WO2025004023A1 - Skin substitutes, methods and uses thereof - Google Patents

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.

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 decellularization methodologies to obtain a highly preserved non- immunogenic dermal-mimetic extracellular matrix, targeting temporary wound coverage.

[0016] The present disclosure also relates to a dermo-epidermal human skin substitute produced obtained by recel lula rizi ng the dRDM using a patient-specific approach. In an embodiment, cells are isolated from full-thickness human skin samples and seeded within the dRDM.

[0017] An aspect of the disclosure comprises a pre-vascularized dermo-epidermal human skin equivalent obtained by re-cellularizing the dRDM using a patient-specific approach. In an embodiment, cells isolated from human skin samples are seeded on the different surfaces of the dRDM. Pre-vascularization is stimulated by microneedle-based technology for microvascular endothelial cell deposition and infiltration into the dRDM. The resulting biofunctional skin equivalent is validated in an in vitro explant model for application in full-thickness burns.

[0018] In an embodiment, the resulting skin substitute is characterized in vitro and biomechanically, to evaluate its feasibility as a skin graft in full-thickness burns.

[0019] The present disclosure also relates to a wound healing kit comprising the dermo- epidermal skin substitute scaffold (i.e. the disclosed decellularized dermal matrix) of the present disclosure, or the composition comprising the decellularized dermal matrix, and at least one of the following components: dressing, bandage, antimicrobial agent, or combinations thereof.

[0020] The present disclosure also relates to the use of the decellularized dermal matrix of the present disclosure as a tissue engineering scaffold for the regeneration of specific tissues, such as skin, bone, cartilage, or other types of soft tissues, wherein the present disclosure provides a supportive and bioactive matrix for cell adhesion, proliferation, and differentiation.

[0021] The present disclosure relates to a method to obtain a decellularized dermal matrix from a rabbit or a hare graft, the method comprises the following steps: removing the hypodermis, preferably using perchloroethylene; removing the epidermis, preferably by contacting the graft with chemical and biological agents, preferably trypsin, collagenase, oxygen peroxide, sodium hydroxide, calcium hydroxide, removing the hypodermis, preferably by mechanical debridement; washing the skin graft; decellularizing the skin graft to obtain the decellularized dermal matrix, preferably by mechanical agitation, immersion in ionic and/or non-ionic detergents, enzymes or combinations thereof.

[0022] In an embodiment, the decellularization step occurs at a temperature ranging from 0 to 40 °C, preferably 20 to 30 °C.

[0023] In an embodiment, the method further comprises a sterilization step. [0024] In an embodiment, the sterilization is a supercritical carbon dioxide sterilization.

[0025] In an embodiment, the detergent is selected from sodium dodecyl sulfate or sodium deoxycholate.

[0026] In an embodiment, the enzymes are selected from DNases.

[0027] An aspect of the present disclosure comprises a decellularized scaffold obtained by the disclosed method.

[0028] The present disclosure relates to a dermo-epidermal skin substitute scaffold comprising: a decellularized dermal matrix obtained by the disclosed method; wherein said decellularized dermal matrix comprises a first part of epidermal side of dermis and a second part of hypodermal side of dermis; epidermal keratinocyte cells; dermal fibroblasts; wherein the epidermal keratinocyte cells are seeded on a first part of the scaffold and the dermal fibroblasts on the second part of said scaffold, wherein the two layers are consecutive from the top to the bottom.

[0029] In an embodiment, the dermo-epidermal skin substitute scaffold further comprises endothelial cells.

[0030] An aspect of the present disclosure comprises a composition comprising a decellularized dermal matrix from rabbit or hare, preferably wherein the decellularized dermal matrix comprises elastin, collagen and glycosaminoglycans.

[0031] In an embodiment, the composition is administered in the form of a scaffold.

[0032] In an embodiment, the composition further comprises a cell, a growth factor, or mixtures thereof.

[0033] In an embodiment, the cell is selected from a list comprising induced pluripotent stem cells, partially differentiated progenitor cells, differentiated functional cell tissue specific cells, peripheral blood cells or a subset thereof, 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.

[0034] In an embodiment, the disclosed composition is for use in medicine or veterinary medicine. [0035] In an embodiment, the composition is for use in regenerative medicine, tissue engineering, pro-angiogenic implantable devices, replacement biomaterials, drug delivery, platforms for 3D cell culture or disease modeling or biomedical or biological applications, cell culture, encapsulation of living cells, drug delivery, cell delivery, cell regeneration, organ development, and tissue growth.

