DE10220472A1 - Bioartificial skin - Google Patents

Abstract

The invention relates to a bioartificial skin, comprising a porous matrix on which isogenic or autologous fibroblasts are located, which express at least one angiogenic protein, a neurotrophic protein and at least one protein stabilizing the epidermo-dermal junction, and a mixture of to form a color-correct epidermis isogenic or autologous generation cells and melanocytes, a process for their preparation and their use.

Description

The invention relates to a bioartificial skin a porous matrix on which isogenic or autologous Fibroblasts and keratinocytes are located at least one angiogenic protein and / or at least one die epidermo-dermal junction stabilizing protein express, a process for their preparation and their Use.

Bioartificial skin replacement is known. For example by Machens et al. in "Cell Tissues Organs" 2000, 167: 88-94 a skin replacement based on a Glycosaminoglycan / chondroitin-6-sulfate matrix. With skin replacement, especially with synthetic or xenogenic, but there are various problems (see e.g. Machens et al. in "Surgeon" 2000, 71: 152-158). On the one hand, these include Vascularization and innervation of the dermis, on the other hand the sufficient molecular stabilization of the epidermo-dermal Junction, i.e. the connecting layer between the epidermis and Dermis. There are no intrinsic growth factors for ingrowth of epidermal cells, blood vessels and Nerve fibers lead so that it is not complete Integration of bioartificial skin in the overall tissue composite comes. An aesthetic problem is also the unnatural one pale shade of bioartificial skin, which differs from that the natural color of the surrounding skin.

The object of the invention is to eliminate this state of the art defects inherent in technology.

According to the invention, therefore, a bioartificial skin provided that corresponds to human skin porous matrix on which isogenic or autologous Fibroblasts are located that have at least one angiogenic Protein (e.g. VEGF165), a neurotrophic protein (e.g. NGF) and at least one the epidermo-dermal junction Express stabilizing protein (e.g. collagen 17). In addition, the isogenic or autologous keratinocytes with isogenic or autologous Melanocytes enriched to the extent that one of the recipient region appropriate coloring of the bioartificial skin is achieved becomes.

The advantage that can be achieved is a better one Vascularization and innervation of the matrix (it is enough for example, if over a period of 2 to 3 weeks an angiogenic and neurotrophic active protein is secreted) as well as a more stable epidermodermal Junction (i.e. junctures between dermis and Epidermis) and an adequate skin color.

A matrix is particularly advantageous, for example an allogeneic acellular dermis from corpse skin. Due to the Cellularity, this dermis is immunocompetent and it comes not to rejection reactions. Such a dermis can for example under the trade name Alloderm from the company Life-Cell Corp., USA, (LifeCell; One millenium way; Branchburg, NJ 08876; UNITED STATES).

Isogenic cells, e.g. B. fibroblasts, in the sense of the invention are genetically identical cells that do not belong to any Immune compatibility after retransplantation into an isogenic Organism. Autologous cells, e.g. B. fibroblasts those body cells in which the donor and recipient are in and are the same organism.

According to one embodiment of the invention, this is at least an angiogenic protein under PDGF-A (platelet derived growth factor A), PDGF-B (platelet derived growth factor B), VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), TGFbeta (tumor growth factor beta), Angiopoetin 1 and Angiopoetin selected.

According to a further embodiment of the invention also isologist on the isologic or autologous fibroblast or autologous keratinocytes and melanocytes in one Amount and applied in such a ratio that a natural skin tone is created.

• a) in einem Körper durch Implantation von biologisch inertem Material die Bildung von isogenen oder autologen Fibroblasten ausgelöst wird,
• b) die in Schritt a) gebildeten Fibroblasten aus dem Körper gewonnen werden,
• c) die in Schritt b) gewonnenen Fibroblasten gentechnisch so verändert werden, daß sie mindestens ein angiogenetisches Protein, ein neurotrophes Protein und mindestens ein die epidermo-dermale Junktion stabilisierendes Protein exprimieren und
• d) die in Schritt c) gewonnenen Fibroblasten auf eine poröse Matrix, beispielsweise Alloderm, aufgebracht werden.

