WO2009013497A1

WO2009013497A1 - Sol-gel bioactive glass

Info

Publication number: WO2009013497A1
Application number: PCT/GB2008/002532
Authority: WO
Inventors: Simon Baker; Xiaobin Zhao
Original assignee: NOVA THERA Ltd
Current assignee: NOVA THERA Ltd
Priority date: 2007-07-26
Filing date: 2008-07-24
Publication date: 2009-01-29
Anticipated expiration: 2010-01-26
Also published as: WO2009013512A1; GB0714602D0

Abstract

The present invention relates to a sol-gel derived bioactive glass comprising less than about 30 ppm of nitrate species. Further claims relate to composites, therapeutic uses and methods of treatment using the same. The invention also relates to a process for preparing a sol-gel derived bioactive glass, said process comprising the steps of: (i) forming a mixture comprising a gelable inorganic base material and at least one calcium component; (ii) subjecting said mixture to a temperature of from about 50 to about 70°C for about 2 to about 36 hours; (iii) drying the material formed in step (ii) by subjecting said material to a temperature of about 80 to about 180°C for about 1 to about 72 hours; (v) stabilising the material formed in step (iii) by subjecting said material to a temperature of about 700 to about 800°C for about 3 to about 24 hours.

Description

SOL-GEL BIOACTIVE GLASS

The present invention relates to calcium containing sol-gel bioactive glass powders, together with optimised processing conditions and methods for the preparation thereof. The powder composites so produced have applications in tissue engineering and other therapeutic areas.

BACKGROUND TO THE INVENTION

The preparation and characterisation of sol-gel glass monoliths and foams have been discussed and reported extensively. The ultimate applications for these sol-gel materials are in the field of biomaterials and tissue engineering. However, to date most biological work undertaken involving ceramics has been on powders rather than bulk materials.

Although processing is very similar to the stages employed in the preparation of monoliths and foams, differences arise during the gelation, aging, drying and stabilising stages in order to reduce the time taken since careful drying/stabilisation is unnecessary.

The first discussion of calcium silicate ceramics as a potential biomaterial was in the early 1990s when Kokubo et al demonstrated that P₂O₅ free wollastonite ceramics (CaO-SiO₂) showed in-vitro and in-vivo bioactivity. They formed a Ca-P rich layer in combination with a Si- rich layer on their surfaces within 3 weeks when implanted into the metaphyses of the tibiae of male rabbits (Ohura K, Nakamura T, Yamamuro T et al. Bone-bonding ability of P₂O₅-free CaO-SiO₂ Glasses. Journal of Biomedical Materials Research 1991; 25: 357-365). Based on the results of this study, they concluded that there was no difference in the bone bonding interface for phosphorous-free and phosphorous containing glasses, once the Ca-P rich layer has formed on their surfaces.

However, wollastonite ceramics have not been used in clinical studies. Instead, a composite of apatite (CaO-P₂O₅) and wollastonite ceramics (commercially known as Cerabone® A-W) has been successfully used in many clinical applications (Kokubo T. In: Hench LL, Wilson J, Eds. Introduction to Bioceramics. World Scientific, Singapore 1993: pp.75.). It has been since established that the index of bioactivity (I_B) for 45S5 Bioglass® is 2.5 time higher than that of Cerabone® A-W (Gross U, Kinne R, Schmitz

HJ, Strunz V. CRC Critical Reviews in Biocompatibility 1998.)-

In 1999, Izquierdo-Barba and Vallet-Regi et al made two publications (Martinez A, Izquierdo-Barba I, Vallet-Regi M. Bioactivity of a CaO-SiO₂ binary glass system. Chem. Mater. 12: 3080-3088, Izquierdo-Barba I, Salinas AJ, Vallet-Regi M. In vitro Ca-P layer formation on sol-gel glasses of the CaO-SiO₂ system. Journal of Biomedical Materials Research. 2000 47: 243-250.) concerning the processing and bioactivity of binary CaO-SiO₂ gel glasses. Although a similar approach with a sol-gel technique is used in the processing, the primary aim was to produce powders of 32 to 63 μm. The powders are then compacted into pellets and sintered at high temperatures to produce what they have termed 'monolith discs'. The first of these papers is based on the 80 mol% SiO₂ composition and concludes that it is bioactive and that phosphorous is taken from the surrounding SBF during the formation of the HCA layer.

The more recent publication is based on binary compositions ranging from 50 mol % SiO₂ to 90 mol % which are processed in the same way as explained earlier. Similar bioactivity is reported and they conclude that lower SiO₂ gel glasses (i.e. gels with 50 mol % to 70 mol % SiO₂) have a higher rate of HCA formation. They relate this to the surface area and pore sizes of the glasses. Although this is found to be true, studies have shown that the lower silica gels (i.e. gels with 50 mol % to 60 mol % SiO₂) degrade at a faster rate (higher rate of dissolution), leading to the resorption of the glasses which hinders the rate of formation of HCA (P. Saravanapavan, Mesoporous Calcium Silicate gel-glasses: Synthesis, Structure and bioactivity 2001).

To date, sol-gel techniques have typically involved extensive gelation, aging, drying and stabilisation programmes to produce monolithic and foamed scaffolds for tissue engineering. However, previously reported stabilisation conditions have been found to be insufficient for the thermal decomposition of calcium nitrate to its oxide form (CaO). As a consequence, bioactive glass materials prepared to date typically contain unacceptably high levels of nitrate species. It is well established that nitrate species are commonly associated with toxicity problems. According to the U.S. Environmental Protection Agency (EPA), the maximum containment level (MCL) for nitrate in drinking water is 10 mg/L nitrate- nitrogen (equivalent to 45 mg/L as nitrate)

Ingested nitrate is reduced to nitrite, which binds to haemoglobin to form methehaemoglobin (MetHb). Methehaemoglobemia occurs when elevated levels of MetHb (exceed about 10%) interfere with the oxygen-carrying capacity of the blood. Infants are particularly susceptible to developing Methehaemoglobemia for several reasons, including their increased capacity to convert nitrate to nitrite and their lower levels of the enzyme cytochrome b5.

Nitrate is a precursor in the formation of N-nitroso compounds (NOC), which are potential animal carcinogens that induce tumours at multiple organ sites including the esophagus, stomach, colon, bladder, lymphatic, and haematopoietic system (Lijinsky W, J Environ Sci Health. 1986; C4: 1-45).

Animal studies also suggest that high doses of nitrate can competitively inhibit iodine uptake and induce hypertrophic changes in the thyroid (Bloomfield RA, Welsch CW, Garner GB, Muhrer ME, Science, 1961; 134: 1690). In a human biomonitoring study in the Netherlands, consumption of water with nitrate levels at or above the MCL was associated with thyroid hypertrophy (van Maanen JM, van Dijk A, Mulder K, de Baets MH, Menheere PC, van der Heide D, et al, Toxicol Lett. 1994; 72: 365-374) and genotoxic effects (van Maanen JM, Welle IJ, Hageman G, Dallinga GW, Mertens PL, Kleinjans JC, Environ Health Perspect. 1996; 104: 522-528). Further animal studies suggest that NOC can damage pancreatic beta cells (Longnecker MP, Daniels JL, Environ Health Perspect. 2001; 109: 871-876).

Three epidemiologic studies (Kostraba JN, Gay EC, Rewers M, Hamman RF, Diabetes Care. 1992; 15: 1505-1508; Parslow RC, McKinney PA, Law GR, Staines A, Williams

R, Bodansky HJ, Diabetologia, 1997; 40: 550-556; van Maanen JMS, Albering HJ, de

Kok TMCM, van Breda SGJ, Curfs DMJ, Vermeer ITM, et al, Environ Health Perspect. 2000; 108:457-461) that were ecologic in design found a positive correlation between drinking-water nitrate levels below MCL and the incidence of type I childhood diabetes, although the association observed by Maanen was not statistically significant.

