Skin patches containing bioabsorbable skin substitutes and related manufacturing processes

Abstract

The subject of the present invention relates to a bioabsorbable skin substitute A) for ulcers and wounds, obtained by subjecting a composition A1) comprising a cellular component a) comprising fibroblasts and a mixture b) comprising hyaluronic acid, fibrin and / or platelet-rich plasma gel (PRP) to a low-temperature procedure comprising the following steps: cooling composition A1) at 4°C and maintaining it at such temperature for a time comprised between 1 and 2 hours, freezing composition A1) at -20°C and maintaining it at this temperature for a time of at least 18 hours.

Description

[Technical Field]

[0001] The field of the invention relates to skin substitutes. More specifically, the invention relates to skin patches comprising bioabsorbable skin substitutes and related manufacturing processes. [Background technology]

[0002] Some epithelial tissue lesions are referred to as "difficult wounds," a composite concept intended to emphasize their characteristics of chronicity, lack of spontaneous closure, and the presence of concomitant systemic biological factors that prevent normal tissue repair. A "difficult wound" is defined as a lesion that, whatever the underlying cause, deviates from the normal process and time frame for resolution.

[0003] In relation to epithelial lesions, chronic cutaneous ulcers (CCU, or CU as an acronym for chronic ulcer) are defined as lesions characterized by parenchymal loss that persist for more than 60 days without showing any tendency to spontaneous healing (Westerhof W., "Leg ulcers: diagnosis and treatment," 1993, Elsevier). The current prevalence of ulcers, including leg ulcers, can be estimated to be between 0.18% and 0.32%, and for venous ulcers, between 0.06% and 1.0%. Studies considering both open and closed ulcers have estimated an overall prevalence of around 1 to 1.3 (Alexander House Group, 1992, "Consensus Paper on Venous Leg Ulcer," Phlebology 7:48-58). Prevalence rates for vascular lesions range from 1.8 to 3.05 per 1000 people, and prevalence increases with age. In Western countries, it is estimated that 10 per 1000 adults will suffer from a leg ulcer at least once in their lifetime (Margolis DJ, Bilker W, Santana J, Baumgarten M., Venous leg ulcer: Mar;46(3):381-6). The incidence is significantly higher in women, with a ratio of 3:1. Venous stasis ulcers are among the most common, with a prevalence of 70-80%, followed by arterial ulcers (15-20%). An epidemiological study conducted by Canonico et al. in the Osservatorio Geriatrico della Regione Campania (Campania Region Elderly Study), Canonico et al. "Prevalence of veins in an Italian elderly population" (Angiology 1998;49:129-3 135), showed that among people over 65 years of age, 42.5% of men and 57.5% of women had varicose vein disease of the lower extremities, and 3% had skin ulcers.More recent analyses have also confirmed the epidemiological importance of venous ulcers (Agale SV, "Chronic Leg Ulcers: Epidemiology, Aetiopathogenesis, and Management", 2013, Ulcers, 5, Volume 2013, Article ID 413604, http: / / dx.doi.org / 10.1155 / 2013 / 413604).

[0004] Although the prevalence of pressure lesions in hospitalized patients ranges from 18% to 29% (AISLe.C. 2010), well-conducted large-scale studies are difficult to find in the residential care population. Projections by the World Health Organization estimate that the number of people with diabetes in 2025 will exceed 300 million, compared with 120 million in 1996. From this, it is easy to intuit the scale of the problem. In fact, it is estimated that approximately 15% of people with diabetes will experience a foot ulcer requiring treatment during their lifetime. Although the diabetic population accounts for approximately 3% of the total population, more than 50% of all major amputations are performed on diabetic patients (http: / / www.iwgdf.org / ).

[0005] The socio-economic impact of CU is considerable. In assessing the cost of this condition, expenses related to pharmaceutical materials, transport time, and medical and nursing staff were taken into account, but other costs directly incurred by the patient (private medical care, ancillary care, lost working days, etc.) were not taken into account, nor were the indirect costs incurred by the National Health Service (hospitalization for surgery, infectious complications, bleeding episodes, neoplastic degeneration, etc.) that would further increase the cost of this condition (Rith-Najaran SJ et al., "Identifying diabetic patients at high risk for lower extremity, amputation in a primary care setting", Diabetes Care 1992;15:1386-9). Anglo-Saxon studies of the cost of dressings for the treatment of chronic ulcers indicate expenditures of approximately £1,300 to £2,500 per patient in the UK, equating to an annual cost of £2 to £3 billion (Rippon et al, "The economic impact of hard-to-heal leg ulcers", Wounds 2007; Vol 3, N2; Posnett, J., Franks, PJ, (2008) "The burden of chronic wounds in the UK", Nursing Times; 104:3, 44-45). The impact of such conditions on healthcare costs has long been known. For example, in Sweden (Baker, S., Stacy, M., Jopp-McKay, A., & Thompson, P. (1991), "Epidemiology of chronic venous ulcers", British Journal of Surgery, 78(7), 864-867), over £120 million is spent annually, which is approximately 1 percent of the national health budget.In the United States, authors Phillips et al. (Phillips T, Stanton B, Provan A, Lew R., "A study of the impact of leg ulcers on quality of life: financial, social and psychologic implications", J Am Acad Dermatol 1994, 31:49-53) reported 2 million lost workdays due to leg ulcers (affecting approximately 2.5 million people). These significant figures can be understood in light of the fact that 45% of skin ulcers result in immobility or a significant reduction in personal autonomy. All this without considering the reduction in quality of life. More recently, the estimated cost per patient with venous ulcers in Germany was approximately 9,500 euros (Purwis S. et al., "Cost-of Illness of Chronic Leg Ulcers in Germany", Int Wound 2010;7:97-102). Similarly, extremely high costs to healthcare budgets continue to be reported (van Gent WB et al, "Management of venous ulcer disease" BMJ 2010;341:c6045).

[0006] This situation is associated with an ever-increasing proportion of affected patients in the future, both due to increased life expectancy and consequent increase in the age groups most at risk, and also due to the increase in pathologies related to aging and / or peripheral vascular disease (e.g., diabetes), resulting in an increasing proportion of lesions that become chronic and / or resistant to available treatments.

[0007] As a result of all this, chronic lower limb ulcers (acronym CLLU) constitute, and will continue to constitute for many years to come, one of the most challenging problems in current medical research, both because of their significant epidemiological importance and because of the high proportion of lesions that do not heal or even recur despite patients receiving the correct therapeutic approach, with consequent impact on the costs of treatment and management (hospitalization, surgery, etc.) as well as societal costs.

