics. The method used should be one that maintains the biological and physical properties of collagen and appropriate collagenous fabrics for use in weaving engineering. In the technique of treating a collagenous tissue to produce essentially a collagenous matrix, detergents and surfactants have commonly been used in the extraction of cells and lipids from tissue. Detergents such as sodium dodecyl sulfate (SDS) are amphipathic molecules in which the hydrophobic region binds to the protein and is believed to increase the negative charge of the protein. When implanted, the increase in loading results in both the increase in tissue volume due to the increased binding by water through the hydrophobic region of the molecule, as well as in the decreased thermal stability in the collagen upon breaking of the hydrogen bond. The increase in volume opens the structure of the collagen molecule making it susceptible to cellular enzymes such as collagenase and destabilizes the collagen matrix to result in weakened construction. (Courtman, et al., Journal of Biomedical Materials Research, ("Gaceta de Inves tigació on Ma teri al es Bi omédi cos") 28: 655-666, 1994). It is further believed that the SDS residues remain attached to the collagen and prevent the cells from migrating towards the implant. (Wilson, GJ et al., Ann Thorac Surg, 60: 3353-8, 1995; Bodnar E, et al. "Damage of aortic valve tissue caused by the surfactant sodium dodecyl sulfate". { "Damage to the tissue of the aortic valve caused by the surfactant of the sodium sulfa to doc") Thorac Cardiovasc Surg, 34: 82-85, 1986). Because the detergents used in a chemical cleaning method can undesirably bind to and alter the bioremodeling capabilities of the collagen in the treated tissue, the inventors have developed a method that eliminates the need for detergents. Chemical cleaning of the tissue with enzymes such as trypsin, pepsin and collagenase is known in the art but its use will result in chemical modification of the natural collagen molecules and will adversely affect the structural integrity of the construct. The treatment of enzymes of collagenous tissues is known in the art for the removal and / or modification of associated proteins of extracellular matrix. Professions such as pepsin, trypsin, dispase or thermolysin are used in the removal of collagen telopeptides to produce atelopeptide collagen. The collagen telopeptides are the triple non-helical portion of the collagen molecule and some researchers have thought that they are weakly antigenic while others think they are responsible for the strong mechanical properties of collagen. Limited digestion of the collagenous tissue will remove the telopeptides without dissociation from the collagen matrix of the tissue, whereas prolonged digestion will dissociate the collagen fibrils into atelopeptide collagen monomers. It is also known in the art to modify and remove nucleic acids from the matrix using enzymes that digest endogenous RNA and DNA through the use of RNase and DNase, respectively. As the treatment with enzymes can affect the structural integrity of the collagen, the present method of the invention avoids its use. Methods for obtaining the tissue and tissue structures from the explanted mammalian tissue, and the processes for constructing prostheses from the tissue, have been extensively investigated for surgical repair or for the replacement of organs and tissues.

The tissue is typically treated to remove non-collagenous and potentially cytotoxic cellular components to leave a natural tissue matrix. Additional processing, such as cross-linking, disinfection, or shaping, has also been investigated. Previous methods for treating collagenous tissue to remove the tissue components of the organized tissue matrix have employed detergents, enzymes or promote the uncontrolled volume increase of the matrix. WO 95/28183 to Jaffe, et al. describes methods to reduce or avoid mineralization of the bioprosthetic valve of the heart after implantation. The described methods provide biological material made acellular by controlled autolysis. The autolysis is carried out in a controllable manner using at least one buffer solution at a preselected pH to allow the autolytic enzymes present in the tissue to degrade the cellular structural components. The U.S. Patent No. 5,007,934 to Stone and, similarly, the US Patent. No. 5,263,984 to Li, et al. both describe a multi-step method for chemical cleaning of the ligamentous tissue. The method uses a detergent to remove the lipids associated with the cell membranes or collagenous tissue. The U.S. Patent No. 5,523,291 to Janzen, et al. describes a pulverized injectable implant composition for gentle augmentation of tissue derived from the igamen tum nuchae. The ligament is treated with a series of impregnations in a strongly alkaline solution of sodium hydroxide followed by hydrochloric acid solution and then sodium bicarbonate. The U.S. Patent No. 5,028,695 to Eckmayer, et al. describes a process for the production of collagen membranes in which the collagen tissue is repeatedly treated with a strong alkali and subsequently with strong acid for several times, then treated further with inorganic saline treatment to contract the membranes and then with solvent for dry them. SUMMARY OF THE INVENTION Bioremodable collagen tissue matrices and methods for chemical cleaning of natural tissue to produce such woven matrices are described. The present invention overcomes the difficulties in obtaining bioremovelable tissue matrices that are substantially collagen.

The invention provides tissue matrices that can be used as a prosthetic device or material for use in the repair, augmentation or replacement of damaged and diseased tissues and organs. The chemical cleaning method of this invention converts the biological material, such as natural tissues and tissue structures, into substantially acellular and substantially free of non-collagenous components while maintaining the structural integrity of the collagenous tissue matrix. Because detergents are not used in chemical cleaning processes, detergent residues that would normally remain attached to the tissue matrix are not present. Because the enzymes are not used, the collagen telopeptides are retained in the collagen molecules. The method comprises contacting a normally cellular natural tissue with a chelating agent at a basic pH, contacting the tissue with saline at an acidic pH, contacting the tissue with a saline solution at a physiological pH and then finally Rinse the resulting chemically clean tissue matrix. This invention is directed to a chemically clean tissue matrix derived from natural, usually cellular, tissues. The clean tissue matrix is essentially intact collagen converted to substantially free of glycoproteins, glycosaminoglycans, protoglycans, lipids, non-collagenous proteins and nucleic acids such as DNA and RNA. Importantly, the bioremolability of the tissue matrix is kept free of bound detergent residues that would adversely affect the bioremodelability of collagen. In addition, collagen is a telopeptide collagen because the telopeptide regions of the collagen molecules remain intact as they do not undergo treatment or modification with enzymes during the cleaning process. The collagenous material generally maintains the general shape of the tissue from which it is derived but must be stratified and bonded to form multilayer sheets, tubes, or prostheses of complex shape. The bonded collagen layers of the invention are structurally stable, flexible, semipermeable, and suturable. When the matrix material is implanted in a mammalian host, biodegradation accompanied by adequate replacement of living cells, or formation of neotejidos is experienced, in such a way that the implanted original material is remodeled and finally replaced by tissues and cells derived from the host. It is therefore an object of this invention to provide a method for cleaning natural tissue that results in a tissue matrix that does not exhibit many of its disadvantages associated with many of the previously developed methods. The method effectively removes the non-collagenous components of the natural tissue without the use of detergents or enzymes to produce a tissue matrix comprising substantially collagen. Another objective is the provision of a bioremoveable tissue matrix material that will allow and facilitate the internal growth of the tissue and / or the regeneration of the organ at the site of implantation. Prosthesis prepared from this material, when grafted onto a recipient or patient host, concomitantly undergo controlled bioremodelation and adequate replacement of living cells so that the implanted original prosthesis is remodeled by the patient's living cells to form a regenerated organ or tissue.

