Bioink

Abstract

The present invention relates to a hydrogel precursor composition and a bioink obtainable from the hydrogel precursor composition, and to a method for preparing and using the same. More specifically, the present invention relates to a bioink containing a cell-containing catechol-grafted spontaneously crosslinked hydrogel, and to its use in 3D bioprinting, and to a method for preparing the same.

Description

[Technical field]

[0001] The present invention relates to bioinks and methods of preparing and using the bioinks. More specifically, the present invention relates to bioinks containing crosslinked hydrogels obtainable from hydrogel precursor compositions and materials of biological origin, such as cells. The present invention also relates to the use of the hydrogels or bioinks in 3D printing and methods of 3D printing the same. [Background technology]

[0002] 3D bioprinting is a powerful technique for the generation of tissue and organ-like structures, offering great potential for the design of artificial tissues. Bioprinting is a form of additive manufacturing, where 3D biological structures are generated layer by layer using computer-aided design. 3D bioprinting allows for precise spatial positioning of cells into patient-specific constructs or implants that mimic the complex and irregular geometry of natural tissues. Despite the potential of this emerging technology, technical challenges remain that limit its more widespread use in tissue engineering and regenerative medicine.

[0003] Bioinks are materials that generally contain single or multi-component matrix components with viable cells or growth factors, and are loaded into 3D bioprinters to produce natural tissue-like constructs. For a bioink to perform well in 3D bioprinting, several requirements are set. The bioink needs to be printable and retain its 3D shape after printing. These properties need to be combined with structural stability, biocompatibility and effective biological performance. Current bioinks are not able to optimize all of these aspects in a single bioink.

[0004] Bioinks can be fabricated from a variety of materials to cater to the specific requirements for each application. Hydrogel compositions that have the ability to be extruded through small-sized nozzles and subsequently form a form-stable gel are considered suitable substrates for bioinks, which can incorporate viable cells. Although there has been progress in the development of hydrogels and bioinks, the incorporation of tissue-adhesive components into hydrogels and bioinks has been somewhat delayed. Catechol has been investigated in tissue engineering applications to increase the tissue-adhesive properties of hydrogels, and to date, many tissue-adhesive hydrogels based on catechol chemistry have been introduced. However, these hydrogels are mostly formed by either autoxidation, self-polymerization, or metal coordination polymerization, and these formation routes may sacrifice cell function and are therefore not biocompatible with cells. In addition, many bioinks require photoinitiators and photocrosslinking for structural stability of the hydrogel substrate, which may act as a significant limitation to the biological functionality of the bioink.

[0005] Click chemistry-based hydrogels are configured to form by spontaneous crosslinking of components after mixing reactive reagents. Such hydrogels have been shown to be capable of encapsulating viable cells with high cell viability. In general, click chemistry reactions are rapid, spontaneous, versatile, and highly selective, and can result in high yields of products when two molecular substances or components are combined. In this regard, crosslinked hydrogels based on hyaluronic acid (HA) have been investigated, and crosslinking of these hydrogels occurs rapidly, resulting in a relatively narrow biofabrication window. For example, Koivusalo et al. 2019 discloses hydrazone crosslinked hydrogels based on HA that have gelation speed and viscosity unsuitable for 3D printing.

[0006] Despite the existence of bioink compositions with tissue adhesive properties, there remains a need for novel bioinks with improved tissue adhesive properties that do not rely on self-polymerization, oxidation, or metal-coordination polymerization of the tissue adhesive components. There is also a need for novel bioink compositions that provide adequate crosslinking rates and therefore optimal biofabrication windows for 3D printing. There is also a need for novel bioink compositions that enable cell proliferation and tissue differentiation and maturation towards more specialized cell phenotypes. Additionally, there is a need for bioinks that do not use photoinitiators and photocrosslinking for improved printability and structural stability. Summary of the Invention

[0007] This application relates to the inventions defined in the attached independent claims and to the embodiments thereof disclosed below. The attached claims define the scope of protection. Any methods, processes, products, or apparatus disclosed in the detailed description or drawings that are not covered by the claims are not embodiments of the claimed invention but are provided as examples useful for understanding the claimed invention.

[0008] Described herein are hydrogel precursor compositions that can be utilized in the preparation of hydrogels and bioinks that can be used in additive manufacturing, such as 3D and / or 4D printing.

[0009] The inventors have surprisingly found, and demonstrate in the Examples below, that crosslinked hydrogels having certain compositions are suitable for additive manufacturing and bioprinting when one or more rheology modifiers are incorporated into the hydrogel composition.

[0010] According to a first aspect, i a. at least one first reactive functional group; and b. at least one catechol and / or phenol group a first component comprising a hyaluronic acid (HA) component conjugated to ii. a second component comprising a hyaluronic acid (HA) component conjugated to at least one second reactive functional group; iii. at least one rheology modifier component; and A hydrogel precursor component comprising: A hydrogel precursor composition is provided in which at least one second reactive functional group is configured to form crosslinks with at least one first reactive functional group.

[0011] According to a second aspect, there is provided a bio-ink obtainable by mixing the hydrogel precursor composition of the first aspect with a cell-containing fluid.

[0012] According to a third aspect, there is provided a use of the hydrogel precursor composition of the first aspect or the bio-ink of the second aspect for additive manufacturing such as 3D printing.

[0013] According to a fourth aspect, there is provided a method for 3D printing a bio-ink comprising: a) mixing a hydrogel precursor composition of the first embodiment with a fluid containing cells; b) crosslinking in the fluid a first component of the hydrogel precursor composition and a second component of the hydrogel precursor composition; c) 3D printing the bioink, where the viscosity of the bioink is between 200 and 2000 Pa·s; and A method is provided, comprising:

[0014] According to a further aspect, there is provided a hydrogel precursor composition comprising: i) a first component comprising a hyaluronic acid (HA) component conjugated to at least one first reactive functional group and at least one catechol and / or phenol group; ii) a second component comprising a hyaluronic acid (HA) component conjugated to at least one second reactive functional group; and iii) at least one rheology modifier component having a molecular weight of 1200 kDa to 1900 kDa, wherein the at least one second reactive functional group is configured to form crosslinks with the at least one first reactive functional group. According to a further aspect, there is provided a hydrogel precursor composition comprising: i) a first component comprising an HA component conjugated to at least one carbohydrazide group (-CONHNH2) and at least one catechol group; ii) a second component comprising an HA component conjugated to at least one aldehyde group (-CHO); and iii) at least one rheology modifier component having a molecular weight of 1200 kDa to 1900 kDa. According to a further aspect, there is provided a hydrogel precursor composition comprising: i) a first component comprising an HA component conjugated to at least one carbohydrazide group (-CONHNH2) and at least one dopamine group (DA); ii) a second component comprising an HA component conjugated to at least one aldehyde group (-CHO); and iii) at least one rheology modifier component comprising HA having a molecular weight of 1200 kDa to 1900 kDa. According to a further aspect, there is provided a hydrogel precursor composition comprising: i) a first component comprising an HA component conjugated to at least one semicarbazide and at least one catechol group; ii) a second component comprising an HA component conjugated to at least one ketone; and iii) at least one rheology modifier component having a molecular weight of 1200 kDa to 1900 kDa.According to a further aspect, there is provided a hydrogel precursor composition comprising: i) a first component comprising an HA component conjugated to at least one semicarbazide and at least one catechol group; ii) a second component comprising an HA component conjugated to at least one aldehyde; and iii) at least one rheology modifier component having a molecular weight of 1200 kDa to 1900 kDa. According to a further aspect, there is provided a hydrogel precursor composition comprising: i) a first component comprising an HA component conjugated to at least one aminooxy and at least one catechol group; ii) a second component comprising an HA component conjugated to at least one ketone; and iii) at least one rheology modifier component having a molecular weight of 1200 kDa to 1900 kDa. According to a further aspect, a hydrogel precursor composition is provided, comprising: i) a first component comprising an HA component conjugated to at least one aminooxy and at least one catechol group; ii) a second component comprising an HA component conjugated to at least one aldehyde; and iii) at least one rheology modifier component having a molecular weight between 1200 kDa and 1900 kDa. According to a further aspect, a hydrogel composition is provided that may be obtained by mixing the hydrogel precursor composition of the first aspect with a fluid. According to a further aspect, a hydrogel composition is provided that may be obtained by spontaneous crosslinking of the components of the hydrogel precursor composition of the first aspect mixed with a fluid. According to a further aspect, a hydrogel composition is provided that may be obtained by spontaneous hydrazone crosslinking of the first and second components of the hydrogel precursor composition of the first aspect when the hydrogel precursor composition is mixed with a fluid. According to a further aspect, a hydrogel composition is provided that may be obtained by mixing the hydrogel precursor composition of the first aspect with a fluid, the hydrogel composition being usable in additive manufacturing.According to a further aspect, there is provided a method for 3D printing a hydrogel composition, the method comprising: a) mixing a hydrogel precursor composition with a fluid; b) crosslinking a first component of the hydrogel precursor composition and a second component of the hydrogel precursor composition in the fluid; and c) 3D printing the hydrogel composition, where the viscosity of the hydrogel composition is between 200 and 2000 Pa·s.

[0015] In one embodiment, the present invention relates to a hydrogel precursor composition for obtaining a printable hydrogel composition. In one embodiment, the present invention relates to a hydrogel composition for 3D printing. In one embodiment, the present invention relates to a bio-ink composition for 3D bioprinting, comprising a hydrogel composition mixed with cells. In one embodiment, the present invention relates to a method of 3D printing.

[0016] The hydrogel precursor compositions of the present invention are advantageous in that they allow for suitable crosslinking rates between the components of the hydrogel composition, allowing for suitable manufacturing times for crosslinked hydrogels to be used in 3D printing.The hydrogel precursor compositions of the present invention are advantageous in that they have high modularity due to the modular linker moieties and reactive functional groups, as well as modularity in the consistency of the rheology modifier components.

[0017] The hydrogel compositions of the present invention that can be obtained from the hydrogel precursor compositions of the present invention are advantageous in that they have improved performance in printability and shape retention after printing. The hydrogel compositions of the present invention that can be obtained from the hydrogel precursor compositions of the present invention are further advantageous in that they have improved shear thinning and viscoelastic properties, allowing 3D printing of the hydrogel. The hydrogel compositions of the present invention that can be obtained from the hydrogel precursor compositions of the present invention are advantageous in that they have an optimized crosslinking rate, allowing the hydrogel to be used in 3D printing. The hydrogel compositions of the present invention are advantageous because the spontaneous biorthogonal crosslinking of the hydrogel compositions is easy to control and therefore different from ionically or photochemically crosslinked hydrogels.

[0018] The bioink of the present invention is advantageous in that it has high biocompatibility with the cells mixed therein, allowing the proliferation of cells, and the differentiation and maturation of cells into more specialized cell phenotypes and mature tissues. The bioink of the present invention is advantageous in that it contains tissue adhesive components that help the printed structure to integrate into the host tissue upon implantation. The bioink of the present invention is also advantageous in that it has improved shear thinning and viscoelastic properties, and the crosslinking rate is optimized, allowing the shape to be retained after printing, allowing the bioink to be used in 3D printing. The bioink of the present invention is also advantageous in that it can be obtained through a crosslinking reaction of the hydrogel precursor composition components that is not harmful to cells. The bioink of the present invention is also advantageous in that it contains only components that are biodegradable in vivo.

[0019] The use of the hydrogel precursor composition of the first aspect of the invention or the bioink of the second aspect of the invention is advantageous in that it allows for use in many applications and many variations of use. For example, the hydrogel precursor composition or bioink can be used with a variety of printing devices or with a wide range of 3D printing / bioprinting techniques. The use of the hydrogel precursor composition or bioink of the invention is advantageous in that it is configured to be used as an in situ injectable for the repair and regeneration of damaged tissue.

[0020] The method for 3D printing of the hydrogel composition of the present invention is advantageous in that it has a high modularity in terms of process conditions.The method for 3D printing of the hydrogel composition of the present invention is advantageous in that it is configured such that the formation of crosslinks between the first and second components of the hydrogel precursor composition is spontaneous, and therefore is not based on ionic crosslinking, nor does it require specific crosslinking inducers such as specific temperatures or wavelengths of light.

