Biofabrication of aligned tissues induced by light projection

Abstract

One aspect of the present invention relates to a method for manufacturing a three-dimensional hydrogel bioimplant characterized by a geometric structure in a size range of less than a millimeter. This method includes providing a first composition that is prone to photocrosslinking within a container. This first composition includes a first polymer that is prone to photocrosslinking, a photoinitiator, and optionally a refractive index matching agent, a light-absorbing dye, and a crosslinking agent. In a first irradiation step, the composition is irradiated with a plurality of spatially coherent light rays, thereby generating a plurality of microcolumns within the composition. Another aspect of the present invention relates to a three-dimensional hydrogel implant comprising, or consisting essentially of, a plurality of microbeams of a photocrosslinked polymer, wherein the three-dimensional hydrogel implant is obtained by the method according to the present invention.

Description

Technical Field

[0001] This application claims the benefit of priority of European application No. 22181705.9, filed on Jun. 28, 2022, which is incorporated herein by reference.

[0002] The present invention relates to a method for manufacturing a three-dimensional hydrogel bioimplant by irradiating a first composition containing a polymer prone to photocrosslinking, a photoinitiator, and optionally a refractive index matching agent, a light-absorbing dye, and a crosslinking agent with a plurality of spatially coherent light rays, thereby generating a plurality of micropillars in the composition. The present invention further relates to a bioimplant manufactured by the method according to the present invention.

Background Art

[0003] Light-based projection techniques are increasingly being used in the fabrication of biomimetic tissues. In recent years, the rapid biofabrication of complex cell architectures has been enabled by tomographic projection of laser light. However, in most light-induced tissue fabrication strategies, there are limited efficient cell alignments achievable for the fabrication of anisotropic tissues such as muscle and tendon. This is because most approaches focus on macroscale features (greater than 100 μm) that lack the topographical cues necessary for the highly aligned cells and extracellular tissues seen in these tissues. In techniques such as two-photon polymerization that can achieve resolutions at the cell scale (less than 30 μm), the translational potential of these approaches is limited by compromises in speed and scalability. In order to promote the fabrication and maturation of anisotropic tissues such as muscle, tendon, and nerve, instructive guidance cues have been widely studied. Topological cues with increasing aspect ratios have been shown to affect the physiology of cells within / on substrates. For example, rod-shaped microgels (aspect ratio 10) fabricated by microfluidics or soft lithography have the ability to enhance cell orientation, which is better realized by the voids between high aspect ratio micro-rods compared to microspheres. Ultra-high aspect ratio (greater than 20:1) topological features created by micropatterning techniques can effectively induce cell adhesion and alignment. In particular, when the confinement dimensions approach the scale of the cell nucleus (less than 10 μm), nuclear deformation due to these longitudinal confinements appears. The elongated shape of the cell nucleus can potentially affect cell differentiation, gene expression, and rejuvenation, where rejuvenation is brought about by chromosomal rearrangement and activation of the DNA repair mechanism, and changes in the nucleus towards a more rounded shape may be associated with disease pathology. At the tissue level, in anisotropic tissues such as tendon, it is possible to observe more elongated cell nuclei (aspect ratio 2.5 - 6) compared to isotropic tissues (1.1 - 1.8).The high aspect ratio of the nuclei of tendon cells contributes to the maintenance of the phenotype of tendon cells and the expression of important genes during aging. However, none of the above techniques provide a topological cue for both the arrangement of cells and nuclear deformation in a three-dimensional environment.

[0004] In this study, the inventors show that the phenomenon of optical modulation instability (OMI) of light rays in a photoresin can be advantageously utilized for the fabrication of highly aligned microbeams (Φ < 30 μm) in hydrogel constructs of macro size (L ≧ 10 mm, Φ ≧ 3 mm), and this can be further utilized for the fabrication of anisotropic tissues. OMI is defined as the spontaneous decomposition of a uniform light ray into smaller light rays of the same diameter that are randomly dispersed while propagating in a photoreactive medium. Photoreactive polymers are well-known optical nonlinear media because the change in their photopolymerization rate, and thus refractive index (RI), depends on the intensity of the cross-linking light. By illuminating a resin-containing vial with a partially spatially coherent light ray, highly aligned beam-like microstructures (microbeams) were generated. When the light ray is incident on the hydrogel interface, individual microbeams are seeded by the local maximum of the intensity of the incident light ray. The cross-linking rate is increased by each local maximum of the light, resulting in a locally maximum RI. As a result, the entire front of the micro-patterned polymerization propagates through the volume of the resin via these self-focusing waveguides, resulting in a permanent record of light filamentation. At the same time, local minima of the light intensity are distributed between these local light rays, which correspond to the formation of voids, where the local intensity is below the threshold required for polymerization. These voids between the hydrogel microbeams result in ultra-high aspect ratio channel-like void spaces (microchannels) after removing the uncrosslinked photoresin.

[0005] Here, the inventors present a biofabrication strategy for anisotropic tissues called Light Beam Projection (LBP). In this strategy, highly aligned microbeams are induced by OMI through the interaction of spatially coherent light beams with various photoresist systems. Cells are rapidly (less than 10 seconds) and safely encapsulated in a hydrogel matrix containing highly aligned microstructures. This approach meets the requirements for designing biomimetic (bio-mimetic) anisotropic tissues, which includes instructive guidance cues at the microscale resolution and fast and cell-friendly processing. In the LBP process, the dimensions of the microbeams and the void space between the microbeams are adjustable and are at the same length scale as the cells, resulting in efficient cell induction properties and facilitating cell migration through the void space between the microbeams.

[0006] The inventors' strategy enables control of the fabricated hydrogel matrix at the micro and macro levels; that is, controlling the dimensions of individual microbeams (2 - 30 μm) and the size and shape of the projected tissue structure (100 μm - 1 cm). Such advantages provide flexibility in the biofabrication of cell-laden hydrogel structures, such as multi-hollow / tubular hydrogel structures that have been shown to improve pre-angiogenic cell survival, for example. Finally, the inventors show that LBP can be efficiently utilized for multi-cell / multi-material biofabrication. By using a multi-step projection method, the (bio)photoresist can be cured at the desired position, enabling the creation of complex tissue constructs that better mimic the hierarchical organization of natural tissues such as muscle.

[0007] Constantini et al. (Biomaterials 2017, 131, 98 - 110) disclose the fabrication of artificial skeletal muscle tissue by three-dimensional bioprinting of hydrogel fibers embedded with muscle progenitor cells.

[0008] Parkatzidis et al. (ACS Biomaterials Sci. Eng. 2019, 5, 6161-6170) disclose the photo-structuring of gelatin methacrylamide-chitosan hydrogels.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0010] Based on the above state of the art, an object of the present invention is to provide means and methods for manufacturing an improved bioimplant having internal structural elements with structural features of less than a millimeter.

Means for Solving the Problems

[0011] This object is achieved by the subject matter of the independent claims of this specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

[0012] Summary of the Invention One aspect of the present invention relates to a method for manufacturing a three-dimensional (3D) hydrogel bioimplant characterized by a geometric structure of a size less than a millimeter. This method includes providing a first composition that is prone to photo-crosslinking in a container. This first composition includes a first polymer that is prone to photo-crosslinking, a photoinitiator, and optionally a refractive index matching agent, a light absorbing dye, and a crosslinking agent. In a first irradiation step, the composition is irradiated with a plurality of spatially coherent light rays, thereby generating a plurality of micro-columns in the composition.

[0013] Another aspect of the present invention relates to a three-dimensional hydrogel implant comprising or consisting essentially of a plurality of microbeams of a photocrosslinked polymer, said three-dimensional hydrogel implant being obtained by the method according to the present invention.

[0014] Terms and Definitions For the purpose of interpreting this specification, the following definitions apply, and terms used in the singular form shall, where appropriate, include the plural form and vice versa. In the event of a conflict between the definitions set out below and the documents incorporated herein by reference, the definitions set out here shall prevail.

[0015] As used herein, the terms "comprising", "having", "containing", "including", and other similar forms, as well as their grammatically equivalent terms, are equivalent in meaning and are not intended to be open-ended in the sense that one or more items following any one of these words do not mean to comprehensively list the one or more items concerned or to be limited to only the one or more items listed. For example, an item "comprising" components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. Thus, "comprising" and its similar forms, as well as their grammatically equivalent terms, are intended and understood to include the disclosure of embodiments of "consisting essentially of" or "consisting of".

[0016] When a range of values is provided, unless the context clearly indicates otherwise, each intervening value between the upper and lower limits of that range, to one tenth of the unit of the lower limit, as well as other recited values or intervening values within the recited range, is understood to be included within the disclosure subject to any specifically excluded limitations of the recited range. When a recited range includes one or both of the limiting values, the range excluding one or both of those included limiting values is also included in the disclosure.

[0017] As used herein, the term “about” in reference to a value or parameter includes (and describes) variations that are directed to that value or parameter itself. For example, a description that refers to “about X” includes the description “X”.

[0018] As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly indicates otherwise.

[0019] 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., cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Standard techniques are used for molecular, genetic, and biochemical techniques (see generally: Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th ed., John Wiley & Sons, Inc.) and chemical techniques.

[0020] As used herein, the term "spatially coherent light beam" relates to a plurality of light beams created from a light source emitting a defined wavelength (e.g., 405 nm) that can trigger photo-crosslinking. These light beams are spatially coherent through generation by an appropriate optical system (e.g., a Köhler standard illumination system) or a spatially coherent laser. Spatial coherence is an essential condition for the strong directivity of laser light beams. A laser can generate light beams with very high spatial coherence. The light beams have a certain phase relationship between the electric fields at different spatial positions across the light beam profile.

[0021] As used herein, the term "thiol-ene photo-crosslinking" relates to a photoinduced sequential reaction between an alkene (-ene) moiety and a thiol group to form a thioether bond. This reaction requires a photoinitiator that generates radical initiating species upon absorption of light. The radicals thus generated abstract hydrogen from the thiol, yielding a thiyl radical, which then attacks the double bond of the -ene group to generate a carbon-centered radical. The carbon-centered radical abstracts the hydrogen of another thiol, leaving behind the formed thioester bond (crosslinking).

[0022] The term "micropillar" in this specification is also referred to as "microfilament" or "microbeam" throughout this specification, and relates to a polymer structure generated by the method of the present invention, which consists of a microbeam-induced photopolymerization product of a polymer that is prone to photocrosslinking as defined in this specification. Typical dimensions of the micropillar are width: 1 - 100 μm and length: 100 μm - 15 cm (i.e., the microfilament extends widely over the entire length of the construct. The term "prevalent" indicates that the micropillar or microfilament extends essentially over the entire length of the construct). Unless otherwise specified, the microstructural dimensions described in this specification are determined by bright-field microscopy and image analysis using ImageJ (public domain, author: Wayne Rasband).

Mode for Carrying Out the Invention

[0023] Detailed Description of the Invention The first aspect of the present invention relates to a method for manufacturing a three-dimensional hydrogel bioimplant. This implant features geometric structures with sub-millimeter sizes that could not be achieved with conventional three-dimensional printing protocols. Such sub-mm structures are important as cues for tissue growth and organization. The method according to this first aspect involves a first composition that is prone to photocrosslinking, and the first composition is provided in a container having a wall suitable for irradiating the first composition with a plurality of light rays of a specified wavelength and coherence length.

[0024] The first composition includes a first polymer that is prone to photocrosslinking and a photoinitiator. Optionally, a crosslinking agent may be present if necessary to crosslink the polymer, and when using a polymer solution with a higher cell count (typically exceeding 10 million cells / mL), a refractive index matching agent and a light-absorbing dye may be applied to improve the quality of projection / multi-step projection.

[0025] In the first irradiation step, the composition is irradiated with a plurality of spatially coherent light beams, thereby generating a plurality of microcolumns in the composition.

[0026] Polymer In principle, any polymer that is easily photopolymerizable can be employed. In certain embodiments, the present composition comprises a polymer in an aqueous solution. Since the method of the present invention is particularly useful for the preparation of cell-containing structures or cell-supporting structures, an aqueous polymerization is the method of choice.

[0027] In certain embodiments, the polymer that easily undergoes photocrosslinking is a biopolymer functionalized by a covalent bond of a carbon-carbon double bond (ene)-containing moiety that can be crosslinked by a thiol-containing crosslinking agent; or a methacrylate-functionalized biopolymer that can be reproducibly prepared by the reaction of a biopolymer and methacrylic anhydride.

[0028] One specific crosslinking agent that has been proven useful by the present inventors is thiol-functionalized poly(ethylene glycol), and one specific example thereof is pentaerythritol-PEG-thiol [C(CH2O(CH2CH2O) n CH2CH2SH)4]. Such linkers are available from Merck-Sigma, and examples include pentaerythritol tetrakis(3-mercaptopropionate) (Sigma 381462), trimethylolpropane tris(3-mercaptopropionate) (Sigma 381489); 2-hydroxymethyi-2-methyl-1,3-propanediol tris(3-mercaptopropionate) (Aldrich S51145), and the like.

[0029] In the case of norbornene or acrylate-modified polymers, a crosslinking agent that provides a thiol group is required. Alternatively, it is also possible to use two polymers, one having a thiol group and the other having a vinyl group. It may also be possible to use a polymer having both a carbon-carbon double bond "ene" functional group and an SH group.

