WO2007003968A1 - Novel morphological form of divalent metal ion phosphates - Google Patents

Abstract

The present invention relates to a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate. The invention also relates to methods for making such tubular assemblies and uses thereof in the biomedical field.

Description

Novel morphological form of divalent metal ion phosphates

The present invention relates to divalent metal ion phosphates existing in the form of tubular assemblies. The invention also relates to methods for making such tubular assemblies, further heat treatments thereof and uses thereof in the biomedical field.

Background of the Invention

Divalent metal ion phosphates, particularly Calcium phosphates, are the most important inorganic constituents of biological hard tissues. In the form of carbonated hydroxyapatite (HA), Calcium phosphates are present in bone, teeth, and tendons to give these organs stability, hardness, and function. Biologically formed calcium phosphates are often in the form of nanocrystals that are precipitated under mild conditions of ambient pressure and at near room temperature.

The biologically relevant calcium phosphates, with regard to natural calcifications and to synthetic biomaterials, are octacalcium phosphate (or OCP) namely Ca_S(HPCXO₂(PCM)₄ 5H₂O; calcium-deficient hydroxyapatite (or CDHA); hydroxyapatite (or HA), namely Cajo(P0₄)e(OH)₂ and β-tricalcium phosphate (or β-TCP), namely Ca₃(PO(_I)₂. The latter two compounds are rarely found in nature, but are commonly used in biomaterials: β-TCP in bone cements and HA as a coating for orthopaedic and dental implants.

The complex phase diagrams of divalent metal ion phosphates are exemplified by some of the calcium phosphates. For example, Octacalcium phosphate (OCP) is often found as an intermediate phase during the precipitation of the thermodynamically more stable CDHA from aqueous solutions. OCP consists of apatitic layers (with atomic arrangements of calcium and phosphate ions similar to those of HA) separated by hydrated layers. OCP plays an important role in the In vivo formation of apatitic biominerals. Unsubstituted CDHA (i.e. containing calcium, phosphate, hydrogen phosphate, and hydroxide ions only) does not exist in biological systems; it only occurs in biological systems with ionic substitutions: Na⁺, K⁺, Mg²⁺, Sr²⁺ for Ca²⁺, carbonate for phosphate; fluoride, chloride and carbonate for hydroxide, and some water, to form "biological" apatite, the main inorganic component of animal and human normal calcifications. Therefore, CDHA is a very promising compound for the manufacture of artificial bone substitutes, for example in bioceramics.

There are further factors of importance in biomaterials other than chemical composition, in view of the strict requirements for biocompatibility under physiological conditions. For example, besides the surface composition (which will dictate the chemical environment) the osteogenic process of bone cells will also depend on the roughness and topography of the surface. An important issue in the case of 3D porous structures (e.g. in scaffolds for tissue engineering) is their morphology and meso/macro- organization.

Given the rich structural chemistry of divalent metal ion phosphates as exemplified by the calcium phosphates, together with their suitability in applications such as biomaterials, the present inventors investigated the relationship between the morphology of a range of divalent metal ion phosphates and various growth conditions.

A change in "morphology" (covering such parameters as porosity, pore diameter, the ability for controlled self-assembly, particle shape, crystallinity, crystal size, directional growth, aspect ratio) has significant implications for the eventual application of the material, as exemplified by the insights we already have on the effects of porosity (in terms of pore dimension distribution and interconnectivity) on bone growth for the calcium phosphates. The ability to control morphology for a wide variety of divalent metal ion phosphates and hence tailor the use of the resulting materials to particular applications is desirable.

Summary of the Invention

Accordingly, in a first main aspect, the present invention provides a morphologically novel form of divalent metal ion phosphates, namely a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate.

The second main aspect of the present invention provides a method for the preparation of a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate.

In accordance with a further aspect of the present invention, there is provided a method for controlling the length of a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate.

The third main aspect of the present invention concerns compositions, methods incorporating/uses of the tubular assemblies defined in the first aspect but wherein Ca²⁺ is the ion present in the greatest concentration and is either the only divalent metal ion present or is present in conjunction with one or more other substituent ions.

In accordance with a further aspect of the present invention, there is provided a method for the incorporation of a tubular assembly of one or more phases of nanocrystalline divalent metal ion phosphate, wherein the divalent metal ion present in the greatest concentration is Ca²⁺ into a biomaterial. In accordance with a further aspect of the present invention, there is provided a method of heat treating the tubular assembly and the products of such a method, which may have a modified crystallinity and, or a modified porosity, as appropriate.

Detailed Description of the Invention

The term "tubular assembly" as used herein is intended to encompass one or a plurality of tubular structures formed of nanocrystalline particles of divalent metal ion phosphate which have formed in a process of self assembly. While typically the assemblies may truly be described as "tubular", there is no intention to exclude assemblies which, in places, are more "spine-like" since they narrow and eventually stop growing at a point. Equally, there is no intention to exclude assemblies which have a contorted morphology, or some which comprise tubular structures having open ends and others which are occluded.

The term "nanocrystalline" as used throughout the specification is intended to encompass a crystalline material which, typically, has one or more dimension(s) of less than 750 ran, preferably less than 500 nm. Often the nanocrystalline material from which the tubes assemble is observed to comprise plate-like or needle-like entities.

Unless specified otherwise the term "divalent metal ion" as used throughout the specification should be interpreted as referring to the divalent metal ion species which is present in the greatest total concentration when a summation is made over the one or more phases of nanocrystalline divalent metal ion phosphate present.

In accordance with a preferred embodiment of this aspect of the invention, the ratio of divalent metal ion: phosphate in the one or more phases of nanocrystalline divalent metal ion phosphate is in the range of from 0.5:1 to 3:1 more preferably in the range of from 1: 1 to 2:1 and most preferably in the range of from 1.6:1 to 1.7:1. It has been possible to isolate and characterize a single tubular structure, for example using Energy Dispersive X-Ray Microanalysis (EDAX). It has also been observed that while sometimes it appears that more than one tube is present, in fact this is a tubular assembly comprising only one tube "body" but wherein multiple "tube mouths" are present. The term "tubular assembly" as used throughout the specification is also intended to cover this possibility.

The inner pores which result in the one or more hollow tubular structures of the assembly typically have a mean pore diameter in the range of from 0.02 microns to 10000 microns, preferably in the range of from 2 to 200 microns, more preferably in the range of from 10 to 100 microns and more preferably still in the range of from 30 to 60 microns.

The one or more tubular structures comprising the assembly have a final tube length which is not limited but instead may be tailored depending upon the final application of the material. For the purpose of many applications however, the final mean tube length is preferably in the range of from 2 mm to 15cm, more preferably in the range of from 5mm to 10cm. Often however, the final mean tube length is in excess of about 15 cm. For example, in bone replacement materials, it is desirable to have tubes which are as long as the projected implant in addition to having shorter tubes to aid porosity.

While use of a range of divalent metal ions has proved suitable, particularly stable morphologies have been observed for the tubular assemblies when the divalent metal ion is selected from the group of alkaline earth metal ions, divalent transition metal ions and when the divalent metal ion is Pb²⁺.

In accordance with a preferred embodiment of this first main aspect of the invention, the divalent metal ion is selected from the group of Ca²⁺, Mg²⁺, Sr⁺, Ba²⁺, Zn²⁺, Pb²⁺, Mn²⁺, Fe²⁺, Ni²⁺, Co³⁺, Cd²⁺ and Cu²ⁱ. In accordance with certain specific embodiments of this first aspect of the invention the tubular assembly will comprise one or more phases in which there is partial ion substitution on either the cation or anion sublattice. For example, when the divalent metal ion is selected from the group of Ca", Mg²", Sr²⁺, Ba²⁺, Zn²⁺, Pb²⁺, Mn*, Fe²", Ni²⁺, Co²⁺, Cd²" and Cu²^ there may be partial substitution on the cation sublattice with one or more other of those ions listed, as well as others, such as the non-metal ammonium ion (NH/). Substitution on the anion sublattice also occurs readily, for example, with halide anions but other anions are not excluded.

Where two or more different cations or anions are present on a particular sublattice, they may either partially occupy the same cry stall ographic site in the one or more phases of phosphate present, or each distinct cation or anion (as appropriate) may fully occupy a particular crystallographic site of a defined phase.

In accordance with a particularly preferred embodiment of this aspect of the invention, the divalent metal ion present in the greatest concentration in the one or more phases of nanocrystalline divalent metal ion phosphate is Ca²⁺ such that the tubular assembly comprises a Calcium phosphate. In a still preferred embodiment, there is a predominant component of apatitic Calcium phosphate, optionally comprising one or more divalent metal ions known to substitute for Ca in the apatite unit cell.

By the term "apatitic" is meant a calcium phosphate wherein the Ca:P ion ratio typically is in the range of from 1,5:1 to 1.7:1. However, while the apatitic phase may be the predominant component, there is no intention to exclude other phases such as phase impure HA, tetra calcium phosphate and hydrated ammonium calcium phosphate, among others, which may also be present.

In accordance with a further preferred embodiment of this aspect of the invention the divalent metal ion is Sr²⁺ such that the tubular assembly comprises a Strontium phosphate. In a still preferred embodiment, there is a predominant component of apatitic Strontium phosphate plus a minority phase of Strontium hydrogen phosphate.

In certain cases where apatitic Calcium and Strontium phosphates have been identified, a minor component present appears to be a hydrated ammonium (Ca or Sr) phosphate.

Further divalent metal ions which have shown to support particularly favourable morphologies are Mn²⁺ and Fe²⁺. Phosphate phases which have been identified comprise, respectively , a predominant component of ammonium manganese phosphate nialiite, NH₁MnPOj-H₂O and a predominant component of ammonium iron phosphate NH₁FePO₄-H₂O. The similarities between the Mn and Fe systems suggest the possibility of generating a range of assemblies of ammonium mixed metal ion phosphate hydrates with Mn and Fe (i.e.NH_t Mn₅Fe PO₄-H₂O) and this does not exclude partial substitution with metal ions of any other valency.

The second main aspect of the present invention provides a method for the preparation of a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate, comprising the steps of

(i) providing an aqueous solution of divalent metal ions;

(ii) providing an organic hydrogel;

(iii) contacting the solution of step (i) with the organic hydrogel of step (ii); and

(iv) incubating the ion-saturated organic hydrogel resulting from step (iii) with an aqueous phosphate salt; whereupon a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate may be isolated.

Inorganic structures have been observed to grow through a process of self assembly from a variety of organic hydrogels placed in a solution providing phosphate ions, following exposure to solutions of a variety divalent metal ions. These divalent metal ions include, but are not limited to, Ca²⁺, Mg²⁺, Ba²⁺, Co ²⁺, Zn²⁺, Ni²⁺, Fe²⁺, Cu²⁺, Pb²⁺, Cd²⁺, Sr ²⁺ and Mn²⁺. The identity of the counterions for these cations does not appear to be critical, however, chloride and nitrate have worked particularly well.

