WO2011139136A1 - Calcium phosphate cement composition and method of making the same - Google Patents

Calcium phosphate cement composition and method of making the same Download PDF

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Publication number
WO2011139136A1
WO2011139136A1 PCT/MY2010/000243 MY2010000243W WO2011139136A1 WO 2011139136 A1 WO2011139136 A1 WO 2011139136A1 MY 2010000243 W MY2010000243 W MY 2010000243W WO 2011139136 A1 WO2011139136 A1 WO 2011139136A1
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Prior art keywords
calcium phosphate
composite
phosphate composite
bone
cement
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PCT/MY2010/000243
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French (fr)
Inventor
Sharif Hussein Sharif Zein
Kah Ling Low
Kean Khoon Chew
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Universiti Sains Malaysia
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Publication of WO2011139136A1 publication Critical patent/WO2011139136A1/en

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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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/42—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having an inorganic matrix
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • the present invention relates to high-strength calcium phosphate cement compositions, and more particularly, to a high-strength calcium phosphate composite material comprising protein and carbon material for use as bone replacing materials.
  • Bone formation or replacement is often a desired therapy for bone loss or defects due to fractures or bone degenerative diseases.
  • a biomaterial for bone formation or replacement should have sufficient mechanical load-bearing and impact strength to maintain structural integrity and provide a suitable environment to induce new bone formation.
  • a potential bone-replacement material would include an organic polymer for mechanical strength and ease-of-use and inorganic particles that participate in the bone mineralization pathway.
  • Phosphate-based hydraulic structural cements are well known (e.g., see Friedman et al "BoneSource hydroxyapatite cement: a novel biomaterial for craniofacial skeletal tissue engineering and reconstruction" Journal of Biomedical Materials Research (1998). 43(4), 428-432). However, these cements do not contain peptide material and carbon nanotubes.
  • CPC Calcium phosphate cement
  • the final cement suffers from a relatively low compressive strength (i.e., limited its use to non- load bearing applications in orthopedics), poor injectability (i.e., resulted in invasive surgical technique which involved open wound surgery) and lack of macroporosity (i.e., limited space available for bone in-growth).
  • compressive strength i.e., limited its use to non- load bearing applications in orthopedics
  • injectability i.e., resulted in invasive surgical technique which involved open wound surgery
  • macroporosity i.e., limited space available for bone in-growth
  • BSA bovine serum albumin
  • the present invention broadly discloses a high-strength and biocompatible calcium phosphate composite material that is suitable for use as bone implants or repairs. It will be understood that those skilled in the art may identify numerous other uses for the composite material of the present invention, even if it is not specifically indicated in the present disclosure. Accordingly, the calcium phosphate composite of the present invention, which may be used as an injectable bone replacing material, comprises at least one calcium phosphate reinforced with a protein concentrate and carbon nanotubes to produce a high compressive strength composite or cement.
  • the calcium phosphate composite of the present invention is preferably an injectable bone replacing material in vivo.
  • the calcium phosphate present in the calcium phosphate composite is comprised of equimolar ⁇ -tri-calcium phosphate ( ⁇ -TCP) and dibasic calcium phosphate anhydrous (DCPA).
  • ⁇ -TCP equimolar ⁇ -tri-calcium phosphate
  • DCPA dibasic calcium phosphate anhydrous
  • the protein concentrate is present in the calcium phosphate composite in an amount of at least 15% of total weight of the calcium phosphate composite.
  • the carbon nanotubes are present in the calcium phosphate composite in an amount of at least 0.5% of total weight of the calcium phosphate composite.
  • the protein concentrate is a serum albumin protein and may be, for example bovine serum albumin (BSA) having 607 amino acid residues and a molecular weight of 66.4 kDa.
  • BSA bovine serum albumin
  • the addition of low concentrations of BSA enhances calcium phosphate crystal growth (being favourable for bone tissue mineralisation), whereas higher concentrations inhibit calcium phosphate crystallisation. Further, the addition of BSA improves cohesiveness of the cement composite.
  • the carbon nanotubes are multi-walled carbon nanotubes that may be hydroxylated or non-hydroxylated. Having the best performance among carbon materials, it is anticipated that carbon nanotubes can potentially enhance the properties of calcium phosphate composite of the present invention.
  • the electrical properties of carbon nanotubes are highly responsive to the changes in the surrounding electrostatic environment and interface charge transfer, causing drastic changes through simple adsorptions of certain molecules or polymer. Thus, the presence of chemical functional groups will induce chemical reaction along the interface, and hence improve the reinforcement efficiency of the cement.
  • the present invention provides a method for preparing calcium phosphate cement, which comprises the steps of: i. providing a calcium phosphate mixture by adding equimolar ⁇ -tricalcium phosphate ( ⁇ -TCP) to dicalcium phosphate anhydrous (DCPA);
  • ⁇ -TCP equimolar ⁇ -tricalcium phosphate
  • DCPA dicalcium phosphate anhydrous
  • step (iii) adding water to the mixture of step (ii) to obtain a calcium phosphate composite material or cement paste.
  • the composite material may also be molded after setting of the cement, for instance by milling or by cutting, into a desired shape.
  • the moulded cement material of the present invention may take any shape desirable.
  • the present invention provides the use of the calcium phosphate composite material according to the present invention as described above for the induction of bone formation in a living organism.
  • the present invention provides the use of the calcium phosphate composite material according to the present invention as described above as an implant material alone or combined with growth factors or/and cells for the production of autologous bone in a non- osseous site.
  • the present invention provides the use of the calcium phosphate composite material according to the present invention as described above for the production of a medical implant or device alone or combined with growth factors or/and cells.
  • Uses of the invention are particularly beneficial for the reconstruction or replacement of bone and/or in dental surgery.
  • Figure 1 is a graph illustrating compressive strength of the calcium phosphate composite (CPC/MWCNTs-OH/BSA) according to the present invention
  • Figure 2 is a scanning electron microscope (SEM) image of the CPC/MWCNTs- OH/BSA according to the present invention
  • Figure 3 is a graph illustrating FTIR patterns of the CPC/MWCNTs-OH/BSA according to the present invention.
  • Figure 4 is a diagram illustrating X-ray diffraction patterns of the CPC/MWCNTs- OH/BSA according to the present invention.
