JP7315943B2 - Porous support containing hydroxyapatite - Google Patents

Description

The present invention relates to a hydroxyapatite-containing porous support.

BACKGROUND ART In recent years, hydroxyapatite, which is the main component of teeth and bones, has attracted attention because it is harmless to the human body and has the ability to adsorb harmful substances.

The application range of hydroxyapatite is expanding to adsorbents, materials for forming teeth and bones, and cell scaffolds, and it is desired to provide new functional materials using hydroxyapatite.

The present inventors have focused on the fact that if such hydroxyapatite can be adhered to a substrate, the adsorption function can be imparted to the substrate. We have invented microfibers containing a film and a dispersion of microfibers, and have already filed an application (Japanese Patent Application No. 2018-075750).
The present inventors have further explored functional materials and found that when a dispersion containing a carrier and hydroxyapatite, or a dispersion containing a carrier to which hydroxyapatite is adhered to the surface, is dried, it is dispersed in the dispersion. The inventors have found that a porous support having a microscopic three-dimensional structure different from that of the previous one can be obtained, which led to the completion of the present invention.

The present invention is as follows [1] to [11].
[1] A step of dispersing a carrier and hydroxyapatite, or a carrier having hydroxyapatite attached to the surface thereof, in a dispersion medium to obtain a dispersion, and a step of drying the dispersion;
A method for producing a hydroxyapatite-containing porous support, comprising:
[2] The method for producing a hydroxyapatite-containing porous support according to [1], wherein the step of drying the dispersion is a step of drying the dispersion by vacuum drying.
[3] The method for producing a hydroxyapatite-containing porous support according to [1], wherein the step of drying the dispersion is a step of drying the dispersion by vacuum freeze-drying.
[4] Any one of claims 1 to 3, wherein the material constituting the carrier is selected from the group consisting of chitin, chitosan, collagen, lignocellulose, cellulose, cellulose nanofibers, cellulose nanocrystals, and mixtures of two or more of these. A method for producing a hydroxyapatite-containing porous support.
[5] A hydroxyapatite-containing porous support obtained by drying a dispersion containing a carrier and hydroxyapatite.
[6] The hydroxyapatite-containing porous support of [5], wherein the drying is vacuum drying.
[7] The hydroxyapatite-containing porous support of [5] or [6], wherein the drying is vacuum freeze-drying.
[8] The material constituting the carrier is selected from the group consisting of chitin, chitosan, collagen, lignocellulose, cellulose, cellulose nanofibers, cellulose nanocrystals, and mixtures of two or more of these [5]-[7] The hydroxyapatite-containing porous support according to any one of
[9] A sintered body obtained by sintering the hydroxyapatite-containing porous support according to any one of [5] to [8].
[10] A cell scaffold comprising the sintered body of [9].
[11] A bone regeneration material comprising the sintered body of [9].

The production method of the present invention makes it possible to obtain an apatite-containing porous support having a novel three-dimensional microscopic structure. In addition, the apatite-containing porous support of the present invention can be a useful material as a cell scaffold or a bone regeneration material as it is or after being baked.

1 is a micrograph of an apatite-containing porous support obtained by vacuum freeze-drying a dispersion containing 0.5 w/w % rheocrysta and 0.5 w/w % apatite. FIG. Magnification is 100 times. 1 is a micrograph of an apatite-containing porous support obtained by vacuum freeze-drying a dispersion containing 0.5 w/w % rheocrysta and 0.5 w/w % apatite. FIG. Magnification is 1000 times. 1 is a micrograph of an apatite-containing porous support obtained by vacuum freeze-drying a dispersion containing 0.5 w/w % rheocrysta and 0.5 w/w % apatite. FIG. The magnification is 3000 times. 1 is a micrograph of an apatite-containing porous support obtained by vacuum freeze-drying a dispersion containing 0.5 w/w % rheocrysta and 0.5 w/w % apatite. FIG. The magnification is 4700 times. FIG. 2 is a micrograph of a porous support obtained by vacuum freeze-drying a 0.5 w/w % suspension of Rheocrysta. The magnification is 5000 times. FIG. 2 is a micrograph of a porous support obtained by vacuum freeze-drying a 0.5 w/w % suspension of Rheocrysta. The magnification is 5000 times.

