Granular hydrogels for promoting lymphatic tube sprouting

Abstract

Described herein are compositions and methods for promoting lymphatic tube formation and sprouting. In some embodiments, the compositions comprise a porous composite granular hydrogel comprising a non-degradable granular gel phase, and a degradable interstitial matrix phase comprising norbornene-modified hyaluronic acid (NorHA) polymer. Also described herein are methods for preparing composite granular hydrogel compositions.

Description

[0001] GRANULAR HYDROGELS FOR PROMOTING LYMPHATIC TUBE SPROUTING

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 554,011 , filed on February 15, 2024, which is incorporated by reference herein in its entirety.

[0004] FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant R35 GM 143055 awarded by the National Institutes of Health (NIH) and grant 2047903 awarded by the National Science Foundation (NSF). The government has certain rights in the invention.

[0006] REFERENCE TO SEQUENCE LISTING

[0007] This application was filed with a Sequence Listing XML in ST.26 XML format accordance with 37 C.F.R. § 1.831 and PCT Rule 13ter. The Sequence Listing XML file submitted in the USPTO Patent Center, “092012-0039-W001_sequence_listing_xml_3-FEB-2025.xml,” was created on February 3, 2025, contains 2 sequences, has a file size of 4.0 kilobytes (4,096 bytes), and is incorporated by reference in its entirety into the specification.

[0008] BACKGROUND

[0009] Granular hydrogels, formed from microgel subunits, enhance cell proliferation by mimicking the extracellular matrix (ECM) and creating a physiologically relevant microenvironment, surpassing traditional 2D cell cultures. These sub-fractioned gels enable tissue constructs at various scales, supporting organoid production, and serving as building blocks for larger constructs. Hence, these materials have been used for producing scaffolds and bioinks in 3D bioprinting processes. Due to their granular structure, these materials offer large interstitial spaces, promoting the free movement of microgel subtractions. This feature improves injectability due to reduced stiffness compared to bulk hydrogels, while facilitating excellent microstructure recovery through self-healing. Moreover, their inherent porous structure enhances endogenous cell invasion, which is crucial for wound healing and tissue repair.

[0010] Moreover, granular hydrogels can be engineered to support various 3D microenvironments by incorporating motifs that enhance cell proliferation, degradability, and therapeutic effects, facilitating improved tissue remodeling. Functionalization of the polymer backbones with RGD has shown enhanced cell adhesion and tissue morphogenesis, while the integration of degradable peptide sequences, such as matrix metalloproteinase (MMP)-sensitive sequences, modifies the scaffold stiffness and promotes remodeling.

[0011] The porosity of granular hydrogels can be controlled by altering microgel size distribution and microgel packing (jamming). There are physical methods, such as centrifugation or vacuum packing, that can modulate the pore interconnectivity. Pore sizes can be further modified through intermolecular and chemical interactions. In this sense, Anderson et al., Acta Biomaterialia 150: 111-127 (2022); Griffin et al., Nature Mater. 737-744 (2015), and Liu et al., Adv. Healthcare Mater. 2300823 (2023), have produced microparticles interconnection via covalent bonding to produce microporous scaffolds. Furthermore, Riley et al., Small 2303466, 2022), demonstrated that by using the same interparticle connectivity strategy, the micropore sizes are influenced by the size distribution of the microgels. Moreover, Widener et al., Biomaterials Sci. 9 2480-2493 (2021) have used guest-host supramolecular assemblies to produce dynamic interparticle interactions to control the gel’s porosity while preserving the gel’s injectability. Additional studies have explored the implementation of interstitial matrices to create composite granular hydrogels for enhancing cell support and attachment in the micropores.

[0012] The variable porosity of granular gels allows for the fabrication of different tissue constructs. For instance, porous architecture supports angiogenesis. Qazi et al., Adv. Mater. 34: 2109194 (2022) demonstrated that embedded human umbilical vein endothelial cells (HUVECs) spheroids can sprout within composite granular hydrogels, and that the length and density of sprouts is driven by the pore size. Similarly, Muir et al., Advanced Science 10: 2206117 (2023) showed that cells undergo sprouting when the interstitial matrix is functionalized with RGD. Also, Ramirez-Calderon et al., ACS Applied Mater. Interf. 13: 29281-29292 (2021) used composite granular hydrogels made with peptide sequences to evaluate angiogenesis in vitro. The authors showed that the cells adapted the shape of the individual microgels and coated them prior to the formation of vessels. However, successful vascularization typically requires matrix support either through interstitial matrix or cell encapsulation within individual microgels. Moreover, typically cells from mesodermal lineage are co-cultured with the endothelial cells to provide additional support to the vasculature. However, there is limited understanding of how growth factors influence lymphatic vascular growth, which can explain the relatively few successful results in lymphatic endothelial cells (LEG) encapsulation in 3D scaffolds. There is also a lack of studies that explore the implementation of granular gels for supporting lymphangiogenesis.

[0013] Lymphatic vessels perform a diverse set of physiological functions such as lymph circulation, inflammation modulation, and wound healing, while also playing a major role in the immune response to several pathologies. Hence, engineering lymphatic vessels is of special interest to produce tissue constructs with physiological fidelity. In this context, Rutsche et al., Adv. Mater. 35: 2209476 (2023) explored how granulated hydrogels systems can sustain lymphatic tube formation. The authors showed that granular hydrogels with different porosities had different outcomes on lymphangiogenesis with lower porosity leading to better tube formation outcomes due to the increased surface contact between microgels and the mechanical support provided to the lymphatic endothelial cells (LECs). However, it is still unclear how the different porosities affect lymphangiogenesis specially during early vascular development, where hyaluronic acid (HA) is made up most of the embryonic ECM.

[0014] What is needed are new hydrogel compositions and methods for promoting lymphatic tube formation and sprouting through lymphatic regeneration and lymphangiogenesis.

[0015] SUMMARY

[0016] One embodiment described herein is a composite granular hydrogel composition comprising: a non-degradable granular gel phase; and a degradable interstitial matrix phase comprising norbornene-modified hyaluronic acid (NorHA) polymer; wherein the composition is porous and comprises a pore density of about 5 » 10'6pores / pm2to about 2.5 x 1Q-5pores / pm2. In one aspect, the NorHA polymer is further functionalized with an arginylglycylaspartic acid (RGD) peptide motif and a matrix metalloproteinase (MMP)-sensitive crosslinker motif. In another aspect, the composition comprises an average pore size of about 1 x 103pm2to about 1 x 105pm2. In another aspect, the composition comprises a porosity of about 5% to about 25%. In another aspect, the composition has a storage modulus of about 200 Pa to about 600 Pa. In another aspect, the composition promotes lymphatic tube formation and sprouting of lymphatic endothelial cells.

[0017] Another embodiment described herein is a method for promoting lymphatic tube formation and sprouting, the method comprising: adding lymphatic endothelial cells to a composite granular hydrogel composition comprising: a non-degradable granular gel phase; and a degradable interstitial matrix phase comprising norbornene-modified hyaluronic acid (NorHA) polymer; wherein the composition is porous and comprises a pore density of about 5 x w6pores / pm2to about 2.5 x 10-5pores / pm2; and incubating the composite granular hydrogel composition containing the lymphatic endothelial cells in cell culture conditions for a period of time sufficient for lymphatic tube formation and sprouting to occur. In one aspect, the method does not include any mesodermal lineage supporting cells. In another aspect, the period of time sufficient for lymphatic tube formation and sprouting to occur is about 1 day to about 10 days. In another aspect, the NorHA polymer of the composite granular hydrogel composition is further functionalized with an arginylglycylaspartic acid (RGD) peptide motif and a matrix metalloproteinase (MMP)-sensitive crosslinker motif to enhance adhesion of the lymphatic endothelial cells and modify the stiffness of the composite granular hydrogel composition. In another aspect, the lymphatic endothelial cells are added to the composite granular hydrogel composition at a density of about 7 x 106cells / mL to about 9 106cells / mL. In another aspect, the method further comprises adding VEGF-C, FGF, or a combination thereof to the composite granular hydrogel composition to stimulate lymphatic tube formation and sprouting. In another aspect, the method produces linear-like lymphatic tube sprouts. In another aspect, the method upregulates the expression of lymphatic biomarkers secreted from the lymphatic endothelial cells into the composite granular hydrogel composition. In another aspect, the lymphatic biomarkers comprise LYVE-1, MMP2, MMP14, PDPN, Proxl, VEGFR3, Reelin, TIMP-1, or combinations thereof. In another aspect, the composite granular hydrogel composition mimics an extracellular matrix environment suitable for lymphatic tube formation and sprouting.