Other studies have found associations between water nitrate exposure and increased blood pressure (Pomeranz A, Korzets Z, Vanunu D, Krystal H, Kidney Blood Press Res. 2000; 23: 400-403) and acute respiratory tract infections in children (Gupta SK, Gupta RC, Gupta AB, Seth AK, Bassin JK, Gupta A, Environ Health Perspect. 2000;108:363-366).

Accordingly, there is a need to provide bioactive materials in which the concentration of nitrate species is reduced to an acceptable level.

The present invention thus seeks to alleviate the toxicity problems associated with prior art materials by providing a bioactive glass having a reduced concentration of nitrate species. The resulting material can be incorporated into degradable and non-degradable polymers to form composites for manipulation into constructs suitable for orthopedic and tissue engineering applications.

STATEMENT OF INVENTION

A first aspect of the invention relates to a sol-gel derived bioactive glass comprising less than about 30 ppm of nitrate species.

Advantageously, the presently claimed sol-gel derived bioactive glasses have a lower concentration of residual nitrate species than those produced by existing methods known in the art. Specifically, the sol-gel powders of the invention are useful as tissue engineering scaffolds, for example, for growing cells, such as osteoblasts to form a bone implant in vitro for ultimate implantation into humans or other animals, or for bone grafts. Additionally powders can be mixed with degradable and non-degradable polymers to form composites for bone fillers, fixations and grafts. A second aspect relates to a process for preparing a sol-gel derived bioactive glass, said process comprising the steps of:

(i) forming a mixture comprising a gelable inorganic base material and at least one calcium component; (ii) subjecting said mixture to a temperature of from about 50 to about 70°C for about 2 to about 36 hours; (iii) drying the material formed in step (ii) by subjecting said material to a temperature of about 80 to about 180⁰C for about 1 to about 72 hours; (iv) stabilising the material formed in step (iii) by subjecting said material to a temperature of about 700 to about 800°C for about 3 to about 24 hours.

A third aspect of the invention relates to sol-gel derived bioactive glass obtainable by a process according to the invention.

Advantageously, the process conditions described herein allow bioactive sol-gel powders to be produced in a more energy efficient manner. The processing stages have been optimised to give reduced ageing, drying and stabilisation for the production of non-toxic bioactive sol-gel powders suitable for cell growth and other tissue engineering applications. This combination of process steps also gives rise to a bioactive sol-gel product containing surprisingly low levels of nitrates.

A fourth aspect relates to a composite comprising a sol-gel derived bioactive glass according to the invention and a biocompatible, biodegradable material.

A fifth aspect relates to the use of a sol-gel derived bioactive glass or composite according to the invention in the preparation of a medicament for treating wounds and/or burns.

A sixth aspect relates to the use of a sol-gel derived bioactive glass or composite according to the invention in the preparation of a medicament for grafting skin. A seventh aspect relates to a method for treating wounds and/or burns, said method comprising contacting a wound with a therapeutically effective amount of a sol-gel derived bioactive glass or composite according to the invention.

An eighth aspect relates to a method for grafting skin, said method comprising applying a sol-gel derived bioactive glass or composite according to the invention to a graft site, donor tissue, or both.

A ninth aspect relates to a wound or burn dressing comprising a bandage comprising a sol-gel derived bioactive glass or composite according to the invention.

A tenth aspect relates to the use of a sol-gel derived bioactive glass or composite according to the invention in tissue engineering.

An eleventh aspect relates to the use of a sol-gel derived bioactive glass or composite according to the invention in orthopedic applications.

DETAILED DESCRIPTION

Sol-gel derived bioactive glass

As mentioned above, a first aspect of the invention relates to a sol-gel derived bioactive glass comprising less than about 30 ppm of nitrate species.

In one preferred embodiment, the sol-gel derived bioactive glass comprises: SiO₂ 40-90%;

CaO 6-50%;

P₂O₅ 0-12%.

Advantageously, the presently claimed sol-gel derived bioactive glasses have a lower concentration of residual nitrate species than those produced by existing methods known in the art. Preferably, the sol-gel derived bioactive glass contains less than about 25 ppm of nitrate species, more preferably, less than about 20 ppm of nitrate species, more preferably still, less than about 15 ppm of nitrate species, even more preferably, less than about 10 ppm of nitrate species. Even more preferably, the sol-gel derived bioactive glass contains less than about 5 ppm of nitrate species.

In one preferred embodiment, the sol-gel derived bioactive glass has the following composition:

SiO₂ 45-86

CaO 6-50

P₂O₅ 0-12

Ag₂O 0-12

Al₂O₃ 0-3

CaF₂ 0-25

B₂O₃ 0-20

K₂O 0-8

MgO 0-5

Na₂O 0-20.

In one preferred embodiment, the sol-gel derived bioactive glass further comprises from about 0.1 to about 12 % Ag₂O.

SiO₂ 45-86

CaO 10-36

P₂O₅ 3-12

Ag₂O 3-12

CaF₂ 0-25

B₂O₃ 0-10

K₂O 0-8

MgO 0-5 Na₂O 0-20.

In one preferred embodiment, the sol-gel derived bioactive glass comprises about 55 to about 80 % by weight of silicon dioxide (SiO₂), from 0 to about 9 % by weight of sodium oxide (Na₂O), about 10 to about 36 % by weight calcium oxide (CaO), and about 0 to about 8 % by weight phosphorus oxide (P₂O₅).

In one preferred embodiment, the sol-gel derived bioactive glass contains about 60 % SiO₂, about 36 % CaO and about 4 % P₂O₅ by weight.

In one preferred embodiment, the sol-gel derived bioactive glass contains about 58 % SiO₂, about 33 % CaO and about 9 % P₂O₅ by weight.

In one preferred embodiment, the sol-gel derived bioactive glass contains about 70 % SiO₂ and about 30 % CaO.

In one preferred embodiment, the sol-gel derived bioactive glass contains about 70 % SiO₂, about 29 % CaO and about 1 % Ag₂O.

In one preferred embodiment, the sol-gel derived bioactive glass is in the form of a powder.

In one preferred embodiment, the sol-gel derived bioactive glass is in particulate form.

In a more preferred embodiment, the particles have a pore size of from about 20 to about 400 Angstroms.

In one preferred embodiment, the particles have a surface area in the range of from about 20 to about 400 m²/g.

In one preferred embodiment, the sol-gel derived bioactive glass has porosity from about 10 to about 80 percent. Process

As described above, one aspect of the invention relates to a process for preparing a sol- gel derived bioactive glass, said process comprising the steps of: (i) forming a mixture comprising a gelable inorganic base material and at least one calcium component;

(ii) subjecting said mixture to a temperature of from about 50 to about 70⁰C for about 2 to about 36 hours; (iii) drying the material formed in step (ii) by subjecting said material to a temperature of about 80 to about 180°C for about 1 to about 72 hours; (iv) stabilising the material formed in step (iii) by subjecting said material to a temperature of about 700 to about 800⁰C for about 3 to about 24 hours.

Processing conditions for previously described sol-gel glasses involve extensive gelation, ageing and drying conditions. Standard gelation is achieved through maintaining the temperature of the sol at 25°C for 72 hours, followed by further ageing at 25°C for another 72 hours. However, these processing conditions are inappropriate for the large scale production of sol-gel powders as the material is ground into a particulate form and does not require the same tensile strength as monolithic and foamed scaffolds. Additionally, standard drying conditions use slow ramp rates and ambient temperature to prevent cracking of the gel monoliths and foams. For the production of powders in the current invention, thermal shock of gels is induced to make later processing and micronisation more efficient. The current invention thus provides optimised gelation, aging and drying conditions for the preparation of sol-gel powders. As a result, complete processing from mixing to drying can be reduced to 96 hours which is more efficient than previously reported processes for bioactive sol-gel glasses.

