WO2013015688A1

WO2013015688A1 - Chitosan-based hydrogels containing enzyme alkaline phosphatase

Info

Publication number: WO2013015688A1
Application number: PCT/NL2012/050534
Authority: WO
Inventors: Sander Cornelis Gerardus Leeuwenburgh; Timothy Douglas; Johannes Arnoldus Jansen; Agata SKWARCZYNSKA; Zofia MODRZEJEWSKA
Original assignee: Stichting Katholieke Universiteit
Priority date: 2011-07-26
Filing date: 2012-07-26
Publication date: 2013-01-31
Also published as: NL2007179C2

Abstract

This invention relates to the temperature-controlled pH dependant in situ formation of biopolymeric gels. More in particular, the invention provides thermogelling systems that can be used, amongst others, for repair and regeneration of hard tissue, such as bone tissue and cartilage, as well as of certain soft tissues. The present inventors have developed new thermogelling systems by adding enzyme alkaline phosphatase solution to thermosensitive solutions of chitosan and beta- glycerophosphate. This approach surprisingly was found to solve shortcomings of the existing systems, in particular of slow or insufficient gelation at body temperature and the lack of a mineral phase within hydrogels.

Description

CHITOSAN-BASED HYDROGELS CONTAINING ENZYME ALKALINE

PHOSPHATASE

Field of the Invention

This invention, generally stated, relates to temperature-controlled pH dependant in situ formation of biopolymeric gels. More in particular, the invention provides thermogelling systems that can be used, amongst others, for repair and regeneration of bone tissue, cartilage and certain soft tissues. The invention also provides methods of aesthetic and/or reconstructive intervention using these systems as well as kits and devices suitable for carrying out the invention.

Background of the Invention

Repair and/or reconstruction of bone and cartilage as well as certain soft tissues has become a hot research topic in the past decade. The high prevalence of degenerative conditions, injuries, surgery related tissue damage, etc., prompted the search for materials that can be used to form scaffolds that support the damaged tissue. Scaffolds are porous artifacts which provide an architectural context in which extracellular matrix, cell-cell and growth factor interaction combine to regenerate tissue. The scaffold materials can be engineered to confer additional properties, such as inducing and guiding cell proliferation, differentiation and new tissue formation at the site of applications. For that purpose, the scaffold materials may contain cells or biomolecules that can attract cells and growth factors

There are significant challenges in the design and manufacture of scaffolds that posses both highly porous structure and the ability to control release kinetics of growth factors over the period of tissue regeneration.

Injectable biomaterials are widely researched and hold great promise as scaffold material in tissue engineering as well as drug delivery. Injectable biomaterials that form scaffolds in situ have the advantage of being able to take the shape of a tissue defect, avoiding the need for patient specific scaffold prefabrication. Injectable scaffolds eliminate the need for surgical interventions for delivery and also eliminate the problems of cell adhesion and bioactive molecule delivery, as they can be easily incorporated in the solution by mixing prior to injection. Thermogelling injectable systems comprise an aqueous polymeric solution that forms a gel upon temperature change. These hydrogels do not require any additional chemical stimulus for their formation and their gelation occurs at body temperature. They are simply injected in a liquid form and solidify inside the body.

Various natural and synthetic thermosensitive polymers are used including natural polymers, such as cellulose derivatives and chitosan, as well as synthetic polymers, such as poly (Msopropylacrylamide) and Poloxamer.

Chitosan and its derivatives represent a particularly attractive group of biocompatible and degradable polymers. Chitosan is biocompatible, non-toxic, and non-immunogenic, allowing its use in the medical, pharmaceutical, cosmetic and tissue construction fields. Moreover, chitosan is cleaved by certain specific enzymes, e.g. lysozyme, and can therefore be considered as bioerodable and biodegradable. Chitosan also promotes wound-healing, as well as acting as an antiadhesive and exhibits antibacterial and anti-fungal effects, and anti-tumor properties.

The preparation of thermosensitive, neutral solutions based on chitosan/polyol salt combinations has been described by Chenite et al., 2000¹. These formulations possess a physiological pH and can be held liquid below room temperature in order to encapsulate living cells and therapeutic proteins; they form monolithic gels at body temperature, without any chemical modification or cross-linking. The addition of polyol salts bearing a single anionic head results in the formation of a gel due to synergistic forces favorable to gel formation, such as hydrogen bonding, electrostatic interactions and hydrophobic interactions. When injected in vivo the liquid formulation turns into gel implants in situ. The system has been used as a container- reservoir for delivery of biologically active growth factors in vivo as well as an encapsulating matrix for living chondrocytes for tissue engineering applications.

An advanced clinical product of such chitosan hydrogels is a hydrogel produced by BioSyntech, described in PCT application WO 99/07416. The thermosensitive chitosan hydrogel of BioSyntech is prepared by neutralizing a commercial chitosan, having a degree of deacetylation of about 80-90%, with mono-phosphate dibasic salts of polyols, particularly β-glycerophosphate. Addition of β- glycerophosphate to chitosan enables the pH to be increased up to about 7 without chitosan precipitation, and to form a hydrogel at that pH, at physiological temperature.

These promising examples also exhibit some limitations. A disadvantages of the chitosan based thermogelling systems described in the prior art resides in the gelation time at body temperature, which is dependent on properties of the chitosan preparation such as degree of deacetylation and may be too slow or not initiated at all at body temperature (Chenite 2000). No method to initiate and enhance gelation is known except altering the pH or degree of deacetylation. This restricts the range of chitosan preparations which can be used. Furthermore, the degree of deacetylation is believed to influence the degradation rate.

The modulation of the properties of the hydrogel, such as gelation time and viscosity, depends on the concentration of glycerophosphate, and is therefore limited by the solubility of glycerophosphate. A high concentration of glycerophosphate is typically required for acceptable gelation time. However, a high concentration of glycerophosphate also decreases the viscosity of the hydrogel. Therefore, the gelation time has to be balanced with the consistency of the hydrogel, and it is not possible to obtain hydrogels that have both low gelation time and high viscosity, which would be a particularly desirable combination of characteristics. Also, a too high concentration of glycerophosphate may induce the precipitation of the hydrogel at its administration site.

