JP6304707B2 - Artificial bone and method for producing the same - Google Patents

Artificial bone and method for producing the same Download PDF

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JP6304707B2
JP6304707B2 JP2014030475A JP2014030475A JP6304707B2 JP 6304707 B2 JP6304707 B2 JP 6304707B2 JP 2014030475 A JP2014030475 A JP 2014030475A JP 2014030475 A JP2014030475 A JP 2014030475A JP 6304707 B2 JP6304707 B2 JP 6304707B2
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low molecular
artificial bone
dex
laminated film
base material
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大塚　英典
英典 大塚
毅知 三盃
毅知 三盃
大輔 松隈
大輔 松隈
袴塚　康治
康治 袴塚
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Tokyo University of Science
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Description

本発明は、人工骨およびその製造方法に関するものである。   The present invention relates to an artificial bone and a method for producing the same.

従来、リン酸カルシウムを主成分とする人工骨が知られている（例えば、特許文献１，２参照。）。骨組織に移植された人工骨が自家骨に置換されるためには、この人工骨を足場として骨を形成する骨芽細胞が必要である。特許文献１，２では、移植前に、デキサメタゾン（ＤＥＸ）のような分化誘導因子を添加した培地を用いて人工骨上で幹細胞を培養して骨芽細胞へ分化させている。   Conventionally, an artificial bone containing calcium phosphate as a main component is known (for example, see Patent Documents 1 and 2). In order to replace the artificial bone transplanted into the bone tissue with the autologous bone, osteoblasts that form bone using this artificial bone as a scaffold are required. In Patent Documents 1 and 2, before transplantation, stem cells are cultured on an artificial bone using a medium to which a differentiation-inducing factor such as dexamethasone (DEX) is added to differentiate into osteoblasts.

特開２００７−２０９２０３号公報JP 2007-209203 A 特開２０１３−２１６６６１号公報JP 2013-216661 A

人工骨を骨組織に移植した後、生体内の血液や骨髄に存在する幹細胞も骨芽細胞へ分化させることができれば、人工骨の自家骨への置換に要する期間の短縮を図ることができる。しかしながら、無機物であるリン酸カルシウムに対して、ＤＥＸのような低分子化合物を化学的に結合させることは難しく、人工骨に低分子化合物を安定的に担持させることが困難であるという問題がある。また、人工骨の表面に単に低分子化合物を吸着させただけでは、生体内に移植後、短時間の内に低分子化合物が人工骨から放出されてしまうために、幹細胞に対する分化誘導効果を得ることが難しいという問題がある。   If the stem cells present in the blood or bone marrow in the living body can be differentiated into osteoblasts after transplanting the artificial bone into the bone tissue, the time required for replacement of the artificial bone with autologous bone can be shortened. However, it is difficult to chemically bind a low molecular weight compound such as DEX to calcium phosphate, which is an inorganic substance, and it is difficult to stably support the low molecular weight compound on an artificial bone. In addition, simply adsorbing a low molecular weight compound on the surface of the artificial bone releases the low molecular weight compound from the artificial bone within a short time after transplantation into the living body. There is a problem that it is difficult.

本発明は、上述した事情に鑑みてなされたものであって、生体内に移植後に低分子化合物を長時間にわたって徐放し続けることができる人工骨およびその製造方法を提供することを目的とする。   This invention is made | formed in view of the situation mentioned above, Comprising: It aims at providing the artificial bone which can continue releasing a low molecular compound over a long time after transplanting in a living body for a long time, and its manufacturing method.

上記目的を達成するため、本発明は以下の手段を提供する。
本発明は、リン酸カルシウムの多孔体からなる基材と、該基材の外表面を被覆する積層膜とを備え、該積層膜は、正に荷電した生体親和性材料からなる正電荷層と、負に荷電した生体親和性材料からなる負電荷層とが交互に積層されてなり、前記正電荷層および前記負電荷層に、低分子化合物が内包されている人工骨を提供する。 In order to achieve the above object, the present invention provides the following means.
The present invention comprises a substrate composed of a porous body of calcium phosphate and a laminated film covering the outer surface of the substrate, the laminated film comprising a positively charged layer made of a positively charged biocompatible material, and a negative film. Provided is an artificial bone in which negatively charged layers made of a biocompatible material charged to each other are alternately laminated, and a low molecular compound is included in the positively charged layer and the negatively charged layer.

