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WO2007003968A1 - Nouvelle forme morphologique de phosphates d'ions metalliques divalents - Google Patents

Nouvelle forme morphologique de phosphates d'ions metalliques divalents Download PDF

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Publication number
WO2007003968A1
WO2007003968A1 PCT/GB2006/050192 GB2006050192W WO2007003968A1 WO 2007003968 A1 WO2007003968 A1 WO 2007003968A1 GB 2006050192 W GB2006050192 W GB 2006050192W WO 2007003968 A1 WO2007003968 A1 WO 2007003968A1
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WO
WIPO (PCT)
Prior art keywords
phosphate
divalent metal
metal ion
tubular assembly
phases
Prior art date
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PCT/GB2006/050192
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English (en)
Inventor
Cait Macphee
David Wright
Original Assignee
Cambridge Enterprise Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0513781A external-priority patent/GB0513781D0/en
Application filed by Cambridge Enterprise Limited filed Critical Cambridge Enterprise Limited
Publication of WO2007003968A1 publication Critical patent/WO2007003968A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/12—Phosphorus-containing materials, e.g. apatite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B25/00—Phosphorus; Compounds thereof
    • C01B25/16—Oxyacids of phosphorus; Salts thereof
    • C01B25/26—Phosphates
    • C01B25/265—General methods for obtaining phosphates
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B25/00—Phosphorus; Compounds thereof
    • C01B25/16—Oxyacids of phosphorus; Salts thereof
    • C01B25/26—Phosphates
    • C01B25/32—Phosphates of magnesium, calcium, strontium, or barium
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B25/00—Phosphorus; Compounds thereof
    • C01B25/16—Oxyacids of phosphorus; Salts thereof
    • C01B25/26—Phosphates
    • C01B25/37—Phosphates of heavy metals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces

Definitions

  • 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.
  • Divalent metal ion phosphates are the most important inorganic constituents of biological hard tissues.
  • HA carbonated hydroxyapatite
  • 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 are octacalcium phosphate (or OCP) namely Ca S (HPCXO 2 (PCM) 4 5H 2 O; calcium-deficient hydroxyapatite (or CDHA); hydroxyapatite (or HA), namely Cajo(P0 4 )e(OH) 2 and ⁇ -tricalcium phosphate (or ⁇ -TCP), namely Ca 3 (PO( I ) 2 .
  • OCP octacalcium phosphate
  • PCM calcium-deficient hydroxyapatite
  • HA hydroxyapatite
  • ⁇ -TCP namely Ca 3 (PO( I ) 2
  • the latter two compounds are rarely found in nature, but are commonly used in biomaterials: ⁇ -TCP in bone cements and HA as a coating for orthopaedic and dental implants.
  • OCP Octacalcium phosphate
  • apatitic layers with atomic arrangements of calcium and phosphate ions similar to those of HA
  • OCP plays an important role in the In vivo formation of apatitic biominerals.
  • Unsubstituted CDHA i.e.
  • CDHA is a very promising compound for the manufacture of artificial bone substitutes, for example in bioceramics.
  • 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.
  • porosity in terms of pore dimension distribution and interconnectivity
  • 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.
  • 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 Ca 2+ 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.
  • 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 Ca 2+ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • EDAX Energy Dispersive X-Ray Microanalysis
  • 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.
  • 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.
  • divalent metal ion is selected from the group of alkaline earth metal ions, divalent transition metal ions and when the divalent metal ion is Pb 2+ .
  • the divalent metal ion is selected from the group of Ca 2+ , Mg 2+ , Sr + , Ba 2+ , Zn 2+ , Pb 2+ , Mn 2+ , Fe 2+ , Ni 2+ , Co 3+ , Cd 2+ and Cu 2i .
  • the tubular assembly will comprise one or more phases in which there is partial ion substitution on either the cation or anion sublattice.
  • the divalent metal ion is selected from the group of Ca
  • substitution on the anion sublattice also occurs readily, for example, with halide anions but other anions are not excluded.
