Hybrid supramolecular biomaterial for bioprinting and cell culture

Abstract

The present invention relates to a hybrid supramolecular biomaterial for bioprinting and cell culture, wherein the composition is comprised of functionalized proteinaceous biopolymer and / or polysaccharide biopolymer. The present invention further relates to a biological scaffold comprised of hybrid supramolecular biomaterial and methods for the production of a hybrid supramolecular biomaterial. The present invention furthermore relates to the use of the hybrid supramolecular biomaterial for cell culture, organoid culture and / or bioprinting.

Description

[0001] HYBRID SUPRAMOLECULAR BIOMATERIAL FOR BIOPRINTING AND CELL

[0002] CULTURE

[0003] Description

[0004] The present invention relates to a hybrid supramolecular biomaterial for bioprinting and cell culture, wherein the composition is comprised of functionalized proteinaceous biopolymer and / or polysaccharide biopolymer. The present invention further relates to a biological scaffold comprised of hybrid supramolecular biomaterial and methods for the production of a hybrid supramolecular biomaterial. The present invention furthermore relates to the use of the hybrid supramolecular biomaterial for cell culture, organoid culture and / or bioprinting.

[0005] Bioprinting has become an important tool for fabricating regenerative implants, in vitro cell culture platforms and biological scaffolds, which are used also to culture organoids either added onto the printed structure or embedded within the printed hydrogel. Organoids, which are miniaturized, organized structures formed by the assembly of multiple cell types, and that mimic the function of a specific organ, have the potential to revolutionize personalized medicine, as they can be used to study mechanisms of disease acting within human tissues. An organoid is a self-organized 3-dimensional (3D) cellular aggregate, often formed in presence of a biomaterial, typically derived from healthy stem cells, induced pluripotent stem cells (iPSCs) or cancer cells that closely mimic the complexity of an organ. Their expansion capacity, differentiation potential and self-assembly properties make them highly interesting structures for patient-specific disease modeling and other tissue engineering applications. The microenvironment regulates and structures the growth of the cell into an organoid. Given the importance of the microenvironment, there have been numerous tissue engineering attempts to imitate the microenvironment using biomaterials.

[0006] Most organoids are grown within an extracellular matrix-based hydrogel obtained from a mixture of proteins derived from the basal membrane of a mouse sarcoma (a type of tumor). This compound is known as Matrigel, Geltrex, Cultrex, or Basal Membrane Extract, among others, depending of the producer. A hydrogel is a biphasic polymer material, a mixture of porous, permeable solids and interstitial fluid comprising a network, that swells but does not dissolve in water. However, basal membrane extracts (i.e. Matrigel) have limited tunability, are difficult to shape into complex structures and exhibit poor reproducibility, and therefore remain constrained in their use. The main disadvantages of Matrigel is that it is of tumor origin and has a poorly defined chemical composition which results in large batch-to-batch variations when used in cell culture and / or tissue engineering and may pose significant safety issues in potential clinical settings. Moreover, conventional brands, such as Matrigel, are non-chemically defined and contain some unknown components, and therefore cannot safely be used in clinical settings. Furthermore, Matrigel is not printable, as it has poor shape fidelity due to its soft mechanical properties. More recently, synthetic Vi hydrogels and natural origin hydrogels (obtained from proteins and polysaccharides) have been engineered for bioprinting, providing a more chemically defined structure and thus exhibiting more controllable properties. The printing of synthetic hydrogels allows for increased control of cell-cell and cell-matrix interactions, for example to induce cell alignment or for controlling cell positioning in 3D. These matrices support the formation of differentiated cell tissues from stem cells and can also be used to grow cancer cells as disease models. Common hydrogels used in bioprinting and 3D tissue culture include polyethylene glycol (PEG) and its derivatives. Another problem arising from conventional hydrogels is the lack of ability to guide cell behavior or cell signaling and to provide bioactive signals recognized by cells. Even though PEG is biocompatible, it does not show any cell attachment sites, and it does not promote any specific or desired cell behavior, unless it is considerably modified by mixing with other materials such as chemically binding bioactive proteins and peptides.

[0007] In bioprinting strategies, cells are mixed with a hydrogel and fabricated into a construct of the desired shape, following a computer design. These constructs may be supplemented with growth factors to create a microenvironment for optimal tissue growth. To enable good shape fidelity however, printable hydrogels require specific viscoelastic and other biomechanical properties that often do not facilitate cell functionality. In regard to performing organoid culture, which requires a matrix that can be remodeled to enable cell assembly and growth, these printable biomaterials have proven to be suboptimal. In fact, cells thrive in hydrogels with low mechanical properties, for example, low stiffness, which in turn have poor structural stability.

[0008] A major challenge in 3D tissue culture, and even more so in 3D printing and bioprinting is that, in terms of mechanical properties, stiff materials are needed so that the printed object can retain its shape after printing and during their subsequent culture. However, embedded (and bio-printed) cells require soft microenvironments (which can be as low as ~ 100 Pa, elastic modulus) in order to thrive, migrate inside the gel and form tissue structures and organoids. Most available hydrogels usually form highly elastic, stiff covalent networks, while gels with low mechanical strength easily undergo deformation and loss of the printed features.

[0009] Considering the above, there is a need in the art for a biomaterial suitable for bioprinting and suitable for cell or organoid culture allowing for high-resolution bioprinting as well as improved post-printing characteristics such as shape fidelity, enhanced cellular interactions, facilitation of cellular self-assembly and biocompatibility for various cell types and organoids and that exhibits low batch-to-batch variability. The biomaterial should also be easy to synthesize at low cost. Moreover, the hydrogel needs to have adjustable mechanical properties with tunable biodegradability to adapt or mimic the tissue microenvironment and to be suitable for a broad range of applications and cell types. It is an object of the present invention, amongst other objects, to address the above need in the art. The object of the present invention, amongst other objects, is met by the present invention as outlined in the appended claims.

[0010] Specifically, the above object, amongst other objects, is met, according to a first aspect by a hybrid supramolecular biomaterial for bioprinting and cell culture, wherein the hybrid supramolecular biomaterial comprises both covalent and non-covalent bonds and is comprised of a proteinaceous biopolymer and / or a polysaccharide biopolymer, wherein said biopolymer comprises one or more free groups selected from the group consisting of amino groups, carboxylic acid groups, alcohol groups and thiol groups, preferably amino groups, wherein the biopolymer is functionalized via said free groups with acrylic and / or alkene moieties, wherein the biopolymer has a degree of covalent modification (DocM) of between 40 to 90%, preferably 50 to 80 %, more preferably 60 to 70%, and wherein the biopolymer comprises at least one other functional moiety providing a degree of is supramolecular modification (DosM), wherein the at least one other functional moiety is non covalently linked to a modified cyclic oligosaccharide with an unsaturated double bond, preferably an acrylated cyclic oligosaccharide with an unsaturated double bond. Cell culture are referred to herein may be 2D or 3D cell culture, preferably 3D cell culture. Suitable modified cyclic oligosaccharide with an unsaturated double bond may be cyclodextrins, such as alpha, beta, and gamma cyclodextrin. However, molecules such as cucurbituril or crown ethers, cryptophanes may also be used.