[0030] In certain embodiments, the biopolymer is selected from the group consisting of gelatin, hyaluronan, alginate, collagen, chitosan, fibrinogen, polyvinyl alcohol, silk fibroin, and cellulose. See Guo et al., ACS Appl. Mater. Interfaces 2021, 13, 6, 7037-7050; Michel et al., ACS Appl. Bio Mater. 2020, 3, 8, 5253-5262. The inventors are also considering decellularized extracellular matrix as a possible biopolymer for functionalization.

[0031] In certain embodiments, the carbon-carbon double bond (ene)-containing moiety is selected from the group consisting of norbornene carboxylic acid or dicarboxylic acid, methacrylic acid ester or -amide, acrylic acid ester or -amide, and vinyl ester.

[0032] In particularly certain embodiments, the carbon-carbon double bond (ene)-containing moiety is conferred by norbornene carboxylic acid or dicarboxylic acid.

[0033] The present invention can be carried out using many derivatives of norbornene or methacrylic acid that can be used to conjugate such reactive groups to polymers (i.e., alkynes, azides, hydrazides, DBCO, etc., and terminal linkers).

[0034] In certain embodiments, the polymers prone to thiol-ene photocrosslinking are selected from the group consisting of norbornene-functionalized gelatin, norbornene-functionalized collagen, norbornene-functionalized chitosan, norbornene-functionalized fibrinogen, norbornene-functionalized polyvinyl alcohol, norbornene-functionalized hyaluronan, gelatin methacryloyl (Gel-MA), hyaluronic acid methacryloyl (HA-MA), methacryloyl alginate (Alg-MA).

[0035] In particular embodiments, a polymer that is prone to thiol-ene photocrosslinking is norbornene-functionalized gelatin; see Gockler et al., Advanced Healthcare Materials 19 June 2021, (https: / / doi.org / 10.1002 / adhm.202100206).

[0036] Similarly, a thiolated derivative of the polymer can be used in combination with an ene-containing crosslinking agent, or a combination of a thiol-containing polymer and an ene-containing polymer can be used, for example, norbornene-functionalized hyaluronic acid and thiolated alginate, or norbornene-functionalized gelatin and thiolated hyaluronic acid.

[0037] In certain embodiments, norbornene-functionalized gelatin is characterized by a degree of substitution of 10% - 90%, particularly 30% - 55%, more particularly 47% - 50%.

[0038] The degree of substitution (DS) of NB (norbornene)-modified gelatin can be defined as the number of millimoles of norbornene moieties per gram of gelatin. Alternatively, it can also be defined as the ratio of lysine groups having norbornene functional groups. In either case, it is determined by 1-H NMR using an internal standard substance (DSS).

[0039] As defined in the Materials and Methods section of Rizzo et al. (op. cit.), "The degree of substitution (DS) of Gel-NB was determined by 1H-NMR (Bruker Ultrashield 400 MHz, 1024 scans). Briefly, 40 mg mL mL -1 of Gel-NB was added to a solution of 3-(trimethylsilyl)-1-propanesulfonic acid (DSS) in D2O (Apollo Scientific) at 0.5 mg -1It was dissolved. Using DSS as an internal standard, the integral values of the nine methyl protons of DSS (≈0.5 to -0.5 ppm) and the two NB-ene protons (≈6.21 to 6.00 ppm) were compared to calculate the NB millimoles per gram of gelatin (n = 3). The DS shown as a percentage was calculated based on the lysine + hydroxylysine content (0.325 mmol g -1 ) of type A porcine skin gelatin estimated by ClaaBen et al. (Biomacromolecules 2018, 19, 42 - 52).

[0040] Neither thiol nor alkene has structural constraints and can react at different reaction rates depending on its structure (for example, norbornene reacts faster than acrylate). This reaction is not restricted by the solvent and can be carried out in aqueous solutions or organic solvents (i.e., DMSO, DMF). At alkaline pH (above 7), since thiol reacts with multiple -ene groups without light through nucleophilic addition reaction (Michael addition), the pH of the solution has a certain influence. In certain embodiments, norbornene derivatives are used as the ene functional group: The advantage of using norbornene is to avoid cross-linking induced by unintended non-photoreactions between the mixed thiol component and the -ene component because this type of reaction is not subject to this kind of reaction.

[0041] Therefore, for most -ene functional groups (i.e., vinyl ether, vinyl sulfone, methacrylate, acrylate), it is recommended to use pH < 7 to avoid unwanted cross-linking (the meaning of thiol-ene reaction occurring without the trigger of light). It is also important to note that when using other -ene moieties (such as methacrylate), a network is partially formed by kinetic chain (chain-growth polymerization in the case of using simple Gel-MA), and thus some advantages of pure photoclick thiol-ene chemistry are lost.

[0042] Another way to classify the polymers that can be employed to practice the present invention is as follows: Resin based on sequential photo-crosslinking: The norbornene-functionalized component is selected from the group consisting of collagen, decellularized matrix, gelatin, hyaluronic acid (HA), polyethylene glycol (PEG), or polyvinyl alcohol (PVA); and the thiolated component for crosslinking is selected from collagen, gelatin, hyaluronic acid, polyethylene glycol (PEG), or polyvinyl alcohol (PVA), or dithiothreitol (DTT). Resin based on chain-growth photopolymerization: Methacrylate or acrylate-functionalized collagen, gelatin, hyaluronic acid, polyethylene glycol (PEG), or polyvinyl alcohol (PVA) or alginate. Resin that does not require modification: Collagen, fibrinogen, decellularized matrix, gelatin, which is used in combination with the Ru-SPS photopolymerization initiator system (details of the photopolymerization initiator will be described later). The Ru-SPS system crosslinks the free tyrosine groups found in these matrices, because the matrices or resins mentioned above do not necessarily require modification. This Ru-SPS photopolymerization initiator system can also crosslink acrylic groups and norbornene groups, so it can also be used for the materials shown in the previous paragraph.

[0043] Exemplary concentrations of polymers Non-limiting values of concentrations that have worked well in the hands of the inventors include the following: collagen: 1 - 30 mg / ml; fibrinogen: 5 - 100 mg / ml; decellularized matrix: 5 - 100 mg / ml; gelatin: 5 - 100 mg / ml; PVA: 1 - 30 mg / ml; PEG: 5 - 200 mg / ml; HA: 1 - 30 mg / ml).

[0044] Photopolymerization initiator In certain embodiments of any photoinitiator, the photoinitiator is selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 2,4,6-trimethylbenzoyldiphenylphosphine oxide (TPO), bis-acylphosphine oxide (BAPO), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), camphorquinone (CAS-No. 10373-78-1), ethyl-dimethylamino benzoate (EDAB), diphenyliodonium hexafluorophosphate (DPIHFP).

[0045] Examples of water-soluble photoinitiators active in the visible region include, but are not limited to, eosin Y, riboflavin, ruthenium(II) chloride hexahydrate [Ru(II)(bpy)3]Cl2, ruthenium sodium persulfate (Ru-SPS), and the like.

[0046] In a particularly specific embodiment, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

[0047] The concentration of the photoinitiator can vary depending on the type of photoinitiator. Enumerating the concentrations of the initiators that have functioned well in the hands of the present inventors, the following are included: LAP (0.1 - 2 mg / ml), Ru (0.1 - 10 mM) + SPS (0.1 - 10 mM), Irgacure 2959 (0.2 - 2 mg / ml).

[0048] Refractive index (RI) matching agent and light absorption dye In any RI matching agent of a particular embodiment, the RI matching agent is selected from propylene glycol, poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(lactic acid) (PLA), bovine serum albumin (BSA), and iodixanol (CAS-No: 92339-11-2). In certain particular embodiments, the RI matching agent is iodixanol.

[0049] Any light-absorbing dye in a particular embodiment is selected from brilliant blue FCF (CAS-No: 3844-45-9), indigotin (CAS-No: 860-22-0), fast green FCF (CAS-No: 2353-45-9), erythrosine (CAS-No: 16423-68-0), tartrazine (CAS-No: 1934-21-0), sunset yellow FCF (CAS-No: 2783-94-0).

[0050] In a particularly specific embodiment, the light-absorbing dye is sunset yellow FCF.

[0051] The amount of refractive index matching agent useful for improving the results of the method depends on the refractive index of the reaction components. In some applications, it can match the refractive index to microbeads or cells that can be embedded in the matrix used for printing.

[0052] The refractive index (RI) matching agent is slowly removed from the matrix by diffusion after making the selected implant by the method.

[0053] The amount of RI integrator is selected based on the RI of the additives in the resin, and the additives include cells or gelatin microbeads, or other particles such as silver microparticles or nanoparticles, or a conductive matrix. For example, in the case of cells, since the RI is about 1.375, to increase the RI of the GelNB / GelSH resin (original RI = 1.34) to 1.375, about 30% (w / v) of iodixanol (RI = 1.384) would be required. This RI matching is essential when it is necessary to print at a high cell density. By RI matching, light scattering from cells can be reduced so that the cells are essentially "transparent" to incident light.

[0054] Optical properties The inventors adopted wavelengths of 405 nm, 450 nm, 473 nm, 488 nm, 520 nm, 638 nm, and 750 nm. Photoinitiators useful for the practice of the present invention are described in detail in Lee et al., Chem. Rev. 2020, 120, 19, 10950 - 1102.

[0055] Other ranges that function well in the hands of the inventors include 360 - 375 nm, 400 - 420 nm, 450 - 475 nm, 550 - 575 nm.

[0056] The diameter of the spatially coherent light beam is characterized by 1 - 100 μm.

[0057] In certain embodiments, the spatially coherent light beam is characterized by a diameter of 3 - 40 μm. In more specific embodiments, the spatially coherent light beam is characterized by a diameter of 5 μm - 15 μm.

[0058] In even more specific embodiments, the spatially coherent light beam is characterized by a diameter of 7.5 μm - 12.5 μm.

[0059] Generally, the diameter of the microcolumns is from 1 μm to 100 μm. In certain embodiments, the diameter of the microcolumns is from 1 μm to 50 μm. In certain embodiments, the diameter of the microcolumns is from 2 μm to 50 μm. In certain embodiments, the diameter of the microcolumns is from 3 μm to 40 μm. In certain embodiments, the diameter of the microcolumns is from 7.5 μm to 12.5 μm.

[0060] In certain embodiments, the average diameter of the microcolumns is from 1 μm to 100 μm.

[0061] In certain embodiments, the average diameter of the microcolumns is from 7.5 μm to 12.5 μm. At a certain diameter, 75% or more, 80% or more, 85% or more, 90% or more, or even 90% or more of the microcolumns are characterized by a diameter of from 7.5 μm to 12.5 μm.

[0062] The optical setup affects the performance of the system. The inventors adopted spatially coherent light rays / coherence lengths in the range of 3.7 to 17.2 μm. In certain embodiments, this coherence length is from 1 to 100 μm.

[0063] In certain embodiments, the composition is irradiated by a plurality of spatially coherent light rays including more than 100 light rays.

[0064] The macroscopic dimensions of the light rays may be changed by changing the projected image via a digital micromirror device or a spatial light modulator.

[0065] In certain embodiments, the dimensions of the cross-section of the light rays are from 2×2 μm 2 (single pixel projection) to about 2×2 cm immediately after light shaping by DMD 2 and can even reach 10×10 cm 2 when the light rays are magnified by a telephoto lens.

[0066] Generation and modulation of the speckle pattern The speckle pattern is a characteristic of the laser light source. The term "speckle pattern" refers to the granular pattern observed when laser light interacts with an optical medium, where light waves interfere constructively or destructively with each other, resulting in bright and dark spots. These bright and dark spots form a random pattern called the speckle pattern.

[0067] The exact speckle pattern observed depends on various factors such as the coherence length of the laser, the homogenization of the laser light, the roughness of the surface, the magnification of the projected image after laser shaping, the size of the laser beam, and the distance between the surface and the observer. If any of these factors change, the speckle pattern also changes.

[0068] In certain embodiments, the homogenization of the laser light can be achieved using a fly-eye homogenizer lens (a lens that converts a round beam with a Gaussian profile into a uniformly illuminated rectangular area) to remove the Gaussian distribution of the light intensity seen in the laser. This is important to achieve a substantially uniform light intensity across the entire projected light after shaping. Homogenization does not remove the individual speckle patterns but only equalizes the light intensity. Importantly, since the FLight projection device still functions without homogenization, this is an optional step, but by homogenizing, it can lead to an improvement in the printing resolution and, since the light intensity becomes uniform across the entire projected image, a more uniformly cross-linked structure may be obtained.

[0069] When the speckle pattern changes, the diameter of the microfilaments / microcolumns and the associated microchannels / microvoids also changes. This can have different effects on cells (e.g., genetic changes due to the confinement of cell nuclei between microfilaments / microcolumns) and also bring about changes in nutrient diffusion characteristics.

[0070] Digital micromirror device / Spatial light modulator A Digital Micromirror Device (DMD) is an optical semiconductor chip consisting of an array of microscopic mirrors that can be individually controlled to reflect or redirect light. It is widely used in display technology, projection systems, and optical communication. A Spatial Light Modulator (SLM) is a device that modulates the phase, intensity, and polarization of light, enabling complex light shaping in applications such as holography, optical trapping, compensating optics, and beam steering.

