JP2009018086A - Fibroblast growth factor sustained release biomaterial - Google Patents
Fibroblast growth factor sustained release biomaterial Download PDFInfo
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- JP2009018086A JP2009018086A JP2007184312A JP2007184312A JP2009018086A JP 2009018086 A JP2009018086 A JP 2009018086A JP 2007184312 A JP2007184312 A JP 2007184312A JP 2007184312 A JP2007184312 A JP 2007184312A JP 2009018086 A JP2009018086 A JP 2009018086A
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Abstract
【課題】生体材料を体内に埋入する際に、骨や皮膚といった周辺組織との固着・接着を促進するために繊維芽細胞成長因子(FGF-2)は効果的であるが、生体材料の埋入箇所に組織再生に適切な量の活性を有するFGF-2を長期間にわたって徐放する材料はこれまで生分解性の有機物に限られていたため、生体材料を被覆しても機械強度の違いから埋入時に剥離することが予想され、さらに生体材料と周辺組織との直接的な接触を妨げることから固着・接着の促進という目的には必ずしも最適ではない。
【解決手段】3種類以上の医療用輸液を混合して作製したリン酸カルシウム過飽和溶液にFGF-2を溶解させた浸漬液を作製し、陽極酸化チタンあるいは水酸アパタイトで出来た生体材料表面上にFGF-2と低結晶質アパタイトの複合層を析出させる。層の厚さやFGF-2含有量は、溶液組成を変化させることで制御可能であり、生体材料表面に直接形成されるため埋入時の剥離の可能性は低い。
【選択図】なしFibroblast growth factor (FGF-2) is effective to promote adhesion and adhesion to surrounding tissues such as bone and skin when biomaterial is embedded in the body. Since materials that release FGF-2, which has an appropriate amount of activity for tissue regeneration at the implantation site, over a long period of time have been limited to biodegradable organic substances, the difference in mechanical strength even when biomaterials are coated It is expected to peel off at the time of implantation, and further, the direct contact between the biomaterial and the surrounding tissue is hindered, so that it is not necessarily optimal for the purpose of promoting fixation / adhesion.
[Solution] An immersion solution in which FGF-2 is dissolved in a calcium phosphate supersaturated solution prepared by mixing three or more kinds of medical infusion solutions is prepared, and FGF is formed on the surface of a biomaterial made of anodized titanium or hydroxyapatite. Precipitate a composite layer of -2 and low crystalline apatite. The thickness of the layer and the FGF-2 content can be controlled by changing the solution composition, and since it is directly formed on the surface of the biomaterial, the possibility of peeling at the time of implantation is low.
[Selection figure] None
Description
本発明は、繊維芽細胞成長因子(FGF-2)−アパタイト複合化層で被覆され、FGF-2を徐放する生体材料に関する。この材料は生体内で複合化層中のアパタイトの吸収に伴いFGF-2を放出することにより周辺組織の再生を促すため、FGF-2担体である生体材料の埋入部周辺組織への早期の固着・接着が可能となる。 The present invention relates to a biomaterial that is coated with a fibroblast growth factor (FGF-2) -apatite composite layer and that releases FGF-2 slowly. This material promotes the regeneration of surrounding tissue by releasing FGF-2 with the absorption of apatite in the composite layer in vivo, so that early fixation to the surrounding tissue of the FGF-2 carrier biomaterial・ Adhesion is possible.
現在、骨欠損部の充填に使用される人工骨材料としてはセラミックや金属などが挙げられる。セラミック人工骨材料としては骨伝導性の水酸アパタイトや吸収性のβ-リン酸三カルシウムなどが用いられることが多い。負荷がかかる大腿骨などの長骨への埋入は、生体内でも化学的に安定なチタンやその合金が用いられる場合が多く、人工骨材料として以外でも人工歯根や創外固定具などの強度を要求される医療材料には広く使用されている。上記のいずれの材料も、高い生体適合性を有するものの、材料自体には埋入部周辺組織の再生を促す作用はなく、挿入後の骨組織との固着・安定に至るまでに時間を要す。 Currently, artificial bone materials used for filling bone defects include ceramics and metals. As the ceramic artificial bone material, osteoconductive hydroxyapatite or resorbable β-tricalcium phosphate is often used. For the embedding of long bones such as the femur where stress is applied, titanium and its alloys, which are chemically stable even in vivo, are often used, and the strength of artificial dental roots and external fixators is not limited to artificial bone materials. It is widely used for medical materials that are required. Although any of the above materials has high biocompatibility, the material itself does not promote the regeneration of the tissue around the implantation site, and it takes time until the bone tissue is fixed and stabilized after insertion.
特に経皮的に骨に埋入される人工歯根や創外固定具といったデバイスに関しては、骨界面との固着が短期間であるほど緩みに起因する埋入初期の感染リスクを低減することが期待できる。反対に、初期の感染が原因で骨髄炎を発症すると完全治癒に至らしめることが難しいため、骨の再生を促し速やかに骨と固着する材料が望まれる。 Especially for devices such as artificial tooth roots and external fixators that are percutaneously implanted in bone, the shorter the fixation to the bone interface, the lower the risk of infection in the initial stage due to loosening. it can. On the other hand, since it is difficult to achieve complete healing when osteomyelitis develops due to the initial infection, a material that promotes bone regeneration and quickly adheres to the bone is desired.
スクリューの骨組織との固着強度増大に関しては、これまでに考案されてきた、人工材料表面を水酸アパタイト(HA)やβ-リン酸三カルシウム(β-TCP)で被覆する方法が有効である。被覆の方法は、例えばプラズマ溶射コーティング法(特許文献１を参照)及びスパッタリングによるHAの製膜法(特許文献２および３を参照)、あるいはカルシウムとリンを含有する材料の塗布熱分解法(特許文献４を参照)などの材料の加熱を伴う方法と、カルシウムイオンとリン酸イオンを含有する過飽和溶液を用いるHAコーティング法(特許文献５〜８を参照)などの材料の加熱を必要としない方法に大別される。特にHAは骨伝導性であることから、プラズマ溶射HA膜を形成した創外固定スクリューや椎弓根スクリュー、あるいは水溶液中でHAコーティングされたチタンスクリューは、未処理のチタンスクリューに比べ骨との固着強度が有意に強くなる(非特許文献１〜３を参照)。しかしHA自体は骨伝導性ではあっても骨組織形成促進効果を持たないことから、スクリューにHAコーティングを施しても埋入初期、あるいは骨形成能が低下している高齢者における速やかな骨固着強度の増大はあまり期待できない。
For increasing the fixation strength of screws to bone tissue, the method of covering the artificial material surface with hydroxyapatite (HA) or β-tricalcium phosphate (β-TCP), which has been devised so far, is effective. . Examples of the coating method include a plasma spray coating method (see Patent Document 1) and a film formation method of HA by sputtering (see
迅速な組織再生には成長因子の投与が効果的である。このうちFGF-2は骨芽細胞のみならず繊維芽細胞、軟骨細胞など様々な細胞の増殖及びそれらが形成する組織の再生に関与し、更に血管新生も促進するため、例えば経皮的に骨に挿入される創外固定用スクリューの骨との固着のみならず、皮膚との接着促進が期待できるなど、用途は非常に幅広い。FGF-2を使用する場合、単独での患部への投与方法は散布や注入が考えられるが、FGF-2を大量に消費せざるを得ないこと、FGF-2の効果が拡散や失活により投与後の短期間に終了してしまうことなどの問題がある。これらの問題解決のため、FGF-2を酸性ムコ多糖、コラーゲン、ゼラチンなどの生分解性材料に含有させて失活を抑制しつつ徐放させる材料も考案されている(特許文献９および１０を参照)。これらはFGF-2の徐放性に優れるものの、基材となるチタンや水酸アパタイトとの機械強度差により挿入時に剥離する可能性、さらに吸収性の高さにより材料と周辺組織との界面に間隙を生じる可能性があり、材料と周辺組織の確実且つ早期の固定実現という目的にはそぐわない。生体材料表面に直接形成される薄層であって、生体活性を有するFGF-2を長期にわたり放出し続ける材料が望ましい。その点、HAコーティングは、基材の表面に化学結合によって直接形成することが可能なため、厚さを調節すれば生体内への挿入時に剥離する可能性は低い。 Administration of growth factors is effective for rapid tissue regeneration. Among them, FGF-2 is involved in the proliferation of various cells such as fibroblasts and chondrocytes as well as osteoblasts and regeneration of tissues formed by them, and also promotes angiogenesis. It can be used for a wide range of applications, including not only fixation of the external fixation screw to the bone but also promotion of adhesion to the skin. When using FGF-2, the administration method alone to the affected area may be spraying or injection, but FGF-2 must be consumed in large quantities, and the effect of FGF-2 may be caused by diffusion or inactivation. There are problems such as termination in a short period after administration. In order to solve these problems, a material has been devised in which FGF-2 is contained in a biodegradable material such as acidic mucopolysaccharide, collagen, gelatin, etc., and sustained release while suppressing inactivation (Patent Documents 9 and 10). reference). Although these are excellent in sustained release of FGF-2, they may be peeled off during insertion due to mechanical strength differences from titanium or hydroxyapatite as the base material, and further due to their high absorbency, the interface between the material and the surrounding tissue There is a possibility of creating a gap, which is not suitable for the purpose of realizing reliable and early fixation of the material and the surrounding tissue. A thin layer directly formed on the surface of the biomaterial, which is a material that continuously releases bioactive FGF-2 over a long period of time is desirable. In that respect, since the HA coating can be directly formed on the surface of the base material by chemical bonding, if the thickness is adjusted, the possibility of peeling at the time of insertion into a living body is low.
