Method for analyzing crystal form of calcium crystals

Abstract

To provide a method for analyzing a crystal form that can more accurately determine a crystal form such as a metastable phase or a stable phase included in calcium crystals.SOLUTION: The presence or absence of at least one of a metastable phase or a stable phase in calcium crystals is identified by irradiating a sample as an analysis target with light in a frequency band of 10 THz or lower and performing spectroscopic analysis. The calcium crystals are, for example, calcium phosphate crystals. If the sample contains octacalcium phosphate, the calcium phosphate crystals contained in the sample are determined to be in a metastable phase. If the sample contains hydroxyapatite, the calcium phosphate crystals contained in the sample are determined to be in a stable phase.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for analyzing the crystalline forms of metastable and stable phases contained in calcium crystals such as calcium phosphate and calcium oxalate. [Background technology]

[0002] Calcium crystals such as calcium phosphate and calcium oxalate exist in the form of a stable phase (also called stable form), a metastable phase (also called metastable form), etc. These calcium crystals are known to be contained in large amounts in objects in the body, such as bones, and in objects formed in the body, such as stones and calcification of blood vessels (arteriosclerosis).

[0003] For example, in the case of calcium phosphate, several phases have been reported, such as ACP (amorphous calcium phosphate), DCPA (dicalcium phosphate anhydride), DCPD (dicalcium phosphate dehydrate), OCP (octacalcium phosphate), β-TCP (β-tricalcium phosphate), α-TCP (α-tricalcium phosphate), CDHA (hydroxyapatite with calcium deficient), HAp (hydroxyapatite), TTCP (TetCP) (tetra calcium phosphate), CPP (β-Ca pyrophosphate), and OAp (oxyapatite) (see Non-Patent Document 1). Among these, in bones and arteriosclerotic tissues in vivo, metastable phase OCP (octacalcium phosphate) is contained in large amounts in the early stages of crystallization, and it has been noted that the process in which this changes to stable phase HAp (hydroxyapatite) by dehydration is important.

[0004] In the living body, OCP (Octacalcium phosphate) contained in the metastable phase is contained in a form in which some of the calcium ions are replaced by sodium ions. In the following, such forms of OCP are also referred to simply as "OCP". In addition, it is known that HAp (Hydroxyapatite) actually exists in the living body as carbonate apatite containing a few percent of carbonate groups (see Non-Patent Document 2). In light of this, HAp is described here as a stable phase, which is a representative phase of apatite.

[0005] It is known that calcium phosphate crystals contained in biological materials such as bones contain a large amount of OCP, which is a metastable phase, in the early stages of their formation, and over time (i.e., as the bone matures), the OCP decreases and the stable phase HAp increases.

[0006] In addition, calcium phosphate crystals have attracted the attention of many researchers and medical professionals as a causative substance of various diseases such as arteriosclerosis (vascular calcification), urinary tract stones, etc. It is known that calcium phosphate crystals in vascular calcified tissues are formed in the form of OCP in the early stage of the formation of arteriosclerosis, and gradually change to HAp and are deposited (see Non-Patent Document 3).

[0007] In this way, by analyzing the crystal state of calcium crystals contained in samples derived from living organisms, it is possible to know the state of tissues and products in the body (e.g., formation process, maturity, etc.). Therefore, the results of the analysis of the crystal state may be effectively used in, for example, research fields related to the developmental process of animals, and in the medical field related to the analysis of various diseases and pathologies such as arteriosclerosis and urinary tract stones. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2008-197081 A [Non-patent literature]

[0009] [Non-Patent Document 1] Liga Berzina-Cimdina and Natalija Borodajenko, Research of Calcium Phosphates Using Fourier Transform Infrared Spectroscopy, Infrared Spectroscopy - Materials Science, Engineering and Technology, Published online 25, April, 2012, 123-148. [Non-Patent Document 2] Kunio Ishikawa, "Bone Composition (Carbonate Apatite) Artificial Bone 46th (Reiwa 3) Inoue Harunari Award Technology," Journal of Industry-Academia Collaboration, January 25, 2022 [Non-Patent Document 3] O. Gourgas, J. Marulanda, P. Zhang, M. Murshed, and M. Cerruti, Multidisciplinary approach to understand medial arterial calcification, Arteriosclerosis, Thrombosis, and Vascular Biology, 38 (2018) 363-372. [Non-Patent Document 4] IA Karampas, CG Kontoyannis, Characterization of calcium phosphates mixtures, Vibrational Spectroscopy, 64 (2013) 126-133. [Non-Patent Document 5] NJ Crane, V. Popescu, MD Morris, P. Steenhuis, MA Ignelzi, Jr., Raman spectroscopic evidence for octacalcium phosphate and other transient mineral species deposited during intramembranous mineralization. Bone 39, 434-442 (2006). Summary of the Invention [Problem to be solved by the invention]

[0010] Conventionally, calcium phosphate crystals have been analyzed using methods such as Raman spectroscopy, infrared absorption spectroscopy in the near-infrared and mid-infrared regions, and X-ray analysis (see, for example, Non-Patent Document 4). However, when calcium phosphate crystals are analyzed using the method of Non-Patent Document 4, it is difficult to distinguish between HAp and OCP based on the similarity of their spectra in Raman spectroscopy and infrared absorption spectroscopy in the near-infrared and mid-infrared regions. Although HAp and OCP can be distinguished using X-ray analysis, there are measurement difficulties, such as the difficulty in obtaining a reflection at 4.7°, which is a clear difference, unless the sample is powdered (see, for example, Figs. 1-3 in Non-Patent Document 4).

