Micropatterned peg composite granular hydrogels, compositions, and their uses

Abstract

Three-dimensional, biocompatible, micropatterned, composite granular hydrogel scaffold compositions are provided. Also provided are methods of their production and use.

Description

MICROPATTERNED PEG COMPOSITE GRANULAR HYDROGELS, COMPOSITIONS,AND THEIR USESGOVERNMENT INTERESTS

[0001] This invention was made with government support under Grant No(s). 2236414 and 1842473, awarded by the National Science Foundation; and Grant No. R01 NS111518, awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0002] Hydrogel biomaterials have been widely used for tissue engineering applications in large part for their high-water content, as it mimics native tissues in the body. Currently, a limiting factor of traditionally formulated bulk hydrogels is that they lack porosity that is characteristic of native tissues. Hydrogel microparticles (HMPs) are micron-scale hydrogels formulated through multiple means like batch emulsions, microfluidics, lithography, or extrusion fragmentation.

[0003] Microgels have found increasing use as components in tissue engineering, drug delivery, and wound healing. The use of microgel building blocks confers certain benefits over traditional bulk hydrogels. Microgels exhibit controllable rheological behavior, including shearthinning, which makes them injectable. Additionally, the granular nature of microgels creates an inherently microporous network upon packing that better mimics native tissues. However, this microporous network is randomly distributed. There remains a need for tissue engineering scaffolds with aligned pores or networks of pores that better facilitate cell infiltration in tissues, enhance molecule diffusion, etc.SUMMARY

[0004] Provided are three-dimensional, biocompatible, micropatterned, composite granular hydrogel scaffolds comprising: (i) microgel particles and (ii) magnetic porogens. In some such scaffolds, the microgels and magnetic porogens are subjected to a magnetic field, such that the magnetic porogens are spatially aligned to form a plurality of columns within the scaffold prior to cross-linking or polymerization of the scaffold. Optionally, the scaffolds further comprise (iii)an interstitial matrix. Optionally, the scaffolds further comprise one or more biomolecules or bioactive agents. In some such scaffolds, the biocompatible scaffold is biodegradable.

[0005] In some such scaffolds, the microgel particles are about 5pm to about 25pm in diameter. Optionally, the microgel particles are about 10pm in diameter. In some such scaffolds, the microgel particles comprise between about 30% w / w and about 45% w / w of the scaffold. In some such scaffolds, the microgel particles comprise between about 36% w / w and about 42% w / w of the scaffold. Optionally, the microgel particles comprise about 39% w / w of the scaffold. In some such scaffolds, the microgel particles were previously lyophilized before being reconstituted and combined with the magnetic porogens.

[0006] In some such scaffolds, the magnetic porogens are about 10pm to about 150pm in diameter. Optionally, the magnetic porogens are about 80pm to about 90pm in diameter. In some such scaffolds, the matrix material of the magnetic porogens has been dissolved and diffused out of the scaffold to produce a plurality of aligned voids and microchannels within the scaffold. Optionally, a portion of the microchannels, a network of interconnected microchannels, or both, extend the length of the scaffold.

[0007] In some such scaffolds, the microgel particles comprise 4-arm polyethylene glycol (PEG) norbornene (PNB) and 4-arm PEG thiol (PTT). Optionally, the ratio of PNB:PTT in the microgel particles is between about 1 :0.5 and about 1 : 1. Further optionally, the ratio of PNB:PTT in the microgel particles is about 1 :0.9. In some such scaffolds, the interstitial matrix comprises PNB and PTT. Optionally, the ratio of PNB:PTT in the interstitial matrix is about 1 : 1. In some such scaffolds, the magnetic porogen comprises iron oxide nanoparticles encapsulated in a biocompatible, dissolvable, alginate hydrogel matrix.

[0008] In some such scaffolds, the scaffold further comprises cells.

[0009] Also provided are methods of preparing a three-dimensional, biocompatible, micropatterned, composite granular hydrogel scaffold, wherein the scaffold comprises: (i) microgel particles and (ii) magnetic porogens.

[0010] Also provided are methods of using a three-dimensional, biocompatible, micropatterned, composite granular hydrogel scaffold in a tissue engineering application, the method comprising: (i) identifying a subject in need of an engineered tissue scaffold, (ii) preparing a suitable scaffold for the application, and (iii) administering the scaffold to the subject.BRIEF DESCRIPTION OF THE FIGURES

[0011] Figure 1 shows fluorescent microscopy images of microgel populations and their respective sizes when prepared according to the homogenization parameters and ratio of continuous phase to droplet phase indicated beneath each representative image. Scale bar = 20pm.

[0012] Figure 2 shows quantification of the average diameters of microgel populations prepared according to the indicated homogenization parameters (left) and scatter plots of the individual diameters of microgels in those populations (right).

[0013] Figure 3 shows the composition and relevant preparation conditions of different versions of magnetic templated, composite granular hydrogel scaffolds, as well as certain physical properties of these scaffolds. Indicated % weights are based on total solid contents only (z.e., water is not included in the weight percentages).

[0014] Figure 4 shows representative images of scaffolds A, B, C, and D, as described in Figure 3, before magnetic templating alignment, after 30 minutes of magnetic alignment, and both top and side views after cross-linking. Scaffold A contains 50% v / v interstitial matrix, scaffold B contains 60% v / v interstitial matrix, scaffold C contains 70% v / v interstitial matrix, and scaffold D contains 80% v / v interstitial matrix.

[0015] Figure 5 shows representative images of optical clearance of templated composite scaffolds over 6 days as the magnetic porogen was dissolved and diffused into EDTA solution. Scaffolds A-D represent decreasing concentration of microgel as described in Figures 3 and 4.

[0016] Figure 6 shows fluorescence microscopy images of composite scaffolds A-D, as described in the previous Figures. Bright spots are the backfilled pores and the strings throughout the interstitial matrix are strands of aggregated microgels that were previously lyophilized.

[0017] Figure 7 shows an illustrated representation of the composition of the various scaffolds and the organization of their components for mechanical testing.

[0018] Figure 8 shows the results of mechanical testing (steady-state modulus) in scaffolds A-D, as described in the previous Figures, measured at 3 different points on each construct. 2- way ANOVA indicated that microgels significantly increased the steady-state modulus of all constructs, while removal of the magnetic porogen significantly reduced the mechanical strength to levels comparable to interstitial matrix alone.

[0019] Figure 9 shows representative top (upper panels) and side views (lower panels) of scaffolds generated using microgel stock that had been packed by centrifugation (i.e., had not been previously lyophilized) prior to addition of interstitial matrix and magnetic porogen. The number of microliters of microgels out of a 125pL total solution volume are shown. All constructs contained 8mg / mL concentration of interstitial matrix (4pL of a 250mg / mL stock solution).

[0020] Figure 10 shows fluorescence microscopy images of composite scaffolds containing 76.9uL of microgels indicating that packing of fresh (i.e., not previously lyophilized) microgels prevents microgel strand aggregation and creates a more granular microgel distribution in the construct. Scale bar = 20pm.

[0021] Figures 11A-11I show magnetic templating of a granular PEG hydrogel composite. Figure 11A shows microgel diameter distributions for varying microgel formulation conditions (I-IV) and corresponding fluorescence images of the Cy3 labeled microgels. Figures 11B-11F show the steps to produce a magnetically templated granular PEG hydrogel composite, starting with mixing of PEG microgels and magnetic porogen with an interstitial polymer in a hydrogel mold (Figure 11B). This is followed by alignment of the magnetic porogens in a magnetic field (Figure 11C), crosslinking of the interstitial PEG precursors using UV light (Figure 11D), and clearance of the porogen in EDTA (Figure HE) to obtain a granular PEG hydrogel composite with aligned porosity (Figure HF). Figure HG shows pre- and post-alignment images of a magnetically aligned and crosslinked granular PEG hydrogel composite. Figure 11H shows side, top, and zoomed view of magnetically aligned porogen chains via nano-CT visualization and Imaris processing. Figure HI shows images of porogen clearance at 0, 2, 4, and 6 days.

[0022] Figures 12A-12H show rheological assessment of granular PEG hydrogel composites with varying microgel content. Figure 12A shows the average frequency sweep. Figure 12B shows the average storage modulus at 1Hz obtained from the frequency sweep. Figure 12C shows the average strain sweep. Figure 12D shows the yield strain at 0.9 G’ of the G’ averaged under 1% strain obtained from the strain sweep. Figure 12E shows the average shear stress vs shear rate obtained from a unidirectional flow curve. Figure 12F shows the average yield stress obtained from a Herschel-Bulkley fit of the unidirectional flow curve data. Figure 12G shows the average viscosity vs shear rate obtained from a unidirectional flow curve. Figure 12H shows the average viscosity at a shear rate of 0.01 1 / s obtained from a unidirectional flow curve, n = 3for all groups. In Figures 12B, 12D, 12F, and 12H, solid lines are the fitted average, and dotted lines represent the standard deviations of the quadratic fit to the data.

[0023] Figure 13 shows qualitative assessment of magnetic templating on granular PEG hydrogel composites with varying microgel concentrations pre- and post-alignment of a porogen. Weight by weight percentages correspond to microgel concentration in the granular hydrogel system. Pre-alignment images correspond to the mixture of PEG microgels, interstitial polymer, and magnetic porogen. Post-alignment images correspond to the same mixtures after alignment of the porogen and crosslink of the hydrogel via photopolymerization. Post-alignment images were taken from both sides of the hydrogel in the mold.

