CA2219399A1

CA2219399A1 - Bulk formation of monolithic polysaccharide-based hydrogels

Info

Publication number: CA2219399A1
Application number: CA 2219399
Authority: CA
Inventors: Amine Selmani; Crystelle Combes; Cyril Chaput; Abdellatif Chenite
Original assignee: Biosyntech Ltd
Current assignee: Biosyntech Canada Inc
Priority date: 1997-10-24
Filing date: 1997-10-24
Publication date: 1999-04-24

Abstract

The present invention relates to compositions and methods for building monolithic massive hydrogels made of ionic polysaccharides such as Chitin, Chitosan, Alginate, Pectin, Hyaluronic Acid by specific in situ uniform pH changes. Low- to high-molecular weight polybase polysaccharide are dissolved at room temperature in acidic aqueous solutions (2% w/v, pH 4-6). An amide such the urea or ureid is added to the acidic polysaccharide solution and the mixture is homogenized and heated to 80-90°C for initiating the hydrolysis of the amide and the in situ delivery of basic products.
The resulting products basify uniformly and continuously the mixture allowing a pH-controlled gelation of the polybase (pH increase from 3-6 to 7-8). Optical signs of the polybase Chitosan gelation at 37°C appear at pH 6.2-6.5. In a similar way, polyacid polysaccharides such as Alginate or Hyaluronan can be gelled from alkaline solutions by hydrolyzing ester or acid anhydride products such the maleic or acetic anhydride, or the beta-esters and liberating acids in situ. In both cases, the resulting materials are hydrogen-bond based monolithic massive hydrogels with good physico-mechanical properties, are easily moulded into complex shaped materials and present limited shrinkages. Polybase or polyacid-based monolithic hydrogels can be obtained with incorporated organic or inorganic components (second polymer or additive).
Ionic polysaccharide may be applied to drug and cell delivery systems, implantable devices or encapsulating materials.

Description

BULK FORMATION OF MQ~-Cr-TTHIC
POLY-C'~C~ARTnR-BAsED ~yl~O~~ c P~CK~ROUND OF THE INVENTION
(a) Field of the Invention The invention relates to the bulk formation of monolithic polysaccharide-based hydrogels. More par-ticularly, polyacidic or polybasic polysaccharide mate-rials are respectively dissolved in basic or acidic media and are gelated through the in si tu uniform neu-tralization and pH-mediated induction of a 3D continu-ous networks of hydrogen bonds.
(b) DescriPtion of Prior Art In recent developments, natural or artificial biodegradable polymers as well as their derivatives have been increasingly selected for satisfying the new challenges that were proposed by recent medical advances. Polypeptides and polysaccharides which repre-sent a large range of natural macromolecules have gen-erally received particular attentions, such as Colla-gen, Gelatin, Fibronectin, Laminin, Tubulin, Fibrin, Haemoglobin, Algar, Alginates, Carrageenan, Chitin, Chitosan, Hyaluronic Acid, Xanthan Gum and the like.
Amino-containing and carboxyl-containing polysaccha-rides, being among either natural or chemically-modi-fied polysaccharides, have been commonly selected, transformed and investigated as biomaterials in the dry or hydrated state.
Among amino-containing polysaccharides, a spe-cial interest has been given to explore the processingand use of D-glycosamine units which are commonly found in Chitosan or Hyaluronic Acid. In Chitosan, D-gly-cosamine units are generated through catalyzed N-deace-tylation of Chitin which in turn is extracted from marine living animals or organisms (shell-fish), par-ticularly from shell crab, or biosynthesized by natural organisms such as the zygomycete fungi. Like other ani-mal polysaccharides, Chitosan is expected to have good viscoelastic properties which combined to its tissue compatibility and biodegradability, makes it ideal for bioactive and bioresorbable implants. In other respects, Chitosan has already been demonstrated a great potential as structural materials for scaffolding or supporting engineered artificial tissues. This poly-D-glucosamine is also known to be able to attach a large number of proteoglycan molecules and coexists with fibrous Collagen to form aqueous gels. It is believed that the role of proteoglycans within the gel is to retain water and supply appropriate viscoelastic-ity. The resulting extracellular matrix is in turn expected to offer compatible environment for cells pro-liferation and tissue formation, particularly for skin or cartilage cells. This characteristic must be viewed with particular attention and may constitute a poten-tial application of Chitosan in the medical or surgical field.
Among Carboxyl-Polysaccharides, the D-man-nuronic acid, L-guluronic acid, D-galacturonic acid and D-glucuronic acid are the well-known units of typical carboxyl-containing Alginate (mannuronic and guluronic), Pectins (galacturonic), Hyaluronic Acid (glucuronic) polysaccharides. These carboxyl-polysac-charides from marine (Alginate), animal (Hyaluronic Acid) and vegetal (Pectins) origin are currently iso-lated, processed and applied in industrial fields, such as the biotechnological, biomedical or food industry.
Alginate and Pectin polysaccharides have been currently gelled through a controlled chelation with bivalent cations such the Barium (Ba2+), Calcium (Ca2+) and Strontium (Sr2+) cations. Alginate and pectin have been reported to form gel structures that can be studied by a ~egg-box~ or dimerisation of chains model. Pectin gels were also processed at low pH by formation of aggregates of chains, and were found to imply non-cova-lent or non-electrostatic bonding forces and to appears as being thermoreversible gels. Alginate gelation is frequently applied to the encapsulation of living bio-logical such as animal or human cells for cellular biology experiments and engineering as well as duplica-tions of organ functions. Pectin gelation found its applications in the manufacture of jellies and jams.
Hyaluronic Acid and its derivatives products are now well-recognized materials in medicine and surgery, as viscoelastic materials, viscosupplementation and thera-peutic products as well as dry or hydrated scaffolding biomaterials of tissues and organs.
Whatever they were microbial or from marine organisms, Chitin, Chitosan and partially-acetylated Chitosan derivatives were extensively investigated as therapeutic substances or biomaterials.
Production of gelled polysaccharide materials has rapidly attracted much interest due to the variety of applications for the expected resulting products, e.g. drug delivery systems, encapsulating, thickening or gelling agents, etc... Many techniques have been proposed for gelling polysaccharides or cross-linking polysaccharides into hydrated materials. Polysaccha-rides can be gelled currently through ionic, physical or chemical pathways. Neutral or ionic polysaccharides were transformed or gelled for forming drug delivery systems, encapsulation of living biological dressing materials or surgical products: linear polysaccharides such the agarose or gelose were gelled with triazine-based products; D-glucose, L-fucose or D-glucuronate containing polysaccharides were gelled by cations, alginate-based gels were obtained by cationic cross-linking in presence of a dialdehyde or diamine or by a pH-controlled complexation with a protein such the Col-lagen and a dehydration or chemical cross-linking.
Hydrogels for surgical uses were equally prepared by blending polysaccharide/protein with acrylic or meth-acrylic. In a same way, carboxyl-containing polysac-charide gels were formed in acidic aqueous media at pH

