WO2020234167A1

WO2020234167A1 - Printable hydrogel, method for generating a printable hydrogel, lyophilizate, printed product and 3d printing method

Info

Publication number: WO2020234167A1
Application number: PCT/EP2020/063653
Authority: WO
Inventors: Marcy Zenobi-Wong; Benjamin KESSEL
Original assignee: Eidgenoessische Technische Hochschule Zurich ETHZ
Current assignee: Eidgenoessische Technische Hochschule Zurich ETHZ
Priority date: 2019-05-17
Filing date: 2020-05-15
Publication date: 2020-11-26
Anticipated expiration: 2021-11-17

Abstract

The invention relates to a printable hydrogel comprising a plurality of microstrands comprising or consisting of a polymer, wherein the microstrands have a diameter of 1 µm to 500 µm and an aspect ratio of at least 100:1, and wherein said microstrands are entangled with each other. A further aspect of the invention is a lyophilizate comprising a plurality of microstrands comprising or consisting of a polymer, wherein the microstrands have a diameter of 1 µm to 500 µm and an aspect ratio of at least 100:1, and wherein said microstrands are entangled with each other. Furthermore, a method for generating a printable hydrogel comprising the step of providing a cross-linked hydrogel, passing said cross-linked hydrogel through a sieve comprising a plurality of openings, such that a plurality of microstrands having a diameter of 1 µm to 500 µm and an aspect ratio of at least 100:1 are generated, and passing said microstrands through a volume, such that the microstrands are entangled with each other, is provided. The invention further relates to a 3D printed product and a 3D printing method using the hydrogel.

Description

Printable hydrogel, method for generating a printable hydrogel, lyophilizate, printed product and 3D printing method

The present invention relates to a printable hydrogel, a method for generating a printable hydrogel, a lyophilizate for generating a printed hydrogel, a printed product which is printed using the printable hydrogel and a 3D printing method using the printable hydrogel.

Background of the Invention

Tissue engineered scaffolds often use hydrogels as the starting bioink materials (El- Sherbiny, I. M., & Yacoub, M. H. (2013). Hydrogel scaffolds for tissue engineering: Progress and challenges. Global Cardiology Science and Practice, 38; Merceron, T. K., & Murphy, S. V. (2015). Hydrogels for 3D bioprinting applications. In Essentials of 3D biofabrication and translation (pp. 249-270). Academic Press.) as they have similar hydration compared to native extracellular matrix and can be rendered more biomimetic through addition of moieties, which confer biological properties to the material. Generally, cell viability is excellent on the surfaces of hydrogels or within thin constructs. However, making larger cellular structures has proven challenging due to the nanometer pore size of most hydrogels. This restricts the access to nutrients and often reduces the cell viability in the material. The mass transport and hence viability of cells within a hydrogel can be greatly improved through the introduction of interconnected macroporosity.

Unfortunately, many current methods for making macroporous materials such as cryogelation gas foaming, electrospinning, porogen and emulsion templating, and phase separation are not fully compatible with cell encapsulation. Further, these methods are often restricted to the use of a single material and need to be post-seeded after fabrication, which prevents spatial organization of cell types and control over final cell density in various regions of the gel. Another significant disadvantage of currently used macroporous hydrogels is the difficulty in producing organization and controlled anisotropy of the pores, which would be highly important for guiding formation of cellular networks.

A cell compatible and relatively new method for producing macroporous materials has utilized the interaction of spherical microbeads, where the interpore space forms a naturally connected pore structure (Riley, L, Schirmer, L, & Segura, T. (2019). Granular hydrogels: emergent properties of jammed hydrogel microparticles and their applications in tissue repair and regeneration. Current opinion in biotechnology, 60, 1-8.; Sheikhi, A., de Rutte, J., Haghniaz, R., Akouissi, O., Sohrabi, A., Di Carlo, D., & Khademhosseini, A. (2019). Microfluidic-enabled bottom-up hydrogels from annealable naturally-derived protein microbeads. Biomaterials, 192, 560-568.; Li, F., Truong, V. X., Fisch, P., Levinson, C., Glattauer, V., Zenobi-Wong, M., ... & Frith, J. E. (2018). Cartilage tissue formation through assembly of microgels containing mesenchymal stem cells. Acta biomaterialia, 77, 48-62.)

Although a promising technology, the current state of granular microgels has several disadvantages, which prevents their easy translation to the clinics. The manufacture of the microbeads involves multiple steps with specialized microfluidic and electrospray equipment, which can limit the material types and/or crosslinking methods as well as scalability. For instance, a material with high viscosity cannot be used for microfludic-based microgel production. In addition, water-in-oil emulsion microfluidic-assisted devices requires additives such as oil and/or surfactants (Jaworek, A. (2007). Micro-and nanoparticle production by electrospraying. Powder technology, 176( 1), 18-35.; Sheikhi, A., de Rutte, J., Haghniaz, R., Akouissi, O., Sohrabi, A., Di Carlo, D., & Khademhosseini, A. (2019). Microfluidic-enabled bottom-up hydrogels from annealable naturally-derived protein microbeads. Biomaterials, 192, 560-568.). Furthermore, structures based on microgel spheres can easily disassemble due to limited interactions between the individual spherical microgel particles (Highley, C. B., Song, K. H., Daly, A. C., & Burdick, J. A. (2019). Jammed microgel inks for 3D printing applications. Advanced Science, 6(1), 1801076.; Riley, L, Schirmer, L., & Segura, T. (2019). Granular hydrogels: emergent properties of jammed hydrogel microparticles and their applications in tissue repair and regeneration. Current opinion in biotechnology, 60, 1-8.), which implies the necessity for additional crosslinking moieties on polymers.

Patent application WO 2018/073235 A1 describes a method for printing crosslinked hyaluronic acid hydrogel scaffolds comprising parallel microfibers, wherein monomers mixed with a cross-linking agent are deposited on a frozen substrate. However, this method requires cooling of the substrate, and thus requires specialized equipment and relatively costly materials.

Therefore, it is an object of the present invention to provide a printable hydrogel, a lyophilizate, a method for generating the printable hydrogel, a 3D printed product and a 3D printing method that is improved in view of the above-stated disadvantages of the prior art.

This object is attained by the independent claims 1 (printable hydrogel), 12 and 13 (lyophilizate), 14 (method for generating a printable hydrogel), 16 (3D printed product) and 18 (3D printing method).

Specific embodiments of the invention are claimed in sub claims 2 to 11 , 15 and 17 and are described hereafter. Summary of the Invention

The invention relates to a printable hydrogel comprising or consisting of a plurality of microstrands, wherein the microstrands comprise or consist of a polymer, and wherein the microstrands have a diameter of 1 pm to 500 pm, particularly 100 pm to 200 pm, and an aspect ratio of at least 100:1 , and wherein the microstrands are entangled with each other.

Due to the entangled microstrands, the resulting hydrogel has mechanical properties which are favorable in use of the hydrogel as an“ink”, particularly“bioink”, for extrusion (3D) printing, especially of tissue products.

The invention further relates to a lyophilizate comprising or consisting of a plurality of microstrands comprising or consisting of a polymer, wherein the microstrands have a diameter of 1 pm to 500 pm, particularly 100 pm to 200 pm, and an aspect ratio of at least 100:1 , and wherein the microstrands are entangled with each other.

Such a lyophilizate can be used as a printability enhancer for aqueous solutions, resulting in the ability to print the solution by extrusion printing.

Moreover, the invention relates to a method for generating a printable hydrogel comprising the steps of providing a cross-linked hydrogel, passing the cross-linked hydrogel through a sieve comprising a plurality of openings, such that a plurality of microstrands having a diameter of 1 pm to 500 pm, particularly 5 pm to 100 pm, and an aspect ratio of at least 100:1 are generated, and passing the microstrands through a volume, such that the microstrands are entangled with each other, thereby generating the printable hydrogel.

Further objects of the invention are a device for generating a printable hydrogel, a 3D-printed product comprising the printable hydrogel and a 3D-printing method using the printable hydrogel.

Brief Description of the Figures

Fig. 1 shows a comparison between jammed granular microgels according to the prior art and entangled microgel strands according to the present invention.

Fig. 2 shows examples of multi-material entangled bioinks based on the printable hydrogel according to the invention to generate a range of functions.

Fig. 3 shows a demonstration of the extrudability of the printable hydrogel according to the invention.

Fig. 4 schematically illustrates a device for generating a printable hydrogel according to the invention. Fig. 5 shows a fluorescence microscopy image of a microstrand from a crosslinked carrageenan hydrogel according to the invention.

Fig. 6 shows a phase contrast microscopy image of a microstrand from a crosslinked i-carrageenan hydrogel according to the invention.

Fig. 7 shows a photograph of a HAMA microstrand according to the invention compared to a human hair.

Fig. 8 shows a phase contrast microscopy image of a segment of a HAMA microstrand according to the invention.

Fig. 9-' show fluorescence microscopy images of fluorescently stained HAMA microstrands according to the invention.

Fig. 12 shows a photographic image of a fluorescently stained HAMA printable hydrogel according to the invention extruded in air.

Fig. 13 shows a photographic image of a fluorescently stained HAMA printable hydrogel according to the invention submerged in PBS.

Fig. 14 shows a photographic image of a 100 pm fluorescently stained HAMA printable hydrogel according to the invention extruded in PBS.

Fig. 15 shows a photographic image of a 200 pm fluorescently stained HAMA microstrand according to the invention extruded in PBS.

Fig. 16 shows data displaying the the stability and macroporosity of entangled microstrands according to the invention.

Fig. 17 shows data displaying the mechanical properties of HA-MA gels.

Fig. 18 shows the results of a rheological characterization of HA-MA entangled microstrands.

Fig. 19 shows data displaying that entangled microstrands according to the invention are printable and align during extrusion.

Fig. 20 shows data displaying that entangled microstrands according to the invention can trigger aligned myotube formation.

Fig. 21 shows data displaying that entangled microstrands according to the invention allow 3D bioprinting with high cell viability.

Fig. 22 shows data indicating that entangled microstrands according to the invention mature into cartilage-like tissue. Fig. 23 show images of 3D printed GelMA and HA-DVS entangled microstrands prepared and printed from HA-DVS (A) and GelMA (B) according to the present invention.

Detailed Description of the Invention Terms and definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell biology, materials science, polymer chemistry and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc.) and chemical methods.

The term“a subject comprises an object” in the context of the present specification includes discrete embodiments where the subject consists of the object, in other words where the term comprise is synonymous with“consist of”. In other discrete embodiments, the object is one of several different comprised in the object.

In the context of the present specification, the term diameter is to be understood as a maximum width of an object in the cross-sectional direction perpendicular to a longitudinal axis along which the object extends.

In the context of the present specification, the term aspect ratio is defined as the ratio between the length and the diameter (or maximum width in the cross-sectional direction) of an object. For instance, an object having an aspect ratio of 10:1 is 10 times as long as its diameter. Therefore, a high aspect ratio indicates a long object compared to its width.

Within the context of the present specification, the term biomolecule designates proteins, carbohydrates, lipids, nucleic acids, primary or secondary metabolites and natural products.

Within the context of the present specification, the term pharmacological compound designates a molecule which exerts a biochemical or physiological effect on human or animal cells.

Within the context of the present specification, the term embedded means that objects are partially or completely positioned within a material.

Within the context of the present specification, the term tissue product designates an artificially engineered three-dimensional object comprising biological cells, for example an artificial organ. Such products are also referred to as tissue scaffolds. In the context of the present specification, the term polymer designates a molecule comprising a plurality of identical subunits ( monomers ) which are covalently linked to form a chain or a branched molecule.

In the context of the present specification, the term sulfated polymer designates a polymer covalently linked to at least one sulfate group (S0₄ ).

Within the context of the present specification, the term pore size designates the average distance between adjacent polymer strands in a microstrand and/or between adjacent microstrands in the printable hydrogel. For example, pore size of internal pores in the microstrands may be determined by measuring the diffusion constant of a fluorescently stained molecule of defined molecular weight in the hydrogel. Such diffusion measurement may be performed for instance by fluorescence recovery after photobleaching (FRAP) as known to the person skilled in the art. The pore size of pores between adjacent microstrands can be determined e.g. by filling up the void space between adjacent microstrands with a fluorescently stained molecule, such as a fluorescein isothiocyanate labeled dextran, and determining the fluorescent volume by fluorescence microscopy (see Sheikhi, A., de Rutte, J., Haghniaz, R., Akouissi, O., Sohrabi, A., Di Carlo, D., & Khademhosseini, A. (2019). Microfluidic-enabled bottom-up hydrogels from annealable naturally-derived protein microbeads. Biomaterials, 192, 560-568.).

Within the context of the present specification, the term macropores describes pores having a pore size from 5 pm to 100 pm.

Within the context of the present specification, the term supermacropores describes pores having a pore size from 100 pm to 500 pm.

Within the context of the present specification, the term transforming growth factor describes a protein selected from human transforming growth factor a (TGF a, UniProtKB P01135) characterized by SEQ ID 1 , human transforming growth factor b1 (TGF b 1 , UniProtKB P01137) characterized by SEQ ID 2, human transforming growth factor b2 (TGF b2, UniProtKB P61812) characterized by SEQ ID 3 and human transforming growth factor b3 (TGF b3, UniProtKB P10600) characterized by SEQ ID 4 or a functional homologue thereof having a sequence identity of at least 70 % to one of SEQ ID 1 to SEQ ID 4.

