WO2007003969A2

WO2007003969A2 - Divalent metal ion phosphates and uses thereof

Info

Publication number: WO2007003969A2
Application number: PCT/GB2006/050193
Authority: WO
Inventors: Cait Macphee; David Wright
Original assignee: Cambridge University Technical Services Limited
Priority date: 2005-07-06
Filing date: 2006-07-06
Publication date: 2007-01-11
Also published as: WO2007003969A3

Abstract

The present invention provides a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate. The invention also relates to methods for making the divalent metal ion phosphates and miscellaneous uses thereof.

Description

Divalent metal ion phosphates and uses thereof

The present invention relates to a novel morphological form of divalent metal ion phosphates. The invention also relates to methods for making the divalent metal ion phosphates, a method for heat treating the new morphological form and products of that method and miscellaneous uses thereof.

Background of the Invention

Divalent metal ion phosphates, particularly Calcium phosphates, typically have complex phase diagrams. Given the rich structural chemistry of divalent metal ion phosphates as exemplified by the calcium phosphates, together with their suitability in a range of applications, 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. 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 therefore 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 nanocry stall ine 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 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.

In accordance with a third main aspect of the present invention, there is provided a range of compositions consisting of or comprising a tubular assembly as defined in the first main aspect of the present invention.

In accordance with a further aspect of the present invention, there is provided a method of heat treating the tubular assembly and products of that method which may have a modified crystallinity and, or 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 nni, 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 Ca^2τ, Mg²⁺, Sr²⁺, Ba²⁺, Zn²% Pb²⁺, Mn²⁺, Fe²⁺, NF, Co²⁺, Cd²⁺ and Cu²⁺.

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 Ca²⁺, Mg²⁺, Sr²", Ba²⁺, Zn²⁺, Pb^"+, Mn²⁺, Fe²⁺, Ni²', Co²⁺, Cd²⁺ and Cu^2"1 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 (NH₄ ⁺). 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 crystallographic 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 nano crystalline 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, hi 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 niahite, NH(MnPO₄J-I₂O and a predominant component of ammonium iron phosphate NH₄Fe, PO₄₁H₂O. 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.NH₄ Mn₃FePO₄H₂O) 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⁺, Nr⁺, Fe^~+, 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⁺, Mn²⁺, Fe²⁺, Ni²⁺, Co²⁺, 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, Ca²⁺ is present in conjunction with one or more of

Mg²⁺, Cu²⁺, Fe²⁺, Zn²⁺, Co²⁺, and Ni²⁺.

The concentration of the divalent metal ion solution is typically in the range of from 0.05M to 12M₅ 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, (NH.,)₂HPO.|, dibasic sodium phosphate, Na₂HPO.,, and dibasic potassium phosphate, K₂HPO₁₁. 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 hose 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, (NRi)₂HPO₄ and the solution providing the divalent metal ions is CaCh

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. In accordance with a third main aspect of the present invention, there is provided a range of compositions consisting of or comprising a tubular assembly as defined the first main aspect of the present invention.

The unusual tubular morphology of the structures comprising nanocrystalline divalent metal ion phosphate, lends itself to a variety of applications, particularly those in which the presence of hollow cavities in the tubular structures may be utilized in some way.

Accordingly, the third (and further) aspect(s) of the invention provide(s) a packaging material for liquid chromatography columns, an ultrafiltration material, a ceramic insulation material, an insulator for microwires, an artificial soil, a slow release fertiliser, an artificial additive for traditional ceramic manufacture (such as bone china, light weight ceramics), finings in fermentation, an air filter, an absorbent (for materials such as paper, nappies, spillages, alternatives to talcum powder), a flux additive in smelting, a hydrogen storage material, a component in fuel cells, carriers for cosmetics (such as moisturisers), sterilistion carriers for antibacterials or antimicrobials, a component in laser or optical applications , a paint/plaster additive textured coating, a component in aerodynamics, a micro flui die component, an abrasive, a fire retardant, a carrier for lubricants, colour additives for glazes and glassware; coral polyps consisting of or comprising a tubular assembly of one or more phases of nanocrystalline divalent metal ion phosphate defined in accordance with the first main aspect of the present invention.

