Photo-erosion of hydrogels for guided growth of cell networks and organoids

Abstract

The invention relates to a method for making a micro-structured hydrogel, comprising the steps of: providing a hydrogel composition comprising a polymer crosslinked by linker moieties comprising a photolabile coumarin moiety, and subjecting the hydrogel composition to structured irradiation with light having a wavelength of ≥700 nm. The invention also relates to a micro-structured hydrogel composition, comprising a hydrogel crosslinked by moieties comprising a photolabile moiety susceptible to cleavage by two-photon irradiation of a wavelength of ≥700 nm, and channels or cavities having a diameter of ≤10 µm. In some embodiments, the hydrogel comprises a plurality of ellipsoid indentations (lacunae) on its surface, and a plurality of channels (canaliculi) departing from each lacuna.

Description

[0001] Photo-Erosion of Hydrogels for Guided Growth of Cell Networks and Organoids

[0002] This application claims the right of priority of EP24188492.3, submitted 13 July 2024, incorporated herein by reference.

[0003] Field

[0004] The present invention relates to a method for making a micro-structured hydrogel by two-photon erosion of microstructures in matrices crosslinked with photolabile linkers, using image-based direct laser writing process. The invention further relates to crosslinkers and the use of such microstructured hydrogels to guide cell network formation and organoid development for organ-on-chip applications provided by the method according to the invention.

[0005] Background

[0006] Molecularly engineered hydrogels are increasingly used in controlled drug delivery, organoid culture, and biomimetic tissue engineering. Among those, photoresponsive hydrogels are promising light-controlled tools since they allow spatial and temporal control of cell fates across different length scales by either single-photon (ultraviolet-visible light (UV-Vis): 300-500 nm) or two- photon (near-infrared laser pulses: 700-900 nm) irradiation. Compared to single-photon patterning, two-photon excitation offers deeper tissue penetration as well as better spatial resolution down to the sub-micrometre scale owing to its non-linear nature. Two-photon polymerization (2PP) has been extensively used for additive manufacturing of 3D micro-objects although direct laser writing via 2PP in the presence of living cells remains challenging due to the use of photoinitiators and resulting reactive oxygen species. Two-photon photodegradation represents a subtractive laser manufacturing process that induces localized gel erosion inside a 3D matrix. This process may be used to fabricate functional hollow structures, such as for directing the extension of motor neurons and vessel networks.

[0007] Photodegradable hydrogels predominantly relied on incorporated photolabile o-nitrobenzyl (oNB) ester derivatives. The speed of two-photon photodegradation of hydrogels is often limited (10-100 pm / s) by the small two-photon absorption cross section (5a) of oNB moieties 5a(<0.1 Goeppert- Mayer (GM), where 1 GM is 10-50 cm4s photon-1). To overcome this limitation, Lunzer et al. [ Angew. Chem. 2018, 130, 15342-15347] reported an elegant approach to enhance the efficiency of two-photon degradation of oNB hydrogels by adding an efficient two-photon sensitizer P2CK[10a] (5a: 176 GM at 800 nm). Very recently, Gehre et al. [Acta Biomater. 2024 174, 141 - 152] reported P2CK-sensitized two-photon ablation of microchannels to guide cell network formation in gelatin methacryloyl hydrogels.

[0008] Recent advances have witnessed the development of efficient chromophores with large 5asuitable for two-photon microprinting [Barner-Kowollik et al., Angew. Chem. Int. Ed. 2017, 56, 15828- 15845], Coumarin derivatives, an important class of photolabile molecules, [Azagarsamy and Anseth, Angew. Chem. 2013, 125, 14048-14052; Azagarsamy et al., ACS Macro Lett. 2014, 3, 515-519, Lin et al., Angew. Chem. Int. Ed. 2018, 57, 3722-3726] have been explored in the design of photocleavable two-photon initiators, hydrogels, phototriggers, [Bao et al., Org. Lett. 2011 , 14, 572-575] and imaging probes. Typically, expanding the ir-conjugation system has been an effective approach to enhance both the 5aand the photolysis rate of coumarin derivatives. Nevertheless, the synthesis of efficient photolabile coumarin linkers and hydrogels continues to be challenging, primarily due to the complexity of tedious multi-step reactions involved.

[0009] Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods facilitating the generation of microstructured hydrogels. Particular embodiments relate to the provision to such hydrogels of cell-guiding physical cues analogous to those present in biological tissues, particularly for generation of 3D tissue and organoid models with cell networks of pre-defined shape, cell culture and generation of implants. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

[0010] Summary of the Invention

[0011] A first aspect of the invention relates to a method for making a microstructured hydrogel. It entails the steps of: a. providing a hydrogel composition, the hydrogel composition comprising a hydrogel forming polymer crosslinked by linker moieties comprising a photolabile coumarin moiety; b. subjecting the hydrogel composition to structured irradiation with light having a wavelength of >700 nm.

[0012] An alternative of this first aspect of the invention relates to a method for making a microstructured hydrogel, comprising the steps of: a. providing a hydrogel composition, the hydrogel composition comprising a crosslinked hydrogel forming polymer susceptible to being sensitized by a photoinitiator; b. subjecting the hydrogel composition to structured irradiation with light having a wavelength of >700 nm.

[0013] Another aspect of the invention relates to a micro-structured hydrogel composition, comprising a hydrogel crosslinked by moieties comprising a photolabile moiety susceptible to cleavage by two- photon irradiation of a wavelength of >700 nm, and channels or cavities having a diameter of <10 pm.

[0014] The invention further provides a linker for crosslinking hydrogels that allows fast, two-photon erosion of hydrogels to obtain the micro-structured hydrogels of the present invention. Terms and definitions

[0015] General

[0016] 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 that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0017] The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of’ or “consisting of.”

[0018] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0019] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

[0020] As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.

[0021] "And / or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0023] Organic Chemistry

[0024] The formulae of the present specification follow the convention of organic chemistry to not show hydrogen atoms on carbon scaffolds. Carbon is tetravalent and bonds not shown are assumed to be hydrogen unless shown otherwise. Hydrogen can be exchanged for deuterium without changing the bulk chemical properties of the molecule; however, in the case of dye or drug molecules, the exchange of hydrogen for deuterium may lead to changes in the spectral properties or receptor interactions of the molecule. Unless explicitly stated otherwise herein, the disclosure of a formula showing, explicitly or implicitly by the convention restated in the first sentence of this paragraph, encompasses molecules in which one or several of the hydrogen atoms are exchanged for deuterium.

[0025] The term alkyl in the context of the present specification relates to a saturated linear, branched or (partially or completely) cyclic hydrocarbon, wherein in certain embodiments one carbon-carbon bond may be unsaturated and one CH2 moiety may be exchanged for oxygen (ether bridge) or nitrogen (NH, or NR with R being methyl, ethyl, or propyl; amino bridge). The term unsubstituted Cnalkyl when used herein in the narrowest sense relates to the moiety -CnH2n- if used as a bridge between moieties of the molecule, or -CnH2n+i if used in the context of a terminal moiety. It may still contain fewer H atoms if used in the context of a cyclical structure. In certain embodiments, alkyl is limited to linear alkyl.

[0026] The term C1-C4 alkyl in the context of the present specification relates to a saturated linear or branched hydrocarbon having 1 , 2, 3 or 4 carbon atoms. Non-limiting examples for a C1-C4 alkyl are methyl, ethyl, propyl, prop-2-enyl, n-butyl, 2-methylpropyl, tert-butyl, cyclo-butyl, cyclo-propyl, methyl-cyclo-propyl. In certain embodiments, a C1-C4 alkyl is a methyl, ethyl, propyl or butyl moiety.

[0027] Where used in the context of chemical formulae, the following abbreviations may be used: Me is methyl CH3, Et is ethyl -CH2CH3, Prop is propyl -(CH2)2CH3 (n-propyl, n-pr) or -CH(CH3)2 (isopropyl, i-pr), but is butyl -C4H9, -(CH2)3CH3, -CHCH3CH2CH3, -CH2CH(CH3)2 or -C(CH3)3.

