MXPA99000085A - Use of oxidized cellulose and complexes of the same for croni wound healing - Google Patents

Abstract

The invention provides the use of oxidized cellulose, preferably oxidized regenerated cellulose (CRO) and complexes thereof with structural proteins, such as collagen, for the preparation of wound healing materials for the treatment of chronic wounds, preferably, the curative material is textile material knitted, interlaced, non-interlaced from CRO suitable for application directly to the surface of a wound, such as a adhering healing material or semisolid ointment for local application containing dispersed CRO fibers or powder, or a collagen sponge / CRO, preferably, the chronic wound is a venous uniulculum, decubital uniulum or diabesti

Description

USE OF OXIDIZED CELLULOSE AND COMPLEXES OF THE SAME FOR THE HEALING OF CHRONIC WOUNDS DESCRIPTIVE MEMORY The present invention relates to the use of oxidized celluloses and complexes thereof with structural proteins, such as collagen, for the healing of chronic wounds. Oxidized cellulose is produced by the oxidation of cellulose, for example with nitrogen tetroxide. This process converts the groups of primary alcohols on saccharide residues to carboxylic acid group, forming uronic acid residues within the cellulose chain. Oxidation does not take place with complete selectivity and as a result the hydroxide groups of carbons 2 and 3 are occasionally converted to the keto form. These ketone units introduce an alkaline labile bond, which at pH 7 or higher initiates the decomposition of the polymer by means of the formation of a lactone and an annular unfolding of sugar. As a result, oxidized cellulose is biodegradable and bioabsorbable under physiological conditions. The preferred oxidized cellulose for practical applications is oxidized regenerated cellulose (CRO) prepared by oxidation of a regenerated cellulose, such as rayon. It has been known for some time that CRO has hemostatic properties. The CRO has been obtainable as a product hemostatic called SURGICEL (registered trademark of Johnson &Johnson Medical, Inc.) since 1950. This product is produced by oxidizing a knitted rayon material to point. Modification of the porosity, density and design of the knitted fabric resulted in the introduction of a second textile product from CRO, INTERCEED (registered trademark - Johnson &Johnson Medical, Inc.) which was shown to reduce the extension of postsurgical lesions of abdominal surgery. US-A-2517772 (Doub et al.) Describes improved hemostatic materials obtained by impregnating CRO textile material with thrombin. EP-A-0437095 discloses a neutralized CRO material which is prepared by contacting a synthesized acid CRO material with a basic salt solution of a weak organic acid, such as sodium citrate. The resulting neutralized product is indicated for hemostasis and prevention of adhesion. Collagen, which is a structural protein of animal origin, is known in various forms for its use as a wound healing material. GB-A-1515963 discloses mixed materials of collagens - interlaced glycol polysaccharides for use in medical and surgical applications, blood vessel grafts and all forms of surgical prostheses. The material mixed contains at least 0.5% by weight of a mucopolysaccharide irreversibly bind to collagen. The mucopolysaccharide is an animal polysaccharide containing hexosamine residues, such as hyaluronic acid, chondroitin sulfate or heparin T-phosphate. It is said that these mixed materials exhibit greater resistance to resorption and better blood compatibility than simple collagen materials. US-A-4614794 describes complexes formed between polyanionic vegetable collagen and polysaccharide, such as sodium alginate. The complexes are preferably formed by combining the protein and the polysaccharide at a pH that is not higher than the isoelectric point of the protein. It is said that the resulting complexes are suitable for a wide variety of medical and surgical applications. including wound healing materials. There is no exposure to the use of oxidized celluloses in place of polyanionic plant polysaccharide. The specification also teaches that proteins other than collagen, such as fibrin or elastin, can be used in the formation of useful protein / polysaccharide complexes. GB-A-2280850 discloses medicated implants for the treatment of periodontal disease, comprising a medicated collagen film, reinforced with a layer of bioresorbable polymer, which may be CRO. The collagen matrix may contain dispersed CRO fibers or fragments.

