WO2016046715A1 - Procédé de fabrication de supports poreux pour des utilisations biomédicales et supports correspondants - Google Patents

Procédé de fabrication de supports poreux pour des utilisations biomédicales et supports correspondants Download PDF

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WO2016046715A1
WO2016046715A1 PCT/IB2015/057196 IB2015057196W WO2016046715A1 WO 2016046715 A1 WO2016046715 A1 WO 2016046715A1 IB 2015057196 W IB2015057196 W IB 2015057196W WO 2016046715 A1 WO2016046715 A1 WO 2016046715A1
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polymer
foaming agent
dispersion
porous
range
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Devid MANIGLIO
Walter Bonani
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Università Degli Studi Di Trento
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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
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • 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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
    • 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
    • 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
    • A61L27/58—Materials at least partially resorbable by the body
    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces

Definitions

  • the present invention concerns a novel method for manufacturing porous scaffolds for biomedical uses and scaffolds thereof.
  • tissue engineering scaffolds play a crucial role for cell sustain and guide to address 3D tissue growth and regeneration.
  • the first factor to consider is the material choice, which is the first constituting interface to the biological environment.
  • a suitable material should have at least the following properties: it should promote cell attachment and function and not provoke any unwanted tissue response from the cells (i.e. it has to be biocompatible) and should degrade in non-toxic products, leaving a scaffold-free living cell construct (i.e. it has to biodegradable).
  • Hydroxyapatite bioactive glasses, calcium phosphate ceramics (inorganic); polyglycolic acid, polylactic acid, ⁇ -polycaprolactone (synthetic organic); collagens, glycosaminoglycan, starch, chitin, chitosan, silk fibroin, hyaluronic acid (natural polymers) are just some examples.
  • porous scaffolds or foams There are many ways to fabricate porous scaffolds or foams. Some of the most used techniques are fiber felts, fiber bonding, phase separation (and sublimation), solvent casting and particulate leaching, membrane lamination, melt molding, polymer/ceramic fiber composite foam (with polymer phase dissolution), high-pressure processing (i.e. supercritical C0 2 ), hydrocarbon templating.
  • the object of the present invention is to provide a method for preparing tridimensional, porous, biomedical scaffolds starting from a polymer in dispersion or melt state in a single manufacturing step. Right after preparation scaffolds are either ready to be used for cell culture or can undergo to different post processing steps with the aim of improving or modifying the scaffold properties.
  • the instant disclosure discloses a novel method for manufacturing a porous, polymeric, biomedical scaffold, the method comprising the following steps:
  • foaming agent is selected among N 2 0, propane, butane and pentane, preferably the foaming agent is N 2 0, and
  • the obtained scaffold is suitable for tissue engineering and regenerative medicine applications.
  • the method allows the production of tunable porous scaffolds constituted by a wide selection of natural and synthetic origin polymers optionally containing additives, such as i.a. drugs, inorganic compounds, bioactive factors, surfactants or emulsifiers.
  • additives such as i.a. drugs, inorganic compounds, bioactive factors, surfactants or emulsifiers.
  • FIG. 2 FT-IR spectrum of a scaffold realized according to the method herein disclosed using a 5.4% silk fibroin dispersion by extrusion foaming through a standard nozzle (10 mm diameter nozzle), large and small diameter needles (4 mm and 2 mm, respectively), compared with the unprocessed 5.4% silk fibroin dispersion after freeze-dry. Amide I peak of the unprocessed dispersion is evidenced.
  • FIG. 3 SEM pictures of silk fibroin scaffolds realized according to the process herein disclosed after freeze-drying; the shown scaffolds have been realized using a standard nozzle ( Figure 3A), a large needle ( Figures 3B and 3C) and a small needle ( Figures 3D and 3E) using different foaming pressures (0.55 or 1.1 MPa). Scale bars refer to distances of 100 ⁇ (pictures on the left, 200x magnification) and 20 ⁇ (pictures on the right, lOOOx magnification).
