Patent Application
20220184274
The present invention relates to a gelatin having improved properties, in particular improved dispensing properties, more particularly improved printing properties. The present invention further relates to a construct produced with the gelatin of the present invention and to a process to produce said construct. Further, the present invention relates to the use of the gelatin of the present invention to solve existing problems encountered with dispensing systems, in particular with 3D printing and more particularly for applications in the medical field. The gelatin of the present invention is particularly suitable for bio-printing in the medical field and may also be used in food applications.
Manufacturing technologies in the medical field and also in the food industry, are in constant evolution. Dispensing techniques, in particular, offer some advantages compared to standard production technologies, particularly in the medical field. Said advantages can be the speed of manufacturing, customization and the like. One very well-known dispensing technique is 3D printing. In particular, in the field of medical research, bioprinting is a powerful tool used for tissue engineering applications due to its capability of directly dispensing biological materials into spatial orientations and geometries. Gelatin is a preferred substrate biomaterial because of its very high biocompatibility with living tissues. Indeed, gelatin shows a natural amino acid sequence of Arginine-Glycine-Aspartate (Arg-Gly-Asp) component which is the main amino acid sequence responsible for cell adhesion in extracellular matrices (ECMs). In biology, an extracellular matrix (ECM) is a three-dimensional network of extracellular macromolecules, such as collagen, enzymes, and glycoproteins, that provide structural and biochemical support to surrounding cells.
An important parameter for dispensing systems is the dispensing time window, i.e. the time during which a material can be dispensed without showing detrimental viscosity increase upon dispensing, in particular, inside the dispensing head or nozzle. It is advantageous to have a longer dispensing time window as this gives more flexibility during the dispensing process. However, an important issue nowadays in dispensing technologies and particular in 3D printing the dispensing time window of materials based on gelatin is short because of the lack of thermostability. Gelatin-based formulations rapidly show an increase in viscosity after the start of the dispensing process, and this increase in viscosity causes the material to block the dispensing head. Dispensing can then only be done for a limited period of time before the dispensing head is blocked and has to be cleaned or changed before dispensing can be continued. This is not convenient at all to make bigger constructs or to make several smaller constructs. Thus with existing gelatin-based dispensing material, the dispensing time window is limited. In particular, gelatin, which as mentioned above is very suitable because of its biocompatibility with living tissues, is known to be a high-risk material for nozzle clogging. Due to the thermal instability of gelatin, a gelatin solution (prepared for printing typically at concentrations above 7.5%) will rapidly show an increase in viscosity at room temperature (20 to 25° C.), and will block dispensing heads, in particular those having a diameter below 0.64 mm (or 22G). Various solutions exist to overcome this problem. One solution is to dispense the material in a temperature-controlled dispensing system. However, such temperature controlled systems are expensive and require great care during operation, which makes the dispensing operation quite complicated. Another solution is to chemically modify the dispensing material or to supplement it with additives. Such currently available bio-printing materials include modified gelatins. Also gelatin can be mixed with other hydrocolloids such as alginates for example. Further, use of only specific raw materials such as fish gelatin may be a solution.
WO 2017216780 A1 relates to a gelatin polymer which is derived from natural sources of cold-adapted marine species, such as salmon gelatin.
U.S. Pat. No. 9,101,681 B2 relates to the use of standard gelatin to make 3D constructs in a temperature-controlled dispensing system.
US 2015/0084232 A1 relates to hydrogel compositions for printing 3D objects, wherein the hydrogel comprises a cross-linked biocompatible polymer. Pre-crosslinking is done before extrusion to ensure stability of the printing material.
There is thus a need to have a dispensing material which is easy to use, e.g. which does not require temperature-controlled systems, which has a longer dispensing time window than existing materials, which may be dispensed at higher concentrations than what is currently possible and so on.
The present invention aims to solve at least the above mentioned problems.
The present invention is based on the findings of a novel gelatin which has improved properties in particular in the field of dispensing systems (or devices), such as 3D printing systems (or devices), in particular for the medical field, in cosmetic applications and food applications, more particularly for the medical field. The gelatin of the present invention allows to have a longer dispensing time window, preferably printing time window, in dispensing systems, such as 3D printing systems. The gelatin of the present invention is thermally stable which is advantageous as it can be dispensed preferably at typical dispensing, or printing concentrations of about 7.5 to 15%, without the need of having complicated temperature controlling systems in place or the need of having complicated formulations with additives or bulking material.
Thus in a first aspect, the present invention relates to a gelatin characterized in that
In a second aspect, the present invention relates to the gelatin of the first aspect of the present invention for use in dispensing devices, preferably 3D printing devices.
In a third aspect, the present invention relates to a gelatin composition comprising the gelatin of the present invention and one or more further ingredients, from the group comprising bulking agents, hydrocolloids and proteins.
In a fourth aspect, the present invention relates to the gelatin composition of the third aspect of the present invention for use in dispensing devices, preferably 3D printing devices.
In a fifth aspect, the present invention relates to an aqueous gelatin composition comprising from 1 to 30 wt % of the gelatin of the present invention or the gelatin composition of the present invention and from 70 to 99 wt % of water.
