Calcium phosphate, method for producing calcium phosphate

Abstract

To provide a method for improving the accumulation of proteins relative to calcium phosphate, and calcium phosphate in which proteins are accumulated.SOLUTION: A buffer solution containing 1 to 5 mM calcium ions and 0.5 to 3 mM inorganic phosphate ions and adjusted to pH of 6 to 8 at 25 to 45°C is prepared. When the calcium ion content of the buffer solution is 1 to 5 mM, proteins are dissolved in the buffer solution in the range of 0.01 to 10 mg mL-1. Calcium phosphate is brought into contact with the protein-containing buffer solution thus obtained.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to calcium phosphate and a method for producing calcium phosphate.

Background Art

[0002] Octacalcium phosphate (OCP, Ca8H2(PO4)6·5H2O) is said to be a precursor of bone apatite crystals. This is based on its structural similarity to hydroxyapatite (HA), solution chemistry, and theoretical solubility analysis (see, for example, Non-Patent Document 1). It is recognized that synthetic OCP has higher osteoconductivity compared to other calcium phosphate materials such as non-sintered HA when synthesized under specific synthetic conditions (see, for example, Non-Patent Document 2).

[0003] The osteoconductivity of OCP is explained not only by its ability to activate the function of osteoblasts but also by promoting the formation of osteoclasts. Its stimulating ability has been proven to enhance not only the differentiation of osteoclasts but also the formation of osteoclasts from precursor cells by direct or indirect contact with OCP materials (see, for example, Non-Patent Documents 10 to 12). It is known that the transformative bone formation after osteoclast resorption in the physiological bone remodeling process involves apatite crystal nucleation and the growth of HA on bone matrix proteins under supersaturated conditions of the surrounding tissue fluid with respect to HA (see, for example, Non-Patent Document 13). It is reasonable to assume that new bone formation on OCP also includes such calcification processes related to crystal growth (see, for example, Non-Patent Document 1).

[0004] Since OCP is considered to be a metastable phase, it can be converted to HA, which is the most thermodynamically stable under supersaturated conditions with respect to HA (see, for example, Non-Patent Document 1). In fact, it has been shown that OCP is gradually converted to the apatite phase over time when implanted into various bone tissues (see, for example, Non-Patent Documents 2 and 3). From the perspective of the interaction between mineral crystals and matrix proteins, the bone mineralization process is generally recognized to proceed with the interaction between mineral crystals and proteins such as tissue-specific cells, osteoblasts, and secreted matrix proteins by circulating serum (see, for example, Non-Patent Document 7). As a result, a mineral / protein complex organized in bone tissue is formed.

[0005] Previous lectin histochemistry and proteome analysis have shown that non-collagenous serum proteins accumulate or adsorb around OCP crystals upon transplantation into mouse calvarial bone or immersion in rat serum in vitro (see, for example, Non-Patent Document 8). Observation using demineralized tissue specimens at the ultrastructural level confirmed that new bone formation in OCP transplantation into mouse calvaria was initiated from structures composed of OCP-non-collagen protein complexes (see, for example, Non-Patent Document 2). Furthermore, observation using non-demineralized tissue specimens at the ultrastructural level demonstrated new nano-crystal precipitation on individual OCP crystals after transplantation into mouse calvaria (see, for example, Non-Patent Document 1). The results suggest that the environment where new bone formation occurs on OCP or where OCP is dissolved can actually initiate new calcium phosphate crystal formation at a certain saturation level regardless of the presence of serum proteins. However, it is insufficient regarding whether the saturation level determined by ion concentrations, mainly calcium and inorganic phosphate ions, can enhance or weaken the serum protein-OCP interaction, which is considered to affect the osteoconductivity of OCP materials.

