EP1504087A2

EP1504087A2 - Bioartificial skin

Info

Publication number: EP1504087A2
Application number: EP03732323A
Authority: EP
Inventors: Hans-Günther Machens
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-05-07
Filing date: 2003-05-07
Publication date: 2005-02-09
Also published as: DE10220472A1; WO2003095630A3; AU2003239844A8; WO2003095630A2; AU2003239844A1

Abstract

The invention relates to a bioartifical skin comprising a porous matrix on which isogenic or autologous fibroblasts can be found, said fibroblasts expressing at least one angiogenetic protein, a neurotrophic protein and at least one protein stabilising the epidermo-dermal junction. The invention also relates to a mixture of isogenic or autologous keratinocytes and melanocytes which is used to form a true-to-colour epidermis, a method for producing said bioartificial skin and the use of the same.

Description

Bioartificial skin

The invention relates to a bioartificial skin, comprising a porous matrix on which isogenic or autologous fibroblasts and keratinocytes are located, which express at least one angiogenic protein and / or at least one protein stabilizing the epidermodermal junction, a process for their production and their use ,

Bioartificial skin replacement is known. For example, Machens et al. in “Cell Tissues Organs” 2000, 167: 88-94, describes a skin replacement based on a glycosaminoglycan / chondroitin-6-sulfate matrix. However, there are still various problems with skin replacement, especially with synthetic or xenogeneic (see, for example, Machens et al. In "Chirurg" 2000, 71: 152-158). On the one hand, this includes vascularization and innervation of the dermis, on the other hand, the sufficient molecular stabilization of the epidermodermal junction, i.e. the connecting layer between the epidermis and dermis. The intrinsic growth factors that lead to the ingrowth of epidermal cells, blood vessels and nerve fibers are missing, so that there is no complete integration of bioartificial skin into the overall tissue composite. An aesthetic problem is also the unnatural one pale shade of bioartificial skin, which differs from the natural shade of the surrounding skin.

The object of the invention is to eliminate these deficiencies inherent in the prior art.

According to the invention, therefore, a bioartificial skin is provided which has a porous matrix corresponding to human skin on which there are isogenic or autologous fibroblasts which contain at least one angiogenic protein (for example VEGF165), a neurotrophic protein (for example NGF) and at least one which is epidermo- Express dermal junction stabilizing protein (eg collagen 17). In addition, the isogenic or autologous keratinocytes to be transplanted are enriched with isogenic or autologous melanocytes to such an extent that a color of the bioartificial skin corresponding to the recipient region is achieved.

The advantage that can be achieved with this is better vascularization and innervation of the matrix (it is sufficient, for example, if an angiogenic and neurotrophic active protein is secreted over a period of 2 to 3 weeks) and a more stable epidermodermal junction (ie connection points between the dermis and epidermis) and a skin color adequate to the environment.

For example, an allogeneic acellular dermis from corpse skin is particularly suitable as a matrix. Due to its cellular nature, this dermis is immunocompetent and there are no 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; USA).

Isogenic cells, e.g. Fibroblasts, in the sense of the invention, are genetically identical cells which do not lead to immune compatibility after retransplantation into an isogenic organism. Autologous cells, e.g. Fibroblasts are cells in the body in which the donor and recipient are one and the same organism.

According to one embodiment of the invention, the at least one angiogenic protein is 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, isologic or autologous keratinocytes and melanocytes are also applied to the isologic or autologous fibroblasts in such an amount and in such a ratio that a natural skin tone is produced.

The invention also provides a process for preparing the bioartifizellen skin according to the invention prepared in which a) in a body by implantation of biologically inert material, the formation of isogenic or autologous fibroblasts is triggered, b) formed in step a) fibroblasts from the ^'body are obtained, c) the fibroblasts obtained in step b) are genetically modified so that they have at least one angiogenic Express protein, a neurotrophic protein and at least one protein stabilizing the epidermo-dermal junction and d) the fibroblasts obtained in step c) onto a porous one

Matrix, for example alloderm, can be applied.