[0011] The hybrid supramolecular biomaterial of the present invention comprises both covalent bonds and non-covalent, supramolecular bonds based on host-guest interactions. These supramolecular interactions tend to be weaker than covalent bonds meaning that these bonds easily break due to, i.e., cell-driven mechanical stresses. A hydrogel relying solely on supramolecular bonds displays limited stability for long-term tissue culture and has poor shape fidelity post-printing. For a biomaterial, such as a hydrogel, to be suitable for bioprinting it should be able to maintain the shape of the cellular structure or tissue and have high post-printing fidelity, i.e. maintain the desired shape as programmed in the bioprinter. In view of this, the hybrid biomaterial of present invention comprises both covalent bonds and non-covalent, supramolecular bonds for improved bioprinting and cell embedding properties. The degree of covalent modification (DocM) used herein defines the % of the free amino acid and / or carboxylic acid groups on biopolymer being functionalized, wherein functionalized refers to the free amino acid and / or carboxylic acid groups being replaced by acrylic and / or alkene moieties, which are groups able to establish a covalent bond, when activated in presence of an initiator, preferably a photo-initiator.

[0012] The degree of supramolecular modification (DosM) as used herein defines the % of the free amino acid and / or carboxylic acid groups on biopolymer being functionalized with moieties able to form non-covalent (supramolecular) bonds, wherein the free amino acid and / or carboxylic acid groups are being replaced by the at least one other functional moiety, preferably an adamantane moiety. Finally, the biopolymer has a degree of functionalization (DoF), which defines the total % of functionalized biopolymer, i.e. the sum of the DosM and DocM, preferably providing a biopolymer being 100% functionalized. The DoF (and related DosM, DocM) of the biopolymer can be determined by using a 2,4,6 - trinitrobenzenesulfonic acid (TNBSA) assay and 'H-NMR, or any other suitable technique known in the art determining functionalization of biopolymers.

[0013] The non-covalent supramolecular interactions are reversible with minimal forces, and can be temporarily broken and reformed, also by forces exerted by the embedded cells. As such, the addition of these supramolecular interactions to the biopolymer functionalized to form covalent interactions, results in the formation of hybrid biomaterials (e.g. hydrogels) that are stiffer than the covalent-only counterpart known in the art. The hybrid supramolecular biomaterial preferably comprises of a proteinaceous biopolymer of gelatin that has been functionalized with methacrylates at 60% (DocM of 60%) and functionalized with adamantane for the remaining 40% (DosM), providing a 100% (DoF) functionalized hybrid supramolecular biomaterial of present invention. The hybrid supramolecular biomaterial comprising a DocM of 60% (comprises 60% covalent bonds, i.e. methacryloyl substitutions and 40% supramolecular reactive functional groups of adamantane) is stiffer than a known gelatin methacryloyl hydrogel with 60% DocM (GelMA60), which is comprised of only 60% functional groups able to form covalent bonds upon (photo)polymerization. The hybrid supramolecular biomaterial of present invention has 40% more side groups forming linking points within the biomaterial. Furthermore, the hybrid biomaterial is more viscoelastic than its GelMA60 counterpart as it comprises 40% functional groups that form supramolecular, reversible bonds that can break and reform, making the biomaterial more mobile so that cells can migrate more easily through the hydrogel. The disengaged supramolecular bonds can reform spontaneously over time.

[0014] The combination of the at least one other functional moiety, for example adamantane, which is non-covalently linked to a modified cyclic oligosaccharide which carries an unsaturated double bond, for example an acrylated cyclodextrin, is essential for increased stiffness, yet allowing for increased flexibility and biocompatibility of the hybrid supramolecular biomaterial of present invention. The supramolecular interactions or bonds (next to the covalent bonds) make the hybrid biomaterial of present invention stiffer and also provide a more marked viscoelastic behavior than its counterpart (which is a covalent gel not comprising supramolecular bonds). The supramolecular bonds present in the hybrid biomaterial of present invention are reversible and able to disengage, thereby making the biomaterial polymer network more mobile such that cells can migrate more easily through the gel. The disengaged supramolecular bonds reform spontaneously over time. Therefore, the combination of covalent interactions and supramolecular interactions is crucial for increased stiffness, while allowing for increased viscoelasticity and improved biocompatibility. According to a preferred embodiment the present invention relates to the hybrid supramolecular biomaterial, wherein said at least one other functional moiety is one or more selected from the group consisting of adamantane, cholesterol, phenyl, alpha cyclodextrin, gamma cyclodextrin, preferably adamantane.

[0015] According to another preferred embodiment the present invention relates to the hybrid supramolecular biomaterial, wherein said acrylic or alkene moieties is selected from the group consisting of acrylate, methacrylate, vinyl or norbornene, preferably methacrylate. Methacrylate is preferred because of the fast reaction kinetics and its ability to conjugate normal acrylates to the forming polymer mesh. Furthermore, no additional compounds are needed using methacrylates to form a covalent network, in contrast to for example norbornenes that need a thiol functionality.

[0016] According to yet a further embodiment, the present invention relates to the hybrid supramolecular biomaterial, wherein the proteinaceous and / or polysaccharide biopolymer(s) is one or more selected from the group consisting of a collagen-based polymer, gelatin, fibrin, silk, keratin, elastin, hyaluronic acid, alginate, laminaran, chitosan, and chitin, preferably gelatin. Gelatin is preferred as biopolymer, since it is derived from collagen, the main organic constituent of the natural extracellular matrix of mammals and has proven to be safe and suitable for cell culture purposes, including organoid culture. Importantly, the unique biocompatibility of the gelatin based hydrogel systems enable the fabrication of tissue constructs, while maintaining high cell viability (>50%). These protein-based polymers with complementary biological and mechanical properties further aid in approaching the functional and structural properties of native cellular microenvironments, using volumetric printing.

[0017] According to yet another preferred embodiment the present invention relates to the hybrid supramolecular biomaterial, wherein the biopolymer has a degree of supramolecular modification (DosM) of between 10 to 60%, preferably 15 to 60%, more preferably 20 to 50% is, even more preferably between 25 to 45%, most preferably between 30 to 40%.

[0018] According to a preferred embodiment the present invention relates to the hybrid supramolecular biomaterial, wherein the hybrid supramolecular biomaterial has a degree of functionalization (DoF) of at least 85%, preferably at least 90%, more preferably at least 95%, most preferably 100% DoF, wherein said biomaterial is covalently functionalized with acrylic and / or alkene moieties able to form covalent bonds and functionalized with the at least one other functional moiety. The biomaterial is functionalized with acrylic and / or alkene moieties as moieties able to form covalent bonds upon polymerization and functionalized with the at least one other functional moiety able to establish non-covalent (supramolecular interactions) with a cyclic oligosaccharide.

