A synthetic bone material
The present invention relates to the field of synthetic bone materials for biomedical applications and, in particular, to silicon-substituted apatite and hydroxyapatite single crystal fibers and a process for their preparation.
The apatite group of minerals are based on calcium phosphate, with naturally occurring apatite having a molar ratio of approximately Ca/P of 1.67.
Hydroxyapatite, which has the chemical formula Ca10 (P04) 6 (OH) 2, is a biomedical ceramic which resembles the mineral component of bone. This similarity has led to the development of the material for biomedical applications, and hydroxyapatite and hydroxyapatite- glass composites have been used as skeletal reconstitution materials.
It has been observed that bone will bond directly to hydroxyapatite in the human body (a property referred to as bioactivity) through a bone-like apatite layer formed in the body environment.
Although the composition of synthetic hydroxyapatite is similar to the mineral component of bone, there are a number of distinct differences between the two materials in terms of their trace element content. In this regard, it is known that the bioactivity of hydroxyapatite can be enhanced by the substitution of suitable elements into the crystal lattice. For example, substitution of low levels of silicon into the lattice has been found to improve the
rate at which bone bonding occurs with implant materials .
PCT/GB97/02325 describes a process for the preparation of an essentially phase pure silicon- substituted hydroxyapatite material.
JP 2691593 and JP 2849686 describe carbonate- containing hydroxyapatite fibers and a process for their preparation. The synthesis process relies on homogeneous precipitation under either normal or hydrothermal conditions.
In an alternative process described in Nihon- kagaku-kaishi , 1988, 1565 (1988) , hydroxyapatite whiskers are synthesized from a calcium phosphate slurry by a hydrothermal crystallization method. The whiskers produced by this process have a long-axis dimension of less than 5 μm.
In another alternative process described in JP 57-117621, JP 61-75817 and JP 61-106166, apatite-like fibers are prepared by spinning melts/slurry using a nozzle. The apatite-like fibers produced by this process are polycrystalline and have inferior mechanical properties compared with single crystal fibers .
In a first aspect, the present invention provides a synthetic silicon-containing apatite single crystal. Apatite as used herein means one of the apatite group of minerals, for example hydroxyapatite. Thus, the present invention is intended to encompass a silicon- containing hyroxyapatite single crystal.
Advantageously, at least some of the silicon in the single crystal is substituted therein. Thus, the present invention also provides a synthetic silicon- substituted apatite single crystal.
By the term silicon-substituted is meant that silicon is substituted into the apatite crystal lattice, rather than being added interstitially into the crystal structure. It is believed that silicon substitutes on or primarily on the phosphate site.
The silicon is thought to exist and/or substitute in the crystal lattice as a silicon ion or as a silicate ion.
The single crystal will typically be in the form of a fibre, which preferably has a long-axis length of
> 5 μm, more preferably > 7' μm, still more preferably
> 10 μm. Advantageously, the long-axis length is in the range of from 5 to 500 μm, typically from 10 to 300 μm, more typically from 20 to 200 μm. The single crystal typically has a short-axis length of < 5 μm, more typically from 0.1 to 5 μm, still more typically from 0.5 to 3 μm.
The aspect ratio of the fibre will typically be from 1 to 5000, more typically from 3 to 600.
With regard to the crystallographic growth direction, the fibre has a preferred orientation to the c-axis direction and develops the a-plane in the apatite hexagonal crystal structure.
The term fibre as used herein is intended to encompass any needle-like, thread-like, filament-like
or acicular three dimensional form of crystal.
The synthetic silicon-containing apatite single crystal will typically comprise up to 5% by weight of silicon, more typically up to 3% by weight. The single crystal will generally comprise at least 0.1 % by weight of silicon. Advantageously, the single crystal comprises from 0.4 to 2.4 % by weight of silicon, more preferably from 0.5 to 1.6%, still more preferably from 0.5 to 1%.
The Ca: (P+Si) molar ratio in the single crystal is preferably from 1:1.4 to 1:2, more preferably from 1:1.6 to 1:1.8, still more preferably from 1:1.65 to 1:1.75. The most preferred molar ratio is approximately 1:1.67.