  • a calcium phosphate material or calcium phosphate composite in accordance with the present invention may be based on any calcium phosphate (CaP), such as a CaP obtained by precipitation from an aqueous solution at low temperature (e.g. 20-80°C) or by a high temperature process (but preferably not higher than 100°C).
  • CaP calcium phosphate
  • Highly preferred calcium phosphates are the calcium orthophosphates.
  • the term "calcium orthophosphate” as used herein refers to a family of compounds, each of which contains a calcium cation, Ca , and a phosphate anion, P0 4 3 ⁇ .
  • calcium orthophosphates including monocalciurh orthophosphate (monobasic), dicalcium orthophosphate (dibasic), tricalcium orthophosphate (tribasic), and hydroxyapatite (penta calcium triphosphate).
  • calcium pyrophosphates e.g., dicalcium diphosphate (Ca 2 P 2 0 7 ), synonym: calcium pyrophosphate), calcium pyrophosphate dihydrate (CPPD, Ca 2 P 2 0 7 .2H 2 0) and calcium dihydrogen diphosphate (CaH 2 P 2 0 ; synonyms: acid calcium pyrophosphate, monocalcium dihydrogen pyrophosphate, and polyphosphate (CaP 2 0 6 )n, n>2; synonyms: calcium metaphosphates, calcium polymetaphosphates, and combinations of the various phosphates.
  • calcium pyrophosphates e.g., dicalcium diphosphate (Ca 2 P 2 0 7 )
  • CPPD calcium pyrophosphate dihydrate
  • CPPD calcium pyrophosphate dihydrate
  • CaH 2 P 2 0 calcium dihydrogen diphosphate
  • CaP 2 0 6 calcium metaphosphates, calcium polymetaphosphates, and combinations
  • Non-limiting examples of the calcium phosphate compound that may be used in aspects of the present invention are as follows:
  • apatite calcium fluoro-phosphate, Ca 5 (F,CI,OH)(P0 4 )3)
  • DCPA dicalcium phosphate anhydrous
  • DCPD dicalcium phosphate dihydrate
  • MCPA monocalcium phosphate anhydrous
  • OCP octacalcium phosphate
  • mixtures of two or more of the above such as mixtures of MCPM or MCPA with another CaP;
  • the calcium phosphates used in methods of the present invention are nanocrystals and are preferably obtained by precipitation from a solution comprising suitable calcium and phosphate sources.
  • suitable calcium source is Ca(N0 3 )2.4H 2 0.
  • a suitable phosphate source is (NH 4 ) 2 HP0 4 .
  • ammonia may be used as a base.
  • calcium phosphates nanocrystals may be obtained by other methods, such as by milling and/or sieving of calcium phosphates microparticles. However, the preparation of calcium phosphates nanocrystals by precipitation is most preferred.
  • the calcium phosphates particularly in case they are derived from natural sources, may be calcined prior to use as used in most of the applications.
  • Preparation of calcium phosphate composite material of the invention which preferably used as an implant in living tissue should mimic the way by which living organs produce mineralized tissues, the calcium phosphate is therefore preferably not sintered or heated.
  • the composite material is preferably both sufficiently compatible and sufficiently biodegradable for use as an implant in living tissue.
  • the calcium phosphate on which the composite material is based is preferably (bio)resorbable, meaning that it exhibits chemical dissolution and cell- mediated resorption when placed in a mammalian body.
  • a composite material according to the invention is preferably based on any calcium phosphates having Ca/P ratio of 1.67 or combinations thereof.
  • the carbon nanotubes are only a few nanometers wide and are comprised of cylindrical carbon molecules with properties that make them potentially useful as mechanical reinforcement materials in accordance with the present invention. These tubes consist of rolled up hexagons, 10,000 times thinner than a human hair. Ideal CNTs can be described as a seamless cylinder of rolled up hexagonal networks of carbon atoms, which is capped with half a fullerene molecule at the end. Their strength is one to two orders of magnitude and weight is six times lighter than steel. Besides that, CNTs are built from sp 2 carbon units and consist of honeycomb lattices and are a seamless structure. They are tubular having a diameter of a few nanometers but lengths of many microns.
  • the multi-walled carbon nanotubes are closed graphite tubules rolled like a graphite sheet. Diameters usually range between 2 and 25 nm and the distance between sheets is about 0.34 nm. Theses tubes have a tendency to form in bundles which are parallel in contact and consist of tens to hundreds of nanotubes. Other possible applications range from semiconductors, electronic memory, drive products, medical delivery systems and in plastics such as automobile body panels, paint, tires, and as flame retardants in polyethylene and polypropylene.
  • Pristine carbon nanotubes CNTs are inherently hydrophobic, therefore the main obstacle in the utilization of CNT in biology and medicinal chemistry is their lack of solubility in most, solvents compatible with the biological milieu (aqueous based).
  • modification of the surface of CNT such as functionalization with different molecules is achieved by adsorption, electrostatic interaction or covalent bonding of different molecules and chemistries that render them more hydrophilic.
  • the water solubility of CNT is improved and their biocompatibility profile is completely transformed.
  • the bundling or aggregation of individual tubes through van der Waals forces is also reduced by the functionalization of their surface.
  • the CNT present in the calcium phosphate composite is hydroxide functionalized multiwalled carbon nanotubes (MWCNTs-OH).
  • BSA bovine. serum albumin
  • the scaffold materials or implant materials in accordance with the present invention may be used in variable forms such as in the form of blocks, foams, sponges, granules, cement, implant coatings, composite components and may for instance be combined with organic or inorganic materials or with ceramics and may be from various origins, natural, biological or synthetic.
  • the various forms may for instance be obtained by injection moulding, extrusion, solvent casting, particular leaching methods, compression moulding and rapid prototyping such as 3D Printing, Multiphase Jet Solidification, and Fused Deposition Modeling (FDM) of the materials.
  • Calcium phosphate cement (CPC) composite in accordance with the present invention may be used as a synthetic injectable (bone) composite paste to fill or conform to the defects in hard tissues and may therefore undergo self-hardening in situ to form hydroxyaptite (HA), which is the putative mineral in teeth and bones.
  • HA hydroxyaptite
  • Such a cement paste may comprise a mixture of ⁇ -tri-calcium phosphate ( ⁇ -TCP) and dibasic calcium phosphate anhydrous (DCPA) in combination with bovine serum albumin (BSA) and hydroxylated multiwalled carbon nanotubes (MWCNTs-OH).
