WO2012059201A1

WO2012059201A1 - Biocompatible and bioactive bone substitute material

Info

Publication number: WO2012059201A1
Application number: PCT/EP2011/005456
Authority: WO
Inventors: Rolf-Dieter Zehbe
Original assignee: Technische Universität Berlin
Priority date: 2010-11-02
Filing date: 2011-10-28
Publication date: 2012-05-10

Abstract

The present invention relates to a method for producing a biocompatible and/or bioactive material, to an apparatus for producing said material, to said material itself and to its uses in medical applications.

Description

BIOCOMPATIBLE AND BIOACTIVE BONE SUBSTITUTE MATERIAL

The present invention relates to a method for producing a biocompatible and/or bioactive material, to an apparatus for producing said material, to said material itself and to its uses in medical applications. BACKGROUND OF THE INVENTION

Bone medicine is becoming increasingly important in the ageing modern society. Up to 55% of over fifty year olds develop osteoporosis or the pre-osteoporosis syndrome osteopenia in the industrialised countries. Osteoporosis is a condition characterised by low bone mass and structural deterioration of bone tissue, leading to bone fragility and an increased susceptibility to fractures, especially of the hip, spine and wrist, although any bone can be affected. In some cases the effect is so serious that bone substitution is required (for a review, see Raisz, J Clin Invest 1 15 (12): 3318-25, 2005). Another field of bone medicine is acute and chronic osteomyelitis, both of which remain difficult to treat (for a review, see Lew and Waldvogel, N Engl J Med, 336:999-1007, 1997). In acute osteomyelitis, rapid bone infection occurs, while the chronic form is characterised by a longer lasting type of bone infection involving low- grade inflammation, sequestra (areas of dead bone), involucra (shells of cortical bone resulting from periosteal elevation due to an inflammatory focus), fistula and bone sclerosis. Infections, often with Staphylococcus aureus, can be difficult to eradicate, and in approximately 15% to 30% acute osteomyelitis cases infection persists. Moreover, chronic osteomyelitis can often only be treated using surgical debridement in combination with antibiotic therapy. Even with surgery, eradication of the disease is not assured. Also, in a considerable amount of cases, affected bone does not recover and requires replacement.

Major difficulties in bone medicine are the limited availability of autologous bone tissue for transplantation and that the risks of rejection and disease transmission in the use of allografts. Therefore, synthetic substitute materials and specific therapeutics such as bisphosphonates or cationic strontium are developed for application. Promising synthetic substitute materials include biocompatible polymeric or polymer-ceramic materials which are formed as a porous matrix. These substitute materials are often designed to be biodegradable, allowing for continuous replacement of the substitute material with regenerated bone tissue. One emerging technology for the production of such materials is taking advantage of the foaming behaviour of polymers contacted with a supercritical gas, e.g. C0₂ (Quirk et al., Current opinion in solid state & materials science, vol. 8(3-4), p. 313-312, 2004).

Due to its chemical similarity to calcium, strontium is readily incorporated into bone tissue (Terra et al., Phys Chem Chem Phys, vol. 1 1(3), p. 568-577, 2009; Wassermann et al., Clin Chem, vol. 44(3), p. 437-439, 1998) and influences bone metabolism. For example, it was shown that the administration of small amounts of strontium to rats increases bone volume (Grynpass et al., Bone, vol. 18, p. 253-259, 1996). Furthermore, many studies demonstrate the inhibition of bone resorption by strontium. While other substances such as bisphosphonates or estrogene exhibit one of these effects, only strontium is known to have both (Kingsley et al., Proc Natl Acad Sci, vol. 104(26), p. 10753-10754, 2007). Strontium compounds have successfully been established as therapeutics, so-called dual action bone agents, which affect both osteosynthesis and bone resorption and which are used to treat osteoporosis (Canalis et al., Bone, vol. 18(6), p. 517-523, 1996; Meunier et al., N Engl J Med, vol. 350(5), p. 459-468, 2004; Servier Austria GmbH, Journal fur Mineralstoffwechsel, vol. 16(3)m p. 160, 2009). More specifically, strontium has a positive effect on the function and differentiation of osteoblasts, while it inhibits the differentiation of osteoclasts (Bonnelye et al., Bone, vol. 42(1), p. 128-138, 2008). Also, strontium has been reported to have angiogenetic effects (Chen et al., Journal of Materials Science: Materials in Medicine, vol. 19(7), p. 2655-2662, 2008), which is another potentially beneficial effect in bone regeneration.

However, the methods for the incorporation of bioactive cations such as calcium and strontium into bone substitute materials leave a lot to be desired, for example in the area of homogeneity. The present invention provides a novel method for the production of bone substitute materials, which allows for the in situ chemical synthesis of relevant inorganic compounds inside a polymeric matrix with a very adaptable composition and distribution of bioactive cations within the bone substitute material. This characteristic is expected to enable a much improved bone tissue growth in the synthetic matrix. SUMMARY OF THE INVENTION

The present invention relates to a method for producing a biocompatible and/or bioactive material, comprising the following steps: (a) contacting one or more biocompatible polymers, one or more salts comprising alkali metal and/or alkaline earth metal cations and a solvent with a supercritical fluid/gas to form a solution or suspension, and

(b) reacting said supercritical fluid/gas with said salt(s) or said salt(s) and said polymer(s), whereby a reaction product with reduced solubility is formed.

Furthermore, the present invention relates to a biocompatible and/or bioactive material producible with this method, its use as a scaffold in tissue engineering, in treating bone loss or injury, for bone implantation, in bone repair and/or for use in treating infections of the bone. Also the present invention relates to an apparatus for generating a biocompatible and/or bioactive material according to the invention.

This summary of the invention does not necessarily describe all features of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise.

The present invention relates to a method for producing a biocompatible and/or bioactive material, comprising the following steps:

(a) contacting one or more biocompatible polymers, one or more salts comprising alkali metal and/or alkaline earth metal cations and optionally a solvent with a supercritical fluid/gas to form a solution or suspension, and

The term "biocompatible" refers to not producing a toxic, injurious, or detrimental immunological response in living tissue. Biocompatible materials or biocompatible polymers can be non-biodegradable, preferably they are partially biodegradable and most preferably they are completely biodegradable. The term "biodegradable" means resorbable in biological tissue, i.e. it refers to the capability of being broken down by cells or the product of cells, e.g. enzymes, preferably within an organism, e.g. a human or animal body.