  • FIG. 4 SEM pictures of a scaffold realized according to the process herein disclosed from a 2% silk fibroin water dispersion by extrusion foaming through a small needle (2 mm diameter) at 1.1 MPa N 2 0 pressure. Scale bars refer to distances of 100 ⁇ (pictures on the left, 200x magnification) and 20 ⁇ (pictures on the right, lOOOx magnification).
  • - Figure 5 SEM pictures of a scaffold realized according to the process herein disclosed using a 15% w/v gelatin dispersion after freeze-drying.
  • Figure 5A is a SEM picture at 200x magnification (scale bar is 100 ⁇ );
  • Figure 5B is a SEM picture at lOOOx magnification (scale bar is 20 ⁇ ).
  • Figure 7 SEM pictures of a scaffold realized according to the process herein disclosed from a 4% w/v Fibroin/gelatin (1: 1 ratio) dispersion after freeze- drying.
  • Figure 7A is a SEM picture at 200x magnification (scale bar is 100 ⁇ );
  • Figure 7B is a SEM picture at lOOOx magnification (scale bar is 20 ⁇ ).
  • FIG. 8 FT-IR spectrum of a scaffold realized according to the process herein disclosed from a 4% w/v silk fibroin-gelatin (1: 1 ratio) dispersion compared with those obtained from extrusion foaming a 2% w/v silk fibroin dispersion or a 2% w/v gelatin dispersion.
  • FIG. 9 SEM picture of a scaffold realized according to the process herein disclosed from a 40% w/v gelatin-hydroxyapatite (1: 1 ratio) dispersion after freeze-drying. Scale bars refer to distances of 100 ⁇ (picture on the left, 200x magnification) and 20 ⁇ (picture on the right, lOOOx magnification).
  • dispersion is intended to refer to a material state comprising more than one phase wherein at least one of the phases consists of finely divided phase domains, often in the colloidal size range, dispersed throughout a continuous phase (IUPAC Recommendations 2011, Terminology of polymers and polymerization processes in dispersed systems, Pure Appl. Chem., Vol. 83, No. 12, pp. 2229-2259, 2011. doi: 10.1351/PAC- REC-10-06-03).
  • dispersions herein we refer to those obtainable mixing a solid or liquid dispersed phase (namely the polymer(s)) into a liquid continuous medium (namely the solvent(s)). This to include the following categories: solutions, sol, colloids, latexes, suspensions and emulsions.
  • the present description concerns a method for manufacturing a porous, polymeric, biomedical scaffold comprising the following steps:
  • foaming agent is selected among N 2 0, propane, butane and pentane, preferably the foaming agent is N 2 0.
  • the method is extremely simple, inexpensive and tunable in term of physical properties (namely, degree of porosity and pore dimensions) of the scaffolds.
  • Porous, polymeric, biomedical scaffolds can be realized from either biodegradable and not degradable polymers in a dispersion or melt state using high pressure foaming agents.
  • the biodegradable polymers suitable to be used for the manufacturing of a porous, biomedical scaffold can be selected from proteins, polysaccharides or lipids (e.g. albumin, collagens, glycosaminoglycan, chitosan, phospholipids, starch, chitin, chitosan, silk fibroin, hyaluronic acid, alginate), biodegradable synthetic polymers (e.g. polyethylene glycol derivates, polyglycolic acid, polylactic acid, poly-DL-lactic acid), or mixtures thereof.
  • proteins polysaccharides or lipids
  • biodegradable synthetic polymers e.g. polyethylene glycol derivates, polyglycolic acid, polylactic acid, poly-DL-lactic acid
  • the preferred solvent is water, either pure, or having at least one salt dissolved therein (such as a saline buffer solution, like PBS), or having a non-neutral pH (in the range of 2 to 10).
  • a saline buffer solution like PBS
  • non-neutral pH in the range of 2 to 10.