[0028] The term substituted alkyl in its broadest sense refers to an alkyl as defined above in the broadest sense, which is covalently linked to an atom that is not carbon or hydrogen, particularly to an atom selected from N, O, F, B, Si, P, S, Cl, Br and I, which itself may be -if applicable- linked to one or several other atoms of this group, or to hydrogen, or to an unsaturated or saturated hydrocarbon (alkyl or aryl in their broadest sense). In a narrower sense, substituted alkyl refers to an alkyl as defined above in the broadest sense that is substituted in one or several carbon atoms by groups selected from amine NH2, alkylamine NHR, imide NH, alkylimide NR, amino(carboxyalkyl) NHCOR or NRCOR, hydroxyl OH, oxyalkyl OR, oxy(carboxyalkyl) OCOR, carbonyl O and its ketal or acetal (OR)2, nitril CN, isonitril NC, cyanate CNO, isocyanate NCO, thiocyanate CNS, isothiocyanate NCS, fluoride F, choride Cl, bromide Br, iodide I, phosphonate PO3H2, PO3R2, phosphate OPO3H2 and OPO3R2, sulfhydryl SH, suflalkyl SR, sulfoxide SOR, sulfonyl SO2R, sulfanylamide SO2NHR, sulfate SO3H and sulfate ester SO3R, with R being defined further in the description. In certain embodiments, R is itself an unsubstituted or substituted Ci to C12 alkyl in its broadest sense, and in a narrower sense, R is methyl, ethyl or propyl unless otherwise specified.

[0029] The term aryl in the context of the present specification relates to a cyclic aromatic C5-C10 hydrocarbon. Examples of aryl include, without being restricted to, phenyl and naphthyl.

[0030] In the context of the present specification, the term aryl encompasses hydrocarbon-based aryl and heteroaryl.

[0031] The term heteroaryl in the context of the present specification relates to a cyclic aromatic C2-C9 hydrocarbon that comprises at least one heteroatom (e.g. N, O, S). Examples for heteroaryl include, without being restricted to, pyrrole, thiophene, furan, imidazole, pyrazole, thiazole, oxazole, pyridine, pyrimidine, thiazin, quinoline, benzofuran and indole.

[0032] The term structured irradiation in the context of the present specification relates to light having a wavelength of >700 nm. In the examples, the inventors used a femtosecond laser operating at 700- 1200 nm and 50-100 MHz, with a pulse length of 50-100 fs. The skilled artisan will be able to derive other workable configurations based thereon.

[0033] The term micro-structured in the context of the present specification relates to structures smaller than 5 pm, particularly smaller than 3 pm. The smallest feature attained in the Examples is 0.9 pm; the inventors estimate that features of 200-300 nm size will be attainable using high numerical aperture (NA) objectives.

[0034] Any patent document cited herein shall be deemed incorporated by reference herein in its entirety.

[0035] Detailed Description of the Invention

[0036] One aspect of the invention relates to a method for making a micro-structured hydrogel. This method for making a micro-structured hydrogel according to the invention comprises the steps of: a. providing a hydrogel composition, the hydrogel composition comprising a hydrogel forming polymer crosslinked by linker moieties comprising a photolabile coumarin moiety; b. subjecting the hydrogel composition to structured irradiation with light having a wavelength of >700 nm.

[0037] Bone and tendon are examples of tissues where a 3D cell network, embedded in lacuno-canalicular network (LCN) cavities is important for physiological function. Another prominent example of tissue organization that could be favourably modelled with the means and methods provide herein is brain tissue. In certain embodiments, the micro-structured hydrogel is characterized by a network of interconnected voids or canals that resemble the canals present in bone. In other embodiments, the micro-structured hydrogel is characterized by a network of interconnected voids that resemble the extracellular space of 3D cell networks consisting of osteocytes, tenocytes and neurons found in bone, tendon and brain respectively. For bone and tendon, these voids are called LCN: an intricate network of fluid-filled tunnels (150-250 nm in diameter). More detailed description of the morphometric features of native osteocyte LCN is summarized in Buenzli and Sims (Bone 2015, 75, 144-50).

[0038] The term “structured irradiation” in this context refers to a laser beam having a wavelength of >700 nm and a focus of <20 pm; particularly a focus of <10 pm, more particularly a focus of <3 pm. One non-limiting example is irradiation provided by a femtosecond laser operating at 780 nm and 80 MHz, with a pulse length of 90 fs. Parameters that influence outcome include gel thickness, glass thickness, the "depth" in which the structures are patterned, working distance of the objective, objective NA, voxel size. The skilled artisan knows how chose these parameters to arrive at the microstructures of the invention.

[0039] The hydrogel forming polymer component:

[0040] A number of polymers offer themselves for practicing the invention. The general requirement is that the polymer may be modified to allow crosslinking of photolabile linker moieties.

[0041] Polyethylene glycol) (PEG) is a polymer often used for the preparation of hydrogels (see for example Kloxin et al. 2009, Science, vol 324, 59-63).

[0042] Polyvinyl alcohol (PVA) is a linear polymer and frequently used for the prepration of hydrogels. Hydrogels formed by norbornene-functionalized PVA (nPVA) has been reported (see Qin et al. 2018, Adv Mater, vol 30, 1705564).

[0043] Hyaluronate or hyaluronic acid (together abbreviated HA throughout the present specification), which is used in the Examples of the present specification, is of advantage because of its compatibility with cell culture and regulatory history in the context of implant production. HA is easily derivatized by covalent coupling of its carboxylic acid moieties, as evidenced in the Examples.

[0044] Another polymer lending itself to the practice of the invention is dextran (see Hiemstra et al., Biomacromolecules 2007, 8(5): 1548-56 for an example of a structure that is adaptable to the concepts of the present invention).

[0045] Yet another polymer useful in the practice of the invention is alginate (see Ooi et al., Biomacromolecules. 2018, 19(8): 3390-3400 for an example of a structure that is adaptable to the concepts of the present invention).

[0046] Also included in the group of polymers that may be employed in practicing the invention is gelatin (see Truong et al., Biomacromolecules 2015, 16 (7), 2246-53 for an example of a structure that is adaptable to the concepts of the present invention). In a particular set of embodiments, the polymer is hyaluronic acid (HA; see the Examples of the present specification).

[0047] In other particular embodiments, the polymer is based on a scaffold of poly (ethyleneglycol), polyvinyl alcohol, gelatin or dextran. Any such compound providing multiple reactive groups can be crosslinked by (thiol)-linkers based to function according to the invention.

[0048] The linker and photolabile moiety

[0049] One key feature that differentiates certain aspects disclosed herein from prior attempts to produce micro-structured hydrogels, is the nature of the photolabile moiety that allows a precise and fast erosion of the hydrogel. Previous approaches were limited to photo-ablation of methacrylate derivates of gelatin that used a soluble photosensitizer (see Gehre et al. Acta Biomaterialia 2024, vol 174: 141-152).

[0050] The present invention provides, in a first aspect, for gels having a photolabile moiety integrated into the gel structure that allows for two-photon erosion of the gel to create microstructures in the micrometre and sub-micrometre range. In the inventors’ practice, coumarin derivatives with increased capture coefficients S fortwo-photon absorption, relative to photolabile groups previously employed for analogous purposes, have proven of particular advantage in increasing the printing speed and decreasing the size of attainable detail to arrive at structures that mimic bone morphology.

[0051] In certain embodiments, the hydrogel is generated by reacting a polymer having a first reactive moiety with a photocleavable linker of the general structure of formula (I): wherein n is an integer from 1 to 150, R is a moiety comprising a coumarin ring and R1is a second reactive moiety capable of creating a covalent link to the first reactive moiety under aqueous conditions at neutral pH (6.5 to 8.0).