[0008] Conventional treatments for ulcers, such as compression bandaging, can only achieve healing over a long period of time. However, complete and more rapid healing is desirable to avoid complications that can often be life-threatening to the patient. Over the past 30 years, tissue engineering techniques have enabled the large-scale production of skin substitutes (acronym SS) (Varkey M at al, J Funct Biomaterials 2015;6:547-563).

[0009] Skin substitutes are classified into the following product types based on the Agency for Healthcare Research and Quality (Maryland, USA) Technology Assessment Program, Report December 12: 1. Products containing autologous or allogeneic human cells; 2. Products made with human donor tissue; 3. Products derived from decellularized animal tissue; 4. Biosynthetic products.

[0010] Although these new treatment options constitute a significant improvement over more traditional approaches, there remains a need to find a definitive cure for chronic ulcers.

[0011] For example, product types 1-4 above have enabled improved care capacity but have not been able to fully meet medical demand. In this regard, it should be noted that CU, and consequently CLLU, fall into the category of conditions with an "unmet medical need." The European Medicines Agency (EMA) includes in this category conditions where current therapeutic approaches do not ensure adequate treatment for patients.

[0012] Due to the characteristics of the wound bed of chronic ulcers, the most suitable skin substitutes for this type of lesion are products containing autologous or allogeneic human cells processed in GMP-certified cell factories for the production of ATMPs (Advanced Therapies Medicinal Products), which are subject to European Regulation 1394 / 2007.

[0013] This type of product also has some drawbacks. There are various factors that can lead to the failure of healing of CUs, especially CLLUs, including the size of the ulcer, the presence of bacterial species within the wound, and the persistence of such ulcers (Slade B et al, Phlebological Rev 2015;2:29-38).

[0014] In addition to this, currently used skin substitutes have several limitations in their use, mostly due to the fact that they are unable to ensure sufficient revascularization, have poor adhesion to the wound bed, and interfere with skin tissue regeneration and therefore wound healing.

[0015] As far as chronic skin lesions are concerned, autologous skin substitutes, i.e., those made from skin samples of the patient being treated, require relatively long manufacturing times and are therefore generally unavailable for the emergency treatment of lesions covering large body surface areas (BSA). Furthermore, autologous skin substitutes are of little use in treating patients suffering from dystrophic epidermolysis bullosa (DEB). Furthermore, cells from patients with infectious diseases present serious difficulties for processing in pharmaceutical factories licensed to manufacture ATMP, making this type of treatment virtually unavailable to these patients (in over 70% of cases).

[0016] Instead, allogeneic skin substitutes, i.e., those made from skin samples from different subjects (living or cadaveric donors) processed in cell factories, are very useful in treating patients with extensive burns with extensive skin loss and can be used in infected wounds after thorough surgical cleaning of the wound bed.

[0017] Recently, several procedures have been proposed for creating skin substitutes based on fibroblasts and keratinocytes as well as for producing skin substitutes from other human cells (peripheral blood monocyte cells - PBMNC), such as those developed by the following companies:

[0018] 1. Organogenesis-Apligraft: This is a product formed from a skin graft containing viable keratinocytes and fibroblasts transplanted onto a bovine collagen type 1 support.

[0019] 2. S&N-HP802-247: This product consists of expanded cultured keratinocytes and fibroblasts implanted onto a fibrin gel. These cells are irradiated and then frozen to stop their growth process.

[0020] 3. CureXcell-Macrocure: This is a product based on PBMNC cells concentrated by centrifugation and activated by a hypotonic shock process (suspension of cells in a solution with low salt concentration). This product is injected directly into the lesion site. The proposed mechanism of action is to stimulate tissue regeneration through the action of growth factors contained in the cells.

[0021] However, such a procedure still has some disadvantages, such as:

[0022] 1. Organogenesis-Apligraft. This is a medical device approved in the US market, but has not been approved in the European market due to an inadequate definition of the mechanism of action of the cellular components. Furthermore, its clinical evidence has been deemed by the US regulatory authority (FDA) to not meet the treatment needs.

[0023] 2. S&N-HP802-247: After expansion, the cells undergo an irradiation process that destroys alarmin proteins and growth factors within the fibroblasts. This results in a product that is safe but lacks the regenerative capabilities necessary for the treatment of LCC. During Phase 3 clinical trials in the United States, the product was found to not meet pre-determined efficacy goals. The product did not demonstrate significant clinical benefit over placebo treatment, and the trial was discontinued, according to a statement on the manufacturer's website (http: / / www.smith-nephew.com / news-and-media / news / top-line-results-of-hp802-247-phase-3-study / ).

[0024] 3. CureXcell-Macrocure. This company conducted a Phase 3 trial of CureXcell to register it as an advanced medicine for treating LCC (EMA trial code - MC-105). The company acknowledged that the product did not meet the clinical efficacy endpoint. Therefore, the registration process has been suspended.

[0025] The skin is a fundamental organ that isolates the organism from the outside world and, to this end, possesses certain features at the molecular level that enable it to act as a physical barrier, to produce antimicrobial substances (at the level of the epidermis) and to perform immunomodulatory functions (at the level of the dermis).

[0026] The repair process in the skin requires the cooperation of several factors, including hyaluronic acid, the most abundant molecule in both the dermis and epidermis. The presence of hyaluronic acid is an essential marker for the early processes of healing and neovascularization, as well as the accompanying inflammatory phenomena necessary for the repair process. The role of hyaluronic acid in the epidermis is crucial, as it allows keratinocyte differentiation and is particularly abundant in the spinous and granular states of the epidermis. The absence of hyaluronic acid is incompatible with life. On the other hand, its overproduction in the dermis is observed in the Shar-Pei dog breed, and recently, the amount of high-molecular-mass hyaluronan has been suggested to be the reason for the long lifespan of naked mole rats (Tian X, Azpurua J, Hine C, Vaidya A, Myakishev-Rempel M, Ablaeva J, Mao Z, Nevo E, Gorbunova V, Seluanov A., "High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat", Nature. 2013 Jul 18;499(7458):346-9). Based on these reports, it seems scientifically justifiable to apply topical preparations containing high-molecular-mass hyaluronan, which have both moisturizing and protective properties, to damaged epidermis.

[0027] Recently, it has been observed that the biological effects of HA fragments are strongly dependent on the size of the oligosaccharide: in fact, the function of HA can change radically depending on its size, sometimes producing opposing effects. Recently, the role of HA fragments has been investigated in neovascularization (Gao F, Yang CX, Mo W, Liu YW, He YQ., "Hyaluronan oligosaccharides are potential stimulators to angiogenesis via RHAMM mediated signal pathway in wound healing", Clin Invest Med.2008;31(3):E106-16) and epidermal repair (Gariboldi S, Palazzo M, Zanobbio L, Selleri S, Sommariva M, Sfondrini L, Cavicchini S, Balsari A, Rumio C., “Low molecular weight hyaluronic acid increases the self-defense of skin 15 epithelium by induction of beta-defensin 2 via TLR2 and TLR4”, J Immunol.2008 Aug 1;181(3):2103-10), and that the presence of oligosaccharides in tissues during tissue repair is a natural phenomenon. HA oligosaccharides are produced by tissues themselves, and although the mechanism is still poorly understood, it involves both enzymatic (endogenous hyaluronidase) and physical (free radical) digestion.