[0006] The composition of the serum is reported to be supersaturated with respect to HA and nearly saturated with respect to OCP (see, for example, Non-Patent Document 9). This is not inconsistent with previous observations that OCP implanted in rat calvarial defects tends to be converted to an apatite phase (see, for example, Non-Patent Document 3). In addition to the possibility that the above osteoclast-like cell resorption affects the saturation level around OCP, the presence of OCP and mesenchymal stem cell-derived cells (ATDC5) has been shown to increase the calcium ion concentration in the medium and dissolve OCP (see, for example, Non-Patent Document 10). OCP implanted in bone defects is also thought to encounter other cells such as mesenchymal-derived cells and inflammatory immune cells during bone formation, thereby regulating cell lysis and reprecipitation. The adsorption of bovine serum albumin (BSA) can be sufficiently explained by a Langmuir-type monolayer adsorption model under saturated conditions with respect to the OCP phase (see, for example, Non-Patent Document 11). Using a similar model, the adsorption of salivary proteins and amino acids to HA was explained (see, for example, Non-Patent Documents 12 and 13).

[0007] However, the stability of the calcium phosphate phase is considered to be determined by the degree of supersaturation (see, for example, Non-Patent Document 1), and other components including proteins are thought to regulate the crystal phase and crystal growth (see, for example, Non-Patent Document 14).

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non - Patent Document 6

Non - Patent Document 7

Non - Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 19

Summary of the Invention

Problems to be Solved by the Invention

[0009] Calcium phosphate, especially OCP, has been shown to promote the formation of new bone in combination with the activities of osteoblasts and osteoclast-like cells and the accompanying HA formation and serum protein adsorption on its surface, as well as the biodegradation of OCP itself. On the other hand, if an arbitrary protein can be accumulated in a necessary amount in advance, it is considered that the usefulness as a biomaterial is improved. Also, in accumulating the necessary amount, a method capable of more efficiently accumulating a larger amount of protein than the conventional method is required.

[0010] In contrast, the present inventors have found a principle that protein adsorption can be promoted under supersaturated conditions in which the same crystal phase (OCP) specifically grows and precipitates on calcium phosphate crystals containing OCP.

[0011] That is, by controlling the supersaturation and the protein concentration within appropriate ranges, crystals having new protein adsorption sites on the crystal surface are formed, and by incorporating the protein into the crystals during precipitation, an amount of protein exceeding the Langmuir equation can be efficiently accumulated on calcium phosphate.

[0012] An object of the present invention is to provide a method capable of improving and controlling the amount of protein accumulated on calcium phosphate and calcium phosphate having the protein accumulated thereon.

Means for Solving the Problems

[0013] The method for accumulating a protein on calcium phosphate of the present invention includes a buffer preparation step of preparing a buffer solution containing 1 to 5 mM of calcium ions and 0.5 to 3 mM of inorganic phosphate ions and adjusted to pH 6 to 8 at 25 to 45°C, and when the calcium ion content of the buffer solution is 1 to 5 mM, the protein is dissolved in the buffer solution within the range of 0.01 to 10 mg·mL -1 to prepare a protein-containing buffer solution, and a calcium phosphate contact step of bringing calcium phosphate into contact with the protein-containing buffer solution.

[0014] In the protein-containing buffer solution preparation step, in the protein-containing buffer solution preparation step, it is preferable to prepare a protein-containing buffer solution by dissolving the protein in the buffer solution within the range of 0.05 to 5 mg·mL -1

[0015] The calcium phosphate of the present invention is characterized in that a protein exceeding the theoretical value of the adsorption isotherm is accumulated.

[0016] ​The present invention was arrived at based on the finding that the degree of supersaturation (DS) with respect to calcium phosphate crystal phases such as OCP affects the accumulation of proteins such as BSA on calcium phosphate in a buffer solution at 25 to 45 °C and pH 6 to 8 by changing the calcium ion concentration. As the DS value increased, the adsorption amount of BSA onto calcium phosphate increased. Furthermore, the newly formed calcium phosphate not only increased the DS value but also promoted it even in a lower equilibrium concentration range of the protein. The increase in the protein accumulation capacity is presumed to be related to the formation of calcium phosphate on calcium phosphate as an adsorbent. The formation of calcium phosphate crystals, which depends not only on the DS value but also on the concentration of the protein to be accumulated, suggests that it controls the adsorption amount of the protein on the calcium phosphate surface when placed under in vivo conditions.

