Cross-linked polysaccharide and protein matrices and methods for production thereof

FIELD: chemistry.

SUBSTANCE: disclosed are versions of a method of producing cross-linked polysaccharides, involving reaction of at least one polysaccharide selected from amino-polysaccharide, amino-functionalised polysaccharide containing one or more amino groups which can be cross-linked by reducing sugar, and combinations thereof, with at least one reducing sugar. The invention also discloses polysaccharides obtained using the disclosed method, a method of producing cross-linked matrices based on polysaccharides and matrices obtained using this method. The obtained matrices may include polysaccharide matrices and composite cross-linked matrices, including polysaccharides cross-linked with proteins and/or polypeptides.

EFFECT: obtained polysaccharides have satisfactory resistance to enzymatic degradation coupled with rheological properties of the preparation for injection, obtained matrices exhibit various physical, chemical and biological properties.

29 cl, 12 dwg, 6 tbl, 11 ex

 

Cross-reference to related applications

In this application claims the priority of provisional patent application U.S. serial number 60/713390, filed September 2, 2005, is incorporated into this description by reference in its entirety.

The scope of the invention

This invention relates primarily to the matrix and drugs on the basis of the transverse cross-linked polysaccharide, and more specifically to a new method of cross-linking amino-polysaccharides and amino-functionalized polysaccharides using reducing sugars and their derivatives as agents of cross-linkage and cross stitched polysaccharide matrices, and drugs obtained using this method.

The level of technology

Characteristics of products based on hyaluronic acid or another amino saccharide depend, on the one hand, from the regulation of their functional longevity in the body-master, and, on the other hand, from the conservation of biological properties of natural hyaluronic acid (or other polysaccharide component. Functional durability or other polysaccharide component depends on their ability to withstand specific enzymatic cleavage hyaluronidase or any other cleave the polysaccharide enzymes is, present in the body of the host. This ability is directly correlated with the number of intramolecular and intermolecular cross-linking in the polymer based ON, or other polysaccharide. Usually the higher the number of cross-links results in a higher resistance to such enzymatic cleavage.

Typical preferred cross linking agents known in the art for cross-linking of polysaccharides and/or derivatives of polysaccharides, and/or artificially functionalized forms of polysaccharides, are bifunctional (or multifunctional) linkers, such as, for example, 1,4-potentialapplications ether, many other synthetic bifunctional cross-linkers and other related non-physiological agents. Such cross linking agents interact with the amino groups or other functional groups of the polysaccharide molecules with the formation of intermolecular cross-linking. However, these hard agents can have a negative impact on the biocompatibility and biological activity of bio-based transverse cross-linked polysaccharide, which may be caused by changes in the conformation of polysaccharide molecules and leaching transversely cross-linking agents. So, polysaccharide products, cross stitched revisionaries what their agents, can show some degree of antigenicity. Moreover, localized inflammatory and more complex systemic reactions, including local swelling, itching, temporary or prolonged erythema, edema, granuloma formation, superficial necrotic urticaria and ugrevidnye damage can be adverse side effects in a small relative number of patients applying for aesthetic purposes existing commercial transversely crosslinked polysaccharide products.

In addition, when the products are prepared for injection in the form of suspensions, gels or emulsions, the use of artificial cross-linkers known in the art, is not always possible to obtain transverse cross-linked products having satisfactory resistance to enzymatic cleavage in combination with the desired rheological properties of the drug for injection.

The invention

Therefore, in accordance with the embodiment of the present invention, a method for receiving a transverse cross-linked polysaccharides. The method involves the interaction of at least one polysaccharide selected from the amino-polysaccharide, and/or amino-functionalized polysaccharide, and/or combinations thereof with at least one regenerating sugar with the formation of the transverse cross-linked polysaccharide.

B is further, in accordance with the embodiment of the invention, at least one polysaccharide selected from a natural amino polysaccharide, a synthetic amino-polysaccharide, amino heteropolysaccharide, amino homopolysaccharide, amino-functionalized polysaccharide and its derivatives and esters and salts, amino-functionalized hyaluronic acid and its derivatives and esters and salts, amino-functionalized hyaluronan and its derivatives and esters and salts, chitosan and its derivatives and esters and salts, heparin and its derivatives and esters and salts, amino-functionalized glycosaminoglycans and their derivatives and esters and salts, and any combinations thereof.

In addition, in accordance with the embodiment of the invention at least one regenerating sugar selected from an aldose, ketose derivative of an aldose derived ketosis, Diaz, TRIZ, tetrose, pentoses, hexose, septate, octose, nansy, dekoze, glycerate, Treaty, erythrose, lyxose, xylose, arabinose, ribose, allose, altrose, glucose, fructose, mannose, gulose, idose, galactose and talose, a reducing monosaccharide, a reducing disaccharide, restorative trisaccharide, reducing oligosaccharide, derivatives of oligosaccharides, producing the forms of monosaccharides, esters of monosaccharides, esters of oligosaccharides, salt, simple sugars, salts oligosaccharides, maltose, lactose, cellobiose, gentiobiose, melibiose, turanose, trehalose, isomaltose, laminaribiose, nanobiosym and kilobyte, glyceraldehyde, sorbose, D-ribose-5-phosphate, glucosamine, and combinations thereof.

In addition, in accordance with the embodiment of the invention, at least one regenerating sugar may be selected from programada form at least one reducing sugar, levogyrate form at least one reducing sugar and mix programada and levogyrate forms at least one reducing sugar.

In addition, in accordance with the embodiment of the invention, the interaction involves the incubation of at least one polysaccharide in the solution containing at least one solvent and at least one regenerating sugar, with the formation of the transverse cross-linked polysaccharide.

In addition, in accordance with the embodiment of the invention, the solution is a buffered solution containing at least one buffer.

In addition, in accordance with the embodiment of the invention, the solvent is an aqueous buffered solvent containing at least one buffer for re is lirovaniya the pH of the solution.

In addition, in accordance with the embodiment of the invention, the solvent is an aqueous solvent containing at least one of an ionisable salt to regulate the ionic strength of the solution.

In addition, in accordance with the embodiment of the invention, the solvent (solvents) includes at least one solvent selected from the group consisting of organic solvent, inorganic solvent, a polar solvent, a nonpolar solvent, a hydrophilic solvent, a hydrophobic solvent, a solvent miscible with water, not mixing with the water solvent, and combinations thereof.

In addition, in accordance with the embodiment of the invention, the solvent includes water and at least one additional solvent selected from the hydrophilic solvent, a polar solvent, a solvent miscible with water, and combinations thereof.

In addition, in accordance with the embodiment of the invention, the solvent is selected from the group consisting of water, phosphate buffered saline, ethanol, 2-propanol, 1-butanol, 1-hexanol, acetone, ethyl acetate, dichloromethane, diethyl ether, hexane, toluene, and combinations thereof.

In addition, in accordance with the embodiment of the invention, the interaction includes the EXT shall implement at least one protein and/or polypeptide, with cross stitched amino group, at least one polysaccharide and at least one regenerating sugar for the formation of composite transverse cross-linked matrix.

In addition, in accordance with the embodiment of the invention, at least one protein and/or polypeptide having transversely stitched amino group selected from collagen, a protein selected from the superfamily of collagen, extracellular matrix proteins, enzymes, structural proteins, isolated from blood proteins, glycoproteins, lipoproteins, natural proteins, synthetic proteins, hormones, growth factors, proteins that stimulate the growth of cartilage, stimulating bone growth proteins, intracellular proteins, extracellular proteins, membrane proteins, elastin, fibrin, fibrinogen, and any combinations thereof.

In addition, in accordance with the embodiment of the invention, the collagen is selected from natural collagen, fibrillar collagen, fibrillar telopeptide collagen containing telopeptide collagen, liofilizirovannogo collagen, collagen obtained from animal sources, collagen, human, mammal collagen, recombinant collagen, pasensyahan collagen, restored collagen, bovine telopeptide collagen, porcine telopeptide collagen, collagen, poluchennogo is from a species of vertebrates, recombinant collagen, genetically engineered or modified collagen, collagen types I, II, III, V, XI, XXIV, fibril-linked collagen types IX, XII, XIV, XVI, XIX, XX, XXI, XXII, and XXVI, of collagen types VIII and X collagens type IV, collagen type VI collagen type VII, collagen types XIII, XVII, XXIII and XXV, collagen type XV and XVIII, the artificially synthesized collagen produced by the genetically modified eukaryotic or prokaryotic cells or genetically modified organisms, purified collagen and the recovered purified collagen particles fibrillar collagen, fibrillar restored telopeptide collagen, collagen isolated from cell culture medium, collagen derived from genetically engineered plants, fragments of collagen, protocollagen and any combinations thereof.

In addition, in accordance with the embodiment of the invention, the interaction includes adding at least one additive to at least one polysaccharide and at least one regenerating sugar for the formation of a transverse cross-linked matrix containing at least one additive.

In addition, in accordance with the embodiment of the invention, at least one additive selected from pharmacological substances, drugs, proteins, polypeptides, anaesthetics is hentov, antibacterial agents, antimicrobial agents, antiviral agents, antifungal agents, antimycotic agents, anti-inflammatory agents, glycoproteins, proteoglycans, glycosaminoglycans, various extracellular matrix components, hormones, growth factors, transforming factors, receptors or receptor complexes, natural polymers, synthetic polymers, DNA, RNA, oligonucleotides, therapeutic agent, morphogenetic proteins, mucoproteins, mucopolysaccharides, matrix proteins, transcription factors, peptides, genetic material for gene therapy, nucleic acids, chemically modified nucleic acids, chimeric constructs, DNA/RNA, DNA or RNA probes, antisense DNA, antisense RNA of the gene of the gene composition comprising natural or artificially synthesized oligonucleotides, plasmid DNA, kosmidou DNA, viral and non-viral vectors needed to stimulate cellular uptake and transcription, chondroitin-4-sulfate, chondroitin-6-sulfate, keratomalacia, dermatosurgery, heparin, heparan sulfate, hyaluronan-enriched lecithin of interstitial proteoglycan, decorin, biglycan, fibromyaliga, lumican, aggrecan, syndecans, beta-glican, versican, tetraglycine, serpitine, fibronectin,fibroplasia, handreaders, fibulins, thrombospondin-5, enzyme, enzyme inhibitor, antibodies and any of their combinations.

In addition, in accordance with the embodiment of the invention, the interaction also includes adding one or more living cells to at least one polysaccharide and at least one regenerating sugar before, during, or after the specified cross-linkage for education transverse cross-linked matrix containing at least one living cell, are included in the matrix.

In addition, in accordance with the embodiment of the invention, living cells are selected from the vertebral chondrocytes, osteoblasts, osteoclasts, stem cells of vertebrates, the embryonic stem cells, stem cells isolated from adult tissue, progenitor cells of vertebrates, vertebrate fibroblasts, cells, genetically engineered for secretion of one or more matrix proteins, glycosaminoglycans, proteoglycans, morphogenetic proteins, growth factors, transcription factors, anti-inflammatory agents, proteins, hormones, peptides, one or more types of living cells, created by genetic engineering for the expression of receptors for one or more molecules selected from the group consisting of proteins, peptides, hormones, glycosaminoglycans, proteoglycans,morphogenetic proteins, growth factors, transcription factors, anti-inflammatory agents, glycoproteins, mucoproteins and mucopolysaccharides, and any combinations thereof.

In addition, in accordance with the embodiment of the invention, the method further includes placing the transverse cross-linked polysaccharide processing selected from drying, freeze-drying, dehydration, drying at the critical point, forming, sterilization, homogenization, handling manual shift, irradiation with ionizing radiation, irradiation of electromagnetic radiation, mixing with a pharmaceutically acceptable carrier, saturation additive, and combinations thereof.

Proposed, in accordance with the embodiment of the invention, a method of obtaining a transverse cross-linked polysaccharides. The method includes the stage of interaction of the polysaccharide with one or more reagents to the formation of the derivative forms of the polysaccharide. Derived form contains one or more amino groups and cross-linking of the derived polysaccharide with at least one regenerating sugar with the formation of the transverse cross-linked polysaccharide.

In addition, in accordance with the embodiment of the invention, the amino group selected from primary amino groups and secondary amino groups.

In addition, in accordance with the embodiment of the invention, one or more of the LCO reagents include carbodiimide.

In addition, in accordance with the embodiment of the invention, one or more reagents include carbodiimide in the presence dihydrazide adipic acid.

In addition, in accordance with the embodiment of the invention, the carbodiimide hydrochloride is 1-ethyl-3-(dimethylaminopropyl)carbodiimide.

In addition, in accordance with the embodiment of the invention, at least one regenerating sugar selected from an aldose, ketose, and their combinations.

In addition, in accordance with the embodiment of the invention, at least one regenerating sugar selected from glyceraldehyde, ribose, erythrose, arabinose, sorbose, fructose, glucose, D-ribose-5-phosphate, glucosamine, Diaz, TRIZ, tetrose, pentoses, hexose, septate, octose, nansy, dekoze, glycerate, Treaty, lyxose, xylose, allose, altrose, mannose, gulose, idose, galactose, talose, a reducing monosaccharide, a reducing disaccharide, restorative trisaccharide, reducing oligosaccharide, derivatives of oligosaccharides derived forms simple sugars, esters of monosaccharides, esters of oligosaccharides, salt, simple sugars, salts oligosaccharides, maltose, lactose, cellobiose, gentiobiose, melibiose, turanose, trehalose, isomaltose, laminaribiose, nanobiosym and kilobyte andtheir combinations.

Proposed, in accordance with the embodiment of the invention, a method of obtaining a composite transverse cross-linked matrix. The method includes transverse cross-linking with at least one regenerating sugar at least one polysaccharide selected from the amino-polysaccharide, amino-functionalized polysaccharide and combinations thereof, in the presence of at least one cross link protein to the formation of the composite transverse cross-linked matrix.

And, finally, also proposed, in accordance with a variant embodiment of the invention, the transverse cross-linked polysaccharides and composite matrix comprising polysaccharides and one or more proteins, obtained by the methods described above.

Brief description of drawings

In order to understand the invention and to understand how it can be implemented in practice, some preferred embodiments will be described only in the form of a non-limiting example with reference to the accompanying drawings:

Fig. 1 is a schematic graph showing the UV-visible spectrum of amino-functionalized hyaluronic acid (AFHA), represented by the dotted curve, and represented by a solid curve range of cross stitched DL-glyceraldehyde AFHA received in accordance with the embodiment of the method according to this image the structure.

Fig. 2 is a schematic graph showing the UV-visible spectrum of cross stitched D(-)-ribose AFHA, represented by the dotted curve, cross stitched D(-)-erythropoi AFHA, represented by a solid curve, and cross stitched D(-)-arabinose AFHA, represented by the dashed curve, obtained in accordance with a variant implementation of the method according to this invention.

Fig. 3 is a schematic graph showing the UV-visible spectrum of seamless cross chitosan, represented by a solid curve, cross stitched D(-)-ribose chitosan, represented by the dotted curve, and cross stitched DL-glyceraldehyde chitosan, represented by the dashed curve, obtained in accordance with a variant implementation of the method according to this invention.

Fig. 4 is a schematic graph representing the Fourier Transform infrared spectrum (FTIR) of hyaluronic acid, represented by the dashed curve, AFHA, represented by the dotted curve, and cross stitched DL-glyceraldehyde AFHA, represented by a solid curve in accordance with a variant implementation of the method according to this invention.

Fig. 5-7 are schematic graphs illustrating the measurement results of the rheological properties of six different compositions of polysaccharides on the basis of AFHA, cross stitched at various concentrations of DL-glyceraldehyde within various who's periods of time in accordance with a variant implementation of the method according to this invention, in comparison with rheological properties of some commercially available matrices based on hyaluronic acid.

Fig. 8 is a schematic graph illustrating the results of measuring the swelling amino-functionalized, cross stitched at various concentrations of DL-glyceraldehyde in accordance with the embodiment of the present invention.

Fig. 9 is a schematic graph illustrating the results of measuring the swelling of chitosan, cross stitched at various concentrations of DL-glyceraldehyde in accordance with the embodiment of the present invention.

Fig. 10 is a schematic graph illustrating the results carbazoles analysis splitting hyaluronidase cross stitched DL-glyceraldehyde amino-functionalized and commercially available Perlane®.

Fig. 11 is a schematic graph illustrating the results carbazoles analysis Fig. 10, where the magnitude of the absorptive capacity for Perlane® were multiplied by ten, to compensate for a 10-fold dilution of the samples Perlane®.

Fig. 12 is a schematic graph illustrating % resistance to splitting of hyaluronidase in vitro typical sample matrix cross stitched D(-)-fructose amino-functionalized and Perlane® as a function of time splitting.

For the detailed description of the invention

Used here is the legend

The following conventions are used in this application.

The termDefinition
álmicroliter
ADHdehydrated adipic acid
AFHAamino-functionalized hyaluronic acid
AGEthe predominant products of glycosylation
CHchitosan
DI waterdeionized water
EDChydrochloride of 1-ethyl-3-(dimethylaminopropyl)carbodiimide
FTIRFourrier Transform Infra-red
GCaliber
HAhyaluronic acid
HzHertz
IRinfrared
Mmolar
MDAmillion daltons
mgmilligrams
mlml
mmmillimolar
Mwmolecular weight
Nnormal
PAPascal
PBSbuffered phosphate saline
rpmrpm

This invention relates to a new method of obtaining new biocompatible matrices based on the transverse cross-linked polysaccharide and preparations having excellent resistance to enzymatic cleavage in vivo and other useful rheological and/or biological properties. The method is based, among other things, cross-linking amino-polysaccharides (such as, but without limitation, chitosan) and/or amino-functionalized polysaccharides (such as, but without limitation, amino-functionalized hyaluronic acid) with reducing sugars such as D(-)-ribose, DL-glyceraldehyde, D(-)-erythrose,D(-)-arabinose and many other types of reducing sugars, known in the art. Also disclosed are examples of such new transverse cross-linked matrix.

This invention also relates to a new method of obtaining composite transverse cross-linked matrices, obtained by cross-linking mixtures of one or more amino-polysaccharides and/or one or more amino-functionalized polysaccharides and/or one or more proteins and/or polypeptides) with one or more reducing sugars (as a cross-linker) to form new composite matrices based on the polysaccharide/protein drugs, which have excellent properties of resistance to enzymatic cleavage in vivo and in vitro, and other useful rheological and/or biological properties.

The terms "polysaccharide" and "polysaccharide" and its related forms are used here to determine any natural and/or artificially obtained (and/or artificially synthesized) polysaccharide or polysaccharides, including any chemically modified forms and/or derivatives of such polysaccharide (polysaccharide) and including, but without limitation, esters and salts of such polysaccharides or their derivatives.

The terms "amino-polysaccharide and amino-polysaccharides and their conjugate forms are used here to define any form of polysaccharide or polysaccharide, is the quiet contain one or more amino groups, can be cross stitched regenerating sugar.

The terms "amino-functionalized polysaccharide and amino - functionalityand polysaccharides and their conjugate forms are used here to determine any polysaccharide which has been chemically modified to attach to it one or more chemical groups, including, among other things, one or more amino groups that can be cross stitched regenerating sugar.

Thus, the methods of cross-linkage described herein may be used, among other things, for the cross-linkage of the natural amino-polysaccharides, synthetic amino-polysaccharides, amino heteropolysaccharides, amino homopolysaccharides, amino-functionalized polysaccharides, hyaluronic acid and its derivatives, hyaluronan and its derivatives, chitosan and its derivatives, heparin and its derivatives, and various combinations thereof. Disclosed methods include cross-stitching any suitable esters and salts of such amino-polysaccharides and amino-functionalized polysaccharides.

Specialists in the chemistry of carbohydrates should be understood that other types containing the amino group of polysaccharides and/or amino-functionalized polysaccharides, which are not described in the specific examples and experiments the Ah here below, can also be cross stitched by the methods disclosed here, with getting a variety of cross stitched products. It should be noted that the cross-linking of such amino-polysaccharides or amino-functionalized polysaccharides with reducing sugars (or derivatives of a reducing sugar) is included in the scope of the methods and products of this invention.

Any suitable regenerating sugar can be used as a cross linking agent in the methods according to this invention. Sugar may be a monosaccharide, a disaccharide, with revitalizing the end, trisaccharides with revitalizing the end, or the like. Suitable sugars include an aldose and a ketose. When the monosaccharide is used as a cross-linker, it can be triazol, tetrazol, pentose, hexoses, heptoses, but monosaccharides with more than seven carbon atoms can also be used. Thus, the number of sugars that can be used in new ways of cross-linking in this invention include glycerate, treasa, erythrose, Lukasa, xylose, arabinose, allose, altose, glucose, mannose, gulose, idose, galactose, fructose, talose or any other diosa, trioza, tetrose, pentose, hexose, septate, octose, Nanase or Dakota and their various suitable derivatives.

In ustanavlivaushee derivatives of these monosaccharides or oligosaccharides, who have active aldehyde or ketogroup, can also be used as cross linking agents in this invention.

The reaction rate cross-linkage may depend on the equilibrium concentration of the aldehyde or catography present in the form of an open ring of the sugar, as it is known in this field. However, there is a possibility to compensate for the slow reaction rate of some specific sugars simple extension of the reaction time, as is well known in this field.

The experiments described here below are non-limiting examples of typical responses such amino-polysaccharides and polysaccharides, synthetically functionalized with amino groups, with selected typical reducing sugars and describes an improved resistance to splitting and rheological properties of the resulting matrices. It should be noted that these experiments are given for example only and are not intended to limit the scope of the claims of this invention. As should be obvious to specialists in this area, polysaccharides, functionalityand polysaccharides, reducing sugar (used as cross-linkers), reaction conditions, composition of the reaction mixture, the reaction temperature, PR is the duration of reaction and chemical, physical, rheological properties and the properties of biological durability of the obtained cross stitched matrices may differ from those specifically described in the experiments disclosed here below.

The term hyaluronic acid (NA) is used in the following text as a generic name to designate and hyaluronic acid as such and its salts or mixtures of salts and, in particular, salts of hyaluronate.

The term amino-functionalized hyaluronic acid is used in the following text as a generic name to refer to hyaluronic acid and its salts or mixtures of salts, which were derivationally so that they contain parts of a molecule with a free amino group. The amino group can be a primary amino groups and/or secondary amino groups. The preferred location for the introduction part of the molecule containing the amino group is carboxyl group of the polysaccharide, but there is the opportunity to enter this part of the molecule containing the amino group, to other sites on the ring (rings) of the saccharide. There is no need to amino-functionalization was full, and some of the carboxyl groups (or other designated derivatization, if used) can be rederivation.

