WO2011121087A1

WO2011121087A1 - A glass ceramic biomaterial

Info

Publication number: WO2011121087A1
Application number: PCT/EP2011/055039
Authority: WO
Inventors: Daniel Boyd; Mark Looney
Original assignee: Cork Institute Of Technology
Priority date: 2010-04-01
Filing date: 2011-03-31
Publication date: 2011-10-06
Also published as: IE20100196A1

Abstract

A glass ceramic biomaterial (1) comprises SrO, ZnO, CaO, SiO₂ and Na₂O. The biomaterial (1) has a crystalline atomic structure. The biomaterial (1) is provided in the form of a porous foam. The biomaterial (1) with the crystalline atomic structure is degradable for release of bio active Sr²⁺ ions and for release of bio active Zn²⁺ ions. The Sr²⁺ ions may assist with bone regeneration, and the Zn²⁺ ions may provide an anti-bacterial function.

Description

A glass ceramic biomaterial Introduction This invention relates to a glass ceramic biomaterial.

Statements of Invention According to the invention there is provided a glass ceramic biomaterial having a crystalline atomic structure, at least part of the biomaterial being degradable for release of bio active ions.

The structure of the biomaterial enables the biomaterial to be employed in load bearing applications without an additional binding agent and/or cement and/or binding gel being required. The biomaterial may also be employed in non load bearing applications.

By releasing bioactive ions, the biomaterial may assist in promoting a therapeutic response in a body tissue and/or in a body bone and/or in a body part.

In one embodiment of the invention the biomaterial comprises strontium (Sr).

Strontium is particularly effective in promoting bone regeneration in bone tissue. Preferably the biomaterial comprises SrO. Ideally the molar percentage of SrO is between 10% and 40%. Most preferably the biomaterial is degradable for release of Sr²⁺ ions. The biomaterial may be degradable for release of Sr²⁺ ions with a level of greater than 5 parts per million. Preferably the biomaterial is degradable for release of Sr²⁺ ions with a level of greater than 50 parts per million. Ideally the biomaterial is degradable for release of Sr²⁺ ions with a level of greater than 100 parts per million. In another embodiment the biomaterial comprises zinc (Zn). Zinc is particularly effective as an anti-bacterial agent to minimise infection. Preferably the biomaterial comprises ZnO. Ideally the molar percentage of ZnO is between 0.1% and 30%. Most preferably the biomaterial is degradable for release of Zn²⁺ ions. The biomaterial may be degradable for release of Zn²⁺ ions with a level of greater than 1.4 parts per million. Preferably the biomaterial is degradable for release of Zn²⁺ ions with a level of greater than 5 parts per million. Ideally the biomaterial is degradable for release of Zn²⁺ ions with a level of greater than 100 parts per million. In one case the biomaterial comprises calcium (Ca). Preferably the biomaterial comprises CaO. Ideally the molar percentage of CaO is between 0.1% and 20%.

In another case the biomaterial comprises silicon (Si). Preferably the biomaterial comprises Si0₂. Ideally the molar percentage of Si0₂ is between 33% and 60%>.

In one embodiment the biomaterial comprises sodium (Na). Preferably the biomaterial comprises Na₂0. Ideally the molar percentage of Na₂0 is between 0.1% and 40%. The biomaterial may comprise crystalline strontium zinc silicate. The biomaterial may comprise crystalline sodium calcium silicate. In one case the biomaterial comprises a blend of crystalline strontium zinc silicate and crystalline sodium calcium silicate.

The biomaterial may comprise crystalline sodium zinc silicate. The biomaterial may comprise crystalline calcium silicate. In one case the biomaterial comprises a blend of crystalline sodium zinc silicate and crystalline calcium silicate.

The biomaterial may comprise crystalline strontium silicate. The biomaterial may comprise crystalline sodium silicate. In one case biomaterial comprises a blend of crystalline calcium silicate and crystalline strontium silicate and crystalline sodium silicate. In another case the biomaterial comprises a blend of crystalline strontium zinc silicate and crystalline strontium silicate and crystalline sodium zinc silicate. In a further case the biomaterial comprises a blend of crystalline strontium silicate and crystalline sodium zinc silicate.

The biomaterial may comprise crystalline zinc silicate.

The biomaterial may comprise crystalline strontium silicon. In another embodiment the crystallisation temperature is between 400° C and 900° C. Preferably the crystallisation temperature is between 500° C and 800° C. Ideally the glass transition temperature is between 400° C and 750° C. Most preferably the glass transition temperature is between 500° C and 650° C. In one case the biomaterial comprises a foam. Preferably at least part of the biomaterial is porous. The porous biomaterial may assist in promoting tissue ingrowth. Ideally the porosity of at least part of the biomaterial is greater than 90%.

The invention also provides in another aspect the use of a glass ceramic biomaterial of the invention for prophylactic treatment at a bone tissue fracture site.

In a further aspect of the invention there is provided the use of a glass ceramic biomaterial of the invention as a bone tissue autograft extender. The invention also provides in another aspect the use of a glass ceramic biomaterial of the invention as a radiopacifier and/or as a coating for a heart tissue.

In another aspect of the invention there is provided a method of manufacturing a glass ceramic biomaterial. In one embodiment of the invention the method comprises the step of sintering a glass powder. Preferably the sintering step is performed prior to crystallisation. The sintering step may comprise the step of performing dilatometry. Preferably the dilatometry step comprises the step of heating at a rate of approximately 5°C per minute. Ideally the dilatometry step comprises the step of heating to approximately 1280°C. The sintering step may comprise the step of performing hot stage microscopy.

The invention provides in another embodiment a method of manufacturing a glass ceramic biomaterial of the invention.