[0021] As shown in the examples provided below, the claimed hydrogel precursor compositions, the resulting printable hydrogel compositions, and bioinks obtained using the hydrogel precursor compositions have improved performance when used in 3D printing as compared to products known in the art.

[0022] Illustrative and non-limiting embodiments of the present invention, both as to structure and method of operation, together with additional objects and advantages thereof, will best be understood from the following description of specific exemplary embodiments when read in connection with the accompanying drawings. [Brief description of the drawings]

[0023] Several illustrative embodiments will now be described with reference to the accompanying drawings. [Figure 1] 1 shows a schematic diagram of the fabrication of an exemplary hydrogel or bioink according to an exemplary embodiment. [Diagram 2] 1 shows the viscosity under continuous flow A) and cyclic flow B) illustrating the shear thinning behavior of a hydrogel composition according to an exemplary embodiment. [Diagram 3] 1 shows strain recovery of a hydrogel composition shown as storage modulus and loss modulus, according to an exemplary embodiment. [Figure 4] 1 shows exemplary 3D printed hydrogel lattices fabricated using hydrogel precursor compositions according to exemplary embodiments A) without any rheology modifiers, B) with sodium hyaluronate and human type I collagen as rheology modifier components according to the present invention, and C) according to the disclosure of Koivusalo et al. 2019. Scale bar 10 mm. [Diagram 5] 1A-1C show exemplary 3D printed hydrogel lattices produced using hydrogel precursor compositions comprising A) 2.92 mg / ml, B) 8.16 mg / ml, and C) 11.66 mg / ml of crosslinking component, i.e., the first and second components of the hydrogel precursor composition, based on final hydrogel composition volumes according to exemplary embodiments. [Figure 6]1A-1C show exemplary 3D printed hydrogel lattices fabricated using hydrogel precursor compositions including type I collagen with hyaluronic acid (HA) as a rheology modifier component at A) 0.18 mg / ml, B) 0.56 mg / ml, and C) 1.09 mg / ml of final hydrogel composition volume, according to exemplary embodiments. [Figure 7] 1A-1C show exemplary 3D printed hydrogel lattices fabricated using hydrogel precursor compositions including HA as a rheology modifier component at A) 0.625 mg / ml, B) 1.25 mg / ml, and C) 3.125 mg / ml of final hydrogel composition volume, according to exemplary embodiments. [Figure 8] 1 shows exemplary 3D printed hydrogel lattices fabricated using hydrogel precursor compositions including A) laminin, B) albumin, or C) fibronectin with HA as a rheology modifier component, according to exemplary embodiments. [Figure 9] 1A) and 1B) show perspective views of exemplary printed 3D hydrogel structures fabricated using hydrogel precursor compositions according to exemplary embodiments. [Figure 10] 1 shows A) the relative filament thickness of an exemplary 3D printed hydrogel lattice as a function of time (D0 indicates the relative filament thickness on day 0 and D7 indicates the relative filament thickness on day 7) and images of the printed hydrogel lattice after B) day 0 and C) 7 days, according to an exemplary embodiment. [Figure 11] 14A-14D show A) 3D printed lines and B) cell viability of hASC and hASC-derived corneal stromal-like cells in exemplary hydrogels at different time points measured as emitted fluorescence indicating cell viability in the 3D printed constructs, as well as phalloidin staining of C) hASC and D) hASC-derived corneal stromal-like cells 7 days after printing in 3D printed lines according to exemplary embodiments. [Figure 12] Figure 1 shows the viscosity after 10 minutes of crosslinking for a bioink with high molecular weight HA and Col I as a rheology modifier (base ink), the same ink without Col I, and a bioink with low Mw HA as a rheology modifier. [Figure 13] Figure 1 shows the viscosity after 1 hour of crosslinking for a bioink with high molecular weight HA and Col I as a rheology modifier (base ink), the same ink without Col I, and a bioink with low Mw HA as a rheology modifier.

[0024] [Figure 14] A) Combining Col I as a rheology modifier with high molecular weight HA results in a stretchable bioink and adhesive material, B) shows that without Col I the same bioink is not stretchable and breaks during pulling. [Figure 15] A) shows that the combination of high molecular weight HA as a rheology modifier with Col I results in an adherent bioink that maintains the printed morphology during culture and does not peel off from the bottom of the dish during incubation, while B) shows that without Col I the same bioink peels off from the dish during incubation and loses the printed structure. [Figure 16] A) shows that the synthesis of high molecular weight HA results in components that are insoluble and cannot be used for printing, and B) shows that the synthesized high molecular weight HA-DA-CDH cannot be analyzed and characterized because the components are not soluble in the reagent solution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The verbs "comprise", "contain" and "include" are used herein as open-ended expressions which neither exclude nor require the presence of unrecited features. As used herein, the term "comprising" includes the broader meanings of "including", "containing" and "comprehending", as well as the narrower expressions "consisting of" and "consisting only of". Features recited in the accompanying dependent claims may be freely combined with one another, unless expressly stated otherwise. Furthermore, it is to be understood that the use of "a" or "an", i.e. the singular, does not exclude a plurality throughout this document.

[0026] As used herein, the term "additive manufacturing" refers to 3D and 4D printing of materials based on computer-guided digital models. The term "3D additive manufacturing" or "3D printing" can refer to applying a material to a surface of an object in a single layer by printing, or forming a single layer coating, for example, of a hydrogel. The term "3D additive manufacturing" or "3D printing" can also refer to printing a material layer by layer to produce a multi-layer (i.e., more than one printed layer) structure of a three-dimensional object. The term "4D additive manufacturing" or "4D printing" can refer to 3D printing in which the 3D printing material can be transformed into different forms after printing. Thus, the term "3D printing" also includes the term "4D printing". The term "3D additive manufacturing" or "3D printing" can also refer to printing on a surface such as a substrate, or on a support bath, including FRESH printing. The term "3D additive manufacturing" or "3D printing" can also refer to printing of materials including materials of biological origin, such as living cells. Thus, the term "3D printing" can also refer to the terms "bioprinting" or "3D bioprinting."

[0027] As used herein, the term "bioprinting" refers to a method of 3D printing of materials, such as bioink, in which the printed material includes materials of biological origin, such as living cells.

[0028] As used herein, the term "hydrazone crosslinking" refers to a spontaneous click-type reaction to conjugate two biocompatible biomolecules or biopolymers, in which the crosslinking reaction occurs between an aldehyde group and a hydrazide group grafted to the biomolecule or biopolymer. A crosslinking containing a hydrazone bond contains a -C(O)-NH-N=C bond.

[0029] As used herein, the term "biofabrication window," in the context of a hydrogel or bioink according to the present invention, refers to the period during which the crosslinked hydrogel or bioink has a consistency that allows for its use in additive manufacturing.

[0030] As used herein, the term "aldehyde group" refers to a functional group that contains or consists of an aldehyde having the chemical formula -CHO, where the carbonyl center is a carbon double-bonded to an oxygen and single-bonded to a hydrogen.

[0031] As used herein, the term "catechol group" refers to a functional group containing or consisting of a catechol in which a benzene nucleus bears two hydroxy substituents in the ortho positions relative to one another.

[0032] As used herein, the term "carbohydrazide group" refers to a functional group that contains or consists of a carbohydrazide in which a carbonyl group (C=O) is adjacent to a hydrazide group (-NH-NH2).

[0033] As used herein, the term "carboxylic acid group" or "carboxyl group" consists of a carbonyl group (C=0) with a hydroxyl group (OH) bonded to a carbon atom.

[0034] As used herein, the term "extracellular matrix protein" refers to any protein found in the large network of proteins, macromolecules, minerals, and other molecules that surround, support, and provide structure from the outside to cells and tissues in vivo.

[0035] As used herein, the term "rheology modifier" refers to an ingredient that, when added to a composition, is capable of modifying the rheological properties of the composition, such as viscosity, viscoelasticity, torque, shear stress, and shear rate.

[0036] As used herein, the term "rheology modifier component" refers to a component configured to improve the rheological properties of a hydrogel composition. The rheology modifier component includes at least one rheology modifier, e.g., two or three rheology modifiers. The rheology modifier component can include one or more polymeric or non-polymeric components, which may be, for example, proteins and / or polysaccharides. The rheology modifier component, when added to a hydrogel precursor composition, can at least modify the rheological properties (e.g., viscoelasticity, viscosity, torque, shear stress and shear rate) of the resulting hydrogel composition. Optionally, the rheology modifier component can also have other effects (e.g., increased cell biocompatibility) when added to a composition.

[0037] As used herein, the term "hyaluronic acid (HA)" refers to a disaccharide polymer composed of D-glucuronic acid and N-acetyl-D-glucosamine pairs as repeating units linked through alternating β-(1→4) and β-(1→3) glycosidic bonds. Hyaluronic acid may be up to about 25,000 disaccharide repeats in length. The term "hyaluronic acid" may also refer to salts and / or solvates of hyaluronic acid, such as sodium hyaluronate. The abbreviation HA refers to hyaluronic acid. As used herein, the term "hyaluronic acid (HA)" may be used to refer to, for example, HA in the HA component (see below) or HA in the rheology modifier component.

[0038] As used herein, the term "disaccharide repeat unit of the HA component" refers to the repeat disaccharide unit of the HA polymer, which is composed of this D-glucuronic acid and N-acetyl-D-glucosamine pair. As used herein, the term "hyaluronic acid (HA) component" refers to a component of the hydrogel precursor composition that includes hyaluronic acid (HA) or a salt or solvate thereof. In one embodiment, the hyaluronic acid component consists entirely of HA. The term "HA component" refers to the hyaluronic acid component. The HA component can refer to the HA component of the first component and / or the second component of the hydrogel precursor composition.

[0039] As used herein, the term "moiety" refers to a portion of a molecule that can be functionally or structurally identified in the structure of the molecule. A moiety may be a functional group or represent a portion of a molecule that has multiple functional groups that share a common structural aspect.

[0040] As used herein, the term "fluid" refers to a liquid or gaseous substance. In hydrogel compositions, a fluid is by definition water or an aqueous solution or suspension, such as cell culture medium, a buffer solution, a saline solution, and / or a sucrose solution.

[0041] As used herein, the term "ingredient" refers to one individual component, which when used in a composition, together with all other ingredients forms the whole composition. An ingredient may include several different compounds, or may consist entirely of one particular compound or molecule.

[0042] As used herein, the term "viscosity" refers to the dynamic viscosity of a fluid and has the unit of measurement Pa·s (Pascal seconds).

[0043] As used herein, the term "conjugate" refers to two moieties or compounds that are joined together by being directly bonded to each other or by being indirectly bonded through another moiety or compound. A conjugate refers to a compound formed by conjugating two or more compounds together.

[0044] As used herein, the term "reactive functional group" refers to a functional group or moiety of a compound that is intended or can reasonably be expected to undergo a chemical reaction in a targeted experimental setting. In one embodiment, a reactive functional group is expected to undergo a chemical reaction when a particular reaction partner (which is also a functional group or moiety) comes into contact with the reactive functional group.

[0045] As used herein, the term "crosslink" means a chemical bond or short sequence of bonds that connects one polymer chain or molecule to another.

[0046] As used herein, the term "linker" refers to a modular region that connects two adjacent moieties in a first or second component of a hydrogel precursor composition.

[0047] As used herein, the term "biocompatibility" describes the property of a material to be compatible with and maintain the viability of living cells or tissues, and the ability to not produce any toxic side effects or immunological responses when exposed to the body or bodily fluids.

[0048] As used herein, the term "wt. %" means percentage by weight. The wt. % of a material in a mixture means the proportion by weight of the material in the total weight of the mixture. The wt. % is calculated by dividing the weight of the test material by the weight of the total mixture.