  • dichloromethane tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, propylene carbonate, formic acid, n-butanol, isopropanol, n-propanol, ethanol, methanol, acetic acid, nitromethane) and non-polar solvents (e.g. diethyl ether, pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, 1,4-dioxane, chloroform) can be successfully employed.
  • non-polar solvents e.g. diethyl ether, pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, 1,4-dioxane, chloroform
  • the process is particularly efficient in the case of aqueous proteins dispersions wherein proteins present both hydrophilic and hydrophobic sites.
  • proteins fold and assemble in water to minimize the hydrophobic sites exposed at the water phase.
  • N 2 0 i.e. a foaming agent
  • the stability of the scaffold itself can be increased.
  • Typical concentrations of the polymer dispersions are between 1% and 50% w/v of polymer, preferably 1 to 20% w/v; lower or higher concentrations may determine the reduction of the porous scaffold mechanical structure for example determining the self-collapsing of the foam or an excessive increase of the viscosity, which hinders the expansion of the dissolved foaming agent, limiting the foam expansion.
  • polymer dispersions extrusion foaming can be realized by increasing the process temperature (i.e. the initial temperature at which the foaming agent is introduced within the extrusion foaming apparatus).
  • Molten state thermoplastic polymers can be processed (e.g. ⁇ - polycaprolactone) according to the instant description by loading them into the extrusion foaming apparatus and pressurizing with the foaming agent.
  • the foaming process occurs thanks to the fact that the foaming agent is capable to dissolve into the at least one polymer, allowing its expansion when pressure is released and the at least one polymer/foaming agent mixture is extruded from the extrusion foaming apparatus.
  • the foaming agent can be selected among N 2 0, pentane, propane, and n- butane, preferably N 2 0.
  • N 2 0 is the preferred foaming agent since it is a non-flammable gas, is highly soluble in hydrophobic polymers and does not induce acidification of the polymer water dispersion (as for example C0 2 does), leading to unwanted side effects, like protein denaturation, accelerated hydrolysis or additives degradation.
  • the use of N 2 0 positively influences the preservation of the integrity of the pressurized dispersions in the time, without causing dispersion instability, polymer degradation or loss of sterility, and reduces the fire risks in the case of use in surgery room or special environments.
  • the initial pressure used during introduction of the foaming agent within the extrusion foaming apparatus is about 0.2 to 30 MPa, preferably 0.5 MPa to 2 MPA, more preferably 0.55 to 1.1 MPa.
  • An initial pressure of about 0.55 to 1.1 MPa provided good results in terms of pores dimensions (considering the desired application) and can be considered a reasonable reference range.
  • the porosity of the obtained porous scaffold depends on the pressure difference between the initial addition pressure of the foaming agent inside the extrusion foaming apparatus and the pressure of the expansion region, wherein higher initial addition pressure determines bigger pore size and pore density (as shown by way of example in Figure 3).
  • Mean pore size of the scaffold obtained according to the method herein disclosed lies typically in the range of 10 to 1,000 ⁇ , but, being pressure related, it can be tuned by increasing/decreasing the initial addition pressure under which the foaming agent is loaded within the extrusion foaming apparatus.
  • the obtained scaffold can be post processed i.a. to reduce solubility in solvents or to remove the solvent used to realize the polymer dispersion (e.g. by freeze drying, exposure to chemical agents such as methanol, ethanol or water vapor, crosslinking for example by UV exposure or by chemical reaction).
  • porous scaffolds by means of a small needle allows the realization of porous wire-like scaffolds that, in some cases, may determine functional changes in the scaffolds themselves making them suitable for specific applications, for example, in combination with a microfabrication apparatus.
  • Microfabrication is a process of fabrication of miniature structures of micrometer scales and smaller.
  • a microfabrication apparatus is typically constituted by an extrusion foaming apparatus provided with a terminal extrusion dies (i.e. a dispensing needle or nozzle) coupled to a 3 axes computer-aided positioning system. This makes it a suitable technique for the realization of meshes, networks or multi-layer 3D structures.
