WO2013045689A1 - Therapeutic use of gelatin hydrogels with a gel-sol transition at body temperature - Google Patents

Abstract

The invention regards the use of gelatin hydrogels that are solid at room temperature and melt or present a gel-sol transition at temperatures near the body temperature, so assuming a liquid state when put contact with body, implanted in the body or heated at temperatures compatible with the body. The gelatin-based hydrogels may contain biologically active agents and/or cells that after hydrogel melting or gel-sol transition, are fast released in situ, thus explicating the therapeutic and biological action. The gelatin-based hydrogels, containing or not containing biologically active agents, may be solid at the temperature of the skin (33°C) and melt when water or aqueous solution at a temperature compatible to the body is poured onto for an easy removal of the hydrogels.

Description

THERAPEUTIC USE OF GELATIN HYDROGELS WITH A GEL-SOL TRANSITION

AT BODY TEMPERATURE

FIELD OF INVENTION

The invention relates to gelatin hydrogels that are solid at room temperature and melt at temperatures near the mammalian body temperature and uses thereof. The hydrogels assume a liquid state when placed in contact with the body, when implanted into the body or when heated at temperatures compatible with the mammalian body temperature. The gelatin hydrogel may be in the form of a membrane, film, fiber, woven and non-woven fabric, foam, microbead or particle. The gelatin hydrogels may also contain biologically active agents and/or cells and/or stem cells and/or conditioned cell medium which are encapsulated and/or entrapped and/or loaded therein. Upon gel melting, such additional components are rapidly released in situ, thus exerting their therapeutic and biological action. BACKGROUND OF INVENTION

Collagens have a tertiary unique structure given by amino acid sequences. The collagen molecules of different sources consist of three polypeptide chains twined around one another as in a three-stranded rope.

Collagen represents the primary structural protein accounting for approximately 30% of all vertebrate body protein. More than 90% of the extracellular protein in the tendon and bone and more than 50% in the skin consist of collagen. Although most of the scaffolding in mammals is composed of collagen, the collagenous spectrum ranges from Achilles tendons to the cornea. Hence, different collagen types are necessary to confer distinct biological features to the various types of connective tissues in the body. Currently at least 13 types have been isolated which vary in the length of the helix and the nature and size of the non-helical portions.

In the manufacture of gelatin, treatment of the animal collagen raw material with dilute acid or alkali results in partial cleavage of these cross-links; the structure is broken down to such an extent that "warm water-soluble collagen", i.e. gelatin, is formed. This chemical hydrolysis can be supplemented or even replaced by the use of enzymes.

The goal of the gelatin manufacturer is to carry out a controlled partial hydrolysis of the crosslinks and peptide bonds of the original collagen structure and to obtain the ideal molecular weight distribution of gelatin for the application envisaged. The viscosity of a gelatin solution, for example, correlates relatively well with the proportion of high molecular weight components. Gelatins are widely used as biomaterials in drug delivery, pharmaceutical applications and regenerative medicine. Moreover, gelatin is biodegradable, bioreabsorbable, no-toxic, and exhibits weak immunogenicity and superior biocompatibility compared to synthetic polymers and to other natural polymers.

The attractiveness of gelatin as a biomaterial rests largely on the view that it is a natural material of low immunogenicity and is therefore seen by the body as a normal constituent rather than foreign matter. Gelatin can be processed into a number of forms such as films, membranes, sheets, tubes, capsules, beads, nets, sponges, powders, fleeces, injectable solutions and dispersions, micro and nano-spheres, single unit devices, or other geometrical forms, all of which have found use in medical practice. Furthermore, gelatin has been applied for drug delivery in a variety of applications, such as ophthalmology, wound and burn dressing, tissue repair and tissue engineering, inserts and physical barrier shields.

Cross-linked gelatins have also been used to confer mechanical firmness and collagenase resistance by introduction of exogenous cross-linking agents into the molecular structure. In fact, to address some of the drawbacks in gelatin based applications due to the poor mechanical strength of gelatin, improvement of their physical, chemical and biological properties has often been needed.

For instance, the patent application WO 2008/076407 discloses a composition comprising gelatin and a non-toxic cross-linking agent such as transglutaminase.

However, a major handicap of chemical cross-linking agents is the potential toxic effect of residual molecules and/or compounds formed during in vivo degradation.

Alternative physical methods have been pursued, including dry heat, exposure to ultraviolet or gamma-irradiation. In these cases, the drawback is that gelatin becomes partially denatured by these physical treatments.

Extensive reviews on collagens and gelatin, their sources, physical and chemical properties, treatments and applications are in Friess W., Eur. J. Pharm. Biopharm. 45, 113-136, 1998, Lee C.H. et al, Int. J. Pharm. 221, 1-22, 2001 , and air et al, JITPS 1(7), 288-304, 2010.

Bioactive molecules may be loaded by different ways into the gelatin matrix which can be both in a solid state or in an hydrogel form. The hydrogel is typically obtained by solidification from a solution or by co-precipitation in suitable conditions. The bioactive molecules are then entrapped in the interstices of the gelatin matrix, which acts as a reservoir.

The release of the bioactive molecules occurs from gelatin hydrogels, prepared with different methods, size and form and then implanted in the human body site. The gelatin hydrogels can also be generated in situ from a solid matrix when placed in contact with an aqueous environment by a swelling mechanism. In both cases, the final aim is to provide a hydrogel, single unit or multi-particulate, from which a bioactive molecule can be released. The release may occur by simple diffusion in the case of cross-linked hydrogels or by a more complex mechanism of diffusion including concurrent erosion by and dissolution in aqueous solutions of the outer layer of the collagen matrix.

Many delivery systems of bioactive molecule have been developed by exploiting the properties of single unit and multi-particulate hydrogels.

Gelatin film, or sheet, or disc has been used for the treatment of tissue infection, such as infected corneal tissue or liver cancer, and wound healing by placing in contact the hydrogel with the part to be treated.

Gelatin sponges have been very useful in the treatment of severe burns and as a dressing for many types of wounds, such as pressure sores, donor sites, leg ulcers and decubitus ulcers as well as for in vitro test systems.

Gelatin gel micro-particles have primarily been used for injectable systems. Gelatin micro- particles containing the bioactive molecules are injected into the tissue site of interest, the molecule then being released in a controlled manner.

Minipellets made of gelatin, typically in rods, have also been developed.

The rod (minipellet) is small enough to be injected into the subcutaneous space through a syringe needle.

Physically resistant gelatin gels, also containing bioactive agents, have been applied more recently as bone substitutes and, more in general, bioengineered tissues.

In all these applications, the gelatin gels must be physically and mechanically sufficiently resistant to stay in situ for a prolonged time period. They must also control the release of the bioactive agents. Consequently, the gel-sol transition temperature of the gelatin hydrogel, also called as the melting temperature of the gel, must be higher than the body temperature, namely at least 43-45°C. EP 0 518 697 discloses single and multiple layer gelatin films to improve the sustained release of pharmaceuticals, specifically of growth factors. For multilayer preparation, the films are attached together by evenly applying pressure. The document describes that platelet derived growth factor was released at constant rate for up to 100 hours and improved wound healing in vivo.

US 4,865,846 discloses an eye treatment system which includes particles of bio-erodible material (i.e. gelatin), which are suspended in a liquid carrier or ointment carrier having at least one drug therein and having a pH acceptable to the eye. The document describes that an administration containing from about 3 to 6 particles of drug loaded cross-linked collagen has provide continuous delivery of tobramycin for about 6 hours, which is a very long time for an ophthalmic delivery.