Within the context of the present specification, the term vascular endothelial growth factor describes a protein characterized by SEQ ID 5 (human vascular endothelial growth factor (VEGF, UniProtKB P49767) or a functional homologue thereof having a sequence identity of at least 70 % to SEQ ID 5.

Within the context of the present specification, the term insulin-like growth factor describes a protein selected from human insulin-like growth factor 1 (IGF 1 , UniProtKB P05019) characterized by SEQ ID 6 and human insulin-like growth factor 2 (IGF 2, UniProtKB P01344) characterized by SEQ ID 7, or a functional homologue thereof having a sequence identity of at least 70 % to SEQ ID 6 or SEQ ID 7.

Within the context of the present specification, the term fibroblast growth factor describes a protein selected from human fibroblast growth factor 1 (FGF 1 , UniProtKB P05230) characterized by SEQ ID 8 and human fibroblast growth factor 2 (FGF 2, UniProtKB P09038) characterized by SEQ ID 9, or a functional homologue thereof having a sequence identity of at least 70 % to SEQ ID 8 or SEQ ID 9.

Within the context of the present specification, the term nerve growth factor describes a protein characterized by SEQ ID 10 (human beta nerve growth factor (NGF, UniProtKB P01138) or a functional homologue thereof having a sequence identity of at least 70 % to SEQ ID 10.

Within the context of the present specification, the term hepatocyte growth factor describes a protein characterized by SEQ ID 11 (human hepatocyte growth factor (HGF, UniProtKB P14210) or a functional homologue thereof having a sequence identity of at least 70 % to SEQ ID 11.

Within the context of the present specification, the term platelet-derived growth factor describes a protein selected from human platelet-derived growth factor A (PDGF A, UniProtKB P04085) characterized by SEQ ID 12, human platelet-derived growth factor B (PDGF B, UniProtKB P01127) characterized by SEQ ID 13, human platelet-derived growth factor C (PDGF C, UniProtKB Q9NRA1) characterized by SEQ ID 14 and platelet-derived growth factor D (PDGF D, UniProtKB Q9GZP0) characterized by SEQ ID 15, or a functional homologue thereof having a sequence identity of at least 70 % to one of SEQ ID 12 to SEQ ID 15.

Within the context of the present specification, the term bone morphogenetic protein describes a protein selected from human bone morphogenetic protein 1 (BMP1 , UniProtKB P13497 characterized by SEQ ID 16, human bone morphogenetic protein 2 (BMP2, UniProtKB P12643) characterized by SEQ ID 17, human bone morphogenetic protein 3 (BMP3, UniProtKB P12645) characterized by SEQ ID 18, human bone morphogenetic protein 4 (BMP4, UniProtKB P12644) characterized by SEQ ID 19, human bone morphogenetic protein 5 (BMP5, UniProtKB P22003) characterized by SEQ ID 20, human bone morphogenetic protein 6 (BMP6, UniProtKB P22004) characterized by SEQ ID 21 , human bone morphogenetic protein 7 (BMP7, UniProtKB P18075) characterized by SEQ ID 22, human bone morphogenetic protein 8A (BMP8A, UniProtKB Q7Z5Y6) characterized by SEQ ID 23, human bone morphogenetic protein 8B (BMP8B, UniProtKB P34820) characterized by SEQ ID 24, human bone morphogenetic protein 9 (BMP9) also termed growth/differentiation factor 2, GDF2, UniProtKB Q9UK05) characterized by SEQ ID 25, human bone morphogenetic protein 10 (BMP10, UniProtKB 095393) characterized by SEQ ID 26, human bone morphogenetic protein 11 (BMP11 , also termed growth/differentiation factor 11 , GDF11 , UniProtKB 095390) characterized by SEQ ID 27, human bone morphogenetic protein 13 (BMP13, also termed growth/differentiation factor 6, GDF6, UniProtKB Q6KF10) characterized by SEQ ID 28, human bone morphogenetic protein 14 (BMP14, also termed growth/differentiation factor 5, GDF5, UniProtKB P43026) characterized by SEQ ID 29 and human bone morphogenetic protein 15 (BMP15, UniProtKB 095972) characterized by SEQ ID 30, or a functional homologue thereof having a sequence identity of at least 70 % to one of SEQ ID 16 to SEQ ID 30.

Within the context of the present specification, the term parathyroid hormone describes a protein characterized by SEQ ID 31 (human parathyroid hormone, PTH, UniProtKB P01270) or a functional homologue thereof having a sequence identity of at least 70 % to SEQ ID 31.

Within the context of the present specification, the term WNT describes a protein selected from human WNT1 , characterized by SEQ ID 32 (UniProtKB P04628), human WNT2, characterized by SEQ ID 33 (UniProtKB P09544), human WNT2B, characterized by SEQ ID 34 (UniProtKB Q93097), human WNT3, characterized by SEQ ID 35 (UniProtKB P56703), human WNT3A, characterized by SEQ ID 36 (UniProtKB P56704), human WNT4, characterized by SEQ ID 37 (UniProtKB P56705), human WNT5A, characterized by SEQ ID 38 (UniProtKB P41221), human WNT5B, characterized by SEQ ID 39 (UniProtKB Q9H1J7), human WNT6, characterized by SEQ ID 40 (UniProtKB Q9Y6F9), human WNT7A, characterized by SEQ ID 41 (UniProtKB 000755), human WNT7B, characterized by SEQ ID 42 (UniProtKB P56706), human WNT8A, characterized by SEQ ID 43 (UniProtKB Q9H1J5), human WNT 8B, characterized by SEQ ID 44 (UniProtKB Q93098), human WNT9A, characterized by SEQ ID 45 (UniProtKB 014904), human WNT9B, characterized by SEQ ID 46 (UniProtKB 014905), human WNT10A, characterized by SEQ ID 47 (UniProtKB Q9GZT5), human WNT10B, characterized by SEQ ID 48 (UniProtKB 000744), human WNT 11 , characterized by SEQ ID 49 (UniProtKB 096014), and human WNT16, characterized by SEQ ID 50 (UniProtKB Q9UBV4) or a functional homologue thereof having a sequence identity of at least 70 % to one of SEQ ID 32 to SEQ ID 50.

Calculations of "homology" or "sequence identity" or "similarity" between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non- homologous sequences can be disregarded for comparison purposes). In a particular embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, particularly at least 40%, more particularly at least 50%, even more particularly at least 60%, and even more particularly at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. In the case of circularly related proteins, the sequence of one of the partners needs to be appropriately split and aligned in two sections to achieve optimal alignment of the functionally equivalent residues necessary to calculate the percent identity.

In the context of the present specification, the terms sequence identity and percentage of sequence identity refer to the values determined by comparing two aligned sequences. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, 1981 , Adv. Appl. Math., 2, 482, by the global alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol., 48, 443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. , 85, 2444 or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).

One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11 , Extension 1 ; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1.- 2; Gap costs: Linear. Unless stated otherwise, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., 1990, J. Mol. Biol., 215, 403-410) using the above identified default parameters for protein and nucleic acid comparison, respectively.

A first aspect of the invention relates to a printable hydrogel comprising or consisting of a plurality of microstrands, wherein the microstrands comprise or consist of a polymer, and wherein the microstrands have a diameter of 1 pm to 500 pm, particularly 100 pm to 200 pm, and an aspect ratio of at least 100:1 , and wherein the microstrands are entangled with each other.

The microstrands are elongated structures which may comprise any cross-sectional shape perpendicular to a longitudinal axis along which the strands extend, i.e. a circular, elliptical or polygonal shape. In case of a non-spherical cross-sectional shape, the term diameter is to be understood as a maximum width in the cross-sectional direction (that is perpendicular to the longitudinal axis). The aspect ratio of the microstrands is defined as the ratio between the length (particularly the arc length, since the microstrands normally do not extend along a straight line), and the diameter (or maximum width in the cross-sectional direction) of the microstrand. For instance, a microstrand having an aspect ratio of 100:1 is 100 times as long as its diameter. Therefore, a high aspect ratio indicates a long strand compared to its width.

The microstrands are entangled with each other. This means that the microstrands form loops (open or closed loops), through which at least one neighboring microstrand extends. In other words, the microstrands do not run parallel, but are engaged with each other.

This results in an increased physical interaction of the microstrands in addition to friction (friction, but not physical interaction due to entanglement, occurs in hydrogels composed of spherical beads). The increased physical interaction gives rise to favorable material properties such as a controlled porosity, increased surface-to-volume ratio, shear thinning properties with a defined yield point, shear recovery properties, high shape retention and minimal swelling of the printable hydrogel. In particular, the shear thinning properties with a defined yield point and the shear recovery properties advantageously improve the ability to be 3D-printed by extrusion printing and are also advantageous for other products generated from the hydrogel, such as mold-cast products.

Furthermore, entangled microstrands, as well as 3D-printed scaffolds based on entangled microstrands, have an inherent stability and do not disintegrate into single microgels in fluids.

Entangled microstrands also result in an inherent macroporosity of the hydrogels, which can be further enhanced by addition of aqueous media. In particular, this macroporosity is based on the gel-free spaces between microstrands.

The hydrogel according to the invention is advantageously printable with improved structural resolution compared to the prior art.

The presented invention allows to produce macroporous, bioactive multimaterial scaffolds for 3D printing in a superior way compared to current approaches.

Furthermore, less manufacturing steps are needed compared to methods of the prior art.

There is no dependency on special equipment and therefore the whole approach is much cheaper and faster compared to prior art methods. Since the process is fast and no expensive equipment is necessary, upscaling of production is easy. No cytotoxic components, such as mineral oils used in microfluidic methods, are involved, resulting in a more cell friendly process, such that the hydrogel can be ideally used in bioinks.

In particular, in use of the hydrogel as a bioink, the physical entanglement of the microstrands allows for wide range of materials and multimaterials and provides inherent mechanical stability. As this approach is universal, any hydrogel system can be utilized in a bioprinting setting and also combined with any other hydrogel. Moreover, it is possible to generate supermacroporous scaffolds with defined pore size and a defined material-to-void- space ratio. There is the possibility to prepare all these entangled inks with or without cells embedded therein.

In certain embodiments, the microstrands are randomly entangled with each other.

This means that the microstrands do not form a regular, defined pattern, but are oriented randomly. Randomly entangled strands are easier to generate compared to regularly entangled strands, in particular by the method described below.

In certain embodiments, the microstrands have an aspect ratio of at least 1000:1 , particularly at least 10000:1 , more particularly at least 100000:1.

A high aspect ratio favorably influences hydrogel stability and mechanical properties.

In certain embodiments, the microstrands have a diameter of 10 pm to 200 pm.

In certain embodiments, the microstrands have a diameter of 5 pm to 100 pm

In certain embodiments, the printable hydrogel comprises macropores, particularly in the interstices of adjacent microstrands, wherein the macropores have a pore size of 5 pm to 100 pm, particularly 10 pm to 100 pm. In particular, the macropores are interconnected.

Macropores are favorable in particular for generating tissue products, since mass transport is improved by the macropores, resulting in better transport of nutrients to cells embedded in the hydrogel structure.

In certain embodiments, the printable hydrogel comprises supermacropores, particularly in the interstices of adjacent microstrands, wherein the supermacropores have a pore size from 100 pm to 500 pm, particularly 100 pm to 300 pm. According to a particular embodiment, the supermacro pores are interconnected.

Supermacropores further improve mass transport into the hydrogel and are therefore especially advantageous when tissue products are printed with the hydrogel. As described in detail below, such supermacropores can be generated by incorporating sacrificial microstrands into the hydrogel and removing the sacrificial microstrands by melting or degradation. Such interconnected porous structures with a wider diameter range (5-500 pm) after their removal in addition to macropores present between entangled microgel strands may facilitate generation of a large vascularized cellular constructs without necrosis due to efficient mass transport. Furthermore, the structure acts as an elongated template along which endothelial cells align to form an endothelialized lumen.

In certain embodiments, the polymer is charged.

In certain embodiments, the polymer is sulfated.

In certain embodiments, the polymer is selected from the group consisting of alginate (CAS- No. 9005-32-7), alginate sulfate (CAS-No. 9010-06-4 for sodium salt), carrageenan (particularly i-carrageenan (CAS-No. 9062-07-01) or k-carrageenan (CAS-No. 11114-20-8)), collagen (9007-34-5) (particularly collagen IV), chitosan (CAS No. 9012-76-4), chitosan sulfate, chondroitin sulfate (CAS No. 9007-28-7), fibrin (CAS No. 9001-31-4), gelatin (CAS No. 9000-70-8), heparin (CAS No. 9041-08-1), heparan sulfate (CAS No. 9050-30-0), hyaluronic acid (9004-61-9), silk, arabic gum (CAS-No. 9000-01-5), cassia gum (CAS-No. 11078-30-1), gellan gum (71010-52-1), sulfated gellan gum, ghatti gum (CAS-No. 9000-28- 6), guar gum (CAS-No. 9000-30-0), konjac gum (CAS-No. 37220-17-0), locust bean gum (CAS-No. 900-40-2), xanthan gum (CAS-No. 11138-66-2), xanthan gum sulfate, and the acrylates or methacryates of the aforementioned polymers.

In certain embodiments, the polymer is selected from hyaluronic acid, hyaluronic acid methacrylate, fibrin, carrageenan (particularly i-carrageenan or k-carrageenan), gelatin, gelatin methacrylate, alginate, alginate methacrylate and silk.