  • the divalent metal ion present in the greatest concentration in the one or more phases of nanocrystalline divalent metal ion phosphate is Ca 2+ such that the tubular assembly comprises a Calcium phosphate.
  • 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.
  • 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.
  • the divalent metal ion is Sr 2+ such that the tubular assembly comprises a Strontium phosphate.
  • the tubular assembly comprises a Strontium phosphate.
  • apatitic Calcium and Strontium phosphates have been identified, a minor component present appears to be a hydrated ammonium (Ca or Sr) phosphate.
  • Mn 2+ and Fe 2+ Further divalent metal ions which have shown to support particularly favourable morphologies are Mn 2+ and Fe 2+ .
  • Phosphate phases which have been identified comprise, respectively , a predominant component of ammonium manganese phosphate nialiite, NH 1 MnPOj-H 2 O and a predominant component of ammonium iron phosphate NH 1 FePO 4 -H 2 O.
  • 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 Mn 5 Fe PO 4 -H 2 O) 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
  • step (iii) contacting the solution of step (i) with the organic hydrogel of step (ii);
  • step (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.
  • divalent metal ions include, but are not limited to, Ca 2+ , Mg 2+ , Ba 2+ , Co 2+ , Zn 2+ , Ni 2+ , Fe 2+ , Cu 2+ , Pb 2+ , Cd 2+ , Sr 2+ and Mn 2+ .
  • the identity of the counterions for these cations does not appear to be critical, however, chloride and nitrate have worked particularly well.
  • Ca 2+ 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 Mg 2+ , Sr + . Ba 2+ , Zn 2+ , Pb 2+ , Mn", Fe 2+ , Ni 2 % Co 2t , Cd 2+ , Cu 2+ and ammonium ion (NH/). There may also be substitution on the anion sublattice, for example, with halide anions, but other anions are not excluded.
  • 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 CaCl 2
  • organic hydrogels 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.
  • 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
  • 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.
  • 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.
  • 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, Na 2 HPO 4 , and dibasic potassium phosphate, K 2 HPO 4 .
  • dibasic phosphate salts such as dibasic ammonium phosphate, (NROzHPO,, dibasic sodium phosphate, Na 2 HPO 4 , and dibasic potassium phosphate, K 2 HPO 4 .
  • tetra-sodium pyrophosphate, Na 4 P 2 O 7 has proved particularly suitable.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • step (iii) contacting the solution of step (i) with the organic hydrogel of step (ii);
  • step (iv) incubating the ion-saturated organic hydrogel resulting from step (iii) with an aqueous phosphate salt;
  • 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.
  • the organic hydrogel will be in contact with the bottom of the vessel in which self assembly occurs.
  • 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.
  • phosphate salt ammonium phosphate
  • (NH 4 ) 2 HPO 4 ammonium phosphate
  • the solution providing the divalent metal ions is CaCl 2.
  • 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.
  • compositions and uses are directed primarily to the technical field of biomaterials.
  • 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.
  • the biomaterial will be any material used to repair, replace or augment bone, teeth, or other mineralised tissues.
  • the biomaterial may also be a composition with a wider general biomedical application, such as toothpaste.
  • 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 Ca 2+ .
  • the biomaterial will be any material used to repair, replace or augment bone, teeth, or other mineralised tissues.
  • the biomaterial further comprises a Calcium Phosphate Cement (CPC).
  • CPC Calcium Phosphate Cement
  • the tubular structures are incorporated primarily to aid osteointegration and fluid transport
  • CPCs Calcium phosphate cements
  • HA hydroxyapatite
  • 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.
  • CPCs 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'");
  • ⁇ -BSM poorly crystalline apatitic calcium phosphate
  • the role of the additive is essentially that of a filler.
  • 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 Ca 2+ .
  • the solid component from which the CPC is derived is selected from ⁇ -TCP, ⁇ -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.
  • Solid cement components which have proved particularly suitable are "Bone Source”, ⁇ - 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.
  • 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 Ca 2+ into a biomaterial which method comprises the steps of
  • 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.
  • 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.