EP 0 069 260 describes gelatin insert containing active ingredient for introduction in bone or soft parts, where collagen has a factor of nitrogen to hydroxy lpro line lower than 3-5 and is made in form of sheet and optionally wound into a rod. The document reports that an insert containing 100 mg gentamycin has been placed in shinbone, where the drug has been released up to the re- absorption of the insert up to three weeks.

US 4,659,572 discloses a burn wound-adherent dressing material composed of a complex with gelatin and a water-soluble resin aimed at controlling water-loss and minimizing the ingress of exogenous micro-organisms. The gel protection remains on the wound seven or more days and is then removed after starting of healing process, when skin grafting is allowed.

In US Patent Application 2005/0036955, a moldable, bioresorbable, biocompatible, non- allergenic cross-linked gelatin derivative dressing for the prevention of post extraction alveolar osteitis pain is disclosed along with methods for use of the gel. The dressing is placed at the time of surgery acting as a bone covering and a physiologic scaffold for the conduction of normal alveolar bone healing sequence of fibroblast ingrowth, blood vessel formation, and reossification of the extraction site defect.

US 5,895,412 discloses an apparatus and method for effecting and enhancing wound closure in tissue by flowing a heated sealant flow, such as gelatin, over the wound.

This creates an effective barrier against further blood leakage and, upon cooling, the flow readily adheres to the tissue to seal the wound. Lin et al. (Material Chemistry and Physics 102(2007) 152-158) discloses a tri-layer (gelatin/hyaluronan/chondroitin-6-sulfate) wound dressing for extensive burn injury that automatically falls off when the wound is completely healed.

EP 1 321 516 discloses a hydrogel of polysaccharide (e.g. alginate), where minimum amount of collagen (gelatin precursor) is added as an excipient for other purposes than to be a matrix. Further agents are added to the hydrogel, such as cryo-protectors, chelating agents and nitric oxide.

For wound protection and skin healing, extensive investigation has been carried out on the hydrophilic and water absorbing properties of gelatin as a clinical wound dressing (Takahashi H. et al, Tokushima. J. Exp MedAO (3-4), 159-67 and 169-75, 1993).

The use of gelatin gels and cross-linked collagen gelatins has been investigated, so far, as "depot" systems which slowly release the biologically active agents for a prolonged release. However, degradation of the gelatin matrix in such gels is observed. The degradation occurs after a long period of time and in any form such as enzymatic, erosion, dissolution. The degradation depends on the site of application (i.e. from 6-8 hours in ophthalmic applications, where the elimination is in general very fast, to 7-10 days in other body districts, such as skin).

The common characteristics of gelatin-based systems are that they are solid at 37 °C and have a melting temperature, i.e. a gel-sol transition temperature, much higher than the body temperature, at least 43-45°C or more. The commercial advertisements for gelatin also stress this point. They report that the melting point of gelatin is to be considered as a quality index. In other words, a higher melting point is advertised as both an index of good gelatin and a sign of good stability. It is also advertised that gelatins will lose activity, denature and die when the temperature is substantially higher than their melting point. Thus, the lost of activity will mainly occur with low melting gelatins. Therefore, the gelatin to be used in the above medical applications is selected to have a melting point as high as possible.

Therefore, there is still the need for an improved gelatin hydrogel which allows:

a. the release of a high amount of biologically active agents in a short period of time (i.e. immediate or almost immediate release); and/or

b. the immediate contact of the biologically active agents with the surface of the organ to be treated, so that the agent can immediately exert its therapeutic and biological function; and/or c. the easy removal of the gelatin gel films, or other forms, from wounded skin (burning, bedsore) without any damage or pain.

Traditional or prior art gels, that are solid at 37 °c, act as a matrix for controlled release but do not act for an almost immediate release.

In topical uses, such as in wound healing, it is better to have the release of a high concentration of the adjuvant agents (such as platelet rich plasma, PRP) in a short time. Further, if the gel melts naturally at body temperature or by pouring or washing with a warm solution (38-40°C), the wound is not affected. By contrast, if the gel is solid on the wound, when it is substituted, it adheres to the wound and its removal provokes both pain and the reopening of the wound.

For instance, there is the need for a gelatin hydrogel allowing the contact between platelet derived components, including, but not limited to platelet rich plasma (PRP) and platelet lysate (PL), platelet poor plasma (PPP), cryoprecipitate (CRYO) alone or together with other therapeutic agents and wounded skin or organ lesion, wherein the platelet components can release growth factors and exert their healing properties. In particular, in skin treatment, an immediate contact of all available platelet components with the skin lesions would help repairing them and would favor the growth of regenerated skin. If the skin lesion is continuously in contact with fresh platelet components (including PRP and PL) by means of a suitable delivery system, rapid regeneration will occur, even when replacing the delivery system.

There is also the need for a delivery system or gelatin hydrogels in which cells and/or stem cells and/or cell conditioned culture medium are encapsulated and/or entrapped and/or loaded into.

There is also the need for a delivery system or gelatin hydrogels, either single unit or multiparticulate, that can be surgically inserted or injected, as a suspension, in the organ to be treated where rapidly release the active principle. Such system would be typically used in anti-cancer and/or anti-microbial and/or antibiotic and/or anti-inflammatory therapy and/or in regenerative medicine and advanced therapies (i.e. cells and/or conditioned culture medium).

For instance, in cancer therapy, depot systems and, sometimes, targeted therapeutics allow the controlled release of the therapeutic drugs preferably in the site of action.

The well-known cytotoxic systemic effects are then avoided. However, the total amount of drug released might be insufficient to properly tackle the tumor mass. Therefore, there is the need for a delivery system which releases the drug only in the target site in order to reduce to a minimum free systemic circulation, but also able to provide enough drug so it can exert its local action.

There is also the need for a gel film that can be easily placed as a protecting barrier against infection on skin wounds or lesions that is not painful to remove.

The gel may contain biologically active agents and may be used on skin wounds or lesions due to burning or to relieve bedsore or metabolic ulcers.

Often, removal of the film is painful given the adhesion of the gel onto the wounds. A hydrogel system which is in a gel state at the temperature of the skin (33°C) but "melts" when water at a temperature compatible to the body (36-38°C) is poured onto it, would be removed easily and without pain when substituted.

Moreover, the hydrogel system needs to be composed of biocompatible, bioresorbable and biodegradable materials, preferably from natural sources (not synthetic) and not chemically modified, such as cross-linked.

Among the materials, pure gelatin (not cross-linked) has proven to possess the characteristics of being biocompatible, bioresorbable and biodegradable. It also has a better biocompatibility than other natural products, such as albumin (Lee C.H. et al., Int. J. Pharm. 221, 1-22, 2001).

The gelatin-based systems taught in the state of the art are not suitable for a medical use requiring an immediate or almost immediate release of the biologically active agents. They are also not suitable for a direct contact with the surface of the organ to be treated and for a rapid bioresorbtion of the gelatin matrix. In fact, in the prior art system, the release is sustained for a long period of time, in general days, and the hydrogel matrix, when not cross-linked, undergoes physical degradation by erosion and/or dissolution of the polymer chains. DESCRIPTION OF THE INVENTION

The above problems have been solved in the present invention by the use of gelatin hydrogels that are solid at room temperature and melt at temperatures near the mammalian body temperature. Thus, the gel assumes a liquid state when placed into contact with the body, when implanted in the body or when heated at temperatures compatible with the mammalian body temperature. The gelatin gel may be in the form of membranes, films, or associated to fibers, woven and non-woven fabrics, foams, micro-beads and particles. The products melt or present gel-sol transition rates varying from minutes to hours depending on the collagen source, on the composition and on preparation methods, as well.