リン酸カルシウム過飽和溶液を用いるHAコーティング法は、適当な生体活性物質、例えばFGF-2、ラミニン、フィブロネクチンやコラーゲン、あるいは非生体活性物質のチトクロームCや牛アルブミンなどを液中に添加することによって、共沈過程で陽極酸化チタン、アパタイトセラミック、生分解性ポリマーなどの様々な基材上に形成されるHAコーティング中に複合化可能である(特許文献１１〜１４、および非特許文献４〜７を参照)。HAコーティング作製に用いるリン酸カルシウム過飽和溶液は、本発明者である伊藤等の研究により、既に医療用に認可されており、しかも滅菌済みである医療用輸液の混合によっても作製可能であるため(特許文献１１を参照)、医療用輸液を使用すれば目的外の毒性物質、例えばエンドトキシンなどを複合化する危険性がない。以上の操作でHAコーティングに取り込まれたチトクロームCは10日間に渡って徐放される(非特許文献７を参照)。つまり、FGF-2を複合化の過程で失活させることがなければ、基材となる物質に数日間作用する特定の生体活性を付与することが可能である。しかしFGF-2は多機能のタンパクで、例えば骨周辺部に投与する場合でも量に応じて骨形成を促進したり軟組織形成を促進したりと効果が異なる(非特許文献８を参照)。そのため、複合化するFGF-2量は埋入部位や目的とする機能に対応して厳密に制御される必要がある。 The HA coating method using a calcium phosphate supersaturated solution involves coprecipitation by adding an appropriate bioactive substance such as FGF-2, laminin, fibronectin or collagen, or non-bioactive substances such as cytochrome C or bovine albumin. It can be compounded in HA coatings formed on various substrates such as anodized titanium, apatite ceramic, biodegradable polymer in the process (see Patent Documents 11-14 and Non-Patent Documents 4-7) . The calcium phosphate supersaturated solution used for HA coating preparation can be prepared by mixing medical infusions that have already been approved for medical use by the inventor Ito et al. 11), there is no risk of complexing non-target toxic substances such as endotoxin if medical infusion is used. Cytochrome C incorporated into the HA coating by the above operation is gradually released over 10 days (see Non-Patent Document 7). In other words, if FGF-2 is not inactivated in the process of conjugation, it is possible to impart a specific biological activity that acts for several days on the base material. However, FGF-2 is a multifunctional protein, and, for example, even when administered to the periphery of bone, the effect is different depending on the amount, such as promoting bone formation or promoting soft tissue formation (see Non-Patent Document 8). Therefore, the amount of FGF-2 to be complexed must be strictly controlled according to the implantation site and the intended function.
本発明は、上記の問題点を考慮して、医療用輸液の混合により得られるリン酸カルシウム過飽和溶液を用いてFGF-2とリン酸カルシウムを含む複合化層で被覆し、その層からのFGF-2の徐放によって埋入周辺部の骨又は皮膚組織の再生を促す陽極酸化チタン又は水酸アパタイト（HA）セラミックを基材とする生体材料である。 In consideration of the above problems, the present invention covers a composite layer containing FGF-2 and calcium phosphate using a calcium phosphate supersaturated solution obtained by mixing medical infusion solutions, and gradually releases FGF-2 from the layer. It is a biomaterial based on anodized titanium or hydroxyapatite (HA) ceramic that promotes the regeneration of bone or skin tissue around the implant by release.
本発明では、3種類以上の医療用輸液のみを混合して得たリン酸カルシウム過飽和溶液に生体活性物質としてFGF-2のみを含む市販の医薬品を添加することによって滅菌済み且つ発熱性物質などが除去されている浸漬液を作成し、滅菌を行った陽極酸化チタンあるいはHAセラミックからなる基材を浸漬してこの表面上にFGF-2及びリン酸カルシウム複合化層を析出させる。さらに、基材上へのFGF-2の担持量はリン酸カルシウム過飽和溶液の組成、あるいはこれに添加するFGF-2の濃度を変化させることによって制御する。 In the present invention, sterilized and pyrogenic substances are removed by adding a commercially available drug containing only FGF-2 as a bioactive substance to a calcium phosphate supersaturated solution obtained by mixing only three or more kinds of medical infusion solutions. An immersion liquid is prepared, and a sterilized base material made of anodized titanium or HA ceramic is immersed to deposit a composite layer of FGF-2 and calcium phosphate on the surface. Furthermore, the amount of FGF-2 supported on the substrate is controlled by changing the composition of the calcium phosphate supersaturated solution or the concentration of FGF-2 added thereto.
すなわち、本発明は以下のとおりである。
[１] 少なくともカルシウム成分を含む医療用輸液と少なくともリン酸成分を含む医療用輸液とを含む少なくとも２種類の医療用輸液並びに繊維芽細胞成長因子（FGF-2)を混合し、１μg/ml以上20μg/ml未満のFGF-2を含有する過飽和リン酸カルシウム溶液を調製し、該FGF-2含有過飽和リン酸カルシウム溶液中に、陽極酸化チタンからなる生体適合性基材を24〜48時間漬浸しFGF-2を含有する低結晶アパタイト層で被覆することを含む、繊維芽細胞成長因子(FGF-2)を0.05〜0.65μg/cm2含有する低結晶質アパタイト層で被覆されたFGF-2徐放性生体材料を製造する方法。
[２] FGF-2を含有する過飽和リン酸カルシウム溶液中のカルシウム濃度が1.4〜3.4 mMであり、リン酸濃度が1.4〜3.1 mMである、[１]のFGF-2徐放性生体材料を製造する方法。
[３] 生体適合性基材が創外固定用基材である、[１]又は[２]のFGF-2徐放性生体材料を製造する方法。
That is, the present invention is as follows.
[1] A mixture of at least two types of medical infusions including a medical infusion solution containing at least a calcium component and a medical infusion solution containing at least a phosphate component, and fibroblast growth factor (FGF-2). Prepare a supersaturated calcium phosphate solution containing less than 20 μg / ml FGF-2, and soak FGF-2 in a supersaturated calcium phosphate solution containing FGF-2 by soaking a biocompatible substrate made of anodized titanium for 24-48 hours. FGF-2 sustained-release biomaterial coated with a low crystalline apatite layer containing 0.05 to 0.65 μg / cm 2 of fibroblast growth factor (FGF-2), including coating with a low crystalline apatite layer containing How to manufacture.
[2] The FGF-2 sustained-release biomaterial according to [1], wherein the calcium concentration in the supersaturated calcium phosphate solution containing FGF-2 is 1.4 to 3.4 mM and the phosphate concentration is 1.4 to 3.1 mM. Method.
[3] The method for producing an FGF-2 sustained-release biomaterial according to [1] or [2], wherein the biocompatible substrate is a substrate for external fixation.
