WO2010046413A2 - Eponge de kératine à usage pharmaceutique - Google Patents

Eponge de kératine à usage pharmaceutique Download PDF

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Publication number
WO2010046413A2
WO2010046413A2 PCT/EP2009/063834 EP2009063834W WO2010046413A2 WO 2010046413 A2 WO2010046413 A2 WO 2010046413A2 EP 2009063834 W EP2009063834 W EP 2009063834W WO 2010046413 A2 WO2010046413 A2 WO 2010046413A2
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WO
WIPO (PCT)
Prior art keywords
keratin
solution
sponge
aqueous
alkaline
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PCT/EP2009/063834
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German (de)
English (en)
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WO2010046413A3 (fr
Inventor
Stephan Reichl
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Technische Universität Braunschweig
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Publication of WO2010046413A2 publication Critical patent/WO2010046413A2/fr
Publication of WO2010046413A3 publication Critical patent/WO2010046413A3/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/425—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges

Definitions

  • the present invention relates to a sponge of keratin and its use for pharmaceutical purposes, a process for its preparation and the use of the keratin sponge for the manufacture of products for medical use, for example for use in wound care or as a support framework for tissue to be formed, e.g. for an implant.
  • the keratin sponge according to the invention is capable of swelling in aqueous compositions and can therefore also be used as wound tamponade. Furthermore, the keratin sponge according to the invention is not cytotoxic and can be populated over the entire cross section of animal cells, so that the keratin sponge is suitable for implantation in the animal or human body, for example as an implant for filling defect sites, optionally with immunocompatible cells, preferably with autologous cells populated. State of the art
  • WO 99/26570 describes open-celled foams of keratin, for example by spraying or freeze-drying a keratin solution.
  • the keratin solution is generated under reducing conditions so that disulfide bridges are reduced to thiol groups.
  • oxidation of the keratin is carried out for crosslinking. Heating of the dry keratin does not take place.
  • Tanaga et al. (Materials Science and Engineering C24, 441-446 (2004)) describe the generation of a film of reduced keratin solution which has been treated with chemical crosslinking agents.
  • the keratin solution is according to Yamauchi et al. (Journal of Biomedical Materials Research 439-444 (1996)) and Yamauchi et al. (Journal Biomaterials Science Polymer Vol. 9, No. 3, 259-270 (1998)) by solubilizing wool under reducing conditions and as described by Yamauchi et al. previously described, including the reducing agent dried, for example by freeze-drying.
  • WO 01/19305 describes the preparation of swellable oxidized keratin in which disulfide bridges have been treated with a peroxide to prevent the formation of disulfide bridges in the finished gel.
  • EP 0 628 573 A1 describes the preparation of keratin films and fibers from a keratin solution prepared in the presence of a reducing agent and subsequently oxidized in the presence of thioglycolic acid to form a mixed disulfide, a carboxymethyl disulfide.
  • the protein obtained is water-soluble.
  • EP 1 470 824 A1 describes in one example the extraction of keratin from skin biopsin and hydrolysis of hair in the presence of a reducing agent, followed by pelleting, resuspension of the pelleted material and its dialysis for use as antigen.
  • keratin sponges Compared to known keratin sponges, it is an object of the invention to provide an alternative keratin sponge and an alternative method for its production.
  • a pharmaceutical i.e.. to provide for medical use suitable keratin sponge which is swellable, and e.g. can be used as an absorber for exudate water.
  • the invention achieves the above object with the features of the claims, in particular by providing a keratin sponge made from a keratin solution prepared under reducing conditions from natural keratin, preferably human hair keratin, and from which reducing compounds are removed, for example by dialysis against an aqueous medium without reducing ingredients.
  • the keratin sponge according to the invention preferably consists of keratin, which is solubilized without additional chemical derivatization in aqueous solution, optionally partially hydrolyzed partially alkaline, and freeze-dried without the presence of reducing and / or oxidizing agent from aqueous solution in the presence of a polyhydroxy compound and then to about 100%. 150 0 C was heated in the air.
