WO2015173206A1

WO2015173206A1 - Artificial dermis, artificial skin, methods for their preparation and their uses

Info

Publication number: WO2015173206A1
Application number: PCT/EP2015/060395
Authority: WO
Inventors: Santos MERINO ÁLVAREZ; Aritz RETOLAZA MUÑOA; Sabino AZCÁRATE LETURIA; José Luis JORCANO NOVAL; Almudena HOLGUÍN FERNÁNDEZ; Blanca DUARTE GONZÁLEZ; Álvaro MEANA INFIESTA; Eva GARCÍA PÉREZ; Sara GÓMEZ LLAMES
Original assignee: Fundacion Tekniker; Centro De Investigaciones Energéticas, Medioambientales Y Tecnológicas (Ciemat); Centro Comunitario De Sangre Y Tejidos De Asturias (Ccst)
Priority date: 2014-05-13
Filing date: 2015-05-11
Publication date: 2015-11-19
Also published as: ES2551143A1; ES2551143B1

Abstract

The present invention relates to an artificial dermis comprising a first gel layer of clotted blood plasma comprising platelets and dermal cells, and a second gel layer of clotted blood plasma comprising platelets and dermal cells, and a polymer net situated between the first and the second gel layers. The invention also relates to an artificial skin comprising said artificial dermis with at least a layer of epithelial cells. The invention discloses methods for the preparation of the artificial dermis and the artificial skin, as well as their uses in the preparation of a medicament, in particular for the treatment of re-epithelialization of a patient in need thereof.

Description

ARTIFICIAL DERMIS. ARTIFICIAL SKIN. METHODS FOR THEIR PREPARATION

AND THEIR USES

FIELD OF THE INVENTION

The present invention is related to an artificial dermis comprising gel layers made from clotted blood plasma comprising platelets, and embedded dermal cells, and a polymer net between gel layers. The invention also relates to an artificial skin comprising said artificial dermis and further comprising epithelial cells which are seeded onto the surface of this artificial dermis. Both the artificial dermis and the artificial skin are especially useful for the treatment for example of major burns, chronic ulcers, in tests for controlling sensitivity to different products, etc. By using genetically altered cells, the artificial dermis and the skin can also be used as a vehicle for gene therapy.

BACKGROUND OF THE INVENTION

The skin is a tissue made up of two parts, the epithelium or external part and the dermis, or the internal part on which the epithelium is positioned. These two parts have clearly different characteristics. There is practically no extracellular tissue in the epidermis, whereas this component is clearly predominant over cells in the dermis.

The skin is a tissue that can be reconstructed by tissue engineering techniques (Parenteau N, Sci Am, 280: 83 84, 1999). In these techniques, in general, the cellular component is generated "ex vivo" by cell cultivation techniques. These techniques start with a small number of cells taken from a small skin biopsy, which are then cultured or "expanded" to obtain a large number of cells in a short time. These "ex vivo" expanded cells can be used to build large areas of artificial skin. The extracellular matrix cannot be produced by cell cultivation, but it is previously designed and manufactured outside the body. The extracellular matrix has to be capable of providing structures that facilitate the adhesion of the previously cultivated dermal cells and stimulating the normal growth of these cells. On this artificial matrix, the cells begin to manufacture the normal proteins that make up the natural dermal matrix, and at the same time, they slowly degrade the original structure, so that over time this artificial matrix is replaced by a true extracellular matrix identical to a natural one. Previously cultivated epithelial cells (keratinocytes in the case of skin) can be seeded onto this artificial matrix, where with new cell cultivation techniques, these cells are capable of generating a structure that is very similar to the normal epithelium from which they originated. In other words, one of the key factors in skin tissue engineering is the design of dermal matrices that imitate the body's natural conditions as much as possible, and where the cells introduced are capable of starting a complex process, the purpose of which is to develop a structure as similar as possible to natural skin.

