EP4125952A1 - Blutplättchenlysatschaum für zellkultur, zelltherapie und geweberegeneration sowie verfahren zur herstellung davon - Google Patents

Blutplättchenlysatschaum für zellkultur, zelltherapie und geweberegeneration sowie verfahren zur herstellung davon

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Publication number
EP4125952A1
EP4125952A1 EP21716804.6A EP21716804A EP4125952A1 EP 4125952 A1 EP4125952 A1 EP 4125952A1 EP 21716804 A EP21716804 A EP 21716804A EP 4125952 A1 EP4125952 A1 EP 4125952A1
Authority
EP
European Patent Office
Prior art keywords
platelet lysate
foam
present
platelet
regeneration
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
EP21716804.6A
Other languages
English (en)
French (fr)
Inventor
Sophie Cazalbou
Thibault CANCEILL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Centre Hospitalier Universitaire de Toulouse
Universite Toulouse III Paul Sabatier
Original Assignee
Centre National de la Recherche Scientifique CNRS
Centre Hospitalier Universitaire de Toulouse
Universite Toulouse III Paul Sabatier
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
Application filed by Centre National de la Recherche Scientifique CNRS, Centre Hospitalier Universitaire de Toulouse, Universite Toulouse III Paul Sabatier filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP4125952A1 publication Critical patent/EP4125952A1/de
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/19—Platelets; Megacaryocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A—HUMAN NECESSITIES
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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    • A61K9/122—Foams; Dry foams
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    • 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
    • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3616—Blood, e.g. platelet-rich plasma
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    • 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
    • A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • 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
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
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    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • A—HUMAN NECESSITIES
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    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/135—Platelet-derived growth factor [PDGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/115—Platelets, megakaryocytes

Definitions

  • the present invention relates to the development of a biomaterial obtained by drying a hydrogel of platelet lysate (LP) by supercritical CO2.
  • Platelet lysate is a blood derivative rich in growth factors. It is used routinely for cell culture and avenues exist for its possible use in human therapy. The platelet lysate obtained by simple destruction of the plasma membrane of circulating blood platelets currently offers new strategies for cell culture, wound healing and tissue regeneration.
  • Platelet lysate hydrogels have been proposed in the prior art. However, the presence of water in the platelet lysate and the gel does not allow good storage or good handling for in vivo implantation.
  • Gelation is a process which causes a solid phase to appear, within a solution, which is organized to form a continuous three-dimensional network which will trap the solvent.
  • a gel is therefore a thermodynamically stable two-phase solid-liquid system consisting of a three-dimensional continuous interpenetrating double network, one solid and the second liquid.
  • foams are alternatives systems to hydrogels.
  • a foam is a dispersion of gases in a condensed phase, in other words, it is a familiar system with complex behavior and ambiguous properties.
  • foams have a very low density, but can sometimes be perfectly rigid, even solid "
  • a foam of a mixture of fibrin and other substances such as thrombin, prothrombin, extracts of blood platelets, protease inhibitors, antibiotics, to absorb biochemicals and substrates for accelerated hemostasis and optimized biochemical control of wound closure.
  • the foam is obtained by lyophilization (US4442655).
  • freeze-drying requires a step of freezing the fiber network which, when poorly controlled, causes the foam to burst and render it unusable.
  • lyophilization unless it is carried out in a clean room, does not allow the manufacture of sterile biomaterials. Freeze-drying in a clean room also imposes additional constraints and costs.
  • Improved controlled delivery fibrin foams and matrices are also described in document US20130183279. Bioactive factors such as growth factors are added before the polymerization of fibrin. These bioactive factors are therefore added and are not naturally present in the precursor composition.
  • the present invention relates to a platelet lysate foam obtained from a blood derivative (allogeneic or autologous) which retains the biological properties of platelet lysates and has properties, in particular mechanical but also of preservation, which are optimal which allow its marketing and facilitate its handling.
  • a blood derivative allogeneic or autologous
  • the platelet lysate foam according to the present invention can be used directly in the dry state thus allowing immediate penetration of cells, growth factors and biological fluids present at the implantation site, or hydrated to regain the gelled form. . It also allows a slow and sustained release of growth factors naturally present in the platelet lysate foam.
  • the platelet lysate foam according to the invention advantageously promotes cell invasion, development and proliferation.
  • the platelet lysate foam according to the present invention is advantageously used for therapeutic purposes, for cell culture and can be envisaged for purposes of cell therapy.
  • the present invention also relates to a process for obtaining a platelet lysate foam by a drying process in a supercritical CO 2 atmosphere and a platelet lysate foam obtainable by this process.
  • the present invention relates to a platelet lysate foam characterized in that it comprises TGF-beta, EGF, PDGF-AB, IGF-1, VEGF and bFGF, within a polymerized fibrin matrix.
  • Platelet lysate foams according to the present invention are obtained directly from platelet lysates and advantageously retain the biological properties of platelet lysates.
  • a foam is a dispersion of gas in a condensed phase.
  • wet foams which contain a high volume fraction of liquid and which can be considered as gas dispersions in a liquid
  • dry foams which contain very little of liquid.
  • the foam according to the present invention is a dry foam.
  • the water content of the foam according to the invention is less than 10% relative to the total weight of the foam, preferably less than 7.5% and preferably, the water content is less than 5 %.
  • the water content of the foam according to the invention is about 4.5%.