In a sixth aspect, the present invention relates to the aqueous gelatin composition of the fifth aspect of the present invention for use in dispensing devices, preferably 3D printing devices.
In a seventh aspect, the present invention relates to a construct which comprises the gelatin of the present invention, or the gelatin composition of the present invention or the aqueous gelatin composition of the present invention, and wherein the gelatin is gelled. Preferably said construct is produced by dispensing, preferably by 3D printing.
In an eight aspect, the present invention relates to a process for making a construct, characterized in that it comprises the steps of
In a ninth aspect, the present invention relates to the use of the gelatin of the present invention or of the gelatin composition of the present invention, or of the aqueous gelatin composition of the present invention to increase the dispensing time window in dispensing systems, preferably to increase the printability time window in 3D printing systems. In other words, the present invention relates to the use of the gelatin of the present invention or of the gelatin composition of the present invention, or of the aqueous gelatin composition of the present invention to obtain a dispensing time window in dispensing devices as defined above, preferably a printing time window in 3D printing devices, of from above 15 minutes to 3 hours, more preferably from 20 minutes to 2 hours and 30 minutes, even more preferably from 30 minutes to 2 hours, yet even more preferably from 40 minutes to 2 hours, yet even more preferably from 40 minutes to 2 hours, yet even more preferably from 50 minutes to 2 hours, yet even more preferably from 1 hour to 2 hours.
In a tenth aspect, the present invention relates to the use of the gelatin of the present invention, or of the gelatin composition of the present invention or of the aqueous gelatin composition of the present invention to reduce or delay the clogging of dispensing systems during dispensing process, preferably to reduce or delay the clogging of 3D printing systems during 3D printing process.
In an eleventh aspect, the present invention relates to the use of the gelatin of the present invention, or of the gelatin composition of the present invention or of the aqueous gelatin composition of the present invention in the production of scaffolds, engineered tissues, devices or micro-devices suitable for therapeutic purposes, diagnostic purposes, research purposes and the like, said production being done by dispensing, preferably by 3D printing.
In a twelfth aspect, the present invention relates to the use of the construct of the present invention as a scaffold for tissue engineering, for regenerative medicine, for diagnostic purposes, for drug testing and the like.
As used in the present description, “gelatin” refers to its well-known definition in the art, i.e. it is a linear polymer resulting from partial hydrolysis of collagen. Said partial hydrolysis may be obtained by chemical, enzymatic and/or heat treatment of collagen. In the case of gelatin, hydrolysis of collagen is said to be partial because gelatin keeps some functional properties (such as the ability to form gels) contrary to collagen hydrolysate, which have lost such functional properties of forming gels. Gelatin may be derived from collagen sourced of any suitable type of connective animal tissue, particularly from bones, teeth and hides, preferably collagen is sourced from bones and/or hides. Collagen may be derived from different animals such as pork, beef, sheep, goat, horse, deer, chicken, fish and the like. Preferably, however, collagen and thus also gelatin, is derived from pork and/or beef. Standard gelatin requires to be heated in order to dissolve in water. Upon cooling standard gelatin forms a gel, the strength of said gel depends on the specific properties of the gelatin such as molecular weight distribution and molecular conformation. Gelatin is mostly characterized by its gel strength, expressed in g Bloom. Further, gelatin is also characterized by a certain viscosity, expressed in mPa·s. Among others, further properties of gelatin are bulk weight or bulk density and particle size.
As used in the present description, “dispensing systems” or “dispensing devices” refer to apparatus able to deposit a composition, typically as a liquid or paste, onto a surface in the form of dots, lines and any other suitable shapes. Simply stated, said composition is pushed from a reservoir onto a surface through a head, which is typically a nozzle. The head may be driven by pressure, piezoelectric motor or laser-assisted jetting. Depending on the apparatus and on the composition, the head may be of different sizes and different openings, also the need for more or less pressure will depend on the apparatus and composition to be dispensed. Dispensing may be done by extrusion, spraying, jetting and the like. Casting where higher shear stress is used, which is a process comprising filling a mold with a composition to create a specific shape is also understood to be a dispensing process. Casting is however not the preferred way of dispensing. Dispensing devices are preferably computer-controlled systems. A suitable software allows the user to enter a pre-determined shape which is then dispensed on a surface by the dispensing device to provide a construct. Thus a dispensing device may be a 3D printer, an ink jet printer, a single jet dispensing device, and the like. Preferably, the dispensing device is a 3D printing system, i.e. a 3D printer. Preferably, in the present description, dispensing comprises 3D printing.
As used in the present description, “3D printing” refers to a specific dispensing system. 3D printing systems allow to produce tridimensional shaped products, typically by depositing a composition layer by layer until the desired shape is obtained. 3D printing is, as many other dispensing systems, a computer-controlled system. Preferably in the present invention, 3D printing systems do not require temperature control during printing.
As used in the present description, “dispensing time window” refers to the time during which the composition to be dispensed can be dispensed without encountering dispensing problems due to, for example, the change in rheology of the composition within the reservoir and/or through the dispensing head of the dispensing system. The dispensing time window starts when dispensing begins and ends when the dispensing head gets clogged by the composition or when the dispensing does no longer happen in a suitable, uniform manner, e.g. undesired lumps are formed, dispensing spots are missed and the like, which is disturbing the desired final shape of the construct.
