Beam projection guided biofabrication of oriented tissue

Abstract

One aspect of the invention relates to a method for preparing a three-dimensional hydrogel bioimplant characterized by a geometry in the sub-millimeter size range. The method includes providing a photo-crosslinking sensitive first composition in a container. The first composition includes a photocrosslinking-sensitive first polymer, a photoinitiator, and optionally an 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 beams, thereby creating a plurality of microcolumns in the composition. Another aspect of the invention relates to a three-dimensional hydrogel implant comprising or consisting essentially of a plurality of microbeams of photo-crosslinked polymers, said three-dimensional hydrogel implant being obtained by the method according to the invention.

Description

[0001] This application claims the benefit of priority of European application 22181705.9 filed on June 28, 2022, which is incorporated herein by reference. Technical Field

[0002] The present invention relates to a method for preparing a three-dimensional hydrogel bioimplant by irradiating a first composition with a plurality of spatially coherent light beams, the first composition comprising a polymer susceptible to photocrosslinking, a photoinitiator and optionally, a refractive index matching agent, a light absorbing dye, a crosslinking agent, thereby generating a plurality of micropillars in the composition. The present invention also relates to a bioimplant produced by the method according to the present invention. Background Art

[0003] Light-based projection techniques are increasingly used for the fabrication of biomimetic tissues. Recently, rapid biofabrication of complex cellular structures has become possible through tomographic projection of laser beams. However, most photoconductive tissue fabrication strategies have limited potential for effective cell orientation when it comes to the creation of anisotropic tissues such as muscle and tendon, because most methods focus on macroscopic features (>100 μm), which lack the topographical cues necessary for the highly oriented cells and extracellular organizations found in these tissues. For techniques such as two-photon polymerization, which can achieve cellular scale (<30 μm) resolution, the trade-off between speed and scalability limits the translational potential of these methods. Instructive guidance cues have been extensively studied to promote the fabrication and maturation of anisotropic tissues such as muscle, tendon, and nerve. Topological cues with increased aspect ratios have been shown to affect the bioactivity of cells in / on the matrix. For example, rod-shaped microgels (aspect ratio of 10) fabricated by microfluidics or soft lithography have the ability to increase cell orientation, which is better achieved by the gaps between high-aspect-ratio microrods compared to microspheres. Topological features with ultrahigh aspect ratios (>20:1) generated by micropatterning techniques can effectively induce cell adhesion and orientation. In particular, when the size of the constraints approaches the scale of the cell nucleus (<10 μm), nuclear deformation caused by these longitudinal constraints becomes obvious. The elongated shape of the nucleus can affect cell differentiation, gene expression, and regeneration, the latter through chromosome reorganization and activation of DNA repair mechanisms, and the change to a more rounded nuclear shape can be associated with disease pathology. At the tissue level, more elongated nuclei can be observed in anisotropic tissues such as tendons (aspect ratios of 2.5–6) compared with isotropic tissues (aspect ratios of 1.1–1.8). The high aspect ratio of the nucleus in tenocytes helps to maintain the tenocyte phenotype and expression of key genes during aging. However, none of the above techniques provide topological cues for cell orientation and nuclear deformation in a 3D environment.

[0004] In this work, the inventors show that the phenomenon of optical modulation instability (OMI) of a light beam within a photosensitive resin can be advantageously used to fabricate highly directional microbeams (Φ<30 μm) within macroscopically sized (L ≥ 10 mm, Φ ≥ 3 mm) hydrogel constructs, which can then be further used to fabricate anisotropic tissues. OMI is defined by the spontaneous splitting of a uniform light beam into randomly distributed smaller beams of the same diameter when propagating in an optically nonlinear medium. Photoreactive polymers are well-known optically nonlinear media because their photopolymerization rate and therefore their refractive index (RI) change depends on the intensity of the cross-linking light. Highly directional bundled microstructures (microbeams) are generated by irradiating a partially spatially coherent light beam into a vial containing the resin. Upon the incident light beam on the hydrogel interface, individual microbeams are seeded by the local intensity maxima of the incident light beam. Each local maximum light intensity provides a faster cross-linking rate and therefore produces a local maximum RI. In turn, the local RI maxima act as optical traps, causing the entire micropattern polymerization front to propagate through the resin volume through these self-focusing waveguides, resulting in a permanent record of the photofilamentation. At the same time, local minima of light intensity are distributed between local beams, corresponding to the formation of void spaces where the local intensity is below the threshold required for polymerization. After removing the uncrosslinked photosensitive resin, these voids between the hydrogel microbeams appear as channel-like void spaces (microchannels) with ultrahigh aspect ratios.

[0005] Here, the inventors propose a biofabrication strategy for anisotropic tissues, termed beam projection (LBP), in which highly directional microbeams are subjected to OMI induction through the interaction of spatially coherent beams with different photoresin systems. Cells are rapidly (<10s) and securely encapsulated in a hydrogel matrix containing highly directional microstructures. This approach meets the requirements of engineering biomimetic anisotropic tissues, including instructive guidance cues with microscale resolution and rapid and cell-friendly processing. During the LBP process, the size of the microbeams and the interstitial spaces between the microbeams are adjustable and at the same length scale as the cells, providing effective cell-guiding properties and supporting cell migration through the interstitial spaces between microbeams.

[0006] The inventors' strategy provides control over the fabricated hydrogel matrix at both the micro- and macro-levels; i.e., control over the size of individual microbeams (2–30 μm) and the size and shape of the projected tissue structures (100 μm–1 cm). These advantages provide flexibility in the biofabrication of cell-laden hydrogel structures, such as multi-hollow / tubular hydrogel constructs, which have been shown to improve cell viability prior to vascularization. Finally, the inventors show that LBP can be effectively used for multi-cellular / multi-material biofabrication. Using multi-step projection, (bio)photosensitive resins can be cured at desired locations to produce complex tissue constructs that better mimic the layered organization of native tissues such as muscle.

[0007] Constantini et al. (Biomaterials 2017, 131, 98–110) disclosed the production of artificial skeletal muscle tissue by 3D bioprinting hydrogel fibers in which muscle precursor cells are embedded.

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

[0009] Based on the above-mentioned prior art, the object of the present invention is to provide means and methods for producing improved bioimplants with internal structural elements having sub-millimeter structural features. This object is achieved by the subject matter of the independent claims of this specification and by further advantageous embodiments described in the dependent claims, examples, drawings and general description of this specification. Summary of the invention

[0010] One aspect of the present invention relates to a method for preparing a three-dimensional hydrogel bioimplant characterized by a geometric structure in the sub-millimeter size range. The method includes providing a first photo-crosslinking sensitive composition in a container. The first composition includes a first photo-crosslinking sensitive polymer, 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 beams, thereby generating a plurality of micropillars in the composition.

[0011] Another aspect of the present invention relates to a three-dimensional hydrogel implant comprising or essentially consisting of a plurality of photo-crosslinked polymer micro-bundles, said three-dimensional hydrogel implant being obtained by the method according to the present invention.

[0012] Terms and Definitions

[0013] For purposes of interpreting this specification, the following definitions will apply and, whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event of a conflict between any definition set forth below and any document incorporated herein by reference, the set forth definition will control.

[0014] As used herein, the terms "comprising," "having," "containing," and "including," and other similar forms and grammatical equivalents thereof are intended to be equivalent in meaning and open ended, in that a term or terms followed by any of these terms is not intended to be an exhaustive list of the term or terms, or to be limited to the listed term or terms. For example, a term "comprising" components A, B, and C may consist of (i.e., contain only) components A, B, and C, or may contain not only components A, B, and C, but may also include one or more other components. Thus, it is intended and understood that "comprising" and its similar forms and grammatical equivalents thereof encompass disclosure of embodiments that "consist essentially of" or "consist of."

[0015] Where a range of values ​​is provided, it is understood that each intervening value to the tenth of the unit of the lower limit, and any other stated or intervening value in that range, is encompassed within the invention, subject to any specifically excluded limits in that range, unless the context clearly dictates otherwise. Where the range includes one or two limits, ranges excluding either or both of those included limits are also encompassed within the disclosure.

[0016] Reference herein to "about" a value or parameter includes (and describes) variations involving the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0017] As used herein, including in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al., Short Protocols in Molecular Biology (2002) 5 th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0019] The term spatially coherent light beam in the context of this specification relates to multiple light beams generated by a light source emitting a defined wavelength (e.g., 405 nm) that can trigger photocrosslinking. The light beams are spatially coherent by being generated by a suitable optical system (e.g., Koehler standard illumination system) or a spatially coherent laser. Spatial coherence is an essential prerequisite for the strong directionality of a laser beam. Lasers have the potential to generate light beams with very high spatial coherence. The light beam has a fixed phase relationship between the electric fields at different spatial positions on the beam profile.

[0020] In the context of this specification, the term thiol-ene photocrosslinking relates to a light-triggered step-growth reaction between an olefin (-ene) moiety and a thiol group to form a thioether bond. This reaction requires a photoinitiator that generates a free radical initiator species upon light absorption. The radical so formed abstracts a hydrogen from the thiol, generating a sulfanyl radical, which then attacks the double bond of the olefin group, generating a carbon-centered radical. The latter abstracts a hydrogen from another thiol, leaving a thioester bond (crosslink) formed.

[0021] In the context of the present specification, the term "microcolumn", also referred to herein as "microwire" or "microbundle", relates to a polymeric structure produced by the method of the present invention, which consists of a microbeam-induced photopolymerization product of a polymer sensitive to photocrosslinking as specified herein. Typical dimensions of a microcolumn are (width): 1-100 μm. Length: 100 μm to 15 cm (i.e., the microwire is distributed over the entire length of the structure). The term "distributed" means that the microcolumn or microwire essentially runs through the entire length of the structure. If not otherwise stated, the microstructure dimensions given herein are determined by bright field microscopy and image analysis with ImageJ (public domain, author: Wayne Rasband). DETAILED DESCRIPTION

[0022] A first aspect of the present invention relates to a method for preparing a three-dimensional hydrogel bioimplant. The implant is characterized by a geometric structure in the sub-millimeter size range, which cannot be obtained by conventional 3D printing schemes. Such sub-millimeter structures are important as clues for tissue growth and organization. The method according to this first aspect includes providing a first photo-crosslinkable sensitive composition in a container having walls suitable for irradiating the first composition by a plurality of light beams having a defined wavelength and coherence length.

[0023] The first composition includes a first polymer that is sensitive to photocrosslinking and a photoinitiator. Optionally, a crosslinking agent may be present, and if crosslinking of the polymer is desired, a refractive index matching agent and a light absorbing dye may be applied to improve the quality of the projection / multi-step projection when using a polymer solution with a higher unit number (typically, >10 million units / mL).

[0024] In a first irradiation step, the composition is irradiated with a plurality of spatially coherent light beams, thereby generating a plurality of microcolumns in the composition.

[0025] polymer

[0026] In principle, any polymer that is susceptible to photopolymerization may be used. In a particular embodiment, the composition comprises the polymer in aqueous solution. As the method of the invention is particularly suitable for preparing cell-containing or cell-supporting structures, the method of choice is aqueous polymerization.

[0027] In certain embodiments, the photocrosslinkable polymer is a biopolymer functionalized by covalent attachment of a carbon-carbon double bond (ene) containing moiety that can be crosslinked by a thiol-bearing crosslinker; or a methacrylate functionalized biopolymer that can be reproducibly prepared by reaction of the biopolymer with methacrylic anhydride.

[0028] One particular cross-linking agent that the inventors have found useful is a thiol-functionalized poly(ethylene glycol), a specific example of which is pentaerythritol-PEG-thiol [C(CH 2 O(CH 2 CH 2 O) n CH 2 CH 2 SH) 4 Such linkers are available from Merck-Sigma and include tetrakis(3-mercaptopropionic acid)pentaerythritol (Sigma 381462), tris(3-mercaptopropionic acid)trimethylolpropane ester (Sigma 381489); tris(3-mercaptopropionic acid)2-hydroxymethyl-2-methyl-1,3-propanediol (Aldrich S51145).

[0029] In the case of norbornene or acrylate modified polymers, a crosslinker that provides thiol groups is required. Alternatively, two polymers can be used, one with thiol and the other with vinyl groups. Polymers with both carbon-carbon double bond "ene" functional groups and SH groups can also be used.

[0030] In certain embodiments, the biopolymer is selected from the group consisting of gelatin, hyaluronic acid, 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 similarly consider 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 at least norbornene carboxylic acids or dicarboxylic acids, methacrylates or amides, acrylates or amides, and vinyl esters.

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

[0033] The present invention can be practiced with a number of norbornene or methacrylic acid derivatives that can be used to graft such reactive groups onto polymers (ie, alkynes, azides, hydrazides, DBCO, etc., as well as end-capping linkers).

[0034] In certain embodiments, the thiol-ene photocrosslinking-sensitive polymer is selected from the group consisting of at least norbornene functionalized gelatin, norbornene functionalized collagen, norbornene functionalized chitosan, norbornene functionalized fibrinogen, norbornene functionalized polyvinyl alcohol, norbornene functionalized hyaluronic acid, methacryloyl gelatin (Gel-MA), methacryloyl hyaluronic acid (HA-MA), and methacryloyl alginate (Alg-MA).

