MXPA06009868A - Method for producing shaped bodies made from crosslinked gelatine - Google Patents

Abstract

The object of the invention is to provide a method for producing shaped bodies made from crosslinked gelatine that can be used as substrates for tissue implants and have individually adjustable decomposition periods. The method comprises the following steps:(a) preparation of an aqueous gelatine solution;(b) partial crosslinking of the dissloved gelatine;(c) production of a shaped body using the gelatine solution containing the partially crosslinked gelatine;and (d) crosslinking the gelatine contained in the shaped body.

Description

METHOD TO PRODUCE CONFIGURED BODIES MADE OF ENTRELATED GELATIN DESCRIPTIVE MEMORY The invention relates to a method for producing shaped bodies based on interlaced gelatin. The invention also relates to shaped bodies based on interlaced gelatin, in particular sheet materials and hollow bodies. The invention also relates to implants that are manufactured using the aforementioned shaped bodies. So-called tissue implants, which are constructions consisting of a carrier material and living cells (tissue engineering), can be used to treat damaged tissues and organs. Such implants are known in the prior art and are used, among other things, for the regeneration of skin or cartilage. The carrier material must be of a type that promotes the growth and proliferation of the cells. In addition, some firmness is desirable in order to protect the cells against mechanical stress during growth in the body. At the same time, the material, however, must be flexible enough to adapt to the shape of the part of the body that is to be treated. Finally, the carrier material must be able to be reabsorbed as completely as possible by the body after the cells have grown to a sufficient degree and have synthesized an extracellular matrix. The materials used to date are unable to meet these multiple requirements to the desired degree. Among others, carriers based on chitosan, alginate, agarose and hyaluronic acid are described in the prior art. The last three mentioned materials present considerable deficiencies in terms of waste-free reabsorption. Another carrier material that is often used is collagen. However, it can not be obtained in a composition and purity that can be reproduced in the desired manner. In addition, collagen obtained from animal sources may contain immunogenic telopeptides that can activate rejection by the body. Moreover, all the aforementioned materials have the disadvantage that the respective reabsorption time after which the material has disintegrated is not individually adjustable. The optimal duration may differ depending on the type of tissue to be treated and the size of the defect. For example, due to the slow growth of chondrocytes, degradation times of four weeks or more are desirable for the regeneration of cartilage defects. The object on which the present invention is based, therefore, is to provide a method for obtaining materials that meet the requirements described above and with which, in addition, the respective degradation time of the material can be adjusted in a specific manner. This object is achieved with the method mentioned at the beginning, according to the invention, which comprises the following steps: a) preparing an aqueous gelatin solution; b) partially intertwining the dissolved gelatine c) producing a shaped body starting from the gelatin solution with the partially interlaced gelatin; and d) interlacing the gelatin contained in the configured body. The use of interlaced gelatin as a starting material for wound dressings and tissue implants has already been described as such in the prior art. Unlike collagen, gelatin is a product with a defined composition, which can also be produced with a very high degree of purity. In addition, materials made of gelatin are optically clear, while products made of collagen mostly have a cloudy, milky appearance. These latter may prove to be disadvantageous in an optical microscope analysis of cell growth. However, the interlaced gelatin materials known to date do not exhibit a stability that is required for long-term applications. For example, an entanglement with 1, 5-pentanodial up to 12 hours, as described in European patent EP 1 053 757, it is not sufficient to obtain a gelatin material suitable for the regeneration of cartilage defects. Nl are interlaced gelatin sponges, like those already used in the treatment of wounds and hemorrhage, suitable, as they are partially disintegrated within a few minutes in the presence of proteases. It has been found that a higher degree of entanglement of the gelatin implies an increased stability. An increase in stability through a higher concentration of entanglement agents or longer duration of the entanglement reaction is limited by a gelatin solution which is no longer capable of being processed and configured when the entanglement is too high. An interlacing of the gelatin only after the production of the shaped body does not produce satisfactory results, since a higher degree of entanglement of the gelatin occurs in the interfaces accessible from the outside than in the internal areas of the shaped body. For example, in the case of bodies configured with a cellular structure, to which reference will be made in detail below, this may result in the walls of cells or gratings between the pores in the interior being only insufficiently intertwined and disintegrably too fast during the last use of the configured bodies. Surprisingly, the above-described method according to the invention, characterized by a two-step entanglement of the gelatin materials, not only makes it possible to produce materials that have a correspondingly long life and are stable in terms of form, without have to give up the advantages of gelatin previously described. The method also allows the desired reabsorption time of the material to be adjusted individually. The shaped bodies produced by the method according to the invention are thus self-supporting, that is, they are sufficiently stable to be handled and used without a carrier element. This is highly advantageous in medical use, since materials that are as uniform as possible have been used here. Gelatin of different origin and quality can be used as starting material for the method. Depending on the method of the invention, the concentration of gelatin in solution (a) may be from 5 to 45% by weight, preferably from 10 to 30% by weight. The shaped body (c) formed after the first entanglement (b) is preferably at least partially dry after the second entanglement (d), preferably up to a residual moisture content of less than 20% by weight, in particular 15% by weight or less. The second entanglement can be carried out by the action of an aqueous solution of an entanglement agent, but the action of a gaseous entanglement agent is preferred. In principle, all the compounds that produce a chemical entanglement of the gelatin can be used as an entanglement agent. Aldehydes, dialdehydes, isocyanates, diisocyanates, carbodiimides and alkyl halides are preferred, and the same or different compounds can be used for the two entanglement steps. Particularly preferred is the use of formaldehyde, especially for the second interlacing step in the gas phase, since the shaped body can be simultaneously sterilized by formaldehyde. The action of formaldehyde on the configured body can be promoted by an atmosphere of water vapor. The properties of the shaped bodies produced in accordance with the method according to the invention can be further improved in terms of their stability by the shaped bodies which are subjected to a subsequent heat treatment under reduced pressure after the second entanglement step. This subsequent treatment is preferably carried out at temperatures of 80 to 160 ° C, since the effects observed are relatively indistinct below 80 ° C, and an undesired discoloration of the gelatin can occur above 160 ° C. Values ranging from 90 to 120 ° C are most preferred. The reduced pressure is to be understood as pressures below atmospheric pressure, with pressure values that are as low as possible, ideally a vacuum, are preferred. Subsequent heat treatment has an advantageous effect in two different aspects. First, under the aforementioned temperature and pressure conditions, an additional dehydrothermal entanglement of the gelatin takes place by different amino acid side chains by reacting with each other and thus removing the water. This is promoted by the eliminated water that is removed from equilibrium by the low pressure. Therefore, due to the subsequent heat treatment, given the same amount of entanglement agents, a greater degree of entanglement can be achieved, or given a comparable degree of entanglement, the amount of entanglement agents can be reduced. The additional advantage of the subsequent heat treatment is that the residual content of interlacing agent that is not used and remains in the shaped body can be significantly reduced. To ensure good biocompatibility of shaped bodies, for example, when used as a carrier material for tissue implants, the excess entanglement agent which has not reacted is preferably removed from the body configured in the method according to the invention. This can be done, for example, by degassing the bodies configured at normal pressure for several days and / or washing with a liquid medium, the latter also requiring a time from one day to a week, depending on the concentration of the entanglement agent, body size set, etc. Since, because of the subsequent heat treatment described above, on the one hand, the amount of entanglement agent that is used can be reduced and, in addition, the excess entanglement agent can be removed from the body formed by the elevated temperature and the At reduced pressure, a significant reduction in residual content of entanglement agent can be achieved within about 4 to 10 hours by this additional method step. Therefore, bodies configured in accordance with the invention, which are preferably substantially free of excess entanglement agent, can be produced with a relatively low expenditure of time due to the subsequent heat treatment. The bodies configured according to the invention preferably have an entanglement agent content of about 0.2% by weight or less than, for example, in the case of the formaldehyde entangling agent represents a limit value for the biocompatibility of carrier materials. This value can not be achieved in the aforementioned duration of 4 to 10 hours by pure washing with liquid medium. Surprisingly, a treatment with reduced pressure heat, in fact, only results in improved stability of the shaped bodies according to the invention when this, as described above, is carried out after the two entanglement steps. A previous treatment of the gelatin used under the corresponding temperature and pressure conditions does not result in any appreciable increase in the life time of the shaped bodies, although the gelatin is chemically modified in this case as well, which is reflected in the ncrement in fluorescence resistance, viscosity and average molecular weight.