[0019] According to another preferred embodiment the present invention relates to the hybrid supramolecular biomaterial, wherein the modified cyclic oligosaccharide with an unsaturated double bond is acrylated cyclodextrin. The modified (preferably acrylated) cyclic oligosaccharide works like a “bucket” (host group) in which at least one other functional moiety, preferably adamantane, can fit (guest). This non-covalent or supramolecular bond (also termed guest-host interaction) is reversible as at least one other functional moiety can be pulled in and out of the “bucket” with minimal forces. A preferred embodiment of the present invention is a gelatin biopolymer network comprising acrylate side groups and adamantane side groups, mixed with acrylated cyclodextrin. Once the mixture is polymerized, it forms a hydrogel (biomaterial) in which covalent bonds are formed by the acrylate side groups and the acrylic group on the cyclodextrin via chain growth polymerization. At the same time adamantane can form the supramolecular bond with the cyclodextrin, thereby forming a hybrid supramolecular biomaterial according to a preferred embodiment of present invention.

[0020] According to yet another preferred embodiment the present invention relates to the hybrid supramolecular biomaterial, wherein the modified cyclic oligosaccharide with an unsaturated double bond is a cyclodextrin comprising 5 to 15 glucose units, preferably 6 to 10 glucose units, more preferably 6 to 8 glucose units, most preferably P-cyclodextrin. Preferably the at least one other functional moiety providing a degree of its supramolecular modification (DosM) should “fit” the modified cyclic oligosaccharide with an unsaturated double bond. Different cyclodextrin sizes have an effect on different guest molecules and in view of using adamantane, the P-cyclodextrin provides the most optimal fit.

[0021] According to a preferred embodiment the present invention relates to the hybrid supramolecular biomaterial, wherein the supramolecular biomaterial is a photo responsive or redox responsive hydrogel. The photo responsive hydrogel changes upon photoirradiation in its physical and / or chemical properties such as elasticity, viscosity, shape, and degree of swelling. Photoirradiation can be used either to stabilize the hydrogel after casting and / or molding, or after bioprinting. Photoirradiation may be an integral part of the bioprinting process, as it is the case of light-based bioprinting, namely volumetric bioprinting, digital light processing printing, stereolithography, or multiphoton polymerization.

[0022] According to another preferred embodiment the present invention relates to the hybrid supramolecular biomaterial, wherein said biomaterial is suitable for bioprinting and / or cell culture of one or more live cell types selected from the group consisting of endothelial cells, mesenchymal stromal cells, breast cancer cells, cholangiocytes and hepatocytes, either included as single cells or as organoids. The hybrid supramolecular biomaterial of present invention provides a more optimal microenvironment for cell culture and cell development and for cells to differentiate into their typical shape, to multiply and develop into more complex structures required for organoid and tissue formation, as compared to comparable covalent biomaterials.

[0023] The present invention according to a second aspect, relates to a biological scaffold comprised of a hybrid supramolecular biomaterial of present invention, as disclosed herein. The biological 1 scaffold is preferably a three-dimensional organized porous structure that is mimicking the cellular micro-environment and morphology of native tissue and / or an organ. Depending on the bioprinting technique used for providing the biological scaffold, the biological scaffold is comprised of a three- dimensional organized porous structure, wherein the porous structure has a pore size of between 1 to 1000 pm, preferably between 10 to 800 pm, more preferably between 20 to 500 pm, most preferably between 50 to 250 pm. The biological scaffold may be cell loaded or embedded with live cells.

[0024] According to another preferred embodiment, the present invention relates to the biological scaffold, wherein the biological scaffold has a negative resolution of at most 140 pm preferably at most 125 pm, most preferably at most 110 pm when printing channels and porous features (herein termed negative features resolution, or negative resolution) and / or a positive resolution of at most 28 pm, preferably at most 25 pm, most preferably at most 23 pm when printing positive features such as pillars, spikes and filaments, for example. Resolution measurements may be performed using stereomicroscopy imaging.

[0025] According to a preferred embodiment, the present invention relates to the biological scaffold, wherein the biological scaffold comprises at least 50%, preferable at least 60%, more preferably at least 70%, most preferably at least 80% viable cells, based on the total percentage of cells present in the scaffold.

[0026] According to a preferred embodiment the present invention relates to the biological scaffold, wherein the scaffold has a Young’s modulus of at least 1.00+0.15 kPa, preferably at least 3+0.15 kPa, most preferably at least 4.00+0.15 kPa compression, as determined by a Dynamic Mechanical Analyzer (DMA) 2980, Q800, TA Instruments. A DMA can be used to determine the compression modulus, stress relaxation kinetics, Tangent of Delta and elasticity ratio of the biomaterial of present invention. (Photo)rheological analysis was applied to study the covalent crosslinking kinetics and to find the storage and loss modulus, as well as the Tangent of Delta, of different bioresin concentrations.

[0027] According to another preferred embodiment the present invention relates to the biological scaffold, wherein said scaffold has a Tangent of Delta (Tan 5) of between 0.01 to 0.4, preferably 0.02 to 0.2, more preferably 0.05 to 0.15, as determined by a rheometer through a frequency sweep between 0.1 and 100 Hz.

[0028] The present invention according to a further aspect, relates to a method for providing a hybrid supramolecular biomaterial according to any one of claim 1 to 10, comprising the steps of a) providing an aqueous medium comprising at least 4% w / v, preferably at least 10% w / v, more preferably at least 15% w / v, even more preferably at least 20% w / v of a proteinaceous biopolymer and / or a polysaccharide biopolymer, wherein said biopolymer comprises one or more free groups selected from the group consisting of amino groups, carboxylic acid groups, alcohol groups and thiol groups, preferably free amino groups, b) functionalizing the biopolymer via said free groups with acrylic and / or alkene moieties, preferably methacrylic anhydride, to obtain a degree of covalent modification (DocM) of between 40 to 90%, preferably 50 to 80%, more preferably 60 to 70%, c) functionalizing the biopolymer with at least one other functional moiety, preferably adamantane isothiocyanate, to obtain a degree of supramolecular modification (DosM) of between 10 to 60%, preferably 15 to 60%, more preferably 20 to 50% is, even more preferably between 25 to 45%, most preferably between 30 to 40%, d) crosslinking the at least one other functional moiety non covalently to a modified cyclic oligosaccharide with an unsaturated double bond, preferably an acrylated cyclic oligosaccharide to provide the hybrid supramolecular biomaterial.

[0029] For example, the biopolymer is a proteinaceous biopolymer and / or a polysaccharide biopolymer comprising free amino groups and / or carboxylic acid groups, and these are functionalized via said free amino acid and / or carboxylic acid groups with acrylic and / or alkene moieties, preferably methacrylic anhydride, to obtain a degree of covalent modification (DocM). The supramolecular hybrid biomaterial is produced by first providing an aqueous medium comprising a proteinaceous biopolymer wherein the proteinaceous biopolymer comprises at least one free amino group. The free amino groups within the proteinaceous biopolymer are functionalized in step b) and c). In step b, preferably methacrylic anhydride is used to obtain a methacrylated proteinaceous biopolymer, for example gelatin methacryloyl (GelMA), having a degree of methacrylation facilitating the formation of covalent bonds between mathacrylic groups upon crosslinking of the biopolymer (DocM). In step c) the methacrylated proteinaceous biopolymer is subsequently preferably further functionalized with adamantane isothiocyanate to obtain a methacrylated proteinaceous biopolymer reacted with adamantane that enables the formation of non-covalent supramolecular bonds upon crosslinking of the biopolymer (DosM) to a modified cyclic oligosaccharide with an unsaturated double bond, such as acrylated cyclodextrin, to provide the hybrid supramolecular biomaterial.

