Sterile autologous, allogenic or xenogenic implants and method for making it
SUBSTANCE: invention refers to medicine. What is described is a sterile dehydrated cell-free implant which when rehydrated by water or body fluids is exposed to anisotropic expansion, and can be used as a substrate for living cell adhesion, migration and growth. Collagen structures of a transplant are at least partially denatured after exposure to heat or organic solvents, such as lower aliphatic alcohols and ketones which are simultaneously preserving and sterilising agents, especially with respect to specific types of viruses. The implant is sterilised by radiation, preferentially by accelerated electrons, practically in the dehydrated condition. The transplant can be produced of various animal tissues, especially of mammalian tissues, such as human or swine tissues. Tissues suitable for the prevent invention can represent, e.g. skin, placenta, pericardium, peritoneum, intestinal wall, tendon, blood vessel, etc.
EFFECT: dehydrated implant is suitable as a temporary coating for a wound or a burn, for recovery, replacement and regeneration of tissues, and also as a substrate for cell cultivation in the laboratory conditions, and easier to flex, and being less fragile.
31 cl, 8 dwg, 6 ex
The scope of the invention
The invention relates to wound healing and tissue regeneration. Specifically it relates to a sterile, digidratirovannogo, the cell-free implant (transplant), which during rehydration with water or body fluids undergoing anisotropic expansion. The product of the specified type can be described as an implant or transplant, depending on the context and rules adopted in different areas of expertise. In this application, both terms are used in accordance with the context; however, it is important to remember that they are interchangeable. The implant is sterilized using irradiation, almost digidratirovannogo condition, preferably with the use of accelerated electrons. The implant can be obtained from tissues of various animals, especially of mammalian tissue, such as tissue of a human or pig, such as, for example, skin, placenta, pericardium, peritoneum, intestinal wall, tendon, blood vessel, etc. the Implant according to the invention is suitable for use in medicine and veterinary, for example, as a temporary bandage on wounds and burns, for repair, replacement or regeneration of tissues and as a substrate for culturing cells in vitro.
The level of technology
Fabric organi a long time and are widely used for transplantation for a variety of indications. One of the well-developed techniques is an autologous transplantation, when the native tissue of the patient (e.g., skin, bone, vein, or adipose tissue) are moved from one place to another for replacement tissue. This is not always possible, however, and in some cases the patient needs a transplant (e.g., heart, kidney, retina and so on) from a suitable donor. The main problems of these so-called allografts are tissue rejection and, increasingly, the shortage of donors because of the greatly increased in their needs. Due to this, trying different ways to replace natural allografts. For example, it is possible the cultivation of autotransplants from cells of a patient using the tissue engineering. These autotransplants easily overcome immune barrier, however, they have several disadvantages: the need for sampling tissue from a patient (biopsy), time-consuming and expensive cultivation and a large amount of time from biopsy to use graft. This method is widely used to replace skin burns 3rd degree, while in the case of other tissues or organs such methods at this point in time is experimental. For example, U.S. patent No. 6878383, 6432710, 5858390, 5665372 and 5660850 (Boss, Jr. et al.) describe the method and means of the implant is of autologous fibroblasts to cause hyperplasia tissue of the patient.
Autologous transplantation using artificially created epidermal layer of the skin is used to treat patients with burns over a number of years. In 1979 Rheinwald and Green (Green H, et al., Proc. Natl. Acad. Sci. USA 1979; 76: 5665-8) have developed a method of serial cultivation of human keratinocytes for autotransplantation. Since 1981 autologous cultured epidermal grafts used in the U.S. for the treatment of extensive burns (O'connor NE, et al., Lancet 1981; 1:75-8). The disadvantage of this method is the long time interval required for culturing autologous keratinocytes, the fragility of cultivate, the difficulty of manipulation, high sensitivity to antibiotics, infections and other stresses, and the difficulty of assessing the assimilation of transplant (Navsaria HA, et al., Trends in Biotechnology 1995; 15: 91-100). Described thus, the following improvements to the method.
U.S. patent No. 4299816 (M.G. Eisinger) describes a modified healing of burns with the use of grafts from artificially cultured epidermal cells. U.S. patent No. 5716411 (Orgill et al.) describes a method of healing, leading to skin regeneration for burns and injuries, using biosynthetic coating, consisting of a collagen matrix and glycosaminoglycans, which allows the penetration of cells and blood vessels of the heal is Asia fabric, on the one hand, and the use of plates of autologous keratinocytes on the other hand. WO 2006/107188 A1 (L. Lurvink et al.) describes non-porous polypeptide film, suitable for cell culture, and its subsequent use for healing wounds and burns. A recent review of these methods can be found in TISSUE ENGINEERING, Vol. 12, No. 9, 2006 Update on Tissue-Engineered Biological Dressings, M. Ehrenreich and Z. Ruszczak.
Not only autologous, but also allogeneic cultured epidermal grafts have a powerful healing effect on deep burns of the dermis, taking biopsies, leg ulcers and other skin defects (Bolivar-Flores J. et al., Burns 1990; 16: 3-8; Matouskova E. Et al., Burns 1993; 19: 118-23.4,5).
The success of the procedure depends on the choice of the donor cells. P. Brychta et al. described in Czech patent No. CZ 282711 cultured epidermal allograft from embryonic or fetal cells for healing defects and wounds of the skin, mainly in accordance with the procedure Rheinwald and Green, but with the use of allogeneic cells, which were well received by the patient.
There are also attempts to increase the mechanical stability and viability of keratinocytes (e.g. by cultivation on synthetic substrate) and to develop a methodology that would allow for permanent assimilation cultivated tissue in burns 3rd degree. One in the am substrate, used for cultivation of keratinocytes, is a membrane-based hyaluronic acid (Laser skin, FIBIA, Italy), various types of collagen matrices combined with fibroblasts, or various substrates made from synthetic polymers (for example, experimental pHEMA in the clinic burn medicine, FNKV in Prague 10). For filling deep burns developed deputies of the dermis, such as Integra (collagen, combined with glycosaminoglycans chondroitin-6-sulfate and allogeneic fibroblasts; Integra LifeSciences Corporation, Plainsboro, New Jersey, USA), Dermagraft (Polyglactin seeded with allogenic dermal fibroblasts; Adbanced Tissue Sciences, La Jolla, CA, USA) or the already mentioned AlloDerm - frozen allogeneic dermis (LifeCell Corporation, The Woodlands, TX, USA). However, all of these deputies of the dermis must be covered with a thin autologous dermo-epidermal graft during the second stage (after 2-3 weeks of vascularization); floor burns 3rd degree with allogenic cultivated so far not been successful.
Another solution to the problems of allotransplantation is the use of tissue or organs from species that are not human, so-called xenotransplantation. In this case it is also necessary to overcome the rejection of foreign tissue by the immune system, and also you pre order to avert the possibility of transmission of disease-causing microbes and viruses from the donor to the patient. Great attention is paid to prevention of prion transmission from animals to humans (for example, the famous "mad cow disease"). On the other hand, a great advantage is that tissues and organs of animals are significantly more affordable compared to tissues and organs.
Well-known examples of xenograft are the heart valves of pigs, which are used to replace the valves of the human heart. Valves pigs subjected to cross-linking using glutaraldehyde (for example, U.S. patent No. 4076468, Liotta et al.; U.S. patent No. 4247292, W.A. Angell), which leads to several desirable results: suppression of rejection reaction in the body, enhances hydrolytic and enzymatic stability of the xenograft, and, in addition, glutaric aldehyde acts as a chemical sterilizing agent. One disadvantage of this method is the change in the mechanical properties of the tissue and, in some cases, even long-term release of toxic glutaraldehyde from insoluble polyarteritis, which may be formed during the process and cannot be removed by simple extraction.
A significant portion of the grafts used in the form of so-called "biological coatings for the dressing on the wound and the resulting the very foundations for healing. Depending on the nature of the wound and other circumstances of the use of biological, synthetic and semi-synthetic coatings. Biological coatings are generally considered to be the most effective. Typical biological coating for healing, for example, burns is the skin of a mammal, but especially a human skin (allograft) or pig skin (xenograft) of different thickness, selected from corpses and stored in fresh form at low temperatures in a short period of time or even over a longer period of time being frozen. Has a large experience in the use of xenografts from pig skin.
Live bandage on the wound (i.e. untreated allografts or xenografts containing all components of living skin) are very effective, however, their disadvantage is the limited shelf life and the possibility of transmission. For a number of patients were offered certain decisions, such as AlloDerm products and XenoDerm from LifeCell Corp., Texas, USA, on the basis of the method of cryopreservation according to U.S. patent No. 4865871 (S. Livesey et al.). This method makes it possible to get frozen and possibly lyophilized tissues and cells without damaging their structure or function.