[0035] In certain specific embodiments, the thiol-ene photocrosslinkable polymer is norbornene functionalized gelatin; see et al., Advanced Healthcare Materials 19June 2021, ( https: / / doi.org / 10.1002 / adhm.202100206 ).

[0036] Similarly, thiolated derivatives of polymers in combination with alkene-bearing crosslinkers, or combinations of thiols and alkene-bearing polymers, such as norbornene-functionalized hyaluronic acid and thiolated alginate, or norbornene-functionalized gelatin and thiolated hyaluronic acid, may be used.

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

[0038] The degree of substitution (DS) of NB modified gelatin can be defined as the number of millimoles of norbornene moieties per gram of gelatin. Alternatively, it can be defined as the percentage of lysine groups bearing norbornene functionalities. In both cases, the determination is made by using an internal standard (DSS). 1 H-NMR was performed.

[0039] As defined in the Materials and Methods section of Rizzo et al. (supra): “The degree of substitution (DS) of Gel-NB is determined by 1H-NMR (Bruker Ultrashield 400 MHz, 1024 scans). 2 0.5 mg mL in O -1 3-(Trimethylsilyl)-1-propanesulfonic acid (DSS) (Apollo Scientific) was added at 40 mg mL -1 DSS was used as an internal standard and the number of millimoles of NB per gram of gelatin was calculated by comparing the integral of the nine methyl protons of DSS (≈0.5 to -0.5 ppm) with the integral of the two olefinic protons of NB (≈6.21–6.00 ppm) (n=3). DS is based on et al. (Biomacromolecules 2018, 19, 42–52) estimated the lysine + hydroxylysine content of porcine skin type A gelatin (0.325 mmol g -1 ) and given as a percentage. ”

[0040] There are no structural restrictions for both thiols and olefins, which can react with different kinetics depending on their structure (i.e., norbornene reacts faster than acrylates). This reaction is not limited by solvent and can be performed in aqueous solution or in organic solvents (i.e., DMSO, DMF). The pH of the solution has some influence because at alkaline pH (>7), thiols react with several olefinic groups in the absence of light via nucleophilic addition reactions ("Michael additions"). In certain embodiments, a norbornene derivative is used as the olefinic functional group; the advantage of using norbornene is that it does not undergo this reaction, thus avoiding unintentional, non-photoreaction-triggered crosslinking of the mixed thiol and olefinic components.

[0041] Therefore, for most ene functionalities (i.e. vinyl ethers, vinyl sulfones, methacrylates, acrylates), it is recommended to use a pH < 7 to avoid unwanted crosslinking (meaning that the thiol-ene reaction occurs without photoinitiation). It is also important to note here that the use of other ene moieties (such as methacrylates) will result in networks that are partially formed by kinetic chains (chain growth polymerization, such as for the use of simple gel MA), thus losing some of the advantages of pure photoclick thiol-ene chemistry.

[0042] Another way to classify polymers useful in practicing the present invention is as follows:

[0043] Resins based on step-growth photopolymerization:

[0044] 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 cross-linking thiolation component is selected from collagen, gelatin, hyaluronic acid, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or dithiothreitol (DTT).

[0045] Resins based on chain-growth photopolymerization:

[0046] Methacrylate or acrylate functionalized collagen, gelatin, hyaluronic acid, polyethylene glycol (PEG), or polyvinyl alcohol (PVA) or alginate.

[0047] No modification of the resin is required:

[0048] Collagen, fibrinogen, decellularized matrix, gelatin (the details of the photoinitiator are mentioned below) used with the Ru-SPS photoinitiator system. The Ru-SPS system crosslinks the free tyrosine groups present in these matrices, so the matrix or resin does not necessarily need to be modified. This Ru-SPS photoinitiator system can also crosslink acryloyl and norbornene groups, so it can also be used with the materials given in the previous paragraph.

[0049] Exemplary concentrations of polymers

[0050] Non-limiting concentration values ​​that have worked well for the inventors include: collagen: 1-30 mg / mL; fibrinogen: 5-100 mg / mL; acellular 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.

[0051] Photoinitiator

[0052] Any photoinitiator In certain embodiments, the photoinitiator is selected from the group consisting of at least lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), oxidized 2,4,6-trimethylbenzoyldiphenylphosphine (TPO), oxidized bisacylphosphine (BAPO), 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), camphorquinone (CAS-No: 10373-78-1), ethyl dimethylaminobenzoate (EDAB), and diphenyliodonium hexafluorophosphate (DPIHFP).

[0053] Water-soluble photoinitiators active in the visible spectrum include, but are not limited to, eosin Y, riboflavin, ruthenium(II) chloride hexahydrate [Ru(II)(bpy) 3 ]Cl 2 , sodium ruthenium persulfate (Ru-SPS).

[0054] In certain specific embodiments, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP).

[0055] The photoinitiator concentration can vary based on the photoinitiator type. A list of initiator concentrations that the inventors have used that work well include: LAP (0.1-2 mg / ml), Ru (0.1-10 mM) + SPS (0.1-10 mM), Irgacure 2959 (0.2-2 mg / ml).

[0056] Refractive Index (RI) Matching Agents and Light Absorbing Dyes

[0057] Any RI matching agent In certain embodiments, 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 specific embodiments, the RI matching agent is iodixanol.

[0058] In some embodiments, the light absorbing dye is selected from Brilliant Blue FCF (CAS-No: 3844-45-9), Indigo (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), and Sunset Yellow FCF (CAS-No: 2783-94-0).

[0059] In certain specific embodiments, the light absorbing dye is Sunset Yellow FCF.

[0060] The amount of refractive index matching that helps improve the results of the method depends on the refractive index of the reaction components. In one application, the refractive index can be matched to microbeads or cells that can be embedded in a matrix for printing.

[0061] After the method has produced the selected implant, the refractive index (RI) matching agent is slowly removed from the matrix by diffusion.

[0062] The amount of RI matching agent is selected based on the RI of the additives in the resin, which contain cells or other particles, such as gelatin microbeads or silver micro- and nanoparticles or conductive matrices. For example, for cells, the RI is about 1.375, so about 30% w / v iodixanol (RI = 1.384) is needed in the GelNB / GelSH resin (original RI = 1.34) to increase its RI to 1.375. Such RI matching is necessary if printing at high cell densities is desired, as RI matching reduces light scattering from the cells, as the cells are essentially "transparent" to incident light.

[0063] Optical properties

[0064] The inventors used wavelengths of 405 nm, 450 nm, 473 nm, 488 nm, 520 nm, 638 nm, and 750 nm. Photoinitiators useful in practicing the present invention are discussed in detail in Lee et al., Chem. Rev. 2020, 120, 19, 10950–1102.

[0065] Other ranges that the inventors have found to work well include 360-375 nm, 400-420 nm, 450-475 nm, and 550-575 nm.

[0066] Spatially coherent light beams are characterized by diameters between 1 and 100 μm.

[0067] In certain embodiments, the spatially coherent light beam is characterized by a diameter of 3 to 40 μm. In certain more specific embodiments, the spatially coherent light beam is characterized by a diameter of 5 to 15 μm.

[0068] In certain even more specific embodiments, the spatially coherent light beam is characterized by a diameter of 7.5 μm to 12.5 μm.

[0069] Typically, the diameter of the microcolumns is between 1 μm and 100 μm. In certain embodiments, the diameter of the microcolumns is between 1 μm and 50 μm. In certain embodiments, the diameter of the microcolumns is between 2 μm and 50 μm. In certain embodiments, the diameter of the microcolumns is between 3 μm and 40 μm. In certain embodiments, the diameter of the microcolumns is between 7.5 μm and 12.5 μm.

[0070] In certain embodiments, the average diameter of the micropillars is between 1 μm and 100 μm.

[0071] In certain embodiments, the average diameter of the micropillars is between 7.5 μm and 12.5 μm. In certain specific diameters, ≥75%, ≥80%, ≥85%, ≥90%, or even ≥90% of the micropillars are characterized by a diameter of 7.5 μm to 12.5 μm.

[0072] The optical setup affects the performance of the system. The inventors have used a spatially coherent beam / coherence length range of 3.7 to 17.2 μm. In certain embodiments, this coherence length varies from 1 to 100 μm.

[0073] In certain embodiments, the composition is illuminated by a plurality of spatially coherent light beams comprising more than 100 light beams.

[0074] The macroscopic size of the beam can be varied by changing the projected image using a digital micromirror device or a spatial light modulator.

[0075] In some embodiments, the cross-sectional size of the beam may be 2×2 μm. 2 Between (single pixel projection), about 2×2 cm immediately after light shaping due to DMD 2 , and can even reach 10×10cm 2 (When the light beam is expanded due to the telephoto lens).

[0076] Speckle pattern generation and modulation

[0077] The speckle pattern is a characteristic property of laser light sources. The term "speckle pattern" refers to the granular pattern observed when laser light interacts with an optical medium, causing the light waves to constructively or destructively interfere with each other, producing bright and dark areas. These bright and dark spots form a random pattern, the speckle pattern.

[0078] The exact speckle pattern observed depends on various factors, including the coherence length of the laser, the homogenization of the laser, 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 will also change.

[0079] In some embodiments, a fly's eye homogenizing lens (a lens that converts a circular beam with a Gaussian distribution into a uniformly illuminated rectangular area) can be used to homogenize the laser to remove the Gaussian distribution of light intensity present in the laser. This is important for achieving substantially uniform light intensity across the entire projected light after shaping. Homogenization does not eliminate individual speckle patterns, but only homogenizes the light intensity. Importantly, this is an optional step, as the FLight projection device can still work without homogenization, but homogenization can result in better print resolution and more uniform cross-linked structures because the light intensity is uniform across the entire projected image.

[0080] When the spot pattern changes, the diameter of the microfilaments / micropillars and the associated microchannels / micropores also changes. This may present different effects on the cells (e.g., genetic changes due to nuclear confinement of the cells between microfilaments / micropillars) and also cause changes in nutrient diffusion properties.

[0081] Digital Micromirror Device / Spatial Light Modulator

[0082] A digital micromirror device (DMD) is an optical semiconductor chip consisting of an array of micromirrors that can be individually controlled to reflect or redirect light. It is widely used in display technology, projection systems, and optical communications. A spatial light modulator (SLM) is a device that changes the phase, intensity, or polarization of light, enabling complex light shaping for applications like holography, optical trapping, adaptive optics, and beam steering.

[0083] In summary, the utility of applying DMD or SLM is to shape the incident light into the final projected light entering the photoresponsive resin. They are both important for obtaining the final desired light projection shape.

[0084] The DMD or SLM may be of commercial origin. In certain cases, the DMD is from Texas Instruments and the SLM is from Thorlabs. A mesh may be introduced into the beam, resulting in microscopic features with sizes between 30 and 150 μm.

[0085] Based on the ratio of microbeams to voids, the material shown in the examples is characterized by about 50% of the space being filled with beam material, so that one cubic centimeter (cm 3 ) contains about 6370 cylindrical microbeams. For theoretical calculations, the microbeams are considered to be cylindrical.

[0086] In certain embodiments, the composition is illuminated by a first plurality of spatially coherent light beams directed along a first direction.

[0087] In certain specific embodiments, the first plurality of spatially coherent light beams form a first pattern.

[0088] Build other structures sequentially

[0089] In certain embodiments, in a subsequent or parallel second irradiation step, the composition is irradiated with a second plurality of spatially coherent light beams directed along a second direction.

[0090] In some embodiments, the second plurality of spatially coherent light beams forms a second pattern.

[0091] In certain embodiments, the first direction and the second direction are parallel.

[0092] According to some embodiments, the first direction and the second direction may be arranged at an angle of 1° to 180°. In certain specific embodiments, the angle ranges from 15° to 180°.

[0093] In certain embodiments, the first and / or second patterns are arranged in cylindrical form, i.e., in bundles, in filled solid cylinders or in hollow cylinders, in sheets, or as parallel lines between anchoring tendon-like support sheets with a geometry similar to that of muscle fiber arrangement (see Fig.19 ).

[0094] The projection can also be varied along different layers of the construction to produce more complex geometries including microbeams.

[0095] In principle, any arbitrary cross-section is possible.

[0096] In certain embodiments, after the first and optional second irradiation steps, a second composition is added to the container, the second composition comprising

[0097] - a second polymer that is sensitive to photocrosslinking, in particular thiol-ene mediated photocrosslinking,

[0098] - Photoinitiators and

[0099] - Optionally, the refractive index matching agent of the photosensitive resin formulation can be increased or decreased (e.g. up to 80% w / v iodixanol)

[0100] -Optionally, a light absorbing dye (e.g., Sunset Yellow or FCF at a concentration of up to 500 μg / mL)

[0101] - Optionally, a cross-linking agent.

[0102] In a third irradiation step, a third plurality of spatially coherent light beams irradiates the composition. This allows the construction of a second structure complementary to the first structure by a different or the same biopolymer.

[0103] This can be repeated again to construct another useful structure. The number of steps is not limited in principle, and the method can be carried out to an infinite number of LBP steps. Any specific projection process can be repeated indefinitely to produce a hydrogel structure with infinite length on the projection axis. Alternatively, by increasing the number of projection steps / reducing the projection dose of each step, the entire projection process can be divided into sub-steps to achieve better projection quality / resolution.

[0104] In certain embodiments, any first composition that is not photo-crosslinked is drained from the container and the container is filled with the second composition.