Brief Description of the Drawings The invention will be more clearly understood from the following description of some embodiments thereof, given by way of example only, with reference to the accompanying drawings, in which:

Fig. 1 is an X-ray tomography image of a glass ceramic biomaterial according to the invention,

Fig. 2 is a graph illustrating the phase transformation of the glass ceramic biomaterial of Fig. 1, Fig. 3 is an X-ray tomography image of four cross sections of the glass ceramic biomaterial of Fig. 1,

Figs. 3(a) to 3(d) are graphs illustrating the phase transformation of four other glass ceramic biomaterials according to the invention, Fig. 4 is a graph illustrating ion release for the glass ceramic biomaterial of Fig. 1 and three of the glass ceramic biomaterials of Figs. 3(a) to 3(d),

Fig. 5 is an image of the glass ceramic biomaterial of Fig. 1 with a precipitate apatite bioactive coating,

Fig. 6 is a graph illustrating a heat treatment program designed for burning-out the polyurethane templates and crystallizing BT glass at T_pi, Fig. 7 is a graph illustrating a XRD trace for BT 110-BT 114 at T_pl ,

Fig. 8 illustrates a XRT image of a fully porous glass-ceramic scaffold,

Fig. 9 are graphs illustrating Zinc release at pH3 over 1, 7 and 30 days with significant differences for (a), BT 110, (b) BT 111 , (c) BT 113 and (d) BT 114,

Fig. 10 are graphs illustrating Zinc release at pH7.4 over 1, 7 and 30 days with significant differences for (a), BT110, (b) BT111, (c) BT113 and (d) BT114, Fig. 1 1 are graphs illustrating Strontium release at pH3 over 1, 7 and 30 days with significant differences for (a), BT110, (b) BT111, (c) BT112, (d) BT113 and (e) BT114,

Fig. 12 are graphs illustrating Strontium release at pH7.4 over 1, 7 and 30 days with significant differences for (a), BT 110, (b) BT 111 , (c) BT 112, (d) BT 113 and (e) BT114,

Fig. 13 are graphs illustrating BT110-BTl 14 showing zinc release at pH3 (a), pH7.4 (b), and strontium release at pH3 (c), pH7.4 (d) over 1, 7 and 30 days with significant differences, Fig. 14 is a graph illustrating the relative length change and rate of length change of sample BT 112,

Fig. 15 is a graph illustrating the relative length change curve, rate of length

st

change curve, DSC curve, TG curve and 1 derivation of the TG curve of sample BT 112,

Fig. 16 is a graph illustrating the relative length change and rate of length change of sample BT 110,

Fig. 17 is a graph illustrating the relative length change and rate of length change of sample BT 111,

Fig. 18 is a graph illustrating the relative length change and rate of length change of sample BT 113,

Fig. 19 is a graph illustrating the relative length change and rate of length change of sample BT 114, and

Fig. 20 is a graph illustrating the comparison of the thermal expansion/shrinkage behaviour of the various glass powders.

Detailed Description

Referring to the drawings, and initially to Figs. 1 to 3 thereof, there is illustrated a glass ceramic biomaterial 1 according to the invention. The biomaterial 1 comprises strontium (Sr), zinc (Zn), calcium (Ca), silicon (Si), and sodium (Na). In particular the biomaterial 1 comprises SrO, ZnO, CaO, S1O₂ and Na₂0. In this case the biomaterial 1 consists of SrO, ZnO, CaO, S1O₂ and Na₂0 and is free of any further binding agents and/or cements and/or binding gels. The molar percentage of SrO may be between 10% and 40%. The molar percentage of ZnO may be between 0.1% and 30%. The molar percentage of CaO may be between 0.1% and 20%. The molar percentage of S1O₂ may be between 33% and 60%. The molar percentage of Na₂0 may be between 0.1% and 40%. In this case the molar percentage of SrO is 20%, the molar percentage of ZnO is 20%, the molar percentage of CaO is 10%, the molar percentage of S1O₂ is 40%, and the molar percentage of Na₂0 is 10%.

The biomaterial 1 has a crystalline atomic structure. In this case the biomaterial 1 comprises a blend of crystalline strontium zinc silicate and crystalline sodium calcium silicate. The biomaterial 1 is provided in the form of a porous foam.

The crystallisation temperature of the biomaterial 1 may be between 400° C and 900° C, and preferably is between 500° C and 800° C. In this case the crystallisation temperature of the biomaterial 1 is 713° C.

Fig. 2 illustrates the phase transformation of the basic glass composition into the biomaterial 1 at the first crystallization point (T_pi). During processing, the basic glass composition converts from an amorphous glass into the blend of crystalline strontium zinc silicate and crystalline sodium calcium silicate at the first crystallization point (T_pi), as illustrated in Fig. 2. This conversion radically alters the structural characteristics and properties of the biomaterial 1 in comparison to the basic glass composition. The temperatures referred to in Fig. 2 are the first crystallization point

(Tpi). Surprisingly the biomaterial 1 with the crystalline atomic structure is degradable for release of bio active ions. In this case the biomaterial 1 is degradable for release of Sr²⁺ ions and for release of Zn²⁺ ions. Fig. 4 illustrates the Zn²⁺ ion release at 7 days and at 30 days maturation under Ph3 and Ph7. It would have been expected that the processing of the basic glass composition would have resulted in a stable inert glass ceramic, and it would have been expected that the resulting atomic structure would not have enabled any ion release. Contrary to what would have been conventionally expected, it has been found unexpectedly that the processing of the basic glass composition results in the biomaterial 1 with the crystalline atomic structure which provides for degradation and release of constituent ions, as illustrated in Fig. 4.

The glass based biomaterial 1 releases ions which may lead to a therapeutic response for example in a bone prosthesis in a human body. The Sr²⁺ ions may assist with bone regeneration, and the Zn²⁺ ions may provide an anti-bacterial function. The biomaterial 1 offers the controlled release of ions which are known to inhibit bacterial colonisation of implants and synergistically release ions which promote osteoblastic bone formation at the expense of osteoclastic bone resorption. The biomaterial 1 offers a synergy of antibacterial and regenerative ion release. In the biomaterial 1, the Zn²⁺ ions are released at levels appropriate to inhibit infection in vivo.

The bioactive glass of the invention comprises Calcium-Strontium-Zinc-Silicate. The glass releases controlled amounts of therapeutic Zn²⁺ and Sr²⁺ ions when placed in normal and extreme physiological conditions. The bioactive glass is suited to utilize as a bone replacement material. The level of Zn²⁺ and Sr²⁺ ions released from the bioactive glass material achieve clinical benefits and therapeutic effects including bone formation in the range of 2.45 to 6.5 parts per million (ppm), and antibacterial efficacy of 3-7 ppm respectively.

The crystalline atomic structure of the biomaterial 1 is a function of the composition of the biomaterial 1, the processing temperature to produce the biomaterial 1, and the length of time which the biomaterial 1 is processed at this temperature. The invention provides a glass ceramics construct with a synergistic composition of Sr²⁺ and Zn²⁺ in which the crystalline atomic structure may be chosen to alter the material properties of the biomaterial 1.

The glass-ceramic 1 is a polycrystalline solid prepared by controlled crystallization of glass. Crystallization is accomplished by subjecting suitable glasses to heat treatments which result in the nucleation and growth of crystal phases within the glass.