A previous heat treatment of the gelatin used, which preferably is carried out under conditions comparable to those of the subsequent heat treatment of the shaped bodies, however, entails other advantages, which depending on the application may be of importance. First, the previous heat treatment results in a higher resistance to detachment of the bodies configured in accordance with the invention in the dry state, particularly in the case of the films described below. In addition, due to the higher viscosity of the gelatin having a previous heat treatment in progress, the concentration of the gelatin solution to be used can be reduced, so that the shaped bodies can be obtained with a lower density and greater flexibility. This applies mainly to bodies configured with a cell structure, which will be described in detail later. A gelatin with a viscosity of 8 mPas or more is preferably used for the method according to the invention. This value refers to the viscosity of 6.7% by weight of aqueous gelatin solution at 60 ° C. The desired strength, in particular, peel strength, and stability, and the lifetime or degradation behavior of the material produced can be adjusted very easily with the method according to the invention, preferably by the specific choice of the low conditions. which occurs. For example, both the resistance and the lifetime, as a rule, can be increased by a higher concentration of the entanglement agent or by the subsequent heat treatment described above. Therefore, surprisingly shaped bodies can be obtained which, on the one hand, under physiological conditions, remain specifically stable, for example, for more than a week, for more than two weeks or for more than four weeks and, on the other hand, satisfy the requirements with respect to the compatibility and reabsorption capacity of the cells. The term stability in this context is understood as the material that substantially maintains its original form both during storage and in the dry state and during the set period under standard physiological conditions and only subsequently being readsorbed to a considerable degree. The standard physiological conditions to which the material is subjected when used to produce implants are mainly characterized by temperature, pH value and ion concentration. The corresponding conditions can be defined in vitro by incubation of the pH regulator material of PBS (pH 7.2, 0.09% by weight of NaCl) at 37 ° C in order to test and compare various materials in terms of their behavior to dependent stability. weather. Also the resistance of the bodies configured to proteases, which are mainly responsible for the degradation of the material, can already be estimated very well in vitro by adding a protease, for example, pepsin, or from colonization with protease-producing cells, for example , fibroblasts. Quantitative details of these can be taken from the modalities given below. Despite the very high degree of interlacing to a certain extent achievable with the method described, the shaped bodies produced however have sufficient flexibility to meet the requirements for use as a tissue implant such as, for example, folding capacity and capacity. of suture. The desired flexibility can preferably be adjusted by adding softeners in the course of the production method. As a general rule, an increase in softener concentration will result in more flexible shaped bodies. Glycerin, oligoglycerins, oligoglycols and sorbite are, for example, suitable as softeners. Various methods can be used to produce the bodies configured from the interlaced gelatin solution, molding or extrusion, optionally combined with foaming, if the purpose is cellular materials. The present invention further relates to shaped bodies made of interlaced gelatin, wherein the degree of entanglement is selected in such a way that under physiological conditions the shaped bodies remain stable for a predetermined time, for example, at least one, two or four weeks A preferred method for producing said shaped bodies is that described above. In a preferred embodiment, the shaped bodies are sheet materials. The sheet materials can be used in many applications as a carrier for tissue implants, for example, in skin regeneration. The sheet material may be cellular, that is, have a cellular structure, or may be in the form of a film (non-cellular). Cell structures such as, for example, sponges or foams, can be obtained by foaming the gelatin solution with a gas, in particular air. Preferred cellular structures are open pores to allow the internal growth of cells and the formation of a three-dimensional tissue structure when used for tissue implants. The density of the bodies configured with a cell structure and the width of the pores can be adjusted within a wide range, preferably by the intensity of the foaming. Moreover, the density can be reduced using a gelatin which has undergone previous heat treatment or a gelatin with high viscosity, as described above. The properties of a body configured with a cellular structure according to the invention can also be influenced by the cellular structure which is modified by mechanical action on the shaped body. The mechanical action includes, for example, pressing or rolling the shaped body to such an extent that some of the walls of the cells or gratings between the pores of the cellular structure are broken. The density of the shaped body is preferably increased by a factor of 2 to 10 by the mechanical action. The flexibility of the bodies configured in the dry state can be increased by the mechanical action without the stability behavior with respect to the time that is markedly influenced. This is advantageous, particularly as flexible sheet materials, when used as a tissue implant, it can be better adapted to body conditions. The pores of the cellular structure preferably have an average diameter of less than 300 μm. In the case of larger average pore diameters, a too low retention capacity is often observed when the cells are introduced into the cellular structure. The preferred lower limit of the pore width complies, in most cases, with the size of the cells that are used and are grown in the three dimensions in the cellular structure. A gelatin solution with a concentration of 5 to 25% by weight, preferably 10 to 20% by weight, can be used to produce bodies configured with a cellular structure. In general, a higher gelatin concentration results in a breaking strength of the shaped bodies. Surprisingly, this works substantially independently of the degree of entanglement, by which the lifetime of the material can be adjusted.