Another way is to store pig skin in glycerol in presets the following silver nitrate at room temperature, as described in the patent application CN 19951010722 (Kai Cao).
Sterilization pig skin (after cleaning and processing using hydrocarbons) in a solution of sodium perchlorate or hydrogen peroxide with the use of gamma radiation from a source of Co60described in the patent application TW 199001117733 (Chang Hong Chi et al.).
Other methods of sterilization pig skin for medical use described in the patent application CN 19921005926 (Guohui Li et al.), which describes sterilization in the wet state using a radiation source of cobalt, with subsequent storage at low temperatures or freeze-dried and then stored in glycerol at room temperature.
Conservation using glycerol is also recommended for human placenta (amnion)used for allografts in Deutches Institut für Zell - und Gewebeersatz gGmbH (Delitzcher St 141, 04128 Leipzig, SRN).
Document UA 12391U (DURING Fistal et al.) describes the healing necrotic wounds after deep burns with the use of freeze-dried pig skin.
Biological dressings on wounds based on collagen for healing of wounds, including burns, also described in documents EN 2185179 and EN 2124354.
The problem of sterility and shelf life can be mitigated by removing cells from the graft, which, thus, become partially or completely acellular. One of the attempts for resolution is whether this problem can be found in the patent document No. CN 20031124306 (Hu Jie), describing the xenograft as a biological dressing for wounds and burns. The animal tissue, such as skin, the wall of the small intestine or the placenta, can partially get rid of the cells, according to the above invention, by using water or a solution of detergent, and it is carried out on a surface that is in contact with the wound. The cellular structure of the other parts, such as the epidermis, will be saved. Then the fabric will be exposed to cross-linking using a suitable agent, such as glutaric aldehyde, washed and stored in a moist condition at a temperature below 4°C.
Another document, CN 20051126108 (Dong Qun Lin), describes a method of removing cells from the skin of a mammal through repeated exposure NaOH solution with a concentration of from 2 H to 5 H, followed by washing in a solution of detergent and water.
Another document, CN 20041022506 (Dai Weihua et al.), describes a method of manufacturing a biodegradable acellular of the dermis using the combined effect of enzymes, alkali and other chemical agents.
Published application US 20050186286 (Yoshihiro Takami), describes a method of removing cells from the skin of a mammal (e.g. human or pig) using the combined effect of proteolytic enzymes and detergents; made so the skin is what to use as allograft or xenograft for healing burns. Sterilization perform subsequent immersion of the dermis acellular in a solution of azide.
Similar cell-free centerpieces matrix is OASIS made AelsLife, which provides the framework for the spatial migration of cells. Specified biological coating for wounds, which, according to the manufacturer, contains important non-cellular connections and patterns that are present in living skin, is produced by lyophilization of the dermis pigs after removal of cells by enzymes and detergents.
In the biosynthetic dressing E*Z DERM from Brennen Medical, Inc. used a xenograft of the dermis pigs treated with cross-linking of collagen with the use of aldehydes.
Patent document JP19900247300 (Koide Mikio) describes the biological coating that uses a matrix of denatured collagen derived from acellular of the dermis cattle through cross-linking and thermal denaturation of collagen structures. This structure, according to the cited invention is suitable for sowing by using autologous keratinocytes for more high healing efficiency.
Other attempts at solving the problem was a different semi-synthetic skin substitutes, for example, the frame of the recovered collagen of cattle, SaaS is regular human fibroblasts (i.e. the above-mentioned dressing INTEGRA).
Another example of a combined transplant is nekombinirovannyh skin" (RK) according to the patent CZ No. 281176. RK is manufactured using a cultivation of human keratinocytes in not containing cells in the dermis pigs. (Burns 1993; 19: 118-23). Dried dermis is used for the cultivation of human keratinocytes and after cultivation of the dermis layer of keratinocytes (or RK) is separated from the Petri dishes and put on the wound. RK is applied so that the keratinocytes in contact with the wound, and the dermis was outside ("upside down"). Compared to the simple epidermal grafts RK demonstrates the advantages of higher strength, detached from the Petri dishes without enzymatic effects and easily manipulated. Advantage compared with cultures of keratinocytes on synthetic substrates and gels on the basis of collagen is that consistency RK is similar to the skin, and it provides excellent adhesion to the wound and hemostatic effect. It is possible to produce RK using both autologous and allogenic keratinocytes. Keratinocytes cultured on the epidermal side of the epidermis, i.e. where the basement membrane separates the dermis from the epidermis. The author cited above inventions notices that do not contain cells of the dermis m who should be sterilized using gamma radiation for longer storage at room temperature and greater security. The disadvantage of sterilized using gamma radiation of the dermis, however, is its partial degradation and loss of strength in the wet state.
Similar combined biological dressing for burns described in the patent application TW 20000118374 (Yang Mei-Ru et al.), in which living human fibroblasts in the dermis acellular pigs combined with human keratinocytes, cultured on the basal side of the membrane cell-free matrix.
The main problem preventing widespread adoption of the use of these biological materials is the inability to use the routine and reliable method of sterilization. Another specific issue that is preventing wider use of the above-mentioned materials is restricted or require special conditions, the retention period and, last but not least, the cost of producing them. A difficult problem is that digidrirovannye materials isotropic swell during rehydration, i.e. the (relative) increase by the same amount across all dimensions of the graft after rehydration. The present invention solves the above problems.
Applicants have found that the presence of transplanted allogeneic or autologous cells is not always necessary for for the ellenia wounds and tissue regeneration if there is a suitable material that will encourage, support and guide the propagation, differentiation, and migration of the patient's own cells. According to the present invention, the specified material is a specially treated acellular collagen matrix derived from autologous, allogeneic, or even xenogeneic biological material. According to the present invention, the matrix is composed mainly of collagen and related proteins, such as elastin, fibrin, or keratin. These matrix components and their concentrations vary depending on the origin of the fabric and how it is processed and for simplicity will be referred to as "collagen"as in all cases, the collagen is the main component of the matrix. In addition to proteins (collagen) matrix also contains some amount of fats and lipoproteins (up to 20 wt.%), some amount of sugar components (polysaccharides, glycoproteins and glycoproteomics) and salt. The protein content is usually from 70% (wt.) to 95% (wt.), preferably, from 80% to 90% (wt.).
According to the invention, a cell-free matrix specifically characterized by the fact that he is almost digidrirovanny and consists mainly of collagen fibrils which demonstrate structural organization, Shodo the structural organization of the original fabric, but, in addition, they are also partially denaturirovannyj and, at least in digidratirovannogo condition are preferably oriented in a selected direction or directions. Partial denaturation is preferred because it increases the resistance to biological degradation, so fibrils provide more time to migrate and attach to the host cells during healing. Too rapid degradation of the implant can be the focus of inflammation, which will not heal and can lead to scarring. Partial denaturation of the collagen fibrils also improves the mechanical strength in the wet state.
The orientation of the collagen fibrils also informs the implant higher strength in the selected direction, and she directs rather, migration and dissemination of cells on the implant surface than their penetration into the implant. This is also confirmed by the fact that the acellular matrix according to the present invention has a low porosity in digidratirovannogo condition compared to liofilizirovannami biological coating on the wound, the porosity of which is usually above 75% (by volume). According to the invention the porosity of the implant is less than 70% (by volume), preferably less than 60% (by volume) and even more edocfile less than 50% (by volume). Low porosity and favorable orientation of the fibrils are important, especially for implants used as biological coatings on wounds, such as burns, which is assumed to be spontaneously separated after healing. Regeneration of the epidermal layer requires the migration of keratinocytes from the wound edges in the area of healing, which represent the contact area between the wound and the surface of the implant. Infiltration of cells into the structure of the implant will not be favorable because it can lead to the re-attachment of the graft to the wound. If, for example, when the graft is subcutaneous, cell migration along the surface will lead to the formation of a thin fibrous cysts, which in many cases is undesirable.
The preservation of orientation of collagen in digidratirovannogo condition also increases the tangent stiffness of the implant (tangent with respect to the orientation of the fibrils), therefore, even digidrirovanny the implant is easy to bend and is less fragile than a similar anisotropic implant. This is of considerable practical importance, because digidrirovanny the implant does not need softeners, and there are no cracks and microarray, which can lead to uncontrolled infiltration of the implant the cells, its destruction and possible calcification.
