WO2001010479A1 - Tissue substitute and method for the production thereof - Google Patents

Tissue substitute and method for the production thereof Download PDF

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Publication number
WO2001010479A1
WO2001010479A1 PCT/EP2000/007567 EP0007567W WO0110479A1 WO 2001010479 A1 WO2001010479 A1 WO 2001010479A1 EP 0007567 W EP0007567 W EP 0007567W WO 0110479 A1 WO0110479 A1 WO 0110479A1
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WIPO (PCT)
Prior art keywords
cells
carrier material
growth factor
polymeric carrier
tissue replacement
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PCT/EP2000/007567
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German (de)
French (fr)
Inventor
Gerhard Björn STARK
Matthias Voigt
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Universitätsklinikum Freiburg
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Priority to AU68355/00A priority Critical patent/AU6835500A/en
Publication of WO2001010479A1 publication Critical patent/WO2001010479A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—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
    • A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial 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
    • A61L27/60—Materials for use in artificial skin
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0629—Keratinocytes; Whole skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]

Definitions

  • the present invention relates to tissue replacement that is suitable for transplantation and methods for its manufacture.
  • Non-healing chronic and extensive wounds on the skin represent a major therapeutic and socio-economic problem.
  • a promising and partially still developing therapeutic option for these problems is the transplantation of cultured keratinocytes. These are used to cover large wounds as autologous or allogeneic grafts.
  • the cells are grown in culture bottles and must be enzymatically detached from the culture bottle before transplantation. In the process, proteolytic adhesive molecules are destroyed, which are important for later adhesion to the wound base. This led to the use of transplantable and at least partially absorbable carrier materials. Examples are plates made of hyaluronic acid, collagen or nylon coated with collagen. However, only a limited number of cells can be applied to the wound. In addition, the material / cell ratio is on the material side, which can cause problems in the formation of the new epidermis.
  • bioabsorbable microspheres for example from dextran, are not broken down, which leads to strong inflammatory reactions which hinder wound healing. Therefore, according to the invention, only materials are used that are completely bioresorbable.
  • the cells can be provided with physiological growth factors for optimal proliferation and timely differentiation.
  • Various methods can be used for this purpose, which introduce plasmids with growth factor genes into the cells to be transplanted.
  • wound healing is accelerated by the expression of growth factors.
  • the method is relatively complicated, however, and there are concerns about transfection efficiency and stability, cell survival, and toxicological hazards. In principle, there is even the possibility of Tumor induction.
  • the growth factors are therefore preferably added as a substance.
  • An object of the invention is to provide an advantageous tissue replacement.
  • the present invention relates to a tissue replacement that is suitable for transplantation and consists of a polymeric carrier material that is populated with cells.
  • the polymeric carrier material has a particle shape so that the cells can be cultivated as a suspension culture after the carrier material has been populated.
  • the carrier material is bioresorbable and loaded with at least one growth factor.
  • the tissue replacement according to the invention has the advantages that it can be transplanted, is well tolerated, provides enough cells, dispenses with enzymatic methods for detaching cells and delivers growth factor in a metered manner so that improved wound healing can be achieved.
  • tissue generally includes epithelial tissue, connective and support tissue, muscle tissue and nerve tissue.
  • fabric in the sense of the application is to be understood further.
  • a tissue replacement should not only replace epithelial or connective tissue, for example, but generally cells and their spatially adjacent cells Intercellular substances. For example, a skin replacement is said to replace both epithelial and connective tissue.
  • the carrier material has a particle shape, and a wide variety of geometric shapes can occur.
  • the particles of the carrier material can have an approximately spherical shape, but can also be elongated, disk-shaped or without a defined geometric shape.
  • the diameter of the particles is usually less than 1 mm.
  • the particles preferably have a diameter of 50 to 1000 ⁇ m, most preferably 100 to 250 ⁇ m. The diameter here is the largest, lengthwise extension of the particle.
  • the particles preferably have a surface that can be well populated by cells.
  • the carrier material is populated with cells.
  • the cells are preferably skin cells, so that the tissue replacement can be used as a skin replacement.
  • the cells are human keratinocytes.
  • the particle shape of the carrier material ensures that after the carrier material has been populated with cells, the cells can be further cultivated in suspension culture. The shape according to the invention thus enables efficient cell multiplication and thus a favorable ratio of cell number to carrier material.
  • the particulate carrier material is also referred to below as microspheres.
  • the carrier material according to the invention is absorbed by the animal or human body after transplantation.
  • the absorption preferably takes place within 50 days and more preferably within 25 days. Due to the biocompatibility of the carrier material and the possibility of absorption, foreign body reactions and inflammation are minimized.
  • Preferred carrier materials are hyaluronic acid, hyaluronic acid derivatives, fibrin, bovine collagen, collagen GAG (glycosaminoglycan) substrates.
  • a particularly preferred polymeric carrier material consists of polylactide.
  • Polylactide is a linear polyester polymer based on lactic acid. It can be made from lactide by polymerization. Lactide is a cyclic ester of two molecules of lactic acid, which can be obtained by high temperature distillation of lactic acid. Like other aliphatic polyesters, which are based on naturally occurring substances, polylactide is biodegradable by the animal and human body and thus bioresorbable.
  • the microspheres are loaded with at least one growth factor. Therefore, at least one growth factor is released while the microspheres are being resorbed.
  • Growth factors are substances that can stimulate cell proliferation and / or differentiation. They are usually proteins, but there are also low-molecular growth factors that are not proteins.
  • the presence of growth factor in the wound fluid stimulates the cells to proliferate and, if necessary, to differentiate, which accelerates wound healing.
  • a growth factor concentration in the wound fluid 100 to 1000 pg / ml can be achieved during the release of the growth factor, preferably a concentration of 200 to 400 pg / ml. The concentration values given were found to be advantageous in the case of EGF.
  • concentrations outside these ranges can also have a stimulating effect and belong to the scope of the invention. It is also clear to the person skilled in the art that different growth factors can be effective in different concentration ranges. An important criterion for this can be, for example, the affinity of the growth factor for its receptor. The specified concentration ranges are therefore not to be regarded as limiting.
  • TGF Transforming Growth Factor beta 1 and 2
  • bFGF basic fibroblast growth factor
  • KGF keratinocyte growth factor
  • PDGF platelet derived growth factor
  • VGF vascular growth factor
  • VEGF vascular endothelial growth factor
  • Epidermal growth factor (EGF) is preferred.
  • the growth factor is preferably not released on the basis of the expression of a growth factor gene which has been introduced into the cells, rather the microspheres are loaded with growth factor protein.
  • the growth factor is preferably homogeneously distributed in the microspheres, so that there is a continuous, uniform release of growth factor during the absorption phase.
  • the tissue replacement according to the invention enables cells from a biopsy to be rapidly replicated and cultivated and to be made available in large numbers.
  • the bioabsorbable and biocompatible polymeric carrier material leads to particularly low foreign body or inflammatory reactions, which promotes wound healing.
  • the release of growth factor (s) during absorption leads to stimulation of the proliferation and / or differentiation of cells in close proximity, which also has a positive effect on wound healing.
  • the present invention provides a tissue replacement, especially a skin replacement, which has the advantageous properties mentioned.
  • Another aspect of the present invention is a method of making a tissue replacement.
  • cells are initially provided with which particulate, bioresorbable, polymeric carrier material is populated.
  • the carrier material was preferably previously loaded with at least one growth factor.
  • the cells are preferably provided from a biopsy.
  • the cells are preferably Skin cells, most preferably around human keratinocytes.
  • the keratinocytes can be obtained, for example, from a groin biopsy.
  • the cells can also be supplied from suitable cell cultures derived from cell lines.
  • the cells used are therefore preferably autologous, but suitable allogeneic cells can also be used.
  • Fibroblasts and endothelial cells are suitable as further cell types, above all to construct composite skin equivalents, for cultivation on the carriers and for transplantation. But also outside of the treatment of chronic wounds, neurocytes can be used to regenerate nerves and adipocytes to fill up fatty tissue defects.
  • the isolated cells can be grown in culture.
  • the polymeric carrier material is preferably produced by in vitro polymerization of monomeric constituents.
  • Possible carrier materials are hyaluronic acid, hyaluronic acid derivatives, fibrin, bovine collagen, collagen GAG (glycosaminoglycan) substrates or mixtures of these materials. Most preferably, polylactide is used.
  • the spheres can be produced, for example, by the so-called aerosol solvent extraction system (ASES).
  • ASES aerosol solvent extraction system
  • an extraction method is used in which the polymer is dissolved in an organic phase. This suspension is sprayed into a supercritical gas phase. Then the organic solvent must be extracted, leaving microspheres of the polymer.
  • the carrier material is preferably loaded with growth factor by adding the growth factor to the carrier material during the polymerization process. This results in a uniform distribution of the growth factor protein in the carrier material.
  • the process conditions must be set so that the growth factor is not inactivated.
  • microspheres that have already been manufactured can be coated with the growth factor.
  • a suitable concentration of the growth factor can be introduced into a suitable coating solution, for example a collagen solution, and brought onto the sphere surface during the coating.
  • the polymeric carrier material can be coated with substances which promote the adhesion of cells to substrates, for example collagen or fibronectin.
