JPWO2021014994A1 - Bone regenerating agent and how to use it - Google Patents

Abstract

Provided is a bone regenerating agent capable of repairing a bone defect while suppressing residual bone. The bone regenerating agent contains zinc carbonate hydroxide containing hydrogen kite. The zinc hydroxide hydroxide preferably has a Zn2 + ion elution amount of 0.1 mass ppm or more after the elution test and a pH of 7.2 or more and less than 8.3 after the elution test. It is preferable that some carbonate ions of the hydrozincite are replaced with sulfate ions or chloride ions. The bone regenerating agent is preferably applied to the bone defect portion.

Description

The present invention relates to a bone regenerating agent and a method of using the same.

Conventionally, a bone regenerating agent containing β-tricalcium phosphate (TCP) is known (see Patent Document 1).

JP-A-2017-61419

The conventional bone regenerating agent (β-TCP) may remain in the bone defect. The remaining bone regenerating agent is a foreign substance to the living body. During bone remodeling, bone resorption (formation of resorption pores) by osteoclasts can become active around the remaining bone remodeling agent. Therefore, the bone regenerating agent is required not to remain in the bone defect portion.
Therefore, an object of the present invention is to provide a bone regenerating agent capable of repairing a bone defect while suppressing residual bone.

As a result of diligent studies, the present inventors have found that the above object can be achieved by adopting the following configuration, and have completed the present invention.
That is, the present invention provides the following [1] to [8].
[1] A bone regenerating agent containing zinc carbonate containing hydrogen kite.
[2] The zinc carbonate hydroxide has a Zn ²⁺ ion elution amount of 0.1 mass ppm or more after the elution test and a pH of 7.2 or more and less than 8.3 after the elution test [1]. The bone regenerating agent described in.
[3] The bone regenerating agent according to the above [1] or [2], wherein a part of the carbonate ion of the hydrozincite is replaced with a sulfate ion.
[4] The bone regenerating agent according to the above [1] or [2], wherein a part of the carbonate ions of the hydrodin kite is replaced with chloride ions.
[5] The bone regenerating agent according to any one of the above [1] to [4], which is applied to a bone defect portion.
[6] The bone regenerating agent according to any one of the above [1] to [5], which further contains a biocompatible polymer and is in the form of a paste.
[7] The bone regenerating agent according to the above [6], wherein the biocompatible polymer is polymethylmethacrylate.
[8] A method for using a bone regenerating agent, wherein the bone regenerating agent according to any one of [1] to [7] above is implanted in a bone defect and covered with a residual periosteum.

According to the present invention, the bone defect can be repaired while suppressing the residual.

It is a graph which shows the XRD pattern of zinc hydroxide containing the hydrogen kite of Examples 1 to 3. FIG.

[Bone regeneration agent]
The bone regenerating agent of the present invention is a bone regenerating agent containing zinc hydroxide (hereinafter, also simply referred to as “zinc hydroxide”) containing hydrogen kite.
In the bone regenerating agent of the present invention, zinc ions (Zn ²⁺ ions) generated by the dissolution of zinc hydroxide hydroxide promote the differentiation of hepatocytes in bone marrow into osteoblasts, and the osteoblasts are produced. It is thought to induce calcification.

<Zinc hydroxide containing hydrozinc kite>
Hydrozinc kites are, for example, Zn ₅ (CO ₃ ) ₂ (OH) ₆ or Zn ₅ (CO ₃ ) ₂ (OH) ₆ · nH ₂ O (n is 0 to 6, preferably 2). expressed.

<< First aspect >>
The zinc hydroxide hydroxide (first aspect) containing such a hydrogen kite is preferably zinc carbonate hydroxide represented by the following formula (1), for example. At this time, the molar ratio of _{Zn to CO 3 (Zn / CO 3} ) is preferably 2.5 to 3.3.
Zn _{4 to 6} (CO ₃ ) _{1 to 3} (OH) _{5 to 6}・ nH ₂ O (1)
However, in the formula (1), n is 0 to 6.

The amount of hydrogen kite contained in zinc carbonate hydroxide is preferably 60% by mass or more, more preferably 80% by mass or more, and further preferably 95% by mass or more.