In certain embodiments, the polymer is selected from the group consisting of poly ethylene glycol (PEG, CAS-No. 25322-68-3), poly (2-methyl-2-oxazoline) (CAS-No. 26375-28-0), poly (2-ethyl-2-oxazoline) (CAS-No. 25805-17-8), poly (2-propyl-2-oxazoline) (CAS-No. 25822-74- 6), poly vinyl alcohol (CAS-No. 9002-89-5), poly (2-hydroxyethyl metacrylate) (P-HEMA, CAS-No. 25249-16-5), poly (1-glycerol methacrylate) (P-GMA, CAS-No. 28474-30-8), poly (2-hydroxypropyl methacrylate) (P-HPMA, CAS-No. 25703-79-1), poly acrylamide (CAS-No. 9003-05-8), poly methacrylamide (CAS-No. 25014-12-4), poly acrylic acid (CAS-No. 9003- 01-4), and poly methacrylic acid (CAS-No. 25087-26-7), particularly wherein the polymer is selected from poly ethylene glycol (PEG), poly (2-methyl-2-oxazoline), poly (2-ethyl-2- oxazoline), poly (2-propyl-2-oxazoline), and poly vinyl alcohol.

In certain embodiments, the polymer is capable of selectively binding or adapted to selectively bind a biomolecule or a pharmacological compound.

Selective binding of the polymer to the biomolecule or pharmacological compound means that the polymer binds to certain biomolecules or pharmacological compounds, whereas other biomolecules or pharmacological compounds are not bound by the polymer. For instance, carrageenans bind to certain growth factors, such as TGF-bI , but not to other growth factors. Furthermore, sulfated polymers bind to all heparin-binding growth factors, but not to certain other proteins.

In certain embodiments, the printable hydrogel comprises at least one biomolecule or pharmacological compound bound or selectively bound to the polymer.

In certain embodiments, the biomolecule or pharmacological compound is a growth factor, particularly a heparin binding growth factor.

In certain embodiments, the biomolecule is selected from the group consisting of transforming growth factor, vascular endothelial growth factor, insulin-like growth factor, fibroblast growth factor, nerve growth factor, hepatocyte growth factor, platelet-derived growth factor, bone morphogenetic protein, WNT and parathyroid hormone.

According to a particular embodiment, the biomolecule is transforming growth factor, more particularly transforming growth factor b1.

In certain embodiments, the microstrands comprise a plurality of first microstrands comprising or consisting of a first polymer and a plurality of second microstrands comprising or consisting of a second polymer. In particular, the microstrands further comprise a plurality of third microstrands comprising or consisting of a third polymer. More particularly, the microstrands further comprise a plurality of fourth microstrands comprising or consisting of a fourth polymer. Even more particularly, the microstrands further comprise at least one plurality of further microstrands comprising or consisting of a further polymer.

The first microstrands have a diameter of 1 pm to 500 pm, particularly 100 pm to 200 pm, and an aspect ratio of at least 100:1. The second microstrands have a diameter of 1 pm to 500 pm, particularly 100 pm to 200 pm, and an aspect ratio of at least 100:1. The third microstrands have a diameter of 1 pm to 500 pm, particularly 100 pm to 200 pm, and an aspect ratio of at least 100:1. The fourth microstrands have a diameter of 1 pm to 500 pm, particularly 100 pm to 200 pm, and an aspect ratio of at least 100:1. The further microstrands have a diameter of 1 pm to 500 pm, particularly 100 pm to 200 pm, and an aspect ratio of at least 100:1.

In certain embodiments, the first microstrands, the second microstrands, the third microstrands, the fourth microstrands and/or the further microstrands have an aspect ratio of at least 1000:1 , particularly at least 10000:1 , more particularly at least 100000:1.

In certain embodiments, the first microstrands, the second microstrands, the third microstrands, the fourth microstrands and/or the further microstrands have a diameter of 10 pm to 200 pm. In certain embodiments, the first microstrands, the second microstrands, the third microstrands, the fourth microstrands and/or the further microstrands have a diameter of 5 pm to 100 pm.

Thus, by the present invention, different microstrands can be mixed to create multimaterial gels behaving as one entity.

The presence of two or more different polymer species in the hydrogel advantageously allows to combine different functionalities in a modular manner. For instance, a polymer providing good mechanical stability to the resulting hydrogel can be combined with a polymer binding compounds or biomolecules or encapsulating cells.

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is charged.

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is sulfated. Sulfated polymers bind to growth factors, which allows to generate tissue products stimulating cells.

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is selected from the group consisting of alginate (CAS- No. 9005-32-7), alginate sulfate (CAS-No. 9010-06-4 for sodium salt), carrageenan (particularly i-carrageenan (CAS-No. 9062-07-01) or k-carrageenan (CAS-No. 11114-20-8)), collagen (9007-34-5) (particularly collagen IV), chitosan (CAS No. 9012-76-4), chitosan sulfate, chondroitin sulfate (CAS No. 9007-28-7), fibrin (CAS No. 9001-31-4), gelatin (CAS No. 9000-70-8), heparin (CAS No. 9041-08-1), heparan sulfate (CAS No. 9050-30-0), hyaluronic acid (9004-61-9), silk, arabic gum (CAS-No. 9000-01-5), cassia gum (CAS-No. 11078-30-1), gellan gum (71010-52-1), sulfated gellan gum, ghatti gum (CAS-No. 9000-28- 6), guar gum (CAS-No. 9000-30-0), konjac gum (CAS-No. 37220-17-0), locust bean gum (CAS-No. 900-40-2), xanthan gum (CAS-No. 11138-66-2), xanthan gum sulfate and the acrylates or methacryates of the aforementioned polymers.

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is selected from hyaluronic acid, hyaluronic acid methacrylate, fibrin, carrageenan (particularly i-carrageenan) gelatin, alginate, alginate methacrylate and silk, particularly wherein the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is selected from hyaluronic acid, hyaluronic acid methacrylate, alginate and alginate methacrylate.

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is selected from the group consisting of poly ethylene glycol, poly (2-methyl-2-oxazoline, poly (2-ethyl-2-oxazoline), poly (2-propyl-2-oxazoline), poly vinyl alcohol, poly (2-hydroxyethyl metacrylate) (P-HEMA), poly (1-glycerol methacrylate) (P-GMA), poly (2-hydroxypropyl methacrylate) (P-HPMA), poly acrylamide, poly methacrylamide, poly acrylic acid, and poly methacrylic acid, particularly wherein the polymer is selected from poly ethylene glycol (PEG), poly (2-methyl-2-oxazoline), poly (2- ethyl-2-oxazoline), poly (2-propyl-2-oxazoline), and poly vinyl alcohol.

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is hyaluronic acid or hyaluronic acid methacrylate (HAMA).

Advantageously, hyaluronic acid or hyaluronic acid methacrylate (HAMA) results in a high mechanical stability of the printable hydrogel.

In particular, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is hyaluronic acid methacrylate (HAMA) and the second polymer is silk or enzymatically crosslinked gelatin.

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is capable of selectively binding or adapted to selectively bind a biomolecule or a pharmacological compound.

In certain embodiments, the printable hydrogel comprises at least one biomolecule or pharmacological compound bound or selectively bound to the first or second polymer.

The incorporation of bioactive drug-loaded strands in entangled inks can locally stimulate a desired cellular function (i.e. differentiation, extracellular matrix production, angiogenesis) in a target tissue. In this manner, the drug release is spatially and temporally resolved, compared to growth factor loading of bulk gels where all seeded cells are exposed to the same concentration. Further, with 3D printing, it is possible to take advantage of combinations of bioactive strands within one printed line as well as deposition of different kinds of printed lines in 3D space. In addition, because of the modular nature of entangled inks, temporally controlled drug release may be achieved by utilizing different types of hydrogel materials retaining different degrees of binding affinity to a growth factor. This may facilitate generation of bioactive strands specifically tailored in terms of drug types, release kinetics, degradation kinetics, etc.

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is i-carrageenan (also termed iota-carrageenan) or K- carrageenan, (also termed kappa-carrageenan, particularly ionically crosslinked iota- carrageenan. i-carrageenan binds certain growth factors, such as TGF-bI

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is meltable or degradable at a temperature of 30°C to 50°C, more particularly 35°C to 45°C, most particularly 37°C. In particular, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is gelatin, more particularly thermal gelatin.

According to a particular embodiment, the printable hydrogel comprises supermacropores having a pore size from 100 pm to 500 pm, particularly 100 pm to 300 pm, wherein the supermacropores are obtained by providing a printable hydrogel, wherein the microstrands of said hydrogel comprise a plurality of first microstrands comprising or consisting of a first polymer and a plurality of second microstrands comprising or consisting of a second polymer, wherein the first polymer or the second polymer is meltable or degradable at a conversion temperature of 30°C to 50°C, more particularly 35°C to 45°C, most particularly 37°C, and wherein the printable hydrogel is incubated at the conversion temperature, such that the first polymer or the second polymer melts or is degraded, thereby generating the supermacropores.

Such supermacropores further improve mass transport into the hydrogel and are therefore especially advantageous when tissue products are printed with the hydrogel.

In certain embodiments, the printable hydrogel comprises biological cells.

In certain embodiments, the biological cells are embedded or encapsulated in the microstrands, particularly wherein the microstrands have a diameter of 10 pm to 500 pm, particularly 10 pm to 200 pm.

That is, the cells can be embedded in all microstrands or in a subset of the microstrands, for example in the first microstrands or the second microstrands.

Therein, the term “embedded” means that the cells are positioned within the polymer material, from which the microstrands are formed. In certain embodiments, the aspect ratio of the first microstrands differs from the aspect ratio of the second microstrands, particularly by a factor of 2 or more, more particularly by a factor of 5 or more, most particularly by a factor of 10 or more.

In certain embodiments, the diameter of the first microstrands differs from the diameter of the second microstrands, particularly by a factor of 2 or more, more particularly by a factor of 5 or more, most particularly by a factor of 10 or more.

In certain embodiments, the microstrands comprise a plurality of kinks. In particular, each of the microstrands comprises a plurality of kinks. In other words, the microstrands deviate from a straight line.

A second aspect of the invention relates to a lyophilizate comprising or consisting of a plurality of microstrands comprising or consisting of a polymer, wherein the microstrands have a diameter of 1 pm to 500 pm, particularly 100 pm to 200 pm, and an aspect ratio of at least 100:1 , and wherein the microstrands are entangled with each other, particularly randomly entangled with each other.

Such a lyophilizate may be mixed with water or an aqueous solution to obtain a printable hydrogel according to the first aspect of the invention. In lyophilized form, the microstrands may be stored at ambient temperature for a long period of time and a hydrogel may be easily prepared from the lyophilizate on demand.

Advantageously, such a lyophilizate may also be used as a printability enhancer by dissolving the lyophilizate in any liquid which is to be made printable. The printability will then be improved by the favorable mechanical properties of the entangled microstrands, such as the shear thinning properties with a defined yield point and the shear recovery properties.

Thus, the lyophilizate provides a universal approach allowing any hydrogel system to become a bioink either as single material, or multimaterial blended with any other hydrogel.

In certain embodiments, the microstrands in the lyophilizate have an aspect ratio of at least 1000:1 , particularly at least 10000:1 , more particularly at least 100000:1.

In certain embodiments, the microstrands in the lyophilizate have a diameter of 10 pm to 200 pm.

In certain embodiments, the polymer is charged.

In certain embodiments, the polymer is sulfated.

In certain embodiments, the polymer is selected from the group consisting of alginate (CAS- No. 9005-32-7), alginate sulfate (CAS-No. 9010-06-4 for sodium salt), carrageenan (particularly i-carrageenan (CAS-No. 9062-07-01) or k-carrageenan (CAS-No. 11114-20-8)), collagen (9007-34-5) (particularly collagen IV), chitosan (CAS No. 9012-76-4), chitosan sulfate, chondroitin sulfate (CAS No. 9007-28-7), fibrin (CAS No. 9001-31-4), gelatin (CAS No. 9000-70-8), heparin (CAS No. 9041-08-1), heparan sulfate (CAS No. 9050-30-0), hyaluronic acid (9004-61-9), silk, arabic gum (CAS-No. 9000-01-5), cassia gum (CAS-No. 11078-30-1), gellan gum (71010-52-1), sulfated gellan gum, ghatti gum (CAS-No. 9000-28- 6), guar gum (CAS-No. 9000-30-0), konjac gum (CAS-No. 37220-17-0), locust bean gum (CAS-No. 900-40-2), xanthan gum (CAS-No. 11138-66-2), xanthan gum sulfate and the acrylates or methacryates of the aforementioned polymers.

In certain embodiments, the polymer is selected from hyaluronic acid, hyaluronic acid methacrylate, fibrin, carrageenan (particularly i-carrageenan), gelatin, alginate, alginate methacrylate and silk.

In certain embodiments, the microstrands in the lyophilizate comprise a plurality of first microstrands comprising or consisting of a first polymer and a plurality of second microstrands comprising or consisting of a second polymer. In particular, the microstrands further comprise a plurality of third microstrands comprising or consisting of a third polymer. More particularly, the microstrands further comprise a plurality of fourth microstrands comprising or consisting of a fourth polymer. Even more particularly, the microstrands further comprise at least one plurality of further microstrands comprising or consisting of a further polymer.