The gelatin hydro gels may contain biologically active agents and/or cells that are rapidly released in situ after melting, thus exerting their therapeutic and biological action.

The gelatin-based hydrogels of the present invention may contain biologically active agents and, when placed in sites which allow the gel-sol transition, they "melt" immediately thus releasing the agents in situ which in turn exert their therapeutic and biological action.

The gelatin-based hydrogels of the present invention containing or not containing biologically active agents, are solid at the temperature of the skin (33°C) and melt when water or an aqueous solution at a temperature compatible to the body (36-38°C) is poured onto, which provide for an easy removal of the hydrogels.

It is therefore an object of the invention a gelatin gel comprising between 5 and 25 % w/w of gelatin in a solvent and having a melting temperature between 34°C and 39°C for medical use. Preferably the solvent is selected from the group consisting of: water, blood and/or plasma derivatives, conditioned cell culture medium, cell suspension containing cell culture medium, therapeutically and/or biologically active agent solution.

Still preferably the blood and/or plasma derivatives are selected from the group consisting of: Platelet-Rich-Plasma (PRP), Platelet-Lysate (PL), and/or Platelet Poor Plasma (PPP), and/or Cryoprecipitate (CRYO). Yet preferably the conditioned cell culture medium is conditioned by human stem cells. Preferably the human stem cells are human amniotic liquid derived stem cells. In a preferred embodiment the therapeutically and/or biologically active agent solution is a growth factor.

Preferably the therapeutically and/or biologically active agent solution is selected from the group of: an anti-tumor agent, an antibacterial and/or an anti-microbial agent, an anti-viral agent, an anti-inflammatory agent.

In a preferred embodiment the gelatin gel of the invention is for use as a tissue regenerating agent. Preferably the tissue is skin, derma and/or connective tissue.

Still preferably the use as a tissue regenerating agent is further to a necrosis or a burning event. In a preferred embodiment the gelatin gel of the invention is for use as a tissue regenerating agent for wound, bed sores or abrasion healing.

In a preferred embodiment the gelatin gel of the invention is for use for topical administration. In a preferred embodiment the gelatin gel of the invention is for use as an anti-adhesion agent and/or as a barrier.

The anti-adhesion characteristic derives from the fact that the gel for instance in the form of a film can act as a barrier in respect of two tissues in contact, preventing their healing. Then, the gel liquefies and disappears.

Preferably the gelatin gel is in a fiat or in a tridimensional shape. Still preferably it is dehydrated or lyophilized.

It is a further object of the invention a process for preparing a flat or tridimensional shaped gelatin gel according to any of previous claims comprising the steps of:

a) dissolving the gelatin powder in a solvent under appropriate temperature to get a gelatin solution;

b) pouring the gelatin solution in a suitable mold and lower the temperature to get a flat or tridimensional shaped gelatin gel;

c) optionally dehydrating or lyophilizing the same.

Preferably the solvent is a blood and/or plasma derivative selected from the group consisting of: Platelet-Rich-Plasma (PRP), Platelet-Lysate (PL), and/or Platelet Poor Plasma (PPP), and/or Cryoprecipitate (CRYO).

Still preferably the solvent is water, step c) is performed and the method further comprises the step of:

d) adsorbing onto the dehydrated or lyophilized gelatin gel a suitable amount of a blood and/or plasma derivative selected from the group consisting of: Platelet-Rich-Plasma (PRP), Platelet-Lysate (PL), and/or Platelet Poor Plasma (PPP), and/or Cryoprecipitate (CRYO), to get a fiat or tridimensional shaped gelatin gel;

e) optionally dehydrating or lyophilizing the same.

The gelatin-based hydrogels can be as a single unit or multi-particulate. The single unit systems are implanted in situ by surgical operation or minimally invasive technique (i.e. sub-cutaneous), or placed on the wounded skin or inserted in any site of therapeutic interest. The multiparticulates are injected in situ by large needle syringes or delivered by other suitable means. The gelatin-based hydrogels possess suitable mechanical properties to be easily handled and manipulated. According to the invention, the gelatin-based hydrogels melt in the range of body temperature in order to exert their action. The melting temperature ranges from 32°C to 42°C, preferably from 35° to 39°C.

According to the invention, the gelatin-based hydrogels have a content of water which depends on the final use, and on the physical resistance to stress needed during manipulation. In fact, the physical resistance of the hydrogels should be higher when they are applied externally, i.e. on the skin surface, and lower when applied in an internal part of the body.

For both the external (skin) and internal use of single unit hydrogels in whatsoever form and size, such as films, sheets and discs, etc., the gelatin/water relative ratio ranges from 5/95 w/w to 50/50 w/w, preferably ranges from 10/90 w/w to 30/70 w/w.

In multi-particulate systems, such as particle, beads, vesicles, etc., the gelatin/water relative ratio ranges from 1/99 w/w to 50/50, preferably ranges from 2/98 w/w to 25/75 w/w.

According to the invention, the source of gelatin can be any organism capable of providing a material with the melting properties required. As a not limitative example, sources are vertebrate animals such as porcine (i.e. pig), bovine (i.e. cow), avian (i.e. chicken), and caprine (i.e. goat), marine fish and invertebrates such as shark, stingray, codfish, salmon, jelly-fish, cattle-fish, squid, octopus.

According to the invention, the gelatin-based single unit hydrogels have different forms and shapes. As a not limitative example, hydrogels are flat, substantially bidimensional forms, such as films, sheets and discs, sponges, porous sponges, or standard tri-dimensional forms, such as tablet, mini-tablet, mini-pellet, beads, nets, or other geometrical forms which are shaped and adapted by the end user (i.e. cut) for the final place of use/implant.

According to the invention, the single unit hydrogels are prepared in different ways. As a not limitative example, the single unit hydrogels are prepared by phase separation from a warm collagen solution (higher than 45-50°C) by mean of temperature cooling or addition of phase- separating agents such as lyotropic salts or addition of water capturing agents, to get a gelatin hydrogel. Such intermediate gelatin hydrogel is then heat dried or lyophilized to get a dry gelatin matrix that after reconstitution (water absorption) gives rise to the final gelatin hydrogel. The dried forms have also the advantage to be more stable and, consequently, have a longer shelf-life. According to the invention, the gelatin-based hydrogels are placed on the wound skin (i.e. lesions, burns, superficial inflammation), or surgically implanted in contact with the target organ(s), or implanted sub-cutaneously, or placed in any place (i.e. buccal delivery) where the systems have to explicate their therapeutic action.

According to the invention, the gel-sol transition of the single unit gelatin hydrogels occurs in less than 96 hours, preferably less than 36 hours, most preferably less than6 hours, as a function of application.

According to the invention, the gelatin multi-particulate systems are, as a not limitative example, micro-spheres, micro-beads, micro-pellets in different forms (i.e. rods), vesicles and nano- composites, and other geometrical forms. They are prepared in different ways. As a not limitative example, the micro-particulates are prepared by adding water, at room temperature or lower, on the gelatin powder, as bulk or previously ground to have a narrower particle size distribution, and stirring the suspension. The micro-particulates can also be prepared by means of micro-fluidic systems through the addition of a separation-inducing oil to a warm collagen gelatin solution and successive removal of the oil.

