Patent Description:
The impact of additive manufacturing, namely 3D printing, in different scientific and technological fields is undeniable. The possibility of fabricating complex structures with precise shape, using the computer-controlled layer-by-layer deposition of materials, has opened the doors for new opportunities in multiple domains, and the pharmaceutical and biomedical sectors are no exception. 3D printing technology may be used in the development of new drug-delivery systems, other biomedical devices, and even in the manufacturing of artificial living tissues. In this latter field, known as 3D bioprinting (one of the younger siblings of 3D printing techniques), it is possible to create living constructs with customized shapes and characteristics, for application in organ transplantation, regenerative medicine, or even drug development.

The materials that are dispensed by the 3D printers are known as inks, and they are a crucial element for the success of the procedure. Therefore, they must be carefully selected considering the printing technique and the desired application. Specifically, the inks should fulfil the physical requirements (and especially the rheological properties) of the 3D printing process. When they are developed for biological endeavors, these materials should also be friendly for living cells, exhibiting biocompatibility and, in some cases, biodegradable nature. In bioprinting, for instance, bioinks containing cells should be able to satisfy the essential conditions for cells to survive the bioprinting procedure, and to proliferate in the newly formed tissue.

There are several 3D printing approaches, but extrusion 3D printing is one of the most explored. This 3D printing technique is based on the application of mechanical or pneumatic pressure forces to promote the ejection of the ink through a nozzle and its deposition. Hydrogel-based inks are one of the most explored biomaterials, and they are frequently applied in extrusion-based processes, as it is disclosed in <NPL>). Hydrogels are polymeric networks that are able to contain large amounts of water, and closely mimic the native 3D microenvironment of living tissues, granting them with great potential for biological endeavors, as it is disclosed in <NPL>). They may be obtained from different polymers, including synthetic options like poly(ethylene glycol) (PEG) and polycaprolactone (PCL), according to <NPL>); or biopolymers like sodium alginate, according to <NPL>; chitosan, according to <NPL>); or gelatin, according to <NPL>).

The use of PEG, for instance, is shown in patent document <CIT>, where the inventors describe a hydrogel from PEG combined with heparin and an immune molecule (i.e., a cytokine or cell-adhesion molecule) for T-cells culture and immunotherapy applications. On the other hand, the use of biopolymers like sodium alginate or gelatin is reported in patent documents like <CIT>, concerning an alginate bioink containing nanocelluloses for 3D bioprinting, and in patent document <CIT>, where the authors describe, for instance, a bioink comprising gelatin methacrylate, collagen methacrylate and a photoinitiator for 3D bioprinting of cardiomyocytes.

<NPL>, discloses a composition for additive manufacturing comprising a composite hydrogel comprising an alginate and cellulose nanocrystals, wherein said cellulose nanocrystals are stiff and spindle-shaped crystalline nanomaterials having high aspect ratios (length <NUM>-<NUM>, width <NUM>-<NUM>).

There is a clear research tendency to focus on natural polymers, also designated as biopolymers, for health-related applications, given their superior biological performance. Among the panoply of natural polymers, sodium alginate, from here on referred to as alginate, is one of the most widely explored biopolymer for 3D printing, envisioning the fabrication of constructs for pharmaceutical and biomedical purposes. Alginate is a linear anionic polysaccharide obtained from brown algae, and its polymeric chains are composed of glucuronate (G) and mannuronate (M) units in different proportions and motif blocks. Alginate hydrogels are easily obtained by crosslinking with divalent cations (e.g., Ca<NUM>+), following an "egg-box" model where Ca<NUM>+ ions are entrapped in cavities formed by the coupling between adjacent G units.

Some publications and patents have also explored the combination of alginate hydrogels with inorganic particles (i.e., hydroxyapatite or calcium phosphate, to promote osteogenesis; and silica to enhance the printability of the hydrogels), or particles obtained from synthetic polymers (i.e. poly(lactic acid), poly(glycolic acid) and polycaprolactone for the delivery of morphogenic growth factors).

The automated manufacturing is a process for the creation of three-dimensional objects from a digital model, for example in computer aided design (CAD), which is converted into standard triangulation (stl) language and subsequently divided in layers by slicing algorithm, thus creating detailed information about each transverse slice.

The creation of a printed component is carried out by means of additive manufacturing processes, wherein the object is created by the deposition of successive layers of material until the product is complete, where the successive layers of raw material can be in the form of hydrogels, pastes or powders. Each one of these layers can be seen as a horizontal transverse section in thin slices.

The interest in using alginate hydrogels for biomedical applications is driven by the interesting features of this polysaccharide, including the simple crosslinking and notable biocompatibility and biodegradability. However, alginate does not possess any cell binding moieties, which often results in low cell adhesion and reduced proliferation in the hydrogels, as it is disclosed in <NPL>). Moreover, alginate-based hydrogels often underperform in terms of rheological and mechanical properties, that are fundamental for 3D printing processes, and show relatively unpredictable degradation rates. These limitations may hamper the long-term stability of the resulting 3D printed constructs, according to <NPL>); and <NPL>).