Yet another objective of this invention is to provide a method for the use of a novel multi-purpose bioremodellable matrix material in indications of autografts, allografts and heteroinjcts. Yet another objective is to provide a novel tissue matrix material that can be implemented using conventional surgical techniques. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for processing natural collagenous tissues for transplantation. The method of manufacture is designed to generate a collapsible, implantable collagenous biological tissue material, an extracellular matrix comprising collagen, which serves as a scaffold that can be bioremodelated by a host in vivo or by living cells in in vitro culture. This invention is further directed to a tissue designed prosthesis formed from processed natural collagenous tissue, which, when implanted in a mammalian host, can serve as a repair, augmentation, or functioning replacement of body parts or tissue structures and will undergo controlled biodegradation occurring concomitantly with remodeling through host cells. The tissue matrix can be used as a prosthetic material for indications of autograft, allograft and heterograft. The prosthesis of this invention, in its various modalities, therefore has dual properties: First, it functions as a substitute part of the body, and second, while continuing to function as a substitute part of the body, it functions as a pattern of remodeling for inner growth of the host cells. Although the prosthesis will be illustrated through the construction of various devices and constructions, the invention is not so limited. It will be appreciated that the design of the device in its material, shape and thickness is selected depending on the last indication for construction. The chemical cleaning method of this invention makes the biological material, such as natural tissues and tissue structures, substantially acellular and substantially free of non-collagenous components while maintaining the structural integrity of the collagenous tissue matrix. Elastin is sometimes present in natural tissue in small quantities and is not removed by the chemical cleaning method. The presence of elastin may be desirable for some applications. As used herein, the term, "substantially acellular" means that it has at least 95% less natural cells and cell structures than the natural state of the biological material. "Cells and cell structures" refer to cells, living or non-living, remnants of cells, cell membranes and membrane structures. The use of the term, "substantially free of non-collagenous components", means that glycoproteins, glycosaminoglycans, protoglycans, lipids, non-collagenous proteins and nucleic acids such as DNA and RNA comprise less than 5% dry weight of the resulting tissue matrix. . Because detergents are not used in the chemical cleaning process, the detergent residues that would normally remain attached to the tissue matrix are not present. Because the enzymes are not used, the collagen telopeptides are retained in the collagen molecules. In addition, the chemical cleaning method makes the biological material sterile and free of endotoxins when processed using sterile equipment, solutions and aseptic techniques. The term, "structural integrity", refers to the ability of the chemically clean collagenous tissue matrix to withstand forces such as tension, compression and support. The structural integrity of the biological material is conserved since the volume increase is minimized in the chemical treatment stages although some volume increase will occur during the treatment. Uncontrolled or excessive volume increase opens the structure of the collagen molecule making it susceptible to cellular enzymes such as collagenase and destabilizes the collagen resulting in weakened construction. Because the increase in volume affects the intramolecular structure of the collagen molecule, affects the general structure of the material at an intramolecular level by breaking the natural crosslinks between the collagen molecules. Together, the structure of the collagen molecule and the crosslinks between the collagen molecules lend structural integrity to the material.

The material of the tissue matrix that maintains much of its natural structural integrity is useful, for example, when it is used as a prosthetic device or as a material to build complex or stratified devices. The integrity of the material is important if a load-bearing function such as a body wall support, a vascular device or an orthopedic device is performed. Related to structural integrity is the term "suturable" which means that the mechanical properties of the material include suture retention which allows needles and suture materials to pass through the prosthetic material when the prosthesis is sutured in sections of natural tissue, a process known as anastomosis. During suturing, the prostheses should not tear as a result of the tension forces applied to them by the suture, nor should they tear when the suture is knotted. The suture capacity of the prosthetic material, ie, the ability of the prosthesis to resist tearing while suturing, is related 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 was tightened. The biological material as defined in the invention includes but is not limited to cultured mammalian tissues, and structures thereof, derived from human, bovine, porcine, canine, ovine, caprine and equine organisms. Tissue structures such as the dermis, artery, vein, pericardium, heart valve, dura mater, ligament, intestine and fascia are preferred tissue structures that are capable of being cleaned by the methods of this invention to produce a matrix of tissue that is substantially acellular and substantially free of non-collagenous components. A preferred source of mammalian tissue is the tunica submucosa from the small intestine, more preferably from the porcine small intestine. In the natural small intestine, the tunica submucosa is the connective tissue layer of the organ and includes both lymphatic cells and blood vessel cells. Methods for obtaining the tunica submucosa are described in WO 96/31157 and incorporated herein. To obtain the submucosa porcine tunic, also called "submucosa", the small intestine of a pig is mechanically cultured and naked, preferably by the use of a bowel cleansing machine (Bitterling, Nottingham, UK). The intestinal cleansing machine forcibly removes the layers of fat, muscle and mucus from the tunica submucosa using a combination of mechanical action and washing with water. The mechanical action can be described as a series of rollers that compress and strip the successive layers of the tunica submucosa when the intact intestine extends between them. Because the tunica submucosa of the small intestine is comparatively harder and stiffer than the surrounding tissue, the softer components coming from the submucosa are removed from the tunica submucosa. The result of the cleaning by machine is such that the mesenteric tissues, the tunica serosa and the muscularis of the tunic coming from the ablumen of the tunica submucosa and as well as the layers of the tunica mucosa coming from the lumen of the tunica submucosa are removed from the submucosa tunic so that only the tunica submucosa layer of the intestine remains. The chemically clean tissue matrix of the tunica submucosa is also called the "intestinal collagen layer" or "ICL". It is observed that in some animal sources, such as carnivores and omnivores, the small intestine includes a stratum compactum which is mostly removed by this mechanical cleansing stage. Other methods for mechanically stripping the layers of the small intestine as described in the U.S. Patent are known in the art. No. 4,902,508 of Badylak, incorporated herein by reference. The method described by this patent includes the soft abrasion of the intestinal tissue to remove the abluminal layers, including the tunica