The preferred shaped bodies with a cellular structure are reversibly compressible. This applies in particular in the hydrated state, with the degree of compression capacity depending, among other things, on the concentration of gelatin used and the width of the pores. By the term "films" is meant thin-film materials without a cellular structure. They can be produced by molding from a gelatin solution preferably substantially degassed. A preferred embodiment refers to flexible films, the flexibility of which can be adjusted, for example, by adding softeners. The compounds already described together with the method according to the invention can be used as softeners. Under standard physiological conditions, the stability of the films remains substantially undisturbed by the use of the softeners. Films with a thickness of 20 to 500 μm are preferred, very preferred 50 to 100 μm. Preferably, gelatin solutions with a concentration of 5 to 45% by weight, preferred in addition to 10 to 30% by weight, are used to produce the films. A further preferred embodiment of the invention relates to a multilayer material comprising a film and a sheet material with a cellular structure. The two layers can be joined directly to one another, which can be carried out, for example, by the sheet material with a cellular structure that is brought into contact with the film, optionally pressed thereon, before the film to dry. Alternatively, the layers can be adhesively bonded together, and a gelatin-based adhesive will preferably be used as an adhesive. In the case of the multi-layer sheet material according to the invention, the film and the sheet material with a cellular structure will preferably be bonded with surface-to-surface contact, in particular, over the entire surface, one to the other. In another preferred embodiment, the shaped bodies may also be in the form of a hollow body, in particular, a hollow section. Said hollow sections are, for example, obtainable by extrusion of the gelatin solution. Alternatively, the hollow sections with a cellular structure described above can be produced by simultaneous extrusion and foaming. However, the hollow sections can also be formed from previously produced sheet materials, in particular films, for example, by lamination. A preferred embodiment refers to hollow cylindrical sections, e.g., small tubes. These can also be produced, inter alia, by rolling the sheet materials described above. In addition to the materials described above, the bodies configured in accordance with the invention can also have any other shape or structure. In particular, shaped bodies that are specially adapted to the tissue defect to be treated can be used as a tissue implant. The invention further relates to the use of shaped bodies described for use in the field of human and veterinary medicine and for the manufacture of implants. A use according to the invention relates to the manufacture of bandages for wounds made of the materials described above. These can be used in the treatment of wounds or internal or external bleeding, for example, during operations. The reabsorption of the material occurs after an individually adjustable time, preferably determined by the manufacturing condition that is selected. It has been found that bodies configured in accordance with the invention are extremely well suited for colonization with mammalian cells, i.e., with human or animal cells. A shaped body can be treated with a suitable nutrient medium and the cells, for example, fibroblasts or chondrocytes, subsequently disseminated thereon. Due to the stability of the material, the cells can grow and proliferate in vitro for several weeks. The invention further relates to imts, in particular tissue imts, comprising a body configured in accordance with the invention and cells cultured thereon, as described above. The imts according to the invention are used to treat tissue defects, for example, skin or cartilage defects, and disseminated cells, for example, can be previously taken from the patient. During the growth phase of the cells, the shaped body protects the tissue against mechanical stress as it is formed, and the formation of the extracellular matrix by the cells becomes possible. The adjustable resorption time according to the invention proves to be of particular advantage. With the use of long-lasting materials in accordance with the invention, which have a resorption time of more than four weeks, defects covering large areas or defects in tissue types with slow cell growth can also be treated. The bodies configured with a cellular structure are particularly preferred for use in imts, since a three-dimensional tissue structure can be developed here by the cells growing in the shaped body. Due to a reversible compression of the configured body, a suspension of cells can be absorbed by the cells homogeneously distributed in the configured body. Depending on the field of application, a sheet material with a cellular structure may be used, for example, to treat extensive lesions or skin burns. However, any other form may also be advantageous, for example, individual, three-dimensional shaped bodies for treating cartilage defects. In a further preferred embodiment of the invention, the imt comprises a multi-layer sheet material described above.

