Multi-purpose heterogeneous collagen matrix applied for implantation and method for its obtaining

FIELD: medicine.

SUBSTANCE: the suggested multi-purpose heterogeneous collagen matrix for implantation is being flexible-elastic mass obtained out of two collagen sources, moreover, one source is a tissue of vertebrates of one and the same class, and another source - that of an animal of another class, moreover, collagen sources are animal tissues of, for example, mammalian class and avian class, matrix consists of two phases: solid phase - as microspheres out of mammalian tissue collagen, and liquid phase - out of denaturated avian tissue collagen at the ratio of phases being (1-10) : (1-10), at microspheres size being 100-300 mcm, as for final products of biodegradation they are represented by CO2 and H2O. Matrix, additionally, contains components of physiological culture media, and, also, additives that favor the growth and differentiation of cells and tissues, antibacterial and/or antiviral components, and, also, antiaggregation preparations in their efficient quantity, for example, additionally, it contains embryonic cells of nervous tissue. Another aspect of the present innovation deals with the method to obtain the matrix due to preparing mammalian collagen solution (MCS) and denaturated avian collagen solution (ACS), moreover, it is necessary to apply 0.3 M acetic acid, at final concentration for MCS being approximately 0.5-1.5%, and for ACS - approximately 3.0-5.0%, then MCS should be treated with γ -irradiation at the dosage of 1.0 Mrad to be further homogenized to obtain microspheres. Then both MCS and ACS should be washed off with distilled water up to pH of not less than 6.0 and with phosphate buffer solution to mix washed off mammalian collagen and avian collagen at 1:1 ratio at obtaining matrix. The matrix obtained should be additionally supplemented with antibacterial and/or antiviral components, and, also, stimulating agents for tissue regeneration and antiaggregation preparations. The matrix obtained should be sterilized due to γ -irradiation at the dosage of 0.5 Mrad/1 ml. The present innovation enables to obtain new heterogeneous collagen matrix which is considered to be a multi-purpose one applied for transplantology and substitution surgery of different tissues and organs in alive body in case of tissue lesions. Moreover, it is distinguished by controlled terms of biodegradation.

EFFECT: higher efficiency of application.

13 cl, 16 ex, 4 tbl

 

The invention relates to the field of biopharmacology and molecular biotransplant, or rather to the creation of a universal plastic matrix based on heterogeneous collagen material for restoration of damage to soft tissues and organs by implantation, and the manner of obtaining such a matrix.

Collagen is the most abundant protein in the human body. He is from 25 to 33% of the total protein and, consequently, about 6% of body weight. The composition of collagen remarkable in the sense that a third of its amino acid residues represented by glycine; the share of Proline in the amount of 3 - and 4-oxoproline accounting for 21% of the residues, while the share of alanine - 11%. The collagens are one of the few proteins containing oxyproline and 5-oxylipin. The study of connective tissue with an electron microscope showed the presence of collagen fibers, which consist of fibrils. Fibrils are in the form of a cylinder with a diameter of 5 to 200 nm (depending on source selection); at a higher resolution, found that the fibrils have a characteristic corrugated structure. Organization of the fibrils in the fiber in different tissues are different. The collagen fibers of the tendon and the skin formed by bundles of parallel fibrils, whereas fibrils in healing the wound aggregated rather chaotic. In the cornea and vitreous body OBN is rivalda narrow band strictly oriented fibrils, this ensures minimum scattering and the maximum transmittance of the incident light.

The extraction of the skin of very young animal cold saline solutions or by prolonged extraction of insoluble collagen diluted acid to obtain a solution forming collagen fibrils units called tropocollagen. Although tropocollagen isolated from different sources differ in composition, but they are all enriched in glycine and contain 5-oxidising (l) and okshikislotu 4-hydroxyproline (Hyp). Some collagens contain 3-hydroxyproline, but considerably less than the 4-hydroxyproline.

Molecules tropocollagen (M~300000) have a thickness of about 1.5 nm and a length of 300 nm. They are formed by three subunits, each of which is a polypeptide chain, containing about 1000 amino acids. Molecules tropocollagen are among the most asymmetric molecules, ever isolated from natural sources. A separate circuit is a solid zero helix containing 3 amino acid residue on the coil, and the three chains form a structure similar to the cable, slightly twisted to the right spiral. The unique structure of tropocollagen due to the high content of glycine and aminocyclo. Pyrolidine ring amino acids are of particular stereochemical with the STS, restrict the flexibility of the chain and contribute to the formation of secondary structure in the form of triplex helixes. This compact triplex structure is possible because every third residue in the sequence chain tropocollagen represented by glycine, α-carbon atom of which is immersed inside the molecule, where the R group of any other amino acids cannot be accommodated. Although collagens from different sources differ in composition, in all cases, the analysis of large polypeptide fragments in every third position of the chain found glycine. Three chains are stabilized by hydrogen bonds between the-C=O...NH-groups of peptide bonds adjacent chains. In tropocollagen in contrast to globular proteins of R-groups of all amino acids on the outer side of the molecule and few involved in the stabilization of the structure, although they probably participate in intermolecular interactions in the formation of fibrils and fibers [1].

