WO2017175229A1

WO2017175229A1 - Polysaccharide compositions and uses thereof

Info

Publication number: WO2017175229A1
Application number: PCT/IL2017/050423
Authority: WO
Inventors: Andrew Pearlman; Smadar Cohen; Emil RUVINOV
Original assignee: B.G. Negev Technologies And Applications Ltd.
Priority date: 2016-04-07
Filing date: 2017-04-06
Publication date: 2017-10-12
Also published as: WO2017175229A9

Abstract

The present invention provides compositions comprising alginate, sulfated alginate, or hyaluronan sulfate for repairing or regenerating damaged tissue and in particular, joint tissue. The invention also provides a multi-layer hydrogel comprising alginate, sulfated alginate, or hyaluronan sulfate in which one of the layers is affinity-bound to one or more bioactive polypeptides and another one of the layers does not comprise a bioactive polypeptide. The present invention also provides compositions comprising a non-biological additive such as hydroxyapatite, calcium phosphate, mannitol beads, and/or magnesium ions; a biological additive such as platelet rich plasma (PRP) and/or bone marrow aspirate; an interleukin-1 receptor antagonist (IL-1RA), or such composition cross-linked using calcium phosphate as a source of calcium ions.

Description

POLYSACCHARIDE COMPOSITIONS AND USES THEREOF

FIELD OF THE INVENTION

[0001] The present invention provides compositions comprising alginate, sulfated alginate, or hyaluronan sulfate for repairing or regenerating damaged tissue and in particular, joint tissue. The invention also provides a multi-layer hydrogel comprising alginate, sulfated alginate, or hyaluronan sulfate in which one of the layers is affinity-bound to one or more bioactive polypeptides and another one of the layers does not comprise a bioactive polypeptide. The present invention also provides compositions comprising a non-biological additive such as hydroxyapatite, calcium phosphate, mannitol beads, and/or magnesium ions; a biological additive such as platelet rich plasma (PRP) and/or bone marrow aspirate; an interleukin- 1 receptor antagonist (IL-1RA), or such composition cross-linked using calcium phosphate as a source of calcium ions.

BACKGROUND OF THE INVENTION

[0002] Controlled-release dosage forms are designed to reduce drug-dosing frequency and to reduce fluctuation in plasma drug concentration, providing a more uniform therapeutic effect. Less frequent dosing is more convenient and may improve patient compliance. These dosage forms are suitable for drugs that otherwise require frequent dosing because elimination half- life and duration of effect are short.

[0003] Man-made controlled release dosage forms, such as hydrogels and solid polymeric implants or microspheres, usually rely on drug release mechanisms that are based on passive diffusion, polymer degradation or passive diffusion coupled with polymer degradation. Examples of these systems include polyester microspheres or alginate hydrogels.

[0004] On the other hand, nature's way of devising controlled release dosage forms is based on principles of biological specificity. A known example of this is the biomolecular interactions between heparin/heparan sulfate and heparin-binding peptides, e.g. growth factors. These interactions form a depot for growth factor storage in the tissues. Upon tissue injury, the growth factors are released and induce processes associated with wound healing. [0005] For years, researchers have been investigating the use of alginate hydrogels for the controlled delivery of drugs and as scaffolds for tissue engineering. Alginate is a polysaccharide derived from brown seaweed. It is an anionic polysaccharide composed of uronic acids (guluronic (G) and mannuronic (M) acids) that undergoes gelation in the presence of bivalent cations, such as Ca²⁺ and Ba²⁺. In the pharmaceutical/medicinal fields, it is used successfully as encapsulation material, mostly for bacterial, plant and mammalian cells. For molecules, it is much less effective, and even macromolecules in size of 250 kDa are rapidly released from alginate hydrogel systems. In particular, biological molecules of interest such as cytokines, growth factors, with sizes ranging between 5 to 100 kDa, are rapidly released.

[0006] Thus, there is a need for modifications in polysaccharides such as alginate for their use in the controlled delivery of drugs.

[0007] The interleukin- 1 receptor antagonist (IL-1RA) is a member of the interleukin 1 cytokine family. IL-1RA is secreted by various types of cells including immune cells, epithelial cells, and adipocytes, and is a natural inhibitor of the pro-inflammatory effect of ILi . This protein inhibits the activities of interleukin 1 , alpha (ILIA) and interleukin 1 , beta (IL1B), and modulates a variety of interleukin 1 related immune and inflammatory responses.

[0008] IL-1RA is used for treating various diseases and conditions. In humans, IL-1RA is used in the treatment of rheumatoid arthritis, an autoimmune disease in which IL-1 plays a key role, diabetes mellitus, and other auto-immune conditions. In animals (e.g., horses), IL- 1RA is used for the treatment of equine lameness secondary to joint injury.

[0009] Despite its outstanding broad spectrum antiinflammatory effects, IL-IRa has short biological half-life (4—6 h). To date, no efficient polysaccharide based system is available for controlled release of IL-1 RA. SUMMARY OF THE INVENTION

[0010] In one embodiment, the present invention provides a multi-compartment hydrogel or scaffold comprising a polysaccharide selected from the group consisting of: an alginate, a sulfated alginate, and a hyaluronan sulfate, wherein at least one compartment of said multicompartment hydrogel or scaffold does not comprise a polypeptide. [0011] In another embodiment, the present invention provides a method for repairing, regenerating, or preventing additional degeneration of a damaged tissue in a subject, the method comprising the step of administering to said subject any one or more of the compositions described hereinabove, thereby repairing or regenerating said damaged tissue in said subject.

[0012] In another embodiment, the present invention provides a method for treating an osteochondral defect in a subject, the method comprising the step of administering to said subject any one or more of the compositions described hereinabove, thereby treating said osteochondral defect in said subject.

[0013] In another embodiment, the present invention provides a method for treating rheumatoid arthritis in a subject, the method comprising the step of administering to said subject any one or more of the compositions described hereinabove, thereby treating said rheumatoid arthritis in said subject.

[0014] In another embodiment, the present invention provides a method for treating a joint injury or its associated condition in a subject, the method comprising the step of administering to said subject any one or more of the compositions described hereinabove, thereby treating said joint injury or its associated condition in said subject.

[0015] In another embodiment, the present invention provides a composition comprising: a polysaccharide and an additive selected from the group consisting of: hydroxyapatite, calcium phosphate, mannitol beads, and magnesium ions, or a combination thereof, wherein said polysaccharide is a sulfated alginate or hyaluronan sulfate.

[0016] In another embodiment, the present invention provides a composition comprising: a polysaccharide and a biological material from a subject, wherein said polysaccharide is a sulfated alginate or hyaluronan sulfate.

[0017] In another embodiment, the present invention provides a composition comprising polysaccharides cross-linked using calcium phosphate to form a matrix, and wherein said polysaccharide is a sulfated alginate or hyaluronan sulfate.

[0018] In another embodiment, the present invention provides a composition comprising: a polysaccharide, and a polypeptide linked to said polysaccharide, wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate, and said polypeptide is interleukin- 1 receptor antagonist (IL-1RA).

[0019] In another embodiment, the present invention provides a method for repairing or regenerating a damaged tissue in a subject, the method comprising the step of administering to said subject any one or more of the compositions described hereinabove.

[0020] In another embodiment, the present invention provides a method for treating an osteochondral defect in a subject, the method comprising the step of administering to said subject any one or more of the compositions described hereinabove.

[0021] In another embodiment, the present invention provides a method of treating rheumatoid arthritis in a subject, the method comprising the step of administering to said subject any one or more of the compositions described hereinabove.

[0022] In another embodiment, the present invention provides a method for treating a joint injury or its associated condition in a subject, the method comprising the step of administering to said subject any one or more of the compositions described hereinabove.

[0023] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 shows affinity binding of IL-1RA to alginate-sulfate (AlgS). SPR sensorgrams of IL-1RA (6 μΜ) binding to alginate-sulfate, alginate and heparin.

[0025] Figure 2 presents representative images of osteochondral defects in the knees of Sinclair minipigs at 6 months post-injury stained with Toluidine Blue and viewed at xlO magnification (bar=200 μπι). One knee received an alginate/alginate sulfate hydrogel scaffold comprising TGF-beta and BMP-4 in separate compartments, while the contralateral knee received just the alginate/alginate sulfate hydrogel scaffold (w/o growth factors).

-A- [0026] Figure 3 presents representative images of osteochondral defects in the knees of Sinclair minipigs at 6 months post- injury stained with Safranin O and viewed at xlO magnification (bar=200 μπι). One knee received an alginate/alginate sulfate hydrogel scaffold comprising TGF-beta and BMP-4 in separate compartments, while the contralateral knee received just the alginate/alginate sulfate hydrogel scaffold (w/o growth factors).

[0027] Figure 4 presents representative images of osteochondral defects in the knees of Sinclair minipigs at 6 months post- injury stained for Collagen Type II and viewed at xlO magnification (bar=200 μπι). One knee received an alginate/alginate sulfate hydrogel scaffold comprising TGF-beta and BMP-4 in separate compartments, while the contralateral knee received just the alginate/alginate sulfate hydrogel scaffold (w/o growth factors).

DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention provides compositions comprising alginate, sulfated alginate, or hyaluronan sulfate for repairing or regenerating damaged tissue and in particular, joint tissue. The invention also provides a multi-layer hydrogel comprising alginate, sulfated alginate, or hyaluronan sulfate in which one of the layers is affinity-bound to one or more bioactive polypeptides and another one of the layers does not comprise a bioactive polypeptide. The present invention also provides compositions comprising a non-biological additive such as hydroxyapatite, calcium phosphate, mannitol beads, and/or magnesium ions; a biological additive such as platelet rich plasma (PRP), bone marrow aspirate, and/or serum; an interleukin-1 receptor antagonist (IL-I RA), or such composition cross-linked using calcium phosphate as a source of calcium ions.

[0029] In one embodiment, the present invention provides a multi-compartment hydrogel or scaffold comprising a polysaccharide selected from the group consisting of: an alginate, a sulfated alginate, and a hyaluronan sulfate, wherein one compartment of said multi- compartment hydrogel or scaffold does not comprise a polypeptide. In another embodiment, one compartment lacks a polypeptide. In another embodiment, one compartment lacks an exogenous polypeptide. In another embodiment, one compartment lacks a recombinant polypeptide. In another embodiment, more than one layer of the multi-compartment hydrogel or scaffold lacks a polypeptide. In another embodiment, all layers of the multi-compartment hydrogel or scaffold lack a polypeptide. In another embodiment, the present invention provides a composition comprising a polysaccharide selected from the group consisting of: an alginate, a sulfated alginate, and a hyaluronan sulfate, wherein said composition lacks a polypeptide. In one embodiment, the polypeptide is a bioactive polypeptide. In another embodiment, the polypeptide is a morphogen. In another embodiment, the polypeptide is a growth factor. The inventors have unexpectedly discovered that a hydrogel made with alginate and alginate sulfate that does not comprise a cytokine or other polypeptide was surprisingly effective in repairing hyaline cartilage and subchondral bone (Example 2).

[0030] In one embodiment, a polysaccharide of the invention can be any suitable polysaccharide that facilitates repair, regeneration, or replacement of a damaged or diseased tissue. Examples of such polysaccharides include, for example, but not limited to, an alginate, a chitosan, and a glycosaminoglycan. In one embodiment, the polysaccharide is a sulfated polysaccharide, which in one embodiment is alginate sulfate and, in another embodiment, is hyaluronan sulfate. The invention also encompasses other polymers that facilitate repair, regeneration, or replacements of a damaged or diseased tissue. These other polymers may include, for example, but not limited to collagen, poly(a-hydroxy acids) (e.g., poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid)), pseudo-poly(amino acids), polyhydroxybutyrate, polyethylene glycol, fibrin, and gelatin.

[0031] In accordance with the present invention, the sulfated polysaccharides forming the bioconjugate may be composed of different recurring monosaccharide units, may be of different lengths, and may have different types of bonds linking said units. The sulfated polysaccharides may be linear as, for example, sulfated cellulose, branched as, for example, sulfated glycogen, and may vary in length; for example, it may be as small as a sulfated tetra- or tri- saccharide. The suitable sulfated polysaccharide may be a homopoly saccharide including, but not limited to, starch, glycogen, cellulose, chitosan, or chitin or a heteropolysaccharide including, but not limited to, alginic acid (alginate) salts and hyaluronic acid.

[0032] According to the present invention and in one embodiment, the sulfated polysaccharides may comprise uronic acid residues such D-glucuronic, D-galacturonic, D- mannuronic, L-iduronic, and L-guluronic acids. Examples of polysaccharides comprising uronic acid residues include, but are not limited to, alginic acid salts, in one embodiment, sodium alginate, pectin, gums and mucilages from plant sources; and glycosaminoglycans (GAGs) from animal sources including hyaluronic acid (hyaluronan). The sulfated polysaccharides comprising uronic acid can be, in one embodiment, chemically sulfated or in another embodiment, may be naturally sulfated polysaccharides.

[0033] In one embodiment, the sulfated polysaccharide is alginate sulfate. In another embodiment, the sulfated polysaccharide is hyaluronan sulfate.

[0034] Alginic acid is a linear polysaccharide obtained from brown algae and seaweed and consists of -l,4-linked glucuronic and mannuronic acid units. As used herein, the term "alginate" refers to a polyanionic polysaccharide copolymer derived from sea algae (e.g., Laminaria hyperborea, L. digitata, Eclonia maxima, Macrocystis pyrifera, Lessonia nigrescens, Ascophyllum codosum, L. japonica, Durvillaea antarctica, and D. potatorum) and which includes β-D-mannuronic (M) and a-L-guluronic acid (G) residues in varying proportions.

[0035] In one embodiment, an alginate suitable for use in the present invention may have a ratio between a-L-guluronic acid and β-D-mannuronic in one embodiment, ranging between 1 :1 to 3:1, and in another embodiment, between 1.5:1 and 2.5:1. In a particular example, the ratio between α-L-guluronic acid and β-D-mannuronic is about 2:1.