  • a further advantage of the method herein disclosed lies in the possibility of direct injection of the extruded scaffold in a body site as filler (e.g. in a critical bone defect) for tissue regeneration support, if the polymer dispersion is biocompatible and sterile.
  • Another advantage of the method herein disclosed lies in the possibility of direct injection of the extruded scaffold in a shape mold to obtain scaffolds of any desired shape (foam injection molding).
  • the disclosed method allows realization of porous, biomedical scaffolds in a single step and with a high degree of reproducibility obtaining the desired degree of porosity and/or pore dimensions necessary for allowing cell migration and proliferation inside the scaffold itself.
  • the method herein disclosed is successful in extrusion foaming polymers particularly difficult to process with other techniques.
  • relevant results according to the instant description have been obtained with silk fibroin water dispersions obtaining compact, highly porous, water stable scaffolds, not obtained with the other known techniques.
  • the method for manufacturing a biomedical, porous, polymeric scaffold herein disclosed allows also use of additives in order to improve the biological and/or the physical/mechanical properties of the scaffold itself.
  • Additives which may be added to the polymer in dispersion or melt state are represented by:
  • - drugs e.g. anti-inflammatory agents or other drugs
  • the scaffold are able to exert their pharmacological effect in the surrounding tissues
  • ligands e.g. surfactants or emulsifiers
  • porous scaffold stability or improve the polymer dispersion stability
  • - not degradable synthetic polymers that incorporated in the scaffold permit to address special physical properties, such as electricity conduction by using conductive polymers (e.g. poly-pyrrole, poly-aniline, polythiophenes, Poly(3,4-ethylenedioxythiophene) Polystyrene sulfonate (PEDOT:PSS),- biomolecules (e.g. antioxidants, hormones, growth factors, etc.), that being released by the scaffold are able to exert their biological function in the surrounding tissues; and/or
  • conductive polymers e.g. poly-pyrrole, poly-aniline, polythiophenes, Poly(3,4-ethylenedioxythiophene) Polystyrene sulfonate (PEDOT:PSS),- biomolecules (e.g. antioxidants, hormones, growth factors, etc.)
  • inorganic compounds preferably inorganic nanoparticles, nanorods, nanowires (e.g. graphene, carbon nanotubes, magnetic nanoparticles, hydroxyapatite or calcium phosphate nanoparticles) that are able to modify the mechanical properties of the scaffold, and to induce some specific biological response.
  • inorganic nanoparticles, nanorods, nanowires e.g. graphene, carbon nanotubes, magnetic nanoparticles, hydroxyapatite or calcium phosphate nanoparticles
  • hydroxyapatite or bio-glass particles added to polymeric scaffolds are reported to improve osteoconductivity and are commonly proposed as filler for scaffolds in bone defect repair.
  • the main features controlling the method herein disclosed are temperature, pressure and polymer composition as well as composition of the polymer dispersion; such features allow to tune the polymer (either in a dispersion or melt state) viscosity, influencing the expansion kinetic and the stability and morphology of the obtained porous scaffold.
  • the initial concentration determines its viscosity, which influences the expansion rate and the final scaffold porosity porous.
  • Polymer dispersions concentration ranges between 1% and 50% w/v in water are here reported, but foaming can occur from low concentrations (0.1 % w/v) up to high concentrations (95% w/v). Longer polymer chains provide better chances to produce stable foams at low concentrations, due to the increase of the number of chain-to-chain interactions.
  • Temperature can favor or contrast the foam expansion and its stability after foaming. Increase of temperature from the environmental condition determines progressive reduction in polymer dispersion viscosity, permitting a faster and higher expansion at constant pressure (at least for dispersions having direct proportionality between viscosity and temperature increase). Fast reduction of temperature after extrusion foaming can block the expansion process, permitting to obtain the desired result in term of bubble size.
