WO2013112491A1

WO2013112491A1 - Tunable hydrogel materials and methods for preparing the same

Info

Publication number: WO2013112491A1
Application number: PCT/US2013/022605
Authority: WO
Inventors: Harald NUHN; Michael HRYNYK; Annelise E. Barron; Keren ZIV
Original assignee: The Board Of Trustees Of The Leland Stanford Junior University
Priority date: 2012-01-23
Filing date: 2013-01-22
Publication date: 2013-08-01
Also published as: WO2013112491A8

Abstract

The disclosure provides hydrogel materials for use in a variety of applications, including cell culturing. The hydrogel materials are controllably crosslinkable, allowing properties of the hydrogel such as elasticity to be tuned as desired. The hydrogel materials contain a protein, a polysaccharide, and a crosslinking material, and can be formed in a mold so as to provide a desired shape.

Description

Tunable Hydrogel Materials and Methods for Preparing the Same

Assignee: The Board of Trustees of the Leland Stanford Junior University

Inventors: Harald Nuhn (Palo Alto, CA], Michael Hrynyk (Kingston, Ontario, Canada], Annelise E. Barron (Palo Alto, CA], and Keren Ziv (Menlo Park, CA]

Cross-Reference to Related Applications

[0001] This application claims priority to US Ser. No. 61/589,784, filed Jan 23, 2012, the disclosure of which is incorporated herein by reference in its entirety.

Introduction

[0002] Hydrogel materials find use in a variety of applications. A common example is the agar hydrogels that are used in electrophoresis and cell culture plates. As the name suggests, hydrogels are water-based materials that contain insoluble, swellable components. The hydrogel structure is generally formed from a hydrophilic polymer that may be nominally water soluble but is rendered insoluble by chemical or physical crosslinking. The crosslinking can occur with or without the use of a separate crosslinking agent. The gelation process varies in speed and complexity depending on the components that form the hydrogel and the environmental conditions in which the hydrogel is formed.

[0003] Given the wide variety of uses for hydrogel materials, improvements in the materials and methods for preparing such materials are scientifically and industrially important.

[0004] Relevant art: US 8,192,760 and Evans et al., European Cells and Materials, Vol. 18, 2009 (pp l-14).

Summary of the Invention

[0005] In some aspects, the disclosure provides a hydrogel composition comprising: (a] a polysaccharide; (b] a non-denatured protein; (c] a crosslinking agent comprising metal ions; and (d] water, wherein the polysaccharide and the protein are physically crosslinked by the metal ions such that the composition forms a hydrogel.

[0006] In embodiments:

[0007] the composition has an elasticity between 1 and 500 kPa; [0008] the polysaccharide is selected from cellulose, hemicellulose, xylan, pectin, alginate, chitin, and hyaluronic acid;

[0009] the protein is selected from elastin, silk, and amyloid precursor protein;

[0010] the metal ions are selected from calcium, barium, magnesium, strontium, nickel, and iron ions;

[0011] the composition does not contain an organic solvent;

[0012] the composition is pliable, and has an elasticity between 1 and 500 kPa;

[0013] the metal ions are present in a predetermined concentration selected such that changes in the concentration of the metal ions causes a change in the elasticity of the composition;

[0014] the protein is non-denatured silk;

[0015] the composition is free-standing;

[0016] the composition further comprises entrapped cells;

[0017] the composition is not immunogenic;

[0018] the composition has long-term physical stability;

[0019] the composition further comprises phosphate ions, and has long-term physical stability over weeks;

[0020] the composition is reversibly crosslinked, wherein a change in the concentration of metal ions causes a change in crosslinked status (i.e., from crosslinked to non-crosslinked, or from non-crosslinked to crosslinked};

[0021] the protein is non-denatured silk and wherein the polysaccharide is alginate;

[0022] the protein is non-denatured silk, and wherein the composition is pliable, and has an elasticity between 1 and 500 kPa;

[0023] the polysaccharide bio degrades in vivo at a rate faster than the

biodegradation rate of the protein;

[0024] the composition further comprises extracellular matrix proteins; and

[0025] the protein is silk and is present in an amount between about 0.1 wt% and 3.5 wt%, and wherein the polysaccharide is alginate and is present in an amount between about 0.1 wt% and 2.0 wt%.

[0026] Furthermore, the invention provides a method for forming the hydrogel composition as above, comprising eroding or dissolving alginate from a precursor hydrogel composition, wherein the precursor hydrogel composition comprises silk, alginate, and the crosslinking agent. [0027] Furthermore, the invention provides a method for forming the hydrogel composition as above, the method comprising combining the protein, the

polysaccharide, and the crosslinking agent in water and in the absence of organic solvents.

[0028] In embodiments:

[0029] the combination forms a physically crosslinked hydrogel, wherein the crosslinking agent provides physical (i.e., non-chemical] crosslinks;

[0030] the combination forms a physical crosslinked hydrogel in less than 60 seconds.

[0031] In another aspect, the disclosure provides a hydrogel composition

comprising: (a] a polysaccharide; (b] a protein, preferably non-denatured; (c] a crosslinking agent comprising metal ions; and (d] water, wherein the composition has an elasticity between 1 and 500 kPa.

[0032] In another aspect, the disclosure provides a hydrogel composition

comprising: (a] alginate; (b] non-denatured silk; and (c] a crosslinking agent comprising metal ions.

[0033] In another aspect, the disclosure provides a hydrogel composition

comprising non-denatured silk and a crosslinking agent, wherein the crosslinking agent comprises metal ions selected from calcium, barium, magnesium, strontium, nickel, and iron.

[0034] In another aspect, the disclosure provides a method for preparing a hydrogel, the method comprising combining a protein, preferably non-denatured, a

polysaccharide, and a crosslinking agent in an aqueous solution and in the absence of organic solvents.