  • the polymeric carrier material can finally be colonized by cells, the colonized carrier material can then be further cultivated so that the cells can proliferate on the surface.
  • the colonized carrier materials are cultivated in suspension culture in so-called "spinner cultures". These are usually vessels that continuously rotate about an axis, or vessels that have a "clapper” that is driven by a magnetic stirrer so that the suspension is continuously mixed.
  • FIG. 1 shows that microspheres loaded with EGF uniformly release EGF for several days (Example 6).
  • FIG. 2 shows that EGF released from microspheres has very good growth factor activity (Example 7).
  • FIG. 3 shows that after transplantation of EGF-loaded microspheres, EGF is continuously released into the Wound fluid occurs so that there is a sufficiently high EGF concentration for at least one week (Example 8).
  • the PLA microspheres are produced using a modified double emulsion solution technique: 4 g of D, L-lactide (RG 202 H, Boehringer Ingelheim, Germany) are mixed in 4 ml of dichloromethane (Merck, Darmstadt, Germany). completely dissolved in a standard test tube.
  • the human recombinant EGF (epidermal growth factor) (Sigma U.S.A.) used for loading is dissolved in 100 ⁇ l PBS and added to the PLA / dichloromethane mixture. Subsequent homogenization on a vortex device results in an even distribution.
  • the placebo microspheres are produced by adding PBS without recombinant EGF.
  • the emulsion is transferred to 500 ml of a 2% isopropanol solution and stirred for 5 hours on a magnetic stirring plate with a 5 cm long stirring rod.
  • the dissolved polymer precipitates and the still soft microspheres solidify.
  • the resulting microspheres are rinsed with 5 liters of distilled water through two series-connected analytical sieves (from Retsch, Germany) with mesh sizes of 250 ⁇ m and 106 ⁇ m.
  • microspheres are then placed in a flask (Duran), deep-frozen in liquid nitrogen and freeze-dried for 36 to 48 hours in a lyophilizer (Alpha 1-4, Christ., FRG). On the one hand, this has the purpose that the microspheres are easier to handle, and on the other hand, the last residues of the cell-toxic dichloromethane evaporate.
  • microspheres are coated shortly before the cells are applied.
  • One gram of the microspheres produced are incubated with 20 ml of a 5% gelatin solution (Bacto Gelatin, Difco) in a sterile 50 ml tube (Greiner) for 30 minutes at 37 ° C.
  • a 5% gelatin solution Bacto Gelatin, Difco
  • a sterile 50 ml tube Greiner
  • the carriers must be kept in suspension at 150 revolutions / minute by means of a shaking table (KS 501 digital, from Ika Labortechnik). Then the gelatin supernatant is carefully pipetted off after the carriers have sedimented.
  • Athymic nude mice are used as experimental animals.
  • a star-shaped skin incision is made on the back of the test animals.
  • the skin is carefully mobilized and a silicone bell 1 cm in diameter open at the bottom is implanted (wound chamber according to Fusenig).
  • the defined wound, which is covered by the chamber is 1 cm in diameter.
  • the edge of the chamber is fixed with four single button threads and thus sealed. Culture medium can be injected into the chamber and liquid can be aspirated for laboratory analysis.
  • the polylactide polymer spheres should first be examined for their biocompatibility and for their degradability.
  • sterile samples of the material can be introduced subcutaneously into a skin incision both in nude mice and as an immunocompetent control in rats.
  • Four experimental animals are used and after 14, 21, 35 and 56 days the site of the implantation is necropsed and examined histologically.
  • microspheres were reabsorbed in two months and only very slight inflammatory reactions could be detected.
  • the material is very well tolerated.
  • Human skin from the plastic surgical operating room is placed in a sterile tube (Greiner) with transport medium (Hank's Buffered Salt Solution, HBSS, Seromed) and gentamycin (Gibco) in a bakericidal concentration of 20 ⁇ g / ml.
  • transport medium Hank's Buffered Salt Solution, HBSS, Seromed
  • gentamycin Gabco
  • a serum-free keratinocyte culture according to Boyce (Boyce 1983) is produced: After extensive cleaning of the tissue samples from the operating room by rinsing with buffer solution and briefly rinsing with 70% alcohol, the piece of skin cleaned from adipose tissue is left at 4 ° C overnight in 10% Dispas solution incubated. After detaching the epidermis and producing a single cell suspension with 0.05% trypsin, a primary culture with keratinocyte medium from Gibco prepared and sown in 75 cm tissue culture bottles. The first trypsin passage is made at 80% confluence.
  • the cells are then incubated in cell culture flasks under the usual conditions.
  • the cells are incubated in keratinocyte medium from Gibco at 37 ° C. and in a standard atmosphere with 5% CO 2 .
  • the cells are trypsinized after renewed subconfluence and added to the microspheres contained in a special spinner system, as prepared above.
  • Preliminary tests with other spherical material have shown a certain stirring mode, which is also observed here (Voigt, 1996).
  • the optimum cell / carrier ratio is about 15 to 1 after detailed preliminary tests.
  • 2 x 10 cells in 75 ml keratinocyte medium are added to a 500 ml spinner bottle (Integra Biosiences) and with stirring intervals optimized in preliminary tests (Cellspinn stirring unit from Integra Biosiences), kept in suspension with the corresponding carrier number.
  • the stirring mode corresponds to a speed of 30 RPM.
  • the direction of rotation was changed during the first stirring phase (6 hours) to give the cells the opportunity to adhere to the carriers, from right to left after a total rotation angle of 1260 °.
  • After 4 hours, all vital cells in the culture are adherent to the carriers and begin to divide on the surface of the beads. After 7 days all spheres were fully covered, the layer on the curved surface is confluent.
  • Collagen-coated polylactide microspheres were made as described in Example 1. The microspheres were loaded with keratinocytes as described in Example 2. Microspheres that were not coated with collagen were used as a control. As a result, the coated microspheres show better adhesion of keratinocytes than uncoated ones.
  • Microspheres colonized with keratinocytes were applied as a sub-confluent culture to a whole skin wound on the back of the nude mouse and covered with a wound chamber according to Fusenig (see Example 2).
  • the experimental animals are kept in special cages and fed with a special antibiotic-containing feed (usually as prophylaxis for athymic nude mice).
  • the biopsy days in this group were 7, 14, 21, 28, 35 and 56.
  • the histology showed a transfer of the keratinocytes to the mouse wound on day 7, an occlusion of the epithelium on day 14 with carriers underneath the epithelium.
  • the carriers with an epithelial cell layer are close to the epidermis and have contact with the surface.
  • recombinant growth factor epidermal growth factor EGF
  • EGF recombinant growth factor
  • EGF concentrations in the different supernatants were determined in an ELISA. The entire supernatant was given for the analysis for the ELISA, so that each individual value corresponds to an EGF release over 24 h. Placebo microspheres in EGF-supplemented medium and placebo microspheres in EGF-free medium were used as controls. The result is shown in FIG. 1. It was possible to achieve such a thorough mixing of the carrier material with the growth factor rhEGF that the protein was released uniformly. The growth factor concentration remained at an effective level. The optimum loading was found to be 2.5 ⁇ g / 100 mg carrier material.
  • Example 6 The bioactivity of the EGF detected in Example 6 was demonstrated by a double-chamber experiment: For this purpose, a cell culture dish with 50,000 keratinocytes was inoculated. The culture dish is manufactured in such a way that it can accommodate an insert, the bottom of which hangs in the cell culture medium about 0.5 cm above the cells. A suitable number (corresponding to experiment in example 6) of growth spheres carrying microspheres was placed in this insert.
  • the proliferation of the cells on the culture dish base was measured by a metabolic test in which a substrate is converted mitochondrially into a dye, which is then determined photometrically (for example at 490 n) (MTS test: 3- (4, 5-dimethylthiazol-2- yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium is converted to formazan).
  • Placebo microspheres in medium ⁇ EGF were used as controls. The result is shown in Figure 2.
  • the protein released by the microspheres significantly influenced the proliferation of the cells compared to the placebo microspheres. Compared to the optimally EGF-supplemented cell medium, the cell proliferation is equally good. It is also noteworthy that the growth factor concentration with which the microspheres are initially loaded is insignificant for the effect on the proliferation of the cells.
  • PLGA microspheres loaded with 2.5 ⁇ g rhEGF / 100 mg carrier material were also overgrown with keratinocytes and transplanted onto nude mouse wounds on the 4th day of the spinner culture.
  • the entire medium in the cell culture chamber (wound chamber according to Fusenig; see Example 2) was renewed every 8 hours and examined for EGF by means of ELISA.
  • the medium in which the microspheres were suspended was free of EGF.
  • the result is shown in FIG. 3.
  • the protein is continuously released to the medium, the concentration reached being about 200 pg / ml every 8 hours from day 3. Sufficient EGF care for the wound can therefore be assumed for the first week.

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Abstract

The invention relates to a tissue substitute which is suitable for transplantation and is composed of a particulate bioresorbable support material. Said material is loaded with at least one growth factor and is colonised with cells. The invention also relates to methods for producing said tissue substitute.