[0018] Another embodiment described herein is a method for preparing a composite granular hydrogel composition, the method comprising: dissolving a norbornene-modified hyaluronic acid (NorHA) polymer in a solution comprising a crosslinker and a photoinitiator; emulsifying the solution with a surfactant to form an emulsified solution; agitating the emulsified solution to form a microdroplet; exposing the microdroplet to ultraviolet (UV) light to form a crosslinked microgel; treating the crosslinked microgel with 1 H,1 H,2H,2H-perfluoro-1-decanol to break emulsions; and centrifuging the crosslinked microgel to form the composite granular hydrogel composition. In one aspect, the crosslinker is dithiothreitol (DTT). In another aspect, the photoinitiator is 2- hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone. In another aspect, agitating comprises pipetting or vortexing the emulsified solution. In another aspect, pipetting comprises about 5 to about 50 repetitive cycles of aspiration and dispensing of the emulsified solution. In another aspect, pipetting comprises a flow rate of about 0.3 pL / s to about 1.0 pL / s. In another aspect, vortexing comprises about 30 s to about 180 s of vortexing. In another aspect, centrifuging comprises about 500 x g to about 7000 x g centrifugation. In another aspect, the method further comprises washing and filtering the composite granular hydrogel composition. In another aspect, the method further comprises functionalizing the NorHA polymer with an arginylglycylaspartic acid (RGD) peptide motif and a matrix metalloproteinase (MMP)-sensitive crosslinker motif. In another aspect, the composite granular hydrogel composition is porous and comprises a pore density of about 5 x 10’6pores / pm2to about 2.5 x 1O’5pores / pm2. In another aspect, the composite granular hydrogel composition is porous and comprises an average pore size of about 1 x 1 o3pm2to about 1 x io5pm2. In another aspect, the composite granular hydrogel composition is porous and comprises a porosity of about 5% to about 25%. In another aspect, the composite granular hydrogel composition has a storage modulus of about 200 Pa to about 600 Pa.

[0019] DESCRIPTION OF THE DRAWINGS

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0021] FIG. 1A-D show graphical representations of granular hydrogels composites fabrication and lymphatic sprouting assays. FIG. 1A shows the reaction mechanism from tetrabutylammonium hyaluronic acid (TBA-HA) to norbornene-modified hyaluronic acid (NorHA). FIG. 1 B shows NorHA crosslinking and functionalization with RGD and FITC using DTT or MMP- sensitive crosslinkers. FIG. 1C shows the procedure for granular gels fabrication via vortexing and pipetting. FIG. 1 D shows the inclusion of a degradable interstitial matrix made with NorHA for supporting LECs sprouting.

[0022] FIG. 2A-E show morphological characterization of the granular gels produced by vortexing and pipetting. FIG. 2A shows an orthogonal view of confocal imaging of FITC labeled granular gels, and porous structure rendering, respectively for pipetting, vortexing 90 s (V90s), and vortexing 180 s (V180s). FIG. 2B shows a scatter plot for the microgels sizes for each one of the granular hydrogels samples. FIG. 2C shows calculated porosity through Imaged. FIG. 2D shows pore cross sectional area. FIG. 2E shows number of pores per 10000 pm2cross sectional area. Scale bar is 200 pm. The error bars represent the mean and Cl (95%) for samples n >30 (FIG. B-C), while for n <30 these represent the mean and standard deviation (FIG. C-D). *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001.

[0023] FIG. 3A-I show rheological behavior evaluated for the granular gels fabricated at loose packing and tight packing conditions. FIG. 3A shows a representation of interparticle surface contact changes with respect to the pore size. FIG. 3B shows amplitude sweep - G' measured from 1% to 130% strain at 10 rad / s. FIG. 3C shows elastic modulus estimated from the linear viscoelastic region (LVR) of the materials measured at 10 rad / s. FIG. 3D shows amplitude sweep - G" measured from 1 % to 130% strain at 10 rad / s. FIG. 3E shows frequency sweep - G' measured from 0.1 to 20 rad / s at 1% strain. FIG. 3F shows frequency sweep - G" measured from 0.1 to 20 rad / s at 1% strain. FIG. 3G shows stress relaxation estimated for the samples at 1% strain. FIG. 3H shows step strain measurements carried out using time ramps for 1 % and 800% strain for loose packed gels, and FIG. 3I shows step strain measurements carried out using time ramps for 1% and 400% for the tight packed gels. For FIG. 3B-G, filled symbols correspond to the tight packing condition while the hollow symbols correspond to the loose packing condition. For FIG. 3H-I, the filled symbols correspond to the storage modulus (G') while the hollow symbols correspond to the loss modulus (G"). All samples were measured by triplicate at 25 °C. The error bars represent the mean and standard deviation. *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001.

[0024] FIG. 4A-E show analysis of LECs embedded in bulk NorHA. FIG. 4A shows Z-projection for the LECs embedded in bulk NorHA and granular hydrogels produced via pipetting, vortexing 90 s, and vortexing 180 s at tight packing conditions. The bulk NorHA and the interstitial matrix of the granular hydrogels were made with 5 mM RGD and 1.2 mM MMP-sensitive crosslinker. Projections were generated using the standard deviation built-in function of Imaged. The images show staining for DAPI (blue), F-actin (red) and VE-Cad (magenta), the green channel corresponds to the FITC-labeled microgels. The scale bars correspond to 250 pm. FIG. 4B shows effective degradable ECM area occupied by lymphatic capillaries. FIG. 4C shows number of branching points. FIG. 4D shows the tube width. FIG. 4E shows vessel skeleton length. The error bars represent the mean and standard deviation. *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001.

[0025] FIG. 5A-D show image analysis. FIG. 5A shows a schematization of the process followed for image analysis. The raw z-max projection for the F-actin channel was binarized and thresholded, then a skeletonization was carried out and finally the images were filtered using a fixed 10 pm segment threshold to remove the noise. FIG. 5B shows lymphatic capillaries curvature distribution. FIG. 5C shows normalized capillaries segments sizes. FIG. 5D shows normalized lymphatic cluster sizes.

[0026] FIG. 6A-C show LEC gene expression analyses. FIG. 6A shows LEC gene expression analysis for LYVE-1, MMP2, MMP14, PDPN, Proxl, and VEGFR3 after culture on the granular hydrogels for 24 h (Day 1) and 120 h (Day 5) with 100 ng-mL-1VEGF-C and 50 ng mL’1FGF. Bulk NorHA was used as reference for applying the AACt method, while GAPDH was used as the housekeeping gene. FIG. 6B shows Reelin and TIMP-1 protein quantification at day 1 and day 5. FIG. 6C shows 3D rendering of V180s gel (green - FITC) exhibiting lymphatic capillaries networks formation (red - F-actin). The error bars represent the mean and standard deviation. *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001.

[0027] FIG. 7 shows a scheme of the automated system used for producing the microgels via pipetting. A sketch of an exemplary system is shown adapting a stepper motor for controlling the aspiration / dispensing of the samples by pulsing a regular micro- pipettor (left). The system includes four push bottoms programed via Arduino to control the stepper motor. A photograph of the exemplary pipetting system set up is shown (center). A representation of the effect of the aspiration / dispensing cycles on the morphology of the produced droplets is shown (right).

[0028] FIG. 8A-C show size distribution analysis for the microgels generated by pipetting. FIG. 8A shows micrographs for the microgels produced by pipetting with different aspiration / dispensing cycles (0, 6, 20 and 30 cycles). The scale bar is 890 pm. FIG. 8B shows scatter plot illustrating microgels sizes vs the number of aspiration / dispensing cycles. FIG. 8C shows scatter plot illustrating microgels sizes vs flow rate. The mean and Cl (95%) are reported for all the results as error bars. *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 .

[0029] FIG. 9A-B show size distribution analysis for the microgels generated by vortexing. FIG. 9A shows micrographs for the microgels produced by vortexing 45 s (V45s), 90 s (V90s), and 180 s (V180s). The scale bar is 300 pm. FIG. 9 B shows a scatter plot illustrating microgels sizes. The error bars represent the mean and Cl (95%).