[0036] In one embodiment, an alginate suitable for use in the present invention has a molecular weight ranging, in one embodiment, between 1 to 300 kDa, in another embodiment, between 5 to 200 kDa, in another embodiment, between 10 to 100 kDa, and in another embodiment, between 20 to 50 kDa.

[0037] In one embodiment, alginate undergoes gelation in the presence of bivalent cations, such as Ca²⁺ and Ba²⁺. In one embodiment, the polysaccharide of the composition forms a supporting matrix. In one embodiment, the polysaccharides are cross-linked using calcium phosphate to form said matrix as was described in: Cardoso et al. J Biomed Mater Res A. 2014 Mar;102(3):808-17; Klein et al. Biomaterials 1987;8(4): 308-310; Rowley et al. Biomaterials 1999;20(l):45-53; Zhang et al. J Mater Sci Mater Med 2003; 14(7) :641-645; Lin et al. J Biomed Mater Res B Appl Biomater 2004;71(l):52-65; Shiraishi et al. J Mater Sci Mater Med 2010;21(3):907-914; and/or Tan et al. J Mater Sci Mater Med 2009;20(6):1245-1253, which are incorporated herein by reference in their entirety. In one embodiment, the calcium phosphate is amorphous calcium phosphate. [0038] In one embodiment, the present invention provides a method of preparing a matrix comprising alginate and sulfated alginate comprising the step of: a) combining said alginate with amorphous calcium phosphate; b) adding said sulfated alginate or sulfated hyaluronan; and c) optionally, adding a bioconjugate; thereby preparing a matrix comprising alginate and sulfated alginate.

[0039] In another embodiment, the present invention provides a method of preparing a matrix comprising alginate and sulfated hyaluronan comprising the step of: a) combining said alginate with amorphous calcium phosphate; b) adding said sulfated alginate or sulfated hyaluronan; and c) optionally, adding a bioconjugate; thereby preparing a matrix comprising alginate and sulfated hyaluronan.

[0040] In one embodiment, amorphous calcium phosphate serves as a source of calcium for cross-linking the alginate, creates void volumes in the matrix, or a combination thereof. In one embodiment, amorphous calcium phosphate causes the matrix to be porous. In one embodiment, the step of adding amorphous calcium phosphate to alginate induces latent cross-linking. In one embodiment, latent cross-linking is a delayed cross-linking. In one embodiment, the delay is due to the dissolving of the cross-linking agent over time before being active. In one embodiment, the time frame for the dissolution of the calcium phosphate is minutes to hours. In one embodiment, cross-linking the matrix increases the rigidity of the matrix.

[0041] In one embodiment, the interaction of calcium phosphate with sulfated alginate or sulfate hyaluronan endows the matrix with distinct physical properties compared to the interaction of calcium phosphate with alginate alone.

[0042] In another embodiment, void volumes or pores are created in the matrix by controlled freezing regime (controlled reduction in sample temperatures) followed by sublimation (lyophilization step), as is known in the art.

[0043] In one embodiment, the method comprises the additional step of adding a second cross-linker, which in one embodiment, is after the step of adding the sulfated alginate or sulfated hyaluronan, and in another embodiment, is after the optional step of adding a bioconjugate. In one embodiment, the cross-linker is calcium phosphate, which in one embodiment, is dissolved when used in the additional step of the method. In another embodiment, the cross-linker is D-gluconic acid hemicalcium salt.

[0044] In another embodiment, the method comprises the additional step of lyophilizing the alginate mixture. In one embodiment, the lyophilization step is prior to the step of adding a second cross-linker.

[0045] In one embodiment, the method comprises the step of combining the alginate mixture with a bioconjugate.

[0046] In another embodiment, the sulfated alginate or sulfated hyaluronan is combined with alginate prior to the addition of amorphous calcium phosphate.

[0047] In another embodiment, the polysaccharides are cross-linked using calcium chloride to form said matrix, as was described in Freeman et al. 2008, which is incorporated herein by reference. In one embodiment, D-gluconic acid hemicalcium salt is used as a source of calcium for cross-linking the polysaccharides.

[0048] Hyaluronic acid is composed of repeating dimeric units of glucuronic acid and N- acetyl glucosamine and forms the core complex proteoglycans aggregates found in the extracellular matrix.

[0049] In one embodiment, the concentration of polysaccharide compared to the total weight of the composition of the present invention may be approximately 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 % (w/w). In some embodiments, the concentration may range approximately 1-5%, 5-95%, 10-90%, 20-80%, 30-70%, or 40-60%

(w/w).

[0050] The present invention also contemplates a mixture of sulfated and unsulfated polysaccharides, for example alginate and alginate sulfate. In one embodiment, the proportion of sulfated polysaccharide may range from about 1% to about 40% of the total polysaccharide by weight, in one embodiment, from about 3% to about 30% of the total polysaccharide by weight, in one embodiment, from about 4% to about 20% of the total polysaccharide by weight, in one embodiment, from about 5% to about 10% of the total polysaccharide by weight. Alternatively, the aforementioned proportions represent percentage by mass. In an alternative embodiment, the rate of binding and release of polypeptides from these bioconjugates can be modulated by adjusting the degree of polysaccharide sulfation and by the extent of sulfated polysaccharide sulfate incorporation into the delivery system.

[0051] In one embodiment, the ratio of sulfated polysaccharide to non-sulfated polysaccharide is 1 :9. In another embodiment, the ratio is 1 :10. In another embodiment, the ratio is 1 :20. In another embodiment, the ratio is 1 :5. In another embodiment, the ratio is 1 :1.5. In another embodiment, the ratio is 1 :2.5. In another embodiment, the ratio is 1 :2. In another embodiment, the ratio is between 1 :1 and 1 :9. In another embodiment, the ratio is 1 :9. In another embodiment, the ratio is 1 :25. In another embodiment, the ratio is 1 :50.

[0052] In one embodiment, the sulfated polysaccharide concentration is 0.1, 0.5, 0.75, 0.9, 1 or 2% w/v.

[0053] Compositions of the present invention may be in any suitable form, for example, a solid, a semi-solid, or a liquid form. The form may be in any form appropriate to the mode of delivery, for example, hydrogel, beads, implants, microspheres (microbeads), hydrogel microcapsules, sponges, scaffolds, meshes, foams, colloidal dispersions, nanoparticles, and suspensions.

[0054] In one embodiment, the composition may be in the form of a hydrogel. The term "hydrogel" as used herein may refer to a network of natural or synthetic hydrophilic polymer chains able to contain water. Examples of compounds able to form such networks are alginate, a partially calcium cross-linked alginate solution, chitosan and viscous hyaluronan. In one embodiment, the bioconjugate of the present invention may be in the form of a flowable gel. In one embodiment, the pore size of a hydrogel of the present invention is in the nanometer-submicron range. In one embodiment, the structure of a hydrogel of the present invention mimics natural extracellular matrix structure.

[0055] In one embodiment, the composition may be in the form of a scaffold, which in one embodiment, is a pre-formed scaffold. The term "scaffold" as used herein refers to any synthetic or organic structure comprising a void. Non-limiting examples of such scaffolds are molds, casts and voids in damaged tissue in a mammal. In one embodiment, the scaffold is a macroporous scaffold. In one embodiment, the macroporous scaffold has pore sizes in the micron range, which in one embodiment, is 50-100 μπι. In one embodiment, the scaffold may be a multi-compartment scaffold. In one embodiment, the scaffold is not hydrated until just prior to use. In one embodiment, the scaffold is highly porous (>90 ). In one embodiment, the scaffold has an interconnected pore structure. In one embodiment, the pore size is 70-100 μπι.

[0056] Thus, in one embodiment, alginate may be used to make hydrogel compositions or macroporous scaffolds, whether single or multi-compartment compositions of the present invention. In one embodiment, said hydrogels and scaffolds may comprise bioactive peptides, additives, biological material, or a combination thereof as described herein.

[0057] In one embodiment, the composition of the present invention is biodegradable. In another embodiment, a composition of the present invention is bioerodable. In one embodiment, a composition of the present invention comprising alginate undergoes dissolution and erosion in the body of a mammal. In another embodiment, the composition of the present invention does not elicit an immune response in a subject. In one embodiment, the composition of the present invention has a half-life of between one and two months.

[0058] In another embodiment, the compositions and methods of the present invention are for repairing or regenerating damaged tissue in a subject. In one embodiment, the damaged tissue is bone, cartilage, or a combination thereof.

[0059] In one embodiment, the composition is homogenous or isotropic. In another embodiment, the composition is denser or less porous in one or more surface portions than its core. In yet another embodiment, the composition is denser or less porous in one or more predetermined portions. In one embodiment, the composition is a multi-compartment hydrogel or scaffold. In another embodiment, the multi-compartment hydrogel or scaffold is denser or less porous in its core than in one or more predetermined surface portions. In one embodiment, a hydrogel of the present invention is permeable to cells and large molecules such as, inter alia, growth factors. In one embodiment, variations in porosity is accomplished using materials that dissolve quickly (in one embodiment, salt, or, in another embodiment, sugar) which then leaves cavities. In another embodiment, degradability is incorporates using chemical modifications. Such methods are well known in the art.

[0060] In some embodiments, a composition of the invention may comprise one or more polypeptides, or one or more additional polypeptides, linked to a polysaccharide. [0061] In one embodiment, the invention relates to a composition comprising an alginate, sulfated alginate, or a hyaluronan sulfate in combination with an IL-1RA for providing a gradual release of IL-1RA.

[0062] Surprisingly and unexpectedly, the inventors of the instant application have found that a sulfated alginate can be used for an effective long-term release of IL-1RA (Example 1).

[0063] In one aspect, provided herein is a composition comprising: a polysaccharide, and a bioactive polypeptide or protein linked to said polysaccharide, wherein said polysaccharide is an alginate or a sulfated alginate and said molecule is interleukin- 1 receptor antagonist (IL- 1RA).

[0064] The term "bioactive polypeptide" or "biologically active polypeptide" as used herein refers to a polypeptide exhibiting a variety of pharmacological activities in vivo. In one embodiment, bioactive peptides include, without being limited to, growth factors, cytokines, chemokines, angiogenic factors, immunomodulators, hormones, and the like.

[0065] In the present application, the terms "polypeptide" and "protein" are used interchangeably. In one embodiment, the polypeptide is a positively charged polypeptide at physiological pH (~7). In another embodiment, the polypeptide is a heparin-binding polypeptide. In another embodiment, the polypeptide is both a positively charged polypeptide and a heparin-binding polypeptide. In another embodiment, the polypeptide is a negatively- charged polypeptide at physiological pH (~7). In one embodiment, the negatively-charged polypeptide has only a partial negative charge. In another embodiment, the polypeptide is neither positively nor negatively charged. In another embodiment, the overall charge of the polypeptide is neutral. In one embodiment, a polypeptide of the present invention is recombinant. In another embodiment, a polypeptide of the present invention is autologous to the subject being treated. In another embodiment, a polypeptide of the present invention is allogeneic to the subject being treated. In another embodiment, a polypeptide of the present invention is heterogenic to the subject being treated. In one embodiment, the polypeptide is produced in vivo. In another embodiment, the polypeptide is produced in vitro.

[0066] According to this aspect and in one embodiment, the compositions of the present invention may comprise a linker to link the polypeptide to the polysaccharide. In one embodiment, the compositions of the present invention may comprise a linker to link a neutral or negatively charged polypeptide to the polysaccharide. In another embodiment, the compositions of the present invention may comprise a linker to link a neutral or negatively charged polypeptide to a sulfated polysaccharide. In another embodiment, the compositions of the present invention may comprise a linker to link a positively charged polypeptide to the a polysaccharide, which in one embodiment, is a sulfated polysaccharide.

[0067] In one embodiment, the bioactive polypeptide is an interleukin-1 receptor antagonist (IL-IRA). IL-IRA is also known as IL-1RN. The present invention encompasses all the known isoforms of IL-IRA, as well as their fragments, mutants, homologs, analogs and allelic variants. Each possibility represents a separate embodiment of the present invention. In one embodiment, IL-IRA is a mammalian IL- IRA. In one embodiment, inclusion of IL- IRA in the compositions (including hydrogels) and methods of the present invention reduces inflammation (such as in post-ACL repair), protects cartilage from further damage, or a combination thereof. In another embodiment, inclusion of IL-IRA in the compositions (including hydrogels) and methods of the present invention prevents or reduces inflammation in the vicinity of a lesion and/or potentially delays progression of degenerative disease.

[0068] In one embodiment, the IL-IRA is isoform 1. In one embodiment, the IL-IRa comprises the sequence as set forth in Genbank Accession No. NP_776214.1 or NP_776214.1. In another embodiment, the IL-IRA is isoform 2. In one embodiment, the IL- lRa comprises the sequence as set forth in Genbank Accession No. NP_776213.1. In another embodiment, the IL-IRA is isoform 3. In one embodiment, the IL-IRa comprises the sequence as set forth in Genbank Accession No. NP_ NP_000568.1 or CAA06730.1. In another embodiment, the IL-IRA is isoform 4. In one embodiment, the IL-IRa comprises the sequence as set forth in Genbank Accession No. NP_001305843.1 ; NP_776215.1. In another embodiment, the IL-IRa comprises the sequence as set forth in Genbank Accession No. CAA36262.1

[0069] In one embodiment, the IL-IRA used in the compositions and methods of the present invention has the following amino acid sequence:

MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHA LFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFES AACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO: 1). In another embodiment, the IL-IRA used in the compositions and methods of the present invention is a homologue of SEQ ID No: 1. In another embodiment, the IL1RA is an isoform of SEQ ID No: 1. In another embodiment, the IL1RA is a variant of SEQ ID No: 1. In another embodiment, the IL1RA is a fragment of SEQ ID No: 1. In another embodiment, the IL1RA is a fragment of an isoform of SEQ ID No: 1. In another embodiment, the IL1RA is a fragment of a variant of SEQ ID No: 1.