本発明によれば、リン酸カルシウムからなる基材の表面は水中において負に荷電するので、基材の外表面上に、該外表面側から順番に正電荷層と負電荷層とが交互に積層されてなる積層膜は、基材の外表面に静電気力によって安定に結合した膜となる。この積層膜に低分子化合物を内包させることによって低分子化合物を基材に安定に担持させることができる。   According to the present invention, since the surface of the substrate made of calcium phosphate is negatively charged in water, the positive charge layer and the negative charge layer are alternately laminated on the outer surface of the substrate in order from the outer surface side. The laminated film is a film that is stably bonded to the outer surface of the substrate by electrostatic force. By encapsulating the low molecular weight compound in the laminated film, the low molecular weight compound can be stably supported on the substrate.

この場合に、人工骨が生体内に移植された後、積層膜が外側から徐々に分解されるにつれて低分子化合物が徐々に放出される。これにより、生体内に移植後に低分子化合物を長時間にわたって徐放し続けることができる。   In this case, after the artificial bone is implanted in the living body, the low molecular weight compound is gradually released as the laminated film is gradually decomposed from the outside. Thereby, the low molecular compound can be continuously released over a long period of time after transplantation into the living body.

上記発明においては、前記低分子化合物が、少なくともデキサメタゾンを含むことが好ましい。
このようにすることで、人工骨を骨組織に移植した後に、人工骨の周囲に存在する幹細胞の骨芽細胞への分化がデキサメタゾンによって誘導され、分化した骨芽細胞によって人工骨を足場とした骨形成が行われる。これにより、人工骨の自家骨への置換を促進することができる。 In the said invention, it is preferable that the said low molecular weight compound contains a dexamethasone at least.
In this way, after transplanting the artificial bone into the bone tissue, the differentiation of the stem cells around the artificial bone into osteoblasts is induced by dexamethasone, and the artificial bone is used as a scaffold by the differentiated osteoblasts. Bone formation is performed. Thereby, the replacement of the artificial bone with the autologous bone can be promoted.

また、本発明は、リン酸カルシウムの多孔体からなる基材を正に荷電した生体親和性材料と低分子化合物とを含む溶液に浸漬する正電荷層形成ステップと、前記基材を負に荷電した生体親和性材料と前記低分子化合物とを含む溶液に浸漬する負電荷層形成ステップとを含み、前記正電荷層形成ステップと前記負電荷層形成ステップとを交互に繰り返すことにより、前記正に荷電した生体親和性材料からなる正電荷層と前記負に荷電した生体親和性材料からなる負電荷層とが交互に積層されてなり前記低分子化合物を内包した積層膜を前記基材の外表面上に形成する人工骨の製造方法を提供する。   The present invention also includes a positively charged layer forming step of immersing a substrate composed of a porous body of calcium phosphate in a solution containing a positively charged biocompatible material and a low molecular weight compound, and a negatively charged biological body A negatively charged layer forming step immersed in a solution containing the affinity material and the low molecular weight compound, and the positively charged layer is formed by alternately repeating the positively charged layer forming step and the negatively charged layer forming step. On the outer surface of the base material is a laminated film in which a positively charged layer made of a biocompatible material and a negatively charged layer made of the negatively charged biocompatible material are alternately laminated and encapsulate the low molecular weight compound. A method for producing an artificial bone to be formed is provided.

本発明によれば、生体内に移植後に低分子化合物を長時間にわたって徐放し続けることができるという効果を奏する。   According to the present invention, there is an effect that a low molecular compound can be continuously released over a long period of time after transplantation into a living body.

本発明の一実施形態に係る人工骨の構成を示す図である。It is a figure which shows the structure of the artificial bone which concerns on one Embodiment of this invention. 図１の人工骨の製造方法を示すフローチャートである。It is a flowchart which shows the manufacturing method of the artificial bone of FIG. 積層膜を備えない人工骨のＤＥＸの放出量を継時的に測定した結果を示す吸光度スペクトルである。It is an absorbance spectrum showing the result of measuring the amount of DEX released from an artificial bone without a laminated film over time. １０層のＰＥＩ層／ゼラチン層からなる積層膜を備える人工骨のＤＥＸの放出量を継時的に測定した結果を示す吸光度スペクトルである。It is an absorbance spectrum showing the result of measurement of the amount of DEX released from an artificial bone having a laminated film composed of 10 PEI layers / gelatin layers over time. ２０層のＰＥＩ層／ゼラチン層からなる積層膜を備える人工骨のＤＥＸの放出量を継時的に測定した結果を示す吸光度スペクトルである。It is an absorbance spectrum showing the result of measuring the amount of DEX released from an artificial bone provided with a laminated film composed of 20 PEI layers / gelatin layers over time. ４０層のＰＥＩ層／ゼラチン層からなる積層膜を備える人工骨のＤＥＸの放出量を継時的に測定した結果を示す吸光度スペクトルである。It is an absorbance spectrum showing the results of measuring the amount of DEX released from an artificial bone with a laminated film composed of 40 PEI layers / gelatin layers over time. 図４から図６の吸光度スペクトルから求めた、ＤＥＸの放出量の時間変化を表わすグラフである。It is a graph showing the time change of the discharge | release amount of DEX calculated | required from the absorbance spectrum of FIGS.