2 to 5 by incorporating multifunctional epoxides and used in the prevention of tissue adhesion.
Ionic polysaccharides such as Chitosan or Algi-nate were gelled at the physiological pH through the use of specific polyoxyalkylene polymers, then applied to the reduction of post-surgical adhesion or as con-tact lens. Chitosan granular gels were cross-linked by polyfunctional agents and reduced by reacted for reduc-ing agents for immobilizing insolubilized active enzymes. Chitosan membrane gels were also formed from acid glycerol-water gels by neutralization and used for medicament carriers. In a same way, Chitosan-based gels were used for immobilized and encapsulated living bio-materials such as cells, bacteria and fungi (U.S. Pat-ent No. 4,647,536 and International Patent Application published under No. WO 93/24,112). Chitosan derivatives were also gelled with poly(N-vinyl lactam) such as polyvinylpyrrolidone for wound dressings, drug delivery dressings or cosmetic products as well as with divalent metal oxides and inorganic additives for bone paste substitutes. Chitin hydrogels were proposed by Drohan et al. for growth factors, plasma proteins or drugs encapsulation (International Patent Application pub-lished under No. WO 94/41,818).
It would be highly desirable to be provided with bulk formation of monolithic polysaccharide-based hydrogels which can gelate in situ.

SUMMARY OF THE INVENTION
One aim of the present invention is to provide bulk formation of monolithic polysaccharide-based hydrogels which can gelate in situ.
The present invention provides a novel method which enables the gelation of ionic polysaccharide solutions into three-dimensionally-shaped monolithic massive hydrogels. This method can be applied on poly-base polysaccharides in acidic aqueous solutions as well as on polyacid polysaccharides in alkaline solu-tions. It is based on an in situ uniform neutralization (or pH variation) carried out through a) the introduc-tion of a hydrolyzable chemical substance into the polysaccharide aqueous solution able to induce pH
changes upon hydrolysis, b) the hydrolysis of the said substance within the polysaccharide aqueous solution such as the pH of the said solution is uniformly modi-fied, and c) the uniform pH modification to a pH level which induces the gelation of the polysaccharide solu-tion, and d) the in situ bulk-gelation of the polysac-charide into a three-dimensionally-shaped monolithic massive hydrogel.
In accordance with the present invention there is provided a monolithic ionic carboxyl-containing or amino-containing polysaccharide hydrogel that is bulk-formed by the in situ uniform modification of the pH
within the solution through the introduction of an acid-releasing or base-releasing hydrolyzable chemical substance and the controlled hydrolysis in solution of the said hydrolyzable chemical substance.
The polysaccharide hydrogel may be charac-terized by a continuous uniform three-dimensional mas-sive structure and obtained by the combination of an in situ chemical hydrolysis of a hydrolyzable substance or a mixture thereof and a progressive uniform pH increase throughout the solution or structure.
The polysaccharide hydrogel may be a polycationic polymer with amino groups on its consti-tutive monomers, such amino groups being free aminegroups (-NH2) or amino groups from acetyls (-NH-).
The polysaccharide hydrogel may contain D-gly-cosamine, N-deacetylated-D-glycosamine, D-galactosamine or N-deacetylated-D-galactosamine units.
The polysaccharide hydrogel may be a Chitin or Chitosan polymer and their derivatives, being essentially made of monomeric beta-(1-4)-D-glucosamine linked units and of monomeric beta-(1-4)-N-acetyl-D-glucosamine linked units, whatever the degree of N-de-acetylation within the said Chitosan.
The polysaccharide hydrogel may be synthetic, or produced biologically, either microbially or by natural marine organisms.
The polysaccharide hydrogel may consists in a pure low-molecular weight polysaccharide or a pure medium-molecular weight polysaccharide or a pure high-molecular weight polysaccharide or a mixture thereof.
The polysaccharide hydrogel may further include a water-soluble chemical component or a mixture of water-soluble components is introduced in the aqueous polysaccharide solution prior to the gelation whatever this said component or mixture of components is rendered later water-nonsoluble within the polysaccharide hydrogel.
Such water-soluble chemical components may include, without limitation, a) dimethyl sulfoxide, glycerin, glycerol, cyclodextrin, sorbitan esters, mannitol or sorbitol and their derivatives; and/or b) poly(vinyl alcohol), poly(vinyl phosphate), poly(ethylene oxide), poly(ethylene glycol), poly(propylene glycol), poly(N-vinyl lactam), dextran, povidone, hydroxyethylcellulose, methylcellulose, polysorbate polymers and their derivatives; and/or c) in inorganic materials or a mixture of inor-ganic materials such as silica or titanium based inorganics.
In accordance with the present invention there is provided a method of preparing an aqueous amino-containing polysaccharide solution capable upon heating up to 80~C and then cooling up to 15~C of bulk-forming a monolithic hydrogel of the present invention, which method comprises:
a) providing the amino-containing polysaccharide normally insoluble in water at pH superior to 6 but soluble in acidic aqueous solution;
b) dissolving the polysaccharide in an acidic aqueous solution at temperatures around the ambient temperature and up to 80~C but lower than the decompo-sition temperature of the polysaccharide to provide a solution thereof; and c) dissolving the desired amount of a hydrolyzable chemical substance in the aqueous polysaccharide solu-tion at temperatures around of the ambient temperature and up to 80~C, and thereafter maintaining the aqueous polysaccharide solution at a high temperature around 50-80~C so as to initiate the hydrolysis of the said hydrolyzable chemical substance; and d) while degasing the aqueous polysaccharide solu-tion, maintaining the said solution at a temperature ranging from 15~C to 80~C so as to hydrolyze completely the hydrolyzable chemical substance and to increase uniformly the pH to 6.4 and higher.

The hydrolyzable chemical substance may be introduced and hydrolyzed through a temperature-controlled and/or acid-controlled process such as the pH of the aqueous polysaccharide solution is increased progressively and uniformly.
The hydrolysis of the chemical substance within the aqueous polysaccharide solution generates enough ammonium by-products to overall basify uniformly the aqueous polysaccharide solution.
The hydrolysis of the chemical substance within the aqueous polysaccharide solution generates ammonium and degasable products.
The degasing of the aqueous polysaccharide solution during the said hydrolysis of the chemical substance controls in part the uniform pH increase within the polysaccharide solution and the bulk-formation of the polysaccharide hydrogel.
The method of Claim 16 wherein the said chemi-cal substance consists in an amide, and specially a carbamide.
The chemical substance consists in urea, thiourea, guanadine, selenourea, ureids, carbamic acid, cyanuric acid and their derivatives or in any low-molecular weight ureathanized substances which are hydrolyzable in an aqueous acidic solution at the selected temperatures.
In accordance with the present invention, there is also provided a polysaccharide hydrogel charac-terized by a continuous uniform three-dimensional mas-sive structure and obtained by the combination of an insitu chemical hydrolysis of a hydrolyzable substance or a mixture thereof and a progressive uniform pH decrease throughout the solution or structure.