  • the keratin sponge comprises a mixture of a first keratin solution prepared under reducing conditions from natural keratin and from which reducing compounds have been removed, for example by dialysis against an aqueous medium without reducing ingredients at neutral to slightly acidic pH, and a portion of a second keratin solution, also preferably made from natural keratin by hydrolysis under reducing conditions, removed from the reducing agent, for example by dialysis partially hydrolyzed by alkaline treatment, preferably by dialyzing the keratin solution against an alkaline aqueous composition followed by removal of the alkaline composition, for example by ultrafiltration, washing and / or dialysis, preferably against water at neutral pH.
  • keratin sponge which has a proportion of partially alkaline hydrolyzed keratin
  • a has higher absorbency for aqueous compositions, eg wound exudate.
  • solubilized hair keratin having a major proportion of about 70 to 80 +/- 10 wt .-% of about 50 to 60 kDa, and a smaller proportion with a MW of> 100 kDa.
  • Alkaline partially hydrolysed keratin suitable for the invention has a molecular weight of ⁇ 50 kDa for at least 50% by weight, preferably at least 75-80% by weight.
  • Partially hydrolyzed keratin produced by alkaline hydrolysis has, according to the preferred alkaline hydrolysis process, a proportion of about 50 to 70% by weight at ⁇ 5 kDa.
  • the portion with a MW of ⁇ 5 kDa is separated, e.g. by ultrafiltration with an exclusion limit of about 5 kDa, and the remaining higher molecular weight fraction is used.
  • the higher molecular weight portion of the partially alkaline hydrolysed keratin used has a MW of at least 5 kDa, e.g.
  • the proportion of alkaline partially hydrolyzed keratin therefore preferably at least 25 to 35 wt .-% with a MW of 5 to 10 kDa, optionally another 3 to 10 wt .-% with a MW of 10 to 30 kDa, further optionally further 5 to 12 wt .-% with a MW of 30 to 50 kDa.
  • the partially alkaline hydrolyzed keratin is obtainable by dialysis of hydrolyzed keratin according to the invention against 0.1 M sodium hydroxide solution at about 4 ° C. for 4 to 24 hours and separation of a fraction of low molecular weight.
  • a low molecular weight fraction of up to 3 kDa, more preferably 5 kDa, or up to 10 kDa or up to 30 kDa is separated off.
  • the keratin sponge of the present invention consists of reduced keratin, which is formed from a solution of reduced keratin which does not contain a reducing agent and no Contains oxidizing agent, followed by oxidation solely by contact with oxygen, for example in admixture with nitrogen, for example air, wherein preferably the keratin solution contains a proportion of reduced keratin, also in the absence of reducing agent, which is partially hydrolyzed alkaline.
  • the keratin sponge according to the invention particularly preferably consists of a skeleton of this keratin or of the mixture of keratin with partially alkaline hydrolyzed keratin, particularly preferably with an addition of a plasticizer which is in particular selected from polyhydroxy compounds, in particular glycerol and propylene glycol.
  • the content of polyhydroxy compound is in particular 0.5 to 3 wt .-% per 15 to 30 mg of keratin, measured as total protein according to Bradford, preferably 1 to 2 wt .-% polyhydroxy compound per 20 mg of keratin measured as a whole protein according to Bradford.
  • the keratin sponges of the present invention may be prepared by conventional methods of forming a porous structure from a solution of keratin, preferably by freeze-drying an aqueous solution of keratin, preferably by freeze-drying an aqueous solution of the mixture of keratin with a proportion of partially alkaline hydrolyzed keratin, preferably has a pH of 9 to 11, preferably 10.
  • partially (partially) alkaline hydrolyzed keratin is also referred to as alkaline partially hydrolyzed keratin.
  • the keratin sponges according to the invention are characterized by a high dimensional stability for a long time within the animal body and are essentially not biodegradable after being implanted in an animal body.
  • these keratin sponges preferably made by the method of the present invention, are for use in the preparation of wound tamponades, e.g. for the absorption of exudate, or for use in the manufacture of implants, since these sponges can be colonized over their entire volume or over their entire inner surface of cells of the implant recipient.
  • the keratin sponges according to the invention are capable of swelling in aqueous compositions, wherein the swelling capacity increases with increasing content of alkaline partially hydrolyzed keratin, so that the swellability is adjustable by the proportion of alkaline partially hydrolyzed keratin on the total keratin.