In accordance with a particularly preferred embodiment of this aspect of the invention Ca²⁺ is the ion present in the greatest concentration in the solution of step (i) and is either the only divalent metal ion present or is present in conjunction with one or more other substituent ions such as Mg²⁺, Sr⁺. Ba²⁺, Zn²⁺, Pb²⁺, Mn", Fe²⁺, Ni²% Co^2t, Cd²⁺, Cu²⁺ and ammonium ion (NH/). There may also be substitution on the anion sublattice, for example, with halide anions, but other anions are not excluded. In a particularly preferred embodiment, Ca" is present in conjunction with one or more of Mg , Cu , Fe , Zn , Co , and Nr .

The concentration of the divalent metal ion solution is typically in the range of from 0.05M to 12M, preferably in the range of from 0.2M to 1OM and most preferably in the range of from 0.5M to IM. The method has proved particularly favourable in preparing tubular assemblies of Calcium phosphate starting with a step (i) solution of IM CaCl₂

A wide variety of organic hydrogels have proved effective. The types of organic hydrogel which have proved particularly suitable in this second aspect of the present invention include, but are not limited to, agar gels, alginate gels, protein gels, acrylamide gels, methyl methacrylate gels, agarose gels, lysozyme gels (such as lysozyme amyloid Fibril gels), insulin gels (such as insulin amyloid fibril gels), Beta- lactoglobulin gels (such as Beta-lactoglobulin amyloid fibril gels), bovine serum albumin (BSA) gels and mixtures thereof.

Typically the organic hydrogels are pre-prepared from stock solutions at a range of temperatures. Incubation with the divalent metal solution typically then leads to ion saturation of the gel. Alternatively, for certain types of organic hydrogel, the gel is formed while in the presence of the divalent metal ion solution, whether by presence in the same solution, through exposure by spraying with the divalent metal ion solution, or by direct contact with the solid divalent metal ion salt. Optionally, the ion saturated gels are partially dried in air before being contacted with the solution of phosphate salt.

A range of phosphate salts has proved suitable as a source of phosphate ion in step (iv) of this aspect of the present invention. Particularly preferred are dibasic phosphate salts such as dibasic ammonium phosphate, (NROzHPO,,, dibasic sodium phosphate, Na₂HPO₄, and dibasic potassium phosphate, K₂HPO₄. Also,tetra-sodium pyrophosphate, Na₄P₂O₇ has proved particularly suitable.

Typically, phosphate salts are used at a concentration in the range of from 0.05 M to a saturated solution, more preferably in the range of 0.5 M to a saturated solution, and most preferably as a saturated solution.

Light microscopy for a variety of hydrogel / ion / phosphate combinations has indicated that the assembly of hollow tubular structures appears to start spontaneously from the interface between the hydrogel and the phosphate solution.

While there is no intention to limit the present aspect of the invention to a particular mechanism, it is thought that tube-like self assembly may partly occur because of the formation and clustering of nano-sized plate-like or needle-like crystals at the surface of the hydrogel. It may be speculated that the main role of the organic hydrogel in tube formation is to act as a reservoir of divalent metal ions and facilitate their slow release into the phosphate ion buffer. It is observed for a variety of gels, that surface imperfections are important for optimal tube growth - a scratched or imprinted gel surface (which then carries a pattern) increases the number of tubes which are observed to grow. This is thought to be due to the surface defects acting as nucleation points. Tubular assemblies longer than 10 cm have been grown from the surface of gels. Tubes tend to grow vertically upwards towards the air-phosphate solution interface where tube growth is then typically converted to the formation of a two-dimensional ribbon at the air- water interface. Approximately 15 cm long tubes tend to take about 2 hours to grow under room temperature conditions. Clusters of very fine crystals can typically be seen diffusing from the mouth of forming tubes and these crystals appear to form "rafts" of material when the tube "mouth" meets the air-phosphate solution interface.

While there is no intention to limit the present aspect of the invention to a particular mechanism, it is thought that there are three possible mechanisms to arrest tube growth, namely either the depletion of divalent metal ions in the gel, the depletion of phosphate ions in the bulk solution or the occlusion of the hollow pores of the tubes.

The tubular assemblies tend to be isolated (or "harvested^") either by gentle mechanical shaking of the vessel in which the tubes are formed (thereby breaking the structures close to the surface of the gel to which they are attached) followed by pouring the resulting suspension into a separate vessel and allowing the tubes to settle. Excess solution is then removed, yielding a slurry of tubes. Alternatively, a wide-tipped pipette may be used to suck up sheaves of tubes directly from the gel surface. Harvesting of tubes typically results in tubes being obtained which are shorter in length than those actually grown.

Phosphate ions in the bulk solution or divalent metal ions in the gel can be constantly or periodically replenished from reservoirs of these ions enabling continuous methods of tube generation and harvesting.

As explained above, with selection of the appropriate experimental arrangement, tubes tend to grow vertically upwards towards the air-phosphate solution interface where tube growth is then typically converted to the formation of a two-dimensional ribbon at the air-water interface. Clusters of very fine crystals can typically be seen diffusing from the mouth of forming tubes and these crystals appear to form rafts of material when tube "mouth" meets the air-phosphate solution interface.

In accordance with a further aspect of the present invention, there is provided a method for controlling the length of a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate, comprising the steps of

(i) providing an aqueous solution of divalent metal ions;

(ii) providing an organic hydrogel;

(iii) contacting the solution of step (i) with the organic hydrogel of step (ii);

(iv) incubating the ion-saturated organic hydrogel resulting from step (iii) with an aqueous phosphate salt;

wherein a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate self assembles and the length of the tubular structures comprising the assembly is determined by the distance from the surface of the organic hydrogel to the solution surface.

Typically, the organic hydrogel will be in contact with the bottom of the vessel in which self assembly occurs. Advantageously, the gel will be located at, and preferably sealed to, the bottom of the vessel in which self assembly occurs. Also, constant recirculation of the phosphate buffer solution to prevent depletion of ions ensures maximum tube growth.

Particularly stable morphologies for tubular structures with a variety of lengths are observed when the phosphate salt is ammonium phosphate, (NH₄)₂HPO₄ and the solution providing the divalent metal ions is CaCl_2.

In accordance with yet further aspects of the present invention, there is provided a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate obtained in accordance with one of the methods described above. The third main aspect of the present invention concerns compositions, methods incorporating/uses of the tubular assemblies defined in the first aspect but wherein Ca^~÷ is the ion present in the greatest concentration and is either the only divalent metal ion present or is present in conjunction with one or more other substituent ions.

In accordance with the third aspect of the invention, compositions and uses are directed primarily to the technical field of biomaterials. For the purpose of the present invention the term "biomaterials" is taken to encompass any material with a biomedical application. This includes, but is not limited to, ceramic or nonceramic materials with a biomedical application. Preferably, the biomaterial will be any material used to repair, replace or augment bone, teeth, or other mineralised tissues. However the biomaterial may also be a composition with a wider general biomedical application, such as toothpaste.

Accordingly, in a third aspect, the present invention provides an additive for a biomaterial which serves to increase the porosity of the biomaterial and the biomaterial comprising that additive. The additive comprises a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate (as defined in the first main aspect of the invention), wherein the divalent metal ion present in the greatest concentration is Ca²⁺.

Preferably, the biomaterial will be any material used to repair, replace or augment bone, teeth, or other mineralised tissues. In accordance with a particularly preferred embodiment of this aspect of the invention, the biomaterial further comprises a Calcium Phosphate Cement (CPC). When the material comprises a CPC, the tubular structures are incorporated primarily to aid osteointegration and fluid transport

Calcium phosphate cements (CPCs) are some of the best known examples of nonceramic biomaterials. They have proved highly effective as hydroxyapatite (HA) bone substitutes. These nonceramic materials are produced by the direct crystallization of HA in vivo, and no heat is required to form a structurally stable implant. The materials allow slow resorption over time and in vivo are replaced by woven bone as the material resorbs. All CPCs are formulated as solid and liquid components that, when mixed in predetermined proportions, react to form a type of nonceramic HA. The final form of HA is important because it determines whether the end product will be nonresorbable, minimally resorbable, or completely resorbable. The powder component usually consists of two or more calcium phosphate compounds, whereas the liquid component is either water, saline solution, or sodium phosphate solution. These materials have been well characterized chemically and have not been reported to cause foreign body reactions or other forms of chronic inflammatory response.

Three examples of commercially available CPCs are: a mixture of tetracalcium phosphate and dicalcium phosphate dihydrate, which are mixed with water in a powder-to-liquid ratio of 4:1 (marketed as "Bone Source'");

a blend of amorphous and crystalline calcium phosphate precursors and saline, which hardens at 37°C to form a poorly crystalline apatitic calcium phosphate (marketed as "α-BSM"); and

a monocalcium phosphate, α-tricalcium phosphate (α-TCP), and calcium carbonate mixture, which is then mixed with sodium phosphate to form a paste (marketed as the "Norian Skeletal Repair System/ Craniofacial Repair System'").

When the biornaterial comprises a calcium phosphate cement (CPC), the role of the additive is essentially that of a filler. In a yet further aspect of the invention there is provided a calcium phosphate cement comprising a tubular assembly of one or more phases of nano crystalline divalent metal ion phosphate, wherein the divalent metal ion present in the greatest concentration in the additive is Ca²⁺ . Preferably, the solid component from which the CPC is derived is selected from α-TCP, β-TCP, monocalcium phosphate, dicalcium phosphate dihydrate, and tetra calcium phosphate or a mixture of two or more of these components. Calcium carbonate will also typically be present in the solid component. Commercially available solid cement components which have proved particularly suitable are "Bone Source", α- BSM, and Norian SRS. The liquid component will typically be water, saline solution or sodium phosphate solution (the latter is used as an accelerating agent to set the cement) but is not restricted thereto.

It has been observed, through the addition of calcium phosphate tubular assemblies to a range of "control" cements, that whilst the presence of the tubular structures provides microscopic porosity to the biomaterial, the assembly remains intact within the set cements and does not significantly alter the mechanical properties of CPCs.

In accordance with a further aspect of the present invention, there is provided a method for the incorporation of a tubular assembly of one or more phases of nanocrystalline divalent metal ion phosphate, wherein the divalent metal ion present in the greatest concentration is Ca²⁺ into a biomaterial, which method comprises the steps of

(i) providing the biomaterial;

(ii) adding the additive comprising a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate, wherein the divalent metal ion present in the greatest concentration is Ca³⁺ to the biomaterial of (i) and

(iii) mixing the additive with the biomaterial; whereupon the additive is incorporated into the biomaterial. In the preferred embodiment in which the biomaterial comprises a CPC, the CPC comprises a calcium phosphate based solid component; and a liquid component, to which the tubular assembly is added.