In certain embodiments, the first microstrands, the second microstrands, the third microstrands, the fourth microstrands and/or the further microstrands have a diameter of 10 pm to 200 pm.

In certain embodiments, the first microstrands, the second microstrands, the third microstrands, the fourth microstrands and/or the further microstrands have a diameter of 5 pm to 100 pm.

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer or the further polymer is charged.

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer or the further polymer is sulfated.

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer or the further polymer is selected from the group consisting of alginate (CAS-No. 9005-32-7), alginate sulfate (CAS-No. 9010-06-4 for sodium salt), carrageenan (particularly i-carrageenan (CAS-No. 9062-07-01) or k-carrageenan (CAS-No. 11114-20-8)), collagen (9007-34-5) (particularly collagen IV), chitosan (CAS No. 9012-76-4), chitosan sulfate, chondroitin sulfate (CAS No. 9007-28-7), fibrin (CAS No. 9001-31-4), gelatin (CAS No. 9000- 70-8), heparin (CAS No. 9041-08-1), heparan sulfate (CAS No. 9050-30-0), hyaluronic acid (9004-61-9), silk, arabic gum (CAS-No. 9000-01-5), cassia gum (CAS-No. 11078-30-1), gellan gum (71010-52-1), sulfated gellan gum, ghatti gum (CAS-No. 9000-28-6), guar gum (CAS-No. 9000-30-0), konjac gum (CAS-No. 37220-17-0), locust bean gum (CAS-No. 900- 40-2), xanthan gum (CAS-No. 11138-66-2), xanthan gum sulfate and the acrylates or methacryates of the aforementioned polymers.

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer or the further polymer is selected from hyaluronic acid, hyaluronic acid methacrylate, fibrin, carrageenan (particularly i-carrageenan), gelatin, alginate, alginate methacrylate and silk, particularly wherein the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is selected from hyaluronic acid, hyaluronic acid methacrylate, alginate and alginate methacrylate.

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is selected from the group consisting of poly ethylene glycol, poly (2-methyl-2-oxazoline), poly (2-ethyl-2-oxazoline), poly (2-propyl-2-oxazoline), poly vinyl alcohol, poly (2-hydroxyethyl metacrylate) (P-HEMA), poly (1-glycerol methacrylate) (P-GMA), poly (2-hydroxypropyl methacrylate) (P-HPMA), poly acrylamide, poly methacrylamide, poly acrylic acid, and poly methacrylic acid, particularly wherein the polymer is selected from poly ethylene glycol (PEG), poly (2-methyl-2-oxazoline), poly (2- ethyl-2-oxazoline), poly (2-propyl-2-oxazoline), and poly vinyl alcohol.

In certain embodiments, the lyophilizate comprises at least one biomolecule or pharmacological compound bound or selectively bound to the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer.

In certain embodiments, the biomolecule is selected from the group consisting of transforming growth factor, vascular endothelial growth factor, insulin-like growth factor, fibroblast growth factor, nerve growth factor, hepatocyte growth factor, platelet-derived growth factor, bone morphogenetic protein, WNT and parathyroid hormone. According to a particular embodiment, the biomolecule is transforming growth factor, more particularly transforming growth factor b1.

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is i-carrageenan (also termed iota-carrageenan), particularly ionically crosslinked iota-carrageenan or k-carrageenan (also termed kappa- carrageenan). i-carrageenan binds certain growth factors, such as TGF-bI .

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymeris meltable or degradable at a temperature of 30°C to 50°C, more particularly 35°C to 45°C, most particularly 37°C. In particular, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is gelatin, more particularly thermal gelatin.

In certain embodiments, the aspect ratio of the first microstrands differs from the aspect ratio of the second microstrands, particularly by a factor of 2 or more, more particularly by a factor of 5 or more, most particularly by a factor of 10 or more.

A third aspect of the invention relates to a lyophilizate obtained by lyophilizing a printable hydrogel according to the first aspect of the invention.

A fourth aspect of the invention relates to a method for generating a printable hydrogel according to the first aspect of the invention, comprising the steps of providing a cross-linked hydrogel, passing the cross-linked hydrogel through a sieve comprising a plurality of openings, such that a plurality of microstrands having a diameter of 1 pm to 500 pm, particularly 100 pm to 200 pm, and an aspect ratio of at least 100:1 are generated, and passing the microstrands through a volume, such that the microstrands are entangled with each other, particularly randomly entangled with each other, thereby generating the printable hydrogel.

Advantageously, this method is easy to implement and requires inexpensive equipment. In particular, no freezing or cooling of substrates is necessary as in methods of the prior art.

The sieve may be manufactured from any suitable material, e.g. metal or nylon, using known manufacturing methods including 3D printing. In particular, the openings have a non-circular cross-section. Such openings may be generated e.g. by 3D printing. Without wishing to be bound by theory, it is believed that non circular openings result in increased entanglement of the microstrands.

In particular, according to the described method, crosslinked bulk hydrogel is mechanically extruded through a sieve, particularly a metal or nylon grid, having openings of known size (particularly a mesh size of 5 pm to 100 pm) to create filament like, microgel particles, which are also referred to as‘microstrands’. The process of creating these microstrands may also be designated as‘sizing’. In particular, the microstrands are microgels based on any bulk hydrogel material with a typical diameter of 5 pm to 100 pm and a high aspect ratio, typically ranging from 1 :100 to 1 :1000 or greater. During the sizing process, hydrogel microstrands entangle and form one bulk entity, which is referred to as entangled microstrands. These entangled microstrands have shear thinning properties with a clear yield point as well as shear recovery properties and are therefore an ideal material for microextrusion based 3D (bio)printing. They also show high shape retention after deposition of the filament and only minimal swelling.

Entangled microstrands are a microgel bioink approach based on high aspect ratio microgels. This can eliminate several drawbacks, microgel bioinks suffer from. Compared to spherical microgels, microstrands have a highly increased surface-to-volume ratio which increases gel-to-gel interaction and also increases the maximum number of microgels, one particular microgel is in contact with. The same is true for gel-to-cell interaction and the maximum number of cells one particular microgel is in contact with.

Another direct consequence of the high aspect ratio is physical entanglement of microstrands which gives rise to a new level of stability. Analog to molecular entanglement that greatly enhances mechanical properties of polymers on a molecular level, entanglement of microstrands does so on a microscopic scale.

The aforementioned properties are still present, even when microstrands are diluted with aqueous media like cell culture media. This allows to create increased porosity of the entangled inks (gel fraction to water fraction). On a second level, this also opens up the possibility to mix entangled inks with cells in solution to create cell-laden entangled bioinks in which cells are in the void space between microstrands.

In certain embodiments of the method, the microstrands have an aspect ratio of at least 1000:1 , particularly at least 10000:1 , more particularly at least 100000:1.

In certain embodiments of the method, the microstrands have a diameter of 10 pm to 200 pm.

In certain embodiments of the method, the microstrands have a diameter of 5 pm to 100 pm. In certain embodiments of the method, the openings of the sieve have a diameter of 1 pm to 500 pm, particularly 100 pm to 200 pm.

In certain embodiments of the method, the openings of the sieve have a diameter of 10 pm to 200 pm.

In certain embodiments of the method, the openings of the sieve have a diameter of 5 pm to 100 pm.

In certain embodiments of the method, the openings of the sieve have a diameter of 30 pm to 500 pm.

In certain embodiments of the method, the polymer is charged.

In certain embodiments of the method, the polymer is sulfated.

In certain embodiments of the method, the polymer is selected from the group consisting of alginate (CAS-No. 9005-32-7), alginate sulfate (CAS-No. 9010-06-4 for sodium salt), carrageenan (particularly i-carrageenan (CAS-No. 9062-07-01) or k-carrageenan (CAS-No. 11114-20-8)), collagen (9007-34-5) (particularly collagen IV), chitosan (CAS No. 9012-76-4), chitosan sulfate, chondroitin sulfate (CAS No. 9007-28-7), fibrin (CAS No. 9001-31-4), gelatin (CAS No. 9000-70-8), heparin (CAS No. 9041-08-1), heparan sulfate (CAS No. 9050-30-0), hyaluronic acid (9004-61-9), silk, arabic gum (CAS-No. 9000-01-5), cassia gum (CAS-No. 11078-30-1), gellan gum (71010-52-1), sulfated gellan gum, ghatti gum (CAS-No. 9000-28- 6), guar gum (CAS-No. 9000-30-0), konjac gum (CAS-No. 37220-17-0), locust bean gum (CAS-No. 900-40-2), xanthan gum (CAS-No. 11138-66-2), xanthan gum sulfate and the acrylates or methacryates of the aforementioned polymers.

In certain embodiments of the method, the polymer is selected from hyaluronic acid, hyaluronic acid methacrylate, fibrin, carrageenan (particularly i-carrageenan), gelatin, alginate, alginate methacrylate and silk.

In certain embodiments, the polymer is selected from the group consisting of poly ethylene glycol (PEG, CAS-No. 25322-68-3), poly (2-methyl-2-oxazoline) (CAS-No. 26375-28-0), poly (2-ethyl-2-oxazoline) (CAS-No. 25805-17-8), poly (2-propyl-2-oxazoline) (CAS-No. 25822-74- 6), poly vinyl alcohol (CAS-No. 9002-89-5), poly (2-hydroxyethyl metacrylate) (P-HEMA, CAS-No. 25249-16-5), poly (1-glycerol methacrylate) (P-GMA, CAS-No. 28474-30-8), poly (2-hydroxypropyl methacrylate) (P-HPMA, CAS-No. 25703-79-1), poly acrylamide (CAS-No. 9003-05-8), poly methacrylamide (CAS-No. 25014-12-4), poly acrylic acid (CAS-No. 9003- 01-4), and poly methacrylic acid (CAS-No. 25087-26-7), particularly wherein the polymer is selected from poly ethylene glycol (PEG), poly (2-methyl-2-oxazoline), poly (2-ethyl-2- oxazoline), poly (2-propyl-2-oxazoline), and poly vinyl alcohol. In certain embodiments, the method comprises the step of cross-linking monomers to provide the cross-linked hydrogel. Cross-linking can be performed by any method known in the art of polymer chemistry, for example by cross-linking agents and/or UV irradiation.

In certain embodiments of the method, the monomers are mixed with biological cells prior to cross-linking the monomers, such that the cells are embedded in the crosslinked hydrogel.

For materials which allow cell encapsulation (e.g. HAMA or Gelatin), cellular microstrands can be created by embedding cells in a bulk gel and subsequently size it in cell-containing microstrands.

In certain embodiments of the method, the monomers are mixed with a biomolecule or a pharmacological compound prior to cross-linking the monomers, such that the biomolecule or the pharmacological compound is selectively bound to the polymer in the crosslinked hydrogel.

In certain embodiments of the method, the cross-linked hydrogel is brought into contact with a solution containing a biomolecule or a pharmacological compound, such that the biomolecule or the pharmacological compound is selectively bound to the polymer in the crosslinked hydrogel

In this manner, entangled microstrands with cells embedded therein can be generated.

In certain embodiments of the method, the polymer is capable of selectively binding or adapted to selectively bind a biomolecule or a pharmacological compound.

In certain embodiments of the method, the printable hydrogel comprises at least one biomolecule or pharmacological compound bound or selectively bound to the polymer.

In certain embodiments of the method, the biomolecule or pharmacological compound is a growth factor, particularly a heparin binding growth factor.

In certain embodiments of the method, the microstrands comprise a plurality of first microstrands comprising or consisting of a first polymer and a plurality of second microstrands comprising or consisting of a second polymer. In particular, the microstrands further comprise a plurality of third microstrands comprising or consisting of a third polymer. More particularly, the microstrands further comprise a plurality of fourth microstrands comprising or consisting of a fourth polymer. Even more particularly, the microstrands further comprise at least one plurality of further microstrands comprising or consisting of a further polymer.

One of the advantages of this method is the possibility for a multimaterial approach: Microstrands of various sizes (length as well as diameter) and based on various materials can be combined in any ratios to achieve custom-tailored combinations for any application.

Since the presented approach is universal and works with any gel, different materials which excel at one specific property can be combined to create comprehensive multimaterial scaffolds, which excel at several properties. Some of these properties are mechanical reinforcement microstrands, charged microstrands, cell-guiding microstrands, bio-active microstrands, cell-embedding microstrands and degradable sacrificial microstrands.

In certain embodiments of the method, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is meltable or degradable at a conversion temperature of 30°C to 50°C, more particularly 35°C to 45°C, most particularly 37°C, wherein the printable hydrogel is incubated at the conversion temperature, such that the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer melts or is degraded, thereby generating supermacropores having a diameter from 100 pm to 500 pm within the printable hydrogel. Therein, in particular, the second polymer is gelatin.

Such microstrands are also referred to as sacrificial strands. After the sacrificial strands are removed by temperature-induced melting or by degradation, supermacropores of 100 pm to 500 pm size remain in the hydrogel. E.g., by selecting the ratio of the first polymer and the second polymer, the number and size of the supermacropores can be advantageously influenced.

Degradable microstrands offer the possibility to create porous scaffolds based on a sacrificial microstrand entangled with a stable microstrand. This way, one can control the size of the pores (diameter of the sacrificial microstrand) as well as the ratio of pores to scaffold material (ratio of unstable to stable material).

