WO1999045972A2

WO1999045972A2 - Skin substitute and a topical composition for skin wounds

Info

Publication number: WO1999045972A2
Application number: PCT/NL1999/000124
Authority: WO
Inventors: Reinier Theodorus Jacobus Van Leeuwen; Esther Middelkoop
Original assignee: Stichting Voor De Technische Wetenschappen
Priority date: 1998-03-09
Filing date: 1999-03-08
Publication date: 1999-09-16
Also published as: WO1999045972A3; NL1008531C2

Abstract

The invention relates to a skin substitute which is suitable for the promotion of the healing of skin wounds. The skin substitute according to the present invention comprises a degradable, hydrophilic matrix, which matrix is permeable for cells, contituted by a collagen and elastin comprising biocompatible matrix material, wherein the matrix of the skin substitute comprises an inhibitor of a protease. Furthermore, the invention relates to a composition showing a protease inhibiting action for the topical application on skin wounds, which composition comprises a protease inhibitor and a pharmaceutically acceptable carrier.

Description

Skin substitute and a topical composition for skin wounds

The present invention relates to a skin substitute suitable for promoting the healing of skin wounds, which skin substitute comprises a degradable and hydrophilic matrix, which matrix is permeable for cells and which matrix comprises a collagen and elastin comprising matrix material .

Such a skin substitute is known in the art and aims to enhance the quality of healing of skin wounds over the full thickness of the skin and covering a large area such as burns (see for instance De Vries et al . , Wound Rep. Reg. 1994; 2; 37-47). The known substitute, based on an artificial matrix of collagen and elastin, may, if so desired, contain bodies' own skin cells, thus to prevent forming of scar tissue because of contraction of the wound (wound contraction) . Thus it is contemplated to obtain a neo-dermis which approaches the natural thickness and structure of the skin and shows decreased wound contraction.

By using such a known skin substitute in for example burns inflammation of the wound and (hypertrophic) scar formation keeps occurring. Also wound contraction still occurs . Thus a known skin substitute results in badly healing burns. Badly healing burns continue to be a big problem. Wound contractions result in a serious disruption of the natural position and of the ease of movement of the patient. Patients suffering from burns are therefore forced to undergo surgery and treatment repeatedly. Moreover, patients with burns are marked for life, and they often become socially isolated. The object of the present invention is to overcome these disadvantages .

This object is achieved according the present invention in that the matrix of the skin substitute comprises an inhibitor of a protease. It appeared that use of such a skin substitute resulted in decreased wound contraction, scar formation and inflammation.

Further it appeared that a skin substitute contain- ing such a protease inhibitor plays a favourable cell migration controlling role, and (myo) fibroblast populations can be restrained.

Preferably the matrix is physically crosslinked (for example by heating and cooling) . Herewith harmful cytotoxic effects caused by chemically crosslinking on the ingrowth of cells is avoided.

Generally the skin substitute according to the present invention may be used for all types of skin wounds , in particular for wounds having a great area . In particular the skin substitute is used for the promotion of the healing of burns, chronical wounds such as leg ulcers or diabetic ulcers and surgical wounds, such as occurring in scar corrections and surgically removing of tattoos . The protease inhibitor may be included in the skin substitute before the application of the skin substitute, but may also be applicated in the form of an ointment, wound dressing, gel or any other appropriate carrier.

According to a further aspect of the present inven- tion the protease inhibitor is an inhibitor of an extracellular protease.

According to a further aspect of the present invention the protease inhibitor is chosen from the group consisting of inhibitors of plasminogen activator and metalloprotease or a mixture thereof.

Useful plasminogen activators to be inhibited are amongst others tissue type plasminogen activator and urokinase; useful metalloproteases to be inhibited are amongst others elastase, gelatinase and collagenase. According to an other aspect of the present invention the protease inhibitor is an inhibitor of a protease degrading the matrix.

Hereby the prematurely degrading of the matrix is prevented. According a further aspect of the present invention the matrix has a pore size in the range of 5 - 100 μm.

Herewith it is obtained that the matrix is permeable for cells, which cells may be added on purpose to the matrix for the promotion of the healing of the skin wound.

According to a further aspect of the present invention the matrix comprises a further cell migration inhibiting compound. Herewith the migration of cells from outside to the wound area is reduced.

According to an other aspect of the present invention the skin substitute comprises autologous cells, preferable derived from the dermis, subcutane fat tissue or from a crust of a burn.