<< Second aspect >>
Hydro Jin zinc carbonate hydroxide containing kite (second embodiment), for example, hydro-Jin kite part of carbonate ions (CO 3 ^_2-) may be replaced by sulfate ions (SO 4 ^_2-). If the sulfur (S) content is not more than a predetermined value, the mineral phase is attributed to hydrozincite.
The zinc hydroxide hydroxide of the second aspect is preferably zinc carbonate hydroxide containing 0.1% by mass or more and less than 1.5% by mass of sulfur (S), and is represented by the following formula (2). More preferably, it is zinc hydroxide. At this time, the molar ratio of Zn to ((1-x) CO ₃ + x (SO ₄ )) (Zn / ((1-x) CO ₃ + x (SO ₄ ))) is 2.5 to 3.3. Is preferable.
Zn _{4 to 6} ((1-x) CO ₃ + x (SO ₄ )) _1-3 (OH) _{5 to 6} · nH ₂ O (2)
However, in the formula (2), n is 0 to 6, and x is 0.005 to 0.1.

<< Third aspect >>
Hydro Jin zinc carbonate hydroxide containing Kite (third embodiment), for example, hydro-Jin kite part of carbonate ions (CO 3 ^_2-) is chloride ion - may be substituted with ^(Cl). If the chlorine (Cl) content is not more than a predetermined value, the mineral phase is attributed to hydrozincite.
The zinc hydroxide hydroxide of the third aspect is preferably zinc carbonate hydroxide containing 0.05% by mass or more and less than 1% by mass of chlorine (Cl), and is represented by the following formula (3). Zinc is more preferred. At this time, Zn and _{((1-x) CO 3} + xCl) the molar ratio of _{(Zn / ((1-x} ) CO 3 + xCl)) is preferably 2.5 to 3.3.
Zn _{4 to 6} ((1-x) CO ₃ + xCl) _{1 to 3} (OH) _{5 to 6}・ nH ₂ O (3)
However, in the formula (3), n is 0 to 6, and x is 0.005 to 0.1.

That is, the "zinc hydroxide containing hydrozincite" (first, second and third aspects) in the present invention can be said to be "zinc hydroxide containing a hydrozincite-like compound", but without distinction. , "Zinc hydroxide containing hydrogen kite" (or simply "zinc hydroxide").
The first aspect or the second aspect is preferable because the residual can be further suppressed.
The second or third aspect is preferable, and the second aspect is more preferable, because the amount ^{of Zn 2+} ions eluted after the dissolution test described later is large.

<< Characteristics after dissolution test >>
^{The amount of Zn 2+} ion eluted after the elution test of zinc carbonate containing hydrogen kite is preferably 0.1 mass ppm or more, and the pH after the elution test is 7.2 or more and less than 8.3. preferable.
^{The amount of Zn 2+} ions eluted after the dissolution test is more preferably 0.5 mass ppm or more, further preferably 1.0 mass ppm or more. ^{The amount of Zn 2+} ions eluted after the dissolution test is more preferably 5.0 mass ppm or more, further preferably 10.0 mass ppm or more, and particularly preferably 15.0 mass ppm or more, for the reason that the bone regeneration area becomes large. It is preferable, and 20.0 mass ppm or more is most preferable.
On the other hand, from the viewpoint of suppressing cytotoxicity, the ^{amount of Zn 2+} ions eluted after the elution test is preferably 80 mass ppm or less, more preferably 50 mass ppm or less, and even more preferably 30 mass ppm or less.
The pH after the dissolution test is more preferably 8.2 or less, further preferably 8.0 or less, and particularly preferably 7.8 or less.

The characteristics after the dissolution test are determined as follows.
First, zinc hydroxide containing hydrogen kite is stirred in physiological saline at 37 ° C. using a rotor at 500 rpm for 3 hours. The concentration of zinc hydroxide containing hydrozinc hydroxide in physiological saline is 20 g / L. This is the dissolution test.
After the dissolution test (that is, after stirring for 3 hours), the ^{amount of Zn 2+} ions eluted in physiological saline (unit: mass ppm) is measured using an ICP emission spectrometer (ICPE-9000, manufactured by Shimadzu Corporation). .. The value obtained by measurement is the amount ^{of Zn 2+} ions eluted after the elution test of zinc hydroxide containing hydrogen kite.
The pH of the physiological saline solution after the dissolution test is measured, and the obtained value is taken as the pH after the dissolution test of zinc hydroxide containing hydrogen hydroxide.

《Average particle size》
The average particle size of the secondary particles of zinc hydroxide contained in the bone regenerating agent of the present invention is preferably 5 to 30 μm, more preferably 10 to 20 μm.
The average particle size of the secondary particles is the particle size (D50) at which the cumulative frequency of the particle size distribution is 50% by volume, which is determined by using a laser diffraction / scattering type particle size distribution meter (CILAS 1064L, manufactured by Cirrus). ..