In certain embodiments of the method, a first cross-linked hydrogel comprising a first polymer is provided in a first chamber and a second cross-linked hydrogel comprising a second polymer is provided in a second chamber adjacent to the first chamber, wherein the first cross-linked hydrogel and the second cross-linked hydrogel are simultaneously passed from the first and second chamber through the sieve, such that first microstrands are generated from the first polymer and second microstrands are generated from the second polymer, wherein the first microstrands and the second microstrands are passed through the volume together, such that the first microstrands are entangled with the second microstrands.

In this manner, entangled first and second microstrands can be generated.

In certain embodiments of the method, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is charged.

In certain embodiments of the method, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is sulfated.

In certain embodiments of the method, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is selected from the group consisting of alginate (CAS-No. 9005-32-7), alginate sulfate (CAS-No. 9010-06-4 for sodium salt), carrageenan (particularly i-carrageenan (CAS-No. 9062-07-01) or k-carrageenan (CAS-No. 11114-20-8)), collagen (9007-34-5) (particularly collagen IV), chitosan (CAS No. 9012-76-4), chitosan sulfate, chondroitin sulfate (CAS No. 9007-28-7), fibrin (CAS No. 9001-31-4), gelatin (CAS No. 9000-70-8), heparin (CAS No. 9041-08-1), heparan sulfate (CAS No. 9050-30-0), hyaluronic acid (9004-61-9), silk, arabic gum (CAS-No. 9000-01-5), cassia gum (CAS-No. 11078-30-1), gellan gum (71010-52-1), sulfated gellan gum, ghatti gum (CAS-No. 9000-28- 6), guar gum (CAS-No. 9000-30-0), konjac gum (CAS-No. 37220-17-0), locust bean gum (CAS-No. 900-40-2), xanthan gum (CAS-No. 11138-66-2), xanthan gum sulfate and the acrylates or methacryates of the aforementioned polymers.

In certain embodiments of the method, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is selected from hyaluronic acid, hyaluronic acid methacrylate, fibrin, carrageenan (particularly i-carrageenan), gelatin, alginate, alginate methacrylate and silk, particularly wherein the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is selected from hyaluronic acid, hyaluronic acid methacrylate, alginate and alginate methacrylate.

In certain embodiments, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is selected from the group consisting of poly ethylene glycol (PEG, CAS-No. 25322-68-3), poly (2-methyl-2-oxazoline) (CAS-No. 26375-28-0), poly (2-ethyl-2-oxazoline) (CAS-No. 25805-17-8), poly (2-propyl-2-oxazoline) (CAS-No. 25822-74- 6), poly vinyl alcohol (CAS-No. 9002-89-5), poly (2-hydroxyethyl metacrylate) (P-HEMA, CAS-No. 25249-16-5), poly (1-glycerol methacrylate) (P-GMA, CAS-No. 28474-30-8), poly (2-hydroxypropyl methacrylate) (P-HPMA, CAS-No. 25703-79-1), poly acrylamide (CAS-No. 9003-05-8), poly methacrylamide (CAS-No. 25014-12-4), poly acrylic acid (CAS-No. 9003- 01-4), and poly methacrylic acid (CAS-No. 25087-26-7), particularly wherein the polymer is selected from poly ethylene glycol (PEG), poly (2-methyl-2-oxazoline), poly (2-ethyl-2- oxazoline), poly (2-propyl-2-oxazoline), and poly vinyl alcohol.

In certain embodiments of the method, the first polymer, the second polymer, the third polymer, the fourth polymer and/or the further polymer is capable of selectively binding or adapted to selectively bind a biomolecule or a pharmacological compound.

In certain embodiments of the method, the printable hydrogel comprises at least one biomolecule or pharmacological compound bound or selectively bound to the first or second polymer.

A fifth aspect of the invention relates to a device for generating a printable hydrogel comprising at least a first chamber for receiving a first cross-linked hydrogel, a pump unit for generating pressure in the first chamber, a sieve comprising a plurality of openings for generating a plurality of microstrands from the cross-linked hydrogel, wherein the sieve is in flow connection with the first chamber, and a volume for entangling the microstrands, wherein the volume is in flow connection with the sieve.

In certain embodiments, the device for generating a printable hydrogel further comprises a second chamber for receiving a second cross-linked hydrogel, wherein the pump unit is adapted to generate pressure in the second chamber, and wherein the second chamber is adjacent to the first chamber and separated from the first chamber, and wherein the second chamber is in flow connection with the sieve, such that the first cross-linked hydrogel and the second cross-linked hydrogel can be simultaneously from the first and the second chamber through the sieve.

In certain embodiments, the device for generating a printable hydrogel comprises at least one further chamber for receiving a further cross-linked hydrogel, wherein the pump unit is adapted to generate pressure in the at least one further chamber, and wherein the at least one further chamber is adjacent to at least one of the first chamber and the second chamber, and wherein the at least one further chamber is separated from the first chamber and the second chamber, and wherein the at least one further chamber is in flow connection with the sieve, such that the first cross-linked hydrogel, the second cross-linked hydrogel and the further cross-linked hydrogel can be simultaneously passed from the first chamber, the second chamber and the at least one further chamber through the sieve.

In certain embodiments, the device for generating a printable hydrogel comprises at least one divider between the first chamber and the second chamber, wherein the divider extends along a straight line, a curved line or a wave-like line when viewed in a cross-section perpendicular to a longitudinal axis, along which said first chamber extends.

Advantageously, different hydrogel materials can be loaded in the first chamber, the second chamber and the further chamber to generate entangled microstrands of mixed species.

A sixth aspect of the invention relates to a 3D-printed product comprising the printable hydrogel according to the first aspect of the invention. In particular, the 3D-printed product is obtained by extrusion printing.

In certain embodiments, the 3D-printed product is a tissue product, particularly a tissue scaffold.

In certain embodiments, the 3D-printed product has been cross-linked after extrusion of the printable hydrogel. Such secondary cross-linking of the polymers in the hydrogel further improve the stability of the hydrogel. Standard methods of cross-linking known in the art of polymer chemistry may be applied for this step, e.g. using cross-linking agents, enzymes, ions and/or UV irradiation.

A seventh aspect of the invention relates to a 3D-printing method comprising extrusion of the printable hydrogel according to the first aspect of the invention, such that a product is 3D- printed from the printable hydrogel.

In certain embodiments, the product is cross-linked after extrusion of the printable hydrogel. Such secondary cross-linking of the polymers in the hydrogel further improve the stability of the hydrogel. Standard methods of cross-linking known in the art of polymer chemistry may be applied for this step, e.g. using cross-linking agents and/or UV irradiation.

Wherever alternatives for single separable features are laid out herein as“embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

Description of the Figures

Fig. 1 shows a comparison between jammed granular microgels according to the prior art and entangled microgel strands according to the present invention. In the line labeled “preparation”, methods for generating granular microgels according to the prior art, namely microfluidics and electrospray, are schematically depicted and compared to the method for generating a printable hydrogel according to the present invention. The high aspect ratio of the microgels according to the invention is expected to confer unique rheological properties to the material allowing excellent printability while simultaneously allowing for an inherently more stable structure than that achievable with jammed microbead based gels.

Fig. 2 schematically depicts the method for generating a printable hydrogel according to the invention and shows examples of multi-material entangled bioinks based on the printable hydrogel according to the invention to generate a range of functions. The depicted embodiment of the method according to the invention (Fig. 2, top) uses shear-thinning entangled microgel strands produced via extrusion of a bulk hydrogel through a metal sieve with openings of a defined diameter. This extrusion process is also called‘sizing’, and the resultant microgel strands which entangle during sizing form a so-called entangled ink. The approach is material- and crosslinking method- independent, and thus, functional strands can be created using any hydrogel(s) of choice. For instance, an ionic crosslinked sulfated iota- carrageenan gel retaining binding affinities to growth factors can be used to prepare drug-loaded strands (Fig. 2, bottom, third column. Supermacroporous structure can be created using thermosensitive gelatin as sacrificial strands (Fig. 2, bottom, second column). The mechanical property and stability of an ink can be enhanced by adding mechanically robust strands (Fig. 2, bottom, fourth column). In addition, cellular strands encapsulating different types of cells allow for the generation of biomimetic tissues comprising of heterogeneous cell populations (Fig. 2, bottom, fifth column).

Fig. 3 shows a demonstration of the extrudability of the printable hydrogel according to the invention. Unexpectedly it was found that the hydrogel according to the invention could be extruded into extremely long strands similar to 3D printed strands.

Fig. 4 schematically illustrates a device for generating a printable hydrogel according to the invention. The device comprises a pump unit 20 comprising a holder 21 for receiving a first barrel 12 of a syringe 10 and a movable plate for moving a plunger 22 of the syringe in the first barrel 12, such that pressure is applied to a substance in the first barrel 12. Furthermore, the device comprises a syringe unit comprising a syringe 10, wherein the syringe 10 comprises a first barrel 12 extending along a longitudinal axis L, a plunger 11 which is movable in a first chamber 121 defined by the first barrel 12 along the longitudinal axis L, a second barrel 14 defining a volume and a sieve 13 comprising a plurality of openings 130, particularly wherein the openings 130 have a diameter D of 30 pm to 500 pm (cross-sectional view depicted in inset A). The sieve 13 is positioned between the first barrel 12 and the second barrel 14, such that the first chamber 121 defined by the first barrel 12 is in flow connection with the volume defined by the second barrel 14 by means of the openings 130 of the sieve 13. The second barrel 14 comprises a hole 15 opposite of the sieve 13. When a cross-linked hydrogel is provided in the first chamber 121 and pressure is applied on the plunger 11 by moving the plate 22 of the pump unit 20 in the direction of the arrow, the cross-linked hydrogel is forced through the openings 130, thereby forming a plurality of microstrands from the hydrogel. The resulting microstrands are passed through the volume defined by the second barrel 14 by further moving the plunger 11 of the syringe unit 10, and are entangled with each other in the volume, thereby generating a printable hydrogel. The printable hydrogel can be removed from the syringe unit, particularly extruded, through the hole 15. Inset B illustrates different embodiments of the first barrel 12 in cross-sectional view perpendicular to the longitudinal axis L. The first barrel 12 shown in a. defines a single first chamber 121 suitable for receiving a single material. In contrast, the first barrels 12 shown in b. to e. additionally comprise at least one divider 120 which separates the interior of the first barrel 12 into at least a first chamber 121 for receiving a first material and a second chamber 122 for receiving a second material. The divider shown in b. is straight in the cross-sectional view, and the dividers shown in d. and e. extend along a waved line in cross- sectional view. The first barrel 12 depicted in c. comprises a cross-shaped divider separating the interior of the first barrel 12 into four chambers which are suitable for receiving up to four different materials. The arrangement of the different hydrogel materials in the first barrel has an influence on the structure of the final printable hydrogel formed by entanglement of the microstrands in the volume defined by the second barrel 14. In particular, such multi compartment barrels with a defined cross-section can be prepared using a DLP printer.

Fig. 5 shows a fluorescence microscopy image of a microstrand from a crosslinked carrageenan hydrogel fluorescently labeled by 5-(4,6-Dichlorotriazinyl) Aminofluorescin (DTAF) extruded through a single opening of 130 pm diameter. The resulting microstrand forms curves and bends, which will result in entanglement of microstrands extruded through parallel openings.

Fig. 6 shows a phase contrast microscopy image of a microstrand from a crosslinked i-carrageenan hydrogel extruded through a single hole of 130 pm diameter.

Fig. 7 shows a photograph of a single microstrand from a crosslinked hyaluronic acid methacrylate (HAMA) hydrogel (2 % w/v HAMA) compared to a human hair (60 pm diameter).

Fig. 8 shows a phase contrast microscopy image of a segment of a microstrand from a crosslinked hyaluronic acid methacrylate (HAMA) hydrogel (2 % w/v HAMA) which has been stretched and fixed on both sides. The filament was drying and therefore shrank to some extent.

Fig. 9 shows a fluorescence microscopy image of a single microstrand from a crosslinked hyaluronic acid methacrylate (HAMA) hydrogel (2 % w/v HAMA) stained with 2 mg/ml Fluorescein isothiocyanate-Dextran 500000-Conjugate.

Fig. 10 shows a fluorescence microscopy image of a segment of a microstrand from a crosslinked hyaluronic acid methacrylate (HAMA) hydrogel (2 % w/v HAMA) stained with 2 mg/ml Fluorescein isothiocyanate-Dextran 500000-Conjugate.

Fig. 11 shows a fluorescence microscopy image of a segment of a microstrand from a crosslinked hyaluronic acid methacrylate (HAMA) hydrogel (2 % w/v HAMA) stained with 2 mg/ml Fluorescein isothiocyanate-Dextran 500000-Conjugate at higher magnification compared to Fig. 10. Fig. 12 depicts a photographic image under UV irradiation of a printable hydrogel comprising entangled microstrands prepared from a crosslinked hyaluronic acid methacrylate (HAMA) hydrogel (2 % w/v HAMA) stained with 2 mg/ml Fluorescein isothiocyanate-Dextran 500000-Conjugate, and extruded through several parallel openings with 100 pm diameter onto a dry microscope slide.

Fig. 13 shows a photographic image under UV irradiation of the printable hydrogel shown in Fig. 12 which has been subsequently submerged in phosphate buffered saline (PBS).

