Systems, articles, and methods related to hydrogels

Abstract

Systems, articles, and methods related to the fabrication microgels with non-spherical shapes and anisotropy are described herein. In one aspect, a technique to fabricate microgels is generally described. In some embodiments, by tuning the aspect ratio of the microgels, it is possible to incorporate anisotropic behavior within the microgels. Such anisotropic behavior can be used in certain embodiments to control and / or influence cellular arrangement in three dimensional space, and can be utilized for biomedical applications ranging from their application as injectable materials to mimicking and / or resembling biological tissues.

Description

[0001] SYSTEMS, ARTICLES, AND METHODS RELATED TO HYDROGELS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 697,370, filed September 20, 2024, entitled “Systems, Articles, and Methods Related to Hydrogels,” by John, et al., incorporated herein by reference in its entirety.

[0004] TECHNICAL FIELD

[0005] Articles, systems, and methods describing hydrogels are generally described.

[0006] BACKGROUND

[0007] Hydrogels are a class of polymeric materials that are relevant to a multitude of biomedical applications ranging from their utilization as 3D cell culture substrates to fabricating full tissues. The ability of hydrogels to resemble cellular microenvironments have led to their translation to products that have improved clinical standards and have provided effective treatment to many diseases and cost-effective drug development. Despite that, when utilized for areas such as cell delivery or as scaffolds for tissue regeneration, the clinical translation of hydrogels is relatively challenging. This may be due to the nanoporous architecture of hydrogels, which leads to problems such as diffusion limitations or delayed anastomosis. Accordingly, hydrogel structures with improved architectures are needed.

[0008] SUMMARY

[0009] Articles, systems, and methods describing hydrogels are generally described. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0010] In one aspect, the present disclosure generally involves an article. In some embodiments, the article comprises the gel defined by a plurality of filaments substantially parallel to each other, the gel having an average aspect ratio of at least 2: 1 and an average length of between 500 micrometers and 1 mm, wherein the gel has an open volume of greater than or equal to 5 vol%.

[0011] In some embodiments, the article comprises a gel comprising a crosslinked-polymer network defining a plurality of channels therein, having an average diameter that is greater than or equal to 200 micrometers and an average length of between 8 mm and 2 cm, wherein the gel has an open volume of greater than or equal to 5%. In another aspect, the present disclosure generally involves a system. In some embodiments, the system comprises a plurality of mirrors; a source of electromagnetic radiation capable of emitting electromagnetic radiation such that at least a portion of the electromagnetic radiation is reflected by at least some of the plurality of mirrors; a liquid reservoir comprising a liquid having a liquid depth of less than or equal to 0.5 mm; and a vessel comprising the liquid reservoir and configured to allow the transmission of at least a portion of the electromagnetic radiation reflected by the plurality of mirrors through a wall of the vessel and through a portion of the liquid.

[0012] In another aspect, the present disclosure generally involves a method. In some embodiments, the method comprises administering a mixture to a subject, wherein the mixture comprises a gel defined by a plurality of filaments substantially parallel to each other, the gel having an average aspect ratio of at least 2: 1 and an average length of between 500 micrometers and 1 mm, wherein the gel has an open volume of greater than or equal to 5 vol%.

[0013] In some embodiments, the method comprises exposing a liquid containing a polymer precursor having a depth of no more than 1 mm to a plurality of light beams having a wavelength greater than or equal to 100 nm and less than or equal to 450 nm, wherein the plurality of light beams causes the polymer precursor to form a plurality of fdaments; and crosslinking at least some of the filaments to form a gel having an open volume of greater than or equal to 20%.

[0014] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale unless otherwise indicated. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures: FIG. 1 is a schematic showing: (FIG. 1A) a bioink composed of varying concentrations of GelMA / PegDA as precursor that can be used to fabricate microgels of varying stiffness; (FIG. IB) a customized DMD setup that can selectively fabricate microgels of different shapes with or without internal architecture; (FIG. 1C) fabricated microgels that can be collected using a cell strainer; and (FIG. ID) application of anisotropic microgels towards fabrication of anisotropic assemblies, according to some embodiments.

[0017] FIG. 2 shows a diagram depicting light-based fabrication of various microgels with internal architecture, according to some embodiments, along with schemes demonstrating that filamented and non-filamented microgels can be fabricated based on aspect ratio (FIG. 2A) and the application of microgel for a multitude of tissue engineering applications (FIG. 2B).

[0018] FIG. 3A depict microscopy images showing the high throughput fabrication of non- spherical micro-gels with internal architecture having difference microgel shapes printed using different hydrogel compositions, according to some embodiments.

[0019] FIG. 3B is a plot depicting a frequency sweep analysis of different hydrogel compositions demonstrating different values of storage modulus, according to some embodiments.

[0020] FIG. 3C is a plot depicting the stiffness of various hydrogel compositions, according to some embodiments.

[0021] FIG. 3D is an image of the injectability of the fabricated migrogels through a 20G needle, according to some embodiments.

[0022] FIG. 4A is a diagram depicting the impact of aspect ratio on the internal structures of fabricated microgels into filamentous or non-filamentous that thereby can effect cellular arrangement, according to some embodiments.

[0023] FIG. 4B shows live-dead staining microscopy images that show high cellular viability over days with microrods providing an elongated cellular arrangement due their filamentous nature whereas in micro- squares cells can spread and form a monolayer type arrangement over days, according to some embodiments.

[0024] FIG. 4C is a plot depicting a presto blue assay demonstrating cellular growth in both shapes over days, according to some embodiments. *p<0.05, **p<0.01.

[0025] FIG. 4D is a high-magnification staining of cytoskeleton demonstrating how internal structures can impact cellular arrangement in microgels with longer shapes promoting alignment whereas in flattened shapes like square cells tend to spread and forms a monolayer arrangement, according to some embodiments. FIG. 5A is a schematic depicting anisotropic micro-annealed particles (MAPs) that can be fabricated using non-spherical microgels including (i) a MAP composed of rods and square having porous architecture that can facilitate cell infiltration (ii) a diagram showing that cells can infiltrate into MAPs due to their micro-porous architecture, according to some embodiments.

[0026] FIG. 5B shows microscopy images depicting HDF cells cultured on MAPs over 5 days, according to some embodiments, wherein cell number seems to increase over days in both MAPs.

[0027] FIG. 5C is a 3D volume rendered image depicting (i) a MAP composed of microrods and respective cell infiltration and (ii) a MAP composed of micro squares and respective cell infiltration, according to some embodiments.

[0028] FIG. 5D is a plot depicting quantified cell infiltration in both MAPs of the previous figures and that cells seems to infiltrate significantly higher on MAPs composed of squares, according to some embodiments.

[0029] FIG. 6A is a schematic illustration demonstrating application of anisotropic microgels for cell-based therapeutics, wherein microrods can help in cellular rearrangement that can be utilized as fillers for treatment of muscle loss, according to some embodiments.

[0030] FIG. 6B is a plot that describes the application of anisotropic microgels for cell-based therapeutics, wherein microrods can help in cellular rearrangement that can be utilized as fillers for treatment of muscle loss, according to some embodiments.

[0031] FIG. 6C includes microscopy images with live / dead and Phalloidin / DAPI staining demonstrating high cellular viability and cellular alignment of cells post and before injection, according to some embodiments. No impact on cellular viability nor cellular alignment was observed post injection. Images were captured at 1-, 3- and 5-days post bio fabrication.

[0032] FIG. 7A is schematic representation depicting microgels (squares) stitched into hydrogel sheets through array of projections can create modular sheets with interconnected channels and demonstrating how such modular scaffolds can be utilized to fabricate hydrogel sheets with patterned vascularization, according to some embodiments.

[0033] FIG. 7B is an image that depicts the gross appearance of microgel sheets and microscopic image showing presence of interconnected channels, according to some embodiments.

