GELLAN GUM BASED INKS, METHOD OF OBTAINING AND USES THEREOF

TECHNICAL FIELD

[0001] The present disclosure relates to bio-instructive gellan gum-based inks, the method of preparation and uses thereof.

BACKGROUND

[0002] 3D bioprinting is a technology capable of building complex engineered 3D living tissues. 3D bioprinting uses a computer-aided design (CAD) to generate 3D models that are then reproduced into an object through layer-by-layer deposition. The object is printed using a biomaterial solution - the ink - or a biomaterial solution containing cells - the bioink. This technology allows the creation of engineered tissues with complexity, efficacy, speed, precision, automation, and reproducibility.

[0003] Extrusion-based printers are commonly used to bioprint 3D living tissues. Extrusion-based printers are composed of one/multiple printhead(s) holding a syringe where the ink/bioink is loaded. When a mechanical force or pneumatic pressure is applied, the ink/bioink is extruded through a needle coupled to the syringe, being dispensed as a fibre into a platform. In order to guarantee this extrusion through the needle without occluding it and causing shear stress to the encapsulated cells, while printing a fiber with shape-fidelity, the ink/bioink must have adequate rheological properties. This also determines the mechanical stability of the whole 3D structure postprinting.

[0004] Inks with cell adhesive features are beneficial as they provide specific sites for encapsulated cells to adhere, further supporting the proliferation, differentiation/maturation of the printed cells and the generation of a matured tissue. Thus, inks composed of biomaterials chemically functionalized with cell adhesive sites are of interest for the printing of cell-laden 3D structures aimed at acting as tissue-like analogues either as in vitro models or as substitutes for tissue regeneration.

[0005] Gellan gum (GG) has been explored as an ink owed to its viscoelastic properties. GG is an exocellular polysaccharide produced by aerobic submerged fermentation of Sphingomonas elodea. GG is a linear and anionic polymer composed of repeating units of a tetrasaccharide (1,3- -D-glucose, 1,4- -D-glucuronic acid, 1,4- -D-glucose, 1,4-a-L- rhamnose). GG is termed thermoreversible as it responds to temperature decrease with a sol-gel transition. In fact, GG has a thermally reversible coil form at high temperatures which, upon temperature decrease down to 37°C, changes to a double-helix form. Then, a structure composed of anti-parallel double helices is self-assembled, forming oriented bundles, called junction zones. The junction zones link untwined regions of extended helical chains, leading to the formation of a hydrogel. In addition, the use of counter ions, specifically monovalent or divalent cations, promotes a physical bonding between cations and carboxylate groups of the GG, particularly strong when involving divalent ions, leading to the formation of the 3D and crosslinked hydrogel. This allows the formation of GG hydrogels at mild and bio-friendly conditions. Gellan gum exists in two different forms, the high-acyl and the low-acyl forms. In high-acyl gellan gum, the acyl residues are located on the periphery of the helix, obstructing the polymer chain association, resulting in soft, elastic and non-brittle gels. In contrast, low-acyl gellan gum produces firm, non-elastic, brittle hydrogels since ions can easily link polymer chains and form a branched polymeric network.

[0006] Document EP2244753A2 discloses the processing and application of gellan gum for regenerative medicine and tissue engineering approaches, focusing on processes for packaging, handling, processing of different structures, controlled anionic crosslinking, as well as its combination with biomolecules and/or live cells in order to reduce the variability in its chemical and physical properties and in the generated biological results, increasing their effectiveness in the regeneration of living tissues in cellular tests before and/ or after implantation in animals and/or humans. This document does not make reference to the printing of gellan gum.

[0007] Document "Peptide modification of purified gellan gum" from Ferris et al. (2015) describes the modification of GG with a G4RGDSY peptide using the EDC/Sulfo-NHS chemistry reaction. The efficiency of conjugation was 40%. Rheological studies revealed that the peptide coupling did not change the gelation behavior. C2C12 cells showed improved attachment on the surface and within the RGD-GG hydrogels, but PC12 cells formed clusters within RGD-GG hydrogels. The modification of gellan gum and the linked peptide described in this document are different from the modification and from the linked peptide described in the present invention. Moreover, this document does not make reference to the printing of the modified gellan gum.

[0008] Document "The effects of peptide modified gellan gum and olfactory ensheathing glia cells on neural stem/progenitor cell fate" from Silva et al. (2012) describes the modification of GG with GRGDS-maleimide peptide using Diels-Alder click chemistry. Proton NMR demonstrated that the degree of furan substitution to GG was 27% and amino acid analysis demonstrated that approximately 300 nmol of maleimide- GRGDS was immobilized to each mg of GG. Neural stem/progenitors adhered, spread and proliferated within the functionalized GG hydrogels. The modification of gellan gum and the linked peptide described in this document are different from the modification and the linked peptide described in the present invention. Moreover, this document does not make reference to the printing of modified gellan gum.

[0009] Document "Gellan Gum Hydrogels with Enzyme-Sensitive Biodegradation and Endothelial Cell Biorecognition Sites" from da Silva et al. (2018) describes the chemical functionalization of gellan gum with divinyl sulfone groups (GGDVS). This allowed the covalent immobilization of thiol terminated peptides to the GG backbone and/or the crosslinking with a bis-thiol peptide crosslinker. GGDVS was post-functionalized with the CTTSWSQCSKS and CGGKAFDITYVRLKF peptides. Proton NMR demonstrated an efficiency of modification of GG to divinyl sulfone (DVS) of 50% and protein quantification analysis identified an efficiency of modification of GGDVS to CTTSWSQCSKS of 100% and to CGGKAFDITYVRLKF of 20%. GGDVS was also crosslinked with the dithiol peptide CRDGPQGIWGQDRC. The peptides linked to the modified gellan gum described in this document are different from the peptides described in the present invention. Moreover, this document does not make reference to the printing of modified gellan gum. [0010] Document CN106474560A discloses a kind of hydrogel material for 3D bioprinting and a preparation method and application of the hydrogel material. The ink comprises the following in percentages by mass: 0.5-10% of gellan gum and/or derivatives, 0.1-20% of polyethylene glycol (PEG) and/or derivatives thereof, 0-1% of a crosslinking initiator, 0-15% of biologically active components and the balance of a solvent. The printed hydrogel is a double-network hydrogel based on the gellan gum and PEG; interpenetrating double-network structures are formed in a physiological environment; and the printed hydrogel has good structure and dimensional stability, and has the advantages of quick gum formation under physiological conditions, excellent cell compatibility, small immunological rejection, high cell encapsulation efficiency, controllable mechanical strength, and biodegradability. This document describes a hydrogel material that contains PEG. The ink described in the present invention does not contain PEG.

[0011] Document WO2018/191244A1 discloses the composition for a polysaccharide hydrogel comprising: one or more water soluble high acyl gellan gum polymers; one or more water soluble low acyl gellan gum polymers; and one or more water soluble chemically modified gellan gum polymers or one or more peptide modified gellan gum polymers. One or more bioactive molecules are added to the gellan gum solution before heating, being then dispersed within the polymer chains but not chemically linked to the polymer, and are selected from the group consisting of: cells, peptides, functional peptide molecules with NH2, COOH and CONH2 group comprising: RGD, IKVAV, REDV, YIGSRY, poly Lysine. The polysaccharide hydrogel is used as a versatile platform for drug discovery and biomedical applications, comprising cell viability assay, live/dead assay, high-throughput screening, fluorescent staining and imaging, histological analysis, and 3D bio-printing. This document describes a polysaccharide hydrogel containing high acyl GG, low acyl GG and chemically modified GG, wherein the chemically modified GG results from the mixing (not chemical tethering) of the GG with a bioactive molecule. The present invention describes an ink containing any GG (low acyl or high acyl) wherein at least 0.1 % (m/v) of the gellan gum is chemically modified with divinyl sulfone and is chemically linked to any cysteine-containing peptide. [0012] Document "3D printing of layered brain-like structures using peptide modified gellan gum substrates" from Lozano et al. (2015) describes a method to bioprint 3D brain-like structures consisting of discrete layers of primary cortical neurons encapsulated in GG-RGD hydrogels. GG was chemically modified with GGGGRGDSY peptide using the EDC/Sulfo-NHS chemistry reaction. The efficiency of conjugation was not determined. Bioprinted primary cortical neurons in GG-RGD hydrogels showed a spread morphology with extended neurites. However, the ink takes a long time to be prepared (approximately 8 days). After the chemical modification, the material needs to be dissolved overnight at 60°C, prior printing. The modification of gellan gum and the linked peptide described in this document are different from the modification and the linked peptide described in the present invention.