  • the water content can be measured by any technique known to those skilled in the art. Typically, mention will be made of infrared balance or thermogravimetry.
  • platelet lysate is understood to mean the product of the lysis of platelets, that is to say the product obtained after disintegration of the cell membrane which leads to the release of molecules such as growth factors and cytokines normally contained inside platelets.
  • the platelet lysate used for the manufacture of the foam can be obtained by purchasing pools of platelet lysates designed from blood samples from several donors or by direct design from samples taken from a patient.
  • the blood, collected in citrated tubes is centrifuged to separate the phases of red blood cells, white blood cells and plasma. Isolation of the plasma concentrated in platelets then makes it possible to subject it to freezing-thawing or sonication cycles to destroy the platelet membrane and result in the platelet lysate.
  • a leukocyte removal phase is applicable to eliminate any leukocyte residue in the solution.
  • the growth factors present in the platelet lysate foam are the growth factors naturally present in the platelet lysate (Fekete et al. “Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components. 2012 May; 14 (5): 540-54. doi:
  • the growth factors present in the platelet lysate foam according to the present invention are present in the composition of the precursor, that is to say in the platelet lysate used to obtain the platelet lysate foam.
  • the method according to the invention advantageously makes it possible to conserve the elements present in the platelet lysate, a precursor of the platelet lysate foam according to the invention.
  • an “added bioactive factor” or “added” designates, in the state of the art, a bioactive factor (for example a growth factor and / or a cytokine and / or bioactive ions) which is not present in the composition.
  • a bioactive factor for example a growth factor and / or a cytokine and / or bioactive ions
  • precursor, the fibrin formulation and / or the fibrin matrix but which is added in the laboratory to the precursor composition and / or to the fibrin formulation and / or matrix.
  • These bioactive factors are therefore “artificially” incorporated into the formulation during the formation of the foam.
  • the presence of growth factors of human origin in natural amounts in the precursor is compatible with the mechanisms of tissue healing and regeneration in humans.
  • the platelet lysate contains between 110 and 150 pg / mL of b FGF (relative standard deviation: 8.09%), between 550 and 600 pg / mL of VEGF (relative standard deviation: 5.03%), between 25 and 29 ng / mL of PDGF-AB (relative standard deviation: 7.77%), between 70 and 75 ng / mL of TGF-beta (relative standard deviation: 4.34%), approximately 2 ng / mL of EGF (relative standard deviation: 6.02%), between 60 and 80ng / mL of IGF-1 (according to the composition of the platelet lysate LP100 marketed by the company MACOPHARMA).
  • the platelet lysate, precursor of the platelet lysate foam according to the invention comprises approximately 2 ng / mL of EGF, 26.5 ng / mL of PDGF-AB, 72.5 ng / mL of IGF -1, 575 pg / mL VEGF, 125 pg / mL b FGF, 70 ng / mL TGF-beta.
  • the growth factor concentrations of platelet lysate foams according to the present invention are proportional to the amount of lysate introduced to create the foam.
  • the concentrations of the growth factors in the final foam are not affected by the production process.
  • the concentration of TGF-beta in the foam according to the present invention is between 1.84.10 3 % by weight and 1.84.10 5 % by weight, and is preferably about 7.10 7 g in 3.8.10 2 g of foam is 1, 84.10 4 % by mass.
  • the EGF concentration in the foam according to the present invention is between 3.63 10 5 and 3.63 10 7 % by weight and is preferably about 1.38 10 9 g in 3.8 10 2 g of foam, ie 3.63 10 6 % by mass.
  • the concentration of PDGF-AB in the foam according to the present invention is between 4.79 10 4 % by weight and 4.79 10 6 % by weight and is preferably about 1.82 10 8 g in 3 , 8 10 2 g of foam or 4.79 10 5 % by mass.
  • the IGF-1 concentration in the foam according to the present invention is between 1.31 10 3 % by weight and 1.31 10 5 % by weight and is preferably about 4.99 10 8 g in 3, 8 10 2 g of foam or 1.31 10 4 % by mass.
  • the VEGF concentration in the foam according to the present invention is between 1.04 10 5 % by weight and 1.04 10 7 % by weight and is preferably about 3.95 10 10 g in 3.8 10 2 g of foam or 1.04 10 6 % by mass.
  • the bFGF concentration in the foam according to the present invention is between 2.26 10 6 % by weight and 2.26 10 8 % by weight and is preferably about 8.6 10 11 g in 3.8 10 2 g of foam or 2.26 10 7 % by mass.
  • the platelet lysate foam according to the present invention further comprises tranexamic acid and / or calcium.
  • the platelet lysate foam according to the present invention further comprises tranexamic acid and / or calcium, and / or chloride, and / or sodium.
  • the platelet lysate foam according to the present invention does not affect the activity of the growth factors and retains the properties of these growth factors as well as the elements introduced to form the lysate hydrogel and in particular the elements preferably introduced are sodium, chloride, tranexamic acid and calcium.
  • the elements introduced into the hydrogel formula which after drying will form the foam are retained in the final dry material. These elements are then likely to be released into the surrounding environment and capable of providing additional activity.
  • Tranexamic acid being a anti-fibrinolytic, it can, among other things, help stabilize the blood clot around the grafted material.
  • Calcium has a non-negligible role in coagulation phenomena (by participating in particular in the activation of factors X and II) up to the stage of transformation of fibrinogen into de-fibrinated monomers ready to polymerize.
