WO2018100580A1 - Procédé et système pour impression en 3d - Google Patents

Procédé et système pour impression en 3d Download PDF

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Publication number
WO2018100580A1
WO2018100580A1 PCT/IL2017/051305 IL2017051305W WO2018100580A1 WO 2018100580 A1 WO2018100580 A1 WO 2018100580A1 IL 2017051305 W IL2017051305 W IL 2017051305W WO 2018100580 A1 WO2018100580 A1 WO 2018100580A1
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WIPO (PCT)
Prior art keywords
hydrogel
layer
gelatinous
scaffold
thermosensitive
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PCT/IL2017/051305
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English (en)
Inventor
Reuven EDRI
Itai Cohen
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Regenesis Biomedical Ltd.
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Publication date
Application filed by Regenesis Biomedical Ltd. filed Critical Regenesis Biomedical Ltd.
Publication of WO2018100580A1 publication Critical patent/WO2018100580A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/10—Processes of additive manufacturing
    • B29C64/106—Processes of additive manufacturing using only liquids or viscous materials, e.g. depositing a continuous bead of viscous material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y10/00—Processes of additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y30/00—Apparatus for additive manufacturing; Details thereof or accessories therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y80/00—Products made by additive manufacturing

Definitions

  • the present disclosure concerns 3D printing, particularly useful for printing 3D biocompatible articles
  • WO 2016/100856 disclosed water dispersion of cellulose nanofibrils that may be used as a bioink for 3D bio-printing of tissue and organs with desired architecture.
  • Ovsianikov et al. (Materials 2011, 4, 288-299), disclosed utilization of a two- photon polymerization technique implementing photosensitive modified gelatin to generate a 3D scaffold with a square cross-section of 250 ⁇ ⁇ 250 ⁇ .
  • current printers based on jetting, extrusion and laser-induced forward transfer cannot produce structures with sufficient size, strength and vascularization to be implanted in the body.
  • US 2017/0217091 disclosed systems, methods, and materials for 3D printing of objects that include a cured hydrogel material, an uncured hydrogel material, and a support material.
  • the cured hydrogel material may define a scaffold for organs or other biological structures.
  • the 3D printing system selectively deposits the hydrogel material and support material, dries the hydrogel material, and selectively applies a catalyst to the hydrogel material to selectively cure the hydrogel material.
  • WO 2016/036275 disclosed a method for printing biological tissues and organs, and in a device for implementing same.
  • WO 2016/194011 discloses a method for preparing cellularized constructs of thermosensitive hydrogels through quick prototyping.
  • WO 2016/090286 disclosed a method or apparatus for 3D-printing.
  • the method may comprise causing a phase change in a region of the first material by applying focused energy to the region using a focused energy source, and displacing the first material with a second material.
  • WO 2015/158718 disclosed a resin composition, in particular suitable for printing, a kit comprising the components of the resin composition, a printing method utilizing the resin composition, a polymer obtained by the printing method, an article comprising or formed from the polymer, and uses thereof.
  • the present disclosure provides a process for generating a 3D scaffold, comprising:
  • thermosensitive hydrogel in (specifically gelatinous) form
  • thermosensitive gelatinous hydrogel (b) directing a focused heat onto portions of the layer of the thermosensitive hydrogel to cause said portions of the thermosensitive gelatinous hydrogel to solidify into a solid scaffold layer within the gelatinous thermosensitive hydrogel, said exposed portions constitute a layer within said 3D scaffold;
  • 3D scaffold obtained or obtainable by the process disclosed herein.
  • the present disclosure provides a system for generation of a 3D scaffold, comprising: a receptacle configured to hold a fluid (gelatinous) thermosensitive hydrogel and optionally including a temperature regulating module; a focused heat source configured to direct a focused heat onto portions of a layer of the thermosensitive hydrogel held within said receptacle and cause solidification of said portions into a solid scaffold layer within the fluid (gelatinous) thermosensitive hydrogel; a dispenser configured for dispensing an amount of said thermosensitive hydrogel within the receptacle to form a layer of fluid (gelatinous) thermosensitive hydrogel within said receptacle; and a control unit for controlling addition, layer by layer, of said thermosensitive hydrogel within the receptacle and/or for controlling the exposure or direction of said focused heat onto said portions of the thermosensitive hydrogel, to cause said thermosensitive gelatinous hydrogel to solidify into a solid scaffold layer within the gelatinous thermosensitive hydrogel.