[0034] FIG. 8A shows a schematic of a janus type modular assembly created by collation of flattened and elongated geometries, and janus type units with capacity to control cellular arrangements, according to some embodiments. FIG. 8B shows microscopy images of tracked cells loaded in two regions of Janus type assembly, according to some embodiments.

[0035] FIG. 8C shows phalloidin / DAPI stained images demonstrating cell alignment on pillars of Janus type assembly and monolayer formation with random orientation on base of Janus type assembly, according to some embodiments.

[0036] FIG. 9A is a schematic depicting subcutaneous implantation of microgels in a mouse model, according to some embodiments.

[0037] FIG. 9B shows hematoxylin and eosin staining demonstrating tissue infiltration through micro-pores of injected microgel plug post 14 days of implantation, according to some embodiments, including micro-rods without QK peptide, (ii) micro-rods with QK peptide, (iii) micro squares without QK peptide, and (iv) micro squares with QK peptide.

[0038] FIG. 9C shows a plot depicting quantified tissue infiltration in subcutaneous cavity, according to some embodiments.

[0039] FIG. 10 is a schematic that depicts a system for fabricating gels comprising a plurality of filaments, wherein the system comprises a plurality of mirrors and a liquid reservoir, according to some embodiments.

[0040] FIG. 11 A shows phase contrast bright field images depicting filamentous and non- filamentous nature of anisotropic microgels with different aspect ratio, including elongated and flattened geometries, according to some embodiments.

[0041] FIG. 11B shows confocal images depicting micro-rods showed presence of microfilaments whereas flattened geometries like squares showed an absence of microfilaments, according to some embodiments.

[0042] FIG. 12A shows images describing the injectability of fabricated microgels including a syringe loaded with microgels (rods), according to some embodiments.

[0043] FIG. 12B shows images of micro-rods passing easily through a 20G channel (microrods were tagged with Rhodamine to enable easy visualization), according to some embodiments.

[0044] FIG. 12C shows an image of microrods post-injection maintaining their structure, according to some embodiments.

[0045] FIGS. 13A-13B shows images of phalloidin stained microrods and micro-squares imaged in brightfield mode with microfilaments in rods causing cellular alignment, according to some embodiments.

[0046] FIG. 14 is a plot depicting a frequency sweep analysis of MAPs composed of rods and square, according to some embodiments. FIG. 15 shows rendered z stacks showing cell infiltration in MAPs composed of rods and squares over 5 days, according to some embodiments.

[0047] FIG. 16 shows low magnification images of live / dead- stained micro-rods before and post injection demonstrating no impact of injection on cellular viability, according to some embodiments.

[0048] FIG. 17 shows phalloidin / DAPI images depicting minimal impact of injection on cellular alignment, according to some embodiments.

[0049] FIG. 18 shows live / dead images of Photoreceptors (PRs) aligning on pillars. High cell viability was observed over 7 days of culture, according to some embodiments.

[0050] FIG. 19A shows non-spherical microgels can be utilized to make MAPs with different cellular infiltration, according to some embodiments, including: a schematic representation of how particle shape can impact cellular infiltration (top left panel); MAPs with different particle shape demonstrating different stiffness (top right panel); and MAPs composed of rods or squares demonstrating different cellular infiltration (bottom panel).

[0051] FIG. 19B depicts the application of anisotropic microrods for treatment of diseases like volumetric muscle loss, according to some embodiments, including: injectable anisotropic microrods helping cellular rearrangement that can be utilized as fillers for treatment of muscle loss (top row); and in the bottom row of the figure, (i) Microrods stained with FITC (ii) C2C12 loaded microrods stained for live / dead wherein high cell viability with alignment of cells can be observed from day 3; and (iii) Actin stained microrods demonstrating aligned C2C12 cells, according to some embodiments.

[0052] FIG. 19C depicts the application of a combination of rods and squares for resembling anisotropic tissues, according to some embodiments, including (i) individual squares and rods that can be stacked on top of each other through multiple projections leading to Janus type tissue constructs with cell spreading and alignment in different regions (top left panel), (ii) Janus type construct composed of non-filamentous base and filamentous pillars (top right panel); cell loaded Janus type tissue construct (bottom leftmost panel); and cell aligning on pillars over 5 days whereas bottom squares cells continue to spread and form monolayer (bottom right panels).

[0053] DETAILED DESCRIPTION

[0054] Systems, articles, and methods related to the fabrication microgels with non-spherical shapes and anisotropy are described herein. In one aspect, a technique to fabricate microgels is generally described. In some embodiments, by tuning the aspect ratio of the microgels, it is possible to incorporate anisotropic behavior within the microgels. Such anisotropic behavior can be used in certain embodiments to control and / or influence cellular arrangement in three dimensional space, and can be utilized for biomedical applications ranging from their application as injectable materials to mimicking and / or resembling biological tissues.

[0055] Microgel based modular scaffolds may be a suitable alternative to bulk hydrogels for certain applications due to factors such as their microporous architecture, injectability, and / or effective host integration upon implantation of microgels. However, it is generally challenging and insufficient to fabricate and use isotropic microparticles for the growth of tissues that have anisotropy. Moreover, to facilitate cell growth, microgels having relatively large amounts of open volume (e.g., porosity) are generally desirable over those that have limited open volume. Modular tissue engineering is a promising approach that can use hydrogels having increased open volume (e.g., porosity). Modular tissue engineering generally involves the concept that tissues are typically modular in nature and accordingly, render bulk hydrogels less desirable than microgels. Microgels may be used as building blocks for certain modular tissue engineering applications. Microgels are relatively small hydrogel particles that can have properties similar to bulk hydrogels, but also exhibit certain advantages, for example, a higher surface area to volume ratio, improved cell infiltration, and / or anastomosis post implantation, etc. When used as cell carriers, microgels may have advantageous outcomes compared to bulk hydrogels, e.g., provide relatively efficient diffusion of nutrients / wastes, which may lead to enhanced cellular viability, proliferation, and / or differentiation, in some instances. When used as tissue scaffolds, implanted microgels can provide effective anastomosis with a host, e.g., owing to their micro-porous nature.

[0056] As discussed herein, light based additive manufacturing techniques may be used to fabricate certain microgels, for example, non- spherical microgels having anisotropy. Certain embodiments allow for the control of shape, sizes, and / or dimensions of non-spherical shapes.

[0057] In certain embodiments, F-light based printing can be used to fabricate macro-scale constructs with anisotropy. In some cases, this may be based on optical modulation instability (OMI) of light as it passes through a non-linear media like hydrogels (e.g., or precursors that form the hydrogel once cured). OMI can break light into filaments, which can be used to fabricate macro-scale tissue constructs that can guide cellular rearrangement in 3D.F-light based bio fabrication techniques may be employed to fabricate non-spherical microgels with internal architecture in certian cases. According to some embodiments, by varying the aspect ratio of micro-particles, the cellular arrangement can be controlled at the micro-level. Microgels such as those discussed herein can be utilized for any of a variety of applications, including tissue engineering applications. The developed fabrication modality can be utilized as an approach towards microgel synthesis in certain cases.

[0058] Certain aspects of this disclosure relate to a gel comprising a plurality of filaments. The plurality of filaments, according to some embodiments, may allow for the gel to advantageously facilitate the growth and proliferation of biological material (e.g., cells). The plurality of filaments may be arranged in some embodiments such that the gel exhibits an anisotropic structure. Without wishing to be bound by any particular theory, it is generally believed that anisotropic gels may provide a desirable substrate for cell growth, as cells may grow along directions associated with the anisotropy. Given that cellular arrangement is related to tissue functionality, such anisotropy may allow the production of tissues with cells having defined and / or controlled arrangements thereby producing tissues with desirable functionality (e.g., muscle tissue). Accordingly, the plurality of filaments, in some embodiments, may facilitate the growth of cells in an anisotropic manner.