[0049] The hydrogel precursor composition i) a) at least one first reactive functional group; and b) at least one catechol and / or phenol group a first component comprising a hyaluronic acid (HA) component conjugated to ii) a second component comprising a hyaluronic acid (HA) component conjugated to at least one second reactive functional group; and iii) at least one rheology modifier component; and Including, The at least one second reactive functional group is configured to form a crosslink with the at least one first reactive functional group.

[0050] Therefore, the object of the present disclosure is to i. a. at least one first reactive functional group; and b. at least one catechol and / or phenol group a first component comprising a hyaluronic acid (HA) component conjugated to ii. a second component comprising a hyaluronic acid (HA) component conjugated to at least one second reactive functional group; iii. at least one rheology modifier component; 1. A hydrogel precursor composition comprising: and providing a hydrogel precursor composition, wherein at least one second reactive functional group is configured to form crosslinks with at least one first reactive functional group, the at least one rheology modifier component comprises hyaluronic acid having a molecular weight of 1200 kDa to 1900 kDa, and the at least one rheology component comprises 2 to 50 weight percent of the total weight of the hydrogel precursor composition.

[0051] In one embodiment, in the hydrogel precursor composition: i) the at least one first reactive functional group is selected from hydrazide, hydrazine, carbohydrazide, aminooxy, amine, semicarbazide, thiosemicarbazide and / or carbazate groups, preferably the at least one first reactive functional group is a carbohydrazide group (-CONHNH2) and the at least one second reactive functional group is an aldehyde group (-CHO); and / or ii) at least one first reactive functional group is semicarbazide or aminooxy and at least one second reactive functional group is a ketone.

[0052] In one embodiment, the hydrogel precursor composition is i) a first component comprising an HA moiety conjugated to at least one first reactive functional group and at least one catechol group, wherein the at least one first reactive group is a carbohydrazide group (-CONHNH2); ii) a second component comprising an HA moiety conjugated to at least one second reactive functional group, wherein the at least one second reactive functional group is an aldehyde group (-CHO); iii) at least one rheology modifier component; and Includes.

[0053] In one embodiment, the HA component of the first component consists entirely of HA polymers. In one embodiment, the HA component of the first component consists entirely of HA polymers having a molecular weight of 100-1000 kDa, preferably 100-300 kDa.

[0054] In one embodiment, the HA component of the second component consists entirely of HA polymers. In one embodiment, the HA component of the second component consists entirely of HA polymers having a molecular weight of 100-1000 kDa, preferably 100-300 kDa.

[0055] In one embodiment, at least one first reactive functional group of the first component is a carbohydrazide group consisting of a -CONHNH2 moiety.

[0056] In one embodiment, the first component comprises at least one catechol group or derivative thereof. In one embodiment, the first component comprises a catecholamine or derivative thereof conjugated to an HA component. In a preferred embodiment, the at least one catechol group of the first component comprises or consists of a dopamine group (DA) conjugated to an HA component. In one embodiment, the hydrogel precursor composition comprising at least one catechol group is configured to produce a non-collapsible hydrogel composition when a fluid is added to the hydrogel precursor composition.

[0057] In one embodiment, the first component comprises at least one phenolic group. In one embodiment, the hydrogel precursor composition comprising the phenolic group is configured to produce a non-crushable hydrogel composition when a fluid is added to the hydrogel precursor composition.

[0058] In one embodiment, at least one second reactive functional group of the second component is an aldehyde group consisting of a -CHO moiety conjugated to an HA component. In one embodiment, at least one aldehyde group of the second component comprises a -CHO moiety conjugated to an HA component.

[0059] In one embodiment, the second component of the hydrogel precursor composition is configured to be spontaneously crosslinkable with the first component of the hydrogel precursor composition. In one embodiment, the second component of the hydrogel precursor composition is configured to be spontaneously hydrazone crosslinkable with the first component of the hydrogel precursor composition. In one embodiment, the hydrogel composition can be obtained by mixing the hydrogel precursor composition with a fluid. In a preferred embodiment, the fluid mixed with the hydrogel precursor composition is a liquid. The fluid mixed with the hydrogel precursor composition can be, for example, water or an aqueous solution (e.g., a cell culture medium, a buffer solution, a saline solution, and / or a sucrose solution).

[0060] In one embodiment, the second reactive functional group(s) of the second component of the hydrogel precursor composition are configured to form crosslinks with the first reactive functional group(s) of the first component of the hydrogel precursor composition. In one embodiment, the second reactive functional group(s) of the second component are configured to form crosslinks with the first reactive functional group(s) of the first component only after the hydrogel precursor composition is mixed with a fluid. In one embodiment, the formation of crosslinks is configured to be spontaneous and does not require an inducer such as a specific temperature or wavelength of light. In one embodiment, the formation of crosslinks is based on a spontaneous click chemistry reaction, such as hydrazone crosslinking the first and second components of the hydrogel precursor composition together.

[0061] In one embodiment, the reactive functional groups of the first and second components of the hydrogel precursor composition are configured to form crosslinks to form a non-crushable hydrogel composition. In one embodiment, at least one first reactive functional group of the first component that forms crosslinks with the second component is a carbohydrazide group. In one embodiment, at least one first reactive functional group of the first component that forms crosslinks with the second component is selected from hydrazide, hydrazine, aminooxy, amine, semicarbazide, thiosemicarbazide and / or carbazate groups.

[0062] In one embodiment, the at least one first reactive functional group of the first component affects the rheological properties of the resulting crosslinked hydrogel composition. In one embodiment, the at least one first reactive functional group of the first component, such as a carbohydrazide group, is configured to form strong crosslinks with the reactive functional group of the second component.

[0063] In one embodiment, at least one second reactive functional group of the second component, such as an aldehyde group, is configured to form strong crosslinks with the reactive functional group of the first component. In one embodiment, at least one second reactive functional group of the second component is reactive with a carbohydrazide group of the first component or with another functional group of the first component. In one embodiment, at least one second reactive functional group of the second component that forms a crosslink with the first component is an aldehyde (-CHO) group.

[0064] In one embodiment, the first and second components of the hydrogel precursor composition are configured to be conjugated by forming a crosslink comprising at least one covalent bond. In some embodiments, the first and second components of the hydrogel precursor composition are configured to form a crosslink comprising a hydrazone (-C(O)-NH-N=C-), an oxime (-C=NO-), an imine (-C=NC-), a semicarbazone (-NH-C(O)-NH-N=C-), a thiosemicarbazone (-NH-C(S)-NH-N=C-), or a carbazone (-OC(O)-NH-N=C-) bond. The crosslinking rate and stability vary depending on the specific crosslink formed between the first and second components of the hydrogel precursor composition. For example, a first component containing a -NHNHCONHNH2 group conjugated to an HA component produces approximately 15 times stronger crosslinks with the reactive functional aldehyde of a second component than a first component containing a -NHNHCO(CH2)4CONHNH2 group conjugated to an HA component.

[0065] The first or second reactive functional group(s) of the first and second components with the lowest conjugation percentage to HA in each component of the hydrogel precursor composition determines the degree of crosslinking in the hydrogel. In one embodiment, the lowest conjugation percentage of HA with either an aldehyde group as the second reactive functional group or a carbohydrazide group as the first reactive functional group determines the degree of crosslinking in the hydrogel. However, if the hydrogel precursor composition contains an excess of one of the reactive functional groups, such as an aldehyde or a carbohydrazide, the stability of the hydrogel matrix increases without affecting the degree of crosslinking. This is due to the dynamic nature of the covalent bonds generated in the hydrazone crosslinking process, where crosslinks are repeatedly formed and broken, and crosslinking partners can quickly find each other if one of the partners is in excess. Excess aldehyde groups in the second component increase the tissue adhesive properties of the hydrogel composition.

[0066] In one embodiment, the rheology modifier component of the hydrogel precursor composition is configured to modify the rheology of a hydrogel composition obtainable from the crosslinked hydrogel precursor composition, hi one embodiment, the rheology modifier component of the hydrogel precursor composition is configured to modify at least the viscoelasticity, viscosity, torque, shear stress, and shear rate of a hydrogel composition obtainable from the crosslinked hydrogel precursor composition.

[0067] In one embodiment, the rheology modifier component of the hydrogel precursor composition is selected from polysaccharides, proteins, synthetic polymers, or combinations thereof.

[0068] In one embodiment, the rheology modifier component of the hydrogel precursor composition is selected from hyaluronic acid, alginate, chitosan, nanocellulose, polyethylene glycol, poloxamer, polyvinyl alcohol, extracellular matrix (ECM) proteins, albumin, or mixtures thereof.

[0069] In one embodiment, the rheology modifier component of the hydrogel precursor composition comprises an extracellular matrix (ECM) protein selected from gelatin, type I collagen, type III collagen, type IV collagen, type V collagen, laminin, fibronectin, vitronectin, and fragments and / or mixtures thereof.

[0070] In one embodiment, the weight percent of the rheology modifier component in the hydrogel precursor composition depends on the molecular weight of the rheology modifier component. In one embodiment, the applied weight percent of the rheology modifier component in the hydrogel precursor composition decreases substantially linearly with increasing molecular weight of the applied rheology modifier component. The rheology modifier component is configured to modify the rheology and viscosity of the hydrogel obtainable from the crosslinked hydrogel precursor composition, and thus, too high a concentration of the high molecular weight rheology modifier component results in a hydrogel composition having an unnecessarily high viscosity. Similarly, too low a concentration of the low molecular weight rheology modifier component results in a hydrogel composition having an unnecessarily low viscosity.

[0071] In one embodiment, the rheology modifier component of the hydrogel precursor composition comprises hyaluronic acid having a molecular weight of 50 kDa to 1900 kDa, preferably 500 kDa to 1900 kDa, more preferably 1200 kDa to 1900 kDa. In one embodiment, the hydrogel precursor composition comprises 2 to 50 wt %, preferably 5 to 40 wt %, more preferably 7 to 17 wt % of HA as a rheology modifier based on the total weight of the hydrogel precursor composition.

[0072] In one embodiment, the rheology modifier component of the hydrogel precursor composition comprises HA having a molecular weight of 50 kDa to 1900 kDa, preferably 500 kDa to 1900 kDa, more preferably 1200 kDa to 1900 kDa. In one embodiment, the rheology modifier component comprises HA, and the weight percent of the rheology modifier component is 2 to 50 weight percent, preferably 5 to 40 weight percent, more preferably 7 to 17 weight percent of the total weight of the hydrogel precursor composition. In one embodiment, the rheology modifier component of the hydrogel precursor composition comprises HA having a molecular weight of 1200 kDa to 1900 kDa, and the weight percent of the rheology modifier component is 7 to 17 weight percent of the total weight of the hydrogel precursor composition. In one embodiment, the molecular weight of the HA of the rheology modifier component is selected based on improved performance in printability and shape retention of the hydrogel composition after 3D printing. In one embodiment, a rheology modifier component comprising HA having a molecular weight between 1200 kDa and 1900 kDa is advantageous for the printing performance and shape retention of the hydrogel composition after 3D printing.

[0073] In one embodiment, the rheology modifier component of the hydrogel precursor composition comprises human collagen having a molecular weight of 250-300 kDa, preferably type I. In one embodiment, the hydrogel precursor composition comprises, as the rheology modifier, 0.05-40 wt %, preferably 2-15 wt %, more preferably 3-8 wt % of collagen based on the total weight of the hydrogel precursor composition.

[0074] In one embodiment, the rheology modifier component of the hydrogel precursor composition comprises human fibronectin having a molecular weight of 440 to 530 kDa. In one embodiment, the hydrogel precursor composition comprises 2 to 50 wt %, preferably 5 to 30 wt %, and more preferably 7 to 12 wt % of human fibronectin as the rheology modifier based on the total weight of the hydrogel precursor composition.

[0075] In one embodiment, the rheology modifier component of the hydrogel precursor composition comprises a laminin having a molecular weight of 400-900 kDa, preferably a human or mouse recombinant laminin, In one embodiment, the hydrogel precursor composition comprises, as a rheology modifier, 0.2-50 wt%, preferably 0.2-5 wt%, more preferably 0.2-2.0 wt% of laminin based on the total weight of the hydrogel precursor composition.