[0028] Nevertheless, skin substitutes based on hyaluronic acid derivatives do not allow for the adequate treatment of LCC.

[0029] Furthermore, it is known that the manufacturing process of skin substitutes, which may also contain fibroblasts, necessarily involves a cryopreservation step. However, this appears to be to obtain a storage reserve and simply serves the function of preserving the skin substitute. The temperatures of the cryopreservation step of known processes are very low, lying in the range comprised between -180°C and -80°C. These low temperatures require a highly complex manufacturing process and high operating costs. In practice, these low temperatures are often obtained by using liquid nitrogen. [Prior art documents] [Non-patent literature]

[0030] [Non-Patent Document 1] Westerhof W., “Leg ulcers: diagnosis and treatment”, 1993, Elsevier [Non-patent document 2] Alexander House Group, 1992, “Consensus Paper on Venous Leg Ulcer”, Phlebology 7:48-58 [Non-patent document 3] Margolis DJ, Bilker W, Santana J, Baumgarten M., Venous leg ulcer:Mar;46(3):381-6 [Non-patent document 4] Canonico et al. “Prevalence of veins in an Italian elderly population”, Angiology 1998; 49:129-3 135 [Non-patent document 5] Agale SV "Chronic Leg Ulcers: Epidemiology, Aetiopathogenesis, and Management" 2013, Ulcers, 5, Volume 2013, Article ID 413604, http: / / dx.doi.org / 10.1155 / 2013 / 413604 [Non-patent document 6] http: / / www.iwgdf.org / [Non-Patent Document 7] Rith-Najaran SJ et al., “Identifying diabetic patients at high risk for lower extremity,amputation in a primary care setting”, Diabetes Care 1992;15:1386-9 [Non-patent document 8] Rippon et al, “The economic 30 impact of hard to heal leg ulcers”, Wounds 2007;Vol 3,N2 [Non-Patent Document 9] Posnett, J., Franks, PJ, (2008) “The burden of chronic wounds in the UK”, Nursing Times;104:3,44-45 [Non-Patent Document 10] Baker, S., Stacy, M., Jopp-McKay, A., & Thompson, P. (1991), "Epidemiology of chronic venous ulcers", British Journal of Surgery, 78(7), 864-867. [Non-Patent Document 11] Phillips T,5 Stanton B, Provan A, Lew R., "A study of the impact of leg ulcers on quality of life: financial, social and psychologic implications", J Am Acad Dermatol 1994, 31:49-53 [Non-Patent Document 12] Purwis S. et al, “Cost-of Illness of chronic leg ulcers in Germany”, Int Wound 2010;7:97-102 [Non-Patent Document 13] van Gent WB et al, "Management of venous ulcer disease", BMJ 2010; 341:c6045

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

[0031] Therefore, there is a need for epithelial tissue substitutes, particularly allogeneic skin substitutes, that overcome the disadvantages of epithelial tissue substitutes known from the background art and / or have better clinical efficacy. In addition, there is a need for a leaner and less complicated manufacturing process and for the advantage of increased production of molecules that can promote partial or total regeneration of injured / wounded or ulcerated epithelium. [Means for solving the problem]

[0032] The present applicant has determined that the first object of the present invention is to a) a cellular component including fibroblasts; b) a physical mixture containing hyaluronic acid, fibrin, and / or platelet-rich plasma gel The composition A1) comprising the following steps: - cooling composition A1) at 4°C and maintaining it at this temperature for a time comprised between 1 and 2 hours, - freezing composition A1) at -20°C and maintaining it at this temperature for a time at least equal to 18 hours developed a bioabsorbable skin substitute A) for ulcers and wounds, which is obtained by subjecting it to a low-temperature procedure including

[0033] The second object of the present invention is to a layer comprising or consisting of a bioabsorbable skin substitute A), an outer protective layer B) comprising an adhesive layer B1 placed in direct contact with the skin substitute A) and an outer support B2) placed on the adhesive layer B1; C) a skin patch comprising:

[0034] A third object of the present invention is a process for preparing a skin patch C), comprising the following steps: i) preparing a cellular component a) comprising fibroblasts; ii) adding the cellular components a) from the previous step i) to a physical mixture b') comprising hyaluronic acid, fibrinogen and / or platelet-rich plasma to obtain composition A2); iii) adding a fibrinogen activator, comprising or consisting of thrombin and an inorganic salt, preferably selected from trisodium citrate, sodium bicarbonate or sodium chloride, to obtain fibrin, and / or a platelet-rich plasma activator, preferably selected from batroxobin and / or calcium salts of organic or inorganic acids, to obtain a PRP gel, to the mixture b') from step ii) to obtain composition A1); iv) transferring the composition A1) from step iii) onto the protective layer B) in contact with the adhesive layer B1) to obtain a patch C1); v) cold treating the patch C1) in a process comprising the following sequential steps: cooling the patch C1) at 4°C and maintaining it at this temperature for a time comprised between 1 and 2 hours, freezing the patch C1) at -20°C and maintaining it at this temperature for a period of at least 18 hours to obtain the patch C),  The process of packaging the patches in a sterile environment. [Effects of the Invention]

[0035] Overall, the invention developed by the applicant is advantageous for the following reasons:

[0036] First, the cold treatment is not aimed at cryopreserving cellular components, but rather at increasing the production activity of substances released by the cells themselves due to the temperature stress experienced by the cells by the aforementioned cold treatment.

[0037] Substances of this type released by cellular components are, for example, growth factors and alarmins, which are beneficial for epithelial regeneration and have anti-inflammatory activity.

[0038] Note how in Examples 1 and 6, cell viability / activity was tested at a temperature of -20°C. Cell viability was drastically reduced or eliminated at this temperature. This condition is aimed at the release of a series of signaling molecules (including alarmins) that can stimulate tissue regeneration.

[0039] In contrast, as shown in Example 5, cells remain viable at -80°C. Therefore, they are not "activated" in the production of alarmins by RNA. Note that in experimental trials performed on cells in suspension at -80°C, it was difficult to quantify the RNA responsible for alarmin production. This demonstrates that the release of these signaling molecules is not possible at temperatures as low as -80°C. Applicant believes that such an inactivating effect at -80°C can also be found at temperatures <80°C.

