JP7370560B2 - Bioactive cement paste and kit for producing bioactive cement, bioactive cement paste and method for producing the same - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Announced at "Poster and product presentations by industry, academia, and government members at the 235th Special Study Group of the New Ceramics Forum" on December 14, 2018

The present invention relates to a bioactive cement paste, a kit for producing a bioactive cement, a bioactive cement paste, and a method for producing the same. More particularly, the present invention provides a method for producing bioactive cement pastes with suitable initial setting times and bioactive cements that are non-disintegrating and have high compressive strength and are absorbed in the body and replaced with bone. The present invention relates to a kit, a bioactive cement paste, and a method for producing the same.

BACKGROUND OF THE INVENTION In recent years, bone grafting materials that undergo hydration and hardening in vivo have been commonly used to repair fractures and bone defects caused by diseases, accidents, and the like. For example, for compression fractures of the vertebral body due to osteoporosis or bone tumors, percutaneous vertebroplasty is performed, in which bone cement as a bone replacement material is percutaneously injected into the vertebral body using a puncture needle to reconstruct the vertebral body. It is being said.

Currently, polymethyl methacrylate (PMMA) cement and calcium phosphate cement (CPC) are mainly used as bone cements.
Although PMMA cement has excellent hardening speed and strength after hardening, it has poor affinity with bone tissue due to its lack of osteoconductivity, and there are problems such as toxicity of unreacted monomers and damage to surrounding tissues due to the generation of polymerization heat. There is.
On the other hand, CPC is inferior to PMMA cement in terms of hardening speed and strength after hardening, but it has excellent osteoconductivity because it converts into bone-like hydroxyapatite in vivo. Many of the calcium phosphate compounds that are the materials for CPC have the property of disappearing naturally by being broken down and absorbed in the body and being replaced with autologous bone, so the development of bone-replacement type CPC is also possible. It's progressing. Furthermore, CPC with improved mechanical properties has been developed by incorporating chitosan, which is a biocompatible substance, into CPC.
In addition, in recent years, porous CPC, which has a large number of microscopic voids called pores, has been developed, and supplementary materials that are mainly composed of calcium phosphate compounds such as hydroxyapatite and that contain substances that are decomposed and absorbed in the living body have also been developed. Proposed.

However, in animal experiments, it has been pointed out that with conventional cement paste that uses an acidic curing liquid prepared by dissolving chitosan in malic acid as a curing agent to be incorporated into CPC, osteolysis occurs near the implantation site. Due to the high viscosity of the cement paste, there was a problem in that it was difficult to dispense with a syringe.
Therefore, the applicant of the present application proposed a cement powder containing calcium hydrogen phosphate dihydrate and tetracalcium phosphate, and a cement powder containing calcium-free phosphate, sodium hydrogen carbonate, and chitosan as a hardening agent. proposed a kit for producing a bioactive cement paste and a bioactive cement characterized by the combination with a neutral aqueous solution (also referred to as a "neutral curing liquid") (Japanese Patent Application Laid-open No. 2018-038590: Patent Document 1).
That is, in Patent Document 1, after dissolving chitosan in an acid, a soluble powder is obtained by salting out, and by using a neutral curing liquid in which the obtained powder is dissolved, the viscosity of the cement paste is reduced, and syringe injection is possible. Made it easy.

Japanese Patent Application Publication No. 2018-038590

However, in animal experiments, the cement paste using a neutral curing liquid containing the salted-out chitosan powder of Patent Document 1 did not cause osteolysis unlike acidic curing liquid, but the chitosan content was still low, and the chitosan content was still low. Further improvement in the compressive strength of the cured product is desired by increasing the content.
On the other hand, it has also been reported that when cement powder hardens, a fibrous film is formed around it. This phenomenon is thought to be due to the fact that as the cement powder hardens, undissolved chitosan in the hardening liquid is expelled around the hardened body, forming a film.
From this point of view, there is a need for a neutral curing liquid that has a high chitosan content and has no undissolved chitosan, and a cement paste containing the same.
Furthermore, there is a need for a neutral curing liquid containing a high content of biodegradable polysaccharide to replace chitosan as a curing agent, and a cement paste containing the same.

Therefore, the present invention provides a neutral curing liquid with improved chitosan content and no undissolved residue, that is, a highly transparent neutral curing liquid, and a neutral curing liquid containing a high content of biodegradable polysaccharide as a curing agent in place of chitosan. A kit for producing a bioactive cement paste having an appropriate initial hardening time and a bioactive cement that is non-disintegrating and has high compressive strength and is absorbed in the body and replaced with bone, and a bioactive cement. An object of the present invention is to provide a paste and a method for producing the same.

As a result of extensive research in order to solve the above problems, the present inventor has found that by blending polyol with chitosan into the curing liquid, the solubility of chitosan is improved, and a uniform medium with high transparency and high transparency can be obtained. By incorporating alginic acid into the curing liquid as a high-content biodegradable polysaccharide in place of chitosan as a curing agent, a highly transparent and uniform neutral curing liquid can be obtained, and the above problems can be solved. This was unexpectedly discovered and the present invention was completed.

Thus, according to the present invention, a powder containing calcium hydrogen phosphate dihydrate and tetracalcium phosphate as a cement powder, and a clear neutral aqueous solution containing a biodegradable polysaccharide and water as a hardening agent. A kit for producing a bioactive cement paste and a bioactive cement characterized in that it is a combination is provided.

Further, according to the present invention, a powder containing calcium hydrogen phosphate dihydrate and tetracalcium phosphate as a cement powder has a biodegradable hardening agent in an amount necessary for hardening the cement powder. A method for producing a bioactive cement paste is provided, which comprises obtaining a bioactive cement paste by adding and mixing a transparent neutral aqueous solution containing a polysaccharide and water.
Furthermore, the present invention provides a bioactive cement paste that is a mixture of the above powder and a neutral aqueous solution.

According to the invention, a kit for producing a bioactive cement paste with a suitable initial hardening time and a bioactive cement that is non-disintegrating and has high compressive strength and is absorbed in the body and replaced with bone; A bioactive cement paste and a method for producing the same can be provided.
Specifically, as described in the examples, a transparent neutral chitosan-containing curing liquid containing polyol (pH 6.71, transmittance 43.73%) was obtained, and a cement paste using it was able to achieve initial curing. Achieved a time reduction of 15.7±1.2 minutes, achieved a static disintegration rate of 0.378±0.108%, which can be said to be almost non-dissolving in the cured product, and achieved osteolysis with acidic curing solution. This solved the problem of fibrous tissue generation around the hardened body.
Furthermore, as described in the Examples, a transparent curing solution containing sodium alginate (pH 7.00, transmittance 56.3%) was obtained, and a cement paste using the same had an initial curing time of 11.5±0. The cured product achieved a static disintegration rate of 0.63±0.30%, which can be said to be almost non-dissolving, and the bone dissolution phenomenon in acidic curing liquid and around the cured product were reduced by 5 minutes. The problem of fibrous tissue generation was solved, and a cured product with excellent osteogenic ability was obtained.

The paste of the present invention is an amorphous mixture, and immediately after mixing cement powder with a neutral aqueous solution containing a hardening agent, it can be changed into any shape or filled into a tube. . The paste of the present invention self-hardens over time, and further hardens through a hydration reaction in water or in vivo to become a hardened product, which is a type of CPC. Therefore, the paste of the present invention can be suitably used as a precursor of a bone grafting material.

Furthermore, the bioactive cement paste and the kit for producing bioactive cement of the present invention further exhibit the above effects when at least one of the following requirements is satisfied.
(1) A transparent neutral aqueous solution containing powder containing calcium hydrogen phosphate dihydrate and tetracalcium phosphate as cement powder, chitosan as a hardening agent, a polyol as a solubilizing component of the chitosan, and water. It is a combination of
(2) The polyol is disodium β-glycerophosphate.
(3) The concentrations of chitosan and polyol in the neutral aqueous solution are 0.5% to 10% by weight and 0.5% to 5% by weight, respectively.
(4) A combination of powder containing calcium hydrogen phosphate dihydrate and tetracalcium phosphate as cement powder, and a transparent neutral aqueous solution containing alginic acid or its salt and water as a hardening agent.
(5) The concentration of alginic acid or its salt in the neutral aqueous solution is 0.2% by weight or more and 20% by weight or less. (6) The blending ratio of calcium hydrogen phosphate dihydrate and tetracalcium phosphate is The molar ratio is 1:1.
(7) The blending ratio P/L of the powder P and the neutral aqueous solution L is 1 to 4 in terms of weight ratio.

Furthermore, the method for producing a bioactive cement paste of the present invention further exhibits the above effects when at least one of the following requirements is satisfied.
(8) Powder containing calcium hydrogen phosphate dihydrate and tetracalcium phosphate as cement powder, chitosan as a hardening agent in an amount necessary for hardening the cement powder, and as a solubilizing component of the chitosan. A clear neutral aqueous solution containing polyol and water is added and mixed to obtain a bioactive cement paste.
(9) A transparent powder containing calcium hydrogen phosphate dihydrate and tetracalcium phosphate as cement powder, and alginic acid or its salt as a hardening agent and water in the amount necessary for the cement powder to harden. A bioactive cement paste is obtained by adding and mixing a neutral aqueous solution.

It is a figure which shows the apparatus used for preparation (kneading) of CPC paste, (a) Petri dish, and (b) medicine spoon, and a state before kneading. FIG. 2 is a diagram showing the instruments used for manufacturing CPC, (a) a Teflon (registered trademark) split mold, (b) a plastic plate, and (c) a clip. It is a figure showing the instruments used to produce CPC, (a) Teflon split mold, (b) Teflon mold fixing plate, (c) flat head screw, (d) nut, and (e) assembly diagram. It is a figure which shows the storage container used for the initial hardening of CPC paste, and the state inside. It is a figure which shows (a) the Vigor needle tester used for measuring the initial hardening time of CPC paste, and (b) the state of the test. FIG. 2 is a diagram showing a tension-compression converter used in a compressive strength test of a cured CPC body. It is a figure which shows (a) the syringe for CPC measurement, and (b) a CPC separation membrane used for the measurement of the static disintegration rate of CPC. It is a figure showing (a) a plastic container and (b) a stainless steel wire mesh stand used for measuring the static disintegration rate of CPC. FIG. 2 is a diagram showing a hole (arrow) made in a rabbit femoral joint in an in vivo test. FIG. 2 is a diagram showing the appearance of a pore distribution measuring device used for specific surface area measurement. Diffraction patterns of (a) produced TTCP and (b) commercially available TTCP, and (c) diffraction diagram of TTCP (ICDD PDF No. 25-1137). (a) Diffraction patterns of DCPD before wet pulverization, (b) DCPD after wet pulverization, and (c) diffraction diagram of DCPD (ICDD PDF No. 11-0293). (a) DCPA before wet milling, (b) diffraction pattern of DCPA after wet milling, and (c) diffraction diagram of DCPA (ICDD PDF No. 09-0080). FIG. 3 shows particle size distributions of (a) produced TTCP, (b) commercially available TTCP, (c) DCPD after wet milling, and (d) DCPA after wet milling. SEM images of (a) produced TTCP, (b) commercially available TTCP, (c) DCPD before wet milling, (d) DCPD after wet milling, (e) DCPA before wet milling, and (f) DCPA after wet milling. FIG. FIG. 3 is a diagram showing (a) an adsorption/desorption isotherm and (b) a BET plot obtained by measuring the amount of gas adsorption of produced TTCP. FIG. 3 is a diagram showing (a) an adsorption/desorption isotherm and (b) a BET plot obtained by measuring the amount of gas adsorption of commercially available TTCP. FIG. 3 is a diagram showing (a) an adsorption/desorption isotherm and (b) a BET plot obtained by measuring the gas adsorption amount of DCPD before wet pulverization. FIG. 3 is a diagram showing (a) an adsorption/desorption isotherm and (b) a BET plot obtained by measuring the gas adsorption amount of DCPD after wet pulverization. The states of (L1) acidic curing liquid containing chitosan (Liquid 1), (L2) neutral curing liquid containing chitosan (Liquid 2), (L3) curing liquid containing polyol-chitosan (Liquid 3), and (L4) chitosan solution (Liquid 4) are shown. It is a diagram. (L2) A diagram showing visible light transmission spectra of a chitosan-containing neutral curing liquid (Liquid 2), (L3) a polyol-chitosan-containing curing liquid (Liquid 3), and (L4) a chitosan solution (Liquid 4). (L3) and (L3-1) to (L3-4) A diagram showing the states of polyol-chitosan-containing curing liquids (Liquid 3 and Liquid 3-1 to Liquid 3-4). (L3) and (L3-5) to (L3-9) A diagram showing the states of polyol-chitosan-containing curing liquids (Liquid 3 and Liquid 3-5 to Liquid 3-9). Samples from an in-vivo test (animal implantation test) using CTC paste using (a) water, (b) chitosan-containing acidic curing liquid (Liquid 1), and (c) chitosan-containing neutral curing liquid (Liquid 2) as curing liquids. This is a micro X-ray CT image. It is a figure which shows the result of the Vigor needle test of cured bodies CPC1, CPC2, and CPC3. It is a figure which shows the result of the Vigar needle test of cured bodies CPC3 and CPC4. It is a figure which shows the compressive strength at each immersion time of hardened|cured body CPC1, CPC2, and CPC3. It is a figure which shows the relationship between the particle size ratio of each component of cement powder of hardened bodies CPC3, CPC5, CPC6, and CPC7 and PA and compressive strength. It is a powder X-ray diffraction pattern at each immersion time of hardened body CPC3 produced using cement powder 1 and hardening liquid Liquid 3. This is a powder X-ray diffraction pattern of cured bodies CPC3, CPC5, CPC6, and CPC7 prepared using cement powders 1 to 4 and hardening liquid Liquid 3 after being immersed for one day. FIG. 2 is a diffraction diagram of TTCP, DCPD, DCPA and HAp (ICDD PDF No. 25-1137, No. 11-0293, No. 09-0080 and No. 74-0566, respectively). It is a figure which shows the state of the static disintegration rate measurement of hardened body CPC3.

It is a figure showing the jig used for preparation (kneading) of CPC paste, and the state before kneading. FIG. 2 is a diagram showing the configuration of a mold (instrument) used for producing CPC (initial curing). It is a figure showing a Teflon split mold, (a) a Teflon mold, (b) a Teflon mold fixing plate, (c) a flat head screw, (d) a nut, and (e) an assembly diagram. It is a figure which shows the container used for initial hardening of CPC. FIG. 2 is a diagram showing a tension-compression converter used in a compressive strength test of a cured CPC body. It is a figure showing (a) the Vigor needle tester used for measuring the initial curing time of a CPC cured product, and (b) the state of the test. It is a figure showing (a) a plastic container and (b) a stainless wire mesh stand used for static disintegration rate measurement of a CPC cured body. It is a figure which shows the syringe with the tip cut off used for the extrusion force measurement of CPC cured body. It is a figure which shows the situation of the extrusion force measurement of CPC cured body. They are (a) a diagram showing the state of the test apparatus and (b) its schematic diagram for an absorbency in vitro test (solubility test) of a CPC cured product. Diffraction patterns of (a) product A, (b) commercially available TTCP and (c) commercially available TTCP (2), and (d) diffraction diagram of TTCP (ICDD PDF No. 25-1137). Diffraction patterns of (a) DCPA before wet milling, (b) DCPA after 30 hours of wet milling, (c) DCPA after 48 hours of wet milling, and (d) DCPA after 96 hours of wet milling, and (e) DCPA ( It is a diffraction diagram of ICDD PDF No. 09-0080). It is a diagram showing the particle size distribution of dry-milled TTCP (milling time 1 hour), (II) dry-milled TTCP (milling time 1.5 hours), (III) commercially available TTCP, and (IV) commercially available TTCP (2). . (V) Wet-milled DCPA (milling time: 30 hours), (VI) Wet-milled DCPA (milling time: 48 hours), and (VII) Wet-milling DCPA (milling time: 96 hours). It is a figure which shows the state of alginic acid containing hardening liquid, (a) Liquid A2, (b) Liquid A3, and (c) Liquid A4. It is a figure which shows the transmittance|permeability of alginic acid containing hardening liquid, (a) Liquid A2, (b) Liquid A3, and (c) Liquid A4. It is a figure which shows the compressive strength in 24-hour immersion of hardened|cured body CPC4, CPC7, and CPC8. It is a figure which shows the compressive strength with respect to each immersion time of hardened body CPC4. It is a figure which shows the compressive strength in 1-day and 7-day immersion of hardened|cured body CPC4, CPC2, and CPC4. It is a figure which shows the compressive strength in 1-day and 7-day immersion of cured bodies CPC10 and CPC6. These are diffraction patterns of cured CPC4 immersed for 1 day and 7 days. It is a diffraction pattern for each immersion time of cured body CPC2 immersed for 1 day, 7 days, and 1 month. It is a diffraction pattern for each immersion time of cured body CPC9 immersed for 1 day and 7 days. It is a diffraction pattern for each immersion time of cured body CPC10 immersed for 1 day and 7 days. It is a diffraction pattern for each immersion time of cured body CPC6 immersed for 1 day and 7 days. FIG. 2 is a diffraction diagram of TTCP, DCPD, DCPA and HAp (ICDD PDF No. 25-1137, No. 11-0293, No. 09-0080 and No. 74-0566, respectively). It is a figure which shows the state of the experiment of the static disintegration rate of hardened body CPC. FIG. 3 is a diagram showing the change over time in the calcium concentration of cured CPC2 in an acetic acid-sodium acetate buffer solution adjusted to pH 5.5. FIG. 3 is a diagram showing the change over time in the calcium concentration of cured CPC6 in an acetic acid-sodium acetate buffer adjusted to pH 5.5. It is a figure which shows the microfocus X-ray CT image of cured body CPC2 after an absorption test. It is a figure showing FE-SEM of cured body CPC2. It is a figure showing FE-SEM of cured body CPC6.