Brief Description of the Drawings

[0017]

Figure 1

Figure 2A

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Figure 3A

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Figure 4A

Figure 4B

Mode for Carrying Out the Invention

[0018] A method for accumulating a cell-activating protein on calcium phosphate according to an embodiment of the present invention includes (1) a buffer preparation step, (2) a buffer preparation step containing a cell-activating protein, and (3) a calcium phosphate contact step.

[0019] (1) In the buffer preparation step, a buffer containing 1 to 5 mM calcium ions and 0.5 to 3 mM inorganic phosphate ions and adjusted to pH 6 to 8 at 25 to 45°C is prepared. (2) In the protein-containing buffer preparation step, when the calcium ion content of the buffer is 1 to 5 mM, the protein is dissolved in the buffer in the range of 0.01 to 10 mg·mL -1 to prepare a protein-containing buffer. (3) In the calcium phosphate contact step, calcium phosphate is brought into contact with the protein-containing buffer.

[0020] In the protein-containing buffer preparation step, it is preferable to prepare a protein-containing buffer by dissolving the protein in the buffer in the range of 0.05 to 5 mg·mL -1 .

[0021] Calcium phosphate according to an embodiment of the present invention has a protein adsorbed thereon that exceeds the theoretical value of the adsorption isotherm.

[0022] "Calcium phosphate" includes calcium dihydrogen phosphate (Ca(H2PO4)2) (MCPA), calcium dihydrogen phosphate monohydrate (Ca(H2PO4)2·H2O) (MCPM), calcium hydrogen phosphate (CaHPO4) (DCPA), calcium hydrogen phosphate dihydrate (CaHPO4·2H2O) (DCPD), tricalcium phosphate (Ca3(PO4)2) (TCP), octacalcium phosphate (Ca8H2(PO4)6·5H2O) (OCP), hydroxyapatite (Ca10(PO4)6(OH)2) (HAP, HA (hydroxyapatite)), fluorapatite (Ca 10(PO4)6F2)(FAP, fluoroapatite), chlorine apatite (Ca 10 (PO4)6Cl2)(chloroapatite), carbonate apatite (carbonate-containing hydroxyapatite), amorphous calcium phosphate (amorphous calcium phosphate, ACP), tetracalcium phosphate (Ca4P2O9)(TTCP) and calcium pyrophosphate (Ca2P2O7) and the like are included.

[0023] "Protein" includes various growth factors such as a series of BMPs such as BMP-2 and BMP-7, TFG-β, bFGF, FGF-2, PDGF, EGF, NGF, BDNF, G-CSF, GM-CSF, EPO, HGF, IGF, SDF-1, VEGF, hormones, cytokines and chemokines. In addition, serum-derived proteins containing albumin are also considered to contribute to the improvement of the biocompatibility of biomaterials such as calcium phosphate. (See, for example, Non-Patent Documents 8 and 15)

[0024] (Examples) The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

[0025] (1. Materials and methods) (1.1 Preparation of OCP) OCP was synthesized by mixing an aqueous solution of calcium acetate and sodium hydrogen phosphate according to the wet synthesis method (see Non-Patent Document 2). The precipitate recovered from the solution was washed with deionized water. After the precipitate was dried at 105 ° C, the precipitate was pulverized and passed through a test sieve (270 mesh) to obtain OCP granules having a diameter of less than 53 μm and a specific surface area of 16 m 2 ·g -1 (See Non-Patent Document 7).

