Patent Publication Number: US-2015064143-A1

Title: Ionically cross-linkable alginate-grafted hyaluronate compound

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims priority to and the benefit of Korean Patent Application No. 10-2013-0106333, filed on Sep. 4, 2013, and No. 10-2014-0113971, filed on Aug. 29, 2014, the disclosure of which is incorporated herein by reference in its entirety. 
     BACKGROUND 
     1. Field of the Invention 
     The present invention relates to an alginate-grafted hyaluronate compound. 
     2. Background of Technique 
     Natural polymer-based hydrogels have been used in various biomedical applications, due to their superior intrinsic biocompatibility compared to synthetic polymer-based hydrogels. Natural polymers have relatively low potential for toxicity, which is crucial with regard to biomaterial design. Among natural polymers, hyaluronate has been used in many biomedical applications, including drug delivery [1,2] and tissue engineering [3,4] applications, due to excellent biocompatibility and biological functionality. This polysaccharide is composed of repeating units of β-1,4-D-glucuronic acid-β-1,3-N-acetyl-D-glucosamine residues [5], and is abundant in synovial fluid and extracellular matrix. Hydrogels prepared from hyaluronate are particularly attractive, due to their excellent biological function, excellent viscoelastic properties, high water content and biodegradable properties. One typical preparation method of hyaluronate gels is chemical cross-linking [6-9]. Chemical cross-linking reagents, however, can induce acute or chronic side effects, such as immune and inflammatory responses[10]. This may cause potential risk, and limit wide biomedical applications of hyaluronate. 
     Physical cross-linking is an alternative approach that can be applied in overcoming toxicity issues brought about by chemical cross-linking. Hydrogels can be physically prepared, by varying external environments around stimulus-responsive polymers (e.g., temperature[11,12] and/or pH[13,14]), or by inducing physical interactions (e.g., ionic[15,16] or hydrophobic interactions[17,18]) to polymers. Alginate forms physically cross-linked structures in the presence of divalent cations (e.g., Ca 2+ ), but in the absence of chemical cross-linkers. Alginate is a linear copolymer composed of blocks of β-D-mannuronate (M) and α-L-guluronate (G) residues [19,20]; it is also a naturally derived biomaterial. It is utilized in biomedical applications owing to its excellent biocompatibility and low toxicity [21,22]. Specifically, alginate modified with cell-adhesive motifs (e.g., RGD peptide) has often been utilized as injectable hydrogels for tissue engineering applications [23,24]. 
     Articular cartilage is important for overall individual well-being. Articular cartilage functions and structures often get interrupted or damaged, due to physical injuries or degenerative diseases, such as osteoarthritis. Because cartilage tissue is both a neural and a vascular, chondrocyte proliferation is slow, and tissue defects are rarely spontaneously recovered[25,26]. Tissue engineering approaches using hydrogels have demonstrated great potential for treating articular cartilage defects through minimally-invasive chondrocyte delivery. Such minimally-invasive chondrocyte delivery may reduce cost, recovery time, and patient pain [27]. Another benefit of hydrogels is their superior viscoelastic properties. Viscoelastic properties of hydrogels are similar to those of cartilage natural extracellular matrix (ECM) materials [28-30]. Hyaluronate is a promising candidate for cartilage regeneration, as it is a main component of the ECM of native cartilage. In addition, hyaluronate interacts with CD44 on the chondrocyte surface, which is a molecule that is important in chondrocyte metabolism [31-33]. Hyaluronate has been extensively used in cartilage regeneration applications [34-36]. 
     Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains. 
     SUMMARY 
     The present inventors have endeavored to develop a method for easily cross-linking hyaluronate, which is a natural polymer, without a chemical cross-linking reagent. As a result, the present inventors have established that alginate-grafted hyaluronate which can be easily cross-linked through only addition of a divalent cation without using an excipient chemical cross-linking reagent can be prepared by grafting alginate to hyaluronate using a covalent linkage, and then completed the present invention. 
     Therefore, an aspect of the present invention is to provide an ionically cross-linkable alginate-grafted hyaluronate compound. 
     Another aspect of the present invention is to provide a composition for cell transplantation. 
     Still another aspect of the present invention is to provide a pharmaceutical composition for cartilage regeneration. 
     Still another aspect of the present invention is to provide a pharmaceutical composition for preventing or treating cartilage-injury disorders or diseases. 
     Still another aspect of the present invention is to provide a method for preparing an ionically cross-linkable alginate-grafted hyaluronate modification. 
     Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  represents schematic description for hyaluronate-g-alginate (HGA) and its hydrogel formation in the presence of calcium ions. Hyaluronate (HA) was initially modified with ethylenediamine (NH 2 -HA). Then, NH 2 -HA was reacted with alginate (AL) via carbodiimide chemistry. 
         FIG. 2   a  represents images of hyaluronate (HA) or hyaluronate-g-alginate (HGA) solution after mixing, in the presence (+) or absence (−) of calcium ions. 
         FIG. 2   b  represents image of HGA gels after injection via syringe needle. 
         FIG. 2   c  represents changes in storage modulus (G′, filled symbols) and loss modulus (G″, open symbols) of HGA1 (circles) and HA (squares) solutions in the presence of calcium ions. 
         FIG. 2   d  represents changes in storage modulus depending on hyaluronate content in hyaluronate-g-alginate gels ([HGA1]=2 wt %). 
         FIG. 2   e  represents changes in storage modulus depending on calcium concentration in hyaluronate-g-alginate gels ([HGA1]=2 wt %). 
         FIG. 2   f  represents gelation time of HGA gels. 
         FIG. 2   g  represents storage modulus of HGA gels depending on alginate molecular weight ([HGA1]=2 wt %, [CaSO 4 ]=29.7 Mm). 
         FIG. 3   a  represents changes in dry weight of either hyaluronate/alginate mixed gels or hyaluronate-g-alginate gels after treated with hyaluronidase for 2 weeks at 37° C. 