Moreover, previously reported methods for the preparation of sol-gel derived glasses have inaccurately described the stabilisation of calcium containing sol-gel glasses. Such glasses can be in the form of particles, fibres and or coatings for implantable devices. Sintering of sol-gel glasses causes the irreversible removal of hydroxyl groups from the surface of the glass and also serves to degrade other species present in the gel, such as nitrates. Previous reports suggest that processing at 561 °C decomposes calcium nitrate, thereby converting all nitrates to their oxide form (CaO). However, contrary to this, studies by the present applicant have shown that these conditions are in fact not sufficient to decompose all the nitrates present. The present invention seeks to alleviate this problem by providing a process for producing purified sol-gel glasses with reduced concentrations, and in particular non-toxic levels, of nitrates.

In one preferred embodiment of the invention, step (ii) of the process comprises: (ii)(a) a gelation phase which comprises subjecting said mixture to a temperature of from about 50 to about 70°C for about 1 to about 12 hours;

(ii)(b) an ageing phase which comprises subjecting said mixture to a temperature of from about 50 to about 70°C for about 1 to about 24 hours;

The sol-gel process is a chemical synthesis technique for the preparation of glasses and ceramics. The chemistry involved in the process is based on inorganic polymerisation reactions of metal alkoxide precursors M(OR)_n, where M represents the network forming element such as Si and R is an alkyl group C_xH_2x+I. These precursors are made to undergo hydrolysis and condensation reactions, usually when dissolved in a solvent, to form soluble metal hydroxides, which ultimately leads to the formation of a continuous metal-oxygen-metal link as in an inorganic network that spans throughout the solvent medium. The particles prior to formation of a continuous phase are known as the 'sol'.

With time, a phase separation occurs when the networked inorganic polymer separates from the solvent to form a weakly bonded colloidal solid. The time at which this occurs is known as the 'gel-point' and the solid is known as the 'gel'. The gel consists of interpenetrating solid and liquid phases: the liquid prevents the solid network from collapsing and the solid network prevents the liquid from escaping. Gelation is a very important stage in the process as the gel-structure formed during this stage is maintained throughout the processing. The point at which gelation occurs can be reduced with an increase in temperature and acid/base concentration. The gel is then preserved for a period known as the 'ageing', where the cross linking and network formation continues and strengthens to give what is known as the 'wet gel'. During the ageing, structural changes occur in order to reduce the free energy via processes known as ripening and syneresis. Ripening is a reconstructive process that reduces the surface area without shrinkage while syneresis is the contraction of the network within the liquid, driven by both a reduction in the solid-liquid interfacial area and continued condensation.

The penultimate stage in the process is known as 'drying' where the wet gel is dried either by evaporation of the solvent to form a 'xerogeF or by forced solvent removal such as freeze-drying, to give an 'aerogel'. During this stage the gel network undergoes considerable shrinkage and weight loss. Cracking occurs due to stresses set up within the gel. These arise from the unequal evaporation rates of the solvent trapped within the pores compared to the evaporation rate from the surface of the gel.

The final stage is the consolidation of the xerogel into dense glasses or ceramics and is known as 'stabilisation'. It is necessary to stabilise xerogels as they have a very large concentration of silanols on the surface of the pores, which renders them unusable at room temperature. Thermal stabilisation involves reducing the surface area sufficiently to enable the material to be used at a given temperature without reversible structural changes.

The main features of the multi-component sol-gel process are that a homogeneous solution is formed before the polymerisation reactions and that the overall processing takes place at lower temperatures than in conventional mixed powder methods. Thus, not only can improved chemical homogeneity be achieved with good control over the final composition and tailoring of the surface characteristics of the product but also phase separation, crystallisation and chemical decomposition can be avoided. The inherent advantage is the elimination of heterogeneities, which limit mechanical properties and are often the source of selective interfacial attack under combined mechanical/chemical environmental stresses. In one preferred embodiment of the invention, the gelable inorganic base material comprises at least one alkoxysilane. More preferably, the alkoxysilane is tetraethoxysilane, tetramethoxysilane (TMOS) or tetrabutoxysilane (TBOS).

In one preferred embodiment of the invention, the mixture formed in step (i) further comprises triethoxyphosphate.

In one preferred embodiment, the calcium component is calcium nitrate.

In another preferred embodiment, the mixture formed in step (i) further comprises at least one silver salt. Preferably, the silver salt is silver nitrate.

In one preferred embodiment, the mixture formed in step (i) further comprises deionised water.

In another preferred embodiment, the mixture formed in step (i) further comprises at least one therapeutic agent. Preferably, said therapeutic agent is selected from a healing promotion agent, a growth factor, an anti-inflammatory agent and a topical anaesthetic.

In one especially preferred embodiment, the therapeutic agent is a topical antibiotic.

More preferably, the topical antibiotic is selected from the group consisting of chloramphenicol, chlortetracycline, clyndamycin, clioquinol, erythromycin, framycetin, gramicidin, fusidic acid, gentamicin, mafenide, murpiroicin, poly-myxin B, bactitracin, silver sulfadiazine, tetracycline, tetracycline, chloroetracycline, and combinations thereof.

Gelation The hydrolysis and condensation reactions discussed in the above sections lead to the growth of clusters that eventually collide and link together into long chains. With time all the chains are linked and the solution looses its fluidity. This point is known as the gel point and the time taken to reach this point is referred to as time of gelation (t_gei).

The chemical reactions that bring about the gelation (i.e. condensation) continue long beyond the gel point permitting gradual changes in the structure and properties of the gel, i.e. the stiffness of the gel increases with time.

The determination of the gel point is an important aspect of the chemistry of sol-gel processing. As expected no latent heat is evolved at the gel-point but the viscosity rises abruptly. This sudden change in the rheological behaviour is generally used to identify tgel.

Generally, the factors that affect the condensation rate also affect t_geι. When the condensation rate is increased, t_geι is decreased. For gels made from silicon alkoxides, time of gelation is much faster in the presence of base catalysts as well as strong acids such as HF. An increase in the R ratio, temperature and the concentration of alkoxide and a decrease in the size of the alkoxide group all decrease the time of gelation.

During the gelation the silicon bonds to form the crosslinked network (semi-solid phase) can be permanent and reversible. Even covalent siloxane bonds in silica gel can be cleaved under certain conditions, allowing the gels to exhibit slow irreversible physical deformation. The gel point represents the moment when a fully networked structure is formed, which can be assessed by monitoring the viscoelasticity change. At that point, there is a cross-over between elasticity modules and viscosity modules. The gelation process is consistently reversible if the sol concentration is not altered by further evaporation-poly-condensation. To date, prior art gelation techniques of sol-gel glasses use ambient temperatures (25°C) with acidic medium (pH 2-4) over a period of 72 hours.

In contrast, the gelation phase (step (ϋ)(a)) of the presently claimed process comprises subjecting the mixture to elevated temperatures between 50-70°C for between 1-12 hours to reduce processing time. Preferably, step (ii)(a) comprises subjecting said mixture to a temperature of from about 55°C to about 65⁰C for about 1 to about 12 hours.

More preferably, step (ii)(a) comprises subjecting said mixture to a temperature of from about 55°C to about 65°C for about 1 to about 3 hours.

In one highly preferred embodiment, step (ii)(a) comprises subjecting said mixture to a temperature of about 60°C for about 12 hours.