Fig. 14 shows a photographic image under UV irradiation of a printable hydrogel comprising entangled microstrands prepared from a crosslinked hyaluronic acid methacrylate (HAMA) hydrogel (2 % w/v HAMA) stained with 2 mg/ml Fluorescein isothiocyanate-Dextran 500000-Conjugate extruded through a nylon disk with several parallel openings of 100 pm diameter directly into phosphate buffered saline (PBS) with 0,5% Tween 20. The surfactant inhibits entanglement of the microstrands to some degree.

Fig. 15 shows a photographic image under UV irradiation of a single microstrand prepared from a crosslinked hyaluronic acid methacrylate (HAMA) hydrogel (2 % w/v HAMA) stained with 2 mg/ml Fluorescein isothiocyanate-Dextran 500000-Conjugate extruded through a 1 mm acrylic plate prepared by laser cutting with a single opening of 200 pm diameter directly into phosphate buffered saline (PBS) with 0,5% Tween 20. The surfactant inhibits self entanglement of the microstrand to some degree.

Fig. 16 shows data displaying the the stability and macroporosity of entangled microstrands according to the invention: (A) crosslinked bulk HAMA hydrogel (B) entangled microstrands prepared from such bulk hydrogel (C) when entangled microstrands are extended, single microstrands become visible (arrows) (D) entangled microstrands are mouldable (E) secondarily crosslinked custom shapes prepared by casting (F) secondary crosslinking tightly anneals microstrands (G) entangled microstrands show long-term stability in aqueous solution even without secondary crosslinking, while granular microgels loose cohesion and disintegrate (H) multiphoton image of entangled microstrands submerged in FITC-dextran (I) the same image after processing with a thresholding algorithm (J) 3D reconstruction of the porous network (K) Void fraction significantly varies with crosslinking density (F (2, 22) = 41.05; P<0.001) as well as mesh size (F (1 , 22) = 48.40; P<0.001). Fig. 17 shows data displaying the mechanical properties of HA-MA gels (A) Photocrosslinking behavior of HAMA with three different time points representing different degrees of crosslinking (Low, Med, High) (B) Storage modulus significantly differs between samples (F (2,24) = 12164, P<0.001) (C) Compression modulus between these samples is significantly different (F (2,12) = 34,P<0.001) (D) Rupture of a dumbbell shaped samples happens after significant different elongation (F (2,6) = 44.24, P<0.001) (E) Swelling ratio is different between samples prepared with different crosslinking degrees.

Fig. 18 shows the results of a rheological characterization of HA-MA entangled microstrands: (A - B) entangled microstrands created by sizing with a grid with aperture size of 40 and 100 pm exhibit shear thinning behavior (C - D) clear flow points can be determined (Crossover points for 40 pm samples: Low = 450 Pa, Med = 409 Pa, High = 141 Pa; 100 pm samples Low = 659 Pa, Med = 559 Pa, High = 225 Pa) (E - F) when subjected to repeated cycles of low and high shear, shear thinning and shear recovery behavior can be observed for all conditions.

Fig. 19 shows data displaying that entangled microstrands according to the invention are printable and align during extrusion: (A) Different HA-MA entangled microstrands printed in a grid structure (B) Entangled microstrands prepared and printed from i-carrageenan (ionic crosslinking), (C) gelatin (thermal crosslinking), (D) HATG (enzymatic crosslinking), (E) Collagen-EDC (carbodiimide crosslinking) (F) 3D model and (G) printing path of a human shaped ear (H) 3D-printed with entangled microstrands prepared from carrageenan (arrows point toward sharp transitions between layers) (I) SEM images of bulk gel, freshly prepared entangled microstrands in random orientation and aligned microstrands after extrusion through a printing nozzle (arrow indicates the direction of extrusion).

Fig. 20 shows data displaying that entangled microstrands according to the invention can trigger aligned myotube formation: (A) C2C12 could be embedded in bulk hydrogel and subsequently sized into cell-laden microstrands without negative impact on viability. Fluorescent images of cell-laden entangled microstrands bioink (green = alive, red = dead) show a healthy population of cells and quantification reveal a significant, but minor drop on viability when cells are encapsulated but not when sized through the grid. (B) After differentiation, cell fusion and aligned myotube formation could be observed. Fig. 21 shows data displaying that entangled microstrands according to the invention allow 3D bioprinting with high cell viability: (A) Chondrocytes could be mixed with already prepared entangled microstrands (outside) and 3D bioprinted with minimal impact on cell viability. (B) Fluorescent images showed high viability of cells after the printing process and cells adhering to the outer surface of entangled microstrands. Chondrocytes occupied and proliferated in the void space between microstrands at Day 7 and Day 21.

Fig. 22 shows data indicating that entangled microstrands according to the invention mature into cartilage-like tissue: (A) In this experiment, bovine chondrocytes were mixed with already prepared HA-MA microstrands (B) appearance of 3D bioprinted discs changed from transparent (day 0) to shiny-white (day 42) (C) comparison of compression modulus of freshly bioprinted entangled microstrands, after in vitro culture and healthy articular cartilage (D) histological staining to highlight the strong deposition of cartilaginous matrix entangled microstrand scaffold directly after printing and after 6 weeks of culture.

Fig. 23 show images of 3D printed GelMA and HA-DVS entangled microstrands prepared and printed from HA-DVS (A) and GelMA (B) according to the present invention.

Example 1 : Cell-laden hyaluronic acid methacrylate (HAMA) entangled microstrands

HAMA was dissolved in an aqueous buffer solution at a concentration of 2% together with 0.1% lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate as a photoinitiator. The solution was sterile filtered and combined with cells. The solution was UV irradiated for 15 seconds to achieve a partial crosslinking of the methacrylate residues. This cell-laden bulk hydrogel was sized into microstrands by pressing it through a nylon grid with a mesh size of 100 pm. The cell containing entangled microstrands were 3D printed into tissue grafts and were either cultured directly in cell culture or alternatively crosslinked a second time by UV irradiation for 15 seconds to give additional mechanical support to the structure.

Example 2: Lyophilized Hyaluronic acid microstrands as an off-the-shelf printability enhancer

Hyaluronic acid was crosslinked into a bulk gel with divinyl sulfone, washed and sized into entangled microstrands. Microstrands were lyophilized to create an off-the-shelf printability and macroporosity enhancer. These lyophilized microstrands can be re-suspended in any aqueous solution to give it printability. Example 3: Growth Factor loaded carrageenan microstrands

Carrageenan is a thermoresponsive polysaccharide that gels at ~50°C and is therefore unsuitable for cell embedding. However, this material is of great interest to incorporate into tissue-engineered constructs due to its high sulfation and natural affinity to proteins like growth factors. With high aspect ratio microgels, such as the printable hydrogels according to the invention, a bioink purely based on native carrageenan and cells can be produced which is not possible using any of the other approached currently utilized in bioprinting. As entangled microstrands tolerate a certain amount of aqueous solution around them, this approach allows to bioprint a suspension of microstrands and cells.

To achieve this, carrageenan was dissolved at 90°C and quiescently cooled down to form a bulk gel. Bulk gel was loaded with a heparin binding growth factor like transforming growth factor b1 (TGF- b 1) by overnight incubation in a concentrated TGF- b 1 solution. Growth factor loaded bulk gel was sized through a nylon grid to fabricate entangled microstrands. Microstrands were mixed with cell solution by using a double barrel syringe in the ratio of 10:1 and a static mixer. Therefore a cell-laden scaffold purely based on carrageenan and cells was created that even allows for increased bioactivity due to growth factor loading.

Example 4: Supermacroporous scaffolds through a sacrificial microstrand

Gelatin was dissolved at 3% and quiescently gelled. Bulk gel was sized into microstrands and combined with 1 % alginate solution at a ratio of 8:2. The gelatin microgels allow the mix to be 3D printed with high printing resolution to create scaffolds. Scaffolds can be crosslinked with calcium ions to stabilize the construct and subsequently incubated at 37°C to dissolve the gelatin out of the scaffold. This results in a supermacroporous scaffold with a porous network respective to the size of the gelatin microstrands and defined pore space of 80% of the total volume.

Example 5: Multimaterial entangled microgel bioinks

Bulk gels were prepared according to example 1 , 2, 3 and 4 and loaded into a multimaterial syringe and sized together to create a multimaterial bioink which contains: cell-laden microstrands of cell (Example 1), mechanical tough support strands (example 2), bioactive and growth factor eluting strands (example 3 without cell addition) and sacrificial strands to form supermacropores (example 4)

Example 6 - 3D bioprinting of microporous materials based on entangled hydrogel microstrands

A robust and versatile method according to the invention was used to prepare‘entangled’ microstrands using hyaluronan-methacrylate (HA-MA). Bulk HAMA hydrogels were mechanically pressed through a sieve with pores ranging from 40 to 100 microns. This process deconstructed the gel into microstrands, which randomly entangled within each other and made up a structured material consisting exclusively of high aspect ratio hydrogels. Passing a 2% bulk HA-MA (degree of substitution 0.28, UV-A exposure) through a 40 micron sieve, resulted in a visibly opaque, macroporous material permeable to dyes (Fig. 16A,B). When entangled microstrands were probed with forceps, single microstrands could be visualized (Fig. 16C). Entangled microstrands were also deformable and moldable (Fig. 16D). Secondary crosslinking of entangled microstrands (UV-A exposure) created a rigid, macroporous structure which could be handled with forceps (Fig. 16E,F).

To investigate the tunability of entangled microstrands for 3D printing, a range of different HAMA bulk gels were prepared. Crosslinking of HA-MA gels was terminated at three different time points, to create bulk hydrogels with a low (Low), medium ( Med) and high (High) degrees of crosslinking (Fig. 17A). These hydrogels had significantly different mechanical properties: storage moduli (F (2,24) = 12164, P<0.001), compression moduli (F (2,12) = 34, P<0.001), maximum elongation until rupture (F (2,6) = 44.2, P<0.001), and swelling behavior (F (2,14) = 463.2, P<0.001 ; Table 1 and Fig. 17B-E). These three distinctively different bulk hydrogels were then sized through nylon meshes of two different apertures (40 and 100 microns) to create a total of 6 different variants of entangled microstrands.

Table 1. Mechanical Properties of Different HA-MA Bulk Gels

Entanglement of microstrands allowed for long-term cohesion in aqueous medium, when compared to repeatedly sized, granular microgels. To compare stability, entangled microstrands and granular microgels were extruded through the grids, submerged in PBS for up to 7 days at 37°C with constant agitation (Fig. 16G). All six entangled microstrand materials (40 and 100 microns, Low, Med, High crosslinking) were stable for the entire period without secondary crosslinking, while all granular microgel materials based on the same hydrogels dissociated within 1 hour of incubation (n = 3).

Porosity is a critical property of materials employed in tissue engineering as this parameter strongly influences transport of nutrients, gas exchange and cell activity. Pore size is also relevant for blood vessel infiltration as well as cell migration. To assess the porosity of HAMA entangled microstrands, freshly prepared entangled microstrands were submerged in a fluorescent high molecular weight dextran dye. Fig. 16H shows a multiphoton image of the dye distribution taken within the central region of the structure. The void space and hydrogel strand could be clearly distinguished. The labeled dextran could enter the space between individual microstrands, but due to the high molecular weight, dextran was unable to penetrate the gel phase of the hydrogel. Since the dye was detectable within the central region and diffusion through the hydrogel microstrands was not possible, the pore space in between microstrands deemed to be interconnected. To calculate the void fraction of entangled microstrands, images were thresholded to achieve distinct transitions between microstrands and pores. An acquired image before and after processing can be seen in Fig. 16H,I. A 3D reconstruction of the interconnected network can be seen in Fig. 16J while a quantification of the void fraction is displayed in Fig. 16K. Calculated void fractions ranged from 2.0±0.8% for the Low (100 pm) condition to 7.4±0.9% for High (40 pm). Void fraction significantly varied with crosslinking density (F (2,22) = 41.05, P<0.001) as well as mesh size (F (1 ,22) = 48.40, P<0.001).

Entangled microstrands exhibited all relevant rheological properties necessary for extrusion 3D (bio)printing. All prepared variants of entangled microstrands showed shear thinning behavior (Fig. 18A,B). To identify the yield stress necessary to induce flow, different methods can be employed. In this example, the crossover of G’ and G” was used to identify the flow point. The required stress to reach crossover was lowest in High samples and highest in Low samples for both mesh apertures (40 pm: Low = 450 Pa, Med = 409 Pa, High = 141 Pa; 100 pm: Low = 659 Pa, Med = 559 Pa, High = 225 Pa, Fig. 18C,D). To simulate the printing process, shear recovery tests based on oscillatory strain sweeps with cycles of high and low strain were conducted. At low strains, microstrands exhibited a solid-like elastic behavior (G’ > G”) that rapidly changed into a viscous liquid-like behavior (G’ < G”) when high strains were applied (Fig. 18E,F). These transitions are crucial for high quality 3D printing as it ensures even material flow during extrusion and shape retention upon deposition on the collector plate.

Two-layered grid structures were printed with entangled microstrands prepared from HA-MA

(Fig. 19A). All six variants were printed with good shape retention and printing resolution.