serosa and muscularis of the tunic and the inner layers consisting of at least the luminal portion of the tunica mucosa. The remaining layers are the tunica submucosa with the attached basilar layer consisting of lamina muscularis mucosa and, if present initially in the harvested mammalian tissue, the stratum compactum. The intestinal material obtained by any method can be implanted or formed first in the body wall or vascular device by various methods including suturing, sewing with staples, adhesive compositions, chemical bonds and thermal bonds. The terms belonging to some operating parameters are defined for the complete specification and the examples for quantities, times and temperatures that can be varied without deviating from the spirit and scope of the invention. As used herein, an "effective amount" refers to the volume and concentration of the composition required to obtain the desired effect A preferred effective amount for chemical cleaning of the fabric is a ratio of 100: 1 v / v of solution to tissue, but larger or smaller volumes can be determined by experts when considering the shape, volume, thickness, density and cellularity of the tissue to be cleaned.The time required for the chemical stages to be effective can be appreciated by those skilled in the art. When considering the cellularity, matrix density and thickness of the material to be cleaned, the larger, thicker or denser materials will take more time for the solutions to penetrate and balance in the fabric, the temperatures for the environment and the solutions used. in the present invention they are preferably at room temperature, of about 25 ° C, but can be found in any range above of the freezing temperatures of the solutions used, up to less than the denaturing temperature of the material of the tissue being treated. Temperatures between approximately 4 ° C and 45 ° C are sufficient for the cleaning treatment to be effective. Agitation means that it is a mechanical agitation or mixture and is used to improve the penetration of the chemical compositions into the tissue and to reduce the time required for the chemical treatment to be effective. The term "buffer solution" refers to an aqueous solution containing at least one agent which retains the concentration of hydrogen ion or the pH of the solution. In the preferred method, the harvested tissue may need to be cleaned manually, such as by ordinary dissection, and / or mechanical cleansing of surplus tissues such as fat and vasculature. Manual cleaning for some tissues may be necessary for the handling of practicability during the procedure or for most of the effective chemical treatment. First, the fabric is treated by contacting the fabric with an effective amount of chelating agent, preferably alkaline, to controllably control the volume increase of the tissue matrix. Chelating agents improve the removal of cells, cell debris and basement membrane structures from the matrix by reducing the concentration of divalent cations. The alkaline treatment dissociates the glycoproteins and glycosaminoglycans from the colnous tissue and saponifies the lipids. Chelating agents known in the art that can be used include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) and ethylenebis (oxyethylene trile) tetraacetic acid (EGTA). EDTA is a preferred chelating agent and can be made more alkaline by the addition of sodium hydroxide (NaOH), calcium hydroxide Ca (OH) 2, sodium carbonate or sodium peroxide. The concentration of EDTA or EGTA is preferably between about 1 to about 200 mM; more preferably between about 50 to about 150 mM; very preferably approximately 100 mM. The concentration of NaOH is preferably between about 0.001 to about 1 M; more preferably between about 0.001 to about 0.10M; more preferably about 0.01M. Other alkaline or basic agents can be determined by someone skilled in the art to bring the pH of the chelating solution within the effective basic pH range. The final pH of the basic chelating solution should preferably be between about 8 and about 12, but more preferably between about 11.1 to about 11.8. In the most preferred embodiment, the tissue is contacted with a solution of 100 mM EDTA / 10 mM NaOH in water. The tissue is preferably contacted by immersion in the alkaline chelating agent while obtaining the most effective treatment by agitating the tissue and the solution together for a time so that the treatment step is effective. The tissue is then contacted with an effective amount of acidic solution, preferably containing salt. Acid treatment also plays a role in the removal of glycoproteins and glycosaminoglycans as well as in the removal of non-collagenous proteins and nucleic acids such as DNA and RNA. The salt treatment controls the increase in volume of the collagenous tissue matrix during acid treatment and is involved with the removal of some glycoproteins and protoglycans from the collagen matrix. Acid solutions known in the art can be used and may include but are not limited to hydrochloric acid (HCl), acetic acid (CH2COOH) and sulfuric acid (H2SO) A preferred acid is hydrochloric acid (HCL) in a concentration preferably between about 0.5 at about 2 M, more preferably between about 0.75 to about 1.25 M, more preferably about 1 M. The final pH of the acid / salt solution is preferably between about 0 to about 1, more preferably between about 0 and 0.75. , and more preferably between about 0.1 to about 0.5 Hydrochloric acid and other strong acids are more effective at breaking nucleic acid molecules while weak acids are less effective.The salts that can be used are preferably inorganic salts and include but are not limited to to chloride salts such as sodium chloride(NaCl), calcium chloride (CaCl2) and potassium chloride (KCl) while other effective salts can be determined by someone skilled in the art. Preferably the chloride salts are used in a concentration of preferably between about 0.1 to about 2 M; more preferably between about 0.75 to about 1.25 M; more preferably about 1 M. A preferred chloride salt for use in the method is sodium chloride (NaCl). In the most preferred embodiment, the tissue is contacted with 1 M HCl / l M NaCl in water. The tissue is preferably contacted by immersion in the acid / salt solution while the effective treatment is obtained by stirring the tissue and the solution together during the time for the treatment step to be effective. The weave is then contacted with an effective amount of saline which is preferably stabilized at about a physiological pH. The stabilized salt solution neutralizes the material while reducing the volume increase. The salts that can be used are preferably inorganic salts and include but are not limited to chloride salts such as sodium chloride (NaCl), calcium chloride (CaCl2) and potassium chloride (KCl); and nitrogen salts such as ammonium sulfate (NH3S04) while other effective salts can be determined by someone skilled in the art. Preferably the chloride salts are used in a concentration preferably between about 0.1 to about 2 M; more preferably between about 0.75 to about 1.25 M; more preferably about 1 M. A preferred chloride salt for use in the method is sodium chloride (NaCl). Stabilizing agents are known in the art and include but are not limited to phosphate and borate solutions while others can be determined by the person skilled in the art for use in the method. A preferred method for stabilizing the saline solution is to add phosphate stabilized salt (BPS) preferably when the phosphate is in a concentration of about 0.001 to about 0.02 M and a salt concentration of about 0.07 to about 0.3 M for the saline solution. A preferred pH for the solution is between about 5 to about 9, more preferably between about 7 to about 8, more preferably between about 7.4 to about 7.6. In the most preferred embodiment, the tissue is contacted with 1 M sodium chloride (NaCl) / 10 mM saline stabilized phosphate (PBS) at a pH between about 7.0 to about 7.6. The weave is preferably contacted by immersion in the stabilized saline solution while the effective treatment is obtained by stirring the tissue and the solution together during the time for the treatment step to be effective. After the chemical cleaning treatment, the fabric is then rinsed preferably free of chemical cleaning agents upon contacting it with an effective amount of rinse agent. Agents such as water, isthonic salt solutions and stabilized pH physiological solutions can be used and contacted with the tissue for a sufficient time to remove the cleaning agents. A preferred rinse solution is pH stabilized physiological saline such as phosphate stabilized saline (PBS). Other means for rinsing the fabric of the chemical cleaning agents can be determined by one skilled in the art. The cleaning steps of contacting the fabric with an alkaline chelating agent and contacting the fabric with an acid solution containing salt can be performed in any order to substantially achieve the same cleaning effect. However, the solutions may not be combined and performed as a single stage. A preferred composition of the invention is a chemically clean tissue matrix derived from natural tissues, usually cellular. The clean tissue matrix is essentially acellular telopeptide collagen, approximately 93% by dry weight, with less than about 5% dry weight of glycoproteins, glycosaminoglycans, proteoglycans, lipids, non-collagenous proteins and nucleic acids such as DNA and RNA. Importantly, the bioremodelability of the tissue matrix is kept free of bound detergent residues that would adversely affect the bioremodelability of collagen. Additionally, the collagen molecules have retained their telopeptide regions since the tissue has not undergone the treatment with enzymes during the impingement process. The tissue matrices are derived from the dermis, arteries, veins, pericardium, heart valves, dura mater, ligaments, intestine and fascia. The most preferred composition is a chemically clean intestinal collagen layer derived from the small intestine. The appropriate sources for the small intestine are mammalian organisms such as the human, the cow, the pig, the sheep, the dog, the goat, or the horse although the pig small intestine is the preferred source. In a preferred embodiment, the collagen layer comprises the tunica submucosa derived from the porcine small intestine. In another embodiment, the collagen layer comprises the tunica submucosa and the basilar layers of the small intestine. The basilar layers consist of mucosa lamina muscularis and, if present in the natural tissue, the stratum compactum. The most preferred composition of the invention is the intestinal collagen layer, cleaned by the chemical cleaning method of the invention, is essentially collagen, mainly Type I collagen, with less than about 5% dry weight of glycoproteins, glycosaminoglycans, protypelicines, lipids, non-collagenous proteins and nucleic acids such as DNA and RNA. The collagen layer is free of bound detergent residues that would adversely affect the bioremodelability of collagen. The collagen layer is substantially free of cells and cell debris, including endogenous nucleic acids such as DNA and RNA and lipids. In addition, the intestinal collagen layer is both sterile and endotoxin-free when processed using sterile equipment, solutions and aseptic techniques. Once the collagenous tissue matrix has become substantially acellular and free of substantially non-collagenous extracellular matrix components, prostheses can be made for implantation or grafting thereof. The collagen layers can be stitched together or joined together using any variety of techniques known in the art. The methods for joining the layers can employ adhesives such as thrombin, fibrin or synthetic materials such as cyanometacrylates or chemical crosslinking agents. Other methods can use heat generated by laser, light or microwave. Convection ovens and baths with heated liquids can also be used. Thermal welding of the collagen layers is the preferred method for joining together the collagen layers of the invention. Methods for thermal welding are described in WO 95/22301, WO 96/31157 and the US Patent. No. 5,571,216, the teachings of which are incorporated herein by reference. The ICL is the first longitudinal cut and flattened on a flat, solid plate. Then one or more successive layers are superimposed on one another, preferably in alternating perpendicular orientation. A second solid flat plate is placed above the layers and the two plates are firmly held together. The entire apparatus, the subject 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 to cause the collagen to become irreversibly denatured. The heating and bonding time will depend on the type of the collagen material layer 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 from 62 ° C. A typical range of times will be from about 7 minutes to about 24 hours, typically about an hour. The degree of heat and the amount of time that heat is applied can easily be verified by routine experimentation by varying the parameters of heat and time. The joining step can be performed in a conventional oven, although other heat appliances or applications may be used including, but not limited to, water baths, laser energy, or electrical heat conduction. Immediately after heating and bonding, the collagen layers are cooled, in air or in a water bath, to a range between room temperature at 20 ° C and 1 ° C. Rapid cooling, called quenching, is required to stop the heating action and to create an effective bond between the collagen layers. To perform this step, the collagen layers can 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 cooling rate may not be sufficient to fix the collagen layers with one another. In the preferred embodiment, the collagenous material will crosslink. The crosslinking imparts increased strength and structural integrity to the prosthetic construction formed while regulating collagen bioremodelation by cells when the construct is implanted in a patient. Collagen cross-linking agents include glutaraldehyde, formaldehyde, carbodimides, hexamethylene diisocyanate, bisimidates, glyoxal, adipyl chloride, dialdehyde starch, and some polyepoxy compounds, such as glycol diglycidyl ether, polyol polyglycidyl ether and biglicidyl ternes of bicarboxylic acid lico Dehydrothermal, UV irradiation, and / or sugar mediated methods can be used. Collagen will also naturally crosslink with rest at room temperature. However, crosslinking agents need not be limited to these examples since other crosslinking agents and methods known to those skilled in the art can be used. The crosslinking agents should be selected to produce a biocompatible material capable of being remodeled by host cells. A preferred crosslinking agent is 1-ethyl-3- (3-dimethylaminopropyl) carbodimide hydrochloride (EDC). The crosslinking solution containing EDC and water may also contain acetone. Cross-linking with EDC has been described in PCT International Publication Nos. WO 95/22301 and WO 96/31157. In some embodiments, additional collagenous layers may be added to any of the outer or inner surfaces of the collagen layers attached, either before or after crosslinking. In tubular constructions, such as in a vascular construct, dense fibrillar collagen can be added to the luminal surface to create a uniform flow surface for ultimate application as described in PCT International Publication No. WO 95/22301, incorporated in the present for reference. This uniform collagenous layer also promotes the binding of the host cell, as in the formation of neointima, which facilitates the internal growth and the bioremodelation of the construction. As described in PCT International Publication No. WO 95/22301, this uniform collagenous layer can be made from fibrillar collagen extracted from acid or non-fibrillar., which