[0105] Rather than draining the contents from the container, the first composition can simply remain while new material is added.

[0106] In certain embodiments, in the first, second, third or any subsequent irradiation step, irradiation with multiple spatially coherent beams is achieved from the bottom side of the container onto the matrix, resulting in the formation of multiple polymer microbeams; under continuous irradiation, the matrix moves upward away from the bottom of the container, thereby forming multiple microbeams protruding from the matrix. This allows the construction of very long structures and is therefore convenient for the production of tendon support implants, nerve guides and other structures that exceed the size limited by the length of the bundle. Tendon-like support structures are particularly described in Nourissat et al., Nature Reviews Rheumatology 11, 223–233 (2015).

[0107] In certain embodiments, the spatially coherent light beam is characterized by one or more of the following parameters:

[0108] - Wavelengths from 360 to 800nm,

[0109] - a coherence length of 1 to 100 μm (in particular 3 to 20 μm);

[0110] -10 to 5000 mJ / cm 2 , especially 90 to 200 mJ / cm 2 The light dose,

[0111] - duration from 0.1s to 100s, in particular from 1s to 4s;

[0112] -1 to 1000mW / cm 2 , especially 1–500 mW / cm 2 , more particularly 50 to 60 mW / cm 2 energy density.

[0113] The coherence length values ​​given here as minimum and maximum values ​​were measured from different microbeams generated using different materials.

[0114] The light dose also depends on the length of the hydrogel structure (i.e., the depth of light penetration). For example, a depth of 6 mm would require 110 mJ / cm 2 The light dose is 195mJ / cm 2 Corresponding to a depth of 15 mm.

[0115] 50 to 60 mJ / cm 2 Corresponds to the light intensity measured before the photosensitive resin container, that is, the light intensity before entering and reaching the photosensitive resin.

[0116] 70 to 250 mJ / cm 2 The light dose is introduced for about 1 to 4 seconds of projection. (100% grayscale)

[0117] The hydrogel prepared by the method of the present invention

[0118] Another aspect of the present invention relates to a three-dimensional hydrogel implant comprising, or essentially consisting of, a first plurality of microbundles of a first photocrosslinked polymer, also referred to herein as microfilaments, obtained by a method according to the present invention as described in any embodiment of the present specification.

[0119] In certain embodiments, a three-dimensional hydrogel bioimplant according to the present invention comprises a second plurality of microbeams of a first photocrosslinked polymer.

[0120] In certain embodiments, the three-dimensional hydrogel bioimplant includes a third plurality of microbeams of a second photocrosslinked polymer.

[0121] In certain embodiments, the first, second and / or third plurality of microbeams is characterized in that each microbeam of the first, second and / or third plurality of microbeams has a diameter ranging from 1 μm to 50 μm. In a specific embodiment, the diameter ranges from 2 to 30 μm. These dimensions allow the microstructures to act as excellent cell guidance cues, resulting in directional cell morphology and extracellular matrix deposition, which is necessary for generating biomimetic anisotropic tissues.

[0122] In certain embodiments, the first, second and / or third plurality of microbeams is characterized in that >75% (particularly >80%, ≥85%, ≥90%, ≥95% or even ≥98%) of the microbeams of the first, second and / or third plurality of microbeams have an orientation that deviates by ≤2° relative to a longitudinal axis common to each of the first, second or third plurality of microbeams (one axis for each).

[0123] In certain embodiments, the first, second and / or third plurality of microbeams is characterized in that >75% (particularly >80%, ≥85%, ≥90%, ≥95% or even ≥98%) of the microbeams in the first, second and / or third plurality of microbeams have a length of 100 μm to 2 cm, particularly a length of 100 μm to 5 cm.

[0124] In some cases, microbeams may fuse with other microbeams that may affect their discrimination capabilities. In certain embodiments, for a 2 cm configuration, the length of a single microbeam may be anywhere between 100 μm and 2 cm, constituting a coherent plurality of microbeams.

[0125] The target length of the construct depends on the tissue structure of the intended application. Anatomically, the longest muscle tissue in the body is greater than 5 cm in length. There is no upper limit to the length of the microbundles produced by the method of the present invention, which has the potential to produce constructs up to the largest major muscle group in the leg, exceeding 27 cm in length.

[0126] 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, in particular a diameter of 400 μm to 600 μm. Of course, larger structures are also possible; the upper limit of the diameter depends only on the size of the photosensitive resin container. The channel structure size can be determined by confocal imaging of fluorescently labeled materials (which can be labeled with rhodamine or FITC) and measuring the microwire / microchannel diameter by manually measuring the diameter of each microwire in the ImageJ length measurement tool.

[0127] In certain embodiments, hydrogel implants include cells. Cells suitable for implantation into the present invention include stem cells (adipose-derived, mesenchymal, induced pluripotent, embryonic) and differentiated cells (myoblasts, fibroblasts, neurons, tenocytes, macrophages, chondrocytes, osteoblasts). Cells will be encapsulated in the implant or seeded after implantation.

[0128] In certain embodiments, the three-dimensional hydrogel bioimplant according to the present invention may include a growth factor or a combination of several growth factors. In a specific embodiment, the growth factor may be selected from nerve growth factor, neurotrophic factor, vascular endothelial growth factor (VEGF), transforming growth factor (TGF-β), etc., all of which have been shown to promote tissue regeneration.

[0129] The present invention also encompasses a filamentary light (FLight) projection system that allows for concomitant and continuous resin supply and filamentary light projection, resulting in long anisotropic constructs with unitary guide filaments present over the entire length of the construct.

[0130] The system according to the present invention uses spatially coherent light from a high intensity laser or a standard Kohler illumination system in a specific FLight projection device. The standard Kohler illumination system is just one of the embodiments using LEDs as the light source. This illumination system introduces speckle in the LED light distribution. Alternatively, and advantageously, the system for generating the source light for implementing the present invention uses a laser, because lasers are inherently characterized by a speckle distribution of the light beam. This laser light can be expanded by a telescope lens and then shaped by a digital micromirror device to obtain the desired beam projection image. If necessary, this image can then be further expanded by another set of telescope lenses and finally projected onto a photosensitive resin container (see Fig.24 ).

[0131] The projected light is characterized by a static noise pattern of intensity, an intrinsic property of lasers, which is then projected onto a cuvette filled with a photosensitive resin. Where the intensity is higher, the resin first crosslinks, which results in a local increase in the refractive index. This causes the light to self-focus only along the crosslinked resin, which further causes the beam to filament into individual microfilaments along the length of the resin. Finally, the resin crosslinks along these microfilaments, producing a porous anisotropic structure. The complete process of gradual filamentation of light into several microbeams within the photoresponsive matrix is ​​called optical modulation instability (OMI). The method is compatible with step-growth and chain-growth polymerization commonly used for photocrosslinkable hydrogel systems.

[0132] To increase compatibility with processes that require fabrication of long structures, the inventors introduced a continuous feeding and FLight projection mechanism in a specific exemplary embodiment of the FLight system ( Fig.23 ; The light path is Fig.24 ). As opposed to stacking multiple projections one on top of the other, the new projection scheme uses continuous resin feeding inside the cuvette and FLight projection from the top, which allows the fabrication of long structures with microwires running through the entire length of the structure ( Fig.25 ). If necessary, by using a light absorber ( Fig.26 ) prevents overexposure of the resin due to light from the top, absorbs light penetrating into the polymer network, and also ensures high resolution of the print by quenching the light intensity in off-target areas (Figure 27). The presence of the light absorber also has no effect on the presence of microfilaments ( Fig.26 ).

[0133] In addition to continuous microfilaments in long cm-scale configurations, the present invention also provides another setup in which the cuvette is attached to a translation and rotation platform. By changing the translation and rotation of the cuvette, the FLight projection from the top or side can be controlled simultaneously ( Fig.29 and Fig.30 ), this device provides the ability to produce a wide range of microfilament orientations (spiral, cross) within the construct. In addition, the pneumatic resin supply mechanism can be used to supply multiple types of resins into the cuvette while FLight projection is ongoing, which can lead to the formation of multi-material tissue interface models (such as tendon junctions).

[0134] Variations in the aperture diameter in a standard Kohler illumination setup can change the size of the microwires, for example between 5–30 μm. Similar effects can be achieved using laser-based illumination systems, where a telescopic lens arrangement (originally used to collimate the beam) can be used to expand / contract the laser speckle pattern by changing the focal length or the distance between the lenses used ( Fig.31 ). Images projected by a digital micromirror device (DMD) or spatial light modulator ( Fig.32) can be used to further increase the porosity of the fabricated anisotropic structures, which can aid in nutrient transport and cell guidance.

[0135] A mesh or grating placed before, between, or after the collimating lens along the optical path ( Fig.33 can be used to further introduce microporosity into the structure ( Fig.33 B, C, and Fig.34 ). In addition, by using two or more gratings with different sizes and different relative orientations with respect to the optical path ( Fig.35 ), which can introduce a moiré pattern in the optical path, which also allows the generation of unique microstructures ( Fig.36 ).

[0136] In summary, the key aspects and advantages of the FLight technology proposed in this paper include but are not limited to:

[0137] - Continuous FLight projection and resin feeding allows fabrication of long constructs up to 20 cm, matching the dimensions of most anisotropic human tissues.

[0138] - Changing the distance between the telescope lenses, or by using lenses with different focal lengths, in the FLight projection device the speckle pattern can be changed, which can change the microfilament diameter. This can allow control of the cellular microenvironment of different tissues.

[0139] - Changing the distance between the telescope lenses in the FLight projection device, or by using lenses with different focal lengths, the size of the projected image can be increased or decreased, which can allow small or large-scale tissue manufacturing.

[0140] - Biocompatible light absorbers (e.g., FD&C Yellow) and free radical inhibitors (e.g., TEMPO) can be added to the photosensitive resin to control the light penetration depth within the photosensitive resin, thereby producing uniformly cross-linked constructs with continuous FLight projection.

[0141] - Switching high / low frequency of FLight projection on / off can lead to better printing resolution within FLight Biofabrication.

[0142] - The present invention provides methods to introduce microstructures within constructs, leading to better tissue maturation and regeneration effects.

[0143] - The present invention provides various systems with or without a digital micromirror device (DMD). If a DMD is not present, inserts can be added along the optical path to change the projected image and speckle pattern. For example, a metal mesh (single or multiple) along the optical path, which produces different micropatterns in the cross-linked structure. Alternatively, a DMD can be present to control the projected image, where image pixelation can produce a porous microstructure within the structure.

[0144] -Multi-directional projection is possible by switching mirrors, resulting in higher degrees of freedom in the orientation of the microwires.

[0145] The position of the printing cuvette can be changed and the cuvette rotated simultaneously to produce complex filament orientations in the construct. This can be achieved, for example, by FLight projection from the top, bottom and side, or by changing the material composition and feed rate, or by changing the light intensity to change the crosslinking rate.

[0146] Available

[0147] Instead of a top-down projection, a bottom-up projection can also be used, in which the substrate is continuously moved upwards.

[0148] Another aspect is that the present invention facilitates continuous printing based on oxygen diffusion (i.e., a concept similar to continuous liquid interface production (CLIP) printing, but the resulting constructs have the characteristics of continuous microfilaments. This is only possible using the limited photo-crosslinking strategy provided herein.

[0149] The 3D shape of the construction produced by the method according to the invention can be varied by:

[0150] a. Changing the projected image from the DMD while executing the continuous FLight projection method.

[0151] b. The projected image is altered by the interference pattern caused by the light passing through multiple screens (which act as diffraction gratings). This operation will not require a DMD.

[0152] c. Alter the projected image by blocking the light path through the template or mask or screen. This operation will not require a DMD.

[0153] The present invention also covers the following:

[0154] [Item 1] A method for preparing a three-dimensional hydrogel bioimplant, the method comprising:

[0155] a. providing a first photo-crosslinkable sensitive composition in a container, the first composition comprising:

[0156] i. a first polymer that is sensitive to photocrosslinking, in particular thiol-ene crosslinking, but also to free radical polymerization chain growth crosslinking;

[0157] ii. Photoinitiator and

[0158] iii. Optionally, the refractive index matching agent (e.g. up to 80% w / v iodixanol) of the photosensitive resin formulation may be increased or decreased

[0159] iv. Optionally, a light absorbing dye (e.g., Sunset Yellow or FCF at a concentration of up to 500 μg / mL)

[0160] v. Optionally, a cross-linking agent and

[0161] b. in a first irradiation step, irradiating the composition with a plurality of spatially coherent light beams,

[0162] Thereby a plurality of micro-pillars are generated in the composition.

[0163] [Item 2] The method according to item 1, wherein the spatially coherent light beam is characterized by a diameter of 1 to 100 μm, in particular 3 to 40 μm, and even more in particular 5 to 15 μm;

[0164] Most particularly the diameter is from 7.5 μm to 12.5 μm.

[0165] [Item 3] A method according to Item 1 or 2, wherein the composition is irradiated with more than 100 light beams.

[0166] [Item 4] A method according to any of the preceding items, wherein the composition is irradiated by a first plurality of spatially coherent light beams directed along a first direction;

[0167] In particular, wherein the first plurality of spatially coherent light beams form a first pattern.

[0168] [Item 5] The method according to Item 4, wherein in the second irradiation step, the composition is irradiated by a second plurality of spatially coherent light beams directed along a second direction;

[0169] In particular, wherein the second plurality of spatially coherent light beams form a second pattern.