[0076] In one embodiment, the rheology modifier component of the hydrogel precursor composition comprises human or bovine albumin having a molecular weight of 66-67 kDa. In one embodiment, the hydrogel precursor composition comprises 0.2-60 wt. % of albumin, preferably 30-60 wt. %, more preferably 54-58 wt. % of the total weight of the hydrogel precursor composition, as the rheology modifier.

[0077] In one embodiment, the rheology modifier component comprises two or more rheology modifiers. In one embodiment, the rheology modifier component comprises at least HA and another rheology modifier selected from alginate, chitosan, nanocellulose, polyethylene glycol, poloxamer, polyvinyl alcohol, extracellular matrix (ECM) proteins, albumin, or mixtures thereof. In one embodiment, the rheology modifier component comprises at least HA and another rheology modifier selected from ECM proteins and / or albumin. In one embodiment, the rheology modifier component comprises HA having a molecular weight of 1200-1900 kDa and another rheology modifier selected from ECM proteins and / or albumin. In one embodiment, the rheology modifier component comprises HA configured to improve the printing performance and shape retention of the hydrogel composition and an ECM protein as another rheology modifier configured to improve the biocompatibility of the hydrogel precursor composition.

[0078] In one embodiment, the rheology modifier component of the hydrogel precursor composition comprises a mixture of HA and collagen, preferably collagen type I. In one embodiment, the hydrogel precursor composition comprises as the rheology modifier component 2-50 wt %, preferably 9-40 wt %, more preferably 11-23 wt % of the mixture of HA and collagen type I based on the total weight of the hydrogel precursor composition.

[0079] In an exemplary embodiment, the hydrogel precursor composition includes 17.6 wt% HA having a molecular weight of 1200-1900 kDa as a rheology modifier component. In another exemplary embodiment, the hydrogel precursor composition includes 5.4 wt% human type I collagen having a molecular weight of 300 kDa and 11.9 wt% HA having a molecular weight of 1200-1900 kDa as a rheology modifier component.

[0080] In one embodiment, at least one first reactive functional group and catechol group of the first component and at least one second reactive functional group of the second component are conjugated to the carboxyl group of the glucuronic acid residue of their respective HA components or to any functional group of their respective HA components. In one embodiment, at least one carbohydrazide group and catechol group of the first component and at least one aldehyde group of the second component are conjugated to the carboxyl group of the glucuronic acid residue of their respective HA components or to any functional group of their respective HA components. Preferably, at least one carbohydrazide group and catechol group of the first component and at least one aldehyde group of the second component are conjugated to their respective HA components through replacement of the hydroxyl group (OH) of the glucuronic acid residue of the HA component, or more preferably, to the carboxyl group of the glucuronic acid residue of their respective HA components. In one embodiment, at least one carbohydrazide group and a catechol group of the first component and at least one aldehyde group of the second component are conjugated directly to their respective HA moieties or via a linker moiety.

[0081] In one embodiment, at least one first reactive functional group is directly conjugated to the HA component in the first component without any linker therebetween. Preferably, at least one first reactive functional group is conjugated to a carboxyl group of a glucuronic acid residue of the HA component or is conjugated to a glucuronic acid residue of the HA component via substitution of an OH group.

[0082] In one embodiment, a hydrogel precursor composition is disclosed in which at least one first reactive functional group is conjugated to an HA component of a first component via a linker L1, which is preferably conjugated to a carboxyl group of the HA component via substitution of a carboxylate hydroxy group of a glucuronic acid residue of the HA component. In one embodiment, a hydrogel precursor composition is disclosed in which at least one first reactive functional group is conjugated to an HA component of a first component via a linker L1, which is preferably conjugated to a carboxyl group of the HA component via substitution of a carboxylate hydroxy group of a glucuronic acid residue of the HA component, and the linker L1 is formed from 1 to 10 moieties, each moiety being independently selected from the group consisting of phenylene (-CH-), oxime (-C=NO-), imine (-C=NC-), alkylene containing 1 to 12 carbon atoms (-CH-), ethynyl (-C≡C-), ethylenediamine ... Disclosed is a hydrogel precursor composition, wherein R' is selected from alkyl (-C=C-), ether (-O-), thioether (-S-), amide (-CO-NH-, -CO-NR'-, -NH-CO- and NR'-CO-), where R' is an alkyl group containing less than 5 carbon atoms, carbonyl (-CO-), ester (-COO- and OOC-), disulfide (-SS-), sulfonamide (-SO2-NH-, -SO2-NR'-), sulfone (-SO2-), phosphate (-O-PO2-O-), diaza (-N=N-), diimine (-NHNH-), secondary amine (-NHNH-) and tertiary amine.

[0083] In one embodiment, a hydrogel precursor composition is disclosed, wherein the first component comprises a plurality of first reactive functional groups, and the first reactive functional groups, or a linker L1 conjugated to the first reactive functional groups, are conjugated to 5-20%, preferably 9-15%, more preferably 12-13% of the disaccharide repeat units of the HA component of the first component.

[0084] In one embodiment, a hydrogel precursor composition is disclosed in which at least one first reactive functional group is a carbohydrazide group and the linker L1 is -NHNH-.

[0085] In a preferred embodiment, at least one first reactive functional group is a carbohydrazide group, which is conjugated to the HA component via a linker L1 in the first component, which is preferably conjugated to the HA component via a carboxyl group of a glucuronic acid residue of the HA component, or alternatively, which is conjugated to another functional group contained in the disaccharide repeat unit of the HA component.

[0086] In one embodiment, at least one carbohydrazide group is conjugated to a carboxyl group of a glucuronic acid residue of HA via a linker L1 according to formula (I), where n is preferably 250-2500, more preferably n is 250-750. [ka]

[0087] In one embodiment, at least one carbohydrazide group is conjugated to a glucuronic acid residue of the HA component via a linker L1 by replacement of the -OH group of the glucuronic acid residue. In one embodiment, the -OH group of the glucuronic acid residue is replaced with a linker L1 that includes an α-substituent such as an amino group or a further hydrazide group.

[0088] In one embodiment, the linker L1 of the first component of the hydrogel precursor composition is preferably -NHNH- and NHNH(CH2)n -, where n is 1 to 12, preferably n is 4. In a preferred embodiment, the linker L1 is -NHNH-, and the linker L1 is conjugated to the HA moiety via the carboxyl group of a glucuronic acid residue of the HA moiety.

[0089] In one embodiment, the hydrogel precursor composition comprising -NHNH- as the linker L1 in the first component comprises -NHNH(CH2) as the linker L1. n - (wherein n is 1-12) is configured to form crosslinks faster. In one embodiment, the hydrogel precursor composition comprising -NHNH- as the linker L1 is configured to form printable hydrogel compositions faster and the formed hydrogel compositions are more stable compared to hydrogel precursor compositions having the four carbon containing linker -NHNH(CH2)4- or adipic acid dihydrazide based linkers. In one embodiment, the hydrogel precursor composition comprising -NHNH- as the linker L1 in the first component is configured to form printable hydrogel compositions with increased stability compared to hydrogel precursor compositions comprising -NHNH(CH2)4 as the linker L1.

[0090] In one embodiment, the structure and length of the linker L1 of the first component is optimized for the conjugation of the carbohydrazide group with the HA component. The optimal structure and length of the linker L1 allows the carbohydrazide group and the HA component of the first component to obtain an optimal configuration, allowing efficient crosslinking of the first component to the second component. Thus, the modular structure and length of the linker L1 of the first component promotes high stability of the crosslinked hydrogel composition with faster reaction kinetics.

[0091] In one embodiment, the first component comprises a plurality of first reactive functional groups that are carbohydrazide groups, and the carbohydrazide groups or the linker L1 conjugated to the carbohydrazide groups are conjugated to 5-20%, preferably 9-15%, more preferably 12-13% of the disaccharide repeat units of the HA component of the first component. In one embodiment, this conjugation percentage of the carbohydrazide groups (or linker L1) to the disaccharide repeat units of the HA component of the first component is beneficial to the printability of the hydrogel composition. In an embodiment where the carbohydrazide groups or the linker L1 conjugated to the carbohydrazide groups are conjugated to more than 20% of the disaccharide repeat units of the HA component of the first component, the crosslinking and stability of the hydrogel is improved, but the printability is reduced.

[0092] In one embodiment, at least one phenolic and / or catechol group is directly conjugated to the HA component in the first component without any linker therebetween. Preferably, at least one phenolic and / or catechol group is conjugated to a carboxyl group of a glucuronic acid residue of the HA component or is conjugated to a glucuronic acid residue of the HA component via substitution of an OH group.

[0093] In one embodiment, a hydrogel precursor composition is disclosed in which at least one phenolic and / or catechol group is conjugated to the HA moiety of the first component via a linker L2, which is conjugated to a carboxyl group of the HA component, preferably via substitution of a carboxylate hydroxy group of a glucuronic acid residue of the HA component. In one embodiment, a hydrogel precursor composition is provided, comprising at least one phenolic and / or catechol group conjugated to an HA component of a first component via a linker, L2, which is conjugated to a carboxyl group of the HA component, preferably via substitution of a carboxylate hydroxy group of a glucuronic acid residue of the HA component, wherein the linker, L2, is formed from 1-10 moieties, each moiety being independently selected from the group consisting of phenylene (-CH), oxime (-C=NO-), imine (-C=NC-), alkylene containing 1-12 carbon atoms (-CH2-), ethynyl (-C≡C-), ethylenediyl (-C= ... In one embodiment, the linker L2 is selected from the group consisting of ether (-O-), thioether (-S-), amide (-CO-NH-, -CO-NR'-, -NH-CO and NR'-CO-), where R' represents an alkyl group containing less than 5 carbon atoms, carbonyl (-CO-), ester (-COO- and OOC-), disulfide (-SS-), sulfonamide (-SO2-NH-, -SO2-NR'-), sulfone (-SO2-), phosphate (-O-PO2-O-), diaza (-N=N-), diimine (-NHNH-), secondary amine (-NH-), and tertiary amine, and preferably the linker L2 is -NH(CH2)2-.

[0094] In one embodiment, a hydrogel precursor composition is disclosed, wherein the first component comprises a plurality of catechol and / or phenol groups, and the catechol and / or phenol groups, or a linker L2 conjugated to the catechol and / or phenol groups, are conjugated to 1-20%, preferably 2-10%, more preferably 3-6%, and even more preferably 3.0-3.6% of the disaccharide repeat units of the HA moiety of the first component.

[0095] In one embodiment, the first component of the hydrogel precursor composition comprises at least one catechol group conjugated to the HA component via a linker L2 in the first component, which is preferably conjugated to the HA component via a carboxyl group of a glucuronic acid residue of the HA component, or alternatively, the linker L2 is conjugated to another functional group contained in the disaccharide repeat unit of the HA component.

[0096] In one embodiment, at least one catechol group is conjugated to a carboxyl group of a glucuronic acid residue of the HA component via a linker L2 according to formula (II), where n is preferably between 250 and 2500, more preferably between 250 and 750. [ka]

[0097] In one embodiment, at least one catechol group is conjugated to the carboxyl group of a glucuronic acid residue of the HA component via a linker, L2, by replacement of the -OH group of the glucuronic acid residue. In one embodiment, the OH group of the glucuronic acid residue is replaced with a linker, L2, that includes an α-substituent, such as an amino group.

[0098] In one embodiment, the linker L2 of the first component of the hydrogel precursor composition is most preferably -NH(CH2)2-, and the linker L2 is preferably conjugated to the HA component via the carboxyl group of the glucuronic acid residue of the HA component. In a preferred embodiment, the linker L2 is -NH(CH2)2-.

[0099] In one embodiment, the first component comprises a plurality of catechol groups, and the catechol groups or the linker L2 conjugated to the catechol groups are conjugated to 1-20%, preferably 2-10%, more preferably 3-6%, even more preferably 3.0-3.6% of the disaccharide repeat units of the HA component of the first component. The linker L2 is preferably conjugated to the carboxyl group of the glucuronic acid unit of the HA component of the first component. In one embodiment, the percentage of catechol groups conjugated to the disaccharide repeat units of the HA component of the first component is advantageous for the tissue adhesive properties, as well as the viscoelastic and shear thinning properties of the final hydrogel composition.

