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WO2013045689A1 - Utilisation thérapeutique d'hydrogels de gélatine avec une transition gel-sol à la température corporelle - Google Patents

Utilisation thérapeutique d'hydrogels de gélatine avec une transition gel-sol à la température corporelle Download PDF

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Publication number
WO2013045689A1
WO2013045689A1 PCT/EP2012/069321 EP2012069321W WO2013045689A1 WO 2013045689 A1 WO2013045689 A1 WO 2013045689A1 EP 2012069321 W EP2012069321 W EP 2012069321W WO 2013045689 A1 WO2013045689 A1 WO 2013045689A1
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WIPO (PCT)
Prior art keywords
gelatin
prp
gelatin gel
platelet
gel according
Prior art date
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PCT/EP2012/069321
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English (en)
Inventor
Claudio Migliaresi
Christian Lorandi
Antonella Motta
Ranieri Cancedda
Maddalena Mastrogiacomo
Anita Muraglia
Original Assignee
BIORIGEN Srl
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from EP11183232A external-priority patent/EP2574349A1/fr
Priority claimed from EP11183233A external-priority patent/EP2574350A1/fr
Application filed by BIORIGEN Srl filed Critical BIORIGEN Srl
Publication of WO2013045689A1 publication Critical patent/WO2013045689A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
    • A61L26/0028—Polypeptides; Proteins; Degradation products thereof
    • A61L26/0038—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/0066—Medicaments; Biocides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/008—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/41—Anti-inflammatory agents, e.g. NSAIDs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/416—Anti-neoplastic or anti-proliferative or anti-restenosis or anti-angiogenic agents, e.g. paclitaxel, sirolimus