The biocompatible polymer can be any polymer or polymer mixture known in the art to be biocompatible. Preferably, it is selected from the group consisting of block copolymers, caprolactones, hydroxybutyric acids, lactide and glycolide polymers, polyanhydrides and polyesters, polyphosphazenes, polyphosphoesters, polyacetals, and polyketals, polycyanoacrylates, polymethacrylates, polyacrylates, polyurethanes, polydioxanons, polyvinylalcohols, polyethyleneglycol, and a mixture of at least two such polymers. Said polyester preferably is a poly-lactide homopolymer, a poly-glycolide homopolymer, a poly- lactide-co-glycolide copolymer, or a mixture of at least two of the foregoing. More preferably, it is a poly-L-lactide-co-glycolide or poly-£-Caprolacton or a mixture thereof. Preferably the biocompatible polymer is capable of forming a scaffold, e.g. solidifies. The biocompatible polymer can also be a naturally occurring polymer or a mixture of at least two naturally occurring polymers, wherein said naturally occurring polymer is preferably selected from the group consisting of collagens, fibrinogen, laminin, fibronectin, albumin, silk fibroin, chitin, chitosan, cellulose, polysugars. The skilled person will choose the component composition depending on the desired application, where a high polymer and/ or optional solvent amount results in a more soft and viscous material, and a high amount of alkaline earth salts and lower amounts of polymer and/ or optional solvent results in a more brittle, harder material. Thus, in a preferred embodiment the polymer exceeds the amount of the salt and the optional solvent. In this preferred embodiment the polymer is present in a range of 50 wt% to 99 wt% based on the total weight of the polymer(s) and the salt(s). More preferably the amount of biocompatible polymer(s) is in the range of 60 wt% to 90wt%, even more preferably in the range of 70 wt% to 90 wt% and most preferably in the range of 80 wt% to 90 wt%. In this embodiment it is preferred that the polymer is selected from the group consisting of polyesters. More preferably the polymer is selected from polyesters with a low glass transition temperature below 50°C and most preferably is polycaprolacton. In another preferred embodiment the salt(s) exceeds the amount of the polymer and the optional solvent. In this preferred embodiment the biocompatible polymer is present in a range of 10 wt% to 50 wt% based on the total weight of the polymer(s) and the salt(s). More preferably the amount of polymer(s) is in the range of 15 wt% to 50 wt%, even more preferably in the range of 20 wt% to 50wt% and most preferably in the range of 25 wt% to 90 wt%. In this embodiment it is preferred that the polymer is selected from the group consisting of polyesters. More preferably the polymer is selected from the group consisting of polylactides, polyglycolides and polylactide-co-glycolides and most preferably is a polylactide-co-glycolide.

The salts of step a) of the method of the present invention are defined by the cations they comprise. It is envisaged that the cations belong to the groups of alkali metal and/or alkaline earth metal cations. It is also envisioned that cations other than alkali metal and/or alkaline earth metal cations are comprised in the salts or that different alkali metal or alkali earth metal cations are comprised, e.g. if the anions have two negative charges, the salt can comprise two different alkali metal cations and if the anions have three negative charges the salt can comprise an alkali metal cation and an alkaline earth metal cation or an alkali metal cation and another cation carrying two positive charges. In a preferred embodiment, the alkali metal cation is selected from lithium cation, sodium cation and potassium cation and the alkaline earth metal cation is selected from calcium cation, strontium cation, and magnesium cation. In a preferred embodiment, the cations are calcium cations, strontium cations, or calcium and strontium cations, i.e. the salts are strontium and/or calcium salts, preferably the salts are a mixture of the two, i.e. strontium and calcium salts. It is preferred that the molar ratio of strontium to calcium cations is between 1 to 1000 and 1 to 10, preferably between 1 to 500 and 1 to 50 most preferably about 1 to 100. Preferably, the salts are of fine grain size to allow even distribution in the solvent or solvent/polymer solution. This is particularly preferred if the salts are poorly soluble in the solvent of solvent polymer solution used.

In the preferred embodiment wherein the polymer(s) exceed the amount of the salt(s) the amount of salt varies in the range of 0.1 wt% to 50 wt% based on the total weight of the polymer(s) and the salt(s). More preferably the amount of salt(s) is in the range o 1 wt% to 18 wt%, even more preferably in the range of 5 wt% to 15 wt% and most preferably in the range of about 10 wt%. In this embodiment it is preferred that the salt(s) are selected from calcium or mixtures of calcium and strontium. In the preferred embodiment wherein the salt(s) exceed(s) the amount of the polymer(s) the salt is present in a range of 50 wt% to 90 wt% based on the total weight of the polymer(s) and the salt(s). More preferably the amount of polymer(s) is in the range of 50 wt% to 80 wt% and most preferably in the range of 50 wt% to 75 wt%. In this embodiment it is preferred that the salt(s) are selected from calcium or mixtures of calcium and strontium.

The salts of step a) are also defined by the anions they comprise. In a preferred embodiment, the anion(s) of the salt(s) is/are pharmaceutically acceptable and include, but are not limited to, hydroxide, oxide, carbonate, bicarbonate, bisulfate, hemisulfate, phosphate, phosphate/diphosphate, halogens, in particular chloride, bromide, iodide, or fluoride, hydrobromide, hydrochloride, hydroiodide, borate, isothionate, nitrate, sulfate, persulfate, ammonium salt, or organic anions including acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bitartrate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, 2-hydroxy- ethanesulfonate, hydroxynaphthoate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2- naphthalenesulfonate, napsylate, nicotinate, N-methylglucamine oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, 3-phenylpropionate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate, valerate, and the like (see, for example, S. M. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 66, pp. 1-19 (1977)).

Preferably, the anions of the salts are chosen in such to form a reaction product with the supercritical fluid/gas that is non-toxic or has a low toxicity, thereby reducing any adverse effects that may results from implanting the biocompatible material of the present invention. Particularly preferred anions are hydroxide, halogenids, or organic anions, which are preferably selected from the group consisting of organic acids, in particular acetate, propionate, lactate, gluconate, lactate gluconate, citrate, anion or a mixture of two or more of said anions. Most preferred anions are halogenids and hydroxide. Hydroxides are the most preferred anions, since they will form water when reacted with a supercritical fluid that provides H⁺; i.e. H₃0⁺. Thus, particularly preferred salts comprising alkali metal and/or alkaline earth metal cations are selected from the group consisting of calcium hydroxide, calcium chloride, calcium bromide, calcium fluoride, calcium iodide, strontium hydroxide, strontium chloride, strontium fluoride, strontium iodide and strontium bromide.