To circumvent these limitations, alginate is commonly combined with other materials, including other biopolymers like cellulose, chitosan, or collagen, to produce novel inks with improved properties. In particular, the mechanical reinforcement of alginate hydrogels with cellulose nanoforms (e.g., cellulose nanofibers, according to <NPL>); and <NPL>), bacterial cellulose, according to <NPL>); and cellulose nanocrystals according to <NPL>); leading to nanocomposite alginate hydrogel-based inks, has been reported in several studies, as appraised in <NPL>). Cellulose, the most abundant biopolymer in nature, can be obtained from plants, tunicates, algae, and some non-pathogenic bacteria, and it has notable mechanical properties. The inks obtained from alginate and cellulose reveal good biocompatibility, given the established non-toxic nature of both biopolymers, while showing enhanced mechanical performance, as disclosed in <NPL>).

This approach has also been reported in several documents, for example the patent document <CIT>, mentioned above, that describes a composite hydrogel containing nanocelluloses (nanofibers and nanocrystals) to reinforce alginate hydrogels for 3D bioprinting of cartilage. A similar approach is explored in the patent document <CIT>, using alginate and cellulose nanofibers obtained from brown seaweed. Additionally, the patent document <CIT>, discloses the use of nanocellulose and a cell-adhesion peptide (RGD)-conjugated alginate for improved 3D bioprinting of human skin (dermis).

In all these studies and documents, cellulose nanoforms are specifically used to reinforce the alginate hydrogels for application in extrusion 3D printing. The incorporation of bioactive compounds into these nanocellulose forms, prior to their incorporation into the hydrogels, could impart them with additional functionalities. However, the loading of these nanostructures, particularly with molecules with low water solubility, is still a challenge.

The present invention solves some of the problems of the state of the art by using spherical particles of cellulose or cellulose derivatives (such as cellulose acetate) for simultaneous reinforcement of alginate hydrogels and to impart additional functionalities. In the preferred embodiments, bioactive compounds are loaded in the particles, to promote the delivery of bioactive compounds from the 3D printed constructs. Additionally, if the inks are loaded with living cells before the printing process, these inks may even show interesting potential for 3D bioprinting of living tissues, due to the known biocompatible nature of these biopolymers.

The present invention constitutes a new strategy to circumvent some of the current limitations of alginate hydrogel inks for 3D printing applications, as the rheological and mechanical properties, while simultaneously imparting 3D printed constructs with new functionalities (drug releasing ability) and improved biological properties.

The cellulose or cellulose derivative-based particles are used to improve the rheological properties of the composition for additive manufacturing comprising a composite hydrogel, including the shear viscosity and the shear rate of said composition, while simultaneously improving the mechanical performance of the final 3D constructs, particularly their compression properties. The incorporation of cellulose or cellulose derivative-based particles into the alginate hydrogels improves their rheological properties for extrusion 3D printing, and the obtained printed constructs display better mechanical performance.

Additionally, the cellulose or cellulose derivative-based particles may be loaded with drugs or bioactive compounds prior to the printing process following different approaches, as the addition of the compounds to the cellulose solution prior to the regeneration step, allowing the final construct to possess drug-releasing potential, or to have improved biological properties and functionalities.

With the purpose of providing an understanding of the principles according to the embodiments of the present invention, reference will be made to the embodiments illustrated in the figures and to the terminology used to describe them. In any case, it must be understood that there is no intention of limiting the scope of the present invention to the content of the figures. Any subsequent alterations or modifications of the inventive characteristics shown herein, as well as any additional applications of the principles and embodiments of the invention shown, which would occur normally to a person skilled in the art having this description in hands, are considered as being within the scope of the claimed invention.

The present invention describes a new biopolymeric composite hydrogel-based ink, composed of sodium alginate and cellulose or cellulose derivative-based spherical particles, for extrusion 3D printing purposes. The composite hydrogel-based ink is designed to be applied as an ink for 3D printing via extrusion techniques, for the layer-by-layer creation of 3D structures for several uses, for instance pharmaceutical and biomedical applications. The composite hydrogel-based ink is identified as a composition for additive manufacturing comprising a composite hydrogel.

The present invention refers, in a first aspect, to a composition for additive manufacturing comprising a composite hydrogel which further comprises:.

In the preferred embodiments according to the present invention, the spherical cellulose or cellulose derivative-based particles have a diameter from <NUM> to <NUM> micrometre.

In the preferred embodiments according to the present invention, the cation of alginate is selected from at least one of the group consisting of magnesium, calcium, sodium or potassium. More preferably, the cation of alginate is sodium.