Patent EP-1-0177064 describes the use of an oxidized knitted cellulose textile material as a surgical hemostat. EP-A-0562862 discloses bioabsorbable sponge materials for use as implants for wounds. The materials comprise a collagen sponge matrix having a substructure oriented thereto. The matrix and / or substructures may comprise oxidized regenerated cellulose. There is no exposure to the use of such materials for the treatment of chronic wounds. GB-A-1006606 discloses haemostatic wound healing materials comprising oxidized cellulose crystallite aggregates, dispersed in a gel matrix. The use of such hemostatic gels in surgery is disclosed. The healing collagen or collagen / polysaccharide wound materials described above provide important advantages. The materials are of natural, biological origin (although sometimes chemically modified) and tend therefore to have low antigenicity. The materials are generally bioabsorbable, which reduces the trauma associated with the removal of healing materials from conventional wounds from the wound surfaces. In addition, some of these materials may have positive therapeutic effects on wound healing. For example, some animal mucopolysaccharide, such as hyaluronic acid, is believed to exert a chemotactic effect in the cells that heal the wound, such as fibroblasts, and thus promote the growth and development of such cells. However, there remains a need for improved wound healing materials of this general type exhibiting even better control of physical properties and biological absorption rates, even better therapeutic effects on wound healing and reduced cost. In an object of the present invention to provide improved wound healing materials, for chronic wounds in mammals and especially in humans, such as venous ulcers, decubital ulcers and diabetic ulcers. Such chronic wounds generally exhibit little or no bleeding or adhesion to other tissues of the body and according to this oxidized cellulose would not have been previously indicated for the treatment of such wounds. It has been discovered that oxidized cellulose is an effective wound healing material for the treatment of chronic wounds. Accordingly, the present invention provides the use of oxidized cellulose for the preparation of a wound healing material for the treatment of a chronic wound. Preferably, the oxidized cellulose is oxidized regenerated cellulose (CRO). Preferably, the oxidized cellulose is in the form of an interlaced, non-interlaced or woven fibrous body that is insoluble in wound fluids. For example, oxidized cellulose may be in the form of one of the SURGICEL (RTM) or (RTM) textile materials known in the art. In other preferred embodiments, the oxidized cellulose is in the form of fibers, such as fiber or shredded powder, preferably dispersed in a suitable medically local vehicle. Preferably, the oxidized cellulose has an average molecular weight greater than 50,000. Such oxidized cellulose is substantially insoluble in wound fluids, but will experience very gradual decomposition to bioresorbable fragments at physiological pH. Preferably, the oxidized cellulose is not neutralized. However, the present invention encompasses the use of partially or completely neutralized materials as described in EP-A-0437095 for the preparation of medicaments for the treatment of chronic wounds as defined hereinbefore. Preferably, the oxidized cellulose forms a complex with a structural protein in said wound healing material. Preferably, the protein (when present) and the oxidized cellulose together constitute at least 75% by weight of the material for pentaimines, more preferably at least 90% by weight of the material. Other components of the material may include 0-25% by weight of one or more biocompatible polysaccharides, for example alginates such as sodium alginate or calcium alginate, starch derivatives such as sodium amino-glycolate, cellulose derivatives such as methylcellulose or carboxymethylcellulose, or glycosaminoglycans such as hyaluronic acid or its salts, chondroitin sulfate or heparin sulfate. The material may also contain up to 20% by weight, preferably up to 10% by weight of water. The material may also contain 0-40% by weight, preferably 0-25% by weight of a plasticizer, preferably a polyhydric alcohol such as glycerol. The material may also contain 0-10% by weight, preferably 0-5% by weight of one or more wound healing therapeutic agents, such as drugs, non-steroidal anti-inflammatories (eg acetaminogel) steroids, antibiotics (ie penicillins) or streptomycins), or growth factors (eg, hydroplast growth factors or platelet-derived growth factor). Preferably, the weight ratio of the protein to the oxidized cellulose is from 1:99 to 99: 99: 1. More preferably, the weight ratio is in the range of 1:10 to 99.9: 1, more preferably still in the range of 2: 1 to 95: 1. The material according to the present invention may be in any convenient form, such as powder, microspheres, flakes, mat or film. However, preferably, the material according to the present invention is in the form of semisolid ointment or gel for local application, or sponge dried by freezing or dried with solvent. Preferably, the wound healing medicament is semisolid ointment or sterile packaged gel for local application to a chronic wound, wherein said ointment contains 0.05% to 50% v / v oxidized cellulose. Preferably, the ointment contains from 0.1% to 20% v / v, more preferably 1% to 10% v / v, of the oxidized cellulose in a pharmaceutically acceptable carrier. Suitable carriers include: hydrogels containing cellulose derivatives, including hydroxyethylcellulose, hydroxymethylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose and mixtures thereof; and hydrogels containing polyacrylic acid (carbopoles). Suitable carriers also include creams / ointments used for local pharmaceutical preparations, for example creams based on cetomacrogol emulsifying ointment. The above vehicles can include arginases (as a thickener or stimulant), preservatives such as benzyl alcohol, regulators for controlling pH such as disodium acid phosphate / sodium diacid phosphate, osmorality adjusting agents such as sodium chloride and stabilizers such as EDTA Preferably, the oxidized cellulose in the ointment is converted to a protein complex, as described herein. Alternatively, the wound healing material is a bioabsorbable sponge dried by freezing or drying with solvent for its application to a chronic wound, the sponge containing