JP2010110404A - Artificial bone material - Google Patents
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Abstract
【課題】ＯＣＰは骨芽細胞の分化を促進する反面、生体内における分解吸収速度が比較的速く、骨芽細胞が再生すべき足場を確保できないという問題があり、ここに本発明が解決せんとする課題がある。
【解決手段】本発明は、上記の如き実情に鑑みこれらの課題を解決することを目的として創作されたものであって、ＯＣＰと、ＡＣＰ又は低結晶性のＯＣＰからなる混合燐酸カルシウムを含むことを特徴とする人工骨材である。この混合燐酸カルシウムは、主に（１）ＯＣＰにＡＣＰ、ＤＣＰ、ＤＣＰＤ、低結晶性のＨＡ又は低結晶性のＯＣＰを析出する、（２）ＯＣＰとＡＣＰ、ＤＣＰ、ＤＣＰＤ、低結晶性のＨＡ又は低結晶性のＯＣＰが同時に製造されるような条件で製造する、（３）ＡＣＰ、ＤＣＰ又はＤＣＰＤを部分的にＯＣＰとなるよう結晶化する、（４）ＯＣＰを部分的に低結晶化する等の方法で製造され得る。
【選択図】図２[Problem] While OCP promotes differentiation of osteoblasts, it has a problem that the rate of degradation and absorption in vivo is relatively fast, and it is impossible to secure a scaffold for osteoblasts to regenerate. There is a problem to do.
The present invention has been made in order to solve these problems in view of the circumstances as described above, and includes OCP and a mixed calcium phosphate composed of ACP or low crystalline OCP. Is an artificial aggregate characterized by This mixed calcium phosphate mainly comprises (1) precipitation of ACP, DCP, DCPD, low crystalline HA or low crystalline OCP on OCP, (2) OCP and ACP, DCP, DCPD, low crystalline HA Or (3) crystallize ACP, DCP or DCPD to partially become OCP, or (4) partially crystallize OCP. Or the like.
[Selection] Figure 2
Description
本発明は、第８燐酸カルシウム（Ｃａ８Ｈ２（ＰＯ４）６・５Ｈ２Ｏ）と、非晶質燐酸カルシウム（Ｃａ３（ＰＯ４）２・ｎＨ２Ｏ）、第２燐酸カルシウム無水和物（ＣａＨＰＯ４）、第２燐酸カルシウム２水和物（ＣａＨＰＯ４・２Ｈ２Ｏ）、低結晶性のハイドロキシアパタイト（Ｃａ１０（ＰＯ４）６（ＯＨ）２）又は低結晶性の第８燐酸カルシウム（Ｃａ８Ｈ２（ＰＯ４）６・５Ｈ２Ｏ）からなる混合燐酸カルシウムを含む人工骨材に関する。 The present invention relates to an eighth calcium phosphate (Ca 8 H 2 (PO 4 ) 6 · 5H 2 O), an amorphous calcium phosphate (Ca 3 (PO 4 ) 2 · nH 2 O), and a dibasic calcium phosphate anhydrate. Product (CaHPO 4 ), dicalcium phosphate dihydrate (CaHPO 4 .2H 2 O), low crystalline hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ) or low crystalline eighth phosphoric acid calcium (Ca 8 H 2 (PO 4 ) 6 · 5H 2 O) artificial bone material including a mixing calcium phosphate consisting of about.