[0030] The crosslinking density of the biomaterial influences the mechanical properties, structure, and porosity, as well as swelling and degradation ability. In step d) of the method of present invention, the acrylated cyclic oligosaccharide bonds covalently to the methacrylates to form a strong backbone within the biopolymer, and bonds via supramolecular, guest-host interactions with adamantane, allowing for flexibility and elasticity. The biomaterial comprises non-covalent bonds, the supramolecular interactions, which are reversible with minimal forces from the embedded cells. The supramolecular interactions form gels that are stiffer than the covalent-only counterpart for more side groups form linking points within the biomaterial. According to another preferred embodiment the present invention relates to the method wherein step d) is done via acryloyl chain growth polymerization of the biopolymer in presence of a photo- or redox-initiator, preferably a photo-initiator. One or more radical initiators may be used including any type of peroxides, N-bromoscuccinimide, diatomic halogens, Azobisisobutyronitrile, Ammonium Persulfate (APS) and / or 1,2-Bis(dimethylamino)ethane (TEMED).

[0031] According to another preferred embodiment the present invention relates to the method wherein the photo-initiator is a free radical photo-initatior, preferably lithium phenyl (2,4,6- trimethylbenzoyl) phosphinate (LAP), Eosin, Riboflavin, Irgacure or Tris(bipyridine)ruthenium(II) salts, preferably LAP.

[0032] According to another preferred embodiment the present invention relates to the method wherein the hybrid supramolecular biomaterial is comprised of metacrylated gelatin functionalized with adamantane (GelMA-ADA) having a DocM of between 60 to 70% and a DosM of between 30 to 40%. Preferably the biomaterial is comprised of 100% functionalized biopolymer, i.e. a DoF of 100%.

[0033] According to a preferred embodiment, the present invention relates to the method for the production of a hybrid supramolecular biomaterial for bioprinting, wherein step b) is performed at a temperature of between 35 to 60°C, preferably 45 to 55°C, more preferably 48 to 52°C.

[0034] According to yet another embodiment, the present invention relates to the method for the production of supramolecular biomaterial for bioprinting, wherein step c) is performed at a temperature of between 45 to 70°C, preferably 55 to 65°C, more preferably 58 to 62°C.

[0035] According to another embodiment the present invention relates to the method for the production of supramolecular biomaterial for bioprinting, wherein the functionalization of steps b) and / or c) are performed in a solvent solution, preferably in a PBS or a dimethyl sulfoxide solution. The solvent solution comprises for example methacrylic anhydride and / or adamantane isothiocyanate, depending on the desired DocM and DosM of the biomaterial being produced. For example, it may be favorable for enhanced adjustment of the mechanical properties of the supramolecular hybrid biomaterial towards matching the stiffness or softness in view of the cells or organoids being used in cell culture with the biomaterials.

[0036] The present invention according to a further aspect, relates to a method for bioprinting of a biological scaffold comprised of a hybrid supramolecular biomaterial as disclosed herein, wherein the bioprinting is selected from the group consisting of volumetric bioprinting (VBP, also known as Volumetric Additive Manufacturing (VAM)), lithography-based 3D printing, stereolithographic printing, digital light processing printing, multiphoton polymerization printing, inkjet bioprinting, laser induced-forward transfer bioprinting, acoustic bioprinting, electrowriting and extrusion-based bioprinting, preferably VBP. The hybrid supramolecular biomaterial of present invention is a versatile and scalable product with optimized synthesis conditions. The ease in tuning the mechanical properties by changing the supramolecular to covalent bond ratio suits bioprinting machines designed for conventional bioprinting, and likely future tissue engineering applications.

[0037] The present invention according to a further aspect, relates to the use of a biomaterial or biological scaffold of present invention as disclosed herein, for cell culture, organoid culture and / or bioprinting of biological scaffolds, immunotherapy assays, clinical studies and / or biological and biomedical research.

[0038] The present invention will be further detailed in the following examples and figures wherein:

[0039] Figure 1: Shows microscope images of fluorescence (green florescent protein, GFP) assay at

[0040] 1, 5 and 7 days after embedding a co-culture of GFP-labelled endothelial colony forming cells (ECFCs; green) and human mesenchymal stromal cells (not fluorescently labeled) within biomaterial scaffolds. Biomaterials as disclosed in Example 1 were included; (hybrid) biomaterial 40, 60 and 80 of present invention and their reference counterparts covalent 40, 60 and 80, as well as covalent 100. Matrigel was included as a positive control condition, which is the bottom right panel of each set of panels, as indicated in Figure 1. Visible stretching and interconnection of cells in all biomaterial was observed. However the longest cell networks are visible for the hybrid biomaterial 40 and 60, especially at days 5 and 7.

[0041] Figure 2: Shows the capillary network formation measurements of ECFCs, more specifically the average vessel length in pm in the various biomaterials as disclosed in Example 1. The hybrid biomaterial of present invention, more specifically the biomaterial 60 comprises the longest vessels, comparable to Matrigel. The hybrid biomaterials outperform the covalent counterpart biomaterials in view of vessel length formation.

[0042] Figure 3: The VBP printing resolution of positive features was measured by printing a star structure with sharp tips, and measuring the width of the tip from the moment it stops being the shape of the stereolithography (STL) design. Figure 3A shows a stereomicroscopy image of a representative VBP-printed star structure using the hybrid biomaterial 60 according to present invention. The image below zooms in on the width of the tip. Figure 3B shows the positive feature resolution measurements for Biomaterial 60 (hybrid) and GelMA 60 (covalent). Samples were printed with a Tomolite vl volumetric bioprinter (Readily3D SA, Lausanne, Switzerland). Samples were imaged with a stereomicroscope, and the size of the features was measured from the microscopy images. The positive resolution of the biomaterial 60 is significantly higher than GelMA, enabling to print smaller features with the hybrid biomaterial of present invention.

[0043] Figure 4: The VBP printing resolution of negative features measured for Biomaterial 60 and

[0044] GelMA 60. Figure 4A shows a stereolithography (STL) design (upper panel) and the lower panel shows a stereomicroscopy image of a representative VBP-printed design using the hybrid biomaterial 60 according to present invention. Figure 4B expresses the negative resolution in pm for Biomaterial 60 and GelMA 60. The negative resolution of the biomaterial 60 is significantly higher than GelMA, enabling to print smaller features with the hybrid biomaterial of present invention.

[0045] Figure 5: Shows the shape fidelity of bioprinted scaffolds comprised of the biomaterial of present invention. Shape fidelity was tested by printing ring-shaped scaffolds (Figure 5 A). A cylinder of 6 mm in width with an inner channel of 3 mm in width was printed with a volumetric printer. The printed scaffolds are put on their side and then the height and width were measured. The shape fidelity is determined by the height divided by the width of the ring structure to obtain a ratio. This ratio can vary between 1 (a perfect circle) and 0.5 (a completely collapsed circle). Biomaterial 40 and 60 and 80 have a similar shape fidelity compared to GelMA 100 (Figure 5B). Biomaterial 40 and 60 show significantly higher shape fidelity than their covalent counterparts.

