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
  • A61L27/20—Polysaccharides
• • 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
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
• • 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
  • A61L27/58—Materials at least partially resorbable by the body
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
  • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
  • B29C48/00—Extrusion moulding, i.e. expressing the moulding material through a die or nozzle which imparts the desired form; Apparatus therefor
  • B29C48/03—Extrusion moulding, i.e. expressing the moulding material through a die or nozzle which imparts the desired form; Apparatus therefor characterised by the shape of the extruded material at extrusion
  • B29C48/05—Filamentary, e.g. strands
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
  • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
  • B29C48/00—Extrusion moulding, i.e. expressing the moulding material through a die or nozzle which imparts the desired form; Apparatus therefor
  • B29C48/03—Extrusion moulding, i.e. expressing the moulding material through a die or nozzle which imparts the desired form; Apparatus therefor characterised by the shape of the extruded material at extrusion
  • B29C48/13—Articles with a cross-section varying in the longitudinal direction, e.g. corrugated pipes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
• • D—TEXTILES; PAPER
  • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
  • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
  • D01F2/00—Monocomponent artificial filaments or the like of cellulose or cellulose derivatives; Manufacture thereof
• • D—TEXTILES; PAPER
  • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
  • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
  • D01F2/00—Monocomponent artificial filaments or the like of cellulose or cellulose derivatives; Manufacture thereof
  • D01F2/02—Monocomponent artificial filaments or the like of cellulose or cellulose derivatives; Manufacture thereof from solutions of cellulose in acids, bases or salts
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
  • B29L—INDEXING SCHEME ASSOCIATED WITH SUBCLASS B29C, RELATING TO PARTICULAR ARTICLES
  • B29L2028/00—Nets or the like

Definitions

• the invention relates to polymer frameworks suitable for the production of artificial tissue, in particular polysaccharide frameworks, processes for their preparation, their use for the production of artificial tissue, and artificial tissues produced on the basis of such polymer frameworks.
• transplantation and mechanical prosthetics, the latter being currently limited to biochemically and electrophysiologically largely inert body parts (eg, joints, ophthalmic lenses, heart valves).
• inert body parts eg, joints, ophthalmic lenses, heart valves.
• tissues or organs with biochemical activity eg, heart, lung, liver, kidney
• transplantation is currently the only possible restorative treatment.
• extracellular matrix refers to the totality of all the structures deposited by the cells but still in contact with them. Both as an attachment point for the cells of the tissue and because of their intrinsic properties, e.g.
• tissue organization Permeability and mechanical stability, the extracellular matrix is important for the stability and functionality of many tissues, and its specificity for specific cell types makes it important for maintenance of tissue organization (morphostasis) and homeostasis.
• tissue organization As a tissue organization here the correct spatial arrangement of the cells is referred to as homeostasis their retention over time, even with changing loads.
• a colonization of preformed synthetic structures with living cells can basically be done in vitro, but post-implantation processes are also possible, eg.
• tissue engineering The production of tissues and organs in vitro is often referred to as "tissue engineering” (see, for example, PL Pabst, "Tissue engineering: a historical review as seen through the United States Patent Office", Expert Opinion Ther. Patents 13 (2003): Griffith and G. Vaughton, "Tissue Engineering - Current Challenges and Expanding Opportunities," Science 295 (2002): 1009-1012; E. Pennisi, “Tending Tender Tenons,” Science 295 (2002): 1011).
• the substance must not induce an inflammatory or rejection reaction, which precludes many of the versatilen proteinaceous materials from the outset.
• the substance should be biodegradable, ideally at a rate equivalent to replacement by biogenic structures in the organ. This should allow subtle replacement of the synthetic matrix by natural tissues / structures while maintaining the shape without critical phases of reduced mechanical stability.
• the degradation should not be swelling / bursting, but eroding, so that the mechanical stability of the synthetic structure remains as long as possible.
• no toxic mono- or oligomers should be formed during this degradation. This places particular demands on both the three-dimensional structure of the preformed synthetic matrix and the material of which it is composed.
• polymeric biomaterials see e.g. B. L. G. Griffith, "Polymeric Biomaterials", Acta mater. 48 (2000), 263-277. Both natural materials such as collagen and fibrin, as well as synthetic polymers such as polyglycolides and polylactide are mentioned.
• a polyglycolide is dipped in a polylactide solution in CHCl 3 and the wetted material is molded in a mold.
• this type of production of three-dimensional frameworks is not very precise and also not applicable to the production of arbitrarily small or complex shaped frameworks.
• the process is limited to materials that have a relatively low softening point or melting point.
• No. 5,328,603 describes a method for the production of cellulose beads in the submillimeter range ("beads") which are to be used in chromatographic methods. In this case, first cellulose is solubilized by chaotropic salts and then the atomized solution is added to a non-cellulose medium.
• WO 03/029329 describes the preparation of a cellulase strand by solubilization of cellulose in an ionic liquid and subsequent extrusion of the solution into an aqueous medium. The production of two-dimensional or three-dimensional cell structure is not described.
• a degree of control over the forming process in the field of precision mechanics enables the miniaturization of CAD / CAM ("computer aided manufacturing” / “computer aided manufacturing”) techniques, today often referred to as “desktop manufacturing” or "rapid prototyping".
• CAD / CAM computer aided manufacturing
• computer aided manufacturing / "computer aided manufacturing”
• rapid prototyping a three-dimensional model of the object to be manufactured is created in the computer, which is then produced without further intermediate stages by an automated tool controlled by the computer.
• the computer typically implements an algorithm which automatically decomposes the three-dimensional model into a number of finite elements suitable for execution and processes them one after the other.
• construction methods are those in which not void spaces are cut out of an originally coherent block of material, but the material is applied stepwise during the shaping process.
• the cavities provided either remain empty, d. H. with the working medium (air or a liquid medium in which the material is not soluble) are filled, or filled with a placeholder substance, which can be removed after completion of the molding process, for example by solvent or heating.
• the working medium air or a liquid medium in which the material is not soluble
• a placeholder substance which can be removed after completion of the molding process, for example by solvent or heating.
• the explanations of the constructive methods can basically be classified in the two categories “printing” (discontinuous) and “plotting” (continuous).
• printing preferably from a phalanx of nozzles
• the material is applied in rasters, while plotting a substantially uninterrupted strand of material is extruded.
• Plotting requires more time, plotting techniques, and control algorithms, but results in more consistent and predictable results.
• Both plotting and printing and their application in the field of tissue enginering are described in the prior art (eg BVL Tsang and SN Bhatia, "Three-dimensional tissue fabrication", Advanced Drug Delivery Reviews 56 (2004): 1635-1647 A.