Herewith a shortage of skin constituents is prevented, whereby scar tissue formation and wound contraction are decreased. The autologous cells which are derived of the crust of a burn will in general be derived from the inwardly facing side of crust of the burn.

The present invention relates moreover to a composition having a protease inhibiting action for the topical application on skin wounds, which composition comprises a protease inhibitor as a cell migration inhibiting compound and, optionally, a pharmaceutical acceptable carrier.

The composition may be applied in the form of an ointment, wound dressing, gel or each other appropriate carrier, but preferably as a fluid and sprayable composition, directly on the skin wound, if so desired provided with a skin substitute. The advantage of the external application of the composition according to the present invention is that the concentration and the interval in which the composition is applied, may be easily adjusted according to the need. Moreover, the healing wound may be easily inspected from time to time.

According to a further aspect of the present invention the protease inhibitor is an extracellular protease inhibitor. According to an other aspect of the present invention the protease inhibitor is chosen from the group consisting of inhibitors of plasminogen activator, tissue type plasminogen activator, urokinase, metalloprotease, elastase, gelatinase and collagenase.

Herewith a satisfactory result is obtained.

The present invention further relates to a method for the production of a skin substitute according to the present invention, wherein autologous cells are added to the skin substitute.

The autologous cells may be added to the skin substitute, directly before the skin substitute is applicated on the wound (for instance by carefully dripping of a cell suspension on one or both sides of the skin substitute) , but may be preceded by a culture period. This culture period ranges in general from 4 hours to 2 weeks .

The present invention relates moreover to the use of a protease inhibitor for cell migration control in a skin substitute, in particular to decrease the number of myofibroblasts .

Hereafter the present invention is further illustrated by several realization examples.

Example 1 The skin of a young Yorkshire pig (± 40 kg) was washed with iodine and ethanol . Subsequently the epidermis (0,2 mm thick) was removed with an electro dermatome (Aesculap GA630) , and the derma (1,2 mm) was harvested with an electro dermatome . This derma was cut into pieces of lxl mm in a sterile way, and each piece was placed in a small dish (Costar TM, 24-wells multiwell tissue culture dishes) . The pieces were dried during 10 minutes (room temperature), whereby they were attached to the wall.

Subsequently a tissue culture medium (Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum, 0,5 ml/well) was added to a series of 12 pieces, the medium containing a protease inhibitor, i.e. an inhibitor of plasminogen activators ( e-aminocaproic acid, eACA; 10 mmol) . As a comparison the same culture medium was added to a series of 12 pieces, but this time without the protease inhibitor ( eACA) . This experiment was repeated with skin material of 3 different pigs.

The number of pieces, wherein distinct fibroblast cells were visible and had migrated from the skin pieces to the bottom of the dishes, was counted every 2 days. In figure 1 the percentage of the number of pieces from which fibroblasts had migrated is plotted against the time; the dotted line is the control and the continuous line shows lO M eACA. It appears from figure 1 that the number of pieces from which fibroblasts had migrated was decreased from an average 96 ± 4% to 72 + 4% on day 8, for the pieces to which the protease inhibitor (eACA) was added (p<0,01; n=3, tested with Student T-test) . A toxicity test (based on LDH-production of cells which are incubated with or without eACA) showed that at the concentration used (10 mmol) eACA was not toxic for the cells. The LDH-activity was 87,8 + 8% of the control (p>5%; n=4 , not differing significantly, tested with Stu- dent T-test) .

Example 2

With an electro dermatome severe wounds (3x3 cm, 2 mm deep) were made on the backs of 3 Yorkshire pigs (20 kg) .

These wounds were covered with a skin substituting matrix consisting of physically crosslinked collagen and elastin and covered with 1:3 wire mesh-like extended autologous epidermis of 0,2 mm thickness. After 1 and 3 weeks the enzyme activity and the mRNA-levels of several extracellular proteases were determined in these wounds; also these were compared with untreated control skin.

The extractions were performed after the wounds or control skin was totally cut out, quickly frozen and subsequently ground. For each pig one wound of one week, one wound of tree weeks and one piece control skin was cut out . The proteases analyzed could be divided in two groups: (1) urokinase plasminogen activator (uPA) and the matrix metalloproteases MMP-1 and MMP-9, which were present in an elevated concentration in the. early phase of the wound healing (after 1 week) ; (2) matrix metallo- protease MMP-2 which was elevated during a longer period compared with untreated control skin.