  • the keratin sponges according to the invention are colonized by their entire volume of cells which adhere within the open pore structure. Accordingly, the sponges according to the invention are suitable for use as builder substance for in vitro cultivation of animal cells in three-dimensional structure and in particular for use as an implant for filling defect sites, for example tissue, in particular soft tissue of the animal body, including man.
  • the preparation method according to the invention comprises a hardening of the keratin, which is hydrolyzed in water, optionally additionally partially alkaline hydrolyzed proportionally, and removed by freeze-drying from aqueous composition, from which reducing agents and / or oxidizing agents are removed and therefore not contained, in the presence of a Polyhydroxy compound by heat treatment, for example at 100 0 C to 160 0 C, preferably to 100 to 110 0 C for 0.5 to 1.5 h.
  • a Polyhydroxy compound by heat treatment for example at 100 0 C to 160 0 C, preferably to 100 to 110 0 C for 0.5 to 1.5 h.
  • the keratin sponge may be subjected to sterilization by ⁇ -irradiation.
  • the keratin sponges according to the invention have no harmful substances for the cell culture, for example no cytotoxic substances and no viruses.
  • tests on the cultivation of animal cells on the sponge showed no contamination of the sponges with cell-damaging agents and the absence of viruses. Therefore, the production method according to the invention, for example, starting from human hair in the production of keratin solution, allows the use for the production of a porous implant structure which is free of viruses and free of cytotoxins.
  • keratin sponges according to the invention have particles of keratin of approximately the same size, eg monomodal by about 50 -250 nm, which promote colonization by cells.
  • the monomodal size distribution of the particles identifiable in inventive keratin sponges is, in particular, a particle size in the size range of 50 up to 250 nm, preferably with a majority of the particle sizes around the mean value, eg around 150 nm in the manner of a Gaussian distribution.
  • the keratin sponges according to the invention have a high structural stability and a better resistance to degradation after implantation in the animal body and / or are populated faster and more uniformly by animal cells, so that when used for the production of an implant, they become an implant resulting in faster formation of natural tissue within the implant structure.
  • Keratin sponges that were prepared without polyhydroxy compound under otherwise identical conditions, a high brittleness, or low structural stability, and a significantly lower water resistance than inventive keratin sponges. It has been found in preliminary experiments that keratin sponges which were produced without a final heating step, for example in accordance with WO 99/26570, or with a heating step, but without a polyhydroxy compound, are not structurally stable in an aqueous medium. In particular, sponges prepared without polyhydroxy compound and without heating step disintegrate in an aqueous medium. In contrast, keratin sponges produced using the method according to the invention are structurally stable even in an aqueous medium. This can e.g. be recognized that electron micrographs of sponges according to the invention show substantially the same structure for sponges, which are analyzed immediately after preparation, or after contacting with aqueous medium and subsequent drying.
  • keratin sponges which are produced under otherwise identical conditions from a keratin solution which, however, still contains reducing agents, or an oxidizing agent, for example a peroxide.
  • the sponges of the present invention have better mechanical and absorption properties over such sponges made from a keratin solution which does not contain a proportion of partially alkaline hydrolyzed keratin, but e.g. essentially only solubilized keratin of> 50 or> 60 kDa.
  • the keratin solution used to make the keratin sponge by removing the solvent, especially water contains no reducing compounds, no oxidizing compounds, and preferably has an approximately neutral pH.
  • the keratin solution used for the production of keratin sponges is particularly preferably an aqueous composition which has reduced keratin, preferably a proportion of alkaline hydrolyzed reduced keratin in water.
  • a keratin solution i. the reduced keratin solution or a reduced keratin reduced keratin solution which is partially alkaline hydrolyzed (also referred to as partially alkaline hydrolyzed) is dialysed against pure water, preferably at pH 6.5-7.5.
  • aggregates of the particles are removed from the aqueous keratin solution, e.g. by ultracentrifugation at 10,000 x g, optionally by filtration.
  • the keratin used to prepare the solution is ⁇ -keratin;
  • the keratin solution is made from hair, such as human hair.
  • an aqueous composition consisting of thiourea, urea and mercaptoethanol in aqueous solution, it being possible for the mercaptoethanol to be replaced by another reducing compound.