  • the process can be tuned to exploit physical transitions of the dispersion, like the case of gelatin which presents a sol/gel transition at about 35°C. In this case the dispersion is foamed at temperature higher than 35°C and temperature dropdown following the expansion allow the gel transition and stabilize the final foam structure.
  • the foaming dispersion temperatures allowed are those at which the dispersion is in liquid state. While in the case of the molten polymer, i.e. when no solvent is required, the practical limit for the process is the temperature at which the polymer chains start degrading.
  • Typical values of the initial temperature in case of foaming polymer(s) in a dispersion state are in the range -20 to 120 °C, preferably 20 to 60 °C.
  • Typical values of the initial temperature in case of foaming polymer(s) in a melt state are in the range 30 to 250 °C, preferably 40 to 200°C.
  • extrusion phase (iv) is carried out at an extrusion temperature that is lower than the initial temperature
  • typical values of the extrusion temperature are in the range -200 to 37 °C.
  • the porous scaffold manufactured according to the method herein disclosed can then be subjected to post-processing, which may allow i.a. to reduce solubility of the scaffold itself in solvents or to remove the solvent still present in the porous scaffold in case the polymer was loaded inside the extrusion foaming apparatus in dispersion state.
  • post-processing can be any of freeze drying, exposure to chemical agents such as methanol, ethanol or water vapor, crosslinking for example by UV exposure or by chemical reaction.
  • the method herein disclosed was successfully tested with different polymer dispersions containing silk fibroin, soy-lecithin, gelatin and alginate and with ⁇ -polycaprolactone (PCL) in melt state.
  • PCL ⁇ -polycaprolactone
  • Porous biomedical scaffolds can be produced starting from high concentration up to low concentration protein dispersion, namely 50% w/v lecithin and 2% w/v silk fibroin water dispersions.
  • the present inventors did not verify specific limitation about the concentrations other than the increase of viscosity that hinders the expansion of the dissolved foaming agent or the reduction of the structural properties, which can lead to self-collapsing scaffolds.
  • the high concentration of hydroxyapatite within the gelatin dispersion allowed the thermal post treatment of the obtained porous scaffold in order remove by pyrolysis the polymeric phase (gelatin), obtaining an inorganic porous scaffold usable for example as bio- absorbable bone graft substitute.
  • the extrusion foaming method was also successfully tested with alginate dispersion to assess the possibility to foam polysaccharides, which generally are prevalently hydrophilic and have high dissolution rates in water. Due to the fact that alginate turns to gel if exposed to divalent cations (which causes ionic crosslink of the macromolecule chains) the foam has been produced in a CaCl solution. In this case the diffusion rate of Ca ++ ions and the N 2 0 expansion are in contrast to each other. This leads to lower homogeneous porosity foams.
  • PCL poly ⁇ -polycaprolactone
  • N 2 0 pressurized N 2 0 at 1.1 MPa.
  • the foaming was realized directly into a dewar flask containing liquid nitrogen in order to suppress bubbles coalescence and to quench the material expansion by fast cooling the foam right after production. This procedure leads to the realization of high-density foams, because of the polymer concentration. Also in this case the foaming process can be tuned by acting on pressure and temperature.
  • the polymers tested herein were constituted by dispersion in water of silk fibroin, pig-skin gelatin, soy-lecithin and alginate.
  • Silk fibroin was obtained from white cocoons produced by polyhybrid
  • Soy lecithin, pig-skin gelatin and alginate were provided by Sigma Aldrich and suspended in warm water (40°C) and gentle stirring to improve dispersion. Tested concentrations for the various dispersions were between 2% and 30% w/v, but there is no specific limitation other than the increase of viscosity that hinders the expansion of the dissolved foaming agent.
  • the foaming agent used is nitrous oxide.
  • foaming agents such as propane, n- butane, pentane.
  • Nitrous oxide has high solubility in the hydrophobic phase of the used polymers, does not induce dispersion acidification and is not flammable, which makes it a preferable foaming agent.