Furthermore, the chitosan-based thermogelling systems known to date have poor bioactivity, such as osseointegrative properties, hindering chemical bonding and integration with surrounding bone. It is known that bioactivity can be improved by addition of a CaP phase. The simplest strategy to introduce a CaP phase into gels would be the incorporation of CaP particles. However, CaP particles tend to aggregate, leading to uneven dispersion and poor reproducibility. Micron-scale CaP granules have been added to solid chitosan scaffolds, as described, for example, in US 2010/0021454. The resulting compositions are not injectable using small needles and are prone to sedimentation.

It is an objective of the present invention to provide novel thermogelling systems that solve one or more of the aforementioned draw-backs of the existing systems.

It is, in particular, a first objective to provide a thermogelling system with improved gelling characteristics at body temperature. It is a further objective to provide a thermogelling system which can be used to prepare scaffolds with improved osseointegrative properties.

Summary of the Invention The present inventors have developed new thermogelling systems by adding enzyme alkaline phosphatase solution to thermosensitive chitosan/beta- glycerophosphate solutions. This approach surprisingly was found to solve the problems associated with the existing systems such as insufficient and/or slow gelation at body temperature and the lack of a mineral phase within hydrogels with associated lack of bioactivity.

It was surprisingly found that the resulting thermogelling system has improved gel formation characteristics, especially a shorter gelation time. Gelation of different chitosan preparations can be accelerated to a speed more suitable from a clinical point of view Furthermore, it was observed that the present invention can yield satisfactory gelation properties with a wider range of chitosan preparations, for instance in terms of deacetylation degrees.

The presence of enzyme alkaline phosphatase was furthermore found to enhance formation of CaP mineral inside the hydrogels. For bone contact applications, the presence of a ceramic phase based on calcium phosphate (CaP) leads to a number of advantages, including increased bioactivity (formation of chemical bonds with surrounding bone after implantation) and affinity for biologically active proteins such as growth factors, which stimulate the natural healing processes of surrounding bone³. Since stiffer ⁴' ⁵ and rougher ⁶ surfaces are known to promote differentiation of cells towards the osteoblastic phenotype, mineralisation is expected to make chitosan more bone-friendly.

Since this invention enables the application of chitosan preparations having a wider range of degrees of deacetylation, which affects degradability, inflammatory response, and antibacterial properties, ALP addition is also of particular interest for themogelling systems intended for non-bone related applications such as for drug delivery.

The present invention thus provides new thermogelling systems, their use in methods of aesthetic and/or reconstructive intervention in a human or mammalian subject, as well as products for carrying out such methods.

Detailed description of the Invention A first aspect of the invention concerns a thermogelling system comprising a combination of chitosan or a salt thereof, an organophosphate and enzyme alkaline phosphatase, which composition is liquid or flowable at ambient temperature.

The term "thermogelling" as used herein refers to the property of a liquid or solution to turn into a gel or to set under the influence of temperature increases.

The term "chitosan" will be understood by those skilled in the art to include all derivatives of chitin, or poly-N-aceryl-D-glucosamine, including all polyglucosamine and oligomers of glucosamine materials of different molecular weights, in which the greater proportion of the N-acetyl groups have been removed through hydrolysis, and all salts thereof.

Synthetically produced beta-l,4-poly-D-glucosamines and derivatives thereof of equivalent structure to chitosan may also be used according to this invention.

Salts with various organic and inorganic acids are suitable. Such suitable salts include, but are not limited to, lactate, citrate, glutamate, nitrate, phosphate, acetate, malate, propionate, ascorbate, formate and the like. Preferred salts are chitosan chloride And chitosan lactate.

Chitosan derivatives are also suitable for use in this invention. Suitable chitosan derivatives include, without limitation, esters, ethers or other derivatives formed by bonding acyl and/or alkyl groups with the hydroxyl groups, but not the amino groups of chitosan. Examples include O-alkyl ethers of chitosan and O-acyl esters of chitosan. Modified chitosans, such as those conjugated to polyethylene glycol may be used in the present invention.

The chitosan or chitosan derivative or salt used preferably has a molecular weight of 4,000 Dalton or more, preferably in the range 25,000 to 2,000,000 Dalton, and most preferably about 50,000 to 300,000 Dalton.

As mentioned previously, the deacetylation degree and molecular weight have been shown to greatly influence the solution properties, enzymatic degradability and biological activity. Chemical modifications, for instance, have been proposed to neutralize or modify chitosan chains by incorporating carboxylic acid, acetate, glutamic acid, carboxymethyl or sulfate groups.

One particularly interesting aspect of the present invention resides in the fact that the choice of the chitosan material, in terms of degree of deacetylation is less critical than it is in conventional chitosan/organophosphate systems. The chitosan or salt thereof used in accordance with this invention may have a degree of deacetylation within broad limits. Preferably, the chitosan is deacetylated to a degree of greater than 30%, preferably greater than 35%, preferably greater than 40%, preferably greater than 50% and more preferably greater than 70%. The degree of deacetylation is preferably less than 99%, preferably less than 98 %, preferably less than 95%, preferably less than 90%.

It is preferable that the chitosan, chitosan derivative or salt used in the present invention is water soluble. By "water soluble" we mean that that the chitosan, chitosan derivative or salt dissolves in water or an aqueous acid at an amount of at least 10 mg/ml at room temperature and atmospheric pressure.

Chitosans suitable for use in the present invention may be obtained from various sources, including Primex, Haugesund, Norway; NovaMatrix, Drammen, Norway; Seigagaku America Inc., MD, USA; Meron (India) Pvt, Ltd., India; Vanson Ltd, Virginia, USA; and AMS Biotechnology Ltd., UK. Suitable derivatives include those that are disclosed in Roberts, Chitin Chemistry, MacMillan Press Ltd., London (1992).

The chitosan or derivative thereof or salt of chitosan or salt of a derivative of chitosan used in this invention is typically used in the form of a finely divided powder. Suitable powders can be prepared by any appropriate method known in the art. Preferred methods for preparing the powder include milling and/or spray drying. The preferred particle size of the chitosan or derivative thereof or salt of chitosan or salt of a derivative of chitosan used in the present invention, expressed as the volume mean diameter, is 25 to 200 μηι.