[0030] FIG. 10A-B show morphological characterization of the granular gels produced by vortexing and pipetting and 1500 x g. FIG. 10A shows orthogonal view of confocal imaging of FITC labeled granular gels, and porous structure rendering, respectively for pipetting, vortexing 90s, and vortexing 180s. FIG. 10B shows pore diameter. The error bars represent the mean and Cl (95%). The scale bar is 200 pm. *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001.

[0031] FIG. 11A-B shows amplitude sweep tests. FIG. 11A shows amplitude sweep tests measured from 1% to 130% strain at 10 rad / s for the loose packed gels. FIG. 11 B shows amplitude sweep tests measured from 1 % to 130% strain at 10 rad / s for the tight packed gels. Filled symbols correspond to the storage modulus (G1) while the hollow symbols correspond to the loss modulus (G"). All samples were measured by triplicate at 25 °C.

[0032] FIG. 12 shows Z projections for the LECs embedded in bulk NorHA and granular hydrogels produced via pipetting, vortexing 90 s, and vortexing 180 s at loose packing conditions. The bulk NorHA and the interstitial matrix of the granular hydrogels were made with 5 mM RGD and 1.2 mM MMP-sensitive crosslinker. Projections were generated using the standard deviation built-in function of Imaged. The images show staining for DAPI (blue), F-actin (red) and VE-Cad (magenta), the green channel corresponds to the FITC-labeled microgels. The scale bar corresponds to 250 pm.

[0033] FIG. 13 shows LEG gene expression analysis for LYVE-1, MMP2, MMP14, PDPN, Proxl, and VEGFR3 after culture on the granular hydrogels for 24 h (Day 1) and 120 h (Day 5) with 100 ng mL'1VEGF-C and 50 ng mL'1FGF. Monolayer LECs were used as reference for applying the AACt method, while GAPDH was used as the housekeeping gene. The error bars represent the mean and standard deviation. *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001. DETAILED DESCRIPTION

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0035] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0036] As used herein, terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of’ the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, “comprising,” is an “open- ended” term that does not exclude additional, unrecited elements or method steps. As used herein, “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim.

[0037] As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

[0038] As used herein, the term “or” can be conjunctive or disjunctive.

[0039] As used herein, the term “and / or” refers to both the conjunctive and disjunctive.

[0040] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0041] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.”

[0042] All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1 , 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points, or as described above in the definition of “about.”

[0043] As used herein, the terms “room temperature,” “RT,” or “ambient temperature” refer to the typical temperature in an indoor laboratory setting. In one aspect, the laboratory setting is climate controlled to maintain the temperature at a substantially uniform temperature or with a specific range of temperatures. In one aspect, “room temperature” refers a temperature of about 15-30 °C, including all integers and endpoints within the specified range. In another aspect, “room temperature” refers a temperature of about 15-30 °C; about 20-30 °C; about 22-30 °C; about 25-30 °C; about 27-30 °C; about 15-22 °C; about 15-25 °C; about 15-27 °C; about 20-22 °C; about 20-25 °C; about 20-27 °C; about 22-25 °C; about 22-27 °C; about 25-27 °C; about 15 °C ± 10%; about 20 °C ± 10%; about 22 °C ± 10%; about 25 °C ± 10%; about 27 °C ± 10%; ~20 °C, ~22 °C, ~25 °C, or ~27 °C, at standard atmospheric pressure.

[0044] As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect.

[0045] As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.

[0046] As used herein, the term “dose” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein. As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.

[0047] As used herein, the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an action, agent, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount may be based on factors individual to each subject, including, but not limited to, the subject’s age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired.

[0048] As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), nonhuman primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.

[0049] As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

[0050] As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0051] As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of” or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a cell, composition, or compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.

[0052] Building upon previous studies, the work described herein aims to employ composite granular hydrogels where the granular component serves as a template to provide mechanical stability to support lymphangiogenesis. Hence, the influence of the granular component morphology on lymphatic vasculature formation was evaluated. This disclosure also presents one of the first successful approaches to 3D in vitro production of lymphatic vessels without mesodermal lineage co-culture (e.g., fibroblasts). NorHA was utilized to fabricate composite granular hydrogels, which include a non-degradable granular phase and a degradable interstitial matrix. Various granular gel morphologies were produced using different fabrication methods and packing degrees, and their rheological behavior and capacity to support lymphatic sprouting were evaluated.

[0053] One embodiment described herein is a composite granular hydrogel composition comprising: a non-degradable granular gel phase; and a degradable interstitial matrix phase comprising norbornene-modified hyaluronic acid (NorHA) polymer; wherein the composition is porous and comprises a pore density of about 5 » 10'6pores / pm2to about 2.5 x 1Q-5pores / pm2. In one aspect, the NorHA polymer is further functionalized with an arginylglycylaspartic acid (RGD) peptide motif and a matrix metalloproteinase (MMP)-sensitive crosslinker motif. In another aspect, the composition comprises an average pore size of about 1 x 103pm2to about 1 x 105pm2. In another aspect, the composition comprises a porosity of about 5% to about 25%. In another aspect, the composition has a storage modulus of about 200 Pa to about 600 Pa. In another aspect, the composition promotes lymphatic tube formation and sprouting of lymphatic endothelial cells.

[0054] Another embodiment described herein is a method for promoting lymphatic tube formation and sprouting, the method comprising: adding lymphatic endothelial cells to a composite granular hydrogel composition comprising: a non-degradable granular gel phase; and a degradable interstitial matrix phase comprising norbornene-modified hyaluronic acid (NorHA) polymer; wherein the composition is porous and comprises a pore density of about 5 x w6pores / pm2to about 2.5 x 10-5pores / pm2; and incubating the composite granular hydrogel composition containing the lymphatic endothelial cells in cell culture conditions for a period of time sufficient for lymphatic tube formation and sprouting to occur. In one aspect, the method does not include any mesodermal lineage supporting cells. In another aspect, the period of time sufficient for lymphatic tube formation and sprouting to occur is about 1 day to about 10 days. In another aspect, the NorHA polymer of the composite granular hydrogel composition is further functionalized with an arginylglycylaspartic acid (RGD) peptide motif and a matrix metalloproteinase (MMP)-sensitive crosslinker motif to enhance adhesion of the lymphatic endothelial cells and modify the stiffness of the composite granular hydrogel composition. In another aspect, the lymphatic endothelial cells are added to the composite granular hydrogel composition at a density of about 7 x 106cells / mL to about 9 106cells / mL. In another aspect, the method further comprises adding VEGF-C, FGF, or a combination thereof to the composite granular hydrogel composition to stimulate lymphatic tube formation and sprouting. In another aspect, the method produces linear-like lymphatic tube sprouts. In another aspect, the method upregulates the expression of lymphatic biomarkers secreted from the lymphatic endothelial cells into the composite granular hydrogel composition. In another aspect, the lymphatic biomarkers comprise LYVE-1, MMP2, MMP14, PDPN, Proxl, VEGFR3, Reelin, TIMP-1, or combinations thereof. In another aspect, the composite granular hydrogel composition mimics an extracellular matrix environment suitable for lymphatic tube formation and sprouting.

[0055] Another embodiment described herein is a method for preparing a composite granular hydrogel composition, the method comprising: dissolving a norbornene-modified hyaluronic acid (NorHA) polymer in a solution comprising a crosslinker and a photoinitiator; emulsifying the solution with a surfactant to form an emulsified solution; agitating the emulsified solution to form a microdroplet; exposing the microdroplet to ultraviolet (UV) light to form a crosslinked microgel; treating the crosslinked microgel with 1 H,1 H,2H,2H-perfluoro-1-decanol to break emulsions; and centrifuging the crosslinked microgel to form the composite granular hydrogel composition. In one aspect, the crosslinker is dithiothreitol (DTT). In another aspect, the photoinitiator is 2- hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone. In another aspect, agitating comprises pipetting or vortexing the emulsified solution. In another aspect, pipetting comprises about 5 to about 50 repetitive cycles of aspiration and dispensing of the emulsified solution. In another aspect, pipetting comprises a flow rate of about 0.3 pL / s to about 1.0 pL / s. In another aspect, vortexing comprises about 30 s to about 180 s of vortexing. In another aspect, centrifuging comprises about 500 x g to about 7000 x g centrifugation. In another aspect, the method further comprises washing and filtering the composite granular hydrogel composition. In another aspect, the method further comprises functionalizing the NorHA polymer with an arginylglycylaspartic acid (RGD) peptide motif and a matrix metalloproteinase (MMP)-sensitive crosslinker motif. In another aspect, the composite granular hydrogel composition is porous and comprises a pore density of about 5 x 10’6pores / pm2to about 2.5 x 1O’5pores / pm2. In another aspect, the composite granular hydrogel composition is porous and comprises an average pore size of about 1 x 1 o3pm2to about 1 x io5pm2. In another aspect, the composite granular hydrogel composition is porous and comprises a porosity of about 5% to about 25%. In another aspect, the composite granular hydrogel composition has a storage modulus of about 200 Pa to about 600 Pa.