Another important result of the anisotropic organization of the collagen fibrils is anisotropic swelling of the implant during rehydration. According to the invention, the implant during rehydration expanding at different speeds in different directions. For example, if the collagen structure is oriented mainly in the longest direction (for example, when using a tendon), the largest expansion is expected in the form of increased diameter of the implant, while the length will vary only slightly or may stay the same, a little increase or even decrease in accordance with the relationship of structural anisotropy and swelling. If the surface of the implant, such as a coating on the wound or burn, the orientation of the fibrils can choose predominantly in the direction perpendicular to the surface of the main plane of the implant. In this case, the extension of hydration will occur mainly or only in terms of increased thickness, while the bearing surface will remain virtually unchanged. Anisotropy of swelling, in addition to the above advantages, has the following practical advantages: the surgeon can better choose the shape and size of the implant for each patient is A. For example, if you want to apply coating on the wound of a certain shape, the implant of the appropriate size and shape can simply be cut out of digidratirovannogo implant, and it will remain unchanged after hydration. In the case of isotropic digidrirovanny implants measurement in digidratirovannogo should be relatively small to compensate for the effect of expansion during hydration. Another advantage may be the way digidrirovanny the implant is attached to the fabric retains its shape after hydration, and thus, the surrounding tissue keeps the tension, which was selected by the surgeon during the surgical intervention. In the case of isotropic implant and the surrounding tissue will lose its natural tension in the hydration expansion of the implant.
Important is also the fact that the anisotropy of the expansion provides for easy discernment of the implant according to the present invention and other implants of similar nature and purpose.
The anisotropy of swelling can be expressed as the ratio between the coefficients of linear expansion in the three selected dimensions. For example, the selected direction along the axis "z" may have a thickness oftand its coefficient of linear expansion of Cz=(thydrate.)/(tdehydrate. ). Similarly, you can choose the length of thelas a dimension in the direction of the axis "x" and to determine the coefficient of linear expansion as Cx=(lhydrate.)/(ldehydrate). And finally, as a dimension in the direction of the y axis, you can choose the width of thewto determine the coefficient of linear expansion as Cy=(whydrate.)/(wdehydrate.), where the signature of "hydrate" means the size (dimension) after hydration, and the signature "dehydrate" means the size (dimension) in the original, digidratirovannogo condition. In the case of isotropic digidratirovannogo material always found that Cz/Cy=Cx/Cz=Cy/Cz=1, no matter what is the value of CxCyand Cz. Anisotropic expansion during hydration is distinguished by the fact that, at least, one of the CxCyand Czhas a value different from 1, and at least one of the coefficients of linear expansion CxCyand Czhas a value less than the others, and its value can be even less than 1. At least one of the coefficients of linear expansion CxCyand Czhas, on the contrary, the amount is significantly more than others, typically, at least 10%, preferably more than 30%. For example, the greater the s linear expansion C xand Cycan have a value less than 1, while Czhas a value of more than 1.2 and preferably more than 1.5.
According to the present invention, the collagen fibrils anisotropic digidrirovanny implant is oriented, preferably in the direction of the lowest expansion coefficient or, depending on circumstances, in the plane perpendicular to the direction in which the value of the coefficient of expansion is the greatest.
Collagen fibers can be oriented mainly in a certain direction even in the fully hydrated state. This orientation can be achieved by partial denaturation of collagen in the oriented state or cross-linking of collagen (which is also a form denaturation). The orientation of the collagen fibrils then remains almost unchanged even after hydration of the implant. The specified orientation can be used for good, to direct the migration and proliferation of cells in a particular direction that is favorable especially for the healing of burns.
Cross-linking of collagen can be performed using well known methods, for example, the influence of aldehydes, such as formaldehyde or glutaric aldehyde, or polyvalent cations, such is AK, for example, Ca2+, Mg2+, Al2+or Cr3+. Cross stitching will also reduce the swelling and increase the strength and resistance to hydrolysis of the collagen matrix. The specified cross-stitching in implantirovannomu condition is often unstable, and gradual reduction of cross linking density will eventually lead to a gradual increase in swelling and change factors of linear expansion and their mutual relations. The kinetics of these processes is controlled, and therefore, these processes can be used, for example, to create a directional pressure or tension on zajavlenij tissues.
According to the invention, the cell-free matrices are highly hydrophilic, and their volume will increase with hydration. Hydration is most often defined as the weight fraction of water in the hydrated state, or as the water content in wt.%. According to the invention the water content of the fully hydrated implant exceeds 33% (wt.) and preferably greater than 50% (wt.). The volume expansion ratio is defined as:
Cv=Cx×Cy×Cz= (hydrated volume)/
According to the invention, the cell-free matrices are Cv>1,1, preferably Cv>1,5. They principles the constraints differ from the predominantly hydrophobic porous structures, which can be formed, for example, covalent cross-linking of tissue using, for example, aldehydes, or they can be formed from synthetic polymers, such as polyurethanes. In this case, during the hydration water fills the pores and increases the weight of the implant, but its volume is not significantly increased, and its Cvapproaching or almost equal to 1.
From the information above we can conclude that the subject of the invention is mainly cell-free, sterile, almost digidrirovanny and at least partially denatured matrix, obtained from the tissues of the animal and containing mainly collagen fibrils which demonstrate structural organization inherent in the original fabric, designed for use as a temporary implant in medicine and veterinary medicine; the specified matrix demonstrates anisotropic change their dimensions during hydration.
According to a preferred implementation of the present invention during hydration acellular matrix when observed anisotropic changes of the measurement matrix, the two largest dimensions remain virtually unchanged or decrease, while the smallest dimension increases with increase in the volume of the matrix is.
According to the invention in another preferred embodiment of the present invention, the temporary implant has an almost flat shape, and its bearing surface defined by the two largest dimensions, while its thickness is determined by the smallest dimension.
According to the invention in another preferred embodiment, cell-free matrix two of the smallest sizes will increase, while the largest dimension remains almost unchanged or decreases.
In yet another preferred embodiment of the present invention, the temporary implant is basically an elongated shape, such as a prism or cylinder, the diameter of which is determined by two smallest dimensions, such as diameter, while its length or height is determined by the largest dimension.
According to the invention the preferred matrix in digidratirovannogo state has a porosity less than 70% (by volume), preferably less than 60% (by volume) and most preferably less than 50% (by volume).
According to the invention in a preferred embodiment, cell-free matrix consists of collagen fibrils, which, at least in digidratirovannogo state are focused mainly in the areas in which ratios are the HT linear expansion during hydration has the smallest value, and generally perpendicular to the direction in which the coefficient of linear expansion of hydration is of the highest importance.
According to the invention in a preferred embodiment, cell-free matrix in digidratirovannogo state has a water content of less than 20% (by weight), preferably less than 10% by mass, and most preferably less than 5% (by weight).
According to the invention in some preferred embodiments, the implementation of the matrix may also contain sleek, preservative or bactericidal additives. Preferred bactericidal additives contain silver, preferably in a colloidal state, and even more preferably in the form of a complex silver-protein. Preferred sleek or preservative additives contain compounds miscible with water, such as DMSO or polyhydroxylated compounds selected from the collections of the glycol or glycerol or derivatives thereof, triethanolamine and sugars.
According to the invention preferred acellular matrix capable of volumetric expansion upon contact with aqueous liquids, the volume expansion ratio of more than 1.1, preferably more than 1.5. In addition, the preferred acellular matrix after exposure to suitable aqueous solutions can take the form, which contains more than 33% water by weight), preferably more than 50% water (by weight).
According to the invention, preferred acellular matrix has a higher coefficient of linear expansion of 10% higher, preferably 30% above the lowest coefficient of linear expansion. Preferred are the cell-free matrices, which have a higher coefficient of linear expansion has a value of more than 1.2, preferably more than 1.5, while the lowest linear expansion coefficient has a value less than 1.1, preferably less than 1,05.
According to the invention, in another preferred embodiment, the solids (dry substance) acellular matrix is composed primarily of proteins, and preferred protein is composed primarily of collagen. Even more preferably, the solids (dry substance) acellular matrix contain from 70% by mass to 95% by weight, preferably from 80% (by weight) to 90% (by weight) proteins collagen type. Preferably, if solids (dry substance) of the matrix contain, in addition to proteins, a lower fraction of lipid compounds, including lipoproteins and phospholipids.
According to the invention, a cell-free matrix is preferred when its protein fraction is at least partially denatured. According to the invention, in yet another options the ante implement at least the protein fraction of the acellular matrix is cross stitched as a result of interaction with aldehydes or polyvalent cations.
According to the invention, preferred acellular matrix obtained from a mammal, preferably from a pig.
According to the invention, the preferred fabric for animal cell-free matrix represents the skin, placenta, pericardium, Dura, intestine, tendon or cartilage.
Temporary implant formed acellular matrix according to the present invention, preferably used as a coating on the wound, more preferably as a coating on the burns.
According to the invention, another preferred variant implementation of the acellular matrix is a matrix that also contains cultured mammalian cells. Preferably the mammal is a pig, and cultured mammalian cells are human autologous or allogeneic keratinocytes.
According to the invention, another preferred variant implementation of the acellular matrix is a matrix that will be subjected to biological degradation after performing its function.