[0046] Figure 6: Shows images of formed cell cluster structures derived of MCF10A cells and primary (patient-derived) human breast cancer cells embedded in Matrigel, in covalent 60 and in Biomaterial 60 after 1 and 7 days. Both MCF10A and the primary (patient-derived) breast cancer cells proliferate in Biomaterial 60.

[0047] Figure 7 : Shows liver organoid proliferation over time in Matrigel and a hybrid supramolecular biomaterial of present invention, more specifically biomaterial 60. The upper row depicts liver organoids in Matrigel during day 3, 5 and 7. Bottom row shows liver organoids in Biomaterial 60 during day 3, 5 and 7.

[0048] Figure 8: Shows measurement of the viscoelastic properties of the materials, expressed as the tan delta (tan 5), also known as phase angle, plotted against frequency in Hz, of the various biomaterials of present invention and their covalent reference counterparts. The stiffness of the biomaterial of present invention (hybrid) 40, 60, 80 and 100, and GelMA (covalent) counterparts was measured using a Discovery HR-2 rheometer (TA Instruments). The results show that increased viscoelasticity is associated with increased supramolecular bonds present in the biomaterial. The tan delta of the hybrid supramolecular biomaterials of the present invention is significantly higher than their covalent counterparts.

[0049] Figure 9: Shows the Young’s Modulus (stiffness) of the biomaterials of present invention

[0050] (hybrid) and their covalent reference counterparts. The Young's modulus describes the relationship between stress in force per unit area, and proportional deformation in the biomaterials. The covalent 100 comprising 100% covalent bonds is significantly the strongest biomaterial in view of the Young’s Modulus value. The Young’s Modulus is positively correlated to the number of covalent bonds of the biomaterial shown (gelatin-based hydrogels). However, the hybrid biomaterial of present invention showed increased Young’s Modulus values in comparison to their covalent counterparts.

[0051] Figure 10: Shows the soluble fraction (sol-fraction; Figure 11 A) and the swelling ratio (figure

[0052] 1 IB) of the biomaterials. The sol-fraction refers to the polymer fraction that remains un-crosslinked within the biomaterial network and is readily washed out after incubation at 37°C. This experiment showed a significantly lower swelling behavior for the hybrid supramolecular biomaterials of present invention, suggesting that a denser crosslinking network is formed than in the covalent counterparts, and therefore less swelling is observed.

[0053] Figure 11: Shows the mass loss of the different biomaterials by enzymatic degradation in % over 60 minutes. The biomaterials were incubated in the presence of enzyme collagenase, which accelerates the breakdown of the gelatin backbone of the biomaterial, and the mass loss of the different conditions was monitored over 60 minutes. This experiment was performed to confirm that the materials are biodegradable also after the different functionalization steps applied to form the hydrogel. It showed a significant decrease in hydrogel stability for the covalent GelMA formulations, as compared to the hybrid biomaterials of present invention. The mass loss is the highest in the GelMA 40 and 60, followed by the biomaterials of present invention. Complete mass degradation was observed for covalent 40 and 60 at 30 minutes and 45 minutes, respectively. All biomaterials, i.e. hydrogels, are completely degraded after 60 minutes. Figure 12: Shows the metabolic activity and cell viability of human mesenchymal stromal cells

[0054] (hMSCs) embedded within the hybrid biomaterials of present invention. Casted cellladen biomaterial samples were cultured for 7 days. Figure 12A shows the metabolic activity (expressed in relative fluorescence units) of cells measured at day 1, 5 and 7 of culture, both in the hybrid biomaterials and their covalent counterparts. Figure 12B shows the cell viability of the cells (expressed as the percentage of live cells out of the total number of cells in the sample) embedded in the hybrid biomaterials and their covalent counterparts, measured at day 1, 5 and 7 of culture.

[0055] Examples

[0056] Example 1 - Synthesis of hybrid biomaterial

[0057] Different hybrid supramolecular biomaterials, more specifically hydrogels comprising both covalent and non-covalent bonds comprised of a biopolymer of gelatin that was functionalized with methacrylates and adamantane (GelMa-ADA), were prepared for use in subsequent experiments below. The prepared biomaterials varied in the degree of covalent modification (DocM) and degree of supramolecular modification (DosM). As reference biomaterial, several hydrogels of metacrylated gelatin, comprising covalent bonds only, were included, having the same degree of covalent modification as compared to the hybrid biomaterials. As additional reference biomaterial a gelatin having 100% DocM was included. The following samples were prepared:

[0058] Hybrid supramolecular biomaterials according to the present invention:

[0059] GelMa-ADA 40 / 60 (gelatin methacrylated for 40% (DocM) and functionalized with adamantane for 60% (DosM)), i.e., having a 100% degree of functionalization (DoF), also referred to herein as Biomaterial 40 or Hybrid 40.

[0060] GelMa-ADA 60 / 40 (60% methacrylated, 40% adamantane), DoF 100%, also referred to herein as Biomaterial 60 or Hybrid 60.

[0061] GelMa-ADA 80 / 20 (80% methacrylated, 20% adamantane), DoF 80%, also referred to herein as Biomaterial 80 or Hybrid 80.

[0062] Reference covalent biomaterials:

[0063] GelMA 40 (gelatin methacrylated for 40%), also referred to herein as covalent 40.

[0064] GelMA 60 (gelatin methacrylated for 60%), also referred to herein as covalent 60.

[0065] GelMA 80 (gelatin methacrylated for 80%), also referred to herein as covalent 80.

[0066] GelMa 100 (gelatin methacrylated for 100%), also referred to herein as covalent 100. Hybrid GelMA-ADA synthesis:

[0067] GelMA with a predetermined DocM was dissolved in DMSO to reach a concentration of 10 w / v%. The solution was heated to 60°C, the temperature was kept constant throughout the synthesis. The amount of adamantane isothiocyanate (AITC) that needs to be added to the reaction was calculated as following: mAITC (mg) = 5 * mGelMA (g) * desired DocM. The calculated amount of adamantane isothiocyanate was added to the reaction after which the reaction was stirred for 5 hours. The reaction was stopped by cooling the solution to room temperature. The solution was centrifuged at 4000 rpm at room temperature for 5 minutes. The supernatant was collected and dialyzed against MilliQ water for 4 days at 4°C. This makes the solution turn white. After the dialysis, the solution was diluted to reach a final concentration of 2.5 w / v% with MilliQ. The solution was centrifuged at 4000 rpm at room temperature for 5 minutes. The supernatant was collected and heated to 50°C, after which the solution was sterile filtered. Following the sterile filtration, the solution is frozen at -80°C and lyophilized to yield the dry product.