  • Fig. 1 is a schematic isometric illustration of a system for generating a 3D scaffold in accordance with some embodiments of the present disclosure.
  • thermosensitive hydrogel s as a supporting environment (supporting hydrogel) for generating 3D scaffold, from the same thermosensitive hydrogel composition.
  • the hydrogel(s) used herein have a dual function, on the one hand, they act as a supporting media for the generated 3D scaffold, and on the other hand, they provide the "building blocks" for the generated 3D scaffold.
  • the 'layer-by-layer' generated 3D scaffold is embedded within the hydrogel environment.
  • the native hydrogel i.e. that has not been affected by the focused heat applied, prevents dispatching or movement of solidified portions and the latter remain intact and static. This allows the projection of a network with high resolution of solid material within its native hydrogel.
  • thermosensitive hydrogel (a) adding into a receptacle a layer of a gelatinous thermosensitive hydrogel
  • thermosensitive gelatinous hydrogel (b) applying onto portions of the layer of the gelatinous thermosensitive hydrogel a focused heat to cause said portions of the thermosensitive gelatinous hydrogel to solidify (e.g. cure or polymerize) into a solid scaffold layer within the gelatinous thermosensitive hydrogel, said exposed portions constitute a layer within said 3D scaffold;
  • a system for generation of a 3D scaffold comprising: - a receptacle configured to hold a gelatinous thermosensitive hydrogel; a focused heat source configured to exposure portions of a layer of thermosensitive hydrogel held within said receptacle to a focused heat and cause solidification of said portions into a solid scaffold layer within the gelatinous thermosensitive hydrogel; a dispenser configured for dispensing an amount of said thermosensitive hydrogel within the receptacle to form a layer of gelatinous thermosensitive hydrogel within said receptacle; and a control unit for controlling addition, layer by layer, of said thermosensitive hydrogel within the receptacle and, for controlling exposure of said focused heat onto said portions of the thermosensitive hydrogel, to cause said thermosensitive gelatinous hydrogel to solidify into a solid scaffold layer within the gelatinous thermosensitive hydrogel.
  • Hydrogels are water insoluble macromolecular polymer gels constructed of a network of crosslinked hydrophilic polymer chains.
  • a principle feature of hydrogels, differentiating them from gels is their inherent crosslinking (which can be chemical or physical) that enables them to swell water while retaining their three dimensional structure. The presence of high concentration of water in the hydrogel makes them more suitable for cell growth and more similar to tissues' extra cellular environment.
  • a gelatinous hydrogel is one having a semi-liquid consistency (jellylike consistency).
  • a gelatinous state can be determined by the material's rheological characteristics. Examples of tests that can be used in this respect include, (1) Time sweep to determine the gelation time of the hydrogel. (2) Strain sweep to determine the linear-viscoelastic region of the hydrogel with respect to strain. (3) Frequency sweep to determine the linear equilibrium modulus plateau of the hydrogel. (4) Time sweep with values obtained from strain and frequency sweeps to accurately report the equilibrium moduli and gelation time and any combination of same.
  • the hydrogel is a biocompatible hydrogel.
  • biocompatible hydrogel may be of natural origin, synthetic or semi-synthetic (e.g. modification of a natural hydrogel).
  • the hydrogels are biological hydrogel, such as, without being limited thereto, peptides, polypeptides, proteins, polysaccharides.
  • hydrogel or hydrogel layer, it is to be understood as one comprising not only the cross-linked macromolecule (forming the swellable polymeric network) but also other components to be incorporated within the 3D scaffold.
  • the hydrogel may comprise growth factors or other elements that would assist in the growth of cells within the scaffold.
  • growth factors include, epidermal growth factor (EGF), fibroblast growth factors (FGFs), sonic hedgehog (SHH), bone morphogenetic proteins (BMPs) and Delta-like 1 ligand (DLL-1) and glycosaminoglycan.
  • EGF epidermal growth factor
  • FGFs fibroblast growth factors
  • SHH sonic hedgehog
  • BMPs bone morphogenetic proteins
  • DLL-1 Delta-like 1 ligand
  • glycosaminoglycan glycosaminoglycan.
  • the hydrogel may comprise viable cells that once a 3D scaffold layer is formed, such cells adhere to the surface of the thus formed solid scaffold.
  • the hydrogel comprises proteins of the extracellular matrix (ECM).
