JPH02182260A - Granular bone prosthetic material having bone forming activity - Google Patents
Granular bone prosthetic material having bone forming activityInfo
- Publication number
- JPH02182260A JPH02182260A JP64000423A JP42389A JPH02182260A JP H02182260 A JPH02182260 A JP H02182260A JP 64000423 A JP64000423 A JP 64000423A JP 42389 A JP42389 A JP 42389A JP H02182260 A JPH02182260 A JP H02182260A
- Authority
- JP
- Japan
- Prior art keywords
- bone
- collagen
- granular
- forming protein
- particle size
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 38
- 239000000463 material Substances 0.000 title claims abstract description 14
- 230000000694 effects Effects 0.000 title description 7
- 108010035532 Collagen Proteins 0.000 claims abstract description 32
- 102000008186 Collagen Human genes 0.000 claims abstract description 32
- 229920001436 collagen Polymers 0.000 claims abstract description 32
- 239000002245 particle Substances 0.000 claims abstract description 30
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims abstract description 14
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims abstract description 14
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 claims description 18
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 claims description 18
- 229940112869 bone morphogenetic protein Drugs 0.000 claims description 18
- 239000000969 carrier Substances 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 11
- 108090000623 proteins and genes Proteins 0.000 abstract description 11
- 239000007864 aqueous solution Substances 0.000 abstract description 10
- 239000000243 solution Substances 0.000 abstract description 10
- 230000035876 healing Effects 0.000 abstract description 8
- 229920002134 Carboxymethyl cellulose Polymers 0.000 abstract description 3
- 239000001768 carboxy methyl cellulose Substances 0.000 abstract description 3
- 235000010948 carboxy methyl cellulose Nutrition 0.000 abstract description 3
- 239000008112 carboxymethyl-cellulose Substances 0.000 abstract description 3
- 238000005507 spraying Methods 0.000 abstract description 3
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 229940105329 carboxymethylcellulose Drugs 0.000 abstract 1
- 238000004587 chromatography analysis Methods 0.000 abstract 1
- 229960000789 guanidine hydrochloride Drugs 0.000 abstract 1
- 238000000034 method Methods 0.000 abstract 1
- 239000002808 molecular sieve Substances 0.000 abstract 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 abstract 1
- 239000002904 solvent Substances 0.000 abstract 1
- 239000000725 suspension Substances 0.000 abstract 1
- 230000007547 defect Effects 0.000 description 7
- 239000008187 granular material Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 108010045569 atelocollagen Proteins 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 230000011164 ossification Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- 102100037011 RNA cytidine acetyltransferase Human genes 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 230000002138 osteoinductive effect Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000002316 cosmetic surgery Methods 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 230000003239 periodontal effect Effects 0.000 description 2
- 238000004904 shortening Methods 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 230000008733 trauma Effects 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 208000018084 Bone neoplasm Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 239000005057 Hexamethylene diisocyanate Substances 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 101710167839 Morphogenetic protein Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 229910052586 apatite Inorganic materials 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000002805 bone matrix Anatomy 0.000 description 1
- 239000000316 bone substitute Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 210000000038 chest Anatomy 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 210000004207 dermis Anatomy 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- RRAMGCGOFNQTLD-UHFFFAOYSA-N hexamethylene diisocyanate Chemical compound O=C=NCCCCCCN=C=O RRAMGCGOFNQTLD-UHFFFAOYSA-N 0.000 description 1
- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- VSIIXMUUUJUKCM-UHFFFAOYSA-D pentacalcium;fluoride;triphosphate Chemical compound [F-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O VSIIXMUUUJUKCM-UHFFFAOYSA-D 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 208000028169 periodontal disease Diseases 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 210000003625 skull Anatomy 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Materials For Medical Uses (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、形成外科、口腔外科、歯周歯科などにおいて
、外傷や骨腫瘍の摘出、あるいは歯周疾患などによる骨
欠損部等に適用する骨補てっ材に関する。[Detailed Description of the Invention] (Industrial Application Field) The present invention is applicable in plastic surgery, oral surgery, periodontal dentistry, etc., for the removal of trauma or bone tumors, or for bone defects caused by periodontal disease, etc. Regarding bone prosthesis materials.