[0170] [Item 6] A method according to Item 5, wherein the first direction and the second direction are parallel.

[0171] [Item 7] The method according to Item 5, wherein the first direction and the second direction are arranged at an angle of 1° to 180°, in particular 15° to 180°.

[0172] [Item 8] A method according to any one of Items 4 to 7 above, wherein the first pattern and / or the second pattern is arranged in a cylindrical form, in a sheet form, or as parallel lines between anchoring rib-like support sheets.

[0173] [Item 9] A method according to any of the preceding items, wherein after the first irradiation step, a second composition is added to the container, the second composition comprising

[0174] i. a second polymer that is sensitive to photocrosslinking,

[0175] ii. Photoinitiator and

[0176] iii. Optionally, the refractive index matching agent (e.g. up to 80% w / v iodixanol) of the photosensitive resin formulation may be increased or decreased

[0177] iv. Optionally, a light absorbing dye (e.g., Sunset Yellow or FCF Yellow at a concentration of up to 500 μg / mL)

[0178] v. Optionally, a cross-linking agent and

[0179] In a third irradiation step, the composition is irradiated with a third plurality of spatially coherent light beams.

[0180] [Item 10] A method according to Item 9, wherein any first composition that is not photo-crosslinked is discharged from the container and the container is filled with the second composition.

[0181] [Item 11] A method according to any of the preceding items, wherein in the first, second, third or any subsequent irradiation step, irradiation is achieved from the bottom side of the container to the substrate; under continuous irradiation, the substrate moves upward away from the bottom of the container, thereby forming a plurality of microbeams protruding from the substrate, in particular wherein the microbeams are distributed throughout the entire length of the hydrogel structure in the longitudinal axis.

[0182] [Item 12] A method according to any of the preceding items, wherein the photocrosslinking sensitive polymer is a biopolymer functionalized by covalent attachment of a carbon-carbon double bond (ene) portion or a methacrylate functionalized biopolymer.

[0183] [Item 13] The method according to Item 12, wherein the biopolymer is selected from the group consisting of at least gelatin, hyaluronic acid, alginate, collagen, chitosan, silk fibroin, and cellulose.

[0184] [Item 14] A method according to Item 12 or 13, wherein the carbon-carbon double bond (ene)-containing portion is selected from the group consisting of at least norbornene carboxylic acid or dicarboxylic acid, methacrylate or amide, acrylate or amide, and vinyl ester.

[0185] [Item 15] A method according to any of the preceding items, wherein the photocrosslinking-sensitive polymer is selected from the group consisting of at least norbornene-functionalized gelatin, norbornene-functionalized collagen, norbornene-functionalized chitosan, norbornene-functionalized hyaluronic acid, methacryloylated gelatin (Gel-MA), methacryloylated hyaluronic acid (HA-MA), and methacryloylated alginate (Alg-MA);

[0186] In particular, among the polymers that are susceptible to thiol-ene photocrosslinking is norbornene-functionalized gelatin.

[0187] [Item 16] A method according to Item 15, wherein the norbornene functionalized gelatin is characterized by a degree of substitution of 10% to 90%, particularly 47% to 50%, and the methacrylate functionalized gelatin is characterized by a degree of substitution of 10% to 90%, particularly 45-60%.

[0188] [Item 17] A method according to any of the preceding claims, wherein the cross-linking agent is a thiol-functionalized poly(ethylene glycol), in particular pentaerythritol-PEG-thiol [C(CH2O(CH2CH2O)nCH2CH2SH)4].

[0189] [Item 18] A method according to any of the preceding items, wherein the photoinitiator is selected from the group consisting of at least lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), oxidized 2,4,6-trimethylbenzoyldiphenylphosphine (TPO), oxidized bisacylphosphine (BAPO), 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), camphorquinone (CAS-No: 10373-78-1), ethyl dimethylaminobenzoate (EDAB), and diphenyliodonium hexafluorophosphate (DPIHFP), in particular wherein the photoinitiator is LAP.

[0190] [Item 19] A method according to any of the preceding items, wherein the refractive index matching agent is selected from propylene glycol, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polylactic acid (PLA), bovine serum albumin (BSA), and iodixanol (CAS-No: 92339-11-2), in particular wherein the refractive index matching agent is iodixanol.

[0191] [Item 20] A method according to any of the preceding items, wherein the light absorbing dye is selected from Brilliant Blue FCF (CAS-No: 3844-45-9), Indigo (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), and Sunset Yellow FCF (CAS-No: 2783-94-0), in particular wherein the light absorbing dye is Sunset Yellow FCF.

[0192] [Item 21] A method according to any of the preceding items, wherein the spatially coherent light beam is characterized by

[0193] a. Wavelength from 360 to 800 nm;

[0194] b. a coherence length of 1 to 100 μm, in particular 3 to 20 μm;

[0195] c.10 to 5000 mJ / cm2 , especially 90 to 200 mJ / cm 2 The light dose,

[0196] d. Duration of 0.1s to 100s, especially 1s to 4s;

[0197] e.1 to 500 mW / cm 2 , especially 50 to 60 mW / cm 2 energy density.

[0198] [Item 22] A three-dimensional hydrogel implant comprising or essentially consisting of a first plurality of microbeams of a first photocross-linked polymer, wherein the three-dimensional hydrogel implant is obtained by a method according to any of the preceding items.

[0199] [Item 23] A three-dimensional hydrogel bioimplant according to Item 22, comprising a second plurality of microbeams of the first photocross-linked polymer.

[0200] [Item 24] A three-dimensional hydrogel bioimplant according to Item 22 or 23, comprising a third plurality of microbeams of a second photocross-linked polymer.

[0201] [Item 25] A three-dimensional hydrogel bioimplant according to any one of Items 22 to 24, wherein the first, second, third and / or any subsequent plurality of microbeams are characterized by

[0202] a. Each of the microbeams has a diameter ranging from 1 μm to 50 μm;

[0203] b. >75% (particularly >80%, ≥85%, ≥90%, ≥95% or even ≥98%) of the microbeams of the plurality have an orientation that deviates by ≤2° relative to the longitudinal axis,

[0204] and / or

[0205] c. >75% (particularly >80%, ≥85%, ≥90%, ≥95% or even ≥98%) of the microbeams in the plurality have a length of ≥2 cm, particularly ≥5 cm.

[0206] d. Microbunches are distributed throughout the entire length of the hydrogel construct in the longitudinal axis.

[0207] [Item 26] A three-dimensional hydrogel bioimplant according to any one of Items 22 to 25, characterized in that the channel structure has a diameter of 100 μm to 10 mm, in particular a diameter of 200 μm to 1000 μm; more particularly a diameter of 400 μm to 600 μm.

[0208] Wherever herein, alternatives of single separable features are set forth as "embodiments", it should be understood that such alternatives may be freely combined to form discrete embodiments of the invention disclosed herein.

[0209] The present invention is further illustrated by the following examples and figures, from which other embodiments and advantages can be derived. These examples are intended to illustrate the present invention but not to limit its scope. BRIEF DESCRIPTION OF THE DRAWINGS

[0210] Figure 1 a) Light beam projection (LBP)—a biofabrication strategy for anisotropic structures containing bundled microstructures with effective cell-guiding properties. The figure shows a schematic diagram of the LBP strategy for arranging microbeams and their cell-guiding properties. b) Long and continuous microbeams and microchannels are generated in anisotropic hydrogel structures, which correspond to local maxima and minima of light intensity.

[0211]

Figure 2

[0212]

Figure 3

[0213]

Figure 4

[0214]

Figure 5

[0215]

【 Figure 6 Figure 1. Advanced beam projection and the potential of multi-material LBP for biofabrication of composite hydrogel constructs. a) Top: Photographs and bright-field images of cell-laden tubular hydrogel constructs fabricated using 2.88% w / v Gel-NB / 4PEG-SH bioresin. b) Middle and bottom: Fluorescence images of tubular constructs from front and side views. Encapsulated NHDFs were labeled with Calcein AM, and RhoD-labeled dextran was added to the tubular hydrogel constructs after 7 days of culture. Scale bar 100 μm. c) Schematic diagram of the two-step LBP for heterogeneous biofabrication of anisotropic tissues. d) Photographs and full-scan fluorescence images of different hydrogel constructs obtained by two-step LBP using 5% w / v FITC and Rhod-labeled Gel-MA photosensitive resin. Scale bar 2 mm in photographs and 1 mm in fluorescence images. e) Fluorescence images of hydrogel constructs fabricated by two-step LBP at day 0 and day 7. NHDFs were labeled with green or red cell tracers and encapsulated in hydrogel constructs made with 5% and 2.88% w / v Gel-NB / 4PEG-SH photosensitive resin, respectively. Side views of cell orientation after 7 days in culture were generated from full scan 3D fluorescence images using Imaris. Scale bars are 200 μm in all fluorescence images.

[0216]

Figure 7

[0217]

Figure 8

[0218]

Fig. 9

[0219]

Fig.10

[0220]

Fig.11

[0221]

Fig.12

[0222]

【 Fig.13Figure 3 shows collagen deposition in hydrogel constructs containing microbeams. a) Negative control of type I collagen staining on a cell-free hydrogel sample after 2 weeks of incubation in PBS. b–d) Immunofluorescence staining of deposited collagen fibers in hydrogel samples containing microbeams after 2 weeks of culture. Microbeams were prepared using 5% w / v Rhod-labeled Gel-MA. Scale bar, 20 μm.

[0223]

Fig.14

[0224]

Fig.15

[0225]

Fig.16

[0226]

Fig.17

[0227]

【 Fig.18 Bottom-up fabrication of long tissue constructs with continuous microbeam structures is shown - The bottom-up strategy can be used to generate long tissue structures. By continuously adding photosensitive resin (automatic feeding) and adjusting the projection image, complex structures can be fabricated. In addition, by feeding different photosensitive resins, multi-z-step projections can be achieved. This allows the fabrication of more complex anisotropic tissue structures, such as muscle-tendon and tendon-bone interfaces.

[0228]

【 Fig.19Demonstration of top-down fabrication of long tissue constructs with continuous microbeam structures - The top-down strategy can be used to generate long tissue structures. By continuously adding photoresin (automatic feeding) and adjusting the projected image, complex structures can be fabricated, just like with DLP. In addition, by feeding different photosensitive resins, multi-z-step projections can be achieved. This allows the fabrication of more complex anisotropic tissue structures, such as muscle-tendon and tendon-bone interfaces.

[0229]

Fig. 20

[0230]

Fig.21

[0231]

Fig. 22

[0232]

Fig.23

[0233]

Fig.24

[0234]

Fig.25

[0235]

Fig.26

[0236] [Figure 27] Light absorbers are needed to control the light penetration depth into the photosensitive resin, which prevents over-crosslinking of previously crosslinked resins. This allows a larger working window at the flow rate of continuous resin feed to achieve a shape index close to 1 (which means high printing fidelity). The scale bar in both figures is 5 mm.

[0237]

【 Fig.28 The use of light-absorbing dyes and the application of dynamic laser intensity improve the printing quality. The shape index of the hydrogel constructs fabricated by different intensity settings and photosensitive resin formulations. For example, intensity 50–75 represents a light intensity curve that starts at 50% of the full intensity and ends at 75% during printing.

[0238]

Fig.29

[0239]

Fig.30

[0240]

【 Fig.31 Changing the distance between the lenses changes the size of the projected image (in this case, it is 1 or 4 times the original size of the projected image (1×1cm 2 ), which in turn affects the diameter of the microfilaments. The higher the image magnification, the larger the diameter of the microfilaments and microchannels (between the microfilaments).

[0241]

【 Fig.32 Image pixelation can introduce unique microstructures into the structure. A. Projected pixelated images (1×1 means black and white pixels alternate, 2×2 means black and white pixels alternate in a set of 2 pixels) can generate unpredictable patterns due to interference effects. Here, the 2×2 pixel arrangement leads to an oblique arrangement of the cross-linked resin rather than a checkered arrangement; B. Cells seeded on top of a hydrogel with a gradient microstructure framework can be arranged along the microstructure imparted using the pixelation strategy.

[0242]

【 Fig.33 Figure 1. Depiction of microstructures in FLight hydrogel constructs using mesh as photomask. A. Metal mesh (grating) added at different positions in front of the cuvette in the FLight setup. B. Photograph of a hydrogel construct consisting of microstructures with a W265D50 mesh (mesh size 265 μm, wire diameter 50 μm), and fluorescent image of the construct after washing of the uncrosslinked photosensitive resin. 5% fluorescently labeled Gel-MA (0.05% LAP) was used in this demonstration, scale bar: 750 μm.

[0243]

【 Fig.34Bright field (BF) image of a wire mesh used as a diffraction grating in the FLight system. Based on the arrangement of the mesh / grating along the light path, microstructures with different sizes can be produced by a series of meshes (W63D36: mesh size 63μm, wire diameter 36μm; W90D36: mesh size 90μm, wire diameter 36μm). The size and shape of the microstructures are highly dependent on the design of the mesh. The scale bar is 750μm in the front view and 300μm in the side view.

