Patent ID: 12221621

DESCRIPTION OF SOME SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to 3D printing of biomaterials, and more particularly, but not exclusively, to a support medium for printing biomaterials based on biocompatible hydrogel particles.

The principles and operation of the present invention may be better understood with reference to the figures and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

The present invention provides a unique support medium that allows stable 3D printing of complex, multilayered structures composed, inter alia, of biomaterials, such as processed ECM and/or ECM derived substances at high resolution, using a fabrication method based on fused-deposition modeling within the unique support medium. At the basis of the present invention is a special formulation and preparation of a hybrid hydrogel composed of calcium alginate (the structural component of the hydrogel) and xanthan gum (a washable additive used to destabilize the calcium alginate gel, making it amenable for further processing such as crushing and grinding, as well as to facilitate and preserve the homogeneity of the grinded material). In addition, it was found that the presence of a soluble polymer in the support medium, such as xanthan gum, enhances the optical clarity of the support medium and improves printing accuracy and precision. Thus, the role of additives such as xanthan gum, being “washable”, has been extended by re-introduction to the medium after the washing steps, so as to become a component in the “final” support medium. In other words, it is optional to preserve the formulation as a “hybrid hydrogel” (e.g., calcium-crosslinked alginate particles supplemented with xanthan gum) in all steps, including in the final step of the process in which the support medium is being generated before using it for printing. The hydrogel is processed into a transparent, μm-sized calcium-alginate uniform and granular support media in which the structure is printed. This granular support media supports the printed biomaterial and preserve its shape during the fabrication process and the following curing phase. When the printed biomaterial is fully cured, the transparent biocompatible, biodegradable, heat stable, non-animal origin support medium can be degraded, releasing a stable, self-supported structure. Of note, this support medium can also be used for printing other curable extruded material, not only biomaterials. Its excellent transparency is especially beneficial in cases when the user needs to inspect the printed construct while fabricated and modify printing parameters accordingly without interrupting the procedure.

As presented hereinabove, the challenges facing the rapidly evolving 3D printing of biomaterial, have been dealt with by various approaches, such as, for example, printing in granular support media composed of gelatin microparticles, as described in WO 2015/017421 and by Hinton et al. (Science Advances,2015). However, these approaches suffer from several major drawbacks. First, the relatively low transparency of the described support media limits real-time monitoring of the printing process. Second, this technology cannot be used to print objects composed of materials that require curing at elevated temperatures in order to achieve stabilized construct conformation, since gelatin is heat-labile and undergoes extensive transformation in its physical properties upon heating (liquefaction), resulting in loss of capability to support the uncured or the not fully cured structure. The heat-lability of gelatin also prevent its use as a pre-heated granulated support media in which the curing of the printed material takes place during the fabrication process. Of note, as gelatin derived from collagen, a major component of the ECM, it can interact adversely with it during fibrillogenesis (when using processed ECM and/or ECM derived substances as printing materials), thus “contaminating” the final structure with the support material. In addition, gelatin is of an animal source, a fact that may rise safety concerns in medical applications. Contrary to these limitations, the presently disclosed support medium is composed of transparent calcium-alginate hydrogel which is heat-stable, inert (do not interact with materials such as processed ECM and/or ECM derived substances) and is of a non-animal origin.

In addition, 3D printing in granular support media composed of Carbopol® (high molecular weight crosslinked polyacrylic acid polymers) microparticles, has been described in Bhattacharjee et al. (Science Advances,2015). However, as Carbopol® is not recognized as a biocompatible and biodegradable product, it is less suitable for tissue engineering and regenerative medicine application. Furthermore, its resolution from the printed construct is complicated and cannot be performed by using mild procedures like chelation and/or enzymatic digestion. Contrary to Carbopol®, the transparent support medium provided herein is made of alginate which is biocompatible, biodegradable and is widely used as a biomaterial for tissue engineering applications. In addition, it can be readily resolved from the printed structure by a mild chemical (chelation) and/or enzymatic treatment.

