WO2005116084A1 - Process - Google Patents

Abstract

The present invention relates to a hydrogel of a polysaccharide cross-linked with a carbodiimide and a succinimide.

Description

PROCESS

FIELD OF INVENTION

The present invention relates to a cross-linked synthetic hydrogel.

A hydrogel is a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open- lattice structure which entraps water molecules to form a gel.

In particular, the present invention relates to a hydrogel of a polysaccharide cross- linked with a carbodiimide and a succinimide. The present invention also relates to inter alia processes, compositions, and uses of the same.

More in particular, the present invention relates to a hydrogel of a carboxyalkyl- containing polysaccharide cross-linked with an aminoalkyl carbodiimide and an hydroxysuccinimide. The present invention also relates to inter alia processes, compositions, and uses of the same.

Even more in particular, the present invention relates to a hydrogel of a carboxymethyl-containing polysaccharide cross-linked with l-Ethyl-(3-3- dimethlyaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The present invention also relates to ter alia processes, compositions, and uses of the same.

BACKGROUND TO THE INVENTION

Over the years, various synthetic and natural polymers and their hybrids have been 1 9 utilised as cell support scaffolds ' . Various hydrogels have been used as extracellular matrices (ECM) for cell immobilisation, transplantation and tissue engineering ^" . Hydrogels with three-dimensional, hydrophilic and polymeric networks could potentially mimic some of the many roles found in vivo¹. ECMs compromising of various amino acids and sugar based macromolecules, as are found in hydrogels, provide a hydrated microenvironment to orientate cells, give mechanical integrity and permit the diffusion of nutrients, metabolites and growth factors. Hydrogels from natural polymers such as collagen, gelatin, fibrin, alginate, agarose and chitosan have already been widely used for tissue engineering applications^8"1 . Previously, reconstituted collagen in the form of gels, sponges or films, has been extensively used to support cell cultures both in vitro and in vivo^11'20. These studies have proved valuable but recent concerns regarding potential viral infection and availability have highlighted a demand for ECMs derived from non-animal sources^21"26.

Hydrogels currently used or with potential applications in drug delivery or tissue engineering are divided into two categories, according to their natural or synthetic origin.

Hydrogels from various synthetic polymers have been widely investigated by using neutral co-monomers - such as 2-hydroxyethyl methacrylate, methyl methacrylate and maleic anhydride (Falamarzian and Varshosaz 1988, Kuo et al. 1988, Brannon-Peppas and Peppas 1990, Khare and Peppas 1993). While considerable effort has been made to synthesize and characterize acrylate-based ionic hydrogels, these materials are not biologically degradable by either hydrolytic or enzymatic mechanisms. As a result, acrylate systems are limited in their potential as biodegradable drag-delivery platforms. To overcome this liability, a range of natural polymers has been used to prepare cross-linked hydrogel networks. For example, pH-sensitive hydrogels based on polypeptides, proteins and polysaccharides have all been produced (Chui et al 1999, Markland et al 1999).

However, limitations of hydrogels from natural polymers have motivated approaches to modify these polymers as well as to use various synthetic polymers. A wide range of synthetic polymers may potentially have suitable chemical and physical properties for these applications.

One example of a synthetic hydrogel is a dextran hydrogel. Several approaches to the preparation of dextran hydrogels have been described. Hydrogels were prepared directly by cross-linking dextran with either 1,6- hexanediisocyanate or glutaraldehyde (Hovgaard and Bronstead 1995, Bronsted et al. 1998). Sequential reactions of dextran with glycidyl acrylate, followed by polymerization of acrylated dextran also led to the formation of the polymer network (Yamamoto et al. 1996). Methacrylation of dextran has been conducted with full control of the degree of substitution by transesterification of glycidyl methacrylate with dextran in dimethyl sulphoxide (Van Dijk- Wolthius et al. 1995, Van Dijk- Wolthius et al. 1997). Kim and Chu (2000) and Kim et al. (1999) obtained dextran hydrogels by UV irradiation of methacrylated and acrylated dextrans that were synthesized by reacting dextran with methacrylic anhydride, and then bromoacetyl bromide and sodium acrylate . Recently, Chiu et al. (2002) have reported dextran hydrogels prepared by radical copolymerization of methacrylate-dextran with acetic anhydride in borate buffer at room temperature, using ammonium peroxydisulfate and N,N,N',N'-tetramethylethylenediamine as an initiation system.

US 4,886,787 describes a crosslinked carboxy-containing polysaccharide that can be used as a degradable implant. The gel is crosslinked with a di- or poly-functional epoxide.

Park et al. (2003) Biomaterials 24, 1631-1641 describe a collagen matrix that is crosslinked with various amounts of hyaluronic acid by l-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC).

Presently, despite many years of research and development, the only commercially available tissue engineered product is a dermal replacement (Dermagraft, Smith and Nephew, USA). Moreover, owing to the limitations of hydrogels from natural polymers, there is a continuing need in the art for new polymers, in particular synthetic polymers, that can be used to prepare hydrogels with improved properties.

The present invention therefore seeks to provide an improved synthetic hydrogel. SUMMARY ASPECTS OF THE PRESENT INVENTION

In its broadest aspect, the present invention relates to a hydrogel of a polysaccharide cross-linked with a carbodiimide and a succinimide.

As indicated above, a hydrogel is a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure which entraps water molecules to form a gel.

Further aspects and preferred embodiments will be apparent from the description and the claims.

PREFERRED ASPECTS OF THE PRESENT INVENTION

In one preferred aspect of the present invention there is provided to a hydrogel of a hydrocarbyl-containing polysaccharide cross-linked with a hydrocarbyl carbodiimide and a succinimide. The present invention also relates to inter alia processes, compositions, and uses of the same.

The term "hydrocarbyl group" means a group that comprises at least carbon and hydrogen. Other atoms may be present such as N or O or S. The hydrocarbyl group may be linear or branched or cyclic. The hydrocarbyl group may be saturated or unsaturated. In some instances, two or more of the hydrocarbyl groups may be fused together - so as to form a single chain - that may be branched and/or saturated or unsaturated.

One or more of the hydrocarbyl groups may be optionally substituted. "Optionally substituted" refers to the replacement of hydrogen with a monovalent or divalent radical. Suitable substitution groups include, for example, hydroxyl, nitro, amino, imino, cyano, halo, thio, thioamido, amidino, imidino, oxo, oxamidino, methoxamidino, imidino, guanidino, sulfonamido, carboxyl, formyl, loweralkyl, haloloweralkyl, loweralkoxy, haloloweralkoxy, loweralkoxyalkyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylthio, aminoalkyl, cyanoalkyl, and the like.

The substitution group can itself be substituted. The group substituted onto the substitution group can be carboxyl, halo ; nitro, amino, cyano, hydroxyl, loweralkyl, loweralkoxy, aminocarbonyl,-SR, thioamido,-SO3H,-SO2R or cycloalkyl, where R is typically hydrogen, hydroxyl or loweralkyl.

When the substituted substituent includes a straight chain group, the substitution can occur either within the chain (e. g., 2-hydroxypropyl, 2-aminobutyl, and the like) or at the chain terminus (e. g., 2-hydroxyethyl, 3-cyanopropyl, and the like). Substituted substitutents can be straight chain, branched or cyclic arrangements of covalently bonded carbon or heteroatoms.

One or more of the hydrocarbyl groups may be or may comprise an alkyl group - which may be saturated or unsaturated (e.g. an alkylene group) - and may be linear or branched. In some embodiments, preferably the alkyl group is a lower alkyl group.

The term "Lower alkyl" as used herein refers to branched or straight chain alkyl groups comprising one to ten carbon atoms that are unsubstituted or substituted, e. g., with one or more halogen, hydroxyl or other groups, including, e. g., methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, neopentyl, trifluoromethyl, pentafluoroethyl and the like.

One or more of the hydrocarbyl groups may be or may comprise an alkylenyl group. The term "alkylenyl" refers to a divalent straight chain or branched chain saturated aliphatic radical having from 1 to 20 carbon atoms. Typical alkylenyl groups employed in compounds are loweralkylenyl groups that have from 1 to about 6 carbon atoms in their backbone." Alkenyl" refers herein to straight chain, branched, or cyclic radicals having one or more double bonds and from 2 to 20 carbon atoms. One or more of the hydrocarbyl groups may be or may comprise an alkynyl group, he term "alkynyl" refers herein to straight chain, branched, or cyclic radicals having one or more triple bonds and from 2 to 20 carbon atoms.

One or more of the hydrocarbyl groups may be or may comprise an alkoxy group - such as a lower alkoxy group. The term "lower alkoxy" as used herein refers to RO- wherein R is loweralkyl. Representative examples of lower alkoxy groups include methoxy, ethoxy, t-butoxy, trifluoromethoxy and the like.

One or more of the hydrocarbyl groups may comprise a halo group. The term "halo" refers herein to a halogen radical, such as fluorine, chlorine, bromine or iodine.

One or more of the hydrocarbyl groups may be or may comprise a haloalkyl group. The term "haloalkyl" refers to an alkyl radical substituted with one or more halogen atoms.

One or more of the hydrocarbyl groups may be or may comprise a haloloweralkyl group. The term "haloloweralkyl" refers to a loweralkyl radical substituted with one or more halogen atoms.

One or more of the hydrocarbyl groups may be or may comprise a haloalkoxy group. The term "haloalkoxy" refers to an alkoxy radical substituted with one or more halogen atoms.

One or more of the hydrocarbyl groups may be or may comprise a haloloweralkoxy group. The term "haloloweralkoxy" refers to a loweralkoxy radical substituted with one or more halogen atoms.

One or more of the hydrocarbyl groups may comprise an amino group. The term "amino" refers herein to the group-NH2. The term "alkylamino" refers herein to the group -NRR' here R and R'are each independently selected from hydrogen or a lower alkyl. The term "arylamino" refers herein to the group-NRR'where R is aryl and R'is hydrogen, a lower alkyl, or an aryl. T he term "aralkylamino" refers herein to the groupNRR'where R is a lower aralkyl and R'is hydrogen, a loweralkyl, an aryl, or a loweraralkyl.

One or more of the hydrocarbyl groups may be or may comprise an alkoxyalkyl group. The term "alkoxyalkyl" refers to the group-alkl-O-alk2 where alkl is alkylenyl or alkenyl, and alk2 is alkyl or alkenyl.

One or more of the hydrocarbyl groups may be or may comprise an loweralkoxyalkyl group. The term "loweralkoxyalkyl" refers to an alkoxyalkyl where alkl is loweralkylenyl or loweralkenyl, and alk2 is loweralkyl or loweralkenyl.

One or more of the hydrocarbyl groups may be or may comprise an alkoxyalkylamino group. The term "alkoxyalkylamino" refers herein to the group-NR- (alkoxylalkyl), where R is typically hydrogen, loweraralkyl, or loweralkyl.