[0035] In another aspect, the disclosure provides a method for preparing a subject hydrogel, the method comprising combining a non-denatured protein, a polysaccharide, and a crosslinking agent in an aqueous solution, wherein the polysaccharide and the protein are physically crosslinked by the metal ions such that the composition forms a hydrogel.

[0036] In another aspect, the disclosure provides a method for culturing cells, the method comprising adding a solution comprising the cells to a subject hydrogel and incubating the cells, wherein the hydrogel comprises a non-denatured protein, a polysaccharide, and a crosslinking agent in an aqueous solution. [0037] These and other aspects will be apparent from the specification, including the examples and claims presented below. The invention includes all combinations of preferred and particular embodiments as though each combination was expressly recited.

Detailed Description of Particular Embodiments

[0038] In some embodiments, the subject hydrogels and hydrogel materials are biocompatible. In some embodiments, the hydrogels are bio-resorbable. In some embodiments, the hydrogels are multi-component materials that comprise a first material comprising a non-denatured protein, a second component comprising a polysaccharide material, and a third component comprising a crosslinking material. In some such embodiments the hydrogels optionally comprise one or more additional materials as described herein. In some embodiments, the hydrogels do not contain an organic solvent. In some such embodiments, the hydrogels do not contain an alcohol solvent such as ethanol or methanol.

Protein

[0039] The protein of the hydrogels has at least a primary structure (i.e., amino acid sequence], and may further comprise a regular secondary structure (i.e., localized substructures such as helix, sheet, etc.], a regular tertiary structure (i.e., the three- dimensional structure of the entire protein], and/or a regular quaternary structure (i.e., an assembly of multiple proteins].

[0040] In some embodiments, at least a portion of the protein component is not denatured prior to formation of the hydrogel, and therefore still retains a regular secondary and regular tertiary structure when the hydrogel is formed. In some such embodiments, the entire protein component is not denatured prior to formation of the hydrogel, and the entire protein component remains non-denatured in the hydrogel. In other such embodiments, a portion of the protein component is denatured prior to or during formation of the hydrogel.

[0041] The protein component of the hydrogels is non-denatured, meaning it comprises non-denatured (i.e. not fully denatured] protein molecules. The protein may comprise molecules having no denatured segments or portions (i.e., are fully non- denatured], and/or molecules with one or more denatured segments (partially non- denatured], and may include molecules that are fully denatured, although most of the protein molecules are not fully denatured. For example, greater than or equal to 75, 80, 85, 90, or 95% of the protein molecules are not fully denatured. Furthermore, in embodiments, greater than or equal to 50, 60, 70, 80, 90, or 95% of the protein molecules have no denatured segments or portions.

[0042] In some embodiments, the overall percentage of non-denatured segments or portions of the protein component (as measured by weight fraction or by number fraction of amino acid monomers] is greater than or equal to 20, 30, 40, 50, 60, 70, 80, or 90%. In embodiments, essentially all of the protein molecules have no denaturation (i.e. no denatured segments or portions].

[0043] In some embodiments, the protein is a silk. The source of the silk is not restricted provided that the silk material forms the hydrogels described herein. Suitable silk materials include silks produced by spiders, worms (e.g., Bombyx mori, etc.], and other insects. In some embodiments modified and synthetic silks, including

recombinant silks, are suitable.

[0044] In some embodiments, the protein is not a silk but is an alternate protein, such as an elastic protein (e.g., elastin], an extracellular matrix protein (e.g., collagen], or a membrane protein (e.g., amyloid precursor protein (APP]]. Such alternative proteins form gels or aggregates due to dehydration.

Polysaccharide

[0045] In some embodiments, the hydrogels include a polysaccharide. Suitable polysaccharides include polymers of L-guluronic acid, D-glucuronic acid, D-galacturonic acid, L-iduronic acid, D-mannuronic acid, L-arabinose, L-rhamnose, L-fucose, D-xylose, and the like, as well as combinations thereof. In some embodiments, the polysaccharide is a block copolymer of such monomers. In some such embodiments, the block copolymer comprises alternating blocks of two or more monomers.

[0046] In some embodiments, the polysaccharide is a naturally-derived material. For example, the polysaccharide is derived from cells walls.

[0047] In some embodiments, the polysaccharide is anionic. In some embodiments the polysaccharide is substituted. Examples of substituents include hydroxyl protecting groups (e.g., alkoxy groups], carboxylic acid protecting groups, amine groups, and the like. In some embodiments, the polysaccharide is in the form of a salt, such as a potassium salt or sodium salt. In other embodiments, the polysaccharide is in the acid (i.e., non-salt] form. It will be appreciated that changes in solution conditions or other environmental factors may cause the polysaccharide to convert between forms.

[0048] In some embodiments, the polysaccharide is selected from cellulose, hemicellulose, xylan, pectin, alginate, chitin, hyaluronic acid, and the like.

[0049] For example, in some embodiments the polysaccharide is alginate, i.e., a copolymer of (l-4]-linked β-D-mannuronate (M-residues] and a-L-guluronate (G- residues]. The relative percentage and relative position of the M-residues and G- residues can be varied as needed in order to tailor the properties of the resulting hydrogel. For example, controlling the G- to M-ratio allows control over various mechanical and morphological properties of the hydrogel, such as stiffness and pore size.

[0050] In some embodiments, the alginate is a block copolymer comprising blocks of G-residues and blocks of M-residues in alternating arrangement. In some embodiments, the alginate is a copolymer with alternating G- and M-residues (i.e., an alternating copolymer]. Combinations of these arrangements are also possible, such as a copolymer comprising homopolymeric G-blocks, homopolymeric M-blocks, and copolymeric MG- blocks.