  • CPC calcium phosphate cement
  • the calcium phosphate cement (CPC) composite of the present invention may be dense or porous, but preferably the composite material is macroporous. Porosity can be easily achieved by the composite itself due to the attempt of using ⁇ -TCP and DCPA as main components that form hydroxyapatite (HA). This mixture results in an excellent combination effect of degrading and promoting bone or HA formation. As a candidate for bone graft material, ⁇ -TCP showed an excellent merit in bone formation. Therefore, the mixture of ⁇ -TCP and DCPA has been used as bone substitute for many years.
  • the solubility of DCPA is roughl eight times higher than ⁇ -TCP and approximately 15 times higher than HA at physiologic pH in vitro.
  • Example 1 Preparation of calcium phosphate cement composite
  • a calcium phosphate powder mixture was prepared by mixing equimolar fractions of ⁇ - tricalcium phosphate, ⁇ -03 3 ( ⁇ 0 4 ) 2 , ( ⁇ -TCP) and dicalcium phosphate anhydrous, CaHPCv, (DCPA) (both supplied by Sigma-Aldrich), which were then mixed with deionised Water.
  • CPC calcium phosphate cement
  • MWCNTs-OH hydroxylated multiwalled carbon nanotubes
  • bovine serum albumin supplied by Fluka
  • the cement paste was blended using a mechanical overhead stirrer at 30-50 rotations per minute for 1 hour and then firmly packed by manual spatulation into a cylindrical stainless steel mould with a diameter of 25 mm.
  • the packed stainless steel mould was wrapped with water-soaked wipe to prevent the sample from drying out and was then stored in a Gyro-Rocker Incubator (Model: S170) at 37°C and 97% humidity for 24 hours. All experiments were carried out under controlled conditions at temperatures of 24-26°C and relative humidity of 50-60%. Once taken out from the incubator, the cylindrical implants were carefully taken out from the mold.
  • the compressive strength of the cylindrical implants was tested using an Instron 3367 universal testing machine at a crosshead speed of 1.0 mm/min. Characterization techniques were used to validate the chemical and physical properties of the composite implants. Scanning electron microscopy (SEM) was performed using a Leo Supra 35VP-24-58 microscope in order to investigate the microstructure and morphology of the composite.
  • FTIR Fourier transform infrared
  • FTIR 2000 spectrometer over the frequency range 4000 to 400 cm "1 in KBr pellets.
  • FTIR spectroscopy was employed to characterize the presence of specific surface functional groups in the composite.
  • X-ray Diffraction (XRD) was used to determine the crystalline structure of the cement composite. The analysis was recorded on a Siemens D5000 diffractometer using a diffraction angle 2 ⁇ in the range 10-70° at a sweep rate of 0.047sec. The qualitative analysis of different characteristic patterns of the materials investigated was achieved by comparing peaks of the XRD spectrum with the standard diffraction patterns of specific compounds based on the International Centre for Diffraction Data (ICDD).
  • ICDD International Centre for Diffraction Data
  • Injectability was qualitatively assessed and evaluated by extruding the paste through a disposable syringe.
  • a 10 ml syringe with a diameter of 16 mm and needle with an inner diameter of 2 mm was filled with the calcium phosphate cement paste, which was then extruded from the syringe manually within a few seconds at a relatively constant speed.
  • the injectability test was carried out in two parts. The objective of the first part was to examine the UP ratio required to produce a workable and injectable calcium phosphate cement paste. Whilst the second part investigated the injectability, which was determined considering the percentage mass of the calcium phosphate cement paste extruded from the syringe divided by the original mass of the paste inside the syringe.
  • Example 4 Effects of the composite
  • Figure 1 shows the effects of multiwalled hydroxylated carbon nanotubes (MWCNTs- OH) on the compressive strength of the calcium phosphate cement (CPC). It was found that with the addition of MWCNTs-OH, the compressive strength of pure CPC composite significantly increased, from 1.0 ⁇ 0.2 MPa to 1.5 ⁇ 0.3 Pa. Moreover, it could be confirmed that when bovine serum albumin (BSA) was added, the compressive strength of CPC/MWCNTs-OH composite significantly increased to 16 ⁇ 3 MPa. Due to the formation of interfacial bonding between hydroxyapatite (HA) nuclei and MWCNTs-OH, HA crystals precipitated on the surface of MWCNTs-OH.
  • BSA bovine serum albumin
  • a strong interfacial bonding is a necessary condition for improving the mechanical properties of composite, in order to achieve load transfer across the MWCNTs matrix interface. This interface favors the load transfer between the MWCNTs-OH and the matrix leading to improved mechanical properties. Furthermore, the improvement in the mechanical properties with addition of BSA can be explained by considering that appropriate amounts of BSA are capable of promoting CPC crystal growth. At low concentrations ( ⁇ 10 g/l), BSA has been hypothesized to stabilize nuclei and promote growth of octacalcium phosphate crystals, while at higher concentrations; crystal growth seems to be impeded by high BSA coverage. Although the net charge on BSA at neutral pH is -17 mV, the protein contains both positively and negatively charged residues.
  • Figure 2 shows SEM images of the composite microstructures.
  • morphologies of the HA crystal structures of CPC/MWCNTs-OH/BSA composites were observed, as shown in Figure 2, respectively.
  • Figure 2 shows that well-packed HA crystals of plate-like shape and clusters are grown in CPC/MWCNTs-OH/BSA composite of the present invention. It is hypothesized that this particular microstructure led to increased compressive strength of the composite of the present invention, as compared with pure calcium phosphate cement.
  • FTIR Fourier Transform Infrared Analysis
  • Figure 3 illustrates the FTIR results on the CPC/MWCNTs-OH/BSA composite of the present invention.
  • the spectra show absorption bands at 3297-3307 cm-1 which correspond to the strong characteristic peak of stretching mode of hydroxyl group (- OH).
  • the peaks pertaining to the HA phase are hydroxyl bands at 3302 cm “1 and 3307 cm “1 .
  • the characteristic bending mode of intercalated H 2 0 can be observed at 1655-1656 cm “1 .
  • the phosphate band derived from the P-0 asymmetric stretching mode (v of the P0 4 3" group was identified in the region 943-1 128 cm "1 , indicating a deviation of phosphate ions from their ideal tetrahedral structure.