[0036] In an embodiment, the composition is for use in the treatment of a skin wound or damage.

[0037] 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, 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.

[0038] In an embodiment, the concentration of the detergent solution ranges from 0.1% (w/v) to 1% (w/v), preferably is 0.5% (w/v).

[0039] In an embodiment, the detergent is selected from ionic detergent, non-ionic detergent, or mixtures thereof.

[0040] In an embodiment, the detergent is selected from sodium dodecyl sulfate, sodium deoxycholate, triton X 100, sodium lauryl sulfate, polysorbate 20, Octyl glucoside, or mixtures thereof

[0041] In an embodiment, 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.

[0042] In an embodiment, the method further comprises a step of sterilization. In preferred embodiment, the sterilisation is a supercritical carbon dioxide sterilisation. [0043] In an embodiment, the method further comprises a step of contacting the skin graft with an enzyme solution. In a preferred embodiment, the enzyme is selected from trypsin, collagenase, DNases, or mixtures thereof.

[0044] In an embodiment, the skin graft is from a rabbit.

[0045] An aspect of the present disclosure relates to a decellularized dermal matrix obtained by the disclosed method.

[0046] The present disclosure also relates to a composition comprising the disclosed decellularized dermal matrix from rabbit, hare or beaver.

[0047] In an embodiment, the decellularized dermal matrix is from a rabbit.

[0048] In an embodiment, the composition further comprises cells, growth factors, active agents, or mixtures thereof.

[0049] In an embodiment, 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.

[0050] In an embodiment, the composition is administered in the form of a three- dimensional scaffold architecture.

[0051] The present disclosure also relates to a composition as disclosed for use in medicine or veterinary medicine.

[0052] In an embodiment, the composition is 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.

[0053] In a further embodiment, the composition is for use in the treatment of a skin wound or damage, in particular burn wounds. [0054] An aspect of the present disclosure relates to a dermo-epidermal skin substitute scaffold comprising: a decellularized dermal matrix obtained by the disclosed method; 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.

[0055] In an embodiment, the dermo-epidermal skin substitute scaffold further comprises endothelial cells. In another embodiment, the dermo-epidermal skin substitute scaffold further comprises a hydrogel, cells, a growth factor, or mixtures thereof.

[0056] In an embodiment, the dermo-epidermal skin substitute scaffold further comprises elastin, collagen and/or glycosaminoglycans.

[0057] The present disclosure relates to the use of a composition as disclosed for the manufacture of a medicament for the treatment of a skin wound or damage.

[0058] The present disclosure also relates to a method for treating a skin wound or damage in a subject, the method comprising administering the disclosed composition to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of the invention.

[0060] Figure 1: Embodiment of a schematic overview of the project.

[0061] Figure 2: Embodiment of a schematic overview of the decellularization protocol for rabbit skin. [0062] Figure 3: Embodiment of a schematic representation of the sterilization and drying of the disclosed decellularized dermal matrix, by using supercritical carbon dioxide (scCCh) and peracetic acid (PAA).

[0063] Figure 4: Embodiment of the morphological differences between the top (epidermis-contacting surface of dRDM) and bottom surfaces (hypodermis-contacting surface of dRDM) of the disclosed decellularized dermal matrix.

[0064] Figure 5: Embodiment of a schematic overview of the study of the influence of pH variations on the matrix conformation and its absorption rate.

[0065] Figure 6: Embodiment of the decellularization protocol.

[0066] Figure 7: Embodiment of the detergent treatment optimization. Differences between sodium dodecyl sulfate (SDS) and Sodium Deoxycholate (SDC) at two different application timings and different initial pH (liming (high pH) and deliming (neutral pH)) were tested.

[0067] Figure 8: Embodiment of DNA quantification results obtained for the different tested conditions.

[0068] Figure 9: Embodiment of scanning electron microscopy (SEM) analysis of the rabbit skin before the decellularization treatment (control).

[0069] Figure 10: Embodiment of SEM analysis of the rabbit skin before the application of the detergents; (i) SEM images obtained after oxidative liming treatment; (ii) SEM images after unliming treatment.