         FIG. 3   b  represents cross-sectional SEM images of alginate/hyaluronate mixed gels after incubation with hyaluronidase over 2 weeks at 37° C. ([polymer]=2 wt %, hyaluronate/alginate (mg/mg)=1, [Ca 2+ ]=29.7 mM, [hyaluronidase]=100 μg/ml). 
         FIG. 3   c  represents cross-sectional SEM image of hyaluronate-g-alginate gels after incubation with hyaluronidase over 2 weeks at 37° C. ([polymer]=2 wt %, hyaluronate/alginate (mg/mg)=1, [Ca 2+ ]=29.7 mM, [hyaluronidase]=100 μg/ml). 
         FIG. 4  represents histological images of tissue section stained with Alcian blue (a-d) and Sirius red (e-h). Immunohistochemical analysis of Matrillin-1 protein (i-l) and S100 protein (m-p) was also carried out. Tissue sections were retrieved from mice after six weeks of transplantation with primary chondrocytes ([cell]=1.0×10 7  cells/ml) and hyaluronate-g-alginate gels. 
         FIG. 5   a  represents quantitative analysis of (a) sulfated GAG formation (**P&lt;0.01). 
         FIG. 5   b  represents quantitative analysis of gene expression levels of SOX-9 (*P&lt;0.05). 
         FIG. 5   c  represents quantitative analysis of gene expression levels of aggrecan (**P&lt;0.01). 
         FIG. 5   d  represents quantitative analysis of gene expression levels of type II collagen in regenerated cartilage tissues (*p&lt;0.05). 
         FIG. 6   a  represents  1 H-NMR spectra of hyaluronate, alginate, and hyaluronate-g-alginate. 
         FIG. 6   b  represents DMMB assay results for quantitative grafting efficiency of hyaluronate-g-alginate. 
     
    
    
     DETAILED DESCRIPTION 
     In one aspect of the present invention, there is provided an ionically cross-linkable alginate-grafted hyaluronate compound containing alginate and hyaluronate, the alginate and the hyaluronate forming a covalent linkage. 
     The present inventors have endeavored to develop a method for easily cross-linking hyaluronate, which is a natural polymer, without using a chemical cross-linking agent. As a result, the present inventors have established that alginate-grafted hyaluronate which can be easily cross-linked only through the addition of a divalent cation without using a chemical cross-linking reagent can be prepared by grafting alginate to hyaluronate using a covalent linkage (see  FIG. 1 ). 
     As used herein, the term “alginate-grafted hyaluronate compound” refers to hyaluronate linked to alginate through a covalent linkage. As used herein, the term “alginate-grafted hyaluronate compound” may be exchangeably used with “alginate-grafted hyaluronate”, “hyaluronate-g-alginate”, “modified hyaluronate”, or “modified compound”, all of which have the same meaning. The alginate-grafted hyaluronate compound of the present invention can be used to easily and conveniently prepare a hydrogel via an ionic cross-linking. For the formation of a hyaluronate hydrogel in the prior art, a method of forming a hydrogel using a chemical cross-linking reagent has been employed. However, the alginate-grafted hyaluronate compound of the present invention can easily induce an ionic cross-linking reaction by addition of divalent cations, such as Ca 2+ , Ba 2+ , Cu 2+ , Fe 2+ , and Mg 2+ , due to cross-linking characteristics of alginate introduced into hyaluronate. This is important in that the possibility that the chemical cross-linking reagent may cause side effects, such as an immune or inflammation response, in the body can be eliminated. Therefore, a divalent cation, preferably Ca 2+ , may be added to the alginate-grafted hyaluronate compound before the injection of the compound into the body, and then the compound may be injected to the body before gelation. After the injection, the compound can form a hydrogel through prompt gelation behavior. In addition, a hydrogel in which alginate and hyaluronate are uniformly dispersed all over can be formed. 
     A hydrogel composition prepared from the modified compound of the present invention can control mechanical properties and gelation behavior according to the degree of introduction of alginate. The present inventors have verified that as the weight ratio of alginate increases from 1:4 (hyaluronate:alginate) to 1:1, the elastic coefficient of the ionic cross-linking hydrogel also increases (see  FIG. 2   d ). The reason seems to be that the number of ionically cross-linkable points increases on the structure. The mechanical properties of the ionically cross-linked alginate-grafted hyaluronate hydrogel are shown to have lower levels than the ionically cross-linked alginate gel, but are sufficient to maintain a gel structure in the body after transplantation. In addition, the hydrogel composition prepared from the modified compound control mechanical properties and gelation behavior according to the amount of divalent cations added. 
     In addition, the alginate-grafted hyaluronate compound of the present invention has many advantages compared with an alginate/hyaluronate mixture. The modified hyaluronate enables the formation of a hydrogel in which alginate and hyaluronate are homogeneously distributed; have a significantly shortened gelation time and thus can be transplanted in the body immediately after the addition of a divalent cation for gelating the modified hyaluronate; and can be gelated in a short time even after the transplantation into the body and thus very useful in application fields including cell transplantation and the like. For example, in the case where chondrocytes are transplanted into the part of articulation, a transplanted subject can behave in a short time without requiring a long waiting time even after the cell transplantation. Further, the modified hyaluronate can be gelated using a divalent cation based on characteristics of introduced alginate, without using a chemical cross-linking reagent, and thus has superior biocompatibility, thereby avoiding various side effects due to the use of the chemical cross-linking agent. Further, the content ratio between alginate and hyaluronate, the content of divalent cations, and the like are controlled, thereby facilitating the control of mechanical properties. 
     According to an embodiment of the present invention, the preferable molecular weight of alginate, which is usable to prepare the alginate-grafted hyaluronate compound of the present invention, is 10,000-1,000,000 g/mol, and the preferable molecular weight of hyaluronate is 10,000-1,000,000 g/mol. 