（従来の技術）
従来、外傷や腫瘍の切除などによる骨欠損部の補てつに
は主に自家骨移植が行なわれている。しかし、自家骨移
植の場合、その採取量に限度があることや、採取のとき
に新たな傷を作ることとなり、患者の負担となることな
どから、最近は他の材料を採用することが検討されてい
る。特に骨の主成分であるハイドロキシアパタイトは良
く検討されており、すでにブロック状や粒状などの形状
で市販され、形成外科や口腔外科、歯周歯科などで使用
さねている。(Prior Art) Conventionally, autologous bone grafting has been mainly used to compensate for bone defects caused by trauma or tumor resection. However, in the case of autologous bone grafting, there is a limit to the amount that can be harvested, and a new wound is created during harvesting, which is a burden on the patient, so recently, consideration has been given to using other materials. has been done. In particular, hydroxyapatite, which is the main component of bone, has been well studied and is already commercially available in block and granular forms, and is used in plastic surgery, oral surgery, periodontal dentistry, etc.
また近年は、骨誘導能を持たないハイドロキシアパタイ
トに代えて、骨誘導能を有する骨形成蛋白質（ｂｏｎｅ
ｍｏｒｐｈｏｇｅｎｅｔｉｃ ｐｒｏｔｅｉｎ、 Ｂ
ＭＰと略称される）を骨補てつ材に用いて治癒効果を向
上させる研究が進められており、この骨形成蛋白質を骨
補てつ材に使用するにあたり、その担体としてハイドロ
キシアパタイト、又はハイドロキシアパタイトと同様骨
の主成分でもあるコラーゲン、又は両者の複合体を用い
ることが検討されている（特開昭６０−２５３４５５号
公報、特開昭６１−２５９６７５号公報、特開昭６２−
１３５４３１号公報）。In addition, in recent years, bone morphogenetic protein (bone morphogenetic protein), which has osteoinductive ability, has been used instead of hydroxyapatite, which does not have osteoinductive ability.
morphogenetic protein, B
Research is underway to improve the healing effect of bone morphogenetic proteins (abbreviated as MP) in bone prosthetic materials. The use of collagen, which is a main component of bone like apatite, or a composite of both is being considered (Japanese Patent Application Laid-Open No. 60-253455, JP-A No. 61-259675, JP-A-62-
135431).
（発明が解決しようとする課題）
本発明は、コラーゲン等の担体に骨形成蛋白質を担持さ
せた骨補てっ材を使用して治療するに当リ、新生骨形成
を早め、治癒期間を短縮することを目的とするものであ
る。(Problems to be Solved by the Invention) The present invention accelerates new bone formation and shortens the healing period when performing treatment using a bone prosthesis material in which bone morphogenetic proteins are supported on a carrier such as collagen. The purpose is to
（課題を解決するための手段）
骨形成蛋白質は骨基質中に存在し、未分化間葉系細胞を
軟骨芽細胞、骨芽細胞に分化させる能力がある。骨補て
っ材に含有させた骨形成蛋白質の能力を十分に発揮させ
て新生骨形成を早め、骨欠損部の治癒を早めるためには
、骨形成蛋白質と間葉系細胞の接触を多くすると良いこ
とを知見した。(Means for Solving the Problems) Bone morphogenetic proteins exist in bone matrix and have the ability to differentiate undifferentiated mesenchymal cells into chondroblasts and osteoblasts. In order to fully demonstrate the ability of the bone morphogenetic proteins contained in the bone grafting material to accelerate new bone formation and speed up the healing of bone defects, increasing the contact between the bone morphogenetic proteins and mesenchymal cells is essential. I learned something good.
そして、骨形成蛋白質と間葉系細胞の接触を多くするに
は、骨形成蛋白質を表面積のできるだけ大きい担体と複
合化させることが必要である。そして、この表面積を大
きくするには、担体をスポンジ状にするか、粒状にする
かが考えられる。しかして、スポンジ状にした場合には
、骨欠損部の形状にあうよう加工する必要があり、使用
に際し煩雑である。その為に本発明では、骨欠損部の形
状と関係なく使用できる粒状にして用いるものである。In order to increase the contact between bone morphogenetic proteins and mesenchymal cells, it is necessary to complex the bone morphogenetic proteins with a carrier having as large a surface area as possible. In order to increase this surface area, it is possible to make the carrier sponge-like or granular. However, if it is made into a sponge shape, it must be processed to fit the shape of the bone defect, which is complicated to use. Therefore, in the present invention, it is used in the form of granules that can be used regardless of the shape of the bone defect.
そして担体を粒状にして用いる場合にはその粒径が大き
く影響し、本発明者等は種々検討の結果、１００〜２０
００μの粒径の粒状にしたときに最も良好な治癒短縮効
果が得られることを見出し、本発明を完成した。When the carrier is used in the form of particles, the particle size greatly affects the carrier, and as a result of various studies, the present inventors found that
The present invention was completed based on the discovery that the best healing shortening effect can be obtained when the granules have a particle size of 00 μm.