[0068] Covalent GelMA synthesis:

[0069] Type A Gelatin was dissolved in a CB-Buffer (carbonate-bicarbonate buffer pH 9, 0.1 M concentration) to reach a 10 w / v% concentration. This solution was heated to 50°C for the Gelatin to dissolve and kept at this constant temperature throughout the synthesis. To reach a desired degree of functionalization (DoF), 0.71 pL of methacrylate anhydride (MA) per gram of gelatin per DoF was used in the reaction. So, MA (pL) = 0.71 * m gelatin (g) * DoF (%). The MA was added every 11 minutes for a total of 6 times starting at t = 0. After every addition of MA the pH of the reaction was stabilized with 5M NaOH to reach a pH of 9. 90 minutes after the first addition of MA, the reaction was stopped by dropping the pH of the reaction to 7.4 using IM HC1. The solution was centrifuged at 4000 rpm at room temperature for 5 minutes. The supernatant was collected and diluted to reach a concentration of 5 w / v%. The solution is then dialyzed against MilliQ water for 4 days at 4°C. After the dialysis the solution was diluted with MilliQ to reach a final concentration of 2.5 w / v%. The solution was then heated to 50°C and sterile filtered. Following the sterile filtration, the solution was frozen at -80°C, and lyophilized to yield the dry product.

[0070] Hydrogel preparation and photopolymerization

[0071] GelMA or GelMA-ADA stock solution was made in PBS at a 10 w / v% concentration. Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) stock solution was made in PBS at a 1 w / v% concentration. A stock solution of acrylated-P-cyclodextrin was made by in PBS at a 35 mM concentration. All stock solutions were heated to 37°C until completely dissolved. Afterwards, the stock solutions were combined and diluted to reach a final concentration of 5 w / v% gelatin-based material, 0.1 w / v% LAP, and a 1: 1 ratio of adamantane to cyclodextrin, which is dependent on the DosM of the material.

[0072] Example 2 - Formation of a network of capillary vessels in Endothelial cells The development of a 3D endothelial cell culture platform is demonstrated using the hybrid biomaterials of example 1. Endothelial cells are cells that line the wall of blood vessels and stretch and connect to form long and interconnected capillary networks and form blood capillaries in the body if these cells are placed in the right microenvironment. These cells grow and develop optimally in soft, low stiffness environments in the order of IkPa or less. Conventional soft hydrogels that possess these properties are most often too unstable post-printing, showing poor shape fidelity and issues in sustaining cell-culture for extended periods of time.

[0073] The endothelial colony forming cells (ECFCs) cells used in this experiment were designed to express green fluorescence protein to easily visualize them within the biomaterial to evaluate vessel formation. ECFCs, in combination with human mesenchymal stromal cells that further support ECFC growth, are embedded within the hybrid supramolecular biomaterials and their covalent counterparts and monitored for 7 days. At day 1, 3 (not shown), 5 and 7 fluorescence microscopy images are taken, Figure 1. The average length of vessels formed by interconnected cells, the number of junctions (connecting points; not shown) between different cells, and the total percentage area occupied by cells in the region (not shown) is measured from the acquired images (Figure 2).

[0074] The hybrid biomaterials of present invention showed an improved ability to form continuous capillary networks compared to their covalent counterparts and Matrigel, which is the current golden standard for vascularization assays and organoid culture. In particular, the best results were observed for Biomaterial 60, showing the largest average vessel length at both day 5 and day 7. The biomaterial of present invention provides an improved biocompatible microenvironment for culture formation of endothelial cells wherein cells can migrate and re-organize in comparison to the covalent reference biomaterials.

[0075] Example 3 - High volumetric printing resolution

[0076] The hybrid supramolecular biomaterial of present invention was used for high-resolution printing. High resolution printing was done with a volumetric bioprinter and demonstrated by measuring the positive and negative features of Biomaterial 60 bioprinted structures. Briefly, GelMA or GelMA- ADA stock solution was made in PBS at a 10 w / v% concentration. Lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) stock solution was made in PBS at a 1 w / v% concentration. A stock solution of acrylated-P-cyclodextrin was made by in PBS at a 35 mM concentration. All stock solutions were heated to 37°C until completely dissolved. Afterwards, the stock solutions were combined and diluted to reach a final concentration of 5 v / N% gelatin-based material, 0.1 v / N% LAP, and a 1 : 1 ratio of adamantane to cyclodextrin, which is dependent on the DosM of the material.

[0077] The solutions were dispensed into cylindrical borosilicate glass vials (0 10 mm), which were then loaded into a commercial volumetric 3D printer (Tomolite VI, Readily3D, Switzerland), equipped with a 405 nm laser, set to deliver an average light intensity of 11.98 mW / cm2within the printing volume. Prior to printing, the samples were cooled to 4°C to achieve physical gelation of the gelatin-based materials. Custom-designed STL files were loaded into the printer software (Apparite, Readily 3D, Switzerland). After the printing process, the vials were heated to 37°C and washed gently with 37°C PBS to retrieve the prints. To ensure homogenous crosslinking, the sample was submerged in 0.1 w / v% solution of LAP in PBS and irradiated for 1 minute in a UV oven in a post-curing step.

[0078] The positive resolution was measured by printing a star structure with sharp tips, and measuring the width of the tip from the moment it stops being the shape of the stereolithography (STL) design, see Figure 3A. The positive resolution of the biomaterial 60 is significantly higher than GelMA, enabling to print smaller features with the hybrid biomaterial of present invention ( Figure 3B).

[0079] The negative resolution was measured by printing small channels of different diameter, and measuring the smallest diameter that could be resolved while maintaining an open lumen (Figure 4A). The negative resolution of the biomaterial 60 is significantly higher than GelMA, enabling to print smaller features with the hybrid biomaterial of present invention (Figure 4B).

[0080] Example 4 — Shape fidelity of bioprinted scaffold

[0081] The shape fidelity of a bioprinted scaffold comprised of the biomaterial of present invention was measured. Shape fidelity was tested by printing ring-shaped scaffolds (Figure 5A). A cylinder of 6 mm in width with an inner channel of 3 mm in width was printed. The printed scaffolds are put on their side and then the height and width were measured. The shape fidelity is determined by the height divided by the width to obtain a ratio. This ratio can vary between 1 (a perfect circle) and 0.5 (a completely collapsed circle). The normalization of this ratio provides the shape fidelity of the printed structure.

[0082] Biomaterial 40 and 60 and 80 have a similar shape fidelity compared to GelMA 100 (Figure 5B). Biomaterial 40 and 60 also show significantly higher shape fidelity than their covalent counterparts, suggesting the added stiffness from the newly introduced host-guest interactions enhances the strength of the gel and aids in maintaining its original shape post-printing. The vessel of GelMA 40 and 60 collapsed hence not retaining shape. The printed vessels of Biomaterial 60 remained in good shape due to high shape fidelity resulting from the higher overall stiffness of the biomaterials. Example 5 Cell proliferation in hybrid supramolecular biomaterial

[0083] Epithelial cells from the mammary duct (breast duct tissue) were encapsulated in different biomaterials (see example 1), to study how the single cell suspension is able to grow into organoids, thanks to the ability of the biomaterial to support cell proliferation, and cell self-organization into organoids. This was tested both with a commercially available cell line derived from healthy breast duct tissue (MCF10A), and with patient-derived human breast cancer cells. The Biomaterial 60 supports proliferation and organoid formation of both cell types (Figure 6).

[0084] Similarly, liver adult progenitor cells were also embedded in the Biomaterial 60, derived from biopsies of human intrahepatic bile ducts and their ability to grow onto Biomaterial 60 was confirmed by formation of small organoids (Figure 7), outperforming the Matrigel.