In a further aspect, the tubular assemblies may be used as a component in a teaching "model" demonstrating inorganic crystallisation.

In a yet further aspect, the tubular assemblies of the first aspect of the invention lend themselves to use as catalysts and / or coated substrates, in view of the high surface area and porosity they possess. Accordingly, there is provided a catalyst and/or coated substrate consisting of or comprising a tubular assembly of one or more phases of nanocry stall ine divalent metal ion phosphate.

In particular, tubular assemblies grown on gels preincubated in BaCl₂ or SrCl₂ are particularly promising candidates as catalyst materials since barium and strontium hydroxyapatites doped with copper catalyse the dehydrogenation of propane. The catalytic high surface area of these materials may be useful in the petrochemical industry.

In a still further aspect, the tubular assemblies of the first aspect of the invention lend themselves to use, when in the form of sintered materials, as toxic metal ion storage materials due to the network of cavities of different sizes which they possess. Accordingly, there is provided a sintered material for the storage of toxic metal ions consisting of or comprising a tubular assembly of one or more phases of nanocrystalline divalent metal ion phosphate.

In a yet further aspect of the present invention, the tubular assemblies in accordance with the first aspect of the invention are useful as precursors for metal deposition. In particular, if a tube material comprising one or more divalent metal ions is exposed to a reducing atmosphere, that metal may be deposited. The resulting metal may be used to make metal microwires, micropipes, microscreening among other applications. Accordingly, there is provided a method for the deposition of a metal, comprising exposure of a tubular assembly comprising one or more phases of nanocrystalline divalent metal ion phosphate to a reducing atmosphere.

Furthermore, the tubular assemblies may be used, when in the form of hydrated metal ion phosphates, as ionic conductors.

In accordance with a yet 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 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 at700°C to 1200⁰C and a variety of temperatures in between, namely 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, 80O₃ 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 applications, including the variety of applications listed above.

FIGURES

Specific embodiments of the various aspects of the present invention are illustrated with reference Io 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 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 5 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 6a represents an apparatus for growing and harvesting tubular metal ion phosphates wherein the numbered features are as follows: 1) phosphate buffer inlet; 2) reaction vessel Hd; 3) rigid plastic sieve ring; 4) plastic mesh; 5) glass plate/petri dish; 6) prepared gel; 7) phosphate buffer outlet.

Figure 6b represents the same arrangement in photographic form.

Figure 7 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 8a to d represent photographic images of calcium phosphate tubular assemblies.

Figures 9a to d represent images of tubular structures formed after lysozyme gel soaked in 1 M SrCl₂ and partially dried is immersed in 1 M (NH₄)IHPO₄ solution.

Figure 10 represents the X-ray diffraction pattern obtained for Strontium Phosphate tubular metal harvested from the surface of a 2% Alginate gel.

Figure 11 represents a comparison of the reflections seen in the X-ray pattern in figure 10 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 CI, 00-016-0666) and d) Strontium Hydrogen Phosphate SrHPO₄, 01-070- 1215.

Figure 12 represents images of tubular structures formed after lysozyme gel soaked in 1 M BaCl? and partially dried is immersed in 1 M (Nl^)₂HPO₄ solution (a) and (b) SEM images; (c) and (d) ESEM images imaged at 3 Torr. Figure 13 represents images of structures formed after lysozyme gel soaked in 1 M CdCl₂ and partially dried is immersed in IM (NH₄)IHPO₄ solution.

Figures 14 a to d represent images of structures formed after lysozyme gel soaked in 1 M MnCl₂ and partially dried is immersed IM (NH4)₂HPO₄ solution.

Figure 15 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 16 gives a comparison of the reflections seen in the X-ray pattern in Figure 15 with known phases on the International Centre for Powder Diffraction database for b) Niahite (NH₄MnPO₄-H₂O, 00-050-0554), c) Dibasic ammonium phosphate (NH₄H₃PO₄, 00-037-1479) and d) Ammonium Hydrogen Phosphate (NH₄J₂HPO₄, 00-009-0391).