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

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

According to a further embodiment of the method according to the invention, after the fibroblasts on the matrix are confluent, isologic or autologous keratinocytes and melanocytes are applied to the isologic or autologous fibroblasts in such an amount and in such a ratio that a natural skin tone is produced.

The invention further specifies the use of a bioartificial skin according to the invention or a bioartificial skin according to the invention produced by a method according to the invention for covering wounds, in particular burns. A biologically inert or metabolically inert material is a material that does not participate in the body's metabolic processes, but can still trigger a cellular reaction (accumulation of fibroblasts). If such a material (eg silastic) is implanted, a connective tissue capsule that contains the desired fibroblasts will form around the implant within a few days (5-7). This capsule together with fibroblasts can be removed and, after trypsinizing the material, the fibroblasts can be isolated and cultivated. These fibroblasts can be isogenic. This means that they come from an isogenic donor animal that has the same genetic properties as the animals, in which the bioartificial skin with the genetically modified isogenic fibroblasts is later used. The alternative (and this makes the most sense clinically) is that the fibroblasts are obtained from the patient, in which one later wants to use these cells after genetic modification in the bioartificial skin. In this case, the cells would be autologous, i.e. cells in which the donor and recipient are identical. In the same way, a skin biopsy for the cultivation of keratinocytes and melanocytes can be carried out on a human donor (who will also be the recipient of the bioartificial skin). The technique of isolating and cultivating these cells does not differ in principle from the process for obtaining the fibroblasts.

In the following, the invention is described in more detail without limitation of the wording of the claims with reference to specific embodiments and working examples. In a series of experimental investigations, the inventor found that, for example after retroviral gene transfer from isogenic fibroblasts of the rat (GMFB), these have a stable integration in vitro, for example of the human gene PDGF-A. In vitro, a concentration of up to 560 times higher, for example, PDGF-AA could be achieved compared to non-genetically modified fibroblasts (NMFB). The inventor has also been able to demonstrate that, after adenoviral gene transfer in fibroblasts, a series of angiogenetically active proteins can be stably expressed for a period of 2-3 weeks and, after retransplantation into an isogenic recipient organism, induce therapeutic angiogenesis there.

material and methods

Cell cultures and their genetic modification Fibroblasts: Fibroblast cultures obtained from autologous Inbred rat strains (female Lewis CRL rats, weight 200-215 grams; Charles River Laboratories)

Virus-producing cell line: amphotrophic psi-CRIP packaging cell line which is derived from murine NIH-3T3 fibroblasts which express the viral gene products gag, pol and env (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. Linden aier, 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 contains the highest virion titers produced (JRMorgan, Shriners Burns Research Laboratories, Cambridge, ass. 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 lOOIU / ml-lOOmicrol / ml (Böhringer), BCS (bovine calf serum = bovine calf serum) 10 % (HtClone / USA)

PBS (phosphate buffered saline) consisting of 138 M NaCl, 2.7 M KC1, 8.1 mM Na2HP04, 1.5 M KH2P04, sterilized via a 0.45 micron filter

ED A solution (called "Versene") (= (ethylenedinitriol) tetraacetic acid disodium salt, Boehringer): 5 mM dissolved in PBS and sterilized using 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 (Dirnethylsulphoxid; Sigma-Aldrich; Irvine / Engländ) Laboratory animals and surgical procedure

400 autologous Lewis rats (Inbred strains; female) from Charles River Laboratories (Pittsfield NH / USA). All animal experiments are carried out strictly according to the protocol arenas provided by the University Hospital Lübeck.