<Other ingredients>
If necessary, the bone regenerating agent of the present invention may further contain a pharmaceutically acceptable carrier (hereinafter, simply referred to as “carrier”).
Examples of the carrier include solvents such as organic solvents and inorganic solvents, and specific examples thereof include water, physiological saline, alcohols, polyhydric alcohols, and mixtures thereof.

For example, when the bone regenerating agent of the present invention contains a solvent as a carrier, a thickener or the like is further added to the bone regenerating agent of the present invention to process the bone regenerating agent of the present invention into a gel or paste. The handleability may be improved.
Examples of the thickener include biocompatible polymers, and specific examples thereof preferably include polymethylmethacrylate (PMMA) and the like.
In addition, as the carrier, for example, the carriers described in paragraphs [0027] to [0030] of International Publication No. 2016/199907 can be used.

The bone regenerating agent of the present invention may contain other additives depending on the actual use.
Examples of other additives include, but are not limited to, moisturizers, antioxidants, preservatives, anti-inflammatory agents, whitening agents, blood circulation promoters, anti-fat leak agents, thickeners, pH adjusters and the like. Specific examples thereof include the components described in paragraphs [0031] to [0034] of International Publication No. 2016/199907.

The form of the bone regenerating agent of the present invention may be a liquid preparation containing a carrier such as physiological saline as a solvent, or a powder form containing no solvent (carrier). Further, it may be in a thickened form (for example, a paste form) or a gelled form, which is intermediate between the two.

[How to use bone regenerating agent]
Examples of the method of using the bone regenerating agent of the present invention include a method of implanting (filling) the bone regenerating agent of the present invention in a bone defect portion. At this time, the site (implanted site) in which the bone regenerating agent of the present invention is implanted may be covered with residual periosteum or the like.
After implanting the bone regenerating agent of the present invention in the bone defect portion, a predetermined period of time elapses.
As a result, the tissue regenerates in the bone defect part and becomes a new bone part. That is, the bone defect is repaired. At this time, the bone regenerating agent of the present invention is unlikely to remain in the bone defect portion.

[Manufacturing method of bone regenerating agent]
Examples of the method for producing the bone regenerating agent of the present invention include a method of obtaining zinc hydroxide containing a hydrozincite and combining it with a carrier or the like, if necessary.
Zinc hydroxide containing hydrogen kite is preferably obtained by a precipitate formation reaction as described below.

<Precipitate formation reaction>
Zinc hydroxide hydroxide containing hydrogen kite is preferably obtained by a precipitate formation reaction (alkali precipitation method) using a zinc source, a carbonic acid source and an alkali.

Examples of the zinc source include zinc sulfate (ZnSO ₄ ), zinc chloride (ZnCl ₂ ), zinc acetate (Zn (CH ₃ COO) ₂ ), zinc nitrate (Zn (NO ₃ ) ₂ ) and the like.
When the zinc hydroxide hydroxide of the first aspect is obtained, it is preferable to use zinc nitrate.
When the zinc hydroxide hydroxide of the second aspect is obtained, it is preferable to use zinc sulfate.
When the zinc hydroxide hydroxide of the third aspect is obtained, it is preferable to use zinc chloride.

Examples of the carbonic acid source include ammonium carbonate ((NH ₄ ) ₂ CO ₃ ), sodium carbonate (Na ₂ CO ₃ ), sodium hydrogen carbonate (NaH (CO ₃ )), and the like, and sodium hydrogen carbonate is preferable.
When obtaining the zinc hydroxide of the second aspect, ammonium sulfate can be used in combination with a carbonate source.
When obtaining zinc hydroxide of the third aspect, ammonium chloride can be used in combination with a carbonate source.

Examples of the alkali include ammonia (NH ₃ ), sodium hydroxide (NaOH) and the like, and sodium hydroxide is preferable.

The zinc source, carbonic acid source and alkali are preferably used in the form of an aqueous solution.

In the precipitate formation reaction, specifically, for example, an aqueous solution of a zinc source (an acidic aqueous solution) is dropped into an aqueous solution of a carbon dioxide source, and during this dropping, an aqueous solution of an alkali is sent to the aqueous solution of the carbon dioxide source to carry out carbon dioxide. It is preferable to maintain the pH of the aqueous solution of the source in a certain range, and after the dropping of the aqueous solution of the zinc source is completed, stir (stirring and curing) for 10 to 30 hours to obtain a reaction solution containing a precipitate.
The pH can vary depending on the type and concentration of the carbonic acid source and zinc source used, but is preferably 6.5 or more and less than 9.5, and more preferably 7.0 or more and less than 9.5. That, ^{Zn 2+} ion, _CO ^{3 2-ion} and OH ^- ions (and, _SO ^{4 2-ionic} or,, Cl ^- may contain an ion), but obtain a precipitate by reaction, pH is within the above range It is preferable to use the precipitate obtained in a controlled reaction field.