[0100] In one embodiment, a hydrogel precursor composition comprising -NH(CH)- as a linker L and a catechol group conjugated to a first component is configured to form a printable hydrogel composition faster compared to a hydrogel precursor composition that does not include the catechol group.

[0101] In one embodiment, at least one catechol group is conjugated to the HA component by reacting an amino group of dopamine with a carboxyl group of the HA component.

[0102] In one embodiment, at least one second reactive functional group is directly conjugated to the HA component in the second component without any linker therebetween. Preferably, at least one second reactive functional group is conjugated to a carboxyl group of a glucuronic acid residue of the HA component or is conjugated to a glucuronic acid residue of the HA component via substitution of an OH group.

[0103] In one embodiment, a hydrogel precursor composition is disclosed in which at least one second reactive functional group is conjugated to the HA component of the second component via a linker L3, which is preferably conjugated to a carboxyl group of the HA component via substitution of a carboxylate hydroxy group of a glucuronic acid residue of the HA component. In one embodiment, at least one second reactive functional group is conjugated to the HA component of the second component via a linker L3, which is preferably conjugated to a carboxyl group of the HA component via substitution of a carboxylate hydroxy group of a glucuronic acid residue of the HA component, and the linker L3 is formed from 1 to 10 moieties, each moiety being independently selected from the group consisting of phenylene (-CH-), oxime (-C=NO-), imine (-C=NC-), alkylene containing 1 to 12 carbon atoms (-CH-), ethynyl (-C≡C-), ethylenediyl (- Disclosed is a hydrogel precursor composition, wherein R' is selected from the group consisting of C=C-), ether (-O-), thioether (-S-), amide (-CO-NH-, -CO-NR'-, -NH-CO- and NR'-CO-), where R' is an alkyl group containing less than 5 carbon atoms, carbonyl (-CO-), ester (-COO- and OOC-), disulfide (-SS-), sulfonamide (-SO2-NH-, -SO2-NR'-), sulfone (-SO2-), phosphate (-O-PO2-O-), diaza (-N=N-), diimine (-NHNH-), and tertiary amine.

[0104] In one embodiment, the second component of the hydrogel precursor composition comprises a plurality of second reactive functional groups, preferably aldehyde groups, and the second reactive functional groups or linkers L3 conjugated to the second reactive functional groups are conjugated to 5-50%, preferably 9-15%, more preferably 11-13% of the disaccharide repeat units of the HA component of the second component.

[0105] In one embodiment, at least one second reactive functional group of the hydrogel precursor composition is an aldehyde group (-CHO) and the linker L3 is -NHCH2-.

[0106] In a preferred embodiment, the at least one second reactive functional group is an aldehyde group conjugated to the HA component via a linker L3 in the second component, which is preferably conjugated to the HA component via a carboxyl group of a glucuronic acid residue of HA, or alternatively, the linker L3 is conjugated to another functional group contained in the disaccharide repeat unit of the HA component.

[0107] In one embodiment, at least one second reactive functional group is an aldehyde group that is conjugated to a carboxyl group of a glucuronic acid residue of the HA component via a linker L3 according to formula (III), where n is preferably between 250 and 2500, more preferably between 250 and 750. [ka]

[0108] In one embodiment, at least one aldehyde group is conjugated to a carboxyl group of a glucuronic acid residue of the HA component via linker L3 by replacement of the -OH group of the glucuronic acid residue. In one embodiment, the -OH group of the glucuronic acid residue is replaced with linker L3 that includes an α-substituent, such as an amino group.

[0109] In one embodiment, the linker L3 of the second component of the hydrogel precursor composition is most preferably -NH(CH2) n -, n is 1 to 12, preferably n is 2, more preferably n is 1. In one embodiment, the linker L3 of the second component of the hydrogel precursor composition is most preferably -NHCH2-, where the linker L3 is conjugated to the HA component via the carboxyl group of the glucuronic acid residue of the HA component. In another embodiment, the linker L3 of the second component of the hydrogel precursor composition is -NHNH-.

[0110] In one embodiment, the structure and length of the linker L3 of the second component is optimized for the conjugation of the second reactive functional group with the HA component. The optimal structure and length of the linker L3 allows the aldehyde group and the HA component of the second component to obtain an optimal configuration, allowing efficient crosslinking of the first component to the second component. Thus, the modular structure and length of the linker L3 of the second component promotes high stability of the crosslinked hydrogel composition.

[0111] In one embodiment, aldehyde groups are generated by NaIO4-mediated oxidation of the C2 and C3 hydroxyls in the glucuronic acid residues of the HA component of up to 5-50%, preferably 9-15%, more preferably 11-13% of the disaccharide repeat units of the HA component of the second component, without the use of linker L3. Aldehyde groups generated in this manner by grafting of aminoglycerol groups followed by NaIO4 oxidation are preferred over traditional backbone oxidation as they minimize polymer fragmentation and preserve the biological activity of the HA. This approach of generating aldehyde groups by grafting of aminoglycerol groups followed by NaIO4 oxidation provides improved control over the extent of aldehyde groups conjugated to the disaccharide repeat units of the HA component of the second component.

[0112] In a preferred embodiment, the carbohydrazide group or linker L1 conjugated to a carbohydrazide group is conjugated to 12-13% of the disaccharide repeat units of the HA component and the catechol group or linker L2 conjugated to a catechol group is conjugated to 3.0-3.6% of the disaccharide repeat units of the HA component in the first component. In a preferred embodiment, the aldehyde group or linker L3 conjugated to an aldehyde group is conjugated to 11-13% of the disaccharide repeat units of the HA component of the second component.

[0113] In one embodiment, the hydrogel precursor composition also includes at least one regulatory signal component selected from growth factors, antibodies, hormones, peptides, polypeptides, nanoparticles, and fragments and / or mixtures thereof. In one embodiment, the regulatory signal component includes heparin, heparin-derived nanoparticles, growth factor-encapsulated nanocarriers, and / or growth factor-immobilized biomaterials adapted to stabilize the growth factor(s). In one embodiment, the regulatory signal component includes nanoparticles that are superparamagnetic iron oxide NPs (SPIONs). In one embodiment, the regulatory signal component includes gold nanoparticles that impart conductive properties when utilizing neurons, cardiomyocytes, or myoblasts in the bioink. The at least one regulatory signal component of the hydrogel precursor composition is selected based on the cell type that is mixed with the hydrogel precursor composition to obtain the printable bioink. In one embodiment, the at least one regulatory signal component mixed with the hydrogel precursor composition is configured to induce or promote differentiation of the cells mixed in the hydrogel precursor composition. In one embodiment, the at least one regulatory signal component admixed with the hydrogel precursor composition is configured to promote cell proliferation, adhesion, cell fate determination, and / or influence the secretome of the cells admixed in the hydrogel precursor composition. In one embodiment, the at least one regulatory signal component admixed with the hydrogel precursor composition is configured to promote tissue structure development of the cells admixed in the hydrogel precursor composition.

[0114] According to a preferred embodiment, therefore, at least one rheology modifier component of the hydrogel precursor components is Hyaluronic acid with a molecular weight of 1200kDa to 1900kDa, Collagen having a molecular weight of 250 to 300, preferably type I; wherein the at least one rheology component comprises 2 to 50 wt %, preferably 9 to 40 wt %, and more preferably 11 to 23 wt % of the total weight of the hydrogel precursor composition.

[0115] The at least one rheology modifier component may also comprise one or more further extracellular matrix proteins, preferably selected from gelatin, laminin, fibronectin, vitronectin, and fragments and / or mixtures thereof.

[0116] According to a preferred embodiment, at least one first reactive group of the hydrogel precursor components is a carbohydrazide and at least one second reactive group is an aldehyde.

[0117] According to another preferred embodiment, at least one first reactive group of the hydrogel precursor components is an aldehyde and at least one second reactive group is a carbohydrazide.

[0118] According to a preferred embodiment, the molecular weight of the first component and the second component is from 100 to 1000 kDa, preferably from 100 to 300 kDa.

[0119] In one embodiment, a bioink is provided that may be obtained by mixing a hydrogel precursor composition with a fluid containing cells.

[0120] In one embodiment, the hydrogel precursor composition and cells are mixed in a fluid that is isotonic with the cells mixed therein. In one embodiment, the selected fluid produces an isotonic hydrogel composition in which the cells are mixed. In a preferred embodiment, the fluid mixed with the cells and hydrogel precursor composition is a liquid. The fluid mixed with the cells and hydrogel precursor composition may be an aqueous solution such as cell culture medium, a saline solution such as phosphate buffered saline (PBS), another buffer solution, and / or a sucrose solution.

[0121] In one embodiment, the cells comprise undifferentiated stem cells and / or differentiated cells. In one embodiment, the cells comprise human or mammalian cells. In one embodiment, the cells comprise proliferative and / or non-proliferative cells. In one embodiment, the cells comprise a mixture of different cell types, preferably originating from the same species. In one embodiment, the cells comprise hASCs, hASC-derived corneal stromal-like cells, human pluripotent stem cell (hPSC)-derived neural cells (NCs) and / or hPSC-derived corneal endothelial cells. In one embodiment, the cells comprise hepatocytes, vascular endothelial cells, lymphatic endothelial cells and / or fibroblasts.

[0122] In one embodiment, the hydrogel precursor composition and / or the bio-ink comprises nutrients that promote cell viability in the bio-ink, where the nutrients are selected from amino acids, proteins, peptides, fatty acids, lipids, trace elements, antibiotics, vitamins, inorganic salts, carbohydrates, and / or serum.

[0123] In one embodiment, there is provided a use of the hydrogel precursor composition or bioink for additive manufacturing, such as 3D printing.

[0124] In one embodiment, a method for 3D printing a bio-ink is disclosed that includes: a) mixing a hydrogel precursor composition with a fluid containing cells; b) crosslinking a first component of the hydrogel precursor composition with a second component of the hydrogel precursor composition in the fluid; and c) 3D printing the bio-ink, where the viscosity of the bio-ink is between 200-2000 Pa·s.

[0125] In one embodiment, a method for 3D printing includes mixing a hydrogel precursor composition with a fluid containing cells to obtain a bioprintable bioink. In one embodiment, the hydrogel may be obtained by spontaneous crosslinking of the components of the hydrogel precursor composition. In one embodiment, the bioink may be obtained by spontaneous crosslinking of the components of the hydrogel precursor composition when the composition is mixed in a fluid containing cells. The hydrogel precursor composition mixed with the fluid is configured to form crosslinks between reactive functional groups of a first component and a reactive functional group of a second component of the hydrogel precursor composition. The resulting crosslinked hydrogel composition is printable by a 3D printing process. The bioink containing the crosslinked hydrogel composition and cells is printable by a 3D printing process.

[0126] In one embodiment, the period during which the crosslinked bioink is printable is limited, and the biofabrication window is determined by the composition of the hydrogel precursor composition, which determines the crosslink formation kinetics of the bioink. Crosslinking of the hydrogel and / or bioink is a continuous process that begins when fluid is added to the hydrogel precursor composition. The hydrogel and / or bioink is printable because the appropriate amount of crosslinks are formed, thereby giving the hydrogel and / or bioink optimal viscosity and shear thinning properties for printing. In one embodiment, stabilizing the printed hydrogel and / or bioink structure, for example by curing, promotes further crosslinking and increases the stability of the printed bioink structure after printing.