Typically, the solid and liquid components of the CPC are mixed prior to the addition of the tubular assembly. The workability of the mixture of solid and liquid components depends upon the nature of and relative amounts of solid and liquid components added (the term ^*'P/L ratio" is used to define the solid (powder) to liquid ratio). Care is taken to ensure there is enough time for the addition of the tubular assembly filler component before the cement sets.

However, alternatively, the tubular assembly may be added to the calcium phosphate solid component prior to the addition of liquid component. When this is the case and the tubular structures are, additionally, dried prior to addition to the solid component, it has been observed that there is a tendency for tube material to agglomerate. This tendency has proved useful in the production of highly porous plugs of material around which cement can be cast. Specimens may also be prepared by gently mixing the solid cement component with tube material deposited as a slurry directly into moulds.

The calcium phosphate tubular assemblies of this aspect of the invention and the biomaterial comprising the same are useful as bone substitutes, for bone repair, implant fixation, bone grafting, fracture fixation, spinal surgery, bone augmentation and dental implants. For bone repair, calcium phosphate tubes of the invention and biomaterial comprising said tubes provide a scaffold on which new bone can grow and remodel. For example, the assemblies can be incorporated into a biomaterial which functions essentially as an artificial osteon, providing directional porosity in the form of sintered shaped monoliths. The invention also relates to the use of calcium phosphate tubular assemblies as a medicament. Furthermore, the invention also relates to a method of treating conditions affecting mineralised tissues by administering a biomaterial comprising calcium phosphate tubular assemblies. Preferably, the assemblies will be incorporated in a CPC. The mineralised tissues include bone and teeth. Various conditions are envisaged particularly those which lead to a need for the repair, replacement or augmentation of mineralised tissues. In a further embodiment, the invention also relates to a method of treating conditions involving the collapse of blood vessels, wherein the calcium phosphate tubular assemblies serve as microstents.

In a further embodiment, the invention relates to the use of calcium phosphate tubular assemblies in the manufacture of a medicament for the repair, replacement or augmentation of mineralised tissues.

In a further embodiment, the calcium phosphate tubular assemblies may be used to facilitate drug delivery by incorporating an "active agent" in the hollow cavities they possess. For example, when the tubular assembly is incorporated into a bone cement, the active agent can be delivered, via the tubular assembly, to the point of use as the bone cement is reabsorbed. Examples of just some of the active agents which may be delivered include antibiotics, antiseptics, growth factors, hormones, cells, or suitable vectors comprising DNA of interest. Accordingly, the invention also relates to a delivery system for active agents comprising calcium phosphate tubular assemblies and an active agent.

In view of the numerous biomedical applications of the calcium phosphate tubular assemblies, cytotoxicity tests have been performed. It has been concluded that the tube materials are no more cytotoxic than inert alumina powder which has been shown to be non toxic through several studies detailed in the literature. In accordance with a yel further aspect of the present invention, the effect of heat treatments, for example sintering at a variety of temperatures and timescales has been investigated.

Interestingly, it has been observed that heat treatment of the tubular assemblies by exposing them to furnace environments can have an effect on the density and crystalline structure of the walls of the tubular structures. Furnace environments may mean any temperature from a few hundreds of degrees Celsius upwards to maximum temperatures achievable by commercially available furnaces and sintering times can vary from minutes to days, with more optimal firing times typically being from 1 to 50 hours, more preferably 2 to 48 hours and more preferably still from 3 to 36 hours and numerous times therebetween. The sintering atmosphere is typically air but there is no intention to exclude other environments such as nitrogen and / or Argon.

A particularly striking effect has been observed by sintering at temperatures of from 600 to 1400⁰C and more preferably at 700⁰C to 1200⁰C and a variety of temperatures in between, for example 800, 900, 1000, 1100, each for 24 hours in air. In particular there is evidence for the densification of the walls of the tubular assemblies. By "densification" is meant what is observed to be the coalescence of the nanoparticles comprising the wall structures such that gradually nanoscale features are lost and one observes formation of larger particles, typically exceeding 0.75 microns in size, and often exceeding approximately 1 micron in size.

X-Ray diffraction data suggest that the densification of the walls is also accompanied by the conversion of the poorly crystalline phases (as characterised by diffuse peaks seen in the x-ray powder diffraction patterns of precipitated material) into more crystalline mixtures.

In accordance with a specific embodiment of this aspect of the present invention, a mixture of phases of Beta-Tricalcium phosphate (βTCP) and Apatite has been identified by X-ray powder diffraction when the divalent metal ion present in the greatest concentration is Ca. This followed sintering at 700, 800, 900, 1000, 1100 and 1200⁰C, each time for 24 hours in air.

An additional observation is that sintering at approximately 1000⁰C and above for times typically of a few hours and more preferably in excess of 3 hours fuses individual tubes together to form a denser material retaining macroporosity. Sintering above approximately 1 100⁰C is often seen to convert the material into Alpha-tricalcium phosphate (αTCP).

These observations have the potential that the porosity on the nanometer to micrometer and potentially tens of micrometer scale for these materials can be controlled by sintering. This ability to control porosity is useful for a range of biomaterial applications.

Significantly, in light of the application described above wherein the tubular assemblies are used as additives for a biomaterial in conjunction with a cement, the above effects of sintering which have been observed imply that the sintered material might potentially be useful as an implant material in its own right, without the need for cement.

FIGURES

Specific embodiments of the various aspects of the present invention are illustrated with reference to the following Figures, which are not intended to limit the scope of the invention in any way.

Figure Ia represents a Light Microscope Image of a tubular structure grown on a coverslip in accordance with a specific embodiment of the first main aspect of the invention.

Figure Ib represents an image of a tubular structure in accordance with a specific embodiment of the first main aspect of the invention which has been grown inside the

ESEM.

Figure 2 represents an X-Ray Powder Diffraction Pattern obtained for harvested tubular structures comprising Calcium Phosphate, in accordance with a specific embodiment of the first main aspect of the invention.

Figure 3 represents a Raman Spectrum for a singular tubular structure comprising Calcium Phosphate, in accordance with a specific embodiment of the first main aspect of the invention.

Figure 4 shows a graphical comparison of the compressive strengths of Calcium Phosphate Cements (CPCs) in accordance with a specific embodiment of the third main aspect of the invention, cast in the presence or absence of Calcium Phosphate tubular

assemblies. P/L 1 and 2 refer to control cements with ratios of 10% (w/w/) CaCO₃ / 90% (w/w) α - TCP to IM(NFLi)₂HPO₄ solution of 1 or 2, respectively. "Plug" refers to the addition of CaP tubes prepared as a dried plug prior to the addition CPC-II cement. "Slurry" refers to the mixing of CaP tubes prepared as an aqueous suspension directly with dried CPC-II to generate a paste which was then transferred to the mould. "AQ" refers to plugs prepared by mixing of an aqueous suspension of CaP tubes with dry CPC-II cement directly within the mould.

Figure 5 shows the porous meshwork of tubular structures comprising Calcium Phosphate present in a dried plug of material within the casting mould, prior to its incorporation into a CPC.

Figure 6 represents a SEM image of a CPC-II cement incorporating a tubular assembly comprising Calcium Phosphate, in accordance with the third main aspect of the invention.

Figure 7 represents a simple mould for casting gel (a) in accordance with a batch processing method wherein the features are numbered as follows: 1 ) petri dish lid; 2) petri dish base; 3) flexible plastic sheet; 4) setting gel; 5) rigid ring; 6) cable tie; 7) optional textured disc.

Figure 8 represents a simple mould for casting gel (b) in accordance with a batch processing method wherein the numbered features are as follows: 1) petri dish lid; 2) petri dish base; 3) flexible plastic sheet; 4) setting gel; 5) rigid ring; 6) optional textured disc.

Figure 9a represents an apparatus for growing and harvesting tubular metal ion phosphates (a) wherein the numbered features are as follows: 1) phosphate buffer inlet; 2) reaction vessel lid; 3) rigid plastic sieve ring; 4) plastic mesh; 5) glass plate/petri dish; 6) prepared gel; 7) phosphate buffer outlet.

Figure 9b represents the same arrangement in photographic form.

Figure 10 represents an apparatus for growing and harvesting tubular metal ion phosphates (b) wherein the numbered features are as follows: 1) phosphate buffer inlet; 2) reaction vessel; 3) reaction vessel lid; 4) rigid mesh with lip; 5) glass plate/petri dish; 6) prepared gel; 7) rigid rod; 8) frictional bearing.

Figures 1 Ia to d represent photographic images of calcium phosphate tubular assemblies.

Figures 12a and b represent the results of cytotoxicity analyses wherein (a) cytotoxicity (%) is plotted as a function of concentration of the species indicated and in (b) viability (%) is plotted as a function of the concentration of the same species.

Figures 13a to d represent images of tubular structures formed after lysozyme gel soaked in 1 M SrCl₂ and partially dried is immersed in 1 M (NRO₂HPO₄ solution.

Figure 14 represents the X-ray diffraction pattern obtained for Strontium Phosphate tubular metal harvested from the surface of a 2% Alginate gel.

Figure 15 represents a comparison of the reflections seen in the X-ray pattern in figure 14 with that on the International Centre for Powder Diffraction database for b) Strontium apatite (Sr₅(PO₄)₃ (OH), 00-033-1348) c) Strontium Chloride Phosphate (Sr₅(PO₄)₃ Cl, 00-016-0666) and d) Strontium Hydrogen Phosphate SrHPO₄, 01-070-

1215.

Figure 16 represents images of tubular structures formed after lysozyme gel soaked in 1 M BaCl₂ and partially dried is immersed in 1 M (NH₄)^HPO₄ solution (a) and (b) SEM images; (c) and (d) ESEM images imaged at 3 Torr.

Figure 17 represents images of structures formed after lysozyme gel soaked in 1 M CdCl₂ and partially dried is immersed in IM (NH^)₂HPO₄ solution.

Figures 18 a to d represent images of structures formed after lysozyme gel soaked in 1 M MnCl₂ and partially dried is immersed IM (NH₄)7HPO₄ solution. Figure 19 represents the X-ray diffraction pattern obtained for Manganese Phosphate Tubular Material Harvested from the surface of a mixed 2% Alginate - 2% Agar gel.

Figure 20 gives a comparison of the reflections seen in the X-ray pattern in Figure 19 with known phases on the International Centre for Powder Diffraction database for b) Niahite (NH₄MnPO₄-H₂O, 00-050-0554), c) Dibasic ammonium phosphate (NH₄H₂PO₄, 00-037-1479) and d) Ammonium Hydrogen Phosphate (NH₄)IHPO₄, 00-009-0391).