[0070] Figure 11: Embodiment of SEM analysis of the rabbit skin after the application of the detergents for different time periods.

[0071] Figure 12: Embodiment of SEM analysis of the rabbit skin after the application of the detergents for different time periods.

[0072] Figure 13: Embodiment of results of the mechanical properties analysis after application of decellularization treatment. [0073] Figure 14: Embodiment of the cytotoxicity assay of the decellularized dermal matrix (dECM) using human dermal fibroblasts (hDFs).

[0074] Figure 15: Embodiment of the direct contact assay using hDFs cultured onto the decellularized dermal matrix (dECM) up to 7 days.

[0075] Figure 16: Embodiment of a schematic illustration of the (a) cell culture set-up for obtaining the dermo-epidermal human skin substitute, and (b) dynamic perfusion platform for validating the cell culture approach under flow conditions.

[0076] Figure 17: Embodiment of a schematic illustration of the three-step cell culture set-up for obtaining the pre-vascularized full-thickness human skin equivalent in the (a) plain dRDM, and (b) microneedling pre-treated dRDM.

[0077] Figure 18: Embodiment of the protocol for the construction of the ex vivo burn wound model.

DETAILED DESCRIPTION

[0078] 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. [0079] The present disclosure describes a method for decellularizing rabbit, hare or beaver skin grafts to produce decellularized dermal matrices. The disclosed preserved decellularized dermal matrices can be used directly as tissue substitutes or further processed into scaffolds for skin regeneration. The decellularized dermal matrix of the present disclosure shows a safer and more versatile option for clinical applications.

[0080] In an embodiment, complete full-thickness skin grafts from rabbit, were decellularized through the combination of green chemical and enzymatic methods.

[0081] In an embodiment, the rabbit skin was processed as follows: i) Rabbit skin was degreased and the hair is mechanically cut. In an embodiment, the graft is degreased using an hydrocarbon solvent, perchloroethylene, modified alcohols supercritical carbon dioxide, or combinations thereof; ii) Oxidative liming is applied for 24h, wherein this step includes application of calcium hydroxide, sodium hydroxide, and hydrogen peroxide in combination with specific enzymes, such as DNAse, resulting in complete hair and epidermis removal and allows the dermis exposure by opening its fibrous structure; iii) Mechanical removal of subcutaneous tissue (fleshing); iv) Neutralization of the pH and elimination of the calcium hydroxide allowing the dermal structure to return to its native conformation (deliming), which can be achieved using methods well known by those skilled in the art.

[0082] In a further embodiment, a detergent-based decellularization protocol was developed and applied to complete the decellularization process. The application of this protocol was tested after steps iii) and iv). Firstly, ethylenediamine tetraacetic acid (EDTA) was applied for lh in agitation, preferably 0,3% (v/v) of EDTA, to break cell-cell and cell-matrix interactions allowing for a more efficient cellular content removal and lysis by the reagents posteriorly applied. A detergent was used to disrupt cell membranes, such as sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), triton X 100, Sodium lauryl sulfate (SLS), polysorbate 20 (Tween 20), Octyl glucoside, or mixtures thereof. For that, the sample treated with EDTA was further incubated in the detergent solution for 30 min to 3h, in particular, 0.5% (w/v) of the detergent. Finally, the samples were rinsed with PBS and washed with ultrapure water in agitation for 24 hours. [0083] Surprisingly, it was possible to obtain a decellularized rabbit dermal matrix with mechanical integrity and a suitable cell content using a short period of treatment with a detergent. Indeed, as compared to protocols known in the art that use detergent treatments of 12 to 24h, the method of the present disclosure, that uses 30 min to 3h, allows to obtain a matrix suitable for use in the treatment of skin wounds using less time.

[0084] In an embodiment, the disclosed decellularization protocol has a minimum impact on the ECM integrity, by combining specific detergents, enzymes, and physical treatments (Fig. 2). A highly preserved dRDM containing an open pore architecture suitable for tissue recellularization and infiltration is obtained.

[0085] Surprisingly, rabbit skin has favorable biomechanics and a thickness close to human skin, making it a possible alternative for developing a decellularized dermal matrix and tissue-engineered skin equivalent.

[0086] In an embodiment, cell lysis and removal efficiency, and structural, mechanical, biochemical, and cytocompatibility properties are compared to the native tissue.