In said implant, the sheet material with a cellular structure serves as a carrier for the cells, while the film offers additional mechanical protection. Said construction may be advantageous, for example, for the regeneration of cartilage tissue that grows very slowly. The invention also relates to nerve guide channels. The implantation of nerve guide channels serves for the regeneration of broken nerve cords. The channel must be of such dimensions that the individual nerve cell can grow in it. This is ensured with a preferred internal diameter of 1 mm. In addition, the nerve guiding channel must be of such a nature that the blood vessels can penetrate it from the sides to allow the nerve cell to be supplied with nutrients. Nerve guide channels that satisfy this requirement can be produced with the method according to the invention. In a preferred embodiment, the rib guiding channel is produced by laminating a laminated material according to the invention described above, in particular, a film. These and further advantages of the invention can be explained in more detail by means of the examples and figures given below. They are shown in: Figure 1: a stress-strain diagram of films according to the invention; figure 2: a stress-strain diagram of additional films according to the invention; Figure 3: Degradation behavior with respect to the time of bodies configured with a cellular structure in accordance with the invention; Figures 4A-4B: photomicrographs of bodies configured with a cellular structure in accordance with the invention; Figure 5: a diagram of resistance to rupture of bodies configured with a cellular structure according to the invention; Figure 6: Protease resistance of bodies configured with a cellular structure in accordance with the invention; Figure 7: degradation behavior with respect to the time of bodies configured with a cellular structure according to the invention; figure 8: an effort-deformation diagram of additional films according to the invention; Figure 9: cellular distribution of chondrocytes in shaped bodies according to the invention; and figure 10: a photographic representation of the colonization of a film according to the invention with fibroblasts.

EXAMPLES EXAMPLE 1 Production and properties of films based on interlaced gelatin Pigskin gelatin (300 fluorescence resistance) was dissolved in four different batches in a mixture of water and glycerin in accordance with the quantities set forth in Table 1 at 60 ° C: After degassing the solutions ultrasonically, the indicated amount in Table 1 of the aqueous formaldehyde solution (1.0 wt.%, room temperature) was added, the mixture was homogenized and applied at about 60 ° C in a thickness of 1 mm with a tongue-swath to a polyethylene base.

TABLE 1 After drying at 30 ° C and a relative humidity of 50% for about a day, the films were removed from the PE base and dried again for about 12 hours under the same conditions. The dried films had a thickness of less than 100 μm and during the second interlacing step they were subjected for two hours to the equilibrium vapor pressure of a 17% aqueous solution of formaldehyde at room temperature in a desiccator. In the case of the film produced in accordance with lot 1-1, the second interlacing step was the only one. The mechanical properties of the various films (in the dry state) are shown in Figure 1: meanwhile, compared to the film 1-1, the film 1-2 presents, due to the interlacing of two steps, a resistance to detachment upper with less elongation at the point of rupture, film 1-3 is considerably more expandable (flexible) due to the increase in glycerin concentration. Due to the higher entanglement agent concentration in film 1-4 compared to film 1-3, a slightly higher strength can be obtained with less elongation to the breaking point in turn. Films were also produced in accordance with lots 1-1 and 1-2. However, these were not subsequently subjected to any interlacing in the aqueous phase (films 1-1 ', non-interlaced and 1-2', entanglement once). The elongation curves at the point of rupture of these films are approximately 140% / 10 N / mm2 (1-1 ') and 115% / 15 N / mm2 (1-2'), respectively, and for reasons of clarity, they are shown in figure 1.

It will be understood that the shapes of the respective curve in relation to laboratory scale production can not be accurately reproduced. However, the relationship of the curves of several films with one another is characteristic. Accordingly, the example shows that with the method according to the invention, the flexibility of the films produced can be adapted in a wide range both by the degree of entanglement and the amount of softener that is varying accordingly. The films that are interlaced twice are distinguished by remaining stable for a considerably longer time under standard physiological conditions: The degradation behavior of the films was measured by placing pieces of film measuring 2 x 3 cm in 500 ml of pH regulator of PBS (pH 7.2, 0.09% by weight of NaCl) in each case and the concentration of the gelatin dissolved in the pH regulator was determined photometrically at a wavelength of 214 nm. While the films that had not been intertwined or had been intertwined once were completely disintegrated after 15 minutes, in the films that had been interlaced twice no changes were observed after one hour.