A method of manufacturing an implant according to the present invention, as described above, includes several basic steps:
1) Collection of the implant, such as pig skin or tendon person. This step is carried out, in the main, as in the case of other modern methods, but with the important advantage that, according to the present invention, the collection of the implant is not as demanding in terms of transportation and rapid subsequent processing, as in the case of implants containing cells. Collagen structures that will represent the final implants are more stable than the cell structure.
2) Removal of cells. According to the invention, various methods for removing cells can be used for the specified implant, including the methods described in the present level of technology. The latter involves the removal of cells using surface-active agents, such as detergents, chemical compounds such as acids and alkalis or enzymes, as described in the following patent applications and documents that are included in this application: CN 200310124306 (Hu Jie); ST (Dong Qun Lin); CN 20041022506 20040512 (Dai Weihua et al.); US 2005 0186286 A1 (Yoshihiro Takami); JP 19900247300 (Koide Mikio) and CZ patent No. 281176 (E. Matouskova).
According to the invention, the two-step method is preferred in which at the first stage, the collected tissue is exposed to a suitable proteolytic enzyme such as trypsin or papain, and the second stage fabric, including possible remaining cells exposed to strong hypotonic the RCM solution, preferably an excess of distilled or deionized water. Deionized water will remove the remaining cells, subjecting them from osmotic shock, which causes rupture of their membranes. The specified second stage of cell removal occurs together with multi-step extraction of the remaining enzyme (such as trypsin) and other compounds. Along with the removal of the remaining enzyme removes the soluble peptides, and polysaccharides, glycoproteins and other compounds with expected biological activity. Remove water-soluble compounds is even more effective because hypotonic solution causes severe swelling of the tissue, improving, thus, the diffusion of the extracts. Applicants have found, however, that even thorough extraction does not remove these water-soluble compounds completely, and new organic compounds can be detected using UV-spectroscopy at the end of each stage. This shows that the new compounds, such as polypeptides and glycoproteins, continue to escape from the collagen structure; thus, a certain level of biological activity is preserved.
3) Dehydration. Dehydration is performed by removing at least a significant fraction of water using evaporation of water present in cell-free page is the established levels, or by extraction using a suitable solvent, such as ethanol. "A significant fraction of water" here refers to the so-called "free water", which represents the fraction of water, structure and thermodynamic properties which are entirely same as those of liquid water (e.g., melting point, vapour pressure or heat capacity). This is usually the largest part of the water in the tissue, with the exception of approximately 20 wt.%, which consist of water, more or less associated with the collagen matrix or other hydrophilic components of the implant. Specified the so-called "bound water" has thermodynamic properties that differ from thermodynamic properties of free water, and acts as a plasticizer of collagen. Completely remove bound water is difficult. Under "digidrirovanny implant" refers to an implant that does not contain free water and has a content of residual humidity less than 20% (wt.), preferably less than 10% (wt.). To increase the shelf life of the product is particularly preferable to maintain the moisture content in the product is less than 5% (wt.). If extraction is carried out using miscible with water solvent, the specified stage is already causing simultaneous partial denaturation of collagen. In order partial denture the Oia was effective, towards the end of the extraction water solvent should contain more than 50 wt.% organic compounds, preferably more than 70 wt.%. Extraction of water can be carried out in several stages, with a gradual increase in the concentration of organic solvent. Suitable solvents are lower aliphatic alcohols from C1to C4, lower aliphatic ketones, such as acetone, ethers such as dimethyl ether, diethyl ether, dioxane or tetrahydrofuran, glycols such as ethylene glycol, 1,2-propylene glycol, diethylene glycol or triethylene glycol, etc. is the Most appropriate ethyl alcohol, which is not only effective denaturing agent for collagen and other proteins, but also preserving and sterilizing agent, effective against, for example, retroviruses. Its advantage is the relatively low toxicity, availability and complete removal of residue by evaporation. If the selected solvent is not volatile, it should be removed by extraction with a volatile solvent, such as methanol, ethanol, acetone or water.
4) Partial denaturation of collagen. Denaturation is performed with the use of heat or a suitable organic agents, such as, for example, alcohols, aldehydes, ketones or p is Rhodesia combination. It is also possible to cause denaturation by partial cross-linking of collagen, for example, with the use of polyvalent cations, such as Ca2+, Mg2+, Al3+or Cr3+. Cross stitching can improve the resistance to biological degradation and, thus, may extend the effective use of the implant.
Partial denaturation of biological coatings, heat treatment, cross-stitching or their combination are described in the following documents included in this application: JP 19900247300 (Koide Mikio) and US 4076468 (Loiotta et al.); US 4247292 (W.A. Angell).
Denaturation of collagen can advantageously be implemented in combination with dehydration, however, both steps can be done individually, in random order. One preferred method is the dehydration by evaporation of water followed by denaturation in digidratirovannogo condition, for example, using heat treatment. Denaturation of the organic solvent can be carried out even by sprinkling or spraying a suitable organic agent on digidrirovanny the implant. Denaturation of solvents can be combined with thermal denaturation by controlled heating of the implant, for example, during the evaporation of water or solvents.
It is important to carry out the dehydration and d is natural acellular matrix under mechanical tension in one or two selected directions, usually in the direction of the greatest dimension. Tension during dehydration and denaturation can be accomplished by maintaining a constant dimension in the desired directions. This can be achieved, for example, by fixing the acellular matrix using fasteners, stretch using elastic bands or rollers, fixing on a suitable frame, pressing on the adhesive substrate or use suction to attach to the substrate using vacuum, etc. Dehydration and denaturation in the stretched state will Orient the collagen structure in the direction in which acellular collagen matrix is stretched (or in which its reduction of at least prevented during dehydration and denaturation).
If the denaturation is carried out in digidratirovannogo anisotropic matrix, it is not necessary to maintain it in an extended condition; thus, digidrirovanny matrix is stabilized by its measurements to a certain temperature, which should not be exceeded during manufacture or storage. The specified temperature limit depends mainly on the residual water content in the matrix, which must not exceed 20% (wt.), preferably 10% (wt.) and most preferably 5% (wt.), from siteline total mass matrix. Denaturation is carried out at a temperature between +15°C to 90°C, preferably from 30°C. to 70°C.
5) Sterilization with ionizing radiation. If the denaturation phase 4 is realized by using suitable solvents such as ethanol, there are two levels of sterilization. The first level of sterilization during the manufacturing process will be primarily to reduce the microbial load for the final sterilization, and, secondly, it will remove even those microorganisms against which the second level of sterilization can be inefficient (e.g., retroviruses).
The final level of sterilization perform location in packaging impermeable to microorganisms and viruses, using radiation. Preferably the minimum level of ionizing radiation used for this microbial load, which reduces degradation of the product. Recommended level below 50 kgray, preferably below 30 kgray. This is important especially in the case of gamma radiation. The preferred sterilization accelerated electrons (e-rays, beta-rays), which is more gentle to the material of the implant and which can more accurately dosed. It is important to understand the differences in the mechanism of degradation when using gamma rays and accelerated electrons. The authors of the present invention with edible the receiving found what implants are sterilized by a combination of ionizing radiation, especially by accelerated electrons, with a chemical sterilizing agents that simultaneously cause denaturation, especially with ethanol, retain their excellent mechanical properties in the wet state and are not cytotoxic even when contact with the patient's cells or with cells cultivated on the implant in the laboratory. Regardless of whatever theories the authors suggest that the beneficial effect of ionizing radiation, such as accelerated electrons, is caused primarily by the release of soluble fragments of peptides and proteoglycans from not soluble in circumstances when the matrix that provides its increased biological activity.
The implant can also be combined with known bactericidal or bacteriostatic agents such as sulfonamides, antibiotics, protein complexes-silver or colloidal silver, distributed according to a collagen matrix. This is especially beneficial during implantation in infected or necrotic wounds. Some supplements at the same time act as softeners such as glycerin and its diacetate or formaldehyde, 1,2-propylene glycol, diethylene glycol, glucose, triethanolamine or dimethylsulfoxide (DMSO). They can Deiss is to participate as a mild preservatives, softeners and weak denaturing agents. Their content can be up to 50% (wt.), preferably less than 30% (wt.). These sleek agents are miscible with water, and preferably will be polyhydroxylated compounds, most preferably glycerol or its derivatives. They can even be used in combination with compounds of silver, as described, for example, in the application CN 199551010722 (Kai Cao), which is incorporated into the present application.
