Skin reconstruction method

Abstract

Some embodiments are directed to a method for preparing a skin substitute, a dermal substitute, to a skin substitute, to a dermal substitute and to a kit for implementing the method. Some other embodiments are directed to a graft that can consist of of a skin substitute and to the use thereof as treating a skin disorder and/or a loss of skin substance.

Claims

1. A method for preparing a skin substitute, comprising the steps of: a. culturing fibroblasts in a fibroblast culture medium M1; b. seeding a matrix including collagen with the fibroblasts obtained in step a; c. culturing the fibroblasts seeded in the matrix including collagen obtained in step b in a fibroblast culture medium M2 that includes ascorbic acid, an ascorbate, or a derivative thereof, whereby the matrix and the cultured fibroblasts form a dermal substitute; d. culturing melanocytes in a melanocyte culture medium M3; e. culturing keratinocytes in a keratinocyte culture medium M4; f. mixing the melanocytes obtained in step d with the keratinocytes obtained in step e; g. seeding the dermal substitute obtained in step c with the mixture of keratinocytes+melanocytes obtained in step f, wherein the seeding is carried out with a (keratinocytes+melanocytes)/fibroblasts ratio equal to or greater than 9/1 and less than or equal to 19/1; and h. culturing the seeded dermal substitute obtained in step g in a skin culture medium M5 thus forming the skin substitute thereof, wherein the medium M5 includes 40 to 60 mg/L of hyaluronic acid, a hyaluronate or a derivative thereof, and 40 to 60 mg/L of ascorbic acid, an ascorbate, a derivative thereof, and the skin substitute provided contains functional basal lamina.

2. The method as claimed in claim 1, wherein the mixing of melanocytes and keratinocytes of step f is carried out with a melanocytes/keratinocytes ratio of 1/20 to 1/15.

3. The method as claimed in claim 1, wherein the seeding in step b is carried out at a density of from 20,000 to 50,000 fibroblasts/cm.sup.2 of surface area of the matrix including collagen.

4. The method as claimed in claim 1, wherein step c comprises: a first culturing step c′ of 18 to 28 hours in the presence of a fibroblast culture medium M21 that includes neither ascorbic acid nor ascorbate, and a second culturing step c″ of at least two days in the presence of a fibroblast culture medium M2 that includes ascorbic acid, an ascorbate, or a derivative thereof.

5. The method as claimed in claim 1, wherein step h comprises: a first culturing step h′ of 6 to 24 hours in the presence of a culture medium M51 that includes neither hyaluronic acid, nor hyaluronate, nor ascorbic acid, nor ascorbate, and a second culturing step h″ of at least 2 days in the presence of a culture medium M52 that includes hyaluronic acid, a hyaluronate, or a derivative thereof, and a third culturing step h′″ of at least two days in the medium M5 that includes 40 to 60 mg/L hyaluronic acid, hyaluronate, or derivative thereof, and 40 to 60 mg/L ascorbic acid, ascorbate, or derivative thereof.

6. The method as claimed in claim 1, wherein the skin substitute is further provided to contain at least one selected from the group consisting of: keratinized pluristratified epithelium with a stratum basal, a stratum spinosum, a stratum granulosum and a stratum corneum.

7. The method as claimed in claim 1, wherein the skin substitute is further provided to contain: basal melanocytes in contact with a dermal substitute containing functional fibroblasts.

8. A skin substitute obtained by implementing the method defined in claim 1.

9. The skin substitute as claimed in claim 8, wherein the fibroblasts are autologous to an intended recipient of a graft constituted of said skin substitute.

10. The skin substitute as claimed in claim 9, wherein the fibroblasts, the melanocytes and the keratinocytes are autologous with respect to said intended recipient.

11. A graft constituted of the skin substitute as claimed in claim 9.

12. The graft as claimed in claim 11, wherein said graft is fabricated for use in treating a skin disorder and/or a loss of skin substance.

13. The graft as claimed in claim 12, wherein the skin disorder and/or a loss of skin substance is chosen from the group including a burn, a healing defect, a chronic wound, a pigmentary disorder, a hemangioma and a skin cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 represents a diagram of the steps for obtaining a skin substitute/equivalent.

(2) FIG. 2 represents optical microscopy photographs of skin (FIG. 2A), and of skin substitutes obtained according to the method of some embodiments with variations in the ratio of cells seeded (FIGS. 2B and 2C).