  • the tubular assembly may be added to the calcium phosphate solid component prior to the addition of liquid component.
  • 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.
  • calcium phosphate tubes of the invention and biomaterial comprising said tubes provide a scaffold on which new bone can grow and remodel.
  • 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.
  • the invention also relates to a method of treating conditions affecting mineralised tissues by administering a biomaterial comprising calcium phosphate tubular assemblies.
  • 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.
  • 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.
  • 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.
  • the calcium phosphate tubular assemblies may be used to facilitate drug delivery by incorporating an "active agent" in the hollow cavities they possess.
  • an active agent 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.
  • the active agents 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.
  • 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 1400 0 C and more preferably at 700 0 C to 1200 0 C and a variety of temperatures in between, for example 800, 900, 1000, 1100, each for 24 hours in air.
  • 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.
  • 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 1200 0 C, each time for 24 hours in air.
  • 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.
  • 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 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
  • P/L 1 and 2 refer to control cements with ratios of 10% (w/w/) CaCO 3 / 90% (w/w) ⁇ - TCP to IM(NFLi) 2 HPO 4 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 SrCl 2 and partially dried is immersed in 1 M (NRO 2 HPO 4 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 (Sr 5 (PO 4 ) 3 (OH), 00-033-1348) c) Strontium Chloride Phosphate (Sr 5 (PO 4 ) 3 Cl, 00-016-0666) and d) Strontium Hydrogen Phosphate SrHPO 4 , 01-070-
  • Figure 16 represents images of tubular structures formed after lysozyme gel soaked in 1 M BaCl 2 and partially dried is immersed in 1 M (NH 4 ) ⁇ HPO 4 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 CdCl 2 and partially dried is immersed in IM (NH ⁇ ) 2 HPO 4 solution.
  • Figures 18 a to d represent images of structures formed after lysozyme gel soaked in 1 M MnCl 2 and partially dried is immersed IM (NH 4 )7HPO 4 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 (NH 4 MnPO 4 -H 2 O, 00-050-0554), c) Dibasic ammonium phosphate (NH 4 H 2 PO 4 , 00-037-1479) and d) Ammonium Hydrogen Phosphate (NH 4 )IHPO 4 , 00-009-0391).
  • Figures 21a to c represent images of structures formed after lysozyme gel soaked in IM ZnCl 2 and partially dried is immersed in IM (NH 4 ) ⁇ HPO 4 solution.
  • Figures 22a to d represent images of structures formed after lysozyme gel soaked in IM Fe(II)Cl 2 and partially dried is immersed in IM (NH 4 )IHPO 4 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 (NH 4 FePO 4 -H 2 O, 00-045-0424).
  • Figures 25a and b represent images of structures formed after lysozyme gel soaked in IM NiCl 2 and partially dried is immersed in 1 M (NFLi) 2 HPO 4 solution.
  • Figures 26a to d represent images of structures formed after lysozyme gel soaked in IM Cu(II)Cl 2 and partially dried is immersed in IM solution.
  • Figures 27a to d represent images of structures formed after lysozyme gel soaked in 1 M CrCl 2 and partially dried is immersed in IM (NH 4 )IHPO 4 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) 800 0 C 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) 900 0 C tubes remain unfused whereas at (b) 1000 0 C the tubes fused together.
  • Figures 30a to f represent images of tube samples sintered in air for 24 hours at (a,b) 1000 0 C; (c, d) 1100 0 C; and (e,f) 1200 0 C.
  • Figures 31 a to i show the X-ray diffraction patterns for tubular calcium phosphates a) as precipitated, b) after drying at 80 0 C for 24 hours and after heating at c) 300 0 C, d) 700°C, e) SOO 0 C, f) 900°C, g) 1000 0 C, h) HOO 0 C and i) 1200 0 C 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 100 0 C) discs containing 2% wt calcium phosphate power, a) as precipitated and material heated to b) 300 0 C 5 c) 700 0 C, d) 800 0 C, e) 900°C, f) 1000°C, g) 1 100 0 C and h) 1200°C for 24 hours.