• the object of the present invention was to provide a process which makes it possible to use two- and / or three-dimensional structures which serve as scaffolds in the formation of reimplantable or researchable models, for example as scaffolding in the field of "
• the method should also allow the use of scaffolds that are beneficial from a biological-medical point of view, but due to their physico-chemical properties, such as melting point, ductility or solubility, conventional processes for scaffolding are not trivial to edit.
• the object has been achieved by a method for producing two- or three-dimensional frameworks from biodegradable and biocompatible polymers, which comprises the following steps:
• Cannula wherein the cannula and the resulting framework move relative to each other in two or preferably three dimensions during the extrusion process;
• the material is basically any biodegradable and biocompatible polymer suitable.
• biodegradable is a polymer which, under the conditions prevailing in the organism, chemically or enzymatically to mono- or oligomers soluble in body fluids within a suitable period of time, eg within one year, preferably within weeks or Months, can be reduced.
• biocompatible in the context of the present invention, a polymer is referred to, if neither the polymer nor its mono- or oligomeric degradation products harmful, z. B.
• the term "scaffold” is understood to mean a spatial structure which comprises at least two straight, bent and / or bent rods or strands, as a rule at least one strand or rod overlap or contact being present. Overlapping here means that the angle between the strands is not equal to 0, while touching also includes the angle 0 (e.g., with strands lying parallel to each other).
• z. B. surface, spiral or circular elements to be included In the framework and non-rod-shaped, z. B. surface, spiral or circular elements to be included.
• rod or "strand” is meant a substantially linear structure in the stretched state (“straight rod / strand”), ie, a three-dimensional shape whose extent is in one dimension
• a "strand” is a rod obtained by extrusion Kinked or bent rods / strands have an overall dimension in two dimensions.
• a structure fulfills a certain dimension if its extent in this dimension is more than one, preferably more than two, strand or bar diameter.
• "beads” according to US 5,328,603 are zero-dimensional and elongated single strands one-dimensional.
• a "three-dimensional framework” is a framework that fulfills three dimensions.
• a "two-dimensional framework” has an extension in two dimensions. Although individual bent or kinked strands are also two-dimensional according to the above definition, in the context of the present invention a two-dimensional framework should be understood as comprising at least two rods / strands which overlap or touch each other in at least one point and their common extension is limited to two dimensions.
• the multi-dimensional structures also include forms without strand overlapping, such as, for example, B. loops or spirals. These do not correspond to those in the frame
• the term "scaffold" used in the present invention may be part of the frameworks according to the invention.
• the term "solubilization" in the context of the present invention refers to a conversion of the framework material (polymer) which can be achieved without significant heating in a flowable, pourable or extrudable state.
• the polymer is converted into a solvated state in which, however, the individual polymer molecules do not have to be completely surrounded by a solvate shell. It is essential that the polymer changes into a liquid state or at least a softening state as a result of the solubilization.
• the term "without significant heating” means that for solubilization temperatures of at most 200 ° C., preferably at most 150 ° C., more preferably at most 120 ° C. and in particular at most 100 ° C. are used.
• Chaotropic refers to substances that are able to dissolve supermolecular associates of macromolecules by disrupting or influencing the intermolecular interactions, without affecting the intramolecular covalent bonds.
• extrusion in the context of the present application is not limited to a specific production technique, but generally refers to the substantially continuous pressing out of a flowable substance through a relatively narrow opening (ie a nozzle in the broadest sense), eg through a cannula.
• Essentially continuous means that the extrusion process can also be interrupted again and again, eg to generate individual polymer strands (for example according to step (ii-b)) or to transfer to another spatial plane in step (ii-a) However, it does not take place with such periodic interruptions that only zero-dimensional structures, such as spheres, arise.
• a "substantially continuous" polymer rod is a one-dimensional polymer structure in the stretched state, that is, a structure that is not formed by joining and / or fusing zero-dimensional structures in a particular arrangement. It is preferable here if the rod is substantially uniform in thickness, especially if it does not show a rhythmic sequence of thicker and thinner portions, and if the molecular structure is substantially uniform in the dimension direction, especially if the molecular structure in the dimension direction does not undergo rhythmic changes shows. Furthermore, it is preferred if the polymer chains in the rod are substantially parallel to each other and to the longitudinal direction of the rod, and in particular if parallel polymer chains overlap in the length dimension, so that contact surfaces between the Molecules are formed.
• the extent of the overlap over the entire length of the rod is substantially uniform.
• the intersection of the polymer chains leads to the formation of partially crystalline regions.
• stabilization by covalent cross-linking in the overlap region is subsequently possible.
• substantially means that usual deviations caused, for example, by the extrusion step are tolerated.
• cannula refers to any type of nozzle through which a solution produced in the first step can be continuously pressed.
• the "relative movement of the cannula and scaffold or polymer strand relative to one another” means that during the extrusion step (ii) both the cannula alone and the framework or the polymer strand or the container which contains the liquid medium into which the polymer extrudes
• the movement takes place over the entire step (ii-a) as viewed in two spatial directions (two-dimensional movement) or preferably in all three spatial directions (three-dimensional movement) or over the entire step (ii-b)
• Moving in one spatial direction produces straight polymer strands, while a two-dimensional relative movement leads to bent or kinked strands.
• a three-dimensional relative movement of the cannula and polymer strand is possible of spiral or circular elements, which may also be part of the framework, but vorzu be built to a minor extent.
• step (ii-a) means that the position of the cannula opening can be varied relative to the framework previously formed in all three space dimensions.
• the extrusion mechanism is displaceable in all three spatial dimensions.
• the extrusion mechanism is displaceable in at least two spatial dimensions, and the framework so far formed is displaceable in at least one spatial dimension, so that the missing space dimensions (degrees of freedom) of the extrusion mechanism are supplemented by displacement of the framework previously formed .
• the extrusion mechanism is displaceable in at least one dimension and the framework so far formed in at least two dimensions, so that the missing degrees of freedom of the extrusion mechanism are complemented by the displaceability of the framework previously formed.
• the extrusion mechanism is in the process of considerably immovable, while the framework thus far formed is displaceable in all three spatial dimensions.
• step (ii-a) or (ii-b) ie either the extrusion mechanism or the resulting polymer strand (or more precisely the container into which it is extruded) is movable.
• the extrusion mechanism moves in one spatial direction and the polymer strand moves in a different spatial direction.
• substantially insoluble means that the polymer has a solubility in the liquid medium of less than 5 g / l, preferably less than 0.5 g / l, and more preferably less than 0.05 g / l.
• a “liquid medium” refers to a medium whose physicochemical properties are mainly determined by those of a liquid solvent.
• the liquid medium may also have a gel-like consistency due to the presence of soluble or swellable macromolecules.
