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WO2025061827A1 - Kit pour la reconstitution d'un dispositif biomédical acellulaire destiné à être utilisé en médecine régénérative, dispositif biomédical ainsi reconstitué et processus de synthèse associé - Google Patents

Kit pour la reconstitution d'un dispositif biomédical acellulaire destiné à être utilisé en médecine régénérative, dispositif biomédical ainsi reconstitué et processus de synthèse associé Download PDF

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Publication number
WO2025061827A1
WO2025061827A1 PCT/EP2024/076222 EP2024076222W WO2025061827A1 WO 2025061827 A1 WO2025061827 A1 WO 2025061827A1 EP 2024076222 W EP2024076222 W EP 2024076222W WO 2025061827 A1 WO2025061827 A1 WO 2025061827A1
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biomaterial
secretome
cell
biomedical device
regenerative medicine
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Cinzia CHINNICI
Pier Giulio CONALDI
Calogero FIORICA
Giovanna Pitarresi
Fabio Salvatore Palumbo
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Istituto Mediterraneo Per I Trapianti E Terapie Ad Alta Specializzazione Ismett
RiMed Foundation
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Istituto Mediterraneo Per I Trapianti E Terapie Ad Alta Specializzazione Ismett
RiMed Foundation
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Publication of WO2025061827A1 publication Critical patent/WO2025061827A1/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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • 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
    • A61L26/0023—Polysaccharides
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • 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
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines

Definitions

  • the present invention refers to a kit for the reconstitution of a cell-free biomedical device for use in regenerative medicine, to a cell-free biomedical device for use in regenerative medicine, and to a process for the synthesis of a cell-free biomedical device for use in regenerative medicine.
  • Secretome means the set of soluble factors secreted by the cells.
  • the secretome of mesenchymal stromal cells is considered a booster of regenerative medicine, a mixture of bioactive factors capable of stimulating endogenous repair processes (Mesenchymal Stem Cells: Time to Change the Name! - Stem Cells Translational Medicine, Volume 6, Issue 6, June 2017, Pages 1445-1451; Mesenchymal Stem Cell Secretome: Toward Cell-Free Therapeutic Strategies in Regenerative Medicine” - Int. J. Mol. Sci. 2017, 18(9), 1852).
  • the release system guarantees “stability” of the secretome by offering protection from clearance and in vivo enzymatic degradation (Polymers for Drug Delivery Systems - Annual Review of Chemical and Biomolecular Engineering Vol. 1:149-173 (March 2010); Hydrogels for protein delivery in tissue engineering - J Control Release 2012 Jul 20; 161 (2) .680-92).
  • hydrogels Due to their biocompatibility and swelling capacity, hydrogels find wide application in regenerative medicine and drug delivery. Generally, hydrogels based on natural polymers are preferred over hydrogels based on synthetic materials and are currently used in the treatment of skin wounds (wound healing) (Functional Hydrogels as Wound Dressing to Enhance Wound Healing - ACS Nano. 2021 Aug 24;15(8):12687-12722; Local injection of high-molecular hyaluronan promotes wound healing in old rats by increasing angiogenesis - Oncotarget.
  • Natural polymers include glycosaminoglycans (GAGs), known for their contribution to the regulation of the biological functions of the extracellular matrix (ECM), including the physical and mechanical regulation of connective tissues and cellular activity. Through specific epitopes, GAGS interact with growth factors and chemokines in the tissue microenvironment, controlling their accumulation and diffusion within cells and maintaining their concentration gradients in the ECM (Regulation of protein function by glycosaminoglycans— as exemplified by chemokines - Annu Rev Biochem - 2005;74:385-410).
  • Hyaluronic acid (HA) is a non-sulfurised GAG and one of the major constituents of the ECM.
  • HA offers a multitude of sites available for chemical crosslinking procedures that create covalent bonds among the macromolecules by stabilizing the structure of the HA itself.
  • Chemical cross-linking was used to reduce the in vivo enzymatic degradation of an HA hydrogel and slow the diffusion of bioactive molecules loaded into the hydrogel itself to control its release (Hyaluronic acid-based hydrogels: from a natural polysaccaride to complex networks - Soft Matter, Volume 8, Issue 12), March 2012, Pages 3280- 3294). Due to its versatility, HA is widely used in tissue engineering and regenerative medicine applications (Molecular engineering of glycosaminoglycan chemistry for biomolecule delivery - Acta Biomaterialia, Volume 10, Issue 4, April 2014, Pages 1705-1719).