  • Figure 35a to c represent a) single X-ray microtomography scan of Ca-P tube approximately 600 ⁇ m in length and 50 ⁇ m 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 ⁇ m 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 ⁇ m in length and 50 ⁇ m 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 lO ⁇ m 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.
  • 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 ⁇ HPO 4 , 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.
  • 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.
  • Insulin amyloid fibril gels were prepared by incubation at 37°C of a 50 mg/ml stock solution of insulin in pH 1.80 H 2 O adjusted with phosphoric acid for 72 lirs. The resulting gel was soaked in 1 M CaCb, partially dried then resuspended in 1 M Na 2 HPO 4 and monitored by light microscopy.
  • Example 2b Preparation of ⁇ -lactoglobulm amyloid fibril gels ⁇ -Lactoglobulin amyloid fibril gels were prepared by incubation at 85°C of a 250 mg/ml stock solution of ⁇ -lactoglobulin in pH 1.96 H 2 O adjusted with HCl for 72 hrs. The resulting gel was soaked in 1 M CaCl 2 , partially dried then resuspended in 1 M Na 2 HPO 4 and monitored by light microscopy.
  • Bovine serum albumin (BSA) gels were prepared by incubation at 7O 0 C of a 250 mg/ml stock solution of BSA in pH 2.08 H 2 O adjusted with HCl for 72 hrs. The resulting gel was soaked in 1 M CaCl 2 , partially dried then resuspended in 1 M Na 2 HPO 4 and monitored by light microscopy.
  • BSA Bovine serum albumin
  • 1% agarose gels were also prepared using 1 M CaCl 2 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 ⁇ ) 2 HPO 4
  • 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 5 ⁇ m to over 100 ⁇ m.
  • 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-Na 2 HPO 4 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 (NH 4 ) I HPO 4 were also sufficient for CaP tube assembly. Tube growth from the lysozyme gel most likely stops when the gel becomes depleted Of Ca 2+ ions since it has been experimentally possible to recycle spent gels and reinstate tube growth by resoaking in IM CaCl 2 .
  • ESEM Environmental Scanning Electron Microscope
  • Example 4 CaP tubular assemblies formed on agarose gels
  • agarose gels (2-10%) were preincubated in 1 M CaCIi and then placed in a solution containing 1 M (NRs) 2 HPO 4 .
  • 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 (NH 4 )IHPO 4 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 CaCl 2 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.
  • 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:
  • 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.
  • Both moulds are designed to fit inside a Petri dish so the gel can be protected from contamination during setting.
  • 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.
  • 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.
  • 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.
  • 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.
  • a ligature plastic cable tie
  • 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(P0 4 )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.
  • 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.
  • 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.
  • self assembly has been observed from gels soaked in molar chloride solutions of Cu(II), Fe(II) , Zn, Co, Ni after immersion in IM (NH 4 )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.
  • FIG. 18 shows a contorted tube grown from a gel soaked in 1 MnCl 2 . Particulates have been observed to emerge from two apertures during the formation of some of these contorted structures.
  • 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.
  • 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 (NH 4 ) 2 HPO 4 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 (NH 4 FePO 4 -H 2 O) (figure 24) suggest that the major phase present is Ammonium Iron Phosphate Hydrate.
  • 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) 2 HPO 4 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.
  • Figure 26 shows the structures formed after lysozyme gel soaked in 1 M Cu(II)Cl 2 and partially dried is immersed in IM (NHJI) 2 HPO 4 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.
  • FIG. 7i Chromium Figure 27 show the structures formed after lysozyme gel soaked in 1 M Cr(II)Cl 2 partially dried is immersed in IM (NHLt) 2 HPO 4 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.
  • 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.
  • Control cements were prepared by adding 1 M (NH ⁇ ) 2 HPO 4 solution to dry cement mixture comprising 10% (w/w) CaCO 3 / 90% (w/w) ⁇ -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
  • 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.
  • Tubes were also harvested in aqueous suspension, which was then added to dry cement powder.
