A method of photoinduced micro-sculpting of a hydrogel

Abstract

The present invention relates to a method of photoinduced micro-sculpting of a hydrogel, said method comprising the steps of: i) provision of a hydrogel having a plurality of reactive groups each selected from the group consisting of a -CH group, a -NH group, an -OH group and a combination of two or more thereof, said hydrogel having a surface to be micro-sculpted; ii) provision of a photo-activator; iii) contacting the surface of the hydrogel of step i) with the photo-activator of step ii); and iv) illumination of said photo-activator on said surface using a digital mask and UV light to photo-excite the photo-activator to induce micro-sculpting of said hydrogel. Moreover, the invention relates to a micro-sculpted hydrogel obtainable or directly obtained by the method according to the invention. In addition, the invention relates to the use of a micro-sculpted hydrogel as a construct for cell studies, materials and biomaterials applications, tissue engineering, and soft robotics.

Description

[0001] TITLE A method of photoinduced micro-sculpting of a hydrogel

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a method of photoinduced micro-sculpting of hydrogels.

[0004] Background

[0005] Hydrogels are a class of soft materials widely used as a substrate or matrix for cell culture to mimic the cell’s native microenvironment. Using a variety of hydrogels, it has been shown that the mechanical properties of the cellular microenvironment play an important role in cell phenotype, organization, and behaviour. In addition, it is known that the topography of the surface of the substrate is also an important factor in cell behaviour. This topography has until now mostly been limited to the use of hard elastomeric materials. Introducing topography in soft hydrogels is technically challenging, labour intensive and often does not provide a flexible design space, due to the high deformability of the material. Current approaches rely on the use of physical moulds, which are inefficient, time consuming, and have high failure rate.

[0006] Different strategies for preparing topographical features used in the prior art employ the transfer of topographical features through the use of a mould or building the features layer by layer on the desired substrate. For example, topographies at the nanoscale can be imprinted on silicon and plastics using electron beam lithography and hot embossing. In addition, alginate and poly(acrylamide) (pAA) hydrogels can be topographically patterned using a poly(dimethoxysiloxane) (PDMS)-based imprinting technique by poured gel mixtures into a PDMS mould containing the desired features and then polymerized. US2020 / 0149000A1 discloses the selective polymerisation to prepare a 3D scaffold. However, releasing the pAA gel from the mould while maintaining the features can be difficult and is only possible within a certain range of stiffness of the hydrogels, with limited resolution. Another disadvantage of the imprinting process is its time-consumption; the time from a digital design to an actual substrate with the features easily takes days. Besides, the mould-based imprinting technique is not flexible in its design space. In order to change the type of topography a new glass imprint with new topographies has to be designed and produced first, after which new negative and positive PDMS moulds can be produced. Another option to produce surface topographies is by using photolithography, a technique that uses masked photopatterning to create surface reliefs, which can either be based on light- induced polymerization, or light-induced degradation of the polymer. However, this method also has its limitations, in particular in the speed of the process. 3D printing technologies can also be used to generate surface topographies with varying stiffnesses by adapting the composition of the ink and printing parameters such as in producing acrylamide / glycerol hydrogel scaffolds with features at the micrometer scale. However, the size of the printed scaffold depends on the nozzle size and thereby restricts the resolution in 3D printing procedures.

[0007] Overall, it is clear that there is a need for a flexible topographical patterning technique that can be applied on a range of materials and stiffnesses, that offers versatile design possibilities of desired topographical features, while feature integrity is preserved during the fabrication process, without being time-consuming.

[0008] Objects

[0009] It is an object of the present invention to provide an improved method to create micro- topographical features on a variety of hydrogels.

[0010] It is a further object of the present invention to provide a method for creating micro- topographical features without the use of physical masks that are required to be in contact with the hydrogel to be sculpted.

[0011] STATEMENT OF THE INVENTION

[0012] In a first aspect, the invention relates to a method of photoinduced micro-sculpting of a hydrogel, said method comprising the steps of: i) provision of a hydrogel having a plurality of reactive groups each selected from the group consisting of a -CH group, a -NH group, an -OH group and a combination of two or more thereof, said hydrogel having a surface to be microsculpted; ii) provision of a photo-activator; iii) contacting the surface of the hydrogel of step i) with the photoactivator of step ii); iv) illumination of said photo-activator on said surface using a digital mask and UV light to photo-excite the photo-activator to induce micro-sculpting of said hydrogel.

[0013] In a second aspect, the invention relates to a micro-sculpted hydrogel obtainable or directly obtained by the method according to the first aspect.

[0014] In a third aspect, the invention relates to a use of a micro-sculpted hydrogel obtainable or directly obtained by the method according to the first aspect as a construct for cell studies, materials and biomaterials applications, tissue engineering, and soft robotics.

[0015] DETAILED DESCRIPTION

[0016] The present invention is elucidated below with a detailed description.