A Process of Preparing Hydrogel-Based Particulate Support Media:

A unique formulation of calcium alginate-xanthan gum hybrid hydrogel, provided herein, was surprisingly found of being capable of being processed into a calcium alginate hydrogel-based fine and uniform particulate media, highly suitable for 3D-printing support media. The support media provided herein is characterized by advantageous heat and mechanical stability, as well as advantageous see-through transparency. While transparent calcium alginate hydrogels are known in the art, it is the novel combination of alginate and an additional water soluble and biocompatible polymer, such has xanthan gum, at specific range of ratios, which has been found to afford the novel fine particulate hydrogel support medium for 3D printing. The combination of two or more polymers in the formation and/or processing of a hydrogel is referred to herein as a “hybrid hydrogel”. Thus, in the context of the present invention, the term “hybrid hydrogel” refers to a hydrogel or a processed hydrogel preparation that comprises at least two types of polymers (at least one of them can form a hydrogel), either by co-jellification or by pre- or in-(pulverizing/grinding) processing introduction or by post-washing introduction/re-introduction a second (or more) polymer into the formed hydrogel network and/or to its surroundings.

Without being bound by any particular theory, it is assumed that the soluble polymer interacts with the crosslinked polymeric network in the hybrid hydrogel in more than one way, regardless of the process by which the two entities are brought in contact and processed. For example, regardless whether the hybrid hydrogel is formed by co-jellification, washing, re-introduction and grinding; or the soluble polymer is introduced into the pre-formed and washed crosslinked polymeric network before, during or after grinding; or a combination of any of the aforementioned or the hydrogel is preformed and only after it formation it is brought in contact with the soluble polymer prior to grinding, the soluble polymer may infiltrate the crosslinked polymeric network, and/or coat the crosslinked polymeric network on accessible surfaces thereof. Without being bound by any particular theory, it has been noted by the inventors that the presence of the two elements in the hybrid hydrogel allows for finer and more uniform grinding of the crosslinked polymeric network, and inhibits the formation of aggregates thereof. The simplistic hypothetic model of how the soluble polymer affects the crosslinked polymeric network (hydrogel) is by integrating inside the hydrogel network and changing the properties of the bulk material. In fact, this is the most intuitive concept of “hybrid hydrogel”. However, the effect of the soluble polymer may also be mediated by other forms of molecular behavior. For example, few studies showed that when mixing xanthan gum and alginate molecules, phase separation may occur. In other words, it is possible that the xanthan gum molecules do not interact directly the alginate molecules, in which case the beneficial effect of the xanthan gum in the instant embodiments may be mediated by a phenomenon in which the xanthan gum and alginate molecules repel each other, thus weakening the bulk hydrogel structure when co-jellified with alginate, and/or envelop the generated alginate particles following pulverization, with or without penetrating the hydrogel network. This may prevent the alginate particles from aggregation and also may act like a lubricant to allow the particles to move freely and smoothly over each other, thus achieving superb printing-supporting capability.

Hence, the term “hybrid hydrogel”, as used herein, is meant to encompass all forms of possible interactions between the soluble polymer molecules (e.g., xanthan gum) and the crosslinked polymeric network (e.g., calcium alginate hydrogel), be it entangled or mechanically bound/entrapped otherwise, infiltrated therein as a leachable, or as an external coating.

According to an aspect of some embodiments of the present invention, there is provided a process of producing the hydrogel-based particulate support medium provided herein, the process includes pulverizing a hybrid hydrogel, which includes a crosslinked polymer, calcium ions and an additional soluble polymer, to thereby obtain hybrid hydrogel particles, and washing the resulting hybrid hydrogel particles in order to substantially remove the remnants of the additional soluble polymer, which is not part of the crosslinked polymeric network, and any leachables and unreacted starting materials, calcium ions and the like. While washes also remove most of the soluble polymer that has not been entangled in the crosslinked polymeric network, a soluble polymer, such as xanthan gum, is optionally (re)introduced into the hydrogel-based particulate support medium in order to enhance the optical clarity of the support medium and improve printing accuracy and precision.