The molar ratio of zinc to carbonic acid (zinc: carbonic acid) in the zinc source and carbonic acid source is preferably 5: 2.
In the second and third aspects, the molar ratio of zinc to anion (zinc: anion) is preferably 5: 2. The molar ratio of this anion (ie, CO ₃ ^2- : SO ₄ ^2- and CO ₃ ^2- : Cl ⁻ ) is appropriately set in consideration of the S content and Cl content of the obtained zinc hydroxide. Will be done.
The concentration of the zinc source aqueous solution is preferably 0.01 mol / L or more, more preferably 0.03 mol / L or more. On the other hand, 3 mol / L or less is preferable, and 1 mol / L or less is more preferable.
The concentration of the aqueous solution of the carbonic acid source is preferably 0.004 mol / L or more, more preferably 0.012 mol / L or more. On the other hand, 1.2 mol / L or less is preferable, and 0.4 mol / L or less is more preferable.
The reaction temperature is preferably 15 ° C. or higher. On the other hand, 60 ° C. or lower is preferable, and 40 ° C. or lower is more preferable.

After the precipitate formation reaction, for example, the reaction solution containing the precipitate is subjected to solid-liquid separation by suction filtration or centrifugation, and the obtained precipitate is washed with pure water or distilled water, and then vacuum dried. As a result, a zinc carbonate powder is obtained. After that, an appropriate particle size may be used by a known method.
The obtained zinc hydroxide powder may contain unreacted substances (raw materials), reaction by-products, impurities mixed from the raw materials, and the like.

Hereinafter, the present invention will be specifically described with reference to Examples. However, the present invention is not limited to the following examples.

<Example 1>
A 0.08 mol / L sodium hydrogen carbonate aqueous solution (500 mL) was prepared in the reaction vessel. Separately, a 0.1 mol / L zinc nitrate aqueous solution (1000 mL) was prepared. Further, a 30% by mass sodium hydroxide aqueous solution was prepared as a pH adjusting liquid.
The pH electrode connected to the pH controller was charged into the aqueous sodium hydrogen carbonate solution in the reaction vessel. A zinc nitrate aqueous solution and a sodium hydroxide aqueous solution were added dropwise to the sodium hydrogen carbonate aqueous solution using a pump controlled on and off by this pH controller. During the dropping, the pH of the aqueous sodium hydrogen carbonate solution was maintained at 7.0. During the dropping, the aqueous sodium hydrogen carbonate solution was stirred with a rotor. After all the zinc nitrate aqueous solution was added dropwise, the mixture was further stirred for 16 hours and cured. The reaction temperature (environmental temperature at the time of dropping and curing) was 25 ° C. In this way, a reaction solution containing a precipitate was obtained.
The obtained reaction solution was separated into solid and liquid by centrifugation. The obtained solid (precipitate) was washed by repeating washing with water and centrifugation three times. The washed precipitate was vacuum dried to obtain the zinc hydroxide powder of the first aspect described above. The obtained powder was used as the bone regenerating agent of Example 1.

The zinc carbonate powder obtained in Example 1 was XRD-measured using an XRD device (D8ADVANCE manufactured by Bruker) (the same applies to Examples 2 and 3 described later). The result of this XRD measurement is shown in FIG. Looking at the XRD pattern in FIG. 1, a peak showing hydrozincite is observed.

<Example 2>
The carbonated water of the second aspect described above was described in the same manner as in Example 1 except that a 0.1 mol / L zinc sulfate aqueous solution (1000 mL) was used instead of the 0.1 mol / L zinc nitrate aqueous solution (1000 mL). A zinc oxide powder was obtained. The obtained powder was used as the bone regenerating agent of Example 2. Looking at the XRD pattern in FIG. 1, a peak showing hydrozincite is observed.

<Example 3>
The carbonated water of the third aspect described above was described in the same manner as in Example 1 except that a 0.1 mol / L zinc chloride aqueous solution (1000 mL) was used instead of the 0.1 mol / L zinc nitrate aqueous solution (1000 mL). A zinc oxide powder was obtained. The obtained powder was used as the bone regenerating agent of Example 3. Looking at the XRD pattern in FIG. 1, a peak showing hydrozincite is observed.