[0011] Patent Document 1 discloses that absorption spectra in the terahertz region are applied to the analysis of substances discharged from living organisms, such as components of stones and substances in urine, and are used for pathological diagnosis. Patent Document 1 discloses that the objects extracted from the body are stones, or body fluids such as joint fluid, or substances reflecting the components of body fluids such as urine, feces, saliva, sweat, and tears, and that the method of analyzing and quantifying the components uses at least one or a combination of absorption lines of hydroxyapatite at 2.9 THz and 5.7 THz, or the ratio of the absorption amounts of a plurality of absorption lines, etc.

[0012] Patent Document 1 describes the identification of hydroxyapatite by applying absorption spectroscopy in the terahertz region, but does not analyze octacalcium phosphate (OCP).

[0013] Thus, while there has been a relatively large amount of analysis of hydroxyapatite (HAp), a form of calcium phosphate crystal, there has been no analysis of OCP (octacalcium phosphate), which is found in large amounts in the early stages of bone formation.

[0014] In addition, Non-Patent Document 5 reports that the OCP was analyzed by Raman spectroscopy and found to be at 955 cm -1 It is described that a characteristic peak is observed in the 100% OCP concentration range. However, when the present inventors performed an analysis using mouse skulls as sample specimens based on the contents of this document, they were unable to accurately identify the OCPs. More specifically, the analysis was performed using two conventional methods for analyzing the proportion of OCPs, leading to mutually contradictory conclusions.

[0015] An object of the present invention is to provide a method for analyzing crystal morphology that can more accurately determine the crystal morphology of metastable and stable phases contained in calcium crystals. [Means for solving the problem]

[0016] In the process of investigating the difference in components (e.g., mineral components) between bones in the early stages of formation and bones with advanced aging, the inventors of the present application noticed that the crystal morphology of calcium phosphate crystals contained in bones changes from the early stages of bone formation to the maturation stage. The inventors of the present application then conducted intensive studies to determine whether there was a method for accurately distinguishing between OCP contained in the metastable phase of calcium phosphate crystals, which is an example of calcium crystals, and HAp contained in the stable phase. As a result, they found that characteristic peaks for OCP and HAp were observed by irradiating a sample to be analyzed with light in a frequency band of 10 THz or less and performing spectroscopic analysis, which led to the completion of the present invention.

[0017] In one aspect of the present invention, a method for analyzing crystal morphology involves irradiating a sample to be analyzed with light in a frequency band of 10 THz or less and performing spectroscopic analysis to identify whether calcium crystals contained in the sample contain at least one of a metastable phase and a stable phase.

[0018] In the above-mentioned crystal morphology analysis method according to one aspect of the present invention, the calcium crystals may be calcium phosphate crystals, in which case, when the sample contains octacalcium phosphate, the calcium phosphate crystals contained in the sample are determined to be in the metastable phase, and when the sample contains hydroxyapatite, the calcium phosphate crystals contained in the sample are determined to be in the stable phase.

[0019] In the above-described method for analyzing a crystal morphology according to one aspect of the present invention, the frequency band may be within a range of 2 THz or more and 9 THz or less.

[0020] The above-mentioned method for analyzing crystal morphology according to one aspect of the present invention may include determining whether or not there is an absorption peak at 2.8 THz in the spectroscopic analysis. Specifically, if there is an absorption peak at 2.8 THz in the spectroscopic analysis, it is determined that the sample contains HAp (hydroxyapatite). On the other hand, if there is no absorption peak at 2.8 THz in the spectroscopic analysis, it is determined that the sample contains OCP (octacalcium phosphate).

[0021] Furthermore, the method for analyzing crystal morphology according to one aspect of the present invention may further include determining whether or not the sample contains a phase less stable than the metastable phase by confirming the presence or absence of an absorption peak at 7.1 THz in the spectroscopic analysis. For example, when the sample contains calcium hydrogen phosphate dihydrate (DCPD), it can be determined that the calcium phosphate crystals contained in the sample contain a phase less stable than the metastable phase. Specifically, when an absorption peak at 7.1 THz is present in the spectroscopic analysis, it can be determined that the sample contains calcium hydrogen phosphate dihydrate (DCPD). Therefore, it can be determined that the sample to be analyzed contains a phase less stable than the metastable phase.