The reaction product with a reduced solubility has a reduced solubility in the supercritical fluid or the supercritical fluid/solvent mixture, if compared to the alkali metal and/or alkaline earth metal salt(s) that is(are) contacted with the supercritical fluid in step (a). Solubility is expressed as a concentration, either by mass (g of solute per kg of solvent, g per dL (100 mL) of solvent), mass concentration, molarity, molality, mole fraction or other similar descriptions of concentration. The maximum equilibrium amount of solute that can dissolve per amount of solvent is the solubility of that solute in that solvent under the specified conditions. Thus, the maximum equilibrium amount of the alkali metal and/or alkaline earth metal salt(s) contacted in step (a) is higher than of the reaction product of step (b). Preferably, the maximum equilibrium amount is reduced at least 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold or at least 1,000-fold. The solubility of ionic compounds in water is indicated by the solubility product K_sp. Thus, in the presence of an aqueous solvent the solubility product is preferably reduced at least 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold or at least 1,000-fold. For example when reacting calcium hydroxide with C0₂ to form CaC0₃ the solubility product is reduced from 4.68 ^χ 10^~6 to 4.8 ^χ 10^~9. As the solubility is affected by temperature, pH, additional ions in the solution etc. it is understood that reduced solubility is observed under identical or like conditions, in particular identical temperature. However, that does not preclude that the solubility differences that are observed under identical conditions are increased or decreased by changing temperature, pressure, pH during the course of step (b) as further outlined below.

The reaction mixture may further comprise poorly soluble calcium salts, preferably calcium phosphate, calcium dihydrogen phosphate, calcium hydrogen phosphate, hydroxyapatite, brushite, tricalciumphosphate, octacalciumphosphate or tetracalciumphosphate, or mixtures thereof. If such further poorly soluble calcium salts are comprised the optional solvent may not dissolve the respective calcium salt and, thus, rather than a solution a dispersion will be contacted with the supercritical fluid.

The solvent can be any solvent or mixture of solvents capable of completely or partly dissolving the biocompatible polymer and/or the salt of step a) of above-described method. Preferably, the solvent dissolves the polymer completely such that a homogeneous reaction mixture is obtained. Such homogeneous reaction mixture entails an even distribution and/or size of pores in the biocompatible and/or bioactive material being produced thereof. In particular, an increased pore size and an improved interconnection of neighbouring pores to each other may be affected. Thereby, a closer resemblance of the structure of naturally occurring bone material is achievable. This improved structure of the biocompatible and/or bioactive material allows for the invasion of cells into the material and their homogenous seeding on the biocompatible and/or bioactive material. Furthermore such structure also contributes to an increased viability of the cells. In this preferred embodiment the salt, preferably the alkaline earth metal salts are completely or partly dissolved in the solution of the biocompatible polymer. If the salt is only poorly soluble, it is preferred that a fine dispersion is produced. This allows a near homogenous or homogenous distribution of the salts in the polymer solution resulting in advantageous properties of the resulting product.

Preferably, the solvent is toxicologically harmless in the amount used in this invention, i.e. it is preferred that such solvent does not have an adverse effect on any living organisms, in particular, it does not have any adverse effect on humans. Adverse effects are harmful and undesired effects leading to a reduction or loss of function of one or more cells, tissues, organs, or the body of a living organism. Adverse effects include but are not limited to toxic effects; effects triggering the immune system, e.g. inflammation, allergic reaction; effects leading to alterations of tissue or organ structure, e.g. scarring, change of local blood flow; and effects causing morbidity and/or death of cells, tissues, organs, or the body. Preferably, any solvent remaining in the biocompatible product is easily metabolised in the human body. Preferably, the solvent is selected from the group consisting of water, ammonia, alcohols, ketons, carboxylic acids, carboxylic acid esters, halogenated hydrocarbons, e.g. acetone, formic acid, acetic acid, ethyl acetate, ethyl lactate, methylenechlorid, chloroform, hexafluoroisopropanol, tetrahydrofuran, dimethylformamide, and preferably is ethyl acetate. It is preferred that the solvent or mixture of solvents does not react with the polymer(s), the salt(s) or the salt(s) and the polymer(s) to form a reaction product as such reaction is occurring with the supercritical fluid. Thus, based on the respective supercritical fluid, salt(s) and polymer(s) chosen the skilled person can select suitable solvents. If the salts are alkali metal or alkali earth metal hydroxides or halides preferred solvents are ethylacetate, acetic acid, mixtures of water and acetic acid, and hexafluoroisopropanol. In particularly preferred embodiments, the solvent is a carboxylic acid ester, preferably ethyl acetate or a mixture of solvents comprising carboxylic acid ester, preferably ethyl acetate. Preferred co-solvents of carboxylic acid ester are water or water and an organic acid, preferably acetic acid.

In preferred embodiments, the solvent(s) or mixture of solvent(s) are added in an amount resulting in a honey-like or syrup-like consistency of the reaction mixture at 65 °C to 80°C. Typically, the viscosity of fluids of such consistency and at that temperature ranged between 10² - 10⁷ mPa*s, preferably 10⁴ - 10⁵ mPa*s. A reaction mixture having such honey- like or syrup-like consistency in this temperature range may be obtained by adding the solvent in an amount constituting between 10 wt% and 100 wt%, preferably between 20 wt% to 80 wt%, more preferably about 50 wt%, calculated on the basis of the weight of the salt(s) and the biocompatible polymer(s).

In further embodiments the solvent(s) may be added in smaller amounts resulting in a different, preferably denser pore structure of the produced biocompatible and/or bioactive material allowing fine-tuning of the material for the particular purpose of use. In such embodiments it is preferred that the solvent(s) are added in an amount constituting between 0.1 wt% and 20 wt%, preferably 0.5 wt% to 10 wt%, more preferably between 1 wt% and 5 wt%. The amounts are calculated on the basis of the weight of the salt(s), preferably alkaline earth salts, and the biocompatible polymer(s) , i.e. the total of biocompatible polymer, salts, solvents and further additives may be higher than 100 wt% as is shown in the following example: a starting composition comprising 3.0 g biocompatible polymer, 1.0 g alkaline earth metal salt and 2 g solvent would be considered to comprise 75 wt% biocompatible polymer, 25 wt% alkaline earth metal salt and 50 wt% solvent.