[0013] Document KR20180035162A discloses a bio-ink for 3D bio-printing and its use, and a method for producing a bio-ink comprising a natural polymer with a mussel adhesive protein or a variant thereof and performing a cross-linking reaction. In the present invention, a mussel adhesive peptide, not a protein, is linked, not crosslinked, to a chemically modified GG, not a natural polymer.

[0014] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

GENERAL DESCRIPTION

[0015] The present disclosure relates to bio-instructive gellan gum-based inks, the method of preparation and uses thereof.

[0016] Bio-instructive gellan gum-based inks comprise a gelifying material and a bio- instructive material. Surprisingly, the combination of cells with the gelifying material and the bio-instructive material resulted in a stable bioink, with suitable properties for bioprinting (e.g. rheologic properties), and lead to an improved biological performance, as showed in Figures 9-13.

[0017] The present disclosure relates to a printable ink comprising 0.5 to 2.5 % (m/vink) of a gellan gum, comprising at least 0.1 % - 2.5 % (m/vink) of divinyl sulfone-modified gellan gum and 0 to 2.4 % (m/vink) of unmodified gellan gum; and 0.0002 to 20 M of a peptide sequence containing cysteine, wherein the peptide is chemically linked to the gellan gum via the divinyl sulfone moieties.

[0018] For the scope and interpretation of the present disclosure it is defined that the divinyl sulfone-modified gellan gum is a gellan gum chemically modified with divinyl sulfone groups. This divinyl sulfone-modified gellan gum can be produced as described in the begin of section 5 of the document "Gellan Gum Hydrogels with Enzyme-Sensitive Biodegradation and Endothelial Cell Biorecognition Sites" from da Silva et al. (2018).

[0019] An aspect of the present disclosure describes an ink comprising:

0.5 to 2.5 % (m/vink) of a gellan gum, wherein at least 0.1 % (m/vink) of the gellan gum is modified with divinyl sulfone; and a peptide sequence containing a cysteine, wherein the peptide is linked to the gellan gum via the divinyl sulfone moieties.

[0020] In an embodiment, the ink comprises 0.75 to 2 % (m/vink) of a gellan gum, wherein 0.25 to 2 % (m/vink) of divinyl sulfone-modified gellan gum; and a peptide sequence containing cysteine, wherein the peptide is linked to the gellan gum via the divinyl sulfone moieties.

[0021] In an embodiment, the ink comprises 1.5 % (m/vink) of a gellan gum, wherein 0.5 % (m/vink) of gellan gum is modified with divinyl sulfone; and a peptide sequence containing cysteine, wherein the peptide is linked to the gellan gum via the divinyl sulfone moieties.

[0022] In another embodiment, the ink comprises 0.75 % (m/vink) of a gellan gum, wherein 0.25% (m/vink) of gellan gum is modified with divinyl sulfone; and a peptide sequence containing cysteine, wherein the peptide is linked to the gellan gum via the divinyl sulfone moieties.

[0023] In another embodiment, the ink comprises 1.125 % (m/vink) of a gellan gum, wherein 0.375% (m/vink) of gellan gum is modified with divinyl sulfone; and a peptide sequence containing cysteine, wherein the peptide is linked to the gellan gum via the divinyl sulfone moieties. [0024] In another embodiment, the ink comprises 1.875 % (m/vink) of a gellan gum, wherein 0.625% (m/vink) of gellan gum is modified with divinyl sulfone; and a peptide sequence containing cysteine, wherein the peptide is linked to the gellan gum via the divinyl sulfone moieties.

[0025] In an embodiment, the ink comprises 1.5 % (m/vink) of a gellan gum, wherein 1.5% (m/vink) of gellan gum is modified with divinyl sulfone; and a peptide sequence containing cysteine, wherein the peptide is linked to the gellan gum via the divinyl sulfone moieties.

[0026] In an embodiment, the peptide sequence comprises at least a sequence 90% identical to the sequences of the following list: SEQ. ID No 1, SEQ. ID No 2, SEQ. ID No 3, SEQ. ID No 4, SEQ. ID No 5, SEQ ID No 6, SEQ. ID No 7, SEQ. ID No 8, SEQ. ID No 9, SEQ. ID No 10, SEQ. ID No 11, SEQ. ID No 12, SEQ. ID No 13, SEQ. ID No 14, SEQ. ID No 15, SEQ. ID No 16, SEQ. ID No 17 or mixtures thereof. Preferably 91% identical, 92% identical, 93% identical, 94% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical or identical.

[0027] In an embodiment, the peptide concentration ranges from 0.0002 to 20M, preferably 0.0007 to 15 M.

[0028] In another embodiment, the degree of modification of the divinyl sulfone- modified gellan gum ranges from 5 to 100%, preferably 25 to 100%.

[0029] In an embodiment, the gellan gum is a high acyl gellan gum, low acyl gellan gum or mixtures thereof.

[0030] In an embodiment, the concentration of unmodified gellan gum ranges from 0.4 to 2.4 % (m/vink), preferably from 0.5 to 1.25 % (m/vink).

[0031] In an embodiment, the concentration of the divinyl sulfone-modified gellan gum ranges from 0.1 to 2.5 % (m/vink), preferably from 0.25 to 1 % (m/vink), more preferably from 0.4 to 0.625 % (m/vink).

[0032] In another embodiment, the volume ratio between the unmodified gellan gum and the divinyl sulfone-modified gellan gum is 0.5:1 to 1:0.5, preferably 1:1. [0033] In an embodiment, the mass ratio between the unmodified gellan gum and the gellan gum-divinyl sulfone is 2:1 to 1:2, preferably 2:1.

[0034] In an embodiment, the ink further comprises an antiseptic agent, an antipyretic agent, an anaesthetic agent, a therapeutic agent, a cell, a growth factor, an antibody, an antibiotic, an anti-microbial agent, an anti-fungi agent, an antimycotic agent, an antiinflammatory agent, an enzyme, a metallic element, a growth hormone, a cytokine, an interleukin, a chemokine, an angiogenic factor, an anti-angiogenic factor, an anticoagulant, a contrasting agent, a chemotherapeutic agent, a signaling pathway molecule, a cell receptor, a cell ligand, or combinations thereof.

[0035] In a further embodiment, the cell is a non-human animal cell, a human cell, a stem cell, a cell line, a primary cell, a progenitor cell or mixtures thereof.

[0036] In an embodiment, the viscosity at 37°C ranges from 0.01 to 300000 Pa.s, preferably from 1.00 - 200 000 Pa.s, more preferably from 100 - 100 000 Pa.s.

[0037] In an embodiment, the shear storage modulus of the ink decreases by increasing the temperature. In a further embodiment, the shear storage modulus of the ink decreases by increasing the temperature above 30°C.

[0038] In an embodiment, the ink is converted into a hydrogel by decreasing temperature, upon contact with a cysteine containing peptide and/or upon contact with aqueous ionic solutions, preferably phosphate buffered saline, cell culture media, or mixtures thereof. In another embodiment, the ink is converted into a hydrogel by decreasing the temperature bellow 90°C, preferably bellow 40°C. In a yet further embodiment, the ink is converted into a hydrogel at a temperature ranging from 4°C to 90°C, preferably from 4°C to 50°C, more preferably 30°C to 50°C.

