Title:  Ionically crosslinked hydrogels with adjustable gelation time

United States Patent:  6,497,902

Issued:  December 24, 2002

Inventors:  Ma; Peter X. (Ann Arbor, MI)

Assignee:  The Regents of the University of Michigan (Ann Arbor, MI)

Appl. No.:  452494

Filed:  December 1, 1999

Biocompatible hydrogels, for: scaffoldings for tissue engineering; cell encapsulation matrices; injectable bulking materials for cosmetic and functional restorations; controlled release matrices; gene delivery vehicles; immunoprotection matrices; immobilization materials; food additives; medical gels; conductive electrode gels; lubricious coatings; film forming creams; membranes; superabsorbents; hydrophilic coatings; and wound dressings. The hydrogels include: at least one water-soluble polymer/copolymer; and at least one slow and/or fast dissolving and/or releasing divalent and/or multivalent cation-containing compound. At least one of the monomers is an acid, and/or contains an acid group or a derivative thereof. Such monomer reacts with the cations to form a three-dimensional ionically crosslinked hydrogel composition. A method for preparing such a composition comprises the step of controlling a rate of gel formation by varying at least one of: solubility of the cation containing compounds; cation concentration; mixture of cation containing compounds; polymer concentration; gelation temperature.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides novel ionically crosslinked hydrogel compositions having adjustably controllable gelation rates, such hydrogels fortuitously useful for a heretofore unexpectedly wide range of applications requiring varied gelation rates.

In aqueous solution, hydrogels may swell to an equilibrium volume but preserve their shape. The hydrophilicity is due to the presence of water-solubilizing groups, such as --OH, --COOH, --CONH₂, --CONH--,--SO₃ H, etc. It is believed that the stability of shape is due to the presence of the present inventive three-dimensional network, which is maintained by crosslinks between polymer chains. These crosslinks can be covalent bonds, ionic bonds, hydrogen bonds, hydrophobic associations, and dipole-dipole interactions.

The inventive ionically crosslinked hydrogels may be very attractive candidates for biomedical applications because of their exceptional biocompatibility without the involvement of harmful chemical crosslinking reagents. For many of these applications, the structural uniformity, mechanical stability, and controllable gelation time are of essential importance. Considerable success, as disclosed further hereinbelow, has been achieved in controlling the structural uniformity and mechanical stability of ionically crosslinked hydrogels in an aqueous environment. See, for example, C. K. Kuo and P. X. Ma, "Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering," Proceedings of the 10th International Conference on Mechanics in Medicine and Biology: 303-306 (Mar. 2-5, 1998), which is incorporated herein by reference in its entirety.

The present invention contemplates a method for tissue engineering in vitro comprising the steps of: a) providing: i) cells, ii) an alginate salt, iii) a source of calcium ions, and iv) a calcium releasing compound; b) mixing the cells, alginate salt, and the source of calcium ions to provide a mixture; c) adding the calcium releasing compound to the mixture to provide a crosslinked gel; and d) culturing the crosslinked gel to provide a three-dimensional crosslinked hydrogel/cell system for growing cells in vitro.

In one embodiment, the alginate salt is selected from the group consisting of sodium alginate and potassium alginate. In another embodiment, the alginate salt is prepared from an alginate source selected from Macrocystis pyrifera and Laminaria hyperborea. In yet another embodiment, the source of calcium ions is selected from the group consisting of calcium carbonate and calcium sulfate. In an alternative embodiment, the calcium releasing compound is D-glucono-.delta.-lactone.

In another embodiment, the method further comprises the step of implanting the three-dimensional crosslinked hydrogel/cell system. In one embodiment, the three-dimensional crosslinked hydrogel/cell system has a thickness of between about 4 mm and about 8 mm, and a diameter of approximately 18 mm.

It is not intended that the present invention be limited for culturing a particular type of cells (or merely one cell type on a scaffold). A variety of cell types (including mixtures of different cells) are contemplated. In one embodiment, the cells are osteoblasts. In another embodiment, the cells secrete a medically useful compound (eg., hormone, cytokine, etc.). Such cells may be (but need not be) cells that have been manipulated by recombinant means to secrete such compounds.

The present invention also contemplates the resulting crosslinked alginate gel as a composition. Moreover, the present invention contemplates the resulting crosslinked gel in combination with other components, such as cells. It is not intended that the cells be limited to particular cell type, or merely one cell type on a scaffold. A variety of cell types, including mixtures of different cells, are contemplated.

As used herein, the term "alginate" refers to any of several derivatives of alginic acid (eg., calcium, sodium, or potassium salts or propylene glycol alginate). These compounds are hydrophilic colloids obtained from seaweed.