is predominantly type I collagen, but may also include other types of collagen. The collagen used can be derived from any number of mammalian sources, typically skin or tendons from cattle, pigs or sheep. The collagen has preferably been processed by acid extraction to result in a high purity fibril dispersion or gel. Collagen can be extracted by acid from the collagen source using a weak acid, such as acetic, citric or formic acid. Once extracted in solution, the collagen can be precipitated by salts 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 the U.S. Patent. No. 5,106,949, incorporated herein by reference. Heparin can be applied to the prosthesis, using a variety of well-known techniques. For illustration, heparin can be applied to the prosthesis in the following three ways. First, benzalkonium heparin (BA-Hep) solution can be applied to the prosthesis by immersing the prosthesis in the solution and then drying it in the air. This process treats collagen with an ion-bound BA-Hep complex. Second, EDC can be used to activate heparin, then to covalently attach heparin to the collagen fiber. Third, EDC can be used to activate the collagen, then covalently bind the protamine to the collagen and then ionically bind the heparin to the protamine. Many other coating, joining, and fastening procedures are well known in the art which can also be used. The treatment of the tissue matrix material can be carried out with agents such as growth factor or pharmaceuticals in addition to or in substitution of heparin. Agents may include, for example, growth factors to promote vascularization and epithelialization, such as growth factor-derived macrophages (MGDF), platelet-derived growth factor (PDGF), vascular endothelial cell-derived growth factor (VEGF). ); antibiotics to combat any potential infection from the surgery implant; or nerve growth factors incorporated into the internal collagenous layer when the prosthesis is used as a conduit for nerve regeneration. In addition to or in substitution of drugs, components such as protoglycans or glycoproteins or glycosaminoglycans may be included within the construct. The collagenose prosthesis thus formed can also be stabilized in a dilute peracetic acid solution with a neutral pH. Methods for sterilizing collagen are described in the U.S. Patent. No. 5,460,962 and are incorporated by reference herein. In the preferred method, the collagen is disinfected with a diluted peracetic acid solution at a neutral pH. The concentration of peracetic acid is preferably between about 0.01 and 0.3% v / v in water at a neutralized pH between about pH6 and pH8. Alternatively, sterilization with gamma irradiation, typically at 2.5 Mrad, or with gas plasma can also be used to sterilize collagen. Other methods known in the art can be used to sterilize collagen. The following examples are provided to better explain 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 deviating from the spirit and scope of the present invention. EXAMPLES Example 1.- Chemical cleaning of mechanically stripped porcine small intestine. A pig's small intestine was harvested and mechanically stripped, using a Bitterling (Nottingham, UK) intestinal cleansing machine which forcibly removes the layers of fat, muscle and mucosal from the tunica submucosa using a combination of action mechanics and washing using water. The mechanical action can be described as a series of rollers that compress and strip the successive layers of the tunica submucosa when the intact intestine extends between them. The submucosa tunica of the small intestine is comparatively harder and more rigid than the surrounding tissue, and the rollers compress the softer components of the submucosa. The result of the mechanical cleaning was such that only the submucosal layer of the intestine remained. The rest of the procedure was carried out under aseptic conditions and at room temperature. The chemical solutions were all used at room temperature. The intestine was then cut longitudinally down the lumen and then cut into sections of 15 cm. The material was weighed and placed in containers in a ratio of approximately 100: 1 v / v of solution to intestinal material. A. To each container containing intestine was added approximately LL of tetrasodium salt and ilenediaminotetracetic salt (EDTA) sterilized by filter 0.22 mm (miera) 100 mM / solution of sodium hydroxide (NaOH) 10 mM. The containers were then placed on a shaker table for about 18 hours at about 200 rpm. After shaking, the EDTA / NaOH solution was removed from each bottle. B. To each vessel was then added approximately 1 L of sterilized hydrochloric acid solution (HCl) by 0.22 mm lM filter / sodium chloride (NaCl) solution. The containers were then placed on a shaker table between about 6 to 8 hours at about 200 r.p.m. After shaking, the HCl / NaCl solution was removed from each container. C. To each vessel was added next approximately 1 L sodium chloride (NaCl) solution sterilized by 0.22 mm filter 1M / 10 phosphate stabilized salt (PBS) 10 mM. The containers were then placed on a shaker table for about 18 hours at 200 r.p.m. After stirring, the NaCl / PBS solution was removed from each container. D. To each vessel was added approximately 1 L of PBS solution sterilized by 0.22 mm filter 10 mM. The containers were then placed on a shaker table for approximately two hours at 200 rpm. After the turmoil, the phosphate-stabilized salt was removed from each container. E. Finally, approximately 1 L of sterilized water per 0.22 mm filter was added to each container. The containers were then placed on a shaker table for 4 hours. about one hour at 200 r.p.m. After stirring, the water was removed from each container. The treated samples were cut and conditioned for histological analysis. Hematoxylin and eosin staining (H & E) and Masson's trichrome were performed on both the cross section and longitudinal sections of both treated and control tissues. Treated tissue samples appear free of cells and cell debris while control samples appear normally and are expected to be cellular. Example 2: Chemical Cleaning of the Porcine Heart Valve. A porcine heart was obtained from a 1 pound piglet and transported in saline at physiological pH on ice. Within 4 hours, the heart valves were removed from the heart mass using scalpel and forceps. Some additional ordinary dissection was performed to remove excess tissue from around the valves. One valve was retained as a control with cut and fixed sample pieces for various histological analyzes while the other valve underwent the chemical cleaning process. He - The rest of the procedure was developed under aseptic conditions and at room temperature. The chemical solutions were all used at room temperature. The valve was placed in the LI solution 100 mM EDTA / 10 mM NaOH for approximately 18 hours while stirring on a shaker platform. The valve was then placed in ITL of HCl IM / NaCl IM and stirred for 8 hours. The valve was then placed in 1 L of lM HCl / 10 mM phosphate stabilized saline (PBS) solution and stirred for approximately 18 hours. The valve was then rinsed in PBS for about 2-4 hours and then finally rinsed in sterilized water for about 1 hour while stirring. The treated sample pieces were then cut and conditioned for various histological analyzes. Hematoxylin and eosin stains (H & E) and Masson's trichrome were performed both on the cross section samples and on the longitudinal section of both treated and control valves. The treated valve samples appear free of cells and cell debris while the control samples appear normally and are expected to be cellular. Example 3: Chemical Cleaning of Porcine Arteries, Pericardium and Fascia. A segment of the femoral artery, the complete pericardium, and the fascia of a sow of 450 pounds The tissues were transported in saline of physiological pH on ice. The tissues were also dissected to remove excess tissue.