[0017] List of definitions

[0018] The following definitions are used in the present description and claims to define the stated subject matter. Other terms not cited below are meant to have the generally accepted meaning in the field.

[0019] “maskless” as used in the present description means: without a physical mask. Physical masks may for example be constructed from quartz with e.g. a chromium coating. The present invention does not require a physical mask but can use a digital mask. This is a mask that does not directly contact the surface to be sculpted and hence is a so-called contactless mask.

[0020] “UV light” as used in the present description means: light in the ultraviolet spectrum, covering wavelengths in the range of 100 to 400 nm. This is typically divided into three separate bands, namely the UV-A band (315-400 nm), the UV-B band (280-315 nm), and the UV-C band (100-280 nm).

[0021] “hydrogel” as used in the present description means: a solid three- dimensional network that forms a structure with a medium within, the medium being an aqueous medium (e.g. water). A hydrogel is able to hold on to large amounts of aqueous medium, e.g. between 90 and 99 wt.% of the hydrogel may be composed of aqueous medium.

[0022] “biological hydrogel” as used in the present description means: hydrogels from naturally occurring biological molecules. These hydrogels are sometimes called natural hydrogels. Typically, biological hydrogels are networks formed of protein-polysaccharide chains. Collagen and gelatin are examples of biological hydrogels.

[0023] “Hybrid hydrogels” as used in the present description means: a hydrogel formed of two or more different hydrogel monomers.

[0024] “micro-sculpting” as used in the present description means: the formation of micro-topographical features.

[0025] “construct” as used in the present description means: a hydrogel that is formed into a specific three dimensional form, either in a container or self- supporting.

[0026] Brief description of drawings

[0027] The present invention is described hereinafter with reference to the accompanying drawings in which embodiments of the present invention are shown and in which like reference numbers indicate the same or similar elements.

[0028] Figure 1A shows an example of an experimental protocol according to the present invention;

[0029] Figure 1 B shows an example of a photo-activator and the possible chemical reaction it undergoes upon illumination, including a possible reaction scheme with the reactive groups (RG-H) of the hydrogel;

[0030] Figure 2 shows optical profilometry results showing the surface topography of the induced micro-topographical features on a variety of hydrogels; A) is digital masked used; (B) results for synthetic hydrogels, pNIPAM - poly(N- isopropylacrylamide, pAA - polyacrylamide, and PEG - polyethyleneglycol), (C) results for biological hydrogel GelMA gel, and (D) results for supramolecular llpy hydrogel; for each of B), C) and D) the chemical structure of the hydrogel used is show, as well as a 3D representation of the micro-topographical features obtained as well as the representative line profile of the indicated sections (dashed lines).

[0031] Figure 3 shows a range of different designs for micro-topographical features; (A) shows different digital masks used, the 3D optical profile and height profile; (B) shows that micro-topographical features with different widths of the invaginations and / or protrusions can be obtained; (C) shows more complicated designs; (D) shows variations in UV dose and the maximum profile height / depth obtained therewith; (E) shows the effect of the concentration of the photo-activator and the influence on the maximum profile height / depth.

[0032] Figure 4 shows the value for the Young’s modulus of pAA, pNIPAM and GelMA hydrogels on the illuminated parts, being the invaginations and / or protrusions (“patterned”) as well as on the non-illuminated parts (“non-patterned”).

[0033] Figure 5 shows grooves prepared according to the present invention and the way they affect fibroblasts orientation.

[0034] DETAILED DESCRIPTION OF EMBODIMENTS

[0035] The present invention provides a new approach that allows the robust creation of micro-scale topographies on hydrogels using a maskless UV-induced patterning approach.

[0036] The present inventors have observed that the present method allows the formation of micro-topographical features on hydrogels that comprise a plurality of reactive groups. Without wishing to be bound by a particular theory, the present inventors believe that the formation of covalent bonds with the reactive groups leads to the desired topography. First, hydrogels are provided having a plurality of reactive groups, thereafter a photo-activator is provided on the surface to be micro-sculpted, and then illumination using a digital mask is used to locally expose the gel with UV light. At the exposed regions, the photo-activator is photoexcited to a reactive state, resulting in local radical coupling and the formation of covalent bonds with reactive -CH, -NH or - OH groups on the surface of the hydrogel. This is clearly shown in Figure 1 , which shows one embodiment of the present invention. In step ia) one or more hydrogel monomers and a solvent are provided, which is subjected to gelation and / or polymerization (step ib)) after which a hydrogel is formed, e.g., in a petri dish. In step ii) a photo-activator is provided and in step iii) applied to the surface of said hydrogel. In step iv), the surface of said hydrogel with the photo-activator is illuminated using a digital mask, in the embodiment of Figure 1a this mask consists of a series of parallel lines. As a consequence, a topography is induced at the exposed regions, corresponding to the applied mask. So, after illumination a micro-sculpted hydrogel is obtained with protrusions. Due to the widespread abundance of -CH, -NH and -OH groups in hydrogel polymers, especially in biological, supramolecular, and synthetic hydrogels, the present inventors have hypothesized that the present photo-activator induced micro-sculpting technique can be applied to create topographies on a variety of hydrogel surfaces. Optical profilometry measurements were used by the present inventors to reveal that indeed anisotropic surface micro-topographies were created on a wide range of hydrogels (synthetic, biological, and supramolecular).