In the preferred embodiments according to the present invention, the cation of a pre-crosslinking salt is selected from at least one of the group consisting of an alkaline-earth metals, for example calcium or magnesium; or zinc; or iron.

In the preferred embodiments according to the present invention, the composition for additive manufacturing comprising a composite hydrogel is configured to be extruded in an additive manufacturing process.

In the preferred embodiments according to the present invention, the composition for additive manufacturing further comprises at least one of the group consisting of an active pharmaceutical ingredient, a bioactive compound, or living cells. The active pharmaceutical ingredient is at least one selected from the group comprising non-steroidal anti-inflammatory drugs (NSAIDs), anti-cancer drugs, and wound healing drugs. The NSAIDs may be selected from one or more compounds, for example, ibuprofen, naproxen, ketoprofen, flurbiprofen, and diclofenac. The anti-cancer drugs may be selected from one or more compounds, for example, doxorubicin, paclitaxel and docetaxel. The wound healing drugs may be selected from one or more drugs, for example, dexpanthenol, hyaluronic acid, and coenzyme Q10. The bioactive compound is at least one selected from the group consisting of phenolic compounds, including but not limited to flavonoids, isoflavonoids, phenolic acids, cinnamic acids, lignans, coumarins and curcuminoids, including the salts, esters, glucosides, and stereoisomers thereof. More preferably, the curcuminoid is curcumin.

In the preferred embodiments according to the present invention, the composition for additive manufacturing comprises cellulose or cellulose derivatives-based particles selected from the group consisting of cellulose, cellulose acetate, methyl cellulose, hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), or hydroxypropylmethyl cellulose (HPMC) particles, or their mixtures. More preferably, the cellulose acetate particles comprise a mixture of diacetate and triacetate cellulose.

The present invention refers, in a second aspect, to a process for obtaining an additive manufacturing composition, as defined in the first aspect of the invention, comprising the following steps:.

wherein the steps i) and ii) are carried out in any order.

In the preferred embodiments according to the present invention, the step iv) is executed from about <NUM> minute to about <NUM> minutes. In the preferred embodiments according to the present invention, the step iv) is executed at a temperature from about <NUM> to about <NUM>.

In the preferred embodiments according to the present invention, the concentration of the cation of a pre-crosslinking salt in the aqueous solution is in the range from <NUM> to <NUM>%, wherein the percentual corresponds to the mass of said cation in relation to the total volume of said solvent.

In the preferred embodiments according to the present invention, the sodium alginate is obtained from brown algae.

In the preferred embodiments according to the present invention, the spherical particles of cellulose or cellulose derivatives are obtained from cellulose acetate via a dissolution step followed by a regeneration step, as it is disclosed in <NPL>), and in <NPL>). Therefore, the particles may be loaded with drugs or bioactive compounds prior to the printing process following different approaches, as it is known by a person skilled in the art, as the addition of the compounds to the cellulose solution prior to the regeneration step, allowing the final construct to possess drug-releasing abilities, or to have improved biological properties and functionalities.

In the preferred embodiments of the process for obtaining an additive manufacturing composition, the active pharmaceutical ingredient or the bioactive compound are included in the spherical particles of cellulose or cellulose derivative-based prior to step iii). A bioactive compound or a drug may be loaded in the cellulose particles previously to their incorporation into the alginate hydrogel, in order to grant them with compound-releasing abilities or improved biological properties. These compounds may be included in the particles during their manufacturing process or by posterior adsorption.

As an example, the pre-crosslinking step is carried out with an aqueous solution of <NUM>% (w/V) of CaCl<NUM>.

The present invention refers, in a third aspect, to a process of additive manufacturing of an object comprising the following steps:.

wherein the step b) comprises an extrusion step of said composition for additive manufacturing through a nozzle comprised in a head of the system configured for the manufacture of objects by additive manufacturing.

In the preferred embodiments of the process of additive manufacturing of an object, the object is selected from the group consisting of a biomedical device or a living tissue. Preferably, the biomedical device is a drug delivery-system, such as a patch, an adhesive, or a bandage. A living tissue may be used as a tissue regeneration material, such as an artificial skin graft.

In the preferred embodiments of the process of additive manufacturing of an object, after the step b) is performed a full-crosslinking stage of an additive manufactured object, wherein said additive manufactured object is immersed into an aqueous solution comprising cations of a full-crosslinking salt, wherein the cation of the full-crosslinking salt is selected from at least one of the group consisting of a divalent or a trivalent cation. The full-crosslinking stage maintain the integrity of the final additive manufactured object.

In the preferred embodiments according to the present invention, the full-crosslinking stage is executed from about <NUM> minutes to about <NUM> minutes. In the preferred embodiments according to the present invention, the full-crosslinking stage is executed at a temperature from about <NUM> to about <NUM>.

In the preferred embodiments according to the present invention, the cation of a full-crosslinking salt is selected from at least one of the group consisting of an alkaline-earth metal, for example calcium or magnesium; or zinc; or iron.