Figures 21a to c represent images of structures formed after lysozyme gel soaked in IM ZnCl₂ and partially dried is immersed in IM (NH₄)^HPO₄ solution.

Figures 22a to d represent images of structures formed after lysozyme gel soaked in IM Fe(II)Cl₂ and partially dried is immersed in IM (NH₄)IHPO₄ solution.

Figure 23 represents the X-ray diffraction pattern obtained for Ion Phosphate Tubular Material harvested from the surface of a 2% Alginate gel.

Figure 24 represents a comparison of the reflections seen in the X-ray pattern in figure 23 with that on the International Centre for Powder Diffraction database for b) Ammonium Iron Phosphate Hydrate (NH₄FePO₄-H₂O, 00-045-0424).

Figures 25a and b represent images of structures formed after lysozyme gel soaked in IM NiCl₂ and partially dried is immersed in 1 M (NFLi)₂HPO₄ solution.

Figures 26a to d represent images of structures formed after lysozyme gel soaked in IM Cu(II)Cl₂ and partially dried is immersed in IM

solution.

Figures 27a to d represent images of structures formed after lysozyme gel soaked in 1 M CrCl₂ and partially dried is immersed in IM (NH₄)IHPO₄ solution.

Figures 28a to f represent images of tubular calcium phosphate material sintered in air for 24 hours at (a,b) 700°C, (c,d) 800⁰C and (e,f) 900°C inside a tube furnace showing the densification of individual grains. Figures 29a and b represent images of tubular calcium phosphate material sintered in air for 24 hours showing that at (a) 900⁰C tubes remain unfused whereas at (b) 1000⁰C the tubes fused together.

Figures 30a to f represent images of tube samples sintered in air for 24 hours at (a,b) 1000⁰C; (c, d) 1100⁰C; and (e,f) 1200⁰C.

Figures 31 a to i show the X-ray diffraction patterns for tubular calcium phosphates a) as precipitated, b) after drying at 80⁰C for 24 hours and after heating at c) 300⁰C, d) 700°C, e) SOO⁰C, f) 900°C, g) 1000⁰C, h) HOO⁰C and i) 1200⁰C for 24 hours.

Figure 32 represents diffraction patterns where the patterns represented in Figure 31 have had peaks assigned to them and identified in comparison with known crystalline materials.

Figure 33 represents an FTIR spectrum obtained from KBr (Oven Dried) discs containing 2% wt calcium phosphate power, a) tubular material as precipitated in comparison to commercially obtained b) hydroxyapatite, c) alpha tricalcium phosphate and d) beta tricalcium phosphate (Plasma Biotal Ltd).

Figure 34 represents an FTIR spectrum obtained from KBr (oven dried for 24 hours at 100⁰C) discs containing 2% wt calcium phosphate power, a) as precipitated and material heated to b) 300⁰C₅ c) 700⁰C, d) 800⁰C, e) 900°C, f) 1000°C, g) 1 100⁰C and h) 1200°C for 24 hours.

Figure 35a to c represent a) single X-ray microtomography scan of Ca-P tube approximately 600 μm in length and 50 μm in diameter, b) 3D reconstruction of central portion of tube constructed from 300 one micrometer cross-sections (voxel size 1 micron), c) 10 sections selected at 60 μm intervals along the tube length demonstrating that the Ca-tubes are hollow.

Figure 36a to c represent a) single X-ray microtomography scan of Ca-P tube approximately 600 μm in length and 50 μm in diameter, b) 3D reconstruction of central portion of tube constructed from 300 one micrometer thick cross- sections (Voxel Size 1 micron), c) 30 sections selected at lOμm intervals along the tube length demonstrating that the Ca-tubes are hollow.

Figure 37a to c: The arrow indicates silver crystals in silver dag polymer. The figure as a whole relates to SEM images as follows: a) images of a single Ca-P phosphate tube embedded in silver dag and sectioned and polished using a 3OkV, 10 nA focused ion beam, b,c) 3D projections of small section of tube reconstructed from sequential SEM images obtained after milling away 250 nm layers with a focused ion beam.

Figure 38 a and b represents a) SEM images of a single Ca-P phosphate tube embedded in silver dag and sectioned and polished using a 3OkV, 10 nA focused ion beam, b) 3D projection of small section of tube reconstructed from sequential SEM images obtained after milling away 100 nm layers with a focused ion beam. Showing that the inner wall of the tube consists of denser material than the outer wall which is comprised of three D interconnected nano-sized grains interpenetrated by nano-sized channels.

EXAMPLES

Specific embodiments of the various aspects of the present invention will now be illustrated with reference to the following examples, which are not intended to limit the scope of the invention in any way.

Example 1

The present example illustrates methods of characterisation of tubular assemblies comprising Calcium phosphate in accordance with the first main aspect of the present invention. These methods are, respectively, X-ray powder diffraction (Example Ia), Raman spectroscopy (Example Ib).

Example Ia: X-ray powder diffraction

Tubes were harvested from the surface of the lysozyme gel by gentle mechanical action (i.e. by shaking the centrifuge tube), washed repeatedly to remove soluble Na^HPO₄, concentrated by centrifugation and allowed to dry in order provide sample material for X-ray powder diffraction. Figure 2 shows the spectrum obtained for sample material deposited on single crystal silicon. The location and intensities the resolvable peaks correspond well to published data for the major peak indexed for hydroxyapatite. However, synthetic hydroxyapatite is rarely stochiometric. Chloride ions are likely to be incorporated into the crystal lattice since calcium chloride is one of the initial ingredients.

The presence of other calcium phosphates, such as Octacalcium phosphate Cag(HPθ₄)₂(Pθ₄)45H₂O(OCP), a transient intermediate in the precipitation of OHAp containing apatite layers interspersed with "hydrated" layers, should not be dismissed at this stage.

Example Ib: Raman spectroscopy

In situ Raman analysis has been performed on single tubes using a laser excitation at 633 ran. The strong peaks observed in the spectrum of Figure 3 are consistent with bands assigned to the vibrational modes of the (PO₄)₃ in hydroxyapatite. However, the breath of the observed peaks does not rule out the presence of other calcium phosphates because the bands associated with the vibrational modes of (PO₄)₃ in other phosphates are in close proximity to these bands in hydroxyapatite. Weaker bands associated with lattice modes of hydroxyapatite are obscured by background. Other, yet to be identified, features are present in the spectrum. These may perhaps be removed rinsing off any residual soluble sodium phosphate adhering to the tubes. X-ray diffraction (XRD) confirms Ca:P ratios in the range of 0.5:1 to 3:1, confirming the presence of a number of phases of Calcium Phosphate.

Examples 2. 3. 4 and 5

Examples 2, 3, 4 and 5 illustrate specific embodiments in accordance with the second main aspect of the present invention.

Examples 2a to 2g illustrate methods of preparation of a variety of organic hydrogels. Examples 3, 4 and 5 illustrate methods of preparation of Calcium phosphate tubular assemblies.

Example 2a: Preparation of insulin amyloid fibril gels

Insulin amyloid fibril gels were prepared by incubation at 37°C of a 50 mg/ml stock solution of insulin in pH 1.80 H₂O adjusted with phosphoric acid for 72 lirs. The resulting gel was soaked in 1 M CaCb, partially dried then resuspended in 1 M Na₂HPO₄ and monitored by light microscopy.

Example 2b: Preparation of β-lactoglobulm amyloid fibril gels β-Lactoglobulin amyloid fibril gels were prepared by incubation at 85°C of a 250 mg/ml stock solution of β-lactoglobulin in pH 1.96 H₂O adjusted with HCl for 72 hrs. The resulting gel was soaked in 1 M CaCl₂, partially dried then resuspended in 1 M Na₂HPO₄ and monitored by light microscopy.

Example 2c: Preparation of bovine serum albumin gels

Bovine serum albumin (BSA) gels were prepared by incubation at 7O⁰C of a 250 mg/ml stock solution of BSA in pH 2.08 H₂O adjusted with HCl for 72 hrs. The resulting gel was soaked in 1 M CaCl₂, partially dried then resuspended in 1 M Na₂HPO₄ and monitored by light microscopy.

Example 2d: Preparation of agarose gels

Solutions containing 2-10% (w/v) agarose were heated at a temperature of 6O⁰C for ~2 minutes, after which 5-2OmL was cast into a petri dish and allowed to cool to room temperature, under which conditions the agarose forms a gel. The surface and the bulk of the gel remained hydrated but there was no additional solvent. The hydrated gel was preϊncubated in 1 M CaCK (as described above) and then placed in a solution containing 1 M (NH₄J₂HPO₄.

1% agarose gels were also prepared using 1 M CaCl₂ solution, rather than pure water. Agarose precipitation was not observed. This allowed thick slices of gel loaded with calcium ions to be prepared without any soaking stage. Ca~*-loaded gels were then transferred to a solution containing 1 M (NH^)₂HPO₄

Example 2e: Preparation of acrylamide gels

5 ml acrylic acid, 0.1 g N₅N' -methylene bis-acryl amide ,0.15 g ammonium peroxosulfate and 5.5 g CaCl₂ were dissolved in 45 mis of water. This solution was heated at 6O⁰C for 2hr in a covered petri dish in order to form a gel. The gel was allowed to dry under ambient conditions (25°C) for 92 hrs until the surface of the gel appeared dry and translucent. After this drying stage the gel was then transferred to a solution containing 1 M (NH₄)₂HPO₄.

Example 2f: Preparation of alginate gels

1-2% (w/v) alginate solutions were poured into petri dishes then spayed mist of IM CaCl₂ solution that caused the surface of alginate solutions to gel. Partially set gels were then left for a period in excess of an hour and up to 24 his in a bath containing a volumetric excess of 1 M CaCl₂ solution to allow them to set fully. Fully-set , CaCl₂- saturated alginate gels were then removed and dried in an oven at ~70°C for -2 hrs until semi-transparent and hard. Dehydrated dried gel was placed in 1 M (NHu)₂HPO₄.

Example 2s;: Preparation of Mixed Gel Systems of Agar and Alginate

2% Sodium Alginate is gradually added to distilled water vigorously stirred as it is heated to boiling. Then 2% Agar is gradually added to the boiling solution. The solution is kept boiling until all the particles of agar powder are completely dissolved. The viscous solution is the poured into moulds. Adding the alginate first makes it easier to dissolve all the polysaccharide. The gel is then saturated with CaC12, dried, textured, and immersed in a saturated phosphate solution. Tube growth is monitored by eye and by light microscopy.

