Bioengineered collagen construct, modified intestinal collagen layer, processed tissue matrix and method of reconstruction or replacement of damaged tissue

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and pharmacology, and represents a bioengineered collagen construct for reconstruction or replacement of damaged tissue, characterised by the fact that it comprises a layer of a purified collagen tissue matrix taken from the small intestinal submucosa, wherein said purified collagen tissue matrix is processed by a pharmaceutically acceptable antimicrobial agent and has the anti-microbial properties.

EFFECT: invention provides the bioengineered collagen construct with the improved antimicrobial properties.

17 cl, 11 ex, 2 tbl

 

The technical field to which the invention relates.

This invention relates to the field of regenerative medicine and tissue engineering. The invention is directed to bioengineered constructs produced from recycled fabric material or matrix, which come from animal sources. Bioengineered constructs of the invention receives the manner in which is stored biocompatibility, cell compatibility, durability of the processed tissue matrix and its bioconstructions. Antimicrobial properties give bioengineered constructs, which are used for engraftment, implantation, repair tissue, wound healing and reconstruction, or used for another purpose in the body of a mammal.

The level of technology

The area of regenerative medicine and tissue engineering combines methods of engineering with the principles of the life Sciences for understanding the structural and functional relationships in normal and pathological mammalian tissues. The aim of regenerative medicine and tissue engineering is the development of biological substitutes and bringing them to the final application in order to restore, maintain or improve tissue functions.

Collagen is the main structural protein in the body of the organism, which sostavlyayushchaya one-third of all proteins of an organism. It is the main part of the organic matter of the skin, tendons, bones and teeth, and occurs as fibrous inclusions in the majority of other body structures. Some of the properties of collagen are high tensile strength, low antigenicity, partly due to the masking of potential antigenic determinants due to the helical structure, and low elongation, polyphonically and solubility. Moreover, collagen is a natural substance for cell adhesion. These and other properties make collagen suitable material for tissue engineering and manufacturing of implantable biocompatible structures and bioconstruction dentures.

Methods of obtaining collagen tissue and tissue sturcture of explantion mammalian tissue and methods design of the prosthesis from these tissues and tissue structures has been widely studied with the aim of applying them in the healing of wounds, surgical recovery and replacement tissues or organs. The ongoing goal of researchers is to develop bioengineered constructs that can be used successfully to improve standards of health care of patients.

Disclosure of inventions

Collagen materials of biological origin, such as submucosal membrane of the intestines, used in restored and tissues or replacement, these materials continue to develop and improve. Nowadays, a new bioengineering, bioconservatism constructs impart antimicrobial properties to improve their performance when used in regenerative medicine, including wound healing, tissue repair and replacement. Disclosed methods of mechanical and chemical processing of proximal porcine jejunum to obtain a single cell-free layer of the intestinal collagen (CCM), derived from intestinal submucosa membrane, which can be used for the formation of layered materials with antimicrobial properties for healing, repair and replacement of tissues. In the processing of cells and products of disintegration of cells are removed, while the matrix structure of native collagen tissue is preserved. The final layer of processed tissue matrix is used to produce single-layer and multi-layer sheet, cross-linked constructs with the desired characteristics. Investigated the effectiveness of a single-layer products for bandaging wounds, multilayer sheet flaps for repair of soft tissues, and the use of inkubiruemykh constructs as a vascular implant. Such fabric material obtained in the processing of the colon, provides the physical support with minimal formation of spikes and able to integrate with the surrounding native tissue, becoming infiltrated by cells of the host. In vivo biorecognition not jeopardize the mechanical integrity of these constructs. Internal and functional properties of the implant, such as modulus of elasticity, the ability to maintain the viability of the seam and ultimate tensile strength are important parameters which can be manipulated for specific requirements by varying the number of layers and cross-stitching. Now these constructs impart antimicrobial properties with the aim of controlling or reducing microbial activity in the treated area, which uses the constructs.

The aim of the invention is bioengineered collagen construct with antimicrobial properties, which contains sheet layer purified collagenous tissue matrix derived from a source tissue, such as submucosal membrane of the small intestine, or processed intestinal collagen layer, derived from the submucosal membrane of the small intestine treated with antimicrobial agent. When using bioengineered collagen construct of the invention, the bandage on the wound in the treatment of wounds of the mammal, the construct is applied on the wounded surface to partially cover the wound so that their own skin tissue of the patient was provided lanai environment, that helps to restore skin tissue, while an antimicrobial agent in the construct regulates, or reduces microbial activity on the wound. The construct is biocompatible, which means that the construct is not cytotoxic, does not cause a decrease in the sensitivity of the skin and does not cause primary skin irritation.

In one embodiment, the bandage on the wound contains a layer of processed intestinal collagen derived from the submucosal membrane of the small intestine, the thickness of which is approximately from 0.05 to 0.07 mm, and an antimicrobial agent. Due to the sheet geometry purified tissue matrix may be covered by layers, and then chemically bind the layers with each other with obtaining a multi-layered construct. So another option is to construct containing two or more than two layers of purified tissue matrix that are already linked together and treated antimicrobial agent. The constructs of the invention can be performed in a grid, perforated or fastreroute or for better coordination with the surface form of a wound or for better drainage of exudates of the wound, or for both purposes.

An additional goal of this aspect of the invention is the treatment of the wound if necessary care or treatment, in particular in the case when think what we antimicrobial intervention and protection, the stripes shall mean any of the following types of wounds: superficial or deep wounds, prolagene, venous ulcers, diabetic ulcers, chronic vascular ulcers, tunneled wounds/wounds with overhanging edges, surgical wounds, donor site wounds with autologous transplantation, wound post-Mohovskoe surgery, wounds, post-laser surgery, wound dehiscence, traumatic wounds, abrasions, lacerations, second-degree burns, skin tears or drainage of the wound.

Another aim of the invention is the provision of restorative surgical devices, such as a flap or grid for processing and recovery of soft tissues and organs, which contains two or more than two layers, for example, from two to ten layers of processed intestinal collagen derived from the submucosal membrane of the small intestine, these layers are linked and cross stitch with each other for forming a multi-layered construct, which is biocompatible and bioconservatism and which implant to the damaged or unhealthy soft tissue undergoes controlled biodegradation, which occurs with an adequate substitution living cells, so that the original implanted prosthesis is remodeled through living cells of a patient. An additional goal of this aspect of the invention is the provision of a JV is soba processing damaged or unhealthy soft tissue in need of repair or antimicrobial intervention, including the implantation of the prosthesis, which contains two or more than two superimposed upon each other chemically related layers of processed intestinal collagen derived from the submucosal membrane of the small intestine and processed antimicrobial agent that, when the implant to the damaged or unhealthy soft tissue undergoes controlled biodegradation occurring with adequate substitution living cells, so that the original implanted prosthesis is reconstructed at the expense of living cells of a patient. Cases when damaged or unhealthy soft tissue needs to be restored include, for example, wounds, defects of the abdominal and chest wall, the need to strengthen the muscle flap, rectal and vaginal prolapse, reconstruction of the pelvic floor hernia, the need to strengthen the seam line and restorative procedures.

The implementation of the invention

The aim of the invention are bioengineered collagen constructs (e.g., dentures, implant), which contains sheet layer purified collagenous tissue matrix, processed tissue material derived from native tissue, for example, processed intestinal collagen layer derived from the submucosal membrane of the small intestine, which is treated with an antimicrobial agent. Bioengineered collagen construct may I is given either a single layer of processed tissue matrix, or several superimposed on each other layers of processed tissue matrix. In the case when bioengineered collagen construct of the invention are used to treat wounds of the patient is a mammal, it is applied to the surface of the wound to partially cover the wound so that their own skin tissue of the patient was provided as a damp environment that helps to restore skin tissue, and antimicrobial composition to regulate or reduce microbial activity in the wound and the edges of the wound. In the case when the bioengineered construct is used as a surgical device, it is implanted at the site of implantation of the organism of a mammal so that it served to restore, heal or replace a body part or tissue structures. Antimicrobial agent regulates, or reduces microbial activity on the wound or the area of the implant by preventing adhesion or proliferation of bacteria on the construct. Antimicrobial constructs of the invention are biocompatible, meaning that the construct is not cytotoxic, does not cause a decrease in the sensitivity of the skin and does not cause primary skin irritation.

The prostheses of the invention are also "bioconservative", which means that they will be subjected to a controlled bio is agradeciy, which accompanies the reconstruction and replacement of new endogenous matrix provided by the cells of the host body or the patient, with the formation of new tissue. Thus, the prosthesis of this invention, when the supply of its antimicrobial agent and when used as replacement tissue, has many properties. First, it functions as a replacement for a body part or as a covering for wounds. Secondly, when it functions as a replaceable part of the body, he performs the role of the reconstructed template for germination inside host cells. Thirdly, it provides an antimicrobial effect locally in the treated location.

The material of the prosthesis in accordance with the present invention is a purified, processed collagenous tissue matrix derived from a collagenous tissue of a mammal, which you can associate with itself or with another recycled, purified tissue matrix, and which can impart antimicrobial properties to obtain a prosthesis for implantation or implantation in the body area of the body that needs treatment.

The invention includes methods of making dunno engineering prostheses from recycled fabric material, where the methods of bonding the layers to each other does not require adhesives, sutures or staples, while str is functioning prostheses biologically reconstructed is stored. The terms "processed tissue matrix" and "recycled fabric material" means native, normal cellular tissue that was extracted from an animal source, preferably from a mammal, and mechanically cleaned from the surrounding tissues, or chemically purified from cells, products of disintegration of cells and presented partly free from Nikolayevich components of the extracellular matrix. Processed tissue matrix, while is partially free and free from Nikolayevich components, essentially retains its native matrix structure, organization, strength and shape. Composition of processed tissue material for the production of bioengineered implants of the invention are obtained from animal tissues containing collagen, such as collagen sources include (but this list is not limited to): the intestines, the dermis, a wide fascia, pericardium, Dura mater, the placenta and other flat or planar-structured tissues that contain collagen tissue matrix. The structure and geometry of these tissue matrices allows you to easily clean them, manipulate them and make them so that you could get bioengineered implants of the invention. Other suitable sources of tissues with similar flat structure is Urai, geometry and matrix composition can be identified, extracted and treated by a qualified specialist from other sources of animal origin in accordance with the invention.

One such processed tissue matrix composition to obtain a bioengineered implant of the invention is the intestinal collagen layer derived from the submucosal membrane of the small intestine. Suitable sources of the small intestine are mammals such as human, cow, pig, sheep, dog, goat, or horse, while the small intestine of the pig is a readily available source. The prostheses of the invention can be obtained from processed intestinal collagen layer (sometimes denoted by the term "intestinal collagen layer" or "CCM"), which is processed tissue material derived from submucosal membrane of the small intestine of the pig. One way to obtain this intestinal collagen layer of the small intestine is removed from a mammal, and associated mesenteric tissue rudely cut off from the intestine. Submucosal membrane separated or prepare the chin from the other layers of the small intestine by means of mechanical compression of the original intestinal material, for example, setting it between the opposing rollers, similar to those used in the apparatus for Vidalia the Oia sausages, to separate the muscle layers (muscle sheath) and membrane (mucosa). Because submucosal membrane of the small intestine is harder and stronger than the surrounding tissue, the rollers squeeze the softer components of the submucosa, in the mechanical cleansing of the tissue matrix. In the following examples, porcine small intestine mechanically cleaned using the apparatus for cleaning Chichkov, and then chemically cleaned by successive digestions, resulting in processed tissue matrix. Such mechanically and chemically cleaned intestinal collagen layer derived from the submucosal membrane of the small intestine, herein referred to as "CCM"this layer is a type of processed tissue matrices or materials, which are antimicrobial constructs of the invention.

In the composition of the recycled CCM fabric material is acellular telopeptide collagen type I, which is about 93% by weight is dry, with less than 5% by weight of dry residue are glycoproteins, glycosaminoglycans, proteoglycans, lipids, collagenase proteins and nucleic acids such as DNA and RNA, this material essentially free from cells and degradation products of cells. Recycled CCM fabric material for the most part retain the structure and strength of his Matri is CA. It is essential that the biocompatibility and bioconstructions tissue matrix is partially retained in the cleaning process, since it is free from binding detergent residues, which could adversely affect bioconstructions collagen. In addition, the collagen molecules retain their telopeptide areas, because the fabric is not subjected to processing enzymes during the cleanup process.

To obtain the processed tissue matrix determine a suitable source of animal and tissue source. The fabric is treated as mechanically and chemically, in order to uninstall the related tissue and to separate collagenase components from the tissue, receiving the processed tissue matrix. As an example, the CCM is one kind of processed tissue matrix used to obtain bioengineering implantable prosthesis of the invention. The following methods are fabric processing to obtain processed tissue matrix and for the manufacture of bioengineering implantable prostheses containing the CCM and antimicrobial agent.