The hybrid hydrogel, according to some embodiments of the present invention, is prepared by co-jellifying calcium ions, alginate and another soluble and compatible polymer, such as xanthan gum, essentially as described in the Examples section that follows below. Without being bound by any particular theory, it is assumed that the addition of an additional soluble polymer, such as xanthan gum, weakens the structure of the calcium alginate hydrogel, as well as facilitating and preserving the homogeneity of the particulate material following pulverizing; hence, while pulverization and homogenization of a calcium alginate hydrogel typically results in large, bulky and irregular flakes and/or aggregates that constitute an inferior support medium, the calcium alginate-xanthan gum hybrid hydrogel can be processed to afford fine, homogenous calcium alginate hydrogel particles (see,FIGS.1A-B).

Hybrid hydrogel particles may be generated by means other than pulverizing a pre-formed, co-jellified hybrid hydrogel. That is to say, they may also be generated by grinding/pulverizing pre-jellified pristine calcium alginate hydrogel (the crosslinked polymeric network) in a solution that contains the soluble polymer, thereby introducing the soluble polymer into the pre-formed calcium alginate network and/or to its surroundings to afford a hybrid hydrogel. Alternatively, the crosslinked polymeric network may be exposed to the soluble polymer before or after the grinding/pulverizing step. However, the co-jellified hybrid hydrogels were found to be easier to pulverize, enabling to shorten the time needed to complete the process and avoid the presence of large, non-homogenous particles and aggregates, especially when processing stiff alginate gels.

It is noted herein that other soluble and compatible polymers, such as low-melting agarose, methylcellulose and the likes, may also be used to form a hybrid hydrogel with calcium alginate structure, making it processable by homogenization to afford a particulate hydrogel media. The inventors have found that xanthan gum is a preferred biopolymer for serving as the additional soluble polymer, which gave the best results in terms of particle size, stability, processability of the homogenate and ease of extraction from the particles after pulverization and before use. As discussed hereinabove, xanthan gum is beneficial in the final preparation of the support medium; however, extraction of alginate and xanthan is essential to eventually release the printed structure form the support medium.

FIGS.1A-Bpresents photographs of two samples of pulverized calcium alginate hydrogels, whereinFIG.1Ashows a pulverized sample of calcium alginate hydrogel prepared without xanthan gum, andFIG.1Bshows a pulverized sample of calcium alginate hydrogel prepared with xanthan gum (scale bar=50 μm).

As can be seen inFIGS.1A-B, calcium alginate hydrogel prepared without xanthan gum breaks into large non-homogeneous flakes and aggregates, while the hybrid hydrogel prepared with xanthan gum, according to some embodiments of the present invention, can be pulverized and homogenized into a fine and homogeneous particulate.

In order to afford highly transparent calcium alginate hydrogel, the jellification process is slowed down. Slowing the jellification process can be achieved, inter alia, by using a water-insoluble source of calcium ions, and further using an acidifying agent that slowly releases calcium ions from the water-insoluble source thereof. In some embodiments, the hybrid hydrogel is prepared using calcium carbonate, which is substantially water-insoluble, with the addition of the acidifying agent glucono delta-lactone (GDL).

FIG.2presents photographs of two samples of pulverized calcium alginate hydrogel support media, showing the transparency of the support media prepared as described herein (left) compared to a preparation of calcium alginate hydrogel composed of microparticles generated by sonication of calcium-alginate in CaCl2solution (right).

As can be inFIG.2, the hydrogel particles prepared with an insoluble calcium ion source and an acidifying agent are transparent, while the calcium alginate hydrogel particles made with calcium chloride are opaque.

After homogenization, the suspended hydrogel particles are incubated and washed repeatedly until all remnants of free calcium ions and the additional soluble polymer are washed away.