[0040] At the same time, lowering the temperature of the low-temperature treatment is also important for the second purpose, namely, reducing or eliminating the proliferation of fibroblast cultures, thereby reducing or eliminating the problem of immune responses (immunogenicity) that may occur in the area of ​​the subject being treated with the allogeneic skin substitute of the present invention.

[0041] Furthermore, the present invention induces clinically significant efficacy that results from the increased controlled release of the aforementioned substances produced by the cells of the cellular component following heat treatment and is associated with the presence of component b), a physical mixture comprising hyaluronic acid, fibrin and / or platelet-rich plasma gel.

[0042] Finally, this manufacturing process is lean and uncomplicated compared to those known in the prior art: in fact, it does not require any special or time-consuming technical measures, such as those that may be associated with reaching relatively low temperatures.

[0043] Other advantages will become apparent in the following detailed description of the invention. [Brief explanation of the drawings]

[0044] [Figure 1] 1 is a graph showing the relative expression levels of alarmins before and after freezing in the test of Example 2 for the first trial. [Figure 2] 10 is a graph showing the relative expression of alarmins before and after freezing from the test of Example 2 for the second trial. [Figure 3] 10 is a graph showing the relative expression of alarmins before and after freezing from the test of Example 2 for the third trial. [Figure 4A] C) A sample of a skin patch of the present invention viewed from above. [Figure 4B] A skin patch C) sample of the present invention seen in perspective. [Figure 5A] 1 is a graph showing the relative expression of biglian as a function of different temperatures [room temperature = RT; -20°C; -80°C]. [Figure 5B] 1 is a graph showing the relative expression of tenascin-C as a function of different temperatures [room temperature = RT; -20°C; -80°C]. [Figure 6A] 1 is a graph showing the relative expression of biglian as a function of different temperatures [room temperature = RT; -20°C; -80°C]. [Figure 6B] 1 is a graph showing the relative expression of tenascin-C as a function of different temperatures [room temperature = RT; -20°C; -80°C]. DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention will be described in more detail below.

[0046] In the present invention, the definition "comprises" does not exclude the presence of elements / steps not expressly listed after such definition, whereas the definitions "constituted by" or "consisting of" exclude the presence of such further elements / steps.

[0047] In the present invention, "physical mixture" means a set of several substances that are not bound together by covalent or ionic bonds, but at most form unstable bonds of the hydrogen bridge type or van der Waals forces.

[0048] In the present invention, platelet-rich plasma or PRP is defined as the supernatant obtained after centrifuging whole blood to separate red blood cells from white blood cells.

[0049] PRP gel is defined as a gel obtained from PRP contacted with an activating agent, such as batroxobin, optionally in the presence of an organic or inorganic calcium salt, more preferably calcium chloride or calcium gluconate.

[0050] The first object of the present invention is to a) a cellular component including fibroblasts; b) a physical mixture containing hyaluronic acid, fibrin, and / or platelet-rich plasma The composition A1) comprising the following steps: - cooling composition A1) at 4°C and maintaining it at this temperature for a time comprised between 1 and 2 hours, - freezing composition A1) at -20°C and maintaining it at this temperature for a period of at least 18 hours A) is a bioabsorbable skin substitute obtained by subjecting it to a low-temperature procedure comprising:

[0051] Preferably, the cellular component a) is allogeneic. In surgery, the term "allogeneic" refers to tissue from another organism, i.e., from a donor. This term is the opposite of the term "autologous."

[0052] Preferably, the cellular component a) is not proliferative. This means that the cellular component a) is capable of being active and / or producing exogenous substances, but at the same time is not capable of growing or replicating or initiating mitotic processes. Thus, the cellular component a) comprising fibroblasts is preferably utilized as a natural source of growth factors and alarmins, which are key players in tissue repair or regeneration processes.

[0053] Preferably, the low temperature procedure comprises the following steps: - cooling composition A1) to 4°C and maintaining it at this temperature for 1.5 hours, - freezing composition A1) at -20°C and maintaining it at this temperature for a time equal to 18 hours.

[0054] The applicant believes that this low temperature procedure (which corresponds to the low temperature treatment of step v) of patch C1) according to the process of the present invention) serves to increase the production of exogenous substances or cellular products by the cellular components a), including fibroblasts, compared to the absence of this low temperature procedure, in other words, compared to when the skin substitute corresponds to A1).

[0055] Preferably, such cellular products are selected from the group consisting of growth factors, alarmins, interleukins, and mixtures thereof.

[0056] Preferably, alarmins (also called damage-associated molecular patterns or DAMPs) have the ability to trigger cellular responses after injury to promote regeneration / repair of the lesion / ulcer. Their antibiotic and immunomodulatory properties allow for faster healing of the lesion, wound or skin ulcer.

[0057] Preferably, the cellular product is selected from the group consisting of high mobility group box 1 (HMGB-1), biglycan, tenascin-C (TNC), fibroblast growth factor 2 (FGF-2), IL-6, IL-8, and mixtures thereof.

[0058] Preferably, the bioabsorbable skin substitute A) is of the allogeneic type, which preferably also constitutes a carrier for the release of proteins with regenerative capacity.

[0059] The cellular component a) of the skin substitute A), including fibroblasts, is prepared by the above-mentioned steps, i.e. - cooling composition A1) at 4°C and maintaining it at this temperature for a time comprised between 1 and 2 hours, - freezing composition A1) at -20°C and maintaining it at this temperature for a period of at least 18 hours It should be noted that the skin substitute A1) is obtained by a low temperature treatment including

[0060] This low temperature treatment corresponds to step v) of the process for preparing the patch C), which is a further object of the present invention, as previously mentioned.

[0061] Compared to intermediate A1), skin substitute A) differs essentially in that the cellular component a) contains substances due to freezing.

[0062] Both components b) of the skin substitute intermediate A1) consist of a physical mixture b) containing hyaluronic acid, fibrin and / or platelet rich plasma gel″.

[0063] Both the intermediate composition A1) and the component b) of the skin substitute, i.e. the physical mixture b) comprising hyaluronic acid, fibrin and / or platelet-rich plasma gel″, are obtained from a physical mixture comprising hyaluronic acid, fibrin and / or platelet-rich plasma in the presence of at least one fibrinogen activator and / or in the presence of at least one platelet-rich plasma activator, as described in step iii) of the process for preparing a skin patch, which is a further subject of the present invention.

[0064] The bioabsorbable skin substitute A) is particularly suitable for the treatment of ulcers and lesions, preferably chronic ulcers and lesions, since it is able to regenerate damaged or ulcerated skin tissue.