[0071] Overall, the usefulness of applying DMD or SLM lies in shaping the incident light into the final projected light that impinges on the light-responsive resin. Both are important for ultimately obtaining the desired light projection shape.

[0072] DMD or SLM is commercially available. In certain cases, the DMD was manufactured by Texas Instruments and the SLM by Thorlabs. By introducing a mesh into the light beam, fine features with dimensions of 30 - 150 μm can be generated.

[0073] Based on the ratio of microbeams to voids, the materials shown in the examples are characterized in that approximately 50% of the space is filled with beam material, and thus, an implant or hydrogel construct of 1 cubic centimeter (cm 3 ) contains approximately 6370 cylindrical microbeams. In theoretical calculations, the microbeams are considered cylinders.

[0074] In certain embodiments, the composition is irradiated by a first plurality of spatially coherent light rays aligned in a first direction.

[0075] Particularly in certain embodiments, the first plurality of spatially coherent light rays form a first pattern.

[0076] Sequential construction of further structures In certain embodiments, the composition is irradiated in a subsequent or parallel second irradiation step with a second plurality of spatially coherent light rays aligned in a second direction.

[0077] In certain embodiments, the second plurality of spatially coherent light rays form a second pattern.

[0078] In certain embodiments, the first direction and the second direction are parallel.

[0079] According to some embodiments, the first direction and the second direction can be arranged at an angle of 1° to 180°. In certain embodiments, the angle is in the range of 15° to 180°.

[0080] In certain embodiments, the first and / or second pattern is arranged as a cylindrical shape, i.e., a beam shape, a filled solid cylindrical shape, or a hollow cylindrical shape, a sheet shape, or parallel strands between anchoring tendon-like support sheets similar to the geometric shape of a muscle fiber arrangement (see FIG. 19). Also, the projection can be varied along different layers of the construct to create complex shapes including microbeams.

[0081] In principle, any arbitrary cross-section is possible.

[0082] In certain embodiments, following the first and optionally the second irradiation step, a second composition is added to the container, and the second composition comprises: - A second polymer that is prone to undergo photocrosslinking, particularly via thiol-ene photocrosslinking, - A photoinitiator, and - Optionally, a refractive index matching agent (e.g., iodixanol at up to 80% (w / v)) that can increase or decrease the refractive index of the photoresin formulation. - Optionally, a photoabsorbing dye (e.g., sunset yellow or FCF at a concentration of up to 500 μg / mL) - Optionally, a crosslinking agent.

[0083] In the third irradiation step, the present composition is irradiated with a third plurality of spatially coherent light beams. Thereby, a second structure that complements the first structure can be constructed by different or the same biopolymers.

[0084] By repeating this again, another useful structure can be constructed. The number of steps is not limited in principle, and this method can proceed up to an infinite number of LBP steps. By infinitely repeating any specific projection process, a hydrogel structure having an infinite length on the projection axis can be created. Alternatively, in order to achieve better projection quality / resolution, the entire projection process can also be divided into sub-steps by increasing the number of projection steps / reducing the projection dose per step.

[0085] In a specific embodiment, the first composition that is not photocrosslinked is discharged from the container, and the container is filled with the second composition.

[0086] It is not always necessary to discharge the contents from the container, and it is also possible to simply leave the first composition and add new materials.

[0087] In a specific embodiment, in the first, second, third or any subsequent irradiation step, irradiation with a plurality of spatially coherent light beams is performed from the bottom side surface of the container onto the substrate, resulting in the formation of a plurality of polymer microbeams; the substrate moves upward from the bottom surface of the container under continuous irradiation, thereby forming a plurality of microbeams protruding from the substrate. This makes it possible to construct a very long structure, facilitating the generation of structures that exceed the dimensions limited by the length of the beam, such as implants supporting tendons and nerve guides. The tendon-like support structure is particularly shown in Nourissat et al., Nature Reviews Rheumatology 11, 223 - 233 (2015).

[0088] In a specific embodiment, the spatially coherent light beam is characterized by one or more of the following parameters: - A wavelength of 360 to 800 nm, - Coherence length of 1 to 100 μm (particularly 3 to 20 μm); - 10 to 5000 mJ / cm 2 、particularly 90 to 200 mJ / cm 2 、light dose; - Duration of 0.1 second to 100 seconds, particularly 1 second to 4 seconds; - 1 to 1000 mW / cm 2 、particularly 1 to 500 mW / cm 2 、more particularly 50 to 60 mW / cm 2 、energy density.

[0089] The minimum and maximum values of the coherence length shown here were measured from different microbeams made of different materials.

[0090] The light dose also depends on the length of the hydrogel structure (i.e., the penetration depth of light). For example, at a depth of 6 mm, a light dose of 110 mJ / cm 2 is required, and 195 mJ / cm 2 corresponds to a depth of 15 mm.

[0091] 50 to 60 mW / cm 2 corresponds to the light intensity measured in front of the photoresin container, i.e., the light intensity before it enters the photoresin and reaches the photoresin.

[0092] A light dose of 70 to 250 mJ / cm 2 is incorporated into a projection of about 1 to 4 seconds (100% gray scale).

[0093] The hydrogel prepared by the method of the present invention Another aspect of the present invention relates to a three-dimensional hydrogel implant comprising, or consisting essentially of, a first plurality of microbeams (also referred to herein as microfilaments) of a first photo-crosslinked polymer, wherein the three-dimensional hydrogel implant is obtained by the method of the present invention as described in any of its embodiments herein.

[0094] In certain embodiments, the three-dimensional hydrogel bioprosthesis according to the present invention comprises a second plurality of microbeams of a first photo-crosslinked polymer.

[0095] In certain embodiments, the three-dimensional hydrogel bioprosthesis comprises a third plurality of microbeams of a second photo-crosslinked polymer.

[0096] In certain embodiments, each of the first, second, and / or third plurality of microbeams is characterized in that the diameter of each of the first, second, and / or third plurality of microbeams ranges from 1 μm to 50 μm. In certain embodiments, the diameter ranges from 2 to 30 μm. With these dimensions, the microstructure functions as an excellent cell guidance cue, leading to an aligned cell morphology and deposition of an extracellular matrix essential for creating a biomimetic anisotropic tissue.

[0097] In certain embodiments, each of the first, second, and / or third plurality of microbeams is characterized in that more than 75% (in particular, more than 80%, 85% or more, 90% or more, 95% or more, or even 98% or more) of the alignment of the first, second, and / or third plurality of microbeams has a deviation of 2° or less from the longitudinal axis (one axis for each plurality) common to each of the first, second, or third plurality of microbeams.

[0098] In certain embodiments, each of the first, second, and / or third plurality of microbeams is characterized in that more than 75% (in particular, more than 80%, 85% or more, 90% or more, 95% or more, or even 98% or more) of the first, second, and / or third plurality of microbeams has a length ranging from 100 μm to 2 cm, and in particular, a length ranging from 100 μm to 5 cm.

[0099] The microbeams may, in some cases, fuse with other microbeams, which may affect their distinguishability. In one embodiment, in a 2 cm construct, the length of an individual microbeam can range from 100 μm to 2 cm anywhere and can form a plurality of coherent microbeams.

[0100] The target length of the structure depends on the target tissue structure for the application. Anatomically, many muscle tissues in the living body are over 5 cm in length. There is no upper limit to the length of the microbeams produced by the method of the present invention, and it is possible to create the structure of the largest primary muscle group of the leg with a length exceeding 27 cm at most.

[0101] In certain embodiments, the three-dimensional hydrogel bioimplant according to the present invention is characterized by a channel structure having a diameter of 100 μm to 1 mm, particularly 400 μm to 600 μm. Of course, larger structures are also possible; the upper limit of the diameter is determined only by the size of the photoresist container. The dimensions of the channel structure can be determined by taking a confocal image of a fluorescently labeled material (the material can be labeled with rhodamine or FITC) and manually measuring the diameter of each microfilament with the length measurement tool of ImageJ to measure the diameter of the microbeam / microfilament / microchannel.

[0102] In certain embodiments, the hydrogel implant contains cells. Suitable cells for transplantation into the implant according to the present invention include, in particular, stem cells (adipose-derived, mesenchymal, induced pluripotent, embryonic) and differentiated cells (myoblasts, fibroblasts, neurons, tendon cells, macrophages, chondrocytes, osteoblasts). The cells are encapsulated in the graft or post-seeded on the graft.

[0103] In certain embodiments, the three-dimensional hydrogel bioimplant according to the present invention may contain a growth factor or a combination of several growth factors. In certain embodiments, the growth factor can be selected from nerve growth factor, neurotropic growth factor, vascular endothelial growth factor (VEGF), transforming growth factor (TGF-β), etc., all of which have been demonstrated to promote tissue regeneration.

[0104] In addition, the present invention encompasses the development of a Filamented Light (FLight) projection system that enables simultaneous and continuous resin supply and projection of filamented light, resulting in long anisotropic constructs having cell-inducing filaments present throughout the length of the construct.

[0105] The system according to the present invention uses spatially coherent light from a high-intensity laser or a standard Keller illumination system in a specific FLight projection setup. A standard Keller illumination system is just one of the embodiments using an LED as a light source. In this type of illumination system, speckles occur in the light profile of the LED. Alternatively, advantageously, the system for generating the light source light for implementing the present invention uses laser light. This is because a laser is essentially characterized by a speckle-like distribution of light rays. This laser light can be expanded through a telescope lens, shaped through a digital micromirror device, and a desired beam projection image can be obtained. This image can be further expanded through another set of telescope lenses if necessary and finally projected onto a photoresin container (see FIG. 24).

[0106] The projected light is characterized by a static noise pattern of intensity, which is a characteristic of the laser, and is projected onto a cuvette filled with photoresin. The resin first crosslinks where the intensity is high, resulting in a local increase in the refractive index. This induces self-focusing of light only along the crosslinked resin, which further causes filamentation of the light rays into individual microfilaments along the length of the resin. Finally, the resin crosslinks along these microfilaments, obtaining a porous anisotropic construct. The complete process of gradual filamentation of light into multiple microbeams within a photo-responsive matrix is called optical modulation instability (OMI). This process is compatible with both sequential polymerization and chain-growth polymerization commonly used in photo-crosslinkable hydrogel systems.

[0107] To enhance compatibility with processes that require the fabrication of long structures, the inventors introduced a continuous supply and a FLight projection mechanism in certain exemplary embodiments of the FLight system (Figure 23; the optical path is illustrated in Figure 24). In the new projection method, instead of stacking multiple projections, a continuous supply of resin within the cuvette is used, and FLight projection is performed from above, enabling the fabrication of long structures with microfilaments running along the entire length of the structure (Figure 25). If necessary, overexposure of the resin by light from above can be prevented by using a light absorber that absorbs the light penetrating the polymer network (Figure 26), which can also ensure high printing resolution by quenching the light intensity in the off-target regions (Figure 27). The presence of the light absorber also has no impact on the presence of the microfilaments (Figure 26).

[0108] In addition to continuous microfilaments within centimeter-scale long structures, the present invention also provides another setup that attaches the cuvette to a translational-rotational stage. By moving and rotating the cuvette while controlling the FLight projection from the top or side surfaces (Figures 29 and 30), this setup provides the ability to generate a wide range of microfilament orientations (helical, cross-shaped) within the structure. Furthermore, by using a pneumatic resin supply mechanism, multiple types of resins can be supplied to the cuvette during FLight projection, enabling the formation of multi-material tissue interface models (such as tendon junctions).

[0109] Changing the aperture diameter in a standard Keller illumination setup allows the size of the microfilaments to be varied, for example, between 5 and 30 μm. A similar effect can be achieved with a laser-based illumination system, and a telescopic lens device (originally used to collimate light rays) can be used to enlarge / reduce the laser speckle pattern by changing the focal length or the distance between the lenses used therein (Figure 31). Pixelation of an image projected through a digital micromirror device (DMD) or a spatial light modulator (Figure 32) can be used to further increase the porosity of the fabricated anisotropic construct, which is useful for nutrient transport and cell guidance.

[0110] A mesh or diffraction grating placed along the optical path before, between, or after the collimating lens (shown in Figure 33) can be used to further introduce micropores into the construct (Figures 33B, C and Figure 34). Furthermore, by using two or more diffraction gratings characterized by different sizes and different relative orientations with respect to the optical path (Figure 35), a moiré pattern can be introduced into the optical path, thereby creating a unique microarchitecture (Figure 36).