[0022] In the above-described method for analyzing a crystal morphology according to one aspect of the present invention, the sample may be a substance derived from a living organism. Effect of the Invention

[0023] According to the method for analyzing crystal morphology according to one aspect of the present invention, the crystal morphologies of metastable and stable phases contained in calcium crystals can be determined more accurately. [Brief description of the drawings]

[0024] [Figure 1] (a) is a schematic diagram showing the chemical structure of HAp (hydroxyapatite), (b) is a schematic diagram showing the chemical structure of OCP (octacalcium phosphate), and (c) is a schematic diagram showing the chemical structure of OCP-Na. [Diagram 2] FIG. 2 is a diagram showing the results of spectroscopic analysis of a sample containing HAp and a sample containing OCP in Example 1. [Diagram 3] FIG. 13 shows the results of spectroscopic analysis of a sample containing HAp, a sample containing OCP, and a sample containing a mixture of HAp and OCP in Example 2. [Figure 4]FIG. 11 is a diagram showing a change in absorbance spectrum when a sample containing HAp is irradiated with terahertz waves for a predetermined period of time in Example 3. [Diagram 5] FIG. 11 is a diagram showing a change in absorbance spectrum when a sample containing an OCP is irradiated with terahertz waves for a predetermined period of time in Example 3. [Figure 6] FIG. 1 shows the results of spectroscopic analysis of a sample containing DCPD in Example 4. [Figure 7] FIG. 13 shows the results of spectroscopic analysis of samples with different elapsed times from the start of the reaction in Example 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention will be described in more detail below, although the present invention is not limited thereto.

[0026] The present invention relates to a method for analyzing the crystal morphology contained in calcium crystals, such as calcium phosphate crystals and calcium oxalate crystals.

[0027] Calcium phosphate crystals are found in relatively large amounts in animal components such as bones, teeth, etc. Calcium phosphate crystals are also found in relatively large amounts in natural components such as soil and rocks.

[0028] Calcium phosphate crystals exist in the form of stable phase (also called stable form) and metastable phase (also called metastable form). The stable phase contains calcium phosphate in the form of HAp (hydroxyapatite), and one of the metastable phases contains calcium phosphate in the form of OCP (octacalcium phosphate).

[0029] Figure 1(a) shows the chemical structure of hydroxyapatite, which is represented by the chemical formula Ca5(PO4)3(OH). Figure 1(b) shows the chemical structure of Ca4HO 12The chemical structure of octacalcium phosphate, represented by the chemical formula P3, is shown in FIG. 1(b). More specifically, the chemical structure shown in FIG. 1(b) is the case where all the ions in the structure are calcium ions (also called "OCP-pure"). FIG. 1(c) shows a schematic diagram of the chemical structure of OCP-pure where some of the calcium ions are replaced by sodium ions. That is, FIG. 1(c) shows the chemical structure of "OCP-Na" contained in the metastable phase of calcium phosphate crystals in vivo. As shown in FIG. 1(c), in "OCP-Na", some of the calcium ions in OCP (octacalcium phosphate) are replaced by sodium ions. In the following, when referring to an OCP in which some of the calcium ions are replaced by sodium ions, the OCP itself will be called "OCP-Na". (Reference: Y. Sugiura and Y. Makita, Sodium induces octacalcium phosphate formation and enhances its layer structure by affecting the hydrous layer phosphate, Crystal Growth & Design, 18 (2018) 6165-6171.)

[0030] HAp is formed by the dehydration reaction of OCP. It is generally known that calcium phosphate crystals exist in the form of a metastable OCP in the early stages of their formation, and as dehydration progresses over time, the amount of HAp in the stable phase increases.

[0031] Calcium oxalate crystals are known to be the main component of stones (e.g., urinary stones) formed in living organisms such as humans. Similar to calcium phosphate crystals, calcium oxalate crystals also exist in different crystalline forms, such as stable and metastable phases. In the body environment, the metastable phase contains calcium oxalate in the form of COD (dihydrate, CaC2O4·2H2O) or COT (trihydrate, CaC2O4·3H2O), while the stable phase contains calcium oxalate in the form of COM (monohydrate, CaC2O4·H2O).

[0032] The method for analyzing crystal morphology according to the present invention is a method for identifying crystal morphology, such as stable and metastable phases, contained in calcium crystals. In this analysis method, a sample to be analyzed is irradiated with light in a frequency band of 10 THz or less, and spectroscopic analysis is performed. Then, it is identified whether or not at least one of the metastable and stable phases is contained in the calcium crystals contained in the sample.

[0033] The light used in spectroscopic analysis is terahertz (THz) waves. 12 Terahertz waves are electromagnetic waves in the 100 THz range. In a broad sense, the frequency band of terahertz waves includes frequencies in the range of 0.1 THz to 100 THz. In terms of wavelength, 1 THz is 0.3 mm. Thus, terahertz waves are electromagnetic waves in the frequency range between light and radio waves.