[0070] Examples of additional bioactive polypeptides include, but are not limited to, transforming growth factor βΐ (TGF- βΐ), bone Morphogenetic Protein 4 (BMP4), BMP2, BMP7, insulin, glatiramer acetate (also known as Copolymer 1 or Cop 1), antithrombin III, interferon (IFN)-y (also known as heparin-binding protein), IGF-1 , somatostatin, erythropoietin, luteinizing hormone-releasing hormone (LH-RH) and interleukins such as IL- 2, IL-6, and IL-10.

[0071] In one embodiment, the additional bioactive polypeptide is a heparin-binding protein or polypeptide. The term "heparin-binding protein or polypeptide" may refer to a protein having clusters of positively-charged basic amino acids and form ion pairs with specially defined negatively-charged sulfo or carboxyl groups on the heparin chain (See Capila and Linhardt, 2002 Angew Chem Int Ed Engl. 2002 Feb 1 ;41(3):391-412, incorporated herein by reference). Examples of heparin-binding proteins include, but are not limited to, thrombopoietin (TPO); proteases/esterases such as antithrombin III (AT III), serine protease inhibitor (SLP1), CI esterase inhibitor (CI INH) and Vaccinia virus complement control protein (VCP); growth factors such as a fibroblast growth factor (FGF, aFGF, bFGF), a FGF receptor, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), transforming growth factor βΐ (TGF- βΐ), a platelet-derived growth factor (PDGF, PDGF-a and PDGF-β), and epidermal growth factor (EGF); chemokines such as platelet factor 4 (PF-4, now called CXC chemokine ligand 4 or CXCL4), stromal cell-derived factor- 1 (SDF-I), IL-6, IL-8, RANTES (Regulated on Activation, Normal T Expressed and Secreted), monocyte chemoattractant protein- 1 (MCP-I), macrophage inflammatory peptide- 1 (MIP-I), lymphotactin, and fractalkine; lipid or membrane-binding proteins such as an annexin, apolipoprotein E (ApoE); pathogen proteins such as human immunodeficiency virus type-1 (HIV- 1) coat proteins e.g. HIV-I gpl20, cyclophilin A (CypA), Tat protein, viral coat glycoprotein gC, gB or gD of herpes simplex virus (HSV), an envelope protein of Dengue virus, circumsporozoite (CS) protein of Plasmodium falciparum, bacterial surface adhesion protein OpaA; and adhesion proteins such as 1- and P-selectin, heparin-binding growth-associated molecule (HB-GAM), thrombospondin type I repeat (TSR), peptide myelin oligodendrocyte glycoprotein (MOG), and amyloid P (AP). In some embodiments of the present invention, the heparin-binding polypeptide is PDGF-β, PDGF-a, bFGF, aFGF, VEGF, TGF i, IL-6, TPO, SDF-I, HGF, EGF or IGF-1 , BMP2, BMP4, BMP7.

[0072] In another example, the additional bioactive polypeptide is an angiogenic factor or a growth factor exhibiting angiogenic activity such as TGF-βΙ, VEGF, bFGF, aFGF, PDGF- β, IGF- 1 , HGF, and a combination thereof. In one embodiment, the angiogenic factor is VEGF, PDGF-β, or a combination of VEGF, PDGF-BB, and TGF-β 1.

[0073] In another example, the bioactive polypeptide or the additional bioactive polypeptide is a positively charged polypeptide. The term "positively charged polypeptide" may refer to a polypeptide or protein that has a positive net charge at physiological pH of about pH = 7.4. The positively charged polypeptides are well known in the art. Examples of a positively charged polypeptide include, but not limited to, insulin, glatiramer acetate (also known as Copolymer 1 or Cop 1), antithrombin III, interferon (IFN)-y (also known as heparin-binding protein), IGF, somatostatin, erythropoietin, luteinizing hormone-releasing hormone (LH-RH) and interleukins such as IL-2 and IL-6.

[0074] In one embodiment, the alginate or sulfated alginate and the polypeptide are each present in an amount effective to repair or regenerate a damaged tissue in a subject.

[0075] The concentration of one or more bioactive polypeptides or proteins in the alginate composition may be approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 μg/ml. In another embodiment, the concentration is approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg/ml. In another embodiment, the concentration is approximately 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μg/ml. In another embodiment, the concentration is approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/ml. In some embodiments, the concentration may range approximately 1-100 μg/ml, 100-1000 μg/ml, or 1-10 mg/ml.

[0076] In one embodiment, the polypeptide of the invention may be linked to the polysaccharide of the invention to form a bioconjugate. In one embodiment, the linkage is non-covalent. In embodiment, the non-covalent linkage is an ionic bond, an electrostatic interaction, a hydrophobic interaction, or a hydrogen bond or a van der Waals force. In one embodiment, a "bioconjugate" is a molecular complex formed by a stably linking two molecules, at least one of which has a biological activity.

[0077] In one embodiment, the bioconjugate formed by the polypeptide and polys acccharide forms a nanoparticle. In one embodiment, the nanoparticle is between 1 and 1000 nm in diameter. In another embodiment, the nanoparticle is between 1 and 100 nm in diameter. In another embodiment, the nanoparticle is between 500 and 1000 nm in diameter. In another embodiment, the nanoparticle is between 1 and 500 nm in diameter. In another embodiment, the nanoparticle is between 20 and 70 nm in diameter. In another embodiment, the nanoparticle is between 100 and 200 nm in diameter.

[0078] In another embodiment, the polypeptide of the invention is linked to the polysaccharide of the invention via a linker. In one embodiment, the linker is a peptide, wherein the first polypeptide is linked to the polysaccharide via a peptide, which serves as a linker. In one embodiment, the linker is a heparin binding peptide (HBP). In another embodiment, the linker is an amino acid sequence that is enriched in positively-charged amino acids. In one embodiment, the linker is a cell-penetrating peptide (CPP). In one embodiment, CPPs have an amino acid composition that contains a high relative abundance of positively charged amino acids such as lysine or arginine.

[0079] In another embodiment, the linker is a linker suitable for binding a negatively charged alginate with a negatively charged molecule (e.g., IL-1RA). In one embodiment, the linker is a HBP. In one embodiment, HBP or other peptides are bound to the alginate by the mechanism described in Sapir et al., 2011, Biomaterials, vol. 32, pages 1838-1847 and Bondalapati et al. Macromol Rapid Commun. 2014 Sep 15, both incorporated herein by reference.

[0080] It has been previously shown that sulfating polysaccharides endows them with properties which allow binding and controlled release of important signal proteins such as various cytokines and growth factors. Alginate sulfate and hyaluronan sulfate were both found to mimic the biological specificities of heparan sulfate and heparin when forming bioconjugates (see e.g. WO 2007/043050, which is hereby incorporated by reference in its entirety). [0081] In one embodiment, a bioactive polypeptide having a positive charge may be reversibly and non-covalently bound to a sulfated polysaccharide, which carries a negative charge due to its sulfur group.

[0082] The methods of manufacturing sulfated polysaccharide bioconjugates have been described (see e.g. US Pat. Pub. 2015/0051148, hereby incorporated by reference in its entirety).

[0083] The composition may be a single compartment composition or a multi-compartment composition. In one embodiment, the multi-compartment composition may comprise a plurality of layers. For example, the multi-compartment composition may comprise a first compartment and a second compartment, and wherein first compartment is a first layer and said second compartment is a second layer. In one embodiment, each compartment may include a distinct polypeptide or a combination of polypeptides. In another embodiment, each compartment may include the same polypeptide(s). In one embodiment, one or more compartments or layers may lack a polypeptide.

[0084] In one embodiment, one compartment is in the bone layer, while a second compartment is in the cartilage layer. In one embodiment, the compartment in the bone layer comprises no polypeptide, and the compartment in the cartilage layer comprises TGF-beta. In one embodiment, the cartilage layer compartment comprises TGF-βΙ.

[0085] In another embodiment, one compartment is implanted in the bone layer, while a second compartment is implanted in the cartilage layer. In one embodiment, the compartment in the bone layer enhances the repair, regeneration, or prevents further degeneration of damaged bone, while the compartment in the cartilage layer enhances the repair, regeneration, or prevents further degeneration of damaged cartilage. In one embodiment, a benefit of the claimed multi-compartment hydrogel or scaffold is that the repaired bone layer is distinct from the repaired cartilage layer.

[0086] In one embodiment, each compartment of the multi-compartment hydrogel or scaffold has distinct properties. In one embodiment, they have distinct physical properties. In one embodiment, each compartment of the multi-compartment hydrogel or scaffold is in intimal contact with at least one other compartment of the hydrogel. [0087] In one embodiment, one compartment of the multi-compartment hydrogel or scaffold has larger pores, while a second compartment of the multi-compartment hydrogel or scaffold has smaller pores. In one embodiment, the compartment that is in the bone layer has larger pores while the compartment that is in the cartilage layer has smaller pores.

[0088] In one embodiment, the bone layer gel has pores in the micron range, while the cartilage layer gel has pores in the submicron range. In one embodiment, the bone layer gel has pores of 50-200 μπι. In another embodiment, the bone layer gel has pores of 50-100 μπι. In another embodiment, the pores are obtained using ACP, beads, etc. In another embodiment, the pores are 80-120μπι, in another embodiment, 150-200μπι and in another embodiment 250-300μπι.

[0089] In one embodiment, a hydrogel as described herein has a pore size that is similar to that of natural extracellular matrix (ECM) in cartilage. In one embodiment, the cartilage layer gel has pores in the submicron range. In one embodiment, the pores are 3-7 nm. In another embodiment, the pores are 1-10 nm. In another embodiment, the pores are 5-20 nm. In another embodiment, the pores are approximately 6 nm. In another embodiment, the hydrogel has a pore size of 1-1000 nm. In another embodiment, the hydrogel has a pore size of 500- 1000 nm. In another embodiment, the hydrogel has a pore size of 5-500 nm. In another embodiment, the hydrogel has a pore size of, 1 -500 nm. In another embodiment, the hydrogel has a pore size of 250-750 nm. In another embodiment, the hydrogel has a pore size of 1-250 nm. In another embodiment, the hydrogel has a pore size of 1-100 nm.

[0090] In another embodiment, the present invention provides a macroporous scaffold. In one embodiment, a macroporous scaffold as described herein has pores in the micron range, which in one embodiment, is a pore size of 50-100 μπι. In another embodiment, the scaffold has a pore size of 1-100 μπι. In another embodiment, the scaffold has a pore size of 50-200 μπι. In another embodiment, the scaffold has a pore size of 1-50 μπι. In another embodiment, the scaffold has a pore size of 25-75 μπι. In another embodiment, the scaffold has a pore size of 1-25 μπι. In another embodiment, the scaffold has a pore size of 1-10 μπι.

[0091] In another embodiment, each compartment of the multi-compartment hydrogel or scaffold has distinct chemical properties. In one embodiment, one compartment has a higher concentration or amount of sulfated alginate than a second compartment of the multicompartment hydrogel or scaffold. In one embodiment, the % w/v of sulfated alginate is 0.6%-0.7% in the cartilage layer and 0.8 -0.9 in the bone layer. In another embodiment, the % w/v of sulfated alginate is 0.65 -0.75 in the cartilage layer and 0.85 -0.95 in the bone layer. In another embodiment, the % w/v of sulfated alginate is 0.4%-0.7% in the cartilage layer and 0.8%- 1.2% in the bone layer. In one embodiment, the % w/v of sulfated alginate is 0.67% in the cartilage layer and 0.87% in the bone layer. As described in Example 2, multi-compartment hydrogel or scaffolds without growth factors comprising 0.67% alginate-sulfate (w/v) in the cartilage layer and 0.87% alginate- sulfate (w/v) in the bone layer showed good defect filling with repaired tissue with cartilage- like properties, good cartilage repair, and prevention of bone outgrowth into the cartilage layer.

[0092] In another embodiment, one compartment has a higher concentration or amount of polypeptide than a second compartment of the multi-compartment hydrogel or scaffold. In another embodiment, one compartment has a higher concentration or amount of TGF-βΙ than a second compartment of the multi-compartment hydrogel or scaffold. In one embodiment, the plypeptide in the compartment in the cartilage layer is TGF-βΙ , while the compartment in the bone layer lacks polypeptide.

[0093] In another embodiment, the different compartments have both distinct physical properties and distinct chemical properties.

[0094] In one embodiment, the composition comprises nanoparticles.

[0095] In some embodiments, the composition may further comprise a supporting matrix. The matrix may serve as support or as a carrier for the bioconjugate and may be made up of particles or porous materials. The matrix material may be flexible and amenable to be fixed in place preventing its migration to an unintended location. In one embodiment, the supporting matrix comprises a polymer selected from the group consisting of a polysaccharide, a protein, an extracellular matrix component, and a synthetic polymer, or a mixture thereof. In one embodiment, the supporting matrix is a macroporous scaffold. In another embodiment, the supporting matrix is solid. In another embodiment, the supporting matrix is preformed. In another embodiment, the supporting matrix is a solid macroporous preformed scaffold.

[0096] In one embodiment, the composition of the present invention, which in one embodiment, is a multi-compartment hydrogel or scaffold, may comprise an additive that is neither a cell nor a polypeptide. In one embodiment, the additive is a scaffold assisting material that facilitates tissue regeneration or repair of a damaged tissue in a subject. Examples of a scaffold assisting material include, for example, but not limited to, hydroxyapatite, calcium phosphate, one or more mannitol beads, one or more magnesium minerals, or a combination thereof.

[0097] In one embodiment, non-physiologically high levels of extracellular Mg concentration obtained, in one embodiment, by the presence of magnesium ions in or on the composition or on the hydrogel, is used to induce chondron formation. In one embodiment, magnesium is used in conjunction with morphogens to induce chondron formation, while in another embodiment, no morphogens are used in conjunction with magnesium. In one embodiment, methods for including magnesium in alginate gels are known in the art (e.g. WO 2005039662 A2, which is incorporated herein by reference). In one embodiment, magnesium may be used instead of calcium to cross-link a polysaccharide. In another embodiment, both magnesium and calcium may be included as cross-linking agents.