The polysaccharide may be a polyanionic polymer with carboxyl (-COOH) groups on its constitutive monomers.
The polysaccharide may contain D-mannuronic acid, L-guluronic acid, D-galacturonic acid or D-glucuronic acid units.
The polysaccharide may be an Alginate or Pectin and their derivatives.
The polysaccharide may alkso be synthetic, or produced biologically.
The polysaccharide consists in a pure low-molecular weight polysaccharide or a pure medium-molecular weight polysaccharide or a pure high-molecu-lar weight polysaccharide or a mixture thereof.
A water-soluble or chemical component or a mixture of water-soluble components may be introduced in the aqueous polysaccharide solution prior to the gelation whatever this said component or mixture of components is rendered later water-nonsoluble within the polysaccharide hydrogel.
Such water-soluble chemical components may include, without limitation, a) dimethyl sulfoxide, glycerin, glycerol, cyclodextrin, sorbitan esters, mannitol or sorbitol and their derivatives; and/or b) poly(vinyl alcohol), poly(vinyl phosphate), poly(ethylene oxide), poly(ethylene glycol), poly(propylene glycol), poly(N-vinyl lactam), dextran, povidone, hydroxyethylcellulose, methylcellulose, polysorbate polymers and their derivatives; and/or c) inorganic materials or a mixture of inorganic materials such as silica or titanium based inorganics.

In accordance with the present invention, there is also provided a method of preparing an aqueous carboxyl-containing polysaccharide solution capable of bulk-forming a monolithic hydrogel within a temperature range from 0~C to 80~C, which method comprises:
a) providing the carboxyl-containing polysaccha-ride soluble in an alkaline aqueous solutions;
b) dissolving the polysaccharide in an alkaline aqueous solution at temperatures around the ambient temperature and up to 80~C but lower than the decompo-sition temperature of the polysaccharide to provide a solution thereofi and c) dissolving the desired amount of a hydrolyzable chemical substance in the aqueous polysaccharide solu-tion at temperatures around 0~C and up to 80~C; and d) maintaining the said solution at a temperature ranging from 0~C to 80~C so as to hydrolyze completely the hydrolyzable chemical substance and to decrease uniformly the pH to 7 and lower.
The hydrolyzable chemical substance may be introduced and hydrolyzed through a temperature-controlled and/or alkali-controlled process such as the pH of the aqueous polysaccharide solution is decreased progressively and uniformly.
The hydrolysis of the chemical substance within the aqueous polysaccharide solution generates enough acidic by-products to overall acidify uniformly the aqueous polysaccharide solution.
The chemical substance includes in an ester, acid anhydride and lactone compounds.
The chemical substance includes acetic anhydride, maleic anhydride, succinic anhydride and butyrolactone.
The chemical substance includes beta-diesters or water-soluble low-molecular weight polyesters.

The monolithic ionic polysaccharide hydrogel of the present invention may be characterized by a three-dimensional moulding and formation of the said hydrogel into specific shapes such as beads, rods, membranes and blocks.
The monolithic ionic polysaccharide hydrogel of the present invention may be processed such as being combined with other materials (textiles, foams, sponges) to form a composite or complex structures.
The monolithic ionic polysaccharide hydrogel of the present invention may have incorporated therein therapeutic substances such as antiviral, antifungal, antibacterial or steroidal and non-steroidal anti-inflammatory agents, growth factors and hormones.
The monolithic ionic polysaccharide hydrogel of the present invention may be implanted in animals or humans for delivering drugs, polypeptides or cells, reconstructing and replacing epithelial, connective, muscular or neural tissues.
The monolithic ionic polysaccharide hydrogel of the present invention may include living animal or human cells from connective tissues are encapsulated for forming biohybrid systems, culturing and engineering biological tissues.
BRIEF DBSCRIPTION OF THE DRAWINGS
Fig. lA shows the chemical structure of the N-acetylated-D-glucosamine linked units (N-ACE-GLU) within the Chitin polysaccharide;
Fig. lB shows the chemical structure of the units linked in the polysaccharide chain, one N-deace-tylated-D-glucosamine unit (N-DEACE-GLU) and one N-ace-tylated-D-glucosamine unit (N-ACE-GLU);
Figs. 2A and 2B shows the possible chemical units in a hyaluronic acid polysaccharide, one N-acety-lated-D-glucosamine unit (N-ACE-GLU) and one Glucuronic acid (GLU. AC.) unit, one N-deacetylated-D-glucosamine unit (N-DEACE-GLU) and one Glucuronic acid unit (GLU.
AC.);
Fig. 3A shows the alpha-L-Guluronic acid unit and 1,4-linked beta-D-Mannuronic acid unit that can be found in Alginate polysaccharidesi Fig. 3B shows the 1,4-linked-alpha-D-Galac-turonic acid units of the pectin polysaccharidesi Fig. 4 shows an example of the potential chemi-cal substances (amides) that can be hydrolyzed in an acidic polysaccharide aqueous solution for producing alkaline moleculesi and Fig. 5 shows an example of the potential chemi-cal substances (acid anhydride, ester) that can be hydrolyzed in an alkaline polysaccharide aqueous solu-tion for producing acidic molecules.

DET~TrRn DESCRIPTION OF THE INVENTION
In the present invention, a general principle is proposed for gelling ionic polysaccharides namely carboxyl-containing and/or amino-containing polysaccha-rides into three-dimensionally-shaped monolithic mas-sive hydrogels. In one of the embodiment of the inven-tion, D-glucos-amine/N-deacetylated-D-glucosamine con-taining Chitin and Chitosan derivatives were gelled by a control-led and progressive induction of an uniform and continuous 3D network of hydrogen bonds. The intro-duction of a hydrolyzable chemical substance into an acidic polysaccharide solution as well as the acid- and temperature-catalyzed hydrolyzing of the said chemical substances result into an uniform in si tu pH increase allowing the bulk-gelation of the (polybasic) polysac-charide into an one-piece hydrogel. In a symmetrical way, the second major embodiment contains the introduc-tion of a hydrolyzable chemical substance into an alka-line polysaccharide solution, the alkali- and tempera-ture-catalyzed hydrolysis of the said chemical sub-stance which result into an uniform in si tu pH decrease allowing the gelation of the (polyacid) polysaccharide into an one-piece hydrogel. This is specially devoted to carboxyl-containing polysaccharides, e.g. D-man-nuronic acid, L-guluronic acid, D-galacturonic acid and D-glucuronic acid, etc., such as the Alginate, Pectin or Hyarulonic Acid based polysaccharides.
The processing techniques of the present inven-tion enable the specific formation and fashioning of 3D-shaped monolithic continuous hydrogel materials made of pure polysaccharides or polysaccharide-based blends.
Monolithic polysaccharide hydrogel materials can be introduced in a wide range of medical or surgical applications such as cell, drug or gene delivery sys-tems, reconstructive or replacement implants and bioen-gineered tissue scaffolding materials.
Of special interest, numerous methods have been proposed for forming polysaccharide gels, either by heating a swollen aqueous mixture of polysaccharide powder into a semi-continuous particulate gel, by heat-ing a polysaccharide dispersion, by acidifying an alka-line solution of polysaccharide, by exposing a polysac-charide solution to acid anhydride vapors such as gase-ous carbon dioxide, by diluting with water a solution of polysaccharide into organic solvent, or by dialyzing alkaline polysaccharide solutions against various solu-tions. A beta-1,3-glucan polysaccharide was gelled thermally through a critical temperature neutralization by reducing the pH of the alkaline polysaccharide solu-tion through a specific temperature range where this reduction does not induce immediately a gelation.