[0024] Figures 14A-14I show quantitative visualization and analysis of chain alignment via nano-CT Top and side views of (Figure 14A) 30%, (Figure 14B) 33%, (Figure 14C) 36%, (Figure 14D) 39%, (Figure 14E) 42%, and (Figure 14F) 45% w / w microgel granular hydrogel composites. (G) Figure 14G shows the orientation angle density of segments in chains obtained from Imaris for each group. Isolated chain segments with orientation angles between 0-20° and 160-180° are shown in Figure 14H for 36% w / w and Figure 141 for 39% w / w microgel granular hydrogel composites.

[0025] Figure 15 shows optical imaging of magnetic porogen clearance, on the indicated days, from granular PEG hydrogel composites placed in 0.1M EDTAthat was changed daily.DEFINITIONS

[0026] As used herein, the term “engineered” indicates that the engineered object is created and / or altered by man. An engineered object may include naturally derived substances, but the object itself is altered in some way by human intervention and design.

[0027] As used herein, the term “scaffold” refers to a three-dimensional tissue engineering composition capable of providing structural / mechanical support and / or various substrates to support the growth of a living biological substance (e.g., cells or organs) in vitro and / or in vivo in a subject. Such scaffolds may be interchangeably referred to as “constructs” throughout, and may comprise additional components, including but not limited to, cells, biomolecules, or other bioactive agents, which are defined elsewhere herein.

[0028] As used herein the term “channels” or “microchannels” refers to tubules or tube-like formations within a scaffold. The channels have a generally elongated and / or cylindrical shape,with a generally circular cross-section. The channels have an open (e.g., hollow or substantially hollow) interior (also referred to herein as a “lumen”) creating a conduit that can act as a template for the growth of cells / tissues and can also facilitate the movement of fluid, cells, and other materials within and / or through the scaffold. These channels or microchannels can be formed between or within any component of the scaffold by various means (e.g., in a space previously occupied by a porogen, in the space between microgel particles and / or interstitial matrix polymers, or within polymers themselves). Accordingly, the diameters of such channels or microchannels can range from nanometer scale (e.g., within polymers) to several micrometers (e.g., l-10pm between microgel particles) or even greater (>50pm in spaces previously occupied by porogens.

[0029] As used herein the term “biocompatible” refers to the ability to co-exist with a living biological substance and / or biological system (e.g., a cell, cellular components, living tissue, organ, etc.) without exerting undue stress, toxicity, or adverse effects on the biological substance or system.

[0030] The term “biocompatible scaffold material” refers to any compound substance with sufficient structural stability to provide a substrate to support the growth of a living biological substance (e.g., living cells). In the present disclosure, the biocompatible scaffold material has a three-dimensional structure (rather than a planar, two-dimensional structure) to support three- dimensional growth of living cells. In some embodiments, the biocompatible scaffold material is made from a liquid / semi-liquid material that can be injected or formed into a mold prior to being cross-linked and / or polymerized into a matrix that provides a more solid (e.g., solid, gel, semisolid, etc.) scaffold.

[0031] The term “matrix material” refers to several different types of semi-solid to solid materials with a gel-like and / or solid consistency and a structure capable of supporting the growth of living biological substances (e.g., living cells). Both synthetic and naturally derived gel matrix materials exist and are in use by those of skill in the art. Gel matrix materials include hydrogels, such as biocompatible naturally derived or synthetic hydrogels, such as, but not limited to polymer-based hydrogels, polyethylene glycol (PEG)-based hydrogels, alginate, cellulose, keratin, elastin, collagen, and the like. Gel matrix materials also include biocompatible polymer or copolymer-based gel materials, such as polymer and copolymer-based hydrogels. Gel matrix materials may also include a gelling agent or cross-linking agent (e.g., formaldehyde,glutaraldehyde, etc.) to increase the structural stability of the gel (i.e., to give it more “solid” characteristics).

[0032] As used herein, the term “solid” shall include “semi-solid” materials, and “liquid” shall include “semi-liquid” materials.

[0033] The term “polymer” includes any compound that is made up of two or more monomeric units covalently bonded to each other, where the monomeric units may be the same or different, such that the polymer may be a homopolymer or a heteropolymer. Representative polymers include polyamides, such as polypeptides, poly-N-substituted glycines (polypeptoids), polysaccharides, polyethylene glycol or polyethylene oxide, plastics (e.g., poly-L-lactic acid, poly-L-glutamic acid, and co-polymers thereof), nucleic acids, and the like. The polymers may be naturally occurring, non-naturally occurring, or synthetic. The term “biopolymer” refers to a polymer made of biologically derived and / or biologically compatible compounds.

[0034] The term “attached” or the phrases “interacts with” and “associated with” refers to a stable physical, biological, biochemical, and / or chemical association. In general, association can be chemical bonding (e.g., covalently or ionically), a biological interaction, a biochemical interaction, and in some instances, a physical interaction. The association can be a covalent bond, a non-covalent bond, an ionic bond, a metal ion chelation interaction, as well as moi eties being linked through interactions such as, but not limited to, hydrophobic interactions, hydrophilic interactions such as hydrogel bonding, charge-charge interactions, 7i-stacking interactions, combinations thereof, and like interactions.

[0035] Use of the phrase “biomolecule” is intended to encompass deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleotides, oligonucleotides, nucleosides, proteins, peptides, polypeptides, selenoproteins, antibodies, protein complexes, peptide nucleic acids, combinations thereof, and the like. The biomolecule can include, but is not limited to, naturally occurring substances such as polypeptides, polynucleotides, lipids, fatty acids, glycoproteins, carbohydrates, fatty acids, fatty esters, macromolecular polypeptide complexes, vitamins, cofactors, whole cells, eukaryotic cells, prokaryotic cells, microorganisms, or combinations thereof. Eukaryotic cells can include cells of any type. For example, the cells can be mammalian cells (e.g., nerve cells, endothelial cells, skeletal muscle cells, mesenchymal stem cells, etc.). The mammalian cells can be human cells. Alternatively, the mammalian cells can be rodent cells. Therodent cells can be mouse cells, such as mouse embryonic stem (ES) cells or induced pluripotent stem (IPS) cells. The rodent cells can also be rat cells, such as rat ES or IPS cells.

[0036] The phrase “bioactive agent” includes a biomolecule or other biocompatible compound (e.g., a drug) that has some activity, use, and / or effect in a biological system or in relation to another biomolecule.

[0037] The terms “polypeptide” and “protein” as used herein refer to a polymer of amino acids of three or more amino acids in a serial array, linked through peptide bonds. The term “polypeptide” includes proteins, protein fragments, protein analogues, oligopeptides and the like. The term “polypeptides” also includes polypeptides, as defined above, that are encoded by nucleic acids, produced through recombinant technology, or chemically synthesized. The term “polypeptides” further includes polypeptides, as defined above, that include chemically modified amino acids or amino acids covalently or non-covalently linked to labeling ligands or with any other useful modification, the likes of which are familiar to those of skill in the art.

[0038] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients. The transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified elements recited in the claim and those that do not materially affect the basic and novel character! stic(s) of the claimed invention. Thus, the term “consisting essentially of’ when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”

[0039] “Optional” or “optionally,” in the context of a method, means that the subsequently described event or circumstance may or may not occur and that the description includes instances in which the event or circumstance occurs and instances in which the event or circumstance does not. “Optional” or “optionally,” in the context of a composition, means that the subsequently described component or aspect of the composition may or may not be present and that the description includes instances in which the component or aspect is present and instances in which the component or aspect is not. Additionally, the presence or absence of such an optional event or circumstance of a method or component or aspect of a composition may or may not have an impact on the outcome of the method or the properties of the composition.

[0040] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range. For example, 5-10 nucleotides is understood as 5, 6, 7, 8, 9, or 10 nucleotides, whereas 5-10% is understood to contain 5% and all possible values through 10%.

[0041] “At least” 17 nucleotides of a 20 nucleotide sequence is understood to include 17, 18, 19, or 20 nucleotides of the sequence provided, thereby providing an upper limit even if one is not specifically provided, as it would be clearly understood. Similarly, up to 3 nucleotides would be understood to encompass 0, 1, 2, or 3 nucleotides, providing a lower limit even if one is not specifically provided. When “at least,” “up to,” or other similar language modifies a number, it can be understood to modify each number in the series.

[0042] As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex region of “no more than 2 nucleotide base pairs” has a 2, 1, or 0 nucleotide base pairs. When “no more than” or “less than” is present before a series of numbers or a range, it is understood that each of the numbers in the series or range is modified.

[0043] Unless otherwise apparent from the context, the term “about” encompasses values ± 5% of a stated value. In certain embodiments, the term “about” is understood to encompass tolerated variation or error within the art, e.g., 2 standard deviations from the mean, or the sensitivity of the method used to take a measurement, or a percent of a value as tolerated in the art. When “about” is present before the first value of a series, it can be understood to modify each value in the series.

[0044] The term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0045] The term “or” refers to any one member of a particular list and also includes any combination of members of that list.

[0046] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a protein” or “at least one protein” can include a plurality of proteins, including mixtures thereof.