[４] 少なくともカルシウム成分を含む医療用輸液と少なくともリン酸成分を含む医療用輸液とを含む少なくとも２種類の医療用輸液並びに繊維芽細胞成長因子（FGF-2)を混合し、１μg/ml以上20μg/ml未満のFGF-2を含有する過飽和リン酸カルシウム溶液を調製し、該FGF-2含有過飽和リン酸カルシウム溶液中に、水酸アパタイトセラミックからなる生体適合性基材を24〜48時間漬浸しFGF-2を含有する低結晶アパタイト層で被覆することを含む、繊維芽細胞成長因子(FGF-2)を0.05〜3.00μg/cm2含有する低結晶質アパタイト層で被覆されたFGF-2徐放性生体材料を製造する方法。
[５] FGF-2を含有する過飽和リン酸カルシウム溶液中のカルシウム濃度が1.6〜3.2 mMであり、リン酸濃度が0.7〜2.2 mMである、[４]のFGF-2徐放性生体材料を製造する方法。
[６] 生体適合性基材が人工骨材料である、[４]又は[５]のFGF-2徐放性生体材料を製造する方法。
[4] Mix at least two types of medical infusions containing at least a calcium component and a medical infusion containing at least a phosphate component, and fibroblast growth factor (FGF-2), 1 μg / ml or more A supersaturated calcium phosphate solution containing less than 20 μg / ml of FGF-2 was prepared, and a biocompatible substrate made of hydroxyapatite ceramic was immersed in the FGF-2 containing supersaturated calcium phosphate solution for 24-48 hours. FGF-2 sustained-release organism coated with a low crystalline apatite layer containing 0.05 to 3.00 μg / cm 2 of fibroblast growth factor (FGF-2), including coating with a low crystalline apatite layer containing A method of manufacturing a material.
[5] The FGF-2 sustained-release biomaterial according to [4], wherein the calcium concentration in the supersaturated calcium phosphate solution containing FGF-2 is 1.6 to 3.2 mM and the phosphate concentration is 0.7 to 2.2 mM. Method.
[6] The method for producing an FGF-2 sustained-release biomaterial according to [4] or [5], wherein the biocompatible base material is an artificial bone material.
[７] [１]〜[３]のいずれかの方法により製造される、陽極酸化チタンからなる繊維芽細胞成長因子(FGF-2)を0.05〜0.65μg/cm2含有する低結晶質アパタイト層で被覆されたFGF-2徐放性生体材料。
[８] 生体活性を有するFGF-2を0.25〜320 ng/cm2含有する、[７]のFGF-2徐放性生体材料。
[９] 生体適合性基材が創外固定用基材である、[７]又は[８]のFGF-2徐放性生体材料。
[１０] [４]〜[６]のいずれかの方法により製造される、水酸アパタイトセラミックからなる繊維芽細胞成長因子(FGF-2)を0.05〜3.00μg/cm2含有する低結晶質アパタイト層で被覆されたFGF-2徐放性生体材料。
[１１] 生体活性を有するFGF-2を0.25〜320 ng/cm2含有する、[９]のFGF-2徐放性生体材料。
[１２] 生体適合性基材がバーホールボタンである、[１０]又は[１１]のFGF-2徐放性生体材料。
[7] A low crystalline apatite layer containing 0.05 to 0.65 μg / cm 2 of fibroblast growth factor (FGF-2) made of anodized titanium produced by the method of any one of [1] to [3] FGF-2 sustained-release biomaterial coated with.
[8] The FGF-2 sustained-release biomaterial according to [7], containing 0.25 to 320 ng / cm 2 of FGF-2 having bioactivity.
[9] The FGF-2 sustained-release biomaterial according to [7] or [8], wherein the biocompatible substrate is a substrate for external fixation.
[10] A low crystalline apatite containing 0.05 to 3.00 μg / cm 2 of fibroblast growth factor (FGF-2) made of a hydroxyapatite ceramic produced by the method of any one of [4] to [6] FGF-2 sustained-release biomaterial coated with a layer.
[11] The FGF-2 having biological activity contained 0.25~320 ng / cm 2, FGF- 2 sustained release biomaterial [9].
[12] The FGF-2 sustained-release biomaterial according to [10] or [11], wherein the biocompatible substrate is a barhole button.
本発明は、滅菌済みの医療用輸液あるいは医薬品を浸漬液の材料として作成される極めて生体適合性の高いFGF-2及びリン酸カルシウム複合層により被覆されており、使用する医療用輸液の混合比や医薬品の溶解量を変化させることにより、ターゲットとなる組織ごとに必要量が異なる複合層中のFGF-2量に応じて変化可能で、複合層に取り込まれたFGF-2を少なくとも10日間に渡って徐放し、徐放されるFGF-2によって埋入部周辺組織の再生を実現する。また、基材として現状でも臨床応用されている医用材料を利用しても作製可能で、特別な材料、試薬や装置を準備する必要なく、50℃以下の低温で簡便に、しかも病院施設内で作製することが可能である。 The present invention is coated with a highly biocompatible FGF-2 and calcium phosphate composite layer prepared by using sterilized medical infusion or medicine as an immersion liquid material. The amount of FGF-2 dissolved in the composite layer can be changed according to the amount of FGF-2 in the composite layer, which varies depending on the target tissue. The tissue around the implantation site is regenerated by FGF-2 that is released slowly. It can also be made using medical materials that are currently used clinically as a base material, and it is not necessary to prepare special materials, reagents or equipment, and it can be easily used at low temperatures of 50 ° C or less, and in hospital facilities. It is possible to produce.
本発明の生体材料は、基材に繊維芽細胞成長因子（FGF-2)を含有する低結晶アパタイト層で被覆された基材からなる。本発明の生体材料は、安全性が高く、生体親和性及び生体適合性が高い。本発明の生体材料からはFGF-2が徐放される。本発明のFGF-2が徐放される生体材料をFGF-2徐放性生体材料という。 The biomaterial of the present invention comprises a base material coated with a low crystalline apatite layer containing fibroblast growth factor (FGF-2). The biomaterial of the present invention has high safety and high biocompatibility and biocompatibility. FGF-2 is gradually released from the biomaterial of the present invention. The biomaterial from which the FGF-2 of the present invention is sustainedly released is referred to as FGF-2 sustained release biomaterial.
本発明で使用する基材としては、表面に低結晶質アパタイトが成長する核となる物質が既に存在する、あるいは不均一核形成を起こしうる加工が施してある基材であって、生体適合性の基材であればどのようなものでも使用可能である。例えば、表面に陽極酸化処理を施したチタン、アルカリ処理とそれに続く加熱処理を施したチタン又はそれらのその合金等の金属材料、水酸アパタイト（HA)セラミック、含カルシウム及び含リン酸溶液に対して交互浸漬処理を施した各種ポリマーなどが挙げられる。陽極酸化処理を施したチタン製の基材は、陽極酸化チタン膜を有する。このため、創外固定に使用される市販の陽極酸化チタンスクリューや脳外科手術後の頭蓋骨の穴を埋める水酸アパタイト製バーホールボタンなどのインプラント材料や創外固定基材を使用して本発明の生体材料を作成することが可能である。これらの基材は滅菌されていないこともあるため、コンタミネーション及びFGF-2の分解を避けるため使用前にはEOGガス滅菌、又は乾熱滅菌等により滅菌処理を行うことが望ましい。 The base material used in the present invention is a base material in which a substance that becomes a nucleus for growing low crystalline apatite already exists on the surface or has been subjected to processing capable of causing heterogeneous nucleation, and is biocompatible. Any substrate can be used. For example, for titanium materials with anodized surfaces, titanium materials with alkali treatment followed by heat treatment, or alloys thereof, hydroxyapatite (HA) ceramics, calcium-containing and phosphoric acid-containing solutions And various polymers subjected to alternate dipping treatment. The base material made of titanium subjected to the anodizing treatment has an anodized titanium film. For this reason, it is possible to use an implant material such as a commercially available anodized titanium screw used for external fixation and a barhole button made of hydroxyapatite that fills a hole in the skull after brain surgery, and an external fixation base material of the present invention. Biomaterials can be created. Since these base materials may not be sterilized, it is desirable to sterilize by EOG gas sterilization or dry heat sterilization before use in order to avoid contamination and decomposition of FGF-2.