In a preferred embodiment wherein the polymer exceeds the amount of salt(s) the composition comprises preferably 50 wt% to 99.9 wt% biocompatible polymer(s) and 0.1 wt% to 50 wt% salts, preferably alkaline earth salts and 0.1% to 10 wt% solvent. Even more preferred 80 wt% to 99 wt% biocompatible polymer and 1 wt% to 20 wt% salts, preferably alkaline earth salts and 1 wt% to 5 wt% solvent.

In another preferred embodiment wherein the biocompatible polymer exceeds the amount of salt(s) the composition comprises preferably 50 wt% to 99.9 wt% biocompatible polymer(s) and 0.1 wt% to 50 wt% salts, preferably alkaline earth salts and 10% to 100 wt% solvent. Even more preferred 80 wt% to 99 wt% biocompatible polymer and 1 wt% to 20 wt% salts, preferably alkaline earth salts and 10% to 100 wt% solvent, more preferably between 20 wt% to 80 wt% solvent. A higher solvent content leads to biocompatible materials with an increased poresize and a lower solvent content leads to biocompatible materials with decreased poresize. An increased amount of salt(s) can lead to biocompatible materials with a higher pH value, meaning, the material will be more alkaline. The pH value can be adjusted using buffers as it is known to persons skilled in the art. These may either be comprised in the reaction mixture and/or be added during or after reaction. In a preferred embodiment the method comprises the further step of rinsing the reaction product in a buffer solution, preferably to adjust the pH of the biocompatible/bioactive material to approximately physiologic pH, e.g. in the range of pH 6.0 to 8.0. Accordingly, it is preferred that the pH of the biocompatible/bioactive material has a pH in the range of 6.0 to 8.0.

In the preferred embodiment wherein the salt(s) exceeds the amount of biocompatible polymer(s) the composition comprises preferably 50 wt% to 90 wt% alkaline earth salts and 10 wt% to 50 wt% biocompatible polymer and 1 wt% to 10 wt% solvent. The preferred weight ratio is 50 wt% to 75 wt% salts, preferably alkaline earth salts and 25 wt% to 50 wt% biocompatible polymer and 1 wt% to 5 wt% solvent.

A supercritical fluid/gas is a substance at a temperature and pressure above its critical point. The critical point, also called critical state, specifies the conditions (critical temperature, critical pressure and sometimes composition) at which a phase boundary ceases to exist. The critical point for a specific substance is known to the skilled person and is readily available from literature. A supercritical fluid/gas can effuse through solids like a gas, and dissolve materials like a liquid. In addition, close to the critical point, small changes in pressure or temperature result in large changes in density, allowing many properties of a supercritical fluid to be fine-tuned. Supercritical fluids are suitable as a substitute for organic solvents. The supercritical fluid in the context of the present invention is chosen in such that it is capable of reacting with the salt, the polymer or the salt and the polymer, preferably with the salt. It is, thus, preferred that the supercritical fluid/gas in its supercritical state forms anions and cations. The supercritical fluid/gas ay be partially of fully ionized. If the supercritical fluid is only partially ionized, the anions of the supercritical fluid/gas will react with the alkali metal and/or alkaline earth metal cations to form a les soluble reaction product and, thus, new anions will be formed due to the removal of anions of the supercritical fluid from the equilibrium. The supercritical fluid can further be selected in such that it is partly or completely miscible with the solvent or it can be immiscible with the solvent. In this context the term "reacting" refers both to the formation of new covalent bonds between the salt and the supercritical fluid, the polymer and the supercritical fluid or the salt and the polymer and the supercritical fluid or any of the former and/or the solvent and to the change of ionic interaction, e.g. the cation(s) of the salt(s) interact with anions formed by the supercritical fluid, e.g. C0₃ ^2", HC0₃\ CHOO^', OH^", H₂PO₄ ^", HPO₄ ^", PO₄ ³\ NH₂\ preferably CO₃ ^2". Another example of a reaction in that sense is trans-salifying, i.e. exchanging the anion of the salt. The reaction between the supercritical fluid and the salt, polymer or salt and polymer leads to a reaction product which exhibits a reduced solubility in the supercritical fluid. It is preferred that the solubility of the reaction product(s) is/are also reduced in the solvent. Preferably the solubility of the reaction product is reduced in such that insoluble material is formed. Insoluble material may form immediately under the conditions of the reaction or may only form once the pressure and/or temperature is further changed, preferably below criticality. Thus, the salts, polymers or salts and polymers as the case may be are chosen in such that they are capable of reacting with each other in that sense. Preferred combinations of supercritical fluids/gas and salts and/or polymers are the following: CO₂ and alkaline earth metal salt(s) with a solubility that is higher than the solubility of the resulting alkaline earth metal carbonate(s) or alkaline earth metal hydrocarbonate(s); carboxylic acid, preferably formic acid and alkali metal salt(s) and/or alkaline earth metal salt(s) with a solubility that is higher than the solubility of the respective alkali metal carboxylic acid salt(s), e.g. formate(s) or alkaline earth metal carboxylic acid salt(s), e.g. formate(s).

It is further preferred that the solvent(s) used in these embodiments are selected from the group consisting of ethyl acetate, acetic acid, mixtures of water and acetic acid, and hexafluoroisopropanol. Thus, in a preferred embodiment, the supercritical fluid/gas is selected from the group consisting of carbon dioxide and/or carboxylic acids, wherein said carboxylic acid is preferably selected from the group consisting of formic acid and/or acetic acid. The critical temperature/pressure for carbon dioxide is 31.04°C/7.38 MPa, for formic acid it is 315°C/5.81 MPa, and for acetic acid it is 320°C/5.78 MPa. More preferably, the supercritical fluid/ gas is carbon dioxide and the preferred temperature is below 40°C for applications comprising temperature critical components like bioactive substances as defined below. It is preferred that the pressure at the respective temperature is as required to render the carbon dioxide supercritical. In another preferred embodiment of this invention, the temperature is chosen to comply with international standards for sterilization (e.g. US Pharmacopoeia, European Pharmacopoeia or others) and usually is at least 160°C. Corresponding to the chosen temperature a minimum time interval for the process is defined and usually is at least 120 min at 160°C and at least 30 min at 180°C. The reaction of step (b) is preferably carried out for a time period of between 5 min and 48 h, more preferably for at least 10 min, 20 min, 30 min, 40 min, 50 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 1 1 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h or 24 h. The skilled person is aware that the length of time required to carry out the reaction of step (b) will depend on the reaction temperature. Thus, if the reaction temperature is decreased the reaction time required will be longer and conversely, if the reaction temperature is increased the reaction time required will be shorter. Preferably, the reaction is carried out to completion. In the preferred embodiment wherein the supercritical fluid is C0₂ the reaction is preferably carried out at a temperature in the range of 25°C to 80°C, preferably at 65°C and for at least 8 h, preferably for at least 12 h.