The object of the present invention is, therefore, a method of manufacturing a cell-free, sterile, almost digidratirovannogo and at least partially denatured matrix, obtained from the tissues of the animal and containing mainly collagen structure, which has already been defined above, on the basis of the tissues of the animal, were processed using a method comprising the following steps:
a) collecting tissue;
b) removing cells using enzymatic effects, surface-active agents, acids, alkalis, hypotonic aqueous solutions or combinations thereof, during the formation of acellular matrix;
C) dehydration acellular matrix by removing the bulk of the water, when the mechanical tension of the matrix in one or more selected directions;
d) partial denatures what I collagen structures in a cell-free matrix by exposure to elevated temperature, organic compounds, polyvalent cations, or combinations thereof, when the mechanical tension of the matrix in one or more selected directions; or while maintaining the dimensions of the matrix at almost a constant level;
(e) sterilization of almost digidratirovannogo and at least partially denatured matrix using ionizing radiation.
According to the invention, the preferred method of manufacture of sterile acellular matrix is a technique in which partial denaturation of collagen structures using organic compounds miscible with water, selected from the group comprising aliphatic alcohols from C1to C4, aliphatic aldehydes, including formaldehyde and glutaric aldehyde, aliphatic ketones, including acetone, and ethers including dimethyl ether, diethyl ether, dioxane and tetrahydrofuran.
According to the invention, the preferred method of partial denaturation of the collagen structures is carried out at temperatures from 15°C to 90°C, preferably from 30°C. to 70°C.
The authors found and confirmed that the implant according to the present invention can be advantageously used as a biological cover for burns, leg ulcers, the place of removal of tissue and other skin defects. Digidrirovanny implant the t can be placed directly on the bleeding or oozing wound, that will hydrate the implant in situ without significantly increasing the coverage area of the wound and to contribute to the reduction of bleeding and exudate from the wound. For this use are particularly suitable implants, softened suitable additives, such as glycerol. Sterile implant can before using be hydrated with sterile saline solution, possibly with the addition of suitable antibacterial agents (such as furantoin, solution of boric acid or protein solution of silver in water (a complex protein-silver)) and place it on the wound. Its biggest advantage is the ability to closely cover all topographic features of the surface of the wound, reduce the pain of the wound and to provide a haemostatic effect. Another great advantage is the fact that the healing of the whole area is observed without changing bandages, which is necessary in the case of other dressings on wounds, is often expensive and is particularly traumatic for the patient (in the case of severe burns, it is necessary to perform even under General anesthesia). Another advantage compared with other biological coatings is the fact that it does not matter which side is in contact with the wound. Acellular dermal coating protects and accelerates wound healing, maintaining biological the th activity, associated with healing, such as migration and proliferation of keratinocytes of the patient. Native keratinocytes are attached to the inner surface of the implant (perhaps the fibrin formed on the implant after contact with blood or plasma of a patient) and to migrate to its surface, so that the implant becomes part of the skin for that period of time when there is an active healing. After healing is complete, and update the epidermal layer of the skin of the patient, the implant will dry out and spontaneously detach, without surgical removal, which is necessary for some other biological coatings and which is also traumatic for the patient.
Another advantage of sterile cell-free implant according to the present invention is that it is well suited as a substrate for culturing cells as autologous and allogeneic. Its surface, thus, can be used for culturing suitable cells, such as keratinocytes, which will form the cellular biological coating, known as "nekombinirovannyh skin" (RK), which can be applied to burns, and other injured areas. The main advantage of this cell biological coating is its ability to combine the stimulating effect of kultivirovan the x keratinocytes with the properties of the membrane substrate, which is an implant according to the present invention. If prevention is deeper skin burns is successful when using the RK within 10 days after the lesion, graft is not necessary, healing will be much faster, and to avoid areas of collection and repeated surgical interventions. RK together with transplanted allogeneic keratinocytes will temporarily stick during healing, keratinocytes will be incorporated in the regenerating epidermis, will proliferate, to migrate, to close the wound and promote healing, induce different growth factors. Xeroderma will protect the wound and to provide a natural substrate for the migration of autologous keratinocytes. Within one week of allogeneic keratinocytes replaced by private keratinocytes. The present invention will be further explained by the examples and the accompanying figures. These examples serve to illustrate certain preferred embodiments of the present invention, and specialist definitely will understand that the scope of the appended claims is not limited to these examples.
Brief description of figures
Fig. 1 is a photomicrograph showing histological section through the treatment is MIS pigs with papillary level of corium.
Fig. 2 is a micrograph of a histological slice of the implant after removal of the cells.
Fig. 3 is a photomicrograph showing histological slice of ploskokletochnogo implant after dehydration.
Fig. 4 is a micrograph showing the staining of collagen structures on van Giesen on histological slice sterile registrationpage implant of the present invention, magnification 400x.
Fig. 5 shows registrationentry sterile implant, prepared for use as a coating on the burns.
Fig. 6 shows the location of the implant of the present invention to burn 2nd degree: Photo left: the adaptation and adhesion of the coating to the wound without external bandages. Photo right: burn 2nd degree after healing and smoothcriminal coverage.
Fig. 7 is a photomicrograph showing histological slice of the samples of the newly formed tissue to burn 3rd degree without necrosis under the floor, made from implant according to the present invention (9 days after application).
Fig. 8 shows a micrograph of histological preparations recombined skin (RK), educated cultured in laboratory conditions by keratinocytes at the implant according to the present invention. Photo left: Recombinative the health of the skin with human keratinocytes, cultured dive on a porcine acellular matrix according to the present invention. Photo right: Nekombinirovannyh skin with human keratinocytes, cultured on the surface section of the air and porcine acellular matrix according to the present invention.
Pig skin as a xenograft
Using a dermatome cut layer clean-shaven and purified porcine epidermis thickness of 300-400 microns, which includes papillary layer of the corium. The histogram of the remote layer is shown in Fig. 1. Remote striped pig skin was immersed 3 times for 20 minutes at 37°C and 12 hours at 4°C in 0.25% trypsin solution, which removed most of the skin cells and separated the epidermis. Received the epidermis was washed 6 times in demineralised water (3 times in 1 hour, 1 every 12 hours, 2 times 0.5 hours) to remove any remaining cells and trypsin. The histogram of Fig. 2 shows that a non-cellular structure was preserved. A strip of the dermis was then attached with glue to a glass Petri dish and dried at room temperature to constant weight. In this state, the dermis contained approximately 18% water. Dried in this way acellular dermis had the same area of coverage as the original hydrated dermis, but its thickness was less than half. Rattanatabtimtong dermis then immersed in 96% ethanol at 15°C for 24 hours. Then the ethanol was decanted, and the dermis was detached from the glass substrate, reinforcing Sisi fasteners in two opposite directions, and dried at 50°C for 1 hour.
Digidrirovanny acellular xenograft containing residual amounts of water to 9.5 wt.%, then put in a bag for sterilization, suitable for sterilization by radiation, sealed by heating and subjected to a dose of 25 kgray gamma radiation. Sterility was confirmed using standard sterility test. After rehydration produced histological section. Fig. 3 shows that the fibrous structure of the connective tissue remained, but was more compact, and the fibers were oriented in the plane. Painting by van gieson in Fig. 4 shows that the implant consists mainly of collagen polymers of the type such as collagen and elastin. The analysis showed that the implant contains approximately 85% (wt.) a mixture mainly of collagen with fewer elastin and fibrin, and the remainder consists of lipids, polysaccharides and glycoproteins.
The porosity of sterile implant was approximately 55% by volume, calculated using density digidratirovannogo condition. The implant rehydratable at 35°C in isotonic NaCl. After the region is ratatsii to constant weight, the water content was 62% (wt.). Repeated measurements in digidratirovannogo and hydrated state was carried out with an accuracy of 0.1 mm and has established the following coefficients of linear expansion:
The ratio of in-plane expansion: Ca=1,05±0,03
The coefficient of volume expansion: Cv=1,63±0,20
Apparent differences in the coefficients of expansion clearly confirm the anisotropic expansion during rehydration of the implant. Specified anisotropy can be further demonstrate the values of correlation coefficients of linear expansion:
After a certain period of storage of the xenograft was used as a coating for deep burn injury 2nd degree on the face. The implant is briefly immersed in a sterile saline solution, as shown in Fig. 5, after which he was magcale and become flexible without any changes to the supporting surface. Then it was placed on the burn, to which he was attached, as seen from the left part of Fig. 6. During healing of burn the implant remained in its original location, according to the invention it gradually began to dry and after about a week he started smoothness from longer healed tissue. H the rez 11 days the wound healed, and the floor was completely ukrepilas, as shown in the right part of Fig. 6.
The implant made according to example 1, was used on the burn 3rd degree
The tissue was carefully dissected. Digidrirovanny the graft was removed from the sterile packaging, were made according to the size and shape of the affected area with scissors, placed on the bleeding wound area and sprayed him sterile antibiotic solution. Xenograft quickly magcale and stick to the surface of the affected, which stopped the bleeding. Implant retained its bearing surface, which took place in digidratirovannogo condition, but its thickness was increased after hydration. It provided a perfect fit to the wound. The implant was covered with a protective layer of gauze bandages for the first 3 days. After 8 days, the implant is dried to a condition similar to a scab, and began to secede from the newly formed skin under the floor. New epidermis is slowly beginning to take shape under the implant, as shown in Fig. 7. After the healing was formed healthy and has a natural structure of the skin, including natural pigmentation and no scarring.