Preferably, step (ii)(a) of the process is carried out at a pH of from about 0.2 to about 2.

Ageing

When a sol reaches gel point, all the liquid (i.e. the water and alcohol produced during hydrolysis and condensation) in the surrounding system is trapped in the gel network. This liquid is known as the pore liquor. When a gel is maintained in its pore liquor, its structure and properties continue to change long after the gel-point. This process is known as ageing. Three processes can occur, singly or simultaneously, during ageing of the silica gels: polycondensation, syneresis and coarsening.

It is described as being formed by connectivity of the gel network via condensation reactions. These condensation reactions create the strength of the material and are a crucial part of the process for making specific monolithic materials. For our process to make non-monolithic material and presented as a powder form for the final product, the strength of the material is not critical and only the physical parameters such as pore sizes, the density and the surface area, which have impact on the dissolution profile.

The requirement for strengthening sol-gel glasses is focused on maintaining monolithic and foamed structures which are not required for sol-gel powders. To date, prior art ageing conditions use temperatures between 25-60°C for durations of from 24 to 72 hours. The current invention uses elevated temperatures between 50-70°C for between 1 -24 hours to reduce the processing time for ageing. In one preferred embodiment, the ageing phase (step (ii)(b) of the process) comprises subjecting the mixture to a temperature of from about 55 to about 65°C for about 1 to about 24 hours, more preferably for about 1 to about 12 hours.

In one preferred embodiment, the ageing phase (step (ii)(b) of the process) comprises subjecting the mixture to a temperature of from about 50 to about 70°C for about 20 to about 24 hours.

In one preferred embodiment, the ageing phase (step (ii)(b) of the process) comprises subjecting the mixture to a temperature of from about 55 to about 65°C for about 20 to about 24 hours.

In one highly preferred embodiment, step (ii)(b) comprises subjecting the mixture to a temperature of about 60°C for about 12 hours.

Drying

When monolithic gels are desired, drying is the most critical and difficult step in the production. This is due to the capillary stresses induced in the emptying pore network that result in cracking of the gels unless the drying conditions and rate of evaporation of pore liquor is carefully controlled.

Investigators who have studied the drying process in great detail have divided it into three stages. Scherer identifies them as: constant rate stage, first falling rate period and second falling rate period. These three stages are illustrated in by the data obtained while drying an alumina gel monolith.

In the first stage, the gel network shrinks as the liquid evaporates from the surface. The forces causing the shrinkage of the gel depend on the surface tension exerted by the liquid vapour (γ_LV ). A corresponding compressive force on the gel network balances this tension and the network contracts to prevent the exposure of the solid phase. The liquid-vapour interface remains at the surface of the gel and the rate of evaporation remains constant as the gel shrinks in response to the fluid loss. As the gel shrinks, the compressive forces build up until the body becomes too stiff to prevent further shrinkage. This point is known as the critical point and is the end of the first stage. Cracking is most likely to occur at this critical point as the pores begin to empty and maximum force is exerted on the network.

After the critical point, further evaporation drives the meniscus into the body. The rate of evaporation decreases, as the liquid must find its way from the interior to the surface of the gel. There are two possible mechanisms by which liquid transport occurs: (a) diffusion of vapour from the remaining filled pores through the empty network to the surface and (b) fluid flow along the funicular surface. During this first falling rate period it is generally conceded that the latter mechanism is responsible. With further evaporation (i.e. during the second falling rate period) the vapour diffusion becomes responsible.

Drying can be achieved in two different ways. When the wet gels are dried by slow evaporation of the pore liquor in an oven in ambient pressure, they are called 'xerogels'. Under these conditions of drying significant reduction in surface area, pore size and pore volume occurs.

An alternative way of drying is supercritical drying. This involves the exchange of pore liquor with a suitable liquid and removal under hypercritical conditions (usually high temperatures and pressures depending on the exchange liquid used) where the surface tension (γ_LV ) is minimised or eliminated. These gels are called 'aerogels'. Because of the absence of the surface tension the texture of the gels remains intact.

These two methods are based on maintaining equal surface tension during the shrinkage of gels during the drying process. They produce crack free monolithic and foamed structures ready for stabilisation. The current invention provides conditions for the production of powders by inducing cracking into the gels by removing the pore liquor before drying and using high ramp rates of heating. This combination provides an efficient drying process for the production of sol-gel powders. The current invention typically involves removing (decanting) the pore liquor, followed by drying at temperatures between 80-180°C, preferably at ramp rates of l-10°C/min for a period of 1-72 hours.

In one preferred embodiment of the invention, the drying phase (step (Ui)) comprises increasing the temperature at a ramp rate of from about 1 to about 10 °C/minute.

In one preferred embodiment of the invention, step (iii) comprises subjecting said material to a temperature of about 80 to about 180°C for about 4 to about 72 hours.

In one preferred embodiment of the invention, step (iii) comprises subjecting said material to a temperature of about 80 to about 180°C for about 36 to about 72 hours.

In one preferred embodiment of the invention, step (iii) comprises subjecting said material to a temperature of about 90 to about 130°C for about 4 to about 72 hours.

In one preferred embodiment of the invention, step (iii) comprises subjecting said material to a temperature of about 90 to about 130°C for about 36 to about 72 hours.

In one particularly preferred embodiment of the invention, step (iii) comprises subjecting said material to a temperature of about 90 ⁰C for about 24 hours.

In another particularly preferred embodiment of the invention, step (iii) comprises subjecting said material to a temperature of about 130 °C for about 36 hours.

In one particularly preferred embodiment of the invention, step (iii) comprises subjecting said material to a temperature of about 90 °C for about 24 hours, followed by a temperature of about 130 ⁰C for about 36 hours.

Stabilisation Stabilisation of a porous gel involves reducing the concentration of silanol groups on the surface to the point where rehydroxylation is inhibited. The purpose is to prevent the porous body from re-adsorbing water and other contaminants. Stabilisation is usually accomplished through thermal or chemical treatments that remove surface silanols and create less reactive surfaces.

Chemical stabilisation of the silica surface is achieved by replacing silanol groups with more hydrophobic species. For example, fluoride catalysed gels have a substantial number of silanols substituted by fluoride and are less hydrophilic making them more stable to atmospheric moisture.

Thermal stabilisation on the other hand consists of heating the silica gel to continue the removal of surface silanol groups. It is known that heat treatments below 500°C result in surfaces that are easily rehydroxylated. Heating above 500⁰C results in an irreversible dehydration of the glass surface due to an increase in the elimination of isolated silanol groups leading to structural relaxation.

In multi-component systems the stabilisation process also serves to degrade other undesirable species that are present in the gel (i.e. nitrates and organics). The decomposition of pure Ca(NOa)₂ occurs at 561° C (Klein LC, Garvey GJ. In: Hench LL, Ulrich DR, Eds. infrastructure Processing of Ceramics, Glasses and Composites. John Wiley and Sons, USA 1984: 88-99). Hence, this temperature must be exceeded during successful stabilisation of binary CaO-SiO₂ gels. Previous reports state that a stabilisation temperature of 561°C must be exceeded, therefore it has become widely accepted that temperatures of 600°C can adequately decompose calcium nitrate to its oxide form (CaO). However, after testing total nitrate concentrations of S70C30 glass stabilised at 600⁰C for an extended duration, it was confirmed that high concentrations of nitrates (>500ppm) still remained within these glasses (Figure 1). Such were the concentration of nitrates present in the glasses that they have been proven to be toxic to humans in concentrations greater than 20ppm in drinking water (Safe Drinking Water Act 1974, Environmental protection agency (EPA), Maximum containment level goals (MCLG) for nitrates in drinking water) and toxic to the proliferation of primary human osteoblasts and embryonic stem cells (Figure 2). Sol-gel S70C30 glasses sintered at 600°C were preconditioned in CDM medium for 3 days prior to seeding. Embryonic stem cells were seeded onto specimens and monitored using SEM. Crystallites were seen to be precipitating on the surface of the glass and were found in some areas of the scaffold after 3 days, and after 6 days, this was much wider spread (Figure 3). This was determined to be calcium and was a result of the rapid dissolution of calcium ions from the glass. Since there was no movement of the surrounding medium, these ions re- precipitated on the surface. It is highly possible that these crystals affect subsequent growth and proliferation of embryonic stem cells on these substrates and contribute to the toxic nature of these substrates in long term culture. In the current invention, processing conditions ensure the concentration of nitrates is preferably reduced to a non-toxic level (e.g. preferably <30 ppm), thereby rendering the material suitable for implantable materials and allowing cell proliferation to take place. The process of the present invention typically uses stabilisation temperatures of 700-800°C for between 3- 24 hours.