The other key factor in skin tissue engineering is the capacity of the dermal matrix to facilitate the growth of the cells that are seeded on it. The development of dermal matrices that encourage the growth of both dermal and epidermal cells would mean that it would be possible to cultivate large areas of artificial skin from a minor biopsy. This is especially important when the artificial skin is to be used for treating major burns, where up to 90 - 95% of the body's total surface area has to be replaced as quickly as possible, using the small areas of healthy skin that remain on the patient. The lack of dermal matrices capable of generating these large areas of artificial skin from minor biopsies is one of the limitations of previously described dermal matrices (Sheridan R and Tompkins, Burns 25: 97 103, 1999).

There are several artificial dermis models. For example, EP 1375647 B1 discloses a dermal matrix for use in generating an artificial dermis. In this model the dermal matrix is prepared as follows: a solution of blood plasma comprising platelets and further containing "ex-vivo" cultured dermal cells is prepared; said solution is then clotted to form a gel layer upon which keratinocytes are seeded and cultured to complete or partial confluence. The so obtained gel layer with the cultivated keratinocytes can be then fixed to a solid support to enable transportation without losing its integrity or breaking. As disclosed in EP 1375647 B1 said solid support is fixed to the upper surface of the artificial dermis where the keratinocytes are seeded. Said support can be in general any solid stand for example of polylactic-polyglycolic acid; or it can be a silicon membrane in which case it may be fixed to the gel layer by an organic glue such as fibrin; or it can also be a gauze, steeped in vaselin or not, which can be fixed by an inert inorganic glue of clinical use or another mechanical system. According to EP 1375647 B1 , the dermal matrix disclosed allows the preparation of an artificial dermis suitable for use in skin grafts, and has been successfully used in the treatment of epidermolysis bullosa.

However the dermal matrix suffers from drawbacks and its application in therapy for example is not fully satisfactory. One of these disadvantages is that it requires extremely careful handling to ensure that the matrix sheet remains intact and suitable for implant. Other artificial skin substitutes include the use of polymer structures as a scaffold for the culture of artificial skin. The Dermagraft™ dermal replacement for use in treatment of diabetic foot ulcers is manufactured by the seeding of fibroblasts onto a polygalactin mesh. Additionally the culture of keratinocytes on a polymer surface is also known, either alone (WO99/064563) or in conjunction with fibroblasts (WO01/066695).

In view of the above, there is still the need in the state of the art of providing an alternative artificial dermis and an alternative artificial skin which overcome at least some of the drawbacks associated to the artificial dermis and skins of the state of the art, which show improved robustness and sturdiness, are easier to manipulate, and enable an efficient, improved treatment of patients requiring re-epithelialization.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the invention relates to an artificial dermis comprising a first gel layer of clotted blood plasma comprising platelets and dermal cells, a second gel layer of clotted blood plasma comprising platelets and dermal cells, and a polymer net situated between the first and the second gel layers.

The artificial dermis, hereinafter also referred to as the artificial dermis of the invention, has a defined thickness from about 0.15 cm to about 5 cm, preferably from about 0.3 cm to about 1 .0 cm. The artificial dermis of the invention may be generated in a wide variety of sizes ranging, for example, between about 1 cm² to about 100 cm², more preferably from about 10 cm² to about 90 cm², more preferably still from about 30 cm² to about 80 cm². The artificial dermis may also present different forms.

The artificial dermis of the invention preferably has one or more substantially linear edges, allowing for the easy alignment of individual sections of artificial dermis together during implant procedures. The dermis has a degree of elasticity, and so the edges need not be exactly linear to allow mutual alignment, as the skilled person readily appreciates.

The polymer net of the artificial dermis consists of a polymeric sheet comprising pores. The pores can present different shapes and sizes depending from the method of their manufacturing. Pore shapes can be very different ranging from square shape to circular shape, including elliptical pores for example, without limitation. Pore sizes can also vary within a broad range, and are typically between 10 μηι and 1000 μηι of diameter in the case of the circular pores, or present a side of between 10 μηι and 1000 μηι in case of square pores.

Since the artificial dermis is intended for its use in the treatment of a patient in need thereof, the polymer is preferably biocompatible and biodegradable. The term "polymer" is intended to include any compound which is comprised of repeating units of monomers, and thus includes polymers comprised of a single monomer unit, and co-polymers comprised of at least two different monomers, for example, 2, 3, 4 or more.