[0087] In an embodiment drying and sterilization are performed using supercritical carbon dioxide (scCO2) (Fig. 3). Efficiency is assessed using standard SAL6, according to 15014937:2009.

[0088] In an embodiment, the obtained results (Fig. 4, Fig. 5, Fig. 8 to Fig. 15) validate the characteristics of rabbit skin as a viable substitute for human skin, both in anatomical and morphological terms, as well as in terms of mechanical properties and thickness.

[0089] In an embodiment, the decellularization protocol was optimized considering what were the best reagents and the optimal pH to apply the detergents (Fig. 6 and Fig. 7). The decellularization efficiency of each condition was evaluated by DNA extraction and quantification (Fig. 8). The integrity and preservation of the extracellular matrix (ECM) structure were assessed through the evaluation of mechanical properties and by SEM analysis (Fig. 9 to 13). [0090] In an embodiment, DNA quantification was performed by PicoGreen assay. The PicoGreen assay is a highly sensitive fluorescent dye-based method for quantifying double-stranded DNA (dsDNA). It involves the use of the PicoGreen dye, which selectively binds to dsDNA, causing a significant increase in fluorescence that can be measured using a fluorometer. This assay is widely used due to its high sensitivity, enabling the detection of very low concentrations of dsDNA.

[0091] In an embodiment, the decellularization protocols proved to be effective in removing cellular content, being able to reach the standard of 50 ng/mg dry tissue, and preservation of the ECM at different extents. After the deliming or deliming step, the skin graft presented a DNA concentration higher than 150 ng/mg dry tissue (Fig. 8, Control sample). Among the protocols studied, SDC applied for 30 minutes shows the best results, as it allows a rapid achievement of lower levels of DNA, near 50 ng/mg dry tissue. The mechanical tests also confirmed the integrity of the tested samples, being the condition "SDS 3h" the least preferred. Morphological analysis of the microstructure, by SEM, reveals no significant matrix degradation, although in SDS 3h treatment samples it is possible to identify a higher level of matrix degradation which corroborates with the mechanical properties analysis. When comparing the application of the detergent protocol of samples with a high pH (Post-Liming) and neutral pH (Post Deliming), it seems that there are no differences, so Deliming with the neutral pH was the one selected.

[0092] In an embodiment, the morphological analysis of the microstructure was performed by Scanning Electron Microscopy (SEM). In another embodiment, the mechanical properties were measured by tensile tests in uniaxial mode.

[0093] Human dermal fibroblasts (hDFs) were used for testing the in vitro cytocompatibility of the preserved dECMs, showing a cell viability superior to 70% (Fig 14). hDFs in direct contact with the dECMs showed an increased metabolic activity up to 7 days of culture into the dermal surfaces (Fig. 15). This shows that the disclosed decellularized scaffold is compatible with skin regeneration. [0094] Cell viability can be measured by different methods well known by those skilled in the art. In an embodiment, cell viability was analyzed by the AlamarBlue assay. Briefly, the AlamarBlue reagent, which contains the blue, non-fluorescent resazurin dye, is added to the cells to be analysed. Viable cells reduce resazurin to resorufin, a pink, fluorescent compound. After an incubation period, the degree of this reduction is measured using a plate reader, either by absorbance at 570 nm and 600 nm for the colorimetric assay or by fluorescence with excitation at 530-560 nm and emission at 590 nm. The results correlate with cell metabolic activity, providing an indirect measure of cell viability and proliferation.

[0095] In an embodiment, the disclosed decellularized dermal matrix was engineered in vitro as a dermo-epidermal skin substitute. Abdominal skin, collected from abdominoplasties, was obtained from Hospital Sao Joao (HSJ) under the authorizations delivered by the Ethical Committee and patient informed consent. The dermis and epidermis were separated enzymatically to isolate dermal fibroblasts (HDFs) and epidermal keratinocytes (HEKa) for expansion (Fig. 16a). Conditioned culture conditions were established, using the media blends provided in Table 2, already known in the literature [22], HDFs are seeded and cultured (5-6 days) on the hypodermis-contacting surface of the dRDM, while HEKa are seeded on the epidermis-contacting surface of the dRDM and left to mature for an additional 6 days [23], A dynamic perfusion platform was designed to validate the cell culture approach under flow conditions that better simulate the diffusion of nutrients in vivo and contribute to accelerating maturation (based on previous works [24], (Fig.16b).