Depending on the heat treatment applied, for example time and temperature, the crystallization process may be taken almost to completion, however it is also possible that a residual glass phase will remain. The properties of the glass ceramic 1 are controlled by the material micro structure and composition. It would not have been possible to predict the evolution of crystalline phases or at what temperatures they occur as a result of processing. No phase diagrams exist for the parent glass systems. It would not have been possible to qualify or quantify which crystalline materials may form under processing. The crystalline phase evolution of the glass 1 is determined by processing of the glass material. Many different parameters may be varied to create crystalline phases for example varying heating rates, holding temperatures and time parameters. Following processing the materials are analysed qualitatively and quantitatively to determine the phases present.

The biomaterial 1 is provided in the form of a solid, load-bearing structure. No further binding agents and/or cements and/or binding gels are required with the biomaterial 1. The biomaterial 1 has the ability to bear loads normal to physiological loading in the skeleton. The biomaterial 1 may also be deployed as a non load-bearing element.

Fig. 1 illustrates an X-ray tomography (XRT) image of the foam biomaterial 1. The porous nature of the biomaterial 1 facilitates tissue in-growth. The glass ceramic biomaterial 1 may be deployed as a fully reticulated foam, a bulk biomaterial or a coating. The biomaterial 1 is suitable for the following fields of use: load-bearing and non load-bearing dental, craniofacial, maxillofacial and/or orthopaedic applications.

Infection may be an ongoing clinical concern, both immediate to introduction of biomaterials in the body and for the long term viability of the in vivo construct. The biomaterial 1 offers an antibacterial solution to reduce infection at the post-operative stage, whilst also offering full porosity for bone in-growth, and load bearing capabilities for increased scope of applications. In respect of patients who suffer from metabolic bone diseases such as osteoporosis, the invention provides for controlled release of therapeutic agents, such as Sr²⁺, from a load bearing construct either foam, bulk or coating. The inclusion of Sr²⁺ in synergy with Zn²⁺ also offers significant advantages in bone regeneration as a function of controlled ion release to mediate specific regenerative responses in bone tissue in a material capable of fomiing a direct bond with bone while retaining bone bonding capabilities, as illustrated in Fig. 5. Fig. 5 illustrates a surface image of the biomaterial 1 immerged in simulated body fluid (SBF) for 7 days showing a precipitate apatite bioactive coating. The biomaterial 1 has numerous advantages from a material and surgical applications standpoint, for example the capability to release ions, applicability to non load-bearing applications as well as applicability to load-bearing applications, capability of being deployed as a reticulated foam, and being a fully crystalline material. The biomaterial 1 may be employed in a variety of applications, for example for controlled drug delivery, and/or for drug delivery in combination with a hydro gel, and/or for stem cell tissue engineering, and/or as a component in a toothpaste for sensitivity control, and/or as a component in a bone cement for improved radiopacity, and/or as a component in a bone cement for improved biocompatibility and/or antibacterial efficacy, and/or as a component of a composite biomaterial for tissue engineering, and/or as a coating on a medical device.

The biomaterial 1 may be used for prophylactic treatment at a bone tissue fracture site, such as the neck of a femur or a vertebra. The biomaterial 1 may be used as a bone tissue autograft extender. The biomaterial 1 may be used as a radiopacifier and/or as a coating for a heart tissue.

The biomaterial 1 may be provided in a variety of shapes, for example as a rod, and/or a plate, and/or a prosthetic bone shape.

Fig. 3 illustrates the XRT montage of the biomaterial 1 of the invention illustrating the interconnected pore structure through multiple cross sections of the biomaterial 1. In a second embodiment of the invention, the biomaterial comprises SrO, ZnO, CaO, Si0₂ and Na₂0. In this case the molar percentage of SrO is 20%, the molar percentage of ZnO is 10%, the molar percentage of CaO is 10%, the molar percentage of Si0₂ is 40%>, and the molar percentage of Na₂0 is 20%>. In this case the biomaterial comprises a blend of crystalline sodium zinc silicate and crystalline calcium silicate.

In this case the crystallisation temperature of the biomaterial is 577° C. In a third embodiment of the invention, the biomaterial comprises SrO, CaO, Si0₂ and Na₂0. In this case the molar percentage of SrO is 20%, the molar percentage of ZnO is 0%), the molar percentage of CaO is 10%>, the molar percentage of Si0₂ is 40%>, and the molar percentage of Na₂0 is 30%. In this case the biomaterial comprises a blend of crystalline calcium silicate and crystalline strontium silicate and crystalline sodium silicate.

In this case the crystallisation temperature of the biomaterial is 525° C.

In a fourth embodiment of the invention, the biomaterial comprises SrO, ZnO, Si0₂ and Na₂0. In this case the molar percentage of SrO is 30%, the molar percentage of ZnO is 20%, the molar percentage of CaO is 0%, the molar percentage of Si0₂ is 40%, and the molar percentage of Na₂0 is 10%.

In this case the biomaterial comprises a blend of crystalline strontium zinc silicate and crystalline strontium silicate and crystalline sodium zinc silicate.

In this case the crystallisation temperature of the biomaterial is 668° C.

In a fifth embodiment of the invention, the biomaterial comprises SrO, ZnO, Si0₂ and Na₂0. In this case the molar percentage of SrO is 30%, the molar percentage of ZnO is 10%), the molar percentage of CaO is 0%>, the molar percentage of Si0₂ is 40%>, and the molar percentage of Na₂0 is 20%.

In this case the biomaterial comprises a blend of crystalline strontium silicate and crystalline sodium zinc silicate.

In this case the crystallisation temperature of the biomaterial is 567° C.

The following table lists the glass compositions in mol. fractions.

Glass designation Si0₂ ZnO CaO SrO Na₂0

First embodiment 0.4 0.2 0.1 0.2 0.1

Second embodiment 0.4 0.1 0.1 0.2 0.2 The following table lists the glass compositions with the crystalline compounds formed at T_pi . T_pi is the first crystallization point, which is the temperature at which the glass converts fully to a crystalline ceramic.

Figs. 3(a) to 3(d) illustrate an XRD trace for each biomaterial 2, 3, 4, 5 after processing at the respective T_pi .