In the preferred embodiments according to the present invention, the concentration of the cation of a full-crosslinking salt in the aqueous solution is in the range from <NUM> to <NUM>%, wherein the percentual corresponds to the mass of said cation in relation to the total volume of said solvent.

Cellulose acetate (composed a mixture of diacetate and triacetate) was acquired from FILTER-LAB (Barcelona, Spain), while sodium alginate from brown algae (viscosity <NUM> - <NUM> cP at <NUM>% in H<NUM>O at <NUM>) was obtained from Sigma-Aldrich (Sintra, Portugal). Calcium chloride anhydrous (<NUM>%) was provided by Carlo Erba Reagents (Barcelona, Spain). Acetone (≥<NUM>%) was supplied by Honeywell (Charlotte, US), and curcumin (<NUM>%) was obtained from Alfa Aesar (Kandel, Germany). Ultrapure water (Type <NUM>, <NUM> MΩ·cm at <NUM>) was obtained by a Simplicity® Water Purification System (Merck, Darmstadt, Germany).

Dulbecco's Modified Eagle's Medium (DMEM) was purchased from PAN-Biotech (Germany). <NUM>-(<NUM>,<NUM>-dimethylthiazol-<NUM>-yl)-<NUM>,<NUM>-diphenyltetrazolium bromide (MTT, <NUM>%), dimethyl sulfoxide (DMSO, ≥<NUM>%), and trypsin-EDTA solution 10x (<NUM>% trypsin, <NUM>% EDTA) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS), phosphate buffer solution (PBS, pH <NUM>), L-glutamine, penicillin/streptomycin and fungizone were obtained from Gibco® (Life Technologies, Carlsbad, CA, USA). L-Glutamine solution <NUM> and Penicillin/Streptomycin solution were purchased from Grisp (Porto, Portugal). Other reagents were of laboratory grade.

Cellulose acetate particles (CAp) were prepared by a water-on-polymer method, as disclosed in <NPL>). Specifically, <NUM> of cellulose acetate were dissolved in <NUM> of acetone, under magnetic stirring at <NUM> rpm. A volume of <NUM> of ultrapure water was then added at a flow rate of <NUM> min-<NUM> using a Harvard Apparatus PHD Ultra syringe pump (Holliston, Massachusetts, EUA), equipped with a syringe with a <NUM> needle gauge, under continuous stirring at <NUM> rpm. The resulting suspension of cellulose acetate particles was washed twice with ultrapure water and stored in the refrigerator.

Curcumin (CUR) loaded particles (CApCUR) were prepared using a similar method as described for the cellulose acetate particles. However, the process was performed in the dark given the photodegradability of CUR. Given so, <NUM> of cellulose acetate were dissolved in <NUM> of acetone containing <NUM> of CUR, under magnetic stirring at <NUM> rpm. <NUM> of distilled water were added at <NUM>/min, using a <NUM> needle, at <NUM> rpm. The resulting suspension of CApCUR was also washed with water twice, protected from light and stored in the refrigerator.

The incorporation of CUR (%) in the cellulose acetate particles was evaluated by ultraviolet-visible (UV-Vis) spectroscopy (Perkin-Elmer FT-IR System Spectrum BX spectrophotometer) based on the determination of the remaining CUR in solution, after centrifugation of CApCUR for <NUM> minutes at <NUM> rpm. Absorbance was measured at <NUM>, and the concentration of CUR was calculated using a calibration curve (y = <NUM>. 0687x + <NUM>, r2 = <NUM>).

In a preferable embodiment of this invention, the alginate-based inks contain <NUM>% (w/V) of alginate, and distinct amounts of CAp or CApCUR, namely <NUM>%, <NUM>% and <NUM>% (wt. %) relative to the mass of ALG (Table <NUM>). To achieve this, <NUM> of ALG and varying amounts of particles were mixed in <NUM> of ultrapure water, using moderate stirring overnight. These formulations were then pre-crosslinked by adding and <NUM> of a <NUM>% (w/V) CaCl2 solution. The obtained inks were left to stabilize overnight at <NUM>.

FTIR-ATR spectra of the cellulose acetate, CAp, CUR and CApCUR samples were obtained using a Perkin-Elmer FT-IR System Spectrum BX spectrophotometer (Perkin-Elmer Inc. , Waltham, MA, USA) equipped with a single horizontal Golden Gate ATR cell (Specac®, London, UK), at a range of <NUM>-<NUM>-<NUM> and a resolution of <NUM>-<NUM> over <NUM> scans.

SEM micrographs of the CAp and CApCUR samples and of the 3D printed constructs were obtained using a HR-FESEM SU-<NUM> Hitachi microscope (Hitachi High-Technologies Corporation, Tokyo, Japan) operating at <NUM> kV. The particle size determination was performed using ImageJ software to analyze at least <NUM> different particles on the SEM micrographs.