from 0.1% to 50% w / w of oxidized cellulose from 50% to 99.9% w / w of one or more structural proteins. Preferably, the average pore size of the sponge is in the region of 10-50 microns, more preferably about 100-300 microns. Suitable proteins for the complex conversion to oxidized cellulose in this invention include structural proteins such as fibronectin, fibrin, laminin, L0 elastin and collagen. Preferably, the protein contains collagen and more preferably consists essentially of collagen. The collagen can be collagen obtained from any natural source, including microbiological sources, but it is preferably collagen obtaining the bovine corium that has been largely released from non-collagenous components, for example fat, non-collagenous proteins, polysaccharides and other carbohydrates as described in US patents Nos. 4614794 and 4320201, the entire contents of which are incorporated herein by reference. The collagen can be type I, II or III collagen, or it can also be chemically modified collagen, for example a pre-collagen that obtains it by separating the immunogenic telopeptides from the natural collagen. The collagen may also contain fragments of solubilized collagen or soluble collagen having molecular weights in the range of 5,000-100,000, preferably 5,000-50,000 obtained by treatment with collagen pepsin. natural in a known way. Preferably, the oxidized cellulose contains oxidized regenerated cellulose (CRO). The oxidized regenerated cellulose (CRO) can be obtained by the process described in the patent of E.U.A. No. 3122479, the entire contents of which are incorporated herein by reference. This material offers numerous advantages including the characteristics of which it is biocompatible, biodegradable, non-immunogenic and easily obtainable in commerce. The CRO is obtainable with degrees of oxidation, and consequently degradation rates, variants. The CRO can be used in the form of insoluble fibers, including interlaced, non-interlaced textile and knitted fabrics. In other preferred embodiments, the CRO is in the form of water-soluble low molecular weight fragments which is obtained by alkaline hydrolysis of CRO. The easy availability of both collagen and CRO having a range of controllable properties means that the properties of the materials used in the present invention can be controlled to the exceptional degree. In particular, the speed of biological absorption, porosity and density of the materials can be controlled. It has also been discovered, surprisingly, that the oxidized protein / cellulose complexes that are used in the preferred aspect of the present invention have an excellent ability to bind to growth factors, in particular, the growth factor derived from platelets. Accordingly, the present invention also provides the use of oxidized cellulose or complexes thereof with a structural protein to bind to one or more cell growth factors. Preferably, the cell growth factor is the platelet derived cell growth factor (FCDP). The present invention further provides a method for separating cell growth factors from a biological sample or organism, the method comprising: (i) contacting the biological sample or organism with a material containing oxidized cellulose or a complex thereof with a structural protein as defined hereinbefore, contact being carried out in vivo or in vitro, to bind the growth factors of the material; and (ii) recover the material the linked growth factors. The present invention further provides a method for preparing an active wound healing material comprising the step of: (i) contacting the oxidized cellulose with a complex thereof with a structural protein as defined hereinabove with a medium biological that contains cell growth factors to bind cell growth factors to the material; (ii) wash and dry the material that the cell growth factors bound thereto to form active wound healing material. Preferably, the cell growth factors include the platelet derived growth factor. In another aspect, the present invention provides a method for treating a chronic wound in a mammal, such as a cuvital ulcer, a venous ulcer or a diabetic ulcer. The method consists in applying to the wound a healing material containing oxidized cellulose or a complex thereof with a structural protein as defined hereinbefore. Preferably, the oxidized cellulose is applied to the chronic wound for a period of at least 1 hour, more preferably at least 6 hours, and more preferably at least 12 hours. The treatment with oxidized cellulose can be prolonged for several days or weeks, with changes of oxidized cellulose healing material as appropriate, if necessary for chronic wounds. This is in contrast to the CRO hemostatic applications, which typically last only a few seconds or minutes. Without wishing to be bound by any theory, it is believed that oxidized cellulose and complexes thereof promote the healing of chronic wounds in at least one of the following manners. First, oxidized cellulose binds to growth factors, such as PDGF, EGF and FGF, to retain these growth factors at the site of growth. the wound. Otherwise, such growth factors tend to be remote from the site of the wound along with the wound exudate. The gradual decomposition of oxidized cellulose at physiological pH results in the gradual release of growth factors back to the wound. A second reason is that the oxidized cellulose is completely bioresorbable and physiologically acceptable. A third reason may be that the fragments of oligosaccharides produced by the decomposition of the oxidized cellulose themselves promote the healing of chronic wounds in vivo. Preferably, the chronic wound of the group consisting of venous ulcers, decubital ulcers and diabetic ulcers is selected. Preferably, the chronic wound is substantially or completely non-bleeding. The term "chronic wound" does not encompass a disorder or periodontal disease. The protein / oxidized cellulose complexes that are used in the present invention can be made by a process consisting of the steps of: providing an aqueous dispersion of a protein; immersing or dispersing oxidized cellulose in the aqueous dispersion; continue extracting water from the aqueous dispersion to leave protein-containing material that forms a complex with oxidized regenerated cellulose. Optional additional components in the materials according to the present invention are preferably included in the aqueous dispersion prior to the extraction of water from the aqueous dispersion.