[0026] (1.2 Adsorption experiment) To obtain solutions having different degrees of supersaturation (DS) with respect to HA and OCP, CaCl2·2H2O, K2HPO4, and tris(hydroxymethyl)aminomethane (Tris) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to ultrapure water, and 1.5 mM (M = mol·L-1 ) calcium ions (Ca 2+ ) and 1.0 mM inorganic phosphate (Pi) ions or 3.0 mM Ca 2+ and 1.0 mM Pi ions were prepared in 150 mM Tris-HCl buffer. The pH of the buffer was adjusted to pH 7.4 (37 °C) using HCl solution. Subsequently, 0, 0.20, 0.25, 0.50, 0.75, 1.0, 1.5 mg·mL -1 of bovine serum albumin (BSA, molecular weight: 66 kDa, manufactured by Sigma-Aldrich) was added (preparation solution).

[0027] To analyze the adsorption behavior of BSA on OCP under supersaturated conditions, 15 mg of OCP granules were immersed in each 3 mL, 37 °C preparation solution and incubated for 1 hour with inversion stirring. The mixture was centrifuged at 3900 rpm for 3 minutes, and the BSA concentration in the supernatant was measured using CBB protein assay solution (manufactured by Nacalai Tesque) (Bradford method). The samples are represented as Ca x P y BSA z where x, y, and z respectively represent the concentrations of Ca 2+ (Ca), Pi ions (P), and BSA in the solution used in the adsorption experiment.

[0028] (Measurement of 1.3 Ca 2+ and Pi ion concentration and calculation of DS) Using Calcium E test Wako and Phospha C test Wako (manufactured by Fujifilm Wako Pure Chemical Corporation) respectively, the concentrations of Ca 2+ and Pi ions in the preparation solution before and after incubation in the presence and absence of OCP were measured.

[0029] Using the analytical values of Ca 2+ and Pi ions in the supernatant, the DS values for HA, OCP, and calcium hydrogen phosphate dihydrate (DCPD) in the preparation solution before and after incubation were calculated, and the thermodynamic stability of each calcium phosphate phase was estimated. The DS value is at 37 °C, pH 7.4 for Ca2+ and Mg 2+ and Pi ions were calculated considering three mass balances (see Non-Patent Documents 16 and 17). In this calculation, it was assumed that HCO 3- was present in the solution, and the presence of ion pairs (CaH2PO4 + , CaHPO4 0 , MgHPO4 0 , CaHCO3 + and MgHCO3 + ) was considered. However, the presence of BSA was not taken into account in the calculation, and the concentration of Mg 2+ was set to be almost zero. Furthermore, a background electrolyte of 150 mM Na + was used in the calculation instead of a tris concentration of 150 mM. The DS value was calculated from the ion activity product (IP) and solubility product (Ksp) for HA, OCP, and DCPD according to the relational expression (1).

[0030] DS = (IP / Ksp) 1 / ν ‥(1).

[0031] Here, ν is the number of ions in calcium phosphate (ν for HA is "9", ν for OCP is "8", and ν for DCPD is "2"). The solubility products of HA, OCP, and DCPD are 7.36 × 10 -60 (mol·L -1 ) 9 , 2.51 × 10 -49 (mol·L -1 ) 8 , and 2.77 × 10 -7 (mol·L -1 ) 2 (see Non-Patent Document 19), respectively. When the DS value is "1.0", "less than 1.0", and "more than 1.0", it indicates "saturated", "unsaturated", and "supersaturated", respectively.

[0032] (Characterization of OCP before and after immersion in a 1.4 supersaturated solution) The OCP particles incubated in the prepared solution were washed several times with ultrapure water and then freeze-dried.

[0033] The OCP before immersion in the preparation solution and the OCP after immersion were analyzed using a powder X-ray diffractometer (XRD, MiniFlex 600 manufactured by Rigaku Corporation). The measurement was performed using monochromatic CuKα radiation, at 40 kV and 15 mA, with a scanning speed of 1.0°·min -1 , and the scanning range was from 2θ = 3° to 60° with a step width of 0.02°.