  • the reducing agent-free aqueous keratin solution is obtained by subsequently removing the reducing agent initially used to prepare the solution, e.g. by replacing the solvent medium with water without reducing agent, in particular by dialysis or ultrafiltration.
  • a further advantage of the keratin sponges according to the invention is that they are at least partially optically transparent, and allow a simple optical control of the colonization with cells, in particular after staining of the cells, e.g. by microscopy in the areas close to the surface of the sponge.
  • keratin sponges having a monomodal size distribution of keratin particles whose major value is in the range of 20 to 5,000 nm, preferably 100 to 1,000 nm, more preferably 100 to 200 nm, are produced from the keratin solution used in the invention for producing the keratin sponges.
  • the invention also relates to methods for culturing animal, in particular human cells in the presence of a sponge according to the invention, or for the use of the sponges according to the invention in the cultivation of such cells. It is currently believed that the significantly better cultivation results are due to the particular structure of the keratin sponges with their nanoparticulate structure.
  • a keratin sponge prepared by the method according to the invention which consists of a mixture of an aqueous keratin solution, which is obtained by hydrolysis of hair in denaturing aqueous solution containing reducing agent, which serves to hydrogenate disulfide bridges of keratin, followed by removal of the reducing agent from the solution, the solution containing no oxidizing agent, with a portion of the aqueous keratin solution thus prepared containing no reducing agent and no oxidizing agent which is partially alkaline hydrolyzed by drying the mixture containing polyhydroxy compound, preferably 1 to 3% by weight per 20 mg of keratin, measured as a total protein according to Bradford, wherein the mixture is converted by the drying to a porous structure.
  • the dried mixture is heated to about 100 to 150 0 C, preferably 100 to 110 0 C, for at least 1 h, preferably 2.5 h without the addition of further compounds in air.
  • the advantageous properties of the keratin sponge thus obtained are particularly evident when using the production process for medical products, for example for implants.
  • Beneficial properties include stability against degradation after implantation in the animal body, ie the high resistance to absorption, the elasticity and stability properties and the good colonization by cells which make the keratin sponges suitable for use as an implant, eg for filling defect sites in the animal body, including man.
  • the advantageous properties include, in particular, swelling capacity, for example for receiving wound exudate.
  • nanostructures in the keratin sponge according to the invention can be made visible, which are also referred to herein as nanoparticles. It is assumed that the mechanical properties and also the suitability of the keratin sponge according to the invention for colonization by animal cells are due to this nanoparticulate structure.
  • the keratin sponge contains essentially no free thiol groups after drying and before the heat treatment. It is believed that thiol groups are already fully oxidized in the dialysate of water-soluble keratin, of which one portion is preferably partially alkaline hydrolyzed, due to the absence of reducing agent.
  • the keratin sponge according to the invention therefore has a nanoparticulate microstructure with a substantially monomodal size distribution of the nanoparticles, and contains no reducing agent and / or oxidizing agent added to the keratin.
  • the keratin is prepared by drying an aqueous keratin solution containing no added reducing agents and / or oxidizing agents and containing solubilized keratin, particularly hair, in aqueous solution, preferably wherein a portion of the solubilized keratin is partially alkaline (partially) hydrolyzed. More preferably, the keratin sponge is made from keratin solution consisting of solubilized keratin in water, i.
  • the keratin sponge according to the invention contains a plasticizer, in particular selected from glycerol and propylene glycol, which is e.g. the aqueous keratin solution is added before freeze-drying and before the subsequent heat treatment.
  • a plasticizer in particular selected from glycerol and propylene glycol, which is e.g. the aqueous keratin solution is added before freeze-drying and before the subsequent heat treatment.
  • keratin sponges according to the invention retain their structural stability even in the swollen state, ie in the presence of aqueous compositions, so that, for example, absorbed liquid is essentially not released even under pressure.
  • swollen sponges give under pressure a small portion of absorbed liquid from, for example, the only capillary bound portion of the liquid, and retain the majority of the absorbed liquid.
  • keratin sponges according to the invention are solid and gel-like, so that bound liquid is essentially not released even under pressure.
  • the elastic resilience is maintained substantially in the swollen state. As the proportion of alkaline hydrolyzed keratin increases, the elastic recovery decreases.