Example 3: CaP tubular assemblies formed on lysozyme gels

Lysozyme amyloid fibril gels were extruded manually into a solution containing 1 M CaCl?. After incubation, the extruded gel was partially dried in air, and placed into a solution of 1 M (NH^HPO^ Light microscopy indicated that the assembly of hollow tubular structures started spontaneously from the sides of the cylindrical extrudate. The hollow cavities within the tubes ranged in diameter from below 5μm to over 100 μm. Tubular assemblies longer than 10 cm were grown from the surface of thick cylindrical gel plugs (1 cm in diameter and 1 cm length) in 15 ml centrifuge tubes. With this experimental arrangement tubes grew vertically upward towards the air-NaiHPCM solution interface where tube growth arrested. 10 cm long tubes take approximately 30 minutes to grow. Clusters of very fine crystals could be seen diffusing from the mouth of forming tubes and these crystals appear to form rafts of material when the tube mouth meets the 3Ir-Na₂HPO₄ solution interface.

Figure Ia shows an optical micrograph of tubes grown on a cover slip with crystal clusters visible on the outer surface of the tubes. Figure Ib shows the cross- section of a tube grown inside an Environmental Scanning Electron Microscope (ESEM), where clusters of loosely packed crystals can be seen decorating a much denser core. Solutions containing 0.5 M (NH₄)_IHPO₄ were also sufficient for CaP tube assembly. Tube growth from the lysozyme gel most likely stops when the gel becomes depleted Of Ca²⁺ ions since it has been experimentally possible to recycle spent gels and reinstate tube growth by resoaking in IM CaCl₂.

Example 4: CaP tubular assemblies formed on agarose gels

Hydrated agarose gels (2-10%) were preincubated in 1 M CaCIi and then placed in a solution containing 1 M (NRs)₂HPO₄. Inorganic CaP tubes, visible by eye, began to grow immediately from the surface of the agarose gel and formed structures whose length was limited only by the volume of the 1 M (NH₄)IHPO₄ solution in which the gels were incubated; tube growth typically ceased when the tubes reached the air- solvent interface.

Agarose gels containing a lower concentration of the polysaccharide (1%) were prepared in 1 M CaCl₂ solution rather than pure water, allowing thick slices of gel loaded with calcium ions to be prepared without any soaking stage, which was otherwise prolonged for thicker gels. For gels cast in Petri dishes relatively few tubes were observed to nucleate on thick gels (~6mm) in comparison to thin gels (<1 mm thick) where a multitude of tubes were observed to nucleate along apparent stress lines locked as the gel sets. Tubes nucleated along scalpel incisions scored on the gel surface and where the surface of the gel was punctured with a needle.

Example 5: CaP tubular assemblies formed on alginate gels.

Semi-transparent and hard CaCb-saturated alginate gels were placed in 1 M (NHL_S)₂HPCM and inorganic CaP tubes, visible by eye, began to grow immediately from the surface of the agarose gel and formed structures whose length was once again limited only by the height of the meniscus above the gel. Alginate gels saturated with CaCl₂ frozen for >24 hrs prior to being added to the solution containing 1 M (NH₄)^HPO₄ appeared to promote the formation of a greater number of tubes from the surface of the gel relative to samples that had not been prefrozen.

Example 6:

The present example describes the Batch Processing Method for the Production of Metal (Calcium) Ion Phosphates Assemblies and also provides a Description of Apparatus using Disposable Plastic components.

The batch method may be summarised as follows:

1) 2% weight agar powder is mixed with water, stirred continuously whilst heated until boiling until and all agar is dissolved. (Typically 4g in 200 ml)

2) 2% weight sodium alginate powder is then added to the agar solution and stirred until completely dissolved.

3) The resultant gel solution is then poured into a cylindrical mould.

4) The hot gel solution is allow to cool and set in the mould

5) Solid metal ion salt is spread evenly over the surface of the gel. The mass of salt added to the surface of the gel is that required to obtain a IM concentration in the volume of the gel after diffusion.

6) The gel is left to stand (typically for 2hours) to allow diffusion of the metal ion salt into the gel.

Moulds which ease the handling of the gel once set can be formed in two ways.

a) By forming drum by stretching flexible plastic sheet over a rigid plastic ring and tying it in place with a ligature (plastic cable tie). The inverted drum then forms the mould. A textured disk can be placed at the bottom of the gel to increase the surface area of one of the gel surfaces. Once set the gel can easily be released from this mould by removing the cable tie.

b) By stretching the flexible plastic sheet over a rigid ring and forcing the plastic sheet into the ring with a rigid plastic disk to form the mould. The disk maybe textured to increase the surface area of one of the gel surfaces. Once set the gel can easily be released from this mould by lifting the ring.

Both moulds are designed to fit inside a Petri dish so the gel can be protected from contamination during setting.

7) The set gel is then extracted from the mould and placed in a 1 M buffer of the divalent salt solution for in excess of 16 hours to allow the distribution of metal ions to equilibrate.

8) The gel is then placed on a petri dish or circular glass plate (roughly the same dimension as the gel) with the textured uppermost surface gently dried with hot air.

It is noteworthy that only drying the uppermost surface of the gel prevents the formation of stalactite like assemblies forming at the base of the gel which may deplete the gel of metal (calcium ions)

9) The gel on the petri dish or glass plate is then placed at the bottom of a large glass beaker and a sieve is then placed in close proximity to the dried surface of the gel.

10) The beaker (reaction vessel) is then filled with I M ammonium phosphate solution.

It is noteworthy that tube nucleation can be enhanced by adding a source of carbonate ions to this solution, (either by saturating the ammonium phosphate solution with the metal ion carbonate (calcium carbonate) or adding 5 g/litre of ammonium, sodium or potassium carbonate to the solution.

11 ) The tubular assemblies are then allowed to grow through (sieve material) mesh. 12) Tubes are then harvested either by draining the solution from the bottom of the beaker or by withdrawing the mesh to the top of the beaker.

Two apparatus have been found useful for minimal manual handling of the tubular assemblies once formed, and are depicted in Figures 7 to 10.

Apparatus a): A perforated drum is made by stretching flexible plastic mesh over a rigid plastic ring and tying it in place with a ligature (plastic cable tie). The perforated drum is placed over the gel with the mesh close but not touching the gel surface. Tubes are then harvested either by draining the solution from the bottom of the beaker.

Apparatus b): A rigid plastic mesh with a lip is attached to a rigid plastic rod which is able to slide through a frictional bearing the lid of the reaction vessel in which tube formation takes place, (this is the reverse coffee press or reverse Cafetiere device). Tubes assemblies are then harvested either by draining the solution from the bottom of the beaker or by withdrawing the mesh to the top of the beaker

Example 7

Examples 7a to 7i inclusive illustrate the range of metal ion substitutions which can take place and for which tube-like self assembled structures are observed.

Tube-like self assembly may partly occur because of the formation and clustering of nano-sized apatite plate-like or needle-like crystals at the surface a gel. If this is the case, it should be possible to grow tubes using chlorides of metals known to substitute for Ca in the apatite unit cell (Caio(P0₄)6(OH)2). To explore this possibility and to determine if other potentially useful materials would self-assemble various metal chlorides have been used in place of calcium chloride as a source of metal ions. Optical microscopy has been used to observe the assembly process using these alternative ions and the resulting structures have been studied using scanning electron microscopy.

7a Strontium

When lysozyme gel is soaked in 1 M SrCl? the structures formed (Figure 13) after immersion in IM (NRs)₂HPO₄ have tube-like morphologies similar to those formed on lysozyme soaked in 1 M CaCb. The internal diameter of the tube apertures is between 10 and 50 micrometers. The tubes are assembled from micrometer sized plate like crystals. Comparison of the reflections seen in the X ray diffraction pattern for tubular strontium tubes harvested from alginate gels, (figure 15), with known diffraction patterns for strontium phosphates in International Centre for powder diffraction data base suggest that the material from suggests the material from which these tubes are assembled is apatitic but may contain other phosphates such as strontium hydrogen phosphate SrHPO₄.

7b Barium

On lysozyme gel soaked in 1 M BaCl₂ the structures formed have a distinct morphology. Tubes seen are up to 100 micrometers in internal diameter (Figure 16a) and are assembled from granules between 2-10 microns in diameter (Figure 16b) that are courser than those found in the calcium phosphate tubular material. These granules are themselves agglomerations of sub~50 nm particles. Some assemblies are better described as spine- like rather than tube-like because they are observed to narrow and eventually stop growing at a point. Figure 16c and 16d are ESEM images of one of these spines illustrating that the material forming the tip is much finer than that closer to the surface of the gel

7c Cadmium

With Cd substituted for Ca the assembled structures are tube-like and consist of plate-like entities giving the surface of the tubes a scaly appearance Figure 17a,b.

7d Manganese

Figure 18 shows that the tube structures formed on Lysozyme gel soaked in manganese chloride have a contorted morphology. Some structures seen have an open ends (Figure 18b) whereas others appear to be occluded. The structures formed appear to consist of micrometer sized plate-like grains. Figure 19 shows the X-ray diffraction pattern obtained for Manganese Phosphate Tubular Material Harvested from the surface of a mixed 2% Alginate- 2%Agar gel. In comparison with reflections seen in the X ray with known phases on the International Centre for powder diffraction data base (Figure 20) these structures are mostly Niahite (NH₄MnPO₄-HjO) hydrated ammonium manganese phosphate with a residue of dibasic and mono basic ammonium phosphate.

Sr, Ba, Cd and Mn are known to completely substitute for Ca in apatite whereas Cu, Ni, Co, Zn and other transition metals are believed to only partially substitute for Ca in apatite. However, self assembly has been observed from gels soaked in molar chloride solutions of Cu(II), Fe(II) _, Zn, Co, Ni after immersion in IM (NH₄)IHPO_4. Implying that apatite formation may not a be prerequisite for tube-like self assembly and that other metal ion phosphates will assemble into tubes. With the gel as a source of Ca, Sr, Ba or Ni ions the direction of tube growth is relatively straight and particulates appear to spew from a single aperture to form tubes. The tube growth path with the gel as a source of Cu(II), Fe(II) , Zn, Mn, Co or Cd ions is contorted. Figure 18 shows a contorted tube grown from a gel soaked in 1 MnCl₂. Particulates have been observed to emerge from two apertures during the formation of some of these contorted structures.

7e Zinc Figure 21 shows an example of cone-like structures seen of a zinc ion rich gel, where tube growth from multiple apertures appears to have halted near the gel surface.

7f Iron

Figure 22a to d show the structures formed after lysozyme gel soaked in 1 M Fe(II)CIi and partially dried is immersed in IM (NH₄)₂HPO₄ solution. Like the structures seen for manganese these assemblies are contorted and assembled from plate- like grains. These structures have external diameters ranging from 5 to 50 micrometers. Some have open apertures whilst others are partially occluded. Figure 23 show the X- ray diffraction pattern obtained for Iron Phosphate Tubular Material Harvested from the surface of a 2% Alginate gel. Comparison with the reflections seen in the X ray pattern with that for Ammonium Iron Phosphate Hydrate (NH₄FePO₄-H₂O) (figure 24) suggest that the major phase present is Ammonium Iron Phosphate Hydrate.