[0059] In some embodiments, the plurality of filaments may be substantially parallel to each other. That is, each filament present in the gel may be parallel to another filament in the gel or within 1 degree, 2 degrees, 3 degrees, 4 degrees, 5 degrees, 7 degrees, 10 degrees, 15 degrees, or 20 degrees of parallel to another filament in the gel. While in some cases each filament as a whole may be substantially parallel to another filament in the gel, in some embodiments only a portion of each filament is substantially parallel to another filament in the gel. Accordingly, throughout the present disclosure, the plurality of filaments that are substantially parallel to one another may involve each filament being substantially parallel to one another in whole or each filament being substantially parallel to one another in part, but in either case, the plurality of filaments are considered to be substantially parallel to each other. In some embodiments, the plurality of filaments are aligned such that the majority of filaments are arranged wherein a long axis parallel to the maximum dimension of each filament is within 20 degrees of parallel to each other. In some cases, the plurality of filaments may be substantially parallel or aligned such that at least 90% of the plurality of filaments is aligned to be at an angle that is less than 20 degrees, less than 15 degrees, less than 10 degrees, less than 7 degrees, less than 5 degrees, less than 4 degrees, less than 3 degrees, less tan 2 degrees, or less than 1 degree of the average alignments of all of the plurality of filaments.

[0060] In some embodiments, at least some of the plurality of filaments are coupled to each other. Some of the plurality of filaments may be bonded and / or connected to one another such that the plurality of filaments form at least a partially interconnected network. In some embodiments, the plurality of filaments may be coupled to each other after exposure to ultraviolet light and / or relatively high temperatures, which may cross-link at least some of the filaments together such that some of the filaments are coupled together. In some embodiments, the plurality of filaments are annealed to each other. In some embodiments, the plurality of filaments and / or the gel comprising the plurality of filaments may be annealed such that the plurality of filaments are coupled to together. Without wishing to be bound by any particular theory, by annealing the plurality of filaments to each other, the gel may have an open volume and / or a porosity that is relatively high and advantageous for cell growth. In some embodiments, gaps between at least some of the plurality of filaments may be present. In some embodiments, a plurality of filaments and / or the gels comprising the filaments may be annealed at elevated temperatures (e.g., at least 25 degrees Celsius, at least 30 degrees Celsius, at least 35 degrees Celsius, at least 40 degrees Celsius, at least 50 degrees Celsius, at least 60 degrees Celsius, at least 70 degrees Celsius, at least 80 degrees Celsius, etc.), for any of variety of suitable durations (e.g., at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 24 hours etc.

[0061] In some embodiments, the plurality of filaments are formed via a bottom-up approach. That is, the plurality of filament may be formed in some embodiments by selectively curing and / or cross-linking a liquid comprising monomers capable of undergoing photopolymerization. The liquid may further comprise one or more photoinitiators to facilitate the initiation of the photopolymerization reaction. Example 1 below describes one example of a process of forming the plurality of filaments. In some embodiments, the plurality of filaments may be fabricated via exposure to collimated electromagnetic radiation (e.g., a plurality of light beams). While any of a variety of methods may be used to produce collimated electromagnetic radiation, one example method involves exposing a reservoir comprising a liquid to electromagnetic radiation (e.g., ultraviolet light) such that the electromagnetic radiation collimates as the electromagnetic radiation propagates through the reservoir thereby selectively curing the liquid exposed to the collimated light. Exposure to collimated light, in some embodiments, may form a filamentous structure associated with the gel.

[0062] In some embodiments, the plurality of filaments may be interconnected, thereby forming a filamentous structure. The filamentous structure, according to some embodiments, may have a relative high porosity that facilities cellular growth. Pores, channels, and / or open volume between each of the filaments may allow for cells and / or other biological material to grow in and / or along such structures such that the resulting tissue has anisotropy that resembles that anisotropy of the filamentous network. Non-limiting examples of such parameters are discussed in more detail herein. The relatively large open volume of the filamentous structure may also for advantageous cell infiltration into the gel.

[0063] In some embodiments, the article comprises a gel. In some embodiments, the gel comprises a first domain comprising a polymeric structure and a second domain comprising a fluid interspersed with the first domain. Accordingly, in some embodiments, the gel may be relatively porous. The relatively porous structure of the gel may facilitate the growth and / or proliferation of biological material (e.g., cells), as the biological material may be capable of infiltrating the gel via the second domain. In some embodiments, the gel is a hydrogel. That is, the gel may be configured to uptake and / or maintain a quantity of liquid (e.g., water) within pores (e.g., the second domain) of the gel. Gels having relatively high porosity and / or capable of holding liquid may be advantageous as a platform and scaffold for cell growth in vivo. In some embodiments, the gel may be a microgel.

[0064] In some embodiments, the gel may have any of a variety of suitable lengths. In some embodiments, the length of the gel is relatively small. That is, the maximum dimension of the gel is relatively small. In some embodiments, the maximum dimension of the gel is less than or equal to 1 mm, less than or equal to 900 micrometers, less than or equal to 800 micrometers, less than or equal to 700 micrometers, less than or equal to 600 micrometers, or less than or equal to 500 micrometers. In some embodiments, the maximum dimension of the gel is greater than or equal to 500 micrometers, greater than or equal to 600 micrometers, greater than or equal to 700 micrometers, greater than or equal to 800 micrometers, greater than or equal to 900 micrometers, or greater than or equal to 1 mm. Combinations of these ranges are possible (e.g., less than or equal to 1 mm and greater than or equal to 500 micrometers). Other ranges are also possible.

[0065] In some embodiments, the gel has a relatively high average aspects ratio. In some embodiments, the average aspect ratio of the gel is equivalent to the average of the ratios of the maximum dimension across the gel to the smallest dimension across gel. In some embodiments, the average aspect ratio of the gel is greater than or equal to 2: 1, greater than or equal to 3: 1, greater than or equal to 4: 1, greater than or equal to 5: 1, greater than or equal to 6: 1, greater than or equal to 7:1, greater than or equal to 8:1, greater than or equal to 9:1, or greater than or equal to 10:1. In some embodiments, the average aspect ratio of the gel is less than or equal to 10:1, less than or equal to 9:1, less than or equal to 8:1, less than or equal to 7:1, less than or equal to 6:1, less than or equal to 5:1, less than or equal to 4:1, less than or equal to 3:1, or less than or equal to 2:1. Combinations of these ranges are possible (e.g., greater than or equal to 2:1 and less than or equal to 2). Other ranges are also possible. In some embodiments, the aspect ratio of the gel may at least partially influence whether the gel comprises the plurality of filaments. In some embodiments, a gel having a relatively low average aspect ratio may comprise a relatively isotropic structure. In some embodiments, a gel having a relatively high average aspect ratio may comprise a relatively anisotropic structure. The structure of the gel and the dependent of aspect ratio on the structure of the gel is described in greater detail below. See also Example 1.

[0066] In some embodiments, the gel comprises a relatively high open volume. In some embodiments, the gel has an open volume greater than or equal to 5 vol%, greater than or equal to 10 vol%, greater than or equal to 15 vol%, greater than or equal to 20 vol%, greater than or equal to 25 vol%, greater than or equal to 30 vol%, greater than or equal to 35 vol%, or greater than or equal to 40 vol%. In some embodiments, the gel has an open volume less than or equal to 40 vol%, less than or equal to 35 vol%, less than or equal to 30 vol%, less than or equal to 25 vol%, less than or equal to 20 vol%, less than or equal to 15 vol%, less than or equal to 10 vol%, or less than or equal to 5 vol%. Combinations of these ranges are possible (e.g., greater than or equal to 5 vol% and less than or equal to 40 vol%). Other ranges are also possible.

[0067] In some embodiments, a plurality of mammalian cells may be contained within at least a portion of the open volume of the gel. As described elsewhere in this disclosure, the gel may in some cases be capable of facilitating the growth of biological materials, and such growth may, in some embodiments, involve biological material (e.g., mammalian cells) located in the open volume of the gel. The relatively high porosity of the gel, as indicated by the relatively high open volume of the gel, may be a desirable environment for cell culture.