[0034] Also, the OCP before and after incubation was analyzed using a Raman spectrometer (Model Aparaman manufactured by LICIR) equipped with an optical microscope to confirm the presence or absence of the spectrum derived from BSA.

[0035] The OCP crystals before and after incubation were observed with a transmission electron microscope (FE-TEM; JEM-2100F manufactured by JEOL Ltd.) to obtain a bright-field image of the TEM.

[0036] (2. Results) (2.1 Measurement of the BSA accumulation capacity on OCP in solutions with different DS) Figure 1 shows the adsorption amount per unit area of OCP with respect to the equilibrium concentration of BSA in each preparation solution. The values are the average values obtained from three independent experiments, and the error bars in the graph indicate the standard deviation values (SD). The dashed line shown in Figure 1 represents the adsorption isotherm of BSA on OCP in the saturated state at 37 °C and pH 7.4 reported by the present inventors, and it is a plot of the results calculated from the adsorption constants obtained based on the Langmuir equation (see relational expression (2)). (See Non-Patent Document 11).

[0037] Q = KQ0C / (1 + KC) ‥(relational expression (2)).

[0038] Here, Q is the adsorption amount of the adsorbate on the adsorbent, C is the equilibrium concentration of the adsorbate, K is the equilibrium constant, and Q0 is the saturation capacity of the adsorbate. The constants of K and Q0 are 1520 mL·μmol -1 and 0.054 μmol·m -2 respectively, and these were obtained from previous adsorption isotherms (see Non-Patent Document 11).

[0039] As can be seen from Fig. 1, the adsorption isotherms of both Ca1.5P1.0 and Ca3.0P1.0 did not follow the Langmuir equation, and particularly showed values far exceeding the theoretical values in the range where the equilibrium concentration of BSA was relatively low.

[0040] (2.2 Measurement of Ca 2+ and Pi ion concentrations and calculation of DS value)

[0041] Table 1 shows the measurement results of the changes in the Ca 2+ and Pi ion concentrations in the solution during the accumulation of BSA on OCP particles. These ion concentrations in the Ca1.5P1.0 and Ca3.0P1.0 solutions decreased after the immersion of OCP particles. The calculated DS values in these solutions decreased after incubation with OCP regardless of the initial DS and BSA concentrations.

[0042]

Table 1

[0043] The order of these DS values is 10 8 ~10 12 for HA, 10 0 ~10 1 for OCP, and 10 -1 for DCPD, respectively, indicating that these solutions after incubation are supersaturated and slightly supersaturated with respect to HA and OCP, respectively. The DS values for HA and OCP tended to be higher for Ca3.0P1.0 than for Ca1.5P1.0.

[0044] (2.3 Characterization of OCP before and after immersion in buffer containing BSA) (2.3.1 XRD analysis) Figure 2A shows the XRD patterns of OCP before and after incubation in the preparation solution of Ca1.5P1.0. Figure 2B shows the XRD patterns of OCP before and after incubation in the preparation solution of Ca3.0P1.0. Peaks corresponding to the (100), (010), and (002) planes of the OCP crystal structure are detected at 2θ = 4.7°, 9.8°, and 26°, respectively. The intensity of the peak corresponding to (100) of OCP decreased slightly, but these characteristic OCP peaks were still detected after incubation regardless of the DS value and BSA concentration of the preparation solution. Also, in the pattern after incubation, no peaks due to crystal phases of other calcium phosphates such as HA were observed.

[0045] (2.3.2 Raman Spectroscopy) Figures 3A and 3B show the Raman spectra of OCP in the range of 1500 - 1800 cm -1 before and after incubation with Ca1.5P1.0 and Ca3.0P1.0, respectively. The spectra after incubation show those of BSA0 and BSA0.50 as representatives. As can be seen from Figures 3A and 3B, in the spectra of OCP immersed in the preparation solution containing BSA, peaks due to amide III of BSA, the amino acids tryptophan and phenylalanine, and amide I are observed at 1548 cm -1 , 1604 cm -1 and 1653 cm -1 respectively (see Non-Patent Document 18).