(3) FIG. 3A is a photograph of a dermal substitute obtained in step c, by optical microscopy after staining of the fibroblasts. FIG. 3B is a photograph of a skin substitute obtained, of small size, namely 0.5 cm.sup.2. FIG. 3C is a photograph of a skin substitute obtained, of medium size, namely 25 cm.sup.2.

(4) FIG. 4 represents photographs after grafting of a matrix including collagen (column A) or of a dermal substitute obtained in step c (column B) on the day of grafting (row 1), 14 days after grafting (row 2) or 24 days after grafting (row 3).

(5) FIG. 5 represents optical microscopy photographs of a sample taken from the graft 24 hours after grafting, after hematoxylin-eosin staining. FIG. 5A shows the substitute colonized by murine fibroblasts. FIG. 5B shows the substitute colonized with human fibroblasts before grafting.

(6) FIG. 6 represents optical microscopy photographs of skin equivalent (FIGS. 6A to D) obtained according to the method with a (keratinocytes+melanocytes)/fibroblasts seeded cell ratio of 13.3 (FIGS. 6A and B); the immunohistochemical labeling of the melanocytes in the basal position (FIG. 6C, light areas) and the production of the basal lamina, labeling of collagen IV (FIG. 6D (2)) and the p63 proliferation marker (FIG. 6 D (1)). FIGS. 6E and 6F represent optical microscopy photographs of skin after immunohistochemical labeling of the melanocytes in the basal position (FIG. 6E, light areas), labeling of collagen IV (FIG. 6F (2)) and of the p63 proliferation marker (FIG. 6F (1)).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

(7) A few inventive aspects of the disclosed embodiments are explained in detail below with reference to the various figures. Exemplary embodiments are described to illustrate the disclosed subject matter, not to limit its scope, which is defined by the claims. Those of ordinary skill in the art will recognize a number of equivalent variations of the various features provided in the description that follows.

EXAMPLES

Example 1: Example of Production of a Skin Substitute

(8) In the present example, the cells used came from a skin biopsy taken from mammoplasties previously carried out on a patient.

(9) The biopsy was taken by a plastic surgeon and the biopsy was placed in a sterile tube containing physiological saline.

(10) The cells were isolated from the biopsy as follows:

(11) 1. Epithelial Cell Isolation

(12) a. Rinsing of the biopsy in sterile HBSS (Hank's Balanced Salt Solution). b. Removal of the adipose tissue by a biologist-technician using a scalpel. c. Incubation with trypsin-EDTA preheated to 37° C., for between 3 and 24 h. d. Neutralization with irradiated FCS (trypsin inhibitor). e. Removal of the epidermis and scraping, with a scalpel, of the basal stratum where the highly proliferative (p63 positive) cells are found. f. Filtration, centrifugation at 1200 revolutions per minute and seeding of the pellet at 100 000 cells per cm.sup.2 in modified* MCDB153 medium including the compounds mentioned in table 4 below in which the concentrations of L-arginine, L-histidine, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, L-valine and choline chloride have been doubled, the NaCl concentration has been reduced to 0.104 M/l, Hepes has been reduced to 2.29×10.sup.−2 M/l and NaHCO.sub.3 has been reduced to 1.19×10.sup.−2 M/l, the pH of the medium being adjusted to 7.4 and antibiotics (penicillin and streptomycin 1%) for the keratinocytes.

(13) For the melanocytes, filtration, centrifugation at 1200 revolutions per minute and seeding of the pellet at 100 000 cells per cm.sup.2 in modified* MCDB153 medium including the compounds mentioned in table 4 below, also including 5.88 g of sodium bicarbonate/5 l, 0.272 g of tyrosine/5 l and 0.157 g of L-methionine/5 l. the pH of the medium being adjusted to 7.4.