The washed calcium alginate particles are thereafter collected, i.e., by centrifugation, and re-suspended in water or another aqueous solution for use as support medium for 3D printing.

According to some embodiments of the present invention, some soluble polymers, such as xanthan gum, may be added (or reintroduced) to the support medium for enhancing the optical clarity of the support medium and improving printing accuracy and precision. In such embodiments, after washing, the washed pellet is supplemented with 1% w/v soluble polymer solution, such as xanthan gum solution, to afford a final concentration of 0.05-0.5% w/w or w/v (0.05-0.5 grams of the soluble polymer such as xanthan gum, in a total weight of 100 gr or 100 ml support medium). The mixture is then vigorously mixed, and incubated at room temperature or at 4° C. for several (1-5) days before use. If needed, the viscosity of the support media can be reduced by addition of aqueous solutions containing the same final concentration of xanthan gum as abovementioned.

Hydrogel-Based Particulate Support Media:

According to an aspect of embodiments of the present invention, there is provided a transparent, stable, safe and (bio)degradable hydrogel-based particulate support medium for 3D printing, comprising a plurality of essentially non-aggregated and substantially discrete calcium alginate hydrogel particles having an average size that ranges from 0.1 μM to 5 μM, and a substantially narrow size distribution. The chemical composition of the hydrogel is mostly, or substantially alginate hydrogel crosslinked network, however, some amounts of the soluble polymer may be present in minute amounts, left after repetitive washes, or present in the hydrogel in amounts/concentration that can be controlled by the process of manufacturing the same. Hence, the presence of the soluble polymer in the hydrogel-based particulate support medium may range from essentially none (minute amounts) to any concentration used in the process of making the hydrogel-based particulate support medium.

In the context of some embodiments of the present invention, the plurality of calcium alginate hydrogel particles is referred to as being essentially or substantially devoid of dissociable elements, namely chains and molecules that are not bound to the hydrogel network by direct, indirect or mechanical association. In some embodiments, the calcium alginate hydrogel particles comprising the support medium are substantially devoid of the soluble polymer (e.g., xanthan gum); in some embodiments the calcium alginate hydrogel particles comprises non-leachable (network-bound) chains of the soluble polymer, and regarded as hybrid hydrogel particles; and in some embodiments that the calcium alginate hydrogel particles comprises chains of the soluble polymer introduced or re-introduced into the hydrogel network and/or to its surroundings by soaking after formation of the network (post-jellification), and are also regarded as hybrid hydrogel particles.

The plurality of calcium alginate hydrogel particles, according to embodiments of the present invention, are characterized by being essentially discrete (non-aggregated) and having a relatively narrow size distribution, or a lower coefficient of variation (CV). CV is also known as relative standard deviation (RSD), which is a standardized measure of dispersion of a probability distribution or frequency distribution, and is often expressed in percent. SRD is defined as the ratio of the standard deviation (sigma, σ) to the mean μ). According to some embodiments of the present invention, the plurality of calcium alginate hydrogel particles is characterized by a particle size distribution of less than 5%, less than 10%, less than 20% or less than 25% RSD.

Moreover, as the fine and homogeneous particles of the support medium provided herein, can easily flow throw very fine needles, and can therefore also serve as a sacrificial material in 3D printing of hollow structures.

The high see-through transparency of the support medium to visible light, according to some embodiments of the present invention, allows the printing process to be monitored at real time: the user can inspect the printed construct while fabricated and modify printing parameters accordingly without interrupting the procedure. The high see-through transparency of the media may also allow automatic calibration and monitoring of the printing process by 3D printers equipped with compatible optical sensors. This enables the user to calibrate and tune the printer conveniently and accurately, what results in a refined printout. Finally, the transparency of the support media may enable curing of light-curable printed materials by external illumination. Thus, according to some embodiments of the present invention, the plurality of calcium alginate hydrogel particles is substantially transparent to visible light.