Description

Gewebeersatz und Verfahren zu seiner Herstellung Tissue replacement and process for its manufacture
Die vorliegende Erfindung betrifft Gewebeersatz, der zur Transplantation geeignet ist, sowie Verfahren zu seiner Herstellung.The present invention relates to tissue replacement that is suitable for transplantation and methods for its manufacture.
Nicht heilende chronische und großflächige Wunden der Haut stellen ein großes therapeutisches und sozioökonomisches Problem dar. Eine erfolgversprechende und zum Teil noch in der Entwicklung befindliche Therapiemöglichkeit dieser Probleme besteht in der Transplantation von kultivierten Keratinozyten. Diese werden zur Deckung großflächiger Wunden als autologe oder allogene Transplantate eingesetzt. Konventionelle Verfahren verwenden mehrschichtige Zellagen als sogenannte "sheets" (Cultured Epithelial Autografts = CEA) .Non-healing chronic and extensive wounds on the skin represent a major therapeutic and socio-economic problem. A promising and partially still developing therapeutic option for these problems is the transplantation of cultured keratinocytes. These are used to cover large wounds as autologous or allogeneic grafts. Conventional methods use multilayer cell layers as so-called "sheets" (Cultured Epithelial Autografts = CEA).
Probleme bei Anwendung der CEA betreffen die spontane Blasenbildung und mangelnde Anheftung der Keratinozyten, ausgedehnte Wundkontraktionen und Mißerfolge bei der Regeneration einer Dermis-äquivalenten Schicht sowie mechanische Instabilität. Das Anwachsen der Transplantate, die sogenannte "take"-Rate, erfüllt nicht die Erwartungen.Problems with the use of the CEA concern spontaneous blistering and insufficient attachment of the keratinocytes, extensive wound contractions and failures in the regeneration of a dermis-equivalent layer, as well as mechanical instability. The growth of the grafts, the so-called "take" rate, does not meet expectations.
In den letzten Jahren hat sich die Grundlagenforschung auf dem Gebiet des "Tissue Engineering" bei Hautersatzverfahren mehr in Richtung der Züchtung von Einzelzellen anstelle von ausdifferenzierten Zellplatten ("sheet grafts") gewandelt. Neuere Entwicklungen bestehen in der Verwendung kultivierter Keratinozytensuspensionen in Fibrinkleber. Die Verwendung von Keratinozyten-Einzelzellsuspensionen in Fibrinklebermatrix kann nachweislich experimentell und klinisch eine Epidermis mindestens der gleichen Qualität rekonstituieren wie "sheet grafts " .In recent years, basic research in the field of tissue engineering for skin replacement procedures has changed more towards the cultivation of single cells instead of differentiated cell plates ("sheet grafts"). Recent developments are the use of cultivated keratinocyte suspensions in fibrin glue. The use of keratinocyte single cell suspensions in a fibrin adhesive matrix can demonstrably experimentally and clinically reconstitute an epidermis of at least the same quality as "sheet grafts".
Die Zellen werden in Kulturflaschen vermehrt und müssen vor der Transplantation enzymatisch von der Kulturflasche abgelöst werden. Dabei werden proteolytisch Haftmoleküle zerstört, die für eine spätere Haftung auf dem Wundgrund wichtig sind. Das führte dazu, daß transplantierbare und zumindest teilweise resorbierbare Trägermaterialien verwendet werden. Beispiele sind Platten aus Hyaluronsäure, Kollagen oder mit Kollagen beschichtetem Nylon. Dabei kann aber nur eine begrenzte Anzahl von Zellen auf die Wunde aufgebracht werden. Außerdem ist das Verhältnis Material/Zelle auf der Seite des Materials, was Probleme bei der Bildung der neuen Epidermis bereiten kann.The cells are grown in culture bottles and must be enzymatically detached from the culture bottle before transplantation. In the process, proteolytic adhesive molecules are destroyed, which are important for later adhesion to the wound base. This led to the use of transplantable and at least partially absorbable carrier materials. Examples are plates made of hyaluronic acid, collagen or nylon coated with collagen. However, only a limited number of cells can be applied to the wound. In addition, the material / cell ratio is on the material side, which can cause problems in the formation of the new epidermis.
Bislang sind jedoch kaum bioresorbierbare Mikrosphären als Träger für die Zellen eingesetzt worden. Nicht-resorbierbare Mikrosphären, beispielsweise aus Dextran, werden nicht abgebaut, was zu starken Entzündungsreaktionen führt, die die Wundheilung behindern. Daher werden erfindungsgemäß nur Materialien verwendet, die vollständig bioresorbierbar sind.So far, however, hardly any bioabsorbable microspheres have been used as carriers for the cells. Non-resorbable microspheres, for example from dextran, are not broken down, which leads to strong inflammatory reactions which hinder wound healing. Therefore, according to the invention, only materials are used that are completely bioresorbable.
Den Zellen können physiologische Wachstumsfaktoren zur optimalen Proliferation und zur zeitgerechten Ausdifferenzierung zur Verfügung gestellt werden. Hierzu können verschiedene Methoden angewendet werden, die Plasmide mit Wachstums faktorgenen in die zu transplantierenden Zellen einschleusen. Versuche haben gezeigt, daß die Wundheilung durch die Expression von Wachstumsfaktoren beschleunigt ist. Die Methode ist jedoch relativ kompliziert, außerdem bestehen Bedenken, was die Transfektionseffizienz und -Stabilität, die Überlebensrate der Zellen und toxikologische Gefahren angeht. Grundsätzlich besteht sogar die Möglichkeit der Tumorinduktion. Erfindungsgemäß werden daher die Wachstumsfaktoren bevorzugt als Substanz zugegeben.The cells can be provided with physiological growth factors for optimal proliferation and timely differentiation. Various methods can be used for this purpose, which introduce plasmids with growth factor genes into the cells to be transplanted. Experiments have shown that wound healing is accelerated by the expression of growth factors. The method is relatively complicated, however, and there are concerns about transfection efficiency and stability, cell survival, and toxicological hazards. In principle, there is even the possibility of Tumor induction. According to the invention, the growth factors are therefore preferably added as a substance.
Gorodetsky et al . (1999) J. Invest. Dermatol. 112, 866-872 offenbaren "Fibrin Microbeads", die mit bestimmten Zelltypen besiedelt werden können. Diese Kügelchen ermöglichen aber keine Besiedelung durch Keratinocyten . Es wird weiterhin der Einsatz von PDGF-BB ( "Platelet-derived growth factor-BB") offenbart, welches zusammen mit den Kügelchen in eine Wunde gegeben wird. Der Wachstumsfaktor wirkt dadurch aber nur kurz und nicht kontinuierlich über längere Zeit.Gorodetsky et al. (1999) J. Invest. Dermatol. 112, 866-872 disclose "fibrin microbeads" that can be populated with certain cell types. However, these beads do not allow colonization by keratinocytes. The use of PDGF-BB ("platelet-derived growth factor-BB") is also disclosed, which is placed in a wound together with the beads. However, the growth factor only works for a short time and not continuously over a long period.
Eine Aufgabe der Erfindung ist es, einen vorteilhaften Gewebeersatz bereitzustellen.An object of the invention is to provide an advantageous tissue replacement.
Die vorliegende Erfindung betrifft einen Gewebeersatz, der geeignet ist zur Transplantation und aus einem polymeren Trägermaterial besteht, das mit Zellen besiedelt ist. Dabei weist das polymere Trägermaterial Partikelform auf, so daß die Zellen nach Besiedelung des Trägermaterials als Suspensionskultur kultiviert werden können. Das Trägermaterial ist bioresorbierbar und mit wenigstens einem Wachstumsfaktor beladen.The present invention relates to a tissue replacement that is suitable for transplantation and consists of a polymeric carrier material that is populated with cells. The polymeric carrier material has a particle shape so that the cells can be cultivated as a suspension culture after the carrier material has been populated. The carrier material is bioresorbable and loaded with at least one growth factor.
Der erfindungsgemäße Gewebeersatz besitzt die Vorteile, daß er transplantierbar ist, gut verträglich ist, genügend Zellen bereitstellt, auf enzymatische Methoden zur Ablösung von Zellen verzichtet und in dosierter Weise Wachstumsfaktor liefert, so daß eine verbesserte Wundheilung erreicht werden kann.The tissue replacement according to the invention has the advantages that it can be transplanted, is well tolerated, provides enough cells, dispenses with enzymatic methods for detaching cells and delivers growth factor in a metered manner so that improved wound healing can be achieved.
Der Begriff "Gewebe" umfaßt im allgemeinen Sprachgebrauch Epithelgewebe, Binde- und Stützgewebe, Muskelgewebe und Nervengewebe. Der Begriff "Gewebe" im Sinne der Anmeldung ist jedoch weiter zu fassen. Ein Gewebeersatz soll somit nicht nur beispielsweise Epithel- oder Bindegewebe ersetzen, sondern allgemein räumlich benachbarte Zellen und ihre Interzellularsubstanzen. Beispielsweise soll ein Hautersatz sowohl Epithel- als auch Bindegewebe ersetzen.The term "tissue" generally includes epithelial tissue, connective and support tissue, muscle tissue and nerve tissue. However, the term "fabric" in the sense of the application is to be understood further. A tissue replacement should not only replace epithelial or connective tissue, for example, but generally cells and their spatially adjacent cells Intercellular substances. For example, a skin replacement is said to replace both epithelial and connective tissue.