EXAMPLE 2 Production and properties of interlaced gelatin-based films Eight batches of a 30% by weight solution of pig skin gelatin (300 fluorescence resistance) in water / glycerin in accordance with the amounts set forth in Table 2 were produced by dissolving the gelatin at 60 ° C. After degassing the solutions ultrasonically, the corresponding amounts of an aqueous solution of formaldehyde (1.0% by weight, room temperature) were added, whereby the final concentration of formaldehyde corresponded respectively to the value given in Table 2. As described, for the rest, in Example 1, the films were produced from the mixtures, dried and, if appropriate, interlaced (see Table 2).

TABLE 2 The stress-strain properties of the eight films are shown in Figure 2. Curves 2-1 to 2-8 are related to the corresponding dry films, curves 2-2A to 2-8A to the hydrated films that were placed during four hours in PBS pH regulator (non-interlaced film 2-1 disintegrates under these conditions to such an extent that the stress-strain properties are not possible to be examined). The vertical marks indicate the end points of the respective curves. It is also clear, from this example, that the peel strength and flexibility, respectively, of the films can be varied over a wide range by means of the different production conditions. Furthermore, it is evident that also the films that are in the dry state are relatively rigid (which may be advantageous for the process) after their hydration, in some cases they can be made very flexible under physiological conditions: Films 2-7 and 2-8 , which in the dry state have almost identical properties, produce after hydration, in one case, an extremely flexible material (2-7A), as required, for example, to be used in the articulation area, in the other case , a more rigid material with superior peel strength (2-8A), can be used, for example in the bone area.

EXAMPLE 3 Production and properties of bodies configured with a cellular structure based on interlaced gelatin Five batches of a 12 wt% solution of pig skin gelatin (300 fluorescence resistance) were produced in water by dissolving the gelatin at 60 ° C, ultrasonically degassed, and the corresponding amount of an aqueous formaldehyde solution (1.0 % by weight, room temperature) was added respectively, which resulted in 1500 ppm formaldehyde (relative to gelatin). No formaldehyde was added to a correspondingly produced reference sample. The homogenated mixtures were annealed after a reaction time of 10 minutes at 45 ° C and mechanically foamed with air. The foaming process that lasted approximately 30 minutes was carried out with a different ratio of air to gelatin solution for the five batches, so that cellular structures with different wet densities and pore sizes were obtained according to Table 3. The foamed gelatin solutions, which showed a temperature of 26.5 ° C, were cast in molds measuring 40 x 20 x 6 cm and dried for about four days at 26 ° C and a relative air humidity of 10%. The dried shaped bodies with cellular structure in the form of a sponge (here referred to as sponges) were cut here in 2 mm thick layers and for the second interlacing step they were subjected for 17 hours at equilibrium vapor pressure to a solution of formaldehyde water at 17% at room temperature in a desiccator. To achieve a uniform degassing of the entire volume of the shaped bodies, the desiccator was evacuated respectively two to three times and re-aerated. The pore structure of the sponges was determined by optical microscopy and could be confirmed by scanning electron microscopy.

TABLE 3 To determine the stability of the sponges, pieces measuring 30 x 30 x 2 mm were weighed, respectively placed in 75 ml of pH buffer of PBS and stored at 37 ° C. After the respective storage time, the pieces were washed for 30 minutes in water, dried and weighed. Figure 3 shows the disintegration characteristics of the sponges 3-1 to 3-5 and the reference sample interlaced once (the sequence of the bars shown is respectively: reference, 3-1, 3-2, 3-3, 3-4, 3-5). While the reference sample is already completely disintegrated after three days, all sponges produced according to the invention will still be maintained above 80% even after 14 days. However, considerable differences are found in the subsequent degradation behavior, which are due to the different foam densities of the materials. The sponge 3-1 is completely disintegrated after 21 days and the sponge 3-2 after 28, while the sponges 3-4 and 3-5 are substantially maintained even after 35 days. This provides the possibility of specifically influencing the degradation behavior of the cellular structure materials independently of other parameters. However, the properties of cell structure materials can also be significantly modified by a change in the concentration of gelatin in the starting solution. Figures 4A-4B show photomicrographs of the cellular structure of two bodies configured in thin sections of 150 μm, starting from 12% by weight (illustration A) and 18% by weight (illustration B) of gelatin solution, were produced under otherwise identical conditions. The higher gelatin concentration results in cell walls or wider (thicker) grids between the individual pores, which is reflected in an increased rupture strength of the corresponding sponges. This is illustrated quantitatively in Figure 5. The three curves, A, B and C respectively represent sponges with three different degrees of entanglement. The breaking strength is constantly increased with an increase in the gelatin concentration of the starting solution from 10 to 18% by weight, with a wide range from about 500 to almost 2000 newtons being covered. At the same time, the deformation until rupture changes only slightly. Surprisingly, the correlation between the breaking force and the gelatin concentration is substantially independent of the degree of entanglement. By means of the degree of entanglement, that is, by the choice of the concentration of the entanglement agent, the stability of the shaped bodies, on the other hand, can be influenced in particular in view of proteolytic degradation. The resistance of various cellular structure materials (sponges) to pepsin depending on the amount of formaldehyde (% by weight relative to gelatin) used in the first step of entanglement is shown in Figure 6. The degradation was performed at 37 ° C in a 1.0% by weight pepsin solution in PBS pH regulator, the pH value of which was adjusted with HCl to 1. With an increase in formaldehyde concentration from 500 to 1500 to 3000 ppm , the degradation time of the sponges increases from 5 minutes to 30 minutes to 75 minutes. The dry density of the materials is substantially independent of the degree of entanglement here. The very drastic degradation conditions chosen here are not to be compared with the considerably lighter physiological conditions, so that considerably longer degradation times are applied under the latter conditions.