FIG.3presents a photograph of two sample vials, comparing the see-through transparency of the support medium provided herein (right), compared to that of a pure cell media (left). As can be seen inFIG.3, the see-through transparency of both almost identical, demonstrating that the alginate particles of the support medium provided herein, according to embodiments of the present invention, have little effect on the overall clarity of the suspension.

The hydrogel-based particulate support medium provided herein is generally a plurality of hydrogel particles hydrated by an aqueous solution in the form of a slurry or a suspension. The amount of the aqueous solution may vary from essentially no excess thereof (a drained slurry) up to a diluted suspension of the particles at a ratio that allows the printed object to be supported and formed adequately. The preferable amount of dilution is within the routine experimental procedures of a person skilled in the art.

According to some embodiments, the slurry is sterile and free of any toxins or other factors that may adversely affect an article of manufacturing being formed therewithin.

A Selling Unit of the Support Medium:

According to an aspect of some embodiments of the present invention, there is provided a selling unit, comprising the support medium provided herein. The selling unit can be in a form of a sealed and oxygen-proof container that contains an amount of the support medium suitable for a specific printing machine or a specific printing bath size.

According to some embodiments, the selling unit contains a drained support medium, wherein the user is instructed to add an aqueous solution thereto in order to arrive at an adequate dilution of the support medium.

According to some embodiments of the present invention, the selling unit includes a packaging material, and identified in print, or on said packaging material, for use as a support medium in a 3D printing machine and process.

Xanthan Gum as Support Medium:

Xanthan gum pose no risk of toxicity and is generally accepted as safe for human consumption (GRAS). It is fully biocompatible and its water solubility enables simple extraction from the fabricated 3D structure post-printing. Interestingly, the present inventors have surprisingly found that even a pure, non-particulate xanthan gum solution (successfully demonstrated below with 1% w/v solution in cell media) can serve as a support medium for 3D printing, resulting in adequate accuracy and printing resolution.

Xanthan gum aqueous solutions can be used as a transparent, easy to prepare, low-cost, temperature insensitive, non-animal support media for 3D printing of biological and other materials.

Thus, according to an aspect of some embodiments of the present invention, there is provided a process of 3D printing in a support medium, wherein the support medium includes an aqueous solution of xanthan gum at a concentration that ranges from 0.5 to 5% w/v, preferably from 0.8 to 2% w/v, or 1% w/v.

According to some embodiments, the xanthan gum used as a support medium, is sterile, and free of any toxins or otherwise any factors that may adversely affect the printing process or the printed article-of-manufacturing being formed thereby.

A demonstration of the usability of pure xanthan gum hydrogel as support medium for 3D printing and other additive structuring methods is presented in the Example section hereinbelow (see, e.g.,FIGS.7A-F).

3D Printed Article:

High-resolution complex 3D printed objects made of biologic or biocompatible printable materials, can be produced using the hydrogel-based particulate support medium provided herein. The end-users of such technology are patients that will benefit from transplantation of engineered cellular or acellular tissues, organs and/or other types of grafts, as well as the pharma industry that may use the printed structures/tissues/organs for drug screening and testing. For non-medical applications, the end-user of such 3D printed objects is any artisan that benefits from rapid-prototyping/3D printing services and/or products.

Thus, according to an additional aspect of embodiments of the present invention, there is provided an article of manufacturing, afforded by 3D printing the article inside the hydrogel-based particulate support medium described herein.

For clinical/medical applications, the hydrogel-based particulate support medium described herein is useful in high-resolution manufacturing of complex cellular or acellular scaffold articles composed of biomaterials, such as processed ECM or ECM-derived substances, developed for tissue engineering and regenerative medicine, as well as for the purpose of drug screening and testing.

For research applications, the hydrogel-based particulate support medium described herein is useful in high-resolution manufacturing of complex cellular or acellular scaffold articles composed of biomaterials such as processed ECM or ECM-derived substances for tissue engineering and regenerative medicine research, as well as for pharmaceutical research, like screening and testing of drugs.