[0034] In the analysis method according to the present invention, a sample is irradiated with terahertz waves in a frequency band of 10 THz or less, and spectroscopic analysis is performed, and it is determined whether or not calcium crystals contained in the sample include at least one of a metastable phase and a stable phase.

[0035] Specifically, when a spectroscopic analysis of an arbitrary sample is performed using the above method, if an absorption peak is present at a frequency corresponding to the stable phase, the sample is determined to contain a stable phase. If an absorption peak is present at a frequency common to the metastable phase and the stable phase, but no absorption peak is present at a frequency corresponding to the stable phase, the sample is determined to contain a metastable phase. If no absorption peak is present at any frequency, the sample is determined to not contain calcium crystals.

[0036] The spectroscopic analysis in this method can be performed using a conventionally known terahertz spectrometer, such as a far-infrared FTIR measurement device, a terahertz time-domain spectroscopy (THz-TDS) device, or a terahertz spectrometer using a single-wavelength terahertz band light source such as photomixing or difference frequency generation / cascade laser.

[0037] The sample to be analyzed may be an object that may contain calcium crystals. The sample may be in the form of, for example, a solid or powder. Of these, it is preferable to use a solid sample. This allows the analysis of objects of biological origin, such as bones and stones, in their original state.

[0038] For example, when the analytical method according to the present invention is used for the purpose of ecological research, examples of samples to be analyzed include animal-derived components such as bones and teeth. When the analytical method according to the present invention is used for the purpose of research into various diseases such as arteriosclerosis and urinary tract stones, examples of samples to be analyzed include samples derived from affected areas taken from patients suffering from arteriosclerosis, urinary tract stones, etc. Thus, samples derived from living organisms are suitable as samples to be analyzed.

[0039] When the present analytical method is for identifying the crystal morphology of calcium phosphate crystals, if the sample being analyzed contains OCP (octacalcium phosphate), the calcium phosphate crystals contained in the sample are determined to be in a metastable phase, and if the sample being analyzed contains HAp (hydroxyapatite), the calcium phosphate crystals contained in the sample are determined to be in a stable phase.

[0040] More specifically, if a sample is irradiated with light in a frequency band of 10 THz or less and a spectroscopic analysis is performed, and an absorption peak at 8.4 THz is confirmed but not at 2.8 THz, the sample is determined to contain OCP (octacalcium phosphate).Furthermore, if a similar spectroscopic analysis is performed and absorption peaks at 8.4 THz and 2.8 THz are confirmed, the sample is determined to contain HAp (hydroxyapatite).

[0041] As described above, by irradiating a sample with light in the terahertz frequency band and performing spectroscopic analysis to confirm the presence or absence of an absorption peak at 2.8 THz, it is possible to determine whether or not the calcium phosphate crystal contains OCP and whether or not it contains HAp.

[0042] For this reason, the frequency band of the terahertz waves irradiated onto the sample is preferably within the range of 2 THz to 9 THz.

[0043] In addition, by referring to each spectrum shown in Figure 3, it may be determined whether the crystal morphology of calcium phosphate crystals contained in the sample to be analyzed contains only a metastable phase or a stable phase, or contains both a metastable phase and a stable phase.

[0044] For example, the crystal morphology of calcium phosphate crystals contained in an arbitrary sample can be determined by performing terahertz wave spectroscopic analysis on the arbitrary sample in the same manner as in Example 2 and comparing the obtained spectrum with each spectrum shown in Fig. 3(a), (b), and (c). Specifically, if the spectrum shape of the arbitrary sample is similar to the spectrum shown in Fig. 3(a), it is determined that the calcium phosphate crystals are formed only of the metastable phase, and if it is similar to the spectrum shown in Fig. 3(c), it is determined that the calcium phosphate crystals are formed only of the stable phase. In addition, if the spectrum shape of the arbitrary sample is close to the spectrum shown in Fig. 3(b), it is determined that the calcium phosphate crystals are formed of a mixed phase of the metastable phase and the stable phase.

[0045] Alternatively, a terahertz wave spectroscopic analysis may be performed on a plurality of samples in the same manner as in Example 2, and the obtained spectra may be compared to determine whether the crystal form of calcium crystals contained in the samples is a metastable phase, a stable phase, or a mixture of these. Specifically, the determination can be made by using the presence or absence of an absorption peak at 8.4 THz, the presence or absence of an absorption peak at 2.8 THz, and the intensities of the absorption peaks at 8.4 THz and 2.8 THz.

[0046] As described above, the method for analyzing crystal morphology according to the present invention involves irradiating a sample to be analyzed with light in a frequency band of 10 THz or less and performing spectroscopic analysis to identify whether or not the calcium crystals contained in the sample contain at least one of a metastable phase and a stable phase.