(14) TABLE-US-00004 TABLE 4 normal composition of the MCDB 153 medium Composition Concentration in g .Math. l.sup.−1 Ammonium Metavanadate 0.000000585 Anhydrous calcium chloride• 0.00333 Cupric Sulfate•5H.sub.2O 0.00000275 Ferrous sulfate•7H.sub.2O 0.00139 Magnesium chloride 0.05713 Manganese Sulfate 0.000000151 Molybdic Acid•4H.sub.2O (ammonium) 0.00000124 Nickel Chloride•6H.sub.2O 0.00000012 Potassium Chloride 0.11183 Sodium Acetate (anhydrous) 0.30153 Sodium Chloride 7.599 Sodium Metasilicate•9H.sub.2O 0.000142 Dibasic Sodium Phosphate 0.284088 (anhydrous) Sodium Selenite 0.0000038 Stannous Chloride•2H.sub.2O 0.000000113 Zinc Sulfate•7H.sub.2O 0.000144 L-Alanine 0.00891 L-Arginine•HCl 0.2107 L-Asparagine•H.sub.2O 0.015 L-Aspartic Acid 0.00399 L-Cysteine•HCl•H.sub.2O 0.04204 L-Glutamic Acid 0.01471 L-Glutamine 0.8772 Glycine 0.00751 L-Histidine•HCl•H.sub.2O 0.01677 L-Isoleucine 0.001968 L-Leucine 0.0656 L-Lysine•HCl 0.01827 L-Methionine 0.00448 L-Phenylalanine 0.00496 L-Proline 0.03453 L-Serine 0.06306 L-Threonine 0.01191 L-Tryptophan 0.00306 L-Tyrosine•2Na 0.00341 L-Valine 0.03513 D-Biotin 0.0000146 Choline Chloride 0.01396 Folic acid 0.00079 myo-Inositol 0.01802 Niacinamide 0.00003663 D-Pantothenic Acid (hemicalcium) 0.000238 Pyridoxine•HCl 0.00006171 Riboflavin 0.0000376 Thiamine•HCl 0.000337 Vitamin B-12 0.000407 Adenine•HCl 0.03088 D-Glucose 1.081 HEPES 6.6 Phenol Red•Na 0.001242 Putrescine•2HCl 0.000161 Pyruvic acid•Na 0.055 Thioctic acid 0.000206 Thymidine 0.000727 g. Incubation at 37° C. at 5% CO.sub.2 for one week with medium changed every three days. h. After approximately one week: differential trypsinization=trypsinization with 0.025% trypsin and 0.0.1 M EDTA (1-2 minutes in order to detach the melanocytes, 10 minutes in order to detach the keratinocytes). The melanocytes detach first, thereby making it possible to purify the cultures. Neutralization with irradiated FCS, centrifugation at 1200 revolutions per minute and seeding of the pellet for amplification in the same medium. i. Incubation at 37° C. at 5% CO.sub.2 for one week with medium changed every three days.
2. Fibroblast Isolation a. Rinsing of the dermal part with HBSS. b. Incubation of the dermis with collagenase at 1% at 37° C. for a maximum of three hours depending on the type of dermis. c. Neutralization with irradiated FCS. d. Filtration via a 40 μm cell sieve, centrifugation at 1200 revolutions per minute with a GR 2022 centrifuge for 5 minutes and seeding of the pellet at 100 000 cells per cm.sup.2 in DMEM including 10% irradiated FCS and penicillin and streptomycin at 1% for 24 hours. e. Incubation in a Jouan IG 150 incubator at 37° C., 5% CO.sub.2, for one week with medium changed every three days.
3. Preparation of a Skin Substitute a. Trypsinization of the fibroblasts with 0.025% trypsin and 0.0.1 M EDTA for 10 minutes, then neutralization with irradiated FCS, centrifugation at 1200 revolutions per minute with a GR 2022 centrifuge for 5 minutes, and seeding in DMEM including 10% FCS on a dermal matrix of sterile collagen origin, namely an Integra matrix (registered trademark) rinsed beforehand with Hank's Balanced Salt Solution (HBSS) three times, in a proportion of 30 000 fibroblasts per cm.sup.2 in a made-to-measure stainless steel incubation chamber. b. After 24 hours of culture at 37° C., 5% CO.sub.2, the incubation chamber was removed from the matrix. C. The seeded matrix was incubated at 37° C., 5% CO.sub.2 in DMEM including 10% of irradiated FCS and penicillin and streptomycin at 1% and 50 mg/mL ascorbic acid, for one week with medium changed every three days. d. Trypsinization of the keratinocytes and of the melanocytes with 0.025% trypsin and 0.0.1 M EDTA for 1 to 2 minutes in order to detach the melanocytes from the melanocyte culture dishes, for 10 minutes in order to detach the keratinocytes from the keratinocyte culture dishes. Neutralization with irradiated FCS and centrifugation and seeding at 400 000 cells per cm.sup.2 in an incubation chamber of a mixture containing 1 melanocyte per 19 keratinocytes. e. Adhesion for 24 hours. f. Submersion for seven days in modified green medium: DMEM/Ham's F12/10% FCS including hyaluronic acid at 50 mg/ml. g. Interface for 7 days in modified Green medium: DMEM/Ham's F12 including 10% FCS, hyaluronic acid at 50 mg/ml and 50 mg/ml ascorbic acid and antibiotics, namely 1% penicillin-streptomycin.