To obtain pork CCM, sub mucosal membrane of the small intestine of a pig used as source material for bioengineering implantable prosthesis of the invention. After removed the I of the small intestine of the pig, accompanying tissue is removed, and then the intestines mechanically cleaned using the apparatus for cleaning the colon, which is energetically separated fatty tissue, muscles and layers of the mucosa from the submucosa membranes using a combination of mechanical impact and water washing. Mechanical action is performed by a set of rolls which compress and squeeze the successive layers of submucosal membrane by passing through these shafts are solid bowel. Submucosal membrane of the small intestine is relatively stronger and harder than the surrounding tissue, and therefore the rollers squeeze the softer components separately from the submucosa. Other means of mechanical treatment used in this technical field, which can determine a qualified technician include other physical manipulation, such as scraping, squeezing, squeezing and rubbing. The result of the mechanical treatment is such that it remains exclusively submucosal layer of the intestine - mechanically cleaned intestines.

After mechanical cleaning using a chemical cleaning procedure for the Department of cellular and matrix components from the mechanically cleaned intestines, preferably holding it under aseptic conditions and at room temperature. Mechanically cleaned intestines cut out in a longitudinal direction down the t cavity, and then cut in lengths of from about 15 cm to 50 cm Material are weighed and placed in containers at the ratio of solution to intestinal material about 100:1 by volume. In the most preferred procedure, chemical cleaning, such as by the method disclosed in U.S. patent No. 5993844 and 6599690 authored by Abraham (these links mean that the information from the above-mentioned patents in full included in this document) collagen fabric is treated with an effective amount of a chelating agent such as Terentieva salt ethylenediaminetetraacetic acid (EDTA) in alkaline conditions, for example, by adding sodium hydroxide (NaOH); followed by treatment an effective amount of acid, where the acid contains salt, for example hydrochloric acid (HCl)containing sodium chloride (NaCl); subsequent treatment an effective amount of buffer salt solution, for example, with a 1 mm solution of sodium chloride (NaCl) concentration of phosphate-saline buffer (PBS). phosphate buffered saline) 10 mm; at the end of the hold phase of the washing water. Each and every stage of processing is preferably performed using oscillating or vibrating platform to improve chemical and drilling fluids. As a result of treatment processes is obtained processed intestinal collagen is Loy, or CCM, mechanically and chemically cleaned, processed tissue matrix derived from the submucosal membrane of the small intestine. After washing, the CCM is removed from the cleaning of containers and moderately wring out or get wet cloth to remove excess water. At this stage it can be stored frozen at -80°C at 4°C in sterile phosphate buffer, either in dry form to the manufacture of the prosthesis. If stored in a dry form, it leaves the CCM smooth on the surface, for example on a flat plate, preferably on a porous plate or membrane, such as a polycarbonate membrane, and any lymph buildup with the treated side of the material removed with a scalpel, then leaves the CCM can be put to dry in the kelp at room temperature and ambient humidity.

CCM is a flat sheet structure, which can be used as a single-layer material for the manufacture of various types of constructs used as prostheses, the shape of the prosthesis depends on the intended use. To obtain a multi-layer prosthesis of the invention leaves the CCM laminate method, which is still biocompatibility and bioconstructions recycled matrix material, in this way can be stored strength and traktornye characteristics of the material, required for efficient operation of it as a replacement tissue. Processed tissue matrix derived from tissue, maintains the structural integrity of the native tissue matrix, i.e. collagen matrix structure of the original fabric remains largely unaffected and retains physical properties so that implantation will occur many functional and its inherent properties. Upon receipt of the multilayer laminates of the CCM, the leaves of the CCM layer for contact with other sheets. The contact area is the area of the binding, where the layers are in contact with each other, or layers directly overlap each other or are partially in contact or overlap with the formation of more complex structures. Ready-made constructs the region of connection must be resistant to the stitches and tension when operating them in a clinical setting, during implantation and during the initial healing phase when the operation of the constructs as a substitute body part. The region of connection must also maintain sufficient strength up until the cells of the patient will not fill and not subsequently bioconstruction prosthesis with the formation of new tissue.

Processed tissue matrix is used as a single layer of the prosthesis or poluchaets him multilayer, the associated prosthesis flat, tubular or of complex shape. In the case where the prosthesis of the invention contain two or more than two layers of processed tissue matrix, the layers are linked by chemical crosslinking using a cross-linking agent (for brevity, also referred to here as the cross-linking agent). When using chemical cross linking for linking multiple layers of processed tissue matrix with one another, the degree of chemical cross-linkage can be varied to modulate the speed of biorecognition throughout the prosthesis, i.e. the speed with which the prosthesis is absorbed and/or replaced by cells of the host and tissue of the host body. In other words, the higher the degree of cross-linkage in the prosthesis, the lower speed dentures will be biorecognition; on the contrary, the lower the degree of cross-linkage, the faster the prosthesis will be bioconstruction. In accordance with the indications for surgical intervention levels will be set and/or speed bioconservative necessary for the prosthesis. For example, when using a single-layer construct as a bandage on the wound prosthesis can chemically to sew or not to sew. For example, when used as a surgical restoring the second flap, or mesh prosthesis is a multi-layered construct, which has a low degree of cross-linkage, so that the prosthesis was biocontrols with higher speed. For example, when using as mahapasana flap to maintain hypermobile bladder to prevent urinary incontinence, the prosthesis is a multi-layered construct, which has a high degree of cross-stitching, so that he did not biocontrols quickly, i.e. it essentially retains the same form he had during implantation, over a long period of time.

Collagen matrix or construct in the case when has the shape of a leaf, usually has two opposite surfaces. For processing recycled collagen matrix antimicrobial agent, this agent is applied by introducing it into contact with any of the parties revised the collagen matrix, or this agent is associated with both sides. Alternatively, you can reach the fiber, the absorption properties of recycled collagen matrix, applying an antimicrobial agent to the inside of processed collagen material into the voids of the fibrous processed tissue matrix, for example, have been recovered by the supply of the collagen matrix in the solution, contains an antimicrobial agent that penetrates into the matrix by means of absorption. Another way of introducing antimicrobial agent inside the multilayer construct is the initial handling of single layers of processed tissue matrix, and then applying layers and link them with each other. In the case of a multilayer configuration, the method includes the following stages: matrix leaves are treated with antimicrobial agent, matrix sheet layer for the formation of compound layers and sew construct cross-linking agent; these stages can be performed in any order, including the following: matrix leaves are treated with antimicrobial agent, matrix sheet layer for the formation of composite layers with subsequent crosslinking by means of a crosslinking agent; matrix leaves are treated with antimicrobial agent, sew through cross-linking agent, followed by the layering of matrix sheets for the formation of compound layers; sew through cross-linking agent, process matrix sheets antimicrobial agent, and then a layer of matrix sheets for the formation of compound layers; sew through cross-linking agent, a layer of matrix sheets for the formation of compound layers, and then treated matrix leaves of proteomically the agent; layer of matrix sheets for the formation of compound layers, sew through cross-linking agent, and then treated matrix sheets antimicrobial agent; or, a layer of matrix sheets for the formation of compound layers, process matrix sheets antimicrobial agent, and then sew through cross-linking agent. In some cases, the binding matrix after coating antimicrobial agent may be impractical because some antimicrobial agents can be washed off with a crosslinking agent.

Raw and processed layers can be layering on each other in different sequence to obtain a prosthesis with a localized antimicrobial agent. Methods of manufacturing such multilayer configurations include layering sheets of processed tissue matrix, which were pre-processed antimicrobial agent, and the raw matrix sheets to each other for making a multilayer construct and the stitching of the layers with each other, for example, at least two of the matrix sheet can be layering, knit and handle antimicrobial agent for making the processed matrix construct, and then one or more of the matrix sheets can be layering or on the top or bottom surface or both the surface is processed matrix construct, and then, the resulting construct can be made to obtain the joint construct. Alternatively, at least two raw matrix sheet can be layering or cross-stitch for a raw matrix of the construct, and then one or more of the matrix sheets can be layering or on the top or bottom surface or both surfaces of the raw matrix construct, after which the resulting construct can then be cross-stitch for obtaining a combined matrix construct. In another example, treated and untreated matrix sheets can be alternately layering, and then cross to sew for the combined construct. In another example, the processed matrix sheets that were pre-treated in two different antimicrobial agents can be arranged in alternating or in a different order to obtain a combined construct. In another example, the orientation of the treated and untreated sheets relative to each other, or relative to processed or unprocessed matrix constructs in kombinirovannom matrix construct may be selected taking into account the best quality CCM material as versatility.

To process only selected portions or areas of the FR is and these parts of the surface of the material can be processed antimicrobial agent by masking machined parts so so when masking blocked contact antimicrobial agent with the material during processing of other areas of the surface. Another way of localization of antimicrobial agent to the collagen matrix is a partial immersion of the collagen material in the tub or tank so that in contact with the antimicrobial agent was only part of the collagen matrix, and the remaining part has not come in contact with. Another way of localization of antimicrobial agent on the surface of the material is a spray, or any other promotion, antimicrobial agent on the surface of the material, in which the opposite surface remains untreated.

Single layer and multilayer constructs treated with antimicrobial agent for imparting antimicrobial properties. At least one antimicrobial agent is applied to the constructs of the invention by introducing it into contact with the antimicrobial agent in whole or only some part. To the preferred antimicrobial agents include antimicrobial agents, silver based and chemical based. Also in the composition may be included antibiotic agent. For treatment of collagenous material in order to give him a wide spectrum of antimicrobial activity can be used is ombinatio agents, for example, a combination of antimicrobial agents, silver based and chemical based; antimicrobial agent chemical based and antibiotic agent; antimicrobial agent based on silver and antibiotic agent; or a combination of all three types of agents.

To impart antimicrobial properties to the prosthesis containing the processed tissue matrix, you can select antimicrobial agents based on silver. Silver can be applied to the collagen constructs in several forms. Antimicrobial agents based on silver containing silver or a compound containing silver, these compounds have some degree of antimicrobial activity and is compatible with both collagen construct, and with the patient's body. Pure silver, also known as elementary or noble silver, relatively chemically inactive and does not react with water or oxygen at normal temperatures and insoluble in dilute acids and bases.

You can also use silver ions. It is assumed that the ionic form of silver is not yet confers resistance to microbes and unlikely to act as well as other antimicrobial agents. Not trying to link it to any theory, the silver in this ionic form is effective against bacteria, is Rogga and fungi, and against extracellular virus through direct effects on respiration and transport of cells and cell membranes, as well as through direct effects on cell reproduction. You can also use silver in different forms, including (but this list is not limited to): silver oxide; silver nitrate; silver sulfadiazine (sulfadiazine (I) contains insoluble polymeric compound which slowly releases silver ions when used as an antimicrobial and antifungal agent, this compound can be used locally in the treatment of severe burns to prevent bacterial infection), imidazole silver, Arglaes® Giltech Limited, UK (Allais, also conventionally referred to as AgKaPO4), colloidal silver, silver crystals, such as silver nanocrystals, also known as "nanocrystalline silver, all these tools are specific to the alternative method of delivery and sustained release of silver cation and its radicals to a wound site or site of implantation.

The composition of the nanocrystalline silver ("nano-silver") are manufactured according to a number of different ways. In the same way - pulse plasma method - get nanocrystals organized structure of the crystal lattice and without newparadize the surface atoms, the particle sizes are distributed in a narrow range and a high degree of homogeneity in their morphology. In a pulsed plasma process using as raw material the two conductive rod. The rods are loaded into the reaction chamber, which is controlled is filled with gas, most likely, an inert gas, such as argon, at atmospheric pressure. The rods connect with a very powerful, pulsed power source (spark gap). Nanomaterial synthesized by rapid electrical discharge from the pulse power source along the rods that are used as raw materials. Powerful discharge removes raw material, creating a high-temperature, metal-containing plasma pressure. Thanks to the unique dynamics of the plasma rapidly expands into the surrounding gas, creating a homogeneous gas-phase suspension of nanoparticles. The obtained nanoparticles were continuously collected using a system with closed circuit. Intake fan recycle gases, transferring the particles to the collection system. In this way we obtain a nanocrystalline silver particles with a diameter from 10 nm to 100 nm depending on the process parameters. Typically, for use in the invention is chosen particle size of from 15 nm to 40 nm, or from 20 nm to 25 nm.

Other ways of making nanoceramic to the of mposite are those that described in U.S. patent No. 6719987 (Barell). In these ways to get crystals, the crystal lattice of which is characterized by disordered atoms. During Barell precipitable material was obtained in the vapor phase, for example by evaporation or sputtering, and is transferred into a large volume, which is regulated by temperature. The atoms of the material collide with atoms of the atmosphere of the working gas, lose energy quickly and condense from the gas phase on the cold substrate, for example on the so-called finger cooled with liquid nitrogen. The disordered atoms is created under conditions which limit diffusion so that the material was maintained sufficient disordered atoms. In the case of silver deposition is carried out at low temperatures of the substrate, from -10°C to 100°C, use the following values of the pressure of the working gas, which is higher than normal, the angle of incidence below approximately 30°and use a higher deposition rate, which creates a higher flux of atoms compared to normal. The disordered atoms can also be achieved with the introduction of different atoms or molecules in a metal matrix in a process called "doping" ("doping"), or through the introduction of chemically active gases (such as oxygen) in the camera. According to this method, oxygen is a compound which process gas.

