WO2024233108A1

WO2024233108A1 - Bone marrow microgels and uses thereof

Info

Publication number: WO2024233108A1
Application number: PCT/US2024/025724
Authority: WO
Inventors: Sangmin Lee; David J. Mooney
Original assignee: President And Fellows Of Harvard College
Priority date: 2023-05-09
Filing date: 2024-04-22
Publication date: 2024-11-14

Abstract

The present disclosure generally provides microgels, e.g., microgel scaffolds, and method for modulating the immune system of a subject. The compositions and methods described herein are useful for enhancing the reconstitution of the immune system of a subject, for example, after allogeneic hematopoietic stem cell transplantation (HSCT).

Description

BONE MARROW MICROGELS AND USES THEREOF

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/465,097, filed on May 9, 2023, the contents of which are hereby incorporated by reference in their entirety.

BACKGROUND

Allogeneic hematopoietic stem cell transplantation (HSCT) is an important and potentially curative treatment for a variety of hematologic malignancies, such as leukemia, lymphoma, and multiple myeloma, as well as for other types of blood and immune system disorders. Typically, prior to HSCT, a patient will be administered a myeloablative conditioning treatment which depletes the immune system, e.g., by destroying hematopoietic cells in the bone marrow, to create space for the engraftment of donor cells.

Notably, the recovery and long-term survival of patients after allogeneic HSCT critically depends on the timely reconstitution of innate and adaptive immunity, e.g., the recovery of normal levels of immune cell subsets and the de novo generation of a new population of T cells and B cells with diverse receptor (TCR and BCR, respectively) repertoires.

However, allogeneic HSCT is associated with a marked delay of immune system recovery which can render patients susceptible to infectious agents, occurrence of graft- versus-host disease (GVHD), and relapse. These complications can be fatal and limit the use of HSCT in settings where it may otherwise be curative. Thus, there is a need in the art for therapeutic strategies to enhance immune reconstitution post-HSCT.

SUMMARY

The present disclosure generally relates to microgels, e.g., microgel scaffolds. The compositions and methods described herein are useful for enhancing the reconstitution of the immune system of a subject, for example, after allogeneic hematopoietic stem cell transplantation (HSCT). Specifically, the compositions and methods described herein are useful for enhancing hematopoiesis at ectopic transplantation sites, e.g., within an ectopic bone marrow niche formed by the microgels, e.g., microgel scaffolds, of the present disclosure. The present disclosure is based, at least in part, on the surprising discovery of a microfluidic technique to prepare microgels, templated using emulsions generated in a microfluidic device, having physicochemical properties (e.g., stiffness, pore size, viscoelasticity, microarchitecture, degradability, ligand presentation, and/or stimulus- responsive properties) that enhance the engraftment of hematopoietic stem and progenitor cells (HSPCs), the recovery of normal levels of immune cell subsets, and/or the de novo generation of a new population of T cells and B cells with diverse receptor (TCR and BCR, respectively) repertoires, e.g., after HSCT. This technique allows for the precise control over the size, uniformity, and degradability of the emulsion templated microgels, to tune the pore size and size distribution of the resulting microgel scaffolds.

By promoting the generation of an ectopic bone marrow nodule, the microgels, e.g., microgel scaffolds, of the present disclosure can serve as alternative sites for hematopoietic stem cell engraftment and differentiation. For example, growth factors and differentiation factors attached to or encapsulated in the microgels, e.g., microgel scaffolds, of the present disclosure work together to synergistically promote the formation of mineralized bone tissue on or around the administered microgel scaffold materials to form a nodule comprising hematopoietic tissue that can function as an ectopic bone marrow niche for the infiltration and/or engraftment of hematopoietic stem cells. In certain embodiments, the ectopic bone marrow niche may recapitulate one or more of the components of the endogenous bone marrow microenvironment, which is characterized by the presence of hematopoietic cells, stromal cells and vasculature, extracellular matrix, and bone.

Accordingly, in one aspect, the present disclosure provides a microgel, comprising: (i) a non-degradable component; and/or (ii) a degradable component, wherein the microgel is spherical in form and is characterized by a diameter of about 10 pm to about 100 pm.

In various embodiments of the above aspects or any other aspect of the invention described herein, the microgel comprises both a non-degradable component and a degradable component. In some embodiments, the non-degradable component comprises a first polymer and a second polymer. In some embodiments, the non-degradable component comprises a third polymer. In some embodiments, the first polymer, the second polymer, and the third polymer are independently selected from the group consisting of alginate, methacrylate alginate, chitosan, polyethylene glycol (PEG), gelatin, hyaluronic acid, collagen, chondroitin, agarose, polyacrylamide, heparin, derivatives thereof, and combinations thereof. In some embodiments, the first polymer and the second polymer are the same polymer. In some embodiments, the first polymer and the second polymer are independently an alginate, optionally wherein the first polymer and the second polymer independently comprise a modified alginate polymer, optionally wherein the first polymer and the second polymer independently comprise oxidized alginate, optionally wherein the first polymer and the second polymer are independently comprise methacrylate alginate, optionally wherein the first polymer and the second polymer independently comprise a click reagent. In some embodiments, the first polymer and the second polymer independently comprise a modified polymer. In some embodiments, the first polymer and the second polymer independently comprise a click reagent. In some embodiments, the click reagent is selected from the group consisting of azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine (Tz), norbomene (Nb), and variants thereof. In some embodiments, the first polymer comprises a tetrazine (Tz) moiety. In some embodiments, the first polymer comprises tetrazine modified alginate (Alg-Tz). In some embodiments, the second polymer comprises a norbomene (Nb) moiety. In some embodiments, the second polymer comprises norbornene modified alginate (Alg-Nb). In some embodiments, the first polymer comprises tetrazine modified alginate (Alg-Tz) and the second polymer comprises norbornene modified alginate (Alg-Nb), optionally wherein: (i) the microgel is about 1% to about 90% covalently crosslinked; and/or (ii) the microgel comprises a ratio of Alg-Tz: Alg-Nb of 1 : 1, 1 :3, or 3 : 1. In some embodiments, the third polymer comprises a modified polymer, optionally wherein the third polymer comprises an oxidized polymer, optionally wherein the oxidized polymer is about 0.1% to about 99% oxidized, optionally wherein the oxidized polymer is about 1% to about 10% oxidized, optionally wherein the oxidized polymer is about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% oxidized. In some embodiments, the third polymer comprises oxidized alginate. In some embodiments, the third polymer degrades in vivo within about 1-day to about 30-days after administration to a subject. In some embodiments, the microgel further comprises an active agent, optionally wherein the active agent is selected from the group consisting of a cell, a biological factor, and/or a small molecule, optionally wherein: (i) the active agent is present at between about 1 ng to about 1000 pg, optionally wherein the active agent is present at between about 1 ng to about 100 pg, optionally wherein the active agent is present at between about 1 pg to about 2 ng per microgel, optionally wherein the active agent is present at about 1 pg per microgel; (ii) the active agent comprises a growth factor, optionally wherein the growth factor is selected from the group consisting of a BMP-2, a BMP-4, a BMP-6, a BMP- 7, a BMP- 12, a BMP- 14, and a combination thereof, optionally wherein the growth factor comprises a BMP -2, optionally wherein the growth factor is present at between about 2 ng to about 500 ng per microgel; (iii) the active agent comprises a differentiation factor, optionally wherein the differentiation factor is selected from the group consisting of a Delta-like 1 (DLL-1), a Delta-like 2 (DLL-2), a Delta-like 3 (DLL-3), a Delta-like 3 (DLL-3), a Delta-like 4 (DLL-4), a Jagged 1, a Jagged 2, and a combination thereof, optionally wherein the differentiation factor comprises DLL-4, optionally wherein the differentiation factor is present at an amount at between about 1 ng to about 100 pg per microgel; (iv) the active agent is covalently and/or non-covalently attached to the microgel, optionally wherein the active agent is covalently attached to the microgel utilizing click chemistry, optionally wherein the active agent is covalently linked to the scaffold utilizing N-hydroxysuccinimide (NHS) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry, NHS and dicyclohexylcarbodiimide (DCC) chemistry, avidin-biotin reaction, azide and dibenzocycloocytne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbomene chemistry, or di-sulfide chemistry; and/or (v) the active agent is released from the microgel within about 1-day to about 30-days after administration to a subject.

In one aspect, the disclosure provides a scaffold composition comprising the microgel of any of the various embodiments of the above aspect or any other aspect of the disclosure described herein.

In another aspect, the disclosure provides a scaffold composition, comprising: a non- degradable microgel comprising a first polymer and a second polymer, wherein the first polymer and the second polymer independently comprise a click reagent; and/or a degradable microgel comprising a third polymer, wherein the third polymer is configured to degrade in vivo within about 1-day to about 30-days after administration to a subject to form pores for the recruitment of immune cells, wherein the non-degradable microgel and the degradable microgel are spherical in form and independently characterized by a diameter of about 10 pm to about 100 pm.

In various embodiments of the above aspects or any other aspect of the invention described herein, the scaffold composition comprises both the non-degradable microgel and the degradable microgel, optionally wherein the non-degradable microgel and the degradable microgel form a three-dimensional scaffold in situ upon administration to a subject, optionally wherein the scaffold comprises pores of a size that permit a eukaryotic cell to traverse into or out of the scaffold, optionally wherein the pores have a diameter of about 1 pm to about 1000 pm. In some embodiments, the first polymer comprises a tetrazine (Tz) moiety. In some embodiments, the second polymer comprises norbornene modified alginate (Alg-Nb). In some embodiments, the third polymer comprises oxidized alginate. In some embodiments, the scaffold composition further comprises an active agent, optionally wherein the active agent is selected from the group consisting of a cell, a biological factor, and/or a small molecule, optionally wherein: (i) the active agent is present at between about 1 ng to about 1000 pg, optionally wherein the active agent is present at between about 1 ng to about 100 pg, optionally wherein the active agent is present at between about 1 pg to about 2 ng per microgel, optionally wherein the active agent is present at about 1 pg per microgel; (ii) the active agent comprises a growth factor, optionally wherein the growth factor is selected from the group consisting of a BMP -2, a BMP -4, a BMP-6, a BMP-7, a BMP-12, a BMP-14, and a combination thereof, optionally wherein the growth factor comprises a BMP-2, optionally wherein the growth factor is present at between about 2 ng to about 500 ng per microgel; (iii) the active agent comprises a differentiation factor, optionally wherein the differentiation factor is selected from the group consisting of a Delta-like 1 (DLL-1), a Delta-like 2 (DLL- 2), a Delta-like 3 (DLL-3), a Delta-like 3 (DLL-3), a Delta-like 4 (DLL-4), a Jagged 1, a Jagged 2, and a combination thereof, optionally wherein the differentiation factor comprises DLL-4, optionally wherein the differentiation factor is present at an amount at between about 1 ng to about 100 pg per microgel; (iv) the active agent is covalently and/or non-covalently attached to the microgel, optionally wherein the active agent is covalently attached to the microgel utilizing click chemistry, optionally wherein the active agent is covalently linked to the scaffold utilizing N-hydroxysuccinimide (NHS) and l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry, NHS and dicyclohexylcarbodiimide (DCC) chemistry, avidin-biotin reaction, azide and dibenzocycloocytne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbomene chemistry, or di-sulfide chemistry; and/or (v) the active agent is released from the microgel within about 1-day to about 30-days after administration to a subject. In some embodiments, the scaffold composition further comprises an aqueous solution, optionally wherein the aqueous solution comprises a saline solution, optionally wherein the aqueous solution comprises phosphate-buffered saline (PBS) optionally wherein the aqueous solution comprises calcium chloride (CaCh) and/or NaCl, optionally wherein the aqueous solution comprises about 2 mM CaCh and/or about 0.8% NaCl, optionally wherein the aqueous solution comprises DMEM media and 10% FBS.

In another aspect, the disclosure provides a method of preparing a microgel, comprising: (i) providing a microfluidics chip; (ii) providing an aqueous phase comprising a first polymer and a second polymer; (iii) providing a continuous oil phase comprising an oil and a surfactant; and (iv) contacting the aqueous phase with the continuous oil phase in the microfluidics chip to form an emulsion, thereby preparing the microgel.

In various embodiments of the above aspects or any other aspect of the invention described herein, the microfluidics chip comprises at least two aqueous inlets, at least one oil inlet, and at least one outlet. In some embodiments, the microfluidics chip comprises at least one junction, wherein the junction permits the aqueous phase to contact the continuous oil phase to form an emulsion. In some embodiments, the first polymer and the second polymer are independently selected from the group consisting of a non-degradable polymer, a degradable polymer, and a combination thereof. In some embodiments, the first polymer and the second polymer are independently selected from the group consisting of alginate, chitosan, polyethylene glycol (PEG), gelatin, hyaluronic acid, collagen, chondroitin, agarose, polyacrylamide, heparin, derivatives thereof, and combinations thereof. In some embodiments, the first polymer and the second polymer are the same polymer. In some embodiments, the first polymer and the second polymer are independently an alginate, optionally wherein the first polymer and the second polymer independently comprise a modified alginate polymer, optionally wherein the first polymer and the second polymer independently comprise oxidized alginate, optionally wherein the first polymer and the second polymer are independently comprise methacrylate alginate, optionally wherein the first polymer and the second polymer independently comprise a click reagent. In some embodiments, the first polymer and the second polymer independently comprise a modified polymer. In some embodiments, the first polymer and the second polymer independently comprise an oxidized polymer, optionally wherein the oxidized polymer is about 0.1% to about 99% oxidized, optionally wherein the oxidized polymer is about 1% to about 10% oxidized, optionally wherein the oxidized polymer is about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% oxidized. In some embodiments, the first polymer and the second polymer independently comprise oxidized alginate. In some embodiments, the first polymer and the second polymer independently comprise a click reagent. In some embodiments, the click reagent is selected from the group consisting of azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine (Tz), norbornene (Nb), and variants thereof. In some embodiments, the first polymer comprises a tetrazine (Tz) moiety. In some embodiments, the first polymer comprises tetrazine modified alginate (Alg-Tz). In some embodiments, the second polymer comprises a norbomene (Nb) moiety. In some embodiments, the second polymer comprises norbomene modified alginate (Alg-Nb). In some embodiments, the first polymer and the second polymer are independently dissolved in deionized water. In some embodiments, the first polymer and the second polymer are independently provided at a concentration of about 0.1% (w/v) to about 10% (w/v). In some embodiments, the first polymer is provided at a concentration of about 0.5% (w/v) to about 1.5% (w/v). In some embodiments, the second polymer is provided at a concentration of about 1.5% (w/v) to about 2.5% (w/v). In some embodiments, the oil comprises mineral oil, silicone, and/or HFE7500. In some embodiments, the surfactant is selected from the group consisting of an amphoteric surfactant, an anionic surfactant, a cationic surfactant, a nonionic surfactant, and a combination thereof, optionally wherein the surfactant comprises fluorosurfactant, optionally wherein the surfactant comprises a nonionic surfactant selected from the group consisting of Brij 93, SPAN 80, AB IL EM90, PGPR, and a combination thereof. In some embodiments, the continuous oil phase comprises about 0.5% (w/v) to about 2% (w/v) fluorosurfactant in HFE7500 solution, optionally wherein the continuous oil phase comprises about 1% (w/v) fluorosurfactant in HFE7500 solution. In some embodiments, the method further comprises injecting the Alg-Nb and the Alg-Tz into the microfluidics chip, optionally wherein the Alg-Nb and the Alg-Tz are injected at a rate of about 25 pl/hour to about 100 pl/hour, optionally wherein the Alg-Nb and the Alg-Tz are injected at a rate of about 50 pl/hour. In some embodiments, the method further comprises injecting the continuous oil phase at a rate of about 175 pl/hour to about 500 pl/hour, optionally wherein the continuous oil phase is injected at a rate of about 200 pl/hour. In some embodiments, the method further comprises allowing the Alg-Nb and the Alg-Tz solutions to form an emulsion when they encounter the continuous oil phase at a junction inside the microfluidics chip, thereby forming an emul si on-templ ated microgel. In some embodiments, the method further comprises collecting the emulsion. In some embodiments, the method further comprises maintaining the emulsion at room temperature for at least about 6 hours to about 24 hours to allow covalent crosslinking between the Alg-Nb and the Alg-Tz polymers. In some embodiments, the method further comprises treating the emulsion with a demulsification and washing process, optionally wherein the treating comprises contacting the emulsion with an about 30-50% (v/v), optionally an about 40% (v/v), 1H,1H,2H,2H- Perfluoro-1 -octanol (PFO) solution, an about 0.1-1 % (v/v), optionally an about 0.5% (v/v) Tween 20 solution, and an about 0.5% (w/v) to about 1.5% (w/v), optionally an about 0.8% (w/v) sodium chloride (NaCl) solution, sequentially. In some embodiments, the method further comprises isolating the microgel. In some embodiments, the method further comprises dispersing the microgel in an aqueous solution, optionally wherein the aqueous solution comprises a saline solution, optionally wherein the aqueous solution comprises phosphate- buffered saline (PBS), optionally wherein the aqueous solution comprises calcium chloride (CaCh) and/or NaCl, optionally wherein the aqueous solution comprises about 2 mM CaCh and/or about 0.8% NaCl, optionally wherein the aqueous solution comprises DMEM media and 10% FBS. In some embodiments, the method further comprises lyophilizing the microgel. In some embodiments, the method further comprises storing the microgel at about 4°C. In some embodiments, the method further comprises contacting the microgel with an active agent, optionally wherein the contacting occurs at about 4°C for about 1 hour to about 5 hours, optionally wherein the contacting occurs at about 4°C for about 3 hours. In some embodiments, the active agent is selected from the group consisting of a cell, a biological factor, and/or a small molecule, optionally wherein: (i) the active agent is present at between about 1 ng to about 1000 pg, optionally wherein the active agent is present at between about 1 ng to about 100 pg, optionally wherein the active agent is present at between about 1 pg to about 2 ng per microgel, optionally wherein the active agent is present at about 1 pg per microgel; (ii) the active agent comprises a growth factor, optionally wherein the growth factor is selected from the group consisting of a BMP-2, a BMP -4, a BMP-6, a BMP-7, a BMP- 12, a BMP- 14, and a combination thereof, optionally wherein the growth factor comprises a BMP -2, optionally wherein the growth factor is present at between about 2 ng to about 500 ng per microgel; (iii) the active agent comprises a differentiation factor, optionally wherein the differentiation factor is selected from the group consisting of a Delta-like 1 (DLL-1), a Delta-like 2 (DLL-2), a Delta-like 3 (DLL-3), a Delta-like 3 (DLL-3), a Delta-like 4 (DLL-4), a Jagged 1, a Jagged 2, and a combination thereof, optionally wherein the differentiation factor comprises DLL-4, optionally wherein the differentiation factor is present at an amount at between about 1 ng to about 100 pg per microgel; (iv) the active agent is covalently and/or non-covalently attached to the microgel, optionally wherein the active agent is covalently attached to the microgel utilizing click chemistry, optionally wherein the active agent is covalently linked to the scaffold utilizing N-hydroxysuccinimide (NHS) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry, NHS and dicyclohexylcarbodiimide (DCC) chemistry, avidin-biotin reaction, azide and dibenzocycloocytne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbomene chemistry, or di-sulfide chemistry; and/or (v) the active agent is released from the microgel within about 1-day to about 30-days after administration to a subject.

In another aspect, the disclosure provides a method for promoting the generation of an ectopic bone marrow niche in a subject in need thereof, comprising administering the microgel or the scaffold composition, as described herein, to the subject, thereby promoting the generation of an ectopic bone marrow niche in the subject.

In another aspect, the disclosure provides a method for promoting the generation of hematopoietic tissue in a subject in need thereof, comprising administering the microgel or the scaffold composition, as described herein, to the subject, thereby promoting the generation of hematopoietic tissue in the subject.

In another aspect, the disclosure provides a method for promoting reconstitution of hematopoietic cells in a subject, comprising administering the microgel or the scaffold composition, as described herein, to the subject, thereby promoting reconstitution of hematopoietic cells in the subject.

In various embodiments of the above aspects or any other aspect of the invention described herein, the microgel forms a three-dimensional scaffold in situ upon administration to the subject, optionally wherein the scaffold comprises pores of a size that permit a eukaryotic cell to traverse into or out of the scaffold, optionally wherein the pores have a diameter of about 1 pm to about 1000 pm. In some embodiments, the method enhances the recruitment, proliferation, and/or differentiation of immune cells in the scaffold material within about 1-week to about 4-weeks after administration, optionally wherein the immune cells comprise hematopoietic stem cells (HSCs) and/or hematopoietic progenitor cells (HPCs), optionally wherein the immune cells comprise myeloid cells and/or lymphoid cells, optionally wherein the immune cells comprise T cells, B cells, and/or natural killer (NK) cells, optionally wherein the immune cells comprise Lin-c-kit+Sca-l+ (LKS) cells, optionally wherein the immune cells comprise CD45.2+ cells. In some embodiments, the method enhances the recovery of a normal absolute lymphocyte count (ALC) and/or immune cell subsets, optionally comprising neutrophils, monocytes, natural killer cells, T cells, and/or B cells. In some embodiments, the subject is a human. In some embodiments, the microgel is administered to the subject via injection, optionally, intravenously, intramuscularly, or subcutaneously. In some embodiments, the method enhances reconstitution of T cells and/or B cells in the subject. In some embodiments, the method enhances T cell and/or B cell diversity in the subject. In some embodiments, the T cell diversity is characterized by an enhanced T cell receptor (TCR) repertoire and/or wherein the B cell diversity is characterized by an enhanced B cell receptor (BCR) repertoire.

In another aspect, the disclosure provides a syringe comprising: (i) a needle; (ii) a reservoir that comprises the microgel of any one of claims 1-19, or the composition of any one of claims 20-27; and (iii) a plunger. In another aspect, the disclosure provides a kit comprising: (i) the microgel of any one of claims 1-19, or the composition of any one of claims 20-27; and (ii) instructions to administer the microgel.

The present disclosure is further illustrated by the following detailed description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an illustration of the study design to evaluate microgels with different degradation kinetics.

FIG. IB shows an illustration of the study design to evaluate subcutaneous injection of BMP -2 adsorbed microgel scaffolds.

FIG. 1C shows an illustration of the microfluidic chip design and focused view of the junction which generates emulsions.

FIG. ID shows a photograph and confocal images of emulsion droplets from PDMS- based microfluidic chip in oil and click-alginate microgels in PBS after crosslinking and washing process, respectively (top), and confocal and cyoSEM images of DLL4 conjugated click microgels and hydrogel network of nanoporous alginate click microgels, respectively (bottom).

FIG. IE shows the microgels size distribution.

FIGs. 2A-2B show release kinetics of the BMP -2 adsorbed to the bone marrow microgels in mass (FIG. 2A) and in percentage (FIG. 2B) from non-degradable microgels (ND) and degradable microgels (SD).

FIG. 3A shows a time series of ultrasound images of ectopic ossification within the microgel scaffolds.

FIG. 3B shows total volume of the microgel scaffolds including bone and microgels in vivo as a function of time.

FIG. 3C shows the ratio of bone volume to total volume on Day 21 and Day 28.

FIGs. 4A-4B shows histological Van Kossa stained sections of the microgels scaffold with mineralization identified (brown color) at Day 28 (FIG. 4A) and quantification of the mineralization area per ROI within these sections (FIG. 4B).

FIGs. 5A-5C shows histological Safranin O stained sections of the degradable- microgels scaffold and non-degradable microgels scaffold with BMP -2 (red color) with ossification (green color) and hematopoietic tissue (purple color) at Day 28 (4 weeks) (FIGs. 5A-5B) and quantification of the hematopoietic tissue, bone, microgels % area per ROI within these sections (FIG. 5C). Fast-D: fast-degradable microgels. Slow-D: slow-degradable microgels. Mix-D: mixture of slow-degrade microgel and fast-degrade microgel.

