WO2018100580A1

WO2018100580A1 - Method and system for 3d printing

Info

Publication number: WO2018100580A1
Application number: PCT/IL2017/051305
Authority: WO
Inventors: Reuven EDRI; Itai Cohen
Original assignee: Regenesis Biomedical Ltd.
Priority date: 2016-12-01
Filing date: 2017-11-30
Publication date: 2018-06-07

Abstract

The present disclosure provides a process and system for generating a 3D scaffold. The process comprises (a) adding into a receptacle (106) a layer of a gelatinous thermosensitive hydrogel; (b) directing a focused heat onto portions of the layer of the gelatinous thermosensitive hydrogel to cause said portions of the thermosensitive hydrogel to solidify into a solid scaffold layer within the gelatinous thermosensitive hydrogel, said exposed portions constitute a layer within said 3D scaffold; and (c) repeating steps (a) and (b) to sequentially form said 3D scaffold. The system comprises a receptacle (106) for holding the gelatinous thermosensitive hydrogel; a focused heat source (116) for directing a focused heat onto portions of a layer of the thermosensitive hydrogel held within said receptacle (106) and for solidifying the portions into a solid scaffold layer within the gelatinous thermosensitive hydrogel; a dispenser (108) configured for dispensing an amount of said thermosensitive hydrogel within the receptacle (106) to form a layer of gelatinous thermosensitive hydrogel within said receptacle; and a control unit (130) for controlling addition, layer by layer, of said thermosensitive hydrogel within the receptacle (106) 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.

Description

METHOD AND SYSTEM FOR 3D PRINTING

TECHNOLOGICAL FIELD

The present disclosure concerns 3D printing, particularly useful for printing 3D biocompatible articles

BACKGROUND ART

References considered to be relevant as background to the presently disclosed subject matter are listed below:

- International Patent Application Publication No. WO 2016/100856

- Ovsianikov et al. Materials 2011, 4:288-299

- US Patent Application Publication No. 2017/0217091

- International Patent Application Publication No. WO 2016/036275

- International Patent Application Publication No. WO 2016/194011

- International Patent Application Publication No. WO 2016/090286

- International Patent Application Publication No. WO 2015/158718

Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

BACKGROUND

The precision of 3 -dimensional (3D) printing makes it a promising method for replicating the body's complex tissues and organs. Several techniques have been developed.

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 250μπι^χ250 μπι. However, 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.

GENERAL DESCRIPTION The present disclosure provides a process for generating a 3D scaffold, comprising:

(a) adding into a receptacle a layer of thermosensitive hydrogel in (specifically gelatinous) form;

(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; and

(c) repeating steps (a) and (b) to sequentially form, layer-by-layer, said 3D scaffold.

Also provided by the present disclosure is a 3D scaffold obtained or obtainable by the process disclosed herein.

In yet a further aspect, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 is a schematic isometric illustration of a system for generating a 3D scaffold in accordance with some embodiments of the present disclosure. DETAILED DESCRIPTION OF EMBODIMENTS

The present disclosure is based on the development of a technology utilizing thermosensitive hydrogel s as a supporting environment (supporting hydrogel) for generating 3D scaffold, from the same thermosensitive hydrogel composition. Thus, 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.

According to the technology disclosed herein, the 'layer-by-layer' generated 3D scaffold, is embedded within the hydrogel environment. At the same time, due to the rheological properties (e.g. viscosity) of the gelatinous hydrogel, 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.

In line with the above, there is disclosed herein a process for generating a 3D scaffold, comprising:

(a) adding into a receptacle a layer of a gelatinous thermosensitive 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; and

Also disclosed herein is 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, by definition, 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.

Thus, in the context of the present disclosure it is to be understood that 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.

In some embodiments, the hydrogel is a biocompatible hydrogel. Such biocompatible hydrogel may be of natural origin, synthetic or semi-synthetic (e.g. modification of a natural hydrogel).

In accordance with some embodiments, the hydrogels are biological hydrogel, such as, without being limited thereto, peptides, polypeptides, proteins, polysaccharides. In this context, it is to be understood that when referring to 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. For example, and without being limited thereto, the hydrogel may comprise growth factors or other elements that would assist in the growth of cells within the scaffold. Non-limiting examples of 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. Similarly, 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.