According to a particular embodiment, said polymer maybe selected from the group consisting of poly(methyl methacrylate), polylactide, including poly (L-lactide) (LPLA) and poly-(dl-lactide) (DLPLA), polyglycolide (PGA), poly(lactic-co-glycolic acid) (PLGA), polycaprolacton, (such as poly(e-caprolactone) (PCL)), polydioxanone (PDO), poly(glycolide-co-trimethylene-carbonate), polyglyconate and their mixtures. According to a preferred embodiment the polymer is PLGA. When PLGA is used, it is more preferred that at least 50% of the monomer units are lactic acid. In an alternative embodiment, the ratio of lactic to glycolic acid monomer units ranges between about 1 :1 to about 17:3. Table 1 provides several examples of polymers, in general, and several examples of PLGA copolymers having suitable ratios of lactic acid to glycolyic acid units, for example 85/15; 75/25; 65/35; and 50/50. These ratios may similarly be applied for other co-polymers.

The artificial dermis of the invention can comprise one or more further gel layers and one or more polymer nets in addition to the artificial dermis as above disclosed of a first gel layer, a polymer net, and a second gel layer. In these embodiments of the artificial dermis of the invention, the alternating disposition of the gel layer/ polymer net/ gel layer is maintained. In this sense examples of possible artificial dermis of the invention, present the following repetitive structures:

(i) gel layer/polymer net/gel layer/polymer net, or

(ii) gel layer/polymer net/gel layer/polymer net/gel layer, or

(iii) gel layer/polymer net/gel layer/polymer net/gel layer/polymer net, and so on and so forth.

Accordingly the artificial dermis of the invention includes 2, 3, 4, 5, or several gel layers. Accordingly the artificial dermis of the invention includes 2, 3, 4, or even more polymer nets.

The artificial dermis of the invention is more robust and sturdy than the dermis disclosed in EP 1375647 B1 , and this is not only due to the mechanical support and consistency provided by the polymer net, but also due to the fact that the cells communicate through the pores of the net.

The gel layers of the artificial dermis may be same or different in respect of their composition, for example in respect of the type and origin of their dermal cells. When the artificial dermis comprises at least two polymeric nets, the polymeric nets can also be of the same or different type, in respect of their composition and their characteristics.

In this sense, various thicknesses of polymer net may be used, and thicknesses may vary between different polymer nets within the same artificial dermis. Examples of suitable thicknesses are those ranging from about 10 micrometres to about 100 micrometres, preferably from about 10 micrometres to about 40 micrometres, and even more preferably from about 25 micrometres to about 35 micrometres.

The gel layers of the artificial dermis of the invention comprise dermal cells such as mesenchymal stem cells (MSCs), endothelial cells, or fibroblasts. According to a preferred embodiment the dermal cells are fibroblasts. According to the invention the dermal cells present in the first and the second gel layer, and in any other additional gel layers, where appropriate, can be of the same or of different kind and origin.

The blood plasma used to prepare the gel layer comprising platelets and the dermal cells may be derived from any animal source. According to a particular embodiment blood plasma and dermal cells are of human origin. Both blood plasma and dermal cells may be derived from a suitable donor subject (i.e. they may be allogeneic with respect to the patient who will be treated with the artificial dermis or the artificial skin of the invention) or they may autologous in origin (i.e. they may be derived from the same patient who is to be treated).

Suitable sources of dermal cells include but are not limited to cell lines and biopsies e.g. skin. The dermal cells are most preferably isolated from donor subject or patient tissue and are then cultured or expanded "ex-vivo" prior to their use in the preparation of the gel layers of the artificial dermis. Methods for the culture or expansion of cells are well known in the art. For example in one embodiment of the invention fibroblasts can be isolated from a dermal sample of a donor subject or a patient in need of treatment. The dermal sample is treated with collagenase thereby releasing the fibroblasts, which are then isolated (e.g. by centrifugation) and then cultivated "ex-vivo". MSCs can be obtained from any suitable source of connective tissue from any suitable animal, but will preferably be of human origin. It is preferred that MSCs are obtained from non-pathological mammalian sources, preferably post-natal (e.g. rodent; primate). In one preferred embodiment, the MSCs are obtained from a source of connective tissue, such as, but not limited to, the stromal fraction of adipose tissue, hyaline cartilage, bone marrow or skin. Preferably, MSCs are obtained from non-pathological, post-natal, human stromal adipose tissue.