The method herein described differs from all the others by the fact that 1~ the gelation can be technically induced in a very similar manner from ionic polyacid or polybase polysaccharides, 2) the gellation is produced in situ through a continuous, progressive and uniform pH modification within the solution volume or gel structure, 3) the pH modification and gelation are ensured by an in situ hydrolysis of an appropriate hydrolyzable chemical substance or mixture thereof, 4) the gelation attributed to a continuous uniform network of hydrogen bonds results in a three-dimensionally-shaped monolithic massive polysaccharide hydrogel, and can be obtained conveniently in any molds.
~Polyacid~ polysaccharide refers to the ionic polysaccharides that contain carboxylic groups and are currently insoluble in acidic media. ~Polybase~ poly-saccharide refers to the ionic polysaccharides that contain essentially amino groups and are currently insoluble in alkaline media.
A ~continuous uniform~ pH modification is said of a pH change which occurs similarly and simultane-ously at every point of the solution or structure. A
continuous uniform pH increase from 4,5 to 6 correspond to a pH change from 4,5 to 6 that can be observed at the same level and at the same time at every point of the solution or structure. In a similar way, a ~continuous uniform~ network of hydrogen bonds is said of a medium or structure wherein hydrogel bonds are homogeneously distributed over the medium or structure, and can be viewed as being present at every point of the said medium or structure.
A ~hydrolyzable~ chemical substance is defined by a chemical substance or compound that reacts with water molecules to give arise to basic or acidic mole-cules. The initial alkali or acid medium as well as the temperature are susceptible to initiate, catalyze or inhibit the hydrolysis reaction.
Herein, ~monolithic~ refers to an one-piece material with its constitutive elements which form a homogeneous rigid system Herein, ~massive~ refers to a solid material that is bulk-formed, occupies the apparent volume, con-taining regular distribution and homogeneous porosity and appears as a compact one-piece material.
Herein, ~three-dimensional shape~ is said of the gelation method which enables any customization of the final hydrogel structure in terms of shape, volume and geometry. The gelation can be performed such as to fashion the hydrogel by molding in any types of solid recipients, e.g. plastic, metallic or glass, or by the processing itself, e.g. dropping in oil.
The presence of (polyacid) carboxyl or (polybase) amino groups which are relatively reactive with a large number of organic functions renders such ionic polysaccharides easily cross-linkable through chemical pathways. Physical gelation can also originate from hydrogen bonds, either intra-chain or inter-chains, involving these carboxyl or amino groups and thus lead to water-insoluble polymeric materials. There exist few available processing methods for the forma-tion of polysaccharide hydrogels that are physically cross-linked into dense continuous networks and fash-ioned in massive pieces with controlled pores and shapes. In particular, the processing and building of monolithic massive hydrogels, one-piece non-particulate hydrogels, that could be modulated in composition, per-formances and properties and molded as required by medical or surgical end-uses would represent a major advance for polysaccharide-based biomaterials. Physical cross-linking by forming three-dimensional networks of hydrogen bonds involving amino or carboxyl groups and the oxygen may help in building the expected monolithic hydrogels.
In one preferred embodiment, amino-containing (polybase) polysaccharides are dissolved in acidic aqueous media such as acetic acid, hydrochloride acid, ascorbic acid, etc. to form a clear polysaccharide solution ranging from 0.1 to 10%, preferentially from 1.0 to 5.0% w/v. For example, an acidic solution of 2.0% w/v partially N-deacetylated Chitosan polysaccha-ride in 0.1 M to 10 M acetic acid solutions, preferen-tially 0.5 M to 6 M acetic acid solution, has a pH
around 3.5-4.5. The pH of the acidic polysaccharide solution can be increased to 5.0 to 6.0 by introducing the necessary amount of an low-concentration alkali, e.g. a 1.0% sodium or potassium hydroxide, but not to a pH level superior to the gelation pH. The gelation pH
of an acidic 2.0% w/v Chitosan solution was found at 37~C to be around 6.2-6.4.
Dissolution of polybase polysaccharides such the Chitin or Chitosan may be obtained with organic or mineral acids such as acetic acid, formic acid, hydro-chloride acid, sulfuric acid, phosphoric acid, maleic acid, tartaric acid, ascorbic acid, etc. or a mixture thereof. The control over, or fine tuning of, the pH of the acidic aqueous solution can be performed by drop-ping organic or mineral alkali such as sodium hydrox-ide, potassium hydroxide or amine in order a pH of the polybase polysaccharide acidic aqueous solution ranging from 3.0 to 6.2.
Any water-soluble chemical components can be added, dissolved and homogenized into the acidic poly-saccharide solutions if they do not break down the homogeneity, uniformity or continuity of the polysac-charide solution or bring non-uniformly or non-progres-sively the pH to a level superior to the gelation pH.
If requested for obtaining a controlled dissolution of the water-soluble components, the said component may be dissolved first in an aqueous solution under specific conditions, then the aqueous solution may be acidified adequately to dissolve the poly-base polysaccharide.
The introduction of thixotropic agents (0.1-20%
w/v, preferentially 0.5-10%) may help in stabilizing the polybase polysaccharide aqueous solution as well as the resulting polysaccharide hydrogels. For example, thixotropic silica agents have been proven to give more consistency and integrity to the polybase polysaccha-ride and to eliminate the prevent shrinking after gela-tion and washing. Suitable inorganic materials include silica, alumina, zinc, zirconia and titanium based inorganics, calcium phosphate, calcium carbonate, apa-tite and the like.
Even in high proportions, the incorporation of water-soluble polymers has been demonstrated that does not prevent the gelation of the polybase polysaccharide even. Mixtures of Chitosan with water-soluble polymers with 70% w/w of the said polymer (70:30 polymer/ Chito-san) gelate similarly to pure Chitosan (100% Chitosan), while the chemical and physicomechanical properties of the resulting hydrogel change with the proportion of the said polymer. Suitable water-soluble polymers include polyvinyl alcohol, polyvinyl phosphate, poly-ethylene oxide, polyethylene glycol, polypropylene gly-col, polyvidone, polyacrylamide, acrylic and meth-acrylic esters, polysorbates, polyester diols, cellu-losic polymers, polypeptides, phospholipids and the like.
In a similar way, water-soluble active agents may be introduced in the polybase polysaccharide hydro-gel prior to the gelation and consist in complexants, surfactants, tonicity preserving or wetting agents.
Suitable agents include beta-cyclodextrin and its derivatives, sorbitan esters, lecithin, sodium chlo-ride, potassium chloride, sorbitol, mannitol and the like.
The incorporation of water-soluble therapeutic or bioactive agents such as the anti-inflammatory, antibacterial, antifungal or antiviral agents, growth factors or other hormones and their derivatives and synthetic analogs. Suitable therapeutic or bioactive agents may include progesterone, norgestrel, estradiol, norethisterone, testosterone, prednisolone, cortisone, dexamethasone, gentamycin, erythromycin, penicillin, neomycin, norfloxacin, methicillin, amphotericin, natamycin, doxorubicin, bleomycin, cisplatin, 5-fluor-ouracil, naltrexon, phenobarbitone, chlorpromazine, methadone, diazepam and the like.
The viscosity of the (amino-containing) poly-base polysaccharide solution will increase with increasing pH levels in the said solution. This is due to the interconversion of ammonium salts of the D-glu-cosamine units of the polysaccharide to free amines, and results in a more water insoluble form of the poly-saccharide. Such an interconversion is an equilibrium process, time-dependent and reversible. This explains that the polybase polysaccharide hydrogel would weaken if placed in acidic media.
The in si tu increase to a certain level of the pH within the polybase polysaccharide aqueous solution would logically result in a water-insoluble polysaccha-ride. As a consequence, an uniform and continuous in situ increase of the pH within the polybase polysaccha-ride solution may result in a uniform and continuous gelation, or bulk-gelation, of the polybase polysaccha-ride, thus producing a monolithic massive polybase polysaccharide gel.
To obtain such an in si tu pH increase and bulk-gelation, the method promotes the addition and dissolu-tion of a hydrolyzable chemical substance within thepolybase polysaccharide aqueous solution, and the homogenization of the said aqueous mixture. The time-dependent, temperature-controlled hydrolysis in situ of the hydrolyzable chemical substance in the said aqueous solution, would result in a pH increase uniformly, con-tinuously and progressively toward the occurrence of a water-insoluble polybase polysaccharide, the monolithic massive polysaccharide hydrogel.
Suitable hydrolyzable chemical substances for gelling polybase polysaccharide contains amides or ure-thanized groups, and may include urea, thiourea, selenourea, ureids, cyclic ureids, guanadine, carbamic acid, cyanuric acid, urethanized products and the like.