[0051] Sources of the polysaccharide are typically not critical to the hydrogel, and include natural and synthetic sources as available. For example, alginate may be obtained from seaweed, bacteria, or other sources.

Crosslinking material

[0052] In some embodiments the hydrogels contain a crosslinking material. The crosslinking material is a small molecule or ionic species that aids in the formation of crosslinks within either or both of the polysaccharide and the protein.

[0053] In some embodiments, the crosslinking material is a metal salt. Anions suitable for the crosslinking salt include CI^", Br, I^", sulfate, sulfonate, nitrate, Gluconate, and the like. Cations suitable for the crosslinking salt include calcium, barium, magnesium, strontium, nickel, iron, and the like. Thus, some examples of suitable crosslinking materials included CaCb, CaBr2, calcium gluconate, MgCb, MgBr2, magnesium gluconate, BaCb, BaBr2, barium gluconate, SrCb, SrBr2, strontium gluconate, NiCb, NiBr2, nickel gluconate, FeCb, FeBr2, iron gluconate, and the like. [0054] The crosslinking material is present in the hydrogel in an amount

appropriate to form physical crosslinks between the polysaccharide and the protein, such that the composition forms a hydrogel. In embodiments, the amount of

crosslinking material is equal to or greater than 0.001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 10, or 15 wt% of the hydrogel (i.e., the composition including water}.

Other materials

[0055] In some embodiments, the hydrogels are aqueous solutions and are free of organic solvents. Accordingly, the hydrogel comprises water as the dispersive phase. For example, in some embodiments no organic solvents are added during the preparation of the hydrogels. Examples of organic solvents that are not added and are not present in the hydrogels include alcohols (e.g., methanol, ethanol, isopropanol, etc.], ethers, sulfoxides (e.g., dimethylsulfoxide], amides, amines, and the like.

[0056] In some embodiments, no organic solvents are added during the

manufacturing of the hydrogels. That is, no organic solvents are present in the materials that are combined to form the hydrogels. In such embodiments, no organic solvents are present in the hydrogel thus formed.

[0057] In some embodiments, they hydrogels contain a polysaccharide as described above, a protein as described above, and one or more additives selected from those described below. In some such embodiments, the hydrogels contain no other organic compounds. For example, the hydrogels contain no inert organic substances such as the organic solvents described above.

[0058] In some embodiments, the hydrogels are free of alcohol solvents. In some embodiments, the hydrogels are prepared using solutions that are free of alcohol solvents.

[0059] In some embodiments, the hydrogels contain one or more functional additives to enhance functionality. For example, in some embodiments the hydrogels can include cell-instructive signaling proteins. For example, in some embodiments, the hydrogels include extracellular matrix (ECM] proteins. Examples of ECM proteins include collagen, elastin, laminin, fibronectin, and the like. The hydrogels may comprise, for example, equal to or more than 0.5, 1, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 wt% of ECM (wherein wt% is measured based on the hydrogel composition including water}. [0060] Also for example, the hydrogels can include pharmaceutically active compounds including small molecule drugs, macromolecular pharmaceutical agents, and other bioactive molecules. Examples of such materials include synthetic drugs, naturally-occurring drugs, hormones, therapeutic peptides, growth factors (e.g., VEGF], enzymes, RNAi, RNA, DNA, toxins, etc. Further examples of additives include inorganic components such as salts, transition metal salts, ligands, ions, and the like.

[0061] In some embodiments, image enhancing agents and other diagnostic agents are added to, and present in, the hydrogels. For example, fluorescent tags or compounds having fluorophores are suitable additives to aid characterization. In some

embodiments, RGD Peptide or polylysine are present in the hydrogels.

[0062] Any of the additives mentioned above, as well as others known in the art, may be used to provide or enhance one or more properties of the hydrogels. For example, in some embodiments where the hydrogel is used as a culture scaffold, an ECM protein (e.g., laminin, collagen, etc.] is used to aid cellular attachment and increase cell acceptance.

Methods of Manufacture

[0063] The hydrogels are prepared by combining the precursor materials (i.e., the protein material, the polysaccharide material, the crosslinking material, and any other additives such as those mentioned herein] in an aqueous solution. In some

embodiments the order of combination is not critical. In some embodiments, the protein and polysaccharide are freshly prepared immediately prior to formation of the hydrogel, whereas in other embodiments one or more component may be stored under appropriate conditions. For example, the protein material may be stored at cryogenic temperatures to avoid denaturation prior to hydrogel formation. Exemplary methods for combining and making the hydrogel materials from the precursors are described herein below in the Examples. After blending the components, the polysaccharide and protein are physically crosslinked via the crosslinking material to form the inventive hydrogel material.

[0064] In embodiments, the invention provides a method for forming the hydrogel composition as above, the method comprising combining the protein, the

polysaccharide, and the crosslinking agent in water and in the absence of organic solvents. [0065] In embodiments, the combination forms a physically crosslinked hydrogel, wherein the crosslinking agent provides physical (i.e., non-chemical] crosslinks.

[0066] In embodiments, the combination forms a physical crosslinked hydrogel in less than 60, 30, 10, 5, 3, 2, 1, 0.5, 0.3, or 0.1 seconds. Within the crosslinking time, the material converts from a liquid to a free-standing gel.

[0067] The amounts of materials used to make the hydrogels may vary according to the specific materials and the desired properties of the hydrogel. For example, when silk is used as the protein, in some embodiments the silk is present in an amount between about 0.1 wt% and 3.5 wt%, or between about 0.5 wt% and 2 wt%. For example, the silk is present in less than about 3.5, or less than about 3, or less than about 2.5, or less than about 2.0, or less than about 1.5, or less than about 1.0, or less than about 0.5 wt%. Also for example, the silk is present in greater than about 0.1, or greater than 0.5, or greater than 1.0, or greater than 1.5, or greater than 2.0, or greater than 2.5, or greater than 3.0 wt%. Such wt% values are based on the hydrogel including water. In some

embodiments, the amount of silk is greater than or equal to 1, 5, 10, 15, 20, 25, 30, 40, or 50 wt% wherein such wt% values are based only on the solid components of the hydrogel.