The expression "organo-phosphates (salt)" refers to mono-phosphate dibasic salts of polyols or sugars, such as polyol-phosphate dibasic salts or sugar-phosphate dibasic salts. The organo-phosphate anions contribute to the cross-linking of chitosan macromolecule chains. The gelation of chitosan/organo-phosphate solution depends on both, the final pH of chitosan/organo-phosphate solution and the temperature. This type of temperature-controlled pH-dependant gelation is specifically induced by organic mono-phosphate dibasic salt in chitosan solution. Some examples of suitable organophosphate salts include Na₂P0₄C₃H₅(OH)₂, Fe₂P0₄C₃H₅(OH)₂, K₂P0₄C₃H₅(OH)₂, MgP0₄C₃H₅(OH)₂, MnP0₄C₃H₅(OH)₂, Ca₂P0₄C₃H₅(OH)₂, Na₂P0₇C₃H₇, Na₂P0₇C₄H₇, K₂P0₇C₄H₇, NaP0₇C₄H₈, K₂P0₇C₄H₈, Na₂P0₈C₅H₉, K₂P0₈C₅H₉, NaP0₈C₅Hio, KPO₈C₅Hi₀, Na₂P0₉C₆Hn, NaP0₉C₆Hi₂, K₂P0₉C₆Hn, KPOC₆Hi2, Na₂P0₈C₆Hi3, K₂P0₈C₆Hi₃, NaP0₈C₆Hi₄, KP0₈C₆Hi₄, Na₂P0₉C₆Hi₂, K₂P0₉C₆Hi₂, NaP0₉C₆Hi₃, KP0₉C₆Hi₃, Na₂PO₈Ci₀Hn, K₂PO₈Ci₀OHn, NaPO₈Ci₀Hi₂, and KP0₈CioHi₂ and the like.

The preferred organo-phosphate salts are selected from mono-phosphate dibasic salts of glycerol, including glycerol-2-phosphate, sn-glycerol 3 -phosphate and 1- glycerol-3 -phosphate salts (alpha-glycerophosphate or beta-glycerophosphate), monophosphate dibasic salts of histidinol, acetol, diethylstilbestrol, indoleglycerol, sorbitol, ribitol, xylitol, arabinitol, erythritol, inositol, mannitol, glucitol, palmitoyl-glycerol, linoleoyl-glycerol, oleoyl-glycerol or arachidonoyl-glycerol, and mono-phosphate dibasic salts of fructose, galactose, ribose, glucose, xylose, rhamnulose, sorbose, erythrulose, deoxy-ribose, ketose, mannose, arabinose, fuculose, fructopyranose, ketoglucose, sedoheptulose, trehalose, tagatose, sucrose, allose, threose, xylulose, hexose, methylthio-ribose or methylthio-deoxy-ribulose. Other mono-salts of interest (sulfate, carboxylate) may be derived from the same polyols or sugars.

In one preferred embodiment of the invention, the organophosphate is glycerophosphate. Glycerophosphoric acids are present under two isomeric structures, alpha-glycerophosphate or beta-glycerophosphate, wherein the beta-glycerophosphoric acid is optically inactive and the alpha-glycerophosphoric acid is optically active. The expression "glycerophosphate" refers herein to both the alpha and beta isomer. Alpha- glycerophosphate is undistinctively referred to as glycerol-3 -phosphate (all optical eniantomers) while beta-glycerophosphate is similarly referred to as glycerol-2- phosphate. A particularly preferred embodiment concerns a thermogelling system as defined herein before, wherein the organophosphate is a glycerophosphate, preferably beta-glycerophosphate.

Gelation of chitosan will occur with any grade or purity of glycerophosphate.

Glycerophosphate salts are precipitated from glycerophosphoric acids which are obtained through the hydrolysis of lecithin, a well-know biological molecule and phosphatides of eggs, soybean and fishes. Glycerophosphoric acid is physiologically active compound, being involved in the catabolism of carbohydrates. Glycerophosphate dehydrogenase was also found active in nerve tissues while glycerophosphate was reported to accelerate the rate of decolonization of methylene blue by guinea pig nerves. Alpha-glycerophosphate interacts with pyruvic acid through oxidation- reduction reactions for producing lactic acid in fresh muscle extracts. Glycerophosphoric acid is available as disodium, calcium, magnesium, dipotassium, strontium and barium salts, having a relatively strong basic character. Both alpha- and beta-glycerophosphate salts are inexpensive readily available sources of organic monophosphate dibasic salts among the polyol or sugar phosphate salts.

Alkaline phosphatases (ALP) are dimeric, zinc-containing, non-specific phosphomono-esterases with enzyme activity optima at alkaline conditions. ALP's occur in prokaryotic and eukaryotic organisms e.g. in E. coli and mammals (McComb et al., 1979 Alkaline Phosphatases Plenum Press, New York). A comparison of the primary structure of various alkaline phosphatases showed that there is a high degree of homology (25-30% homology between E. coli and mammalian AP; Millan, 1988 Anticancer Res. 8, 995-1004; Harris, 1989 Clin. Chim. Acta 186, 133-150). In humans and higher animals the AP family consists of four members which are coded in different gene loci (Millan, 1988 Anticancer Res. 8, 995-1004; Harris 1989 Clin. Chim. Acta 186, 133-150). The family of alkaline phosphatases includes the tissue-specific APs (placental AP (PALP), germ cell ALP (GCALP) and intestinal ALP (IALP)) and the non-tissue-specific ALPs (TnAP) which are mainly located in the liver, kidney and bones. All forms are useful in this invention. The use of the non-tissue specific ALP is preferred. Several types of ALP's are commercially available, such as ALP's originating from animals, e.g. bovine ALP's. The invention is not particularly limited in this respect. In a preferred embodiment recombinant human ALP is used.

The thermogelling system according to the invention preferably is a water based system, preferably it takes the form of an aqueous liquid, preferably an aqueous solution. In a preferred embodiment of the invention an composition is provided comprising chitosan, organophosphate and enzyme alkaline phosphatase dissolved in an aqueous phase or aqueous liquid.