  • the absorption bands appearing at about 400 to 600 cm “1 can be attributed to the (v 4 . mode) 548, 587 and 603 (v 4 mode) and double (v 2 )-degenerated fundamental bending mode of the P0 4 3 functional group.
  • the bands observed at 1543 cm “1 (v 3 mode) and 1546 cm “ (v 3 mode) can be assigned to the C0 3 2" group.
  • the XRD pattern of the CPC/MWCNTs-OH/BSA composite of the present invention is shown in Figure 4. Diffraction peaks corresponding to HA crystalline phase were detected at 2 ⁇ angles of 26, 29, 32, 40 and 53°. It is therefore evident that it is possible to obtain self-setting injectable HA by mixing ⁇ -TCP and DCPA with de- ionized water. The sharp and narrow diffraction peaks observed in the regions of relevance to HA suggest that the HA formed is crystalline, which can be correlated with the crystal morphology observed by SEM. As a whole, the XRD, SEM and FTIR results showed that the investigated CPC composites developed a crystalline HA phase, which is in its chemical and crystallographic composition similar to the mineral phase of bone.
  • the injectability test was performed with the CPC/MWCNT-OH/BSA composite of the present invention.
  • the desired physical condition of workable CPC/MWCNTs- OH/BSA composite paste was found at an UP ratio of 0.27 ml/g, resulting in an injectability of 97%, i.e., 97% of the calcium phosphate cement (CPC) paste could be extruded.
  • CPC calcium phosphate cement
  • the present invention demonstrated the possibility of developing high compressive strength calcium phosphate cement (CPC) by reinforcement with hydrxylated multiwalled carbon nanotubes (MWCNTs-OH) and bovine serum albumin (BSA) for the use as injectable bone substitute.
  • CPC calcium phosphate cement
  • MWCNTs-OH hydrxylated multiwalled carbon nanotubes
  • BSA bovine serum albumin

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Abstract

The present invention discloses a high-strength and biocompatible calcium phosphate composite material that is suitable for use as bone implants or repairs. Accordingly, the calcium phosphate composite of the present invention, which may be used as an injectable bone replacing material, comprises at least one calcium phosphate reinforced with a protein concentrate and carbon nanotubes to produce a high compressive strength composite or cement.

Description

CALCIUM PHOSPHATE CEMENT COMPOSITION AND METHOD OF MAKING
THE SAME
The present invention relates to high-strength calcium phosphate cement compositions, and more particularly, to a high-strength calcium phosphate composite material comprising protein and carbon material for use as bone replacing materials.
BACKGROUND QF THE INVENTION" Bone formation or replacement is often a desired therapy for bone loss or defects due to fractures or bone degenerative diseases. A biomaterial for bone formation or replacement should have sufficient mechanical load-bearing and impact strength to maintain structural integrity and provide a suitable environment to induce new bone formation. A potential bone-replacement material would include an organic polymer for mechanical strength and ease-of-use and inorganic particles that participate in the bone mineralization pathway. However, it is difficult to maintain the bone- replacement material as a homogenous mixture because the organic polymer and the inorganic particles cannot be homogeneously mixed together. Phosphate-based hydraulic structural cements are well known (e.g., see Friedman et al "BoneSource hydroxyapatite cement: a novel biomaterial for craniofacial skeletal tissue engineering and reconstruction" Journal of Biomedical Materials Research (1998). 43(4), 428-432). However, these cements do not contain peptide material and carbon nanotubes.
Calcium phosphate cement (CPC) is the most attractive bone substitute material with many desirable properties such as biocompatible, bioactive, non-toxic, osteoconductive, self-hardened in situ, resorbed by osteoclasts in vitro, and able to be integrated as well as remodeled into healthy bone in vivo. However, the final cement suffers from a relatively low compressive strength (i.e., limited its use to non- load bearing applications in orthopedics), poor injectability (i.e., resulted in invasive surgical technique which involved open wound surgery) and lack of macroporosity (i.e., limited space available for bone in-growth). Thus, a great deal of recent researches have been carried out to incorporate various types of reinforcement materials and resulted in substantially increase in the compressive strength. For instance, Dos Santos et al., 2000 incorporated polyamide fibers into their cement formulation; Fujishiro et al., 2001 added gelatin gel to tricalcium phosphate; and other authors used a number of polymers blended with CPC. All of these researchers noted a modest improvement over the neat cement, but yet, some experience the problem of reduction in cement workability as well as setting time. Having the best performance among carbon materials, it is anticipated that carbon nanotubes can potentially enhance the properties of calcium phosphate cement. In addition, the electrical properties of carbon nanotubes are highly responsive to the changes in the surrounding electrostatic environment and interface charge transfer, causing drastic changes through simple adsorptions of certain molecules or polymer.
Another strategy to provide improved calcium phosphate ceramics is the incorporation with proteins, such as bovine serum albumin (BSA). For example, the addition of low concentrations of bovine serum albumin (BSA) has shown to enhance calcium phosphate crystal growth (being favourable for bone tissue mineralisation), whereas higher concentrations inhibit calcium phosphate crystallisation.
Accordingly, there exists a need for cement having improved mechanical properties, in particular high overall compressive strength, rapid setting time, good expansiveness to offset shrinkage, as well as having biocompatible and bioactive properties, so as to be useful for bone implants or repairs applications in the field of orthopedics.
SUMMARY OF THE INVENTION The present invention broadly discloses a high-strength and biocompatible calcium phosphate composite material that is suitable for use as bone implants or repairs. It will be understood that those skilled in the art may identify numerous other uses for the composite material of the present invention, even if it is not specifically indicated in the present disclosure. Accordingly, the calcium phosphate composite of the present invention, which may be used as an injectable bone replacing material, comprises at least one calcium phosphate reinforced with a protein concentrate and carbon nanotubes to produce a high compressive strength composite or cement.
The calcium phosphate composite of the present invention is preferably an injectable bone replacing material in vivo.
In a preferred embodiment of the present invention, the calcium phosphate present in the calcium phosphate composite is comprised of equimolar β-tri-calcium phosphate (β-TCP) and dibasic calcium phosphate anhydrous (DCPA).
In a preferred embodiment of the present invention, the protein concentrate is present in the calcium phosphate composite in an amount of at least 15% of total weight of the calcium phosphate composite.
In a preferred embodiment of the present invention, the carbon nanotubes are present in the calcium phosphate composite in an amount of at least 0.5% of total weight of the calcium phosphate composite.