One or more of the hydrocarbyl groups may be or may comprise an aminoloweralkoxyalkyl group. The term "aminoloweralkoxyalkyl" refers herein to an aminoalkoxyalkyl in which the alkoxyalkyl is a loweralkoxyalkyl.

One or more of the hydrocarbyl groups may be or may comprise an aminocarbonyl group. The term "aminocarbonyl" refers herein to the group-C (O)-NH2.

One or more of the hydrocarbyl groups may be or may comprise a substituted aminocarbonyl group. The term "substituted aminocarbonyl" refers herein to the group-C (O)-NRR'where R is loweralkyl and R'is hydrogen or a loweralkyl.

One or more of the hydrocarbyl groups may be or may comprise an aminosulfonyl group. The term "aminosulfonyl" refers herein to the group-S (0) 2-NH2."Substituted aminosulfonyl" refers herein to the group-S (O) 2-NRR' where R is loweralkyl and R'is hydrogen or a loweralkyl. The term "aralkylaminosulfonlyaryl" refers herein to the group -aryl-S (O) 2-NH-aralkyl, where the aralkyl is loweraralkyl. One or more of the hydrocarbyl groups may comprise a carbonyl group. The term "carbonyl" refers to the divalent group-C (O)-.

One or more of the hydrocarbyl groups may comprise a carbonyloxy group. The tenn "carbonyloxy" refers generally to the group-C (O)-O-,. Such groups include esters, -C (O)-O-R, where R is loweralkyl, cycloalkyl, aryl, or loweraralkyl.

One or more of the hydrocarbyl groups may comprise a sulfonyl group. The tenn "sulfonyl" refers herein to the group-S02-.

One or more of the hydrocarbyl groups may comprise an alkylsulfonyl group. The term "alkylsulfonyl" refers to a substituted sulfonyl of the structure-SO2R-in which R is alkyl. Alkylsulfonyl groups employed in XD compounds are typically loweralkylsulfonyl groups having from 1 to 6 carbon atoms in its backbone structure. Thus, typical alkylsulfonyl groups employed in XD compounds include, for example, methylsulfonyl (i. e., where R is methyl), ethylsulfonyl (i. e., where R is ethyl), propylsulfonyl (i. e., where R is propyl), and the like.

One or more of the hydrocarbyl groups may comprise a carbonylamino group. The term "carbonylamino" refers to the divalent group-NH-C (O)in which the hydrogen atom of the amide nitrogen of the carbonylamino group can be replaced a loweralkyl, aryl, or loweraralkyl group. Such groups include moieties such as carbamate esters (- NH-C (O)-O-R) and amides-NH-C (O)-O-R, where R is a straight or branched chain loweralkyl, cycloalkyl, or aryl or loweraralkyl.

One or more of the hydrocarbyl groups may comprise a loweralkylcarbonylamino group. The term "loweralkylcarbonylamino" refers to alkylcarbonylamino where R is a loweralkyl having from 1 to about 6 carbon atoms in its backbone stracture.

One or more of the hydrocarbyl groups may comprise a guanidino group or a guanidyl group. The terms "guanidino"or"guanidyl" refers to moieties derived from guanidine, H2N-C (=NH)-NH2. Such moieties include those bonded at the nitrogen atom carrying the formal double bond (the"2"-position of the guanidine, e. g., diaminomethyleneamino, (H2N) 2C=NH-) and those bonded at either of the nitrogen atoms carrying a formal single bond (the"l-"and/or"3"-positions of the guandine, e. g, H2N-C (-NH)-NH-). The hydrogen atoms at any of the nitrogens can be replaced with a suitable substituent, such as loweralkyl, aryl, or loweraralkyl.

One or more of the hydrocarbyl groups may comprise an amidino group. The term "amidino" refers to the moieties R-C (=N)-NR'- (the radical being at the"Nl"nitrogen) and R (NR') C=N- (the radical being at the"N2" nitrogen), where R and R'can be hydrogen, loweralkyl, aryl, or loweraralkyl.

Preferably, at least one of the hydrocarbyl groups comprises just C and H.

Preferably, the at least one of the hydrocarbyl groups which comprises just C and H is an alkyl group.

Preferably, the at least one of the hydrocarbyl groups which comprises just C and H is a C1-C6 alkyl group.

Preferably, the at least one of the hydrocarbyl groups which comprises just C and H is a linear alkyl group.

Preferably, the at least one of the hydrocarbyl groups which comprises just C and H is a linear C1-C6 alkyl group.

Preferably, the at least one of the hydrocarbyl groups which comprises just C and H is a saturated alkyl group.

Preferably, the at least one of the hydrocarbyl groups which comprises just C and H is a saturated C1-C6 alkyl group. Preferably, the at least one of the hydrocarbyl groups which comprises just C and H is a linear and saturated alkyl group.

Preferably, the at least one of the hydrocarbyl groups which comprises just C and H is a linear and saturated C1-C6 alkyl group. An example of an unsaturated alkyl group is an alkylene group.

Preferably, the at least one of the hydrocarbyl groups which comprises just C and H is a C3 alkyl group.

Preferably, the at least one of the hydrocarbyl groups which comprises just C and H is a linear C3 alkyl group.

Preferably, the at least one of the hydrocarbyl groups which comprises just C and H is a saturated C3 alkyl group.

Preferably, the at least one of the hydrocarbyl groups which comprises just C and H is a linear and saturated C3 alkyl group.

In one preferred aspect of the present invention there is provided a hydrogel of a carboxyalkyl-containing polysaccharide cross-linked with an aminoalkyl carbodiimide and an hydroxysuccinimide. The present invention also relates to inter alia processes, compositions, and uses of the same.

In one preferred aspect of the present invention there is provided a hydrogel of a carboxymethyl-containing polysaccharide cross-linked with l-Ethyl-(3-3- dimethlyaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The present invention also relates to inter alia processes, compositions, and uses of the same.

ADVANTAGES The present invention has a number of advantages. These advantages will be apparent in the following description.

By way of example, the present invention is advantageous since it provides inter alia commercially useful hydrogels, processes for making the same and uses of the same.

By way of further example, the present invention is advantageous since the hydrogel can be prepared by a sample and rapid process.

By way of further example, the present invention is advantageous since the availability of carboxymethyl-containing polysaccharides - such as carboxymethyl dextran - possessing a range of different molecular weights, its solubility in polar organic solvents (which enables chemical modification) and its biocompatibility make this natural polysaccharide immediately attractive.

By way of further example, the present invention is advantageous since it provides the potential benefits of: biocompatibilty; rapid gelation; malleabilty; controllable degradation; controllable porosity; ability to be chemically modified; transparent for clinical evaluation and microscopic examination.

By way of further example, the present invention is advantageous since the hydrogel remains intact when steam sterilised post-gelation.

By way of further example, the present invention is advantageous since the variation of carboxylic group density and crosslinking reagent concentration permits control of the charge density and the degree of crosslinking. This means that the balance between the degradation rate and mechanical properties can be easily controlled.

By way of further example, the present invention is advantageous since EDC/NHS does not chemically bind to dextran molecules, in contrast with conventional cross- linl ng agents (Nakajima and Ikada 1995), but instead reacts to form a water-soluble urea derivative, which has far lower cytotoxity and is easily washed out. Importantly for cell culture, the EDC/NHS does not chemically bind to the carboxymethyl- containing polysaccharide - such as carboxymethyl dextran - in contrast with conventional crosslinking agents, and the chemicals are changed into water soluble urea derivatives which are less cytotoxic and can be removed by adequate washing.

By way of further example, the present invention is advantageous since the pH- sensitive hydrogel membranes as described herein can be produced by including an excess of carboxylic groups, which changes their porosity reversibly in response to changes in environmental pH. Because of this nature, these materials can be used in a wide variety of applications - such as separation membranes, biosensors, drag delivery devices and tissue engineering.

FIGURE LEGENDS

Figure 1

Synthesis of a dextran hydrogel according to the present invention.

Figure 2

The dextran hydrogel was characterized by FTLR. The peak resulting from the carbonyl (C=O) stretching of the non-sensitive and pH sensitive CM-dextran gels appeared at ca.1734 cm^"1 and 1580 cm^"1. The carbonyl group of the gel ester bond displayed apeak at ca.1775 cm^"1.

Figure 3

Light micrographs showing attachment of primary human dermal fibrob lasts onto CM- dextran after post-seeding. Cellular attachment was present on all non-sensitive hydrogels whether possessing additional adhesion factors or not (A- CM-dextran only, B- with fibronectin, C- with poly L-lysine, D- with concanavadin A, E- All pH sensitive gels showed no attachment). Figure 4

Light microscopy demonstrating the confluent sheets of primary human dermal fibroblasts that have formed on CM-dextran hydrogels after 5 days culture. The gels shown are non-sensitive and contained no additional adhesion factors.

Figure 5

Schematic of the composite hydrogel (bold lines denote covalent links) a) Gel porosity reduced by cross-links in pH 5.5 that exclude protein, e.g. lysozyme. b) The porosity increases in high pH leading to ionization of the carboxylic groups. c) Protein enters the more highly porous gel. d) Protein diffuses through the membrane.

Figure 6

SEM showing a structure comparison of hydrogel kept in pH 5.0 and 7.4 phosphate buffer. Magnification x 2000.

Figure 7

Reversible of hydrogel response to pH change. pH 7.4 phosphate buffer plus 2.0 mg/ml lysozyme was added at the down-arrows. pH 5.0 phosphate buffer plus 2.0 mg/ml lysozyme was used at the beginning and restored at the up-arrows . (Temperature 25°C, fixed ionic strength 0.1M ).

Figure 8

Effect of pH on lysozyme diffusion. Lysozyme 2.0 mg/ml, temperature 25°C. 20 mM phosphate buffer of constant ionic strength of 0.1 M (adjusted using NaCl). Figure 9

The profile of rate of Lysozyme diffusion rate as a function of ionic strength at pH 7.4 and 5.5. All other conditions as for Figure 7.

Figure 10

The pH and ionic strength effect on 2mg/ml lysozyme through non-pH-sensitive hydrogel membrane at room temperature (25 °C). The system was inititally operated in pH 5.5 phosphate buffer. At 100 minutes, either pH 7.4 phosphate buffer 20 mM (ionic strength 0.1M) or pH 5.5 phosphate buffer (ionic strength 0.2M) added as shown by the arrow. The baseline was conducted at pH 7.4, ionic strength 0.1 M. Ionic strength adjustments were made with NaCl.

DETAILED DESCRIPTION OF THE INVENTION

HYDROGEL

In one aspect, the present invention relates to a hydrogel of a polysaccharide cross- linked with a carbodiimide and a succinimide.

Preferably, the carbodiimide is aminopropyl carbodiimide.