[0127] An exemplary process for obtaining hydrogels and / or bioinks is shown in Figure 1. An exemplary hydrogel precursor composition includes a first component 1, HA-DA-CDH (hyaluronic acid-dopamine-carbohydrazide), a second component 2, HA-Ald (hyaluronic acid-aldehyde), and a third component 3, which includes two rheology modifier components, collagen type I and HA (step (I)). When the hydrogel precursor composition is mixed with a fluid, such as a buffer or cell culture medium (step (II)), a spontaneous hydrazone cross-linking reaction occurs along with strong secondary interactions with dopamine and collagen, forming a double network structure, thereby forming the hydrogel composition (step (III)). As also shown in Figure 1, the bioink is obtained by mixing the hydrogel precursor composition with a fluid containing cells. Mammalian cells, such as human adipose tissue-derived stem cells (hASC) and hASC-derived corneal stromal-like cells, are mixed with the hydrogel before cross-linking, resulting in the formation of the bioink. The resulting hydrogel and / or bioink is suitable for a pre-determined time period (biofabrication window) for additive manufacturing. The bioink can be 3D bioprinted within the biofabrication window, resulting in a viable bioprinted scaffold.

[0128] Hydrogels and bioinks containing hydrogels must be printable but also sufficiently stable. Stability is affected by the degree of crosslinking of the hydrogel, which is in part affected by the concentration of the first and second components of the hydrogel precursor composition used in the hydrogel. In one embodiment, the combined concentration of the first and second components of the hydrogel precursor composition in the hydrogel composition is preferably 2.5-15.5 mg / ml. In one embodiment, the combined concentration of the first and second components of the hydrogel precursor composition in the bioink is preferably 2.5-15.5 mg / ml. The degree of crosslinking of the hydrogel is also affected by the available reactive functional groups of the first and second components of the hydrogel precursor composition. The degree of crosslinking is determined by the lower of the limiting amount of either the first reactive functional group in the first component or the second reactive functional group in the second component attached to the disaccharide repeat unit of each HA component. The degree of crosslinking refers to the percentage of the disaccharide repeating units of the first and second HA components that are conjugated to the first or second reactive functional groups that are crosslinked to the second or first reactive functional groups, respectively. The degree of crosslinking of the bioink can be further influenced by the composition of the linkers L1, L2 and / or L3 of the first and second components of the hydrogel precursor composition. The extent of hydrazone crosslinking formed between the first and second components should be less than 15% of the total amount of disaccharide repeating units of the first and second HA components. If the degree of hydrazone crosslinking exceeds 15% of the combined amount of disaccharide repeating units of the HA components of the first and second components, the crosslinking rate will be too fast, limiting the printability of the hydrogel.

[0129] In one embodiment, the crosslinked hydrogel composition and / or bioink is printable when the viscosity of the hydrogel composition is between 100-5000 Pa·s, preferably between 200-2000 Pa·s. In one embodiment, the method for 3D printing comprises crosslinking a first component and a second component of a hydrogel precursor composition until the viscosity of the mixture reaches between 100-5000 Pa·s, preferably between 200-2000 Pa·s. In one embodiment, the method for 3D printing comprises 3D printing the hydrogel composition when the viscosity of the hydrogel composition is between 100-5000 Pa·s, preferably between 200-2000 Pa·s.

[0130] In one embodiment, the first and second components of the hydrogel precursor composition are configured to crosslink at a temperature between 1 and 38° C. In one embodiment, the hydrogel crosslinking temperature is room temperature in the range of 18-24° C., preferably 19-22° C. In one embodiment, the optimal temperature for crosslinking the hydrogel and / or bioink is between 1 and 38° C., preferably 18-24° C. In one embodiment, the optimal temperature for crosslinking the hydrogel and / or bioink is between 1 and 38° C., preferably 18-24° C., more preferably 19-22° C.

[0131] In a preferred embodiment, crosslinking of the hydrogel and / or bioink is allowed to proceed until the viscosity of the hydrogel and / or bioink reaches approximately 200 Pa·s, after which the hydrogel and / or bioink can be used for printing. In one embodiment, crosslinking of the hydrogel and / or bioink is allowed to proceed for up to 60 minutes, preferably 45-60 minutes, at 20° C. until the viscosity of the hydrogel and / or bioink reaches 200 Pa·s.

[0132] In one embodiment, the period during which the hydrogel and / or bioink can be printed, i.e., processed, depends on the concentration of the crosslinking component, i.e., the first and second components of the hydrogel precursor composition used. In one embodiment, for a hydrogel / bioink with 5.83 mg / ml of crosslinking component, the biofabrication window is at least 150 minutes, or preferably at least 90 minutes. In one embodiment, the printing pressure for printing the hydrogel / bioink depends on the size of the printing nozzle used.

[0133] In one embodiment, the 3D printed hydrogel composition is preferably allowed to form further crosslinks after printing to achieve sufficient stability after printing. In one embodiment, the bioink is stabilized and allowed to form further crosslinks after 3D printing before immersing the 3D printed bioink in an isotonic liquid for further processing. In one embodiment, the 3D printed hydrogel composition and / or bioink is preferably cured for a period of time before immersing the printed hydrogel in an isotonic liquid for further processing to achieve sufficient stability of the hydrogel after printing. In one embodiment, the 3D printed hydrogel composition and / or bioink is cured for 15-60 minutes. EXAMPLES

[0134] Example 1. Synthesis of dopamine-modified hyaluronic acid as part of the first component of the hydrogel precursor composition of the present invention 1 mmol of HA (400 mg, 1 eq.) was dissolved in 60 mL of deionized water, to which was then added 1 mmol of HOBt (153 mg, 1 eq.) and 1 mmol of dopamine (190 mg, 1 eq.). The pH of the reaction solution was adjusted to 5.5 with 1 M HCl and 1 M NaOH. 0.25 mmol of EDC (48 mg, 0.25 eq.) was then added in two batches at 30 min intervals. The pH of the solution was maintained at 5.5 for 6 h and then stirred overnight. The reaction mixture was loaded into a dialysis bag and dialyzed against dilute HCl (pH=3.5) containing 100 mM NaCl (4×2 L, 24 h), followed by dialysis in dilute HCl (pH 3.5, 2×2 L, 24 h), and then against deionized water (2×2 L, 24 h). The solution was then freeze-dried. Dopamine is conjugated via its α-amino group to the carboxyl group of the HA component, so the linker L2 is -NH(CH2)2-. The extent of dopamine conjugation was determined by NMR spectroscopy ( 1 1 H NMR, 300 MHz) was 3.6% (relative to the disaccharide unit of HA).

[0135] Example 2. Synthesis of dopamine and carbohydrazide modified hyaluronic acid (HA-DA-CDH) as the first component of the hydrogel precursor composition of the present invention The first component was synthesized, containing a carbohydrazide group (-CONHNH2) as the first reactive group and a hyaluronic acid (HA) component conjugated to a catechol group. The conjugation of carbohydrazide (CDH) onto dopamine-modified hyaluronic acid (HA-DA) was carried out by carbodiimide coupling chemistry. Briefly, 0.5 mmol of HA-DA (200 mg, 1 equiv.) was dissolved in 120 mL of deionized water. Then, 0.375 mmol of carbodihydrazide (34 mg, 0.75 equiv.) and 0.5 mmol of HOBt (76.5 mg, 1 equiv.) were added to the HA-DA aqueous solution. The pH of the reaction mixture was adjusted to 4.7. Finally, 0.1 mmol of EDC·HCl (20 mg, 0.2 equiv.) was added and stirred overnight. The reaction mixture was loaded into a dialysis bag and dialyzed against dilute HCl (pH = 3.5) containing 100 mM NaCl (4 x 2 L, 24 h), followed by dialysis in dilute HCl (pH 3.5, 2 x 2 L, 24 h), and then against deionized water (2 x 2 L, 24 h). The mixture was then lyophilized to obtain HA-DA-CDH. The degree of hydrazide modification was found to be 13.2% (with respect to the disaccharide repeat unit of HA), as determined using the TNBS assay. Carbodihydrazide (NH2NHCONHNH2) was able to react with the COOH group of HA-DA, so the linker L1 formed is -NHNH-.

[0136] Example 3. Synthesis of aldehyde-modified hyaluronic acid (HA-Ald) as the second component of the hydrogel precursor composition of the present invention A second component was synthesized that included a hyaluronic acid (HA) component conjugated to an aldehyde group (-CHO) as the second reactive group. The title compound was synthesized as disclosed in Biomacromolecules 2013. The aldehyde group (-CHO) was conjugated to the HA component as disclosed in this document, and the linker L3 formed was -NHCH2-. The percentage of aldehyde modification in HA was: 1It was found to be 9% (relative to the disaccharide unit) as determined by H NMR spectroscopy. The extent of aldehyde modification was estimated by reacting tert-butyl carbazate with aldehyde-modified HA, followed by NaCNBH3 reduction and integration of the tert-butyl signal at 1.4 ppm relative to the N-acetyl signal at 2.0 ppm of HA.

[0137] Example 4. Preparation of hydrogel compositions The hydrogel compositions were prepared using hydrazone crosslinking chemistry. The synthesized HA-DA-CDH (first component of the hydrogel precursor composition) and HA-Ald (second component of the hydrogel precursor composition) components were sterilized by UV for 20 min and dissolved in sterile PBS at a concentration of 10 mg / ml (w / v). The M wSodium hyaluronate (Pharma Grade 150) (Novamatrix) having the formula: was used as the primary rheology modifier component, dissolved in sterile 5×PBS containing 0.4 M NaCl at a concentration of 10 mg / ml (w / v). OptiCol™ human type I collagen (3.1 mg / ml) (Cell Guidance Systems Ltd, Cambridge, UK) was introduced into the hydrogel precursor composition as a secondary rheology modifier component to increase biocompatibility and viscoelasticity. Human type I collagen was neutralized to pH 7.4 with 1 M sodium hydroxide (NaOH) in the presence of 10× Dulbecco's phosphate buffered saline (DPBS, Carl Roth, Karlsruhe, Germany). The concentration of the crosslinking components in the final hydrogel composition, i.e., the first and second components of the hydrogel precursor composition, was 5.83 mg / ml. The final hydrogel composition consisted of HA-DA-CDH 29.2% (v / v), HA-Ald 29.2% (v / v), HA 12.5% ​​(v / v), neutralized human type I collagen 18.2% (v / v), and cell culture medium 10.9% (v / v). Mixing of the components was performed using a dual syringe system with two syringes combined with a female-female luer lock. After thorough mixing, the mixture was placed into a 30cc Nordson EFD syringe barrel and the cartridge piston was immediately placed into the barrel. The crosslinked components of the mixture were allowed to crosslink and cure at room temperature for 45-60 minutes to form a printable hydrogel. Crosslinking allows the hydrogel to obtain optimal consistency and viscosity before using the hydrogel for printing.

[0138] Example 5. 3D printing 3D printing was performed with the hydrogel disclosed in Example 4. Extrusion-based 3D printing with a 3D-Bioplotter® Manufacturer Series by Envisiontec (Gladbeck, Germany) was used. After a pre-crosslinking period, a biofabrication window of 90 minutes for 3D printing was obtained. The barrel with the hydrogel composition was loaded into the low-temperature print head of the 3D bioprinter. A 32G blunt needle with a length of 0.50 inches and an inner diameter of 100 μm was used for printing. The print head temperature was adjusted to 20° C. and printing was performed at room temperature. 3D models in .stl format were created with Perfactory RP Software and internal parameters including the printing pattern were adjusted with Visuals Machine. A slice spacing of 80 μm was used for all printed structures and the needle height was adjusted to 0.07 mm. For 3D printing with the cell-laden hydrogel, i.e. bioink, a printing pressure of 1.0 bar and a printing speed of 6.0 mm / s were used. The 3D printing parameters used for the bioink were otherwise the same as those disclosed above.