[0056] It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0057] Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

[0058] Clause 1 . A composite granular hydrogel composition comprising: a non-degradable granular gel phase; and a degradable interstitial matrix phase comprising norbornene-modified hyaluronic acid (NorHA) polymer; wherein the composition is porous and comprises a pore density of about 5 x 10'6pores / pm2to about 2.5 1O’5pores / pm2.

[0059] Clause 2. The composition of clause 1 , wherein the NorHA polymer is further functionalized with an arginylglycylaspartic acid (RGD) peptide motif and a matrix metalloproteinase (MMP)-sensitive crosslinker motif.

[0060] Clause 3. The composition of clause 1 or 2, wherein the composition comprises an average pore size of about 1 x 1Q3pm2to about 1 x 105pm2. Clause 4. The composition of any one of clauses 1-3, wherein the composition comprises a porosity of about 5% to about 25%.

[0061] Clause 5. The composition of any one of clauses 1-4, wherein the composition has a storage modulus of about 200 Pa to about 600 Pa.

[0062] Clause 6. The composition of any one of clauses 1-5, wherein the composition promotes lymphatic tube formation and sprouting of lymphatic endothelial cells.

[0063] Clause 7. A method for promoting lymphatic tube formation and sprouting, the method comprising: adding lymphatic endothelial cells to a composite granular hydrogel composition comprising: a non-degradable granular gel phase; and a degradable interstitial matrix phase comprising norbornene-modified hyaluronic acid (NorHA) polymer; wherein the composition is porous and comprises a pore density of about 5 x 10'6pores / pm2to about 2.5 x 10'5pores / m2; and incubating the composite granular hydrogel composition containing the lymphatic endothelial cells in cell culture conditions for a period of time sufficient for lymphatic tube formation and sprouting to occur.

[0064] Clause 8. The method of clause 7, wherein the method does not include any mesodermal lineage supporting cells.

[0065] Clause 9. The method of clause 7 or 8, wherein the period of time sufficient for lymphatic tube formation and sprouting to occur is about 1 day to about 10 days.

[0066] Clause 10. The method of any one of clauses 7-9, wherein the NorHA polymer of the composite granular hydrogel composition is further functionalized with an arginylglycylaspartic acid (RGD) peptide motif and a matrix metalloproteinase (MMP)- sensitive crosslinker motif to enhance adhesion of the lymphatic endothelial cells and modify the stiffness of the composite granular hydrogel composition.

[0067] Clause 11. The method of any one of clauses 7-10, wherein the lymphatic endothelial cells are added to the composite granular hydrogel composition at a density of about 7 x 106cells / mL to about 9 x 1O6cells / mL.

[0068] Clause 12. The method of any one of clauses 7-11 , further comprising adding VEGF-C, FGF, or a combination thereof to the composite granular hydrogel composition to stimulate lymphatic tube formation and sprouting. Clause 13. The method of any one of clauses 7-12, wherein the method produces linear-like lymphatic tube sprouts.

[0069] Clause 14. The method of any one of clauses 7-13, wherein the method upregulates the expression of lymphatic biomarkers secreted from the lymphatic endothelial cells into the composite granular hydrogel composition.

[0070] Clause 15. The method of any one of clauses 7-14, wherein the lymphatic biomarkers comprise LYVE-1, MMP2, MMP14, PDPN, Proxl, VEGFR3, Reelin, TIMP-1, or combinations thereof.

[0071] Clause 16. The method of any one of clauses 7-15, wherein the composite granular hydrogel composition mimics an extracellular matrix environment suitable for lymphatic tube formation and sprouting.

[0072] Clause 17. A method for preparing a composite granular hydrogel composition, the method comprising: dissolving a norbornene-modified hyaluronic acid (NorHA) polymer in a solution comprising a crosslinker and a photoinitiator; emulsifying the solution with a surfactant to form an emulsified solution; agitating the emulsified solution to form a microdroplet; exposing the microdroplet to ultraviolet (UV) light to form a crosslinked microgel; treating the crosslinked microgel with 1 H,1 H,2H,2H-perfluoro-1-decanol to break emulsions; and centrifuging the crosslinked microgel to form the composite granular hydrogel composition.

[0073] Clause 18. The method of clause 17, wherein the crosslinker is dithiothreitol (DTT).

[0074] Clause 19. The method of clause 17 or 18, wherein the photoinitiator is 2-hydroxy-4'-(2- hydroxyethoxy)-2-methylpropiophenone.

[0075] Clause 20. The method of any one of clauses 17-19, wherein agitating comprises pipetting or vortexing the emulsified solution.

[0076] Clause 21. The method of any one of clauses 17-20, wherein pipetting comprises about 5 to about 50 repetitive cycles of aspiration and dispensing of the emulsified solution.

[0077] Clause 22. The method of any one of clauses 17-21 , wherein pipetting comprises a flow rate of about 0.3 pL / s to about 1.0 pL / s.

[0078] Clause 23. The method of any one of clauses 17-22, wherein vortexing comprises about 30 s to about 180 s of vortexing. Clause 24. The method of any one of clauses 17-23, wherein centrifuging comprises about 500 x g to about 7000 x g centrifugation.

[0079] Clause 25. The method of any one of clauses 17-24, further comprising washing and filtering the composite granular hydrogel composition.

[0080] Clause 26. The method of any one of clauses 17-25, further comprising functionalizing the NorHA polymer with an arginylglycylaspartic acid (RGD) peptide motif and a matrix metalloproteinase (MMP)-sensitive crosslinker motif.

[0081] Clause 27. The method of any one of clauses 17-26, wherein the composite granular hydrogel composition is porous and comprises a pore density of about 5 x 10’6pores / pm2to about 2.5 x 10-5pores / pm2.

[0082] Clause 28. The method of any one of clauses 17-27, wherein the composite granular hydrogel composition is porous and comprises an average pore size of about 1 x 103pm2to about 1 x 105pm2.

[0083] Clause 29. The method of any one of clauses 17-28, wherein the composite granular hydrogel composition is porous and comprises a porosity of about 5% to about 25%.

[0084] Clause 30. The method of any one of clauses 17-29, wherein the composite granular hydrogel composition has a storage modulus of about 200 Pa to about 600 Pa.

[0085] EXAMPLES

[0086] Example 1

[0087] NorHA Synthesis

[0088] NorHA polymer was synthesized following established procedures. See e.g., Muir et al., Adv. Sci. 10: 2206117 (2023). Briefly, hyaluronic acid (HA) in its tetrabutylammonium (HA-TBA) form was dissolved in anhydrous dimethyl sulfoxide (DMSO). Dimethyl aminopyridine (DMAP), norbornene-2-carboxylic acid, and di-terf-butyl dicarbonate (Boc2O) were added to the solution and were allowed to react for 20 h. The NorHA product was dialyzed for two days with a NaCI solution in Dl-water, and two days more with DI water alone. The polymer solution was then frozen at -80 °C, lyophilized for one day, and stored at -20 °C prior to usage. To quantify the degree of substitution (DS), lyophilized polymer was dissolved in deuterium oxide (D2O) and analyzed via1H-NMR (Bruker AVANCE III HD 400 Nanobay).

[0089] Fabrication of Granular Hydrogels

[0090] For the fabrication of the granular gels, first NorHA was dissolved (2% w / v) in 1 x Dulbecco’s phosphate-buffered saline (DPBS) (Corning Life Sciences, MA, USA) with DL- dithiothreitol (DTT) (Sigma-Aldrich, LO, USA) and FITC (GenScript, NJ, USA) at a ratio with respect to the norbornene groups of 0.8 and 0.05, respectively. The water soluble photoinitiator 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) was used at 0.05% (w / v).

