WO2016194011A1 - Procédé de préparation de constructions cellularisées sur la base d'hydrogels thermosensibles - Google Patents

Procédé de préparation de constructions cellularisées sur la base d'hydrogels thermosensibles Download PDF

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Publication number
WO2016194011A1
WO2016194011A1 PCT/IT2016/000142 IT2016000142W WO2016194011A1 WO 2016194011 A1 WO2016194011 A1 WO 2016194011A1 IT 2016000142 W IT2016000142 W IT 2016000142W WO 2016194011 A1 WO2016194011 A1 WO 2016194011A1
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WO
WIPO (PCT)
Prior art keywords
cellularized
gel
hydro
temperature
cells
Prior art date
Application number
PCT/IT2016/000142
Other languages
English (en)
Inventor
Valeria Chiono
Susanna SARTORI
Monica BOFFITO
Emilia GIOFFREDI
Marcella Trombetta
Pamela MOZETIC
Alberto Rainer
Sara Maria GIANNITELLI
Original Assignee
Politecnico Di Torino
Universita' Campus Bio-Medico Di Roma
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 Politecnico Di Torino, Universita' Campus Bio-Medico Di Roma filed Critical Politecnico Di Torino
Publication of WO2016194011A1 publication Critical patent/WO2016194011A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells

Definitions

  • the present invention is related to the sector of tissue engineering and refers to a method for preparing cellularized constructs based on hydro- gels by using a quick prototyping technique.
  • the invention deals with preparing cellularized constructs based on heat-sensitive hydro-gels, which are particularly stable under physiologic conditions of the human body.
  • Tissue engineering identifies procedures for regenerating human body tissues by seeding cells on structures (scaffolds) made of suitable materials and features and their cultivation in suitable reactors (bio-reactors) till the scaffold is colonized and a new tissue is produced (through deposition of an Extra Cellular Matrix, ECM, by the cells) .
  • the scaffold is generally made of biodegradable or bio-re-absorbable materials. Both terms, even if not equivalent, define the phenomena for which the scaffold, after a certain period passed in contact with the biologic environment, is subjected to chemical modifications which brings about its progressive "disappearance".
  • tissue engineering has more and more frequently used quick prototyping techniques, in order to obtain two- or three-dimensional cellularized scaffolds having a controlled geometry and incorporating cells inside the obtained structure.
  • Quick prototyping is an innovative technology which makes it possible to produce, in a few hours and without the use of tools, objects with an even complex three-dimensional geometry, based on a "file" containing information about their geometry.
  • quick prototyping is also designated with the terms “bio-printing” or “cell printing”, in order to point out a process for generating structures with controlled geometry incorporating cells, wherein cell functionality and vitality are preserved inside the printed construct (scaffold) .
  • bio-printing or “cell printing”
  • “printed” tissues and organs can be made available for their implant on a patient.
  • the “cell printing” technique uses cells and hydro-gels as printing materials, which, when mixed together in suitable amounts, form the so-called bio-ink.
  • a 3D printer is equipped with one or more heads which depose the bio-ink one layer at a time, embedding the cells into the polymeric hydro-gel which operates as support.
  • the three-dimensional object is obtained based on a computerized model, prepared with dedicated software, which is then "printed", deposing one layer of material above the other in the desired shape.
  • the object can be made of a single material or of a combination of mutually different materials: whichever the adopted solution, there is always a progressive addition, one layer above the other, of new material modelled in the desired shape through the relative movement between delivery nozzle and motored piece-holder table, together with material dispensing. This latter one can occur, for example, by extruding the material shaped as filaments organized according to the desired geometry, conveying the bulk of material through one or more holes (nozzle) with suitable shape and sizes.
  • cellularized scaffolds having a controlled structure and incorporating cells inside the filaments obtained by extrusion.
  • Such cellularized scaffolds can be obtained starting from suspensions of cells in aqueous solutions based on hydro-gels precursors, both of natural and of synthetic origin.
  • Hydro-gels are three-dimensional polymeric structures composed of hydrophilic homopolymers or copolymers, made insoluble in water through cross- linking (chemical or physical cross-linking) , which guarantee the shape stability of the structure.
  • cross-linking agents for example salts
  • ionic cross-linking use of low-molecular- weigh chemical agents (forming covalent links) and unsaturated cross-linkers in combination with a initiator and a physical irradiation stimulation (photo-cross-linking with formation of covalent links )
  • photo-cross-linking with formation of covalent links photo-cross-linking with formation of covalent links
  • WO 2010/030964 discloses a method to obtain three-dimensional multi-layer, cellularized or non- cellularized hydro-gels. The method is based on the nebulization of a thin layer of cross-linker on a substrate; on the following deposition of an hydro- gel precursor for its partial cross-linking; on the nebulisation of another thin layer of cross-linker to complete the cross-linking; finally, on the repetition of the above described processes for the desired number of times.
  • the method for embedding the cells between the various hydro-gel layers provides for nebulising a thin layer of cross- linker on a substrate; afterwards depositing an hydro-gel precursor for its partial cross-linking; depositing a layer of cells; depositing a layer of hydro-gel precursor; nebulising another thin layer of cross-linker to complete the cross-linking; finally, repeating the above described processes for the desired number of times.
  • Such description does not deal with a "cell printing" method like in the present patent application.
  • WO 2010/060080 disclosed necessary method and materials to obtain three-dimensional cellularized constructs with smooth muscle cells.
  • drops of collagen solution which embed smooth muscle cells are deposited inside the scaffold under construction.
  • the scaffold can be based on collagen, hyaluronic acid, fibrin and kitosan, synthetic polymers (poly glycol acid PGA, poly caprolactone PCL, poly (lactic acid) PLA) and carbon nanotubes.
  • Such method does not use heat-sensitive hydro-gels and is based on the cellular printing technique as drops of a cellular dispersion in a collagen solution.