The equilibrium reaction of the hydrolysis of some products may be controlled by a degassing if one resulting products is gaseous, for example, such the carbon dioxide formed during the hydrolysis of urea. By eliminating the gaseous carbon dioxide, the equilibrium is displaced to favor the hydrolysis reaction, the ammonium production in situ which accelerates the gela-tion of the polybase polysaccharide. Degassing also occurs naturally during the hydrolysis of urea within the polybase polysaccharide aqueous solution.
The second embodiment concerns the carboxyl-containing polysaccharides (polyacid) which dissolve inalkaline aqueous media to form clear polysaccharide solutions ranging from 0.1 to 10%, preferentially from 1.0 to 5.0% w/v.
The interconversion of carboxylate salts of the polyacid units of the polysaccharide to free carboxyls results in a more water insoluble form of the polysac-charide. Thus an in si tu decrease to a certain level of the pH within the polyacid polysaccharide aqueous solu-tion would logically result in a gelated polysaccha-ride. As a consequence, an uniform and continuous insi tu decrease of the pH within the polyacid polysaccha-ride solution may result in an uniform and continuous gelation, or bulk-gelation, of the polyacid polysaccha-ride, thus producing a monolithic massive polyacid polysaccharide gel.
To obtain such an in si tu pH decrease and bulk-gelation, the method promotes the dissolution and homogenization of a hydrolyzable chemical substance within the polyacid polysaccharide aqueous solution.
The time-dependent, temperature-controlled in situ hydrolysis within the resulting homogeneous aqueous solution would lead to a pH decrease uniformly, con-tinuously and progressively toward the occurrence of a water-insoluble polyacid polysaccharide, the said mono-lithic massive polysaccharide hydrogel.
Typical example consists of an Alginate poly-saccharide solution ( 2.0% W/V) obtained by dissolving either an Alginate salts in water or an Align acid in alkaline aqueous media (e.g. potassium hydroxide 0.01 mM to 100 mM) If necessary, the pH of the alkaline polysaccharide solution is regulated (decreased) by dropping the necessary amount of an low-concentration acid, e.g. a 1.0% acetic acid, but not to a pH level inferior to the gelation pH.
As for polybase polysaccharides, any water-soluble chemical components can be added, dissolved and homogenized into the alkaline polysaccharide solutions if they do not inhibit or impair the gellation process or the hydrogel structure. If requested for obtaining a controlled dissolution of the water-soluble components, the said component may be dissolved first in an aqueous solution under specific conditions, then the aqueous solution may be basified adequately to dissolve the polyacid polysaccharide.
As for polybase polysaccharides, the introduc-tion of thixotropic agents (0.1-20% W/V, preferentially 0.5-10%) may help in stabilizing the polyacid polysac-charide aqueous solution as well as the resulting poly-saccharide hydrogels. Suitable inorganic materials include silica, alumina, zinc, zirconia and titanium based inorganics, calcium phosphate, calcium carbonate, apatite and the like.
As for polybase polysaccharides, water-soluble polymers has been demonstrated that does not prevent the gelation of the polyacid polysaccharide and may be mixed to the polyacid polysaccharide aqueous solution.
Suitable water-soluble polymers include polyvinyl alco-hol, polyvinyl phosphate, polyethylene oxide, polyeth-ylene glycol, polypropylene glycol, polyvidone, poly-acrylamide, acrylic and methacrylic esters, polysor-bates, polyester diols, cellulosic polymers, polypep-tides, phospholipids and the like.
In a similar way, water-soluble active agents may be introduced in the polyacid polysaccharide hydro-gel prior to the gelation and consist in complexants,surfactants, tonicity preserving or wetting agents.
Suitable agents include beta-cyclodextrin and its derivatives, sorbitan esters, lecithin, sodium chlo-ride, potassium chloride, sorbitol, mannitol and the like.
Anti-inflammatory, antibacterial, antifungal or antiviral agents, growth factors or other hormones and their derivatives and synthetic analogs may be incorpo-rated as well within the solution. Suitable therapeutic 3 5 or bioactive agents may include progesterone, norg-estrel, estradiol, norethisterone, testerone, predniso-lone, cortisone, dexamethasone, gentamycin, erythromy-cin, penicillin, neomycin, norfloxacin, methicillin, amphotericin, natamycin, doxorubicin, bleomycin, cis-platin, 5-fluorouracil, naltrexon, phenobarbitone, chloro-promazine, methadone, diazepam and the like.
Suitable hydrolyzable chemical substances for gelling polyacid polysaccharide contains acid anhy-drides, beta-diesters and esters-containing chains that hydrolyzed into acids. It may include acetic anhydride, tartaric anhydride, malonic anhydride, maleic anhy-dride, polyester diols products and the like.
For both polyacid and polybase polysaccharides, the gelation is induced through an uniform in situ pH
change by hydrolyzing in situ a selected hydrolyzable chemical substances. The hydrolysis of the said chemi-cal substance in aqueous solutions is time-dependent, temperature-controlled and acid or alkali-catalyzed.
The hydrolysis rate in situ of the introduced hydrolyz-able chemical substance may be controlled, lowered orincreased, within the range of temperature going from 0~C to 100~C, preferably from 4~C to 80~C. For polyacid polysaccharides, acid anhydrides hydrolyze quite spon-taneously and a lowering of the temperature may help in controlling the hydrolysis rate in situ. For polybase polysaccharide, amide hydrolysis may be initiated or stimulated by increasing the temperature.
The present invention will be more readily un-derstood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLBasic formulation and method for processinq monolithic Chitosan hydlu~els.
Typical experiment is carried out by dissolving 0.2 g of chitosan in 10 ml of aqueous solution of ace-tic acid (O. 5M). The pH measurements indicate an aver-age value around 5.0 for a such solution. Urea (8 g) is then added to previous solution, followed by heating the resulting solution up to 85~C, in order to acceler-ate the hydrolysis of urea, and therefore increase thepH level. When the pH level reaches 6.0 (around the starting point of gelation, pH 6.2-6.4, of a 2% Chito-san solution at 37~C), the mixture is cooled down to 37~C and maintained at this temperature for 24 hours, enough time to achieve the gelation process. The resulting hydrogel is immersed in renewed baths of dis-tilled water in order to remove the excess in urea and ammonium salts. A limited but visible shrinkage of the monolithic massive Chitosan hydrogel is observed ( 1/4-1/3).
Table 1 Chemical constituentsProportions Water 10 ml Acetic Acid, 99.9% 0.30 g Chitosan 0.2 9 Urea 8 9 Adiusting the PH of the Chitosan solution prior to gelformation The process of Example II is similar to the one presented in the Example I, except that the pH of the acidic Chitosan aqueous solution is increased by adding drops of a 1.0% potassium or sodium hydroxide solution in order to reach a pH level ranging from 5.0 to 6Ø
Once the pH of the polysaccharide aqueous solution is stabilized at 37-40~C, the urea is added and the mix-ture is maintained at this temperature until the gela-tion process is accomplished. The resulting hydrogel isimmersed in renewed baths of distilled water in order to remove the excess in urea and ammonium salts.
Table 2 Chemical constituents Proportions Water 10 ml Acetic Acid, 99.9% 0.30 g Chitosan 0.2 g Sodium/Potassium hydroxide s.q. pH 6.0 Urea 49 EXAMPLE III
Processing monolithic Silica particles modified Chitosan gels The process of the Example III is the same as in the Example I, but a fumed silica AEROSIL~ 300 was added to the acidic Chitosan aqueous solution t5-10%
w/w Chitosan). AEROSIL solid inorganic is coagulated silicon dioxide with spherical particles of 10-20 nm.
AEROSIL 300 is a fluffy white powder and contains par-ticles of 7 nm average diameter. A 4.0% AEROSIL 300aqueous dispersion has a pH varying from 3.6 to 4.3.
Once the AEROSIL 300 particles are homogeneously dis-persed in the Chitosan aqueous solution, the gelation is initiated as previously described in Example I. The resulting hydrogel is immersed in renewed baths of dis-tilled water in order to remove the excess in urea and ammonium salts. The resulting monolithic massive Chito-san hydrogel seems to be stabilized structurally, more compact and does not shrink at all. It seems that the silica particles allow to retain water within the Chi-tosan hydrogel.
Table 3 Chemical constituents Proportions Water 10 ml Acetic Acid, 99.9% 0.30 9 Chitosan 0.2 g Fumed silica particles, AEROSIL~ 300 0.012 g Urea 8g BXAMPLB IV
Monolithic PVA/Chitosan hydrogels with high PVA content A 0.2 g of 99+% hydrolyzed polyvinyl alcohol is dissolved in 5 ml of a lM acetic acid solution by heat-ing to 60~C. When the PVA is dissolved and the PVAsolution is clear, the heating is stopped, then 0.1 g of medium M.W. Chitosan is added to the PVA aqueous solution. When the Chitosan is dissolved, the PVA/Chitosan aqueous mixture is generally a clear vis-cous mixture. A 5.0 g of urea is added to the said mix-ture and dissolved by heating which results in a less viscous mixture. The PVA/Chitosan/urea mixture is homogenized and maintained at a high temperature (80-85~C~ for some minutes, then placed in thermal bath at 40~C. The PVA/Chitosan/urea mixture is maintained at 40~C until the urea totally hydrolyzes and progres-sively induces a complete gelation of the system.
The PVA/Chitosan system is a 66.7:33.3 (% w/w) hydrogel. The resulting hydrogel is immersed in renewed 25 baths of distilled water in order to remove the excess in urea and ammonium salts.