As used in the present description, “crosslinking” refers to the formation of a stable network structure, in particular stable network gel structure, via covalent bonding. Typically, crosslinking is obtained by the action of an external factor (crosslinking factor) such as exposure to heat, exposure to UV light (photo-initiated chain-growth polymerization or crosslinking) and the like, on functionalized molecules. Thus molecules are first functionalized such that crosslinking can happen upon subjection to the right conditions. During functionalization, functional groups are added to the chemical structure of (some of) the molecules by a chemical reaction (chemical functionalization) or by a biochemical reaction (biochemical functionalization) such as an enzymatic reaction for example (enzymatic functionalization). Chemical functionalization may be for example an acrylation reaction, i.e. introducing acrylate substitution groups; a methacrylation reaction, i.e. introducing methacrylate substitution groups, diels-alder, azide-alkyne cycloaddition reaction, thiol-ene chemistry (click chemistry) or a succinylation reaction. Other chemical or biochemical functionalizations are not excluded. Molecules may also be previously treated to add reactive groups in their chemical structure in order to give access to new functional chemical groups which will facilitate crosslinking. Alternatively, crosslinking may also be obtained for example by the reaction of certain components present in a composition with specific reactants, for example, alginate present in a composition will react and crosslink when exposed to calcium ions, for example when the composition is put in a bath comprising calcium ions. For a composition to be crosslinked, it is sufficient that at least one of its components is crosslinked. For example, a composition comprising gelatin and alginate may be crosslinked when it is exposed to calcium ions, in that case only the alginate is in fact crosslinked, however it may be said that the composition is crosslinked.
As used in the present description, “crosslinkable” refers to the ability of a composition to react to an external crosslinking factor causing at least two components of the composition to be crosslinked. A composition can be made crosslinkable by adding functional groups to the chemical structure of (some of) the component(s) of the composition by a chemical or biochemical reaction (see above: functionalization). Alternatively, a composition can be made crosslinkable by adding crosslinking agents (or crosslinking inducers) such as formaldehyde, glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl), carbodiimide/N-hydroxysuccinimide, 1,4-Butanediol Diglycidyl Ether, epoxy compounds and the like. Natural crosslinking agents are for example enzymes (for example transglutaminase) and genipin (an agent extracted from gardenia fruit). Such natural crosslinking agents can provide lower cytotoxicity levels. Crosslinking agents like e.g. genipin containing more than one reactive group may react with reactive groups on more than one protein molecule and chemically crosslink the gelatin molecules; the crosslink inducers then become part of the crosslinked structure chemically. Enzymes, e.g. transglutaminases or tyrosinases catalyse the chemical reaction between reactive groups on the protein molecule to form chemical bonds and inter- and intra-molecular crosslinks; the enzyme does not become part of the crosslinked network chemically.
The invention will further be illustrated with different embodiments. It is understood that, unless expressly mentioned, all embodiments/aspects may be combined with one another.
In a first aspect, the present invention relates to a gelatin characterized in that
The gelatin comprises from 95 weight % (wt %) to 100 wt %, preferably at least 96 wt %, more preferably at least 97 wt %, yet even more preferably at least 98 wt %, yet even more preferably at least 99 wt % of gelatin on dry basis of gelatin (wt % db). The gelatin may indeed comprise some minor amounts of ashes and/or salts which typically result from the processing of collagen into gelatin.
The gelatin of the present invention is soluble in an aqueous medium. In this context, solubility in an aqueous medium means that when 10 g of the gelatin is stirred in 100 ml of demi-water at 60° C. during 1 hour, a clear solution is obtained with no particles visible to the naked eye.
FTIR is a well-known method to characterize the structure of organic components. The FTIR-ATR spectrum of a gelatin sample is obtained with a resolution of 4 cm−1, accumulating 16 scans per spectrum. Spectral measurements are performed within the mid-infrared range of 4500-600 cm−1. Measurement is performed with a diamond triple-bounce ATR accessory (Bruker, Tensor 27). Background air spectrum is collected before each sample measurement. A sample is directly compressed on attenuated total reflectance (ATR) crystal prior to analysis without any preliminary preparation. After measurement of a gelatin sample, ethanol and distilled water are used to clean the diamond ATR crystal, which has to dry before performing a next measurement. Between each sample measurement a blank measurement is done (i.e. measurement without presence of sample). The spectra is depicted in transmittance mode.
OriginPro8.5 is used to calculate the first derivative of the spectra within the 1440-1355 cm−1 wavenumber range. The signal is smoothed by applying a quadratic Savitzky-Golay filter with 5-points.
The gelatin of the present invention may be obtained by a process of flash hydrolysis followed by rapid evaporation of water.
Preferably the gelatin is further characterized in that it shows a shift of the peak in the region of the Amide I band (C═O stretch) (1660-1600 cm−1) of about 1 cm−1, preferably of about 0.5 cm−1 towards the high ranges of wavenumber, compared to the gelatin starting material.
More preferably, the gelatin is further characterized in that it shows a shift of the peak in the region of the C—N—H bending band (1565-1500 cm−1) of about 9 cm−1, preferably of about 7 cm−1, more preferably about 5 cm−1 towards the lower ranges of wavenumber, compared to the gelatin starting material.