The term amino-functionalized polysaccharide is used in the following text to the to the generic term, denoting any polysaccharide which contains amino groups that can interact with aldehyde or catography transversely cross-linking a reducing sugar. Amino groups may be primary and/or secondary amino groups. The amino group can be placed directly on sacharides ring structure (as in chitosan), but can also be part of a group that covalently associated with one or multiple sites or chemical groups on the sugar rings polysaccharide chain.

Thus, the amino group may be located directly on the sugar ring, as in the case of chitosan (as disclosed in detail hereafter), which does not require functionalliteracy and can be directly cross stitched with regenerating sugar through the amino group of the ring base. It should be noted that polymers based on partially acetylated chitin can also be cross stitched, using the method of cross-linking sugar according to this invention, as can be sewn and any polysaccharide having a free amino group (primary or secondary).

It is noted that in accordance with the results of the experiments disclosed here, adding a cross linking reaction mixture is miscible with the polar water solvent, such as, but without limitation, ethanol, may be significant is but to increase the efficiency of cross-linkage and results in improved resistance to decomposition products of the reaction in comparison with the cross-linking in the presence of a buffered aqueous solution without any the polar solvent.

Although the actual mechanisms of the reaction of cross-linking and chemical nature of the resulting transverse cross-linked polysaccharides currently not fully understood, it is assumed that the reaction can be something like (though not necessarily identical) classical glycosylation reactions, in which the regenerating sugar used for cross-linking of protein molecules on the basis of the reaction of the aldehyde or catography sugars with amino groups of amino acids of proteins as, for example, with the free amino group of a lysine or arginine or other amino acids in the protein chain.

Such transversely protein cross-linking reaction of reducing sugars is well known in this field. It is likely that this reaction is the cross-linkage of the protein occurs at least partially through the rearrangement of Amadori the initial product of the reaction leading to the formation of yellowish or brown glycosylation end products.

Although the authors of the present invention is not fully describe the structure of the transverse cross-linked polysaccharides, obtained in the experiments disclosed in this application, the characteristic absorption peaks in the range 225-235 and 285-355 nanometers obtained transverse cross-linked polysaccharides may be evidence of the presence of products of glycosylation by nature what about the somewhat similar (but not necessarily identical) products glycosylation of proteins and end products of protein glycosylation (AGE).

The materials used in the experiments

Sodium salt of heparin EP (Batch No. 9818030) received from JUK Kraeber GmbH & Co, Hamburg, Germany.

Restylane® (lot # 7349) and Restylane-Perlane (lot number 7064) commercially available from Q-Med AB, Uppsala Sweden. Hylaform® Plus (lot number R0409) commercially available from Genzyme Biosurgery (a division of Genzyme Corporation) through their agent for the sale of INAMED AESTHETICS, Ireland.

The Turrax homogenization was performed using model ULTRA TURRAX® T-25 (main), commercially available from IKA®-WERKE, Germany, unless otherwise stated.

All procedures lyophilization was performed using the model of the dryer FD 8 Freeze, commercially available from Heto Lab Equipment, Denmark. The condenser temperature was -80°C. storage Temperature during pre-freezing was -40°C. storage Temperature for lyophilization was +30°C. the pre-freeze was 8 hours and the time lyophilization was 24 hours. The vacuum during lyophilization was approximately 0.01 bar.

In table 1 below lists commercial sources of materials used in the experiments described in this application.

Table 1
MaterialProviderCat. No.
Hydrochloride, N-ethyl-N'-(3-dimethylaminopropyl)ka is bodiimide SigmaE7750
Dehydrated adipic acidSigmaA0638
Chitosan, the average molecular weightAldrich448877
D(-)-ArabinoseSigmaA3131
D(-)-ErythroseFluka18934
DL-GlyceraldehydeSigmaG5001
Cytochrome C from bovine heartSigmaC2037
The dihydrate salt of D-ribose-5-phosphate disodiumSigma83875
D(-)FructoseFlukaF0127
D(-)RiboseSigmaR7500
D(+)GlucoseSigma49159
D(+)SorboseSigmaS4887
L(-)SorboseSigmaS3695
L(+)FructoseSigma31140
Hydrochloride D(+)glucosamineSigmaG4875
The maltose monohydrateSigmaM5885
Monohydrate, D(+)lactoseSigma61340
1-ButanolFluka19430
1-HexanolFluka52840
2-PropanolAldrich32,047-1
AcetoneFluka00585
The ethyl acetateFluka45770
DichloromethaneSigma-Aldrich443484
Diethyl etherRiedel de Haaen32203
Ethanol (absolute) Merk100983
HexaneFluka52770
TolueneFluka89682

Procedure I amino-functionalization of HA

400 mg HA 150 (commercially available as product No. 2222003, sodium hyaluronate Pharma Grade 150 from NovaMatrix FMC Biopolymer, Oslo, Norway), having a molecular weight in the range of 1.4 to 1.8 MDA, or HA 80 (commercially available as product No. 2222002, sodium hyaluronate Pharma Grade 150 from NovaMatrix FMC Biopolymer, Oslo, Norway), having a molecular weight in the range of 0.62-1,15 MDA, was dissolved in 350 ml of DI water, 7 grams dihydrazide adipic acid (ADH) was added to the mixture. the pH of the resulting solution was brought to 4.75 and the solution was stirred for two hours. 764 mg of the Hydrochloride of 1-ethyl-3-(dimethylaminopropyl)carbodiimide were dissolved in 2.0 ml of DI water was added to the mixture and the pH was again brought to 4.75 at room temperature. The reaction was monitored by change in pH and constantly brought him to 4.75. When it was impossible to detect any change in pH, the reaction mixture was left for an additional hour or overnight. After that, the solution was transferred into a dialysis tube and dialyzed against DI water until, when there is no ADH was not found during dialysis. Subjected to dialysis solution bore is 3.5 liters of 100% ethanol, added 2 grams of NaCl and the mixture was stirred for one hour. In order to separate the precipitated modified HA, the solution was centrifuged for 20 minutes at 7000 rpm and supernatant was removed. The obtained amino-functionalized HA (AFHA) kept at 4°C until use.

It should be noted that in all experiments the cross-linkage using AFHA described below, the amino-functionalized HA obtained by functionalization of HA 80, respectively referred to as AFHA80 hereafter, and amino-functionalized HA obtained by functionalization of 150 HA, respectively referred to as the AFHA I 150 here next. These two materials amino-functionalized HA (AFHA80 and AFHA I 150) was used for all experiments the cross-linkage HA, disclosed here below).

Procedure cross-linking HA

In all experiments described here further, stirring was performed using a rotary mixer vortex™. All centrifugation (unless specifically stated otherwise) was performed using a gas centrifuge RC5C with the rotor SORVALL SS-34, commercially available from SORVALL® Instruments DU PUNT, USA.

Each of the following experiments, described below, was set so that the first number referring to the number of experimental series) follows the slash (/), and then specify the range is actually what's experiments, conducted in series. For example, EXPERIMENTAL SERIES 32/1-3, below, includes the following three experiments: experiment 32/1, experiment 32/2 and experiment 32/3. These designations are respectively used throughout the description.

EXPERIMENTAL SERIES 32/1-3

Approximately 5 mg AFHA80 was dissolved in 1 ml of DI water and added to 5 ml of 100% ethanol and in which for 1 minute, after which the following different amounts of glyceraldehyde was added to the mixture AFHA80 as follows:

a) 2 mg glyceraldehyde dissolved in 100 ál of DI water (Experiment 32/1);

b) 4 mg glyceraldehyde dissolved in 200 ál of DI water (Experiment 32/2);

c) 6 mg glyceraldehyde dissolved in 300 ál of DI water (Experiment 32/3).

The resulting reaction mixture which was within 1 minute and placed in an incubator and rotated for 24 hours at 37°C. Then the solution was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 1 ml of DI water was added to the remaining precipitate. After 30 minutes at room temperature, the mixture was centrifuged again at 6000 rpm for 20 minutes. Received transversely sewn products had the following characteristics:

a) Experiment 32/1): 500 ál solid gel.

b) (Experiment 32/2): Soft opaque gel without phase separation between the crosslinked HA and water (after 3 hours repeated centrifugation received 500 μl of p is ozracing gel).

(C) (Experiment 32/2): 800 ál of gel.

Experiment 33/1

Approximately 25 mg AFHA80 was dissolved in 5 ml of DI water was added to 25 ml of 100% ethanol and which was within 1 minute. A solution of 10 mg of DL-glyceraldehyde dissolved in 500 μl of DI water was added to the mixture, and the resulting mixture which was within 1 minute, placed in an incubator and rotated for 24 hours at 37°C. After 6 hours of rotation in the incubator for an additional 5 mg of glyceraldehyde dissolved in 250 ál of DI water was added to the reaction mixture, and the mixture was returned to the incubator to complete the incubation period. After 24-hour incubation the solution was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 40 ml of DI water and 2 ml of PBS buffer (10 mm) was added to the precipitate and left at room temperature for 6 hours. The mixture was then centrifuged again at 6000 rpm for 20 minutes. The obtained product was 500 µl solid opaque gel.

EXPERIMENTAL SERIES 35/1-4

Approximately 5 mg AFHA80 was dissolved in 1 ml of DI water and added to 12 ml of 100% ethanol and in which for 1 minute, after which the following different amounts of DL-glyceraldehyde were added as follows:

a) 1.5 mg DL-glyceraldehyde dissolved in 75 μl of DI water (Experiment 35/1);

b) 3.0 mg DL-glyceraldehyde dissolved in 150 ál of DI water (Experiment 35/2);

C) 4.5 mg DL, dissolved in 225 μl of DI water (Experiment 35/3);

d) 6.0 mg DL-glyceraldehyde dissolved in 300 ál of DI water (Experiment 35/4).

The resulting reaction mixture which was within 1 minute, placed in an incubator and rotated for 24 hours at 37°C. After 24 hours, the solution was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 5 ml of DI water was added to each draught. After 30 minutes at room temperature the mixture was centrifuged again at 6000 rpm for 20 minutes. Received transversely sewn products had the following characteristics:

a) (Experiment 35/1) 3.4 ml of a transparent gel.

b) (Experiment 35/2) 3.5 ml transparent gel.

(C) (Experiment 35/3) 4,0 ml transparent gel.

d) (Experiment 35/4) 4,0 ml transparent gel.

EXPERIMENTAL SERIES 37/4-6

Approximately 5 mg AFHA80 was dissolved in 1 ml of DI water and added to 10 ml of 100% ethanol. The mixture was which for 1 minute, after which the following different amounts of DL-glyceraldehyde were added to the mixture as follows:

a) 8 mg DL-glyceraldehyde dissolved in 400 μl of DI water (Experiment 37/4);

b) 10 mg of DL-glyceraldehyde dissolved in 500 μl of DI water (Experiment 37/5);

c) 12 mg DL-glyceraldehyde dissolved in 600 μl of DI water (Experiment 37/6).

The resulting reaction mixture which was within 1 minute and placed in an incubator and rotated the 24 hours at 37°C. At the end of the incubation period, the solution was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 5 ml of DI water was added to each draught. After 30 min at room temperature, the mixture was centrifuged again at 6000 rpm for 20 minutes. Received transversely sewn products had the following characteristics:

a) (Experiment 37/4) 2.5 ml transparent gel;

b) (Experiment 37/5) 1.9 ml transparent gel;

c) (Experiment 37/6) 1.5 ml transparent gel.

EXPERIMENTAL SERIES 38/1-3

Approximately 5 mg AFHA80 was dissolved in 1 ml of DI water and added to 10 ml of 100% ethanol and in which for 1 minute, after which the following different amounts of DL-glyceraldehyde were added to the mixture as follows:

a) 14 mg DL-glyceraldehyde dissolved in 700 μl of DI water (Experiment 38/1);

b) 16 mg of DL-glyceraldehyde dissolved in 800 μl of DI water (Experiment 38/2);

c) 18 mg of DL-glyceraldehyde dissolved in 900 ml DI water (Experiment 38/3).

The resulting reaction mixture which was within 1 minute, placed in an incubator and rotated for 24 hours at 37°C. After 24 hours, the solution was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 5 ml of DI water was added to each of rainfall. After 30 minutes at 37°C. the mixture was centrifuged again at 6000 rpm for 20 minutes. Received transversely sewn products the s had the following characteristics:

a) (Experiment 38/1) 1.0 ml transparent gel.

b) (Experiment 38/1) 0.75 ml transparent gel.

(C) (Experiment 38/1) and 0.50 ml of a transparent gel.

EXPERIMENTAL SERIES 41/1-4

Approximately 5 mg AFHA I 150 was dissolved in 5 ml of DI water was added to 10 ml of 100% ethanol and in which for 1 minute, after which the following different amounts of DL-glyceraldehyde were added to the mixture as follows:

a) 6 mg DL-glyceraldehyde dissolved in 300 ál of DI water (Experiment 41/1);

b) 8 mg DL-glyceraldehyde dissolved in 400 μl of DI water (Experiment 41/2);

(C) 10 mg of DL-glyceraldehyde dissolved in 500 μl of DI water (Experiment 41/3);

d) 12 mg DL-glyceraldehyde dissolved in 600 μl of DI water (Experiment 41/4).

The resulting reaction mixture which was within one minute, placed in an incubator and rotated for 24 hours at 37°C. After 24 hours, the solution was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 20 ml of DI water was added to each of rainfall. After 30 minutes at 37°C. the mixture was centrifuged again at 6000 rpm for 20 minutes. No phase separation was not observed in cases a (Experiment 41/1), b (Experiment 41/2) and with (Experiment 41/3); 5 ml of the supernatant could be removed if d (Experiment 41/4). 50 ml of 1 n NaOH Solution was then added to all samples and the samples centrifuged and washed DV is GDI 40 ml of PBS buffer (10 mm, the pH of 7.36). Diluted transverse cross-linked products were centrifuged at 6000 rpm for 20 min after each stage and the supernatant was removed. The results were as follows: sample a (Experiment 41/1), b (Experiment 41/2) and with (Experiment 41/3) no gel was left. In sample d (Experiment 41/4) 5 ml transparent gel was obtained.

EXPERIMENTAL SERIES 42/1-3

Approximately 5 mg AFHA I 150 was dissolved in 5 ml of DI water was added to 40 ml of 100% ethanol and which for 1 min, after which the following different amounts of DL-glyceraldehyde were added to the mixture as follows:

a) 16 mg of DL-glyceraldehyde dissolved in 800 μl of DI water (Experiment 42/1);

b) 20 mg of DL-glyceraldehyde dissolved in 1000 μl of DI water (Experiment 42/2);

c) 40 mg DL-glyceraldehyde dissolved in 2000 ál DI water (Experiment 42/3).

The resulting reaction mixture which was within 1 minute and placed in an incubator and rotated for 24 hours at 37°C. After 24 hours, the solution was centrifuged at 6000 rpm for 20 minutes, supernatant was removed and 40 ml of DI water was added to each of the received precipitation. After 30 minutes at room temperature, each of the mixtures was centrifuged at 6000 rpm for 20 minutes. The obtained transverse cross-linked gels were found increasing the viscosity of the gel from a) after b) before c) (i.e., the gel obtained in the experiment 42/1, it is l the low viscosity of the three, the gel obtained in the experiment 42/3, had a higher viscosity of the three, and obtained in the experiment 42/2 had a viscosity between highest and lowest of the three samples).

EXPERIMENTAL SERIES 44/1-2

Approximately 5 mg AFHA80 was dissolved in 5 ml of DI water was added to 40 ml of 100% ethanol and which for 1 min, after which the following different amounts of various reducing sugars as cross-linkers were added to the mixture as follows:

a) 44 mg of DL-glyceraldehyde dissolved in 2 ml DI water (Experiment 44/1);

b) 44 mg of D(-)-ribose dissolved in 2 ml DI water (Experiment 44/1).

The reaction mixture which was within 1 minute, placed in an incubator and rotated for 24 hours at 37°C (Experiment 44/1), and within eleven (11) days at 37°C (Experiment 44/2). At the end of the incubation period, each of the reaction mixtures were centrifuged at 6000 rpm for 20 min, the supernatant was removed and 40 ml of physiological NaCl solution (0.9 percent) was added to each of the received precipitation. The mixture was left for 30 minutes at room temperature and centrifuged at 6000 rpm for 20 minutes. The results were as follows: (a) (Experiment 44/1) received soft transparent gel, (b) (Experiment 44/2) the obtained gel is not quite white, yellowish hue.

EXPERIMENTAL SERIES 53/1-3

Approximately 50 mg AFA I 150 was dissolved in 2 ml DI water. 100 mg of DL-glyceraldehyde was dissolved in 2 ml DI water. The two solutions were mixed and extrudible five times through a syringe without needle and twice through a needle of 18G. The mixture at the end was extrudible through a needle 18G in the following amounts of ethanol:

a) 20 ml 100% ethanol (Experiment 53/1);

b) 30 ml of 100% ethanol (Experiment 53/2);

C) 40 ml of 100% ethanol (Experiment 53/3).

Each of the resulting reaction mixtures for the cross-linkage was placed in an incubator and rotated for 24 hours at 37°C. after the incubation period, each of the solutions were centrifuged at 6000 rpm for 20 minutes. Supernatant was removed and 20 ml of physiological NaCl solution (0.9 percent) was added to each of the received precipitation. The mixture is then left for three hours at 37°C and centrifuged at 6000 rpm for 20 minutes.

EXPERIMENT 54/1

Approximately 50 mg AFHA I 150 was dissolved in 4 ml of DI water containing 150 mg of DL-glyceraldehyde. The mixture was re extrudible through a needle 22G (six times) and then extrudible through a needle 18G in 40 ml of 100% ethanol were placed in an incubator and rotated for 24 hours at 37°C. After incubation the solution was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 20 ml of physiological NaCl solution (0.9 percent) was added to the precipitate. The resulting mixture was left for two hours at 37°C and the mixture was then centrifuged at 6000 rpm for 20 minutes.

The EXPO IS IMENT 55/1

Approximately 50 mg AFHA I 150 was dissolved in 4 ml of DI water containing 150 mg of DL-glyceraldehyde. The mixture was re extrudible through a needle 22G (six times) and then extrudible through a needle 18G in a mixture of 35 ml of 100% ethanol and 5 ml of DI water. The reaction mixture was placed in an incubator and rotated for 24 hours at 37°C. After an incubation period, the mixture was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 20 ml of physiological NaCl solution (0.9 percent) was added to the precipitate. After two hours at 37°C, the mixture was centrifuged at 6000 rpm for 20 minutes. The obtained white material did not absorb water.

EXPERIMENT 60/1

Approximately 50 mg AFHA I 150 was dissolved in 4 ml of DI water containing 300 mg of DL-glyceraldehyde, and was shaken for 30 minutes at 50°C. the Reaction mixture was then extrudible through a syringe (without needle) in 40 ml of 100% ethanol and the resulting mixture was placed in a water bath and which for six hours at 50°C. the Mixture was then centrifuged at 6000 rpm for 20 min, the supernatant was removed and 40 ml of physiological NaCl solution (0.9 per cent) together with 2 ml of PBS buffer solution (10 mm, pH of 7.36) was added to the precipitate. The mixture was then centrifuged at 6000 rpm for 20 minutes. The obtained reaction product was of 2.8 ml of gel.

EXPERIMENT 61/1

Approximately 50 mg AFHA I 150 was dissolved in 4 ml of DI water containing 300 mg DL-glycerol is degidi, and the mixture was which for 60 minutes at 50°C. the Mixture was then extrudible through a syringe (without needle) in 40 ml of 100% ethanol and the resulting mixture was placed in a water bath and which for five hours at 50°C. After incubation the mixture was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 40 ml of physiological NaCl solution (0.9 per cent) together with 2 ml of PBS buffer solution (10 mm, pH of 7.36) was added to the precipitate. The mixture was then centrifuged at 6000 rpm for 20 minutes.

EXPERIMENT 62/1

Approximately 50 mg AFHA I 150 was dissolved in 4 ml of DI water containing 300 mg of DL-glyceraldehyde, and which was for 10 minutes at room temperature. The mixture is produced through a syringe in 40 ml of 100% ethanol were placed in a water bath and which was within 24 hours at 50°C. Then the solution was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 40 ml of physiological NaCl solution (0.9 per cent) together with 2 ml of PBS buffer solution (10 mm, pH of 7.36) was added to the precipitate. The mixture was then centrifuged at 6000 rpm for 20 minutes. The obtained product was 1 ml opaque gel.

EXPERIMENTAL SERIES 65/3-5

Approximately 50 mg AFHA I 150 was dissolved in 4 ml of DI water containing:

(a) 100 mg of DL-glyceraldehyde (Experiment 65/3);

b) 200 mg of DL-glyceraldehyde (Experiment 65/4);

c) 300 mg DL-glyceraldehyde (Experiment 65/5).

The resulting reaction is haunted mixture was extrudible four times through a needle 2OG and each of the reaction mixtures were extrudible through a syringe without a needle in 40 ml of 100% ethanol and placed in a heating bath and which were within three (3) hours at 50°C and then were placed in an incubator and rotated for sixteen (16) hours at 37°C. Each of the resulting reaction mixtures were then centrifuged at 6000 rpm for 20 minutes, supernatant was removed and 40 ml of physiological NaCl solution (0.9 per cent) together with 2 ml of PBS buffer solution (10 mm, pH of 7.36) was added to each of the received precipitation. The mixtures were then centrifuged again at 6000 rpm for 20 minutes. The resulting reaction products had the following form:

a) (Experiment 65/3) 3.5 ml opaque gel;

b) (Experiment 65/3) 2.5 ml opaque gel;

(C) (Experiment 65/3) 2.0 ml opaque gel.

EXPERIMENTAL SERIES 67/1-2

Approximately 50 mg AFHA I 150 was dissolved in 4 ml of DI water containing 50 mg of DL-glyceraldehyde. The material was mixed in a syringe and was released in 40 ml of 100% ethanol. The mixture was placed in an incubator and rotated within the following periods:

a) 2 days at 37°C. (Experiment 67/1);

b) 3 days at 37°C. (Experiment 67/2).