The synthetic silicon-containing apatite single crystal may be phase pure (or at least essentially phase-pure) , containing substantially no other calcium phosphate forms or phases, such as octacalcium phosphate, calcium hydrogen phosphate, calcium oxide, tricalcium phosphate and tetracalcium phosphate. Thus, the present invention also provides a single phase (or essentially single phase) silicon-containing apatite single crystal. In this connection, the phase purity, as measured by x-ray diffraction, may be at least 98%, preferably at least 99%, more preferably approximately 100%.
The silicon-containing apatite single crystal as herein described may, however, contain carbonate ions depending on the method of synthesis. At least some of the carbonate ions (C03 2~) are believed to
substitute in the P04 and/or OH sites in the crystal lattice.
The present invention also provides a synthetic bone material (which term is intended to encompass dental materials) comprising one or more synthetic silicon-containing apatite single crystal (s) as herein described.
The present invention also provides a composition which comprises a synthetic bone material as herein described, together with a pharmaceutically acceptable carrier .
The present invention also provides a bone implant, bone graft, bone scaffold, hydroxyapatite- polymer composite material, filler, coating (for a metallic implant for example) or cement which comprises a synthetic bone material as herein described or a composition as herein described. The bone implant, graft, scaffold, composite material, filler, coating or cement may be porous.
The present invention also provides a synthetic bone material, bone implant, bone graft, bone scaffold, hydroxyapatite-polymer composite material, filler or cement comprising a plurality of synthetic silicon-containing apatite single crystals as herein described in a biocompatible matrix (for example a polymer matrix such as PLLA, PGA or copolymer thereof) . In this case,, at least some of said single crystals are aligned in said matrix in substantially the same direction.
The present invention also provides for the use of synthetic silicon-containing apatite single crystals as herein described as a fibre reinforcement in a synthetic bone material, bone implant, bone graft, bone scaffold, hydroxyapatite-polymer composite material, filler or cement.
In a further aspect, the present invention provides a process for the preparation of silicon- containing apatite single crystals as herein described, which process comprises: (i) reacting a calcium source with a phosphorus source to form a precipitate comprising octacalcium phosphate and/or calcium hydrogen phosphate; and (ii) heating said precipitate in the presence of a silicon source to form one or more silicon-containing apatite single crystals.
The single crystals produced by this process are advantageously in the form of fibers, which preferably have a long-axis length in excess of 5 μm, more preferably in excess of 10 μm. For example, the average long-axis length may be in the range of from .20 to 200 μm. In this case, the precipitate formed in step (i) will also typically be in the form of single crystal fibers, which preferably have a long-axis length in excess of 5 μm, more preferably in excess of 10 μm. The average long-axis length of the precipitate fibers will typically be in the range of from 100 to 300 μm.
The calcium source preferably comprises a calcium salt and may be selected, for example, from one or more of calcium nitrate, a calcium carboxylate (for
example calcium acetate) , a calcium halide (for example calcium chloride), and a calcium alkoxide.
The phosphorus source preferably comprises phosphoric acid or a salt thereof and may be selected, for example, from one or more of ammonium-dihydrogen phosphate, diammonium hydrogen phosphate, phosphoric acid and an organic phosphate such as trimethyl phosphate .
The silicon source preferably comprises a silicate and may be selected, for example, from one or more of a silicon carboxylate (for example silicon acetate) , a silicon halide (for example silicon chloride) , tetraethyl orthosilicate and tetramethyl orthosilicate .
Step (i) of the process typically involves forming a solution comprising the calcium source and the phosphorus source. The solution may also comprise the silicon source. In this case, the silicon source is present in both steps (i) and (ii) .
The solution will typically comprise a polar solvent, for example water.
Advantageously, the solution further comprises a pH regulator, for example urea.
Advantageously, the solution further comprises an acid, for example nitric acid or hydrochloric acid.
A preferred solution comprises water, calcium nitrate, diammonium hydrogen phosphate , tetraethyl
orthosilicate (TEOS) , urea and nitric acid.
The reaction to form the precipitate in step-(i) is preferably carried out at a temperature Tl, where Tl is in the range of from 50 to 90°C, preferably from 70 to 90°C, more preferably from 75 to 85°C, still more preferably approximately 80°C. Heating in step (ii) is preferably carried out at a temperature T2, where T2 > Tl. T2 will typically be in the range of from 90 to 150°C, preferably from 95 to 130°C.