Table 2 - Media blends used for co-culture.

Cells or type of culture Culture medium Culture period

HDFs+HMECs Dermal submersion media (1:1 1 week in 3D culture to mature HDFs media/HDMECs media) dermis with supplements

HEKa Dermal submersion media 3 days in 3D culture to epidermal with CaCk and supplements maturation

ALI Dermal submersion media Lift to ALI and maintain for 8 with CaCk and Selenium weeks HDFs - Human dermal fibroblasts; HDMECs - Human dermal microvascular endothelial cells; HEKa - Human epidermal keratinocyte cells; ALI - Air Liquid Interface

[0096] In an embodiment, the culture-generated dermo-epidermal skin substitute is evaluated according to its morphology, physicochemical and mechanical properties.

[0097] In a further embodiment, the disclosed dermo-epidermal skin substitute is engineered as a vascularized skin equivalent for full-thickness burn wound healing and regeneration (Fig. 17). A three-step cell-culture approach is implemented, using cells isolated from human skin, under conditioned culture conditions (Table 2) [22]:

(1) Dermal seeding and maturation, using co-culture of HDFs and human dermal microvascular endothelial cells (HDMECs). The HDFs are pre-seeded on the hypodermiscontacting surface (hypodermal side of dermis) of the dRDMs until adherence, followed by HDMECs seeding until maturation. Microchannels can stimulate pre-vascularization by guiding endothelial cells towards a tubular structure [25],

(2) Epidermal seeding is performed using the HEKa cells on the epidermis-contacting surfaceof the dRDM and let to mature for additional 3 days.

(3) The establishment of an Air Liquid Interface (ALI) allows for inducing epidermal stratification and culture maturation for additional 8 weeks [27], ALI is a long-standing method for culturing keratinocytes under physiological conditions [28], A dynamic perfusion platform is also used to validate under flow conditions the three-step cell culture approach under flow conditions on both plain and microneedling treated dRDM.

[0098] In an embodiment, the dermo-epidermal skin substitute is tested in a human ex vivo burn wound model for the validation of the dRDM and dRDM-derived skin equivalent in burn wounds. Skin explants are obtained from abdominoplasties provided by Hospital de Sao Joao under informed consent, according to approved ethical guidelines. The protocol for the ex vivo burn wound model is based on previous works [29,30], and is schematically represented in Fig. 18. Briefly, skin samples, after subcutaneous fat removal, are washed in PBS and soaked in DMEM with antibiotics, cut into equal pieces, mounted on a metal grid preventing retraction and placed in Petri dishes containing complete medium at 37°C with 5% CO2, in an ALI. The burn wounds are done in triplicate by placing a heated 5mm diameter metal for 5 seconds for a superficial wound or 10 seconds to form a second-degree burn wound. The debridement of the wound is performed on day 1 after the burn. Methodology is explained in Fig.16.

[0099] In an embodiment, the ex vivo model's viability and proliferation, is evaluated.

[00100] In another embodiment, the dermo-epidermal skin substitute, with or without cells, is implanted in the ex vivo burn wound model and screened for tissue regeneration, following standard histological staining protocols and comparative immunohistochemical analysis.

[00101] In an embodiment it is possible to use green methods to decellularize fullthickness rabbit skin while maintaining the integrity and mechanical performance of the ECM. A large sample volume can be used for decellularization, allowing to obtain dermal allografts with a set of biological, structural and biomechanical properties to cover large areas of the human body while promoting the regeneration of skin.

[00102] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[00103] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above-described embodiments are combinable.

[00104] The following dependent claims further set out particular embodiments of the disclosure.

References:

[1] D. Greenhalgh, "Managment of Burns," The New England Journal of Medicine, vol. 380, pp. 2349-2359, 2019. [2] T. Lang, R. Zhao, A. Kim, A. Wijewardena and e. al, "A Critical Update of the Assessment and Acute Management of Patients with Severe Burns," Wound Healing Society, vol. 8, no. 12, 2019.

[3] Y. Yao, A. Zhang, C. Yuan, X. Chen and Y. Liu, "Recent trends on burn wound care: hydrogel dressings and scaffolds," Royal Society of Chemistry - Biomaterials Science, vol.

9, pp. 4523-4540, 2021.