[Carrier]
The carrier used in the present invention may be any carrier that allows hydroxyapatite to adhere to its surface and that is dispersible in a dispersion medium such as water. The carrier material is preferably made of a biocompatible material. Examples of carrier materials include chitin, chitosan, collagen, lignocellulose, cellulose, cellulose nanofibers, cellulose nanocrystals, and mixtures thereof. Preferred materials are collagen, cellulose, and cellulose nanofibers. Cellulose nanofiber (CNF) is a fine cellulose fiber with a fiber width of nanometer size. The resulting nanometer-sized (1 to 20 nm fiber width) very thin and long fibers are exemplified.

A carrier may be used in the form of a suspension. For example, when the carrier is cellulose nanofibers, hydroxyapatite may be added to the cellulose nanofiber suspension and stirred to obtain a hydroxyapatite dispersion, or a cellulose nanofiber suspension and a hydroxyapatite suspension may be obtained. may be mixed to obtain a hydroxyapatite dispersion.

The structure of the carrier is not particularly limited. Examples include structures such as microparticles, microfibers, and flakes. Microfibers are not only thin, long, thread-like linear ones, but also those in which two or more of these thin, thread-like linear ones are combined or entangled, fine fiber pieces, and nanofibers. Also includes Examples of nanofibers include the cellulose nanofibers described above. When the carrier is microfibers, the aspect ratio is, for example, preferably in the range of 1:5 to 1:1000, more preferably 1:5 to 1:500, still more preferably 1:5 to 1:200. In the present disclosure, "aspect ratio" means "average diameter of microfiber pieces":"average length of microfiber pieces".
The size of the carrier is not particularly limited, but when the carrier is a microfiber, for example, its average length is preferably 5 nm to 500 μm, more preferably 10 nm to 100 μm, and still more preferably 30 nm to 20 μm. . Scanning electron microscopy can be used to measure the length of microfibers. The average length of microfibers can be measured, for example, by measuring the length of each of 10 randomly selected microfibers and calculating the average.
The average diameter of the microfibers is, for example, preferably 1 nm to 100 nm, more preferably 2 nm to 50 nm, even more preferably 5 nm to 30 nm.
The average diameter of the microfibers can be determined by scanning electron microscopy.

[Hydroxyapatite]
Any known hydroxyapatite can be _{used as} the _{hydroxyapatite} used in the present _invention . Also included are so-called deficient apatites in which (for example, one or more calcium elements) are deficient, and so-called substitutional apatites in which some elements in the structural formula are replaced with other elements. Examples of such deficient or substituted apatites include _Ca9 ( _PO4 ) ₆ ( _OH ) ₂ , Ca10( _PO4 ) _6F , and _Ca9Mg ( _PO4 ) ₆ (OH) _2. etc. Moreover, hydroxyapatite derived from biomaterials such as eggshells and seashells is also preferable.
Either before or after the step of drying the dispersion, or both, the hydroxyapatite may cover the entire surface of the carrier in the form of particles or a film, or may cover only part of the surface of the fiber portion. good. When the hydroxyapatite particles and/or film cover only a part of the carrier, there is no particular limitation on the area ratio. The ratio of the area of the hydroxyapatite particles and/or coating when only a part of the carrier is covered is, for example, preferably 1 to 100%, more preferably 5 to 60%, relative to the total surface area of the carrier. More preferably 10 to 40%.
The size of the hydroxyapatite particles is not particularly limited. The size of the hydroxyapatite particles, in the form of primary particles or secondary particles, is, for example, preferably 1 nm to 10 μm, more preferably 1 nm to 1 μm, even more preferably 5 nm to 100 nm. The size of hydroxyapatite particles can be determined by scanning electron microscopy.
In the present invention, hydroxyapatite may be used in the form of a suspension. That is, a hydroxyapatite suspension may be prepared, mixed with a carrier to obtain a dispersion, and dried.
The method for obtaining the hydroxyapatite suspension is described below. A phosphoric acid solution (additive liquid) is added to a calcium oxide suspension (additive liquid), or a calcium oxide suspension (additive liquid) is added to a phosphoric acid solution (additive liquid) to obtain the hydroxyapatite suspension. get the liquid