[0139] Example 6. Viscosity and Shear Thinning of Hydrogel Compositions The injectability and shear thinning properties of the encapsulated hydrogel composition in Example 4 were measured. The viscosity and flow properties of the hydrogel were measured under both continuous flow (0.01-10 s-1) and cyclic flow. The shear rates used for cyclic flow were 0.01 and 10 s-1 in a TA instruments DHR-II rheometer using a 12 mm stainless steel parallel plate geometry for up to 7 cycles with a hold time of 60 seconds for each cycle. Figure 2 discloses the viscosity of the hydrogel composition in Pa·s under A) continuous flow and B) cyclic flow, and discloses that the hydrogel composition has excellent injectability and shear thinning properties. To further evaluate the strain recovery of the crosslinked hydrogel of Example 4, its storage and loss moduli were measured. Measurements were performed using a 12 mm diameter stainless steel parallel plate geometry under flow alternating between low (1% strain) and high (50% strain) oscillatory strain conditions at 25° C. and an oscillatory frequency of 1 Hz for 7 cycles with a 60 second hold period at each step. The results, which disclose the storage modulus (Pa) and loss modulus (Pa) of the hydrogel composition in FIG. 3, indicate that the hydrogel composition is shear thinning under strain.

[0140] Example 7. 3D printing using hydrogel compositions with and without a rheology modifier component Hydrogel compositions with and without rheology modifiers were tested for 3D printing. The composition disclosed in Example 4 was used in this experiment as the hydrogel with rheology modifier. A hydrogel corresponding to the hydrogel disclosed in Example 4 was prepared without any rheology modifier. Both hydrogels contained 5.83 mg / ml of crosslinking moiety. A lattice structure with 6 layers and a distance between strands of 2.5 mm was printed. A photograph of the printed structure was taken immediately after printing. The hydrogel was crosslinked at room temperature for 1 hour before printing and then printed through a 32G nozzle. For both hydrogels, printing parameters of 1.0 bar and 6.0 mm / s were used.

[0141] The printability of these hydrogel compositions was further compared to a hydrogel composition prepared as previously disclosed by Koivusalo et al. 2019. The hydrogel composition disclosed by Koivusalo et al. 2019 was printed immediately after mixing the crosslinking components, as the gelation of the material was very rapid compared to the other two hydrogel compositions. A lattice structure with two layers and a line spacing of 2.5 mm was used. A larger nozzle of 27 G was used, and a higher pressure of 1.5-2.0 bar was required for the hydrogel material to flow through the nozzle.

[0142] An image showing a 3D printed hydrogel composition without any rheology modifier components is shown in FIG. 4A, an image showing a 3D printed hydrogel composition with hyaluronic acid and human type I collagen as rheology modifiers is shown in FIG. 4B, and an image showing the 3D printed hydrogel composition of Koivusalo et al. 2019 is shown in FIG. 4C. These results show that the hydrogels with the exemplary rheology modifiers form hydrogels with good print quality. These results also show that the hydrogels without the rheology modifiers do not form printable filaments, cannot be patterned into specific structures, and as such cannot be used for 3D printing. Furthermore, the hydrogel composition disclosed by Koivusalo et al. 2019 formed clumps and printed unevenly, rather than being printed as a continuous filament, as the hydrogel composition shown in FIG. 4C. This indicates that the hydrogel composition described in Koivusalo et al. 2019, which contains a higher concentration of crosslinking components and does not contain rheology modifiers, is not suitable for 3D printing.

[0143] Example 8. 3D printing using hydrogel compositions containing different concentrations of crosslinking components Different concentrations of cross-linking moieties in the hydrogel composition were studied to observe the effect on the printability of the hydrogel. The same components of the hydrogel precursor composition as in Example 4 were used. Hydrogels with cross-linking moiety concentrations of 2.92 mg / ml, 8.16 and 11.66 mg / ml were prepared and studied. A 20×20 mm grid structure with two layers and a line distance of 1.00 mm was printed with hydrogels with cross-linking moiety concentrations of 2.92 mg / ml and 8.16 mg / ml, and a 15 mm×15 mm grid with a line distance of 2.50 mm was used for the hydrogel with cross-linking moiety concentration of 11.66 mg / ml. The hydrogel with cross-linking moiety concentration of 2.92 mg / ml was printed using a 34 G nozzle at a pressure of 0.2 bar and a speed of 18 mm / s. For the hydrogel with cross-linking moiety concentration of 8.16 mg / ml, a 32 G nozzle was used with printing parameters of 1.6 bar pressure and 5 mm / s speed. Hydrogels with a cross-linking moiety concentration of 11.66 mg / ml were printed using a 27 G nozzle at 1.3 bar and 6 mm / s. Cross-linking times were 1 h for the first two concentrations and 30 min for the latter. Figure 5 shows representative images of hydrogels with cross-linking moieties of 2.92 mg / ml (Figure 5A), 8.16 mg / ml (Figure 5B), and 11.66 mg / ml (Figure 5C). Successful printing with good filament formation and finely tuned patterning was observed for all three hydrogel compositions, indicating that the hydrogel compositions with the cross-linking moieties at the concentrations studied are suitable for use in 3D printing.

[0144] Example 9. 3D printing using hydrogel compositions containing different concentrations of type I collagen as a rheology modifier The combined effect of HA as a rheology modifier component and collagen concentration on the printability of hydrogels was evaluated. Hydrogel compositions as disclosed in Example 4 containing modified concentrations of collagen type I as the second rheology modifier component were used for testing. Three hydrogel compositions were prepared containing the same concentration of HA as the composition disclosed in Example 4 as the rheology modifier. All three hydrogel compositions also contained a crosslinking component of 5.83 mg / ml. The tested neutralized human collagen type I component concentrations in the hydrogels were 0.18 mg / ml, 0.56 mg / ml, and 1.09 mg / ml. For all investigated hydrogel compositions, printing parameters of 0.9 bar and 6.00 mm / s were used, and all samples were printed in two layers and 20 mm x 20 mm lattice structures with a line distance of 1.0 mm. A 32G nozzle was used for printing. Exemplary images showing 3D printed hydrogel compositions containing three different concentrations of human type I collagen as the secondary rheology modifier component are shown in Figure 6. Figure 6 presents representative images of hydrogels with human type I collagen concentrations of 1 mg / ml (Figure 6A), 3.1 mg / ml (Figure 6B), and 6.0 mg / ml (Figure 6C). Increased cross-linking and improved shape fidelity were observed in the hydrogel compositions with increasing collagen concentration.

[0145] Example 10. 3D printing using hydrogel compositions containing different concentrations of HA as a rheology modifier component The hydrogel crosslinking component ratios and reagents included in Example 4 were used. Hydrogels with different concentrations of HA as the rheology modifier component were prepared and their printability was examined. No type I collagen was added as a rheology modifier to these hydrogels. Final HA concentrations of 0.625mg / ml, 1.25mg / ml and 3.125mg / ml in the hydrogel were tested. Hydrogels were printed using 1.3 bar, 8mm / s to 3D print 6 layered lattices with a strand distance of 2.50mm.

[0146] The results shown in Figure 7 show that the concentration of HA has a clear effect on the printing quality and shape fidelity of the hydrogel. A concentration of 0.625 mg / ml of HA in the hydrogel (Figure 7A) is not optimal and appears to be too low for the multilayer hydrogel to retain its shape. The hydrogel with a HA concentration of 3.125 mg / ml (Figure 7C) is stiffer compared to the other tested concentrations, but the filaments spread during printing of the multilayer 3D structure, and therefore this concentration is not optimal. The hydrogel with 1.25 mg / ml of HA as a rheology modifier (Figure 7B) showed the best printability and shape fidelity quality of the tested concentrations.

[0147] Example 11. 3D printing using hydrogel compositions containing various rheology modifiers Several different combinations of rheology modifier components in the hydrogel composition were investigated and their printability was tested. The hydrogel composition of Example 4 was used with modifications to replace the human collagen type I component with other proteins. Human fibronectin in PBS with a final concentration of 0.8 mg / ml in the hydrogel was studied. Additionally, human recombinant laminin 521 (Biolamina) with a final concentration of 0.02 mg / ml in the hydrogel, and human albumin (Sigma) with a final concentration of 9.1 mg / ml in the hydrogel were investigated. After mixing the components, the hydrogel was allowed to crosslink for 1 hour at room temperature. All prepared hydrogels were printed at 0.9 bar and 6.0 mm / s using a 32G needle.

[0148] Figure 8 presents representative hydrogel lattices generated using hydrogel precursor compositions with fibronectin and HA (Figure 8A), laminin and HA (Figure 8B), and albumin and HA (Figure 8C) as the rheology modifier components at the concentrations indicated. The hydrogel composition containing laminin and albumin exhibited rapid crosslinking and behaved very similarly to the hydrogel containing human type I collagen as the rheology modifier. The hydrogel containing fibronectin was printable but showed increased filament spreading and reduced printability compared to the other two investigated hydrogel compositions containing laminin and albumin. These results indicate that there are several potential combinations of rheology modifiers that can be used in hydrogel precursor compositions and that will result in good crosslinking and shear thinning properties in the hydrogel.

[0149] Example 12. Observation of shape fidelity of 3D printed hydrogel compositions in 3D hydrogel cylinder structures To observe the shape fidelity of 3D printed hydrogels, the hydrogel composition of Example 4 was used.

[0150] 3D cylinders with a diameter of 15 mm and a height of 1 mm were printed. The line distance used was 400 μm, and the alternating layers were at a 90° angle. Figure 9 shows two exemplary perspective views A and B of the printed 3D structure with 12 printed layers using a 100 μm print nozzle immediately after printing. These results show that the hydrogel composition prints well layer by layer and has good shape fidelity when printed in a 3D cylindrical shape.

[0151] Example 13. Observation of Shape Fidelity of 3D Printed Hydrogel Compositions as a Function of Time To further investigate the shape fidelity of the hydrogel composition, the filament thickness of the hydrogel was measured as a function of time. The hydrogel composition of Example 4 was also used in this experiment. The hydrogel was 3D printed in a grid with a 6-layer crisscross pattern with dimensions of 15 mm x 15 mm. A distance of 2.50 mm was used between the printed lines, with alternating layers at a 90° angle. Samples were imaged immediately after printing and after immersion in PBS at +37°C for 7 days. The thickness of the printed hydrogel filaments was quantified using Image J. Results are presented as relative filament thickness, calculated as the ratio of the filament thickness at the time the sample was taken to the filament thickness immediately after printing. Figure 10A shows the calculated relative filament thickness of the 3D printed hydrogel as a function of time, where D0 indicates the relative filament thickness at day 0 and D7 indicates the relative filament thickness at day 7. Corresponding images of the printed grids at day 0 (D0) and day 7 are shown in Figures 10B and 10C, respectively. These results demonstrate that the shape fidelity of the hydrogel does not change significantly over the observed period. All holes (36 / 36) of the printed hydrogel lattice remained open throughout the 7-day observation period from day 0 (Figure 10B) to day 7 (Figure 10C). Only a 12% increase in relative filament thickness was observed over the 7-day culture period.

[0152] Example 14. Cells and cell culture Human adipose-derived stem cells (hASCs) were mechanically and enzymatically isolated from subcutaneous adipose tissue samples as disclosed in the art. hASCs were then cultured in medium containing DMEM / F-12 supplemented with 5% human serum (AB male, from BioWest, Nuaille, France, HIV tested), 1% GlutaMAX™ and 1% penicillin / streptomycin. Human ASCs were passaged at confluence using TrypLE™ and used for bioprinting at passages 4-5. For bioprinting, hASCs were detached by the enzymatic action of TrypLE™, centrifuged and resuspended in culture medium for counting. hASCs were then centrifuged, the supernatant removed and the cells resuspended in culture medium and cultured at 1.1×10 6 It was mixed with the hydrogel precursor composition at a cell density of cells / ml to obtain a printable bioink for use in bioprinting.

[0153] In another experimental setup, hASCs were cultured for 7 days to differentiate into corneal stromal-like cells before being used for bioprinting. For differentiation, hASCs were cultured at 7000 cells / cm in medium containing Advanced DMEM (Gibco™), 1% GlutaMAX™, 1% penicillin / streptomycin, supplemented with 10 ng / mL basic fibroblast growth factor (bFGF), 0.1 mM ascorbic acid-2-phosphate, and 1 μM retinoic acid. 2 Cells were plated onto T75 cell culture flasks at a cell density of 1000 x 1000. For printing, pre-differentiated cells were prepared as described for hASCs.