  • 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.
  • 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).
  • HOS Human Causian Osteosarcoma
  • Ground tube material 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 M ⁇ ) for at least 3 days before being drained and washed three times in ultra pure water and sterilized in 96% ethanol.
  • Cells were plated out in 96 well plates with a density of 5X10 4 cells per well and grown to confluency.
  • 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 ⁇ 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 ⁇ M 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.
  • 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.
  • Tubular Calcium Phosphate material heated to 700 0 C, 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 800 0 C 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 900 0 C, figure 28e,f however the grains themselves remain distinct but have lost features at the nanoscale.
  • Figure 29a shows that tubes heated to 900 0 C 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 0 C and above has fused into a solid conglomerate but retains porosity on at the tens of micrometer scale.
  • Figure 29b illustrates that at 1000 0 C 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 100 0 C (figure 30) with the boundary between grains becoming less distinct.
  • Figure 31 shows the X-ray diffraction patterns for tubular calcium phosphates a) as precipitated, b) after drying at 8O 0 C for 24 hours and after heating at c) 300 0 C, d) 700 0 C 5 e) 800 0 C f) 900 0 C g) 1000 0 C, h) 1100 0 C and i) 1200 0 C for 24 hours.
  • Samples heated below 300 0 C have very broad peaks associated with poorly crystalline nano- sized calcium phosphates. The sharp peak seen at 7° is associated with the presence of NH 4 CaPO 4 -H 2 O and is absent from the oven dried sample perhaps due to the loss of water of crystallisation.
  • 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 ⁇ TCP. 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.
  • the 0210 reflection for ⁇ TCP at 31° becomes increasingly intense being a weak peak at 700 0 C pattern (d) to become the most intense peak in the 1100 0 C pattern (h).
  • the principal 211 reflection of HA at 32° strong in the X- ray diffraction patterns at 700 and 800 0 C diminishes with increasing temperature and is absent the pattern at 1 100 0 C.
  • the predominance of the ⁇ TCP phase seen in the sample sintered for 24 hours at 1100 0 C suggests that the ratio of calcium to phosphate in the tube material as precipitated is close to 1.5.
  • the reflections assigned to ⁇ TCP have almost completely disappeared to be replaced with those of ⁇ TCP 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 11O 0 C) containing 2% by weight of a) tubular calcium phosphate and commercial obtained HA (b), ⁇ TCP (c) and ⁇ TCP (d) from 400 to 4000 cm '1 .
  • the tubular calcium phosphate material as precipitated contains peaks corresponding to the j? 4 PO 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 .
  • the peaks seen at 1043 cm-1 could correspond to U 3 modes of either PO 3" 4 in ⁇ TCP or HA.
  • the peak at 1108 cm '1 corresponds to a v$ mode of PO 3" 4 reported for ⁇ TCP.
  • a band in the region 1440-1550 corresponds to CO 3 2" bands (v 3 ) (but a spectrometer that can be purged with nitrogen will be needed to confirm this).
  • 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 0 C 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 P 2 O 7 4" known to appear when ACP is heated to 650 0 C.
  • the CaP material is heated above 1100 0 C the double band between 500 and 700 cm " ' becomes a single peak indicating a phase change from ⁇ TCP to ⁇ TCP.

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Abstract

La présente invention concerne un ensemble tubulaire comprenant une ou plusieurs phases de phosphate de ion métallique divalent nanocristallin. L'invention concerne également des méthodes destinées à la fabrication de ces ensembles tubulaires et leurs applications dans le domaine biomédical.
PCT/GB2006/050192 2005-07-06 2006-07-06 Nouvelle forme morphologique de phosphates d'ions metalliques divalents WO2007003968A1 (fr)

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CN109350766A (zh) * 2018-11-15 2019-02-19 无锡中科光远生物材料有限公司 一种琼脂糖醋酸酯纳米纤维膜的制备方法
CN113912034A (zh) * 2021-11-22 2022-01-11 河南佰利新能源材料有限公司 一种磷酸铁及其制备方法

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