(1) Kit for producing bioactive cement paste and bioactive cement The kit for producing bioactive cement paste and bioactive cement of the present invention (hereinafter also simply referred to as "kit") is a method for producing bioactive cement paste and bioactive cement. It is characterized by a combination of powder containing calcium hydrogen phosphate dihydrate and tetracalcium phosphate, and a transparent neutral aqueous solution containing water and a biodegradable polysaccharide as a hardening agent.
In the present invention, "bioactive cement paste (hereinafter also simply referred to as "paste")" means that the powder as the cement powder described above is mixed with a neutral aqueous solution containing a hardening agent, and then immediately after it is filled into at least a cylinder and extruded. Refers to mixtures of up to As will be described later, the paste gradually begins to initially harden immediately after being mixed, and further undergoes a hydration reaction in vivo to become a hardened product (bioactive cement).

The kit of the present invention is preferably a two-reagent type kit in which a powder containing cement powder and a neutral aqueous solution containing a hardening agent are stored in separate containers.
The shape of these containers is not particularly limited, and for example, the powder may be placed in a kneading syringe that can be mixed with a neutral aqueous solution, and the neutral aqueous solution may also be placed in the syringe. In this case, the kit of the present invention may further include an injection needle and water for adjusting the viscosity of the resulting composition.
The biodegradable polysaccharide used in the present invention is not particularly limited as long as it can act as a hardening agent in combination with cement powder and exhibit excellent osteogenic function.
Such biodegradable polysaccharides include chitosan, alginic acid, starch, glycogen, dextran, inulin, chondroitin, etc. Among these, chitosan and alginic acid are particularly preferred.

Therefore, the kit of the present invention
A combination of a powder containing calcium hydrogen phosphate dihydrate and tetracalcium phosphate as a cement powder, chitosan as a hardening agent, a polyol and water as a solubilizing component of the chitosan, and a transparent neutral aqueous solution. (Embodiment 1), and a powder containing calcium hydrogen phosphate dihydrate and tetracalcium phosphate as cement powder, alginic acid or a salt thereof as the hardening agent, and a transparent neutral aqueous solution containing water. (Embodiment 2)
It is preferable that
Details of each component of the powder and the neutral aqueous solution and their blending ratio will be explained in (2) Method for producing bioactive cement paste and (3) Bioactive cement paste below.
The above (2) will be explained separately in Embodiment 1 and Embodiment 2.

(2) Method for producing bioactive cement paste (2-1) Embodiment 1
The method for producing bioactive cement paste of the present invention includes adding a hardening agent to a powder containing calcium hydrogen phosphate dihydrate and tetracalcium phosphate as cement powder in an amount necessary for hardening the cement powder. It is characterized in that a bioactive cement paste is obtained by adding and mixing chitosan, a polyol as a solubilizing component of the chitosan, and a transparent neutral aqueous solution containing water.

[Cement powder]
Cement powder includes calcium hydrogen phosphate dihydrate (DCPD) and tetracalcium phosphate (TTCP).
By blending DCPD and TTCP, curing proceeds through a hydration reaction after self-curing, and in the resulting cured product, both react and gradually convert into hydroxyapatite.

[Calcium hydrogen phosphate dihydrate (DCPD)]
As DCPD, a commercially available one may be used, or one manufactured by oneself by a known method may be used.
Further, instead of DCPD, its anhydride calcium hydrogen phosphate (DCPA) can also be used. A commercially available DCPA may be used, or one manufactured by oneself using a known method may be used.
The particle size of DCPD and DCPA is not particularly limited, but from the viewpoint of hydration reaction and conversion formation of hydroxyapatite with TTCP, it is preferably in the range of 0.2 to 10 μm, more preferably in the range of 0.5 to 5 μm. In addition, when the particle size of DCPD and DCPA powder is not preferable, the particle size may be adjusted by pulverizing by a known method such as wet pulverization.
In addition, calcium hydrogen phosphate anhydrous can be used instead of calcium hydrogen phosphate dihydrate, and although it depends on other conditions, the hardening obtained with the anhydrous is better than that with the dihydrate. It may be good for improving body strength.

[Tetracalcium phosphate (TTCP)]
As TTCP, a commercially available one may be used, or one produced by oneself by a known method may be used.
For example, TTCP powder is produced by wet mixing calcium carbonate powder and calcium hydrogen phosphate dihydrate powder in an aqueous medium, and then heating the resulting mixture at a temperature of 1200 to 1700°C for 3 to 6 hours. It can be manufactured by firing and pulverizing the obtained fired product.
The particle size of TTCP is not particularly limited, but from the viewpoint of hydration reaction and conversion formation of hydroxyapatite with DCPD, it is preferably less than 150 μm, and if it exceeds 75 μm, TTCP may remain in the cured product. More preferably, it is less than 32 μm. In addition, when the particle size of the TTCP powder is not preferable, the particle size may be adjusted by screening by a known method such as sieving.
[Ratio between DCPD and TTPC]
The molar ratio of DCPD and TTPC is preferably 1:1.
By using cement powder with such a mixing ratio, hydroxyapatite is generated as shown in the formula below, and a uniform composition (paste) can be obtained.
2Ca ₄ O(PO ₄ ) ₂ +2CaHPO ₄ →10Ca ²⁺ +6PO ₄ ^3- +2OH ^-
→Ca ₁₀ (PO ₄ ) ₆ (OH) ₂

[Neutral aqueous solution]
The neutral aqueous solution (also referred to as "neutral curing solution") contains chitosan as a curing agent, a polyol as a solubilizing component of the chitosan, and water.
"Transparent" in the "transparent neutral aqueous solution" of the present invention is an index indicating that there is no undissolved chitosan contained therein, and as described in Examples, in visible light transmittance spectrum measurement, 40% % or more.
If the transmittance is less than 40%, undissolved chitosan may exist in the aqueous solution, and the effect of the added amount of chitosan may not be obtained.
Moreover, "neutral" means that the pH is 6 to 7.5.
If the pH is less than 6, osteolysis may occur around the sclerotic body in the living body. On the other hand, if the pH exceeds 7.5, the strength may decrease due to non-uniform curing or bubbles may be generated during the preparation of the cured liquefied liquid.

[Polyol]
The polyol has the function of increasing the chitosan content in the neutral aqueous solution, and thus the function of providing a neutral and transparent curing liquid.
Polyols are aliphatic compounds, i.e. polyhydric alcohols, having multiple alcoholic hydroxy groups, such as glycols (diols), such as ethylene glycol (C ₂ H ₄ (OH) ₂ ), 3 having 2 hydroxy groups. Examples include triols such as glycerol (C ₂ H ₅ (OH) ₃ ), which have 2 hydroxy groups.
In the present invention, from the viewpoint of affinity with cement powder, those in which any one of a plurality of hydroxy groups is phosphoric acid esterified and salts thereof are preferable. Here, examples of the salt include alkali metal salts such as sodium.

Examples of such phosphoric acid esterified polyols include glycerol phosphoric acid and its salts in which any of the three hydroxy groups of glycerol are phosphoric esterified, and among these, the viscosity of the curing liquid is Disodium β-glycerophosphate (NaGP: C ₃ H ₇ Na ₂ O ₆ P) is particularly preferred from the viewpoint of injection of paste.

NaGP is a compound used in osteoblast culture media, and has been shown to induce ossification and exhibit good mineralization, and while it rapidly absorbs water in a solid state, it releases water slowly in a solvent, so it absorbs moisture. It is known to show gender.
It is also known that by adding NaGP to a chitosan solution, an injectable, neutral, and biodegradable solution can be obtained. This neutral solution is presumed to be obtained from the synergistic forces of hydrogen bonding, electrostatic interaction, and hydrophobic interaction between chitosan and NaGP, and is used to retain living cells and therapeutic proteins. Furthermore, it is known that blending NaGP with cement powder leads to a reduction in extrusion force during pouring, that is, an improvement in fluidity. However, there is no known example of using NaGP together with chitosan and cement powder as in the present invention.

NaGP has a chemical structure as shown below.

[Chitosan]
Chitosan has the function of improving the hardening speed of the paste and the compressive strength of the cured product, and also functions as a gelling agent for the paste, as well as the ability to improve the hardened paste after the cured product is injected or implanted into a living body. It has the function of making the cured product porous by being eluted from it.
Chitosan is known as a natural polymer obtained by deacetylating chitin, which is contained in the exoskeleton of crustaceans such as crabs and shrimps and the cell walls of fungi such as mushrooms, with concentrated alkali. The origin of chitosan is not particularly limited.
The form of chitosan is not particularly limited, and it may be in either powder or solution form.
In the present invention, the degree of deacetylation of chitosan is not particularly limited, but it is preferably 70 to 100% because biodegradation is easier.

Chitosan has a chemical structure as shown below.

It is known that the molecular weight of chitosan is generally proportional to the viscosity of a chitosan solution.
In the present invention, the average molecular weight of chitosan is preferably from 10,000 to 310,000 based on viscosity.
A paste containing chitosan in the above range with a relatively medium average molecular weight tends to have a larger fracture energy of a cured product obtained by a hydration reaction than a paste containing chitosan with a relatively small average molecular weight of less than 190,000. It is preferable because it is in

In this specification, the viscosity of a chitosan solution is determined by dissolving chitosan in a 1% acetic acid aqueous solution to a concentration of 1% by weight, and measuring the obtained chitosan solution using a B-type rotational viscometer at a solution temperature of 20°C. say. Then, the intrinsic viscosity of chitosan is calculated based on the measured viscosity, and the molecular weight of chitosan is calculated using the Mark-Houwink-Sakurada equation.

[Other ingredients]
The neutral aqueous solution may contain components known in the art as long as they do not impede the effects of the present invention.
Calcium-free phosphates such as potassium dihydrogen phosphate are thought to have the function of supplementing the phosphorus component of the phosphorus compound constituting cement powder.
Calcium-free means that the phosphate does not contain calcium, ie is not a calcium salt. During the hardening process of cement powder, calcium reacts with phosphoric acid to precipitate needle-like or rod-like crystals that grow predominantly in the long axis direction, and these crystals intersect three-dimensionally and act on the initial hardening. The present invention excludes calcium salts of phosphoric acid because of their harmful effects.
Preferably, the calcium-free phosphate is selected from alkali metal salts of orthophosphoric acid and phosphorous acid, and ammonium hydrogen phosphate, the alkali metals including sodium, potassium, etc. Trisodium phosphate (Na ₃ PO ₄ ), disodium hydrogen phosphate (Na ₂ HPO ₄ ); tripotassium phosphate (K ₃ PO ₄ ), potassium dihydrogen phosphate (KH ₂ PO ₄ ), dipotassium hydrogen phosphate (K ₂ HPO ₄ ); Sodium phosphite (Na ₂ PHO ₃ ), Sodium hydrogen phosphite (NaHPHO ₃ ); Potassium phosphite (K ₂ PHO ₃ ), Potassium hydrogen phosphite (KHPHO ₃ ); Phosphorus Examples include ammonium dihydrogen acid (NH ₄ H ₂ PO ₄ ) and diammonium hydrogen phosphate ((NH ₄ ) ₂ HPO ₄ ). Among these, dipotassium hydrogen phosphate and disodium hydrogen phosphate are particularly preferred since they inhibit chitosan redeposition. The amount relative to chitosan is about 150 to 200% by weight.

Dicarboxylic acids such as citric acid are believed to affect the dissolution of chitosan. The amount relative to chitosan is about 30 to 200% by weight.
It is thought that a polymer compound such as dextran sodium sulfate acts as a gelling agent on the above-mentioned chitosan solution and works to form a chitosan-containing salt. The amount relative to chitosan is about 30 to 200% by weight.
Sodium chloride and the like are thought to act auxiliary to salt formation. The amount relative to chitosan is about 30 to 800% by weight.

[Composition of neutral aqueous solution]
The concentrations of chitosan and polyol in the neutral aqueous solution of the present invention are preferably 0.5% to 10% by weight and 0.5% to 5% by weight, respectively.

If the concentration of chitosan is less than 0.5% by weight, the effect of creating porosity after curing is poor and the fracture energy may not increase. On the other hand, if the concentration of chitosan exceeds 10% by weight, the viscosity of the curing liquid may increase, making injection (injectability) difficult. It may not dissolve completely.
The concentration of chitosan is more preferably 1% by weight or more and 10% by weight or less, and even more preferably 1.5% by weight or more and 2% by weight or less.

If the concentration of the polyol is less than 0.5% by weight, gelation will occur, making injection difficult and may not result in neutrality. On the other hand, if the polyol concentration exceeds 5% by weight, curing may not proceed.
The concentration of the polyol is more preferably 1% by weight or more and 4% by weight or less, and even more preferably 2% by weight or more and 3% by weight or less.

[Preparation of neutral aqueous solution]
The neutral aqueous solution of the present invention can be obtained, for example, by adding and mixing a polyol solution to a chitosan solution in which chitosan is dissolved in an acid.

[Ratio between powder powder P and neutral aqueous solution L]
The blending ratio (powder-liquid ratio) P/L (or PL ratio) of the powder P and the neutral aqueous solution L is preferably 1 to 4 in terms of weight ratio.
If the PL ratio is less than 1, the curing strength may be insufficient. On the other hand, if the PL ratio exceeds 4, uniform kneading may become difficult.
A more preferable PL ratio is 2 to 4, and an especially preferable PL ratio is 3 to 4.

[Preparation of paste]
The paste of the present invention is produced by adding a transparent neutral aqueous solution containing chitosan as a hardening agent, a polyol as a solubilizing component of the chitosan, and water in an amount necessary for hardening the cement powder to the above powder. It can be obtained by mixing (mixing or kneading).
The addition/mixing means is not particularly limited, and may be appropriately selected from known mixing means depending on the amount of addition (mixing amount).
After mixing the powder and the neutral aqueous solution, the paste of the present invention usually completes self-hardening in about 3 to 20 minutes (initial hardening). The temperature and humidity conditions at this time are not particularly different from the general CPC preparation conditions, but usually the temperature and humidity are 20 to 40° C. and 50 to 100%.

(2-2) Embodiment 2
The method for producing bioactive cement paste of the present invention includes adding a hardening agent to a powder containing calcium hydrogen phosphate dihydrate and tetracalcium phosphate as cement powder in an amount necessary for hardening the cement powder. A bioactive cement paste is obtained by adding and mixing a transparent neutral aqueous solution containing alginic acid or its salt and water.
In Embodiment 2, the curing agent component alginic acid and its salt does not require a solubilizer such as a polyol like the curing agent component chitosan in Embodiment 1, and more curing agent is added to the neutral aqueous solution. be able to.
In addition, when alginic acid and its salts are used as the hardening agent in Embodiment 2, similar to the case where chitosan is used as the hardening agent in Embodiment 1, it is possible to "eliminate the bone dissolution phenomenon in acidic hardening liquid" and "harden The effect of "resolving fibrous tissue generation around the body" is obtained, and compared with Embodiment 1, it is superior in bone formation.

[Cement powder]
According to Embodiment 1.
[Calcium hydrogen phosphate dihydrate (DCPD)]
According to Embodiment 1.
[Tetracalcium phosphate (TTCP)]
According to Embodiment 1.
[Ratio between DCPD and TTPC]
According to Embodiment 1.