Definitions

  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • improvement of their physical, chemical and biological properties has often been needed.
  • the patent application WO 2008/076407 discloses a composition comprising gelatin and a non-toxic cross-linking agent such as transglutaminase.
  • 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.
  • 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.
  • the rod (minipellet) is small enough to be injected into the subcutaneous space through a syringe needle.
  • 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.
  • a liquid carrier or ointment carrier having at least one drug therein and having a pH acceptable to the eye.
  • 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.
  • 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.
  • Lin et al. 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.
  • polysaccharide e.g. alginate
  • collagen gelatin precursor
  • agents such as cryo-protectors, chelating agents and nitric oxide.
  • gelatin gels and cross-linked collagen gelatins have been investigated, so far, as "depot” systems which slowly release the biologically active agents for a prolonged release.
  • 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).
  • gelatin-based systems 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.
  • 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.
  • PRP platelet rich plasma
  • PL platelet lysate
  • PPP platelet poor plasma
  • CYO cryoprecipitate
  • 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.
  • 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).
  • depot systems and, sometimes, targeted therapeutics allow the controlled release of the therapeutic drugs preferably in the site of action.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • gelatin hydrogels that are solid at room temperature and melt at temperatures near the mammalian body temperature.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the conditioned cell culture medium is conditioned by human stem cells.
  • the human stem cells are human amniotic liquid derived stem cells.
  • the therapeutically and/or biologically active agent solution is a growth factor.
  • 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.
  • the gelatin gel of the invention is for use as a tissue regenerating agent.
  • tissue is skin, derma and/or connective tissue.
  • tissue regenerating agent is further to a necrosis or a burning event.
  • gelatin gel of the invention is for use as a tissue regenerating agent for wound, bed sores or abrasion healing.
  • 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.
  • the gelatin gel is in a fiat or in a tridimensional shape. Still preferably it is dehydrated or lyophilized.
  • 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).
  • PRP Platelet-Rich-Plasma
  • PL Platelet-Lysate
  • PPP Platelet Poor Plasma
  • Cryoprecipitate Cryoprecipitate
  • step c) is performed and the method further comprises the step of:
  • 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;
  • 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)
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the source of gelatin can be any organism capable of providing a material with the melting properties required.
  • 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.
  • the gelatin-based single unit hydrogels have different forms and shapes.
  • 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.
  • the single unit hydrogels are prepared in different ways.
  • 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.
  • phase- separating agents such as lyotropic salts or addition of water capturing agents
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the gelatin multi-particulate systems are delivered in situ by injection using a large hollowed needle.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • PreOP Laser Doppler Imaging of flaps before operation
  • ACM gelatin membrane
  • 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 Human bone marrow derived mesenchymal stem cells
  • 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;
  • ALP alkaline phosphatase
  • 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.
  • ALP alkaline phosphatase
  • 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 ( ⁇ 1), 3 months ( ⁇ 3), 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.
  • the solutions are prepared by pouring under stirring the gelatin powder into water at 50°C.
  • the solutions are filtered at 0,22 ⁇ 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.
  • 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.
  • 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.
  • the two processes do not affect the melting temperatures of the reconstituted gel, compared to a gel not undergoing drying or lyophilization.
  • the two processes 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.
  • 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 lxlO 6 and lOxlO 6 platelets ⁇ L. Water is used for reconstitution from the lyophilized form.
  • 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: ⁇ 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 10 6 AFSC. This concentration (1 ml medium conditioned by 800.000 AFSC during a 16 hour culture) was used.
  • 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.
  • 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.
  • 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.
  • a drug paclitaxel
  • 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 ⁇ 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.
  • AFSCs Amniotic Fluid Stem Cells
  • ACM conditioned medium
  • 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.
  • Table VIII Melting temperature and dissolution rate of gelatin microcapsules.
  • 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.
  • hydrogels here in form commonly defined as “membranes” are implanted in mice with a stimulated necrosis model, as detailed in Example 14.
  • 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.
  • 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 12 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).
  • hydrogel membranes (30 x 30 mm) according to the invention are poured onto the skin every 24 hours for 7 consecutive days.
  • 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 .
  • Table X Burning grades in PRP/gelatin hydrogels after 7 days.
  • 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).
  • ACM amniotic liquid derived stem cells
  • 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.
  • ACM conditioned medium
  • 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.
  • 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
  • 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 xlO 6 to lOxlO 6 platelets/ ⁇ ).
  • Cryoprecipitate is produced by slow thawing of frozen PPP, at low temperature (4°C).
  • PL platelet lysate
  • PRP platelet lysate
  • 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.
  • 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 ⁇ 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.
  • 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.
  • the gelatin is dissolved in PRP at 37°C at a 15% concentration (w/v) under constant agitation for 1-2 hours.
  • 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.
  • the gelatin-PRP membrane i.e. from 0,5 mm to 2 cm thickness
  • the gelatin-PRP membrane 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
  • Human MSC 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.
  • 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 5x10 4 cells/well. At confluence cells were treated with an osteogenic inductive medium containing 50 ⁇ g/ml ascorbic acid, 10 mM ⁇ -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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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 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.
  • 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 ( ⁇ 3), and 6 months ⁇ 6) 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 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 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.
  • 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.
  • 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.
  • PRP-gelatin membrane according to the invention showed better biological performances (less necrosis and inflammation) than those of the reference.
  • 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

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Abstract

L'invention concerne l'utilisation d'hydrogels de gélatine qui sont solides à température ambiante et fondent ou présentent une transition gel-sol à des températures proches de la température corporelle, passant ainsi à l'état liquide lorsqu'ils sont mis en contact avec le corps, implantés dans le corps ou chauffés à des températures compatibles avec le corps. Les hydrogels à base de gélatine peuvent contenir des agents et/ou des cellules biologiquement actifs qui, après fusion de l'hydrogel ou transition gel-sol, sont rapidement libérés in situ, permettant leur action thérapeutique et biologique. Les hydrogels à base de gélatine, contenant ou ne contenant pas des agents biologiquement actifs, peuvent être solides à la température de la peau (33 °C) et fondent lorsque de l'eau ou une solution aqueuse à une température compatible avec le corps est versée pour une élimination aisée des hydrogels.
PCT/EP2012/069321 2011-09-29 2012-10-01 Utilisation thérapeutique d'hydrogels de gélatine avec une transition gel-sol à la température corporelle WO2013045689A1 (fr)

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CN116251227A (zh) * 2023-03-06 2023-06-13 江西博恩锐尔生物科技有限公司 一种可吸收止血流体明胶材料的制备方法
CN116251227B (zh) * 2023-03-06 2024-02-02 江西博恩锐尔生物科技有限公司 一种可吸收止血流体明胶材料的制备方法
CN116196467A (zh) * 2023-03-21 2023-06-02 南京鼓楼医院 一种纳米纤维增强负载ADSCs自修复水凝胶及其制备方法

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