本発明で用いるリン酸カルシウム過飽和溶液は、公知の方法(特開2004−173795、特開2005−237632号公報等参照)に従い、少なくともカルシウム成分を含有する医療用輸液又は製剤、少なくともリン酸成分を含有する医療用輸液又は製剤及びpH補正用の適当な酸性輸液又はアルカリ性輸液を混合することで調製できる。医療用輸液とは、体液又はその成分の欠乏に際して、それを補充するための溶液をいい、水、電解質、ブドウ糖等を含み、細胞外液と類似した電解質組成を有する。例えば、０〜５％程度のブドウ糖、10〜150mEq/Lのナトリウム、０〜20mEq/L程度のカリウムを含む。また、カルシウム成分として例えば塩化カルシウム、乳酸カルシウム、酢酸カルシウム、グルコン酸カルシウム、クエン酸カルシウム等が挙げられ、リン酸成分としてはリン酸、リン酸水素二カリウム、リン酸二水素カリウム、リン酸水素二ナトリウム、リン酸二水素ナトリウム等が挙げられる。医療用輸液又は製剤としては、例えば、医療用電解質輸液剤、透析・腹膜灌流液、輸液の補正用製剤、カルシウム製剤、透析・腹膜灌流液の補充剤、補正用電解質輸液剤等が挙げられる。本発明で用いる輸液又は製剤は、カルシウム成分又はリン酸成分の少なくとも1つを含んでいればよく、両方を含んでいてもよい。最終的に複数の輸液又は製剤を混合したときにカルシウム成分とリン酸成分の両方が含まれていればよい。また、混合する輸液又は製剤の種類数に限定はなく少なくとも２種類、好ましくは３種類以上の輸液又は製剤を混合すればよい。少なくともカルシウム成分を含有する医療用輸液の例としては市販のリンゲル液(大塚製薬)やコンクライトCa(大塚製薬)、少なくともリン酸成分を含有する医療用輸液の例としては市販のクリニザルツB(アイロム製薬)、ソリタT2(大塚製薬)やコンクライトPK(大塚製薬)、pH補正用電解質輸液の例としては市販のメイロン(大塚製薬)や透析専用炭酸水素ナトリウム補充液のバイフィル(味の素ファルマ)などのアルカリ化剤が挙げられる。 The calcium phosphate supersaturated solution used in the present invention contains at least a phosphoric acid component, a medical infusion solution or preparation containing at least a calcium component, according to a known method (see JP 2004-173795 A, JP 2005-237632 A, etc.). It can be prepared by mixing a medical infusion or preparation and an appropriate acidic or alkaline infusion for pH correction. Medical infusion refers to a solution for replenishing body fluid or its components when deficient, and includes water, electrolyte, glucose, etc., and has an electrolyte composition similar to that of extracellular fluid. For example, it contains about 0 to 5% glucose, 10 to 150 mEq / L sodium, and about 0 to 20 mEq / L potassium. Examples of the calcium component include calcium chloride, calcium lactate, calcium acetate, calcium gluconate, and calcium citrate. Examples of the phosphate component include phosphoric acid, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and hydrogen phosphate. Examples include disodium and sodium dihydrogen phosphate. Examples of the medical infusion or preparation include medical electrolyte infusions, dialysis / peritoneal perfusate, infusion correction preparations, calcium preparations, dialysis / peritoneal perfusion replenishers, correction electrolyte infusions, and the like. The infusion solution or preparation used in the present invention only needs to contain at least one of a calcium component and a phosphate component, and may contain both. When a plurality of infusions or preparations are finally mixed, both the calcium component and the phosphate component may be contained. Moreover, there is no limitation in the number of types of infusions or preparations to be mixed, and at least two kinds, preferably three or more kinds of infusions or preparations may be mixed. Examples of medical infusions containing at least a calcium component include commercially available Ringer's solution (Otsuka Pharmaceutical) and Concrete Ca (Otsuka Pharmaceutical), and examples of medical infusions containing at least a phosphate component include commercially available Clinizarz B (Irom Pharmaceutical). ), Solita T2 (Otsuka Pharmaceutical), Concrite PK (Otsuka Pharmaceutical), and examples of electrolyte infusion solutions for pH correction include alkalis such as commercially available Meyron (Otsuka Pharmaceutical) and dialysis sodium bicarbonate replenisher bifil (Ajinomoto Pharma). An agent.
混合後の溶液中のカルシウムイオン濃度は、１mM以上、好ましくは1.4〜3.4 mM、混合後の溶液中のリン酸成分の量は、リン酸イオン濃度で、0.7〜3.1mMが好ましい。また、Ca/Pモル比は、0.5以上、好ましくは1.0〜2.0である。混合溶液中のカルシウムイオン濃度およびリン酸イオン濃度は生体適合性材料により変えることができる。例えば、陽極酸化チタン膜を有する生体適合性基材を用いる場合、好ましくはカルシウムイオン濃度が1.4〜3.4 mMであり、リン酸イオン濃度が1.4〜3.1 mMである。また、水酸アパタイトセラミック基材を用いる場合、好ましくはカルシウムイオン濃度が1.6〜3.2 mMであり、リン酸イオン濃度が0.7〜2.2 mMである。 The calcium ion concentration in the solution after mixing is 1 mM or more, preferably 1.4 to 3.4 mM, and the amount of the phosphate component in the solution after mixing is preferably 0.7 to 3.1 mM in terms of phosphate ion concentration. The Ca / P molar ratio is 0.5 or more, preferably 1.0 to 2.0. The calcium ion concentration and the phosphate ion concentration in the mixed solution can be changed depending on the biocompatible material. For example, when a biocompatible substrate having an anodized titanium film is used, the calcium ion concentration is preferably 1.4 to 3.4 mM and the phosphate ion concentration is 1.4 to 3.1 mM. Moreover, when using a hydroxyapatite ceramic base material, Preferably calcium ion concentration is 1.6-3.2 mM, Phosphate ion concentration is 0.7-2.2 mM.
繊維芽細胞成長因子-2（FGF-2）は、天然のもの、リコンビナントのものいずれも用いることができる。FGF-2の配列情報は、GenBankアクセッション番号A26642、X04431等で入手することができる。また、市販のフィブラストスプレー(科研製薬)のキットの内、凍結乾燥品の有効成分として含まれており、これを使用することが出来る。この凍結乾燥品は直接上記の医療用輸液、例えばリンゲル液などに溶解することも可能であるし、場合によっては市販の生理食塩水(大塚製薬等)に溶解した溶液を上記医療用輸液の混合液に添加することも可能である。 Fibroblast growth factor-2 (FGF-2) can be either natural or recombinant. The sequence information of FGF-2 can be obtained from GenBank accession numbers A26642, X04431, and the like. Moreover, it is contained as an active ingredient of a freeze-dried product in a kit of commercially available fiblast spray (Kaken Pharmaceutical Co., Ltd.), and this can be used. This lyophilized product can be directly dissolved in the above-mentioned medical infusion solution, such as Ringer's solution, and in some cases, a solution dissolved in commercially available physiological saline (Otsuka Pharmaceutical Co., Ltd.) can be mixed with the above-mentioned medical infusion solution. It is also possible to add to.
FGF-2含有リン酸カルシウム過飽和溶液(浸漬液)の組成は、混合する医療用輸液の体積比、あるいは添加するフィブラストスプレーキットの凍結乾燥品の量によって自由に変化させることが可能で、基材表面の性質や必要となるFGF-2量によって適宜調節する。好ましくは、医療用輸液の混合液中に、FGF-2が１μg/ml以上20μg/ml未満、好ましくは2〜10μg/ml、さらに好ましくは3〜5μg/ml含まれるように調製する。 The composition of the FGF-2-containing calcium phosphate supersaturated solution (immersion solution) can be freely changed depending on the volume ratio of the medical infusion solution to be mixed or the amount of the lyophilized product of the fiblast spray kit to be added. The amount of FGF-2 is adjusted as appropriate according to the nature of the substance and the amount of FGF-2 required. Preferably, it is prepared so that FGF-2 is contained in a mixture of medical infusion solution at 1 μg / ml or more and less than 20 μg / ml, preferably 2 to 10 μg / ml, more preferably 3 to 5 μg / ml.