Preferably, the gelatin is in powder form, particle size may vary but is preferably 70 to 8 mesh, more preferably 60 to 15 mesh, even more preferably 60 to 20 mesh, yet even more preferably 50 to 30 mesh, yet even more preferably 40 to 30 mesh.
Preferably, the gelatin has a gel strength of above 50 g Bloom, preferably from 50 g to 360 g Bloom, preferably 80 g to 350 g Bloom, more preferably 100 g to 350 g Bloom, yet even more preferably 120 g to 340 g Bloom, yet even more preferably 150 g to 340 g Bloom, yet even more preferably 180 g to 330 g Bloom, yet even more preferably 200 g to 330 g Bloom, yet even more preferably 250 g to 330 g Bloom, yet even more preferably 270 g to 320 g Bloom, yet even more preferably 280 g to 320 g Bloom.
In one embodiment, the gelatin of the present invention is substantially not functionalized. Substantially not functionalized means that the degree of functionalization, in particular the degree of chemical functionalization is less than 5%, more preferably from 3% to 0%, yet even more preferably from 1% to 0%, most more preferably from 0.1% to 0%. Functionalization is as defined above.
In another embodiment, the gelatin of the present invention is functionalized, meaning that the gelatin of the present invention is subjected to a functionalization reaction. Functionalization is as defined above. It is an advantage of the present gelatin that due to its high amounts of gelatin, a functionalization reaction is easier to perform, compared to other dispensing material where the composition renders a functionalization reaction more difficult to perform, or makes it more difficult to find the right reaction conditions. Preferably the degree of functionalization is from 30% to 95%, more preferably from 35 to 90%, yet even more preferably from 40 to 85%, yet even more preferably from 45 to 80%, yet even more preferably from 50 to 75%, yet even more preferably from 55 to 70%, yet even more preferably from 60 to 65%. The levels of functionalization will depend on the application. Higher degrees of functionalization is indicated for applications that require a more stable structure in physiological conditions of pH and temperature. Preferably the gelatin is chemically functionalized, more preferably the gelatin is acrylated or methacrylated, more preferably the gelatin is methacrylated.
Thus the present invention further relates to a functionalized gelatin, preferably a methacrylated gelatin characterized in that it is prepared by providing a gelatin according to the present invention and subjecting the gelatin of the present invention to a functionalization reaction, preferably to a methacrylation reaction.
In a further embodiment, the gelatin comprises functionalized gelatin of the present invention and substantially non functionalized gelatin of the present invention. Thus from 1 to 99 wt %, preferably from 5 to 90 wt %, more preferably from 10 to 80 wt %, even more preferably from 20 to 60 wt %, yet even more preferably from 30 to 50 wt % yet even more preferably from 40 to 50 wt % of the gelatin is functionalized and from 1 to 99 wt %, preferably from 5 to 90 wt %, more preferably from 10 to 80 wt %, even more preferably from 20 to 60 wt %, yet even more preferably from 30 to 50 wt % yet even more preferably from 40 to 50 wt % is substantially not functionalized. The degree of functionalization is as described here above.
During methacrylation, methacrylate groups are introduced on the gelatin molecules through a well-known process: typically gelatin is dissolved in a solvent, typically phosphate buffer saline (PBS) or dimethyl sulfoxide solvent (DMSO), methacrylic anhydride is then directly added to said gelatin solution, the methacrylation reaction is conducted at about 50° C. The degree of methacylation can be controlled by the amount of methacrylic anhydride added. The gelatin of the present invention may be methacrylated and may have a degree of methacrylation as mentioned above.
During acrylation, acrylate groups are introduced on the gelatin molecules through a well-known process. Reference is made for example to Billiet, I. T. (2014) “Gelatin Functionalizations for Cell Embedding in 3D Geometries Using Rapid Prototyping Technology”.
Preferably further, the gelatin of the present invention comprises less than 100 Endotoxin Units (EU), more preferably less than 80 EU, even more preferably less than 60 EU, yet even more preferably less than 40 EU, yet even more preferably less than 30 EU, yet even more preferably less than 10 EU per gram of gelatin, expressed on as is basis. Endotoxins may be removed by clearance filters for endotoxins removal such as charged filters. Such charged filters are available and can be for example Mustang E® membrane and Mustang Q® membrane (from Pall), Sartobind® filter (from Sartorious). The LAL test (acronym for Limulus Amebocyte Lysate) is a test for the determination of bacterial endotoxins, which uses an Amebocyte Lysate of the Limulus crab.
Further preferably the gelatin of the present invention is purified by using one or more of the following techniques: ultrafiltration, diafiltration, filtration with depth filters. These filtration techniques are useful to (partially) remove for example undesired toxins, bacteria, product fractions and the like.