Heating at temperatures between 800° C and 1100° C (depending on the initial porosity, interconnectivity, atmosphere and composition) will density the gel to become a consolidated glass with a density equivalent to that of glasses made by conventional melting and casting.

In one preferred embodiment, the stabilisation phase (step (iv)) comprises subjecting the material formed in step (iii) to a temperature of about 700°C to about 750°C for about 3 to about 24 hours.

In one preferred embodiment, step (iv) comprises subjecting the material formed in step (iii) to a temperature of about 700°C to about 800 °C for about 10 to about 25 hours.

More preferably, step (iv) comprises subjecting the material formed in step (iii) to a temperature of about 700°C to about 750 °C for about 10 to about 25 hours.

In one particularly preferred embodiment, step (iv) comprises subjecting the material formed in step (iii) to a temperature of about 700°C for about 24 to about 72 hours. More preferably, step (iv) comprises subjecting the material formed in step (iii) to a temperature of about 700⁰C for about 24, 48 or 72 hours.

In another particularly preferred embodiment, step (iv) comprises subjecting the material formed in step (iii) to a temperature of about 800⁰C for about 3 to about 24 hours. More preferably, step (iv) comprises subjecting the material formed in step (iii) to a temperature of about 700°C for about 6 or about 10 hours.

A further aspect of the invention relates to a sol-gel derived bioactive glass obtainable by a process according to the invention.

Applications of sol-gel derived bioactive glass

The non-toxic bioactive sol gel powders of the present invention have numerous applications in the field of tissue engineering and other therapeutic areas such as wound healing. Specifically, the sol-gel powders of the invention may be useful as tissue engineering scaffolds, for example, for growing cells, such as osteoblasts to form a bone implant in vitro for ultimate implantation into humans or other animals, or for bone grafts. Additionally powders can be mixed with degradable and non-degradable polymers to form composites for bone fillers, fixations and grafts.

One aspect of the invention thus relates to a sol-gel derived bioactive glass as described above for use in medicine.

Another aspect of the invention relates to the use of a sol-gel derived bioactive glass as described above in the preparation of a medicament for treating wounds and/or burns.

Yet another aspect of the invention relates to the use of a sol-gel derived bioactive glass as described above in the preparation of a medicament for grafting skin.

A further aspect of the invention relates to a method for treating wounds and/or burns, said method comprising contacting a wound with a therapeutically effective amount of a sol-gel derived bioactive glass as described above. A further aspect of the invention relates to a method for grafting skin, said method comprising applying a sol-gel derived bioactive glass as described above to a graft site, donor tissue, or both. More preferably, the method further comprises applying a topical antibiotic to the graft site, the donor tissue, or both.

Another aspect of the invention relates to a wound or burn dressing comprising a bandage comprising a sol-gel derived bioactive glass as described above. Preferably, the wound or burn dressing further comprises a topical antibiotic as described above.

Yet another aspect of the invention relates to a composite comprising a sol-gel derived bioactive glass as described above and a biocompatible, biodegradable material. As used herein, the term "biocompatible" means that the material does not elicit a substantial detrimental response in the host.

Preferably, the biocompatible, biodegradable material is a degradable polymer, more preferably a thermoplastic polymer.

The therapeutic applications described above apply mutatis mutandis to the composites of the invention.

Preferably, the thermoplastic polymer is plasticizable within a temperature range of - 20°C to +80°C and may be to a varying degree biodegradable or even bioactive. As used herein, the term "biodegradable" covers all plastics which are not inert and includes all bioresorbable plastics (degrading under the action of cells) and biodegradable plastics (degrading under the effect of moisture). The intended use of the composite will determine the choice of plastic. In one preferred embodiment, the thermoplastic polymer is biodegradable. Preferably, the thermoplastic polymer is biologically nearly stable, thereby promoting tissue contact with the bioactive component. In this particular embodiment, the plastic of the composite keeps the particles of the bioactive component in place but does not necessarily prevent the bioactive material from coming into contact with tissue fluid. Since the plastic component gradually decomposes, the water of the tissue fluid comes into contact with the bioactive component through diffusion. Likewise, ions and any active additives released from the bioactive material can diffuse through the plastic and affect their surroundings. The surrounding and/or contact surface tissue grows, filling the void formed by the degradation of the plastic. Ultimately, the plastic component completely decomposes and releases all remaining bioactive component.

Alternatively, the plastic component may be nearly inert. If such a composite breaks, it can repair itself in a physiological environment under the mineralizing affect of the bioactive component.

The plasticization temperatures of the plastics used in the composite of the invention are again determined on the basis of the intended use of the composite.

In one preferred embodiment, the plastics have a plasticization temperature in the vicinity of body temperature. The application of a product in a plastic state will therefore not cause any thermal damage to the tissue. Moreover, other additives that may be admixed with the composite remain undamaged during preparation and application of the product. If the product implanted in a tissue is required to be soft, it is possible to select a plastic component having a plasticization temperature lower than body temperature. Such a product can be applied in a hardened form, and will soften thereafter in the tissue.

An especially suitable plastic is a copolymer which is based on structural units such as a hydroxy acid; a hydroxy acid derivative such as a cyclic ester of a hydroxy acid, i.e. lactone; or a cyclic carbonate, such as trimethyl carbonate. L-, D- and DL-lactic acids;

L-, D- and DL-lactides; and epsilon-caprolactone are highly suitable structural units.

A plastic component which is a copolymer based on L-lactide and epsilon-caprolactone structural units is especially suitable for this use. The composition of the copolymer typically varies within the range 30-90% and the molar mass M of the copolymer is within the range 10,000 to 1,000,000 g/mol, suitably within the range 30,000 to 300,000 g/mol.

In one highly preferred embodiment, the thermoplastic polymer is poly-(ε- caprolactone) (PCL) or L-lactide/glycolide copolymer.

The control of the melting temperature of the plastic component, i.e. the polymer material, intended for the composite according to the invention is based on one hand on the selection of the monomer ratio in the initial substances and on the other hand on the control of the molar mass in the copolymerization. Both of these factors together affect the melting temperature of the copolymer obtained, and thus only certain combinations produce the desired result.

In many applications, it is desired that the implant material degrades in a controlled manner, or conversely, has mechanical properties that remain stable for at least a certain period. The first stage in the biodegradability of the polymers of the present type is hydrolysis which cuts down the polymer chains until the molecule size is at a level at which the enzymatic functions of the body are capable of converting the degradation products into compounds natural for the body.

In terms of the degradation rate, the hydrophilicity of the polymer is crucial. Thus, in the copolymers being discussed it is possible to control their hydrolytic degradation rate by controlling the monomer composition, and thereby also hydrophilicity, and this, in accordance with what has been stated above, directly affects the degradation of the material in the body. In terms of the invention, it is important that, if the composition of the material is solely or almost solely bioactive glass, the balance being, for example, L-lactide, DL-lactide, D-lactide or dimethyl carbonate, the polymer is almost stable in the body, or degrades very slowly, typically in the course of a number of years.