[0068] In some embodiments, the gel comprises a plurality of gel subunits. The plurality of gel subunits may in some cases be annealed and / or coupled together to form the gel, as described elsewhere in the disclosure. In some embodiments, the plurality of gel subunits comprises microgels. In some embodiments, each of the plurality of gel subunits may have any of a variety of a cross-sectional shapes. In some embodiments, the plurality of gel subunits may have a cross-section that is and / or resembles a polygon, a rectangle, a square, a triangle, a circle, and / or other geometric pattern. In some embodiments, the plurality of gel subunits may be non- spherical. Each of the gel subunits may be referred to as a gel, and therefore, the gel described throughout this disclosure may involve a single gel subunit and / or a plurality of gel subunits as both structures can be considered gels and have any of the properties described in the present disclosure.

[0069] In some embodiments, the gel comprises a plurality of gel subunits comprising the plurality of filaments. The plurality of filaments within at least some of the plurality of gel subunits may in some cases provide an anisotropic structure that may advantageously promote and / or direct cell growth along directions associated with each of the filaments (e.g., substantially parallel to each of the filaments). It should be noted that, in certain embodiments, not all of the plurality of gel subunits of the gel comprise the plurality of filaments. In some embodiments, at least some of the plurality of gel subunits do not comprise the plurality of filaments, but rather have a structure that is relatively isotropic. That is, at least some of the plurality of gel subunits may be capable of facilitating the growth and proliferation of biological material (e.g., cells) in a relatively isotropic manner. Accordingly, the gel described throughout the present disclosure may comprise a first portion having anisotropy and a second portion having isotropy (see, for example, FIG. 19C).

[0070] In some embodiments, the gel comprises a cross-linked polymer network. The crosslinked polymer network, in some embodiments, defines a plurality of channels. That is, the cross-linked polymer network of the gel may be arranged such that a plurality of channels are present between portions of the cross-linked polymer network. In some embodiments, the plurality of gel subunits that make up the gel may be arranged such that the plurality of channels are present between portions of the cross-linked polymer network. The channels may advantageously allow for the growth and / or proliferation of biological material (e.g., cells) along at least some of the channel. Growth along such channels may allow for the formation of vasculature, and / or other biological structures having anisotropy.

[0071] In some embodiments, some or all of the plurality of channels may have any of a variety of suitable channels widths. Different channels may independently have the same or different channel widths. In some embodiments, at least some of the plurality of channels have a channel width of greater than or equal to 200 micrometers, greater than or equal to 300 micrometers, greater than or equal to 400 micrometers, greater than or equal to 500 micrometers, greater than or equal to 600 micrometers, or greater than or equal to 700 micrometers. In some embodiments, at least some of the plurality of channels have a channel width of less than or equal to 700 micrometers, less than or equal to 600 micrometers, less than or equal to 500 micrometers, less than or equal to 400 micrometers, less than or equal to 300 micrometers, and less than or equal to 200 micrometers. Combinations of these ranges are possible (e.g., greater than or equal to 200 micrometers and less than or equal to 700 micrometers). Other ranges are also possible.

[0072] In some embodiments, each of the plurality of channels may have any of a variety of suitable channel lengths. Different channels may independently have the same or different channel lengths. In some embodiments, at least some of the plurality of channels have a channel length of greater than or equal to 8 mm, greater than or equal to 1 cm, greater than or equal to 1.2 cm, greater than or equal to 1.4 cm, greater than or equal to 1.6 cm, greater than or equal to 1.8 cm, greater than or equal to 2 cm. In some embodiments, at least some of the plurality of channels have a channel length of less than or equal to 2 cm, less than or equal to 1.8 cm, less than or equal to 1.6 cm, less than or equal to 1.4 cm, less than or equal to 1.2 cm, less than or equal to 1 cm, or less than or equal to 8 mm. Combinations of these ranges are possible (e.g., greater than or equal to 8 mm and less than or equal to 2 cm). Other ranges are also possible.

[0073] In some embodiments, the gel comprises any of a variety of polymeric materials. In some embodiments, the gel comprises polymeric materials suitable for cell growth. In some embodiments, the gel comprises polymeric material capable of undergoing and / or have undergone a photopolymerization process. In some embodiments, the gel comprises and / or is derived from gelatin methacryloyl and / or polyethylene glycol diacrylate. In some embodiments, the gel comprises and / or is derived from gelatin methacryloyl, polyethylene glycol diacrylate, and / or Lithium Phenyl (2,4,6-Trimethylbenzoyl) Phosphinate (LAP). In some embodiments, the gel comprises and / or is derived from a thiolated gelatin, a PEG crosslinker, and LAP. In some embodiments, any suitable photo-initiator capable of crosslinking GelMA and PEGDA may be used in lieu or in combination with LAP (e.g., Irgacure 1929). In some embodiments, the gel may be derived from reactions involving thiol-ene click chemistry and / or free radical polymerization.

[0074] In some embodiments, as mentioned above, the gel is a hydrogel. That is, the gel may be at least partially saturated with a fluid, such as water and / or a cell culture medium (e.g., saline solutions such as phosphate buffer saline (PBS)). Accordingly, in some embodiments, the gel may be hydrophobic such that the gel can absorb, adsorb, and / or otherwise retain fluids. Hydration of the gel may be desirable prior to administering the gel and / or a mixture comprising the gel to a subject, as noted below.

[0075] Certain aspects of the present disclosure generally relate to systems for fabricating gels. In some embodiments, the system comprises one or more mirrors. The mirrors may, in some embodiments, allow for gels having any of a variety of cross-sectional shapes, e.g., as described above, to be fabricated in a bottom-up manner. The mirrors may reflect light that is emitted and modulated to create the desired cross-sectional shape of the gel. The emitted light (e.g., electromagnetic radiation) may be directed to a liquid, as described below, which may cure the liquid to form the gel. In some embodiments, the plurality of mirrors may be a digital micromirror device. The plurality of mirrors may allow for relatively precise control of the cross-sectional shape of the gels. In some embodiments, the system comprises a source of electromagnetic radiation. In some embodiments, the source of electromagnetic radiation is configured to emit radiation (e.g., light) having any of a variety of suitable wavelengths. In some embodiments, the source of electromagnetic radiation emits radiation having wavelengths greater than or equal to 100 nm and less than or equal to 450 nm (e.g., ultraviolet light). It should be noted that the source of electromagnetic radiation may also directly and / or indirectly emit radiation having other wavelengths. For example, in some embodiments, the source of electromagnetic radiation may emit radiation having wavelengths in the infrared regime of the electromagnetic spectrum (e.g., wavelengths greater than or equal to 780 nm to 1 mm).

[0076] In some embodiments, the source of electromagnetic radiation is positioned relative to the plurality of mirrors such that at least some of the emitted radiation is reflected by the plurality of mirrors. That is, the plurality of mirrors may be capable of controlling and / or directing at least some of the emitted radiation from the source of electromagnetic radiation. The emitted radiation may be reflected by the plurality of mirrors such that the emitted radiation initiates a photopolymerization reaction to form the gel. In some embodiments, the plurality of mirrors may allow for gels having intricate geometries (e.g., star-shaped, or other shapes such as any of those described herein) to be formed.

[0077] In some embodiments, the system comprises a liquid reservoir. The liquid reservoir may contain a liquid comprising one or more monomers (e.g., gelatin methacryloyl and / or polyethylene glycol diacrylate), a photoinitiator, and / or a solvent such that, when the liquid is exposed to electromagnetic radiation of a suitable wavelength, the liquid polymerizes to form the gel. The liquid reservoir, accordingly, may be configured to, not only act as a source of the liquid, but also in some cases transmit at least a portion of the emitted electromagnetic radiation through the reservoir.