[0111] In summary, the main aspects and advantages of the FLight technology presented herein include, but are not limited to, the following: - By continuous FLight projection and resin supply, constructs up to 20 cm in length can be fabricated, which corresponds to the scale of most anisotropic human tissues. - It is possible to change the speckle pattern by changing the distance between the telescopic lenses in the FLight projection setup or by using lenses with different focal lengths, thereby making it possible to change the diameter of the microfilaments. This allows control over the cellular microenvironment of various tissues. - In the FLight projection setup, it is possible to increase or decrease the size of the projected image by changing the distance between the telescopic lenses or by using lenses with different focal lengths. This will enable tissue fabrication on a small or large scale. - By adding a biocompatible light absorber (e.g., FD&C Yellow) and a free radical inhibitor (e.g., TEMPO) to the photoresin, the light penetration depth within the photoresin can be controlled, thereby enabling the obtaining of a uniformly cross-linked construct in continuous FLight projection. - By increasing / decreasing the on / off frequency of the FLight projection, it is possible to achieve better printing resolution in FLight bioprinting. - The present invention provides various methodologies for introducing fine structures into the construct, thereby bringing about better tissue maturation and regeneration effects. - The present invention provides various systems regardless of the presence or absence of a digital micromirror device (DMD). In the case where there is no DMD, it is possible to add inserts along the optical path to change the projected image and the speckle pattern. For example, by arranging a metal mesh (single or multiple) along the optical path, different micro patterns can be generated within the cross-linked construct. Alternatively, a DMD may be present to control the projected image, and in this case, a porous fine structure can be formed within the construct by pixelation of the image. - By switching the mirror, multi-directional projection becomes possible, thereby increasing the degree of freedom in the orientation of the microfilaments.

[0112] To create a complex filament orientation within the construct, it is possible to change the position of the printing cuvette and simultaneously rotate the cuvette. This can be achieved, for example, by FLight projection from above, below, and the sides, or by changing the material composition and supply rate, or by changing the light intensity to change the cross-linking rate.

[0113] Bottom-up projection can also be used instead of top-down projection, in which case the substrate moves continuously upward.

[0114] Another aspect is to facilitate continuous printing based on oxygen diffusion (i.e., a concept similar to Continuous Liquid Interface Production (CLIP) printing, but the resulting construct is characterized by continuous microfilaments). This is only possible with the limited photocrosslinking strategy as provided herein.

[0115] The three-dimensional shape of the construct produced by the method according to the present invention can be changed by: a. Changing the projection image from the DMD during the execution of the continuous FLight projection method. b. Utilizing the interference pattern generated by light passing through a plurality of wire meshes (which act as diffraction gratings) to change the projection image. This procedure may not require a DMD. c. Changing the projection image by blocking the light path through a stencil or mask or wire mesh. This procedure may not require a DMD.

[0116] The present invention further includes the following items: Item 1. A method for manufacturing a three-dimensional hydrogel bioimplant, the method comprising: a. Providing a first composition in a container that is prone to photocrosslinking, the first composition comprising: i. A first polymer that is prone to photocrosslinking, particularly thiol-ene crosslinking, and is also prone to free radical polymerization chain growth crosslinking; ii. A photoinitiator, and iii. Optionally, a refractive index matching agent (e.g., iodixanol up to 80% (w / v)) that can increase or decrease the refractive index of the photoresin formulation iv. Optionally, a light-absorbing dye (e.g., sunset yellow or FCF at a concentration of up to 500 μg / mL) v. arbitrarily, crosslinking agent comprising, and b. irradiating the composition with a plurality of spatially coherent light rays in a first irradiation step, thereby generating a plurality of microcolumns in the composition comprising, the method.

[0117] Item 2. The spatially coherent light rays are characterized by having a diameter of 1 to 100 μm, particularly 3 to 40 μm, and more particularly 5 μm to 15 μm; The method according to Item 1, most particularly characterized by having a diameter of 7.5 μm to 12.5 μm.

[0118] Item 3. The composition is irradiated with more than 100 light rays, the method according to Item 1 or 2.

[0119] Item 4. The composition is irradiated with a first plurality of spatially coherent light rays aligned in a first direction; In particular, the first plurality of spatially coherent light rays form a first pattern, the method according to any one of Items 1 to 3.

[0120] Item 5. The composition is irradiated with a second plurality of spatially coherent light rays aligned in a second direction in a second irradiation step; In particular, the second plurality of spatially coherent light rays form a second pattern, the method according to Item 4.

[0121] Item 6. The first direction and the second direction are parallel, the method according to Item 5.

[0122] Item 7. The first direction and the second direction are arranged at an angle of 1° to 180°, particularly 15° to 180°, the method according to Item 5.

[0123] Item 8. The method according to any one of Items 4 to 7, wherein the first pattern and / or the second pattern are arranged as a cylindrical shape, a sheet shape, or parallel strands between anchor ring tendon-like support sheets.

[0124] Item 9. Following the first irradiation step, a second composition is added to the container, and the second composition comprises: i. A second polymer that is prone to photocrosslinking, ii. A photoinitiator, and iii. Optionally, a refractive index matching agent (e.g., up to 80% (w / v) iodixanol) that can increase or decrease the refractive index of the photoresin formulation iv. Optionally, a light-absorbing dye (e.g., sunset yellow or FCF yellow at a concentration of up to 500 μg / mL) v. Optionally, a crosslinking agent and In a third irradiation step, the composition is irradiated with a third plurality of spatially coherent light rays. The method according to any one of Items 1 to 8.

[0125] Item 10. Any non-photocrosslinked first composition is discharged from the container, and the container is filled with the second composition. The method according to Item 9.

[0126] Item 11. In the first, second, third, or any subsequent irradiation step, irradiation is performed from the bottom side of the container onto the substrate; the substrate is moved upward away from the bottom of the container under continuous irradiation, thereby forming a plurality of microbeams protruding from the substrate, and in particular, the microbeams extend widely over the length of the longitudinal axis of the hydrogel construct. The method according to any one of Items 1 to 10.

[0127] Item 12. The polymer that is prone to photocrosslinking is a biopolymer functionalized by a covalent bond of a carbon-carbon double bond (ene) - containing moiety or a methacrylate-functionalized biopolymer. The method according to any one of Items 1 to 11.

[0128] Item 13. The method according to item 12, wherein the biopolymer is selected from the group consisting of gelatin, hyaluronan, alginate, collagen, chitosan, silk fibroin, and cellulose.

[0129] Item 14. The method according to item 12 or 13, wherein the carbon-carbon double bond (ene)-containing moiety is selected from the group consisting of norbornene carboxylic acid or dicarboxylic acid, methacrylic acid ester or -amide, acrylic acid ester or -amide, and vinyl ester.

[0130] Item 15. The polymer that is prone to photocrosslinking is selected from the group consisting of norbornene-functionalized gelatin, norbornene-functionalized collagen, norbornene-functionalized chitosan, norbornene-functionalized hyaluronan, gelatin methacryloyl (Gel-MA), hyaluronic acid methacryloyl (HA-MA), and methacryloyl alginate (Alg-MA); In particular, the method according to any one of items 1 to 14, wherein the polymer that is prone to thiol-ene photocrosslinking is norbornene-functionalized gelatin.

[0131] Item 16. The method according to item 15, wherein norbornene-functionalized gelatin is characterized by a degree of substitution of 10% to 90%, particularly 47% to 50%, and methacrylate-functionalized gelatin is characterized by a degree of substitution of 10% to 90%, particularly 45 to 60%.

[0132] Item 17. The crosslinking agent is thiol-functionalized poly(ethylene glycol), particularly pentaerythritol-PEG-thiol [C(CH2O(CH2CH2O) n CH2CH2SH)4], and the method according to any one of items 1 to 16.

[0133] Item 18. The photoinitiator is selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 2,4,6-trimethylbenzoyldiphenylphosphine oxide (TPO), bisacylphosphine oxide (BAPO), 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), camphorquinone (CAS-No: 10373-78-1), ethyldimethylaminobenzoate (EDAB), diphenyliodonium hexafluorophosphate (DPIHFP), and in particular the photoinitiator is LAP, the method according to any one of Items 1 to 17.

[0134] Item 19. The refractive index matching agent is selected from propylene glycol, poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(lactic acid) (PLA), bovine serum albumin (BSA), and iodixanol (CAS-No: 92339-11-2), and in particular the refractive index matching agent is iodixanol, the method according to any one of Items 1 to 18.

[0135] Item 20. The light-absorbing dye is selected from brilliant blue FCF (CAS-No: 3844-45-9), indigotin (CAS-No: 860-22-0), fast green FCF (CAS-No: 2353-45-9), erythrosine (CAS-No: 16423-68-0), tartrazine (CAS-No: 1934-21-0), sunset yellow FCF (CAS-No: 2783-94-0), and in particular the light-absorbing dye is sunset yellow FCF, the method according to any one of Items 1 to 19.

[0136] Item 21. The spatially coherent light beam is: a. a wavelength of 360 to 800 nm, b. a coherence length of 1 to 100 μm, particularly 3 to 20 μm; c. a light irradiation amount of 10 to 5000 mJ / cm 2 and in particular 90 to 200 mJ / cm 2 and, d. A duration of 0.1 second to 100 seconds, particularly 1 second to 4 seconds; e. 1 to 500 mW / cm 2 , particularly 50 to 60 mW / cm 2 , of energy density A method according to any one of items 1 to 20, characterized by the above.

[0137] Item 22. A three-dimensional hydrogel implant comprising, or consisting essentially of, a first plurality of microbeams of a first photo-crosslinked polymer, obtained by the method according to any one of items 1 to 21.

[0138] Item 23. The three-dimensional hydrogel bioimplant according to item 22, comprising a second plurality of microbeams of a first photo-crosslinked polymer.

[0139] Item 24. The three-dimensional hydrogel bioimplant according to item 22 or 23, comprising a third plurality of microbeams of a second photo-crosslinked polymer.

[0140] Item 25. The first, second, third and / or any subsequent plurality of microbeams are: a. Each microbeam having a diameter in the range of 1 μm to 50 μm; b. More than 75% (particularly more than 80%, 85% or more, 90% or more, 95% or more or even 98% or more) of the alignment of the plurality of microbeams having a deviation of 2° or less from the longitudinal axis, and / or c. More than 75% (particularly more than 80%, 85% or more, 90% or more, 95% or more or even 98% or more) of the length of the plurality of microbeams being 2 cm or more, particularly 5 cm or more, d. The microbeams being widely distributed over the length of the longitudinal axis of the hydrogel construct, A three-dimensional hydrogel bioimplant according to any one of items 22 to 24, characterized by the above.

[0141] Item 26. The three-dimensional hydrogel bioimplant according to any one of Items 22 to 25, characterized by having a channel structure with a diameter of 100 μm to 10 mm, particularly a diameter of 200 μm to 1000 μm, and more particularly a diameter of 400 μm to 600 μm.

[0142] In this specification, when alternative forms of a single separable feature are described as "embodiments", it should be understood that such alternative forms can be freely combined to form separate embodiments of the invention disclosed in this specification.

[0143] The present invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be derived. These examples are for the purpose of illustrating the present invention and do not limit its scope.

Brief Description of the Drawings

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Example

[0145] Example 1: Optical modulation instability and self-convergence induce ray-like microstructures in photoresin Figure 1a shows the physical phenomenon underlying the ability of LBP (light beam projection) to form cell - instructive microbeams from a photosensitive resin. The projected light beam is characterized by an intensity noise pattern that is converted into an array of microbeams constituting the hydrogel structure. First, the inventors clarify the physical factors that bring about the formation of these microbeams within the photoresin. The image projected onto a transparent photoresin - containing cuvette is characterized by the intensity noise of a speckle pattern originating from the spatial coherence of the input light source. This small intensity noise is non - linearly amplified by the cross - linking of the photoresin, and as a result, fine resin waveguides are formed through which the front of the filamentized light beam propagates through the resin volume (i.e., the inner thickness within the cuvette). More particularly, the maximum value of the initial local intensity noise locally cross - links the photoresin from the cuvette - photoresin interface, inducing a local increase in the refractive index (RI) of the photoresin. As a result, the RI of the photoresin changes, and a z - direction self - focusing effect occurs at the interface between the cross - linked and uncross - linked photosensitive materials. The formation of local RI maxima forms local micro - waveguides, and the waveguides optically capture the front of the incident light beam and propagate over long distances within the photoresin. In other words, the captured and focused light beam further cross - links the photoresin, and as a result, the self - focusing effect is continuously induced, and long microbeams are generated within seconds. Due to the cumulative effect of multiple local intensity maxima in close proximity to each other, an array of aligned microbeams is produced in the created hydrogel construct. Interestingly, the formation of hydrogel microfilaments has also been observed in previous volume 3D printing studies. However, these microstructures are considered to be defects in volume 3D printing, and their potential value in the engineering of anisotropic tissue constructs has not yet been realized. Furthermore, the uncross - linked photoresin between the microbeams exists due to local minima of intensity noise, and high aspect ratio microchannels remain between the microbeams after washing. Figure 1b shows the self - focusing and self - propagation of microbeams within the photoresin.While the spatial coherence of the incident light can be optically adjusted, the generation and propagation of the microbeams depend on the optical nonlinear properties of the resin (i.e., the change in RI during photo-crosslinking). As a result, it is important to note that the properties of the microbeams change. In the next section, the inventors will describe how to adjust the dimensions of the microbeams from different photoresins.

[0146] Example 2: Microbeams for cell guidance are highly aligned and their dimensions can be adjusted LBP was performed with several photosensitive resins. Due to the excellent kinetics of the norbornene-thiol reaction, microbeams could be formed within seconds, minimizing potential cell damage by photo-crosslinking and allowing the use of low concentrations of the polymer. The inventors first prepared a hydrogel sample using Gel-NB / 4PEG-SH photoresin. The critical light exposure dose for projection was determined by conducting a light exposure dose test based on previous research. For the 2.88% (w / v) Gel-NB / 4PEG-SH photoresin, an LBP hydrogel structure with high fidelity to the designed dimensions was created using a light exposure dose of 195 mJ / cm 2 (Figure 7, Table 1). A hydrogel cylinder with a diameter of 1 mm was projected (Figure 2a), and it was confirmed that microbeams (diameter 9.8 ± 2.5 μm) were formed inside the cylinder (Figure 2b). Approximately 88% of the microbeams were parallel (-1° to 1°) to the projection direction (Figure 2c).