Calcium Oxide Suspension Calcium oxide is used as a starting material in preparing the hydroxyapatite suspension. In the above method, a suspension obtained by adding calcium oxide to a dispersion medium is used.
Commercially available calcium oxide can be used as the calcium oxide used in preparing the calcium oxide suspension. A calcium oxide suspension is obtained by adding calcium oxide to a dispersion medium. The amount of calcium oxide to be added is, for example, preferably 0.2 to 9.0 mol, more preferably 1.0 to 5.0 mol, more preferably 1.5 to 4.0 mol per liter of dispersion medium. It is more preferable to add 0.0 mol. By setting the amount to be added within the above range, better production efficiency of the hydroxyapatite dispersion can be achieved. As the dispersion medium, a dispersion medium selected from an alcohol dispersion medium such as methanol or ethanol and water can be used. From the viewpoint of production efficiency and work safety, it is more preferable to use water as the dispersion medium.

Phosphoric acid solution The phosphoric acid solution used in the method for obtaining the hydroxyapatite suspension can be prepared by dissolving commercially available phosphoric acid in a solvent selected from alcoholic solvents such as ethanol or methanol and water. From the viewpoint of production efficiency and work safety, it is preferable to use water as the solvent. The concentration of the phosphoric acid solution is preferably, for example, 0.5 to 10.0 M, more preferably 1.0 to 7.0 M, from the viewpoint of achieving better production efficiency of the hydroxyapatite suspension. More preferably, it is more preferably 2.0 to 5.0M.

Method for producing hydroxyapatite suspension As described above, the method for producing the hydroxyapatite dispersion comprises adding a phosphoric acid solution (addition liquid) to a calcium oxide suspension (addition liquid) or adding a phosphoric acid solution (addition liquid). liquid) to obtain the hydroxyapatite suspension by adding a calcium oxide suspension (addition liquid).
When adding the additive liquid to the additive liquid, any usual addition method can be used. A specific method includes, for example, a method in which the liquid to be added is placed in a container, and the liquid to be added is dropped into the container using a device such as a dropping funnel. When a calcium oxide suspension is used as the additive liquid, it is preferable to drop the calcium oxide suspension in a dropping funnel or the like while stirring.

The addition rate of the additive solution is, for example, 0.01 to 8.0 mol/h in terms of calcium oxide or phosphoric acid contained in the additive solution per 1 mol of calcium oxide or phosphoric acid contained in the additive solution. is preferably 0.05 to 5.0 mol/h, and even more preferably 0.1 to 2.0 mol/h. By setting the addition rate within the above numerical range, the formation of calcium phosphate compounds other than hydroxyapatite can be further suppressed.
The ratio of the total amount of calcium oxide in the calcium oxide suspension to the total amount of phosphoric acid in the phosphoric acid solution is preferably such that the molar ratio of calcium ions to phosphate ions is 10:6. Of course, it is also possible to change the above ratio depending on the reaction conditions, the structure of the target apatite (for example, the defect-type hydroxyapatite as described above), and the like. The molar ratio can be adjusted by adjusting the concentrations and amounts of the additive liquid and the additive liquid.
Further, an additive or the like may be added to the liquid to be added and/or the additive liquid for the purpose of obtaining the defective hydroxyapatite or substituted hydroxyapatite as described above.