[0068] Also for example, when alginate is used as the polysaccharide, in some embodiments the alginate is present in an amount between about 0.1 wt% and 2.0 wt%, or between about 0.5 wt% and 1.5 wt%. For example, the alginate is present in less than about 2.0, or less than about 1.5, or less than about 1.2, or less than about 1.0, or less than about 0.5 wt%. Also for example, the alginate is present in greater than about 0.1, or greater than 0.5, or greater than 1.0, or greater than 1.5, or greater than 2.0 wt%. Such wt% values are based on the hydrogel including water. In some embodiments, the amount of alginate is greater than or equal to 1, 5, 10, 15, 20, 25, 30, 40, or 50 wt% wherein such wt% values are based only on the solid components of the hydrogel.

[0069] The relative ratio of protein to polysaccharide can also vary depending on the materials and desired properties. Using silk and alginate as an example, the ratio can vary between 1:20 to 35:1 (silk: alginate}. For example, in some embodiments the silk:alginate ratio is between 1:10 and 20:1, or between 1:5 and 10:1, or between 1:5 and 5:1, or between 1:2 and 2:1. In some embodiments the ratio is approximately 1:1, whereas in some embodiments the ratio is greater than 1:1 and in other embodiments the ratio is less than 1:1. [0070] The hydrogels can be formed to take on any suitable shape or configuration. For example, the precursor materials can be placed in a mold with a desired shape, such as disc-shaped, cylinder-shaped, cube-shaped, or the like and then crosslinked to retain the shape of the mold. In some embodiments, because of the rapid onset of gelation for the hydrogels, at least one of the three precursor components (i.e., protein,

polysaccharide, or crosslinking material] is not combined with the other two until just the other two are in position to be crosslinked (e.g., present in a suitable mold, etc.}. In some embodiments, for example, an aqueous solution of the crosslinking material is added to a mold containing an aqueous solution of the protein and polysaccharide. In some such embodiments, gelation of the hydrogel upon addition of the crosslinking material is extremely rapid (as described herein], and in some embodiments is limited only by diffusion kinetics of the crosslinking material through the precursor solution.

[0071] In some embodiments, the crosslinking agent is combined with the protein in a precursor solution. In such embodiments, a solution of the polysaccharide is added to the protein/crosslinking agent solution in order to form the hydrogel. Alternatively, in some embodiments, the polysaccharide and protein are combined into a single solution, and the crosslinking agent is added to the combined solution in order to form the hydrogel. Alternatively, the polysaccharide, protein, and crosslinking agents are prepared in three separate aqueous solutions and combined at once to form the hydrogel.

Uses and properties

[0072] The hydrogels are suitable for a variety of uses, and the examples provided here are not intended to be limiting. In some embodiments, the hydrogels are suitable as a biological scaffold material. For example, the hydrogels are suitable as a scaffold for culturing cells. Cells that can be cultured on the hydrogels include stem cells, such as adipose stem cells, mesenchymal stem cells, endothelial stem cells, hematopoietic stem cells, mammary stem cells, intestinal stem cells, neural stem cells, olfactory stem cells, neural crest stem cells, testicular stem cells, and the like. Cells that can be cultured on the hydrogels also include fibroblasts and epithelial cells. In some embodiments, the hydrogels can be used as an implantable scaffold for the in vivo generation of cells and tissue. In some embodiments, the hydrogels are biocompatible, bioerodible, biodegradable, or a combination thereof. [0073] In some embodiments, the hydrogels are suitable for use as a biocompatible matrix. For example, the hydrogels can be used to prepare a matrix for physically encapsulating biological or pharmaceutical material such as cells (e.g., stem cells], drugs (e.g., active pharmaceutical agents, pharmaceutical compositions], fluorophores, enzymes, proteins, nucleic acids (e.g., DNA, RNA], liposomes, and the like. For example, the hydrogels can be used to encapsulate nucleic acids in micro fluidic devices. In some embodiments as described above, additional components are included (such as ECM proteins, drugs, etc.] and the hydrogels can be used as a tailored material.

[0074] In some embodiments, when used as a scaffold for cell culturing, the hydrogels can be prepared prior to introduction of the cells to be cultured. That is, the cells can be cultivated on top of the pre-formed hydrogel. Alternatively, or in addition, cells to be cultivated can be mixed with the hydrogel starting materials prior to crosslinking. Subsequent crosslinking then entraps the cells within the hydrogel network. Thus, in embodiments, the disclosure provides a hydrogel with entrapped cells (e.g., bone, blood, muscle, nerve, or other cells].

[0075] The hydrogels have a number of desirable properties that cause them to be particularly suited for one or more of the applications . For example, the blending of materials to form the inventive hydrogels results in a self-supporting, biocompatible hydrogel, which forms with ultrafast crosslinking kinetics, and shows excellent stability over time in a variety of buffer conditions.

[0076] For example, in some embodiments the hydrogels exhibit rapid crosslinking. For example, upon combination of the protein, polysaccharide, and crosslinking agent, the hydrogel forms a crosslinked gel in less than 30 sec, or less than 20 sec, or less than 15 sec, or less than 10 sec, or less than 8 sec, or less than 5 sec, or less than 3 sec, or less than 2 sec, or less than 1 sec, or less than 0.5 sec, or less than 0.1 sec. The crosslinking time is measured from the time that the three components (protein, saccharide, and crosslinking agent] are combined to the time that a gel is formed.