[0147] [Table 1]

[0148] The generation of microbeams depends on the spatial coherence of the light beam and the nonlinear medium. Therefore, the inventors tested LBP using a higher concentration of Gel-NB / 4PEG-SH (5% (w / v)) and other common photosensitive biomaterials (gelatin methacryloyl (Gel-MA), hyaluronic acid methacrylate (HA-MA), and methacryloyl alginate (Alg-MA)) (Figure 2d). Microbeam structures were formed using all photoresins, and highly aligned microbeams were formed in all cases (exceeding 86% at -1 to 1°). The projection dose was varied between 180 and 5650 mJ / cm 2 by the resin, and the diameter of the microbeams was also varied (8.1 ± 1.4 to 11.1 ± 3.1 μm) (Figures 8-9, Table 1).

[0149] High aspect ratio structures can provide cell guidance, and due to the ability to adjust their dimensions, LBP can be applied to a wide range of tissue architectures with ECM fibers in the range of 1 to about 100 μm. Here, microbeams with various diameter ranges were fabricated by adjusting the spatial coherence length using a standard Keller illumination system consisting of a 405 nm LED light source. In particular, this photoinduced self-organization depends on the spatial coherence of the light source and the strength of the RI nonlinearity of the photoreactive material. The more spatially coherent the light beam is, that is, the spatial coherence length l of the light beam cThe longer it is, the larger the diameter of the microbeam becomes. In the Keller illumination system of the present inventors, by adjusting the aperture diameter (AD), the spatial coherence length of the light beam could be extended to the range of 3.7 to 25.8 μm in theory (Fig. 10). With this range of spatial coherence length, the present inventors were able to control the diameter of the microbeam between 8.3 and 17.8 μm using 5% (w / v) Gel-NB / 4PEG-SH photoresin (Fig. 2e). Using 5% (w / v) Gel-MA, 2% (w / v) HA-MA, and 2% (w / v) Alg-MA photoresins, the average diameters of the microbeams made of methacryloyl resin were 5.0 to 13.6 μm, 2.4 to 8.3 μm, and 4.7 to 13.7 μm, respectively. However, it was found that the average diameter of the microbeam did not depend on the field diameter of the Keller system of the present inventors. This further demonstrates that the spatial coherence of the light beam induced by OMI controls microbeam formation and that the microbeam diameter can be adjusted by adjusting the spatial coherence length of the light beam (Fig. 10).

[0150] Due to the strengthening effect of the fiber components aligned in height, the hydrogel construct composed of microbeams exhibits ideal mechanical properties for tissue engineering applications. Mechanical stimuli can be applied to such hydrogel constructs, which further induces cell alignment and promotes tissue maturation. Bulk hydrogel and microbeam-containing hydrogel samples were prepared using Gel-NB / 4PEG-SH or Gel-MA photoresin, and tensile and compression tests were performed (Figs. 2f, g). The yield stress of the hydrogel sample prepared using 2.88% (w / v) Gel-NB / 4PEG-SH photoresin increased from 2.7 kPa (bulk hydrogel) to 6.0 kPa (microbeam-containing hydrogel), and the yield strains were 64% and 122%, respectively. Similar results were obtained for hydrogel samples fabricated using 5% (w / v) Gel-MA photoresin: for the bulk hydrogel sample, the yield stress was 1.8 kPa and the yield strain was 36%. However, due to the presence of microbeams, the yield stress and yield strain increased to 7.8 kPa and 105%, respectively. The compressive modulus of the cylindrical LBP hydrogel construct (diameter 5 mm, height 4 mm) increased from 1.8 kPa to 40.7 kPa when the Gel-NB / 4PEG-SH photoresin concentration was increased from 1.44% to 5%. Similarly, in the compression of the LBP hydrogel sample, when the test was performed along the microbeam direction, it was revealed that the modulus was higher compared to the bulk hydrogel sample: 8.4 kPa vs. 3.0 kPa for 2.88% (w / v) Gel-NB / 4PEG-SH and 15.8 kPa vs. 4.0 kPa for 5% (w / v) Gel-MA photoresin. These differences in the compressive mechanical properties between the bulk hydrogel and the microbeam-reinforced hydrogel demonstrated the reinforcing properties of the microbeams in the hydrogel construct again. Furthermore, as shown in Fig. 2h, the deformation recovery of the hydrogel sample was investigated by repeated load-unload tests. No residual strain was generated after load-unload during stretching at 50% tensile strain for 10 minutes in air. This indicates that the hydrogel containing microbeams did not undergo plastic deformation under tensile load.

[0151] Example 3: Channel-like voids induce anisotropic cell migration The nanoscale pore sizes of conventional hydrogel networks impede the diffusion of oxygen and nutrients and lack cell - guiding cues. An ideal tissue - engineering matrix should have interconnected void spaces in the micron range, providing a three - dimensional space for cell migration, proliferation, angiogenesis, and extracellular matrix (ECM) deposition. Interestingly, most of the three - dimensional matrix fabricated by LPB is occupied by microchannels with an ultra - high aspect ratio (exceeding 700:1). These microchannels provide a three - dimensional space for cells to move, resulting in cell and nucleus alignment. Fluorescently labeled microbeams were fabricated using 5% (w / v) fluorescently labeled Gel - MA photoresin and then the uncrosslinked photoresin was washed. In Figure 3a, numerous microchannels were observed between the microbeams highlighted by white arrows. The void fraction was found to be approximately 50% of the entire hydrogel volume and was found to be independent of the spatial coherence length of the light beam (Figure 3b). The microchannels were uniformly distributed within the hydrogel volume portion by measuring the ratio of microbeams / microchannels in each layer in a three - dimensional image (Figure 3c, Figure 11).

[0152] Physical limitations imposed by pores smaller than 3 μm have been shown to significantly restrict cell migration in a three-dimensional tissue matrix. This size is considered a threshold that cells cannot pass through because of the limited deformability of the nucleus. Here, by adjusting the spatial coherence length of light, we adjusted the diameter of the microchannels and ensured effective migration of cells through the microchannels. When the coherence length was decreased from 17.2 μm to 3.7 μm using 5% (w / v) fluorescently labeled Gel-MA, the average diameter of the microchannels increased from 2.7 μm to 5.8 μm (Figure 3e). On the other hand, the average aspect ratio of the microchannel structure decreased from 1540:1 to 700:1 with an increase in the spatial coherence length (Figure 3f). To achieve a high aspect ratio for cell migration and efficient cell guidance, normal human dermal fibroblasts (NHDF) were encapsulated in an LBP hydrogel containing microchannels with an average diameter of 3.5 μm and an aspect ratio of 1178:1. After 1 hour, 1 day, 3 days, and 7 days of culture, NHDF were stained with Calcein-AM and their migratory ability was evaluated. Surprisingly, already after 1 hour, NHDF were observed to move into the microchannels from the original encapsulation site, with nuclear deformation (highlighted by the yellow arrow in Figure 3d). The average aspect ratio of the cells increased from 1.24 to 15.8 after 7 days of culture (Figure 3g), indicating that the cells elongated while moving through the microchannels. As evidence that the microchannels in the hydrogel matrix are interconnected (e.g., cells can move across multiple microchannels), it was observed that fibroblasts seeded on the microbeam surface moved into the hydrogel matrix from this surface 1 day after incubation, but not from the bulk hydrogel surface. Since there was a significant difference in the depth of cell migration, it can be seen that these microchannels are interconnected to allow cell migration across the microbeam (Figure 12). Overall, the above results indicate that continuously interconnected microchannels can support cell migration not only in the microchannel direction but also across the microchannel direction.Such cell migration is essential for establishing cell-cell contact in a three-dimensional matrix, such as the three-dimensional network of tendon cells that requires cell-cell communication via gap junctions. Furthermore, microchannels can induce nuclear elongation and support nuclear movement, which contributes to an important role in maintaining the cell phenotype and promoting myoblast fusion for cell reconstruction after muscle physiological injury.

[0153] The control of gene expression due to mechanical confinement-induced nuclear reorganization has shown interesting results in the field of tissue engineering. For example, nuclear confinement can induce the redifferentiation and reprogramming of cell fate by activating DNA repair pathways. At the tissue level, under dynamic mechanical loading, the expression of ECM-related genes is upregulated in cells with elongated nuclei. In this study, the inventors demonstrated that ultra-high aspect ratio microchannels have unique advantages in creating highly aligned tissues by inducing mechanical confinement. Using the same bioresin formulation (2.88% (w / v) Gel-NB / 4PEG-SH photo-resin containing NHDF), various tissue microenvironments were created, including three-dimensional microbeams, three-dimensional bulk hydrogels, and two-dimensional (2D) microbeams. Cells were encapsulated in hydrogel constructs containing three-dimensional microbeams, in bulk hydrogels, or seeded later on the microbeam surface (two-dimensional microbeams). After 1 hour, 1 day, 3 days, and 7 days of culture, phalloidin staining was used to determine cell alignment under different conditions (Figure 4a). Cells began to align along the microbeams 1 day after incubation under both three-dimensional and two-dimensional conditions, but did not align in bulk hydrogels, thus demonstrating again the cell-inducing properties of microbeams as physical cues. Compared to two-dimensional microbeams, NHDF encapsulated within the three-dimensional microbeam matrix showed a lower proliferation rate (ratio of ki67-positive cells) on the first day. However, after 3 days of culture, it was found that approximately 76% of NHDF were ki67-positive cells, while this ratio was approximately 19% on the microbeam surface (Figure 4b). Similar results have also been reported for the activation and proliferation state of fibroblasts after static compressive force and lateral confinement culture. The mechanical stimuli obtained from hydrogel matrices may offer a new way to induce cell proliferation, especially for senescent cells with growth deficiencies such as tendon cells and tendon stem / progenitor cells.

[0154] Next, the inventors demonstrated that the ultra-high aspect ratio microchannels and the resulting cell confinement can control gene expression (Figures 4c and 13). Upregulation of the mechanosensitive ion channel Piezo1 was seen at the early stage (1 hour and 1 day) under the 3D microbeam condition compared to the 3D bulk hydrogel and 2D microbeam. Similarly, the inventors observed upregulation of the expression of lamin A / C protein (encoded by the LMNA gene) after 1 hour in the 3D microbeam; this gene plays an essential role in the transmission of mechanical signals from the cell membrane to the nucleus and the nuclear stiffness. Furthermore, the expression of COL1A1 was upregulated at the start of incubation, similar to the expression of PIEZO1. Previous studies have shown the association between the expression of Piezo1 and ECM-related genes. For example, the deficiency of Piezo1 results in a decrease in the expression of COL1A1, and Piezo1 plays an important role in ECM deposition in tissue maturation under mechanical stimulation. Also, the deficiency of micron-sized voids in the bulk hydrogel increases the expression level of MMP2, which helps cell migration by remodeling the hydrogel matrix. However, the decrease in PIEZO1 expression was seen from day 3, which can be explained as the loss of mechanical stimulation. This may be due to the degradation of the microbeam and the deformation of the microbeam caused by cell contraction. In the future, it is expected that the loss of mechanical stimulation can be offset by applying static / dynamic mechanical loads, which is promising for the biofabrication of mature anisotropic tissues.

[0155] Example 4: LBP for the Biofabrication of Highly Aligned Tissue Constructs The inventors demonstrated the ability of LBP to biofabricate highly aligned tissues using 2.88% (w / v) Gel-NB / 4PEG-SH photoresin in four cell types: NHDF, human tenocytes (HT), human umbilical vein endothelial cells (HUVEC), and mouse myoblasts (C2C12). The cell viability 1 hour after LBP was found to be over 85% for all cell types. In contrast, low cell viability (less than 70%) of HUVEC was observed in bulk hydrogels prepared using the same light dose as LBP (Figure 14). However, when the incubation was extended up to 21 days, the viability of C2C12 in the hydrogel samples of LBP was about 74%. To overcome this limitation, a series of hollow microstructures with diameters of several hundred microns were created in the hydrogel construct by blocking the light from the designed projection image. The encapsulated C2C12 cells showed higher cell viability (about 89%) in the hydrogel construct containing multiple hollow channels after 3 weeks of culture. Therefore, in subsequent experiments, multi-hollow microstructures (diameter 216 μm) were adopted to obtain higher bioactivity in cell-laden hydrogel constructs.