[0039] In an embodiment, the cysteine containing peptide is selected from the following list: SEQ. ID No 8, SEQ. ID No 9, SEQ. ID No 10, SEQ. ID No 11, SEQ. ID No 12, SEQ. ID No 13, SEQ. ID No 14, SEQ. ID No 15, SEQ. ID No 16, SEQ. ID No 17, or mixtures thereof.

[0040] In an embodiment, the ink is converted into a stable hydrogel by contact with aqueous ionic solutions, preferably phosphate buffered saline, cell culture media, or mixtures thereof. [0041] In an embodiment, the shear storage modulus after the conversion of the ink into a stable hydrogel ranges from 10 to 10000 Pa, preferably from 150 to 10000 Pa.

[0042] An aspect of the present disclosure comprises the use of the ink as a 3D printable ink and/or bioink.

[0043] The present disclosure also relates to an article comprising a stable hydrogel obtainable from the ink described in any of the previous embodiments, preferably an article obtained by 3D printing.

[0044] In an embodiment, the article is a hydrogel or a three-dimensional dried polymer matrix.

[0045] In an embodiment, the peptide sequence can be an artificial sequence.

[0046] In an embodiment, the article is for use in tissue engineering or regenerative medicine. In another embodiment, the ink is for use in skin repair or skin regeneration. In yet another embodiment, the ink is for use in the therapy or treatment of skin wounds.

[0047] In an aspect, the present disclosure also relates to a kit comprising the ink described in any of the previous embodiments. In an embodiment, the ink can be in a reservoir configured to be used in a 3D printer.

[0048] A method of preparing the ink is also described, comprising the following steps: (i) dissolving a divinyl sulfone-modified gellan gum in distilled water or a suitable buffer with pH above 8.2, preferably in distilled water containing 0.5M of sucrose; (ii) reacting the dissolved divinyl sulfone-modified gellan gum with a peptide sequence for 1 hour at 20-25°C, preferably 25°C; (iii) optionally, dissolving an unmodified gellan gum in a suitable buffer, preferably in distilled water, at 90°C under stirring for 15-60 minutes, preferably for 30 minutes, and mixing the dissolved gellan gum with the dissolved divinyl sulfone-modified gellan gum reacted with a peptide sequence, in a 1:1 (v:v) ratio, at a temperature between 4-50°C.

[0049] In an embodiment, the method further comprises the step of mixing the peptide- containing gellan gum mixture with a cell pellet or a solution containing cells, at a temperature between 4-45°C, preferably between 4 to 37°C, more preferably at 37°C. In a yet further embodiment, the cell pellet or the solution containing cells comprises non-human animal cells, human cells, stem cells, cell lines, primary cells, progenitor cells or mixtures thereof.

[0050] In an embodiment, the suitable buffer is selected from the following list: Triethanolamine, Tris-acetate-EDTA, Tris base, tricine, bicine, diglycine, 4-(2- Hydroxyethyl)-l-piperazinepropanesulfonic acid, 4-(2-Hydroxyethyl)piperazine-l- propanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N'-(3-propanesulfonic acid) (HEPES), N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, [(2-Hydroxy-l,l- bis(hydroxymethyl)ethyl)amino]-l-propanesulfonic acid (TAPS), 2-Amino-2-methyl-l,3- propanediol, Ammediol (AMPD), N-(l,l-Dimethyl-2-hydroxyethyl)-3-amino-2- hydroxypropanesulfonic acid (AMPSO), 2-(Cyclohexylamino)ethanesulfonic acid (CHES), 3-(Cyclohexylamino)-2-hydroxy-l-propanesulfonic acid (CAPSO).

[0051] In an embodiment, the bio-instructive gellan gum-based ink alone, or in combination with cells, can be used to print bio-instructive biomaterials or tissue-like substitutes for the repair/regeneration of tissue defects or/and of diseased tissues or/and organs, such as for the treatment of wounds.

[0052] In another embodiment, the bio-instructive gellan gum-based ink alone, or in combination with cells, can be used to print tissues or/and organ models for the in vitro study of diseases or/and drug screening.

BRI EF DESCRI PTION OF THE DRAWI NGS

[0053] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0054] Figure 1 shows the rheological behaviour of different GG/GGDVS, GG/GGDVS- SEQ. ID No 2 and GGDVS inks, which composition is described in Table 1 and Table 2. Representative shear rate ramps of a) GG/GGDVS, c) GG/GGDVS-SEQ. ID No 2 and e) GGDVS inks at 10 and 15 mg/mL are shown. The shear viscosity was monitored for shear rates between 0.1 and 100 s’ ¹ , at 37°C. Representative shear stress ramps of b) GG/GGDVS inks, d) GG/GGDVS-SEQ. ID No 2 and f) GGDVS inks are shown. [0055] Figure 2 shows the rheological behaviour of formulations a) A, b) B, c) C and d) D of GG/GGDVS inks, which composition is described in Table 1, at decreasing temperatures. The shear storage (G') and loss (G") moduli were recorded within a temperature ramp ranging from 60°C to 5°C (2°C min ^-1 ), with constant shear of 1.6 Hz and 0.5 % target shear strain.

[0056] Figure 3 shows the rheological behavior of GGDVS inks crosslinked with the SEQ. ID No 14 peptide, which composition is described in Table 8 and 9. Representative time sweeps of GGDVS inks are shown. The storage modulus was recorded in a time sweep at 37°C, with constant shear of 1.6 Hz and 0.5 % target shear strain. G" modulus values ranged between 1 and 5 Pa.

[0057] Figure 4 plots an embodiment of the force needed to extrude GG/GGDVS inks at 37°C using a a) 20-gauge and b) 27-gauge needle. Similar results were achieved with the peptides of the present disclosure.

[0058] Figure 5 shows an embodiment of fibres printed with GG/GGDVS inks in an extrusion-based printer. Similar results were achieved using inks containing the peptides of the present disclosure.

[0059] Figure 6 shows an embodiment of 3D discs printed with GG/GGDVS inks in an extrusion-based printer. Similar results were achieved using inks containing the peptides of the present disclosure.

[0060] Figure 7 shows the swelling ratio of GG/GGDVS, GG/GGDVS-SEQ. ID No 2, GG/GGDVS-SEQ. ID No 3, GG/GGDVS-SEQ. ID No 4, GG/GGDVS-SEQ. ID No 5 discs after immersion in a) phosphate buffered saline solution, b) calcium chloride, c) a-minimum essential medium (a-MEM), d) keratinocyte serum free medium (K-SFM), e) EndoGRO- IVIV medium, f) a-MEM for 48h at 37°C.

[0061] Figure 8 shows the mechanical properties of GG/GGDVS, GG/GGDVS-SEQ. ID No 2, GG/GGDVS-SEQ. ID No 3, GG/GGDVS-SEQ. ID No 4, GG/GGDVS-SEQ. ID No 5 discs measured after immersion in a) phosphate buffered saline solution, b) calcium chloride, c) a-minimum essential medium (a-MEM), d) keratinocyte serum free medium (K-SFM), e) EndoGRO-MV medium, f) a-MEM for 48h at 37°C. [0062] Figure 9 shows the phenotype of human dermal microvascular endothelial cells within different formulations of GG/GGDVS-SEQ. ID No 2 inks.

[0063] Figure 10 shows the morphology of human dermal fibroblasts within different formulations of GG/GGDVS-SEQ. ID No 2 inks.

[0064] Figure 11 shows the morphology of human dermal fibroblasts within GG/GGDVS- SEQ. ID No 2 ink (formulation C) in co-culture medium.

[0065] Figure 12 shows human adipose stem cells within GG/GGDVS-SEQ. ID No 1 inks.