[0085] Example 6 — Mechanical analysis hybrid supramolecular biomaterial

[0086] Photorheology experiments on hydrogel precursor solutions to determine the crosslinking kinetics were assessed using a DHR2 rheometer (TA Instruments, The Netherlands). Time sweep experiments were performed at a frequency of 1.0 Hz, angular frequency of 6.283 rad / s, with 5.0% constant strain at 21 °C (n = 3 independent samples). 30 seconds after the start of the measurement, the light source was activated (1200mha, AOMEES, China, X = 365 nm, intensity of 24 mW / cm2 for the remaining 2.5 minutes). Subsequently, frequency sweep experiments were performed at a frequency range from 0.1 Hz to 100 Hz with 5.0% constant strain at 21°C to calculate the Tangent of Delta (Tan 5). Subsequently, amplitude sweep experiments were performed at a frequency of 1.0 Hz at a strain rate from 0.1% to 100% strain at 21°C. A volume of 100 pF of gel was used with a gap size of 300 pm. A 20.0 mm parallel EHP stainless steel plate was used as geometry.

[0087] The results show that increased stiffness is associated with increased covalent bonds present in the biomaterial. The Tan 5 (loss modulus divided by storage modulus) of the hybrid supramolecular biomaterials of the present invention is significantly higher than their covalent counterparts, suggesting that the hybrid hydrogels have a higher liquid behavior, while being stiffer allowing for biocompatibility and adaptive microenvironment (Figure 8).

[0088] The stiffness of the following compositions Biomaterial 40, GelMA 40, Biomaterial 60 and GelMA 60 was measured using a dynamic mechanical analysis (DMA), (DMA 2980, Q800, TA Instruments). Briefly, hydrogel solutions were casted in a cylindrical mold (6 mm diameter, 2 mm height), and crosslinked for 10 minutes (Cl-1000, Ultraviolet Crosslinker, X = 365 nm, 1= 8 mW / cm2 UVP, USA). Samples were washed in PBS at 37°C overnight to reach equilibrium swelling.

[0089] The Young’s modulus of the biomaterials and their covalent counterparts is determined using a dynamic mechanical analysis (DMA). The Young's modulus describes the relationship between stress in force per unit area, and proportional deformation in an object. To assess the I S compressive properties, the samples were subjected to a strain ramp at 20% min- 1 strain rate until 30% deformation using a dynamic mechanical analyzer (DMA Q800, TA Instruments, The Netherlands). The compression modulus was calculated as the slope of the stress / strain curve in the 10-15% linear strain range (Figure 9). GelMA 100, comprising 100% covalent bonds is significantly the strongest. The Young’s Modulus is positively correlated to the number of covalent bonds of the biomaterial (i.e. hydrogel).

[0090] The soluble fraction (sol-fraction; Figure 10A) and swelling ratio (Figure 10B) of the biomaterials was measured using the wet and dry weights of the samples during a series of freeze- drying and rehydration steps, as described previously (M. Falandt et al., 2023, Advanced material Technologies, Spatial-Selective Volumetric 4D Printing and Single-Photon Grafting of Biomolecules within Centimeter-Scale Hydrogels via Tomographic Manufacturing). The solfraction refers to the polymer fraction that remains un-crosslinked within the biomaterial network and is readily washed out after incubation at 37°C.

[0091] Briefly, to assess sol-fraction of the hydrogel formulation, cylindrical samples (6 mm diameter, 3 mm height, n = 5 independent samples) were weighed immediately after crosslinking for their initial mass. Next, samples were placed in PBS and placed in the incubator at 37°C overnight. The next day, the hydrogel samples were weighed again, and their mass was measured as masswet,to- Subsequently the hydrogels were lyophilized, and the dry mass (massdry, to) was measured. The samples were stored in PBS again to ensure swelling of the dry gels and placed in the incubator at 37°C overnight. The wet mass of the hydrogels was measured as masswet,ti. The samples were lyophilized, and the mass of the dry samples was measured as massdry, u. The sol-fraction of the hydrogel formulations was calculated with the following formula:

[0092] The swelling ratio of the hydrogel formulations was calculated with the following formula:

[0093] Results showed a significantly lower swelling behavior for the hybrid supramolecular biomaterials of present invention, suggesting that a denser crosslinking network is formed than in the covalent counterparts, and therefore less swelling is observed. For Bioresin 80, a significantly higher swelling was observed than in GelMA 80. Furthermore, there was no significant difference between the different hybrid biomaterials.

[0094] Next, the biomaterials were monitored in view of their enzymatic degradation rate. The biomaterials were incubated in the presence of enzyme collagenase, which accelerates the breakdown of the gelatin backbone of the biomaterial, and the mass loss of the different conditions was monitored over 60 minutes (Figure 11). It showed a significant decrease in hydrogel stability for the covalent GelMA formulations, as compared to the hybrid biomaterials of present invention. The hybrid biomaterials are significantly more stable under enzymatic degradation conditions than the covalent GelMA controls. Where the hybrid mixes were all stable up to an hour of accelerated enzymatic degradation, the covalent GelMA conditions showed complete degradation starting at 30 minutes for the covalent 40 hydrogels. Proving an increase in stability for the hybrid hydrogel mixtures. It is important to notice that, under physiological tissue culture condition, all gels remained stable over culture times longer than 2 weeks (even 4 weeks for the breast tissue organoids).

[0095] Example 7 - Metabolic activity and cell viability of human mesenchymal stromal cells (hMSCs) embedded within the hybrid biomaterials

[0096] The metabolic activity and cell viability of human mesenchymal stromal cells (hMSCs) in a 3D cell culture platform is demonstrated using the hybrid biomaterials of example 1. hMSCs are stem cells that have the potential to differentiate into multiple cell lineages, and are therefore a highly interesting cell type for tissue engineering applications. These cells are also easy to obtain from patient samples and can be expanded to high yields in vitro. Therefore, hMSCs were used to assess the overall effect of the hybrid biomaterials on fundamental cellular behaviors.

[0097] HMSCs are embedded within the hybrid supramolecular biomaterials of present invention and their covalent counterparts and monitored for 7 days. At days 1 and 7 their metabolic activity was measured using a resazurin salt assay, which demonstrates high fluorescence levels when cells are in a metabolic state and are capable of reducing the reasazurin compound and indicating that they are functional within the biomaterials. Briefly, the embedded hMSCs were incubated in a resazurin sodium salt solution (1:10 resazurin to culture media ratio) for 4 hours, and the fluorescence of the medium was measured afterwards using a fluorescence plate reader.

[0098] While the metabolic activity (represented as relative fluorescence units (RFU)) increases in both the hybrid and covalent biomaterials over time, all hybrid biomaterials exhibit higher metabolic activity than their covalent counterparts, and the covalent 100 control, suggesting cells perform more optimally from a metabolic standpoint in the more viscoelastic environment provided by the hybrid biomaterials (Figure 12A).