  • the sulfurised GAG eparan sulphate (ES) is also known for its use in regenerative medicine thanks to its ability to mimic characteristics of the ECM (Materials Science and Design Principles of Growth Factor Delivery Systems in Tissue Engineering and Regenerative Medicine - Advanced Healthcare Materials - 06.11.2018- Citations: 108).
  • Heparin (EP) is a poorly abundant constituent of the ECM and is considered an analogue of the ES.
  • EP and ES have a similar ability to interact with various soluble proteins, including growth factors and chemokines, influencing numerous pathophysiological processes.
  • FGF fibroblast growth factor
  • HBP heparin binding proteins
  • HA-based hydrogels integrated with MSC secretome for regenerative medicine applications are described in the literature (Hyaluronic Acid Hydrogel Integrated with Mesenchymal Stem Cell-Secretome to Treat Endometrial Injury in a Rat Model of Asherman's Syndrome — Advanced Healthcare materials Volume 8, issue 14, 25/07/2019; A nanocomposite hydrogel delivery system for mesenchymal stromal cell secretome - Stem Cell Research & Therapy - Article number: 205 (2020); Hyaluronic Acid Hydrogel Microspheres for Slow Release Stem Cell Delivery- ACS Biomater Sci Eng. 2021 Aug 9;7(8):3754-3763).
  • Chronic skin wounds represent a major medical problem. In the US alone, about 6.5 million patients are affected at a cost of about $25 billion per year. In general, chronic wounds arise as a result of pathologies such as diabetes or vasculitis, or as complications of surgical wounds.
  • the task of the present invention is thus to provide a kit for the reconstitution of a cell-free biomedical device for use in regenerative medicine, to provide a cell-free biomedical device for use in regenerative medicine, and to provide a process for the synthesis of a cell-free biomedical device for use in regenerative medicine that enable the described drawbacks of the prior art to be overcome.
  • an object of the present invention is to provide a kit for the reconstitution of a cell-free biomedical device that is ready-to-use and easy to manage.
  • Another object of the present invention is to provide a kit for the reconstitution of a cell-free biomedical device that allows it to be preserved and stored until it is used.
  • a further object of the present invention is to provide a biomedical device that is easy to apply and inexpensive.
  • Another object of the present invention is to provide a biomedical device that is safe.
  • Yet another object of the invention is to provide a process for the synthesis of a cell- free biomedical device that allows the release of the bioactive factors contained in the secretome to be effectively modulated based on the type of treatment to be made.
  • the secretome is separately harvested, processed and stored for immediate use, avoiding any additional processing that could increase risks of instability and denaturation. Subsequently, the secretome is added to the dried biomaterial just before administration, thereby avoiding the need for additional manipulation.
  • biomaterial in lyophilised form and the secretome are sterilized.
  • the kit formulation comprises a sterile and dried biomaterial and a sterile secretome which can be reconstituted just before their application on a patient.
  • the innovative aspect of the present invention lies in the ability to prepare the reconstituted formulation extemporaneously.
  • Packaging the biomaterial in a dried state which could even be sterilized in its final packaging, and reconstituting the SECR-hydrogel just before application, represents a novelty in the field of regenerative therapies based on secretome-laden biomaterials, and offers an advantage over the existing examples.
  • the present invention also refers to a process for the synthesis of a cell-free biomedical device for use in regenerative medicine comprising the following steps:
  • the process for the synthesis of a cell- free biomedical device for use in regenerative medicine comprises the following steps:
  • Figure 1 shows a 12-well plate containing the biomaterial based on secretome-rehydrated hyaluronic acid (HA) and heparin (EP).
  • HA secretome-rehydrated hyaluronic acid
  • EP heparin
  • Figures 2 A, 2B and 2C respectively show the kinetics of the release from the hydrogel of the growth factors VEGF-A and HGF (Fig. 2A) and chemokines IL-6, IL-8, SDF-la (Fig. 2B), GRO- a and MCP-1 (Fig. 2C) contained in the secretome.
  • the concentrated secretome was loaded into the dehydrated biomaterial.
  • the assay was carried out at 37 °C in a humidified atmosphere with 5% CO2.
  • the amount of soluble factors released at each time-point quantified with Luminex and expressed in pg/ml as a function of time (mean ⁇ SD, n 3).
  • Grey curve release of the factors from the EPi% biomaterial
  • black curve release of the factors from the EP2i% biomaterial.