FIG. 6A shows ectopic ossification within the microgel scaffolds with different degradation property at Day 28 recorded by high frequency ultrasound.

FIG. 6B shows the volumetric change of the microgel scaffolds in vivo as a function of time by ultrasonography imaging.

FIG. 6C shows one volume (left) and the ratio of bone volume to total volume (right) at Day 28.

FIGs. 7A-7B shows histological Van Kossa stained sections of the microgels scaffold with different degradation property with mineralization identified (brown color) at Day 28 (FIG. 7A) and quantification of the mineralization area per ROI within these sections (FIG. 7B).

FIG. 8 shows quantification of the hematopoietic tissue, bone, microgels % area per ROI from microgels scaffold within Safaranin O stained sections.

FIGs. 9A-9D shows microgel scaffolds with different BMP-2 dose at Day 35 (FIG. 9 A), high frequency ultrasound image (FIG. 9B), microCT rendering image identified calcified region (red) and microgels scaffold (white) (FIG. 9C), and micro CT reconstruction 3D image (FIG. 9D).

FIG. 10A shows volume of gel and calcified region within microgel scaffolds with different BMP -2 dose measured by microCT.

FIG. 10B shows the ratio of bone volume to total volume at microgels scaffolds with different BMP -2 dose.

FIGs. 11A-11B shows total cell (FIG. 11 A) and lineage negative cell (FIG. 11B) from microgels scaffold with different BMP -2 dose at days 28.

FIGs. 12A-12B shows LKS (Lin, c-kit, Seal) cell (FIG. 12A) and hematopoietic cell (CD45.2 expressed) (FIG. 12B) from microgels scaffold with different BMP -2 dose at Day 28.

FIG. 13 shows hematopoietic cell population including myeloid cells, B cells, and T cells from microgels scaffold with different BMP-2 dose at Day 28. DETAILED DESCRIPTION

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the content clearly dictates otherwise.

The articles “a” and “an” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.

As used herein, the term “about,” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

As used herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

As used herein, “comprise,” “comprising,” “comprises,” and “comprised of’ are meant to be synonymous with “include,” “including,” “includes,” or “contain,” “containing,” “contains” and are inclusive or open-ended terms that specifies the presence of what follows, e.g., component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein. By way of example, the term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to.”

As used herein, the terms “such as,” “for example,” and the like are intended to refer to exemplary embodiments and not to limit the scope of the present disclosure.

As used herein, the term “contacting” includes the physical contact of at least one substance to another substance, either directly or indirectly.

As used herein, the term “sufficient amount” and “sufficient time” includes an amount and time needed to achieve the desired result or results. As used herein the terms “preventing” or “prevention” refer to a reduction in risk of acquiring a disease or disorder (z.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).

As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more symptoms, diminishing the extent of infection, stabilized (z.e., not worsening) state of infection, amelioration or palliation of the infectious state, whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

As used herein, the term “prophylactically effective amount,” is intended to include the amount of an active agent that, when administered to a subject who does not yet experience or display symptoms of a condition, disease, and/or disorder, but who may be predisposed to the condition, disease, and/or disorder, is sufficient to prevent or ameliorate the condition, disease, and/or disorder or one or more symptoms of the condition, disease, and/or disorder. Ameliorating the condition, disease, and/or disorder includes slowing the course of the condition, disease, and/or disorder or reducing the severity of later-developing condition, disease, and/or disorder. The “prophylactically effective amount” may vary depending on the active agent, how the active agent is administered, the degree of risk of condition, disease, and/or disorder, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A “therapeutically effective amount” or “prophylactically effective amount” also includes an amount of an active agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. Active agents employed in the methods of the present disclosure may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

As used herein, the term “administering” to a subject includes dispensing, delivering or applying a composition as described herein to a subject by any suitable route for delivery of the composition to the subject, including delivery by injection. Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion. In preferred embodiments, the compositions are administered by injection, e.g., subcutaneous injection.

As used herein, the term “immune cells” generally refer to resting and/or activated cells of the immune system involved in defending a subject against both infectious disease and foreign materials. Examples of immune cells include, without limitations, white blood cells including, e.g., neutrophils, eosinophils, basophils, lymphocytes (e.g., B-cells, T-cells, and natural killer cells), monocytes, macrophages (including, e.g., resident macrophages, resting macrophages, and activated macrophages); as well as Kupffer cells, histiocytes, dendritic cells, Langerhans cells, mast cells, microglia, and any combinations thereof. In some embodiment, immune cells include derived immune cells, for example, immune cells derived from lymphoid stem cells and/or myeloid stem cells. In some embodiment, immune cells include white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) and/or hematopoietic progenitor cells (HPC). In some embodiment, immune cells include hematopoietic stem cells (HSC) and/or hematopoietic progenitor cells (HPC). In some embodiment, immune cells include lymphocytes (T cells, B cells, natural killer (NK) cells) and/or myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells).

As used herein, the term “T cell” refers to all types of immune cells expressing CD3 including, without limitation, T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), T- regulatory cells (Treg), and gamma-delta T cells. As used herein, the term “cytotoxic cell” refer, without limitation, to cells capable of mediating cytotoxicity responses, such as CD8+ T cells, natural-killer (NK) cells, and neutrophils. As used herein, the term “stem cell” generally includes pluripotent or multipotent stem cells. “Stem cells” includes, e.g., embryonic stem cells (ES); mesenchymal stem cells (MSC); induced-pluripotent stem cells (iPS); and committed progenitor cells (hematopoietic stem cells (HSC); bone marrow derived cells, neural progenitor cells, etc.).

As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is composed of a heterodimer of an alpha (a) and beta (P) chain, although in some cells the TCR consists of gamma and delta (y/5) chains. TCRs may exist in alpha/beta and gamma/delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain. In some embodiments, the TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell.

As used herein, the term “hematopoietic stem cells” or “HSC” refers to stem cells that can differentiate into the hematopoietic lineage and give rise to all blood cell types such as white blood cells and red blood cells, including myeloid (c.g, monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (e.g., T-cells, B-cells, K-cells). Stem cells are defined by their ability to form multiple cell types (multipotency) and their ability to selfrenew. Hematopoietic stem cells can be identified, for example by cell surface markers such as CD34-, CD133+, CD48-, CD150+, CD244-, cKit+, Scal+, and lack of lineage markers (negative for B220, CD3, CD4, CD8, Macl, Grl, and Teri 19, among others).

As used herein, the term “hematopoietic progenitor cells” or “HPC” encompasses pluripotent cells which are committed to the hematopoietic cell lineage, generally do not selfrenew, and are capable of differentiating into several cell types of the hematopoietic system, such as granulocytes, monocytes, erythrocytes, megakaryocytes, B-cells and T-cells, including, but not limited to, short term hematopoietic stem cells (ST-HSCs), multi-potent progenitor cells (MPPs), common myeloid progenitor cells (CMPs), granulocyte-monocyte progenitor cells (GMPs), megakaryocyte-erythrocyte progenitor cells (MEPs), and committed lymphoid progenitor cells (CLPs). The presence of hematopoietic progenitor cells can be determined functionally as colony forming unit cells (CFU-Cs) in complete methylcellulose assays, or phenotypically through the detection of cell surface markers (e.g., CD45-, CD34+, Teri 19-, CD16/32, CD127, cKit, Seal) using assays known to those of skill in the art.

The term “reduced” or “reduce” or “decrease” as used herein generally means a decrease of at least 5%, for example a decrease by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. substantially absent or below levels of detection), or any decrease between 5- 100% as compared to a reference level, as that term is defined herein, and as determined by a method that achieves statistical significance (p <0.05).

The term “increased” or “increase” as used herein generally means an increase of at least 5%, for example an increase by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase (i.e., substantially above levels of detection), or any increase between 5-100% as compared to a reference level, as that term is defined herein, and as determined by a method that achieves statistical significance (p <0.05). In some embodiments, the methods described herein can result in a greater number of immune cells (e.g., hematopoietic stem cells (HSCs) and/or hematopoietic progenitor cells (HPCs)), such as primitive Lin- Kit+ Sca+ hematopoietic cells (HSCs), localized in the scaffold material in vivo as compared to a reference, optionally, by at least about 5%, or, at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99%, or up to and including a 100% increase, or any increase between about 5 and about 100%.

As used herein, the term “standard” or “reference” refers to a measured biological parameter including, but not limited to, the level (e.g., concentration) of a cell, e.g., an immune cell, in a known sample against which another sample is compared; alternatively, a standard can simply be a reference number that represents an amount of the measured biological parameter that defines a baseline for comparison. The reference number can be derived from either a sample taken from an individual, or a plurality of individuals or cells obtained therefrom. That is, the “standard” does not need to be a sample that is tested, but can be an accepted reference number or value. A series of standards can be developed that take into account an individual's status, e.g., with respect to age, gender, weight, height, ethnic background etc. A standard level can be obtained, for example, from a known sample from a different individual (e.g., not the individual being tested). A known sample can also be obtained by pooling samples from a plurality of individuals (or cells obtained therefrom) to produce a standard over an averaged population. Additionally, a standard can be synthesized such that a series of standards are used to quantify the biological parameter in an individual's sample. A sample from the individual to be tested can be obtained at an earlier time point (presumably prior to the onset of treatment) and serve as a standard or reference compared to a sample taken from the same individual after the onset of treatment. In such instances, the standard can provide a measure of the efficacy of treatment. Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 100 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96- 99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.

I. Compositions

The present invention is directed to hydrogels fabricated as microscale particles, known as microgrels, with tailored size, morphology and mechanics, providing a highly tunable, modular and biocompatible system.

According to one aspect, the present disclosure provides compositions comprising microgels, e.g., bone marrow microgels. Such microgels may be advantageously used to address known limitations associated with hematopoietic stem cell transplantation (HSCT), which can damage the endogenous bone marrow niche and limit its ability to support the engraftment of hematopoietic stem and progenitor cells (HSPCs) and restore the diversity of the T-cell and B-cell receptor (TCR and BCR, respectively) repertoires. By promoting the generation of an ectopic bone marrow nodule, the microgel scaffolds of the present disclosure can serve as alternative sites for hematopoietic stem cell engraftment and differentiation. Without wishing to be bound by theory, growth factors and differentiation factors attached to or encapsulated in the microgel scaffolds of the present disclosure can work together to synergistically promote the formation of mineralized bone tissue on or around the administered microgel scaffold materials to form a nodule comprising hematopoietic tissue that can function as an ectopic bone marrow niche for the infiltration and/or engraftment of HSPCs. In certain embodiments, the ectopic bone marrow niche may recapitulate one or more of the components of the endogenous bone marrow microenvironment, which is characterized by the presence of hematopoietic cells, stromal cells and vasculature, extracellular matrix, and bone. Use of the microgels, e.g., bone marrow microgels, described herein can permit recapitulation of hematopoiesis at ectopic transplantation sites.

As used herein the term “microgel” refers to a hydrogel fabricated as microscale particles, for example, a three-dimensional hydrogel particle that is about 0.001 pm to about 500 pm in diameter. The microgels may be formed of any suitable biomaterial, e.g., a non- degradable component and/or a degradable component. The microgels may be of any shape, including, e.g., spheres, spheroids, ovals, ovoids, ellipsoids, discs, capsules, rectangles, polygons, toroids, cones, pyramids, rods, cylinders, and fibers, or any other suitable shape. In some embodiments, the microgel is spherical in form and is characterized by a diameter of about 10 pm to about 100 pm. In some embodiments, the microgel may comprise a diameter of about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 21 pm, about 22 pm, about 23 pm, about 24 pm, about 25 pm, about 26 pm, about 27 pm, about 28 pm, about 29 pm, about 30 pm, about 31 pm, about 32 pm, about 33 pm, about 34 pm, about 35 pm, about 36 pm, about 37 pm, about 38 pm, about 39 pm, about 40 pm, about 41 pm, about 42 pm, about 43 pm, about 44 pm, about 45 pm, about 46 pm, about 47 pm, about 48 pm, about 49 pm, about 50 pm, about 51 pm, about 52 pm, about 53 pm, about 54 pm, about 55 pm, about 56 pm, about 57 pm, about 58 pm, about 59 pm, about 60 pm, about 61 pm, about 62 pm, about 63 pm, about 64 pm, about 65 pm, about 66 pm, about 67 pm, about 68 pm, about 69 pm, about 70 pm, about 71 pm, about 72 pm, about 73 pm, about 74 pm, about 75 pm, about 76 pm, about 77 pm, about 78 pm, about 79 pm, about 80 pm, about 81 pm, about 82 pm, about 83 pm, about 84 pm, about 85 pm, about 86 pm, about 87 pm, about 88 pm, about 89 pm, about 90 pm, about 91 pm, about 92 pm, about 93 pm, about 94 pm, about 95 pm, about 96 pm, about 97 pm, about 98 pm, about 99 pm, or about 100 pm.

In certain embodiments, the microgels may be configured to form a three-dimensional scaffold in situ upon administration to a subject. Such three-dimensional scaffolds may comprise pores of a size that permit a eukaryotic cell, e.g., an immune cell, to traverse into or out of the scaffold. The pores may have a diameter of about 1 pm to about 1000 pm (e.g., about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm, about 100 pm, about 105 pm, about 110 pm, about 115 pm, about 120 pm, about 125 pm, about 130 pm, about 135 pm, about 140 pm, about 145 pm, about 150 pm, about 155 pm, about 160 pm, about 165 pm, about 170 pm, about 175 pm, about 180 pm, about 185 pm, about 190 pm, about 195 pm, about 200 pm, about 205 pm, about 210 pm, about 215 pm, about 220 pm, about 225 pm, about 230 pm, about 235 pm, about 240 pm, about 245 pm, about 250 pm, about 255 pm, about 260 pm, about 265 pm, about 270 pm, about 275 pm, about 280 pm, about 285 pm, about 290 pm, about 295 pm, about 300 pm, about 305 pm, about 310 pm, about 315 pm, about 320 pm, about 325 pm, about 330 pm, about 335 pm, about 340 pm, about 345 pm, about 350 pm, about 355 pm, about 360 pm, about 365 pm, about 370 pm, about 375 pm, about 380 pm, about 385 pm, about 390 pm, about 395 pm, about 400 pm, about 405 pm, about 410 pm, about 415 pm, about 420 pm, about 425 pm, about 430 pm, about 435 pm, about 440 pm, about 445 pm, about 450 pm, about 455 pm, about 460 pm, about 465 pm, about 470 pm, about 475 pm, about 480 pm, about 485 pm, about 490 pm, about 495 pm, about 500 pm, about 505 pm, about 510 pm, about 515 pm, about 520 pm, about 525 pm, about 530 pm, about 535 pm, about 540 pm, about 545 pm, about 550 pm, about 555 pm, about 560 pm, about 565 pm, about 570 pm, about 575 pm, about 580 pm, about 585 pm, about 590 pm, about 595 pm, about 600 pm, about 605 pm, about 610 pm, about 615 pm, about 620 pm, about 625 pm, about 630 pm, about 635 pm, about 640 pm, about 645 pm, about 650 pm, about 655 pm, about 660 pm, about 665 pm, about 670 pm, about 675 pm, about 680 pm, about 685 pm, about 690 pm, about 695 pm, about 700 pm, about 705 pm, about 710 pm, about 715 pm, about 720 pm, about 725 pm, about 730 pm, about 735 pm, about 740 pm, about 745 pm, about 750 pm, about 755 pm, about 760 pm, about 765 pm, about 770 pm, about 775 pm, about 780 pm, about 785 pm, about 790 pm, about 795 pm, about 800 pm, about 805 pm, about 810 pm, about 815 pm, about 820 pm, about 825 pm, about 830 pm, about 835 pm, about 840 pm, about 845 pm, about 850 pm, about 855 pm, about 860 pm, about 865 pm, about 870 pm, about 875 pm, about 880 pm, about 885 pm, about 890 pm, about 895 pm, about 900 pm, about 905 pm, about 910 pm, about 915 pm, about 920 pm, about 925 pm, about 930 pm, about 935 pm, about 940 pm, about 945 pm, about 950 pm, about 955 pm, about 960 pm, about 965 pm, about 970 pm, about 975 pm, about 980 pm, about 985 pm, about 990 pm, about 995 pm, or about 1000 pm in diameter). In certain embodiments, the pores may be formed by the complete or partial degradation of a component of the microgel, e.g., a degradable component.

In one aspect, the present disclosure provides a microgel, comprising: (i) a non- degradable component; and/or (ii) a degradable component. The non-degradable component may comprise a first polymer and a second polymer, and the non-degradable component may comprise a third polymer. In some embodiments, the microgel may comprise both a non- degradable component and a degradable component.

The first polymer, the second polymer, and the third polymer may be independently selected from the group consisting of alginate, chitosan, polyethylene glycol (PEG), gelatin, hyaluronic acid, collagen, chondroitin, agarose, polyacrylamide, heparin, methacrylated alginate, derivatives thereof, and combinations thereof. In some embodiments, the first polymer and the second polymer are the same polymer. In some embodiments, the first polymer and the second polymer are independently an alginate, optionally wherein the first polymer and the second polymer independently comprise a modified alginate polymer, optionally wherein the first polymer and the second polymer independently comprise oxidized alginate, optionally wherein the first polymer and the second polymer are independently comprise methacrylate alginate, optionally wherein the first polymer and the second polymer independently comprise a click reagent.

In some embodiments, the first polymer and the second polymer independently comprise a modified polymer. In some embodiments, the first polymer and the second polymer independently comprise methacrylated alginate. In some embodiments, the first polymer and the second polymer independently comprise a click reagent. The click reagent may be selected from the group consisting of azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine (Tz), norbornene (Nb), and variants thereof. In some embodiments, the first polymer comprises a tetrazine (Tz) moiety. In some embodiments, the first polymer comprises tetrazine modified alginate (Alg-Tz). In some embodiments, the second polymer comprises a norbornene (Nb) moiety. In some embodiments, the second polymer comprises norbornene modified alginate (Alg-Nb). In some embodiments, the first polymer comprises tetrazine modified alginate (Alg-Tz) and the second polymer comprises norbomene modified alginate (Alg-Nb).

In some embodiments, the microgel may be about 1% to about 90% covalently crosslinked (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, or about 90% covalently crosslinked).

In some embodiments, the microgel may independently comprises about 1% to about 100% Alg-Tz (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% Alg-Tz).

In some embodiments, the microgel may comprise about 1% to about 100% Alg-Nb (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% Alg-Nb).

In some embodiments, the microgel may comprise a ratio of norbomene (Nb)/tetrazine (Tz) of about 0.1 to about 10 (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about

4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, or about 10).

In some embodiments, the microgel may comprise a ratio of Alg-Tz: Alg-Nb of 1 : 1, 1 :3, or 3: l.

In some embodiments, the third polymer may comprise a modified polymer. In some embodiments, the third polymer may comprise an oxidized polymer. In some embodiments, the oxidized polymer is about 0.1% to about 99% oxidized (e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% oxidized). In some embodiments, the oxidized polymer is about 1% to about 10% oxidized, optionally wherein the oxidized polymer is about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% oxidized. In some embodiments, the third polymer may comprise oxidized alginate. In some embodiments, the third polymer may degrade in vivo within about 1-day to about 30-days after administration to a subject.

In some embodiments, the microgel may comprise an active agent. In some embodiments, the active agent may be selected from the group consisting of a cell, a biological factor, and/or a small molecule.

In some embodiments, the active agent may be present at between about 1 ng to about 1000 pg. In some embodiments, the active agent may be present at between about 1 ng to about 100 pg. In some embodiments, the active agent may be present at between about 1 pg to about 2 ng per microgel. In some embodiments, the active agent may be present at about 1 pg per microgel. In some embodiments, the active agent may comprise a growth factor. The growth factor may be selected from the group consisting of a BMP-2, a BMP -4, a BMP-6, a BMP-7, a BMP- 12, a BMP- 14, and a combination thereof. In some embodiments, the growth factor may comprise a BMP-2. In some embodiments, the growth factor may be present at between about 2 ng to about 500 ng per microgel.

In some embodiments, the active agent may comprise a differentiation factor. In some embodiments, the differentiation factor may be selected from the group consisting of a Deltalike 1 (DLL-1), a Delta-like 2 (DLL-2), a Delta-like 3 (DLL-3), a Delta-like 3 (DLL-3), a Delta-like 4 (DLL-4), a Jagged 1, a Jagged 2, and a combination thereof. In some embodiments, the differentiation factor may comprise DLL-4. In some embodiments, the differentiation factor may be present at an amount at between about 1 ng to about 100 pg per microgel.

In some embodiments, the active agent is covalently and/or non-covalently attached to the microgel. The active agent may be covalently attached to the microgel utilizing click chemistry. For example, the active agent may be covalently linked to the scaffold utilizing N- hydroxysuccinimide (NHS) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry, NHS and dicyclohexyl carbodiimide (DCC) chemistry, avidin-biotin reaction, azide and dibenzocycloocytne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbornene chemistry, or di-sulfide chemistry. In some embodiments, the active agent may be released from the microgel within about 1-day to about 30-days after administration to a subject.

IL Microgel Scaffolds

The composition of the present disclosure comprise a microgel, e.g, a microgel scaffold. The microgel, e.g, microgel scaffold, can comprise one or more biomaterials. Preferably, the biomaterial is a biocompatible material that is non-toxic and/or non- immunogenic. As used herein, the term "biocompatible material" refers to any material that does not induce a significant immune response or deleterious tissue reaction, e.g., toxic reaction or significant irritation, over time when implanted into or placed adjacent to the biological tissue of a subject.

The microgel, e.g., microgel scaffold, can comprise biomaterials that are non- biodegradable or biodegradable. In certain embodiments, the biomaterial can be a non- biodegradable material. In certain embodiments, the polymer scaffold comprises a biodegradable material. The biodegradable material may be degraded by physical or chemical action, e.g., level of hydration, heat, oxidation, or ion exchange or by cellular action, e.g., elaboration of enzyme, peptides, or other compounds by nearby or resident cells. In certain embodiments, the polymer scaffold comprises both non-degradable and degradable materials.

In some embodiments, the microgel, e.g., microgel scaffold, can degrade at a predetermined rate based on a physical parameter selected from the group consisting of temperature, pH, hydration status, and porosity, the cross-link density, type, and chemistry or the susceptibility of main chain linkages to degradation. Alternatively, the microgel, e.g., microgel scaffold, can degrade at a predetermined rate based on a ratio of chemical polymers. For example, a high molecular weight polymer comprised of solely lactide degrades over a period of years, e.g., 1-2 years, while a low molecular weight polymer comprised of a 50:50 mixture of lactide and glycolide degrades in a matter of weeks, e.g., 1, 2, 3, 4, 6, or 10 weeks. A calcium cross-linked gels composed of high molecular weight, high guluronic acid alginate degrade over several months (1, 2, 4, 6, 8, 10, or 12 months) to years (1, 2, or 5 years) in vivo, while a gel comprised of low molecular weight alginate, and/or alginate that has been partially oxidized, will degrade in a matter of weeks.

In certain embodiments, one or more active agents (e.g., the growth factors, the differentiation factors, and the homing factors), disclosed herein, may be attached to or encapsulated in the microgel, e.g., microgel scaffold. In some embodiments, one or more active agents disclosed herein may be covalently or non-covalently linked or attached to the microgel, e.g., microgel scaffold. In various embodiments, one or more active agents disclosed herein may be incorporated on, into, or present within the structure or pores of, the scaffold composition.

In some embodiments, the microgels, e.g., microgel scaffolds, comprise biomaterials, such as polymers, that are modified. In some embodiments, the modified polymer comprises an oxidized polymer. In some embodiments, the modified polymer comprises a reduced polymer. In some embodiments, the modified polymer comprises a polymer modified with a click reaction moiety. Exemplary click reaction moieties include, but are not limited to, an azide moiety, a dibenzocyclooctyne (DBCO) moiety, a transcyclooctene moiety, a tetrazine (Tz) moiety, a norbornene (Nb) moiety, and variants thereof.