[4] X. Zhang, X. Chen, H. Hong, R. Hu, J. Liu and C. Liu, "Decellularized extracellular matrix scaffolds: Recent trends and emerging strategies in tissue engineering," Bioactive Materials, vol. 10, pp. 15-31, 2022.

[5] M. Dussoyer, A. Michopoulou and P. Rousselle, "Decellularized Scaffolds for Skin Repair and Regeneration," Applied Sciences, vol. 10, no. 10, 2020.

[6] M. Martinez-Ortiz, A. Hernandez-Fuentes, D. Pimentel-Gonzalez and e. al, "Extraction and characterization of collagen from rabbit skin: partial characterization," CyTA-Journal of Food, vol. 13, no. 2, p. 253-258, 2015.

[7] R. Parenteau-Bareil, R. Gauvin, S. Cliche, C. Gariepy, L. Germain and F. Berthod, "Comparative study of bovine, porcine and avian collagens for the production of a tissue engineered dermis," Acta Biomaterialia, vol. 7, no. 10, pp. 3757-3765, 2011.

[8] B. Dearman, S. Boyce and J. Greenwood, "Advances in skin tissue bioengineering and the challenges of clinical translation," Frontier in Surgery, vol. 8, p. 640879, 2021.

[9] W. Chua, Y. Khoo, B. Tan, K. Tan, F. C. and S. Chong, "Skin tissue engineering advances in severe burns: review and therapeutic applications," Regenerative Biomaterials, vol. 6, no. 6, pp. 437-447, 2019.

[10] S. Choudhury and A. Das, "Advances in generation of three-dimensional skin equivalents: pre-clinical studies to clinical therapies," Biotechnology Letters, vol. 40, no. 6, pp. 931-942, 2018.

[11] J. Yang, H. Dang and Y. Xu, "Recent advancement of decellularization extracellular matrix for tissue engineering and biomedical application," Artificial Organs, no. 46, p. 549-567, 2021. [12] D. Hsieh, P. Srinivasan, K. Yen, Y. Yeh, Y. Chen, H. Wang and Y. Tarng, "Protocols for the preparation and characterization of decellularized tissue and organ scaffolds for tissue engineering," Biotechniques, vol. 70, no. 2, 2020.

[13] T. Rajab, T. O'Malley and V. Tchantchaleishvili, "Decellularized scaffolds fortissue engineering: Current status and future perspective," Artificial Organs, vol. 44, no. 10, pp. 1015-1106, 2020.

[14] B. Kim, J. Kim, K. So and N. Hwang, "Supercritical Fluid-Based Decellularization Technologies for Regenerative Medicine Applications," Macromolecular Biosciences, vol. 21, no. 8, 2021.

[15] S. Badylak, R. Tullius, K. Kokini and e. al, "The use of xenogeneic small intestinal submucosa as a biomaterial for Achille's tendon repair in a dog model," Journal of Biomedical Materials Research, vol. 29, no. 8, pp. 977-985, 1995.

[16] M. Duarte, I. Silva, A. Eisenhut, N. Bionda, A. Duarted and A. Oliveira, "Contributions of supercritical fluid technology for advancing decellularization and postprocessing of viable biological materials," Materials Horizons, vol. 9, no. 3, pp. 864- 891, 2022.

[17] P. Bornstein and R. Ness, "The Comparative Biochemistry of Collagen: The Structure of Rabbit Collagen and Its Relevence to Immunochemical Studies of Collagen," Archives of Biochemistry and Biophysics, no. 138, pp. 443-450, 1970.

[18] P. Crapo, T. Gilbert and S. Badylak, "An overview of tissue and whole organ decellularization processes," Biomaterials, vol. 32, no. 12, pp. 3233-3243, 2011.

[19] A. Zambon, M. Vetralla, L. Urbani, M. Pantano, G. Ferrentino, M. Pozzobon, N. Pugno, P. Coppi, N. Elvassore and S. Spilimbergo, "Dry acellular oesophageal matrix prepared by supercritical carbon," The Journal of Supercritical Fluids, 2016.

[20] J. Wehmeyer, S. Natesan and R. Christy, "Development of a Sterile Amniotic Membrane Tissue Graft Using Supercritical Carbon Dioxide," Tissue Engineering, 2015.