  • the diameter of the predominantly present pores of the platelet lysate foam according to the invention is between 0.1 and 100 ⁇ m.
  • This pore size discriminates from the method of obtaining the platelet lysate foam.
  • the drying process in a supercritical CO2 atmosphere makes it possible to obtain a foam having a diameter of the predominantly present pores of between 0.1 and 100 ⁇ m.
  • the diameter of the predominantly present pores is between 1 ⁇ m and 10 ⁇ m, preferably between 2 and 7 ⁇ m, and preferably between 3.2 and 4 ⁇ m.
  • the diameter of the predominantly present pores is around 3.5 ⁇ m.
  • the diameter of the majority pores present can be measured by any technique known to those skilled in the art. Typically, mention will be made of mercury porosimetry or mercury porometry, which is an instrument for investigating porous media, known to those skilled in the art.
  • This method consists of using pressure to penetrate the mercury (non-wetting liquid) inside the porous network of the material and to measure the rate of intrusion in relation to the pressure applied. This method makes it possible to determine the percentage of porosity measured between 3nm and 360pm as well as the size of the pores which constitute the network (brochure “AutoPore TM IV Sériés, Automated Mercury Porosimeters, from the company Micromeritics®”).
  • diameter of the predominantly present pores is meant the diameter of the pores for which the mercury porosimeter records the highest mercury intrusion rates.
  • the diameter of the predominantly present pores is therefore measured from the volume of mercury introduced.
  • a person skilled in the art could, for example, use the AutoPore IV device, from the company Micromeritics® (see for example the user manual Autopore IV Operator's manual, Micromeritics 2004) to measure the diameter of the predominantly present pores. .
  • this predominantly present pore diameter allows colonization of the material by cells as well as diffusion of fluids, ions and surrounding molecules to the core of the biomaterial.
  • the foam according to the invention has pores whose diameters vary from 7 nm (allowing the fluids to diffuse) to 100 ⁇ m (allowing the passage of cells and blood vessels).
  • the diameter of the pores as well as the predominantly present pores are shown in Figure 4.
  • the platelet lysate foam according to the invention has an average porosity of between 70% and 95%, preferably between 75% and 90%, even more preferably between 75% and 82%, more preferably between 79% and 89%, and even more preferably, between 77 and 89%.
  • the foam has an average porosity of about 80%.
  • average porosity means the volume of the average porous network of the material corresponding to the volume not occupied by the material which constitutes the material. It indicates the spaces in which fluids, molecules and later cells can enter between the fibers of the network. A rate of porosity that is too low will limit the phenomena of diffusion and colonization by the cells of the foam and / of the gel corresponding to the hydrated foam.
  • the porosity of the foam can be measured by any technique known to those skilled in the art. Mercury porosimetry will also be mentioned by way of illustration.
  • a person skilled in the art could, for example, use the AutoPore IV device, from the company Micromeritics ® (see for example the Autopore IV user manual Operator's manual, Micromeritics 2004) to measure the average porosity of the foam.
  • the platelet lysate foam according to the present invention advantageously retains the three-dimensional arrangement of its fibrin network and makes it possible to release growth factors in the medium over time. Growth factors are indeed embedded in the fibrin network (i.e. within the fibrin matrix). This network will allow the growth factors to be released for a long time.
  • the platelet lysate foam according to the present invention is a solid foam due to polymerization.
  • the foams of platelet lysate according to the invention therefore have superior mechanical properties and can easily be handled with the forceps or by hand without disintegrating.
  • the present invention also relates to a process for obtaining a platelet lysate foam comprising the steps:
  • the platelet lysate hydrogel is obtained by polymerization of a platelet lysate or by polymerization of fibrinogen, the platelet lysate or the fibrinogen being combined with at least one element chosen from a polymerization initiator, a polymerization promoting factor , a coagulation stabilizer, an agent for maintaining isotonicity and swelling of the gel, an agent for promoting network degradation, an agent for promoting bonds in the network.
  • a polymerization initiator e.g., a polymerization promoting factor
  • a coagulation stabilizer e.g., an agent for maintaining isotonicity and swelling of the gel
  • an agent for promoting network degradation e.g., thrombin and genepin.
  • the calcium chloride will also have a gelling power.
  • factor XIII 1 - ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • 1 -ethyl-3- (3-dimethylaminopropyl) carbodiimide also promotes the creation of bonds within the network.
  • tranexamic acid which is a coagulation stabilizer by anti-fibrinolytic action
  • amino-caproic acid which is a stabilizer by anti-degradation action of the fiber network
  • fibronectin which is a coagulation stabilizer by adhesion of cells to the extracellular matrix.
  • NaCl sodium chloride
  • N-hydroxysuccinimide Among the agents promoting bonds in the network, N-hydroxysuccinimide will be mentioned.
  • factors making it possible to give elasticity to the network of fibers factors having stimulating properties of the host's cells and / or antibacterial and / or anti-inflammatory, factors making it possible to stimulate host cells, factors that induce crystal precipitation and create structures similar to that of natural mineralized tissues, additional growth factors or cytokines, coagulation factors, clot stabilizing factors can be combined with the platelet lysate to obtain a platelet lysate foam.
  • bioactive ions Among the factors having stimulating properties of host cells and / or antibacterial and / or anti-inflammatory), we can mention bioactive ions.