• alkyl is a linear or branched alkyl radical
• alkyl is C 1 -C 6 -alkyl
• C 1 -C 6 -alkyl is a linear or branched alkyl radical having 1 to 6 carbon atoms, examples of which are methyl, ethyl, propyl, isopropyl, n- Butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl and constitutional isomers thereof
• C 1 -C 4 -alkyl is a linear or branched alkyl radical having 1 to 4 carbon atoms, examples being methyl, ethyl, propyl, isopropyl , n-butyl, sec-butyl, isobutyl and tert-butyl.
• C- ⁇ -C 6 -alkoxy 6 alkyl group for a bonded via an oxygen atom dC examples thereof are methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy and constitutional isomers thereof.
• -C 4 -alkoxy 4 alkyl group for a bonded via an oxygen atom dC examples of these are methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy and tert-butoxy.
• C 1 -C 6 -alkoxy-C 1 -C 6 -alkyl is a C 1 -C 6 -alkyl radical in which one or more
• Hydrogen atoms are substituted by a dC 6 alkoxy. Examples of these are methoxymethyl, ethoxymethyl, propoxymethyl, 1- and 2-methoxyethyl, 1- and 2-ethoxyethyl, 1- and 2-propoxyethyl and the like.
• C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl is a C 1 -C 4 -alkyl radical in which one or more hydrogen atoms are substituted by a C 1 -C 4 -alkoxy radical. Examples of these are the abovementioned radicals.
• Aryl is a carboaromatic radical having preferably 6 to 14 carbon atoms.
• Suitable substituents are, for example, halogen, C 1 -C 6 -alkyl, NO 2 , OH and CN.
• Aryl is preferably phenyl or substituted phenyl, such as ToIyI, XyIyI, nitrophenyl or chlorophenyl.
• Aryl-C 1 -C 6 -alkyl is an aryl radical bonded via C 1 -C 6 -alkyl, preferably C 1 -C 2 -alkyl, such as benzyl or 2-phenylethyl.
• Aryloxy is an oxygen-bonded aryl radical, such as phenoxy.
• Aryl-C 1 -C 6 -alkoxy represents a C 1 -C 6 -alkoxy radical, preferably C 1 -C 2 -alkoxy radical in which a hydrogen atom is substituted by an aryl group, for example benzoxy.
• Aryloxy-C 1 -C 6 -alkyl is a C 1 -C 6 -alkyl radical, preferably C 1 -C 2 -alkyl radical, in which a hydrogen atom is substituted by an aryloxy group.
• Halogen is fluorine, chlorine, bromine or iodine, in particular fluorine or chlorine.
• Acid anions of CrC ⁇ monocarboxylic acids are the acid anions of aliphatic C-i-Ce monocarboxylic acids. Examples of these are acetate, propionate, butyrate, isobutyrate, pentanoate, hexanoate and the like.
• Mono- and dianions of C 2 -C 6 -dicarboxylic acids are the simple anions or the dianions of aliphatic C 2 -C 6 -dicarboxylic acids, for example the mono- or dianions of oxalic acid, malonic acid, succinic acid, adipic acid and the like.
• the polymeric framework material is an organic polymer.
• An organic polymer is understood here to mean a polymer whose monomers are essentially organic molecules, eg. As alcohols, in particular di- and polyalcohols, carboxylic acids, in particular hydroxydicarboxylic acids and amino acids, amines, in particular di- and polyamines and amino acids, and saccharides, in particular glucose and Fructo- sevenezen. "Substantially organic molecules” means that they are also anor- may contain ganic components, eg metal cations or halide ions, but the nature of the molecule is overall organic.
• the polymer is a biopolymer.
• a biopolymer is here understood a polymer whose monomers occur in nature, for. As saccharides and amino acids, and in particular a polymer whose total structure occurs in nature.
• examples of biopolymers are proteins, e.g. Silk protein, and polysaccharides, e.g. Cellulose, cellulose derivatives, chitin, chitosan, dextran, hyaluronic acid, chondroitin sulfate, xylan and starch.
• the polymer is selected from polysaccharides and modified polysaccharides, and more particularly from polysaccharides. These not only meet the chemical and mechanical requirements of suitable materials in the area of "tissue engineering", but are also immunologically harmless, in contrast to many other proteins.
• suitable polysaccharides are, for example, cellulose, cellulose derivatives, chitin, Chitosan, dextran, hyaluronic acid, chondroitin sulfate, xylan and starch.
• cellulose or a cellulose derivative is used in the process according to the invention.
• Suitable cellulose derivatives are, for. Methylcellulose, ethylcellulose, propylcellulose, hydroxyethylcellulose and
• Hydroxypropyl cellulose In particular, cellulose is used. As cellulose any known form of cellulose can be used, for. As cellulose, cotton, paper-derived cellulose or bacterial cellulose.
• the polymer is mechanically comminuted prior to solubilization, e.g. B. by grinding and / or shredding.
• the polymer can be used in step (i) as such or together with further components.
• Preferred additional components are those which advantageously influence the framework construction and / or the subsequent use of the framework.
• suitable components are inorganic particles, eg. Hydroxyapatite particles, and non-structural, d. H. different from the scaffold polymer, biopolymers, eg. As proteins, protein fragments, peptides or certain carbohydrates.
• biopolymers eg. As proteins, protein fragments, peptides or certain carbohydrates.
• non-structural biopolymers are additional
• suitable non-structural biopolymers are matrix proteins, e.g. Fibronectin, vitronectin, collagen, laminin, lectins, tissue extracts, growth factors, e.g. As VEGF, or fusion proteins or other derivatives of said proteins.
• suitable biopolymers are Proteins or peptides containing the amino acid motif RGD, further adhesion promoting carbohydrates such as sialyl Lewis x or fragments thereof or otherwise bioactive carbohydrates such as heparin or fragments thereof.
• the corresponding molecules can each be covalently or noncovalently linked to polymer molecules.
• step (i) contains one or more of said biopolymers, these are present in an amount of preferably 0.1% by weight to 5% by weight, in particular from 1% by weight to 2% by weight. %, based on the total weight of the backbone polymer.
• the backbone polymer contains inorganic particles such as hydroxyapatite, they are contained in an amount of preferably 1 to 20% by weight, more preferably 5 to 10% by weight, based on the weight of the backbone polymer.
• the chaotropic liquid is substantially anhydrous.
• “Substantially anhydrous” means that the chaotropic liquid contains less than 5% by weight of water, preferably less than 2% by weight of water, more preferably less than 1% by weight of water, based on the total weight of the chaotropic liquid , contains.
• the chaotropic liquid is substantially free of nitrogenous bases.