[0078] In some embodiments, the reservoir of liquid is configured to hold the liquid such that liquid depth is relatively shallow. The relatively shallow depth of the liquid may allow for gels having a relatively small maximum dimension to be formed. Moreover, sufficient depth may be needed to allow for light transmitted through the liquid to collimate within the liquid to thereby produce the plurality of filaments within the gel. In some embodiments, the liquid has a depth that is less than or equal to 1 mm, less than or equal 0.5 mm, less than or equal 0.4 mm, less than or equal 0.3 mm, less than or equal 0.2 mm, or less than or equal 0.1 mm. In some embodiments, the liquid has a depth that is greater than or equal to 0.1 mm, greater than or equal to 0.2 mm, greater than or equal to 0.3 mm, greater than or equal to 0.4 mm, greater than or equal to 0.5 mm, or greater than or equal to 1 mm. Combinations of these ranges are possible (e.g., less than or equal to 1 mm and greater than or equal to 0.1 mm). Other ranges are also possible.

[0079] In some embodiments, the system comprises a vessel. In some embodiments, the vessel is configured to hold and / or house the reservoir of liquid. In some embodiments, the vessel has one or more walls configured to transmit electromagnetic radiation from the source of electromagnetic radiation into the reservoir of liquid. In some embodiments, the one or more walls may be at least partially transparent to wavelengths of electromagnetic radiation capable of curing and / or crosslinking the liquid into the gel.

[0080] In some embodiments, the system is capable of forming gels comprising the plurality of filaments. The electromagnetic radiation emitted by the source of electromagnetic radiation may reflect off of at least some of the mirrors such that the reflected radiation is transmitted through the vessel into the reservoir of liquid (see, e.g., FIGS. 1, 2, and 10). Without wishing to be bound by any particular theory, when electromagnetic radiation enters the liquid, the electromagnetic radiation may form collimated beams of radiation (e.g., a plurality of light beams) that can selectively cure the liquid into gels having a plurality of filaments. That is, a portion of the electromagnetic radiation transmitted through the liquid may be partially split into distinct optical paths capable of forming the gel comprising the plurality of filaments. In addition to the advantages described regarding the filamentous structure, the system itself may be also desirable in some embodiments, as the system may be capable producing such gels with relatively high throughput and / or efficiency.

[0081] As mentioned above, the plurality of light beams may be at least partially responsible for forming the plurality of filaments within the gel. However, as the plurality of light beams only form upon transmission through the liquid, reservoirs of liquid having a relatively low depth may not have sufficient liquid to collimate the emitted electromagnetic radiation from the source. Accordingly, the gels fabricated from exposing a reservoir of liquid having a low depth may have a relatively isotropic structure, as opposed to the relatively anisotropic structure of gels fabricated using reservoirs of liquid with relatively large depths.

[0082] In some embodiments, the system is capable of fabricating the gel at a relatively high throughput. In some cases, the gel may be formed after being exposed to the electromagnetic radiation for less than or equal to 60 seconds, less than or equal to 45 seconds, less than or equal to 30 seconds, less than or equal to 20 seconds, less than or equal to 15 seconds, less than or equal to 10 seconds, or less than or equal to 5 seconds. In some embodiments, the gel may be formed after being exposed to the electromagnetic radiation for greater than or equal to 5 seconds, greater than or equal to 10 seconds, greater than or equal to 15 seconds, greater than or equal to 20 seconds, greater than or equal to 30 seconds, greater than or equal to 45 seconds, greater than or equal to 60 seconds. Combinations of these ranges are possible (e.g., less than or equal to 60 seconds and greater than or equal to 5 seconds). Other ranges are also possible.

[0083] Certain aspects of the present disclosure generally relate to methods for fabricating gels. In some embodiments, the method comprises exposing the liquid comprising a polymer precursor (e.g., such as those described above) to a plurality of light beams (e.g., collimated light) such that the plurality of light beams causes the polymer precursor to form a plurality of filaments. In some embodiments, exposing the liquid to the plurality of light beams may occur using the system described elsewhere in this disclosure. In some embodiments, the exposing the liquid to the plurality of light beams may involve the plurality of light beams forming after transmitting at least partially through the liquid precursor. Accordingly, the precursor solution may be initially exposed to electromagnetic radiation without a plurality of light beams, but after the electromagnetic radiation is transmitted through a portion of the precursor solution, the plurality of light beams form, thereby exposing the liquid precursor solution to the plurality of light beams.

[0084] In some embodiments, the liquid comprising the polymer precursor may be exposed to electromagnetic radiation have any of a variety of wavelengths. In some embodiments, the electromagnetic radiation described throughout this disclosure involves ultraviolet light. As mentioned above, ultraviolet light may have a wavelength greater than or equal to 100 nm and less than or equal to 450 nm. In some embodiments, the source of electromagnetic radiation exposes the liquid comprising the prepolymer precursor to ultraviolet light.

[0085] In some embodiments, after exposure of the liquid precursor to the plurality of light beams to form the plurality of filaments, the method comprises cross-linking at least some of the filaments to form a gel. By cross-linking at least some of the filaments together, some of the filaments may be coupled and / or bonded together to form the gel while having a relatively porous structure. In some embodiments, the gel, after the cross-linking, has an open volume of greater than or equal to 5 vol%, or other open volumes such as those described herein.

[0086] In some embodiments, the method comprises administering the gel and / or a mixture comprising the gel defined by a plurality of filaments to a subject. Since the gel may be capable of facilitating the growth and / or proliferation of biological material (e.g., cells), the gel may administered to a subject such that the gel may act as a scaffold and / or substrate for cell growth. Accordingly, the subject, in some embodiments, may be in need of the administration of a gel to facilitate the growth of tissue and / or closure of a wound. In some embodiments, the subject is a living organism. According to certain embodiments, the subject is a human. The subject may also be a non-human mammal in some cases. The subject may be in need for the administration of the mixture to treat a physiological ailment.

[0087] The mixture may be administered to the subject using any of a myriad of devices. The mixture may be administered using a syringe, or more than one syringe in some cases. In some embodiments, the mixture is administered using a catheter, such as a double-lumen catheter, a mixing tip, and / or a needle, or via spraying of the mixture. In some embodiments, the gel may be administered to the subject via injection such that the ability of the gel to facilitate the growth and proliferation of biological material is not harmed (see FIGS. 16, 17, 19A-19B). That is, the filamentous structure associated with the gel and / or the aspect ratio associated with the gel may not be altered or otherwise disrupted after administration to the subject.

[0088] U.S. Provisional Patent Application Serial No. 63 / 697,370, filed September 20, 2024, entitled “Systems, Articles, and Methods Related to Hydrogels,” by John, et al., is incorporated herein by reference in its entirety.

[0089] The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

[0090] EXAMPLE 1

[0091] Certain articles, systems, and methods related to microgel fabrication are demonstrated in this example. The microgel fabrication is based on optical modulation instability (OMI) of light as it passes through non-linear solvents like hydrogel. OMI can cause the light to break into filaments. As this phenomenon is dependent on aspect ratios, it allows for some freedom to fabricate microgels with or without fibrillated architecture. Such microgels can control cellular arrangement in 3D.

[0092] Materials and methods

[0093] Synthesis of GelMA

[0094] GelMA was synthesized as follows: 10 gm of Gelatin Type A (Sigma Aldrich, US) was dissolved in carbonate buffer (pH: 9.0) at 40 degrees under constant magnetic stirring. 2 hours after complete dissolution, methacrylic anhydride (1ml, Sigma Aldrich, US) was added dropwise and the reaction was carried out for 4 hours. The reaction was stopped by adjusting the pH to 7.0 and the solution was dialyzed for 3 days (14 kDA dialysis bag) with water changes in every 12 hours. Thereafter the solution was lyophilized for 2 days to obtain GelMA flakes that were stored at -20 degrees until used further.