[0244]

【 Fig.35 Unique patterns of light transmitted through a grating system (e.g., a moiré pattern) can produce different hydrogel structures. For example, two screens can be inserted into the optical setup of the FLight device to produce a moiré pattern, thereby forming a hydrogel structure with the same shape as the moiré pattern input. In this system, the two or more gratings can be rotated (i.e., the angle between the meshes is offset), and / or the distance between the gratings can be adjusted to produce a varying pattern along the light path ( Fig.36 The results shown in ).

[0245]

Fig.36

[0246] Example

[0247] Example 1: Optical modulation instability and self-focusing induce bundled microstructures in photosensitive resin

[0248] The physics behind the ability of LBP to form cell-instructive microbeams from photosensitive resins is shown in Figure 1a. The projected beam is characterized by an intensity noise pattern that translates into an array of microbeams that compose the hydrogel structure. First, the inventors highlight the physics that causes these microbeams to form in the photosensitive resin. The image projected onto a transparent cuvette containing the photosensitive resin is characterized by a speckle pattern intensity noise originating from the spatial coherence of the input light source. This small intensity noise is then nonlinearly amplified by the cross-linking of the photosensitive resin, thereby generating a microscopic resin waveguide that propagates the filamentary beam through the resin volume (i.e., the thickness of the cuvette interior). More specifically, the initial local intensity noise maximum locally cross-links the photosensitive body from the cuvette-photosensitive body interface, thereby inducing a local increase in RI in the photosensitive resin. In turn, the change in the RI of the photosensitive resin leads to a self-focusing effect at the interface between the cross-linked and uncross-linked photosensitive materials in the z-direction. The formation of the local RI maximum also generates a local micro-waveguide that optically captures the incident beam front and propagates over a long distance into the photosensitive resin. In other words, the trapped and focused beam further cross-links the photosensitive resin, thereby continuously inducing a self-focusing effect and generating long microbeams within seconds. The cumulative effect of multiple local intensity maxima close to each other produces an array of oriented microbeams in the resulting hydrogel construct. Interestingly, the formation of hydrogel microfilaments has also been observed in previous volumetric 3D printing studies. However, these microstructures are considered a defect of volumetric 3D printing, and their potential value in the engineering of anisotropic tissue constructs has not been realized. In addition, due to local minima of intensity noise, there are uncross-linked photosensitive resins between microbeams, leaving high aspect ratio microchannels between microbeams after washing. Figure 1b The self-focusing and self-propagation of microbeams within a photoresin are illustrated. It is important to note that the spatial coherence in the incident light can be optically tuned, while the generation and propagation of microbeams depends on the optical nonlinear properties of the resin (i.e., the RI changes upon photocrosslinking), thereby changing the properties of the microbeams. In the next section, the inventors discuss how to tune the size of microbeams from different photosensitive resins.

[0249] Example 2: Microbeams for cell guidance are highly directional and dimensionally adjustable

[0250] LBP was demonstrated to be useful for several photosensitive resins. Due to the excellent kinetics of the norbornene-thiol reaction, microbeams can be formed within seconds, thus minimizing potential cell damage from photocrosslinking and allowing the use of low polymer concentrations. The inventors first prepared hydrogel samples using Gel-NB / 4PEG-SH photosensitive resin. The critical light dose for projection was determined by performing light dose tests based on previous work. For 2.88% w / v Gel-NB / 4PEG-SH photosensitive resin, 195 mJ / cm 2 The light dose of 200 μm produced high-fidelity LBP hydrogel structures with designed dimensions ( Figure 7, Table 1). Projection of a 1 mm diameter hydrogel cylinder ( Figure 2 a) and confirmed the formation of microbeams (diameter 9.8 ± 2.5 μm) in the cylinder ( Figure 2 b). ~88% of the microbeams are parallel to the projection direction (-1° to 1°) ( Figure 2 c).

[0251] Table 1: Figure 2 Characterization of the changed photosensitive resin and microbeam structure depicted in e.

[0252]

[0253] Since the generation of microbeams depends on the spatial coherence of the light beam and the nonlinear medium, the inventors used a higher concentration of Gel-NB / 4PEG-SH (5% w / v), as well as other common photosensitive biomaterials (methacryloyl gelatin (Gel-MA), methacryloyl hyaluronic acid (HA-MA), and methacryloyl alginate (Alg-MA)) to test LBP ( Figure 2 d). Microbeam structures were formed using all photosensitive resins, with highly directional microbeams in all cases (>86%, -1 to 1°). Projection dose ranged from 180 to 5650 mJ / cm 2 varies between 8.1±1.4 and 11.1±3.1 μm, depending on the resin. Figure 8 –9, Table 1).

[0254] High-aspect-ratio structures can provide cell guidance, and the ability to tune their size will make LBPs suitable for a wide range of directional organizations with ECM fibers ranging from 1 to ~100 μm. Here, microbeams with different diameter ranges were produced by tuning the spatial coherence length using a standard Köhler illumination system consisting of a 405 nm LED light source. Specifically, this light-induced self-organization depends on the spatial coherence of the light source and the strength of the RI nonlinearity of the photoresponsive material. The higher the spatial coherence of the beam - in other words, the longer the spatial coherence length l of the beam. c The longer the microbeam, the wider its diameter. In the inventors' Kohler illumination system, by adjusting the aperture diameter (AD), the spatial coherence length of the beam can span the theoretical range of 3.7 to 25.8 μm ( Fig.10 This range of spatial coherence lengths allowed the inventors to control the diameter of the microbeams between 8.3 and 17.8 μm using 5% w / v Gel-NB / 4PEG-SH photosensitive resin ( Figure 2e). The average diameters of the microbeams made from methacryloyl resins ranged from 5.0 to 13.6 μm, 2.4 to 8.3 μm, and 4.7 to 13.7 μm using 5% w / v Gel-MA, 2% w / v HA-MA, and 2% w / v Alg-MA photosensitive resins, respectively. However, it was found that the average diameter of the microbeams was independent of the field diameter of the inventors' Kohler system, which further demonstrated that the spatial coherence of the beam induced by OMI controls the microbeam formation and that the microbeam diameter can be adjusted by adjusting the spatial coherence length of the beam ( Fig.10 ).

[0255] Due to the reinforcement of the highly oriented fiber components, hydrogel constructs composed of microbeams exhibit ideal mechanical properties for tissue engineering applications. Mechanical stimulation can be applied to such hydrogel constructs to further induce cell orientation and promote tissue maturation. Block hydrogels and hydrogel samples containing microbeams were prepared using Gel-NB / 4PEG-SH or Gel-MA photosensitive resins for tensile and compression testing ( Figure 2 f, g). The yield stress of the hydrogel samples prepared using 2.88% w / v Gel-NB / 4PEG-SH photosensitive resin increased from 2.7 kPa (block hydrogel) to 6.0 kPa (hydrogel containing microbeams), and the yield strains were 64% and 122%. Similar results were found in the hydrogel samples prepared using 5% w / v Gel-MA photosensitive resin: the yield stress and yield strain in the block hydrogel samples were found to be 1.8 kPa and 36%. However, due to the presence of microbeams, the yield stress and yield strain increased to 7.8 kPa and 105%. When the concentration of Gel-NB / 4PEG-SH photosensitive resin increased from 1.44% to 5%, the compressive elastic modulus of the cylindrical LBP hydrogel structure (diameter 5 mm and height 4 mm) increased from 1.8 kPa to 40.7 kPa. Likewise, when tested along the microbeam direction, the compression of the LBP hydrogel samples showed higher elastic moduli compared to the bulk hydrogel samples; 8.4 vs. 3.0 kPa for 2.88% w / v Gel-NB / 4PEG-SH and 15.8 vs. 4.0 kPa for 5% w / v Gel-MA photoresist. These differences in compressive mechanical properties between bulk hydrogels and microbeam-enhanced hydrogels again demonstrate the reinforcing properties of microbeams in hydrogel construction. Furthermore, by Figure 2 The deformation recovery of the hydrogel samples was investigated by cyclic loading-unloading test shown in h. During the stretching at 50% tensile strain for 10 min under air conditions, no residual strain was generated after loading-unloading, indicating that the hydrogel containing microbeams did not undergo plastic deformation during tensile loading.

[0256] Example 3: Channel-like gaps guide anisotropic cell migration

[0257] The nanoscale pores of conventional hydrogel networks hinder the diffusion of oxygen and nutrients and lack cell guidance cues. In an ideal tissue engineering matrix, there should be micrometer-scale interconnected void spaces to provide 3D space for cell migration, proliferation, vascularization, and ECM deposition. Interestingly, most of the 3D matrix fabricated by LPB is occupied by microchannels with ultra-high aspect ratios (>700:1). These microchannels provide 3D spaces into which cells can migrate and cause the orientation of cells and their nuclei. Fluorescently labeled microbeams were fabricated using 5% w / v fluorescently labeled Gel-MA photosensitive resin, and the uncross-linked photosensitive resin was subsequently washed. A large number of microchannels were observed between the microbeams, Figure 3 The void fraction is found to be approximately 50% throughout the hydrogel volume and is independent of the spatial coherence length of the beam ( Figure 3 b) By measuring the ratio of microbeams / microchannels in each layer in the 3D image, the microchannels are evenly distributed within the hydrogel volume ( Figure 3 c. Fig.11 ).

[0258] The physical confinement provided by pores smaller than 3 μm has been shown to significantly limit cell migration in 3D tissue matrices. Due to the limited deformability of the cell nucleus, this size is considered to be the threshold at which cells cannot pass. Here, the diameter of the microchannel is adjusted by adjusting the spatial coherence length of the light beam to ensure efficient cell migration through the microchannel. When the coherence length is reduced from 17.2 μm to 3.7 μm using 5% w / v fluorescently labeled Gel-MA, the average diameter of the microchannel increases from 2.7 μm to 5.8 μm ( Figure 3 e). At the same time, the average aspect ratio of the microchannel structure decreases from 1540:1 to 700:1 as the spatial coherence length increases ( Figure 3 f). In order to obtain cell migration and a higher aspect ratio for efficient cell guidance, normal human dermal fibroblasts (NHDF) were encapsulated in LBP hydrogels 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, the migration of NHDF was evaluated by staining them with Calcein AM. Surprisingly, after 1 hour, migration of NHDF from the original encapsulation site into the microchannels was observed with a simultaneous deformation of the nucleus (at Figure 3 Highlighted by yellow arrows in d). The average aspect ratio of cells increased from 1.24 to 15.8 after 7 days of culture ( Figure 3g), indicating elongation of cells during migration within the microchannels. As evidence of interconnected microchannels in the hydrogel matrix (e.g., cells can migrate through the microchannels), fibroblasts post-seeded on the microbeam surface were observed to migrate from the surface into the hydrogel matrix after 1 day of incubation, but not from the surface of the block hydrogel. The significant difference in cell migration depths suggests that these microchannels are interconnected to allow cell migration through the microbeams ( Fig.12 ). Taken together, these results suggest that these continuous, interconnected microchannels can support cell migration in and across the microchannel direction. Such cell migration is essential for establishing cell-cell contacts in 3D matrices, such as the 3D network of tendon cells that requires gap junction-mediated intercellular communication. In addition, microchannels can induce nuclear elongation and support nuclear migration, contributing to the key role of maintaining cell phenotype and accelerating myoblast fusion for cell reconstruction after physiological muscle injury.

[0259] The regulation of gene expression produced by nuclear reorganization during mechanical constraint has shown interesting results in the field of tissue engineering. For example, nuclear restriction can induce redifferentiation and reprogramming of cell fate by activating DNA repair pathways. At the tissue level, the expression of ECM-related genes is upregulated in cells with elongated nuclei under dynamic mechanical loads. In this study, the inventors demonstrated that ultra-high aspect ratio microchannels have the unique advantage of producing highly oriented tissues by inducing mechanical constraints. Different tissue microenvironments were produced using the same bioresin preparation (2.88% w / v Gel-NB / 4PEG-SH photosensitive resin with NHDF), including 3D microbeams, 3D block hydrogels, and 2D microbeams. Cells were encapsulated in a hydrogel structure containing 3D microbeads, in a block hydrogel, or post-seeded on the microbeam surface (2D microbeam). After culturing for 1 hour, 1 day, 3 days, and 7 days, the orientation of cells under different conditions was determined by phalloidin staining ( Figure 4 a). After 1 day of incubation in both 3D and 2D conditions, cells began to orient along the microbeads, but not in the block hydrogels, again demonstrating the cell-guiding properties of the microbeads as physical cues. NHDFs encapsulated in the 3D microbead matrix showed lower proliferation levels (proportion of Ki67-positive cells) on the first day compared to 2D microbeads. However, after 3 days of culture, approximately 76% of the NHDFs were found to be Ki67-positive cells, while this proportion was approximately 19% on the microbead surface ( Figure 4 b) Similar results have been reported for the regeneration and proliferation status of fibroblasts after static compressive forces and lateral confinement culture. The mechanical stimulation obtained from the hydrogel matrix may provide a new way to induce cell proliferation, especially for aged cells with proliferation defects, such as tenocytes and tendon stem / progenitor cells.