In a highly preferred embodiment of the present invention, the aminopropyl carbodiimide is l-Ethyl-(3-3-dimethlyaminopropyl) hydrochloride (EDC).

Preferably, the succinimide is hydroxysuccinimide.

In a highly preferred embodiment of the present invention, the hydroxysuccinimide is N-hydroxysuccinimide (NHS). The hydrogel may be in the form of a sheet, a coating, a membrane, a composite, a laminate or the like.

Advantageously, the hydrogel of the present invention is able to degrade controllably. The rate of degradation may be altered by introducing network defects resulting in the fonnation of soft hydrogels with longer degradation times then stiffer, more cross- linked gels.

The cross-linked hydrogel may release a bioactive substance over several days up to several months.

The cross-linked hydrogel may release the bioactive substance at a constant or substantially constant rate.

The hydrogel may degrade and eventually disintegrate and/or dissolve. The amount of degradation may be modulated by varying the crosslink densities.

Preferably, the polysaccharide is a carboxymethyl-containing polysaccharide.

The carboxymethyl-containing polysaccharide may include and is not limited to carboxymethyl starch, carboxymethyl dextran, carboxymethyl cellulose and glucosaminoglycans - such as heparin, heparan sulphate, chondroitin sulphate and hyaluronic acid. The polysaccharide may either contain carboxyl groups from the outset or may be derivatised so as to then contain such groups.

In a highly preferred embodiment of the present invention, the carboxymethyl- containing polysaccharide is carboxymethyl dextran.

Dextran is a bacterial polysaccharide consisting essentially of 1,6-linked d- glucopyranoside residues with a small percentage of a 1,3 -linked side chains which has low tissue-toxicity and high enzymatic degradability at desired sites. Dextran possesses a range of different molecular weights, is soluble in polar organic solvents (which enable chemical modification) and is biocompatible.

Preferably, the dextran that is used in accordance with the present invention is dextran having a molecular weight of less than about 1000 kDa, preferably less than about 800 kDa, preferably less than about 600 kDa.

Preferably, the dextran that is used in accordance with the present invention is dextran having a molecular weight of from 300 kDa to 600 kDa.

Preferably, the dextran that is used in accordance with the present invention is dextran having a molecular weight of from 400 kDa to 550 kDa.

Preferably, the dextran that is used in accordance with the present invention is dextran having a molecular weight of from 450 kDa to 550 kDa.

More preferably, the dextran that is used in accordance with the present invention is dextran has a molecular weight of about 480 kDa.

The low tissue toxicity, high enzymatic degradability at desired sites and ease of chemical modification mean that dextran hydrogels have been frequently considered as a potential matrix system for cell culture²⁷.

Preferably, the CM-dextran is prepared by mixing dextran and sodium chloroacetate.

The carboxymethylation reaction may be initiated of a base - such as NaOH.

Carboxymethylation is then allowed to proceed for about 15 minutes at about 62°C for non-sensitive gels and for about 1 hour at about 70°C for pH-sensitive control gels.

Termination of the reaction may be achieved by adjusting the solution pH to 7, using a suitable acid - such as HCl. The CM-dextran may then be precipitated and allowed to stand overnight, the sediments dissolved in distilled water and subsequently dialysed.

The final solution may then rapidly frozen in liquid nitrogen and lyophilised. Advantageously, the composition of the hydrogel, in particular, the pH sensitivity of the hydrogel may be altered by using a carboxymethyl-containing polysaccharide - such as carboxymethyl dextran - that has a different degree of carboxylic group substitution.

Accordingly, the pH-sensitive hydrogel membranes can be produced by including an excess of carboxylic groups.

If the hydrogel is pH sensitive then typically, the ratio of COOH groups per dextran will be in the region of 1 -COOH: 50 to 100 glucose.

If the hydrogel is pH sensitive then more typically, the ratio of COOH groups per dextran will be in the region of 1-COOH:50 to 70 glucose.

If the hydrogel is pH sensitive then even more typically, the ratio of COOH groups per dextran will be in the region of 1-COOH:60 to 70 glucose.

By way of one example, if the hydrogel is pH sensitive then the ratio of COOH groups per dextran can be l-COOH:63-glucose.

By way of another example, if the hydrogel is pH sensitive then the ratio of COOH groups per dextran can be l-COOH:70-glucose.

If the hydrogel is non pH sensitive then typically, the ratio of COOH groups per dextran will be in the region of l-COOH:l to 25 glucose.

If the hydrogel is non pH sensitive then more typically, the ratio of COOH groups per dextran will be in the region of l-COOH:5 to 20 glucose.

If the hydrogel is non pH sensitive then even more typically, the ratio of COOH groups per dextran will be in the region of 1 -COOH: 10 to 15 glucose. By way of example, if the hydrogel is non pH sensitive then the ratio of COOH groups per dextran can be l-COOH:13-glucose.

By way of another example, if the hydrogel is non pH sensitive then the ratio of COOH groups per dextran can be l-COOH:10-glucose.

The number of COOH groups may be calculated using acid titration.

Advantageously, the porosity of the hydrogel of the present invention increases in response to changes in the pH and the ionic strength of the external medium. Without wishing to be bound by theory, it appears that the pH-dependent swelling behaviour arises from the acidic pendant groups in the polymer network. As a result, the conformation in the hydrogel changes from an expanded free-draining matrix to that of a more compact and non-free-draining matrix, so the diffusion rate will dramatically drop with increasing ionic strength.

Advantageously, the pH-sensitive response is reversible.

The CM-dextran may also offer the ability to enhance implantation by the inclusion of additional dermal components as additional solutions or compounds pre-gelation.

CELLS

The hydrogel of the present invention may be used with multiple cell types - such as cells that are or are derived from a human or animal body (eg. artery, bladder, skin, cartilage, bone, ligament and tendon).

The cells may be or may be derived from differentiated or undifferentiated cells - such as stem cells.

Preferably, the cells are or are derived from primary human cell lines. The cells may or may be derived from skin - such as the epidermal and/or dermal layers of skin (eg. primary human dermal fibroblasts).

The cells may be or may be derived from human skin biopsy material, in particular the epidermal and dermal layers of human skin biopsy material.

The cells may be genetically altered cells.

The cells may be obtained from, for example, a donor, from cell culture of cells from a donor, or from established cell culture lines.

If the cells are obtained directly from a donor, they may be washed and implanted directly in combination with the polymeric material. Cellular material may be dissociated single cells, minced tissue (i.e., clumps of aggregated cells) or cell aggregates generated in vitro from dissociated cells. The cells may be cultured using techniques known to those skilled in the art of tissue culture.

Cell persistence and viability may be assessed using scanning electron microscopy, histology, and quantitative assessment with radioisotopes.

PROCESS

In a further aspect, the present invention relates to a process for preparing a hydrogel comprising crosslinking a polysaccharide with at least one carbodiimide and at least one succinimide (NHS).

To prepare the hydrogel, the lyophilised dextran may be dissolved in a suitable medium - such as fibroblast medium and typically sterilised.

Separately, the carbodiimide - such as EDC - and succinimide - such as NHS - are dissolved and the solution added to the polysaccharide. The hydrogel is incubated until gelation and then washed several times using, for example, a buffer - such as 0.1M phosphate buffer solution (PBS) - followed by fibroblast media.

Hydrogels may be formed using pH-sensitive gels and non-sensitive CM-dextran gels.

If the gel is to be used for tissue culture, then it may be pipetted into well tissue culture plates.

OTHER ADDITIVES

Additional polymer(s) may be incorporated into the polymerisation reaction mixture. Such polymer(s) may include polyacrylamide, poly-NaAMPS, polyethylene glycol (PBG), polyvinylpyrrolidone (PVP) or carboxymethyl cellulose.

Additional functional ingredients may also be incorporated in the reaction mixture used in the invention - such as antimicrobial agents (e.g. citric acid, stannous chloride) and, for drag delivery applications, pharmaceutically active agents, the latter being designed to be delivered either passively (e.g. transdennally) or actively (e.g. iontophoretically) through the skin. For this purpose, penetration enhancing agents may also be presents in the reaction mixture and resultant hydrogel.

Initiators may also be added as additional functional ingredients.

The hydrogels may be additionally modified by the inclusion of additional adhesive factors - such as poly (L-lysine), fibronectin, concanavadin A and/or cell adhesion molecules (CAMs).

ADMINISTRATION The hydrogel of the present invention may be administered alone but will generally be administered as a pharmaceutical or a cosmetic composition - eg. when the components are in admixture with a suitable pharmaceutical or cosmetic excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical or cosmetic practice.

The routes for administration (delivery) include, but are not limited to, topical or parenteral (e.g. by an injectable form).

If a component is administered parenterally, then examples of such administration include one or more of: intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the component; and/or by using infusion techniques.

For parenteral administration, the hydrogel is best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

The hydrogel solution may be injected directly into a site in a patient, where the hydrogel forms an implant and induces a fibrotic response from the surrounding tissues. The loose connective tissue formed by the fibrotic response holds the hydrogel particles in place, and tissue formation is accompanied by vascularisation, producing a vascularised mass of soft tissue consistency occupying the volume established during implantation.

By way of example, the liquid components of the hydrogel to the desired site for in situ formation using a multi-chamber syringe. The multi-chamber syringe may be attached to a multi-lumen catheter or needle such that the high molecular weight components that form the cross-linked hydrogel do not interact until injected into the site inside the body where the matrix is needed.

Another method involves the use of a multi-chamber syringe with a single lumen catheter or needle containing a static mixing element where the components remain separated until injection into the site, but the high molecular weight components actually contact one another within the lumen of the catheter or needle during injection into the specified site. Additional methods of delivery of the hydrogel components for in situ formation would be readily apparent to one skilled in the art.

Alternatively, cells may be included in the hydrogel composition of the present invention and injected directly into a site in a patient, where the hydrogel hardens into a matrix having cells dispersed therein.

The hydrogel may be applied topically. For application topically to the skin, the hydrogel may be suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, it may be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

If the hydrogel is to be used as a pharmaceutical delivery device, the compositions may optionally contain topical, transdermal or iontophoretic agents and excipients. The compositions may contain penetration-enhancing agents to assist the delivery of water of active agents into the skin. Non-limiting examples of penetration-enhancing agents for use in such applications include methyl oleic acid, isopropyl myristate, Azone(R) Transcutol(R) and N-methyl pyrrolidone.

Alternatively, cells may be suspended in a biopolymer solution which is poured or injected into a rigid or inflatable mould having a desired anatomical shape, then hardened to form a matrix having cells dispersed therein which can be implanted into a patient. After implantation, the hydrogel ultimately degrades, leaving only the resulting tissue. Such hydrogel-cell mixtures may be used for a variety of reconstructive procedures, including custom moulding of cell implants to reconstruct three dimensional tissue defects, filling preinserted inflatable moulds or scaffolds, as well as implantation of tissues generally.