[0047] Statistically significant means p <0.05. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.DETAILED DESCRIPTIONI. Overview

[0048] Hydrogel biomaterials have been widely used for tissue engineering applications in large part for their high-water content, as it mimics native tissues in the body. Currently, a limiting factor of traditionally formulated bulk hydrogels is that they lack porosity that is characteristic of native tissues. Hydrogel microparticles (HMPs) are micron-scale hydrogels formulated through multiple means like batch emulsions, microfluidics, lithography, or extrusion fragmentation. Such hydrogel microparticles, or “microgels,” can also comprise multiple formulation chemistries. Granular hydrogels are microgel-based systems composed of individual microgels packed together. These granular hydrogels exhibit packing-dependent rheological behavior that allows injectability, and upon cross-link or annealing, they form a bulk scaffold. Microgel composites are microgel-based systems that comprise microgels suspended in a secondary interstitial matrix material (e.g., a hydrogel).

[0049] Microgels have found increasing use as components in tissue engineering, drug delivery, and wound healing. The use of microgel building blocks confers benefits over traditional bulk hydrogels. As mentioned above, they exhibit controllable rheological behavior, including shear-thinning, which makes them injectable. Additionally, the granular nature of microgels creates an inherently microporous network upon packing that better mimics native tissues (z.e., the generally spherical microgels, unless ultra-closely packed under deforming pressure, naturally leave a network of spaces between these spheres when packed together). However, this microporous network is randomly distributed, and in some cases, an aligned pore is preferred, as disorganized porosity may not be enough to induce the desired response in the scaffold (e.g., cellular organization). The benefits of granular materials (e.g., microgels or microgel composites) have been leveraged to improve cell infiltration in tissues, enhance molecule diffusion, and even modulate the immune response of the host into which the scaffold is implanted. Assembly of granular composite materials (e.g., microgel composites) can also be leveraged for their material properties, e.g., increased mechanical strength of a scaffold.

[0050] Provided herein are compositions comprising a granular polyethylene glycol (PEG) composite hydrogel comprising PEG microgels and a magnetic porogen (also referred to interchangeably as “micropatterned, composite granular hydrogel scaffolds” or “granular PEG hydrogel composites”). Using the magnetic, sacrificial porogen (z.e., the porogen is dissolved and removed from the scaffold to create a void), aligned pores (e.g., channels or microchannels) are created within the scaffold. Also provided are scaffolds further comprising an additional interstitial matrix. Such templated, composite hydrogel scaffolds provide significant advantages over current bioengineered tissue scaffolds, particularly because the aligned pores (e.g., channels or microchannels) better mimic the native environment into which cells can migrate (e.g., ingrowth of nerve cells or vasculature) and facilitate movement of fluid, cells, and other materials within and / or through the scaffold molecules through interstitial fluid flow.II. Magnetic Templated, Micropatterned PEG Composite Granular Hydrogels

[0051] Magnetic fields to control nano- and micro-topography, or to direct the assembly of cells or tissue engineering constructs can be used in various applications. The alignment of collagen and fibrin fibers through magnetic fields has been studied to direct cell growth, but these collagen and fibrin scaffolds suffer practical limitations because fiber alignment relaxes once the magnetic field is removed. Magnetic alignment of anisotropic dissolvable particles has been used to create bone cement scaffolds with anisotropic porosity, but the reported materials lack gap-spanning, aligned microchannels. Magnetic hydrogels have been reported that respond to magnetic fields because of magnetic nanoparticles retained within the hydrogel, but these constructs lack the tubular features required for directed cellular growth (e.g., nerve cells or vasculature) or interstitial fluid flow. Magnetic fields have also been used to direct 2D patterning of dissolvable magnetic sugar particles, leaving behind a scaffold with patterned 2D porosity, but the pores are too large to effectively direct cell growth and do not form continuous tubular guidance conduits that span dimensions that would be relevant in a physiologic setting such as nerve or vascular repair. Magnetic fields have also been used to direct assembly of cells and hydrogels, but not with the topographic features, nor at the size scales needed for complex tissue regeneration settings.

[0052] Provided herein are hydrogel / microgel granular PEG composite scaffolds with magnetic templated, patterned porosity. The scaffolds comprise modular PEG microgels andmagnetic alginate porogens. Also provided are scaffolds further comprising an interstitial matrix. Variations in the ratios of microgels, magnetic porogens, and / or interstitial matrix allow tunability of the alignment of the magnetic porogens, tunability of cross-linking within the scaffold to alter mechanical properties, and clearance of the magnetic porogen to leave aligned pores and / or networks of pores (e.g., channels or microchannels or networks thereof) for potential tissue engineering applications. Alignment of the pores in the scaffold is achieved through the application of a magnetic field to a mold containing a mixture of a cross-linkable and / or polymerizable biocompatible microgel particles, dissolvable / sacrificial magnetic porogen particles, and / or interstitial matrix material (e.g., a biocompatible, polymerizable polymer, a cross-linkable or photopolymerizable hydrogel of naturally derived biomaterial, etc.). If interstitial matrix is present, the microgel particles can be of similar composition to the interstitial matrix or can be of a different composition from the interstitial matrix. The magnetic field causes the magnetic porogens to align and form a plurality of lines / columns of adjacent porogens, where the columns are also substantially aligned with each other (e.g., substantially oriented in the same direction, substantially parallel, etc.). These porogen columns can be aligned in any desired orientation (e.g., longitudinally, crosswise, diagonally, etc.) within the scaffold, depending upon orientation of the magnetic field across the scaffold. Following alignment, and while still applying the magnetic field, or optionally, immediately after removal of the magnetic field, the scaffold is appropriately stimulated to activate cross-linking or polymerization of the microgels, porogens, and / or interstitial matrix (e g., application of UV light for photopolymerizable materials, addition of a chemical cross-linker, heat activation, etc.). Accordingly, the magnetic porogens are fixed in their columnar organization in the substantially solidified scaffold.

[0053] In some such scaffolds, the biocompatible scaffold is also biodegradable. Those of skill in the art will recognize that any number of biocompatible and / or biodegradable compounds capable of cross-linking and / or polymerization can be selected for use in the formulation of the microgels, interstitial matrix, and magnetic porogens in the scaffolds of the present disclosure. In some such scaffolds, the scaffold materials can include a matrix of a synthetic or naturally derived resorbable or non-resorbable materials. Examples of naturally derived biomaterials include, but are not limited to, such natural scaffolds as extracellular matrix (ECM)-based scaffolds and the like. Examples of biocompatible materials that can be used toform the scaffolds of the present disclosure include, but are not limited to, hyaluronic acid, collagen, polyethylene glycol (PEG), fibrin, and the like. Any of the naturally derived biomaterials or biocompatible materials described herein can be further modified, chemically or otherwise (e.g., addition of other bioactive agents) to promote a desired property in the scaffold (e g., greater cross-linking capability to increase mechanical strength, growth factors to guide cell invasion, etc.). In some such scaffolds, the biocompatible material is in a liquid or semiliquid form prior to cross-linking / polymerization into a gel / solid / semi-solid matrix that forms the biocompatible scaffold to facilitate the movement and alignment of magnetic porogens under a magnetic field. In some such scaffolds, the biocompatible scaffolding material is formed in a mold to provide shape and support to the biocompatible scaffolding material prior to cross- linking / polymerizing. In some such scaffolds, the mold is a sacrificial material that is later removed using known techniques. In other such scaffolds, the mold is also a biocompatible material that remains integral to the engineered construct.

[0054] In some such scaffolds, the microgel particles comprise 4-arm polyethylene glycol (PEG) norbornene (PNB) and 4-arm PEG thiol (PTT). In some such scaffolds, the ratio of PNBPTT in the microgel particles is about 1 :0.1, about 1 :0.2, about 1 :0.3, about 1:0.4, about 1 :0.5, about 1 :0.6, about 1 :0.7, about 1 :0.8, about 1 :0.9, or about 1 : 1. In some such scaffolds, the ratio of PNB TT in the microgel particles is about 0.1: 1, about 0.2: 1, about 0.3:1, about 0.4:1, about 0.5: 1, about 0.6:1, about 0.7: 1, about 0.8: 1, about 0.9: 1, or about 1 : 1. In a particular example, the ratio of PNB PTT in the microgel particles is about 1 : 1. In another example, the ratio of PNB PTT in the microgel particles is about 1:0.5.

[0055] In some such scaffolds, the interstitial matrix comprises 4-arm polyethylene glycol (PEG) norbornene (PNB) and 4-arm PEG thiol (PTT). In some such scaffolds, the ratio of PNBPTT in the interstitial matrix is about 1 :0.1, about 1:0.2, about 1:0.3, about 1 :0.4, about 1 :0.5, about 1 :0.6, about 1 :0.7, about 1 :0.8, about 1 :0.9, or about 1 : 1. In some such scaffolds, the ratio of PNBPTT in the interstitial matrix is about 0.1 :1, about 0.2: 1, about 0.3: 1, about 0.4:1, about 0.5: 1, about 0.6:1, about 0.7: 1, about 0.8: 1, about 0.9: 1, or about 1 : 1. In a particular example, the ratio of PNBPTT in the interstitial matrix is about 1 : 1.