[Neutral aqueous solution]
The neutral aqueous solution contains alginic acid and water as a hardening agent.
"Transparent" in the "transparent neutral aqueous solution" of the present invention is an indicator indicating that there is no undissolved alginic acid contained therein. Similar to Form 1, this means that it has a transmittance of 40% or more.
However, in Embodiment 2, even if the transmittance is a few percent or more and less than 40%, no change such as gelation is observed even if it is kept at room temperature for a certain period of time, and it is stable and can be used as a neutral aqueous solution.
Moreover, "neutral" means that the pH is 6 to 7.5.
If the pH is less than 6, osteolysis may occur around the sclerotic body in the living body. On the other hand, if the pH exceeds 7.5, the strength may decrease due to non-uniform curing or bubbles may be generated during the preparation of the cured liquefied liquid.

[Alginic acid]
Alginic acid has the function of improving the hardening speed of the paste and the compressive strength of the cured product, and also functions as a gelling agent for the paste, and the cured product of the paste after being injected or implanted into a living body. It has the function of making the cured product porous by being eluted from it.
Alginic acid is a plant-derived polysaccharide found in brown algae and coralline algae, and is white or pale yellow in color and has a fibrous, granular, or powdery form.
Examples of alginates include sodium alginate, ammonium alginate, potassium alginate, calcium alginate, and the like, which are soluble salts.

Alginic acid has a chemical structure as shown in the following formula (left: β-D-mannuronic acid, right: α-L-guluronic acid).

It is known that the viscosity of alginic acid is generally proportional to its degree of polymerization (molecular weight), and becomes more pronounced as the degree of polymerization increases.
In the case of sodium alginate, the alginic acid used in the present invention includes those with a low molecular weight (viscosity of 20 to 50 mPa·s) and medium molecular weight (viscosity of 80 to 120 mPa·s), which can be added to the curing liquid in a large proportion. Low molecular weight ones are preferred because the curing liquid can be made to have a low viscosity.

[Other ingredients]
According to Embodiment 1.

[Composition of neutral aqueous solution]
The concentration of alginic acid in the neutral aqueous solution of the present invention is preferably 0.2% by weight or more and 20% by weight or less.

If the concentration of alginic acid is less than 0.2% by weight, the effect of creating porosity after curing is poor and the fracture energy may not increase. On the other hand, if the concentration of alginic acid exceeds 20% by weight, the viscosity of the curing liquid increases, and injection (injectability) may become difficult. May not dissolve completely.
The concentration of alginic acid is more preferably 2% by weight or more and 20% by weight or less, further preferably 8.8% by weight or more and 16.3% by weight or less, and still more preferably 10% by weight or more and 15% by weight or less.

[Preparation of neutral aqueous solution]
The neutral aqueous solution of the present invention can be obtained, for example, by adding and mixing alginic acid to water.
[Ratio between powder powder P and neutral aqueous solution L]
According to Embodiment 1.
[Preparation of paste]
According to Embodiment 1.

(3) Bioactive cement paste The paste of the present invention is a mixture of the above powder and a neutral aqueous solution.
[Application]
The paste of the present invention obtained by the above mixing has shapeability and can be molded into a complex shape, and as described above, can be used in living bodies as a bone replacement material. Then, the paste of the present invention becomes a hardened product (bioactive cement) through a hydration reaction from the above-mentioned initial hardening in vivo.
If the extrusion force of the paste from immediately after mixing until it is filled into a cylinder and extruded is high and fluidity is high, it will be easier to inject into living organisms, but the powder-liquid ratio (PL ratio) of the paste will be low and the hardened product will be Since the strength will be reduced, it is preferable to define the paste by its powder/liquid ratio. On the other hand, if the extrusion force of the paste exceeds 2000 g (1.2 MPa), it becomes difficult to extrude it from the cylinder without using both hands.
A preferred paste extrusion force is 200 g or less.
The cured product obtained by the hydration reaction exhibits a compressive strength of at least 10 MPa.

Furthermore, the cured product becomes porous in vivo due to the elution of chitosan contained therein. Since such porous formation of the cured product increases the surface area and facilitates bone replacement in vivo, the paste of the present invention can be suitably used as a bone replacement material.
Note that the cured body can be made porous by immersing the cured body in physiological saline. Therefore, after the cured product is immersed in physiological saline, the presence or absence of porosity and its state (disintegration rate) can be confirmed by weight measurement or observation using a scanning electron microscope.

The present invention will be specifically explained with reference to the following Examples and Comparative Examples, but the present invention is not limited by the Examples.
In the examples and comparative examples, unless otherwise specified, "water" was purified using an ultrapure water production device (Merck K.K., ultrapure water system Smart series, model: Direct-Q UV 3). Milli-Q® water means "ultra pure water".

[Embodiment 1]
The abbreviations described below are valid only within the first embodiment.
[Preparation of cement powder]
(1) Preparation of tetracalcium phosphate (TTCP) Using an electronic balance (manufactured by As One Co., Ltd., model: sefi IBA-200: below, use the raw materials (used for weighing the reagent) to adjust the Ca/P ratio to 2. In addition, calcium hydroxide (Ca(OH) ₂ , MW 74.0927, manufactured by Nacalai Tesque Co., Ltd., grade: special grade) and orthophosphoric acid (H ₃ PO ₄ , MW 98.00, manufactured by Wako Pure Chemical Industries, Ltd. (currently Fuji 31.32 g (0.4 mol) and 23.06 g (0.2 mol) of film manufactured by Wako Pure Chemical Industries, Ltd., grade: special grade, content 85%) were weighed, respectively, and each of them was weighed using a measuring cylinder. In addition to a 500 mL beaker containing 400 mL of water and a 100 mL beaker containing 50 mL of water, a hot stirrer (manufactured by As One Co., Ltd., model: RSH-1DN) using a stirrer was set at a rotation speed of 200 rpm and a temperature of 25°C. ) to obtain a Ca(OH) ₂ solution and a H ₃ PO ₄ solution.
Next, a water jacket was attached to the beaker containing the obtained Ca(OH) ₂ solution, and water at a temperature of 5°C was circulated through the water jacket from a low-temperature constant temperature water bath (manufactured by As One Corporation, model: LTB-125A) to dissolve the solution. While maintaining the temperature below 10°C and stirring the solution using a tornado (manufactured by As One Co., Ltd., model: PM-203), adjust the stirring power appropriately to create a slight vortex, and stir the solution with a glass cock. Using a burette (manufactured by As One Corporation), a fixed amount of the H ₃ PO ₄ solution was added dropwise to the Ca(OH) ₂ solution over 2 hours.
After the dropwise addition was completed, the mixture was kept at room temperature for 24 hours to age.

Then, using a centrifuge (manufactured by Tomy Seiko Co., Ltd., model: Suprema 21), the resulting mixed solution was centrifuged at a rotation speed of 5000 rpm for 10 minutes, the supernatant liquid was removed by decantation, and the resulting mixture was further centrifuged at a rotation speed of 5000 rpm for 7 minutes. Centrifuged for minutes. The separated precipitate was placed in a Petri dish, placed in a program constant temperature dryer (As One Corporation, model: DOV-450P), and dried at a temperature of 110° C. for 24 hours.
The dried sample was placed in an alumina boat (manufactured by Nikkato Co., Ltd., model: SSA-H2B), and was heated using an electric furnace (manufactured by Marusho Denki Co., Ltd., high-performance small high-temperature electric furnace SUPER MINI, model: SPM). It was fired for 5 hours in the air at a temperature of 1500°C and a temperature increase/decrease rate of 10°C/min.
After firing, the sample was taken out of the electric furnace, ground in a super hard steel mortar (Ito Seisakusho Co., Ltd., model: WD), and then dry ground in an agate mortar. Furthermore, using a mini sieve shaker (manufactured by As One Corporation, model: MVA-1) and a stainless steel sieve (size φ75 μm), the product was classified to 75 μm or less at a rotation speed of 2500 rpm to obtain produced TTCP.

(2) Preparation of calcium hydrogen phosphate dihydrate (DCPD) Calcium hydrogen phosphate dihydrate (CaHPO ₄ 2H ₂ O, MW 172.09, Wako Pure Chemical Industries, Ltd.) weighed using an electronic balance. 15.00 g (0.087 mol) of ethanol (C ₂ H ₅ OH, manufactured by Kishida Chemical Co., Ltd., grade: special grade) weighed in a measuring cylinder using a measuring cylinder. A pot mill (manufactured by Nikkato Co., Ltd., model: HD-A3, outer diameter: φ90 mm, capacity :400 mL). Next, the pot mill was rotated for 48 hours at room temperature and rotation speed of 110 rpm using a tabletop pot mill rotary table (manufactured by As One Corporation, model: ANZ-51S), and the contents were wet-pulverized.
The obtained wet-pulverized product was suction-filtered using a Buchner funnel, filter paper (JIS, type: 5C), and an aspirator (manufactured by Tokyo Rika Kikai Co., Ltd. (EYELA), model: A-3S), and the solid matter was placed in a Petri dish. and dried for 24 hours in a programmed constant temperature dryer (manufactured by As One Corporation, model: DOV-450A) set at a temperature of 50°C. The obtained sample was dry ground in an agate mortar to obtain DCPD.

(3) Preparation of calcium hydrogen phosphate (DCPA) Using 5.00 g (0.11 mol) of calcium hydrogen phosphate (CaHPO ₄ , MW 136.06, manufactured by Taihei Chemical Industry Co., Ltd.) weighed using an electronic balance, DCPA was obtained by wet grinding for 48 hours in the same manner as in (2).

(4) Mixing of cement powder Using an electronic balance, 6.80 g (0.019 mol) of produced TTCP (CaHPO ₄ , MW 366.26) obtained in (1) and 3.20 g (0.019 mol) of DCPD obtained in (2) 0.019 mol) was weighed out. Put them into a 50 mL test tube screw cap with Maruem scale (manufactured by As One Corporation, model: NX-50), set it in a shaker (manufactured by As One Corporation, model: MALTI SHAKER MS-300), and adjust the number of revolutions. Mixing was performed by shaking at 1300 rpm for 100 minutes. The resulting mixture was dry mixed in an agate mortar for 5 minutes to obtain cement powder 1.
7.33 g (0.020 mol) of generated TTCP, 2.72 g (0.020 mol) of DCPA obtained in (3), and 6.80 g (0.019 mol) of commercially available TTCP (manufactured by Taihei Chemical Industry Co., Ltd., commercially available TTCP) and DCPD 3.20 g (0.019 mol), commercially available TTCP (manufactured by Taihei Kagaku Kogyo Co., Ltd.) 7.33 g (0.020 mol) and DCPA 2.72 gg (0.020 mol) were used in the same manner as above, Cement powders 2, 3 and 4 were obtained (Table 1-1).

[Preparation of hardening liquid]
(1) Preparation of chitosan-containing acidic curing solution Add 10 mL of water measured using a graduated cylinder to a 50 mL beaker, and add chitosan ((C ₆ H ₁₁ NO ₄ ) _n , Aldrich) weighed using an electronic balance (same as above). 1.25 g (manufactured by Co., Ltd., medium molecular weight: 50,000 to 190,000, degree of deacetylation: about 81.3%) was added. Next, malic acid (C ₄ H ₆ O ₅ (HOOC-CH(OH)-CH ₂ -COOH), manufactured by Wako Pure Chemical Industries, Ltd. (currently Fuji Film Wako Pure Chemical Industries, Ltd.), grade: special grade) 1. 25 g was added, stirred and dissolved to obtain a chitosan-containing acidic curing liquid (also referred to as "Liquid 1").

(2) Preparation of chitosan-containing neutral curing liquid Add 5 mL of water measured with a measuring cylinder to a 50 mL beaker, add 0.13 g of chitosan (same as above) weighed with an electronic balance, and add 0.13 g of chitosan (same as above) weighed with an electronic balance. A suspension was obtained by stirring at a rotational speed of 500 rpm using a stirrer (manufactured by the company, model: RSHJ-1DN) and a stirrer. There, citric acid (C ₆ H ₈ O ₇ (C(OH)(CH ₂ COOH) ₂ COOH), manufactured by Wako Pure Chemical Industries, Ltd. (currently Fuji Film Wako Pure Chemical Industries, Ltd.), grade: special grade) 0 .17 g was added and stirred until uniformly dissolved. In addition, 5 mL of water measured using a graduated cylinder was added to a 50 mL beaker, and dextran sodium sulfate ((C ₆ H ₇ Na ₃ O ₁₄ S ₃ ) _n , Wako Pure Chemical Industries, Ltd. (currently 0.21 g (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., grade: special grade) was added and stirred. The latter was added to the former, stirred and mixed, and 1.0 g of sodium chloride (NaCl, manufactured by Kishida Chemical Co., Ltd., grade: special grade) was added to form a coagulated product by salting out.

The mixture was further stirred for 3 hours, and the formed coagulate was centrifuged, and the supernatant was removed. The obtained coagulated product was washed by adding 10 mL of water, and this process was repeated three times in total. 10 mL of water was added to the washed coagulum to suspend it, cooled to -30°C using a low-temperature constant-temperature water bath, and dried with a freeze dryer (manufactured by As One Corporation, model: FDU-12AS). After drying, it was ground in an agate mortar to obtain a chitosan-containing powder.
Add 10 mL of water measured with a graduated cylinder to a 50 mL beaker, and add 0.16 g of chitosan-containing powder weighed with an electronic balance, 0.40 g of sodium hydrogen carbonate (NaHCO ₃ , manufactured by Kishida Chemical Co., Ltd., grade: special grade), and 0.50 g of dipotassium hydrogen phosphate (K ₂ HPO ₄ , manufactured by Kishida Chemical Co., Ltd., grade: special grade) was added and stirred to obtain a chitosan-containing neutral curing liquid (also referred to as "Liquid 2").

(3) Preparation of polyol-chitosan-containing curing solution 8.8 mL of hydrochloric acid (HCl, MW 36.46, 35% content, d = 1.18 g/cm ³ , manufactured by Kishida Chemical Co., Ltd., grade: special grade) measured in a graduated cylinder (0.10 mol) was placed in a volumetric flask with a volume of 1000 mL, and water was added to make the volume 1000 mL to obtain a 0.1M-HCl solution.
Add 9 mL of 0.1M-HCl solution measured with a measuring cylinder to a 50 mL beaker, add 0.20 g of chitosan (same as above) weighed with an electronic balance, dissolve, and further stir to obtain a chitosan solution. Ta. Next, 1 mL of water measured using a graduated cylinder was added to a 50 mL beaker, and β-glycerophosphate disodium tetrahydrate (C ₃ H ₇ Na ₂ O ₆ P.4H ₂ O, MW 216.04, Tokyo Kasei Co., Ltd.) was added thereto. 0.56 g (0.0259 mol) (manufactured by Kogyo Co., Ltd.) was added and dissolved by stirring to obtain a disodium β-glycerophosphate solution. While stirring the chitosan solution, a disodium β-glycerophosphate solution was added dropwise using a measuring pipette, and the mixture was stirred and mixed to obtain a polyol-chitosan-containing curing liquid (also referred to as "Liquid 3").

[Evaluation of hardening liquid]
(1) Transmittance measurement Using a UV-visible spectrophotometer (manufactured by JASCO Corporation, model: V-500), the spectrum and fixed wavelength of the prepared curing liquids (Liquid 1 to 3) were measured under the following measurement conditions. The transmittance of each curing liquid was investigated.
(Measurement condition)
・Cell used: Square quartz cell (10mm, manufactured by JASCO Corporation)
・Bandwidth: 1.0nm
・Scanning speed: 200nm/min
・Measurement wavelength: 380-780nm
・Fixed wavelength: 550nm

(2) pH measurement The pH of the prepared curing liquid was measured.
The pH of the prepared curing liquids (Liquids 1 to 3) was measured using a pH meter (manufactured by Horiba Seisakusho Co., Ltd., model: D-51). In the measurement, three-point calibration was performed using pH standard solutions (pH 4.01, 6.86, and 9.18, manufactured by Wako Pure Chemical Industries, Ltd. (currently Fuji Film Wako Pure Chemical Industries, Ltd.)).

[CPC hardening test]
(1) Preparation of CPC paste The following was carried out in accordance with JIS T0330-4.
The prepared cement powders 1 to 4 were weighed using an electronic balance so that the powder-liquid ratio (blending ratio P/L of powder P and neutral aqueous solution L) was achieved. The hardened liquid was placed in a petri dish used for kneading and weighed using an electronic balance.
As shown in Figure 1-1, (a) Cement powder was placed in a petri dish, and a spatula (bottom left of the drawing) prepared by appropriately cutting an OHP sheet was used to spread it out to crush the lumps. Next, the cement powder and hardening liquid were mixed using a spatula and a stainless steel spoon (b). Specifically, in the atmosphere (temperature 25°C), cement powder is divided into three equal parts, first 1/3 amount of cement powder and hardening liquid are kneaded for 20 seconds, then 1/3 amount of cement is mixed with hardening liquid. CPC paste was obtained by adding powder and kneading for 30 seconds, and finally adding 1/3 amount of cement powder and kneading for 40 seconds, and kneading for a total of 90 seconds.