Figure 2 shows the relative activity of two matrix metalloproteases MMP-2 and MMP-9 in untreated skin (c) and in severe wounds on the backs of Yorkshire pigs, one (1) and three (3) weeks after transplantation of a skin substituting matrix in these wounds. The activity was determined by homogenating the skin tissue in a Tris (100 mmol, pH 7, 5) /Tween-80 (0,1% v/v) buffer and by electrophorating a fixed amount (corresponding with 50 μg protein) on a gelatin containing polyacrylamide gel and by aftercolour- ing with Coomassie-Blue. In the presence of MMP-2 or MMP-9 gelatin is degraded and the remaining blue colour is a measure for the activity of the respective metallo- proteases, and is expressed in dimensionless units.

Figure 3 shows the relative amounts of mRNA, uPA, MMP-1 and MMP-2 in untreated skin (c) and in severe wounds on the backs of Yorkshire pigs after 1 and 3 weeks after transplantation of a skin substituting matrix in these wounds. RNA was extracted from the skin or wounds with guanidium isothiocyanate buffer, and a fixed amount (corresponding with 10 μg total RNA) was applied on an agarose gel and then blotted on a Hybond nylon membrane . The mRNA' s were detected with ³²P-labelled probes on a phosphor screen scanner. The measured signal is normalized for ribosomal RNA and is expressed in dimensionless units.

Example 3

With an electro dermatome severe wounds (2 x 2 cm, 2 mm deep) were made on the backs of 2 Yorkshire pigs

(+ 20 kg) (2 x 3 wounds) . These wounds were treated with a 100 μl solution of e-amino caproic acid (eACA) (100 mM Dulbecco's Modified Eagle Medium, without bovine serum) . For comparison 2 x 3 wounds were also treated with solution only, thus without eACA. After 2, 3 and 6 weeks the contraction of the wounds was determined, corrected for the growth of the laboratory animal , by comparing the area of the wounds with a raster of lines that was tattooed in the skin of the pigs .

The wounds were appropriately closed, both in those cases wherein eACA-containing solvent was used as in those cases wherein only the solvent was used; the epidermis was expanded from the edges of the wound over the area. The remaining wound area was 130 ± 20 mm (control) compared to 148 ± 14 mm² (p<0,05; n=6 with Student T-test). Thus, the contraction of the wounds treated with e-aminocaproic acid (eACA), was significantly decreased compared to wounds treated with solvent only. This appears from the greater remaining wound area in figure 4 (dotted line is control) , wherein the remaining wound area in mm is plotted against the time.

Claims

1. Skin substitute suitable for promoting the healing of skin wounds, which skin substitute comprises a degradable and hydrophilic matrix, which matrix is permeable for cells and which matrix comprises a collagen and elastin comprising biocompatible matrix material, characterized in that the matrix of the skin substitute comprises an inhibitor of a protease.

2. Skin substitute according to claim 1, characterized in that the protease inhibitor is an inhibitor of an extracellular protease inhibitor.

3. Skin substitute according to claim 1 or 2 , char- acterized in that the protease inhibitor is chosen from the group consisting of inhibitors of plasminogen activator and metalloprotease or a mixture thereof.

4. Skin substitute according to one of the preceding claims, characterized in that the protease inhibitor is an inhibitor of a protease degrading the matrix.

5. Skin substitute according to one of the preceding claims, characterized in that the matrix has a pore size in the range of 5 - 100 ╬╝m.

6. Skin substitute according to one of the preceding claims, characterized in that the matrix comprises a further cell migration inhibiting compound.

7. Skin substitute according to one of the preceding claims, characterized in that the skin substitute com- prises autologous cells.

8. Skin substitute according to claim 7, characterized in that the autologous cells are derived from the dermis .

9. Skin substitute according to claim 7, character- ized in that the autologous cells are derived from subcutane fat tissue.

10. Skin substitute according to claim 7, characterized in that the autologous cells are derived from a crust of a burn.

11. Composition having a protease inhibiting action for topical application on skin wounds, comprising a protease inhibitor and a pharmaceutical acceptable carrier.

12. Composition according to claim 11, characterized in that the protease inhibitor is an extracellular protease inhibitor.

13. Composition according to claim 11 or 12, characterized in that the protease inhibitor is chosen from the group consisting of inhibitors of plasminogen activator and metallo protease.

14. Method for the production of a skin substitute according to one of the claims 7 - 10, wherein autologous cells are added to the skin substitute.

15. Use of a protease inhibitor for cell migration control in a skin substitute, in particular to decrease the number of myofibroblasts .