[0077] Furthermore, the hydrogel may be destabilized as desired by the removal or dilution of the crosslinking agent, or by enzymatic degradation. Entrapped materials or material supported by the hydrogel scaffold can be released via such degradation or destabilization. This allows, for example, controlled release of pharmaceutical agents, controlled release of cultured cells, and other controlled release applications. [0078] Gelation time can be modified by adjusting the ion concentration present in the crosslinking solution. A higher ion concentration results in faster crosslinking. To maintain a desired stiffness of the gel, but to increase gelation time, the crosslinking time can be increased using an ion-chelating system, which releases ions over time, such as calcium gluconate (CG]. CG releases calcium ions over time, and the release kinetics are pH dependent.

[0079] In some embodiments, another desirable property exhibited by the hydrogels is long-term stability. For example, in some embodiments the hydrogels are stable in vivo for greater than 1 day, or greater than 3 days, or greater than 1 week, or greater than 2 weeks, or greater than 1 month. In some embodiments the hydrogels are stable ex vivo (e.g., at room temperature in water buffer] for greater than 1 day, or greater than 3 days, or greater than 1 week, or greater than 2 weeks, or greater than 1 month, or greater than 2 months. By "stable" in this context is meant that the hydrogel decreases in mass or volume by no more than 40%, or no more than 35%, or no more than 30%, or no more than 25%, or no more than 20%, or no more than 15%, or no more than 10%, or no more than 5% over the specified time. Alternatively, by "stable" is meant that the hydrogel decreases in elasticity (as measured by Young's modulus] by no more than 50%, or nor more than 40%, or no more than 30%, or no more than 20%, or no more than 15%, or no more than 10%, or no more than 5% over the specified time. For example, in some embodiments, the hydrogels suffer a decrease in elasticity of no more than 50% over 10 days in vivo. Alternatively, by "stable" is meant that the hydrogels substantially retain their original shape over the specified time. Such long-term stability is achieved using the ionic-based gelation chemistry described herein rather than irreversible chemical crosslinking methods. The stability is achieved despite the presence, in embodiments, of ions (e.g., phosphate ions], divalent ions, and/or acids.

[0080] Without wishing to be bound by theory, in some embodiments it is believed that the protein and polysaccharide are present in separate domains in the hydrogels . As an analogy, the two materials phase separate, with overlap between the phases ranging from extensive to moderate to minimal.

[0081] In some embodiments, when the hydrogel is implanted in vivo, the polysaccharide material biodegrades or bioerodes and is removed from the hydrogel at a higher rate than the protein material. Accordingly, the composition of the hydrogel changes over time, with the amount of protein increasing relative to the amount of polysaccharide. In some such embodiments, after a period of time in vivo, the hydrogel comprises mostly or entirely protein with little or no polysaccharide remaining (e.g. greater than 75% protein, or greater than 80% protein, or greater than 85% protein, or greater than 90% protein, or greater than 95% protein, or greater than 98% protein, or greater than 99% protein, or greater than 99.9% protein as measured by weight}. Nevertheless, in some such embodiments, the hydrogel remains stable as defined herein with respect to elasticity and shape. It will be appreciated that the time required to reach a hydrogel containing substantially no polysaccharide will vary depending, for example, on the relative amounts of materials in the starting hydrogel as well as the conditions in which the hydrogel are place.

[0082] Another desirable properly is that, in some embodiments, the hydrogels have tunable mechanical properties over a wide range. For example, the hydrogels have an elasticity that can range between 1-500 kPa, or between 2-400 kPa, or between 2- 200 kPa, or between 2-100 kPa. For example, the hydrogels have an elasticity equal to or more than 1, 2, 3, 4, 5, 10, 25, 50, 100, 200, 300, or 400 kPa. Tuning the elasticity of the hydrogel allows the hydrogels to provide an ideal environment, for example, for cultured cells. In some embodiments, for example, the hydrogel can be prepared having an elasticity to mimic heart tissue at 18-19 kPa, or to mimic muscle tissue at approximately 12 kPa.

[0083] Another desirable properly is that, in some embodiments, no organic solvents are involved in the preparation of the hydrogels . This reduces the amount of organic solvent waste and also eliminates toxicity concerns associated with organic solvents. Furthermore, in some embodiments, the hydrogels are not immunogenic.

[0084] In some embodiments, as mentioned herein, the elasticity and other properties of the hydrogels are variable based on the concentration of crosslinking agent present. In some embodiments where alginate (or a derivative thereof) is the polysaccharide, another factor that affects such properties is the presence and concentration of phosphate ion, which tends to increase stiffness in the alginate material. Increased phosphate ion concentration relative to crosslinking agent causes a corresponding increase in the stiffness of the hydrogel. Thus, in such embodiments the stiffness of the hydrogel can be tuned by adjusting the relative concentration of crosslinking agent and phosphate ion. [0085] In some embodiments, it is important to be able to adjust the stiffness and other properties of the gels in order to guide stem cell differentiation during

development into a desired linage (similarly to, e.g., heart tissue stiffening during pre and post natal development}. See, e.g., Evans et al., "Substrate Stiffness Affects Early Differentiation Events in Embryonic Stem Cells," European Cells and Materials, Vol. 18 2009 (pp. 1-14], the contents of which are incorporated herein by reference.

[0086] In some embodiments, the hydrogel can be designed to maintain elasticity over time. For example, the hydrogel can incorporate a biocompatible, degradable polymer (e.g., poly(lactic-co-glycolic acid] (PLGA], ε-caprolactone, etc.] that entraps crosslinking agent and releases the crosslinking agent over time.