以下に、本発明の一実施形態に係る人工骨１とその製造方法について図１および図２を参照して説明する。
本実施形態に係る人工骨１は、図１に示されるように、リン酸カルシウムの多孔体からなる基材２と、該基材２の外表面を被覆する積層膜３とを備えている。 Below, the artificial bone 1 which concerns on one Embodiment of this invention, and its manufacturing method are demonstrated with reference to FIG. 1 and FIG.
As shown in FIG. 1, the artificial bone 1 according to the present embodiment includes a base material 2 made of a calcium phosphate porous body and a laminated film 3 that covers the outer surface of the base material 2.

基材２は、その移植部位に適した形状および寸法を有している。図１には、微細な顆粒状の基材２が示されているが、基材２は、例えば、ブロック状であってもよい。
積層膜３は、基材２の外表面上に正電荷層３ａと負電荷層３ｂとが交互に積層されてなる多層構造を有している。積層膜３のうち、基材２に隣接する最内層は正電荷層３ａである。正電荷層３ａは、正に荷電した生体親和性材料（以下、生体親和性材料（＋）と言う。）、好ましくはポリエチレンイミン（ＰＥＩ）のような高分子材料からなる。負電荷層３ｂは、負に荷電した生体親和性材料（以下、生体親和性材料（−）と言う。）、好ましくはゼラチンのような高分子材料からなる。 The base material 2 has a shape and dimensions suitable for the transplant site. Although FIG. 1 shows a fine granular base material 2, the base material 2 may be in a block shape, for example.
The laminated film 3 has a multilayer structure in which positive charge layers 3 a and negative charge layers 3 b are alternately laminated on the outer surface of the substrate 2. Of the laminated film 3, the innermost layer adjacent to the substrate 2 is a positively charged layer 3a. The positively charged layer 3a is made of a positively charged biocompatible material (hereinafter referred to as a biocompatible material (+)), preferably a polymer material such as polyethyleneimine (PEI). The negatively charged layer 3b is made of a negatively charged biocompatible material (hereinafter referred to as a biocompatible material (−)), preferably a polymer material such as gelatin.

基材２の外表面とその内部に形成された多数の細孔の内表面とには、低分子化合物４が略均一に分散して吸着している。また、正電荷層３ａおよび負電荷層３ｂにも、低分子化合物４が内包されている。低分子化合物４は、人工骨１の移植部位の治癒に寄与する化合物であり、少なくともデキサメタゾン（ＤＥＸ）を含む。また、低分子化合物４は、正電荷層３ａおよび負電荷層３ｂのいずれにも安定に担持されるように、電気的に略中性であることが好ましい。   On the outer surface of the base material 2 and the inner surfaces of a large number of pores formed therein, the low molecular compound 4 is adsorbed in a substantially uniform manner. Further, the low molecular compound 4 is also included in the positive charge layer 3a and the negative charge layer 3b. The low molecular weight compound 4 is a compound that contributes to the healing of the graft site of the artificial bone 1, and includes at least dexamethasone (DEX). Moreover, it is preferable that the low molecular weight compound 4 is electrically substantially neutral so that it can be stably supported on both the positive charge layer 3a and the negative charge layer 3b.

次に、上述した人工骨１の製造方法について説明する。
本実施形態に係る人工骨１の製造方法は、図２に示されるように、基材２に低分子化合物４を担持させる低分子化合物担持ステップＳ１と、正電荷層３ａを形成する正電荷層形成ステップＳ２と、正電荷層３ａを形成した基材２を洗浄する洗浄ステップＳ３と、負電荷層３ｂを形成する負電荷層形成ステップＳ４と、負電荷層３ｂを形成した基材２を洗浄する洗浄ステップＳ５と、ステップＳ２〜ステップＳ５を繰り返す繰り返しステップＳ６とを含む。 Next, a method for manufacturing the above-described artificial bone 1 will be described.
As shown in FIG. 2, the manufacturing method of the artificial bone 1 according to this embodiment includes a low molecular compound supporting step S1 for supporting a low molecular compound 4 on a base material 2, and a positive charge layer for forming a positive charge layer 3a. Forming step S2, washing step S3 for washing the substrate 2 on which the positive charge layer 3a is formed, negative charge layer forming step S4 for forming the negative charge layer 3b, and washing the substrate 2 on which the negative charge layer 3b is formed Cleaning step S5 to be performed, and step S6 to repeat step S2 to step S5 are included.