[0046] (2.3.3 TEM Observation) Each of Figures 4A and 4B shows OCP before incubation and 0.50 mg·mL -1The bright-field image of OCP after incubation in Ca3.0P1.0 containing BSA is shown. As shown in the low-magnification image of Fig. 4A, particles having a plate-like structure, which is a typical form of OCP crystals, were observed. As shown in the low-magnification image of Fig. 4B, this plate-like structure was maintained even after incubation. As shown in the high-magnification image of Fig. 4A, the OCP before incubation had a smooth surface. In contrast, as shown in the high-magnification image of Fig. 4B, a large number of irregularities were observed on the surface of the OCP incubated in the preparation solution.

[0047] (2.3.4 Characterization) From the results of the decrease in calcium ion and inorganic phosphate ion concentrations in the preparation solution after the above incubation, the XRD pattern, and the TEM image, it is suggested that the large number of irregularities on the surface seen in the TEM image are fine OCP crystals. This crystal is considered to have grown through nucleation, either from the original crystal or from a newly precipitated crystal.

[0048] (2.3.5 Protein Adsorption) It is considered that the present invention can efficiently accumulate proteins beyond the adsorption isotherm shown by the Langmuir equation because new protein adsorption sites appear as described above, or because proteins are incorporated during precipitation.

Claims

1. comprising a first crystal and a plurality of second crystals formed on the surface of the first crystal and having a size smaller than that of the first crystal; wherein the second crystals form irregularities on the surface of the first crystal; wherein the first crystal and the second crystals are calcium phosphate composed of octacalcium phosphate.

2. The calcium phosphate according to claim 1, wherein a protein is accumulated via the second crystals.

3. a protein is accumulated in an amount exceeding the amount predictable from the Langmuir equation, wherein the amount of protein adsorbed, calculated from the adsorption constant obtained based on the Langmuir equation represented by the following relational expression (2), exceeds the amount of protein adsorbed to calcium phosphate in a saturated state at 37° C. and pH 7.4 for the calcium phosphate. The calcium phosphate according to claim 2. Q = KQ0C / (1 + KC) (Relational expression (2)) (where Q is the amount of adsorbate adsorbed to the adsorbent, C is the equilibrium concentration of the adsorbate, K is the equilibrium constant, and Q0 is the saturation capacity of the adsorbate. The constants of K and Q0 are 1520 mL·μmol−1 and 0.054 μmol·m−2, respectively.)

4. A method for producing calcium phosphate with a protein accumulated thereon, comprising: a buffer solution preparation step of preparing a buffer solution containing 1 to 5 mM calcium ions and 0.5 to 3 mM inorganic phosphate ions and adjusted to pH 6 to 8 at 25 to 45° C.; When the calcium ion content of the buffer solution is 1 to 2 mM, it is in the range of 0.01 to 0.5 mg / mL, and when the calcium ion content is 2 to 5 mM, it is in the range of 0.01 to 1.5 mg / mL. Dissolve the protein in the buffer solution to prepare a protein-containing buffer solution, and a protein-containing buffer solution preparation step; －１ When the calcium ion content is in the range of 2 to 5 mM, it is in the range of 0.01 to 1.5 mg / mL. Dissolve the protein in the buffer solution to prepare a protein-containing buffer solution, and a protein-containing buffer solution preparation step; －１ In the range, a protein-containing buffer solution preparation step of dissolving the protein in the buffer solution to prepare a protein-containing buffer solution; a calcium phosphate contact step of bringing a first crystal composed of octacalcium phosphate into contact with the protein-containing buffer solution to form a second crystal composed of octacalcium phosphate on the surface of the first crystal composed of octacalcium phosphate.

5. The method for producing calcium phosphate according to claim 4, wherein in the calcium phosphate contact step, a protein is adsorbed to the second crystal composed of octacalcium phosphate.

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