Figures 17a to c represent images of structures formed after lysozyme gel soaked in 1 M ZnCl₂ and partially dried is immersed in IM (NH,§)₂HPO4 solution.

Figures 18a to d represent images of structures formed after lysozyme gel soaked in IM Fe(II)Cl₂ and partially dried is immersed in IM (NH₄)^HPO₄ solution.

Figure 19 represents the X-ray diffraction pattern obtained for Ion Phosphate Tubular Material harvested from the surface of a 2% Alginate gel.

Figure 20 represents a comparison of the reflections seen in the X-ray pattern in figure 19 with that on the International Centre for Powder Diffraction database for b) Ammonium Iron Phosphate Hydrate (NH₄FePO₄-H₂O, 00-045-0424).

Figures 21a and b represent images of structures formed after lysozyme gel soaked in IM NiCl₂ 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 Cu(II)CI₂ and partially dried is immersed in IM (NH₄)₂HPO₄ solution.

Figures 23a to d represent images of structures formed after lysozyme gel soaked in IM CrCl₂ and partially dried is immersed in IM (NFL_f)₂HPCM solution.

Figures 24a to f represent images of tubular calcium phosphate material sintered in air for 24 hours at (a,b) 700⁰C, (c,d) 800⁰C and (e,f) 900°C inside a tube furnace showing the densification of individual grains.

Figures 25a 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 26a to f represent images of tube samples sintered in air for 24hours at (a,b) 1000⁰C; (c, d) HOO⁰C; and (e,f) 1200⁰C.

Figures 27 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) 700⁰C₅ e) 800°C, 0 900⁰C, g) 1000°C, h) 1 100⁰C and i) 1200⁰C for 24 hours.

Figure 28 represents diffraction patterns where the patterns represented in figure 27 have had peaks assigned to them and identified in comparison with known crystalline materials.

Figure 29 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 30 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) 300⁰C, c) 700⁰C, d) 800⁰C, e) 900°C, f) 1000°C, g) HOO⁰C and h) 1200⁰C for 24 hours.

Figure 31 a 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 32a 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 33a 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 34 a and b represents 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) 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 1 a: 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 Na₂HPO₄, 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

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 run, The strong peaks observed in the spectrum of Figure 3 are consistent with bands assigned to the vibrational modes of the (POj)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 (PO^ 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 hrs. The resulting gel was soaked in 1 M CaCl₂, partially dried then resuspended in 1 M Na₂HPO₄ and monitored by light microscopy.

Example 2b: Preparation of β-lactoglobulin amyloid fibril gels β-Lactoglobulin amyloid fibril gels were prepared by incubation at 85^αC 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 insulin 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 preincubated in 1 M CaCl₂ (as described above) and then placed in a solution containing

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. Ca²⁺-loaded gels were then transferred to a solution containing 1 M

Example 2e: Preparation of acrylamide gels

5 ml acrylic acid, 0.1 g N, N' -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)^HPO₄.

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 hrs 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 -7O⁰C for ~2 hrs until semi-transparent and hard. Dehydrated dried gel was placed in 1 M (NH4₂HPO₄.

Example 2g: 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 lysozvme gels

Lysozyme amyloid fibril gels were extruded manually into a solution containing 1 M CaCl₂. After incubation, the extruded gel was partially dried in air, and placed into a solution of 1 M (NI-Ls)₂HPO₄. 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-Na?HPO4 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 air-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 (NHLi)₂HPO₄ were also sufficient for CaP tube assembly. Tube growth from the lysozyme gel most likely stops when the gel becomes depleted of Ca"⁺ ions since it has been experimentally possible to recycle spent gels and reinstate tube growth by resoaking in IM CaCh.

Example 4: CaP tubular assemblies formed on agarose gels

Hydrated agarose gels (2-10%) were preincubated in 1 M CaCl₂ and then placed in a solution containing 1 M (NH₄)IHPO₄. 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 CaCh-saturated alginate gels were placed in 1 M (NI-LJ^HPCM 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 (NI-Li)₂HPC^ appeared to promote the formation of a greater number of tubes from the surface of the gel relative to samples that had not been pre frozen.