The hydrogel-based particulate support medium, according to some embodiments of the present invention, is useful in the development of new biomaterials with desirable features, which behavior can be directly monitored during printing by virtue of the high see-through transparency of the support media.

It is expected that during the life of a patent maturing from this application many relevant hydrogel-based particulate support media will be developed and the scope of the term hydrogel-based particulate support media is intended to include all such new technologies a priori.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the phrases “substantially devoid of” and/or “essentially devoid of” in the context of a certain substance, refer to a composition that is totally devoid of this substance or includes less than about 5, 1, 0.5 or 0.1 percent of the substance by total weight or volume of the composition. Alternatively, the phrases “substantially devoid of” and/or “essentially devoid of” in the context of a process, a method, a property or a characteristic, refer to a process, a composition, a structure or an article that is totally devoid of a certain process/method step, or a certain property or a certain characteristic, or a process/method wherein the certain process/method step is effected at less than about 5, 1, 0.5 or 0.1 percent compared to a given standard process/method, or property or a characteristic characterized by less than about 5, 1, 0.5 or 0.1 percent of the property or characteristic, compared to a given standard.

The term “exemplary” is used herein to mean “serving as an example, instance or illustration”. Any embodiment described as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments and/or to exclude the incorporation of features from other embodiments.

The words “optionally” or “alternatively” are used herein to mean “is provided in some embodiments and not provided in other embodiments”. Any particular embodiment of the invention may include a plurality of “optional” features unless such features conflict.

As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the terms “process” and “method” refer to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, material, mechanical, computational and digital arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental and/or calculated support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

Example 1

A Hydrogel-Based Printing Support

A proof of concept of some embodiments of the present invention was carried out by forming a hybrid calcium alginate and xanthan gum hydrogel.

Briefly, a hybrid hydrogel, composed of 0.25-0.5% (w/v) sodium alginate (tested with PROTANAL LF 10/60 FT and PROTANAL LF200 FTS, FMC BioPolymer, or KIMIKA ALGIN I-3G by KIMICA corporation), 0.125-0.5% (w/v) xanthan gum (tested with Xantural 180, CP Kelco, Sigma G1253, xanthan gum fromXanthomonas campestris), 7.5-15 mM calcium carbonate and 15-30 mM D-(+)-Gluconic acid δ-lactone (GDL) was made by mixing the following ingredients:25-50% v/v of 1% (w/v) sodium-alginate solution (0.22 μm filtered);12.5-25% v/v (or centrifuged pellet) of 60 mM suspension of calcium carbonate (CaCO3) in deionized water, sonicated to reduce the size of the calcium-carbonate particles (autoclaved);0-50% v/v of deionized water (autoclaved or 0.22 μm filtered); and12.5-50% v/v of 1% (w/v) xanthan gum in 150 mM NaCl solution (autoclaved).

It is noted that a higher percentage of xanthan (up to 0.5%) may be required if using an alginate batch that form stronger gels.

The above ingredients were mixed for at least 30 minutes, and thereafter a fresh GDL solution (0.22 μm filtered) was add to a final concentration of 15-30 mM.

An embodiment of a specific formulation, preferred in terms of printout support capacity and see-through optical clarity is given below:

37.5% (v/v) of 1% (w/v) sodium alginate solution (PROTANAL LF 10/60 FT, FMC biopolymer); 25% (v/v) of 1% (w/v) xanthan gum (Xantural 180, CP Kelco) in 150 mM NaCl solution; 18.75% (v/v) of 60 mM suspension of calcium carbonate, 18.75% (v/v) deionized water, 22.5 mM GDL.

The mixture was vigorously stirred for 3 minutes, after which the stirring speed was lowered to generate a fine turbulence that bends the surface of the liquid. When the bend was no longer visible due to the increase in the viscosity of the reaction mixture, the stirring was stopped and the mixture was incubated uninterrupted, at room temperature, for 24 hours.