[0095] Preparation of photo resins Photo resins were prepared either by using pure GelMA (7.5% w / v) or blending GelMA with different concentrations of Polyethylene Glycol Diacrylate (PegDA, Mn750 Da, Sigma Aldrich, US). Briefly, GelMA, PegDA and 0.1% Lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP, Sigma Aldrich, US) were dissolved at 40 degrees in PBS for 30 min. to obtain homogenous solution. Three types of photo resins were prepared for fabrication of microgels (GelMA 7.5% w / v; GelMA 7.5%, PegDA 2.5% w / v, P2.5G7.5; and GelMA 7.5%, PegDA 5% w / v, P5G7.5).

[0096] F -light based fabrication ofmicrogels

[0097] Microgels were fabricated using a projection-based system that can transmit digital shapes using DMDs onto a cubical vial (51 mm X 51 mm) containing photo resins. Briefly, different shapes of microgels were designed on PowerPoint. Gray scaling format was used wherein individual shapes represent 100% light intensity. The designed images were converted to jpeg format and were used for projections. Photo resins were poured into cubical vials, kept at 4 degrees for physical gelation and were then placed at the platform where projector selectively crosslinks the desired micro areas representing different microparticle shapes. After 50 sec. of exposure, cubical vials were stored at 37 degrees to melt the uncross linked photo resin and microgels were collected on a cell strainer (100 micrometers). For fabrication with cells, all protocols were followed under a sterile environment and photo resin was mixed with cells before pouring it into cubical vial. 7.5% GelMA based bioink was utilized for all cell experiments.

[0098] Rheological evaluation of Photo Resins

[0099] For rheological analysis, photo resin discs (8 mm diameter, 1 mm thickness) were fabricated using projections. Rheological properties were evaluated using frequency sweep analysis between 0.1 to 10 Hz at 1 mm plate gapping using Instron Parallel Plate rheometer. G’ values were utilized for stiffness calculations.

[0100] Injectability evaluation

[0101] To evaluate injectability of microgels, microrods of around 500 micrometers were fabricated and collected using cell strainer. Collected microrods were transferred to a 5 ml syringe and were injected through a 20 Gauge blunt nozzle. To visualize the microrods inside the channel, transparent 20 Gauge glass channels were utilized and images were captured using a fluorescent microscope. Fabrication of MAPs

[0102] For fabrication of MAPs, collected microgels (rods and squares) were resuspended in 0.1% LAP solution, centrifuged at 10,000 rpm for packing and were placed in PDMS molds (2 mm thick, 8mm diameter). The microgels were further stabilized by exposure to 405 nm light and the fabricated MAP discs were then utilized for cell seeding. All procedures were performed in sterile environment. Rhodamine solution (0.01%) was used for microgel fabrication for observation under confocal microscope.

[0103] MAP characterization

[0104] To evaluate the stiffness of MAPs composed of rods and squares, MAP discs were synthesized as stated above. Rheological properties were evaluated using frequency sweep analysis between 0.1 to 10 Hz at 1 mm plate gapping using Instron Parallel Plate rheometer. G’ values were utilized for stiffness calculations.

[0105] For porosity calculations, 2D slices from z-stacked images were obtained and the porosity was calculated as reported previously.

[0106] For cell infiltration in MAPs, cell seeded MAPs (microparticle tracked with FITC, cells tracked green) were imaged at different time points. 3D images were utilized to calculate depth of penetration. A minimum of 3 samples were utilized at each time points and a minimum 3 independent experiments were performed.

[0107] Fabrication of modular hydrogel sheets

[0108] For modular hydrogel sheet fabrication, individual micro squares were placed in an array before projection. Multiple projection of microgel arrays was then utilized to obtain modular sheet (2 cm X 2 cm).

[0109] Fabrication of Janus assembly

[0110] For fabrication of Janus assembly, individual squares were fabricated using projections. Fabricated squares were then seeded with cells (HDF) and were allowed to stay in incubator for 24 hours to allow adherence of first cells (Green tracked). Following 24 hours, pillars were bio fabricated on top of previously constructed squares. Cell loaded micropillars (red tracked HDF) constructed on top of squares were then kept in culture and were monitored over 5 days.

[0111] Cell culture Human dermal fibroblasts (HDF) were cultured in Dulbecco’s Modified Eagle Medium + GlutaMAX™ (Gibco) with 10% fetal bovine serum (Gibco) and 1% penicillin streptomycin (Gibco) at 37 °C with 5% CO2 humidified atmosphere. Once the cells reached the confluency, they were trypsinized using TrypLE™ (Gibco) and utilized for future experiments. Similar protocols were adapted for C2C12 cells. For bio fabrication of microgels, both cells were used at concentration of 1.5 million / ml. of photo resin. For cell seeding, 20,000 cells were utilized. For certain experiments, cell were tracked either using red cell tracker (CellTracker™ Red CMTPX, Thermo Fisher Scientific, US) or green cell tracker (CellTracker™ Green CMFDA, Thermo Fisher Scientific, US) following the manufacturer’s protocol.

[0112] Cell viability. Proliferation, and cytoskeleton staining

[0113] The viability of cells was evaluated using calcein AM (1 micromolar, Invitrogen™) and ethidium homodimer (1 micromolar, Invitrogen™) staining at room temperature for 20 min. Then fluorescent stained samples were washed with IX DPBS and visualized under confocal microscopy (Zeiss ESM 700) at 488 nm for calcein AM (green color, viable cells) and 561 nm for ethidium homodimer (red color, dead cells).

[0114] The proliferation of cells was quantified using PrestoBlue™ cell viability reagent at different time points. In short, PrestoBlue™ (Invitrogen™) working concentration solution (1:10 dilution with complete media) was prepared as per the manufacturer’s instructions. Cell culture complete media was removed from respective samples followed by the addition of PrestoBlue™ solution and incubated for 3 h at 37 °C with 5% CO2 incubator. After that, the solution was collected from each sample and absorbance was measured at 570 nm and 600 nm using Varioskan Eux plate reader (Thermo Fisher Scientific). The cell proliferation rate was directly proportional to the dye reduction percentage was calculated from the absorbance data and presented as a bar graph with standard deviation (triplicate samples were used for each time point).

[0115] The morphology of the cells was visualized using actin staining. At indicated time points, cells were fixed with 4% formaldehyde solution overnight at 4 °C then washed with IX DPBS followed by 0.1% Triton X-100 treatment for 10 min. These permeabilized samples were washed with IX DPBS thrice and blocked with 1% bovine serum albumin for 30 min then washed with IX DPBS. Then, Alexa Fluor™ 594 phalloidin (Invitrogen™) fluorescent dye was added and incubated for 30 min at room temperature. These fluorescently stained microgels were washed with IX DPBS and finally, nuclei were stained with DAPI (Invitrogen™) for 5 min. Fluorescently stained microgels were imaged under confocal microscopy (Zeiss LSM 700) for fluorescent channels 594 nm (cytoskeleton, red) and 405 nm (nuclei, blue).

[0116] Statistical analysis

[0117] All the cell culture experiments were performed in triplicate samples (n=3) and the data were expressed as mean + / - standard deviation. GraphPad Prism 9 software was used for performing statistical analysis. For determining statistical differences among the groups one-way ANOVA with Tukey test was performed and two-way ANOVA was used for comparing among and within groups. Statistical significance was set at * p<0.05, ** p<0.01.

[0118] Results and Discussion

[0119] Microgels were fabricated using GelMA (Gelatin Methacryloyl, Degree of methacrylation 90%) / PegDA (Polyethylene Glycol Diacrylate, Mn 750 Da) based photo resin wherein fraction of two components were varied to fabricate microgels of different stiffness. A 2D projector-based system which can focus collimated beams of designed geometries using DMDs on a photo resin was used. By tuning the aspect ratio of the microparticles (elongated geometries vs flattened geometries), the internal architectures (micro-filaments) can be incorporated into these fabricated microgels. The concept is based on optical modulation instability (OMI) of uniform light beam as it passes through a non-linear medium, breaking it into micro-filaments. This can result in macroscale constructs which are highly filamentous and can be utilized for controlling cellular arrangement in 3D. Such behavior of light, without wishing to be bound by any particular theory, can be utilized at micro-scales. By varying the aspect ratio of fabricated micro units, the phenomenon can be suppressed or enhanced leading to micro-units with or without internal architectures. Such fabricated units have the capacity to control cellular behavior at micro-scales leading to a applications in tissue engineering and regenerative medicine as demonstrated in FIGS. 1-2.