After an incubation period, the solution was centrifuged at 9000 rpm for 20 minutes, the supernatant was removed and 40 ml of physiological NaCl solution (0.9 per cent) together with 2 ml of PBS buffer solution (10 mm, pH of 7.36) was added to each of rainfall. The mixtures were then centrifuged again at 9000 rpm 5 for 20 minutes. The obtained reaction products were: a) 2 ml opaque gel (Experiment 67/1) and (b) 1.6 ml opaque gel (pilot phase of the UNT 67/2).

EXPERIMENTAL SERIES 67/4-6

Approximately 50 mg AFHA I 150 was dissolved in 4 ml of DI water containing:

a) 300 mg of D(-)-ribose (Experiment 67/4);

b) 300 mg of D(-)-arabinose (Experiment 67/5);

c) approximately 150 mg of D(-)-erythrose (Experiment 67/6).

Three mixtures, each, mixed in syringe extrusion several times through a needle 20G and then each was extrudible through a needle 2OG in 40 ml of 100% ethanol. The mixture was then placed in an incubator and rotated for 15 days at 37°C. After incubation the mixture was centrifuged at 9000 rpm for 20 minutes, the supernatant was removed and 40 ml of physiological NaCl solution (0.9 per cent) together with 2 ml of PBS buffer solution (10 mm, pH of 7.36) was added to the precipitation and the mixture was centrifuged again at 9000 rpm for 20 minutes. The resulting reaction products showed the following properties:

a) (Experiment 67/4) No water absorption - yellowish fibers.

b) (Experiment 67/5) Transparent gel.

(C) (Experiment 67/6) No water absorption - white fibers.

EXPERIMENT 72/1

Approximately 100 mg AFHA I 150 was dissolved in 4 ml of DI water containing 100 mg of DL-glyceraldehyde. The resulting reaction mixture was extrudible four times through a needle 18G and then extrudible twice through a 21G needle. The mixture was divided into two equal portions. Each of the two portions was extrudible through a needle 21G in 40 ml of 100% ethanol. The resulting mixture was placed in an incubator eversale for three days at 37°C. After an incubation period, 5 ml of physiological NaCl solution (0.9 percent) was added to each of the two mixtures and the solution was centrifuged at 9000 rpm for 20 minutes. Supernatant was removed and 40 ml of physiological NaCl solution (0.9 to%)together with 2 ml of PBS buffer solution (10 mm, pH of 7.36) was added to each of the received precipitation. The mixtures were then centrifuged at 9000 rpm for 20 minutes and the resulting precipitates were combined. The experiment was the result of the total (combined) volume of 2.1 ml opaque gel.

EXPERIMENTAL SERIES 75/1,2

100 mg of DL-glyceraldehyde was dissolved in 7 ml of DI water. A suspension of 100 mg AFHA I 150 1.5 ml of 100% ethanol was added to the prepared solution of DL-glyceraldehyde. The resulting mixture was homogenized by extrusion (three times) through a needle 18G and was divided into two equal portions. Each of the two portions was extrudible in 40 ml of 100% ethanol. The mixture was then placed in an incubator and rotated for 3 days at 37°C. Then 5 ml of physiological NaCl solution (0.9 percent) was added to each of the two mixtures and solutions were centrifuged at 9000 rpm for 20 minutes. Supernatant was removed and 40 ml of physiological NaCl solution (0.9 per cent) together with 2 ml of PBS buffer solution (10 mm, pH of 7.36) was added to each of the received precipitation. Two mixtures are then centrifuged again at 9000 rpm for 20 minutes and two sediment together.

EXPERIMENTAL BEHOLD THE OIA 75/3,4

80 mg of DL-glyceraldehyde was dissolved in 7 ml of DI water. A suspension of 100 mg AFHA I 150 in 2 ml of 100% ethanol was added to the prepared solution of DL-glyceraldehyde. The resulting mixture was homogenized by extrusion (three times) through a needle 18G and was divided into two equal portions. Each of the two portions separately was extrudible in 40 ml of 100% ethanol. The mixture was then placed in an incubator and rotated for 3 days at 37°C. Then 5 ml of physiological NaCl solution (0.9 percent) was added to each of the two reaction mixtures and the mixture was centrifuged at 9000 rpm for 20 minutes. Supernatant was removed and 40 ml of physiological NaCl solution (0.9 per cent) together with 2 ml of PBS buffer solution (10 mm, pH of 7.36) was added to each of the received precipitation. Precipitation and then centrifuged again at 9000 rpm for 20 minutes and the resulting precipitates were combined.

EXPERIMENT 77/1

90 mg of DL-glyceraldehyde was dissolved in 14 ml of DI water. A suspension of 100 mg AFHA I 150 in 5 ml of 100% ethanol was added to the prepared solution of DL-glyceraldehyde. The resulting reaction mixture is homogenized by extrusion (three times) through a needle 18G and was divided into two equal portions. Each of the portions was added to 40 ml of 100% ethanol. The resulting mixture was placed in an incubator and rotated for 3 days at 37°C. Then 5 ml of physiological NaCl solution (0.9 percent) was added to each of the mixtures and the mixture was centrifuged at 9000 rpm for 20 minutissima was removed and 40 ml of physiological NaCl solution (0.9 per cent) together with 2 ml of PBS buffer solution (10 mm, the pH of 7.36) was added to each of the received precipitation and mixed. The mixture was centrifuged again at 9000 rpm for 20 minutes and the resulting precipitates were combined.

Procedure cross-linking of chitosan:

The buffer fibrillatory used in the experiments were prepared as follows: 6.5 liters of DI water were placed in a 10-liter glass vessel. 11.3 Grams of NaOH (final concentration of 0.04 M) and 252 grams of Na2HPO4·2H2O (for a final concentration of 0.2 M) was dissolved in DI water. the pH was Brought to 11.2 10 N. NaOH. The volume of solution was added to 7 liters of DI water. The final pH was brought (using NaOH) to pH range 11,20-11,30.

EXPERIMENT 9/1

Approximately 181,5 mg of chitosan was dissolved in 9.6 ml of 0.1 G. of HCl. 14 mg of DL-glyceraldehyde were dissolved in 2.5 ml of DI water and mixed with a solution of chitosan. The mixture was which within 1 minute and 1 ml of buffer fibrillatory and 9.6 ml of 100% ethanol was slowly added to a mixture of chitosan/DL-glyceraldehyde with constant stirring. The reaction mixture was placed in an incubator and rotated for 24 hours at 37°C. At the end of the incubation solution containing 28 mg of DL-glyceraldehyde dissolved in 1.4 ml of DI water was added to the mixture and the resulting mixture was left in an incubator and rotated for an additional 24 hours at 37°C. the Mixture was then centrifuged at 7000 rpm for 15 minutes, supernatant was removed and the resulting OS the dock was washed with 30 ml of 1 N. HCl and then 30 ml of DI water. Each stage of the washing consisted of centrifuging the sample to remove excess liquid. The precipitate had the consistency of a dense gel.

EXPERIMENT 12/1

Six different solutions of glyceraldehyde was prepared as follows:

a) 20 mg of DL-glyceraldehyde 2.5 ml of DI water;

b) 40 mg of DL-glyceraldehyde 2.5 ml of DI water;

C) 60 mg of DL-glyceraldehyde 2.5 ml of DI water;

d) 80 mg of DL-glyceraldehyde 2.5 ml of DI water;

e) 100 mg of DL-glyceraldehyde 2.5 ml of DI water.

Each of the obtained solutions of DL-glyceraldehyde (a-e were separately mixed with a solution prepared by dissolving 196 mg of chitosan in 10 ml of 0.1 G. of HCl. Each of the resulting six reaction mixtures which were within 1 minute. For each of the six mixtures were added to 1 ml of buffer fibrillatory slowly with constant stirring followed by the addition of 15 ml of a mixture of 70% ethanol/DI water (about./about.) also slowly with constant stirring. The reaction mixture was then placed in an incubator and rotated for 24 hours at 37°C. After 24 hours of incubation to the reaction mixture was slowly added 5 ml of PBS buffer and 2.5 ml of buffer fibrillatory, accompanied by agitation. The mixtures were then re-incubated at 37°C. During the second incubation, the mixture was removed from the incubator twice, which was then returned to the incubator (one and two hours the donkey adding PBS and buffer fibrillatory). The mixture was then left in the incubator and rotated. The total time of incubation at 37°C was 48 hours. After incubation, six samples (a-e) was centrifuged at 7000 rpm for 15 minutes. Supernatant was removed and the product washed with 30 ml of 1 N. HCl, and then 30 ml of DI water. Each stage of the washing consisted of centrifuging the sample to remove excess solvent. All five of the samples had a yellowish color (which was stronger than the original yellowish color of unreacted solution of chitosan), probably due to the formation of the product glycosylation. A clear phase separation observed in the gel phase was detected only in samples a and b.

EXPERIMENTAL SERIES 35/1-4

The following four different solution of DL-glyceraldehyde were prepared:

a) 15 mg of DL-glyceraldehyde dissolved in 75 μl of PBS (Experiment 35/1);

b) 30 mg of DL-glyceraldehyde dissolved in 150 μl of PBS (Experiment 35/2);

c) 45 mg DL-glyceraldehyde dissolved in 225 μl of PBS (Experiment 35/3);

d) 60 mg of DL-glyceraldehyde dissolved in 300 μl of PBS (Experiment 35/4).

Each of these four solutions of DL-glyceraldehyde (a-d separately mixed with a solution of approximately 20 mg of chitosan dissolved in 1 ml of HCl 0.1 N., and neutralized (to pH 7.0) with 0.1 G. of HCl. Each of the resulting four reaction the mixtures which were within 1 minute and 12 ml of 100% ethanol was added to each agitated reaction mixture with constant stirring. The resulting reaction mixture was then placed in an incubator and rotated for 24 hours at 37°C. After incubation period, the reaction mixture was centrifuged at 7000 rpm for 15 minutes. Supernatant was removed and the resulting precipitation, each, washed with 10 ml of 1 N. HCl followed by washing with 5 ml of DI water. Each stage of the washing consisted of centrifuging the sample to remove excess solvent. No phase separation between water and chitosan gel was not observed in any of the four samples a-d (experiments 37/1, 37/2, 37/3 and 37/4, respectively).

EXPERIMENTAL SERIES 38/1-6 and 39/1-4

Approximately 20 mg of chitosan was dissolved in 1 ml of 0.1 G. of HCl and neutralized with 0.1 G. of NaCl. Ten different solutions of DL-glyceraldehyde were prepared as follows:

a) 80 mg of glyceraldehyde was dissolved in 400 μl of PBS (Experiment 38/1);

b) 100 mg of DL-glyceraldehyde were dissolved in 500 μl of PBS (Experiment 38/2);

c) 120 mg of DL-glyceraldehyde were dissolved in 600 μl of PBS. (Experiment 38/3);

d) 160 mg DL-glyceraldehyde was dissolved in 800 μl of PBS (Experiment 38/4);

e) 200 mg of DL-glyceraldehyde were dissolved in 1000 μl of PBS (Experiment 38/5).

f) 240 mg of DL-glyceraldehyde were dissolved in 1200 μl of PBS (Experiment 38/6);

g) 300 mg of DL-glyceraldehyde were dissolved in 1500 µl PBS
(Experiment 39/1);

h) 350 mg of DL-glyceraldehyde was dissolved in 1750 μl PBS (e is speriment 39/2);

i) 400 mg of DL-glyceraldehyde were dissolved in 2000 µl PBS (Experiment 39/3);

j) 500 mg of DL-glyceraldehyde were dissolved in 2500 ál PBS (Experiment 39/4).

Each of the solutions of DL-glyceraldehyde (a-j then separately mixed with 1 ml of chitosan solution containing approximately 20 mg of chitosan dissolved in 1 ml of HCl 0.1 N., and neutralized (to pH 7.0), using 0.1 G. of HCl. Each reaction mixture which was within 1 minute and 10 ml of 100% ethanol was added to each of the reaction mixtures with constant stirring. The reaction mixture was then placed in an incubator and rotated for 24 hours at 37°C. After incubation period, the reaction mixture was centrifuged at 7000 rpm for 15 minutes, supernatant was removed and the resulting precipitation, each, washed with 10 ml of 1 N. HCl followed by washing with 5 ml DI water (for experiments 38/1-6, above) or 10 ml of DI water (for experiments 39/1-4, above). Each stage of the washing consisted of centrifuging the sample to remove excess solvent. In the final reaction products of experiments 38/1-6 no phase separation between water and transversely crosslinked chitosan gel was not observed. In the final reaction products 39/1-4 phase separation of the supernatant and chitosan gel was observed after centrifugation. Observed color change of the reaction products of experiments 38/1-6 and 39/14 from not quite white to yellowish, with concomitant reduction in water absorption of the obtained cross crosslinked chitosan gel with increasing concentrations of cross-linker (DL-glyceraldehyde).

EXPERIMENTAL SERIES 40/1-3

In these experiments, the conditions were exactly the same experiment 39, described above, except that only part g, i and j were carried out with the following concentrations of DL-glyceraldehyde:

g) 300 mg of DL-glyceraldehyde were dissolved in 1500 μl of PBS (Experiment 40/1);

i) 400 mg of DL-glyceraldehyde were dissolved in 2000 µl PBS (Experiment 40/2);

j) 500 mg of DL-glyceraldehyde were dissolved in 2500 ál PBS (Experiment 40/3).

Other reaction conditions were carried out exactly as in the EXPERIMENTAL SERIES 39/1-4, which is described above.

The obtained precipitation was used to measure the swelling of the products (results shown in Fig. 9).

EXPERIMENTAL SERIES 44/3-4

a) 56 mg of DL-glyceraldehyde were dissolved in 500 μl of DI water (Experiment 44/3).

b) 56 mg of D(-)-ribose was dissolved in 500 μl of DI water (Experiment 44/4).

Each of solutions a and b, above, were separately mixed with a solution of chitosan, prepared by dissolving approximately 100 mg of chitosan in 5 ml of HCl 0.1 N. and neutralization solution using 0.1 G. of NaCl. Each of the two resulting reaction mixtures which were within 1 minute and was mixed with 40 ml of 100% ethanol. The reaction mixture was placed in an incubator and rotated for 24 hours at 37°C (Experiment 44/3) or for 12 days at 37°C (Experiment 44/4). After completion of the two different who's periods of incubation of the reaction mixture was centrifuged at 7000 rpm for 15 minutes, supernatant was removed and the precipitates were washed with 40 ml of 1 N. HCl followed by washing with 10 ml DI water. Each stage of the washing consisted of centrifuging the sample to remove excess solvent. Both experiments were the result of a soft gel. Obtained in the experiment 44/3 cross stitched DL-glyceraldehyde gel had a more vivid yellowish color than the color of cross stitched D(-)-ribose gel obtained in the experiment 44/4.

Spectroscopic characterization of the transverse cross-linked polysaccharides

Now in relation to Fig. 1-4. In the graphs presented in Fig. 1-3, the vertical axis is given absorption capacity of the sample and the horizontal axis is wavelength in nm. On the graph shown in Fig. 4, the vertical axis is given absorption capacity of the sample and the horizontal axis is given wave number (in units of cm-1).

Fig. 1 is a schematic graph representing the visible UV spectrum of amino-functionalized hyaluronic acid (AFHA) (represented by the dashed curve 10) and cross stitched DL-glyceraldehyde AFHA (represented by a solid curve 20)received in accordance with the embodiment of the method according to this invention. The dotted curve represents the spectrum of a sample of amino-functionalized HA (AFHA I 150 obtained, as disclosed in detail here above), and the solid curve PR is dstanley range of cross stitched DL-glyceraldehyde product, obtained in the experiment 72/1, which is described here above. In contrast to the sample AFHA I 150 sample transverse cross-linked polysaccharide detects a strong absorptive capacity in the range 225-235 nm and 285-355, which may indicate the formation of the products of glycosylation in the reaction of cross-linking.

Fig. 2 is a schematic graph representing the visible UV spectrum of cross stitched D(-)-ribose AFHA (represented by the dashed curve 30), cross stitched D(-)-erythropoi AFHA (represented by a solid curve 32) and cross stitched D(-)-arabinose AFHA (represented by the dotted curve 34)received in accordance with a variant implementation of the method according to this invention. Sample cross stitched D(-)-ribose HA was obtained from a sample experiment 67/1. Sample cross stitched D(-)-erythropoi HA was obtained from a sample experiment 67/6. Sample cross stitched D(-)-arabinose HA was obtained from a sample experiment 67/2.

The shifts of the peaks are probably the result of various reducing sugars, which are used for cross stitching. Each sugar has its own specific chain length and conformation, which has an impact on the final advanced glycosylation products (AGE), formed in the reaction.

Fig. 3 is a schematic graph representing the visible UV spectrum Nessi the CSOs transversely of chitosan (represented by a solid curve 40), cross stitched D(-)-ribose chitosan (represented by the dashed curve 42) and cross stitched DL-glyceraldehyde chitosan (represented by the dotted curve 44), obtained in accordance with a variant implementation of the method according to this invention. Sample unstitched transversely of chitosan (curve 40) was obtained from Aldrich, as indicated in table 1 here above. Sample cross stitched D(-)-ribose chitosan (curve 42) was obtained from a sample experiment 44/4. Sample cross stitched DL-glyceraldehyde chitosan (curve 44) was obtained from a sample experiment 44/3.

In contrast to the sample with unmodified (unstitched cross) two samples of chitosan chitosan, cross stitched D(-)-ribose and DL-glyceraldehyde, discovered an improved absorbent capacity of about 290 nm (possibly indicating a typical absorptive capacity proposed products glycosylation and possibly AGE).

Fig. 4 is a schematic graph representing the infrared spectrum (FTIR) (Fourrier Transform Infra-red spectra) of hyaluronic acid (represented by the dotted curve 46), AFHA, represented by the dashed curve 48, and cross stitched DL-glyceraldehyde AFHA, represented by a solid curve 50, in accordance with a variant implementation of the method according to this invention.

The dotted curve represents the IR spectrum of hyaluronan is howling acid (HA150). Dashed curve illustrates the IR spectrum of the amino-functionalized HA (AFHA I 150). The solid curve represents the spectrum of the sample cross stitched DL-glyceraldehyde HA obtained in the experiment 33/1. The IR spectrum in Fig. 4 shows the range of absorptive capacity carboxyl groups (COO) and the overlap of this absorption in the range 1610-1550 cm-1and 1420-1300 cm-1absorptive capacity amide and amine groups (1650 cm-1and 1560 cm-1, respectively), which may change to a higher or lower wave numbers depending on their environment. In addition, the absorption capacity in the range 1740-1700 cm-1appears after cross-linking. Usually absorption of carboxylic acids, as well as absorbing capacity of amides and amino acids are in the specified wavelength range (1740-1700 cm-1). Because these functional groups present in the final cross sewn ON, it is difficult to distinguish between them. As a rule, significant changes in the absorption spectrum can be observed for the products of the reaction cross-linkage disclosed here that strongly confirms the modification and chemical reaction during procedures amino-functionalization and cross-linking and the formation of the products of glycosylation.

The physical nature of the stick transverse cross-linked polysaccharides

All the characteristics of a transverse cross-linked polysaccharides, obtained in the experiments described here above were obtained using standard methods, on the torsion viscometer model HAAKE RheoStress 600, commercially available from Thermo Electron Corporation GmbH, Germany. For all measurements used the rotor model PP 20 Ti PR with a top plate dimensions model MPC20/S QF. The measurements were carried out at a temperature of 23°C.

All rheological tests were carried out by the method of oscillatory measurements. 400 µl of the test material was placed between two different plate viscometer. A sinusoidal load was applied to all samples at a frequency in the range between 0.01 and 10 Hz. The obtained values of the complex viscosity [η*] are given in table 2 below. Sample results of the rheological measurements are also illustrated in more detail in Fig. 5-7.

Table 2
No. of experiment
or product name
Complex viscosity [η*] in PA
when the generation frequency of 0.01 Hz
53/1248 (extrodianry)
387 (extruded through a needle 20G)
53/2526 (extrodianry)
643 (e is studiowany through a needle 20G)
53/3929 (extrodianry)
483 (extruded through a needle 20G)
54/11094
653 (extruded through a needle 20G)
60/12197
61/11554
62/12765
65/32689
65/41663
65/5623
67/11333 (extrodianry)
2391 (extruded 4 times through a 27G needle)
67/25599 (extrodianry)
7036 (extruded 5 times through a 27G needle)
6727 (when extruded 2 times through a needle 30G)
72/12279
3873 (20 hours at 37°C)
75/18926
75/26606
75/36721
75/44415
77/1227
5226 (24 hours at 37°C)
5436 (48 hours at 37°C)
6863 (20 days at 37°C)
Restylane® lot: 73492216
Perlane® lot: 70642419
Hylaform® lot: R0409614

It should be noted that, when multiple measured values [η*] is presented in the right column of table 2, the first specified value represents the measurement result of the sample of the final reaction product, as taken directly from the final precipitate (or taken directly from a syringe commercial product for samples Restylane®, Perlane® and Hylaform®). Other measured values [η*]specified (within the same row) for the same sample in the same experiment, the results obtained from the same sample, which was further processed or extrusion of the sample through a needle (as described in detail in parentheses after the numeric value), or an additional incubation of the sample for a specific period of time at 37°C (the exact time period of incubation is indicated in parentheses after the numeric value).

With regard to Fig. 5-7, they are schematic graphs illustrating the measurement results of the rheological properties of various compositions of polysaccharide-based AFHA, cross schitov the various concentrations of DL-glyceraldehyde during different periods of time, in comparison with rheological properties of some commercially available matrices based on hyaluronic acid. In Fig. 5-7 vertical axis represents the complex viscosity [η*] in PA, and the horizontal axis represents the frequency in Hz.

In Fig. 5 by dashed circles plotted the experimental data obtained for the transverse cross-linked matrix of experiment 53/3 (which was cross stitched 100 mg of glyceraldehyde for 24 hours at 37°C). The shaded squares represent the experimental data obtained for the transverse cross-linked matrix of experiment 54/1 (which was cross stitched 150 mg DL-glyceraldehyde for 24 hours at 37°C). The shaded triangles represent the experimental data obtained for the transverse cross-linked matrix of experiment 62/1 (which was cross stitched 300 mg of DL-glyceraldehyde for 24 hours at 37°C). Where there's no shading circles represent the experimental data obtained for commercially available Perlane® (lot: 7064) (valid numeric value see table 2).