Suitable sources of fibroblast cells include, but are not limited to, cell lines and biopsies e.g. fibroblasts cultivated from foreskins obtained from phimosis operations and/or dermal fibroblasts from healthy adults, obtained, for example, from a skin biopsy; autologous fibroblasts may, for example, be obtained from a skin biopsy extracted from the same subject who is to be treated with the artificial dermis or artificial skin of the invention.

By way of an example different lines of human fibroblasts will be obtained from human foreskins obtained after programmed phimosis surgery or from a skin biopsy. The sample is collected in a transport medium (Dulbecco's modified eagle medium (DMEM), 10% bovine fetal serum albumin, 100u/ml penicillin, 100μg/ml streptomycin). In the laboratory, the sample is washed, preferably three times in sterile phosphate buffered saline (PBS), and carefully cut into pieces. It can be introduced into 30 ml of 0.05% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) solution while stirring at 37 ⁵C. Every 30 minutes, the trypsin is collected and changed for fresh trypsin. The trypsin is then neutralized by adding complete culture medium (e.g. DMEM, 10% bovine fetal serum). The operation is repeated until no more cells are obtained. The cells obtained are placed in a culture dish at a density of 100,000 cells per cm² of culture surface. The medium is changed every 72 hours until the cells are confluent. Upon confluence, these cells may be tripsinated and secondary cultures may be made in a proportion of two culture dishes for each culture dish in the previous phase. When the cells show a single layer of cells similar to fibroblasts, part of them may be frozen, using a common technique, and stored in cryovials in liquid nitrogen. The ideal passes for the use of these fibroblasts are between the 4^th and the 12^th. When human fibroblasts from the same patient who is to be treated are used in the artificial dermis, it is possible to proceed in the same way. For example, a patient's skin biopsy may be processed as described above. Once the cells are obtained, part of them may be cultivated, for example, in DMEM 10% bovine fetal serum at a density of about 100,000 cells per cm². The corresponding sub-cultures can be propagated until a sufficient number of human fibroblasts are obtained to manufacture the artificial dermis that the patient needs.

The cultivated human fibroblasts may then be tripsinated, counted and re-suspended in culture medium, preferably, for immediate use in the preparation of the artificial dermis of the invention.

Preferably, the dermal cells are provided in each of the gel layers as a confluent or partially confluent layer of cells. The degree of confluence may vary between 50% and 100% confluence, for example, at least 60%, or at least 70%, or at least 80%, or at least 90% or 100% confluence. The initial concentration of dermal cells within the gel can vary considerably. In general a concentration of not less than 500 dermal cells/cm2 of gel surface is recommended, although it can be greater, but preferably less than 4,000 cells/cm2.

The blood plasma comprising platelets for use in the preparation of the gel layers of the artificial dermis of the present invention may be prepared by any means known in the art, as the skilled reader will appreciate, including the preparation from total or whole blood. Blood may be collected by standard means, such as, vein puncturing. It is preferred that the collection of whole blood is carried out in the presence of anti-clotting agents suitable for use in clinical settings, preferably agents that act by means of the chelation of the ionized calcium concentration in total blood (for example, sodium citrate, EDTA, or sodium heparin). The blood may be collected and/or stored in containers known in the art for such purposes, such as haemotherapy bags or, for small amounts, small containers of the Vacutainer type. Blood plasma comprising platelets may then be isolated from the extracted whole blood by means known in the art such as centrifugation at low speed (approximately 1500 rpm, preferably for 5 minutes; around 160g) to obtain a blood plasma that is very rich in platelets, or at high speed (between 2900-3000rpm, preferably for 10 minutes; around 400g) to obtain a product with a lower concentration of such platelet cells. In one particular embodiment of the invention, the blood plasma may be enriched for growth factors, such as those of platelet origin. The person skilled in the art is able to enrich for platelet factors during plasma preparation by selecting appropriate centrifugation techniques, using his common general knowledge. Alternatively, the blood plasma can also be extracted from total blood by means of plasmapheresis.