[0120] Firstly, the capacity of this approach to fabricate microgels of varying shapes and sizes ranging from as small as 300 micrometers to 900micrometers was demonstrated with a range of photo resin formulations as shown in FIG. 3A. Different photo resins were utilized composed of GelMA (7.5% w / v, G7.5) or GelMA blended with different concentration of PegDA (P2.5G7.5, P5G7.5, where numbers represent w / v concentration of each polymer). These formulations were demonstrated to have mechanical properties in a window of 10-50 kPa (FIGS. 3B-3C) thereby capable of targeting tissues of varying stiffness. Moreover, these microparticles were injectable and allow for applications in minimal invasive surgeries (FIGS. 3D and 11A-11B). Internal architectures were shown to be incorporated by varying aspect ratios as demonstrated in (FIG. 12). Elongated geometries like micro-rods appear fibrillated, whereas flattened geometry like square showed absence of fibrillation. Such microgels can provide anisotropic environment to cells.

[0121] Cellular arrangement defines tissue functionality, and therefore, microgel units that can control cellular arrangement in 3D may be advantageous. The effectiveness of the bio-fabricated micro-units to control cellular arrangement was demonstrated (FIG. 4A). The filamentous nature of elongated geometries like micro-rods can provide directional cues for aligning cells whereas the isotropic nature of flattened geometries like squares cells growth was less oriented (mostly spreading). Microgels encapsulating cells (Human dermal fibroblasts) were fabricated with different aspect ratios (rods represent elongated geometries whereas squares represent flattened geometries) to evaluate cellular arrangement in the fabricated micro-units over time. As seen from live / dead staining in FIG. 4B, cells were highly viable in microgels (rods and square), however morphology of cells (cellular arrangement) looked different in the two micro-units. In rods cells tend to align over 5 days whereas square shape microgels cells seems to demonstrate a random orientation. An alamar blue assay over 5 days showed that cells showed an increase in metabolic activity over days (FIG. 4C), demonstrating no toxicity from the developed bioink formulation and light exposure during fabrication. It should be noted that all the experiments were performed using 7.5% GelMA based photo-resin given that the target was soft tissue engineering. Further high-resolution confocal images of cytoskeleton demonstrated ability of the fabricated micro-units to control cellular arrangement at micro-scales (FIGS. 4D and 13). The developed microgel system was anisotropic in terms of its (non-spherical units) shape, and the microgel’s internal architecture provided freedom to tune cellular arrangements in different shapes.

[0122] As demonstrated, anisotropic microgels can be fabricated with high cellular viability and an ability to define cellular response. Several different applications of microgels are also described within this example. Due to their micro-porous architectures, modular scaffolds like micro annealed particles (MAPs) are a promising platform for cell growth. The fabrication modality described in these examples provides versatility to fabricate non-spherical shapes with ease. By controlling particle shape, cellular behaviour including cell infiltration and migration into such microporous scaffolds can be controlled.

[0123] To demonstrate anisotropic cell infiltration in MAPs, MAPs were fabricated out of two microgels configurations: squares representing flattened geometries and rods represent elongated geometries. As shown in FIG. 5 A, although MAPs composed of squares and rods showed no significant difference in overall porosity, the internal architecture of 2D stacks appeared different with more interconnected pores on MAPs composed of squares compared to MAPs composed of rods. Both MAPs demonstrated different mechanical properties, as indicated by their G’ values in FIG. 5A. This difference in pore architecture and stiffness can impact cellular infiltration. Human dermal fibroblasts (HDFs) were cultured on MAPs for 5 days and the cells were monitored. As seen from FIG. 5B, cells on both MAPs increased in number from day 1 to 5, with a relatively large covering of cells by day 5. Also, cells can be seen infiltrating through pores on both MAPs (white arrows). To quantify infiltration, volumetric rendered Z stack images were captured. As seen from FIG. 5C and FIGS. 14-15, cells begin to infiltrate into both MAPs from day 1 itself, with highest infiltration on day 5. However, depth of infiltration was dependent on particle shape. More infiltration can be seen on MAPs composed of squares compared to rods (FIG. 5C and FIG. 14). Further quantified values of cells infiltration presented in FIG. 5D shows that MAPs composed of squares allows higher cell infiltration probably due to presence of interconnected micropores. Overall, this example demonstrates how properties of bulk scaffolds can be altered by utilizing micro-modules as building blocks. By altering shapes of building blocks, scaffold mechanical properties, micro-porosities as well as cell-material interactions can be modified without modifying pre-polymers.

[0124] Encapsulating cells in microgels can promote their long-term survival and improved therapeutics when applied in regenerative medicine. Moreover, such systems offer minimal invasive surgeries making them a suitable candidate for cell delivery. Since the fabricated microrods were fibrillated, the microrods may serve as micro-fillers. As shown in FIG. 6A, the microrods can be utilized for treatment of degenerative muscular diseases, wherein they can be injected in a minimally invasive manner at the damage site. Due to the microrod’s biocompatible nature and ability to align cells, 3D bio fabricated rods carrying cells of muscle origin can be utilized in regenerative medicine for cell delivery. To demonstrate this, micro-rods of approximately 300 pm were fabricated and the ability to align cells of muscle origin was evaluated. C2C12 loaded micro-rods were checked for their biocompatibility and their capability to provide anisotropic environment to the encapsulated cells. As shown in FIG. 6B, cells were highly viable and demonstrated an aligning morphology over 5 days of culture (live-dead staining). Cytoskeleton staining (FIG. 6C) further revealed that cells begin to align from day 1 itself and populated the rods over 5 days with fused cytoskeleton observed at 5 days thereby demonstrating the formation of myotubes. Such micromodules may be promising in cell based therapeutics where the micromodules can enhance the regeneration process by providing proper niche for cells. The capacity of the fabrication modality was explored by combining these modules to fabricate different type of modular assemblies. Although tissue engineered scaffolds have shown promising results in various areas of regenerative medicine, performance upon implantation has typically been severely limited due to delayed anastomosis which can reduced their therapeutic efficacy. Most tissues in the body have a well-organized patterned vasculature that supports tissue functioning and maturation. A modular hydrogel sheet composed of array of microsquares with micro-channels embedded between them was demonstrated. As shown in FIG. 7A, micro-channel embedded modular sheets may be selectively cultured with patient derived endothelial cells within channels, providing scaffolds with patterned or well-organized vascular network surrounding micromodules that can be utilized to hold tissue specific cells. FIG. 7B shows the gross appearance and microscopic images of micro-modular assembly wherein the micro-squares can be seen connected and presence of micro-channels in between. The potential application of such micro-modular units for patterned cellular arrangement was also explored. Patient derived endothelial cells (iECs) seeded on micro-modular scaffolds showed that cells can restrict themselves to channels and by day 6, cells can form patterned architectures with high cellular viability and high expression for CD31. Such micro modular hydrogel sheets mimic the collation of micro-units connected by vasculature as seen in native tissues. Such sheets may facilitate quicker anastomosis and cell survival when implanted at some defect site.

[0125] FIGS. 9A-C describe results demonstrating the regenerative capacity of microgels upon implantation in subcutaneous cavity in animals. Both microgels (rods and squares) were loaded with QK (angiogenic peptide) to evaluate their angiogenic potential and neo-tissue formation. 200 microliters of each shape were administered using a 21G needle. Cells may infiltrate through the micro-pores of injected microgels. The microgels generally mimic the migration of surrounding cells into damaged areas where microgels were loaded. This may be considered similar to positioning microgels, having different shapes, in injured tissue. For example, it may be advantageous for squares to be used as wound fillers whereas rods may be used for injuries involving muscle tissue due to the general alignment of muscle tissue. These microgels can attract the cells from surroundings helping facilitate a relatively quick recovery.