     According to an embodiment of the present invention, the weight ratio between alginate and hyaluronate contained in the alginate-grafted hyaluronate compound of the present invention is 10:1 to 1:10. The weight ratio between alginate and hyaluronate is specifically 5:1 to 1:8, more specifically 2:1 to 1:6, more specifically 1:1 to 1:4, more specifically 1:1.2 to 1:3, still more specifically 1:1.5 to 1:2.5. 
     According to an embodiment of the present invention, the covalent linkage between alginate and hyaluronate is achieved via a linker capable of forming a covalent linkage between a carboxyl group of alginate and a carboxyl group of hyaluronate. As the linker forming a covalent linkage between hyaluronate and alginate, any linker that has at least two functional groups capable of reacting with carboxyl groups to form a covalent linkage and is known in the art can be used without limitation. 
     According to an embodiment of the present invention, the linker of the present invention which links alginate and hyaluronate through a covalent linkage is selected from diamine, divinylsulfone, 1,4-butanediol diglycidyl ether (BDDE), glutaraldehyde, carbodiimide, hydroxysuccinimide, imidoester, maleimide, haloacetyl, disulfide hydroazide, and alkoxy amine. According to another embodiment of the present invention, the linker is diamine, and preferably ethylene diamine. 
     In order to link alginate and hyaluronate through a covalent linkage by using the linker, any one selected from alginate and hyaluronate is preferentially treated, and here, it is important to use a large amount of linker to prevent self-assembling. As used herein, the term “self-assembling” refers to the linkage of alginate molecules or hyaluronate molecules, that is, homologous molecules, via one linker, but not the linkage of alginate and hyaluronate molecules, that is, heterogeneous molecules via one linker, and includes the linkage of several carboxyl groups in one molecule with one linker. 
     As used herein, alginate or hyaluronate which first reacts with a linker is called a “first reactant”, and a compound which is later added in order to be linked to the linker linked to the first reactant is called a “second reactant”. That is, when the first reactant is alginate, the second reactant is hyaluronate, and when the first reactant is hyaluronate, the second reactant is alginate. Before the first reactant linked to the linker reacts with the second reactant, a linker that is not linked to the first reactant is removed by filtration, for example, a dialysis procedure. After that, through additional reaction with hyaluronate or alginate, which is a second reactant, an alginate-grafted hyaluronate compound is finally obtained. 
     According to an embodiment of the present invention, a cell adhesive peptide is linked to at least one of alginate and hyaluronate of the present invention. Any one cell adhesive peptide that is known in the art may be used. 
     According to an embodiment of the present invention, the cell-adhesive peptide of the present invention is at least one selected from the group consisting of RGD(Arg-Gly-Asp), RGDS(Arg-Gly-Asp-Ser) (SEQ ID NO: 1), RGDC(Arg-Gly-Asp-Cys) (SEQ ID NO: 2), RGDV(Arg-Gly-Asp-Val) (SEQ ID NO: 3), RGES(Arg-Gly-Glu-Ser) (SEQ ID NO: 4), RGDSPASSKP(Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro) (SEQ ID NO: 5), GRGDS(Gly-Arg-Gly-Asp-Ser) (SEQ ID NO: 6), GRADSP(Gly-Arg-Ala-Asp-Ser-Pro) (SEQ ID NO: 7), KGDS(Lys-Gly-Asp-Ser) (SEQ ID NO: 8), GRGDSP(Gly-Arg-Gly-Asp-Ser-Pro) (SEQ ID NO: 9), GRGDTP(Gly-Arg-Gly-Asp-Thr-Pro) (SEQ ID NO: 10), GRGES(Gly-Arg-Gly-Glu-Ser) (SEQ ID NO: 11), GRGDSPC(Gly-Arg-Gly-Asp-Ser-Pro-Cys) (SEQ ID NO: 12), GRGESP(Gly-Arg-Gly-Glu-Ser-Pro) (SEQ ID NO: 13), SDGR(Ser-Asp-Gly-Arg) (SEQ ID NO: 14), YRGDS(Tyr-Arg-Gly-Asp-Ser) (SEQ ID NO: 15), GQQHHLGGAKQAGDV (Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val) (SEQ ID NO: 16), GPR(Gly-Pro-Arg), GHK(Gly-His-Lys), YIGSR(Tyr-Ile-Gly-Ser-Arg) (SEQ ID NO: 17), PDSGR(Pro-Asp-Ser-Gly-Arg) (SEQ ID NO: 18), CDPGYIGSR(Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg) (SEQ ID NO: 19), LCFR(Leu-Cys-Phe-Arg) (SEQ ID NO: 20), EIL(Glu-Ile-Leu), EILDV(Glu-Ile-Leu-Asp-Val) (SEQ ID NO: 21), EILDVPST(Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr) (SEQ ID NO: 22), EILEVPST(Glu-Ile-Leu-Glu-Val-Pro-Ser-Thr) (SEQ ID NO: 23), LDV(Leu-Asp-Val), and LDVPS(Leu-Asp-Val-Pro-Ser) (SEQ ID NO: 24). More specifically, RGD(Arg-Gly-Asp) may be used. The cell adhesive peptide serves to improve an interaction between cells and a hydrogel formed by the modified hyaluronate. 
     In another aspect of the present invention, there is provided a method for cell transplantation, comprising: 
     (a) preparing a composition comprising the alginate-grafted hyaluronate compound and cells for transplantation; and 
     (b) administering to a subject in need thereof the composition of step (a). 
     The alginate-grafted hyaluronate compound may be provided as a biocompatible support for cells for transplantation by forming a hydrogel in the body. As used herein, the term “composition” of step (a) refers to a composition which serves as a support for cells in need of transplantation into the body and thus can be injected into the body together with cells at the time of cell transplantation. The kind of cells as an object of cell transplantation is not particularly limited. The composition for cell transplantation may be mixed with cells for transplantation, and then injected into a desired site using for example a syringe. When the composition of the present invention mixed with cells is mixed with a divalent cation before being injected into the body, the injected composition may promptly form a hydrogel at the transplantation site, so that the cells can be transplanted into the body. The cells for transplantation may be contained at 1×10 5  to 5×10 8  cells/ml within 0.1 wt % to 10 wt % of a polymer solution for the composition for cell transplantation. 