The concentration of calcium ions is preferably from 0.0167 to 1.67 moldm-3, more preferably 0.08 to 0.25 moldm-3, still more preferably approximately 0.167 moldm"3.
The concentration of phosphorous-containing ions is preferably from 0.01 to 1.00 moldm-3, more preferably 0.05 to 0.14 moldm-3, still more preferably approximately 0.09547 moldm-3.
The Ca/P molar ratio is preferably from 1:1.4 to 1:2, more preferably from 1:1.6 to 1:1.8, still more preferably from 1:1.65 to 1:1.75.
Similarly, the Ca/(P+Si) molar ratio is preferably from 1:1.4 to 1:2, more preferably from 1:1.6 to 1:1.8, still more preferably from 1:1.65 to 1:1.75. The most preferred molar ratio is approximately 1:1.67.
The pH Of the reaction mixture during heating (step (ii) ) is preferably maintained substantially at a pH of from 7 to 10, although the pH of the starting
solution prior to heating is typically from 1 to 3.
The single crystals produced by the process according to the present invention are preferably free or essentially free of any of the (precursor) precipitate material. The single crystals may be phase pure, or at least essentially phase pure.
The process is preferably a homogeneous precipitation process, which may be conducted under hydrothermal conditions if desired. The process involves forming a precursor precipitate for the silicon-containing apatite single crystals. The precipitate is believed to comprise octacalcium phosphate. The precipitate may be formed by heating the starting solution at a temperature typically in the range of from 70 to 90°C for from 12 to 24 h. In the early stages of heating, after typically about 2 h, a white compound is formed on the walls of the glass reaction flask. This compound is believed to be calcium hydrogen phosphate (CaHP04) . Nucleation is believed to occur on the surface of the calcium hydrogen phosphate and octacalcium phosphate (Ca8H2 (P04) 6.xH20) single crystal fibers grow with an average long-axis size of typically 100 to 300 μm
(after 24 h heating) . Thus, the (precursor) crystal fibers are formed by a process of nucleation and growth. A pH regulator is preferably present (for example urea) to control the pH in the reaction system. The resulting octacalcium phosphate crystal fibers are transformed into silicon-containing apatite crystal fibers in a further heating step in the presence of the silicon source. Under normal conditions (i.e. non-hydrothermal conditions), the
further heating step is preferably conducted at a temperature of from 90 to 98°C, typically for from 96 to 240 h. Under hydrothermal conditions, the further heating step is preferably conducted at a temperature of from 100 to 150°C, typically for from 6 to 48 h. During the further heating step, the long-axis dimension of the octacalcium phosphate fibers tends to decrease. The hydrothermal treatment is typically performed at a pressure of from 2 to 4 bar, more typically from 2.5 to 3.5 bar. The Si content in the final fibers will typically be up to 3 weight % (up to 5 weight % in the case of the hydrothermal treatment) and may be controlled by adjusting the concentration of silicate.
In a further aspect, the present invention provides a process for the preparation of silicon- containing apatite single crystals as herein described, which process comprises heating a precipitate comprising octacalcium phosphate and/or calcium hydrogen phosphate in the presence of a silicon source to form one or more silicon-containing apatite single crystals.
As will be appreciated, the features described herein in relation to the first mentioned process are applicable either singularly or in combination to this further aspect of the present invention.
In a yet a further aspect, the present invention provides a process for the preparation of silicon- containing apatite single crystals as herein described, which process comprises: (a) providing a solution comprising a calcium source
selected from one or more of calcium nitrate, calcium acetate and calcium chloride, a phosphorus source selected from one or more of ammonium-dihydrogen phosphate, diammonium hydrogen phosphate and phosphoric acid, and a silicon source selected from one or more of a silicon carboxylate, silicon chloride, tetraethyl orthosilicate and tetramethyl orthosilicate, wherein the concentration of calcium ions is from 0.0167 to 1.67 moldm-3, the concentration of phosphorous-containing ions is from 0.01 to 1.00 moldm-3, and the Ca/ (P+Si) molar ratio is from 1:1.6 to 1:1.8; and
(b) heating the solution at a temperature in the range of from 50 to 90°C to form a precursor precipitate comprising calcium and phosphorous, followed by further heating at a temperature in the range of from 90 to 150°C to form one or more silicon- containing apatite single crystals.