  • Bioactive ions correspond to cations known for their biological activity such as Sr2 +, Mg2 +, Cu2 +, Zn2 +, Ag +.
  • Recombinant BMP-2 is a factor that stimulates host cells.
  • Factors such as recombinant BMP-2 will make it possible to confer on the biomaterial a function of stimulating bone regeneration.
  • these factors precipitate crystals of calcium phosphate similar to the crystals that make up the mineral phase of natural bone.
  • the growth factors or additional cytokines are chosen from members of the TGF superfamily (transforming growth factor b), the isoforms of the growth factor of platelet origin (the platelet-derived growth factor or PDGF), the factors growth factors of the EGF (epithelial growth factor) and VEGF (vascular endothelial growth factor) family.
  • TGF superfamily include members of the activin subfamily such as inhibin A and inhibin B, members of the Drosophila Decapentaplegic (dpp) gene subfamily which includes genes encoding factors of bone morphogenesis, factor BMP4, osteogenetic factors BMP3, BMP5, BMP6, BMP7, BMP8 of the 60A subfamily. All of these factors have an inducing activity on the formation of cartilage and bone.
  • EGF amphiregulin
  • TGF-a transforming growth factor a
  • epigen EPG
  • betacellulin BTC
  • I ⁇ B-RGF heparin- binding EGF
  • EPR epiregulin
  • neuregulins NGF
  • PDGF-AA isoforms of PDGF
  • PDGF-BB isoforms of PDGF
  • PIGF vascular endothelial growth factor
  • VEGF-C vascular endothelial growth factor-B
  • thrombin thrombin
  • alpha-1 antitrypsin serine protease inhibitor
  • aprotinin anti-fibrinolytic
  • amino-caproic acid plasmin inhibitor
  • the platelet lysate hydrogel is obtained by polymerization of a platelet lysate, the platelet lysate being combined with at least one element chosen from calcium chloride (CaCl2), sodium chloride ( NaCI), thrombin, amino-caproic acid, factor XIII, fibronectin, plasminogen, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, genepin, tranexamic acid ( or 4- (methylamino) cyclohexanecarboxylic acid).
  • CaCl2 calcium chloride
  • NaCI sodium chloride
  • thrombin amino-caproic acid
  • factor XIII factor XIII
  • fibronectin plasminogen
  • plasminogen 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • N-hydroxysuccinimide genepin
  • tranexamic acid or 4- (methylamin
  • the platelet lysate hydrogel is obtained from fibrinogen combined with at least one of the elements chosen from calcium chloride (CaCl2), sodium chloride (NaCl), thrombin , amino-caproic acid, factor XIII, fibronectin, plasminogen, 1-ethyl-3- (3- dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, genepin, tranexamic acid,
  • the elements chosen from calcium chloride (CaCl2), sodium chloride (NaCl), thrombin , amino-caproic acid, factor XIII, fibronectin, plasminogen, 1-ethyl-3- (3- dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, genepin, tranexamic acid,
  • the hydrogel is obtained by polymerization of a platelet lysate, said platelet lysate being combined with calcium chloride, sodium chloride, and tranexamic acid.
  • the platelet lysate represents between 60 and 80% by volume
  • the calcium chloride represents between 2 and 3% by volume
  • the sodium chloride represents between 20 and 30% by volume
  • the acid. tranexamic represents between 0.1 and 0.5% by volume.
  • the hydrogel thus obtained makes it possible to obtain a three-dimensional network of fibrin which has a tight mesh in which it has been shown that human mesenchymal stromal cells could proliferate and differentiate.
  • the hydrogel polymerization time is between 10 minutes and 12 hours, preferably 15 minutes and 1 hour, and more preferably, the polymerization time is about 30 minutes, the polymerization being carried out at room temperature.
  • this polymerization time makes it possible to obtain a quality hydrogel and the formation of the fibrous network.
  • the platelet lysate hydrogels thus obtained are used to obtain platelet lysate foams capable of providing the same biological properties as platelet lysates while demonstrating superior qualities for commercialization.
  • the present invention also relates to a process for obtaining a platelet lysate foam comprising the steps:
  • the polar solvent is a polar solvent miscible with CO2, chosen from ethanol, acetone, benzene, butane, dioxane, ethane, ethylacetoacetate, isopropanol .
  • the polar solvent is acetone or ethanol.
  • the hydrogel will be soaked in a bath of polar solvent in order to remove the water contained in the platelet lysate hydrogel.
  • the hydrogel is soaked in the bath of polar solvent for a period of between 24 hours and 96 hours, preferably 36 hours and 72 hours. According to one embodiment, the hydrogel is soaked in the organic solvent bath for a period of about 48 hours.
  • the hydrogel After the soaking step, the hydrogel is separated from its support before being placed in the closed reactor of the dryer for drying by supercritical CO2. Drying process in a supercritical CO2 atmosphere
  • the step of drying with supercritical CO 2 comprises a preliminary rinsing step, this step advantageously comprising between 1 and 5 rinses with liquid CO 2 or with CO 2 in the supercritical state.
  • the CO2 rinsing step allows the removal of the polar solvent trapped in the hydrogel and its substitution with liquid CO2, or in the supercritical state.
  • the CO2 rinses make it possible to remove all solvent residues and prevent the retraction of the three-dimensional fibrous network obtained. The architecture of the hydrogel is thus maintained.