[0087] In one example, silk fibroin is selected as the protein and is blended with alginate (as the polysaccharide component] since both materials share a common feature during gelation - local dehydration. Alginate gels are stabilized due to the chelation of ions by several /-guluronic (G] acid blocks of different molecules. Thereby G-blocks form the so called egg-box domain, being dehydrated. Silk fibroin shows hydrophobic domains, when dehydrated assemble into stable β-sheets, known to stabilize a silk fibroin formed gel or solid. In contrast to alginates ability to gel immediately, silk fibroin gelation occurs over hours to days. However, the silk-alginate blends form self-supporting gels in milliseconds to minutes. Without wishing to be bound by theory, it is suspected that, during gelation of the blend, local dehydration of the alginate immediately induces dehydration of entrapped silk fibroin blocks, significantly accelerating the crosslinking process of the silk fibroin.

[0088] 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 this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are described herein. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0089] It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0090] The term "typically" is used to indicate common practices of the invention. The term indicates that such disclosure is exemplary, although (unless otherwise indicated] not necessary, for the materials and methods of the invention. Thus, the term "typically" should be interpreted as "typically, although not necessarily." Similarly, the term "optionally," as in a material or component that is optionally present, indicates that the invention includes instances wherein the material or component is present, and also includes instances wherein the material or component is not present.

[0091] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties. However, where a patent, patent application, or publication containing express definitions is incorporated by reference, those express definitions should be understood to apply to the incorporated patent, patent application, or publication in which they are found, and not to the remainder of the text of this application, in particular the claims of this application.

[0092] It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the foregoing description and the examples that follow are intended to illustrate and not limit the scope of the invention. It will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention, and further that other aspects, advantages and modifications will be apparent to those skilled in the art to which the invention pertains.

[0093] Example 1: Hydrogel Preparation

[0094] Materials: Freshly prepared Bombyx mori silkworm silk solutions (7.4 - 7.8 w/v% in distilled water] were kindly provided by D. Kaplan (Tufts University] and if not otherwise indicated, used without further treatment. Alginate (PROTANAL

LF10/60LS] was kindly provided by FMC Biopolymers and used without further treatment. Mouse laminin was obtained from Roche and used as indicated by the manufacturer. Lyophilized Collagen I, bovine was obtained from MD Bioproducts (St. Paul, MN]. Fibronectin, Calcium chloride, Bis(2-hydroxyethyl]amino- tris(hydroxymethyl] methane (Bis-tris; BioReagent], were obtained from Sigma-Aldrich. Dialysis tubing (50.000 MWCO] was purchased from Spectra/Por (Houston, TX].

[0095] Solutions and storage: Alginate solutions were prepared freshly at 4 °C, stored at 4 °C and used within 24 h to prevent degradation. Silk solutions were aliquoted and stored at -80 °C to prevent degradation. Frozen samples were allowed to return to RT without heating and used immediately. Unused silk was discarded.

[0096] Hydrogel precursor formation: A hydrogel precursor mixture was prepared consisting of alginate (4 % stock in distilled water, or alternatively prepared in a compatible buffer] and silk (7.4 - 7.8 w/v% solution in distilled water, or alternatively prepared in a compatible buffer]. To enhance cell compatibility, and depending on the experiment, laminin (0.5 mg/ml as received], fibronectin (0.5 mg/ml] or collagen I (1 mg/ml], respectively, were added in the stated concentration. Concentrations were adjusted using distilled water or the selected buffer. Hydrogel precursor solutions were mixed until they appeared homogeneous.

[0097] Example A - preparation of a precursor solution: Balanced 9.6 g of distilled water. Balanced 0.4 g of alginate. Alginate was added to the water and stirred using a magnetic stir bar at 4 °C. The mixture was stirred until completely dissolved (or overnight]. This resulted in a 4 % (w/w] solution. 30 min prior to gel-precursor preparation, an aliquot of silk was thawed at room temperature.

[0098] Preparing 1 mL of a 1.5 % (w/w] silk and 1 % (w/w] alginate solution: 0.25 mL of the alginate stock solution was added to a reaction vial (1.5 mL]. 0.192 mL of the silk solution (assuming the stock solution has a concentration of 7.8 % (w/w]] was added to the same reaction vial. 0.557 mL of distilled water was added. The solution was pipetted up and down until a homogeneous, greyish, milky solution was obtained. Bubbles were removed by a quick spin using a table-top centrifuge. The solution was stored at 4 °C and used within 24 h.

[0099] Example B - making a precursor solution containing laminin or any other component. Balanced 9.6 g of distilled water. Balanced 0.4 g of alginate. The alginate was added to the water and stirred using a magnetic stir bar at 4 °C. The solutions were stirred until completely dissolved (or overnight.]. This resulted in a 4 % (w/w] solution. 30 min prior to gel-precursor preparation, an aliquot of silk was thawed at room temperature. [00100] Preparing 1 mL of a 1.5 % (w/w} silk and 1 % (w/w} alginate solution: 0.25 mL of the alginate stock solution was added to a reaction vial (1.5 mL}. 0.192 mL of the silk solution (assuming the stock solution has a concentration of 7.8 % (w/w}} was added to the same reaction vial. 0.2 mL of Laminin (stock solution 0.5 mg/mL} was added, followed by 0.357 mL of distilled water. The solution was pipetted up and down until a homogeneous, greyish, milky solution was obtained. Bubbles were removed by a quick spin using a table-top centrifuge. The solution was stored at 4 °C and used within 24 h. By adding a new component, interactions between the added and existing components can change the elasticity of the resulting hydrogel.

[00101] Example 2: Hydrogel Characterization

[00102] Gelation and Variation of Gelation time and gel strength. Gelation of alginate can be induced by, among others, calcium, barium, magnesium, strontium, nickel and iron ions.