(2) Preparation of CPC Immediately place the CPC paste obtained in (1) into the (a) Teflon split mold (hole inner diameter φ6 mm x length 12 mm) shown in Figure 1-2 using a stainless steel pharmaceutical spoon (not shown). and a glass rod (outer diameter φ5.6 mm, not shown). Specifically, (a) a Teflon split mold is placed on one (b) plastic plate (thickness 2 mm), and using a spoon, insert CPC paste into the hole of the (a) Teflon split mold. It was packed and held down with a glass rod from above to prevent air bubbles from entering. After filling, the top surface of the Teflon split mold was held down with another (b) plastic plate (2 mm thick), and the upper and lower (b) plastic plates were further held together using (c) clips.
Figure 1-3 shows the instruments used to make CPC: (a) Teflon split mold, (b) Teflon mold fixing plate (30 mm x 12 mm x 2.3 mm, inner diameter of center hole φ5 mm), and (c) flat head screw. (M5 x 33mm), (d) nut and (e) assembly diagram shown.

Next, a sponge soaked in water was placed at the bottom of an airtight container (storage container) with a capacity of approximately 100 mL, the mixture was filled with the mixture, and the top and bottom were sandwiched between (b) plastic plates and (c) clips (a). A Teflon split mold was placed on top. In this state, the humidity was set to 100%, and the storage container was placed in a cool incubator (manufactured by As One Co., Ltd., model: CN-40A), and maintained at a temperature of 37° C. for 1 hour to allow initial hardening.
Thereafter, as shown in FIG. 1-4, the kneaded product (a) was immersed in the Teflon split mold (a) in water that was weighed (100 mL) using a graduated cylinder and kept at a temperature of 37°C. Furthermore, the sample was kept warm and immersed in a cool incubator at a temperature of 37° C., and then demolded to obtain a sample of a cured CPC body.
Table 1-2 shows the combinations of cement powder and hardening liquid. In addition. The hardened products obtained by kneading Cement Powder 1 and Liquids 1 to 3 are also referred to as CPCs 1 to 3.

(3) Initial curing time measurement In accordance with JIS T0330-4, the initial curing time of the CPC paste was measured using a Vigor needle tester (Nippon MEC Co., Ltd., model: tester A-004).
Specifically, as shown in Figure 1-5, 90 seconds after the start of kneading, the CPC paste was put into a Teflon-type syringe (φ10 mm x 5 mm), and a Vigor needle (weight (load) 300 g, tip) was placed on the surface of the CPC. A specimen with a cross-sectional area of 2 mm ² ) was gently dropped from directly above the sample surface, and the time until no impression remained on the surface was recorded. Measurements were made three times per condition, and the average value was taken as the initial curing time (minutes).
During the measurement period, the Vigor needles other than those used during measurement were placed in a storage container lined with a water-soaked sponge at the bottom under the condition (2), and stored at a temperature of 37° C. in a cool incubator.

(4) Compressive strength test The compressive strength of the CPC cured product was measured in accordance with JIS T0330-4.
According to the procedure (2), five compression test pieces of CPC cured bodies were prepared for each immersion condition. During production, care was taken to ensure that the upper and lower surfaces of the cured CPC body were parallel to each other to prevent stress from concentrating on the protruding portions and resulting in lower measured values than originally intended. After immersion, the water on the surface of the cured CPC body was wiped off and subjected to a test.
For compressive strength measurement, a universal testing machine equipped with a 5kN load cell (manufactured by Shimadzu Corporation, model: 87394) using a tension-compression converter that changes the tension direction to the compression direction is used as shown in Figure 1-6. (manufactured by Shimadzu Corporation, model: AUTGRAPH AG-10) was used. A water-wetted filter paper was sandwiched between the tension-compression transducer and the sample, and when a load of 1N was applied, the displacement was set to 0 mm and the zero point was set. Even if there was a sudden decrease in compressive strength due to microfracture during the test, the test was not interrupted and continued until the displacement reached 2 mm. The measurement conditions were a head speed of 0.500 mm/sec, a sampling interval of 100 msec, and measurement was performed using control and analysis software (Shimadzu Corporation, Tranpezium).

(5) Static disintegration rate measurement The static disintegration rate of CPC was measured in accordance with JIS T0330-4.
As shown in Figure 1-7, the tip of the φ9 mm was cut off by 5 mm, and (b) the separation membrane (sheet, φ10 mm) was inserted into a plastic (a) CPC measuring syringe. 0.5 mL of CPC paste (kneaded product) was filled within 3 minutes from the start of kneading. In order to prevent inclusion of air bubbles, the extrusion rod was positioned near the tip, and the CPC paste was filled from the tip while pulling the extrusion rod little by little.
In advance, 80 mL of physiological saline was placed in a plastic container (inner diameter φ70 mm x depth 40 mm) as shown in FIG. 1-8 (a) and heated in a cool incubator set at a temperature of 37°C. For physiological saline, 9.00 g of sodium chloride (NaCl, manufactured by Wako Pure Chemical Industries, Ltd. (currently Fuji Film Wako Pure Chemical Industries, Ltd.), grade: special grade) weighed using an electronic balance was placed in a volumetric flask with a capacity of 1000 mL. , water was added to make 1000 mL.
Five minutes after the start of kneading, the CPC paste was gently extruded from the syringe onto a (b) stainless steel wire mesh stand (50 mm x 50 mm, 2 mm x 2 mm mesh) as shown in Figure 1-8. The (b) stainless steel wire mesh stand on which the sample was placed was gently placed into the (a) plastic container that had been filled with physiological saline and heated, and submerged in the physiological saline. The CPC (residue) thus collapsed falls from the (b) stainless wire mesh stand. Thereafter, (a) the lid of the plastic container was closed, and the container was allowed to stand for 72 hours in a cool incubator at a temperature of 37° C. to be cured.

After standing still, (a) the CPC and (b) the stainless wire mesh stand were taken out from the plastic container and transferred to another plastic container. (a) The physiological saline in the plastic container was discarded using a dropper to prevent the residue at the bottom of the plastic container from flowing out, and the separation sheet was removed. The (a) plastic container containing the sample, wire mesh stand, and collapsed CPC was dried in a constant temperature dryer at a temperature of 50°C for 24 hours, and the weights of a to d below were measured using an electronic balance, and the following formula was used: The decay rate was determined.
Decay rate (%) = [(c-d)/((a-b)+(c-d))]×100
a: Weight of dried CPC and stainless wire mesh stand (g)
b: Weight (g) of stainless steel wire mesh stand after removing CPC, washing and drying
c: Weight of dried residue and plastic container (g)
d: Weight of plastic container after removing residue, washing and drying (g)

(6) In vivo test As shown in Figure 1-9, the skin of the forefoot of a New Zealand domestic rabbit (male, weight: 3.0-3.5 kg) was incised, and a diameter of 4.2 mm was placed on the femoral condyle. A hole with a depth of 5.0 mm was made. The hole was filled with the CPC paste (knead) obtained by the procedure (1) and sutured. Three weeks after implantation of the CPC paste sample, the rabbit was killed under anesthesia, and the filled bone was taken out and used as a sample.
The obtained sample was photographed and observed using a micro X-ray CT (manufactured by Shimadzu Corporation, model: inspeXio SMX-90CT Plus) under the following conditions.
(shooting conditions)
・Tube voltage: 90kV
・Tube current: 110μA
・Number of views: 600
・Average number: 2
The obtained images were analyzed using three-dimensional image analysis software (Japan Visual Science Co., Ltd., product name: ExFact VR).
To prepare the CPC paste, cement powder 1 was used as the cement powder, water was used as the curing liquid, Liquid 1 (acidic curing liquid containing chitosan), and Liquid 2 (neutral curing liquid containing chitosan) were used, and the powder-liquid ratio was set to 3.

[Sample analysis]
(1) Identification of sample by powder X-ray diffraction 2 kW, wavelength: 1.790 Å) under the following measurement conditions, powder X-ray diffraction was performed on the produced TTCP, DCPD and DCPA before and after wet milling, and the peak of the crystal phase of the sample was investigated.
(Measurement condition)
・Measurement angle: 10~50°
・Sampling angle: 0.02°
・Scan speed: 1.0° (min ^-1 )
・Tube voltage: 40kV
・Tube current: 20mA
・Slit: DS=1°, ss=1°, RS=0.3mm

(2) Particle size distribution measurement Using a laser diffraction/dispersion measuring device (manufactured by Horiba Seisakusho Co., Ltd., model: LA920), synthesized and classified TTCP of 75 μm or less, DCPD and DCPA after wet grinding, and commercially available TTCP were measured. Particle size distribution was measured.
Ultrapure water was used as the dispersion medium, and one drop of a synthetic kitchen detergent (manufactured by P&G) containing a surfactant was used as the dispersant. Immediately before the measurement, the dispersion liquid was subjected to ultrasonic treatment for 10 minutes to disperse the sample. The refractive index of calcium phosphate with respect to water was set to 1.24.

(3) Specific surface area measurement As shown in Figure 1-10, using a pore distribution measuring device (manufactured by Yuasa Ionics Co., Ltd. (currently Spectris Co., Ltd., model: AUTOSORB-1), The specific surface areas of the classified produced TTCP, wet-milled DCPD and DCPA, and commercially available TTCP were measured.
Nitrogen gas was used as the adsorption gas, and the specific surface area was calculated from the adsorption/desorption isotherm obtained by measurement.
Approximately 300 mg of the weighed sample was placed in an empty cell whose weight had been measured before measurement, the cell was set in a measuring device, and the cell was surrounded by a mantle heater. Next, the sample was degassed under vacuum and kept at a temperature of about 300° C. for 1 hour to remove molecules attached to the sample surface. After the pretreatment was completed, the cell containing the sample was cooled to room temperature and its weight was measured. The weight of the empty cell measured first was subtracted from this weight to obtain the weight of the sample.
Thereafter, the cell containing the sample was placed in the measuring device again, and the cell was surrounded by liquid nitrogen to keep the temperature inside the cell constant (liquid nitrogen temperature -196°C) during the measurement. Then, nitrogen gas was gradually blown into the cell from a vacuum state and measurements were taken. The amount of adsorption was measured until the relative pressure p/p ₀ between the adsorption equilibrium pressure p and the saturated equilibrium pressure p ₀ became 1. Next, after the relative pressure reached 1, the pressure was lowered until the relative pressure became 0, and the nitrogen gas molecules adsorbed on the sample were desorbed. Note that the number of measurement points was 79.

(4) Consideration of the crystalline phase of the cured CPC body by powder X-ray diffraction (1) In the same manner as in the sample identification by powder X-ray diffraction, the produced cured CPC body was dried and ground in an agate mortar to form a measurement sample powder. X-ray diffraction was performed to examine the peak of the crystal phase of the sample.

(5) Observation of sample by FE-SEM Using a field emission scanning microscope (manufactured by JEOL Ltd., model: FE-SEM JSM-6500F), synthesized and classified TTCP of 75 μm or less, wet-pulverized DCPD and DCPA as well as commercially available TTCP were observed.
Each powder sample was fixed to an aluminum plate using conductive tape, and unfixed powder was removed using an air duster. Next, using an osmium plasma coater (manufactured by JEOL Ltd., model: OPC60A Filgen), the sample was coated with OsO ₄ to a thickness of 12 nm to obtain a SEM sample. The acceleration voltage during observation was 15 kV, and the degree of vacuum was 5.00×10 ⁻⁴ Pa or less.

[Analysis of cement powder]
(1) Identification of samples by powder X-ray diffraction Figure 2-1 shows the diffraction patterns of (a) produced TTCP and (b) commercially available TTCP, and (c) the diffraction diagram of TTCP (ICDD PDF No. 25-1137). be.
According to Figure 2-1, the peak positions and intensities of (a) the diffraction pattern of the produced TTCP and (c) the diffraction diagram of the TTCP almost match, and no other peaks are observed in particular, so the produced TTCP is It is considered to be a single phase of TTCP. Moreover, when comparing the diffraction pattern of (a) the produced TTCP and (b) the diffraction pattern of the commercially available TTCP, no obvious difference is observed.

FIG. 2-2 is a diffraction pattern of (a) DCPD before wet pulverization, (b) DCPD after wet pulverization, and (c) a diffraction diagram of DCPD (ICDD PDF No. 11-0293).
According to FIG. 2-2, (b) the intensity of each peak of DCPD after wet pulverization has decreased and the half-width has increased. It is thought that the former is due to amorphization, and the latter is due to a decrease in crystallite diameter. Furthermore, the strength of the (020) and (040) planes is greatly reduced. This is thought to be because in DCPD, the {010} plane grows the most and exhibits a plate-like crystal, but this growing plane is crushed and homogenized.

FIG. 2-3 is a diffraction pattern of (a) DCPA before wet pulverization, (b) DCPA after wet pulverization, and (c) a diffraction diagram of DCPA (ICDD PDF No. 09-0080).
According to FIG. 2-3, (b) the intensity of each peak of DCPA after wet pulverization has decreased and the half-width has increased. However, in the relative relationship of diffraction peak intensities, no change like DCPD is observed.

(2) Particle size distribution Figure 2-4 is a diagram showing the particle size distribution of (a) produced TTCP, (b) commercially available TTCP, (c) DCPD after wet grinding, and (d) DCPA after wet grinding. 2-1 shows the arithmetic mean diameter and median diameter of (a) to (d).
According to FIG. 2-4, (a) the generated TTCP has an asymmetric particle size distribution with a particle size ranging from less than 1 μm to about 70 μm. This is thought to be due to the fact that it contains parts that have been made fine by crushing and parts that have not been made fine.
On the other hand, (b) commercially available TTCP has a sharp particle size distribution with a peak particle size of 6.72 μm. (c) DCPD after wet pulverization has a particle size distribution with a peak particle size of 2.98 μm, and has a distribution of particle sizes of approximately 1 μm or less, but some particles of 1 μm or more are present. (d) DCPA after wet pulverization has a particle size distribution with a peak particle size of 0.77 μm, and (c) although not as sharp as DCPD after wet pulverization, the distribution has a particle size of approximately 1 μm or less. .

(3) SEM image Figure 2-5 shows (a) produced TTCP, (b) commercially available TTCP, (c) DCPD before wet grinding, (d) DCPD after wet grinding, (e) DCPA before wet grinding, and (f) It is a figure which shows the SEM image of DCPA after wet grinding. The scale bar at the bottom right of each figure indicates 10 μm in (a) to (e) and 1 μm in (f).
According to FIG. 2-5, it can be seen that (a) the generated TTCP is agglomerated particles of about 1 to 10-odd μm, and (b) the commercially available TTCP is uniform and fine particles. Furthermore, it can be seen that (c) DCPD before wet pulverization is a relatively large plate-like crystal, but DCPD after wet pulverization (d) is fine. Similarly, (e) DCPA before wet pulverization is plate-like and intertwined with each other, but (d) DCPD after wet pulverization has a particle size of 1 μm or less. Recognize.

(4) Surface area measurement Figures 2-6 to 2-9 show (a) adsorption/desorption isotherms and (b) gas adsorption isotherms of produced TTCP, commercially available TTCP, DCPD before wet grinding, and DCPD after wet grinding, respectively. ) is a diagram showing a BET plot.
In addition, the slope (C-1)/Cν _m and the intercept 1/Cν _m of the BET equation obtained from the BET plot, the constant C calculated from them and the monomolecular layer adsorption amount ν _m , and further calculated from the following formula The specific surface area A _s is shown in Table 2-2.
A _s = ν _m × (n/m) × a _m [m ² /g]
N: Avogadro's constant (=6.022×10 ²³ )
M: Molar mass of nitrogen (=28.0134 [g/mol])
a _m : Adsorption occupied area of one nitrogen molecule (=0.162 [nm ² ]

As shown in Table 2-1, the arithmetic mean diameter of the produced TTCP is 21.41 μm, which is more than three times larger than the commercially available TTCP, which is 6.14 μm, but its specific surface area is 0.89 m ² /g, There is no difference from TTCP's 0.87 m ² /g.
Further, the surface areas of DCPD before and after wet pulverization are 16.8 m ² /g and 27.8 m ² /g, respectively, which are relatively large. Furthermore, in these adsorption/desorption isotherms, hysteresis occurs from around the point where the relative pressure exceeds 0.4. Such hysteresis is usually seen when particles have mesopores, but in the measured DCPD, the gaps between particles are wedge-shaped pores, and it is thought that the pores appear as hysteresis.