[0098] In one embodiment, hydroxyapatite and calcium phosphate are osteogenic.

[0099] In some embodiments, the composition of the present invention, which in one embodiment, is a multi-compartment hydrogel or scaffold, may comprise a biological fluid or biological material from a subject. Examples of a biological fluid or biological material include, for example, but not limited to platelet rich plasma (PRP), bone marrow aspirate, and serum. In another embodiment, the biological material is a cell. In one embodiment, the cell may be any of the cells described hereinbelow. In one embodiment, the biological material is autologous to the person whom is receiving or will be administered the multi-compartment hydrogel or scaffold. In another embodiment, the biological material is heterologous to the person whom is receiving or will be administered the multi-compartment hydrogel or scaffold. In one embodiment, the biological material is platelet rich plasma (PRP), bone marrow aspirate, serum or a combination thereof. In another embodiment, the biological material is autologous platelet rich plasma (PRP), autologous bone marrow aspirate, autologous serum or a combination thereof.

[00100] In one embodiment, the biological fluid or material is an autologous fluid or material. In another embodiment, the biological fluid or material is a heterologous fluid or material. In another embodiment, the biological fluid or material is an allogenic fluid or material.

Accordingly, in one embodiment, the composition may further comprise a biological fluid or material from a patient in need of said composition for regenerating or repairing a damaged tissue in the patient.

[00101] In one embodiment, methods of harvesting, processing, activation, and administering PRP are known in the art (e.g. Sakata et al. Tissue Eng Part B Rev. 2015 Oct;21(5):461-73. doi: 10.1089/ten.TEB.2014.0661. Epub 2015 Jul 14, incorporated herein by reference in its entirety, page 465 column 2). In one embodiment, methods used to prepare PRP concentrate the platelets in some amount of autologous serum, induce the platelets to release their morphogens prior to administration, whether by chemical or by physical stimulus ex vivo, or a combination thereof.

[00102] In one embodiment, compositions of the present invention, including hydrogels, comprise one or more cells. In one embodiment, the cells are stem cells. In one embodiment, the stem cells are mesenchymal stem cells. In one embodiment, the cells are tissue precursor cells. In one embodiment, the tissue precursor cells are chondrocytes. In another embodiment, the tissue precursor cells are osteoblasts. In another embodiment, the tissue precursor cells can include any of the following: epidermal cells, chondrocytes and other cells that form cartilage, macrophages, dermal cells, muscle cells, hair follicles, fibroblasts, organ cells, osteoblasts and other cells that form bone, endothelial cells, mucosal cells, pleural cells, ear canal cells, tympanic membrane cells, peritoneal cells, Schwann cells, corneal epithelial cells, gingiva cells, neural cells, neural stem cells such as central nervous system (CNS) stem cells, e.g., spinal cord or brain stem cells, as well as autonomic nervous system (ANS) stem cells, e.g., post-ganglionic stem cells from the small intestine, bladder, liver, lung, and heart, (for engineering sympathetic or parasympathetic nerves and ganglia), tracheal epithelial cells, hepatocytes, pancreatic cells, and cardiac cells. The tissue precursor cells can also be neuroendocrine stem cells.

[00103] The composition of the invention may be administered via any suitable method known to one of skilled in the art. Examples of such method include, but is not limited to, intraliver, intradermal, transdermal (e.g. in slow release formulations), intramuscular, intraperitoneal, intravenous, intracoronary, subcutaneous, oral, epidural, topical, intraarticular, and intranasal routes.

[00104] The administration of the invention also encompasses surgically administering, implanting, inserting, or injecting the implant (or sections thereof) into a subject. The implant (or section) can be located subcutaneously, intramuscularly, or located at another body location which allows the implant to perform its intended function. Generally, implants (or sections) are administered by subcutaneous implantation at sites including, but not limited to, the upper arm, back, or abdomen of a subject. In one embodiment, a composition of the present invention is implanted into a damaged joint. Other suitable sites for administration may be readily determined by a medical professional. Multiple implants or sections may be administered to achieve a desired dosage for treatment. Any other therapeutically efficacious route of administration can be used. Administration may also include systemic or local administration of the composition of the invention.

[00105] The present invention provides sustained release of polypeptides that is maintained over a period of about 10 days, 15 days, 30 days, 60 days, 90 days, 180 days, 270 days, or 365 days. In one embodiment, the sustained release period is about 10 days. In another embodiment, the sustained release period is about 15 days. In yet another embodiment, the sustained release period is about 30 days. In a further embodiment, the sustained release period is about 60 days. In another embodiment, the sustained release period is about 90 days. In yet another embodiment, the sustained release period is about 180 days. In a further embodiment, the sustained release period is about 270 days. In yet further embodiment, the sustained release period is about 365 days.

[00106] In some embodiments, the invention provides for the localized release of the polypeptide(s). In other embodiments, the invention provides for the systemic release of the polypeptide(s).

[00107] The present invention further contemplates adding a pharmaceutically acceptable carrier to the polysaccharide/polypeptide bioconjugate as described herein. The term "pharmaceutically acceptable carrier" refers to a vehicle which delivers the active components to the intended target and which will not cause harm to humans or other recipient organisms. As used herein, "pharmaceutical" will be understood to encompass both human and veterinary pharmaceuticals. Useful carriers include, for example, water, acetone, ethanol, ethylene glycol, propylene glycol, butane- 1 , 3-diol, isopropyl myristate, isopropyl palmitate, mineral oil and polymers composed of chemical substances like polyglycolic acid or polyhydroxybutyrate or natural polymers like collagen, fibrin or polysaccharides like chitosan and alginate. The carrier may be in any form appropriate to the mode of delivery, for example, solutions, colloidal dispersions, emulsions (oil-in-water or water-in-oil), suspensions, creams, lotions, gels, foams, mousses, sprays and the like. Methodology and components for formulation of pharmaceutical compositions are well known and can be found, for example, in Remington's Pharmaceutical Sciences, Eighteenth Edition, A. R. Gennaro, Ed., Mack Publishing Co. Easton Pa., 1990. In one embodiment of the invention, the carrier is an aqueous buffer. In another embodiment, the carrier is a polysaccharide and is in one embodiment, an alginate or in another embodiment, hyaluronic acid. In one embodiment, the carrier is a hydrogel.

[00108] Examples of a disease or disorder treated by the composition of the invention include, but are not limited to, rheumatoid arthritis, osteoporotic fracture, microfracture, osteochondral defect, osteoarthritis, post-trauma arthritis, anterior cruciate ligament (ACL) injury, meniscal tear, articular fracture, high-limb ischemia, myocardial infarction, heart failure, spinal cord injury, stroke, and a joint injury associated disease or condition (e.g., equine lameness secondary to joint injury). In one embodiment, the compositions described herein are used as an adjunct therapy to microfracture or microdrilling, which in one embodiment, is an arthroscopic surgical procedure for the repair of damaged articular cartilage that involves debriding loose and calcified cartilage and drilling small holes in the bone beneath so that mesenchymal stem cells are released from marrow in the underlying bone to permeate the damaged cartilage area.

[00109] Additional examples of a disease or disorder treated by the composition of the invention include, but are not limited to, autoimmune disorder (e.g., multiple sclerosis, psoriasis, or type I diabetes), allograft rejection, multiple sclerosis, psoriasis, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, Hashimoto thyroiditis, primary biliary cirrhosis, active chronic hepatitis, adrenalitis/ Addison's disease, polymyositis, dermatomyositis, autoimmune haemolytic anaemia, myocarditis, myopericarditis, scleroderma, uveitis (including phacouveitis and sympathetic ophthalmia) pemphigus vulgaris, pemphigoid, pernicious anaemia, autoimmune atrophic gastritis, Crohn's disease, and colitis ulcerosa.

[00110] In one embodiment, the methods of the present invention may be used to treat a disease, condition or disorder described herein. In another embodiment, the methods of the present invention may be used to prevent a disease, condition or disorder described herein. In another embodiment, the methods of the present invention may be used to suppress a disease, condition or disorder described herein. In another embodiment, the methods of the present invention may be used to inhibit a disease, condition or disorder described herein.

[00111] In one embodiment, "treating" as used herein refers to therapeutic treatment. In one embodiment, "preventing", "suppressing" or "inhibiting" as used herein refers to prophylactic or preventative measures, wherein the object is to prevent or lessen the targeted condition or disorder as described herein. Thus, in one embodiment, "treating" refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof. In one embodiment, "preventing" refers, inter alia, to delaying the onset of symptoms, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, or a combination thereof. In one embodiment, "suppressing" or "inhibiting", refers inter alia to reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.

[00112] The composition of the invention can also be used for enhancing an immunotolerant response. Examples of immunotolerant responses include, but are not limited to, allograft success, lack of allograft rejection, suppression of autoimmune disorder, suppression of an immune response to an allocell transplantation, suppression of allocell apoptosis, an increase in allocell survival, stimulation of vascularization of allocell transplant, prolonged presentation of said bioactive polypeptide, suppression of inflammatory signaling, suppression of dendritic cell maturation, suppression of CD8+ T cell cytotoxicity response, and stimulation of regulatory T cell differentiation.

[00113] In another aspect, the invention relates to a method for repairing or regenerating a damaged tissue in a subject, the method comprising: administering to said subject a composition comprising: a polysaccharide, and a bioactive polypeptide or a protein molecule linked to said polysaccharide, wherein said polysaccharide is an alginate or a sulfated alginate and said molecule is interleukin-1 receptor antagonist (IL-1RA), thereby repairing or regenerating said damaged tissue in said subject. [00114] In another aspect, the invention relates to a method for preventing degeneration of a tissue in a subject, the method comprising: administering to said subject a composition comprising: a polysaccharide, and a bioactive polypeptide or a protein molecule linked to said polysaccharide, wherein said polysaccharide is an alginate or a sulfated alginate and said molecule is interleukin-1 receptor antagonist (IL-IRA), thereby preventing degeneration of said tissue in said subject. In one embodiment, the degeneration is disease-associated degeneration.

[00115] In another aspect, the invention relates to a method for treating an osteochondral defect in a subject, the method comprising: administering to said subject a composition comprising: a polysaccharide, and a bioactive polypeptide or protein molecule linked to said polysaccharide, wherein said polysaccharide is an alginate or a sulfated alginate and said polypeptide or molecule is interleukin-1 receptor antagonist (IL-IRA), thereby treating said osteochondral defect in said subject.

[00116] In one embodiment, a condition that may be treated using a composition comprising: a polysaccharide, and a bioactive polypeptide linked to said polysaccharide, wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate and said polypeptide is IL-IRA is a condition characterized by inflammation. In another embodiment, the condition is characterized by IL-lbeta expression.

[00117] In another aspect, the invention relates to a method for treating a rheumatoid arthritis in a subject, the method comprising: administering to said subject a composition comprising: a polysaccharide, and a bioactive polypeptide or a protein molecule linked to said polysaccharide, wherein said polysaccharide is an alginate or a sulfated alginate and said molecule is interleukin-1 receptor antagonist (IL-IRA), thereby treating said rheumatoid arthritis in said subject.

[00118] In another aspect, the invention relates to a method for treating a joint injury or its associated condition in a subject, the method comprising: administering to said subject a composition comprising: a polysaccharide, and a bioactive polypeptide or a protein molecule linked to said polysaccharide, wherein said polysaccharide is an alginate or a sulfated alginate and said molecule is interleukin-1 receptor antagonist (IL-IRA), thereby treating said joint injury or its associated condition in said subject. In an exemplary embodiment, said joint injury associated condition is an equine lameness secondary to joint injury. [00119] In one embodiment, the present invention provides a method for repairing or regenerating a damaged tissue in a subject, the method comprising the step of administering to said subject a composition of the present invention as described herein. In one embodiment, the present invention provides a method for repairing damaged tissue in a subject, the method comprising the step of administering to said subject a composition of the present invention as described herein. In one embodiment, the present invention provides a method for regenerating a tissue in a subject, the method comprising the step of administering to said subject a composition of the present invention as described herein.

[00120] Thus, in one embodiment, the present invention provides a method for repairing or regenerating a damaged tissue in a subject comprising the step of administering to said subject a composition comprising a polysaccharide, and a polypeptide linked to said polysaccharide, wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate, and said polypeptide is interleukin-1 receptor antagonist (IL-1RA). In another embodiment, the present invention provides a method for repairing or regenerating a damaged tissue in a subject comprising the step of administering to said subject a composition comprising a polysaccharide and an additive selected from the group consisting of: hydroxyapatite, calcium phosphate, mannitol beads, and magnesium ions, or a combination thereof, wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate. In another embodiment, the present invention provides a method for repairing or regenerating a damaged tissue in a subject comprising the step of administering to said subject a composition comprising a polysaccharide and a biological material from a subject, wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate. In another embodiment, the present invention provides a method for repairing or regenerating a damaged tissue in a subject comprising the step of administering to said subject a composition comprising a polysaccharide, wherein said polysaccharides are cross-linked using calcium phosphate to form a matrix, and wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate.

[00121] In one embodiment, the present invention provides a method for treating an osteochondral defect in a subject comprising the step of administering to said subject a composition of the present invention as described herein. Thus, in one embodiment, the present invention provides a method for treating an osteochondral defect in a subject comprising the step of administering to said subject a composition comprising: a polysaccharide, and a polypeptide linked to said polysaccharide, wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate, and said polypeptide is interleukin- 1 receptor antagonist (IL- 1RA). In another embodiment, the present invention provides a method for treating an osteochondral defect in a subject comprising the step of administering to said subject a composition comprising a polysaccharide and an additive selected from the group consisting of: hydroxyapatite, calcium phosphate, mannitol beads, and magnesium ions, or a combination thereof, wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate. In another embodiment, the present invention provides a method for treating an osteochondral defect in a subject comprising the step of administering to said subject a composition comprising a polysaccharide and a biological material from a subject, wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate. In another embodiment, the present invention provides a method for treating an osteochondral defect in a subject comprising the step of administering to said subject a composition comprising a polysaccharide, wherein said polysaccharides are cross-linked using calcium phosphate to form a matrix, and wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate.