As you can see in the graph of Fig. 5, the increase in the concentration of DL-glyceraldehyde under similar reaction conditions greatly increases the viscosity of the obtained transverse cross-linked polysaccharide. In addition, the transverse cross-linked polysaccharide obtained in the experiment 62/1 (what oterom 300 mg of DL-glyceraldehyde were used in transverse cross-linking of the mixture), is the magnitude of the complex viscosity is significantly higher than that of commercially available Restylane®-Perlane® - based hyaluronic acid for values of frequencies below 0.1 Hz.

In Fig. 6 the shaded circles represent the experimental data for transverse cross-linked matrix of experiment 67/1 (which was cross stitched 50 mg of DL-glyceraldehyde within 48 hours at 370C). The shaded squares represent the experimental data obtained for the transverse cross-linked matrix of experiment 67/2 (which was cross stitched 50 mg of DL-glyceraldehyde within 72 hours at 37°C). Where there's no shading circles represent the experimental data obtained for commercially available Perlane® (lot: 7064) (valid numeric value see table 2).

As can be seen from Fig. 6, when the incubation time of the reaction cross-linkage AFHA increase from 48 hours to 72 hours, the magnitude of the complex viscosity obtained transverse cross-linked polysaccharide-based ON significantly increased with the increase in the length of the cross-linkage reaction. Moreover, the transverse cross-linked polysaccharide obtained in the experiment 67/2 (which was carried out 72-hour reaction cross-linkage using 50 mg of DL-glyceraldehyde in transverse cross-linking of the mixture)has a magnitude of complex viscosity significantly higher than that of the com the Cesky available Restylane®-Perlane® - based hyaluronic acid for values of frequencies below 0.1 Hz.

In Fig. 7 shaded rhombic symbols represent experimental data obtained for the transverse cross-linked matrix of experiment 77/1 (which was cross stitched 40 mg DL-glyceraldehyde for three days at 37°C). Where there's no shading circles represent the experimental data obtained for commercially available Perlane® (lot: 7064). Where there's no shading squares represent the experimental data obtained for commercially available Restylane® (lot: 7349). Where there's no shading triangles represent the experimental data obtained for commercially available Hylaform® Plus lot number R0409 (valid numeric value see table 2).

As can be seen from Fig. 7, when the magnitude of the complex viscosity for the three commercially available extruding from a syringe gels based on hyaluronic acid and polysaccharide-based cross stitched DL-glyceraldehyde HA of experiment 77/1 compare measured values of complex viscosity consistently and significantly higher for cross stitched material obtained in the experiment 77/1 in the range of frequencies of 0.1-0.01 Hz. For example, at 0.01 Hz complex viscosity cross stitched material obtained in the experiment 77/1, equal to more than double complex viscosity measured for Restylane®-Perlane® lot No. 7064, more than triple komplekteyuschee, measured for Restylane® lot No. 7349, and more than nine times the complex viscosity, measured for Hylaform® Plus lot No. R0409.

The person skilled in the art will appreciate that such an increase in the viscosity values can advantageously be correlated with improvement in firming and strengthening the ability of the filler, which may be especially desirable in the materials used for plastic surgery and cosmetic purposes. Although disclosed here advanced gels have such excellent value viscosity, however, they are still able to be extruded through the needle is so small as needle 3OG.

Tests of resistance to enzymatic cleavage

Tests of resistance to splitting was performed using the splitting of hyaluronidase and analysis method with the use of uronic acid and carbazole, which is described in Carbohydrate Analysis: A Practical Approach, 2nd ed.: M.F. Chaplin and 20 J.F. Kennedy, IRL Press at Oxford University Press, UK, 1994, (ISBN 0-19-963449-1P) pp. 324, which is included in the present description by reference in its entirety for all purposes.

The results of the experiments cleavage hyaluronidase samples from some of the above experiments are given in Fig. 10, below. Two experiments were performed:

The FIRST EXPERIMENT CLEAVAGE

1a) Splitting transversely stitched HA

Five samples of 200 µl of the cross school is the amino-functionalized HA, obtained in the experiment 75/3, each, were mixed with 250 μl of NaCl (0.9 per cent) and 60.8 units hyaluronidase dissolved in 50 μl of DI water. All samples were incubated at 37°C. the Samples were taken sequentially at hourly intervals after the start splitting, homogenized by agitation of the material within 1 minute and centrifuged at 13,000 rpm for 5 min in the same centrifuge Heraeus biofuge pico" cat. No. 75003280 using the rotor Heraeus # 3325B (centrifuge and rotor commercially available from Kendro Laboratory Products, Germany). 250 μl of the obtained supernatants were used to conduct carbazoles analysis.

1b) Splitting Perlane® lot No.7064

Five samples of 200 µl Perlane® (lot No. 7064), each, were mixed with 250 μl of NaCl (0.9 per cent) and 60.8 units hyaluronidase dissolved in 50 μl of DI water, and the samples were incubated at 37°C. the Samples were taken sequentially in 1-hour intervals after the onset of cleavage. The confiscated samples of the homogenized by agitation of the material within 1 minute and centrifuged at 13,000 rpm for 5 min in the same centrifuge Heraeus biofuge pico". 250 μl of the obtained supernatants were used to conduct carbazoles analysis.

According to the procedure carbazoles analysis, absorptive capacity was measured at 525 nm for each sample. Because of too intense a color reaction in the case of Perlane® sample needed is asbait to 1:10, using Borisovna sulfuric acid. Samples after two hours of incubation deteriorated in both cases (experimental product 75/3 and Perlane®) because of too high temperature during carbazole procedures and are therefore not represented in the graph of Fig. 10.

Now, with regard to Fig. 10, it is a schematic graph illustrating the results carbazoles analysis splitting hyaluronidase cross stitched DL-glyceraldehyde amino-functionalized HA and commercially available Perlane®.

The vertical axis of the graph in Fig. 10 represents the absorptive capacity of the tested samples at a wavelength of 525 nm, and the horizontal axis represents the time in hours from the beginning of test cleavage. Given the fact that the samples Perlane® (data are represented by dashed squares in Fig. 10) were diluted ten times before reading absorptive capacity (because of the unexpectedly high absorption capacity of undiluted samples), while the absorptive capacity of other samples of amino-functionalized HA obtained in the experiment 75/3 (data which is represented by dashed circles in Fig. 10), read with samples, such as they were (without dilution), it is obvious that the samples are cross stitched amino-functionalized HA obtained in the experiment 75/3, had significant is considerable higher (at least seven times higher) resistance to splitting hyaluronidase, than the resistance manifested Perlane®.

With regard to Fig. 11, it is a schematic graph illustrating the results carbazoles analysis Fig. 10, in which the magnitude of the absorptive capacity Perlane® (schematically represented by dashed squares in Fig. 11) were multiplied by ten, to compensate for a 10-fold dilution of the test samples Perlane®. The magnitude of the absorption capacity of samples of amino-functionalized HA obtained in the experiment 75/3, schematically represented by dashed circles in Fig. 11. The magnitude of the absorptive capacity of the samples Perlane®, obtained in the experiment 75/3, represented by dashed circles in Fig. 11.

Although it is well known that it is usually impossible to obtain the exact value of absorptive capacity by simple multiplication absorptive capacity obtained for the diluted sample, this was done simply to give a rough idea of the difference between the values of the capacity of absorption cross stitched amino-functionalized HA obtained in the experiment 75/3, and those for Perlane®. Thus, the values shown in Fig. 11, are a very rough approximation only for the purpose of explanation and may not give an accurate representation of the true difference absorbing abilities of the two materials.

The SECOND EXPERIMENT RASS THE CRIME

0,1439 mg cross stitched HA or 0,1507 mg Perlane® (lot No. 7064) separately mixed with 250 μl of NaCl (0.9 per cent) and 60.8 units hyaluronidase dissolved in 50 μl of DI water. Two of the mixture for the cleavage reaction was incubated at 37°C. After 4 hours incubation, two samples homogenized by agitation of the material within 1 minute and centrifuged at 13,000 rpm for 5 min in a centrifuge Heraeus Biofuge pico. Supernatant removed from both samples and the rest (split) the mass of the sample was determined for each subject to splitting samples. 99.5% of the cross stitched amino-functionalized HA obtained in the experiment 75/3, and 9.3% Perlane® (Lot No. 7064), remained in the sediment after centrifugation and removal of supernatant. These results strongly confirm a significantly higher in vitro resistance to splitting hyaluronidase amino-functionalized HA obtained in the experiment 75/3, in comparison with a commercial sample Perlane®.

High resistance to splitting hyaluronidase in vitro cross stitched sugar polysaccharide material obtained according to the disclosed here are methods of cross-linking indicates that the material in this way can exhibit a high resistance to biodegradation in vivo, which is very useful in the matrices that are used as fillers Il the volumetric agents for the increment of tissues in normal and used in aesthetic procedures, treatment, in particular, when this can increase the life expectancy of the implant and may reduce the frequency used in aesthetic procedures, thus reducing the overall cost of treatment and the number and/or frequency of necessary procedures or injections, with the improved comfort for the patient.

Tests swelling sample

Due to the presence of excessive amounts of ethanol during the cross-linkage of the cross stitched sugar amino-functionalized HA appears in its dehydrated form. Compared with its hydrated form, the volume of the dehydrated forms of cross stitched ON a minor. After the above washing procedures (in which the reaction products again hydratious in the final rinse (for example, 5 ml of DI water in experiment 35)) the obtained gel was transferred into a standard tubes for testing and determined the volume of gel (ml).

Now, with regard to Fig. 8-9. Fig. 8 is a schematic graph illustrating the results of measuring the swelling aminobenzoylamino HA, cross stitched using various concentrations of DL-glyceraldehyde in accordance with the embodiment of the present invention.

Schematic bar chart of Fig. 8 shows the results of water absorption (swelling) of the samples p is pepper stitched DL-glyceraldehyde amino-functionalized hyaluronic acid, obtained in experiments 35/2, 35/4, 37/4, 37/5, 37/6, 38/1, 38/2 and 38/3 (represented by columns 52, 54, 56, 58, 60, 62, 64, and 66 of Fig. 8, respectively). The leftmost column 50 of the graph of Fig. 8 represents the result of hydration AFHA80 in number, similar to the number used in the experiments, cross-stitching, but without using DL-glyceraldehyde (this result is a sample of seamless cross AFHA80). For each column of the graph of Fig. 8 quantity (in mg) of DL-glyceraldehyde, used in cross-linking of each sample is indicated on the horizontal axis of the graph. Volume (in ml) of the test sample after hydration (lavage) and centrifugation is represented on the vertical axis as a quantitative display caused by the water swelling of the sample after removal of ethanol from the reaction mixture by washing. The results in Fig. 8 show successive higher swelling of the sample, which is maximum at the sample unstitched transversely AFHA80 consistently and clearly decreases with increasing cross-linker (DL-glyceraldehyde) in the reaction mixture.

Fig. 9 is a schematic graph illustrating the results of measuring the swelling of chitosan, cross stitched at various concentrations of DL-glyceraldehyde in accordance with the embodiment of the present invention. Schematic hundred is bcata chart of Fig. 9 shows the results of water absorption (swelling) of the samples cross stitched DL-glyceraldehyde chitosan obtained in experiments 40/1, 40/2 and 40/3 (represented by the bars 62, 64 and 66, Fig. 9, respectively). The leftmost column 60 of the graph of Fig. 9 represents the result of hydration unstitched transversely of chitosan in number, similar to the number used in the EXPERIMENTAL SERIES 40/1-3 cross-stitching, but without using DL-glyceraldehyde (sample unstitched transversely of chitosan). Quantity (in mg) of DL-glyceraldehyde, used for cross-linking chitosan samples, indicated on the horizontal axis of the graph. Volume (in ml) of the sample after hydration (lavage) and centrifugation is represented on the vertical axis as a quantitative display caused by the water swelling of the sample after removal of ethanol from the reaction mixture by washing. Results Fig. 9 show successive higher swelling of the sample, which is the maximum for the sample unstitched transversely of chitosan and explicitly consistently reduced when the amount of cross-linker (DL-glyceraldehyde) in the reaction mixture increases (from 300 mg 400 mg 500 mg in the examples illustrated in Fig. 9).

Composite matrix, obtained by cross-linking of chitosan and collagen

Fibrillar collagen was received, a description is about in detail in U.S. patent 6682760, included in this description by reference in its entirety for all purposes. Fibrillated collagen was concentrated by centrifugation (4500 rpm).

EXPERIMENT 1

Three different test tubes for testing were prepared so that each contained 140 mg fibrillated collagen added to a mixture of 14.5 ml of 10 mm PBS buffer (pH of 7.36), 5 ml of buffer fibrillatory (prepared as disclosed in detail here above), 35 ml of 100% ethanol and 100 mg of D(-)-ribose dissolved in 500 μl of PBS buffer. The reaction mixture was which.

Three different solution of chitosan (a, b and C were prepared as follows:

a) 13.5 mg of chitosan was dissolved in 2.5 ml HCl 0.1 N.;

b) 27 mg of chitosan was dissolved in 5.0 ml HCl 0.1 N.;

C) 54 mg of chitosan was dissolved in 10.0 ml HCl 0.1 N..

Each of solutions a, b and C dropwise slowly added to one of the mixtures of collagen/D(-)-ribose in test tubes for tests with constant stirring. The reaction mixture which was within 1 minute and rotated in an incubator at 37°C for 12 days. At the end of the incubation period, the mixture was centrifuged for 15 minutes at 5000 rpm All the obtained reaction products had a pasty consistency. With increasing concentrations of chitosan yellowish color products also gradually changed from not quite white to deep yellow. In the case of) conglomerates transverse cross-linked chitosan is found, weeny inside the paste.

EXPERIMENT 2

Three different test tubes for testing were prepared so that each contained 140 mg fibrillated collagen added to a mixture of 17.5 ml of 10 mm PBS buffer, 5 ml of buffer fibrillatory and 17.5 ml of 100% ethanol and 33 mg of DL-glyceraldehyde dissolved in 300 μl of 10 mm PBS buffer. The reaction mixture was which.

Three different solution of chitosan (a, b and C were prepared as follows:

a) 13.5 mg of chitosan was dissolved in 2.5 ml HCl 0.1 N.;

b) 27 mg of chitosan was dissolved in 5.0 ml HCl 0.1 N.;

C) 54 mg of chitosan was dissolved in 10.0 ml HCl 0.1 N..

Each of the chitosan solutions a, b and C, above, is added dropwise slowly added to one of the reaction mixtures collagen/DL-glyceraldehyde in test tubes for tests with constant stirring. After additional stirring for 1 minute, the reaction mixture was rotated in the incubator at 37°C for 24 hours. At the end of the incubation period, the mixture was centrifuged for 15 minutes at 5000 rpm All the obtained reaction products had a pasty consistency. When the concentration of chitosan increased, the yellowish color of the products gradually increased from the not-quite-white to bright yellow. In case c) conglomerates transverse cross-linked chitosan detected within the paste.

EXPERIMENT 7/1

Five different test tubes for testing were prepared so that each contains 108 mg of fibrillarin the frame of collagen, added to a mixture of 9.8 ml of 100% ethanol and 14 mg DL-glyceraldehyde dissolved in 700 μl of buffer fibrillatory, and which. Five mixtures of collagen/DL-glyceraldehyde was rotated for 6 hours in an incubator at 37°C.

The following five chitosan solutions were also prepared:

a) 53 mg of chitosan was dissolved in 3.2 ml Hcl 0.1 N.;

b) to 75.6 mg of chitosan was dissolved in 4.5 ml of Hcl 0.1 N.;

(C) to 107.5 mg of chitosan was dissolved in 6.4 ml of Hcl 0.1 N.;

d) 141 mg of chitosan was dissolved in an 8.4 ml Hcl 0.1 N.;

e) 161,3 mg of chitosan was dissolved in 9.6 ml of Hcl 0.1 N..

Each of solutions a, b, c, d and e was slowly added dropwise in one of the five test tubes for testing, containing a mixture of collagen/DL-glyceraldehyde described here above. After additional stirring for 1 minute, the mixture was again placed in the incubator and rotated for 24 hours at 37°C. After this second incubation period, the mixture was centrifuged for 15 min at 5000 rpm

All received transversely sewn products had a pasty consistency. When increasing concentrations of chitosan yellowish color of the products gradually increased from the not-quite-white to bright yellow. Sample c) were incubated at 50°C for 6 hours in excess of the number 6 N. NaOH to dissolve seamless cross collagen. After three stages washes (in DI water) and centrifugation, the sample was subjected to hydro is studied Hcl and analyzed by the instrument for analysis of amino acids (commercial laboratory), to detect hydroxyproline (representing collagen), covalently linked with chitosan. Hydroxyproline was detected in the samples.

EXPERIMENT 7/2

Three different test tubes for testing were prepared so that each contains 108 mg fibrillated collagen added to a mixture of 9.8 ml of 100% ethanol and 14 mg DL-glyceraldehyde dissolved in 700 μl of buffer fibrillatory, and was which.

The following three of chitosan solution a, b and C were also prepared:

a) 53 mg of chitosan was dissolved in 3.2 ml Hcl 0.1 N.;

b) to 107.5 mg of chitosan was dissolved in 6.4 ml of Hcl 0.1 N.;

C) 161,3 mg of chitosan was dissolved in 9.6 ml of Hcl 0.1 N..

Each of solutions a, b and c was slowly added dropwise into one of three test tubes for testing, containing a mixture of collagen/DL-glyceraldehyde, with constant stirring. After additional stirring for 1 minute the mixture was rotated for 24 hours in an incubator at 37°C. After incubation period, the mixture was centrifuged for 15 min at 5000 rpm All the obtained reaction products had a pasty consistency. With increasing concentrations of chitosan yellowish color of the products gradually increased from the not-quite-white to bright yellow.

Amino-functionalization of heparin

500 mg of sodium salt of heparin EP (Batch No. 9818030) was dissolved in 300 ml DI water. 3.0 g Dihydrazide adipic key is lots (ADH) was added to the mixture. the pH of the resulting solution was brought to 4.75 and the solution was stirred until then, until he received the homogeneous solution. 400 mg of the Hydrochloride of 1-ethyl-3-(dimethylaminopropyl)carbodiimide were dissolved in 2.0 ml of DI water was added to the mixture and the pH was again brought to 4.75 at room temperature. The reaction was monitored by monitoring the pH, which is constantly brought to 4.75. The reaction mixture was left overnight under stirring. The solution is then transferred into a dialysis tube and subjected to alternating dialysis against DI water and against a mixture of DI water/ethanol (4:1 vol./about.) until then, when there is no ADH was not found during dialysis. The obtained sodium salt of amino-functionalized heparin EP (heparin-M) was stored at 4°C until use.

Procedure II amino-functionalization

2.4 g 150 HA (sodium hyaluronate Pharma Grade 150, commercially available as product No. 2222003 from NovaMatrix FMC Biopolymer, Oslo, Norway), having a molecular weight in the range of 1.4 to 1.8 MDA, was dissolved in 2.0 l of DI water and to the mixture was added 7.0 g dihydrazide adipic acid (ADH). the pH of the resulting solution brought to a pH of 4.75, and the solution was stirred until then, until he received the homogeneous solution. 760 mg of the Hydrochloride of 1-ethyl-3-(dimethylaminopropyl)carbodiimide were dissolved in 10.0 ml DI water was added to the mixture and the pH was again brought to 4.75 at room temperature. The reaction was monitored by change in pH, which is just the NGOs brought up to a pH of 4.75. When no change in pH was not observed, the reaction mixture was left for an additional hour or overnight. The solution is then transferred into a dialysis tube and subjected to alternating dialysis against DI water and against a mixture of DI water/ethanol (4:1 vol./about.) until then, when there is no ADH was not found during dialysis. Subjected to dialysis the solution was transferred into 3.5 liters of 100% ethanol, 5 g such as NaCl was added and the mixture was stirred for one hour. In order to separate the precipitated modified HA, the solution was centrifuged for 20 minutes at 7000 rpm and supernatant was removed. The obtained amino-functionalized HA (AFHA II) kept at 4°C until use.

Procedure III amino-functionalization

2.5 g 150 HA (sodium hyaluronate Pharma Grade 150, commercially available as product No. 2222003 from NovaMatrix FMC Biopolymer, Oslo, Norway), having a molecular weight in the range of 1.4 to 1.8 MDA, was dissolved in 2.0 l of DI water and to the mixture was added 3.4 g dihydrazide adipic acid (ADH). the pH of the resulting solution brought to a pH of 4.75, and the solution was stirred until then, until he received the homogeneous solution. 400 mg of the Hydrochloride of 1-ethyl-3-(dimethylaminopropyl)carbodiimide were dissolved in 10.0 ml DI water was added to the mixture, and the pH was again brought to 4.75 at room temperature. The reaction was monitored by change in pH, which is constantly brought up to a pH of 4.75. When no change of pH is not what was lodales, the reaction mixture was left for an additional hour or overnight. The solution is then transferred into a dialysis tube and subjected to alternating dialysis against DI water and against a mixture of DI water/ethanol (4:1 vol./about.) until then, when there is no ADH was not found during dialysis. Subjected to dialysis the solution was transferred into 3.5 liters of 100% ethanol, 5 g such as NaCl was added and the mixture was stirred for one hour. In order to separate the precipitated modified HA, the solution was centrifuged for 20 minutes at 7000 rpm and supernatant was removed. The obtained amino-functionalized HA (AFHA III) kept at 4°C until use.

Procedure IV amino-functionalization

2.4 g 150 HA (sodium hyaluronate Pharma Grade 150, commercially available as product No. 2222003 from NovaMatrix FMC Biopolymer, Oslo, Norway), having a molecular weight in the range of 1.4 to 1.8 MDA, was dissolved in 350 ml of DI water, to the mixture was added 5.0 g dihydrazide adipic acid (ADH). the pH of the resulting solution brought to a pH of 4.75, and the solution was stirred until then, until he received the homogeneous solution. 500 mg of the Hydrochloride of 1-ethyl-3-(dimethylaminopropyl)carbodiimide were dissolved in 10.0 ml DI water was added to the mixture, and the pH was again brought to 4.75 at room temperature. The reaction was monitored by change in pH and constantly brought it up to a pH of 4.75. When no change in pH was not observed, the reaction mixture was left to fill in the nutrient hours or overnight. Then the solution was transferred into a dialysis tube and subjected to alternating dialysis against DI water and against a mixture of DI water/ethanol (4:1 vol./about.) until then, when there is no ADH was not found during dialysis. Subjected to dialysis the solution was transferred into 3.5 liters of 100% ethanol, 5 g such as NaCl was added and the mixture was stirred for 1 hour. In order to separate the precipitated modified HA, the solution was centrifuged for 20 minutes at 7000 rpm and supernatant was removed. The obtained amino-functionalized HA (AFHA IV) kept at 4°C until use.