Biochemical studies helped to create a complete concept of intracellular biosynthesis and extracellular modification of collagen. On ribosomes, fixed to the membranes of the granular cytoplasmic network is built from amino acids single spiral α-chains, and with the help of propyl - and ligilghedaselt formed specific number is of Agen hydroxyproline and oxylipin. Then the three α-circuit are connected in trehserijnuju molecule procollagen, which is characterized by the presence on both ends of the nonhelical peptides, preventing intracellular aggregation of molecules. After the release of procollagen from the cell terminal N - and C-propeptide hatshepsuts procellariidae, but at the ends of the molecules are small naselenie remains - telopeptide. Molecules tropocollagen aggregate into fibrils, and then in the presence of the enzyme lysyloxidase and copper, the formation of covalent cross-linking. Protofibril (diameter 3-5 nm), in turn, are combined into fibrils with a typical periodicity of 64 nm, which is explained by the shift of the molecules by a quarter length at the lateral aggregation. Fibrils form (optical level) fibers, and those in turn - fiber bundles. At all levels of the spiral torsion elements in structures, of which the following, which gives collagen formations of particular strength and resistance to stretching. The scheme of fibrillogenesis refers to collagens I-III types. However, currently known for 13 types of collagen (see table 1), differing from each other by a combination of α-chains, with differences in amino acid sequence, quantity, and location (at the ends or internally and molecules) nonhelical areas (domains), the length of the macromolecule and localization in tissues.

Table 1
TypeLocalization in tissues
ILoose and dense connective tissue, bone, ligaments, tendons, dermis, dentin
IICartilage, vitreous body, heart valves
IIILoose and dense connective tissue, skin, spleen, lymph nodes, bone marrow, dermis of human embryos
IVThe basal membrane of blood vessels
VLoose and dense connective tissue, skin, eye lens
VISame, the vitreous body
VIIThe skin, lungs, sclera of the eyes
VIIIEndothelium
IXCartilage
XHypertrophic cartilage
XICartilage
XIICartilage
XIIIThe basal membrane

Most types of connective tissue consists of collagens I and III types (as maturation ratio is measured predominance of type I) with a mixture of collagens V and VI types. The basis of reticular fibers, and t is the train immature argyrophilic collagen fibers is type III collagen. In cartilage and heart valves, the predominant collagen type II mixed with IX-XII types in the basal membrane collagen type IV admixture XIII type. Furthermore, the molecules of type IV collagen does not form fibrils, as they are connected in the original network structure.

Results ultrastructural immunohistochemistry studies show that collagen type I forms a typical periodic fibrils, collagen type III presents thinner fibrils with a diameter of 10-15 nm, which weave in the form of a network of fibrils of type I. Very thin filaments (diameter of 6-10 nm), closely associated with fibril collagens I and III types consist of collagens V and VI types, which, in addition, cover the surface of the fibrils of the first type, reinforcing the pattern periodicity, and collagen V type - even and the surface of the cells. Fibronectin is also associated with the surface of the fibrils, although detected in the form of globules and filaments in mifepriston space [2].

Accounting support (stromal) part of the majority of soft organs and tissues, collagen is used in biocomposite materials for reconstructive surgery, accounting for the main competition synthetic biopolymer substances used for such purposes.

In particular, for therapeutic purposes, a series of hydrogels based on acrylamide or methacrylamide derived from the Pach the surrounding agents (see U.S. patent No. 5863557, 1999, patent RF №2205034, may 2003), as well as other substances, for example, the material for the plastic fabrics, the Russian Federation No. 2137441, 1999, which is a porous, durable and super-elastic material, nickelide titanium.

Although these substances and have low tissue reaction to implantation of such substances, but they can't stimulate the repair of damaged tissue and are not intended for subsequent resorption, remaining in the body foreign body. In some cases, the polymeric biocompatible materials for plastic purposes, together with collagen matrix (see RF patent №2033090, 1995).

Analogs of the present invention can be considered different collagen substance and biocomposite that can be used for bioplastic purposes: for example, wound-healing coating (patent RF №2085217, 1997), a method of improving quality silk surgical thread (patent RF №2076740, 1997), the material for the plastic tissue (RF patent No. 2137441, 1999), the composition for injection in arthroplasty (RF patent No. 2034465, 1995), the composition for datatranslator containing collagen-gel, methylcellulose and alcohol extract of propolis (RF patent No. 2106781, 1998), method of preservation functions of the intervertebral segment with osteochondrosis (RF patent No. 2171649, 2001), reinforced graft DL is scleroplastic operations (patent RF №2140242, 1999), release agent (patent RF №2155592, 2000), biocompatible polymer material and method thereof (patent RF №2162343, 2001), the transplant for scleroplasty containing neuteralise collagen (RF patent No. 2157159, 2000), collagen containing material to keratinolytic (U.S. patent No. 6197330, 2001), ═ and cardiopatici (U.S. patent 2002094573, 2002), a method for surgical repair of cicatricial deformities of the skin (patent RF №2201146, March 2003).