  • WO 2011/119607 defines the composition of a polymeric ink for "quick prototyping" based on a polymer added with a photo-polymerizing monomer or a monomer having photo-polymerizing functionality.
  • Ink can include a cross-linking agent, a photo- initiator and water as solvent.
  • the polymer is solubilized in water at a concentration grater than 5-100 times with respect to a critical concentration (namely the transition concentration from a diluted to a semi-diluted solution) .
  • Viscous-elastic ink can include a photo- polymerizing monomer, such as acryl-amide or hydroxyl ethyl methacrylate for inks based on poly acryl-amide (pAM) or poly ( 2-hydroxyl ethyl methacrylate (pHEMA) .
  • pAM poly acryl-amide
  • pHEMA 2-hydroxyl ethyl methacrylate
  • US 2012/0282448 discloses a method for preparing three-dimensional structures through quick prototyping starting from a complex ink containing various ingredients: a compound cross- linkable with a cationic mechanism, a cationic photo-initiator, a compound cross-linkable with a radical mechanism, a radical photo-initiator and a gelling material.
  • the gelling material is used as semi-solid gel and is extruded in combination with the other materials.
  • the gelling material is then used to avoid the structure collapse after its deposition and before its chemical cross-linking.
  • Such patent application does not deal with a "cell printing" method, and provides for a chemical cross-linking of the structure, after its deposition by extrusion.
  • Hydro-gel for printing three-dimensional constructs.
  • Hydro-gel is composed of a three-block ABA polymer in combination with a second three-block Ama-B-Ama polymer, where Ama is a polymer based on A units functionalized with methacrylate functionality (ma) .
  • the obtained material is heat-sensitive and photo-cross-linkable and is solubilised in de- ionized water.
  • the solution is afterwards extruded through the nozzle of a quick prototyping machine on a substrate heated above the gelling point. Under the selected process conditions, the sol-gel transition quickly occurs. Afterwards, the material is stabilized by irradiation (photo-cross-linking) . Also in this case, the above listed inconveniences can occur, related to the stabilization of hydro- gels due to photo-induced cross-linking.
  • the process consists in preparing a cellular suspension in a solution based on a mixture of the "first hydro-gel precursor” (component capable of gelling when the temperature decreases and having a transition temperature from solution phase to gel phase included between 10°C and 30 °C) and of the "second hydro-gel precursor” (chemically cross-linkable component) .
  • the material is extruded as a solution or as a gel on a substrate with temperature lower than the gelling point of the first component, in order to stabilize the shape of the extruded structure.
  • the construct is then chemically cross-linked, possibly by immersion in a medium containing the cross-linking agent.
  • the second component of the mixture is cross-linked.
  • the first component of the system therefore, has the function of ensuring the structure stability in short times, while the second component is the structural material of the construct.
  • the first component usually is quickly solubilised and moved away from the system.
  • the use of heat-sensitive hydro-gels with gel-sol transition upon increasing the temperature as disclosed in EP 2679699, has several negative aspects, such as:
  • US-A1-2015/084232 discloses a prior art method for producing a scaffold which includes cells.
  • EP-A1-2679669 discloses a prior art process for preparing three-dimensional hydro-gel objects comprising living cells.
  • O-A2-2005/016114 discloses a prior art method of manufacturing hydro-gels tissue scaffolds loaded with cells.
  • the Applicant has discovered the chance of preparing cellularized constructs based on hydro- gels having high stability, using heat-sensitive hydro-gels having a transition from the sol phase to the gel phase upon increasing the temperature.
  • the present invention deals with a method for preparing cellularized constructs through quick prototyping comprising the following steps:
  • step B) dispersing cells inside the polymeric solution prepared in step A) ;
  • step A wherein the polymeric solution prepared in step A) has a transition from the "sol” phase to the "gel” phase upon increasing the temperature.
  • the method for preparing cellularized constructs according to the present invention uses a polymeric dispersion as heat-sensitive hydro-gel, having a transition from the "sol” phase to the "gel” phase which is performed through heating, and occurs at a temperature included between 20 °C and 30°C.
  • the chemical nature of the biocompatible and biodegradable hydro-gel material consists in a natural or synthetic polymer, or in a mixture of natural polymers or of synthetic polymers, or in a mixture of natural and synthetic polymers, possibly embedding one or more pharmaceuticals for promoting the cellular survival (for example, anti-oxidizing and/or anti-inflammatory pharmaceuticals) and/or other agents (for example, inorganic calcium phosphate nanoparticles in case of applications in bone regeneration) .
  • pharmaceuticals for promoting the cellular survival for example, anti-oxidizing and/or anti-inflammatory pharmaceuticals
  • other agents for example, inorganic calcium phosphate nanoparticles in case of applications in bone regeneration
  • the synthetic polymers which can be used in step A) of the preparation of the polymeric solution are preferably chosen among poloxamers, polyurethanes and polyester-based amphiphilic copolymers (for example, block copolymers composed of poly (caprolactone) and of poly (ethylene glycol) units) .