[0052] With regard to the size of the photocleavable linker, the inventors used a 5000 Da PEG derivate in their Examples; this corresponds roughly to n=110. Other PEG structures, such as 4-arm or 8-arm PEG, are expected to work similarly. The skilled artisan is aware that depending on the amount of linker used, relative to other gel-forming polymer components, both size and amount of the linker can vary across a range that allows to attain the general objective, which is a gel that is stable, yet can be eroded to yield microstructures resembling the native architecture in bone or other highly structured organs. Moieties R1for coupling the linker to the polymer component:

[0053] R1in (I) can be adapted to suit the polymer that is to be crosslinked. The Examples use an ethylene moiety CH2=CH (acrylates, methacrylates, vinyl sufones, vinyl esters, vinyl carbonates, vinyl carbamates), thus offering the linker to Michael-addition type crosslinking with thiol-functionalized HA (HA-SH), through the carboxylic acid groups comprised in the HA, with a thiol-containing moiety (see “Synthesis of HA-SH, Examples).

[0054] Other options for R1in (I) include variations of the ethylene to render other acrylic acid derivatives, thus R1may be, for example, CH3HC=CH, CH3CH2HC=CH, and the like.

[0055] In other embodiments, R1may be an hydroxy(N)succinimide moiety, making the linker reactive to amino groups of a base polymer. In yet other embodiments, R1may comprise a terminal maleimide moiety for conjugation to thiol groups.

[0056] In particular embodiments, the photocleavable linker is described by general formula (la):

[0057] The photolabile moiety R renders the linker susceptible to degradation by two-photon absorption. Typical capture coefficients S for two-photon absorption, working for the present invention are in the range of 1 -1000 GM at 800 nm.

[0058] The photolabile moiety R is a conjugated coumarin

[0059] In certain embodiments, R is a conjugated coumarin derivate of general structure (II): wherein L is the bond to the linker, and Rccomprises at least one double bond conjugated to the Ti electron system of the coumarin scaffold of (II). Rcmay comprise an aryl or heteroaryl moiety having a K electron system in conjugation with the K electron system of the coumarin scaffold of (II). Furthermore, Rcmay comprise, in addition or in lieu of the aryl or heteroaryl system, an oxygen or nitrogen group, particularly an ORNand NRN2 moiety where RNis a lower alkyl, such as Me or Et.

[0060] In a more limited set of embodiments described by general structure, R is described by the general structure (Ila):

[0061] In particular embodiments, and Rcis selected from (CH=CH)P-X and (CH=CH)n-Ay-Z wherein p is an integer selected from 0, 1 , 2 and 3, Ay is an aryl or heteroaryl, and Z is selected from ORNand NRN2, with each RNindividually being selected from H, C1-C4 alkyl.

[0062] In more particular embodiments described by general structure II or Ila, Rcis (CH=CH)P-NRN2 with RNbeing methyl or ethyl, and p being 1 or 2.

[0063] In other more particular embodiments described by general structure, Rcis (CH=CH)n-Ay-Z with p being 0, 1 or 2, Ay being phenyl or halogen-monosubstituted phenyl, and Z being selected from the group of ORNand NRN2 , with each RNindividually being selected from H, C1-C4 alkyl.

[0064] In other more particular embodiments described by general structure, Rcis (C^C)-Ay-Z with Ay being phenyl or halogen-monosubstituted phenyl, and Z being selected from the group of ORNand NRN2 , with each RNindividually being selected from H, C1-C4 alkyl. C=C) relates to an ethynyl moiety, i.e. a carbon-carbon triple bond.

[0065] In particular embodiments, R is described by general structure (lib):

[0066] In particular embodiments of (lib), Z is bismethylamino-4-phenyl. In other particular embodiments of (lib), Z is bisethylamino-4-phenyl. In yet other particular embodiments of (lib), Z is methoxylphenyl.

[0067] Other options for R include the following:

[0068]

[0069] The advantages of long-conjugated coumarin include a high two-photon absorption cross-section (ca. 100-300 GM at 800 nm), and thus higher efficiency for photodegradation at low laser dosage, offering more cell-compatible conditions for direct laser bioprinting in the presence of cells.

[0070] In one particular embodiment, the linker employed to crosslink SH-modified HA is: wherein n is an integer from 1 to 150. R’ is a second reactive moiety capable of creating a covalent link to the first reactive moiety present in the polymer, under aqueous conditions at neutral pH (6.5 to 8.0).

[0071] Where Rccreates issues of solubility or miscibility in water, it may be of advantage to add a component to the composition alleviating such issues. The inventors have found that adding cyclodextrin to the composition alleviated issues observed when using long conjugated coumarins. In their compositions, the inventors used gamma cyclodextrin. In particular embodiments, the ratio of cyclodextrin to photocleavable linker is selected from 1 :0.2 to 1 :5.0, particularly from 1 :0.5 to 1 :2.0. Other workable ratios of cyclodextrin to photocleavable linker may be selected from the following: 1 :0.2 to 1 :1 ; 1 :0.2 to 1 :0.8; 1 :0.8 to 1 :1.2; 1 :0.8 to 1 : 2.0.

[0072] Concentrations of polymer in the composition

[0073] Generally, there is no specific restriction on the concentration of polymer, except that the gel should be made to fit the general purpose for which it is intended.

[0074] In particular embodiments, the polymer concentration in the composition ranges from 1% to 10% (w / v). In more articular embodiments, the polymer concentration in the composition ranges from 2.5% to 8% (w / v).

[0075] In particular embodiments, the hydrogel composition does not comprise a soluble photosensitizer agent.

[0076] In other particular embodiments, a soluble photosensitizer agent is present in the composition, particularly at a concentration of 0.1 mmol / L-5 mmol / L.

[0077] Ablation of hydrogels by photosensitizer

[0078] A method for making a micro-structured hydrogel, comprising the steps of: c. providing a hydrogel composition, the hydrogel composition comprising a crosslinked hydrogel forming polymer susceptible to being sensitized by a soluble photoinitiator; d. subjecting the hydrogel composition to structured irradiation with light having a wavelength of >700 nm.

[0079] Cell containing hydrogels, systems and organoids

[0080] Among the outstanding advantages of the hydrogels provided by the present invention is their suitability to accommodate live mammalian cells, for example by means of top-seeding or 3D embedding. The cells may be present at the time of the hydrogels being structured by two-photon laser erosion in situ, or may be seeded onto or into the micro-structured hydrogels.

[0081] The inventors’ initial cell experiments focused on guiding cells seeded on patterned substrates for establishing the principle, however the final objective of this work will be to translate the same method also to cell-containing gels for 3D patterning of guidance cues to direct organoid formation. The inventors have already shown this using cell clusters as well as using single cells. The lability to erosion by two-photon degradation is a result entirely of the photolabile group comprised in the crosslinker, i.e. it is part of the hydrogel structure prior to irradiation. The inventors have shown that combining other types of hydrogels without photolabile moities with two-photon ablation with higher level of laser dose allows subtractive hydrogel structuring, where two-photon ionization produces sufficient electron excitation to cause direct ablation of the hydrogel.

[0082] Live mammalian cells that can be accommodated by the hydrogel compositions of the present invention include, but are not limited to, fibroblasts, osteoblasts, stem cells, osteocytes, neurons, and tenocytes.

[0083] Peptide modification of the hydrogel structure:

[0084] Several peptide sequences are known to facilitate the attachment of specific cells to biomaterials and implants. An RGD peptide useful for cellular attachment in biomaterials is a short chain of amino acids that includes the specific sequence Arg-Gly-Asp (R-G-D). This sequence is recognized by cell surface receptors called integrins, which are proteins that facilitate cell adhesion to the extracellular matrix and to biomaterial surfaces. Integrins bind to the RGD sequence, promoting cell attachment, spreading, and signalling. This interaction is crucial for various cellular processes, including cell migration, proliferation, and differentiation, making RGD peptides essential components in the design of biomaterials for tissue engineering, wound healing, and regenerative medicine.