However, these developments include a description of the use of collagen, often in combination with other polymers, for individual organs and tissues.

Well-known works can be found a description of some of the substances used as a universal collagen framework for transplantation. For example, collagen matrix, selected from the submucosal tissue of the shell, which can be used as the growth prosthesis (WO 98/22158, 1999). Allograft used as a layer between the fabric and the transfer material representing ethylpropane collagen with anti-bacterial properties (patent RF 2143868, 2000). There is also known a method of processing collagen containing tissue, which is taken from the donor and transplanted to a recipient, the method is aimed at suppressing the immunogenicity of the tissue or organ in a recipient (U.S. patent 2002119437, 2002).

As a researcher who is named as the closest analogue we can consider the RF patent №2188206, published in August 2002, where we are talking about the use of native collagen or such in the crushed and solubilization condition gradually heated with certain physico-chemical properties. This drug can be used in the pharmaceutical, medical-surgical, ophthalmic and cosmetic compositions. The main disadvantage of collagen gels is a high rate of biodegradation. The result of the application is only a partial restoration of tissue with scar formation.

The technical task of the present invention is the creation of a universal matrix based on natural collagen with adjustable term biodegradation, is able to stimulate damaged tissue axonal regeneration and neovascularization, as well as ways of obtaining such a matrix.

The problem is solved in that a versatile heterogeneous collagen matrix for implantation, which represents the elastic mass, obtained from two sources of collagen, one source is the tissue of vertebrate animal of the same class, and the second animal of another class. Sources of collagen, for example, are a tissue of animals of the class Mammalia and the class Aves. In physico-chemical terms, the matrix consists of two phases: solid - mikros the EP of the collagen tissue of mammals, and liquid from denatured collagen tissue of birds at the ratio of the phases (1-10) parts on solid (1-10) parts of the liquid phase. In our tests we used the matrix (collagen mammal + collagen birds) at a ratio of phase 1:1. The matrix has a size of 100-300 microns microspheres and the end products of biodegradation are the CO2and H2O. Optionally, the matrix may contain physiological components of culture media and supplements that promote growth and differentiation of cells and tissues, antibacterial and/or anti-virus components, and antiaggregatory agents.

When using a matrix for implantation of specific tissues it can optionally contain, for example, embryonic cells of the nervous tissue.

The invention is illustrated by the following example:

EXAMPLE 1. For our studies used the collagen of the connective tissue of farm animals (cattle), representing transperancy protein with a molecular weight of 300,000 as a solid phase. for the liquid phase used denatured collagen birds in the ratio of 1:1, the immunogenicity of the resulting collagen represents approximately types VIII-XIII.

In General, as the solid phase, it is preferable to use collagen from vertebrate animals of a higher class than for the liquid phase, for Example, mammal animal (solid phase) - bird (liquid phase), bird (solid phase) - reptile (liquid phase) and so the resulting matrix has a relatively low immunogenicity.

As additional components of the matrix may contain antibiotics, antiseptics, stimulants regeneration, Universiada drugs and other antiplatelet agents.

For example, antibiotics: penicillins - penicillin, cloxacillin, ampicillin, cephalosporins - tsefaloridin, cefuroxime, cefotetan, ceftazidime, carbapenem - Meropenem, carbapenems - aztreonam, aminoglycosides - streptomycin, gentamicin, amikacin, tetracycline is a tetracycline, minocycline, macrolides - erythromycin, azithromycin, lincosamides - lincomycin, an antibiotic peptide group - polymyxin B, polymyxin M, ristomycin, bacitracin.

Antiseptics, for example, sangviritrin, acterized or tomized. Stimulators of cell regeneration, for example, methyluracil stronger, ATP, pentoksil, potassium orotate, Riboxin, etagen.

The benefits of the new heterogeneous collagen matrix for implantation in damage to soft tissues and organs are:

- versatility (reference and trophic function for cell cultures stimulates axonal regeneration and neovascularization)

- high biocompatibility on the protein and cellular level as the finished product, t is K and the products of biodegradation

- the ability to stimulate proliferation and differentiation of cells

- adjustable biodegradation time from several weeks to several months (the end products of biodegradation are carbon dioxide and water)

- ability to porosity directly in contact with biological media, ensuring the processes of neovascularization

- sterilization without changing the medico-technical properties.

At room temperature heterogeneous matrix is an elastic mass, stable during prolonged storage and are not prone to the phenomenon of syneresis (separation of the liquid phase). In this state, the matrix is not miscible with water and hydrophobic liquids. At a temperature of 37°With the viscosity of the biopolymer matrix due to poorly sewn liquid component, decreases sharply, while the physical condition of globular (solid) component is not changed.