Example 1

Glass Synthesis Five glass formulations as listed in Table 1 were synthesised. Glasses were prepared by weighing out the appropriate amounts of analytical grade reagents obtained from Sigma Aldrich, Wicklow, Ireland; silicon dioxide, zinc oxide, calcium carbonate, strontium carbonate and sodium carbonate into a plastic container. Each formulation was thoroughly mixed in the closed container for 30 mins. Compositions were then fired at 1480°C for 1 hour in platinum crucibles and the glass melts shock quenched into water. The resulting frit was dried in an oven at 120°C for 1 day.

Glass Si0₂ ZnO CaO SrO Na₂0

BT110 0.4 0.2 0.1 0.2 0.1

BT111 0.4 0.1 0.1 0.2 0.2

BT112 0.4 0 0.1 0.2 0.3

BT113 0.4 0.2 0 0.3 0.1

BT114 0.4 0.1 0 0.3 0.2 Table 1 : Glass composition (mol. fraction). Attrition Milling

The dried frit was ground in an attrition mill obtained from NETZSCH,

Lebenschauerschmierung, Germany using iso-propanol and Zirconia beads. The frit was processed in 15 g batches, which were placed in a ceramic crucible, zirconia beads with a diameter ranging 2-4 mm were placed in the crucible, and iso-propanol was added to the level of the beads. The frit was milled by means of two shaft mounted blades rotating at a speed of 1500 rpm for one hour. The resulting ground powder was placed in a sieve and washed with iso-propanol in order to separate it from the Zirconia beads and to capture any glass particles attached to the beads. The resulting slurry was placed in a round bottomed flask, dried in a water bath at 100°C, and stored in a dessicator for subsequent use.

Particle Size A MALVERN MS 2000 LF particle size analyzer, with the ability to detect particles in the range of 0.02μηι - 2000μηι, equipped with a Hydro 2000 wet dispersion module was used to determine the particle size distribution using the ISO 13320-1 protocol with a fixed wavelength λ=0.63, and result accuracy ± 1%. The glass was prepared for particle size analysis by the following means. 0.5g of the attrition milled glass powder was placed into a 40ml beaker and 30 ml of a 0.1% Sodium

Pyrophosphate, which acts as a dispersant was added, as the MALVERN MS 2000 LF uses a wet technique. The beaker was placed on a magnetic stirrer and stirred for a minimum of 1 minute to ensure that the powder and dispersant formed a suspension. Following a debubbling step, 20 ml of the suspension was taken, by means of a pipette, from between the middle of the vortex and edge of the glass at half the depth of the liquid. The suspension was placed in the MS2000 sample tank and ultrasonic vibrations applied for two minutes. Differential Scanning calorimetry

The identification of glass transition temperatures (T_g) and first the crystallization temperatures (T_pi) for each glass using differential scanning calorimetry (DSC) was performed using a Labsys 1600 obtained from SETARAM Instruments, Caluire, France. The results will be used herein and are presented in Table 3.

Scaffold fabrication

The slurry for the impregnation of the polyurethane foam was prepared using the following recipe. Polyvinyl alcohol (PVA) having 87-89% hydrolyzed, and Average Mw 13,000-23,000, was dissolved in water, the ratio being 0.01 mol/L. Then BT glass powder was added to 100 ml PVA- water solution up to concentration of 40 wt%. Each procedure was carried out under vigorous stirring using a magnetic stirrer for 1 hour. The polyurethane foams cut to shape, with dimensions 5 x 5mm, were immersed in the above prepared slurry and remained in it for 15 minutes. The foams were manually retrieved from the suspension as quickly as possible, and the extra slurry was completely squeezed out. The samples called green bodies were then placed on a smooth surface and dried at ambient temperature for at least 12 hours. Post-forming heat treatments for the burnout of the polymer template structure were programmed, as shown in Fig. 6. The burning condition of the polymer template was identical for all samples. The polyurethane foam exhibits both thermoplastic and thermoset characteristics and when heated partial decomposition occurs resulting in vaporization and the formation of a melt, which occurs at approximately 260°C. All foams were subsequently heated to their respective T_pi values and held for 2 hours.

Fig. 6 illustrates a heat treatment program designed for burning-out the polyurethane templates and crystallizing BT glass at T_pi .

X-ray Diffraction

X-ray diffraction (XRD) analysis was performed using a Philips X'pert MPD Pro 3040/60 -ray Diffraction (XRD) Unit obtained from Philips, Netherlands. Zero background nickel coated sample holders were used for analysis of the 90-710 lm glass particles with Cu ka radiation at 40 kV and 35 mA. Diffractograms were collected in the range 5°<θ<80°, at a scan step size 0.0083° and a step time of 10 s. Where significant crystalline activity occurred above the glass transition temperature the phases present were identified using Joint Committee for Powder Diffraction Studies (JCPDS) standard diffraction patterns using X'pert Highscore Plus software obtained from PANalytical and graphed using Origin lab 8 version 8.1.

X-Ray Tomography

The X-ray tomography examination of the samples was carried out using a phoenix|x- ray Nanotom obtained from General Electric Company with a tungsten transmission target and a 2 megapixel high contrast flat panel digital detector. The maximum accelerating voltage used was 50 kV. 1440 projections in 360° views were obtained. A voxel resolution of 10μ was employed to obtain a 3D image of the glass ceramic foam. Analysis of reconstructed 3D data sets was carried out using Volume Graphics VGStudioMax Software. Dissolution experiments

In order to simulate physiological conditions of normal and extreme environments within the body, as pH 7.4 is representative of the physiological environment and pH 3.0 representative of extreme physiological conditions and the acidic environment produced by osteoclasts, TRIS-HCL buffer and Citric acid buffer solutions were prepared to have a pH of 7.4 ± 0.1 and 3.0 ± 0.2, respectively at a temperature of 37°C ± 1°C, according to ISO 10993-14. The TRIS-HCI buffer solution was freshly prepared as follows:

13.25 g of tris (hydroxymethyl) aminomethane were dissolved in 500 ml of water (ISO 3696, grade 2). The pH was adjusted with an appropriate amount of 1 mol/1 hydrochloric acid to pH 7.4 ±0.1 at a temperature of (37 ±1) °C. The solution was topped up to 1000 ml with water (ISO 3696, grade 2).