[00122] In another embodiment, the present invention provides a method of treating arthritis in a subject comprising the step of administering to said subject a composition of the present invention as described herein. In one embodiment, the arthritis is rheumatoid arthritis. In one embodiment, the arthritis is osteoarthritis. Thus, in one embodiment, the present invention provides a method of treating arthritis in a subject comprising the step of administering to said subject a composition comprising a polysaccharide, and a polypeptide linked to said polysaccharide, wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate, and said polypeptide is interleukin-1 receptor antagonist (IL- 1RA). In another embodiment, the present invention provides a method of treating arthritis in a subject comprising the step of administering to said subject a composition comprising a polysaccharide and an additive selected from the group consisting of: hydroxyapatite, calcium phosphate, mannitol beads, and magnesium ions, or a combination thereof, wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate. In another embodiment, the present invention provides a method of treating arthritis in a subject comprising the step of administering to said subject a composition comprising a polysaccharide and a biological material from a subject, wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate. In another embodiment, the present invention provides a method of treating arthritis in a subject comprising the step of administering to said subject a composition comprising a polysaccharide, wherein said polysaccharides are cross-linked using calcium phosphate to form a matrix, and wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate.

[00123] In another embodiment, the present invention provides a method for treating a joint injury in a subject comprising the step of administering to said subject a composition of the present invention as described herein. In another embodiment, the present invention provides a method for treating a condition associated with a joint injury in a subject with a joint injury, comprising the step of administering to said subject a composition of the present invention as described herein. In one embodiment, the condition associated with a joint injury is equine lameness.

[00124] Thus, in one embodiment, the present invention provides a method for treating a joint injury in a subject comprising the step of administering to said subject a composition comprising a polysaccharide, and a polypeptide linked to said polysaccharide, wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate, and said polypeptide is interleukin- 1 receptor antagonist (IL-1RA). In another embodiment, the present invention provides a method for treating a joint injury in a subject comprising the step of administering to said subject a composition comprising a polysaccharide and an additive selected from the group consisting of: hydroxyapatite, calcium phosphate, mannitol beads, and magnesium ions, or a combination thereof, wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate. In another embodiment, the present invention provides a method for treating a joint injury in a subject comprising the step of administering to said subject a composition comprising a polysaccharide and a biological material from a subject, wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate. In another embodiment, the present invention provides a method for treating a joint injury in a subject comprising the step of administering to said subject a composition comprising a polysaccharide, wherein said polysaccharides are cross-linked using calcium phosphate to form a matrix, and wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate. [00125] In one embodiment, in the methods of the present invention, the step of administering a hydrogel or a multi-compartment hydrogel or scaffold comprises applying a hydrogel or a multi-compartment hydrogel or scaffold to a joint or to a portion of a joint. In one embodiment, the first hydrogel layer is first applied to the bone layer in a joint of a subject and then a second hydrogel layer is then applied to the cartilage layer in the same joint of the same subject, thereby creating a multi-compartment hydrogel or scaffold. In one embodiment, the second hydrogel layer is applied after the first hydrogel layer forms a distinctive physical interface, surface, or "floor" to which the second hydrogel layer may be applied. In one embodiment, the first hydrogel provides a solid surface against which the second layer is then formed. In another embodiment, the first hydrogel provides a semi-solid surface against which the second layer is then formed. In one embodiment, the hydrogel layer applied to the bone layer does not comprise a morphogen and the hydrogel layer applied to the cartilage layer comprises TGF-beta, which in one embodiment, is TGF-betal.

[00126] In one embodiment, the cross-linking agent is applied to the defect surface prior to applying the first layer of gel. In one embodiment, additional cross linking agent is applied to the first gel layer after it is applied to the defect surface in the bone. In one embodiment, additional cross linking agent is applied to the second gel layer after it is applied to the defect surface in the cartilage. In one embodiment, the cross-linking agent is calcium chloride.

[00127] In one embodiment, the hydrogel or matrix layers can be distinguished for at least 1 week. In another embodiment, the hydrogel or matrix layers can be distinguished for at least

2 weeks. In another embodiment, the hydrogel or matrix layers can be distinguished for at least 3 weeks. In another embodiment, the hydrogel or matrix layers can be distinguished for at least 1 month.

[00128] In one embodiment, the hydrogel or matrix layers remain substantially separate for at least 1 week. In another embodiment, the hydrogel or matrix layers remain substantially separate for at least 2 weeks. In another embodiment, the hydrogel or matrix layers remain substantially separate for at least 3 weeks. In another embodiment, the hydrogel or matrix layers remain substantially separate for at least 1 month. In one embodiment, there is some diffusion between the layers when they are substantially separate. In another embodiment, there is no diffusion at all between the layers when they are substantially separate. [00129] In one embodiment, the step of administering comprises implanting said composition as part of a solid macroporous preformed scaffold.

[00130] In another embodiment, a preformed scaffold of the present invention made with cross-linked alginate and/or alginate sulfate or hyaluronan sulfate, optionally comprising a bioactive polypeptide, is pre-molded to fit into a repository or cage such as that described in US5984967, which is incorporated herein by reference, and other similar osteogenic fusion devices known in the art. In one embodiment, the pre-formed scaffold is shaped to fit into an Infuse® Bone Graft/LT-Cage® Lumbar Tapered Fusion Device. In one embodiment, the bioactive polypeptide in the pre-formed scaffold is BMP-2. In another embodiment, the bioactive polypeptide in the pre-formed scaffold is BMP-4.

[00131] In one embodiment, the scaffold preformed to fit into a fusion device, which in one embodiment, is a cage, is used for bone regeneration. In one embodiment, the fusion device is metal. In another embodiment the fusion device is plastic. In one embodiment, the preformed scaffold provides improved manufacturability compared to other osteogenic materials (such as collagen). In another embodiment, the preformed scaffold provides increased safety compared to other osteogenic materials. In another embodiment, a scaffold of the present invention reduces the overall dose of bioactive polypeptide that is needed, which in one embodiment, is due to greater retention of the bioactive polypeptide in the matrix. In one embodiment, BMPs such as BMP-4 and BMP-2 bind to a scaffold of the present invention at physiological nanomolar concentrations vs milligrams for collagen. In one embodiment, BMP-2 may be administered to a subject at lOOOx lower dose compared to other osteogenic materials. In another embodiment, the presence of the pre-formed alginate/alginate sulfate or alginate/hyaluronan sulfate scaffold inside a cage prevents leakage of the bioactive polypeptides outside of the implant volume. In one embodiment, prevention of leakage reduces the likelihood of ectopic bone formation. In another embodiment, the lower dose of

BMP-2 reduces the likelihood of adverse events due to less BMP-2 that may reach circulation.

[00132] In one embodiment, the present invention provides an osteogenic fusion device comprising a pre-formed scaffold comprising cross-linked alginate sulfate or hyaluronan sulfate, wherein said scaffold is designed with dimensions so as to fit into said osteogenic fusion device and wherein said scaffold maintains its shape. In one embodiment, the scaffold is an alginate scaffold. In one embodiment, the scaffold is cross-linked using amorphous calcium phosphate. In another embodiment, the osteogenic fusion device is made from metal. In another embodiment, the osteogenic fusion device is a LT-Cage® Tapered Fusion Device. In another embodiment, the pre-formed scaffold comprises a bioactive polypeptide. In one embodiment, the bioactive polypeptide is bone morphogenetic protein (BMP)-2. In another embodiment, the bioactive polypeptide is BMP-4.

[00133] In another embodiment, the present invention provides a method of facilitating bone regeneration, intervertebral disc regeneration, bone growth, vertebrate fusion, spinal fusion, or a combination thereof comprising the step of implanting in a subject the osteogenic fusion device as described herein, wherein the osteogenic fusion device comprising a pre-formed scaffold comprising cross-linked alginate sulfate or hyaluronan sulfate, wherein said scaffold is designed with dimensions so as to fit into said osteogenic fusion device and wherein said scaffold maintains its shape.

[00134] In another embodiment, the present invention provides methods of facilitating bone regeneration, intervertebral disc regeneration, bone growth, vertebrate fusion, spinal fusion, or a combination thereof using compositions, including hydrogels and scaffolds, described herein. In another embodiment, the present invention provides methods of treating, inhibiting or suppressing joint disease, osteoarthritis, craniofacial diseases, periodontal diseases, or a combination thereof using compositions, including hydrogels and scaffolds, described herein. Other applications of the present invention include cardiac indications such as hind-limb ischemia, myocardial infarction, and spinal cord repair.

[00135] In one embodiment, the present invention provides a hydrogel lacking morphogens for treating, preventing, suppressing, or inhibiting osteochondral defects and osteoarthritis. In another embodiment, the present invention provides a hydrogel comprising TGF-beta for treating osteochondral defects and osteoarthritis. In one embodiment, the composition is adjunct to microfracture or microdrilling for hyaline cartilage regeneration in patients with osteochondral defects and/or osteoarthritis (OA).

[00136] In another embodiment, the present invention provides a hydrogel lacking cytokines for treating, preventing, suppressing, or inhibiting post-trauma arthritis or OA. In another embodiment, the present invention provides a hydrogel comprising IL-1RA for treating, preventing, suppressing, or inhibiting post-trauma Arthritis or OA. In one embodiment, post- trauma arthritis comprises ACL tear, meniscal tear, and articular fracture. In one embodiment, the present invention provides compositions and methods for early treatment of osteochondral defect or OA to prevent or reduce inflammation in the vicinity of the lesion and potentially delay progression of degenerative disease. In one embodiment, compositions of the present invention provide sustained delivery of ILl-Ra (increased half-life), increase efficacy while reducing total protein administered to patient (increased safety), or a combination thereof.

[00137] In one embodiment, the polysaccharides in the multi-compartment hydrogel or scaffold are cross-linked by calcium. In one embodiment, the polysaccharides in the multi- compartment hydrogel or scaffold are cross-linked using calcium phosphate, calcium chloride, calcium gluconate or a combination thereof. In one embodiment, the calcium cross- linked polysaccharide is in the form of solid macroporous scaffold.

[00138] In one embodiment, a composition of the present invention is injectable, biodegradable/bioerodable, or a combination thereof. In one embodiment, the composition is a multi-compartment hydrogel or scaffold.

[00139] In one embodiment, the multi-compartment hydrogel or scaffold comprises multiple compartments that lack polypeptides or bio active polypeptides. In another embodiment, the multi-compartment hydrogel or scaffold comprises at least one compartment comprising a polypeptide or a bioactive polypeptide. In one embodiment, the polypeptide present in at least one compartment of the multi-compartment hydrogel or scaffold is present in an effective amount. In one embodiment, the effective amount is effective to repair or regenerate a damaged tissue in a subject. In one embodiment, the polypeptide is biologically active. In one embodiment, the polypeptide is linked to said alginate or said sulfated alginate to form a bioconjugate. In one embodiment, the bioconjugate forms a nanoparticle. In one embodiment, the polypeptide is linked to said polysaccharide by a reversible non-covalent binding involving ionic bonds, an electrostatic interaction, a hydrophobic interaction, or hydrogen bonds or van der Waals forces. In one embodiment, the polypeptide is linked to said polysaccharide via a linker. In one embodiment, the linker is a peptide. In one embodiment, the linker is a second polypeptide. In one embodiment, the linker is heparin binding peptide.

[00140] In one embodiment, the polypeptide is TGF-beta. In one embodiment, the polypeptide is a heparin-binding polypeptide. In one embodiment, the heparin-binding polypeptide is transforming growth factor βΐ (TGF-βΙ), TGF- 3, antithrombin III (AT III), thrombopoietin (TPO), serine protease inhibitor (SLP1), CI esterase inhibitor (CI INH), Vaccinia virus complement control protein (VCP), a fibroblast growth factor (FGF), a FGF receptor, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), a platelet-derived growth factor (PDGF), epidermal growth factor (EGF), CXC chemokine ligand 4 (CXCL4), stromal cell-derived factor-l (SDF-l), interleukin-6 (IL-6), interleukin-8 (IL-8), Regulated on Activation, Normal T Expressed and Secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory peptide-1 (MIP-1), lymphotactin, fractalkine, an annexin, apolipoprotein E (ApoE), immunodeficiency virus type-1 (HIV-1) coat protein gpl20, cyclophilin A (CypA), Tat protein, viral coat glycoprotein gC, gB or gD of herpes simplex virus (HSV), an envelope protein of Dengue virus, circumsporozoite (CS) protein of Plasmodium falciparum, bacterial surface adhesion protein OpaA, 1-selectin, P-selectin, heparin-binding growth-associated molecule (HB-GAM), thrombospondin type I repeat (TSR), peptide myelin oligodendrocyte glycoprotein (MOG), amyloid P (AP) PDGF-BB, PDGF-AA, bFGF, aFGF, VEGF, IL-6, IL- 10, TPO, SDF-1, HGF, EGF, MOG, or IGF-1, BMP2, BMP4, BMP7. In one embodiment, the heparin-binding polypeptide is a polypeptide exhibiting angiogenic activity. In one embodiment, the polypeptide exhibiting angiogenic activity is VEGF, bFGF, aFGF, PDGF- ββ, IGF, or a combination thereof. In one embodiment, the polypeptide is interleukin- 1 receptor antagonist (IL- 1 RA) .

[00141] In one embodiment, the multi-compartment hydrogel or scaffold is for use in repair or regeneration of a damaged tissue in a subject. In one embodiment, the multi-compartment hydrogel or scaffold is for use in treating rheumatoid arthritis, osteochondral defect, osteoarthritis, microfracture, post-trauma arthritis after anterior cruciate ligament (ACL) injury, meniscal tear, or articular fracture. In one embodiment, the multi-compartment hydrogel or scaffold is for use in high-limb ischemia, myocardial infarction, heart failure, stroke, or spinal cord injury. In one embodiment, the multi-compartment hydrogel or scaffold is for use in enhancing an immunotolerant response. In one embodiment, the multicompartment hydrogel or scaffold described herein is used as an adjunct therapy to surgical microfracture or microdrilling. [00142] In one embodiment, the present invention provides a multi-compartment hydrogel or scaffold comprising any one or more of the compositions described herein in one or more of the compartments of said multi-compartment hydrogel or scaffold.