• “Substantially free of nitrogenous bases” means that the chaotropic liquid is less than 5% by weight, preferably less than 2% by weight, more preferably less than 1% by weight of nitrogenous bases, based on the total weight of the Nitrogen-containing bases are, for example, ammonia, amines and aromatic or nonaromatic heterocycles having at least one basic nitrogen atom as ring member.
• the chaotropic liquid is at a temperature of at most 150 ° C, e.g. in the temperature range from -100 ° C to +150 ° C or from 0 to +150 ° C or from 50 to +150 ° C, more preferably from at most 120 ° C, z.
• the solubilization process can also be supported by ultrasound.
• the heating is effected by microwave irradiation.
• the solubilization occurs at temperatures of at most 200 ° C, e.g. from 0 to 200 ° C, or preferably from 20 to 200 ° C, or more preferably from 50 to 200 ° C, or more preferably from 100 to 200 ° C, more preferably at most 150 ° C, e.g. from 0 ° C to +150 ° C or preferably from 20 to 150 ° C or more preferably from 50 to 150 ° C or more preferably from 100 to 150 ° C, more preferably from at most 120 ° C, e.g.
• the chaotropic liquid is selected from liquid salts.
• Liquid salts are also referred to as "ionic liquids.”
• ionic liquids are understood to mean salts in which the ions are only weakly coordinated, so that these salts are stable at relatively low temperatures, for example below 150 ° C. or below 100 ° C. or even at room temperature, in which case the charge is delocalized in at least one of the ions and at least one of the ions is organic, preventing the formation of stable crystal lattices.
• the liquid salt has the formula Het + A x " 1 / X.
• Het + is a positively charged N-alkylated, N-arylated, N-arylalkylated, N-alkoxylated, N-aryloxylated, N-arylalkoxylated, N-alkoxyalkylated and / or N-aryloxyalkylated nitrogen-containing heterocycle.
• Het + stands for a positively charged nitrogen-containing heterocycle in which formally a ring nitrogen atom carries an alkyl radical, aryl radical, arylalkyl radical, alkoxy radical, aryloxy radical, arylalkoxy radical, alkoxyalkyl radical and / or an aryloxyalkyl radical via its free electron pair
• a positive charge is generated, ie, the positive charge of the heterocycle is due to the substitution of the free electron pair of a ring nitrogen atom.
• Alkyl in the radicals mentioned is preferably C 1 -C 6 -alkyl.
• Alkoxy in the radicals mentioned is preferably C 1 -C 6 -alkoxy.
• Aryl in the radicals mentioned is preferably phenyl.
• Arylalkyl in the radicals mentioned preferably represents aryl-C 1 -C 6 -alkyl, such as benzyl or phenylethyl.
• Aryloxy is in the radicals mentioned preferably for an oxygen-bonded phenyl radical, for example phenoxy.
• Arylalkoxy in the radicals mentioned preferably represents an aryl-C 1 -C 6 -alkoxy radical, for example benzoxy.
• Alkoxyalkyl in said radicals preferably for a -C 6 - alkoxy-C- ⁇ -C6 alkyl.
• Aryloxyalkyl in the radicals mentioned preferably represents an aryloxy-C 1 -C 6 -alkyl radical, in particular a phenoxy-C 1 -C 6 -alkyl radical.
• Het + is an aromatic heterocycle or an alicyclic heterocycle in which the ring nitrogen atom is not part of a double bond
• the nitrogen atom which formally generates the positive charge is either monosubstituted or disubstituted by the above-mentioned radicals.
• a x " 1 / X represents an anion in which x is 1, 2 or 3.
• Het + is selected under
• R a and R a independently of one another are C 1 -C 6 -alkyl, aryl, C 1 -C 6 -alkoxy, aryloxy, C 1 -C 6 -
• R b is hydrogen, C r C 6 alkyl, aryl, C r C 6 alkoxy, aryloxy, Ci-C 6 -alkoxy-CrC 6 - alkyl or aryloxy -C 6 alkyl and preferably -C 6 alkyl or -C 6 -
• Alkoxy-C 1 -C 6 -alkyl; wherein the alicyclic or aromatic heterocycles or the benzene rings may be at the latter of which is condensed may carry up to 5 substituents 1 are selected from C r C 6 alkyl, C r C 6 alkoxy and Ci-C 6 -alkoxy-C C 6 alkyl.
• Het + is particularly preferably selected from compounds of the formulas Het.1 to Het.15:
• R 1 and R 2 are independently dC 6 alkyl or Ci-C 6 alkoxy-CrC 6 alkyl
• R 3 to R 9 are each independently hydrogen, CrC 6 alkyl, Ci-C 6 alkoxy-C 6 - alkyl or d-C6-alkoxy, with hydrogen being particularly preferred.
• both R 1 and R 2 for dC 4 alkyl or Ci-C4-alkoxy-Ci-C 4 - alkyl where, it is particularly preferred if one of these groups is methyl.
• both R 1 and R 2 are C 1 -C 4 -alkyl.
• one of the radicals R 1 or R 2 is methyl and the other is C r C 4 alkyl, for example ethyl.
• R 3 to R 9 are preferably H.
• Het + is monocyclic. Accordingly, Het + is preferably selected from the compounds of formulas Het.1 to Het.13. Het + is more preferably a monocyclic five-membered ring. Accordingly, Het + is particularly preferably selected from the compounds of the formulas Het.5 to Het.1 1 and Het.13.
• Het + is more preferably an imidazolium ion of the formula Het.5, a pyrazolium ion of the formula Het.6, an oxazolium ion of the formula Het.7, a 1,2,3-triazolium ion of the formula Het.8 or Het.9, a 1,2,4-triazolium ion of the formula Het.10 or a thiazolium ion of the formula Het.11, where R 1 to R 5 are as defined above.
• both R 1 and R 2 are preferably C 1 -C 4 -alkyl or C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl and particularly preferably C 1 -C 4 - Alkyl, wherein it is particularly preferred if one of these groups is methyl.
• one of the radicals R 1 or R 2 is methyl and the other is C 1 -C 4 -alkyl, for example ethyl.
• R 3 to R 5 are preferably H.
• Het + is an imidazolium ion of the formula Het.5, wherein R 1 to R 5 are as defined above.
• R 1 to R 5 apply here correspondingly, ie, both R 1 and R 2 preferably upstream for -C 4 -alkyl or Ci-C 4 -alkoxy-CrC 4 -alkyl and particularly preferably -C 4- alkyl, wherein it is particularly preferred if one of these groups is methyl.
• R 3 to R 5 are preferably H.
• Het + stands in particular for an imidazolium ion of the formula Het.5 which carries a methyl group on a ring nitrogen atom and on the second ring nitrogen atom a C 1 -C 4 -alkyl group or C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl group
• R 3 , R 4 and R 5 stand for H.