Table 4 Chemical constituents Proportions Water 5 ml Acetic Acid, 99.9% 0.30 g Polyvinyl alcohol 0.20 g Chitosan 0.10 9 Urea 5.0 9 Example V
Monolithic PVA/Chitosan hydrogels The processing method of the Example V is strictly identical to the one described in the Example IV, except 0.15 g of PVA is dissolved, then 0.15 g of Chitosan is incorporated such as the resulting PVA/Chitosan system is a 50:50 (% w/w) hydrogel. The PVA/Chitosan/urea mixture with 50:50 proportions is the less viscous system. The resulting hydrogel is immersed in renewed baths of distilled water in order to remove the excess in urea and ammonium salts.
Table 5 Chemical constituents Proportions Water 5 ml Acetic Acid, 99.9% 0.30 9 Polyvinyl alcohol 0.15 g Chitosan 0.15 g Urea 5.0 9 Monolithic PVA/Chitosan gels h~ o~els with low PVA
content The processing method of the Example VI is strictly identical to the one described in the Example IV, except 0.10 g of PVA is dissolved, then 0. 20 g of Chitosan is incorporated such as the resulting PVA/Chitosan system is a 33.3:66.7 (% w/w) hydrogel.
The resulting hydrogel is immersed in renewed baths of distilled water in order to remove the excess in urea 5 and ammonium salts.
Table 6 Chemical constituents Proportions Water 5 ml Acetic Acid, 99.9% 0.30 g Polyvinyl alcohol 0.10 9 Chitosan 0.20 9 Urea 5.0 9 While the invention has been described in con-nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia-tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
Example VII
Basic formulation and method for processing Monolithic Alqinate hy~loyels Typical experiment was carried out by gelling a 2% (w/w) aqueous solution of alginate. The preparation consisted on the dissolution of 0.2g of alginate sodium salt in 10 mL of distilled water. The pH value of such a solution was in the vicinity of 8.2. This resulting solution was then cooled down to about 2~C, after which ~0.2g of a finely ground maleic anhydride was slowly added and carefully dispersed in the viscous aqueous solution of alginate. Once a quite homogeneous dispersion was reached, the mixture was returned back at room temperature and left for a spontaneous and progressive hydrolysis of anhydride, which caused a progressive pH decrease and therefore allowed the gel formation. In the final step, the alginate gel formed was washed with a large volume of distilled water in order to remove any excess of maleic acid.
Table 7 Chemical constituents Proportions Water 10 ml Alginate 0.2 g Maleic anhydride 0.2 g Example VIII15 Basic formulation and method for processing Monolithic Pectate or polygelacturonic acid hydroqels Typical experiment was carried out by gelling a 2% (w/w) aqueous solution of polygalacturonic acid.
The preparation consisted on the dissolution of 0.2g of Polygalacturonic acid in 10 mL of distilled water. The pH value of such a solution was in the vicinity of 8.2.
This resulting solution was then cooled down to about 2~C, after which ~0.2g of a finely ground maleic anhydride was slowly added and carefully dispersed in the viscous aqueous solution of polygalacturonic acid.
Once a quite homogeneous dispersion was reached, the mixture was returned back at room temperature and left for a spontaneous and progressive hydrolysis of anhydride, which caused a progressive pH decrease and therefore allowed the gel formation. In the final step, the polygalacturonic acid gel formed was washed with a large volume of distilled water in order to remove any excess of maleic acid.
Table 8 Chemical constituents Proportions Water 10 ml Poly9~ ctllronic acid 0.2 9 Maleic anhydride 0.2 9 While the invention has been described in con-nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia-tions, uses, or adaptations of the invention following,in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