[0091] The microgels were fabricated using two different alternatives (1) vortexing, and (2) pipetting. In all cases, microgels were produced via emulsification using a 008-FluoroSurfactant (1 % by mass) dissolved in HFE7500 (RAN Biotechnologies, Inc, MA, USA). For vortexing, the prepared polymer solutions were mixed in a 1 : 1 ratio with the oil and stirred at 60 s, 90 s, and 180 s at 2500 rpm. For pipetting, a home-made pipette microfluidic device was used for the microdroplets generation. Elliptical pipette tips were fabricated following established procedures. Tips were deformed with a torque screwdriver using 20 inch-pounds and equipped to a multichannel pipettor. A stepper motor-controlled system was used to dispense the polymer solution inside the oil (FIG. 7). Different aspiration / dispensing cycles and flow rates were used for generating the microgels.

[0092] Upon collection, the microdroplets were exposed to UV light (Omnicure S2000, Excelitas Canada Inc., Canada) with a power of 20 mW cm2for 4 minutes to prompt their gelation via thiolene reaction. Moreover, 30% 1 H,1H,2H,2H-Perfluoro-1 -decanol (Sigma-Aldrich, LO, USA) in HFE7500 was mixed in a 1 :1 ratio with the suspended microgels for breaking the emulsion. The suspension was centrifuged at 600 * g for 5 minutes and the oil phase was retrieved afterwards. The microgels were then washed and centrifuged at 1500 * g two times with ethanol (70% purity) and seven times with DPBS to extract the remaining oil. The microgels were filtered using a 37 pm reversible cell strainer (STEMCELL Technologies, Canada) to separate the excess DPBS. Finally, loose and tightly packed granular gels were fabricated by centrifuging the microgels at 1500 x g and 6000 g, respectively. All the materials were sterilized prior to their implementation in cell culture.

[0093] Morphological Characterization of Granular Hydrogels

[0094] Fluorescence microscopy (ECHO Revolve, San Diego, CA-USA) was utilized to take micrographs of a monolayer of microgels suspended in 008-FluoroSurfactan. Confocal microscopy was performed using a Nikon AX-R (Nikon Instruments Inc, NY, USA) to obtain randomized 3D stacks of the granular gels. A 10x objective was used for a Z-stack range of roughly 400 pm with an interslice spacing of ~7 pm. The stacks were smoothened and thresholded, while the porosity and pores cross section were estimated by counting the binary pixels using a Python code. Imaged was used to determine the size distribution of the microgels with the analyze particles built-in function. Similarly, the pore size and diameter were estimated. The 3D stacks were then rendered using the Nixon-NIS Elements Software to obtain the orthogonal view of the granular gel.

[0095] Rheological Characterization

[0096] Dynamic rheological measurements were carried out using a Discovery H -2 Rheometer (TA Instruments, DE, USA) equipped with a solvent trap. A 20 mm plate-plate geometry for all the experiments. Silicon oil was placed along the edge of the plate after sample trimming. Amplitude sweep tests were performed at 10 rad / s with a strain ranging from 0.1 % to 130%. After determining the linear viscoelastic region (LVR) frequency sweep tests from 0.1 to 20 rad / s at a constant 1% strain, were performed. Additionally, stress relaxation experiments were carried out for 3 min under 1% strain. Finally, cyclic step strain analyzes were performed by repeatedly cycling the strain for 60 s and 30 s from 1 % to 800% for the loose packing gels, and 1% to 400% for the tight packing gels, to evaluate the self-healing behavior of the granular gels. A 600 pm GAP was used for all the rheological measurements and no wall-slip was evidenced while carrying out the tests. All the rheological measurements were carried out in triplicate at 25 °C and the collected data were smoothed using the Savitzky-Golay filter before plotting.

[0097] Cell Culture

[0098] Human juvenile LECs derived from the foreskin of four donors (C-12216, PromoCell, Heidelberg, Germany) were expanded and used for experiments between passages 4 and 8. Briefly, LECs were grown in endothelial cell growth media MV2 (EGM-MV2, C-22022, PromoCell, Heidelberg, Germany) and incubated at 37 °C with 5% CO2. To keep the cell passaging constant throughout experiments, cells were passaged every 5 days at a 1 :3 ratio. Human LECs were characterized for the positive expression of CD31, LYVE-1, Proxl, and PDPN throughout the experiments. Cell lines were routinely tested for mycoplasma contamination and were negative throughout the present study.

[0099] Lymphatic Sprouting Assays

[0100] A degradable NorHA polymer solution was used to generate an interstitial matrix that was then mixed with each one of the non-degradable granular gels. The polymer solution used as the interstitial matrix precursor was prepared using 2% w / v of NorHA alongside 5 mM thiolated RGD (GCGYGRGDSPG (SEQ ID NO: 1), 1025.1 Da, GenScript, 1.2 mM thiolated MMP-sensitive crosslinker (GCRDGPQGjJWGQDRCG (SEQ ID NO: 2), 1754.0 Da; down arrow indicates the site of proteolytic cleavage, GenScript), and 0.05% w / v Irgacure 2959 (2-Hydroxy-4'-(2- hydroxyethoxy)-2-methylpropiophenone). Cells were added to the polymer solution and suspended by pipetting at a density of 8 * 106cells / mL. The cell-laden polymer solution was mixed with the granular gels through pipetting with wide bore tips. The volumetric ratio of the NorHA solution added to the granular gels was adjusted to match their calculated porosity. The mixtures were placed in a glass bottom 96 well plate where these were exposed to UV light with a power of 10 mW cm2for 1 min to prompt the interstitial matrix gelation. Bulk NorHA gels fabricated utilizing the same composition used for the degradable interstitial matrix were used as experimental control. The constructs were cultured using MV2 media with 100 ng / mL VEGF-C (R&D Systems, MN, USA) and 50 ng / mL FGF (R&D Systems). Constructs were cultured for 3 days with media changes every day. The porosity of the granular gels was recalculated to assess the effect of the filling matrix addition.

[0101] Immunostaining and Imaging

[0102] Constructs were washed with DPBS and fixed with 3.7% PFA. Furthermore, the samples were blocked with 1 % BSA, permeabilized with 0.1% Triton-X 100 and stained for F-actin (Phalloidin-iFluor 594, Abeam, MA, USA) DAPI, and VE-Cadherin (conjugated with Alexa Fluor 647, Santa Cruz Biotechnology, TX, USA) to visualize lymphatic tube formation (Table 1). Confocal microscopy was performed to obtain 3D stacks of the lymphatic vessels. 10x and 20x objectives were used for a Z-stack range of roughly 400 pm with an interslice spacing of ~10 pm.

[0103] Table i . Commercial Antibodies

[0104] Reagent Company Host Species Catalog Number Dilution Factor

[0105] Phalloidin Thermo Fisher iFluor 594 ab176757 1 :1 ,000

[0106] VECAD Santa Cruz Alexa Fluor 647 sc-9989 AF647 1 :50

[0107] Lymphatic Networks Analysis and Quantification

[0108] The AutoTube Software was used to analyze and quantify the lymphatic networks in terms of occupied area, skeleton size, tube width, and number of branches. For this, the Z-stacks obtained with the 10x objective were preprocessed by first obtaining the Z-max intensity projection, and then by smoothing and denoising. Finally, thresholding was applied to exclude cells that were not forming vessels from the quantification. The calculated network area was normalized according to the degradable portion of each of the gels. Finally, the Z-projections of the 20x Z-stacks were rendered using the Imaged standard deviation built-in function after smoothing, denoising, and color thresholding. Expression of Lymphatic Related Genes

[0109] Granular hydrogels were collected at day 1 and 5 to be mechanically homogenized for RNA extraction and purification. RNA was reverse transcribed using a High-Capacity cDNA reverse transcription toolkit (Thermo Fisher, MA, USA). TaqMan Universal PCR Master Mix was used with the cDNA to determine the gene expression levels for the genes of interest (Table 2). GAPDH was used as endogenous control for the relative expression which was analyzed through the AACt method. Both monolayer cultured LECs and Bulk NorHA were used as controls and reference in the AACf estimations. All the samples were prepared in triplicate.