すなわち、本発明は、粒径１００〜２０００μのコラー
ゲン粒状担体に骨形成蛋白質を担持させた粒状骨補てつ
材である。That is, the present invention is a granular bone prosthesis material in which bone morphogenetic proteins are supported on collagen granular carriers having a particle size of 100 to 2000 microns.
骨形成蛋白質は、一般に新鮮な牛骨から４Ｍグアーシン
塩酸溶液で抽出される。この抽出物を５ｅｐｈａｃｒｙ
ｌ Ｓ−２００で（ファルマシア社製）を用いた分子ｆ
”６ ’１１で分離し、得られる活性画分を更にカルボ
キシメチルセルローズ、最後にハイドロキシアパタイト
カラム、クロマ１−クラフィーを用いて精製した。精製
はハイドロキシアパタイトカラムのみでも行なえる。ま
た、上記抽出物も骨誘導性を有している。Bone morphogenetic proteins are generally extracted from fresh bovine bone with a 4M guarcine hydrochloride solution. 5ephacry this extract
Molecule f using l S-200 (manufactured by Pharmacia)
The active fraction obtained was further purified using carboxymethyl cellulose, and finally using a hydroxyapatite column and chroma 1-craphy. Purification can also be performed using a hydroxyapatite column alone. It also has osteoinductive properties.
コラーゲンは、生体の主要タンパク質の一つであり、生
体の細胞間マトリックスを構成しているものであって、
生体材料として既に多方面で研究応用されており、その
安全性については既に確立されている。また、コラーゲ
ン自身は抗原性の非常に低い蛋白質であるが、その主要
な抗原部位は分子末端のごく一部である。本発明におい
ては、この分子末端を除いた、所Ｊｉ７アテロコラーゲ
ンを用いるのが好ましい。粒径１００〜２０００μのコ
ラーゲンの粒状担体（粒子）は、例えば、コラーゲン水
溶液を水と混和しない有機溶媒中に０〜２５°Ｃの温度
で多数の小滴として分散させて乳濁液を形成し、この乳
濁液の温度を３０〜４０’Ｃに上昇させて前記小滴を固
化させてつくる。固化したコラーゲン粒子をヘキサメチ
レンジイソシアナー１〜、ゲルタールアルデヒド等で架
橋処理して、耐熱変性、貯蔵安定性を改善して使用して
も良い。Collagen is one of the main proteins in living organisms and constitutes the intercellular matrix of living organisms.
It has already been researched and applied as a biomaterial in many fields, and its safety has already been established. Furthermore, although collagen itself is a protein with very low antigenicity, its main antigenic site is only a small part of the terminal end of the molecule. In the present invention, it is preferable to use Ji7 atelocollagen with this molecular terminal removed. Granular carriers (particles) of collagen with a particle size of 100 to 2000 μ can be prepared, for example, by dispersing an aqueous collagen solution as a large number of droplets in a water-immiscible organic solvent at a temperature of 0 to 25° C. to form an emulsion. , by raising the temperature of the emulsion to 30-40'C to solidify the droplets. The solidified collagen particles may be crosslinked with hexamethylene diisocyaner 1 or more, geltaraldehyde, etc. to improve heat resistance and storage stability before use.
本発明でコラーゲン粒状担体の粒径を１００〜２０００
μとするのは、後述の実施例１で明らかなように、この
範囲の粒径のものが治癒期間短縮効果に最も優れている
からである。In the present invention, the particle size of the collagen granular carrier is 100 to 2000.
The reason why μ is used is because, as will be clear from Example 1 below, particles with a particle size within this range are most effective in shortening the healing period.
コラーゲン粒子を担体にし、これに骨形成蛋白質を担持
させるには、精製した骨形成蛋白質の水溶液をコラーゲ
ン粒子にスプレーしたり、或は該水溶液にコラーゲン粒
子を浸漬したりすることにより行なう。また、コラーゲ
ン溶液に混合してから造粒することもできる。Collagen particles are used as carriers to support bone morphogenetic proteins by spraying the collagen particles with an aqueous solution of purified bone morphogenetic proteins or by immersing the collagen particles in the aqueous solution. Alternatively, it can be mixed into a collagen solution and then granulated.