[0037] The invention provides in a first aspect, a method of photoinduced micro-sculpting of a hydrogel comprising steps i), ii), iii) and iv) as discussed above under summary of invention. Each of these steps will be discussed in more detail below.

[0038] Step i)

[0039] Step i) is the provision of a hydrogel having a plurality of reactive groups each selected from the group consisting of a -CH group, a -NH group, an -OH group and a combination of two or more thereof, said hydrogel having a surface to be microsculpted. As discussed above, the presence of these reactive groups will lead to the desired micro-sculpting.

[0040] In an embodiment, in step i) the hydrogel is selected from the group consisting of synthetic hydrogels, biological hydrogels, and supramolecular hydrogels or a combination of one or more thereof. With “a combination of one or more thereof” is meant a hybrid hydrogel formed by gelation and / or polymerization of a combination of two or more hydrogel monomers. A person skilled in the art is aware of the different methods for preparing a hydrogel. Specific examples are provided in the experimental section. Polymerization may be induced by illumination, e.g. by light in the UV range, such as 365 nm. This illumination differs from the illumination in step iv) since no photo-activator is present. Other polymerization methods include free-radical polymerization and temperature-driven polymerization. In step iv) hardly any to none monomers are present.

[0041] In an embodiment, in step i) the hydrogel is selected from the group consisting of polyacrylamide (pAA), poly(N-isopropylacrylamide) (pNIPAM), poly(ethyleneglycol) (PEG)), methacrylated gelatin (GelMA), gelatin, collagen, and supramolecular UPy- poly(ethyleneglycol)) hydrogels or a combination of two or more thereof. With “a combination of one or more thereof” is meant a hybrid hydrogel formed by gelation and / or polymerization of a combination of two or more hydrogel monomers. The chemical structures for each of these are depicted in Figure 2.

[0042] In an embodiment, in step i) comprises sub steps ia) and ib), being: ia) provision of one or more type monomers and a solvent, and ib) gelation and / or polymerization of said monomers into said hydrogel. Examples of suitable solvents are water, PBS, medium for collagen, standard DMEM culture medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin.

[0043] In an embodiment, in step i) the hydrogel is formed within a container. It is possible that the hydrogel is a standalone, as long as during the process the hydrogel does not move its position. However, it is preferred that a container is used since that also assists in containing the aqueous liquid of the photo-activator.

[0044] Step ii)

[0045] Step ii) is the provision of a photo-activator. The photo-activator will provide the required radicals that are needed in the present method to induce the formation of the topographical features. Any type of photo-activator may be used.

[0046] In an embodiment, in step ii) as photo-activator ((4-benzoylbenzyl)trimethylammonium chloride (PLPP) (see formula A below), benzophenone (see formula B below) or a derivative thereof, 2-Hydroxy-2-methylpropiophenone (see formula C below), Tetra-n- butylammonium decatungstate (see formula D below), or a combination of one or more thereof is used.

[0047] A B C D Step iii)

[0048] Step iii) is contacting the surface of the hydrogel of step i) with the photo-activator of step ii).

[0049] In an embodiment, in step iii) said photo-activator is applied to the surface of the hydrogel by first preparing a solution of said photo-activator in a solvent and secondly applying said solution to the surface of said hydrogel.

[0050] The application of the photo-activator in the form of a solution is a convenient way to apply the photo-activator over the surface. This allows an even distribution of the photo-activator over the surface of the hydrogel to be micro-sculpted.

[0051] In an embodiment, the solvent used in the photo-activator solution is an aqueous solution, for example water or a mixture of acetonitrile with water.

[0052] In an embodiment, the photo-activator solution has a concentration of photo-activator of between 2 and 30 mg / ml, such as between 3 and 28 mg / ml. This can be determined on a case by case basis depending on the type of hydrogel used, the type of photoactivator used, the amount of the solution of photo-activator that is added to the surface and the dosage of light to be used. For example, if a more dilute solution is used, a larger volume of the solution per surface area of the hydrogel may be used then if a more concentrated solution is used.

[0053] In an embodiment, in step iii) said solution of said photo-activator is applied to the surface in such a manner that said solution contacts at least 80 %, preferably at 90 %, more preferably at least 95 %, at least 99% or even 100 % of said surface of said hydrogel. For the method according to the inventor to perform optimally it is preferred that the surface onto which the micro-topographical features are to be sculpted is fully provide with photo-activator since that locally will provide the radicals needed. If only part of a surface is to be provided with micro-topographical features, it will be sufficient if only that part of the surface is provided with photo-activator.