[0260] Next, the inventors demonstrated that ultrahigh aspect ratio microchannels and the resulting cell confinement can regulate gene expression ( Figure 4 c. Fig.13 ). Compared with 3D block hydrogels and 2D microbeams, the mechanically sensitive ion channel Piezo1 was found to be upregulated in the initial stages (1 hour and 1 day) under 3D microbeam conditions. Similarly, the inventors observed an upregulation of Lamin A / C protein expression (encoded by the LMNA gene) in 3D microbeams at 1 hour; this gene has an integral role in mechanical signaling from the cell membrane to the nucleus and nuclear stiffness. In addition, COL1A1 expression was upregulated at the beginning of incubation, similar to PIEZO1 expression. Earlier studies have demonstrated the correlation between Piezo1 and ECM-related gene expression. For example, the lack of Piezo1 leads to reduced expression of COL1A1, which plays a key role in ECM deposition during tissue maturation under mechanical stimulation. In addition, the lack of these micron-sized voids in block hydrogels leads to higher MMP2 expression levels to help cell migration by remodeling the hydrogel matrix. However, PIEZO1 expression was found to be reduced from day 3, which can be explained by the loss of mechanical stimulation. It may be caused by the degradation of microbeams and deformation of microbeams due to cell contraction. In the future, it is expected that the loss of mechanical stimulation will be able to be offset by applying additional static / dynamic mechanical loading, which is promising for the biofabrication of mature anisotropic tissues.

[0261] Example 4: LBP for biofabrication of highly oriented tissue constructs

[0262] The inventors demonstrated the ability of LBP to biofabricate highly oriented tissues using 2.88% w / v Gel-NB / 4PEG-SH photosensitive resin for four cell types: NHDF, human tenocytes (HT), human umbilical vein endothelial cells (HUVEC), and mouse myoblasts (C2C12). Cell viability was found to be above 85% for all cell types 1 hour after LBP. In contrast, low cell viability (<70%) was observed for HUVEC in block hydrogels that were prepared using the same light dose as LBP ( Fig.14 ). However, when the culture was extended to 21 days, the viability of C2C12 in the LBP hydrogel sample was about 74%. To overcome this limitation, a series of hollow microstructures with a diameter of several hundred microns were generated in the hydrogel structure by blocking the light from the designed projected image. In the hydrogel structure containing multiple hollow channels, the encapsulated C2C12 cells showed higher cell viability (~89%) after 3 weeks of culture. Therefore, multiple hollow microstructures (with a diameter of 216 μm) were used in subsequent experiments to achieve higher bioactivity in the cell-loaded hydrogel structure.

[0263] The potential of LBP in engineering anisotropic tissues is based on the ability of the microstructures to guide cell orientation and the orientation of the ECM (i.e., self-aggregation into oriented collagen fibers). Using NHDF, the cell-guiding properties of the microbeams were tested in structured samples with a diameter of approximately 1 mm (exposure 2.8 s, containing multiple hollow microstructures). Phalloidin staining was used to observe the ability of the microbeams to guide cell orientation ( Figure 5 a). After 14 days of culture, 99% of the NHDFs were elongated and oriented within +30° of the projection direction ( Figure 5 e). Using anti-type I collagen antibody, the formation of oriented type I collagen was confirmed from immunofluorescence images ( Fig.15 On the other hand, due to the lack of cell guidance cues, no cell-oriented and oriented collagen fibers were found in the block hydrogels ( Fig.16 ). Similarly, Figure 5 f, more collagen-positive areas were found in the hydrogel sample containing microbeams (~48%) than in the bulk hydrogel (~28%). Interestingly, the nuclei of fibroblasts in the microbeam-containing material were also elongated and oriented ( Figure 5 g). Recent studies have shown that physical cues can regulate cell and nuclear shape, ultimately affecting phenotype, cell behavior, and gene expression. For example, the presence of fibrous components in the tissue matrix can induce fibroblasts to switch their phenotype to a myofibroblast state, characterized by an elongated shape. In addition, lateral confinement culture of fibroblasts induces dedifferentiation and reprogramming, leading to the regeneration of aged fibroblasts.

[0264] Next, the inventors studied the HT orientation in the hydrogel matrix made from LBP, which is of great significance for the biofabrication of tendon tissue models. After 2 weeks of culture, highly oriented filamentous actin (F-actin) was found in the HTS. Immunofluorescence detection of type I collagen fibers once again demonstrated that the microbeams provided effective guidance for cells and their deposited ECM ( Figure 5 b). In addition, by introducing a fibroblast-HUVEC co-culture system, the inventors showed that the formation of highly oriented capillaries can also be guided by LPBs, as detected by CD31 immunostaining in two-week cultures ( Figure 5 c). Interestingly, the inventors found that continuous capillaries were oriented in the projection direction and observed some vasculature with a length > 1.4 mm. From the cross-sectional view of the 3D confocal image, it can be seen that the capillaries are confirmed to have an inner lumen ( Fig.17 ).

[0265] Finally, C2C12 cells were encapsulated in 2.88% w / v Gel-NB / 4PEG-SH photosensitive resin and subjected to LBP to further demonstrate its potential in the biomanufacturing of muscle tissue. After exposure to differentiation medium containing 2% v / v horse serum, the inventors observed the fusion of myoblasts and the orientation of myotubes by immunofluorescence staining using anti-mouse myosin heavy chain (MyHC) antibody and phalloidin after 3 weeks of culture ( Figure 5 d). In LBP tissue samples, 95% of myotubes were oriented along the direction of the microfascicles (-30 to 30°). Figure 5 h). As expected, myotubes showed random orientation in the bulk hydrogels. Similarly, more highly oriented contractile myotubes with higher fusion index were found in the LBP samples compared to the bulk hydrogels ( Figure 5 i). It is interesting to observe that more than 76% of myotubes in LBP-made hydrogel constructs had more than 6 nuclei per myotube ( Figure 5 j), but not found in bulk hydrogel samples.

[0266] In summary, the inventors have demonstrated that LBP is an effective strategy for biofabrication of anisotropic tissues with rapid fabrication time and excellent cellular biocompatibility. Cell-guiding physical cues, microbeams, lead to the orientation of cells required to produce anisotropic tissues. LBP offers key advantages over conventional strategies, which typically rely on post-seeding of cells onto fiber-based scaffolds and matrices. The fact that these techniques only provide 2D microenvironments and uneven cell distribution further limits their applications. For extrusion-based bioprinting strategies, high concentrations of fibril components and smaller nozzles are typically required to achieve effective cell orientation, which can generate significant shear stress on the cells.

[0267] Example 5: Biofabricated LBP for Composite Hydrogel Construction

[0268] Finally, the inventors investigated the potential of LBP in generating anisotropic tissues that mimic in vivo tissue architecture. Using 2.88% w / v Gel-NB / 4PEG-SH photosensitive resin, various hydrogel constructs with detailed features were produced, including hollow structures ( Figure 6 a. Fig.18 ) and centimeter-scale hydrogel QR codes with a characteristic size of ~220 μm. Similarly, cell-loaded tubular hydrogel structures were generated and encapsulated NHDFs were stained with calcein AM after 7 days of culture ( Figure 6b). A monolayer of NHDF was observed on the inner and outer walls of the tubular structures. Subsequent filling of the channels with fluorescent marker dye (red) showed that these tubular structures were maintained after 7 days of culture. The above results demonstrate that LBP is a promising method for generating anisotropic hydrogel constructs with freely designed cross-sectional structures for anisotropic tissue engineering.

[0269] By performing multiple sequential projections and exchanging bioresins between projections, a multicellular / multimaterial biofabrication strategy was established. LBP allows the generation of hierarchical organization of tissues, control of cell and material placement, and material stiffness. Figure 6 c, a multi-material structure was obtained using two complementary projection images. For the first image, the bioresin was spatially solidified to produce 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, the use of two fluorescently labeled photosensitive resins to generate acellular hydrogel structures ( Figure 6 d). NHDF labeled with CellTracker green was mixed with 5% w / v Gel-NB / 4PEG-SH photosensitive resin and the central cylindrical hydrogel structure was projected. The peripheral tissue structure loaded with NHDF labeled with CellTracker red was projected with 2.88% w / v Gel-NB / 4PEG-SH photosensitive resin. It was confirmed that the two fluorescently labeled cells were successfully encapsulated in the designed hydrogel structure ( Figure 6 e). As expected due to the presence of microbundles in the hydrogel constructs, orientation of cells in both projected hydrogel constructs was observed after 7 days of incubation.

[0270] Example 6: Multi-directional projection strategy

[0271] The inventors have developed a projection strategy that allows biofabrication of hydrogel constructs containing microbeams in multiple directions. This is achieved by repeating the projection process from different directions. Briefly, after the first projection, the photosensitive resin container is rotated by a specific angle (e.g., 30 degrees, 45 degrees, 90 degrees) for the second projection ( Fig. 20 ). This approach will meet the biofabrication of anisotropic tissues that require specific topological guidance cues, such as myocardial tissue.

[0272] Next, a method for making ultra-long hydrogel structures is described. This allows the production of tissue structures with a length of ~cm and containing a continuous microwave beam. This enables the production of infinitely long hydrogel constructs. The beam comes from the top of the container ( Fig.21 ), or the bottom of the container ( Fig. 22). When the light beam comes from the bottom, the bottom surface of the container is treated to prevent the solidified hydrogel structure from adhering. The light projected from below will cross-link the photosensitive resin between the bottom surface of the container and the matrix, thereby adhering the hydrogel structure to the matrix. The matrix will then move to the next position to allow new photosensitive resin to be filled, followed by projection to produce a long hydrogel structure.

[0273] Finally, the inventors established a hybrid projection method that combines the horizontal multi-directional projection strategy with the top projection method ( Fig.23 ). This allows the production of hydrogel structures containing horizontally multidirectionally oriented and vertically oriented microbeams.

[0274] Example 7: Hybrid biofabrication strategy for hydrogel constructs (multi-material / multi-light source)

[0275] The inventors have demonstrated that the light source ( Fig.24 ), and novel designs of optical setups, allowing our biofabrication strategy to be applicable to a wider range of photosensitive materials (e.g., photoresponsive systems that require different wavelengths to trigger a response). Different optical setups further provide a wider range of control over the size, porosity, etc. of microbeams / microchannels, which will allow the fabricated hydrogel matrices to support many or more complex tissue types. For example, smaller microbeams drive better cell orientation, but larger (or more) porosity effectively helps cells migrate and establish cell-cell communication.

[0276] Example 8: Fabrication of long microbeam-loaded tissue constructs at high cell density

[0277] Light scattering induced by high cell density (>5 million cells / ml) during projection typically limits the formation of microbeams to a few millimeters (typically 2 mm) along the length of the hydrogel construct. The inventors have demonstrated that the use of a refractive index matching agent (e.g., 30% w / v iodixanol in the resin) to increase the refractive index of the resin (e.g., the refractive index of 5% Gel-NB / Gel-SH photosensitive resin increased from 1.345 to 1.46 after adding iodixanol to a final concentration of 30% w / v in the Gel-NB / Gel-SH photosensitive resin). This approach can reduce light scattering and allow the formation of long and continuous microbeams in cell-loaded hydrogel constructs with higher cell densities.

[0278] In addition, for the preparation of long hydrogel constructs, the inventors used a bottom-up projection method (as previously described in Fig.21), where a first layer is added and crosslinked by the aforementioned projection method, followed by the addition of a subsequent photosensitive resin and repeated photocrosslinking. This can be performed in an almost unlimited number of steps to obtain long (>20 cm long) hydrogel constructs. However, the inventors have also observed that the projection of any layer will also expose the underlying layers to receive additional light doses, thereby increasing the degree of crosslinking in the underlying layers. This will result in uneven crosslinking over the entire length of the construct, with the bottom layer typically having high hardness and features of merged microbeams, demonstrating suboptimal cell orientation and anisotropic matrix organization. The inventors have demonstrated that the photosensitive resin can be supplemented with a light absorbing dye (such as Sunset Yellow or FCF), resulting in finely tuned penetration of light into the photosensitive resin ( Fig.25 ). For example, the inventors calibrated the amount of photosensitive resin added for each layer and the associated light dose intensity and duration so that the penetration depth of each projection matched the projection depth of the new layer. This allowed the formation of an overall construct without excessive photocrosslinking of each layer, resulting in cell orientation and anisotropic matrix organization over the entire length of the structure.

[0279] For example, in a 5% w / v Gel-NB / Gel-SH photosensitive resin 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 405 nm light) to achieve a penetration depth of 5 mm per layer projection in the presence of a microbeam. Fig.26 ). The resulting construct (made by 6 projections, ∼3 cm long) is characterized by microfascicles throughout its length and oriented myotube formation throughout its length. Here, the concentrations of light absorbing dye (1 μg / mL to 500 μg / mL) and iodixanol (5–80% w / v) can be varied according to experimental needs.

[0280] Example 9: Conclusion

[0281] The inventors have demonstrated a rapid biofabrication method for generating 3D hydrogel constructs containing cell-guiding microbeams. Orientation of four types of cells / deposited ECM was achieved by LBP. The ultrahigh aspect ratio topological cues provided by the microchannels had a potent effect on cell and nuclear morphology. By performing multiple sequential projection processes, a multicellular / multimaterial biofabrication strategy was established that was able to mimic the hierarchical organization of tissues.

[0282] Example 10A: Grafts for nerve repair

[0283] Nerve damage can lead to loss of sensation, muscle function, and overall function of the affected limb or body part. Traditional treatments for nerve damage, such as nerve autotransplantation, have limitations and may cause donor site morbidity. Grafts made using filamentous light (FLight) biofabrication technology can provide a promising alternative for nerve injury repair due to their ability to promote directional axonal growth, as well as tissue regeneration and integration. FLight grafts with directional microfilaments can be used for nerve injury repair, for example, to produce nerve conduits (acellular grafts of nerve conduits) that provide a scaffold for directional axonal regeneration; or as nerve wraps for the treatment of compressive injuries.