  • ECM proteins are selected from the group consisting of collagen, laminin, fibronectin and elastin or any combination thereof.
  • the hydrogel is glycosaminoglycan -based hydrogel comprising ECM proteins.
  • the thermosensitive hydrogel is MatrigelTM, which is a gelatinous protein mixture secreted by Engelbreth -Holm- Swarm (EHS) mouse sarcoma cells or any other similar product (basal membrane products) (produced and marketed by Corning Life Sciences and BD Biosciences or can be obtained under the tradename Cultrex BME, marketed by Trevigen, Inc.).
  • the main components of Matrigel are structural proteins such as laminin, entactin, collagen and heparan sulfate proteoglycans which present cultured cells with the adhesive peptide sequences that they would encounter in their natural environment.
  • thermosensitive hydrogels and in particular, biocompatible thermosensitive hydrogels, are known and those versed in the art would know how to pick and choose a hydrogel suitable for use in accordance with the present disclosure.
  • the hydrogel used is a thermosensitive hydrogel.
  • a thermosensitive hydrogel is to be understood as encompassing any hydrogel or combination of hydrogels that solidify at a temperature above 24°C or, at times, above 30°C.
  • the thermosensitive hydrogel solidifies at the temperature above 35°C or above 37°C.
  • the thermosensitive hydrogel solidifies at the temperature between about 30°C to about 40°C, or between about 35°C to about 37°C.
  • synthetic hydrogels that can be used in the generation of a 3D scaffold according to the present disclosure.
  • examples include, without being limited thereto, Poly-N- vinyl caprolactam (PNVC), Poly-N-isopropylacrylamide, (PNIPAM), Poly-silamine, Poly-vinyl-methyl ether (PVME) Poly-propylene glycol (PPG), Poly- lactic-co-glycolic-acid: Poly-ethylene glycol: Poly-lactic-co-glycolic acid (PLGA-PEG- PLGA), triblock copolymers of poly(ethylene glycol)-poly(epsilon-caprolactone-co- glycolide)-poly(ethylene glycol) [PEG-P(CL-GA)-PEG], triblock copolymers of polyethylene glycol and partially methacrylated poly[N-(2-hydroxypropyl) methacrylamide mono/dilactate] and others.
  • PNVC Poly-N- vinyl caprolactam
  • the hydrogel is added to the receptacle at each process cycle (a cycle being defined by a single addition of hydrogel and subsequent exposure to heat) to form a thin layer of gelatinous hydrogel on top of a previously added hydrogel layer.
  • the layer is added selectively onto specific portions that are associated with the formation of the 3D scaffold, namely a low resolution of deposition of the hydrogel and subsequently exposing the desired portion of the newly added hydrogel layer to a focused heat to obtain a high resolution solid formation that constitutes a layer of the 3D scaffold.
  • Layer by layer the 3D scaffold is formed, within the hydrogel medium that has not been exposed to heat.
  • it may be a continuous layer or a patterned layer (corresponding to a slice of the 3D scaffold) having the desired thickness.
  • the addition of the hydrogel can be by any dispensing means known in the art, such as injecting, extruding, and pouring.
  • the addition of the hydrogel is by the use of a dispenser, as described with respect to the disclosed system.
  • the dispenser may include a single dispensing unit, such as a syringe or an extruder, or a set of dispensing units, e.g. a set of syringes or set of extruders.
  • the receptacle for receiving the hydrogel layer has a top end, that is at least partially open, to allow at least the dispensing of hydrogel into the receptacle above a previously added layer.
  • the gelatinous thermosensitive hydrogel is added to the receptacle so as to form a layer over any previously placed hydrogel matter a new layer with a vertical depth (VD).
  • the vertical depth VD of the newly added hydrogel layer is related to a solidification depth sd of the hydrogel into which solidification is effected by the applied heat. For example, given that solidification of the hydrogel layer is effected by the applied heat to a solidification depth sd within the layer of gelatinous hydrogel, the vertical depth VD may be adapted to be similar or smaller than solidification depth sd.
  • the gelatinous hydrogel layer above the generated solid scaffold layer may solidify throughout its entire thickness, and the resulting new payer of (solidified) scaffold layer can merge, during solidification, with the previous generated scaffold layer.
  • VD ⁇ sd a sequential series of generated scaffold layers will typically merge together into an integral 3D scaffold.