まず、低分子化合物担持ステップＳ１において、基材２を、低分子化合物４を含む低分子化合物水溶液に浸漬させる。これにより、低分子化合物水溶液が多孔質の基材２の内部にまで浸透し、低分子化合物水溶液中の低分子化合物４が、基材２の外表面のみならず細孔の内表面にも吸着する。   First, in the low molecular compound loading step S <b> 1, the substrate 2 is immersed in a low molecular compound aqueous solution containing the low molecular compound 4. Thereby, the low molecular compound aqueous solution penetrates into the porous substrate 2 and the low molecular compound 4 in the low molecular compound aqueous solution is adsorbed not only on the outer surface of the substrate 2 but also on the inner surface of the pores. To do.

次に、正電荷層形成ステップＳ２において、低分子化合物４を担持させた基材２を、生体親和性材料（＋）と低分子化合物４とを含む水溶液（以下、水溶液（＋）と言う。）に浸漬させる。水溶液（＋）中において、リン酸カルシウムからなる基材２の表面は、負に荷電する。したがって、基材２と生体親和性材料（＋）との間に働く静電気力によって、生体親和性材料（＋）は基材２の外表面全体を覆うように該外表面上に安定に吸着して正電荷層３ａを形成する。この正電荷層３ａの形成過程において、水溶液（＋）に含まれている低分子化合物４が正電荷層３ａに取り込まれることによって、正電荷層３ａに低分子化合物４が内包される。   Next, in the positive charge layer forming step S2, the base material 2 carrying the low molecular compound 4 is referred to as an aqueous solution containing the biocompatible material (+) and the low molecular compound 4 (hereinafter referred to as an aqueous solution (+)). ). In the aqueous solution (+), the surface of the substrate 2 made of calcium phosphate is negatively charged. Therefore, the biocompatible material (+) is stably adsorbed on the outer surface so as to cover the entire outer surface of the substrate 2 by the electrostatic force acting between the base material 2 and the biocompatible material (+). Thus, the positive charge layer 3a is formed. In the formation process of the positive charge layer 3a, the low molecular compound 4 contained in the aqueous solution (+) is taken into the positive charge layer 3a, whereby the low molecular compound 4 is included in the positive charge layer 3a.

次に、洗浄ステップＳ３において、水溶液（＋）から抽出した基材２を洗浄液に浸漬することによって、過剰な生体親和性材料を（＋）を除去する。この後、洗浄液から抽出した基材２を乾燥させる工程をさらに行った後に、次のステップに移ることが好ましい。このようにすることで、正電荷層３ａを安定化することができ、構造的により安定した積層膜３を形成することができる。   Next, in the cleaning step S3, the base material 2 extracted from the aqueous solution (+) is immersed in the cleaning liquid, thereby removing the excess biocompatible material (+). Thereafter, it is preferable to move to the next step after further performing a step of drying the substrate 2 extracted from the cleaning liquid. By doing in this way, the positive charge layer 3a can be stabilized and the laminated film 3 more structurally stable can be formed.

次に、負電荷層形成ステップＳ４において、正電荷層３ａを形成した基材２を、生体親和性材料（−）と低分子化合物４とを含む水溶液（以下、水溶液（−）と言う。）に浸漬させる。これにより、正電荷層３ａと、負に荷電した生体親和性材料（−）との間に働く静電気力によって、生体親和性材料（−）は、正電荷層３ａの外表面全体を覆うように該外表面上に安定に吸着して負電荷層３ｂを形成する。この負電荷層３ｂの形成過程においても、水溶液（−）に含まれている低分子化合物４が負電荷層３ｂに取り込まれることによって、負電荷層３ｂに低分子化合物４が内包される。   Next, in the negative charge layer forming step S4, the base material 2 on which the positive charge layer 3a is formed is an aqueous solution containing the biocompatible material (-) and the low molecular compound 4 (hereinafter referred to as an aqueous solution (-)). Soak in. Accordingly, the biocompatible material (−) covers the entire outer surface of the positively charged layer 3a by the electrostatic force acting between the positively charged layer 3a and the negatively charged biocompatible material (−). The negative charge layer 3b is formed by being stably adsorbed on the outer surface. Also in the formation process of the negative charge layer 3b, the low molecular compound 4 contained in the aqueous solution (-) is taken into the negative charge layer 3b, whereby the low molecular compound 4 is included in the negative charge layer 3b.