[0110] Table 2. Commercial Primers

[0111] Genes Catalog Number

[0112] LYVE-1 Thermo Hs00272659_m1

[0113] PDPN Thermo Hs00366766_m1

[0114] Prox-1 Thermo Hs00896294_m1

[0115] MMP2 Thermo Hs01548727_m1

[0116] MMP14 Thermo Hs01037003_g1

[0117] VEGFR3 Thermo Hs01047677_m1

[0118] GAPDH Thermo Hs02786624_g1

[0119] Reelin and TIMP-1 Protein Quantification

[0120] To carry out the Reelin and TIMP-1 protein quantification, gel culture supernatants were collected at day 1 and 5 and centrifuged and filtered through 0.2 pm filters. Then, Bradford assays (Thermo Fisher, MA, USA) were performed to quantify the protein content in each sample. Abeam Elisa kits were used to quantify Reelin (ab284620 - Abeam, MA, USA) and TIMP-1 (ab100651). The experiments were carried out following the manufacturer’s suggested procedure. Each sample was run at least in triplicate.

[0121] Statistical Analysis

[0122] Data visualization and analysis were carried out using GraphPad Prism software. The reported experiments were repeated at least three times. Shapiro-Wilk test was used to test the normality of sample sizes <50 n, while Kolmogorov-Smirnov test was used for n >50. Welch’s t test was utilized to compare differences between two sample groups, while one-way Brown- Forsythe and Welch ANOVA tests were carried out to compare differences between more than two groups. The corresponding levels of significance were *p < 0.05, **p < 0.01, ***p < 0.001 , ****p < 0.0001. Example 2

[0123] NorHA Synthesis / Characterization and Granular Hydrogels Fabrication

[0124] As reported in previous studies, NorHA (FIG. 1A) was used to fabricate granular gels. NorHA was specifically chosen for this study to take advantage of its HA backbones for LECs binding via their lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), an interaction which has been shown in the past to upregulate key lymphatic markers and preserve lymphatic phenotypes. The degree of substitution (DS) of norbornene groups on the HA backbone was estimated via1H-NMR, obtaining 22% DS. Norbornene allows for an easy crosslinking process via thiol-ene reactions with DTT and MMP-sensitive crosslinkers modified with thiol groups (FIG. 1 B). At the same time, the norbornene-modified HA backbone can be easily functionalized with other motifs for gel visualization purposes (i.e., FITC), and for enhancing cells attachment to the ECM (i.e., RGD). In this sense, the polymer solution formulation was adjusted to use 80% of norbornene groups for crosslinking, while 16% of these were occupied by RGD, and the remaining 4% by FITC.

[0125] Different methods were utilized for fabricating the granular gels (FIG. 1C), namely vortexing and pipetting methods. By using a pipette with an elliptical cross-section to enhance the destabilizing azimuthal curvature and capillary pressure, the pinching action becomes more robust and periodic resulting in uniformly sized droplets. Diverse granular gel morphologies were expected by using these different methods, which would give insights on their effect on different properties of the hydrogels such as their rheological behavior, cell invasion, and lymphatic tube formation. The abilities of the different granular hydrogels to support lymphatic sprouting were also evaluated. Human LECs were suspended in an interstitial matrix precursor produced with NorHA, which would provide microgels interconnectivity while supporting the lymphatic sprouts (FIG. 1 D). 3D lymphatic tube formation was finally evaluated after five days of culture. Further results reported in this study are mainly divided into five sections: (I) morphological characterization of the granular gels, (II) the effect of the gels’ morphology on their rheological behavior, (III) lymphatic sprouting assays performed in granular hydrogels composites, (IV) the evaluation of the lymphatic capillaries’ connectivity, and (V) the influence of the gel morphology on the lymphatic gene expression.

[0126] Example 3

[0127] Morphological Characterization of Granular Hydrogels Granular hydrogels were produced using different alternatives to test their effects on the morphological characteristics. For the pipetting alternative, an automated system incorporating a stepper motor for controlling solution dispensing was used (FIG. 7). Dispensing flow rate and dispensing / aspiration cycles were varied to determine their effect on the microgels’ uniformity. Results showed that for the evaluated range, flow rate had no significant effect on the droplet size distribution, whereas higher number of cycles resulted in higher uniformity (FIG. 8). Hence, a flow rate of 0.66 pL / s and 20 dispensing / aspiration cycles were chosen for fabricating the granular gel at scale. Similarly, the effect of the vortexing duration on the size distribution of the microgels was studied (FIG. 9). Interestingly, by increasing the vortexing time, the size distribution became more uniform with lower average. Based on this, two different vortexing times (90 s and 180 s) were used for producing gel at scale, as the wider size distributions of gels may lead to tight microgels packing without changing the microgels jamming conditions.

[0128] Hydrogels were produced by microgels jamming via centrifugation at 1500 x g (loose packing) and 6000 x g (tight packing). Visually, the hydrogels produced by vortexing and pipetting had different sizes for the microgels subunits and different porous morphologies (FIG. 2A, and FIG. 10A). This may affect the interconnectivity within the granular gels and the kind of tissue structures that can be obtained. Moreover, it was also observed that each one of the samples had different size distributions (FIG. 2C), with the granular gel prepared by vortexing for 90 s having the widest distribution, while the pipetting method resulted in the most uniform distribution.

[0129] Additional analyses of the porous structure of the granular hydrogels were conducted. It was observed that there are significant differences in the porosity depending on the degree of packing of the microgels after the jamming process (FIG. 2C). Additionally, the porosity of the vortexing samples was statistically similar, with significant differences with the pipetting-produced granular gel for both the loose and tight packing scenarios. The latter had the lowest porosity (-22% loose packing and -9% for tight packing); although, due to the different size dispersion of the microdroplets, each sample had a different pore size distribution for the loose packing condition (FIG. 2D) which also resulted in larger pore diameter (FIG. 10B). Conversely, for the tightly packed gels, those differences were not significant. This suggests that the packing process (i.e. , centrifugation) had greater influence on the porous structure of the granular gels than the fabrication method and size distribution of the microgels. Moreover, the pores’ density was estimated (FIG. 2E). As expected, lower pore counts per sectional area (10000 pm2) were obtained for the pipetting sample due to its larger particles size. However, marginal differences were observed between the loose and tight packing scenarios, suggesting that higher centrifugation velocity affects the porosity but not the pores density. These different interstitial properties can affect the mechanical properties of the granular gels, as with larger pores it is expected to have less interparticle friction. Hence, the rheological properties of the gels produced by both methods should be significantly different. Moreover, previously it has been described that the interstitial structure of the granular gels can act as a physical cue, affecting the phenotype of embedded cells and tissue development. Therefore, variations in the LEG sprouting morphologies were expected based on the granular gel type.

[0130] Example 4

[0131] Effect of Morphology on the Rheological Behavior of the Hydrogels

[0132] It was expected that granular hydrogels with different morphological characteristics have different rheological behaviors as their elastic and viscous components are governed by their interparticle surface contact (i.e., friction) and deformation. Hence, for granular hydrogels composed of microgels with smooth surfaces, not only the porosity, but also the size of the pores plays an important role on their rheological behavior as these morphological properties determine the microgels’ contact (FIG. 3A). Therefore, the effect of the granular hydrogels packing on their oscillatory rheological behavior was evaluated.

[0133] By evaluating the materials via amplitude sweep, the linear viscoelastic region (LVR) for the loose packing conditions was shorter for the pipetting-produced gel than that of both vortexing- produced gels (FIG. 11A). These results are in agreement with the pore sizes calculated for the materials, as with a higher packing the interparticle surface contact for the vortexing hydrogels is greater than for the pipetting gel due to their smaller pore sizes. Nonetheless, while at low strains the vortexing gels had a notably higher elastic behavior, their elastic modulus decayed faster than for the pipetting gel which is demonstrated by their crossover points indicating the shift towards a viscous-like behavior at a strain of -43% for the V90s and -91 % for V180s. Interestingly, the crossover point was not observed for the pipetting gel within the experimental range, meaning that its predominant elastic behavior is preserved over a larger strain range. This effect is presumably due to the dominance of the microgels’ mechanical response over the bulk material’s response due to their size distribution which is much larger than the hydrogel pores. Nonetheless, at tight packing conditions, the crossover point is clearly observed for all the samples, with the pipetting-produced gel remaining higher (FIG. 11 B).