In one preferred embodiment, the thermogelling system is a water based composition, preferably an aqueous solution, comprising chitosan or a salt thereof in an amount of more than 0.1 % w/v, based on the total weight of the thermogelling system, preferably more than 0.25 % w/v, preferably more than 0.5 % w/v, preferably more than 0.75 % w/v, preferably more than 1 wt.%, preferably more than 1.25 % w/v, preferably more than 1.5 % w/v, preferably more than 1.75 % w/v, based on the total weight of the thermogelling system. In one preferred embodiment, the thermogelling system is a water based composition, preferably an aqueous solution, comprising chitosan or a salt thereof in an amount of less than 7.5 % w/v based on the total weight of the thermogelling system, preferably less than 6.5 % w/v, preferably less than 6 % w/v, preferably less than 5.5 % w/v, preferably less than 5 % w/v, preferably less than 4.5 % w/v, preferably less than 4 % w/v, preferably less than 3 % w/v, preferably less than 2.75 % w/v, preferably less than 2.5 % w/v, preferably less than 2.25 % w/v, based on the total weight of the thermogelling system.

In one preferred embodiment, the thermogelling system is a water based composition, preferably an aqueous solution, comprising the organophosphate or glycerophosphate, in an amount of more than 0.5 % w/v, based on the total weight of the thermogelling system, preferably more than 1 % w/v, preferably more than 2 % w/v, preferably more than 3 % w/v, preferably more than 4 % w/v, preferably more than 5 % w/v, preferably more than 5.5 % w/v, preferably more than 5.75 % w/v, preferably more than 6 % w/v, preferably more than 6.25 % w/v, based on the total weight of the preservative system, based on the total weight of the thermogelling system.

In one preferred embodiment, the thermogelling system is a water based composition, preferably an aqueous solution, comprising the organophosphate or glycerophosphate, in an amount of less than 15 % w/v based on the total weight of the thermogelling system, preferably less than 12 % w/v, preferably less than 10 % w/v, preferably less than 9 % w/v, preferably less than 8.5 % w/v, preferably less than 8 % w/v, preferably less than 7.75 % w/v, preferably less than 7.5 % w/v, preferably less than 7.25 % w/v, preferably less than 7 % w/v, preferably less than 6.75 % w/v, based on the total weight of the thermogelling system.

In one preferred embodiment, the thermogelling system is characterized by a ratio of chitosan : organophosphate of less than 5/1, preferably less than 3/1, preferably less than 2/1, preferably less than 1/1, preferably less than 0.5/1. In one preferred embodiment, the thermogelling system is characterized by a ratio of chitosan : organophosphate of more than 0.05/1, preferably more than 0.1/1, preferably more than 0.2/1, preferably more than 0.25/1, preferably more than 0.3/1.

In one preferred embodiment, the thermogelling system is a water based composition, preferably an aqueous solution, comprising the enzyme alkaline phosphatase in an amount of more than 0.00 lmg per gram of chitosan or salt thereof, preferably more than 0.0025 mg/ml, preferably more than 0.005 mg/ml, preferably more than 0.01 mg/ml, preferably more than 0.025 mg/ml, preferably more than 0.05 mg/ml, preferably more than 0.1 mg/ml, preferably more than 0.15 mg/ml, preferably more than 0.175 mg/ml, preferably more than 0.2 mg/ml, preferably more than 0.21 mg/ml, preferably more than 0.22 mg/ml, based on the total weight of the thermogelling system.

In one preferred embodiment, the thermogelling system system is a water based composition, preferably an aqueous solution, comprising the enzyme alkaline phosphatase in an amount of less than 5 mg/ml based on the total weight of the thermogelling system, preferably less than 2.5 mg/ml, preferably less than 1.5 mg/ml, preferably less than 1.25 mg/ml, preferably less than 1 mg/ml, preferably less than 0.75 mg/ml, preferably less than 0.5 mg/ml, preferably less than 0.4 mg/ml, preferably less than 0.3 mg/ml, preferably less than 0.275 mg/ml, preferably less than 0.26 mg/ml, preferably less than 0.25 mg/ml, preferably less than 0.24 mg/ml, based on the total weight of the thermogelling system.

In one preferred embodiment, the thermogelling system comprises enzyme alkaline phosphatase in an amount of more than 12.5 mg / g of chitosan, preferably more than 25 mg / g of chitosan, preferably more than 50 mg / g of chitosan, preferably more than 75 mg / g of chitosan, preferably more than 100 mg / g of chitosan. In one preferred embodiment, the thermogelling system comprises enzyme alkaline phosphatase in an amount of less than 500 mg / g of chitosan, preferably less than 400 mg / g of chitosan, preferably less than 300 mg / g of chitosan, preferably less than 200 mg / g of chitosan, preferably less than 150 mg / g of chitosan.

Solubilization of chitosan in aqueous solutions requires the protonation of the amine groups of the chitosan chains which is reached within acidic aqueous solutions having a pH ranging from 3.0 to 5.0. When solubilized, chitosan remains soluble until a pH of about 6.2. Neutralization of acidic chitosan solutions by alkali results in a pH increase as well as a de-protonation of the amine groups. Neutralization of acidic chitosan solutions to a pH above the pKa of chitosan at about 6.3-6.4 results in OH-HN and O-HN interchains and water-chitosan hydrogen bonds which induce a hydrated three-dimensional network, a chitosan gel. At pH above 6.3-6.4, chitosan solutions result systematically into chitosan gels at a normal temperature range (0-60 °C). However, admixing of an organo-phosphate to a chitosan aqueous solutions increases the pH of the chitosan/organo-phosphate solutions which remain ungelled and liquid for long periods of time even at pH above 6.5, and up to 7.2.

Depending on their final pH, the chitosan/glycerophosphate/ALP solutions are expected to lead either to thermally reversible or irreversible gel. Reversible gels arise from solutions having a pH comprising between 6.5 and 6.9, while the irreversible gels originate from solutions having a pH above 6.9. The nature of the acids that are used for the acidic chitosan solutions does not influence fundamentally the sol to gel transition of the system. The final pH is dependent upon the pH of the water/acid solution as well as the chitosan and glycerophosphate concentrations. As chitosan and glycerophosphate are two alkaline components, they tend to increase the pH of the acidic solution wherein they are dissolved. Concentrations in chitosan and glycerophosphate can be balanced to reach the appropriate pH of the chitosan/glycerophosphate solution, while taking into consideration the solubility limit of both components, and particularly the one of chitosan. The pH of the aqeous solution can be adjusted with conventional agents, especially acids such as hydrochloric acid.