  • FIG. 1 shows the measurement result of the photon correlation spectroscopy of the keratin solution according to Comparative Example 1 (Y axis shows intensity in percent),
  • FIG. 2 shows the measurement result of the photon correlation spectroscopy of the keratin solution according to Example 1 (Y axis shows intensity in percent),
  • FIG. 3 shows a scanning electron micrograph of a keratin sponge according to the invention with about 10% hydrolyzed keratin
  • FIG. 4 shows an enlarged scanning electron micrograph of a keratin sponge according to the invention from FIG. 3,
  • FIG. 5 shows an enlarged scanning electron micrograph of a keratin sponge according to the invention from FIG. 3,
  • FIG. 6 shows a scanning electron micrograph of a keratin sponge according to the invention with about 20% hydrolyzed keratin and
  • FIG. 7 shows a scanning electron micrograph of a keratin sponge according to the invention with about 30% hydrolyzed keratin.
  • a keratin sponge was prepared from a keratin solution by freeze-drying, which according to Yamauchi et al. (J. Biomater, Polym. Ed 9: 259-270 (1998)), but without SDS in the keratin solution.
  • human hair (20 g) was washed in 0.5% SDS solution, dried and degreased overnight in n-hexane after removal of the hexane in 360 mL of 7 M urea with 32 mL of 2-mercaptoethanol in a closed glass bottle at 50 Shake 0 C for 24 h and then filter.
  • the filtrate was dialyzed three times against 12 L of distilled water with 0.2% by weight of 2-mercaptoethanol overnight. A turbid solution of approx. 480 mL was obtained.
  • the analysis of the particle size distribution with photon correlation spectroscopy is shown in Figure 1 and gave a multimodal size distribution with a Z-average value around a particle size of about 125 nm and a polydispersity index of 0.41 due to a highly multimodal distribution.
  • the distribution curve shows three peaks at approximately 115 nm, 700 nm and 4770 nm.
  • the solution was used either directly for the production of sponges or after removal of aggregates by ultracentrifugation.
  • the solution was frozen at -20 0 C and then freeze-dried.
  • the analysis showed a lower seeding efficiency and a lower growth of animal cells in culture, a lower mechanical structural strength of the dry and water-hydrated sponge, and a low swelling capacity in connection with a small increase in volume, e.g. when taking wound exudate.
  • aqueous keratin solution to be used according to the invention, 20 g of human hair were washed with 0.5% SDS solution, dried and degreased overnight by incubation with n-hexane. After removal of the hexane, 400 ml of a 25 mM Tris solution (pH 8.5) 2 M thiourea, 5 M urea and 5% mercaptoethanol in water were added. After sealing the vessel with parafilm was stirred at 50 0 C for 72 h. Undissolved constituents were removed by centrifugation at approximately 4,500 ⁇ g (laboratory centrifuge), 10 min, 5,000 rpm).
  • the supernatant was additionally filtered through a filter having a pore size of 2.5 ⁇ m.
  • the filtrate could be stored at 4 0 C or frozen in aliquots without significant changes in the properties.
  • the filtrate was dialyzed against distilled water, eg with a Spectrapore 1 membrane (exclusion limit 6,000-8,000 Da), usually per 100 mL filtrate against 5 L water over 72 h with 6-fold replacement of the water.
  • the dialysate was centrifuged in an ultracentrifuge at 15,000 rpm for 10 min to remove aggregates.
  • the protein content of this keratin solution was about 20 mg / mL (Bradford).
  • the centrifugate can be used directly for the production of keratin sponges, preferably by freezing the solution and subsequent freeze-drying.
  • a portion of the keratin solution described above was hydrolyzed alkaline, for example by dialysis against 0.1 M sodium hydroxide solution at 4 0 C.
  • the resulting alkaline dialysate was purified by ultrafiltration (exclusion limit 5000 Da, filter available under the name Vivaspin) and with 0.1 mM sodium hydroxide solution, the remaining pH was 10.
  • the dissolved keratin was partially hydrolyzed.
  • the protein content was determined to Bradford to 25 to 30 mg / mL.
  • the mixtures of the keratin solution shown in the table below were prepared with the alkaline hydrolyzed keratin solution and freeze-dried after freezing.
  • the polyhydroxy compound in each case 1% by weight of glycerol per ml of keratin solution was added to the keratin solution.