[0284] The material of the implant may be based on one or a combination of the resins described above, in particular a resin based on step-growth photopolymerization or a resin based on chain-growth photopolymerization.

[0285] A combination of cells may be used (eg Schwann cells, human mesenchymal stem cells, human adipose stem cells or induced pluripotent stem cells), which have been shown to promote axonal regeneration. The cells will be encapsulated in the graft.

[0286] Growth factors that have been shown to promote axonal regeneration (eg, nerve growth factor, neurotrophic factor) may be present.

[0287] In a specific embodiment, at least one material component of the matrix is ​​based on collagen or acellular matrix.

[0288] The dosing regimen for the neural graft is as follows: The graft is essentially cylindrical, with a diameter between 1 and 7 mm and a length between 5 and 50 mm. The graft can be made with different microarchitectural orientations, such as bundle arrangement of microfilaments (φ microfilament = 1–30 μm) and mesh structure (φ mesh = 30–150 μm). In addition, by changing the grayscale of the projected image in the FLight system, grafts with different core and shell stiffness can be produced. Here, the shell can be produced at a higher light intensity so that it has a high yield strength to allow suturing / anastomosis, while the core is made at a lower intensity so that it has a lower stiffness (<10 kPa) to promote axonal growth. Both the core and shell parts will be made with uniaxially oriented microfilaments to allow axonal growth. In addition to increasing the light exposure of the shell region, the strength of the shell region can be further increased by secondary crosslinking mechanisms, such as chemical crosslinking (for gelatin or collagen-based resins) or ionic crosslinking (for alginate-based resins) upon brief exposure to bacterial transglutaminase.

[0289] The yield strength required for suturing a nerve graft can depend on a variety of 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 should be high enough to resist the tension applied during suturing without breaking or tearing. The detachment force of a nerve graft determined using a tensile test will be about 20-60N, which translates to a yield strength of 10-100 MPa.

[0290] The yield strength required for suturing nerve grafts varies depending on the application. However, studies have shown that nerve grafts with a yield strength of at least 30–50N are suitable for suturing. Additionally, nerve grafts with higher yield strengths provide better results because they are more resistant to stretching or deformation that may occur during suturing or post-operative manipulation.

[0291] Experiments with dorsal root ganglia (DRGs) encapsulated in FLight gels have shown that softer gels with mesh (Young's modulus 1–3 kPa) lead to optimal axon growth in terms of axon length and axon number. Both axon length and the number of axons normalized to the size of the DRG were higher (~2–3 kPa) in softer constructs featuring a mesh (preferably 63 μm mesh). The microfilaments of the FLight hydrogels can guide axon growth bidirectionally (front-to-back).

[0292] Example 10B Graft for muscle repair:

[0293] Several types of muscle lesions are suitable for treatment with FLight grafts. For example, skin matrix allografts have been used to treat a large number of irreparable rotator cuff tears, which may involve significant muscle damage. Similarly, using cells or acellular grafts to treat volumetric muscle loss is a highly abundant research and development field. In the case of muscle damage, the graft provided herein has the ability to support the attachment, orientation and proliferation of cells and promote the formation of new extracellular matrix. Here, the graft characterized by different combinations of resin and material components (preferably, one of the components is based on collagen or acellular matrix) described in the neural graft section will be ideal.

[0294] Potential applications of the FLight graft include:

[0295] 1. Muscle strains and tears - Acellular grafts can be used to repair and regenerate muscle tissue that has been damaged by strain or tear. The grafts provide support and scaffolding for new tissue growth and help improve muscle function.

[0296] 2. Muscle Atrophy - Muscle atrophy is the loss of muscle mass and strength that can occur due to aging, injury, or disease. Acellular grafts can be used to promote muscle regeneration and prevent further muscle loss in these conditions.

[0297] 3. Muscular Dystrophy - Muscular dystrophy is a genetic disease that causes progressive muscle weakness and degeneration. Although acellular transplants may not cure the disease, they may be used to improve muscle function and quality of life for patients.

[0298] 4. Volumetric muscle loss (VML) - VML occurs when a significant portion of muscle tissue is lost due to injury or surgery. In these cases, acellular grafts can be used to promote muscle regeneration and prevent the development of scar tissue.

[0299] Example 10C: Graft for tendon repair:

[0300] FLight grafts can also be used to treat a variety of tendinopathies that have not responded to conservative treatment. Here, the oriented microarchitecture of the FLight graft is important for treating tendons using acellular grafts. Tendons have a highly organized structure in which collagen fibers are aligned in parallel directions to resist tension and transmit forces. Some of the tendinopathies that can be repaired with FLight grafts include:

[0301] 1. Achilles tendinopathy

[0302] 2. Rotator cuff tendinopathy

[0303] 3. Patellar tendinopathy

[0304] 4. Tennis elbow (lateral epicondylitis)

[0305] 5. Golfer's elbow (medial epicondylitis)

[0306] In these cases, FLight grafts can be used to repair or augment already damaged or weakened tendon tissue. They help improve tendon strength, reduce pain, and promote healing. The mechanical properties (yield strength) required of grafts used in tendinopathy repairs can vary depending on the specific application and the individual needs of the patient. In general, the graft should have sufficient strength to support the damaged or weakened tendon and allow proper healing. For Achilles tendon repair, acellular grafts with a yield strength of at least 250 kPa have been reported to provide good clinical results. For rotator cuff repairs, grafts with a yield strength of at least 200 kPa are typically used. For patellar tendinopathy, acellular grafts with a yield strength of at least 150 kPa have been used successfully. For tennis elbow and golfer's elbow, grafts with a yield strength of at least 100 kPa have been reported to provide good results.

[0307] Example 10D: Articular cartilage graft:

[0308] In some pathologies, articular cartilage repair is essential because this tissue has limited self-repair capabilities due to limited vascular supply and the highly dynamic growth environment of the joint. Cartilage damage can occur due to a variety of reasons, including traumatic injury, degenerative joint diseases such as osteoarthritis, or genetic disorders such as osteochondritis dissecans.

[0309] Articular cartilage damage can range from small surface defects to full-thickness cartilage loss. If left untreated, these defects can progress to more severe degenerative joint disease, leading to pain, loss of joint function, and reduced quality of life.

[0310] To perform articular cartilage repair using acellular grafts, the damaged cartilage is first removed and smoothed to create a stable surface. The graft is then prepared and sized to fit the defect. The graft will be anchored in place using sutures or tissue glue. Over time, the graft will incorporate into the surrounding tissue, stimulating the infiltration and growth of new chondrocytes and promoting tissue regeneration.

[0311] Materials and methods

[0312] Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich and cell culture reagents were purchased from Gibco.

[0313] Synthesis of Gel-NB

[0314] The synthesis process and properties of Gel-NB were described in the inventors' previous studies: [5] Briefly, gelatin type A from porcine skin was dissolved in 0.1 M carbonate-bicarbonate buffer, pH 9, at 50°C at a concentration of 10% w / v. Then, 1 / 5 of the total cis-5-norbornene-endo-2,3-dicarboxylic anhydride (carbonic anhydride, CA) required to obtain the desired Gel:CA ratio was added to the solution. The reaction was allowed to proceed for 10 min under stirring, and then the pH was adjusted to 9 with 0.5 M NaOH solution. The sequential pH adjustment and CA addition were repeated five times every 10 min. The solution was then diluted two-fold with Milli-Q water preheated to 40°C, and the pH was adjusted to 7.4 with 0.5 M HCl solution. After centrifugation at 3000 rcf for 15 min, the supernatant was dialyzed against Milli-Q water at 40°C with frequent water changes for 3–4 days and finally freeze-dried.

[0315] For photosensitive resin preparation, lyophilized Gel-NB was dissolved in PBS and kept at 40°C for 30 min. 4PEG-SH (10 kDa, JenKem Technology) was added to the Gel-NB solution to obtain the desired SH:NB ratio (1:1). Next, a 2% w / v stock solution of photoinitiator (PI) lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in PBS was mixed with the Gel-NB / 4PEG-SH solution to a final concentration of 0.05% w / v. The photosensitive resin was filtered through a 0.45 μm filter (Filtropur S 0.45, SARSTEDT AG) and stored in the dark.

[0316] Synthesis of Gel-MA and Fluorescently Labeled Gel-MA

[0317] Gel-MA was synthesized as described above. The degree of substitution (DS) was expressed as 1 H-NMR (Bruker Ultrashield 400 MHz, 1024 scans) was performed on D 2 O (Apollo Scientific). The Gel-MA lysine integration signal (2.95–3.05 ppm) was compared to 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 ~45%.

[0318] Fluorescently labeled Gel-MA was prepared by modifying fluorescein-5-isothiocyanate (FITC) or rhodamine B isothiocyanate (RBITC) onto Gel-MA. Briefly, 10% w / v of Gel-MA was first dissolved in 100 mM sodium bicarbonate solution. 0.1% w / v FITC-DMF or rhodamine solution was then 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, followed by freeze drying to obtain fluorescently labeled Gel-MA.

[0319] For photosensitive resin preparation, freeze-dried Gel-MA was dissolved in PBS and kept at 40°C for 30 minutes. Then, 2% w / v LAP stock solution was mixed with Gel-MA solution to obtain a final concentration of 5% w / v Gel-MA and 0.05% w / v LAP. The photosensitive resin was filtered through a 0.45 μm filter and stored in the dark.

[0320] Synthesis of HA-MA

[0321] We purchased low molecular weight HA-MA (M w =50,000–70,000).

[0322] For photosensitive resin preparation, HA-MA was dispersed in PBS and stirred at 4°C until completely dissolved, then mixed with 2% w / v LAP stock solution to prepare 2% w / v HA-MA and 0.05% w / v LAP solution. The photosensitive resin was filtered through a 0.45 μm filter and stored in the dark.

[0323] Synthesis of Alg-MA

[0324] Sodium alginate (M w >200kDa,PRONOVA UP MVG, ) was dissolved in 25 mL of Milli-Q water to produce a 1% w / v solution with continuous stirring. Methacrylic anhydride (20 equivalents of hydroxyl, 52 mmol, 7.7 mL) was distilled and added before use; the resulting emulsion was stirred vigorously at room temperature for 24 hours. The pH was checked frequently and adjusted to 7–8 with 0.5 M NaOH solution. The 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 into ethanol, and the precipitate was filtered through a fritted glass funnel (S4 porosity) and dried under high vacuum overnight. The dried Alg-MA was dissolved in Milli-Q water and then dialyzed at room temperature with frequent water changes for 3–4 days. Alg-MA was obtained after freeze drying. Use D 2 The DS was determined using 1% w / v Alg-MA in O on a Bruker spectrometer operating at 400 MHz proton frequency. 1 H-NMR spectrum. DS -51%.

[0325] To prepare the photosensitive resin, freeze-dried Alg-MA was dissolved in PBS. The LAP stock solution was then mixed with the Alg-MA solution to achieve a final concentration of 2% w / v Alg-MA and 0.05% w / v LAP. Before use, the photosensitive resin was filtered through a 0.45 μm filter.

[0326] Cell culture

[0327] Tenocytes were kindly provided by Professor Lee Ann Applegate and have been described previously.

[66] Human fetal progenitor tenocytes were isolated from the Achilles tendon of a 14-week male fetal organ donor according to a protocol approved by the ethics committee. Lausanne University Hospital (CHUV), Ethics Committee Protocol No. 62 / 07: 14-week fetal organ donation, registered with the Federal Transplantation Program and its Department of Musculoskeletal Medicine (DAL) Biobank, and in compliance with legal and regulatory requirements. NHDF were isolated from juvenile foreskin skin biopsies. The biopsy was obtained with informed consent from the parents and its use for research purposes was approved by the Ethics Committee of the Canton of Zurich (BASEC-Request-Nr. 2018-00269). Tenocytes and normal human dermal fibroblasts (NHDF) were isolated from the 14-week male fetus. Cell culture multi-layer bottle (TC 5 layers, 875cm 2 ) and cultured in DMEM+GlutaMAX TM -I + 10% w / v fetal bovine serum (FBS) + 10 μg mL -1 Antibiotics and antifungals (Anti-Anti). Myoblasts (C2C12) were obtained from ATCC and grown in DMEM medium + 10% v / v FBS + 10 μg mL -1 Anti-Anti. Cells were passaged at 90% confluence and detached using 0.25% trypsin / EDTA. For C2C12 differentiation, cell-loaded hydrogels were incubated in DMEM + 2% v / v horse serum + 1% v / v insulin-transferrin-selenium (ITS+, Corning) + 10 μg mL -1 Incubate in differentiation medium composed of gentamicin. The culture medium was frequently changed in a 35 mm culture dish (PS 60 / 15MM, Greiner Bio-One) to culture the sample. 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 structure, HUVEC and NHDF were mixed in a photosensitive resin at a ratio of 1:2 to prepare a biophotosensitive resin. The prepared cell-loaded hydrogel sample was transferred to a 6-well plate and cultured in a mixed culture medium (DMEM: EGM-2 is 1:1).