The buffered citric acid solution was freshly prepared as follows:

21 g of citric acid monohydrate were dissolved in 500 ml water (ISO 3696, grade 2) in a 1000 ml volumetric flask. 200 ml of 1 mol/1 sodium hydroxide solution were added and diluted to the mark with water (ISO 3696, grade 2). 40.4 ml of this solution were mixed with 59.6 ml of 0.1 mol/1 hydrochloric acid yielding the buffered citric acid solution. Each glass-ceramic foam was immersed in 10 ml of TRIS buffer with a sample size = 5 for each designate, and citric acid buffer with a sample size = 5 for each designate, and maintained at 37°C in a thermally controlled oven. Specimens were stored for 1, 7 and 30 days. After each storage period, specimens were removed via filtration and filtrates retained for ionic content analysis. The Sr²⁺ and Zn²⁺content of each filtrate was analysed using a SpectrAA 220 Fast Sequential, using flame atomic absorption spectroscopy in an acetylene-air flame. Zn and Sr hollow cathode lamps were used at wavelengths 213.9 and 460.7 nm, respectively. In order to eliminate interferences when measuring strontium levels 0.5 g KCL was added to each filtrate. Results are expressed as mean of the mean of triplicate determinations. Analysis of the results was carried out using Students 's t-test, and then one way analysis of variance followed by Bonferroni post-hoc test for multiple comparison between different groups. Each experiment was performed in triplicate and analysed using Graphpad prism 5 software obtained from Graphpad software Inc. with a significance level of P<0.05.

Results

Attrition milling and Particle Size analysis

The particle size analysis carried out shows that attrition milling the glass powders produced a multimodal particle size distribution including sub micron particles as listed in Table 2. Particle size distribution ranges from a d90 of 5.53μιη to 18.23 μιη. The inclusion of particle sizes in the sub micron range and larger is preferred for the fabrication of reticulated glass-ceramic foams utilizing the replication technique.

Particle Size Distribution (μιη)

Glass dlO d50 d90

BT110 0.35 0.61 5.48

BT111 0.42 6.71 7.41

BT112 2.98 5.59 18.23

BT113 0.32 0.47 5.53

BT114 0.40 3.09 17.68

Table 2: Particle size distribution for each glass (post-attrition milling).

Differential scanning calorimetry The results of the differential scanning calorimetry are shown in Table 3. The glass transition temperatures recorded range from 522°C to 623°C, and the first crystallization temperatures range from 525°C to 713 °C.

Glass: T_g (°C): pi (°C):

BT1 10 603 713

BT1 1 1 546 577

BT1 12 522 525

BT1 13 598 668

BT1 14 542 567

Table 3 : Glass transition temperature (T_g) and first crystallization peak (T_pi) for each glass.

Table 3 illustrates T_g and T_pi for each glass composition; it was observed, as one would expect, that both T_g and T_pi decrease with increasing Na to Zn ratio. The atomic radii of Zn²⁺ and of Na⁺ are 74 pm and 95 pm respectively, thus as Na₂0 content increases, the glass network is expanded resulting in a reduction of both glass density and glass oxygen density accounting for the reduction of T_pi as Na₂0 content is increased. Another factor in the reduction of T_g is the Sr to Ca ratio. A substitution of 0.1 mole fraction of strontium for calcium, where all calcium has been substituted in BT1 13 and BT1 14 shows a marked reduction in T_g. Sr²⁺ is slightly bigger that the Caseation, (1 13 and 99 pm respectively) again resulting in the expansion of the network and a reduced glass density.

X-ray diffraction

The following diffraction pattern, Fig. 7 illustrates the phase for each glass-ceramic at T_pi. Table 4 identifies diffraction peak positions for each of the crystal systems identified during analysis. Table 4 is intended to guide the interpretation of Fig. 7. Peak Identifier JCPDS card no. Phase Identifier. Chemical Composition

A 00-037-0407 Sodium Zinc Silicate Na₂ZnSi0₄

B 00-039-0235 Strontium Zinc Sr₂ZnSi₂0₇

Silicate

C 00-033-0302 Larnite Ca₂Si0₄

(Calcium Silicate)

D 00-031-0299 Calcium Silicate Ca₂Si0₄

E 00-016-0818 Sodium Silicate Na₂Si0₃

F 00-039-1256 Strontium Silicate Sr₂Si0₄

G 00-037-1485 Zinc Silicate Zn₂Si0₄

H 00-038-0271 Strontium Silicate Sr₂Si0₄

I 00-038-1366 Strontium Silicon SrSi₂

Table 4: Detailed Legend for Fig. 7. Fig. 7 illustrates a XRD trace for BT 110-BT 1 14 at T_pl .

X-ray tomography

Fig. 8 illustrates a XRT image of a fully porous glass-ceramic scaffold. An XRT image of a glass-ceramic scaffold produced from the Sr-doped zinc-silicate glass- ceramic may be seen in Fig. 8. The XRT image obtained shows an integrated porous network. The porosity of three BTl 13 scaffolds was measured in a 250x250x250 voxel cube and was found to have an average porosity of 95%.

Dissolution experiments

Zinc Release The Zn²⁺ release profiles at pH 3 (extreme physiological condition) are shown in Figs. 9 and 13. The Zn²⁺ release profiles at pH 7.4 (normal physiological condition) are shown in Figs. 9 and 13. Strontium Release

The Sr²⁺ release profiles at pH 3 are shown in Figs. 11 and 13. The Sr²⁺ release profiles at pH 7.7 are shown in Figs. 12 and 13.

Fig. 9 illustrates Zinc release at pH3 over 1, 7 and 30 days with significant differences for (a), BTl 10, (b) BTl 11, (c) BTl 13 and (d) BTl 14. Fig. 10 illustrates Zinc release at pH7.4 over 1, 7 and 30 days with significant differences for (a), BTl 10, (b) BTl 11, (c) BTl 13 and (d) BTl 14. Fig. 11 illustrates Strontium release at pH3 over 1, 7 and 30 days with significant differences for (a), BTl 10, (b) BTl 11, (c) BTl 12, (d) BTl 13 and (e) BTl 14. Fig. 12 illustrates Strontium release at pH7.4 over 1, 7 and 30 days with significant differences for (a), BT 110, (b) BT 111 , (c) BT 112, (d) BT 113 and (e) BTl 14. Fig. 13 illustrates BTl 10-BTl 14 showing zinc release at pH3 (a), pH7.4 (b), and strontium release at pH3 (c), pH7.4 (d) over 1, 7 and 30 days with significant differences. Discussion