[0085] Other notable examples of peptides guiding cellular attachment in biomaterials include:

[0086] RGDS (Arg-Gly-Asp-Ser; SEQ ID NO: 002): This is a modified version of the RGD sequence that can enhance cell adhesion.

[0087] YIGSR (Tyr-lle-Gly-Ser-Arg; SEQ ID NO: 003): This peptide sequence from laminin promotes adhesion and migration of endothelial cells and some types of epithelial cells.

[0088] IKVAV (lle-Lys-Val-Ala-Val; SEQ ID NO: 004): Another laminin-derived sequence, IKVAV, promotes neuronal adhesion and neurite outgrowth, making it particularly useful for neural tissue engineering.

[0089] PHSRN (Pro-His-Ser-Arg-Asn; SEQ ID NO: 005): This sequence from fibronectin works synergistically with the RGD sequence to enhance cell adhesion and spreading.

[0090] REDV (Arg-Glu-Asp-Val; SEQ ID NO: 006): Derived from fibronectin, REDV specifically promotes the adhesion of endothelial cells, making it useful for vascular tissue engineering.

[0091] DGEA (Asp-Gly-Glu-Ala; SEQ ID NO: 007): This collagen-derived peptide sequence promotes the adhesion of osteoblasts and is often used in bone tissue engineering. QGDV (Gln-Gly-Asp-Val; SEQ ID NO: 008): A variant used for promoting adhesion of smooth muscle cells.

[0092] These peptide sequences can be incorporated into biomaterials via covalent bonds to enhance their biocompatibility and functionality by guiding the attachment and behaviour of specific cell types, thus optimizing the performance of implants and tissue-engineered constructs.

[0093] The sequence of the RGD peptide used in the examples is CGRGDSP (SEQ ID NO: 001), which contains one thiol group. The thiol group can easily react with the acrylate groups of photo-linker at 37°C by Michael addition reaction to be grafted into the gel network.

[0094] In a particular set of embodiments, the peptide comprises an RGD peptide of SEQ ID NO: 001 .

[0095] In particular embodiments, cell adhesive peptides (e.g. SEQ ID NO: 001) is immobilized to the micro-structured hydrogel matrices at a concentration of 0.05-5 mmol / L and particularly 0.2- 2 mmol / L.

[0096] In other embodiments, cell adhesive peptides can be site-specifically patterned to the microstructured hydrogel niche to achieve spatially controlled cell attachment. Photo-induced conjugation such as radical-mediated thiol-ene reaction can be applied (see Qin et al. 2018, 30, 1705564).

[0097] Micro-structured hydrogels obtained by the methods of the present invention

[0098] Another aspect of the invention relates to a micro-structured hydrogel composition, comprising a hydrogel crosslinked by moieties comprising a photolabile moiety susceptible to cleavage by two- photon irradiation of a wavelength of >700 nm, and channels or cavities having a diameter of <10 pm.

[0099] In particular embodiments, the channels or cavities are characterized by a diameter of <5 pm. In more particular embodiments, the channels or cavities are characterized by a diameter of <3 pm.

[0100] In particular embodiments of the micro-structured hydrogel composition according to invention, the hydrogel is HA. The hyaluronic acid may be modified by attaching a thiol-bearing linker through amidation of the HA carboxylic acid moiety, which is capable of crosslinking through Michael addition of a linker moiety as described above.

[0101] Another aspect of the invention relates linker for crosslinking a polymer, characterized by the general formula: wherein

[0102] R1is selected from CH2=CH, CH3HC=CH, CH3CH2HC=CH and CH2=C(CH3), and R is described by (Ila): wherein Rcis selected from (CH=CH)P-X and (CH=CH)n-Ay-Z, with p being selected from 0, 1 , 2 and 3, Ay being aryl or heteroaryl, and Z being selected from ORNand NRN2, with each RNindividually being selected from H, C1-C4 alkyl.

[0103] Image-based micro-structuring of hydrogels

[0104] The microstructure is in accordance with computer-aided design (CAD) models of imaging data of native tissues, such as from focused ion beam scanning electron microscopy (FIB-SEM) with resolution of 5-30 nm, confocal laser scanning microscopy (cLSM) with resolution of 100-200 nm, and Ptychographic X-ray CT with resolution of <100 nm.

[0105] One application of the compositions and methods provided herein is to make image-based lacuno- canalicular network (LCN) models for printing osteocyte-specific morphologies and functions for bone organoids. The advantages of these models allow independent control of topological features of lacunae and canaliculi such as lacunar shape (round vs ellipsoid), canalicular density (i.e., number of canaliculi per cell), cell-to-cell spacing, and network connectivity to grow 3D cell networks of prescribed shape, recapitulating healthy / aged tissue environments.

[0106] Application of the image-based two-photon patterning as provided herein provides the structural complexity observed in tissues such as bone LCN, in artificial models.

[0107] Using image-based computer models, the methods and compositions according to the invention provide access to microprinted cell networks (artificial lacuno-canalicular networks) which enable user-defined construction of cell networks and (bone) tissue niches. The inventors foresee great potential for translation and commercialization.

[0108] Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for a polymer may be combined with any of the alternative embodiments of photolabile moiety and these combinations may be combined with any R’ mentioned herein.

[0109] The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope. Description of the Figures

[0110] Fig. 1 shows (a) Schematic of the synthetic strategy for photolabile linkers and hydrogels: multicomponent reaction (MCR) via the incorporation of bis-isocyanide PEG precursor, an aldehyde-bearing chromophore and a carboxylic acid component. Chemical structures of the photocleavable moieties (R) such as nitrobenzyl (NB), coumarin (C) and long-conjugated coumarin (LC) as well as end groups (R’) such as acrylate and maleimide for thiol-Michael click reaction, (b) Schematic of hydrogel formation by thiol-Michael reaction and subsequent photocleavage under two- photon excitation.

[0111] Fig. 2 shows (a) UV-vis spectra of three photolabile linkers (PEG-NB-A, PEG-C-A, PEG- LC-A). Single-photon absorption at 390 nm, corresponding to half of the wavelength for two-photon lithography, is marked with a dash line, (b) Photodegradation of PEG-NB-A and PEG-C-A hydrogels under irradiation of 365 nm at 50 mW / cm2: The influence of irradiation time on G’ (normalized to G’O) was monitored by photorheology. (c) The influence of light intensity on single-photon photodegradation in a PEG-C-A hydrogel, (d) Single-photon photodegradation of PEG-C-A hydrogels by either continuous (black line) or periodic irradiation (blue line) with 365 nm at 25 mW / cm2. The light was shut down at the period of I, II, III, IV.

[0112] Fig. 3 shows (a) Schematic of two-photon subtractive patterning of microchannels in a preformed hydrogel at 780 nm and visualization of microchannels by soaking the samples in a solution of FITC-dextran (Mw ~ 2000 kDa). (b) Confocal images of microchannels fabricated with varying laser power at constant speed of 300 mm / s in three different hydrogels derived from acrylated linkers (PEG-NB-A, PEG-C-A and PEG-LC-A). Scale bar, 50 pm. (c) Screening of the smallest feature size in a PEG-LC-A gel. A custom model with different cross-section size was used (left) and confocal image (right) after staining with FITC-dextran. (d) A complex pattern was fabricated with laser power of 70 mW and speed of 300 mm / s. Scale bar, 20 pm. (e) Confocal images (right) of the FITC-dextran stained sample and model of a bone-mimicking microfluidic network (left bottom). Scale bar, 50 pm. (f) Florescence recovery after photobleaching (FRAP) validation of the void space: Confocal images before bleaching (t = 0 s), just after bleaching (t = 35 s), and after florescence recovery (t = 120 s). Scale bars, 20 pm. (g) Recovery of the fluorescence intensity after bleaching. Control: unbleached region. Bleached: bleached region marked with dashed circle.