Since the gels are intended for use in the physiological environment it is preferred that the thermogelling system has a pH of 6.5-8. As mentioned before, the present thermogelling systems which also contains enzyme alkaline phosphatase can be gelled more rapidly under the same pH and temperature conditions as compared to the same system without enzyme alkaline phosphatase. In one embodiment a thermogelling system as previously described is provided, wherein the system forms a gel when exposed to the physiological environment, especially when exposed to a temperature of about 37 °C and at a pH of 7.4. Typically under such conditions gelation is observed within 10 minutes, e.g. within 6 minutes.

Another important characteristic is related to the injectability and in vivo gelation of the thermogelling systems. Hence in a preferred embodiment of the invention the thermogelling system is liquid or flowable or syringable at ambient temperature and at a pH within the range of 6.5-7.5, meaning that it is a liquid possessing a viscosity of from 25 to 1000 mPas and in some embodiments, preferably 50 to 1000 mPas, measured at 20 oC.

The thermogelling system as previously described is an ideal material for drug delivery purposes, as will be recognized by those skilled in the art. Such a in situ gel forming vehicle, wherein a solid particulate or water-soluble drug or biologically active compound is incorporated prior to the gelation, can be administrated where upon delayed controlled and/or slow release of the drug or biologically active compound, directly to the body site to be treated. Hence, one preferred embodiment of the invention entails a thermogelling system as previously defined, further comprising at least one biologically active component other than organophosphate and enzyme alkaline phosphatase.

For the purposes of the present invention the term, "biologically active substance" generally refers to a molecule that cause(s) a biological effect when administered in vivo to mammals, especially humans and which can typically be of any nature. For instance synthetic organic compounds, peptides, proteins, carbohydrates (including monosaccharides, oligosaccharides, and polysaccharides), steroids, nucleic acids, nucleotides, nucleosides, oligonucleotides, genes, lipids and hormones may be incorporated in the present thermogelling systems.

The biologically active substance, broadly speaking, can be any substance which can exert a desired therapeutic effect at the implant site. Examples include thrombin inhibitors, antithrombogenic agents, thrombolytic agents, cell growth promoting factors, osteogenic agents, angiogenesic factors, lipogenic factors, fibrinolytic agents, antimicrobial agents, antibiotics, antiplatelet agents, anti secretory agents, actin inhibitors, remodeling inhibitors, anti-inflammatory steroids or non-steroidal antiinflammatory agents, immunosuppressive agents, radiotherapeutic agents, extracellular matrix components, ACE inhibitors, growth factors, enzymes, cytokines, chemokines, cell adhesive sequences, anti-bacterial agents, anti-fungal agents, anti-cancer agents, anti-fibrosis agents, anti-viral agents, anti-glucoma agents, miotic and anti-cholinergic agents, anti-histaminic and decongestant agents, anesthetic and anti-parasitic agents.

Since, a particularly preferred embodiment of the invention concerns thermogelling agents for repair or reconstruction of bone and cartilage tissue, especially bone, one particularly preferred embodiment provides a thermogelling system comprising a biologically active agent other than organophosphate and enzyme alkaline phosphatase, which biologically active agents is an osteogenic agent. An "osteogenic agent," as used herein, generally refers to an agent capable of inducing and/or supporting the formation, development and growth of new bone, and/or the remodeling of existing bone. The osteogenesis process typically involves the deposition of new bone by cells called osteoblasts. Many osteogenic agents function, at least in part, by stimulating or otherwise regulating the activity of osteoblast and/or osteoclasts.

Agents that can be employed for supporting the formation, development and growth of new bone, and/or the remodeling thereof, include extracellular matrix- associated bone proteins such as, osteocalcin, bone sialoprotein (BSP) and osteocalcin in secreted phosphoprotein (SPP)-l . Other suitable examples of osteogenic agents include type I collagen, fibronectin, osteonectin, thrombospondin, matrix-gla-protein, SPARC, osteopontin, osteogenin, Insulin-like Growth Factor (IGF)-l, TGFpi, TGFP2, TGF.beta.3, TGFP4, TGFP5, osteoinductive factor (OIF), basic Fibroblast Growth Factor (bFGF), acidic Fibroblast Growth Factor (aFGF), Platelet-Derived Growth Factor (PDGF), vascular endothelial growth factor (VEGF), Growth Hormone (GH), osteogenic protein-1 (OP-1) and any one of the many known bone morphogenic proteins (BMPs), including but not limited to BMP-1, BMP-2, BMP-2A, BMP-2B, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-8b, BMP-9, BMP- 10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15.

The thermogelling systems of the present invention are also suited for producing cell-loaded artificial matrices that are applied to the engineering and culture of bioengineered hybrid materials and tissue equivalents. Living microorganisms, plant cells, animal cells or human cells may be entrapped identically within the polysaccharide gel by introduction prior to the gelation. The loaded cells may be selected from the group consisting of osteoblasts, osteocytes, osteoclasts, osteoprogenitor cells, (bone tissue), chondrocytes (articular cartilage), fibrochondrocytes (meniscus), ligament fibroblasts (ligament), skin fibroblasts (skin), tenocytes (tendons), myofibroblasts (muscle), mesenchymal stem cells and keratinocytes (skin).

In another embodiment, the thermogelling system of the invention may comprise a particulate material, which particulate material may become entrapped within the scaffold upon gellification so as to obtain a composite material, which upon gel formation may have enhanced stiffness and rigidity. Such materials may be inorganic, e.g. calcium phosphate and calcium carbonate, as well as organic, e.g. gelatin.

As will be understood by those skilled in the art, it is also an important characteristic that the thermogelling system is physiologically acceptable, meaning that a small volume of it can be injected directly into a mammal without inducing undesired pathological changes, such as an undesired immune response or undesired metabolic alterations due to toxicity. Furthermore, in some embodiments, the thermogelling system is sterile. Sterilization of the chitosan/organophosphate gels can be accomplished using conventional techniques such as gamma-irradiation, sterility filters or by using strictly sterile procedures

Another aspect of the invention provides the use of the polysaccharide gel for producing biocompatible degradable materials used in cosmetics, pharmacology, medicine and/or surgery. The present invention includes methods of forming different gelated materials, those materials being either molded (customized shapes, tubes, membranes, films, etc.) or formed in situ within biological environments (filling of tissue defects). This polysaccharide gel can be used as substituting materials for tissues and organs, as a carrier for drugs or as a therapeutics delivery system.