The resulting hydrogel was manually broken into chunks to which sterile deionized water at a volume of 4 times the volume of the hydrogel was added to a total volume of 5 times the volume of the hydrogel. The water-suspended hydrogel chunks were thereafter homogenized into fine particles using an electric homogenizer, and the homogenate was incubated over-night at 4° C. to allow dissolution of remaining traces of CaCO3.

The suspension was centrifuged at 15,777 G for 5-20 minutes (depending on the stability of the pellet) at room temperature. Thereafter the pellet was washed 2-5 times with sterile deionized water, added at a volume equal to the discarded supernatant, by repeated centrifugation at 15,777 G for 5-20 minutes at room temperature and re-suspension by vigorous mixing. Lastly, the pellet was washed 1-2 times with cell media, added at a volume equal to the discarded supernatant, by repeated centrifugation at 15,777 G for 5-20 minutes at room temperature and re-suspension by vigorous mixing. Excess calcium ions and xanthan gum are washed away during these washing steps, leaving transparent granular media composed of calcium-alginate particles in the form of a pudding-like pellet.

In some samples, after washing, a soluble polymer, such as xanthan gum, was added to the pudding-like pellets in order to enhance the optical clarity of the support medium and improve printing accuracy and precision.

In the embodiments in which a soluble polymer (xanthan gum) was introduced into the support medium, the procedure further included:

After washing, the washed pellet was supplemented with 1% w/v xanthan gum solution to reach a final concentration of 0.05-0.5% (w/w or w/v; 0.05-0.5 gr xanthan gum in a total weight of 100 gr or 100 ml support medium), vigorously mixed, and incubated at room temperature or at 4 centigrade for 1-5 days before use.

The pudding-like pellet was then transferred to a chamber large enough to contain the printed structure, and manually stirred using a spatula for about 10 seconds. Optionally the pellet can be homogenized using an electric homogenizer and/or vortexed before use.

Example 2

3D Printing into the Hydrogel-Based Printing Support

Printable biologic inks, composed of biomaterials (supplemented or not supplemented with cells) such as processed ECM or ECM-derived substances (or other extrudable materials) were printed within the support media described hereinabove, using a 3D printer equipped with microextrusion tools.

The printing chamber was incubated at 37° C. in order to cure (solidify) the printed structure, considering that incubation at 37° C. is most suitable for processed ECM or ECM-derived substances that incorporate mammalian cells. It is noted herein that incubation at temperatures higher or lower than 37° C. may be used for other ink materials.

The support media was then supplemented with a solution containing a chelating agent, such as citrate and/or EDTA and/or EGTA, and/or depolymerizing enzyme, such as alginate lyase, in order to degrade the alginate particles and release the printed construct.

Example 3

Hybrid Hydrogel Effect

As discussed hereinabove, the support medium, which is composed of specially prepared alginate particles in cell growth medium, may optionally be supplemented after washing with concentrated xanthan gum solution (e.g., 1% w/v in aqueous cell growth medium) to reach a final xanthan gum concentration of 0.05%-0.5% (w/v or w/w) in the support media. It was found that the presence of xanthan gum in the support medium enhances its optical clarity and greatly improves and maintains printing accuracy and precision, especially as time passes following vortexing/stirring/homogenizing of the support medium. It was also found that when supplementing the support medium with low xanthan gum concentrations in the rage of 0.05-0.2% w/v, the mixture requires longer incubation times (e.g., about 72 hours) for the xanthan gum to endow is beneficial effect. This finding was supported by a microscopic analysis, showing that xanthan supplementation improves particles' dispersion and prevent aggregation over time.

FIGS.4A-Bpresent microscopic images of alginate particles in a drop of supporting medium without xanthan gum (FIG.4A) and the same supplemented with final concentration of 0.5% (w/v) xanthan gum after the washing steps (FIG.4B).

As can be seen inFIGS.4A-B, the presence of xanthan gum in the support medium enhances its optical clarity.