Das Trägermaterial weist Partikelform auf, dabei können verschiedenste geometrische Formen vorkommen. Die Partikel des Trägermaterials können annähernd Kugelform aufweisen, aber auch langgestreckt, scheibenförmig oder ohne definierte geometrische Form sein. Der Durchmesser der Partikel ist in der Regel kleiner als 1 mm. Bevorzugt haben die Partikel einen Durchmesser von 50 bis 1000 μm, am bevorzugtesten 100 bis 250 μm. Als Durchmesser ist hier die größte, längenmäßige Ausdehnung des Partikels zu verstehen. Bevorzugt weisen die Partikel eine Oberfläche auf, die gut von Zellen besiedelt werden kann .The carrier material has a particle shape, and a wide variety of geometric shapes can occur. The particles of the carrier material can have an approximately spherical shape, but can also be elongated, disk-shaped or without a defined geometric shape. The diameter of the particles is usually less than 1 mm. The particles preferably have a diameter of 50 to 1000 μm, most preferably 100 to 250 μm. The diameter here is the largest, lengthwise extension of the particle. The particles preferably have a surface that can be well populated by cells.
Erfindungsgemäß ist das Trägermaterial mit Zellen besiedelt. Vorzugsweise handelt es sich bei den Zellen um Hautzellen, so daß der Gewebeersatz als Hautersatz eingesetzt werden kann. Am bevorzugtesten sind die Zellen menschliche Keratinozyten. Die Partikelform des Trägermaterials gewährleistet, daß nach Besiedelung des Trägermaterials mit Zellen die Zellen in Suspensionskultur weiterkultiviert werden können. Die erfindungsgemäße Form ermöglicht somit eine effiziente Zellvermehrung und damit ein günstiges Verhältnis von Zellzahl zu Trägermaterial. Das partikelförmige Trägermaterial wird im folgenden auch als Mikrosphären bezeichnet.According to the invention, the carrier material is populated with cells. The cells are preferably skin cells, so that the tissue replacement can be used as a skin replacement. Most preferably, the cells are human keratinocytes. The particle shape of the carrier material ensures that after the carrier material has been populated with cells, the cells can be further cultivated in suspension culture. The shape according to the invention thus enables efficient cell multiplication and thus a favorable ratio of cell number to carrier material. The particulate carrier material is also referred to below as microspheres.
Das erfindungsgemäße Trägermaterial wird nach Transplantation vom tierischen oder menschlichen Körper resorbiert. Bevorzugt findet die Resorption innerhalb von 50 Tagen statt und noch bevorzugter innerhalb von 25 Tagen. Durch die Bioverträglichkeit des Trägermaterials und die Möglichkeit der Resorption werden Fremdkörperreaktionen und Entzündungen minimiert. Bevorzugte Trägermaterialien sind Hyaluronsäure, Hyaluronsäurederivate, Fibrin, Rinderkollagen, Kollagen-GAG (Glycosaminglycan) Substrate. Ein besonders bevorzugtes polymeres Trägermaterial besteht aus Polylactid. Polylactid ist ein lineares Polyester-Polymer auf Basis von Milchsäure. Es kann durch Polymerisation aus Lactid hergestellt werden. Lactid ist ein zyklischer Ester aus zwei Molekülen Milchsäure, der durch Hochtemperatur-Destillation von Milchsäure erhältlich ist. Polylactid ist - wie andere aliphatische Polyester, die auf natürlich vorkommenden Substanzen beruhen - vom tierischen und menschlichen Körper abbaubar und damit bioresorbierbar.The carrier material according to the invention is absorbed by the animal or human body after transplantation. The absorption preferably takes place within 50 days and more preferably within 25 days. Due to the biocompatibility of the carrier material and the possibility of absorption, foreign body reactions and inflammation are minimized. Preferred carrier materials are hyaluronic acid, hyaluronic acid derivatives, fibrin, bovine collagen, collagen GAG (glycosaminoglycan) substrates. A particularly preferred polymeric carrier material consists of polylactide. Polylactide is a linear polyester polymer based on lactic acid. It can be made from lactide by polymerization. Lactide is a cyclic ester of two molecules of lactic acid, which can be obtained by high temperature distillation of lactic acid. Like other aliphatic polyesters, which are based on naturally occurring substances, polylactide is biodegradable by the animal and human body and thus bioresorbable.
Die Mikrosphären sind mit wenigstens einem Wachstumsfaktor beladen. Deshalb wird wenigstens ein Wachstumsfaktor freigesetzt, während die Mikrosphären resorbiert werden. Wachstumsfaktoren sind Substanzen, die die Proliferation und/oder Differenzierung von Zellen stimulieren können. In der Regel handelt es sich um Proteine, es gibt jedoch auch niedermolekulare Wachstumsfaktoren, die keine Proteine sind. Die Anwesenheit von Wachstumsfaktor in der Wundflüssigkeit stimuliert die Zellen zur Proliferation und gegebenenfalls zur Ausdifferenzierung, was die Wundheilung beschleunigt. Gemäß der vorliegenden Erfindung kann während der Freisetzung des Wachstumsfaktors eine Wachstumsfaktorkonzentration in der Wundflüssigkeit von 100 bis 1000 pg/ml erreicht werden, vorzugsweise eine Konzentration von 200 bis 400 pg/ml. Die angegebenen Konzentrationswerte wurden als vorteilhaft im Fall von EGF gefunden. Auch Konzentrationen außerhalb dieser Bereiche können aber stimulierende Wirkung ausüben und gehören zum Bereich der Erfindung. Dem Fachmann ist auch klar, daß verschiedene Wachstumsfaktoren in unterschiedlichen Konzentrationsbereichen wirksam sein können. Ein wichtiges Kriterium dafür kann beispielsweise die Affinität des Wachstumsfaktors zu seinem Rezeptor sein. Somit sind die angegebenen Konzentrationsbereiche nicht als limitierend anzusehen.The microspheres are loaded with at least one growth factor. Therefore, at least one growth factor is released while the microspheres are being resorbed. Growth factors are substances that can stimulate cell proliferation and / or differentiation. They are usually proteins, but there are also low-molecular growth factors that are not proteins. The presence of growth factor in the wound fluid stimulates the cells to proliferate and, if necessary, to differentiate, which accelerates wound healing. According to the present invention, a growth factor concentration in the wound fluid of 100 to 1000 pg / ml can be achieved during the release of the growth factor, preferably a concentration of 200 to 400 pg / ml. The concentration values given were found to be advantageous in the case of EGF. However, concentrations outside these ranges can also have a stimulating effect and belong to the scope of the invention. It is also clear to the person skilled in the art that different growth factors can be effective in different concentration ranges. An important criterion for this can be, for example, the affinity of the growth factor for its receptor. The specified concentration ranges are therefore not to be regarded as limiting.
Als Wachstumsfaktoren können alle Wachstumsfaktoren, die für die Wundheilung von Bedeutung sind, eingesetzt werden, insbesondere TGF (Transforming Growth Factor )-beta 1 und 2, bFGF (basic Fibroblast Growth Factor), KGF (Keratinocyte Growth Factor), PDGF (Platelet derived Growth Factor)-AB, VGF (Vascular Growth Factor), VEGF (Vascular Endothelial Growth Factor) . Bevorzugt ist epidermaler Wachstumsfaktor (EGF) .All growth factors that are important for wound healing can be used as growth factors, especially TGF (Transforming Growth Factor) beta 1 and 2, bFGF (basic fibroblast growth factor), KGF (keratinocyte growth factor), PDGF (platelet derived growth factor) -AB, VGF (vascular growth factor), VEGF (vascular endothelial growth) Factor). Epidermal growth factor (EGF) is preferred.
Die Freisetzung des Wachstumsfaktors erfolgt bevorzugt nicht aufgrund der Expression eines Wachstumsfaktorgens, das in die Zellen eingebracht worden ist, vielmehr sind die Mikrosphären mit Wachstumsfaktor-Protein beladen. Vorzugsweise ist der Wachstumsfaktor in den Mikrosphären homogen verteilt, so daß es während der Resorptionsphase zu einer kontinuierlichen, gleichmäßigen Wachstumsfaktorfreisetzung kommt.The growth factor is preferably not released on the basis of the expression of a growth factor gene which has been introduced into the cells, rather the microspheres are loaded with growth factor protein. The growth factor is preferably homogeneously distributed in the microspheres, so that there is a continuous, uniform release of growth factor during the absorption phase.