According to the invention, the gelatin multi-particulate systems are delivered in situ by injection using a large hollowed needle.

According to the invention, the gel-sol transition of the gelatin multi-particulate systems occurs in less than 48 hours, preferably less than 24 hours, most preferably less than 6 hours.

According to the invention, the gelatin-based systems are suitable for many therapies which need an immediate or almost immediate release of the therapeutically and biologically active agents in situ, in contact with the organ where they explicate the action. As a not limitative example, suitable therapies includes the field of skin lesions and skin repair, such as burning and wounds, oncology, anti-inflammatory, antibacterial, anti-infectious, and anti-microbial.

The biologically active agents can be included into a gelatin hydrogel also having a temporarily function of physical barrier, i.e. between two or more organs. The gelatin hydrogels would have in this case both the functions of biologically active agent carrier and separation barrier between organs.

According to the invention, the therapeutically and biologically active agents to be loaded in and carried by the gelatin-based systems are, as a not limitative example, blood and plasma derivatives, such as platelet rich plasma, also including co-agents like growth factors; anti-tumor drugs, such as bleomycins, anthraquinone (anthracycline) series carcinostatics such as adriamycin (doxorubicin), daunomycin (daunorubicin), aclarubicin, amrubicin, idarubicin, epirubicin, pirarubicin, and mitoxantrone, mitomycins, actinomycins, camptothecines such as irinotecan, cisplatins, streptozotocin, 5-fluorouracil (5-FU) and derivatives thereof, pirarubicin, dacarbazine and pharmacologically acceptable salts, alkeran, hydrea, avastin, busulfan, cis-platinum, carbo- platinum, methotrexate, Cytoxan, xelodan, taxol, paclitaxel, docetaxel, erlotinib, lapatinib, leustatin, genticabine, fiudarabine, herceptin, rituxan, ifosfamide, navelbine, topotecan, velban, vincristine; anti-bacterial (antibiotic) and microbial drug, such as oxophosphoric acid, ormetoprim, trimethoprim, sulfonamids, phosphomycin, penicillin-series antimicrobial drugs, cephalosporin-series antimicrobial drugs, vancomycin, tetracycline-series antimicrobial drugs, rifampicin, fiuoroquinone-series, furazoldone, gentamycin, lincomycin; anti-infectious drugs, such as antifungal (itraconazole, ketoconazole, myconazole, imidazole, triazole, clotrimazole, butoconazole, ciclopirox), anti-viral (acyclovir, famciclovir, ritonavir, oseltamivir, valacyclovir, amprenavir, lopinavir, nelfinavir, atanazavir, indinavir, saquanavir), local anti-infective (chlorexidine); anti-inflammatory drugs, such as acetylsalicylic acid, ibuprofen, ketoprofen, , naproxen, fenbufen, fenoprofen, flurbiprofen, etodolac, ketorolac, paracetamol (acetaminophen), diclofenac, piroxicam, meloxicam, tenoxicam, nimesulide, COX2 -inhibitors (celecoxib, rofecoxib, etoricoxib, valdecoxib), indomethacin, azapropazone, phenylbutazone, nabumetone, diflunisal, tiaprofenic acid, mefenamic, sulindac, tolfenamic acid. The biologically active agents may also be growth factors or other biologically active molecules which exert an action of protection and/or stimulation on wounds and/or the surgically operated organs.

The biologically active agents may also be cells and/or stem cells and/or cell conditioned medium which are encapsulated and/or entrapped and/or loaded in the gelatin hydrogels.

The invention is now described by reference to the following non-limiting examples referring to the following figures.

Fig. 1 : Laser Doppler Imaging of flaps before operation (PreOP) and at different times after ligation of the epigastric bundle for the conditioned medium containing gelatin membrane (ACM) treated group of animals.

Fig. 2: A macroscopic view of flaps in the conditioned medium containing gelatin membrane treated and in the control (untreated) group of animals at day 7 after ligation of the epigastric bundle.

Fig 3: A) Human bone marrow derived mesenchymal stem cells (hMSC) doublings at different time of culture. Cells were expanded with media supplemented with platelet products PRP, PL, PPP at 5% concentration. Control cultures were maintained in 10% FCS or 10% FCS + 1 ng/mL FGF-2 (Fibroblast Growth Factor-2); B) In vitro osteogenic differentiation of hMSC expanded with different media containing 10% FCS or 10% FCS + 1 ng/ml FGF-2 or 5% PPP or 5% PL or 5% PRP. hMSC were induced with an osteogenic medium and after 12 days were stained for alkaline phosphatase (ALP), a marker of osteogenic differentiation, and Alizarin Red S to reveal calcium deposition; C) Proliferation rate (population doublings) of human fibroblasts expanded in different media containing 10% FCS or 5% CRYO or 5% PRP; D) PDGF-BB (Platelet Derived Growth Factor-BB) and VEGF-A (Vascular Endothelial Growth Factor-A) quantification in PRP/CRYO samples. The growth factors content was determined by Elisa test in fresh platelet rich plasma or cryoprecipitate samples (PRP F and CRYO F), in frozen platelet rich plasma or cryoprecipitate samples (PRP C and CRYO C) and in lyophilized platelet rich plasma or cryoprecipitate samples (PRP L and CRYO L).

Fig. 4: Average colony number at different times after treatment of bone marrow derived mesenchymal stem cells cultured with lyophilized PRP normalized to frozen PRP -80°C condition. Different aliquots of the same freeze-dried PRP preparation were stored at room temperature (RT), 4°C and -20°C. For each of these conditions, PRP preparations were evaluated for clonogenic potential immediately after preparation (TO), 1 month (Γ1), 3 months (Γ3), 6 months (T6), 24 months (T24) of storage;

Fig. 5: Macroscopic view of the PRP gelatin membranes (prepared as described in examples 16 and 17) implanted in CD1 mice and retrieved at different times.

EXAMPLES

Example 1

20 mL solutions of gelatin (Sigma Aldrich, G8150)/water at a ratio of

a. 5/95 w/w (5% gelatin)

b. 10/90 w/w (10% gelatin)

c. 25 c. 15/85 w/w (15% gelatin)

d. 25/75 w/w (25% gelatin)

are prepared by pouring under stirring the gelatin powder into water at 50°C. The solutions are filtered at 0,22 μπι to remove the suspended particles and for sterilization. The filtered solution is then poured at 50°C into a suitable mold (3-4 mm thickness) and cooled at 25°C to induce gelification.

After cooling, half of the samples for each preparation (from 5 to 25 % as indicated above as a. to d.) is also aged at 29°C for 24 hours. The remaining half is kept at room temperature (20-23°C).

Example 2

A 20 mL solution of 15/85 w/w gelatin water is prepared as in example 1 at 50°C and poured into a suitable mold. The solution is then dried in a ventilated oven at 60°C for 24 hours.

The heat dried sample is then reconstituted to hydrogel (15% w/w gelatin) by addition 5 of 17 mL water and used 1 hour (a.) or 24 hours (b. aged) after the addition of water.

Example 3

A 15% w/w gelatin hydrogel is prepared as in example 1 into a suitable mold. The hydrogel is then lyophilized at -50°C under vacuum (0,3 mbar) for 48 hours.

The lyophilized sample is then reconstituted to hydrogel (15% w/w gelatin) by addition of 17 mL water and used 1 hour (a.) or 24 hours (b. aged) after the addition of water.