[00143] In one embodiment, the present invention provides a method for repairing or regenerating a damaged tissue in a subject, the method comprising the step of administering to said subject a multi-compartment hydrogel or scaffold as described herein.

[00144] In another embodiment, the present invention provides a method for treating an osteochondral defect in a subject, the method comprising the step of administering to said subject a multi-compartment hydrogel or scaffold as described herein.

[00145] In another embodiment, the present invention provides a method for treating a rheumatoid arthritis in a subject, the method comprising the step of administering to said subject a multi-compartment hydrogel or scaffold as described herein.

[00146] In another embodiment, the present invention provides a method for treating a joint injury or its associated condition in a subject, the method comprising the step of administering to said subject a multi-compartment hydrogel or scaffold as described herein. In one embodiment, the joint injury associated condition is an equine lameness, secondary to said joint injury. In one embodiment, the joint injury is from surgical microfracture, microdrilling, or other deliberate surgical damage to the subchondral bone.

[00147] In one embodiment, the step of administering a multi-compartment hydrogel or scaffold in the methods of the present invention comprises applying a first hydrogel layer to the bone layer in the joint of said subject and subsequently applying a second hydrogel layer to the cartilage layer of the joint of said subject. In one embodiment, the second hydrogel layer is applied after the formation of a distinct physical interface by the first hydrogel layer. In one embodiment, the hydrogel layer applied to the bone layer does not comprise a morphogen, and the hydrogel layer applied to the cartilage layer comprises TGF-betal.

[00148] In one embodiment, the cross-linking agent is applied to the defect surfaces prior to applying the first layer of gel. In one embodiment, additional cross linking agent is applied to the first gel layer after it is applied to the defect surface in the bone. In one embodiment, additional cross linking agent is applied to the second gel layer after it is applied to the defect surface in the cartilage. In one embodiment, the cross-linking agent is calcium chloride. [00149] In one embodiment, one hydrogel or scaffold layer does not comprise a sulfated polysaccharide. In one embodiment, a second hydrogel or scaffold layer does comprise a sulfated polysaccharide. In one embodiment, the layer that does not comprise a sulfated polysaccharide also does not comprise a bioactive polypeptide. In one embodiment, the layer that comprises a sulfated polysaccharide also comprises a bioactive polypeptide. In one embodiment, the hydrogel or scaffold layer applied to the bone layer does not comprise a sulfated polysaccharide, while the hydrogel or scaffold layer applied to the cartilage layer does comprise a sulfated polysaccharide.

[00150] In another embodiment, the step of administering comprises implanting said multi- compartment hydrogel or scaffold as a solid macroporous preformed scaffold. In one embodiment, a preformed scaffold comprises a polypeptide of interest. In one embodiment, a scaffold is stored dry until use.

[00151] In one embodiment, the methods of the present invention comprise the step of applying a first layer of a polysaccharide or polysaccharide/sulfated polysaccharide mixture in fluid form to a damaged bone, which in one embodiment, is damaged due to microdrilling, microfracture or other deliberate surgical damage to the subchondral bone. In one embodiment, the first layer is applied to completely fill the void in the bone but does not enter the cartilage layer. In one embodiment, the methods comprise the step of adding a cross-linker to the defect surface prior to applying the first layer of gel. In one embodiment, the methods comprise the step of adding a cross-linker to the first layer. In one embodiment, the methods comprise the step of waiting for the first solution to gel partially. In one embodiment, the wait is 1-5 minutes, in another embodiment, 5-10 minutes, in another embodiment, 10-15 minutes. In another embodiment, the methods comprise the step of waiting for the first solution to gel completely. In one embodiment, the determination of the gelling of the solution is made using visible signs of gelling such as color changes from clear to cloudy, etc as is known in the art. In one embodiment, the methods comprise the step of then applying a second layer of polysaccharide or polysaccharide/sulfated polysaccharide mixture in fluid form to the gelled first layer, where the second layer completely fills the void in the damaged cartilage. In one embodiment, a second cross-linking solution is applied to the second layer. As described herein, in one embodiment, the bone layer will have micron-size pores, while the cartilage layer will have sub-micron-size pores. In one embodiment, the cartilage layer will comprise a bioactive polypeptide, while the bone layer will not comprise a bioactive polypeptide. In one embodiment, one or both layers will comprise additives and autologous biological fluids to promote bone and/or cartilage regeneration.

[00152] In one embodiment, the present invention provides a composition comprising: a polysaccharide and an additive selected from the group consisting of: hydroxyapatite, calcium phosphate, mannitol beads, and magnesium ions, or a combination thereof, wherein said polysaccharide is a sulfated alginate or hyaluronan sulfate. In one embodiment, the composition further comprises a polypeptide linked to the polysaccharide. In another embodiment, the composition does not comprise a polypeptide, morphogen or growth factor.

[00153] In another embodiment, the present invention provides a composition comprising: a polysaccharide and a biological material from a subject, wherein said polysaccharide is a sulfated alginate or hyaluronan sulfate. In one embodiment, the composition further comprises a polypeptide linked to the polysaccharide. In another embodiment, the composition does not comprise a polypeptide, morphogen or growth factor.

[00154] In another embodiment, the present invention provides a composition comprising: a polysaccharide, wherein said polysaccharides are cross-linked using calcium phosphate to form a matrix, and wherein said polysaccharide is a sulfated alginate or hyaluronan sulfate. In one embodiment, the composition further comprises a polypeptide linked to the polysaccharide. In another embodiment, the composition does not comprise a polypeptide, morphogen or growth factor.

[00155] In another embodiment, the present invention provides a composition comprising: a polysaccharide, and a polypeptide linked to said polysaccharide, wherein said polysaccharide is an alginate, a sulfated alginate, or hyaluronan sulfate, and said polypeptide is interleukin- 1 receptor antagonist (IL-1RA).

[00156] In one embodiment, the term "subject" includes, but is not limited to, a human. The methods of treatment described herein can be used to treat any suitable mammal, including primates, such as monkeys and humans, horses, cows, sheep, pigs, goats, cats, dogs, rabbits, birds such as turkey, chickens, and ducks, and rodents such as rats, mice, guinea pigs, and hamsters. In a particular embodiment, the mammal to be treated is human. Other subjects include species that are commonly used in scientific research, animal husbandry or as human companions.

[00157] All patents, patent applications, and scientific publications cited herein are hereby incorporated by reference in their entirety.

[00158] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES EXAMPLE 1

SPR evaluation of IL-1 receptor antagonist binding to alginate sulfate

Experiment objective:

[00159] Study the affinity of alginate-sulfate (VLVG-SO3) to Interleukin-1 Receptor Antagonist (IL-1RA) and determine the rate and equilibrium constants.

Experimental Materials and Methods:

[00160] 1) IL-1RA protein (#200-01RA, Peprotech); 2) Polysaccharides: VLVG - Sodium alginate (VLVG, >65 guluronic acid monomer content, FMC Biopolymers), Heparin (Sigma, #H3149), VLVG-S03 (alginate-sulfate, AlgS), synthesized from VLVG as described previously in the art. See e.g., Freeman et al., 2008, Biomaterials, vol. 29, pages 3260-3268, incorporated herein by reference.

[00161] The molecular interactions of sulfated alginate with IL-1RA were evaluated using Surface Plasmon Resonance (SPR) instrument BIACORE 3000 (Pharmacia, Uppsala, Sweden), operated using BIA evaluation version 3.2 Software.

[00162] Biotinylation of polysaccharides was done as previously described in the art. See e.g., Polyak et al. , 2004, Biomacromolecules, vol. 5, pages 389-396, incorporated herein by reference. Biotinylated polysaccharides (Heparin, VLVG, VLVG-SO3) were immobilized onto a streptavidin (SA) chip (#BR10003, General Electric) according to manufacturer's instructions. IL-1RA was diluted with HBS buffer to concentration of 6 μΜ and injected over the channels. Between assays, the sensor chip surface was regenerated (1 M NaCl and 1 mM NaOH). The real-time curve obtained from the reference channel (FC-1) was subtracted from the binding curves obtained from the flow channels containing the immobilized polysaccharides. Association and dissociation rate and equilibrium constants were calculated by nonlinear curve fitting of the sensorgrams using the Langmuir binding model with respect to mass transfer and drifting baseline, available in the BIA evaluation 3.2 Software.

Results:

[00163] The SPR assay showed moderate-low affinity to IL-1 Ra protein tested. Flow of high (6 μΜ) IL-IRA concentration showed weak, but specific, affinity binding by Alg-S, but not by alginate or heparin (Fig. l).

[00164] The constants obtained from fitting this single sensorgram to Langmuir 1 :1 binding model also indicate an existent, yet weak affinity binding of IL-IRA to AlgS with a relatively large K_D of 2.08 mM (Table 1).

Table 1. Alg-S-IL-IRA equilibrium rate and binding constants.

[00165] Association and dissociation rate constants (ko_n and koff, respectively) and equilibrium binding constants (KA and KD, respectively) were calculated from the interactions of AlgS with IL-IRA. Goodness of fit to a 1 :1 Langmuir biomolecular reaction model is indicated by χ2 values.

[00166] The results indicate a moderate, yet specific, interaction between the IL-IRA and Alg- S, which is not observed between alginate and IL-IRA.

EXAMPLE 2

Treatment with gel alone induces hyaline cartilage repair Materials and animals:

[00167] Sodium alginates (VLVG, >65 guluronic acid monomer content) were from FMC Biopolymers (Drammen, Norway). Alginate- sulfate was synthesized from sodium alginate (VLVG) as previously described (Freeman I. et al., 2008). Human recombinant transforming growth factor βΐ (TGF-βΙ) and human bone morphogenetic protein-4 (BMP -4) were from Peprotech (Rocky Hill, NJ). All chemicals, unless specified otherwise, were from Sigma Aldrich, and were analytical- grade.

[00168] Adult Sinclair mini-pigs (male, 13 months, -40 kg) were acquired from Harlan laboratories (Jerusalem, Israel). The experiments were conducted in Lahav CRO facility under an ethic committee approved protocol in accordance with local legislation and guidelines. Routine blood examination was performed prior to operation, 3 months after operation, and at euthanasia.

Methods:

Preparation of injectable growth factor-loaded affinity-binding alginate hydrogel:

[00169] The bioconjugates were prepared by mixing of reconstituted TGF-βΙ or BMP-4 solutions (reconstituted under manufacturer's instructions to a concentration of 500 μg/ml) with alginate sulfate solution (3%, w/v) for lh, at 37°C, to allow equilibrium binding of the factor. Stock solutions of sodium alginate (VLVG), and D-gluconic acid/hemicalcium salt were prepared by dissolving the materials in DDW and stirring at room temperature. Each solution was filtered separately through a sterile 0.2-μπι filter membrane into a sterile container in a laminar flow cabinet. Equal volumes from each stock solution (5.3% and 3% (w/v) for VLVG alginate and D-gluconic acid, respectively) were combined by extensive homogenization for several minutes to facilitate homogenous distribution of the calcium ions and cross-linking of alginate chains. Finally, the TGF-βΙ and BMP-4/alginate-sulfate bioconjugates were mixed with the cross-linked alginate solution to yield an injectable, affinity-bound TGF-βΙ or BMP4-containing, alginate solutions.

[00170] The composition of the alginate solution for the cartilage layer (w/v) was: Alginate: 1.82%; Ca-gluconate (D-gluconic acid/hemicalcium salt): 1.03%; and Alginate-sulfate: 0.67%. The composition of the alginate solution for the bone layer (w/v) was: Alginate: 1.78%; Ca-gluconate (D-gluconic acid/hemicalcium salt): 1.00%; and Alginate-sulfate: 0.87%.

[00171] The bilayer hydrogels were formed in-situ in the subchondral defect by injecting, layer-by- layer, first the BMP-4/affinity-bound alginate solution followed by the TGF- βΐ/affinity-bound alginate solution, as described in WO2013124855, which is incorporated herein by reference in its entirety. The amount of loaded TGF-βΙ or BMP-4 in each layer was 10 μ&

Subchondral defect model in Sinclair minipigs:

[00172] Using general anesthesia and sterile technique an anteromedial mini-arthrotomy of ~3 cm was performed and the patella was laterally dislocated. Standard OATS core punch (Arthrex) was used to create a subchondral defect (6 mm in diameter, 8 mm deep) in the weight bearing zone of the medial femoral condyle. The defect was cleaned and rinsed with sterile saline, and then washed with calcium chloride solution. The defect was first filled with -500 μΐ of BMP-4/affinity-binding alginate hydrogel and gelation was induced by addition of 1 M CaCb. After the instant gelation of the bottom layer, the top layer of TGF-βΙ /affinity- binding alginate was similarly constructed (-200 μΐ). Contralateral knees were treated with empty (w/o GFs) affinity-binding alginate hydrogel. After hydrogel application and gelation, the joint capsule was closed, and the wound was sutured in layers with bioabsorbable stitches. The animals were allowed to move freely in their cages with full load-bearing and no external support. The minipigs were killed 6 months after operation and treatment.