  • Additives can be either soluble or not soluble, or partially soluble (like surfactants or emulsifiers) in the polymer in its dispersion or melt state, this determining how the additives will be available in the obtained biomedical scaffold or interfere with the biomedical scaffold structuring.
  • hydroxyhapatite nanoparticles (Sigma Aldrich) were loaded into a gelatin dispersion. Hydroxyhapatite powder was added to the gelatin dispersion at 50°C under gentle stirring until complete dispersion.
  • a stainless steel 0.5 liters (ICO, Whip it) siphon was used as extrusion foaming apparatus.
  • the purging valve was implemented with different extrusion dies to obtain different biomedical scaffold physical structure.
  • a standard nozzle having 50 mm length and 10 mm inner diameter, as well as two different needles, both 100 mm long and having 5 mm inner diameter (large needle), or 2 mm inner diameter (small needle) were used.
  • Foaming method The method consist in four main steps (as shown in Figure 1):
  • An extrusion foaming apparatus is loaded with a polymer (in the form of a polymer liquid dispersion or melt material) with the residual volume occupied by a low solubility aeriform (typically air);
  • the extrusion foaming apparatus is pressurized with the foaming agent at a predetermined pressure, typically between 0.5 and 2.0 MPa;
  • the polymer is allowed to absorb the foaming agent obtaining a polymer/foaming agent mixture (gas dissolution can be speed up by shaking or stirring);
  • the polymer/foaming agent mixture is expelled out of the extrusion foaming apparatus by releasing the pressure inside the extrusion foaming apparatus by opening a valve. Foaming occurs as polymer/foaming agent mixture flows outside the apparatus through a nozzle or a needle because of the foaming agent expansion.
  • the obtained scaffold underwent to freeze drying.
  • the obtained porous scaffolds were analyzed by optical imaging and electron microscopy (after freeze drying). FT-IR spectra were collected to assay molecular rearrangement.
  • Silk fibroin, gelatin, lecithin, alginate dispersions were tested at different concentrations and different foaming conditions (needle/nozzle diameter, foaming agent pressure) to assess the limits of the disclosed method.
  • the obtained porous scaffolds have been analyzed by FT-IR ( Figure 2) to check different protein structuring, compared with that obtained from the unprocessed dispersion.
  • the spectrum reveals a slight shift of the Amide I peak towards 1500 cm "1 , which is addressable to a ⁇ -sheet organization of the protein structure, conferring to the obtained foams higher stability to water.
  • the resulting scaffold reveals evident fiber-like structures, aligned along the extrusion direction ( Figure 4), thus maintaining a wide porosity, with pores size ranging between 10 and 100 ⁇ .
  • the organization in fibrils or fiber-like structures is evidence of a higher molecular organization, which prevents the foam to solubilize in water, even after long time.
  • the dispersion was heated at 50°C to liquefy gelatin inside the extrusion foaming apparatus.
  • the extruded polymer/foaming agent mixture was deposited on a metal plate floating on a liquid nitrogen bath to suppress bubbles coalescence permitting gelatin to structure and become solid. Similar results can be obtained by putting the metal plate into a refrigerator right after the foaming process.
  • Figure 5 shows the SEM structure of the gelatin scaffold after freeze- drying, which presents a closed porosity in the range of 50 to 250 ⁇ . Pores result homogeneously distributed and present a roughly spherical shape inherited by the foaming gas bubbles expansion.
  • Results are shown in Figure 7.
  • the produced foams are compact and stable.
  • the internal structure from the SEM pictures reveals an open porosity ranging from 50 to 250 ⁇ .
  • a second order, lamellar structure porosity (measuring few microns) is also present and fills the gaps between the bigger pores. This is addressable to the freeze-drying process and is supposed to widely swell once the foam is rehydrated.