Example 4

The physical resistance (elastic modulus E) of samples from the examples 1-3 is assessed on a 0,5 inch diameter gel by means of an Instron 4502 at a testing speed of 1 ,3 mm/min at 23°C and 33°C. The results are reported in Table I.

Table I: Physical resistance of gel samples obtained according to Examples 1-3

Preparation Collagen E (KPa) E (KPa)

(example) (% w/w) at 23°C at 33°C

1 a. 5 6,9 2,7

1 a. aged 5 7,0 2,7

1 b. 10 34,9 2,9

1 b. aged 10 34,7 2,9

1 c. 15 79,6 23,2

1 c. aged 15 79,9 22,9 1 d. 25 188,4 75,7

1 d. aged 25 188,6 75,0

2 a. 15 78,8 22,9

2 b. aged 15 79,4 23,4

3 a. 15 80,0 23,5

3 b. aged 15 79,4 23,1

The results show that aging and the two processes (heat-drying, Example 2 or lyophilization, Example 3) do not affect the mechanical properties of the reconstituted gel compared to a gel not undergoing drying or lyophilization. As expected, the physical resistance of the hydrogels increases by increasing the gelatin concentration.

Example 5

Melting temperature (gel-sol transition) of samples from the examples 1-3 is determined by means of Differential Scanning Calorimetry (DSC) at l°C/min scan rate using a Mettler DSC30 calorimeter equipped with a StarE v.6 software. The results are reported in Table II.

Table II: Melting temperature of gel samples obtained according to Examples 1 -3

Preparation Collagen Melting Temperature

(example) (% w/w) (°C)

1 a. 5 34,5

1 a. aged 5 37,1

1 b. 10 34,5

1 b. aged 10 37,8

1 c. 15 36,2

1 c. aged 15 37,9

1 d. 25 37,5

1 d. aged 25 38,6

2 a. 15 36,0

2 b. aged 15 37,8

3 a. 15 36,2 3 b. aged 15 38,0

The results show that the aging slightly increase (by 1 -2°C) the melting temperature of the reconstituted gel. The slight difference is statistically meaningful, since DSC is a very precise and discriminating method especially at the used experimental scan rate. The results will drive the inventors to select the most suitable hydrogel and its preparation process depending on the use and the targeted organ.

The two processes (heat-drying, Example 2 or lyophilization, Example 3) do not affect the melting temperatures of the reconstituted gel, compared to a gel not undergoing drying or lyophilization.

Example 6

Samples having 35 mm diameter and 4 mm thickness from the examples 1-3 are poured in water at 37°C to determine the dissolution rate. The results are reported in Table III.

Table III: Dissolution rate of samples obtained according to Examples 1 -3

Preparation Collagen Dissolution rate

(example) (% w/w)

1 a. 5 <20 sec

1 a. aged 5 <1 min

1 b. 10 <20 sec

1 b. aged 10 <1 min

1 c. 15 <20 sec

1 c. aged 15 <1 min

1 d. 25 <1 min

1 d. aged 25 <2 min

2 a. 15 <10 min

2 b. aged 15 <2 min

3 a. 15 <10 min

3 b. aged 15 <2 min Compared to the reference samples, aging allows a faster dissolution, therefore a faster release of the biologically active substances, or a comparable dissolution rate.

The two processes (heat-drying, Example 2 or lyophilization, Example 3) slightly decrease the dissolution, compared at the same gelatin concentration in the hydrogel.

A modulation on the release rate (immediate or almost immediate) is therefore possible by selecting the process variables.

Example 7

A 15% w/w gelatin hydrogel is prepared according to the example 3 (gel formation + lyophilization + reconstitution). Platelet Rich Plasma (PRP) is used instead of water to form the gel by cooling from a solution at 42°C. PRP is prepared from pooling buffy coats preparations which are centrifuged at low speed in order to separate the platelet poor plasma (PPP) from the platelet pellet. After the centrifugation the PPP fraction is removed while the platelet pellet is diluted with a defined volume of PPP in order to have a PRP preparation with a platelet concentration between lxlO⁶ and lOxlO⁶ platelets^L. Water is used for reconstitution from the lyophilized form. The physical resistance, melting temperature and dissolution rate in water at 37°C are determined 1 hour or 24 hours (aged) after the addition of water according to the methods reported in examples 4, 5 and 6, respectively. Results are reported in Table IV. Table TV: Physical resistance, melting temperature and dissolution rate of PRP/gelatin

The addition of PRP in the system does not modify the characteristics of the reference hydrogel, such as physical resistance, melting temperature and dissolution rate. Example 8

A 15% w/w gelatin hydrogel is prepared according to the example 3. A human Amniotic Fluid Stem Cells (AFSCs) conditioned medium (ACM) was used instead of water. Culture medium was: αΜΕΜ medium (Gibco, Milan, Italy) containing 15% ESI 5 FBS, 1% glutamine and 1% penicillin/streptomycin (Gibco), supplemented with 18% Chang B and 2% Chang C (Irvine Scientific, Santa Ana, CA, USA). Conditioned medium (ACM) was collected and stored in 5 milliliter aliquots equivalent to the medium conditioned by 4 X 10⁶ AFSC. This concentration (1 ml medium conditioned by 800.000 AFSC during a 16 hour culture) was used.

The physical resistance, melting temperature and dissolution rate in water at 37°C are determined 1 hour or 24 hours (aged) after the addition of water according to the methods reported in examples 4, 5 and 6, respectively. Results are reported in Table V. Table V: Physical resistance, melting temperature and dissolution rate of conditioned medium (ACM) /gelatin gel

The addition of a cell conditioned culture medium in the system does not modify the characteristics of the reference hydrogel, such as physical resistance, melting temperature and dissolution rate.

Example 9

A 15% w/w gelatin hydrogel is prepared according to the example 3. A 2% w/w acyclovir (Yung Zip Chemical Co. Ltd) aqueous solution is used instead of water. The physical resistance, melting temperature and dissolution rate in water at 37°C are determined 1 hour or 24 hours (aged) after the addition of water according to the 10 methods reported in examples 4, 5 and 6, respectively. Results are reported in Table VI.

Table VI: Physical resistance, melting temperature and dissolution rate of acyclovir/gelatin gel.

Preparation Gelatin E (KPa) E (KPa) Melting Dissolution

(example) in acyclovir at 23°C at 33°C Temperature rate

(% w/w) (°C) (min) 3.a 15 78,8 22,9 36,0 <10

3.b. aged 15 79,3 23,4 36,2 <2

The addition of a drug (acyclovir) in the system does not modify the characteristics of the reference hydrogel, such as physical resistance, melting temperature and dissolution rate. Example 10

A 15% w/w gelatin hydrogel is prepared according to the example 3. A 5% w/w paclitaxel (Yung Zip Chemical Co. Ltd) acetone/water 60/40 v/v solution is used instead of PRP. The physical resistance, melting temperature and dissolution rate in water at 37°C are determined 1 hour or 24 hours (aged) after the addition of water according to the methods reported in examples 4, 5 and 6, respectively. Results are reported in Table VII.

Table VII: Physical resistance, melting temperature and dissolution rate of paclitaxel/gelatin gel.

The addition of a drug (paclitaxel) in the system does not modify the characteristics of the reference hydrogel, such as physical resistance, melting temperature and dissolution rate.

Example 1 1 - Microcapsules

Gelatin microcapsules, having an average size of 300 urn, (15% w/w gelatin) are prepared by adding gelatin powder, previously finely grounded to an average size of 50 μηι in a mortar, in a suitable amount of water (from 2 to 10 g of water per gram of 10 gelatin) at 20°C under stirring. At 20°C, the gelatin does not dissolve but only swell in contact with water.