Histology and immunohistochemistry:

[00173] Explants were fixed in 4% paraformaldehyde (v/v, in PBS, pH 7.4) for 7 d. After embedding and polymerization in methyl-methacrylate (Techno vit 9100 Newl, Heraeus-

Kulzer, Hanau, Germany), thin sections (5μιη thick) were cut using RM 2155 microtome (Leica, Bensheim, Germany). Prior to performing staining, sections were deacrylated in xylol (2 x 15 min) and 2-methoxyethylacetate (2 x 10 min), cleared in a decreasing ethanol series (2 x isopropyl alcohol, 2 x 96% ethanol, 2 x 70% ethanol, 2 min each), and rehydrated in distilled water. Rehydrated sections were incubated in 0.1 % Toluidine blue or Safranin O for 20 s, washed in distilled water, dehydrated in ethanol, and mounted in Eukitt (Labonord, Monchengladbach, Germany). For type II collagen immunohistochemistry, the rehydrated sections were first mildly digested for antigen retrieval with 2% (v/v) proteinase K (Dako) in Tris-buffered saline (TBS) for 20 min at 37 °C, followed by a short wash in TBS and a decalcification step in EDTA buffer for 60 min at 37 °C. After washing in TBS, sections were pre-incubated with a solution of 3% (v/v) H₂O₂ in TBS for 30 min to block endogenous peroxidase activity, followed by an incubation for 30 min in a solution of 10% (v/v) normal goat serum (Vector/Linaris, Wertheim-Bettingen, Germany) in TBS to block unspecific binding. The primary mouse- anti-collagen II antibody was applied for 60 min at room temperature. The sections were washed three times in TBS (5 min each), followed by reacting with peroxidase-labelled secondary antibody for 30 min at room temperature. Peroxidase activity is visualized using the liquid DAB substrate chromogen system (Dako). Sections are washed twice with TBS and distilled water, and finally mounted with Aquatex (Merck, Darmstadt, Germany). Photomicrographs were taken with a Zeiss Axioskop 40 microscope equipped with a Zeiss AxioCam Mrc digital camera and Zeiss Axio Vision software, or Olympus light microscope (BX61 , Motorized System Microscope) connected to an Olympus (DD71) digital capture system and Olympus Cell^p software.

Results:

[00174] Figures 2-4 compare osteochondral defects treated with gel/cytokines in each subject versus its counterpart treated with gel only. Figure 2 and 3 show staining for cartilage proteoglycans, and Figure 4 shows staining for Collagen Type II, a prominent hyaline cartilage marker.

[00175] Treatment with cytokine-loaded gel induced hyaline cartilage regeneration, with typical cell organization, which was similar between the defect zone and distant healthy area.

This was associated with marked expression of Collage Type II. Importantly, however, gel only treatment (w/o cytokines) resulted in significant degree of staining for cartilage proteoglycans (as seen in Safranin O and Toluidine Blue stainings), as well as for Collagen Type II. While cellular organization in this group was different from healthy area (note hypertrophy and difference in cell morphology), the results clearly show good defect filling with repaired tissue with cartilage-like properties. The data therefore demonstrates that the alginate sulfate/alginate hydrogel can induce cartilage repair, even in the absence of cytokines.

[00176] Data from the study (Figs. 2-4) suggest that newly formed bone remained in the intended bone area and did not invade the cartilage area in both treatment formulations tested: with no morphogens in either gel, or with TGFbetal in the cartilage area, and BMP-4 in the bone area. These data support the suggestion that the multi-compartment hydrogel model contributed to the independent and separate development of bone and cartilage layers in this model.

EXAMPLE 3

Treatment with a multi-compartment hydrogel with no morphogen in the bone compartment and TGF-betal with alginate sulfate in the second compartment induces safe and effective hyaline cartilage repair

[00177] A bone gel layer comprising alginate and alginate sulfate in fluid form is applied to fill in the volume of bone where it is damaged, and/or where the surgeon has drilled or punched holes (microdrilling or microfracture), or has otherwise created a damaged volume on the bone surface. The bone gel layer does not include any polypeptide (though it might have non-protein additives).

[00178] Once the fluid gel layer is in place, it is cross linked in situ by addition of a cross- linker (calcium chloride) and allowed to gel at least partially, sufficient to establish a relatively firm surface, onto which the next layer (cartilage gel) can then be applied as a fluid.

[00179] The gel composition of the cartilage gel layer may be the same or different from that of the bone gel layer - in chemical makeup, and/or in density or size of pores.

[00180] Additives will be included in the bone gel layer which create large pores in the gel (typically in the 50-250 micron diameter range) if the bone defect is more than a couple mm deep.

[00181] Next, a cartilage gel layer is applied as a fluid on top of the gelled (or partially gelled) bone gel layer. The cartilage gel layer fills in the volume of damaged cartilage, which in some cases is pre- "trimmed" by the surgeon. The cartilage gel layer includes a polypeptide such as TGF-betal . The submicron pore size and associated web appearance of the fibers in the cartilage layer is similar to that of natural extracellular matrix (ECM) in cartilage.

[00182] The hydrogel plug created as described is flexible and sticky, adhering to the surrounding tissue surfaces of the defect volume it fills. The step-wise method of administration allows the plug can be shaped in order to match the shape of the missing healthy tissue. Flexing the joint during the procedure, so that the surrounding tissues rub against the plug, forces it into the defect volume and shapes its outer contours to precisely fill in the missing tissue and mate with the surroundings. Flexing the joint during the procedure does not tear out the newly formed plug, because the plug is adherent to the tissue. Flexing the joint during the procedure does not break the plug due to its flexibility.

[00183] The joint can bear weight and undergo moderate exercise starting soon after the procedure, without fear of tearing or damaging the newly treated area. The ability to apply weight and thus high mechanical pressure to the joint area promotes the differentiation of stem cells into chondrocytes to form cartilage. The ability to perform exercise early after the procedure further promotes the early return to normal function. Other methods typically require immobilizing the joint area for weeks in order to avoid tearing or damaging the newly treated area, thus delaying the repair process and return to normal function.

[00184] The cartilage layer provides a unique scaffold whose physical properties are similar to ECM, thus providing a scaffold that closely mimics that of natural cartilage, and whose fibers are affinity loaded with the relevant polypeptide cytokine (typically TGF-betal ) to promote stem cell migration into the scaffold and stimulate differentiation towards becoming chondrocytes to form hyaline cartilage. The stem cells migrate into the scaffold to encounter close-webbed fibers that carry the cytokine which triggers their differentiation towards chondrocytes to become hyaline cartilage. This is accomplished either by action on the relevant receptor(s) on the stem cells, whether via direct contact of the affinity-bound polypeptide with the receptor once the stem cells are attached to gel fibers, or via free polypeptide released in the scaffold volume.

[00185] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications that are within the spirit and scope of the invention, as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A multi-compartment hydrogel or scaffold comprising a polysaccharide selected from the group consisting of: an alginate, a sulfated alginate, and a hyaluronan sulfate, wherein at least one compartment of said multi-compartment hydrogel or scaffold does not comprise a polypeptide.

2. The multi-compartment hydrogel or scaffold of claim 1 , wherein one compartment of said multi-compartment hydrogel or scaffold is in a bone layer, while a second compartment is in a cartilage layer.

3. The multi-compartment hydrogel or scaffold of claim 2, wherein said compartment in said bone layer does not comprise a polypeptide, and said compartment in said cartilage layer comprises a polypeptide.

4. The multi-compartment hydrogel or scaffold of claim 3, wherein said polypeptide is TGF-betal .

5. The multi-compartment hydrogel or scaffold of any one of claims 1-4, wherein said composition further comprises an additive that is not a cell or polypeptide.

6. The multi-compartment hydrogel or scaffold of claim 5, wherein said additive is hydroxyapatite.

7. The multi-compartment hydrogel or scaffold of claim 5, wherein said additive is calcium phosphate.

8. The multi-compartment hydrogel or scaffold of claim 5, wherein said additive is mannitol beads.

9. The multi-compartment hydrogel or scaffold of claim 5, wherein said additive is magnesium ions.

10. The multi-compartment hydrogel or scaffold of any one of claims 1-9, wherein said composition further comprises a biological material from a subject.

11. The multi-compartment hydrogel or scaffold of claim 10, wherein said biological material is autologous to said subject.

12. The multi-compartment hydrogel or scaffold of any one of 10-11 wherein said biological material is platelet rich plasma (PRP).

13. The multi-compartment hydrogel or scaffold of any one of claims 10-11, wherein said biological material is bone marrow aspirate.

14. The multi-compartment hydrogel or scaffold of any one of claims 1-13, wherein said polysaccharides are cross-linked using calcium phosphate to form said hydrogel.

15. The multi-compartment hydrogel or scaffold of any one of claims 1-13, wherein said polysaccharides are cross-linked using calcium chloride or calcium gluconate to form said hydrogel.

16. The multi-compartment hydrogel or scaffold of any one of claims 1-15, wherein said hydrogel is a solid macroporous preformed scaffold.

17. The multi-compartment hydrogel or scaffold of any one of claims 1-16, wherein said hydrogel is homogenous or isotropic.

18. The multi-compartment hydrogel or scaffold of any one of claims 1-16, wherein said hydrogel is denser or less porous in one or more surface portions than its core.

19. The multi-compartment hydrogel or scaffold of any one of claims 1-16, wherein said hydrogel is denser or less porous in its core than in one or more predetermined surface portions.

20. The multi-compartment hydrogel or scaffold of any one of claims 1-19, wherein said hydrogel is an injectable composition.

21. The multi-compartment hydrogel or scaffold of any one of claims 1-20, wherein said hydrogel is bioerodable.

22. The multi-compartment hydrogel or scaffold of any one of claims 1-21, wherein a second compartment of said multi-compartment hydrogel or scaffold comprises a polypeptide.

23. The multi-compartment hydrogel or scaffold of any claim 22, wherein said polypeptide is present in an amount effective to repair or regenerate a damaged tissue in a subject.

24. The multi-compartment hydrogel or scaffold of any one of claims 22-23, wherein said polypeptide is biologically active.

25. The multi-compartment hydrogel or scaffold of any one of claims 22-24, wherein said polypeptide is linked to said alginate or said sulfated alginate to form a bioconjugate.

26. The multi-compartment hydrogel or scaffold of claim 25, wherein said bioconjugate forms a nanop article.

27. The multi-compartment hydrogel or scaffold of any one of claims 22-26, wherein said polypeptide is linked to said polysaccharide by a reversible non-covalent binding involving ionic bonds, an electrostatic interaction, a hydrophobic interaction, or hydrogen bonds or van der Waals forces.

28. The multi-compartment hydrogel or scaffold of any one of claims 22-26, wherein said polypeptide is linked to said polysaccharide via a linker.

29. The multi-compartment hydrogel or scaffold of claim 28, wherein said linker is a peptide.

30. The multi-compartment hydrogel or scaffold of any one of claims 28-29, wherein said linker is a heparin binding peptide.

31. The multi-compartment hydrogel or scaffold of any one of claims 22-30, wherein said polypeptide is TGF-beta.

32. The multi-compartment hydrogel or scaffold of any one of claims 22-30, wherein said polypeptide is a heparin-binding polypeptide.

33. The multi-compartment hydrogel or scaffold of claim 29, wherein said heparin- binding polypeptide is transforming growth factor βΐ (TGF-βΙ), antithrombin III (AT III), thrombopoietin (TPO), serine protease inhibitor (SLP1), CI esterase inhibitor (CI INH), Vaccinia virus complement control protein (VCP), a fibroblast growth factor (FGF), a FGF receptor, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), a platelet-derived growth factor (PDGF), epidermal growth factor (EGF), CXC chemokine ligand 4 (CXCL4), stromal cell-derived factor-l (SDF-l), interleukin-6 (IL-6), interleukin-8 (IL-8), Regulated on Activation, Normal T Expressed and Secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory peptide-1 (MIP-1), lymphotactin, fractalkine, an annexin, apolipoprotein E (ApoE), immunodeficiency virus type-1 (HIV-1) coat protein gpl20, cyclophilin A (CypA), Tat protein, viral coat glycoprotein gC, gB or gD of herpes simplex virus (HSV), an envelope protein of Dengue virus, circumsporozoite (CS) protein of Plasmodium falciparum, bacterial surface adhesion protein OpaA, 1-selectin, P-selectin, heparin-binding growth-associated molecule (HB-GAM), thrombospondin type I repeat (TSR), peptide myelin oligodendrocyte glycoprotein (MOG), amyloid P (AP) PDGF-BB, PDGF-AA, bFGF, aFGF, VEGF, IL-6, TPO, SDF-1 , HGF, EGF,MOG, or IGF-1, BMP2, BMP4, BMP7.

34. The multi-compartment hydrogel or scaffold of claim 30, wherein said heparin- binding polypeptide is a polypeptide exhibiting angiogenic activity.

35. The multi-compartment hydrogel or scaffold of claim 34, wherein said polypeptide exhibiting angiogenic activity is VEGF, bFGF, aFGF, PDGF- ββ, IGF-1 , HGF, or a combination thereof.

36. The multi-compartment hydrogel or scaffold of any one of claims 22-26, 28-29, wherein said polypeptide is interleukin-1 receptor antagonist (IL-1RA).

37. The multi-compartment hydrogel or scaffold of any one of claims 1-36, wherein said multi-compartment hydrogel or scaffold is for use in repair, regeneration, or prevention of further degeneration of a damaged tissue in a subject.

38. The multi-compartment hydrogel or scaffold of any one of claims 1-36, wherein said multi-compartment hydrogel or scaffold is for use as an adjunct treatment to surgical microfracture or microdrilling in a subject with an osteochondral defect, osteoarthritis, or both.

39. The multi-compartment hydrogel or scaffold of any one of claims 1-36, wherein said multi-compartment hydrogel or scaffold is for use in treating rheumatoid arthritis, osteochondral defect, osteoarthritis, microfracture, post-trauma arthritis, anterior cruciate ligament (ACL) injury, meniscal tear, or articular fracture.

40. The multi-compartment hydrogel or scaffold of any one of claims 1-36, wherein said multi-compartment hydrogel or scaffold is for use in high-limb ischemia, myocardial infarction, or spinal cord injury.

41. The multi-compartment hydrogel or scaffold of any one of claims 1-36, wherein said multi-compartment hydrogel or scaffold is for use in enhancing an immunotolerant response.

42. Use of the multi-compartment hydrogel or scaffold of any one of claims 1-41 in a method of repairing, regenerating, or preventing additional degeneration of a damaged tissue in a subject.