Example 6:

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

1 ) 2% weight agar powder is mixed with water, stirred continuously whilst heated until boiling until and all agar is dissolved.(Tyρically 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 1 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.

1 1 ) 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 4 to 7.

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 factional 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 7 a 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 (Ca_Io(PO₄)_S(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 SrCl₂ the structures formed (Figure 9) after immersion in IM (NH^)₂HPO₄ have tube-like morphologies similar to those formed on lysozyme soaked in 1 M CaCl₂. 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 11, 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 upto 100 micrometers in internal diameter (Figure 12a) and are assembled from granules between 2-10 microns in diameter (Figure 12b) that are courser than those found in the calcium phosphate tubular material. These granules are themselves agglomerations of sub-50 nrn 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 12c and 12d 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 13a,b.

7d Manganese

Figure 14 shows that the tube structures formed on Lysozyme gel soaked in manganese chloride have a contorted morphology. Some structures seen have an open ends whereas others appear to be occluded. The structures formed appear to consist of micrometer sized plate-like grains. Figure 15 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 16) these structures are mostly Niahite (NH₄MnPCW-HiO) 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)₂HPO₄. 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) _v Zn₅ Mn, Co or Cd ions is contorted. Figure 14 shows a contorted tube grown from a gel soaked in 1 MnCI₂. Particulates have been observed to emerge from two apertures during the formation of some of these contorted structures. 7e Zinc

Figure 17 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 lron

Figure 18a to d show the structures formed after lysozyme gel soaked in 1 M Fe(II)C12 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 19 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 20) suggest that the major phase present is Ammonium Iron Phosphate Hydrate.

This similarity to manganese system suggests the possibility of generating a range 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 (this does not exclude partial substitution with metal ions of any other valency)

7g Nickel

Figure 21 shows a tube-like assembly formed when lysozyme gel soaked in 1 M NiC12 and partially dried is immersed in IM (NH₄)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 22 shows the structures formed after lysozyme gel soaked in 1 M Cu(O)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

Figures 23 a to d show the structures formed after lysozyme gel soaked in 1 M Cr(II)Cl₂ partially dried is immersed in IM (NHiI)₂HPO₄ solution. Most of the structures formed are highly contorted and have the appearance of split tubes (figures 23a,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: Heat Treatment

Example 8 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 ⁰C₅ 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 24a,b, shows little signs of sintering and is composed like unheated material, of sub 500 nrn grains that are agglomerates of much smaller nano-scale particulates. Material heated to 800⁰C figure 24c_;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 24e,f, however the grains themselves remain distinct but have lost features at the nanoscale.

Figure 25 a shows that tubes heated to 900 ⁰C are not fused and do not lose their porosity at the tens of micrometer scale (rnacroporosity) 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 25b illustrates that at 1000 ⁰C 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 26) with the boundary between grains becoming less distinct. At 1100 ⁰C, the temperature often used to density hydroxyapatite. there is an interesting effect where growth of micron-sized grains produces pores in the tube wall. Tubes heated to 1200°C 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 27 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 ⁰C, 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-HjO 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 26=25° to 35° is expanded in figure 28 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 29 shows the FTIR spectrum obtained from discs pressed from of potassium bromide KBr (oven dried at 1 10⁰C) containing 2% by weight of a) tubular calcium phosphate and commercial obtained IiA (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 y₄PO ^"4 double band at 571 and 601 cm-1 in hydroxyapatite but lacks a band corresponding to the OH liberation band at 630 cm^"! of HA. A peak that corresponds to the y ₁ mode of PO^3" ₄ can be seen at around 950 cm^'1. A peak 88O^"1 cm around suggests the presence of HPO^2" ₄. The peaks seen at 1043 cm-1 could correspond to y₃ modes of either PO^3" ₄ in β TCP or HA. Whereas, the peak at 1108 cm^'1 corresponds to a U₃ 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 KBr disks to HO⁰C for 16 hours. There is no feature corresponding to the OH- stretching band of HA at 3572 cm^"1.

Figure 30 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 1 100 ⁰C the double band between 500 and 700 cm^"1 becomes a single peak indicating a phase change from βTCP to αTCP.