The methods of the present invention permit the formation and preparation of structurally homogeneous and mechanically strong alginate gels with defined dimensions, which can be used to incorporate living cells. The three dimensional gel structure with incorporated cells can be maintained in an in vitro tissue culture environment by adjusting calcium ion concentration in the culture medium. Ionically crosslinked alginate gels with defined three dimensional structure can be reliably used as a tissue engineering scaffold.

In addition to the advantages stated immediately hereinabove, it would further be advantageous to control the gelation rate of hydrogels, in that for many applications such as in biomedical, pharmaceutical, food and cosmetic formulations, the gelation rate may be critical. For some applications, a slower gelation rate is preferred (hours to days); whereas for others, a faster gelation rate is preferred (instant, or seconds to minutes); whereas for still others, an intermediate gelation rate is preferred (minutes to hours).

For example, the gel-forming solution or paste (alone or with other ingredients) can be used as an injectable material to cast into a three-dimensional shape with structural uniformity and superior mechanical properties. A slower gelation rate is preferred because it can result in uniform gel formation and better mechanical properties. For another application, such as filling materials to block a leakage in a blood vessel or intestines, a fast gelation rate may be essential to ensure a gel formation before being diluted or flushed away. In another example, the gels can be used as filling materials (with cells or not, with biological agents or not) to repair a complex tissue/organ defect(s) in situ by a reconstructive/plastic surgeon. The surgeon needs enough working time to shape the material before it gels (forms three-dimensional structures). However, the gelation time should also be reasonably short so that the structure "solidifies" after the shaping procedure without prolonged patient waiting and shape-maintaining time.

As such, it can be seen that, for a particular end-use for hydrogels, the necessary/preferred rate of gelation falls within relatively narrow parameters. Thus, for the hydrogels to be useful, their gelation rate should fall within such parameter(s). To be far more useful, the gelation rate of the hydrogels should be controllable so as to fall within such parameter(s) for a wide range of particular end-uses (which end-uses prefer rates of gelation ranging from fast to slow). The present invention, in meeting this need, is based upon the unexpected and fortuitous discovery that the gelation rate of ionically crosslinked hydrogels may be selectively varied and controlled to advantageously meet a wide range of relatively narrow end-use parameter(s) (eg. rates of gelation).

The present invention provides ionically crosslinked hydrogels with controlled gelation time. Both the exemplary compositions and the methods of preparing such hydrogels are disclosed. The hydrogels are made of one or more synthetic and/or natural water-soluble polymers (macromolecules), and one or more divalent or multivalent cation containing or releasing compounds. The polymers can be either homopolymers or copolymers (with two or more types of structural units). The copolymers can be random copolymers, block copolymers, or graft copolymers. At least one of the structural units (monomers) is an acid (e.g., carboxylic acid, sulfonic acid and phosphonic acid), or contains an acid group or a derivative of an acid (such as its salt, ester, or anhydride) that can react with divalent and/or multivalent cations to form ionic crosslinks intermolecularly among polymer chains. A cation containing compound can either directly dissolve in an aqueous solution to produce free cations or react with one or more other reactants to release the cations. Such reactants are defined herein as "cation releasing compounds."

The cation releasing compound need simply cause the cation source to release cations, thereby initiating gelation. It is to be understood that any suitable cation releasing compound may be used in conjunction with the present invention. In one embodiment, the cation releasing compound comprises D-glucono-.delta.-lactone (C₆ H₁₀ O₆) (GDL), and causes release of calcium cations. Without being bound to any theory, it is believed that the GDL functions in the following manner. The GDL slowly hydrolyzes into an acid, thereby lowering the pH in its vicinity. This causes the CaCO₃ to dissolve (which is generally insoluble in a neutral solution), presumably due to the now-mildly acidic solution. As such, it is believed that the GDL may be useful to cause release of cations from any cation containing compound which is generally insoluble in a neutral solution.

Further, in lieu of the GDL, after a generally insoluble cation containing compound is suspended with the water soluble polymer, it is within the purview of the present invention to slowly add an acid to the suspension in order to lower the pH and cause release of the cation.

The inventor of the present methods of preparing an ionically crosslinked gel has unexpectedly found that utilizing one or more of: the solubility of the cation containing compounds; cation concentration; mixture/ratio of cation containing compounds; polymer concentration; gelation temperature; and so forth controls the rate of gel formation.

The divalent or multivalent cation(s) contained or released from the source compounds are selected from the group consisting of calcium, magnesium, beryllium, strontium, barium, radium, aluminum, copper, zinc, osmium or any other divalent or multivalent cations that can form ionic bonds with the acid(s) or its derivatives contained in the water-soluble polymers, and mixtures thereof.