  • Figure 3 shows the functionality of the secretome released from the hydrogel obtained after reconstitution of the heparinised biomaterials HA/EPi% and HA/EP21 % with the secretome.
  • In vitro angiogenesis assay (tube formation assay). Representative images of HUVEC (human umbilical vein endothelial cells) plated on the Matrigel acquired after 6 hours in culture.
  • Figure 4 shows the functionality of the secretome released from the hydrogel.
  • Real-time cell migration assay recorded with the XCELLigence for 7 hours.
  • Secretome prior to addition to the biomaterial and serum-free alpha-MEM culture medium were used as a positive and negative control.
  • A Fibroblast migration induced by the secretome released from thei% HA/EP biomaterial measured at different time-points.
  • B Absence of fibroblast migration performed with a secretome released from the2i% HA/EP biomaterial. Abbreviations: Pos Ctrl, positive control; neg Ctrl, negative control; EP, heparin.
  • Figure 5A shows the lyophilisedi% HA/EP biomaterial prior to secretome addition.
  • Figure 5B shows the experimental plan with the treatments.
  • Figure 6 shows the treatment of the pressure ulcer.
  • the images are representative of the ulcers on day 3, 12, 21 and 28 after the injury.
  • On day 12 an evident reduction of erythema can be observed in the ulcers treated with hydrogel/secretome compared to hydrogel alone and vehicle groups.
  • Figure 7A shows a graph representing the residual open area on day 28 of the treatment referred to in figure 6.
  • Figure 7B shows a graph representing the residual de-epithelialised area on day 28 of the treatment referred to in figure 6.
  • Figure 8 shows an image of the residual open area of figure 7A and the residual de-epithelialised area of figure 7B.
  • Figure 9 shows the number of open ulcers at the end of the treatment (day 28) referred to in Figure 6.
  • Figure 10 A, 10 B, 10 C show the histological analysis of the treated ulcers made on day 28.
  • A Representative photomicrographs of H&E stained ulcers taken at the equator of the lesion.
  • the arrows in the vehicle group indicate the granulation step in the central area of the lesion.
  • B The same images taken at a higher magnification.
  • Figures 11A and 11B show the neo- vascularization of the treated skin area analysed by immunofluorescence and anti-lamin-IR antibody.
  • A Representative photomicrographs of the treated ulcers and the vehicle corresponding to the equator of the lesion. The arrows indicate capillaries.
  • B Morphometric evaluation of the anti-laminin antibody positive area (immunoreactive area), expressed as a fraction of the immunoreactive area with respect to the total area considered (capillary density).
  • the present invention relates to a newkit for the reconstitution of a cell-free biomedical device for use in regenerative medicine, characterised in that it comprises a biomaterial in lyophilised form based on hyaluronic acid (HA) and heparin (EP) that is rehydrated by secretome containing growth factors and chemokines, collected from cultured human mesenchymal stromal cells (MSC).
  • HA hyaluronic acid
  • EP heparin
  • biomaterial means a lyophilised (dried) material
  • hydrogel means a hydrated biomaterial
  • the lyophilised biomaterial is frozen until the time of reconstitution.
  • the secretome is also frozen until the time of reconstitution.
  • the cell- free biomedical device is ready to use after reconstitution.
  • the biomaterial in lyophilised form and the secretome are sterilized.
  • the weight content of heparin in the lyophilised biomaterial is modulated for a controlled release of the growth factors and chemokines present in said secretome.
  • the heparin is contained in a concentration range of 1-37.5% by weight in the lyophilised biomaterial, more preferably in a concentration range of 1-21% by weight in the lyophilised biomaterial.
  • the hyaluronic acid is present in a concentration range of 62.5-99% by weight in the biomaterial, more preferably in a concentration range of 79-99% by weight in the lyophilised biomaterial.
  • the present invention further relates to a cell-free biomedical device ready to use in regenerative medicine characterised in that it is reconstituted with the kit described above.
  • the present invention also relates to a process for the synthesis of a cell-free biomedical device ready to use in regenerative medicine.
  • the present invention provides two different processes for the synthesis of the cell-free biomedical device ready to use in regenerative medicine.
  • the process for the synthesis of a cell-free biomedical device ready to use in regenerative medicine comprises the following steps:
  • the process provides, between the step of heparinising said hydrogel and the step of integrating said biomaterial, for a further step of sterilizing said lyophilised biomaterial.