Figure 1 shows a photograph obtained by scanning electron microscope, a new matrix, conventionally designated as “Spherogel-e.

2 shows the micrograph of the matrix with the stem cells of the bone marrow of rats.

The product is packaged in syringes of different capacities, sterilized γ-radiation. Enter the matrix directly into the area of the defect. Eventually replaced recognize the Noah fabric without the formation of scar tissue.

Another aspect of the present invention is a method for heteroclitus matrix described above.

Known methods for producing collagen matrices for plastic purposes: in particular, in the patent of Russian Federation №2174410, 2001, describes a method for collagen lamellae, which allows you to get collagen materials in the form of powders, granules, plates, RF patent №2118176, 1998, which describes a method of obtaining a collagen material with its saturation water vapor, which is then dried, structure in pairs of formaldehyde, in the material you can enter a variety of useful supplements.

However, the methods of obtaining heterogeneous collagen matrix on the basis of vertebrate collagens of different classes in the available literature is not found.

The technical objective is to provide a method for obtaining such a heterogeneous collagen material.

The method is as follows.

The method of obtaining generic heterogeneous collagen matrix for implantation includes the preparation of a solution of collagen mammal (RCM) and denatured solution of collagen birds (RCP), while using a 0.3 M acetic acid at a final concentration for RCM of ~0.5-1.5%for RCP ~3,0-5,0%, then the RCM process γ-irradiation at a dose of 1 Mrad and homogenized to obtain m is crognolo, next washed RCM and RCP with distilled water to a pH not less than 6.0, optionally washed with phosphate buffer solution and mix the washed collagen mammals and collagen birds in the ratio of 1:1 with the receiving matrix.

In the matrix can be also added antibacterial, antiviral components and or stimulants regeneration and antiaggregatory drugs, then it is sterilized γ-irradiation at a dose of 1 ml of 0.5 Mrad.

Obtained as described above, the matrix with the properties described in example 1 were subjected to biological tests. Here are the results of such tests.

EXAMPLE 2. Tests in vitro.

Cytotoxicity: 1) extract on suspension culture of motile cells (the seed of cattle) was not detected. Toxicity index was 100% for allowable values 70-120%; 2) extract on fibroblasts mouse line T clone SC-1. The morphology of the cells was similar to control, the number of cells differed from the control in the margin of error. Toxic effects were not identified.

Hemolytic activity of the extracts was tested on isolated human erythrocytes. The degree of hemolysis was 0.02% at an acceptable value not more than 2%.

EXAMPLE 3. Tests in vivo.

Irritating effect in a single installer the and into the conjunctival eye bag rabbit no. On the scale of the reaction corresponded to zero degrees.

Study of the reactions of General anaphylaxis and active cutaneous anaphylaxis in Guinea pigs showed no allergic reactions anaphylactic type.

Sensitizing effect on white rats is not detected, the reaction degranulation of mast cells is negative.

Implantation performed on rats (intramuscularly). The observation period of 3 months. Morphological studies of the pathological changes in the surrounding tissue was not detected.

Sterility: the samples Tested sterile.

Progenote: Extracts prepared with 0.9% sodium chloride for injection, pyrogenic reactions when administered intravenously to rabbits did not show. The total temperature rise is not exceeded 1.4°C.

The resulting product (matrix) can be placed in disposable syringes, for example, a volume of 1 ml, 3 ml, 5 ml, 20 ml, etc. syringe sterile pack, provide instructions for use and labels, which indicate the contents of the package, the date of manufacture.

The priority scope of the proposed heterogeneous biopolymer hydrogel is used for cultivation of stem cells and their subsequent differentiation into neural cells, cardiomyocytes, endothelial and epithelial cells to create biosyst is the R coatings for artificial heart blood vessels and skin, for cell therapy of diseases of the cardiovascular system.

The invention is illustrated in the following laboratory test matrix.

Trials of a new drug, provisionally designated as Spherogel-e, was conducted under conditions of acute experience in laboratory animals for the purposes of recovery of spinal cord injury.

EXAMPLE 4. For experimental spinal cord injury were used adult outbred rats-females weighing not less than 300 GRS. In accordance with the objectives of the study in rats was developed technique of surgical injury to the spinal cord in rats (acute model of spinal cord injury) by deleting the section of the spinal cord after dissection at the level of 10 vertebrae of the thoracic spine, S. Woerly et al. [3, 4].

The day before surgery, the rats were administered prophylactic dose of antibiotic enroflon 5% solution of enrofloxacin, LLC VIC animal health”, Russian Federation), 0.1 ml) and menadione 0.02 ml intramuscularly (I/m) once. Anesthesia was carried out in a/m the introduction of 1 ml per animal of a mixture consisting of 2% solution of xylazine - of 0.18 ml +10% of a solution of ketamine (Ketamine, alfasan, Woerden-Holland) - 0,36 ml of 0.5% solution of novocaine - 0,46 Jr. In the two experimental groups after complete hemostasis defect of the spinal cord was filled with drug Spherogel-e or SFU is ogel-e + embryonic stem cells (neurons and glial cells). The implant Spherogel-e formed so that it matches the size and shape of the damaged area of the spinal cord and fills the cavity of the wound, ensuring full contact of the polymer surface with the surfaces of the transected spinal cord. The top area of the injection and the adjacent areas of the spinal cord isolated from the surrounding tissue subcutaneous adipose tissue (Agrocomplex), as in the control. In the third (experimental) group Spherogel-e was located in a channel formed from a polymer spinal shell (CMD).