  • polyester-based amphiphilic copolymers for example, block copolymers composed of poly (caprolactone) and of poly (ethylene glycol) units
  • kitosan and gelatine can be preferably used.
  • step A) of the invention consists in preparing a polymeric solution comprising one or more polymers in a culture medium.
  • the culture medium is a saline solution, generally isotonic and at physiologic pH, enriched with nutrient substances for the cells, such as for example amino-acids, proteins (among which hormones) and, optionally, colorimetric indicators of pH and antibiotics, which is used for the in vitro culture of cells.
  • Such solution is usually referred to as "culture medium” and can be purchases ready made, or can be prepared in a laboratory.
  • culture medium There are several types of culture mediums according to the type of cell that has to be embedded, which are differentiated for the type of uses nutritive substances. Therefore, once having selected the type of cells that have to be embedded in the polymeric dispersion, the most appropriated culture medium for them will be used.
  • the polymeric solution prepared in step A) of the inventive method can optionally comprise one or more bioactive molecules or pharmaceuticals suitable for promoting a particular cellular response.
  • step B) of the method of the present invention the dispersion of the cells to be embedded in the polymeric solution is performed, namely step B) of the method of the present invention.
  • the obtained cellular dispersion is then subjected to gelling through a temperature increase, such as to take the dispersion to its gel status.
  • the operating temperature is then increases from a starting value, suitable to prepare the polymeric solution (step A) included between 1°C and 20°C, preferably between 1°C and 5°C, till the value provided in step C) , namely between 36°C and 38 °C, thereby determining the gelling of the cellular dispersion obtained in step B) .
  • step C The necessary time to obtain the gelling of the polymeric dispersion comprising the cells (step C) is generally included between 1 and 30 minutes, preferably between 5 and 15 minutes.
  • step D) of the invention is performed, namely extruding the cellularized hydro- gel on a thermostated support at a temperature included between 36°C and 38 °C, according to a controlled geometry.
  • the thermostat support is composed of a plate for the cellular culture, which is kept at a temperature around 37 °C through a suitable heating system (for example, due to contact with a metallic support heated through resistances) . Keeping the temperature at a value as much as possible near 37 °C is above all functional to the viability of cells dispersed inside the hydro-gel to be printed.
  • step (D) of extrusion printing is repeated for a number n of times for depositing n two-dimensional layers, in order to obtain a three- dimensional cellularized construct according to the desired geometry.
  • the cellularized construct obtained with the method of the invention remains stable in a physiologic medium at 37 °C for some days, even without the use of cross-linking agents (its stability depends on the chemical nature of the used material) .
  • the cells remain viable during the process for gelling and producing the cellularized constructs as described above.
  • the proposed system is based on heat-sensitive hydro-gels with sol-gel transition upon increasing the temperature, since heat-sensitive hydro-gels having an inverse behaviour (sol-gel transition upon decreasing the temperature) , if processed according to a similar method, show various critical points.
  • the cells should ideally be embedded in a polymeric solution prepared at high temperature (>37°C), and such methodology could result aggressive for the cells.
  • the method of the invention specifically deals with heat-sensitive hydro-gels with sol-gel transition upon increasing the temperature.
  • bioactive molecules such as pharmaceuticals and growth factors
  • the diameter of the extruded structures can drop down to 50 microns, being 200 microns the preferred value in order not to induce cellular stress.
  • the diameter of the extruded structures can drop down to 50 microns, being 200 microns the preferred value in order not to induce cellular stress.
  • the circularity parameter computed through ImageJ software on scaffold sections with 200- ⁇ fibres is equal to 0.9986 +/- 0.0011.
  • the method of the invention finds application in the biomedical field within tissue engineering, mainly for making models of human/animal tissues for in vitro studies and for making cellularized structures for reconstructing tissues/organs with in vitro or in vivo techniques.
  • Another field of application is the possibility of performing a printing of the construct directly onto the injuried tissue (for example, injuried skin) .
  • injuried tissue for example, injuried skin
  • the described method could be particularly advantageous for treating severe burns. Burnt patients, in fact, can be subjected to deadly infections unless timely treated (as an average within two weeks) .
  • the current treatment consists in implanting heterologous skin (skin graft) , but this treatment is painful and implies the formation of scars.
  • the direct printing technique onto the patient has the advantage of being able to be timely applied, avoiding the danger of having potentially deadly infections, and is a promise for the regeneration of a completely functional skin.