The aim of this example was to fabricate a fully integrated porous Sr-doped zinc- silicate glass-ceramic scaffold, whose structure was permissive for osseous integration and to investigate the ion release rate with respect to the divalent ions of zinc and strontium. It is clear from the results that a fully porous material, where the porosity is between 93-96% has been created, hence providing a suitable scaffold to enhance bone ingrowth, and to allow proper vascularisation. It is appropriate to examine the XRD findings in corporation with the ion release results as these are intrinsically linked, by the fact that the structure of the material governs its chemical properties. Detailed analysis based on inter glass ceramics and the statistically significant differences that occur between glass ceramics at similar time timepoints, as outlined in Fig. 13, is difficult to carry out as the relative percentages of each crystalline phase have not been determined and therefore requires further investigation. There are four materials that are designed to release zinc, namely BT110, BT111, BT113 and BT114. Each glass ceramic as outlined in Fig. 7 and Table 4 comprise a unique grouping of crystalline species. BT110 comprises two crystalline phases, sodium zinc silicate (Na₂ZnSi0₄) and strontium zinc silicate (Sr₂ZnSi₂07). Both phases are noted to contain zinc, where the level of zinc in the parent glass is 20 molar percent. The range of Zn²⁺ released from the glass ceramic under pH3 conditions is between 74 and 142 ppm whilst at pH7.4 range from 2 to 6.2 ppm based on the mean released at each timepoint, the mean released at each timepoint being the values that will be mentioned throughout the discussion. No significant difference is found between the three timepoints at either pH level indicating that the level of Zn²⁺ released is not time dependant but is most noticeably dependant on its environment. BT111 contains one crystalline phase containing zinc namely, sodium zinc silicate (Na₂ZnSi0₄). The parent glass for BT111 includes 10 molar percent zinc, which is half that of BT110. The range of Zn²⁺ released from the glass ceramic under pH3 conditions is between 140 and 300 ppm whilst at pH7.4 range from 10.2 to 18.6 ppm. The levels of zinc detected plateaux at 7 days at pH 3 and at 30 days at pH7 clearly indicating from Fig. 10 that at a neutral pH, the release of zinc ions appears to be time dependant. BT113 contains 30 molar percent zinc. The crystalline phases observed include sodium zinc silicate (Na₂ZnSi0₄), strontium zinc silicate (Sr₂ZnSi₂07) and zinc silicate (Zn₂SiC"4). The range of Zn²⁺ released from the glass ceramic has been detected at 154-600 ppm and 8-14 ppm at pH3 and pH7.4 respectably. At pH3 the levels detected plateaux at 7 days and statistically significant differences occur between all timepoints. At pH7.4 the level of Zn²⁺ detected is greatest at day 30, indicating the release is linked to incubation time. BT114 like BT110 and BT111 comprises one zinc containing crystalline phase namely sodium zinc silicate (Na₂ZnSi0₄). At pH 3 the highest level of zinc ion released was detected at day 7, where the range at the extreme pH level was 76-172 ppm. At pH 7 the levels detected were 1.4-5ppm where the highest level was detected at 30 days. There were no significant differences observed between the timepoints at their respective pH levels.

The dissolution mechanism for many glass-ceramics has been shown to be an initial attack on a residual glass phase followed by degradation of the crystalline material. However as we can see from the XRD analysis in Fig. 7, the glass-ceramics appear to be fully crystalline with no residual glassy phase. In considering the durability of glass- ceramics, the materials must be viewed as multiphased composition dependant systems, where each phase possesses individual corrosion characteristics. This example is particularly interested in the degradation products Zn²⁺ and Sr²⁺ and whether the levels released are within identified therapeutic ranges. Figs. 9(a&b) and 10(a&b) demonstrate the range of Zn²⁺ released from the zinc containing glass ceramics where the levels were found to be in the range of 1.4 to 600 ppm (parts per million) . Zn²⁺ levels associated with clinical benefits on bone formation range from 2.45 to 6.5 ppm. Zinc deficiency is associated with retardation and failure of bone growth in animals and humans. Zinc is also associated with increasing osteoblast proliferation, osteoclast inhibition, biomineralization and bone formation. A range of 3-7 ppm of Zn²⁺ has been associated with antibacterial efficacy against S. mutans and A. viscosus both associated with infected hip joints. As is evident from the results presented above, glass-ceramics BT110 and BT114 fall within the range known to be of therapeutic benefit without exhibiting cytotoxic effects, with BT111 and BT113 being marginally outside the range at 10.2 and 8 ppm respectively. The Sr²⁺ released from the glass ceramics BT110 to BT114 ranged from 0 to 583 ppm. Strontium induces osteoblast activity in the range of 8.7 to 87.6 ppm and reduce bone resorption by inhibiting osteoclast action from 8.7 to 2102.8 ppm. It is thus evident that the glass ceramics BT110 to BT114 may indeed have the desired effect on bone turnover.

Example 2 Synthesis of materials In order to achieve a dense material, sintering of the glass powder compact takes place prior to crystallization. Analysis of materials, for example utilising dilatometry or hot stage microscopy, are employed to establish the optimal sintering temperatures. The optimum sintering temperature of a material is controlled by the materials chemical composition; it is not possible to predict such temperatures without analytical evaluation. The processing parameters for dilatometry included heating the BT glass at a rate of 5°C/min up to 1280°C. This heating regime produced optimum sintering temperatures where the BT glass-ceramics are at their most dense. The range can be found in Table 5.

Optimum sintering study

The results for the thermal expansion shown in a red solid line and the length

st

change rate shown in a red dashed line, 1 derivation of the length change curve of sample BT 112 are shown in Fig. 14. The sample length seems to decrease continuously, starting with a small step at 131 °C - the peak temperature of the derivation, followed by bigger step at 806°C and a sharp drop down at appro x 1100°C - also peak temperatures of the derivation. The comparison with an STA measurement illustrated in Fig. 15 reveals that there is a permanent mass change of approx 18% in total. Especially the two steps at 131°C and 806°C are caused by mass changes, also reflected by corresponding DTG and DSC peaks of 131°C/802°C and 145°C/805°C. The two further DSC peaks at 940°C and 1054°C do not have significant counterparts in the DTG curve, just minor peaks are visible. Therefore perhaps melting is the reason. The increasing of the length change signal as shown in the extra window in Fig. 14 may perhaps be a consequence of partial melting at peak temperature of 940°C. If the big step with a length change of more than 60% - the peak temperature of the derivation:

1100°C - is again caused by melting of material components or if sintering plays a role, too, may not be decided. The length change data of sample BT 112 are summarized in Table 5.

The shape of the sample after finishing the measurement was not rod-like, as expected; it was more like a molten lump of material.