[0066] Figure 13 shows human keratinocytes on GG/GGDVS-SEQ. ID No 3 inks.

DETAILED DESCRIPTION

[0067] The present disclosure relates to bio-instructive gellan gum-based inks, the method of preparation and uses thereof. Particularly, the present disclosure relates to the development of a bio-instructive gellan gum-based ink for 3D printing, preferably for 3D bioprinting.

[0068] The term "ink" refers to materials of synthetic or natural origin whose properties, particularly the rheological and mechanical properties, are suitable for 3D printing. When the ink is combined with cells, it is referred as "bioink".

[0069] The terminology "cells" refers to a variety of cells from human or animal origin, such as, but not limited to, cell lines, primary cells, progenitor cells, pluri and multipotent stem cells.

[0070] The terminology "biological performance" refers to the cellular activity that includes, but is not limited to, cell viability, proliferation, adhesion, spreading, differentiation, signaling, and tissue repair and regeneration including processes such as, but not limited to, inflammation, angiogenesis, extracellular matrix remodeling.

[0071] The terminology "biological moiety" refers to any molecule that has any beneficial or adverse effect on a cell, tissue or living organism. The molecules include, but are not limited to, an antiseptic agent, an antipyretic agent, an anesthetic agent, a therapeutic agent, growth factors, antibodies, antibiotics, anti-microbial, anti-fungi, antimycotic, anti-inflammatory factors, enzymes, metallic elements, growth hormones, cytokines, interleukins, chemokines, angiogenic factors, anti-angiogenic factors, anticoagulants, contrasting agents, chemotherapeutic agents, signaling pathway molecule, a cell receptor, cell ligand.

[0072] The terminology "viscosity" as used herein refers to the mechanical property of a polymeric solution that represents its resistance to flow or, in other terms, is the ratio between applied shear stress and deformation.

[0073] The terminology "crosslinking" refers to the process of forming bonds between polymeric chains. The crosslinking may be defined as covalent (covalent bonds between the chains) and physical (physical bonds between the chains). Covalent crosslinking can be activated by a chemical reagent or light (photocrosslinking). Physical crosslinking can be activated by a change in temperature (thermal crosslinking) or by the addition of ions (ionic crosslinking). The term "crosslinker" refers to the agent used to promote the crosslinking process. For the scope of the present disclosure, the crosslinking process results in the conversion of the ink into a hydrogel i.e. the ink is convertible into a hydrogel.

[0074] The term "3D printing" refers to the process of building a three-dimensional object from a computer-aided design (CAD) model. It involves the successive deposition of an ink, layer by layer, in an automated and reproducible manner. The terminology "3D bioprinting" or simply "bioprinting" refers to the process of 3D printing involving the successive deposition of a bioink, a particular form of ink.

[0075] The term "3D printer" refers to the equipment used for 3D printing, and the term "3D bioprinter" refers to the equipment used for 3D bioprinting. The 3D printer can also be used for 3D bioprinting. In the present disclosure, the term "3D printer" will be used to refer to "3D printer" and "3D bioprinter".

[0076] The term "extrusion-based" associated to "printing" or "bioprinting" refers a specific process of 3D printing/3D bioprinting that involves the deposition of continuous inks/bioinks that together can make up individual layers and layered constructs. [0077] The term "co-axial-based" associated to "printing" or "bioprinting" refers to a specific process of 3D printing/3D bioprinting that involves the separate dispensing of two or more inks/bioinks through different syringes, that are united in a final (co-axial) nozzle, leading to the extrusion of a single fiber.

[0078] In an embodiment, the bio-instructive gellan gum-based ink comprise a gelifying material and a bio-instructive material.

[0079] In an embodiment, the gelifying material is gellan gum as this material is able to crosslink and form hydrogels at mild conditions, at a temperature ranging from 4°C to 50°C, and with the addition of mono- or divalent cations (e.g. calcium, sodium, potassium).

[0080] In an embodiment, gellan gum is selected from low-acyl gellan gum, high acyl gellan gum, or a mixture of these.

[0081] In an embodiment, the concentration of gellan gum ranges from 0.4 to 2.4 % (m/vink), preferably from 0.5 to 1.25 % (m/vink).

[0082] In an embodiment, the bio-instructive material is a chemically-modified gellan gum linked to a biological moiety that instructs a biological performance.

[0083] In an embodiment, the biological moiety can be a peptide, a combination of peptide(s), a combination of peptide(s) and other components, a protein, a combination of proteins, or a combination of proteins and other components. Other components can be polymers, polysaccharides, glycosaminoglycans, or proteoglycans.

[0084] In an embodiment, the bio-instructive material comprises one or more biological moieties or mixtures thereof.

[0085] In the present disclosure, a chemically-modified gellan gum is used to allow the link of biological moieties. In a preferred embodiment, gellan gum is chemically modified with divinyl sulfone allowing the chemical binding of any cysteine-containing peptide. In other embodiments, gellan gum can be chemically modified with carbodiimides or periodate, that also allow the binding between GG and one or more peptides. Regardless the nature of the chemical modification, the chemically-modified gellan gum with the cysteine-containing peptide can induce a biological performance, and therefore be used as a bio-instructive material.

[0086] In an embodiment, the degree of modification of the divinyl sulfone-modified gellan gum ranges from 5 to 100%, preferably 25 to 100%.

[0087] In an embodiment, the peptide sequences are selected from proteins comprising extracellular matrix proteins, growth factors, enzymes, growth hormones, cytokines, antibodies, anti-inflammatory factors, angiogenic factors, anti-angiogenic factors, signalling pathway molecules, cell receptors and cell ligands.

[0088] In an embodiment, peptide sequences from extracellular matrix proteins such as SEQ. ID No 2 (peptide sequence derived from fibronectin protein), SEQ. ID No 1 (peptide sequence derived from fibronectin protein), SEQ. ID No 3 (peptide sequence derived type IV collagen protein), SEQ. ID No 4 (peptide sequence derived from fibronectin protein), SEQ. ID No 5 (peptide sequence derived from laminin 1-derived) are chemically bound to chemically modified gellan gum to promote cell adhesion. The composition of the peptide sequences is described in Table 2.

[0089] In a preferred embodiment, peptide sequences from extracellular matrix proteins like SEQ. ID No 6 (peptide sequence derived from the Mefp3 protein from mussels); SEQ. ID No 7 (peptide sequence derived from the Mefp5 protein from mussels) are chemically bound to chemically modified gellan gum to improve the adhesive properties of the material. The composition of the peptide sequences is described in Table 2.

[0090] In an embodiment, peptide sequences from extracellular matrix proteins like SEQ. ID No 13 (peptide sequence derived from the resilin protein), SEQ. ID No 14 (peptide sequence derived from the resilin protein), SEQ. ID No 15 (peptide sequence derived from the elastin protein), SEQ. ID No 16 (peptide sequence derived from the elastin protein), SEQ. ID No 17 (peptide sequence derived from the elastin protein) are chemically bound to chemically modified gellan gum for crosslinking and to improve the elastic properties of the material. The composition of the peptide sequences is described in Table 9. [0091] In an embodiment, peptide sequences comprising active sites for metalloproteinases degradation, like SEQ. ID No 8 (peptide sequence derived containing a MMP-1 cleavage site), SEQ. ID No 9 (peptide sequence derived containing a MMP-2 cleavage site), SEQ. ID No 10 (peptide sequence derived containing a MMP-13 cleavage site), SEQ. ID No ll(peptide sequence derived containing a MMP-14 cleavage site), SEQ. ID No 12 (peptide sequence derived containing an elastase cleavage site) are chemically bound to chemically-modified gellan gum to add biodegradability to the material. The composition of the peptide sequences is described in Table 9.

[0092] In an embodiment, peptide sequences containing at least two cysteines are used to crosslink the chemically-modified gellan gum. The crosslinker material allows the crosslinking of the gellan gum at mild conditions, forming a hydrogel.