次に、洗浄ステップＳ５において、上述したステップＳ３と同様に、水溶液（−）から抽出した基材２を洗浄液に浸漬することによって、過剰な生体親和性材料（−）を除去する。このときも、負電荷層３ｂを安定化するために、洗浄液から抽出した基材２を乾燥させる工程をさらに行った後に、次のステップに移ることが好ましい。   Next, in the cleaning step S5, as in step S3 described above, the excess biocompatible material (-) is removed by immersing the base material 2 extracted from the aqueous solution (-) in the cleaning liquid. Also at this time, in order to stabilize the negative charge layer 3b, it is preferable to move to the next step after further performing a step of drying the substrate 2 extracted from the cleaning liquid.

以上のステップＳ２からステップＳ５までが繰り返しステップＳ６において繰り返されることにより、多層構造を有する積層膜３が形成され、本実施形態に係る人工骨１が製造される。
ここで、積層膜３は、上述したように、基材２と正電荷層３ａとの間、および、正電荷層３ａと負電荷層３ｂとの間に働く静電気力によって、基材２の外表面に安定に結合して存在することができる。 By repeating the above steps S2 to S5 repeatedly in step S6, the laminated film 3 having a multilayer structure is formed, and the artificial bone 1 according to the present embodiment is manufactured.
Here, as described above, the laminated film 3 is formed on the outside of the base material 2 by the electrostatic force acting between the base material 2 and the positive charge layer 3a and between the positive charge layer 3a and the negative charge layer 3b. It can exist stably bound to the surface.

次に、このように構成された人工骨１の作用について説明する。
本実施形態に係る人工骨１が生体内の骨組織の欠損部に移植された後、積層膜３が外側から徐々に分解されるにつれて、積層膜３に内包されていたＤＥＸが徐々に周囲へ放出される。放出されたＤＥＸは、人工骨１の周囲に存在する幹細胞に作用し、該幹細胞の骨芽細胞への分化を誘導する。そして、移植前から骨組織に存在していた骨芽細胞と新たに分化した骨芽細胞とが基材２を足場として骨を形成することによって、基材２が徐々に自家骨に置換されて骨欠損部が治癒する。 Next, the effect | action of the artificial bone 1 comprised in this way is demonstrated.
After the artificial bone 1 according to the present embodiment is transplanted to a bone tissue defect in a living body, as the laminated film 3 is gradually decomposed from the outside, the DEX contained in the laminated film 3 gradually moves to the surroundings. Released. The released DEX acts on the stem cells existing around the artificial bone 1 and induces differentiation of the stem cells into osteoblasts. The base material 2 is gradually replaced with autogenous bone by forming bone with the osteoblasts that have been present in the bone tissue before transplantation and the newly differentiated osteoblasts using the base material 2 as a scaffold. The bone defect is healed.

このように、本実施形態によれば、基材２の外表面上に安定に結合した積層膜３にＤＥＸのような低分子化合物４を内包させることによって、リン酸カルシウムからなる基材２に対して低分子化合物４を安定的に担持させることが可能となる。また、積層膜３に内包された低分子化合物４は、積層膜３が分解されるまで該積層膜３に留まる。これにより、人工骨１を生体内に移植後に低分子化合物４を長時間にわたって徐放し続け、ＤＥＸによる幹細胞の分化誘導効果を十分に得ることができるという利点がある。   Thus, according to this embodiment, the low molecular weight compound 4 such as DEX is encapsulated in the laminated film 3 that is stably bonded on the outer surface of the base 2, so that the base 2 made of calcium phosphate is included. It becomes possible to carry the low molecular compound 4 stably. Further, the low molecular compound 4 included in the laminated film 3 remains in the laminated film 3 until the laminated film 3 is decomposed. Accordingly, there is an advantage that the low molecular compound 4 can be continuously released over a long period of time after the artificial bone 1 is transplanted into the living body, and the effect of inducing stem cell differentiation by DEX can be sufficiently obtained.

なお、本実施形態においては、ＤＥＸを分化誘導剤として使用することとしたが、これに代えて、抗炎症剤として使用してもよい。生体内に移植後のＤＥＸの作用は、その濃度によって異なる。すなわち、ＤＥＸを抗炎症剤として使用するときは、人工骨１に担持させるＤＥＸの濃度を、分化誘導剤として使用するときよりも高くすればよい。   In the present embodiment, DEX is used as a differentiation-inducing agent, but instead, it may be used as an anti-inflammatory agent. The action of DEX after transplantation in vivo varies depending on its concentration. That is, when using DEX as an anti-inflammatory agent, the concentration of DEX supported on the artificial bone 1 may be higher than when using it as a differentiation inducer.