1. A monolithic ionic carboxyl-containing or amino-containing polysaccharide hydrogel that is bulk-formed by the in situ uniform modification of the pH
within the solution through the introduction of an acid-releasing or base-releasing hydrolyzable chemical substance and the controlled hydrolysis in solution of the said hydrolyzable chemical substance.

2. A monolithic polysaccharide as described in Claim 1 wherein a polysaccharide hydrogel is characterized by a continuous uniform three-dimensional massive structure and obtained by the combination of an in situ chemical hydrolysis of a hydrolyzable substance or a mixture thereof and a progressive uniform pH increase throughout the solution or structure.

3. A monolithic polysaccharide hydrogel as described in Claim 2 wherein the polysaccharide is a polycationic polymer with amino groups on its constitutive monomers, such amino groups being free amine groups (-NH2) or amino groups from acetyls (-NH-).

4. A monolithic polysaccharide as described in Claim 3 wherein the polysaccharide contains D-glycosamine, N-deacetylated-D-glycosamine, D-galactosamine or N-deacetylated-D-galactosamine units.

5. A monolithic polysaccharide as described in Claim 4 wherein the polysaccharide is a Chitin or Chitosan polymer and their derivatives, being essentially made of monomeric beta-(1-4)-D-glucosamine linked units and of monomeric beta-(1-4)-N-acetyl-D-glucosamine linked units, whatever the degree of N-deacetylation within the said Chitosan.

6. A monolithic polysaccharide as described in Claim 5 wherein the polysaccharide is synthetic, or produced biologically, either microbially or by natural marine organisms.

7. A monolithic polysaccharide as described in Claim 5 wherein the polysaccharide consists in a pure low-molecular weight polysaccharide or a pure medium-molecular weight polysaccharide or a pure high-molecular weight polysaccharide or a mixture thereof.

8. A monolithic polysaccharide as described in Claim 5 wherein a water-soluble chemical component or a mixture of water-soluble components is introduced in the aqueous polysaccharide solution prior to the gelation whatever this said component or mixture of components is rendered later water-nonsoluble within the polysaccharide hydrogel.

9. A polysaccharide hydrogel as obtained in Claim 8 wherein the water-soluble chemical components consists in:
a) dimethyl sulfoxide, glycerin, glycerol, cyclodextrin, sorbitan esters, mannitol or sorbitol and their derivatives; and/or b) poly(vinyl alcohol), poly(vinyl phosphate), poly(ethylene oxide), poly(ethylene glycol), poly(propylene glycol), poly(N-vinyl lactam), dextran, povidone, hydroxyethylcellulose, methylcellulose, polysorbate polymers and their derivatives; and/or c) inorganic materials or a mixture of inorganic materials such as silica or titanium based inorganics.

10. A method of preparing an aqueous amino-containing polysaccharide solution capable upon heating up to 80°C and then cooling up to 15°C of bulk-forming a monolithic hydrogel as described in Claim 2, which method comprises:
a) providing the amino-containing polysaccharide normally insoluble in water at pH superior to 6 but soluble in acidic aqueous solution;
b) dissolving the polysaccharide in an acidic aqueous solution at temperatures around the ambient temperature and up to 80°C but lower than the decomposition temperature of the polysaccharide to provide a solution thereof; and c) dissolving the desired amount of a hydrolyzable chemical substance in the aqueous polysaccharide solution at temperatures around of the ambient temperature and up to 80°C, and thereafter maintaining the aqueous polysaccharide solution at a high temperature around 50-80°C so as to initiate the hydrolysis of the said hydrolyzable chemical substance; and d) while degasing the aqueous polysaccharide solution, maintaining the said solution at a temperature ranging from 15°C to 80°C so as to hydrolyze completely the hydrolyzable chemical substance and to increase uniformly the pH to 6.4 and higher.