今日、ハイドロキシアパタイト（Ｃａ１０（ＰＯ４）６（ＯＨ）２：以下「ＨＡ」と略記する）の前駆体である第８燐酸カルシウム（Ｃａ８Ｈ２（ＰＯ４）６・５Ｈ２Ｏ：以下「ＯＣＰ」と略記する）は、生理的環境下で準安定相であり、骨形成にとって適度な溶解度を有することで、優れた骨伝導能を示すこと（非特許文献１）、破骨細胞による吸収性を有すること（非特許文献２）、そして用量依存的に骨芽細胞の分化を促進すること（非特許文献３）が知られている。また、ＨＡの前駆体である非晶質燐酸カルシウム（Ｃａ３（ＰＯ４）２・ｎＨ２Ｏ：以下「ＡＣＰ」と略記する）についても、準安定相であるゆえ一部溶解してＯＣＰに結晶化することから、ＯＣＰと同様の性質があることが報告されている（非特許文献１及び非特許文献４参照）。さらにまた、同じくＨＡの前駆体である第２燐酸カルシウム無水和物（ＣａＨＰＯ４：以下「ＤＣＰ」と略記する）、第２燐酸カルシウム２水和物（ＣａＨＰＯ４・２Ｈ２Ｏ：以下「ＤＣＰＤ」と略記する）についてもＯＣＰと同様の性質があることが報告されている（非特許文献１及び非特許文献５参照）。 Today, hydroxyapatite (Ca 10 (PO 4) 6 (OH) 2: hereinafter abbreviated as "HA") Eighth calcium phosphate is a precursor of (Ca 8 H 2 (PO 4 ) 6 · 5H 2 O: less (Abbreviated as “OCP”) is a metastable phase in a physiological environment and exhibits an excellent osteoconductivity by having an appropriate solubility for bone formation (Non-patent Document 1). It is known to have resorbability (Non-patent document 2) and to promote osteoblast differentiation in a dose-dependent manner (Non-patent document 3). Also, amorphous calcium phosphate (Ca 3 (PO 4 ) 2 .nH 2 O: hereinafter abbreviated as “ACP”), which is a precursor of HA, is partially metastable and dissolved in OCP. Since it crystallizes, it has been reported that it has properties similar to OCP (see Non-Patent Document 1 and Non-Patent Document 4). Furthermore, the second calcium phosphate anhydrate (CaHPO 4 : hereinafter abbreviated as “DCP”) and the second calcium phosphate dihydrate (CaHPO 4 .2H 2 O: hereinafter “DCPD”) which are also precursors of HA. (Hereinafter abbreviated as ")" has been reported to have the same properties as OCP (see Non-Patent Document 1 and Non-Patent Document 5).
ところで、ＯＣＰは骨芽細胞の分化を促進する反面、生体内における分解吸収速度が比較的速く、ラット頭蓋冠骨欠損では生体内吸収に合わせ、骨と置換していく傾向があるが、大腿骨のような長管骨の骨髄内では、生体内吸収に合わせ、形成された新生骨も吸収を受けてしまう（非特許文献２参照）。骨置換性の観点から整形外科領域における骨再生を目標とした場合には、現状の骨再生能を維持したまま、あるいは、より自家骨に近い骨再生能を獲得した新規な性質を有するＯＣＰの開発が渇望されてきた。しかし、現状では、骨髄内では骨芽細胞が再生すべき足場を確保できないという問題がある。しかし、材料の吸収性が小さいと骨再生能に劣ることが一般的であり（非特許文献２参照）、ここに本発明が解決せんとする課題がある。 By the way, OCP promotes osteoblast differentiation, but has a relatively high rate of degradation and resorption in vivo. In rat calvarial bone defects, there is a tendency to replace bone with the resorption in vivo. In the bone marrow of such long bones, the formed new bone is also absorbed in accordance with in vivo resorption (see Non-Patent Document 2). When bone regeneration in the orthopedic field is targeted from the viewpoint of bone replacement, the OCP has a novel property that maintains the current bone regeneration ability or has acquired bone regeneration ability closer to that of autologous bone. Development has been craving. However, at present, there is a problem that a scaffold for the regeneration of osteoblasts cannot be secured in the bone marrow. However, when the resorbability of the material is small, the bone regeneration ability is generally inferior (see Non-Patent Document 2), and there is a problem that the present invention does not solve.
本発明は、上記の如き実情に鑑みこれらの課題を解決することを目的として創作されたものであって、請求項１の発明は、ＯＣＰと、ＡＣＰ、ＤＣＰ、ＤＣＰＤ、低結晶性のＨＡ又は低結晶性のＯＣＰからなる混合燐酸カルシウムを含むことを特徴とする人工骨材である。
請求項２の発明は、混合燐酸カルシウムは、ＯＣＰにＡＣＰ、ＤＣＰ、ＤＣＰＤ、低結晶性のＨＡ又は低結晶性のＯＣＰを析出して製造され得るものであることを特徴とする請求項１に記載の人工骨材である。
請求項３の発明は、混合燐酸カルシウムは、ＯＣＰと、ＡＣＰ、ＤＣＰ、ＤＣＰＤ又は低結晶性のＨＡが同時に製造されるような条件で製造され得るものであることを特徴とする請求項１に記載又は低結晶性のＯＣＰの人工骨材である。
請求項４の発明は、混合燐酸カルシウムは、ＡＣＰ、ＤＣＰ、ＤＣＰＤを部分的にＯＣＰとなるよう結晶化して製造され得るものであることを特徴とする請求項１に記載の人工骨材である。
請求項５の発明は、混合燐酸カルシウムは、ＯＣＰを部分的に低結晶化して製造され得るものであることを特徴とする請求項１に記載の人工骨材である。
The present invention has been created in view of the above circumstances and has been created for the purpose of solving these problems. The invention of claim 1 is directed to OCP, ACP, DCP, DCPD, low crystalline HA or An artificial aggregate comprising mixed calcium phosphate made of low crystalline OCP.