[0134] Similarly, the storage modulus for the tightly packed gels was significantly higher than the one of their loose packing counterparts (FIG. 3B). Additionally, the storage modulus was similar across all the granular hydrogels fabricated under tight packing conditions. These findings are presumably related to the reduction in the porosity of the materials when using a higher centrifugation velocity, as the interparticle surface contact is increased, thereby increasing the contribution from the bulk material. It is anticipated that these differences in the rheological behavior with the materials’ packing may have implications on lymphatic morphogenesis, as at loose packing conditions the mechanical support provided by the granular hydrogels would be lower. Moreover, as at static conditions the mechanical properties of the tight packed granular hydrogels are similar. Hence, it is anticipated that the differences in the lymphatic sprouting can be attributed to the distinct materials morphology.

[0135] The additional rheological characterization shows that the G' of the frequency sweep tests for the loose packing condition (FIG. 3D) suffers little to no variations with increasing the rate of deformation, while for the tight packing there is a small build-up at 12 rad / s. For all the samples the loss modulus (G") slightly reduces with the rate of deformation (FIG. 3F). Furthermore, the samples show typical stress relaxation behavior with the initial modulus value following similar trends as from the previous tests (FIG. 3G). The self-healing behavior of the gels was also evaluated. For all the loose packing samples (FIG. 3H) there is a full mechanical recovery after being subjected to 800% strain. Conversely, at strains higher than 400% no reliable data for the storage modulus of the tightly packed sample was collected due to the sensitivity of the instrument. However, the materials experienced similar mechanical recovery after decreasing the strain (FIG. 3I). As shown by the amplitude sweep tests, the difference between the elastic and viscous modulus at 800% (loose packing) and 400% (tight packing) strain for the pipetting- produced gel is smaller compared to the shift observed for the vortexing-produced gels, which again is in agreement with a predominant microgels deformation contribution over the pore structure re-shaping contribution at high strains.

[0136] Example 5

[0137] Lymphatic Tube Formation Within Composite Granular Hydrogels

[0138] As reported before, the morphology of granular gels can affect the type of tissue-like architecture of constructs obtained when embedding cells in these interstitial spaces. Porosity can be especially important during the early stage of lymphatic vessel formation as the connectivity between tube fragments may be affected by microgels’ surface contact. Hence, lymphatic tube formation was tested on the granular hydrogels by seeding human LECs on a degradable interstitial matrix precursor as shown in FIG. 1 D. The interstitial matrix formulation included 5 mM of RGD and 1.2 mM of MMP-sensitive crosslinkers. Such formulation was chosen based on a previous study in which the bulk NorHA composition was optimized for supporting functional and mature lymphatic networks. 100 ng / mL VEGF-C and 50 ng / mL FGF were also used in the culture media to help stimulate lymphatic tube formation.

[0139] LECs were embedded within pipetting, V90s and V180s granular hydrogels at a density of 8 x 106cells / mL, based on previous reports. The gels were fixed and stained for DAPI, F-actin and VE-Cad after 5 days under culture to be further analyzed via confocal imaging. Z-stacks for the stained gels were captured every 10 pm and the images were rendered using ImageJ. From the confocal images, no tube formation was observed for the loose packing samples (FIG. 12) as there was no formation of continuous cellular structures as indicated by the VE-Cad staining showing a lack of cell-cell adhesion. Instead, it was observed that the cells tend to attach to the microgels adapting to their shapes. Conversely, for the tight packing conditions (FIG. 4A), the formation of lymphatic capillaries with cell-cell attachment was observed. Visually, the percentage of cells included in these networks is higher for the granular hydrogels than for the bulk NorHA, with the V180s sample having the highest vessels density. Importantly, the tube width / diameter is about 20 microns (FIG. 4D) for all granular gels, which is comparable to the mean pore size of V180 sample and much smaller than the mean pore size of the pipetted gel (FIG. 2D). For this reason, the gel supported capillaries wrap around the droplet and assume the curvature of the droplets, which is clearer in the vortexing samples.

[0140] To verify this, the capillary-like structures were quantified using the AutoTube Software. First, the area occupied by the lymphatic networks was analyzed (FIG. 4B) and normalized based on the degradable portion of the gel (effective tube area). The results showed that the V180s and pipetting gels had the largest effective area occupied by the lymphatic networks but with a higher experimental consistency for the V180s, as observed from the error bars. Other features such as the number of branching points (FIG. 4C) and the skeleton length (FIG. 4E) exhibited no significant differences between the granular hydrogels and the bulk NorHA control. However, the mean skeleton size appeared substantially higher for the pipetting condition. This effect may be related to other morphological differences that can result in higher capillary connectivity, and therefore higher complexity of vessel clusters.

[0141] To further demonstrate this effect, additional image analyses were carried out to evaluate the capillary clusters and connectivity.

[0142] Example 6

[0143] Evaluation of Lymphatic Capillaries Curvature and Clusters Connectivity

[0144] While effective area occupied by lymphatic vessels is larger for the granular gel samples (FIG. 4B), conventional vessel structure quantification does not account for the connectivity of capillary structures. Therefore, additional image analysis was performed to address the connectivity of the vessel constructs.

[0145] The F-actin channel was utilized to create max intensity projections of the gel samples z stacks (FIG. 5A). Afterwards, the images were binarized and thresholded for carrying out a skeletonization rendering of the lymphatic capillaries with a fixed 1-pixel width. Finally, the skeletonized images were filtered to remove structures smaller than 10 pm and decrease the noise. The results showed that the lymphatic vessels had greater curvature in the vortexing gels whereas for the pipetting samples the capillaries had a straighter shape (FIG. 5B). Interestingly, the mean capillaries segment size was ~17 microns for all the samples (FIG. 5C) which is around the persistence length of F-actin. These two observations might suggest that in tight packing templates with small droplets, the vessels have a higher tendency to adapt to the curvature of the microgels due to the persistence length of F-actin which influence the cells to bend and wrap around the gels surface.

[0146] Additionally, how the curvature and segment size of the capillaries might impact their connectivity was evaluated (FIG. 5D). The results showed that the pipetting gel had bigger vessel clusters whereas the vortexing samples had more satellite small vessel constructs. These results suggest that lymphatic capillaries with lower curvature are less prompt to interact with the granular hydrogel support and wrap around individual microgels. This causes the formation of straighter vessels where the cells could form connected constructs by bridging between several microgels.

[0147] Example 7

[0148] Effect of Granular Hydrogels Morphology on Key Lymphatic Markers

[0149] The gene expression levels for key lymphatic markers were analyzed using RT-qPCR and quantified using the AACr method with GAPDH as housekeeping gene, and monolayer LECs and bulk NorHA as reference. Gels were collected at day 1 and 5 to be mechanically homogenized for RNA isolation. After cDNA synthesis, the samples were assessed using RT-qPCR. Although some of the lymphatic markers were upregulated in the monolayer cultured LECs compared to all the gel samples (FIG. 13), as the LECs cultured in tissue culture plastic surface are unable to form lymphatic capillaries, bulk NorHA was chosen as the control sample (FIG. 5A).

[0150] L.YVE-1 exhibited no significant changes across all the gel samples. Nonetheless, PDPN was upregulated in all the granular gel samples at day 1 , and at day 5 except for the V90s hydrogel. L.YVE-1 and PDPN are important lymphatic markers, responsible for leukocyte trafficking and LEC recognition, respectively. Remarkably, the key receptor to vascular endothelial growth factor-C (VEGF-C), VEGFR3, is upregulated just for V180s sample on day 1 , but reaches a similar level as the bulk NorHA control on day 5. Similarly, prospero homeobox 1 (Proxl) shows a ~2-fold upregulation for the V180s gel on day 1 and on day 5, whereas the pipetting gel experiences upregulation on day 5. Since Proxl is the master regulator of lymphatic vasculature, these results indicate lymphatic vessel phenotype.

[0151] These results, combined with a similar late upregulation of MMP14 in the pipetting gel compared to the V180s gel, account for the faster maturation time in the V180s gel compared to the other gel samples. This is also supported by the 3-fold and ~2-fold upregulation of MMP2 and MMP14 early on, which plays a major role in angiogenesis. It is worth mentioning that Proxl expression levels follow a similar trend as the MMP14. This suggests that the upregulation of MMP14 derived from the ECM remodeling and vessel formation could trigger Proxl upregulation to maintain lymphatic homeostasis.