As explained in the foregoing, the thermogelling composition of this invention will gelate when exposed to an adequate temperature. The time of gelation is also controlled by the temperature. For example, a chitosan/organophosphate/ ALP solution will gelate in about 5-10 minutes at 37 °C. This property makes the present thermogelling system particularly attractive for applications involving in situ gelation in human or mammalian tissue, where the gelation is induced by the physiological temperature.

The gels can be formed in situ within defects or cavities in biological tissues, , typically by injecting the system within the specific sites. Gelation in situ allows for complete and precise filling of such tissue defects or body cavities. The liquid or flowable thermogelling system fills the cavity or defect and sets to form a solid within the cavity or defect.

Hence another aspect of the present invention concerns a method of aesthetic or reconstructive intervention, typically aesthetic or reconstructive surgery, in a human or mammalian subject employing the present thermogelling system. In a particularly preferred embodiment of the invention, a method is provided for reconstructing or repairing a defect or cavity in a human or mammalian tissue comprising filling the defect or cavity with a thermogelling composition as previously described. In one preferred embodiment a method of aesthetic or reconstructive intervention in a subject is provided, said method comprising i) providing a thermogelling system as defined herein previously; ii) applying the thermogelling composition to a defect or cavity in a tissue of said subject so as to fill said defect or cavity; and iii) allowing the composition to form a gel under the influence of the physiological environment.

As explained previously, one particular advantage of the present invention resides in the fact that mineralization, especially the formation of calcium phosphate, within the gelled or set compositions (scaffolds) is significantly enhanced as compared to existing chitosan/organophosohate systems without enzyme alkaline phosphatase. As is generally understood by those skilled in the art, calcium phosphate is a promotor of bone growth. Hence the gels formed typically favor the ingrowth of hard-tissue, especially bone.

For this reason, the thermogelling systems of the invention are particularly suitable for repairing or reconstruction of defects or cavities in hard tissues, in particular bone tissue and cartilage, preferably bone. The hydrogels described herein are useful in the treatment of what may be termed "orthopedic tissues". The medical specialty of orthopedics is concerned with the preservation, restoration and development of the form and function of the musculoskeletal system, extremities, spine and associated structures by medical, surgical and physical methods. Orthopedic tissues include bone, cartilage, and related structures, for example, meniscus, bursa, synovial membranes, and other structures of the joints. Tissues with similar mechanical properties such as teeth can also be treated effectively with the hydrogel materials.

Other Embodiments are envisaged wherein the present thermogelling composition is used for repairing and/or reconstructing defects and/or cavities in soft tissues, such as fat, skin and muscle. In one embodiment, the present thermogelling composition may be used to fill wrinkles for purely aesthetic purposes.

The present thermogelling composition can suitably include any type of pharmaceutical or biological agent, which will be released gradually after application. Hence, further embodiments are envisaged wherein the thermogelling composition is used for the pro-longed and/or controlled and/or sustained release of an active agent at the site of administration, within soft or hard tissues.

The polysaccharide gel solution may be introduced within an animal or human body by injection or endoscopic administration using techniques and equipment generally known by those skilled in the art. Injection of the system may be limited by the viscosity thereof which controls the injectability or syringeability of the solutions. A needle having a gauge of 20 and below are ideal materials for injection of such gel solution.

In the methods of the invention described herein previously a thermogelling composition may be prepared in any suitable way.

In one exemplary embodiment Chitosan in powder form is dissolved in an aqueous acidic solution having a pH ranging from 4.5 to 5.5. Aqueous chitosan solutions can be sterilized either by filtration with in-line sterile filters or by steam- autoclaving. Glycerophosphate felt in fine powder form is added to, and dissolved within, the aqueous chitosan solution at a temperatures ranging from 4°C to 22°C. When a clear homogeneous aqueous solution with a pH ranging from 6.5 to 7.2 is attained, enzyme alkaline phosphatase may be added. Typically, the enzyme alkaline phosphatase will be added in the form of a solution or dispersion, but the invention is not particularly limited in this respect. The liquid or flowable thermogelling system thus obtained may be stored, typically at temperature below 10 °C, e.g. below 5 °C, or they may be used instantaneously.

In situ gelation of the thermogelling system can be conducted by dispensing the solution from a hypodermic syringe or the like. If needed, the solution may be pre- gelated (initiate the thermal gelation) by keeping the syringe and chitosan/glycerophosphate solution at desired temperature, ideally 37 °C, until the first signs of gelation appear. The ready-to-gel mixture is then administrated so as to fill tissue defects or cavities and complete in situ the gelation process.

According to one embodiment of the invention the thermogelling system is prepared only shortly before using it in a method of the invention. Typically, this will be accomplished by combining a first composition comprising the chitosan or salt thereof and organophosphate and a second composition comprising enzyme alkaline phosphatase. Precursor solutions, kits and dispensing devices that are particularly suitable in the such methods are described herein below.

Another aspect of the invention provides, kits and dispensing devices for producing a thermogelling system of the invention. One embodiment provides a kit of parts or dispensing device comprising a first and a second reservoir, each holding distinct compositions containing one or more substances selected from chitosan and salts thereof, organophosphates and enzyme alkaline phosphatase. Preferably, said first reservoir holds a composition comprising chitosan or a salt thereof, preferably a combination of chitosan or a salt thereof and organophosphate, and said second reservoir holds a composition comprising enzyme alkaline phosphatase.

As will be understood on the basis of the foregoing products for applying the present invention preferably comprise ready to use liquids, while it is also envisaged that dry solid forms of one or more of the components are provided, which are to be dissolved or resuspended before use.

In case the kit or dispensing device comprises reservoirs holding liquid solutions or dispersion of the components making up the thermogelling system, an embodiment is particularly preferred wherein the kit contains a first and second reservoir each holding distinct liquids containing one or more substances selected from chitosan and salts thereof, organophosphates and enzyme alkaline phosphatase. In one preferred embodiment such a kit comprises a first reservoir holding a liquid containing the chitosan or salt thereof as well as the organophosphate and another reservoir holding the enzyme alkaline phosphatase.

In case the kit comprises a reservoir holding one or more of the components selected from chitosan or a salt thereof, organophosphate and enzyme alkaline phosphatase in solid/dry form, it is preferred that the kit also comprises a reservoir holding an aqueous liquid for dissolving or resuspending said components in solid/dry form. Said aqueous liquid may contain one or more of the components of the present thermogelling system, as will be understood by those skilled in the art.