COMPOSITIONS

In a further aspect, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a hydrogel and a pharmaceutically acceptable carrier, diluent and/or excipient (including combinations thereof).

In a further aspect, the present invention relates to a cosmetic composition comprising a cosmetically effective amount of a hydrogel and a cosmetically acceptable carrier, diluent and/or excipient (including combinations thereof).

The compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically or cosmetically acceptable diluent, carrier, or excipient.

Acceptable carriers or diluents for therapeutic use are well known in the art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of carrier, excipient or diluent may be selected with regard to the intended route of administration. The compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilising agent(s).

Preservatives, stabilisers and dyes may also be provided in the compositions. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

FORMULATION

The hydrogel of the present invention may be formulated into a composition - such as by mixing with one or more of a suitable carrier, diluent or excipient, by using techniques that are known in the art.

In particular, the hydrogel may be formulated, for example, as sterile components at a manufacturing site and supplied to a physician either as separate units, which may be combined to form the final gel material, or as a pre-loaded syringe containing the final cross-linked product.

Additional components may be included in the formulation.

By way of example, growth factors - such as VEGF, bFGF, EGF and/or BMP may be included. By way of further example, plasmid DNA containing the gene(s) encoding angiogenic proteins may be included to enhance vascular network formation in engineered tissues. By way of even further example, co-transplantation of endothelial cells along with the primary cell type of interest may allow, or provide for, the formation of blood vessels in an engineered tissue

DOSE LEVELS

Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the age, body weight, general health, diet, mode and time of administration, rate of excretion, drag combination, the severity of the particular condition, and the individual undergoing therapy. APPLICATIONS

Advantageously, the hydrogel of the present invention has a multiplicity of applications, as described below.

To aid research into cell growth

As described herein, the hydrogel of the present invention has applications in cell culture, in particular, as a matrix to support cells (eg. clinically relevant cells).

In standard cell culture, cells are typically recovered with proteases. However, the culture of cells on hydrogels enables one to easily recover intact cell sheets without damage by simply decreasing the temperature and/or modulating the hydrophobicity of the gel.

The hydrogel may be used as an ex vivo culture scaffold for the development of small diameter vascular grafts, valves, or other complex tissue-engineered constructs prior to implantation in a patient. In this case, the cross-linked hydrogel serves as an organising template directing cell growth in vitro, and can be used to develop complex organ or tissue structures through a sequence of culture steps.

A delivery system

Stimuli-responsive hydrogels can exhibit dramatic changes in their swelling behaviour, network stracture, permeability or mechanical strength in response to changes in the pH or ionic strength of the surrounding fluid, temperature, bimolecular and applied electrical or magnetic fields (Peppas, 2000). Because of their nature, these materials can be used as pharmaceutical delivery devices for the delivery of pharmaceuticals or other active agents to or through mammalian skin. As described herein, the porosity of the hydrogel of the present invention increases in response to changes in the pH and the ionic strength of the external medium. Without wishing to be bound by theory, it appears that the pH-dependent swelling behaviour arises from the acidic pendant groups in the polymer network. As a result, the conformation in the hydrogel changes from an expanded free-draining matrix to that of a more compact and non-free-draining matrix, so the diffusion rate will dramatically drop with increasing ionic strength.

A tissue engineering scaffold.

The replacement or repair of damaged or diseased tissues or organs by implantation has been, and continues to be, a long-standing goal of medicine towards which tremendous progress has been made. Working toward that goal, there is an increasing interest in tissue engineering techniques where biocompatible, biodegradable materials are used as a support matrix, as a substrate for the delivery of cultured cells, or for three-dimensional tissue reconstruction.

Accordingly, the hydrogel of the present invention also has application as a tissue engineering scaffold.

The hydrogel may be used as a wound healing device to protect open wounds during healing and also to promote healing by administration of the cross-linked hydrogel to the wound.

The hydrogel may be used as an adhesive (such as a tissue sealant) in wound repair. The use of adhesives in wound repair is known in the art. Wound adhesives provide a popular alternative for wound closure over standard methods, such as sutures, staples, and adhesive strips, because they offer ease of use, decreased pain, reduced application time, and no follow-up for removal. The historically first wound adhesive made available, and the one still used most often today, is a type of cyanoacrylate. The use of cyanoacrylate wound adhesives, however, has several drawbacks that limit its use, such as, allergic reactions, presence of residual solvents, and migration of chemicals to other parts of the body. Further, cyanoacrylate adhesives should not be used in pregnant women or patients with a history of peripheral vascular disease, diabetes mellitus, or prolonged corticosteroid use, or on patients who have puncture wounds or bite or scratch wounds (animal or human in origin). Cyanoacrylate wound adhesives may only be used on the surface of the skin and on regularly shaped wounds with even surfaces that are easily pushed back together. It is highly desirable to ensure that none of the cyanoacrylate touches raw skin or enters the wound because it may cause severe irritation and can actually function to impair epithelialization within the wound.

The hydrogel may also be used in a range of skin contact or covering applications where the composition is brought into contact either with skin or with an intermediary article which interfaces between the composition and the skin.

The composition may be unsupported or supported on a backing stracture.

The compositions may suitably be in the form of sheets, coatings, membranes, composites or laminates and the like.

Such applications may include patches, tapes, bandages, devices and dressings of general utility or for specific uses, including, for example, biomedical, skin care, personal and body care, palliative and veterinary uses - such as skin electrodes; wound and burn healing; wound and burn management; skin cooling; skin moisturising; skin warming; aroma release or delivery; decongestant release or delivery; phannaceutical and drag release or delivery; perfume release or delivery; fragrance release or delivery, scent release or delivery; adhesive use, e.g. in skin contacting devices, ostomy and related incontinence devices, and the like. Instrumentation

Since the hydrogel of the present invention can change porosity and/or its dimensions under conditions of, for example, changing pH, the hydrogel also has applications in various types of instrumentation- such as biosensors.

The hydrogel described herein may also be used in the preparation of separation membranes.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in can-ying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES

PART A

For applications in cell and tissue culture it may also be beneficial to incorporate coupled proteins in order to facilitate cellular adhesion and morphology. Using MA- dextran, the proteins or attachment ligands can be grafted onto the surface. Synthesising gels using the method described here for CM-dextran with carbodiimide chemistry permits protein distribution throughout the hydrogel.

The ability of the CM-dextran to support cells would highlight a potential application in tissue engineering. Presently, despite many years of research and development, the only commercially available tissue engineered product is a dermal replacement (Dermagraft, Smith and Nephew, USA). It is for the same flat sheet application that we initially examine the potential of CM-dextran and seeded cells. The preferential use of autologous split-thickness skin grafting to cover wounds is severely restricted by donor availability. Therefore, the ability to rapidly and efficiently create a suitable sterile replacement is conducive to optimal healing, since it minimises infection, shock, desiccation, wound contraction and eventual scar formation at the earliest stage . Replacement dermis functions as a scaffold for local cells migrating from the wound periphery and supports subsequent grafts of partial thickness or cultured epithelial sheets³⁸'³⁹. Presence of a dermal component has long been shown to improve epithelial acceptance, function and aesthetic appearance, in addition to providing strength and ease of clinical handling⁴⁰'⁴¹. For the following study, the potential of CM-dextran to perform the task of a dermal replacement or cell delivery system was examined by using flat sheets for the maintenance of human dermal fibroblasts. To maintain clinical relevance, primary human cells were used.

Example 1

Materials & Methods

Tissue Culture

Human skin biopsy material (obtained with ethical consent) was manipulated to separate the epidermal and dermal layers. The dermis was then minced and incubated in 625 U/mL collagenase (Sigma, Poole, UK) in Dulbecco's modified Eagle's media (DMEM) (Gibco Life Technologies, Paisley, UK) for 2 hours. The resulting primary cell solution was resuspended in fibroblast media [DMEM supplemented with 10% fetal calf serum (FCS) (Globepharm, USA), penicillin (100 U/mL) and streptomycin (100 μg/mL) (Gibco)]. The cells were cultured at 37°C with a 95% air, 5% CO₂ gaseous composition and 100%) humidity, and used between passage 2 and 4.

Hydrogel membrane synthesis

Dextran (480 kDa) was obtained from Sigma-Aldrich, Poole, UK. All other chemicals were of reagent grade and obtained from Lancaster Synthesis Ltd., UK.

The chemistry of the CM-dextran hydrogel synthesis is shown in Figure 1. Briefly, to prepare the CM-dextran, 5g of dextran (480 kDa) was dissolved in 75ml of distilled water and to this was added 5g of sodium chloroacetate. The carboxymethylation reaction was initiated by the addition of 25ml of 8M NaOH, which was further diluted to 100ml total volume and stirred. Carboxymethylation was allowed to proceed for 15 minutes at 62°C for non-sensitive gels and 1 hour at 70°C for pH- sensitive control gels. Termination of the reaction was achieved by adjusting the solution pH to 7, using 6M HCl. The CM-dextran was then precipitated using 300ml absolute ethanol and allowed to stand overnight. The sediments were dissolved in distilled water and subsequently dialysed for two days. The final solution was then rapidly frozen in liquid nitrogen and lyophilised. The amount of COOH groups present was calculated using acid titration and the ratio for pH-sensitive gels was 1 COOH: 13 glucose compared to 1 COOH: 63 glucose for non-sensitive gels.

Preparing gels for tissue culture

All methods for preparation were carried out in sterile conditions. The lyophilised dextran (3g) was dissolved in 20ml of fibroblast medium and sterilised using a 0.2μm filter. Separately, 1.5g of EDC and 300mg NHS were dissolved in 5ml of distilled water and also filtered. The EDC/NHS solution was added to the CM-dextran and pipetted into 12 well tissue culture plates. Some hydrogels were additionally modified by the inclusion of additional adhesive factors: 1ml of either poly (L-lysine), fibronectin (50 μg ml^"1) or concanavadin A was added into the CM-dextran solution and mixed.

Hydrogel were incubated at 37°C until gelation (approximately 1 hour), and then washed several times using 0. IM phosphate buffer solution (PBS) at pH 7.4 followed by fibroblast media. Hydrogels were formed using pH-sensitive gels and non-sensitive CM- dextran gels. Finally, all gels were soaked overnight in fibroblast media. Early passage primary human dermal fibroblasts were seeded onto the gels at a density of 5x10⁴ cells cm^"2 and maintained in a humidified incubator at 37°C and 5% CO₂/95% air. Media was replenished every second day and samples were observed at various time points during culture up to 7 days. Analysis of samples

Images were obtained using a light microscope (Carl Zeiss 68040) utilising a colour video camera (JVC 3-CCD KY-F55B) fitted with an imaging system (KS 300, Imaging Associates Ltd, UK).