According to some embodiments, the hydrogel comprises proteins of the extracellular matrix (ECM). According to some embodiments the ECM proteins are selected from the group consisting of collagen, laminin, fibronectin and elastin or any combination thereof.

According to some embodiments, the hydrogel is glycosaminoglycan -based hydrogel comprising ECM proteins.

According to some embodiments, the thermosensitive hydrogel is Matrigel™, 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. Also present in Martigel are growth factors like TGF-beta and EGF that prevent differentiation and promote proliferation of many cell types. A growth-factor-reduced Matrigel is also available. Alternatives to Martigel are also known, such as Cultrex^® natural extracellular matrix (ECM) membrane extracts (http://vv^vw.amsbio >in cultTex.aspx). Generally, 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.

In accordance with the present disclosure, the hydrogel used is a thermosensitive hydrogel. In accordance with some embodiments, 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. According to some embodiments, the thermosensitive hydrogel solidifies at the temperature above 35°C or above 37°C. According to some further embodiments, the thermosensitive hydrogel solidifies at the temperature between about 30°C to about 40°C, or between about 35°C to about 37°C.

There is a variety of 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.

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. In some embodiments 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. As such, when referring to a layer of hydrogel, 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. In some embodiments, the addition of the hydrogel is by the use of a dispenser, as described with respect to the disclosed system. In the context of the present disclosure, 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. To this end, 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. In some embodiments, 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). According to some embodiments, 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. When VD < 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. In other words, when VD≤ sd , a sequential series of generated scaffold layers will typically merge together into an integral 3D scaffold.

In some embodiments, the vertical depth VD is between ΙΟμπι to 1mm, at times, between 10 μπι to about 100 μπι, or between about 20 μπι to about 80 μπι, or between about 30 μπι to about 70 μπι, or between about 40 μπι to about 60 μπι or between about 50 μπι deep.

There is a correlation between the vertical depth of the added layer and the thickness/vertical height (aligned with said VD) of the generated solid 3D scaffold layer. Therefore, for VD of between ΙΟμπι to 1mm, it is expected and desirable to generate within each gelatinous layer, a solid scaffold having a vertical height of also between ΙΟμπι to 1mm.

In some embodiments, prior to adding a new layer of hydrogel, 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.

In accordance with some embodiments, the focused heat comprises electromagnetic radiation.

In some particular embodiments, the electromagnetic radiation is or comprises infrared (IR) radiation. In some embodiments, 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.

Without being bound be theory, it is believed that the selection of 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. As such, the use of IR radiation may have some advantages over using other energy sources, such as UV.

In some embodiments, 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. Subsequently 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.

At each process cycle, the radiation applied may comprise a single laser beam or a plurality of beams. According to some embodiments, the laser is femto-second laser beam capable of leading to multiphoton absorption. According to yet some other embodiments, the laser beam is a single uniformly split beam forming a network of laser beams.

In some embodiments, the laser beam has a diameter of between about 1 μιη to about ΙΟΟμπι at the surface of the exposed hydrogel layer. According to some embodiments, the laser beam diameter is between about 5 μιη to about 50 μπι, at times, between about 10 to about 20 μιη. 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/e² intensity level that is focused with a single lens is defined with:

4 * f * λ

diameter_min = -7- * ₂ where f is a focal length of a lens, λ is the wavelength, D' is the input beam size and 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). In some embodiments, 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 μΐ to about 100 μΐ (depending on the average power and the repetition rate).

In yet some additional embodiments, 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/cm² to 10 J/cm², at times, between about 0.5 and 5 J/cm², or at times between about 1 and 3 J/cm². 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.

According to some embodiments, 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. Where the deflector compensate quicker for the angular deflection error, thus producing a higher scan speed. Thermal penetration of the laser beam, preferably, IR 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/CT¹/² where k is the thermal diffusivity and τ is the pulse duration.