This similarity to manganese system suggests the possibility of generating a range of assemblies of ammonium mixed metal ion phosphate hydrates with Mn and Fe (LcNH₄ Mn,Fe PO₄-H₂O) any other divalent metal ion (tills does not exclude partial substitution with metal ions of any other valency).

7g Nickel

Figure 25 shows a tube-like assembly formed when lysozyme gel soaked in 1 M NiC12 and partially dried is immersed in IM (NX-Lt)₂HPO₄ solution. This assembly is about 50 microns in diameter. It is assembled for particles exceeding 5 micrometers in size some of which appear orthorhombic in shape.

7h Copper

Figure 26 shows the structures formed after lysozyme gel soaked in 1 M Cu(II)Cl₂ and partially dried is immersed in IM (NHJI)₂HPO₄ solution. These structures range from 2 to 20 micrometers in diameter and consist of plate-like particles. Though contorted, these structures appear though their apertures appear partially occluded.

7i Chromium Figure 27 show the structures formed after lysozyme gel soaked in 1 M Cr(II)Cl₂ partially dried is immersed in IM (NHLt)₂HPO₄ solution. Most of the structures formed are highly contorted and have the appearance of split tubes (figures 27 a,b ) However some intact tubes less than 2 microns in internal diameter were found and appear to be assembled from plate-like submicron particles.

Example 8

Examples 8a and 8b illustrate specific embodiments in accordance with the third aspect of the present invention, namely compositions and uses of the tubular assemblies directed primarily to the technical field of biomaterials.

Example 8a: Preparation of cements

Control cements were prepared by adding 1 M (NH^)₂HPO₄ solution to dry cement mixture comprising 10% (w/w) CaCO₃/ 90% (w/w) α-TCP in various powder to liquid (P/L) ratios. With a P/L ratio of 1 or 2 the control cements were initially watery and remained workable for about 15 minutes before setting. With a powder to liquid ratio of 4 the control cement had the consistency of a thick and dry paste, and remained workable for only 5 minutes. Specimens of cements formed in the presence or absence of CaP tubes as filler material were crushed between the plates of a screw-riven mechanical testing machine at a cross head speed ofl mm min^" in order to compare their compressional strength. Samples were tested at least 48 hours after casting to allow complete curing of the cements and were tested under ambient conditions. The compressional strength of the control specimens having P/L ratios of 1 or 2 is shown in Figure 4.

Example 8b: Incorporation of Calcium Phosphate Tubular Assemblies into Bone Cement

Various methods were explored for the incorporation of CaP tubes as a filler material within the bone cement. Addition of dried tubes to the CPC-II cement prior to addition of solvent significantly affected the workability of the cement. Addition of 10-20% dried tubes weight for weight to dry cement powder reduced the P/L required to produce a workable paste from 4 to 1. Drying was also found to cause the tubes to agglomerate, perhaps due to the high surface energy of their nanoparticulate assembly. Mixing of the dried tube material with the cement also appeared to cause significant fragmentation of the tube structures, and initial analysis using SEM suggests that little tube material remains intact when dried tubes were manually mixed with the cement.

The tendency of tube material to agglomerate may be perhaps be utilized to produce highly porous plugs of material (which in future may contain cells) around which cement can be cast. To explore this possibility, tube material was harvested from gels using the modified pipette and deposited directly into the 6 *12 mm mould to create porous plugs. Figure 5 illustrates the porous meshwork of tubes formed by drying this plug of material. The dried plug was then removed, and the mould filled with a liquid CPC-II cement mixture with a P/L of 1. The plug was then returned to the filled mould to generate a composite which was allowed to set in the mould. The fourth column in Figure 1 shows that the mechanical properties of the group of samples is approximately 1/10 th of that of the P/L=2 control group and 1/5 of the P/L=l group. Moreover, both control specimens were observed to fracture parallel to the axis of the cylindrical plug, whereas the specimens formed around the porous plugs failed perpendicular to the axis of the plug.

Tubes were also harvested in aqueous suspension, which was then added to dry cement powder. The compressional strength of these specimens lies between the two control groups P/L-l, PfL=¹I. Therefore the addition the CaP tubes as filler by this aqueous route may not affect the mechanical properties of the resultant cements too adversely. Like the control specimens these samples failed forming cracks in the direction parallel to the long axis of the cylindrical test specimen. Figure 6 shows an SEM image of the fracture surface and incorporation of tubes into the cement. Similarly, the preparation of specimens by gently mixing dry CPC-Il cement with tube material deposited as a slurry directly into the moulds yielded samples with compressional strength comparable to that of the P/L=l control.

It should be borne in mind that the medical practitioner (dentist, maxiofacial or orthopaedic surgeon) may in practice prefer to mix dried powder to their own sterile source of water over mixing a wet slurry of tubes to an existing branded cement. Example 9

Example 9 illustrates the results of Cytotoxicity tests carried out for unsintered calcium phosphate tube materials. Reference should also be made to Figure 12.

The cytotoxicity of the tube material has been assessed against control materials and toxins using a Human Causian Osteosarcoma (HOS) cell line (MNNG/HOS (TE85, Clone F-5). Ground tube material (GTM) was used instead of intact material because as a powder it could easily be suspended in tissue culture medium and added to cells. Prior to grinding tube material was suspended in excess ultra pure water (18 MΩ) for at least 3 days before being drained and washed three times in ultra pure water and sterilized in 96% ethanol. CPC II cement powders were prepared by grinding material (P:L=2) previously soaked in synthetic body fluid (SBF) for in excess of a week and sterilizing with ethanol before drying. 0.3 micron alumina powder, used as a negative control, was also sterilized in this way. Cells were plated out in 96 well plates with a density of 5X10⁴ cells per well and grown to confluency.

Powders suspended in tissue culture media, with a range of concentrations 2, 0.2, 0.02, 0.002 and 0.0002 mg/ml were then added to the wells. The apoptosis inducing agent staurosporin (STAU) and the cell membrane permeablizϊng agent DMSO were used as positive controls. Cytotoxicity was access using the (lactate dehyrodgenase) assay LDH which quantifies the number of dead cells by comparing the amount of lactate released by cells treated with an agent into the tissue culture to that release when permeablising cells using the surfactant (Sodium dodecyl sulfate) SDS. Cell viability was accessed using the MTT ((3~(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)~2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay that quantifies the number of cells able to metabolize the MTT agent. Both assays were performed after 24 and 48 hour incubations.

Figure 12a and Table I show the results of the LDH assay after 48 hrs. Below a concentration of 0.1 mg/ml the measure of cytotoxicity, in wells containing powders is the same as that only containing cells in which no agent has been added. Below 0.1 mg/ml tube material and cement are therefore non-toxic. This contention is supported by the results of the MTT assay Figure 12b, table ϊϊ which shows that below a concentration of 0.1 mg/ml cells are greater than 80% viable. I.e. the measure of cell viability is approximately twice for cells treated with 1 μM STAU and four times that of cells treated with 5% DMSO. Above 0.1 mg/ml the addition of the powder appears to be killing cells. This however is a smothering effect where the "apparent toxicity" is caused by material preventing access to nutrients in the tissue culture media and not by any inherent toxicity of the material. This assertion is supported by the observation that at concentrations of 0.2 mg/ml or cells appear almost completely covered by powder though, where gaps occur in this covering apparently health cells can be seen. The alumina powder has nearly the same concentration dependant effect on the both LDH and MTS assays as the cement powder and the ground tube material. It is therefore reasonable to conclude that they are no more cytotoxic than inert alumina powder which has been shown to be non toxic by more detailed scientific studies of particulate toxicity in the literature.

Table I Summary of LDH assay after 48 hours

Cone Tubes alumina CPCII

Cytotoxicity Cytotoxicity Cytotoxicity Mg/ml % % %

2 64.0 2.3 67.6 2.3 72.0 2.4

0.2 16.5 0.9 9.5 1.0 15.7 3.5

0.02 5.8 0.7 5.3 0.6 6.1 2.4

0.002 4.9 0.6 5.1 0.7 5.3 2.1

0.0002 5.1 0.7 4.8 0.4 5.0 1.3

Controls

Cells only 0 6 0.8

STAU - 38 1.5

DMSO - 43 2.7 Table II Summary of MTS assay after 48 hours

Cone Tubes alumina CPCII

Viability Viability Viability Mg/ml % % %

2 21.9 4.3 64.1 16.6 67.1 8.9

0.2 79.2 3.8 82.1 6.2 83.5 4.3

0.02 83.1 4.1 85.0 8.7 88.4 7.4

0.002 84.4 3.0 83.6 6.0 86.8 10.0

0.0002 89.3 3.8 98.1 7.3 94.9 10.3

Controls (%) ± cells only 0 100 3.5

STAU - 56.5 2.3

DMSO - 22.1 1.2

Example 10

Example 10 details the effects observed on sintering the tubular assemblies obtained in accordance with the methods describing the 'wet' chemistry such as those detailed in Examples 2 to 5 inclusive.

To investigate the effect of heat treatment between 700 ⁰C and 1200 ⁰C two types of furnace were used to heat washed, dried tubular calcium phosphate material contained in alumina crucibles in air. One crucible was placed at the hot zone of a tube furnace set at 900 ⁰C whilst two others were placed at positions set distances from the hot zone where the temperature was calibrated to be 700⁰C and 800⁰C with a thermocouple.

To heat treat material at higher temperatures between 1000 ⁰C and 1200 ⁰C a brick type furnace set at temperatures of 1000 ⁰C, 1100 ⁰C and 1200 ⁰C was used to heat material. All samples were placed in furnaces at a set temperature for 24 hours then removed and allowed to cool in air.

Tubular Calcium Phosphate material heated to 700⁰C, figure 28a,b, shows little signs of sintering and is composed like unheated material, of sub 500 nm grains that are agglomerates of much smaller nano-scale particulates. Material heated to 800⁰C figure 28c,d shows signs of sintering with the amalgamation of the nano-scale particulates within the grains. The amalgamation is more apparent in material heated to 900⁰C, figure 28e,f however the grains themselves remain distinct but have lost features at the nanoscale.

Figure 29a shows that tubes heated to 900 ⁰C are not fused and do not lose their porosity at the tens of micrometer scale (macroporosity) despite losing porosity on the nanometer scale. Tubular material heated to 1000 ⁰C and above has fused into a solid conglomerate but retains porosity on at the tens of micrometer scale. Figure 29b illustrates that at 1000⁰C that grains have grown to over a micron in size and have fused removing porosity in the tube well. Grain size has doubled for material heated tol 100⁰C (figure 30) with the boundary between grains becoming less distinct. At 1 100 ⁰C, the temperature often used to densify hydroxyapatite, there is an interesting effect where growth of micron-sized grains produces pores in the tube wall. Tubes heated to 1200⁰C the tubes have fused into a single monolithic mass although the mouths of individual tubes are visible in this mass which is also riddled with micron sized pores.