[0113] Fig. 4 shows (a) Cell viability exposed to different photolabile linker solutions for 24 h examined by MTS cell proliferation assay. Data presented as mean ± S.D. Statistical analysis was evaluated with one-way ANOVA. ns, not significant (n > 3). (b) Bright-field image and confocal image of live-dead stained HDF spheroids. The white dash line in the right image presents the printed microchannels. Scale bar, 100 pm. (c) Laser-guided cell outgrowth with varying laser power: the schematic (left) and microscopy images of the sample after 16 h (middle) and 40 h (right) of cell culture. Scale bar, 50 pm.

[0114] Fig. 5 shows chemical structures of all photolabile linkers (PEG-C-A, PEG-C-M, PEG-NB- A, PEG-LC-A).

[0115] Fig. 6 shows (a) Chemical structure of y-cyclodextrin (r-CD) and the schematic of the interaction between y-cyclodextrin (y-CD) and long-conjugated coumarin, (b) Rheology plot (Elastic modulus - G’) of a PEG-LC-A hydrogel supplemented with y-CD.

[0116] Fig. 7 shows CAD-models with lacunae and canaliculi dimensions, (a) Bottom view of a semisynthetic lacuna with varying lengths on the XY plane. While an ellipsoid shape is preferable to model the bone cell environment, its dimensions can be altered to mimic various conditions. The lacuna shell thickness can be adapted to accommodate for differing cell sizes and printing resolution, (b) Side view of a semisynthetic lacuna across the XZ plane. Dendrite shape and thickness depend on the desired model and available printer resolution, (c) Lacuna depicted with increasing number of dendrites (25, 35, and 50 dendrites per lacuna) to model disease and aging phenotypes. Orientation and position of the dendrites allow for the creation of a seamless array of connected lacunae.

[0117] Fig. 8 shows the work-flow of biomimetic two-photon printing of bone niches. A photosensitive hydrogel is integrated with a two-photon printer or similar (1). A tightly focused NIR laser enables subtractive printing of sub-micron tunnels (e.g. LCN) on the hydrogel. By seeding cells atop these substrates (2), the LCN cues physically guide cells to form a cell network with native-like morphologies (3) followed by differentiation (4).

[0118] Examples

[0119] Example 1:

[0120] The inventors present a modular approach to prepare a set of coumarin-based photodegradable hydrogels (Figure 1) using a one-pot Passerini multicomponent reaction (MCR). Compared to conventional photolabile oNB hydrogels, the reported coumarin-based photolabile linkers and hydrogels exhibit much higher efficiency for two-photon degradation at 780 nm and thus enabled fast subtractive two-photon 3D microprinting at speeds up to 300 mm / s and at low laser power down to 10 mW. Accordingly, complex microfluidic networks that mimic bone microarchitecture were rapidly fabricated within 2 min. Additionally, the cell-compatibility of these hydrogels and the feasibility of laser-guided cell migration in cell culture were studied. The use of Passerini MCR as shown in Figure 1a could help overcome the current challenges in developing efficient two-photon photolabile hydrogels in a modular fashion. In MCR, a carboxylic acid group, an aldehyde group, and an isocyanide react to form an a-acyloxyamide, which could assemble different functions into one linker in a one-pot procedure. Importantly, the aldehyde substrate determines the photolysis of the linker. Thanks to its mild reaction conditions and high efficiency, MCR has been widely used in the preparation of polymers, bioactive molecules, and photocaged compounds. However, so far, only a single study by Truong et al. (ACS Appl. Mater. Interfaces 2017, 9, 32441-32445) applied MCR to prepare photolabile hydrogels for selective cell release under UV-Vis light.

[0121] Four PEG-based photolabile linkers: nitrobenzyl-modified PEG acrylate (PEG-NB-A), coumarin- modified PEG-acrylate / maleimide (PEG-C-A / PEG-C-M), long-conjugated PEG-acrylate (PEG-LC- A) were synthesized by reacting PEG bis-isocyanides with respective aldehyde and carboxylic acid components, respectively (Fig. 5). The PEG bis-isocyanides precursor was prepared through the amidation of PEG diamine followed by hydrolysis. Three aldehyde-modified photolabile chromophores (nitrobenzyl (NB), 7-diethylcoumarin (C) and ir-conjugated coumarin (LC)) were selected to tune the photochemical properties of the corresponding photolabile linkers. Except for the LC aldehyde, which needs four synthetic steps, all carboxylic acid components with pendant acrylate / maleimide groups are commercially available. The chemical structures of the synthesized linkers were confirmed by1H-NMR.

[0122] The UV-vis absorbance spectra of linkers was measured in phosphate-buffered saline (1x PBS, pH 7.4). PEG-NB-A exhibits negligible absorption at the wavelength above 350 nm, whereas PEG- C-A and PEG-LC-A display a maximum absorption at 403 nm and 395 nm, respectively. Notably, the one-photon absorption at ~390 nm correlates with two-photon absorption at 780 nm (approximately twice wavelength). To form a hydrogel, thiol-functionalized hyaluronic acid (HA-SH) was introduced to react with PEG linkers via thiol-Michael addition (Lunzer et al., Angewandte Chemie 2018, 57, 15122-27). Gel precursor solutions were prepared by mixing stock solutions of acrylated PEG linkers (15%, w / v) and HA-SH (5%, w / v) in PBS at a thiokene stoichiometric ratio of 9:10. Stable and transparent gels were obtained after 1 h crosslinking at 37°C.

[0123] The kinetics of gel formation and subsequent single-photon photodegradation of various formulations were evaluated using in-situ photo-rheology. Notably, the gelation of a mix containing coumarin-functionalized PEG maleimides (PEG-C-M) and HA-SH was too fast (< 5 seconds) to be suitable for cell culture applications. The inventors thus only used acrylate-bearing coumarin PEG linkers in the following study. The storage moduli (G’) of PEG-C-A and PEG-NB-A hydrogels shows no significant difference after 1 h of crosslinking, indicating that the variations in chromophores do not influence the crosslinking process. Notably, initial attempts to form PEG-LC-A hydrogels were unsuccessful. This formulation took much longer time to reach the onset of gelation and exhibited lower reactivity compared to the others. Besides, remarkable structural defects were observed in this hydrogel, presumably due to the hydrophobic nature of the long-conjugated PEG-LC-A linker upon dispersion in water. To address this issue, we hypothesized that adding cyclodextrin (CD) might facilitate host-gust interactions between the linker and CD, thereby weakening the TT-TT stacking of the long-conjugated coumarin moieties. The PEG-LC-A linker was dissolved in a y-CD solution at a 1 :1 molar ratio. The in-situ rheology measurement indicated that the G’ reached approximately 8 kPa after 1 h crosslinking, which is comparable to other groups (Fig. 6). Importantly, the structural defects disappeared after the inclusion of y-CD. Subsequently, the photodegradation of hydrogels under 365 nm irradiation was assessed by photo-rheology. The PEG-C-A hydrogel completely degraded after 25 min, whereas the PEG-NB-A hydrogel needed almost 150 min to reach >90% degradation (Fig. 2b). This difference is consistent with previous findings.

[0020] As expected, reducing the light intensity slows down the photodegradation process as reflected by the increased time to reach complete gel erosion (Fig. 2c). Figure 2d shows that the process of photodegradation is tightly controlled by light. When the irradiation is ceased, the degradation is arrested; degradation is recommenced upon further irradiation. Surprisingly, the PEG-LC-A hydrogel cannot degrade under 365 nm nor 405 nm light. The inertness of PEG-LC-A hydrogel to UV and visible light allows it to be safely handled without the need for special protection.

[0124] Next, we investigated the printability of different hydrogels in terms of efficiency for two-photon degradation at 780 nm. A commercial two-photon printing system (NanoOne 1000) with a femtosecond-pulsed NIR laser (780 nm) was employed. Leveraging the printing accuracy and speed, a 20x objective was chosen. To facilitate tracer diffusion and visualization of microprinted hollow structures, we first created a rectangle access block (x-y-z: 600 pm x 200 pm x 100 pm) at the edge of preformed hydrogels with high laser power (150 mW) and speed of 300 mm / s. Then, arrays of rectangular microchannels (x-y-z: 200 pm x 20 pm x 20 pm) were printed at the border of the access block with varying laser power (10-100 mW) but constant scanning speed of 300 mm / s (Fig. 3a). Note that this speed is 2-3 orders of magnitude higher than the speed of conventional hydrogels (< 0.1 mm / s) reported for two-photon patterning.