[0065] Skin patch C) The second object of the present invention is to an inner layer comprising or consisting of a bioabsorbable skin substitute A), an outer layer B) arranged on the skin substitute A), comprising an adhesive B1) arranged in contact with the skin substitute A) and an outer layer B2) arranged on the adhesive layer B2); C) a skin patch comprising:

[0066] The support B2) is a sterile support, preferably transparent, more preferably a highly permeable polyurethane film, and the adhesive layer B2 is a hypoallergenic, latex-free pressure-sensitive adhesive (PSA). This allows for optimal exchange of oxygen and water vapor, for example, maintaining adequate moisture in contact with the wound / ulcer. Furthermore, it is impermeable to liquids, bacteria, and viruses, thus protecting the insertion site from external contamination.

[0067] Preferably, the skin patch C) may also contain active ingredients useful for the purposes of the present invention that are not produced by the cellular components a), including fibroblasts, which may be contained, for example, in the adhesive layer B1.

[0068] Use of skin patch C) The skin patch C) is preferably used in the regeneration of tissue, preferably epithelial or cutaneous tissue, in particular in the treatment of wounds, ulcers or skin lesions.

[0069] Preferably, such ulcers are of the chronic type.

[0070] Preferably, the skin patch C) is applied with the skin substitute A) side to the wounded lesion or ulcer and the protective layer is removed after a period comprised between 15 and 23 days, preferably between 17 and 23 days, preferably between 17 and 21 days, preferably a period equal to 21 days.

[0071] Preparation process of skin patch C) A further object of the present invention is a process for preparing a skin patch C), comprising the following steps: i) preparing a cellular component a) comprising fibroblasts; ii) adding the cellular components a) from the previous step i) to a physical mixture b') comprising hyaluronic acid, fibrinogen and / or platelet-rich plasma to obtain composition A2); iii) adding a cross-linking agent comprising or consisting of thrombin to composition A2) from step ii) to obtain composition A1); iv) transferring the composition A1) from step iii) onto the outer adhesive layer B) to obtain a patch C1); v) subjecting the patch C1) to a low temperature treatment in a process comprising the following steps: cooling the patch C1) at 4°C and maintaining it at this temperature for a time comprised between 1 and 2 hours, Freezing the patch C1) at -20°C and maintaining it at this temperature for a period of at least 18 hours.

[0072] Step i) - Preparation of the cellular component a) containing fibroblasts Preferably, step i) of preparing the cellular component a) comprising fibroblasts comprises the following steps: i-1) preparing at least one human skin fragment obtained by biopsy from a skin donor; i-2) separating the epidermis from the dermis in a conventional manner; i-3) enzymatically treating the dermis to isolate fibroblasts; i-4) culturing fibroblasts in a suitable culture medium until confluent to obtain a cell culture of fibroblasts; i-5) Freezing the cell culture of fibroblasts obtained in the previous step.

[0073] Step i-1)—Providing at least one human skin fragment obtained by biopsy from a skin donor Step i-1) consists of providing at least one human skin fragment obtained by biopsy from a skin donor.

[0074] Preferably, first select skin donor.For example, this skin can be the skin waste obtained from weight loss surgery.Skin donor selection is carried out in accordance with the European Directive on the donation, procurement, management, processing, preservation, storage and distribution of human tissue and cells (2004 / 23 / EC, 2006 / 17 / EC and subsequent amendments and national legislation).

[0075] Preferably, once a skin donor is identified, a biopsy is performed to obtain at least one fragment of human skin.

[0076] Preferably, said at least one skin fragment is preferably stored in a suitable transport medium and then sent by an authorized courier in controlled transport at a temperature comprised between 2°C and 8°C to an approved pharmaceutical laboratory for the manufacture of a cell product.

[0077] Preferably, about 36 cm 2 of human skin waste is collected, but by keeping the same geometry, more or fewer flasks can be seeded after the digestion step.

[0078] Step i-2)—Separating the epidermis from the dermis The separation of the epidermis from the dermis is preferably carried out by incubation in the presence of a neutral protease. The operating conditions are preferably as follows: 6 DMCU / cm in 7 ml of HBSS. 2 -Tissue + 14 μl of 1 M CaCl2 solution, preferably at 4°C for 18 hours. After enzymatic digestion with neutral protease, the skin fragments are washed with DPBS (Dulbecco's phosphate-buffered saline). They are then transferred to a Petri dish, and the epidermis is separated from the dermis with sterile tweezers. The dermal fragments are then minced into small pieces with a scalpel and transferred to a 15 mL tube. DMCU is the following compound: (3-(3,4-dichlorophenyl)-1,1-dimethylurea). HBSS is Hank's balanced salt solution.

[0079] Step i-3)—Enzymatically treating the dermis to isolate fibroblasts The dermis is enzymatically treated, preferably with collagenase. The preferred operating conditions are: 0.3 PZU / ml in 10 ml of HBSS + 20 μl of 1 M CaCl, preferably for 2 hours at 37° C. At the end of the digestion, the solution is filtered to remove coarse debris and centrifuged to obtain a pellet of cells extracted from the tissue.

[0080] Step i-4)—To obtain a cell culture of fibroblasts, fibroblasts are cultured in an appropriate culture medium until confluent. Preferably, the fibroblasts are culture-expanded, preferably in flasks, in an appropriate medium until confluent, preferably 70-80% confluent.

[0081] Preferably, the appropriate culture medium or media consists of Dulbecco's Modified Eagle's Medium (DMEM), glutamine, sodium pyruvate, and fetal bovine serum (FBS) (irradiated and certified by the European Directorate for the Quality of Medicines (EDQM)). The formula for complete culture medium or media (CM) is as follows: 500 mL DMEM, 10 mL 200 mM glutamine, 50 mL FBS, 5 mL 1 M sodium pyruvate.

[0082] Preferably, the culture expansion process scheme according to step i-4) is as follows: - Seed six T25 flasks -Expansion in 6 T150 flasks -Seeding into one 5-layer cell stack - Seeding density is approximately 5000 cells / cm 2 Further divisions are made in 5-layer cell stacks depending on the required batch size while maintaining the value of

[0083] All media components must be sterile.

[0084] Preferably, culture expansion is performed under a laminar flow hood (grade A) located in a grade B area, and components are aseptically added to bottles containing DMEM.

[0085] Preferably, the medium is changed every 2 to 4 days, and cell growth is checked under a microscope after each medium change.

[0086] At each split, the medium is removed from the flask, the flask is then washed with Dulbecco's phosphate buffered saline (DPBS), recombinant trypsin is added, and the cells are then seeded into flasks or cell stacks.

[0087] Step i-5) - Freezing the fibroblast cell culture obtained in the previous step Preferably, at the end of the expansion of the previous step i-4), the cell culture of fibroblasts is collected and separated into containers or vials or cryovials. Preferably, each vial or each cryovial contains 10 x 10 6 Contains cells.