EXAMPLE 4 Production and properties of bodies configured with a cellular structure based on interlaced gelatin with subsequent heat treatment A 12 wt.% Solution of pig skin gelatin (fluorescence resistance 300) was produced as in Example 3, ultrasonically degassed, and mixed with the corresponding amount of an aqueous formaldehyde solution (1.0 wt.%, Temperature environment), so that 1500 ppm of formaldehyde was obtained (in relation to gelatin). The homogenized mixture was warmed after a reaction time of 5 minutes at 45 ° C and mechanically foamed with air for about 30 minutes. The foamed gelatin solution was cast into molds and dried as described in example 3, and a body configured with a cellular structure in the form of a sponge (sponge) with a wet density of 121 mg / cm 3, a density was obtained dry at 18 mg / cm3 and an average pore size of 250 μm. The configured body was already firm after the first entanglement step. Four samples measuring 30 x 30 x 2 mm were cut from the sponge and each was subjected to a second interlacing step in the gas phase at an equilibrium vapor pressure of a 10% formaldehyde aqueous solution as in the method described in example 3. However, unlike example 3, the time during which formaldehyde acted was significantly shorter in this case, namely 2 hours in the case of samples 4-1 and 4-3 and 5 hours in the case of samples 4-2 and 4-4. All four samples were degassed under vacuum after the second entanglement step, and samples 4-3 and 4-4 were then subjected to a subsequent heat treatment. By means of a rotary evaporator, the sponges in question were kept for 6 hours at a vacuum of about 14 mbar at 105 ° C. The stability of the various samples in the pH regulator of PBS was determined as described in example 3. Figure 7 shows the disintegration behavior of samples 4-1 to 4-4 (the sequence of the bars illustrated is respectively: 4-1, 4-2, 4- 3, 4-4).

It became evident that the lifetime of the sponges under physiological conditions can be significantly prolonged by the subsequent heat treatment. While sample 4-1 that was not treated subsequently is already completely disintegrated after 14 days, sample 4-3 that was treated with heat subsequently remains at almost 50% of this point in time. A corresponding difference has also been seen between samples 4-2 (not treated subsequently) and 4-4 (treated with heat subsequently), which were each interlaced for 5 hours in the gas phase. After 35 days, sample 4-2 is completely disintegrated and sample 4-4 is still maintained at more than 70%. In addition to the increase in the degree of entanglement and the stability of the sponges, the subsequent heat treatment under vacuum has the additional advantage that the residual amount of entanglement agent remaining in the shaped body can be effectively reduced, so that the Washing for a long time before use can be avoided or at least shortened. This applies particularly to mechanically relatively firm sponges that are produced on the basis of high gelatin concentrations. In order to measure this effect, two batches of an 18 wt% solution of pig skin gelatin (300 fluorescence resistance) in water were produced by dissolving the gelatin at 60 ° C, degassing ultrasonically, and each mixed with the corresponding amount of an aqueous formaldehyde solution (1.0% by weight, room temperature), to yield 2000 ppm formaldehyde (relative to gelatin). The homogenized mixtures were annealed after a reaction time of 5 minutes at 45 ° C and mechanically foamed with air. Due to a different ratio of air to gelatin solution in the two batches, cellular structures with different densities and pore sizes were obtained according to table 4. The casting and drying of the foamed gelatin solution were carried out as described above, similarly the cut was carried out on discs of 2 mm thickness and the second step of entanglement under the action of formaldehyde vapor. The interlacing time was 17 hours.

TABLE 4 After determining the content of excess formaldehyde in the sponges, the samples were subjected to subsequent heat treatment at 105 ° C and a vacuum of approximately 14 mbar for a duration of 4 hours (sample 4-5) and 10 hours (sample 4-6), respectively.

The residual content of the free formaldehyde was determined again afterwards. The results are shown in table 5.

TABLE 5 In the case of sponge 4-5, the formaldehyde content could already be reduced by approximately 30% after 4 hours of subsequent heat treatment. In the case of the considerably denser sponge 4-6, a subsequent treatment of 10 hours resulted in a reduction of the residual content of approximately 40%. In many medical applications, a residual content of approximately 0.2% by weight represents the upper physiological limit for formaldehyde. It is evident that, in many cases, this value can be achieved by the subsequent heat treatment, so the time required to wash the sponges can be shortened considerably.

EXAMPLE 5 Production of bodies configured from gelatin that has undergone previous heat treatment For comparison, the pig skin gelatin (fluorescence resistance 300) used to produce the bodies configured in Examples 1 to 4 was subjected to prior heat treatment as in the subsequent heat treatment of the bodies configured in Example 4 The gelatin was kept for 6 hours at a vacuum of about 14 mbar at 105 ° C. As a result, the fluorescence resistance increased from 300 to 310, the viscosity increased from 5.92 mPas to 9.04 mPas (measured in a 6.7% by weight solution at 60 ° C) and the average molecular weight from 172 kDa to 189 kDa. As in the method described in example 1, four different films were produced from untreated gelatin and gelatin which has undergone previous heat treatment, respectively. The quantities for the various batches were set out in Table 6. In a different manner to Example 1, an aqueous solution at 2.0% by weight of formaldehyde was used for the first interlacing step, and the second interlacing step was carried out during 2 hours at an equilibrium vapor pressure of a 10% aqueous solution of formaldehyde.