Samples of unseeded CM-dextran were characterised when hydrated at pH 7.4 in PBS, using cryo-SEM. Samples were observed in the field emission SEM (JSM-6210, JEOL, Japan) operated at 1.5-30kV and maintained at a temperature of 148-163K.

Infrared spectroscopy was performed using a Bruker-equinox 55 FT-LR spectrometer. Lyophilised samples were mixed with KBr powder and pressed under vacuum to form tablets. For each sample, 100 scans were conducted in the range 400 to 4000 cm^"1, using a resolution of 2 cm^"1.

Example 2

Characterisation of blank CM-dextran matrices

It was demonstrated that COOH groups could be grafted into dextran in the presence of sodium chloroacetate. The extent of grafting can be controlled by adjusting reaction temperature and time. The successful incorporation of the COOH groups was demonstrated by the presence of a carboxylic FT-LR band from the carbonyl (C=O) stretching of CM-dextran at ca. 1734 cm^"1 and 1580 cm^"1 (Figure 2). The representative absorption peak in the gel is assigned to an ester bond at ca. 1775 cm^"1. The relative peaks of COOH in the hydrogel are also confirmed at 1734 cm^"1 and 1580 cm^" . The above finding indicates that activated carboxyl groups can react with hydroxyl groups in CM-dextran to form ester linkages.

The internal structure of the hydrogel has a high voidage and should allow the unrestricted passage of nutrients throughout the nascent tissue construct. In addition, the stracture should also provide suitable for maintenance of cells in a three- dimensional orientation as tissue develops. Fibroblast cultures served to demonstrate the increased biological interaction that is possessed by the covalently modified (non- sensitive) CM-dextran. Hydrogels that were additionally modified by the inclusion of adhesive factors (fibronectin, conA or poly(L-lysine)) showed the beginnings of attachment and spreading of cells by 4 hours post-inoculation on all samples (Figure 3). However, qualitatively there appeared to be no improvement over samples of the CM-dextran possessing no additional adhesive factors. Control pH-sensitive dextran hydrogels with a ratio 1 COOH: 13 glucose demonstrated no cellular attachment by the same time (4 hours), nor at any subsequent time period. Cells were maintained upon the hydrogels for 7 days. An increasing number of cells were observed during this period indicating that cells were proliferating. This occurred on all non-sensitive CM- dextran hydrogels. By the end of this 7 day period entire confluent cell sheets had formed upon the surface of the gels. Cells showed an affinity for alignment along natural cracks and cavities that had formed in the gel upon gelation (Figure 4).

Summary

The hydrogel described herein could be beneficial in three dimensional cell culture as it would permit some microenvironmental response to the changing culture conditions. This will have implications for cellular migration, timed release of growth factors, and facilitation of diffusion of nutrients and toxic metabolites (such as lactic acid).

Carbodiimide chemistry was employed to crosslink carboxylic and hydroxyl groups in CM-dextran. The expected mechanism would result in EDC reacting with the carboxyl of the CM-dextran and form activated O-urea. This is an intermediate and may subsequently react with available hydroxyl to form ester linkages or release a soluble urea derivative. The extent of crosslinking of the carboxyl groups has implications for the reactivity of the gel to physicochemical conditions such as pH, and also to the cell affinity for the matrix. Similar methods have been reported for hyaluronic acid hydrogels, which were prepared utilising intermolecular formation of ester bonds between hydroxyl and carboxyl groups. Cell culture experiments using primary human dermal fibroblasts were utilised to illustrate the ability of the matrix to support clinically relevant cells and demonstrate the potential to extend its use as a delivery system or tissue engineering scaffolding. The failure of fibroblasts to attach to the pH-sensitive CM-dextran hydrogels may have been due to the negative influence of the ionic charge present in these gels. The absence of fibroblasts on these gels suggested that it was the ratio of COOH to glucose that predicted whether attachment would occur. The presence of fibroblasts on all forms of the non-sensitive CM-dextran was encouraging. This demonstrated that the presence of additional adhesion molecules were not a prerequisite for improving cell attachment, spreading or survival. This makes the synthesis of matrices more cost effective, rapid and simple.

The CM-dextran gels were successfully prepared by chemically crosslinking CM- dextran using EDC and NHS. These crosslinking agents introduce "zero length" ester linkages between carboxylic acid and hydroxyl groups in CM-dextran. When the CM- dextran is compared to the commonly used methylacrylated form crosslinked with the same agents, then the former has the following advantages. Firstly, the procedure for preparation is simple and rapid. Secondly, variation of carboxylic group density and crosslinking reagent concentration permits control of the charge density and the degree of crosslinking. This means that the balance between the degradation rate and mechanical properties can be easily controlled. Importantly for cell culture, the EDC/NHS does not chemically bind to the dextran molecules, in contrast with conventional crosslinking agents, and the chemicals are changed into water soluble urea derivatives which are less cytotoxic and can be removed by adequate washing. The presence of cells upon the non-sensitive CM-dextran suggested that the only cytotoxicity present was due to variations in the number of COOH groups. The observation that cell numbers increased over 7 days also indicated that the dermal fibroblasts were not merely remaining attached or surviving. The proliferation would also suggest that no toxicity was present due to leachables from the process leaving the hydrogels. The ester bond linkages were confirmed by FT-LR (Figure 2). From these, it was shown that the chemically reactive groups responsible for crosslinking CM-dextran were the carboxyl and hydroxyl groups.

The ability of the matrix to support human cells may have an immediate application as a flat sheet supporting dermal fibroblasts for the tissue engineering replacement of: venous/arterial, decubitus, diabetic ulceration; burns; or loss of tissue due to accident or disease. In the example of ulcers, chronic wounds require significant maintenance and cost. The Global wound care market in 2002 was estimated at $15 billion⁴⁵ and venous ulcers had a reported worldwide incidence of 2.8 million due to venous insufficiency⁴⁶. A matrix synthesised from dextran could be easily sourced and inexpensive. The matrix also offers the additional potential benefits: biocompatible; rapid gelation; malleable; controllable degradation; controllable porosity; ability to be chemically modified; transparent for clinical evaluation and microscopic examination. In addition, although constituent solutions were filter sterilised in this study, we have found that the hydrogel remains intact when steam sterilised post-gelation.

Previously, implantation of collagen dennal replacements have been enhanced by the inclusion of additional dermal components, such as the glycosaminoglycans^{47" 9}. The CM-dextran also offers the ability to combine these factors easily as additional solutions or compounds pre-gelation. Longer-term studies will be required to examine whether cells have the ability to infiltrate the matrix.

This research has shown that a CM-dextran hydrogel is capable of maintaining primary human dermal fibroblasts over a period of 7 days without any apparent adverse effects in vitro. Based upon these findings, the CM-dextran hydrogel offers some possibility for development as an extracellular matrix substrate for use in cell culture or tissue engineering applications. PART B

Stimuli-responsive hydrogels can exhibit dramatic changes in their swelling behaviour, network stracture, permeability or mechanical strength in response to changes in the pH or ionic strength of the surrounding fluid, temperature, bimolecular and applied electrical or magnetic fields (Peppas, 2000). Because of their nature, these materials can be used in a wide variety of applications, such as separation membranes, biosensors, drag delivery devices and tissue engineering (Lee and Mooney, 2001).

All pH-sensitive polymers are produced by adding pendant acidic or basic functional groups to the polymer backbone; these either accept or release protons in response to appropriate pH and ionic strength changes in aqueous media (Qui and Park, 2001). The network porosity in these hydrogels is a result of electrostatic repulsion. For example, ionic hydrogels containing carboxylic or sulfonic acid show either sudden or gradual changes in their dynamic and equilibrium swelling behaviour as a result of changing the external pH (Katchalsky and Mitchaeli, 1955, Brannon-Peppas and Peppas, 1991). The degree of ionisation of these hydrogels depends on the number of pendant acidic groups in the hydrogel, which results in increased electrostatic repulsions between negatively charged carboxyl groups on different chains, as shown in Figure 1. This, in turn, results in an increased hydrophilicity of the network, and greater swelling ratios. On the other hand, a hydrogel containing basic pendant groups, such as amines, ionises extensively at low pH, also causing increased electrostatic repulsions.

Previously we reported the synthesis of a D-glucose-sensitive dextran hydrogel and demonstrated that the release of proteins (insulin, lysozyme, and BSA) from this hydrogel varied with D-glucose concentration (Tang et al. 2003). We have also shown that hydrogels synthesized by grafting Cibacron Blue and lysozyme to dextran can yield a reversible and specific controlled release of both cytochrome C and haemoglobin in response to changes in environmental NAD concentration (Tang et al. 2002). We now describe the synthesis of a novel pH-responsive dextran hydrogel produced by the intermolecular cross-linking of carboxymethyl dextran (CM-dextran) using 1- Ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS). In view of the toxicity of the crosslinking agent in the work reported here, EDC was used for preference as it is not incorporated into the cross-linked stracture, but is simply changed to a water-soluble urea derivative. The cytotoxity of the urea derivative has been found to be quite low compared with that of EDC (Benslimane et al. 1988). Our hydrogel is formed by ester bonds between hydroxyl and carboxyl in CM-dextran in the presence of EDC and NHS.

Example 1

Materials & Methods

Materials

Dextran and lysozyme were obtained from Sigma-Aldrich, UK. All other chemicals were of reagent grade and obtained from Lancaster Synthesis Ltd., UK.

Hydrogel membrane synthesis

The synthesis procedure was as follows, and is summarised in Figure 1 : 5 g 480KD dextran was dissolved in 75ml of distilled water, followed by the addition of 5g- sodium chloroacetate. The carboxymethylation reaction was initiated by the addition of 25mL of 8M NaOH. The reaction mixture was diluted to 100 ml total volume. Carboxymethylation was allowed to proceed for 1 hour at 70°C with stirring to make a pH-sensitive hydrogel membrane and 15 minutes at 62 °C with stirring to make a non- sensitive hydrogel membrane. The reaction was terminated by lowering the solution pH to 7 with 6 M HCl. The product was then precipitated with 300 ml absolute ethanol and allowed to stand overnight. The sediments were dissolved using distilled water and then packed into a dialysis membrane. This solution was dialysed for two days with many changes of distilled water. The solution was rapidly frozen using liquid nitrogen and then was lyophilised to give a white power.

The amount of COOH groups in the above two CM-dextrans were calculated by means of acid titration. The ratio of COOH groups per dextran is l-COOH:13-glucose for the pH-sensitive hydrogel and l-COOH:63-glucose for the non-pH-sensitive hydrogel.