[0125] The fidelity of microchannels was examined by incubating the samples in a solution of fluorescein- modified dextran (FITC-dextran) with an average molecular weight of 2000 kDa (Fig. 3a). Owing to its large hydrodynamic size, FITC-dextran can only diffuse into the printed hollow microchannels, but not into the bulk matrix. Therefore, the fidelity of microchannels was visualized via the fluorescence of FITC-dextran. Notably, applying vacuum during the incubation step with FITC- dextran greatly facilitated the staining process. By this means, the threshold laser power required for two-photon photodegradation was determined. As shown in Figure 3b, there are no visible microchannels in the PEG-NB-A hydrogel due to its poor efficiency for photodegradation. By contrast, well-defined microchannels were observed in the coumarin-based hydrogels (PEG-C-A and PEG-LC-A). For printing at 300 mm / s, the threshold power for the PEG-LC-A hydrogel is as low as 30 mW, while the PEG-C-A hydrogel requires 60 mW. Nevertheless, this gap decreased when the scanning speed was reduced to 10 mm / s. At this speed, the PEG-LC-A remains printable with a threshold power as low as 10 mW, while PEG-C-A is not printable at such low power levels. Compared to P2CK-sensitized PEG-NB-A hydrogels, our coumarin hydrogels exhibit higher efficiency for two-photon degradation. This allows faster printing at lower laser power, which is desirable for biofabrication. These findings stress the importance of incorporating efficient photolabile chromophores for two-photon photodegradation of hydrogels at high writing speeds.

[0126] To test the feasibility of printing even smaller features, a custom array of microchannels with decreasing width from 20 pm to 1 pm was fabricated using the PEG-LC-A hydrogels (Fig. 3c). The results suggest that microchannels of 1 pm can be printed with high scanning speed. Further, high spatial resolution of two-photon subtractive microprinting allows the creation of highly complex patterns with laser power of 70 mW and high scanning speed at 300 mm / s (Fig.3d). For potential applications in bone tissue engineering, we implemented a custom image-based computer model mimicking the lacuno-canalicular network in bone. We successfully fabricated a microarray of this model in the PEG-C-A hydrogels. Figure 3e depicts the confocal image of FITC-dextran stained LCNs following subtractive two-photon microprinting. Furthermore, the permeability of the printed void space to FITC-dextran tracers was confirmed by a fluorescence recovery after photobleaching (FRAP) experiment (Fig. 3f-g).

[0127] Next, the biocompatibility of the photolabile linkers was assessed using a MTS cell proliferation assay (Fig. 4a). The results suggest that these linkers are essentially non-cytotoxic. Considering the versatility of PEG-C-A hydrogel in both single-photon and two-photon applications, we employed this group for proof-of-concept laser-guided cell migration experiments. Spheroids of human dermal fibroblasts (HDF) were prepared and encapsulated in a cell adhesive hydrogel, which was functionalized with fibronectin-derived RGD peptide motifs. After incubation in cell culture medium for 3 hours, simplified microchannels (200 pm x 20 pm x 40 pm) were printed in close proximity to the edge of spheroids at different laser power ranging from 30 mW to 100 mW (Fig. 4b). Live-dead staining evidenced that both the procedure of in situ crosslinking and subtractive two-photon microprinting processes are cell-compatible (Fig. 4b). Importantly, cell death is negligible in the samples (Fig.4b). Over time, cells migrate into the microchannels printed at a laser power of 60 mW and above (Fig. 4c). This suggests that under these conditions the photo-eroded matrices are sufficiently permissive to support cell migration. This is consistent with the print fidelity analysis (Fig. 3b): the FITC-dextran tracers only permeate the channels at a laser power of 60 mW and above. After 40 h, cells almost filled up the void space in the microchannels (Fig. 4c). In addition to these straight channels, a branch channel was produced and connected to the spheroid. After printing and cultivation for 40 h, cells could spread into the branch channels.

[0128] We then investigated the mechanism of cell migration in photodegradable hydrogels. Qin et al. (Adv. Mater. 2018, 30) reported that 3D migration of HDF relies on both matrix degradability to matrix metalloproteinases (MMP) and the presence of cell adhesive arginyl-glycyl-aspartic acid (RGD) motifs in a synthetic MMP-sensitive polyvinyl alcohol hydrogel. Guided 3D cell migration was achieved by site-specific photografting of a thiolated peptide (CGRGDSP; SEQ ID NO: 001). Here, a control experiment shows that cell migration in our photodegradable hydrogels (no RGD) was prohibited, suggesting the necessity of RGD adhesive motifs in laser-guided migration.

[0129] In summary, we developed efficient coumarin-based photodegradable hydrogels that allow fast subtractive two-photon microprinting at 780 nm in the presence of cells. By using the Passerini MCR, we demonstrate a modular approach to introduce coumarin-based chromophores with high efficiency for photolysis for the preparation of two-photon photolabile hydrogels. The established hydrogels are suitable for complex patterning and enable biocompatible laser-guided cell growth and migration. We anticipate that these hydrogels will open new avenues in a few exciting bioapplications, such as spatiotemporal delivery of therapeutics under two-photon excitation and on- demand control of organoid development.

[0130] Example 2: Materials and Methods:

[0131] Synthesis of aldehyde-modified chromophores

[0132] 7-(diethylamino)-2-oxo-2H-chromene-4-carbaldehyde.

[0133] 7-diethylamino-4-methylcoumarin (2.03 g, 8.78 mmol) and selenium dioxide SeO2 (1.89 g, 17.03 mmol) were mixed in p-xylene (30 mL). The mixture was heated to reflux for 12 h. After cooling the reaction to room temperature, the solution was filtrated and evaporated in vacuum. Then the crude product was purified using chromatography (petroleum ether / ethyl acetate, 5:1) yielding 430 mg dark red solid (yield 20%).1H NMR (400 MHz, CDCI3) 5 10.05 (s, 1 H), 8.32 (d, J = 9.2 Hz, 1 H), 6.65 (dd, J = 9.2, 2.6 Hz, 1 H), 6.54 (d, J = 2.6 Hz, 1 H), 6.47 (s, 1 H), 3.44 (q, J = 7.2 Hz, 4H), 1 .24 (t, J = 7.1 Hz, 6H).

[0134] The aldehyde-modified long conjugated coumarin, 7-((4-(dimethylamino)phenyl)ethynyl)-2-oxo- 2H-chromene-4-carbaldehyde (LC), was synthesized based on our published procedures. (Qiu et al., 2019) Compound 3 (400.2 mg, 1 .33 mmol), Pd(PPh3)2CI2 (11 .6 mg, 0.016 mmol), Cui (7.3 mg, 0.038 mmol) and 4-ethynyl-N,N-dimethylaniline (250.5 mg, 1.73 mmol) were dissolved in 8 mL of degassed dry THF. 2 mL of degassed dry triethanolamine was then added. The reaction mixture was stirred at 53°C for 5 h. After cooling to room temperature, the mixture then poured into water and extracted with dichloromethane (5 x 15 mL). The combined organic layers were dried with Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, PE: CH2CI2 = 1 :3), giving 245 mg of LC as a red solid (yield: 57%).1H NMR (400 MHz, CDCI3) 5 10.14 (s, 1 H), 8.54 (d, J = 8.3 Hz, 1 H), 7.50 - 7.44 (m, 4H), 6.86 (s, 1 H), 6.72 - 6.68 (m, 2H), 3.05 (s, 6H). 13C NMR (101 MHz, CDCI3) 5 191.26, 160.17, 154.35, 150.61 , 143.37, 133.19, 129.27, 127.98, 125.99, 124.75, 118.95, 113.67, 111.77, 96.12, 86.54, 40.14.