Preferably the gelatin of the present invention comprises 5 wt % or less than 5 wt %, more preferably 4 wt % or less than 4 wt %, even more preferably 3 wt % or less than 3 wt %, yet even more preferably 2 wt % or less than 2 wt %, yet even more preferably 1 wt % or less than 1 wt %, yet even more preferably 0.5 wt % or less than 0.5 wt %, yet even more preferably 0.1 wt % or less than 0.1 wt % of additives, on dry basis of the gelatin. More preferably, the gelatin of the present invention comprises substantially no additives. Additives may be rheological enhancers, flowability enhancers and the like, such as for example glycols, starches, alginates and the like. It is an advantage of the present gelatin to comprises such low amounts of additives in the ranges mentioned above, in particularly to comprise substantially no additives, in particular when the gelatin is used to produce scaffolds for use as ECM with living cells as the additives have a negative effect on cell survival rates. Many current material for producing scaffolds for use as ECM nowadays comprise additives in order to allow the materials to be dispensed, in particular to be 3D printed into said scaffolds.
The gelatin of the present invention is alternatively characterized in that when dispensed
Preferably, dispensing is done at around the gelation temperature of the 10% solution of gelatin, i.e. at a temperature in the range of about 10° C. less to 10° C. more than the gelation temperature, preferably 5° C. less to 5° C. more than the gelation temperature. The possibility to print at said temperature is an advantage of the present invention, indeed, existing gelatin-based printing material cannot be printed at a temperature around the gelation temperature unless specific measures are taken such as for example the addition of specific additives. Also as mentioned above, existing gelatin-based printing material are often printed in heated system, more than at least 10° C., at least 15° C. or even at least 20° C. above the gelation temperature.
Advantageously also, at equal concentration, dispensing can be done at lower pressure with the gelatin of the present invention, compared to existing gelatin-based printing material.
Preferably, said solution keeps a substantially consistent flow behavior for a time of from above 15 minutes to 3 hours, more preferably from 20 minutes to 2 hours and 30 minutes, even more preferably from 30 minutes to 2 hours, yet even more preferably from 40 minutes to 2 hours, yet even more preferably from 40 minutes to 2 hours, yet even more preferably from 50 minutes to 2 hours, yet even more preferably from 1 hour to 2 hours.
In a second aspect, the present invention relates to the gelatin of the first aspect of the present invention for use in dispensing devices, preferably for use in a 3D printer. It has been found that said gelatin is particularly suitable for dispensing, in particular for 3D printing, such as for use as a bio-ink in 3D printing. The present invention also relates to the gelatin of the first aspect of the present invention for use in the construction of scaffold for medical applications, preferably for tissue engineering, for regenerative medicine, for implants, for medical devices, for drug discovery and drug testing; or for cosmetic applications, preferably a scaffold for testing of cosmetic products; and the like. The gelatin can be dispensed, without the need of having a temperature controlled dispensing system and without the need of having other ingredients added, or additives added or other solutions that are typically used in the art to be able to dispense gelatin. Preferably dispensing is done under pressure, more preferably at room temperature. Dispensing, and particularly printing, is done by putting the material into an aqueous solution. The solvent may be a phosphate buffer, bovine serum albumin (BSA), water, preferably the solvent is a phosphate buffer or BSA, more preferably a phosphate buffer. Advantageously, the gelatin is put in solution at a temperature of from 30 to 60° C., more preferably from 40 to 55° C., yet even more preferably from 40 to 50° C. and allowed to cool down to room temperature before dispensing. It is preferable to start the dispensing process before a period of maximum 3 hours after cool down.
Further advantage of the present gelatin is that it can be dispensed as a highly concentrated aqueous solution, i.e. aqueous solution containing up to 30 wt % of the gelatin of the present invention, preferably the gelatin of the present invention is used in dispensing devices as a solution comprising from 1 to 30 wt %, more preferably from 5 to 25 wt %, even more preferably from 7.5 to 20 wt %, yet even more preferably from 7.5 to 15 wt %, yet even more preferably from 10 to 15 wt % of the gelatin of the present invention.
In a third aspect, the present invention relates to a gelatin composition comprising the gelatin of the first aspect of the present invention and further ingredients.
Preferably the gelatin composition is in powder form.
The gelatin composition comprises one or more further ingredients chosen from bulking agents, hydrocolloids, proteins, anticaking agents and the like. Preferably the gelatin composition of the present invention comprises from 40 wt % to 95 wt %, more preferably from 45 wt % to 95 wt %, even more preferably from 50 wt % to 95 wt %, yet even more preferably from 55 wt % to 95 wt %, yet even more preferably from 60 wt % to 95 wt %, yet even more preferably from 65 wt % to 95 wt %, yet even more preferably from 70 wt % to 95 wt %, yet even more preferably from 75 wt % to 95 wt %, yet even more preferably from 80 wt % to 95 wt %, yet even more preferably from 85 wt % to 95 wt % of the gelatin of the present invention, on dry basis of the gelatin composition. Thus preferably the gelatin composition of the present invention comprises from 5 wt % to 60 wt %, more preferably from 5 wt % to 55 wt %, even more preferably from 5 wt % to 50 wt %, yet even more preferably from 5 wt % to 45 wt %, yet even more preferably from 5 wt % to 40 wt %, yet even more preferably from 5 wt % to 35 wt %, yet even more preferably from 5 wt % to 30 wt %, yet even more preferably from 5 wt % to 25 wt %, yet even more preferably from 5 wt % to 20 wt %, yet even more preferably from 5 wt % to 15 wt % of the further ingredients as defined herein.