Anomalies arose for lower crosslinked samples when sharp edges were printed. This phenomenon was especially pronounced in Low (40 pm) samples where sharp edges of the model printed with a very rounded appearance and the filament was dragged away during printing. This problem could be explained by the higher mechanical strength of the microstrands compared to that of a typical polymer solution. In typical bioinks, deposited filaments and the reservoir within the printing nozzle are separated as soon as the printing head retracts. In Low microstrand samples, however, microstrands spanned the distance between the printed construct and the printing nozzle. This connection did not rupture right away, but instead the deposited filaments were slightly dragged to the new printing position. Since the more crosslinked samples Med and High were more brittle and ruptured at lower elongation distances, the printing accuracy was higher in these samples.

Since all variants of microstrands prepared from HA-MA were successfully printed, the versatility of the approach was investigated by using other hydrogel systems commonly used in tissue engineering and 3D culture. The tested systems included gelatin, a thermoresponsive denatured form of collagen together with its photoresponsive derivative, gelatin-methacrylol (gelMA), iota-carrageenan, a highly sulfated polysaccharide that forms ionic crosslinks upon addition of monovalent as well as divalent cations, and enzymatically or chemically crosslinked hyaluronan hydrogels. Finally, collagen, a material prevalent in tissue engineering because of its abundance in the extracellular matrix of many tissues, was crosslinked with carbodiimide chemistry. Although derived from a wide range of materials and crosslinking methods, all bulk hydrogels were successfully sized and the entangled microstrands could be 3D printed according to the grid model used for the HA-MA microstrands (Fig. 19B-E).

In a next step, the potential of microstrands by bioprinting large, complex constructs was explored. A biologically relevant structure was printed with entangled microstrands prepared from bulk carrageenan (ear, Fig. 19F-H). This 3D model represents a human ear printed at 50% size and demonstrates the power of this approach to create macro-sized scaffolds (28 x 14 x 7 mm). Printed structures were stable and no flow of the bioink was observed, even after 15 layers were stacked in z-direction. Moreover, individual layers and deposited filaments remained visible and clearly reproduced the printing path created by the slicing software (Fig. 19H).

Anisotropy in tissues like muscle, tendon or nerves is difficult to replicate with conventional tissue engineering approaches. Several studies have shown the possibility to align (nano-) fibers and anisotropic particles in the direction of flow. Extruded microstrands showed alignment in the 3D printing direction, as observed after HA-MA microstrands were extruded through a 410 micron conical printing nozzle and imaged with scanning electron microscopy (Fig. 191). Clear differences between bulk gel and microstrands (pre- and post-printed) were apparent. Bulk hydrogels had an even surface, whereas microstrands before printing were randomly entangled within each other and had no clear orientation. Extrusion through a nozzle oriented the microstrands and post- printing, microstrands were aligned in the direction of printing.

To explore the potential for 3D bioprinting with entangled microstrands, two possible cell delivery approaches were investigated. Firstly, cells were embedded inside the bulk hydrogel before the hydrogel was sized into microstrands to create cell-laden microstrands (Inside). Alternatively, entangled microstrands were mixed with a cell suspension, leaving cells to occupy the void space between microstrands (Outside).

For anisotropic tissues like muscles, the Inside approach enables to confine cells within the gel microstrand and gives orientation during tissue maturation. As a first proof of principle, C2C12 cells were embedded within a bulk gel of 2% gelatin and 2% GelMA, and subsequently sized (Cells inside gel phase). A significant, but minor drop of viability was observed for cells embedded in bulk hydrogel compared to freshly trypsinized cells (Fig. 20A, 2D 98.2±0.6%; Bulk = 92.6±1.6%; T (4) = 4.6, P<0.01). The viability of cells in hydrogels that have been sized into entangled microstrands was high (40 pm = 93.2±0.8%, 100 pm = 94.1 ±0.7%) and no significant difference in viability was found between either of the sized samples and the bulk gel controls (F (2,6) = 0.96, P=0.443). Differentiation media was added to trigger myotube formation of cultured C2C12 cells and subsequent cytoplasmic Calcein AM staining revealed cell fusion and formation of myotubes aligned with the orientation of entangled microstrands (Fig. 20B).

In the outside approach, cells were present within the void fraction of the entangled microstrands and not embedded within the polymer network of the hydrogel itself. In this case, cells were less confined, since they were able to utilize the porous network to migrate, proliferate and deposit extracellular matrix. The outside approach was used to engineer de novo cartilage tissue. Bovine chondrocytes were combined with HA-MA microstrands and were 3D bioprinted into cylindrical discs. Cell viability was above 90% for the entire duration of the experiment (Fig. 21A, Pre-printing = 95.3±0.5%; Day 1 = 90.1±0.6%; Day 7 =

92.3±1.1 %, Day 21 = 92.6±2%). Even though there was a significant drop in cell viability when bioprinted cells were compared to the original cell population (T (4) = 9.6, P < 0.001), cell viability decreased by only 5.2±1.2% (Pre-Printing to Day 0). Cell viability remained high and no statistical difference in viability was found between printed cells at day 1 , 7 and 21 (F

(2,6) = 1.873, P=0.233). In the fluorescent Live/Dead staining, chondrocytes showed a rounded phenotype after 3D bioprinting, but proliferated and displayed a more elongated phenotype after 7 and 21 days in culture (Fig. 21 B). Immediately after fabrication, bioprinted scaffolds were transparent and slightly opaque. After 6 weeks of culture, the discs appeared cartilage-like and white, indicating deposition of a dense extracellular matrix (Fig. 22B). To confirm this, cultured tissue constructs were fixed and histologically stained for cartilage specific markers. A representative sample ( n = 6) is depicted in Fig. 22D. Staining with

Safranin O showed an increased intensity with time, indicating strong proteoglycan content in the samples. Staining with Hematoxylin & Eosin (H&E) resulted in a contrast between stained cells and unstained entangled microstrands. While high levels of collagen type I were detected 3 weeks after fabrication, collagen type I staining was reduced after 6 weeks of maturation. Collagen type II was present after 3 weeks but restricted to the void space in between microstrands. After 6 weeks of culture, collagen II staining intensified and showed deposition inside the void space, as well as the hydrogel network of the entangled microstrands. The difference in staining between time-points was particularly striking in the outer -400 pm of the sample. After 3 weeks, staining for cartilage-ECM markers as well as abundance of cells in the outer area of the sample was lower compared to the central part of the scaffold. This distribution was reversed after 6 weeks of culture as there was a very dense deposition of ECM as well as cells in the outermost part of the scaffold. At the week 6 timepoint, cells in the outermost part did not show the initial pattern of thin lines and small clusters also found in Live/Dead staining anymore but had a more homogeneous distribution. This suggests that cells were able to migrate into the space previously occupied by the hydrogel microstrands.

The mechanical properties of cartilage tissue are highly important for its functions, especially the crucial ability to sustain load. Compression modulus of freshly bioprinted samples was very low (2.7±0.3 kPa) but showed a significant increase over time (F* (2,15) = 44.38, P < 0.001), reaching 212±83.7 kPa after 3 weeks and 780.2±218.4 kPa after 6 weeks (Fig. 22C). While this was still significantly lower when compared to native articular bovine cartilage which has a compression modulus of 1829.8±72 kPa (T (10) = 10.2, P < 0.001), this is a remarkable increase. Since the material used in this study had very little resistance to compression on its own, change in compression modulus could exclusively attributed to the abundant deposition and maturation of extracellular matrix.

Materials and Methods

All chemicals were purchased from Sigma-Aldrich unless stated otherwise.

Hyaluronic Acid Methacrylol (HA-MA) Hyaluronic acid (1 gram, HTL Biotechnology) was dissolved in ultrapure water (400 ml) and kept at 4°C overnight to ensure complete dissolution. Ice-cold DMF (267 ml) was added under continuous stirring. To start the reaction methacrylic anhydride (2370 pi) was added and the pH kept between 8 - 9 through the addition of 10 M NaOH for 4 hours. Solid sodium chloride was dissolved in the solution to achieve a concentration of 0.5 M and the polymer was subsequently precipitated with ethanol (Merck). The precipitate was washed with ethanol, dried and dissolved in ultrapure water. Solution was purified by diafiltration (Akta 3, 10 NMWC hollow fiber). The purified product, hyaluronic acid methacrylate (HA-MA) was lyophilized dissolved in deuterium (Cambridge Isotope Laboratories) and characterized by 1 H NMR spectroscopy and stored at -20°C in the dark until used.

NMR spectra were recorded at room temperature on a Bruker AV-NEO 600 MHz spectrometer equipped with a TCI cryo probe. Spectra were obtained with 1024 scans using a 5 s recycle delay. To determine the degree of substitution, the ratio of the sum of the integrated peaks was compared of the methacrylate protons (peaks at ~6.1 and ~5.6) and the integrated peak of the methyl protons of HA (~1.9 ppm).

For gel preparation, HA-MA was dispersed in PBS and kept at 4°C until complete dissolution. HA-MA solution was mixed with a 1 % lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (l_AP) stock solution to create a 2% HA-MA and 0.05% l_AP solution and crosslinked by controlled photoexposure in the UV-A range (Omnicure Series 100, 400 nm wavelength, 9.55 mW cm ²).

Gelatin Methacrylol (GelMA) Gelatin type A was dissolved in PBS at pH 7.4 and warmed up to 50°C under vigorous stirring. Total used MA volume was split into five and after every addition, pH was adjusted with NaOH and the solution left to react for 30 minutes. After the last addition, reaction was diluted 2 fold and left to react for another 30 minutes. Product was cleaned by subsequent dialysis (10-12 kDa Cutoff) against ultrapure water for 4 days. Solution was filtered, lyophilized and stored at -20°C until use.

For gel preparation, GelMA and gelatin was dissolved in 70°C hot PBS. GelMA solution was mixed with LAP stock solution (1 %) to achieve a final concentration of 2% GelMA, 2% gelatin and 0.05% LAP. Bulk gel was formed through thermoreversible gelation and sized into microstrands. To ensure stability in cell culture at 37°C, microstrands were photocrosslinked by controlled photoexposure in the UV-A range.

Hyaluronic acid transglutaminase (HA-TG) For HA-TG hydrogel precursors, two different batches of HA were substituted with reactive glutamine (HA-TG/GIn) and lysine (HA-TG/Lys) residues respectively following published protocols.

For gel preparation, HA-TG/Lys and HA-TG/GIn were dissolved in TBS buffer (150 mM NaCI, 40 mM CaCI2, 50 mM TRIS, pH 7.6) and combined at equal volume to form HA-TG solution. To initiate gelation, a solution of thrombin (Baxter, 500 U ml ¹) and factor XIII (Fibrogammin, CSL Behring, 200 U ml ¹) was added to form a gel with final concentrations of 3% HA-TG. i-carrageenan (i-CRG): 300 mg of /-carrageenan particles (Genuvisco CG-131 , GP Kelco) were added to 4°C cold buffer solution (10 ml, 150 mM KCI, 20 mM HEPES, pH 7.4) to allow hydration of particles. Dispersion was then heated to 80°C, stirred until complete dissolution, and transferred into a 10 ml syringe. Solution was cooled down and stored at 4°C to form a 3% (w/v) gel.

Gelatin: Gelatin particles from porcine skin (type A, 300 mg) were added to 4°C cold PBS (10 ml) and left to hydrate for 15 minutes with subsequent heating to 70°C until complete dissolution. The solution was transferred into a 10 ml syringe and cooled down to 4°C to form a 3% (w/v) bulk gel. To ensure reproducible results, gelatin solution was stored at 4°C for 24 h to minimize variances due to the hardening of gelatin gels. Hyaluronic acid divinyl sulfone (HA-DVS) A solution of 3% (w/v) hyaluronic acid, 3% (w/v) NaCI and 0.2 M NaOH was prepared and stirred vigorously until complete dissolution of hyaluronic acid. Double the amount of divinylsulfone was added to the hyaluronic acid (w/w). Solution was mixed to ensure a homogeneous distribution and left to gel for 3 hours at room temperature. Gel was then washed for 2 days in deionized water and used for experiments.

Carbodiimide crosslinked collagen (Collagen-EDC) Two different concentrations of Type I collagen solution (5 mg ml ¹ , Symatese; 80 mg ml ¹ , 3dbio) were mixed on ice to achieve a final concentration of 20 mg ml-1. An equal amount (w/w) of 3,3'- Dithiobis(propionohydrazide) (DTPHY) was directly dissolved in this solution and 6 times excess of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was first dissolved in MES buffer and subsequently added to the solution. Solution was mixed and left to react at 4°C over night to form a stable gel.

Deconstruction of Bulk Gels into Microstrands (sizing) To prepare entangled microstrands, bulk hydrogels were prepared inside a 10 ml syringe and manually pressed through a nylon sieve (Millipore, Filter code: NY41 for 40 pm, NY1 H for 100 pm) and directly used for experiments.

Rheology To assess rheological properties of the samples, all measurements were conducted on an Anton Paar MCT 301 rheometer equipped with a 20 mm parallel plate geometry at 25°C in a humid atmosphere with a gap distance of 1 mm. Rheological properties of samples were examined by oscillatory shear sweeps (1% strain, 1-100 Hz), ramped shear rate (0.01 - 50 s-1) and strain sweeps (1 Hz, 0.01-1 % strain) to evaluate storage and loss modulus, yield point and shear thinning behavior. To investigate shear recovery properties, samples were exposed to repeating cycles of alternating phases of strain (1 Hz, 1% and 500% strain).