Again, the features described herein in relation to the first mentioned process are applicable either singularly or in combination to this further aspect of the present invention.
As will be appreciated, the thus produced single crystals may be heated to effect drying, calcination and/or sintering.
As will also be appreciated, the processes may further comprise collecting the silicon-containing apatite single crystals, and forming a synthetic bone material, bone implant, bone graft, bone scaffold, hydroxyapatite-polymer composite, filler or cement comprising a plurality of the single crystals, for
example at least 5 to 10 crystals. This may involve aligning at least some of the crystals in substantially the same direction. The single crystals may be intertwined and a woven-like material may also be formed from the single crystals. A sinter step may be performed after forming the desired product.
A preferred process for producing silicon- containing apatite single crystals fibers according to the present invention will now be described further by way of example.
The process generally relies on homogeneous precipitation (in an aqueous medium) of a precursor precipitate, which is subsequently transformed into the desired silicon-containing apatite single crystal. The homogeneous precipitation may be carried out under normal conditions or under hydrothermal conditions.
Homogeneous Precipitation
Single crystal fibers may produced by: 1) forming a starting solution; 2) carrying out a first heating step to precipitate a precursor (for example octacalcium' phosphate, Ca8H2 (P04) 65H20) of the silicon- substituted hydroxyapatite; and 3) carrying out a second heating step for conversion of the precursor precipitate into the silicon-containing apatite single crystal fibre.
The starting solution may be prepared using a calcium salt, a phosphate, a silicate, a pH regulator (for example urea, (NH2)2C0) and an acid (for example nitric acid, HN03) . The calcium salt may be selected
from calcium nitrate tetrahydrate (Ca (N03) 24H20) , calcium acetate (Ca (CH3COO) 2) and calcium chloride (CaCl2) , including- mixtures of two or more thereof. The preferred calcium salt is calcium nitrate tetrahydrate. The phosphate may be selected from ammonium dihydrogen phosphate (NH4H2P04) , diammonium hydrogen phosphate ((NH4)2HP04) and phosphoric acid (H3P04) , including mixtures of two or more thereof. The preferred phosphate is diammonium hydrogen phosphate. The silicate may be selected from both inorganic and organic silicon compounds, for example, silicon acetate (Si (CH3C00) 4) , silicon chloride (SiCl4), tetraethyl orthosilicate (TEOS) (Si(OC2H5)4) and tetramethyl orthosilicate (TMOS) (Si(OCH3)), including mixtures of two or more thereof. The preferred silicate is TEOS owing to its hydrolysis reaction rate.
The concentrations of Ca2+ and P04 3- ions in the starting solution are preferably in the ranges of from 0.0167 to 1.67 moldm-3 and 0.01 to 1.00 moldm-3, respectively. The Ca/(P+Si) molar ratio is preferably fixed at approximately 1.67. The concentration of .urea is preferably from 0.05 to 5.00 moldm-3. The most preferred concentrations in the starting solution are approximately: 0.167 moldm-3 Ca2+ ions, 0.09547 moldm-3 P04 3- ions, 4.57 x 10-3 moldm-3 of Si04 4- ions and 0.50 moldm-3 of urea (in the case of the addition of 0.8 weight % Si as a nominal composition).
The process involves forming a precursor precipitate for the silicon-containing apatite fiber. The precipitate is believed to comprise or consist of octacalcium phosphate. The precipitate is formed by
heating the starting solution at a temperature typically in the range of from 70 to 90°C for from 12 to 24 h. The preferred heating conditions are approximately 80°C for 24 h. In the early stages of heating (after about 2 h) , a white compound is formed on the wall of the glass reaction flask. On the basis of XRD analysis, this compound is believed to be calcium hydrogen phosphate (CaHP04) . Nucleation is believed to occur on the surface of the calcium hydrogen phosphate and octacalcium phosphate single crystal fibers grow with a long-axis size of typically 100 to 300 μm (after 24 h heating) . Thus, the crystal fibers are formed by process of nucleation and growth. The urea in the starting solution controls the pH in the reaction system.