  • the vertical depth VD is between ⁇ to 1mm, at times, between 10 ⁇ to about 100 ⁇ , or between about 20 ⁇ to about 80 ⁇ , or between about 30 ⁇ to about 70 ⁇ , or between about 40 ⁇ to about 60 ⁇ or between about 50 ⁇ deep.
  • the surrounding gelatinous hydrogel may be replaced, or supplemented with other components that may be required for maintaining the integrity of the 3D scaffold layer(s) generated, and/or for supporting cell growth on the 3D scaffold, etc.
  • the layer of gelatinous hydrogel is exposed to focused heat.
  • the focused heat comprises electromagnetic radiation.
  • the electromagnetic radiation is or comprises infrared (IR) radiation.
  • the electromagnetic radiation comprises a wavelength or wavelength range and within the IR spectrum, for example, a single wavelength or a band of between about 700 to about 1300 nm, or of between about 800 to about 1200 nm or of between about 900 to about 1100 nm.
  • this low energy wavelength does not cause ionization of water molecules and therefore cannot cause the formation of free radicals that in turn may harm the proteins or the cells thereafter.
  • the use of IR radiation may have some advantages over using other energy sources, such as UV.
  • the focused heat is applied in a form of a laser beam or as an array of laser beams or a single laser beam spatially split into several beams.
  • the laser beam may be split by any known method e.g. using sets of half mirrors, prisms, lenses, beam splitters, liquid crystal gratings etc.
  • the power of the laser can be adapted for each of the beams projected on the hydrogel layer. According to one embodiment the power is split equally between the beams.
  • the radiation applied may comprise a single laser beam or a plurality of beams.
  • the laser is femto-second laser beam capable of leading to multiphoton absorption.
  • the laser beam is a single uniformly split beam forming a network of laser beams.
  • the laser beam has a diameter of between about 1 ⁇ to about ⁇ at the surface of the exposed hydrogel layer.
  • the laser beam diameter is between about 5 ⁇ to about 50 ⁇ , at times, between about 10 to about 20 ⁇ .
  • the beam diameter may be controlled by the quality of the laser beam, optical system set-up (for example by expansion of the beam diameter and focusing with the lens smaller beam diameter could be produced), beam shape, etc. Beam diameter at the aperture and the diffractive limit depends on the beam profile.
  • the minimum beam diameter of a Gaussian beam at 1/e 2 intensity level that is focused with a single lens is defined with:
  • diameter min -7- * 2
  • f is a focal length of a lens
  • ⁇ is the wavelength
  • D' is the input beam size
  • 2 is the value of the input laser beam (usually due to set-up limitation, the actual beam size is 1.5-5 times bigger than the diffraction-limited spot size).
  • the energy per pulse of a single beam or each sub-beam of the split beam depends on the average power (related to the average energy flow, the pulse width and the repetition rate, as should be understood by the person skilled in the art).
  • the focused heat preferably, but not exclusively, applied in a form of a laser beam, preferably, IR laser beam, has an energy per pulse of between InJ to lOmJ, at times, between about 100 nJ to 1 mJ, or at times, between about 1 ⁇ to about 100 ⁇ (depending on the average power and the repetition rate).
  • the focused heat or laser beam has an average energy flow of a single beam or each sub-beam of a split beam for heat, and preferably IR induced solidification, of between about 0.1 J/cm 2 to 10 J/cm 2 , at times, between about 0.5 and 5 J/cm 2 , or at times between about 1 and 3 J/cm 2 .
  • the pulses When applied in a form of pulsed beams, the pulses have, in accordance with some embodiments, a repetition rate between 1 Hz to 80MHz, at times, between 10 Hz to 80MHz.
  • Femto-second (fs) laser may preferably be used for IR curing, whereas thermal equilibrium after excitation with a fs laser pulse may take a few hundreds of fs to few ps.
  • a laser beam when using a laser beam as the focused heat, it may be controlled by an optical deflection system or a scanning system comprising mirrors and/or prisms and/or lenses.
  • a scanning system may comprise a galvanoscanner controlled by the servo circuits that drives the galvo and controls the position of mirrors. Long travel servo galvanoscanner could be synchronized with the linear stage to move the workpiece underneath the high speed scanner. In this case an actual field of view of lens in not limiting factor, therefore a desired beam diameter of could be achieved.
  • Another possible scanning mechanism is polygon scanner, which can provide higher speed than galvanoscanner.