11. The method of Claim 10 wherein the said polysaccharide is defined as in Claim 3.

12. The method of Claim 10 wherein the said polysaccharide is defined as in Claim 4.

13. The method of Claim 10 wherein the said polysaccharide is defined as in Claim 5.

14. The method of Claim 10 wherein the said polysaccharide is defined in Claim 6.

15. The method of Claim 10 wherein the said polysaccharide is defined in Claim 7.

16. The method of Claim 10 wherein the said monolithic polysaccharide hydrogel is defined as in Claim 8.

17. The method of Claim 10 wherein the hydrolyzable chemical substance is introduced and hydrolyzed through a temperature-controlled and/or acid-controlled process such as the pH of the aqueous polysaccharide solution is increased progressively and uniformly.

18. The method of Claim 17 wherein the said hydrolysis of the chemical substance within the aqueous polysaccharide solution generates enough ammonium by-products to overall basify uniformly the aqueous polysaccharide solution.

19. The methods of Claim 18 wherein the said hydrolysis of the chemical substance within the aqueous polysaccharide solution generates ammonium and degasable products.

20. The method of Claim 19 wherein the degasing of the aqueous polysaccharide solution during the said hydrolysis of the chemical substance controls in part the uniform pH increase within the polysaccharide solution and the bulk-formation of the polysaccharide hydrogel.

21. The method of Claim 17 wherein the said chemical substance consists in an amide, and specially a carbamide.

22. The method of Claim 17 wherein the said chemical substance consists in urea, thiourea, guanadine, selenourea, ureids, carbamic acid, cyanuric acid and their derivatives or in any low-molecular weight ureathanized substances which are hydrolyzable in an aqueous acidic solution at the selected temperatures.

23. A monolithic polysaccharide as described in Claim 1 wherein a polysaccharide hydrogel is characterized by a continuous uniform three-dimensional massive structure and obtained by the combination of an in situ chemical hydrolysis of a hydrolyzable substance or a mixture thereof and a progressive uniform pH decrease throughout the solution or structure.

24. A monolithic polysaccharide hydrogel as described in Claim 23 wherein the polysaccharide is a polyanionic polymer with carboxyl (-COOH) groups on its constitutive monomers.

25. A monolithic polysaccharide as described in Claim 24 wherein the polysaccharide contains D-mannuronic acid, L-guluronic acid, D-galacturonic acid or D-glucuronic acid units.

26. A monolithic polysaccharide as described in Claim 25 wherein the polysaccharide is an Alginate or Pectin and their derivatives.

27. A monolithic polysaccharide as described in Claim 26 wherein the polysaccharide is synthetic, or produced biologically.

28. A monolithic polysaccharide as described in Claim 26 wherein the polysaccharide consists in a pure low-molecular weight polysaccharide or a pure medium-molecular weight polysaccharide or a pure high-molecular weight polysaccharide or a mixture thereof.

29. A monolithic polysaccharide as described in Claim 26 wherein a water-soluble or chemical component or a mixture of water-soluble components is introduced in the aqueous polysaccharide solution prior to the gelation whatever this said component or mixture of components is rendered later water-nonsoluble within the polysaccharide hydrogel.

30. A polysaccharide hydrogel as obtained in Claim 29 wherein the water-soluble chemical components include:
a) dimethyl sulfoxide, glycerin, glycerol, cyclodextrin, sorbitan esters, mannitol or sorbitol and their derivatives; and/or b) poly(vinyl alcohol), poly(vinyl phosphate), poly(ethylene oxide), poly(ethylene glycol), poly(propylene glycol), poly(N-vinyl lactam), dextran, povidone, hydroxyethylcellulose, methylcellulose, polysorbate polymers and their derivatives; and/or c) inorganic materials or a mixture of inorganic materials such as silica or titanium based inorganics.

c) inorganic materials or a mixture of inorganic materials such as silica or titanium based inorganics.

31. A method of preparing an aqueous carboxyl-containing polysaccharide solution capable of bulk-forming a monolithic hydrogel within a temperature range from 0°C to 80°C as described in Claim 23, which method comprises:
a) providing the carboxyl-containing polysaccharide soluble in an alkaline aqueous solutions;
b) dissolving the polysaccharide in an alkaline aqueous solution at temperatures around the ambient temperature and up to 80°C but lower than the decomposition temperature of the polysaccharide to provide a solution thereof; and c) dissolving the desired amount of a hydrolyzable chemical substance in the aqueous polysaccharide solution at temperatures around 0°C and up to 80°C; and d) maintaining the said solution at a temperature ranging from 0°C to 80°C so as to hydrolyze completely the hydrolyzable chemical substance and to decrease uniformly the pH to 7 and lower.

32. The method of Claim 30 wherein the said polysaccharide is defined as in Claim 24.

33. The method of Claim 30 wherein the said polysaccharide is defined as in Claim 25.

34. The method of Claim 30 wherein the said polysaccharide is defined as in Claim 26.

35. The method of Claim 30 wherein the said polysaccharide is defined in Claim 27.

36. The method of Claim 30 wherein the said polysaccharide is defined in Claim 28.

37. The method of Claim 30 wherein the said monolithic polysaccharide hydrogel is defined as in Claim 29.

38. The method of Claim 30 wherein the hydrolyzable chemical substance is introduced and hydrolyzed through a temperature-controlled and/or alkali-controlled process such as the pH of the aqueous polysaccharide solution is decreased progressively and uniformly.

39. The method of Claim 38 wherein the said hydrolysis of the chemical substance within the aqueous polysaccharide solution generates enough acidic by-products to overall acidify uniformly the aqueous polysaccharide solution.

40. The method of Claim 38 wherein the said chemical substance consists in an ester, acid anhydride or lactone compounds.

41. The method of Claim 38 wherein the said chemical substance consists in acetic anhydride, maleic anhydride, succinic anhydride or butyrolactone.

42. The method of Claim 38 wherein the said chemical substance consists in beta-diesters or water-soluble low-molecular weight polyesters.

43. A monolithic ionic polysaccharide hydrogel as defined in Claim 1 which is characterized by a three-dimensional moulding and formation of the said hydrogel into specific shapes such as beads, rods, membranes and blocks.

44. A monolithic ionic polysaccharide hydrogel as defined in Claim 1 which is processed such as being combined with other materials (textiles, foams, sponges) to form a composite or complex structures.

45. A monolithic ionic polysaccharide hydrogel as defined in Claim 1 wherein are incorporated therapeutic substances such as antiviral, antifungal, antibacterial or steroidal and non-steroidal anti-inflammatory agents, growth factors and hormones.

46. A monolithic ionic polysaccharide hydrogel as defined in Claim 45 which is implanted in animals or humans for delivering drugs, polypeptides or cells, reconstructing and replacing epithelial, connective, muscular or neural tissues.

47. A monolithic ionic polysaccharide hydrogel as defined in Claim 1 wherein living animal or human cells from connective tissues are encapsulated for forming biohybrid systems, culturing and engineering biological tissues.