Preferably, the pH of the dispersion is adjusted to pH 3-4.5. This pH range is less than the isoelectric pH of the collagen. The water can be extracted from the aqueous dispersion by evaporation, for example by evaporation of the dispersion in a tray to leave a film of material. However, preferably, the water is extracted by drying by freezing (lyophilizing) or drying with solvent to produce the material in the form of a sponge. Preferably, the aqueous dispersion contains 5-30 mg / ml of collagen. Preferably, the collagen-based sponge is formed by lyophilization substantially as described in US-A-2157224. Preferably, the method further comprises treating the protein and the polysaccharide in the dispersion or in the dried material with an interlacing collagen, such as carbodiimide, hexamethylene diisocyanate (HMDI) or glutaraldehyde. Alternatively, it can be carried out in entanglement dehydrothermally. The entanglement method can significantly affect the final product. For example, HMDI intertwines the primary amino groups on the protein within the complex, while the carbodiimide binds the carbohydrate that is on the CRO to the primary amino groups on the protein. Oxidized cellulose can be added to the aqueous dispersion of protein in the form of suspension or solution of the oxidized cellulose, preferably at a pH comparable to the collagen suspension, followed by mixing by agitation or homogenization. Alternatively, fibers or oxidized cellulose fabric can be immersed in the aqueous collagen dispersion. More specific embodiments of the present invention will now be described more extensively, by way of example, with reference to the accompanying drawings, in which: Figure 1 shows a graph of the growth of fibroblast cells (in arbitrary units) in films treated with serum consisting of (a) collagen solubilized with pepsin, (b) collagen solubilized with pepsin that form a complex with fragments of CRO having an average molecular weight of 8,000, (c) collagen solubilized with pepsin that form a complex with fragments of CRO having an average molecular weight of about 20,000, and (d) collagen solubilized with pepsin that form a complex with heparin sulfate (by comparison). Figure 2 shows a graph of the percentage of binding of platelet derived growth factor (FCDP) to the following materials: (a) plastics, collagen sponge, (c) collagen sponge / solubilized CRO, (d) CRO INTERCEED ( RTM) and the ease of extracting this factor from the growth of materials; and Figure 3 shows a graph of the relative amounts of MMP bond measured for collagen sponge and textile material from CRO SURGICEL (RTM) (comparative measurements) and for collagen sponges that form complexes with 10% by weight, 20% by weight and 30% by weight of fibrous CRO.