The invention also provides a method for producing the bioartificial skin according to the invention, in which

• a) the formation of isogenic or autologous fibroblasts is triggered in a body by implantation of biologically inert material,
• b) the fibroblasts formed in step a) are obtained from the body,
• c) the fibroblasts obtained in step b) are genetically modified such that they express at least one angiogenic protein, one neurotrophic protein and at least one protein stabilizing the epidermodermal junction and
• d) the fibroblasts obtained in step c) are applied to a porous matrix, for example alloderm.

After about 1 week the fibroblasts are on the matrix, e.g. B. Alloderm, confluent, i.e. H. grown together.

According to one embodiment of the method according to the invention the fibroblasts obtained in step c) become an under PDGF-A (platelet derived growth factor A), PDGF-B (platelet derived growth factor B), VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), TGFbeta (tumor growth factor beta), angiopoetin 1 and angiopoetin Express selected angiogenic protein.

According to a further embodiment of the invention Procedures are also performed after the fibroblasts on the Matrix are confluent on the isologists or autologists Fibroblasts isologic or autologous keratinocytes and Melanocytes in such an amount and in such an amount Ratio applied that a natural skin tone arises.

The invention further provides the use of a bioartificial skin according to the invention or one after a inventive methods produced bioartificial skin for covering wounds, in particular Burns, on.

A biologically inert or metabolically inert material a material that is not related to the metabolic processes of the Body participates, but still a cellular response can trigger (accumulation of fibroblasts). If such Material (e.g. Silastic) is implanted, then forms a connective tissue capsule within a few days (5-7) around the implant, which is the desired fibroblast contains. This capsule together with fibroblasts can be removed and after trypsinization of the material, the fibroblasts isolate and cultivate. These fibroblasts can be isogenic his. That means they come from an isogenic donor animal, which has the same genetic traits as that Animals in which the bioartificial skin with the genetically modified isogenic fibroblasts is used. The alternative (and that makes the most sense clinically) consists of getting the fibroblasts out of the patient wins when you later genetic these cells Want to use modification in bioartificial skin. In this case it would be autologous cells that is, cells in which the donor and recipient are identical. Likewise, with a human donor (who also at the same time be the recipient of the bioartificial skin a skin biopsy for the cultivation of keratinocytes and Melanocytes are made. The technique of isolation and Cultivation of these cells differs in principle not from the process of obtaining the fibroblasts.

In the following the invention is carried out without limitation of the Claim wording with reference to concrete Embodiments and working examples in more detail described.

In a series of experimental studies, the Inventors found that after, for example, retroviral Gene transfer from isogenic fibroblasts from the rat (GMFB) this a stable integration in vitro, for example the human Show gene PDGF-A. As a result, up to 560 times higher concentration of, for example, PDGF-AA, compared to non-genetically modified fibroblasts (NMFB) can be achieved. The inventor has also demonstrated can do that after adenoviral gene transfer in fibroblasts a series of angiogenetically active proteins for a Time of 2-3 weeks can be stably expressed and after Retransplantation into an isogenic recipient organism there induce therapeutic angiogenesis.