To make a pH-sensitive hydrogel membrane, 0.5 g CM-dextran was weighed and dissolved using 3 ml distilled water while stirring thoroughly. 160 mg EDC and 25 mg NHS were weighed and dissolved in 1 ml distilled water. If no bubbles were found in the CM-dextran solution, 1 ml of the EDC/NHS mixture was added and stirred for 30 minutes. The solution was then cast on a nylon gauze mechanical support (pore size 0.1 mm, thickness of 0.05 mm) between two glass plates using spacers to give the required total membrane thickness.

Non-pH sensitive hydrogels were produced by a similar procedure using a CM-dextran with a lower degree of carboxylic group substitution (l-COOH:63-Glucose; see above)

Lysozyme diffusion experiments

The trans-membrane transport of lysozyme was studied using a diffusion cell consisting of donor and receptor chambers of equal volumes of 4.4 ml, as described by Tang et al. (2003). Hydrogel membranes with a surface area of 4.58 cm² were mounted between the two chambers. Once the membranes were mounted, both chambers were filled with 20 mM phosphate buffer. The donor chamber was connected to the lysozyme reservoir via a pump. The receptor chamber was connected to a UV- Visible spectrophotometer, to allow lysozyme diffusion across the membrane to be automatically monitored and logged from optical density changes. The amount of lysozyme diffusing across to the receptor chamber was calculated using calibration data for lysozyme at 280 nm. The effect of pH and ionic strength was investigated by varying pH and ionic strength levels in the lysozyme solution feed to the donor chamber and following the resultant changes in transport rate. Lysozyme (2 mg/ml) was dissolved in 20 mM phosphate buffer containing 50 mM NaCl. Transport was monitored at room temperature.

Morphology of the pH-sensitive hydrogel membrane in different pH environments

The morphology of the hydrogel in different pH phosphate buffers was examined using a Jeol 6310 SEM equipped with a cryo-stage and energy-dispersive X-ray (EDX). A sample of the hydrogel was clamped between two pieces of aluminium sheet and rapidly frozen in liquid nitrogen then introduced into the SEM-chamber pre-cooled to a temperature of ca. -160 °C. The stage was the heated to a temperature of ca-80 °C to sublime the surface water. After cooling to -160 °C, the sample was gold sputtered for 3 minutes. The sample was scanned at a magnification of 2000x.

Physicochemcal Properties of the hydrogel

Infrared spectroscopy was performed on a Braker-equinox 55 FT-LR spectrometer. Freeze-dried samples were mixed with potassium bromide powder and pressed into tablets under vacuum. For each sample 100 scans were recorded from 4000 to 400 cm^{" 1} with a resolution of 2 cm^"1.

Example 2

Hydrogel characterization

As shown in Figure 1, the carboxylic groups can be grafted into dextran in the presence of sodium chloroacetate. Adjusting both reaction temperature and time can control the degree of carboxylic substitution. In this experiment, two CM-dextran preparations were made. The first, containing one COOH group per 13 glucose residues, was produced using a reaction temperature of 65 °C for 90 minutes. The second, containing one COOH per 65 glucose residues was attained at 50 °C for 15 minutes. Carbodiimide chemistry was employed to cross-link carboxylic groups and hydroxyl groups in CM-dextran. According to the expected cross-linking mechanism, EDC can react with the carboxyl groups in CM-dextran to form an activated O-urea. This intermediate may either react with available hydroxyl to form an ester linkage or release a soluble urea derivative. To get a pH-sensitive hydrogel, not all carboxyl groups should be crosslinked; the extra hydroxyl group will play the key role in the response to environmental pH changes. Therefore, the degree of sensitivity will be decided by the amount of extra carboxyl groups in the hydrogel. In the method reported by Tomihata and Ikada (1997), hyaluronic acid (HA) hydrogel was prepared by the means of intermolecular formation of ester bonds between hydroxyl and carboxyl groups.

The successful incorporation of the COOH group into dextran is demonstrated by the presence of a carboxylic FT-LR band from the carbonyl (C=O) stretching of CM- dextran at ca .1734 cm^"1 and 1580 cm^"1 as shown in Figure 2. This indicates that activated carboxyl groups can react with hydroxyl groups in CM-dextran to form ester linkages.

The cross-sectional interior stracture of swollen hydrogels kept in pH 7.4 and 5.5 phosphate buffer were revealed by the SEM. Two different structures were observed as shown in Figure 6. The porosity of the hydrogel in pH 5.5 is seen to be compact compared with that in pH 7.4. The pH-responsive feature of the hydrogel is thought to exist in the pore walls. We ascribe the relatively loose stracture at pH 7.4 to expanded pore walls, which results from the response of the hydrogel to the pH change.

Example 3

Reversibility ofthepH response

The reversibility of the pH effect was studied by switching between lysozyme solutions in pH 5.5 and 7.4 phosphate buffer (20 mM) while maintaining the same ionic strength (0.1M) using sodium chloride. In the experiment shown in Figure 7, lysozyme (2.0 mg/ml in a 100ml reservoir) was used for the first 200 minutes at pH 5.5, providing the baseline diffusion curve. At 200 minutes, the pH 5.5 solution was replaced by protein solution at pH 7.4. As the total amounts of lysozyme in the reservoir were much higher than the corresponding amounts in the receptor side of the diffusion cell the effective driving concentration can be assumed constant throughout the run. A significant increase in the rate of lysozyme diffusion occurred about 20 minutes after the switch to the high-pH solution. This cycle was repeated: at 350 minutes protein solution in pH 5.5 was reintroduced. The curve of diffusion became stable after 50 minutes. Repetition of this procedure gave a similar response. The results obtained with lysozyme show that the pH-sensitive membrane exhibits a reversible response in protein diffusion in response to pH changes under the condition of the same ionic strength (0.1M).

Effect ofpH on diffusion rate

2mg/ml lysozyme was used to examine the diffusion rate across the hydrogel membrane in response to pH changes adjusted by phosphate buffer of 20 mM with the same ionic strength as 0.1M (attained by the addition of sodium chloride). Figure 8 shows lysozyme transport through the membrane when pH was varied from 5.0 to 9.0. All these experiments were conducted with a single membrane. To avoid artefacts arising from diffusion lags after pH changes a pre-incubation period was allowed before data collection took place. The results show that the transport rate of lysozyme across the hydrogel increased as the pH increased as the acidic moieties of the CM- dextran became increasingly ionised. Similar results have been observed with pH- sensitive polypeptide hydrogels, methacrylated dextran hydrogels, pofyacrylamide-g- guar gum microgels and chitosan-polyvinyl pyrrolidone hydrogels (Markland et al 1999, Risbud et al 2000, Soppimath et al 2001, Chfu et al 2002). We therefore assume that the pH-sensitive hydrogel grafted carboxyl groups are similar to polyvalent weak acids. At high pH, the COOH groups may be ionised or dissociate, which induces the electrostatic repulsion depicted in Figure 5. As a consequence, the distance between chains increases and the hydrogel becomes swollen. The inset curve in Figure 8 shows that the rate/pH response fits to a titration curve giving a pKa value of 6.1.

The effect of ionic strength on diffusion rate

The ionic strength characteristics of the hydrogels were investigated by measuring the diffusion rate of lysozyme across the membrane under a range of ionic strengths between 0.045 M and 0.3 M at pH 5.5 and 7.4 respectively. Considering the hydrogel as a polyelectrolyte suggests porosity should decrease as ionic strength increases. As shown in Figure 9, at pH 7.4 the diffusion rate of lysozyme firstly increased with increasing ionic strength up to 0.15M; however after that, it decreased with higher ionic strengths. A similar result was attained at pH 5.5, with a shift of the concentration of ionic strength to 0.2 M at which the diffusion rate reaches its maximal point. Ju et al. (2002) have also reported similar results in alginate/PNiPAAm-NH copolymer graft hydrogels.

Osmotic pressure effects

Diffusion experiments were conducted using a 0.5mm membrane with different osmotic pressures induced by exposure to sucrose at room temperature (25°C) in pH 7.4 phosphate buffer of 20 mM (1=0. IM). Sucrose at 0.2M and 0.5M were introduced after 100 minutes. In both cases there was no observable effect on transport rate, suggesting that the diffusion-rate fluctuation seen with changes in pH and salt concentration are not the result of changes in osmotic pressure.

pH and ionic strength effects on control hydrogels

The operation mechanism of our pH hydrogel membrane is based on extra COOH groups in the hydrogel. As seen in Figure 5, the hydrogel membrane can work only in ionised carboxylic group after exposure to high pH. In contrast, hydrogels with a lower carboxylic acid substitution would not be expected to show a significant pH response. From the curve in Figure 10, the diffusion rate of lysozyme across a hydrogel made from CM-dextran with 1 COOH per 65 glucose units shows no change as external pH increased form 5.5 to 7.4. However, ionic strength effects are still apparent as 0.2 M NaCl is added to the buffer used.

Summary

pH-sensitive hydrogels were successively prepared by chemical cross-linking of carboxy methyl dextran (CM-dextran) using l-Ethyl-(3-3-dimethylami-nopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). These cross- linking agents introduce 'zero length' ester-crosslinks between carboxylic acid groups and hydroxyl groups in CM-dextran. When compared with commonly used methacrylated dextran hydrogels, prepared by radical polymerization of methacrylated dextran in the presence of cross-linking agents, hydrogels based on CM- dextran with EDC and NHS have several advantages. Firstly, the preparation procedure is simple and rapid. More importantly, variation of carboxylic group density and cross-linking reagent concentration allow control of both charge density and degree of cross-linking in the hydrogel so that the balance between degradation rates and mechanical properties can be easily controlled. Lastly EDC/NHS does not chemically bind to dextran molecules, in contrast with conventional cross-linking agents (Nakajima and Ucada 1995), instead reacting to form a water-soluble urea derivative, which has far lower cytotoxity and is easily washed out.

The formation of an ester bond as the cross-link bridge was confirmed by FT-IR. Without wishing to be bound by theeory, the chemically reactive groups responsible for the cross-linking of CM-dextran molecules are hydroxyl and carboxyl from Figure 2. Proof of the presence of COOH groups in the hydrogel is also provided by the evidence from FT-LR.

The prominent transition of the hydrogel stracture in response to pH is shown in Figure 6. Although the size of pores in the hydrogel as shown by the SEM is much larger than the protein's diameter, we assume that pores shown by the SEM are water- filled. The functional properties stem from the polymer layer comprising the pore wall, where ionic components will produce changes in electrostatic repulsion with pH change. As explained above, the pores should be regarded as water reservoirs that can be measured from the swelling ratio of the hydrogel. In addition, the COOH content of the hydrogel determines whether the hydrogel is sensitive to pH. As seen in Figure 8, the hydrogel will lose sensitivity to external pH change when there are few or no COOH groups in the hydrogel. It follows that the sensitivity of this hydrogel can be predicted from the substituted degree of carboxymethyl in the dextran.