     The composition of step (a) of the present invention may be used for transplantation of various stem cells. The kind of stem cell to which the present invention is applied is not limited, and the present invention may be applied to cells that have characteristics of stem cells, that is, undifferentiation, indefinite proliferation, and differentiation ability into particular cells. The stem cells to which the present invention is applied are largely classified into two: pluripotent stem cells including embryonic stem cells (ES) and embryonic germ cells (EG) and multipotent stem cells. The embryonic stem cells are derived from inner cell mass (ICM) of the blastocyst, and the embryonic germ cells are derived from primordial germ cells present in 5-10 week aged gonadal ridge. Meanwhile, the multipotent stem cells are found in embryonic tissues, fetus tissues, or adult tissues, and include adult stem cells. The pluripotent stem cells are indefinitely proliferated in vitro and have the capability to differentiate into various cells derived from all three germ layers (ectoderm, mesoderm, and endoderm). Meanwhile, the multipotent stem cells have capability to differentiate into particular tissues derived therefrom, and their self-renewal potency is conventionally restricted to the lifetime of organisms. The source of multipotent stem cells includes all type of tissues, and is mainly isolated from, particularly, bone marrow, blood, liver, skin, intestine, spleen, brain, skeletal muscle, and dental pulp. In the case where the composition of the present invention is used in the transplantation of stem cells, the therapeutic composition of the present invention further includes a stem cell differentiation inducer. Preferable examples of the stem cell differentiation inducer include retinoic acid, ascorbic acid, melatonin, or several growth factors (e.g., glial cell line-derived neurotrophic factor (GDNF), epidermal growth factor (EGF), and nerve growth factor (NGF)). The composition for cell transplantation of the present invention has superior biocompatibility since a chemical cross-linking agent for forming a hydrogel is not used. In addition, the use of hydrogel containing a cell adhesive peptide can improve an interaction with cells and thus promote higher tissue regeneration efficiency. 
     According to an embodiment of the present invention, the cells for transplantation of the present invention are selected from the group consisting of chondrocytes, myoblasts, hepatocytes, osteoblasts, embryonic stem cells, embryonic germ cells, adult stem cells, mesenchymal stem cells, neural stem cells, endothelial stem cells, hematopoietic stem cells, liver stem cells, cardiac stem cells, pancreatic stem cells, endothelial progenitors, outgrowth endothelial cells, hematopoietic stem cells, human neural stem cells, satellite cells, intestinal epithelial cells, smooth muscle cells, and fibroblasts. 
     In another aspect of the present invention, the present invention provides a method for cartilage regeneration, comprising administrating to a subject in need thereof a pharmaceutical composition comprising a pharmaceutically effective amount of the alginate-grafted hyaluronate compound. The pharmaceutical composition for cartilage regeneration comprising a pharmaceutically effective amount of the alginate-grafted hyaluronate compound. The modified hyaluronate of the present invention forms a hydrogel in the body and thus acts as a support for primary chondrocytes, stem cells, or the like, thereby can be transplanted into the site in the body in need of cartilage regeneration. The pharmaceutical composition for cartilage regeneration of the present invention can be transplanted into the body together with cells for transplantation, through injection, and can be easily and promptly gelated after being transplanted. In the pharmaceutical composition of the present invention, the cells for transplantation may be contained at 1×10 5  to 5×10 8  cells/ml within 0.1 wt % to 10 wt % of a polymer solution for the composition for cell transplantation. 
     The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the therapeutic agent composition of the present invention is conventionally used at the time of formulation of medicines, and examples thereof may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The therapeutic agent composition of the present invention may further include, in addition to the above components, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspension, a preservative, and the like. Pharmaceutically acceptable carriers and agents are described in detail in  Remington&#39;s Pharmaceutical Sciences  (19th ed., 1995). 
     The pharmaceutical composition of the present invention may be parenterally administered, and local injection is the most preferable manner of administration. 
     The suitable dose of the composition of the present invention may vary depending on various factors, such as method of formulation, manner of administration, age, body weight, sex, and morbidity of the patient, diet, route of administration, excretion rate, and response sensitivity. Meanwhile, the dose of the composition of the present invention is 1×10 5  to 5×10 8  cells/ml per a single dose, and the dose may be adjusted as needed. 
     The composition of the present invention may be formulated into a unit dosage form or a multidose container, using a pharmaceutically acceptable carrier and/or excipient according to the method easily conducted by a person having ordinary skills in the art to which the present invention pertains. Here, the dosage form may be a solution in an oily or aqueous medium, a suspension, or an emulsion, and may further include a dispersant or a stabilizer. 
     The pharmaceutical composition of the present invention enables the remarkable formation of intercellular networking to sufficiently exert functions of the cells for transplantation, thereby actually exhibiting improved therapeutic efficacy in disease animals. 
     According to an embodiment of the present invention, the pharmaceutical composition of the present invention further includes primary chondrocytes or stem cells for cartilage regeneration. The pharmaceutical composition of the present invention serves as a support for primary chondrocytes or stem cells for cartilage regeneration, and remarkably increases the success rate of cell transplantation. 
     In another aspect of the present invention, there is provided a method for preventing or treating cartilage-injury disorders or diseases, comprising administrating to a subject in need thereof a pharmaceutical composition comprising a pharmaceutically effective amount of the alginate-grafted hyaluronate compound. 
     According to an embodiment of the present invention, the cartilage-injury disorders are selected from the group consisting of osteoarthritis, rheumatoid arthritis, meniscus Injury, costochondritis, relapsing polychondritis, and chondrosarcoma. The method for preventing or treating cartilage-injury disorders or diseases is associated with the method for cell transplantation and the method for cartilage regeneration, and descriptions of overlapping contents thereof are omitted to avoid excessive complication of the specification. 