  • Another possibility is to combine mirror based scanner (galvoscanner) with an optical deflector.
  • Thermal penetration of the laser beam depends on the reflectivity of the hydrogel and pulse length, which depends on thermo-physical properties such as conductivity, density and heat capacity. In general, thermal penetration will be lower in IR region than, for example, UV region.
  • the thermal penetration length is given by the equation:
  • L th 2/CT 1 / 2 where k is the thermal diffusivity and ⁇ is the pulse duration.
  • the depth of focus and the precision (spatial resolution) of thermal effect may be controlled by various parameters.
  • the laser beam profile may be spatially optimized.
  • a top-hat profile is preferred, because it can produce sharper edges than a Gaussian beam profile, creating more localized heating effect.
  • Conversion to the top-hat profile can be accomplished by refractive or diffractive optics. Is it possible to shape a Gaussian beam into Bessel-Gaussian beam thus according to other embodiments, shaping of a Gaussian beam into Bessel-Gaussian beam may be effected. Bessel beam possesses a micron-sized focal spot and it can be generated by using axicon lens or diffractive optics. Additionally, the depth of focus may be controlled by choosing a suitable optical set up.
  • a precise laser beam is achieved by ultra-short laser pulses, precision of positioning stages and suitable optics.
  • Femtosecond laser pulses cause minimal heat-affected zone and maximize spatial precision and some such lasers can be characterized by the following specifications.
  • the operation of such laser can be controlled, for example, by SCA software.
  • the focused heat can be applied by directly contacting a heated tip or probe (e.g. heated by electric current as short as about ⁇ sec) with the hydrogel portions that are to be solidified.
  • a heated tip or probe e.g. heated by electric current as short as about ⁇ sec
  • the focused heat is applied onto portions of the hydrogel layer so as to cause heating of the hydrogel (at these portion) to a temperature at which the exposed hydrogel solidifies.
  • solidify encompasses "curing” or “polymerization” or any other form of interaction within the hydrogel that causes the hydrogel to convert, at the heated portions, from its gel state into a solid state.
  • solidification of the hydrogel refers to increase in the young's modulus of the hydrogel and/or maximum elastic modulus thereof as compared to the respective value before exposure to the focused heat (i.e. when in fluid/gelatinous state).
  • the heating temperature is to a temperature above 25°C.
  • the selection of the focused heat parameters may depend inter alia on the type of hydrogel used, e.g. such that the applied heating induces heating above the solidification temperature of the hydrogel. Such parameters can be easily determined, if such data is not available in the art.
  • thermosensitive hydrogels such as MATRIGEL® may solidify upon the application of a focused beam of laser in the infrared, heating the hydrogel locally to 25°C or above.
  • high spatial resolution may be achieved by employing a pulsed laser, such as a nano-second or pico-second or even femto-second laser, with a high peak power, thereby affecting temperature rise which is practically limited to the region of focus of the laser beam.
  • a pulsed laser such as a nano-second or pico-second or even femto-second laser
  • an additional layer of gelatinous hydrogel is preferably added. Equilibrium can be determined when the surface of the solidified layer is horizontal and essentially planar.
  • the hydrogel layers may be introduced into a receptacle having temperature/climate control module, including a heat exchanger, temperature sensor, humidity sensor to determine and control/adjust the temperature and humidity of the hydrogel surroundings within the receptacle.
  • temperature/climate control module including a heat exchanger, temperature sensor, humidity sensor to determine and control/adjust the temperature and humidity of the hydrogel surroundings within the receptacle.
  • the addition of layers and solidification (process cycles) continues until the desired complete 3D scaffold is formed within the supporting hydrogel.
  • the remaining of the hydrogel that is not used for the formation of the 3D scaffold e.g. that is used as a support, can be rinsed during the process of the formation of the 3D scaffold such that mainly only the solidified gel remains in the receptacle in an intermediate phase of the formation of the 3D scaffold.
  • the remaining of the hydrogel are being left in the receptacle during the formation of the 3D scaffold and being rinsed at the end of the process.
  • process cycles can be manual but are typically controlled by a control unit that is operably linked (via wire or wirelessly) to the system's components, i.e. the dispenser, the heat source, the climate control module.
  • the control unit is thus configured and operable to receive input data indicative of one or more process parameters and analyze the same (by a dedicated analyzer therein) to produce an output comprising operational data/instruction for actuating at least the focused heat source and said dispenser (with optionally the climate control module).