Human cartilage as allograft
Cartilage taken from the hip deceased donor, delivered from the cells is re-soak it in a solution of trypsin and in distilled water, he was then attached using a vacuum, with the concave side to the convex side of a porous substrate suitable form, made of pittulongu glass. On the specified substrate was placed in an excess of a solution of 20 parts (wt.) methanol and 5 parts (wt.) dimethyl sulfoxide (DMSO) at 35°C for 48 hours. Thus the cell-free implant dehydrational, and at the same time, the collagen contained in it, was partially denaturiruet in flatness-oriented state. After a specified period of time acellular allograft was removed from the solution and the methanol evaporated at room temperature using air flow. By the end of this stage, the graft contained approximately 13% (wt.) DMSO, approximately 4% (wt.) water and less than 0.5% (wt.) of methanol. After removal from the porous substrate, the graft was placed in waterproof sterilization pouch and sterilized by using a dose of beta radiation 45 kgra of the electron accelerator. Sterility was confirmed using standard sterility test.
Manufactured in this way the implant is appropriate for the experimental replacement of cartilage in the hip joint in dogs, where he was pulled over the damaged cartilage and attached by means of a loop of surgical suture material around the neck of votes is key. The implant will hydrogenate itself in situ without changing the supporting surface, so the entire period of time he remains in a stable position relative to the joint of the patient. The implant protects the cartilage from ankylosis and, thus, from the persistent loss of mobility. In addition, the implant supports and accelerates the healing of cartilage. When healing is complete, the implant is gradually degraded and resorbed, while natural cartilage do not heal and function of the joint is restored.
Pig skin as a scaffold for culturing keratinocytes for the manufacture of recombined skin)
Sterile acellular matrix from example 1 was removed from the sterile packaging, was placed on a Petri dish used for cultivation of cell cultures, and carefully poured a small excess of distilled water. Cellfree xeroderma was gidratirovana without changing the supporting surface, only its thickness was approximately doubled as a result of hydration. Excess water was then carefully aspirated to prevent any deformation of hydrated dermis, and the remainder water was allowed to evaporate in a fume hood with laminar flow at room temperature. Dried up the cell-free xeroderma then used for the cultivation of human keratinocytes irradiated on the death who enoy dose T fibroblasts (they are not bred, but kept in metabolism and has produced important growth factors), which formed the so-called nekombinirovannyh skin (RK)containing allogenic keratinocytes, cultured on xenogenic acellular-dermis of the present invention. The structure of the layer of keratinocytes was determined by cultivation conditions, as follows from Fig. 8. When the cultivation was carried out on the surface of the implant under immersion, got a smooth, regular layer of keratinocytes. When the cultivation was carried out at the interface of the implant and the air layer keratinocytes was graded with the formation of a form similar to natural epidermis, including orogovevshi layerstratum corneumand the basement membrane,stratum basale. Made thus RK can be used to heal burns, leg ulcers and other poorly healing skin defects.
RK is applied by keratinocytes at the wound and dermis outward ("upside down"). According to the invention, xeroderma, which serves as a substrate for cultivation during the phase of cultivation, a support structure to move keratinocytes during use of the graft; it protects the wound from infection, desiccation and mechanical damage. The advantage compared to a simple cultured grafts is higher strength,separation from Petri dishes without the influence of enzymes (using only 2 of tweezers) and easy manipulation. Advantage compared with cultures of keratinocytes on the gels on the basis of collagen is the consistency of RK, which is similar to the consistency of normal skin, as well as excellent adhesion to the wound and hemostatic effect. After extraction from Petri dishes transplant is not reduced, the keratinocytes on the substrate is not exposed to during extraction, as it would be in the case of enzymes. RK with allogeneic keratinocytes stimulates the healing fields collection of the grafts and the deep healing of skin burns (2nd degree).
The main advantage of the specified cell biological coating is the combination of stimulating effects of cultured keratinocytes with the properties of biological membranes. In cases when it is possible to prevent the deepening of deep burns of the skin by the application of RK within 10 days after injury, there is no need for transplantation, the healing process is considerably accelerated, and the collection will be reduced, and you can do without repeated surgical interventions. RK together with transplanted allogeneic keratinocytes will temporarily stick during healing, keratinocytes will be incorporated in the regenerating epidermis, will proliferate, to migrate, to close the wound and stimulate they healed the group producing various growth factors. Xeroderma will protect the wound and to provide a natural substrate for the migration of autologous keratinocytes. Within one week of allogeneic keratinocytes replaced by private keratinocytes of the patient.
Of tendon taken from Turkey legs, removing the cells, as described in the method of example 1, and then recorded in the apparatus, placed in the container in which it was situated on the roller and stretched by means of a cord attached to a weight of 12 kg, It was immersed in a 1% solution of aluminum chloride in the specified state and left for 16 hours at 37°C. Then a solution of 3 times changed using pyrogen-free water, then was replaced with a mixture of 20 parts (wt.) acetone and 10 parts (wt.) glycerol and 0.05 parts (wt.) sodium chloride, and the structure remained in the specified solution at 25 hours when the tension created 12 kg weight and stretching apparatus. Then the structure was dried and together with the stretching apparatus was moved into the chamber for vacuum drying, pre-heated to 70°C., where it was removed the remaining solvent within two hours, and, in addition, had a partial denaturation of collagen-induced heating. The denaturation process was completed by heating to 88°C for 10 minutes under mechanical tension in the atmosphere azo is and. Dried cell-free implant with a residual water content of 3% (wt.) were placed in plastic packaging for sterilized and sterilized by beta-radiation at a dose of 15 kgray using an electron accelerator. Sterility was confirmed using standard sterility test.
Cell-free implant remained strong during dehydration, and its diameter increased, while the length was decreased. After the implantation, it was additionally gidratirovana by reducing Poperechnaya density, which can be defined, for example, as the molar fraction of the groups linking the two chains in the polymer. It also led to a gradual reduction in the length and increase the tension surrounding tissues, which can profitably be used, for example, or reconstructive orthopedic surgery.
Small intestine of a pig
The small intestine was removed which had just slaughtered a pig, washed with water, turned inside out and re-soaked in excess of 3% solution dodecylsulfonate sodium at 45°C. After this step, one end of the intestine was closed by the clamp and the other end connected with a source of demineralized water under pressure to 30 mm Hg (4 kPa). Excess pressure is maintained in the bath with demineralized water at 40°C for 24 hours. Then the Demin is ilizovano water was replaced with a solution of isopropyl alcohol and tert-butyl alcohol (1:1, wt.) and maintained a temperature of 70°C With internal overpressure alcohol solution for a further 6 hours. Finally, the mixture of alcohols was replaced with methanol three times at room temperature. Cell-free and digidratirovannogo oriented membrane was then pumped with nitrogen and the outer surface was dried using a stream of clean air in a fume hood with laminar flow, and then her flat was spread between two polypropylene plates were sealed in a waterproof bag for sterilization. Then it was sterilized in two stages: the first stage of its sterilized using gamma radiation at a dose of 5 kgray, then accelerated electrons at a dose of 15 kgray. Sterile cell-free membrane is intended to fill a suspension of allogeneic fibroblasts and subcutaneous implantation the patient for the regeneration of the connective tissue of the patient.
1. Cell-free sterile almost digidrirovanny and at least partially denatured matrix, obtained from the tissues of the animal and containing mainly collagen fibrils which have the same structural organization as in the original fabric, designed for use as a temporary implant in medicine and veterinary, characterized in that it undergoes anisotropic change is of its measurements during hydration.
2. Acellular matrix according to claim 1, characterized in that during the anisotropic change their dimensions during the hydration of the two largest dimensions remain virtually unchanged or decrease, while the smallest dimension increases with increase in the volume of the matrix.
3. Acellular matrix according to claim 2, characterized in that the temporary implant is mostly flat shape, and its bearing surface is defined by two of its greatest dimensions, while its thickness is determined by the lowest of its dimension.
4. Acellular matrix according to claim 1, characterized in that during the hydration of the two smallest dimensions increase, while the largest dimension remains almost unchanged or decreases.
5. Acellular matrix according to claim 4, characterized in that the temporary implant is mostly an elongated shape, such as a bar or cylinder, the cross section of which is defined by two smallest dimensions, such as diameter, while its length or height is determined by the largest dimension.
6. Acellular matrix according to claim 1, characterized in that the matrix in digidratirovannogo state has a porosity less than 70 vol.%, preferably, less than 60 vol.%, and, most preferably, less than 50%vol.