The resulting octacalcium phosphate crystal fibers are transformed into silicon-containing apatite single crystal fibers by heating at a temperature in the range of from 90 to 98°C for from 96 h to 240 h in the presence of the silicon source (which has always been in solution) . The preferred heating conditions are approximately 95°C for 144 h. The long-axis dimension of the fibre will generally decrease during the second heating stage and so the resulting single crystal fibers typically have an average long-axis dimension of 30 to 200 μm.
The Si content in the final single crystal fibers will typically be up to 3 weight % and may be controlled by adjusting, the concentration of silicate. However, the actual Si content in the final product tends to be lower than the nominal composition of starting solution.
Homogeneous Precipitation Under Hydrothermal Conditions
The octacalcium phosphate fibers prepared in the manner described above are hydrothermally transformed into silicon-containing apatite fibers by heating at a temperature in the range of from 100 to 150°C for from 6 to 48 h in the presence of the silicon source (which has always been in solution) . The preferred heating conditions are approximately 120°C for 24 h. The long-axis dimension of the fibers tend to decrease during the hydrothermal treatment and so the resulting silicon-containing apatite single crystal fibers have a long-axis dimension of typically 10 to 200 μm. The Si content in the final single crystal fibers will typically be up to 5 weight % and may be controlled by adjusting the concentration of silicate. The hydrothermal treatment is preferably performed at a pressure of from 2 to 4 bar, more preferably from 2.5 to 3.5 bar, still more preferably approximately 3 bar.
The present invention will now be described further, by way of example, with reference to the following drawings in which :-
Figure 1 shows XRD patterns of silicon-containing hydroxyapatite single crystal fibers synthesized by a homogeneous precipitation method according to the present invention ((a) Run #6 and (b) Run #8);
Figure 2 shows IR spectra of silicon-containing hydroxyapatite single crystal fibers synthesized by a
homogeneous precipitation method according to the present invention ( (a) Run #6 and (b) Run #8) ;
Figure 3 shows SEM micrographs of silicon- containing hydroxyapatite single crystal fibers synthesized by a homogeneous precipitation method according to the present invention ( (a) Run #6 and (b) Run #8) ;
Figure 4 shows XRD patterns of silicon-containing hydroxyapatite single crystal fibers synthesized by a hydrothermal homogeneous precipitation method according to the present invention ( (a) Run #11 and (b) Run #13-5) ;
Figure 5 shows IR spectra of silicon-containing hydroxyapatite single crystal fibers synthesized by a hydrothermal homogeneous precipitation method according to the present invention ( (a) Run #11 and (b) Run #13-5) ; and
Figure 6 shows SEM micrographs of silicon- containing hydroxyapatite single crystal fibers synthesized by a hydrothermal homogeneous precipitation method according to the present invention ( (a) Run #11 and (b) Run #13-5) .
Examples
The following experiments are provided by way of example and were performed to synthesize silicon- containing apatite single crystal fibers with an essentially single apatite phase and a controlled silicon content. The experiments are classified into
two categories: (1) The effect of Si content on the synthesis of fibers by homogeneous precipitation; and (2) The effect of hydrothermal conditions and the Si content on the synthesis of fibers by homogeneous precipitation (under hydrothermal conditions) .
(1) The effect of Si content on the synthesis of fibers by homogeneous precipitation
Synthesis of fibers was carried out using a starting solution based on a Ca(N03)2 - (NH4)2HP04 - TEOS - (NH2)2CO - HN03 system. In order to obtain single phase apatite, the conditions (in the second heating stage) were fixed at approximately 95°C for 144 h.
Five starting solutions were prepared with various Si contents. The preparation conditions are shown in Table 1, together with the phase composition and morphology of the resultant products. The starting solution included 0.50 moldm-3 (NH2)2CO and 0.1 moldm-3 HN03.
The starting solutions were first heated at approximately 80°C for 24 h and then at approximately 95°C for 144 h to form the single crystal fibers.
Table 1
1) Determined using XRD results of the crushed fibers. Conversion of OCP into HAP (%) = I
HAP(
2ID / (I
HAP ID +
The XRD patterns of the products (Runs #7 to #9) prepared from the starting solutions with TEOS showed that the (100), (200) and (300) reflections of the apatite fibers were more intense than those of a typical hydroxyapatite as listed in the JCPDS card (#9-432) , as well as the XRD pattern of the fibers without TEOS (Run #6) . The results of IR spectroscopy showed that the fibers were composed of a carbonate-containing apatite. The C03 2- group is believed to substitute on both the P04 and OH sites in the lattice structure (Type A + B) . The formation of a carbonate-containing apatite may be due to the generation of C02 through the hydrolysis of the urea:
(NH2)2CO + 2H20 --> 2NH3 + C02
SEM observations showed that the final product was composed of fiber-shaped crystals with long-axis sizes of from about 30 to about 200 μm. The presence of silicon in the individual fibers was evidenced by EDX analysis .