[Analysis of curing liquid]
(1) Characteristics of the curing liquid The compositions of the acidic curing liquid containing chitosan (Liquid 1), the neutral curing liquid containing chitosan (Liquid 2), and the curing liquid containing polyol-chitosan (Liquid 3) are shown in Table 2-3.
In the table, Liquid 4 is the chitosan solution used in the preparation of Liquid 3, and was used as a comparison to examine the influence of polyol blending.
In addition, Figure 2-10 shows (L1) chitosan-containing acidic curing liquid (Liquid 1), (L2) chitosan-containing neutral curing liquid (Liquid 2), (L3) polyol-chitosan-containing curing liquid (Liquid 3), and (L4) chitosan-containing curing liquid (Liquid 3). It is a figure which shows the state of a solution (Liquid4).
According to the observation of each liquid shown in FIG. 2-10, Liquid 1 was gel-like and had poor fluidity, and Liquids 2 to 4 were sol-like and had fluidity, but were slightly viscous.

(2) Translucency of curing liquid Figure 2-11 shows (L2) chitosan-containing neutral curing liquid (Liquid 2), (L3) polyol-chitosan-containing curing liquid (Liquid 3), and (L4) chitosan solution (Liquid 4). It is a figure showing a visible light transmission spectrum.
Table 2-4 also shows the pH, transmittance, and chitosan content of the acidic curing liquid containing chitosan (Liquid 1), the neutral curing liquid containing chitosan (Liquid 2), the curing liquid containing polyol-chitosan (Liquid 3), and the chitosan solution (Liquid 4). Indicate quantity.

Although the chitosan-containing acidic curing liquid (Liquid 1) has a high chitosan content, it is strongly acidic, gel-like, and has a high viscosity, making it difficult to fill the transmittance measurement cell without introducing air. , transmittance measurement was not possible. Further, undissolved chitosan was visually observed, and the solution had the lowest transparency among all the solutions.
The chitosan-containing neutral curing liquid (Liquid 2) exhibited weak basicity similar to body fluids, but had the lowest chitosan content. Moreover, although no undissolved chitosan was observed by visual observation, the transmittance was 13.48%.
The polyol-chitosan-containing curing liquid (Liquid 3) of the present invention was approximately neutral, and its chitosan content was 4.6 times that of Liquid 2. Furthermore, while the chitosan solution (Liquid 4) used to prepare Liquid 3 is a strong acid with a pH of 2.05, it is neutral with a pH of 6.71, and the transmittance of Liquid 4 is 24.9%. The transmittance was high at , 43.7%, and almost no undissolved chitosan was observed.
In Liquids 1, 2, and 4, the transmittance increases from low to high wavelengths, probably because it is less susceptible to confusion on the long wavelength side, whereas in Liquid 3 of the present invention, the transmittance increases in the wavelength range of 380 to 780 nm. High transmittance in all visible light regions.

[Preparation of polyol-chitosan-containing curing solution]
Among the neutral curing liquids, the polyol-chitosan-containing curing liquid (Liquid 3) shows high transmittance but has a low chitosan content. It had excellent mechanical properties.
Therefore, in order to improve the chitosan content, various amounts of chitosan were dissolved using an acid other than a 0.1M HCl solution and a polyol, and the curing liquid Liquid 3-1 to Liquid 3 was prepared with CPC P/L = 3. -4 was prepared. The composition and pH of Liquid 3 and each curing solution are shown in Table 2-5.
Further, FIG. 2-12 is a diagram showing the states of (L3) and (L3-1) to (L3-4) polyol-chitosan-containing curing liquids (Liquid 3 and Liquid 3-1 to Liquid 3-4).

The results of Liquid 3-1, Liquid 3-2, and Liquid 3-4 show that it is possible to increase the chitosan content compared to Liquid 3 by using 1M-HCl or 1M-acetic acid. However, the acidity became stronger, and even when the same amount of polyol as Liquid 3 was added, it showed a strong acidity.
Liquid 3-3, in which chitosan was added so that the chitosan content was 2.5 wt%, the same amount as the chitosan-containing acidic curing liquid (Liquid 1), became gel-like. This is considered to be due to the addition of excessive chitosan to the components of the curing liquid.
Comparing Liquid 3-1, Liquid 3-2, and Liquid 3-3, the pH shifts to neutrality as the chitosan content increases. This is thought to be due to the amino groups that chitosan has.

As will be described later, the initial curing time of a CPC paste using a polyol-chitosan-containing curing liquid (Liquid 3) as a curing liquid is long.
Therefore, in order to shorten the initial curing time of CPC paste, potassium dihydrogen phosphate (KH ₂ PO ₄ ), which is acidic, and dipotassium hydrogen phosphate (K ₂ HPO ₄ ), which is weakly basic, were added to Liquid 3 as appropriate. In addition, curing liquids Liquid 3-5 to Liquid 3-9 were prepared.
Specifically, in the preparation of Liquid 3-5 to Liquid 3-8, phosphate was added after Liquid 3 was prepared. Furthermore, in the preparation of Liquid 3-9, phosphate was dissolved and added to the polyol solution used in the preparation process of Liquid 3.
Other components of the curing liquid were 0.20 g of chitosan, 1.0 g of water, 0.56 g of polyol, and 9.0 g of 0.1M HCl solution, and P/L of CPC was set to 3.
The composition and pH of each curing liquid are shown in Table 2-6.
Further, FIG. 2-13 is a diagram showing the states of (L3) and (L3-5) to (L3-9) polyol-chitosan-containing curing liquids (Liquid 3 and Liquid 3-5 to Liquid 3-9).

The pH of all the curing solutions did not deviate significantly from neutrality.
In Liquid 3-5, the added K ₂ HPO ₄ remained as a white precipitate and did not dissolve. After that, Liquid 3-6 was obtained by adding NaHCO ₃ , but neither K ₂ HPO ₄ nor NaHCO ₃ was dissolved. Furthermore, the cured liquid became cloudy due to KH ₂ PO ₄ to which Liquid 3-7 was also added. Thereafter, Liquid 3-8 was obtained by adding NaHCO ₃ , but KH ₂ PO ₄ and NaHCO ₃ reacted and foamed. This is considered to be because a neutralization reaction occurred because KH ₂ PO ₄ is acidic and NaHCO ₃ is weakly basic.
On the other hand, with Liquid 3-9, a transparent cured liquid with no undissolved residue was obtained. Air bubbles mixed during kneading were observed on the surface of the solution. When K ₂ HPO ₄ and KH ₂ PO ₄ were added to the β-glycerophosphate disodium solution, no reaction occurred and the solution remained transparent.
From these results, it was found that Liquid 3-9 was the most neutral and highly transparent curing liquid even though it contained phosphate.

[Analysis of cured body]
(1) In-vivo test Figure 2-14 shows CTC pastes using (a) water, (b) chitosan-containing acidic curing liquid (Liquid 1), and (c) chitosan-containing neutral curing liquid (Liquid 2) as curing liquids. This is a micro-X-ray CT image of a sample from an in-vivo test (animal implantation test). A CTC paste was prepared with P/L=3.
Only when (b) chitosan-containing acidic curing liquid (Liquid 1) was used as the curing liquid, a state that appeared to be osteolysis was observed at the interface between CPC and living bone. This is because the pH of Liquid 1 is 3.06, so it is considered that there is a high possibility that the living bone at the boundary where it contacts the CPC was dissolved.
When (c) chitosan-containing neutral curing liquid (Liquid 2) was used as the curing liquid, no osteolysis was observed, but precipitation of fibrous tissue was observed around the CPC. This is considered to be because the undissolved chitosan in the hardening liquid was discharged around the hardened body as the cement powder hardened and covered the CPC.
For comparison, when (a) water was used instead of the hardening liquid containing a hardening component, it was observed that bone cells came into direct contact with the hardened body. This is considered to be due to the osteoconductivity of CPC.
From these results, it is considered that in order for CPC to be absorbed in vivo and replaced by living bone, a transparent neutral curing liquid with no undissolved chitosan is required.

(2) Curing time FIG. 2-15 is a diagram showing the results of the Vigor needle test of the cured products CPC1, CPC2, and CPC3. The number of test specimens n is 5. In the figure, "p<0.01" means that there is a significant difference of less than 1%.
In addition, the initial hardening time of each hardened product is shown in Table 2-7 along with the cement powder and hardening liquid used to prepare the CPC paste. In the table, cement powder 1 is a mixture of produced TTCP and DCPD in a molar ratio of 1:1, and a CTC paste was prepared with P/L=3.

The initial curing time of CPC3 of the present invention using Liquid 3 as a curing liquid was 47.7 minutes, which was longer than the conventional CPC1 and CPC2 using Liquid 1 and Liquid 2 as curing liquids. This is thought to be because disodium β-glycerophosphate as a polyol has hygroscopic properties, which delays the supply of water in the curing liquid to the curing reaction field.
The initial hardening time of CPC paste is usually about 8 to 15 minutes, as it is necessary to secure time for operations from mixing the cement powder and hardening liquid to injection (replenishment) into the vertebral body. . From these test results, the initial curing time of CPC1 is somewhat short, and the initial curing time of CPC2 is almost desirable. In CPC1, the curing time is thought to be shortened because the curing liquid Liquid 1 contains malic acid, which has a chelating effect with dicarboxylic acids, and in CPC2, the curing reaction is shortened because the curing liquid Liquid 2 contains phosphate. It is thought that phosphate ions were supplied to the field, causing early curing. However, when Liquid 2 is used, there is a problem that fibrous tissue is precipitated around the CPC, as shown in FIG. 2-14.

Therefore, for the purpose of shortening the initial curing time of CPC3, a Vigor needle test was conducted on a cured product CPC4 prepared using a curing liquid Liquid 3-9 in which a phosphate was added to the curing liquid Liquid 3.
The obtained results are shown in Figure 2-16 together with the results of CPC3 for comparison. In the figure, "p<0.01" means that there is a significant difference of less than 1%.
In addition, the initial curing time of the cured product is shown in Table 2-8 along with the cement powder and curing liquid used to prepare the CPC paste. In the table, cement powder 1 is a mixture of produced TTCP and DCPD at a molar ratio of 1:1, and a CTC paste was prepared with P/L=3.
According to the obtained results, the initial curing time of CPC4 was 15.7 minutes, which was significantly shortened compared to CPC3, and was confirmed to be suitable as the time required for operations from kneading to replenishment. .

(3) Compressive strength FIG. 2-17 is a diagram showing the compressive strength of the cured bodies CPC1, CPC2, and CPC3 at each immersion time (left: immersed for 1 day, center: immersed for 3 days, and right: immersed for 7 days). In the figure, "p<0.01" means that there is a significant difference of less than 1%.
In addition, the compressive strengths of the cured bodies CPC1, CPC2, and CPC3 at each immersion time (1 day immersion, 3 day immersion, and 7 day immersion) are shown in Table 2-9, along with the chitosan content.

CPC1 had the highest compressive strength, and exhibited a strength close to 30 MPa after being immersed for one day. As described above, although CPC1 has a short curing time and exhibits high compressive strength, it exhibits acidity, so as shown in the results of in vivo tests, osteolysis may occur, which is not preferable.
Although CPC2 and CPC3 were lower than CPC1 and varied, they exhibited a strength of about 10 MPa, that is, a strength comparable to that of cancellous bone, which has strong variation because it is a biomaterial and is heterogeneous and has strong anisotropy. Furthermore, in CPC2 and CPC3, a decrease in strength over time was observed. This is thought to be due to the calcium phosphate being dissolved after being cured by immersion.
The compressive strength of CPC3 tends to be higher than that of CPC2. This is thought to be due to the difference in chitosan content, and further improvement in compressive strength can be expected by increasing the chitosan content.

FIG. 2-18 is a diagram showing the relationship between the particle size ratio and compressive strength of each component of cement powder of hardened bodies CPC3, CPC5, CPC6, and CPC7 and PA.
That is, the compressive strength of the hardened bodies CPC3, CPC5, CPC6 and CPC7 obtained by mixing the hardening liquid Liquid 3 and any one of cement powders 1 to 4 at P/L = 3 and soaking for one day is as follows. The relationship between the particle ratio of TTCP and DCPD or DCPA of each cement powder was investigated. The results obtained are shown in Table 2-10.
In the figure, PA is a literature value described in K. Ishikawa, "Bioactive Ceramics: Cements", Comprehensive Biomaterials, 2011, Vol. 1, p. 267-283, as a reference.
As shown in Table 2-10, the compressive strength of the cured bodies CPC3, CPC5, CPC6 and CPC7 was 13.0 to 20.2 MPa, which was comparable to that of cancellous bone.

(4) Analysis of crystal phase by powder X-ray diffraction Figure 2-19 shows each immersion time (1-day immersion, 3-day immersion, and 7-day immersion) of cured product CPC3 prepared using cement powder 1 and hardening liquid Liquid 3. This is a powder X-ray diffraction pattern of .
The peak of hydroxyapatite (Hap, "H" in the figure) was confirmed in all the diffraction patterns, and it was confirmed that HAp was precipitated and hardened. Similarly, in all the diffraction patterns, the peak of TTCP ("T" in the figure) of the raw material cement powder 1, that is, the residual TTCP was confirmed.
In order to match the dissolution rate of DCPD and TTCP, which are raw materials for cement powder, attempts were made to wet-pulverize DCPD, which has a low solubility, to reduce the particle size and thereby increase the surface area and increase the dissolution rate. It is thought that the dissolution rate exceeded the dissolution rate of TTCP, resulting in DCPD being completely consumed first and TTCP remaining.

FIG. 2-20 shows powder X-ray diffraction patterns of cured bodies CPC3, CPC5, CPC6 and CPC7 prepared using cement powders 1 to 4 and hardening liquid Liquid 3 after being immersed for one day.
When CPC3 and CPC5 were compared, there was almost no difference in the peaks that appeared, and the peaks of TTCP, which is a raw material for cement powder, and HAp, which is a precipitate, were confirmed. Looking at the characteristic (032) and (040) plane peaks of TTCP, CPC5 is stronger than CPC3.
Since DCPA has a higher solubility than DCPD and is easier to dissolve, it is thought that more DCPA was consumed and the remaining amount of TTCP increased, resulting in a stronger peak.
When CPC6 and CPC7 are compared, there is almost no difference in the peaks that appear, and when they are compared with the diffraction patterns of CPC3 and CPC5, most of the peaks other than the strongest of TTCP disappear, and the peak of HAp appears largely. These are considered to be due to the fact that the particle size of TTCP decreased, so that consumption of TTCP progressed, and the conversion to HAp progressed. Furthermore, since the HAp peaks of CPC6 and CPC7 are wider than those of CPC3 and CPC5, it is thought that a HAp phase with lower crystallinity and lower crystallinity than that of CPC3 and CPC5 is precipitated. .

Figure 2-21 shows the diffraction diagrams of the reference compounds of the present invention, TTCP, DCPD, DCPA, and HAp (ICDD PDF No. 25-1137, No. 11-0293, No. 09-0080, and No. 74, respectively). -0566).

(5) Static disintegration rate The static disintegration rate of the cured body CPC3 prepared using Cement Powder 1 as the cement powder and Liquid 3 as the hardening liquid was measured under the conditions and method described above.
FIG. 2-22 is a diagram showing how the static disintegration rate of the cured body CPC3 is measured.
The obtained results are shown in Table 2-11, each measured value in 5 measurements, the disintegration rate calculated from them, and their average value.
The disintegration rate of CPC3 was 0.378±0.108%, and it was confirmed that CPC3 hardly disintegrated. That is, it can be seen that Liquid 3 imparts non-disintegrability to CPC and is useful as a curing liquid for CPC.

The cement paste of the present invention, which uses a polyol-containing neutral transparent chitosan-containing curing liquid (pH 6.71, transmittance 43.73%), shortens the initial curing time, and the cured product has almost no dissolution. It is possible to achieve a static disintegration rate that can be said to be almost zero, and to solve the problems of osteolysis in acidic hardening fluids and fibrous tissue generation around the hardened bodies.

[Embodiment 2]
Embodiment 1 is partially included for comparison.
The abbreviations described below are valid only within the second embodiment.
[Preparation of cement powder]
(1) Preparation of tetracalcium phosphate (TTCP) Commercially available calcium hydroxide (Ca(OH) ₂ , MW 74.0927, grade: special grade, manufactured by Nacalai Tesque Co., Ltd.) and orthophosphoric acid (H ₃ PO ₄ , MW 98.0927). 00, content 85%, grade: special grade, manufactured by Wako Pure Chemical Industries, Ltd.) was used as a raw material. 31.32 g (0.4 mol) of Ca(OH) ₂ and 23.06 g (0.2 mol) of H ₃ PO ₄ so that the Ca/P ratio is 2, an analytical balance (model: sefi IBA-200, AS ONE) Co., Ltd.), and added the former to a 500 mL beaker containing 400 mL of water weighed out using a measuring cylinder, and the latter to a 100 mL beaker containing 50 mL of water. : RSH-1DN, manufactured by As One Corporation) to obtain a Ca(OH) ₂ solution and a H ₃ PO ₄ solution.
A fixed amount of H ₃ PO ₄ solution was added dropwise to the obtained Ca(OH) ₂ solution over 2 hours using a burette with a glass cock (manufactured by As One Corporation), and then a Tornado (model: PM-203, As One Co., Ltd.) Co., Ltd.) was used to stir the mixture. At that time, the rotation speed was appropriately adjusted to such an extent that the solution was slightly swirled by stirring. Water at a temperature of 5°C is circulated from a low-temperature water tank (model: LTB-125A, manufactured by AS ONE Co., Ltd.) through the water jacket, and the 500 mL beaker containing the Ca(OH) ₂ solution is maintained at a temperature of 10°C or less in the water jacket. did. After dropping, the mixture was kept at room temperature for 24 hours to age.