EXPERIMENTAL SERIES 03/105/1-6

Two separate identical suspensions, each of which contains 152 mg AFHA I 150 in 6 ml of 100% ethanol was prepared.

A solution containing 900 mg of D(-)-sorbose dissolved in 9 ml of DI water was added to the first suspension AFHA I 150 by introducing a solution of cross-linker (D(-)-sorbose) under suspension AFHA I 150 and stirring to obtain a homogeneous mixture. The resulting reaction mixture was divided into three equal portions 1, 2 and 3, and each of the received portions were placed in an incubator and rotated at 37°C as follows:

In the experiment 03/105/1 portion 1 were incubated for six (6) days.

In the experiment 03/105/2 portion 2 is incubated for a period of twelve (12) days.

In the experiment 03/105/3 portion 3 were incubated for eighteen (18) days.

A solution containing 900 mg of D(-)-FR is ktoz, dissolved in 9 ml of DI water was added to the second suspension AFHA I 150 by introducing a solution of cross-linker (D(-)-fructose) under suspension AFHA I 150 and stirring to achieve a homogeneous mixture. The resulting reaction mixture was divided into three equal portions 4, 5 and 6 and each of the received portions were placed in an incubator and rotated at 37°C as follows:

In the experiment 03/105/4 portion 4 were incubated for six (6) days.

In the experiment 03/105/5 portion 5 is incubated for a period of twelve (12) days.

In the experiment 03/105/6 portion 6 were incubated for eighteen (18) days.

After incubation, reaction mixtures 1-6 above 40 ml of DI water was added to each of the reaction mixtures 1-6 and the mixture was centrifuged at 9000 rpm for 20 minutes. Supernatant was removed and 40 ml of physiological solution, such as NaCl (0.9 per cent) together with 2 ml of PBS buffer solution (10 mm, pH of 7.36) was added to each of the received precipitation and mixed. The mixture was centrifuged again at 9000 rpm for 20 minutes. The measured values of the complex viscosity of precipitation obtained in experiments 03/105/1, 03/105/1, 03/105/2, 03/105/3, 03/105/4, 03/105/5 and 03/105/6, and a brief description of some of the observed characteristics of the sediment are summarized in table 5 hereafter.

EXPERIMENTAL SERIES 03/114/1-4

Experiment 03/114/1

To prepare a suspension containing 50 mg AFHA I 150 in 2 ml of 100% ethanol. Rast is the PR 50 mg of D(-)-fructose in 1.5 ml DI water was added to the suspension by introducing a solution of cross-linker under suspension AFHA I 150 and which to achieve a homogeneous mixture. The resulting mixture was poured into 40 ml of 100% ethanol. The reaction mixture was placed in an incubator and rotated for two (2) days at 37°C. After incubation to the reaction mixture were added 40 ml of DI water and the mixture was centrifuged at 9000 rpm for 10 minutes.

Experiment 03/114/2

The experiment was carried out as described for experiment 03/114/1 above, except that the reaction mixture was incubated with rotation for four (4) days.

Experiment 03/114/3

The experiment was carried out as described for experiment 03/114/1 above, except that 50 mg of D(-)-sorbose used instead of D(-)-fructose.

Experiment 03/114/4

The experiment was carried out as described for experiment 03/114/3 above, except that the reaction mixture was incubated with rotation for four (4) days instead of two days.

The magnitude of the complex viscosity, determined for the obtained precipitation of experiments 03/114/1, 03/114/2, 03/114/3 and 03/114/4, and a brief description of some of the observed characteristics of the sediment are summarized in table 5 hereafter.

EXPERIMENTAL SERIES 03/140/1-4

Experiment 03/140/1

To prepare a suspension containing 50 mg AFHA I 150 in 1 ml of 100% ethanol. The solution of cross-linker was prepared by dissolving 300 mg of D(-)-ribose in 2.0 ml of DI water. The solution of cross-linker was placed under suspension of AFFA I 150 and the test tube for testing of vesbaltarve and before formation of a homogeneous mixture. The mixture was then poured into 40 ml of 100% ethanol. The resulting reaction mixture was placed in an incubator and rotated for 5 days at 37°C. after the incubation period, 40 ml of DI water was added to the reaction mixture and the resulting mixture was centrifuged at 7000 rpm for 20 minutes. The precipitate was washed with 40 ml of physiological solution, such as NaCl (0.9 per cent), mixed with 2 ml of PBS buffer solution (10 mm, pH of 7.36), and centrifuged at 7000 rpm for 20 minutes. The precipitate is then homogenized by extrusion once through the needle 18G and then once through the needle 21G and maintained in 40 ml of physiological solution, such as NaCl (0.9 percent) for 6 hours, and then centrifuged at 7000 rpm for 30 minutes. The precipitate was filtered using filter paper Whatman® No. 4 (commercially available as cat. room 1004 320 from Whatman, USA), and incubated at 37°C for three days.

Experiment 03/140/2

The experiment was carried out as described for experiment 03/140/1 above, except that the solution of cross-linker consisted of 50 mg of D(+)-sorbose dissolved in 2.0 ml of DI water.

Experiment 03/140/3

The experiment was carried out as described for experiment 03/140/1 above, except that the solution of cross-linker consisted of 50 mg L(+)-fructose dissolved in 2.0 ml of DI water.

Experiment 03/140/4

The experiment was carried out as described for experiment 03/140/1 above, except that the solution of cross-linker included 300 mg of D(+)glucose, dissolved in 2.0 ml of DI water, and sediment were not filtered using filter paper.

The magnitude of the complex viscosity, determined for the obtained precipitation of experiments 03/140/1, 03/140/2, 03/140/3 and 03/140/4, and a brief description of some of the observed characteristics of the sediment are summarized in table 5 hereafter.

EXPERIMENT 03/140/6

To prepare a suspension containing 50 mg AFHA I 150 in 1 ml of 100% ethanol. The solution of cross-linker was prepared by dissolving 300 mg of the dihydrate disodium salt of D-ribose-5-phosphate in 2.0 ml of DI water. Suspension AFHA I 150 was mixed with a solution of cross-linker by introducing a solution of cross-linker under a layer of suspension AFHA I 150 and stirring to achieve a homogeneous mixture. The resulting mixture was poured into 40 ml of 100% ethanol. The reaction mixture was then placed in an incubator and rotated for 5 days at 37°C. At the end of the incubation period to the reaction mixture were added 40 ml of DI water and the resulting mixture was which and centrifuged at 7000 rpm for 5 minutes. The supernatant was removed and the precipitate was washed twice with 40 ml of physiological solution, such as NaCl (0.9 per cent), mixed with 2 ml of PBS buffer solution (10 mm, pH of 7.36), and centrifuged at 7000 rpm for 5 minutes. The magnitude of the complex viscosity, specific for precipitation received, and a brief description of some of the observed characteristics of the sediment are summarized in table 5 hereafter.

The results of the s experiment 03/140/6 demonstrate the use of reducing sugars for cross-linking amino-functionalized polysaccharides is not limited to the use of simple reducing sugars, and that various derivatives of reducing sugars can also be successfully used to obtain cross crosslinked polysaccharide matrix and composite matrix according to this invention.

EXPERIMENTAL SERIES 03/110/1-4

Experiment 03/110/1

Preparing a suspension containing 50 mg AFHA I 150 in 5 ml of 100% ethanol. The suspension was mixed with a solution of cross-linker containing 160 mg of DL-glyceraldehyde dissolved in 8 ml of DI water, by introducing a solution of cross-linker under suspension and stirring to achieve a homogeneous mixture. 6,5 ml of the mixture was poured into 40 ml of 100% ethanol and was which. The resulting reaction mixture was placed in an incubator and rotated for one day at 37°C. after the incubation period, to the mixture was added 40 ml of DI water and 2 ml of PBS buffer solution (10 mm, pH of 7.36) with stirring and the resulting mixture was centrifuged at 9000 rpm for 20 minutes to obtain a precipitate.

Experiment 03/110/2

The experiment was carried out as described for experiment 03/110/1 above, except that 40 ml of 1-hexanol used instead of 40 ml of 100% ethanol.

Experiment 03/110/3

The experiment was carried out as described for the ex is eriment 03/110/1 above, except that 40 ml of 1-butanol was used instead of 40 ml of 100% ethanol.

Experiment 03/110/4

The experiment was carried out as described for experiment 03/110/1 above, except that 40 ml of 2-propanol was used instead of 40 ml of 100% ethanol.

The magnitude of the complex viscosity, determined for the obtained precipitation of experiments 03/110/4, 03/110/4, 03/110/4 and 03/110/4, and a brief description of some of the observed characteristics of the sediment are summarized in table 5 hereafter.

EXPERIMENTAL SERIES 03/131/2-5

Experiment 03/131/2

Preparing a suspension containing 50 mg AFHA I 150 in 2 ml of 100% ethanol. The suspension was mixed with a solution of cross-linker containing 140 mg of DL-glyceraldehyde dissolved in 6 ml of DI water, by introducing a solution of cross-linker under suspension and stirring to achieve a homogeneous mixture. 3.5 ml of the mixture was poured into 40 ml of ethyl acetate and the resulting reaction mixture was placed in an incubator and rotated for one day at 37°C. after the incubation period, to the mixture was added 40 ml of physiological solution, such as NaCl (0.9 per cent) with stirring and the resulting mixture was centrifuged at 7000 rpm for 20 minutes and the supernatant was removed to obtain a precipitate.

Experiment 03/131/3

The experiment was carried out as described for experiment 03/131/2 above, except that 40 ml of acetone (dimethylketone) used the place 40 ml of ethyl acetate.

Experiment 03/131/4

The experiment was carried out as described for experiment 03/131/2 above, except that 40 ml of 1-hexanol used instead of 40 ml of ethyl acetate.

Experiment 03/131/5

Preparing a suspension containing 50 mg AFHA I 150 in 2 ml of 100% ethanol. The suspension was mixed with a solution of cross-linker containing 140 mg of DL-glyceraldehyde dissolved in 6 ml of DI water, by introducing a solution of cross-linker under suspension and stirring to achieve a homogeneous mixture. to 8.0 ml of the mixture was poured into 40 ml of toluene, and the resulting reaction mixture was placed in an incubator and rotated for six (6) days at 37°C. after the incubation period, the toluene was washed by adding 30 ml of 100% ethanol to the reaction mixture, by mixing and centrifugation at 7000 rpm for 20 minutes. Washing with ethanol and centrifugation was repeated two or more times. The precipitate was washed three times with a mixture of 25 ml of DI water and 20 ml of physiological solution, such as NaCl (0.9%) and centrifugation at 7000 rpm for 20 minutes the Final stage of washing was carried out by repeated suspendirovanie sludge in 40 ml of physiological solution, such as NaCl (0.9 per cent), mixed with 2 ml of PBS buffer solution (10 mm, pH of 7.36), and centrifugation at 7000 rpm for 20 minutes. The supernatant was removed and the residue was kept for testing.

The magnitude of the complex viscosity, specific DL is obtained precipitation of experiments 03/1310/2, 03/131/3, 03/131/4 and 03/131/5, and a brief description of some of the observed characteristics of the sediment are summarized in table 5 hereafter.

EXPERIMENT 03/146/2

Preparing a suspension of 100 mg AFHA I 150 in 2 ml of 100% ethanol. 100 mg of DL-glyceraldehyde were dissolved in 40.0 ml of PBS buffer solution (10 mm, pH of 7.36). Suspension AFHA I 150 was mixed with a solution of cross-linker by introducing a solution of cross-linker under suspension AFHA I 150 and stirring to achieve a homogeneous mixture. The resulting mixture was placed in an incubator and rotated for 1 day at 37°C. after the incubation period, the mixture was centrifuged at 10,000 rpm for 15 minutes, supernatant was removed, the residue re-suspended in 40 ml of DI water and the resulting suspension was left for 12 hours at room temperature. The mixture was then centrifuged at 7000 rpm for 10 minutes and the precipitate was washed twice by repeated suspendirovanie in 40 ml of physiological solution, such as NaCl (0.9 per cent), mixed with 2 ml of PBS buffer solution (10 mm, pH of 7.36), mixing and centrifugation at 7000 rpm for 10 minutes. The precipitate was placed in an incubator for 3 days at 37°C. the Magnitude of the complex viscosity, specific to the obtained precipitate, and a brief description of some of the observed characteristics of the sediment are summarized in table 5 hereafter.

EXPERIMENTAL SERIES 05/08/2-4

Experiment 05/08/2

Preparing suspen the Oia, containing 50 mg AFHA I 150 in 2 ml of 100% ethanol. 100 mg of DL-glyceraldehyde was dissolved in 3 ml of DI water. Suspension AFHA I 150 was mixed with a solution of cross-linker by introducing a solution of cross-linker under suspension AFHA I 150 and stirring to achieve a homogeneous mixture. 5.0 ml of the mixture was poured into 40 ml of dichloromethane and the reaction mixture was placed in an incubator and rotated for one (1) day at 37°C. after the incubation period, the material was added 35 ml of PBS buffer solution (10 mm, pH of 7.36) and mixed, and the resulting suspension was centrifuged at 7000 rpm for 15 minutes and supernatant was removed. The precipitate was retained for testing.

Experiment 05/08/3

The experiment was carried out as described for experiment 05/098/2 above, except that 40 ml of hexane was used instead of 40 ml of dichloromethane.

Experiment 05/08/4

Preparing a suspension containing 50 mg AFHA I 150 in 2 ml of 100% ethanol. 100 mg of DL-glyceraldehyde was dissolved in 3 ml of DI water. Suspension AFHA I 150 was mixed with a solution of cross-linker, placing the solution of cross-linker under suspension AFHA I 150 and shaking to achieve a homogeneous mixture. 5.0 ml of the mixture was poured into 40 ml of diethyl ether and the resulting reaction mixture was placed in a water bath and which was for 2 days at 30°C. At the end of the period of incubation in a water bath to the resulting material was added 35 ml buffer the solution of PBS (10 mm, the pH of 7.36) and mixed to suspended material. The resulting suspension was centrifuged at 7000 rpm for 15 minutes and supernatant was removed. The final stage of washing was performed with 40 ml of PBS buffer solution (10 mm, pH of 7.36). The mixture was then centrifuged at 20,000 rpm for 45 minutes and supernatant was removed.

The magnitude of the complex viscosity, specific for precipitation obtained in experiments 05/08/2, 05/08/3 and 05/08/4, and some observed characteristics of precipitation are summarized in table 5 hereafter.

Additional examples of composite matrices, including HA

Pork fibrillar collagen was prepared as described in detail in U.S. patent 6682760.

EXPERIMENT 03/94/2

Preparing a suspension of 80 mg AFHA I 150 in 5 ml of 100% ethanol. The solution of cross-linker consisted of 40 mg of DL-glyceraldehyde dissolved in 2.5 ml of DI water. Suspension AFHA I 150 was mixed with a solution of cross-linker by introducing a solution of cross-linker under suspension AFHA I 150. Addition of 0.4 ml source fibrillated collagen (having a concentration of 35 mg/ml buffer fibrillatory) was added to the mixture and the resulting mixture was which to obtain a homogeneous mixture. The resulting mixture was added 40 ml of 100% ethanol. The resulting reaction mixture was placed in an incubator and rotated for 3 days at 37°C. after the incubation period, the mixture was centrifuged pri rpm for 20 minutes and supernatant was removed. The remaining precipitate was washed with 40 ml of DI water and centrifuged at 20000 rpm for 30 minutes. The precipitate was combined with 25 ml of physiological NaCl solution (0.9% NaCl) and homogenized using a turrax at 24000 rpm for 1 minute. Gomogenizirovannogo the mixture was centrifuged at 9000 rpm for 20 minutes and supernatant was removed. The precipitate was retained for testing. The magnitude of the complex viscosity, specific to the obtained precipitate, and a brief description of some of the observed characteristics of the sludge obtained composite matrix are summarized in table 5 hereafter.

EXPERIMENTAL SERIES 04/37/23-29

Suspension AFHA I 150 100% ethanol was prepared according to table 3. DL-glyceraldehyde was dissolved in 1.0 ml of DI water in the amount indicated in table 3 below. Suspension AFHA I 150 was slowly added with continuous agitation in 2.0 ml of solution fibrillated porcine collagen (having a concentration of 35 mg of collagen per ml of buffer fibrillatory), the total amount of collagen in each experiment is given in table 3. After that, 1 ml of the solution of cross-linker was added to a mixture of collagen/AHFA and the combined mixture is homogenized using a turrax at 24000 rpm for 0.5 minutes to obtain a homogeneous mixture. Gomogenizirovannogo the mixture was added 40 ml of 100% ethanol. The resulting reaction mixture was placed in incubat the p and rotated overnight at 37°C. At the end of the incubation period the supernatant was removed. The material was washed once with 40 ml of DI water and centrifuged at 6000 rpm for 15 minutes and twice with 40 ml of PBS buffer solution (10 mm, pH of 7.36) with centrifugation at 6000 rpm for 15 minutes. The actual quantity of materials used in the preparation of various reaction mixtures for experiments 04/37/23, 04/37/24, 04/37/25, 04/37/26, 04/37/27, 04/37/28 and 04/37/29, and experimentally defined values of the complex viscosity are summarized in table 3 below.

EXPERIMENTS 04/39/30, 04/41/31, 04/44/32, 04/48/34, 04/52/35 and 04/52/36

Prepared suspension AFHA I 150 100% ethanol. The composition of the suspension for each experiment in detail is given in table 3 hereafter. DL-glyceraldehyde (used as cross-linker) was dissolved in 1.0 ml of DI water in the amount indicated in table 3. Suspension AFHA I 150 were mixed with the amount of fibrillated porcine collagen suspended in 2 ml PBS buffer solution (10 mm, pH of 7.36)by slow addition with stirring of a solution of collagen to the solution AHFA. The amount of fibrillated collagen used in each experiment are listed in table 3. The solution of cross-linker (DL-glyceraldehyde) was then added to a mixture of collagen/AHFA with stirring. The resulting reaction mixture for all experiments homogenized using a turrax at 24000 rpm for 05 minutes to obtain a homogeneous mixture. 40 ml of 100% ethanol was added to each of the reaction mixtures. The resulting mixture was placed in an incubator and rotated overnight at 37°C. After incubation period supernatant removed.

Each of the obtained precipitation was washed once with 40 ml of DI water and centrifuged at 6000 rpm for 15 minutes and then washed twice with 40 ml of PBS buffer solution (10 mm, pH of 7.36) and centrifuged at 6000 rpm for 15 minutes. Each of rainfall washed and then mixed with 25 ml of physiological NaCl solution and homogenized using a turrax at 24000 rpm for 0.5 minutes. After homogenization using a turrax each gomogenizirovannogo the mixture was centrifuged at 6000 rpm for 15 minutes and supernatant was removed. The obtained precipitation was experienced. The complex viscosity of each of the obtained precipitation was determined, as disclosed in detail hereafter. The amount of material used to obtain different reaction mixtures, and experimentally defined values of the complex viscosity are summarized in table 3.

Table 3
The number of experimentThe number of AFHA I 150 [mg]The number of ethanol [ml]The number to which lagena [mg] The number of DL-glyceraldehyde [mg]Complex viscosity at 0.01 Hz [PA·s]
04/37/236,00,250,050,01382
04/37/2410,00,450,050,02313
04/37/2518,50,750,050,01403
04/37/2633,31,350,050,03023
04/37/27of 56.42,350,050,02041
04/37/2886,83,550,050,02738
04/37/29112,6 4,550,050,02154
04/44/32160,04,040,0240,03478
04/41/31100,03,011,1100,04744
04/48/34185,31,3the 9.7278,04035
04/52/35100,02,00,0150,06451
04/52/36100,02,042,3150,05417

EXPERIMENT 04/55/1

Preparing a suspension of 150 mg AFHA IV 150 in 2 ml of 100% ethanol. 150 mg of D(-)-fructose was dissolved in 5.0 ml fibrillated porcine collagen (having a concentration of approximately 3 mg of collagen per ml of buffer solution fibrillatory). Suspension AFHA I 150 was mixed with a suspension of F. brilliancy collagen/D(-)-fructose with continuous agitation. The resulting mixture was homogenized using a turrax at 24000 rpm for 0.5 minutes to obtain a homogeneous mixture. Gomogenizirovannogo the mixture was added 35 ml of 100% ethanol and 0.5 ml of acetic acid (10% vol./vol.). The resulting reaction mixture was placed in an incubator and rotated for 6 hours at 37°C. after the incubation period, the supernatant was removed. The remaining residue was mixed with 25 ml of physiological NaCl solution and homogenized using a turrax at 24000 rpm for 0.5 minutes. Gomogenizirovannogo the mixture was centrifuged at 6000 rpm for 15 minutes and supernatant was removed. The precipitate was washed once with 40 ml of DI water and centrifuged at 6000 rpm for 15 minutes and then washed twice with 40 ml of PBS buffer solution (10 mm, pH of 7.36) and centrifuged at 6000 rpm for 15 minutes. Precipitates were incubated for 3 days at 37°C.

The magnitude of the complex viscosity was experimentally determined for the obtained precipitate, and a brief description of some of the observed characteristics of the sediment are summarized in table 5 hereafter.

EXPERIMENT 03/145/2

Preparing a suspension containing 150 mg AFHA I 150 in 2 ml of 100% ethanol. 105 mg of DL-Glyceraldehyde and 5.0 mg of cytochrome C from bovine heart was dissolved in 3.0 ml of DI water. Suspension AFHA I 150 was mixed with a solution of cross-linker and protein by introducing a solution of cross-linker under suspen the s AFHA I 150 and stirring to achieve a homogeneous mixture. Shaken up, the mixture was added to 40 ml of ethanol. The resulting mixture was placed in an incubator and rotated for 2 days at 37°C. At the end of the incubation period, supernatant was removed and the resulting material was washed three times by repeated suspendirovanie in 40 ml of physiological solution, such as NaCl, shaking and centrifugation at 7000 rpm for 10 minutes. The precipitate homogenized sequential extrusion (once through a needle) through the needle 18G, 22G and 25G. After the extrusion, the material was washed with 40 ml of physiological solution, such as NaCl (0.9%) and centrifuged at 7000 rpm for 10 minutes. The magnitude of the complex viscosity, specific to the obtained precipitate, and a brief description of some of the observed characteristics of the sediment are summarized in table 5 hereafter.