[0156] The potential of LBP in designing anisotropic tissues is based on the ability of the microstructure to induce cell alignment and the alignment of the ECM (i.e., self - aggregation into aligned collagen fibers). The cell - inducing properties of micro - beams were tested in structured samples of approximately 1 mm in diameter (2.8 - second light irradiation, containing multi - hollow microstructures) using NHDF. Phalloidin staining was used to visualize the ability of the micro - beams to induce cell alignment (Figure 5a). After 14 days of culture, 99% of the NHDFs extended and aligned within ±30° of the projection direction (Figure 5e). The formation of aligned type I collagen was confirmed from immunofluorescence images using anti - type I collagen antibody (Figure 15). On the other hand, in bulk hydrogels, no cell alignment and aligned collagen fibers were observed due to the lack of cell - inducing cues (Figure 16). Similarly, as shown in Figure 5f, in hydrogel samples containing micro - beams, more collagen - positive regions (about 48%) were observed compared to bulk hydrogels (about 28%). Interestingly, cell nuclei were also elongated and oriented in the micro - beam - containing materials compared to the nuclei of randomly oriented fibroblasts in bulk hydrogel samples (Figure 5g). Recent studies have shown that physical cues can control the shape of cells and nuclei and ultimately affect phenotype, cell behavior, and gene expression. For example, the presence of fiber components in the tissue matrix can induce fibroblasts to switch their phenotype to a myofibroblast - like state characterized by an elongated shape. Furthermore, culturing fibroblasts with lateral restriction induces dedifferentiation and reprogramming, leading to the activation of senescent fibroblasts.

[0157] Next, the inventors investigated the arrangement of HTs within the hydrogel matrix fabricated by LBP, which has important implications for the biofabrication of tendon tissue models. In HTs after 2 weeks of culture, highly aligned filamentous actin (F-actin) was observed. Immunofluorescent detection of type I collagen fibers again demonstrated that microbeams can efficiently induce cells and the ECM in which they are deposited (Figure 5b). Furthermore, by introducing a fibroblast-HUVEC co-culture system, the inventors showed that highly aligned blood capillary formation can also be induced by LPB, as detected by CD31 immunostaining in 2-week cultures (Figure 5c). Interestingly, the inventors found that continuous capillaries are aligned in the projection direction and observed that some vessels exceed 1.4 mm in length. Blood capillaries were confirmed to have lumens as seen in the cross-sectional views of 3D confocal images (Figure 17).

[0158] Finally, C2C12 cells were encapsulated in 2.88% (w / v) Gel-NB / 4PEG-SH photoresin and LBP was performed, further demonstrating its potential in the biofabrication of muscle tissue. When exposed to differentiation medium containing 2% (v / v) horse serum, the inventors observed myoblast fusion and myotube alignment as seen by immunofluorescent staining using anti-mouse myosin heavy chain (MyHC) antibody and phalloidin after 3 weeks of culture (Figure 5d). In LBP tissue samples, it was found that 95% of the myotubes were aligned along the orientation of the microbeams (-30 to 30°) (Figure 5h). As expected, myotubes showed a random orientation in the bulk hydrogel. Similarly, in LBP samples, more highly aligned contractile myotubes with a higher fusion index were observed compared to the bulk hydrogel (Figure 5i). Interestingly, it was observed that more than 76% of the myotubes in the hydrogel constructs fabricated by LBP had more than 6 nuclei per myotube, which was not seen in the bulk hydrogel samples (Figure 5j).

[0159] Overall, the inventors demonstrated that LBP is an effective strategy for the biofabrication of anisotropic tissues that can be manufactured in a short time and has excellent biocompatibility with cells. The microbeams, which are physical cues for inducing cells, result in efficient alignment of the cells required to create anisotropic tissues. LBP offers decisive advantages over conventional strategies that often rely on post-seeding of cells onto fiber-based scaffolds and substrates. These techniques simply provide a two-dimensional microenvironment, and the fact that the cell distribution is non-uniform further limits these applications. In extrusion-based bioprinting strategies, high concentrations of fibrous components and smaller nozzles are often required to achieve effective cell alignment, which can generate significant shear stress on the cells.

[0160] Example 5: LBP for the Biofabrication of Complex Hydrogel Constructs Finally, the inventors explored the potential of LBP to create anisotropic tissues mimicking in vivo tissue structures. Using 2.88% (w / v) Gel-NB / 4PEG-SH photoresin, various hydrogel constructs with detailed features were fabricated, such as hollow structures (Figure 6a, Figure 18) and centimeter-scale hydrogel QR codes with a size of approximately 220 μm. Also, a tubular hydrogel structure carrying cells was fabricated, and after 7 days of culture, the encapsulated NHDF was stained with Calcein-AM (Figure 6b). A monolayer of NHDF was observed on the inner and outer walls of the tubular structure. Subsequently, since the channels were filled with the fluorescently labeled dye (red), it was shown that these tubular constructs were maintained even after 7 days of culture. From the above results, it was shown that LBP is a promising method for fabricating anisotropic hydrogel constructs with a freely designed cross-sectional structure for anisotropic tissue engineering.

[0161] A multi - cell / multi - material bioprinting strategy was established by performing multiple projections continuously while exchanging the bioresin between projections. LBP enables the creation of hierarchical tissue organization by controlling the placement of cells and materials, as well as the stiffness of the materials. As seen in Figure 6c, a multi - material structure was realized with two complementary projection images. In the first image, the bioresin was spatially cured to create the first part of the hydrogel structure. After removing the uncrosslinked bioresin, the second bioresin was loaded into the vial and the second complementary image was projected. As demonstrated, cell - free hydrogel structures were fabricated using two types of fluorescently labeled photoresins (Figure 6d). NHDF labeled with Cell Tracker Green was mixed with 5% (w / v) Gel - NB / 4PEG - SH photoresin and a central cylindrical hydrogel structure was projected. A peripheral tissue structure loaded with NHDF labeled with Cell Tracker Red was projected using 2.88% (w / v) Gel - NB / 4PEG - SH photoresin. It was confirmed that the encapsulation of two fluorescently labeled cells into the designed hydrogel structure was successful (Figure 6e). As expected, due to the presence of the microbeam structure constructed in the hydrogel construct, cell alignment was observed after 7 days of incubation in both of the projected hydrogel constructs.

[0162] Example 6: Multi - direction Projection Strategy The inventors developed a projection strategy that enables the multi - directional bioprinting of hydrogel constructs containing microbeams. This is achieved by repeating the projection process from different directions. Briefly, the photoresin container is rotated by a specific angle (e.g., 30 degrees, 45 degrees, 90 degrees) for the second projection after the first projection (Figure 20). This method will satisfy the bioprinting of anisotropic tissues that require specific topological cues such as myocardial tissue.

[0163] Next, a method for fabricating an ultra-long hydrogel structure was implemented. As a result, a tissue structure approximately cm in length containing continuous micro-light rays can be fabricated. This enables the fabrication of hydrogel constructs with infinite length. The light rays are irradiated from the upper part (Fig. 21) or the lower part (Fig. 22) of the container. When the light rays are irradiated from the lower part, the bottom surface of the container is treated so that the cured hydrogel construct does not adhere. The light projected from below crosslinks the photoresin between the bottom surface of the container and the substrate, thereby causing adhesion of the hydrogel construct to the substrate. Subsequently, the substrate moves to the next position, enabling it to be filled with fresh photoresin, and then being projected, a long hydrogel construct is formed.

[0164] Finally, the inventor established a hybrid projection method that combines a horizontal multi-directional projection strategy and a top (upper) projection approach (Fig. 23). As a result, the fabricated hydrogel construct can include microbeams aligned in multiple directions horizontally and vertically aligned microbeams.

[0165] Example 7: Hybrid Biofabrication Strategy for Hydrogel Constructs (Multi-Material / Multi-Light Source) The inventors implemented a new design of the light source (Fig. 24) and the optical setup. As a result, the inventors' biofabrication strategy can be adapted to a wider range of photosensitive materials (e.g., photoreactive systems that require different wavelengths to induce reactions). With various optical setups, it is further possible to control the dimensions of microbeams / microchannels, the porosity, etc. over a wider range, enabling the fabricated hydrogel matrix to support more or more complex types of tissues. For example, when the microbeams are small, cell alignment is better, but when the voids are large (or numerous), cell migration is effectively promoted and cell-cell communication is established.

[0166] Example 8: Fabrication of a Long Microbeam-Supported Tissue Construct at High Cell Density Due to light scattering induced by high cell density (exceeding 5 million cells / ml) in projection, the formation of microbeams is often limited to several millimeters (typically 2 mm) along the length of the hydrogel construct. The inventors have demonstrated increasing the refractive index of the resin using a refractive index matching agent (e.g., 30% (w / v) iodixanol in the resin), such that after adding iodixanol to Gel-NB / Gel-SH photoresin to a final concentration of 30% (w / v), the refractive index of 5% Gel-NB / Gel-SH photoresin increases from 1.345 to 1.46. This method reduces light scattering and enables the formation of long, continuous microbeams in cell-laden hydrogel constructs with high cell density.

[0167] Furthermore, to fabricate long hydrogel constructs, the inventors use a bottom-up projection approach (described above in Figure 21), where the first layer is added and crosslinked via the aforementioned projection approach, followed by addition of subsequent photoresin and repeated photocrosslinking. This can be carried out, in effect, infinitely to obtain long (exceeding 20 cm in length) hydrogel constructs. However, the inventors have also observed that projection of any layer can irradiate the underlying layer such that it receives additional light exposure, thereby increasing the degree of crosslinking in the underlying layer. This results in non-uniform crosslinking across the entire length of the construct, with the lower layers generally characterized by high rigidity and coalesced microbeams, which will likely result in near-optimal cell alignment and anisotropy of matrix organization. The inventors have demonstrated that it is possible to add a light-absorbing dye (e.g., sunset yellow or FCF) to the photoresin, thereby enabling fine tuning of light transmission through the photoresin (Figure 25). For example, the inventors calibrated the amount of photoresin added to each layer, and the associated light irradiation intensity and irradiation time, such that the penetration depth of each projection matches the projection depth of the new layer. This results in the formation of an integrated construct without over-photocrosslinking in each layer, thereby resulting in cell alignment and organization of an anisotropic matrix across the entire length of the construct.

[0168] For example, in a 5% (w / v) Gel-NB / Gel-SH photoresin containing 0.05% (w / v) LAP, 30% (w / v) iodixanol, and 10 million cells / mL of C2C12 cells, the inventors used 50 μg / ml of sunset yellow dye (known to absorb light at 405 nm) to achieve a penetration depth of 5 mm per projection layer where the microbeams were widely distributed (Figure 26). The resulting construct (about 3 cm in length made with 6 projections) is characterized by microbeams throughout its length and the formation of aligned myotubes throughout its length. Here, the concentration of the light-absorbing dye (from 1 μg / mL to 500 μg / mL) and the concentration of iodixanol (5 - 80% (w / v)) can be varied according to the needs of the experiment.

[0169] Example 9: Conclusion The inventors demonstrated a rapid biofabrication method for creating 3D hydrogel constructs containing cell-induced microbeams. Through LBP, alignment of four types of cells / deposited ECM was achieved. The ultra-high aspect ratio topological cues provided by the microchannels have a strong influence on cell and nuclear morphology. By performing multiple consecutive projection processes, a multi-cell / multi-material biofabrication strategy was established that enables mimicking the hierarchical organization of tissues.

[0170] Example 10A: Graft for Nerve Repair Nerve injury can lead to loss of sensation, muscle function, and overall function of the limb or body part on the affected side. Conventional treatments for nerve injury, such as nerve autografting, have limitations and can cause complications at the donor site. Grafts fabricated using filamented light (FLight) bioprinting technology can provide a promising alternative for nerve injury repair due to their ability to promote directed axonal growth and tissue regeneration and integration. FLight grafts with unidirectionally oriented microfilaments can be used for nerve injury repair, for example, creating nerve conduits (acellular grafts for nerve conduits) that provide a scaffold for directional axonal regeneration, or nerve wraps for the treatment of crush injuries.

[0171] The graft material can be based on one or a combination of the resins as described above, particularly resins based on sequential photopolymerization or chain-growth photopolymerization.

[0172] Combinations of cells that have been shown to promote axonal regeneration (such as Schwann cells, human mesenchymal stem cells, human adipose stem cells, or induced pluripotent stem cells) can also be employed. These cells would be encapsulated within the graft.

[0173] Growth factors that have been demonstrated to promote axonal regeneration (such as nerve growth factor, neurotropic growth factors, etc.) may be present.

[0174] In certain embodiments, at least one of the material components in the matrix will be based on collagen or a decellularized matrix.

[0175] The implementation scheme of the nerve graft is shown below: The graft is substantially cylindrical, with a diameter of 1 - 7 mm and a length of 5 - 50 mm. The graft can be made to feature different microstructural configurations such as a filamentous arrangement of microfilaments (microfilament diameter (φ) = 1 - 30 μm) and a mesh structure (mesh diameter (φ) = 30 - 150 μm). Furthermore, by changing the gray scale of the image projected in the FLight system, a graft can be created that features different rigidities in the core and shell. Here, the shell can be created at a high light intensity to have a high yield strength to enable suturing / anastomosis, while the core part is created at a low intensity to have a low rigidity (less than 10 kPa) to promote axonal growth. Both the core and shell parts will be made to feature uniaxially aligned microfilaments to enable axonal growth. In addition to increasing the exposure to light in the shell region, the strength of the shell region can be further enhanced by secondary cross-linking mechanisms such as short-time exposure to bacterial transglutaminase for chemical cross-linking (in the case of gelatin- or collagen-based resins) or ionic cross-linking (in the case of alginate-based resins).