     In another aspect of the present invention, there is provided a method for preparing an ionically cross-linkable alginate-grafted hyaluronate compound, the method including: 
     (a) reacting alginate or hyaluronate as a first reaction material with a linker to covalently link the linker to a carboxyl group of the alginate or the hyaluronate; and 
     (b) reacting the reaction product obtained in step (a) with hyaluronate or alginate as a second reactant to obtain an alginate-grafted hyaluronate compound in which the alginate is linked to the hyaluronate via the linker. 
     According to an embodiment of the present invention, the linker of step (a) is used at such an amount as to be linked to all the carboxyl groups of the alginate or hyaluronate as the first reactant. 
     The method for preparing an alginate-grafted hyaluronate compound of the present invention is associated with a method for preparing “an alginate-grafted hyaluronate compound” according to another aspect of the present invention, and thus descriptions of overlapping contents thereof are omitted to avoid excessive complication of the specification. 
     EXAMPLES 
     Materials and Methods 
     Synthesis of Hyaluronate-g-Alginate 
     All alginate samples were originally modified with RGD peptides in order to enhance cellular interactions. Briefly, 1 g of sodium alginate (molecular weight=200,000-300,000; FMC Biopolymer) was dissolved in 100 ml of 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES; Sigma-Aldrich) buffer solution (pH 6.5, 0.3 M NaCl). Then, 16.7 mg of RGD peptide with the sequence of (glycine)4-arginine-glycine-aspartic acid-serine-proline (G4RGDSP; Anigen) was added to the alginate solution in the presence of 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC; Sigma-Aldrich) and N-hydroxysulfosuccinimide (Sulfo-NHS; Thermo), in conjunction with vigorous solution stirring. Reaction was allowed to take place for 20 h at room temperature. Resultant solution was dialyzed for four days (molecular weight cut off=3,500), and then treated with charcoal. After filtration with a 0.22-μm filter for sterilization, solution was frozen and lyophilized. Hyaluronate (molecular weight=600,000-850,000; Lifecore) was first reacted with ethylenediamine (Sigma-Aldrich), prior to conjugation with alginate. Synthesis of NH 2 -hyaluronate was carried out with the same procedure with EDC and NHS, as described above. A 10-fold molar ratio excess of ethylenediamine was added to inhibit intermolecular cross-linking between hyaluronate chains during the reaction. The reaction was allowed to proceed for 20 h at room temperature. The solution was then dialyzed, treated with charcoal, filtered with a 0.22-μm filter for sterilization, and lyophilized. Alginate was coupled to NH 2 -hyaluronate via carbodiimide chemistry according to the above-described procedure ( FIG. 1 ). 
     Nuclear Magnetic Resonance Spectroscopy 
     Hyaluronate, alginate and hyaluronate-g-alginate were analyzed by 1H nuclear magnetic resonance (NMR) spectroscopy (Bruker Avance500 MHz) at 70° C. Samples were dissolved in D2O at 3 mg/ml. 
     Dimethyl Methylene Blue (DMMB) Assay 
     DMMB assays were performed to quantify alginate/hyaluronate content in hyaluronate-g-alginate. Briefly, 16 mg of DMMB was dissolved in 25 ml of ethanol and filtered with filter paper, and 100 ml of 1 M guanidine hydrochloride containing 0.17 M of sodium formate and 1 ml of formic acid was mixed with filtered DMMB. The solution was mixed with deionized water to a total volume of 500 ml. Each sample was diluted with deionized water, to yield a solution of 0.1 wt %. One milliliter of DMMB solution was added to 100 μl of each sample and mixed vigorously for 30 min. Incubated samples were centrifuged at 12,000 g for 10 min to precipitate the complex. Supernatant was removed and dried for 30 min at room temperature. Pellets were dissolved with 1 ml of decomplexation solution. Decomplexation solution was prepared with 50 mM of sodium acetate buffer (pH 6.8), containing 10% 1-propanol and 4 M guanidine hydrochloride. After 30 min of mixing, 100 μl of each sample was transferred to a 96-well plate. Absorbance was measured at 656 nm using a spectrophotometer (SpectraMax M2; Molecular Devices). 
     Enzymatic Stability Test 
     Hyaluronate-g-alginate (HGA1) was dissolved in PBS and mixed with calcium sulfate to form gels ([polymer]=2 wt %, [CaSO 4 ]=29.7 mM) using two glass plates with a spacer (1 mm thickness). Gel disks were obtained using a punch (10 mm diameter) and placed into PBS solution containing hyaluronidase in the concentration range of 0 to 100 μg/ml, before being incubated at 37° C. for 24 h. Gel disks were frozen and lyophilized, before dry weight was measured. A physical mixture of hyaluronate and alginate was also used as a control ([polymer]=2 wt %, [CaSO 4 ]=29.7 mM). Cross-sectional images of the gels were observed after treatment with hyaluronidase by scanning electron microscopy (S-4800 UHR FE-SEM; Hitachi). 
     Rheological Measurement 
     Viscoelastic properties of ionically cross-linked hyaluronate-g-alginate gels were measured using a rotational rheometer with a cone-and-plate (20 mm diameter plate, 4 degree cone angle) fixture (Bohlin Gemini 150; Malvern). A 150 m gap opening was set at the apex of the cone and plate, and operating temperature was set to be constant at 37±0.1° C. 
     Cell Isolation and Culture 
     Primary chondrocytes were isolated from the articular cartilage of New Zealand white rabbits (four-week-old; Samtako). The rabbits were sacrificed, and cartilage tissue fragments were obtained from hind leg knee joints. After fragments were washed with cold PBS, they were minced and digested with 4.5 mg/ml collagenase type II (Worthington) in DMEM/F-12 containing 10% FBS and 1% penicillin-streptomycin over a 6-h period. Digested cell suspension was passed through a cell strainer (40 μm; SPL Life Science) to remove undigested tissue fragments. Cells were collected using a centrifuge, washed twice with PBS, and suspended in DMEM/F-12 media containing 10% FBS and 1% penicillin-streptomycin. Isolated cells were cultured using standard culture procedures, and cells corresponding to passage number 2 or below were used for in vivo transplantation. 