The protein concentrate is a serum albumin protein and may be, for example bovine serum albumin (BSA) having 607 amino acid residues and a molecular weight of 66.4 kDa. The addition of low concentrations of BSA enhances calcium phosphate crystal growth (being favourable for bone tissue mineralisation), whereas higher concentrations inhibit calcium phosphate crystallisation. Further, the addition of BSA improves cohesiveness of the cement composite.
The carbon nanotubes are multi-walled carbon nanotubes that may be hydroxylated or non-hydroxylated. Having the best performance among carbon materials, it is anticipated that carbon nanotubes can potentially enhance the properties of calcium phosphate composite of the present invention. In addition, the electrical properties of carbon nanotubes are highly responsive to the changes in the surrounding electrostatic environment and interface charge transfer, causing drastic changes through simple adsorptions of certain molecules or polymer. Thus, the presence of chemical functional groups will induce chemical reaction along the interface, and hence improve the reinforcement efficiency of the cement.
It is an advantage of the present invention to provide a composite or cement composition having a significant increase of compressive strength, which makes it suitable for higher load bearing bone implant or repair applications.
It is another advantage of the present invention to provide a composite or cement that can be prepared in the form of a paste, which makes it suitable for use as injectable bone replacement materials in bone implant or repair applications.
In another aspect, the present invention provides a method for preparing calcium phosphate cement, which comprises the steps of: i. providing a calcium phosphate mixture by adding equimolar β-tricalcium phosphate (β-TCP) to dicalcium phosphate anhydrous (DCPA);
ii. mixing the calcium phosphate mixture with 15 wt% of serum albumin protein and 0.5 wt % of carbon nanotubes; and
iii. adding water to the mixture of step (ii) to obtain a calcium phosphate composite material or cement paste.
The skilled person will understand that the composite material may also be molded after setting of the cement, for instance by milling or by cutting, into a desired shape. In such instances the moulded cement material of the present invention may take any shape desirable.
In another aspect, the present invention provides the use of the calcium phosphate composite material according to the present invention as described above for the induction of bone formation in a living organism. In another aspect, the present invention provides the use of the calcium phosphate composite material according to the present invention as described above as an implant material alone or combined with growth factors or/and cells for the production of autologous bone in a non- osseous site. In yet another aspect, the present invention provides the use of the calcium phosphate composite material according to the present invention as described above for the production of a medical implant or device alone or combined with growth factors or/and cells.
Uses of the invention are particularly beneficial for the reconstruction or replacement of bone and/or in dental surgery.
The foregoing and other objects, features and advantages will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph illustrating compressive strength of the calcium phosphate composite (CPC/MWCNTs-OH/BSA) according to the present invention;
Figure 2 is a scanning electron microscope (SEM) image of the CPC/MWCNTs- OH/BSA according to the present invention;
Figure 3 is a graph illustrating FTIR patterns of the CPC/MWCNTs-OH/BSA according to the present invention; and
Figure 4 is a diagram illustrating X-ray diffraction patterns of the CPC/MWCNTs- OH/BSA according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
A calcium phosphate material or calcium phosphate composite in accordance with the present invention may be based on any calcium phosphate (CaP), such as a CaP obtained by precipitation from an aqueous solution at low temperature (e.g. 20-80°C) or by a high temperature process (but preferably not higher than 100°C). Highly preferred calcium phosphates are the calcium orthophosphates. The term "calcium orthophosphate" as used herein refers to a family of compounds, each of which contains a calcium cation, Ca , and a phosphate anion, P04 3~ . Under this definition, there are multiple calcium orthophosphates, including monocalciurh orthophosphate (monobasic), dicalcium orthophosphate (dibasic), tricalcium orthophosphate (tribasic), and hydroxyapatite (penta calcium triphosphate).
Although the present invention is described mainly in terms of calcium orthophosphate, other suitable materials useful herein include for instance calcium pyrophosphates (e.g., dicalcium diphosphate (Ca2P207), synonym: calcium pyrophosphate), calcium pyrophosphate dihydrate (CPPD, Ca2P207.2H20) and calcium dihydrogen diphosphate (CaH2P20 ; synonyms: acid calcium pyrophosphate, monocalcium dihydrogen pyrophosphate, and polyphosphate (CaP206)n, n>2; synonyms: calcium metaphosphates, calcium polymetaphosphates, and combinations of the various phosphates.
Non-limiting examples of the calcium phosphate compound that may be used in aspects of the present invention are as follows:
• amorphous calcium phosphate (ACP, Ca3(P04)2.nH20, n = 3 - 4.5, Ca/P ratio = 1.5);
apatite (calcium fluoro-phosphate, Ca5(F,CI,OH)(P04)3);
• calcium dihydrogen phosphate (Ca(H2P04)2);
calcium dihydrogen phosphate hydrate (Ca(H2P04)2.H20);
calcium hydrogen phosphate hydrate (CaHP04.2H20);
calcium hydrogen phosphate, anhydrous (CaHP04);
calcium-deficient hydroxyapatite or precipitated hydroxyapatite (PHA) Ca 0- x(HPO4)x(P04)6-x(OH)2-x (0≤x>1 ) with Ca/P ratio varying from 1.5 to 1.67;
carbonate apatite (Ca5(P04, C03)3F;
dicalcium phosphate anhydrous (DCPA, CaHP04);
dicalcium phosphate dihydrate (DCPD, CaHP04.2H20);
monocalcium phosphate anhydrous (MCPA, Ca(H2P04)2);
• monocalcium phosphate monohydrate (MCPM, Ca(H2P04)2.H20);
octacalcium phosphate (OCP, Ca8H2(P04)6.5H20);
mixtures of two or more of the above such as mixtures of MCPM or MCPA with another CaP; and
composites of two or more of the above having Ca/P ratio of 1.0 - 2.0. The calcium phosphates used in methods of the present invention are nanocrystals and are preferably obtained by precipitation from a solution comprising suitable calcium and phosphate sources. The skilled person is well aware that precipitation of calcium phosphates from such a solution will occur depending on the pH of the solution. Suitably, the precipitation occurs in the presence of a base. A suitable calcium source is Ca(N03)2.4H20. A suitable phosphate source is (NH4)2HP04. As a base, ammonia may be used.