Antimicrobial agents are chemical based and can be applied to the collagen constructs to provide antimicrobial properties. Chemical antimicrobial agents can be selected from the following list (but the list is not limited to, hydrochloride, poly(hexamethylenebiguanide) (RNPS from English. poly(hexamethylene biguanide) hydrochloride, chlorhexidine gluconate, bis-amido polybiguanides, for example, those described in U.S. patent No. 6316669 (link stipulates that the information included in this document), honey, benzalkonium chloride, triclosan (2,4,4'-trichloro-2'-hereditarily ether and dimethyl-octadecyl-(3-trimethoxysilylmethyl)azanium chloride.

In addition to the antimicrobial agent collagen construct may additionally contain antibiotic agent. The antibiotic agent is a tool that is produced by microorganisms to destroy or suppress the growth of other microorganisms. Typically, antibiotics (non-protein) are molecules with low molecular weight that are produced as secondary metabolites, mainly by microorganisms living in the soil. Most of these microorganisms form some type of dispute or other types of dormant cells, and suppose that there is some relationship between the production of antibiotics and processes cpaaob is adowanie. Among the mold notable producers of antibiotics are a genus of fungi Penicillium and Cephalosporium, which are a major source of beta-lactam antibiotics, which include penicillin and related compounds. In the case of bacteria, actinomyces, in particular Streptomyces species produce different types of antibiotics, including aminoglycosides, such as streptomycin macrolides, such as airtronics, and tetracyclines. Barcelonie species that form endospores, produce polypetide antibiotics, such as polymyxin and bacitracin. Antibiotics can have sideline action, i.e. the "mortifying" action, or static effect, i.e. the "inhibitory" effect on the number of microbes. Action spectrum antibiotic agent covers a range of bacteria and other microorganisms, smitten by him. Antibiotics that are effective against prokaryotes, which destroy or inhibit a wide range of gram-positive and gram-negative bacteria are called antibiotics "broad spectrum"; those that are effective mainly against gram-positive and gram-negative bacteria are called antibiotics "narrow spectrum"; and those that are effective against certain organisms or diseases, antibiotics called the "limited range" actions. Preferably used antibiotic with whom unity is a broad-spectrum antibiotic. Antibiotic agents provided in collagen constructs can be combined, for example in combination gram-positive connection narrow spectrum and gram-negative compounds narrow spectrum; however, you can use any combination of antibiotics with different spectrum of activity - broad, narrow and limited.

To antibiotics, intended for use in the invention include: beta-lactams (penicillins and cephalosporins), such as penicillin G, cephalothin; a semisynthetic penicillin (which may also include clavulanic Kyoto), such as ampicillin, amoxicillin, methicillin; carbapenems, such as aztreonam; carboxybenzene, for example, imipenem; aminoglycosides, such as streptomycin, gentamicin; glycopeptides, such as vancomycin; lincomycin, such as clindamycin; macrolides, such as eritromicin; polypeptides, such as polymyxin, bacitracin; polyene, such as amphotericin; nystatin; rifamycins such as rifampin, tetracyclines for example tetracycline; semisynthetic tetracycline, such as doxycycline, and chloramphenicol.

Processed tissue matrix can only be used as a single layer in single layer prosthesis or may be used for prosthesis having two or more than two layers. If it is used as a single is Loy, the antimicrobial agent is introduced into contact with the processed tissue matrix to impart antimicrobial properties to the matrix. Before or after contact with the antimicrobial agent of processed tissue matrix can chemically bind to regulate the speed of biorecognition and biodegradation of the material. In other words, a single-layer antimicrobial constructs include: single layer and processed antimicrobial agent; single layer, chemically cross-linked cross-linking agent, and then processed antimicrobial agent; or a single layer treated with an antimicrobial agent, and then chemically cross-linked cross-linking agent.

Methods of making multi-layer prostheses containing two or more than two layers of processed tissue matrix, are described using the CCM.

One variant of the invention is directed to a flat sheet prostheses and methods of making and using flat sheet prosthesis consisting of two or more than two layers of the CCM, which is connected and cross stitched, as an implantable biomaterial, which has the ability to bioconservatism cells of the patient. Thanks to the flat sheet form CCM, the prosthesis is easily made in the form, containing any number of layers, preferably from 2 to 10 layers, bol is e preferably from 2 to 6 layers, their number depends on the strength and volume necessary for the final intended use of the construct. The CCM has a structural matrix fibers, which extend in the same General direction. When layering the orientation of the layers can vary in order to achieve the overall orientation of the tissue fibers in recycled tissue layers. The sheets can be layering so that the orientation of their fibers were parallel or that they were located at different angles. Layers can overlay each other with obtaining construction with continuous layers over the entire area of the prosthesis. Alternatively, because of the finite size of the execution of the overlay is limited to the circumference of the bowel, layers can be arranged in a checkerboard pattern, this location is done in order to form a sheet construct with a surface area larger than the source material, but without a continuous layer along the area of the prosthesis. For example, you can enter a complex part such as a pipe or network of pipes and channels passing between the layers or crossing them.

In the manufacture of multilayer construct containing the CCM, preferably using aseptic environment and sterile conditions to maintain the sterility of the construct in the case, when you start with STERI inogo CCM material. To obtain a multilayer construct of the CCM, the first sterile stationary support element, for example a fixed plate made of polycarbonate, placed in a sterile area chamber laminar. If, after the processes of mechanical and chemical cleaning sheets of the CCM are not yet in the hydrated state, their hydratious in aqueous solution, for example in water or in phosphate-buffered saline. The leaves get wet CCM sterile absorbent tissue to absorb the excess water from the material. If this is not done, then the CCM material smooth out, cutting off any lymph buildup on the serosal surface, from the washed side. The first sheet is peeled CCM put on the polycarbonate plate and manually smooth on polycarbonate plate to remove any air bubbles, creases and folds. The second sheet is peeled CCM is placed on the upper part of the first sheet again manually removing air bubbles, folds, and bends. This procedure is repeated until then, until you get the desired number of layers for specific applications, preferably until it has received from 2 to 10 layers.

The CCM has such as versatility, from his native tubular condition in which the internal mucous surface facing the intestinal cavity in the native state, while the opposite outer serous is again facing the treated side. It was found that these surfaces have characteristics that can affect the postoperative performance of the prosthesis, but it is also possible to achieve improved performance of the device. Currently using a synthetic devices is the formation of adhesions can cause the need for re-operation for separating adhesions from the surrounding tissue. During the formation of pericardial graft or prosthesis for hernia repair, consisting of two layers of the CCM, prefer the region of connection of the two layers was between serous surfaces, as shown, that the mucous surfaces have the ability to counteract the formation of postoperative adhesions after implantation. In other embodiments implement prefer one surface CCM patchwork prosthesis was non-adhesive, non-sticky, and the other surface had affinity (sticky) to the tissue of the host body. In this case, one surface of the prosthesis will be mucous, and other serous. In addition, in other embodiments, prefer on the opposite surfaces had the opportunity to create adhesions for splicing with each other fabrics, these spikes are in contact with the tissues on each side, thus the prosthesis contains serous surfaces on both side of the construct. Since only the two outer sheet potentially come in contact with other body structures during implantation, the orientation of the inner layers in the case where the construct comprises more than two layers, becomes less important because they probably will not contribute to the formation of postoperative spikes.

After stratifying the required number of sheets of the CCM, they are tied by dehydration together in their binding, namely, where the sheets are in contact. Not trying to associate it with theory, dehydration causes the collagen fibers of the CCM layers are joined together when removing water from the area between the fibers of the matrix CCM. Layers can be degidratiruth or open layer on the first substrate or between the first element substrate and the other control substrate, for example, the second plate is made of polycarbonate, available before drying on the top layer of the CCM and fixed on the first support element to hold all the layers in plasmaplasma location in the application or no application of a small pressure. To facilitate the flow of dehydration, the element substrate may be porous, allowing air and moisture to pass through digidrirovannye layers. Layers can be dried in air, under vacuum or by chemical is of rest, such as acetone or alcohol, for example ethyl alcohol or isopropyl alcohol. Dehydration can hold up to room humidity values in the range of from about 10% to 20% relative humidity or less; or the moisture content of approximately 10% to 20% by mass or less. Dehydration can be performed easily by turning the holder fixing polycarbonate sheet and the CCM layers up to turn his face to the oncoming stream of air laminar, at least for 1-24 hours at ambient temperature, which constitutes approximately 20°C and at room humidity.

Although this is not necessary, in one embodiment, digidrirovannye layers again hydratious before stitching. Digidrirovannye CCM layers together is removed from the porous support link and re-hydratious water the hydrating agent, preferably water, by transferring them to a container containing water the hydrating agent, and keeping it around for 10-15 minutes at about 4°C - 20°C in order to re-hydrate layers without division or separation.

Digidrirovannye or digidrirovannye and re-hydrated, link layers are then sewn with each other in binding by introducing obrabatyvaemogo CCM cross-linking agent, preferably the chemical cross-linking agent, which preserves the ability of the CCM to bioconstruction. As mentioned above, dehydration of collagen fiber matrices of adjacent layers of the CCM stick together, and when the stitching of these layers with each other are formed chemical bonds between the components, whereby the layers are linked. Staple bound prosthetic device gives the strength and durability of the device, improving its performance characteristics. Various types of cross-linking agents known in the art and may be used, such agents are ribose and other sugars, oxidants and dihydroceramides (DHT). dehydrothermal methods. Preferred cross-linking agent is the hydrochloride of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). In another preferred method of sulfo-N-hydroxysuccinimide add to the EDC cross-linking agent as described in Staros, J.V., Biochem. 21, 3950-3955, 1982. In addition to chemical cross-linking agents, the layers can be connected to each other by means of adhesives, fibrin or medical adhesives such as polyurethane, vinyl acetate or polyepoxide. In the most preferred method, the EDC dissolved in water, preferably to a concentration approximately in the range from 0.1 mm to 100 mm, more preferably in the range from 1.0 mm to 10 mm, Naib is more preferably to a concentration of 1.0 mm. Besides water, you can use phosphate-saline buffer, or a buffer of 2-[N-morpholine]econsultancy acid (MESC) to dissolve the EDC. The solution is to add other agents, such as acetone or alcohol, until the volume concentration of 99% in water, usually 50%, in order to make the stitching more evenly and efficiently. These agents remove the water layer, bringing together the matrix of fiber, which promotes binding between the fibers. By regulating the ratio of these agents to the water in the cross-linking agent can be adjusted transverse stitching. The EDC solution for stitching receive immediately before use, since the EDC loses its activity over time. For the introduction of a cross-linking agent into contact with the CCM, hydrated associated CCM layers is transferred into a container, such as a flat cell, and a crosslinking agent carefully decanted into a cuvette, after ascertaining that the CCM layers as covered, and are free-floating state, and that under the layers of the CCM constructs and inside them there are no air bubbles. The container cover, and the layers of the CCM enable cross tsitsa for about 4-24 hours, more preferably for about 8-18 hours at a temperature of from 4°C to 20°C. Cross-linking can be adjusted by variation of the temperature: at low temperatures the priest is acnee stitching runs more efficiently since the reaction is slowed; at higher temperatures the cross-linking proceeds less efficiently, because the EDC thus less stable.

After crosslinking crosslinking agent is decanted and removed and constructs washed in a cell by introducing them into contact with proryvnym agent to remove residual cross-linking agent. Preferred proryvnym agent is water or another aqueous solution. Preferably, effective rinsing is achieved at three times the introduction of chemically bound construct into contact with an equal volume of sterile water for five minutes each wash. As described in this document, constructs impart antimicrobial properties by introducing them into contact with the antimicrobial agent either by introducing a contact, or processing, each layer of processed tissue matrix or by introducing a contact, or process, a multilayer intermediate construct. The method of processing the processed tissue matrix in single-layer and multi-layer form will vary depending on the type of antimicrobial agent, but should remain bioconstruction, biomechanical and biocompatible properties of processed tissue matrix.

In the case where as an antimicrobial AG the NTA choose nanocrystalline silver, it put on a collagen matrix material by introducing into contact with the collagen matrix material with a nanocrystalline silver. The antimicrobial agent can be applied by coating the processed matrix material by means of a suspension agent in solution. Nanocrystalline silver is dispersed in water, aqueous solution or in a solvent to form solution dispersed nanocrystalline silver. CCM immersed in a tray with a certain amount of solution of nanocrystalline silver so that when immersed CCM nanocrystalline silver stick to the CCM. The solution can also be shaken or stirred during the dive.

After that CCM is removed from the solution and placed in an environment in which water or solvent are given the opportunity to evaporate from the CCM with an associated silver, which results in obtaining the CCM construct with a layer of nanocrystalline silver. Other methods of coating the CCM dispersion containing nanocrystalline silver include spraying and providing solvent opportunities for evaporation of the CCM.

In the case when the antimicrobial agent is chosen RNPS, RNPS added to the solution, using his 0,09%and 0.5% (by volume) solution in water, in which the processed tissue matrix is immersed so that the RNM is the solution saturated matrix. After the passage of time sufficient to saturate the solution processed tissue matrix, the matrix is removed from the solution and give it a chance to dry out so that during the evaporation of a solution of the matrix remains RNPS.