Without wishing to be bound by any theory, it is believed that the reaction under supercritical conditions occurs by dissolution of C0₂ in the reaction mixture until equilibrium is reached. Subsequently the one or more salts comprising alkali metal and/or alkaline earth metal cations, preferably a mixture of calcium and strontium salts, more preferably calcium hydroxide and strontium hydroxide, react with the supercritical fluid to the respective less soluble salt, preferably less soluble carbonate, most preferably calcium carbonate and strontium carbonate, thereby releasing as a reaction product the alkali metal and/or alkaline earth metal reaction product with a reduced solubility and the anion of the reaction product the less soluble. Thus, an example of a particularly preferred reaction can be depicted as follows:

X(OH)₂ + C0₂→ XC0₃ + H₂0

wherein X is preferably = Ca and/or Sr, preferably in above indicated ratios.

It is further believed that the process is improved with respect to the homogeneity of the microemulsion of the less soluble reaction product formed in the biocompatible polymer, if the supercritical fluid is present in an excess, since this - also in conjunction with the solvent that is present or with water formed in the course of the reaction, will increase the solubility of the reaction product with reduced solubility and, thus, lead to a more even distribution of the less soluble reaction product throughout the polymer. An example of a preferred further reaction can be depicted as follows: XC0₃ + H₂0 + C0₂→ X²⁺ (aq) + 2 HC0₃ ^' (aq) wherein X is preferably = Ca and/or Sr, preferably in above indicated ratios.

Additionally it is possible that further components can permeate from the polymer phase into the supercritical fluid or supercritical fluid/gas solvent phase or vice versa. Thus, it is preferred that the reaction comprises an excess of the supercritical fluid/gas, preferably at least a two-fold, three-fold or four-fold excess.

In a preferred embodiment the salt(s), biocompatible polymer(s) or salt(s) and biocompatible polymer(s) are dissolved in the solvent prior to being contacted in step (a) with the supercritical fluid/gas. In a further preferred embodiment only one of the components is dissolved in the solvent, i.e. the salt(s) or the polymer(s), and the other component is dispersed in the solvent.

In another preferred embodiment the salt(s), biocompatible polymer(s) or salt(s) and biocompatible polymer(s) are dissolved when contacted with the supercritical fluid/gas and the solvent.

Said solution optionally further comprises one or more bioactive substances, wherein said bioactive substances have a positive effect on bone growth, on the biocompatibility of the material and/or on post-implantation effects within the body (e.g. infections). Preferably, the one or more bioactive substances are selected from the group consisting of bone growth promoting substances, e.g. calcium phosphates, proteins, e.g insulin, peptides, amino acids, antibiotics, immunomodulators, growth factors, anti-tumour agents, angiogenesis factors, and cell differentiation factors. Bioactive substances comprised in said solution may in particular encompass substances promoting the invasion, seeding, growth and/or viability of cells. Preferably, these substances are selected from the group consisting of carbohydrates (e.g. glucose phosphates such as a-D-glucose 1 -phosphate or a-D-glucose 6-phosphate, glucuronic acid, and salts thereof), essential amino acids, proteins and minerals.

Furthermore, said solution may also comprise components buffering the pH value of the solution in order to adjust the pH value to a pH of about 7 and thereby, increase the biocompatibility of the produced material. Such buffering compounds are known to the person skilled in the art and include but are not limited to sodium phosphate or kalium phosphate containing buffer systems and mixtures thereof, e.g. salts from buffer systems based on formulations by R. Dulbecco, W.R. Earle and J.H. Hanks (Earle, W.R. et al., J. Nat. Cancer Inst. 4, 165, 1943; Hanks, J. H. and R.E. Wallace, Proc. Soc. Exp. Biol. Med. 71 , 196, 1949; Dulbecco, R. and M. Vogt, J. Exp. Med. 99, 167, 1954). Preferably, for 5 g to 6g reaction mixture an addition of 0.0838g NaCl, 0.0021 g KC1, 0.0120g Na₂HP0₄, 0.0021 g KH₂P0₄ is made.

It is envisaged that the method of the invention can comprise further optional steps, which can be added alone or in combination with any number of other additional steps. In one embodiment, the method further comprises the step of cooling said polymer(s) and/or said salt(s) and said solvent prior to step (a) to or below freezing temperature of the solvent, preferably to about -192°C, to about -78°C, to about -20°C, or to about 0°C. In another embodiment, the method comprised the step of raising the temperature of said polymer(s), salt(s), optional solvent, supercritical gas/fluid and/or said solution above the supercritical temperature of said supercritical fluid/gas during step (a) or (b), preferably (b). In one embodiment in which the method comprises both this step and the afore-described step of cooling, the step of cooling is performed first. In preferred embodiments, the temperature/pressure is raised in that step to a temperature suitable for sterilisation (e.g. 100°C to 180°C, depending on time and pressure), to a temperature with a specific miscibility or immiscibility according to the phase diagram of said polymer(s) and/or salt(s) and/or solvent(s), wherein said specific temperature with a specific miscibility or immiscibility regulates the crystalline structure of precipitated compounds, to a temperature which is a specific reaction temperature for said polymer(s) and/or salt(s), and/or to a temperature at which a specific conformation of said polymer(s) and/or salt(s) is prevalent. Also, the temperature and/or the pressure can be raised to a point at which the solution of said salt(s), polymer(s) or polymer(s) and salt(s) is homogenous and/or at which the precipitation of said salt(s) is triggered. In embodiments wherein the temperature/pressure is raised to a temperature suitable for sterilisation (e.g. 100°C to 180°C, depending on time and pressure) it is particularly preferred that the produced biocompatible and/or bioactive material is foamed directly into a sterile bag such that the produced material remains sterile as long as needed. This can be achieved by lining the reaction chamber wherein the biocompatible and/or bioactive material is foamed with a sterile membrane that is suitable to serve as a microbial barrier and thereby, keeping the biocompatible and/or bioactive material inside sterile. Sterile membranes suitable in the context of the present invention include but are not limited to Tyvek membranes or PTFE based membranes. Tyvek is a nonwoven product consisting of spunbond polyethylene fibers.