例えば、整形外科分野において、滑膜の炎症の治療として、患部にＤＥＸを注射する方法が用いられている。このような用途において、微小な顆粒状の基材２を用いた人工骨１を注射で患部に投与することで、ＤＥＸの抗炎症効果を従来に比べて長時間にわたって持続させることができる。   For example, in the field of orthopedics, a method of injecting DEX into an affected area is used as a treatment for synovial inflammation. In such a use, the anti-inflammatory effect of DEX can be maintained for a long time compared to the conventional art by administering the artificial bone 1 using the fine granular base material 2 to the affected part by injection.

また、本実施形態に係る人工骨１およびその製造方法においては、低分子化合物４として、ＤＥＸに代えて、またはＤＥＸに加えて、他の薬剤も使用可能である。他の薬剤を使用した場合にも、生体内に移植後に積層膜３が外側から徐々に分解されるにつれて薬剤が長時間にわたって徐放され続けることにより、薬剤の効果を長時間にわたって持続させることができる。なお、骨以外の組織に移植された基材２は、自家骨化することなく分解されて消失するので、生体に影響を及ぼすことはない。   Further, in the artificial bone 1 and the method for producing the same according to the present embodiment, as the low molecular weight compound 4, other drugs can be used instead of or in addition to DEX. Even when other drugs are used, the effect of the drug can be maintained for a long time by continuing to release the drug for a long time as the laminated film 3 is gradually decomposed from the outside after transplantation into the living body. it can. In addition, since the base material 2 transplanted to tissues other than bone is decomposed and lost without becoming autologous bone, it does not affect the living body.

次に、上述した人工骨１の実施例について、図３から図７を参照して以下に説明する。
以下の材料を使用して本発明の一実施例に係る人工骨を製造した。
基材として、βリン酸三カルシウム（β−ＴＣＰ）多孔体ブロックを使用した。
低分子化合物水溶液として、０．２％のＥｔＯＨを含むＰＢＳに、０．５×１０−４ＭのＤＥＸを添加したＤＥＸ溶液を使用した。
水溶液（＋）として、０．５×１０−４ＭのＮａＣｌを含む０．１ＭのＰＢＳに、０．５×１０−４ＭのＤＥＸと１ｍｇ／ｍＬのＰＥＩ（分子量１００００）とを添加したＰＥＩ溶液を使用した。 Next, an example of the above-described artificial bone 1 will be described below with reference to FIGS.
An artificial bone according to an embodiment of the present invention was manufactured using the following materials.
As a substrate, β-tricalcium phosphate (β-TCP) porous body block was used.
As a low molecular weight compound aqueous solution, a DEX solution in which 0.5 × 10 −4 M DEX was added to PBS containing 0.2% EtOH was used.
PEI obtained by adding 0.5 × 10 −4 M DEX and 1 mg / mL PEI (molecular weight 10,000) to 0.1 M PBS containing 0.5 × 10 −4 M NaCl as an aqueous solution (+) The solution was used.

水溶液（−）として、０．５×１０−４ＭのＮａＣｌを含む０．１ＭのＰＢＳ（ｐＨ７．４）に、０．５×１０−４ＭのＤＥＸと１．０ｍｇ／ｍＬのゼラチンとを添加したゼラチン溶液を使用した。
洗浄液として、１ＭのＮａＣｌを含む０．１ＭのＰＢＳ（ｐＨ７．４）に、０．５×１０−４ＭのＤＥＸを添加した洗浄液を使用した。 As an aqueous solution (−), 0.5 × 10 −4 M DEX and 1.0 mg / mL gelatin were added to 0.1 M PBS (pH 7.4) containing 0.5 × 10 −4 M NaCl. The added gelatin solution was used.
As a washing solution, a washing solution in which 0.5 × 10 −4 M DEX was added to 0.1 M PBS (pH 7.4) containing 1 M NaCl was used.

上記の材料を使用し、次の（１）〜（６）の手順に沿ってβ−ＴＣＰ多孔体ブロックを処理することにより、本実施例に係る人工骨を製造した。
（１）４５ｍｇのβ−ＴＣＰ多孔体ブロックに１ｍＬのＤＥＸ溶液を混合し、２時間静置した。これにより、β−ＴＣＰ多孔体ブロックにＤＥＸを担持させた。
（２）（１）でＤＥＸを担持させたβ−ＴＣＰ多孔体ブロックを、４ｍＬのＰＥＩ溶液に５分間浸漬させた。
（３）（２）でＰＥＩ層を形成したβ−ＴＣＰ多孔体ブロックを、洗浄液に１分間浸漬させた。
（４）（３）で洗浄したβ−ＴＣＰ多孔体ブロックを、４ｍＬのゼラチン溶液に５分間浸漬させた。
（５）（４）でゼラチン層を形成したβ−ＴＣＰ多孔体ブロックを、洗浄液に１分間浸漬させた。
（６）（２）〜（５）を、１０回、２０回または４０回繰り返した。 The artificial bone according to the present example was manufactured by processing the β-TCP porous body block according to the following procedures (1) to (6) using the above materials.
(1) 1 mL of DEX solution was mixed with 45 mg of the β-TCP porous body block and allowed to stand for 2 hours. As a result, DEX was supported on the β-TCP porous body block.
(2) The β-TCP porous body block carrying DEX in (1) was immersed in 4 mL of PEI solution for 5 minutes.
(3) The β-TCP porous body block on which the PEI layer was formed in (2) was immersed in a cleaning solution for 1 minute.
(4) The β-TCP porous body block washed in (3) was immersed in 4 mL of a gelatin solution for 5 minutes.
(5) The β-TCP porous body block on which the gelatin layer was formed in (4) was immersed in a cleaning solution for 1 minute.
(6) (2) to (5) were repeated 10 times, 20 times or 40 times.