Der erfindungsgemäße Gewebeersatz ermöglicht es, daß schnell Zellen aus einer Biopsie vermehrt und kultiviert werden können und in großer Anzahl zur Verfügung gestellt werden können. Das bioresorbierbare und bioverträgliche polymere Trägermaterial führt zu besonders niedrigen Fremdkörper- bzw. Entzündungsreaktionen, was die Wundheilung fördert. Schließlich führt die Freisetzung von Wachstumsfaktor (en) während der Resorption zu einer Stimulation der Proliferation und/oder Differenzierung von Zellen in räumlicher Nähe, was die Wundheilung ebenfalls positiv beeinflußt. Die vorliegende Erfindung stellt zum ersten Mal einen Gewebeersatz, vor allem einen Hautersatz zur Verfügung, der die genannten vorteilhaften Eigenschaften aufweist.The tissue replacement according to the invention enables cells from a biopsy to be rapidly replicated and cultivated and to be made available in large numbers. The bioabsorbable and biocompatible polymeric carrier material leads to particularly low foreign body or inflammatory reactions, which promotes wound healing. Finally, the release of growth factor (s) during absorption leads to stimulation of the proliferation and / or differentiation of cells in close proximity, which also has a positive effect on wound healing. For the first time, the present invention provides a tissue replacement, especially a skin replacement, which has the advantageous properties mentioned.
Ein weiterer Aspekt der vorliegenden Erfindung ist ein Verfahren zur Herstellung eines Gewebeersatzes. Erfindungsgemäß werden dabei zunächst Zellen bereitgestellt, mit denen partikelförmiges , bioresorbierbares , polymeres Trägermaterial besiedelt wird. Das Trägermaterial wurde bevorzugt zuvor mit wenigstens einem Wachstumsfaktor beladen.Another aspect of the present invention is a method of making a tissue replacement. According to the invention, cells are initially provided with which particulate, bioresorbable, polymeric carrier material is populated. The carrier material was preferably previously loaded with at least one growth factor.
Die Bereitstellung der Zellen erfolgt vorzugsweise aus einer Biopsie. Bei den Zellen handelt es sich vorzugsweise um Hautzellen, am bevorzugtesten um menschliche Keratinozyten. Die Keratinozyten können beispielsweise aus einer Biopsie der Leistengegend gewonnen werden. Alternativ hierzu können die Zellen auch von geeigneten Zellkulturen, die von Zellinien abstammen, geliefert werden. Die eingesetzten Zellen sind daher bevorzugt autolog, aber auch geeignete allogene Zellen können verwendet werden.The cells are preferably provided from a biopsy. The cells are preferably Skin cells, most preferably around human keratinocytes. The keratinocytes can be obtained, for example, from a groin biopsy. Alternatively, the cells can also be supplied from suitable cell cultures derived from cell lines. The cells used are therefore preferably autologous, but suitable allogeneic cells can also be used.
Als weitere Zelltypen, vor allem um zusammengesetzte Hautäquivalente zu konstruieren, sind zur Anzüchtung auf den Carriern und zur Transplantation Fibroblasten und Endothelzellen geeignet. Aber auch außerhalb der Therapie von chronischen Wunden können Neurozyten zur Regeneration von Nerven und Adipozyten zur Auffüllung von Fettgewebsdefekten eingesetzt werden.Fibroblasts and endothelial cells are suitable as further cell types, above all to construct composite skin equivalents, for cultivation on the carriers and for transplantation. But also outside of the treatment of chronic wounds, neurocytes can be used to regenerate nerves and adipocytes to fill up fatty tissue defects.
Vor der Besiedelung des Trägermaterials können die isolierten Zellen in Kultur vermehrt werden.Before the support material is colonized, the isolated cells can be grown in culture.
Die Herstellung des polymeren Trägermaterials erfolgt bevorzugt durch in vitro-Poly erisation von monomeren Bestandteilen. Mögliche Trägermaterialien sind Hyaluronsäure, Hyaluronsäurederivate, Fibrin, Rinderkollagen, Kollagen-GAG (Glycosaminglycan) Substrate oder Mischungen dieser Materialien. Am bevorzugtesten wird Polylactid eingesetzt.The polymeric carrier material is preferably produced by in vitro polymerization of monomeric constituents. Possible carrier materials are hyaluronic acid, hyaluronic acid derivatives, fibrin, bovine collagen, collagen GAG (glycosaminoglycan) substrates or mixtures of these materials. Most preferably, polylactide is used.
Die Sphären können beispielsweise durch das sogenannte Aerosol solvent extraction System (ASES) hergestellt werden. Hierbei macht man sich eine Extraktionsmethode zunutze, bei der das Polymer in einer organischen Phase gelöst ist. Diese Suspension wird in eine superkritische Gasphase gesprayt. Dann muß das organische Lösungsmittel extrahiert werden, wonach Mikrosphären des Polymers übrigbleiben.The spheres can be produced, for example, by the so-called aerosol solvent extraction system (ASES). Here, an extraction method is used in which the polymer is dissolved in an organic phase. This suspension is sprayed into a supercritical gas phase. Then the organic solvent must be extracted, leaving microspheres of the polymer.
Vorzugsweise wird das Trägermaterial mit Wachstumsfaktor beladen, indem der Wachstumsfaktor während des Polymerisationsvorganges dem Trägermaterial zugegeben wird. Dadurch wird eine gleichmäßige Verteilung des Wachstumsfaktor- Proteins in dem Trägermaterial erreicht. Die Verfahrensbedingungen müssen so eingestellt werden, daß der Wachstumsfaktor nicht inaktiviert wird.The carrier material is preferably loaded with growth factor by adding the growth factor to the carrier material during the polymerization process. This results in a uniform distribution of the growth factor protein in the carrier material. The process conditions must be set so that the growth factor is not inactivated.
Alternativ können bereits gefertigte Mikrosphären mit dem Wachstumsfaktor beschichtet werden. Hierzu kann eine geeignete Konzentration des Wachstumsfaktors in eine geeignete Beschichtungslösung, beispielsweise eine Kollagenlösung eingebracht werden und bei der Beschichtung mit auf die Sphärenoberfläche gebracht werden.Alternatively, microspheres that have already been manufactured can be coated with the growth factor. For this purpose, a suitable concentration of the growth factor can be introduced into a suitable coating solution, for example a collagen solution, and brought onto the sphere surface during the coating.
Um die Anheftung der Zellen zu verbessern, kann das polymere Trägermaterial mit Substanzen beschichtet werden, die die Adhäsion von Zellen an Substraten begünstigen, beispielsweise Kollagen oder Fibronektin.In order to improve the attachment of the cells, the polymeric carrier material can be coated with substances which promote the adhesion of cells to substrates, for example collagen or fibronectin.
Das polymere Trägermaterial kann schließlich durch Zellen besiedelt werden, das besiedelte Trägermaterial kann daraufhin weiterkultiviert werden, so daß die Zellen auf der Oberfläche proliferieren können. Die Kultivierung der besiedelten Trägermaterialien erfolgt in Suspensionskultur in sogenannten "Spinnerkulturen". Dabei handelt es sich meist um Gefäße, die um eine Achse kontinuierlich rotieren, oder um Gefäße, die einen "Klöppel" aufweisen, der von einem Magnetrührgerät angetrieben wird, so daß die Suspension kontinuierlich durchmischt wird.The polymeric carrier material can finally be colonized by cells, the colonized carrier material can then be further cultivated so that the cells can proliferate on the surface. The colonized carrier materials are cultivated in suspension culture in so-called "spinner cultures". These are usually vessels that continuously rotate about an axis, or vessels that have a "clapper" that is driven by a magnetic stirrer so that the suspension is continuously mixed.
Figur 1 zeigt, daß mit EGF beladene Mikrosphären mehrere Tage gleichmäßig EGF freisetzen (Beispiel 6).FIG. 1 shows that microspheres loaded with EGF uniformly release EGF for several days (Example 6).
Figur 2 zeigt, daß von Mikrosphären freigesetztes EGF sehr gute Wachstumsfaktor-Aktivität besitzt (Beispiel 7).Figure 2 shows that EGF released from microspheres has very good growth factor activity (Example 7).
Figur 3 zeigt, daß nach Transplantation EGF-beladener Mikrosphären eine kontinuierliche Abgabe von EGF in die Wundflüssigkeit erfolgt, so daß für mindestens eine Woche eine ausreichend hohe EGF-Konzentration vorliegt (Beispiel 8).FIG. 3 shows that after transplantation of EGF-loaded microspheres, EGF is continuously released into the Wound fluid occurs so that there is a sufficiently high EGF concentration for at least one week (Example 8).
Die nachfolgenden Beispiele sollen die Erfindung näher erläutern:The following examples are intended to explain the invention in more detail:
Beispiel 1example 1
Herstellung, Beladung und Beschichtunσ der Mikrosphären:Production, loading and coating of the microspheres:
Die Herstellung der PLA-Mikrosphären erfolgt mittels einer modifizierten Doppel-Emulsion-Lösungs Technik: 4 g D,L-Lactid (RG 202 H, Fa. Boehringer Ingelheim, BRD) werden in 4 ml Dichlormethan (Fa. Merck, Darmstadt, BRD) in einem handelsüblichen Reagenzglas vollständig aufgelöst. Das zur Beladung verwendete humane rekombinante EGF (epidermal growth factor) (Fa. Sigma U.S.A.) wird in 100 μl PBS aufgelöst und dem PLA-Dichlormethan-Gemisch beigemengt. Durch anschließendes Homogenisieren auf einem Vortex-Gerät erreicht man eine gleichmäßige Verteilung.The PLA microspheres are produced using a modified double emulsion solution technique: 4 g of D, L-lactide (RG 202 H, Boehringer Ingelheim, Germany) are mixed in 4 ml of dichloromethane (Merck, Darmstadt, Germany). completely dissolved in a standard test tube. The human recombinant EGF (epidermal growth factor) (Sigma U.S.A.) used for loading is dissolved in 100 μl PBS and added to the PLA / dichloromethane mixture. Subsequent homogenization on a vortex device results in an even distribution.