EXAMPLE 1 Preparation of a collagen sponge / fibrous CRO The lyophilized collagen, prepared as described in the US patent, is resuspended. No. 4614794 of 4320201, in cold acetic acid at 0.05 M at a concentration of 10 mg / ml. Ground CRO powder (ground Surgicel textile material) is added to the suspension in a 1: 3 ratio of CRO: collagen and homogenized using a Waring blender at low speed for 3 x 30s. The complex suspension is degassed in a vacuum oven for 10 minutes and then poured to a depth of 3 mm and frozen by burst. The frozen suspension is then dried by freezing and cross-linked dehydrothermally using a programmable Edwards freeze dryer with a means to rapidly raise the temperature or dried using a solvent drying process as described in US-A-2157524.

EXAMPLE 2 Preparation of a collagen / oliqosaccharide sponge from CRO Soluble collagen is prepared by the published method of E.J. Miller and R.K. Rhodes, "Preparation and Characterization of the Different Types of Collagen", Methods Enzimol Vol. 82, pages 33-64 (1982). The CRO oligosaccharide is prepared as described in the copending patent application filed on the same date as this application and commonly assigned herein. Briefly, the CRO oligosaccharides are prepared by treating CRO commercially available with 6M sodium hydroxide solution at 37 ° C for 45 minutes, followed by neutralization and dialysis to extract fragments and impurities having molecular weight less than 1000. The oligosaccharides The resulting soluble CROs are slowly added while stirring to soluble collagen (.075 mg / ml) (both in acetic acid cooled to 0.05M) until the precipitation of the complex no longer occurs. The complex precipitated product is isolated by centrifugation, the phosphate buffer is washed at pH 7.2 and resuspended by homogenization at 30% v / v in the same regulator. The suspension is poured to a depth of 3 mm, frozen by burst at -30 ° C and freeze-dried.

EXAMPLE 3 Preparation of a collagen film / CRO A collagen / CRO film is made for application to a wound, by methods described in any of the first two examples, except that the suspensions are not precipitated, but are air dried, instead of frozen and dried by freezing. To prepare flexible films, a small amount of glycerol can be added to the suspensions.

EXAMPLE 4 Preparation of a collagen sponge / CRO using a pre-chelation procedure of the CRO CRO (SURGICEL) textile material (4% v / v) is suspended in dilute alkali (NaHC? 3) for a time that is sufficient to convert the textile material into gelatinous mass. The diluted collagen paste is added to the same pH to give a final solids content in both the Surgicel® and the 1% v / v collagen. The diluted paste is stirred and the pH is adjusted to pH 3.0-4.0 using acetic acid. The final diluted paste is molded, frozen and dried by freezing under vacuum.

EXAMPLE 5 A suitable wound healing material is prepared for application to a venous ulcer, a decubital ulcer or a diabetic ulcer, cutting 5 cm by 5 cm of INTERCEED textile material (RTM) obtained from Johnson & Johnson Medical, Inc.