     Cartilage Regeneration In Vivo 
     All animal procedures were performed in accordance with Hanyang University guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of Hanyang University (HY-IACUC-12-060A). BALB/c nude mice (six-week-old; Nara Biotech) were anesthetized with intraperitoneal injections of Zoletil (Virbac)/Rompun (Bayer) solution (9:1). Primary chondrocytes (1.0×10 7  cells/ml) were mixed with either RGD-alginate solution (AL) or RGD-alginate-grafted hyaluronate solution (HGA1, HGA2 or HGA4), before calcium sulfate slurries were mixed in ([polymer]=2 wt %). 100 μl of each cell-polymer mixture was subcutaneously injected into the dorsal region of the mouse (n=6 mice, per group). Six weeks after transplantation, mice were sacrificed, and the regenerated tissues were retrieved. 
     Histological and Immunohistochemical Analysis 
     Retrieved tissue samples were washed with cold PBS, and volume was measured. Samples were transferred to 4% formaldehyde solution overnight for fixation, and then dehydrated by immersion in a series of increasing concentrations of ethanol solution. Following immersion into xylene and paraffin, samples were embedded with paraffin. The specimens were cross-sectioned to 5 μm thickness with a microtome (RM2145; Leica). Tissue sections were stained with Alcian blue or Sirius red. For visualization of Matrillin-1 protein expression, samples were treated with HRP-conjugated anti-Matrillin-1 primary antibody (1/100 diluted; Bioss) and visualized with a diaminobenzidine peroxidase substrate kit (Vector). Images were obtained using optical microscopy (Axioskop 40; Carl Zeiss). Tissue sections were treated with anti-S100 primary antibody (1/25 diluted; Abcam) and FITC-conjugated secondary antibody (1/100 diluted; Jackson) in a humid box at 4° C. to visualize S100 protein expression. The stained slide glass was mounted with VECTASHIELD® Mounting Medium with DAPI (Vector Laboratories). Fluorescent images were obtained by fluorescence microscopy (ECLIPSE TE2000-E; Nikon). 
     Quantification of Sulfated Glycosaminoglycan (GAG) 
     For evaluation of GAG content in the regenerated tissues, a BlyscanTMsGAG assay kit (Biocolor) was used. Briefly, the retrieved tissues were lyophilized and weighted. The tissue fragments were put into 70 mM EDTA solution and incubated for 30 min at 37° C. to eliminate alginate from the tissues in order to exclude a potentiality of GAG detection from alginate in the hydrogels. After centrifugation, supernatant was removed. For extraction of GAGs, 500 μl of papain extraction solution (10 μl/ml) dissolved in 50 mM phosphate buffer (pH 6.5) containing 1 M NaCl, 5 mM cysteine HCl, and 1 mM EDTA was added to each sample and incubated at 60° C. overnight. A 1,9-dimethylmethylene blue reagent (1 ml) was then added, and samples were allowed to react over 30 min with gentle shaking. Following centrifugation at 10,000 rpm over 10 min, supernatant was drained to eliminate unbound dye, and the pellet was dried. A dissociation reagent was added to the sample pellet, and vigorous mixing took place. Spectrophotometer measurements were taken to observe samples and chondroitin 4-sulfate standards at 646 nm (SpectraMax M2; Molecular Devices). 
     Gene Expression 
     Total RNA was isolated from retrieved tissues using an RNA isolation kit (RNA iso plus; Takara). Isolated RNA was reverse transcribed to cDNA using a reverse transcription master mix (ELPIS Biotech). Chondrogenic marker genes (SOX-9, aggrecan, and type II collagen) and housekeeping gene (GAPDH) expression was investigated by an ABI Prism 7500 real-time PCR system (Applied Biosystems), in conjunction with the use of SYBR® Premix Ex Taq™. The sequences of primers (IDT) were as follows: SOX-9, 5′-CTTCATGAAGATGACCGACGAG-3′ (SEQ ID NO: 25), 5′-CTCTTCGCTCTCCTTCTTGAGG-3′ (SEQ ID NO: 26); aggrecan, 5′-GTGAAAGGTGTTGTGTTCCACT-3′ (SEQ ID NO: 27), 5′-TGGGGTACCTGACAGTCTGAT-3′ (SEQ ID NO: 28); type II collagen, 5′-AAGAGCGGTGACTACTGGATAG-3′ (SEQ ID NO: 29), 5′-TGCTGTCTCCATAGCTGAAGT-3′ (SEQ ID NO: 30); GAPDH, 5′-GACATCAAGAAGGTGGTGAAGC-3′ (SEQ ID NO: 31), 5′-CTTCACAAAGTGGTCATTGAGG-3′ (SEQ ID NO: 32). 
     Statistical Analysis 
     All data are presented as mean± standard deviation (n=6). Statistical analyses were performed using a Student&#39;s t-test. *P-values&lt;0.05 and **P&lt;0.01 were considered statistically significant. 
     Results 
     Characterization of Alginate-Grafted Hyaluronate 
     Hyaluronate-g-alginate (HGA) was designed and synthesized in the preparation of ionically cross-linkable hyaluronate. In brief, hyaluronate (HA) was first modified with ethylenediamine (NH 2 -HA), followed by conjugation with alginate (AL) by carbodiimide chemistry (HA-g-AL;  FIG. 1 ). Various alginate-g-hyaluronate samples were synthesized. Weight ratios of hyaluronate to alginate were 1, 2, and 4 (hereinafter referred to as HGA1, HGA2, and HGA4, respectively). RGD peptides were coupled to alginate to enhance cellular interactions of resultant HGA gels. 