The cytotoxicity of CAp and CApCUR was assessed in human keratinocytes (HaCaT cell line) using the MTT assay, as it is disclosed in <NPL>). To achieve this, cells were cultivated in complete DMEM supplemented with <NUM>% FBS, <NUM> L-glutamine, <NUM> U mL-<NUM> penicillin/streptomycin and <NUM>µg mL-<NUM> fungizone at <NUM> in a <NUM>% CO<NUM> humidified atmosphere. Cells were observed daily using an inverted phase-contrast Eclipse TS100 microscope (Nikon, Tokyo, Japan). All the essays were performed in triplicate.

Suspensions of particles were sterilized by ultraviolet irradiation, and then incubated in DMEM at <NUM> with <NUM>% CO<NUM> for <NUM> hours. Different amounts of particles were used to obtain analogue concentrations to the seen in the inks' formulations. As negative controls, HaCaT cells were treated identically as described for the samples but exposed only to DMEM.

In the meantime, one <NUM>-well plate was prepared with <NUM>×<NUM> wells filled with <NUM> cells/well, and then incubated for <NUM> hours for adhesion. After that time, the cell culture medium in the wells was replaced by <NUM>µL of each sample, and cells were further incubated for <NUM> hours.

At the end of the incubation time, a <NUM>µL aliquot of MTT (<NUM> L-<NUM>) was added to each well and incubated for <NUM> hours at <NUM> in a <NUM>% CO<NUM> humidified atmosphere. After that, the culture medium was replaced with <NUM>µL of DMSO and the plate was placed in an orbital shaker for <NUM> hours in the dark. The absorbance of the samples was measured with a BioTek Synergy HT plate reader (Synergy HT Multi-Mode, BioTeK, Winooski, VT) at <NUM> with blank corrections. The cell viability was calculated with respect to the controls by the formula: <MAT> where Abs sample is the absorbance of the samples, Abs DMSO is the absorbance of DMSO and Abs control is the absorbance of the control.

The rheological properties of the inks and fully crosslinked hydrogels were evaluated by rotational and oscillatory tests, performed using a Kinexus Pro Rheometer (Malvern Instruments Limited, Malvern, United Kingdom) with a cone-plate geometry (cone angle of <NUM>° and diameter of <NUM>). Rotational tests were performed on the hydrogel inks using a measurement gap of <NUM> and a shear rate of <NUM>-<NUM>-<NUM>. The oscillatory tests used to determine the storage (G') and loss (G") moduli of the fully crosslinked hydrogels were performed at <NUM> frequency and a shear strain of <NUM>-<NUM>%, using cylinder-shaped (<NUM> diameter, <NUM> height) samples of the hydrogels crosslinked overnight in a <NUM>% (m/V) CaCl<NUM> solution.

The recovery rate of the inks was evaluated using a <NUM>-step oscillatory test: (i) measurement of G' in relaxation, at <NUM> Pa for <NUM>; (ii) measurement of the G' under stress, at <NUM> Pa for <NUM>; and (iii) a second measurement of the G' in a relaxation stage, at <NUM> Pa for <NUM>. The recovery (%) was calculated according to the equation: <MAT> where G' Initial is the average storage modulus in the initial relaxation phase, and G' Recovered corresponds to the storage modulus measured <NUM> after the shear stress reduced from <NUM> Pa back to <NUM> Pa.

All the rheological measurements were performed at <NUM>, using a water lock to prevent dehydration of the samples and a Peltier module for temperature control.

Mechanical compression tests were performed on at least five similar cylindrical samples of fully crosslinked hydrogels obtained from the ALG, ALG:CAp and ALG:CApCUR inks. The samples, with a diameter of <NUM> and height of <NUM>, were tested using a uniaxial Instron <NUM> machine (Instron Corporation, USA) in the compression mode, using a static load cell of <NUM> N at <NUM>. All tests were performed up to <NUM>% of strain, and the results obtained were calculated using the Bluehill <NUM> Software.

All printing essays were performed on a 3D-Bioplotter from EnvisionTEC (Germany). The printing parameters (pressure and printing speed) were first optimized by printing straight filaments with around <NUM> length using a <NUM> nozzle (inner diameter) at different speeds (<NUM>-<NUM>. s-<NUM>) and air pressures (<NUM> bar (<NUM> kPa) to <NUM> bar (200kPa)).

Then, the design of the 3D printed structures was previously developed using appropriate 3D-modelling software to model grid-like structures with dimensions of <NUM> × <NUM><NUM> and a spacing of <NUM> between filaments.

Structures with varying layers were 3D printed at <NUM> using the ALG, ALG:CAp and ALG:CApCUR inks, and fully crosslinked with an aqueous solution of <NUM>% (m/V) of CaCl<NUM>.