Figure 31 shows the X-ray diffraction patterns for tubular calcium phosphates a) as precipitated, b) after drying at 8O⁰C for 24 hours and after heating at c) 300 ⁰C, d) 700 ⁰C₅ e) 800 ⁰C f) 900 ⁰C g) 1000 ⁰C, h) 1100 ⁰C and i) 1200 ⁰C for 24 hours. Samples heated below 300 ⁰C have very broad peaks associated with poorly crystalline nano- sized calcium phosphates. The sharp peak seen at 7° is associated with the presence of NH₄CaPO₄-H₂O and is absent from the oven dried sample perhaps due to the loss of water of crystallisation. This reflection is seen in samples heated to higher temperatures perhaps because the samples stored in air before obtaining the diffraction patterns may have regained this water. All the other peaks in the X-ray diffraction patterns a) to i) are associated with either hydroxyapatite (HA), beta-tricalcium phosphate(βTCP) or alpha- tricalcium phosphate (αTCP) .

The region from 29-25° to 35° is expanded in figure 32 to show the major peaks associated with these phases and assignment of some of the observed reflections. The diffused peak seen in the oven dried sample (curve b) around 26° can be associated with either the 002 reflection of HA or the 1010 and 122 reflections of βTCP. This peak becomes sharper when heated to progressively higher temperatures due to the increases in particle size (reported in the scanning electron images) and in crystallinity. There are many reflections that can be assigned to either HA or βTCP in the range of the broad peak seen between 30 and 35°. For samples heated to higher temperatures this broad peak splits to reveal sharper reflections that can be designated either to HA or βTCP. As the tubular material is heated the 0210 reflection for βTCP at 31° becomes increasingly intense being a weak peak at 700 ⁰C pattern (d) to become the most intense peak in the 1100 ⁰C pattern (h). In contrast the principal 211 reflection of HA at 32° strong in the X- ray diffraction patterns at 700 and 800 ⁰C diminishes with increasing temperature and is absent the pattern at 1 100 ⁰C. The predominance of the βTCP phase seen in the sample sintered for 24 hours at 1100 ⁰C suggests that the ratio of calcium to phosphate in the tube material as precipitated is close to 1.5. In the sample heated to 1200 ⁰C the reflections assigned to βTCP have almost completely disappeared to be replaced with those of αTCP indicating a change in phase from the beta to alpha form.

Figure 33 shows the FTIR spectrum obtained from discs pressed from of potassium bromide KBr (oven dried at 11O⁰C) containing 2% by weight of a) tubular calcium phosphate and commercial obtained HA (b), α TCP (c) and β TCP (d) from 400 to 4000 cm^'1. In the range 500 to 700 cm^"1 the tubular calcium phosphate material as precipitated contains peaks corresponding to the j?₄PO^3" ₄ double band at 571 and 601 cm-1 in hydroxyapatite but lacks a band corresponding to the OH liberation band at 630 cm^"1 of HA. A peak that corresponds to the V _\ mode of PO^3" ₄ can be seen at around 950 cm .A peak 880^' cm around suggests the presence of HPO^~" ₄. The peaks seen at 1043 cm-1 could correspond to U₃ modes of either PO^3" ₄ in β TCP or HA. Whereas, the peak at 1108 cm^'1 corresponds to a v$ mode of PO^3" ₄ reported for βTCP. A band in the region 1440-1550 corresponds to CO₃ ^2" bands (v₃) (but a spectrometer that can be purged with nitrogen will be needed to confirm this). The broad peak between 2400 and 3800 cm^"1 is probably associated with water loosely bound to the tubular CaP since this feature is removed by reheating the ICBr disks to HO⁰C for 16 hours. There is no feature corresponding to the OH- stretching band of HA at 3572 cm^"1.

Figure 34 shows the effect of heat treatment on the FTIR. The complex spectrum seen in the region 900-1300 cm^"1 for samples heated between 700 and 900 ⁰C in the region 900-1300 cm^"1 suggests the presence of multiple apatitic phases. The multitude of peaks observed in this region particularly that around 1210 cm^"1 would also indicate the presence of pyrophosphate P₂O₇ ^4" known to appear when ACP is heated to 650 ⁰C. When the CaP material is heated above 1100 ⁰C the double band between 500 and 700 cm^"' becomes a single peak indicating a phase change from βTCP to αTCP.

Claims

CLAIMS:

1. A tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate.

2. A tubular assembly according to claim 1 wherein the one or more phases of nanocrystalline divalent metal ion phosphate are in the form of a plurality of tubular structures.

3. A tubular assembly according to claim 1 or claim 2 wherein the plurality of tubular structures has formed in a process of self-assembly.

4. A tubular assembly according to any of claims 1 to 3 wherein the one or more phases of nanocrystaliine divalent metal ion phosphate comprise a crystalline material which has one or more dimension(s) of less than about 750 nm, preferably less than 500 run.

5. A tubular assembly according to any one of the preceding claims wherein the ratio of divalent metal ion:phosphate in the one or more phases of nanocrystalline divalent metal ion phosphate is in the range of from 0.5:1 to 3:1.

6. A tubular assembly according to claim 5 wherein the ratio of divalent metal ion:phosphate in the one or more phases of nanocrystalline divalent metal ion phosphate is in the range of from 1: 1 to 2: 1.

7. A tubular assembly according to claim 5 or claim 6 wherein the ratio of divalent metal ion:phosphate in the one or more phases of nanocrystalline divalent metal ion phosphate is in the range of from 1.5:1 to 1.7:1.

8. A tubular assembly according to any of the preceding claims wherein the tubular structure(s) comprising the assembly have inner pores with a mean diameter in the range of from 0.02 to 10000 microns.

9. A tubular assembly according to claim 8 wherein the inner pores have a mean diameter in the range of from 2 to 200 microns.

10. A tubular assembly according to claim 8 or claim 9 wherein the inner pores have a mean diameter in the range of from 10 to 100 microns.

1 1. A tubular assembly according to any of claims 8 to 10 wherein the inner pores have a mean diameter in the range of from 30 to 60 microns.

12 A tubular assembly according to any of the preceding claims wherein the tubular structure(s) comprising the assembly have a mean length in the range of from 2 mm to 15 cm.

13 A tubular assembly according to claim 12 wherein the tubular structure(s) comprising the assembly have a mean length in the range of from 5 mm to 10 cm.

14 A tubular assembly according to any of claims 1 to 1 1 wherein the tubular structure(s) comprising the assembly have a mean length in excess of about 15 cm.

15. A tubular assembly according to any one of the preceding claims wherein the divalent metal ion present in the greatest concentration in the one or more phases of nano crystalline divalent metal ion phosphate is selected from the group of alkaline earth metal ions and divalent transition metal ions.

16. A tubular assembly according to claim 15 wherein the divalent metal ion is selected from the group of Ca²⁺, Mg²⁺, Sr⁺, Ba²⁺, Zn²⁺, Mn²⁺, Fe^2", Ni²⁺, Co²⁺, Cd²^ and Cu²\

17. A tubular assembly according to any one of claims 1 to 16 wherein the divalent metal ion is Ca²⁺.

18. A tubular assembly according to any one of claims 1 to 14 wherein the divalent metal ion present in the greatest concentration is Pb²⁺.

19. A tubular assembly according to any one of claims 15 to 18 wherein, in addition to one divalent metal ion being present in greatest concentration in the one or more phases of nanocrystalline divalent metal ion phosphate, there are substituent ions present on either the cation or anion sublattice.

20. A tubular assembly according to claim 19 wherein the divalent metal ion present in the greatest concentration in the one or more phases of nanocrystalline divalent metal ion phosphate is Ca²⁺ and there are substituent ions present on either the cation or anion sublattice.

21. A tubular assembly according to claim 20 wherein the one or more phases of nanocrystalline divalent metal ion phosphate in which Ca²⁺ is present in the greatest concentration comprise an apatitic Calcium phosphate,

22. A tubular assembly according to claim 21 wherein the one or more phases of nanocrystalline divalent metal ion phosphate comprising an apatitic Calcium phosphate have a Ca:phosphate ion ratio in the range of from 1.5:1 to 1.7:1.

23. A tubular assembly according to any of claims 19 to 22 wherein the substituent ions present on either the cation or anion sublattice are selected from the group of Ca²⁺, Mg²⁺, Sr⁺, Ba^{2^} ₅ Zn²⁺, Pb²⁺, Mn²⁺, Fe²⁺, Ni²⁺, Co²⁺, Cd²⁺, Cu^, NH₁ ⁺ and halide ions.

24. A tubular assembly according to claim 16 wherein the divalent metal ion is Sr²⁺.

25. A tubular assembly according to claim 24 wherein the divalent metal ion present in the greatest concentration in the one or more phases of nanocrystallme divalent metal ion phosphate is Sr⁺ and there are substituent ions present on either the cation or anion sublattice.

26. A tubular assembly according to claim 25 wherein the one or more phases of nanocrystalline divalent metal ion phosphate in which Sr²⁺ is present in the greatest concentration comprise an apatitic Strontium phosphate.

27. A tubular assembly according to any of claims 24 to 26 wherein the substituent ions present on either the cation or anion sublattice are selected from the group of Ca²⁺, Mg²⁺, Ba²⁺, Zn²⁺, Pb²⁺, Mn²⁺, Fe²⁺, Ni²⁺, Co²⁺, Cd²⁺, Cu²⁺, NH/ and halϊde ions.

28. A tubular assembly according to claim 16 wherein the divalent metal ion present in the greatest concentration is Mn²⁺.

29. A tubular assembly according to claim 28 wherein the one or more phases of nanocrystalline divalent metal ion phosphate comprise a predominant component of ammonium manganese phosphate niahite, (NH₁MnPO^H₂O).

30. A tubular assembly according to claim 16 wherein the divalent metal ion present in the greatest concentration is Fe²⁺.

31. A tubular assembly according to claim 30 wherein the one or more phases of nanocrystalline divalent metal ion phosphate comprise a predominant component of ammonium iron phosphate niahite, (NH₁FePCvH₂O).

32. A method for the preparation of a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate, comprising the steps of:

(i) providing an aqueous solution of divalent metal ions;

(ii) providing an organic hydrogel;

(iii) contacting the solution of step (i) with the organic hydrogel of step (ii); and

(iv) incubating the ion-saturated organic hydrogel resulting from step (iii) with an aqueous phosphate salt, whereupon a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate may be isolated.

33. A method according to claim 32 wherein the solution of step (i) is a divalent metal ion solution comprising alkaline earth metal ions and / or divalent transition metal ions.