  • a 40% water dispersion obtained by mixing equal parts of gelatin and hydroxy apatite nanometric particles (particle size ⁇ 200 nm) was extruded using a 6 mm diameter nozzle.
  • Dispersion (and extrusion foaming apparatus) temperature was set at 50°C to keep the gelatin liquid, 0.55 MPa N 2 0 pressure was used.
  • the polymer/foaming agent mixture was extruded on an aluminum plate floating on a liquid nitrogen bath, in order to quick cool the obtained porous scaffold, to preserve the porous structure by accelerating the gelatin sol to gel transition.
  • FIG. 9 An example of the resulting foam is shown in Figure 9 where the porosity is appreciable and ranges from about 50 to 250 ⁇ , with a combination of closed and open cells structure.
  • the resulting foam appears relatively compact, but, even at so high protein concentration, the foaming occurs thus indicating that better results could be obtained by increasing the dissolved foaming agent volume (and increasing the pressure).
  • the resulting material present a closed cells porosity structure, with a pore size ranging from few microns to about 150 ⁇ . From the higher magnification images it is possible to evidence the presence of micrometric and sub-micrometric cavities, reasonably due to the expansion limited by the dispersion viscosity.
  • ⁇ -polycaprolactone 80000 MW was loaded into the pressurized extrusion foaming apparatus, filled with N 2 0 (1.1 MPa) and heated at 90°C. After complete melting and foaming agent dissolution inside the polymer, the polymer/foaming agent mixture was extruded using a standard nozzle (10mm inner diameter) into a dewar flask containing liquid nitrogen. The contact with the cold liquid permits to stop the expansion, quickly solidify the foam and thus to obtain the desired solid porous scaffold.

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  • Materials For Medical Uses (AREA)

Abstract

L'invention concerne un procédé de fabrication d'un support biomédical, poreux, polymère, le procédé comprenant les étapes suivantes, consistant à : i) charger, à l'intérieur d'un appareil d'extrusion-moussage, au moins un polymère dans un état de dispersion ou de masse fondue à une température initiale; ii) introduire, à l'intérieur de l'appareil d'extrusion-moussage, un agent moussant à une pression initiale supérieure à la pression atmosphérique, l'agent moussant étant soluble dans ledit au moins un polymère; iii) permettre, à l'agent moussant, de se dissoudre dans ledit au moins un polymère de manière à former au moins un mélange comprenant ledit au moins un polymère et l'agent moussant; iv) extruder ledit au moins un mélange à travers une filière d'extrusion dans une zone d'expansion présentant une pression d'expansion inférieure à la pression initiale de telle sorte que ledit au moins un mélange subit une chute de pression et se dilate, ce qui permet d'obtenir le support biomédical, polymère, poreux.
PCT/IB2015/057196 2014-09-25 2015-09-18 Procédé de fabrication de supports poreux pour des utilisations biomédicales et supports correspondants WO2016046715A1 (fr)

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ITTO20140759 2014-09-25
ITTO2014A000759 2014-09-25

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WO2016046715A1 true WO2016046715A1 (fr) 2016-03-31

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109289091A (zh) * 2018-10-09 2019-02-01 温州医科大学 一种用于装载骨髓间充质干细胞的复合支架及其制备方法
TWI729999B (zh) * 2016-08-30 2021-06-11 艾爾生技有限公司 生醫支架及其製造方法