  • the supercritical CO2 rinsing step is carried out by circulating CO2 in the supercritical state through the reactor.
  • liquid CO2 rinsing steps are carried out at a temperature of 5 "Celsius and a pressure of 40 to 50 bars, the duration of each rinsing being about 1 hour.
  • the supercritical atmosphere is reached by a rise in temperature beyond 39 ° C and in pressure beyond 90 bars, then maintained between 10 min and 12 h, preferably between 30 min and 10 h, preferably between 1 h and 8 h, preferentially between 2 h and 6 h, more preferably between 3 h and 5 h, preferentially for 4 hours.
  • the maintenance of the supercritical atmosphere allows the maintenance of the three-dimensional structure and the drying to the heart of the network.
  • the maintenance of the supercritical atmosphere is carried out at a temperature of about 40 ° Celsius and a pressure of about 90 bars.
  • the depression gradient is between 1 bar / s and 20 bar / min, and is preferably 1 bar / s.
  • the rapid degassing of 1 bar / s makes it possible to obtain the porous structure of the platelet lysate foam.
  • the foam will be frozen. Too rapid degassing, i.e., greater than 1 bar / s, causes the foam to burst. Too slow degassing, greater than 20 bars / min causes a loss of volume of the foam which will retract and settle.
  • the supercritical CO2 drying step advantageously allows the three-dimensional structure of the hydrogel to be maintained during the drying operation and makes it possible to obtain a platelet lysate foam having mechanical properties superior to those of the hydrogel. initial hydrogel.
  • the material thanks to this process, is advantageously sterile without recourse to a clean room, unlike the lyophilization process used in the state of the art.
  • the present invention also relates to a platelet lysate foam capable of being obtained by the method of the invention.
  • the platelet lysate foam obtained advantageously retains its three-dimensional fibrous arrangement and also its growth factors, making it possible to obtain biological properties identical to those of platelet lysates and its major elements such as tranexamic acid, sodium, chlorine. , and calcium.
  • the platelet lysate foam according to the invention are a support which allows targeted and prolonged release of growth factors in situ and thus promotes the repair or regeneration of damaged tissues.
  • VEGF Vascular Endothelium Growth Factor
  • PDGF growth factor derived from platelets
  • EGF Epidermal growth factor
  • TGF-b Transforming Growth Factor
  • Smad pathway hi Y, Massagué J. Mechanisms of TGF-b Signaling from Cell Membrane to the Nucleus. Cell 2003; 113 (6): 685-700.
  • IGFI insulin-like growth factor
  • bFGF basic fibroblast growth factor or FGF2
  • FGF2 basic fibroblast growth factor
  • the present invention relates to the use of platelet lysate foam according to the invention for cell culture.
  • FCS fetal calf serum
  • hLP pooled human platelet lysates
  • the culture of stem cells in media enriched with platelet lysate makes it possible on the one hand to validate the therapeutic use in humans of these cells and on the other hand it has been shown that in general the mesenchymal stromal cells present better proliferation rates and greater metabolic activity in the presence of platelet lysate (Ma J, et al. Osteogeny capacity of human BM-MSCs, AT-MSCs and their co-cultures using HUVECs in FBS and PL supplemented media. J Tissue Eng Regen Med 2015; 9 (7): 779-788).
  • the present invention also relates to a platelet lysate foam for its use in a method of cell therapy.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention in a method of cell therapy.
  • the present invention also relates to a method of cell therapy comprising administering, to a patient in need thereof, a platelet lysate foam according to the present invention.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the manufacture of a medicament for a method of cell therapy.
  • spongy dressings based on chitosan glutamate and sodium hyaluronate (Rossi S, Faccendini A, Bonferoni MC, Ferrari F, Sandri G , Del Fante C, et al. “Sponge-like” dressings based on biopolymers for the delivery of platelet lysate to skin chronic wounds. Int J Pharm 2013; 440 (2): 207-215), porous silica microparticles (Fontana F, Mori M, Riva F, Màkilà E, Liu D, Salonen J, et al.
  • the present invention therefore relates to a platelet lysate foam according to the present invention for its use in a method for promoting skin healing, regeneration of the dermis and tissue regeneration.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention to promote skin healing, regeneration of the dermis and tissue regeneration.
  • the present invention also relates to a method of treatment for promoting skin healing, dermal regeneration and tissue regeneration, comprising the administration, to a patient in need thereof, of a platelet lysate foam according to the present invention. .
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the manufacture of a medicament intended to promote skin healing, regeneration of the dermis and tissue regeneration.
  • promote is not an absolute term, and, when applied to skin healing, dermis regeneration and tissue regeneration, it refers to a designed procedure or plan of action, even with a low probability of success, but having to induce an overall beneficial effect such as reduction in the severity of one or more symptoms or stabilization.
  • the platelet lysate foam according to the present invention is used for its use in the treatment of chronic skin ulcer.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the treatment of chronic skin ulcer.
  • the present invention also relates to a method of treating chronic skin ulcer comprising administering, to a patient in need thereof, a platelet lysate foam according to the present invention.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the manufacture of a medicament for the treatment of chronic skin ulcer.
  • the present invention also relates to a platelet lysate foam according to the present invention for its use in a method for promoting osteogenesis and bone regeneration.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention to promote osteogenesis and bone regeneration.