[0154] Example 15. Biocompatibility of 3D Printed Hydrogel Compositions The biocompatibility of the hydrogel composition disclosed in Example 4 was evaluated using hASC and hASC-derived corneal stromal-like cells derived from 3D printed lines and cylinders. Hydrogel compositions were prepared and mixed with either hASC or hASC-derived corneal stromal-like cells to obtain two different bioinks. The bioinks were 3D printed in two layers of parallel lines. The dimensions of the printed area were 20 mm x 20 mm, with a line distance of 1.00 mm. 3D cylinders with a diameter of 15 mm and a height of 800 μm were also 3D printed. The line distance used was 400 μm. The alternating layers were at a 90° angle. After 3D printing, the printed lines and cylinders were further crosslinked and stabilized at +37°C, 5% CO2 for 20 minutes before immersing in cell culture medium or sterile PBS. For printing of higher 3D cylinder samples, a stabilization period of 1 hour was used before immersing the printed hydrogel in medium or sterile PBS.

[0155] Cell viability after printing was determined using PrestoBlue™ cell viability reagent (Invitrogen) 1, 3 and 7 days after printing. For PrestoBlue® analysis, three samples of each time point from both cell types were washed once with DPBS (Lonza) and PrestoBlue® reagent diluted 1:10 (v / v) in cell culture medium was added to the samples. After 1 h incubation at 37° C., 100 μL aliquots of PrestoBlue® medium were collected in triplicate from each sample on a 96-well plate and their fluorescence was measured using a Viktor 1420 Multilabel Counter (Wallac, Turku, Finland) at excitation and emission wavelengths of 544 nm and 590 nm, respectively.

[0156] Fluorescence values ​​for the media samples are shown in Figure 11. D0 indicates fluorescence on day 0, D3 indicates fluorescence on day 3, and D7 indicates fluorescence on day 7. Figure 11A shows fluorescence in a 3D printed bilayer bioink line, and Figure 11B shows fluorescence in a 3D printed solid 3D cylinder with this cell type. These results indicate that the hydrogel used with cells to produce the printable bioink is biocompatible with multiple cell types and is capable of supporting cell viability after printing. The increase in fluorescence over time indicates that the hydrogel used with cells to produce the printable bioink is also capable of promoting cell proliferation with multiple cell types after printing. Cell morphology for both printed cell types was assessed with IF staining of actin filaments of cells after D7. Briefly, printed lines were fixed with 4% PFA for 30 min, washed, and permeabilized with 0.1% Triton-X-100 in PBS for 15 min at room temperature, followed by blocking with 5% BSA in PBS for 1 h at room temperature. Phalloidin (Sigma) was diluted in a 5% BSA-PBS solution at a ratio of 1:100 and incubated for 1 h at room temperature. The samples were washed three times with PBS and immunofluorescence images were taken with an Olympus IX 51 fluorescent microscope. IF images of hASC and hASC-derived corneal stromal-like cells are shown in Figure 11C and D, respectively (scale bar 500 μm). Both cell types had elongated cell morphology, indicating good biocompatibility and cell and tissue maturation in the bioink.

[0157] Example 16. Observation of the effect of rheology modifier Mw on hydrogel properties The effect of the composition and Mw of the rheology modifier for the hydrogel composition disclosed in Example 4 was evaluated. A hydrogel composition with high Mw HA (Mw=1200-1900 kDa) and human type I collagen as the rheological component disclosed in Example 4 was used as a control (basic bioink). A similar hydrogel composition to that disclosed in Example 4 without human type I collagen (collagen-free bioink) was also investigated. Finally, the rheological component of the hydrogel disclosed in Example 4 was replaced with low Mw HA (Mw 100-300 kDa). The injectability and shear thinning properties of these hydrogel compositions were evaluated using a TA instruments DHR-II rheometer under continuous flow (0.01-10 s) after 10 min and 1 h of crosslinking at room temperature. -1 ) were determined. The base bioink composition showed the highest viscosity after 10 minutes (FIG. 12) and 1 hour (FIG. 13) of crosslinking. The viscosity of the base bioink was higher compared to a similar hydrogel composition without collagen. The hydrogel composition with low Mw HA showed the lowest viscosity values ​​at both time points. These results indicate that the Mw of HA used as a rheology modifier has a significant effect on the viscosity and printability of the developed hydrogels. Furthermore, the combined effect of using both high Mw HA and human type I collagen as rheology modifiers is beneficial to the printability of the hydrogel composition.

[0158] Example 17. Effect of human type I collagen on hydrogel properties The effect of human collagen type I on the hydrogel composition disclosed in Example 4 was evaluated. To this end, the hydrogel composition disclosed in Example 4 was compared to a similar hydrogel composition without the human collagen type I component. Both hydrogels were crosslinked at room temperature for 1 hour. Elasticity and filament formation were then evaluated by slightly pushing the hydrogel out of the syringe and pulling the hydrogel. The hydrogel composition containing human collagen type I showed excellent filament formation and elasticity (FIG. 14A). The hydrogel containing collagen was stretched into long fibers. The hydrogel containing collagen was sticky and did not detach from the substrate even after multiple stretches. The hydrogel without collagen type I was unable to form fibers and showed poor elasticity (FIG. 14B). Both hydrogels were 3D bioprinted in two layers of parallel lines as included in Example 15. The printed lines were placed in cell culture medium and examined for adhesion to the substrate and shape fidelity. The hydrogel composition containing collagen I remained attached to the printed substrate and maintained the morphology of the printed lines (FIG. 15A). In contrast, the hydrogel composition without type I collagen peeled off from the printing substrate in cell culture medium and lost the printed organized line pattern (Figure 15B). These results indicate that the combination of human type I collagen and high Mw HA produces a unique hydrogel composition with excellent printability, elasticity, adhesion and filament formation.

[0159] Example 18. Effect of Mw on HA-DA-CDH synthesis and hydrogel composition HA-DA-CDH was synthesized as described in Example 2, with the modification of using high Mw HA (Mw=1200-1900 kDa). The synthesized HA-DA-CDH was examined for preparing the hydrogel composition included in Example 4. For this, HA-DA-CDH was dissolved in 1xPBS. The solution was incubated at +37°C for 5 hours with continuous shaking. HA-DA-CDH was not soluble in 1xPBS up to a concentration of 10 mg / ml (Figure 16A). TNBS assay was used to determine the degree of synthesis of the prepared HA-DA-CDH components. The synthesized components were not dissolved in the reagent solvent (Figure 16B), and quantification by TNBS assay could not be performed. These results indicate that high Mw HA is not suitable for the synthesis of HA-DA-CDH for preparing hydrogel compositions.

[0160] The foregoing description provides a complete and informative description of the best mode currently contemplated by the inventors for carrying out the invention, as non-limiting examples of specific implementations and embodiments. However, it will be apparent to those skilled in the art that the invention is not limited to the details of the above-described embodiments, and that it may be implemented in other embodiments using equivalent means, or in different combinations of embodiments, without departing from the characteristics of the invention.

[0161] Moreover, some of the features of the exemplary embodiments disclosed above may be used to advantage without the corresponding use of other features. Thus, any suitable combination of embodiments or aspects may be made. Any combination of aspects or embodiments disclosed herein may also be made without at least one non-essential feature disclosed in the aspect or embodiment.

[0162] Different non-binding exemplary aspects and embodiments have been described above. The aforementioned embodiments are merely used to illustrate selected aspects or steps that may be utilized in different implementations. Some embodiments may be presented with respect to certain exemplary aspects only. It should be understood that the corresponding embodiments may also be applied to other exemplary aspects. The appended claims define the scope of protection. Any method, process, use, product or apparatus disclosed in the description or drawings and not covered by the claims should not be understood as an embodiment of the claimed invention, but is provided as an example useful for understanding the claimed invention.

[0163] References Biomacromolecules 2013,14,7,2427-2432,Published: May 30, 2013,https: / / doi.org / 10.1021 / bm400612h Koivusalo L. et al.Biomaterials 2019,225, Published September 23, 2019, https: / / doi.org / 10.1016 / j.biomaterials.2019.119516

Claims

1. A hydrogel precursor composition for bioink, comprising: i. a. A plurality of carbohydrazide groups (-CONHNH2), wherein the carbohydrazide groups are conjugated to 5-20% of the disaccharide repeating units of the HA component of the first component via a linker L1, where the linker is -NHNH-. And b. A plurality of catechol groups, wherein the catechol groups are conjugated to 1-20% of the disaccharide repeating units of the HA component of the first component via a linker L2, where the linker is -NH(CH2)2-. A first component comprising hyaluronic acid (HA) conjugated thereto, ii. A second component comprising hyaluronic acid (HA) conjugated to a plurality of aldehyde groups (-CHO), wherein the aldehyde groups are conjugated to 5-50% of the disaccharide repeating units of the HA component of the second component via a linker L3, where the linker is -NHCH2-. And iii. A rheology modifier component, comprising, The aldehyde groups are configured to form a crosslink with the carbohydrazide groups, the rheology modifier component comprises hyaluronic acid and collagen having a molecular weight of 1200 kDa to 1900 kDa, and the rheology component constitutes 2-50% by weight of the total weight of the hydrogel precursor composition. A hydrogel precursor composition characterized by this.

2. The hydrogel precursor composition according to claim 1, wherein the rheology modifier component of the hydrogel precursor composition further comprises human fibronectin having a molecular weight of 440-530 kDa.

3. The hydrogel precursor composition according to claim 1 or 2, wherein the rheology modifier component of the hydrogel precursor composition further comprises laminin.

4. The hydrogel precursor composition according to claim 1, wherein the at least one rheology modifier component comprises one or more extracellular matrix proteins selected from gelatin, laminin, fibronectin, vitronectin, and fragments and / or mixtures thereof.

5. A bioink that can be obtained by mixing the hydrogel precursor composition according to claim 1 with a fluid containing cells.

6. Use of the hydrogel precursor composition according to claim 1 or the bioink according to claim 5 for additive manufacturing.

7. A method for 3D printing the bioink according to claim 5, comprising: a) mixing the hydrogel precursor composition according to claim 1 with a fluid containing cells; b) crosslinking the first component and the second component of the hydrogel precursor composition in the fluid; c) 3D printing the bioink when the viscosity of the bioink is 200 to 2000 Pa·s. A method comprising the above steps.

8. The carbohydrazide group is conjugated to 9 to 15% of the disaccharide repeating units of the HA component of the first component via a linker L1, where the linker is -NHNH-; The catechol group is conjugated to 2 to 10% of the disaccharide repeating units of the HA component of the first component via a linker L2, where the linker is -NH(CH2)2-; The aldehyde group is conjugated to 9 to 15% of the disaccharide repeating units of the HA component of the second component via a linker L3, where the linker is -NHCH2-; and The rheology modifier component constitutes 9 to 40% by weight of the total weight of the hydrogel precursor composition, the hydrogel precursor composition according to claim 1. **Claim 9**: The carbohydrazide group is conjugated via a linker L1 to 12 - 13% of the disaccharide repeating units of the HA component of the first component, where the linker is -NHNH-. The catechol group is conjugated via a linker L2 to 3 - 6% of the disaccharide repeating units of the HA component of the first component, where the linker is -NH(CH2)2-. The aldehyde group is conjugated via a linker L3 to 11 - 13% of the disaccharide repeating units of the HA component of the second component, where the linker is -NHCH2-, and The rheology modifier component constitutes 11 - 23% by weight of the total weight of the hydrogel precursor composition, the hydrogel precursor composition according to claim 1. **Claim 10**: The collagen is type I collagen having a molecular weight of 250 - 300 kDa, the hydrogel precursor composition according to claim 1. **Claim 11**: The collagen is type I collagen having a molecular weight of 250 - 300 kDa, the hydrogel precursor composition according to claim 8. **Claim 12**: The collagen is type I collagen having a molecular weight of 250 - 300 kDa, the hydrogel precursor composition according to claim 9. **Claim 13**: A bioink obtainable by mixing the hydrogel precursor composition according to claim 8 with a fluid containing cells. **Claim 14**: A bioink obtainable by mixing the hydrogel precursor composition according to claim 9 with a fluid containing cells.

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