The results of the experiment 03/145/2 demonstrate that proteins other than collagen, can be successfully cross stitched with amino-functionalized polysaccharides by glycosylation. They also demonstrate that proteins, which differ significantly from collagen, can be used in the formation of composite cross crosslinked matrix according to this invention.

The formation of such glycosylated matrix protein/amino-polysaccharide can be useful not only for modifying the rheological properties of the obtained composite matrices by using the RA of different proteins, but may be also applicable for pre-emptive administration of biologically active proteins (such as, but without limiting the above, enzymes and growth stimulating or inhibiting growth of proteins, various signaling proteins and peptides, and the like) in the matrix.

EXPERIMENT 03/146/1

Preparing a suspension containing 150 mg AFHA I 150 in 2 ml of 100% ethanol. 105 mg of DL-glyceraldehyde and 3 ml of heparin-M (approximately 40 mg) was dissolved in 3.0 ml of DI water. Suspension AFHA I 150 was mixed with a solution of cross-linker containing heparin, by introducing a solution of cross-linker under suspension AFHA I 150 and stirring to achieve a homogeneous mixture.

The mixture was added 40 ml of 100% ethanol. The resulting reaction mixture was placed in an incubator and rotated for 2 days at 37°C. At the end of the incubation period, supernatant was removed and the resulting material was washed twice by mixing with 40 ml of physiological solution, such as NaCl (0.9 per cent), combined with 2 ml of PBS buffer solution (10 mm, pH of 7.36), stirring and centrifugation at 7000 rpm for 10 minutes. The precipitate homogenized sequential extrusion through a needle 18G, 22G and 25G (once on the needle) and placed in an incubator at 37°C for 3 days. The resulting material was creamy but firm opaque gel.

The magnitude of the complex viscosity, specific for precipitation received, and a brief op is a description of some of the observed characteristics of the sediment are summarized in table 5 hereafter.

The results of the experiment 03/146/1 demonstrate that cross-linking of reducing sugars may be applicable for different mixtures of different amino-polysaccharides and amino-functionalized polysaccharides that contain amino groups that can be cross stitched reducing sugars and their derivatives. Such mixed cross stitched matrix can be useful, because it is possible to control and modify the physical, chemical and biological properties of such mixed membranes by regulating specific types and/or correlation of various polysaccharides crosslinked matrix.

EXPERIMENTAL SERIES 05/02/1-2

Experiment 05/02/1

Preparing a suspension containing 150 mg AFHA II 150 in 2 ml of 100% ethanol. A solution containing 50 mg of DL-glyceraldehyde dissolved in 10 ml DI water was mixed with a solution of AFHA II 150 by introducing a solution of cross-linker under suspension and stirring to achieve a homogeneous mixture. The resulting mixture was poured into 40 ml of 100% ethanol. The resulting reaction mixture was placed in an incubator and rotated for 5 hours at 37°C. after the incubation period, supernatant was removed and the remaining residue was washed with 35 ml of DI water and centrifuged at 7000 rpm for 10 minutes. The precipitate was washed twice with 40 ml of physiological solution, such as NaCl, smeshannoj is with 2 ml of PBS buffer solution (10 mm, the pH of 7.36), re-suspended and centrifuged at 7000 rpm for 10 minutes. The result was about 30 ml soft transparent gel. This gel was washed four (4) times repeated suspendirovanie in 15 ml of 100% ethanol and centrifugation at 10,000 rpm for 30 minutes. The precipitate was transferred into a 35 ml of 100% ethanol was mixed with 0.5 ml of a solution of acetic acid (10% in DI water) and the mixture was placed in an incubator at 37°C and rotated within 24 hours. At the end of incubation, the supernatant was removed. The remaining material was washed in DI water and left at 37°C for one hour. The sample is then centrifuged at 10,000 rpm for 30 minutes. The precipitate was washed with 40 ml of physiological solution, such as NaCl, mixed with 2 ml of PBS buffer solution (10 mm, pH of 7.36), and which was centrifuged at 10,000 rpm for 15 minutes. Sediment homogenized successive punching through a needle 18G, 2OG, 22G, 25G, 27G and 3OG, washed with 40 ml of physiological solution, such as NaCl, mixed with 2 ml of PBS buffer solution (10 mm, pH of 7.36), and which was centrifuged at 10,000 rpm for 5 minutes. The precipitate was transferred into a syringe and incubated for 3 days at 37°C. After incubation, the material was tested to determine the complex viscosity.

Experiment 05/02/2

The experiment was carried out as described for experiment 05/02/1 here above, except h is used on the solution of cross-linker included 100 mg of D(-)-fructose, dissolved in 10 ml of DI water. The first stage of incubation were given 40 ml of soft transparent gel.

The magnitude of the complex viscosity, specific end-precipitation obtained in experiments 05/02/1 and 05/02/2, and a brief description of some of the observed characteristics of the sediment are summarized in table 5 hereafter.

EXPERIMENT 05/15/1

Preparing a suspension of 150 mg AFHA III 150 in 2 ml of 100% ethanol. The solution of cross-linker consisted of 150 mg of D(-)-fructose dissolved in 10 ml DI water.

Suspension AFHA III 150 was mixed with a solution of cross-linker by introducing a solution of cross-linker under suspension AFHA III 150 and stirring to achieve a homogeneous mixture. The resulting mixture was poured into 40 ml of 100% ethanol. The reaction mixture was then placed in an incubator and rotated for 12 hours at 37°C and then for an additional two (2) days at room temperature. At the end of incubation, the supernatant was removed. The remaining material was washed with 40 ml of DI water mixed with 2 ml of PBS buffer solution (10 mm, pH of 7.36), and centrifuged at 8000 rpm for 15 minutes. The precipitate was washed with 30 ml of physiological solution such as NaCl, mixed with 2 ml of PBS buffer solution (10 mm, pH of 7.36), and which was centrifuged at 10,000 rpm for 15 minutes. The sample was filtered using filter paper Whatman® No. 113 (cat. room 1113 320). After filtration of the sample incubated at 37°C during the course the e 3 days and experienced. The experiment gave around 4.0 ml slightly opaque gel. The magnitude of the complex viscosity, specific end-precipitation obtained in the experiment 05/15/1, and a brief description of some of the observed characteristics of the sediment are summarized in table 5 hereafter.

EXPERIMENT 05/18/1

Preparing a suspension of 150 mg AFHA III 150 in 2 ml of 100% ethanol.

As the cross-linker used 150 mg of D(-)-fructose dissolved in 7 ml of DI water. Suspension AFHA III 150 was mixed with a solution of cross-linker by introducing a solution of cross-linker under suspension AFHA III 150 and stirring to achieve a homogeneous mixture. The mixture was poured into 40 ml of 100% ethanol, mixed with 0.5 ml of acetic acid (10% in DI water). The resulting mixture was placed in an incubator and rotated for 12 hours at 37°C and then within two (2) additional days at room temperature. After incubation, the supernatant was removed. The remaining material was washed with 40 ml of DI water mixed with 2 ml of PBS buffer solution (10 mm, pH of 7.36), and centrifuged at 8000 rpm for 15 minutes. The precipitate was washed with 30 ml of physiological solution such as NaCl, mixed with 2 ml of PBS buffer solution (10 mm, pH of 7.36), and which was centrifuged at 10,000 rpm for 15 minutes. The sample was filtered using filter paper Whatman® No. 113, and homogenized by pushing through an 18-gauge needle. After the homogenization treatment is the EC were incubated at 37°C for 3 days. The magnitude of the complex viscosity, specific for precipitation received, and a brief description of some of the observed characteristics of the sediment are summarized in table 5 hereafter.

EXPERIMENTAL SERIES 05/22/1-4

Been prepared by the four suspension (suspension 1-4), each of which contained 75 mg AFHA IV 150 in 2 ml of 100% ethanol. Two different solution of cross-linker was then prepared as follows:

A. 220 mg of D(-)-fructose was dissolved in 7 ml of DI water.

B. 140 mg of D(-)-fructose was dissolved in 7 ml of DI water.

Suspension AFHA IV 150 samples 1 and 3 (from experiments 05/22/1 and 05/22/3, respectively), each, were mixed with 3.5 ml of a solution of cross-linker A, and suspension AFHA IV 150 samples 2 and 4 (from experiments 05/22/2 and 05/22/4, respectively) was mixed with 3.5 ml of a solution of cross-linker B.

Mixing for all four samples was carried out by adding a solution of cross-linker to the suspension AFHA IV 150 with continuous agitation to achieve a homogeneous mixture.

Samples 1 and 2 (experiment 05/22/1 and 05/22/2, respectively) homogenized using a turrax at 24000 rpm for 0.5 minutes. Each of the four mixtures were separately poured into 40 ml of 100% ethanol. Received four reaction mixture was placed in an incubator and rotated for 2 days at 37°C. After incubation supernatant removed. The remaining precipitation, each, washed with 40 ml of physiological NaCl solution, with Sanogo with 2 ml of PBS buffer solution (10 mm, the pH of 7.36), and centrifuged at 3000 rpm (centrifuge: Kubota KS-8000, swinging bucket rotor RS 3000/6, Stainless steel bucket 53592) for 5 minutes. Received four sediment, each, washed with 40 ml of physiological NaCl solution mixed with 2 ml of PBS buffer solution (10 mm, pH of 7.36), which was and centrifuged at 3000 rpm for 15 minutes. Received four sediment incubated at 37°C for 3 days. The magnitude of the complex viscosity, specific for precipitation obtained in experiments 05/22/1, 05/22/2, 05/22/1 and 05/22/2, and a brief description of some of the observed characteristics of the sediment are summarized in table 5 hereafter.

EXPERIMENTAL SERIES 05/23/1,2

Prepared two suspension 75 mg AFHA IV 150 in 2 ml of 100% ethanol. 70 mg of D(-)-fructose was dissolved in 5 ml of DI water.

Each suspension AFHA IV 150 was combined with 2.5 ml of a solution of cross-linker added a solution of cross-linker during the stirring suspension AFHA IV 150 to obtain a homogeneous mixture.

Sample number 2 homogenized using a turrax at 24000 rpm for 0.5 minutes. Each mixture was added 40 ml of ethanol. The mixture was transferred into an incubator and rotated for 2 days at 37°C. Then the supernatant was removed. The remains were washed in 40 ml of physiological NaCl solution with 2 ml of PBS buffer solution (10 mm, pH of 7.36) and centrifuged at 3000 rpm (centrifuge: Kubota KS-8000, swinging bucket rotor RS 3000/6, Stainless steel bucket 53592) during the 5 minutes. The precipitate was washed with 40 ml of physiological NaCl solution with 2 ml of PBS buffer solution (10 mm, pH of 7.36), which was and centrifuged at 3000 rpm for 15 minutes. The samples then were incubated at 37°C for 3 days. The magnitude of the complex viscosity, specific for precipitation received, and a brief description of some of the observed characteristics of the sediment are summarized in table 5 hereafter.

Tests of resistance to enzymatic cleavage

Tests of resistance to splitting was performed using splitting hyaluronidase and method of analysis of uronic acid carbazole, which is described in: Carbohydrate Analysis: A Practical Approach, 2nd ed.: M.F. Chaplin and J.F. Kennedy, IRL Press at Oxford University Press, UK, 1994, (ISBN 0-19-963449-1P) pp.324, which is included in the present description by reference in its entirety for all purposes.

The results of the experiments cleavage hyaluronidase of some materials disclosed above experiments are given in Fig. 10. Conducted two experiments cleavage:

1a) Splitting transversely stitched HA

Five samples of approximately 100 μl cross stitched amino-functionalized HA obtained in the experiment 05/02/02 (having a concentration of 25.6 mg AFHA II 150, cross stitched D(-)-fructose, ml)each, were mixed with 500 μl of PBS buffer solution (10 mm, pH of 7.36) and 10 units of hyaluronidase dissolved in 10 μl of DI water. All treatment the samples were incubated at 37°C. The exact volumes of the samples are given in the second column of table 4A below. The samples were taken from the incubation stage via a serial one-hour intervals after the start splitting, each of the remote sample is homogenized by agitation of the material within one minute and centrifuged at 13,000 rpm for 15 min in a centrifuge Heraeus biofuge pico" (cat. No. 75003280, using rotor Heraeus # 3325B, centrifuge and rotor commercially available from Kendro Laboratory Products, Germany). 25 μl of the Obtained supernatant and 225 μl of PBS buffer solution (10 mm, pH of 7.36) were used to conduct carbazoles analysis. The results of tests of fission cross stitched HA are summarized in table 4A.

1b) Splitting Perlane® lot no.7576 an

Five samples of approximately 100 ál of Perlane® (lot No. 7576)having a concentration of 20 mg/ml each, were mixed with 500 μl of PBS buffer solution (10 mm, pH of 7.36) and 10 units of hyaluronidase dissolved in 10 μl of DI water, and the samples were incubated at 37°C. the Exact amount of samples are given in the second column of table 4B, below. The samples were taken from the incubation stage via a serial one-hour intervals after the onset of cleavage. Each of remote samples homogenized by agitation of the material within one minute and centrifuged at 13,000 rpm for 15 min in the same centrifuge Heraeus biofuge pico". 25 μl of the Obtained supernatant and 225 μl of b is atmospheric PBS solution (10 mm, the pH of 7.36) were used to conduct carbazoles analysis. According to the procedure carbazoles analysis of absorptive capacity was measured at 525 nm for each sample.

With regard to Fig. 12, it is a schematic graph illustrating % resistance to splitting of hyaluronidase in vitro typical sample matrix cross stitched D(-)-fructose amino-functionalized HA of experiment 05/02/02 and commercially available sample Perlane® as a function of time splitting (in hours). The vertical axis of the graph of Fig. 12 represents the resistance to splitting hyaluronidase (number HA, remaining after the specified time of the splitting of the initial amounts of HA at time 0, expressed as a percentage of the original number), and the horizontal axis represents the time splitting in hours. In Fig. 12, the curve 70 represents the results of the splitting for the matrix obtained in the experiment 05/02/02, and the curve labeled 72 presents the results of the splitting for Perlane® (lot No. 7576). From the graph of Fig. 12 you can see that the matrix obtained in the experiment 05/02/02 has a resistance to splitting hyaluronidase, which is far superior to the resistance experienced commercial sample Perlane®.

For example, after 5 hours splitting almost all Perlane® is cleaved, while approximately 68% of the sample matrix, obtained in the experimental is the 05/02/02, remain unsplit. The results of tests of splitting Perlane® are also summarized in table 4B.

Table 4A
Time splitting [clock]Sample volume [ál]Weight of HA in the sample [mg]The number of cleaved HA [mg]Number
residual HA [mg]
Resistance to splitting hyaluronidase [%]
097,52,50,02,5100,0
194,52,40,02,4100,0
2to 102.32,60,22,493,7
3103,42,60,42,285,1
496, 2,50,71,871,2
599,02,50,81,768,3

td align="center"> 3
Table 4B
Time and ful-fill of [clock]Sample volume Perlane [ál]Weight Perlane in the sample [mg]The number of split-tion Perlane [mg]Number
residual Perlane [mg]
Resistance
to ful-fill
of hyaluronidase [%]
0117,52,40,51,979,4
1111,52,21,80,520,5
291,71,81,70,15,0
108,12,22,20,00,0
498,52,01,90,01,8
5108,12,22,30,00,0

___
Table 5
Room
experiment
Complex viscosity [η*] PA
(when the generation frequency of 0.01 Hz)
The form of the obtained material
03/105/14673Thick gel
03/105/28288Yellowish thick gel
03/105/3___Yellowish, solid granules
03/105/4___Dense material
03/105/5Dense material
03/105/6___Dense material
03/114/1as follows 7069Opaque gel
03/114/26436Opaque gel
03/114/3Opaque gel
03/114/46761Opaque gel
03/140/172Transparent gel (1.5 ml)
03/140/23691Yellowish gel (1.5 ml)
03/140/32384Yellowish gel (1.5 ml)
03/140/49Soft gel (3,7 ml)
03/140/63135Soft opaque gel (1.2 ml)
03/110/1___Soft gel (5.5 ml)
03/110/2___Solid yellow plate
03/110/3___Very dense gel (0.5 ml)
03/110/4___Thick gel
03/131/2___Dense yellow gel (0.5 ml)
03/131/334Gel (3.2 ml)
03/131/4___Inhomogeneous yellow gel (0.8 ml)
03/131/5543Gel (4.5 ml)
03/146/2100Soft gel (0.9 ml)
05/08/211Soft gel with opaque particles (5 ml)
05/08/36Soft gel with opaque particles (1 ml)
05/08/424,8Soft gel (10 ml)
03/94/27740/td> Dense gel (0.8 ml)
04/55/11524Homogeneous white solid gel (5 ml)
03/145/220230Red-brown dense gel (3,7 ml)
03/146/117870Opaque dense gel (2.7 ml)
05/02/17810Light yellow opaque gel (4.5 ml)
05/02/211540Light yellow transparent gel (4.5 ml)
05/15/11326Slightly turbid gel (4.5 ml)
05/18/145Soft gel (7.5 ml)
05/22/121Soft gel (6 ml)
05/22/283Soft gel (5 ml)
05/22/355Soft gel (6 ml)
05/22/46 Soft gel (5 ml)
05/23/110Soft gel
05/23/10,2Liquid gel
05/22/121Soft gel (6 ml)
05/22/283Soft gel (5 ml)
05/22/355Soft gel (6 ml)
05/22/46Soft gel (5 ml)
05/23/110Soft gel
05/23/20,2Liquid gel
09/95/12220No white gel
09/95/22098No white gel
09/95/32562No white gel
09/95/44496No white is spruce
09/102/1573No white gel
09/102/2253Opaque gel
09/102/347Opaque gel
09/102/46934No white gel
09/102/52321Opaque gel
09/102/61038Opaque gel

Experiment 05/82/1

150 mg AFHA II 150 was dissolved in 440 ml of DI water and the solution was transferred into a round bottom flask. 10 mg of D(-)-fructose was dissolved in 10 ml of saline solution. The resulting solution was mixed with a solution of AFHA II 150 and the resulting mixture was slowly evaporated while rotating the flask in a vacuum. A concentrated mixture (approximately 2 ml) were incubated for 2 days at 37°C in a light vacuum. At the end of the incubation period, 30 ml of saline solution was added to the contents of the flask was rotated for 1 hour without vacuum. The obtained gel was removed, filtered through filter paper Whatman No. 113) and brought to a final volume of 6 ml by diluting the gel with what the combat solution. The material then was extrudible through needle 16G, 18G, 2OG, 21G and 22G. Each stage of the extrusion process was repeated three times. The obtained particles were yellowish and had a dense texture.

EXPERIMENTAL SERIES 09/95/1-4

Experiment 09/95/1

Preparing an aqueous sample solution AFHA II 150 (1 mg/ml)containing the total number of 200 mg AFHA II 150. 1.2 ml of a Solution fibrillated porcine collagen (16,5 mg/ml) was added to the sample. 100 mg of D(-)-fructose dissolved in 10 ml of saline solution was then added to a mixture of collagen/AFHA II 150. The resulting mixture was stirred for 1 min at 800 rpm turbine stirrer (model R 1312, commercially available from IKA®-Werke GmbH & Co., Germany), was transferred to a tray of stainless steel and liofilizirovanny. After lyophilization, the sample was covered with a mixture of ethanol/DI water (90:10./about.) and incubated at 37°C for 6 hours. After incubation, the material was washed three times with a mixture of ethanol/DI water (90:10./vol.), the solvent was removed by drying the sample and the sample was dried by lyophilization. 2 ml of Saline solution was added to liofilizovannye material and the mixture is incubated for 3 days at 37°C. after the incubation period, the sample was extrudible through a needle 16G, was added 4 ml of saline solution and the material was extrudible again through a needle 18G and 20G.

Experiment 09/95/2

The experiment was carried out as described for the experiment is enta 09/95/1 above, except that the number D(-)-fructose was 130 mg

Experiment 09/95/3

The experiment was carried out as described for experiment 09/95/1 above, except that the amount of D(-)-fructose was 160 mg

Experiment 09/95/4

The experiment was carried out as described for experiment 09/95/1 above, except that the amount of D(-)-fructose was 100 mg and the collagen was not added (this experiment was a control for AFHA II 150, cross stitched without collagen).

EXPERIMENTAL SERIES 09/102/1-6

An aqueous solution AFHA II 150 (at a concentration to 2.85 mg/ml DI water) was used to obtain a sample containing the total amount of 300 mg AFHA II 150. 1.8 ml of a Solution fibrillated collagen (having a concentration of 16.5 mg of collagen per ml of buffer fibrillatory) was added to the samples 2-5 (from experiments 09/102/2-5, respectively). 1.8 ml buffer fibrillatory was added to the sample 1 instead of the solution of collagen (control without collagen - experiment 09/102/1). 5.0 ml of Solution D(-)-fructose in saline solution (having a concentration of 40 mg of D(-)-fructose per ml saline) was added to each of the six samples and the samples were mixed. All the resulting reaction mixture was stirred for 1 minute at 800 rpm turbine stirrer, was transferred to a separate trays made of stainless steel and lyophil zerouali. After lyophilization, the samples 1, 2 and 3 (from experiments 09/102/1, 09/102/2 and 09/102/3, respectively) were covered with a mixture of ethanol/DI water (90:10./about.) and incubated at 37°C for 6 hours. Each of the samples l, 2 and 3 (from experiments 09/102/1, 09/102/2 and 09/102/3, respectively) were washed three times with a mixture of ethanol/DI water (90:10./vol.), the solvent was removed by drying the samples and the samples were dried by lyophilization. Samples 4, 5 and 6 (from experiments 09/102/4, 09/102/5 and 09/102/6, respectively) were not washed.

2 ml of saline solution was added to each of samples 1-6 and all samples were incubated for 3 days at 37°C. After incubation, all samples were extrudible once through the needle 16G. 4 ml of Saline solution was added to each of the extruded samples and each of the samples was consistently extrudible once through the needle 18G and once through a 20G needle. Detailed materials and reaction conditions used in each of the experiments the EXPERIMENTAL SERIES 09/102/1-6, are given in table 6 below.