Bulking agents are well-known in the art and refer to material which do not possess any functionality, inert products, such as sugars, mono-, di- and tri-saccharides, maltodextrins, fibers, protein hydrolysates (such as collagen hydrolysates for example) and the like.
Hydrocolloids may be one or more of alginate, pectin, carrageenan, gellan, agar, starch, xanthan gum, locust bean gum, guar gum, gum karaya, gum tragacanth, gum Arabic, cellulose derivatives, chitosan and the like. Preferably the hydrocolloids are alginate or carrageenan, more preferably alginate.
Proteins may be one or more of keratin, elastin, fibroin, thrombin, albumin, and the like. Protein may also be derived from collagen raw materials.
In an embodiment, the gelatin composition comprises functionalized gelatin of the present invention and regular gelatin (i.e. not according to the present invention) which is not functionalized. The gelatin composition preferably comprises from 1 to 99 wt %, preferably from 5 to 90 wt %, more preferably from 10 to 80 wt %, even more preferably from 20 to 60 wt %, yet even more preferably from 30 to 50 wt % yet even more preferably from 40 to 50 wt % of functionalized gelatin of the present invention (the degree of functionalization is as described above), and from 1 to 99 wt %, preferably from 5 to 90 wt %, more preferably from 10 to 80 wt %, even more preferably from 20 to 60 wt %, yet even more preferably from 30 to 50 wt % yet even more preferably from 40 to 50 wt % of regular gelatin, non-functionalized.
Further preferably the gelatin composition of the present invention has been purified by using one or more of the following techniques: ultrafiltration, diafiltration, filtration with depth filters. These filtration techniques are useful to remove or at least substantially lower the amount of bacteria, endotoxins, fats, non-collagen proteins, fibres and the like.
In a fourth aspect the present invention relates to the gelatin composition of the third aspect of the present invention for use in dispensing devices, preferably in 3D printer. It has been found that said gelatin composition is particularly suitable for dispensing, in particular 3D printing, such as for use as a bio-ink in 3D printing. The present invention also relates to the gelatin composition of the third aspect of the present invention for use in the construction of scaffold for medical applications, preferably for tissue engineering, for regenerative medicine, for implants, for drug discovery and drug testing, or for cosmetic applications, preferably a scaffold for testing of cosmetic products and the like, or for food applications, such as for example personalized meals, reconstituted meals and the like.
The gelatin composition can be dispensed when put into solution without the need of having a temperature-controlled dispensing system and without the need of having additives added or other solutions that are typically used in the art to be able to dispense gelatin composition. Preferably dispensing is done under pressure, more preferably at room temperature. Dispensing, and particularly printing, is done with the gelatin composition put into an aqueous solution. The solvent may be a phosphate buffer, BSA, water, preferably it is a phosphate buffer or BSA, more preferably a phosphate buffer. Advantageously, the gelatin composition is put in solution at a temperature of from 30 to 60° C., more preferably from 40 to 55° C., yet even more preferably from 40 to 50° C. and allowed to cool down to room temperature before dispensing. It is preferable to start the dispensing process maximum before a period of 3 hours after cool down.
A further advantage of the present gelatin composition is that it can be dispensed as a highly concentrated aqueous solution, i.e. aqueous solution containing up to 30 wt % of the gelatin composition, preferably the present gelatin composition is used in dispensing devices as a solution comprising from 1 to 30 wt %, more preferably from 5 to 25 wt %, even more preferably from 7.5 to 20 wt %, yet even more preferably from 7.5 to 15 wt %, yet even more preferably from 10 to 15 wt % of the gelatin of the present invention.
In a fifth aspect, the present invention relates to an aqueous gelatin composition comprising from 1 to 30 wt %, preferably from 5 to 25 wt %, even more preferably from 7.5 to 20 wt %, yet even more preferably from 7.5 to 15 wt %, yet even more preferably from 10 to 15 wt % of the gelatin of the first or second aspect of the present invention or of the third or fourth aspect of the present invention and from 70 to 99 wt %, preferably from 95 to 75 wt %, even more preferably from 80 to 92.5 wt %, yet even more preferably from 85 to 92.5 wt %, yet even more preferably from 85 to 90 wt % of an aqueous medium.
The aqueous medium may be a phosphate buffer, bovine serum albumin (BSA), water, preferably the solvent is a phosphate buffer or BSA, more preferably a phosphate buffer.
Preferably, the aqueous gelatin composition of the present invention comprises various components, preferably cell growth components, i.e. components which have a beneficial effect on the growth of living plant, human or animal cells. Such components may be cytokines (Adiponectin Human) cell growth promoters or growth factors, such as (EGF, FGF, NGF, PDGF, VEGF, IGF, GMCSF, GCSF, TGF, Erythropieitn, TPO, BMP, HGF, GDF, Neurotrophins, MSF, SGF, GDF); nutrients, such as glucose, yeast extract; salts, such as potassium chloride, sodium chloride and the like; proteins, such as thrombin transferrin, albumin, peptones and the like.
Preferably, the aqueous gelatin composition of the present invention further comprises living organisms, preferably human, animal and/or plant cells. Said living organisms may be encapsulated or non-encapsulated.