Abstract

La présente invention concerne un procédé de préparation de constructions cellularisées par prototypage rapide comprenant les étapes suivantes : (A) préparation d'une solution polymère comprenant un ou plusieurs polymères naturels et/ou synthétiques dans un milieu de culture à une température comprise entre 1 °C et 20 °C ; (B) dispersion de cellules dans la solution polymère ; (C) gélification de la dispersion polymère contenant lesdites cellules en augmentant la température à une valeur comprise entre 36 °C et 38 °C afin d'obtenir un hydrogel cellularisé ; et (D) extrusion de l'hydrogel cellularisé par l'intermédiaire d'une buse adaptée sur un support thermostaté à une température comprise entre 36 °C et 38 °C.
PCT/IT2016/000142 2015-06-05 2016-06-01 Procédé de préparation de constructions cellularisées sur la base d'hydrogels thermosensibles WO2016194011A1 (fr)

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ITUB20151326 2015-06-05
IT102015000020718 2015-06-05

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018100580A1 (fr) 2016-12-01 2018-06-07 Regenesis Biomedical Ltd. Procédé et système pour impression en 3d

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005016114A2 (fr) 2003-05-05 2005-02-24 Massachusetts Eye & Ear Infirmary Production de tissu vivant 'in vivo' au moyen d'un moule
WO2010030964A2 (fr) 2008-09-12 2010-03-18 The Brigham And Women's Hospital, Inc. Hydrogels multicouches tridimensionnels et leurs procédés de préparation
WO2010060080A1 (fr) 2008-11-24 2010-05-27 Immunotrex Corporation Génération de tissu tridimensionnel
WO2011119607A2 (fr) 2010-03-24 2011-09-29 The Board Of Trustees Of The University Of Illinois Encre viscoélastique pour l'écriture directe de structures d'hydrogel
US20120282448A1 (en) 2011-05-03 2012-11-08 Xerox Corporation Methods for fabricating three-dimensional objects
EP2679669A1 (fr) 2012-06-26 2014-01-01 ETH Zurich Fabrication d'hydrogels tridimensionnels
EP2679699A2 (fr) 2005-03-31 2014-01-01 Kabushiki Kaisha Kobe Seiko Sho Feuille d'acier laminée à froid à haute résistance mécanique et pièces d'automobiles en acier ayant d'excellentes propriétés d'adhésion de film de revêtement, maniabilité et résistivité face à la fragilisation par l'hydrogène
US20140179822A1 (en) 2012-07-27 2014-06-26 Gongyao Zhou Thermosensitive and Crosslinkable Polymer Composite for Three-Dimensional Soft Tissue Scaffold Printing
US20150084232A1 (en) 2013-09-26 2015-03-26 Northwestern University Poly(ethylene glycol) cross-linking of soft materials to tailor viscoelastic properties for bioprinting

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005016114A2 (fr) 2003-05-05 2005-02-24 Massachusetts Eye & Ear Infirmary Production de tissu vivant 'in vivo' au moyen d'un moule
EP2679699A2 (fr) 2005-03-31 2014-01-01 Kabushiki Kaisha Kobe Seiko Sho Feuille d'acier laminée à froid à haute résistance mécanique et pièces d'automobiles en acier ayant d'excellentes propriétés d'adhésion de film de revêtement, maniabilité et résistivité face à la fragilisation par l'hydrogène
WO2010030964A2 (fr) 2008-09-12 2010-03-18 The Brigham And Women's Hospital, Inc. Hydrogels multicouches tridimensionnels et leurs procédés de préparation
WO2010060080A1 (fr) 2008-11-24 2010-05-27 Immunotrex Corporation Génération de tissu tridimensionnel
WO2011119607A2 (fr) 2010-03-24 2011-09-29 The Board Of Trustees Of The University Of Illinois Encre viscoélastique pour l'écriture directe de structures d'hydrogel
US20120282448A1 (en) 2011-05-03 2012-11-08 Xerox Corporation Methods for fabricating three-dimensional objects
EP2679669A1 (fr) 2012-06-26 2014-01-01 ETH Zurich Fabrication d'hydrogels tridimensionnels
US20140179822A1 (en) 2012-07-27 2014-06-26 Gongyao Zhou Thermosensitive and Crosslinkable Polymer Composite for Three-Dimensional Soft Tissue Scaffold Printing
US20150084232A1 (en) 2013-09-26 2015-03-26 Northwestern University Poly(ethylene glycol) cross-linking of soft materials to tailor viscoelastic properties for bioprinting

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018100580A1 (fr) 2016-12-01 2018-06-07 Regenesis Biomedical Ltd. Procédé et système pour impression en 3d

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