The release of the model bioactive compound (CUR) from the fully crosslinked hydrogels was evaluated by dipping them in <NUM> of PBS at <NUM>, under moderate agitation for <NUM> hours. To achieve this, hydrogels were obtained by using <NUM> of the ink formulation with higher curcumin content (ALG:CApCUR_10 ink). The essay was performed in triplicate. This ink was chosen given its higher content of CApCUR, which would result in higher CUR-release.

Aliquots of <NUM> were collected at selected time points, and the collected medium was replaced with fresh medium, preheated at <NUM>. The amount of CUR released into the media was measured by UV-Vis spectroscopy (using a Thermo Scientific Evolution UV-Vis <NUM>, Thermo Fisher Scientific) at <NUM>. The cumulative release was calculated using the formula: <MAT> where Cn and Cn-<NUM> are the concentrations of CUR in solution at times n and n-<NUM>.

The SEM micrographs of the obtained cellulose acetate particles (CAp) confirm that the used water-on-polymer method originates spherical, individualized, and smooth particles (<FIG>). The size measurements (using ImageJ software) revealed that these spherical cellulose derivative-based particles display an average diameter of <NUM> ± <NUM>.

Moreover, as observed in the SEM micrographs (<FIG>), the particles produced using a cellulose acetate solution containing curcumin (CApCUR) have similar morphological features when compared to the cellulose acetate counterparts, with sizes of <NUM> ± <NUM>. The CApCUR particles are equally spherical, smooth, and well dispersed and individualized. In sum, the incorporation of CUR in the process has no significant impact on the morphology and size distribution of the cellulose acetate particles.

The FTIR-ATR spectroscopic analysis (<FIG>) of the raw materials (cellulose acetate and curcumin) and of the obtained particles (CAp and CApCUR) confirms that the production process and the incorporation of curcumin does not affect the structure of this cellulose derivative, given the close resemblance of the spectra of the particles with the one of pristine cellulose acetate, with main vibrations at <NUM>-<NUM> (-OH stretching), <NUM>-<NUM> (symmetric C-H stretching), <NUM>-<NUM> (C=O stretching), <NUM>-<NUM> (C-H bending of the CH<NUM> in the acetyl group) and <NUM>-<NUM> (C-O stretching). In the spectrum of the CApCUR particles, the emergence of a peak at <NUM>-<NUM>, characteristic of CUR, is an indication of the successful incorporation of this compound in the cellulose acetate particles. Furthermore, the presence of curcumin in the cellulose acetate particles (CApCUR) is indeed confirmed by the vibrant yellow colour of the spheres under suspension (<FIG>). An incorporation rate of <NUM>% relative to the initial mass of curcumin added was determined by UV-Vis spectroscopy analysis of the curcumin content in the supernatant solution obtained after the fabrication step. This incorporation is similar to the results obtained for the incorporation of CUR in particles from other materials, and corroborates the potential of cellulose acetate for the production of particles incorporating CUR.

The determination of the cytotoxicity of the CAp and CApCUR was assessed against HaCaT cells using the MTT assay after <NUM> hours of exposure. This cell line was selected considering the potential applications of the developed inks for the 3D printing of constructs for topical/transdermal drug delivery or skin regeneration, for instance. As displayed in <FIG>, the CAp particles showed no significant cytotoxic effect, with cell viabilities of <NUM> ± <NUM>%, <NUM> ± <NUM>% and <NUM> ± <NUM>% for concentrations of <NUM>, <NUM> and <NUM>% particles, respectively, when compared with the control. Similarly, the CApCUR counterparts reveal no cytotoxic effect in the concentrations tested, as seen in <FIG>, with <NUM> ± <NUM>%, <NUM> ± <NUM>% and <NUM>% ± <NUM>% viabilities after <NUM> hours for <NUM>, <NUM> and <NUM>% concentrations. All these cell viabilities are higher than the threshold of <NUM>% cell viability, and therefore, the particles can be considered non-cytotoxic for HaCaT cells in these concentrations, as defined by the ISO <NUM>-<NUM>:<NUM> for the evaluation of the cytotoxicity of the materials.

The rheologic properties have a very important role on the extrusion printability of hydrogel-based inks. Given so, the behaviour of the hydrogels under stress was studied, to understand how these materials would perform along the 3D printing process.

First, the impact of the pre-crosslinking step on the characteristics of these hydrogel inks was assessed. The viscosity and shear stress (as a function of shear rate) were initially evaluated for the mixtures of alginate and CAp without pre-crosslinking. The results shown in <FIG>, reveal a slight increase on these parameters with the increasing content of cellulose acetate particles, suggesting an improvement of the rheological properties with the addition of these cellulose derivative-based spherical particles. However, these rheological properties are still not ideal for extrusion printing. Yet, the results confirm that all inks show a shear-thinning behaviour, viz. a decrease of shear viscosity with the increase of the shear rates applied.

