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EP-4735067-A1 - SKIN SUBSTITUTES, METHODS AND USES THEREOF

EP4735067A1EP 4735067 A1EP4735067 A1EP 4735067A1EP-4735067-A1

Abstract

The present disclosure relates to a method to obtain a decellularized dermal matrix from a rabbit, hare or beaver skin graft, the method comprising the following steps: degreasing the skin graft using a hydrocarbon solvent, perchloroethylene, modified alcohols or supercritical carbon dioxide, or combinations thereof; liming the degreased graft by immersing the graft in a solution comprising calcium hydroxide, sodium hydroxide, hydrogen peroxide, or mixtures thereof; fleshing the skin graft; optionally, deliming the skin graft; incubating the skin graft in a solution of ethylenediamine tetraacetic acid; treating the skin graft with a detergent solution for 30 minutes to 3 hours to obtain the decellularized dermal matrix. The disclosure also relates to a decellularized dermal matrix obtained by the method described; to a composition comprising said decellularized cell matrix for use in medicine or veterinary medicine; and to dermo-epidermal skin substitute scaffold comprising the disclosed decellularized dermal matrix.

Inventors

  • LEITE DE ALMEIDA MONTEIRO DE OLIVEIRA, Ana
  • PINTO RIBEIRO, Viviana
  • OLIVEIRA VASCONCELOS ROSADAS, Marta
  • VASCONCELOS MARTINS SILVA, Inês
  • DA CONCEIÇÃO FERREIRA DE SOUSA, Alda
  • DA SILVA LOPES RODRIGUES DOS REIS, Ana Helena
  • SOARES TERRA DE OLIVEIRA FIGUEIREDO, Ricardo Nicolau

Assignees

  • Universidade Católica Portuguesa - UCP
  • Cortadoria Nacional De Pelo, SA

Dates

Publication Date
20260506
Application Date
20240701

Claims (20)

  1. 1. Method to obtain a decellularized dermal matrix from a rabbit, hare or beaver skin graft, the method comprising the following steps: degreasing the skin graft using a hydrocarbon solvent, perchloroethylene, modified alcohols or supercritical carbon dioxide, or combinations thereof; liming the degreased graft by immersing the graft in a solution comprising calcium hydroxide, sodium hydroxide, hydrogen peroxide, or mixtures thereof; fleshing the skin graft; optionally, deliming the skin graft; incubating the skin graft in a solution of ethylenediamine tetraacetic acid, preferably a solution of 0.1 to 0.3% (v/v) ethylenediamine tetraacetic acid; treating the skin graft with a detergent solution for 30 minutes to 3 hours to obtain the decellularized dermal matrix.
  2. 2. The method according to the previous claim wherein the concentration of the detergent solution ranges from 0.1% (w/v) to 1% (w/v), preferably is 0.5% (w/v).
  3. 3. The method according to any of the previous claims wherein the detergent is selected from ionic detergent, non-ionic detergent, or mixtures thereof.
  4. 4. The method according to any of the previous claims wherein the detergent is selected from sodium dodecyl sulfate, sodium deoxycholate, triton X 100, sodium lauryl sulfate, polysorbate 20, Octyl glucoside, or mixtures thereof
  5. 5. The method according to any of the previous claim wherein the step of treating the skin graft with a detergent solution occurs at a temperature ranging from 0 to 40 °C, preferably 20 to 30 °C.
  6. 6. The method according to any of the previous claims further comprising a sterilization step.
  7. 7. The method according to the previous claim wherein the sterilisation is a supercritical carbon dioxide sterilisation.
  8. 8. The method according to any of the previous claims further comprising a step of contacting the skin graft with an enzyme solution.
  9. 9. The method according to the previous claim wherein the enzyme is selected from trypsin, collagenase, DNases, or mixtures thereof.
  10. 10. The method according to any of the previous claims wherein the skin graft is from a rabbit.
  11. 11. A decellularized dermal matrix obtained by the method described in any of the previous claims.
  12. 12. A composition comprising a decellularized dermal matrix from rabbit, hare or beaver as described in any of the preceding claims.
  13. 13. The composition according to the previous claim wherein the decellularized dermal matrix is from a rabbit.
  14. 14. The composition according to any of the previous claims 12-13 further comprising cells, growth factors, active agents, or mixtures thereof.
  15. 15. The composition according to the previous claim wherein the cell is selected from a list comprising, differentiated functional cell tissue specific cells, tissue-derived stem cells or progenitor cells or a subset thereof, mesenchymal stem cells or a subset thereof, multipotent adult progenitor cells or a subset thereof, and mixtures thereof.
  16. 16. The composition according to any of the previous claims 12-15 wherein the composition is administered in the form of a three-dimensional scaffold architecture.
  17. 17. A composition as described in any of the previous claims 12-16 for use in medicine or veterinary medicine.
  18. 18. The composition according to the previous claim for use in regenerative medicine, tissue engineering, pro-angiogenic implantable device, replacement biomaterial, drug delivery, platforms for 3D cell culture, disease modelling, biomedical or biological applications, cell culture, encapsulation of living cells, drug delivery, cell delivery, organ development and tissue growth.
  19. 19. The composition according to any of the previous claims 17-18 for use in the treatment of a skin wound or damage, in particular burn wounds.
  20. 20. A dermo-epidermal skin substitute scaffold comprising: a decellularized dermal matrix obtained by the method disclosed in any of the previous claims 1-10; wherein said decellularized dermal matrix comprises a first layer of an epidermal side of dermis and a second layer of a hypodermal side of dermis; epidermal keratinocyte cells; dermal fibroblasts; wherein the epidermal keratinocyte cells are seeded on the first layer of the decellularized dermal matrix and the dermal fibroblasts are seeded on the second layer of the decellularized dermal matrix; and wherein the two layers are arranged consecutive from the top to the bottom.