Thereafter, it was centrifuged for 10 minutes at a rotation speed of 5000 rpm using a centrifuge (model: Suprema 21, manufactured by Tomy Seiko Co., Ltd.), the supernatant liquid was removed, and further centrifuged for 7 minutes at a rotation speed of 5000 rpm. The separated precipitate was placed in a petri dish and dried for 24 hours at a temperature of 110° C. in a programmed constant temperature dryer (model: DOV-450P, manufactured by As One Corporation). The dried sample was placed in an alumina boat (model: SSA-H2B, manufactured by Nikkato Co., Ltd.) and heated in the air at a temperature of 1500°C in a hearth elevating electric furnace (model: Super Boy, manufactured by Marusho Denki Co., Ltd.). , and baked for 5 hours. The temperature increase/decrease rate was 10°C/min.
After baking, the sample was taken out of the electric furnace, ground in a mini blender (model: Select Grind CG-II, manufactured by Melita Japan Co., Ltd.) for 5 minutes, and then dry ground in an agate mortar for 1 hour or 1.5 hours. Furthermore, using a mini sieve shaker (model: MVA-1, manufactured by AS ONE Co., Ltd.) and a stainless steel sieve (size φ75 μm), the product was classified to 75 μm or less at a rotation speed of 2500 rpm, and a product A was obtained.

Commercially available TTCP (Ca ₄ (PO ₄ ) ₂ O, MW 366.26, in-house standard product, manufactured by Taihei Kagaku Sangyo Co., Ltd.) was ground with a high-speed mill (model: HS-15, manufactured by Labnect Co., Ltd.) to make a 20 wt% slurry. Using a measuring cylinder, add 2-propanol (isopropyl alcohol (IPA), grade: special grade, manufactured by Wako Pure Chemical Industries, Ltd.) to a zirconia ball with a diameter of 3 mm (model: YTZ-3, diameter 3 mm, manufactured by Nikkato Co., Ltd.). 900 g was weighed out using a personal electronic balance (model: EK600i, manufactured by As One Corporation), and both were added to a pot mill (model: HD-B-104, manufactured by Nikkato Corporation). Wet pulverization was carried out using a tabletop pot mill rotating table (Universal Ball Mill, model: UBM-2, manufactured by Masuda Rika Kogyo Co., Ltd.) at room temperature and rotation speed of 490 rpm for 5 hours. The obtained product was dried at a temperature of 50° C. for 48 hours in a programmed constant temperature dryer (model: DOV-450P, manufactured by As One Corporation), and dry ground in an agate mortar to obtain commercially available TTCP (2).

(2) Preparation of calcium hydrogen phosphate (DCPA) 15.00 g (0.11 mol) of calcium hydrogen phosphate anhydrate (CaHPO ₄ , MW136.06, manufactured by Taihei Chemical Industry Co., Ltd.) was weighed using an analytical balance, Add 180 mL of ethanol (C ₂ H ₅ OH, grade: special grade, manufactured by Kishida Chemical Co., Ltd.) in a measuring cylinder, and add a 10 mm diameter zirconia ball (model: YTZ-10, 500 g of a sample (diameter: 10 mm, manufactured by Nikkato Co., Ltd.) was weighed out, and both were added to a pot mill (model: HD-A-3, manufactured by Nikkato Co., Ltd.). Wet milling was carried out at room temperature and rotation speed of 110 rpm for 30 hours, 48 hours, and 96 hours using a tabletop pot mill rotary table (model: ANZ-51S, manufactured by As One Corporation). The obtained wet pulverization product was suction filtered using filter paper (5C, manufactured by AS ONE Co., Ltd.), a Buchner funnel, and an aspirator (model: A-3S, manufactured by EYELA Tokyo Rikakikai Co., Ltd.), placed in a petri dish, and programmed. It was dried in a constant temperature dryer at a temperature of 50° C. for 24 hours. Further, it was dry ground in an agate mortar. As described below, the particle size does not change even if the grinding time is changed, so the product obtained by this method is hereinafter referred to as DCPA.

(3) Mixing of cement powder Cement powder was prepared as follows. 6.00 g (0.0164 mol) of TTCP and 2.23 g (0.0164 mol) of DCPA were weighed using an analytical balance. Place the 50 mL test tube with Maruem scale screw cap (model: NX-50, manufactured by As One Corporation) in a shaker (model: MALTI SHAKER MS-300, manufactured by As One Corporation), and rotate at 1600 rpm for 100 minutes. Mixing was performed by shaking. The resulting mixture was dry mixed in an agate mortar for 10 minutes to obtain cement powder.
As described below, product A, commercially available TTCP, was a TTCP single phase, and commercially available TTCP (2) was also almost a TTCP single phase. In addition, as will be described later, the average particle diameters of product A after dry grinding in an agate mortar for 1 hour and 1.5 hours were 25-30 μm and 10-15 μm, respectively, and commercially available TTCP and commercially available TTCP(2) were 7 .2 μm and 2.0 μm. In order to distinguish each particle size, the product A obtained after dry grinding in an agate mortar for 1 hour and 1.5 hours was classified into TTCP (25-30), TTCP (10-15), commercially available TTCP, and commercially available TTCP (2). ) is referred to as TTCP (7.2) and TTCP (2.0), and the cement powders of each combination are hereinafter referred to as shown in Table 3-1.

[Preparation of hardening liquid]
(1) Preparation of hardening solution containing sodium alginate Put 10 mL of water weighed in a measuring cylinder into a 50 mL beaker, and add medium molecular weight sodium alginate ((NaC ₆ H ₇ O ₆ ) _n , grade: first class, 80 to 120 mPa・s, Wako pure 0.20 g of dipotassium hydrogen phosphate (K ₂ HPO ₄ , MW136.086, grade: special grade, manufactured by Kishida Chemical Co., Ltd.), 0.20 g of potassium dihydrogen phosphate (KH ₂ PO 4 , manufactured by Kishida Chemical Co., Ltd.) ₄ , MW 174.2, grade: special grade, manufactured by Kishida Chemical Co., Ltd.) was weighed out using an analytical balance and added. This solution is called Liquid A1. In addition, instead of 0.20 g of medium-molecular-weight sodium alginate, 1.0 g and 1.5 g of low-molecular-weight sodium alginate ((NaC ₆ H ₇ O ₆ ) _n , 20-50 mPa・s, manufactured by Kimika Co., Ltd.) were added using an analytical balance. , 2.0g were weighed and added, and the solutions were designated as Liquid A2, A3, and A4, respectively. The solution was mixed using a stirring bar and a hot stirrer (model: REXIM RSH-1DN, manufactured by As One Corporation) at room temperature and a rotational speed of 300 rpm for 15 minutes to uniformly dissolve the solution.

(2) Preparation of polyol-chitosan-containing curing solution Add 8.828 mL of hydrochloric acid (HCl, MW 36.46, grade: special grade, 35% content, d = 1.18 g/cm ³ , manufactured by Kishida Chemical Co., Ltd.) using a graduated cylinder ( 0.10 mol) was weighed out, and using a 1 L graduated cylinder, water was added to make 1000 mL to prepare a 0.1 M-HCl solution. 9 mL of the prepared 0.1M-HCl was weighed out using a graduated cylinder, 0.20 g of low molecular weight chitosan weighed using an analytical balance was added, and the mixture was stirred with a glass rod to obtain a chitosan solution.
Next, β-glycerophosphate disodium tetrahydrate (C ₃ H ₇ Na ₂ O ₆ P・4H ₂ O, MW 216.04, manufactured by TCI Tokyo Kasei Kogyo Co., Ltd.) was added to 1 mL of water weighed using a graduated cylinder. Weigh and add 0.56 g (0.0259 mol) using an analytical balance, stir with a glass rod to dissolve, and then add potassium dihydrogen phosphate (KH ₂ PO ₄ , MW 136.086, manufactured by Wako Pure Chemical Industries, Ltd.). Basic dipotassium hydrogen phosphate (K ₂ HPO ₄ , MW 174.2, manufactured by Wako Pure Chemical Industries, Ltd.) was added to obtain a disodium β-glycerophosphate solution. 1 mL of disodium β-glycerophosphate solution was added drop by drop to the chitosan solution using a measuring pipette and stirred, and the resulting solution was used as a polyol-chitosan-containing curing liquid. This is hereinafter referred to as LiquidC1.

[Evaluation of hardening liquid]
(1) Transmittance measurement Spectrum measurement and fixed wavelength measurement were performed using an ultraviolet-visible spectrophotometer (model: V-500, manufactured by JASCO Corporation) to examine the transmittance of the prepared cured liquid. The measurement conditions are shown in Table 3-2 below. The prepared curing liquid was placed in a square quartz cell (10 mm, manufactured by JASCO Corporation) and measured.

(2) pH measurement The pH of the prepared hardening solution was measured using a pH meter (model: D-51, manufactured by Horiba Seisakusho Co., Ltd.). Before use, prepare borate mild pH standard solution (pH 9.18), neutral phosphate pH standard solution (pH 6.86), and phthalate pH standard solution (pH 4.01) (all from Wako Pure Chemical Industries, Ltd.). A three-point calibration was performed using the

(3) Evaluation of sodium alginate The viscosity of alginic acid was measured. Osteoclasts produce protons and chloride ions and dissolve bone in the body. Osteoclasts are immersed in physiological saline, Tris-HCl aqueous solution, and simulated body fluid (SBF) with different chloride ion concentrations, and changes in the molecular weight of alginic acid are measured by viscosity. It was estimated from
For the physiological saline, 9.000 g of sodium chloride (NaCl, MW 58.44, grade: special grade, manufactured by Wako Pure Chemical Industries, Ltd.) was weighed using an analytical balance, placed in a 1 L volumetric flask, and water was added to make the volume constant. Prior to the preparation of SBF and Tris-HCl aqueous solution, a 1N-HCl solution was prepared using hydrochloric acid (HCl, 35.0-37.0 mass/mass%, grade: special grade, manufactured by Wako Pure Chemical Industries, Ltd.) in a measuring cylinder. It was obtained by weighing and diluting 12 times with water.

SBF is SB Cho, K. Nakanishi, T. Kokubo, N. Soga, C. Ohtsuki, T. Nakamura, T. Kitsugi, T. Yamamuro, J. Am. Ceram. Soc., Vol. 78, No. 7 , p. 1769-1774 (1995) as follows. Weigh and pour 700 mL of water into a 2L plastic bottle using a measuring cylinder, and while stirring at a rotation speed of 500 rpm using a stirrer and a hot stirrer, add the reagents listed in Table 3-3 (grade: special grade, manufactured by Wako Pure Chemical Industries, Ltd.). ) were added and dissolved in order using an analytical balance, and then the next reagent was weighed and dissolved in the same manner. The temperature was adjusted to 36.5±0.5°C using a hot stirrer and confirmed using an alcohol thermometer. Granular CaCl ₂ was added one by one one by one, and after one granule was dissolved, the next one was added. Once the Na ₂ SO ₄ was added, the pH meter electrode was immersed in the solution. After adding another 200 mL of water to make the liquid volume 900 mL, add tris (tris-hydroxymethylaminomethane) while watching the pH meter until the pH is below 7.45. When the pH rises to 7.45, 1M- HCl solution was added dropwise to lower the pH to 7.42. Next, tris was added, and when the pH rose to 7.45, the operation of adding 1M HCl solution was repeated until tris was used up, and the pH was finally adjusted to 7.40. The adjusted aqueous solution was transferred to a 1000 mL volumetric flask and cooled with water. After the liquid temperature reached 20° C. or less, water was added to adjust the volume to a constant volume. SBF was used within one month after preparation. The chlorine concentration of SBF is 148.8 mmol. pH measurement was performed using a pH meter (model: D-51, manufactured by Horiba Seisakusho Co., Ltd.).

Tris-HCl was prepared as follows. 900 mL of water was weighed in a measuring cylinder and adjusted to a temperature of 36.5±0.5°C using a stirrer and a hot stirrer. 39 mL of 1.0M HCl was weighed and added using a graduated cylinder, and the pH meter electrode was immersed. Tris weighed using an analytical balance was added, and when the pH rose to 7.45, a 1.0 M HCl solution was added, and this was repeated until 6.118 g of Tris was used up, and the pH was finally adjusted to 7.40. The adjusted aqueous solution was transferred to a 1000 mL volumetric flask, cooled with water, and allowed to stand until the liquid temperature became 20° C. or lower. Thereafter, the volume was adjusted to volume with water.
The chlorine concentrations of physiological saline, Tris-HCl, and simulated body fluid (SBF) are 145 mM, 50 mM, and 170 mM. 200 mL of each was weighed using a graduated cylinder and placed in a beaker, and 2.00 g of sodium alginate (grade: special grade, manufactured by Wako Pure Chemical Industries, Ltd.) powder weighed using an analytical balance was added, and the mixture was allowed to stand at room temperature for 24 hours. A B-type rotational viscometer (model: Brookfield DV-I Prime, manufactured by Hideko Seiki Co., Ltd.) was used to measure the viscosity. The rotation speed was 60 rpm, and a rotor of No. 62 (φ19×7 mm) was used. In addition, for measurement, water was poured into a double-tube reaction container (φ150 x 100 mm, manufactured by Sansho Co., Ltd.), and water was circulated from a low-temperature thermostatic water tank (model: LTB-400A, manufactured by AS ONE Co., Ltd.) at a temperature of 20°C (room temperature). ) was held. The data was read and recorded from the displayed values.

[CPC hardening test]
(1) Preparation of CPC paste CPC was prepared in accordance with JIS T0330-4. Cement powders 1 to 4 were weighed using an analytical balance to achieve the set powder-liquid ratio. The hardened liquid was placed in a petri dish used for kneading and weighed using an analytical balance. Kneading was performed using the jig shown in Figure 3-1. Cement powder was placed in a Petri dish without touching the hardening solution, and if there were any lumps, they were crushed by pressing them with a plastic plate (Figure 3-1, (a)) before mixing. The cement powder was divided into three parts and kneaded together with the curing liquid (Liquid C2 in the figure) using a plastic plate and a stainless steel pharmaceutical spoon (Figure 3-1, (b)). The mixture was kneaded in the air (temperature 25°C) for 70 seconds to obtain a kneaded product.

Immediately after kneading, the kneaded product was placed in a φ6 x 12 mm Teflon (registered trademark) split mold (Figure 3-2, (a)) placed on a single plastic plate with a stainless steel pharmaceutical spoon and a glass rod (φ5 .6mm). At this time, to prevent air bubbles from entering, push it into the center hole of the Teflon split mold with a stainless steel pharmaceutical spoon, and press it down from above with a glass rod as appropriate. Furthermore, both sides were fastened with plastic plates (Figure 3-2, (b)) and clips (Figure 3-2, (c)). The configuration of the mold is shown in Figure 3-2.
In a cool incubator (model: CN-40A, manufactured by Mitsubishi Electric Engineering Co., Ltd.) maintained at a temperature of 37°C in advance, a container containing a sponge soaked in water and a screw capped screw containing 50 mL of water measured using a graduated cylinder. I put in a tube bottle. The kneaded product was placed on a sponge together with a Teflon split mold, and the container was covered with a lid and held for 1 hour to obtain an initial hardened product. Thereafter, the kneaded product was immersed in water kept at a temperature of 37° C. in its split form. The CPC cured product obtained by incubating and immersing the product in a cool incubator at a temperature of 37°C and then demolding was used as a sample.
Figure 3-3 shows a Teflon split mold, (a) Teflon mold, (b) Teflon mold fixing plate (30mm x 12mm x 2.3mm, center hole diameter φ5mm), (c) flat head screw (5M x 33mm) , (d) nut and (e) assembly diagram.
FIG. 3-4 is a diagram showing a container used for initial curing of CPC.