[0176] The yield strength required for a sutured nerve graft can depend on various factors such as the type of nerve, the diameter of the nerve, and the tension applied during suturing. Generally, the yield strength of a nerve graft must be high enough to resist breaking or tearing under the tension applied during suturing. When determining the peel force of a nerve graft using a tensile test, it will be approximately 20 - 60 N, which would correspond to a yield strength of 10 - 100 MPa.

[0177] The yield strength required for a sutured nerve graft varies depending on the application. However, studies have shown that nerve grafts with a yield strength of at least 30 - 50 N are suitable for suturing. Furthermore, nerve grafts with a higher yield strength may be more resistant to stretching or deformation that can occur during suturing or postoperative procedures, and thus may lead to better results.

[0178] According to experiments using dorsal root ganglion (DRG) encapsulated in FLight gel, it has been shown that a softer gel with a mesh (Young's modulus 1 - 3 kPa) results in the best axon growth in terms of axon length and number of axons. Both axon length and number of axons normalized to the size of the DRG are greater in softer constructs (about 2 - 3 kPa) characterized by a mesh (preferably 63 μm mesh). The microfilaments of the FLight hydrogel can direct axon growth in both (forward and backward) directions.

[0179] Example 10B: Grafts for muscle repair: Several types of muscle lesions are suitable for treatment with FLight grafts. For example, dermal matrix grafts have been used for the treatment of irreparable massive aponeurotic ruptures that can be involved in large muscle injuries. Similarly, the use of cell grafts or acellular grafts for the treatment of muscle mass loss is a very active area of research and development. In the case of muscle injury, the grafts provided herein have the ability to support cell adhesion, alignment, proliferation, and to promote the formation of a new extracellular matrix. Here, grafts characterized by different combinations of the resins and material components described in the section on nerve grafts (preferably one of the components is based on collagen or decellularized matrix) would be ideal.

[0180] Possible uses of FLight grafts include the following: 1. Muscle strain (muscle detachment) and rupture - Acellular grafts can be used for the repair and regeneration of muscle tissue damaged by strain or rupture. The graft provides a support and scaffold for the growth of new tissue and helps to improve muscle function. 2. Muscle atrophy - Muscle atrophy refers to the loss of muscle mass and muscle that can occur due to aging, injury or disease. In such cases, acellular grafts can be used to promote muscle regeneration and prevent further muscle loss. 3. Muscular dystrophy - Muscular dystrophy is a genetic disease that causes progressive muscle weakness and degeneration. Although acellular grafts may not be able to cure the disease, they can be used to improve the muscle function and quality of life of patients. 4. Volumetric muscle loss (VML) - VML occurs when most of the muscle tissue is lost due to injury or surgery. In such cases, acellular grafts can be used to promote muscle regeneration and prevent the formation of scar tissue.

[0181] Example 10C: Graft for tendon repair: The FLight graft can also be used to treat various tendon disorders where conventional treatments have been ineffective. Here, the aligned microstructure of the FLight graft would be important for tendon treatment using acellular grafts. Tendons have a highly organized structure where collagen fibers are arranged in a parallel orientation to resist tension and transmit force. Some of the tendon disorders that can be repaired with the FLight graft include the following: 1. Achilles tendon disorder 2. Rotator cuff tendinopathy 3. Patellar tendinopathy 4. Tennis elbow (lateral epicondylitis) 5. Golfer's elbow (medial epicondylitis)

[0182] In such cases, a FLight graft can be used to repair or augment damaged or weakened tendon tissue, thereby increasing tendon strength, reducing pain, and promoting healing. The mechanical properties (yield strength) required of the graft used for tendon repair can vary depending on the specific application and the individual needs of the patient. Generally, the graft needs to support the damaged or weakened tendon and have sufficient strength to allow for proper healing. It has been reported that acellular grafts with a yield strength of at least 250 kPa result in good clinical outcomes for Achilles tendon repair. For rotator cuff repair, grafts with a yield strength of at least 200 kPa are commonly used. For knee tendon injuries, success has been achieved using acellular grafts with a yield strength of at least 150 kPa. For tennis elbow and golfer's elbow, it has been reported that grafts with a yield strength of at least 100 kPa result in good outcomes.

[0183] Example 10D: Articular cartilage: Repair of articular cartilage is required in some conditions because the self-repair ability of this tissue is limited due to restricted vascularization and the very dynamic joint growth environment. Cartilage damage can occur for various reasons, such as trauma, degenerative joint diseases such as osteoarthritis, or genetic diseases such as osteochondritis dissecans.

[0184] Articular cartilage damage can range from small surface defects to full-thickness cartilage defects. If these defects are left untreated, they can progress to more severe degenerative joint diseases, leading to pain, loss of joint function, and a decrease in quality of life.

[0185] To use an acellular graft for articular cartilage repair, first, the damaged cartilage is excised and smoothed to create a stable surface. Then, the graft is prepared and sized to fit the defect. The graft is fixed with sutures or tissue adhesives. Over time, the graft becomes incorporated into the surrounding tissue, stimulating the infiltration and proliferation of new chondrocytes and promoting tissue regeneration.

[0186] Materials and Methods Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich and cell culture reagents were purchased from Gibco.

[0187] Synthesis of Gel-NB The synthesis process and properties of Gel-NB are described in the inventors' previous research: Briefly, type A gelatin derived from porcine skin was dissolved at 10% (w / v) in 0.1 M pH 9 carbonate-bicarbonate buffer at 50 °C. Next, 1 / 5 of the total cis-5-norbornene-end-2,3-dicarboxylic anhydride (Carbic anhydride: CA) required to obtain the desired Gel:CA ratio was added to the solution. The reaction was allowed to proceed for 10 minutes with stirring before adjusting the pH to 9 with 0.5 M NaOH solution. The pH adjustment and successive addition of CA were repeated 5 times at 10-minute intervals. Next, the solution was diluted 2-fold with pre-warmed Milli-Q water at 40 °C and the pH was adjusted to 7.4 with 0.5 M HCl solution. For centrifugation at 3000 rcf for 15 minutes, the supernatant was then dialyzed against Milli-Q water at 40 °C with frequent water changes for 3 - 4 days and finally lyophilized.

[0188] For photoresin preparation, lyophilized Gel-NB was dissolved in PBS and held at 40 °C for 30 minutes. 4PEG-SH (10 kDa, JenKem Technology) was added to the Gel-NB solution to obtain the desired SH:NB ratio (1:1). Next, a lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) stock solution of 2% (w / v) photoinitiator (PI) in PBS was mixed with the Gel-NB / 4PEG-SH solution such that the final concentration was 0.05% (w / v). The photoresin was filtered through a 0.45 μm filter (Filtropur S 0.45, SARSTEDT AG) and stored in the dark.

[0189] Synthesis of Gel-MA and Fluorescently Labeled Gel-MA Gel-MA was synthesized as described above. The degree of substitution (DS) was estimated by 1 1 1H-NMR (Bruker Ultrashield 400 MHz, 1024 scans) in D2O (Apollo Scientific). The Gel-MA lysine integration signal (2.95 - 3.05 ppm) was compared with the unmodified gelatin lysine integration signal (2.95 - 3.05 ppm). The phenylalanine signal (7.2 - 7.5 ppm) was used as an internal reference. The DS was found to be approximately 45%.

[0190] Fluorescently labeled Gel-MA was prepared by modifying Gel-MA with fluorescein-5-isothiocyanate (FITC) or rhodamine B isothiocyanate (RBITC). Briefly, first, 10% (w / v) Gel-MA was dissolved in 100 mM sodium bicarbonate solution. Then, 0.1% (w / v) FITC-DMF or rhodamine solution was added, and the mixture was stirred at 40 °C in the dark for 6 hours. After the reaction, the mixture was dialyzed against deionized water at 30 °C for 4 days to remove unreacted monomers, and then lyophilized to obtain fluorescently labeled Gel-MA.

[0191] For photoresin preparation, lyophilized Gel-MA was dissolved in PBS and held at 40 °C for 30 minutes. Next, 2% (w / v) LAP stock solution was mixed with the Gel-MA solution to obtain a final concentration of 5% (w / v) Gel-MA and 0.05% (w / v) LAP. The photoresin was filtered through a 0.45 μm filter and stored away from light.

[0192] Synthesis of HA-MA Low molecular weight HA-MA (M w = 50,000 - 70,000) with a DS of 20 - 50% was purchased.

[0193] For the preparation of the photoresin, HA-MA was dispersed in PBS and stirred at 4 °C until completely dissolved. After that, it was mixed with a 2% (w / v) LAP stock solution to prepare a 2% (w / v) HA-MA and 0.05% (w / v) LAP solution. The photoresin was filtered through a 0.45 μm filter and stored in the dark.

[0194] Synthesis of Alg-MA Sodium alginate (M w > 200 kDa, PRONOVA UP MVG, NovaMatrix (registered trademark)) was dissolved in 25 mL of Milli-Q water and made into a 1% (w / v) solution while stirring continuously. Methacrylic anhydride was distilled before use (20 equivalents (eq) relative to the hydroxyl group, 52 mmol, 7.7 mL) and added; the resulting emulsion was vigorously stirred at room temperature for 24 hours. The pH was checked and adjusted frequently to 7 - 8 with a 0.5 M NaOH solution. This solution was transferred to a Falcon tube and centrifuged at 3000 rcf for 15 minutes to remove excess methacrylic anhydride. The aqueous phase was precipitated in ethanol, and the precipitate was filtered through a glass funnel with frit (S4 porosity) and dried under high vacuum overnight. The dried Alg-MA was dissolved in Milli-Q water and dialyzed at room temperature (RT) for 3 - 4 days while frequently changing the water. Alg-MA was obtained after lyophilization. For 1 The 1H-NMR spectrum for determining DS was obtained using a Bruker spectrometer operating at a proton frequency of 400 MHz with 1% (w / v) Alg-MA in D2O. It was found that DS was approximately 51%.

[0195] To prepare the photoresin, lyophilized Alg-MA was dissolved in PBS. Next, the LAP stock solution was mixed with the Alg-MA solution to obtain a final concentration of 2% (w / v) Alg-MA and 0.05% (w / v) LAP. The photoresin was filtered through a 0.45 μm filter before use.

[0196] Cell culture Tendon cells were provided by Professor Lee Ann Applegate and have been described previously. Human fetal progenitor tendon cells were isolated from the Achilles tendon of a male organ donor at 14 weeks of gestation according to a protocol approved by the Ethics Committee. University Hospital of Lausanne (CHUV), Ethics Committee Protocol No. 62 / 07: Organ donation at 14 weeks of gestation. This is registered with the Federal Transplantation Program and its DAL (Department of Musculoskeletal Medicine) biobank and complies with the law. NHDFs were isolated from skin biopsies of juvenile foreskins. Biopsies were taken under the informed consent of the guardians, and their use for research purposes was approved by the Ethics Committee of the Canton of Zurich (BASEC-Request-Nr. 2018-00269). Tendon cells and normal human dermal fibroblasts (NHDFs) were cultured in DMEM + GlutaMAX™-I + 10% (w / v) fetal bovine serum (FBS) + 10 μg mL -1 antibiotic–antimycotic (Anti-Anti) in Falcon® cell culture multi-flasks (TC5 layer, 875 cm 2 2). Myoblasts (C2C12) were obtained from ATCC and cultured in DMEM medium + 10% (v / v) FBS + 10 μg mL -1 Anti-Anti for cell proliferation. Cells were passaged at 90% confluence and detached using 0.25% trypsin / EDTA. For C2C12 differentiation, the cell-laden hydrogels were prepared in DMEM + 2% (v / v) horse serum + 1% (v / v) insulin–transferrin–selenium (ITS+, Corning) + 10 μg mL -1It was incubated in a differentiation medium composed of gentamicin. The samples were cultured in 35 mm Petri dishes (PS 60 / 15MM, Greiner Bio-One) with frequent medium changes. Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from Lonza and cultured in Endothelial Cell Growth Medium-2 BulletKit (EGM-2, Lonza). For the HUVEC-NHDF co-culture system in the hydrogel construct, HUVEC and NHDF were mixed at a ratio of 1:2 in the photoresin to prepare the biophotoresin. The prepared cell-loaded hydrogel samples were transferred to 6-well plates and cultured in a mixed medium (DMEM:EGM-2 at 1:1).

[0197] Preparation of Biophotoresin The photoresin was prepared as described above. The photoresin was sterilized using a 0.2 μm filter (Filtropur S 0.2, SARSTEDT AG) to remove potentially scattering particles. NHDF and HT were resuspended in the photoresin at a concentration of 1 million cells / mL -1 . C2C12 was mixed with the photoresin at a concentration of 2 million cells / mL -1 . For the co-culture of NHDF-HUVEC, the two types of cells were mixed in the photoresin at a concentration of 1 million cells / mL -1 at a ratio of 2:1.

[0198] Design of Projection Images The projection images were created using Affinity Photo (AffinitySuite 1.9, Serif Europe Ltd.) at a fixed resolution of 1024×768 pixels. The images were in grayscale, and the pure white color corresponded to a light intensity of 100% (about 62.5 mW / cm 2 ). The width of each pixel in the projection image was approximately 27 μm. The pattern was corrected by 90° during projection by rotating the vial and cuvette by 90°. The projection images were then exported as PNG files.