• one of the radicals R 1 or R 2 is methyl and the other is C 1 -C 4 -alkyl, for example ethyl.
• a x " 1 / X is preferably chosen from coordinating anions, ie those which are in principle capable of coordination eg to a metal center.
• a x " 1 / X is selected from halides, pseudohalides, perchlorate, the acid anions of dC 6 -monocarboxylic acids and the mono- and dianions of C 2 -C 6 - dicarboxylic acids, wherein the mono- and dicarboxylic acids mono-, di-or trihydric Halogen and / or hydroxy may be substituted
• a preferred acid anion is acetate.
• a x " 1 / X selected from halides, pseudohalides and acetate.
• Pseudohalides are z. Cyanide (CN “ ), cyanate (OCN “ ), isocyanate (CNO “ ), thiocyanate (SCN “ ), isothiocyanate (NCS “ ) and azide (N 3 " ).
• a x " 1 / X is chloride, bromide, cyanate, thiocyanate or acetate.
• a x" 1 / X is chloride or acetate.
• Het + A x " 1 / X stands for an imidazolium chloride Het.5-Cl " or an imidazolium acetate Het.5- (CH 3 COO " ), the imidazolium ion preferably being substituted as described above.
• the chaotropic liquid is selected from solutions of chaotropic salts in polar aprotic solvents.
• the inorganic salts are preferably selected from alkali halides, alkaline earth halides, ammonium halides, alkali metal pseudohalides, alkaline earth metal halides. halogenides, ammonium pseudohalides, alkali perchlorates, alkaline earth perchlorates and ammonium perchlorates and mixtures thereof.
• the inorganic salt is particularly preferably selected from lithium chloride, calcium thiocyanate, sodium iodide, sodium perchlorate and mixtures thereof.
• Preferred polar aprotic solvents are dimethylformamide, dimethylacetamide, dimethylsulfoxide and diethylamine and mixtures thereof.
• the chaotropic liquid is selected from the ionic liquids described above.
• the statements made above on the preferred embodiments of the ionic liquid are hereby incorporated by reference.
• step (i) of the process according to the invention is generally carried out by mechanically mixing the optionally previously comminuted polymer with the chaotropic liquid and stirring until complete dissolution.
• the mixture is heated to accelerate the dissolution and homogenization process during or after mixing, e.g. B. by microwave irradiation, but preferably not to a temperature of more than 150 ° C, preferably not more than 120 ° C, in particular not more than 100 ° C.
• the concentration of the solubilized polymer in the chaotropic liquid is 5% by weight to 35% by weight, preferably 5% by weight to 25% by weight and in particular 10% by weight to 25% by weight. %.
• step (N) Upon introduction of the solubilized polymer into the liquid medium (step (N)), it precipitates within a very short period of time, e.g. In less than 1 s.
• the introduction takes place by extrusion, d. H. Press out the solubilizate through a cannula.
• the chaotropic solution is introduced into the liquid medium in which the chaotropic components are soluble but the polymer material is substantially insoluble, the polymer precipitates.
• the extrusion of the polymer into a liquid medium takes place by means of a movable cannula, which is preferably part of an automated device.
• the cannula or the container in which the liquid medium is, or both are in this case moved so that the extrudate in variant (ii-a) takes the form of a three-dimensional framework, network or lattice and in variant (ii-b) the Shape of a straight, bent or bent polymer strand.
• the liquid medium used in step (ii) is on the one hand miscible with the chaotropic liquid of step (i), on the other hand, the polymer used is substantially insoluble therein.
• Preferred liquid media are protic solvents such as water and alkanols, cyclic ethers such as tetrahydrofuran and dioxane, ketones such as acetone and ethyl methyl ketone, and nitriles such as acetonitrile, and mixtures thereof.
• Preferred liquid media are protic solvents such as water and alkanols, as well as mixtures thereof.
• Suitable alkanols are C 1 -C 4 -alkanols, such as, for example, methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol and tert-butanol.
• the liquid medium is aqueous, ie it contains at least 10% by weight of water.
• the liquid medium particularly preferably contains at least 50% by weight of water, in particular at least 80% by weight of water.
• the remaining constituent of the aqueous medium is preferably selected from C 1 -C 3 -alkanols, such as methanol, ethanol, n-propanol and isopropanol. Specially used water.
• the points of intersection between various elements of the scaffold, grid or network are stabilized by polyelectrolytes.
• Polyelectrolytes refers to polymers whose repeat units carry a group capable of accepting or releasing protons, and which can thus take up and release charges in a protic, in particular aqueous medium, and these can be positive and / or negative within a molecule , A negatively charged in the aqueous medium group is z.
• the carboxyl group, a group positively charged in the aqueous medium is e.g. the amino group.
• all common polyelectrolytes are suitable. Suitable polyelectrolytes are, for example, compounds used as additives for increasing the wet strength of paper in papermaking, such as polycarboxylic acids, e.g. Polyacrylic acid, polyamines, e.g.
• Polyvinylamine, polyimines, copolymers of amide-part unsaturated carboxylic acid amides and unsaturated carboxylic acids e.g. N-vinylformamide / acrylic acid copolymers, polymerizable basic heterocycles, e.g. N-vinylpyrrolidone, reaction products of polyamines with epichlorohydrin, epoxidized polyamides, urea resins, melamine resins, polyurethanes and the like.
• Such wet strength enhancers are described, for example, in EP 01 1 18 439, to which reference is hereby fully made.
• polyelectrolytes are polycarboxylic acids, such as. For example, polyacrylic acid, monotone aliphatic polyamines such.
• polyvinylamine and polymerizable basic heterocycles, ie heterocycles having an exocyclic ethylenic double bond, such as polyvinylpyrrolidone.
• the polyelectrolytes are part of the liquid medium into which the solubilized polymer is extruded. It is preferred if the liquid medium contains up to 20 wt .-% of polyelectrolyte, in particular from 5 wt .-% to 10 wt .-%, based on the total weight of the liquid medium.
• single or all parts of the scaffold are provided with signal or growth factors that act on living cells.
• Corresponding factors eg. As VEGF or NGF, are familiar to the expert. Depending on their individual physical or chemical properties, these factors may preferably be added either to the material to be extruded in step (i) as previously described ("doping") or during the extrusion process in step (ii) or thereafter using a suitable cannula be applied as a layer on the surface of the resulting strand.
• the resulting polymer backbone may be homogeneous with respect to the distribution of the factors, but preferably a heterogeneous distribution that leads to the formation of signal factor gradients and thus tissue regeneration, z. B. the ingrowth of blood vessels and nerve fibers, an orientation pretending.