material and methods Cell cultures and their genetic modification

Fibroblasts: Fibroblast cultures obtained from autologous Inbred rat strains (Lewis CRL female rats, weight 200-215 grams; Charles River Laboratories)
Virus-producing cell line: psi-CRIP packaging amphotrophic cell line derived from murine NIH-3T3 fibroblasts that express the gag, pol and env viral gene products (R. Mulligan, Whitehead Institute of Biomedical Research, Cambridge, Mass.). For the adenoviral gene transfer, a padcos46 RESeGFP (39155 bp) is used as the cosmid vector fragment and equipped with the corresponding desired gene sequence (W. Lindenmaier, Society for Biotechnological Research / Baunschweig). Transduction of the psi-CRIP packaging cell line with MFG plasmid DNA, cloning of the same and screening of the cell line which produces the highest virion titers (JR Morgan, Shriners Burns Research Laboratories, Cambridge, Mass. And W. Lindenmaier, Society for Biotechnological Research / Baunschweig)
Fibroblast cell culture medium: Dulbecco's modified Eagle medium (DMEM, high-percentage glucose, L-glutamine, sodium pyruvate 110 mg / L from Gibco BRL / USA), FBS (fetal bovine serum = fetal bovine calf serum) 10% (HtClone / USA), Penicillin-streptomycin 100 IU / ml-100 microl / ml (Böhringer), BCS (bovine calfserum = bovine calf serum) 10% (HtClone / USA)
PBS (phosphate buffered saline) consisting of 138 mM NaCl, 2.7 M KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, sterilized via a 0.45 micron filter EDTA solution (called "Versene") (= (Ethylenedinitriol) - tetraacetic acid disodium salt, from Boehringer): 5 mM dissolved in PBS and sterilized via a 0.45 micron filter
Trypsin solution (Trypsin 1-300, ICN Biochemicals), consisting of 0.1% D-dextrose (w / v) and trypsin 0.1% (w / v) in PBS at a pH of 7.5
Storage bottle for trypsin (25 ml, Wheaton Scientific)
Polybrene (Sigma / USA)
DMSO (dimethylsulphoxide; Sigma-Aldrich; Irvine / England) Experimental animals and operative procedure 400 autologous Lewis rats (Inbred strains; female) from the Charles River Laboratories (Pittsfield NH / USA). All animal experiments are carried out strictly according to the protocol arenas provided by the University Hospital of Lübeck.
Silica film (0.0127 mm diameter; PharmElast, SF Medical Hudson MA / USA)
Neck collar for rats as protection against car cannibalism (Kent Scientific / Litchfield, CT / USA)
Surgical instruments including micro cutlery
Ethyl ether (Fisher Scientific, Fair Lawn, NJ / USA)
Ketamine (Ketanest 100 mg / ml; Fort Dodge Laboratories, Iowa / USA)
Xylazine (Rampun 20 mg / ml; Bayer Corporation, Kansas / USA).
Betadine immunoassays and ELISA ELISA (Enzyme Linked Immunosorbent Assay) (R&D Systems, Minneapolis / USA).
ELISA for VEGF-156, sFLT-1, PDGF-B and bFGF from the laboratory of Dr. Weich / GBF Braunschweig Histology stains and immunohistochemistry haematoxylin-eosin
Anti-Human by Willebrand Factor, IgG Fraction (Sigma, St. Louis, MO / USA)
FITC Conjugate, Anti-IgG (Sigma, St. Louis, MO / USA)

VEGF165 mRNA analysis

The methods for VEGF mRNA quantification are in themselves known (competitive RT-PCR, Northern blot).

fibroblast production

Autologous rat fibroblasts are found in 5 animals of the above mentioned rat strain bred as later carrier cells to express the desired gene. The animals are used for this anesthetized by intraperitoneal injection e.g. B. one Combination of 0.05 mg / gm ketamine (Ketanest 100 mg / ml; cont Dodge Laboratories, Iowa / USA) and 0.0013 mg / gm xylazine (Rampun 20 mg / ml; Bayer Corporation, Kansas / USA). The spontaneous Breathing animals are from the xyphoid to the groin region shaved and placed on an operating table.

The body temperature is measured during each experiment measured using a digital rectal thermometer and over a heating mat kept constant at 36-37 degrees Celsius. After the surgical area has been washed sterile, the Xyphoid along the linea alba caudally cut with the scalpel of 10, whereby only the skin and the subcutaneous Adipose tissue is cut and the fascia of the rectus muscles is left to then with careful care of the epigastric skin vessels laterally on both sides to create an approximately 4 × 5 cm wound pocket.

An equally large silica membrane is placed in this pocket (Silastic is just one example of a suitable material, that after the implantation the formation of isogenic Fibroblasts and no other side effects hat) (PharmElast; SF Medical) and subcutaneous Corner seams (6-0 Ethilon) fixed. This membrane has a thickness from Z. B. 0.0127 mm, is particularly soft, extremely flexible and is delivered sterile packed. Before implanting the film it is washed sterile. After fixing the film in situ the wound is closed by an intracutaneous 6-0 Ethilon seam with sinking of the corner knots.

The animals are observed daily for 7 days in the open Access to water and food to help prevent problems in the early stages To be able to uncover wound healing. After that period will be the animals are anesthetized as previously described and the Silastic membrane that acts as a foreign body to the local Should stimulate fibroblast production, along with that formed scar tissue removed. The animals will euthanized by an intraperitoneal overdose of called anesthetic mixture.