In some embodiments, the microgel may comprise a polymer modified with norbomene (Nb) and/or tetrazine (Tz). In some embodiments, the microgel may comprise a ratio of norbornene (Nb)/tetrazine (Tz) of about 0.1 to about 10 (c.g, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about

2.9, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about

3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about

4.7, about 4.8, about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about

5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about

6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about

7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about

8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9, about 9.1, about

9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, or about 10).

The degree of modification, such as oxidation, can be varied from about 1% to about 100%. As used herein, the degree of modification means the molar percentage of the sites on the biomaterial that are modified with a functional group. For example, the degree of modification can be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,

30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,

46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,

62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,

78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. It is intended that values and ranges intermediate to the recited values are part of this disclosure.

The degree of substitution (DS) of a polymer is the (average) number of substituent groups attached per base unit (in the case of condensation polymers) or per monomeric unit (in the case of addition polymers). In the context of alginate, for example, the degree of substitution (DS) may be given as the ratio of substituted alginate residues to the total number of alginate residues in percent (mol/mol). In some embodiments, the degree of substitution of a polymer, e.g., an alginate polymer, can be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,

26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,

42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,

58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,

74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,

90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In some embodiments, a polymer may be modified to achieve an average degree of substitution (DS) of between about 5 to about 15 (e.g., about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, or about 15) functional groups per polymer chain.

In some embodiments, a polymer may be modified with a click reaction moiety to achieve an average degree of substitution (DS) of between about 5 to about 15 (e.g., about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, or about 15) click reaction moi eties per polymer chain.

In some embodiments, an alginate polymer may be modified with norbomene (Alg- Nb) or tetrazine (Alg-Tz), e.g., by carbodiimide coupling, to achieve an average degree of substitution (DS) of between about 5 to about 15 (e.g., about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, or about 15) functional groups (e.g., Nb or Tz) per alginate chain. Exemplary modified biomaterials, e.g., hydrogels, include, but not limited to, reduced-alginate, oxidized alginate, MA-alginate (methacrylated alginate), MA-gelatin (methacryl ated gelatin), hyaluronic acid, norbornene modified alginate (Alg-Nb), or tetrazine modified alginate (Alg-Tz).

In some embodiments, the microgel may comprise an polymer, e.g., a modified polymer, at a weight percent (wt%) of about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt%, or about 10 wt%.

In some embodiments, the microgel may comprise a norbornene (Nb) modified polymer and/or a tetrazine (Tz) modified polymer at a weight percent (wt%) of about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt%, or about 10 wt%.

Exemplary biomaterials suitable for use as microgels, e.g., microgel scaffolds, in the present disclosure include glycosaminoglycan, silk, fibrin, MATRIGEL®, polyethyleneglycol (PEG), polyhydroxy ethyl methacrylate, polyacrylamide, poly (N-vinyl pyrolidone), (PGA), poly lactic-co-glycolic acid (PLGA), poly e-carpolactone (PCL), polyethylene oxide, poly propylene fumarate (PPF), poly acrylic acid (PAA), polyhydroxybutyric acid, hydrolysed polyacrylonitrile, polymethacrylic acid, polyethylene amine, esters of alginic acid; pectinic acid; and alginate, fully or partially oxidized alginate, hyaluronic acid, carboxy methyl cellulose, heparin, heparin sulfate, chitosan, carboxymethyl chitosan, chitin, pullulan, gellan, xanthan, collagen, gelatin, carboxymethyl starch, carboxymethyl dextran, chondroitin sulfate, cationic guar, cationic starch, and combinations thereof. In certain embodiments, the biomaterial is selected from the group consisting of alginate, fully or partially oxidized alginate, and combinations thereof.

The microgels, e.g., microgel scaffolds, of the present disclosure may comprise an external surface. Alternatively, or in addition, the scaffolds may comprise an internal surface. External or internal surfaces of the microgels, e.g., microgel scaffolds, of the present disclosure may be solid or porous. Pore size of the scaffolds can be less than about 10 nm, between about 100 nm-20 pm, or greater than about 20 pm, e.g., up to and including 1000 pm in diameter. For example, the pores may be nanoporous, microporous, or macroporous. For example, the diameter of nanopores is less than about 10 nm; the diameter of micropores is in the range of about 100 nm-20 pm; and, the diameter of macropores is greater than about 20 m, e.g., greater than about 50 pm, e.g., greater than about 100 pm, e.g., greater than about 400 pm, e.g., greater than 600 pm or greater than 800 pm. In some embodiment the diameter of the pore is between about 50 pm and about 80 pm.

In some embodiments, the microgels, e.g., microgel scaffolds, of the present disclosure may be organized in a variety of geometric shapes (e.g., spheres, discs, beads, pellets), niches, planar layers (e.g., thin sheets). For example, discs of about 0.1-200 millimeters in diameter, e.g., 5, 10, 20, 40, or 50 millimeters may be implanted subcutaneously. The disc may have a thickness of 0.1 to 10 millimeters, e.g., 1, 2, or 5 millimeters. The discs are readily compressed or lyophilized for administration to a patient. An exemplary disc for subcutaneous administration has the following dimensions: 8 millimeters in diameter and 1 millimeter in thickness.

In some embodiments, the scaffolds may comprise multiple components and/or compartments. In certain embodiments, a multiple compartment device is assembled in vivo by applying sequential layers of similarly or differentially doped gel or other scaffold material to the target site. For example, the device is formed by sequentially injecting the next, inner layer into the center of the previously injected material using a needle, thereby forming concentric spheroids. In certain embodiments, non-concentric compartments are formed by injecting material into different locations in a previously injected layer. A multiheaded injection device extrudes compartments in parallel and simultaneously. The layers are made of similar or different biomaterials differentially doped with pharmaceutical compositions. Alternatively, compartments self-organize based on their hydro-philic/phobic characteristics or on secondary interactions within each compartment. In certain embodiments, multicomponent scaffolds are optionally constructed in concentric layers each of which is characterized by different physical qualities such as the percentage of polymer, the percentage of crosslinking of polymer, chemical composition of the hydrogel, pore size, porosity, and pore architecture, stiffness, toughness, ductility, viscoelasticity, the growth factors, the differentiation factors, and/or homing factors incorporated therein and/or any other compositions incorporated therein.

Hydrogel and Cryosel Scaffolds

In certain embodiments, the microgels, e.g., microgel scaffolds, of the present disclosure comprise one or more hydrogels. A hydrogel is a polymer gel comprising a network of crosslinked polymer chains. A hydrogel is usually a composition comprising polymer chains that are hydrophilic. The network structure of hydrogels allows them to absorb significant amounts of water. Some hydrogels are highly stretchable and elastic; others are viscoelastic. Hydrogel are sometimes found as a colloidal gel in which water is the dispersion medium. In certain embodiments, hydrogels are highly absorbent (they can contain over 99% water (v/v)) natural or synthetic polymers that possess a degree of flexibility very similar to natural tissue, due to their significant water content. In certain embodiments, a hydrogel may have a property that, when an appropriate shear stress is applied, the deformable hydrogel is dramatically and reversibly compressed (up to 95% of its volume), resulting in injectable macroporous preformed scaffolds. Hydrogels have been used for therapeutic applications, e.g., as vehicles for in vivo delivery of therapeutic agents, such as small molecules, cells and biologies. Hydrogels are commonly produced from polysaccharides, such as alginates. The polysaccharides may be chemically manipulated to modulate their properties and properties of the resulting hydrogels.

The hydrogels of the present disclosure may be either porous or non-porous. Preferably the compositions of the disclosure are formed of porous hydrogels. For example, the hydrogels may be nanoporous wherein the diameter of the pores is less than about 10 nm; microporous wherein the diameter of the pores is preferably in the range of about 100 nm-20 pm; or macroporous wherein the diameter of the pores is greater than about 20 pm, more preferably greater than about 100 pm and even more preferably greater than about 400 pm. In certain embodiments, the hydrogel is macroporous with pores of about 50-80 pm in diameter. In certain embodiments, the hydrogel is macroporous with aligned pores of about 400-500 pm in diameter. Methods of preparing porous hydrogel products are known in the art. (See, e.g., U.S. Pat. No. 6,511,650, incorporated herein by reference).

The hydrogel may be constructed out of a number of different rigid, semi-rigid, flexible, gel, self-assembling, liquid crystalline, or fluid compositions such as peptide polymers, polysaccharides, synthetic polymers, hydrogel materials, ceramics (e.g., calcium phosphate or hydroxyapatite), proteins, glycoproteins, proteoglycans, metals and metal alloys. The compositions are assembled into hydrogels using methods known in the art, e.g., injection molding, lyophilization of preformed structures, printing, self-assembly, phase inversion, solvent casting, melt processing, gas foaming, fiber forming/processing, particulate leaching, microfluidics, or a combination thereof. The assembled devices are then implanted or administered, e.g., by injection, to the body of an individual to be treated.

The composition comprising a hydrogel may be assembled in vivo in several ways. The hydrogel is made from a gelling material, which is introduced into the body in its ungelled form where it gels in situ. Exemplary methods of delivering components of the composition to a site at which assembly occurs include injection through a needle or other extrusion tool, spraying, painting, or methods of deposit at a tissue site, e.g, delivery using an application device inserted through a cannula. In some embodiments, the ungelled or unformed hydrogel material is mixed with at least one pharmaceutical composition prior to introduction into the body or while it is introduced. The resultant in vivo/in situ assembled device, e.g, hydrogel, contains a mixture of the at least one pharmaceutical composition.

In situ assembly of the hydrogel may occur as a result of spontaneous association of polymers or from synergistically or chemically catalyzed polymerization. Synergistic or chemical catalysis is initiated by a number of endogenous factors or conditions at or near the assembly site, e.g., body temperature, ions or pH in the body, or by exogenous factors or conditions supplied by the operator to the assembly site, e.g., photons, heat, electrical, sound, or other radiation directed at the ungelled material after it has been introduced. The energy is directed at the hydrogel material by a radiation beam or through a heat or light conductor, such as a wire or fiber optic cable or an ultrasonic transducer. Alternatively, a shear-thinning material, such as an amphiphile, is used which re-cross links after the shear force exerted upon it, for example by its passage through a needle, has been relieved.

In certain embodiments, the microgels, e.g., microgel scaffolds, may be configured to form a three-dimensional scaffold in situ upon administration to a subject. Such three- dimensional scaffolds may comprise pores of a size that permit a eukaryotic cell, e.g., an immune cell, to traverse into or out of the scaffold. The pores may have a diameter of about 1 pm to about 1000 pm (e.g., about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm, about 100 pm, about 105 pm, about 110 pm, about 115 pm, about 120 pm, about 125 pm, about 130 pm, about 135 pm, about 140 pm, about 145 pm, about 150 pm, about 155 pm, about 160 pm, about 165 pm, about 170 pm, about 175 pm, about 180 pm, about 185 pm, about 190 pm, about 195 pm, about 200 pm, about 205 pm, about 210 pm, about 215 pm, about 220 pm, about 225 pm, about 230 pm, about 235 pm, about 240 pm, about 245 pm, about 250 pm, about 255 pm, about 260 pm, about 265 pm, about 270 pm, about 275 pm, about 280 pm, about 285 pm, about 290 pm, about 295 pm, about 300 pm, about 305 pm, about 310 pm, about 315 pm, about 320 pm, about 325 pm, about 330 pm, about 335 pm, about 340 pm, about 345 pm, about 350 pm, about 355 pm, about 360 pm, about 365 pm, about 370 pm, about 375 pm, about 380 pm, about 385 pm, about 390 pm, about 395 pm, about 400 pm, about 405 pm, about 410 pm, about 415 pm, about 420 pm, about 425 pm, about 430 pm, about 435 pm, about 440 pm, about 445 pm, about 450 pm, about 455 pm, about 460 pm, about 465 pm, about 470 pm, about 475 pm, about 480 pm, about 485 pm, about 490 pm, about 495 pm, about 500 pm, about 505 pm, about 510 pm, about 515 pm, about 520 pm, about 525 pm, about 530 pm, about 535 pm, about 540 pm, about 545 pm, about 550 pm, about 555 pm, about 560 pm, about 565 pm, about 570 pm, about 575 pm, about 580 pm, about 585 pm, about 590 pm, about 595 pm, about 600 pm, about 605 pm, about 610 pm, about 615 pm, about 620 pm, about 625 pm, about 630 pm, about 635 pm, about 640 pm, about 645 pm, about 650 pm, about 655 pm, about 660 pm, about 665 pm, about 670 pm, about 675 pm, about 680 pm, about 685 pm, about 690 pm, about 695 pm, about 700 pm, about 705 pm, about 710 pm, about 715 pm, about 720 pm, about 725 pm, about 730 pm, about 735 pm, about 740 pm, about 745 pm, about 750 pm, about 755 pm, about 760 pm, about 765 pm, about 770 pm, about 775 pm, about 780 pm, about 785 pm, about 790 pm, about 795 pm, about 800 pm, about 805 pm, about 810 pm, about 815 pm, about 820 pm, about 825 pm, about 830 pm, about 835 pm, about 840 pm, about 845 pm, about 850 pm, about 855 pm, about 860 pm, about 865 pm, about 870 pm, about 875 pm, about 880 pm, about 885 pm, about 890 pm, about 895 pm, about 900 pm, about 905 pm, about 910 pm, about 915 pm, about 920 pm, about 925 pm, about 930 pm, about 935 pm, about 940 pm, about 945 pm, about 950 pm, about 955 pm, about 960 pm, about 965 pm, about 970 pm, about 975 pm, about 980 pm, about 985 pm, about 990 pm, about 995 pm, or about 1000 pm in diameter). In certain embodiments, the pores may be formed by the complete or partial degradation of a component of the microgel, e.g., a degradable component.

In some embodiments, the hydrogel may be assembled ex vivo. In some embodiments, the hydrogel is injectable. For example, the hydrogels are created outside of the body as macroporous scaffolds. Upon injection into the body, the pores collapse causing the gel to become very small and allowing it to fit through a needle. See, e.g., WO 2012/149358; and Bencherif et al., 2012, Proc. Natl. Acad. Sci. USA 109.48:19590-5, the content of which are incorporated herein by reference).

Suitable hydrogels for both in vivo and ex vivo assembly of hydrogel devices are well known in the art and described, e.g., in Lee et al., 2001, Chem. Rev. 7: 1869-1879. The peptide amphiphile approach to self-assembly assembly is described, e.g., in Hartgerink et al., 2002, Proc. Natl. Acad. Sci. USA 99:5133-5138. A method for reversible gellation following shear thinning is exemplified in Lee et al., 2003, Adv. Mat. 15: 1828-1832. In certain embodiments, exemplary hydrogels are comprised of materials that are compatible with attachment and/or encapsulation of materials including polymers, nanoparticles, active agents, polypeptides, and cells . Exemplary hydrogels are fabricated from alginate, polyethylene glycol (PEG), PEG-acrylate, agarose, hyaluronic acid, or synthetic protein (e.g., collagen or engineered proteins (ie., self-assembly peptide-based hydrogels)). For example, a commercially available hydrogel includes BD™ PuraMatrix™. BD™ PuraMatrix™ Peptide Hydrogel is a synthetic matrix that is used to create defined three dimensional (3D) micro-environments for cell culture.

In some embodiments, the hydrogel is a biocompatible polymer matrix that is biodegradable in whole or in part. Examples of materials which can form hydrogels include alginates and alginate derivatives, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA) polymers, gelatin, collagen, agarose, hyaluronic acid, hyaluronic acid derivative, natural and synthetic polysaccharides, polyamino acids such as polypeptides particularly poly(lysine), polyesters such as polyhydroxybutyrate and poly-epsilon- caprolactone, polyanhydrides; polyphosphazines, poly(vinyl alcohols), poly(alkylene oxides) particularly poly(ethylene oxides), poly(allylamines)(PAM), poly(acrylates), modified styrene polymers such as poly(4-aminom ethyl styrene), pluronic polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone), and copolymers of the above, including graft copolymers. Synthetic polymers and naturally-occurring polymers such as, but not limited to, collagen, fibrin, hyaluronic acid, agarose, and laminin-rich gels may also be used. The term “derivative,” as used herein, refers to a compound that is derived from a similar compound by a chemical reaction. For example, oxidized alginate, which is derived from alginate through oxidization reaction, is a derivative of alginate,

The implantable composition can have virtually any regular or irregular shape including, but not limited to, spherical, spheroid, cubic, polyhedron, prism, cylinder, rod, disc, or other geometric shape. Accordingly, in some embodiments, the implant is of cylindrical form from about 0.5 to about 10 mm in diameter and from about 0.5 to about 10 cm in length. Preferably, its diameter is from about 1 to about 5 mm and its length from about 1 to about 5 cm.

In some embodiments, the compositions of the disclosure are of spherical form. When the composition is in a spherical form, its diameter can range, in some embodiments, from about 0.5 to about 50 mm in diameter. In some embodiments, a spherical implant’s diameter is from about 5 to about 30 mm. In an exemplary embodiment, the diameter is from about 10 to about 25 mm. In some embodiments, the microgel is spherical in form and is characterized by a diameter of about 10 pm to about 100 pm. In some embodiments, the microgel may comprise a diameter of about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 21 pm, about 22 pm, about 23 pm, about 24 pm, about 25 pm, about 26 pm, about 27 pm, about 28 pm, about 29 pm, about 30 pm, about 31 pm, about 32 pm, about 33 pm, about 34 pm, about 35 pm, about 36 pm, about 37 pm, about 38 pm, about 39 pm, about 40 pm, about 41 pm, about 42 pm, about 43 pm, about 44 pm, about 45 pm, about 46 pm, about 47 pm, about 48 pm, about 49 pm, about 50 pm, about 51 pm, about 52 pm, about 53 pm, about 54 pm, about 55 pm, about 56 pm, about 57 pm, about 58 pm, about 59 pm, about 60 pm, about 61 pm, about 62 pm, about 63 pm, about 64 pm, about 65 pm, about 66 pm, about 67 pm, about 68 pm, about 69 pm, about 70 pm, about 71 pm, about 72 pm, about 73 pm, about 74 pm, about 75 pm, about 76 pm, about 77 pm, about 78 pm, about 79 pm, about 80 pm, about 81 pm, about 82 pm, about 83 pm, about 84 pm, about 85 pm, about 86 pm, about 87 pm, about 88 pm, about 89 pm, about 90 pm, about 91 pm, about 92 pm, about 93 pm, about 94 pm, about 95 pm, about 96 pm, about 97 pm, about 98 pm, about 99 pm, or about 100 pm.

In certain embodiments, the microgel, e.g., microgel scaffold, comprises clickhydrogels and/or click-cryogels. A click hydrogel or cryogel is a gel in which cross-linking between hydrogel or cryogel polymers is facilitated by click reactions between the polymers. Each polymer may contain one of more functional groups useful in a click reaction. Given the high level of specificity of the functional group pairs in a click reaction, active compounds can be added to the preformed device prior to or contemporaneously with formation of the hydrogel device by click chemistry. Non-limiting examples of click reactions that may be used to form click-hydrogels include Copper I catalyzed azide-alkyne cycloaddition, strain-promoted assize-alkyne cycloaddition, thiol-ene photocoupling, Diels- Alder reactions, inverse electron demand Diels-Alder reactions, tetrazole-alkene photo-click reactions, oxime reactions, thiol-Michael addition, and al dehy de-hydrazide coupling. Nonlimiting aspects of click hydrogels are described in Jiang et al., 2014, Biomaterials, 35:4969- 4985, the entire content of which is incorporated herein by reference.

In various embodiments, a click alginate is utilized (see, e.g., PCT International Patent Application Publication No. WO 2015/154078 published October 8, 2015, hereby incorporated by reference in its entirety).

In some embodiments, the concentration of crosslinks (e.g., noncovalent and/or covalent crosslinks) per hydrogel is at least about 10% (w/w), e.g., at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (w/w). In some embodiments, the concentration of crosslinks per hydrogel is about 10% to about 100% (w/w), e.g., about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (w/w). In some embodiments, the concentration of crosslinks per hydrogel is about 25% to about 50% (w/w). In some embodiments, the concentration of crosslinks per hydrogel is about 25% to about 75% (w/w). In some embodiments, the concentration of crosslinks per hydrogel is about 50% to about 75% (w/w). In some embodiments, the concentration of crosslinks per hydrogel is about 75% to about 100% (w/w).

In some embodiments, the click-hydrogel devices and scaffold materials include a hydrogel comprising a first polymer and a second polymer. The first polymer and the second polymer can be the same or different. In some embodiments, the first polymer and the second polymer are the same type of polymer. In some embodiments, the first polymer and/or the second polymer are independently selected from the group consisting of alginate, chitosan, polyethylene glycol (PEG), gelatin, hyaluronic acid, collagen, MATRIGEL®, chondroitin, agarose, polyacrylamide, and heparin. In some embodiments, the first polymer and the second polymer are the same polymer independently selected from the group consisting of alginate, chitosan, polyethylene glycol (PEG), gelatin, hyaluronic acid, collagen, MATRIGEL®, chondroitin, agarose, polyacrylamide, and heparin. In some embodiments, the hydrogel is an interpenetrating polymer network (IPN) hydrogel.

In some embodiments, polymers, e.g., alginate polymers, are modified with tetrazine or norbornene groups that can subsequently be covalently cross-linked to form click- crosslinked hydrogels, e.g., click alginate hydrogels. In some embodiments, the first polymer and the second polymer may be formulated for specific applications by controlling the molecular weight, degree of modification (e.g., % oxidation and/or % crosslinking), rate of degradation, and method of scaffold formation.

Such scaffolds and scaffold materials, as well as methods for producing such scaffolds, are described in PCT International Patent Application Publication No. WO 2015/154078 published October 8, 2015, the entire content of which is incorporated herein by reference. For example, a click hydrogel may be prepared in a process: a) providing a first polymer comprising a first click reaction moiety and a second polymer comprising a second click reaction moiety. In non-limiting examples, the first click reaction moiety and the second click reaction moiety may be react with each other in a copper I catalyzed azide-alkyne cycloaddition, strain-promoted assize-alkyne cycloaddition, thiol-ene photo coupling, a Diels- Alder reaction, an inverse electron demand Diels-Alder reaction, a tetrazole-alkene photo-click reaction, a oxime reaction, a thiol-Michael addition, or via aldehyde-hydrazide coupling. In an embodiment, the first click reaction moiety is a diene moiety and the second click reaction moiety is a dienophile moiety. In an embodiment, the first click reaction moiety is a tetrazine moiety and the second click reaction moiety is a norbornene moiety. As used herein, the terms "tetrazine" and "tetrazine moiety" include molecules that comprise 1, 2,4,5- tetrazine substituted with suitable spacer for linking to the polymer (c.g, alkylamines like methylamine or pentylamine), and optionally further substituted with one or more substituents at any available position. Exemplary tetrazine moieties suitable for the compositions and methods of the disclosure are described in Karver et al. Bioconjugate Chem. 22(2011):2263-2270, and WO 2014/ 065860, both incorporated herein by reference). As used herein, the terms "norbornene" and "norbomene moieties" include but are not limited to norbomadiene and norbomene groups further comprising suitable spacer for linking to the polymer (c.g, alkylamines like methylamine or pentylamine), and optionally further substituted with one or more substituents at any available position. Such moieties include, for example, norbomene-S-methylamine and norbomadienemethylamine.

In certain embodiments, a hydrogel (e.g., cryogel) system can deliver one or more agent (e.g., a growth factor such as BMP -2, and/or a differentiation factor, such as a DLL-4, while creating a space for cells (e.g., stem cells such as hematopoietic stem cells (HSC) infiltration and trafficking). In some embodiments, the hydrogel system according to the present disclosure delivers BMP -2, which acts as a hematopoietic stem cell (HSC) and/or hematopoietic progenitor cell enhancement/recruitment factor, and DLL-4 as a differentiation factor, which facilitates T cell lineage specification of hematopoietic stem cell and/or hematopoietic progenitor cells.