  • control unit comprises input/output utilities and a memory utility and an analyzer.
  • Fig. 1 providing, in a non-limiting manner, a system
  • System 100 includes a frame 102 including a stage 104 having fixed at its bottom portion, a receptacle 106 and a set of dispensers 108 , in a form of syringes, each connected to a respective pump 110, for pumping hydrogel from a central reservoir (not shown) into said dispensers, via injection tubes 112.
  • the dispensers may each be equipped with a dedicated replaceable/rechargeable hydrogel containing cartridge as a direct source of hydrogel. While not illustrated, the dispenser may also have other forms, such as, an inlet tube connected to the hydrogel reservoir and by means of a pump, introducing hydrogel into the receptacle 106.
  • Dispensers 108 are mounted above receptacle 106 that is open at its top 114 so as to allow introducing of hydrogel into the receptacle 106.
  • An IR laser 116 is mounted on a laser holder 118 and is configured for directing a laser beam onto portions of hydrogel held within receptacle 106.
  • Laser 116 can be directed by guided movement of laser holder 118 to allow heat induced solidification according to a predefined pattern, or alternatively laser 116 can be directed by an optical arrangement such as a galvo optical system.
  • stage 104 is movable with respect to dispensers 108 and/or laser 116 and the exposure to heat is dictated by the controlled movement of stage 104
  • the system also comprise a temperature/climate control module 126 for regulating temperature of the gelatinous hydrogel within receptacle 106.
  • System 100 also comprises a control unit 130 that is connected, either by physical connection or by wireless connection to the various components, including one or more of the dispensers, laser, moveable elements, climate control module etc.
  • Control unit 130 thus comprises, at least input/output utilities 132, memory utility 134 and a programmable analyzer 136 and operates to receive the input data regarding the process parameters, such as the hydrogel used and/or required IR parameters, type of beam to be applied, amount of hydrogel to be introduced, time interval between process cycles etc., and analyze the same so as to respectively operate the system's components.
  • Memory utility 134 may store or may be inputted with a data indicative to a desired 3D scaffold formation to be generated and / or with a series of virtual planar patterns of slices of the 3D scaffold which when combined together form the 3D map of the scaffold.
  • Each of the planar patterns is associated and correlates with one layer of the scaffold (a solid scaffold layer), so that the pattern identifies the regions to which the focused heat should be delivered to solidify the hydrogel and generate a scaffold layer.
  • the control unit is thus configured and operable to receive input data and to produce an output comprising operational data/instruction for actuating at least the focused heat source and said dispenser (with optionally the climate control module).
  • control unit is configured to control amount of hydrogel dispensed into receptacle 106.
  • the dispensing of hydrogel may be accommodated by a dedicated pump or valve (not shown).
  • the system also comprises a scanning system (not illustrated) configured to direct, for example, mechanically or optically, the focused laser beam applied onto the desired portions of the hydrogel within the receptacle.
  • a scanning system (not illustrated) configured to direct, for example, mechanically or optically, the focused laser beam applied onto the desired portions of the hydrogel within the receptacle.
  • the scanning system may comprise an x-y scanning table capable and operable to direct the movement of the focused heat source/laser above the receptacle.
  • the scanning system may also have a z displacement capability to approach towards the hydrogel or to distance therefrom.
  • the scanning system may have an x-y-z scanning capability, enabling the system to scan an x-y plane at any desired point within a section along the z-axis.
  • the receptacle is place on an x-y movement system capable and operable to move the receptacle according to a desired pattern, while the laser remains static.
  • control unit 130 can iteratively actuate hydrogel dispensers 108 to release an amount of hydrogel into receptacle 106.
  • the released amount is determined so that, when being spread within receptacle 106, a uniform hydrogel layer is formed with a pre-determined layer thickness VD (vertical depth).
  • VD vertical depth
  • the amount of hydrogel may be released into the receptacle from a bottom thereof, e.g. through an inlet at a bottom portion of the receptacle. Consequently hydrogel level rises in the container so that a new layer of hydrogel supersedes and covers the scaffold to generate, above the most-recent solidified layer of the scaffold, a new hydrogel layer ready to be solidified.
  • control unit may actuate the laser to cause solidification of the hydrogel and specifically selected portions of the layered hydrogel, the selected portions being determined by an associated virtual planar pattern of the scaffold as described above.
