WO2010046413A2

WO2010046413A2 - Pharmaceutical keratin sponge

Info

Publication number: WO2010046413A2
Application number: PCT/EP2009/063834
Authority: WO
Inventors: Stephan Reichl
Original assignee: Technische Universität Braunschweig
Priority date: 2008-10-26
Filing date: 2009-10-21
Publication date: 2010-04-29
Also published as: DE102008053114B4; DE102008053114A1; WO2010046413A3

Abstract

The invention relates to a keratin sponge manufactured from a keratin solution prepared under reducing conditions from natural keratin, preferably keratin from human hair, from said solution reducing compounds are extracted, for example through dialysis against an aqueous medium without reducing contents. The keratin sponge according to the invention preferably comprises keratin that is solubilized in an aqueous solution without chemical derivatization, or that is optionally partially alkalinically hydrolyzed, and that is freeze-dried from an aqueous solution in the presence of a polyhydroxy compound without any reduction and/or oxidation agents present, and then heated in air to about 100 - 150°C.

Description

Pharmaceutical Keratin Sponge

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. After the formation of a film or foam from the reduced keratin solution, which still contains a reducing agent, after oxidation, 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.

Reichl (5th World Meeting of Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology Geneva, 27-30 March 2006) describes coating a carrier with a reduced keratin solution by precipitation with trichloroacetic acid.

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. Object of the invention

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. In particular, it is an object of the invention to provide 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.

General description of the invention

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.

In a preferred embodiment, 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. Because it has been shown that keratin sponge, which has a proportion of partially alkaline hydrolyzed keratin, a has higher absorbency for aqueous compositions, eg wound exudate. Preferably, the proportion of alkaline partially hydrolyzed keratin in its aqueous mixture with solubilized keratin 10 to 100 wt .-%, in particular 20 to 80 wt .-%.

The partial alkaline hydrolysis of a portion of the water-solubilized keratin, which has no reducing agent and no oxidizing agent, results in a lowering of the molecular weight. Thus, according to the invention 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. Preferably, 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. In the preferred embodiment, the higher molecular weight portion of the partially alkaline hydrolysed keratin used has a MW of at least 5 kDa, e.g. from 25 to 35 wt .-%, in particular about 30 wt .-% with a MW of 5 to 10 kDa, 3 to 10 wt .-%, in particular about 6 wt .-% with a MW of 10 to 30 kDa , 5 to 12 wt .-%, in particular about 9 wt .-% with a MW of 30 to 50 kDa and 45 to 65 wt .-%, in particular about 55 wt .-% with a MW of> 50 kDa composed , According to the invention, 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.

Preferably, 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. Preferably, 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.

Preferably, 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, preferably measured before the heat treatment, 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. In the present case 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. In particular, 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. The combination of the absence of reducing agents from the aqueous composition of solubilized keratin with the presence of a polyhydroxy compound with the heat treatment results in a keratin sponge having high structural stability, elasticity, water absorbency and resistance to degradation in the animal body. Optionally, the keratin sponge may be subjected to sterilization by γ-irradiation. The keratin sponges according to the invention have no harmful substances for the cell culture, for example no cytotoxic substances and no viruses. Thus, 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.

The particular suitability of the keratin sponges according to the invention for cell culture and for use in the production of implants for medical purposes, in particular as a scaffold for filling defect sites of soft tissue, is attributed to the fact that 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.

In comparative experiments it can be shown that 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.

Thus, e.g. 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.

For comparison, it is also possible, for example, to use 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. In particular, 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.

In the following, the preferred embodiment for the production of keratin sponges from keratin solution is described, which is used to form nanoparticles of keratin in about monomodal size distribution leads. 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.

Preferably, in the method of the invention, 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.

More preferably, aggregates of the particles are removed from the aqueous keratin solution, e.g. by ultracentrifugation at 10,000 x g, optionally by filtration. Preferably, the keratin used to prepare the solution is α-keratin; Most preferably, the keratin solution is made from hair, such as human hair. For the solubilization of keratin from hair, it is preferable to use 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.

The analysis of the size distribution of solubilized keratin in the solutions used according to the invention for the preparation of the keratin sponges, which can also be referred to as nanoparticulate suspension of keratin, aqueous composition of keratin or solubilized keratin, shows that the method according to the invention for dissolving or Solubilizing the keratin from hair produces a very narrow, monomodal size distribution around 50 to 250 nm. By means of photon correlation spectroscopy with dynamic laser light scattering a polydispersity index can be analyzed which is significantly smaller than that for prior art keratin solutions. Accordingly, 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.

Furthermore, when comparing the keratin sponges according to the invention with other porous keratin-containing materials, it has been shown that increased seeding efficiency and / or better growth on the keratin sponges according to the invention is obtained, at least for some cell types and immortalized cell lines, as well as for primary cells. Accordingly, 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.

It has been found that 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. Subsequently, 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. In addition to the elasticity and the structural resistance in the body, the advantageous properties include, in particular, swelling capacity, for example for receiving wound exudate.

By electron microscopic analysis, 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. without reducing agent and / or oxidizing agent. According to the invention, 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.

It has been shown that 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. Thus, 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. In the swollen state, for example, 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.

Detailed description of the invention

The invention will now be described in more detail by way of example with reference to the figures in which:

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.

Comparative example: Preparation of a sponge from a keratin solution containing reducing compounds

As a comparison, 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. Specifically, 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.

Example: Production of Keratin Sponges

To prepare an 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 μm. The filtrate could be stored at 4 ⁰ C or frozen in aliquots without significant changes in the properties. To remove reducing agents, 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.