Once the blood plasma has been isolated, it may be used directly to form a gel layer according to the invention or it may be frozen, for example, at -20 ⁵C for later use. Before being stored, the plasma can also be treated to ensure that any microorganisms are killed e.g. by treatment with methylene blue. This is particularly important if the plasma is to be used in the preparation of an artificial dermis or skin for the treatment of a non-donor (i.e. allogeneic) subject. The blood plasma used in the preparation of artificial dermis of the present invention comprises fibrinogen. This is preferably at a minimum level of about 1 .67 mg/ml, more preferably of at least about 2 mg/ml. However, the level may also be as low as 0.4 mg/ml, or as high as 4 mg/ml. As an example, between about 2 ml to about 30 ml of blood plasma may be utilized in the preparation of a gel layer having a surface area of about 70 to about 90 square centimeters.

A detailed description of methods for the manufacture of dermal layers from blood plasma and dermal cells is contained in EP 1375647 B1 from paragraph [0007] to [0044] which content is herein incorporated by reference.

In a further aspect the invention relates to a method for the preparation of the artificial dermis of the invention, which comprises the following steps:

(0 Providing a first and optionally also a second reaction solution comprising blood plasma with platelets and dermal cells, Clotting at least a portion of the first reaction solution to render a first gel layer of clotted blood plasma comprising platelets and dermal cells,

(iii) Applying a polymer net onto said first gel layer,

(iv) Clotting a further portion of the first reaction solution or at least a portion of the second reaction solution comprising blood plasma with platelets and dermal cells, onto the surface of the said polymer net to render a second gel layer of clotted blood plasma comprising platelets and dermal cells.

In the first step of the method, a first reaction solution is prepared comprising blood plasma with platelets and dermal cells. Optionally a second reaction solution is prepared if an artificial dermis is desired with different gel layers. A reaction solution may be prepared by combining or mixing blood plasma comprising platelets, obtained as above disclosed, with dermal cells obtained and cultured as above described, such that the dermal cells are dispersed or suspended within the plasma. Different dermal cells in respect of their type and origin may be used to prepare a reaction solution as well.

The reaction solution may be diluted with appropriate agents for example but not limited to saline solution.

In the second step of the method a first gel layer is formed from a portion of the first reaction solution. This portion of the reaction solution is preferably placed within a container or mold that is suitable for the formation of an essentially planar gel layer, and is then clotted by treatment under suitable conditions such that the solution is converted into a gel. Methods for the preparation of a gel from a reaction solution are well known in the art, and include treatment with clotting agents, such as, but not limited to calcium salts (for example, a 1 % calcium chloride, dissolved for example in 0.9% sodium chloride) and exogenous thrombin.

The gel is formed from the fibrinogen present in the plasma, by the action of exogenous human or bovine thrombin and calcium ions. In an alternative embodiment, the clotting process may be enabled by the addition of non-protein agents.

Blood derived plasma, unlike fibrinogen concentrates well known in the state of the art, provides all the components of the clotting cascade, including the thrombin that is present in the form of its (inactive) precursor, prothrombin. The gellif ication, and therefore the production of the gel layer of the artificial dermis, can be performed by stimulating the intrinsic clotting pathway by the addition of calcium in the presence of phospholipids of platelet origin. It is particularly preferred that the gel is formed at least in part by the conversion of fibrinogen into fibrin that then provides the main structural component of the gel. Other structural components of the gel may include, for example, plasmatic fibronectin.

Furthermore, other proteins that are present in blood plasma e.g. albumin, globulins, growth factors, plasminogen, etc., are involved in the gel formation and stability and in the growth of the cells that are cultivated in the gel.

Optionally, the formation of the gel by means of clotting agents may be aided by the addition of anti-fibrinolytic agents to the reaction solution, such as, but not limited to aprotonin, tranexamic acid (TXA), and ε-aminocaproic acid (EACA). The use of such agents is preferred wherein the reaction solution comprises 2 mg/ml or less of fibrinogen.