[0047] According to the above-mentioned analysis method, the crystal forms of metastable and stable phases contained in calcium crystals can be determined by using terahertz waves with lower photon energy compared to X-rays. By using terahertz waves with lower photon energy, the crystal forms contained in the sample can be analyzed while reducing damage to the sample to be analyzed. Therefore, it is possible to avoid the characteristics of the sample (e.g., the components contained, the crystal form, etc.) from changing during the analysis due to irradiation with light, and the crystal forms of metastable and stable phases contained in the sample can be determined more accurately.

[0048] In addition, in the conventional method for analyzing the crystal morphology of calcium crystals using X-rays, it was necessary to prepare the sample in a powder form. In contrast, in the analysis method of the present invention, for example, solid samples such as bones and stones can be analyzed in their original state. Therefore, the crystal morphology can be easily analyzed without time-consuming sample preparation or destruction of the sample.

[0049] In this way, the analytical method of the present invention makes it possible to analyze the crystal morphology of calcium crystals in a minimally invasive manner, and is therefore expected to be useful in the diagnosis and treatment of various diseases, such as arteriosclerosis and urinary tract stones.

[0050] In an analytical method according to one embodiment of the present invention, the calcium crystals contained in a sample are calcium phosphate crystals, and when the sample contains octacalcium phosphate (OCP), the calcium phosphate crystals contained in the sample are identified as the metastable phase, and when the sample contains hydroxyapatite (HAp), the calcium phosphate crystals contained in the sample are identified as the stable phase.

[0051] OCP is a substance (crystalline form) that is generated in the early stages of bone formation, and by knowing the presence, content, and ratio of this component, it is expected that the mechanism involved in bone mineralization will be elucidated. Understanding the mechanism involved in bone mineralization is essential in research on bone tissue engineering and bone diseases.

[0052] Natural calcium crystals such as bones contain a mixture of stable and metastable phases, but the peaks of both are very similar in Raman spectroscopy and infrared absorption spectroscopy in the near-infrared and mid-infrared regions. Therefore, it is very difficult to determine the proportion of stable and metastable phases in natural calcium crystals. In addition, the metastable phase formed by OCP is unstable, and if spectroscopic analysis is performed using light with high photon energy, it may undergo a phase transition to HAp during analysis. Therefore, it is difficult to accurately analyze the crystal morphology contained in natural samples.

[0053] Using the analysis method according to the present embodiment to determine the presence or absence of a metastable phase containing OCP in calcium phosphate crystals is highly useful in research on bone formation, stone formation, etc. The analysis method according to the present embodiment can be used to measure the maturity of bones, stones, rocks, fossils, etc. In addition, the analysis method according to the present embodiment is expected to be applied in fields such as food and agriculture, and geology, ecology, and oceanography.

[0054] Furthermore, the inventors of the present application have found that it is possible to determine whether or not a phase that is more unstable than OCP, which is a type of metastable phase, is present by using the analysis method according to the present embodiment.

[0055] As mentioned above, octacalcium phosphate (OCP) and hydroxyapatite (HAp) are the main calcium phosphates reported to be present in bones in vivo, but calcium hydrogen phosphate dihydrate (CaHPO4·2H2O, also known as DCPD), which is even more unstable than OCP, is sometimes found in vivo. It is believed that DCPD changes over time to the more stable octacalcium phosphate (OCP), and then to the even more stable hydroxyapatite (HAp).

[0056] The inventors of the present application irradiated a sample containing DCPD with terahertz waves in a frequency band of 10 THz or less and performed spectroscopic analysis, and found that an absorption peak was observed at a frequency different from that of OCP and HAp. By utilizing this result, it is possible to determine whether or not a phase more unstable than the metastable phase is contained using the analysis method according to the present embodiment.

[0057] For example, a sample to be analyzed is irradiated with terahertz waves in a frequency band of 10 THz or less, and spectroscopic analysis is performed to confirm whether or not an absorption peak at 7.1 THz exists. If an absorption peak at 7.1 THz exists, it can be determined that the sample contains calcium hydrogen phosphate dihydrate (DCPD). If it is confirmed that the sample contains calcium hydrogen phosphate dihydrate (DCPD), it can be determined that the calcium phosphate crystals contained in the sample contain a phase that is less stable than the metastable phase.

[0058] By using the above analytical method, it is possible to analyze the crystal morphology of calcium phosphate contained in bones and other tissues in a minimally invasive manner and confirm the presence or absence of phases with morphologies that are even more unstable than OCP (specifically, phases containing DCPD).

[0059] [Example] The present invention will be described in detail below with reference to examples of the present invention, but the present invention is not limited to these.

[0060] Example 1 In Example 1, the difference in absorption characteristics between a sample containing HAp and a sample containing OCP was confirmed by using terahertz spectroscopy. In this example, a sample containing OCP in which some calcium ions were replaced with sodium ions (i.e., "OCP-Na") was used.