[0093] In an embodiment, peptide sequences comprise at least a sequence 90% identical to the sequences of the following list: SEQ. ID No 1, SEQ. ID No 2, SEQ. ID No 3, SEQ. ID No 4, SEQ. ID No 5, SEQ ID No 6, SEQ. ID No 7, SEQ. ID No 8, SEQ. ID No 9, SEQ. ID No 10, SEQ. ID No 11, SEQ. ID No 12, SEQ. ID No 13, SEQ. ID No 14, SEQ. ID No 15, SEQ. ID No 16, SEQ. ID No 17 or mixtures thereof. Preferably 96% identical, 97% identical, 98% identical, 99% identical or identical. The composition of the peptide sequences is described in Table 2 and 9.

[0094] Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (over the whole the sequence) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul 10; 4:29. MatGAT is an application that generates similarity/identity matrices using protein or DNA sequences). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. The sequence identity values, which are indicated in the present subject matter as a percentage were determined over the entire amino acid sequence, using BLAST with the default parameters.

[0095] In an embodiment, the peptide concentration ranges from 0.0002 to 15 M, preferably 0.0007 to 10 M.

[0096] In an embodiment, the rheological properties of bio-instructive gellan gumbased inks are only slightly modified by the alteration of the cysteine-containing peptide. This allows the use of similar printing settings for the same ink formulation independently of the peptide used.

[0097] In an embodiment, the viscosity of the bio-instructive gellan gum-based ink at 37°C ranges from 0.01 to 300000 Pa.s.

[0098] In an embodiment, the shear storage modulus of the bio-instructive gellan gumbased ink decreases by increasing the temperature.

[0099] In an embodiment, the bio-instructive gellan gum-based ink is able to encapsulate and/or support cells, forming bioinks that can be used for bioprinting.

[00100] An aspect of the present disclosure describes an ink comprising:

0.5 to 2.5 % (m/vink) of a gellan gum, wherein at least 0.1 % (m/vink) of gellan gum is modified with divinyl sulfone; and a peptide sequence containing a cysteine, wherein the peptide is linked to the gellan gum via the divinyl sulfone moieties

[00101] A method of preparing the bio-instructive gellan gum-based ink is also described, comprising the following steps: (i) dissolving a divinyl sulfone-modified gellan gum in distilled water or a suitable buffer with pH above 8.2, preferably in distilled water containing 0.5M of sucrose; (ii) reacting the dissolved divinyl sulfone-modified gellan gum with a peptide sequence for 1 hour at 20-25°C, preferably 25°C; (iii) optionally, dissolving an unmodified gellan gum in a suitable buffer, preferably in distilled water, at 90°C under stirring for 15-60 minutes, preferably for 30 minutes, and mixing the dissolved gellan gum with the dissolved divinyl sulfone-modified gellan gum reacted with a peptide sequence, in a 1:1 (v:v) ratio, at a temperature between 4-50°C.

[00102] In an embodiment, the gelifying material and the bio-instructive material are mixed at physiological mild conditions (5-37°C) at a preferred volume:volume ratio of 1:1. In a further embodiment, the gelifying material and the bio-instructive material are mixed at concentrations that can vary from 0.5% to 2.5% (w/vink) at a preferred ratio of 2:1 (mass:mass) of gelifying material to bio-instructive material.

[00103] In an embodiment, the concentration of the divinyl sulfone-modified gellan gum ranges from 0.1 to 2.5 % (m/vink), preferably from 0.25 to 1 % (m/vink). In a further embodiment, the volume ratio between the unmodified gellan gum and the divinyl sulfone-modified gellan gum is 0.5:l-l:0.5, preferably 1:1. In a yet further embodiment, the mass ratio between the unmodified gellan gum and the gellan gum- divinyl sulfone is 2:1-1:2, preferably 2:1.

[00104] In an embodiment, the method further comprises the step of mixing the bio-instructive gellan gum-based ink with a cell pellet or a solution containing cells, at a temperature between 4-37°C. In a yet further embodiment, the cell pellet or the solution containing cells comprises non-human animal cells, human cells, stem cells, cell lines, primary cells, progenitor cells or mixtures thereof.

[00105] In an embodiment, the suitable buffer used in the preparation of the ink can be selected from the following list: Triethanolamine, Tris-acetate-EDTA, Tris base, tricine, bicine, diglycine, 4-(2-Hydroxyethyl)-l-piperazinepropanesulfonic acid, 4-(2- Hydroxyethyl)piperazine-l-propanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N'-(3- propanesulfonic acid) (HEPES), N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, [(2-Hydroxy-l,l-bis(hydroxymethyl)ethyl)amino]-l-propanesulf onic acid (TAPS), 2- Amino-2-methyl-l,3-propanediol, Ammediol (AMPD), N-(l,l-Dimethyl-2-hydroxyethyl)- 3-amino-2-hydroxypropanesulfonic acid (AMPSO), 2-(Cyclohexylamino)ethanesulfonic acid (CHES), 3-(Cyclohexylamino)-2-hydroxy-l-propanesulfonic acid (CAPSO). [00106] In an embodiment, the ink crosslinks into a hydrogel by decreasing temperature, upon contact with a peptide containing at least two cysteines, and/or upon contact with aqueous ionic solutions.

[00107] In an embodiment, the ink crosslinks into a hydrogel by decreasing the temperature bellow 90°C. In a yet further embodiment, the gelation temperature ranges from 4°C to 90°C, preferably from 4°C to 50°C.

[00108] In an embodiment, the ink crosslinks into a stable hydrogel by contact with aqueous ionic solutions, preferably phosphate buffered saline, cell culture media, or mixtures thereof.

[00109] In an embodiment, the ink crosslinks into a hydrogel upon contact with a peptide containing at least two cysteines, preferably a peptide selected from the following list: SEQ. ID No 8, SEQ. ID No 9, SEQ. ID No 10, SEQ. ID No 11, SEQ. ID No 12, SEQ. ID No 13, SEQ. ID No 14, SEQ. ID No 15, SEQ. ID No 16, SEQ. ID No 17, or mixtures thereof. The composition of the peptide sequences is described in Table 9.

[00110] In the present disclosure, a "stable hydrogel" is a hydrogel with defined structure, and with a shear storage modulus higher than the shear loss modulus.

[00111] In the present disclosure, the "gelation temperature", or "sol-gel temperature" is the temperature where the hydrogel crosslinking occurs, thus converting the ink into a stable hydrogel.

[00112] In an embodiment, after crosslinking the shear storage modulus ranges from 10 to 10000 Pa.

[00113] In an embodiment, the bio-instructive gellan gum-based ink is printable using a standard 3D printer.

[00114] In an embodiment, the bio-instructive gellan gum-based ink is printable using an extrusion-based 3D printer. In a further embodiment, the bio-instructive gellan gum-based ink is printable using a co-axial 3D printer.

[00115] In an embodiment, the bio-instructive gellan gum-based ink can be used to produce an article, preferably an article obtained by 3D printing. The article is a three- dimensional or two-dimensional polymeric network, in the dried or wet state, that can be a product for Tissue Engineering and Regenerative Medicine purposes.

[00116] In an embodiment, the bio-instructive gellan gum-based ink alone, or in combination with cells, can be used to print bio-instructive biomaterials or tissue-like substitutes for the repair/regeneration of tissue defects or/and of diseased tissues or/and organs, such as for the treatment of wounds.

[00117] In another embodiment, the bio-instructive gellan gum-based ink alone, or in combination with cells, can be used to print tissues or/and organs models for the in vitro study of diseases or/and drug screening.

[00118] The present disclosure also relates to a kit comprising the ink described in any of the embodiments of the present disclosure. In an embodiment, the ink can be in a reservoir configured to be used in a 3D printer.

Example 1:

[00119] As a first example, the method to prepare GG/GGDVS inks is described and their main properties are described.