Table 6
A number of experiments-
Menta
Number
AFHA II
150 [mg]
Volume
added fibrillin-cell
collagen [ml]
Number
D(-)fructose in saline solution (40mg/ml)
Incubation
at 37°C
in the mix
ethanol and saline solution (90:10)
3 flush
in the mix
ethanol and saline solution (90:10)
Incubation at 37°C in 2 ml of saline solution (days)
09/102/11500,0 ml200 mg6 hoursYes3
09/102/21501.8 ml200 mg6 hoursYes3
09/102/31503.6 ml200 mg6 hoursYes3
09/102/41500,0 ml200 mg-no3
09/102/51501.8 ml200 mg- no3
09/102/61503.6 ml200 mg-no3

Some properties of the gels obtained in experiments 09/102/1-6, are given in table 5 above.

EXPERIMENTAL SERIES 11/40/1,2

Chitosan base, used in experiments 11/40/1 and 11/40/2 described below, commercially available as Protasan UP B 80/200 from NovaMatrix FMC Biopolymer, Oslo, Norway.

Experiment 11/40/1

Preparing an aqueous solution AFHA II 150 (1.0 mg/ml)containing 300 mg AFHA II 150. A solution containing 30 mg of chitosan dissolved in 0.1 M Hcl (pH 5 - brought the addition of a buffer of fibrillatory), 330 mg of D(-)-fructose dissolved in 10 ml of saline solution was added to a solution of AFHA II 150 with stirring. The mixture was stirred for 1 minute at 800 rpm turbine stirrer, was transferred to a tray of stainless steel and has liofilizovane. After lyophilization, the sample was covered with a mixture of ethanol/DI water (90:10./about.) and incubated at 37°C for 6 hours. The resulting material was washed three times with a mixture of ethanol/DI water (90:10./vol.), the solvent was removed by draining and the sample was dried by lyophilization. 4 ml of Saline solution was added to liofilizovannye material and the material was incubated for 3 days the ri 37°C. At the end of the incubation the sample was extrudible through a needle 16G, 8 ml of saline solution was added and the mixture is again consistently extrudible through a needle 18G and 20G.

Experiment 11/40/2

The experiment was carried out as described for experiment 11/40/1 here above, except that the solution AFHA II 150 was mixed with 60 mg of chitosan dissolved in 0.1 M Hcl (pH 5 - brought the addition of a buffer of fibrillatory), and 360 mg of D(-) fructose dissolved in 10 ml of saline solution. The material obtained was gel having a dense texture and no white/yellow color.

EXPERIMENTAL SERIES 11/40/3-5

Experiment 11/40/3

Preparing an aqueous solution AFHA II 150 (1.0 mg/ml)containing 300 mg AFHA II 150. A solution of 1.1 millimoles (237 mg) of the hydrochloride of D(+)-glucosamine was dissolved in 10 ml of saline solution was mixed with an aqueous solution AFHA II 150. The mixture was stirred for 1 minute at 800 rpm turbine stirrer, was transferred to a tray of stainless steel and has liofilizovane. After lyophilization, the sample was covered with a mixture of ethanol/DI water (90:10./about.) and incubated at 37°C for 6 hours. The resulting material was washed three times with a mixture of ethanol/DI water (90:10./vol.), the solvent was removed by draining and the sample was dried by lyophilization. 4 ml of saline solution was added to liofilizovannye material and the material was incubated for 3 days at 37°C. At the end of incubation, the sample e which was studioware through a needle 16G, 8 ml of saline solution was added and the mixture is again consistently extrudible through a needle 18G and 20G. The material obtained was gel having a dense texture and no white/yellow color.

Experiment 11/40/4

The experiment was performed as described for experiment 11/40/3 here above, except that as a reducing sugar used 396 mg (1.1 millimoles) of maltose monohydrate (instead hydrochloride glucosamine). The material obtained was gel having a dense texture and no white/yellow color.

Experiment 11/40/5

The experiment was performed as described for experiment 11/40/3 here above, except that as a reducing sugar used 396 mg (1.1 millimoles) of monohydrate, D(+)-lactose (instead of hydrochloride of D(+)-glucosamine). The material obtained was gel having a dense texture and no white/yellow color.

It should be noted that, although a limited number of types of reducing sugars used in typical experiments disclosed here above, many other types of reducing sugars and/or derivatives of reducing sugars can be used as cross-linkers to obtain transverse cross-linked matrix according to this invention. Such reducing sugars may include, but without limiting the above, aldose, keto is, dioso, trios, tetrose, pentose, hexose, septos, octose, nanaso, dekoze, glycerate, treasu, erythrose, lyxose, xylose, arabinose, ribose, allose, altrose, glucose, fructose, mannose, gulose, idose, galactose, talose, reducing monosaccharide, reducing disaccharide, inviting trisaccharide, reducing oligosaccharide, maltose, lactose, cellobiose, gentiobiose, melibiose, turanose, trehalose, isomaltose, laminaribiose, manobos and kilobyte, glyceraldehyde, sorbose and their combinations.

Other types of reducing sugars, which can be used to obtain transverse cross-linked matrices of this invention are reducing sugars and derivatives of reducing sugars disclosed among others in U.S. patent 5955438, 6346515 and 6682760 and in published international patent application WO 2003/049669, which is incorporated into this description by reference in their entirety and for all purposes.

In addition, it should be noted that in accordance with the embodiment of the invention suitable derivatives of reducing sugars can also be used for cross-linking of the matrix according to this invention, such derivatives may include, but are not limited to, D-ribose-5-phosphate, glucosamine and any other type other derivatives regenerating sa is Ares, known in the art. Esters and salts of these reducing sugars and their derivatives can also be used separately or in any suitable combination with the disclosures provided above types of reducing sugars.

It should be noted that in accordance with additional options of implementation of the present invention used revitalizing sugar (sugar) can be programada, levogyrate and the mixture programada and levogyrate forms. Racemic mixtures of one or more reducing sugars can also be used. In addition, reducing sugar, which contain one or more asymmetric (chiral) carbon atoms, can also be used in the methods and matrices in this invention, including the various optically active isomeric forms (enantiomers) and/or any mixtures and combinations thereof.

The specialist should be clear that, in accordance with an additional embodiment of the present invention more than one revitalizing sugar can be used for cross-linking amino-polysaccharides and/or amino-functionalized polysaccharides, and/or any mixtures of different amino-polysaccharides and/or amino-functionalized polysaccharides, and/or any mixture of amino-polysaccharides and/or amino-functionality avannah polysaccharides with one or more protein (and/or any desired additives). For example, in accordance with non-limiting example, AHFA 150 I cross stitched a mixture of D(-)-ribose and D(+)-sorbose. Similarly, in accordance with another embodiment of the invention, a mixture of chitosan and HFA I 150 can be cross stitched in a mixture containing maltose, glucose and fructose. In another typical embodiment, the mixture AHFA, collagen and heparin can be cross stitched a mixture of cross-linkers, including ribose, glucosamine and D-ribose-5-phosphate. These implementation options are given for purposes of example, and many other variants and modifications are possible by changing the number and types of reducing sugars included in transverse cross-linking reaction mixture.

The specialist should be understood that although specific reactions the cross-linkage as disclosed here above, used a limited range of common solvents and mixtures of solvents, many modifications and changes may be made in the system of solvents used in the reactions of cross-linkage in this invention. Thus, the reaction of the cross-linkage used to generate matrices in this invention, can be carried out in aqueous solutions, buffered aqueous solutions, solutions containing water and/or aqueous buffered solution and one or more organic solvents, non-aqueous rest the arts, includes one or more non-aqueous solvents, and the like. As you can see from the practical experiments disclosed here above, used non-aqueous solvents may be polar and/or hydrophilic and/or miscible with water, solvents, but may also include various non-polar, negitiable solvents and solvent (solvents), which is essentially miscible with water. In principle, any system type solvents include any solvent or combination of solvents may be used for the implementation of cross-linkage reactions of these reactions provided a reasonable approach to the selection of solvents.

For example, the solvents preferably (but not necessarily) should not be too chemically reactive group or parts of molecules, which may adversely affect or interfere with the reactions of cross-linking (except when confounding adverse reactions are undesirable or are actually acceptable or even desirable). Similarly, one should be careful with the choice of the used solvent (solvents)to avoid unwanted denaturirovannyj any proteins and/or polypeptides that have to be cross stitched together with amino-polysaccharides and/or amino-functionalis Rovaniemi polysaccharides. Having in mind these precautions, almost any type of solvent or solvent mixtures may be used for the implementation of cross-linkage reactions according to this invention.

Thus, the matrix according to this invention can be formed by cross-linking of any suitable combination of amino-polysaccharides and/or amino-functionalized polysaccharides, and/or any mixtures of different amino-polysaccharides and/or amino-functionalized polysaccharides, and/or any mixture of amino-polysaccharides and/or amino-functionalized polysaccharides with one or more proteins in any desirable combination of reducing sugars and/or derivatives of reducing sugars. All such combinations and changes, of course, included in the scope of this invention. The use of these different combinations can be advantageous for fine adjustment of the chemical and/or physical and/or rheological, and/or biological properties of the transverse cross-linked matrices, in order to adapt the matrix for any desired application. Properties of the resulting matrices may therefore depend, among other things, on the number and properties of the used amino-polysaccharides and/or amino-functionalized polysaccharides, the number and type of proteins used (if used), the number and type of cross linking in ostanavlivaya sugars and properties, any other additives, included in the matrix. It should also be noted that the matrix properties can also be affected by reaction conditions, the reaction temperature, pH, the type of solvent or solvents and the presence or absence of any additives presented in the reaction mixture and/or added to the matrix after cross-linking.

It should be noted that the solvent (solvents)used in cross-linking of the reaction mixture may include at least one of an ionisable salt (such as, but not limited to, such as NaCl, used in salt solutions in the experiments 09/95/1 in experiments 09/102/1-6, or PBS used in experiments 2, 12/1 and 37/1-3 in the other experiments, which are disclosed in detail here above). An ionisable salt (salt) can be used to regulate the ionic strength of the specified solution and can be useful for implementing the methods of forming the composite matrices, including proteins, in cases where proteins are sensitive to the ionic strength of the reaction solution. It should be noted that any suitable an ionisable salt(salt), known in the art, may be used to adjust ionic strength of the reaction solution, as is well known in the art. Some non-limiting examples of an ionisable salts that may be used include razlichnyei alkali metals, the halides of alkali metals, various sulfates and/or phosphates of metals, various ammonium salts and the like, as is known in the art. However, any other suitable type of an ionisable salts (salts), known in the art, can also be used in reactions of cross-linkage in this invention.

It should be noted that products new cross-linkage reactions described here above can be used to obtain a variety of matrices on the basis of different transverse cross-linked polysaccharides and composite matrices based on the polysaccharide/protein. Such matrices can be obtained as such or can be adequately handled by the appropriate use of templates and/or compression and/or drying and/or freeze-drying, and/or any other method known in the art for the formation of solid or semi-solid products of such matrices)in order to provide a solid form of the matrix in any desired configuration and/or any form of drug injection equipment, including, but without limitation, suitable for injection and not entered by the injection of a suspension of matrix particles, microspheres, microparticles of any desired size and shape. Solid forms of the matrices may include, but are not limited to, plates, tubes, membranes, sponges, flakes, gels, beads, microspheres, microparticles and other Rhodes is ment geometric shapes, made from all types of matrices based on the polysaccharide disclosed here above (including, but without limitation, the composite matrix polysaccharide/protein), which can be obtained by cross-linking using the methods of glycosylation in this invention.

It should be noted that products new cross-linkage reactions described here above (including cross stitched sugar polysaccharides, and cross stitched sugar composition of the matrix protein/polysaccharide), can be further processed and/or processed and/or modified by exposure to cross stitched matrices additional processing and/or one or more process stages. Such processing and/or modifications may include, but are not limited to, drying, lyophilization, dehydration, drying at the critical point, the molding form (to obtain molded products), sterilization, homogenization (to modify or improve the properties of fluidity and suitability for injection matrices), handling manual shift (to modify the rheological properties and to facilitate the introduction of injection), irradiation with ionizing radiation for sterilization and/or implementation of additional cross-linking and/or for other purposes), exposure to electromagnetic radiation (releaserelease and/or to implement additional cross-linking and/or for other purposes), mixing with pharmaceutically acceptable carrier (such as, for example, to obtain the drug for injection to increase the amount of tissue and/or for the increment of the tissue and/or other purposes), thermal sterilization means (autoclaving, and the like), sterilized by chemical means (such as, but without limitation, sterilization using hydrogen peroxide, ozone, ethylene oxide, and the like), the saturation of the additive and/or any combination of these process steps.

In addition, any suitable combination disclosed above, additional processing and process steps can be used in any suitable sequence, to provide any desired modified, and/or dried, and/or molded articles and/or drugs new cross stitched sugar matrices disclosed here. All of the above processing methods well known in the art and therefore not described in detail here.

Additionally it should be noted that the composite matrix according to this invention is not limited to any particular type of collagen. Rather, any desired type of collagen, including, but not limited to, natural collagen, fibrillar collagen, fibrillar telopeptide collagen containing telopeptide collagen, liofilizovane the collagen, collagen obtained from animal sources, the collagen is human, mammal collagen, recombinant collagen, ipsilaterally collagen, restored collagen, bovine telopeptide collagen, porcine telopeptide collagen, collagen derived from vertebrate species, genetically engineered or modified collagen, collagen types I, II, III, V, XI, XXIV, fibril-associated collagen types IX, XII, XIV, XVI, XIX, XX, XXI, XXII and XXVI, collagen types VIII and X, collagen type IV, collagen type VI collagen type VII, collagen type XIII, XVII, XXIII and XXV, collagen type XV and XVIII, the artificially synthesized collagen produced by the genetically modified eukaryotic or prokaryotic cells or genetically modified organisms, purified collagen and restored purified collagen particles fibrillar collagen, fibrillar restored telopeptide collagen, collagen isolated from cell culture medium, collagen, isolated from genetically engineered plants, fragments of collagen, protocollagen and any combination of the above types of collagen may be used to form the composite matrix according to this invention, as disclosed here above.

The specialist should be understood that the composite matrix disclosed in this application, not ogran who receive use of collagen and cytochrome C, as experimentally demonstrated here above. Rather, the composite matrix according to this invention may include a matrix that includes, in addition to the amino-polysaccharides and/or amino-functionalized polysaccharides any suitable type protein (proteins and/or polypeptides (natural or synthetic)that can be cross stitched with amino-polysaccharides and/or amino-functionalized polysaccharides by one or more cross-linkers reducing sugars and/or cross-linker derived from a reducing sugar. This cross stitched protein or polypeptide may include, but is not limited to, collagen, protein, selected from the superfamily of collagen, extracellular matrix proteins, enzymes, structural proteins, isolated from blood proteins, glycoproteins, lipoproteins, natural proteins, synthetic proteins, hormones, growth factors, proteins that stimulate the growth of cartilage proteins, stimulating bone growth, intracellular proteins, extracellular proteins, membrane proteins, elastin, fibrin, fibrinogen, and various combinations thereof.

In accordance with one aspect of the present invention, cross stitched polysaccharide matrix according to this invention can be prepared in the form of suitable compositions for injection with suitable pharmaceutical additives and/or pharmaceutical preparations is automatic acceptable carrier (carriers) or without them. Such injections can be Packed into an appropriate syringe (with a suitable needle or without it). Such pre-filled, pre-sterilized syringes can be used for various cosmetic and medical purposes, such as, but not limited to, use for wrinkles, increment tissue, the increase in the volume of tissue and the like.

According to an additional variant of the invention, the matrix according to this invention can be chemically and/or physically and/or biologically modified agents and substances, such as, but not limited to, drugs, drugs, proteins, polypeptides, anesthetics, antibacterial agents, antimicrobial agents, antiviral agents, antifungal agents, antinicotine agents, anti-inflammatory agents, glycoproteins, proteoglycans, glycosaminoglycans, the various components of the extracellular matrix, hormones, growth factors, transforming factors, receptors or receptor complexes, natural polymers, synthetic polymers, DNA, RNA, oligonucleotides, therapeutic agents, morphogenetic proteins, mucoproteins, mucopolysaccharides, matrix proteins, transcription factors, peptides, genetic material for gene therapy, NUS is ainova acid, chemically modified nucleic acid chimeric structure of DNA/RNA, DNA or RNA probes, antisense DNA, antisense RNA, gene, part of a gene, the composition comprising natural or artificially synthesized oligonucleotides, plasmid DNA, cosmina DNA, viral and non-viral vectors, it is necessary for stimulation of cellular uptake and transcription, chondroitin-4-sulfate, chondroitin-6-sulfate, Kermanshah, dermatooncology, heparin, heparansulfate, hyaluronan-enriched lecithin interstitial proteoglycan, decorin, biglycan, fibronodular, lumican, aggrecan, syndecan, beta-glycan, versican, centralian, serpitine, fibronectin, FibroGen, handreader, fibulin, thrombospondin-5, an enzyme, an enzyme inhibitor, an antibody, and any combination of the above materials and/or any other modifying properties of an agent or substance that is known in the art. Such agents or substances can be added to the matrix after cross-linking. Additionally or alternatively, the agent(s) or substance (substances) can be added to the reaction mixture before cross-linking, and the cross-linkage reaction can then be carried out in the presence of an agent (agents) or substance (s)to embed and/or link cross-link agent(s) or substance (substance) is formed inside transverse cross-linked matrix, to change the properties of the matrix.

Such added substances can be covalently linked to the polysaccharide matrix by any suitable transverse cross-linking agents, as is well known in the art. Alternatively or additionally, such modifying agents may be included in the reaction mixture during the processes of cross-linkage, described here, and can thus be captured by the matrix, or included in a matrix based on the transverse cross-linked polysaccharides or composite matrix.

The specialist should be clear that the described here transversely stitched matrix can be further modified by exposure to matrices of any chemical or biological modifiers known in the art. For example, some or all of the available functional groups such as, but without limitation specified, amino groups, and/or carboxypropyl, and/or hydroxyl groups remaining on the components transverse cross-linked matrix after cross-linking, can be chemically or enzymatically treated to chemically introduce other chemical groups or parts of molecules (such as, but without limitation specified, amino groups, and/or carboxypropyl, and/or hydroxyl groups and/or nitro, and/or chlorine-and/or bromo - and/or joagroupe, and or pericarp, and/or prodgroup, and/repercharge, and/or any other chemical group and/or chemical part of the molecule, and the like)to further modify such groups in order to further adjust the properties of the matrix. Examples of such possible modifications after cross-linkage may include, but are not limited to, the etherification of the free hydroxyl or carboxylate present on the main chain of the polysaccharide cross stitched matrix or on the main chain of the protein including any cross stitched protein or polypeptide composition of the matrix, acetylation any free amino groups on the main chain of the polysaccharide or polypeptide, or any other type known in the art of chemical reactions or enzymatic modification of functional groups. The chemistry of such modifications are well known in the art and therefore not disclosed in detail here.

Such modifications of functional groups may be applicable for additional modifications and fine regulation of matrix properties (such as, but without limitation specified, hydrophobicity, hydrophilicity, the resulting charge at various selected values of pH, porosity of the matrix, the ability of the matrix to absorb water, resistance to enzymatic cleavage in vivo and/or in vitro, and the like), in order to adapt the matrix for a particular desired application the response. You should take into account that if a matrix that modifies, are intended for applications that require biocompatibility, care must be taken when selecting chemical groups exposed modification, and to consider the nature of any chemical groups, which enter into the structure of the matrices to ensure a sufficient degree of biocompatibility. However, in other applications of matrices that do not require a high degree of biocompatibility, many of the groups listed above, and any other chemical groups known in the art (such as, but without limitation specified, isopropy, sidegroup, nitrosopropane and the like), can be introduced into the matrix structure, to provide additional modification of the structure and properties of the matrix.

In accordance with an additional embodiment of the matrix according to this invention, living cells can be added to any cross stitched matrices described herein above, or obtained by applying the disclosed here. Living cells can be added during or after cross-linking, to form a transversely stitched matrix containing one or more types of cells that are included or embedded in the matrix.

In accordance with an additional embodiment of the matrix according to this invention, alive the mi cells, included in the matrix may be a vertebral chondrocytes, osteoblasts, osteoclasts, stem cells of vertebrates, the embryonic stem cells, stem cells isolated from adult tissue, precursor cells of vertebrates, vertebrate fibroblasts, cells, genetically engineered for secretion of one or more matrix proteins, glycosaminoglycans, proteoglycans, morphogenetic proteins, growth factors, transcription factors, anti-inflammatory agents, proteins, hormones, peptides, one or more types of living cells, designed for the expression of receptors for one or more molecules selected from the group consisting of proteins, peptides, hormones, glycosaminoglycans, proteoglycans, morphogenetic proteins, growth factors, transcription factors, anti-inflammatory agents, glycoproteins, mucoproteins and mucopolysaccharides. The combination of several different types of cells can also be included in the matrix according to this invention.

In accordance with the variations in implementation of the present invention, the transverse cross-linked matrix based on the polysaccharide obtained by means of this application, can be suitable for various applications, such as, but not limited to, matrix decks applicable in tissue engineering (forin vivo in vitro applications), systems with controlled release of pharmacological substances and biological agents and biologically active proteins, genes, genetic vectors, and the like), membranes for directed regeneration of bone and soft tissue, administered by injection and/or implantable bulk agents, and/or prosthetic devices for the increment of the tissue and/or cosmetic use (such as, but not limited to, injections for wrinkles and other cosmetic and aesthetic purposes), shell to secure it in place of natural and/or reconstructed, and/or artificial organs, filling material for artificial tissues or organs, such as, but not limited to, artificial breast, and as a component of composite materials containing transverse cross-linked polysaccharides according to this invention, combined with other natural or synthetic polymeric structures, materials and/or matrices, or with other natural or synthetic organic and inorganic compounds and/or polymers and/or combinations of all the above substances.

The specialist should be clear that, although the buffer used in many reactions and preparations of the samples disclosed above, was phosphate buffered saline R is the target (PBS), it is not mandatory for the implementation in practice of the invention. Thus, many different types of buffers and/or buffered solutions and buffered solvents can be used to implement the procedures for obtaining material and/or cross-linkage reactions to obtain matrices based on the polysaccharide and/or composite matrices based on the polysaccharide/protein according to this invention. For example, other typical buffers that can be used in the preparations and reactions of cross-linking according to this invention, may include, but are not limited to, the buffer is citric acid/citrate, 2-(N-morpholino)econsultancy acid (MES), 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propandiol (BIS-TRIS), piperazine-N,N'-bis(2-econsultancy acid) (PIPES), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)piperazine-1-econsultancy acid (HEPES), and many other types of buffers known in the art. However, when choosing a buffer compositions it is necessary to ensure that the buffers are not included active chemical groups or parts of molecules that can interfere in the reaction cross-linkage described here above. Such buffers and considerations are accounted for when they are selected for use, is well known in the art and widely described in the literature, and therefore not disclosed in detail here.