[0054] Step iv) Step iv) is illumination of said photo-activator on said surface using a digital mask and UV light to photo-excite the photo-activator to induce micro- sculpting of said hydrogel.

[0055] Figure 1 B shows the mechanism that is hypothesised by the present inventors (without wishing to be bound by a particular theory) that when as photo-activator ((4- benzoylbenzyl)trimethylammonium chloride (PLPP) is used. Upon illumination (hv) a radical ketone is formed of the PLPP which will transfer its radical to a reactive group (RH) thereby forming a radical thereof. This radical of the reactive group may partake in a variety of transformations (e.g. radical-radical coupling, radical conjugate addition, fragmentations, etc.) that may be at the origin of the observed morphological variations. These follow-up reactions are specific to specific hydrogels and can provide different magnitude of the topographical features for different hydrogels.

[0056] In an embodiment, in step iv) said illumination forms micro-topographical features comprising invaginations and / or protrusions in the surface of said hydrogel. These features may have invaginations and / or protrusions with a depth of between 0.1 and 3 micrometer. These features may have invaginations and / or protrusions with a width of at least 1 micrometer, such as at least 2 micrometer.

[0057] In an embodiment, in step iv) light with a wavelength in the range of 315 to 400 nm, being the UV-A range. In a specific embodiment, light with a wavelength of 375 nm is used, more in particular when a photo-activator according to formula A, B, C or D above is used.

[0058] In an embodiment, in step iv) the digital mask is obtained using a digital micromirror device (DMD).

[0059] In an embodiment, in step iv) illumination is carried out with a UV dose of between 500 and 1500 mJ / mm2.

[0060] In a second aspect, the invention relates to a micro-sculpted hydrogel obtainable or directly obtained by the method according to the first aspect.

[0061] Use of the invention In a third aspect, the invention relates to a use of a micro-sculpted hydrogel obtainable or directly obtained by the method according to the first aspect as a construct for cell studies, materials and biomaterials applications, tissue engineering, and soft robotics.

[0062] The in vivo cellular microenvironment of cells provides these cells with multiple biochemical, physical and mechanical cues. For example, the presence of growth factors and cytokines are required for tissue development and homeostasis. Besides, the extracellular matrix presents topographical cues on various length scales. Additionally, tissues display a range of stiffnesses, which can also change during pathologies. Together, these environmental cues influence biological processes such as cell adhesion, proliferation, migration, and differentiation. The utilization of environmental cues in cell behaviour studies is a fascinating topic that has garnered much interest in the scientific community. To answer mechanobiological research questions, engineered platforms are created using different strategies. Efforts have been made to create multi-cue in vitro cell culture platforms. The micro-sculpted hydrogels (or hydrogel constructs) of the present invention provide such platforms. The present inventors have shown that dermal fibroblasts sense and respond to topographically patterned hydrogels of different stiffnesses, paving the way to perform systematic investigations into the combined effect of stiffness and topography on cell behaviour.

[0063] Effect of the invention

[0064] The induced topographies produced via the inventive method are highly adaptable: the features can be designed using any digital drawing software, while the dimension and resolution of the topographical features can be easily tuned by changing the UV dose or photo-activator concentration. The inventive approach is also versatile, as demonstrated by the creation of topographies on a wide range of hydrogels with distinct chemical characters (biological, synthetic, supramolecular) and mechanical properties (for example between 0.5 Pa to 30 kPa). As a biological proof of concept, the present inventors have shown that cells on softer hydrogels align better to topographical cues than on stiffer hydrogels, underlining the importance of studying cell response in physiologically more relevant multi-cue environments. The present invention is a fast, flexible and user-friendly photoactivated sculpting technique that allows the introduction of surface topographies with desired microscale geometries, on a variety of gels. The inventors have demonstrated that that a broad spectrum of hydrogels (biological, synthetic and supramolecular) can be provided with micro-topographical features in a contact-free way by using the photoinduced activation of a photo-activator.

[0065] Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used to advantage. The scope of the present invention is defined by the appended claims. One or more of the objects of the invention are achieved by the appended claims.

[0066] EXAMPLES

[0067] The present invention is further elucidated based on the Examples below which are illustrative only and not considered limiting to the present invention.