[0056] In some such scaffolds, the magnetic porogens comprise magnetic alginate microparticles (MAMs), including biocompatible iron oxide nanoparticles embedded in a crosslinked calcium alginate matrix. Iron oxide nanoparticles are one component in various FDAapproved magnetic resonance imaging (MRI) contrast agents and are believed to be biocompatible and bioabsorbable. Furthermore, in magnetic templating to create aligned pores in the scaffold, the majority, if not all, of the iron oxide nanoparticles are removed during the MAM dissolution step. Alginate is highly biocompatible, is used to encapsulate viable cells for culture, and its dissolution can be achieved under mild conditions that do not affect other biomolecules or cell viability, such as may be optionally included in the disclosed scaffold and as described in the following references, which are herein incorporated by reference in their entirety for all purposes (Gombotz et al. (2012) Advanced Drug Delivery Reviews 64: 194-205; Tonnesen et al. (2002) Drug Development and Industrial Pharmacy 28:621-630; and Bucke (1987) Methods in Enzymology 135: 175-189). These qualities of alginate encapsulated microparticles are compatible with such scaffolds where cells or biomolecules are incorporated into the microgel particles, interstitial matrix, and / or MAMs to provide chemical and biological cues.

[0057] In addition to tuning of the scaffold through composition of the components described above, various methods of producing the components and scaffolds themselves, described in detail elsewhere herein, can be employed to produce various sizes of microgels and magnetic porogens, which can also affect the material properties and alignment of the porogens within the resultant scaffolds. In some such scaffolds, the microgel particles are about 1pm, about 2pm, about 3pm, about 4pm, about 5pm, about 6pm, about 7pm, about 8pm, about 9pm, about 10pm, about 11pm, about 12pm, about 13pm, about 14pm, about 15pm, about 16pm, about 17pm, about 18pm, about 19pm, about 20pm, about 21pm, about 22pm, about 23pm, about 24pm, or about 25pm in diameter. In some such scaffolds, the microgel particles are about 5pm to about 25pm in diameter. In some such scaffolds, the microgel particles are about 5pm to about 15pm in diameter. In a particular example, the microgel particles are about 10pm in diameter.

[0058] In some such scaffolds, the microgel particles comprise between about 5% w / w and about 70% w / w, between about 10% w / w and about 60% w / w, between about 20% w / w and about 50% w / w, between about 30% w / w and about 50% w / w, between about 30%> w / w and about 45% w / w, or between about 35% w / w and about 45% w / w of the scaffold. In some such scaffolds, the microgel particles comprise about 5% w / w, about 10% w / w, about 15% w / w, about 20% w / w, about 25% w / w, about 30% w / w, about 31% w / w, about 32% w / w, about 33% w / w, about 34% w / w, about 35% w / w, about 36% w / w, about 37% w / w, about 38% w / w, about39% w / w, about 40% w / w, about 41% w / w, about 42% w / w, about 43% w / w, about 44% w / w, about 45% w / w, about 50% w / w, about 60% w / w, or about 70% w / w of the scaffold.

[0059] The relative size (e.g., diameter) of microgel particles as compared to the magnetic porogens can also affect the material properties and alignment of the porogens within the resultant scaffolds. In some such methods, the microgel particles are about 2 times smaller, about 3 times smaller, about 4 times smaller, about 5 times smaller, about 6 times smaller, about 7 times smaller, about 8 times smaller, about 9 times smaller, about 10 times smaller, about 15 times smaller, or about 20 times smaller than the magnetic porogens in the scaffold. In some such methods, the microgel particles are about 2 times to about 5 times, about 5 times to about 10 times, or about 10 times to about 20 times smaller than the magnetic porogens in the scaffold. In a particular example, the microgel particles are about 3 times to about 5 times smaller than the magnetic porogens in the scaffold.

[0060] In some such scaffolds, the magnetic porogens are about 5pm, about 10pm, about 15pm, about 20pm, about 25pm, about 30pm, about 35pm, about 40pm, about 45pm, about 50pm, about 55pm, about 60pm, about 65pm, about 70pm, about 75pm, about 80pm, about 85pm, about 90pm, about 95pm, about 100pm, about 105pm, about 110pm, about 115pm, about 120pm, about 125pm, about 130pm, about 135pm, about 140pm, about 145pm, or about 150pm in diameter. In some such scaffolds, the magnetic porogens are about 10pm to about 150pm, about 10pm to about 100pm, or about 10pm to about 50pm in diameter. In some such scaffolds, the magnetic porogens are about 50pm to about 150pm, about 50pm to about 100pm, about 70pm to about 100pm, or about 80pm to about 90pm in diameter. In a particular example, the magnetic porogens are about 80pm in diameter. In another example, the magnetic porogens are about 90pm in diameter.

[0061] One of the principal functions of a biocompatible scaffold is to direct cell behavior such as migration, proliferation, differentiation, maintenance of phenotype, etc. by facilitating the sensing and responding of the cell to the environment via cell-matrix and cell-cell communications. As described above, the magnetic alginate particles (MAMs) described can be employed to provide aligned tracks of biomolecules or bioactive agents (e.g., growth factors or drugs to promote cell migration or provide other cues from the construct) or cells (e.g., mesenchymal stems cells to mimic columnar chondrocyte organization). In some scaffolds, the MAMs can also be porogens that confer voids that can mimic the native environment into whichcells can migrate (e.g., in-growth of cells) and otherwise facilitate movement of fluid, cells, and other materials within and / or through the scaffold. In some such scaffolds, the biocompatible composite material substantially solidifies (e.g., into a gel, solid / semi-solid) to form a three- dimensional (3D) scaffold around the aligned porogens. After formation of the scaffold, the matrix material of the microparticles (e.g., alginate) is dissolved / sacrificed, and the dissolved material and the magnetic nanoparticles diffuse / leach out of the scaffold through the pores (e.g., microchannels) left behind by the porogen microparticles when the scaffold substantially solidified. In some such scaffolds, additional biomolecules (e.g., cells, proteins, carbohydrates, nucleic acids, etc.) and / or bioactive agents (e.g., drugs) may be included in the biocompatible scaffold material of the microgels, interstitial matrix, and / or matrix material of the porogen microparticles, and all of the polymerizing / cross-linking / dissolution steps are carried out in biocompatible conditions that are non-toxic / non-harmful to any such biomolecules (e.g., they do not interfere with the intended purpose / activity of the biomolecules). These voids and channels may then direct growth, including directional growth, of cells in the scaffold. The voids and channels may also be modified during or post-preparation with cell adhesion factors and other desirable biomolecules or bioactive agents.III. Methods of Preparing Magnetic Templated, Micropatterned PEG Composite Granular Hydrogels

[0062] Also provided herein are methods of preparing or making a three-dimensional, biocompatible, micropatterned composite granular hydrogel scaffold comprising (i) microgel particles and (ii) magnetic porogens, and optionally (iii) an interstitial matrix, as described in detail above. Methods of preparation of hydrogel microparticles, or microgels, are familiar to those of skill in the art and include such processes as batch emulsions, microfluidic emulsions, various forms of lithography (e.g., imprint lithography, photolithography, flow lithography), electrohydrodynamic spraying, or mechanical fragmentation of larger hydrogels. A skilled artisan will readily appreciate that the selected method of microgel preparation will be informed by the desired size, shape, and consistency thereof that is necessary for use in the intended biocompatible scaffold.

[0063] In one example, batch emulsion can be used to produce microgels for use in such biocompatible scaffolds. In some such methods, a droplet phase comprising the microgelcomponents is added to an excess of continuous phase (e.g., ratio of continuous phase to droplet phase of 10: 1; interchangeably expressed as 10X, or 10 times the amount of continuous phase as compared to the droplet phase). The ratio of continuous phase to droplet phase can also be modified to tune the size of microgels. In some such methods, the ratio of continuous phase to droplet phase is about 2:1, about 3:1, about 4: 1, about 5:1, about 6: 1, about 7:1, about 8:1, about 9: 1, about 10:1, about 15: 1, about 20: 1, about 25: 1, about 30:1, about 40: 1, or about 50:1. In some such methods, the ratio of continuous phase to droplet phase is about 2: 1 to about 5:1, about 5:1 to about 10: 1, about 5: 1 to about 15: 1, about 10: 1 to about 15: 1, about 10: 1 to about 20: 1, about 10: 1 to about 30: 1, or about 20: 1 to about 50: 1. In a particular example, the ratio of continuous phase to droplet phase is about 5: 1 to about 20: 1. In another example, the ratio of continuous phase to droplet phase is about 20: 1. In another example, the ratio of continuous phase to droplet phase is about 5:1. In another example, the ratio of continuous phase to droplet phase is about 10: 1.

[0064] The mixture is then homogenized to create the emulsion. In some such methods, homogenization is performed at about 1,000 RPM to about 15,000 RPM. In a specific example, homogenization is performed at 2,500 RPM. In another example, homogenization is performed at 10,000 RPM. The duration of homogenization can be from about 10 seconds to about 10 minutes. In a specific example, the duration of homogenization is about 3 minutes. In another example, the duration of homogenization is about 1 minute. In another example, the homogenization is performed at 10,000 RPM for 1 minute. In another example, the duration of homogenization is about 30 seconds. In another example, the homogenization is performed at 10,000 RPM for 30 seconds. Following creation of the microgel emulsion, the microgel particles are appropriately stimulated to activate cross-linking or polymerization (e.g., application of ultraviolet (UV) light for photopolymerizable materials, addition of a chemical crosslinker, heat activation, etc.). In a specific example, the microgel emulsion is stirred at 200 RPM for 10 minutes while exposed to 20 mW / cm2UV light (365nm wavelength). The cross- linked / polymerized microgels are then centrifuged to pellet and washed as appropriate for their composition. In the specific example of the PEG microgels described herein, the microgels are centrifuged at 7,000 RCF to form a pellet prior to removal of the supernatant. The pellet is then washed 3 times in 0.3% triton in water (or until the supernatant is clear), followed by 4 washes with water. Optionally, the prepared and washed microgels can then be lyophilized to later createa reconstituted / re-swelled stock. A skilled artisan will readily understand that any suitable method of preparation, based on the desired microgel components and physical properties can be employed.