以上の手順により、β−ＴＣＰ多孔体ブロックの表面上に、ＰＥＩ層／ゼラチン層が１０層、２０層または４０層積層されてなる積層膜を形成し、本実施例に係る人工骨を製造した。また、本実施例の比較例に係る人工骨として、β−ＴＣＰ多孔体ブロックに（１）の処理のみを施すことにより、積層膜を備えない人工骨も製造した。   By the above procedure, a laminated film formed by laminating 10 layers, 20 layers or 40 layers of PEI layer / gelatin layer was formed on the surface of the β-TCP porous body block, and an artificial bone according to this example was manufactured. . In addition, as the artificial bone according to the comparative example of the present example, only the treatment (1) was performed on the β-TCP porous body block, thereby manufacturing an artificial bone that does not include a laminated film.

次に、本実施例に係る人工骨と比較例に係る人工骨とのＤＥＸの放出機能を比較するために、以下の実験を行った。
本実施例および比較例に係る人工骨をそれぞれ１ｍＬのＰＢＳに浸漬し、所定時間毎に上澄みを回収するとともに等量のＰＢＳを残りのＰＢＳに添加し、回収した上澄みの吸光度を測定することにより、上澄みに含まれるＤＥＸの量を測定した。吸光度測定においては、ＰＢＳの測定値をブランクとして用いた。これにより、各人工骨からＰＢＳ中に放出されたＤＥＸの量を評価した。なお、上記の吸光度測定においては、８．００ｇ／ＬのＮａＣｌ、０．２０ｇ／ＬのＫＣｌ、１．４４ｇ／ＬのＮａ２ＨＰＯ４および０．２４ｇ／ＬのＫＨ２ＰＯ４を含むＰＢＳを使用した。 Next, in order to compare the DEX release function between the artificial bone according to this example and the artificial bone according to the comparative example, the following experiment was performed.
By immersing the artificial bones according to this example and the comparative example in 1 mL of PBS, collecting the supernatant every predetermined time, adding an equal amount of PBS to the remaining PBS, and measuring the absorbance of the collected supernatant The amount of DEX contained in the supernatant was measured. In the absorbance measurement, the measured value of PBS was used as a blank. This evaluated the amount of DEX released from each artificial bone into PBS. In the above absorbance measurement, PBS containing 8.00 g / L NaCl, 0.20 g / L KCl, 1.44 g / L Na 2 HPO 4 and 0.24 g / L KH 2 PO 4 was used. used.

図３から図６に、本実施例に係る人工骨と比較例に係る人工骨の上澄みから得られた吸光度スペクトルを示す。各人工骨について、ＰＢＳに浸漬してから０、３０、６０、…１８０、３００、３３０分間、および２４時間経過時に上澄み液を回収し、吸光度測定を行った。ＤＥＸの最大吸収波長は、２４０ｎｍである。図７は、図３から図６のそれぞれについて、波長２４０ｎｍにおける吸光度を時系列に並べたグラフである。   FIG. 3 to FIG. 6 show absorbance spectra obtained from the supernatant of the artificial bone according to the present example and the artificial bone according to the comparative example. For each artificial bone, the supernatant was collected at 0, 30, 60,..., 180, 300, 330 minutes, and 24 hours after immersion in PBS, and the absorbance was measured. The maximum absorption wavelength of DEX is 240 nm. FIG. 7 is a graph in which the absorbance at a wavelength of 240 nm is arranged in time series for each of FIGS.