  • the weight gain was measured as drained weight after immersion in water.
  • For the sponge formation about 1 mL of the mixture of Keratinrawen with 1 wt% glycerol was frozen in cylindrical tubes at -20 0 C, freeze-dried and then heated at 110 0 C for about 2.5 hours.
  • FIG. 3 shows an edge length of the image of 1,800 ⁇ m) a sponge which is produced from a keratin solution with 10% by volume of alkaline partially hydrolyzed keratin solution,
  • Figure 4 shows a detail of Fig. 3, edge length of the figure 450 microns
  • Figure 5 shows a detail of Fig. 3 with an edge length of the image of 9 microns.
  • Figures 3, 4 and 5 show that the structure of the sponge is very uniform, and the pore walls are smooth and closed in itself, wherein in Figure 5, the nanoparticulate surface structure is clear.
  • FIG. 6 shows a scanning electron micrograph of a sponge produced from a mixture of keratin solutions containing 20% by volume of alkaline hydrolyzed keratin solution
  • FIG. 7 shows a scanning electron micrograph of a sponge consisting of a mixture with 30% by volume. alkaline partially hydrolyzed keratin solution was prepared.
  • the images of Figures 6 and 7 each have an edge length of 1,800 microns.
  • FIGS. 6 and 7 show that a higher proportion of alkaline partially hydrolysed keratin solution produces larger pores in the keratin sponge, but the self-contained surface of the pore walls is retained.
  • the sponges shown in Figures 3-7 were analyzed without intermediate contact with water. Equally prepared sponges showed in the REM analysis in Substantially identical structures, if the sponges were previously introduced in aqueous medium and dried after complete wetting and swelling, for example at ambient temperature or at the drying temperature.
  • the cultivation of cells can be carried out in vitro with the sponge according to the invention by incubation of the sponge in cell culture medium under cell culture conditions with added animal cells.
  • approximately 100,000 human stromal keratocytes were incubated with a sponge prepared according to the invention (10% by volume of alkaline partially hydrolyzed keratin) in standard medium for three days.
  • a keratin sponge was treated the same, but without adding cells.
  • MTT yellow tetrazolium salt
  • FIG. 8 shows a photograph, namely the control without cells on the left in the image, and on the right in the image the sponge incubated with cells after conversion of MTT. It becomes clear that the livid staining occurs through the entire volume of the sponge according to the invention, which indicates a complete colonization of the keratin sponge by its entire volume.
  • the sponge of the present invention is colonized by its total volume of cells, preferably faster and / or more uniformly than sponges made from keratin solution containing a reducing or oxidizing agent or prepared without heating the dried structure.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Materials Engineering (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Dispersion Chemistry (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Materials For Medical Uses (AREA)
  • Manufacture Of Porous Articles, And Recovery And Treatment Of Waste Products (AREA)

Abstract

L'invention porte sur une éponge de kératine, qui est fabriquée à partir d'une solution de kératine, laquelle est préparée dans des conditions réductrices à partir de kératine naturelle, de préférence de la kératine de cheveux humains, et dont on a éliminé les composés réducteurs, par exemple, par dialyse contre un milieu aqueux sans constituants réducteurs. L'éponge de kératine selon l'invention est, de préférence, constituée de kératine, qui sans transformation en dérivé chimique est solubilisée en solution aqueuse, et en option est en partie hydrolysée dans des conditions alcalines, et sans la présence de réducteurs et/ou d'oxydants provenant de la solution aqueuse a été lyophilisée en présence d'un composé polyhydroxylé, puis chauffée à l'air à une température d'environ 100 à 150°C.
PCT/EP2009/063834 2008-10-26 2009-10-21 Eponge de kératine à usage pharmaceutique WO2010046413A2 (fr)

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DE102008053114.6 2008-10-26
DE200810053114 DE102008053114B4 (de) 2008-10-26 2008-10-26 Pharmazeutischer Keratinschwamm

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CN102492166A (zh) * 2011-11-24 2012-06-13 东华大学 一种利用羽毛制备羽毛角蛋白海绵的方法

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WO2008073376A2 (fr) * 2006-12-11 2008-06-19 Keratec, Ltd. Construction kératinique poreuse et procédé de fabrication de celle-ci

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