Silica film (0.0127 mm diameter; PharmElast, SF Medical Hudson MA / USA)

Neck collar for rats as autocannibalism protection (Kent Scientific / Litchfield, CT / USA)

Surgical instruments incl. micro cutlery

Aechylacher (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-165, sFLT-1, PDGF-B and bFGF from Labor Dr, Weich / GBF Braunschweig Histology staining and immunohistochemistry

Hematoxylin-eosin

Anti-Human from Willebrand Factor, IgG Fraction (Sigma,

St.Louis, MO / USA)

FITC Conjugate, Anti-IgG (Sigma, St. Louis, MO / USA)

VEGFl65 mRNA analysis

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

fibroblast production

Autologous rat fibroblasts are grown in 5 animals of the above-mentioned rat strain as later carrier cells for the expression of the desired gene. The animals are anesthetized for this by intraperitoneal injection e.g. a combination of 0.05 mg / gm ketamine (Ketanest 100 mg / ml; Fort Dodge Laboratories, Iowa / USA) and 0.0013 mg / gm xylazine (Rampun 20 mg / ml; Bayer Corporation, Kansas / USA). The spontaneously breathing animals are shaved from the xyphoid to the groin region and placed on an operating table.

Body temperature is measured during each experiment using a digital rectal thermometer and kept constant at 36 - 37 degrees Celsius using a warming mat. After sterile washing of the surgical area, a cut is made with the scalpel from the xyphoid along the linea alba towards the caudal end, whereby only the skin and the subcutaneous fatty tissue are severed and the fascia of the rectus muscles are left, followed by careful care of the epigastric skin vessels to create an approximately 4 x 5 cm wound pocket laterally on both sides. An equally large silastic membrane is placed in this pocket (silastic is only one example of a suitable material that causes the formation of isogenic fibroblasts after implantation and has no other side effects) (PharmElast; SF Medical) and through subcutaneous corner sutures (6 -0 Ethilon) fixed. This membrane has a thickness of 0.0127 mm, for example, is particularly soft, extremely flexible and is supplied sterile packed. Before the film is implanted, it is washed sterile. After the film has been fixed in situ, the wound is closed by an intracutaneous 6-0 ethilon suture with countersinking the corner knots.

The animals are observed daily for 7 days with free access to water and food in order to be able to uncover problems in wound healing at an early stage. After this period, the animals are anesthetized as described above and the silastic membrane, which should stimulate local fibroblast production as a foreign body, together with the scar tissue formed around it, are removed. The animals are euthanized by means of an intraperitoneal overdose of the anesthetic mixture mentioned.

Fibroblast separation and cultivation

The surgically obtained fibroblast conglomerate is immediately stored in DMEM at 4 degrees Celsius and processed as quickly as possible. The material is detached from the silica membrane under sterile conditions. All work with the fibroblast culture takes place in a laboratory with genetic engineering level 2 instead of with air extraction at every sterile workplace. The material is now subjected to extensive washing in a total of 10 plastic containers, each with 10 ml PBS (phosphate-buffered table salt 0.9%). Each container can be closed sterile so that the fibrocyte conglomerate can be shaken vigorously in the PBS for 10 x 60 seconds. After washing twice, the tissue is removed again in order to remove any remaining connective tissue residues and portions of silica that have been demarcated under sterile cauldrons.

The material is then placed in a sterile 25 ml monovette and enzymatically treated with 5 ml trypsin and 5 ml EDTA for 5 minutes in order to detach the fibroblasts from the collagen dressing. The cells are filtered through sterile gauze and then washed in DMEM with 20% FBS to neutralize the trypsin activity. The suspension obtained in this way is centrifuged at 800 rpm for 5 minutes. The fibroblasts settle on the bottom 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 a floor area of 75 cm ² with a seeding of 5 × 10 ⁴ cells / cm ² . The cultivation is carried out in a medium of DMEM, mixed with 100 micrograms / ml streptomycin, 100 IU / l 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. When the fibroblasts almost 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. As a protective protective agent, the substance DMSO is added to the medium in a mixing ratio of 1:10. Between 1 x 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 deep-frozen at - 80 degrees Celsius, in order to remain in liquid nitrogen at - 196 degrees Celsius 24 hours later. This gradual freezing prevents 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 designated security level 1 and 2 premises in accordance with the relevant genetic engineering laboratory protocols and is approved by the competent authority for genetic engineering safety.