Claims

CLAIMS:

1. A tubular assembly comprising one or more phases of noncrystalline 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 nanocrystalline 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 nm.

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 11 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 nanocrystalline 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⁺, Sr²⁺, Ba²⁺, Zn²⁺, Mn²⁺, Fe²⁺, 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 nano crystalline 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^2*, Ba²⁺, Zn²⁺, Pb²⁺, Mn²⁺, Fe²⁺, Ni²⁺, Co²⁺, Cd²^₅ Cu²⁺, NH₁ ⁺ and halide ions.

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

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 nanocrystalline 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 Ca^2'*, Mg²⁺, Ba²⁺, Zn²⁺, Pb²⁺, Mn²⁺, Fe²⁺, Ni²⁺, Co²⁺, Cd²⁺, Cu²⁺, NH/ and halide 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 nialiite, (NHj(Mn, Mg, Ca)(PO.,).H₂0).

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, (NR₁(Fe, Mg, Ca)(PO₄). H₃O).

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;

(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⁺, Sr²⁺, Ba²⁺, Zn²⁺, Mn²', Fe²⁺, Ni²⁺, Co²⁺, Cd²⁺ and Cu²⁺.

35. A method according to claim 34 wherein the solution of step (i) comprises Ca²⁴ 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 Ca²⁴, Mg²⁺, Sr²⁺, Ba²⁺, Zn²⁺, Pb²⁺, Mn²⁺, Fe²⁺, Ni²⁺, Co²⁺, Cd²⁺, Cu²⁺,and NH₁ ⁺-

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 Ca²⁴.

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 (π) 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 (iϋ) 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, (NH₄)₂HPO₄, dibasic sodium phosphate, Na₂HPC₁, or dibasic potassium phosphate, K₂HPO.s.

55. A method according to claim 54 wherein the dibasic phosphate salt is ammonium phosphate, (NHi)₂HPO,, 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;

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 nanocrystalline 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 crystallinity 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 300°C to 1300⁰C.

71. A method according to claim 70, wherein the tubular assembly is sintered at temperatures of 700°C, SOO⁰C, 900⁰C, 1000⁰C, 1100⁰C and 1200⁰C 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 80⁰C 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 1200⁰C.

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-tricalcium phosphate.

83. A method according to any one of claims 76 to 82 wherein the heat treatment comprises sintering at approximately 1000⁰C 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 1100⁰C.

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. A packaging material for liquid chromatography columns consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

91. An ultrafiltration material consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

92. A ceramic insulation material consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

93. An insulator for microwires consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

94. An artificial soil consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

95. A slow release fertiliser consisting of or comprising a tubular assembly defined in accordance with aiy one of claims 1 to 31 and claim 89.

96. An artificial additive for traditional ceramic manufacture such as bone china or light weight ceramics, consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

97.Finings in fermentation consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

98. An air filter consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

99.An absorbent for materials such as paper, nappies, alternatives to talcum powder, consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

100. A flux additive in smelting consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

101.A hydrogen storage material consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

102. A component in fuel cells consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

103.A carrier for a cosmetic such as a moisturiser, consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

104. A sterilistion earner for an antibacterial or an antimicrobial, consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

105. A component in laser or optical applications, consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

106. A paint or plaster additive for providing a textured coating, consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

107. A component in aerodynamics, consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

108. A microfiuidic component consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

109. An abrasive consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

110. A fire retardant consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

11 1. A carrier for lubricants consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

1 12. A colour additive for glazes and glassware consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

1 13. Coral polyps consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

114. A catalyst and/or coated substrate consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

1 15. A catalyst and/or coated substrate as claimed in claim 1 14, wherein the divalent metal ion is Ba²' or Sr²⁺ .

116. A sintered material for the storage of toxic metal ions consisting of or comprising a tubular assembly defined in accordance with any one of claims 1 to 31 and claim 89.

117. A method for the deposition of a metal, comprising exposure of a tubular assembly defined in accordance with any one of claims 1 to 31 and 89 to a reducing atmosphere.