[0061] 1. Sample Preparation The synthesized OCP and HAp powders were prepared. Figure 1(a) shows the chemical structure of HAp. Figure 1(c) shows the chemical structure of OCP-Na. Each of the prepared powders was mixed with a transparent matrix material powder to obtain a mixture containing 5% by weight of OCP or HAp. After homogenizing each powder mixture, it was heated at 200 kgf / cm 2 The mixture was pressed at a pressure of 0.01 mm for 20 seconds to form pellet samples. The diameter of each pellet sample was 10 mm. The thickness of the pellet sample containing HAp was 0.344 mm, and the thickness of the pellet sample containing OCP was 0.310 mm.

[0062] 2. Spectroscopic analysis using the device Each pellet sample was subjected to spectroscopic analysis using Fourier transform infrared (FTIR) spectroscopy in the far infrared region. Specifically, a far infrared Fourier transform infrared spectrophotometer (manufactured by JASCO Corporation, "FT / IR-660Plus") was used as the measuring device, and measurements were performed in a transmission configuration. The diameter of the aperture used was 8 mm, and the spectral range of the measurement was 1.5 to 19.0 THz (50 to 635 cm -1 ) The sample chamber was evacuated to minimize THz absorption by water vapor.

[0063] 3.Results Figure 2 shows the terahertz band absorbance spectrum of each pellet sample. The upper part (a) of Figure 2 shows the terahertz band absorbance spectrum of the sample containing HAp, and the lower part (b) of Figure 2 shows the terahertz band absorbance spectrum of the sample containing OCP. The numerical values ​​shown in each spectrum diagram are the center frequencies of the absorption peaks.

[0064] Comparing the spectra in Figure 2, it was confirmed that both HAp and OCP have broad peaks centered at 8.4 THz.

[0065] On the other hand, apart from the peak at 8.4 THz, clear differences were confirmed between the samples containing HAp and the samples containing OCP. That is, the sample containing HAp was confirmed to have characteristic absorption peaks at 2.8 THz, 5.4 THz, 6.8 THz, and 10.4 THz. In contrast, no peak was observed around 2.8 THz in the sample containing OCP. Furthermore, the peaks at 5.4 THz, 6.8 THz, and 10.4 THz were significantly smaller than those of HAp.

[0066] The above results show that the samples containing HAp and the samples containing OCP can be distinguished from each other by comparing the terahertz band absorbance spectrum shapes of the samples containing HAp and the samples containing OCP, in particular by the presence or absence of a characteristic absorption peak at 2.8 THz.

[0067] Example 2 In Example 2, terahertz spectroscopy was used to confirm the difference in absorption characteristics between a sample containing a mixture of HAp and OCP and a sample containing HAp or OCP alone. In this example, an OCP in which some calcium ions are replaced with sodium ions (i.e., "OCP-Na") was used.

[0068] 1. Sample Preparation The synthesized OCP and HAp powders were prepared, and each of the prepared powders was mixed with a transparent matrix material powder to obtain a mixture containing 5 wt% of OCP or HAp.

[0069] In addition, the OCP powder and the HAp powder were mixed at a weight ratio of 50%:50%. Then, the mixture of OCP and HAp was mixed with a transparent matrix material powder to obtain a mixture containing the mixture of OCP and HAp at a content of 5% by weight.

[0070] After homogenizing each powder mixture, it was subjected to a pressure of 200 kgf / cm 2 The mixture was pressed at a pressure of 1000 psi for 20 seconds to form a pellet-shaped sample.

[0071] 2. Spectroscopic analysis using the device Each pellet sample was subjected to spectroscopic analysis using Fourier transform infrared (FTIR) spectroscopy in the far infrared region. Specifically, a far infrared Fourier transform infrared spectrophotometer (manufactured by JASCO Corporation, "FT / IR-660Plus") was used as the measuring device, and measurements were performed in a transmission configuration.

[0072] 3.Results The terahertz band absorbance spectrum of each pellet sample is shown in Figure 3. The upper row (a) of Figure 3 is the absorbance spectrum of the sample containing HAp, the middle row (b) of Figure 3 is the absorbance spectrum of the sample containing a mixture of OCP and HAp, and the lower row (c) of Figure 3 is the absorbance spectrum of the sample containing OCP.

[0073] In the sample containing only HAp, characteristic absorption peaks were observed at 2.8 THz, 5.4 THz, 6.8 THz, 8.4 THz, and 10.4 THz (see Figure 3(a)).

[0074] Similarly, in the sample in which HAp and OCP were mixed in a 1:1 weight ratio, characteristic absorption peaks were observed at 2.8 THz, 5.4 THz, 6.8 THz, 8.4 THz, and 10.4 THz; however, the peak intensities were lower than in the sample containing HAp alone (see Figure 3 (b)).

[0075] In the HAp sample and the mixed sample of HAp and OCP, the peak intensities at 2.8 THz, 5.4 THz, and 8.4 THz were extracted by curve regression analysis using the Lorentzian function.