The first step in gene transfer is the production of a recombinant virus that encodes the gene to be transferred. In these experiments, a cDNA that encodes the protein of interest is aplicated by means of PCR (polymerase chain reaction). The corresponding primers produce, for example, a BspHl locus at the translation starter codon and, for example, a BamHl locus at the translation stop codon. The product of the PCR can then be isolated by separating the gene product at the sites mentioned and inserting the gene obtained into the Ncol / BamHl loci of a viral vector called MFG. Successful transmission is then checked by DNA sequencing.

This vector (MFG plasmid DNA) comes, for example, from the murine Moioney leukemia virus and does not itself contain any viral genes other than those which are necessary for the transcription, packaging, reverse transcription, integration and expression of the viral vector with the modifying gene therein. In order to be able to produce virions that can genetically anchor this vector in target cells, the vector must be integrated into a special packaging cell line, which comes, for example, from murine 3T3 fibroblasts. The transduction of the vector into the corresponding packaging cell line is facilitated by the addition of calcium phosphate, since this makes the cell membranes of the packaging cell lines temporarily porous and gives the viral vector easier access to the cell. This packaging cell line (Psi-CRI) was specially produced to deliver the viral proteins pol, env and gag, which in turn can produce virions that code and transmit the vector with the modifying gene therein. The packaging cell line itself cannot produce viruses that correspond to "wild type" replicants and are therefore virulent. Instead, she transcribes them. PNS of the recombinant viral vector in RNA, which is then integrated into the RNA of the virion. The Psi-CRP packaging cell line then excretes the virion, ie the recombinant virus together with the modifying gene, into the cell medium. A transfection of the target cells is effective with a quantity of 1.0 - 10.0 million virions / ml medium. Each transfected cell line is therefore checked for the highest titer production in order to then select it for gene transfer. A padcos46 RESeGFP is used as a cosmid vector fragment for adenoviral gene transfer

(39155 bp) used and equipped with the corresponding desired gene sequence. Furthermore, the vector can be constructed in such a way that gene expression takes place only with the simultaneous administration of another substance. This enables a time-controlled expression. 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 / l amphotericin and 5% FBS. The fibroblasts are sown in a low density (5 x 10 ⁵ cells) in order to achieve the greatest possible effectiveness of the transduction.

The following day, when all fibroblasts have come into contact with the bottom of the brood chamber and are slowly spreading, a medium change is carried out with medium from the Psi-CRIP cell line. This medium contains 1.0-10.0 million virions / ml medium, which are freshly pipetted off from the medium of the packaging cell line (Psi-CRIP). For this purpose, the medium is first filtered through a pore filter with a pore diameter of 0.45 micrometers in order to free it from cell debris and possible contaminants. The substance polybrene is then added to the medium in a concentration of 8 micrograms / ml. Like protamine and DEAE-dextran, polybrene is a cationic polymer consisting of 1, 5-dimethyl-l, 5-diaxadecamethylene polymethobromide and exerts its transfection-supporting effect by adsorbing it on the virus particles and also on the surface of the target cell, so as to weaken the electrostatic repulsive forces of these two negatively charged substances. In order to obtain sufficiently large quantities of virus particles, the cells of the Psi-CRIP cell line must already be confluent and the medium changed on the day before the transduction in order to ensure the highest possible number of active viruses / ml medium. The viruses have an average half-life of 6-8 hours at 37 degrees Celsius in the incubator. The lifespan of the viruses can be increased tenfold by lowering the incubator temperature to 32 degrees Celsius, thus considerably increasing the effectiveness of the transduction.

When the recombinant viruses come together with target cells in the medium, the virion is bound to the cell surface of the fibroblasts by special receptors and releases the packaged RNA genome into the interior of the cell. This is reverse transcribed and the resulting DNA reaches the cell nucleus, where it is stably integrated into the genome of the target cell. This integrated copy of the recombinant viral vector with the modifying gene is passed on to the daughter cells like any other autosomal gene. In addition, the gene is stable and regularly expressed, so that the target cells now secrete large amounts of the desired protein. When using adenoviral vectors, only temporary gene expression can be expected, which will be eliminated from the cell genome again after a few cell generations. The medium should be left with the target cells for 24 hours to complete the transduction process in all cells.