Additionally, calcium phosphates nanocrystals may be obtained by other methods, such as by milling and/or sieving of calcium phosphates microparticles. However, the preparation of calcium phosphates nanocrystals by precipitation is most preferred. The calcium phosphates, particularly in case they are derived from natural sources, may be calcined prior to use as used in most of the applications. Preparation of calcium phosphate composite material of the invention which preferably used as an implant in living tissue should mimic the way by which living organs produce mineralized tissues, the calcium phosphate is therefore preferably not sintered or heated. Moreover, the composite material is preferably both sufficiently compatible and sufficiently biodegradable for use as an implant in living tissue. Thus, the calcium phosphate on which the composite material is based is preferably (bio)resorbable, meaning that it exhibits chemical dissolution and cell- mediated resorption when placed in a mammalian body.
A composite material according to the invention is preferably based on any calcium phosphates having Ca/P ratio of 1.67 or combinations thereof.
The carbon nanotubes (CNTs) are only a few nanometers wide and are comprised of cylindrical carbon molecules with properties that make them potentially useful as mechanical reinforcement materials in accordance with the present invention. These tubes consist of rolled up hexagons, 10,000 times thinner than a human hair. Ideal CNTs can be described as a seamless cylinder of rolled up hexagonal networks of carbon atoms, which is capped with half a fullerene molecule at the end. Their strength is one to two orders of magnitude and weight is six times lighter than steel. Besides that, CNTs are built from sp2 carbon units and consist of honeycomb lattices and are a seamless structure. They are tubular having a diameter of a few nanometers but lengths of many microns. The multi-walled carbon nanotubes (MWCNTs) are closed graphite tubules rolled like a graphite sheet. Diameters usually range between 2 and 25 nm and the distance between sheets is about 0.34 nm. Theses tubes have a tendency to form in bundles which are parallel in contact and consist of tens to hundreds of nanotubes. Other possible applications range from semiconductors, electronic memory, drive products, medical delivery systems and in plastics such as automobile body panels, paint, tires, and as flame retardants in polyethylene and polypropylene.
Pristine carbon nanotubes CNTs (as prepared, non-functionalized) are inherently hydrophobic, therefore the main obstacle in the utilization of CNT in biology and medicinal chemistry is their lack of solubility in most, solvents compatible with the biological milieu (aqueous based). To overcome this problem the modification of the surface of CNT such as functionalization with different molecules is achieved by adsorption, electrostatic interaction or covalent bonding of different molecules and chemistries that render them more hydrophilic. Through such modifications, the water solubility of CNT is improved and their biocompatibility profile is completely transformed. Moreover, the bundling or aggregation of individual tubes through van der Waals forces is also reduced by the functionalization of their surface. However, severe limitations persist as the production of structurally and chemically reproducible batches of CNT with identical characteristics, high quality control and minimal impurities is still a challenge to the pharmaceutical and clinical application of these nanomaterials. In a preferred embodiment of the present invention, the CNT present in the calcium phosphate composite is hydroxide functionalized multiwalled carbon nanotubes (MWCNTs-OH).
The improvement in the mechanical properties with addition of bovine. serum albumin (BSA) can be explained by considering that appropriate amounts of BSA are capable of promoting calcium phosphate cement crystal growth. At low concentrations (< 10 g/l), BSA has been hypothesized to stabilize nuclei and promote growth of octacalcium phosphate crystals. While at higher concentrations, crystal growth seems to be impeded by high BSA coverage. Although the net charge on BSA at neutral pH is -17 mV, the protein contains both positively and negatively charged residues. The arrangement of these charges, as well as the complementarities between the charged groups on the protein and the growing apatite surfaces, may influence crystal growth behavior and also lead to more cohesive cements for higher BSA contents. BSA will be negatively charged in the physiological solution with a pH of 7.4, thus tends to bind positive ions like Ca2+ in the solution. This strongly affects the available Ca2+ ions for nucleation and growth of apatite. In this study, it is suggested that BSA promotes hydroxyapatite (HA) crystal growth and enhances the mechanical properties of the calcium phosphate cement composite. The scaffold materials or implant materials in accordance with the present invention may be used in variable forms such as in the form of blocks, foams, sponges, granules, cement, implant coatings, composite components and may for instance be combined with organic or inorganic materials or with ceramics and may be from various origins, natural, biological or synthetic. The various forms may for instance be obtained by injection moulding, extrusion, solvent casting, particular leaching methods, compression moulding and rapid prototyping such as 3D Printing, Multiphase Jet Solidification, and Fused Deposition Modeling (FDM) of the materials.
Calcium phosphate cement (CPC) composite in accordance with the present invention may be used as a synthetic injectable (bone) composite paste to fill or conform to the defects in hard tissues and may therefore undergo self-hardening in situ to form hydroxyaptite (HA), which is the putative mineral in teeth and bones. Such a cement paste may comprise a mixture of β-tri-calcium phosphate (β-TCP) and dibasic calcium phosphate anhydrous (DCPA) in combination with bovine serum albumin (BSA) and hydroxylated multiwalled carbon nanotubes (MWCNTs-OH). The injectability of the calcium phosphate cement (CPC) is significant in clinical applications, particularly bone fractures that deal with irregular defects with limited accessibility or narrow cavities, or when there is a requirement for precise placement of the paste to perfectly adapt to the defect geometry; or when using minimally invasive surgical techniques.
The calcium phosphate cement (CPC) composite of the present invention may be dense or porous, but preferably the composite material is macroporous. Porosity can be easily achieved by the composite itself due to the attempt of using β-TCP and DCPA as main components that form hydroxyapatite (HA). This mixture results in an excellent combination effect of degrading and promoting bone or HA formation. As a candidate for bone graft material, β-TCP showed an excellent merit in bone formation. Therefore, the mixture of β-TCP and DCPA has been used as bone substitute for many years. The solubility of DCPA is roughl eight times higher than β-TCP and approximately 15 times higher than HA at physiologic pH in vitro. The similar orders of magnitude applied for the resorption of the materials in vivo; new bone forms at the space left by the resorption of the DCPD matrix, with β-TCP acted as the guiding structure. The slower the resorbing granules were surrounded by newly grown bone, thus providing an inverse scaffold for bone regeneration. Therefore, the overall resorption rate of the cement can be tailored to specific needs and to control the bone formation rate with the addition of β-TCP.
The present invention will now be illustrated by way of the following non-limiting examples.