[0088] The fibroblast cell culture is then preferably frozen in a suitable medium and in the presence of a cryopreservative or cryoprotectant.Preferably, the suitable medium is fetal bovine serum (FBS or PBS).Preferably, the cryopreservative is dimethyl sulfoxide (DMSO), preferably the latter being equal to 10% of the total content.

[0089] Preferably, the cellular components a) are thawed prior to step ii) of adding component a) from the previous step i) to the physical mixture b') comprising hyaluronic acid, fibrinogen, and platelet-rich plasma. Preferably, for purposes of the present invention, the volume of the thawed cellular components a) is 1 ml. Preferably, the thawed cellular components a) are placed in a 15 ml tube.

[0090] Preferably, after thawing, an appropriate medium, saline or PBS (phosphate buffered saline), preferably 9 ml of PBS, is added to the cellular component a), then the appropriate medium is added and the cellular component a) is centrifuged, the supernatant is then removed and the resulting solid (or pellet) is resuspended in NaCl saline, preferably 200 μl of NaCl.

[0091] Step ii) - Adding the cellular component a) from step i) above to the physical mixture containing hyaluronic acid, fibrinogen and / or platelet-rich plasma. First, a physical mixture b') containing hyaluronic acid, fibrinogen and / or platelet-rich plasma is prepared.

[0092] The hyaluronic acid used in this step is first sterilized by filtration through a 0.2 micron filter and then, in step ii) of the process of the present invention, aseptically added to fibrinogen or PRP in a sterile solution in an ISO 7 clean room within an ISO 5 biosafety facility to give physical mixture b').

[0093] Preferably, the amount of hyaluronic acid added in step ii) is comprised between 0.5 mg / ml and 5 mg / ml, preferably between 1 mg / ml and 4 mg / ml, by weight relative to the total volume of the physical mixture, and exhibits a weight average molecular weight comprised between 1000 kDa and 3000 kDa.

[0094] The cellular components from the previous step, suspended in an appropriate medium, are then added to the physical mixture b').

[0095] Step iii) - adding a cross-linking agent comprising or consisting of thrombin to the primary mixture obtained in step ii) to obtain composition A1). In step iii), an activator for activating fibrinogen and converting it to fibrin, and / or comprising or consisting of thrombin and at least one inorganic salt, batroxobin and / or a calcium salt of an organic or inorganic acid for precipitating the PRP gel, is added to the mixture b') from step ii) to obtain composition A1).

[0096] The organic or inorganic salts present in the aqueous thrombin solution are preferably selected from trisodium citrate, sodium bicarbonate, sodium chloride, calcium chloride.

[0097] The thrombin is added as a dilute aqueous solution, preferably a 1:40 thrombin / calcium chloride solution, obtained by adding 10 μl of 1000 U / ml thrombin to 390 μl of 1 M CaCl2.

[0098] Preferably, in step iii), 134 μl of thrombin is added to the primary mixture obtained from step ii) to initiate polymerization with fibrin glue formation. Preferably, gentle mixing is used during this step iii) to avoid the formation of air bubbles.

[0099] Preferably, the concentration of sodium bicarbonate is comprised between 1 mg / ml and 3 mg / ml, preferably 2 mg / ml.

[0100] Preferably, the concentration of sodium chloride is comprised between 7 mg / ml and 10 mg / ml, preferably between 8 mg / ml and 9 mg / ml.

[0101] Preferably, the formation of the skin substitute A1) is carried out at a temperature comprised between 35°C and 40°C, preferably between 36°C and 38°C, preferably equal to 37°C.

[0102] The organic or inorganic calcium salt added to batroxobin to precipitate the PRP gel is selected from calcium chloride and calcium gluconate.

[0103] This combination of hyaluronic acid-fibrin and / or PRP gel obtained in step iii) and contained in the skin substitute A that is the subject of the present invention and, consequently, in the patch C, makes it possible to obtain a safe and stable product. Indeed, the mixture of hyaluronic acid-fibrin and / or PRP gel combines the viscoelastic, protective and anti-inflammatory properties specific to hyaluronic acid with those of a fibrin-based matrix and / or PRP gel to generate a unique biocompatible environment that stimulates the physiological regeneration processes of the skin.

[0104] Step iv) - Transferring the composition A1) from step iii) onto the outer adhesive layer B) to obtain a patch C1) Preferably, a protective layer B) comprising a support B1 based on a transparent polyurethane film is first prepared, to which an adhesive layer B2 comprising a hypoallergenic and therefore latex-free pressure-sensitive adhesive (PSA) is applied.

[0105] Preferably, such a protective layer B) comprising both the support B2 and the adhesive layer B1 is a commercially available product. Preferably, the size of the protective adhesive layer B) is 10 x 12 cm.

[0106] Preferably, the packaging of the protective adhesive layer B) is opened in a laminar flow hood and the protective adhesive layer B) is then placed in a capsule or petri dish, preferably rectangular and of appropriate size.

[0107] Composition A1) is preferably immediately transferred from adhesive layer B1 onto the surface of outer protective layer B) placed inside a Petri dish, to obtain a patch C1), which is preferably covered with a suitable lid.

[0108] Preferably, the patch C1) is left at room temperature (RT) for a time comprised between 15 and 25 minutes, preferably 20 minutes, where room temperature is understood to mean a temperature comprised between 18°C ​​and 25°C, preferably not exceeding 25°C.

[0109] Step v) - Cold treatment of patch C1) Then, after the required time has elapsed, a suitable freezing solution is prepared and placed on the patch C1), preferably covering it with at least one layer (veil) of freezing solution.

[0110] Preferably, the freezing solution consists of 90% NaCl saline and 10% dimethyl sulfoxide (DMSO). Preferably, the freezing solution placed on the patch C1) is equal to 1 ml.

[0111] Preferably, the low-temperature treatment of the patch C1) is carried out in a low-temperature process comprising (or consisting of) the following successive substeps: cooling the patch C1) at 4°C and maintaining it at this temperature for a time comprised between 1 and 2 hours, Freezing the patch C1) at -20°C and maintaining it at this temperature for a time equal to at least 18 hours and until final use.

[0112] Step vi) Packaging the patch under a sterile atmosphere The patch C1) is transferred from inside the Petri dish to inside a suitable container, preferably inside an aluminum bag, which is then finally sealed (preferably heat sealed) and labeled. [Example]

[0113] The following examples are provided for illustrative purposes only and not for limitation.