[0065] The fabricated microgels can then be combined in the desired ratios with the magnetic alginate microparticles (MAMs), the production of which is described in the Examples below. Optionally, an interstitial matrix solution can also be included in the biocompatible scaffolding material mixture. The biocompatible scaffolding material mixture can be, optionally, formed in a mold to provide shape and support to the biocompatible scaffolding material, as appropriate for the application of the biocompatible scaffold. Such a mold can be of any size and shape as required by the desired application. For example, the length of the scaffold can be less than 1 cm, about 1 cm, about 2 cm, about 3 cm, about 4 cm, about 5 cm, about 6 cm, about 7 cm, about 8 cm, about 9 cm, about 10 cm, about 11 cm, about 12 cm, about 13 cm, about 14 cm, about 15 cm, or greater than 15 cm. In some such methods, the mold produces a cylindrical or tubular scaffold (e.g., suitable for use in repairing a nerve gap). In some such methods, the mold produces a spherical or cuboid scaffold. Those skilled in the art will recognize that the size and shape of the mold, and the resulting scaffold, will vary widely based on the application, and may also include irregular shapes that are custom designed for the application (e.g., tissue repair of a 3D mapped blast injury site). The mixture can then be exposed to a magnetic field (e.g., 90 mT) to align the MAMs within the mixture in any desired orientation. In a specific example, the magnetic field is applied for 30 minutes, such that the MAMs align in vertical columns, surrounded by the microgel particles. Secondary cross-linking can then be performed to freeze the aligned MAMs in their orientation within the scaffold. In a specific example of the scaffolds described above, this secondary cross-linking is performed using UV photoactivation at 20 mW / cm2UV light (365 nm wavelength) for 30 minutes.

[0066] If desired, the MAMs can then be dissolved / sacrificed (e.g., through exposure to a solution that can dissolve the matrix material of the MAMs) to produce voids in the scaffold as described above. In a specific example, such clearance comprises placing the constructs in 0.1 M ethylenediaminetetraacetic acid (EDTA) on a shaker in an environmental chamber (e.g., benchtop cabinet capable of maintaining a temperature of 37°C) shaking at 70 RPM, with the EDTA solution changed once every 24 hours until the scaffold construct clears (e.g., after 6 days). Alternatively, the MAMs can be dissolved / sacrificed with PBS. MAMs can bedissolved / sacrificed with any effective solution based on the composition of the matrix material of the MAMs and associated components of the scaffold (i.e., a solution that can effectively degrade the MAM matrix without negatively impacting the desired properties of the remaining scaffold and / or any bioactive agents or biomolecules that may be present within the scaffold). The skilled artisan will recognize that the scaffold can be modified as described elsewhere herein, at various points during preparation, to comprise additional biomolecules or bioactive agents, etc.IV Methods of Use of Magnetic Templated, Micropatterned PEG Composite Granular Hydrogels

[0067] One of the principal functions of a biocompatible scaffold is to direct cell behavior such as migration, proliferation, differentiation, maintenance of phenotype, etc., in tissue engineering applications, by facilitating the sensing and responding of the cell to the environment via cell-matrix and cell-cell communications. As such, the biocompatible scaffolds described herein, and prepared by the methods also described herein, can be used in vitro or in vivo to such end. For example, cells of any type can be combined within the scaffold construct as it is produced, and then further cultured in vitro. Optionally, this construct may serve as an in vitro organoid model system. Further optionally, this construct could be administered to a subject in need of a cellular engineered tissue construct. The subject can be a human, a non-human primate, or any other non-human animal, including but not limited to rodents, rabbits, sheep, goats, dogs, etc. In some such methods, the biocompatible scaffold is administered to or introduced into the subject in vivo (e.g., surgically implanted or injected) without a cellular component or prior in vitro culturing. A skilled artisan will recognize that the means of use is highly dependent upon the intended environment of use (e.g., stimulating nerve regeneration, bone regeneration, vascular regeneration, or muscle regeneration) and can adjust such a construct and use thereof (e.g., selection of additional bioactive agents, porosity, mechanical properties, cells, etc.) as required.

[0068] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at differenttimes, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated.

[0069] Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.EXAMPLESExample 1. Microgel Formulation and Production

[0070] Batch emulsion was used to produce microgels for further use in the production of biocompatible composite scaffolds, as described in Examples 2 and 3 below. Briefly, a droplet phase comprising the microgel components (4-arm polyethylene glycol (PEG) norbornene (PNB) and 4-arm PEG thiol (PTT) in a ratio of 1 :0.5 PNB:PTT or 1 : 1 PNB:PTT, as well as lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) at a ratio of 10.75: 1 LAP:PTT, tris(2- carboxyethyl)phosphine (TCEP) at a ratio of 1 : 1 TCEP:PTT, fluorophore at a ratio of 1 :2 fluorophore:PNB or 1 :4 fluorophore PNB, and 0.6 mL of water was added to an excess of continuous phase containing mineral oil and 2% v / v Span-80 at a ratio of continuous phase to droplet phase of 10: 1. The mixture was homogenized to create the emulsion for either (i) 2,500 RPM for 3 minutes, at a ratio of 5: 1 continuous phase to droplet phase; (ii) 10,000 RPM for 30 seconds, at a ratio of 10: 1 continuous to droplet phase; (iii) 10,000 RPM for 3 minutes, at a ratio of 10: 1 continuous to droplet phase; or (iv) 10,000 RPM for 3 minutes, at a ratio of 20: 1 continuous to droplet phase. Following creation of the microgel emulsions, the microgel particles were stirred at 200 RPM for 10 minutes while exposed to 20mW / cm2UV light (365nm wavelength). The cross-linked / polymerized microgels were then centrifuged at 7,000 RCF to form a pellet prior to removal of the supernatant. The pellet was then washed 3 times in 0.3% triton in water (or until the supernatant was clear), followed by 4 washes with water. Themicrogels were then lyophilized to later create a reconstituted / re-swelled stock for addition to the composite scaffold mixture prepared in Example 2.

[0071] Figure 1 shows fluorescent microscopy images of the resulting microgel populations and their respective sizes when prepared according to the procedure described above and the homogenization parameters indicated beneath each representative image. Figure 2 shows quantification of the average diameters of microgel populations prepared according to the indicated homogenization parameters (left) and scatter plots of the individual diameters of microgels in those populations (right). As demonstrated, simply altering the homogenization of the microgel emulsion can produce a variety of sizes of microgels.Example 2. Composite Scaffold Formulation and Production with Lyophilized Microgels

[0072] MAMs were produced by water-in-oil microfluidic emulsion cross-linking of sodium alginate and iron oxide nanoparticle mixtures using calcium chloride, as described in detail in Rivera-Llabres et al. (2023) Part. Part. Syst. Charact. 40:2300026, which is herein incorporated by reference in its entirety for all purposes. Briefly, the droplet phase contained lOmg / mL sodium alginate, 90mg / mL iron oxide (PBG300, Ferrotec USA), and 6.25mM CaEDTA. The continuous phase contained FLUORINERT™-70 fluorocarbon oil with 5% perfluoro polyether (PFPE)-PEG surfactant. The droplet and continuous phases were flowed through a 3D flowfocusing microfluidic device, and droplets were pinched at the junction of the device and collected downstream in 0.1% v / v acidified oil to perform an initial crosslink upon CaEDTA dissociation at acidic pH. MAMs were then washed with acetone three times before performing a secondary crosslink in calcium chloride. The MAMs were then finally washed with water. MAMs were resuspended in water and stored at 4°C until use.

[0073] Microgels utilized in all described scaffold compositions were those that were emulsified at 10,000 RPM for 30 seconds, at a 10: 1 ratio of continuous phase to droplet phase, and were approximately 10pm in diameter. Multiple ratios of microgel to interstitial matrix were effectively micropatterned with the MAM porogen and led to a cross-linked composite hydrogel. The complete details of the composition of each composite scaffold prepared are provided in Figure 3. Lyophilized microgels were re-swelled in water for 48 hours prior to addition to the composite scaffold mixture, and both microgel stock and interstitial matrix stock were at 20mg / mL concentration in all testing scaffold compositions. Following combination of themicrogels, interstitial matrix, and MAM porogens, the various scaffold solutions were exposed to a 90 mT magnetic field for 30 minutes to facilitate alignment of the magnetic porogens. The scaffolds were then cross-linked with 20 mW / cm2UV light (365nm wavelength) to fix the position of the aligned porogens within the composite scaffolds.