  • the process for the synthesis of a cell- free biomedical device ready to use in regenerative medicine comprises the following steps: Synthesis of an intermediate product starting from at least one amino derivative of hyaluronic acid, with average molecular weight comprised between 50 and 1000 kDa with functionalisation in amino groups ranging from 25 to 50 mol% with respect to the repeating units of hyaluronic acid;
  • the process also in the second embodiment, provides, between the step of heparinising said hydrogel and the step of integrating said biomaterial, for a further step of sterilizing said lyophilised biomaterial
  • the heparinisation step is performed by modulating the content by weight of EP added to said intermediate product for a controlled release of the growth factors and chemokines present in said secretome.
  • the cross-linking step in accordance with the second embodiment of the synthesis process, is performed by modulating the weight content of EP added to said intermediate product for a controlled release of the growth factors and chemokines present in said secretome.
  • the heparinisation step preferably comprises a step of lyophilising the heparinised biomaterial prior to the integration step.
  • the biomedical device used in particular for regenerative medicine applications and consisting of a biomaterial based on HA and EP integrated with secretome has been designed so that it can be reconstituted at the time of application by adding an appropriate volume of secretome to the biomaterial that will be in its lyophilised form. After reconstitution, the device will absorb the entire volume of secretome and can be placed on the tissue to be repaired.
  • the innovation of the present invention lies in the disclosure of a ready-to-use secretome-loaded hydrogel, formulated prior to its application onto the wound bed.
  • the cell-free biomedical device obtained by means of the kit is a single-use device that is reconstituted at the time of application by adding the right volume of secretome to the lyophilised biomaterial.
  • the secretome is in the form of an aqueous dispersion.
  • Example 1 hyaluronic acid/heparin (HA/EP) device synthesis procedures
  • HA with an average molecular weight comprised between 50-1000 kDa
  • ethylenediamine 2.5 ml or 5.0 ml
  • the pH of the solution is brought to 6.8, then 1.4 g of hydroxybenzotriazole (HOBt) (solubilised in 10 ml of water: dimethylsulfoxide 1:1 mixture) and 1.6 g of l-ethyl-3-[3- (dimethylamino)propyl] -carbodiimide (EDC) are added.
  • the reaction is carried out at a temperature of 25 °C or 40 °C for 24 or 72 hours while maintaining the pH at 6.8.
  • the product is purified by dialysis (cut-off 12-14 kDa) against a 5% w/v NaCl solution for 48 hours, then against water for a further 72 hours and isolated by lyophilisation.
  • the variation in temperature and reaction time allows different derivatives to be obtained with a functionalisation in amino groups ranging from 25 to 50 mol% with respect to the HA repeating units.
  • lyophilised HA-amine derivative 25 or 50 mol% functionalisation
  • 20 mg of 1000 kDa HA (HMW) are mixed and dispersed in 4 ml of MES buffer pH 5.5 at 40 °C for at least 24 hours.
  • EDC and NHS in varying amounts depending on the amino derivative used (Table 1) are added to the dispersion obtained and the same is placed in a Petri dish and incubated at 37 °C for 24 hours.
  • the obtained hydrogel is frozen at -80 °C and lyophilised to obtain a spongy structure which is rehydrated, washed with water and dried again by lyophilisation.
  • the EP is solubilised in MES buffer pH 5.5 at a concentration of 2% w/v in the presence of EDC/NHS (Table 2). After 2 hours of incubation at 37 °C, the solution obtained is used to rehydrate the biomaterial based on the amine derivative of HA. After 24 hours of incubation at 37 °C, the biomaterial is washed with water and lyophilised.
  • the amount of bound EP will vary depending on the amine derivative used for the preparation of the biomaterial, the degree of cross-linking of the biomaterial and the degree of heparinisation to be obtained.
  • Table 2 shows the amounts of EP used to treat 100 mg of biomaterial obtained starting from the two amino derivatives of HA. Table 2. Amount of EP, EDC and NHS used for the heparinisation of the biomaterial based on the amine derivative of HA
  • HA-HMW 1000 kDa HA
  • MES buffer pH 5.5 pH 5.5
  • EP and EDC/NHS in MES pH 5.5
  • gelling is carried out at 40 °C for at least 24 hours.
  • Table 3 The various compositions of the hydrogelforming dispersions are shown in Table 3. The hydrogel obtained is frozen, freeze-dried, washed with water and dried again.
  • Example 3 HA/EP biomaterial production with 1% EP with respect to the total weight of the device
  • HA 200 kDa amine derivative and 10 mg of HA 1000 kDa are dispersed in 1.5 mL of 0.5M MES buffer (pH 5.5).