Pre-crosslinked hydrogel inks were then evaluated for the same parameters. As shown in <FIG>, the pre-crosslinked inks reveal higher shear viscosities and shear stress, given the pre-crosslinking of the hydrogels, and a very clear shear-thinning behaviour. Additionally, there seems to be an impact of the presence of CAp in these parameters, with an increase on both shear viscosity and shear stress in ALG:CAp inks, when compared with the single ALG counterpart.

Afterwards, the recovery rate of the formulations was assessed in order to understand how the rheological properties of the inks is recovered after being subjected to the forces applied in the printing process. The data, summarized in <FIG>, shows that pristine alginate hydrogels show a recovery rate of <NUM> ± <NUM>%, and that the composite inks containing cellulose acetate particles have recovery rates of <NUM> ± <NUM>%, <NUM> ± <NUM>% and <NUM> ± <NUM>% for the ALG:CAp_1%, ALG:CAp_5% and ALG:CAp_10%, respectively. Therefore, all inks present very high recovery rates (above <NUM>%), and the recovery rate does not seem to be affected by the presence and different contents of CAp for the investigated range. Considering all these data, the viscosity, shear rate and recovery rate of all of these inks seems adequate for extrusion 3D printing applications.

The subsequent evaluation of the G' and G'' moduli of fully crosslinked hydrogels obtained from these hydrogel inks was performed to enlighten their viscoelasticity. The values obtained for the elastic modulus are significantly higher than the viscous counterpart (G'>G'') for all samples (<FIG>), indicating that all fully crosslinked hydrogels have a solid-like behaviour, as desired for integer and robust 3D printed constructs.

All the rheological properties assessed for the CAp particles were also evaluated for the CApCUR counterparts, aiming to understand how the incorporation of CUR could impact the characteristics of the inks. As may be seen in <FIG>, for the formulations without pre-crosslinking, the tendency observed above for the CAp inks was confirmed, with increasing amounts of CApCUR particles leading to higher shear viscosities and shear stress. The shear-thinning behaviour is also observable for these mixtures.

The effect of the pre-crosslinking step on the rheological characteristics of these mixtures is also very relevant (<FIG>) with the values of shear stress and shear viscosity at max shear rate increasing from around <NUM> Pa and <NUM> Pa. s to above <NUM> Pa and <NUM> Pa. s, respectively. Once more, the shear thinning behaviour is more evident from the pre-crosslinked hydrogels.

Additionally, the recovery rates are not affected by the incorporation of CApCUR in the hydrogels (<FIG>), with the ALG:CApCur_1% showing <NUM> ± <NUM>%, ALG:CApCUR_5% with <NUM> ± <NUM>% and ALG:CApCUR_10% with <NUM> ± <NUM>%. Again, the recovery rates of these inks are higher than <NUM>%, and very similar to those of the hydrogels obtained using cellulose acetate particles without curcumin.

The viscoelastic properties of the corresponding fully crosslinked hydrogels (<FIG>) also show an analogous solid-like behaviour, with all ink samples displaying G' values above the G''. Interestingly, the incorporation of CUR-loaded CA particles in these hydrogels is easily noticed by the substantial change in their colour (<FIG>), with the yellow colour increasing notoriously with the increasing concentrations of CApCUR.

Considering these data, the incorporation of curcumin in the cellulose acetate particles did not affect the rheological properties of the alginate hydrogel inks with or without pre-crosslinking, and therefore does not compromise the potential of the pre-crosslinked inks for 3D extrusion bioprinting.

The mechanical properties of fully crosslinked hydrogels obtained from ALG:CAp and ALG:CApCUR inks were assessed using compression assays. To understand how the presence of both types of particles (CAp and CApCUR) would impact these properties, a hydrogel composed exclusively of ALG was also tested. The results of these assays, indicate that that the Young's Modulus (<FIG>) of the hydrogels seems to increase with the content of particles. In fact, ALG hydrogels show values of <NUM> ± <NUM> MPa, while CAp containing hydrogels show values of <NUM> ± <NUM> MPa (CAp_1%), <NUM> ± <NUM> MPa (CAp_5%) and <NUM> ± <NUM> MPa (CAp_10%). A similar increase is seen for CApCUR composite hydrogels with <NUM> ± <NUM> MPa, <NUM> ± <NUM> MPa and <NUM> ± <NUM> MPa, for CApCUR concentrations of <NUM>%, <NUM>% and <NUM>% respectively.

The evaluation of the compressive stress of these hydrogels at <NUM>% strain also seems to show an increase with the presence (and increasing concentrations) of particles in the inks (<FIG>, with ALG showing the lowest value at <NUM> ± <NUM> MPa, and CAp / CApCUR samples revealing higher values and analogue results for the same concentrations. Specifically, CAp_1% and CApCUR_1% at <NUM> ± <NUM> MPa and <NUM> ± <NUM> MPa, respectively; CAp_5% and CApCUR_5% with <NUM> ± <NUM> MPa and <NUM> ± <NUM> MPa respectively; and finally, CAp_10% and CApCUR_10% with <NUM> ± <NUM> MPa and <NUM> ± <NUM> MPa.