Recycled tissue matrices and constructs can be treated or modified, either physically or chemically, before or after fabrication of multilayer associated implantable prosthesis. You can conduct a physical modification to the treated tissue matrices and constructs, such as molding, air conditioning through stretching and relaxation, or perforation in the form of a grid or fenestration. Air conditioning reduces the total deformation of the material, while the perforation, making hole or fenestration provides better conformity to the shape of the surface of the wound and the best passage or drainage of exudates, or provides both mentioned properties. You can also spend a chemical modification, such as immobilization of growth factors, some components of the extracellular matrix, genetic material and other agents that could affect biorecognition or healing part of the body that heal, restore, or replace.

Methods physical modification of the tissue matrix and constructs of the invention can be chosen so that anyone versed in the art, when taking into account the operational characteristics required from the construct. The constructs can be performed with reportnow perforation, which permeates the entire layer construct, through the use of press stamped with needles, blades, or pins, made rapporto on the working surface of the stamp. The construct is placed on the platform and press the stamp so that the needles, blades, or pins were being squeezed through the construct to the platform, when this construct is punctured. In the method of performing slots or mesh structure is used by the plant for the production of holes in the skin, similar to that typically used in procedures autotransplantation of the skin. One such installation to make holes in the skin is the device Zimmer. The constructs can be drilled with a laser to create pores of micron size in the finished prosthesis to help the cells to grow inside, using an excimer laser (for example, when the wavelength KrF or ArF). The constructs can punch, drill, laser, or to make holes in them at any stage of the process of the origin of the process, but it is better to do this before disinfection or heat sterilization. For some indications prefer perforations or holes drilled by the laser passed through all the layers is ratesa to facilitate the passage of cells or fluid drainage. In the case of other indications prefer that they not passed through all the layers so that the holes provided access cells to the interior of the multilayer construct or contributed to the formation of new blood vessels construct.

Other physical modifications of single-layer construct are perforation and fenestration that are cross-cutting. Other physical modification in relation to the single construct is the manufacturer of the construct grid, i.e. the execution of regularly spaced slots in the construct so that it becomes similar to a grid. The slits do with the ratio of the size of the cell, similar to that obtained using the installation by making the skin holes, such as manufactured by Zimmer. The ratio of the size of the cell can be set to a value of 1:1.5 or 2:1. For the manufacture of single-layer CCM construct with antimicrobial properties, the CCM level mucosal side down on a smooth polycarbonate plate, ensuring the elimination of wrinkles, air bubbles and visible lymph buildup. Levelling the CCM polycarbonate plate is conducted to optimize the size. The material is optionally dried sufficiently across its surface. The material is applied at least one antimicrobial agent. Material which does not have to physically modify by punching or manufacturing cells and then cut to a certain size, Packed and finally sterilized according to the technical requirements of sterilization. In an alternative embodiment, the antimicrobial treatment material conduct after he was physically modified through execution in a grid, and making perforations and fenestrate.

After preparation of processed tissue matrix and covering antimicrobial agent constructs shorten to the desired size. For illustration, a practical size is about 6 square inches (about 15.2 cm2), but you can get and use for implantation into a patient constructs of any size.

Anti-microbial-treated collagen matrix is then packaged in a container that is sealed for final sterilization, storage, and distribution. Anti-microbial-treated collagen matrix can be Packed in the container in a dry or wet state. Preferred packaging materials compatible with antimicrobial treatment, collagen matrix, and in the packaging in the wet state is also compatible with any agents that keep the product moist. For constructs that are processed light-sensitive antimicrobial agents, e.g. the antimicrobial agents based on silver, for product packaging use packaging materials that prevent the passage of light or filter it in order to prevent the reduction of antimicrobial activity and discoloration constructs.

At the end of the constructs are sterilized using means known in the field of sterilization of medical devices. The preferred method of sterilization is the introduction of constructs into contact with sterile 0.1% solution of peracetic acid followed by neutralization sufficient quantity of 10 normal sodium hydroxide solution (NaOH), according to U.S. patent No. 5460962, the disclosure of which is included in this document. Disinfection is carried out in a container on a vibrating platform, for example in 1-liter containers Nalge, about 18 hours + 2 hours. The constructs are then washed by introducing them into contact with three volumes of sterile water for 10 minutes for each wash. In a more preferred embodiment, the CCM constructs are sterilized using gamma irradiation in the range of 25-37 GSR. Gamma irradiation significantly, but not fatally, reduces the young's modulus, tensile strength and shrinkage temperature. Mechanical properties after gamma-irradiation is still sufficient for use in a range of applications, as well as gamma-irradiation is prefer inim means for sterilization, because it is widely used in the field of implantable medical devices. Provides for the use of monitoring when conducting each sterilization to ensure that the radiation dose is within the specified range. The constructs are packaged using the packaging material and the model, which is compatible with the composition of the construct and ensures sterility during storage. Preferred packaging means are double-layer legkouswaivaemye packaging where the packaging is termoklina, blister pack consisting of polyethylene terephthalate modified with glycol (PETG from English. polyethylene terephtalate, glycol modified) tray with lid, covered with foil with paper surface, which is placed in the second melt the bag, consisting of a polyethylene/polyethylene terephthalate (PET) film. All of this together, i.e. both main and secondary packaging and the CCM construct contained therein are sterilized using gamma radiation.

The prosthesis of this invention can be flat, tubular, or to have a complex geometry. The shape of the resulting prosthesis will depend on the intended use. Thus, when receiving a binding layer of the prosthesis of the present invention, the elements of the template and the substrate can be configured under the desired f is the NDB. Flat multi-layer prosthesis can be implanted to repair, build or replace diseased or damaged organs, such as the abdominal wall, pericardium, hernia and other various organs and structures, including (but this list is not limited to, bone, periosteum, perichondrium, intervertebral disc, articular cartilage, dermis, intestine, ligaments and tendons. In addition, a flat multi-layer prostheses can be used as a vascular or intracardiac flap, or as a replacement heart valve.

Flat sheet prostheses, either single layer or multi-layer, used in the healing of wounds to cover the wound of the patient for education waterproofing strip, to provide peace and comfort to the wounded area and to provide antimicrobial activity, since the area of the wound stabilize before beginning treatment.

Flat sheets can also be used to support bodies, for example to support loose or hypermobile bodies, through the use of leaves as a support bandage for organs such as the bladder or uterus. Tubular prostheses can be used, for example, to replace the cross sections of the tubular bodies, such as the vasculature, esophagus, trachea, intestine and fallopian tubes. These organs are the main tubular shape sudeshna surface and internal luminale surface. In addition, a flat sheet and tubular structures can be linked together to form complex structures intended to replace or increase cardiac or venous valves.

Materials CCMS used in the manufacture of antimicrobial constructs of the invention are biocompatible. Biocompatibility test conducted on the prostheses made from CCM, according to the Tripartite and ISO 10993 guidelines for the biological evaluation of medical devices. The term "biocompatible" means that the prosthesis of recitation, gemosovmestimosti, apirogenny, free of endotoxins, regeneracion, neoantigen and do not cause dermal sensitization reaction, do not cause acute systematic toxicity. These biocompatible quality are discussed further in more detail. The test items of the constructs derived from the CCM, show no biological reactivity (Grade 0) or cytotoxicity observed in L929 cells after a certain period of time, when the tested product was subjected to exposure using a test called "Test L929 agar overlay on cytotoxicity in vitro. The observed cellular response to positive control product (Grade 3) and negative control product (Grade 0) confirmed the reliability of the system under test. Testing and evaluation was performed according to the ACLs guidelines by the U.S. Pharmacopoeia. It is believed that the prosthesis of the invention recitation and meet the requirements of the Test L929 agar overlay on cytotoxicity in vitro.

Test hemocompatibility tests (in vitro hemolysis using in vitro modified extraction method : ASTM) prostheses of the invention was carried out according to the modified extraction method : ASTM. In terms of research value hemolytic index to extract fixtures amounted to 0%, with positive and negative controls are represented as receiveready. The results of the study indicated that the prosthesis of the invention phenolation and gemosovmestimosti.

The prostheses of the invention was tested on progenote according to the current USP Protocol for testing on progenote in rabbits. In terms of studying the total temperature rise of the rabbits during the period of observation is within acceptable according to USP. The results confirmed that the prosthesis of the invention apirogenny. The prostheses of the invention are free from endotoxins, preferably at the level of ≤0,06 units/ml (cm2product). Endotoxin refers to individual pyrogen, which is part of the cell membrane of gram-negative bacteria, it is thrown away bacteria and contaminates materials.

The prostheses of the invention do not cause a skin reaction when debilizatsii. In the literature there are no reports that would indicate that the chemicals used for purification of porcine intestinal collagen, cause a sensitization reaction, or may modify the collagen, causing a reaction. The results of the test prosthesis of the invention derived from chemically purified CCM indicates that the prosthesis does not cause a sensitization reaction.

The prostheses of the invention does not cause the reaction of primary skin irritation. The results of tests of chemically purified CCM on irritancy indicate that the prosthesis of the invention, composed of chemically purified CCM does not cause the reaction of primary skin irritation.

Testing for acute systemic toxicity, intracutaneous toxicity was conducted on chemically cleaned CCM used to obtain the prostheses of the invention, the test results show no toxicity among the tested prostheses. Additionally, in the study of animal implants are not found evidence that chemically purified porcine intestinal collagen causes of acute systemic toxicity.

Tests on the subacute toxicity of the prosthesis of the invention containing the porcine intestinal collagen, confirmed the absence of the device subacute toxicity.

In the literature there are no messages that would indicate that the chemist is s, used for purification of porcine intestinal collagen, could potentially be genotoxic attention, or could modify the collagen, causing such a reaction. Tests for genotoxicity prostheses of the invention containing the porcine intestinal collagen, confirmed the absence of the device genotoxicity.

The aim of the process of chemical cleaning porcine intestinal collagen used to obtain the prostheses of the invention is the minimization of antigenicity due to the removal of cell residues, Nikolayevich and inelastic matrix components. Confirmed that the prosthesis of the invention containing the porcine intestinal collagen, there is no such drawback as the antigenicity of the device, it is confirmed by implantation studies conducted using chemically purified porcine intestinal collagen.

CCM constructs of the invention preferably possess antiviral activity. In the manufacturing process tested the effectiveness of two treatments chemical treatment, using chelating alkaline solution of NaOH/EDTA (pH 11-12) and acidic salt solution of HCl/NaCl (pH 0-1), to inactivate the four relevant and model viruses. Model viruses were selected on the basis of the source of porcine material and to provide a broad range of physico-chemical the ski properties (DNA, RNA, enveloped and bezobolochnye viruses). Viruses included virus pseudoleskeella, virus viral diarrhea bull, reovirus-3 and porcine parvovirus. Studies were conducted on the basis of instructions by the FDA and ICH, including the following documents: CBER/FDA "Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993)"; ICH Note for Guidance on Quality of Biotechnological Products: Viral Safety Evaluation of Biotechnology Products derived from Cell Lines of Human or Animal Origin (CPMP/ICH/295/95); and document CPMP biotechnology working group "Note for Guidance on Virus Validation Studies: The Design, Contribution and Interpretation of Studies Validating the Inactivation and Removal of Viruses" (CPMP/BWP/268/95). The results show that the total viral inactivation of two-stage chemical treatment has a coefficient of purification above 106for all four model viruses. The data indicate that the chemical cleaning procedures are robust and effective process that has the potential to inactivate a wide variety of viral agents.

The prostheses of the invention are bioconservatism. When functioning as a substitute body part or support the prostheses of the invention also function as bioconservative matrix scaffold for ingrowth inside the cells of the host body. The term "bioconstruction" is used in this document to refer to the production of endogenous structural collagen, in which scolarizarii and secondary settling cells by growing inside host cells with the speed approximately equal to the rate of biodegradation, transformation, and substitution matrix components of an implantable prosthesis cells masters and enzymes. Implantable prostheses retain their structural features while reconstructing the organisms in which they are implanted in all, or almost all, the fabric of the host body, and essentially function as similar cloth, which they recover or heal. In addition to these bioconservatism qualities get the prostheses of the invention are derived from two or more than two layers of processed tissue matrix, to give them the desired biomechanical properties.

The young's modulus (MPa) is defined as a constant coefficient linear relationship between load and deformation. The tensile strength (N/mm) is a measure of the strength throughout the prosthesis. Both of these properties depend on the number of layers of the CCM in the prosthesis. When used as a load bearing or supporting device, the prosthesis must be able to withstand the uneven physical activity during the initial healing phase and throughout the reconstruction.

The strength of the stratification binding regions is measured using the test for exfoliation. Immediately after surgical implantation, it is important that the layers are not of otstaiva is of n during exercise. Animal studies explantion materials did not show any signs of delamination. Before implantation, the adhesion strength between the opposing layers is about 8.1+2.1 N/mm for multilayer construct, made in 1 mm solution of EDC.

Shrinkage temperature (°C) gives a measure of the degree of crosslinking of the matrix. The higher shrinkage temperature, the more is sewn matrix in the material. Unstitched, gamma-irradiated CCM has a shrinkage temperature of about 60,5±1,0°C. In a preferred embodiment, the prosthesis is sewn EDC will preferably have a temperature approximately in the range from 64,0±0.2°C up to 72.5±1.1°C in the case of devices that are sewn together in a solution of EDC in 50% acetone with a concentration of EDC from about 1 mm to 100 mm, respectively.