15% w/w gelatin microcapsules are also prepared as above using PRP or a human Amniotic Fluid Stem Cells (AFSCs) conditioned medium (ACM) or a 2% w/w acyclovir aqueous solution or a 5% w/w paclitaxel acetone/water solution instead of water. The melting temperature and the dissolution rate in water at 37°C are determined according to the methods reported in examples 5 and 6, respectively.

Results are reported in Table VIII. Table VIII: Melting temperature and dissolution rate of gelatin microcapsules.

The dissolution rate of the microcapsules is immediate and comparable to that of similar single unit hydrogels. Example 12 - In vivo effect

A 15% w/w gelatin hydrogel is prepared according to the example 1. PRP is used instead of water to form the gel by cooling from a solution at 42°C. The solution is poured into a 20 x 20 mm casting container for gelling to have a final thickness of 2-3 mm.

A 15% gelatin hydrogel reference (Gelatin & Protein Co., Ltd, GP type), having a gelsol transition temperature higher than 42°C, is prepared in the same way using PRP.

Both the hydrogels, here in form commonly defined as "membranes", are implanted in mice with a stimulated necrosis model, as detailed in Example 14.

The mice are sacrificed 6 hours, 24 hours and 7 days after implantation. The recovery 10 from necrosis and the presence of inflammation in the surrounding area are evaluated by means of histology. The results are reported in the Table DC.

Table DC: Necrosis and inflammation grades in PRP/gelatin hydrog

Time Reference membrane Hydrogel membrane according to example 1

% necrosis Inflammation % necrosis Inflammation

6 hours 100 Poor 90 No

24 hours 95 Poor 75 No

7 days 70 No 50 No

The hydrogels according to invention showed a better in vivo performance (lower necrosis and no inflammation) compared to those with hydrogels having a higher melting temperature. Example 13 - In vivo skin regeneration

The same hydrogel preparation and reference membranes of Example 12 are evaluated in hairless mice with severe burning on the skin (experimental reference: J.M. Stevenson, R.L. Gamelli, R. Shankar, Mouse Model of Burn Wounding and Sepsis, Methods in Molecular Medicine, 1 , Volume 78, Wound Healing, I, Pages 95-105).

The hydrogel membranes (30 x 30 mm) according to the invention are poured onto the skin every 24 hours for 7 consecutive days.

While the reference membrane remain intact, the hydrogel membrane according to the invention partially melts in 24 hours. The remaining part of the partially melted membrane which remains onto the burned skin after 24 hours is then removed by 30 pouring warm water at 38-40°C; the lesion is left to dry and then a new membrane is poured onto it.

The recovery from the burning is evaluated by determining the grade (%) of remaining burning. The burning extension is evaluated optically, by making a picture of the burned wound, evaluating the area still burned and normalizing this value to the area at time zero . The recovery is the complementary value to the burning (higher 5 recovery = lower burning).

The results are reported in Table X.

Table X: Burning grades in PRP/gelatin hydrogels after 7 days.

Hydrogel according to example 1 Reference

% Burning 45 75 The hydro gels according to invention showed a better recovery (lower burning grade) compared to the hydrogels having a higher melting temperature.

Example 14

Mirabella T et al. (Mirabella T, Hartinger J, Lorandi C, Gentili C, van Griensven M, Cancedda R. Proangiogenic soluble factors from amniotic fluid stem cells mediate the recruitment of endothelial progenitors in a model of ischemic fasciocutaneous flap. Stem Cells Dev. 2012; 21(12): 2179-88) recently reported the pro-angiogenic properties of the conditioned medium obtained from human amniotic liquid derived stem cells (ACM).

The capability of the lyophilized gelatin membrane loaded with this conditioned medium to promote new blood vessel formation, to restore the normal perfusion levels in an ischemic damaged area and to reduce the necrotic area was tested in a rat model of ischemic full-thick skin flap elevated on the epigastric region (Ref. Michlits W, Mittermayr R, Schafer R, Redl H, Aharinejad S. Fibrin-embedded administration of VEGF plasmid enhances skin flap survival. Wound Repair Regen. 2007; 15(3): 360-367).

In the experimental animal group the lyophilized gelatin membrane is reconstituted before the use with the cell culture conditioned medium while in the control animal group the lyophilized gelatin membrane is reconstituted before the use with distilled water.

The authors determined the vascular perfusion rate, the vessel distribution and the survival of flaps treated with membranes containing conditioned medium (ACM) (ACM-treated flaps) and demonstrated the ACM -mediated recruitment of endothelial-like progenitors.

The Ischemic Sectors of ACM treated flaps showed at Day 7 a perfusion level 50% higher than the pre-operation baseline (Figure 1), while the not treated control group never recovered the initial perfusion.

The consequent necrosis developed in the Ischemic Sector was delayed and significantly lower in the ACM group (Figure 2). The histology of the ACM-treated flaps revealed a thin dead stratum corneum, a normal arrangement of epidermal and dermal structures and a high density of vessels in subcutaneous tissues. The authors also found that ACM recruited endothelial progenitors (CD31+/VEGFR2+ and CD31+/CD34+ cells) into the ischemic subcutaneous tissues.

Example 15 - PRP, PPP, PL preparation for lyophilization Products derived from blood platelet fractions PRP and PPP can be prepared in different ways. As a not limiting example, PRP and PPP derivatives were prepared from pooling buffy coats derived from high speed centrifuged whole blood. After the centrifugation, the plasma and buffy layer were carefully removed, transferred to a fresh tube and centrifuged at a revolution speed higher than 850 rpm in order to recover the platelets.

The upper phase, represented by the PPP, was carefully transferred to a fresh tube to derive the cryoprecipitate (CRYO). The platelet pellet was recovered and diluted with an appropriate volume of PPP in order to obtain a PRP preparation with a defined platelet concentration (from 1 xlO⁶ to lOxlO⁶ platelets/μΐ). Cryoprecipitate is produced by slow thawing of frozen PPP, at low temperature (4°C).

PL (platelet lysate) is obtained from a PRP preparation at a platelet concentration between lxlO⁶ and 10χ10⁶/μ1 which has been frozen at -80°C in polypropylene tubes. The tubes are immersed into liquid nitrogen for 1 min and then immediately transferred to a 37°C water bath for 6 min. This freeze-thaw cycle is repeated three times in order to break the platelet membranes and to release their growth factor content (platelet activation). The activated PRP is centrifuged in high speed centrifuge tubes at 19000xg for 3 min to precipitate the broken platelet membranes (pellet). The supernatant represents the PL which is collected and stored at -80°C.

Each 4 platelet product (PRP, PPP, CRYO, PL) is then frozen at -80°C for at least overnight and then lyophilized for about 15 hours.

Reconstitution of the lyophilized platelet fraction derived products is done by adding distilled sterile water in the same amount of the lyophilized volume of the product.

Example 16 - Preparation of lyophilized PRP gelatin membrane I

The gel is formed by adsorption of PRP on a dehydrated gelatin membrane, previously prepared by freeze-drying of a gelatin based hydrogel. The hydrogel has been previously prepared by dissolving the gelatin in water. Gelatin powder (gelatin from porcine skin, Sigma G8150) is poured under stirring into water at 40-50°C. The solution is filtered at 0,22 μηι to remove the suspended particles and for sterilization. The filtered solution is then poured into a suitable mold and cooled at room temperature.