• the heterogeneity of the signal factors is combined in a suitable manner with a structural heterogeneity of the skeleton, lattice or network, for example by B. for blood vessels or nerves larger, heavily doped recesses are kept free or the conditions for the formation of more complex organ structures are created.
• the extrusion is carried out in such a way that the framework is built up essentially in layers, so that a framework with a layer structure is formed, ie the vast majority of the bars or strands lie in each other parallel planes, wherein the stabilizing contacts between the respective adjacent layers come about mainly by intersections of strands, while the contribution of not extending in a plane bars or strands for interactions between the layers and thus for the three-dimensional stability of the scaffold is irrelevant.
• Substantially layered construction means that the framework may also contain strand arrangements which do not belong to these layers, but the framework mainly, for example at least 60%, preferably at least 80% and in particular at least 90%, based on the length of the polymer strands that make up the framework as a whole, is composed of stratified polymer strands.
• each layer consists mainly of extrudate strands running parallel to one another, in particular of bustrophedone, d. H. alternating from neighbor strand to neighbor strand in the direction and opposite direction, extending extrudate strands.
• bustrophedone d. H.
• the stabilization of the strands of each individual layer is achieved primarily by the contact with the strands of the neighboring layer or neighboring layers, provided that a grid-like, permeable structure is provided in the relevant area of the plane.
• planes or parts of planes can also be rendered opaque by placing the parallel strands in the area in question so that they touch each other.
• the strands of adjacent planes are neither parallel nor antiparallel to each other.
• the angles of the strands between adjacent layers are 90 °, 60 ° or 45 °.
• FSS curves, FASS space-filling, self-avoiding, simple and self-similar
• Substantially means that the layers are at least 60%, preferably at least 80% and especially at least 90%, based on the total length of the polymer strands of which the respective layers are constructed of polymer strands in the form of two-dimensional space-filling curves
• a FASS curve is a path that leads over a surface made up of a number of uniform fields or through a three- or more-dimensional space made up of a number of "chambers" so that each field or chamber is touched without the path intersecting with itself, resulting in a more uniform structure in both dimensions of the plane than in building the plane of parallel strands.
• Preferred special forms of FASS curves are Peano curves, Hilbert curves, and Sierpi ⁇ ski. curves.
• each plane is preferably decomposed into a number of surfaces, each of which is substantially independently filled by the other surfaces of the same plane.
• adjacent planes are divided into different surface groups.
• each plane is decomposed in such a way that the largest possible proportion of square surfaces is created, each of which is filled by a FASS curve. It is particularly preferred in this case if the decomposition of the surfaces takes place in such a way that as continuous a extrusion as possible is possible.
• the decomposition takes place in such a way that the plane essentially, for. B. at least 50%, preferably at least 75%, in particular at least 90% consists of FASS curves.
• FASS curves can be generated using recursive algorithms. Corresponding methods are familiar to the person skilled in the art and, for example, in V. Batagelj: Logo to PostScript. Paper prepared for Eurologo'97, Ljubljana 1997; AJ. CoIe: A note place filling curves. Software - Practice and Experience, 13 (1983), 181-1189; AJ. CoIe: Note Peano Polygons and Gray Codes. International Journal of Computer Mathematics, 18 (1985), 3-13; C. Davis, D.E. Knuth: Number Repre- sentations and Dragon Curves, / - //. Journal of Adventureal Mathematics, 3 (1970), 66-81; 3 (1970), 133-149; M.
• the polymer backbone comprises helical, spiral or circular elements, which may be round or angular, continuous or stepwise, single helices or multiple helices.
• the helical, spiral or circular elements are different from the latticed elements in terms of strand thickness and / or doping.
• the structure of the polymer skeleton is substantially three-dimensionally homogeneous, ie the strands or rods in all spatial dimensions make quantitatively and qualitatively comparable contributions.
• the polymer backbone consists essentially of an extrudate strand in the form of a three-dimensional FASS curve and in particular a three-dimensional Peano curve.
• “Substantially” means that the framework to at least 60%, preferably at least 80% and in particular at least 90%, based on the total length of the polymer strands that make up the skeleton as a whole, of polymer strands in the form of three-dimensional FASS curves is constructed.
• At least 25% of the total volume of the polymer backbone accounts for through channels.
• a "through-channel" is a cavity whose length is at least half the length of the dimension of the entire polymer framework parallel to it and which communicates with the outer surface of the framework.
• step (ii) To isolate the polymer backbone, it is either taken from the container used in step (ii) or first the liquid medium into which it has been extruded is removed.
• An alternative method of isolation particularly when using water or aqueous mixtures as the liquid medium in step (ii), is to freeze the medium and isolate the framework from the frozen medium by suitable methods, e.g. by mechanical removal of the frozen medium or by its sublimation.
• the framework can subsequently be freed from residues of the liquid medium, e.g. by drying in air, in a drying oven or in a vacuum oven or by lyophilisation.
• the solubilizate obtained in step (i) is extruded in such a way that individual straight, bent or bent polymer strands are formed.
• the desired shape is created by the relative movement of cannula to container and / or by informing the strand after extrusion, e.g. by stretching, bending and / or buckling.
• all common mechanical aids such as staples, tweezers, rods, etc., or also dipped into the liquid medium matrices with the desired shape, which are subsequently removed, can be used.
• the polymer strand is isolated from the liquid medium before processing to the scaffold, optionally (after) molded and / or dried. Isolation and drying can be carried out as described above.
• the (After) molding can be done after or preferably before drying.
• the (post) molding may include, for example, stretching, bending and / or buckling of the polymer strand, eg with the aid of the abovementioned auxiliaries.
• the polymer strands can be linked to the desired framework structure.
• either only identical or different polymer fibers can be linked together.
• these may differ, for example, in their diameter, in their nature and / or in their production process.
• polymer fibers may be used which differ in that they have been produced by extrusion with needles of different shape and / or diameter and / or that they have been prepared from various biodegradable and biocompatible polymers and / or that they by means of different methods wherein at least one polymer strand type must have been produced by the process according to the invention.
• Joining can be accomplished by known techniques for bonding / bonding such polymers, e.g. by means of customary biodegradable and biocompatible adhesives.
• the linking is preferably carried out by applying a small amount of the obtained in step (i) or another solubilizate from a biocompatible and biodegradable polymer in a chaotropic liquid at the desired linking points and then adding a liquid medium, in which the polymer is not soluble. As the polymer precipitates, it simultaneously bonds the individual polymer strands together.
• step (i) it is in principle also possible to link the polymer strands together in the liquid medium, for example by applying a small amount of the solubilizate obtained in step (i) to the desired attachment points.
• the scaffolds prepared in the liquid medium can then be isolated as described above and, if desired, dried.