     Hyaluronate-g-alginate synthesis was confirmed by  1 H-NMR spectroscopy ( FIG. 6   a ). A peak area of δ=4.2-4.4 ppm increased after modification with ethylenediamine, indicating successful introduction of ethylenediamine to hyaluronate (NH 2 -HA). Specific peaks of alginate and hyaluronate are concurrently shown on spectra at δ=4.2-4.7 and δ=2.5-2.6 (NCOCH 3 ), respectively, after NH 2 -hyaluronate modification with alginate. Graft efficiency of HA-g-AL was determined by calculating the polymer content from the spectra (HGA1=93.7%, HGA2=82.6, HGA4=94.7%). Graft efficiency was also evaluated with dimethylmethylene blue (DMMB) assays ( FIG. 6   b ). Hyaluronate does not bind to DMMB dye; thus, from this assay, only the alginate content in HA-g-AL was determined (HGA1=90.4±4.9%, HGA2=86.0±7.9%, HGA4=83.6±9.0%). The results found here were quite consistent with those obtained from  1 H-NMR spectroscopy. 
     Hydrogel Formation of Hyaluronate-g-Alginate 
     Hydrogels were not formed upon mixture of hyaluronate solution with calcium ions ([hyaluronate]=2 wt %, [CaSO 4 ]=30 mM). In contrast, in the presence of calcium ions, hyaluronate-g-alginate was able to form gels ([HGA1]=2 wt %, [CaSO 4 ]=30 mM) ( FIG. 2   a ). Most toxicity-related issues in hyaluronate-based hydrogels are derived from use of chemical cross-linkers, rather than stemming from issues related to the polymers[37,38]. Toxicity-related issues in typical hyaluronate gels, as caused by chemical cross-linking reagents after transplantation into the body, may be circumvented by use of ionically cross-linkable, hyaluronate-based hydrogels. Ionically cross-linkable alginate-g-Hyaluronate gels were able to be injected via a syringe with a 23-G needle ( FIG. 2   b ). This is also attractive in terms of achieving minimally-invasive delivery of drugs and/or cells to the body. 
     Characterization of Hyaluronate-g-Alginate Gels 
     Viscoelastic properties and gelation behaviors of hyaluronate-g-alginate gels were investigated through use of rotational rheometry ( FIG. 2   c - g ). When HGA1 solution was mixed with calcium ions, the storage modulus (G′) was higher than the loss modulus (G″) at various frequencies ( FIG. 2   c ), indicating that the mixture constructs a hydrogel structure via ionic cross-linking. In contrast, hyaluronate solutions did not form hydrogels upon calcium addition. We then investigated the effects of alginate content on the mechanical properties of ionically cross-linked hyaluronate-g-alginate gel ( FIG. 2   d ). Hydrogels were prepared at constant polymer and calcium concentrations for all samples tested ([polymer]=2 wt %, [CaSO 4 ]=30 mM). Weight ratios between hyaluronate and alginate were varied. As weight ratio of hyaluronate-to-alginate increased from 1 to 4, gel mechanical properties changed significantly from 2.5±0.2 kPa to 0.075±0.001 kPa. We then monitored how changes in elastic properties of ionically cross-linked hydrogels depended on calcium concentrations ( FIG. 2   e ). As calcium sulfate concentration increased from 7.4 mM to 30 mM, G′ values of hyaluronate-g-alginate gels increased from 0.3±0.1 kPa to 2.7±0.6 kPa. However, no further increase was observed when calcium sulfate concentration was increased to 60 mM. Gelation time of hyaluronate-g-alginate gels was not significantly influenced by the alginate content in the gels ( FIG. 2   f ). Shear modulus of hyaluronate-g-alginate gels was dependent on alginate molecular weight ( FIG. 2   g ). As alginate molecular weight increased from 50,000 to 250,000 g/mol, G′ of ionically cross-linked gels also increased from 0.04±0.003 to 2.4±0.2 kPa. Alginate-g-hyaluronate gels prepared with alginate of either 50,000 g/mol or 100,000 g/mol showed poor mechanical properties, even in the presence of calcium ions. It is very important to regulate the mechanical properties of polymer scaffold with regard to controlling cellular behaviors (e.g., adhesion, proliferation and migration) [39-41]. In addition, mechanical properties are also crucial in the modulation of stem cell differentiation, which has an immediate influence on tissue regeneration [41-44]. The mechanical properties of hyaluronate-g-alginate gels were easily adjustable through control of polymer composition and calcium ion concentration, which may be very attractive for use in tissue engineering. 
     Enzymatic Stability of Alginate-g-Hyaluronate Gels 
     Alginate-grafted hyaluronate gels were treated with hyaluronidase to test stability against enzyme reactions in vitro. At 2 weeks incubation time, slight reductions in gel weights were observed. Gel weights depended on enzyme concentration ( FIG. 3   a ). Hydrogels maintained approximately 80% of their initial weight after treatment with 100 μg/ml of hyaluronidase for 2 weeks compared with control conditions (no enzyme). Hyaluronate-g-alginate gels maintained well-packed, porous structures following enzymatic treatment ( FIG. 3   c ). In contrast, gels prepared from simple mixtures of hyaluronate and alginate exhibited significant reduction of gel weight even in the presence of 1 μg/ml of hyaluronidase during incubation. Approximate weight reduction of 40% was observed, independent of hyaluronidase content. This weight reduction is similar to that expected based on hyaluronate content in the gel. A cross-sectional image of hyaluronate/alginate mixed gel following treatment with hyaluronidase showed substantial gel loss, as caused by enzymatic degradation ( FIG. 3   b ). 