[0114] Example 1 - In vitro testing of cell viability The cell viability of fibroblasts inserted into the skin patch of the present invention (C) was assessed after freezing using the reagent Alamar Blue and a corresponding colorimetric assay, which provides an indication of the metabolic activity of the cells by calculating the percentage reduction of the Alamar Blue reagent.

[0115] 1.1 First attempt (April 27, 2021) Freezing included a 1.5 hour portion at 4°C and an 18 hour portion at -20°C.

[0116] At each time point, the metabolic activity of the fibroblasts was assessed, which correlates with cell viability. [Table 1]

[0117] The data in Table 1 highlight the 4% residual metabolic activity at -20°C. These results therefore indicate that freezing leads to a high mortality rate of fibroblasts. This condition is essential for the cells to comprehensively release a range of signaling molecules (including alarmins) that can stimulate tissue regeneration.

[0118] 1.2 Second attempt (November 24, 2021) In this trial, the metabolic activity of three different batches of fibroblasts (FB batch 1, FB batch 2 and FB batch 3) was analyzed after leaving the scaffolds at −20°C for 18 hours, 7 days, 14 days and 28 days. [Table 2]

[0119] The data in Table 2 highlight that the residual metabolic activity after 18 hours at -20°C was 4% for FB batch 3, confirming the data from the first trial, 11% for FB batch 1, and 9% for FB batch 2.

[0120] After 7 days at -20°C: activity for FB batch 2 is negative, but appears to increase slightly for FB batch 1 and FB batch 3, to 17% and 11%, respectively.

[0121] After 14 days at -20°C, the metabolic activity of FB Batch 1 is 0, FB Batch 2 is at a percentage of 4% and FB Batch 3 is at 12%.

[0122] After 28 days at -20°C, metabolic activity becomes negative in FB batch 1, decreases to 7% in FB batch 2 and to 8% in FB batch 3.

[0123] Considering the results obtained, we conclude that the residual metabolic activity of less than 12% is not related to cell viability but rather to the presence of enzymes within the cell debris that are still able to reduce the Alamar Blue reagent used in the assay.

[0124] Thus, we confirmed that freezing at -20°C leads to high mortality of fibroblasts as early as 18 hours, a condition essential for the cells to release a comprehensive set of signaling molecules (including alarmins) that can stimulate tissue regeneration.

[0125] Example 2 - In vitro assessment of alarmin expression levels after the freezing process The analysis focused on the production of alarmins by fibroblasts inserted into the scaffolds.

[0126] RT-PCR (Real Time-PCR) assays were performed to evaluate the expression levels of alarmins HMBG1, biglycan and tenascin-C, fibroblast growth factor (FGF2) and interleukins (IL-6 and IL-8).

[0127] Genes observed in real time: High Mobility Group Box 1 (HMGB1) is a non-histone nuclear protein. It is ubiquitously present under physiological conditions and regulates DNA transcription, replication, and repair. Under stress conditions, it is secreted in a hyperacetylated form and supports tissue regeneration by stimulating cell activation, differentiation, and migration (fibroblasts, monocytes / macrophages, dendritic cells, and endothelial cells). Biglycan (BGN) is a proteoglycan present on the cell membrane, where it can interact with various extracellular molecules (TNF-alpha, TGF-beta, cytokines, growth factors, etc.). Following stress, cells increase the production of biglycan, which is released extracellularly. In its soluble form, it acts as an alarmin by interacting with cells of the immune system (macrophages and neutrophils) and activating the immune response. Tenascin-C (TNC) is an extracellular matrix glycoprotein with a regulatory role in inflammation. Following tissue injury or cellular stress, its expression is upregulated within the first 24 hours, leading to the resolution of the inflammatory state. -Fibroblast growth factor 2 (FGF2) is a growth factor expressed by fibroblasts and is used as a cell marker. Both IL-6 and IL-8 are cytokines that mediate inflammation. In particular, IL-8 has pro-inflammatory activity.

[0128] The analysis is -Before freezing (immediately after polymerization) and -After freezing (18 hours at -20°C) This was done by comparing the relative expression levels of

[0129] 2.1 First Attempt (March 31, 2021 and April 14, 2021) Figure 1 shows that for this first trial, dermal-derived fibroblasts express the alarmins tested. The heat shock, performed by shifting to 4°C and subsequent freezing at -20°C, led to changes in the expression of the alarmins considered, with the exception of HMGB1. It can further be seen that the freezing gradient used favors an increase in the expression of biglycan and tenascin-C, as well as a decrease in the inflammatory cytokines IL-6 and IL-8.

[0130] As our interest was in alarmins, in the next two trials (second and third), we analyzed only biglycan, tenascin-C, HMGB1 and the growth factor FGF2 as a marker of fibroblast-like cells.

[0131] 2.2 Second attempt (June 8, 2021) The results confirm the previous analysis, as shown in Figure 2. The freezing process resulted in increased expression levels of the alarmins biglycan and tenascin-C, proving to be a critical step for the presence of pro-regenerative factors in the final product.

[0132] 2.3 Third attempt (October 20, 2022) As can be seen in Figure 3, the data again confirm the results of the other two trials: the freezing process leads to an increase in the expression of the alarmins biglycan and tenascin-C. In addition, an increase in HMGB1 alarmin levels is also observed.

[0133] Example 3 - In vitro evaluation of alarmin production and release after freezing process We first proceeded to quantify the total proteins released by fibroblasts inserted into the scaffolds after the freezing process.

[0134] The samples considered were: - a scaffold or skin patch containing fibroblasts (referred to as "gel + cells"), and -Scaffolds or skin patches without cells (referred to as "gel only" or "blank sample"), which served as blanks (fibrinogen / fibrin, HA, and thrombin were present inside the scaffold) is.

[0135] BCA (microbicinchoninic acid (BCA) protein assay) data are summarized in Table 2 below. [Table 3]

[0136] Next, we aimed to verify and quantify the presence of alarmins produced and released by fibroblasts inserted into the scaffolds after freezing using an Elisa assay.

[0137] The targets tested were HMBG1, biglycan, tenascin-C and fibroblast growth factor (FGF2).

[0138] Table 3 summarizes the data (units: pg / ml) obtained during the two trials performed. The samples considered were fibroblast-containing scaffolds ("gel + cells") after a controlled freezing ramp and 18 hours at -20°C, as well as scaffolds without cells ("gel only") used as a negative control (no alarmins should be present in the gel only sample). [Table 4]

[0139] These analyses show that fibroblasts release proteins within the scaffold, and therefore the skin patch product of the present invention will be enriched in these regenerative factors useful for ulcer healing.

[0140] It can be seen that even in the presence of "gel only," certain levels of, for example, FGF2 can be observed, due to possible cross-reactivity or non-specific binding of the secondary antibody in the ELISA assay to proteins present in the gel only sample.