Advantageously, the aqueous gelatin composition of the present invention is prepared by bringing all components in solution at a temperature of from 30 to 60° C., preferably from 40 to 55° C., yet even more preferably from 40 to 50° C. and letting the aqueous composition cool down to room temperature before further use.
Further preferably the aqueous gelatin composition of the present invention has been purified by using one or more of the following techniques: ultrafiltration, diafiltration, filtration with depth filters. These filtration techniques are useful to remove or at least substantially lower the amount of bacteria, endotoxins (removal of e.g. from 10 to 90% of endotoxins), fats, non-collagen proteins, fibres and the like.
In a sixth aspect, the present invention relates to the aqueous gelatin composition of the present invention for use in dispensing devices, preferably in 3D printer. It has been found that said aqueous gelatin composition is particularly suitable for dispensing, in particular for use as a bio-ink in 3D printing. The present invention also relates to the aqueous gelatin composition of the present invention for use in dispensing devises, preferably in 3D printers, in the construction of scaffold for medical applications, preferably for tissue engineering, for regenerative medicine, for implants, for drug discovery and drug testing, or for cosmetic applications, preferably a scaffold for testing of cosmetic products, or food applications and the like. Also the present aqueous gelatin composition is very suitable for dispensing, preferably 3D printing.
The aqueous gelatin composition can be dispensed without the need of having a temperature-controlled dispensing system and without the need of having additives added or other solutions that are typically used in the art to be able to dispense aqueous gelatin compositions. Preferably dispensing is done under pressure, more preferably at room temperature.
Advantageously, the aqueous gelatin composition is heated to a temperature of from 30 to 60° C., more preferably from 40 to 55° C., yet even more preferably from 40 to 50° C. and allowed to cool down before dispensing. It is not desired to keep the cooled aqueous gelatin composition for a period of time of more than 3 hours at room temperature before starting the dispensing process.
In a seventh aspect, the present invention relates to a construct, comprising the gelatin of the present invention, the gelatin composition of the present invention and/or the aqueous gelatin composition of the present invention, wherein the gelatin is gelled. Preferably said construct being produced by dispensing, more preferably by 3D printing.
As mentioned above, the gelatin of the present invention may or may not be functionalized. When functionalized, the construct is crosslinkable, and crosslinking may be done by application of the right crosslinking conditions (such as applying a photoinitiator for example). When crosslinking reaction has been performed, it may be said that the construct is crosslinked. Crosslinked constructs, especially comprising gelatin which is a natural ingredient that melts at body temperature, are very stable and can be introduced in the body or used at temperatures above the melting temperature of gelatin, without the fear that the construct would melt away or show deformations.
Preferably crosslinking takes place immediately after dispensing (filament deposition) to ensure self-supporting properties of the 3D-construct, to avoid collapsing of the 3D construct after printing.
The construct may further comprise one or more of other components such as carbohydrates, salts, living cells, cell growth promoters and enzymes.
The construct may comprise the gelatin, the gelatin composition and/or the aqueous gelatin composition of the present invention (‘gelatin comprising compositions’) and other further components due to for example dispensing the construct with multiple headed nozzles, where one or more nozzles dispensing the gelatin comprising composition and the other one or more nozzles dispensing another type of suitable composition which can be dispensed and is preferably biocompatible, such as a composition comprising hydrocolloids, for example, alginate; proteins; carbohydrates and the like.
In an eight aspect, the present invention relates to a process for making a construct, characterized in that the process comprises the steps of
The construct is as defined herein.
Operating the dispensing device may be done at a controlled temperature such as at a temperature of from above 25 to 40° C., however advantageously with the present aqueous gelatin composition, dispensing is done without controlled temperature system, at room temperature (18 to 25° C., preferably 20 to 25° C.). Preferably, dispensing is is done at around the gelation temperature of the 10% solution of gelatin, i.e. at a temperature in the range of about 10° C. less to 10° C. more than the gelation temperature, preferably 5° C. less to 5° C. more than the gelation temperature. The possibility to print at said temperature is an advantage of the present invention, indeed, existing gelatin-based printing material cannot be printed at a temperature around the gelation temperature unless specific measures are taken such as for example the addition of specific additives. Also as mentioned above, existing gelatin-based printing material are often printed in heated system, more than at least 10° C., at least 15° C. or even at least 20° C. above the gelation temperature.
Preferably the construct is made by dispensing, preferably by printing, layer above layer or dot above dot of the aqueous gelatin composition. If needed the construct may also be a single layer or dot of the aqueous gelatin composition. In fact, any suitable construct may be printed, in accordance with the possibilities of the used dispensing system, preferably of the used 3D printer software.
Once the construct is made, it may be crosslinked, provided either that the aqueous gelatin composition was functionalized to be crosslinked or provided that enzymes have been added which can cause crosslinking. Preferably crosslinking takes place right after dispensing (filament deposition), i.e. the moment of cross-linking depending on the viscoelastic properties of the dispended material, to ensure self-supporting properties of the 3D-construct, thus cross-linking is done on time after dispensing to avoid collapsing of the 3D construct after dispensing or printing.
It is also possible to start the crosslinking reaction whilst dispensing, either directly from the start of the dispensing step or at any suitable time after the start of the dispensing step, before the end of the dispensing step.