• the first approach i. however, isolation of the polymer strands and subsequent linking to a scaffold is preferred, but is more easily feasible.
• step (ii) especially when only small amounts of the liquid medium are used, gel-shaped products often form, which can be isolated relatively easily.
• the conversion into the solid state takes place by drying. It can also be convenient to leave the scaffolds formed in gelled form until their use, to increase their shelf life, and to dry them just before use.
• the scaffolds are not yet (completely) dry, they may, if desired, be formed (post), which may be done as described above.
• variant (N-a) is preferred for step (ii).
• this variant allows a simple and reproducible access to otherwise not trivially produced three-dimensional frameworks.
• a method according to variant (ii-b) is also suitable for meshes which are sufficient, for example, for the construction of flat tissue such as skin.
• the resulting scaffolds may then be treated as described further below, e.g. by coating or doping with living cell effective signal and / or growth factors or by colonization with living cells.
• two- and three-dimensional scaffolds from biodegradable and biocompatible polymers which are generally difficult to process, such as cellulose or cellulose derivatives, are easy to prepare.
• These frameworks which can also take on highly complex forms, can be used as shaping structures in the construction of artificial tissue.
• Another object of the present invention is a polymer backbone obtainable by the process according to the invention.
• the polymer skeleton With regard to the preferred embodiments of the polymer skeleton, reference is also made to the above statements.
• a "negative scaffold” is formed by pouring the voids of the final primary scaffold with another, meltable or gellable polymer that differs in degradability in vitro from the first polymer, followed by degradation of the first polymer.
• living cells are bound to it. These are preferably eukaryotic cells, in particular mammalian cells, eg. B. human cells.
• the living cells are preferably prokaryotic cells, in particular the cells of socially organized bacteria, e.g. B. biofilm-forming or mycelium-growing bacteria.
• the preparation of the finished polymer backbone on the colonization by living cells for example by washing one or more times with an aqueous medium, eg.
• physiological saline solution As water, physiological saline solution ("Ringer solution”) or phosphate-buffered saline (“phosphate buffered saline", PBS) take place. Multiple washes are particularly appropriate if the polymer used and / or the polyelectrolyte used contain a significant proportion of low molecular weight substances.
• the polymer backbone may be dried by live cells prior to colonization, e.g. By rapid freezing followed by freeze-drying. In this case, it is preferable if the drying parameters are selected such that the dried polymer backbone is storable. Shelf life in this context means that the polymer backbone does not show damage to the structure which can be detected by light or electron microscopy over a period of preferably at least one week, more preferably at least one month.
• the dried scaffold is equilibrated with an aqueous medium prior to colonization by living cells, wherein the equilibration step may be a washing step or may be followed by one or more washing steps.
• the equilibration may further comprise impregnation of the polymer backbone with substances which specifically or non-specifically bind to the surface of the polymer strands. Such impregnation can also take place without preceding drying steps.
• Preferred molecules for soaking are those which modulate or affect the colonization and / or function of the living cells, but are not compatible with the extrusion process of the present invention, e.g. due to lack of stability to the chaotropic substances used.
• either the desired distribution of the substance to be grown on the polymer strands is substantially homogeneous or the various polymer strands are designed so that they have a different affinity for the substance to be absorbed, so that there is a differential distribution of the substance to be absorbed results.
• the equilibration / tendrils are suitably carried out especially when molecules bound to the surface of the polymer strands are to be activated, e.g. By cleavage of protective groups, activating proteolytic cleavage of proenzymes and / or renaturation of polypeptide chains denatured as a result of treatment with chaotropic substances, for example by treatment of the backbone with proteins or protein mixtures with chaperone activity under slightly reducing conditions. conditions, eg B. by incubation with a 10 wt .-% serum albumin and 1 mM ß-mercaptoethanol containing physiological saline buffer at + 37 ° C.
• a mechanical preparation of the polymer backbone can be carried out, for. B. by stretching or biasing.
• Such processes are familiar from polymer engineering; without wishing to be bound by theory, it is believed that application of low mechanical forces to a polymer strand results in an improvement in the supermolecular ordering, and thus enhancement of intermolecular interactions and increase in the mechanical stability of the strand.
• the colonization of the prepared polymer scaffold by living cells is basically in vitro, whereby degradation of the polymer scaffold, formation of extracellular matrix, etc. may possibly continue beyond the implantation.
• adherent or adhesive cells are used, which have been previously removed from their natural association, z. B. by treatment with proteases, preferably trypsin, and / or chelating agents such.
• proteases preferably trypsin
• chelating agents such as ethylenediaminetetraacetic acid (EDTA).
• EDTA ethylenediaminetetraacetic acid
• the colonization is conveniently carried out by incubation of the prepared and optionally equilibrated with the cell growth medium polymer backbone with the cells in a growth medium under generally permissive conditions.
• Typical conditions for colonization by human cells are z.
• DMEM Dulbecco's Modification of Eagle's Medium
• fetal calf serum and appropriate antibiotics at + 37 ° C under an atmosphere of 5% CO 2 .
• Such media and conditions are familiar to those skilled in the art.
• Control of colonization and tissue buildup can be done in several ways, e.g. B. in situ by light microscopy. Prior to use, further washes as well as adjustment of the medium to more body-like conditions can be made.
• Preformed structures suitable for biochemically-physiologically active tissues must have a three-dimensional fine structure that allows them to be colonized by cells in vitro, allows these cells sufficient oxygen and nutrient supply, and later allows blood vessels to enter (vascularization) and possibly nerves from the organism , For this purpose and to build up a complex structured organ or organ part (eg of a nephron) It is further desirable to be able to "dope" individual portions of the preformed synthetic structure with suitable growth and signaling factors to thereby organize the self-assembly of cells into functional bandages.
• a complex structured organ or organ part eg of a nephron
• Another object of the invention relates to the use of a polymer backbone to which living cells are bound as described above for the preparation of an implant for restoring, measuring or altering biological functions in the organism to be treated.
• the implant is selected from artificial bone tissue, artificial skin, artificial blood vessels and hollow organs.
• the implant serves as a carrier in a "drug delivery" system or an implantable sustained release formulation.
• Another object of the invention is an artificial tissue, which is constructed on a polymer scaffold according to the invention.
• the polymer scaffold at the time of implantation or other use may still be substantially completely preserved, partially degraded and / or replaced by extracellular matrix or substantially completely degraded and / or replaced by extracellular matrix.
• the implant serves as a "nerve guide" for the restoration of broken nerve fibers. It is preferred in this case if, at the time of implantation, the polymer scaffold has not yet completely degraded.