The diffusion data for lysozyme across the hydrogel at different pH or ionic strengths exhibit the porosity response of the hydrogel to its external environment. The results in Figure 6 indicate that by increasing pH from 5.5 to 7.4, a considerable increase in diffusion rate results. The apparent pKa of 6.1 observed for the CM-dextran, as determined by the bulk titration method, is basically in agreement with the values observed for the center of the relationship curve between diffusion rates against pH in Figure 6 (inset). As far as the ionic strength effect is concerned, the dissociation of COOH in CM-dextran may be enhanced as the ionic strength increases up to a fixed value; but with ionic strength continuously increasing, the anionic groups in the hydrogel are screened by Na⁺ ions. As a result, the conformation in the hydrogel changes from an expanded free-draining matrix to that of a more compact and non- free-draining matrix, so the diffusion rate will dramatically drop with increasing ionic strength. Another possible contribution to this result is that chloride ions present in the external solution might have swamped the carboxylic (COO-) group as described by Soppimath et al. (2001).

In addition, this response to pH is reversible as seen in Figure 5. The shorter lag time of diffusion at pH 7.4 than that at pH 5.5 results from the time taken for the system pH to reach equilibrium after the new buffer is introduced. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry or related fields are intended to be within the scope of the following claims.

REFERENCES A

I. Hansbrough, J.F. and Franco, E.S. (1998). Skin replacements. Clinics in Plastic Surgery. 25: 3. 2. Pomahac et al, 1998

3. Jon A. Rowley, Gerard Madlambayan, David J. Mooney, Alginate hydrogels as synthetic extracellular matrix materials, Biomaterials 20 (1999) 45-53

4. Lim F, Sun AM. Microencapsulated islets as bioartificial endocrine pancreas. Science 1980;210:908-10. 5. Atala A, Kim W, Paige KT, Vacanti CA, Retik AB. Endoscopic treatment of vesicoureteral reflux with a chondrocyte—alginate suspension. J Urol 1994;152:641-3.

6. Jen AC, Wake MC, Mikos AG. Review: hydrogels for cell immobilization. Biotechnol Bioeng 1996;50:357-64.

7. Kuen Yong Lee and David J. Mooney, 2001. Hydrogels for Tissue Engineering Chemical Reviews, 101: 1868-1879

8. Putnam AJ, Mooney DJ. Tissue engineering using synthetic extracellular matrices. Nature Med 1996;2:824-6.

9. J.-K. Francis Suh, Howard W.T. Matthew, Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review, Biomaterials 21 (2000) 2589-2598

10. J. Tradela,b, S.P. Massia, Assessment of the cytotoxicity of photocrosslinked dextran and hyaluronan-based hydrogels to vascular smooth muscle cells Biomaterials 23 (2002) 3299-3307

II. Jean-Pierre Draye, Bernard Delaey, Andre' Van de Voorde, An Van Den Bulcke, Bepke De Reu, Etienne Schacht, In vitro and in vivo biocompatibility of dextran dialdehyde cross-linked gelatin hydrogel films, Biomaterials 19 (1998) 1677- 1687

12. Cees J. De Groot, Marja J.A. Van Luyn, Welmoed N.E. Van Dijk-Wolthuis, Jenny A. CadeH e, JoseH e A. Plantinga, Willem Den Otter, Wim E. Hennink. In vitro biocompatibility of biodegradable dextran-based hydrogels tested with human fibroblasts. Biomaterials 22 (2001) 1197-1203 13. A. J. Kuijpers, G. H. M. Engbers, and J. Feijen, Characterization of the Network Stracture of Carbodiimide Cross-Linked Gelatin Gels Macromolecules 1999, 32, 3325-3333

14. Kenji Tomihata and Yoshito Lkada Crosslinking of hyaluronic acid with water-soluble carbodiimide, J Biomed Mater Res, 37, 243-251, 1997.

15. J.S. Pieper, T. Hafinans, J.H. Veerkamp, T.H. van Kuppevelt, Development of tailor-made collagen glycosammoglycan matrices: EDC/NHS crosslinking, and ultrastructural aspects, Biomaterials 21 (2000) 581-593

16. M.J.B. Wissink, R. Beeniink, J.S. Pieper, A.A. Poot, G.H.M. Engbers, T. Beugeling, W.G. van Aken, J. Feijen, Immobilization of heparin to EDC/NHS- crosslinked collagen. Characterization and in vitro evaluation Biomaterials 22 (2001) 151-163

17. Bell, E., Ehrlich H., Buttle D., Nakatsuji T. (1981). Living tissue formed in vitro and accepted as skin-equivalent tissue of full thickness. Science. 211:1052-4. 18. Burke JF, Yannas IV, Quinby WC, Bondoc CC, Jung WK, Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury, Annals of Surgery, 194 (4): 413-428 1981

19. Wilkins, L. M., Watson, S. R., Prosky, S. J., Meunier, S. F., And Parenteau, N. L. (1994). Development Of A Bilayered Living Skin Construct For Clinical Applications. Biotech. Bioeng. 43; 747-756.

20. B. M. Kim, S. Suzuki, Y. Nishimura, S. C. Urn, K. Morota, T.Maraguchi, Y. Ikada, Cellular artificial skin substitute produced by short period simultaneous culture of fibroblasts and keratinocytes, Brit J Plastic Surg. 52 (7) (1999) 573-578.

21. Kreis RW, Vloemans AFPM, Hoekstra MJ, Mackie DP, Hermans RP, The use of non-viable glycerol-preserved cadaver skin combined with widely expanded autografts in the treatment of extensive 3rd-degree burns, Journal of Trauma-Injury Infection and Critical Care 29 (1): 51-54, 1989

22. M. Heenan, C. Graef, D. Parent, G. De Dobbeleer, P. Galand, Renewal and differentiation of keratinocytes cultured non dead de-epidermalized dermis, Cell Prolif. 25 (1992) 311-319. 23. Wainwright, D. (1995). Use of an acellular allograft dermal matrix (Alloderm) in the management of full thickness bums. Burns. 21; 243-248.

24. Srivastava A, Jennings LJ, Hanumadass M, Sethi S, DeSagun E, Pavlis N, Reyes HM, Walter RJ, Xenogeneic acellular dermal matrix as a dermal substitute in rats , Journal Of Burn Care & Rehabilitation, 20 (5): 382-390 SEP-OCT 1999

25. Horch, R., Debus, M., Wagner, G., and Stark, G. (2000). Cultured human keratinocytes on type i collagen membranes to reconstitute the epidermis. Tissue Engineering, 6, 53.

26. Lee, D., Ahn, H., Cho, K. (2000). A new skin equivalent model: dermal substrate that combines de-epidermized dermis with fibroblast populated collagen matrix. J. Dermatol. Sci. 23, 132-137.

27. Lino Ferreiraa, Maria H. Gila, Jonathan S. Dordickb, Enzymatic synthesis of dextran-containing hydrogels, Biomaterials 23 (2002) 3957-3967

28. H. -C. Chiu et al, 2002. Effects of acrylic acid on preparation and swelling properties of pH-sensitive dextran hydrogels, Biomaterials 23: 1103-1112

29. Hovgaard L, Brondsted H. 1995, Dextran hydrogels for colon-specific drag delivery. J Contr Rel, 36:159 -66

30. Brondsted H, Anderson C, Hovgaard. 1998, Crosslinked dextran F new capsule material for colon targeting of drags.J Contr Rel, 53:7 -13. 31. N. Yamamoto, M. Kurisawa, N. Yui, 1996. Double stimuli-responsive degradation based on phase separation of interpenetrating polymer networks consisting of gelatin and dextran, Macromol. Rapid Commun. 17: 313-318

32. Van Dijk-Wolthuis WNE, Franssen O, Talsma H, Van Steenbergen MJ, Kettenes-Van Den BoschJJ, Hennink WE. 1995. Synthesis, characterization and polymerization of glycidyl methacrylate derivatized dextran. Macromolecules, 28:6317 -6322.

33. Van Dijk-Wolthuis WNE, Kettenes-Van Den Bosch JJ, Van Der Kerk-Van Hoof A, Hennink WE. 1997. Reaction of dextran with glycidyl methacrylate: an unexpected transesteri .cation. Macromolecules, 30:3411 -3413. 34. Kim S H, Chu C C. 2000. Synthesis and characterization of dextran- methacrylate hydrogels and structural study by SEM. J Biomed Mater Res, 49:517 -27 35. Kim S H, Won C Y, Chu C C. 1999. Synthesis and characterization of dextran- based hydrogels prepared by photocrosslinking. Carbohydr Polym, 40: 183 -90

36. H.-C. Chiu, Lin Y.F. Hsu. Y. H., 2002. Effects of acrylic acid on preparation and swelling properties of pH-sensitive dextran hydrogels Biomaterials 23: 1103- 1112

37. I. V. Yannas and J. F. Burke, Design of an artificial skin. I. Basic design principles, J. Biomed. Mater. Res., 14, 65- (1980).

38. Cuono, C, Langdon, R., Mcguire, J. (1986). Use of cultured epidermal autografts and dermal allografts as skin replacement after burn injury. Lancet. 1123. 39. Rheinwald, J., and Green, H. (1975). Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinising colonies from single cells. Cell. 6, 331.

40. Billingham, R. and Reynolds, J. (1952). Transplantation studies on sheets of pure epidermal epithelium and on epidermal cell suspensions. Br. J. Plast. Surg. 5:25- 36.

41. Kangesu T, Navsaria HA, M anek S, Fryer PR, Leigh LM, Green CJ, Kerato- dermal grafts - the importance of dermis for the in-vivo growth of cultured keratinocytes, British Journal of Plastic Surgery, 46 (5): 401-409 Jul 1993

42. M. Tang, R. Zhang, A. Bowyer, R. Eisenthal, J. Hubble, 2002. Smart membranes for delivery of macromolecules, ICOM Toulouse

43. M. Tang, R. Zhang, A. Bowyer, R. Eisenthal, J. Hubble, 2002. An NAD- sensitive hydrogel for the release of macromolecules, ICOM Toulouse

44. R. Zhang, M. Tang, A. Bowyer, R. Eisenthal, J. Hubble, A Novel pH and Ionic Strength-sensitive Carboxy Methyl Dextran Hydrogel, (submitted) 45. New Developments in wound Care. Clinica Reports. 2000. PJB Publications Ltd., CBS836).

46. Baker and Stacey, 1994. Epidemiology of chronic leg ulcers in Australia. Autralian and New Zealand Journal of Surgery, 64, 4, 258).

47. Yannas, I. and Burke, J. (1980). Design of an artificial skin - basic design principles. J.Biomed.Mat.Res. 14, 65. 48. Butler, C, Yannas, I., Compton, C, Correia, C, and Orgill, D. (1999). Comparison of cultured and uncultured keratinocytes seeded into a collagen-gag matrix for skin replacements. British Journal Of Plastic Surgery. 52, 127. 49. Compton, C, Butler, C, Yannas, I., Warland, G., and Orgill, D. (1998). i Organized skin stracture is regenerated in vivo from collagen-gag matrices seeded with autologous keratinocytes. Journal Of Investigative Dermatology. 110, 908.