Die Herstellung der Placebo Mikrosphären erfolgt durch Zugabe von PBS ohne rekombinantes EGF.The placebo microspheres are produced by adding PBS without recombinant EGF.
Dieser Lösung wird 10 ml einer 0,5%igen wäßrigen Polyvinylalkohollösung (PVA 25/40, Molekulargewicht 80000, Fa. Serva) zugesetzt und für 30 Sekunden auf einem Vortex-Gerät homogenisiert. Durch diese Homogenisationsdauer erhält man ein Maximum an Trägern mit der zur Zellkultivierung optimalen Größe von 100 bis 250 μm.10 ml of a 0.5% aqueous polyvinyl alcohol solution (PVA 25/40, molecular weight 80,000, from Serva) are added to this solution and homogenized for 30 seconds on a vortex device. This homogenization period gives a maximum of carriers with the optimal size for cell cultivation of 100 to 250 μm.
Die Emulsion wird in 500 ml einer 2%igen Isopropanollösung überführt und für 5 Stunden auf einer Magnetrührplatte mit einem 5 cm langen Rührstab gerührt. Durch das Auswaschen des Dichlormethans in die äußere alkoholische Phase präzipitiert das aufgelöste Polymer und die noch weichen Mikrosphären verfestigen sich. Um Träger zu entfernen, die größer oder kleiner als die gewünschte Größe sind, spült man die entstandenen Mikrosphären mit 5 1 destilliertem Wasser durch zwei hintereinandergeschaltete Analysesiebe (Fa. Retsch, BRD) mit der Maschenweite 250 μm und 106 μm.The emulsion is transferred to 500 ml of a 2% isopropanol solution and stirred for 5 hours on a magnetic stirring plate with a 5 cm long stirring rod. By washing out the dichloromethane in the outer alcoholic phase, the dissolved polymer precipitates and the still soft microspheres solidify. In order to remove supports that are larger or smaller than the desired size, the resulting microspheres are rinsed with 5 liters of distilled water through two series-connected analytical sieves (from Retsch, Germany) with mesh sizes of 250 μm and 106 μm.
Anschließend werden die Mikrosphären in ein Kolbenglas (Duran) gegeben, in flüssigem Stickstoff tiefgefroren und für 36 bis 48 Stunden in einem Lyophilisator (Alpha 1-4, Fa. Christ, BRD) gefriergetrocknet. Dies hat zum einen den Zweck, daß die Mikrosphären einfacher zu handhaben sind, zum anderen verdampfen die letzten Reste des zelltoxischen Dichlormethans .The microspheres are then placed in a flask (Duran), deep-frozen in liquid nitrogen and freeze-dried for 36 to 48 hours in a lyophilizer (Alpha 1-4, Christ., FRG). On the one hand, this has the purpose that the microspheres are easier to handle, and on the other hand, the last residues of the cell-toxic dichloromethane evaporate.
Die Beschichtung der Mikrosphären erfolgt kurz vor Aufbringen der Zellen. Ein Gramm der hergestellten Mikrosphären werden mit 20 ml einer 5% Gelatine Lösung (Bacto Gelatin, Fa. Difco) in einem sterilen 50 ml Röhrchen (Fa. Greiner) für 30 Minuten bei 37 °C inkubiert. Um eine mögliche Verklu pung oder Verklebung der Sphären zu vermeiden und eine gleichmäßige Beschichtung zu erhalten, müssen die Träger durch einen Schütteltisch (KS 501 digital, Fa. Ika Labortechnik) bei 150 Umdrehungen/Minute ständig in Suspension gehalten werden. Danach pipettiert man den Gelatineüberstand nach dem Absedimentieren der Träger vorsichtig ab.The microspheres are coated shortly before the cells are applied. One gram of the microspheres produced are incubated with 20 ml of a 5% gelatin solution (Bacto Gelatin, Difco) in a sterile 50 ml tube (Greiner) for 30 minutes at 37 ° C. In order to avoid possible jamming or sticking of the spheres and to obtain a uniform coating, the carriers must be kept in suspension at 150 revolutions / minute by means of a shaking table (KS 501 digital, from Ika Labortechnik). Then the gelatin supernatant is carefully pipetted off after the carriers have sedimented.
Beispiel 2Example 2
Nachweis der Bioverträαlichkeit und Resorbierbarkeit der Träqermaterialien (Tierversuch)Proof of the biocompatibility and absorbability of the carrier materials (animal experiment)
Als Versuchstiere werden athymische Nacktmäuse verwendet.Athymic nude mice are used as experimental animals.
Auf dem Rücken der Versuchstiere wird eine sternförmige Hautinzision angebracht. Die Haut wird vorsichtig mobilisiert und eine nach unten offene Siliconglocke von 1 cm Durchmesser implantiert (Wundkammer nach Fusenig) . Die definierte Wunde, die von der Kammer bedeckt wird, ist 1 cm im Durchmesser. Der Rand der Kammer wird mit vier Einzelknopffäden fixiert und somit abgedichtet. In die Kammer kann Kulturmedium über eine Injektion eingebracht und Flüssigkeit zur laborchemischen Untersuchung abgesaugt werden.A star-shaped skin incision is made on the back of the test animals. The skin is carefully mobilized and a silicone bell 1 cm in diameter open at the bottom is implanted (wound chamber according to Fusenig). The defined wound, which is covered by the chamber is 1 cm in diameter. The edge of the chamber is fixed with four single button threads and thus sealed. Culture medium can be injected into the chamber and liquid can be aspirated for laboratory analysis.
Die Polylactidpolyπier Sphären sollen zunächst auf ihre Bioverträglichkeit und auf ihre Abbaubarkeit hin untersucht werden. Hierzu können sterile Proben des Materials sowohl in Nacktmäuse als auch als immunkompetente Kontrolle in Ratten subcutan über eine Hautinzision eingebracht werden. Es werden je vier Versuchstiere eingesetzt und nach 14, 21, 35 und 56 Tagen die Stelle der Implantation nekropsiert und histologisch untersucht .The polylactide polymer spheres should first be examined for their biocompatibility and for their degradability. For this purpose, sterile samples of the material can be introduced subcutaneously into a skin incision both in nude mice and as an immunocompetent control in rats. Four experimental animals are used and after 14, 21, 35 and 56 days the site of the implantation is necropsed and examined histologically.
Die Mikrosphären wurden in zwei Monaten resorbiert, es konnten nur sehr geringe Entzündungsreaktionen nachgewiesen werden. Das Material weist sehr gute Verträglichkeit auf.The microspheres were reabsorbed in two months and only very slight inflammatory reactions could be detected. The material is very well tolerated.
Beispiel 3Example 3
Kultivierung von Keratinozyten auf Mikrosphären:Cultivation of Keratinocytes on Microspheres:
Humane Haut aus dem plastisch-chirurgischen Operationssaal wird in ein steriles Röhrchen (Fa. Greiner) mit Transportmedium (Hank's Buffered Salt Solution, HBSS, Fa. Seromed) und Gentamycin (Fa. Gibco) in bakerizider Konzentration von 20 μg/ml gegeben.Human skin from the plastic surgical operating room is placed in a sterile tube (Greiner) with transport medium (Hank's Buffered Salt Solution, HBSS, Seromed) and gentamycin (Gibco) in a bakericidal concentration of 20 μg / ml.
Dann wird eine serumfreie Keratinozytenkultur nach Boyce (Boyce 1983) hergestellt: Nach ausgiebiger Reinigung der Gewebeproben aus dem OP durch Spülung mit Pufferlösung und kurzem Abspülen mit 70%igem Alkohol wird das von Fettgewebe gereinigte Hautstück bei 4°C über Nacht in 10%iger Dispaselösung inkubiert. Nach Ablösung der Epidermis und Herstellen einer Einzelzellsuspension mit 0,05% Trypsin, wird eine Primärkultur mit Keratinozytenmedium der Fa. Gibco angesetzt und in 75 cm Gewebekulturflaschen ausgesäht. Bei 80% Konfluenz wird die erste Trypsinpassage hergestellt.Then a serum-free keratinocyte culture according to Boyce (Boyce 1983) is produced: After extensive cleaning of the tissue samples from the operating room by rinsing with buffer solution and briefly rinsing with 70% alcohol, the piece of skin cleaned from adipose tissue is left at 4 ° C overnight in 10% Dispas solution incubated. After detaching the epidermis and producing a single cell suspension with 0.05% trypsin, a primary culture with keratinocyte medium from Gibco prepared and sown in 75 cm tissue culture bottles. The first trypsin passage is made at 80% confluence.