Mechanical properties include mechanical integrity under which it is understood that the prosthesis must be resistant to slipping during bioconservative, and, in addition, flexible and suitable for suturing. The term "flexible" means good performance properties in terms of ease of use in the clinical setting.

The term "suitable for suturing" means that the mechanical layer properties include the ability to hold the seam, allowing the needle and suture material to pass through the material of the prosthesis and twisting during the sewing of the prosthesis to the sections of the native tissue. During closing such prostheses should not break in the tension, which has the seam, and they should not break when the closure seam in the form of a node. The suitability of the prosthesis to the stitches, i.e. the ability of the prosthesis to resist rupture when stitching, refers to its own mechanical strength of the prosthetic material, the thickness of the implant, the tensile force applied to the seam, and the speed with which close the site. The ability to keep the seam flat, 6-layer prosthesis with a high degree of crosslinking, chemical crosslinking in 100 mm EDC and 50% acetone solution, is about 6.7±1.6 km N. The ability to hold the seam for 2-layer prosthesis, made in 1 mm solution of EDC in water, is about 3.7 N ± 0.5 N. The preferred lower limit for the strength to hold the seam is about 2 H for stitched, flat, 2-layer prosthesis as surgical force at the overlap seam is about 1.8 N.

Used herein, the term "responsewe" means that the biomechanical properties of the prosthesis impart wear resistance so that the prosthesis is not stretched, swells or expands above the normal limits after implantation. As described below, the total elongation of the implantable prosthesis of the present invention is within acceptable limits. The prosthesis of this izaberete the Oia acquires resistance to stretching, as a function of post-implantation cell bioconservative by replacing the structural collagen cells-owners, with a higher speed than is the loss of mechanical strength of implantable materials due to biodegradation and biorecognition.

Recycled fabric material of the present invention is "semi-permeable", even if its layer and bind. Polyphonically allows for ingrowth inside host cells for biorecognition and deposition agents and components that could affect bioconstruction, cellular ingrowth, preventing or promoting adhesion, or the bloodstream. "Non-porous" as prosthesis prevents the flow of liquids, intended to hold the implantable prosthesis. On the contrary, pores, perforations, fenestration, notches and holes can be formed in the prosthesis, if its use requires porous and perforated property.

The mechanical integrity of the prosthesis of the present invention also depends on its ability to drape or shape, as well as from cutting or grinding with a clean face without peeling or abrasion facets of the construct.

The sheet of processed intestinal collagen derived from the submucosal membrane of the small intestine, the usual has a thickness in the range of about from 0.05 to 0.07 mm. Giving sheet geometry purified tissue matrix, it can be layering, and then chemically bind the sheets together to produce a multi-layered construct with greater thickness. Processed tissue matrix layers prosthetic device of the invention can originate from the same collagen material, for example, two or more than two layers of the CCM, or from different collagen materials, for example, one or more layers of the CCM and one or more layers of wide fascia.

Constructs with antimicrobial properties can be used for the treatment of wounds, including: superficial and deep wounds, pressure ulcers, venous ulcers, diabetic ulcers, chronic vascular ulcers, tunneled wounds, wounds with overhanging edges, surgical wounds (e.g., donor site wounds with autologous transplantation, wound post-Mohovskoe surgery, wounds, post-laser surgery, wound dehiscence), trauma wounds (such as abrasions, lacerations, second-degree burns, and skin tears) and draining wounds. The dressing on the wound is a single layer sheet or a multilayer construct of mechanically and chemically cleaned intestinal collagen pigs, which contains at least one antimicrobial agent, each sheet of processed tissue matrix has a thickness in the range of about from 0.05 is about 0.07 mm, contain the fenestration, which establish end-to-end communication between both sides of the sheet. The product contains primary porcine collagen type I (approximately the number of >95%) in its native form, the lipids in an amount of about less than 0.7%, glycosaminoglycans on non-detectable level (approximately <0,6%) and DNA (approximately at a concentration of <0.1 ng/µl). Porcine intestinal collagen are essentially free from cells and cell remnants. Invented the bandage on the wound can be chemically crosslinked or may not be crosslinked, but in the case of stitching, sew it to such an extent that it was possible to regulate and control the biodegradation, bioconstruction or replacement headbands cells of the patient. Physical modifications you can spend on the bandage to the wound in order to ensure the possibility of leakage of liquids. The dressing on the wound provides a moist healing environment, a barrier to control and minimize bacterial contamination, and eases the pain of a patient by covering the sensitive nerve endings. The antimicrobial agent provides effective protection against microbial contamination in the area of the wound and around it.

The dressing on the wound applied to the patient who has a wound that needs treatment. Before applying the size of the wound can be proteinpilot and clean away the dead tissue, using standard IU Tiki applying a bandage to the wound. Preferably, the reorganization of the wound cavity is where the wound edges contain viable tissue. For the application of dressings of the invention, it is cut on the contour area of the wound. If the wound more piece bandage on the wound, you can use a few pieces, partially overlaid on each other to cover the entire wound. If the bandage is dry, it can re-hydrate, using sterile saline or other isotonic solution. The edge of the bandage should be in contact with intact tissue, after that its smooth area in order to ensure that the dressing is in contact with the underlying layer of the wound. If the sheet is collected excess exudate, then cut small slits in the sheet to allow exudate to flow.

Invented antimicrobial dressing on the wound can be used as the interface between the wound and traditional, or secondary dressing on the wound. Alternatively, the antimicrobial dressing on the wound of the invention can be used as the outer layer to replace the skin, such as autograft, allograft or construct artificially grown skin. When used as the outer layer of the bandage to the wound provides moisture environment RA is s so, the cells of any skin replacement can colonize the wound surface for faster wound closure.

After application is preferably applied with a non-sticky, secondary dressings to maintain a moist wound environment. Optimal secondary dressing is determined by the location of the wound, its size and depth, as well as user preference. If necessary, the secondary dressing can be replaced to maintain the area of the wound in a moist, clean condition. The frequency of changing dressings will vary depending on the type, size and depth of the treated wounds, the amount of exudates and the type of dressing. During the flow of healing you may need to replace the bandage on the wound, in this case it is possible to carry out additional imposition of bandages on the wound until then, until you achieve complete healing.

Other embodiments of the invention relates to a multilayer constructs, endowed with antimicrobial properties. Prosthetic device of the present invention contains two or more than two overlapping collagen layer, which are connected together. Used herein, the term "linked collagen layers" means that the construct consists of two or more than two layers of the same or different collagen material, which is processed so that the layers overlap one on the other and are held together sufficiently by clonaslee and formation of chemical cross-links. More specifically, the prosthetic device is a surgical mesh or implant intended for use in implantation in order to enhance soft tissue, including (but this list is not limited to): defects of the abdominal and chest wall, strengthen the muscular valve, rectal and vaginal prolapse, the restoration of the pelvic floor hernia, bridge gaps in fascial defects, gapping and restorative procedures. One of the prosthetic mesh, and the implant of the invention contains a five-layer sheet pork CCM and an antimicrobial agent, its thickness is from about 0.20 mm to 0.25 mm Product consists primarily of porcine collagen (approximately the number of >95%) in its native form of lipids in an amount of about less than 0.7%, contains glycosaminoglycans on non-detectable level (approximately <0,6%) and DNA (approximately at a concentration of <0.1 ng/µl). Porcine intestinal collagen are essentially free from cells and cell remnants. Provides a prosthesis in a sterile form with sizes ranging from 5×5 cm to 12×36 cm in the two-layer legkootkryva packaging. Denaturation temperature of the prosthesis is approximately 58±5°C; tensile strength greater than 15 N; strength retention suture above 2 N when using 2-0 braided silk suture; and the level of endotoxin is <a 0.06 units/ml (cm2product). In castnet is, the prosthesis from a flat sheet construct consists of five layers of the CCM and antimicrobial agent, his tie and sew a 1 mm solution of the hydrochloride of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in water. To generate this construct, the first sheet CCM distribute mucosal side down on a smooth polycarbonate plate; ensuring the absence of wrinkles, air bubbles and visible lymph buildup. Smoothing the CCM is performed to optimize the size. Three sheets of the CCM layer (mucosal side down) on top of the first sheet, ensuring the absence of wrinkles, air bubbles and visible lymph buildup when layering of each sheet. The fifth sheet layer so that the mucous side was the front, ensuring the absence of wrinkles and air bubbles. Visible lymph growths removed before layering this fifth sheet. Layers together dried for 24±8 hours. Now the layers are dried together, and then sew a 1 mm solution of EDC in water for 18±2 hours cross-linking solution is taken in a quantity of 500 ml per 30 cm five-layer sheet. Each product is washed with sterile water, and then cut to the desired final size, while it is in a hydrated state.

In an alternative embodiment, the prosthetic device is a selected minimal the practical belt, at least one antimicrobial agent, which is intended for implantation to enhance and support soft tissue, where there is a need for this, including (but the list is not limited to) the following procedures: the fold-urethral support, recovery prolapse (urethral, vaginal, rectal, and relating to the colon), reconstruction of the pelvic floor, mahapasana bearing, keeping in uterosacral vaginal area, recovery and tissue repair. In another most preferred embodiment, the prosthetic device is a surgical belt, consisting of three to five layers associated, cross stitched CCM processed, at least one antimicrobial agent. For making double-wall device CCM level mucosal side down on a smooth polycarbonate plate, ensuring the absence of wrinkles, air bubbles and visible lymph buildup. The CCM leveling is performed to optimize the size. The second, third and fourth sheets of the CCM layer (mucosal side down) on top of the first sheet, ensuring the absence of wrinkles, air bubbles and visible lymph buildup when layering of each sheet. The fifth sheet layer so that the mucous side was the front ensuring the absence of wrinkles and air bubbles. Visible lymph growths should be removed before layering this fifth sheet (three-layer construct is made, levelling the first sheet CCM mucosal side down on a smooth polycarbonate plate; ensuring the absence of wrinkles, air bubbles and visible lymph buildup; a second sheet of the CCM layer (mucosal side down) on top of the first sheet and the third sheet layer on the upper part of the second sheet so that the mucous side was the front). Layers dried for 24±8 hours and after drying sew in 90% acetone solution with a concentration of 10 mm EDC for 18±2 hours cross-linking solution is taken in a quantity of 500 ml per 30 cm five-layer sheet. Each linked, cross stitched construct washed with sterile water, and then cut to the desired final size, while it is in a hydrated state. By providing pubic-urethral support strap can be used for the treatment of urinary incontinence resulting from urethral hypermobility or lack of own sphincter. Surgical belt consists of a five-layer laminated sheet porcine intestinal collagen, its thickness is from about 0.20 mm to 0.25 mm, and an antimicrobial agent. The device sew the hydrochloride of 1-ethyl-3-(3-dimethylaminopropyl is) carbodiimide (EDC). The device consists mainly of porcine collagen type I (approximately the number of >95%) in its native form of lipids in an amount of about less than 0.7%, contains glycosaminoglycans on non-detectable level (approximately <0,6%) and DNA (approximately at a concentration of <0.1 ng/µl). Porcine intestinal collagen are essentially free from cells and cell remnants. Denaturation temperature of the prosthesis about above 63°C, tensile strength greater than 15 N, the strength retention of the seam above 2 N when using 2-0 braided silk suture, and the final level of endotoxin is ≤0,06 units/ml (cm2product). While bioconservative aspects of the belt can be varied and used to achieve the goal, dressing prosthesis of the invention can not be a replacement for parts of the body, but is a supporting device for a body implantable as an auxiliary structure. Prefer the CCM layers of strap sewed more longevity highly crosslinked to reduce bioconstructions belt (band). Dressing prosthesis is biocompatible, flexible, its collagen structure during implantation supports the necessary structural support and strength when functioning as a support device for a body.

In yet another alternative embodiment, the prosthetic device is the Wallpaper flap for repair of the brain, which is intended for implantation to restore the Dura mater, a dense membrane, which protects the Central nervous system. The device of the invention for recovery of the brain consists of four layers associated, cross stitched CCM, and contains an antimicrobial agent. For manufacturing a four-layer device, the CCM level mucosal side down on a smooth polycarbonate plate, ensuring the absence of wrinkles, air bubbles and visible lymph buildup. The CCM leveling is performed to optimize the size. The second and third sheet of the CCM layer (mucosal side) on the upper part of the first, ensuring the absence of wrinkles, air bubbles and visible lymph buildup when layering of each sheet. The fourth sheet layer so that the mucous side was the front, ensuring the absence of wrinkles and air bubbles. Visible lymph growths should be removed before layering this fourth sheet. Layers dried for 24±8 hours, and after they become dry, they cross stitched in a buffer solution of 2-[N-morpholine]econsultancy acid (CIE) with EDC concentration from about 0.1 mm to 1 mm for 18±2 hours cross-linking solution is taken in a quantity of 500 ml per 30 cm four-layer sheet. Each linked, the cross is sewn on the construct washed with sterile water, and then cut to the desired final size, while it is in a hydrated state. The device for repair of Dura mater consists of a four-layer laminated sheet porcine intestinal collagen, its thickness is from about 0.14 mm to 0.21 mm, and an antimicrobial agent. The device sew the hydrochloride of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The device consists mainly of porcine collagen type I (approximately the number of >95%) in its native form of lipids in an amount of about less than 0.7%, contains glycosaminoglycans on non-detectable level (approximately <0,6%) and DNA (approximately at a concentration of <0.1 ng/µl). Porcine intestinal collagen are essentially free from cells and cell remnants. Denaturation temperature of the prosthesis about above 63°C; its tensile strength is greater than 15 N, its strength retention suture above 2 N when using 2-0 braided silk suture, and the final level of endotoxin is ≤0,06 units/ml (cm2product). The device for repair of the Dura biocomercio and bioconstruction so that upon implantation in the body of the patient, if necessary, repair of the Dura, it functions as a replacement for Dura over time, during which p is oshodi biorecognition cells masters, which destroy and replace the device so that the new fabric of the host body over time replaces the device.