[0076] As a result, the peak intensity at 2.8 THz and 5.4 THz were 49.3% and 48.9%, respectively, of the HAp sample alone. These values ​​are close to the weight ratio of HAp in the mixture sample, which is 50%. Meanwhile, the peak intensity at 8.4 THz was only 86.7% of the HAp sample alone, showing little change.

[0077] Therefore, it is considered that the peak intensities at 2.8 THz and 5.4 THz reflect the mixture ratio of HAp and OCP, whereas the peak intensity at 8.4 THz more strongly reflects the total amount of HAp and OCP.

[0078] These results suggest that by comparing the absorption peak intensities measured by terahertz spectroscopy, it is possible to distinguish a mixture of calcium phosphate crystals from a material consisting of a single crystalline phase and to estimate the mixture ratio.

[0079] Example 3 In Example 3, changes in absorbance spectrum were confirmed when a sample containing HAp and a sample containing OCP were irradiated with terahertz waves for a long period of time. In this example, a sample containing OCP in which some calcium ions were replaced with sodium ions (i.e., "OCP-Na") was used.

[0080] 1. Sample Preparation The synthesized HAp and OCP powders were prepared. Each of the prepared powders was mixed with a transparent matrix material powder to obtain a mixture containing HAp or OCP at a content of 1 wt%. After homogenization of each powder mixture, it was subjected to a pressure of 200 kgf / cm. 2 The mixture was pressed at a pressure of 1000 psi for 20 seconds to form a pellet-shaped sample.

[0081] 2. Spectroscopic analysis using the device Each sample was subjected to spectroscopic analysis using Fourier transform infrared (FTIR) spectroscopy in the far infrared region. Specifically, a far infrared Fourier transform infrared spectrophotometer (manufactured by JASCO Corporation, "FT / IR-660Plus") was used as a measuring device, and measurements were performed in a transmission configuration.

[0082] Specifically, first, immediately after starting irradiation of the HAp sample with terahertz waves, the terahertz band absorbance spectrum of the sample was measured. After that, the sample was continuously irradiated with terahertz waves for one hour, and then the terahertz band absorbance spectrum of the sample was measured again. Similarly, immediately after starting irradiation of the OCP sample with terahertz waves, the terahertz band absorbance spectrum of the sample was measured. After that, the sample was continuously irradiated with terahertz waves for one hour, and then the terahertz band absorbance spectrum of the sample was measured again.

[0083] 3.Results The results are shown in Figures 4 and 5. Figure 4 shows the results for the HAp sample, and Figure 5 shows the results for the OCP sample. In Figures 4 and 5, the absorbance spectrum measured immediately after the start of irradiation is shown by a solid line, and the absorbance spectrum measured after one hour of irradiation is shown by a dashed line. As shown in Figures 4 and 5, it was confirmed that the spectrum shape of each sample hardly changed immediately after the start of terahertz wave irradiation and after one hour of irradiation.

[0084] This result shows that the characteristics of the calcium phosphate crystal sample do not change even when the sample is continuously irradiated with terahertz waves, and supports the idea that terahertz wave measurements cause very little damage to the sample.

[0085] Example 4 In Example 4, the absorption characteristics of a sample containing DCPD were confirmed by using terahertz spectroscopy.

[0086] 1. Sample Preparation A commercially available DCPD (dibasic calcium phosphate dihydrate) powder (Fujifilm Wako Pure Chemical Industries, Ltd., 036-19915, Lot WTL6929) was prepared. The prepared DCPD powder was mixed with a transparent matrix material powder to obtain a mixture containing DCPD at a content of 3 wt%. After homogenizing this powder mixture, it was subjected to a pressure of 200 kgf / cm. 2 The mixture was pressed at a pressure of 1000 psi for 20 seconds to form a pellet-shaped sample.

[0087] 2. Spectroscopic analysis using the device The obtained pellet sample was subjected to spectroscopic analysis using Fourier transform infrared (FTIR) spectroscopy in the far infrared region. Specifically, a far infrared Fourier transform infrared spectrophotometer (manufactured by JASCO Corporation, "FT / IR-660Plus") was used as a measuring device, and measurements were performed in a transmission configuration.

[0088] 3.Results The terahertz band absorbance spectrum of a pellet sample containing DCPD is shown in Figure 6. The numerical values ​​shown in the spectrum diagram in Figure 6 are the center frequencies of the absorption peaks.

[0089] As shown in Figure 6, in the sample containing DCPD, a strong peak was observed at 7.1 THz (the largest absorption peak in the frequency band below 10 THz). In addition to 7.1 THz, characteristic absorption peaks were also observed at 3.8 THz, 5.1 THz, 6.2 THz, and 8.4 THz.