The invention of claim 2 is characterized in that the mixed calcium phosphate can be produced by precipitating ACP, DCP, DCPD, low crystalline HA or low crystalline OCP on OCP. It is the described artificial aggregate.
The invention of claim 3 is characterized in that the mixed calcium phosphate can be produced under conditions such that OCP and ACP, DCP, DCPD or low crystalline HA are produced simultaneously. Description or low crystalline OCP artificial bone.
The invention according to claim 4 is the artificial bone material according to claim 1, wherein the mixed calcium phosphate can be manufactured by crystallizing ACP, DCP, and DCPD to partially become OCP. .
The invention according to claim 5 is the artificial bone material according to claim 1, wherein the mixed calcium phosphate can be manufactured by partially crystallizing OCP.
本発明の人工骨材は、骨芽細胞の分化促進というＯＣＰの特性を有するだけでなく、その機能をさらに向上させ、その上で、生体内における耐分解吸収性を有し、骨芽細胞が再生すべき足場を確保することができる。 The artificial bone material of the present invention not only has the OCP characteristic of promoting osteoblast differentiation, but further improves its function, and further has decomposition and absorption resistance in vivo. A scaffold to be regenerated can be secured.
本発明の発明者等は、ＯＣＰと、ＡＣＰ、ＤＣＰ、ＤＣＰＤ、低結晶性のＨＡ又は低結晶性のＯＣＰからなる混合燐酸カルシウムを人工骨材として使用したところ、骨芽細胞の分化促進というＯＣＰの特性を有するだけでなく、その機能をさらに向上させ、その上で、生体内における耐分解吸収性を有し、骨芽細胞が再生すべき足場を確保することができたことを見出し、本発明を完成するに至った。 The inventors of the present invention used OCP and mixed calcium phosphate composed of ACP, DCP, DCPD, low crystalline HA or low crystalline OCP as an artificial bone material. In addition to having the above characteristics, it was found that the function was further improved, and furthermore, it was resistant to decomposition and absorption in vivo, and a scaffold to be regenerated by osteoblasts could be secured. The invention has been completed.
本発明の人工骨材は、ラットの脛骨骨幹端内部に埋植した場合、埋植５６日目における骨形成率は約３０％である一方、生体内におけるこの燐酸カルシウムの残存率は約４０％と比較的多く残っていた（図３参照）。つまり、本発明の人工骨材は、骨芽細胞の分化促進というＯＣＰの特性を有しつつも、生体内における耐分解吸収性を有し、骨芽細胞が再生すべき足場を確保することができるのである。 When the artificial aggregate of the present invention is implanted in the metaphysis of a rat tibia, the bone formation rate on the 56th day after implantation is about 30%, while the residual rate of this calcium phosphate in vivo is about 40%. A relatively large amount remained (see FIG. 3). In other words, the artificial bone material of the present invention has OCP characteristics of promoting osteoblast differentiation, has anti-degradable absorbability in vivo, and can secure a scaffold for osteoblasts to regenerate. It can be done.