For example, the multilayer construct of the CCM is used to restore the structures of the walls of the body. It can also be used, such as pericardial graft, myocardial graft, vascular graft, flap to the bladder wall, or device for repair of a hernia (as a flap, free from stress, or tube), or to use as a belt to support hypermobile and falling bodies (at loss of rectal cancer, vault prolapse, hernia of the bladder). Multilayer construct suitable for the treatment of connective tissue in the muscles responsible for rotation of the shoulders or the recovery capsule. Multilayer construct suitable for repair of Dura mater in cranial defects after procedures craniotomy or to restore cerebral channels along the spinal cord. The material is suitable for annular repair fibrous ring in the case, when it goes beyond the cavity (i.e. when the herniated intervertebral disk), as well as the material used as the tube to deepen formed slipped disc or as a cover for the recess, or both. The material is suitable in proced the arts of plastic surgery, such as mastopexy, abdominal surgery and in plastic surgery of the face (eyebrows and cheeks). Both single-layer and multi-layer CCM materials can be used as coatings or dressings to wounds to promote wound healing. Moreover, it can also be implanted in a flat, collapsed or folded form to increase the volume and capacity of the fabric. Several layers of the CCM can be included in the construct in the case when there are indications for an increase in volume and strength. Before implantation layers can additionally be treated or coated with collagen or other extracellular matrix components such as hyaluronic acid, heparin, growth factors, peptides or cultured cells.

The following examples are provided to better explain the practical part of the present invention, and should not be interpreted in any way as limiting the scope of the present invention. It is worth estimate that the scheme of arrangement in terms of its composition, shape, and thickness is selected depending on the final guidance on the application of the construct. Those who are well-versed in this field of medicine, recognize various modifications that can be made on the methods described in this document without derogation from the essence and scope of the present invention.

Examples

Example 1. Chemical cleaning m is a mechanical purified small intestine of the pig

The small intestine of the pig seize and mechanically cleaned using a heated cleaning device Bitterling (Nottingham, UK), which forcibly separates the fat, muscle and mucous layers from submucosal membrane, using a combination of mechanical and water washing. Mechanical action can be described as a set of rollers which compress and squeeze the successive layers of submucosal membrane by passing the whole of the intestine between them. Submucosal membrane of the small intestine is relatively firmer and stronger in comparison with the surrounding tissue, so the rollers squeeze the softer components of the submucosa. The result of the cleaning machine is that solely remains the submucosal layer of the intestine.

The remainder of the treatment chemical treatment according to international PCT application number WO 98/49969 authored by Abraham, et al., the disclosure of which are incorporated into this document by reference, is carried out in aseptic conditions and at room temperature. All chemical solutions used at room temperature. The intestine is then cut in a longitudinal direction down from the cavity, and then cut into 15 cm segments. The material is weighed and placed in containers with a volume ratio of solution to intestinal material 100:1.

In each container that contains the intestines, add approximately 1 l of p is the target, sterilized through a filter with a size of 0.22 μm (microns) and contains TETRANITRATE salt ethylenediaminetetraacetic acid (EDTA) at a concentration of 100 mm sodium hydroxide (NaOH) - 10 mm. The containers are then placed on a vibrating table, soaking for 18 hours at a frequency of about 200 revolutions per minute. After shaking the EDTA/NaOH removed from each flask.

In each container, then add approximately 1 l of solution, sterilized through a filter with a size of 0.22 μm and containing hydrochloric acid (HCl) in a concentration of 1 M sodium chloride (NaCl) - 1 M Containers are then placed on a vibrating table, soaking for 6-8 hours at a frequency of about 200 revolutions per minute. After shaking the solution with HCl/NaCl removed from each container. In each container, then add approximately 1 l of solution, sterilized through a filter with a size of 0.22 μm and containing sodium chloride (NaCl) at a concentration of 1 M phosphate-saline buffer (PBS) 10 mm. The containers are then placed on a vibrating table, soaking for 18 hours at a frequency of about 200 revolutions per minute. After shaking the NaCl/PBS removed from each container.

In each container, then add approximately 1 l of 10 mm PBS solution, sterilized through a filter with a size of 0.22 μm. The containers are then placed on a vibrating table, aderiva for 2 hours at a frequency of about 200 revolutions per minute. After shaking phosphate-saline buffer removed from each container. In conclusion, for each container, add approximately 1 l of water, sterilized through a filter with a size of 0.22 μm. The containers are then placed on a vibrating table, soaking for 1 hour at a frequency of about 200 revolutions per minute. After shaking the water removed from each container.

Recycled CCM samples cut and fixed for histological analysis. Spend staining hematoxylin-eosin (H&E) and trichrome staining Masson as the cross-section and longitudinal section of the samples of both the control and the treated tissue. Samples processed CCM fabrics look free of cells and products of disintegration of cells, whereas untreated control samples look normal and as expected, are highly expressed cellular structure.

This single layer of material, the CCM can be used as a single layer or be used for reporting related multilayer constructs, tubular constructs or constructs with complex tubular structure and with flat sides.

Example 2. A method of manufacturing a multi-layer CCM construct CCM processed according to the method of Example 1, are used for composing the multilayer construct containing 2 layers of the CCM. CTE is strong sheet of porous polycarbonate lay in the sterile area laminar. CCM soak a sterile cloth TEXWIPES (LYM-TECH Scientific, Chicopee, MA) to absorb the excess water from the material. CCM material cleaned from his lymph buildup with the treated side, and then cut into pieces of a length of about 6 inches (about 15.2 cm). The first sheet is peeled CCM put on the polycarbonate plate, mucosal side down, manually remove any air bubbles, bends and folds. The second sheet is peeled CCM is placed on the upper face, or purified by the party of the first sheet, with the treated side of the second sheet is in contact with the treated side of the first sheet; again remove any air bubbles, bends and folds. Polycarbonate plate with the CCM layers turn, paying the CCM layers against the oncoming air stream of laminar. Layers give you the ability to be dried for about 18±2 hours in the cockpit at room temperature of approximately 20≤C. the Dried layers of the CCM together then remove from polycarbonate plate without separation or peeling, and is transferred at room temperature in a water bath, soaking in it for about hydration layers. Chemically cross-linking a 100 mm solution of EDC in 50% acetone receive immediately before the stitching, because the EDC will eventually lose its activity. Hydrated layers then transferring the flat in a ditch, and a crosslinking agent carefully decanted into a cuvette, making sure that the layers are covered and swim freely in the fact that under the constructs or within them there are no air bubbles. The cell cover and leave in a fume hood for about 18±2. Cross-linking solution is decanted and discarded. Constructs washed in the cuvette three times with sterile water for five minutes for each wash. Using a scalpel and ruler constructions sanded to the desired size.

Constructs sterile sterilized by treatment with 0.1% solution of peracetic acid (NAA), neutralizing its 10-normal sodium hydroxide solution NaOH according to U.S. patent No. 5460962, the disclosure of which is included in this document. Constructs sterilized in 1 l containers Nalge on a vibrating platform for 18±2 hours. The constructs are then washed with three volumes of sterile water for 10 minutes for each wash, and watching the activity NUS through test Minncare on the tissue sample in the form of a tape, in order to ensure that this activity constructs.

The constructs are then Packed in plastic bags using a vacuum sealer, which, in turn, placed in sealed bags for gamma radiation in the range from 25.0 to 35.0 rag.

Example 3. Analysis of zone of inhibition

A is Y. this Example is to illustrate how to determine an effective amount of an antimicrobial agent for single-layer constructs derived from recycled fabric material - derived submucosal membrane of porcine intestine (CCM)obtained by the method of Example 1, which creates a microbial barrier, and kills germs. Several antimicrobial agents applied to the CCM either by dissolution or by suspension in a solution, as set forth below:

1. 0.5% solution of silver nitrate;

2. 1% solution of silver nitrate;

3. 1-2% solution of nanocrystalline silver in isopropyl alcohol (IPA);

4. 1-2% solution of nanocrystalline silver in a single PBS;

5. 1-2% solution of poly(hexamethylenebiguanide) (RNPS) in phosphate-buffered saline;

6. 1-2% solution of poly(hexamethylenebiguanide) (RNPS) in purified water.

Chemically cleaned CCM cut into pieces the size of 4 cm × 6 cm, and each piece is placed as in hydrated and digidratirovannogo state, approximately 100 ml of each solution and leave overnight. Recycled CCM then removed from solution and dried. As control conditions using the raw samples of the CCM. Cut out round pieces from each piece of the sample. On agar plates stripe bacteria. The samples are placed on the plates and incubated them for 24 hours with C is poured observation.

Nanocrystalline silver (AUC) fully displaytouch in the solutions prepared in IPA, and leave in solution, whereas the AUC in a single PBS does not remain in solution and requires shaking before adding to the CCM. IPA, one stripped off during the night, looks smooth and uniform on both the hydrated and digidrirovanny, the pieces CCM digidrirovanny piece retains a clearer form and it is easier to handle. Both pieces return color gas black. Party CCM, which is facing down in the tray, has the distinction of parties, despite the lack of evidence of impact on the floor of the CCM or the effectiveness of the material when creating a large zone of inhibition (ZI -). zone of inhibition). AUC products create the best ZI and also create ZI, which will not allow bacteria to grow in 24 hours ZI after 4 days, as it happens with several other samples. Although the samples of silver nitrate are ZI, which is compared with the result for the AUC, but large compared with other samples, the message on the toxicity of silver nitrate does not allow you to use it as the ideal additive to the CCM. Known antimicrobial properties of silver nitrate make it suitable for comparison with the AUC, and, as installed, AUC exceeds the efficiency of nitrate of silver.

Both solution PH IS IN the look equal, while the CCM after the soaking does not appear so. PBS solutions leave the CCM at the level that was observed for the uneven balance of salt, while WFI solutions leave CCM without visible changes. They both give similar ZI. They are smaller and do not create them in the same way as in the case of the CR BS.

Found that nanocrystalline silver and RNPS are effective antimicrobial agents that can be used on CCM constructs, as evidenced by their bactericidal effect found in this method of analysis.

Example 4. Treatment 2-layer collagen constructs containing antimicrobial nanocrystalline silver or RNPS

Get laminated 2-layer CCM constructs (as laminated and cross stitched), and then treated with an antimicrobial agent to obtain a two-layer constructs with antimicrobial properties.

Twelve 2-layer CCM constructs, each approximately the size of 9 cm × 9 cm, receive according to Example 2. When receiving these CCM constructs cross stitch, using a buffer of 10 mm EDC / 0.1 M CIE [2-(N-morpholine)econsultancy acid] (Pierce, Rockford, IL)for 16-20 hours and washed three times in sterile filtered water for 30 minutes. The resulting construct is then treated PR is tuomikoski agent.

Antimicrobial agents are given either in the form of solutions or dispersions. Five dispersions of nanocrystalline silver get by mixing 10, 1,0, 0,1, of 0.01, 0.001 gram of nanocrystalline silver (nano technologies (Ag-20) or equivalent, Austin, TX) in 1 liter of dispersing agent, which is isopropyl alcohol (sterile filtered water is also acceptable dispersing agent). 0,2%, 0,1%, 0,02%, 0,002% the solution RNPS get by mixing Cosmocil CQ (20% solution RNPS, ArchChemicals, Inc., Norwalk, CT) with RODI/WFI.

To cover laminated and cross stitched constructs antimicrobial agent is hydrated constructs are placed in trays size 9.5 cm × 9.5 to see 50 ml each agent is decanted into the trays, and the trays are placed on a vibrating table. The time intervals of the coating is 10 seconds, 1 hour, 3 hours and overnight (about 18 hours). Upon completion of the preset time of the coating samples provide an opportunity to be dried on polycarbonate plates under sterile air stream biologically safe cabin laminar to the value of the relative humidity of 10-20%.

The resulting constructs are 2-layer CCM constructs that are already laminated, stitched and processed antimicrobial agent in order to give the constructs antimicrobial properties.

Use the 5. Processing a single layer of collagen matrix antimicrobial nanocrystalline silver or RNPS, which is used for manufacturing 2 ply antimicrobial constructs

Get a single-layer CCM constructs, cross sew them treated with antimicrobial agent, and then applied for the compilation of two constructs with the aim of obtaining a double layer of processed tissue-matrix constructs with antimicrobial properties. A greater quantity of antimicrobial agent is introduced between the CCM layers of processed tissue matrix by laminating constructs after treatment of their antimicrobial agent.