100 mg lyophilized gelatin membrane is loaded with PRP drop by drop by means of a pipette about 3-4 times its volume for 2 hours at room temperature on an orbital shaker. The excess liquid is removed, the membrane is frozen at -80°C for at least 16 hours, then 20 lyophilized for 15 hours. During the freezing-drying process to avoid membrane folding, a sterilized stainless steel grid is used to maintain the membrane fiat. After freeze-drying, the lyophilized gelatin membrane loaded with PRP is sterilized by gamma radiation.

Example 17 - Preparation of lyophilized PRP gelatin membrane II

In this example gelatin powder (gelatin from porcine skin, Sigma G8150) is directly dissolved in PRP in order to obtain a gelatin-PRP membrane, then lyophilized and sterilized.

Briefly, the gelatin is dissolved in PRP at 37°C at a 15% concentration (w/v) under constant agitation for 1-2 hours. When the gelatin-PRP solution is homogeneous, it is transferred to a sterile plate and maintained for at least one hour at room temperature for the gelification. Then the gelatin-PRP membrane (i.e. from 0,5 mm to 2 cm thickness) is frozen in the plate at -80°C for at least 16 hours, lyophilized, poured in a sealant bag and sterilized by gamma radiation. Example 18 - Biological activity of the blood platelet fraction derived products on human cells proliferation

The biological activity of the PRP, PL, and PPP on human cells proliferation was evaluated in vitro. Human MSC were obtained from bone marrow aspirate of patient undergoing orthopaedic surgery after informed consent. The nucleated cells were plated in 10% FCS containing medium for 24 hours, then the cells were transferred to a serum free medium supplemented with 5% PRP, or 5% PL, or 5% PPP. 20 IU/ml heparin was added to the cultures containing the platelet products in order to avoid gel formation. Standard culture conditions 10% FCS and 10% FCS+FGF-2 were performed as control cultures. At 80% confluence, cells were enzymatically detached and replated for several passages in order to determine number of doublings and to establish the cell life span. At each passage, the number of doublings was calculated according to the formula: "Log2 of cells obtained/cells plated" and plotted against time in culture (Fig. 3 A). The number of cell doublings was much higher, from 3 to 5 times, with supplements of the different platelet products (in the rank PRP>PL>PPP) than using the standard culture conditions. The "in vitro" osteogenic potential of hMSC cultured in the presence of the different platelet products was investigated. The cells isolated from the bone marrow sample were expanded until 80% confluence. After trypsinization, the MSC were replated in 24-well culture plates at a density of 5x10⁴ cells/well. At confluence cells were treated with an osteogenic inductive medium containing 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate and 10^"7 M dexamathasone.

Negative control cultures were maintained in the corresponding medium without osteogenic inducers. The medium was changed three times weekly and osteogenic differentiation observed for 12 days. The osteogenic differentiation was verified by the alkaline phosphatase histochemical staining of the mineralized matrix and with the Alizarin Red S staining of the deposited calcium.

Fig. 3B shows that cells expanded with PRP or PL or PPP presented a similar osteogenic differentiation as compared to the cells grown in standard conditions, i.e FCS or FCS + FGF-2. The proliferation of human skin fibroblasts cultured with different platelet products was also evaluated. Cells were isolated from surgical specimens which were minced and plated in medium containing 10% FCS. Skin fragments were held to the bottom of the culture dishes by sterile cover glasses until a significant cell growth was reached. After confluence, cells were detached with trypsin and replated in dishes for several passages in serum free medium containing 5% PRP or 5% CRYO in order to establish the cell life span. Control culture was maintained in medium with 10% FCS. At each passage, the number of doublings was calculated according to the formula: "Log2 of cells obtained/cells plated" and plotted against time in culture. Compared to the FCS expansion condition, the proliferation rate was exalted by PRP (about 3 times higher than the standard FCS) while the CRYO supplement had similar effect to the standard culture condition (10% FCS) (Fig. 3C).

The PDGF-BB and VEGF-A content in fresh, frozen and lyophilized PRP and CRYO preparations was evaluated by Elisa test. As shown in Fig. 3D, PDGF-BB and VEGF-A concentrations did not significantly differ between the frozen and lyophilized samples showing that the freeze-drying process does not alter the concentration of the analyzed growth factors.

Example 19 - Biological activity of the lyophilized PRP

Different aliquots of the same freeze-dried sterilized PRP preparations were stored at room temperature (RT), 4°C and -20°C. The control culture condition was represented by the frozen PRP maintained at -80°C. Three different PRP preparations were tested. For each of the storage conditions reported above, the biological activity of the lyophilized PRP was verified immediately after production (TO) and after 1 month (Tl), 3 months (T3), 6 months (T6), and 24 (T24) months of storage with the CFU-f (Colony Forming Unit-fibroblasts) clonogenic assay by hMSC. Bone marrow sample was plated at low cellular density in 10% FCS medium and after 72 hours the medium was replaced with serum-free medium containing 5% of the lyophilized PRP stored at the different temperatures. After 10-14 days of culture, colonies derived from adherent stem cells were stained with methylene blue and counted with the ImageJ software.

As shown in Fig. 4, cultures performed with lyophilized PRP preparations stored at 4°C display a good stability after 1 month, but showed a significant decrease in colony number after 3 and 6 months of storage vanishing completely at later time points. Cultures grown with PRP preparations stored at RT showed a strong significant decrease in colony number after 3 months of storage and no more colonies are formed at 6 month RT storage.

The cell cultures treated with the lyophilized PRP preparations stored at -20°C up to 24 months showed the best performance and the colony number was comparable to the control condition (frozen PRP -80°C).

Example 20 - Biological activity of the lyophilized gelatin PRP bio-membrane

The biological activity of the lyophilized gelatin membrane (produced according to the example 2) loaded with PRP (example 16) versus the control gelatin bio-membrane was assessed in vitro. The lyophilized bio-membranes were stored at either 4°C or -20°C for 1 week before use and their activity was tested by determining the proliferation kinetics of human MSC with the MTT (3-(4,5-Dimethylthiazolil-2-yl)-2,5-diphenyltetrazolium bromide) assay. hMSC were cultured in 10% FCS until 80% confluence, then detached and re -plated in a 24-well culture plate at a density of 5x10 cells/well in triplicate. After 24 hours, the medium was removed and replaced with serum- free medium supplemented with different preparations at 5% concentration: 1) eluate derived from the melting of a gelatin membrane loaded with PRP fragment at 42°C; 2) gelatin membrane loaded with PRP as solid fragment; 3) eluate derived from the melting of a gelatin membrane fragment at 42°C; 4) gelatin membrane as solid fragment. Control cultures were performed with 10% FCS and 5% frozen PRP. Growth kinetics was measured for 5 days and the absorbance readings were performed on a spectrophotometer using test and reference wavelengths of 570 and 670 nm respectively. Cultures supplemented with the gelatin membrane loaded with PRP, either as fragment or eluate (conditions 1) and 2) above) promoted cell proliferation similarly to standard culture medium (10% FCS). On the contrary, in cultures supplemented with membrane non loaded with PRP (conditions 3) and 4) above), no cell proliferation was observed. Cells cultured in the presence of 5% PRP showed the highest and more immediate proliferation compared to the other samples, in agreement with a controlled and progressive release of the absorbed factors by the gelatin membranes.