Samples were taken from each tissue without cleaning for control samples and conditioned for various histological analyzes while the rest of the tissues underwent the chemical cleaning process. The rest of the procedure was carried out under aseptic conditions and at room temperature. The chemical solutions were all used at room temperature. The tissues were placed separately in 1 L of 100 mM / 10 mM EDTA solution and shaken on a shaker platform for about 18 hours. The tissues were then placed separately in .lL of HCl / MCl NaCl solution and shaken for 8 hours. Next, the fabrics were separately placed in 1 L of lM HCl / 10 mM phosphate stabilized saline (PBS) solution and stirred for 18 hours. The tissues were rinsed separately in PBS for about 2 to 4 hours and then finally wiped in sterilized water for about 1 hour while stirring. The treated sample pieces were cut and conditioned for the various histological analyzes. Hematoxylin and eosin stains (H & E) and Masson's trichrome were performed on both the cross section and longitudinal sections of both treated and control tissues. The Tissue samples treated appear cell-free and cellular debris while the control samples appear normally and are expected to be cellular. Example 4: Chemical Cleaning Ordered Differently. This procedure was carried out under aseptic conditions and at room temperature and the chemical solutions were all used at room temperature. The mechanically stripped porcine intestine was cut into five 15 cm sections as described in example 1. To each vessel was added after approximately 1 liter of hydrochloric acid solution - (HCl) sterilized by 0.22 mm lM filter / sodium chloride (NaCl) ÍM. The containers were then placed on a shaker table for about 6 to 8 hours at about 200 r.p.m. After stirring, the HCl / NaCl solution was removed from each container. To each container containing the intestine was added approximately 1 μL of sterile-labeled ileal-diamine-electronetatraetic acid (EDTA) solution 0.22 mm (miera) 100 mM / 10 mM sodium hydroxide solution (NaOH). The containers were placed on a shaker table for about 18 hours at about 200 r.p.m. After stirring, the EDTA / NaOH solution was removed from each bottle. To each vessel was added approximately 1 L sodium chloride (NaCl) solution sterilized by 0.22 mm lM filter / 10 mM phosphate stabilized salt (PBS). The containers were then placed on a shaker table for about 18 hours at 200 r.p.m. After stirring, the NaCl / PBS solution was removed from each container. To each vessel was added approximately 1 L of PBS solution sterilized by 0.22 mm 10 M filter. The containers were then placed on a shaker table for about 1 hour at 200 r.p.m. After stirring, the phosphate-regulated salt from each container was removed. Finally, approximately 1 L of sterilized water per 0.22 mm filter was added to each vessel. The containers were then placed on a shaker table for about one hour at 200 rpm. After stirring, the water was removed from each container. The treated sample pieces were cut and conditioned for the various histological analyzes. Hematoxylin and eosin stains (H & E) and Masson's trichrome were performed on both the cross section and longitudinal sections of both treated and control tissues. The treated woven samples appear free of cells and cell debris while the control samples appear normally and are expected to be cellular. Example 5: Various alkaline and chelating agents. The cleaning of mechanically stripped porcine intestinal submucosa was carried out according to example 1. This procedure was carried out under aseptic conditions and at room temperature and all the chemical solutions were used at room temperature. The chemical cleaning process of Example 1 was followed but with the substitution of the alkaline chelating agent from step A which was replaced by other alkaline chelating agents of a similar nature: A. To each container containing intestine was added approximately 1 L of sterilized solution. by 0.22 mm filter (miera) of either ethylenebis (oxyethylenitrile) tetraacetic acid (EGTA) 100 mM / 10 mM NaOH; 100 mM EDTA / Ca (OH) 2 (calcium hydroxide) 10 mM; or 100 mM EDTA / 10 mM K2C03 (potassium carbonate) solution. The containers were then placed on a shaker table for about 18 hours at about 200 rpm. After stirring, the alkaline chelating agents were removed from each bottle. B. To each vessel was then added approximately 1 L of sterilized hydrochloric acid solution (HCl) by 0.22 mm lM filter / sodium chloride (NaCl) solution. The containers were then placed on a shaker table for about 6 to 8 hours at about 200 r.p.m. After stirring, the HCl / NaCl solution was removed from each container. C. To each vessel was added next approximately 1 L of sodium chloride solution (NaCl) sterilized by 0.22 mm lM filter / 10 mM phosphate stabilized salt (PBS). The containers were then placed on a shaker table for about 18 hours at 200 r.p.m. After stirring, the NaCl / PBS solution was removed from each container. D. To each vessel was added approximately 1 L of PBS solution sterilized by 0.22 mm filter 10 mM. The containers were then placed on a shaker table for about one hour at 200 rpm. After the turmoil, the phosphate-stabilized salt was removed from each container. E. Finally, approximately 1 L of sterilized water per 0.22 mm filter was added to each container. The containers were then placed on a shaker table for about one hour at 200 rpm. After stirring, the water was removed from each container. Samples were conditioned for histological analysis Hematoxylin and eosin stains (H & amp; amp;; E) and Masson's trichrome was performed both on the cross section samples and on the longitudinal section of both treated and control tissues. Treated tissue samples appear free of cells and cell debris while control samples appear normally and are expected to be cellular. Example 6: Various acid and saline agents. The mechanically stripped porcine intestinal submucosa of Example 1 was chemically cleaned using an acidic substitute agent or a substitute saline agent in step B. This procedure was performed under aseptic conditions and at room temperature and all chemical solutions were used at room temperature. ambient. A. To each container containing intestine was added approximately 1 L of tetrasodium salt and ilenodiaminotetraacetic salt (EDTA) sterilized by filter 0.22 mm (miera) 100 mM / solution of sodium hydroxide (NaOH) 10 mM. The containers were then placed on a shaker table for about 18 hours at about 200 rpm. After stirring, the EDTA / NaOH solution was removed from each bottle. B. To each container that was added approximately LL of sterilized solution by 0.22 mm filter of either CH3COOH (acetic acid) MM / 1M NaCl or H2SO4 (sulfuric acid) MM / NaCl solution ÍM. The containers were then placed on a shaker table for about 6 to 8 hours at about 200 r.p.m. After stirring, the solution was removed from each container. C. To each vessel was added approximately 1 L sodium chloride (NaCl) sterilized by 0.22 mm lM filter / 10 mM phosphate stabilized salt (PBS). The containers were then placed on a shaker table for about 18 hours at 200 r.p.m. After stirring, the NaCl / PBS solution was removed from each container. D. To each vessel was added after approximately 10 μM of 10 mM PBS sterilized by 0.22 mm filter. The containers were then placed on a shaker table for about one hour at 200 rpm. After stirring, the phosphate-stabilized salt from each container was then removed.