Fibroblast separation and cultivation

The surgically obtained fibroblast conglomerate is immediately in Preserves DMEM at 4 degrees Celsius and one as quickly as possible Subjected to processing. The material is under sterile Conditions detached from the silica membrane. All Work with the fibroblast culture takes place in a laboratory Genetic security level 2 instead of with air extraction at everyone sterile workplace.

The material is now subjected to extensive washing in a total of 10 plastic containers with 10 ml PBS each (Phosphate buffered table salt 0.9%). Any container can be sealed sterile so that the fibrocyte conglomerate Can be shaken vigorously in the PBS for 10 × 60 seconds. After washing twice, the fabric becomes one more time Taken to last connective tissue remnants and portions of silasik have demarcated themselves to remove under sterile cauldrons. Then the material is placed in a sterile 25 ml Monovette given and with 5 ml trypsin and 5 ml EDTA over 5 minutes enzymatically treated to remove the fibroblasts from the Loosen collagen bandage. The cells are covered by sterile gauze filtered and then in DMEM with 20% FBS washed to neutralize trypsin activity. The so The suspension obtained is at 800 rpm for 5 minutes centrifuged. The fibroblasts settle on the floor and are mixed with 10 ml DMEM.

20 microliters of the suspension are counted in a cell counter (hemocytometer) and the supernatant is then pipetted off. The cells are then grown in brood chambers with 75 cm ² floor area with a seeding of 5 × 104 cells / cm ² . The cultivation is carried out in a medium from DMEM, mixed with 100 micrograms / ml streptomycin, 100 IU / ml penicillin, 3 microliters / ml amphotericin, 5% FBS and 10 micrograms / ml ascorbic acid, which is added daily to the nutrient medium. The nutrient medium itself is changed every 3 days because the fibroblasts divide on average once every 16-18 hours and have a corresponding energy metabolism.

If the fibroblasts nearly conflate, another cell separation is performed. The cells obtained in this way can then be re-sown for further cultivation of new cells or can also be preserved and frozen. This is done by cell separation and subsequent suspension in DMEM with 10% FBS and appropriate antibiotic addition. If CRIP cells are preserved, they are suspended with BCS medium. The substance DMSO is added to the medium as a cryoprotective agent in a mixing ratio of 1:10. Between 1 × 10 ⁶ cells should then be contained per ml of medium. Then 1-2 ml of medium / container are filled and frozen for 24 hours at -20 degrees Celsius. The following day, the containers are then frozen to -80 degrees Celsius to remain in liquid nitrogen at -196 degrees Celsius 24 hours later. This gradual freezing avoids the formation of ice crystals in the suspension with consequent cell damage.

Production of recombinant retro and adenoviruses and Gene transfer to the fibroblast cultures

All genetic engineering work is carried out in the planned premises of security level 1 and 2 the corresponding genetic engineering laboratory protocols and are carried out by the responsible authority for genetic safety approved.

The first step in gene transfer is production of a recombinant virus which carries the gene to be transferred encoded. In these experiments, a cDNA that does this Protein of interest encoded using PCR (polymerase Chain Reaction) amplified. The corresponding primers produce z. B. a BspH1 locus at the translational Starter Codon and z. B. a BamH1 locus at the translational Stop codon. The PCR product can then be isolated by separating the gene product at the points mentioned and insertion of the gene obtained into the Nco1 / BamH1 loci of a viral vector called MFG. The successful one Transmission is then carried out through DNA sequencing checked.