Of all operated rats 20 control animals (group I) were transplanted drug Spherogel-e in the area of spinal cord injury. In 25 cases (group II) Spherogel-e insulated from the surrounding tissue subcutaneous adipose tissue, 12 rats after spinal cord injury has created a channel made of polymer spinal meninges, in which were placed Spherogel-E. 8 rats injected Spherogel-e to the stem cells in the channel formed polymer spinal sheath (group III).

During the observations were recorded changes in muscle tone and motor activity of the hind limbs of rats (table 2).

Table 2
GroupHypertone the flexor (DAYS) The reaction of repulsion on tactile stimulation (DAYS)Attempts to use the hind limbs during locomotion (DAYS)Other changes
Control3 of 12 (18, 24, 55)1 of 12 (30)--
Spherogel-e (isolation Agrocomplex)11 of 21 (14-49)*8 of 21 (21-40)4 of 21 (35-54)4 of 21 - weakly trying to rely on the feet when walking; 2 of 21 feet forward deployed, wiggles fingers
Spherogel-e (isolation JI)5 out of 9 (12-54)4 out of 9 (22)1 of 9* (25)1 of 9 - feet forward deployed, with 42 days rat uses its hind legs when walking, trying to scratch their head
Spherogel-e + embryonic stem cells2 of 3 (21)3 of 3 (21 to 41)--

In parentheses table 2 shows the early signs of changes in motor activity of the hind limbs (the “/” Character in some tables replaces the preposition “from” (for readability of the table).

Test results matrix on laboratory animals, the following examples:

EXAMPLE 5. In the control group in 9 of 12 rats (75%) paraplegia hind limbs. 3 of 12 (25%) W the animal identified hypertonicity of the muscles of the hind limbs, namely:

In the cell 13, in rat No. 2 - 55 days after surgery revealed hypertonicity right hind limb

In the cell 31, the rat No. 2 on the 24th day after the operation marked hypertonicity of the left hind limb

In the cell 32, the rat No. 1 - on the 18th day after surgery was observed hypertonicity right hind limb, 40 day rat weakly tries to push off with his paw when tactile stimulation.

EXAMPLE 6. In the experimental group (I) 48% (10 of 21) of cases there was hind limb paraplegia, atony and muscular dystrophy. In 11 of 21 rats (52%) identified changes in muscle tone and motor activity of hind legs, namely:

In the cell 7, the rats No. 1 - 49 day experience was observed hypertonicity of the flexors of both limbs, the reaction of repulsion on tactile stimulation. 54 day foot rear legs forward deployed, rat tries to rely on them.

In the cell 7, the rats No. 2 on the 49th day experience was observed hypertonicity of the flexors of both limbs, the reaction of repulsion on tactile stimulation, which increased slightly in the later periods of observation.

In the cell 9 in rat No. 1 on the 34th day after surgery was observed hypertonicity of the flexor tendons of the hind limbs, weak reaction of repulsion on tactile irritation persisting with further observation.

In the cell 10, in rat No. 2 on day 21 after perceivable hypertonicity of the flexor tendons of the hind limbs, weak reaction of repulsion on tactile stimulation. In subsequent periods is unchanged.

In the cell 14, the rats No. 1 - 30 day experience was marked hypertonicity of the flexors of the left paw. The speakers are not.

In the cell 16, in rat No. 1 - 30 day experience revealed hypertonicity of the flexors of the right paw. The speakers are not.

In the cell 17, in rat No. 1 on the 24th day after the operation, the tone of the muscles of the hind limbs increased, the rat was well off the legs at their tactile stimulation. In subsequent periods (35 day of observation) is a rare weak attempts to rely on the legs when moving.

In the cell 17, in rat No. 2 on the 24th postoperative day was celebrated in a strong repulsive movement of the rear legs when tactile stimulation. 30 day rat tries to use his hind legs when moving.

In the cell 18, in rat No. 1 - 14 days after surgery was noted hypertonicity of the muscles of the hind limbs. On the 20th day the rat was well off the legs at their tactile stimulation. 35 day paws forward deployed, rat weakly tried to rely on them when walking. Wiggles his fingers.

In the cell 19, in rat No. 1 - on the 20th day after the operation marked hypertonicity flexor one paw. 28 day rat actively repelled paws when tactile stimulation.

In the cell 19, in rat No. 2 - on the 20th day after the operation revealed hypertonicity flexor one paw. If further monitoring is NII - involuntary pushing movement of the legs when they are tactile stimulation.