EXAMPLES
Example 1 : Preparation of calcium phosphate cement composite
Pristine hydroxylated multiwalled carbon nanotubes (MWCNTs-OH) with diameter of 30-50 nm and length of = 30 μι were provided by Chinese Academic of Science. A calcium phosphate powder mixture was prepared by mixing equimolar fractions of β- tricalcium phosphate, β-033(Ρ04)2, (β-TCP) and dicalcium phosphate anhydrous, CaHPCv, (DCPA) (both supplied by Sigma-Aldrich), which were then mixed with deionised Water. Next, the calcium phosphate cement (CPC) powder mixture was mixed with 0.5 wt% of hydroxylated multiwalled carbon nanotubes (MWCNTs-OH) and 15 wt% of bovine serum albumin (supplied by Fluka) to produce calcium phosphate cement composite.
The final solution volume was determined by the amount required to produce a workable paste, i.e., viscous cement with UP ratio of 0.27 ml/g was prepared. Example 2: Preparation of implants
The cement paste was blended using a mechanical overhead stirrer at 30-50 rotations per minute for 1 hour and then firmly packed by manual spatulation into a cylindrical stainless steel mould with a diameter of 25 mm. The packed stainless steel mould was wrapped with water-soaked wipe to prevent the sample from drying out and was then stored in a Gyro-Rocker Incubator (Model: S170) at 37°C and 97% humidity for 24 hours. All experiments were carried out under controlled conditions at temperatures of 24-26°C and relative humidity of 50-60%. Once taken out from the incubator, the cylindrical implants were carefully taken out from the mold.
Example 3: Physical and bioactivity tests
The compressive strength of the cylindrical implants was tested using an Instron 3367 universal testing machine at a crosshead speed of 1.0 mm/min. Characterization techniques were used to validate the chemical and physical properties of the composite implants. Scanning electron microscopy (SEM) was performed using a Leo Supra 35VP-24-58 microscope in order to investigate the microstructure and morphology of the composite.
Sample fracture fragments were mounted on conducting carbon tape and observed using an accelerating voltage of 5 keV. Fourier transform infrared (FTIR) spectroscopy was carried out on a Perkin-Elmer FTIR 2000 spectrometer over the frequency range 4000 to 400 cm"1 in KBr pellets. FTIR spectroscopy was employed to characterize the presence of specific surface functional groups in the composite. X-ray Diffraction (XRD) was used to determine the crystalline structure of the cement composite. The analysis was recorded on a Siemens D5000 diffractometer using a diffraction angle 2Θ in the range 10-70° at a sweep rate of 0.047sec. The qualitative analysis of different characteristic patterns of the materials investigated was achieved by comparing peaks of the XRD spectrum with the standard diffraction patterns of specific compounds based on the International Centre for Diffraction Data (ICDD).
Injectability was qualitatively assessed and evaluated by extruding the paste through a disposable syringe. A 10 ml syringe with a diameter of 16 mm and needle with an inner diameter of 2 mm was filled with the calcium phosphate cement paste, which was then extruded from the syringe manually within a few seconds at a relatively constant speed. The injectability test was carried out in two parts. The objective of the first part was to examine the UP ratio required to produce a workable and injectable calcium phosphate cement paste. Whilst the second part investigated the injectability, which was determined considering the percentage mass of the calcium phosphate cement paste extruded from the syringe divided by the original mass of the paste inside the syringe. Example 4: Effects of the composite
Compressive strength
Figure 1 shows the effects of multiwalled hydroxylated carbon nanotubes (MWCNTs- OH) on the compressive strength of the calcium phosphate cement (CPC). It was found that with the addition of MWCNTs-OH, the compressive strength of pure CPC composite significantly increased, from 1.0 ± 0.2 MPa to 1.5 ± 0.3 Pa. Moreover, it could be confirmed that when bovine serum albumin (BSA) was added, the compressive strength of CPC/MWCNTs-OH composite significantly increased to 16 ± 3 MPa. Due to the formation of interfacial bonding between hydroxyapatite (HA) nuclei and MWCNTs-OH, HA crystals precipitated on the surface of MWCNTs-OH. A strong interfacial bonding is a necessary condition for improving the mechanical properties of composite, in order to achieve load transfer across the MWCNTs matrix interface. This interface favors the load transfer between the MWCNTs-OH and the matrix leading to improved mechanical properties. Furthermore, the improvement in the mechanical properties with addition of BSA can be explained by considering that appropriate amounts of BSA are capable of promoting CPC crystal growth. At low concentrations (< 10 g/l), BSA has been hypothesized to stabilize nuclei and promote growth of octacalcium phosphate crystals, while at higher concentrations; crystal growth seems to be impeded by high BSA coverage. Although the net charge on BSA at neutral pH is -17 mV, the protein contains both positively and negatively charged residues. The arrangement of these charges, as well as the complementarities between the charged groups on the protein and the growing apatite surfaces, may influence crystal growth behavior and also lead to more cohesive cements for higher BSA contents. In this study, it is suggested that BSA promotes HA crystal growth and enhances the compressive strength, as it was found that the compressive strength of CPC/MWCNTs-OH/BSA composite of the present invention is significantly higher than that of CPC/ MWCNTs-OH composite.
Scanning Electron Microscopy (SEM) Characterization
Figure 2 shows SEM images of the composite microstructures. In general, morphologies of the HA crystal structures of CPC/MWCNTs-OH/BSA composites were observed, as shown in Figure 2, respectively. Referring to the morphologies of HA crystals obtained by Xu et al. (2006 & 2008), it can be confirmed that the HA crystals are grown in CPC/MWCNTs-OH/BSA composites.
Figure 2 shows that well-packed HA crystals of plate-like shape and clusters are grown in CPC/MWCNTs-OH/BSA composite of the present invention. It is hypothesized that this particular microstructure led to increased compressive strength of the composite of the present invention, as compared with pure calcium phosphate cement. Fourier Transform Infrared Analysis (FTIR)
Figure 3 illustrates the FTIR results on the CPC/MWCNTs-OH/BSA composite of the present invention. The spectra show absorption bands at 3297-3307 cm-1 which correspond to the strong characteristic peak of stretching mode of hydroxyl group (- OH). The peaks pertaining to the HA phase are hydroxyl bands at 3302 cm"1 and 3307 cm"1. The characteristic bending mode of intercalated H20 can be observed at 1655-1656 cm"1. The phosphate band derived from the P-0 asymmetric stretching mode (v of the P04 3" group was identified in the region 943-1 128 cm"1, indicating a deviation of phosphate ions from their ideal tetrahedral structure. The absorption bands appearing at about 400 to 600 cm"1 can be attributed to the (v4. mode) 548, 587 and 603 (v4 mode) and double (v2)-degenerated fundamental bending mode of the P04 3 functional group. The bands observed at 1543 cm"1 (v3 mode) and 1546 cm" (v3 mode) can be assigned to the C03 2" group. As a result, all the bands discussed above and also their positions in the FTIR spectra confirm the formation of HA in CPC/MWCNTs-OH/BSA composite of the present invention.