43. Use of the multi-compartment hydrogel or scaffold of any one of claims 1-41 in a method for treating an osteochondral defect in a subject

44. Use of the multi-compartment hydrogel or scaffold of any one of claims 1-41 in a method for treating a rheumatoid arthritis in a subject.

45. Use of the multi-compartment hydrogel or scaffold of any one of claims 1-41 in a method for treating a joint injury or its associated condition in a subject.

46. The use of claim 45, wherein said joint injury associated condition is an equine lameness, secondary to said joint injury.

47. The use of claim 45, wherein said joint injury is from surgical microfracture or microdrilling.

48. The use of any one of claims 42-47, wherein a first hydrogel layer is applied to the bone layer in the joint of said subject and subsequently a second hydrogel layer is applied to the cartilage layer of the joint of said subject.

49. The use of claim 48, wherein said second hydrogel layer is applied after the formation of a distinct physical interface by said first hydrogel layer.

50. The use of any one of claims 48-49, wherein said first hydrogel layer does not comprise a morphogen, and said second hydrogel layer comprises a polypeptide.

51. The use of claim 50, wherein said polypeptide is TGF-betal.

52. The use of any one of claims 42-51, wherein said multi-compartment hydrogel or scaffold is a solid macroporous preformed scaffold.

53. A composition comprising: a polysaccharide and an additive selected from the group consisting of: hydroxyapatite, calcium phosphate, mannitol beads, and magnesium ions, or a combination thereof, wherein said polysaccharide is a sulfated alginate, or hyaluronan sulfate.

54. A composition comprising a polysaccharide and a biological material from a subject, wherein said polysaccharide is a sulfated alginate or hyaluronan sulfate.

55. A composition comprising polysaccharides cross-linked using calcium phosphate to form a matrix, and wherein said polysaccharide is a sulfated alginate or hyaluronan sulfate.

56. The composition of claim 55, wherein said calcium phosphate is amorphous calcium phosphate.

57. A composition comprising a polysaccharide, and a polypeptide linked to said polysaccharide, wherein said polysaccharide is an alginate, a sulfated alginate or hyaluronan sulfate, and said polypeptide is interleukin- 1 receptor antagonist (IL-1RA).

58. The composition of any one of claims 54-57, wherein said composition further comprises an additive that is not a cell or polypeptide.

59. The composition of claim 58, wherein said additive facilitates tissue regeneration or repair of a damaged tissue in a subject.

60. The composition of any one of claims 58-59, wherein said additive is hydroxyapatite, calcium phosphate, mannitol beads, magnesium ions, or a combination thereof.

61. The composition of any one of claims 53 and 56-60, wherein said composition further comprises a biological material from a subject.

62. The composition of any one of claims 54 and 61, wherein said biological material is autologous to said subject.

63. The composition of any one of claims 54 and 61, wherein said biological material is platelet rich plasma (PRP).

64. The composition of any one of claims 54 and 61, wherein said biological material is bone marrow aspirate.

65. The composition of any one of claims 53-56 and 58-64, wherein said composition further comprises a polypeptide linked to said polysaccharide.

66. The composition of any one of claims 57 and 65, wherein said alginate or sulfated alginate and said polypeptide are present in an amount effective to repair or regenerate a damaged tissue in a subject.

67. The composition of any one of claims 57 and 65-66, wherein said polypeptide is biologically active.

68. The composition of any one of claims 57 and 65-67, wherein said polypeptide is linked to said alginate or said sulfated alginate to form a bioconjugate.

69. The composition of claim 67, wherein said bioconjugate forms a nanoparticle.

70. The composition of any one of claims 65-69, wherein said polypeptide is linked to said polysaccharide by a reversible non-covalent binding involving ionic bonds, an electrostatic interaction, a hydrophobic interaction, or hydrogen bonds or van der Waals forces.

71. The composition of any one of claims 57 and 65-70, wherein said polypeptide is linked to said polysaccharide via a linker.

72. The composition of claim 71, wherein said linker is a peptide.

73. The composition of any one of claims 71-72, wherein said linker is a heparin binding peptide.

74. The composition of any one of claims 57 and 65-73, wherein said composition further comprises an additional polypeptide.

75. The composition of claim 74, wherein said polypeptide or said additional polypeptide is a heparin-binding polypeptide.

76. The composition of any claim 75, wherein said heparin-binding polypeptide is transforming growth factor βΐ (TGF-βΙ), antithrombin III (AT III), thrombopoietin (TPO), serine protease inhibitor (SLP1), CI esterase inhibitor (CI INH), Vaccinia virus complement control protein (VCP), a fibroblast growth factor (FGF), a FGF receptor, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), a platelet-derived growth factor (PDGF), epidermal growth factor (EGF), CXC chemokine ligand 4 (CXCL4), stromal cell-derived factor-l(SDF-l), interleukin-6 (IL-6), interleukin-8 (IL-8), Regulated on Activation, Normal T Expressed and Secreted (RANTES), monocyte chemoattractant protein- 1 (MCP-1), macrophage inflammatory peptide- 1 (MIP-1), lymphotactin, fractalkine, an annexin, apolipoprotein E (ApoE), immunodeficiency virus type-1 (HIV-1) coat protein gpl20, cyclophilin A (CypA), Tat protein, viral coat glycoprotein gC, gB or gD of herpes simplex virus (HSV), an envelope protein of Dengue virus, circumsporozoite (CS) protein of Plasmodium falciparum, bacterial surface adhesion protein OpaA, 1-selectin, P-selectin, heparin-binding growth-associated molecule (HB-GAM), thrombospondin type I repeat (TSR), peptide myelin oligodendrocyte glycoprotein (MOG), amyloid P (AP) PDGF-BB, PDGF-AA, bFGF, or aFGF, BMP2, BMP4, BMP7.

77. The composition of claim 75, wherein said heparin-binding polypeptide is a polypeptide exhibiting angiogenic activity.

78. The composition of claim 77, wherein said polypeptide exhibiting angiogenic activity is VEGF, bFGF, aFGF, PDGF- ββ, IGF-1 , HGF, or a combination thereof.

79. The composition of any one of claims 65-69 and 71-73, wherein said polypeptide is inter leukin-1 receptor antagonist (IL-1RA).

80. The composition of any one of claims 53-79, wherein said composition is a multicompartment composition.

81. The composition of claim 80, wherein said IL-1RA is in a first compartment and an additional polypeptide is in a second compartment.

82. The composition of claim 81 , wherein said first compartment is a first layer and said second compartment is a second layer.

83. The composition of any one of claims 53-54 and 57-82, wherein the polysaccharide of said composition forms a supporting matrix.

84. The composition of claim 83, wherein said polysaccharides are cross-linked using calcium phosphate to form said matrix.

85. The composition of claim 83, wherein said polysaccharides are cross-linked using calcium chloride or calcium gluconate to form said matrix.

86. The composition of any one of claims 53-54 and 57-82, wherein said composition further comprises a supporting matrix.

87. The composition of claim 86, wherein said supporting matrix is a polymer selected from the group consisting of a polysaccharide, a protein, an extracellular matrix component, a synthetic polymer, and a mixture thereof.

88. The composition of any one of claims 53-87, wherein said composition is homogenous or isotropic.

89. The composition of any one of claims 53-87, wherein said composition is denser or less porous in one or more surface portions than its core.

90. The composition of any one of claims 53-87, wherein said composition is denser or less porous in its core than in one or more predetermined surface portions.

91. The composition of any one of claims 53-90, wherein said composition is a hydrogel.

92. The composition of any one of claims 53-90, wherein said composition is a solid macroporous preformed scaffold.

93. The composition of any one of claims 53-91 , wherein said composition is an injectable composition.

94. The composition of any one of claims 53-93, wherein said composition is bioerodable.

95. The composition of any one of claims 53-94, wherein said composition is for use in repair or regeneration or prevention of degeneration of a damaged tissue in a subject.

96. The composition of any one of claims 53-94, wherein said composition is for use as an adjunct treatment to surgical microfracture or microdrilling in a subject with an osteochondral defect, osteoarthritis, or both.

97. The composition of any one of claims 53-94, wherein said composition is for use in treating rheumatoid arthritis, osteochondral defect, osteoarthritis, microfracture, post-trauma arthritis, anterior cruciate ligament (ACL) injury, meniscal tear, or articular fracture.

98. The composition of any one of claims 53-94, wherein said composition is for use in high-limb ischemia, myocardial infarction, heart failure, stroke, or spinal cord injury.

99. The composition of any one of claims 53-94, wherein said composition is for use in enhancing an immuno tolerant response.

100. A multi-compartment hydrogel or scaffold comprising the composition of any one of claims 53-94 in one or more of the compartments of said multi-compartment hydrogel or scaffold.

101. A method for repairing or regenerating a damaged tissue in a subject, the method comprising: administering to said subject the composition of any one of claims 53 and 61- 100, thereby repairing or regenerating said damaged tissue in said subject.

102. A method for repairing or regenerating a damaged tissue in a subject, the method comprising: administering to said subject the composition of any one of claims 54, 58-60 and 65-100, thereby repairing or regenerating said damaged tissue in said subject.

103. A method for repairing or regenerating a damaged tissue in a subject, the method comprising: administering to said subject the composition of any one of claims 55, 58-82, and 88-100, thereby repairing or regenerating said damaged tissue in said subject.

104. A method for preventing further degeneration of an injured tissue in a subject, the method comprising: administering to said subject the composition of any one of claims 56-78 and 80-100, thereby preventing further degeneration of said injured tissue in said subject.

105. A method for treating an osteochondral defect in a subject, the method comprising: administering to said subject the composition of any one of claims 53 and 61-100, thereby treating said osteochondral defect in said subject.

106. A method for treating an osteochondral defect in a subject, the method comprising: administering to said subject the composition of any one of claims 54, 58-60 and 65-100, thereby treating said osteochondral defect in said subject.

107. A method for treating an osteochondral defect in a subject, the method comprising: administering to said subject the composition of any one of claims 55, 58-82, and 88-100, thereby treating said osteochondral defect in said subject.

108. A method for treating an osteochondral defect in a subject, the method comprising: administering to said subject the composition of any one of claims 56-78 and 80-100, thereby treating said osteochondral defect in said subject.

109. A method for treating rheumatoid arthritis in a subject, the method comprising: administering to said subject the composition of any one of claims 53 and 61-100, thereby treating said rheumatoid arthritis in said subject.

110. A method for treating rheumatoid arthritis in a subject, the method comprising: administering to said subject the composition of any one of claims 54, 58-60 and 65-100, thereby treating said rheumatoid arthritis in said subject.

111. A method for treating rheumatoid arthritis in a subject, the method comprising: administering to said subject the composition of any one of claims 55, 58-82, and 88-100, thereby treating said rheumatoid arthritis in said subject.

112. A method for treating rheumatoid arthritis in a subject, the method comprising: administering to said subject the composition of any one of claims 56-78 and 80-100, thereby treating said rheumatoid arthritis in said subject.

113. A method for treating a joint injury or its associated condition in a subject, the method comprising: administering to said subject the composition of any one of claims 53 and 61- 100, 24-26, 28-41 , thereby treating said joint injury or its associated condition in said subject.

114. A method for treating a joint injury or its associated condition in a subject, the method comprising: administering to said subject the composition of any one of claims 54, 58-60 and 64-99, thereby treating said joint injury or its associated condition in said subject.

115. A method for treating a joint injury or its associated condition in a subject, the method comprising: administering to said subject the composition of any one of claims 55, 58-82, and 88-100, thereby treating said joint injury or its associated condition in said subject.

116. A method for treating a joint injury or its associated condition in a subject, the method comprising: administering to said subject the composition of any one of claims 56-78 and 80- 100, thereby treating said joint injury or its associated condition in said subject.

117. The method of any one of claims 113-116, wherein said joint injury associated condition is an equine lameness, secondary to said joint injury.

118. The method of any one of claims 113-116, wherein said joint injury is from surgical microfracture or microdrilling.

119. The method of any one of claims 101-118, wherein said step of administering comprises implanting said composition as a solid macroporous preformed scaffold.

120. A method for preparing a matrix comprising alginate and i) sulfated alginate or ii) sulfated hyaluronan comprising the step of: a) combining said alginate with amorphous calcium phosphate and b) adding said sulfated alginate or sulfated hyaluronan, thereby preparing a matrix comprising alginate and i) sulfated alginate or ii) sulfated hyaluronan.

121. The method of claim 120, wherein said method further comprises the step of adding a bioconjugate to the alginate solution.

122. The method of any one of claims 120-121 , further comprising the step of adding a second cross-linker after the completion of the prior steps.

123. The method of any one of claims 120-122, wherein said second cross-linker is dissolved amorphous calcium phosphate.

124. An osteogenic fusion device comprising a pre-formed scaffold comprising cross- linked alginate sulfate or hyaluronan sulfate, wherein said scaffold is designed with dimensions so as to fit into said osteogenic fusion device and wherein said scaffold maintains its shape.

125. The osteogenic fusion device of claim 124, wherein said scaffold is an alginate scaffold.

126. The osteogenic fusion device of any one of claims 124-125, wherein said scaffold is cross-linked using amorphous calcium phosphate.

127. The osteogenic fusion device of any one of claims 124-126, wherein said osteogenic fusion device is a metal device.

128. The osteogenic fusion device of any one of claims 124-127, wherein said osteogenic fusion device is a LT-Cage® Tapered Fusion Device.

129. The osteogenic fusion device of any one of claims 124-128, wherein said scaffold comprises a bioactive polypeptide.

130. The osteogenic fusion device of claim 129, wherein said polypeptide is bone morphogenetic protein (BMP)-2.

131. The osteogenic fusion device of claim 129, wherein said polypeptide is BMP-4.

132. The osteogenic fusion device of any one of claims 124-131 , for use for bone regeneration, bone growth, vertebrate fusion, or a combination thereof.

133. A method of facilitating bone regeneration, bone growth, vertebrate fusion, or a combination thereof comprising the step of implanting in a subject the osteogenic fusion device of any one of claims 124-131 , thereby facilitating bone regeneration, bone growth, vertebrate fusion, or a combination thereof.