By the selection of the monomer composition and the simultaneous control of the average molar mass of the copolymer by means of the preparation parameters, it is possible to exploit the previously known good biocompatibility of polyhydroxy acids. On the other hand, a waxy version of the copolymer material according to the invention can be rendered very rapidly degradable by controlling of the average molar mass and the monomer composition, as presented above. In this case the degradation period in the body is typically from a few days to a few weeks.

In another preferred embodiment, the biocompatible composite material consists of an elastomeric material and the herein described bioactive glass particles. The bio-active ceramic or glass particles can be dispersed through the matrix of the elastomeric material which has the predetermined shape. Alternatively, the elastomeric material having the predetermined shape may have the bio-active glass particles coated on its surface by any known method, such as spraying using a compressed gas propellant.

The elastomeric material is preferably a silicone elastomer. However, other materials such as polyurethane and its derivatives, hydrogels such as polyvinyl pyrrolidone and its derivatives and polyhema, C-Flex.RTM., etc., may be used. The elastomeric material may be in the form of an open- or closed-cell foam.

The proportion of bio-active glass particles and elastomeric material used in the prosthetic device of the present invention varies depending upon the intended end use. In general, however, with respect to the prosthetic device of the present invention wherein the bio-active glass particles are dispersed throughout a matrix of elastomeric material, the bioactive glass particles are preferably contained in the matrix in an amount of 10 to 70 weight % based on the total amount of bio-active glass particles and elastomeric material. Above 70 weight %, the strength of the composite decreases. When the bio-active glass particles are coated on the surface of an elastomeric material having a predetermined shape, the amount of bioactive glass particles is such that nearly the entire exposed surface is coated with bioactive glass so as to increase tissue adhesion. However, if desired, exposure may be reduced to decrease tissue adhesion. The elastomeric material can be substantially coated with larger sized particles and finer particles may be used to fill spaces between the larger particles.

The prothesis of the present invention can also comprise a base material of predetermined shape, e.g., a conventional prosthetic device, and a layer of elastomeric material provided on the base material, wherein the layer of elastomeric material has distributed therein or provided thereon bio-active glass particles.

In one preferred embodiment of the invention, the composite is in the form of a coating or powder.

Another aspect of the invention relate to the use of a sol-gel derived bioactive glass or composite as described above in tissue engineering.

In one preferred embodiment, the sol-gel derived bioactive glass or composite is incorporated into a bone graft substitute.

In another preferred embodiment, the sol-gel derived bioactive glass or composite is incorporated into an implantable device. Preferably, the implantable device is selected from the group consisting of prosthetic implants, sheets, pins, valves, screws, plates, and tubes.

Yet another aspect of the invention relate to the use of a sol-gel derived bioactive glass or composite as described above for orthopedic applications.

The present invention is further described by way of non-limiting example, and with reference to the accompanying figures, wherein: Figure 1 shows the total nitrate concentration of 70S:30C sol-gel glass after 600°C stabilisation for extended periods of time.

Figure 2 shows a SEM image of S70C30 (600°C) glass leading to death of all cells which were attached to the surface.

Figure 3 shows a SEM image of S70C30 (600°C) glass containing calcium crystallites inhibiting cell proliferation.

EXAMPLES

Preparation of low nitrate containing sol- gel derived bioactive glasses Bioactive sol-gel glasses with varying composition have been prepared by a modified sol-gel process to reduce nitrate levels to an acceptable level. The textural characteristics of the materials (surface area, pore volume, and average pore diameter) were measured by gas-sorption. The biocompatibility of the resulting materials was compared to high nitrate containing materials using standard ISO 10993-5 cytotoxicity testing.

Materials preparation

A bioactive gel-glass of the two components SiO₂-CaO (namely "70S30C") was prepared in which 70% (molar) of silica was mixed with 30% (molar) of calcium oxide. In the case of the silver containing sol-gel glass system (namely "70SlAg"), 1% (molar) CaO was replaced by 1% Ag₂O. The mix compositions of materials, as well as the non-silver containing 58S are listed in Table 1. The 70SlAg specimen was always handled in the dark, using safe light, and stored in a black-box to preserve it in its oxidised state. Table 1: Compositions of bioactive glasses in mol % (in weight %)

Hydrolysis and copolymerisation

In a PMP container calcium nitrate tetrahydrate were dissolved in water for injection, and the pH adjusted to -0.6 through the addition of 2N nitric acid. The mixture was stirred using an overhead mixer at a speed of 450 rpm. Tetraethyloxysilane (TEOS) was added whilst monitoring temperature to ensure complete hydrolysis of the alkoxysilane. Silver containing sol-gel glasses were prepared by adding silver nitrate to the reaction solution and stirring for thirty minutes. Sol-gel glasses containing phosphates were prepared by adding TEP and stirring for an additional thirty minutes. After of the completion of the stirring, the mixture was transferred into a perfluoroalkoxyethylene (PFA) screw lid jar and sealed tightly.

The foregoing compositions were prepared from reaction mixtures prepared as follows:

Table 2: Sample 70S30C:

Table 3: Sample 70SlAg:

X = Any factor to increase batch size.

Comparison of regular sol-gel processing with nitrate free processing schedules:

Gelation and ageing The moulds containing the reaction mixture were placed in a programmable oven for gelation and aging. The oven is set to an optimised temperature of 60°C for a period of 24 hours. Gelation at this accelerated temperature occurs within 2 hours and the remainder of the programme constitutes the ageing of the gel.

Drying

The PFA jar is then opened to release the water and ethanol vapours and the material is dried using temperatures of 9O⁰C for 24 hours followed by 130°C for 36 hours. The high ramp rates (5°C/min) induce thermal shock and the resulting material is in the form of small fragments ready for nitrate removal. Stabilisation

The stabilisation was carried out in a programmable muffle furnace at temperatures and dwell times shown in Table 5. All temperature monitoring is carried out using calibrated thermocouples to ensure the material reach the set stabilisation temperatures for the required time.

Table 5: Process conditions used for the production of multiple composition, nitrate free sol-gel glasses

Without being bound to any particular theory or mechanism we believe that the sol-gel glasses produced using the process described in the current invention exhibit enhanced purity. Textural characteristics are displayed in Table 6, showing the affect that variations in stabilisation temperatures and duration have on the overall surface area and pore size distribution. Table 6:

The resulting sol-gel glasses were micronized using a PMlOO ball mill and zirconium oxide grinding media under conditions of 600 rpm for 20 minutes. The resulting powders were sieved using a 50μm sieve and analysed for nitrate concentration particle size distribution.

Determination of inorganic anions by anion exchange chromatography

Each of the sol-gel bioactive glasses fabricated using the optimised conditions stated above were testing for total nitrate content using the following conditions. Samples of bioactive glass powder were weighed accurately (1Og) and saturated in 100ml of deionised water. The mixture was then centrifuged for 12 hours to ensure all soluble nitrates had been dissolved. The sample was then diluted such that the ion content falls within a predetermined calibration range. The sol-gel glass was then filtered through a 1.2 μm and/or 0.2 μm ministart filter collecting the final filtrate in a chromatography vial (Stock solution). Each sample was accompanied by a blank (deionised water) and a QC sample (certified anion standard solution, 5.12 to 5.15 at 30mg/litre).