EXAMPLE 7. In group (II) in 2 of 9 rats (22%) paraplegia hind limbs, atony and muscular dystrophy. 7 of 9 rats (78%) were observed changes in muscle tone and motor activity of hind legs:

In the cell 20, in rat No. 1 - 48 day experience increased muscle tone of the hind limbs.

In the cell 22, in rat No. 1 - 12 day experience was observed hypertonicity of muscles, with 27 days - slight pushing motion of the legs at their tactile stimulation.

In the cell 22, in rat No. 2 - 12 day experience was observed hypertonicity of the flexor tendons of the hind limbs, with 25 days - weak repulsive movement of the legs when they are tactile stimulation (right > left). From day 29 response to tactile irritation increased.

In the cell 23, a rat No. 2 at 54 days after surgery the rats slightly increased tone of the muscles of the hind limbs. The speakers are not.

In the cell 24, the rat No. 2 - 40 day experience rat weakly repelled left paw when tactile stimulation. Further speakers are not.

In the cell 24, the rat No. 3 - 22 days after surgery revealed hypertonicity of the flexor tendons of the hind legs. The speakers are not.

In the cell 29, the rat No. 1 - 7 days after surgery was observed hypertonicity of the flexor tendons of the hind legs. On the 22nd day rat repelled paws when they are tactile sec is hagenii (right > left). With 25 days hind legs rotated forward, the rat was trying to rely on them when walking. On the 42nd day after the operation revealed a positive trend: the rat actively moved around the cage, leaning on his hind legs, paws in flattened condition, the pelvic part of the body is significantly elevated, tried to use the hind limb scratching their heads.

EXAMPLE 8. In the third experimental group in all 3 surviving animals after surgery revealed a recovery of tactile sensitivity and the emergence of motor activity of the hind limbs.

In the cell 25, the rat No. 1 on day 21 after surgery, the rat well pushes the hind legs (left > right) during tactile stimulation.

In the cell 26, the rat No. 3 - 40 days after surgery, the rat weakly repelled the right paw with tactile stimulation.

In the cell 28, the rats No. 2 on day 21 after surgery revealed hypertonicity of the rear limbs, weak repulsion of the hind limbs during tactile stimulation, and on day 41 rat actively pushes both arms during their compression.

Thus, the increased tone of the muscles of the hind limbs (weakly expressed) was diagnosed in 25% of the control rats from 18 to 55 days, while hypertonicity was determined in 2 rats only at one extremity, and 1 rat - on both limbs (table 2). 40 day 1 of 3 rats paws began to respond to tactile the e irritation. Both of these characteristics were observed in one rat.

When used for transplantation Spherogel-e, 52% of the rats of the experimental group was detected hypertonicity of the muscles of the hind limbs of varying severity (from 14 to 49 days after surgery). The reaction of repulsion hind limbs on tactile irritation was observed in 38% of the animals from 21 to 40 day experience. It was noted weak sporadic attempts to use the movement of the hind limbs in 4 rats (19%) 35-54 days after surgery. Hypertonicity of the muscles and the reaction of repulsion irritation manifested simultaneously in 7 rats.

When using Spherogel-e in the second experimental group hypertonicity of the muscles of the hind limbs began to register with 12-54 days in 56% of rats, the reaction of repulsion hind limbs on tactile irritation was observed from 22 days in 44% of cases. 25 day 1 rat tried to rely on its hind legs when walking. Upon further observation showed positive dynamics: the soles of the feet forward deployed, the movement of the rat relied on them, the legs are more flattened than in the normal pelvis elevated. On day 42 the rat tried to scratch your head. These data allow us to make an assumption on the partial recovery of nerve conduction. Hypertonicity and the reaction of repulsion irritation was evident in this group of animals simultaneously in 3 rats.

If the COI is the whether as a transplant Spherogel-e to the stem cells on day 21 after surgery was observed muscle hypertonicity in 21 out of 3 rats. With 21-41 day rats reacted with repulsion upon stimulation of the hind limbs. Hypertonicity and the reaction of repulsion irritation manifested simultaneously 2/3 of rats.

EXAMPLE 9. Trophic changes in rats.

All groups (except III experienced) on the background of vitamin therapy in rats was developed hypovitaminosis. Rats became geodinamica dramatically lost weight, was observed loss of hair, down to the bare patches in the area of the shoulders and waist, the coat is dull, unkempt. 1 of 12 rats in the control developed bedsores in the hips (table 3). In rats with applied Spherogel-e (isolated subcutaneous fatty tissue) pressure ulcers developed in 2 of 21 rats, while isolating Spherogel-e polymer spinal sheath - 2/9 rats.

EXAMPLE 11. Function urination

In the control and experimental groups after surgery, laminectomy at the level of T10 vertebra in all animals there was no function reflex voiding the entire period of observation.

EXAMPLE 12. Histological examination.