In the third step a polymer net is applied onto said first gel layer by manual means. The polymer net has been previously obtained by conventional methods of the art. In a particular embodiment the net is prepared by hot-embossing. In this particular case a silicon stamp fabricated as referenced in (1 ) ["The influence of stamp deformation on residual layer homogeneity in thermal nanoimprint lithography". S. Merino, A. Retolaza, A. Juarros, H. Schift. Microelectronic Engineering 85 (2008) 1892] is used presenting the inverse of the 3D patterning to be stamped onto the polymer sheet. The stamp and polymer sheet are heated at a temperature above the glass transition temperature of the polymer and a force is applied to the stack at this temperature. The polymer flows, and occupies the 3D mold motifs. The stack is then cooled to room temperature and demolded (2) ["The use of automatic demoulding in Nanoimprint Lithography processes". S. Merino, H. Schift, A. Retolaza, T. Haatainen. Microelectronic Engineering 84 (2007) 958]. A polymer structure is obtained with a stamped 3D pattern. The sheet with the 3D pattern is then introduced in a vacuum chamber. An oxygen-plasma is then applied to the polymer sheet so as to achieve holes through the polymer sheet while the patterning is kept on the whole surface. The artificial dermis and artificial skin of the present invention may be removed from the mould and applied directly to a patient.

It is particularly preferred that the polymer net is applied prior to the completion of the previous gellif ication step, but subsequent to the addition of any gellif ication or clotting agents (i.e. the polymer net is so applied to a semi-solidified surface before completion of the gellif ication process). In one embodiment of the method, wherein an exogenous agent or agents (e.g. thrombin and/or calcium) is added to the reaction solution to induce the formation of a gel, the polymer net is applied between about 5 minutes and 30 minutes after to the addition of said agent or agents (for example, 10, 15, 20, or 25 minutes beforehand). In the fourth step a second gel layer is formed either from a further portion of the same reaction solution employed in the first gel layer or from a different reaction solution. This portion of a reaction solution is applied to the surface of the polymer net and is then clotted by treatment under suitable conditions such that the solution is converted into a second gel layer. Clotting may be carried out by any of the means above disclosed.

According to a particular embodiment of the method, step (iv) and optionally also step (iii) may be repeated in this order to provide an artificial dermis with the structures above disclosed. Steps (iii) and (iv) can be thus repeated 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 times to provide an artificial gel comprising a plurality of gel layers and polymer nets.

Further to step (iv) the method for preparing the artificial dermis of the invention may comprise storing the artificial dermis under appropriate conditions until confluence or pre-confluence of the dermal cells is achieved within said artificial dermis. In one embodiment of the method, this may take between about 8 to about 12 days. Accordingly the present invention provides an artificial dermis of the invention having dermal cells within having at least 60%, 70%, 80%, 90% or even 100% confluence.

The artificial dermis of the present invention may be stored in an appropriate culture medium at a maximum of 37 ⁵C prior to application to a patient.

In a further aspect the invention relates to an artificial skin, hereinafter also referred to as the skin of the invention. The skin of the invention comprises the artificial dermis of the present invention and at least one layer of epithelial cells.

Said at least one layer of epithelial cells may be in principle situated at any location between any gel layer and a contiguous polymer net of the artificial dermis. According to a preferred embodiment, said at least one layer of epithelial cells is placed on the outside surface of the artificial dermis. According to a particular embodiment said outside surface of the dermis is of a gel layer. According to another particular embodiment, said outside surface is of a polymer net.

The epithelial cells of the layer may be derived from any suitable animal source but are most preferably of human origin. The epithelial cells may be derived from a suitable donor subject (i.e. they may be allogeneic with respect to the patient treated with the artificial dermis or the artificial skin of the invention) or they may autologous in origin (i.e. they may be derived from the same patient who is to be treated).

The epithelial cells are most preferably isolated from donor subject or patient subject tissue and cultured or expanded "ex-vivo" prior to their use in the preparation of the artificial skin or epidermis as disclosed below. Suitable sources of epithelial cells include but are not limited to cell lines and biopsies. Methods for the culture of epithelial cells are well known in the art and any suitable method may be used. This may include, for example, cell culture in the presence of feeder cells (such as, but not limited to, irradiated 3T3-Swiss cells), or using specialized cell culture media such as, but not limited to, low calcium media.