(15) FIG. 1 represents a diagram of the steps for obtaining a skin substitute.

(16) In the present example, two skin substitutes obtained had (keratinocytes+melanocytes)/fibroblasts ratios of 13.3. For the ratio of 13.3, during the steps of depositing the fibroblasts and the keratinocytes/melanocytes mixture, the amounts of cells seeded were respectively 15 000 for the fibroblasts, 10 000 for the melanocytes and 190 000 for the keratinocytes.

(17) Once the substitute had been obtained, it was fixed in 4% formol, embedded in paraffin, then a 4 μm section was cut, and then hematoxylin-eosin staining was performed in order to label the various layers of the skin. Observation under an optical microscope and at a magnification of ×40 was carried out. Since the microscope was coupled to a CCD camera (Nikon, software NIS element Br), photographs of the observations were taken. FIG. 2B represents an optical microscopy photograph of a skin substitute obtained according to the method in which the (keratinocytes+melanocytes)/fibroblasts ratio was 13.3. FIG. 2C represents an optical microscopy photograph of a skin substitute obtained according to the method in which the (keratinocytes+melanocytes)/fibroblasts ratio was 6.7 and FIG. 2A represents an optical microscopy photograph of a normal skin biopsy. FIGS. 6A and 6B also represent optical microscopy photographs of a skin equivalent obtained according to the method in which the (keratinocytes+melanocytes)/fibroblasts ratio was 13.3.

(18) Moreover, immunohistochemical labeling of the substitute obtained according to the method in which the (keratinocytes+melanocytes)/fibroblasts ratio was 13.3 and of an in vivo skin was carried out according to the method described in Salducci, M., André, N., Guéré, C., Martin, M., Fitoussi, R., Vié, K., and Cario-André, M. (2014). Factors secreted by irradiated aged fibroblasts induce solar lentigo in pigmented reconstructed epidermis. Pigment Cell Melanoma Res. 27, 502-504 [12] or Simon, D., Daubos, A., Pain, C., Fitoussi, R., Vié, K., Taieb, A., de Benetti, L., and Cario-André, M. (2013). Exposure to acute electromagnetic radiation of mobile phone exposure range alters transiently skin homeostasis of a model of pigmented reconstructed epidermis. Int. J. Cosmet. Sci. 35, 27-34 [13] in order to identify in the substitute the presence of melanocytes, the production of the basal lamina including in particular collagen IV (FIG. 6D (2)), the proliferative capacity of the cells in the basal lamina (FIG. 6D (1)) and the presence of melanocytes (FIG. 6C). FIGS. 6E and 6F represent optical microscopy photographs of skin in vivo after immunohistochemical labeling of the melanocytes in the basal position (FIG. 6E, light areas), labeling of collagen IV (FIG. 6F (2)) and labeling of the p63 proliferation marker (FIG. 6F (1)). It is clearly apparent on FIGS. 6C and 6D that the skin substitute according to some embodiments includes melanocytes, and a basal lamina as demonstrated by the presence of collagen IV at the level of which the cells present are highly proliferative as for the skin in vivo (FIGS. 6E and 6F).

(19) As represented on these photographs, the substitute obtained by the method has a structure identical to that of the skin in vivo.

Example 2: Grafting of a Dermal Substitute According to Some Embodiments onto Mice

(20) In this example, the skin substitute used was the substitute obtained as described in example 1 with cells obtained from mammoplasties with the (keratinocytes+melanocytes)/fibroblasts ratio of 13.3. The mice used were swiww nu/nu nude mice from Jackson Lab.

(21) In this example, grafts were performed on ten mice in parallel.