本発明の人工骨材は、ＯＣＰと、ＡＣＰ、ＤＣＰ、ＤＣＰＤ、低結晶性のＨＡ又は低結晶性のＯＣＰからなる混合燐酸カルシウムを含んでなる。本発明の混合燐酸カルシウムは、ＨＰＯ４／Ｐ値で約２５〜３５％、ＣａとＰのモル比で約１．２０〜１．４０の値をとる。ちなみに、化学量論的ＯＣＰはＨＰＯ４／Ｐ値が約３３％、Ｃａ／Ｐモル比が約１．３３であるが、本発明の実施例で用いているＯＣＰは、非化学量論組成を持ち、ＨＰＯ４／Ｐ値は約３８．３％であり、Ｃａ／Ｐモル比は約１．２６を有する。また、化学量論的ＨＡのＨＰＯ４／Ｐ値は１０．６％であり、Ｃａ／Ｐモル比は約１．６７であるが、本発明の実施例で用いているＨＡのＨＰＯ４／Ｐ値は約１０．６％、Ｃａ／Ｐモル比は約１．５５である。 The artificial aggregate of the present invention comprises OCP and mixed calcium phosphate composed of ACP, DCP, DCPD, low crystalline HA or low crystalline OCP. The mixed calcium phosphate of the present invention has an HPO 4 / P value of about 25 to 35% and a Ca to P molar ratio of about 1.20 to 1.40. Incidentally, the stoichiometric OCP has an HPO 4 / P value of about 33% and a Ca / P molar ratio of about 1.33, but the OCP used in the examples of the present invention has a non-stoichiometric composition. And the HPO 4 / P value is about 38.3% and the Ca / P molar ratio is about 1.26. The stoichiometric HA has an HPO 4 / P value of 10.6% and a Ca / P molar ratio of about 1.67, but the HA used in the examples of the present invention has an HPO 4 / P value. The value is about 10.6% and the Ca / P molar ratio is about 1.55.
この混合燐酸カルシウムは、主に下記（１）〜（４）の方法により製造することができる。
（１）ＯＣＰにＡＣＰ、ＤＣＰ、ＤＣＰＤ、低結晶性のＨＡ又は低結晶性のＯＣＰを析出する。
（２）ＯＣＰとＡＣＰ、ＤＣＰ、ＤＣＰＤ、低結晶性のＨＡ又は低結晶性のＯＣＰが同時に製造されるような条件で製造する。
（３）ＡＣＰ、ＤＣＰ又はＤＣＰＤを部分的にＯＣＰとなるよう結晶化する。
（４）ＯＣＰを部分的に低結晶化する。
This mixed calcium phosphate can be produced mainly by the following methods (1) to (4).
(1) ACP, DCP, DCPD, low crystalline HA or low crystalline OCP is deposited on OCP.
(2) Manufacture under conditions such that OCP and ACP, DCP, DCPD, low crystalline HA or low crystalline OCP are simultaneously produced.
(3) ACP, DCP or DCPD is crystallized so as to be partially OCP.
(4) Partially crystallize OCP.
尚、低結晶性のＯＣＰとは、非特許文献１で得た結晶性の比較的高いＯＣＰの構造が示すＸ線回折（ＸＲＤ）の結果よりもピーク強度の低い状態を有するＯＣＰであって、下記（Ａ）〜（Ｄ）のいずれか１つの状態のＯＣＰをいう。
（Ａ）結晶性が低い
（Ｂ）部分的に加水分解している
（Ｃ）低結晶性又は非晶質のＨＡが一部混在する
（Ｄ）ＡＣＰ、ＤＣＰ又はＤＣＰＤが一部混在する
本発明において低結晶性のＯＣＰは、ＯＣＰから完全にＡＣＰ、ＤＣＰ、ＤＣＰＤ又は低結晶性のＨＡに構造転移したものでない場合もあるため、本発明者らはこのような名称で特定している。尚、低結晶性のＯＣＰは、上記方法においては、例えば、（４）の方法により得ることができると考えられる。
The low crystalline OCP is an OCP having a lower peak intensity than the result of X-ray diffraction (XRD) indicated by the structure of OCP having a relatively high crystallinity obtained in Non-Patent Document 1, The OCP in any one of the following (A) to (D) states.
(A) Low crystallinity (B) Partially hydrolyzed (C) Partially mixed with low crystalline or amorphous HA (D) Partially mixed with ACP, DCP or DCPD In this case, the low crystalline OCP is not completely transferred from OCP to ACP, DCP, DCPD, or low crystalline HA. In addition, it is thought that low crystalline OCP can be obtained by the method (4), for example.