このようにして市販の医療用輸液や医薬品を滅菌的に混合して作成された、カルシウム成分、リン酸成分及びFGF-2を含む浸漬液に滅菌された基材を浸漬することにより、基材上にFGF-2及び低結晶質アパタイト複合層を形成することが出来る。この際、溶液中のリン酸イオンとカルシウムイオンを主成分として、FGF-2が共沈しリン酸カルシウム沈殿が生じる。FGF-2の失活を極力抑えるため、浸漬時間は長くとも2日間以内、浸漬温度は体温と同等の37℃以下に設定することが望ましい。浸漬時間は、好ましくは数時間〜２日間（48時間）、さらに好ましくは12〜48時間、特に好ましくは24〜48時間である。また、浸漬温度は好ましくは20〜37℃である。加えて浸漬液のpHは、FGF-2の変性を防止できれば特に制限はないが、浸漬液調製直後のpHは6.5〜8.5の中性〜弱塩基性領域が好ましい。FGF-2は、低結晶質アパタイト表面への単なる吸着だけではなく、低結晶質アパタイト内に捕捉され、低結晶質アパタイト内部にも存在する。すなわち、FGF-2が低結晶質アパタイト層中に化学的あるいは物理的に保持される。本明細書において、FGF-2が低結晶質アパタイトに「担持」されるという。 By immersing the sterilized base material in the immersion liquid containing calcium component, phosphate component and FGF-2, which is prepared by sterilizing commercially available medical infusions and pharmaceuticals in this way, An FGF-2 and low crystalline apatite composite layer can be formed thereon. At this time, FGF-2 co-precipitates mainly with phosphate ions and calcium ions in the solution, resulting in calcium phosphate precipitation. In order to suppress the deactivation of FGF-2 as much as possible, it is desirable to set the immersion time within 2 days at the longest and the immersion temperature to 37 ° C or less, which is the same as the body temperature. The immersion time is preferably several hours to 2 days (48 hours), more preferably 12 to 48 hours, and particularly preferably 24 to 48 hours. The immersion temperature is preferably 20 to 37 ° C. In addition, the pH of the immersion liquid is not particularly limited as long as FGF-2 can be prevented from being denatured, but the pH immediately after preparation of the immersion liquid is preferably in the neutral to weakly basic region of 6.5 to 8.5. FGF-2 is not only simply adsorbed on the surface of the low crystalline apatite, but also trapped in the low crystalline apatite and exists inside the low crystalline apatite. That is, FGF-2 is chemically or physically held in the low crystalline apatite layer. In this specification, FGF-2 is said to be “supported” by low crystalline apatite.
また、浸漬液からの炭酸の放出過程で層形成が行われ、炭酸ガスが生成されるため、浸漬容器からガスが放出されるようにしておくのが好ましい。例えば浸漬容器のふたをコンタミネーションが起こらない程度に緩めておいてもよいし、あるいは浸漬容器に小孔を設けてもよい。 Moreover, since layer formation is performed in the process of releasing carbonic acid from the immersion liquid and carbon dioxide gas is generated, it is preferable that the gas is released from the immersion container. For example, the lid of the immersion container may be loosened to such an extent that contamination does not occur, or a small hole may be provided in the immersion container.
浸漬後は浸漬液から基材を取り出してそのまま使用可能であるが、複合層に取り込まれず基材表面に吸着しているFGF-2が一緒に投与されることを避ける場合には、浸漬液を作成する際に使用した生理的食塩水やリンゲル液などの輸液で洗浄して使用することが出来る。 After immersion, the substrate can be taken out of the immersion solution and used as it is.However, in order to avoid administration of FGF-2 adsorbed on the substrate surface without being incorporated into the composite layer, the immersion solution can be used. It can be used after being washed with an infusion solution such as physiological saline or Ringer's solution used in preparation.
本発明で基材上に形成されるFGF-2を含む低結晶質アパタイト複合層の厚さは特に制限されないが、あまり厚さが増すと埋入時の剥離が起こりやすくなるため、300μm以下、好ましくは80〜200μmに制御されることが望ましい。基材表面に形成された被覆の厚さは、例えば、電子顕微鏡で計測することができる。 The thickness of the low crystalline apatite composite layer containing FGF-2 formed on the substrate in the present invention is not particularly limited, but if the thickness increases too much, peeling at the time of embedding tends to occur. It is desirable that the thickness is controlled to 80 to 200 μm. The thickness of the coating formed on the substrate surface can be measured, for example, with an electron microscope.
このように作製された生体材料が、低結晶質アパタイトを無機主成分とする層で覆われていることは、当業者に既知の方法で確認できる。例えば、混合層を乾燥、より好ましくは凍結乾燥して、さらに好ましくは混合層を材料表面から分離すれば、粉末X線回折で構成成分の相同定が可能である。但し、基材にHAセラミックを選択した場合は、材料表面からの分離が困難なため有効な確認手段ではない。また、走査型電子顕微鏡や光学顕微鏡等の当業者に既知の方法で、浸漬液への浸漬前後の基材表面の変化を観察することにより、被覆状態を確認することもできる。この場合、表面に成長している結晶形状の観察により、経験則ではあるが層の無機主成分の同定が可能である。すなわち、結晶形状がいわゆる骨類似アパタイト様であるか否かによって複合層構成物が低結晶質アパタイトであると判断できることから、HAセラミックを基材にした場合の有用な分析方法といえる。 It can be confirmed by a method known to those skilled in the art that the biomaterial produced in this way is covered with a layer containing low crystalline apatite as an inorganic main component. For example, if the mixed layer is dried, more preferably freeze-dried, and more preferably the mixed layer is separated from the surface of the material, the phase of the constituent components can be identified by powder X-ray diffraction. However, when HA ceramic is selected as the substrate, separation from the material surface is difficult, so it is not an effective confirmation means. The coating state can also be confirmed by observing changes in the surface of the substrate before and after immersion in the immersion liquid by a method known to those skilled in the art such as a scanning electron microscope and an optical microscope. In this case, it is possible to identify the inorganic main component of the layer by observing the shape of the crystal growing on the surface, as a rule of thumb. In other words, the composite layer composition can be judged to be low crystalline apatite depending on whether the crystal shape is so-called bone-like apatite-like, so it can be said that this is a useful analysis method when HA ceramic is used as a base material.
本発明の生体材料上に形成される低結晶質アパタイトとは、浸漬液中のリン酸イオンとカルシウムイオンを主成分として化学反応の結果生成した塩のことであるが、浸漬液は医療用輸液を混合して作製するため、医療用輸液に含まれるナトリウム、カリウム、マグネシウムなどの陽イオン、炭酸、酢酸などの陰イオンあるいは水や糖類などの成分が少量含まれることがあり、その点についての規定は特にない。詳細な化学組成を必要とする場合、FGF-2及び低結晶質アパタイト複合層を材料から分離し、必要ならば酸で溶解して当業者に既知の種々の方法で化学分析することも可能である。 The low crystalline apatite formed on the biomaterial of the present invention is a salt produced as a result of a chemical reaction mainly composed of phosphate ions and calcium ions in the immersion liquid. In order to produce a mixture of these substances, there may be small amounts of cations such as sodium, potassium and magnesium, anions such as carbonic acid and acetic acid, or water and saccharides contained in medical infusions. There are no regulations. If a detailed chemical composition is required, the FGF-2 and low crystalline apatite composite layer can be separated from the material and dissolved with acid if necessary and analyzed by various methods known to those skilled in the art. is there.
このように生体材料を覆っている低結晶質アパタイトを無機主成分とする層には0.05〜0.65μg/cm2のFGF-2が含有されることが望ましい。その分析は、定性的にはX線光電子分光分析、定量的には浸漬液あるいは抽出液のタンパク定量キットによる分析が適用できる。本材料の作製に用いられる医療用輸液、医薬品、基材の内、窒素を含有するものは医薬品中のFGF-2のみとすることが可能なので、作製された材料表面のX線光電子スペクトルを測定し、窒素の存在を確認することで、材料表面に形成された低結晶質アパタイト層にFGF-2が取り込まれていることが確認できる。また、基材の浸漬前後での溶液中のFGF-2濃度変化を調べることにより、基材上へのFGF-2の担持量を見積もることが出来る。あるいは、表面に形成された層を、1〜2時間程度の短時間、室温、pH5〜6に調製した滅菌済みのクエン酸バッファで溶解し、抽出したFGF-2を直接定量する方法も有効である。定量方法としては例えばBradford法や、Biuret法にBicinchonic Acidを組み合わせた比色分析が挙げられる。 Thus, it is desirable that 0.05 to 0.65 μg / cm 2 of FGF-2 be contained in the layer mainly composed of low crystalline apatite covering the biomaterial. The analysis can be applied qualitatively by X-ray photoelectron spectroscopic analysis, and quantitatively by using a protein quantification kit of immersion liquid or extract. Of the medical infusions, pharmaceuticals, and base materials used to make this material, nitrogen-containing ones can only be FGF-2 in the pharmaceutical, so measure the X-ray photoelectron spectrum on the surface of the material By confirming the presence of nitrogen, it can be confirmed that FGF-2 has been incorporated into the low crystalline apatite layer formed on the material surface. Further, the amount of FGF-2 supported on the substrate can be estimated by examining the change in the concentration of FGF-2 in the solution before and after the substrate is immersed. Alternatively, a method of directly quantifying the extracted FGF-2 by dissolving the layer formed on the surface with a sterilized citrate buffer prepared at room temperature and pH 5-6 for a short time of about 1-2 hours is also effective. is there. Examples of the quantification method include Bradford method and colorimetric analysis in which Bicretonic acid is combined with Biuret method.