A few examples of published reference materials to which a skilled artisan may look to determine if a cation containing/releasing compound would be a fast or slow dissolving/releasing compound include, but are not limited to the "Solubility Product Constants" table from the CRC Handbook of Chemistry and Physics, available, for example, from Knovel Engineering & Scientific Online References at www.knovel.com; the "Aqueous Solubility of Inorganic Compounds at Various Temperatures" table, also from the CRC Handbook of Chemistry and Physics; and The Merck Index, An Encyclopedia of Chemicals, Drugs, and Biologicals, Budavari, O'Neil and Smith, Editors, 11th Edition, published by Merck & Co., Inc. (1989).

Some exemplary suitable acid-containing monomers that may constitute the polymers include but are not limited to the following: 1) Monomers containing carboxyl: D-glucopyramuronic acid, D-manopyranuronic acid, D-galactopyranuronic acid, 4-O-methyl-D-glycopyranuronic acid, L-idopyranuronic acid, L-idopyranuronic acid, L-gulopyranuronic acid, sialic acids, acrylic acid, methacrylic acid, 4-vinylbenzoic acid, crotonic acid, oleic acid, elaidic acid, itaconic acid, maleic acid, fumaric acid, acetylenedicarboxylic acid, tricarbollylic acid, sorbic acid, linoleic acid, linolenic acid, eicosapentenoic acid, other unsaturated carboxylic acids, and their derivatives such as salts, anhydrides, and esters; 2) Monomers with other acids such as sulfonic acid, or phosphonic acid replacement of the carboxyl group of the above listed monomers and their derivatives.

It is to be understood that any polymers that are made from one or more of the above monomers with or without other monomers may be suitable to form hydrogels according to the present invention. Some exemplary other monomers (not the acid or acid derivative containing monomers) include but are not limited to the following: D-xylopyranose, L-arabinopyranose, L-arabinofuranose, D-glucopyranose, D-mannopyranose, D-galactopyranose, L-galactopyranose, D-fructofuranose, D-galactofuranose D-glucosamine, D-galactosamine, methacrylates (e.g., methyl methacrylate), ethylene, propylene, tetrafluoroethylene, styrene, vinyl chloride, vinylidene chloride, vinyl acetate, acrylonitrile, 2,2-bis[4-(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl]propane(BisGMA), ethyleneglycol dimethacrylate (EGDMA), triethyleneglycol dimethacrylate (TEGDMA), bis(2-methacrylyoxyethyl)ester of isophthalic acid (MEI), bis(2-methacrylyoxyethyl)ester of terephthalic acid (MET), bis(2-methacrylyoxyethyl)ester of phthalic acid (MEP), 2,2-bis-(4-methacrylyoxy phenyl)propane(BisMA), 2,2-bis[4-(2-methacrylyloxyethoxy)phenyl]propane (BisEMA), 2,2,-bis[4-(3-methacrylyloxypropoxy)phenyl]propane (BisPMA), hexafluoro-1,5-pentanediol dimethacrylate (HFPDMA), bis-(2-methacrylyloxyethoxyhexafluoro-2-propyl)benzene [Bis(MEHFP).phi.], 1,6-bis(methacrylyloxy-2-ethoxycarbonylamino)-2,4,4-trimethylhexan (UEDMA), spiro orthocarbonates, and the derivatives of these monomers.

An exemplary list of some polymers that can be made into ionically crosslinked hydrogels with controlled gelation time includes but is not limited to the following: alginic acid, pectin, hyaluronic acid, heparin, proteins, proteoglycans, poly(methacrylic acid), poly(acrylic acid), poly(maleic anhydride), poly(maleic acid), poly(methyl methacrylate-methacrylic acid), poly(methyl acrylate-acrylic acid), poly(methyl methacrylate-acrylic acid), poly(ethyl acrylate-acrylic acid), poly(ethyl methacrylate-methacrylic acid), poly(butyl acrylate-acrylic acid), poly(ethylene-acrylic acid), poly(ethylene-methacrylic acid), poly(acrylonitrile-maleic anhydride), poly(butadiene-acrylonitrile-acrylic acid), poly(butadiene-maleic acid), poly(butadiene-maleic anhydride), poly(acrylamide-acrylic acid), poly(2-hydroxyethyl methacrylate-methacrylic acid), poly(propylene-acrylic acid), poly(propylene-ethyleneacrylic acid), poly(vinyl chloride-vinyl acetate-maleic acid), and derivatives of the polymers (salts, anhydrides, esters, etc.).

It is also to be understood that the above listed polymers can be used together with other polymers, including but not limited to water soluble polymers such as gelatin, agar, agarose, chitin/chitosan, cellulose, collagen, poly(vinyl alcohol), poly(ethylene oxide), Pluronics (block copolymers of ethylene oxide and propylene oxide), poly(2-hydroxyethyl methacrylate), and poly(N-vinyl-pyrrolidinone).

It is to be understood that this invention is conceptually suitable for all the aforementioned polymer systems, and as such, the supporting experimental data hereinbelow are not intended to be exhaustive. Instead, they are collected primarily from alginates as representative ionically crosslinked hydrogels.