E. Finally, approximately 1 L of sterilized water per 0.22 mm filter was added to each container. The containers were then placed on a shaker table for about one hour at 200 rpm. After stirring, the water was removed from each container. The treated sample pieces were then cut and conditioned for various histological analyzes. Hematoxylin and eosin stains (H & E) and Masson's trichrome were performed on both the cross section and the longitudinal sections of both treated and control tissues. Treated tissue samples appear free of cells and cell debris while control samples appear normally and are expected to be cellular. Example 7: Glycosaminoglycan (GAG) Content of ICL Determined by Cellulose Acetate Gel Electrophoresis and Blue Alcian Test. To determine the GAG content of ICL, cellulose acetate gel electrophoresis was performed with the subsequent blue Alcian dye in chemically clean ICL extracts. The ICL samples underwent the chemical cleaning regime described in Example 1, cut into 0.125 cm2 pieces and placed in eppendorf tubes. To assimilate the samples, 100 μl of papain (0.1 Mg / ml papain in O.lM sodium phosphate, O.lM sodium chloride, 0.005 M EDTA, 0.9 mg / ml cystaine, pH 5.8) was added to each tube, and they were allowed to incubate for approximately 18 hours at 60 ° C. The standard containing known quantities of GAG (heparin) was prepared in parallel. Dowex (0.4 g of the HCl form) and 3 ml of water were added. After rotating it to remove the Dowex resin, it was removed and lyophilized 1 ml. The samples were then rehydrated in 100 μl of purified water and centrifuged for approximately 5 minutes. The samples were separated into cellulose acetate sheets using the method of Newton, et al. (1974). The cellulose acetate sheets were impregnated in 0.1M lithium chloride / EDTA stabilizer (pH 5.8) and blotted gently. The samples (5 μl each) were applied to the leaves at the end of the cathode and electrophoresed for 30 minutes at 5 mA. Following the electrophoresis, the leaves were immersed immediately in a solution of blue Alcian dye (0.2% Alcian blue 8GX, 0.05 M magnesium chloride, 0.025 M sodium acetate stabilizer (pH 5.8) in 50% alcohol of ethylene) and placed on a shaker platform for about 30 minutes at room temperature. The leaves were then destained in at least three washes of decolorizing solution (magnesium chloride 0.05 M, stabilizer of 0.025 M sodium acetate (pH 5.8) in 50% ethylene alcohol) for a total of about 30 minutes on a shaker platform . No detectable GAG dye was observed for ICL assimilated from papain while a standard as small as 0.005 microgame of heparin was detectable. These results showed that the total amount of GAG remaining in the chemically clean ICL is less than 1% (dry weight). Example 8: Lipid Content of ICL Determined by Extraction of Methylene Chloride. The ICL was spread flat on plastic plates and air-dried for two hours. Once dry, the ICL was cut into smaller pieces of approximately 1 cm2 of which 1100 g were transferred to a filter element of soxhlet paper. To a round flask of Kontes 24/40 brand 90 ml of methylene chloride were added. The soxhlet was assembled in the ventilation hood with the bottom of the flask in a hot water bath and ice-cooled water running through the distiller. The extraction was allowed to proceed for four hours after which the soxhlet was disassembled. The round flask containing the solvent and the extracted material was left in the hot water bath until the methylene chloride evaporated until they remained. 5 ml. The methylene chloride was then transferred to an 11x13 glass culture tube and the remaining solvent was boiled. To the tube was added 2 ml of methylene chloride and the tube was immediately capped and the tube was placed in a freezer at -20 ° C. Then the weight of the extracted material was determined. The glass tube was placed in an ice bath. The weight of a 1.12 ml aluminum weight can Ludiag was packed in a microbalance (Spectrum Supermicro). 10 μl of resuspended extraction was added to the weight pot and the solvent was boiled by placing the weight pot on a hot plate for 45 seconds. The weight pot was allowed to cool for approximately 190 seconds and was placed in the microbalance. The procedure was then repeated to extract volumes of 20 μl and 30 μl. The results indicate that the percentage of lxpido is less than about 0.7% lipid per dry weight of chemically clean ICL. By contrast, clean ICL does not chemically contain a higher fraction of lipids; at least about 1.5% by dry weight of ICL that was not chemically cleaned by the method of the invention. Example 9: Amino Acid Analysis of the ICL. Collagens are proteins characterized by their triple helical regions which have a repeating triplet of glycine-X-Y amino acids, where X is frequently proline and Y is frequently hydroxyproline. Hydroxyproline is often used as an amino acid to identify and quantify collagens. (Udenfriend, Science., 152: 1335-1340, 1966). To determine the complete amino acid analysis of the ICL, PICO-TAG HPLC was performed in mechanically cleaned porcine ICL (clean non-chemically) and chemically clean ICL. The content of the hydroxyproline was measured for both materials and compared. The ICL sample pieces of each condition weighing above 0.31 to about 0.36 g were further dried using a CEM AVC80 oven (CEM corp, Matthews, NC). The smaller samples were cut from these pieces of dry ICL weighing about 9.5 to about 13.1 mg. The samples were placed in screw cap culture tubes and the samples were then hydrolyzed (n = 3 for each condition) in 1% phenol in 6M HCl at 110 ° C for approximately 16 hours. The ICL hydrolysates were then diluted in O.lM HCl to normalize the material concentrations to 1mg / ml. For labeled glass tubes (6 X 55), 20 ml of hydrolysates and 8 ml of L-norleucine 1.25 mmol / ml as an internal standard. The samples were then frozen and lyophilized. The samples were then resected by adding 20 ml of 2: 2: 1 ethanol: water: triethylamine to the tubes, freezing and freezing. The samples were then derived for 20 minutes at room temperature by adding 20 ml of reagent (7: 1: 1: 1 ethanol: gua: triethylamine: PITC) followed by freezing and lyophilization. The samples were finally suspended in 200 ml of PICO-TAG Samples Diluent and aliquoted in HPLC bottles. The amino acid standards were prepared as follows: 0.1M1 of amino acid standard (Product #: A-9531, Sigma) was added to 1.9 ml of O.lM HCl. Five solutions were made in series at 1: 1 using O.lM HCl. Volumes of 100 ml for each serial solution and 8 ml of L-norleucine 1.25 mmol / ml were added together to glass tubes (6 X 55) and then prepared in the same manner as the ICL samples. Samples and standards were run on a 3.9 x 150 mm PICO-TAG Amino Acid column (Part # 88131; Waters Corp., Milford, MA). The injections of 10 ml for the samples and 20 ml for the standards were analyzed in triplicate for each one. The results indicate that the chemically clean ICL material, the content of the major collagenous amino acids in the material approach the purified collagen preparations. When using hydroxyproline as a measure of - 51 Collagen content, the percentage of collagen by weight in the ICL is calculated to be at least about 93% collagen by dry weight. In contrast, clean ICL does not chemically contain a high fraction of non-collagenous amino acids; between about 11 to 25% by dry weight of ICL is non-collagenous material. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious to one skilled in the art that some changes and modifications may be practiced within the scope of the appended claims.

Claims (27)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and therefore the property described in the following claims is claimed as property. 1. A detergent-free and enzyme-free method for removing the non-collagenous components from natural mammalian tissue to produce an essentially collagenous tissue matrix, comprising: (a) contacting the tissue with an effective amount of agent Chelating agent in alkaline solution; (b) contacting the tissue with an effective amount of acidic solution containing a salt; (c) contacting the tissue with an effective amount of stabilized salt solution at approximately a physiological pH; Y, (d) contacting the fabric with an effective amount of rinsing agent.
2. 2. The method according to claim 1 characterized in that the chelating agent of the stage (a) is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA) and ethylenebis (oxyethylene trile) tetraacetic acid (EGTA).
3. 3. The method according to claim 2 characterized in that the concentration of the chelating agent is between about 1 to about 200 mM.
4. 4. The method according to claim 2 characterized in that the concentration of the chelating agent is between about 50 to about 150 mM. 5. The method according to claim 1 characterized in that the alkaline agent of the stage (a) is selected from the group consisting of sodium hydroxide, calcium hydroxide, sodium carbonate or sodium peroxide. 6. The method according to claim 5 characterized in that the concentration of the alkaline agent is between about 0.001 to about. 7. The method according to claim 1 characterized in that the acidic solution of the stage (b) is selected from the group consisting of hydrochloric acid (HCl), acetic acid (CH3COOH) and sulfuric acid (H2S04). The method according to claim 7 characterized in that the concentration of the acidic solution is between about 0.5 to about 2 M. 9. The method according to claim 7 characterized in that the concentration of the acidic solution is between about 0.95 to about 1.25. M. The method according to claim 7, characterized in that the concentration of the acidic solution is approximately around IM. The method according to claim 1 characterized in that the pH of the acidosal solution is between 0 to about 1. 12. The method according to claim 1 characterized in that the pH of the acid-salt solution is between 0 and 0.75. . The method according to claim 1 characterized in that the pH of the acid-salt solution is between 0.1 to about 0.
5. 5. The method according to claim 1 characterized in that the salt of either step (b) or (c) is an inorganic salt. 15. The method according to claim 1 characterized in that the salt of either step (b) or (c) is selected from the group consisting of sodium chloride (NaCl), calcium chloride (CaCl2), potassium chloride (KCl) ) and ammonium sulfate (NH3S04). 16. The method according to claim 1 characterized in that the salt of either step (b) or (c) is at a concentration between about 0.1 to about 2M. 17. The method according to claim 1 characterized in that the salt of either step (b) or (c) is at a concentration of 0.75 to about 1.25M. 18. The method according to claim 1 characterized in that the salt of either step (b) or (c) is in a concentration of about MM. 19. The method according to claim 1 characterized in that such tissue is selected from the group consisting of dermis, artery, vein, pericardium, heart valve, dura mater, ligament, bone, cartilage, fascia and intestine. 20. The method according to claim 19, characterized in that said intestinal tissue comprises the tunica submucosa of the small intestine. The method according to claim 19, characterized in that said tissue is derived from human, bovine, porcine, ovine, canine, caprine, or equine. 22. The method according to claim 1, characterized in that the rinsing agent is selected from the group consisting of physiologically stabilized water or salt. 23. The method according to claim 1, characterized in that step (b) is carried out before step (a). 24. A detergent-free and enzyme-free method for the removal of the non-collagenous components of natural mammalian tissue to produce an essentially collagenous tissue matrix, characterized in that it comprises: (a) contacting the tissue with a basic acid solution ethylenediaminetetraacetic acid at a pH between about 8 and about 12; (b) contacting the tissue with an acidic sodium chloride solution at a pH between about 0 and about 1; (c) contacting the tissue with a solution of sodium chloride at a pH between; and, (d) rinsing the tissue. 25. A bioremovelable collagenous tissue matrix obtained by the method of claim 1 or 24. 26. A bioremoveable collagenous tissue matrix composition derived from natural mammalian tissue rendered essentially free of non-collagenous and non-elastin components found in such natural tissue. and free of detergent residues and enzymatic modification for its implantation for the repair or replacement of a diseased or damaged body part comprising collagen and elastin. 27. A bioremoveable collagenous tissue matrix composition free of detergent residues derived from the natural tunica submucosa of the small intestine where the tissue matrix comprises: telopeptide collagen; elastin, where elastin is less than 10% of the total composition; and non-collagenous and non-elastinous components where such components are less than 5% of the total composition.