図１（ａ）は、本発明を実施した混合燐酸カルシウム（後述する実施例１）の粉末Ｘ線測定（ＭｉＮｉＦｌｅｘ，株式会社リガク）の結果である。比較対象としてＯＣＰの粉末Ｘ線測定（ＭｉＮｉＦｌｅｘ，株式会社リガク）の結果も示す（図１（ｂ））。回折角４．７度にＯＣＰ特有のピークがあり（図中矢印）、さらに回折角２５〜３５度辺りにＡＣＰに起因するベースラインの隆起が伺える（図中破線丸枠）。ちなみに、図１（ｂ）は、ＯＣＰの粉末Ｘ線測定の結果であるが、回折角４．７度におけるＯＣＰ特有のピークは、図１（ａ）よりも大きいことが伺える。 Fig.1 (a) is a result of the powder X-ray measurement (MiNiFlex, Rigaku Corporation) of the mixed calcium phosphate (Example 1 mentioned later) which implemented this invention. As a comparison object, the results of OCP powder X-ray measurement (MiNiFlex, Rigaku Corporation) are also shown (FIG. 1 (b)). There is a peak peculiar to OCP at a diffraction angle of 4.7 degrees (arrows in the figure), and a baseline bulge caused by ACP can be observed around a diffraction angle of 25 to 35 degrees (dotted line circles in the figure). Incidentally, FIG. 1 (b) shows the result of the powder X-ray measurement of OCP. It can be seen that the peak specific to OCP at a diffraction angle of 4.7 degrees is larger than that in FIG. 1 (a).
以下、前記（１）〜（４）の製造方法を順に説明する。尚、下記（１）〜（４）の中では、製造条件の設定が容易である観点から、（１）の方法が好ましいが、本発明は（１）の方法に必ずしも限定されるものではない。 Hereinafter, the production methods (1) to (4) will be described in order. In the following (1) to (4), the method (1) is preferable from the viewpoint of easy setting of manufacturing conditions, but the present invention is not necessarily limited to the method (1). .
第１の方法は、ＯＣＰに、ＡＣＰ、ＤＣＰ、ＤＣＰＤ、低結晶性のＨＡ又は低結晶性のＯＣＰを析出する方法である。より詳細には、ＯＣＰをＡＣＰ、ＤＣＰ、ＤＣＰＤ、低結晶性のＨＡ又は低結晶性のＯＣＰが製造される条件下でシードする方法である。ＡＣＰ、ＤＣＰ、ＤＣＰＤ、低結晶性のＨＡ又は低結晶性のＯＣＰが製造される条件は、当業者により適宜設定されるものであるが、例えば、ＡＣＰを製造する場合は、燐酸カルシウム濃度が自発沈殿を生じる程度まで高い過飽和条件（highly supersaturated condition）であればよい。原材料となるＯＣＰは、当業者により適宜製造できるものであるが、例えば、非特許文献１や、Ｙ． Ｈｏｎｄａ ｅｔ． ａｌ． Ｊｏｕｒｎａｌ ｏｆ Ｂｉｏｍｅｄｉｃａｌ Ｍａｔｅｒｉａｌｓ Ｒｅｓｅａｒｃｈ Ｐａｒｔ Ｂ，ｖｏｌ．８０（２）．ｐａｇｅ ２８１−２８９（２００７）を参考にすることができる。また、ＯＣＰにＤＣＰＤを析出する方法としては、非特許文献１の方法において、ＯＣＰが析出た直後の懸濁液を、さらに約４５〜８５℃で１００時間以上の長時間で撹拌する方法も挙げられる。 The first method is a method of depositing ACP, DCP, DCPD, low crystalline HA or low crystalline OCP on OCP. More specifically, OCP is seeded under conditions where ACP, DCP, DCPD, low crystalline HA or low crystalline OCP is produced. Conditions for producing ACP, DCP, DCPD, low crystalline HA or low crystalline OCP are appropriately set by those skilled in the art. For example, when ACP is produced, the calcium phosphate concentration is spontaneous. Any supersaturated conditions that are high enough to cause precipitation may be used. OCP used as a raw material can be appropriately manufactured by those skilled in the art. Honda et. al. Journal of Biomedical Materials Research Part B, vol. 80 (2). Reference can be made to page 281-289 (2007). In addition, as a method for precipitating DCPD on OCP, in the method of Non-Patent Document 1, a method in which the suspension immediately after the OCP is precipitated is further stirred at about 45 to 85 ° C. for 100 hours or more. It is done.