Stock, blank and QC samples (1.0, 2.0, 3.0, 4.0 and 5.0 ml) were pipetted into separate 100ml volumetric flasks and dilute to volume with deionised water. The standard solutions contained 10.0, 20.0, 30.0, 40.0 and 50.0 mg/litre of each of the inorganic ions. From each solution a 3.0ml solution was transferred into a 100ml volumetric flask and diluted to volume with deionised water. Using a Pasteur pipette each standard solution was transferred into a chromatography vial ready for analysis.

The following instrumental settings were used for anion analysis: Column: IonPac AS 15 analytical (5μm, 3x150mm) and AGl 5 guard (5μm, 3x30mm) or equivalent,

Injection volume: lOμl, Oven temperature: 30°C, Detection: Conductivity and UV,

Eluent flow rate: As appropriate to achieve optimum separation (nominally 0.60 ml/min), Eluent: EG40 generated potassium hydroxide, following the appropriate manufacturer's instructions for making a run. Replicate injections of the standard were performed until satisfactory reproducible peak areas were obtained.

The sequence was run on a Dionex DX500 chromatography system with the reference 20 mg/litre standard run after calibration standards and after every ten injections. The slopes of the calibration curves were then calculated by plotting concentration (mg/L) against area.

A_spi x (1 /slope) x D = mg/L of ion

where: A_spi = Peak Area of sample D = Dilution

Bioactive glasses processed using the subject invention show minimal levels of nitrates (Table 7). Using the above method nitrate concentrations can be reduced to 3-9ppm, well below the level that induces a toxic response in humans (20ppm). Extending the stabilisation duration have shown this to be the minimum level of nitrate reduction, process at temperatures of 700°C for 48 and 72 hours have not shown any significant reduction in nitrate concentration. Table 7: Nitrate level of sol-gel glasses produced using optimised process conditions

Optimised sol-gel processing with stabilisation temperatures of 800°C was carried out to verify the range of temperatures that nitrates could be reduced to acceptable levels. However an increase in processing temperature shows extensive densification of the bioactive glass, therefore mimicking the physical characteristics and activity of melt derived glasses (lower activity). Binary and ternary sol-gel glasses must maintain their high surface areas to achieve a comparable bioactive response to melt derived glass such as Bioglass^®, therefore temperatures must not exceed 800°C.

Nitrate concentrations of materials processes in the prior art have not addressed the issue of nitrate concentrations when using calcium nitrate precursors for sol-gel processing. Nitrate concentrations were measured in bioactive glasses synthesised using prior art methods for processing (Table 8).

Table 8: Prior art sol-gel processing conditions and total nitrate concentrations

US 6,482,444 documents an example of a bioactive glass synthesised using a calcium nitrate precursor with gelation, aging and drying stages totalling a 2 week processing time, followed by stabilisation conditions of 450⁰C for 19 hours. Using the above method, the nitrate concentration was measured and reveals nitrate concentrations >20,000ppm, highly unsuitable for tissue engineering applications.

The majority of reported methods for synthesising sol-gel glasses use processing times for gelation, aging and drying which exceeding five days not making them favourable for manufacturing. These conditions are followed by inadequate stabilisation conditions that produce sol-gel glasses with unacceptable levels of nitrate concentrations.

Cytotoxicity Testing

Materials with the same composition were selected for standard cytotoxicity testing to evaluate the cellular response of sol-gel glasses that contain high levels of nitrates compared to those fabricated using the process stated in the current invention.

Method: Monolayer cultures of mouse L929 cells were treated, in triplicate, with an extract prepared from the test material, at the following dilutions N, N/2, N/4, N/8 and N/16 (N = Neat extract). The extract was prepared by incubating the test material in tissue culture medium for 24 hours at 37⁰C. The extract ratio was 0.2 g/ml. The cultures were examined 24 and 48 hours after exposure and graded for cytotoxicity/reactivity (Table 9). Table 9: Numerically graded system for cytotoxicity observed in each well

Samples tested for cytotoxicity using the above methodology are listed in Table 10. The extract of the negative control (polypropylene filters) was non-cytotoxic to L929 cells under the conditions of this test. The extract of the positive control (tin- impregnated PVC strips) was cytotoxic to L929 cells under the conditions of this test showing a level 4 grading. The extract of the test material with high levels of nitrates (l l,782ppm) exhibits a cytotoxic response under the test conditions (Grade 3). Current regulatory guideline state that materials for implantation must exhibit a cytotoxicity level of 2 or below, therefore this nitrate containing sol-gel glass would not be suitable for implantation. The extract of the test material with low nitrate (7.3ppm) shows a reduction in cytotoxic response to level 2 which shows a significant advantage of the current invention.

Table 10: Cytotoxicity studies

Preparations of the nitrate free sol-gel glass composites PCL-Nitrate free sol-gel glass composite

An amount of 60Og of Poly-(ε-caprolactone) (PCL, Sigma Aldrich) polymer was combined with 40Og of nitrate free sol-gel glass (type 70S30C) having an average particle size of 23 μm. The components were poured into the hopper of a Rondol High Torque 13-6 Conical twin screw extruder, where the rotation speed was set between 34- 40 rpm and mixed for ten minutes. The temperature of the extrusion zones was set at 90°C to achieve a molten free flowing extruded composite. The composite was extruded through a 2mm die.

PLGA-Nitrate free sol-gel glass composite An amount of 60Og of 85/15 L-lactide/Glycolide copolymer (Purac BioChem) was combined with 40Og of nitrate free sol-gel glass (type 70S30C) having an average particle size of 23 μm. The components were poured into the hopper of a Rondol High Torque 13-6 Conical twin screw extruder, where the rotation speed was set between 34- 40 rpm and mixed for ten minutes. The temperature of the extrusion zones was set at 160⁰C to achieve a molten free flowing extruded composite. The composite was extruded through a 2mm die.

The final products obtained were homogeneous composite materials having a nitrate free (<10ppm) sol-gel glass content of 40% by weight. The composite was stored in an air and light restricting vacuum package until further processing. Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be covered by the present invention.

Claims

1. A sol-gel derived bioactive glass comprising less than about 30 ppm of nitrate species.

2. A sol-gel derived bioactive glass according to claim 1 comprising:

SiO₂ 40-90%;

CaO 6-50%;

P₂O₅ 0-12%.

3. A sol-gel derived bioactive glass according to claim 1 or claim 2 which contains less than about 25 ppm of nitrate species.

4. A sol-gel derived bioactive glass according to any preceding claim which contains less than about 20 ppm of nitrate species.

5. A sol-gel derived bioactive glass according to any preceding claim which contains less than about 15 ppm of nitrate species.

6. A sol-gel derived bioactive glass according to any preceding claim which contains less than about 10 ppm of nitrate species

7. A sol-gel derived bioactive glass according to any preceding claim which has the following composition:

SiO₂ 45-86

CaO 6-50

P₂O₅ 0-12

Ag₂O 0-12

Al₂O₃ 0-3

CaF₂ 0-25

B₂O₃ 0-20

K₂O 0-8 MgO 0-5

Na₂O 0-20

8. A sol-gel derived bioactive glass according to any preceding claim which further comprises from about 0.1 to about 12 % Ag₂O.

9. A sol-gel derived bioactive glass according to any preceding claim which has the following composition:

SiO₂ 45-86

CaO 10-36

P₂O₅ 3-12

Ag₂O 3-12

CaF₂ 0-25

B₂O₃ 0-10

K₂O 0-8

MgO 0-5

Na₂O 0-20

10. A sol-gel derived bioactive glass according to any one of claims 1 to 7 which comprises about 55 to about 80 % by weight of silicon dioxide (SiO₂), from 0 to about 9 % by weight of sodium oxide (Na₂O), about 10 to about 36 % by weight calcium oxide (CaO), and about 0 to about 8 % by weight phosphorus oxide (P₂O₅).