In another embodiment, the method of the invention comprises a further step of adding one or more of above-mentioned bioactive substances, wherein the temperature at said further step preferably is at or below 37°C and which preferably is sufficiently low to avoid adverse temperature-related alterations of said bioactive substance(s). Said further step can be carried out at any stage of the method of the invention, provided that the subsequent temperatures are sufficiently low to avoid adverse temperature-related alterations of said bioactive substance(s) In yet another embodiment, the method of the invention further comprises a step of reducing the pressure at a rate of 0.001 bar/s (thereby preferably establishing a very fine pore network, possibly an aerogel) to 50 bar/s (thereby preferably establishing a very coarse pore network), preferably 0.01 to 10.0 bar/s, more preferably 0.05 to 0.1 bar/s, and/or reducing the temperature at a rate of 0.001 to 30 °C/s, preferably 0.5 to 3 °C/s , more preferably 1 to 2 °C/s during and or after step (c) or reducing the pressure and/or temperature of the supercritical gas/fluid to ambient pressure/temperature within 0.5 minutes to 48 hours, preferably 1 minute to 1 hour, more preferably 10 minutes to 1 hour. Further preferred temperature reduction rates comprise a reduction of 0.001 to 30 °C/s, preferably 0.01 to 3 °C/s , more preferably 0.1 to 0.3 °C/s during and/or after step (c) or reducing the pressure and/or temperature of the supercritical gas/fluid to ambient pressure/temperature within 0.5 minutes to 48 hours, preferably, 1 minute to 1 hour, more preferably 3 minutes to 10 minutes. These rates are particularly preferred in the context of using C0₂ as supercritical fluid.

The present invention further relates to a biocompatible and/or bioactive material producible with the method of the invention. This biocompatible and/or bioactive material preferably is solid or viscous. Said material when solid is preferably in a monolithic form. In a preferred embodiment, said material is porous, i.e. a porous matrix. In a preferred embodiment, the pore size is between 10 to 1000 μιη, preferably 100 to 500 μπι and 1 to 1000 μ^ηι, preferably after solidification. In further preferred embodiment, the pore size is between 10 to 5000 μπι, preferably 100 to 3000 μηι and more preferably 200 to 1000 μπι, preferably after solidification. In another embodiment, some or all pores are interconnected or not interconnected, preferably they are interconnected. In yet another embodiment, said material is sterile.

Typically, in the method of the present invention the molar concentration of Ca and/or Sr initially comprised in the reaction mixture is tuned into an equivalent molarity of CaC0₃ and/or SrC0₃. Thus, in further preferred embodiments the biocompatible and/or bioactive material of the present invention comprises a molar concentration of CaC0₃ and/or SrC0₃ which corresponds to the molarity of Ca and/or Sr initially added in the reaction mixture.

It is anticipated that the biocompatible and/or bioactive material of the invention is used as a scaffold in tissue engineering. Hence, the present invention also relates to said material comprising cells, preferably osteoblasts and/or osteoclasts. The biocompatible and/or bioactive material of the invention can also be used in treating bone loss or injury, for bone implantation, in bone repair and/or for use in treating infections of the bone. Thereby, it is envisaged that said material can be implanted in a solidified or viscous form or a mixture of both. For example, a solidified material may be used to substitute for larger bone structures, whereas a viscous material may be used for the minimally invasive repair of bone defects. Accordingly, the present invention also relates to a method of treating a patient in need of bone implantation, bone repair, bone regeneration or treatment of an infection using the biocompatible and/or bioactive material according to the invention, wherein said matrix is implanted in solid or viscous form. For example, the bone substitute material of the invention loaded with antibiotics may be used for implantation into a patient suffering from osteomyelitis. Controlled local release from the implant achieves high local antibiotic concentrations to combat the infection locally while maintaining low systemic levels.

The present invention further relates to an apparatus for generating a biocompatible and/or bioactive material according to the invention. The general assembly of said apparatus comprises a vessel for holding a supercritical fluid/gas or a gas/fluid to be rendered supercritical, and which is connected to a pressure control and/ or temperature control for delivering and/or exposing said gas/fluid to a reaction chamber with the polymer(s) and/or salt(s) and/or solvent(s), and an optionally sealable nozzle for controlled release of the fluidic components or for releasing said matrix from said apparatus. Optionally, said apparatus further comprises a means of heating and/or cooling said reaction chamber, a sealable outlet connected to said reaction chamber for reducing the pressure in the reaction chamber and/or releasing the supercritical gas/fluid in a supercritical and/or post-supercritical state, and/or at least one separate reagent chamber for feeding one or more of said polymer(s) and/or said salt(s) and/or the solvent into the reaction chamber (4). In a preferred embodiment the reaction chamber (4) is lined with a sterile membrane such as but not limited to a Tyvek membrane or a PTFE based membrane allowing for the simultaneous sealing of the biocompatible and/or bioactive material into a sterile bag during foaming. In a further preferred embodiment the apparatus comprises one or more separate reagent chambers, which comprise a solution or dispersion of the salt(s) and the solvent(s), the polymer(s) and the solvent(s) or of the salt(s), the polymer(s) and the solvent(s). If the apparatus is configured to inject a solution or dispersion into the reaction chamber (4) from one or more, preferably one reagent chamber, the mixing of these components is obviated. It is preferred that this separate reagent chamber is removably attached to the apparatus of the invention. This allows separate storage of the solution or dispersion under appropriate conditions and the attachment of the reagent chamber to the apparatus immediately prior to use. In these embodiments it is preferred that the solution or dispersion in the separate reagent chamber is sterile and that the reagent chamber is sealed with a fluid tight closure that opens upon attachment of the reagent chamber to the apparatus. For injection of the solution or dispersion into the reaction chamber the reagent chamber may further comprise a plunger, which is manually or automatically actuated. Alternatively, the reaction chamber may comprise a pressurized part, from which the pressure can be released into the solution or dispersion comprising part to inject the solution or dispersion into the reaction chamber. It is also preferred that the apparatus comprises a heating element to heat the reaction chamber. This is particular preferred, if the supercritical fluid used in the reaction is C0₂. In a preferred mode of operation dry ice, i.e. frozen C0₂, is placed into the reaction chamber, the solution or dispersion of the salt, polymer and solvent is injected and then the reaction chamber is heated to form the supercritical fluid in situ. The reaction chamber has to be configured to withstand the pressure and temperatures that are required to transform the supercritical fluid, e.g. C0₂, to its supercritical state.