Die Zellen werden dann in Zellkulturflaschen unter den üblichen Bedingungen inkubiert. Beispielsweise werden die Zellen in Keratinozytenmedium der Fa. Gibco bei 37°C und einer Standardatmosphäre mit 5% CO2 bebrütet. Zur Kultur der Zellen auf den Mikrosphären werden die Zellen nach erneuter Subkonfluenz trypsinisiert und zu den in einem speziellen Spinnersystem enthaltenen, wie oben angefertigten, Mikrosphären gegeben. Vorversuche mit anderem Sphärenmaterial haben einen bestimmten Rührmodus ergeben, der auch hier eingehalten wird (Voigt, 1996).The cells are then incubated in cell culture flasks under the usual conditions. For example, the cells are incubated in keratinocyte medium from Gibco at 37 ° C. and in a standard atmosphere with 5% CO 2 . For the culture of the cells on the microspheres, the cells are trypsinized after renewed subconfluence and added to the microspheres contained in a special spinner system, as prepared above. Preliminary tests with other spherical material have shown a certain stirring mode, which is also observed here (Voigt, 1996).
Das optimale Zell/Carrier Verhältnis ist nach eingehenden Vorversuchen etwa 15 zu 1. Damit werden in eine 500 ml Spinnerflasche (Fa. Integra Biosiences) 2 x 10 Zellen in 75 ml Keratinozytenmedium gegeben und mit in Vorversuchen optimierten Rührintervallen (Cellspinn Rühreinheit der Fa. Integra Biosiences), mit der entsprechenden Carrierzahl in Suspension gehalten. Der Rührmodus entspricht einer Geschwindigkeit mit 30 RPM. Die Drehrichtung wurde während der ersten Rührphase (6 Stunden) verändert, um den Zellen die Möglichkeit zu geben auf den Carriers zu haften, und zwar von rechts nach links nach einem Rotationswinkel von insgesamt 1260°. Nach 4 Stunden sind alle vitalen Zellen in der Kultur an den Carriern adhärent und beginnen sich auf der Kügelchenoberflache zu teilen. Nach 7 Tagen waren alle Sphären voll bewachsen, die Schicht auf der gebogenen Oberfläche ist konfluent . Beispiel 4The optimum cell / carrier ratio is about 15 to 1 after detailed preliminary tests. 2 x 10 cells in 75 ml keratinocyte medium are added to a 500 ml spinner bottle (Integra Biosiences) and with stirring intervals optimized in preliminary tests (Cellspinn stirring unit from Integra Biosiences), kept in suspension with the corresponding carrier number. The stirring mode corresponds to a speed of 30 RPM. The direction of rotation was changed during the first stirring phase (6 hours) to give the cells the opportunity to adhere to the carriers, from right to left after a total rotation angle of 1260 °. After 4 hours, all vital cells in the culture are adherent to the carriers and begin to divide on the surface of the beads. After 7 days all spheres were fully covered, the layer on the curved surface is confluent. Example 4
Eignung von beschichteten Träqermaterialien alsSuitability of coated carrier materials as
Kulturoberflächeculture surface
Kollagen-beschichtete Polylactid-Mikrosphären wurden hergestellt wie in Beispiel 1 beschrieben. Die Mikrosphären wurden mit Keratinozyten beladen wie in Beispiel 2 beschrieben. Als Kontrolle wurden Mikrosphären eingesetzt, die nicht mit Kollagen beschichtet waren. Im Ergebnis zeigen die beschichteten Mikrosphären eine bessere Anhaftung von Keratinozyten als unbeschichtete.Collagen-coated polylactide microspheres were made as described in Example 1. The microspheres were loaded with keratinocytes as described in Example 2. Microspheres that were not coated with collagen were used as a control. As a result, the coated microspheres show better adhesion of keratinocytes than uncoated ones.
Tabelle 1:Table 1:
Zellanhaftung bei Polyactidcarriern mit und ohne Beschichtung mit Rinderkollagen Typ 1. Es wurden drei unterschiedlichen Versuchsansätze ausgewertet und die Mittelwerte angegeben.Cell adhesion in polyactide carriers with and without coating with bovine collagen type 1. Three different experimental approaches were evaluated and the mean values were given.
Figure imgf000015_0001
Figure imgf000015_0001
Beispiel 5Example 5
Mit Keratinozyten besiedelte Mikrosphären als Hautersatz (Tierversuch)Microspheres populated with keratinocytes as a skin replacement (animal experiment)
Mit Keratinozyten besiedelte Mikrosphären wurden als subkonfluente Kultur auf eine Vollhautwunde am Nacktmausrücken aufgebracht und mit einer Wundkammer nach Fusenig (siehe Beispiel 2) bedeckt. Die Versuchstiere werden in speziellen Käfigen gehalten und mit speziellem antibiotikahaltigem Futter (üblich als Prophylaxe bei athymischen Nacktmäusen) ernährt. Die Biopsietage waren in dieser Gruppe 7, 14, 21, 28, 35 und 56.Microspheres colonized with keratinocytes were applied as a sub-confluent culture to a whole skin wound on the back of the nude mouse and covered with a wound chamber according to Fusenig (see Example 2). The experimental animals are kept in special cages and fed with a special antibiotic-containing feed (usually as prophylaxis for athymic nude mice). The biopsy days in this group were 7, 14, 21, 28, 35 and 56.
Die Histologie zeigte einen Transfer der Keratinozyten auf die Mauswunde an Tag 7, einen Verschluß des Epithels an Tag 14 mit unterhalb des Epithels reizlos liegenden Carriern. Teilweise sind die Carrier mit einer Epithelzellschicht dicht an der Epidermis und haben Kontakt zur Oberfläche.The histology showed a transfer of the keratinocytes to the mouse wound on day 7, an occlusion of the epithelium on day 14 with carriers underneath the epithelium. In some cases, the carriers with an epithelial cell layer are close to the epidermis and have contact with the surface.
Hiermit konnte gezeigt werden, daß die Transplantation von Keratinozyten und die Rekonstitution einer Epidermis entsprechend den alternativen, etablierten Transplantationsverfahren in diesem System gelingt.It could be shown that the transplantation of keratinocytes and the reconstitution of an epidermis are possible according to the alternative, established transplantation procedures in this system.
Beispiel 6Example 6
Nachweis der EGF-Freisetzunα in vitroEvidence of EGF release in vitro
Der Polylactidrohsubstanz wurden verschiedene Konzentrationen rekombinanten Wachstumsfaktors (epidermal growth factor EGF) (Fa. Sigma) zugesetzt. Die Mikrosphären wurden dann wie in Beispiel 1 hergestellt. Zur Überprüfung der Freisetzung wurden dann 2 ml in PBS resuspendierte Sphären in eine 6 Lochplatte verteilt. Der Überstand wurde 7 Tage lang alle 24 h komplett gewonnen und durch 2 ml PBS erneuert.Various concentrations of recombinant growth factor (epidermal growth factor EGF) (from Sigma) were added to the polylactide raw substance. The microspheres were then made as in Example 1. To check the release, 2 ml spheres resuspended in PBS were then distributed into a 6-well plate. The supernatant was completely recovered every 24 hours for 7 days and renewed with 2 ml of PBS.
In einem ELISA wurden die EGF-Konzentrationen in den verschiedenen Überständen bestimmt. Zur Untersuchung für den ELISA wurde der gesamte Überstand gegeben, so daß jeder einzelne Wert einer EGF-Freisetzung über 24 h entspricht. Als Kontrollen wurden Placebo-Mikrosphären in EGF-supplementiertem Medium und Placebo-Mikrosphären in EGF-freiem Medium eingesetzt. Das Ergebnis ist in Figur 1 dargestellt. Es ist gelungen, eine solche Durchmischung des Trägermaterials mit dem Wachstumsfaktor rhEGF zu erreichen, daß eine gleichmäßige Freisetzung des Proteins erfolgte. Die Wachstumsfaktorkonzentration blieb in einer wirksamen Höhe. Als optimale Beladung wurde 2,5 μg/100 mg Trägermaterial gefunden .The EGF concentrations in the different supernatants were determined in an ELISA. The entire supernatant was given for the analysis for the ELISA, so that each individual value corresponds to an EGF release over 24 h. Placebo microspheres in EGF-supplemented medium and placebo microspheres in EGF-free medium were used as controls. The result is shown in FIG. 1. It was possible to achieve such a thorough mixing of the carrier material with the growth factor rhEGF that the protein was released uniformly. The growth factor concentration remained at an effective level. The optimum loading was found to be 2.5 μg / 100 mg carrier material.
Beispiel 7Example 7
Nachweis der Bioaktivität des freigesetzten EGFEvidence of the bioactivity of the released EGF
Die Bioaktivität des in Beispiel 6 nachgewiesenen EGF wurde durch einen Doppelkammerversuch gezeigt: Dazu wurde eine Zellkulturschale mit 50000 Keratinozyten inoculiert. Die Kulturschale ist so gefertigt, daß sie einen Einsatz aufnehmen kann, dessen Boden im Medium der Zellkultur ca. 0,5 cm über den Zellen hängt. In diesen Einsatz wurde eine geeignete Zahl (entsprechend Versuch in Beispiel 6) wachstumsfaktortragender Mikrosphären gegeben. Die Proliferation der Zellen auf dem Kulturschalengrund wurde durch einen Stoffwechseltest gemessen, bei dem ein Substrat mitochondrial in einen Farbstoff umgesetzt wird, der dann photometrisch (z.B. bei 490 n ) bestimmt wird (MTS Test: 3- ( 4 , 5-Dimethylthiazol-2-yl ) -5- ( 3-carboxymethoxyphenyl) -2- ( 4-sulfophenyl ) -2H-tetrazolium wird in Formazan umgewandelt).The bioactivity of the EGF detected in Example 6 was demonstrated by a double-chamber experiment: For this purpose, a cell culture dish with 50,000 keratinocytes was inoculated. The culture dish is manufactured in such a way that it can accommodate an insert, the bottom of which hangs in the cell culture medium about 0.5 cm above the cells. A suitable number (corresponding to experiment in example 6) of growth spheres carrying microspheres was placed in this insert. The proliferation of the cells on the culture dish base was measured by a metabolic test in which a substrate is converted mitochondrially into a dye, which is then determined photometrically (for example at 490 n) (MTS test: 3- (4, 5-dimethylthiazol-2- yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium is converted to formazan).