FIGS.5A-Ddemonstrate the difference in accuracy of printing in a non-supplemented particulate support medium or in support medium supplemented with final concentration of 0.05% (w/v) xanthan gum following the washing steps, whereinFIG.5Ashows a 3D element printed 3 minutes after vortexing a support medium without xanthan gum,FIG.5Bshows a 3D element printed 3 minutes after vortexing a support medium supplemented with xanthan gum,FIG.5Cshows a 3D element printed 3 hours after vortexing a support medium not supplemented with xanthan gum, andFIG.5Dshows a 3D element printed 3 hours after vortexing a support medium supplemented with xanthan gum.

As can be seen inFIGS.5A-D, the presence of xanthan gum in the support medium greatly improved and maintained printing accuracy and precision, especially as time passes following vortexing/stirring/homogenizing of the support medium.

FIGS.6A-Bpresent microscopic images of alginate particles in a smear of a five-fold diluted supporting medium without xanthan gum (FIG.6A) or supplemented with final concentration of 0.05% w/v xanthan gum (FIG.6B), wherein the images were taken 30 minutes after vortexing.

As can be seen inFIGS.6A-B, supplementation with as low as 0.05% (w/w or w/v) final concentration of xanthan gum improved particles' dispersion and prevented aggregation over time.

Example 4

Xanthan Gum Solution as Support Medium

As discussed hereinabove, it was surprisingly found that a pure xanthan gum solution (e.g., 1% w/v solution in cell media) can serve as a support medium for 3D printing, resulting in adequate accuracy and printing resolution. Xanthan gum at a concentration of about 1% or higher can be used as a transparent, easy to prepare, low-cost, temperature insensitive, non-animal-originate support media for 3D printing of biological and other materials.

As xanthan gum is a polysaccharide derived from fermentation of simple sugars by the gram-negative bacteria (Xanthomonas campestris), commercially available xanthan gum preparations may contain endotoxins that are known to elicit in-vivo immunogenic/pyrogenic reaction. In order to eliminate this possible adverse effect, a method for destruction of endotoxins in xanthan gum preparations has been developed without damaging the xantan's beneficial properties. To this end, concentrated xanthan gum solution was treated with sodium hydroxide (NaOH) that eliminate the pyrogenicity and adverse immunogenic activity of the endotoxins. The NaOH was then extracted from the xanthan by washing with ethanol:water solutions.

Specifically, xanthan gum was solubilized in 150 mM NaCl aqueous solution to reach a final concentration of 4% w/v. The resulting viscous solution was then sterilized by autoclaving. Thereafter, 5 M NaOH solution is added (at 1:4 v/v ratio) and mixed with the xanthan gum solution to reach a final concentration of 1 M NaOH in the mixture. After 24 hours, the mixture was transferred into a 50% (v/v, in water) ethanol solution that was 10 times larger in volume. Under these conditions, the xanthan gum gel remains stable (as an alcoholic gel—“alcogel”); but the solutes (NaOH and NaCl) diffuse and solubilized in the 50% ethanol solution that was exchanged every 24 hours. When the solution has reached a neutral pH, the xanthan gum was washed twice with 70% ethanol (v/v, in water), and twice with pure ethanol, with exchange every 24 hours. Finally, the insoluble xanthan was separated from the ethanol. Ethanol residues were then evaporated under a jet of a sterile, dry air, followed by vacuum drying at room temperature. The resulting sterile, depyrogenated pellets were stored dry for future use, or solubilized in aqueous media to be used in the preparation of particulate alginate-based printing support medium as described above, or as a “pure”, non-particulate, xanthan-based support medium.

FIGS.7A-Fpresent microscopic images of a simple crisscross 2-layered pattern printed in two types of support media, whereinFIGS.7A-Cshow the pattern as printed in a particulate alginate support medium supplemented with 0.05% w/v xanthan gum, andFIGS.7D-Fshow a similar pattern printed into a support medium consisting of 1% (w/v) xanthan gum in cell growth medium.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.