[0152] To further confirm how the differences in vessel maturation may affect the LECs function, Reelin and Tissue Inhibitors of Metalloproteinase (TIMP-1) secretions were quantified through ELISA (FIG. 5B). Reelin is a key lymphangiocrine, which plays a major role in lymphangiogenesis and promote the health of many vital organs. Reelin is an indicator of tight junctions of LECs with functional architecture, as its secretion depends on the presence of VE-cadherin. Quantification of reelin secretion showed higher levels for the granular hydrogels, suggesting that the encapsulated LECs have the desired functionality with the formation of junctions which are necessary for the generation of functional and mature lymphatic capillaries. Moreover, TIMP-1 functions to inhibit the activity of MMPs, which indicate vessel maturation and stabilization. TIMP- 1 quantification showed an increase in TIMP-1 secretion over time, with the V180s gel having a slightly higher earlier production compared to the pipetting gel. Similarly to the trends observed in the gene expression levels, this trend shifts at day 5, suggesting once more an earlier vessel formation in the V180s gel.

[0153] This study presents novel insights into factors influencing lymphangiogenesis within granular hydrogels, enabling the generation of early-stage lymphatic sprout formation without the need for any supporting cells. The granular hydrogels morphology and their effects on lymphatic capillaries and key lymphatic markers was assessed. The various microgel generation methods (pipetting and vortexing) using NorHA polymer resulted in distinct morphologies. Vortexing- produced gels exhibited higher porosity but wider microgel size distribution, leading to tighter packing compared to pipetting, resulting in smaller pores. This morphology resulted in a higher storage modulus and wider LVR ranges due to increased interparticle contact at loose packing conditions. Such differences were drastically reduced when the granular gels were produced at tight packing conditions. Hydrogel morphological variances significantly affected the lymphatic development. While no lymphatic sprouting was observed for the loose packing samples, at tight packing conditions the pipetting and vortexing gels exhibited different vessel formation and maturation patterns, with the V180s gel having earlier development and maturation. Nonetheless, it was found that the lymphatic capillaries have a higher connectivity in the pipetting gels with a lower number of satellite small vessel constructs. Remarkably, this enhanced connectivity might be derived from the lower mean curvature of the pipetting templates compared to the vortexing ones which reduce the cell-microgels contact. Quantitative RT-PCR data supported these observations, indicating an early MMP2 and MMP14 upregulation in the V180s gel with a higher presence of TIMP-1 protein, which indicates lymphatic vessel early maturation and stabilization. Moreover, the secretion of Reelin, a key lymphangiocrine, indicates lymphatic vessel functionality for all the granular gel samples, that can be further used to support co-culture of other vital organs for tissue engineering applications.

[0154] Overall, this study provides new insights into 3D in vitro lymphatic tube formation, which contributes to the understanding of lymphatic biology and may lead to novel approaches to lymphatic regeneration. Importantly, lymphatic tube networking seems to exhibit some innate curvature and length scales such that robust capillary network is promoted by a templating granular gel that exhibits the same length scales and curvatures.

[0155] The gel droplets generated by the pipetting methods described herein were found to be uniform in size, and this size uniformity permitted a densely packed granular gel, which is not possible with other techniques that produce gel droplets having large variations in size. The densely packed granular gels of certain sizes (curvatures) were much more favorable towards lymphatic vessel formation and sprouting than granular gels having large size variation which cannot be densely packed. The densely packed, uniform in size, granular gels exhibited a specific granular support curvature and pore structure that significantly enhanced lymphatic vessel interconnectivity. These results were both surprising and unexpected.

Claims

CLAIMSWhat is claimed:

1. A composite granular hydrogel composition comprising: a non-degradable granular gel phase; and a degradable interstitial matrix phase comprising norbornene-modified hyaluronic acid (NorHA) polymer; wherein the composition is porous and comprises a pore density of about 5 x 10'6pores / pm2to about 2.5 x 1O'5pores / pm2.

2. The composition of claim 1, wherein the NorHA polymer is further functionalized with an arginylglycylaspartic acid (RGD) peptide motif and a matrix metalloproteinase (MMP)- sensitive crosslinker motif.

3. The composition of claim 1 , wherein the composition comprises an average pore size of about 1 x 103pm2to about 1 x 105pm2.

4. The composition of claim 1 , wherein the composition comprises a porosity of about 5% to about 25%.

5. The composition of claim 1 , wherein the composition has a storage modulus of about 200 Pa to about 600 Pa.

6. The composition of claim 1, wherein the composition promotes lymphatic tube formation and sprouting of lymphatic endothelial cells.

7. A method for promoting lymphatic tube formation and sprouting, the method comprising: adding lymphatic endothelial cells to a composite granular hydrogel composition comprising: a non-degradable granular gel phase; and a degradable interstitial matrix phase comprising norbornene-modified hyaluronic acid (NorHA) polymer; wherein the composition is porous and comprises a pore density of about 5 x 1O'6pores / pm2to about 2.5 x 1O’5pores / pm2; andincubating the composite granular hydrogel composition containing the lymphatic endothelial cells in cell culture conditions for a period of time sufficient for lymphatic tube formation and sprouting to occur.

8. The method of claim 7, wherein the method does not include any mesodermal lineage supporting cells.

9. The method of claim 7, wherein the period of time sufficient for lymphatic tube formation and sprouting to occur is about 1 day to about 10 days.

10. The method of claim 7, wherein the NorHA polymer of the composite granular hydrogel composition is further functionalized with an arginylglycylaspartic acid (RGD) peptide motif and a matrix metalloproteinase (MMP)-sensitive crosslinker motif to enhance adhesion of the lymphatic endothelial cells and modify the stiffness of the composite granular hydrogel composition.

11. The method of claim 7, wherein the lymphatic endothelial cells are added to the composite granular hydrogel composition at a density of about 7 x 106cells / mL to about 9 x 106cells / mL.

12. The method of claim 7, further comprising adding VEGF-C, FGF, or a combination thereof to the composite granular hydrogel composition to stimulate lymphatic tube formation and sprouting.

13. The method of claim 7, wherein the method produces linear-like lymphatic tube sprouts.

14. The method of claim 7, wherein the method upregulates the expression of lymphatic biomarkers secreted from the lymphatic endothelial cells into the composite granular hydrogel composition.

15. The method of claim 14, wherein the lymphatic biomarkers comprise LYVE-1, MMP2, MMP14, PDPN, Proxl, VEGFR3, Reelin, TIMP-1, or combinations thereof.

16. The method of claim 7, wherein the composite granular hydrogel composition mimics an extracellular matrix environment suitable for lymphatic tube formation and sprouting.

17. A method for preparing a composite granular hydrogel composition, the method comprising: dissolving a norbornene-modified hyaluronic acid (NorHA) polymer in a solution comprising a crosslinker and a photoinitiator; emulsifying the solution with a surfactant to form an emulsified solution; agitating the emulsified solution to form a microdroplet; exposing the microdroplet to ultraviolet (UV) light to form a crosslinked microgel; treating the crosslinked microgel with 1 H,1 H,2H,2H-perfluoro-1-decanol to break emulsions; and centrifuging the crosslinked microgel to form the composite granular hydrogel composition.

18. The method of claim 17, wherein the crosslinker is dithiothreitol (DTT).

19. The method of claim 17, wherein the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2- methylpropiophenone.

20. The method of claim 17, wherein agitating comprises pipetting or vortexing the emulsified solution.

21. The method of claim 20, wherein pipetting comprises about 5 to about 50 repetitive cycles of aspiration and dispensing of the emulsified solution.

22. The method of claim 20, wherein pipetting comprises a flow rate of about 0.3 pL / s to about 1.0 pL / s.

23. The method of claim 20, wherein vortexing comprises about 30 s to about 180 s of vortexing.

24. The method of claim 17, wherein centrifuging comprises about 500 * g to about 7000 x g centrifugation.

25. The method of claim 17, further comprising washing and filtering the composite granular hydrogel composition.

26. The method of claim 17, further comprising functionalizing the NorHA polymer with an arginylglycylaspartic acid (RGD) peptide motif and a matrix metalloproteinase (MMP)- sensitive crosslinker motif.

27. The method of claim 17, wherein the composite granular hydrogel composition is porous and comprises a pore density of about 5 x w6pores / pm2to about 2.5 1C)-5pores / pm2.

28. The method of claim 17, wherein the composite granular hydrogel composition is porous and comprises an average pore size of about 1 x 103pm2to about 1 x 105pm2.

29. The method of claim 17, wherein the composite granular hydrogel composition is porous and comprises a porosity of about 5% to about 25%.

30. The method of claim 17, wherein the composite granular hydrogel composition has a storage modulus of about 200 Pa to about 600 Pa.

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