EXAMPLE 6 A gel is prepared for treating wounds, for local application to a wound, as follows. SURGICEL textile material (RTM) is crushed through a 1 mm screen and the resulting fibers are dispersed to a concentration of 3.0% w / w in a 3.0% w / w aqueous carboxymethyl cellulose (CMC) gel. The gel is packaged in a metallized polymer bag and sterilized by gamma-irradiation.

EXAMPLE 7 The neutralized CRO wound healing material suitable for application to a venous ulcer, ulnar ulcer or diabetic ulcer was prepared as described in FIG.

EP-A-0437095. In short, a quantity of 71.9 grams of oxidized regenerated cellulose textile material (CRO) with a carboxylic acid content of 15.2% obtainable as INTERCEED (RTM) from Johnson S. Johnson Medical, Inc., around a drilled core and placed in a reactor equipped with a circulating pump. The reactor was filled with a mixture of 3 liters of water and 2 liters of methyl alcohol. A Brookfield EX-100 for constant temperature bath was lit, as a built-in pump for the external circulation of solutions, to pump the solvent through the center of the perforated core and through the fluff of textile material, wrapped around the core. The solvent flowed through the outlet line and recirculated again through the pump and back to the core. A stocheometric amount of sodium acetate trihydrate equal to the number of moles of carboxylic acid on the textile was added to the solution, i.e. 71.9 x 15.2% = 10.92 grams of carboxylic acid, equivalent to 33.05 grams of sodium acetate trihydrate. 33.05 grams of sodium acetate trihydrate was added to the aqueous alcohol solution and circulated by pump around and through the textile material for 30 minutes. After 30 minutes, the pH of the circulating solution reached a constant value (pH 4.6). The textile material was then removed from the reactor and placed in 600 ml of metal for 10 minutes. The methanol was removed and replaced with another 600 ml of fresh methanol. After the second wash, the textile material was hung and dried with air. A piece of fabric of 5 cm x 5 cm was cut for use as wound healing material according to the present invention. The advantageous properties of the materials according to the present invention, prepared as above, were determined as follows: PROCEDURE 1 Promotion of fibroblast cell growth An alkali-soluble collagen / CRO complex film was prepared in a petri dish as described in Examples 2/3 and serum was poured onto the film and covered at 37 ° C overnight. The serum was removed and the effects of fibroblast cell growth were measured (Figure 1). Cell growth was observed for a film (comparison standard) of collagen solubilized with pepsin (CSP), the CSP oligosaccharides having more CRO average molecular weights of about 8000 to about 20,000, prepared as described above and CSP plus heparin was included as a pattern of positive cooperation. The results show that the collagen film / CRO fragments appear to bind to serum factors that stimulate cell growth.

PROCEDURE 2 Linkage of platelet-derived growth factor Studies were carried out on the FCDP link, as follows: Two small actions of test material were weighed (approximately 1 cm of CRO INTERCEED (RTM) cloth and sections of 1 cm x 0.5 cm x 0.4 cm sponge of collagen) and soaked in dibasic regulator of 100 mM sodium phosphate containing 150 mM sodium chloride (total volume of 1 ml) for at least 1 hour at room temperature. Samples were then incubated with 2% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 2 hours at room temperature. 22 ng of FCDP were then added to each sample in 250 μl of PBS containing 2% ASB and then the samples were incubated for another hour at 37 ° C. Each sample was then washed three times with 250 μl of PBS, followed by increasing concentrations of sodium chloride. Finally, each sample was washed with urea at 4.0 M. FCDP ELISA analysis of the original preparation of FCDP and the various washes were carried out. The data shown in Figure 2 indicate that the growth factor can be fully recovered from the mixed body of collagen and CRO, whereas the individual components do not appear to release the entire growth factor. The link characteristics are also singularly different for the collagen / CRO complex compared to the individual components. These observations indicate that the complex has unique FCDP binding which can be used appropriately for both exogenous binding and for endogenous binding and the release of growth factor.