[0135] Synthesis of isocyanide functionalized PEG

[0136] Amine difunctionalized PEG (2 g, 0.4 mmol, 5000 Da, Laysan Bio Inc. USA) was dissolved in ethyl formate (18 mL) and the solution was heated under refluxing for 48 h. Excessive ethyl formate was removed under reduced pressure, and the amide product (PEG-2NHCHO) was obtained. Next, the intermediate product (1 .2 g, 0.24 mmol) and triethylamine (TEA, 1 .2 mL, 8.66 mmol) were dissolved in anhydrous dichloromethane (6 mL) and cooled under an ice-bath. The phosphoryl chloride (POCI3, 0.36 mL, 3.88 mmol) in 6 mL of dichloromethane was added dropwise. Afterwards, the reaction mixture was warmed to room temperature and stirred for 4 h. The reaction was quenched by adding K2CO3 (20% w / w, 4 mL). The aqueous layer was extracted with dichloromethane (3 x 10 mL). The combined organic phase was washed with saturated NaCI (2 x 15 mL), and dried over Na2SC>4, and concentrated with rotary evaporator. The concentrated solution was precipitated into the cold diethyl ether. The precipitate was centrifuged and dried in vacuum. Finally, the isocyanide product (PEG-2NC) was obtained.

[0137] General procedure of the Passerini reaction

[0138] Aldehyde (0.15 mmol), carboxylic acid (0.15 mmol) and PEG isocyanate (0.036 mmol) were added into dichloromethane (1 mL). The resulting solution was stirred for 72 h at room temperature. The reaction mixture was precipitated into the cold diethyl ether. The precipitate was centrifuged, and washed for five times with cold diethyl ether, and dried in vacuum. The degree of substitution of all designed products is above 90% based on1H NMR data.

[0139] Synthesis of HA-SH

[0140] Hyaluronic acid (HA, 320 mg, Mw 50 kDa, Lifecore Biomedical) was dissolved in MilliQ water at a concentration of 8 mg / mL. Dihydrazide linker was added to the HA solution at a reagent to HA disaccharide molar ratio of 3:10. N-hydroxybenzotriazole (HOBt) was separately dissolved in a 1 :1 (v / v) mixture of acetonitrile-water at a concentration of 0.16 M and added to the solution of HA at a molar ratio of HOBt to HA disaccharide of 1 :1. The resultant solution was adjusted to 4.7 after which the coupling reaction was initiated by addition of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 0.5 molar equivalents per HA disaccharide units) to the reaction mixture. The reactant solution was stirred for 24 h, and then was basified to pH 8.5. DL-Dithiothreitol (DTT, 680 mg) was added to the above solution to cleave the disulfide bonds. The mixture was stirred for another 24 h. The solution then was transferred to a dialysis tube (Mw cutoff = 3.5 kDa) and dialyzed against dilute HCI (pH 3.5) containing 0.1 M NaCI, followed by two dialysis steps against dilute HCI (pH 3.5) and MilliQ water, respectively. After lyophilization of the dialyzed solution thiol-modified HA (302 mg) was obtained as a white solid. The degree of substitution of HA-SH was ~38% using 1 H NMR analysis (D2O) by comparing the integrals of the -CH2CH2SH side chain methylene peaks at 2.62 and 2.77 ppm with the N-acetyl moiety of HA at 1 .92 ppm.

[0141] Rheology

[0142] PEG crosslinker and HA-SH were dissolved in PBS to form a stock solution of 15%w / v and 5% w / v, respectively. As for PEG-LC-A, it was dissolved with the y-cyclodextrin (7.2% w / v in PBS) solution with assistant of ultrasonic (10 min). Hydrogels were formed in situ by pipetting 10 ul of monomer solutions between a glass bottom and 8 mm parallel plate on a shear rheometer (MCR- 302, Anton-Paar). All tests were performed at 25°C. Mineral oil was loaded around the plate to prevent the samples from drying during measurement. Oscillatory measurements were performed in triplicates at 1 % shear rate and 1 Hz frequency with a gap of 0.1 mm.

[0143] Evaluation of two-photon degradation

[0144] The two-photon degradation was evaluated under a commercial two-photon printer (UpNano, NanoOne 1000). The setup is based on a femtosecond laser operating at 780 nm and 80 MHz, with a pulse length of 90 fs. The drop casted photodegradable gels on confocal dishes (35 pm, Ibidi GmbH, Germany) were used for the patterning. Using a 20* water immersion objective (NA = 0.7), the access channels (rectangular, 600 pm x 200 pm x 100 pm) Firstly, they were created at the edge of the gel with high laser power 150 mW (scanning speed, 300 mm / s). Parallel channels with rectangular cross sections (length = 200 pm, A = 20 x 20 pm) were fabricated next to the access channels with varied mean laser power per channel (10-100 mW, 10 mW steps) and a constant scanning speed of 300 mm / s. The samples were then soaked in a solution of FITC-dextran (Mw = 2000 kDa, 1 mg / mL) in PBS and were put in a vacuum for 5 minutes so that the FITC-dextran solution could diffuse into the printed channels via the access channel quickly. Channels were visualized by confocal microscopy (Lecia, SP8). Furthermore, to evaluate the permissiveness of printed construct, fluorescence recovery after photobleaching (FRAP) measurement was employed. FRAP was conducted in the gel which contains complex patterning constructs and incubated in FITC-dextran. The FRAP was executed on Lecia (SP8) confocal microscope by using 488 nm laser for imaging and bleaching. A region of interested in the center of complex patterning was bleached for 10 seconds after an initial waiting period of 25 seconds and imaged until a steady fluorescence signal was reached. The positive control was chosen in the same construct.

[0145] Design of lacuno-canalicular network (LCN)

[0146] The LCN models were designed with serial focused ion beam scanning electron microscopy (Fl B- SEM) images of lacuno-canaliculi from mice. We used a CAD software (SolidWorks) to create 3D models of the individual canaliculi from the image data. In said CAD software, it was possible to manipulate the shape and length of the canaliculi and add them to ellipsoids that function as osteocyte lacunae. The canaliculi had varying diameters ranging from 780 to 1580 nm and the lacunae had a short axis length of 15.5 pm and long axis length of 27.9 pm. These models allowed us to assemble 2D and 3D arrays of interconnected lacuno-canaliculi with modular distances between lacunae and various canalicular densities.

[0147] Cell culture and encapsulation of spheroids

[0148] Human dermal fibroblasts (HDFs) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% anti-anti. Cells were maintained in an incubator (5% CO2, 37°C) and the medium was changed every two days. Confluent HDFs were trypsinized. Cells were suspended in corresponding culture medium containing 0.2% methocel, seeded in round-bottom 96-well plates with a concentration of 1000 cells per well. After centrifugating at 300 g, under 4°C for 10 min and cultivated for 2 days, HDF spheroids were generated. The prepared spheroids were resuspended in HA-SH solution, meanwhile, PEG crosslinker reacted with RGD-SH (CGRGDSP, (SEQ ID NO: 001) final concentration 1 mM) for at least 10 minutes. The above two mixtures then were combined by pipetting up and down with a total polymer concentration of 8% w / v. Aliquots (10 pL) of the suspension were dropped onto confocal dishes. After a short reaction (~5 min), the samples were maintained in the incubator for 1 h to allow hydrogel formation before adding cell culture medium. Thereafter, channels were eroded with varying laser intensities (30-100 mW, 10 mW steps) around spheroids in a star-sharped manner into the bulk hydrogel at a constant writing speed of 300 mm / s. Live / dead was conducted post printing to evaluate the biocompatibility of printing process.