In another aspect the invention relates to a method for preparing the artificial skin of the invention. This method comprises the following steps:

(i) preparing an artificial dermis according to the present invention,

(ii) seeding epithelial cells on at least the surface of one gel layer or polymer net; and

(iii) cultivating the cells until a layer of epithelial cells is produced.

Preferably the epithelial cells are seeded at a density of between about 1 ,500 and about 15,000 cells per square centimeter of surface area of the artificial dermis (e.g. 2,500; 5,000; 7500; 10,000; 12,500 cell per cm²).

Epithelial cells placed on the surface of the artificial dermis will adhere and proliferate under appropriate cultivating conditions, as well as the dermal cells within the artificial dermis gel, during step (iii), the ephitelial cells forming a layer.

Accordingly in a particular embodiment, step (iii) is carried out by storing the artificial skin obtained from step (ii), under conditions suitable for the cultivation of the epithelial cells, preferably in a culture medium or other suitable liquid. The person skilled in the art will be aware of suitable conditions for the cultivation of these cells and may include the use of appropriate culture media and/or a controlled environment for putting step (iii) in practice.

Storing is carried out under appropriate conditions until confluence or pre-confluence of the dermal cells and the epithelial cells is achieved on the surface of and/or within said artificial dermis. In one embodiment of the method, this may take between about 8 to about 12 days. Accordingly the present invention provides an artificial skin of the invention having epithelial cells and dermal cells on the surface and/or within the artificial dermis having at least 60%, 70%, 80%, 90% or even 100% confluence. The artificial dermis and the artificial skin of the present invention may be stored at a maximum of 37 ⁵C in an appropriate culture medium prior to application to a patient in need thereof. The artificial dermis and artificial skin of the present invention can be used in therapy. In this respect one aspect of the invention relates to the use of the artificial dermis or the artificial skin of the invention for the preparation of a medicament. In the present context a medicament refers to a preparation comprising the artificial skin or the artificial dermis of the invention, which is suitable for its application as a graft on a patient in need of a treatment therewith.

According to a particular embodiment the use of the artificial dermis or artificial skin is for the preparation of a medicament for the treatment of re-epithelialization of a patient in need thereof.

In a further particular embodiment said re-epithelialization is of a mucosal, dermal or epidermal tissue of a patient.

Thus, the artificial dermal and artificial skin of the invention, are for use in the treatment of a defect or disorder of the dermal, epidermal or mucosal tissues. The artificial dermis and artificial skin of the present invention may be used as a replacement or substitute for patient mucosal, dermal or epidermal tissues, including, but not limited to, those listed in Table 2. The artificial skin used as such a replacement or substitute preferably comprises a simple non-stratified epithelium but may alternatively comprise an appropriate epithelium as described in Table 2.

The invention encompasses the artificial dermis or artificial skin of the invention for use as a surgical graft, for example in the replacement of mucosal, dermal or epidermal tissues including, but not limited to, those disclosed in Table 2. In some embodiments, the invention encompasses a method of using the artificial dermis or artificial skin in a surgical procedure, such that the surgical graft is applied directly to the graft location of the subject.

The artificial dermis and artificial skin of the present invention are also for use in repairing, treating, and/or preventing skin conditions, disorders or diseases, such as symptoms of burns, including but not limited to first-degree burns, second-degree burns and third degree burns; symptoms of wounds, including but not limited to epidermal wounds, skin wounds, chronic wounds, acute wounds, external wounds, internal wounds and congenital wounds (e.g., epidermolysis bullosa); and symptoms of ulcers including, but not limited to, pressure ulcers and diabetic ulcers.

The process of the present invention is illustrated below by reference to the examples which are intended to be only illustrative and are not construed to limit the present invention in any way.

EXAMPLES

Table 1 : Polymers suitable for use in artificial dermis of the present invention.

a: Degradation time in vivo. This time depends on the final substrate size. b: PLGA refers to the copolymer poly(lactic-co-glycolic acid) and ab/cd shows the composition of each polymer in the copolymer polylactic/polyglycolic.

c: Both polymers are usually referred as poly-lactide polymers.