Results for the fibers derived from Run #6 and Run #8 are shown in Figure 1 (XRD) , Figure 2 (IR) and Figure 3 (SEM) .
Table 2 shows the XRF results of the fibers derived from Run #6 and Run #8. The actual Si content in the fibers is lower than that in the nominal composition.
Table 2
(2) The effect of hydrothermal conditions and the Si content on the synthesis of fibers by homogeneous precipitation
While single crystal fibers can be synthesized using homogeneous precipitation at 95°C for 144 h, longer heating periods may be required to obtain a single apatite phase. The following experiments rely on homogeneous precipitation under hydrothermal conditions to more rapidly synthesize the single crystal fibers. The second heating stage is carried out at approximately 120°C for from 2 to 24 h.
Five starting solutions were prepared with various Si contents. The preparation conditions are shown in Table 3, together with the phase composition and
morphology of the resultant products. The starting solution included 0.50 moldm-3 (NH2)2CO and 0.1 moldm-3 HN03.
Table 3
1) Determined using XRD results of the crushed fibers Conversion of OCP into HAP (%) = IHAp(2ιi) / (IHAP(2ii) + -LOCPIOIO) ) X 10U
The Ca/(P+Si) ratio in the starting solution was fixed at approximately 1.67. The starting solutions were first heated at approximately 80°C for 24 h and then at approximately 120°C for 2 to 24 h to form the single crystal fibers.
Five experiments (Runs #13-1 to #13-5) were performed in order to determine the hydrothermal period required to obtain an apatite single phase. Large amounts of octacalcium phosphate and trace amounts of hydroxyapatite and calcium hydrogen phosphate were present in the product in the case of Run #13-1 before
the hydrothermal treatment. The octacalcium phosphate was transformed into hydroxyapatite with increasing hydrothermal treatment period. A single apatite phase was obtained after a 24 h hydrothermal treatment. The products derived from Runs #13-1 to #13-5 were composed of fiber-shaped crystals. The long-axis dimension decreased with increasing hydrothermal treatment period.
The effect of the Si content on the synthesis of single crystal fibers was assessed under hydrothermal ' conditions fixed at approximately 120°C for 24 h. These experiments correspond to Runs #11, #12, #13-5, #14 and #15.
The XRD patterns of the products (Runs #12, #13-5, #14, #15) prepared from the starting solutions comprising TEOS showed that the (100), (200) and (300) reflections of the apatite fibers were more intense than those of a typical hydroxyapatite listed in the JCPDS card (#9-432) , as well as the XRD pattern of the fibers without silicon (Run#ll) . The results from the IR spectra indicated that the fibers were composed of a carbonate-containing apatite (Type A + B) similar to that produced by the homogeneous precipitation process. SEM observations showed that the final product was composed of fiber-shaped crystals with long-axis sizes of from about 30 to about 200 μm. The presence of silicon in the individual fibers was evidenced by EDX analysis. The long-axis size of the fibers decreased with increasing Si content in the starting solution.
The results for the single crystal fibers derived from Run #11 and Run #13-5 are shown in Figure 4 (XRD) , Figure 5 (IR) and Figure 6 (SEM) .
Table 4 shows the XRF results in respect of the single crystal fibers derived from Run #11 and Run #13- 5. The actual Si content in the fibers agrees with that in the nominal composition.
Table 4
The present invention provides a silicon-containing apatite ceramic material (for example silicon-containing hydroxyapatite) having enhanced bioactivity compared with pure hydroxyapatite. At least some of the silicon is preferably substituted in the apatite crystal lattice. Moreover, the provision of the material in the form of single crystal fibers results in enhanced mechanical properties over polycrystalline equivalents. The single crystal fibers may be used, for example, in the production of fibre-reinforced biomedical composite materials for bone grafts and scaffolds.