The compositions in the first and second reservoir, upon admixing, should yield the thermogelling compositions described above. This means, as will be understood by those skilled in the art, that the amounts, concentrations and relative ratios of the components in each composition and in each reservoir should be chosen so as to obtain the values defined above in the final thermogelling system, i.e. after admixing the individual components and/or compositions provided in the kit.

In some embodiments, the kits comprise an additional reservoir holding a further biologically active substance, tissue cells, particulate materials, a detectable label, a contrast agent, etc. as defined herein previously. In some embodiments, the kits comprise a further biologically active substance, tissue cells, particulate materials, etc. included in the reservoir comprising the chitosan and organophosphate and/or in the reservoir comprising the enzyme alkaline phosphatase.

Kits preferably contains instructions for use. In some embodiments, the kits comprise a mixing device. Mixing devices may be integrated as part of a reservoir or reservoir system. In some embodiments, the mixing device comprises a valve system which allows for passage of the dispersion from one reservoir to a different reservoir to facilitate mixing.

In some embodiments, the kits comprise a dispensing device. The dispensing device may be an applicator in communication with a mixing device and/or a reservoir adapted for containing the dispersion. In some embodiments, the dispensing device comprises a catheter. In some embodiments, the dispensing device comprises a syringe.

In accordance with the invention the afore described reservoirs may be separate reservoirs or they may be compartments of an integrated reservoir system or dispensing device. In some embodiments a single device is provided a first and second reservoir each holding distinct liquids containing one or more substances selected from chitosan and salts thereof, organophosphates and enzyme alkaline phosphatase. In one preferred embodiment such a device comprises a first reservoir holding a liquid containing the chitosan or salt thereof as well as the organophosphate and another reservoir holding the enzyme alkaline phosphatase. Preferably, the dispensing device further comprises a means for simultaneously expelling the compositions from the reservoirs, as well as an interconnecting part interconnecting an outlet from the first reservoir and an outlet from the second reservoir, said interconnecting part being arranged in such a way that the compositions from the first and second reservoir are mixed when they are expelled from the respective reservoirs.

A further aspect of the invention concerns the use of enzyme alkaline phosphatase for accelerating thermal gelation of a chitosan organophosphate thermogelling composition and/or for enhancing the osseointegrative properties of a chitosan organophoshate gel. The details and preferred embodiments of this aspect of the invention will be readily understood by those skilled in the art based on the foregoing.

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any of the methods, objects, compositions and uses of the invention, and vice versa. It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

Furthermore, for a proper understanding of this invention and its various embodiments it should be understood in this document and the appending claims, the verb "to comprise" and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The following examples describe various new and useful embodiments of the present invention. These examples are for illustrative purposes and do not limit the scope of the invention.

Example

The study aimed to investigate the effect of ALP incorporation on gelation properties, such as gelation speed and temperature of induction of gelation. The amount and nature of mineral formed due to ALP incorporation was also investigated.

Materials and Methods

Production of hydrogels and ALP incorporation

Chitosan hydrogels were produced according to a protocol based on that of

Chenite et al.¹. Briefly, 0.4 g chitosan was dissolved in 16 ml 0.1 HC1. 10 g Sodium glycerophosphate (Na-GP) were dissolved in 14 ml water. ALP was dissolved in water at concentrations of 0, 1.25 and 2.5 mg/ml. 3.6 ml chitosan solution was mixed with 0.4 ml Na-GP solution and 0.4 ml ALP solution. This yielded gels with the following final concentrations:

Chitosan: 20.5 mg/ml

Na-GP: 64.9 mg/ml

ALP: 0.227, 0.114 or 0 mg/ml

Investigation of gelation speed and rheological properties by Rheometry Rheological meaturements were carried out on a AR2000 rheometer, TA instruments, using a 20 mm flat plate geometry. Temperature sweeps, time sweeps and stress sweeps were performed.

Time sweeps

Time sweeps were performed at 37 °C at an oscillatory stress of 5 Pa and frequency of 1 Hz for 45 minutes.

Temperature sweeps

Temperature sweeps were performed at an oscillatory stress of 5 Pa and a frequency of 1 Hz. Temperature was raised from 15 °C to 45 °C at a heating rate of 1 °C/min.

Mineralization of gels

Gel mineralization was induced by incubation in a mineralization medium consisting of 0.1 M calcium glycerophosphate (Ca-GP) (aq). Mineralization medium was changed every day. After conclusion of mineralization, gels were rinsed three times in Milli-Q water and subsequently incubated in Milli-Q for 1 day to remove residual Ca-GP.

Calculation of mass change due to mineralization

The dry mass percentage, i.e. the gel weight percentage not consisting of water, was calculated as: (weight after mineraization and subsequent freeze-drying/weight after mineralization)* 100. This served as a measure of mineral formed. Freeze-drying was performed for 24 hours.

Physicochemical characterization: FTIR

The molecular structure of gels was examined after mineralization experiments using Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy (ATR- FTIR, Spectrum One). Samples were freeze-dried for 24 hours prior to measurements.

Results

Influence of ALP incorporation on gelation speed

ALP acelerated gelation, as demonstrated by temperature and time sweeps. Temperature sweeps (Figure 1) showed that gelation took place in the presence of ALP, was already initiated at 15 °C was accelerated from 30 °C and complete by 45 °C. In the absence of ALP gelation appeared to be initiated at 30 °C but was not complete by 45 °C. Since temperature was raised by 1 °C/minute, this indicates that gelation in the presence of ALP was quicker.

Time sweeps at 37 °C demonstrated that gelation was induced after approx. 25 minutes in the absence of ALP, but after only approx. 6 minutes in the presence of 0.23 mg/ml ALP. Experiments were repeated 3 times and average gelation times ± standard deviation are presented. Differences were significant according to t-test (p < 0.01).

Influence of ALP on dry mass ofhydrogel

The dry mass percentage, i.e. the gel weight percentage not consisting of water, serves as a measure of amount of mineral formed. It increased with increasing ALP concentration and duration of incubation in mineralization medium (Figure 3).

Experiments were repeated 3 times and average dry mass percentages ± standard deviation are presented. Differences between ALP concentrations of 0 and 0.23 mg/ml were significant. Differences were significant according to t-test (p < 0.01).