[0068] Materials and methods

[0069] Bind-silane glass functionalization: A bind-silane solution was made consisting of 4.286 mL absolute ethanol, 357 pL PlusOne Bind-Silane (3- (trimethoxysilyl)propyl methacrylate, Scientific Laboratory Supplies), 357 pL acetic acid (VWR), and used within 7 days. Microscopy glass slides were incubated with the bind-silane solution (30 min, room temperature), washed with absolute ethanol and dried under a flow of nitrogen. Functionalized glass slides were used immediately. pAA hydrogels’. Hydrogels were made consisting of 40% acrylamide, 2% bis acrylamide, ammonium persulfate (Bio-Rad) and tetramethylethylenediamine (Bio-Rad) in PBS. Droplets (12 pL) were pipetted on bind-silane functionalized glass slides and a coverslip (0 13 mm) was placed on top. After 60 min, PBS was added and incubated at room temperature. After 1 h, the coverslip was removed and gels were washed with phosphate buffered saline (PBS, Sigma-Aldrich). Gels were stored in PBS at 4°C until use. pNIPAM hydrogels: Hydrogels were made using a 2:1 dioxane-water monomer cocktail (0.44 mg / pL), consisting of 87 mol% N-isopropylacrylamide (NIPAM, Sigma-Aldrich), 10 mol% acrylic acid (Sigma-Aldrich), 1 mol% N,N'- methylenebis(acrylamide) (MBIS, Sigma-Aldrich) and 1 mol% 2-hydroxy-2-methyl-1- phenyl-1-propanone (Sigma-Aldrich). Droplets (12 pL) were pipetted on bind-silane functionalized glass slides and a coverslip (0 13 mm) was placed on top. After 5 min. exposure to UV-light (365 nm, 3.44 mW / cm2, J110619 Analytik Jena), the hydrogels were polymerized and covalently attached to the coated glass slides. Gels were soaked in PBS and stored at 4°C until use.

[0070] 2% PEG hydrogels: To create hydrogels, 4-arm poly(ethyleneglycol)- vinyl sulfone (PEG-VS, JenKem Technology) and 4-arm PEG thiol (PEG-SH, JenKem Technology) were diluted to 20 mg / mL PEG-VS and 10 mg / mL PEG-SH in PBS. After mixing the two solutions droplets of 12 pL were pipetted onto a microscopy glass slide. Then, a 0 13 mm coverslip was placed a on top and let it sit at room temperature for 1-2 hours. After soaking the gels in PBS and removing the coverslips, the hydrogels were stored at 4°C until use.

[0071] 10% GelMA hydrogels: Droplets (12 pL) containing 10% w / v gelatin methacryloyl monomers with 80% degree of substitution 900496, Sigma-Aldrich) in PBS and lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (1 mg / mL final concentration, Merck Life Science) were pipetted on bind-silane functionalized glass slides with a coverslip (0 13 mm) on top, and were exposed with UV (365 nm, 5 min, 3.44 mW / cm2, J110619 Analytik Jena) to enable polymerization. Gels were soaked in PBS, stored at 4°C and used within 24 hours.

[0072] Gel fraction experiments: pAA and pNIPAM hydrogels were prepared as described above. Directly after polymerization, hydrogels were weighted (W0). Subsequently, gels incubated 5 days with PBS or tetrahydrofuran, were dried in an oven (48h, 65°C) and weighted (Wt). The gel fraction (%) was determined as (Wt / WO) x 100.

[0073] Topographical sculpting: The photo-sculpting machinery is calibrated according to the protocol showed in C. van der Putten et al.; ACS Appl. Mater. Interfaces 2021 , 13, 25589. The hydrogel is placed on the stage and its surface is covered with 40 pL of 4-benzoylbenzyl-trimethylammonium chloride in milliQ water. The grayscale design of the pattern is uploaded in Leonardo software (Alveole, version 4.13, Paris) and projected on the gel surface using UV light (375 nm, 1000 mJ / mm2). The focal plane should be focused on the surface where the features should be sculpted.

[0074] Optical profilometry: An optical surface profiler (Sensofar Pip 2300 with 20x / 0.45 NA Nikon and 50x / 0.8 NA objectives) was used to measure height profiles. Measurements were processed with PLp 2300 software version 2.41 and plotted in GraphPad Prism 9.

[0075] Atomic force microscopy (AFM): The Young’s modulus of the gels was characterized using an AFM (Nanowizard 4, Bruker, USA) in the force spectroscopy mode. A qp-BioAC-CI probe (Nanosensors, Switzerland) with a nominal spring constant of 0.06 N / m was used. The sensitivity and spring constant of the probe were calibrated using the thermal noise method. 20 force-distance curves were acquired in each region of interest of the analysed samples with a set-point force of 1 nN. The Young’s modulus was calculated by fitting the force-distance curves to the Hertz- Sneddon model considering a paraboloid tip shape with a radius of 30 nm (nominal value).

[0076] Cell culture: Normal human dermal fibroblasts, nhDF (Lonza, CC- 2511), were cultured in Dulbecco's Medium Eagle’s Medium (DMEM 41966-029, Thermofisher) supplemented with 10% fetal bovine serum (Biochrom AG) and 1% penicillin-streptomycin (Biochrom AG). Cells were cultured in T25 flasks in a humidified atmosphere (5% CO2, 37 °C) until passage nr. 18.