In some embodiments, a cryogel composition, e.g., formed of MA-alginate, can function as a delivering platform by creating a local niche, such as a specific niche for enhancing T-lineage specification. In some embodiments, the cryogel creates a local niche in which the encounter of cells, such as recruited stem cells or progenitor cells, and various exemplary agent of the disclosure, such as the growth factor and/or differentiation factor can be controlled. In certain embodiments, the cells and the exemplary agents of the present disclosure are localized into a small volume, and the contacting of the cells and the agents can be quantitatively controlled in space and time.

In certain embodiments, the hydrogel (e.g., cryogel) can be engineered to coordinate the delivery of both growth factor and differentiation factor in space and time, potentially enhancing overall immune modulation performance by adjusting the differentiation and/or specification of recruited cells, such as hematopoietic stem cells or progenitor cells. In certain embodiments, the cells and growth factor/differentiation factor are localized into a small volume, and the delivery of factors in space and time can be quantitatively controlled. As the growth/differentiation factors are released locally, few systemic effects are anticipated, in contrast to systemically delivered agents, such as growth factors.

Examples of polymer compositions from which the cryogel or hydrogel is fabricated are described throughout the present disclosure, and include alginate, hyaluronic acid, gelatin, heparin, dextran, carob gum, PEG, PEG derivatives including PEG-co-PGA and PEG-peptide conjugates. The techniques can be applied to any biocompatible polymers, e.g., collagen, chitosan, carboxymethylcellulose, pullulan, polyvinyl alcohol (PVA), Poly(2-hydroxyethyl methacrylate) (PHEMA), Poly(N-isopropylacrylamide) (PNIPAAm), or Poly(acrylic acid) (PAAc). For example, in a particular embodiment, the composition comprises an alginate- based hydrogel/cryogel. In another example, the scaffold comprises a gelatin-based hydrogel/cryogel.

Cryogels are a class of materials with a highly porous interconnected structure that are produced using a cryotropic gelation (or cryogelation) technique. Cryogels also have a highly porous structure. Typically, active compounds are added to the cryogel device after the freeze formation of the pore/wall structure of the cryogel. Cryogels are characterized by high porosity, e.g., at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% pores with thin pore walls that are characterized by high density of polymer crosslinking. As used herein, the term “porosity” refers to the percentage of the volume of pores to the volume of the scaffold. It is intended that values and ranges intermediate to the recited values are part of this disclosure. The walls of cryogels are typically dense and highly cross-linked, enabling them to be compressed through a needle into a subject without permanent deformation or substantial structural damage.

In various embodiments, the pore walls comprise at least about 10, 15, 20, 25, 30, 35, or 40% (w/v) polymer. It is intended that values and ranges intermediate to the recited values are part of this disclosure. In other embodiments, the pore walls comprise about 10-40% polymer. In some embodiments, a polymer concentration of about 0.5-4% (w/v) (before the cryogelation) is used, and the concentration increases substantially upon completion of cryogelation. Non-limiting aspects of cryogel gelation and the increase of polymer concentration after cryogelation are discussed in Beduer el al.. 2015 Advanced Healthcare Materials 4.2: 301-312, the entire content of which is incorporated herein by reference.

In certain embodiments, cryogelation comprises a technique in which polymerizationcrosslinking reactions are conducted in quasi-frozen reaction solution. Non-limiting examples of cryogelation techniques are described in U.S. Patent Application Publication No. 20140227327, published August 14, 2014, the entire content of which is incorporated herein by reference. An advantage of cryogels compared to conventional macroporous hydrogels obtained by phase separation is their high reversible deformability. Cryogels may be extremely soft but can be deformed and reform their shape. In certain embodiments, cryogels can be very tough, can withstand high levels of deformations, such as elongation and torsion and can also be squeezed under mechanical force to drain out their solvent content. The improved deformability properties of alginate cryogels originate from the high crosslinking density of the unfrozen liquid channels of the reaction system.

In the cryogelation process, during freezing of the macromonomer (e.g., methacrylated alginate) solution, the macromonomers and initiator system (e.g., APS/TEMED) are expelled from the ice concentrate within the channels between the ice crystals, so that the reactions only take place in these unfrozen liquid channels. After polymerization and, after melting of ice, a porous material is produced whose microstructure is a negative replica of the ice formed. Ice crystals act as porogens. Desired pore size is achieved, in part, by altering the temperature of the cryogelation process. For example, the cryogelation process is typically carried out by quickly freezing the solution at -20 °C. Lowering the temperature to, e.g., -80° C , would result in more ice crystals and lead to smaller pores. In some embodiments, the cryogel is produced by cryo-polymerization of at least methacrylated (MA)-alginate and MA-PEG. In some embodiments, the cryogel is produced by cryo-polymerization of at least MA-alginate, the growth factor, the differentiation factor, and MA-PEG.

In some embodiments, the disclosure also features gelatin scaffolds, e.g., gelatin hydrogels such as gelatin cryogels, which are a cell-responsive platform for biomaterialbased therapy. Gelatin is a mixture of polypeptides that is derived from collagen by partial hydrolysis. These gelatin scaffolds have distinct advantages over other types of scaffolds and hydrogel s/cryogels. For example, the gelatin scaffolds of the disclosure support attachment, proliferation, and survival of cells and are degraded by cells, e.g., by the action of enzymes such as matrix metalloproteinases (MMPs) (e.g., recombinant matrix metalloproteinase-2 and -9).

In certain embodiments, prefabricated gelatin cryogels rapidly reassume their approximately original shape ("shape memory") when injected subcutaneously into a subject (e.g., a mammal such as a human, dog, cat, pig, or horse) and elicit little or no harmful host immune response (e.g., immune rejection) following injection.

In some embodiments, the hydrogel (e.g., cryogel) comprises polymers that are modified, e.g., sites on the polymer molecule are modified with a methacrylic acid group (methacrylate (MA)) or an acrylic acid group (acrylate). Exemplary modified hydrogel s/cryogels are MA- alginate (methacryl ated alginate) or MA-gelatin. In the case of MA-alginate or MA-gelatin, 50% corresponds to the degree of methacrylation of alginate or gelatin. This means that every other repeat unit contains a methacrylated group. The degree of methacrylation can be varied from about 1% to about 100%. Preferably, the degree of methacrylation varies from about 1% to about 90%.

In certain embodiments, polymers can also be modified with acrylated groups instead of methacrylated groups. The product would then be referred to as an acrylated-polymer. The degree of methacrylation (or acrylation) can be varied for most polymers. However, some polymers (e.g., PEG) maintain their water-solubility properties even at 100% chemical modification. After crosslinking, polymers normally reach near complete methacrylate group conversion indicating approximately 100% of cross-linking efficiency. As used herein, the term “cross-linking efficiency” refers to the percentage of macromonomers that are covalently linked. For example, the polymers in the hydrogel are 50-100% crosslinked (covalent bonds). The extent of crosslinking correlates with the durability of the hydrogel. Thus, a high level of crosslinking (90-100%) of the modified polymers is desirable.

For example, the highly crosslinked hydrogel/cryogel polymer composition is characterized by at least about 50% polymer crosslinking (e.g., about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%; it is intended that values and ranges intermediate to the recited values are part of this disclosure). The high level of crosslinking confers mechanical robustness to the structure. Preferably, the percentage of crosslinking is less than about 100%. The composition is formed using a free radical polymerization process and a cryogelation process. For example, the cryogel is formed by cryopolymerization of methacrylated gelatin, methacrylated alginate, or methacrylated hyaluronic acid. In some embodiments, the cryogel comprises a methacrylated gelatin macro monomer or a methacrylated alginate macromonomer at concentration of about 1.5% (w/v) or less (e.g., about 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or less; it is intended that values and ranges intermediate to the recited values are part of this disclosure). In some embodiments, the methacrylated gelatin or alginate macromonomer concentration is about 1% (w/v).

In certain embodiments, the cryogel comprises at least about 75% (v/v) pores, e.g., about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (v/v) or more pores. It is intended that values and ranges intermediate to the recited values are part of this disclosure. In some embodiments, the pores are interconnected. Interconnectivity is important to the function of the hydrogel and/or cryogel, as without interconnectivity, water would become trapped within the gel. Interconnectivity of the pores permits passage of water (and other compositions such as cells and compounds) in and out of the structure. In certain embodiments, in a fully hydrated state, the hydrogel (e.g., cryogel) comprises at least about 90% water (volume of water / volume of the scaffold) (e.g., between about 90-99%, at least about 92%, 95%, 97%, 99%, or more). For example, at least about 90% (e.g., at least about 92%, 95%, 97%, 99%, or more) of the volume of the cryogel is made of liquid (e.g., water) contained in the pores. It is intended that values and ranges intermediate to the recited values are part of this disclosure. In certain embodiments, in a compressed or dehydrated hydrogel, up to about 50%, 60%, 70% of that water is absent, e.g, the cryogel comprises less than about 25% (e.g., about 20%, 15%, 10%, 5% or less) water.

In certain embodiments, the cryogels of the disclosure comprise pores large enough for a cell to travel through. For example, the cryogel contains pores of about 20-500 pm in diameter, e.g., about 20-30pm, about 30-150pm, about 50-500 pm, about 50-450 pm, about 100-400 pm, about 200-500 pm. In some embodiments, the hydrated pore size is about 1- 500 pm (e.g., about 10-400 pm, about 20-300 pm, about 50-250 pm). In certain embodiments, the cryogel contains pores about 50-80 pm in diameter.

In some embodiments, injectable hydrogels or cryogels are further functionalized by addition of a functional group selected from the group consisting of: amino, vinyl, aldehyde, thiol, silane, carboxyl, azide, or alkyne. Alternatively or in addition, the cryogel is further functionalized by the addition of a further cross-linker agent (e.g., multiple arms polymers, salts, aldehydes, etc.). The solvent can be aqueous, and in particular, acidic or alkaline. The aqueous solvent can comprise a water-miscible solvent (e.g., methanol, ethanol, DMF, DMSO, acetone, dioxane, etc).

For cryogels, the cryo-crosslinking may take place in a mold and the cryogels (which may be injected) can be degradable. The pore size can be controlled by the selection of the main solvent used, the incorporation of a porogen, the freezing temperature and rate applied, the crosslinking conditions (e.g. polymer concentration), and also the type and molecule weight of the polymer used. The shape of the cryogel may be dictated by a mold and can thus take on any shape desired by the fabricator, e.g., various sizes and shapes (disc, cylinders, squares, strings, etc.) are prepared by cryogenic polymerization.

Injectable cryogels can be prepared in the micrometer-scale to centimeter-scale. Exemplary volumes vary from a few hundred pm³ (e.g., about 100-500 pm³) to about 10 cm³. In certain embodiment, an exemplary scaffold composition is between about 100 pm³ to 100 mm³ in size. In various embodiments, the scaffold is between about 10 mm³ to about 100 mm³ in size. In certain embodiments, the scaffold is about 30 mm³ in size.

In some embodiments, the cryogels are hydrated, loaded with compounds and loaded into a syringe or other delivery apparatus. For example, the syringes are prefilled and refrigerated until use. In another example, the cryogel is dehydrated, e.g., lyophilized, optionally with a compound (such as a growth factor or differentiation factor) loaded in the gel and stored dry or refrigerated. Prior to administration, a cryogel -loaded syringe or apparatus may be contacted with a solution containing compounds to be delivered. For example, the barrel of the cryogel pre-loaded syringe is filled with a physiologically- compatible solution, e.g., phosphate-buffered saline (PBS). Alternatively, the cryogel may be administered to a desired anatomical site followed by administration of the physiologically- compatible solution, optionally containing other ingredients, e.g., a growth factor and/or a differentiation factor or together with one or more compounds disclosed herein. The cryogel is then rehydrated and regains its shape integrity in situ. In certain embodiments, the volume of PBS or other physiologic solution administered following cryogel placement is generally about 10 times the volume of the cryogel itself.

The cryogel also has the advantage that, upon compression, the cryogel composition maintains structural integrity and shape memory properties. For example, the cryogel is injectable through a hollow needle. For example, the cryogel returns to its approximately original geometry after traveling through a needle (e.g., a 16 gauge (G) needle, e.g., having a 1.65 mm inner diameter). Other exemplary needle sizes are 16-gauge, an 18-gauge, a 20- gauge, a 22- gauge, a 24-gauge, a 26-gauge, a 28-gauge, a 30-gauge, a 32-gauge, or a 34- gauge needle. Injectable cryogels have been designed to pass through a hollow structure, e.g., very fine needles, such as 18-30 G needles. In certain embodiments, the cryogel returns to its approximately original geometry after traveling through a needle in a short period of time, such as less than about 10 seconds, less than about 5 seconds, less than about 2 seconds, or less than about 1 second.

The cryogels may be injected to a subject using any suitable injection device. For example, the cryogels may be injected using syringe through a needle. A syringe may include a plunger, a needle, and a reservoir that comprises compositions of the present disclosure. The injectable cryogels may also be injected to a subject using a catheter, a cannula, or a stent.

The injectable cryogels may be molded to a desired shape, in the form of rods, square, disc, spheres, cubes, fibers, foams. In some cases, the cryogel is in the shape of a disc, cylinder, square, rectangle, or string. For example, the cryogel composition is between about 100 pm³ to 10 cm³ in size, e.g., between 10 mm³ to 100 mm³ in size. For example, the cryogel composition is between about 1 mm in diameter to about 50 mm in diameter (e.g., about 5 mm). Optionally, the thickness of the cryogel is between about 0.2 mm to about 50 mm (e.g., about 2 mm).

Three exemplary cryogel materials systems are described below. a) Methacrylated gelatin cryogel (CryoGelMA) - An exemplary cryogel utilized methacrylated gelatin and the results are described in detail in U.S. Patent Application Publication No. 2014-0227327, published August 14, 2014, the entire contents of which are incorporated herein by reference. b) Methacrylated alginate cryogel (CryoMAAlginate) - An exemplary cryogel utilized methacrylated alginate and the results are described in detail in U.S. Patent Application Publication No. 2014-0227327, published August 14, 2014, the entire contents of which are incorporated herein by reference. c) Click Alginate cryogel with Laponite nanoplatelets (CryoClick) - The base material is click alginate (PCT International Patent Application Publication No. WO 2015/154078 published October 8, 2015, hereby incorporated by reference in its entirety). In some examples, the base material contains laponite (commercially available silicate clay used in many consumer products such as cosmetics). Laponite has a large surface area and highly negative charge density which allows it to adsorb positively charged moieties on a variety of proteins and other biologically active molecules by an electrostatic interaction, thereby allowing drug loading. When placed in an environment with a low concentration of drug, adsorbed drug releases from the laponite in a sustained manner. This system allows release of a more flexible array of various agents, e.g., growth factors, compared to the base material alone. Various embodiments of the present subject matter include delivery vehicles comprising a pore-forming scaffold composition. For example, pores (such as macropores) are formed in situ within a hydrogel following hydrogel injection into a subject. Pores that are formed in situ via degradation of a sacrificial porogen hydrogel within the surrounding hydrogel (bulk hydrogel) facilitate recruitment and trafficking of cells, as well as the release of any composition or agent of the present disclosure, for example, a growth factor, such as BMP -2, a differentiation factor , or a homing factor, or any combination thereof. In some embodiments, the sacrificial porogen hydrogel, the bulk hydrogel, or both the sacrificial porogen hydrogel and the bulk hydrogel may comprise any composition or agent of the present disclosure, for example, a growth factor, a differentiation factor, and/or, a homing factor, or any combination thereof.

In various embodiments, the pore-forming composition becomes macroporous over time when resident in the body of a recipient animal such as a mammalian subject. For example, the pore-forming composition may comprise a sacrificial porogen hydrogel and a bulk hydrogel, wherein the sacrificial porogen hydrogel degrades at least about 10% faster (e.g., at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% faster) than the bulk hydrogel. It is intended that values and ranges intermediate to the recited values are part of this disclosure. The sacrificial porogen hydrogel may degrade leaving macropores in its place. In certain embodiments, the macropores are open interconnected macropores. In some embodiments, the sacrificial porogen hydrogel may degrade more rapidly than the bulk hydrogel, because the sacrificial porogen hydrogel (i) is more soluble in water (comprises a lower solubility index), (ii) is cross-linked to protease-mediated degradation motifs as described in U.S. Patent Application Publication No. 2005-0119762, published June 2, 2005 (incorporated herein by reference in its entirety), (iii) comprises a shorter polymer that degrades more quickly compared to that of a longer bulk hydrogel polymer, (iv) is modified to render it more hydrolytically degradable than the bulk hydrogel (e.g., by oxidation), and/or (v) is more enzymatically degradable compared to the bulk hydrogel.

In various embodiments, a scaffold is loaded (e.g., soaked with) with one or more active compounds after polymerization. In certain embodiments, device or scaffold polymer forming material is mixed with one or more active compounds before polymerization. In some embodiments, a device or scaffold polymer forming material is mixed with one or more active compounds before polymerization, and then is loaded with more of the same or one or more additional active compounds after polymerization. In some embodiments, pore size or total pore volume of a composition or scaffold is selected to influence the release of compounds from the device or scaffold. Exemplary porosities (e.g., nanoporous, microporous, and macroporous scaffolds and devices) and total pore volumes (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more of the volume of the scaffold) are described herein. It is intended that values and ranges intermediate to the recited values are part of this disclosure. Increased pore size and total pore volume increases the amount of compounds that can be delivered into or near a tissue, such as bone marrow. In some embodiments, a pore size or total pore volume is selected to increase the speed at which active ingredients exit the composition or scaffold. In various embodiments, an active ingredient may be incorporated into the scaffold material of a hydrogel or cryogel, e.g., to achieve continuous release of the active ingredient from the scaffold or device over a longer period of time compared to active ingredient that may diffuse from a pore cavity.

Porosity influences recruitment of cells into devices and scaffolds, growth of cells embedded in devices and scaffolds, and/or the release of substances from devices and scaffolds. Pores may be, e.g., nanoporous, microporous, or macroporous. For example, the diameter of nanopores is less than about 10 nm. Micropores are in the range of about 100 nm to about 20 pm in diameter. Macropores are greater than about 20 pm (e.g., greater than about 100 pm or greater than about 400 pm). Exemplary macropore sizes include about 50 pm, about 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, about 500 pm, about 550 pm, and about 600 pm. Macropores are those of a size that permit a eukaryotic cell to traverse into or out of the composition. In one example, a macroporous composition has pores of about 400 pm to about 500 pm in diameter. In some embodiments, the pore diameter can be about 0.5 pm to about 10 pm (e.g., about 0.5 pm, about 1 pm, about 1.5 pm, about 2 pm, about 2.5 pm, about 3 pm, about 3.5 pm, about 4 pm, about 4.5 pm, about 5 pm, about 5.5 pm, about 6 pm, about 6.5 pm, about 7 pm, about 7.5 pm, about 8 pm, about 8.5 pm, about 9 pm, about 9.5 pm, or about 10 pm). The preferred pore size depends on the application.

In various embodiments, the composition is manufactured in one stage in which one layer or compartment is made and infused or coated with one or more compounds. Exemplary bioactive compositions comprise polypeptides or polynucleotides. In certain embodiments, the composition is manufactured in two or more (3, 4, 5, 6, .... 10 or more) stages in which one layer or compartment is made and infused or coated with one or more compounds followed by the construction of second, third, fourth or more layers, which are in turn infused or coated with one or more compounds in sequence. In some embodiments, each layer or compartment is identical to the others or distinguished from one another by the number or mixture of bioactive compositions as well as distinct chemical, physical and biological properties. Polymers may be formulated for specific applications by controlling the molecular weight, rate of degradation, and method of scaffold formation. Coupling reactions can be used to covalently attach bioactive agent, such as the differentiation factor to the polymer backbone.

In some embodiments, one or more compounds is added to the scaffold compositions using a known method including surface absorption, physical immobilization, e.g., using a phase change to entrap the substance in the scaffold material. For example, a growth factor is mixed with the scaffold composition while it is in an aqueous or liquid phase, and after a change in environmental conditions (e.g., pH, temperature, ion concentration), the liquid gels or solidifies thereby entrapping the bioactive substance. In some embodiments, covalent coupling, e.g., using alkylating or acylating agents, is used to provide a stable, long term presentation of a compound on the scaffold in a defined conformation. Exemplary reagents for covalent coupling of such substances are provided in the table below.

Table 1: Methods to Covalently Couple Peptides/Proteins to Polymers

a] EDC: l-ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride;

DCC: di cyclohexylcarbodiimide Alginate Scaffolds

In certain embodiments, the composition of the disclosure comprises an alginate hydrogel, e.g., an alginate microgel. Alginates are versatile polysaccharide based polymers that may be formulated for specific applications by controlling the molecular weight, rate of degradation and method of scaffold formation. Alginate polymers are comprised of two different monomeric units, (l-4)-linked P-D-mannuronic acid (M units) and a L-guluronic acid (G units) monomers, which can vary in proportion and sequential distribution along the polymer chain. Alginate polymers are polyelectrolyte systems which have a strong affinity for divalent cations (e.g., Ca⁺², Mg⁺², Ba⁺²) and form stable hydrogels when exposed to these molecules. See Martinsen A., et al., 1989, Biotech. & Bioeng., 33 : 79-89). For example, calcium cross-linked alginate hydrogels are useful for dental applications, wound dressings chondrocyte transplantation and as a matrix for other cell types. Without wishing to be bound by theory, it is believed that G units are preferentially crosslinked using calcium crosslinking, whereas click reaction based crosslinking is more indiscriminate with respect to G units or M units (i.e., both G and M units can be crosslinked by click chemistry). Alginate scaffolds and the methods for making them are known in the art. See, e.g., International Patent Application Publication No. WO 2017/075055 Al, published on May 4, 2017, the entire contents of which are incorporated herein by reference.

In some embodiments, the microgel may comprise an alginate polymer, e.g., a modified alginate polymer, at a weight percent (wt%) of about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt%, or about 10 wt%.

In some embodiments, the microgel may comprise a norbornene modified alginate (Alg-Nb) and/or a tetrazine modified alginate (Alg-Tz) at a weight percent (wt%) of about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt%, or about 10 wt%.

The alginate polymers useful in the context of the present disclosure can have an average molecular weight from about 20 kDa to about 500 kDa, e.g., from about 20 kDa to about 40 kDa, from about 30 kDa to about 70 kDa, from about 50 kDa to about 150 kDa, from about 130 kDa to about 300 kDa, from about 230 kDa to about 400 kDa, from about 300 kDa to about 450 kDa, or from about 320 kDa to about 500 kDa. In one example, the alginate polymers useful in the present disclosure may have an average molecular weight of about 32 kDa. In another example, the alginate polymers useful in the present disclosure may have an average molecular weight of about 265 kDa. In some embodiments, the alginate polymer has a molecular weight of less than about 1000 kDa, e.g, less than about 900 KDa, less than about 800 kDa, less than about 700 kDa, less than about 600 kDa, less than about 500 kDa, less than about 400 kDa, less than about 300 kDa, less than about 200 kDa, less than about 100 kDa, less than about 50 kDa, less than about 40 kDa, less than about 30 kDa or less than about 25 kDa. In some embodiments, the alginate polymer has a molecular weight of about 1000 kDa, e.g, about 900 kDa, about 800 kDa, about 700 kDa, about 600 kDa, about 500 kDa, about 400 kDa, about 300 kDa, about 200 kDa, about 100 kDa, about 50 kDa, about 40 kDa, about 30 kDa or about 25 kDa. In one embodiment, the molecular weight of the alginate polymers is about 20 kDa.

Coupling reactions can be used to covalently attach bioactive agent, such as an atom, a chemical group, a nucleoside, a nucleotide, a nucleobase, a sugar, a nucleic acid, an amino acid, a peptide, a polypeptide, a protein, or a protein complex, to the polymer backbone.