CCM receive according to Example 1, distributing on polycarbonate plates and removing the lymph buildup. Cut out pieces approximately the size of 10 cm × 9 cm Each piece CCM then cross stitched in a buffer of 10 mm EDC / 0.1 M CIE [2-(N-morpholine)econsultancy acid] (Pierce, Rockford, IL), taking it to 2 litres of 26 pieces, and shaken on a vibrating table, setting up to 4 samples for 16-20 hours and then washed three times, at least 2 l of sterile filtered water for at least 20 minutes for each wash.

Antimicrobial agents are given either in the form of solutions or dispersions. Three dispersion NANOKRISTALLIChESKOGO the silver get by mixing 1,0, of 0.1, 0.01 gram of the composition of the nanocrystalline silver (nano technologies (Ag-20) or equivalent, Austin, TX) in 1 liter of dispersing agent, such as isopropyl alcohol (sterile filtered water is also acceptable dispersing agent). 0,2%, 0,1%, 0,02% solutions RNPS get by mixing Cosmocil CQ (20% solution RNPS, ArchChemicals, Inc. Norwalk, CT) with RODI/WFI.

To cover the CCM antimicrobial agent pieces are placed in a square 125 ml sterile containers (Nalgene), in calculating the 4 piece to the container. 100 ml of solution or dispersion containing an antimicrobial agent, decanted into each container to dive constructs. CCM leave immersed for 3-6 hours, in this time, the container is shaken on a rotating vibrating platform.

Anti-microbial-treated CCM then layer for making a two-layer constructs, placing one machined piece CCM exactly on the polycarbonate plate. The second processed piece CCM then placed directly on top and spread over the first layer so that between the layers, there were no air bubbles. Polycarbonate plate with constructs are then placed under a sterile air flow biological safety cab laminar flow for drying them up to 10-20% of relative humidity, at least for 12 hours.

the resulting constructs are 2-layer CCM constructs already stitched and processed antimicrobial agent in order to give the constructs antimicrobial properties, and then layered in order to implement an antimicrobial agent not only on the outer surface of the constructs, but also between the layers of constructs.

Example 6. Treatment 2-layer collagen constructs containing antimicrobial nanocrystalline silver

Get laminated 2-layer CCM constructs (as laminated and cross stitched), and then treated with antimicrobial agent to obtain a two-layer constructs with antimicrobial properties.

Get twelve 2-layer CCM constructs according to Example 2, the size of each is approximately 35-40 cm × 9 see During their receipt of these CCM constructs cross stitch, using a buffer solution of 10 mm EDC/0.1 M CIE [2-(N-morpholine)econsultancy acid] (Pierce, Rockford, IL) in water, for 16-20 hours and washed three times in sterile filtered water for 30 minutes. The resulting constructs and then treated with an antimicrobial agent.

Antimicrobial agents receive in the form of dispersions. Three dispersion of nanocrystalline silver get by mixing of 10.0, 5.0 and 1.0 grams of nanocrystalline silver (nano technologies (Ag-20) or equivalent, Austin, TX) in 1 l of isopropyl alcohol, vystupum the th as a dispersing agent for the nanocrystalline silver (sterile filtered water is also acceptable dispersing agent).

To cover the constructs antimicrobial agent constructs are placed in 1-liter containers (Nalgene) in the ratio of 4 to construct the container. Decanted into each container 200 ml of a solution or dispersion containing an antimicrobial agent, for dipping constructs. Constructs leave immersed for 3-6 hours, and the containers are shaken on a rocking vibration platform. The constructs that were introduced in contact with the dispersion of the nanocrystalline silver, washed once in 1 l of sterile filtered water. Anti-microbial-treated constructs are then placed exactly on the polycarbonate plate, placing them under a sterile air flow biological safety cab laminar flow for drying to a value of 10-20% relative humidity, at least for 12 hours.

The resulting constructs are 2-layer CCM constructs that are already laminated, stitched and processed antimicrobial agent in order to give the constructs antimicrobial properties.

Example 7. Processing a single layer of collagen matrix antimicrobial nanocrystalline silver for the manufacture of 2-layer antimicrobial constructs

Get custom made single-layer CCM constructs, sew, treated with an antimicrobial agent, and C is the layer for making a two-layer constructs with the aim of obtaining a two-layer constructs with antimicrobial properties. By laminating constructs after treatment antimicrobial agent, you can embed a larger quantity of antimicrobial agent in the constructs by coating the external surfaces and due to the presence of the agent between the layers of the construct.

CCM receive according to Example 1. Then the CCM distribute polycarbonate plates with a length of 35-40 cm and a width of 9 cm, and remove the lymph buildup. Each piece CCM then cross stitched in a buffer of 10 mm EDC/ 0.1 M CIE [2-(N-morpholine)econsultancy acid] (Pierce, Rockford, IL), taking him at the rate of 3 liters per 30 pieces, shaken on a vibrating table, setting up to 4 samples for 16-20 hours and then washed three times in 3-5 l of sterile filtered water for 30 minutes for each wash.

Antimicrobial agents receive in the form of dispersions. Four dispersion of nanocrystalline silver get by mixing of 10.0, 5.0 and 1.0 grams of nanocrystalline silver (nano technologies (Ag-20) or equivalent, Austin, TX) in 1 liter of dispersing agent, such as isopropyl alcohol. Prepare also a solution of 5.0 g of the nanocrystalline silver in 1 l RODI.

To cover the CCM antimicrobial agent pieces CCM placed in a 250 ml sterile containers (Nalgene), in calculating the 4 piece to the container. Decanted into each container 200 ml of a solution or dispersion containing the second antimicrobial agent, the purpose of dive constructs. CCM leave immersed for 3-6 hours, in this time, the container is shaken on a rotating vibrating platform. The CCM, which were put in contact with the dispersion of the nanocrystalline silver, washed once in 1 l of sterile filtered water.

Each anti-microbial-treated CCM then layer for making a two-layer constructs, placing one machined piece CCM exactly on the polycarbonate plate. The second processed piece CCM then placed directly on top and spread over the first layer so that between the layers, there were no air bubbles. Polycarbonate plates together with constructs then placed under a sterile air flow biological safety cab laminar flow for drying up to 10-20% of relative humidity, at least within 12 hours.

The resulting constructs are 2-layer CCM constructs that cross stitched and processed antimicrobial agent in order to give the constructs antimicrobial properties, and then layered with the aim of introducing more antimicrobial agent between the layers of the construct.

Example 8. Floor after cross-linkage and before lamination

Get cross stitched single-layer CCM con is trusty, sew, and then treated with an antimicrobial agent, after which the layer for making a two-layer constructs with the aim of obtaining a two-layer constructs with antimicrobial properties. By laminating constructs after treatment of their antimicrobial agent introducing a higher amount of antimicrobial agent between the layers of the CCM.

CCM receive according to Example 1. Pieces CCM then distribute polycarbonate plates, removing the lymph buildup, then their sizes are about 35-40 cm in length and 9 cm in width. Each piece CCM then cross stitch using a 10 mm solution of EDC in 0.1 M buffer CIE [2-(N-morpholine)econsultancy acid] (Pierce, Rockford, IL), taking him at the rate of 3 liters per 30 pieces, shaken on a vibrating table, setting up to 4 samples for 16-20 hours and then washed three times in 3-5 l of sterile filtered water for 30 minutes for each wash.

Antimicrobial agents are given either in the form of solutions or dispersions. Dispersion of nanocrystalline silver get by mixing 5.0 g of nanocrystalline silver (nano technologies (Ag-20) or equivalent, Austin, TX) in 1 liter of dispersing agent, RODI. 0.1% solution RNPS get by mixing 5.0 ml Cosmocil CQ (20% solution RNPS) in 1000 ml of RODI/WFI.

To cover the CCM with SIP is utilized RNPS agent placed 28-30 pieces CCM 5-liter clean flask of Pyrex glass. Add 3000 ml of 0.1% solution RNPS. CCM leave immersed for 3-6 hours, in this time, the container is shaken on a rotating vibrating platform.

To cover the CCM dispersion of nanocrystalline silver add 200 ml of the dispersion in a 250 ml sterile containers (Nalgene). Four piece CCM placed in each container and shaken for 3-6 hours. The CCM, which were put into contact with a dispersion of nanocrystalline silver, washed once in 250 ml of RODI for 15 minutes and once in 500 ml of RODI, at least for 5 minutes before laminating. The CCM, which were put into contact with a solution RNPS not washed.

Anti-microbial-treated CCM then layer for making a two-layer constructs, placing one machined piece CCM exactly on the polycarbonate plate. The second processed piece CCM then placed directly on top and spread over first layer, between the layers, there were no air bubbles. Polycarbonate plate with constructs are then placed under a sterile air flow biological safety cab laminar for drying up to 10-20% of relative humidity, at least for 12 hours.

The resulting constructs are 2-layer CCM constructs that cross stitched and treated proteomika the m-agent to make the constructs antimicrobial properties, and then layered with the aim of introducing a higher amount of antimicrobial agent between the layers of the construct.

Example 9. Evaluation of three antimicrobial dressings on the proliferation of meticillin resistant Staphylococcus aureus (MRSA) in superficial wounds

Evaluate the antibacterial activity of three antimicrobial dressings for superficial wounds, which are colonised by methicillin-resistant Staphylococcus aureus (MRSA from English. methicilline resistant staphylococcus aureus).

Use the same pig as an experimental investigational animal, because pig skin morphologically similar to human skin. The pig weighed, and its weight is approximately 25-30 kg, kept in the home for two weeks before beginning of the experiment. This pig fed on diet, appropriate caloric main exchange, ad libitum and kept in isolation in the favorable for animal conditions (satisfying the requirements of the Ministry of agriculture, USA) at a controlled temperature (19-21°C) and light (12 h/12 h light per day). Pig anaesthetize Telezoom (5 mg/kg), Xylazine (2 mg/kg), Atropine (0.05 mg/kg) I.M. and inhalation with isoflurane in combination with oxygen. The hair on the back of the pig shear off the standard scissors for animals. The skin on both sides of the back of the pig cook, p is wash her needlebearing soap (Neutrogena®) and sterile water. Animal impregnated with dry sterile gauze. Make thirty-six (36) surface wounds (10×7×0.3 mm) on the dorsal skin using a specialized elektrotrauma. Then wound infect methicillin resistant Staphylococcus aureus ADS 33593.

For the preparation of bacterial contamination using a fresh culture of pathogenic strains obtained from the American type culture collection (ATSS), Rockville, Maryland. The inoculum is Methicillin resistant, Staphylococcus aureus was ATSS 33593. Freeze-dried bacterial culture restore the standard ATSC Protocol recovery. All suspension inoculum is prepared by scraping over night growth in cultural tablet in 5 ml of isotonic up until the turbidity of the suspension is not equivalent to the turbidity standard MacFarland #8. This will result in the fact that the suspension concentration of approximately 108colony forming units/ml (CFU/ml). Suspension with a concentration of 108serially diluted to prepare the inoculum suspension with a concentration of approximately 106CFU/ml. a Small amount of inoculum suspension is coated with a thin layer of cultural environments to measure the exact concentration of viable organisms. Suspension Ino is uleta used directly to infect each wounded area. of 0.025 ml (25 μl) aliquot of the precipitated suspension in sterile glass cylinder (diameter 22 mm) in the center of each wound. The suspension is slightly soskrebajut in each test plot within ten seconds, using a sterile Teflon spatula and leave to dry for 3 minutes. Within 10 minutes of inoculation in all infected wound covered with a bandage made of polyurethane film for 24 hours before treatment in order to give the bacteria time to check the wounds.

Create and infect three additional wounds to obtain baseline CFU/ml before treatment. The inoculate: Log 4,24 CFU/ml; estimates baseline: Log 5,38 CFU/ml Six RAS to determine the various treatment groups, as shown by the following experimental scheme. The body of the animal monitorium daily for any observable indications of pain or discomfort. In order to help minimize any discomfort use a painkiller (Duragesic - fentanyl transdermal system, which elute at a rate of 25 mcg/h) in the beginning of the experiment.

1) the Bandage And is a 2-layer construct, processed RNPS and prepared according to Example 8.

2) Dressing In is a 2-layer construct, treated with nanocrystalline silver and prepared according to Example 8.

3) Stand the lubricant is a 1-layer construct, processed nanocrystalline silver and prepared according to Example 8.

4) Control (tissue oil-based or polyurethane film only).

5) Positive control (competitive antimicrobial bandage or ointment Bactroban).

6) Raw, unprotected from air control.

All group processing, except for the untreated control, cover with a polyurethane film. Processing groups of randomly assign any region 1, 2, 3, 4, 5 or 6. Estimate the amount of 3 wounds on the processing group in the day.

Three wounds were cultured from each group treatment for 24 or 72 h after treatment. At each time of selection of the sample plots cultivated quantitatively. The area covered with a sterile glass cylinder (diameter 22 mm), kept in its place by means of two handles. One ml of the leaching solution is pipetted into a glass cylinder, and the area cleaned with a sterile Teflon spatula for 30 seconds. Spend serial dilution to determine the amount of the leaching solution, using a system of spiral electroplating, which brings a small amount (40 µl) of the suspension on the surface of a rotating agar plate.