[0074] Figure 4 shows representative images of scaffolds A, B, C, and D, as described in Figure 3, before magnetic templating alignment, after 30 minutes of magnetic alignment, and both top and side views after cross-linking. Scaffold A contains 50% v / v interstitial matrix, scaffold B contains 60% v / v interstitial matrix, scaffold C contains 70% v / v interstitial matrix, and scaffold D contains 80% v / v interstitial matrix. The aligned porogens were then sacrificed by placing the constructs in 0.1M ethylenediaminetetraacetic acid (EDTA) in an environmental chamber at 37°C, shaking at 70 RPM, with the EDTA solution changed once every 24 hours until the scaffold construct cleared. As shown in Figure 5, all representative composite scaffolds (A-D) cleared in less than 6 days. Fluorescent imaging confirmed the presence of an interstitial polymer, dispersed (but strand aggregated) microgels, and empty pores in the composite hydrogel scaffolds, representative images of which are provided in Figure 6.

[0075] We next compared the mechanical properties of scaffolds that contained: (i) interstitial matrix alone; (ii) interstitial matrix and microgels; (iii) interstitial matrix, microgels, and unaligned magnetic porogens; and (iv) interstitial matrix, microgels, and magnetically aligned magnetic porogens, as illustrated in Figure 7. These tests were performed using a 3mm round tip probe on a Bruker BioScan system with 3 random locations probed on each scaffold. Indentation was performed at a rate of 20 pm / second to a depth of 100 pm (5% of total composite scaffold thickness) and held for 40 seconds before release. As shown in Figure 8, 2- way ANOVA indicated that microgels significantly increased the steady-state modulus of all constructs, while removal of the magnetic porogen significantly reduced the mechanical strength to levels comparable to interstitial matrix alone.Example 3. Composite Scaffold Formulation and Production with Fresh Packed (Non- Lyophilized) Microgels

[0076] Having noted stickiness when handling re-swelled lyophilized microgels in preparing the composite scaffold solutions described in Example 2, it was contemplated that the lyophilization and re-swelling process might be the cause of the aggregation of microgels andformation and microgel strands within the composite scaffolds that was evident in Figure 6. Accordingly, this was directly tested by preparing several scaffolds using centrifuge-packed microgels that had not been previously lyophilized. These microgels were otherwise produced as described in Example 1, but they were centrifuged at 30,000 RPM for 20 minutes prior to the addition of interstitial matrix and magnetic porogen as specified in the table below.

[0077] Figure 9 shows representative top (upper panels) and side views (lower panels) of these scaffolds generated using microgel stock that had been packed by centrifugation (i.e., had not been previously lyophilized) prior to addition of interstitial matrix and magnetic porogen. Particularly good porogen alignment was observed in those scaffolds that contained between 60pL and 76.9pL of 20mg / mL microgel stock. As shown in Figure 10, fluorescence microscopy images of composite scaffolds containing 76.9pL of microgels confirmed that packing of fresh (i.e., not previously lyophilized) microgels prevents microgel strand aggregation and creates a more granular microgel distribution in the construct.

[0078] The constructs described in the Examples above possess several key features that are particularly advantageous as compared to other current composite scaffolds. First, these scaffolds possess aligned porosity of 80-90pm in diameter channels that span from hundreds of micrometers to millimeters in distance. As described elsewhere herein, the diameter of the magnetic porogens and the resultant channels are highly tunable and can be made smaller (e.g., 10pm), depending on the requirements of the application. Moreover, these scaffolds possess multiple porosity length scales, including the highly oriented 80-90pm diameter pores suitable for providing physical migratory cues to cells, a potential interstitial pore that is less than 10pm and suitable for allowing adequate nutrient diffusion, and nanoscale porosity in each individual microgel that is suitable for controlling the release of a small molecule or drug in the finalcomposite hydrogel. As with the magnetic porogen, the size of the microgel is also highly tunable to permit variation of interstitial pore size in the construct (i.e., a smaller microgel will result in smaller interstitial pores) as required by the application of the construct. Second, the use of a PEG-norbomene and PEG-thiol (thiol-ene) click chemistry allows for high tunability of the bioactivity of the scaffold as thiol groups are found in peptide and proteins and can be leveraged to add adhesive peptide sequences, or cell-degradable sequences as cross-linkers in the network. Third, the use of microgels in the disclosed scaffolds is inherently modular, as we could include multiple microgel particles that can vary in size, chemistry, drugs loaded, stiffness, etc.

[0079] By contrast, typical micropattemed bulk hydrogels still lack an aligned porosity and are highly homogeneous, which hinders modularity of the biomaterial. Further, common granular biomaterials possess high, but randomly organized porosity and typically use larger microgels in their formulation (greater than 30pm, with some even greater than 100pm). Composite hydrogel biomaterials commonly made also typically only add microgels as a means to increase the mechanical strength of the hydrogel, not to add modularity or load a drug. While some have used microgels as porogens to add randomly distributed pores, it is at the cost of mechanical integrity of the final hydrogel, unlike the composite scaffolds.Example 4. Fabricating Magnetically Templated Granular PEG Hydrogel CompositesMaterials and Methods (in Example 4)Materials

[0080] 4-arm PEG Norbornene (PSB-4112) (20kDa) and 4-arm PEG Thiol (20kDa) (PSB- 445) were obtained from Creative PEGworks. Lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) (900889-1G), light mineral oil (330779-1L), and Tris(2- carboxyethyl)phosphine hydrochloride (TCEP) (C4706-2G) were purchased from Sigma- Aldrich. Sorbitan Monooleate (Span-80) (S0060) was acquired from TCI America. Cy3-PEG- Thiol (2k) (FL167003-2K) and Cy5-PEG-Thiol (2k) (FL078003-2K) were obtained from Biopharma PEG. Ethylenediamine tetraacetic acid (EDTA) (SS412-1) was obtained from Fisher Chemical. Triton X-100 (Electrophoresis) (BP151-5OO) was obtained from Fisher Bioreagents. Hydrogel molds (8 mm x 1 .7 mm) silicon molds acquired from Grace Bio-labs (664201).Magnetic porogen formulation

[0081] Magnetic porogens were made by forming magnetic alginate microparticles (MAMs) utilizing flow focusing microfluidics as previously described [see, e.g., Rivera-Llabres, et al. (2023) Part. Part. Syst. Charact. 40, 2300026], Briefly, a droplet phase composed of sodium alginate (10 mg / mL), iron oxide nanoparticles (90 mg FesO mL), and calcium ethylenediamine tetraacetic acid (6.25 mM) at 12 pL / min was made to flow against a carrier phase composed of HFE Novec 7500 (1% w / w PFPE-PEG) at 15 pL / min through a microfluidic device with a 100 pm junction to form droplets. Droplets were then left to crosslink for 15 minutes in an acidified Novec (0.1% v / v acetic acid) bath. Afterwards, the oil was siphoned out with a needle and the crosslinked microparticles were washed with acetone 3 times, followed by an additional calcium chloride crosslink. Finally, the microparticles were washed with DI water and stored at 4°C until use.Microgel formulation

[0082] Microgels were formulated through a batch emulsion process. The continuous phase was composed of well-mixed light mineral oil and 5% v / v SPAN-80. The droplet phase was composed of 4-arm PEG Norbornene (4PNB) (126.25 mg / ml), 4-arm PEG thiol (4PT)(110.47 mg / mL), LAP (17.5 mg / mL), Cy5-PEG thiol, and TCEP in deionized water. Microgels were formed by mixing a 20X volume ratio of continuous to droplet phase ratio. The resulting solution was homogenized at 10,000 RPM for 1 minute (Silverson L5M-A High Shear Laboratory Mixer from Silverson Machines) then poured into a beaker and placed under a UV lamp (UVP High Intensity UV Lamp B-100AP model from Analytik Jena, an Endress+Hauser Company) (365 nm) at 20 mW / cm2for 10 minutes while being stirred at 200 RPM. Microgels were then poured into a 50 mL centrifuge tube and spun down (Centrifuge 5430R from Eppendorf) and the supernatant oil removed. Microgels were then washed using 0.3% Triton-X 100 three times and with deionized water 3 times. The microgels were pelleted at 15°C and 7,000 RCF for 5 minutes between washes. Microgels were stored in deionized water at 4°C until use.Methods of preparing composite solutions and the magnetic templating of granular PEG hydrogel composites

[0083] To prepare templated composites, the microgel solution was centrifuged at 30,000 RPM (Sorvall WX Ultra Series Centrifuge, Thermo Scientific) for 20 minutes at 4°C.Meanwhile, the interstitial solution was prepared by mixing 4PNB (250 mg / mL), 4PT (250mg / mL), LAP (40 mg / mL), a trace amount of Cy3-PEG-thiol, and deionized water. After centrifugation, the packed microgels were mixed, and aliquots were weighed and transferred into separate 1.5 mL tubes, ensuring they remained hydrated with their respective water content. The interstitial solution was then added to the microgels for a final 8 mg / mL 4PNB concentration and thoroughly mixed using a positive displacement pipette. Magnetic porogens were incorporated into the mixture and carefully combined. The resulting solution was transferred into a cylindrical mold of dimensions 1.8 mm height and 9 mm diameter, ensuring no air bubbles are trapped, and a glass cover was applied to the mold. The particles were aligned vertically in the presence of a magnetic field for 5 minutes. The composite was removed from the magnetic field and crosslinked under UV light (365 nm) at 20 mW / cm2for 30 minutes. After crosslinking, the composites were imaged via nano-CT, then removed from the mold, placed in EDTA solution, and incubated on a shaker inside of an environmental chamber at 37°C. The EDTA was replaced daily for six days, after which the solution was switched to IX PBS and the composites stored at 4°C until further use.Optical imaging