34. A method according to claim 32 or claim 33 wherein the solution of step (i) is a divalent metal ion solution comprising one or more of Ca²⁺, Mg²⁺, Sr⁺, Ba²⁺, Zn²⁺, Mn²⁺, Fe²⁺, Ni²⁺, Co²⁺, Cd²⁺ and Cu²⁺.

35. A method according to claim 34 wherein the solution of step (i) comprises Ca²⁺ in the greatest concentration.

36. A method according to claim 32 wherein the solution of step (i) is a divalent metal ion solution comprising Pb²⁺.

37. A method according to any one of claims 32 to 36 wherein the solution of step (i) further comprises one or more substituent cations or anions.

38. A method according to any one of claims 32 to 37 wherein the solution of step (i) further comprises one or more ions selected from Ca²⁺, Mg²', Sr³', Ba²', Zn²⁺, Pb²⁺, Mn²⁺, Fe²⁺, Ni²⁺, Co²⁺, Cd²⁺,

NH₄"

39. A method according to any one of claims 32 to 38 wherein the solution of step (i) is a chloride or nitrate solution.

40. A method according to any of claims 32 to 39 wherein, in the solution of step (i), Ca²⁺ is the divalent metal ion present in the greatest concentration.

41. A method according to any one of claims 32 to 40 wherein the concentration of the divalent metal ion solution is in the range of from 0.05M to 12M.

42. A method according to claim 41 wherein the concentration of the divalent metal ion solution is in the range of from 0.2M to 1OM.

43. A method according to claim 41 or claim 42 wherein the concentration of the divalent metal ion solution is in the range of from 0.5M to IM.

44. A method according to any one of claims 32 to 43 wherein the solution of step (i) comprises CaCl₂ at a concentration of IM.

45. A method according to any one of claims 32 to 44 wherein the organic hydrogel provided in step (ii) is selected from the group of agar gels, alginate gels, protein gels, acrylamide gels, agarose gels, lysozyme gels (such as lysozyme amyloid fibril gels), insulin gels (such as insulin amyloid fibril gels), Beta-lactoglobulin gels (such as Beta- lactoglobulin amyloid fibril gels) and bovine serum albumin (BSA) gels or mixtures thereof.

46. A method according to claim 45 wherein the organic hydrogel is an alginate gel, an agarose gel or a lysozyme gel.

47. A method according to claim 45 or claim 46 wherein the solution of step (i) comprises Ca²⁺.

48. A method according to claims 45 to 47 wherein the organic hydrogel is pre- prepared from a stock solution.

49. A method according to any of claims 32 to 48 wherein, in step (iii) of the method, the solution of step (i) is contacted with the organic hydrogel of step (ii) following preparation of the organic hydrogel.

50. A method according to claim 49 wherein the organic hydrogel is extruded directly into the solution of step (i).

51. A method according to any one of claims 32 to 47 wherein, in step (iii) of the method, the solution of step (i) is contacted with the organic hydrogel of step (ii) during the preparation of the organic hydrogel.

52. A method according to any one of claims 32 to 51 wherein the ion-saturated organic hydrogel is partially dried before being contacted with the solution of phosphate salt in step (iv).

53. A method according to any of claims 32 to 52 wherein, in step (iv) of the method, the aqueous phosphate salt is a dibasic phosphate salt.

54. A method according to claim 53 wherein the dibasic phosphate salt is one of dibasic ammonium phosphate,

dibasic sodium phosphate, Na₂HPOj, or dibasic potassium phosphate, K₂HPO.!.

55. A method according to claim 54 wherein the dibasic phosphate salt is ammonium phosphate, (NHO₂HPO., and the solution of step (i) is CaCl₂

56. A method according to any of claims 32 to 52 wherein, in step (iv) of the method, the aqueous phosphate salt is tetra-sodium pyrophosphate, Na₄P₂O₇.

57. A method according to any one of claims 32 to 56 wherein the aqueous phosphate salt is present as a saturated solution.

58. A method for controlling the length of a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate, comprising the steps of

(i) providing an aqueous solution of divalent metal ions;

(ii) providing an organic hydrogel;

(iii) contacting the solution of step (i) with the organic hydrogel of step (ii);

(iv) incubating the ion-saturated organic hydrogel resulting from step (iii) with an aqueous phosphate salt; wherein a tubular assembly comprising one or more phases of nanocry stall ine divalent metal ion phosphate self assembles and the length of the tubular structures comprising the assembly is determined by the distance from the surface of the organic hydrogel to the solution surface.

59. A method according to claim 58 wherein during self assembly, the organic hydrogel is sealed to the bottom of the vessel in which self assembly occurs.

60. A method according to claim 58 or 59 wherein, during self assembly, the phosphate solution is recirculated.

61. A method according to any of claims 58 to 60 wherein the difference in heights of the organic hydrogel and the solution surface in the vessel in which self assembly occurs is selected such as to provide a tubular assembly of mean length in excess of 10 cm.

62. A method according to any one of claims 58 to 61 wherein the tubular assembly comprises one or more phases of nanocry stall ine Calcium phosphate.

63. A method of modifying the crystallinity of one or more phases of nanocrystalline divalent metal ion phosphate which exist in the form of a tubular assembly.

64. A method of modifying the porosity of a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate.

65. A method according to claim 64 wherein the change in porosity of the tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate is independent of a change in crystallmity of one or more phases of nanocrystalline divalent metal ion phosphate obtainable from the method of claim 63.

66. A method according to claim 64 wherein the change in porosity of a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate is related to a change in crystallinity of one or more phases of nanocrystalline divalent metal ion phosphate obtainable from the method of claim 63.

67. A method according to claim 66 wherein the change in porosity of a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate and a change in crystallinity of one or more phases of nanocrystalline divalent metal ion phosphate occur simultaneously.

68. A method according to any one of claims 63 to 67 which comprises heat treating the tubular assembly.

69. A method according to any one of claims 63 to 68 wherein the tubular assembly is as defined in any one of claims 1 to 31 or is obtainable from any one of the methods of claims 32 to 62.

70. A method according to claim 68 or claim 69 wherein the heat treatment comprises sintering the tubular assembly at a temperature of from 300⁰C to 1300⁰C.

71. A method according to claim 70, wherein the tubular assembly is sintered at temperatures of 700⁰C, 800°C, 900⁰C, 1000⁰C, HOO⁰C and 1200°C in air.

72. A method according to claim 71, wherein the tubular assembly is sintered for equal time periods at each of these temperatures.

73. A method according to claim 72 wherein the sintering time is approximately 24 hours.

74. A method according to any one of claims 68 to 73 wherein the tubular assembly material undergoes drying in air prior to the heat treatment.

75. A method according to claim 74 wherein the tubular assembly is dried at approximately 8O⁰C for approximately 24 hours.

76. A method according to any one of claims 68 to 75 wherein sintering is accompanied by densification of the walls of the tubular assemblies.

77. A method according to claim 76 wherein sintering is carried out at successively increasing temperatures between 300⁰C and 1200°C.

78. A method according to claim 76 or claim 77 wherein the densification is accompanied by the coalescence of the nanoparticles comprising the wall structures of the tubular assemblies.

79. A method according to claim 78 wherein the mean particle size of the crystalline material comprising the divalent metal ion phosphate exceeds 0.75 microns.

80. A method according to claim 78 or claim 79 wherein the mean particle size of the crystalline material comprising the divalent metal ion phosphate exceeds 1 micron.

81. A method according to any one of claims 63 to 80 wherein the divalent metal ion present in the greater concentration is Calcium.

82. A method according to claim 81 , wherein the heat treatment results in a reduction of the concentration of beta-tricalcium phosphate and increase in the concentration of alpha-trϊcalcium phosphate.

83. A method according to any one of claims 76 to 82 wherein the heat treatment comprises sintering at approximately I CK)O⁰C and above for a time in excess of 3 hours.

84. A method according to claim 83 wherein the heat treatment involves sintering above approximately 1100°C.

85. A method according to claim 84 wherein individual tubes comprising the tubular assemblies are observed to fuse together.

86. A method according to claim 85 wherein the fused material has a higher density than the unfused material.

87. A method according to claim 85 or claim 86 wherein the fused material retains macroporosity.

88. A method according to claim 87 wherein the macroporosity may be due, in part, to the presence of pores in the tube walls.

89. A tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate obtained in accordance with any of the methods defined by claims 32 to 88.

90. An additive for a biomaterial, which additive comprises a tubular assembly comprising one or more phases of nanocry stall ine divalent metal ion phosphate as defined in claim 17 or any of claims 20 to 23 or claim 89.

91. A biomaterial consisting of or comprising the additive of claim 90.

92. A biomaterial according to claim 91 which is a material suitable for use in the repair, replacement or augmentation of bone, teeth, or other mineralised tissues.

93. A biomaterial according to claim 92 which comprises Calcium Phosphate Cement (CPC).

94. A biomaterial according to claim 93 wherein the CPC component is derived from a calcium phosphate based solid component and a liquid component.

95. A biomaterial according to claim 94 wherein the calcium phosphate based solid component comprises α-TCP, β-TCP, monocalcium phosphate, dicalcium phosphate dihydrate, and tetra calcium phosphate or a mixture of two or more of these components.

96. A biomaterial according to claim 94 wherein the liquid component is water, saline solution or sodium phosphate sodium.

97. A method for the incorporation of an additive as defined in claim 90 into a biomaterial, which method comprises the steps of

(i) providing the biomaterial;

(ii) adding the additive comprising a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate, as defined in claim 17 or any one of claims 20 to 23 or claim 89 to the biomaterial of (i) and

(iii) mixing the additive with the biomaterial; whereupon the additive is incorporated into the biomaterial.

98. A method according to claim 97 wherein the biomaterial comprises a Calcium Phosphate Cement (CPC).

99. A tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate as defined in claim 17 or any one of claims 20 to 23 or claim 89 for use as a medicament.

100. A method of treating conditions affecting mineralised tissues comprising administering a biomaterial comprising a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate as defined in claim 17 or any one of claims 20 to 23 or claim 89.

101. A method according to claim 100 wherein the condition is one which leads to a need for the repair, replacement or augmentation of mineralised tissues.

102. A method according to claim 100 or claim 101 wherein the tubular assembly is incorporated in a Calcium Phosphate Cement (CPC).

103. The use of a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate as defined in claim 17 or any one of claims 20 to 23 or claim 89 for the manufacture of a medicament for the repair, replacement or augmentation of mineralised tissues.

104. Use of a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate as defined in claim 17 or any one of claims 20 to 23 or claim 89 as a vehicle for the delivery of an active agent.

105. A use according to claim 104 wherein the active agent comprises an antibiotic, an antiseptic, a growth factor, a hormone, a vector comprising DNA, or mixtures thereof.

106. A use according to claim 104 or claim 105 wherein the tubular assembly is incorporated into a bone cement and the active agent is delivered to the point of use as the cement is reabsorbed.