PROCEDURE 3 Matrix metalloproteinase bond The effect of the formation of a complex between collagen and CRO on the binding of matrix metolaproteinase (MPM) was evaluated as follows. Collagen / CRO fibrous sponges containing 0%, 20% and 30% by weight of fibrous CRO were prepared by the procedure described in Example 1. A non-CRO collagen sponge was prepared for comparison purposes. A sample of CRO SURGICEL (RTM) fabric was also prepared, as a comparison. Briefly, 50 mg of each material was placed in a 15 ml plastic vessel containing 2.5 ml of an acute wound fluid, diluted 1:50 in a proteolysis buffer (tris / HCl at 50 mM at pH 7.8, CACI2 at 50 mM, NaCl at 0.5 M) and incubated at 37 ° C on a shaking water bath for 3 hours. The acute wound filter contains several proteinases, including matrix metalloproteinases and many of these enzymes will be linked to various materials of the healing material. The excess fluid absorbed by each material was mechanically expressed, using a metal spatula, and discarded. The remaining healing materials were approximated in pre-packaged 2 ml syringes (each syringe contained 0.5 ml volume of 2.5 ml glass beads). 4 ml of the proteolysis buffer was passed through the syringe into a 1 ml aliquot that was discarded. In this washing step, all the unbound proteinases and the proteinase that were only weakly bound to the healing material had been removed from the healing material leaving the linked forms more compactly. The rinsed curative materials with pH buffer were then removed to another 15 ml plastic vessel. 1 ml of non-denatured sample pH buffer (6.3 ml of tris-HCl, ca 0.05 M at pH 6.8, 2.5 ml of glycerol, 0.5 grams of SDS, 16.2 ptl of water and bromophenol blue) was added to each sample. was placed on an orbital shaker in graduation 6 for 2 hours. The sample pH agitator after the tightly bound preteinases of the materials that are present in the same sample pH regulator. After this time, 20 μl of pH regulator was taken from the sample of each container and subjected to SDS-polyacrylamide gel electrophoresis with gelatin substrate (simography), as described by Heussen C and Dowdle E.B., Anal. Biochem.102: 196-202 (1980). The areas of the individual free space zones on the gels, which are due to the activity of the proteinase, were measured exactly by the Optilab system. This was achieved by repeating each link experiment (n = 3) and results were statistically analyzed by student T-test, where P < 0.05. The analysis was regarding the controls of pure collagen. The results, shown in Figure 3, show a surprising synergistic improvement in MPM binding for collagen complexes with CRO. The 0 data were presented for the proenzyme forms (PR02 and PR09) of the matrix metalloproteinase 2 (gelatinase A) and the matrix metalloproteinase 9 (gelatinase B). Without saying to be limited by the theory, it is thought that the improvement may be related to the neutralization of opposite electrostatic changes on the collagen and the CRO by the formation of complexes.

I PROCEDURE 4 The link of the purified growth factors to the natural CRO and naturalized CRO fabric was investigated as follows. 250 μm samples of FCDP, EGF and FGF were added to 1 cm x 1 cm squares of INTERCEED (RTM) and neutralized Interceed (from example 2) and the amount of the factor was measured. of growth not linked by CLAR. The study investigated both the effect of ionic intensity and the effect of carboxylation in the growth factor binding. The CRO materials were incubated with growth factor for 1 hour at 37 ° C, then washed with 2 x 1 ml of NaCl at 0.05 or 0.03 M for 5 minutes. The solutions were collected, pooled and valued for the growth factor by CLAR. The results are shown in table 1.

TABLE 1 1 1 1 | Interceed ™ PH | %% EGF% FGF% FCDP | 1 1 COOH linked linked linked | I 1 Low High Low High Low High | content content content content content content | of salt of salt of salt of salt of salt of salt | 1 1 | Interceed ™ 2.0 | 15% 97 87 100 100 97 95 | 1 1 | Interceed ™ 2.0 | 6% 97 33 91 89 98 64 | | nTC7 5.4 | 6% 0 or or 100 84 100 84 | 1 l l The results show that between 85 and 100% of FCDP, FGF and EGF is linked to INTERCEED. However, only FGF and EGF bind to neutralized INTERCEED. It can be postulated that EGF does not bind due to the difference in the isoelectric points of the growth factors FCPD = 9.6, FGF = 10 and EFG = 4.8). The above examples are designated for the purpose of illustration only. Many other modalities that fall within the scope of the accompanying claims will be evident to the expert reader.