To extrude the viscous material from the apparatus, the apparatus may be configured to comprise a plunger that is connected to the reaction chamber.

In a preferred embodiment, the material produced with said apparatus is viscous. Preferably, the released viscous matrix is replenished continuously or said material is produced in batches.

The above-described method of the invention for the production of a bone substitute material is novel in that the reactive properties of supercritical fluids are used to form bioactive components (e.g. calcium carbonate) from chemical precursors (e.g. calcium hydroxide) during the production procedure instead of integrating said bioactive components directly. Since chemical precursors with a higher solubility than the bioactive components are used, the method of the invention achieves a far better control of the desired granularity and a more even distribution of the bioactive components in the bone substitute material, allowing for a much improved and more homogenous bone tissue growth within the substitute material.

BRIEF DESCRIPTION OF THE FIGURES

The following figures are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way.

Fig. 1 : Example for a disc-shaped porous bone substitute material based on polycaprolactone according to the invention which has been cultivated with an osteoclast cell line and which has been stained with a specific fluorescence stain (fluoresceindiacetate/ ethidiumbromide) demonstrating high cell vitality and homogeneous cell seeding (a, b). Square shaped and cell free bone substitute material based on poly-lactide-co- glycolide in SEM imaging (c, e), poly-lactide-co-glycolide scaffold with CaC0₃ cultivated with an osteoblast cell line and stained with fluoresceindiacetate/ ethidiumbromide (d). Macroscopic comparison of a pure poly-lactide-co-glycolide scaffold (diameter 6 mm) and one poly-lactide-co-glycolide scaffold with additional CaCC-3 (f).

Fig. 2: General assembly of an apparatus for producing a bone substitute material according to the invention. (1) reaction chamber, (2a) C0₂ (dry ice, optional), (2b) C0₂, (3a) component mixture (before process), (3b) synthesized material (post process), (4) vessel, (5) high pressure sealing, (6a) high pressure valve (closed), (6b) high pressure valve (open), (7) pressure gauge (manometer), (8a) outlet (closed), (8b) outlet (open, relaxation).

Fig. 3:General assembly of an injection apparatus for producing a viscous bone substitute material according to the invention. (1) C0₂ container, (2) first valve (e.g. needle valve), (3) first valve (lever), (4) component container, (5) component mixture, (6) heater/cooler (e.g. electrical), (7) outlet screw (used to open or exchange component container), (8) second valve (lever), (9) second valve (e.g. needle valve), (10) outlet nozzle Fig. 4: General assembly of an apparatus for producing a sterile bone substitute material according to the invention as described in Fig. 2, with the component mixture (before process), (3b) in a Teflon reaction chamber (4b) with porous plates (1 la and 1 l b) and Tyvek membranes (12a and 12b) on both sides of the vessel. Fig. 5: Macroscopic comparison of an example of a disc-shaped porous bone substitute material based on polycaprolacton according to the invention foamed (a) with or (b) without solvent comprising ethyl acetate, water and acetic acid.

Fig. 6: Osteoblast cells seeded on disc-shaped porous bone substitute material were stained with fluoresceindiacetate/ ethidiumbromide (b) demonstrating high cell vitality and homogeneous cell seeding. Influence of buffer addition to the vitality of cells in a MTT-Assay (a) on seeded samples of the material when modified with buffer substance (left) or without buffer substance (right). Material with buffer substance shows improved biocompatibility properties due to the adjusted and more physiological pH. EXAMPLES

The following examples are for illustrative purposes only and do not limit the invention described above in any way.

Example 1:

0.1 g poly-lactide-co-glycolide (polymer) and 0.01 g Ca(OH)₂ (alkaline earth salt) are mixed with 50 μΐ of ethylacetate (solvent) inside a 1.0 ml polypropylene reaction tube. This reaction tube is placed in a high pressure autoclave filled with dry ice. The autocalve is sealed pressure tight and heated to 45°C and kept in the pressurized state for 12 hours.

Subsequently, the autoclave is depressurized over the course of 10 minutes. The reaction tube is removed obtaining the foamed bone substitute material which is composed of CaC0₃ and poly-lactide-co-glycolide. To obtain a disc-shaped material, the material can be cut with a suitable knife or scalpel. A photographic image and fluorescencemicroscopic images closely corresponding to this example are shown in Figure Id and f.

Example 2:

1.0 g polycaprolacton (polymer) and 0.1 g Ca(OH)₂ (alkaline earth salt 1) and 0.001 g Sr(OH)₂ (alkaline earth salt 2) and 100 μΐ 100% acetic acid (solvent) are mixed inside a 15 ml polypropylene reaction vessel and cast into a semi porous shape mould. This mould is rapidly cooled in liquid nitrogen before placing it inside a high pressure autoclave filled with dry ice. The autoclave is sealed pressure tight and heated to 60°C. It is kept in the pressurized state for 24 hours.

Thereafter, the autoclave is depressurized at a rate of 0.5 bar/s. The mould is removed obtaining the foamed bone substitute material which is composed of CaC0₃, SrC0₃ and polycaprolacton and which has the shape of the semi porous mould.

Example 3:

0.1 g poly-lactide-co-glycolide (polymer) and 0.00 lg Ca(OH)₂ (alkaline earth salt) are mixed inside a 1.0 ml polypropylene reaction tube with a 10 μΐ saturated solution of insulin (optional bioactive substance) in hexafluoroisopropanol (solvent). This reaction tube is placed in a high pressure autoclave filled with dry ice. The autoclave is sealed pressure tight and is heated to 32°C and kept in the pressurized state for 12 hours. The autoclave is then depressurized at a rate of 1.0 bar/s. The insulin modified foamed bone substitute material is otherwise prepared similar to examples 1 or 2.