Regarding the temperature conditions during addition, for example, the temperature of the liquid to be added and the liquid to be added are preferably in the range of 5 to 90°C, more preferably in the range of 15 to 60°C, and more preferably in the range of 20 to 40°C. It is more preferable that By setting the temperatures of the liquid to be added and the liquid to be added within the above range, the effects of suppressing the crystallization of hydroxyapatite as much as possible and allowing the reaction for obtaining hydroxyapatite to proceed more smoothly can be obtained.
It is also possible to add the additive liquid while stirring the liquid to be added.

The hydroxyapatite suspension obtained by the above production method has a crystallite size of 10 to 200 Å, preferably 30 to 150 Å, more preferably 30 to 150 Å, at a peak appearing at 2θ of 31.500 to 32.500° in X-ray structural analysis. contains 50-120 Å of hydroxyapatite. By including hydroxyapatite having a crystallite size within the above range, when the dispersion of the microfiber pieces and the hydroxyapatite is stirred, the hydroxyapatite contained in the hydroxyapatite dispersion is more strongly adhered to the fiber surface. adhere to. The crystallite size represents the size of crystal grains, and is a numerical value that serves as a measure of crystallinity. A larger crystallite size value means a higher crystallinity of the substance to be measured. The hydroxyapatite having a crystallite size within the above range is only non-crystallized (amorphous) hydroxyapatite or a mixture of non-crystalline hydroxyapatite and low-crystallized (low-crystalline) hydroxyapatite. means Here, "amorphous hydroxyapatite" means a substance from which a chart such as that shown in FIG. 1 can be obtained by X-ray analysis. In addition, the “low crystalline hydroxyapatite” in the present disclosure means that the chart obtained by X-ray analysis has a relatively high degree of peak separation compared to the chart obtained by X-ray analysis of crystalline hydroxyapatite. It is a low substance.

Amorphous hydroxyapatite and low-crystalline hydroxyapatite have a slight stickiness due to their low crystallinity. Amorphous hydroxyapatite and low-crystalline hydroxyapatite easily adhere to the surface of the carrier and between hydroxyapatites. Therefore, by using the hydroxyapatite suspension, hydroxyapatite particles and/or films adhering to the surface of the carrier can be formed without using an adhesive such as a resin. Since the surface of such hydroxyapatite particles and/or film is not coated with a resin, the adsorption performance of hydroxyapatite is not significantly impaired.

Amorphous hydroxyapatite tends to crystallize gradually over time. However, with the passage of time, it may become a mixture of amorphous hydroxyapatite and low-crystalline hydroxyapatite. The crystallite size can be measured, for example, by an X-ray analyzer model number: RINT2200V/PC manufactured by Rigaku Corporation.
The dispersion medium for the hydroxyapatite suspension may be any dispersion medium provided that it does not dissolve hydroxyapatite. However, the dispersion medium is selected from an alcohol dispersion medium such as methanol or ethanol and water, and water is particularly preferred as the dispersion medium from the viewpoint of safety.

The amount of hydroxyapatite contained in the hydroxyapatite suspension is, for example, preferably 0.01 to 0.8 mol, preferably 0.03 to 0.5 mol, per liter of the hydroxyapatite suspension. more preferably 0.05 to 0.25 mol. By setting the amount of hydroxyapatite contained in the hydroxyapatite suspension within the above range, a more appropriate amount of non-crystalline hydroxyapatite and/or low-crystalline hydroxyapatite can be adhered to the surface of the carrier, and the can further suppress the falling off of the amorphous hydroxyapatite and/or the low-crystalline hydroxyapatite. The amount of hydroxyapatite in the hydroxyapatite suspension is adjusted by adjusting the amount of calcium oxide in the calcium oxide suspension, the phosphoric acid concentration in the phosphoric acid solution, and the amounts of the calcium oxide suspension and the phosphoric acid solution. can be adjusted by Alternatively, the hydroxyapatite suspension obtained by the above method can be adjusted to the above range by diluting it with a dispersion medium such as water or by evaporating the dispersion medium to concentrate it.