REFERENCES B

S. Benslimane, R. Guidoin, D. Marceau, Y. Merhi, M.W. King, M.F. Sigo-Luizar, Characteristics of polyester arterial grafts coated with albumin, the role and importance of the crosslinking chemicals. Eur Surg Res 20 (1988) 18.

L. Brannon-Peppas, N.A. Peppas, Dynamic and equilibrium swelling behaviour of pH- sensitive hydrogels containing 2-hydroxyethyl methacrylate, Biomaterials 11 (1990) 635.

L. Brannon-Peppas, N.A. Peppas, Equilibrium swelling behavior of pH-sensitive hydrogels, Chem. Eng. Sci. 46 (1991) 715.

H. Brondsted, C. Anderson, L. Hovgaard, Crosslinked dextran F new capsule material for colon targeting of drags, J Contr Rel, 53 (1998) 7.

H.C. Chiu, G.H. Hsiue, Y.P. Lee, L.W. Huang, Synthesis and characterization of pH- sensitive dextran hydrogels as a potential colon-specific drag delivery system, J. Biomater. Sci. Poly. Ed. 10 (1999) 591.

H. C. Chiu, Y.F. Lin, Y.H. Hsu, Effects of acrylic acid on preparation and swelling properties ofpH-sensitive dextran hydrogels, Biomaterials 23 (2002) 1103.

M. Falamarzian, J. Varshosaz, The effect of stractural changes on swelling kinetics of polybasic /hydrophobic pH sensitive hydrogels, Drag Dev. hid. Pharm. 24 (1998) 667.

L. Hovgaard, H. Brondsted, Dextran hydrogels for colon-specific drug delivery. J Contr Rel, 36 (1995) 159. H. K. Ju, S. Y. Kim and Y. M. Lee, pH/temperature-responsive behaviors of semi-LPN and comb-type graft hydrogels composed of alginate and polyfN- isopropylacrylamide), Polymer, 42 (, 2002) 6851.

A. Katchalsky, I. Michaeli, Polyelectrolyte gels in salt solution, J Polym. Sci. 15 (1955) 69.

A.R. Khare, N.A. Peppas, Release behavior of bioactive agents from pH-sensitive hydrogels, J. Biomater. Sci. Poly. Ed. 4 (1993) 275, (published erratum appears in J. Biomater. Sci. Polym. Ed. 6 (1994) 598).

S. H. Kim, C.C. Chu, Synthesis and characterization of dextran-methacrylate hydrogels and stractural study by SEM. J Biomed Mater Res, 49 (2000)517.

S. H. Kim, CY. Won, C.C. Chu, Synthesis and characterization of dextran-based hydrogels prepared by photocrosslinl ing. Carbohydr Polym, 40 (1999) 183. J.H. Kou, G.L. Amidon, P.I. Lee, pH-dependent swelling and solute diffusion characteristics of polyfhydroxyethyl meth-acrylate-co-methacrylic acid) hydrogels, Pharm. Res. 5 (1988) 592. K. Y. Lee, D. J. Mooney, Hydrogels for Tissue Engineering, Chemical Reviews, 101 (2001) 1868.

P. Markland, Y. Zhang, G.L. Amidon, V.C. Yang, A pH- and ionic strength- responsive polypeptide hydrogel: synthesis, characterization, and preliminary protein release studies, J. Biomed. Mater. Res. 47 (1999) 595.

N. Nakajima and Y. Ikada, Mechanism of amide formation by carbodiimide for bioconjugation in aqueous media, Bioconjugate Chem., 6 (1995) 123. N.A. Peppas, Hydrogels in phannaceutical formulations, European Journal of Pharmaceutics and Biopharmaceutics 50 (2000) 27.

Y. Qiu, K. Park, Environment-sensitive hydrogels for drag delivery, Advanced Drag Delivery Reviews 53 (2001) 321.

MN. Risbud , A. A. Hardikar , SN. Bhat , R. R. Bhonde, pH-sensitive freeze-dried chitosan-polyvinyl pyrrolidone hydrogels as controlled release system for antibiotic delivery, Journal of Controlled Release 68 (2000) 23. K .S. Soppimath, A. R. Kulkami, T. M. Aminabhavi, Chemically modified polyacrylamide-g-guar gum-based crosslinked anionic microgels as pH-sensitive drag delivery systems: preparation and characterization, Journal of Controlled Release 75 (2001) 331. M. Tang, R. Zhang, A. Bowyer, R. Eisenthal, J. Hubble, An ΝAD-sensitive hydrogel for the release of macromolecules, ICOM Toulouse (2002) (Supplementary book of abstracts page 40).

M. Tang, R. Zhang, A. Bowyer, R. Eisenthal, J. Hubble, "A reversible hydrogel membrane for controlling the delivery of macromolecules" Biotechnology & Bioengineering, 82(1) (2003) 47.

K. Tomihata, Y, Ikada, Crosslinking of hyaluronic acid with water-soluble carbodiimide. J Biomed Mater Res. 37 (1997) 243.

W.Ν.E. Van Dijk-Wolthuis, O. Franssen, H. Talsma, M. J. Van Steenbergen , Kettenes- J.J. Van Den Bosch, W.E. Hennink, Synthesis, characterization and polymerization of glycidyl methacrylate derivatized dextran. Macromolecules, 28 (1995) 6317. W.Ν.E. Van Dijk-Wolthuis, J.J. Kettenes-Van Den Bosch , A. Van Der Kerk-Van Hoof, W.E. Hennink, Reaction of dextran with glycidyl methacrylate:an unexpected transesterication. Macromolecules, 30 (1997) 3411. N. Yamamoto, M. Kurisawa, N. Yui, Double stimuli-responsive degradation based on phase separation of interpenetrating polymer networks consisting of gelatin and dextran, Macromol. Rapid Coimnun. 17 (1996) 313.

Claims

1. A hydrogel of a polysaccharide cross-linked with a carbodiimide and a succinimide.

2. A hydrogel according to claim 1, wherein the carbodiimide is aminoalkyl carbodiimide, preferably wherein the carbodiimide is aminopropyl carbodiimide.

3. A hydrogel according to claim 1 or claim 2, wherein the succinimide is hydroxysuccinimide.

4. A hydrogel according to claim 2, wherein the aminopropyl carbodiimide is 1- Ethyl-(3-3-dimethlyaminopropyl) hydrochloride (EDC).

5. A hydrogel according to claim 3, wherein the hydroxysuccinimide is N- hydroxysuccinimide (NHS).

6. A hydrogel according to any of the preceding claims, wherein the polysaccharide is a carboxyalkyl-containing polysaccharide, preferably wherein the polysaccharide is a carboxymethyl-containing polysaccharide.

7. A hydrogel according to claim 6, wherein the carboxymethyl-containing polysaccharide is carboxymethyl-dextran.

8. A hydrogel according to any of the preceding claims, wherein the ratio of COOH groups per dextran is l-COOH:13-glucose.

9. A hydrogel according to any of claims 1-7, wherein the ratio of COOH groups per dextran is l-COOH:63-glucose.

10. A hydrogel according to claim 9, wherein the hydrogel shows a pH-sensitive response.

11. A hydrogel according to claim 10, wherein the pH-sensitive response is reversible.

12. A hydrogel according to any of the preceding claims, wherein the hydrogel is in the form of a sheet, a coating, a membrane, a composite or a laminate.

13. A process for preparing a hydrogel comprising crosslinking a polysaccharide with a carbodiimide and a succinimide.

14. A process according to claim 13, wherein the hydrogel has any of the features of claims 1-12.

15. A process for preparing a non-pH sensitive hydrogel comprising crosslinking a polysaccharide with a low degree of carboxylic group substitution with a carbodiimide and a succinimide.

16. A process according to claim 15, wherein the hydrogel has any of the features of claims 1-8 and 12.

17. A process for preparing a pH sensitive hydrogel comprising crosslinking a polysaccharide with a high degree of carboxylic group substitution with carbodiimide and succinimide.

18. A process according to claim 17, wherein the hydrogel has any of the features of claims 1-7 and 9-12.

19. A hydrogel obtainable or obtained by the process according to any of claim 13- 18.

20. A hydrogel according claim 19, wherein the hydrogel is in the form of a sheet, a coating, a membrane, a composite or a laminate.

21. A process for preparing an extracellular matrix for cell culture comprising the steps of:

(i) performing the process according to any of claims 13-18; and

(ii) contacting the hydrogel with one or more cells.

22. A process according to claim 21, wherein the hydrogel is in the form of a flat sheet.

23. A process according to claim 21 or claim 22, wherein the cells are human cells.

24. A process according to claim 23, wherein the cells are primary human dermal fibroblasts.

25. An extracellular matrix obtainable or obtained by the process of any of claims 21-24.

26. A pharmaceutical composition comprising a therapeutically effective amount of a hydrogel according to any of claims 1-12 or claim 19 and a pharmaceutically acceptable carrier, diluent and/or excipient.

27. A cosmetic composition comprising a cosmetically effective amount of a hydrogel according to any of claims 1-12 or claim 19 and a cosmetically acceptable carrier, diluent and/or excipient.

28. A method for regenerating and/or repairing a diseased, damaged and/or injured tissue comprising administering a hydrogel according to any of claims 1 to 12 or claim 19 and/or a pharmaceutical composition according to claim 26 wherein said hydrogel and/or said pharmaceutical composition is capable of causing a beneficial therapeutic effect.

29. A method according to claim 28 wherein the diseased, damaged and/or injured tissue is selected from: venous/arterial tissue, decubitus tissue, diabetic ulcerated tissue; burnt tissue; or loss of tissue.

30. A cosmetic method for regenerating and/or repairing tissue comprising administering a hydrogel according to any of claims 1 to 12 or claim 19 and/or a pharmaceutical composition according to claim 26, wherein said hydrogel and/or said pharmaceutical composition is capable of causing a beneficial cosmetic effect.

31. A hydrogel according to any of claims 1-12 or claim 19 for use in the treatment of disease.

32. Use of a hydrogel according to any of claims 1-12 or claim 19 as a scaffold or matrix for tissue engineering.

33. Use of a hydrogel according to any of claims 1-12 or claim 19 in the manufacture of a composition for drag delivery.

34. Use of a hydrogel according to any of claims 1-12 or claim 19 as an extracellular matrix in cell culture.

35. Use of a hydrogel according to any of claims 1-12 or claim 19 as a sensor.

36. Use of a hydrogel according to any of claims 1-12 or claim 19 in wound repair.

37. Use of a hydrogel according to any of claims 1-12 or claim 19 as a separation membrane.