[0090] This was in contrast to the results for HAp and OCP, both of which showed prominent peaks at 8.4 THz (see Figure 3). Based on this, it is believed that the presence or absence of a characteristic absorption peak at 7.1 THz can distinguish samples containing DCPD from other samples (e.g., samples containing HAp, samples containing OCP, or samples containing HAp and OCP).

[0091] Example 5 In Example 5, the phase transition over time of a sample containing DCPD was confirmed by the following method.

[0092] 1. Sample Preparation A commercially available DCPD (calcium hydrogen phosphate dihydrate) powder (Fujifilm Wako Pure Chemical Industries, Ltd., 036-19915, Lot WTL6929) was prepared. The prepared DCPD powder was added to a solution containing 2 mM calcium ions and 2 mM phosphate ions in 200 mM acetate buffer, and the mixture was stirred at 70°C. The phase change reaction was allowed to proceed by continuing this stirring.

[0093] Samples were taken out 60 minutes, 120 minutes, 1 day, 2 days, and 3 days after the start of the reaction, and each sample was dried at 37°C for 1 day.

[0094] Each dried sample was mixed with a transparent matrix material powder to obtain a mixture containing the sample at a content of 3% by weight. After homogenizing the powder mixture, it was subjected to a pressure of 200 kgf / cm 2 The mixture was pressed at a pressure of 1000 psi for 20 seconds to form a pellet-shaped sample.

[0095] 2. Spectroscopic analysis using the device Each of the pellet-shaped samples obtained was subjected to spectroscopic analysis using Fourier transform infrared (FTIR) spectroscopy in the far infrared region. Specifically, a far infrared Fourier transform infrared spectrophotometer (manufactured by JASCO Corporation, "FT / IR-660Plus") was used as a measuring device, and measurements were performed in a transmission configuration.

[0096] 3.Results The results are shown in Figure 7. From top to bottom, Figure 7 shows the terahertz band absorbance spectra of each sample before the reaction (a), 60 minutes after the start of the reaction (b), 120 minutes after the reaction started (c), 1 day after the reaction started (d), 2 days after the reaction started (e), and 3 days after the reaction started (f). Each absorbance spectrum shown in Figure 7 has been normalized so that the maximum value within the displayed frequency range is 1, making it easier to compare the changes in shape.

[0097] Sixty minutes after the start of the reaction, the 7.1 THz peak that was present in the sample before the reaction (i.e., the sample containing DCPD) was no longer observed (see Figure 7(b)). In Figure 7(b), it was confirmed that the strongest peak shifted to 8.4 THz and the spectrum became similar to that of the sample containing OCP (see Figure 2(b)). This suggests that at this point, most of the calcium phosphate crystals in the sample had changed from DCPD to OCP.

[0098] After more than two days from the start of the reaction, the peaks at 2.8 THz and 5.4 THz became more clearly observed. These peaks were not clear in the sample containing OCP (see Figure 2 (b)), but were prominent in the sample containing HAp (see Figure 2 (a)). This suggests that the calcium phosphate crystals contained in the samples were gradually changing from OCP to HAp.

[0099] As described above, by measuring the shape of the terahertz band absorbance spectrum, we were able to observe the process in which DCPD changes into OCP and then into HAp.

[0100] By performing spectroscopic analysis of terahertz waves using the method of this embodiment and comparing the obtained spectra, it is possible to determine whether the crystalline form of the calcium crystals contained in the sample contains a more unstable phase, a metastable phase, or a stable phase.

[0101] In addition, it is expected that the results of this example will enable a wider range of confirmation of the state of calcium phosphate crystals contained in the sample being analyzed, which will enable prediction of the maturity of calcium phosphate-containing biomaterials such as bones.

[0102] The embodiments disclosed herein should be considered to be illustrative and not restrictive in all respects. The scope of the present invention is defined by the claims, not the above description, and is intended to include all modifications within the meaning and scope of the claims.

Claims

1. By irradiating a sample to be analyzed with light in a frequency band of 10 THz or less and performing spectroscopic analysis, A method for analyzing the crystal morphology of calcium crystals, which identifies whether or not the calcium crystals contained in the sample contain at least one of a metastable phase and a stable phase.

2. The calcium crystals are calcium phosphate crystals, When the sample contains octacalcium phosphate, the calcium phosphate crystals contained in the sample are identified as the metastable phase; When the sample contains hydroxyapatite, calcium phosphate crystals contained in the sample are identified as the stable phase. The analytical method according to claim 1.

3. The analysis method according to claim 2 , wherein the frequency band is within a range of 2 THz to 9 THz.

4. The spectroscopic analysis includes determining whether or not there is an absorption peak at 2.8 THz. The method of analysis according to claim 2.

5. The analytical method according to claim 2, further comprising determining whether or not the sample contains a phase that is less stable than the metastable phase by confirming the presence or absence of an absorption peak at 7.1 THz in the spectroscopic analysis.

6. The analytical method according to claim 1 , wherein the sample is a substance of biological origin.

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