[Dispersion medium]
In the present invention, any dispersion medium capable of dispersing the carrier and hydroxyapatite can be used as the dispersion medium for dispersing the carrier and hydroxyapatite. Those that do not dissolve hydroxyapatite and carriers are preferred. Examples of the dispersion medium include water, ethanol, methanol, and other alcohol dispersion mediums, which are listed as the dispersion medium usable for the hydroxyapatite suspension.

[Dispersion]
The dispersion used in the present invention is obtained by dispersing the above-mentioned hydroxyapatite and the above-mentioned carrier in a dispersion medium. The content of hydroxyapatite in the dispersion is, for example, preferably 0.01 to 0.8 mol, more preferably 0.03 to 0.5 mol, more preferably 0.01 to 0.8 mol, per liter of the dispersion. More preferably 05 to 0.25 mol.
The content of the carrier in the dispersion is, for example, preferably 0.1 to 50 g, more preferably 0.5 to 30 g, still more preferably 1 to 20 g, relative to 1 liter of dispersion medium.
The dispersion may contain a resin or the like for adhering the hydroxyapatite particles to the carrier. Examples of such resins include epoxy resins, urethane resins and acrylic resins. Moreover, the dispersion may further contain other components. Examples of such ingredients include, for example, dispersants, defoamers, leveling agents, and the like.

[Dispersion step]
The method for producing a hydroxyapatite-containing porous support of the present invention comprises a step of dispersing a carrier and hydroxyapatite or a carrier having hydroxyapatite attached to the surface thereof in a dispersion medium to obtain a dispersion.
The dispersion method is not particularly limited, and generally any device for stirring liquids can be used. Specific examples of such equipment include dispersers, homogenizers and mill mixers. However, in order to mix the hydroxyapatite particles and the fine fiber pieces in the dispersion liquid more uniformly and efficiently, and to easily apply the hydroxyapatite particles and/or film to the surface of the fine fiber pieces, a homogenizer is used. is more preferably used as the stirring means. The time required for stirring may vary depending on the amount of hydroxyapatite particles and microfiber pieces contained in the dispersion, the type of hydroxyapatite, the material of the fiber pieces, the means of stirring, and the like. For example, the treatment time with a homogenizer is preferably 5 to 180 minutes, more preferably 10 to 90 minutes, still more preferably 10 to 60 minutes. The stirring speed is, for example, 10-10000 rpm, preferably 100-1000 rpm, more preferably 100-500 rpm.

[Drying process]
The method for producing a hydroxyapatite-containing porous support of the present invention has a step of drying the dispersion. The drying step is a step of evaporating and removing the dispersion medium in the dispersion to obtain a porous support. Although the drying method is not particularly limited, vacuum drying and vacuum freeze-drying are preferred. Vacuum drying is performed using a commercially available vacuum drying apparatus such as System FDU1200 manufactured by Tokyo Rikaki. In the case of vacuum freeze-drying, drying is performed using a vacuum drying apparatus while cooling with liquid nitrogen or the like. The drying time may be appropriately adjusted depending on the amounts of hydroxyapatite and carrier.