第２の方法は、ＯＣＰと、ＡＣＰ、ＤＣＰ、ＤＣＰＤ、低結晶性のＨＡ又は低結晶性のＯＣＰが同時に製造されるような条件で製造する方法である。より詳細には、横軸を溶液のｐＨ、縦軸を燐酸又はカルシウム濃度とした相図において、ＯＣＰとＡＣＰ、ＤＣＰ、ＤＣＰＤ、低結晶性のＨＡ又は低結晶性のＯＣＰとの臨界領域付近で製造する方法である。例えば、ＯＣＰとＡＣＰの混晶を製造する方法としては、ＯＣＰの種結晶を、水に分散させ、２５℃のスラリー状態とした水溶液に、硝酸カルシウム塩と水酸化アンモニウムの水溶液、および、燐酸水素アンモニウム塩と水酸化アンモニウムの水溶液を迅速に混合・攪拌する方法等が挙げられる。 The second method is a method in which OCP and ACP, DCP, DCPD, low crystalline HA, or low crystalline OCP are manufactured at the same time. More specifically, in the phase diagram where the horizontal axis is the pH of the solution and the vertical axis is the phosphoric acid or calcium concentration, in the vicinity of the critical region between OCP and ACP, DCP, DCPD, low crystalline HA or low crystalline OCP. It is a manufacturing method. For example, as a method for producing a mixed crystal of OCP and ACP, an OCP seed crystal is dispersed in water to form a slurry state at 25 ° C., an aqueous solution of calcium nitrate salt and ammonium hydroxide, and hydrogen phosphate. Examples thereof include a method of rapidly mixing and stirring an ammonium salt and an aqueous solution of ammonium hydroxide.
第３の方法は、ＡＣＰ、ＤＣＰ又はＤＣＰＤを部分的にＯＣＰとなるよう結晶化する方法である。具体的には、ＡＣＰ、ＤＣＰ又はＤＣＰＤからＯＣＰへの構造転移が生じる条件（主に温度処理）を短時間行う方法である。例えば、ＡＣＰを部分的にＯＣＰとなるよう結晶化する方法としては、ＯＣＰの種結晶を、水に分散させ、２５℃のスラリー状態とした水溶液に、硝酸カルシウム塩と水酸化アンモニウムの水溶液、および、燐酸水素アンモニウム塩と水酸化アンモニウムの水溶液を迅速に混合・攪拌して析出させた後、酢酸Ｎａ緩衝液（ｐＨ４．８〜５．６）に適当な時間分散浸漬する方法等が挙げられる。 The third method is a method of crystallizing ACP, DCP or DCPD so as to partially become OCP. Specifically, this is a method in which a condition (mainly temperature treatment) for causing a structural transition from ACP, DCP or DCPD to OCP is performed for a short time. For example, as a method of crystallizing ACP to partially become OCP, an OCP seed crystal is dispersed in water to form a slurry state at 25 ° C., an aqueous solution of calcium nitrate salt and ammonium hydroxide, and Examples thereof include a method in which an aqueous solution of ammonium hydrogenphosphate and ammonium hydroxide is rapidly mixed and stirred to precipitate, and then dispersed and immersed in an Na acetate buffer solution (pH 4.8 to 5.6) for an appropriate time.