These results confirm that the incorporation of CApCUR enhances the mechanical properties of the fully crosslinked alginate-based hydrogels.

The optimization of the extrusion printing conditions was performed initially by printing straight filaments of the inks. The obtained results show that the inks with CAp and CApCUR could be successfully printed in the same conditions: at <NUM>, using a printing speed of <NUM>. s-<NUM> and a pressure of <NUM> bar (<NUM> kPa), the process originated an integer and defined filament and dispensed moderate amounts of the inks, as illustrated for the CApCUR inks in <FIG>.

With this in mind, grid-like 3D structures with various numbers of layers were printed using ALG, ALG:CAp and ALG:CApCUR inks. The structures printed using ALG:CApCUR (<FIG>), show the lively yellow colour of CUR, with higher concentration of particles leading to a more notorious yellow tone, when compared with structures obtained exclusively from ALG or ALG:CAp. Additionally, all the 3D printed constructs with the composite hydrogel inks (ALG:CAp and ALG_CApCUR) demonstrated better resolution, with a higher definition of the grid-like structure, when compared with the structures obtained from the ALG ink. Moreover, the SEM micrographs of the 3D structures obtained from ALG (<FIG>) and ALG:CApCUR_10% (<FIG>) show that the structures obtained with the composite hydrogels possess spherical particles on the surface and embed in the matrix (which are absent in the ALG counterpart), confirming the presence of the CUR-loaded cellulose acetate particles in these structures, even after the extrusion 3D printing process.

Therefore, the use of ALG:CApCUR and ALG:CApCUR reinforced inks improves the outcome of the 3D printing procedure, and these inks may be used for the enhanced printing of detailed multi-layered 3D structures.

The release of curcumin from the fully crosslinked hydrogels was evaluated for a period of <NUM> hours through their emersion in PBS, at <NUM>. As depicted in <FIG>, after an initial burst, the cumulative release of CUR reaches a plateau after around <NUM> hours, achieving a final cumulative release of nearly <NUM>%.

These data confirm the possibility of using CApCUR to provide ALG hydrogels with the capability of releasing bioactive compounds with low water solubility to the media.

As used in this description, the expressions "composite hydrogel ink" or "composite hydrogel-based ink" refer to a "composition for additive manufacturing comprising a composite hydrogel".

As used in this description, the expression "3D printing" refers to a "process of additive manufacturing".

As used in this description, the pressure values refer to values above the atmospheric pressure.

As used in this description, the expressions "around" and "approximately" refer to an interval of values of more or less <NUM>% of the number specified.

Further, as used herein, the term "exemplary" is intended to mean serving as an illustration or example of something and is not intended to indicate a preference.

As used in this description, the expression "substantially" means that the real value is within the interval of around <NUM>% of the desired value, variable or related limit, particularly within around <NUM>% of the desired value, variable or related limit or specially within the <NUM>% of the desired value, variable or related limit.

As used in this description, the spherical cellulose or cellulose derivative-based particles comprise substantially <NUM>% of the polymer.

The subject matter described above is provided as an illustration of the present invention and, therefore, cannot be interpreted so as to limit it. The terminology used herein with the purpose of describing preferred embodiments of the present invention, must not be interpreted to limit the invention. As used in the specification, the definite and indefinite articles, in their singular form, aim at the interpretation of also including the plural forms, unless the context of the description indicates, explicitly, the contrary. It will be understood that the expressions "comprise" and "include", when used in this description, specify the presence of the characteristics, the elements, the components, the steps and the related operations, however, they do not exclude the possibility of other characteristics, elements, components, steps and operations also being contemplated.

Claim 1:
A composition for additive manufacturing comprising a composite hydrogel characterized by comprising:
a salt of alginic acid, referred to as alginate, in a concentration from <NUM> to <NUM>%, wherein the percentual corresponds to the mass of alginate in relation to the volume of said composition; and
spherical cellulose or cellulose derivative-based particles in a concentration from <NUM> to <NUM>%, wherein the percentual corresponds to the mass of said spherical cellulose or cellulose derivative-based particles in relation to the mass of said alginate; and
a cation of a pre-crosslinking salt in a concentration from <NUM> to <NUM>%, wherein the percentual corresponds to the mass of said cation in relation to the total volume of the composition;
wherein the cation of alginate is selected from at least one of the group consisting of an alkali metal or an alkaline-earth metal; and
wherein the spherical cellulose or cellulose derivative-based particles have a diameter from <NUM> to <NUM> micrometres; and
wherein the cation of a pre-crosslinking salt is selected from at least one of a divalent or a trivalent cation.