The term “alginate,” used interchangeably with the term “alginate polymers,” includes unmodified alginate or modified alginate. Modified alginate includes, but not limited to, oxidized alginate (e.g., comprising one or more algoxalate monomer units), reduced alginate (e.g., comprising one or more algoxinol monomer units), MA-alginate (methacrylated alginate), hyaluronic acid, norbornene modified alginate (Alg-Nb), and/or tetrazine modified alginate (Alg-Tz).

In some embodiments, oxidized alginate comprises alginate comprising one or more aldehyde groups, or alginate comprising one or more carboxylate groups. In other embodiments, oxidized alginate comprises highly oxidized alginate, e.g., comprising one or more algoxalate units. Oxidized alginate may also comprise a relatively small number of aldehyde groups e.g., less than 15%, e.g., 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% aldehyde groups or oxidation on a molar basis). It is intended that values and ranges intermediate to the recited values are part of this disclosure.

In some embodiments, an alginate polymer may be modified to achieve an average degree of substitution (DS) of between about 5 to about 15 (e.g., about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about

11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, or about 15) functional groups per alginate chain.

In some embodiments, an alginate polymer may be modified with a click reaction moiety to achieve an average degree of substitution (DS) of between about 5 to about 15 (e.g., about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, or about 15) click reaction moieties per alginate chain.

In some embodiments, an alginate polymer may be modified with norbomene (Alg- Nb) or tetrazine (Alg-Tz), e.g., by carbodiimide coupling, to achieve an average degree of substitution (DS) of between about 5 to about 15 (e.g., about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about

7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, or about 15) functional groups (e.g., Nb or Tz) per alginate chain.

In some embodiments, the microgel may comprise an alginate polymer modified with norbomene (Nb) and/or tetrazine (Tz). In some embodiments, the alginate microgel may comprise a ratio of norbomene (Nb)/tetrazine (Tz) of about 0.1 to about 10 (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about

3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about

4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about

5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about

6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, or about 10).

In certain embodiments, alginate microgels may be fabricated using microfluidic emulsion, which can provide defined size and shape by controlled droplet formation. Alginate polymer may be first modified with norbomene (Alg-Nb) or tetrazine (Alg-Tz), e.g., by carbodiimide coupling, to achieve an average degree of substitution (DS) of about 13 or about 11.5 functional groups per alginate chain, respectively, as quantified, e.g., by proton nuclear magnetic resonance spectra. Stock solutions of Alg-Nb and Alg-Tz may then be mixed at a final concentration of 2 wt% in a microfluidic device and injected to form microdroplets by emulsion, which may then be crosslinked, e.g., overnight, to generate microgels with a diameter of about 77 ± 2 pm.

The term “alginate” or “alginate polymers” may also include alginate, e.g., unmodified alginate, oxidized alginate or reduced alginate, or methacrylated alginate or acrylated alginate. Alginate may also refer to any number of derivatives of alginic acid (e.g., calcium, sodium or potassium salts, or propylene glycol alginate ). See, e.g., WO1998012228A1, hereby incorporated by reference.

Hyaluronic Acid

In certain embodiments, the composition of the present disclosure comprises a hyaluronic acid hydrogel, e.g., a hyaluronic acid microgel. Hyaluronic acid (HA; conjugate base hyaluronate), is an anionic, nonsulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues. One of the chief components of the extracellular matrix, hyaluronic acid contributes significantly to cell proliferation and migration. Natural hyaluronic acid is an important component of articular cartilage, muscular connective tissues, and skin.

Hyaluronic acid is a polymer of disaccharides, composed of D-glucuronic acid and N- acetyl-D-glucosamine, linked via alternating P-(l — >4) and P-(l — 3) glycosidic bonds. Hyaluronic acid can be 25,000 disaccharide repeats in length. Polymers of hyaluronic acid can range in size from 5,000 to 20,000,000 Da. Hyaluronic acid can also contain silicon.

Hyaluronic acid is energetically stable, in part because of the stereochemistry of its component disaccharides. Bulky groups on each sugar molecule are in sterically favored positions, whereas the smaller hydrogens assume the less-favorable axial positions.

Hyaluronic acid can be degraded by a family of enzymes called hyaluronidases, which are present in many mammals, e.g., a human. Hyaluronic acid can also be degraded via non-enzymatic reactions. These include acidic and alkaline hydrolysis, ultrasonic disintegration, thermal decomposition, and degradation by oxidants.

Due to its high biocompatibility and its common presence in the extracellular matrix of tissues, hyaluronic acid is used to form hydrogels, e.g., cryogels, as a biomaterial scaffold in tissue engineering research. Hyaluronic acid hydrogels are formed through crosslinking. Hyaluronic acid can form a hydrogel, e.g, cryogel, into a desired shape to deliver therapeutic molecules into a host. Hyaluronic acids, for use in the present compositions, can be crosslinked by attaching thiols, methacrylates, hexadecylamides, and tyramines. Hyaluronic acids can also be crosslinked directly with formaldehyde or with divinylsulfone.

The term “hyaluronic acid,” includes unmodified hyaluronic acid or modified hyaluronic acid. Modified hyaluronic acid includes, but is not limited to, oxidized hyaluronic acid and/or reduced hyaluronic acid. In some embodiments, the modified hyaluronic acid comprises a hyaluronic acid modified with a click reaction moiety. Exemplary click reaction moieties include, but are not limited to, an azide moiety, a dibenzocyclooctyne (DBCO) moiety, a transcyclooctene moiety, a tetrazine (Tz) moiety, a norbornene (Nb) moiety, and variants thereof.

The term “hyaluronic acid” or “hyaluronic acid polymers” may also include hyaluronic acid, e.g., unmodified hyaluronic acid, oxidized hyaluronic acid or reduced hyaluronic acid, or methacrylated hyaluronic acid or acrylated hyaluronic acid. Hyaluronic acid may also refer to any number of derivatives of hyaluronic acid.

Porous and Pore-forming Scaffolds

The microgels, e.g., microgel scaffolds, of the present disclosure may be nonporous or porous. In certain embodiments, the microgels, e.g., microgel scaffolds, of the present disclosure are porous. Porosity of the scaffold composition influences migration of the cells through the device. Pores may be nanoporous, microporous, or macroporous. For example, the diameter of nanopores is less than about 10 nm. Micropores are in the range of about 100 nm to about 20 pm in diameter. Macropores are greater than about 20 pm (e.g, greater than about 100 pm or greater than about 400 pm) in diameter. Exemplary macropore sizes include about 50 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 550 pm, and 600 pm in diameter. It is intended that values and ranges intermediate to the recited values are part of this disclosure. Macropores are of a size that permits a eukaryotic cell to traverse into or out of the composition. In certain embodiments, a macroporous composition has pores of about 400 pm to 500 pm in diameter. The size of pores may be adjusted for different purpose. For example, for cell recruitment and cell release, the pore diameter may be greater than 50 pm. In certain embodiments, a macroporous composition has pores of about 50 pm - about 80 pm in diameter. In some embodiments, the scaffolds contain pores before the administration into a subject. In some embodiments, the scaffolds comprise a pore-forming scaffold composition. Pore-forming scaffolds and the methods for making pore-forming scaffolds are known in the art. See, e.g., U.S. Patent Publication US2014/0079752A1, the content of which is incorporated herein by reference. In certain embodiments, the pore-forming scaffolds are not initially porous, but become macroporous over time resident in the body of a recipient animal such as a mammalian subject. In certain embodiments, the pore-forming scaffolds are hydrogel scaffolds. The pore may be formed at different time, e.g., after about 12 hours, or 1, 3, 5, 7, or 10 days or more after administration, i.e., resident in the body of the subject.

In certain embodiments, the pore-forming scaffolds comprise a first hydrogel and a second hydrogel, wherein the first hydrogel degrades at least about 10% faster (e.g., at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% faster, at least about 2 times faster, or at least about 5 times faster) than the second hydrogel. It is intended that values and ranges intermediate to the recited values are part of this disclosure. In certain embodiments, the first hydrogel comprises a porogen that degrades leaving a pore in its place. For example, the first hydrogel is a porogen and the resulting pore after degradation in situ is within 25% of the size of the initial porogen, e.g., within 20%, within 15%, or within 10% of the size of the initial porogen. Preferably, the resulting pore is within 5% of the size of the initial porogen. It is intended that values and ranges intermediate to the recited values are part of this disclosure. The first hydrogel may degrade faster than the second hydrogel due to the difference in their physical, chemical, and/or biological properties. In certain embodiments, the first hydrogel degrades more rapidly than the second hydrogel, because the first hydrogel is more soluble in water (comprises a lower solubility index). In certain embodiments, the first hydrogel degrades more rapidly because it is cross-linked to protease-mediated degradation motifs as described in U.S. Patent Publication US2005/0119762A1, the content of which is incorporated herein by reference.

In certain embodiments, the molecular mass of the polymers used to form the first hydrogel composition (a porogen) is approximately 50 kilodaltons (kDa), and the molecular mass of the polymers used to form the second hydrogel composition (bulk) is approximately 250 kDa. A shorter polymer (e.g., that of a porogen) degrades more quickly compared to that of a longer polymer (e.g., that of the bulk composition). In certain embodiments, a composition is modified to render it more hydrolytically degradable by virtue of the presence of sugar groups (e.g., approximately 3-10% sugar of an alginate composition). In certain embodiments, the porogen hydrogel is chemically modified, such as oxidized, to render it more susceptible to degradation. In some embodiments, the porogen hydrogel is more enzymatically degradable compared to the bulk hydrogel. The composite (first and second hydrogel) composition is permeable to bodily fluids, e.g., containing an enzyme which is exposed to the composition and degrades the porogen hydrogel. In some embodiments, the second hydrogel is cross-linked around the first hydrogel, z.e., the porogens (first hydrogel) are completely physically entrapped in the bulk (second) hydrogel.

The click reagents disclosed herein can be provided in the bulk hydrogel or the porogen hydrogel. In exemplary embodiments, the click reagents, e.g., polymers or nanoparticles, are provided in the bulk hydrogel.

In certain embodiments, hydrogel micro-beads (“porogens”) are formed. Porogens are encapsulated into a “bulk” hydrogel that is either non-degradable or which degrades at a slower rate compared to the porogens. Immediately after hydrogel formation, or injection into the desired site in vivo, the composite material lacks pores. Subsequently, porogen degradation causes pores to form in situ. The size and distribution of pores are controlled during porogen formation, and mixing with the polymers which form the bulk hydrogel.

In some embodiments, the polymer utilized in the pore-forming scaffolds is naturally- occurring or synthetically made. In one example, both the porogens and bulk hydrogels are formed from alginate.

In certain embodiments, the alginate polymers suitable for porogen formation have a molecular weight from 5,000 to 500,000 Daltons. The polymers are optionally further modified (e.g., by oxidation with sodium periodate, (Bouhadir et al. , 2001, Biotech. Prog. 17:945-950, hereby incorporated by reference), to facilitate rapid degradation. In certain embodiments, the polymers are crosslinked by extrusion through a nebulizer with co-axial airflow into a bath of divalent cation (for example, Ca²⁺ or Ba²⁺) to form hydrogel microbeads. Higher airflow rate leads to lower the porogen diameter.

In some embodiments, the porogen hydrogel microbeads contain oxidized alginate. For example, the porogen hydrogel can contain about 1-50% (w/v) oxidized alginate. In exemplary embodiments, the porogen hydrogel can contain about 1-10% oxidized alginate. In one embodiment, the porogen hydrogel contains about 7.5% oxidized alginate.

In certain embodiments, the concentration of divalent ions used to form porogens may vary from about 5 to about 500 mM, and the concentration of polymer from about 1% to about 5% by weight/volume. However, any method which produces porogens that are significantly smaller than the bulk phase is suitable. Porogen chemistry can further be manipulated to produce porogens that interact with host proteins and/or cells, or inhibit interactions with host proteins and/or cells.

The alginate polymers suitable for formation of the bulk hydrogel have a molecular weight from about 5,000 to about 500,000 Da. The polymers may be further modified (for example, by oxidation with sodium periodate), to facilitate degradation, as long as the bulk hydrogel degrades more slowly than the porogen. The polymers may also be modified to present biological cues to control cell responses (e.g., integrin binding adhesion peptides such as RGD). Either the porogens or the bulk hydrogel may also encapsulate bioactive factors such as oligonucleotides, growth factors or drugs to further control cell responses. The concentration of divalent ions used to form the bulk hydrogel may vary from about 5 to about 500 mM, and the concentration of polymer from about 1% to about 5% by weight/volume. The elastic modulus of the bulk polymer is tailored for its purpose, e.g., to recruit stem cells or progenitor cells.

Methods relevant to generating the hydrogels described herein include the following. Bouhadir et al., 1999, Polymer, 40: 3575-84 (incorporated herein by reference in its entirety) describes the oxidation of alginate with sodium periodate, and characterizes the reaction. Bouhadir et al., 2001, BiotechnoL Prog., 17: 945-50 (incorporated herein by reference in its entirety) describes oxidation of high molecular weight alginate to form alginate dialdehyde (alginate dialdehyde is high molecular weight (M_w) alginate in which a certain percent, e.g., 5%, of sugars in alginate are oxidized to form aldehydes), and application to make hydrogels degrade rapidly. Kong et al., 2002, Polymer, 43: 6239-46 (incorporated herein by reference in its entirety) describes the use of gamma-irradiation to reduce the weight-averaged molecular weight (M_w) of guluronic acid (GA) rich alginates without substantially reducing GA content (e.g., the gamma irradiation selectively attacks mannuronic acid, MA blocks of alginate). Alginate is comprised of GA blocks and MA blocks, and it is the GA blocks that give alginate its rigidity (elastic modulus). Kong et al., 2002, Polymer, 43: 6239-46 (incorporated herein by reference in its entirety) shows that binary combinations of high M_w, GA rich alginate with irradiated, low M_w, high GA alginate crosslinks with calcium to form rigid hydrogels, but which degrade more rapidly and also have lower solution viscosity than hydrogels made from the same overall weight concentration of only high M_w, GA rich alginate. Alsberg et al., 2003, J Dent Res, 82(11): 903-8 (incorporated herein by reference in its entirety) describes degradation profiles of hydrogels made from irradiated, low M_w, GA- rich alginate, with application in bone tissue engineering. Kong et al., 2004, Adv. Mater,

16(21): 1917-21 (incorporated herein by reference) describes control of hydrogel degradation profile by combining gamma irradiation procedure with oxidation reaction, and application to cartilage engineering.

Techniques to control degradation of hydrogen biomaterials are well known in the art. For example, Lutolf MP et al., 2003, Nat BiotechnoL , 21 : 513-8 (incorporated herein by reference in its entirety) describes poly(ethylene glycol) based materials engineered to degrade via mammalian enzymes (MMPs). Bryant SJ et al., 2007, Biomaterials, 28(19): 2978-86 (US 7,192,693 B2; incorporated herein by reference in its entirety) describes a method to produce hydrogels with macro-scale pores. A pore template (e.g., polymethylmethacrylate beads) is encapsulated within a bulk hydrogel, and then acetone and methanol are used to extract the porogen while leaving the bulk hydrogel intact. Silva et al., 2008, Proc. Natl. Acad. Sci USA, 105(38): 14347-52 (incorporated herein by reference in its entirety; US 2008/0044900) describes deployment of endothelial progenitor cells from alginate sponges. The sponges are made by forming alginate hydrogels and then freeze- drying them (ice crystals form the pores). Ali et al., 2009, Nat Mater (incorporated herein by reference in its entirety) describes the use of porous scaffolds to recruit dendritic cells and program them to elicit anti-tumor responses. Huebsch et al., 2010, Nat Mater, 9: 518-26 (incorporated herein by reference in its entirety) describes the use of hydrogel elastic modulus to control the differentiation of encapsulated mesenchymal stem cells.

In some embodiments, the scaffold composition comprises open interconnected macropores. Alternatively or in addition, the scaffold composition comprises a pore-forming scaffold composition. In certain embodiments, the pore-forming scaffold composition may comprise a sacrificial porogen hydrogel and a bulk hydrogel, wherein the pore-forming scaffold composition lacks macropores. For example, the sacrificial porogen hydrogel may degrade at least 10% faster than the bulk hydrogel leaving macropores in its place following administration of said pore-forming scaffold into a subject. In some embodiments, the sacrificial porogen hydrogel is in the form of porogens that degrade to form said macropores. For example, the macropores may comprise pores having a diameter of, e.g., about 10-400 pm.

Active Agents

The compositions of the present disclosure can comprise an active agent.

As used herein, the term “active agent” refers to an active ingredient that is intended for use in a particular application. In some embodiments, the term “active agent” refers to an agent that possesses therapeutic, prophylactic, or diagnostic properties in vivo, for example when administered to a human subject or an animal, including mammals and domestic animals.

Examples of active agents include, but are not limited to, amino acids, proteins, peptides, antibodies, growth factors, nucleic acids, vectors, sugars, antigens, vaccines, viruses, enzymes, cells, small molecules, drugs, and any combination thereof. In some embodiments, the active agent may be selected from the group consisting of a growth factor, a differentiation factor, a homing factor, and a combination thereof.

In some embodiments, the active agent may be present at between about 1 ng to about 1000 pg. In some embodiments, the active agent may be present at between about 1 ng to about 100 pg. In some embodiments, the active agent may be present at between about 1 pg to about 2 ng per microgel. In some embodiments, the active agent may be present at about 1 pg per microgel. In some embodiments, the active agent may be present at between about 1 ng to about 500 ng. In some embodiments, the active agent may be present at between about 1 ng to about 100 ng (e.g., about 1 ng, about 2 ng, about 3 ng, about 4 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 11 ng, about 12 ng, about 13 ng, about 14 ng, about 15 ng, about 16 ng, about 17 ng, about 18 ng, about 19 ng, about 20 ng, about 21 ng, about 22 ng, about 23 ng, about 24 ng, about 25 ng, about 26 ng, about 27 ng, about 28 ng, about 29 ng, about 30 ng, about 31 ng, about 32 ng, about 33 ng, about 34 ng, about 35 ng, about 36 ng, about 37 ng, about 38 ng, about 39 ng, about 40 ng, about 41 ng, about 42 ng, about 43 ng, about 44 ng, about 45 ng, about 46 ng, about 47 ng, about 48 ng, about 49 ng, about 50 ng, about 51 ng, about 52 ng, about 53 ng, about 54 ng, about 55 ng, about 56 ng, about 57 ng, about 58 ng, about 59 ng, about 60 ng, about 61 ng, about 62 ng, about 63 ng, about 64 ng, about 65 ng, about 66 ng, about 67 ng, about 68 ng, about 69 ng, about 70 ng, about 71 ng, about 72 ng, about 73 ng, about 74 ng, about 75 ng, about 76 ng, about 77 ng, about 78 ng, about 79 ng, about 80 ng, about 81 ng, about 82 ng, about 83 ng, about 84 ng, about 85 ng, about 86 ng, about 87 ng, about 88 ng, about 89 ng, about 90 ng, about 91 ng, about 92 ng, about 93 ng, about 94 ng, about 95 ng, about 96 ng, about 97 ng, about 98 ng, about 99 ng, or about 100 ng).

Growth Factors

The compositions of the present disclosure can comprise a growth factor. The term “growth factor,” as used herein, refers to an agent that is capable of stimulating cellular growth, proliferation, healing, and/or cellular differentiation. In certain embodiments, growth factors are polypeptides. Growth factor polypeptides typically act as signaling molecules. In certain embodiments, the growth factor polypeptides are cytokines.

In certain embodiments, the growth factor can recruit a cell to the scaffold following the administration of the composition to a subject. The recruited cell may be autologous. For example, the recruited cell may be a stromal cell from the subject. In certain embodiments, the autologous cell may be a stem cell (e.g., umbilical cord stem cells) of the subject. The recruited cell may also be syngeneic, allogeneic or xenogeneic. As used herein, the term “syngeneic” refers to genetically identical, or sufficiently identical and immunologically compatible as to allow for transplantation. For example, syngeneic cells may include transplanted cells obtained from an identical twin. As used herein, the term “allogeneic” refers to cells that are genetically dissimilar, although from individuals of the same species. As used herein, the term “xenogeneic” refers to cells derived from a different species and therefore genetically different.

For example, the recruited cell may be a donor cell in a transplantation. In certain embodiments, the transplantation is a hematopoietic stem cell transplantation (HSCT). As used herein, HSCT refers to the transplantation of multipotent hematopoietic stem cells or hematopoietic progenitor cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood -HSCT may be autologous (the patient's own stem cells or progenitor cells are used), allogeneic (the stem cells or progenitor cells come from a donor), syngeneic (from an identical twin) or xenogenic (from different species).

The growth factors of the present disclosure may induce the formation of a tissue or organ within or around the administered composition. In certain embodiments, the tissue or organ is a bony tissue or hematopoietic tissue. The tissue formation may be restricted to the scaffold of the composition.

Methods of incorporating polypeptides (e.g., growth factor and/or differentiation polypeptides) are known in the art. See, US Patent Nos.: 8,728,456; 8,067,237; and 10,045,947; US Patent Publication No.: US20140079752; International Patent Publication No.: WO 2017/136837; International Patent Application Publication No.: WO 2020/131582; incorporated herein by reference in their entirety. The release of the growth factor polypeptides may be controlled. The methods of controlled release of polypeptides (e.g., growth factor polypeptides) are known in the art. See, US Patent Nos.: 8,728,456; 8,067,237; 10,045,946, incorporated by reference in their entirety. In certain embodiments, the growth factors (e.g, BMP-2) may be released over an extended period of time, such as 7-30 days or longer. The controlled release of the growth factors may affect the timing of the formation of the tissue or organ within the scaffold. In certain examples, the release of the growth factors is controlled with the goal of creating a functional, active bone nodule or tissue within one to two weeks after subcutaneous injection of the compositions of the present disclosure.

In certain embodiments, the growth factors retain their bioactivity over an extended period of time. The term “bioactivity,” as used herein, refers to the beneficial or adverse effects of an agent, such as a growth factor. The bioactivity of the growth factor may be measured by any appropriate means. For example, the bioactivity of BMP-2 may be measured by its capacity to induce the formation of bone nodule or tissue and/or recruit cells into the scaffold. In certain example, the growth factors retain their bioactivity for at least 10 days, 12 days, 14 days, 20 days, or 30 days after the incorporation of the growth factors into the scaffold.

Exemplary growth factors include, but are not limited to, bone morphogenetic proteins (BMP), epidermal growth factor (EGF), transforming growth factor beta (TGF-P), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nerve growth factor (NGF), neurotrophins, Platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF9), acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), and interleukins.

In some embodiments, the growth factor comprises a protein belonging to the transforming growth factor beta (TGF-P) superfamily. As used herein, TGF-P superfamily is a large group of structurally related cell regulatory proteins. TGF-P superfamily includes four major subfamilies: the TGF-P subfamily, the bone morphogenetic proteins and the growth differentiation factors, the activing and inhibin subfamilies, and a group encompassing various divergent members. Proteins from the TGF-P superfamily are active as homo- or heterodimer, the two chains being linked by a single disulfide bond. TGF-P superfamily proteins interact with a conserved family of cell surface serine/threonine-specific protein kinase receptors, and generate intracellular signals using a conserved family of proteins called SMADs. TGF-P superfamily proteins play important roles in the regulation of basic biological processes such as growth, development, tissue homeostasis and regulation of the immune system.

Exemplary TGF-P superfamily proteins include, but are not limited to, AMH, ARTN, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2, MSTN, NODAL, NRTN, PSPN, TGF-pi, TGF-P2, TGF-P3, and TGF-P4. In a particular embodiment, the growth factor is BMP2.