本発明のFGF-2及び低結晶質アパタイト複合層で被覆された、FGF-2を担持した生体材料からFGF-2が徐放される。上記の使用する医療用輸液の混合比やFGF-2の溶解量を変化させることにより、ターゲットとなる組織ごとに必要量が異なる複合層中のFGF-2量を応じて変化可能で、複合層に取り込まれたFGF-2を少なくとも10日間に渡って徐放し、徐放されるFGF-2によって埋入部周辺組織の再生を実現する。 FGF-2 is gradually released from a biomaterial carrying FGF-2, which is coated with the FGF-2 and low crystalline apatite composite layer of the present invention. By changing the mixing ratio of the medical infusion solution used above and the amount of FGF-2 dissolved, the amount of FGF-2 in the composite layer can be changed according to the target tissue depending on the target tissue. The FGF-2 taken in is gradually released for at least 10 days, and the tissue around the implantation site is regenerated by the slowly released FGF-2.
FGF-2及び低結晶質アパタイト複合層で被覆された生体材料中のFGF-2が徐放される期間は、作製された材料を、体液に類似の組成を有する無機溶液、例えば生理食塩水などを体温とほぼ同等の37℃に保った状態で浸漬し、一定期間ごとにこれを採取して溶液中のタンパク濃度変化曲線を作成することで調査可能である。 During the period in which FGF-2 in the biomaterial coated with FGF-2 and the low crystalline apatite composite layer is slowly released, the prepared material can be used as an inorganic solution having a composition similar to body fluid, such as physiological saline. Can be investigated by immersing the sample in a state maintained at 37 ° C., which is almost equal to the body temperature, and collecting the sample at regular intervals to create a protein concentration change curve in the solution.
このようにFGF-2を徐放するFGF-2及び低結晶質アパタイト複合層には、細胞増殖効果を含む生体活性を保持するFGF-2は0.25〜320 ng/cm2の密度で存在することが望ましい。FGF-2及び低結晶質アパタイト複合層で被覆された材料中のFGF-2が活性を維持しているか否かについては、材料自体、あるいは複合層の溶解により得られるFGF-2の抽出液を、NIH3T3などの繊維芽細胞の無血清培養系に浸漬、あるいは添加して72時間培養した後の細胞数変化を調べることによりin vitroで検査可能である。活性を持つFGF-2の量は、FGF-2濃度既知に調製された無血清培養液中でNIH3T3を72時間培養した後の細胞数変化を基に検量線を作成すれば、試料浸漬後の細胞増殖数から算出可能である。材料自体を浸漬する場合は、細胞と材料が直接触れ合わないように適当なスペーサーを使用して培養することにより徐放性についても検討可能である。抽出液を使用する場合は、1〜2時間程度の短時間、室温、pH5〜6に調製した滅菌済みのクエン酸バッファ中といった極力穏やかな条件で複合層を溶解しFGF-2を抽出する。 Thus, in the FGF-2 and low crystalline apatite composite layer that slowly releases FGF-2, FGF-2 that retains biological activity including cell proliferation effects should be present at a density of 0.25 to 320 ng / cm 2 Is desirable. Whether FGF-2 in the material coated with FGF-2 and the low crystalline apatite composite layer maintains its activity or not depends on whether the material itself or the FGF-2 extract obtained by dissolution of the composite layer is used. It is possible to test in vitro by examining the change in the number of cells after immersing or adding to a serum-free culture system of fibroblasts such as NIH3T3 and culturing for 72 hours. The amount of FGF-2 with activity can be determined by preparing a calibration curve based on the change in the number of cells after culturing NIH3T3 for 72 hours in a serum-free medium prepared with a known FGF-2 concentration. It can be calculated from the cell proliferation number. When the material itself is immersed, the sustained release can be examined by culturing using an appropriate spacer so that the cell and the material do not directly touch each other. When using an extract, FGF-2 is extracted by dissolving the composite layer under mild conditions such as in a sterilized citrate buffer prepared at room temperature and pH 5-6 for a short time of 1-2 hours.
本発明のFGF-2及び低結晶質アパタイト複合層で被覆された生体材料は、骨組織等の組織へ埋込むことにより、周辺組織にFGF-2が徐放され、周辺組織の再生を促す。例えば、創外固定用スクリュー等の創外固定用器具として骨折等により骨欠損部に埋込むことにより、周辺骨組織の再生を促し、スクリューの骨組織に対する固着強度を増すことができる。ここで、スクリューとは骨折部位の固定を行うための器具をいう。特に基材が陽極酸化チタン膜を有する生体適合性基材である場合、好適に創外固定用器具として用いることができる。また、バーホールボタン等の人工骨材料をFGF-2及び低結晶質アパタイト複合層で被覆した後に、骨欠損部に埋入することで周辺骨組織と人工骨材料との早期固着が促進される。ここで、バーホールボタンとは、頭蓋骨に空けた穴に挿入する人工骨をいう。特に基材が水酸アパタイトセラミックである場合、好適に人工骨材料として用いることができる。 When the biomaterial coated with the FGF-2 and low crystalline apatite composite layer of the present invention is embedded in a tissue such as a bone tissue, the FGF-2 is gradually released into the surrounding tissue, and the regeneration of the surrounding tissue is promoted. For example, as an external fixation device such as an external fixation screw, it is embedded in a bone defect part by fracture or the like, thereby promoting the regeneration of surrounding bone tissue and increasing the fixing strength of the screw to the bone tissue. Here, the screw refers to an instrument for fixing a fracture site. In particular, when the substrate is a biocompatible substrate having an anodized titanium film, it can be suitably used as an external fixation device. In addition, after artificial bone materials such as barhole buttons are covered with FGF-2 and a low crystalline apatite composite layer, early fixation between the surrounding bone tissue and the artificial bone material is promoted by embedding in the bone defect part. . Here, the barhole button refers to an artificial bone that is inserted into a hole formed in the skull. In particular, when the base material is a hydroxyapatite ceramic, it can be suitably used as an artificial bone material.
FGF-2及び低結晶質アパタイト複合層で被覆された材料中のFGF-2が組織再生能を保持しているか否かは動物実験によって確認が可能である。骨組織再生に関しては、骨埋入型の場合、ラットの頭蓋骨に欠陥を作製し、そこに複合層で覆われた欠陥と同じサイズの生体材料を埋入し、4週間飼育後の生体材料周辺部の新生骨形成状態を組織学的に観察することで評価できる。経皮的挿入型の場合、白色家ウサギの脛骨近位骨幹端内側に埋入し、4週間飼育後の材料周辺部の新生骨形成状態を組織学的に観察すると同時に、抜去トルクを測定すれば、生体材料の骨固着強度を数値として評価することが可能である。皮膚組織の再生に関しては、経皮的に埋入された試料周辺部の4週間飼育後の皮膚を組織学的に観察することで評価できる。更に、炎症反応を観察することで、生体材料と皮膚界面における封鎖状態を判定することが出来る。 Whether or not FGF-2 in the material coated with the FGF-2 and the low crystalline apatite composite layer retains the tissue regeneration ability can be confirmed by animal experiments. Regarding bone tissue regeneration, in the case of the bone-embedded type, a defect is created in the rat's skull, and a biomaterial of the same size as the defect covered with the composite layer is embedded therein, and around the biomaterial after 4 weeks of breeding It can be evaluated by histologically observing the new bone formation state of the part. In the case of the percutaneous insertion type, it should be implanted inside the proximal tibia of the white rabbit and observed histologically for the formation of new bone around the material after 4 weeks of breeding. For example, it is possible to evaluate the bone fixation strength of the biomaterial as a numerical value. Regarding the regeneration of skin tissue, it can be evaluated by histologically observing the skin after feeding for 4 weeks in the periphery of the sample embedded percutaneously. Furthermore, by observing the inflammatory reaction, it is possible to determine the blocking state at the interface between the biomaterial and the skin.
以下、本発明を実施例により更に具体的に説明するが、本発明の範囲は下記の実施例に限定されることはない。 EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.