[0084] Microgels were imaged using a Keyence BZ-X710 benchtop microscope from Keyence Corporation and a 40X objective for determination of their diameter distribution. Images were then imported into ImageJ for analysis. Images were made binary and filtered for circularity by diving four times the area by the perimeter squared as is set in the analyze particles tool, with values ranging from 0.0 to 1.0, with 1.0 being a perfectly circular object. Areas were obtained from circular objects, and effective diameters were calculated from obtained areas. Templated hydrogels were imaged after alignment of the magnetic porogen using optical microscopy. Stitched images were obtained using a 4X objective to get a full view of the hydrogel area. Clearance of templated hydrogels was assessed using optical microscopy (AmScope SF-2 Series Trinocular Stereo Microscope on Track Stand with Dual-illumination and 9.7" Touchscreen Imaging System). Hydrogels were placed under a benchtop microscope with a camera attached to the objective for imaging.Rheological assessment

[0085] All rheological measurements were performed on a 702 MC rheometer from Anton Paar Instruments, fitted with a 20 mm sandblasted parallel plate at a 0.1 mm gap height and 25°C, following the protocol developed by Qazi et al. [see, e.g. Qazi et al. (2022) ACS Biomater.Sci. Eng. 8(4): 1427-1442], Approximately 100 pL of microgels were placed on the bottom plate and the top plate was lowered to fill the gap with microgels. Excess sample was trimmed. Oscillatory shear strain amplitude sweeps were performed at 1 Hz between strains of 0.01 - 500%. Storage modulus (G’) and loss modulus (G”) were determined from frequency sweeps performed at 1 % strain from 10 - 0.01 Hz using the same geometric configuration. Unidirectional shear rate (y) sweeps were performed by shearing the sample from 100 - 0.01 s’1while measuring shear stress (G) and viscosity (p). An effective viscosity was determined from the ratio of shear stress to shear rate in these measurements. Shear stress versus shear-rate data was fitted to a Herschel-Bulkley model to determine yield stress on GraphPad Prism 10.4.1 (Dotmatics, Boston, MA) using the following equation:T = Ty+ Kyf (1) where T is the shear-stress in Pa, ryrepresents the yield stress in Pa, K is the consistency index in Pa s, y is the shear rate in s’1, and n is the flow index (dimensionless).Characterization of chain formation on templated granular PEG hydrogel composites via nano computed tomography (nano-CT)

[0086] Nano computed tomography (nano-CT) was used to assess the formation of porogen chains in magnetically templated granular PEG hydrogel composites prior to porogen clearance. Scans were conducted in an Xradia 620 Versa (Carl Zeiss Microscopy, Oberkochen, Baden- Wiirttemberg, Germany) at a voltage of 70 kV, a current of 121 pA for a power of 8.5 W and an air filter. The voxel size in the x, y, and z dimension was ~12 pm. Post-processing of reconstructed scans was conducted using VGStudio Max. Reconstructed scans were exported as TIFF files for further analysis.Imaris quantification of chain alignment

[0087] Imaris 10.1.1 software (from Oxford Instruments, Santa Barbara, CA, US) was used to analyze imported TIFF stacks from nano-CT data. TIFF stacks were converted into Imaris native files (.ims) and regions of interest were segmented using the cropping function. Surfaces were created around chains or porogens based on contrast differences between the objects and the background using the Imaris machine learning module for surface identification.Subsequently, a masked intensity channel was created from the generated surfaces and this masked channel was used to form the filaments using the filament tracing module in Imaris,resulting in volume objects representative of the chains. Chain orientation angles were obtained from the results.Statistical analysis

[0088] All plots were made with Graph Pad Prism 10.4.1. Statistical analysis was performed in R. First, one-way ANOVA was employed to determine statistically significant differences in the rheology of different microgel solutions. Then, rheological properties were fit to a second order polynomial and each model was evaluated using an ANOVA to determine significant trend correlations.Results

[0089] Microgels were formulated through batch emulsion of the precursor hydrogel in light mineral oil (5% v / v SPAN 80, surfactant) as described in the methods above. Processing parameters like batch size (total volume), homogenizing time, and molar ratio of reactants were studied. Figure 11A illustrates a minimal effect of batch size and reactant molar ratio on the average microgel diameter for microgels prepared according to the procedures described in this Example. However, increased homogenization time resulted in a narrower size distribution, and as such the conditions from group I in Figure 11A were used for the remainder of the experiments of this Example. The sacrificial MAM porogen was made to be much larger than the microgels, with average size of ~84 pm and formulated using 75 mg FesO mL of iron oxide.

[0090] Upon formation of a microgel building block, we could now perform magnetic templating in a granular hydrogel, following the procedure outlined in Figures 11B-11F. Briefly, the interstitial matrix precursor was mixed with a known weight of pre-packed microgels and a known concentration of magnetic porogen via ultracentrifugation to make a final 2.2% v / v porogen mixture. The combined solution was exposed to a magnetic field, the porogen was aligned, and the whole system exposed to UV light to photo-crosslink the interstitial matrix and form a granular PEG hydrogel composite. First, we aimed to identify potential microgel composition ranges and centrifugation speeds that led to a packed microgel pellet that enabled both alignment of the porogen and crosslinking of the hydrogel. From these experiments, we identified ultracentrifugation speeds of 30,000 RPM as the optimal packing for the process. We then took the templated gel and imaged it using an optical microscope to assess changes in alignment by comparing it to a pre-aligned image (Figure 11G). We observed a qualitative change in the alignment of the particles, as many were in a single plane or concentrated beforealignment, and the particles seemed more homogeneously distributed in the field of view postalignment. The composites were also evaluated using a nano-CT instrument to generate a 3D image of the whole hydrogel (Figure 11H). In the side view, generated using Imaris, we can see overall alignment of the porogen chains in the hydrogel. The top view shows that chains are homogeneously distributed throughout the hydrogel. Zoomed in images of the chain morphology suggest most of the objects captured with the nano-CT are in fact aligned and possess a column or chain-like shape. Finally, Figure 111 demonstrates that porogen clearance can be accomplished within 6 days, as in prior work using hyaluronic acid and collagen to form the hydrogel.Influence of microgel fraction on the rheology of granular PEG hydrogel composite solutions and alignment of magnetic porogens

[0091] Having explored the roles of microgel packing and interstitial polymer composition that yielded conditions suitable for magnetic porogen alignment, composite crosslinking, and porogen clearance, we identified microgel packing as one of the principal process parameters and conducted a systematic study of its influence on granular hydrogel composite rheology and magnetic porogen alignment. We explored packing at multiple speeds, with centrifugation or ultracentrifugation, but found significant variability in magnetic porogen alignment and granular hydrogel composite rheology between microgel samples, even when packed under similar conditions. Further, we observed variability even in triplicate samples prepared from a single packed batch. After ruling out the possible effects of microgel batch-to-batch variability, we determined that consistency in microgel packing is itself a source of variability, possibly due to variations in the total packed pellet volume or non-uniform packing distribution in the centrifuge tube. To control these potential effects, we formulated all the samples used in this study from a single large batch of microgels (1.2 mb of droplet phase at 126.25 mg / mL) that were packed, thoroughly mixed to avoid pellet inhomogeneity, and immediately apportioned into the required samples, followed by refrigerated storage until use. Briefly, a batch of microgels was placed in an ultracentrifuge and spun down at 30,000 RPM for 20 minutes. The supernatant was removed with a pipette and the pellet was mixed thoroughly. An appropriate mass of pellet was weighed, split into microcentrifuge tubes, and stored in 4°C until use in two sets of experiments. In one set of experiments, microgels with interstitial polymer were used for rheology assessment. In another set of experiments, microgels were later mixed with the corresponding interstitialpolymer and porogen solutions and magnetically templated. After tempi ati ng, some gels were imaged using nano-CT and others were imaged for porogen clearance.

[0092] With a standardized method for reducing variability in microgel preparation, we tested the rheology of granular solutions with varying microgel concentrations. We were specifically interested in yield stress, yield strain, and viscosity, as these could play a role in the magnetic porogen alignment process. Medium viscosity is important as it opposes magnetic attraction, slowing down the process of chain formation. We also reasoned that yield stress and strain properties of the suspension would be relevant in opposing gravitational settling of the dense magnetic porogens. However, these same properties, in excess, may oppose chain formation by setting a threshold magnetic attraction below which chains may not form. Yield strain may be relevant as it could correlate to the minimum deformation needed for the porogen to flow past the microgels, potentially correlating with the magnetic field force. Additionally, yield stress could represent a force against gravity and hinder any potential settling of the porogen.