[0077] Cell seeding on pAA hydrogels’, prior to cell seeding, pAA hydrogels were functionalized by adding 1.0 mg / mL Sulfo-SANPAH (Pierce) on the gel surface and exposing it to UV-light (365 nm, 5 min). After washing with PBS, gels were coated with fibronectin (bovine plasma fibronectin, 33010-018, Invitrogen, 15 pg / mL in PBS) and incubated 30 min at room temperature. Meanwhile, cells were harvested using 0.05% trypsin / Ethylenediaminetetraacetic acid (EDTA) with phenol red (25300054, Gibco). A total of 25,000 cells was seeded per gel.

[0078] Immunofluorescence staining’. Cells were fixed with 3.7% paraformaldehyde (104033.1000, Merck) for 15 min at room temperature, washed with PBS, and stained with Phalloidin-Atto 488 (1 :200, 49409, Sigma-Aldrich) for 1h at room temperature. After washing with PBS, nuclei were stained using 4',6-diamidino- 2-phenylindole dihydrochloride (D9542, Sigma-Aldrich). Samples were imaged using a Zeiss Axio Observer 7-Apotome epifluorescence microscope with 20x / 0.4 NA and 40x / 0.6 NA objectives. Images were analyzed using a custom pipeline in CellProfiler and the data were processed using a custom made Python library which follows FAIR requirements.

[0079] Statistical analysis’. Data are plotted as mean with standard deviation (SD). Data were checked for normality using the Shapiro-Wilk test, and experimental groups were subsequently tested for significant differences using the test indicated in the caption. Statistical analyses were performed using GraphPad Prism 910.

[0080] Graphics were created with BioRender.com

[0081] Different types of hydrogels tested

[0082] In pAA hydrogels, invaginations of 1.158 ± 0.185 pm were created. Also in PEG hydrogels invaginations of 0.2858 ± 0.04306 pm were induced (Fig. 2Bii). On the contrary, in pNIPAM gels extrusions of 2.140 ± 0.4255 pm high (Fig. 2B) and in 10% GelMA gels extrusions of 2.877 ± 0.405 pm were formed (Fig. 2Cii). In supramolecular UPy gels extrusions of 1.255 ± 0.3804 pm were sculpted (Fig. 2Dii).

[0083] The process of creating topographies on various hydrogels relies heavily on the properties of the used material. Depending on the chemical makeup of the hydrogel, either an invagination or extrusion on the surface will be created (as seen in Figure 2B). Profile measurements reveal a correlation between the effectiveness of the photoreaction induced and the chemical stability of the hydrogel (Fig. 2E). For instance, SBS (styrene-butadiene-styrene) and PEG (polyethyleneglycol) have a lower depth in topographies compared to pNIPAM and pAA (Fig. 2ii).

[0084] Without wishing to be bound by a particular theory, the present inventors believe that the observed phenomenon can be linked to a variation of internal chemical architecture of the polymer, upon light irradiation of PLPP. The variation in invagination and / or protrusions may possibly be further explained by looking at the chemical composition of the polymer (i.e. its monomeric unit) and the hydrogen atom that can be reasonably considered to be involved in the second step of the mechanism in Figure 1 B. Monomers having an aromatic (SBS) or an ether moiety (PEG) are best at stabilizing the resulting species RG- , followed by pNIPAM and lastly pAA. It could be hypothesized that a low energy radical will be less prone to react, leading to a smaller change in morphology. On the other hand, a less stabilized radical will display a more powerful reactivity, causing a more pronounced variation.

[0085] Figure 2 shows that a wide variety of hydrogels can be micro-sculpted with grooves by using a digital mask of alternating white and black lines (A). Optical profilometry reveals the generation of surface topography on a variety of hydrogels: (B) Synthetic (pNIPAM - poly(N-isopropylacrylamide, pAA - polyacrylamide PEG - polyethyleneglycol), (C) biological GelMA gel and (D) supramolecular UPy hydrogels, i) 3D representation, ii) representative line profile of the indicated sections (dashed lines). Note that panel C has a different length scale (y-axis) to ensure clarity.

[0086] Different designs of topographical features

[0087] The present inventors also tested topography design flexibility and printing fidelity. To do so, pAA hydrogels were sculpted using different digital masks (square, circles, lines, crossbows, gradient) and the sculpted profiles were measured with optical profilometry (see Fig. 3). pAA hydrogels were selected for this specific test because of their easy production process and high reproducibility in mechanical properties. Optical profilometry confirmed that a wide range of patterns were sculpted in the hydrogel surface with high fidelity, resulting in a resolution of ca. 2 pm (Fig. 3B). Additionally, by incorporating grey values in the digital mask, a gradient in topography was created (Fig. 3A). Moreover, even more complicated designs could be printed on the hydrogel (Fig. 3C).