(2) Compressive strength test The compressive strength test was conducted in accordance with JIS T0330-4. Five cured CPC bodies prepared as described above were produced for each immersion condition. Care was taken to ensure that the upper and lower surfaces of the cured CPC body were parallel. This is because if there are protruding parts that are not parallel, stress will be concentrated and a lower value than originally expected will be obtained. After immersion, the water on the surface was wiped off and the sample was tested. To measure the compressive strength, a universal testing machine (model: AUTOGRAPH AGI-20kN, manufactured by Shimadzu Corporation) and a 5kN load cell (model: 87394, manufactured by Shimadzu Corporation) were used. - Using a compression converter (Figure 3-5). A water-wetted filter paper was sandwiched between the tension-compression transducer and the sample, and when a load of 1N was applied, the displacement was set to 0 mm and the zero point was set. Even if there was a sudden decrease in compressive strength due to microfracture during the test, the test was not interrupted and continued until the displacement reached 2 mm. The measurement conditions were a head speed of 0.500 mm/sec, a sampling interval of 100 msec, and measurement was performed using control and analysis software (Tranpezium, manufactured by Shimadzu Corporation).

(3) Initial curing time measurement Initial curing time measurement was performed in accordance with JIS T0330-4. The CPC paste kneaded as described above was put into a Teflon mold (φ10 x 5 mm) within 90 seconds after the start of kneading, and placed in a cool incubator kept at a temperature of 37°C, as in the preparation of the initial hardening product. Place a sponge in a container and use a Vigor needle tester (model: A-004, manufactured by Nippon MEC Co., Ltd.) to gently drop a Vigar needle (300 g, tip area 2 mm ² ) from directly above to make an impression. After checking, if any indentation remained, it was returned to the container repeatedly, and the time when no indentation appeared was defined as the initial curing time. The test situation is shown in Figure 3-6. The test was performed three times for each condition, and the average value was taken as the initial curing time.

(4) Static disintegration rate measurement Static disintegration rate measurement was performed in accordance with JIS T0330-4. Physiological saline (0.9% NaCl aqueous solution) was prepared by adding sodium chloride (NaCl, grade: special grade, manufactured by Kishida Chemical Co., Ltd.) to water weighed using a graduated cylinder and weighed using an analytical balance. Place a stainless steel wire mesh stand (Figure 3-7(b), 30 x 30 mm) in a plastic container (Figure 3-7(a), φ40 x 30 mm) and physiological saline adjusted as above and weighed with a measuring cylinder. 45 mL was added and preheated to 37°C in a cool incubator. 1 mL of the CPC paste obtained by the above procedure was filled into a syringe (φ10 mm, 40 mL), and gently extruded onto a stainless steel wire mesh stand inside a plastic container. Five minutes after the start of kneading, the lid of the container was closed, and the mixture was left standing in a cool incubator at a temperature of 37° C. for 72 hours. After 72 hours, carefully remove the physiological saline inside the container, dry the plastic container together with the wire mesh stand in a programmed constant temperature dryer for 24 hours at a temperature of 37°C, weigh it using an analytical balance, and calculate the disintegration rate using the following formula. I asked for it.
Decay rate (%) = [(c-d)/((a-b)+(c-d))]×100
a: Weight of the cured sample and wire mesh stand (g)
b: Weight of the wire mesh stand from which the cured sample was removed and dried (g)
c: Weight of disintegrated sample and plastic container (g)
d: Weight of the plastic container from which the disintegrated sample was removed and dried (g)

(5) Extrusion force measurement In order to evaluate the injectability of the CPC paste, the following measurements were performed instead of the consistency test of JIS T0330-4.
CPC was kneaded for 70 seconds, and 0.2 mL was measured within 90 seconds using a CPC measuring syringe (Figure 3-8, φ4.6 mm). After 120 seconds, as shown in Figure 3-9, place the cylinder tip side up on an electronic balance (model: EK-4100i, manufactured by A&D Co., Ltd.), hold the outer cylinder with your hand, and apply a load vertically downward. The maximum load required until the syringe was completely ejected from the tip was measured. In addition, when measuring the load, a video was taken of the liquid crystal part of the electronic balance, and the maximum load was confirmed from the video. Measurements were repeated three times, and their average values and standard deviations were obtained.

[Sample analysis]
(1) Identification of crystal phase by powder X-ray diffraction Product A prepared above, commercially available TTCP, commercially available TTCP (2), DCPA and the prepared cured CPC (placed in a petri dish and dried at 50°C in a programmed constant temperature dryer) powder X-ray diffraction was performed. An X-ray diffractometer (model: RINT2200, manufactured by Rigaku Corporation), analysis software (JADE6, manufactured by Rigaku Corporation), and an enclosed tube (target Co, 2 kW) were used for X-ray generation. The measurement conditions are as follows. Measurement angle 10° to 70°, sampling angle 0.02°, scan speed 2.0° min ^-1 , tube voltage 40 kV, tube current 20 mA, slit DS = 1°, SS = 1°, RS = 0.3 mm, The wavelength of Co-Kα is 1.790 Å.

(2) Particle size distribution measurement The particle size distribution of the synthesized and classified TTCP (product A) was measured. The measurement was carried out using a laser diffraction/dispersion measuring device (model: LA920, manufactured by Horiba, Ltd.). The dispersion medium was ultrapure water, and no dispersant was used. Each sample was dispersed using an ultrasonic cleaner and then measured. The refractive index of calcium phosphate with respect to water was 1.24.

(3) Absorption in vitro test A solubility test was conducted in accordance with JIS T0330-3:2012. The amount of calcium ions eluted from the sample and the amount of mass loss were measured in a solution with a pH of 5.5, which is an environment created by osteoclasts. After kneading the sample in the same manner as above, the sample was initially cured at a temperature of 37°C, and after being immersed in 50 mL of physiological saline at a temperature of 37°C for 7 days, it was dried in a programmed constant temperature dryer (model: DOV-450P, manufactured by As One Corporation). A sample dried at a temperature of 50° C. for 24 hours was used as an absorbency test sample.

Preparation of acetic acid-sodium acetate buffer (pH 5.5) Weigh out 4.804 g of acetic acid (0.08 mL, CH ₃ COOH, MW 60.05, grade: special grade, manufactured by Kishida Chemical Co., Ltd.) using an analytical balance, and place it in a 1 L volumetric flask. and water was added to adjust the volume to 1 L to prepare a 0.08 mol/L acetic acid solution.
6.562 g of sodium acetate (0.08 mol, CH ₃ COONa, MW82.0343, grade: special grade, manufactured by Kishida Chemical Co., Ltd.) was weighed in the same manner, and 0.08 mol/L was similarly weighed using a 1 L volumetric flask and water. It was made into a sodium acetate solution. This operation was repeated twice to produce 2 L of 0.08 mol/L sodium acetate.
Add 0.08 mol/L sodium acetate solution to 20.0 mL of 0.08 mol/L acetic acid solution weighed in a measuring cylinder, and adjust the pH to 5.50± using a pH meter (model: D-51, manufactured by Horiba Estec Co., Ltd.). It was adjusted to 0.02. The pH became 5.49 to 5.50 with 142 mL of 0.08 mol/L sodium acetate solution. Therefore, an acetic acid-sodium acetate buffer solution having a pH of 5.50±0.02 was prepared by mixing a 0.08 mol/L acetic acid solution and a 0.08 mol/L sodium acetate solution at a mixing ratio of 1:7.

Preparation of 1 mg/L, 10 mg/L, and 100 mg/L Ca standard solutions for calibration A 1000 ppm Ca ²⁺ standard solution was prepared according to the following procedure.
Weigh out 9.132 g of nitric acid (0.1 mol, HNO ₃ , 69-70 mass/mass%, grade: special grade, manufactured by Kishida Chemical Co., Ltd.) using an analytical balance, put it in a 1 L volumetric flask, and add water to adjust the volume to 1000 mL. A 0.1 mol/L nitric acid solution was prepared.
Weigh 2.502 g of calcium carbonate (CaCO ₃ , MW100.0869, grade: special grade, manufactured by Wako Pure Chemical Industries, Ltd.) using an analytical balance, put it in a 1 L volumetric flask, and add 0.1 mol/L nitric acid solution to make the volume 1000 mL. The volume was determined to be 1000 ppm Ca ²⁺ standard solution.
The 1000 ppm Ca ²⁺ standard solution was serially diluted using a 100 mL volumetric flask and a 10 mL whole pipette to prepare a 100 ppm Ca ²⁺ standard solution and a 10 ppm Ca ²⁺ standard solution. Weigh out 10 mL of each Ca ²⁺ standard solution and 10 mL of 0.8 mol/L acetic acid-sodium acetate buffer using a 10 mL whole pipette, put them into a 100 mL volumetric flask, add water to make a constant volume of 100 mL, and make 0.08 mol/L. L-acetic acid-sodium acetate-100 mg/L, 10 mg/L, and 1 mg/L Ca ²⁺ standard solutions were prepared.

(3) Measurement of Ca ion elution amount Before measurement, a Ca ion electrode (model: CH-9101, manufactured by Metrohm Japan Co., Ltd.) was conditioned as follows.
It was immersed in water for 2 hours, and then immersed in a 0.08 mol/L acetic acid-sodium acetate buffer using a stirring bar and a hot stirrer (model: REXIM RSH-1DN, manufactured by As One Corporation) at a rotation speed of 430 rpm for 30 minutes at room temperature. It was immersed while stirring.
A calibration curve was created using the calibration Ca standard solution prepared above. Using a stirrer and a hot stirrer (model: REXIM RSH-1DN, manufactured by As One Corporation), Ca ions were added to 1, 10, and 100 mg/L Ca ^{2+ standard solutions in the order of 1, 10, and 100 mg/L Ca 2+} standard solution while stirring and immersing at a rotation speed of 430 rpm at room temperature. The electrode was immersed and the ion concentration was measured.

As shown in Figure 3-10, the test piece was placed under a resin stirrer stand (made by the laboratory), a stirrer was placed on top, a Ca ion electrode was set, and 0.08 mol/L acetic acid-acetic acid 100 mL of sodium buffer solution was put into a container, and the ion concentration was measured by stirring at a rotation speed of 430 rpm using a stirring bar and a hot stirrer (model: REXIM RSH-1DN, manufactured by As One Corporation). Measurements were started within 1 minute after adding the acetic acid-sodium acetate buffer. Measurements were taken every 2 minutes until 20 minutes after the start of measurement, every 15 minutes from 30 to 150 minutes after that, and every 30 minutes thereafter, and ended after 300 minutes. After the measurement, the pH of the buffer solution was measured and confirmed to be pH 5.50. Measurements were performed three times for each condition, and the average was taken as the experimental result. In addition, the sample that had been immersed in physiological saline was taken out and dried, and the sample after the solubility test was dried at a temperature of 50°C for 24 hours, and then placed in a desiccator (vacuum polycarbonate desiccator, model: 240G type, manufactured by As One Corporation). After cooling for 2 hours, the weight was measured using an analytical balance, and the difference was taken as the amount of mass loss.

(4) Tomographic imaging Tomographic imaging of the cured CPC body was performed using a tabletop microfocus X-ray CT (model: SMX-90CT Plus, inspeXio, manufactured by Shimadzu Corporation).
A sample was attached to a sample stage, and a tomographic image was taken at a tube voltage of 90 kV and a tube current of 110 μA. The data were visualized in 3D by volume rendering of tomographic images using 3D image processing software (ExFact VR, manufactured by Japan Visual Science Co., Ltd.). The LUX table was adjusted and the 3D cross section of the sample was observed.

(5) Observation of sample with FE-SEM The sample after the absorption in vitro test was coated with 12 nm of OsO ₄ using an Osmium Plasma Coater (model: OPC60A, manufactured by Philgen Co., Ltd.) to prepare a SEM sample. Each sample was observed using a field emission scanning microscope (FE-SEM, model: JSM-6500F, manufactured by JEOL Ltd.). The acceleration voltage during observation was 15 kV, and the degree of vacuum was 5.00×10 ⁻⁴ Pa or less.

[Analysis of cement powder]
(1) Identification of samples by powder X-ray diffraction (XRD) Diffraction patterns of (a) Product A, (b) Commercially available TTCP and (c) Commercially available TTCP (2) and (d) TTCP (ICDD PDF No. 25- 1137) is shown in Figure 4-1.
According to Figure 4-1, the peak positions and intensities of the diffraction patterns of (a) product A and (b) commercially available TTCP and (d) the diffraction pattern of TTCP by ICCD almost match, and other than these, there are no particular peaks. Almost never seen. Therefore, product A and commercial TTCP are considered to be a single phase of TTCP. In addition, commercially available TTCP (2) obtained by wet-pulverizing commercially available TTCP has different intensity ratios between the two strongest peaks, and the peak separation is somewhat unclear, suggesting the presence of low crystalline apatite.

Diffraction patterns of (a) DCPA before wet milling, (b) DCPA after 30 hours of wet milling, (c) DCPA after 48 hours of wet milling, and (d) DCPA after 96 hours of wet milling, and (e) DCPA ( The diffraction diagram of ICDD PDF No. 09-0080) is shown in Figure 4-2.
According to FIG. 4-2, focusing on the three strongest peaks, the peak of the (112) plane decreased after 30 hours of wet milling. However, the relative peak intensities between the (020) plane and the (220) plane hardly change even when the grinding time is changed from 48 hours to 96 hours. DCPA is insoluble in ethanol, but if it is slightly dissolved by the water contained in ethanol, it is thought that it will locally become weakly acidic due to the ratio of Ca and phosphoric acid. Under weak acidity, apatite has the lowest solubility and is stable, but no peak corresponding to apatite is observed. This is thought to be due to the fact that the morphology of the powder in the c-axis direction changed due to the pulverization effect, which caused a change in the crystal orientation of the glass sample plate filled into the glass sample plate for X-ray diffraction measurement.

(2) Particle size distribution (I) Dry-milled TTCP (milling time 1 hour), (II) Dry-milled TTCP (milling time 1.5 hours), (III) Commercially available TTCP and (IV) Commercially available TTCP (2) Figure 4-3 shows the particle size distribution of (V) wet-milled DCPA (milling time: 30 hours), (VI) wet-milled DCPA (milling time: 48 hours), and (VII) wet-milled DCPA (milling time: 96 hours). ) is shown in Figure 4-4, and their arithmetic mean system and median diameter are shown in Table 4-1.
According to FIG. 4-3, when comparing (I) and (II), it is clear that the particle size of TTCP becomes smaller when the grinding time is increased from 1 hour to 1.5 hours. However, even in (II), the distribution is asymmetrical, with many distributions having a diameter of several μm or less. After pulverizing in a mini blender, pulverization was performed in an agate mortar, which suggests the possibility of further refinement. On the other hand, when comparing (III) and (IV), the commercially available TTCP and the pulverized commercially available TTCP (2) show a symmetrical particle size distribution with a sharp peak, and the refinement due to pulverization can be clearly seen.
Furthermore, according to FIG. 4-4, when comparing (V) to (VII), there is almost no change in the particle size distribution. Commercially available DCPA was wet-pulverized using ethanol as a medium for up to 30, 48, and 96 hours, but as suggested by It is thought that the development has not progressed very much.

[Analysis of curing liquid]
(1) Composition of hardening liquid The compositions of the prepared alginic acid-containing hardening liquids (Liquid A1, Liquid A2, Liquid A3, Liquid A4) are shown in Table 4-2.
When medium molecular weight sodium alginate (80 to 120 mPa·s) was dissolved in water up to 2%, it was possible to dissolve more medium molecular weight sodium alginate, but it became slightly gel-like and fluidity decreased. When 2% medium molecular weight sodium alginate was dissolved in water and used as a hardening liquid, the initial hardening time became longer, and phosphate was added to promote the dissolution of TTCP in the cement powder. As a result of using potassium dihydrogen phosphate (KH ₂ PO ₄ ), which is acidic, and dipotassium hydrogen phosphate (K ₂ HPO ₄ ), which is weakly basic, to adjust the pH, the initial curing time for LiquidA1 was 8 to 15 minutes. It was a minute. Furthermore, in order to increase the amount of sodium alginate dissolved, when low molecular weight sodium alginate (20 to 50 mPa·s) was used in Liquid A2, Liquid A3, and Liquid A4, it was possible to dissolve up to nearly 20%. In addition, a decrease in viscosity was observed compared to the medium molecular weight.

The compositions of the prepared chitosan-containing curing liquids (Liquid C1, Liquid C2, Liquid C3, Liquid C4) are shown in Table 4-3.
In addition, Table 4-4 shows the types of chitosan used and the curing liquids classified accordingly.
Chitosan is protonated under acidic conditions below pH 3 and dissolves as its molecular weight decreases, but in 0.1M-HCl at a pH of about 1, medium molecular weight chitosan can reach up to 2%, while dichitosan, which has a high degree of deacetylation, can dissolve up to 2%. There was some undissolved material at 2%, so only 1% was added. Diquitosan was a flake-like mass, did not penetrate well with acid, and had a tendency to be difficult to dissolve. It is expected that solubility will be improved by further crushing the flaky lumps. It is also believed that protonation is further promoted by using a higher concentration of acid. Also, to make the curing liquid neutral, sodium glycerophosphate, which has many hydroxyl groups, was added, but glycerophosphoric acid has a somewhat high viscosity, and it seems possible to reduce the viscosity by using sodium glycolate. A decrease in viscosity was observed by using low molecular weight chitosan.