Fig. 14 illustrates the relative length change (red, solid line) and rate of length change (red, dashed line) of sample BT 112. Fig. 15 illustrates the relative length change curve (red, solid line), rate of length change curve (red, dashed line), st

DSC curve (blue), TG curve (green, solid line) and 1 derivation of the TG curve (DTG curve, green, dashed line) of sample BT 112.

Fig. 16 illustrates the relative length change (violet, solid line) and rate of length change (violet, dashed line) of sample BT 110. In contrast to sample BT 112, sample BT 110 illustrated in Fig. 16 shows up to approx 460°C an expansion of about 0.7%. At 597°C - the extrapolated onset temperature - a shrinkage step of about 15% takes place, followed by two further steps between 950°C and 1200°C with extrapolated onset temperatures of 1002°C and 1124°C. The corresponding peak temperatures in the rate of length change curve are 625°C, 1030°C and 1155°C, respectively.

The length change data of sample BT 110 may be found in Table 5.

Fig. 17 illustrates the relative length change (blue, solid line) and rate of length change (blue, dashed line) of sample BT 111. The thermal behaviour of sample BT 111 is depicted in Fig. 17. The sample shrinks almost from the beginning of the measurement. During heating up to 1100°C three major steps occur: at 508°C, 970°C and 1017°C - the extrapolated onset temperatures.

The corresponding length change data are listed in Table 5. Fig. 18 illustrates the relative length change (green, solid line) and rate of length change (green, dashed line) of sample BT 113. The thermal length change profile of sample BT 113 illustrated in Fig. 18 is more complex than the previous ones. There is almost no change in length of the sample until 563°C - the extrapolated onset temperature of the first step. But then several other steps are following, the biggest ones are at 811°C, 961°C and 1143°C - the extrapolated onset temperatures.

The corresponding length change data are presented in Table 5.

Fig. 19 illustrates the relative length change (dark blue, solid line) and rate of length change (dark blue, dashed line) of sample BT 114. Sample BT 114 illustrated in Fig. 19 starts to decrease in length at about 100°C. Up to 470°C a shrinkage of 0.9% takes place. In the following three steps occur: at 492°C, at 942°C and at 1015°C - the extrapolated onset temperatures. The length change profile itself, except the absolute temperature values, is similar to sample BT 111.

The corresponding length change data can be found in Table 5. A comparison between the length change curves of the various samples is illustrated in Fig. 20. Fig. 20 illustrates the comparison of the thermal expansion/shrinkage behaviour of the various glass powders.

Temperature[°C] Relative Length Change / [%]

BT 110 BT 111 BT 112 BT 113 BT 114

40 1.01E-02 -6,90E-03 -2,50E-02 UOE-02 -4,25E-03

60 1.89E-02 -3,46E-02 -4,63E-02 1.90E-02 -6,57E-03

80 2,46E-02 -6,97E-02 -0,13903 2,56E-02 -1.29E-02

100 2,80E-02 -0,11892 -0,33851 2,96E-02 -4.21E-02 120 I 3,09E-02 I -0,18663 -0,65796 I 2.98H-02 I -0,10817

140 3,26E-02 -0,22898 -1,16986 2.25 H-02 -0,16211

160 3.5 1 H-02 -0,30527 I -1,61234 I 1.80H-02 I -0,20686

180 3J9E-02 -0,38947 -1,94222 1.63 H-02 -0,2446

200 4.17H-02 I -0,44373 I -2,19813 1.56H-02 -0,27427

220 4.62 H-02 -0,48051 -2,39412 1.5 1 H-02 -0,29978

240 5,17E-02 -0,53155 I -2,56394 I 1.64 H-02 I -0,32571

260 5.72H-02 -0,59387 -2,71445 1.86H-02 -0,352

280 I 6,16E-02 I -0,67451 I -2,86361 I 2.15 H-02 -0,37853

300 6,59E-02 -0,71435 -3,00127 2.46H-02 -0,4068

320 I 6,88E-02 I -0,75312 I -3,11965 2.71 H-02 I -0,43695

340 7,18E-02 -0,78642 -3,23344 2. 1 H-02 -0,46784

360 7,45E-02 I -0,8218 -3,35155 I 2.69H-02 -0,50458

380 7,67E-02 -0,86506 -3,48056 2.37H-02 -0,54828

400 7,71E-02 I -0,91001 -3,5787 I 1.90 H-02 I -0,60186

420 7.80H-02 -0,96625 -3,65397 1.37 H-02 -0,6716

440 7J9E-02 I -1,03186 I -3,71918 I 9.14H-03 I -0,76524

460 7,40E-02 -1,11274 -3,78738 5.24H-03 -0,89094

480 I 6,38E-02 I -1,21918 -3,86715 I 1.69H-03 I -1,09132

500 3,64E-02 -1,45067 -3,96473 -2.13H-03 -1,52828

520 -7,28E-03 I -2,2561 I -4,09908 -8.76H-03 -2,42226

540 -8,88E-02 -3,72063 -4,22891 -2.80H-02 -3,22345

560 I -0,29646 I -4,17867 I -4,28067 I -0,1114 I -3,45407

580 -0,86778 -4,45359 -4,30408 -0,54602 -3,58499

600 I -2,93152 I -4,53094 I -4,32215 I -0,91106 I -3,68212

620 -8,25511 -4,53745 -4,34781 -0,97884 -3,83705

640 I -14,39519 I -4,52136 I -4,38459 I -1,20867 I -3,97364

660 -14,99596 -4,47685 -4,41691 -1,43434 -4,09378

680 -15,19991 -4,43337 -4,44894 -1,45939 -4,20155

700 -15,23652 -4,42049 -4,53522 -1,45096 -4,29706

720 -15,24554 -4,43589 -4,80118 -1,43794 -4,37461

740 -15,24559 -4,46571 -5,3859 -1,41147 -4,429 Table 5: Length change values of the various glass powders.

Figs. 14 to 20 illustrate the shrinkage of each glass ceramic BT 110 to BT 114. Table 5 outlines the shrinkage levels found with the corresponding increase in temperature.

The invention is not limited to the embodiments hereinbefore described, with reference to the accompanying drawings, which may be varied in construction and detail.

Claims

1. A glass ceramic biomaterial having a crystalline atomic structure, at least part of the biomaterial being degradable for release of bioactive ions.

2. A biomaterial as claimed in claim 1 wherein the biomaterial comprises

strontium (Sr).