This vector (MFG plasmid DNA) comes from e.g. B. from the murine Moloney Leukemia Virus, itself contains no viral genes except those that reverse for transcription, packaging Transcription, integration and expression of the viral vector with the modifying gene contained therein are. In order to be able to produce virions that The vector must be able to anchor genetically in target cells Vector in a special packaging cell line, which, for. B. from murine 3T3 fibroblasts. The Transduction of the vector into the corresponding one Packing cell line is made by adding calcium phosphate relieved, because it the cell membranes of the Packaging cell lines become temporarily porous and the viral Vector to provide easy access to the cell. This Packaging cell line (Psi-CRI) was specially produced to to deliver the viral proteins pol, env and gag, which in turn can produce virions that match the vector with the encode the modifying gene therein and transfer. The packaging cell line itself cannot contain viruses produce that correspond to "wild type" replicants and thus are virulent. Instead, it transcribes the DNA of the recombinant viral vector in RNA, which is then in the RNA of the Virion is integrated. The Psi-CRP packaging cell line then separates the virion, i.e. the recombinant virus together with the modifying gene in the cell medium. A Target cells can be transfected effectively with a lot from 1.0-10.0 million virions / ml medium. It will therefore be everyone transfected cell line after the highest titer production checked in order to then select them for gene transfer. For adenoviral gene transfer, is used as Cosmid vector fragment a padcos46 RESeGFP (39155 bp) used and with the corresponding desired gene sequence stocked. Furthermore, the vector can be constructed that gene expression is only possible with simultaneous administration of a further substance takes place. This is a timed one Expression possible. Suitable vectors with so-called "on / off" - Gene functions are known in the prior art.

For the transduction, the fibroblasts obtained are sown in brood chambers with a floor area of 75 cm ² and with a medium of DMEM, mixed with 100 micrograms / ml streptomycin, 100 IU / ml penicillin, 3 microliters / ml amphotericin and 5% FBS. The fibroblasts are sown in a low density (5 × 10 ⁵ cells) in order to achieve the greatest possible effectiveness of the transduction.

The next day when all fibroblasts contact the Floor area of the brood chamber and get slow spread, a medium change is made with medium the Psi-CRIP cell line. Are in this medium 1.0-10.0 million virions / ml medium, which are fresh from the medium of Packaging cell line (Psi-CRIP) are pipetted off. The medium is first done with a pore filter with a Pore diameter of 0.45 microns filtered to get it from Free cell debris and possible contaminants.

The substance is then polybrene in a medium Concentration of 8 micrograms / ml added.

Like protamine and DEAE dextran, polybrene is a cationic Polymer consisting of 1,5-dimethyl-1,5-diaxadecamethylene Polymethobromide, and unfolds its transfection-supporting effect in that it is attached to the Virus particles adsorbed and equally to the Surface of the target cell, so the electrostatic Repulsive forces of these two negatively charged substances mitigate. To obtain large enough quantities of Virus particles must pass the cells of the Psi-CRIP cell line already be confluent and a change of medium the day before Transduction take place to the highest possible number of ensure active viruses / ml medium. The viruses have An average of 37 degrees Celsius in the incubator half life of 6-8 hours The lifetime of the Viruses can be reduced by lowering the incubator temperature to 32 degrees Celsius extended up to ten times and thus the Effectiveness of transduction can be increased significantly.

If the recombinant viruses in the medium with target cells come together, the virion on the cell surface of the Fibroblasts bound and released by special receptors the packaged RNA genome into the cell interior. This will be reversed transcribed and the resulting DNA arrives in the Cell nucleus where it is stable in the genome of the target cell is integrated. This integrated copy of the recombinant viral vector with the modifying gene is attached to the Daughter cells are passed on like any other autosomal gene. In addition, there is a stable regular expression of the gene, so that the target cells now have large amounts of the desired Secrete protein.

If you use adenoviral vectors, you only become one can expect temporary gene expression, which after some Cell generations can be eliminated from the cell genome becomes. The medium should be with the target cells for 24 hours left together to complete the transduction process to have completed all cells.

The virus-containing medium can also be preserved to it at a later time for transduction of To use fibroblasts. This is the medium pipetted off and snap frozen on dry ice until there is a yellowish color. Then the medium is at -80 degrees Celsius stored. You have to go with one Expect loss of 30-50% viruses through this process.

The growth of the genetically modified compared to the untreated fibroblasts is examined by sowing 5 × 105 cells each on 60 mm diameter petri dishes and counting the cells 12 hours apart a total of 4 days according to the above procedure. From this conclusions can be drawn about the biological activity of the secreted protein pull because of the substances used with the exception of sFLT1 D1-D6 also have an autocrine mitogenic effect, so the fibroblasts, which themselves secrete the protein, stimulates cell division. The number of fibroblasts will then under the fluorescence microscope using a Counting chamber measured and the vitality of the cells by evans Blue determined and with the controls, which out untreated fibroblasts exist.