[00120] GG/GGDVS inks are prepared by combining different amounts of GG and GGDVS functionalized with peptide(s) (Table 1 and 2).

[00121] GGDVS is prepared by reacting GG with divinyl sulfone (with an excess molar ratio of divinyl sulfone) for lh, under stirring, at room temperature (20-25°C), at pH 12. After reaction, the GGDVS is purified through precipitation in diethyl ether and dialysis against dHzO. Dried GGDVS is obtained by freeze-drying.

[00122] GGDVS is dissolved in dHzO containing 0.5M of sucrose (pH=9) at room temperature (20-25°C) under stirring for 20 min. The resulting material is then reacted with 800 pM of a peptide (Table 2) for lh at room temperature (20-25°C). GG is dissolved in dHzO at 90 C under stirring for 30 min. After this period, both solutions are mixed at 1:1 ratio (v:v) at a temperature between 4-40 'C to achieve the final concentrations described on Table 1. Table 1 - Examples of GG/GGDVS Ink Formulations

Table 2 - Examples of Peptide Sequences

[00123] In an embodiment, the amount of peptide conjugated to the GGDVS can be quantified by the micro bicinchoninic acid (micro BCA) protein assay. For this analysis, peptide standard dilutions are prepared in Milli-Q® H2O at different dilutions 200, 40, 20, 10, 5, 2.5 and 1 pg/ml and the GGDVS-peptide is prepared in Milli-Q® H2O at 1 mg/mL. Each sample is then reacted with a working reagent for 2h at 37°C, and the absorbance measured at 562 nm with a microplate reader (BioTek, USA). The percentage of conjugation efficiency is given by the ratio between the peptide amount calculated from the micro-BCA assay and the peptide amount used to react with GGDVS multiplied by 100. The amount of peptide conjugated to the GGDVS was quantified by the micro bicinchoninic acid (micro BCA) protein assay and is presented in Table 3. Table 3: Efficiency of peptide conjugation to GGDVS, determined by micro-BCA.

[00124] In an embodiment, the rheological properties of the GG/GGDVS ink can be determined using a Kinexus Pro from Malvern (serial number MAL1097376), coupled with a conical geometry. The rheological behaviour of the GG/GGDVS and GG/GGDVS- SEQ. ID No 2 inks was determined and is plotted in Figure 1. Shear viscosity was recorded along a range of 0.1-100 s ¹ of shear rate, at 37°C (Figure la and lc). Inks showed a nonNewtonian behaviour as the viscosity of the inks was dependent on the shear rate, as well as, a shear-thinning behaviour as the viscosity of the inks decreased with increasing shear rates. In addition, it is evident that the shear viscosity of inks increased with the amount of polymer existing in the ink formulation, i.e. increasing from the formulation A to D. These results validate the possibility of extruding inks at higher shear rates, where the viscosity is lower. No significant differences were observed between the behaviour of GG/GGDVS and GG/GGDVS-SEQ. ID No 2 inks. Shear viscosity was recorded along a range of 0.1-100 s ¹ of shear stress, at 37°C (Figure lb and Id). Whereas the viscosity of formulation A was constant for increasing shear stresses in the tested range of 0.1 - 100 Pa, the viscosity of formulations B, C and D was high at low shear stress and a breakdown of the inner structural skeleton and drop of viscosity occurred at increasing shear stress. In addition, it is evident that the shear viscosity increased with the amount of polymer existing in the ink formulation, i.e. increasing from the formulation A to D. These results validate the possibility of extruding inks at higher shear stresses, where the viscosity is lower. No significant differences were observed between the behaviour of GG/GGDVS and GG/GGDVS-SEQ. ID No 2 inks. [00125] Table 4 presents the average values of yield stress, flow consistency index and flow behaviour index of GG/GGDVS inks and GG/GGDVS-SEQ. ID No 2 inks. The Yield stress was calculated by the intersection of the curve fitted to the plateau region and the curve fitted to the steeply declining region of shear stress/viscosity curves. The shear stress at the point of intersection corresponds to the yield stress. The shear thinning coefficients (flow consistency index and flow behaviour index) were derived from power law regression of the shear-thinning curves, according to the following equation r| = Ky ^71-1 ; where q is viscosity, y is the shear rate, K is the flow consistency index and n is the flow behaviour index.

Table 4: Average values of yield stress, flow consistency index and flow behaviour index of GG/GGDVS inks and GG/GGDVS-SEQ. ID No 2 inks.

[00126] In an embodiment, the rheological behaviour of the GG/GGDVS inks was further assessed at decreasing temperatures (Figure 2). The shear storage (G') and shear loss (G") moduli were recorded within a temperature ramp from 60°C to 5°C (2°C min- ¹ ), with constant shear of 1.6 Hz and 0.5 % target shear strain (Figure 2). The sol-gel transition temperature was identified by the intersection of G' and G", temperature at which occurs the transition of the inks from the liquid (G' < G") to the solid (G' > G") state. A sol-gel transition was only detected for the formulation A and B, as intersection of G' and G" only occurred within the tested temperature range for these two formulations. For both formulations C and D, the storage modulus was higher than the loss modulus at all temperatures tested, indicating that the sol-gel transition occurs at temperatures above 60°C.

[00127] Table 5 presents the viscosity at 20°C and 37°C, the gelation temperature and the shear storage modulus of GG/GGDVS inks. The gelation temperature was calculated from the temperature ramps, at the intersection of G' and G". The shear storage modulus was calculated from the time sweeps, at the plateau when G'>G". Before each measurement, ink mechanics were standardized by a 5 min initial shearing at 100 s ¹ followed by 10 min of resting. The values are displayed as mean 1 standard deviation.

Table 5: Average values of viscosity at 20°C and 37°C, gelation temperature and shear storage modulus of GG/GGDVS inks.

[00128] In an embodiment, the force needed to extrude GG/GGDVS inks can be measured using a Legato® 100 syringe pump (KD Scientific, Holliston, USA). The force needed to extrude GG/GGDVS inks was measured at 37°C using either a 20-gauge or 27- gauge needle (Figure 4). Dispensable HSW SOFT-JECT® 5 ml single-use plastic luer-lock syringe (Henke-Sass, Wolf GmbH; Tuttlingen, Germany) was loaded with the GG/GGDVS- based ink, connected to precision dispensing tips gauge 20 (I D=0.58 mm) or 27 (I D=0.2 mm) (SmoothFlow Tapered Tips, Nordson EFD, USA) and mounted on the Legato® 100 syringe pump (KD Scientific, Holliston, USA). The flow rate (mL.min ^-1 ) was set according to the needle diameter. Force measurements were performed in triplicate during 1 min of extrusion and the mean force during that time interval was used for analysis. For the needle 20G, the force used to extrude was nearly 10 N, independently of the ink formulation (A, B, C or D) and independently of the printing speed (10 or 20 mmsec ^-1 ). For the needle 27G, the force used to extrude was dependent on the ink formulation, as it increased from 10 N (formulation A and B) to nearly 20-25 N (formulation C and D). No significant effects were detected at different printing speeds (10 or 20 mmsec ^-1 ).

[00129] In an embodiment, the GG/GGDVS inks can be printed using 3D printers. GG/GGDVS inks were printed using an extrusion-based printer. One dispensable HSW SOFT-JECT® 5 ml single-use plastic luer-lock syringe (Henke-Sass, Wolf GmbH; Tuttlingen, Germany) was loaded with the GG/GGDVS ink, connected to precision dispensing tips gauge 20 (ID=0.61 mm), 22 (ID=0.41 mm) and 27 (ID=0.2 mm) (SmoothFlow Tapered Tips, Nordson EFD, USA) and mounted on the extrusion-based printer. The extrusion velocity was set to 5 mm sec ^-1 . The temperature of the printer was settled at 20°C for inks A and B, and at 37°C for inks C and D. GG/GGDVS inks were sprayed with a CaC solution (100 mM) during printing for ionic crosslinking to occur and then immersed in cell culture medium for 24h for stabilization.