[0126] This example also generally describes the impact of collating two different units (anisotropic rods and isotropic squares) together into one module. Such units combined into single scaffolds can provide two different regions for cells. A region composed of flattened geometry (e.g., such as a shape resembling a square), which can be utilized for cell seeding stacked on top with a region embedded with cells that can align cells, was fabricated using multiple projections. Such an assembly can be referred to as a Janus Type Assembly as shown in FIG. 8A. Cells tracked with green and red trackers were utilized to show cell loaded regions in the Janus type assembly as shown in FIG. 8B. Cells in different regions showed different orientations (FIG. 8C), with cells showing a more aligned orientation on pillars (see FIG. 18) and a monolayer with random orientation on base. Such Janus type modular scaffolds can be utilized for different areas in regenerative medicine.

[0127] Conclusion

[0128] An F-light based bioprinting technique was demonstrated in this example for fabrication of non-spherical microgels with anisotropy for biomedical applications. Based on the OMI of light as it travels through hydrogels, it was demonstrated how such phenomenon can be controlled based on aspect ratios. Such control allowed for the fabrication of microgels with internal architectures, that can be utilized for controlling cellular arrangement in 3D. Through a series of in-vitro studies, it was shown how aspect ratios can be utilized at micro-scales to target different areas of regenerative medicine. Overall, the above microfabrication approach may improve modular tissue engineering due to its ability to fabricate anisotropic microgels at high throughput.

[0129] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0130] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0131] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0132] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0133] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0134] As used herein, “wt%” is an abbreviation of weight percentage. As used herein, “at%” is an abbreviation of atomic percentage.

[0135] Some embodiments may be embodied as a method, of which various examples have been described. The acts performed as part of the methods may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include different (e.g., more or less) acts than those that are described, and / or that may involve performing some acts simultaneously, even though the acts are shown as being performed sequentially in the embodiments specifically described above.

[0136] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0137] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMSWhat is claimed is:

1. An article, comprising: the gel defined by a plurality of filaments substantially parallel to each other, the gel having an average aspect ratio of at least 2: 1 and an average length of between 500 micrometers and 1 mm, wherein the gel has an open volume of greater than or equal to 5 vol%.

2. The article of claims 1, further comprising a plurality of mammalian cells contained within the open volume of the gel.

3. A method, comprising: administering the article of any one of claims 1-2 to a subject.

4. The article of any one of claims 1-3, wherein at least some of the plurality of filaments are coupled to each other.

5. The article of any one of claims 1-4, wherein at least some of the plurality of filaments are annealed to each other.

6. The article of any one of claims 1-5, wherein the gel has an aspect ratio of at least 5:1.

7. The article of any one of claims 1-6, wherein the gel has average length less than or equal to 800 micrometers.

8. The article of any one of claims 1-7, wherein the gel has an open volume greater than or equal to 10 %vol.

9. The article of any one of claims 1-8, wherein the plurality of channel have a diameter greater than or equal to 300 micrometers.

10. The article of any one of claims 1-9, wherein a cross-section of the gel has a polygonal shape.

11. The article of any one of claims 1-10, wherein the plurality of filaments are interconnected such that gaps are present between at least some of the filaments.

12. The article of any one of claims 1-11, wherein at least some of the filaments are within 20 degrees of parallel to each other.

13. The article of any one of claims 1-12, wherein the gel is configured to facilitate the proliferation of biological cells.

14. The article of any one of claims 1-13, wherein the gels comprise a polymer derived from monomers comprising gelatin methacryloyl and / or polyethylene glycol diacrylate.

15. An article, comprising: a gel comprising a crosslinked-polymer network defining a plurality of channels therein, having an average diameter that is greater than or equal to 200 micrometers and an average length of between 8 mm and 2 cm, wherein the gel has an open volume of greater than or equal to 5%.

16. The article of claim 15, wherein the plurality of channels have a diameter greater than or equal to 300 micrometers.

17. The article of any one of claims 15-16, wherein the plurality of channels have a length less than or equal to 1 cm.

18. The article of any one of claims 15-17, wherein the gel has an aspect ratio of at least 5:

119. The article of any one of claims 15-18, wherein the gel has average length less than or equal to 800 micrometers.

20. The article of any one of claims 15-19, wherein the gel has an open volume greater than or equal to 10 %vol.

21. The article of any one of claims 15-20, wherein a cross-section of the gel has a polygonal shape.

22. The article of any one of claims 15-21, wherein the gel is configured to facilitate the proliferation of biological cells.

23. The article of any one of claims 15-22, wherein the particles comprise a polymer derived from monomers comprising gelatin methacryloyl and / or polyethylene glycol diacrylate.

24. A system, comprising: a plurality of mirrors; a source of electromagnetic radiation capable of emitting electromagnetic radiation such that at least a portion of the electromagnetic radiation is reflected by at least some of the plurality of mirrors; a liquid reservoir comprising a liquid having a liquid depth of less than or equal to 0.5 mm; and a vessel comprising the liquid reservoir and configured to allow the transmission of at least a portion of the electromagnetic radiation reflected by the plurality of mirrors through a wall of the vessel and through a portion of the liquid.

25. The system of claim 24, wherein the liquid reservoir has a liquid depth of less than or equal to 0.4 mm.

26. The system of any one of claims 24-25, wherein a portion of electromagnetic radiation transmitted through the liquid is at least partially split into distinct optical paths capable of forming filamentous structures in the particle.

27. The system of claims 24-26, wherein the plurality of mirrors is provided by a digital micromirror device (DMD).

28. A method, comprising: administering a mixture to a subject, wherein the mixture comprises a gel defined by a plurality of filaments substantially parallel to each other, the gel having an average aspect ratio of at least 2: 1 and an average length of between 500 micrometers and 1 mm, wherein the gel has an open volume of greater than or equal to 5 vol%.

29. The method of claim 28, wherein the gel has an aspect ratio of at least 5:1.

30. The method of any one of claims 28-29, wherein the gel has average length less than or equal to 800 micrometers.

31. The method of any one of claims 28-30, wherein the gel has an open volume greater than or equal to 10 %vol.

32. The method of any one of claims 28-31, wherein a cross-section of the gel has a polygonal shape.

33. The method of any one of claims 28-32, wherein the plurality of filaments are interconnected such that gaps are present between at least some of the filaments.

34. The method of any one of claims 28-33, wherein at least some of the filaments are within 20 degrees of parallel to each other.

35. The method of any one of claims 28-34, wherein the gel is configured to facilitate the proliferation of biological cells.

36. A method, comprising: exposing a liquid containing a polymer precursor having a depth of no more than 1 mm to a plurality of light beams having a wavelength greater than orequal to 100 nm and less than or equal to 450 nm, wherein the plurality of light beams causes the polymer precursor to form a plurality of filaments; and cross-linking at least some of the filaments to form a gel having an open volume of greater than or equal to 20%.

37. The method of claim 36, wherein the gel has an aspect ratio of at least 5:1.

38. The method of any one of claims 36-37, wherein the gel has average length less than or equal to 800 micrometers.

39. The method of any one of claims 36-38, wherein the gel has an open volume greater than or equal to 10 %vol.

40. The method of any one of claims 36-39, wherein a cross-section of the gel has a polygonal shape.

41. The method of any one of claims 36-40, wherein the plurality of filaments are interconnected such that gaps are present between at least some of the filaments.

42. The method of any one of claims 36-41, wherein at least some of the filaments are within 20 degrees of parallel to each other.43 The method of any one of claims 36-42, wherein the gel is configured to facilitate the proliferation of biological cells.

44. The method of any one of claims 36-43, wherein the administering the mixture to the subject comprises injecting the mixture into a subject.

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