The depth of focus and the precision (spatial resolution) of thermal effect may be controlled by various parameters. According to some embodiments, the laser beam profile may be spatially optimized. According to some embodiments, a top-hat profile is preferred, because it can produce sharper edges than a Gaussian beam profile, creating more localized heating effect. In a beam shaping process, there is a transform of the irradiance and the phase profiles of the original beam. Conversion to the top-hat profile (super-Gaussian 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.

In some embodiments, 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.

- Wavelengths: 1064nm;

- Pulse length of 280 femoseconds;

- Power up to 10W;

- Pulse rate between 44KHz and 200KHz;

- Stage Precision ±1 micron;

- Spot size 2μπι at 355nm, 30μπι micron at 1064nm;

- Travel XY: 200mm Z: 100mm.

The operation of such laser can be controlled, for example, by SCA software. In some other embodiments, 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. In yet some other embodiments,

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. In this context, it is to be understood that the term "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. According to some embodiments, 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).

In some embodiments, 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.

For example, 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.

According to some embodiments, high spatial resolution (high degree of locality of solidification) 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. Once the exposed portions solidify, and the solid scaffold layer reaches a thermal equilibrium, 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.

At times, e.g. to assist in reaching a stable thermal equilibrium, 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.

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. Though, in other embodiments 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.

These 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).

At times, the control unit comprises input/output utilities and a memory utility and an analyzer. Reference is now made to Fig. 1, providing, in a non-limiting manner, a system

100 according to one embodiment of the present disclosure. As will be appreciated, this embodiment does not intended to be limiting but rather as an illustration of the broader scope of this disclosure as described above.

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. Similarly, 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.

In some embodiments, 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. Thus, for generation of a specific layer of the 3D scaffold, portions of the layer of the hydrogel are solidified, the portions being determined by the pattern associated with that specific 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).

In some embodiments, 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).

In some embodiments, 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.

According to some embodiments 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. According to some embodiments the scanning system may also have a z displacement capability to approach towards the hydrogel or to distance therefrom.

According to some embodiments 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. According to some other embodiments, 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.

For layer-by-layer generation of the 3D scaffold, 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). In other words, the volume of the released amount is equal to the thickness VD multiplied by the surface area of the receptacle.

In some embodiments, 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.

Following the release of the hydrogel amount, and following an optional waiting period for allowing the hydrogel to reach stable equilibrium characterized by having a planar, horizontal top surface of the hydrogel, the 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.

According to the principles of the present disclosure, 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. In accordance with some embodiments, 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.

According to some embodiments, the 3D scaffold can be used as an analog or precursor of an animal organs. According to some embodiments, the scaffold is a precursor of an animal tissue, upon which cells are grown in order to obtain an animal viable tissue analog. According to some other embodiments the tissue analog comprises the 3D scaffold and any one or combination of epithelial, connective tissue, muscle tissue and nerve tissue. According to some embodiments, the scaffold is a part or a fragment of an organ or a biological system such as part of an intestine, trachea, skin etc.

According to some embodiments, the scaffold comprises structures corresponding to blood vessels within said organ or tissue. According to some embodiments, the animal is a mammal. In some other embodiments, 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. According to other embodiments the mammal is human.

In some embodiments, 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.

According to some embodiments, the organ or tissue analog is biocompatible and transplantable.

According to some embodiments, the transplantable organ is selected from liver, kidney, heart, lung, bladder, intestine or pancreas.

According to some embodiment, the tissue analog is selected from muscle, adipose, neural, epithelial, connective, glandular, and mesenchymal tissue.

In some embodiments, in order to obtain the tissue or organ analog, 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.

According to some embodiments the stem cells are selected from embryonic, somatic, induced pluripotent and generalized nonspecific stem cells.

According to some embodiments the progenitor cells are selected from neural, epithelial, connective tissue, and muscle cell. In one embodiment, the progenitor cells are selected from stem cell derived cells, hepatic cells, epithelial cells, pancreatic cells and muscle cells.