Table 2: Uses of artificial dermis or artificial skin.

System ^Tissue iJ Epithelium* ^{, (}

Digestive ducts of submandibular Stratified columnar

alands

attached ainaiva Stratified squamous, keratinized dorsum of tonaue Stratified squamous, keratinized hard palate Stratified squamous, keratinized oesophaqus Stratified squamous, non- keratinised

small intestine Simple columnar, non-ciliated larae intestine Simple columnar, non-ciliated rectum Simple columnar, non-ciliated anus Stratified squamous,

Integumentary skin Stratified squamous, keratinized sweat aland ducts Stratified cuboidal

mesothelium of bodv Simple squamous

cavities

Female cervix (endocervix) Simple columnar

reproductive cervix (ectocervix) Stratified squamous, non- keratinised

vaaina Stratified squamous, non- keratinised

labia maiora Stratified squamous, keratinised

Respiratory oropharynx Stratified squamous, non- keratinised

larvnx Pseudostratified columnar, ciliated larvnx - True vocal cords Stratified squamous, non- keratinised

trachea Pseudostratified columnar, ciliated

Visual cornea Stratified squamous, non- keratinised

Nasal nose Pseudostratified columnar

Urinary ureter Transitional

membranous urethra Pseudostratified columnar, non- ciliated

penile urethra Pseudostratified columnar, non- ciliated

external urethral orifice Stratified squamous

Claims

1 . An artificial dermis comprising a first gel layer of clotted blood plasma comprising platelets and dermal cells, and a second gel layer of clotted blood plasma comprising platelets and dermal cells, and a polymer net situated between the first and the second gel layers.

2. An artificial dermis according to claim 1 wherein the polymer net comprises pores having an average diameter of between 10 μηι and 1000 μηι.

3. An artificial dermis according to claim 2, wherein the polymer net is made from interwoven polymer strands each strand having an average diameter of 10 μηι to 1000 μπι.

4. An artificial dermis according to any one of claims 1 to 3, wherein said polymer is biocompatible and biodegradable.

5. An artificial dermis according to claim 4 wherein said polymer is selected from the group consisting of poly(methyl methacrylate), polylactide, polyglycolide, poly(lactic- co-glycolic acid), polycaprolacton, polydioxanone, poly(glycolide-co-trimethylene- carbonate), polyglyconate and their mixtures.

6. An artificial dermis according to claim 5, wherein said polymer is poly(lactic-co- glycolic acid).

7. An artificial dermis according to any one of previous claims, wherein the dermal cells of the first and the second gel layer can be of the same or of different kind.

8. A method for the preparation of an artificial dermis according to any one of the previous claims, comprising the steps of:

(i) Providing a first reaction solution and optionally a second reaction solution both comprising blood plasma with platelets and dermal cells,

(ii) Clotting at least a portion of the first reaction solution to render a first gel layer of clotted blood plasma comprising platelets and dermal cells,

(iii) Applying a polymer net onto said first gel layer,

(iv) Clotting a further portion of the first reaction solution or at least a portion of a second reaction solution comprising blood plasma with platelets and dermal cells, onto the surface of the said polymer net to render a second gel layer of clotted blood plasma comprising platelets and dermal cells.

9. An artificial skin comprising an artificial dermis according to any one of claims 1 to 7, and at least a layer of epithelial cells.

10. An artificial skin according to the previous claim, wherein the at least one layer of epithelial cells is placed on the outside surface of the artificial dermis.

1 1 . A method for preparing an artificial skin according to claims 9-10, which comprises:

(iv) preparing an artificial dermis according to claims 1 to 7,

(v) seeding epithelial cells on at least one gel layer or polymer web; and

(vi) cultivating the cells until a layer of epithelial cells is produced.

12. Use of an artificial dermis or artificial skin according to the previous claims 1 to 7 or 9-10, for the preparation of a medicament.

13. Use of the artificial dermis or artificial skin according to claim 12, for the preparation of a medicament for the treatment of re-epithelialization of a patient in need thereof.

14. Use of the artificial dermis or artificial skin according to claim 13, wherein the re- epithelialization is of a mucosal, dermal or epidermal tissue.