(22) An incision of the mice was made using a scalpel on an area of 5 cm.sup.2 in order to eliminate the dermis and the epidermis, and the scarified area was cleaned with physiological saline. A skin substitute previously prepared was applied to the scarified area in order to cover it (FIG. 4 A, row 1) and then the grafted area was closed with the skin of the mouse that was sutured (FIG. 4 B, row 1).

(23) The mice were kept in a specialized animal house one per cage for the time required for the graft to take and then five per cage in cages of appropriate size.

(24) After two weeks, an observation was made of how the graft had taken by removing the flap of skin from the mouse (FIG. 4 A, row 2). In other words, the sutures were removed from the mouse skin that had been sutured, said skin was removed and the grafted dermal substitute was revealed for visual observation (FIG. 4 B, row 2).

(25) After three weeks, visual observation of how the graft had taken was carried out (FIGS. 4B and B, row 3).

(26) As demonstrated in FIG. 4, it is clearly apparent that the dermal substitute can advantageously be applied, in particular for a graft without induction of side effects.

(27) In addition, in order to study the structural condition of the dermal substitute after three weeks post-application, a sample was taken by cutting the substitute with a scalpel. The substitute was fixed in 4% formol, embedded in paraffin, then a 4 μm section was cut, and then hematoxylin-eosin staining was performed in order to label the various layers of the skin. Observation under an optical microscope at a magnification of ×40 was carried out. Since the microscope was coupled to a CCD camera (Nikon, software NIS element Br), photographs of the observations were taken.

(28) FIG. 5 represents the optical microscopy photographs of grafted substitutes. In particular, FIG. 5A shows the substitute colonized by the murine fibroblasts of which there are not very many and which have not begun to reorganize the collagen. FIG. 5B shows the substitute colonized with the human fibroblasts before grafting. A large number of fibroblasts and reorganization of the matrix, in particular in the superficial area with thicker bunches of collagen, are observed. The present example thus clearly demonstrates that the presence of all or most of the cells makes it possible to obtain an easily integratable substitute, which does not degrade over time and which exhibits maturation after grafting, which is accelerated in the presence of fibroblasts in the matrix.

LIST OF REFERENCES

(29) 1. Pendaries V et al., siRNA-mediated allele-specific inhibition of mutant type VII collagen in dominant dystrophic epidermolysis bullosa.JID 2012, June; 132(6):1741-3. 2. Petek L M et al., “Efficient KRT14 targeting and functional characterization of transplanted human keratinocytes for the treatment of epidermolysis bullosa simplex”. Mol ther 2010, September; 18 (9):1624-32. 3. Kogut et al., “Differentiation of human induced pluripotent stem cells into a keratinocyte lineage” Methods Mol Biol 2014, 1195:1-12. 4. Ohta et al., “Generation of human melanocytes from induced pluripotent stem cells” Methods Mol Biol, 2013; 989:193-215. 5. Revilla et al., “Current advances in the generation of human iPS cells: implications in cell-based regenerative medicine.” J Tissue Eng Regen Med, 2015, Mar. 11. 6. Bell et al., 1979 7. Boyce S T et al., “Structure of a collagen-GAG dermal skin substitute optimized for cultured human epidermal keratinocytes”, 1988 October; 22 (10):939-57. 8. Hafemann et al., “Use of a collagen/elastin-membrane for the tissue engineering of dermis.” Burns 1999, August; 25(5):373-84. 9. Wonhye Lee et al., “Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication.” Biomaterials, 2009, March; 30(8):1587-95 10. Pena, and al., J Oral and Maxillofacial Surgery, 70:10 10, 2012 11. E. Dantzer, F. Braye “Reconstructive surgery using an artificial dermis (Integra): results with 39 grafts.” Br J Plast Surg, 54:8 8, 2001. 12. Salducci, M., André, N., Guéré, C., Martin, M., Fitoussi, R., Vié, K., and Cario-André, M. (2014). Factors secreted by irradiated aged fibroblasts induce solar lentigo in pigmented reconstructed epidermis. Pigment Cell Melanoma Res. 27, 502-504 13. Simon, D., Daubos, A., Pain, C., Fitoussi, R., Vié, K., Taieb, A., de Benetti, L., and Cario-André, M. (2013). Exposure to acute electromagnetic radiation of mobile phone exposure range alters transiently skin homeostasis of a model of pigmented reconstructed epidermis. Int. J. Cosmet. Sci. 35, 27-34