X-Ray Diffraction analysis
The XRD pattern of the CPC/MWCNTs-OH/BSA composite of the present invention is shown in Figure 4. Diffraction peaks corresponding to HA crystalline phase were detected at 2Θ angles of 26, 29, 32, 40 and 53°. It is therefore evident that it is possible to obtain self-setting injectable HA by mixing β-TCP and DCPA with de- ionized water. The sharp and narrow diffraction peaks observed in the regions of relevance to HA suggest that the HA formed is crystalline, which can be correlated with the crystal morphology observed by SEM. As a whole, the XRD, SEM and FTIR results showed that the investigated CPC composites developed a crystalline HA phase, which is in its chemical and crystallographic composition similar to the mineral phase of bone.
Injectability Test
The injectability test was performed with the CPC/MWCNT-OH/BSA composite of the present invention. The desired physical condition of workable CPC/MWCNTs- OH/BSA composite paste was found at an UP ratio of 0.27 ml/g, resulting in an injectability of 97%, i.e., 97% of the calcium phosphate cement (CPC) paste could be extruded. It is important to note here that the maximum percentage of cement paste extruded can never achieve 100 %, due to small amounts of residual cement paste inside the syringe. It is clear that the injectability of cement pastes can be influenced by varying the UP ratio. The injectability of cement pastes with an UP ratio < 0.25 ml/g was not tested because the specimen was not workable (too viscous). The injectability of cement pastes with an UP ratio > 0.28 ml/g was not tested because the resulting cement paste was too liquid. For example, Bohner and Baroud (2005) suggested that a well-injectable cement paste should have the capacity to stay homogeneous during injection, independently of the injection force. They suggested that this approach can be achieved by increasing the cement UP ratio. As a result, the ability of cement paste to harden in aqueous condition will be reduced because the viscosity of the cement paste is reduced at the same time. This reduced stability will cause a total degradation of the cement paste. Summarising the above, an UP ratio of CPC/MWCNTs-OH/BSA composite paste of 0.27 ml/g yielded mechanically strong and injectable CPCs with injectability of 97 %. This material is thus suitable for bone repair applications as an injectable bone substitute.
Conclusion
The present invention demonstrated the possibility of developing high compressive strength calcium phosphate cement (CPC) by reinforcement with hydrxylated multiwalled carbon nanotubes (MWCNTs-OH) and bovine serum albumin (BSA) for the use as injectable bone substitute. Drawing on the results from the compressive strength tests, the CPC/MWCNTs-OH/BSA composite of the present invention exhibited substantially improved compressive strength (= 16 MPa) compared to pure cement (= 1 MPa). Of all MWCNTs studied, functionalized MWCNTs-OH were found to be the most effective to increase the compressive strength of CPC. It was suggested that hydroxyl functional groups on the surface of MWCNTs improved the reactivity and wettability of MWCNTs leading to strong interfacial bonding. In addition, the effective attraction of both Ca2+ and P04 3" by the functional groups of MWCNTs-OH is expected to promote the nucleation and growth of HA crystals. The XRD, SEM and FTIR analyses confirmed the formation of crystalline HA during the synthesis of CPC. SEM observations demonstrated that the addition of MWCNTs-OH modifies the morphology of HA crystallites.

Claims

1. A calcium phosphate composite that is capable of being used as bone implants or repairs, characterized in that the composite comprising at least one calcium phosphate reinforced with a protein concentrate and carbon nanotubes, thereby producing a high compressive strength composite or cement.
2. A calcium phosphate composite according to claim 1 , characterized in that the composite is an injectable bone replacing material in vivo.
3. A calcium phosphate composite according to claim 1 , characterized in that the calcium phosphate present in the composite is comprised of equimolar β-tri- calcium phosphate (β-TCP) and dibasic calcium phosphate anhydrous (DCPA).
4. A calcium phosphate composite according to claim 1 , characterized in that the protein concentrate is present in the calcium phosphate composite in an amount of at least 15 wt % of total weight of the calcium phosphate composite.
5. A calcium phosphate composite according to claim 1 , characterized in that the carbon nanotubes are present in the calcium phosphate composite in an amount of at least 0.5 wt % of total weight of the calcium phosphate composite.
6. A calcium phosphate composite according to claim 1 , characterized in that the protein concentrate is serum albumin protein.
7. A calcium phosphate composite according to claim 6, characterized in that the serum albumin protein is having 607 amino acid residues and a molecular weight of 66.4 kDa.
8. A calcium phosphate composite according to claim 1 , characterized in that the carbon nanotubes are multi-walled carbon nanotubes that are hydroxylated.
9. A method for preparing a calcium phosphate composite, which comprises the steps of: i. providing a calcium phosphate mixture by adding equimolar β-tricalcium phosphate (β-TCP) to dicalcium phosphate anhydrous (DCPA);
ii. mixing the calcium phosphate mixture with 15 wt % of serum albumin protein and 0.5 wt % of carbon nanotubes; and
iii. adding water to the mixture of step (ii) to obtain a calcium phosphate composite material or cement paste.
10. Use of the calcium phosphate composite material according to any one of claims 1 to 9 for the induction of bone formation in a living organism.
11. Use of the calcium phosphate composite material according to any one of claims 1 to 9 as an implant material alone or combined with growth factors or/and cells for the production of autologous bone in a non-osseous site.
12. Use of the calcium phosphate composite material according to any one of claims 1 to 9 as a medical implant or device alone or combined with growth factors or/and cells.
13. Use of the calcium phosphate composite material according to any one of claims 11 to 13 for the reconstruction or replacement of bone.
14. Use of the calcium phosphate composite material according to any one of claims 11 to 13 in dental surgery.
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