These hydrogels might be used in a variety of biomedical, pharmaceutical, food, cosmetic and other applications. They could be used as scaffoldings for tissue engineering, cell encapsulation matrices, injectable bulking materials for cosmetic and functional restorations, controlled release matrices, gene delivery vehicles, immunoprotection matrices, immobilization materials, food additives, medical gels, conductive electrode gels, lubricious coatings, film forming creams, membranes, superabsorbents, hydrophilic coatings, wound dressings, and so forth. It is to be understood that the term "biocompatible hydrogel" as used herein is intended to include, but not be limited to all of the uses enumerated immediately hereinabove, as well as throughout the present disclosure.

It is further contemplated as being within the purview of the present invention to include other minor components in the ionically crosslinked hydrogels of the present invention. For example, inert components and bioactive agents (such as, for example, growth factors and hormones) may be incorporated thereinto if desired, without substantially affecting the methods and/or compositions of the present invention.

Although alginates from various sources such as Laminaria hyperborea, Laminaria digitata, Eclonia maxima, Macrocystis pyrifera, Lessonia nigrescens, Ascophyllum codosum, Laminaria japonica, Durvillaea antarctica, and Durvillaea potatorum in a variety of salt forms can be used, two sodium alginates from Laminaria hyperborea (LH) and Macrocystis pyrifera (MP) are used in the exemplary preferred embodiments.

It is to be understood that various cations from countless compounds can potentially be used as ionic crosslinkers. However, in the preferred embodiments, calcium ions from calcium carbonate (CaCO₃) and calcium sulfate dihydrate (CaSO₄.2H₂ O) are used in the exemplary embodiments as representative slow dissolving and fast dissolving calcium containing compounds, respectively. For example, water soluble CaCl₂ or other cation containing compounds can be used instead of CaSO₄.2H₂ O. Although calcium sulfate may be used, the calcium sulfate dihydrate is preferred in that the dihydrate is its naturally occurring form, and is water soluble.

It is to be understood that the calcium ion to carboxyl molar ratio of the present invention may range between about 0.05 and about 2.0, and the ratio of CaCO₃ to CaSO₄.2H₂ O may range between about 98:2 and about 2:98. In a preferred embodiment, the calcium ion to carboxyl molar ratio of the present invention may range between about 0.18 and about 0.9, and the ratio of CaCO₃ to CaSO₄.2H₂ O may range between about 90:10 and about 50:50. In a more preferred embodiment, the calcium ion to carboxyl molar ratio of the present invention may range between about 0.27 and about 0.54, and the ratio of CaCO₃ to CaSO₄.2H₂ O may range between about 65:35 and about 85:15.

It is to be understood that various aqueous solutions can be used to make the hydrogels (water, saline solution, buffer solutions, tissue culture mediums, etc.). However, in the preferred embodiments, water is used.

Sodium alginate prepared from Laminaria hyperborea (LH) is commercially available under the trade name PROTANAL LF200 from Pronova Biopolymer in Drammen, Norway. High viscosity sodium alginate prepared from Macrocystis pyrifera (MP), calcium carbonate (CaCO₃), calcium chloride dihydrate (CaCl₂.2H₂ O), calcium sulfate dihydrate (CaSO₄.2H₂ O), and D-glucono-delta-lactone (C₆ H₁₀ O₆) (GDL) are commercially available from Sigma Chemical Company in St. Louis, Mo.

Claim 1 of 29 Claims

What is claimed is:

1. A hydrogel composition, comprising:

at least one water-soluble polymer composed of one or more monomers, the polymer being present in a predetermined concentration;

at least one of: a first divalent or multivalent cation-containing compound having a K_sp at or less than 3.36x10^-9 ; and a first divalent or multivalent cation-releasing compound, the at least one first divalent or multivalent cation-containing compound and the first divalent or multivalent cation-releasing compound being present in a predetermined concentration; and

at least one of a second divalent or multivalent cation-containing compound having a K_sp at or greater than 3.14x10^-5 ; and a second divalent or multivalent cation-releasing compound, the at least one second divalent or multivalent cation-containing compound and the second divalent or multivalent cation-releasing compound being present in a predetermined concentration, wherein the K_sp of the second cation-containing compound is greater than the K_sp of the first cation-containing compound;

wherein at least one of the monomers is selected from the group consisting of acids, monomers containing an acid group, monomers containing a derivative of an acid, and mixtures thereof, wherein the at least one monomer reacts with the divalent or multivalent cations to form ionic cross-links inter-molecularly among polymer chains to form an ionically cross-linked hydrogel composition;

and wherein the first and second divalent or multivalent cations are selected from the group consisting of calcium, beryllium, strontium, barium, radium, aluminum, copper, zinc, osmium, and mixtures thereof.
 

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