Spine length 2-2 .5 cm, containing the damaged area of the spinal cord was fixed in 10% neutral buffered formalin. Decalcomania samples was carried out using formic acid, then the samples consisted of paraffin. rodolia slices at the level of the spinal cord 4-5 microns thick were stained haematoxylin-eosin.

EXAMPLE 13. Immunohistochemical study was performed on sections from paraffin blocks of 4-5 microns thick using standard immunohistochemical reactions. Immunohistochemistry was carried out using a protein characteristic of the nervous tissue and involved in the process of regeneration.

1. GAP-43 (neuromodulin) is a neuron-specific protein 43, associated with the growth of neurons. This intracellular phosphoprotein specifically localized in the peripheral and Central nervous system. GAP-43 is the main protein of axonal growth during axonogenesis and regeneration (GAP-43, Sigma, 1:160).

2. GFAP - glial fibrillar acidic protein found in astrocytes and glial cells (GFAP, Sigma 1:80).

3. Neurofilament 200 - one of the representatives of the interim neurofilaments molecular weight of 200 kDa, localized in cells and tissues of neural origin (NF200, Sigma, 1:100).

Paraffin sections were deparaffinization and rehydratable by the standard method. For damascenone" antigens were warm slices on a water bath pre-heated to 95-99°With 10 mm citrate buffer (pH 6.0) for 30 minutes. Then, the slides were cooled at room temperature for 15-20 minutes, and transferred to phosphate buffer for 5 minutes. To block nonspecific binding of antibodies cf the PS were incubated 15 minutes with a 1% solution of bovine serum albumin. Incubation with primary antibodies was carried out at 37°C for 1 hour. After the primary antibody, the slides were washed 2 times for 10 minutes in phosphate buffer. Immunological response was demonstrated using fluorescent staining or indirect, streptavidin-Biotin peroxidase reaction.

Immunofluorescence staining. Incubation with the second antibody labeled with FITC [Sigma], was carried out at 37°C for 30 minutes, and then the sections were washed 2 times for 10 minutes. The sections consisted of 50% glycerol. Evaluation of the results was performed using a fluorescent microscope Opton [Carl Zeiss].

Indirect peroxidase staining. Incubation with the second antibody [LSAB®+kit, DAKO] was carried out at room temperature for 20 minutes, and then the sections were washed 2 times for 5 minutes. Incubation with antibodies labeled with streptavidin, [LSAB®+kit, DAKO] was carried out at room temperature for 20 minutes, and then the sections were washed 3 times for 5 minutes. For visualization of immunohistochemical reactions used the DAB + system [DAKO]. The reaction was carried out in the dark for 5-10 minutes. The slices they finished painting areas with hematoxylin Mayer and concluded in canadian balsam.

Studied 39 samples taken 2 months after surgery. Of these 25 samples were subjected to histological and immunohistochemical evaluation (table 4).

Table 4
GroupThe number of samplesNote
Control5In one case, the area of damage to the spinal cord is located on the border of the sample. Case in the analysis not included.
Spherogel-e (isolation Agrocomplex)15In three cases the area of the injury to the spinal cord is located on the border of the sample. These cases in the analysis not included.
Spherogel-e (isolation JI)5In one case, the area of damage to the spinal cord is located on the border of the sample. Case in the analysis not included.

EXAMPLE 14. Control samples (number - 4).

In the defect spongy bone and tissue of the spinal cord in all cases is determined by the granular fabric of varying degrees of maturity with angiomatosis and accumulation of hemosiderin containing large amounts of hemosiderin. In the center of granular tissue is determined by the multi-cystotomy defect tissue, the walls of which epithelioid cells, macrophages and multinucleated giant cells of the type absorbable foreign body - painting nonspecific chronic granulomatous reaction. In 2 cases it was observed the formation of kislotoobrazutmi cavity similar structure, filled with the th foreign substance.

In the three sample areas the regeneration of nerve tissue underneath cystosonography the defect is insignificant. Immunohistochemical study showed very little staining of cells with antibodies to neuron-specific proteins (GFAP, GAP and neurofilament 200). In the sample cell 32 h under multi-cavity without foreign body defined areas of tissue loose structure with the absence of large neurons and cells of type ganglion. In this area you will find separate components of microglia, represented by a small number of small nerve cells and randomly branched loosely arranged nerve fibers. Cells very weakly stained with antibodies to GFAP and GAP.

EXAMPLE 15. Spherogel-e, isolated Agrocomplex (number 12)

In the area of the injury in 1 case observed the formation of a single chamber kitsopoulos cavity, the walls of which epithelioid cells, macrophages. At the intersection of the spinal cord and the introduction of Spherogel-e is determined by the dense region in comparison with the control drugs in 6 cases. There is a higher number of nerve cells and their processes, as well as the plethora of blood vessels without the output of red blood cells outside the vascular bed. Some medications (7 samples) the micro - cavernous dilated, thin-walled, anachronia. In most of the samples have extensive extravasation and fresh hemorrhage, possibly traumatic nature. The tissue has a large number of hemosiderosis surrounding areas of hemorrhage. Noticed that when using Spherogel-e, isolated Agrocomplex, the diameter of the blood microvessels, their full-blooded and the number of extravasation more than in the control or use of isolation JI.