  • the present invention also relates to a method for promoting osteogenesis and bone regeneration comprising administering, to a patient in need thereof, a platelet lysate foam according to the present invention.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the manufacture of a medicament intended to promote osteogenesis and bone regeneration.
  • promoting is not an absolute term, and, when applied to osteogenesis and bone regeneration, it refers to a procedure or course of action designed, even with a low probability of success, but having to induce an overall beneficial effect such as reduction in the severity of one or more symptoms or stabilization.
  • to promote osteogenesis and bone regeneration means the capacity of the platelet lysate foam to improve the differentiation of MSCs into cells of the osteoblastic line, by the constant and progressive release of growth factors. and cytokines and thus generate bone formation, to increase the amount of bone and promote its mineralization.
  • intra-articular injections of autologous platelet lysates have been carried out in osteoarthritis horses thus significantly improving the physical performance of the animals (Tyrnenopoulou P, Diakakis N, Karayannopoulou M, Savvas I, Koliakos G .
  • the present invention also relates to a platelet lysate foam according to the present invention for its use for the treatment of osteoarthritis.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the treatment of osteoarthritis.
  • the present invention also relates to a method of treating osteoarthritis comprising administering, to a patient in need thereof, a foamed platelet lysate according to the present invention.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the manufacture of a medicament for the treatment of osteoarthritis.
  • the proposed biomaterials are intended to serve as a reservoir of biomolecules impregnated at the time of use and capable of supporting / promoting the activity of cells present in situ or that of human tendon-derived cells (hTDC) associated at the time of implantation.
  • hTDC human tendon-derived cells
  • the present invention also relates to a platelet lysate foam according to the present invention for its use in a method for promoting cartilage regeneration.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention to promote cartilage regeneration.
  • the present invention also relates to a method of treatment for promoting cartilage regeneration comprising administering, to a patient in need thereof, a platelet lysate foam according to the present invention.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the manufacture of a medicament intended for cartilage regeneration.
  • promote is not an absolute term, and, when applied to cartilage regeneration, it denotes a procedure or course of action designed, even with a low probability of success, but before induce an overall beneficial effect such as reduction in the severity of one or more symptoms or stabilization.
  • promoting cartilage regeneration refers to the ability of platelet lysate foam to promote differentiation of mesenchymal stromal cells to a chondroblast phenotype and thereby promote repair / regeneration of deficient or damaged cartilage.
  • the devices In the treatment of chronic corneal wounds, the devices must make it possible to increase the pre-corneal persistence time of the growth factors contained in the platelet lysate reduced due to a significant drainage by the tear flow triggered by tearing. .
  • Heat-sensitive and mucoadhesive eye drops obtained based on sodium chondroitin sulfate (CS) and hydroxypropylmethylcellulose (HPMC) (Sandri G, Bonferoni MC, Rossi S, Ferrari F, Mori M, Del Fante C, et al. Thermosensitive eyedrops containing platelet lysate for the treatment of corneal ulcers. Int J Pharm 2012; 426 (1 - 2): 1 - 6), chitosan gels or polyacrylic acid carriers (Sandri G, Bonferoni MC, Rossi S, Ferrari F , Mori M, Del Fante C, et al.
  • the present invention also relates to a platelet lysate foam according to the present invention for its use for the treatment of corneal lesions, such as chronic lesions of the cornea.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the treatment of corneal lesions, such as chronic lesions of the cornea.
  • the present invention also relates to a method of treating corneal damage, such as chronic corneal damage, comprising administering, to a patient in need thereof, a platelet lysate foam according to the present invention.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the manufacture of a medicament for the treatment of corneal damage, such as chronic corneal damage.
  • treatment is not an absolute term, and, when applied to the treatment of corneal injury, it refers to a procedure or course of action designed, even with a low probability of success, but should induce an overall beneficial effect such as the delay in onset of the pathology or the reduction in the severity of one or more symptoms or stabilization.
  • the treatment of a corneal lesion refers to the ability of the platelet lysate foam to maintain the platelet lysate instilled in the eye and to increase the pre-corneal persistence time of the growth factors contained in the platelet lysate. due to extended release.
  • Parkinson's disease with its high morbidity, has recently been studied in cell models treated by exposure to pooled / pooled human platelet lysates (hLP).
  • hLP pooled / pooled human platelet lysates
  • the results confirm that such therapies could be used to prevent neuronal loss in vivo because the platelet lysate exhibits protective properties against cell death pathways and certain inducers of oxidative stress (Gouel F, Do Van B, Chou ML, Jonneaux A , Moreau C, Bordet R, et al.
  • the protective effect of human platelet lysate in models of neurodegenerative disease involvement of the Akt and MEK pathways. J Tissue Eng Regen Med 2017; 11 (11): 3236-3240).
  • the present invention also relates to a platelet lysate foam according to the present invention for its use for the treatment of neurodegenerative disorders such as Parkinson's disease.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the treatment of neurodegenerative disorders such as Parkinson's disease.
  • the present invention also relates to a method of treating neurodegenerative disorders such as Parkinson's disease, comprising administering, to a patient in need thereof, a platelet lysate foam according to the present invention.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the manufacture of a medicament for the treatment of neurodegenerative disorders such as Parkinson's disease.
  • treatment is not an absolute term, and, when applied to the treatment of neurodegenerative disorders and more particularly of Parkinson's disease, it denotes a procedure or a plan of action devised, even with a low probability of success, but having to induce an overall beneficial effect such as the delay in the onset of the pathology or the reduction in the severity of one or more symptoms or stabilization.