Description

D E S C R I P T I O N SKI N SUBSTITUTES, M ETHODS AND USES THEREOF TECHNICAL FIELD [0001] The present disclosure relates to a skin substitute prepared from full-thickness rabbit skin graft, the method of preparation and its uses. Methods for preparing decellularized dermal matrices from rabbit, hare or beaver skin grafts and their application as dermo-epidermal skin substitutes. The present disclosure aims to produce decellularized dermal matrices that are biocompatible, have minimal immunogenicity, and retain the necessary biomechanical properties for medical use in humans. BACKGROUND [0002] In full-thickness skin lesions, including in burn patients, dermal reconstruction may be difficult through classic plastic surgery. Burns affect 11 million people annually worldwide, causing severe scarring and morbidity without proper treatment. [0003] The use of autologous substitutes may be an alternative, however for extensive burn areas it can be a limitation1. Indeed, skin autografts, the standard of care, result in poor functional and aesthetic outcomes, while being painful and limited for extensive burns. [0004] Artificial dermal substitutes can be used, composed of extracellular matrix components (i.e., collagen, glycosaminoglycans, and hyaluronic acid) and covered by autologous split-thickness skin grafts2. However, this is a two-step procedure in which the scaffolds are first incorporated to obtain neovascularization (up to 21 days) and then the autologous skin grafts are implanted, able to be blood supplied by the dermal substitutes. [0005] More recently, skin xenografts evolved as skin bio-substitutes, in which decellularized tissues with an intact extracellular matrix (ECM) were proposed for skin regeneration3. Decellularized extracellular matrices (dECM) have promising potential in tissue engineering, providing cues for restoring damaged tissues and organs. However, the decellularization process may damage tissue integrity. [0006] Commercially available full-thickness skin equivalents like Apligraf, OrCel, and denovoSkin, composed of living dermal and epidermal components, have made important progress in clinical practices (Table 1) [9] [10], Some products are made from bovine or porcine collagen which may carry the risk of disease transmission or pose religious restrictions [7], Furthermore, these animal-origin collagen skin substitutes may not provide the ideal anatomy, microarchitecture, or mechanical properties for optimal tissue regeneration. Table 1 - List of commonly used bioengineered skin substitutes. [0007] Human-based Decellularized Dermal Matrices (DDMs), such as FlexHD, AlloMax, or AlloDerm, can stimulate natural dermis production and re-epithelialization, but they have limited availability and tissue integrity is often not preserved due to damaging decellularization processes. [0008] Decellularization can be achieved through various physical, chemical, enzymatic, or combined treatments, [11] [4] [12] [13] [14] [15] [16], and more recently, the utilization of supercritical CO2 [14] [16], However, there is no established standard decellularization technique as it depends on factors such as tissue density, shape, architecture, and cell and fat content [4], [0009] Rabbit skin is an unexplored source of ECM for wound healing and regeneration. It has collagen type I with a similar amino acid sequence to human skin [17] and is deprived of religious and public health concerns [6] [7], This ECM source is generated at a rate of 500 Kg/day just in one Portuguese company. This represents a unique opportunity for the valorization of this collagen source into advanced products for skin tissue regeneration. [0010] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure. GENERAL DESCRIPTION [0011] The present disclosure relates to a method for the decellularization of fullthickness rabbit skin grafts in order to obtain extensive and preserved dermal matrices applied as skin bio-substitutes, preferably in humans. [0012] The present disclosure provides a novel method for the decellularization of fullthickness skin grafts from rabbits, hares or beavers to produce preserved and extensive dermal matrices suitable for use as skin bio-substitutes in humans and other animals. The disclosure further relates to dermo-epidermal skin substitute scaffolds comprising the decellularized dermal matrix prepared by the disclosed methods, and a composition comprising the decellularized dermal matrix. [0013] In an embodiment, rabbit dermis was used as an extracellular matrix for the development of a skin substitute to repair and regenerate burn wounds. [0014] In an embodiment, different processing approaches were used for the creation of a decellularized rabbit dermal matrix (dRDM) with full integrity, that was further recellularized to generate a functional human skin equivalent. [0015] In an embodiment, a highly preserved dRDM was developed, by using the disclosed decel