(3) Characteristics of hardening liquid The pH of the prepared alginic acid-containing hardening liquids (LiquidA1, LiquidA2, LiquidA3, LiquidA4) and chitosan-containing hardening liquids (LiquidC1, LiquidC2, LiquidC3, LiquidC4) is shown in Table 4-5.

Furthermore, the conditions of the alginic acid-containing curing liquids, (a) Liquid A2, (b) Liquid A3, and (c) Liquid A4 are shown in FIG. 4-5, and their transmittances are shown in FIG. 4-6.
According to FIG. 4-5, as the alginic acid concentration increases (a) Liquid A2, (b) Liquid A3, and (c) Liquid A4, some coloring is observed. However, even after preparing the hardening liquid and leaving it at room temperature for two weeks, no change in quality such as gelation occurred, so it is thought that there is no undissolved residue.

(4) Evaluation of sodium alginate The results of the viscosity test are shown in Table 4-6.
Compared to the case of immersion in water, the viscosity was almost the same for Tris-HCl, and the viscosity was lower in the order of physiological saline and SBF, and the results were similar to those for chitosan. When the Cl ion content was high, the viscosity tended to be low. A decrease in viscosity means a decrease in molecular weight, and alginic acid may be degraded and absorbed in the body by chloride ions produced by osteoclasts. On the other hand, since alginic acid forms a chelate bond with Ca ions, it may not be degraded and absorbed as much as chitosan in the osteoclast environment in the body.

[Analysis of cured body]
(1) Curing time The results of the Vigor needle test of the cured products CPC1 to 5 are shown in Table 4-7.
Including phosphate in the curing solution can speed up the initial curing time, but if it hardens too quickly in the field of bone formation surgery, it will be difficult to perform operations from kneading to injection. Curing time is required. The preferred initial curing time varies depending on the application site and operator, and it is difficult to determine an appropriate initial curing time, but it is thought that a period of several minutes to 20 minutes is desirable. With the exception of cured product CPC5, it is almost within this range. Although there was almost no difference in initial curing time between different sodium alginate contents and particle sizes, CPC5 with a lower powder/liquid ratio resulted in a longer curing time.
In Table 4-7, the curing liquids Liquid A1, Liquid A2, and Liquid A4 each contain 10 mL of water, 0.2 g of KH ₂ PO ₄ , 0.1 g of K ₂ HPO ₄ , and alginic acid (number of grams in parentheses), and Liquid C2 contains: Contains 1 mL of water, 9 mL of 0.1M HCl, 0.56 g of polyol, and chitosan (grams in parentheses).

(2) Compressive strength Table 4-8 shows the conditions of the cured CPC subjected to the compression test, and Figure 4-7 shows the compressive strength of the cured bodies CPC4, CPC7, and CPC8 after 24-hour immersion.
In Table 4-8, the curing liquids Liquid A2, Liquid A3, and Liquid A4 each contain 10 mL of water, 0.2 g of KH ₂ PO ₄ , 0.1 g of K ₂ HPO ₄ , and alginic acid (the number of grams in parentheses).

The compressive strength of the cured product CPC8 was the highest, exhibiting a strength of 50 MPa. Addition of sodium alginate at an addition rate of 3.2% or more did not contribute to improving compressive strength, but it has been reported that compressive strength increases due to an increase in polysaccharide content.Yuki Yamada, Master's degree, Graduate School of Engineering, Osaka City University Thesis, 2013 and consistent with H. H.K. The difference between the cured products CPC7 and CPC8 is thought to be due to the difference in the content of sodium alginate, which is a polysaccharide.

Table 4-9 and Figure 4-8 show the compressive strength of the cured product CPC4 for each immersion time.
There was a tendency for the compressive strength to decrease as the immersion time increased. As will be described later, this is thought to be because cement powder remained in the CPC hardened body, and the cement powder that did not contribute to hydration hardening was eluted, reducing the density of the hardened body. If Ca or phosphoric acid is supersaturated, such as in body fluids rather than water, there is a possibility that the eluted content will be replenished, but since the bone replacement part is loaded from multiple directions, there is a risk of collapse, and improvement is necessary. I think that the. Furthermore, although low molecular weight alginic acid reduces viscosity and easily dissolves in water, alginic acid that does not contribute to curing may be eluted from the cured product.

Cement powder is composed of two types of phosphates, and since calcium tetraphosphate tends to remain, TTCP (7.2) and TTCP (2.0) with small particle sizes were used to avoid this.
Table 4-10 shows the conditions of the cured CPC under which the compression test was conducted, and Figure 4-9 shows the compressive strength of the cured CPC4, CPC2, and CPC9 after immersion for 1 day and 7 days.
In Table 4-10, the curing liquid Liquid A4 contains 10 mL of water, 0.2 g of KH ₂ PO ₄ , 0.1 g of K ₂ HPO ₄ and alginic acid (number of grams in parentheses).

In CPC2 using TTCP (7.2), the compressive strength increased as the immersion time increased. On the other hand, in CPC9 using TTCP (2.0) with a smaller particle size, the powder/liquid ratio was small, so the compressive strength was reduced.
When CPC2, whose strength improved with the immersion time, was immersed for one month, the compressive strength was 22.488±2.076 MPa, which decreased after 7 days. Although TTCP disappeared in the XRD described below and the hydration hardening reaction was completed, it is thought that the elution of alginic acid and the like caused a decrease in compressive strength.

Next, Table 4-11 shows the conditions of the chitosan-containing cured product CPC subjected to the compression test, and Figure 4-10 shows the compressive strength of the cured products CPC10 and CPC6 after immersion for 1 day and 7 days.
In Table 4-11, the curing liquid Liquid C2 contains 1 mL of water, 9 mL of 0.1M-HCl, 0.56 g of polyol, and chitosan (g number in parentheses).

No change was observed in compressive strength due to cement powders with different particle sizes. The chitosan content is lower than the alginic acid content, and the strength improvement seems to be lower. After 7 days of immersion, the strength decreased slightly, and in CPC6 that was immersed for 1 month, it decreased to 8.935±1.563 MPa.

(3) Analysis of crystal phase by powder X-ray diffraction The diffraction patterns of the cured product CPC4 immersed for 1 day and 7 days are shown in Figure 4-11.
Although the peak of HAp can be confirmed in both diffraction patterns, the peak of TTCP, which is a type of cement powder, was also observed. This is thought to be due to the difference in solubility between TTCP and DCPA.

Figure 4-12 shows the diffraction pattern for each immersion time of the cured product CPC2, in which the particle size of TTCP is further reduced, the surface area is increased, and the dissolution rate is increased.
Compared to the peaks of the (300) plane and (221) plane of HAp, the peaks of the (040) plane and (032) plane of TTCP are smaller. Furthermore, after one month of immersion, the TTCP peak almost disappeared, indicating that the hydration curing reaction was completed. Since the strength of CPC2 decreased after being immersed for one month, this seems to be due to the elution of alginic acid and the like.

The diffraction patterns of the cured product CPC9 immersed for 1 day and 7 days are shown in FIG. 4-13.
In CPC9 using TTCP (2.0) with a reduced particle size, no peak of TTCP was observed, and only a peak of HAp was observed. However, as mentioned above, the compressive strength decreased. In the diffraction pattern of TTCP (2.0), the peak separation is somewhat unclear, suggesting the presence of amorphous calcium phosphate. As the particle size became smaller, TTCP also reacted, but since amorphous calcium phosphate was generated, TTCP contributing to hydration hardening decreased by that amount, leading to a decrease in strength.

Next, the diffraction patterns of CPC10 and CPC6 containing chitosan for each immersion time are shown in FIGS. 4-14 and 4-15, respectively.
Although there was no decrease in strength, it is thought that TTCP disappeared and the hydration hardening reaction was completed.
Figure 4-16 shows the diffraction diagrams of the reference compounds of the present invention, TTCP, DCPD, DCPA, and HAp (ICDD PDF No. 25-1137, No. 11-0293, No. 09-0080, and No. 74, respectively). -0566).

(4) Extrusion force Table 4-12 shows the combinations and results of cured CPC for which extrusion force was measured.
The extrusion forces of the cured bodies CPC2 and CPC6 were 169.52 kPa and 77.05 kPa, respectively. The extrusion force increased according to the P/L ratio (powder-liquid ratio) of CPC2. All of them can be pushed out with one hand. Chitosan had a lower curing liquid content than alginic acid, resulting in a smaller value. It seems that CPC6 can raise the P/L ratio to about 2.6 to 2.8.
In Table 4-12, the curing liquid Liquid A2 contains 10 mL of water, 0.2 g of KH ₂ PO ₄ , 0.1 g of K ₂ HPO _{4 and alginic acid (number of grams in parentheses), and Liquid C2 contains 1 mL of water, 0.2 g of KH 2 PO 4} , and 0.1 g of K 2 HPO 4 . Contains 9 mL of 1M HCl, 0.56 g of polyol, and chitosan (grams in parentheses).

(5) Static disintegration rate The results of the static disintegration test of cured bodies CPC2 and CPC6 are shown in Tables 4-13 and 4-14, respectively.
The decay rate of CPC2 was 2.003±1.258%, which was a slightly higher value than that of CPC6, 0.629±0.303%. CPC2 had a high P/L ratio and high strength, but on the other hand, it seems that kneading was partially insufficient. Figure 4-17 shows the static collapse rate experiment.

(6) Absorption in vitro test [Calcium ion elution amount/mass loss amount]
Cured products CPC2 and CPC6 were prepared and an in vitro absorption test was conducted. In accordance with JIS T0330-4, the changes in calcium concentration of CPC2 and CPC6 in an acetic acid-sodium acetate buffer solution adjusted to pH 5.5, which is the environment created by osteoclasts, are shown in Figure 4-18 and Figure 4-18, respectively. Shown in 4-19.
Table 4-15 shows the amount of calcium ions eluted per 100 mL of solution, CPC mass loss, and the difference after the absorption test of cured bodies CPC2 and CPC6.
No change was observed in the pH of the solution before and after the test. Although the amount of calcium ion elution did not change much, the result was that CPC2 had a larger weight loss than CPC6. Since phosphoric acid is also thought to be eluted along with calcium, the difference in weight loss is thought to be due to the difference in alginic acid and chitosan added to the curing solution. It is thought that if the polysaccharide is eluted too quickly, it will cause a decrease in the strength of the cured CPC. During hydration and hardening, it is desirable for polysaccharides to bind cement powder to each other like "glue." Strength can be improved by optimizing the amount added, and bone replacement can be achieved by creating porosity while maintaining a certain strength after hardening. An ideal CPC paste is expected.

[Sample observation after absorption test]
A microfocus X-ray CT image (3D image) of the cured product CPC2 after the absorption test is shown in Figure 4-20.
Many areas with different shading from the matrix can be seen. CPC is filled into a Teflon mold hole with a diameter of 6 mm and a depth of 12 mm, but what is visible in the center may be air bubbles due to uneven filling of the Teflon mold.
FE-SEM of cured bodies CPC2 and CPC6 are shown in FIGS. 4-21 and 4-22, respectively. The magnification of each figure for CPC2 and CPC6 is 5000x and 70x, respectively.
It appears that a larger number of fine pores are visible in the cured product CPC6 using the chitosan hardening liquid than in the cured product CPC2 using the alginic acid hardening liquid.

From the above results, the following can be understood.
(1) When low molecular weight sodium alginate is used, the proportion that can be added to the curing liquid is 10 times that of medium molecular weight sodium alginate, and the viscosity of the CPC paste is reduced. (2) Alginic acid is immersed in solutions with different amounts of chlorine. After that, the viscosity was measured, and it was found that the higher the amount of chlorine in the soaked solution, the lower the viscosity and the lower the degree of polymerization, and the possibility that osteoclasts promote porosity and absorb it in the body. (3) When potassium dihydrogen phosphate and dipotassium hydrogen phosphate were added to an alginic acid-containing curing solution and the initial curing time was measured, the amount of alginic acid did not affect the curing time, but the amount of phosphate was large. (4) After adjusting the particle size of the cement powder and measuring the compressive strength of the prepared CPC, it was found that although the initial strength was inferior, it prevented the strength from decreasing due to water immersion, and Considering the number of days required for replacement with living bone, it can be predicted that a cement powder with adjusted particle size that does not lose its initial strength over time would be more useful.

(5) When the crystal phase of the hardened product was examined using powder X-ray method, the peak of TTCP, which is a raw material for cement powder, remained even after hardening, but this is thought to be due to the relatively high solubility of DCPA and its ease of dissolution. (6) Although chitosan-containing CPC and sodium alginate-containing CPC show a slightly higher static disintegration rate, which is thought to be due to the elution of alginic acid, the latter hardly disintegrates as a whole, and CPC containing polysaccharides does not disintegrate into CPC. In particular, alginic acid-containing curing liquid Liquid A4 and chitosan-containing curing liquid Liquid C2 are considered to be useful as curing liquids for CPC. Although the weight loss corresponding to the elution of chitosan and chitosan is large, the amount of calcium eluted from the cured CPC body is small. (8) According to surface observation using micro fine pores can also be observed

Claims (12)

Cement powder containing calcium hydrogen phosphate dihydrate or anhydride and tetracalcium phosphate, and 8.8% to 20% by weight of alginic acid or its salt , dipotassium hydrogen phosphate, dihydrogen phosphate A kit for producing a bioactive cement paste , comprising a curing liquid with a pH of 6 to 7.5 containing potassium and water , the bioactive cement paste meeting the requirements of "(5) Extrusion force measurement" in the specification. A kit that exhibits an extrusion force of 1.2 MPa or less or 2000 g or less when measured by the method described in the section . The kit according to claim 1, wherein the alginic acid is a low molecular weight alginic acid. The kit according to claim 1 or 2, wherein the salt of alginic acid is sodium alginate. A cement powder containing calcium hydrogen phosphate dihydrate or anhydride and tetracalcium phosphate, and a curing liquid containing chitosan, polyol , dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and water , A kit for producing a bioactive cement paste , comprising a curing liquid having a pH of 6 to 7.5 and having a transmittance of 550 nm light of 40% or more, wherein the polyol is β-glycerophosphoric acid or a salt thereof. The kit according to claim 4, wherein the polyol is disodium β-glycerophosphate. The kit according to claim 4 or 5, wherein the concentration of chitosan in the curing liquid is 1.5 % by weight or more and 10% by weight or less . Any of claims 4 to 6, wherein the bioactive cement paste exhibits an extrusion force of 1.2 MPa or less or 2000 g or less when measured by the method described in "(5) Extrusion force measurement" section of the specification. or the kit described in item 1. Any of claims 4 to 7, wherein the bioactive cement paste exhibits an extrusion force of 0.12 MPa or less or 200 g or less when measured by the method described in "(5) Extrusion force measurement" section of the specification. or the kit described in item 1. Any one of claims 1 to 7, wherein the bioactive cement paste, after hardening, exhibits a compressive strength of at least 10 MPa when measured by the method described in section "(4) Compressive strength test" of the specification. Kits listed in section. Cement powder containing calcium hydrogen phosphate dihydrate or anhydride and tetracalcium phosphate, and 8.8% to 20% by weight of alginic acid or its salt , dipotassium hydrogen phosphate, phosphoric acid 1.2 MPa or less or 2000 g or less when mixed with a hardening liquid containing potassium dihydrogen and water and having a pH of 6 to 7.5 , and measured by the method described in "(5) Extrusion force measurement" section of the specification. A method for producing a bioactive cement paste to obtain a bioactive cement paste exhibiting an extrusion force of . A cement powder containing calcium hydrogen phosphate dihydrate or anhydride and tetracalcium phosphate, and a hardening liquid containing chitosan , polyol , dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and water, The polyol is glycerol phosphoric acid or a salt thereof, and is mixed with a curing liquid having a pH of 6 to 7.5 and having a transmittance of 40% or more for light at a wavelength of 550 nm, and the method described in "(5) Extrusion force measurement" in the specification is carried out . A method for producing a bioactive cement paste, which obtains a bioactive cement paste exhibiting an extrusion force of 1.2 MPa or less or 2000 g or less when measured by the method described in the section . A bioactive cement paste which is a mixture of the cement powder according to any one of claims 1 to 9 and the hardening liquid according to any one of claims 1 to 9 .