  • Parkinson's disease refers to the ability of the platelet lysate foam to prevent and / or decrease the loss. neuronal in vivo to reduce the progression of Parkinson's disease and its sequelae.
  • Ischemic stroke models are common in rats to assess neurological deficits or motor function after occlusion of blood vessels.
  • platelet lysate shows favorable results on post-attack neuro-motor functions (Yamauchi T, Saito H, Ito M, Shichinohe H, Houkin K, Kuroda S. Platelet lysate and granulocyte-colony stimulating factor serve safe and accelerated expansion of human bone marrow stromal cells for stroke therapy.
  • the present invention also relates to a platelet lysate foam according to the present invention for its use for promoting neuromotor functions following a cerebrovascular accident (stroke).
  • stroke cerebrovascular accident
  • the present invention also relates to the use of a platelet lysate foam according to the present invention to promote neuromotor functions following a cerebrovascular accident (stroke).
  • stroke cerebrovascular accident
  • the present invention also relates to a method of treatment for promoting neuromotor functions following a cerebrovascular accident (stroke), comprising the administration, to a patient in need thereof, of a platelet lysate foam according to the following. present invention.
  • stroke cerebrovascular accident
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the manufacture of a medicament intended to promote neuromotor functions following a cerebrovascular accident (stroke).
  • stroke cerebrovascular accident
  • the present invention also relates to a platelet lysate foam according to the present invention for its use in a method for promoting the regeneration of periodontal tissues.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention to promote the regeneration of periodontal tissue.
  • the present invention also relates to a method of treatment for promoting regeneration of periodontal tissue, comprising administering, to a patient in need thereof, a platelet lysate foam according to the present invention.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the manufacture of a medicament intended to promote the regeneration of periodontal tissues.
  • peripheral tissue regeneration means the ability of the platelet lysate foam to stabilize in contact with the dental root proteins contained in the platelet lysate and thus increase the quantity and density of periodontal tissue and more particularly to restore the periodontium to an original structure based on the presence of cement on the surface of the tooth, alveolar bone and periodontal ligament between the two.
  • lysate was capable of activating anagen pathways promoting hair growth (Dastan M, Najafzadeh N, Abedelahi A, Sarvi M, Niapour A. Human platelet lysate versus minoxidil stimulates hair growth by activating anagen promoting signaling pathways. Biomed Pharmacother Biomedecine Pharmacother 2016; 84: 979-986).
  • the present invention also relates to a platelet lysate foam according to the present invention for its use for the treatment of alopecia.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the treatment of alopecia.
  • the present invention also relates to a method of treating alopecia, comprising administering, to a patient in need thereof, a foamed platelet lysate according to the present invention.
  • the present invention also relates to the use of a platelet lysate foam according to the present invention for the manufacture of a medicament for the treatment of alopecia.
  • FIG. 2 shows the degradation kinetics in aqueous medium of the platelet lysate foam according to the invention
  • FIG. 3 shows the release of the growth factor VEGF (in pg / ml) over time (in days) according to the different forms (platelet lysate foam according to the invention, platelet lysate hydrogel and control liquid);
  • Example 1 obtaining hvdroqel from platelet lysate
  • Platelet lysate hydrogels were obtained from platelet lysate combined with the various elements in liquid form according to the proportions as summarized in Table 1 below:
  • the hydrogels thus obtained have optimal fibrous and porous structures, in particular for promoting cell proliferation, migration and differentiation.
  • these platelet lysate hydrogels are used to obtain platelet lysate foams capable of providing the same properties. than platelet lysates while demonstrating superior qualities for commercialization.
  • the use of platelet lysate foams is facilitated and may be suitable for all pathologies treated by tissue or cell engineering.
  • Example 2 Process for obtaining a platelet lysate foam
  • the platelet lysate hydrogel obtained is then dried in the reactor of a supercritical CO2 dryer.
  • This type of reactor advantageously allows the three-dimensional structure of the hydrogel to be maintained during the drying operation.
  • the platelet lysate hydrogel In order to remove the water contained in the platelet lysate hydrogel, the latter is soaked for 48 hours in an acetone bath and then separated from its support before being placed in the closed reactor of the dryer.
  • the hydrogel is soaked in a glass container or a metal container.
  • the temperature of the reactor chamber is lowered to a temperature below 10 ° C to allow the entry of CO2 in the liquid state.
  • the reactor with liquid CO2 is filled until the samples are submerged and then the whole is left to soak for 45 minutes to allow the liquid CO2 to penetrate the porous network of the gel.
  • Rinsing is then carried out by emptying the CO2 present in the chamber and entering the same new quantity of liquid. This soaking / rinsing operation is repeated three times.
  • the tank is again filled to mid-height, the reactor closed and then its temperature is gradually raised to 40 ° C and the pressure to 90 bar.
  • the supercritical state which corresponds to the 4th state of matter, is reached when the temperature is above 31 ° C and the pressure above 74 bars.
  • the reactor is maintained at this temperature and at this pressure for 4 hours then degassed and depressurized rapidly in 90 seconds.
  • the platelet lysate foam obtained advantageously retains its three-dimensional fibrous arrangement (example 3) and the major elements such as sodium, chlorine, phosphorus, sulfur and calcium (example 4). .
  • the platelet lysate foam also has mechanical properties superior to those of the initial hydrogel (Example 5).