7. Acellular matrix according to claim 1, great for the present, however, that fibrils of collagen, at least in digidratirovannogo state, focused mainly in the areas in which the coefficient of linear expansion of hydration has the smallest value, and generally perpendicular to the direction in which the coefficient of linear expansion of hydration is of the highest importance.
8. Acellular matrix according to claim 1, characterized in that the matrix in digidratirovannogo state has a water content of less than 20 wt.%, preferably, less than 10 wt.% and, most preferably, less than 5 wt.%.
9. Acellular matrix according to claim 1, characterized in that it also contains a sleek, preservative or bactericidal additives.
10. Acellular matrix according to claim 9, characterized in that the bactericidal additives contain silver, preferably in a colloidal state, and, most preferably, in the form of complex silver-protein.
11. Acellular matrix according to claim 9, characterized in that these sleek or preservative additives contain compounds miscible with water, such as DMSO or polyhydroxylated compounds selected from the group of glycol, of glycerol or derivatives thereof, triethanolamine and sugars.
12. Acellular matrix according to claim 1, characterized in that upon contact with a suitable aqueous liquid matrix capable of volumetric expansion, with coefficie is that volume expansion of more than 1.1, preferably, more than 1.5.
13. Acellular matrix according to claim 1, characterized in that upon contact with a suitable aqueous liquid matrix is able to hydrogenate itself to a state where it contains more than 33 wt.% water, preferably more than 50 wt.% water.
14. Acellular matrix according to any one of claims 1 to 5, characterized in that the higher the coefficient of linear expansion of more than 10% higher, preferably more than 30% above the lowest coefficient of linear expansion.
15. Acellular matrix according to any one of claims 1 to 5, characterized in that the higher the linear expansion coefficients have a value of more than 1.2, preferably more than 1.5, while the lowest linear expansion coefficients have a value of less than 1.1, preferably, less than 1,05.
16. Acellular matrix according to claim 1, characterized in that it is a solid material (dry matter) is composed primarily of proteins.
17. Acellular matrix according to item 16, wherein the proteins comprise mainly of collagen.
18. Acellular matrix according to item 16, characterized in that its material (dry matter) contains from 70 wt.% to 95 wt.%, preferably, from 80 wt.% up to 90 wt.% proteins collagen type.
19. Acellular matrix according to item 16, characterized in that its material (dry matter) contains, in addition to proteins, is also a small fraction of openacademy, including lipoproteins and phospholipids.
20. Acellular matrix according to item 16, characterized in that it is the protein component is at least partially denatured.
21. Acellular matrix according to item 16, characterized in that at least part of its protein component is cross stitched as a result of interaction with aldehydes or polyvalent cations.
22. Acellular matrix according to claim 1, wherein the animal is a mammal.
23. Acellular matrix according to claim 4, characterized in that the mammal is a pig.
24. Acellular matrix according to claim 1, wherein the animal tissue is a skin, placenta, pericardium, Dura, intestine, tendon or cartilage.
25. Acellular matrix according to claim 2, characterized in that the temporary implant is a covering for the wound such as a burn.
26. Acellular matrix according to claim 1, characterized in that it further comprises mammalian cells.
27. Acellular matrix according to claim 1, characterized in that upon completion of its function, the implant destroys itself through biological degradation.
28. Acellular matrix on p. 25 and/or 26, characterized in that the animal is a pig, and cultured mammalian cells are celoveceskoe autologous or allogenic keratinocytes.
29. A method of manufacturing a cell-free sterile, almost digidratirovannogo matrix and at least partially denatured matrix, obtained from the tissues of the animal, and includes, mainly, the collagen structure according to any one of claims 1 to 26, wherein the animal tissue is treated using a method comprising the following steps:
a) collecting tissue;
b) removing cells using effects enzymes, surface-active agents, acids, alkalis, hypotonic aqueous solutions or combinations thereof, during the formation of acellular matrix;
c) dehydration acellular matrix by removing significant parts of water, when the mechanical tension of the matrix in one or more selected directions;
d) partial denaturation of the collagen structures in a cell-free matrix by exposure to elevated temperature, organic compounds, polyvalent cations, or combinations thereof, when the mechanical tension of the matrix in one or more selected directions; or while maintaining the dimensions of the matrix in the selected directions at almost a constant level;
e) sterilization of almost digidratirovannogo and at least partially denatured matrix using ionizing radiation.
30. The method according to clause 29, from which causesa fact, partial denaturation of collagen structures is performed with the use of organic compounds miscible with water, selected from the group consisting of aliphatic alcohols from C1to C4, aliphatic aldehydes, including formaldehyde and glutaric aldehyde, aliphatic ketones, including acetone, and ethers including dimethyl ether, diethyl ether, dioxane and tetrahydrofuran.
31. The method according to clause 29, wherein the partial denaturation of collagen structures is carried out at temperatures from 15°C to 90°C, preferably from 30°C. to 70°C.
SUBSTANCE: skin equivalent is produced that represents extracellular protein matrix, with mesenchymal cells within it and epidermal cells on its surface. As a matrix collagen or fibrin gel is used. As epidermal cells previously selected keratocyte population with stem cell character is used. Application of the invention allows producing skin equivalent, containing cells in active initial growth stage. Skin equivalent is intended for cutaneous cover regeneration in patients with extensive burns, trophic ulcers, bedsores, and other kinds of wound.
EFFECT: prolonged stimulating effect on patient's cells migration and proliferation.
4 cl, 4 ex
SUBSTANCE: means include biopolymer carrier and cells, applied on carrier mentioned, and characterised as follows: the biopolymer carrier represents collagen-chitosan film, or crumbs of collagen-chitosan film, or high polymer gel in form of 5% macrogol-1500 solution; the cells are diploid human lung strain "ФЭЧ" 16-1 cells, or diploid human musculo-cutaneous tissue strain "ФЭЧ" 16-2 cells in concentration 2.0-3.0×105 to 1 cm2 of film or crumb carrier, or 1.0×106 to 1 ml carrier in form of 5% macrogol solution high polymer gel.
EFFECT: novel means production for replacement therapy based on standard certified cell material, possibility of optimal means form selection, reducing therapy duration, and widening field of application in replacement therapy.
3 cl, 17 ex, 3 tbl
FIELD: medicine, experimental and clinical surgery, combustiology, transplantology, cosmetology.
SUBSTANCE: the present innovation deals with restoring artificial matrix of dermal-epidermal skin equivalent. The method, also, deals with mixing both collagen and chitosan by adding a surface-active substance and a binding agent followed by pouring the mixture obtained, its freezing followed by freeze drying and gamma-sterilization, moreover, before obtaining polyelectrolytic complex of collagen and chitosan one should successively supplement chitosan with aqueous solutions of 1.8 g/g dry chitosan of ascorbic acid, 5-100 mg/g dry chitosan of chondroitin sulfuric acid, 10-100 mg/g dry chitosan of hyaluronic (D-glucuronic) acid, 11-220 mcg/g dry chitosan of cattle serumal growth factor and 2.5-5 mg/g dry chitosan of heparin. The method provides to obtain a structure similar to skin that substitutes skin defect perfectly.
EFFECT: higher efficiency.
6 dwg,1 tbl
SUBSTANCE: group of inventions refers to medicine. The group of inventions consists of two objects: a method of processing and a biological tissue. The method is characterised by taking the biological tissue stabilised by glutaric dialdehyde and presented by both animal and human biological tissue, e.g. swine or bovine pericardium or human cadaver mitral valves wherein physical plasma processing, particularly of collagen tissue is performed.
EFFECT: group of inventions provides higher biocompatibility of biological tissue, cell colonisation and prolonged service life.
SUBSTANCE: group of inventions refers to medicine, and can be used for production of transplantable tissues. A method for cell-free organic tissue preparation for living cell reimplantation involves cell-free tissue preparation on a substantially flat surface and creation of a number of holes on the surface, spaced along the whole surface mentioned above and arranged so that they penetrate deep down at least a portion of thickness of said tissue with the holes designed for keeping the reimplanted living cells. Said number of the holes is created by means of one or more metal needles connected to a power supply which on each needle point induces current flow of such intensity and of such waveform to provide adequate energy for destruction of molecular couplings comprising organic tissue near to the needle point mentioned above. Each hole is formed by current flow and is great enough that the needle point mentioned above could penetrate into a space formed by opening of the molecular couplings. The group of inventions also refers to cell-free organic tissue for living cell reimplantation produced by said method.
EFFECT: group of inventions enables reducing time of producing transplantation tissue and provides living cell penetration along the whole thickness of cell-free tissue.