Example 21 - Chemical physical characteristics of the lyophilized gelatin PRP biomembrane The chemical and physical characteristics of the lyophilized PRP preparation made according to Example 17 were assessed. The evaluations were performed on lyophilized biomembrane immediately after production (TO) and after 1 month (Tl), 3 months (Γ3), and 6 months Γ6) of storage at -80°C.

The physical resistance (elastic modulus E) was determined on a 0,5 inch diameter gel disc by means of an Instron 4502 (Instron Italia) at a 1 ,3 mm/min testing speed and a temperature of 23°C and 33°C.

The melting temperature (gel-sol transition) was determined by means of Differential 5 Scanning Calorimetry (DSC) using a Mettler DSC30 equipped with a StarE v.6 software at l °C/min scan rate. Results are reported in Table XI.

Table XI: Melting temperature and elastic modulus of lyophilized PRP bio-membrane.

The results showed that the physical characteristics (melting temperature and elastic modulus) were maintained in the storage conditions up to 6 months. Moreover, the absolute values of E indicated that the lyophilized PRP bio-membrane can be easily handled during the different process steps.

Example 22 - In vivo resorption of the lyophilized membranes

The lyophilized PRP gelatin membranes produced according to the methods reported in example 16 and example 17 were implanted (1 sample for condition) subcutaneously in six CD1 mice. Each mouse was implanted with one sample of gelatin membrane alone (CTRL), one sample of gelatin membrane loaded with PRP (prepared as described in example 16), one sample of gelatin- PRP membrane (prepared as described in example 17).

The implanted samples were retrieved after 6 h, 24 h and 7 days.

The resorption of the membrane was evaluated by visual inspection, as clearly reported in the pictures of Fig. 5. The gelatin membrane alone (CTRL) was resorbed within 24 hours, the gelatin membrane loaded with PRP was resorbed within 7 days, the gelatin-PRP membrane was still present at 7 days.

As reported in the method section, the membrane prepared according to example 16 releases PRP faster than the membrane made according to example 17. Compared to the control, the erosion/resorption rate was slower. This result suggests a strengthened interaction between PRP and gelatin, thus allowing PRP to exert properly its functions in vivo. Example 23 - In vivo properties of lyophilized PRP membrane

The in vivo performances of the lyophilized PRP membrane made according to the Example 16 were investigated.

A PRP gelatin hydrogel, which did not undergo to the lyophilization process, was taken as 10 the gold standard (reference hydrogel).

In vivo resorption properties

Both the hydrogels were implanted subcutaneously in mouse as in Example 22. The mice were sacrificed after 15 days and the resorption of PRP gelatin hydrogel membranes was evaluated visually, observing if the membrane is still present and, if yes, by weighing the residual amount and normalize it to the membrane before implant. The results are reported in Table XII. Table XII: Resorption grade of PRP-gelatin hydrogel membranes.

PRP-gelatin membrane according to the invention (example 2) showed a full bioresorption in 15 days, while the reference was still present in a residual amount. The bioresorption of the PRP- gelatin membrane according to the invention is therefore better.

In vivo effect on necrosis and inflammation

The capability of the lyophilized gelatin membrane loaded with this conditioned medium to promote new blood vessel formation, to restore the normal perfusion levels in an ischemic damaged area and to reduce the necrotic area was tested in a rat model of ischemic full-thick skin flap elevated on the epigastric region (Michlits W, et al., Wound Repair Regen. 2007; 15(3): 360- 367).

The rats were sacrificed after 6 hours, 24 hours and 7 days. The recovery from necrosis and the presence of inflammation (biocompatibility) in the surrounding area were evaluated by means of histology. The results are reported in Table XIII.

Table XIII: Grade of necrosis and inflammation in PRP-gelatin hydrogel membranes

PRP-gelatin membrane according to the invention (example 2) showed better biological performances (less necrosis and inflammation) than those of the reference.

In vivo effect on skin regeneration after burning Both the hydrogels were evaluated in hairless mice with severe burning on the skin (J.M. Stevenson, et al, Methods in Molecular Medicine, 1(78), Wound Healing, I: 95-105).

The recovery from the burning was evaluated by determining the grade (%) of remaining burning by histological examination at 1, 2, 5 and 10 days. The remaining burning area was measured and normalized to that at time zero. The results are reported in the Table XIV.

Table XIV: % burning at different time after application of PRP gelatin biomembranes

A marked improvement of burning recovery (faster healing) was found after application of the PRP gelatin bio-membrane according to the invention.

Claims

1- A gelatin gel comprising between 5 and 25 % w/w of gelatin in a solvent and having a melting temperature between 34°C and 39°C for medical use.

2- The gelatin gel according to claim 1 wherein the solvent is selected from the group consisting of: water, blood and/or plasma derivatives, conditioned cell culture medium, cell suspension containing cell culture medium, therapeutically and/or biologically active agent solution.

3- The gelatin gel according to claim 2 wherein the blood and/or plasma derivatives are selected from the group consisting of: Platelet-Rich-Plasma (PRP), Platelet-Lysate (PL), and/or Platelet Poor Plasma (PPP), and/or Cryoprecipitate (CRYO).

4- The gelatin gel according to claim 2 wherein the conditioned cell culture medium is conditioned by human stem cells.

5- The gelatin gel according to claim 4 wherein the human stem cells are human amniotic liquid derived stem cells.

6- The gelatin gel according to claim 2 wherein the therapeutically and/or biologically active agent solution is a growth factor.

7- The gelatin gel according to claim 6 wherein the therapeutically and/or biologically active agent solution is selected from the group of: an anti-tumor agent, an antibacterial and/or an antimicrobial agent, an anti-viral agent, an anti-inflammatory agent.

8- The gelatin gel according to any of previous claims for use as a tissue regenerating agent.

9- The gelatin gel according to claim 8 wherein the tissue is skin, derma and/or connective tissue.

10- The gelatin gel according to claims 8 or 9 wherein the use as a tissue regenerating agent is further to a necrosis or a burning event.

11 - The gelatin gel according to claims 7 or 8 for wound, bed sores or abrasion healing.

12- The gelatin gel according to any of previous claims for topical administration.

13 - The gelatin gel according to claim 1 for use as an anti-adhesion agent and/or as a barrier.

14- The gelatin gel according to any of previous claims being in a flat or in a tridimensional shape.

15 - The gelatin gel according to any of previous claims being dehydrated or lyophilized.

16- A process for preparing a flat or tridimensional shaped gelatin gel according to any of previous claims comprising the steps of: a) dissolving the gelatin powder in a solvent under appropriate temperature to get a gelatin solution;

b) pouring the gelatin solution in a suitable mold and lower the temperature to get a flat or tridimensional shaped gelatin gel;

c) optionally dehydrating or lyophilizing the same.

17- The process according to claim 16 wherein the solvent is a blood and/or plasma derivative selected from the group consisting of: Platelet-Rich-Plasma (PRP), Platelet-Lysate (PL), and/or Platelet Poor Plasma (PPP), and/or Cryoprecipitate (CRYO).

18- The process according to claim 16 wherein the solvent is water, step c) is performed and further comprising the step of:

d) adsorbing onto the dehydrated or lyophilized gelatin gel a suitable amount of a blood and/or plasma derivative selected from the group consisting of: Platelet-Rich-Plasma (PRP), Platelet-Lysate (PL), and/or Platelet Poor Plasma (PPP), and/or Cryoprecipitate (CRYO), to get a fiat or tridimensional shaped gelatin gel;

e) optionally dehydrating or lyophilizing the same.