<Characteristics after dissolution test>
For the zinc hydroxide powder (bone regenerating agent) of Examples 1 to 3, the Zn ²⁺ ion elution amount and pH after the elution test were determined by the above-mentioned method. The results are shown in Table 1 below.

<Evaluation>
Animal experiments were carried out using the zinc carbonate powder (bone regenerating agent) of Example 2.
Further, as the bone regenerating agent of Comparative Example 1, β-TCP (Osferion (registered trademark), manufactured by Olympus Terumo Biomaterials), which is a commercially available bone filling material, was used.

In animal experiments, a bone regenerating agent was implanted in the rib defect of Zenno Premium Pig. Zenno Premium Pig was under complete anesthesia, and the implant site was locally anesthetized with dental xylocaine.
More specifically, both ventral sides of the Zenno Premium Pig were shaved to expose the ribs, and a bone defect portion of about 2 mm × 14 mm was prepared on the exposed ribs using Volver Vmax. 0.005 g of a bone regenerating agent was implanted in the bone defect, covered with the residual periosteum, and then closed. The burial period was 4 weeks.
After 4 weeks, Zenno Premium Pig was anesthetized with isoflurane, exsanguinated, and then a part of the ribs (implantation site and its surroundings) was removed. Bone regeneration was evaluated after trimming the soft tissue attached to the removed ribs.

《Microscopic evaluation》
The implantation site (bone defect) of the excised rib was observed using a system microscope (BX53 manufactured by Olympus Corporation).
As a result, in Comparative Example 1, although tissue regeneration was confirmed in the bone defect portion, a large amount of bone regeneration agent (β-TCP) remained when the bone defect portion was cross-sectionally viewed.
On the other hand, in Example 2, tissue regeneration was confirmed in the bone defect portion, and no residual bone regenerating agent (zinc hydroxide) was observed.

《Histological evaluation》
Hematoxylin and eosin (HE) staining and Masson's trichrome (MT) staining were performed on the implanted site (bone defect) of the excised ribs.
As a result, in Comparative Example 1, the bone defect portion in which the bone regenerating agent (β-TCP) was implanted was calcified, but a large number of absorption holes were present and inflammatory cells were observed.
On the other hand, in Example 2, no residual bone regenerating agent (zinc hydroxide) was observed in the bone defect, and thick calcified bone was formed.

When the bone regenerating agents of Examples 1 and 3 were used, the same results as when the bone regenerating agent of Example 2 was used were obtained. However, while the bone regenerating agent of Example 3 remained in an extremely small amount, the bone regenerating agent of Examples 1 and 2 did not remain at all.
There was also a difference in the bone regeneration area. That is, the area of tissue regeneration in the bone defect portion was larger in the order of Comparative Example 1, Example 1, Example 3, and Example 2 (the bone regeneration area of Example 2 was the largest).

<Summary of evaluation results>
From the above results, the bone regenerating agent (β-TCP) of Comparative Example 1 remained in the rib defect portion, whereas the bone regenerating agent of Examples 1 to 3 remained in the rib defect while suppressing the residual. It became clear that the part could be repaired.
Since zinc hydroxide containing hydrogen kite is eluted by contact with body fluid, it is considered that all of it is eluted in the ribs having abundant bone marrow. Therefore, zinc hydroxide containing hydrogen kite is suitable as a bone regenerating agent.

Claims (8)

A bone regenerating agent containing zinc carbonate containing hydroxyzine kite. The bone according to claim 1, wherein the zinc carbonate hydroxide has a Zn ²⁺ ion elution amount of 0.1 mass ppm or more after the elution test and a pH of 7.2 or more and less than 8.3 after the elution test. Regenerative agent. The bone regenerating agent according to claim 1 or 2, wherein a part of the carbonate ion of the hydrozincite is replaced with a sulfate ion. The bone regenerating agent according to claim 1 or 2, wherein a part of the carbonate ion of the hydrozincite is replaced with a chloride ion. The bone regenerating agent according to any one of claims 1 to 4, which is applied to a bone defect portion. In addition, it contains a biocompatible polymer,
The bone regenerating agent according to any one of claims 1 to 5, which is in the form of a paste. The bone regenerating agent according to claim 6, wherein the biocompatible polymer is polymethylmethacrylate. A method for using a bone regenerating agent, wherein the bone regenerating agent according to any one of claims 1 to 7 is implanted in a bone defect and covered with a residual periosteum.

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