[0099] The cell viability over 7 days of the embedded hMSCs was evaluated using a LIVE / DEAD assay (ThermoFischer Scientific, The Netherlands), according to manufacturer’s protocol. Live (stained with Calcein-AM) and dead cells (stained with ethidium homodimer- 1) within the hybrid biomaterials and their covalent counterparts were imaged using a fluorescent microscope. Cells were counted using image analysis software. The cell viability was calculated as the percentage of live cells over the total cell number of the acquired images. All hybrid and covalent biomaterials exhibited a decrease in cell viability over 7 days in culture, but at day 7, the hybrid biomaterials showed higher cell viability than their covalent counterparts, but not higher than the covalent 100 biomaterial (Figure 12B). Cell viability remained above 75% for all biomaterials.

Claims

Claims1. A hybrid supramolecular biomaterial for bioprinting and cell culture, wherein the hybrid supramolecular biomaterial comprises both covalent and non-covalent bonds and is comprised of a proteinaceous biopolymer and / or a polysaccharide biopolymer, wherein said biopolymer comprises one or more free groups selected from the group consisting of amino groups, carboxylic acid groups, alcohol groups and thiol groups, preferably free amino groups, wherein the biopolymer is functionalized via said groups with acrylic and / or alkene moieties, wherein the biopolymer has a degree of covalent modification (DocM) of between 40 to 90%, preferably 50 to 80 %, more preferably 60 to 70%, and wherein the biopolymer comprises at least one other functional moiety providing a degree of supramolecular modification (DosM), wherein the at least one other functional moiety is non-covalently linked to a modified cyclic oligosaccharide with an unsaturated double bond, preferably an acrylated cyclic oligosaccharide with an unsaturated double bond.

2. The hybrid supramolecular biomaterial according to claim 1, wherein said at least one other functional moiety is one or more selected from the group consisting of adamantane, cholesterol, phenyl, alpha cyclodextrin, gamma cyclodextrin, preferably adamantane.

3. The hybrid supramolecular biomaterial according to claim 1 or 2, wherein said acrylic or alkene moieties are selected from the group consisting of acrylate, methacrylate, vinyl or norbornene, preferably methacrylate.

4. The hybrid supramolecular biomaterial according to any one of the claims 1 to 3, wherein the proteinaceous and / or polysaccharide biopolymer(s) is one or more selected from the group consisting of a collagen-based polymer, gelatin, fibrin, silk, keratin, elastin, hyaluronic acid, alginate, laminaran, chitosan, and chitin, preferable gelatin.

5. The hybrid supramolecular biomaterial according to any one of claims 1 to 4, wherein the biopolymer has a degree of supramolecular modification (DosM) of between 10 to 60%, preferably 15 to 60 %, more preferably 20 to 50% is, even more preferably between 25 to 45%, most preferably between 30 to 40%.

6. The hybrid supramolecular biomaterial according to one of the claims 1 to 5, wherein the hybrid supramolecular biomaterial has a degree of functionalization (DoF) of at least 85%, preferably at least 90%, more preferably at least 95%, most preferably 100% DoF, wherein said biomaterial iscovalently functionalized with acrylic and / or alkene moieties able to form covalent bonds and functionalized with the at least one other functional moiety.

7. The hybrid supramolecular biomaterial according to any one of claim 1 to 6, wherein the modified cyclic oligosaccharide with an unsaturated double bond is acrylated cyclodextrin.

8. The hybrid supramolecular biomaterial according to any one of claim 1 to 7, wherein the modified cyclic oligosaccharide with an unsaturated double bond is a cyclodextrin comprising 5 to 15 glucose units, preferably 6 to 10 glucose units, more preferably 6 to 8 glucose units, most preferably P-cyclodextrin.

9. The hybrid supramolecular biomaterial according to any one of claims 1 to 8, wherein the supramolecular biomaterial is a photo responsive or redox responsive hydrogel.

10. The hybrid supramolecular biomaterial according to any one of the claims 1 to 9, wherein said biomaterial is suitable for bioprinting and / or cell culture of one or more live cells selected from the group consisting of endothelial cells, mesenchymal cells, breast cancer cell, cholangiocytes and hepatocytes.

11. A biological scaffold comprised of a hybrid supramolecular biomaterial according to any one of the claims 1 to 10.

12. The biological scaffold according to claim 11, wherein said scaffold has a Young’s modulus of at least 1.00+0.15 kPa, preferably at least 3+0.15 kPa, most preferably at least 4.00+0.15 kPa compression, as determined by a DMA 2980, Q800, TA Instruments.

13. The biological scaffold according to claim 11 or 12, wherein said scaffold has a Tangent of Delta (Tan 5) of between 0.01 to 0.4, preferably 0.02 to 0.2, more preferably 0.05 to 0.15, as determined by a Discovery HR-2 rheometer (TA Instruments) through a frequency sweep between 0.1 and 100 Hz.

14. A method for providing a hybrid supramolecular biomaterial according to any one of claim 1 to 10, comprising the steps of a) providing an aqueous medium comprising at least 4% w / v, preferably at least 10% w / v, more preferably at least 15% w / v, even more preferably at least 20% w / v of a proteinaceous biopolymer and / or a polysaccharide biopolymer, wherein said biopolymer comprises one or more free groupsselected from the group consisting of amino groups, carboxylic acid groups, alcohol groups and thiol groups, preferably free amino groups, b) functionalizing the biopolymer via said free groups with acrylic and / or alkene moieties, preferably methacrylic anhydride, to obtain a degree of covalent modification (DocM) of between 40 to 90%, preferably 50 to 80%, more preferably 60 to 70%, c) functionalizing the biopolymer with at least one other functional moiety, preferably adamantane isothiocyanate, to obtain a degree of supramolecular modification (DosM) of between 10 to 60%, preferably 15 to 60%, more preferably 20 to 50% is, even more preferably between 25 to 45%, most preferably between 30 to 40%, d) crosslinking the at least one other functional moiety non covalently to a modified cyclic oligosaccharide with an unsaturated double bond, preferably an acrylated cyclic oligosaccharide to provide the hybrid supramolecular biomaterial.

15. Method according to claim 14, wherein step d is done via acryloyl chain growth polymerization of the biopolymer in presence of a photo- or redox-initiator, preferably a photoinitiator.

16. Method according to claim 15, wherein the photo-initiator is a free radical photo-initatior, preferably lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP), Eosin, Riboflavin, Irgacure orTris(bipyridine)ruthenium(II) salts, preferably LAP.

17. Method according to any one of the claims 14 to 16, wherein the hybrid supramolecular biomaterial is comprised of metacrylated gelatin functionalized with adamantane (GelMA-ADA) having a DocM of between 60 to 70% and a DosM of between 30 to 40%.

18. Method for bioprinting of a biological scaffold of any one of the claims 11 to 13, wherein the bioprinting is selected from the group consisting of volumetric bioprinting (VBP, also known as Volumetric Additive Manufacturing (VAM)), lithography-based 3D printing, stereolithographic printing, digital light processing printing, multiphoton polymerization printing, inkjet bioprinting, laser induced-forward transfer bioprinting, acoustic bioprinting, and electrowriting and extrusionbased bioprinting, preferably VBP.

19. A use of a biomaterial or biological scaffold according to any one of claim 1 to 13 for cell culture, organoid culture and / or bioprinting of biological scaffolds, immunotherapy assays, clinical studies and / or biological and biomedical research.

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