  • the foam thus obtained is a dry material capable of keeping and rehydrating easily (example 6), which promotes the rapid penetration of biological fluids and cells but also cell activity by the release of growth factors and other proteins ( example 7).
  • Example 3 characterization of the microstructure
  • the network of fibers of the platelet lysate foam was observed by environmental scanning electron microscopy with metallization before and after drying with supercritical CO2.
  • the process makes it possible to obtain a fibrous network such as a 3D matrix.
  • the fibrin network retains its three-dimensional fibrous arrangement.
  • the mesh of the fibrous network is wider after drying, which makes it possible to control the porosity. It is thus possible, by modifying the average porosity and the average diameter of the pores mainly present in the three-dimensional network, to modify the diffusion phenomena inside the porous material.
  • the porosity of the platelet lysate foam was quantified and the porous network was characterized.
  • the method used is mercury porosimetry (apparatus: Autopore III, Micromeritics).
  • the method consists in making the mercury penetrate into the pores of the foam of platelet lysate under increasing pressure.
  • a sample of platelet lysate foam will be weighed in a conductance cell before and after filling with mercury.
  • An analysis of the mercury pressure differential will be performed in order to quantify the porosity and characterize the porous network.
  • the platelet lysate foams according to the invention have an average porosity of about 80%.
  • an average porosity of around 80% allows fluids, molecules, ions and cells to penetrate between the fibers of the network and thus promote their penetration.
  • the diameter of the predominantly present pores of the platelet lysate foam is 3.5 ⁇ m.
  • this diameter of the predominantly present pores allows fluids, ions, molecules and surrounding cells to penetrate to the heart of the network.
  • Example 4 characterization of the mechanical properties
  • TAX T2 compression tests were carried out in order to characterize the mechanical properties of platelet lysate foams dried with supercritical CO2. These mechanical properties were compared with those of the initial hydrogels (“hydrogels” in FIG. 1).
  • the foams of platelet lysate according to the invention therefore exhibit mechanical properties which are superior to those of the initial hydrogel. These foams can thus be easily handled with the pliers or by hand without disintegrating as hydrogel does.
  • Example 5 determination of the rehydration rate after drying
  • the calculated average rehydration rate is 804.9%.
  • the platelet lysate foam thus has a high degree of rehydration. Also advantageously, and in the absence of water, the platelet lysate foam exhibits conservation favorable to its marketing. Indeed, and in the absence of water, the dry material does not deteriorate over time.
  • the method used is that of weighing.
  • the platelet lysate foam according to the invention is disintegrated after 5 days. The growth factors are therefore released in a prolonged manner and not immediately as is the case with the platelet lysate liquid.
  • VEGF was assayed in order to assess its release.
  • the method used is that of the Human VEGF Pre-Coated ELISA Kit ELISA Test, Biogems.
  • the release of VEGF from the foamed platelet lysate according to the invention was compared with the release kinetics from the platelet lysate hydrogel.
  • a liquid was used as a control, as shown in Figure 3.
  • the platelet lysate hydrogel was prepared by the method described in Example 1 and the foamed platelet lysate was prepared by the method described in Example 2.
  • VEGF is released gradually over 5 days until it reaches its maximum concentration. The prolonged release of VEGF continues over 25 days.
  • the platelet lysate foam according to the present invention initially composed based on platelet lysate rich in growth factors, releases VEGF over time. This demonstrates that growth factors are embedded in the fibrin network and are accessible to cells.
  • the platelet lysate foams according to the invention can be used in numerous biological applications such as the regeneration and repair of damaged tissues.
  • growth factors and cytokines such as VEGF, PDGF, EGF and TGF which will be released during the implantation of the platelet lysate foam according to the invention in the medium, thus contributing to tissue growth and organ development, constitutes an important argument for the biomedical use of platelet lysate foams according to the invention.
  • the platelet lysate foams according to the invention are furthermore more easily handled and have improved storage and mechanical properties.

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EP21716804.6A 2020-03-23 2021-03-15 Blutplättchenlysatschaum für zellkultur, zelltherapie und geweberegeneration sowie verfahren zur herstellung davon Pending EP4125952A1 (de)

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FR2002800A FR3108253B1 (fr) 2020-03-23 2020-03-23 Mousse de lysat plaquettaire pour la culture cellulaire, la thérapie cellulaire et la régénération tissulaire et procédé d’obtention
PCT/FR2021/050427 WO2021191525A1 (fr) 2020-03-23 2021-03-15 Mousse de lysat plaquettaire pour la culture cellulaire, la thérapie cellulaire et la régénération tissulaire et procédé d'obtention

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US4442655A (en) 1981-06-25 1984-04-17 Serapharm Michael Stroetmann Fibrinogen-containing dry preparation, manufacture and use thereof
US20130183279A1 (en) 2007-12-28 2013-07-18 Kuros Biosurgery Ag Fibrin Formulations for Wound Healing
WO2015031347A2 (en) * 2013-08-27 2015-03-05 Mayo Foundation For Medical Education And Research Cross-linked platelet material
FR3036707B1 (fr) * 2015-05-29 2019-05-17 Maco Pharma Procede de sterilisation d'un lysat plaquettaire
US20170252411A1 (en) * 2016-03-03 2017-09-07 Emory University Compositions Derived from Platelet Lysates and Uses in Vascularization

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