TABLE 6 The mechanical properties of films 5-1 to 5-4 in the dry state are shown in figure 8. It is evident that the peel strength of films 5-2 and 5-4 produced from the gelatin which had suffered prior heat treatment is considerably higher compared to the corresponding films 5-1 and 5-3 produced from untreated gelatin. The handling ability of the films in the medical application context is therefore improved. Alternatively, it is possible to produce thinner films with comparable peel strength from gelatin which has undergone previous heat treatment. With regard to their long-term stability under physiological conditions, films produced from pre-treated gelatin have the same advantageous properties as films made from untreated gelatin. The gelatin which has undergone previous heat treatment is also suitable for producing bodies configured with a cellular structure as in example 3. In this case, also, comparable long-term stabilities are observed as in the use of untreated gelatin. Due to the higher viscosity of the pre-treated gelatin (in this case 9.04 mPas compared to 5.92 mPas), it is possible to significantly reduce the gelatin concentration of the solutions used to produce the sponges. Since the viscosity of the gelatin solution increases in a linear to quadratic ratio with the concentration, a 5 to 8% solution of gelatin that has gone through previous heat treatment can be used instead of a 12% solution of gelatin not treated. The bodies configured with a cellular structure produced in this way are distinguished by thinner gratings between the pores and a lower density, so that in turn the flexibility of the sponges is increased. A lower density also means that, in total, a smaller amount of gelatin is required to be used as a carrier material for tissue implants.

EXAMPLE 6 Production of multi-layer sheet materials In accordance with the method described in Example 1, a film of 33 g of pig skin gelatin (fluorescent resistance 300), 53.25 g of water, 15.5 g of glycerin and 8.25 g of a 2.0% solution was produced. of formaldehyde, and the film applied with a tongue ablaze was maintained for 2 hours at 40 ° C before drying.

A sponge 2 to 3 mm thick was produced according to example 3, lot 3-2. Prior to the second entanglement, the two sheet materials were adhesively bonded to one another by means of a solution made of bone gelatin (fluorescence resistance 160). As described in example 2, the multi-layer sheet material was then entangled by the formaldehyde vapor action. Alternatively to the method described herein, the film and the sponge may each be separately subjected to the second entanglement step before joining. Instead of using a gelatin solution as an adhesive, the two sheet materials can also be bonded by the sponge when they are partially pressed into the film that has been applied with a tongue swath and has not dried. A joining of the sheet materials preferably over the entire surface is obtained with both alternative methods.

EXAMPLE 7 Colonization of sponges with chondrocytes The sponges produced in accordance with batches 3-1 to 3-3 and the interlaced reference sample once from example 3 were used, each as a sheet material with a thickness of 2 mm, for colonization with chondrocytes. DMEM / 10% FCS / glutamine / pen / strep, which is a standard medium for culturing mammalian cells, was used as a culture medium. Before colonization, excess formaldehyde must be removed from the sponges, for example, by washing the sponges with culture medium or ethanol. One million pork chondrocytes, suspended in 150 μl of culture medium, were disseminated per cm 2 in the starting materials. The distribution of cells within the sponges observed after one hour is shown in Figure 9. The percentile indicates in percent the proportion of all the cells that are distributed to the respective depth of colonization in the material. Due to the open pore structure, the distribution of cells is substantially uniform throughout the thickness of the sponges and is not altered by the highest degree of entanglement of sponges 3-1 to 3-3 as compared to the reference sample R .

EXAMPLE 8 Culture of fibroblasts on films Films produced in accordance with lots 2-3 to 2-6 were used for colonization with fibroblasts.

DMEM / 10% FCS / glutamine / pen / strep was used again as a culture medium.

Before colonization with cells, the films should also be washed to remove any residual formaldehyde. Human fibroblasts from the anterior skin (0.5 million cells / cm2) were disseminated on the films and cultured for 6 weeks in the medium at 37 ° C. The vitality of the cells was examined microscopically twice a week. It was found that the fibroblasts in all the films were viable for at least four weeks. In addition, after four weeks of protease production of the cells none of the films had disintegrated yet. Films 2-5 and 2-6 with a higher degree of entanglement were even stable for up to six weeks. Figure 10 shows a photomicrograph of the fibroblasts in film 2-5 after a culture time of 14 days. Since the disintegration of the material has not yet started, the edge of the film is clearly recognizable. Although pig skin gelatin (fluorescence resistance 300) was used in all the above examples, it is understood that comparable results can be obtained with other types and grades of gelatin.

Claims (54)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for producing shaped bodies based on interlaced gelatin, comprising the following steps: a) preparing an aqueous solution of gelatin; b) partially entangle the dissolved gelatin; c) producing a shaped body starting from the gelatin solution containing the partially interlaced gelatin; and d) interlacing the gelatin contained in the configured body.

2. The method according to claim 1, further characterized in that the shaped body is at least partially dried before step d).

3. The method according to claim 1 or 2, further characterized in that the entanglement in step d) is carried out by the action of an entanglement agent in aqueous solution.

4. The method according to any of claims 1 to 3, further characterized in that the entanglement in step d) is carried out by the action of a gaseous phase interlacing agent.

5. The method according to any of claims 1 to 4, further characterized in that the crosslinking agents used in steps b) and d) are the same or different and each is selected from aldehydes, dialdehydes, isocyanates, diisocyanates, carbodiimide and alkyl dihalides.

6. The method according to claim 5, further characterized in that the entanglement agent in steps b) and / or d) is formaldehyde.

7. The method according to any of claims 1 to 6, further characterized in that the excess entanglement agent is removed from the body configured after interlacing.