Physicochemical and morphological characterization: FTIR, SEM

FTIR revealed characteristic peaks for CaP at 1030 cm^"1 and between 600 and

520 cm^"1 in samples containing ALP, especially at an ALP concentration of 0.23 mg/ml, which were absent in samples without ALP. (Figure 4)

Conclusions

It can be concluded that incorporation of ALP into thermosensitive chitosan/Na-

GP gels accelerated gelation and stimulates formation of CaP in gels after incubation in Ca-GP solution. Amount of CaP formed increases with ALP concentration and incubation time in Ca-GP solution. Description of the Figures

Figure 1: gelation properties of chitosan gels as a function of temperature increase without ALP (A) and with ALP (B). When the line representing storage modulus is above the line representing the loss modulus the system is a gel.

Figure 2: gelation properties of chitosan gels at 37 °C as a function of time without ALP (A) and with ALP (B). When the line representing storage modulus is above the line representing the loss modulus the system is a gel. Graph (C) shows the gelation time of gels with and without ALP. Figure 3: dry mass percentage of gels with different amounts of ALP after incubation in mineralization medium for 6 or 10 days.

Figure 4: Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy of gels containing different amounts of ALP after incubation in mineralization medium for 6 days. Peaks characteristic for calcium phosphate are marked.

References

1. Chenite, A.; Chaput, C; Wang, D.; Combes, C; Buschmann, M. D.; Hoemann, C. D.; Leroux, J. C; Atkinson, B. L.; Binette, F.; Selmani, A., Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials 2000, 21, (21), 2155-61.

2. Patois, E.; Osorio-da Cruz, S.; Tille, J. C; Walpoth, B.; Gurny, R.; Jordan, O., Novel thermosensitive chitosan hydrogels: in vivo evaluation. J Biomed Mater Res A 2009, 91, (2), 324-30.

3. Ruhe, P. Q. ; Boerman, O. C; Russel, F. G.; Spauwen, P. H.; Mikos, A. G;

Jansen, J. A., Controlled release of rhBMP-2 loaded poly(dl-lactic-co-glycolic acid)/calcium phosphate cement composites in vivo. J Control Release 2005, 106, (1- 2), 162-71.

4. Rowlands, A. S.; George, P. A.; Cooper-White, J. J., Directing osteogenic and myogenic differentiation of MSCs: interplay of stiffness and adhesive ligand presentation. Am J Physiol Cell Physiol 2008, 295, (4), C1037-44.

5. Evans, N. D.; Minelli, C; Gentleman, E.; LaPointe, V.; Patankar, S. N.;

Kallivretaki, M.; Chen, X.; Roberts, C. J.; Stevens, M. M., Substrate stiffness affects early differentiation events in embryonic stem cells. Eur CellMater 2009, 18, 1-13; discussion 13-4.

6. Boyan, B. D.; Lossdorfer, S.; Wang, L.; Zhao, G.; Lohmann, C. H.; Cochran, D. L.; Schwartz, Z., Osteoblasts generate an osteogenic microenvironment when grown on surfaces with rough microtopographies. Eur CellMater 2003, 6, 22-7.

7. Filmon, R.; Basle, M. F.; Barbier, A.; Chappard, D., Poly(2-hydroxy ethyl methacrylate)-alkaline phosphatase: a composite biomaterial allowing in vitro studies of bisphosphonates on the mineralization process. J Biomater Sci Polym Ed 2000, 11, (8), 849-68. 8. Spoerke, E. D.; Anthony, S. G.; Stupp, S. I, Enzyme Directed Templating of Artificial Bone Mineral. Adv. Mater. 2009, 21, 425-430.

Claims

1. Thermogelling system comprising a combination of chitosan or a salt thereof, an organophosphate and enzyme alkaline phosphatase, which composition is liquid or flowable at ambient temperature.

2. Thermogelling system according to claim 1, wherein the organophosphate is a glycerophosphate, preferably beta-glycerophosphate.

3. Thermogelling system according to claim 1 or 2, wherein chitosan, organophosphate and alkaline phosphatase are dissolved in an aqueous phase.

4. Thermogelling system according to claim 3, wherein the system forms a gel when exposed to the physiological environment.

5. Thermogelling system according to any one of the preceding claims, comprising an aqueous phase comprising at least 0.5 % w/v of chitosan, preferably 0.5-5 % w/v; and/or at least 1 % w/v of the glycerophosphate, preferably 1-10 % w/v and/or at least 0.02 mg/ml of the enzyme alkaline phosphatase, preferably 0.02-0.5 mg/ml.

6. Thermogelling system according to any one of the preceding claims, comprising at least one biologically active component other than organophosphate and enzyme alkaline phosphatase.

7. Thermogelling system according to any one of the preceding claims, for use in a method of aesthetic and/or reconstructive intervention in a subject in need thereof.

8. Kit of parts or dispensing device comprising a first and a second reservoir, said first reservoir holding a composition comprising chitosan, preferably a combination of chitosan and organophosphate, and said second reservoir holding a composition comprising enzyme alkaline phosphatase.

9. Kit of parts or dispensing device according to claim 8, wherein said compositions in said first and said second reservoir comprise aqueous compositions.

10. Dispensing device according to claim 8 or 9, further comprises a means for simultaneously expelling the compositions from the reservoirs, as well as an interconnecting part interconnecting an outlet from the first reservoir and an outlet from the second reservoir, said interconnecting part being arranged in such a way that the compositions from the first and second reservoir are mixed when they are expelled from the respective reservoirs

11. Method of aesthetic or reconstructive intervention in a subject, said method comprising:

i) providing a thermogelling system as defined in any one of claims 1-7, which composition is liquid or flowable at ambient temperature;

ii) applying the thermogelling composition to a defect or cavity in a tissue of said subject so as to fill said defect or cavity; and

iii) allowing the composition to form a gel under the influence of the

physiological environment.

12. Method according to claim 11, wherein i) comprises combining a first composition comprising chitosan and organophosphate and a second composition comprising enzyme alkaline phosphatase.

13. Method according to claim 11 or 12, wherein i) and ii) are performed in a single step using a dispensing device as defined in claim 10.

14. Use of enzyme alkaline phosphatase for accelerating thermal gelation of a chitosan organophosphate thermogelling composition and/or for enhancing the osseointegrative properties of a chitosan organophoshate gel.