第４の方法は、ＯＣＰを部分的に低結晶化する方法である。具体的には、ＯＣＰを短時間加水分解する。加水分解の方法は、固液比および顆粒径を適切に調節して、ＯＣＰを熱水に浸漬・撹拌して実施できるが、反応時間の短縮を目的として、酸又はアルカリ条件下としたものであってもよい。但し、生体内における安全性を考慮すると、熱水における撹拌が望ましいと考える。より詳細には、例えば、ＯＣＰを４５〜８５℃の熱水に分散し、１〜３６時間撹拌することにより製造され得るものが挙げられる。もちろん、水温を上記温度範囲より高くしたり、加水分解の環境を酸又はアルカリ環境とすれば反応時間を短くすることができ、逆に、水温を上記温度範囲より低くすれば、反応時間は長くすることができる。尚、２００メッシュアンダーの乾燥粉体あるいは合成後のスラリーの、７０℃の熱水による加水分解の条件においては、約４８時間撹拌することによりＯＣＰは、結晶構造が転移しＨＡになる。従って、ＯＣＰがＨＡになる最小反応時間を明らかにすれば、その１／８〜１／２程度の反応時間を採用し、かつ反応時間以外の反応条件を変えないことにより、本発明の燐酸カルシウムを得ることができるものと考える。 The fourth method is a method of partially crystallizing OCP. Specifically, OCP is hydrolyzed for a short time. The hydrolysis method can be carried out by appropriately adjusting the solid-liquid ratio and the granule diameter, and immersing and stirring OCP in hot water. However, for the purpose of shortening the reaction time, it is under an acid or alkaline condition. There may be. However, in view of safety in the living body, stirring in hot water is considered desirable. More specifically, for example, one that can be produced by dispersing OCP in hot water at 45 to 85 ° C. and stirring for 1 to 36 hours. Of course, the reaction time can be shortened if the water temperature is higher than the above temperature range, or if the hydrolysis environment is an acid or alkali environment, and conversely, if the water temperature is lower than the above temperature range, the reaction time is long. can do. Under the conditions of hydrolysis of the dry powder of 200 mesh or the slurry after synthesis with hot water at 70 ° C., the OCP becomes HA with the crystal structure transitioned by stirring for about 48 hours. Therefore, if the minimum reaction time when OCP becomes HA is clarified, the reaction time of about 1/8 to 1/2 is adopted, and the reaction conditions other than the reaction time are not changed, so that the calcium phosphate of the present invention is used. Think that you can get
以下、本発明を実施例により具体的に説明する。但し、本発明はこれらの実施例に限定して解釈してはならない。 Hereinafter, the present invention will be specifically described by way of examples. However, the present invention should not be interpreted as being limited to these examples.
＜実施例１＞ 第４の方法により製造した混合燐酸カルシウムを含む人工骨材
ＯＣＰ０．５ｇを、５ｍｇ／ｍＬの比で７０度の熱水に６時間撹拌し、その後に回収した燐酸カルシウムを人工骨材とした。図１（ａ）は、本実施例により得られた混合燐酸カルシウムの粉末Ｘ線回折測定の結果である。
<Example 1> Artificial aggregate OCP containing mixed calcium phosphate manufactured by the fourth method was stirred in hot water of 70 degrees at a ratio of 5 mg / mL for 6 hours, and then recovered calcium phosphate was artificially used. Aggregate. FIG. 1A shows the results of powder X-ray diffraction measurement of the mixed calcium phosphate obtained in this example.
＜比較例１＞ ＯＣＰのみからなる人工骨材
ＯＣＰをそのまま骨再生材料として用いた。
<Comparative example 1> Artificial bone material OCP which consists only of OCP OCP was used as a bone regeneration material as it was.
＜比較例２＞ ＨＡのみからなる人工骨材
ＨＡをそのまま骨再生材料として用いた。
<Comparative example 2> Artificial bone material HA which consists only of HA was used as a bone regeneration material as it was.
＜実験例＞ 埋植実験
実施例１、比較例１及び２の人工骨材をそれぞれラットの脛骨骨幹端内部に埋植した。埋植１４、２８及び５６日後のＨＥ染色の結果から、組織形態計測による新生骨の定量により求めた骨形成率を図２（ａ）に、人工骨材の残存率を図２（ｂ）に示す。実施例１の人工骨材で埋植したラットの骨形成率は約３０％と比較例よりも高い値を示した。一方、それにも関わらず生体内における人工骨材の残存率は約４０％と比較的多く残っていた。つまり、本発明の人工骨材は、骨芽細胞の分化促進というＯＣＰの特性を有しつつも、生体内における耐分解吸収性を有し、骨芽細胞が再生すべき足場を確保することができると考えられる。
<Experimental Example> Implantation Experiment The artificial bone materials of Example 1 and Comparative Examples 1 and 2 were implanted in the tibia metaphysis inside a rat, respectively. FIG. 2 (a) shows the bone formation rate determined by quantification of new bone by histomorphometry based on the results of HE staining after 14th, 28th, and 56th implantation, and FIG. 2 (b) shows the remaining rate of artificial aggregate. Show. The bone formation rate of the rat implanted with the artificial aggregate of Example 1 was about 30%, which was higher than that of the comparative example. On the other hand, the survival rate of the artificial aggregate in the living body remained relatively high at about 40%. In other words, the artificial bone material of the present invention has OCP characteristics of promoting osteoblast differentiation, has anti-degradable absorbability in vivo, and can secure a scaffold for osteoblasts to regenerate. It is considered possible.