MRSA is grown on selective nutrient media obtained using the agar Mueller Hinton, 4% NaCl and oxacillin with a concentration of 6 MK is/ml. Oxacillin is used instead of methicillin, because it is more sustainable. After the incubation period (24 hours), count colonies on plates and calculate the value of colony forming units per ml (CFU/ml).

The results of this study are presented in Table 1. The results show that 2-layer constructs, treated RNPS and nanocrystalline silver, and 1-aloynoe construct, treated with nanocrystalline silver, are effective to release the wounds from bacteria.

Table 1
Table 1. Recovery of MRSA (log CFU/ml)
Evaluation timeProcessinglog CFU/mlthe average (log CFU/ml)STD. deviation
24 hours2-layer using RNPS5,1350,63
or 4.31
5,56
2-layer nanocrystalline silver5,465,99 0,57
6,6
to 5.93
1-layer nanocrystalline silver3,974,220,48
3,92
4,78
Acticoat5,65,270,89
5,97
4.26 deaths
The positive control6,276,610,36
6,56
7
Raw available for airto 6.586,410,14
6,33
6,33

72 hours2-layer using RNPSis 3.083,17 0,92
4,15
2,3
2-layer nanocrystalline silverthe 5.455,610,38
6,05
5,34
1-layer nanocrystalline silver5,045,080,17
5,27
is 4.93
Acticoatto 5.57to 5.93to 0.480
6,48
5,76
The positive controlof 5.81of 5.810,25
the 6.06
5,56
Raw available for air5,264,670,54
4,19
4,56

Example 10. Met the Dickey mechanical testing and mechanical properties of multilayer CCM dentures

Testing preferred options multi-layer CCM miscellaneous constructs. The constructs of 2, 4, and 6 layers of the CCM made a 100 mm solution of EDC in 50% acetone (100/50), and 6-layer constructs made of 7 mm solution of EDC in 90% (by volume) solution of acetone in water (7/90), and 1 mm solution of EDC in water (1/0) is estimated in accordance with the number of dimensions. The results are summarize in Table 2.

The failure test tensile carried out using a servo-hydraulic MTS testing system software TestStar-SX. The band width of 1.25 cm stretch to failure in uniaxial tension at a constant speed of deformation of 0.013-1. Calculate the slope of the linear region (EY) and tensile strength (UTS) of curve deformation strain.

The adhesion strength between the layers is tested using a standard Protocol for testing adhesives (ASTM D1876-95). The adhesion strength is the average force required to remove two layers of laminated CCM with a constant speed of 0.5 cm/s

Differential scanning calorimeter is used to measure the heat flow towards and from the sample under thermally controlled conditions. Shrink temperature is defined as the temperature of appearance of the peak denaturation chart temperature-energy. The ability to hold the seam is not shown for 2 - or 4-layer design is s, sewn in 100 mm solution of EDC in 50% acetone, whereas the ability to hold the seam (3,7 N+0.5 N) for 2-layer construction made of 1 mm solution of EDC and without acetone (which reduces the degree of crosslinking), is good and exceeds the minimum limit in 2 H. The strength of the lamination between the CCM layers and shrinkage temperature depends on the concentration of crosslinking agent and adding acetone to a greater extent than on the number of layers in the construct.

Table 2
Table 2. Mechanical properties of multilayer CCM constructs
Mechanical analysis2-layer, 100 mm EDC/50% acetone4-ply, 100 mm EDC/50% acetone6-ply, 100 mm EDC/50% acetone6-ply, 70 mm EDC/50% acetone6-layer, 1 mm EDC in water (without acetone)
The tensile strength (N/mm)0,6±0,13,1±0,22,0±0,22,7±0,21,3±0,4
The young's modulus38,0±5,849,5±4,035,9±2,6 43,0±1,214,5±7,8
The strength of the lamination (N/m)39,7±6,163,1±24,48,1±2,1
The strength retention of the weld (H)not testednot tested6,6±1,610,6±2,210,9±2,8
The shrinkage temperature (°C)72,5+1,169,5±0,164,0±0,2

Example 11. Method of treatment of wounds anti-microbial-treated construct

For the treatment of deep wounds of the skin using either a single layer of the CCM in Example 1, or a linked multi-layered sheet construction of the CCM prepared according to the method of Example 2. In the plate bore hole or fenestration to create small slits, which allow to infiltrate the exudates of the wound.

With skin injuries, including second degree burns, lacerations, fractures and abrasions; surgical excision wounds arising from the removal of cancerous tumors, or autotransplantation areas to the and of the donor; skin ulcers, such as venous, diabetic and pressure ulcers, and other chronic ulcers accessed using the CCM in a single layer or multilayer form. Collagen CCM matrix protects the surface of the wound, maintaining moisture and giving her the opportunity to drain freely from the wound. Before application of the CCM to the wound, the wound prepare for its implementation.

Select patients with burn wounds, which required transplantation. CCM is placed either directly on isecheno the surface of the wound or on the net autograft, unstretched or stretched in a ratio of 2:1 or more. Test plots (CCM) and control plots (autograft) when using have the same ratio of dimensions of the cell. The transplanted areas of burn wounds prepare, for example, through rehabilitation of the wound cavity, primary processing according to the conventional practice so that the burnt area of skin is completely unobservable. Clipped areas look clean and clinically clean.

Patients who undergo surgical excision, injected locally anesthetic. Preoperative area disinfected antimicrobial/antiseptic disinfectant for skin (Hibielens®) and washed with isotonic. Do incomplete deep wounds on the skin and about the W ill result transplantation of skin from another place, if it is not malignant. The CCM applied to the surface of the wound using a sterile bandage.

In the case of any wounds in the assessment, cleaning, replacement of bandages processed RAS patient provide appropriate post-operative care. Spend a full account of the conditions treated plots, writing in documents all procedures, medications, frequency of changing dressings and any observations done. The wound remains protected from the external environment and wet, contributing to the treatment of the wound and its healing.

The dressing on the wound to check the model of the animal organism. Invented the construct for bandaging wounds is either single layered or multi-layered sheet construction made from CCM made according to the methods of Examples 1 and 2. Use the model of healing the deep wounds of rats (a model that is widely used for dressing products on the wound) to assess performance of the dressing construct on the wound, made from a single layer of material CCM. All 20 animals, four on the estimated time, have two (2) full then wound the size of 2 cm × 2 cm, which was created on their back. Test and control articles cut in pieces, slightly larger than the periphery, and is applied in a dry form on any wound, following the RAS is alizirovannoj scheme application. Headbands re hydratious liquid wounds and sterile saline if necessary. Secondary dressing from petrolatum Marley put on each test and control product and replace weekly or during each assessment. Wounds evaluated at 3, 7, 14, 28 and 42 days after treatment. Evaluate parameters such as the rate of wound closure and the percentage of closed wounds (based on the records of the condition of the wound), erythema, exudate and histology explantion areas of the wound.

According to the results of the analysis of the percentage and rate of wound closure, the plots treated with the construct for bandaging wounds, show slightly faster wound closure, although statistically meaningless, compared to control plots. When analyzing the time of complete wound closure is not detected differences between the tested and the control and treated plots. The results of the analysis of erythema, exudation and histological analysis of the same for the two products. Histological analysis shows that the construct for bandaging wounds, made of a single layer of the CCM, shows the required therapeutic characteristics over time, such as re-epithelialization of the wound and the resorption of the collagen material. There is no evidence that the test organisms occurs adverse is the first reaction to the construct.

Although the foregoing invention has been described in some detail in the form of illustration and example for a more clear explanation and understanding, one who is proficient in this field of medicine, it will be obvious that in practice it is possible to make certain changes and modifications within the scope of the attached claims.

1. Bioengineered collagen construct for the repair or replacement of damaged tissue, characterized in that it includes a layer of purified collagen tissue matrix derived from the submucosal membrane of the small intestine, these purified collagenous tissue matrix is processed pharmaceutically acceptable antimicrobial agent and has antimicrobial properties.

2. The construct according to claim 1, characterized in that it is pedestrianism, perforated or made in the form of a grid.

3. The construct according to claim 2, characterized in that it has a ratio of cell sizes of 1:1.5.

4. The construct according to claim 1, wherein the antimicrobial agent is selected from the group comprising nanocrystalline silver, silver oxide, silver nitrate, silver sulfadiazine, imidazole silver, Arglaes®, hydrochloride, poly(hexamethylenebiguanide), chlorhexidine gluconate, bis-amido polybiguanides, honey, benzalkonium chloride, triclosan and dimethyloctadecyl-(3-triethoxysilylpropyl)azanium chloride.

5. The construct according to claim 1, characterized in that the layer is chemically bound using the hydrochloride of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

6. The method of repair or replacement of damaged tissue, including the implantation of the prosthesis into the patient, which is a mammal, characterized in that use subject to the controlled biodegradation of the prosthesis, which is reconstructed by an adequate substitution living cells and comprising two or more than two overlapping interconnected layers purified collagenous tissue matrix, processed pharmaceutically acceptable antimicrobial agent and having antimicrobial properties.

7. The method according to claim 6, characterized in that the use of prosthesis, comprising from two to five layers of purified collagen matrix derived from the submucosal membrane of the small intestine, which bind and chemically bound to each other by means of a solution of the hydrochloride of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide having a concentration of 0.1÷100 mm.

8. The method according to claim 6, characterized in that the use of prosthesis, comprising from two to ten layers of purified collagen matrix derived from the submucosal membrane of the small intestine, which bind and chemically bound to each other by means of a solution of the hydrochloride of 1-ethyl-3-(3-d is methylaminopropyl)carbodiimide, having a concentration of 0.1÷100 mm.

9. The method according to claim 6, characterized in that the use of prosthesis selected from the group comprising a flap for repair of a hernia, tampon for repair of femoral hernia, pericardial flap, strap to support the bladder, strap to support the uterus, intracardiac flap, replacement heart valve, vascular graft, a tampon for the recovery of the fibrous ring, the flap for the recovery of the fibrous ring, the prosthesis is to restore the muscles responsible for rotation of the shoulder flap for the repair of Dura mater, the device for repair of a hernia of the bladder, the device for repair of rectal prolapse, the strap for prolapse repair vaginal vault, the implant for plastic surgery.

10. A layer of purified collagen tissue matrix to restore or replace damaged tissue, characterized in that it is derived from the submucosal membrane of the small intestine, these layer derived from the submucosal membrane of the small intestine is treated pharmaceutically acceptable antimicrobial agent and has antimicrobial properties.

11. The layer of claim 10, wherein the antimicrobial agent is selected from the group comprising nanocrystalline silver, silver oxide is, silver nitrate, silver sulfadiazine, imidazole silver, Arglaes®, hydrochloride, poly(hexamethylenebiguanide), chlorhexidine gluconate, bis-amido polybiguanides, honey, benzalkonium chloride, triclosan and dimethyl-octadecyl-(3-triethoxysilylpropyl)azanium chloride.

12. Layer according to claim 11, characterized in that the atoms of nanocrystalline silver are not disordered.

13. Purified collagen tissue matrix for repair or replacement of damaged tissue, including tissue cell of a mammal, which is essentially free from Nikolayevich components, characterized in that it is processed pharmaceutically acceptable antimicrobial agent and has antimicrobial properties.

14. The matrix according to item 13, wherein the antimicrobial agent is selected from the group comprising nanocrystalline silver, silver oxide, silver nitrate, silver sulfadiazine, imidazole silver, Arglaes®, hydrochloride, poly(hexamethylenebiguanide), chlorhexidine gluconate, bis-amido polybiguanides, honey, benzalkonium chloride, triclosan and dimethyl-octadecyl-(3-triethoxysilylpropyl)azanium chloride.

15. The matrix according to 14, characterized in that the atoms of nanocrystalline silver are not disordered.



 

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FIELD: medicine.

SUBSTANCE: invention refers to polymer composites with the special characteristics used as medical implants, transformed structures, thermal activators and the other commercial structures. The composite comprises a polymer matrix with working temperatures exceeding fibre configuration recovery temperature, and tensile extension min. 2%, reinforced with the elements presented in the form of fibres extended in one or more directions with the fibre length min. 3 times more than the in-between distance, made of a thermal shape memory alloy.

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7 ex, 9 dwg, 1 tbl, 6 cl

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EFFECT: improved durability and biocompatibility of bioprostheses.

3 ex, 7 tbl, 3 dwg

FIELD: medicine.

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5 ex, 4 tbl

FIELD: medicine.

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1 ex

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SUBSTANCE: bone-and-mineral product contains porous bone mineral particles produced from natural bone and having crystalline structure practically corresponding to natural bone structure and practically containing no endogenous organic material. The particles have fibers of physiologically compatible type II resorbable collagen at least on their surface. Mass proportion of type II collagen fibers and porous bone mineral is at least equal to approximately 1:40.

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8 cl, 6 dwg

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17 cl, 7 dwg, 1 ex

FIELD: medicine.

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12 cl, 21 dwg, 12 ex

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