EXAMPLE 16. Spherogel-e, isolated JI (quantity - 4). In the damaged area on the edge of the spinal cord there are foreign bodies, similar in appearance to the film. The area of introduction of Spherogel-e on the structure of dense, contains a large number of small nerve cells and nerve fibers. The number of cellular elements, the length and number of shoots comparable results with the introduction of Spherogel-e, isolated Agrocomplex. The blood vessels are thin-walled, red-blooded, cavernous dilated vessels are absent.

Immunohistochemistry showed the presence of proteins GFAP, GAP and neurofilament 200 in germination.

In one case observed multi cystotomy defect tissue foreign body, the walls of which epithelioid cells, macrophages and multinucleated giant cells of the type absorbable foreign body. Below the cavity observed in the area of high energy density is ness, similar in structure to the fabric intact spinal cord.

Thus, with the introduction of Spherogel-e in the area of acute injury to the spinal cord in 54% of cases in isolation Agrocomplex and in 75% of cases in the isolation of Spherogel-e JI are seeing the emergence of cellular elements: the individual components of microglia, represented by a small number of small nerve cells and nerve fibers. These cells positively stained with antibodies to neuron-specific proteins such as GAP, GFAP and neurofilament 200.

In addition to neurosurgery declared the matrix can be used in orthopedics; when creating bioisosteres liver and pancreas, as well as to restore other soft organs and tissues when they are damaged.

The creation of new heteroclitus matrix for bioplastics is a significant advancement in the improvement of materials for transplantation and replacement surgery of various tissues and organs in a living organism.

LITERATURE

1. Await and other “Fundamentals of biochemistry”, Ed. by Ovchinnikov, Moscow, Mir, 1981, 786 S.

2. “Inflammation,” a Guide for clinicians, edited by VVV, Vsaugov, Moscow, “Medicine”, 1995, 640 S.

3. Burdenko I.A., and Some other aspects of the pathophysiology of traumatic injury and regeneration of the spinal cord, the Issues of neurosurgery, 2000, No. 2, pp.28-31.

4. 7. S.Woerly, V..Doan, F.Evans-Martin, C.G.Paramore, J.D.Peduzzi, “Spinal cord reconstruction using NeuroGel™ implants and functional recovery after chronic injury”, J. of Neurosciensce Res. 2001. Vol.66, P.1187-1197.

1. Universal heterogeneous collagen matrix for implantation, which represents the elastic mass, obtained from two sources of collagen, one source is the tissue of vertebrate animal of the same class, and the second animal of another class.

2. The matrix according to claim 1, characterized in that the sources of collagen are the animal tissues of mammals and class of birds.

3. The matrix according to claim 1, 2, characterized in that it consists of two phases: solid, in the form of microspheres of collagen tissue of mammals, and liquid from denatured collagen tissue of birds at the ratio of the phases (1-10):(1-10).

4. The matrix according to claims 1 to 3, characterized in that the ratio of solid and liquid phases is 1:1.

5. The matrix according to claim 2 to 4, characterized in that it has a size of 100-300 microns microspheres, and the end products of biodegradation are the CO2and H2O.

6. The matrix according to claims 1 to 5, characterized in that it further comprises physiological components of culture media and supplements that promote growth and differentiation of cells and tissues in an effective amount.

7. The matrix according to claims 1 to 6, characterized in that it further contains antibacterial and/or anti-virus components, as well as Integra is sure its preparations in effective amounts.

8. The matrix according to claims 1 to 7, characterized in that it further comprises embryonic stem cells of the nervous tissue.

9. The method of obtaining generic heterogeneous collagen matrix for implantation, comprising preparing a solution of collagen mammal (RCM) and denatured solution of collagen birds (RCP), use 0,3M acetic acid at a final concentration for RCM of ~0.5-1.5%for RCP ~3,0-5,0%, and then the RCM process γ-irradiation at a dose of 1.0 Mrad and homogenized to obtain microspheres, then washed RCM and RCP with distilled water to a pH not less than 6.0, optionally washed with phosphate buffer solution and mix the washed collagen mammals and collagen birds in the ratio of 1:1 with the receiving matrix.

10. The method according to claim 9, characterized in that the resulting matrix addition bring antibacterial and/or anti-virus components, as well as stimulators of tissue regeneration and antiaggregatory agents.

11. The method according to claim 10, characterized in that the matrix is sterilized γ-irradiation.

12. The method according to claim 11, characterized in that the matrix is sterilized γ-irradiation at a dose of 0.5 Mrad to 1 ml.

13. The method according to claim 9 to 12, characterized in that the resulting matrix additionally make embryonic stem cells of the nervous tissue.



 

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