[0088] Since topography formation depends on photo-activator UV-induced activation, the present inventors reasoned that by altering the UV-dose or photo-activator concentration, it would be possible to have control over the height profiles. Optical profilometry measurements showed that reducing the UV dose led to shallower invaginations compared to the control dose of 1000 mJ / mm2. On the other hand, increasing the UV dose to 1500 mJ / mm2resulted in a clearer and more distinct pattern in pAA gels (Fig. 3D). For this specific hydrogel it was found that a dose of 1500 mJ / mm2did not result in deeper invaginations, suggesting that the PLPP UV-activated micro-sculpting technique for pAA gels has a maximum limit in height profile generation. On the contrary, GelMA gels showed a linear UV-dose dependency; a higher UV-dose (up to 1500 mJ / mm2) resulted in higher extrusions. Next, the present inventors have investigated the influence of photo-activator (PLPP) concentration on topography formation in pAA hydrogels by varying the PLPP concentration in the presented UV-induced micro-sculpting technique while keeping the UV-dose constant (1000 mJ / mm2). The present inventors observe an optimum concentration (7 mg / mL) of PLPP-induced profile generation, and patterning below or above this concentration resulted in smaller maximum height profiles (Fig. 3E).

[0089] The present inventors also have tested other types of photo-activators and found similar results, but the generation of patterns can be less efficient for certain gel types than with PLPP. For example, with Tetra-n-butylammonium decatungstate pAA gels could be sculpted with the same pattern fidelity as with PLPP, but patterns on pNIPAM hydrogels were less sharp. Similarly, PEG and UPy gels could be less efficiently patterned with 2-Hydroxy-2-methylpropiophenone than with PLPP, whereas GelMA, gelatin, pAA and pNIPAM can be patterned using both PLPP and 2-Hydroxy-2- methylpropiophenone with equal sculpting outcomes.

[0090] Figure 3 shows that a range of designs can be micro-sculpted on hydrogels, using different digital masks. In (A) representative surface profiles are depicted. Note that for clarity patterns are depicted as black lines on a white background, while the digital mask used for patterning is inverted for the square, circles, lines and crossbow design. (B) Lines with different widths (2,5, 10, and 30 pm wide) show that lines as small as 2 pm can be sculpted on pAA hydrogels. Note that for clarity the pattern depicted as black lines on a white background, while the digital mask used for patterning is inverted. (C) More complicated designs can be successfully sculpted on a pNIPAM hydrogel. Artwork: girl with the pearl of Johannes Vermeer. (D) UV dose determines the maximum profile (z) of micro-sculpted topographies. Left: Average surface profile of pAA hydrogels (n>4). Right: maximum generated height (z) in various hydrogels depends on the UV dose. Each dot represents one measurement, with at least 3 independent samples. (E) Concentration of the photo-activator (PLPP) influences maximum profile inscription. Left: Average surface profile of 4 pAA hydrogels sculpted with squares using varying PLPP concentrations. Right: Maximum induced height (z) of synthetic hydrogels depends on the PLPP concentration. Mean with SD, n>4.

[0091] Mechanical characterization To gain a deeper understanding about the effect of sculpting a surface topography on material properties, synthetic and natural hydrogels were patterned with a square (1.5 mm x 1.5 mm ) and Young’s moduli were measured with atomic force microscopy (Fig. 4). pAA hydrogels display no significant difference in Young’s moduli in and outside of the patterned region (E = 20 kPa). On the contrary, pNIPAM and GelMA hydrogels are ca. 1.5-fold stiffer inside the patterned region than outside the region. Without wishing to be bound to a particular theory, the present inventors hypothesize that the change in hydrogel stiffness could be explained by a change in water content between the patterned and nonpatterned regions. Radical formation in the polymer backbone by PLPP changes the local chemical environment, resulting in diffusion of water molecules away from the exposed area, thereby causing an increase in hydrogel stiffness. This is supported by studies investigating water diffusion mechanisms in hydrogels with different properties, highlighting the role of polymer network length, crosslinking densities, and inter- and intramolecular interactions on water diffusion.

[0092] Figure 4 shows the Young’s modulus of pAA, pNIPAM and GelMA hydrogels in- and outside the pattern as measured with AFM N > 18 indentations per condition. Unpaired t-test with Welch’s correction.