[00130] Figure 5 shows the GG/GGDVS fibres printed using an extrusion-based printer and the settings defined before. The thickness of the fibres was measured by ImageJ software at different points of the fibres and is displayed as mean 1 standard deviation on Table 6.

Table 6: Thickness of the printed GG/GGDVS fibres.

N/P - Not printable

[00131] Figure 6 shows the GG/GGDVS discs (10 mm of diameter; 5 mm of height) printed using an extrusion-based printer and the settings defined before. The dimensions of the discs were determined after swelling in cell culture medium for 24h and are described in Table 7. Table 7: Dimensions of the printed GG/GGDVS discs after 24h of swelling

[00132] In an embodiment, the swelling of hydrogel discs (5 mm diameter, 3 mm height) was determined by weighting the discs prior (Wi) and after (Wf) immersion in distinct solutions for 48 h at 37°C. The swelling of hydrogel discs was then calculated according to the following equation: Swelling = Wf/Wi. Results are depicted in Figure 7.

[00133] In an embodiment, the mechanical properties of hydrogel discs (5mm diameter, 3mm height) were measured using the Instron 5543 (Instron Int. Ltd., USA) mechanical testing device, after immersion in distinct solutions for 48h at 37°C. Hydrogel discs were placed on the base platform of the device, compressed at 1 mm.s ⁴ , and the ratio of compressive stress to strain was measured. The Young's Modulus was calculated in the loglO linear region (0 - 5 % compressive strain). Results are plotted in Figure 8.

Example 2:

[00134] As a second example, GG/GGDVS inks have been defined to be used for skin engineering. The human skin is composed by two main layers: the dermis and the epidermis. The dermis is mainly composed by fibroblasts and the epidermis composed of keratinocytes. One of the main proteins of the extracellular matrix existing in the dermis is the fibronectin and in the epidermis is the collagen IV.

[00135] A human skin equivalent can be engineered by using two GG/GGDVS inks that specifically represent the cells and extracellular matrix proteins of each layer: the dermis using human dermal fibroblasts loaded in GG/GGDVS-SEQ. ID No 2 (formulation C, from previous Table 1) inks (bioinks) and the epidermis using human keratinocytes on GG/GGDVS-SEQ. ID No 3 (formulation A, from previous Table 1) inks (bioinks). The peptide SEQ. ID No 2 encloses a RGD cell adhesive sequence existing in the fibronectin and the peptide SEQ. ID No 3 is a cell adhesive sequence existing in collagen IV. GG/GGDVS-SEQ. ID No 2 ink loaded with human dermal fibroblasts is used to print the dermis (bottom part) of the skin equivalent and the GG/GGDVS-SEQ. ID No 3 ink loaded with human keratinocytes is used to print the epidermis (top) of the equivalent. After printing, the engineered skin equivalents can be used as skin models for high- throughput screening or for skin wound repair/regeneration.

[00136] A dermis equivalent was obtained using human dermal fibroblasts loaded in GG/GGDVS-SEQ. ID No 2 (formulation C, from previous Table 1) inks (bioinks) after 21 days of culture in standard culture medium. Human dermal fibroblasts were isolated from the human skin of adult healthy donors who underwent abdominoplasties under standard protocols. 1 mL of GG/GGDVS-SEQ. ID No 2 ink was homogeneously and gently mixed with cell pellets containing 1 x 10 ⁶ cells at 37°C. Cell-laden hydrogels (according to Table 1) were formed after dispensing the bionk into the cell culture plate at 25°C. Cell-laden hydrogels were further cultured up to 21 days in a-MEM cell culture medium (Thermo Fisher Scientific, USA) supplemented with 10 % of fetal bovine serum (GibcoTM, Thermo Fisher Scientific, USA) and 10 % of antibiotics/antimycotics (Life Technologies Corporation, USA). Cell morphology (Figure 10) was visualized under a confocal microscope after F-actin staining with Phalloidin-TRITC (0.01 mg/mL, SIGMA, USA) and nuclei counter-staining with 4',6-diamidino-2-phenylindole (DAPI, 0.02 mg/mL). As showed, formulation C (Ink C) promoted better cell adhesion and spreading than formulations A, B and D (Ink A, B and D). The cells in the formulation C (Ink C) proliferated along the time.

[00137] A dermis equivalent was obtained using human dermal fibroblasts loaded in GG/GGDVS-SEQ. ID No 2 (formulation C, from previous Table 1) inks (bioinks) after 21 days of culture in endothelial cell culture medium. 1 mL of GG/GGDVS-SEQ. ID No 2 ink (formulation C, as stated in Table 1) was homogeneously and gently mixed with cell pellets of human dermal fibroblasts containing 1 x 10 ⁶ cells at 37°C. Hydrogels were formed after dispensing the cell-laden ink into the cell culture plate at 25°C. Cell-laden hydrogels were further cultured up to 14 days in 1:1 (v:v) mixture of a-MEM cell culture medium (Thermo Fisher Scientific, USA) supplemented with 10 % of fetal bovine serum (GibcoTM, Thermo Fisher Scientific, USA) and 10 % of antibiotics/antimycotics (Life Technologies Corporation, USA) and endothelial growth medium EGM-2 MV (Lonza, USA). Cell morphology (Figure 11) was visualized under a confocal microscope after F- actin staining with Phalloidin-TRITC (0.01 mg/mL, SIGMA, USA) and nuclei counterstaining with 4',6-diamidino-2-phenylindole (DAPI, 0.02 mg/mL). As showed, cells proliferated along the time.

[00138] An epidermis equivalent layer was obtained using human keratinocytes on GG/GGDVS-SEQ. ID No 3 (formulation A, from previousTable 1) inks (bioinks). Human keratinocytes were isolated from the human skin of adult healthy donors who underwent abdominoplasties under standard protocols. GG/GGDVS-SEQ. ID No 3 (formulation A, from previous Table 1) ink was homogeneously and gently mixed at 37°C. Hydrogels were formed after dispensing the ink into the cell culture plate at 25°C. Cells were added on top of hydrogels at a density of 20.000 cells/cm ² and the cell-loaded hydrogels were further cultured up to 7 days in Keratinocyte-serum-free medium (SFM) supplemented with 10 % of antibiotics/antimycotics (Life Technologies Corporation, USA). Cell morphology (Figure 13) was visualized under a confocal microscope after F- actin staining with Phalloidin-TRITC (0.01 mg/mL, SIGMA, USA) and nuclei counterstaining with 4',6-diamidino-2-phenylindole (DAPI, 0.02 mg/mL). As showed, cells adhered on the ink.

Example 3:

[00139] As a third example, GG/GGDVS inks are defined to be used for tissue repair and regeneration. Accomplishment of tissue regeneration greatly depends on a fast neovascularization post-implantation. Stem cells and endothelial cells present a great angiogenic potential owed to their angiogenic secretome. Endothelial cells have also the ability to pre-vascularize engineered tissue-like constructs which is an added potential as it can benefit inosculation with the host tissue.

[00140] In an embodiment, a pre-vascularized tissue-like construct can be engineered combining two bioinks: human dermal microvascular endothelial cells loaded in GG/GGDVS-SEQ. ID No 2 (formulation C, as described in Table 1) bioink and human adipose stem cells loaded in GG/GGDVS-SEQ. ID No 1 (formulation D, as described in Table 1) bioink. The ink GG/GGDVS-SEQ. ID No 2 (formulation C) loaded with human dermal microvascular endothelial cells can be printed in a tree-like network in between the GG/GGDVS-SEQ. ID No 1 (formulation D) ink loaded with human adipose stem cells. After printing, the engineered constructs can be transplanted to wounded tissues for repair or regeneration.