1. Pic is b receiving transverse cross-linked polysaccharides, including the interaction of at least one polysaccharide selected from the amino-polysaccharide, amino-functionalized polysaccharide containing one or more amino groups that can be cross stitched regenerating sugar, and combinations thereof with at least one regenerating sugar with the formation of the transverse cross-linked polysaccharide.

2. The method according to claim 1, where the specified at least one polysaccharide selected from a natural amino polysaccharide, a synthetic amino-polysaccharide, amino heteropolysaccharide, amino homopolysaccharide, amino-functionalized polysaccharides and their derivatives and esters and salts, amino-functionalized hyaluronic acid and its derivatives and esters and salts, amino-functionalized hyaluronan and its derivatives and esters and salts, chitosan and its derivatives and esters and salts, amino-functionalized heparin and its derivatives and esters and salts, amino functionalized glycosaminoglycans and their derivatives and esters and salts, and any combinations thereof.

3. The method according to claim 1, where the specified at least one regenerating sugar selected from an aldose, ketose derivative of an aldose derived ketosis and any combinations thereof.

4. The method according to claim 1, where specified on ENISA least one regenerating sugar selected from Diaz, Treaty, tetrose, pentoses, hexose, septate, octose, nansy, decoz and their combinations.

5. The method according to claim 1, where the specified at least one regenerating sugar selected from glycerate, Treaty, erythrose, lyxose, xylose, arabinose, ribose, allose, altrose, glucose, fructose, mannose, gulose, idose, galactose and talose.

6. The method according to claim 1, where the specified at least one regenerating sugar selected from a reducing monosaccharide, a reducing disaccharide, restorative trisaccharide, reducing oligosaccharide, derivatives of oligosaccharides, derivatives of monosaccharides, esters of monosaccharides, esters of oligosaccharides, salt, simple sugars, salts oligosaccharides and any combinations thereof.

7. The method according to claim 6, where the specified regenerating a disaccharide selected from the group consisting of maltose, lactose, cellobiose, gentiobiose, melibiose, turanose, trehalose, isomaltose, laminaribiose, nanobiosym and kilobyte.

8. The method according to claim 1, where the specified at least one regenerating sugar selected from glyceraldehyde, ribose, erythrose, arabinose, sorbose, fructose, glucose, D-ribose-5-phosphate, glucosamine, and combinations thereof.

9. The method according to claim 1, where the specified at least one regenerating sugar selected from programada the specified form at least one Voss is analivia sugar, levogyrate specified form at least one reducing sugar and mix programada and levogyrate forms specified at least one reducing sugar.

10. The method according to claim 1, where the specified interaction involves incubation of the specified at least one polysaccharide in the solution containing at least one solvent and the at least one regenerating sugar, before the formation of the specified transverse cross-linked polysaccharide.

11. The method according to claim 10, where this solution is a buffered solution containing at least one buffer.

12. The method according to claim 10, where the specified at least one solvent is an aqueous buffered solvent containing at least one buffer to regulate the pH of the specified solution.

13. The method according to claim 10, where the specified at least one solvent is an aqueous solvent comprising at least one of an ionisable salt to regulate the ionic strength of the specified solution.

14. The method according to claim 10, where the specified at least one solvent comprises at least one solvent selected from the group consisting of organic solvent, inorganic solvent, a polar solvent, a nonpolar solvent, a hydrophilic solvent, a hydrophobic dissolve the La, solvent miscible with water, not mixing with the water solvent, and combinations thereof.

15. The method according to claim 10, where the specified at least one solvent contains water and at least one additional solvent selected from the hydrophilic solvent, a polar solvent, a solvent miscible with water, and combinations thereof.

16. The method according to claim 10, where the specified at least one solvent selected from the group consisting of water, phosphate buffered saline, ethanol, 2-propanol, 1-butanol, 1-hexanol, acetone, ethyl acetate, dichloromethane, diethyl ether, hexane, toluene, and combinations thereof.

17. The method according to claim 1, where this interaction also includes adding at least one protein selected from collagen and cytochrome C to the specified at least one polysaccharide and the specified at least one regenerating sugar for the formation of composite transverse cross-linked matrix.

18. The method according to 17, where the specified collagen selected from natural collagen, fibrillar collagen, fibrillar telopeptide collagen containing telopeptide collagen, liofilizirovannogo collagen, collagen obtained from animal sources, collagen, human, mammal collagen, recombinant collagen, pasensyahan collagen, Voss is set out collagen, bullish telopeptide collagen, porcine telopeptide collagen, collagen obtained from vertebrate species, genetically engineered or modified collagen, collagen types I, II, III, V, XI, XXIV, fibril-linked collagen types IX, XII, XIV, XVI, XIX, XX, XXI, XXII, and XXVI, of collagen types VIII and X collagens type IV, collagen type VI collagen type VII, collagen types XIII, XVII, XXIII and XXV, collagen type XV and XVIII, the artificially synthesized collagen produced by the genetically modified eukaryotic or prokaryotic cells or genetically modified organisms, purified collagen and restored purified collagen particles fibrillar collagen, fibrillar restored telopeptide collagen, collagen isolated from cell culture medium, collagen derived from genetically engineered plants, fragments of collagen, protocollagen and any combinations thereof.

19. The method according to claim 1, where this interaction includes adding at least one additive to the specified at least one polysaccharide and the specified at least one regenerating sugar for the formation of a transverse cross-linked matrix containing the specified at least one additive.

20. Transverse cross-linked polysaccharide, obtained by the method according to claim 1.

21. With the persons receiving the transverse cross-linked polysaccharides, contains stage:
the interaction of the polysaccharide with one or more reagents to the formation of the derivative of the specified form of the polysaccharide, and specified derivative form is an amino-polysaccharide or amino-functionalized polysaccharide containing one or more amino groups that can be cross stitched regenerating sugar, and
the cross-linkage specified derived polysaccharide with at least one regenerating sugar to the formation of the transverse cross-linked polysaccharide.

22. The method according to item 21, where these amino groups selected from primary amino groups and secondary amino groups.

23. The method according to item 21, where the specified one or more reagents contain carbodiimide.

24. The method according to item 21, where the specified one or more reagents contain carbodiimide in the presence dihydrazide adipic acid.

25. The method according to item 23, where the specified carbodiimide is the hydrochloride of 1-ethyl-3-(dimethylaminopropyl)carbodiimide.

26. The method according to item 21, where the specified at least one regenerating sugar selected from an aldose, ketose, and their combinations.

27. The method according to item 21, where the specified at least one regenerating sugar selected from glyceraldehyde, ribose, erythrose, arabinose, sorbose, fructose, glucose, D-ribose-5-phosphate, glucosamine, Diaz, TRIZ, tetrose, p is ntozi, hexose, septate, octose, nansy, dekoze, glycerate, Treaty, lyxose, xylose, allose, altrose, mannose, gulose, idose, galactose, talose, a reducing monosaccharide, a reducing disaccharide, restorative trisaccharide, reducing oligosaccharide, derivatives of oligosaccharides, derivatives of monosaccharides, esters of monosaccharides, esters of oligosaccharides, salt, simple sugars, salts oligosaccharides, maltose, lactose, cellobiose, gentiobiose, melibiose, turanose, trehalose, isomaltose, laminaribiose, nanobiosym and kilobyte and their combinations.

28. A method of obtaining a composite transverse cross-linked matrix comprising cross-linking with at least one regenerating sugar at least one polysaccharide selected from the amino-polysaccharide, amino-functionalized polysaccharide containing one or more amino groups that can be cross stitched regenerating sugar, and combinations thereof, in the presence of at least one cross stitched protein selected from collagen and cytochrome C, before the formation of the specified composite transverse cross-linked matrix.

29. Cross stitched composite matrix obtained by the method according to p.



 

Same patents:

FIELD: medicine.

SUBSTANCE: claimed is application of conjugate of heparin with lysine and ibuprofen of formula as anti-inflammatory medication with anticoagulant, antitumour and antimetastatic activity. Conjugate of heparin with lysine and ibuprofen, where R=So3H/Ac (30:70).

EFFECT: conjugate possesses anti-inflammatory activity, which is 1,4 times higher than ibuprofen activity, anticoagulant activity is comparable with pharmacopeia heparin, its antitumour activity being comparable with that of doxorubicin, cyclophosphane, vincristine, prednisolone with demonstration of antimetastatic activity.

6 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: there is offered a method for preparing low-molecular heparins by enzymatic degradation. For enzymatic depolymerisation, immobilised lysozyme is used.

EFFECT: prepared low-molecular heparin shows higher inhibitor activity with respect to blood coagulation factor Xa and lower inhibitor activity with respect to thrombin in comparison with initial heparin.

2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, more exactly to drug technology intended for treatment of thrombotic conditions. There is offered method for making low-molecular heparin with using enzyme depolymerisation, characterised by adding dry lysozyme to 1% heparin in 0.1 M NaCl in the weight relation 1:100, mixing at 50°C within 3 hours, desalting with sephadex column and lyophilised.

EFFECT: invention ensures production of low-molecular heparin with using ovalbumin lysozyme enzyme.

3 ex, 2 tbl

FIELD: chemistry.

SUBSTANCE: method of producing sulphated glycosaminoglycans from N-acetylheparosan involves a) N-deacetylation and N-sulphation of polysaccharide of N-acetylheparosan, obtained from natural or recombinant bacterial strains, preferably E. coli K5, b) enzymic epimerisation using glucoronyl-C5-epimerase, c) partial O-sulphation with subsequent partial O-desulphation, d) partial 6-O-sulphation, e) N-sulphation and an intermediate stage of controlled depolymerisation, characterised by the fact that, both O-sulphations (O-sulphation and 6-O-sulphation) are partial. The invention also relates to products obtained in accordance with the method, which exhibit ratio between anti-Xa and anti-IIa-activity equal to 1 or greater than 1, and to antithrombotic and anticoagulant pharmaceutical compositions, containing the said products combined with suitable and pharmaceutically acceptable fillers and/or solvents.

EFFECT: increased effectiveness of composition and method of treatment.

61 cl, 18 dwg, 6 tbl, 18 ex

FIELD: chemistry.

SUBSTANCE: mixtures of oligosaccharides with antithrombotic activity, with general structure of constitutive polysaccharides of heparin, have the following characteristics: average molecular weight from 1800 to 2400 Daltons, anti-Xa activity, from 190 M.units./mg to 450 M.units/mg, anti-IIa activity equals zero or practically equal to zero. Constitutive oligosaccharides of these mixtures: contain 2-16 saccharide links, have a 2-0-sulphate-4,5-unsaturated uronic acid link on one of their ends. The mixtures contain from 30 to 60% hexasaccharide fraction, and the hexasaccharide fraction contains from 20 to 70% hexasaccharide with the following formula: in form of a salt of alkali or alkali-earth metal. A method is given of obtaining oligosaccharide mixtures and antithrombotic pharmaceutical compositions containing them, as well as a method of determining activity of oligosaccharide mixtures.

EFFECT: obtaining oligosaccharides with antithrombotic activity.

28 cl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention describes a method of epi-K5-N-sulfate oversulfation for obtaining epi-K5-amine-O-oversulfate with very high sulfation degree, which produces new epi-K5-N,O-oversulfate derivatives with sulfation degree of 4-4.6 on following N-sulfation, the derivatives being almost inactive to fibrillation parametres and applicable in pharmaceutical compositions with antidermatitis and antiviral effect. The invention also describes new low-molecular epi-K5-N-sulfates applicable as transit products in obtaining the respective low-molecular epi-K5-N,O-oversulfate derivatives.

EFFECT: method of epi-K5-N-sulfate oversulfation for obtaining epi-K5-amine-O-oversulfate with extremely high sulfation degree.

55 cl, 4 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to the mixture of sulfated oligosaccharide having common structure of polyose that is included in heparin composition with average molecular mass ranging from 1500 to 3000 Da and proportion of anti-Xa/anti-IIa more than 30, to the method of their production and antithrombotic pharmaceutical compositions containing them.

EFFECT: production of the pharmaceutical compositions containing sulfated oligosaccharide that has antithrombotic activity.

31 cl, 12 ex

FIELD: medicine, biochemical technology.

SUBSTANCE: invention proposes a method for preparing low-molecular heparins using enzymatic cleavage. For enzymatic depolymerization the method involves using papain, chymotrypsin or complexes of hydrolases as components of preparations "Protease C" and celloviridine immobilized on silochrome or polyvinyl alcohol. Method provides preparing low-molecular heparins by effect of immobilized hydrolases with the yield of the end product about 70-80%.

EFFECT: improved preparing method.

8 ex

FIELD: bioorganic chemistry, chemical technology.

SUBSTANCE: invention describes N-deacylated N-sulfatized derivative of polysaccharide K5 epimerized up to at least to 40% of the content of L-iduronic acid with respect to the total content of uronic acids and with molecular mass 2000-30000 Da and comprising 25-50% by the chain weight showing the high affinity to ATIII and possessing anti-coagulant and anti-thrombosis activity with the ratio of abovementioned activities of HCII/antiXa from 1.5 to 4. Also, invention describes a method for preparing a derivative of polysaccharide K5 comprising isolation of polysaccharide K5 from Escherichia coli cells, N-deacylation and N-sulfatization, C-5-epimerization of D-glucuronic acid to L-induronic acid, supersulfatization, selective O-desulfatization, selective 6-O-sulfatization and N-sulfatization. The reaction of C-5-epimerization is carried out by using the enzyme glucuronosyl-C-5 epimerase in the soluble or immobilized form in the presence of bivalent cations chosen from the group comprising Ba, Ca, Mg and Mn.

EFFECT: improved preparing method, valuable medicinal properties of substances.

12 cl, 3 tbl, 12 dwg, 12 ex

FIELD: medicine.

SUBSTANCE: chitosan is dissolved in an organic acid: 4-6% citric acid or 2-8% lactic acid in the relation of the ingredients chitosan: the organic acid 1:2-1:4 to prepare a forming solution. Chitosan has molecular weight 80-500 kDa. The forming solution is added with vitamin B1 in the amount of max. 0.5 wt %. The prepared forming solution is applied on a substrate in the amount of 0.2-0.25 ml/cm2 and kept to achieve a film structure. Said method is used to form the chitosan film coating having the thickness of 50-250 mcm and the breaking elongation of 42 to 470%.

EFFECT: group of inventions allows preparing high-elastic chitosan citrate or lactate films possessing bactericidal action.

2 cl, 1 tbl, 13 ex

FIELD: chemistry.

SUBSTANCE: method involves preparation of material for enzymatic hydrolysis. Alkaline hydrolysis is carried out with proteolytic enzyme preparations with neutralisation of the obtained solution to pH=7. A salt is added to the obtained enzymatic hydrolysate to a value of not less than 0.1 mol/l. Successive ultrafiltration is carried out, first on a membrane with maximum retention of 50 kD with separation of high-molecular weight impurities, and then on a membrane with maximum retention of 5 kD with separation of low-molecular weight substances. The chondroitin sulphate solution retained at the membrane is washed on the same membrane with distilled water until complete removal of salts. Final washing with distilled water is carried out on a membrane with maxim retention of 50 kD.

EFFECT: invention enables to obtain a chondroitin sulphate preparation with weight ratio of the basic substance.

7 ex

FIELD: chemistry.

SUBSTANCE: method involves activation of hyaluronic acid using a cross-linking agent and an auxiliary cross-linking agent. The activated hyaluronic acid then reacts with a nucleophilic cross-linking agent. The pH of the reaction medium ranges from 8 to 12. The nucleophilic cross-linking agent contains at least 50 wt % oligopeptide or polypeptide. Further, pH of the reaction medium is regulated to 5-7 and cross-linked hyaluronic acid is precipitated in the organic solvent. The invention also relates to use of the cross-linked hyaluronic acid obtained using this method in plastic surgery to make implants and to a hedrogel containing said cross-linked hyaluronic acid in a buffer aqueous solvent.

EFFECT: invention enables to obtain cross-linked hyaluronic acid in dry form, having high resistance to decomposition factors such as temperature, free radicals and enzymes.

18 cl, 3 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of determining antibacterial properties of chitosan by estimating its minimum bacteriostatic and/or bactericidal concentration. Complex buffer solutions based on three organic acids MES, ACES and TES with different pH values are prepared. The ready buffer solutions are poured into a vessel. Double dilutions of chitosan are then prepared in vessels with the buffer solutions. Aliquots of a bacterial suspension in a fluid medium are added to the chitosan solutions in the buffer. The solutions are incubated for 24 hours at temperature which is optimum for bacterial growth. The minimum bacteriostatic and/or minimum bactericidal concentration of chitosan is then determined after incubation by determining growth of the culture or a drop in the number of living cells, respectively.

EFFECT: invention enables to determine antibacterial properties of chitosan in a wide pH range from 5,50 to 8,00 without the need to use buffers of different chemical composition.

5 dwg, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a method for preparing sodium salt of hyaluronic acid modified by boron compounds with no fluid medium added. The method consists in the fact that powdered sodium salt of hyaluronic acid together with a modifying agent and mixed modifying agents is pre-homogenised in a mixer at temperature ranging within 20° to 50°C; thereafter the prepared homogenous powder mixture is simultaneously exposed to pressure and shearing deformation in a mechanochemical reactor at temperature ranging within 20° to 50°C and pressure 5-1000 MPa.

EFFECT: invention provides preparing boron-containing sodium salt of hyaluronic acid applied in boron neutron capture therapy in one-stage process parameters with no fluid medium added which requires low power, labour and water consumptions.

13 cl, 15 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, more specifically to producing chitosan oligomers possessing biological activity and applicable in food industry and medicine. In a method for producing chitosan oligomers, a chitosan solution is taken in the concentration of 0.025-0.075% (weight/volume) and exposed to low-frequency ultrasound of the intensity of 92-460 Wt/cm2 for 5-30 minutes.

EFFECT: reduction in price of the chitosan oligomers production combined with promotion of higher medium viscosity molecular weight of the product within the range 25 ÷ 120 kDa.

3 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: method involves preliminary acetylation of chitin with acetic anhydride, washing and drying the acetylated chitin in order to reduce degree of deacetylation thereof and, as a result, increase output of the desired product - D(+)-glucosamine hydrochloride when obtaining said product through hydrolysis of acetylated chitin with concentrated hydrochloric acid while heating, followed by evaporation, crystallisation, separation, washing and drying the desired product.

EFFECT: high output of the desired product while maintaining its high quality; method is more environmentally friendly since pre-treatment of chitin reduces the amount of processing wastes.

1 cl, 2 ex

FIELD: chemistry.

SUBSTANCE: method of producing chitosan chromate involves reaction of soluble chitosan salts with metal chromates in ratio of 2 moles of the chitosan cation to 1 mole of chromate anion or with metal bichromates in ratio of 4 moles of the chitosan cation to 1 mole of the bichromate anion. The solid chitosan chromate residue formed is then separated and dried at temperature not higher than 150°C. The invention discloses an energy-intensive composition based on chitosan dodecahydro-closo-dodecaborate containing an effective amount of chitosan chromate. The quantitative ratio in the energy-intensive composition is by the required combustion mode: the higher the content of chitosan chromate, the higher the activity of the composition.

EFFECT: invention enables to obtain a chemical compound having sufficiently high oxidative properties and suitable for use in energy-intensive compositions which burn without emitting harmful gaseous products.

3 cl, 5 ex

FIELD: chemistry.

SUBSTANCE: method involves taking a certain weighed amount of chitosanium chromate which is first purified from extraneous impurities and reduced to constant weight. The weighed amount is then turned into a stable weighted form through thermal treatment on air at temperature 800-900°C to form chromium oxide Cr2O3. The weight of the formed chromium oxide is then determined. Content of chromic acid in the initial weighed amount of chitosanium chromate is then calculated from the weight of chromium oxide. The degree of deacetylation of chitosan is calculated using defined formulae.

EFFECT: invention enables to increase accuracy of determining degree of deacetylation of chitosan.

2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of extracting and stabilising ultra low-molecular aminoglycans from eggshell wastes. Aminoglycan extract is used to produce cosmetic creams with skin moisturising and anti-wrinkle properties. The method of extracting low-molecular aminoglycan compound of formula I from a natural source of eggshell wastes, which consists of alternating glucuronic acid and N-acetylglucosamine units, where M can be one or more of Na, Ca, K, Mg; and n is a whole number from 20 to 40, involves the following steps: (a) preparing eggshell wastes for extraction of embryonic low-molecular aminoglycan compound of formula I using a polar organic solvent in water, (b) extracting low-molecular aminoglycan compound of formula I in form of a water-soluble salt, for which the eggshell from step (a) is vigorously shaken with aqueous polar salt solution at 10°C - 35°C for 6-12 hours, then filtered or centrifuged in order to collect an aqueous layer containing a dissolved aminoglycan compound of formula I; (c) extracting a purified low-molecular aminoglycan compound of formula I by forming a gel from an aqueous mixture of salts using a polar organic solvent, for which the solution from step (b) is successively and step-by-step mixed with an organic solvent mixed with water while gently stirring and then cooled to maintain temperature from 20°C to 25°C, and the formed gel is left for 2-24 hours for complete precipitation, then filtered or centrifuged in order to extract a semidry aminoglycan compound of formula I; (d) the extracted aminoglycan compound of formula I from step (c) is stabilised via gradual addition of organic oils to the semidry gel to form aminoglycan compound of formula I. In order to prepare a composition having anti-wrinkle properties, at least one pharmaceutically acceptable filler is added to the stabilised aminoglycan compound of formula I obtained at step (d).

EFFECT: method enables to obtain an aminoglycan compound of formula I with the necessary viscosity and skin penetrating properties for reducing skin wrinkles, as well as excellent softening and moisturising effects.

8 cl, 9 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of producing fullerene adducts by mixing a dispersion of fullerene C60 with hydrophilic compounds, where the hydrophilic compound used is an amino acid or an amino sugar or polyhydroxylamine or a protein, which are first converted to a trimethylsilyl (TMS) derivative by mixing with a trimethyl silylating reagent (N,O-bis(trimethylsilyl)-acetamide in the medium of a polar aprotic solvent, after which the obtained solution of trimethylsilyl (TMS) derivative is mixed with a dispersion of fullerene in a polar aprotic solvent and the mixture is stirred at room temperature for 2-24 hours.

EFFECT: improved method.

5 cl, 7 dwg, 7 ex

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