In certain embodiments, the growth factor comprises a bone morphogenetic protein (BMP). As used herein, a BMP is a protein belonging to a group of growth factors also known as cytokines and as metabologens. BMPs can induce the formation of bone and cartilage and constitute a group of important morphogenetic signals, orchestrating tissue architecture throughout the body. Absence or deficiency of BMP signaling may be an important factor in diseases or disorders.

In certain embodiments, the BMP is selected from a group consisting of a BMP-2, a BMP -4, a BMP-6, a BMP-7, a BMP- 12, a BMP- 14, and any combination thereof. In certain embodiments, the BMP is BMP -2. BMP-2 plays an important role in the development of bone and cartilage. BMP -2 can potently induce osteoblast differentiation in a variety of cell types.

In certain embodiments, the growth factor comprises a TGF-P subfamily protein. As used herein, TGF-P subfamily protein or TGF-P is a multifunctional cytokine that includes four different isoforms (TGF-pi, TGF-P2, TGF-P3, and TGF-P4). Activated TGF-P complexes with other factors to form a serine/threonine kinase complex that binds to TGF-P receptors, which is composed of both type 1 and type 2 receptor subunits. After the binding of TGF-P, the type 2 receptor kinase phosphorylates and activates the type 1 receptor kinase that activates a signaling cascade. This leads to the activation of different downstream substrates and regulatory proteins, inducing transcription of different target genes that function in differentiation, chemotaxis, proliferation, and activation of many immune cells.

In certain embodiments, the growth factor comprises a TGF-pi. TGF-pi plays a role in the induction from CD4+ T cells of both induced Tregs (iTregs), which have a regulatory function, and Thl7 cells, which secrete pro-inflammatory cytokines. TGF-pi alone precipitates the expression of Foxp3 and Treg differentiation from activated T helper cells.

The growth factors, (e.g., BMP-2 or TGF-pi), may be isolated from endogenous sources or synthesized in vivo or in vitro. Endogenous growth factor polypeptides may be isolated from healthy human tissue. Synthetic growth factor polypeptides are synthesized in vivo following transfection or transformation of template DNA into a host organism or cell, e.g., a mammalian or human cell line. Alternatively, synthetic growth factor polypeptides are synthesized in vitro by cell free translation or other art-recognized methods Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), herein incorporated by reference.

In certain embodiments, growth factor (e.g., BMP-2 or TGF-pi) polypeptides may be recombinant. In some embodiments, growth factor polypeptides are humanized derivatives of mammalian growth factor polypeptides. Exemplary mammalian species from which growth factor polypeptides are derived include, but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate. In some embodiments, the growth factor is a recombinant human protein . In some embodiments, the growth factor is a recombinant murine (mouse) protein. In some embodiments, the growth factor is a humanized derivative of a recombinant mouse protein.

In certain embodiments, the growth factor polypeptides may be modified to increase protein stability in vivo. In certain embodiments, the growth factor polypeptides may be engineered to be more or less immunogenic. The terms “immunogenic” and “immunogenicity” refer to the ability of a particular substance, such as a protein, an antigen, or an epitope, to provoke an immune response in the body of a human and other animal.

In certain embodiments, the growth factors may be present at between about 0.001 nmol and about 1000 nmol per scaffold, or about 0.001 and about 100 nmol per scaffold, or about 0.001 nmol and about 1 nmol per scaffold.

In some embodiments, the growth factors may be present at between about 1 ng to 1000 micrograms per scaffold. For example, the growth factors may be present at an amount between about 1 pg and about 1000 pg, between about 1 pg and 500 pg, between about 1 pg and about 200 pg, between about 1 pg and about 100 pg, between about 1 pg and about 50 pg, or between about 1 pg and 10 pg.

In some embodiments, the growth factor may be present at between about 1 ng to about 1000 pg. In some embodiments, the growth factor may be present at between about 1 ng to about 100 pg. In some embodiments, the growth factor may be present at between about 1 pg to about 2 ng per microgel. In some embodiments, the growth factor may be present at about 1 pg per microgel. In some embodiments, the growth factor may be present at between about 1 ng to about 500 ng. In some embodiments, the growth factor may be present at between about 1 ng to about 100 ng (e.g., about 1 ng, about 2 ng, about 3 ng, about 4 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 11 ng, about 12 ng, about 13 ng, about 14 ng, about 15 ng, about 16 ng, about 17 ng, about 18 ng, about 19 ng, about 20 ng, about 21 ng, about 22 ng, about 23 ng, about 24 ng, about 25 ng, about 26 ng, about 27 ng, about 28 ng, about 29 ng, about 30 ng, about 31 ng, about 32 ng, about 33 ng, about 34 ng, about 35 ng, about 36 ng, about 37 ng, about 38 ng, about 39 ng, about 40 ng, about 41 ng, about 42 ng, about 43 ng, about 44 ng, about 45 ng, about 46 ng, about 47 ng, about 48 ng, about 49 ng, about 50 ng, about 51 ng, about 52 ng, about 53 ng, about 54 ng, about 55 ng, about 56 ng, about 57 ng, about 58 ng, about 59 ng, about 60 ng, about 61 ng, about 62 ng, about 63 ng, about 64 ng, about 65 ng, about 66 ng, about 67 ng, about 68 ng, about 69 ng, about 70 ng, about 71 ng, about 72 ng, about 73 ng, about 74 ng, about 75 ng, about 76 ng, about 77 ng, about 78 ng, about 79 ng, about 80 ng, about 81 ng, about 82 ng, about 83 ng, about 84 ng, about 85 ng, about 86 ng, about 87 ng, about 88 ng, about 89 ng, about 90 ng, about 91 ng, about 92 ng, about 93 ng, about 94 ng, about 95 ng, about 96 ng, about 97 ng, about 98 ng, about 99 ng, or about 100 ng). In some embodiments, the growth factor may be present at greater than about 2 ng.

In certain embodiments, the composition of the present disclosure comprises nanogram quantities of growth factors (e.g., about 1 ng to about 1000 ng of BMP-2). For example, the growth factors may be present at an amount between about 5 ng and about 500 ng, between about 5 ng and about 250 ng, between about 5 ng and about 200 ng, between about 10 ng and about 200 ng, between about 25 ng and about 200 ng, between about 50 ng and 200 ng, between about 100 ng and 200 ng, and about 200 ng. Nanogram quantities of the growth factor are also released in a controlled manner. The nanogram quantities of the growth factors and/or the controlled release can contribute to reduced toxicity of the compositions and methods of the present disclosure as compared to other delivery system, which uses high dose of growth factors and has suboptimal release kinetics.

In various embodiments, the amount of growth factors present in a scaffold may vary according to the size of the scaffold. For example, the growth factor may be present at about 0.03 ng/mm³ (the ratio of the amount of growth factors in weight to the volume of the scaffold) to about 350 ng/mm³, such as between about 0.1 ng/mm³ and about 300 ng/mm³ , between about 0.5 ng/mm³ and about 250 ng/mm³, between about 1 ng/mm³ and about 200 ng/mm³, between about 2 ng/mm³ and about 150 ng/mm³, between about 3 ng/mm³ and about 100 ng/mm³, between about 4 ng/mm³ and about 50 ng/mm³, between about 5 ng/mm³ and 25 ng/mm³, between about 6 ng/mm³ and about 10 ng/mm³, or between about 6.5 ng/mm³ and about 7.0 ng/mm³.

In some embodiments, the amount of growth factors may be present at between about 300 ng/mm³ and about 350 pg/mm³, such as between about 400 ng/mm³ and between about 300 pg/mm³, between about 500 ng/mm³ and about 200 pg/mm³, between about 1 pg/mm³ and about 100 pg/mm³, between about 5 pg/mm³ and about 50 pg/mm³, between about 10 pg/mm³ and about 25 pg/mm³.

Differentiation Factors

The composition of the present disclosure can comprise a differentiation factor. As used herein, a differentiation factor is an agent that can induce the differentiation of a cell, for example, a recruited cell. In certain embodiments, the differentiation factor is a polypeptide. As used herein, “differentiation,” “cell differentiation,” “cellular differentiation,” or other similar terms refer to the process where a cell changes from one cell type to another. In certain embodiments, the cell changes to a more specialized type, e.g., from a stem cell or a progenitor cell to a T cell progenitor cell. Differentiation occurs numerous times during the development of a multicellular organism as it changes from a simple zygote to a complex system of tissues and cell types. Differentiation continues in adulthood as adult stem cells divide and create fully differentiated daughter cells during tissue repair and during normal cell turnover. Differentiation may change a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals. These changes may be due to highly controlled modifications in gene expression.

Among dividing cells, there are multiple levels of cell potency, the cell's ability to differentiate into other cell types. A greater potency indicates a larger number of cell types that can be derived. A cell that can differentiate into all cell types, including the placental tissue, is known as totipotent. A cell that can differentiate into all cell types of the adult organism is known as pluripotent. In mammals, e.g., human being, a pluripotent cell may include embryonic stem cells and adult pluripotent cells. Induced pluripotent stem (iPS) cells may be created from fibroblasts by induced expression of certain transcription factors, e.g., Oct4, Sox2, c-Myc, and KIF4. A multipotent cell is one that can differentiate into multiple different, but closely related cell types. Oligopotent cells are more restricted than multipotent, but can still differentiate into a few closely related cell types. Finally, unipotent cells can differentiate into only one cell type, but are capable of self-renewal.

In certain embodiments, the differentiation factors of the present disclosure induce the differentiation of stem cells or progenitor cells into T-cell progenitor cells. As used herein, the term “T cell progenitor cell” refers to a progenitor cell that ultimately can differentiate to a T lymphocyte (T cell). The term “lymphocyte,” as used herein, refers to one of the subtypes of white blood cell in a vertebrate’s (e.g., human being) immune system. Lymphocytes include natural killer cells, T cells, and B cells. Lymphocytes originate from a common lymphoid progenitor during hematopoiesis, a process during which stem cells differentiate into several kinds of blood cells within the bone marrow, before differentiating into their distinct lymphocyte types.

In some embodiments, the T cell progenitor cell comprises a common lymphoid progenitor cell. The term “common lymphoid progenitor cell,” as used herein, refers to the earliest lymphoid progenitor cells, which give rise to lymphocytes including T-lineage cells, B-lineage cells, and natural killer (NK) cells. In various embodiment, the T cell progenitor cell comprises a T cell competent common lymphoid progenitor cell. The term “T cell competent common lymphoid progenitor cell,” as used herein, refers to a common lymphoid progenitor cell that differentiates into T-lineage progenitor cell. A T cell competent common lymphoid progenitor is usually characterized by lacking of biomarker Ly6D. The composition of the present disclosure can create an ectopic niche that mimics important features of bone marrow and induces the differentiation of stem cells or progenitor cells into T cell progenitor cells.

In certain embodiments, the lymphocytes comprise T cells. In some embodiments, the T cells are naive T cells. As used herein, a naive T cell is a T cell that has differentiated in bone marrow. Naive T cells may include CD4⁺ T cells, CD8⁺ T cells, and regulatory T Cells (Treg).

In certain embodiments, the differentiation factors induce the differentiation of the recruited cells into T cell progenitor cells. In certain embodiments, the differentiation factors induce the differentiation of the recruited cells into T cell progenitor cells through the Notch signaling pathway. The Notch signaling pathway is a highly conserved cell signaling system present in many multicellular organisms. Mammals possess four different Notch receptors, referred to as Notchl, Notch2, Notch3, and Notch4. Notch signaling plays an important role in T cell lineage differentiation from common lymphoid progenitor cells. In certain embodiments, the differentiation factors bind to one or more Notch receptors and activates the Notch signaling pathway. In certain embodiments, the differentiation factor is selected from a group consisting of a Delta-like 1 (DLL-1), a Delta-like 2 (DLL-2), a Delta-like 3 (DLL-3), a Delta-like 3 (DLL-3), a Delta-like 4 (DLL-4), a Jagged 1, a Jagged 2, and any combination thereof. In certain embodiments, the binding of the differentiation factor to one or more Notch receptors activates the Notch signaling pathway and induces T cell lineage differentiation. In certain embodiments, the differentiation factor is a Delta-like 4 (DLL-4). DLL-4 is a protein that is a homolog of the Drosophila Delta protein. The Delta protein family includes Notch ligands that are characterized by a DSL domain, EGF repeats, and a transmembrane domain.

In certain embodiments, the differentiation factor polypeptides are isolated from endogenous sources or synthesized in vivo or in vitro. Endogenous differentiation factor polypeptides may be isolated from healthy human tissue. Synthetic differentiation factor polypeptides are synthesized in vivo following transfection or transformation of template DNA into a host organism or cell, e.g., a mammal or cultured human cell line. Alternatively, synthetic differentiation factor polypeptides are synthesized in vitro by cell free translation or other art-recognized methods Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), herein incorporated by reference).

In certain embodiments, differentiation factor polypeptides may be recombinant. In some embodiments, the differentiation factor polypeptides are humanized derivatives of mammalian differentiation factor polypeptides. Exemplary mammalian species from which the differentiation factor polypeptides are derived include, but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate. In some embodiments, the differentiation factor is a recombinant human protein . In some embodiments, the differentiation factor is a recombinant murine (mouse) protein. In some embodiments, the differentiation factor is a humanized derivative of a recombinant mouse protein.

In certain embodiments, the differentiation factor polypeptides may be modified to achieve a desired activity, for example, to increase protein stability in vivo. In certain embodiments, the differentiation factor polypeptides may be engineered to be more or less immunogenic.

In certain embodiments, the differentiation factor (e.g., DLL-4) may be covalently linked to the scaffold of the present disclosure. For example, rather than being released from a scaffold material, a differentiation factor may be covalently bound to polymer backbone and retained within the composition that forms following implantation of the composition in the subject. By covalently binding or coupling a differentiation factor to the scaffold material, such differentiation factor will be retained within the scaffold that forms following administration of the composition to a subject, and thus will be available to promote the differentiation of stem cells or progenitor cells, as contemplated herein. In certain embodiments, the differentiation factors are conjugated to the scaffold material utilizing N- hydroxysuccinimide (NHS) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry. Any methods of covalently binding or coupling differentiation factors known in the art may be used and are not limited. See "Bioconjugate Techniques Bioconjugate Techniques (Third Addition)", Greg T. Hermanson, Academic , Greg T. Hermanson, Academic Press, 2013 Press, 2013. In some embodiments, the differentiation factor may be covalently linked to the scaffold utilizing click chemistry. The methods of covalently binding or coupling differentiation factors include, but are not limited to, avidin-biotin reaction, azide and dibenzocycloocytne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbornene chemistry, or di-sulfide bond.

In certain embodiments, the differentiation factors (e.g., DLL-4) of the present disclosure further comprise a tether (e.g., PEG, PEG2k) and a methacrylate group (MA). In certain embodiments, the differentiation factor is methacrylated DLL-4-PEG2k.

In certain embodiments, the covalent linking retains the differentiation factors within the scaffold to provide the differentiation signal to the recruited cells in the scaffold. For example, less than 1 % of the total differentiation factor is detected outside of the scaffold. The bioactivity of the differentiation factor may be retained for an extended period of time, such as at least three months after incorporation to the scaffold. The bioactivity of the differentiation factors may be measured by any appropriate methods, such as a colorimetric assay for DLL-4.

In certain embodiments, the differentiation factors may be present at between about 0.01 nmol and 1000 nmol, about 0.1 nmol and 100 nmol, or about 1 nmol and 10 nmol per scaffold.

In some embodiments, the differentiation factors may be present at between about 1 ng and 1000 micrograms per scaffold. For example, the differentiation factor may be present at between about 10 ng and about 500 pg, between about 50 ng and about 250 pg, between about 100 ng and about 200 pg, between about 1 pg and about 100 pg, between about 1 pg and about 50 pg, between about 1 pg and about 25 pg, between about 1 pg and about 10 pg, between about 2 pg and about 10 pg, or about 6 pg.

In some embodiments, the differentiation factor may be present at between about 1 ng to about 1000 pg. In some embodiments, the differentiation factor may be present at between about 1 ng to about 100 pg. In some embodiments, the differentiation factor may be present at between about 1 pg to about 2 ng per microgel. In some embodiments, the differentiation factor may be present at about 1 pg per microgel. In some embodiments, the differentiation factor may be present at between about 1 ng to about 500 ng. In some embodiments, the differentiation factor may be present at between about 1 ng to about 100 ng (e.g., about 1 ng, about 2 ng, about 3 ng, about 4 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 11 ng, about 12 ng, about 13 ng, about 14 ng, about 15 ng, about 16 ng, about 17 ng, about 18 ng, about 19 ng, about 20 ng, about 21 ng, about 22 ng, about 23 ng, about 24 ng, about 25 ng, about 26 ng, about 27 ng, about 28 ng, about 29 ng, about 30 ng, about 31 ng, about 32 ng, about 33 ng, about 34 ng, about 35 ng, about 36 ng, about 37 ng, about 38 ng, about 39 ng, about 40 ng, about 41 ng, about 42 ng, about 43 ng, about 44 ng, about 45 ng, about 46 ng, about 47 ng, about 48 ng, about 49 ng, about 50 ng, about 51 ng, about 52 ng, about 53 ng, about 54 ng, about 55 ng, about 56 ng, about 57 ng, about 58 ng, about 59 ng, about 60 ng, about 61 ng, about 62 ng, about 63 ng, about 64 ng, about 65 ng, about 66 ng, about 67 ng, about 68 ng, about 69 ng, about 70 ng, about 71 ng, about 72 ng, about 73 ng, about 74 ng, about 75 ng, about 76 ng, about 77 ng, about 78 ng, about 79 ng, about 80 ng, about 81 ng, about 82 ng, about 83 ng, about 84 ng, about 85 ng, about 86 ng, about 87 ng, about 88 ng, about 89 ng, about 90 ng, about 91 ng, about 92 ng, about 93 ng, about 94 ng, about 95 ng, about 96 ng, about 97 ng, about 98 ng, about 99 ng, or about 100 ng). In some embodiments, the differentiation factor may be present at greater than about 2 ng-

In various embodiments, the amount of differentiation factor present in a scaffold may vary according to the size of the scaffold. For example, the differentiation factor may be present at about 0.03 ng/mm³ (the ratio of the amount of differentiation factor in weight to the volume of the scaffold) to about 350 pg/mm³, such as between about 0.1 ng/mm³ and about 300 pg/mm³, between about 1 ng/mm³ and about 250 pg/mm³, between about 10 ng/mm³ and about 200 pg/mm³, between about 0.1 pg/mm³ and about 100 pg/mm³, between about 0.1 pg/mm³ and 50 about pg/mm³, or between about 0.1 pg/mm³ and about 20 pg/mm³, between about 0.1 pg/mm³ and about 10 pg/mm³, between about 0.1 pg/mm³ and about 5 pg/mm³, between about 0.1 pg/mm³ and about 1 pg/mm³, between about 0.1 pg/mm³ and 0.5 pg/mm³, or about 0.2 pg/mm³.

In certain embodiments, the DLL-4 may be present at about 6 pg per scaffold.

Homing Factors

In certain embodiments, the composition of the present disclosure may further comprise a homing factor. As used herein, the term “homing factor” refers to an agent that is capable of inducing directed movement of a cell, e.g., a stem cell or a progenitor cell. In certain embodiments, the homing factors of the present disclosure are signaling proteins that can induce directed chemotaxis in nearby responsive cells. In various embodiments, the homing factors are cytokines and/or chemokines.

In certain embodiments, the inclusion of such homing factors in the compositions of the present disclosure promotes the homing of cells (e.g., transplanted stem cells and/or progenitor cells) to the scaffold composition administered to a subject. In certain aspects, such homing factors promote the infiltration of the cells (e.g., transplanted stem cells or progenitor cells) to the scaffold composition administered to the subject. In some embodiments, the homing factors comprise stromal cell derived factor (SDF-1). In certain embodiments, the homing factors are encapsulated in the material. In certain embodiments, the homing factors are released from the material over an extended period of time (e.g., about 7-30 days or longer, about 17-18 days).

In certain embodiments, the homing factors retain their bioactivity over an extended period of time. The bioactivity of the growth factor may be measured by any appropriate means. In certain example, the homing factors retain their bioactivity for at least 10 days, 12 days, 14 days, 20 days, or 30 days after the incorporation of the homing factors into the scaffold.

In some embodiments, the homing factors may be present at between about 0.01 nmol and 1000 nmol, about 0.1 nmol and 100 nmol, or about 1 nmol and 10 nmol per scaffold.

In some embodiments, the homing factors may be present at between about 1 ng and 1000 micrograms per scaffold. For example, the homing factor may be present at between about 10 ng and about 500 pg, between about 50 ng and about 250 pg, between about 100 ng and about 200 pg, between about 1 pg and about 100 pg, between about 1 pg and about 50 pg, between about 1 pg and about 25 pg, between about 1 pg and about 10 pg, between about 2 pg and about 10 pg, or about 6 pg.

In some embodiments, the homing factor may be present at between about 1 ng to about 1000 pg. In some embodiments, the homing factor may be present at between about 1 ng to about 100 pg. In some embodiments, the homing factor may be present at between about 1 pg to about 2 ng per microgel. In some embodiments, the homing factor may be present at about 1 pg per microgel. In some embodiments, the homing factor may be present at between about 1 ng to about 500 ng. In some embodiments, the homing factor may be present at between about 1 ng to about 100 ng (e.g., about 1 ng, about 2 ng, about 3 ng, about 4 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 11 ng, about 12 ng, about 13 ng, about 14 ng, about 15 ng, about 16 ng, about 17 ng, about 18 ng, about 19 ng, about 20 ng, about 21 ng, about 22 ng, about 23 ng, about 24 ng, about 25 ng, about 26 ng, about 27 ng, about 28 ng, about 29 ng, about 30 ng, about 31 ng, about 32 ng, about 33 ng, about 34 ng, about 35 ng, about 36 ng, about 37 ng, about 38 ng, about 39 ng, about 40 ng, about 41 ng, about 42 ng, about 43 ng, about 44 ng, about 45 ng, about 46 ng, about 47 ng, about 48 ng, about 49 ng, about 50 ng, about 51 ng, about 52 ng, about 53 ng, about 54 ng, about 55 ng, about 56 ng, about 57 ng, about 58 ng, about 59 ng, about 60 ng, about 61 ng, about 62 ng, about 63 ng, about 64 ng, about 65 ng, about 66 ng, about 67 ng, about 68 ng, about 69 ng, about 70 ng, about 71 ng, about 72 ng, about 73 ng, about 74 ng, about 75 ng, about 76 ng, about 77 ng, about 78 ng, about 79 ng, about 80 ng, about 81 ng, about 82 ng, about 83 ng, about 84 ng, about 85 ng, about 86 ng, about 87 ng, about 88 ng, about 89 ng, about 90 ng, about 91 ng, about 92 ng, about 93 ng, about 94 ng, about 95 ng, about 96 ng, about 97 ng, about 98 ng, about 99 ng, or about 100 ng). In some embodiments, the homing factor may be present at greater than about 2 ng.

III. Methods of Making

The present disclosure provides methods of making microgels, e.g., microgel scaffolds.

The microgels of the present disclosure may be formed by a method comprising forming emulsions. Without wishing to be bound by theory, the size of the resulting microgels can be determined, at least in part, by the size of the emulsion. Thus, adjusting the size of the emulsion at the emulsion formation step can be used to tune the physicochemical properties (e.g., size) of the microgels. Such adjusting can be achieved, for example, by controlling the flow rate and/or dimensions of the microfluidic chip. Formation of emulsion- templated microgels can also include a hydrophobic treatment, e.g., in which the walls of the microfluidic chip are contacted with a predetermined amount of a surfactant.

In one aspect, the present disclosure provides a method of preparing a microgel, comprising: (i) providing a microfluidics chip; (ii) providing an aqueous phase comprising a first polymer and a second polymer; (iii) providing a continuous oil phase comprising an oil and a surfactant; and (iv) contacting the aqueous phase with the continuous oil phase in the microfluidics chip to form an emulsion, thereby preparing the microgel.