24 cl, 4 dwg
SUBSTANCE: acellular transplant is described comprising (i) a connected skeletal matrix with openness of pores of biologically and pharmaceutically usable material and (ii) human serum. In accordance with a particularly preferred embodiment, the matrix also contains a gel. Also the method of manufacturing such acellular transplant is described when the matrix and the gel contact with human blood serum. If necessary, the transplant and the serum can be dried. Alternatively, the matrix and the gel can be presented in a dry form prior to contacting. Use of acellular transplant for regeneration of tissues and in particular for regeneration of cartilage and/or bones is also described.
EFFECT: transplant is easy to manufacture, long stored and easy to use.
16 cl, 2 dwg, 2 ex
SUBSTANCE: there is offered method for chemical treatment of xenopericardium that involves chemical stabilisation of xenopericardium with 0.625% glutardialdehyde and following processing with 1% sodium dodecyl sulphate; chemically stabilised xenopericardium is additionally processed with 0.05÷0.25% aqueous solution of chitosan or metal-containing chitosan with deacetylation degree 50÷98% and molecular weight 4÷140·103 at pH 3÷5; upon termination of processing, xenopericardium is fixed in 70% aqueous solution of ethanol; then modified xenopericardium is kept in 0.10÷0.50% aqueous solution of chitosan N-sulphosuccinate with molecular weight 10÷166·103 or chitosan 3,6-O-disulphate with molecular weight 7÷180·103 at pH 4÷8 during 20÷60 mines at temperature 20÷30°C with following fixation in absolute ethanol.
EFFECT: improved durability and biocompatibility of bioprostheses.
3 ex, 7 tbl, 3 dwg
SUBSTANCE: method involves chemical stabilisation of biotissue with 0.625 % aqueous solution of glutaraldehyde, pH 7.4, followed with preparation with a surface-active substance and quadruple change of a working solution. Immediately ahead of implantation, bioprostheses are thoroughly washed with sterile physiologic saline sixfold changed, at 500 ml of the solution for 100 g of biotissue. Then it is processed with 0.05-0.5% aqueous solution of chitosan N-sulphosuccinate of molecular weight 50-150 kDa in intensive stirring during 0.5-2 h with pH within 5 to 8, and to temperature 22±2°C. Further, it is fixed in sterile absolute ethanol and put in sterile physiologic saline, and stored at temperature 6-8°C before implantation.
EFFECT: improved durability of bioprostheses.
5 ex, 4 tbl
SUBSTANCE: bone of natural origin is cleaned, sawed up to 0.2-2.0 cm thick plates, washed with heated to 65°C 0.1 M pH 5.8-6.0 phosphate buffer, digested in 0.1-0.4% activated papain solution at 65°C during 24 hours, then washed in five volumes of water at 40-80°C, treated with 0.4 N alkali at room temperature during 10-24 hours, rinsed in running water, degreased in ethanol/chloroform mixtures in ratio 1:2 firstly, and 2:1 secondly, decalcified in 0.4-1 N hydrochloric acid, treated with 1.5-3% hydrogen peroxide during 4 hours, washed with purified water, then with ethanol, dried at room temperature, packed up and sterilised. Material for osteoplasty and tissue engineering represents compound, in which native collagen matrix space structure and natural bone mineral component are preserved, containing 25% collagen and 75% mineral matter. According to dry material analysis it includes less than 1% non-collagen proteins.
EFFECT: method improvement.
3 cl, 5 ex
SUBSTANCE: autograft is made as mixture of minced muscular auto-issue with the concentrated serum autofibronectin in proportion 1:(0.2-0.5).
EFFECT: autograft reduces the treatment and prevents the post-operational complications.
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.
EFFECT: enhanced effectiveness in recovering combined injuries of cartilage and bone tissue in articulations having defects.
8 cl, 6 dwg
SUBSTANCE: invention relates to experimental medicine, in particular to biopolymeric matrix for proliferation of cells and regeneration of nervous tissues. Biopolymeric matrix is developed on the basis of heterogeneous collagen-containing structure, which consists of microparticles of collagen with size 30-300 mcm, dispersed in homogeneous gel of collagen, and containing neutral stem cells and stimulating additive in form of silicon nanoparticles with size 50-150 nm in quantity 1·10-3-1·10-2 wt %. Additive in form of silicon nanoparticles in applied for stimulation of cells proliferation and regeneration of nervous tissues. Nanoparticles of silicon are obtained by method of laser-induced pyrolysis of monosilane SiH4. Assessment in vitro of biological action of biopolymeric matrix (influence on viability and proliferation of cells) in carried out with application of neutral stem cells.
EFFECT: biopolymeric matrix stimulates proliferation of cells and regeneration of nervous tissues and has biocompatible properties.
3 cl, 1 dwg, 1 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to pharmaceutics and medicine and concerns a method for making a composite of collagen and nanohydroxyapatite for bone tissue defect repair by mixing restructured collagen and aqueous glucose, keeping at room temperature to swelling and heating to 35-45°C; the prepared suspension is homogenised and added with a nanohydroxyapatite suspension in aqueous glucose at weight proportions of collagen to nanohydroxyapatite 5:1 to 1:5; it is followed by homogenising and sterilising the prepared composite for bone tissue defect repair to be used in bone tissue defect repair.
EFFECT: material has a distinct architecture close to a native analogue, and possesses high mechanical strength.
7 cl, 4 dwg, 1 ex
SUBSTANCE: cortical plate of mesh compact osteotomy is perforated in an alveolar bone within a defect. A paste osteoplastic composite containing bone growth and regeneration factors is introduced into each perforation. A bone frame made of xenogenic cortical bone of the shape and size formed so that the frame adding the alveolar bone within the defect to a normally developed alveolar bone is introduced into the bone frame. After the introduction, the frame is filled with said osteoplastic material, and the bone defect is filled with a resorbed membrane, and the wound is closed.
EFFECT: method provides a process of permanent osteoinductivity within the bone defect, limited distribution of the osteoplastic materials outside of the bone build-up zone that leads to higher effectiveness of replacement with reduced length of treatment, number of injuries.
2 cl, 1 ex
SUBSTANCE: invention relates to medicine, namely to combustiology, and can be used for treatment of infected burn wounds of IIIA degree. For this purpose on wound surface cleaned from damaged tissues and serous-purulent discharge, for 2-4 days applied is bandage representing construction from organosilicon polymer, covered with type I human collagen with platelet growth factor -PDGF-BB and pectin, for obtaining which collagen in form of thin layer of collagen sponge about 1-1.5 mm, obtained by method of lyophilisation of 1-2% collagen solution on perforated substrate from polysiloxane-polycarbonate film, is moistened with 2% solution of apple pectin with realisation of re-lyophilisation.
EFFECT: method makes it possible to increase treatment efficiency due to reduction of quantity and species diversity of microflora without influencing activity of proliferation process with simultaneous acceleration of wound healing.
SUBSTANCE: group of inventions refers to medicine, and concerns a vascular ageing inhibitor which contains as a basic ingredient collagen of low molecular weight approximately 3000 Da of fish skin prepared by enzymatic decomposition with using anacanthe or flatfish skin. The anti-ageing compositions contain the vascular ageing inhibitor in the granulated form.
EFFECT: invention provides better elasticity of the whole vascular wall and prevented adhesion of thrombocytes and cholesterol.
4 cl, 2 ex, 1 tbl, 3 dwg
SUBSTANCE: invention refers to the area of biotechnology, namely to production of short peptides - stimulators of production of extracellular matrix protein in the skin, and can be used in the medicine. The peptides produced consist of four amino-acid residues that can be used separately or in combination in the method of stimulation of collagen production using fibroblast.
EFFECT: invention provides for effective collagenesis stimulation in fibroblast cells.
25 cl, 3 dwg, 7 tbl, 6 ex
SUBSTANCE: injection heterogeneous elastically-resilient biodegradable biopolymer hydrogel for substitutional and regenerative surgery, obtained from hydrolysate of embryonic or postnatal collagen-containing tissues of animal origin, excluding human, which consists of two constituents: hard - microparticles from linked hydrolysate and liquid - from initial hysrolysate, taken in specified ratio, with particle size not exceeding 100 mcm. Method of obtaining injection hydrogel includes crush of initial raw material, freezing obtained mass under definite conditions, following defrosting, processing with solution of icy acetic acid, separation of supernatant liquid, washing with water, processing with sodium hydroxide solution, centrifuging obtained hydrolysate of animal tissues in form of hydrogel mass with its further filtration, part of obtained hydrolysate is processed by γ-irradiartion in dose 1.0 kGy in gaseous medium and homogenised until particles with size not larger than 100 mcm are obtained, washed with phosphate buffer solution and mixed with remaining volume of initial hydrolysate of animal tissues, obtaining injection heterogeneous elastically-resilient biodegradable biopolymer hydrogel.
EFFECT: hydrogel possesses immunogenicity, prolonged effect of biostimulating properties.
5 cl, 4 dwg, 3 tbl, 6 ex