[00141] A tissue containing endothelial cells was obtained using human dermal microvascular endothelial cells loaded in GG/GGDVS-SEQ. ID No 2 (formulation C, as described in Table 1) bioink. Human dermal microvascular endothelial cells were isolated from the human skin of adult healthy donors who underwent abdominoplasties under standard protocols. 1 mL of GG/GGDVS-SEQ. ID No 2 ink (as described in Table 1) was homogeneously and gently mixed with cell pellet containing l x 10 ⁶ cells at 37°C. Cell-laden hydrogels were formed after dispensing the bioink into the cell culture plate at 25°C. Cell-laden hydrogels were further cultured up to 21 days in EndoGRO-MV-VEGF Complete Media (Millipore, U.S.A.) supplemented with 10 % of antibiotics/antimycotics (Life Technologies Corporation, USA). Cell morphology (Figure 9) was visualized under a confocal microscope after immunostaining with CD31 antibody (1:50, DAKO, Denmark) and nuclei counter-staining with 4',6-diamidino-2-phenylindole (DAPI, 0.02 mg/mL). As showed, formulation C and D (Ink C and D) promoted better cell adhesion and spreading than formulation A and B (Ink A and B).

[00142] A tissue containing stem cells was obtained using human adipose stem cells loaded in GG/GGDVS-SEQ. ID No 1 (formulation D, as described in Table 1) bioink. Human adipose stem cells were isolated from the human adipose tissue of adult healthy donors who underwent abdominoplasties under standard protocols. 1 mL of GG/GGDVS SEQ. ID No 1 (formulations A and D, as described in Table 1) was homogeneously and gently mixed with cell pellets containing 1 x 10 ⁶ cells at 37°C. Hydrogels were formed after dispensing the bioink into the cell culture plate at 25°C. Cell-laden hydrogels were further cultured up to 14 days in a-MEM cell culture medium (Thermo Fisher Scientific, USA) supplemented with 10% of fetal bovine serum (GibcoTM, Thermo Fisher Scientific, USA) and 10 % of antibiotics/antimycotics (Life Technologies Corporation, USA). Cell morphology (Figure 12) was visualized under a confocal microscope after F-actin staining with Phalloidin-TRITC (0.01 mg/mL, SIGMA, USA) and nuclei counter-staining with 4', 6- diamidino-2-phenylindole (DAPI, 0.02 mg/mL). As showed, formulation D (Ink D) promoted better cell adhesion and spreading than formulation A (Ink A). Example 4:

[00143] In an embodiment, GGDVS inks can be prepared by combining divinyl sulfone modified gellan gum (GGDVS) (component A) and a peptide crosslinker sequence (component B, Table 9) to attain hydrogels with different percentages of crosslinking densities (Table 8). A hydrogel is formed when both components A and B are mixed.

• Component A: GGDVS (70 % in volume) is dissolved in Triethanolamine buffer (0.2 M, pH=8.3) containing 0.25 IVI of sucrose, at 37°C under stirring for 10 min. A peptide sequence (10% in volume, Table 2) is linked to GGDVS by adding it to the GGDVS solution and let to react under stirring for lh at room temperature (20-25°C). In the present example the amount of peptide used was 0.8 M.

• Component B: the peptide crosslinker sequence (20 % in volume; Table 9) is dissolved in Triethanolamine buffer (0.2 M, pH=8.3) containing 0.25 IVI of sucrose at room temperature (20-25°C). Table 8 shows examples of GGDVS ink formulations, by altering the crosslinking density. To have a crosslinking density of 100%, the quantity of the peptide crosslinker sequence (mol) used is half of the moles of GGDVS. The other crosslinking densities are calculated from the latest, i.e. the quantity of the peptide crosslinker to crosslink 50% of GGDVS crosslinker is half of the quantity needed of the peptide crosslinker to crosslink 100% of GGDVS.

Table 8 - Examples of GGDVS Ink Formulations

Table 9 - Examples of Peptide Crosslinker Sequences

[00144] In an embodiment, the rheological properties of GGDVS inks can be determined using a Kinexus Pro from Malvern (serial number MAL1097376), coupled with a conical geometry. The alteration of the behaviour of the ink from liquid to solid was followed using rheological measurements (Figure 3), using formulations e, f and g (table 8) crosslinked with SEQ ID No 14. The shear storage (G') and loss (G") moduli were recorded for 800 secs at 37°C, with constant shear of 1.6 Hz and 0.5 % target shear strain. G" modulus values ranged between 1 and 5 Pa (not visible in the graphic). The sol-gel transition occurred immediately after mixing component A and B, and hence it is not evident in the graphics. The shear storage modulus increased along the time, indicating that the process is still occurring, until it reaches a plateau, indicating that the process of crosslinking finished. It is evident that formulations with higher crosslinking density (from e to g) present higher shear storage modulus.

[00145] The rheological behaviour of GGDVS inks (component A) was further analysed to understand the ability of the ink to be extruded by a printer (Figure le and If). Independently of GGDVS concentration (10 or 15 mg/mL), the shear viscosity was low validating its extrusion.

[00146] GGDVS inks can be printed using an extrusion co-axial printer. Two dispensable HSW SOFT-JECT® 5 ml single-use plastic syringes (Henke-Sass, Wolf GmbH; Tuttlingen, Germany) were loaded with the GGDVS ink components: A) one syringe is loaded with the GGDVS solution (5 to 15 mg/ml) with/without cells and B) another syringe is loaded with the peptide crosslinker solution, according to the crosslinking densities described on Table 8. Both syringes are connected to precision dispensing tips gauge 20 (I D= 0.58 mm) or 27 (ID=0.2 mm) (SmoothFlow Tapered Tips, Nordson EFD, USA) and mounted on an extrusion-based printer with a co-axial system. The mixer is settled to print 1 part of the component A per 4 parts of the component B. The extrusion velocity is set to 5 mm/sec and the temperature of the printer is settled at 37°C.

Example 5:

[00147] As a fifth example, GGDVS inks have been defined to be used for printing hydrogels for tissue repair and regeneration. The delivery of growth factors and cytokines is recognized as an appealing strategy in tissue repair and regeneration. A biomaterial containing chemical mediators can be engineered to present 1) different gradients of degradation or 2) different degradation specificity, in order that the encapsulated chemical mediator is released in pair with the biomaterial degradation.

[00148] In an embodiment, constructs with different gradients of biodegradation are formed by combining a bioactive molecule of interest (such as epidermal growth factor), the GGDVS ink (1%, m/vink) and the peptide SEQ. ID No 10 as crosslinker sensitive to MMP-13 cleavage. To create a degradation gradient within the construct, the amount of the crosslinker is altered. GGDVS ink (formulation e from previous Table 7) is used to print the first layer, GGDVS ink (formulation f, from previous Table 7) is used to print the second layer, GGDVS ink (formulation g, from previous Table 7 is used to print the third layer), GGDVS ink (formulation h, from previous Table 7 is used to print the fourth layer, in order to have a construct with different levels of degradation by metalloproteinase (Formulation e > Formulation h; Lower degradation > Higher degradation).

[00149] In an embodiment, constructs with different biodegradation sensitivities are formed by combining the bioactive agent of interest, the GGDVS ink (1%, m/vink) and the peptide SEQ. ID No 10 as crosslinker sensitive to MMP-13 cleavage and the peptide SEQ. ID No 9 as crosslinker sensitive to MMP-2 cleavage. To create a construct with two compartments sensitive to two different metalloproteinases, the type of crosslinker is altered. GGDVS ink (formulation g, from previous Table 7) and the crosslinker sensitive to MIVIP-13 cleavage is used to print the first two layers, GGDVS ink (formulation g, from previous Table 7) and the crosslinker sensitive to MMP-2 cleavage is used to print the second two layers, in order to have a construct with different specificities of degradation by metalloproteinase.

[00150] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[00151] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[00152] The above described embodiments are combinable.