[0328] Preparation of bio-photosensitive resin

[0329] The photosensitive resin was prepared as described above. A 0.2 μm filter (Filtropur S 0.2, SARSTEDT AG) was used to sterilize the photosensitive reflective protein and remove potential scattering particles. NHDF and HT were mixed at 1×10 6 Cells·mL -1C2C12 cells were mixed with photoresist at a concentration of 2 million cells / mL-1. For NHDF-HUVEC co-culture, the two cell types were mixed at a ratio of 2:1 and the concentration in photoresist was 1 million cells / mL -1 .

[0330] Projection image design

[0331] Projected images were generated using Affinity Photo (AffinitySuite 1.9, Serif Europe Ltd.) at a fixed resolution of 1024 × 768 pixels. Images are grayscale, with pure white corresponding to 100% light intensity (approximately 62.5 mW / cm 2 ). The width of each pixel in the projected image is equal to about 27 μm. When projecting, the pattern is corrected by 90°, where the vial and the cuvette are rotated by 90°. The projected image is then output as a PNG file.

[0332] Standard Kohler lighting system assembly

[0333] If not otherwise stated, all parts used to assemble a standard Kohler illumination system were from Thorlabs, including the light source (M405L4). The assembly and orientation schemes were described in previous studies (J. Madrid-Wolff et al., Biophys. 2020, 1, 10.). The light intensity in front of the cuvette was measured after each adjustment of the field diameter and / or aperture diameter using a power meter.

[0334] Beam projection

[0335] (Bio) photosensitive resins are prepared as described above and transferred to a sterilized colorimetric trough or glass vial. Gel-NB / 4PEG-SH and Gel-MA (bio) photosensitive resins are allowed to heat gel at 4°C for 15 minutes. For highly viscous photosensitive resins or non-thermoreversible photosensitive resins (i.e., HA-MA, Alg-MA), the above steps are skipped. Projection is then performed using the "Advanced" built-in function on a commercially available volumetric printer (Tomolite Ver.1.0, Readily3D SA). The projection time is calculated based on the light intensity and light dose required to crosslink each photosensitive resin. The projected image is loaded into the software (Apparite, Readily3D SA) before starting the projection. Uncrosslinked (bio) photosensitive resins are washed away using PBS preheated to 37°C. The projected structure is removed using a sterile scraper and transferred to PBS or culture medium.

[0336] Compression modulus measurement

[0337] All samples were prepared as cylindrical molds 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. Block hydrogel samples (control group) were produced by filling photosensitive resin into PDMS molds (5 mm inner diameter and 4 mm height) before UV cross-linking. The samples were tested by unconstrained uniaxial compression using a TA.XTplus Texture Analyzer (Stable Microsystems). A 500 g load cell and a flat plate probe (15 mm) were used. A 0.2 g preload was applied to ensure full contact between the sample and the plate. The samples were compressed at a compression rate of 0.01 mm s-1 to a final strain of 50%. The elastic compression modulus was calculated by linear fitting of the initial linear region (0.5–5%) of the stress-strain curve. The tests were performed at 25 °C and repeated 3 times.

[0338] Tensile test

[0339] All samples were prepared as wires with a diameter of 1 mm. A circular matrix (8×8) image with a diameter of 37 pixels was loaded onto the LBP. Block hydrogel samples were produced by extruding the block gel through a grid (1 mm, micron-sized holes) after thermoreversible gelation of the photosensitive resin. The microwires were then cross-linked under a UV lamp using the same light dose as the LBP method. Tensile tests were performed using a micro tension clamp using a structural analyzer equipped with a 500 g force sensor, with a 0.1 g preload applied before stretching. All samples were loaded at 0.1 mm s -1 The tensile strain was 0.1 mm s at a rate of 0–50% until fracture. -1 The loading-unloading curves were measured within 12 cycles at a tensile rate.

[0340] Cell viability

[0341] After 0, 3, and 7 days of culture, the hydrogel structure was washed three times with PBS and then incubated in FluorobuteTM DMEM supplemented with 1:2000 CalceinAM (Invitrogen), 1:1000 Hoechst33342 (Invitrogen), and 1:500 Propidium Iodide (PI, Fluka) for 45 minutes. After washing three times in the culture medium, fluorescence imaging was performed using a confocal laser scanning microscope (Fluoview 3000, Olympus). Z stack scanning was performed starting from the surface of the structure, with a step length of 5 μm to 200 μm deep into the structure. Z projection images were analyzed with Fiji ImageJ. The above experiment was repeated three times, and the vitality of each sample was averaged on three images of randomly selected areas.

[0342] CellTracker Dye Loading

[0343] CellTracker green dye CMFDA (Invitrogen) and CellTracker red dye CMTPX were applied to NHDF 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.

[0344] Immunofluorescence staining

[0345] After 7 days of culture, the cell-laden hydrogel threads were washed three times with PBS and fixed in 4% paraformaldehyde for 30 min at 25°C. The threads were permeabilized with 0.2% Triton-X100 in PBS for 30 min and then blocked with 1% v / v BSA in PBS for 1 h. They were then incubated with anti-human collagen I primary antibody (ab138492, Abcam) diluted 1 / 500 in BSA-PBS for 12 h at 4°C. Next, the samples were washed three times with PBS and then incubated with 1 / 200 diluted secondary antibody (goat anti-rabbit Alexa Fluor 488, Invitrogen), Hoechst 33342, and pre-prepared phalloidin-tetramethylrhodamine B isothiocyanate working solution (0.13 μg mL-1, P1951) in BSA-PBS for 2 h at 4°C. C2C12 constructs were incubated with anti-human myosin heavy chain antibody (MF-20, DSHB) at 1 / 20 dilution and collagen I antibody at 1 / 500 dilution for 12 h at 4°C. Samples were then incubated with secondary antibodies (goat anti-rabbit Alexa Fluor 647, goat anti-mouse Alexa Fluor Plus488) at 1 / 200 dilution, phalloidin-tetramethylrhodamine B isothiocyanate working solution, and Hoechst at 4°C for 2 h. Samples were washed with PBS before imaging on the CLSM. NHDFs encapsulated in tubular wires, encapsulated in fluorescent GelMA, or seeded on the surface were stained with CalceinAM for 1 h. Tubular constructs were then perfused with 40 kDa tetramethylrhodamine isothiocyanate-dextran (TRITC-dextran) for 10 min before confocal imaging.

[0346] RNA isolation and qRT-PCR

[0347] NHDF was encapsulated in a hydrogel sample made using 2.88% w / v Gel-NB / 4PEG-SH photosensitive resin. 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 sample, and the mixture was centrifuged at 12000rcf for 10 minutes. The supernatant was mixed with 70% EtOH and transferred to a RNeasy mini kit (Qiagen) column to extract total RNA. A260 / 280 ratios between 1.8 and 2.1 were accepted as sufficient quality for RNA samples. The isolated RNA was transcribed into complementary DNA according to the instructions of the GoScript reverse transcriptase kit (Promega). Relative gene expression levels were measured using SYBR Green PCR Master Mix (Promega) on a real-time PCR system (QuantStudio 5, AppliedBiosystems). The GAPDH housekeeping gene was used as an internal control for RNA level standardization.

[0348] 3D Image Reconstruction

[0349] The 3D images obtained using confocal microscopy were imported into Imaris 9.2.1 to reconstruct the 3D model. The measurements in this work were performed by surface and statistical functions.

[0350] Statistical analysis

[0351] Statistical analysis was performed using GraphPad Prism (x64, v9.2.0) and unpaired t-test. α was set to 0.05, and the difference between the two experimental groups was considered statistically significant when *p<0.05, *p<0.05, **p<0.01, and ***p<0.005; ns represents “no significant difference” between the two groups.

[0352] Prior art documents cited:

[0353] et al., Advanced Healthcare Materials 19June 2021;

[0354] Guo et al.,ACS Appl.Mater.Interfaces 2021,13,6,7037–7050;

[0355] Michel et al., ACS Appl. Bio Mater. 2020, 3, 8, 5253–5262;

[0356] M.Veettikazhy et al.,Opt.Express 2021,29,11819;

[0357] M.Lee,et al.,Chem.Rev.2020,120,10950.

[0358] J.Madrid-Wolff,M.Forero-Shelton,Biophys.2020,1,10.

Claims

1. A method for preparing a three-dimensional hydrogel bioimplant, the method comprising: a. providing a first photo-crosslinkable composition in a container, the first composition comprising i. a first polymer that is sensitive to photocrosslinking, ii. Photoinitiator and iii. Optionally, the refractive index matching agent of the photosensitive resin formulation may be increased or decreased (e.g. up to 80% w / v, particularly 10% to 50% iodixanol) 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 cross-linking agent and b. in a first irradiation step, irradiating the composition with a plurality of spatially coherent light beams, Thereby a plurality of micro-pillars are generated in the composition. 2 . The method according to claim 1 , wherein the spatially coherent light beam is characterized by a diameter of 1 μm to 100 μm, in particular a diameter of 3 μm to 40 μm.

3. The method according to any one of claims 1 and 2, wherein a. the composition is illuminated by a first plurality of spatially coherent light beams directed in a first direction, and b. wherein in the second irradiation step, the composition is irradiated by a second plurality of spatially coherent light beams directed along a second direction. 4 . The method according to claim 3 , wherein the first direction and the second direction are arranged at an angle of 1° to 180°, in particular 15° to 180°.

5. A method according to any one of claims 3 or 4, wherein - the first plurality of spatially coherent light beams form a first pattern; - said second plurality of spatially coherent light beams form a second pattern; and - The first and / or the second pattern is arranged in cylindrical form, in sheet form, or as parallel lines between anchoring rib-like support sheets.

6. A method according to any one of the preceding claims, wherein after the first irradiation step, a second composition is added to the container, the second composition comprising i. a second polymer that is sensitive to photocrosslinking, ii. Photoinitiator and iii. Optionally, the refractive index matching agent (eg, up to 80% w / v iodixanol) of the photosensitive resin formulation may be increased or decreased; 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 cross-linking agent and In the third irradiation step or any subsequent irradiation step, the composition is irradiated with a plurality of spatially coherent light beams.

7. A method according to any of the preceding claims, wherein in the first, second, third or any subsequent irradiation step, irradiation is achieved onto the substrate from the top side or bottom side of the container; continuous irradiation is performed from the top of the container, or under continuous irradiation, the substrate moves upward away from the bottom of the container, thereby forming a plurality of microbeams protruding from the substrate, wherein the microbeams are distributed in the longitudinal axis over the entire length of the hydrogel structure.

8. A method according to any one of the preceding claims, wherein the photo-crosslinking sensitive polymer is a biopolymer functionalised with covalent attachment of a carbon-carbon double bond (ene) containing moiety or a methacrylate functionalised biopolymer.

9. The method according to claim 8, wherein the biopolymer is selected from the group consisting of gelatin, hyaluronic acid, alginate, collagen, fibrinogen, polyvinyl alcohol, chitosan, silk fibroin, cellulose.

10. The method according to claim 8 or 9, wherein the carbon-carbon double bond (ene) containing moiety is selected from the group consisting of at least norbornene carboxylic acids or dicarboxylic acids, methacrylates or amides, acrylates or amides, and vinyl esters.

11. The method according to any one of claims 8 to 10, wherein the photocrosslinking-sensitive polymer is norbornene-functionalized gelatin, characterized in that A degree of substitution of 10% to 90%, in particular a degree of substitution of 47% to 50%.

12. The method according to any one of claims 8 to 10, wherein the photocrosslinking-sensitive polymer is methacrylate-functionalized gelatin, characterized in that A degree of substitution of 10% to 90%, in particular 45-60%.

13. A method according to any preceding claim, wherein the spatially coherent light beam is characterised by a wavelength of 360 to 800 nm.

14. A method according to any preceding claim, wherein the spatially coherent light beam is characterised by a coherence length of 1 to 100 μm.

15. A method according to any preceding claim, wherein the spatially coherent beam is characterized by 10 to 5000 mJ / cm 2 , especially 90 to 200 mJ / cm 2 of light dose.

16. The method according to any of the preceding claims, wherein the spatially coherent light beam is characterized by a duration of 0.1 s to 100 s, in particular 1 to 4 s.

17. A method according to any preceding claim, wherein the spatially coherent light beam is characterized by 1 to 500 mW / cm 2 , especially 50 to 60 mW / cm 2 energy density.

18. A three-dimensional hydrogel implant comprising or consisting essentially of a first plurality of micropillars of a first photocrosslinked polymer, wherein said three-dimensional hydrogel implant is obtained by the method according to any one of the preceding claims.

19. The three-dimensional hydrogel bioimplant of claim 18, wherein the first plurality of micropillars is characterized in that each of the micropillars has a diameter ranging from 1 μm to 100 μm.

20. The three-dimensional hydrogel bioimplant of claim 18 or 19, wherein the first plurality of micropillars is characterized in that each of the microbunches has a diameter ranging from 2 μm to 30 μm.

21. A three-dimensional hydrogel bioimplant according to any one of claims 18 to 20, wherein >75% (particularly >80%, ≥85%, ≥90%, ≥95% or even ≥98%) of the plurality of microbeams have an orientation that deviates by ≤2° relative to the longitudinal axis.

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

23. The three-dimensional hydrogel bioimplant of any one of claims 18 to 21, wherein the micro-bundles extend throughout the entire length of the hydrogel construct in the longitudinal axis.

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

Images