[0141] Example 4 - In vitro assessment of alarmin levels before and after freezing Following the results obtained, we wanted to understand whether this freezing process (1 hour 30 minutes at 4°C followed by -20°C) could stimulate an increase in the production of the alarmins biglycan, tenascin-C and HMGB1.

[0142] Two cell-containing gels ("gel + cells") were prepared: after polymerization, one was used to extract proteins before freezing ("before freezing" sample), and the other was first frozen and then used for protein extraction ("after freezing" sample).

[0143] Total Protein Quantification by BCA: [Table 5]

[0144] The values ​​obtained from the ELISA assays performed are summarized in Table 5 below (pg / ml). [Table 6]

[0145] Comparing the pre-freezing and post-freezing samples, we found that HMGB1 increased by 40% and biglycan increased by 80%, but tenascin-C did not increase and remained at the same level. The inventors point out that low-temperature treatment is essential for concentrating regeneration-promoting factors such as alarmins.

[0146] Note that in Example 4, HMGB1 is increased but TNC is not increased compared to the run in Example 2. This is due to the type of assay performed: Example 2 evaluates the expression of messenger RNA in cells that encodes a certain protein, whereas Example 4 quantifies protein extracted from gel + cells.

[0147] Example 5 - Evaluation of alarmin expression at different temperatures A set of experiments was performed to identify freezing processes that can cause increased levels of alarmins produced by fibroblasts seeded within hyaluronic acid ("HA") and fibrin scaffolds.

[0148] All cell and gel samples were prepared and processed in the same way: RNA was extracted before freezing, then at -20°C and -80°C. RNA from these samples was subjected to reverse transcription and then RT-PCR (real-time PCR) assays to assess the expression levels of the alarmins biglycan and tenascin-C.

[0149] As can be seen in Figure 5A, biglycan expression increases after freezing at -20°C and then decreases at -80°C. Similarly, as can be seen in Figure 5B, tenascin-C expression also increases after freezing at -20°C but decreases at -80°C.

[0150] Temperature stress on the cells showed that keeping samples at -20°C induced an increase in alarmin RNA expression, whereas at -80°C their expression was similar to that obtained at room temperature.

[0151] These results were confirmed by a second set of experiments performed using the same procedure (see Figures 6A and 6B).

[0152] Example 6 - Stability of fibroblast cellular components Stability assays of dermal fibroblasts were performed to demonstrate that their viability remained high over time and was limited to the scaffold preparation process, and that transferring the HA-Fb ("hyaluronic acid-fibrin") and cell mixture to -20°C resulted in cell death, with the beneficial consequence of stimulating alarmin release by the fibroblasts. Note that before mixing with HA-Fb, the cells were thawed in saline, centrifuged, and the pellet resuspended in 0.2 ml of saline.

[0153] The prepared dermal fibroblasts were frozen in cryovials in the appropriate freezing solution (saline and 10% DMSO). At the time of freezing, the viability was 96%. For stability testing, cryovials prepared at time 0 were kept in liquid nitrogen until various thawing times.

[0154] Table 6 shows the cell stability data in saline and 10% DMSO. [Table 7]

[0155] The data obtained show that the cells have a high viability of over 85% after thawing and before being placed into the HA-Fb scaffolds, and that it is the freezing process of the final product at -20°C that induces programmed fibroblast death, which leads to the release of alarmins.

[0156] This finding was confirmed by assays of fibroblast metabolic activity, which correlates with cell viability (Table 7). [Table 8]

[0157] These results therefore indicate that freezing leads to high mortality of fibroblasts, a condition essential for the cells to comprehensively release alarmins, a suite of signaling molecules that can stimulate tissue regeneration.

Claims

1. a) a cellular component comprising fibroblasts; b) a physical mixture comprising hyaluronic acid, fibrin, and / or platelet-rich plasma gel A composition A1) comprising the following sequential steps: - cooling said composition A1) at 4°C and maintaining it at this temperature for a time comprised between 1 and 2 hours, - freezing said composition A1) at -20°C and maintaining it at this temperature for a period of at least 18 hours; A) Bioabsorbable skin substitute for ulcers and wounds obtained by subjecting the skin to a low temperature procedure comprising:

2. The skin substitute of claim 1 , wherein the cellular component a) is allogeneic.

3. a layer comprising or consisting of a bioabsorbable skin substitute A) according to claim 1 or claim 2, an outer protective layer B) arranged on the skin substitute A), comprising an adhesive layer B1) arranged in direct contact with the skin substitute A) and an outer support B2) arranged on the adhesive layer B1); C) a skin patch containing

4. A skin patch, wherein the adhesive layer B) is a polyurethane film containing a non-allergenic, latex-free pressure sensitive adhesive (PSA).

5. A process for preparing a skin patch C) according to claim 3 or claim 4, comprising the following steps: i) preparing said cellular component a) comprising fibroblasts; ii) adding said cellular components a) from the previous step i) to a physical mixture b') comprising hyaluronic acid, fibrinogen and / or platelet-rich plasma to obtain composition A2); iii) adding a fibrinogen activator and / or a rich plasma gel activator to said composition A2 from step ii) to obtain composition A1 according to claim 1, wherein said activators comprise or consist of thrombin and at least one organic or inorganic salt of an alkali metal or alkaline earth metal to precipitate said fibrin, and / or comprise or consist of a calcium salt of an organic or inorganic acid to precipitate said platelet gel. iv) transferring said composition A1) from step iii) onto said outer layer B) in contact with said adhesive layer B1) to obtain a patch C1); v) subjecting the patch C1) to a cold treatment process comprising the following steps: cooling the patch C1) at 4°C and maintaining it at this temperature for a time comprised between 1 and 2 hours, freezing the patch C1) at -20°C and maintaining it at this temperature for a period of at least 18 hours, vi) packaging said patch in a sterile environment.

6. 6. The process of claim 5, wherein step i) comprises the steps of: i-1) providing at least one human skin fragment obtained by biopsy from a skin donor; i-2) Separating the epidermis from the dermis; i-3) enzymatically treating the dermis to isolate fibroblasts; i-4) culturing the fibroblasts in a suitable medium until confluent to obtain a cell culture of said fibroblasts; i-5) Freezing the cell culture of said fibroblasts obtained in the previous step.

7. 7. The process of claim 5 or claim 6, wherein prior to step ii), the cellular components a) are thawed.

8. The process according to any one of claims 5 to 7, wherein step v) is carried out in the presence of a cryopreservative, preferably dimethyl sulfoxide.

9. 5. A patch according to claim 3 or claim 4 for use in the treatment of wounds, ulcers or skin injuries.

10. The patch for use according to claim 9, wherein the ulcer is a chronic ulcer.

Images