CN114077094A (zh) * 2020-08-13 2022-02-22 颖台科技股份有限公司 光扩散板及其制造方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000275A1 (fr) * 2000-06-24 2002-01-03 Victrex Manufacturing Limited Materiaux polymeriques biocompatibles
WO2002019947A1 (fr) * 2000-09-08 2002-03-14 Ferro Corporation Fabrication de parties orthopediques au moyen de traitement par fluide supercritique
EP1405649A1 (fr) * 2002-09-27 2004-04-07 Ethicon, Inc. Matrice d'échafaudage composite ensemencées de cellules de mammifères
EP1676591A2 (fr) * 2004-11-24 2006-07-05 Lifescan, Inc. Compositions et méthodes pour la création d'un environnement vascularisé pour la transplantation cellulaire
EP1797909A2 (fr) * 2005-11-28 2007-06-20 Lifescan, Inc. Compositions et procédés pour créer un environnement vascularisé pour la transplantation cellulaire

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000275A1 (fr) * 2000-06-24 2002-01-03 Victrex Manufacturing Limited Materiaux polymeriques biocompatibles
WO2002019947A1 (fr) * 2000-09-08 2002-03-14 Ferro Corporation Fabrication de parties orthopediques au moyen de traitement par fluide supercritique
EP1405649A1 (fr) * 2002-09-27 2004-04-07 Ethicon, Inc. Matrice d'échafaudage composite ensemencées de cellules de mammifères
EP1676591A2 (fr) * 2004-11-24 2006-07-05 Lifescan, Inc. Compositions et méthodes pour la création d'un environnement vascularisé pour la transplantation cellulaire
EP1797909A2 (fr) * 2005-11-28 2007-06-20 Lifescan, Inc. Compositions et procédés pour créer un environnement vascularisé pour la transplantation cellulaire

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Terminology of polymers and polymerization processes in dispersed systems", PURE APPL. CHEM., vol. 83, no. 12, 2011, pages 2229 - 2259
CONG CHEN ET AL: "In Vitro and In Vivo Characterization of Pentaerythritol Triacrylate-co-Trimethylolpropane Nanocomposite Scaffolds as Potential Bone Augments and Grafts", TISSUE ENGINEERING PART A, vol. 21, no. 1-2, 19 September 2014 (2014-09-19), US, pages 320 - 331, XP055223886, ISSN: 1937-3341, DOI: 10.1089/ten.tea.2014.0018 *
E SACHOLS ET AL: "MAKING TISSUE ENGINEERING SCAFFOLDS WORK. REVIEW ON THE APPLICATION OF SOLID FREEFORM FABRICATION TECHNOLOGY TO THE PRODUCTION OF TISSUE ENGINEERING SCAFFOLDS", EUROPEAN CELLS AND MATERIALS, vol. 5, 24 July 2003 (2003-07-24), pages 29 - 40, XP055193654 *
PETRIE ARONIN C E ET AL: "Osteogenic differentiation of dura mater stem cells cultured in vitro on three-dimensional porous scaffolds of poly(epsilon-caprolactone) fabricated via co-extrusion and gas foaming", ACTA BIOMATERIALIA, ELSEVIER, AMSTERDAM, NL, vol. 4, no. 5, 1 September 2008 (2008-09-01), pages 1187 - 1197, XP023611081, ISSN: 1742-7061, [retrieved on 20080318], DOI: 10.1016/J.ACTBIO.2008.02.029 *
TELEDYNE ISCO ET AL: "Extrusion Foaming Using Teledyne Isco Syringe Pumps Syringe Pump Application Note AN21", 28 September 2012 (2012-09-28), pages 1 - 5, XP055193152, Retrieved from the Internet <URL:http://www.isco.com/WebProductFiles/Applications/105/Application_Notes/Extrusion_Foaming.pdf> [retrieved on 20150602] *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI729999B (zh) * 2016-08-30 2021-06-11 艾爾生技有限公司 生醫支架及其製造方法
CN109289091A (zh) * 2018-10-09 2019-02-01 温州医科大学 一种用于装载骨髓间充质干细胞的复合支架及其制备方法
CN109289091B (zh) * 2018-10-09 2021-04-13 温州医科大学附属第一医院 一种用于装载骨髓间充质干细胞的复合支架及其制备方法
CN114077094A (zh) * 2020-08-13 2022-02-22 颖台科技股份有限公司 光扩散板及其制造方法
CN114077094B (zh) * 2020-08-13 2024-01-30 颖台科技股份有限公司 光扩散板及其制造方法

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