[00103] Time stability. Silk alginate gels as prepared in Examples 1 and 2 become softer over time when stored in crosslinking ion free solutions, such as cell media. However, by adding e.g. calcium to a cell culture media in a concentration tolerated by the cells, stiffness can be maintained or altered during cell culture.

[00104] Preparing discs. Bubble-free precursor solution as prepared in Example 1 was injected into a disc caster enabling preparation of 6 disc shaped gel samples in parallel (8 mm diameter, 1.56 mm thickness}. The mold was covered on both sides by a dialysis membrane (50.000 MWCO, Spectra/Por}, allowing calcium ions to enter and induce gelling. Gelation was induced by immersing the mold into a buffered 25 mM CaCb solution (10 mM Bis-tris, 100 mM NaCl, pH 6.5}. Gelation time was in the range of 20-60 min.

[00105] Example C - making a hydrogel disc using the disc caster. The disc caster was rinsed with distilled water and sterilized using 70 % ethanol. The disc caster was dried using a N2 air stream or allowed to air dry. 40 mm of dialysis membrane (50.000

MWCO} was cut along the long side to yield two single membranes. The membranes were rinsed using distilled water. Excess water was removed by shaking. The first membrane was placed into the bottom part of the caster. The insert was placed into the bottom part of the caster. The second membrane was placed on top of the insert, and the top piece on the frame. Clips were used to keep the frame assembled. The hydrogel precursor solution was loaded into a 1 mL syringe, without making bubbles. [00106] Casting. Using a 21G ^χ 1½" needle attached to the syringe, the air and any bubbles were removed. The needle was slowly inserted into the frame via the loading slits until needle tip was in the center of the lowest pocket, but without piercing the membrane. Solution was slowly pushed into the lowest pocket without bubble formation. When the lowest pocket was filled, solution entered the center pocket. The needle was pushed to the center pocket when half filled, but the tip kept within the already injected solution. Precursor solution injection was continued, repeating the last step until all pockets were filled. Added a little extra volume until solution was leaving the loading slit, thus preventing bubble formation in the first pocket. Repeat steps with the remaining discs.

[00107] Making the Gel. The frame was submerged into a CaCb solution of desired concentration. No bubbles were present on the membranes, as such could have preventing the calcium ions from entering the gel cast device. Incubation time varied from 20 minutes to 3 hours, depending on the desired stiffness.

[00108] Gel discs were removed from the device by taking off the clips, shearing off the top piece, and pushing out the center piece using a pipette tip. The membranes were removed, and the gel was pushed out into a solution of: (f) CaCb or (if) cell media or (iif) any other solution.

[00109] Preparing gel films. 100 μΐ of the precursor solution were transferred in the insert of a transwell plate. For gelation, the insert was immersed into a 25 mM CaCb solution (1.5 ml per well}. After gelation, the CaCb solution was replaced by cell media and cell media was changed 3 times prior to cell culture.

[00110] Example 3: Embryonic Stem Cell Survival

[00111] Mouse embryonic stem cells (D3 cells], stably expressing luciferase, were transplanted in the lower back of nude mice with matrigel or the silk:alginate scaffold. Bioluminescence imaging (BLI] was used in order to estimate cell survival in-vivo. Cells were cultured on a silk:alginate scaffold prepared according to Examples 1 and 2, and the entire scaffold was transplanted. At day 1 after transplantation, D3 cells

transplanted with matrigel gave a stronger signal comparing to cells that were transplanted on top of the scaffold. In contrast, five days after transplantation cells transplanted using the silk:alginate scaffold gave a significantly stronger signal compared to cells transplanted using matrigel. [00112] Example 4: The effect of additives on cell culturing

[00113] Silk:alginate hydrogels were prepared according to Examples 1 and 2. D3 cells did not adhere to the silk:alginate scaffolds and formed clusters. Therefore, several ECM components were added to the scaffold in order to induce cells' adhesion (B-E}. The following additives were used: collagen I, RGD, fibronectin, and laminin. D3 cells which express firefly luciferase (Flue] were plated on top of the scaffolds, and cultured for a week. Bioluminescence imaging (BLI} was used in order to estimate cell growth and proliferation. The scaffold supplemented with laminin yielded the best cell adhesion. The scaffold supplemented with Collagen I yielded the least cell adhesion, and was only slightly better than the non-supplemented control. The RGD- and Fibronectin- supplemented scaffolds yielded intermediate adhesion.

[00114] Example 5: Effect of hydrogel composition on stiffness

[00115] Variation of the silk:alginate ratio allows tuning of the scaffold's stiffness. Stiffness values for various tissues are known to vary from fat and marrow (about 0.5- 1.5 kPa} to teeth and bone (about 15000-25000 kPa}. Given this variation, the hydrogel scaffold's elasticity is a potent regulator of stem cells differentiation. Variation of the silk:alginate ratio allowed preparation of hydrogels having a broad range of stiffness values; this was useful in order to recapitulate biophysical features of the stem cell niche. Stiffness (elasticity] values ranged from 6 kPa (for a silk:alginate of 2:2} up to 49 kPa (silk:alginate of 7:4}. Additional values included 10 (silk: alginate of 3:2 and also 4:2}, 18 (silk:alginate of 4.66:2.66}, 22 (silk: alginate of 2:3}, and 26 kPa (silk:alginate of 2:4}.

[00116] Example 6: Hydrogel characterization

[00117] Crosslinking of the silk:alginate blend in preparation of a hydrogel as described in Examples 1 and 2 above resulted in β-sheet formation of the silk. A brightfield image was recorded for a Congo red stained, crosslinked silk:algnate gel (ratio 3.5:2}. A second image of the same sample was viewed under polarized light. The observed birefringence indicates the presence of β-sheet. It is believed that, due to sol- gel transition, the secondary structure of the silk gets rearranged towards β-sheets.