[0093] Response of cells to micro-sculpted hydrogels

[0094] Finally, pAA hydrogels with high (~ 30 kPa) and low (~ 9 kPa) stiffness were sculpted with microgrooves of varying widths (20, 50, 100 pm) and coated with fibronectin to show the potential of the presented UV-activated micro-sculpting technique for mechanobiological applications. Normal human dermal fibroblasts (nhDF) were seeded on the surface topographies and cultured for 24 hours. According to Fig. 5, nhDFs were able to sense the microgrooves and aligned anisotropically with the pattern. Besides, hydrogel stiffness was found to affect cell orientation. In order to compute cell orientation FOA tool (see Tromp I., Journal of orthopaedic research, 2021 :39, pages 1955-1964) has been used to define cytoskeleton orientation. The results show a clear trend of fibroblasts to follow the -sculpted micro-topographies. Additionally, softer hydrogels (9 kPa) seem to increase the sensitiveness of fibroblasts to the sculpted grooves since better cell alignment is detected (Fig. 5B). Moreover, the highest degree of alignment has been observed on 50 pm wide topographies. Due to the user-defined control over hydrogel stiffness and surface topography, the presented UV-activated micro-sculpting technique will allow to systematically investigate the combined effect of material properties on cellular behaviour in future studies.

[0095] Figure 5 shows grooves prepared according to the present invention and the way they affect fibroblasts orientation. (A) sculpted grooves on pAA hydrogels with different stiffness showing cell alignment according to the topographies with respect to the flat substrate. (B) Quantification of cytoskeleton alignment according to the microtopographies. *** p<0.005, **** p< 0.0005. The scope of the present invention is defined by the appended claims.

Claims

CLAIMS1. A method of photoinduced micro-sculpting of a hydrogel, said method comprising the steps of: i) provision of a hydrogel having a plurality of reactive groups each selected from the group consisting of a -CH group, a -NH group, an -OH group and a combination of two or more thereof, said hydrogel having a surface to be microsculpted; ii) provision of a photo-activator; iii) contacting the surface of the hydrogel of step i) with the photoactivator of step ii); iv) illumination of said photo-activator on said surface using a digital mask and UV light to photo-excite the photo-activator to induce micro- sculpting of said hydrogel.

2. The method according to claim 1 , wherein in step i) the hydrogel is selected from the group consisting of synthetic hydrogels, biological hydrogels, and supramolecular hydrogels or a combination of one or more thereof.

3. The method according to claim 1 or 2, wherein in step i) the hydrogel is selected from the group consisting of polyacrylamide (pAA), poly(N- isopropylacrylamide) (pNIPAM), poly(ethyleneglycol) (PEG)), methacrylated gelatin (GelMA), gelatin, collagen, and supramolecular UPy-poly(ethyleneglycol)) hydrogels or a combination of two or more thereof.

4. The method according to any one of the preceding claims, wherein in step i) comprises sub steps ia) and ib), being: ia) provision of one or more type monomers and a solvent, and ib) gelation and / or polymerization of said monomers into said hydrogel.

5. The method according to any one of the preceding claims, wherein in step i) the hydrogel is formed within a container.

6. The method according to any one of the preceding claims, wherein in step ii) as photo-activator ((4-benzoylbenzyl)trimethylammonium chloride (PLPP), benzophenone or a derivative thereof, 2-Hydroxy-2-methylpropiophenone, Tetra-n- butylammonium decatungstate, or a combination of one or more thereof is used.

7. The method according to any one of the preceding claims, wherein in step iii) said photo-activator is applied to the surface of the hydrogel by first preparing asolution of said photo-activator in a solvent, preferably water, and secondly applying said solution to the surface of said hydrogel, preferably wherein said solution has a concentration of photo-activator of between 2 and 30 mg / mL, such as between 3 and 28 mg / mL.

8. The method according to any one of the preceding claims, wherein in step iii) said solution of said photo-activator is applied to the surface in such a manner that said solution contacts at least 80 %, preferably at 90 %, more preferably at least 95 %, at least 99% or even 100 % of said surface of said hydrogel.

9. The method according to any one of the preceding claims, wherein in step iv) said illumination forms micro-topographical features comprising invaginations and / or protrusions in the surface of said hydrogel.

10. The method according to any one of the preceding claims, wherein in step iv) micro-topographical features are obtained having invaginations and / or protrusions with a depth of between 0.1 and 3 micrometer.

11. The method according to any one of the preceding claims, wherein in step iv) micro-topographical features are obtained having invaginations and / or protrusions with a width of at least 1 micrometer, such as at least 2 micrometer.

12. The method according to any one of the preceding claims, wherein in step iv) light with a wavelength in the range of 315 to 400 nm , e.g. 375 nm is used.

13. The method according to any one of the preceding claims, wherein in step iv) the digital mask is obtained using a digital micromirror device (DMD).

14. The method according to any one of the preceding claims, wherein in step iv) illumination is carried out with a UV dose of between 500 and 1500 mJ / mm2.

15. A micro-sculpted hydrogel obtainable or directly obtained by the method according to any one of claims 1-14.

16. A use of a micro-sculpted hydrogel obtainable or directly obtained by the method according to any one of claims 1-14 as a construct for cell studies, materials and biomaterials applications, tissue engineering, and soft robotics.

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