8. The method according to any of claims 1 to 7, further characterized in that the shaped body is subjected to treatment with subsequent heat under reduced pressure after step d).

9. The method according to claim 8, further characterized in that the subsequent heat treatment is carried out at a temperature of 80 to 160 ° C.

10. The method according to any of claims 1 to 9, further characterized in that step a) is carried out on the basis of a gelatin that has previously been subjected to heat treatment prior to reduced pressure.

11. The method according to claim 10, further characterized in that the previous heat treatment is carried out at a temperature of 80 to 160 ° C.

12. - The method according to any of claims 1 to 11, further characterized in that step a) is carried out on the basis of a gelatin having a viscosity of 8 mPas or more, measured in an aqueous solution at 6.7% in weight at 60 ° C.

13. The method according to any of claims 1 to 12, further characterized in that the body further configured contains a softener.

14. The method according to claim 13, further characterized in that the softener is selected from glycerin, oligoglycerin, oligoglycols and sorbite.

15. A shaped body produced in accordance with a method of claims 1 to 14.

16. The body configured according to claim 15, further characterized in that it is substantially free of excess entanglement agent.

17. The body configured in accordance with the claim 16, further characterized in that the shaped body has an excess content of crosslinking agent of about 0.2% by weight or less.

18. A configured body based on interlaced gelatin, the degree of interlacing being selected in such a way that under physiological conditions the configured body is stable for at least one week.

19. - The body configured according to claim 18, further characterized in that the degree of entanglement is selected in such a way that under physiological conditions the shaped body is stable for at least two weeks.

20. The body configured in accordance with the claim 18, further characterized in that the degree of entanglement is selected in such a way that under physiological conditions the shaped body is stable for at least four weeks.

21. The body configured according to any of claims 15 to 20, further characterized in that the configured body is self-supporting.

22. The body configured according to any of claims 15 to 21, further characterized in that the configured body is free of a carrier element.

23. The body configured according to any of claims 15 to 22, further characterized in that the configured body is flexible.

24. The body configured according to any of claims 15 to 23, further characterized in that the body further configured contains a softener.

25. The body configured according to claim 24, further characterized in that the softener is selected from glycerin, oligoglycerins, oligoglycols and sorbite.

26. - The body configured according to any of claims 15 to 25, further characterized in that the shaped body is a sheet material.

27. The sheet material according to claim 26, further characterized in that the sheet material has a cellular structure.

28.- The sheet material according to claim 27, further characterized in that the cellular structure is of open pores.

29. The sheet material according to claim 27 or 28, further characterized in that the cellular structure is modified by mechanical action on the configured body.

30. The sheet material according to claim 29, further characterized in that some of the walls of the cells between the pores of the cellular structure are broken.

31.- The sheet material in accordance with the claim 29 or 30, further characterized in that the density of the shaped body is increased by the mechanical action by a factor of 2 to 10.

32.- The sheet material according to any of claims 27 to 31, further characterized in that the pores have an average diameter of less than 300 μm.

33. The sheet material according to claim 26, further characterized in that the sheet material is a film.

34.- The sheet material according to claim 33, further characterized in that the film has a thickness of 20 to 500 μm, preferably 50 to 100 μm.

35.- The sheet material in accordance with the claim 26, further characterized in that the sheet material has a multilayer structure, comprising a film according to claim 33 or 34 and a sheet material with a cellular structure according to any of claims 27 to 32.

36. - The sheet material in accordance with the claim 35, further characterized in that the sheet material with a cellular structure is directly attached to the film.

37.- The sheet material in accordance with the claim 36, further characterized in that the joining is carried out by pressing the sheet material with a structure in the film.

38.- The sheet material according to claim 35, further characterized in that the sheet material with a cellular structure is attached to the film by means of an adhesive.

39.- The sheet material according to claim 38, further characterized in that the adhesive comprises gelatin.

40.- The body configured according to any of claims 15 to 25, further characterized in that the configured body is a hollow body.

41.- The hollow body according to claim 40, further characterized in that the hollow body is a hollow section.

42. - The hollow body according to claim 41, further characterized in that the hollow section is a hollow cylinder.

43.- The hollow body according to any of claims 40 to 42, further characterized in that the hollow body has a cellular structure.

44. The hollow body according to any of claims 40 to 43, further characterized in that the hollow body is produced by extruding a gelatin solution.

45.- The hollow body according to any of claims 41 to 43, further characterized in that the hollow section is produced from a starting material according to any of claims 26 to 39.

46.- The use of a body configured as claimed in any of claims 15 to 45 to produce a resorbable material for covering wounds or internal or external bleeding in the field of human or veterinary medicine.

47.- The use of a body configured as claimed in any of claims 15 to 42 as a carrier for culturing mammalian cells in vitro.

48. The use as claimed in claim 47, further characterized in that the mammalian cells are fibroblasts.

49. The use as claimed in claim 47, further characterized in that the mammalian cells are chondrocytes.

50. An implant comprising a body configured according to any of claims 15 to 42 and mammalian cells cultured in the shaped body.

51. The implant according to claim 50, further characterized in that it is suitable for treating damage, injuries and / or burns to the skin of humans or animals.

52. The implant according to claim 50, further characterized in that it is suitable for treating damage and / or injuries to the cartilage tissue of humans or animals. 53.- A rib guiding channel, comprising a hollow cylinder according to any of claims 42 to 45. 54.- The rib guiding channel according to claim 53, further characterized in that the hollow cylinder has a internal diameter of approximately 1 mm.