3. A biomaterial as claimed in claim 2 wherein the biomaterial comprises SrO.

4. A biomaterial as claimed in claim 3 wherein the molar percentage of SrO is between 10% and 40%.

5. A biomaterial as claimed in any of claims 2 to 4 wherein the biomaterial is degradable for release of Sr²⁺ ions.

6. A biomaterial as claimed in claim 5 wherein the biomaterial is degradable for release of Sr²⁺ ions with a level of greater than 5 parts per million.

7. A biomaterial as claimed in claim 6 wherein the biomaterial is degradable for release of Sr²⁺ ions with a level of greater than 50 parts per million.

8. A biomaterial as claimed in claim 7 wherein the biomaterial is degradable for release of Sr²⁺ ions with a level of greater than 100 parts per million.

9. A biomaterial as claimed in any of claims 1 to 8 wherein the biomaterial comprises zinc (Zn).

10. A biomaterial as claimed in claim 9 wherein the biomaterial comprises ZnO.

A biomaterial as claimed in claim 10 wherein the molar percentage of ZnO is between 0.1% and 30%.

A biomaterial as claimed in any of claims 9 to 11 wherein the biomaterial is degradable for release of Zn²⁺ ions.

A biomaterial as claimed in claim 12 wherein the biomaterial is degradable for release of Zn²⁺ ions with a level of greater than 1.4 parts per million.

A biomaterial as claimed in claim 13 wherein the biomaterial is degradable for release of Zn²⁺ ions with a level of greater than 5 parts per million.

A biomaterial as claimed in claim 14 wherein the biomaterial is degradable for release of Zn²⁺ ions with a level of greater than 100 parts per million.

A biomaterial as claimed in any of claims 1 to 15 wherein the biomaterial comprises calcium (Ca).

A biomaterial as claimed in claim 16 wherein the biomaterial comprises CaO.

A biomaterial as claimed in claim 17 wherein the molar percentage of CaO is between 0.1% and 20%.

A biomaterial as claimed in any of claims 1 to 18 wherein the biomaterial comprises silicon (Si).

A biomaterial as claimed in claim 19 wherein the biomaterial comprises S1O₂.

21. A biomaterial as claimed in claim 20 wherein the molar percentage of S1O₂ is between 33% and 60%.

22. A biomaterial as claimed in any of claims 1 to 21 wherein the biomaterial comprises sodium (Na).

23. A biomaterial as claimed in claim 22 wherein the biomaterial comprises Na₂0.

24. A biomaterial as claimed in claim 23 wherein the molar percentage of Na₂0 is between 0.1% and 40%.

25. A biomaterial as claimed in any of claims 1 to 24 wherein the biomaterial comprises crystalline strontium zinc silicate.

26. A biomaterial as claimed in any of claims 1 to 25 wherein the biomaterial comprises crystalline sodium calcium silicate.

27. A biomaterial as claimed in claims 25 and 26 wherein the biomaterial

comprises a blend of crystalline strontium zinc silicate and crystalline sodium calcium silicate.

28. A biomaterial as claimed in any of claims 1 to 27 wherein the biomaterial comprises crystalline sodium zinc silicate.

29. A biomaterial as claimed in any of claims 1 to 28 wherein the biomaterial comprises crystalline calcium silicate.

30. A biomaterial as claimed in claims 28 and 29 wherein the biomaterial

comprises a blend of crystalline sodium zinc silicate and crystalline calcium silicate. A biomaterial as claimed in any of claims 1 to 30 wherein the biomaterial comprises crystalline strontium silicate.

A biomaterial as claimed in any of claims 1 to 31 wherein the biomaterial comprises crystalline sodium silicate.

A biomaterial as claimed in claims 29, 31 and 32 wherein the biomaterial comprises a blend of crystalline calcium silicate and crystalline strontium silicate and crystalline sodium silicate.

A biomaterial as claimed in claims 25, 31 and 28 wherein the biomaterial comprises a blend of crystalline strontium zinc silicate and crystalline strontium silicate and crystalline sodium zinc silicate.

A biomaterial as claimed in claims 31 and 28 wherein the biomaterial comprises a blend of crystalline strontium silicate and crystalline sodium zinc silicate.

A biomaterial as claimed in any of claims 1 to 35 wherein the biomaterial comprises crystalline zinc silicate.

A biomaterial as claimed in any of claims 1 to 36 wherein the biomaterial comprises crystalline strontium silicon.

A biomaterial as claimed in any of claims 1 to 37 wherein the crystallisation temperature is between 400° C and 900° C.

A biomaterial as claimed in claim 38 wherein the crystallisation temperature is between 500° C and 800° C.

40. A biomaterial as claimed in any of claims 1 to 39 wherein the glass transition temperature is between 400° C and 750° C.

41. A biomaterial as claimed in claim 40 wherein the glass transition temperature is between 500° C and 650° C.

42. A biomaterial as claimed in any of claims 1 to 41 wherein the biomaterial comprises a foam.

43. A biomaterial as claimed in any of claims 1 to 42 wherein at least part of the biomaterial is porous.

44. A biomaterial as claimed in claim 43 wherein the porosity of at least part of the biomaterial is greater than 90%.

45. A glass ceramic biomaterial substantially as hereinbefore described with reference to the accompanying drawings.

46. Use of a glass ceramic biomaterial as claimed in any of claims 1 to 45 for prophylactic treatment at a bone tissue fracture site.

47. Use of a glass ceramic biomaterial as claimed in any of claims 1 to 45 as a bone tissue autograft extender.

48. Use of a glass ceramic biomaterial as claimed in any of claims 1 to 45 as a radiopacifier and/or as a coating for a heart tissue.

49. A method of manufacturing a glass ceramic biomaterial.

50. A method as claimed in claim 49 wherein the method comprises the step of sintering a glass powder.

51. A method as claimed in claim 50 wherein the sintering step is performed prior to crystallisation.

52. A method as claimed in claim 50 or 51 wherein the sintering step comprises the step of performing dilatometry.

53. A method as claimed in claim 52 wherein the dilatometry step comprises the step of heating at a rate of approximately 5°C per minute.

54. A method as claimed in claim 52 or 53 wherein the dilatometry step comprises the step of heating to approximately 1280°C.

55. A method as claimed in claim 50 or 51 wherein the sintering step comprises the step of performing hot stage microscopy.

56. A method of manufacturing a glass ceramic biomaterial as claimed in any of claims 1 to 45.

57. A method of manufacturing a glass ceramic biomaterial substantially as

hereinbefore described with reference to the accompanying drawings.