Als Kontrollen wurden Placebo-Mikrosphären in Medium ± EGF eingesetzt. Das Ergebnis ist in Figur 2 dargestellt. Das von den Mikrosphären freigesetzte Protein beeinflußte die Proliferation der Zellen deutlich verglichen mit den Placebo- Mikrosphären. Verglichen mit dem optimal EGF-supplementierten Zellmedium findet man eine gleich gute Proliferation der Zellen. Beachtenswert ist auch, daß die Wachstumsfaktorkonzentration, mit der die Mikrosphären anfänglich beladen werden, für die Wirkung auf die Proliferation der Zellen unerheblich ist. Beispiel 8Placebo microspheres in medium ± EGF were used as controls. The result is shown in Figure 2. The protein released by the microspheres significantly influenced the proliferation of the cells compared to the placebo microspheres. Compared to the optimally EGF-supplemented cell medium, the cell proliferation is equally good. It is also noteworthy that the growth factor concentration with which the microspheres are initially loaded is insignificant for the effect on the proliferation of the cells. Example 8
Es wurden außerdem mit 2,5 μg rhEGF/100 mg Trägermaterial beladene PLGA-Mikrosphären mit Keratinocyten bewachsen und am 4. Tag der Spinnerkultur auf Nacktmauswunden transplantiert .PLGA microspheres loaded with 2.5 μg rhEGF / 100 mg carrier material were also overgrown with keratinocytes and transplanted onto nude mouse wounds on the 4th day of the spinner culture.
An den darauffolgenden 7 Tagen wurde alle 8 Stunden das gesamte Medium in der Zellkulturkammer (Wundkammer nach Fusenig; siehe Beispiel 2) erneuert und mittels ELISA auf EGF untersucht. Das Medium, in dem die Mikrosphären suspendiert waren, war EGF-frei. Das Ergebnis ist in Figur 3 dargestellt. Es findet eine kontinuierliche Abgabe des Proteins an das Medium statt, wobei die erreichte Konzentration ab dem 3. Tag bei etwa 200 pg/ml alle 8 Stunden liegt. Es kann somit von einer ausreichenden Versorgung der Wunde mit EGF für die erste Woche ausgegangen werden. On the following 7 days, the entire medium in the cell culture chamber (wound chamber according to Fusenig; see Example 2) was renewed every 8 hours and examined for EGF by means of ELISA. The medium in which the microspheres were suspended was free of EGF. The result is shown in FIG. 3. The protein is continuously released to the medium, the concentration reached being about 200 pg / ml every 8 hours from day 3. Sufficient EGF care for the wound can therefore be assumed for the first week.

Claims

Patentansprüche ; Claims;
1. Gewebeersatz, der geeignet ist zur Transplantation, umfassend ein polymeres Trägermaterial, das mit Zellen besiedelt ist, dadurch gekennzeichnet, daß1. tissue replacement which is suitable for transplantation, comprising a polymeric carrier material which is populated with cells, characterized in that
a) das polymere Trägermaterial Partikelform aufweist, so daß die Zellen nach Besiedelung des Trägermaterials als Suspensionskultur kultiviert werden können,a) the polymeric carrier material has a particle shape so that the cells can be cultivated as a suspension culture after the carrier material has been populated,
b) das Trägermaterial bioresorbierbar ist,b) the carrier material is bioresorbable,
c) das polymere Trägermaterial mit wenigstens einem Wachstumsfaktor beladen ist.c) the polymeric carrier material is loaded with at least one growth factor.
2. Gewebeersatz nach Anspruch 1, dadurch gekennzeichnet, daß das polymere Trägermaterial hauptsächlich aus Polylactid besteht .2. Tissue replacement according to claim 1, characterized in that the polymeric carrier material consists mainly of polylactide.
3. Gewebeersatz nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß die Partikel des polymeren Trägermaterials einen Durchmesser von etwa 100 bis etwa 250 μm aufweisen.3. Tissue replacement according to claim 1 or 2, characterized in that the particles of the polymeric carrier material have a diameter of about 100 to about 250 microns.
4. Gewebeersatz nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, daß die Zellen Hautzellen sind.4. Tissue replacement according to one of claims 1 to 3, characterized in that the cells are skin cells.
5. Gewebeersatz nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, daß die Hautzellen hauptsächlich Keratinozyten sind.5. Tissue replacement according to one of claims 1 to 4, characterized in that the skin cells are mainly keratinocytes.
6. Gewebeersatz nach einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, daß die Zellen menschliche Zellen sind. 6. Tissue replacement according to one of claims 1 to 5, characterized in that the cells are human cells.
7. Gewebeersatz nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, daß das polymere Trägermaterial mit epidermalem Wachstumsfaktor (EGF) beladen ist.7. Tissue replacement according to one of claims 1 to 6, characterized in that the polymeric carrier material is loaded with epidermal growth factor (EGF).
8. Gewebeersatz nach einem der Ansprüche 1 bis 7, dadurch gekennzeichnet, daß durch die Freisetzung eines oder mehrerer Wachstumsfaktoren während der Bioresorption des Trägermaterials eine Wachstumsfaktorkonzentration von 200 bis 400 pg/ml in der Wundflüssigkeit erreicht werden kann.8. Tissue replacement according to one of claims 1 to 7, characterized in that a growth factor concentration of 200 to 400 pg / ml in the wound fluid can be achieved by the release of one or more growth factors during the bioresorption of the carrier material.
9. Verfahren zur Herstellung eines Gewebeersatzes, das folgende Schritte umfaßt:9. A method of making a tissue replacement comprising the steps of:
a) Bereitstellung von Zellen,a) provision of cells,
b) Bereitstellung eines partikelförmigen, bioresorbierbaren, polymeren Trägermaterials, das mit wenigstens einem Wachstumsfaktor beladen ist,b) provision of a particulate, bioresorbable, polymeric carrier material which is loaded with at least one growth factor,
c) Besiedelung des polymeren Trägermaterials mit den Zellen.c) colonization of the polymeric carrier material with the cells.
10. Verfahren nach Anspruch 9, dadurch gekennzeichnet, daß die Zellen aus einer Biopsie gewonnen werden.10. The method according to claim 9, characterized in that the cells are obtained from a biopsy.
11. Verfahren nach Anspruch 9 oder 10, dadurch gekennzeichnet, daß die Zellen vor der Besiedelung des polymeren Trägermaterials in Zellkultur vermehrt werden.11. The method according to claim 9 or 10, characterized in that the cells are grown in cell culture before the settlement of the polymeric carrier material.
12. Verfahren nach einem der Ansprüche 9 bis 11, dadurch gekennzeichnet, daß die Zellen Hautzellen sind.12. The method according to any one of claims 9 to 11, characterized in that the cells are skin cells.
13. Verfahren nach einem der Ansprüche 9 bis 12, dadurch gekennzeichnet, daß die Zellen menschliche Hautzellen sind.13. The method according to any one of claims 9 to 12, characterized in that the cells are human skin cells.
14. Verfahren nach einem der Ansprüche 9 bis 13, dadurch gekennzeichnet, daß die Zellen menschliche Keratinozyten sind. 14. The method according to any one of claims 9 to 13, characterized in that the cells are human keratinocytes.
15. Verfahren nach einem der Ansprüche 9 bis 14, dadurch gekennzeichnet, daß das polymere Trägermaterial hauptsächlich aus Polylactid besteht.15. The method according to any one of claims 9 to 14, characterized in that the polymeric carrier material consists mainly of polylactide.
16. Verfahren nach einem der Ansprüche 9 bis 15, dadurch gekennzeichnet, daß die Beladung des polymeren Tr germaterials mit Wachstumsfaktor dadurch erfolgt, daß der oder die Wachstumsfaktoren bei der Polymerisierung des Trägermaterials anwesend sind.16. The method according to any one of claims 9 to 15, characterized in that the loading of the polymeric carrier material with growth factor takes place in that the growth factor or growth factors are present during the polymerization of the carrier material.
17. Verfahren nach einem der Ansprüche 9 bis 16, dadurch gekennzeichnet, daß das polymere Trägermaterial vor der Besiedelung durch Zellen mit Kollagen beschichtet wird. 17. The method according to any one of claims 9 to 16, characterized in that the polymeric carrier material is coated with collagen before colonization by cells.
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