Claims (24)

NOVELTY OF THE INVENTION CLAIMS

1. - Use of oxidized cellulose for the preparation of a medication for the treatment of a chronic wound.

2. Use according to claim 1, further characterized in that the oxidized cellulose is in the form of an interlaced fibrous body, not interlaced or knitted, which is insoluble in the wound fluids.

3. Use according to any preceding claim, further characterized in that the oxidized cellulose is in the form of dispersed fibers or powders.

4. Use according to claim 3, further characterized in that said fibers or powder are dispersed in a semi-solid vehicle for local application.

5. Use according to any preceding claim, further characterized in that the oxidized cellulose has an average molecular weight greater than 50,000.

6. Use according to any preceding claim, further characterized in that the oxidized cellulose consists of oxidized regenerated cellulose (CRO).

7. Use according to any preceding claim, further characterized in that the oxidized cellulose forms a complex with the structured protein.

8. Use according to claim 7, further characterized in that the oxidized cellulose and the structural protein (if present) together constitute at least 75% by weight of the material.

9. Use according to claim 8, further characterized in that the oxidized cellulose and the structural protein (if present) together constitute at least 90% by weight of the material.

10. Use according to claims 7, 8 or 9, further characterized in that the material is a sponge dried by freezing or drying with solvent.

11. Use according to claims 7, 8 or 9, further characterized in that the material is a solid film.

12. - Use according to copy of claims 7 to 11, further characterized in that the weight ratio of the protein to the oxidized cellulose is 1:99 to 99.99: 1.

13. Use in accordance with copy of claims 12, further characterized in that the weight ratio of the protein to the oxidized cellulose is from 1:10 to 99.9: 1.

14. - Use in accordance with copying claims 7 to 13, further characterized in that the protein consists of collagen, fibronectin, fibrin, laminin or elastin.

15. Use according to claim 14, further characterized in that the protein consists essentially in collagen.

16. Use according to claim 14 or 15, further characterized in that the protein consists of soluble collagen, partially hydrolyzed, having molecular weights in the range of 5,000-100,000.

17. Use according to claim 14 or 15, further characterized in that the collagen is substantially insoluble, fibrous collagen.

18. Use according to any preceding claim, further characterized in that the oxidized cellulose consists of water-soluble oxidized cellulose fragments, having molecular weights in the range of 5,000-50,000.

19. Use according to any preceding claim, further characterized in that said chronic wound is selected from the group consisting of venous ulcers, decubital ulcers and diabetic ulcers.

20. A method for separating cell growth factors from a biological sample or organism, said method consisting of: (i) contacting said sample or biological organism with a material consisting of oxidized cellulose with a structural protein, taking place contact in vivo or in vitro, to enhance growth factors to the material; (ii) recover the linked growth factors of the material. 21.- A method to prepare a material with active pair wounds that consists of the steps of: (i) putting in contact a material consisting of oxidized cellulose or a complex of oxidized cellulose with a structural protein with a biological medium containing cell growth factors to enhance cell growth factors to the material: and (ii) washing and drying the material that has the factors of cell growth bound thereto to form said active wound healing material. 22. - A method according to claim 20 or 21, further characterized because the cell growth factors consist of the platelet derived growth factor. 23. Use according to claim 1, further characterized in that said drug is semisolid ointment or sterile packaged gel for local application to a chronic wound, wherein said ointment contains 0.05% to 50% w / v of said cellulose rusted 24. Use according to claim 1, further characterized in that said medicament is a bioabsorbable sponge for application to a chronic wound, said sponge containing from 0.1% to 50% w / w of said oxidized cellulose and from 50% to 99.9 % of one or more structural proteins.