[0199] Assembly of the Standard Keller Lighting System All components for assembling a standard Keller illumination system, including the light source (M405L4), were obtained from Thorlabs unless otherwise clearly stated. The assembly and alignment protocols are described in a previous study (J. Madrid-Wolff et al., Biophys. 2020, 1, 10.). The light intensity in front of the cuvette was measured using a power meter each time the field diameter and / or aperture diameter was adjusted.

[0200] Light projection (Bio)photoresins were prepared as described above and transferred to sterile cuvettes or glass vials. Gel-NB / 4PEG-SH and Gel-MA (bio)photoresins were thermally gelled at 4 °C for 15 minutes. For highly viscous photoresins or non-thermally reversible photoresins (i.e., HA-MA, Alg-MA), the above steps were omitted. Projection was performed using the built-in function "Advanced" of a commercially available volumetric printer (Tomolite Ver. 1.0, Readily3D SA). The projection time was calculated based on the light intensity and light exposure required for crosslinking each photoresin. The projection image was loaded into the software (Apparite, Readily3D SA) before starting the projection. Uncrosslinked (bio)photoresin was rinsed away using pre-warmed PBS at 37 °C. The projected constructs were removed using a sterile spatula and transferred to PBS or medium.

[0201] Measurement of compressive elastic modulus All samples were prepared as cylindrical models with a diameter of 5 mm and a height of 4 mm. LBP hydrogel samples were prepared using circular projection images with a diameter of 185 pixels. Bulk hydrogel samples (control group) were generated by filling PDMS molds (inner diameter 5 mm, height 4 mm) with photoresin before UV crosslinking. The samples were tested by unconfined uniaxial compression using a TA.XTplus texture analyzer (Stable Micro systems). A 500 g load cell and a flat plate probe (15 mm) were used. A preload of 0.2 g was applied to ensure complete contact of the sample with the plate. The samples were compressed at a speed of 0.01 mm s -1 to a final strain of 50%. The compressive elastic modulus was calculated by linear fitting of the initial linear region (0.5 - 5%) of the stress-strain curve. The tests were conducted at 25 °C and repeated three times.

[0202] Tensile test All samples were prepared as strands with a diameter of 1 mm. Circular matrix (8×8) images with a diameter of 37 pixels were loaded into LBP. Bulk hydrogel samples were prepared by thermally reversibly gelling photoresin and then pressing the bulk gel through a grid (1 mm, micron-sized aperture). Subsequently, the microstrands were crosslinked under a UV lamp using the same light irradiation dose as the LBP process. The tensile test was performed using a texture analyzer equipped with a 500 g load cell, a small tensile grip, and a preload of 0.1 g was applied before tension. All samples were stretched at a speed of 0.1 mm s -1 until rupture. The load-unload curve was measured at a tensile strain of 0 - 50% and a tensile speed of 0.1 mm s -1 for 12 cycles.

[0203] Cell viability The hydrogel constructs were washed three times with PBS after 0, 3, and 7 days of culture, and incubated for 45 minutes in FluoroBrite™ DMEM supplemented with 1:2000 CalceinAM (Invitrogen), 1:1000 Hoechst 33342 (Invitrogen), and 1:500 Propidium Iodide (PI, Fluka). After washing three times with the medium, fluorescence imaging was performed with a confocal laser scanning microscope (Fluoview 3000, Olympus). Z-stack scans were acquired from the surface of the construct to 200 μm in 5-μm steps into the construct. Z-projected images were analyzed with Fiji ImageJ. The above experiments were repeated three times, and the viability of each sample was averaged over three images from randomly selected regions.

[0204] Loading of CellTracker Dye CellTracker Green dye CMFDA (Invitrogen) and CellTracker Red dye CMTPX were applied to NHDFs in serum-free medium at a working concentration of 10 μM. The cells were transferred to a 37 °C incubator for 1 hour and then washed three times with PBS for subsequent experiments.

[0205] Immunofluorescence Staining Cell-laden hydrogel strands were washed three times with PBS after 7 days of culture and fixed with 4% paraformaldehyde at 25 °C for 30 minutes. The strands were permeabilized with 0.2% Triton-X100 in PBS for 30 minutes and then blocked with 1% (v / v) BSA in PBS for 1 hour. The strands were incubated with a primary anti-human collagen I antibody (ab138492, Abcam) diluted 1 / 500 in BSA-PBS at 4 °C for 12 hours. Next, the samples were washed three times with PBS and incubated with a secondary antibody (goat anti-rabbit Alexa Fluor488, Invitrogen) diluted 1 / 200, Hoechst33342, and a phalloidin-tetramethylrhodamine B isothiocyanate working solution (standard solution) prepared in advance in BSA-PBS (0.13 μg mL -1, (P1951) was incubated at 4 °C for 2 hours. The C2C12 constructs were incubated with 1 / 20 diluted primary anti-human myosin heavy chain antibody (MF-20, DSHB) and 1 / 500 diluted collagen I antibody at 4 °C for 12 hours. The samples were then incubated with 1 / 200 diluted secondary antibodies (goat anti-rabbit Alexa Fluor647, goat anti-mouse Alexa Fluor Plus488), phalloidin-tetramethylrhodamine B isothiocyanate working solution, and Hoechst at 4 °C for 2 hours. Before imaging by CLSM, the samples were washed with PBS. NHDF encapsulated in tubular strands, NHDF encapsulated in fluorescent GelMA, or NHDF seeded on the surface were stained with CalceinAM for 1 hour. The tubular constructs were perfused with 40 kDa tetramethylrhodamine isothiocyanate-dextran (TRITC-dextran) for 10 minutes and then confocal imaging was performed.

[0206] RNA Isolation and qRT-PCR NHDF was encapsulated in hydrogel samples prepared using 2.88% (w / v) Gel-NB / 4PEG-SH photoresin. The tissue samples were homogenized with a tissue grinder and incubated with NucleoZOL (MACHEREY-NAGEL) at room temperature for 10 minutes. DNase-free water was added to the samples and the mixture was centrifuged at 12000 rcf for 10 minutes. The supernatant was mixed with 70% EtOH, transferred to a column of the RNeasy mini kit (Qiagen), and total RNA was extracted. If the A260 / 280 ratio was between 1.8 and 2.1, the quality of the RNA sample was considered appropriate. The isolated RNA was transcribed into complementary DNA according to the instructions of the GoScript reverse transcriptase kit (Promega). SYBR Green PCR master mix (Promega) was used to determine the relative gene expression levels with a real-time PCR system (QuantStudio 5, Applied Biosystems). The GAPDH housekeeping gene was used as an internal control for normalizing the RNA amount.

[0207] 3D Image Reconstruction The three-dimensional image obtained using a confocal microscope was imported into Imaris 9.2.1, and the three-dimensional model was reconstructed. The measurements in this operation were performed using surface functions and statistical functions.

[0208] Statistical analysis Statistical analysis was performed using GraphPad Prism (x64, v.9.2.0) and an unpaired t-test. α was set to 0.05, and the difference between the two experimental groups was * p < 0.05, * p < 0.05, *** p < 0.01, *** judged to be statistically significant at p < 0.005; ns represents "no significant difference" between the two groups.

[0209] JPEG2025523581000002.jpg77132

Claims

1. A method for manufacturing a three-dimensional hydrogel bioimplant, the method comprising: a. Providing in a container a first composition that is prone to photocrosslinking, the first composition comprising: i. A first polymer that is prone to photocrosslinking, ii. A photoinitiator, and iii. Optionally, a refractive index matching agent (e.g., iodixanol up to 80% (w / v), particularly 10% - 50%) capable of increasing or decreasing the refractive index of the photoresist formulation, iv. Optionally, a light-absorbing dye (e.g., sunset yellow or FCF yellow at a concentration of up to 500 μg / mL), v. Optionally, a crosslinking agent and b. Irradiating the composition with a plurality of spatially coherent light rays in a first irradiation step, thereby generating a plurality of microcolumns in the composition The method comprising the above.

2. The method according to claim 1, wherein the spatially coherent light rays have a diameter of 1 - 100 μm, particularly 3 μm - 40 μm.

3. a. The composition is irradiated by a first plurality of spatially coherent light rays aligned in a first direction, and b. The composition is irradiated by a second plurality of spatially coherent light rays aligned in a second direction in a second irradiation step, The method according to claim 1 or 2.

4. The method according to claim 3, wherein the first direction and the second direction are arranged at an angle of 1° - 180°, particularly 15° - 180°.

5. - The first plurality of spatially coherent light rays form a first pattern; - The second plurality of spatially coherent light rays form a second pattern; and - The first and / or the second pattern is arranged as a cylindrical shape, a sheet shape, or parallel strands between an anchor-like tendon support sheet, The method according to claim 3 or 4.

6. After the first irradiation step, a second composition is added to the container, the second composition comprising: i. A second polymer that is prone to photocrosslinking, ii. A photoinitiator, and iii. Optionally, a refractive index matching agent (e.g., iodixanol up to 80% (w / v)) capable of increasing or decreasing the refractive index of the photoresist formulation; iv. Optionally, a light-absorbing dye (e.g., sunset yellow or FCF yellow at a concentration of up to 500 μg / mL), v. Optionally, a crosslinking agent and In the third irradiation step or any subsequent irradiation step, irradiate the composition with a plurality of spatially coherent light beams. The method according to any one of claims 1 to 5. **Claim 7** In the first, second, third or any subsequent irradiation step, irradiation is carried out on the substrate from the upper side or the bottom side of the container; continuous irradiation is carried out from the upper part of the container, or the substrate is moved away from the bottom of the container and upward under continuous irradiation, thereby forming a plurality of microbeams protruding from the substrate, where the microbeams extend widely over the length of the longitudinal axis of the hydrogel construct. The method according to any one of claims 1 to 6. **Claim 8** The polymer prone to photocrosslinking is a biopolymer or a methacrylate-functionalized biopolymer functionalized by a covalent bond of a carbon-carbon double bond (ene) - containing moiety. The method according to any one of claims 1 to 7. **Claim 9** The biopolymer is selected from the group consisting of gelatin, hyaluronic acid, alginate, collagen, fibrinogen, polyvinyl alcohol, chitosan, silk fibroin, and cellulose. The method according to claim 8. **Claim 10** The carbon-carbon double bond (ene) - containing moiety is selected from the group consisting of norbornene carboxylic acid or dicarboxylic acid, methacrylic acid ester or methacrylamide, acrylic acid ester or acrylamide, and vinyl ester. The method according to claim 8 or 9. **Claim 11** The polymer prone to photocrosslinking is norbornene-functionalized gelatin characterized by a degree of substitution of 10% to 90%, particularly 47% to 50%. The method according to any one of claims 8 to 10. **Claim 12** The polymer prone to photocrosslinking is methacrylate-functionalized gelatin characterized by a degree of substitution of 10% to 90%, particularly 45 to 60%. The method according to any one of claims 8 to 10. **Claim 13** The spatially coherent light beam is characterized by a wavelength of 360 to 800 nm. The method according to any one of claims 1 to 12. **Claim 14** The spatially coherent light beam is characterized by a coherence length of 1 to 100 μm. The method according to any one of claims 1 to 13. **Claim 15** The spatially coherent light beam has a light irradiation dose of 10 to 5000 mJ / cm ２ , particularly 90 to 200 mJ / cm ２ , and is a method according to any one of claims 1 to 14, characterized by such a light irradiation dose. **Claim 16** The spatially coherent light beam is characterized by a duration of 0.1 second to 100 seconds, particularly 1 second to 4 seconds. The method according to any one of claims 1 to 15. **Claim 17** A spatially coherent light beam has an energy density of 1 to 500 mW / cm ２ , in particular 50 to 60 mW / cm ２ , and the method according to any one of claims 1 to 16, characterized thereby. **Claim 18** A three-dimensional hydrogel implant comprising, or consisting essentially of, a first plurality of microcolumns of a first photo-crosslinked polymer, said three-dimensional hydrogel implant obtainable by the method according to any one of claims 1 to 17.

19. The three-dimensional hydrogel bioimplant according to claim 18, wherein each microbeam of the first plurality of microcolumns has a diameter in the range of 1 μm to 100 μm.

20. The three-dimensional hydrogel bioimplant according to claim 18 or 19, wherein each microbeam of the first plurality of microcolumns has a diameter in the range of 2 μm to 30 μm.

21. The three-dimensional hydrogel bioimplant according to any one of claims 18 to 20, wherein more than 75% (in particular more than 80%, 85% or more, 90% or more, 95% or more, or even 98% or more) of the plurality of microbeams have an alignment deviation of 2° or less from the longitudinal axis.

22. The three-dimensional hydrogel bioimplant according to any one of claims 18 to 21, wherein more than 75% (in particular more than 80%, 85% or more, 90% or more, 95% or more, or even 98% or more) of the plurality of microbeams have a length of 2 cm or more, in particular 5 cm or more.

23. The three-dimensional hydrogel bioimplant according to any one of claims 18 to 21, wherein the microbeams are widely distributed over the length of the longitudinal axis of the hydrogel construct.

24. The three-dimensional hydrogel bioimplant according to any one of claims 18 to 23, wherein the plurality of microbeams form a channel structure having a diameter of 100 μm to 10 mm, in particular 400 μm to 600 μm.

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