また、Ｒｅａｌ−ｔｉｍｅ ＰＣＲ法により各ラットのＩＬ−１βの発現量を評価した。その結果を図３に示す。実施例１のラットのＩＬ−１βの発現量は、埋植６日後以降は比較例１よりも抑制されていることが明らかとなった。つまり、ＯＣＰそのものよりも緩慢な条件で骨再生がなされていることが明らかとなった。 Further, the expression level of IL-1β in each rat was evaluated by Real-time PCR method. The result is shown in FIG. It became clear that the expression level of IL-1β of the rat of Example 1 was suppressed more than that of Comparative Example 1 after 6 days after implantation. In other words, it was revealed that bone regeneration was performed under a slower condition than OCP itself.
＜実施例２＞ 第１の方法により製造した混合燐酸カルシウムを含む人工骨材
１０ｇのＯＣＰの種結晶を、５００ｍＬの水に分散させ、２５℃、３００ｒｐｍで回転させてスラリー状態とした水溶液を調整する。次に、０．２５ｍｏｌの硝酸カルシウム塩と０．１７ｍｏｌの水酸化アンモニウムの水溶液８００ｍＬ、及び、０．１６ｍｏｌの燐酸二水素アンモニウム塩と１．１ｍｏｌの水酸化アンモニウムの水溶液８００ｍＬを、迅速に混合・攪拌する。反応後、迅速に濾過して、洗浄中にＡＣＰを安定化させるためにＭｇ２＋を含む水溶液で数回洗浄して、最後にアセトンで置換洗浄して、凍結乾燥する。
<Example 2> Artificial aggregate containing mixed calcium phosphate produced by the first method 10 g of OCP seed crystal is dispersed in 500 mL of water and rotated at 25 ° C. and 300 rpm to prepare an aqueous solution in a slurry state. To do. Next, 800 mL of an aqueous solution of 0.25 mol of calcium nitrate and 0.17 mol of ammonium hydroxide and 800 mL of an aqueous solution of 0.16 mol of ammonium dihydrogen phosphate and 1.1 mol of ammonium hydroxide were rapidly mixed. Stir. After the reaction, it is quickly filtered, washed several times with an aqueous solution containing Mg 2+ in order to stabilize ACP during washing, and finally washed by substitution with acetone and lyophilized.
＜実施例３＞ 第２の方法により製造した混合燐酸カルシウムを含む人工骨材
非特許文献１の手法でＯＣＰを製造した直後の状態で、さらに、１２０ｒｐｍ、７０．０℃で３６０分熟成し、濾過、乾燥させた。図４（ａ）は、本実施例により得られた混合燐酸カルシウムの粉末Ｘ線回折測定装置（メーカー名：理学電機、型式：ＲＩＮＴ２５００ＶＨＦ）により測定した結果である。回折角４．７度にＯＣＰ特有のピークがあり、かつ、回折角１１．６度及び２９．２６度にＤＣＰＤ特有のピークがあることがわかる。また、図４（ｂ）は、ＯＣＰの粉末Ｘ線測定（メーカー名：理学電機、型式：ＲＩＮＴ２５００ＶＨＦ）の結果である。
<Example 3> Artificial aggregate containing mixed calcium phosphate produced by the second method In the state immediately after the OCP was produced by the method of Non-Patent Document 1, it was further aged at 120 rpm and 70.0 ° C for 360 minutes, Filtered and dried. FIG. 4 (a) shows the results of measurement with a mixed calcium phosphate powder X-ray diffraction measurement apparatus (manufacturer name: Rigaku Denki, model: RINT2500VHF) obtained in this example. It can be seen that there are peaks peculiar to OCP at a diffraction angle of 4.7 degrees, and peaks peculiar to DCPD at diffraction angles of 11.6 degrees and 29.26 degrees. FIG. 4B shows the results of OCP powder X-ray measurement (manufacturer name: Rigaku Denki, model: RINT2500VHF).
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| JP2016158827A (en) * | 2015-02-27 | 2016-09-05 | 国立大学法人東北大学 | Bone regeneration material |
| US10064979B2 (en) | 2013-12-02 | 2018-09-04 | Tohoku University | Bone regeneration material |
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