陽極酸化チタンスクリュー（AO Ti スクリュー、SYNTHES(株)製、φ4.0、300 mm長）を15 mLのコニカルチューブに入れ、FGF-2添加リン酸カルシウム過飽和溶液10 mlに浸漬した。この浸漬液は創傷治療薬であるフィブラスト（商標）を、試薬を溶解させることにより作製した医療用輸液であるリンゲル液、クリニザルツB、バイフィルの炭酸ナトリウム補充液(以下本文中バイフィル)、コンクライトCa、コンクライトPKと同じ化学成分を含む溶液を混合して作製した（表１）。浸漬液はCa/Pモル比が2.0、カルシウム濃度が1.83 mM、炭酸ナトリウム濃度が15.09 mMとなるよう設定し、FGF-2は0μg・mL-1 (以下、浸漬液名F0)、4μg・mL-1 (同F4)、10μg・mL-1 (同F10)及び20μg・mL-1 (同F20)の濃度となるように添加し調製した（表２）。 An anodized titanium screw (AO Ti screw, manufactured by SYNTHES Co., Ltd., φ4.0, 300 mm length) was placed in a 15 mL conical tube and immersed in 10 ml of FGF-2-added calcium phosphate supersaturated solution. This soaking solution is a wound healing agent Fiblast (trademark), Ringer's solution which is a medical infusion solution prepared by dissolving a reagent, Clinizart B, sodium carbonate supplement solution of bifil (hereinafter bifil in the text), Concrite Ca A solution containing the same chemical components as Concrete PK was mixed (Table 1). The immersion liquid is set so that the Ca / P molar ratio is 2.0, the calcium concentration is 1.83 mM, and the sodium carbonate concentration is 15.09 mM, FGF-2 is 0 μg · mL -1 (hereinafter referred to as immersion liquid name F0), 4 μg · mL -1 (same as F4), 10 μg · mL −1 (same as F10) and 20 μg · mL −1 (same as F20) and added (Table 2).
AO Tiスクリューの浸漬時間は2日間（48h）とし、その際の処理温度は体温とほぼ等しい37℃に設定した。浸漬後、AO Tiスクリューを浸漬液から抜き取り、超純水で3回洗浄した後、室温にて乾燥させた。浸漬後のAO Tiスクリューの表面形態を走査電子顕微鏡(SEM)で観察し、X線光電子分光法(XPS)により表面の定性分析も行った。更に表面の析出物をAO Tiスクリューから掻き落とし、無反射シリコン板に塗布して粉末X線回折測定(XRD)を行った。また、浸漬後の浸漬液に残留したタンパク量を定量し、AO Tiスクリューに担持されたFGF-2量を見積もった。 The immersion time of the AO Ti screw was 2 days (48 hours), and the treatment temperature at that time was set to 37 ° C., which was almost equal to the body temperature. After immersion, the AO Ti screw was removed from the immersion liquid, washed 3 times with ultrapure water, and dried at room temperature. The surface morphology of the AO Ti screw after immersion was observed with a scanning electron microscope (SEM), and the surface was qualitatively analyzed by X-ray photoelectron spectroscopy (XPS). Further, the precipitate on the surface was scraped off from the AO Ti screw, applied to a non-reflective silicon plate, and powder X-ray diffraction measurement (XRD) was performed. In addition, the amount of protein remaining in the immersion liquid after immersion was quantified, and the amount of FGF-2 supported on the AO Ti screw was estimated.
表２に混合直後(Initial)及び浸漬後(Final)の浸漬液のpHを示す。混合直後のpHは7.4よりも高く、アパタイトなどのリン酸カルシウムが析出しやすいpHを示している。一方で浸漬後はF20を除くすべての条件でpHが低下している。これは、アパタイトの析出時に特徴的な傾向である。 Table 2 shows the pH of the immersion liquid immediately after mixing (Initial) and after immersion (Final). The pH immediately after mixing is higher than 7.4, indicating a pH at which calcium phosphate such as apatite is likely to precipitate. On the other hand, after immersion, the pH is lowered under all conditions except F20. This is a characteristic tendency when apatite is precipitated.
図１〜４は、各組成の浸漬液で処理したAO Tiスクリュー表面を走査電子顕微鏡(SEM)で観察した結果である。F0、F4及びF10処理AO Tiスクリュー表面は0.3〜2.5μm程度の球状粒子で覆われている様子が確認できた。一方で、F20処理後は球状粒子の析出はほとんど見られなかった。析出した球状粒子をAO Tiスクリューから掻き落としてXRD測定を行ったところ、図５に示すようにいずれも低結晶質のアパタイトであると同定された。アパタイト層が析出していることが明らかとなったF0、F4及びF10処理スクリューについて、さらにXPSによる表面分析を行うと、ピーク面積で0.00 ± 0.00、2241 ± 657及び2092 ± 1103の窒素が存在することが確認された。窒素はFGF-2のみに含まれていることから、F4及びF10処理AO Tiスクリュー上にはFGF-2が存在していることが分かった。浸漬液中の残留FGF-2量の定量結果から、F4処理するとAO Tiスクリュー上に0.62±0.22μg/cm2、F10では0.67±0.19μg/cm2の FGF-2が担持されることが分かった。 FIGS. 1-4 are the results of observing the surface of the AO Ti screw treated with the immersion liquid of each composition with a scanning electron microscope (SEM). It was confirmed that the surface of the F0, F4 and F10 treated AO Ti screw was covered with spherical particles of about 0.3 to 2.5 μm. On the other hand, almost no precipitation of spherical particles was observed after F20 treatment. The precipitated spherical particles were scraped off from the AO Ti screw and subjected to XRD measurement. As shown in FIG. 5, they were all identified as low crystalline apatite. When F0, F4 and F10 treated screws with apatite layer deposited are further analyzed by XPS, there are 0.00 ± 0.00, 2241 ± 657 and 2092 ± 1103 nitrogen in peak areas. It was confirmed. Since nitrogen was contained only in FGF-2, it was found that FGF-2 was present on the F4 and F10 treated AO Ti screws. From the residual FGF-2 of quantification results of the immersion liquid, found that FGF-2 of AO Ti screw on the 0.62 ± 0.22μg / cm 2, F10 in 0.67 ± 0.19μg / cm 2 is carried when F4 process It was.
本実施例では、AO Tiスクリュー上にFGF-2と低結晶質アパタイトからなる複合層の形成が可能で、浸漬液中のFGF-2濃度は、少なくとも10μg・mL-1の濃度まで上昇させることが可能であり、20μg・mL-1以上の濃度にすると低結晶質アパタイト形成阻害効果が顕著になることを示している。 In this example, a composite layer composed of FGF-2 and low crystalline apatite can be formed on the AO Ti screw, and the concentration of FGF-2 in the immersion liquid should be increased to a concentration of at least 10 μg · mL −1. It is shown that the effect of inhibiting the formation of low crystalline apatite becomes remarkable when the concentration is 20 μg · mL −1 or more.
AO Tiスクリュー上に担持されたFGF-2の活性の有無を調べるため、FGF-2・低結晶質アパタイト複合層からFGF-2を抽出し、それを無血清の細胞培養系に添加して増殖効果を調べた。 In order to investigate the activity of FGF-2 supported on the AO Ti screw, FGF-2 was extracted from the FGF-2 / low crystalline apatite composite layer and added to a serum-free cell culture system to proliferate. The effect was investigated.
AO Tiスクリュー上への複合層の形成はF0処理及びF4処理で行った（表２）。これを10 mM、pH5.43に調製したクエン酸バッファ3 mLに2時間浸漬し、複合層を溶解することによってFGF-2を抽出した。一方、繊維芽細胞様細胞NIH3T3を1×104個含む無血清培地1mLを24ウェルプレートの各ウェルに注ぎ、1時間予備培養を行った。無血清培地はDMEMにL-グルタミン(0.3 mg・mL-1)、牛血清アルブミン(1.0 mg・mL-1)、インスリン(5.0μg・mL-1)及びトランスフェリン(1.0μg・mL-1)を添加して作製した。予備培養後、先の抽出液50 μLを各ウェルに添加し、更に72時間の本培養を行った後、細胞数を位相差顕微鏡下でカウントした。コントロールは、抽出液の代わりにクエン酸バッファのみを50μL加える系とした。更に、抽出液ではなく、既知のFGF-2量を添加したウェルも用意し、72時間後の細胞数を求めることによって検量線を作製し、活性を持つFGF-2量の定量も行った。 Formation of the composite layer on the AO Ti screw was performed by F0 treatment and F4 treatment (Table 2). This was immersed in 3 mL of citrate buffer adjusted to 10 mM and pH 5.43 for 2 hours, and FGF-2 was extracted by dissolving the composite layer. On the other hand, 1 mL of serum-free medium containing 1 × 10 4 fibroblast-like cells NIH3T3 was poured into each well of a 24-well plate, and precultured for 1 hour. Serum-free medium is DMEM with L-glutamine (0.3 mg ・ mL -1 ), bovine serum albumin (1.0 mg ・ mL -1 ), insulin (5.0 μg ・ mL -1 ) and transferrin (1.0 μg ・ mL -1 ). It was prepared by adding. After the preliminary culture, 50 μL of the previous extract was added to each well, and after further main culture for 72 hours, the number of cells was counted under a phase contrast microscope. The control was a system in which only 50 μL of citrate buffer was added instead of the extract. Furthermore, a well to which a known amount of FGF-2 was added was prepared instead of the extract, and a calibration curve was prepared by determining the number of cells after 72 hours, and the amount of active FGF-2 was quantified.