The microfluidics chip may comprise at least two aqueous inlets, at least one oil inlet, and at least one outlet. In some embodiments, the microfluidics chip may comprise at least one junction, wherein the junction permits the aqueous phase to contact the continuous oil phase to form an emulsion. Exemplary microfluidics chips are known in the art (see, e.g., PCT International Patent Application Publication No. WO 2015/069634, herein incorporated by reference in its entirety.

In various embodiments, the first polymer and the second polymer may be independently selected from the group consisting of a non-degradable polymer, a degradable polymer, and a combination thereof. In some embodiments, the first polymer and the second polymer may be independently selected from the group consisting of alginate, chitosan, polyethylene glycol (PEG), gelatin, hyaluronic acid, collagen, chondroitin, agarose, polyacrylamide, heparin, derivatives thereof, and combinations thereof. In some embodiments, the first polymer and the second polymer can be the same polymer. In some embodiments, the first polymer and the second polymer can be independently an alginate, optionally wherein the first polymer and the second polymer independently comprise a modified alginate polymer, optionally wherein the first polymer and the second polymer independently comprise oxidized alginate, optionally wherein the first polymer and the second polymer are independently comprise methacrylate alginate, optionally wherein the first polymer and the second polymer independently comprise a click reagent.

In some embodiments, the first polymer and the second polymer independently may comprise a modified polymer. For example, the first polymer and the second polymer may independently comprise an oxidized polymer. The oxidized polymer may be about 0.1% to about 99% oxidized (e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% oxidized). The first polymer and the second polymer may independently comprise oxidized alginate.

In some embodiments, the first polymer and the second polymer may independently comprise a click reagent. For example, the first polymer and the second polymer may independently comprise a click reagent selected from the group consisting of azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine (Tz), norbomene (Nb), and variants thereof. In some embodiments, the first polymer may comprise a tetrazine (Tz) moiety. In some embodiments, the first polymer may comprise tetrazine modified alginate (Alg-Tz). In some embodiments, the second polymer may comprise a norbomene (Nb) moiety. In some embodiments, the second polymer may comprise norbomene modified alginate (Alg-Nb).

In some embodiments, the first polymer and the second polymer are independently dissolved in deionized water. In some embodiments, the first polymer and the second polymer are independently provided at a concentration of about 0.1% (w/v) to about 10% (w/v). In some embodiments, the first polymer is provided at a concentration of about 0.5% (w/v) to about 1.5% (w/v). In some embodiments, the second polymer is provided at a concentration of about 1.5% (w/v) to about 2.5% (w/v).

In some embodiments, the oil comprises HFE7500. In some embodiments, the oil comprises mineral oil. In some embodiments, the oil comprises silicone.

In some embodiments, the surfactant is selected from the group consisting of an amphoteric surfactant, an anionic surfactant, a cationic surfactant, a nonionic surfactant, and a combination thereof. In some embodiments, the surfactant comprises a nonionic surfactant selected from the group consisting of Brij 93, SPAN 80, AB IL EM90, PGPR, and a combination thereof. In some embodiments, when mineral oil and/or silicone oil is included, the surfactant may be a nonionic surfactant such as Brij 93, SPAN 80, AB IL EM90, and/or PGPR.

In some embodiments, the surfactant comprises fluorosurfactant. In some embodiments, the continuous oil phase comprises about 0.5% (w/v) to about 2% (w/v) fluorosurfactant in HFE7500 solution, optionally wherein the continuous oil phase comprises about 1% (w/v) fluorosurfactant in HFE7500 solution.

In some embodiments, the methods described herein may further comprise injecting the Alg-Nb and the Alg-Tz into the microfluidics chip, optionally wherein the Alg-Nb and the Alg-Tz are injected at a rate of about 25 pl/hour to about 100 pl/hour, optionally wherein the Alg-Nb and the Alg-Tz are injected at a rate of about 50 pl/hour. In some embodiments, the methods described herein may further comprise injecting the continuous oil phase at a rate of about 175 pl/hour to about 500 pl/hour, optionally wherein the continuous oil phase is injected at a rate of about 200 pl/hour. In some embodiments, the methods described herein may further comprise allowing the Alg-Nb and the Alg-Tz solutions to form an emulsion when they encounter the continuous oil phase at a junction inside the microfluidics chip, thereby forming an emul si on-templ ated microgel. In some embodiments, the methods described herein may further comprise collecting the emulsion. In some embodiments, the methods described herein may further comprise maintaining the emulsion at room temperature for at least about 6 hours to about 24 hours to allow covalent crosslinking between the Alg-Nb and the Alg-Tz polymers. In some embodiments, the methods described herein may further comprise treating the emulsion with a demulsification and washing process, optionally wherein the treating comprises contacting the emulsion with an about 30- 50% (v/v), optionally an about 40% (v/v), 1H,1H,2H,2H-Perfluoro-1 -octanol (PFO) solution, an about 0.1-1 % (v/v), optionally an about 0.5% (v/v) Tween 20 solution, and an about 0.5% (w/v) to about 1.5% (w/v), optionally an about 0.8% (w/v) sodium chloride (NaCl) solution, sequentially. In some embodiments, the methods described herein may further comprise isolating the microgel. In some embodiments, the methods described herein may further comprise dispersing the microgel in an aqueous solution, optionally wherein the aqueous solution comprises a saline solution, optionally wherein the aqueous solution comprises phosphate-buffered saline (PBS), optionally wherein the aqueous solution comprises calcium chloride (CaCh) and/or NaCl, optionally wherein the aqueous solution comprises about 2 mM CaCh and/or about 0.8% NaCl, optionally wherein the aqueous solution comprises DMEM media and 10% FBS. In some embodiments, the methods described herein may further comprise lyophilizing the microgel. In some embodiments, the methods described herein may further comprise storing the microgel at about 4°C.

In some embodiments, the methods described herein may further comprise contacting the microgel with an active agent, optionally wherein the contacting occurs at about 4°C for about 1 hour to about 5 hours, optionally wherein the contacting occurs at about 4°C for about 3 hours. In some embodiments, the active agent is selected from the group consisting of a cell, a biological factor, and/or a small molecule, optionally wherein: (i) the active agent is present at between about 1 ng to about 1000 pg, optionally wherein the active agent is present at between about 1 ng to about 100 pg, optionally wherein the active agent is present at between about 1 pg to about 2 ng per microgel, optionally wherein the active agent is present at about 1 pg per microgel; (ii) the active agent comprises a growth factor, optionally wherein the growth factor is selected from the group consisting of a BMP-2, a BMP-4, a BMP-6, a BMP-7, a BMP- 12, a BMP- 14, and a combination thereof, optionally wherein the growth factor comprises a BMP-2, optionally wherein the growth factor is present at between about 2 ng to about 500 ng per microgel; (iii) the active agent comprises a differentiation factor, optionally wherein the differentiation factor is selected from the group consisting of a Deltalike 1 (DLL-1), a Delta-like 2 (DLL-2), a Delta-like 3 (DLL-3), a Delta-like 3 (DLL-3), a Delta-like 4 (DLL-4), a Jagged 1, a Jagged 2, and a combination thereof, optionally wherein the differentiation factor comprises DLL-4, optionally wherein the differentiation factor is present at an amount at between about 1 ng to about 100 pg per microgel; (iv) the active agent is covalently and/or non-covalently attached to the microgel, optionally wherein the active agent is covalently attached to the microgel utilizing click chemistry, optionally wherein the active agent is covalently linked to the scaffold utilizing N-hydroxysuccinimide (NHS) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry, NHS and dicyclohexylcarbodiimide (DCC) chemistry, avidin-biotin reaction, azide and dibenzocycloocytne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbomene chemistry, or di-sulfide chemistry; and/or (v) the active agent is released from the microgel within about 1-day to about 30-days after administration to a subject.

Exemplary bone marrow microgels may be made from PDMS-based microfluidics which have two aqueous inlets, one oil inlet, and one outlet. Norbomene modified alginate (Alg-Nb) and Tetrazine modified alginate (Alg-Tz) was dissolved in a deionized water (1.2% wt/vol and 1.8% wt/vol, respectively) and used as dispersed prepolymer aqueous phase in the microfluidics. 1% (wt/vol) fluorosurfactant in HFE7500 solution was prepared and used as a continuous oil phase in the microfluidics. Alg-Nb and Alg-Tz solutions were injected at 50 pl/hour and the continuous oil phase was injected at 200 pl/hour. Alg-Nb and Alg-Tz solutions were form emulsion when they encounter oil phase at the junction and form emul si on-templ ated microgels. The emulsion was collected in a tube and left at room temperature for overnight to allow covalent crosslinking between alginate polymers. After that, the emulsion go through demulsification and washing process by adding 40% vol/vol 1H,1H,2H,2H-Perfluoro-1 -octanol (PFO) solution, 0.5% vol/vol Tween 20 solution, and 0.8% wt/vol sodium chloride solution, sequentially. The microgels were redispersed in saline solution and stored at 4°C until further use. BMP -2 was added to the microgels and incubated at 4°C for 3-hours right before in vivo experiment.

Exemplary alginate microgels may be fabricated using microfluidic emulsion, which can provide defined size and shape by controlled droplet formation. Alginate polymer may be first modified with norbornene (Alg-Nb) or tetrazine (Alg-Tz), e.g., by carbodiimide coupling, to achieve an average degree of substitution (DS) of about 13 or about 11.5 functional groups per alginate chain, respectively, as quantified, e.g., by proton nuclear magnetic resonance spectra. Stock solutions of Alg-Nb and Alg-Tz may then be mixed at a final concentration of 2 wt% in a microfluidic device and injected to form microdroplets by emulsion, which may then be crosslinked, e.g., overnight, to generate microgels with a diameter of about 77 ± 2 pm.

IV. Methods of Use

In one aspect, the present disclosure provides methods of modulating the immune system of a subject. In certain embodiments of the present disclosure, the methods of modulating the immune system of the subject comprise administering to the subject one or more compositions of the present disclosure.

In another aspect, the present disclosure provides methods for promoting the generation of an ectopic bone marrow niche in a subject in need thereof. In certain embodiments of the present disclosure, the methods for promoting the generation of an ectopic bone marrow niche in a subject comprise administering to the subject one or more compositions of the present disclosure.

In another aspect, the present disclosure provides methods for promoting the generation of hematopoietic tissue in a subject in need thereof. In certain embodiments of the present disclosure, the methods for promoting the hematopoietic tissue in a subject comprise administering to the subject one or more compositions of the present disclosure.

In another aspect, the present disclosure provides methods for promoting reconstitution of hematopoietic cells in a subject. In certain embodiments of the present disclosure, the methods for promoting reconstitution of hematopoietic cells in a subject comprise administering to the subject one or more compositions of the present disclosure.

In certain embodiments, the methods comprise administering a microgel as described herein. In some embodiments, the microgel forms a three-dimensional scaffold in situ upon administration to the subject. In some embodiments, the scaffold comprises pores of a size that permit a eukaryotic cell to traverse into or out of the scaffold. In some embodiments, the pores have a diameter of about 1 pm to about 1000 pm.

In certain embodiments, the methods enhance the recruitment, proliferation, and/or differentiation of immune cells in the scaffold material within about 1-week to about 4-weeks after administration, optionally wherein the immune cells comprise hematopoietic stem cells (HSCs) and/or hematopoietic progenitor cells (HPCs), optionally wherein the immune cells comprise myeloid cells and/or lymphoid cells, optionally wherein the immune cells comprise T cells, B cells, and/or natural killer (NK) cells, optionally wherein the immune cells comprise Lin-c-kit+Sca-l+ (LKS) cells, optionally wherein the immune cells comprise CD45.2+ cells.

In certain embodiments, the methods enhance the recovery of a normal absolute lymphocyte count (ALC) and/or immune cell subsets, optionally comprising neutrophils, monocytes, natural killer cells, T cells, and/or B cells.

In certain embodiments, the methods enhance reconstitution of T cells and/or B cells in the subject. In certain embodiments, the methods enhance T cell and/or B cell diversity in the subject. In some embodiments, the T cell diversity is characterized by an enhanced T cell receptor (TCR) repertoire. In some embodiments, the B cell diversity is characterized by an enhanced B cell receptor (BCR) repertoire.

In certain embodiments, the subject is a human. In certain embodiments, the microgel is administered to the subject via injection, optionally, intravenously, intramuscularly, or subcutaneously.

The composition may include a porous scaffold, a growth factor present in an amount effective for inducing formation of a tissue or an organ within the scaffold and recruiting a cell into the scaffold, and a differentiation factor that induces the differentiation of the recruited cell into a T cell progenitor cell. In a particular embodiment, the composition includes a porous scaffold; a growth factor present at between about 1 ng to about 1000 ng per scaffold and in an amount effective for inducing formation of a tissue or an organ within the scaffold and recruiting a cell into the scaffold; and a differentiation factor that induces the differentiation of the recruited cell into a lymphocyte.

In certain embodiments, the methods further comprise administering to the subject a hematopoietic stem cell or a hematopoietic progenitor cell.

In certain embodiments, the cells are stem cells or progenitor cells. As used herein, the term “stem cell” refers to a biological cell that can differentiate into other types of cells and can divide to produce more of the same type of stem cells. Stem cells include embryonic stem cells, which are isolated from the inner cell mass of blastocysts, and adult stem cells, which are found in various tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing adult tissues. In certain embodiments, the stem cells are embryonic stem cells, fetal stem cells, amniotic stem cells, umbilical cord stem cells, adult stem cells, or induced pluripotent stem cells. In certain embodiments, the stem cells are hematopoietic stem cells. Hematopoietic stem cells are the stem cells that give rise to other blood cells, including both myeloid and lymphoid lineage of blood cells.

As used herein, the term “progenitor cell” refers to a biological cell that can differentiate into a specific type of cell. Progenitor cells are generally more differentiated than stem cells. Typically, progenitor cells can only divide a limited number of times.

In certain embodiments, the progenitor cells are blast cells, such as thymocytes, lymphoblasts, myeloid, or bone marrow precursor cells. In certain embodiments, the progenitor cells are cells that are capable of differentiating into T cell progenitor cells. In certain embodiments, the lymphocytes include T cells, such as naive T cells.

In certain embodiments, the recruited cells are hematopoietic bone marrow cells, or mobilized peripheral blood cells.

In certain embodiments, the cells may be recombinant cells. The term “recombinant cell,” as used herein, refers to a cell into which a genetic modification has been introduced. The genetic modification may be at chromosomal level or extra-chromosomal. “Genetic modification at chromosomal level” refers to the genetic modification in the genome of the cell, e.g., insertion, deletion, and/or substitution on the chromosome of the cell. Extra- chromosomal genetic modification refers to the genetic modification not located in the genome of the cell. For example, a plasmid containing a protein encoding gene may be introduced to the cell. The plasmid may replicate and transmit from parental cells to offspring cells.

In various embodiments, the genetic modification introduces a gene into the cell. The introduced gene may compensate for the function of a defective gene of the cell. For example, the cell may contain a mutant defective gene. The genetic modification may introduce a wild type functional gene into the cell to restore the function of the gene. In some embodiment, the genetic modification may increase or decrease the expression of certain gene. For example, the genetic modification may introduce a small interfering RNA (siRNA) specific to a gene to inhibit the expression of the gene.

The methods to genetically modify a cell are commonly known in the art such as the methods described in Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), herein incorporated by reference.

In certain embodiments, the genetic modification may be introduced through gene editing, also known as genome editing. Gene editing is a group of technologies that give skilled artisans the ability to change an organism's DNA. These technologies allow genetic material to be added, removed, or altered at particular locations in the genome. Gene editing technologies include, but are not limited to, meganucleases system, Zinc finger nucleases (ZFN) system, transcription activator-like effector nucleases (TALENs) system, and CRISPR-Cas system. CRISPR-Cas systems, which is short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein systems, in particular CRISP-Cas9, is faster, cheaper, more accurate, and more efficient than other existing genome editing methods.

In certain embodiments, the present disclosure features methods that modulate the immune system of a subject after the subject receives a transplantation. For example, the subject may receive a hematopoietic stem cell transplantation. In certain embodiments, the compositions of the present disclosure are administered to the subject concurrently with, or after, the hematopoietic stem cell transplantation.

In certain embodiments, at least two compositions are administered to the subject. The compositions can be of similar size.

In certain embodiments, the methods of the present disclosure modulate the immune response of a human over 30 years of age. For example, the human may be over 40, over 50, over 60, over 70, over 80 years of age.

In some embodiments, one or more compositions of the present disclosure (e.g., bone marrow microgels) may be administered in conjunction with stem cell mobilization techniques. Stem cell mobilization is a process by which certain cell mobilization agents are used to cause the movement of stem cells from the bone marrow into the blood, such as described in Hopman and DiPersio, Advances in Stem Cell Mobilization, Blood Rev., 2014, 28(1): 31-40, the content of which is incorporated herein by reference. Such techniques may also be used for mobilization of progenitor cells.

Accordingly, in certain embodiments, a subject is administered a stem and/ or progenitor cell mobilization agent in an amount effective to induce the movement of stem/progenitor cells from bone marrow into the blood. Released stem and/or progenitor cells are subsequently recruited to the composition of the present disclosure (e.g., a bone marrow microgel) to differentiate into T cell progenitor cells. The stem and/or progenitor cell mobilization agent may be administered prior to, concurrently with, or following the administration of the composition (e.g., a bone marrow microgel).

In various embodiments, the composition of the present disclosure (e.g., a bone marrow microgel) may be administered to a subject in conjunction with a stem and/or progenitor cell mobilization agent. In particular embodiments, the subject is a human with advanced age, for example, the human may be over 30, 40, 50, 60, 70, or 80 years old. The stem and/or progenitor cell mobilization agent may mobilize the subject’s own stem and/or progenitor cells out of the bone marrow so these cells can home to the composition of the present disclosure, thereby enhancing generation of T cells in the subject without involving other conditioning or stem cell transplant.

In certain embodiments, the composition of the present disclosure (e.g., a bone marrow microgel) may be administered to a subject in conjunction with stem and/or progenitor cell mobilization techniques and stem cell transplantation. The transplantation may be autologous, allogeneic, or xenogeneic.

In some embodiments, a therapeutically-effective amount of one, or more cell mobilization agents that can stimulate mobilization into the peripheral bloodstream, production and/or improve function of one or more cell types is administered. The agent(s) could be given through any desired route of administration, including orally, rectally, intravenously, intramuscularly, subcutaneously, or an aerosol. Some non-limiting embodiments of an agent that can stimulate mobilization into the peripheral bloodstream, production of and/or improve function of a cell type include IL-1, IL-2, IL-3, IL-6, GM-CSF, G-CSF, plerixafor, PDGF, TGF-beta, NGF, IGFs, growth hormone, erythropoietin, thrombopoietin, and the like. In addition to naturally occurring growth factors, growth factor analogs and growth factor derivatives such as fusion proteins can be used as well. In some embodiments, the method involves administration of a therapeutically-effective amount of G- CSF and a therapeutically-effective amount of electromagnetic radiation. In some embodiments, the method comprises administering a combination of a therapeutically- effective amount of plerixafor and a therapeutically-effective amount of electromagnetic radiation. In some embodiments, a therapeutically-effective amount of electromagnetic radiation is combined with another agent that, in some embodiments, could be a hematopoietic stem cell mobilizer. In some embodiments, a therapeutically-effective amount of electromagnetic radiation is combined with combinations of two or more of G-CSF, GM- CSF, plerixafor, IL-1, IL-2, IL-3, IL-6, PDGF, TGF-beta, NGF, IGFs, growth hormone, erythropoietin, thrombopoietin or another agent.

In another aspect, the present disclosure provides methods that lead to a balanced reconstitution of T cells in a subject by administering to the subject one or more compositions of the present disclosure. The term “balanced reconstitution of T cells,” as used herein, refers to the reconstitution of T cells that is characterized by CD4⁺: CD8⁺ ratio in a normal range within a certain period of time, such as 30 days. For example, the reconstitution of CD4⁺ cells is usually delayed in a HSCT recipient. The methods of the present disclosure may accelerate the reconstitution of CD4⁺ T cells and lead to a balanced reconstitution of T cells.

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for multiple disorders, but allogeneic HSCT is limited by deficiency and dysregulation of T-cells. In a subject that receives allogeneic HSCT, CD4⁺ T-cell recovery is usually delayed, leading to an inversion of the normal CD4/CD8 ratio, which is about 0.9 to about 2.5 in periphery blood. The ratio may be different in other tissue or organ.

In certain embodiments, the methods of the present disclosure stabilize the CD4⁺ : CD8⁺ ratio to a normal range, while CD4⁺ T-cell compartment in a subject receiving HSCT only has not fully reconstituted. In certain embodiments, a balanced T cell reconstitution is characterized by homeostatic CD4⁺ : CD8⁺ ratio in a normal range in 30 days or less after the transplantation of the hematopoietic stem cells and the administration of the composition of the present disclosure.

In certain embodiments according to the present disclosure, a subject, such as a human, receives between about 1 x 10⁵ and about 50 x 10⁶ hematopoietic stem cells or progenitor cells per kilogram of the subject’s weight in a hematopoietic stem cell transplantation. In certain embodiments, the subject receives about 1 x 10⁵ hematopoietic stem cells per kilogram of the subject’s weight.

The methods of the present disclosure result in similar or better curative and/or therapeutic effects when compared to a subject that receives hematopoietic stem cell or T-cell progenitor infusion alone (z.e., without receiving the treatment of the compositions of the present disclosure). For example, treatment with the compositions of the present disclosure may result in a higher number of T-cell progenitors and functional T-cells in the thymus and the periphery, for example, even when used with a lower dose relative to T-cell progenitor infusion alone. In some embodiments, similar or better curative and/or therapeutic effects can be achieved when less than ten percent (10%) of hematopoietic stem cells or progenitor cells used in a HSCT alone are administered to a subject in combination with the compositions of the present disclosure.

In various embodiments, the balanced reconstitution of T-cells is also characterized by enhanced T-cell neogenesis. The term “neogenesis,” as used herein, refers to the generation of new cells. In various embodiments, the enhanced T-cell neogenesis is characterized by enhanced T-cell receptor excision circles (TRECs). In certain embodiments, T-cell neogenesis using the compositions and methods of the present disclosure achieves a baseline or normal number of TRECs. The term “baseline number of TRECs,” as used herein, refers to the subject’s TRECs number before the subject receives any treatment that impairs the subject’s immune system. The term “normal number of TRECs,” as used herein, refers to the number of TRECs of an individual with uncompromised immune system. The normal number of TRECs may be within certain range. In certain embodiments, TRECs may be assessed in a quantitative and noninvasive fashion in human by estimating TRECs in peripheral blood cells.

V Kits

The disclosure includes various kits which comprise a microgel, e.g., a bone marrow microgel, of the disclosure.

Although exemplary kits are described below, the contents of other useful kits will be apparent to the skilled artisan in light of the present disclosure. Each of these kits is included within the disclosure.

In some embodiments, the kit further comprises an applicator useful for administering the microgel, e.g., bone marrow microgel. The particular applicator included in the kit will depend on, e.g., the method used to administer the microgel, e.g., bone marrow microgel, and such applicators are well-known in the art and may include, among other things, a pipette, a syringe, a dropper, a needle, and the like.

Moreover, in some embodiments the kit further comprises an instructional material which describe the use of the kit to perform the methods described herein. These instructions simply embody the disclosure provided herein.

In some embodiments, the kit includes a pharmaceutically-acceptable carrier. The composition is provided in an appropriate amount as set forth elsewhere herein. Further, the route of administration and the frequency of administration are as previously set forth elsewhere herein.