     Hyaluronate is cleaved by action of hyaluronidase into smaller oligosaccharide fragments [45,46]. However, hyaluronate degrades rapidly in vivo in the absence of chemical modification or cross-linking caused by endogenous hyaluronidase [47]. Fast gel degradation may be unfavorable in regeneration of many types of tissues, as gels should supply 3-D structural environments and maintain their integrity as a scaffold during new ECM development from delivered cells. Hyaluronate/alginate mixed hydrogels do not form complete interpenetrating polymer network structures, because only alginate in mixtures participate in gel formation when calcium ions are added. Hyaluronate in mixed gels was almost entirely degraded by the action of hyaluronidase ( FIG. 3 ). On the other hand, degradation of hyaluronate in hyaluronate-g-alginate gels was restricted by covalent conjugation of alginate chains, leading to slow gel degradation by hyaluronidase. This might make these gels be more appropriate for in vivo applications than mixed gels. 
     In Vivo Cartilage Regeneration Using Hyaluronate-g-Alginate Gels 
     Ionically cross-linkable hyaluronate hydrogels with primary chondrocytes were injected into the dorsal region of mice to verify the efficacy of the gels in cartilage regeneration in vivo. RGD peptides were initially introduced to all hyaluronate-g-alginate gels tested in vivo. RGD-alginate (AL) was also used as a control. Six weeks post-transplantation, tissue volume for each group had not significantly changed as compared with initial injection volumes. This indicates that these gels were able to maintain their 3-D volumetric structures during tissue formation. Tissue sections were stained with Alcian blue ( FIG. 4   a - d ) and Sirius red ( FIG. 4   e - h ) to visualize proteoglycan and collagen, respectively, which probe new ECM formation in regenerated cartilage. ECM formation is essential for transplanted cells with regard to maintenance of viability and functional activity. Homogeneous distributions of lacunae structures were observed in the tissue sections in response to use of HGA2 and HGA4 gels. On the other hand, sparse distributions of lacunae structures were found in AL- and HGA1-treated groups. Interestingly, ECM formation and cartilage lacunae development were more abundant for the HGA2 group, as compared with other groups ( FIG. 4   c ,  4   g ). HGA4 gels were less efficient than HGA2 gels, likely due to weaker mechanical properties of the HGA4 gels as compared with those of HGA2 gels ( FIG. 2   d ). AL and HGA1 gels were not appropriate for cartilage regeneration in vivo. 
     Matrillin-1 ( FIGS. 4   i - l ) and S100 proteins ( FIG. 4   m - p ) were visualized with antibodies for immunohistochemical analysis of regenerated tissue sections. Matrillin-1 plays an important role in linking type VI collagen microfibrils and type II collagen fibrils involved in constructing ECM in cartilage tissues [48]. RemarkableMatrillin-1 signals were observed in HGA2 gels ( FIG. 4   k ), as compared with other groups, which is evident in construction of collagen matrices. AL and HGA1 gels did not sufficiently induce protein production ( FIG. 4   i, j ); a finding that was consistent with results obtained from histological analysis. S100 protein is an intracellular calcium binding protein expressed in chondrocytes, adipocytes, Schwann cells and neural cells [49]. S100 protein is expressed at early stages during the chondrogenesis process, and expression fades in conjunction with phenotypic changes in chondrocytes, such as those caused by hypertrophy [50-52]. Positive S100 fluorescent signals were observed in HGA2 and HGA4 groups ( FIG. 4   o, p ), indicating that the transplanted cells maintained their chondrogenic phenotypes in vivo when these hyaluronate-g-alginate gels were injected. 
     Quantitative Analysis of GAG Formation and Gene Expression 
     Sulfated glycosaminoglycan (GAG) content was determined and normalized by dried tissue-weight ( FIG. 5   a ). GAGs were rarely formed for the 1HA (3.2±1.1 μg/ml) and AL (4.6±1.3 μg/ml) groups. Significantly more GAGs were secreted when HGA2 gels were used (52.5±7.7 μg/ml) compared with the other groups. Gene expression of typical chondrogenic markers was next investigated ( FIG. 5   b - d ). Gene expression informs cell functionality, which regulates protein production, morphogenesis, cellular differentiation and phenotype. Chondrogenic and ECM-formation processes can be described as related to expression of chondrogenic marker genes such as SOX-9, aggrecan, and type II collagen. SOX-9 is expressed in early development of chondrogenesis, and is critical for chondrocyte differentiation and function [53,54]. Aggrecan and type II collagen are important ECM components of cartilage tissues [55]. In the current study, little expression of chondrogenic marker genes for AL was observed, and enhanced gene expression with hyaluronate was found. Prominent expression of SOX-9, aggrecan and type II collagen was found upon transplantation of primary chondrocytes in HGA2 gels. Relative expression levels of SOX-9, aggrecan, and type II collagen were 7.1±1.1, 8.0±1.6, and 31.8±2.5 times higher than those for the AL group. While HGA4 gels contained more hyaluronate than HGA2 gels, the gels did not enhance in vivo chondrogenic gene expression. In particular, type II collagen expression levels were lower in the HGA4 groups than in the HGA2 groups. These results were consistent with those obtained from histological and immunohistochemical analyses. 
     In this study, alginate-grafted hyaluronate formed cross-linked structures in the presence of calcium ions, and showed great potential with regard to in vivo cartilage regeneration. In previous studies, we reported that RGD-modified alginate hydrogels could successfully regenerate cartilage tissues in vivo [56,57]. However, approximately 5×10 7  cells/ml were required for successful cartilage tissue regeneration. This study found that hyaluronate-g-alginate was able to reduce cell concentration (with [cell]=1×10 7  cells/ml). We also learned that hyaluronate proportion in hyaluronate-g-alginate was an important parameter in cartilage regeneration. The optimal weight ratio between hyaluronate and alginate was found to be two (HGA2), as gel stiffness was found to deteriorate upon addition of hyaluronate (860±70 Pa for HGA2 and 74.6±0.1 Pa for HGA4;  FIG. 3   d ). Thus, harmonization between polymer content and mechanical stiffness should be carefully considered in hopes of obtaining successful in vivo cartilage engineering. Hyaluronate-g-alginate may be a promising candidate for this approach. 
     Having described a specific embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents. 
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