[0093] We performed oscillatory rheology and flow curve measurements for each of the precursor composite solutions without the porogen (Figure 12). Frequency sweeps show typical viscoelastic material behavior, with the average G” of three independent replicates sharply increasing above 10 Hz (Figure 12A) and the data suggest that intermediate microgel concentrations tend to have lower storage moduli (Figure 12B). Oscillatory strain sweeps showed the expected viscoelastic behavior of granular hydrogel materials with an overshoot of G” over G’ at higher strain deformations, indicating a transition from elastic dominated deformation to a more significant viscous component (Figure 12C). This overshoot in G” was not as sharp nor as defined in the 36% w / w microgel group compared to the others. The yield strain of these materials was determined to occur at 0.9 G’ of the averaged G’ in the linear viscoelastic region (< 1% strain). Again, the data suggests that intermediate microgel concentrations have lower yield strains (Figure 12D). Unidirectional flow curve measurements were performed to obtain the stress vs shear rate curve, which was fitted to a Herschel-Bulkley model (Figure 12E) to estimate the yield stress exhibited by these materials (Figure 12F), with the results also suggesting that intermediate microgel concentrations tend to have lower yield stress. Finally, viscosity vs shear rate curves were obtained from unidirectional flow curves, demonstrating shear-thinning behavior (Figure 12G). The average viscosity at a shear rate of0.01 s'1suggests that intermediate microgel concentrations tend to have lower viscosity (Figure 12H).

[0094] We applied statistical analysis to determine if differences in microgel concentration led to differences in rheological properties. First, a one-way Welch ANOVA test was performed to evaluate the effect of microgel concentration on each property. However, it was found that there was no statistically significant difference between concentration levels. Despite this, inspection of Figures 12B, 12D, 12F, and 12H illustrate a consistent trend in which the mean values reach a minimum at an intermediate microgel concentration close to 36% w / w and tend to increase with higher and lower concentrations. A standard ANOVA will not sufficiently capture this trend by evaluating the data as unordered categorical data, rather than the reality of concentration being a continuous variable. Because of this, each of the rheological properties was fitted to a second order polynomial to capture the non-monotonic behavior of the data with the minimal number of additional fitting terms. Figures 12B, 12D, 12F, and 12H illustrate the fitted regression and 95% confidence interval for each of the properties. The regression broadly follows the observed trends despite relatively low R2values in the range of 0.3 - 0.45, which is likely attributable to the high variance among replicates of each concentration. An ANOVA was performed on each regression model to evaluate the variance captured by the linear and quadratic terms. The linear terms have a p-value above 0.05, suggesting that there is not a significant linear relationship between any of the evaluated properties, while the quadratic term is significant for all models, except in the case of the yield strain (p = 0.058), indicating that there is a significant nonlinear trend between each property and the concentration which is explained by the quadratic model.

[0095] Magnetic templating was performed on samples with the previously tested granular hydrogel rheology and alignment was qualitatively assessed by imaging the gels in molds pre- and post-alignment (Figure 13). There is a discernible difference between the pre- and postalignment images for composites formed at microgel concentrations between 30 - 42% w / w. The lowest microgel concentration (30%) shows a slanted orientation of chains, suggesting that viscosity and yield stress was low enough to allow chain formation but not high enough to maintain chains against gravitational settling. At the other extreme of concentration (45% w / w microgel content) there is little to no change in alignment between pre- and post- images, suggesting that the rheological properties of this higher microgel content in composites supportporogens in place after magnetic field removal but do now allow for much alignment or chaining of the magnetic porogen. A greater degree of alignment was qualitatively observed with the 36, 39, and 42% w / w microgel solutions. This qualitative assessment and the rheological measurements support the notion that microgel solution rheology plays an important role in MAM alignment under a magnetic field.

[0096] The granular PEG hydrogel composites with magnetically aligned porogens were analyzed via nano-CT to obtain 3D images of the chains (Figure 14). Nano-CT images were processed in Imaris to create volume objects of the chains and obtain quantitative information, including orientation angle distribution and number of chains formed. Figures 14A-14F show representative images of the chains generated for each gel composition in a side view and top view. At the lower microgel concentration of 30% w / w, the chains aligned but appear slanted with respect to the vertical axis (Figure 14A). At a microgel concentration of 33% w / w, there is better alignment with the vertical axis (Figure 14B), but the porogen is not evenly distributed in the hydrogel, with more chains at the edges. This is likely due to variance in the magnetic forces arising from magnetic field gradients at the edges of the mold. We observe what can be described as “peak alignment” in the samples of 36% w / w (Figure 14C) and 39% w / w (Figure 14D), where most chains appear vertically aligned and the magnetic porogen appears to be homogeneously distributed throughout the hydrogel volume. In Figure 14E, we see that the 42% w / w microgel sample still has mostly aligned porogen, but the alignment is not fully continuous as chains appear more tortuous and / or shorter. Finally, the porogen did not align in the 45% w / w microgel sample, consistent with our visual observations (Figure 14F).

[0097] Based on the samples that supported porogen alignment, we determined the orientation angle of the chains with respect to the vertical axis in which the magnetic field was applied (90°). Figure 14G shows the orientation angle distribution of the chains. Most groups showed a normal distribution of the chains with peak orientation angle density increasing with microgel content up to the 36% w / w microgel composite. The peak of the normal distribution was a function of microgel content. The orientation angle distributions of the 30, 33, 36, 39, and 42% microgel groups were fitted to a normal distribution, and we obtained average orientation angles of 65, 83, 109, 80, and 91°, respectively. The 36% group exhibited the most non-normal distribution.

[0098] Owing to the observed differences in chain alignment and rheological properties, the 36% w / w hydrogels were further investigated using Imaris. Figure 14H and 141 show isolated views of chain segments with orientations between 0-20° and 160-180° — which would correspond to almost horizontal segments — for 36% and 39% w / w composites, respectively. There are appreciably more segments with almost horizontal orientation angles in the 36% w / w hydrogels. These segments seem to be “buckling” under the weight of the chain during alignment. In contrast, the nearly horizontal segments in the 39% w / w composites tend to be part of small chains that likely did not align due to the higher yield stress and viscosity of the solution prior to polymerization.Evaluation of magnetic porogen clearance from magnetically templated granular PEG hydrogel composites

[0099] Finally, we evaluated porogen clearance from these granular hydrogel composites, as in our prior work with hyaluronic acid and collagen hydrogels, by placing the templated composites in an EDTA clearing solution. Images were acquired every 24 hours prior to an EDTA solution change. The images in Figure 15 show that porogen clearance is achieved in all hydrogels, regardless of microgel content, in a span of 6 days. This is comparable to our observations in bulk microgels, where clearance is observed in under a week.

Claims

We claim:

1. A three-dimensional, biocompatible, micropatterned, composite granular hydrogel scaffold comprising:(i) microgel particles, and(ii) magnetic porogens, wherein the microgels and magnetic porogens are subjected to a magnetic field, such that the magnetic porogens are spatially aligned to form a plurality of columns within the scaffold prior to cross-linking or polymerization of the scaffold.

2. The scaffold of claim 1, further comprising (iii) an interstitial matrix.

3. The scaffold of claim 1 or 2, further comprising one or more biomolecules or bioactive agents.

4. The scaffold of any one of claims 1-3, wherein the biocompatible scaffold is biodegradable.

5. The scaffold of any one of claims 1-4, wherein the microgel particles are about 5pm to about 25pm in diameter.

6. The scaffold of claim 5, wherein the microgel particles are about 10pm in diameter.

7. The scaffold of any one of claims 1-6, wherein the microgel particles comprise between about 30% w / w and about 45% w / w of the scaffold.

8. The scaffold of claim 7, wherein the microgel particles comprise between about 36% w / w and about 42% w / w of the scaffold.

9. The scaffold of claim 8, wherein the microgel particles comprise about 39% w / w of the scaffold.

10. The scaffold of any one of claims 1-9, wherein the microgel particles were previously lyophilized before being reconstituted and combined with the magnetic porogens.

11. The scaffold of any one of claims 1-10, wherein the magnetic porogens are about 10pm to about 150pm in diameter.

12. The scaffold of claim 11, wherein the magnetic porogens are about 80pm to about 90pm in diameter.

13. The scaffold of any one of claims 1-12, wherein the matrix material of the magnetic porogens has been dissolved and diffused out of the scaffold to produce a plurality of aligned voids and microchannels within the scaffold.

14. The scaffold of claim 13, wherein a portion of the microchannels, a network of interconnected microchannels, or both, extend the length of the scaffold.

15. The scaffold of any one of claims 1-14, wherein the microgel particles comprise 4-arm polyethylene glycol (PEG) norbornene (PNB) and 4-arm PEG thiol (PTT).

16. The scaffold of claim 15, wherein the ratio of PNB:PTT in the microgel particles is between about 1:0.5 and about 1 : 1.

17. The scaffold of claim 16, wherein the ratio of PNB:PTT in the microgel particles is about 1 :0.9.

18. The scaffold of claim 2, wherein the interstitial matrix comprises PNB and PTT.19 The scaffold of claim 18, wherein the ratio of PNB:PTT in the interstitial matrix is about 1 : 1.

20. The scaffold of any one of claims 1-19, wherein the magnetic porogen comprises iron oxide nanoparticles encapsulated in a biocompatible, dissolvable, alginate hydrogel matrix.

21. The scaffold of any one of claims 1-20, further comprising cells.

22. A method of preparing a three-dimensional, biocompatible, micropatterned, composite granular hydrogel scaffold, wherein the scaffold comprises:(i) microgel particles, and(ii) magnetic porogens.

23. A method of using a three-dimensional, biocompatible, micropatterned, composite granular hydrogel scaffold in a tissue engineering application, the method comprising:(i) identifying a subject in need of an engineered tissue scaffold,(ii) preparing a suitable scaffold for the application, and(iii) administering the scaffold to the subject.

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