The virus-containing medium can also be preserved for later use for transduction of fibroblasts. For this purpose, the medium is pipetted off and snap-frozen on dry ice until it takes on a yellowish color. The medium is then stored at - 80 degrees Celsius. However, you have to expect a loss of 30 - 50% viruses through this process.

The growth of the genetically modified compared to the untreated fibroblasts is examined by sowing 5 × 10 ⁵ cells in each case into Petri dishes with a diameter of 60 mm and counting the cells at intervals of 12 hours over a total of 4 days according to the above-mentioned method. This allows conclusions to be drawn about the biological activity of the secreted protein, since the substances used, with the exception of sFLTl D1-D6, also have an autocrine mitogenic effect, i.e. the fibroblasts that secrete the protein themselves stimulate cell division. The number of fibroblasts is then measured under the fluorescence microscope using a counting chamber and the vitality of the cells is determined by Evans-Blue and compared with the controls, which consist of untreated fibroblasts.

Protein expression will also be determined in vitro using ELISA techniques. It becomes essential higher protein production expected from the genetically modified fibroblasts.

The production of autologous cells takes place according to exactly the same methodological specifications as described above.

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 design that corresponds exactly to the patient's needs. When using AlloDer, the dermis is initially rehydrated with its basement membrane upwards and covered with DMEM with 10% FBS and 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 taken simultaneously with the fibroblasts have since been separated and cultivated and can be sown on the matrix if the fibroblasts are confluent using the same technique as the fibroblasts (2 x 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 11) 1 x 10 ⁴ melanocytes / cm ² dermis are calculated, while for a rather dark European type '(skin type IV) it is 3 x 10 ⁴ melanocytes / cm ² dermis. After the confluence of the keratinocytes in a Patients (consisting of acellular matrix, genetically modified fibroblasts, keratinocytes and melanocytes from the patient) are transplanted. 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 bandaged with Cuticerin soaked in Lavasept. The entire region, like any other skin graft, must be immobilized by splints for 10 days.

Claims

claims

1. Bioartificial skin, comprising a porous matrix on which isogenic or autologous fibroblasts are located, which express at least one angiogenic protein, one neurotrophic protein and one protein stabilizing the epidermo-dermal junction.

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 claim 1 or 2 with fibroblasts that

- VEGF165 as an angiogenic protein and / or

- NGF as a neurotrophic protein and / or

- Express collagen, especially collagen 17, as an epidermo-dermal junction stabilizing protein.

4. Bioartificial skin according to one of the preceding claims, which additionally 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 is produced in relation to the skin environment.

5. Bioartificial skin according to claim 4 with 0.1 to 10 x 10 ⁴ and in particular about 1 x 10 ⁴ melanocytes / cm ² dermis

Chew type II.

6. A method for producing bioartificial skin according to one of claims 1 to 5, 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) from the body c) the fibroblasts obtained in step b) are genetically modified so that they express at least one angiogenic protein, a neurotrophic protein and at least one protein stabilizing the epidermo-dermal junction, and d) the fibroblasts obtained in step c) to one porous matrix can be applied.

7. The method according to claim 6, wherein the fibroblasts obtained in step c) include PDGF-A (platelet derived growth factor A), PDGF-B (platelet derived growth factor B), VEGF (vascular endothelial growth factor), bFGF (basic express fibroblast growth factor), TGFbeta (tumor growth factor beta), angiopoetin 1 and angiopoetin selected angiogenic protein.

8. The method according to any one of the preceding claims, wherein in addition to the isogenic or autologous fibroblasts - isogenic or autologous keratinocytes and melanocytes - in such an amount and in such a ratio that a natural skin tone is produced.

9. Use of a bioartificial skin according to one of claims 1 to or of a bioartificial skin produced by a method according to one of claims 6 to 8 for covering wounds.