Protein expression is also assessed in vitro using ELISA Techniques are determined. It becomes essential higher protein production due to the genetically modified Fibroblasts expected.

The production of autologous cells takes place according to exactly that same methodological guidelines as described above were.

The transfected fibroblasts are applied to a matrix which has previously been cut in vitro according to the size of the graft to be used. This results in a construction that corresponds exactly to the needs of the patient. When using AlloDerm, the dermis is initially rehydrated with its basement membrane upwards and covered with DMEM with 10% FBS and the corresponding antibiotic addition for 15 minutes. The modified fibroblasts are then applied to the dermis with medium (1 × 10 ⁵ cells / cm ² ) and preserved in the incubator for 24 hours. The confluence of the fibroblasts is waited for and is complete after approx. 2-3 days. During this time, the medium is changed every 48 hours. The keratinocytes and melanocytes removed simultaneously with the fibroblasts have since been separated and cultured and, if the fibroblasts are confluent, can be sown on the matrix using the same technique as the fibroblasts (2 × 10 ⁵ keratinocytes / cm ² ). The amount of melanocytes to be transplanted with the keratinocytes from suspension depends on the desired color of the recipient region. For a rather light type (skin type II) 1 × 10 ⁴ melanocytes / cm ² dermis are calculated, while for a rather dark European type (skin type IV) it is 3 × 10 ⁴ melanocytes / cm ² dermis. The bio-composite skin produced in this way can be transplanted after the confluence of the keratinocytes in a patient (consisting of acellular matrix, genetically modified fibroblasts, keratinocytes and melanocytes of the patient). For this purpose, the wound areas to be transplanted are surgically subtly debrided again under sterile conditions and then the graft is precisely placed on the wound area. A liquid-permeable layer of cuticerin (Beiersdorff) is spread over the graft. A layer of 1 cm thick sterile sponge is placed over it. This sponge contains DMEM with 10% FBS and a corresponding antibiotic additive for the primary nutrition of the upper graft layers). The entire wound area is then covered with a further layer of cuticerin and fixed with tie-on pads. After 2 days, the entire bandage can be removed and the transplanted wound can be moistly bonded with Lavasept-soaked cuticerin. The entire region, like any other skin graft, must be immobilized by splints for 10 days.

Claims (7)

1. Bioartificial skin, having a porous matrix who are isogenic or autologous fibroblasts who at least one angiogenic protein (e.g. VEGF165) neuroptrophic protein (e.g. NGF) and an epidermo-dermal Junction stabilizing protein (e.g. collagen 17) express. 2. Bioartificial skin according to claim 1, wherein the at least one angiogenic protein under PDGF-A (platelet derived growth factor A), PDGF-B (platelet derived growth factor B), VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), TGFbeta (tumor growth factor beta), angiopoetin 1 and angiopoetin is selected. 3. Bioartificial skin according to one of the preceding claims, which also has isologic or autologous keratinocytes and melanocytes on the isologic or autologous fibroblasts in such an amount and in such a ratio that a natural skin tone arises in relation to the skin surrounding (for skin type II z. B. 1 × 10 ⁴ melanocytes / cm ² dermis) 4. A method for producing bioartificial skin according to one of claims 1 to 3, in which a) the formation of isogenic or autologous fibroblasts is triggered in a body by implantation of biologically inert material, b) the fibroblasts formed in step a) are obtained from the body, c) the fibroblasts obtained in step b) are genetically modified such that they express at least one angiogenic protein, one neurotrophic protein and at least one protein stabilizing the epidermodermal junction and d) the fibroblasts obtained in step c) are applied to a porous matrix. 5. The method of claim 4, wherein the in step c) Fibroblasts obtained under PDGF-A (platelet derived growth factor A), PDGF-B (platelet derived growth factor B), VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), TGFbeta (tumor growth factor beta), Angiopoetin 1 and Angiopoetin selected express angiogenic protein. 6. The method according to any one of the preceding claims, wherein also isogenic to isogenic or autologous fibroblasts or autologous keratinocytes and melanocytes in one Amount and applied in such a ratio that a natural skin tone is created. 7. Use of a bioartificial skin according to one of the Claims 1 to 3 or one according to a method according to one of claims 4 to 6 produced bioartificial skin for Covering wounds.