For the preferred embodiment, 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. By this alkaline treatment, the dissolved keratin was partially hydrolyzed. The protein content was determined to Bradford to 25 to 30 mg / mL.

To prepare a sponge, the mixtures of the keratin solution shown in the table below were prepared with the alkaline hydrolyzed keratin solution and freeze-dried after freezing. As the polyhydroxy compound, in each case 1% by weight of glycerol per ml of keratin solution was added to the keratin solution.

Table: Swelling capacity and water absorption

The weight gain was measured as drained weight after immersion in water. For the sponge formation about 1 mL of the mixture of Keratinlösungen 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.

Also shown in the table are the values for the increase in volume and the weight increase of the sponges for storage in water (20 ^° C., 30 minutes).

The results show that with a higher proportion of alkaline partially hydrolyzed keratin the swelling capacity increases.

Scanning electron micrographs of the keratin sponges show a uniform open-pore structure. In detail, FIG. 3 shows an edge length of the image of 1,800 μm) 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, and 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, and 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.

In comparison with FIG. 3, 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. In the present case, 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. As a control, a keratin sponge was treated the same, but without adding cells.

Following culturing, live staining was performed with MTT (yellow tetrazolium salt), which is converted by the mitochondrial activity of viable cells to blue formazan.

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.

Accordingly, even when used as an implant, 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.

Claims

claims

A method of making a keratinous sponge having an open cell pore structure comprising the steps of:

Solubilization of hair keratin under reducing conditions in denaturing, aqueous solution containing reducing agent,

Removing the reducing agent from the aqueous solution of keratin

Preparation of an aqueous keratin solution which does not contain a reducing agent and / or

Contains oxidizing agent,

Adding a polyhydroxy compound,

Drying the mixture into a porous structure,

Heating the dried porous structure to a temperature of at least 100

⁰ C.

2. The method according to claim 1, characterized in that the aqueous keratin solution consists of solubilized keratin and water.

3. The method according to any one of the preceding claims, characterized by the partial alkaline hydrolyzing a portion of the aqueous keratin solution for producing a partially hydrolyzed keratin solution whose keratin to 85 to 35 wt .-% has a molecular weight of <50 kDa and the no reducing agent or oxidizing agent containing and mixing the aqueous keratin solution with the portion of the alkaline partially hydrolyzed keratin solution, adding a polyhydroxy compound, drying the mixture to a porous structure and heating the dried porous structure to a temperature of at least 100 ° C.

4. The method according to any one of the preceding claims, characterized in that are separated from the alkaline partially hydrolyzed keratin solution constituents with a MW less than 5 kDa.

5. The method according to any one of the preceding claims, characterized in that the drying is freeze-drying.

6. The method according to any one of the preceding claims, characterized in that aggregates are separated from the keratin solution.

7. The method according to any one of the preceding claims, characterized in that the polyhydroxy compound is glycerol, propylene glycol or a mixture thereof.

8. The method according to any one of the preceding claims, characterized in that the polyhydroxy compound to 0.5 to 3 wt .-% per 20 mg of keratin, measured as total protein according to Bradford added.

9. The method according to any one of the preceding claims, characterized by the use of the method for producing a medical implant or a Wundtamponade.

10. The method according to any one of the preceding claims, characterized in that the proportion of alkaline partially hydrolyzed keratin solution in the mixture with aqueous keratin solution is 10% to 100%.

A keratin sponge for use in the manufacture of a medical implant, characterized by a nanoparticulate structure of the pore cell walls with monomodal size distribution of the keratin particles contained in the pore cell walls, wherein the pore cell walls of solubilized keratin of natural origin are in admixture with a polyhydroxy compound, the solubilized keratin being only the chemical modification produced by the presence of a reducing agent and / or an oxidizing agent and by the heating of the dried keratin in admixture with the polyhydroxy compound.

12. keratin sponge according to claim 11, characterized in that the solubilized keratin comprises a mixture of solubilized keratin of natural origin and an alkaline partially hydrolyzed portion of the solubilized keratin of natural origin, wherein the partially hydrolyzed portion to 85 to 35 wt .-% keratin with a Molecular weight of <50 kDa.

13. Keratin sponge according to claim 11 or 12, characterized in that the keratin sponge is obtainable by

Hydrolysis of hair in denaturing aqueous solution containing

Reducing agent,

Removing the reducing agent from the aqueous solution of keratin

Preparation of an aqueous keratin solution which does not contain a reducing agent or

Contains oxidizing agent,

Adding a polyhydroxy compound,

Drying the mixture to produce a porous structure and

Heating the dried porous structure to a temperature of at least 100

⁰ C.

14. keratin sponge according to claim 13, characterized in that a portion of the aqueous keratin solution for the preparation of an alkaline partially hydrolyzed keratin solution containing no reducing agent or oxidizing agent, is partially hydrolyzed by contacting with an aqueous alkaline solution.

15. keratin sponge according to one of claims 11 to 14, characterized in that the alkaline partially hydrolyzed keratin to at least 85 to 35 wt .-% has a molecular weight of <50 kDa and contains no reducing agent or oxidizing agent.

16. Keratin sponge according to one of claims 11 to 15, characterized in that the polyhydroxy compound is glycerol, propylene glycol or a mixture thereof.

17. Keratin sponge according to claim 16, characterized in that the polyhydroxy compound is added to 0.5 to 3% by weight per 20 mg of keratin, measured as a total protein according to Bradford.