  • the 3D scaffold is built layer-by-layer, i.e. one layer of a hydrogel is added, and then a surface of said layer or a portion thereof is exposed to the heat, e.g. IR radiation.
  • the resulting 3D scaffold may contain, according to some embodiments, voids corresponding to vascular cavities.
  • the generated 3D scaffold may have a variety of applications.
  • the scaffold provides a structure suitable for adherence and/or attachment and/or incorporation and/or maturation and/or differentiation and/or proliferation of cells.
  • the scaffold may further provide mechanical stability and support the cells.
  • the scaffold may be in a particular shape, composition or form so as to influence or delimit a three-dimensional shape or form assumed by a population of proliferating cells.
  • the 3D scaffold can be used as an analog or precursor of an animal organs.
  • the scaffold is a precursor of an animal tissue, upon which cells are grown in order to obtain an animal viable tissue analog.
  • the tissue analog comprises the 3D scaffold and any one or combination of epithelial, connective tissue, muscle tissue and nerve tissue.
  • the scaffold is a part or a fragment of an organ or a biological system such as part of an intestine, trachea, skin etc.
  • the scaffold comprises structures corresponding to blood vessels within said organ or tissue.
  • the animal is a mammal.
  • the mammal is a non-human mammal selected from livestock animals, such as cattle, pigs, sheep, goats, horses, mules, donkey, buffalo, or camels, domestic pets, and primates.
  • livestock animals such as cattle, pigs, sheep, goats, horses, mules, donkey, buffalo, or camels, domestic pets, and primates.
  • the mammal is human.
  • the scaffold is a precursor or analog of non-animal organ or tissue. According to some embodiments, the scaffold is a precursor or analog of plant tissue.
  • the organ or tissue analog is biocompatible and transplantable.
  • the transplantable organ is selected from liver, kidney, heart, lung, bladder, intestine or pancreas.
  • the tissue analog is selected from muscle, adipose, neural, epithelial, connective, glandular, and mesenchymal tissue.
  • propagation of cells onto the 3D scaffold is in order. At times, propagation of stem cell or progenitor cells into the scaffold takes place. According to some embodiments, the propagation is performed during generation of the scaffold. According to some other embodiments, the propagation is performed after the 3D scaffold is completed.
  • the stem cells are selected from embryonic, somatic, induced pluripotent and generalized nonspecific stem cells.
  • the progenitor cells are selected from neural, epithelial, connective tissue, and muscle cell.
  • the progenitor cells are selected from stem cell derived cells, hepatic cells, epithelial cells, pancreatic cells and muscle cells.

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Abstract

La présente invention concerne un processus et un système de production d'un échafaudage en 3D. Le processus comprend (a) l'ajout dans un réceptacle (106) d'une couche d'un hydrogel thermosensible gélatineux ; (b) l'orientation d'une chaleur concentrée sur des parties de la couche de l'hydrogel thermosensible gélatineux pour amener lesdites parties de l'hydrogel thermosensible à se solidifier dans une couche d'échafaudage solide à l'intérieur de l'hydrogel thermosensible gélatineux, lesdites parties exposées constituant une couche à l'intérieur dudit échafaudage en 3D ; et (c) la répétition des étapes (a) et (b) pour former séquentiellement ledit échafaudage en 3D. Le système comprend un réceptacle (106) pour contenir l'hydrogel thermosensible gélatineux ; une source de chaleur concentrée (116) pour diriger une chaleur concentrée sur des parties d'une couche de l'hydrogel thermosensible maintenue à l'intérieur dudit réceptacle (106) et pour solidifier les parties dans une couche d'échafaudage solide à l'intérieur de l'hydrogel thermosensible gélatineux ; un distributeur (108) conçu pour distribuer une quantité dudit hydrogel thermosensible à l'intérieur du réceptacle (106) pour former une couche d'hydrogel thermosensible gélatineux à l'intérieur dudit réceptacle ; et une unité de commande (130) pour commander l'ajout, couche par couche, dudit hydrogel thermosensible à l'intérieur du réceptacle (106) et, pour commander l'exposition de ladite chaleur concentrée sur lesdites parties de l'hydrogel thermosensible, pour amener ledit hydrogel gélatineux thermosensible à se solidifier dans une couche d'échafaudage solide à l'intérieur de l'hydrogel thermosensible gélatineux.
PCT/IL2017/051305 2016-12-01 2017-11-30 Procédé et système pour impression en 3d WO2018100580A1 (fr)

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