[0149] Compatibility of synthesized PEG crosslinkers

[0150] To test the cytotoxic effect of all the synthesized PEG crosslinkers in cells for over a 24 h period, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Abeam, lot: 1028026-3) cell proliferation assay was used. Human dermal fibroblasts (HDF) were seeded in 96-well plate with a concentration of 10 k cells / well 24 h before treatment. The cells then were treated by replacing the medium with PEG crosslinker solution (0.1 % in cell medium) for 24 h at 37°C, 5% CO2. Subsequently, the treatment solution was replaced with MTS (1 :10 dilution) testing solution and incubate for 4 h at 37°C, 5%CO2 and read the absorbance at 490 nm by using a plate reader. The cell viability was calculated according to the following equation: VR = A / A0 x 100%, where A and A0 are the absorbance of the experimental groups and control group, respectively.

[0151] Actin-nuclei Staining

[0152] Cell morphology after printing was evaluated with the actin nuclei staining. Fixed samples (4% PFA) were blocked for 1 h with 1% bovine serum albumin (BSA) in PBS and cell membranes were permeabilized with 0.2% Triton-X100 in PBS (containing 0.1 % BSA) for 10 min. Samples were washed with 0.1 % BSA in PBS 3 times. Cell nuclei and the actin cytoskeleton were stained with Hoechst 33342 (2 pg / mL) and Phalloidin-647 (2 pM) in PBS supplemented with 0.1 % BSA for 2 h at room temperature. Samples were washed by PBS and imaged on a confocal microscope (Leica, SP8) equipped with a water immersion 25x objective.

[0153] Statistical analysis

[0154] Statistical analyses were conducted using GraphPad Prism 10.1.0. Student’s t-test was applied to analyze the statistical significance between two groups. One-way ANOVA was used to analyze the statistical significance between three groups.

[0155] Cited references:

[0156] Azagarsamy and Anseth, Angew. Chem. 2013, 125, 14048-14052;

[0157] Azagarsamy et al., ACS Macro Lett. 2014, 3, 515-519

[0158] Bao et al., Org. Lett. 2011 , 14, 572-575 Gehre et al., Acta Biomater. 2024 174, 141-152

[0159] Kloxin et al. 2009, vol 324, 59-63

[0160] Lin et al., Angew. Chem. Int. Ed. 2018, 57, 3722-3726

[0161] LeValley et al., J. Am. Chem. Soc. 2020, 142, 4671-4679;

[0162] Lunzer et al., Angew. Chem. 2018, 130, 15342-15347

[0163] Truong, Li, and Forsythe, ACS Appl. Mater. Interfaces 2017, 9, 32441-32445

[0164] Truong, Tsang, Forsythe, Biomacromolecules 2017 Mar 13;18(3):757-766

[0165] Qiu et al., ChemPhotoChem 2019, 3, 1090-1094.

[0166] All scientific publications and patent documents cited in the present specification are incorporated by reference herein.

[0167] SEQUENCES:

[0168] In the event of discrepancies between the sequences shown in the present specification and those of the enclosed sequence protocol according to WIPO standard ST.26, the sequences shown herein shall prevail.

[0169] CGRGDSP (SEQ ID NO: 001)

[0170] RGDS (SEQ ID NO: 002)

[0171] YIGSR (SEQ ID NO: 003)

[0172] IKVAV (SEQ ID NO: 004)

[0173] PHSRN (SEQ ID NO: 005)

[0174] REDV (SEQ ID NO: 006)

[0175] DGEA (SEQ ID NO: 007)

[0176] QGDV (SEQ ID NO: 008)

Claims

Claims1 . A method for making a micro-structured hydrogel, comprising the steps of: a. providing a hydrogel composition, the hydrogel composition comprising a polymer crosslinked by linker moieties comprising a photolabile coumarin moiety; b. subjecting the hydrogel composition to structured irradiation with light having a wavelength of >700 nm.

2. The method according to claim 1 , wherein the polymer is hyaluronic acid (HA).

3. The method according to any one of the preceding claims, wherein the hydrogel is generated by reacting a polymer having a first reactive moiety with a photocleavable linker of the general structure (I)wherein n is an integer from 1 to 150, R is a moiety comprising a coumarin ring and R1is a second reactive moiety capable of creating a covalent link to the first reactive moiety under aqueous conditions at neutral pH (6.5 to 8.0).

4. The method according to claim 1 or 2, wherein the coumarin moiety is described by the general structure (II):whereinL is the bond to (I), andRcis selected from (CH=CH)P-X and (CH=CH)n-Ay-Z, wherein p is an integer selected from 0, 1 , 2 and 3,Ay is an aryl or heteroaryl, andZ is selected from ORNand NRN2, with each RNindividually being selected from H, C1-C4 alkyl.

5. The method according to any one of the preceding claims, wherein the composition further comprises cyclodextrin.

6. The method according to claim 5, wherein the cyclodextrin is gamma cyclodextrin.

7. The method according to claim 5 or 6, wherein the ratio of cyclodextrin to photocleavable linker is selected from 1 :0.2 to 1 :5, particularly from 1 :0.5 to 1 :2.

8. The method according to any one of the preceding claims, wherein the polymer concentration in the composition ranges from 1 % to 10% (w / v), particularly from 2.5% to 8% (w / v).

9. The method according to any one of the preceding claims, wherein the hydrogel composition does not comprise a soluble photosensitizer agent.

10. The method according to any one of the preceding claims 1 to 8, wherein the hydrogel composition comprises a soluble photosensitizer agent.11 . The method according to any one of the preceding claims, wherein the hydrogel composition comprises live mammalian cells.

12. The method according to any one of the preceding claims, wherein an RGD peptide is selected from a sequence of the group consisting of CGRGDSP (SEQ ID NO: 001), RGDS (SEQ ID NO: 002), YIGSR (SEQ ID NO: 003), IKVAV (SEQ ID NO: 004), PHSRN (SEQ ID NO: 005), REDV (SEQ ID NO: 006), DGEA (SEQ ID NO: 007), QGDV (SEQ ID NO: 008).

13. The method according to claim 12, wherein the peptide sequence is RGD peptide is SEQ ID NO: 001.

14. A micro-structured hydrogel composition, comprising a hydrogel crosslinked by moieties comprising a photolabile moiety susceptible to cleavage by two-photon irradiation of a wavelength of >700 nm, and channels or cavities having a diameter of <10 pm, particularly having a diameter of <5 pm, more particularly having a diameter of <3 pm.

15. The micro-structured hydrogel composition according to claim 14, wherein the hydrogel is HA.

16. A micro-structured hydrogel obtained by a method according to any one of claims 1 to 13.

17. The micro-structured hydrogel according to claim 16, characterized by a plurality of ellipsoid indentations (lacunae) on its surface, and a plurality of channels (canaliculi) of 0.5 to 3 pm in width departing from each lacuna into the micro-structured hydrogel.

18. The micro-structured hydrogel according to claim 17, wherein the ellipsoid indentations are characterized by a longitudinal diameter of 30 to 40 pm, and a latidudinal diameter of 10 to 30 pm.

19. The micro-structured hydrogel according to claim 17 or 18, wherein the plurality of channels comprises 10 to 150 channels per lacuna, particularly 15 to 100, more particularly 25 to 50.

20. The micro-structured hydrogel according to any one of claims 17 to 19, wherein the position of lacunae and canaliculi reflects an image-based computer model of network structures found in native tissues.

21. A method for generating an image-based cell network, comprising providing a microstructured hydrogel according to any one of claims 17 to 20 and positioning live mammalian cells into each of the lacunae under conditions of mammalian cell culture.

22. An organoid cell culture model comprising an image-based cell network generated by the method according to claim 21 .

23. Use of an organoid cell culture model, or a micro-structured hydrogel, according to any one of the preceding claims in a method of disease modeling or drug testing.

24. A linker for crosslinking a polymer, characterized by the general formula (I):whereinR1is selected from CH2=CH, CH3HC=CH, CH3CH2HC=CH and CH2=C(CH3), and R is described by (Ila):wherein RC is selected from (CH=CH)P-X and (CH=CH)n-Ay-Z, with p being selected from 0, 1 , 2 and 3, Ay being aryl or heteroaryl, and Z being selected from ORNand NRN2, with each RNindividually being selected from H, C1-C4 alkyl.

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