11. A sol-gel derived bioactive glass according to claim 7 which contains about 60 % SiO₂, about 36 % CaO and about 4 % P₂O₅ by weight.

12. A sol-gel derived bioactive glass according to claim 7 which contains about 58 % SiO₂, about 33 % CaO and about 9 % P₂O₅ by weight.

13. A sol-gel derived bioactive glass according to claim 7 which contains about 70 % SiO₂ and about 30 % CaO.

14. A sol-gel derived bioactive glass according to claim 7 which contains about 70 % SiO₂, about 29 % CaO and about 1 % Ag₂O.

15. A sol-gel derived bioactive glass according to any preceding claim which is in the form of a powder.

16. A sol-gel derived bioactive glass according to any preceding claim which is in particulate form.

17. A sol-gel derived bioactive glass according to claim 16, wherein the particles have a pore size of from about 20 to about 400 Angstroms.

18. A sol-gel derived bioactive glass according to claim 16 or claim 17, wherein the particles have a surface area in the range of from about 20 to about 400 m²/g.

19. A sol-gel derived bioactive glass according to any preceding claim which has porosity from about 10 to about 80 percent.

20. A process for preparing a sol-gel derived bioactive glass, said process comprising the steps of:

(i) forming a mixture comprising a gelable inorganic base material and at least one calcium component; (ii) subjecting said mixture to a temperature of from about 50 to about 70°C for about 2 to about 36 hours; (iii) drying the material formed in step (ii) by subjecting said material to a temperature of about 80 to about 180°C for about 1 to about 72 hours; (iv) stabilising the material formed in step (iii) by subjecting said material to a temperature of about 700 to about 800°C for about 3 to about 24 hours.

21. A process according to claim 20 wherein step (ii) comprises:

(ii)(a) a gelation phase which comprises subjecting said mixture to a temperature of from about 50 to about 70°C for about 1 to about 12 hours; (ii)(b) an aging phase which comprises subjecting said mixture to a temperature of from about 50 to about 70°C for about 1 to about 24 hours;

22. A process according to claim 21 wherein step (ii)(a) is carried out at a pH of from about 0.2 to about 2.

23. A process according to claim 21 or claim 22 wherein step (ii)(a) comprises subjecting said mixture to a temperature of from about 55°C to about 65⁰C for about 1 to about 3 hours.

24. A process according to any one of claims 21 to 23 wherein step (ii)(b) comprises subjecting said mixture to a temperature of from about 55°C to about 65°C for about 20 to about 24 hours.

25. A process according to any one of claims 20 to 24 wherein the gelable inorganic base material comprises at least one alkoxysilane.

26. A process according to claim 25 wherein the alkoxysilane is tetraethoxysilane, tetramethoxysilane (TMOS) or tetrabutoxysilane (TBOS).

27. A process according to any one of claims 20 to 26 wherein the mixture formed in step (i) further comprises triethoxyphosphate.

28. A process according to any one of claims 20 to 27 wherein the calcium component is calcium nitrate.

29. A process according to any one of claims 20 to 28 wherein the mixture formed in step (i) further comprises at least one silver salt.

30. A process according to claim 29 wherein said silver salt is silver nitrate.

31. A process according to any one of claims 20 to 30 wherein the mixture formed in step (i) further comprises deionised water.

32. A process according to any one of claims 20 to 31 wherein step (iii) comprises increasing the temperature at a ramp rate of from about 1 to about 10 °C/minute.

33. A process according to any one of claims 20 to 32 wherein step (iii) comprises subjecting said material to a temperature of about 80 to about 180°C for about 4 to about 72 hours.

34. A process according to any one of claims 20 to 33 wherein step (iii) comprises subjecting said material to a temperature of about 90 to about 13O⁰C for about 36 to about 72 hours.

35. A process according to any one of claims 20 to 34 wherein step (iv) comprises subjecting the material formed in step (iii) to a temperature of about 700°C to about 750°C for about 3 to about 24 hours.

36. A process according to any one of claims 20 to 35 wherein step (iv) comprises subjecting the material formed in step (iii) to a temperature of about 700°C to about 750°C for about 10 to about 24 hours.

37. A process according to any one of claims 20 to 36 wherein the mixture formed in step (i) further comprises at least one therapeutic agent.

38. A process according to claim 37 wherein said therapeutic agent is selected from a healing promotion agent, a growth factor, an anti-inflammatory agent and a topical anaesthetic.

39. A process according to claim 37 wherein said therapeutic agent is a topical antibiotic.

40. A sol-gel derived bioactive glass obtainable by a process according to any one of claims 20 to 39.

41. A sol-gel derived bioactive glass according to any one of claims 1 to 19 or claim 40 for use in medicine.

42. A composite comprising a sol-gel derived bioactive glass according to any one of claims 1 to 19 or claim 40 and a biocompatible, biodegradable material.

43. A composite according to claim 43 wherein said biocompatible, biodegradable material is a degradable polymer.

44. A composite according to claim 43 wherein the degradable polymer is a thermoplastic polymer.

45. A composite according to claim 44 wherein the thermoplastic polymer comprises subunits selected from L-, D- and DL-lactic acids; L-, D- and DL-lactides; and epsilon-caprolactone, and mixtures thereof.

46. A composite according to claim 44 or claim 45 wherein the thermoplastic polymer is is poly-(ε-caprolactone) (PCL) or L-lactide/glycolide copolymer.

47. A composite according to any one of claims 42 to 46 which further comprises a therapeutically active agent.

48. A composite according to any one of claims 42 to 47 which is in the form of a coating or powder.

49. Use of a sol-gel derived bioactive glass according to any one of claims 1 to 19 or claim 41, or a composite according to any one of claims 42 to 48, in the preparation of a medicament for treating wounds and/or burns.

50. Use of a sol-gel derived bioactive glass according to any one of claims 1 to 19 or claim 41, or a composite according to any one of claims 42 to 48, in the preparation of a medicament for grafting skin.

51. A method for treating wounds and/or burns, said method comprising contacting a wound with a therapeutically effective amount of a sol-gel derived bioactive glass according to any one of claims 1 to 19 or claim 41, or a composite according to any one of claims 42 to 48.

52. A method for grafting skin, said method comprising applying a sol-gel derived bioactive glass according to any one of claims 1 to 19 or claim 41, or a composite according to any one of claims 42 to 48, to a graft site, donor tissue, or both.

53. A method according to claim 52 which further comprises applying a topical antibiotic to the graft site, the donor tissue, or both.

54. A wound or burn dressing comprising a bandage comprising a sol-gel derived bioactive glass according to any one of claims 1 to 19 or claim 41, or a composite according to any one of claims 42 to 48.

55. A wound or burn dressing according to claim 54 which further comprises a topical antibiotic.

56. Use of a sol-gel derived bioactive glass according to any one of claims 1 to 19 or claim 41, or a composite according to any one of claims 42 to 48, in tissue engineering.

57. Use according to claim 56 wherein the sol-gel derived bioactive glass is incorporated into a bone graft substitute.

58. Use of according to claim 56 wherein the sol-gel derived bioactive glass is incorporated into an implantable device.

59. Use according to claim 58 wherein the implantable device is selected from the group consisting of prosthetic implants, sheets, pins, valves, screws, plates, and tubes.

60. Use of a sol-gel derived bioactive glass according to any one of claims 1 to 19 or claim 41, or a composite according to any one of claims 42 to 48, for orthopedic applications.

61. A process, sol-gel derived bioactive glass, wound or burn dressing, use, method or composite substantially as described herein.

62. A sol-gel derived bioactive glass according to any one of claims 1 to 19 or claim 41, or a composite according to any one of claims 42 to 48, for treating wounds and/or burns and/or grafting skin.