The kit may further encompass an additional agent comprising a wide plethora of molecules, such as, but not limited to, the active agents as set forth elsewhere herein. However, the skilled artisan armed with the teachings provided herein, would readily appreciate that the disclosure is in no way limited to these, or any other, combination of molecules. Rather, the combinations set forth herein are for illustrative purposes and they in no way limit the combinations encompassed by the present disclosure. EXAMPLES

Example 1. Characterization of Bone Marrow Microgel Scaffolds

Developing artificial hematopoietic tissue may enable one to strengthen the host immune system after hematopoietic stem cell transplantation. To evaluate the ability of biomaterial scaffolds to provide controlled release of morphogens and activate recruited cells to enable the ectopic formation of bone marrowlike structures, injectable, sacrificial microgel scaffolds were designed to offer space for both osteogenesis and hematopoietic cell infiltration.

The microgels were designed to have different levels of degradation, and evaluated for differences in volume, ossification, and hematopoietic compartment across the microgel scaffolds. BMP-2 dosage was also evaluated using the microgels. The study design is shown in FIGs. 1A-1B

Microgel scaffolds were prepared using water-in-oil emulsions as template and BMP- 2 was used as a morphogen. Non-degradable microgels were prepared from norbomene- alginate and tetrazine-alginate. Fast-degradable microgels were prepared from gelatin. Slow- degradable microgels were prepared from oxidized alginate. Microgel scaffolds were subcutaneously injected. Mice underwent weekly ultrasonography imaging for volume quantification and were euthanized on Day 28 to harvest microgel scaffolds for bone and hematopoietic compartment, total cell infiltration, the number and population of hematopoietic cells quantification.

The 1.8% Alg-Tz, 1.2% Alg-Nb with TCO solution, and HFE7500 with 1% surfactant was injected separately into the microfluidic chip as shown at FIG. 1C. Collected emulsions were washed with PBS after one day. Microgels were incubated with DLL4-Nb for 1 day. To check DLL4-Nb conjugation onto microgels, biotinylated anti-mouse DLL4 antibody and streptavidin-Dylight 488were added sequentially and the DLL4 presence under Confocal microscope was observed as shown at FIG. ID. FIG. 1C shows an illustration of the microfluidic chip design and focused view of the junction which generates emulsions. FIG. ID shows a photograph and confocal images of emulsions in oil and microgels in PBS, respectively (top), and confocal and cyoSEM images of DLL4 conjugated click microgels and hydrogel network of click microgels, respectively (bottom). FIG. IE shows the microgels size distribution.

The microgels were incubated with BMP-2 for 3 hours at 4°C and were moved to a 37°C incubator to start the release study. The released BMP -2 was collected from supernatant of microgels at different time point and was quantified with ELISA as shown at FIGs 2A-2B. For tracking the volumetric changes over time in vivo, a high frequency ultrasound probe with motor was used and a series of 2D images collected. The microgel volume and bone volumes from the 2D stacked images was analyzed, and the percentage area per ROI was measured using imaged. FIGs. 2A-2B show release kinetics of the adsorbed BMP-2 in mass (FIG. 2A) and in percentage (FIG. 2B) from non-degradable microgels (ND) and degradable microgels (SD).

For histological analysis, the microgel scaffolds were harvested on week 4 and fixed with 4% PFA and incubated with 70% ethanol. The microgel scaffolds underwent paraffin embedding and were sectioned in 5 pm thickness for histological staining process.

FIG. 3A shows a time series of ultrasound images of ectopic ossification within the microgel scaffolds. FIG. 3B shows total volume of the microgel scaffolds including bone and microgels in vivo as a function of time. FIG. 3C shows the ratio of bone volume to total volume on Day 21 and Day 28.

FIGs. 5A-5C shows histological Safranin O stained sections of the microgels scaffold (red color) with ossification (green color) and hematopoietic tissue (purple color) at Day 28 (FIGs. 5A-5B) and quantification of the hematopoietic tissue, bone, microgels % area per ROI within these sections (FIG. 5C).

FIG. 6A shows ectopic ossification within the microgel scaffolds with different degradation property at Day 28 recorded by high frequency ultrasound. FIG. 6B shows the volumetric change of the microgel scaffolds in vivo as a function of time by ultrasonography imaging. FIG. 6C shows one volume (left) and the ratio of bone volume to total volume (right) at Day 28.

FIG. 10A shows volume of gel and calcified region within microgel scaffolds with different BMP -2 dose measured by microCT. FIG. 10B shows the ratio of bone volume to total volume at microgels scaffolds with different BMP -2 dose.

FIGs. 11A-11B, FIGs. 12A-12B, and FIG. 13 show the quantification of infiltrated cells, specifically total cell number, hematopoietic cell number, and population of hematopoietic cells in the microgel scaffolds across the different BMP-2 dosages at Week 5.

FIGs. 11A-11B shows total cell (FIG. 11 A) and lineage negative cell (FIG. 11B) from microgels scaffold with different BMP -2 dose at days 28. FIGs. 12A-12B shows LKS (Lin, c-kit, Seal) cell (FIG. 12A) and hematopoietic cell (CD45.2 expressed) (FIG. 12B) from microgels scaffold with different BMP -2 dose at Day 28.

FIG. 13 shows hematopoietic cell population including myeloid cells, B cells, and T cells from microgels scaffold with different BMP-2 dose at Day 28.

Overall, these data demonstrate that microgel scaffolds containing biodegradable components led to more ossification and a higher fraction of hematopoietic compartment than non-degradable microgel scaffolds. This injectable sacrificial microgel scaffolds, desderibed herein, thus offer an efficient and scalable approach to create hematopoietic tissue.

Materials

UP MVG sodium alginate was purchased from ProNova; Gelatin from porcine skin, 1H,1H,2H,2H-Perfluoro-1 -octanol (PFO), TWEEN 20, Sodium chloride (NaCl), Hydrochloric acid (HC1), Calcium chloride (CaC12), Ethylenediaminetetraacetic acid tetrasodium salt dihydrate(EDTA), FITC-BSA, were purchased from Sigma Aldrich; HFE 7500 engineered fluid was purchased from 3M Novec; 5 weight % 008 FluoroSurfactant in HFE7500 was purchased from RAN biotechnologies; Polyethylene Tubing and 26G PrecisionGlide Needle were purchased from BD biosciences; D-PBS, Dulbecco’s Modified Eagle Medium (DMEM) were purchased from Thermo Fisher; Cy3 trans-cyclooctene(Cy3 TCO) was purchased from AAT Bioquest; Fetal Bovine Serum, Recombinant Human/Mouse/Rat BMP -2 Protein and Human BMP-2 DuoSet ELISA were purchased from RnD systems; Hematoxylin and Eosin kit was purchased from Abeam, Method

Bone marrow microgels were made from PDMS-based microfluidics which have two aqueous inlets, one oil inlet, and one outlet. Norbomene modified alginate (Alg-Nb) and Tetrazine modified alginate (Alg-Tz) was dissolved in a deionized water (1.2% wt/vol and 1.8% wt/vol, respectively) and used as dispersed prepolymer aqueous phase in the microfluidics. 1% (wt/vol) fluorosurfactant in HFE7500 solution was prepared and used as a continuous oil phase in the microfluidics. Alg-Nb and Alg-Tz solutions were injected at 50 pl/hour and the continuous oil phase was injected at 200 pl/hour. Alg-Nb and Alg-Tz solutions were form emulsion when they encounter oil phase at the junction and form emul si on-templ ated microgels. The emulsion was collected in a tube and left at room temperature for overnight to allow covalent crosslinking between alginate polymers. After that, the emulsion go through demulsification and washing process by adding 40% vol/vol 1H,1H,2H,2H-Perfluoro-1 -octanol (PFO) solution, 0.5% vol/vol Tween 20 solution, and 0.8% wt/vol sodium chloride solution, sequentially. The microgels were redispersed in saline solution and stored at 4°C until further use. BMP -2 was added to the microgels and incubated at 4°C for 3-hours right before in vivo experiment.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS What is claimed is:

1. A microgel, comprising:

(i) a non-degradable component; and/or

(ii) a degradable component, wherein the microgel is spherical in form and is characterized by a diameter of about 10 pm to about 100 pm.

2. The microgel, comprising both a non-degradable component and a degradable component.

3. The microgel of claim 1 or 2, wherein the non-degradable component comprises a first polymer and a second polymer.

4. The microgel of any one of claims 1-3, wherein the non-degradable component comprises a third polymer.

5. The microgel of claim 3 or 4, wherein the first polymer, the second polymer, and the third polymer are independently selected from the group consisting of alginate, methacrylate alginate, chitosan, polyethylene glycol (PEG), gelatin, hyaluronic acid, collagen, chondroitin, agarose, polyacrylamide, heparin, derivatives thereof, and combinations thereof.

6. The microgel of any one of claims 3-5, wherein the first polymer and the second polymer are the same polymer.

7. The microgel of any one of claims 3-6, wherein the first polymer and the second polymer are independently an alginate, optionally wherein the first polymer and the second polymer independently comprise a modified alginate polymer, optionally wherein the first polymer and the second polymer independently comprise oxidized alginate, optionally wherein the first polymer and the second polymer are independently comprise methacrylate alginate, optionally wherein the first polymer and the second polymer independently comprise a click reagent.

8. The microgel of any one of claims 3-7, wherein the first polymer and the second polymer independently comprise a modified polymer.

9. The microgel of any one of claims 3-8, wherein the first polymer and the second polymer independently comprise a click reagent.

10. The microgel of claim 9, wherein the click reagent is selected from the group consisting of azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine (Tz), norbomene (Nb), and variants thereof.

11. The microgel of claim 10, wherein the first polymer comprises a tetrazine (Tz) moiety.

12. The microgel of claim 11, wherein the first polymer comprises tetrazine modified alginate (Alg-Tz).

13. The microgel of any one of claims 10-12, wherein the second polymer comprises a norbomene (Nb) moiety.

14. The microgel of claim 13, wherein the second polymer comprises norbornene modified alginate (Alg-Nb).

15. The microgel of any one of claims 9-14, wherein the first polymer comprises tetrazine modified alginate (Alg-Tz) and the second polymer comprises norbornene modified alginate (Alg-Nb), optionally wherein:

(i) the microgel is about 1% to about 90% covalently crosslinked;

(ii) the microgel comprises a ratio of Alg-Tz: Alg-Nb of 1 : 1, 1 :3, or 3: 1; and/or

(iii) the microgel comprises a ratio of norbomene (Nb)/tetrazine (Tz) of about 0.1 to about 10.

16. The microgel of any one of claims 4-15, wherein the third polymer comprises a modified polymer, optionally wherein the third polymer comprises an oxidized polymer, optionally wherein the oxidized polymer is about 0.1% to about 99% oxidized, optionally wherein the oxidized polymer is about 1% to about 10% oxidized, optionally wherein the oxidized polymer is about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% oxidized.

17. The microgel of claim 16, wherein the third polymer comprises oxidized alginate.

18. The microgel of any one of claims 4-17, wherein the third polymer degrades in vivo within about 1-day to about 30-days after administration to a subject.

19. The microgel of any one of claims 1-18, further comprising an active agent, optionally wherein the active agent is selected from the group consisting of a cell, a biological factor, and/or a small molecule, optionally wherein:

(i) the active agent is present at between about 1 ng to about 1000 pg, optionally wherein the active agent is present at between about 1 ng to about 100 pg, optionally wherein the active agent is present at between about 1 pg to about 2 ng per microgel, optionally wherein the active agent is present at about 1 pg per microgel;

(ii) the active agent comprises a growth factor, optionally wherein the growth factor is selected from the group consisting of a BMP -2, a BMP -4, a BMP-6, a BMP-7, a BMP- 12, a BMP-14, and a combination thereof, optionally wherein the growth factor comprises a BMP- 2, optionally wherein the growth factor is present at between about 2 ng to about 500 ng per microgel;

(iii) the active agent comprises a differentiation factor, optionally wherein the differentiation factor is selected from the group consisting of a Delta-like 1 (DLL-1), a Deltalike 2 (DLL-2), a Delta-like 3 (DLL-3), a Delta-like 3 (DLL-3), a Delta-like 4 (DLL-4), a Jagged 1, a Jagged 2, and a combination thereof, optionally wherein the differentiation factor comprises DLL-4, optionally wherein the differentiation factor is present at an amount at between about 1 ng to about 100 pg per microgel;

(iv) the active agent is covalently and/or non-covalently attached to the microgel, optionally wherein the active agent is covalently attached to the microgel utilizing click chemistry, optionally wherein the active agent is covalently linked to the scaffold utilizing N- hydroxysuccinimide (NHS) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry, NHS and dicyclohexyl carbodiimide (DCC) chemistry, avidin-biotin reaction, azide and dibenzocycloocytne chemistry, tetrazine and transcyclooctene chemistry, tetrazine and norbornene chemistry, or di-sulfide chemistry; and/or

(v) the active agent is released from the microgel within about 1-day to about 30-days after administration to a subject.

20. A scaffold composition, comprising the microgel of any one of claims 1-19.

21. A scaffold composition, comprising: a non-degradable microgel comprising a first polymer and a second polymer, wherein the first polymer and the second polymer independently comprise a click reagent; and/or a degradable microgel comprising a third polymer, wherein the third polymer is configured to degrade in vivo within about 1-day to about 30-days after administration to a subject to form pores for the recruitment of immune cells, wherein the non-degradable microgel and the degradable microgel are spherical in form and independently characterized by a diameter of about 10 pm to about 100 pm.

22. The composition of claim 21, comprising both the non-degradable microgel and the degradable microgel, optionally wherein the non-degradable microgel and the degradable microgel form a three-dimensional scaffold in situ upon administration to a subject, optionally wherein the scaffold comprises pores of a size that permit a eukaryotic cell to traverse into or out of the scaffold, optionally wherein the pores have a diameter of about 1 pm to about 1000 pm.

23. The composition of claim 21 or 22, wherein the first polymer comprises a tetrazine (Tz) moiety.

24. The composition of any one of claims 21-23, wherein the second polymer comprises norbomene modified alginate (Alg-Nb).

25. The composition of any one of claims 21-24, wherein the third polymer comprises oxidized alginate.

26. The composition of any one of claims 21-25, further comprising an active agent, optionally wherein the active agent is selected from the group consisting of a cell, a biological factor, and/or a small molecule, optionally wherein:

27. The composition of any one of claims 21-26, further comprising an aqueous solution, optionally wherein the aqueous solution comprises a saline solution, optionally wherein the aqueous solution comprises phosphate-buffered saline (PBS), optionally wherein the aqueous solution comprises calcium chloride (CaCh) and/or NaCl, optionally wherein the aqueous solution comprises about 2 mM CaCh and/or about 0.8% NaCl, optionally wherein the aqueous solution comprises DMEM media and 10% FBS.

28. A method of preparing a microgel, comprising:

(i) providing a microfluidics chip;

(ii) providing an aqueous phase comprising a first polymer and a second polymer; (iii) providing a continuous oil phase comprising an oil and a surfactant; and

(iv) contacting the aqueous phase with the continuous oil phase in the microfluidics chip to form an emulsion, thereby preparing the microgel.

29. The method of claim 28, wherein the microfluidics chip comprises at least two aqueous inlets, at least one oil inlet, and at least one outlet.

30. The method of claim 28 or 29, wherein the microfluidics chip comprises at least one junction, wherein the junction permits the aqueous phase to contact the continuous oil phase to form an emulsion.

31. The method of any one of claims 28-30, wherein the first polymer and the second polymer are independently selected from the group consisting of a non-degradable polymer, a degradable polymer, and a combination thereof.

32. The method of any one of claims 28-31, wherein the first polymer and the second polymer are independently selected from the group consisting of alginate, chitosan, polyethylene glycol (PEG), gelatin, hyaluronic acid, collagen, chondroitin, agarose, polyacrylamide, heparin, derivatives thereof, and combinations thereof.

33. The method of any one of claims 28-32, wherein the first polymer and the second polymer are the same polymer.

34. The method of any one of claims 28-33, wherein the first polymer and the second polymer are independently an alginate, optionally wherein the first polymer and the second polymer independently comprise a modified alginate polymer, optionally wherein the first polymer and the second polymer independently comprise oxidized alginate, optionally wherein the first polymer and the second polymer are independently comprise methacrylate alginate, optionally wherein the first polymer and the second polymer independently comprise a click reagent.

35. The method of any one of claims 28-34, wherein the first polymer and the second polymer independently comprise a modified polymer.

36. The method of any one of claims 28-35, wherein the first polymer and the second polymer independently comprise an oxidized polymer, optionally wherein the oxidized polymer is about 0.1% to about 99% oxidized, optionally wherein the oxidized polymer is about 1% to about 10% oxidized, optionally wherein the oxidized polymer is about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% oxidized.

37. The method of any one of claims 28-36, wherein the first polymer and the second polymer independently comprise oxidized alginate.

38. The method of any one of claims 28-37, wherein the first polymer and the second polymer independently comprise a click reagent.

39. The method of claim 38, wherein the click reagent is selected from the group consisting of azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine (Tz), norbomene (Nb), and variants thereof.

40. The method of claim 39, wherein the first polymer comprises a tetrazine (Tz) moiety.

41. The method of claim 40, wherein the first polymer comprises tetrazine modified alginate (Alg-Tz).

42. The method of any one of claims 39-41, wherein the second polymer comprises a norbomene (Nb) moiety.

43. The method of claim 42, wherein the second polymer comprises norbornene modified alginate (Alg-Nb).

44. The method of any one of claims 28-43, wherein the first polymer and the second polymer are independently dissolved in deionized water.

45. The method of any one of claims 28-44, wherein the first polymer and the second polymer are independently provided at a concentration of about 0.1% (w/v) to about 10% (w/v).

46. The method of any one of claims 28-45, wherein the first polymer is provided at a concentration of about 0.5% (w/v) to about 1.5% (w/v).

47. The method of any one of claims 28-46, wherein the second polymer is provided at a concentration of about 1.5% (w/v) to about 2.5% (w/v).

48. The method of any one of claims 28-47, wherein the oil comprises mineral oil, silicone, and/or HFE7500.

49. The method of any one of claims 28-48, wherein the surfactant is selected from the group consisting of an amphoteric surfactant, an anionic surfactant, a cationic surfactant, a nonionic surfactant, and a combination thereof, optionally wherein the surfactant comprises fluorosurfactant, optionally wherein the surfactant comprises a nonionic surfactant selected from the group consisting of Brij 93, SPAN 80, AB IL EM90, PGPR, and a combination thereof.

50. The method of any one of claims 28-49, wherein the continuous oil phase comprises about 0.5% (w/v) to about 2% (w/v) fluorosurfactant in HFE7500 solution, optionally wherein the continuous oil phase comprises about 1% (w/v) fluorosurfactant in HFE7500 solution.

51. The method of any one of claims 28-50, further comprising injecting the Alg-Nb and the Alg-Tz into the microfluidics chip, optionally wherein the Alg-Nb and the Alg-Tz are injected at a rate of about 25 pl/hour to about 100 pl/hour, optionally wherein the Alg-Nb and the Alg-Tz are injected at a rate of about 50 pl/hour.

52. The method of any one of claims 28-51, further comprising injecting the continuous oil phase at a rate of about 175 pl/hour to about 500 pl/hour, optionally wherein the continuous oil phase is injected at a rate of about 200 pl/hour.

53. The method of any one of claims 28-52, further comprising allowing the Alg-Nb and the Alg-Tz solutions to form an emulsion when they encounter the continuous oil phase at a junction inside the microfluidics chip, thereby forming an emul si on-templ ated microgel.

54. The method of any one of claims 28-53, further comprising collecting the emulsion.

55. The method of any one of claims 28-54, further comprising maintaining the emulsion at room temperature for at least about 6 hours to about 24 hours to allow covalent crosslinking between the Alg-Nb and the Alg-Tz polymers.

56. The method of any one of claims 28-55, further comprising treating the emulsion with a demulsification and washing process, optionally wherein the treating comprises contacting the emulsion with an about 30-50% (v/v), optionally an about 40% (v/v), 1H,1H,2H,2H- Perfluoro-1 -octanol (PFO) solution, an about 0.1-1 % (v/v), optionally an about 0.5% (v/v) Tween 20 solution, and an about 0.5% (w/v) to about 1.5% (w/v), optionally an about 0.8% (w/v) sodium chloride (NaCl) solution, sequentially.

57. The method of any one of claims 28-56, further comprising isolating the microgel.

58. The method of any one of claims 28-57, further comprising dispersing the microgel in an aqueous solution, optionally wherein the aqueous solution comprises a saline solution, optionally wherein the aqueous solution comprises phosphate-buffered saline (PBS), optionally wherein the aqueous solution comprises calcium chloride (CaCh) and/or NaCl, optionally wherein the aqueous solution comprises about 2 mM CaCh and/or about 0.8% NaCl, optionally wherein the aqueous solution comprises DMEM media and 10% FBS.

59. The method of any one of claims 28-58, further comprising lyophilizing the microgel.

60. The method of any one of claims 28-59, further comprising storing the microgel at about 4°C.

61. The method of any one of claims 28-60, further comprising contacting the microgel with an active agent, optionally wherein the contacting occurs at about 4°C for about 1 hour to about 5 hours, optionally wherein the contacting occurs at about 4°C for about 3 hours.

62. The method of claim 61, wherein the active agent is selected from the group consisting of a cell, a biological factor, and/or a small molecule, optionally wherein:

63. A method for promoting the generation of an ectopic bone marrow niche in a subject in need thereof, comprising administering the microgel of any one of claims 1-19, or the composition of any one of claims 20-27, to the subject, thereby promoting the generation of an ectopic bone marrow niche in the subject.

64. A method for promoting the generation of hematopoietic tissue in a subject in need thereof, comprising administering the microgel of any one of claims 1-19, or the composition of any one of claims 20-27, to the subject, thereby promoting the generation of hematopoietic tissue in the subject.

65. A method for promoting reconstitution of hematopoietic cells in a subject, comprising administering the microgel of any one of claims 1-19, or the composition of any one of claims 20-27, to the subject, thereby promoting reconstitution of hematopoietic cells in the subject.

66. The method of any one of claims 63-65, wherein the microgel forms a three-dimensional scaffold in situ upon administration to the subject, optionally wherein the scaffold comprises pores of a size that permit a eukaryotic cell to traverse into or out of the scaffold, optionally wherein the pores have a diameter of about 1 pm to about 1000 pm.

67. The method of claim 66, wherein the method enhances the recruitment, proliferation, and/or differentiation of immune cells in the scaffold material within about 1-week to about 4-weeks after administration, optionally wherein the immune cells comprise hematopoietic stem cells (HSCs) and/or hematopoietic progenitor cells (HPCs), optionally wherein the immune cells comprise myeloid cells and/or lymphoid cells, optionally wherein the immune cells comprise T cells, B cells, and/or natural killer (NK) cells, optionally wherein the immune cells comprise Lin-c-kit+Sca-l+ (LKS) cells, optionally wherein the immune cells comprise CD45.2+ cells.

68. The method of any one of claims 63-67, wherein the method enhances the recovery of a normal absolute lymphocyte count (ALC) and/or immune cell subsets, optionally comprising neutrophils, monocytes, natural killer cells, T cells, and/or B cells.

69. The method of any one of claims 63-68, wherein the subject is a human.

70. The method of any one of claims 63-69, wherein the microgel is administered to the subject via injection, optionally, intravenously, intramuscularly, or subcutaneously.

71. The method of any one of claims 63-70, wherein the method enhances reconstitution of T cells and/or B cells in the subject.

72. The method of any one of claims 63-71, wherein the method enhances T cell and/or B cell diversity in the subject.

73. The method of claims X, wherein the T cell diversity is characterized by an enhanced T cell receptor (TCR) repertoire and/or wherein the B cell diversity is characterized by an enhanced B cell receptor (BCR) repertoire.

74. A syringe comprising:

(i) a needle;

(ii) a reservoir that comprises the microgel of any one of claims 1-19, or the composition of any one of claims 20-27; and

(iii) a plunger.

75. A kit comprising:

(i) the microgel of any one of claims 1-19, or the composition of any one of claims 20-27; and

(ii) instructions to administer the microgel.