  • the dispersion is placed in an orbiting incubator at 37 °C overnight before adding 1.3 mg of heparin solubilised in 200 pL of 0.5M MES buffer (pH 5.5).
  • 24 mg of EDC in 500 pL of 0.5M MES buffer (pH 5.5) and 14 mg of NHS are added to the polysaccharide dispersion and the same is placed in a Petri dish at room temperature for 12 hours to complete the gelling process.
  • the obtained hydrogel is lyophilised to obtain a spongy structure which is rehydrated, washed with distilled water and dried again by lyophilisation.
  • Example 4 cell source; secretome collection, concentration, quantification and storage
  • the MSCs used in the study are derived from the cell bank of the “Fondazione Ri.MED and IRCCS ISMETT” and were isolated from the foetal dermis (DF-MSC) of skin biopsies resulting from therapeutic abortions according to a protocol approved by the Institutional Research Review Board of IRCCS ISMETT (IRRB/00/15) and following an informed consent signed by the donor (Small Extracellular Vesicles from Human Fetal Dermal Cells and Their MicroRNA Cargo: KEGG Signaling Pathways Associated with Angiogenesis and Wound Healing. Stem Cells Int. 2020 Aug 13;2020:888937).
  • umbilical cord MSCs (CO-MSC) were obtained according to a protocol approved by the Institutional Research Review Board of IRCCS ISMETT (IRRB/18/14) and following informed consent signed by the donors (Extracellular Vesicle-Derived microRNAs of Human Wharton 's Jelly Mesenchymal Stromal Cells May Activate Endogenous VEGF-A to Promote Angiogenesis. Int J Mol Sci. 2021 Feb 19;22(4):2045).
  • the MSCs were cultured in DMEM medium with 10% foetal bovine serum (FBS) and used at established passages (p2-p8).
  • FBS foetal bovine serum
  • the soluble factors were dosed by Luminex xMAP technology (simultaneous detection of multiple analytes) and considering a panel of growth factors and chemokines with a role in wound healing including vascular endothelial growth factor (VEGF)-A, hepatocyte growth factor (HGF), interleukin-6 (IL- 6), interleukin-8 (IL-8), derived stromal factor (SDF)-l alpha, growth-related oncogene (GRO)- alpha and monocyte chemoattractant protein (MCP-1).
  • VEGF vascular endothelial growth factor
  • HGF hepatocyte growth factor
  • IL-6 interleukin-6
  • IL-8 interleukin-8
  • SDF derived stromal factor
  • GRO growth-related oncogene
  • MCP-1 monocyte chemoattractant protein
  • Example 5 kinetics of the release of the secretome from the HA/EPi% and HA/EP2i% materials
  • the dried devices EPi% and EP2i% were placed on sterile 12-well plastic plates and loaded with 200 pl of concentrated secretome, while 600 pl of PBS were added to the outside (Fig. 1).
  • the device was maintained at 37 °C in a humidified atmosphere with 5% CO2.
  • the secretome released was collected at different timepoints (24 hours, 48 hours and then every 3 days for a total of 30 days). To ensure the same volume, the secretome collected at each time-point was immediately restored with an equal volume of PBS.
  • Example 6 functionality of the secretome released from the HA/EPi% and HA/EPzi% biomaterials
  • the secretome released from the hydrogel was tested in vitro for functionality (ability to induce biological responses in the target cells).
  • the individual aliquots released at each time-point were tested in an angiogenesis assay (“tube formation assay”) using human umbilical vein endothelial cells (HUVEC) as target cells.
  • tube formation assay human umbilical vein endothelial cells
  • HUVEC human umbilical vein endothelial cells
  • about 10,000 HUVEC were kept (cultured in the absence of serum) for 3 hours, plated on the Matrigel and maintained at 37 °C in a humidified atmosphere with 5% CO2 (Small Extracellular Vesicles from Human Fetal Dermal Cells and Their MicroRNA Cargo: KEGG Signaling Pathways Associated with Angiogenesis and Wound Healing. Stem Cells Int. 2020 Aug 13;2020:888937).
  • the formation of the “tubes” (capillary-like structures) induced by the secretome was monitored under the inverted microscope by assigning a score from 0 to 5 to each pattern according to the specifications of the assay (Millipore).
  • the positive control (HUVEC resuspended in the secretome before being integrated into the hydrogel reached the “close polygons” pattern (score 4) (Fig. 3A), same as the pattern reached by the HUVECs resuspended in the secretome released from thei% HA/EP biomaterial at early time points (Fig. 3B, representative time-point day 6).