Compression modulus measurements Disc shaped test specimen with 8 mm diameter and 2 mm height were stamped out of the bulk gel for acellular samples. For cellular samples, bioprinted constructs were used and exact dimension measured before testing. Samples were tested by unconfined compression using a texture analyzer (TA.XTplus, Stable Microsystems). A 500 g load cell and a flat plate probe with a diameter of 15 mm were used. Samples were compressed to a final strain of 15% at a rate of 0.01 mm s-1. The compression modulus was calculated from the slope of the linear first 3% of the stress-strain curve.

Elastic Modulus Bulk gels with defined crosslinking were prepared and dumbbell shaped specimens according to ISO 527-2-5B were stamped out. Specimen were attached in a custom clamp system and elongated until failure at a rate of 0.01 mm s ¹. Swelling Bulk hydrogel discs were prepared and weighted before immersion in PBS to induce swelling. For each time point, samples were removed from the PBS, blotted with a tissue to remove excess PBS and weighted. The degree of swelling was calculated using following equation.

(mass of swollen gel ) — (mass of dried gel )

degree of swelling [%] = 100

(mass of dried gel )

3D Printing Entangled microstrands were loaded into printing cartridges (Nordson EFD) and printed through a 410 pm conical needle (Nordson EFD) with a pneumatic driven extrusion 3D bioprinter (3D Discovery, RegenHU). 3D models for grid and disc structures were created with OpenSCAD version 2015.03-2. 3D models were processed with Slic3r version 1.3.0 dev to create machine code (G-Code).

Scanning electron microscopy (SEM) Entangled microstrands were extruded as straight lines through a 410 pm conical needle and collected on a glass plate. Bulk HA-MA hydrogel and freshly prepared entangled microstrands were also prepared as control. All samples were frozen in liquid nitrogen and lyophilized. For SEM analyses, the lyophilized samples were coated using Pt/Pd (80/20) at a thickness of 10 nm by a sputter coater (CCU-010 HV, Safematic). The imaging was performed using a SEM instrument (JSM-7100, JEOL).

Macroporosity Entangled inks were prepared as described and submerged in PBS containing a high molecular weight, fluorescent FITC-dextran (average molecular weight of 500 kDa). Entangled microstrands were then imaged by two-photon microscopy (SP8, Leica).

Stability Entangled microstrands were prepared as described, cut into cylinders and transferred into well plates. Samples were submerged into PBS for up to 7 days and PBS was removed and exchanged after 5 min, 1 hour, 24 hours and 7 days.

Cell laden microstrands (inside) C2C12 mouse immortalized myoblasts were obtained from ATCC. Cells were cultured in a humidified atmosphere (5% C02, 37°C) in Dulbecco’s modified Eagle’s medium (DMEM GlutaMAX, Gibco) with fetal bovine serum (FBS, 10%, Gibco) and gentamycin sulfate (10 pg ml ¹, Gibco). Cells were passaged at 90% confluence and detached by Tripsin/EDTA (0.25%, Gibco).

Encapsulation solution (2% gelatin, 2% GelMA and 0.05% LAP) was prepared and kept at 37°C to avoid solidification. Freshly detached C2C12 were added to the solution and gently mixed by continuous pipetting to achieve a final concentration of 10 x 10⁶ cells ml ¹. After homogeneous distribution was achieved, solution was transferred into a syringe and cooled in an ice bath for 1 hour, while the syringe was constantly rotated for the first 5 minutes to avoid sedimentation. After gelation period, cell containing bulk gel was pressed through a nylon grid (sized), secondarily crosslinked by photoexposure in the UV-A range and kept in culture media. Cells were then cultured in differentiation medium composed of high glucose DMEM, insulin (1%), transferrin and selenium mix (ITS+, Corning) and horse serum (2%, Gibco) and gentamycin sulfate (10 pm ml ¹). Medium was changed thrice a week.

Bioprinting of entangled microstrands (outside) Primary articular chondrocytes were isolated from the femoral cartilage of 6 month old calves obtained from the local slaughterhouse. Cartilage from the medial and lateral condyle was harvested, minced and digested by collagenase solution (0.1%, from Clostridium histolyticum) over night. Cells were cultured in a humidified atmosphere (5% C0₂ at 37°C) and high glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with FBS (10%), Lascorbic acid 2-phosphate sesquimagnesium salt hydrate (50 pg ml-1) and gentamycin sulfate (10 pg ml ¹). Cells were passaged at 90% confluence by detachment with trypsin/EDTA (0.25%) and used for experiments at passage 3.

To prepare bioink, entangled microstrands and dense solution of bovine chondrocytes (100 x 106 cells ml ¹) were loaded in separate chambers of a double barrel syringe with a chamber ratio of 10:1 (Medmix). The two components were mixed by extrusion through a static mixing element (Medmix) to prepare cell-laden entangled microstrands. Bioink was transferred into a printing cartridge (Nordson EFD) and printed into discs (d = 5 mm, h = 2 mm). To ensure longterm shape fidelity, microstrands were annealed by UV-A exposure (15 s). Constructs were cultured in high glucose DMEM supplemented with ITS liquid media supplement (1%, Fisher Scientific), proline (40 pg ml ¹), ascorbic acid (50 pg ml ¹), gentamycin sulfate (10 pg ml ¹) and TGF-bO (10 ng ml ¹, Preprotech) for up to 6 weeks with full media change three times a week.

Live/Dead Staining Bioprinted constructs were cut in half, washed with phenol-free DMEM (Gibco) and stained with propidium iodide (0.5 pg ml ¹), calcein AM (0.008 mM) and Hoechst 33342 (5 pg ml ¹) for 20 minutes and imaged with fluorescent light microscopy (ZEIS, Axio Observer Z1). Zstack images spanning 100 pm were acquired from the center of the scaffold and analyzed with FIJI. Experiment was done in triplicates, with the viability of each sample averaged over three pictures of randomly chosen positions inside the center of the hydrogel.

Histological evaluation Samples for histology were fixed in paraformaldehyde for 2 hours, dehydrated and paraffinized (LogosJ, Milestone). Paraffin blocks were cut with a microtome in 5 pm thick sections, dried deparaffinized and hydrated. Tissue sections were stained with SafraninO, hematoxylin and eosin (H&E), Picosirius red and Alizarin Red according to standard protocols. For colorimetric, immunohistochemical stainings of collagen type I and II, sections were first digested in a hyaluronidase solution (1200 U ml ¹, from Streptococcus equi) for 30 minutes at 37°C. Sections were then washed and blocked with normal goat serum (NGS, 5%) in PBS for 1 hour at room temperature. Subsequently, slides were blotted and primary antibody in NGS (1 %) was added and left overnight in humidified atmosphere to avoid drying. Anti-collagen type I antibody (mouse, Abeam #ab6308) was used at 1 :1500 dilution, while anti-collagen type II antibody (mouse, DSHB #l l-l I6B3) was used at 1 :200 dilution. On the following day, sections were washed with PBS and treated with 0.3% H₂0₂ to quench any endogenous peroxidase or pseudoperoxidase activity to prevent non-specific signals. After an additional washing step, secondary antibody (goat, anti-mouse IgG (HRP), Abeam #ab6789) in NGS (1 %) solution was added and left under humidified atmosphere for 1 hour. Secondary antibody was removed by three washes in PBS and DAB substrate (ab64238, Abeam) was added and left to react for precisely 3 minutes. Sections were washed again and counterstained by Mayer’s hematoxylin solution. All samples were mounted and coverslipped with resinous mounting media (Eukitt) before imaging with a Pannoramic 250 histology slide scanner from 3D Histech.

Statistical analysis Statistical analysis was conducted with GraphPad Prism (v. 8.2.0 (425)) and statistical significance was assumed for P < 0.05. For acellular samples, storage modulus, elongation, compression and shear thinning between samples were compared with one-way analysis of variance (ANOVA) with a Tukey post hoc test. For swelling of samples, a mixed-effects model with Geisser Greenhouse correction was used.

The influence of mesh size and crosslinking degree on the formation of macroporosity was investigated with a two-way ANOVA. To analyze the viability of encapsulated and bioprinted cells, one-way ANOVA was used. To compare viability of freshly trypsinized cells with encapsulated ones, an unpaired, two-tailed T-Test was conducted. Mechanical properties of cellular, tissue engineered constructs were compared with a Brown-Forsythe and Welch ANOVA test due to unequal standard deviations. Additionally, Day 42 samples were compared to native cartilage with an unpaired, two tailed T-Test. Mechanical properties of cellular, tissue engineered constructs were compared with a Brown- Forsythe and Welch ANOVA test due to unequal standard deviations. Additionally, Day 42 samples were compared to native cartilage with an unpaired, two tailed T-Test.

Claims

1. A printable hydrogel comprising a plurality of microstrands comprising or consisting of a polymer, wherein the microstrands have a diameter of 1 pm to 500 pm, particularly 100 pm to 200 pm, and an aspect ratio of at least 100:1 , characterized in that said microstrands are entangled with each other.

2. The printable hydrogel according to claim 1 , characterized in that said microstrands are randomly entangled with each other.

3. The printable hydrogel according to claim 1 or 2, characterized in that said microstrands have an aspect ratio of at least 1000:1.

4. The printable hydrogel according to any one of the preceding claims, characterized in that said printable hydrogel comprises macropores, particularly interconnected macropores, having a pore size from 5 pm to 100 pm and/or supermacropores, particularly interconnected supermacropores, having a pore size from 100 pm to 500 pm.

5. The printable hydrogel according to any one of the preceding claims, characterized in that said microstrands comprise a plurality of first microstrands comprising or consisting of a first polymer and a plurality of second microstrands comprising or consisting of a second polymer.

6. The printable hydrogel according to claim 5, characterized in that said first polymer or said second polymer is capable of selectively binding a biomolecule or a pharmacological compound, particularly wherein said printable hydrogel comprises at least one biomolecule or pharmacological compound selectively bound to said second polymer, more particularly wherein said biomolecule or pharmacological compound is a growth factor, most particularly wherein said second polymer is Carrageenan.

7. The printable hydrogel according to claim 5 or 6, characterized in that said first polymer or said second polymer is meltable or degradable at a conversion temperature of 30°C to 50°C, particularly 35°C to 45°C, more particularly wherein said first polymer or said second polymer is gelatin.

8. The printable hydrogel according to any one of the claims 1 to 7, characterized in that said polymer or said first polymer or said second polymer is selected from the group consisting of alginate, alginate sulfate, carrageenan (particularly i-carrageenan or K-carrageenan), collagen (particularly collagen IV), chitosan, chitosan sulfate, chondroitin sulfate, fibrin, gelatin, heparin, heparan sulfate, hyaluronic acid, silk, arabic gum, cassia gum, gellan gum, sulfated gellan gum, ghatti gum, guar gum, konjac gum, locust bean gum, xanthan gum, xanthan gum sulfate and the acrylates or methacryates of the aforementioned polymers.

9. The printable hydrogel according to any one of the claims 1 to 7, characterized in that said polymer or said first polymer or said second polymer is selected from the group consisting of poly ethylene glycol, poly (2-methyl-2-oxazoline), poly (2-ethyl-2- oxazoline), poly (2-propyl-2-oxazoline), poly vinyl alcohol, poly (2-hydroxyethyl metacrylate), poly (1-glycerol methacrylate), poly (2-hydroxypropyl methacrylate), poly acrylamide, poly methacrylamide, poly acrylic acid, and poly methacrylic acid, particularly wherein the polymer is selected from poly ethylene glycol, poly (2-methyl- 2-oxazoline), poly (2-ethyl-2-oxazoline), poly (2-propyl-2- oxazoline), and poly vinyl alcohol.

10. The printable hydrogel according to any one of the preceding claims, characterized in that said printable hydrogel comprises cells, particularly wherein said cells are embedded in said microstrands.

11. The printable hydrogel according to any one of the preceding claims, characterized in that said microstrands comprise a plurality of kinks.

12. A lyophilizate comprising a plurality of microstrands comprising or consisting of a polymer, wherein the microstrands have a diameter of 1 pm to 500 pm, particularly 100 pm to 500 pm, and an aspect ratio of at least 100:1 , and wherein said microstrands are entangled with each other, particularly randomly entangled with each other.

13. A lyophilizate obtained by lyophilizing a printable hydrogel according to any one of the claims 1 to 11.

14. A method for generating a printable hydrogel according to any one of the claims 1 to 11 , comprising the steps of

a. providing a cross-linked hydrogel,

b. passing said cross-linked hydrogel through a sieve comprising a plurality of openings, such that a plurality of microstrands having a diameter of 1 pm to 500 pm and an aspect ratio of at least 100:1 are generated,

c. passing said microstrands through a volume, such that the microstrands are entangled with each other, thereby generating said printable hydrogel.

15. The method according to claim 14, wherein said microstrands comprise a plurality of first microstrands comprising or consisting of a first polymer and a plurality of second microstrands comprising or consisting of a second polymer, wherein said first polymer or said second polymer is meltable or degradable at a conversion temperature of 30°C to 50°C, particularly 35°C to 45°C, and wherein said printable hydrogel is incubated at said conversion temperature, such that said first polymer or said second polymer is melted or degraded, thereby generating supermacropores having a pore size from 100 pm to 500 pm within said printable hydrogel.

16. A 3D-printed product comprising the printable hydrogel according to any one of the claims 1 to 11.

17. The 3D-printed product according to claim 16, characterized in that said 3D-printed product is a tissue product.

18. A 3D-printing method comprising extrusion of the printable hydrogel according to any one of the claims 1 to 11 , such that a product is 3D-printed from said printable hydrogel.