Claims

CLAIMS:

1. A process for generating a 3D scaffold, comprising:

(a) adding into a receptacle a layer of a gelatinous thermosensitive hydrogel;

(b) exposing portions of the layer of the gelatinous thermosensitive hydrogel to a focused heat to cause said portions of the thermosensitive hydrogel to solidify into a solid scaffold layer within the gelatinous thermosensitive hydrogel, said exposed portions constitute a layer within said 3D scaffold; and

2. The process of claim 1, wherein said layer of gelatinous thermosensitive hydrogel has a thickness of between ΙΟμιη to 1mm.

3. The process of claim 1 or 2, wherein said thermosensitive hydrogel is a biocompatible hydrogel.

4. The process of any one of claims 1 to 3, wherein said thermosensitive hydrogel comprises macromolecules selected from the group consisting of proteins and polysaccharides, and any combination thereof.

5. The process of any one of claims 1 to 4, wherein said focused heat comprises an electromagnetic radiation.

6. The process of claim 5, wherein said focused heat is a laser beam or an array of laser beams.

7. The process of any one of claims 1 to 6, wherein said focused heat comprises an electromagnetic radiation of a wavelength or a wavelength range within the infrared spectrum.

8. The process of claim 7, wherein said focused heat comprises radiation of a wavelength or wavelength range within the wavelength spectrum of 700 to about 1300 nm.

9. The process of any one of claims 1 to 8, wherein said exposing of the thermosensitive gelatinous hydrogel is to a temperature of at least 25°C.

10. The process of any one of claims 1 to 9, wherein when said focused heat is by a laser beam, said beam has an average energy flow of between about 0.1 J/cm² to 10 J/cm².

11. The process of any one of claims 1 to 9, wherein when said focused heat is by a laser beam, said beam has an energy per pulse of between 1 nJ to 10 mJ.

12. The process of any one of claims 1 to 11, wherein when said focused heat is by exposure to pulses of a laser beam, said pulses have a repetition rate of between 1 Hz to 80MHz.

13. The process of any one of claims 1 to 12, wherein addition of a further layer of the gelatinous thermosensitive hydrogel is after the solid scaffold layer reaches a thermal equilibrium.

14. The process of any one of claims 1 to 13, for generating an analog of mammal tissue or organ.

15. A 3D scaffold obtained or obtainable by the process of any one of claims 1 to 14.

16. A system for generation of a 3D scaffold, comprising: a receptacle configured to hold a gelatinous thermosensitive hydrogel; 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 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.

17. The system of Claim 16, wherein said receptacle has an open top for allowing at least the dispensing of hydrogel into the receptacle and the directing of focused heat onto portions of the hydrogel.

18. The system of claim 16 or 17, wherein said focused heat source comprises an electromagnetic radiation source.

19. The system of claim 18, wherein said focused heat source is configured to apply a laser beam or an array of laser beams.

20. The system of any one of claims 16 to 19, wherein said focused heat source is configured to apply an electromagnetic radiation at a wavelength or a wavelength range within the infrared spectrum.

21. The system of claim 20, wherein said focused heat source is configured to apply radiation at a wavelength or wavelength range within the wavelength spectrum of 700 to about 1300 nm.

22. The system of any one of claims 16 to 21, wherein said focused heat source is selected to apply a beam at an average energy flow of between about 0.1 J/cm² to 10 J/cm².

23. The system of any one of claims 16 to 22, wherein said focused heat source is configured to apply pulses of heat at an energy per heat pulse of between 1 nJ to 10 mJ.

24. The system of any one of claims 16 to 23, wherein said focused heat source is configured to apply pulses of heat at a repetition rate of between 1 Hz to 80MHz.

25. The system of any one of claims 16 to 21, wherein said focused heat source is configured to heat the thermosensitive gelatinous hydrogel within the receptacle to a temperature of at least 25°C.

26. The system of any one of claims 16 to 25, wherein said dispenser is a syringe or an array of syringes.

27. The system of any one of claims 16 to 26, comprising temperature control module for controlling temperature of gelatinous hydrogel when held within said receptacle.

28. The system of any one of claims 16 to 27, wherein said control unit is configured and operable to receive input data indicative of one or more process parameters and process the same to produce an output comprising operational data/instruction for at least said focused heat source and said dispenser.

29. The system of any one of claims 16 to 28, wherein said control unit comprises input/output utilities and a memory utility.