  • the “individual cells, well separated” pattern was observed for all late time-points (Fig.
  • the functionality of the secretome was also assayed by means of cell migration measured with the xCELLigence Real-Time Cell Analyzer (RTCA) (Small Extracellular Vesicles from Human Fetal Dermal Cells and Their MicroRNA Cargo: KEGG Signaling Pathways Associated with Angiogenesis and Wound Healing - Stems Cells Int. 2020 Aug 13; 2020:8889379)and using fibroblasts as target cells.
  • RTCA Real-Time Cell Analyzer
  • the system allows to record in real-time the active passage of cells (chemotaxis) from the upper chamber to the lower chamber which are separated from each other by an electrode (all three constituting the CIM plate) that detects the electrical impedance generated by the passage of cells.
  • the secretome (chemoattractant) was loaded in the lower chamber, while 30,000 starved fibroblasts were resuspended in serum-free medium and loaded in the upper chamber.
  • the CIM plate was assembled to the instrument and maintained at 37 °C in a humidified atmosphere with 5% CO2 and cell migration was recorded for 7 hours.
  • the analysis was performed by means of the RTCA Software 1.2 coupled with the xCELLigence and the results obtained expressed as a cell index (CI), figure 4A shows that the aliquots of secretome released from thei% HA/EP biomaterial from 1 hour to day 6 (early time-points) induce a migratory response reaching CI values similar to the positive control (secretome-induced migration before being added to the biomaterial).
  • the functionality of the medical device was tested in an efficacy proof-of-concept on a mouse model of pressure ulcer. Lyophilised biomaterial discs and secretome batches were prepared in advance and frozen until the time of shipment to the IRET Foundation (Tecnopolo in Bologna) to which the study was commissioned. The study was conducted in accordance with the directives of the European Community Council (2010/63/EU) and approved by the Ministry of Health (authorisation no. 391/2017-PR).
  • mice were killed, the left ulcers were collected for future molecular biology analyses and the right ulcers were collected for morphological analysis, half ulcer for histology and half for immunohistochemistry.
  • the samples were fixed in 4% paraformaldehyde, included in paraffin and sections (4 pm) stained with haematoxylin eosin (H&E).
  • H&E haematoxylin eosin
  • the analysis was carried out with the Nis- Elements AR 3.2 software, by applying the same procedure to all the images considered.
  • the immunoreactive area was calculated as area/fraction (percentage of the laminin-positive area considered on a total area of 400 x 300 pm).
  • the analyses were performed blindly. Immunofluorescence with anti-laminin antibody showed a significant increase in capillary density in the ulcers treated with hydrogel/secretome of both MSCs compared to hydrogel alone and vehicle groups (Fig. 11 A). The result was confirmed by the relative quantification of the immunoreactive area (laminin-positive area) (Fig. 1 IB).
  • the results support the use of the proposed biomedical device as a pro-healing agent of diabetic skin ulcers.
  • the device was well tolerated by the animals.
  • the semi-permanent consistency of the hydrogel and the possibility of easily removing it from the wound bed facilitated serial applications (three applications in total).

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  • Medicinal Chemistry (AREA)
  • Dermatology (AREA)
  • Engineering & Computer Science (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Materials Engineering (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicinal Preparation (AREA)

Abstract

Kit pour la reconstitution d'un dispositif biomédical acellulaire destiné à être utilisé en médecine régénérative, caractérisé en ce qu'il comprend un biomatériau sous forme lyophilisée à base d'acide hyaluronique et d'héparine réhydraté par un secrétome contenant des facteurs de croissance et des chimiokines, collectés à partir de cellules souches mésenchymateuses (MSC) humaines cultivées.
PCT/EP2024/076222 2023-09-21 2024-09-19 Kit pour la reconstitution d'un dispositif biomédical acellulaire destiné à être utilisé en médecine régénérative, dispositif biomédical ainsi reconstitué et processus de synthèse associé Pending WO2025061827A1 (fr)

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IT102023000019479 2023-09-21
IT102023000019479A IT202300019479A1 (it) 2023-09-21 2023-09-21 Kit per la ricostituzione di un dispositivo biomedico cell-free ad uso in medicina rigenerativa, dispositivo biomedico così ricostituito e relativo procedimento di sintesi.

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WO2025061827A1 true WO2025061827A1 (fr) 2025-03-27

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* Cited by examiner, † Cited by third party
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