• the tissue is selected from artificial bone tissue, artificial skin, artificial blood vessels and hollow organs. If the tissue is an artificial blood vessel or hollow organ, it is preferred if it comprises helical elements, since these have a geometry suitable for producing the inner and outer surfaces.
• Another object of the present invention relates to the use of an artificial tissue based on a polymer scaffold according to the invention for the diagnosis ex vivo and in vitro.
• Another object of the invention relates to the use of a polymer scaffold according to the invention, in which living cells have bound to the scaffold, in a bioreactor.
• cells are kept under "steady state" conditions, for. B. using a countercurrent exchange.
• the cells secrete soluble products, and particularly preferred are hybridomas or stable transfectants which form a soluble protein.
• three-dimensional polymer scaffolds provide a more robust alternative to the hollow-fiber systems known in the art (see, e.g., BTL Evans and RA Miller, "Large-scale production of murine monoclonal antibodies using hollow fiber bioreactors” Biotechniques 1988, Sep. 6 (US Pat. 8): 762-767).
• eukaryotic cells are used according to the invention in a bioreactor, so are hybridomas and other antibody-forming cells, eg. As quadromas, preferred. Also preferred are stable transfected cells, e.g. CHO or NIH3T3 cells, e.g. With a transgene integrated into the genome, it being particularly preferred if the cells secrete a soluble protein.
• prokaryotic cells are used according to the invention in a bioreactor, socially organized formers of low molecular weight metabolites are preferred, in particular formers of antibiotics, eg. B. Streptomycetes, z. B. Streptomyces caelicolor.
• Another object of the present invention relates to an apparatus for carrying out the method according to the invention, in particular for producing a polymer skeleton by extrusion.
• the device comprises a relative to the resulting grid three-dimensionally movable extrusion onskanüle, a mechanical positioning mechanism and a computer unit suitable for controlling the positioning mechanism. It is particularly preferred if the computer unit comprises a program for the automatic generation of the structures.
• the extrusion mechanism and / or the framework so far formed can be rotated about a fixed or variable axis.
• Corresponding mechanical devices for relative three-dimensional positioning of the cannula are generally known to the person skilled in the art (see, for example, BTH Ang et al., "Fabrication of 3D chitosan-hydroxyapatite scaffolds using a robotic dispensing system", Materials Science and Engineering C 20 (2000). : 35-42), as well as the principles of extrusion of polymers.
• the three-dimensional movement takes place mainly in parallel to the three axes of the resulting framework extending steps.
• the cannula is held parallel to none of the three axes, and in particular if it forms a maximum angle to all three axes (arctan ⁇ / 2 ⁇ 55 °, corresponds to point 11 in the Cartesian coordinate system).
• the cannula is parallel to one of the axes of the framework.
• the framework can be moved in one dimension, the extrusion mechanism in the other two, and the cannula is kept parallel to the movement dimension of the framework ("Z-axis").
• the cross section of the cannula is round.
• the cannula is oval in cross-section, polygonal, serrated or irregular shaped.
• multiple needles with the same or different diameter and cross-section similar or different geometry can be used.
• the device according to the invention basically comprises three component groups:
• the first component comprises those parts which come into direct contact with the solution of the polymer in a chaotropic solvent.
• the corresponding parts are therefore expediently made of materials which are resistant to the chaotropic agents used, which, inter alia, have a corrosive effect.
• the parts of the first component can be handled aseptically, e.g. B. by being sterilized by strained steam ("autoclavable").
• the reservoir consists of silicate materials or corrosion-resistant metal, for. As glass, ceramic or stainless steel.
• the cannula is made of corrosion resistant metal, e.g. B. stainless steel.
• the line leading from the reservoir to the movable cannula usually comprises flexible as well as in a particular embodiment also rigid parts.
• the flexible parts of the conduit are made of corrosion resistant polymeric material, e.g. As silicone. If rigid parts are used as line elements, these can basically be made out the same materials as the reservoir or made of the same materials as the flexible parts.
• the storage container serves to receive the polymer dissolved in a chaotropic agent according to the invention.
• it is provided with an agitator to ensure the homogeneity of the polymer solution.
• the storage container is tempered, it being preferred if the contents of the storage container can be kept at a temperature at which the solution of polymer in chaotropic solution is liquid. Both agitator and tempering device can be independently connected to the control system of the second component or independent thereof.
• the reservoir can basically have any suitable shape.
• the second component comprises mechanical components and preferably software for controlling the relative movement between cannula and extrudate.
• the mechanical components are known in principle.
• the second component group comprises a number of stepper motors, which drive mutually orthogonally arranged precision drives, which serve to adjust the cannula.
• the second component preferably comprises as many stepper motors / precision drives as the cannula has degrees of freedom of lateral movement.
• the second component group further comprises a device for adjusting the angle of the cannula.
• the second component group comprises a device for rotating the cannula.
• the second component group comprises a device for automatically changing between different cannulas of different diameters and / or different geometry, for. B. after the turret principle.
• the stepper motors and optional components of the second group are controlled from a computer by means of a D / A converter.
• the valves or pumps of the first group and in particular the agitator and temperature control of the first group are controlled accordingly.
• the computer uses commercially available hardware as well as software suitable for three-dimensional control of the cannula.
• the software is able to automatically convert a given spatial form into an arrangement of extrudate strands according to the invention, in particular a FASS curve, arrangement of extrudate strands comprising especially peano curves and to guide the cannula accordingly.
• the control computer also controls the pump associated with the first group of components which regulates the flow of dissolved polymer to the cannula, in accordance with the movement of the cannula.
• stepper motors fine-drive systems, D / A converters and suitable computer hardware components are generally familiar to the person skilled in the art.
• the third group of components comprises the container for the liquid medium in which the extrusion of the dissolved polymer material takes place.
• the water basin is made of glass or ceramic.
• the water basin is also mounted on a system of stepping motors and fine drives, which can supplement the missing degrees of freedom of the cannula in lateral displacement and / or rotation.
• the stepper motors and precision drives of the third group of components are controlled by the same hardware and software as the second, so that a uniform control of all movements within the system for precise shaping is possible.
• the parts of the third component can be handled aseptically, for. B. by being sterilized by strained steam ("autoclavable").
• the figure shows a cellulose net generated according to the example from two viewing directions.
• the pictured 1 cent coin illustrates the size of the net. example
• Cellulose was added to i-ethyl-3-methylimidazolium acetate and dissolved by stirring at 90 ° C for 2 hours.
• the cellulose content of the solution was 1% by weight of cellulose, based on the total weight of the solution.
• This solution was injected by means of a surgical needle into a water bath at a rate of 70 ml / hr and with stretching of the resulting polymer fiber.
• a gel was obtained which shrank on drying and formed free fibers.
• the dry fibers had a diameter of 70 ⁇ m on average.

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