Example 4:

3 g polycaprolactone (PCL) and 0.2 g polyethylene glycol (PEG), 0.3 g Ca(OH)₂ and

0,0108 Sr(OH)₂ (alkaline earth salts), 0,1 g a-D-glucose 1 -phosphate (optional bioactive substance) and 0.1 g Instamed (0.0838g NaCl, 0.0021g KC1, 0.0120g Na₂HP0₄, 0.0021g KH₂P0₄) are mixed inside a Teflon or polypropylene reaction tube with or without a mixture of 1.5 ml ethyl acetate, 0.1 ml acidic acid and 0.2 ml water (solvent). This reaction mixture is moderatly heated to 80°C and homogenised in the closed reaction tube. Subsequently it is cooled down to -20°C and homogenised until the reaction mixture obtains a powdery consistency. The resulting reaction material is placed in a Teflon form comprising porous plates and Tyvek membranes on both sides, and is compressed therein (see Fig. 4). The complete Teflon form is weld in Tyvek membrane and placed in a high pressure autoclave filled with dry ice. The autoclave is sealed pressure tight and is heated to 65°C and kept in the pressurized state for 12 hours. Subsequently, the autoclave is depressurized over the course of 3 minutes.

Macroscopic comparison of an example of a disc-shaped porous bone substitute material produced as described above with or without solvent is shown in Fig. 5 (a) and (b), respectively. The porous bone substitute material produced with solvent exhibits an increased pore size and a more evenly distribution of the pores compared to the bone substitute material produced without solvent. Furthermore, an improved interconnectivity of neighbouring pores can be observed in bone substitute material produced with solvent. Overall, the bone substitute material produced with solvent features a close resemblance of the naturally occurring bone structure.

Porous bone substitute material disc with a diameter of about 2 cm were produced with solvent as described above and which was directly welted into a sterile bag during the foaming process. The osteoblast cell line Cal-72 was seeded on such disc-shaped porous bone substitute material. Cells were stained with a specific fluorescence stain (fluoresceindiacetate/ ethidiumbromide) after 10 and 14 days demonstrating high cell vitality and homogeneous cell seeding (see Fig. 6(b)).

The influence of buffer addition to the vitality of cells was assessed in a MTT- Assay (see Fig. 6(a)) using a material extract and a corresponding vitality assay using fluorescein diacetate and ethidium bromide (FDA & EB) on seeded samples of the material when modified with buffer substance (left) or without buffer substance (right).

Using buffer substances reduces significantly the pH value to near neutral values and shows higher vitality in the MTT-assay (pH 8.1 and 84% vitality) than the sample without buffer (pH 10.7 and 21% vitality). In seeding experiments the impact of pH is not as significant due to intrinsic pH buffering and dilution with cell culture medium.

Claims

A method for producing a biocompatible and/or bioactive material, comprising the following steps:

(a) contacting one or more biocompatible polymers, one or more salts comprising alkali metal and/or alkaline earth metal cations and a solvent with a supercritical fluid/gas to form a solution or suspension, and

The method of claim 1 , wherein the alkali metal cation is selected from lithium cation, sodium cation and potassium cation and the alkaline earth metal cation is selected from calcium cation, strontium cation, and magnesium cation.

The method of claim 2, wherein the salts are strontium and calcium salts wherein the molar ratio of strontium and calcium cations is between 1 to 1000 and 1 to 10, preferably 1 to 500 and most preferably 1 to 100.

The method of claims 1 to 3, wherein the anion of said salt(s) is selected from the group consisting of hydroxide, chloride, acetate, propionate, lactate, gluconate, lactate gluconate, oxide, citrate, carbonate and phosphate anion or a mixture of two or more of said anions.

The method of claims 1 to 4, wherein said salt(s) is/are selected from the group consisting of calcium phosphate, calcium dihydrogen phosphate, calcium hydrogen phosphate, hydroxyapatite, brushite, tricalciumphosphate, octacalciumphosphate and tetracal c i umpho sphate .

The method of claims 1 to 5, wherein said supercritical fluid/gas is selected from the group consisting of carbon dioxide, dinitrogen monoxide, nitrogen, carboxylic acid and/or carboxylic acids esters.

The method of claims 1 to 6, wherein said biocompatible polymer(s) is/are selected from the group consisting of block copolymers, caprolactones, hydroxybutyric acids, lactide and glycolide polymers, polyanhydrides and polyesters, polyphosphazenes, polyphosphoesters, polyacetals, and polyketals, polycyanoacrylates, polymethacrylates, polyacrylates, polyurethanes, polydioxanons, polyvinylalcohols, polyethyleneglycol, and a mixture of at least two such polymers.

8. The method of claims 1 to 7, wherein said solvent is selected from the group consisting of water, ammonia, alcohols, ketons, carboxylic acids, carboxylic acid esters, halogenated hydrocarbons, e.g. acetone, formic acid, acetic acid, ethyl acetate, ethyl lactate, methylenechlorid, chloroform, hexafluoroisopropanol, tetrahydrofuran, dimethylformamide, and preferably is ethyl acetate.

9. The method of claims 1 to 8, further comprising the step of raising the temperature of said polymer, salt(s), optional solvent, supercritical gas/fluid and/or said solution above the supercritical temperature of said supercritical fluid/gas during step (a), (b), or (c).

10. The method of claims 1 to 9, wherein said solution further comprises one or more bioactive substances selected from the group consisting of bone growth promoting substances, calcium phosphates, insulin, antibiotics, immunomodulators, growth factors, anti-tumour agents, angiogenesis factors, pH buffering substances and cell differentiation factors.

1 1. The method of claim 10, wherein said bioactive substance is a derivative of glucose, preferably glucose 1 -phosphate, glucose 6-phosphate or glucuronic acid and most preferably is glucose 1 -phosphate.

12. A biocompatible and/or bioactive material producible with the method of claims 1 to 1 1 , optionally comprising cells, preferably osteoblasts and/or osteoclasts.

13. Use of the biocompatible and/or bioactive material of claim 12 as a scaffold in tissue engineering.

14. A biocompatible and/or bioactive material according to claim 12 for use in treating bone loss or injury, for bone implantation, in bone repair and/or for use in treating infections of the bone. An apparatus for generating a biocompatible and/or bioactive material according to claim 12, comprising a vessel for holding a supercritical fluid/gas or a gas/fluid to be rendered supercritical (1), connected to a pressure control inlet valve (2) for delivering said gas/fluid into a reaction chamber (4), and an optionally sealable nozzle (10) for releasing said matrix from said apparatus, and optionally further comprising a means of heating and/or cooling said reaction chamber (12), a sealable outlet (7) connected to said reaction chamber (4) for reducing the pressure in the reaction chamber and/or releasing the supercritical gas/fluid in a supercritical and/or post-supercritical state, and/or at least one separate reagent chamber for feeding one or more of said polymer(s) and/or said salt(s) and/or the solvent into the reaction chamber (4).