[00118] Example 7: Hydrogel characterization [00119] Hydrogel scaffolds were prepared according to Examples 1 and 2 above. Scaffold porosity was measured by variable pressure SEM. Images were taken from a freshly prepared sample. Structural changes and porosity of the scaffold were determined from the images. Due to the structural changes over time, it is believed that the ribbon-like structures are composed of silk. Such structures remain in place after one week of incubation. In contrast, the small porous spaces between the ribbon-like structures represent the alginate, which diffuses out over time.

[00120] Scaffold pore sizes were also evaluated using E-SEM. Analysis of the pore size was done using the evaporated sample. 86% of the pores had an average size under 1 μιη. The silk:alginate scaffold has small pore size, allowing it to provide good support to the transplanted cells and also allowing nutrient diffusion.

[00121] Example 8: Variations in Elasticity

[00122] Hydrogel scaffolds prepared according to the procedure provided in

Examples 1 and 2. Scaffolds were incubated in 95% FBS and 95% FBS with 25mM CaCb for 20-360 min. Rheology measurements were taken at various times during the incubation and following incubation in order to test the scaffold's elasticity. In the absence of calcium ions, the Young's modulus declined by 66% (18 kPa to 6 kPa] within the first 20 minutes, reaching 3.6 kPa after 6 hours. In the presence of 25 mM CaCb, the Young's modulus increased from 15 kPa to 25 kPa. Thus, it is possible to adjust the scaffold elasticity prior to or during cell culture as desired.

[00123] Example 9: In vivo Stability of Hydrogels

[00124] Fluorescent scaffolds were transplanted in mice with a window chamber. Intravital microscopy (I VM] was used to track the scaffold's degradation for 10 days following transplantation. Images were taken from such measurements on days 3, 7, and 10. Elasticity of the hydrogel scaffold was measured prior to implantation. Ten days after transplantation, the scaffold was retrieved from the mouse and elasticity was again measured. The scaffold elasticity decreased by 2/3, but the scaffold maintained its shape after 10 days in vivo.

[00125] Example 10: Hydrogel Characterization

[00126] Different tissues have different stiffness, therefore the scaffold's elasticity is a potent regulator of stem cells differentiation. The stiffnesses of several hydrogels having different silk:alginate ratios were examined in order to recapitulate biophysical niche features. By varying the silk:alginate ratio, the scaffold elasticity was tuned. For example, a ratio of 2:3 produces the approximate elasticity of heart tissue. The following Young's Moduli (in kPa} were observed (silk: alginate ratios in parentheses}: 23 (0:2}; 16 (0:4}; 26 (2:4}; 25 (4:4}; 49 (7:4}; 22 (2:3}.

[00127] Example 11 : Modulation of Elasticity

[00128] Tissue stiffness varies with age or during development. Therefore, it is important to be able to adjust the stiffness of a scaffold during stem cell differentiation to guide their development into the desired linage. The ability to modulate the stiffness of the scaffold as function of cross linker concentration (25 mM or 0 mM} was shown by experiment, with the data provided in the table below. The gels elasticity increases or decreases after the addition or removal of a crosslinking agent. Thus, Young's modulus was modified in the presence (+} and absence (-} of CaCb (25 mM} in cell culture medium (DMEM}. Incubation time for the first data point: 60 min (gel formation}. For every other time point, every 30 minutes samples have been immersed in CaCb containing or free cell culture medium.

[00129] Example 12: Stability

[00130] To show stability, a gel prepared according to the disclosure was imaged using SEM directly after preparation as well as 7 days after preparation, The SEM images showed little or no change over the time period.

Claims

What is Claimed is:

\. A hydrogel composition comprising:

(a] a polysaccharide;

(b] a non-denatured protein;

(c] a crosslinking agent comprising metal ions; and

(d] water,

wherein the polysaccharide and the protein are physically crosslinked by the metal ions such that the composition forms a hydrogel.

2. The hydrogel composition of claim 1, wherein the composition has an elasticity between 1 and 500 kPa.

3. The hydrogel composition of claim 1, wherein the polysaccharide is selected from cellulose, hemicellulose, xylan, pectin, alginate, chitin, and hyaluronic acid.

4. The hydrogel composition of claim 1, wherein the protein is selected from elastin, silk, and amyloid precursor protein.

5. The hydrogel composition of claim 1, wherein the metal ions are selected from calcium, barium, magnesium, strontium, nickel, and iron ions.

6. The hydrogel composition of claim 1, wherein the composition does not contain an organic solvent.

7. The hydrogel composition of claim 1, wherein the composition is pliable, and has an elasticity between 1 and 500 kPa.

8. The hydrogel composition claim 1, wherein the metal ions are present in a predetermined concentration selected such that changes in the concentration of the metal ions causes a change in the elasticity of the composition.

9. The hydrogel composition of claim 1, wherein the protein is non-denatured silk.

10. The hydrogel composition of claim 1, wherein the protein is non-denatured silk and wherein the polysaccharide is alginate.

11. The hydrogel composition of claim 1, wherein the protein is non-denatured silk, and wherein the composition is pliable, and has an elasticity between 1 and 500 kPa.

12. The hydrogel composition of claim 1, wherein the polysaccharide biodegrades in vivo at a rate faster than the biodegradation rate of the protein.

13. The hydrogel composition of claim 1, further comprising extracellular matrix proteins.

14. A method for forming the hydrogel composition of claim 1, comprising eroding or dissolving alginate from a precursor hydrogel composition, wherein the precursor hydrogel composition comprises silk, alginate, and the crosslinking agent.

15. A method for forming the hydrogel composition of claim 1, the method comprising combining the protein, the polysaccharide, and the crosslinking agent in water and in the absence of organic solvents.