Typically, accurate time control during the dispensing process is important when crosslinking agents are present in the aqueous gelatin composition because the crosslinking reaction may cause detrimental viscosity increase inside the nozzle while dispensing. In this case, thermostability of the gelatin of the present invention is advantageous as it allows to prevent the combined effect of physical gel formation together with increments of viscosity caused by (chemical) crosslinking, making it possible to have a more flexible time control during dispensing process.
The present invention further relates to the use of the gelatin of the present invention, the gelatin composition of the present invention or the aqueous gelatin composition of the present invention to increase the dispensing time window in dispensing systems, preferably to increase the printability time window in 3D printing systems.
This effect is due to the fact that said gelatin, gelatin composition and aqueous gelatin compositions show little to no viscosity increase during printing, also at room temperature, in fact even at around the gelation temperature of the gelatin, as defined above , for a period of time of up to 3 hours, preferably of above 15 minutes to 3 hours, more preferably from 20 minutes to 2 hours and 30 minutes, even more preferably from 30 minutes to 2 hours, yet even more preferably from 40 minutes to 1 hour and 30 minutes, yet even more preferably from 40 minutes to 1 hour. In comparison, previous compositions comprising gelatin typically only allowed a dispensing time window of about 15 minutes or less. As a results, clogging of the dispensing head or nozzle, preferably printing nozzle does not happen until after 3 hours of dispensing, preferably printing, preferably not until after a period of above 15 minutes to 3 hours, more preferably from 20 minutes to 2 hours and 30 minutes, even more preferably from 30 minutes to 2 hours, yet even more preferably from 40 minutes to 2 hours, yet even more preferably from 40 minutes to 2 hours, yet even more preferably from 50 minutes to 2 hours, yet even more preferably from 1 hour to 2 hours.
Further, the present invention relates to the use of the gelatin, gelatin composition and aqueous gelatin compositions of the present invention in the preparation, preferably by dispensing, more preferably by 3D printing, of a scaffold for medical applications, preferably for tissue engineering, for regenerative medicine, for implants, for drug discovery and drug testing, or for cosmetic applications, preferably a scaffold for testing of cosmetic products, and the like. Also, the present invention relates to the use of the gelatin, gelatin composition and aqueous gelatin compositions of the present invention in the preparation of food applications such as customized meals, reconstituted foods, bakery applications and the like.
Further, the present invention relates to the use of the construct of the present invention in the preparation of a scaffold for medical applications, preferably for tissue engineering, for regenerative medicine, for implants, for drug discovery and drug testing, or for cosmetic applications, preferably a scaffold for testing of cosmetic products, and the like.
Further, the present invention relates to a process for identifying a gelatin suitable for use in dispensing device, preferably for use in 3D-printing devices, said process comprises the steps
The present invention will be further illustrated in the non-limiting examples.
A solution of acid treated porcine gelatin of high bloom (300 g) (T=60° C.) at a concentration of 35 wt % is submitted to flash hydrolysis by heating to achieve a product temperature of 120° C. for 60 seconds. The heated gelatin is then submitted to evaporation by roll-coating the heated gelatin solution on the surface of a rotating cylinder heated by steam at 4.5 bar. The adhering thin layer of product on the surface of the cylinder is rapidly dried and scratched off by the action of a sharp knife. The gelatin so produced is soluble in water.
The FTIR-ATR spectrum of the gelatin so produced is measured. The first derivative and the transmittance is depicted in
FTIR-ATR spectrum of following material are also depicted in
As depicted in
Comparative experiment demonstrating the printing behavior of gelatin from example 1 in comparison with regular gelatin was conducted. Both gelatins are derived from porcine source and present bloom higher than 280 g, about 300 g. On a Cellink Inkredible 3D printer, constructs are is prepared with the gelatin according to example 1 and with the regular gelatin.
The gelatin of example 1 and the regular gelatin are each dispersed in a glass vial containing PBS (phosphate buffer solution, pH7.4) to produce a solution at a concentration of 10 w/w %. The dispersion of the powder in solution is performed using a vortex mixer (3000 rpm). The vials containing 10 mL of solution are then placed in a water bath at 60° C. for 1 hour. While warm, the solutions are then each poured into a 3 mL syringe from the 3D printer.
The 2 syringes are placed on the bench and allowed to cool at ambient temperature conditions (about 20° C.).
The syringe containing the gelatin according to example 1 is placed in the printhead 1 (PH1) and the syringe containing the regular gelatin is placed in the printhead 2 (PH2). Printing of 6 lattice structures was initiated simultaneously by PH1 and PH2.
The printing settings applied on the Slic3r software for printing of a lattice structure are adjusted as follows:
The gelatin according to example 1 requires extrusion pressure of 85-100 kPa while the regular gelatin requires a pressure of 130-140 kPa in order to have filament formation.
With the regular gelatin, the printing of a third lattice construct is not possible due to complete blockage of the nozzle. With the gelatin according to example 1, it is possible to print up to 6 lattice constructs, printing can be done up to 105 minutes without any blockage of the nozzle and uniform filament production, the flow behavior remains uniform during the entire printing operation.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19167094.2 | Apr 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/059669 | 4/3/2020 | WO | 00 |