図６〜図９はそれぞれクエン酸のみを添加した系、及びF0、F4あるいはF10処理で形成された層の抽出液を添加した各系の、それぞれの無血清培地中のNIH3T3細胞の72時間培養後の様子を示したものである。F0処理で形成した層の抽出液を添加してもクエン酸のみ添加の場合と細胞形態に明瞭な差異はなかったが、F4あるいはF10処理で形成した層の抽出液を添加すると形状が明瞭となり、細胞数も増加していることが分かる。図１０に示す細胞カウントの結果の通り、確かにF4及びF10処理物の抽出液添加によって有意に細胞数が増加していることが明らかとなった。すなわち、F4あるいはF10処理で形成された複合相中のFGF-2は完全には失活していないことが示された。検量線(図１１)から、F4処理で形成した層中には活性のあるFGF-2が少なくとも3.74 ng/cm2 (総FGF-2担持量の約0.6 %)存在し、F10処理の場合は少なくとも387 ng/cm2(総FGF-2担持量の約58 %)が存在することが明らかとなった。 FIGS. 6 to 9 show cultures of NIH3T3 cells in serum-free medium for 72 hours, respectively, in a system to which only citric acid was added and in each system to which an extract of a layer formed by F0, F4 or F10 treatment was added. It shows what happened later. Even when the extract of the layer formed by F0 treatment was added, there was no clear difference in cell morphology from the case of adding citric acid alone, but the shape became clear when the extract of the layer formed by F4 or F10 treatment was added. It can be seen that the number of cells is also increasing. As shown in the cell count results shown in FIG. 10, it was clarified that the number of cells was significantly increased by adding the extract of F4 and F10 treated products. That is, it was shown that FGF-2 in the composite phase formed by F4 or F10 treatment was not completely inactivated. From the calibration curve (FIG. 11), there is at least 3.74 ng / cm 2 of active FGF- 2 (about 0.6% of the total amount of FGF-2 supported) in the layer formed by F4 treatment. It was revealed that there was at least 387 ng / cm 2 (about 58% of the total amount of FGF-2 supported).
実施例１では、FGF-2の担持処理方法について議論したが、担持処理中のFGF-2の失活については検討していなかった。実施例２によって、担持したFGF-2は少なくとも完全に失活していないことが示された。 In Example 1, the method for supporting FGF-2 was discussed, but the deactivation of FGF-2 during the supporting process was not examined. Example 2 showed that the supported FGF-2 was not at least completely inactivated.
未処理及びF0処理、F4処理あるいはF10処理により複合層を形成した4種類のAO TiスクリューをEOG滅菌後に動物実験に供した。体重約3.0 kgの成熟日本白色家兎の脛骨近位骨幹端内側に経皮的にスクリューを挿入し、挿入時の最大トルクを測定した(n=5)。術後4週でスクリュー挿入部の肉眼観察を行い、炎症レベルをGrade 1〜Grade 3の三段階に分類した。更に屠殺した後のスクリューの抜去時の最大トルクを測定し(n=19)、抜去後に組織学的に観察・評価を行った。
Four types of AO Ti screws with composite layers formed by untreated and F0, F4 or F10 treatment were subjected to animal experiments after EOG sterilization. A screw was inserted percutaneously inside the proximal tibia of a mature Japanese white rabbit weighing approximately 3.0 kg, and the maximum torque at the time of insertion was measured (n = 5). Visual observation of the screw insertion part was performed 4 weeks after the operation, and the inflammation level was classified into three stages of
術後4週でのスクリュー周辺部の炎症の様子を例示すると、図１２はGrade 1で、皮膚組織に異常がなく、スクリューは脛骨に固着していた例、図１３はGrade 2で、皮膚に発赤が認められるもののスクリューが脛骨と固着していた例であり、図１４はGrade 3で皮膚の発赤を認め、更に骨髄炎発症によりスクリューの脛骨との固着が起こらなかった例である。すなわちGradeの数値が大きいほど重度の感染を起こしているといえる。各検体についてn=16で炎症レベルを分類したところ図１５に示すようになった。重度の炎症であるGrade 3は、未処理(UN)及びF0処理スクリューを挿入した場合に確認されたが、F4及びF10処理スクリュー挿入では確認されなかった。一方で、炎症を認めないGrade 1の検体数が最も多いのはF4処理スクリュー挿入した場合であり、F10処理スクリューの場合は却って炎症を認めない検体数は減少した。以上の結果はAO Tiスクリューに上に形成された複合層から徐放されるFGF-2の働きによってスクリュー−皮膚界面の封鎖性が最も向上するのはF4処理を施した場合であることを示している。
Explaining the state of inflammation around the
スクリューの挿入トルクはいずれのスクリューでも0.12 Nmでほぼ一定であり、複合層が形成されたことによる摩擦などの物理的要因がトルク測定に影響を及ぼすことがないことが確かめられた(図１６)。術後4週になると、F0処理及びF4処理スクリューの抜去トルクは未処理スクリューの抜去トルクに比べて35〜58%も高い値を示し、骨固着強度が上昇することが分かった。しかし、活性を保持したFGF-2が最も多く担持されているF10処理スクリューを用いると、抜去トルクは未処理スクリューのそれとほぼ同等程度となった(図１６)。FGF-2は過剰投与により骨組織の形成を抑制するとともに軟組織の形成を促進するが、この結果は、複合層が存在することによって上昇するスクリューと骨の界面の固着強度がF4処理でFGF-2を担持しても低下せず、骨組織形成に悪影響を及ぼさないことを示している。
The insertion torque of the screw was almost constant at 0.12 Nm in any screw, and it was confirmed that physical factors such as friction due to the formation of the composite layer did not affect the torque measurement (FIG. 16). . At 4 weeks after the operation, the extraction torque of the F0-treated and F4-treated screws showed a
スクリュー挿入部周辺の軟組織を観察すると、Grade 0の場合炎症反応が見られたとしても非常に穏やかなものであることが図１７より分かる。更に血管新生を伴う皮膚の再生も認められた。一方で、図１８に示すようにGrade 1やGrade 2の場合は軟組織に壊死を伴う明瞭な炎症反応を認めた。スクリュー挿入部周辺の骨組織の脱灰標本の観察結果は図１９〜２２に示す。未処理のスクリュー周辺部では皮質骨との界面の空隙にのみ新生骨が形成されるのに対して、F0及びF4処理スクリュー周辺部では皮質骨との界面の空隙だけでなく、髄腔に突き出たスクリュー表面にも新生骨の形成が認められた。F10スクリュー表面には骨様の組織の形成は認められなかった。これらの事から、抜去トルクがF0及びF4処理スクリューで高い値を示すのは、スクリュー周辺部での新生骨形成量の増大によるものと推察される。
When observing the soft tissue around the screw insertion part, it can be seen from FIG. 17 that
実施例３の結果により、複合層中のFGF-2は生体内でも生理活性を示し、特に皮膚組織の再生に有効であることが確かめられた。更に、複合層に含まれる活性FGF-2には骨組織・皮膚組織再生に有効となる最適量(少なくとも3.74 ng/cm2)が存在し、逆に多すぎると(例えば387 ng/cm2)効果が低減することが示された。 From the results of Example 3, it was confirmed that FGF-2 in the composite layer showed physiological activity even in vivo and was particularly effective for the regeneration of skin tissue. Furthermore, the active FGF-2 contained in the composite layer has an optimum amount (at least 3.74 ng / cm 2 ) effective for bone tissue / skin tissue regeneration, and conversely if it is too much (eg 387 ng / cm 2 ) It has been shown that the effect is reduced.
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