[Porous support containing hydroxyapatite]
The hydroxyapatite-containing porous support of the present invention is a hydroxyapatite-containing porous support obtained by drying a dispersion containing a carrier and hydroxyapatite. In other words, it is a hydroxyapatite-containing porous support obtained by drying the dispersion, or a hydroxyapatite-containing porous support obtained by removing the dispersion. A dispersion containing a carrier and hydroxyapatite is a dispersion obtained by dispersing the carrier and hydroxyapatite in a dispersion medium.
As is clear from FIGS. 1 to 4, the hydroxyapatite-containing porous support of the present invention has a structure in which fine particles derived from hydroxyapatite adhere to the surface of a fine three-dimensional network structure formed by the carrier. As far as the inventors of the present invention know, no hydroxyapatite-containing porous support having such a structure has been reported.
The hydroxyapatite-containing porous support of the present invention has a large surface area, a complicated three-dimensional microstructure, and hydroxyapatite adhered to the surface, so that it has a high substance adsorptive power. In addition, since cells and the like enter into the three-dimensional fine structure, it is suitable as a cell scaffold, a bone regeneration material, and the like.

[Firing]
The hydroxyapatite-containing porous support of the present invention becomes a more useful material by firing. The firing conditions are preferably 100 to 2000°C, more preferably 300 to 1500°C, still more preferably 500 to 1500°C. Firing can be performed using a known device such as a rotary kiln. The sintered body obtained by sintering the hydroxyapatite-containing porous support of the present invention is excellent in terms of substance adsorption and porosity, can contain many cells, and is close to the original living body. Since it can provide a growth environment for cells and has high biocompatibility, it is suitable for cell scaffolds and bone regeneration materials.

Hereinafter, the contents of the present disclosure will be described in more detail with reference to examples. It goes without saying that the examples do not limit the scope of the present disclosure.

[Example 1]
1. Materials used Carrier: Rheocrysta C-2SP manufactured by Daiichi Kogyo Seiyaku Co., Ltd. (suspension with a cellulose nanofiber concentration of 2%)
Hydroxyapatite: Eggshell-derived hydroxyapatite (20 w/w%)

2. Step of dispersing hydroxyapatite to obtain a dispersion (1) Add water to 250 g of Rheocrysta to make 800 g, and put it in a bead mill (bead mill: Nitto Kagaku Co., Ltd. desktop pot mill rotary table ANZ-51D, beads: zirconia beads 8 mm ). The stirring conditions were 300 rpm for 5 hours.
(2) Then, 25 g of hydroxyapatite (dispersed in water; concentration: 20 w/w %) was added to (1) to make 200 g by adding water, and the mixture was stirred with a bead mill. The stirring conditions were 300 rpm for 10 hours.

3. Drying Step (3) The stirred dispersion was subjected to vacuum freeze-drying for 20 hours in a vacuum dryer (system FDU1200 manufactured by Tokyo Rika Kikai Co., Ltd.). As a result, a white porous support was obtained.

[Comparative Example 1]
After adding Rheocrysta to water and stirring in the same manner as in Example 1, freeze-drying was performed without adding the hydroxyapatite suspension.

The obtained dry porous support was observed with a scanning electron microscope. Electron micrographs taken are shown in FIGS. 1 to 6. FIG.

As is clear from the comparison between FIGS. 1 to 4 (photographs of Example 1) and FIGS. 5 to 6 (photographs of Comparative Example 1), a dispersion containing a carrier composed of hydroxyapatite and cellulose nanofibers The hydroxyapatite-containing porous support of the present invention obtained by freeze-drying was observed to have a peculiar structure in which fine particles were attached to the surface of a complicated three-dimensional network.

Claims (3)

A step of dispersing a carrier (excluding collagen) and hydroxyapatite or a carrier (excluding collagen) having hydroxyapatite attached to the surface thereof in a dispersion medium to obtain a dispersion, and a step of drying the dispersion;
A method for producing a hydroxyapatite-containing porous support comprising and a method for producing a hydroxyapatite-containing porous support containing one or more selected from the group consisting of mixtures of two or more of these . 2. The method for producing a hydroxyapatite-containing porous support according to claim 1, wherein the step of drying the dispersion is a step of drying the dispersion by vacuum drying. 2. The method for producing a hydroxyapatite-containing porous support according to claim 1, wherein the step of drying the dispersion is a step of drying the dispersion by vacuum freeze-drying.