[21] "ISO 14937:2009 Sterilization of health care products — General requirements for characterization of a sterilizing agent and the development, validation and routine control of a sterilization process for medical devices," 9 mar$o 2023. [Online], Available: https://www.iso.org/standard/44954.html. [22] M. Sanchez, T. Tonmoy, B. Park and J. Morgan, "Development of a Vascularized Human Skin Equivalent with Hypodermis for Photoaging Studies," Biomolecules, vol. 12, no. 12, 2022.

[23] C. Oostendorp, S. Meyer, M. Sobrio, J. Arendonk, E. Reichmann, W. Daamen and T. Kuppevelt, "Evaluation of cultured human dermal- and dermo-epidermal substitutes focusing on extracellular matrix components: Comparison of protein and RNA analysis," Burns, vol. 43, no. 3, pp. 520-530, 2017.

[24] H. Yan, H. Tang, W. Qiu, R. Tan, W. Zhang, G. Yang and J. Wu, "A new dynamic culture device suitable for rat skin culture," Cell and Tissue Research, vol. 371, pp. 723- 731, 2019.

[25] S. Dikici, F. Claeysses and S. MacNeil, "Bioengineering Vascular Networks to Study Angiogenesis and Vascularization of Physiologically Relevant Tissue Models in Vitro," ACS Biomaterials Science Engineering, vol. 6, no. 6, pp. 3513-3528, 2020.

[26] H. Chang, S. Chew, M. Zheng, D. Lio, C. Wiraja, Y. Mei, X. Ning, M. Cui2, A. Than, P. Shi, D. Wang, K. Pu, P. Chen, H. Liu and C. Xu, "Cryomicroneedles for transdermal cell delivery," Nature - Biomedical Engineering, pp. 1008-1018, 2021.

[27] S. Choudhury and A. Das, "Advances in generation of three-dimensional skin equivalents: pre-clinical studies to clinical therapies," International Society for Cell and Gene Therapy, vol. 23, no. 1, pp. 1-9, 2020.

[28] M. Prunieras, M. Regnier and D. Woodley, "Methods for cultivation of keratinocytes with an air-liquid interface," The Journal of Investigative Dermatology, vol. 81, no. 1, pp. 28-33, 1983.

[29] O. Gross-Amat, M. Guillen, D. Salmon, S. Nataf and C. Auxenfans, "Characterization of a Topically Testable Model of Burn Injury on Human Explants," International Journal of Molecular Sciences, vol. 21, 2020.

[30] N. Coolen, M. Vlig, A. Bogaerdt, E. Middelkoop and M. Ulrich, "Development of an in vitro burn wound model," Wound Repair and Regeneration, vol. 16, p. 559-567,

Claims

C L A I M S

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. 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. The method according to any of the previous claims wherein the detergent is selected from ionic detergent, non-ionic detergent, or mixtures thereof.

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. 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. The method according to any of the previous claims further comprising a sterilization step.

7. The method according to the previous claim wherein the sterilisation is a supercritical carbon dioxide sterilisation.

8. The method according to any of the previous claims further comprising a step of contacting the skin graft with an enzyme solution.

9. The method according to the previous claim wherein the enzyme is selected from trypsin, collagenase, DNases, or mixtures thereof.

10. The method according to any of the previous claims wherein the skin graft is from a rabbit.

11. A decellularized dermal matrix obtained by the method described in any of the previous claims.

12. A composition comprising a decellularized dermal matrix from rabbit, hare or beaver as described in any of the preceding claims.

13. The composition according to the previous claim wherein the decellularized dermal matrix is from a rabbit.

14. The composition according to any of the previous claims 12-13 further comprising cells, growth factors, active agents, or mixtures thereof.

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. 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. A composition as described in any of the previous claims 12-16 for use in medicine or veterinary medicine.

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. 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. 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.

21. The dermo-epidermal skin substitute scaffold according to the previous claim further comprising endothelial cells.

22. The dermo-epidermal skin substitute scaffold according to any of the previous claims further comprising a hydrogel, cells, a growth factor, or mixtures thereof.

23. The dermo-epidermal skin substitute scaffold according to any of the previous claims further comprising elastin, collagen and/or glycosaminoglycans.

24. Use of a composition as described in any of the previous claims 12-16 for the manufacture of a medicament for the treatment of a skin wound.

25. A method for treating a skin wound in a subject, the method comprising administering the composition as described in any of the previous claims 12-16 to the subject.