図３から図６から分かるように、人工骨からＰＢＳ中へ放出されたＤＥＸの量を示す２４０ｎｍにおけるピークが、比較例の０層の場合と比べて、１０、２０および４０層の場合には顕著に大きくなっていた。これにより、積層膜にＤＥＸが担持されることが確認された。さらに、図７からより明確に分かるように、１０、２０および４０層においては、ＤＥＸの十分な量の放出が２４時間経過時にも確認された。積層数とＤＥＸの放出量との関係に着目すると、１０層と２０層との間では、放出量が積層数に比例して２倍となったが、２０層と４０層との間では、積層数と放出量との間に比例関係は認められなかった。   As can be seen from FIGS. 3 to 6, the peak at 240 nm indicating the amount of DEX released from the artificial bone into PBS is 10, 20, and 40 layers compared to the 0 layer of the comparative example. It was significantly larger. This confirmed that DEX was supported on the laminated film. Furthermore, as can be seen more clearly from FIG. 7, in the 10, 20 and 40 layers, a sufficient release of DEX was also observed after 24 hours. Paying attention to the relationship between the number of stacks and the amount of DEX released, between 10 and 20 layers, the amount of release was doubled in proportion to the number of layers, but between 20 and 40 layers, There was no proportional relationship between the number of layers and the amount released.

以上の実験結果から、本実施例に係る人工骨は、その外表面に形成された積層膜によってＤＥＸを安定的に担持し、ＤＥＸを長時間にわたって放出し続けることが確認された。生体内におけるＤＥＸの放出は、上記の試験管内でのそれよりも緩やかに進むと考えられる。したがって、本実施例の人工骨は、生体内に移植後、２４時間以上ＤＥＸを徐放し続けることができると期待される。   From the above experimental results, it was confirmed that the artificial bone according to the present example stably supported DEX by the laminated film formed on the outer surface thereof and continued to release DEX for a long time. The release of DEX in vivo is thought to proceed more slowly than that in the test tube. Therefore, it is expected that the artificial bone of this example can continue to release DEX for 24 hours or more after implantation in the living body.

１ 人工骨
２ 基材
３ 積層膜
３ａ 正電荷層
３ｂ 負電荷層
４ 低分子化合物
Ｓ１ 低分子化合物担持ステップ
Ｓ２ 正電荷層形成ステップ
Ｓ３，Ｓ５ 洗浄ステップ
Ｓ４ 負電荷層形成ステップ DESCRIPTION OF SYMBOLS 1 Artificial bone 2 Base material 3 Laminated film 3a Positive charge layer 3b Negative charge layer 4 Low molecular compound S1 Low molecular compound carrying step S2 Positive charge layer formation step S3, S5 Washing step S4 Negative charge layer formation step

Claims (3)

リン酸カルシウムの多孔体からなる基材と、
該基材の外表面を被覆する積層膜とを備え、
該積層膜は、基材の外表面上に該外表面側から順番に、正に荷電した生体親和性材料からなる正電荷層と、負に荷電した生体親和性材料からなる負電荷層とが交互に積層されてなり、
前記正電荷層および前記負電荷層に、電気的に中性である低分子化合物が内包されている人工骨。 A base material composed of a porous body of calcium phosphate;
A laminated film covering the outer surface of the substrate,
The laminated film has, on the outer surface of a base material, in order from the outer surface side, a positively charged layer made of a positively charged biocompatible material and a negatively charged layer made of a negatively charged biocompatible material. Alternately stacked,
An artificial bone, wherein the positively charged layer and the negatively charged layer contain an electrically neutral low molecular weight compound. 前記低分子化合物が、少なくともデキサメタゾンを含む請求項１に記載の人工骨。   The artificial bone according to claim 1, wherein the low molecular compound contains at least dexamethasone. リン酸カルシウムの多孔体からなる基材を正に荷電した生体親和性材料と低分子化合物とを含む溶液に浸漬する正電荷層形成ステップと、
前記基材を負に荷電した生体親和性材料と前記低分子化合物とを含む溶液に浸漬する負電荷層形成ステップとを含み、
前記正電荷層形成ステップと前記負電荷層形成ステップとを交互に繰り返すことにより、前記正に荷電した生体親和性材料からなる正電荷層と前記負に荷電した生体親和性材料からなる負電荷層とが交互に積層されてなり前記低分子化合物を内包した積層膜を前記基材の外表面上に形成する人工骨の製造方法。 A positively charged layer forming step of immersing a base material composed of a porous body of calcium phosphate in a solution containing a positively charged biocompatible material and a low molecular weight compound;
A negative charge layer forming step of immersing the base material in a solution containing a negatively charged biocompatible material and the low molecular weight compound,
By alternately repeating the positive charge layer forming step and the negative charge layer forming step, the positive charge layer made of the positively charged biocompatible material and the negative charge layer made of the negatively charged biocompatible material A method for producing an artificial bone, wherein a laminated film in which the low molecular weight compound is contained is formed on the outer surface of the base material.
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