またコラーゲンとハイ１くロキシアパタイトを併用して
担体に用いることもできる。ハイドロキシアパタイトは
歯科関連の骨補てつ材として常用されているが、新生骨
が結合組織を介さず直接ハイドロキシアパタイトに結合
するなど骨親和性が高い。更に、これをコラーゲンと併
用することによって治癒のより一層の短期化が期待でき
る。この併用した担体は１例えば、１００〜２０００μ
の粒状のハイドロキシアパタイトにコラーゲンをスプレ
ー浸漬などにより塗布して調製する。ハイドロキシアパ
タイトは合成したもの又は天然骨を焼成したものを粉砕
などにより粒状にしたものでよいが、天然骨から得たも
のが好ましい。Further, collagen and high-loxyapatite can be used in combination as a carrier. Hydroxyapatite is commonly used as a dental prosthetic material, and has high bone compatibility, with new bone directly bonding to hydroxyapatite without the use of connective tissue. Furthermore, by using this in combination with collagen, it is expected that the healing time will be further shortened. The carrier used in combination is 1, for example, 100 to 2000μ
It is prepared by applying collagen to granular hydroxyapatite by spray dipping or the like. Hydroxyapatite may be synthesized or calcined from natural bone and pulverized into granules, but hydroxyapatite obtained from natural bone is preferred.
実施例１
（１，）骨形成蛋白質の調製
新鮮牛骨を液体窒素で凍結し、約０．５ｍｍに粉砕する
。これをクロロホルムとメタノールとの１：１混合溶剤
で脱脂し、０．５Ｍ ＨＣＱで脱灰した。この残渣を蛋
白質分解酵素阻害剤を含む４Ｍグアニジン−塩酸及び０
．０５Ｍ ＋−リス−塩酸のＰＩ−１７，４の水溶液で
抽出し、遠心分離後透析、凍結乾燥を行ない粗管形成蛋
白質を得た。Example 1 (1.) Preparation of bone morphogenetic protein Fresh bovine bone is frozen in liquid nitrogen and ground to approximately 0.5 mm. This was degreased with a 1:1 mixed solvent of chloroform and methanol, and deashed with 0.5M HCQ. This residue was mixed with 4M guanidine-hydrochloric acid containing a protease inhibitor and 0%
．． It was extracted with an aqueous solution of PI-17,4 in 05M+-Lis-HCl, centrifuged, dialyzed, and freeze-dried to obtain crude tube-forming protein.
この粗管形成蛋白質を４阿グアニジン−塩酸及び０．０
５Ｍ ｌ−リス−塩酸を含むｐ Ｈ７、４の水溶液に溶
解し、５ｅｐｈａｃｒｙ１． Ｓ−２００カラム（４Ｘ
１５０ｃｍ）に添加し、同溶液にて溶出した。５ｅｐ
ｈａｃｒｙｌ Ｓ−２００で得られた活性画分を、６Ｍ
尿素及び０．０５Ｍ酢酸ナトリウムを含むＰＩ、８の水
溶液に溶解し、ＣＭ５２（カルボキシメチルセルロース
）カラム（１、６Ｘ ６ｃｍ）に添加し、これに吸着す
る蛋白質を０〜０．５ＭのＮａＣＱ濃度勾配（１００ｍ
Ω）をかけ分画した。さらに、ＣＭ５２で得られた活性
画分を６Ｍ尿素及び０．０１Ｍリン酸二水素カリウムを
含むｐｕ６０ｇの水溶液に溶解し、ハイドロキシアパタ
イトカラムに添加し、これに吸着する蛋白質を０．０１
〜０．４００ＭのＫＨ２ＰＯ４濃度勾配（１００ｍ Ｑ
）をかけ分画した。This rough tube forming protein was dissolved in 4-guanidine-hydrochloric acid and 0.0
5 ephacry1. S-200 column (4X
150 cm) and eluted with the same solution. 5ep
The active fraction obtained with hacryl S-200 was added to 6M
It was dissolved in an aqueous solution of PI, 8 containing urea and 0.05 M sodium acetate and applied to a CM52 (carboxymethylcellulose) column (1,6X 6 cm), and the protein adsorbed thereon was subjected to a 0-0.5 M NaCQ concentration gradient ( 100m
Ω) and fractionated. Furthermore, the active fraction obtained with CM52 was dissolved in an aqueous solution of 60 g of pu containing 6 M urea and 0.01 M potassium dihydrogen phosphate, and added to a hydroxyapatite column, and the protein adsorbed thereon was reduced to 0.01 g.
~0.400M KH2PO4 concentration gradient (100m Q
) and fractionated.
（２）コラーゲン粒子の調製
新鮮な仔牛の真皮を無菌的に取り出し細断し、コラーゲ
ンに対し１％のペプシンを加え溶解し、精製して得た無
菌で発熱性物質を含まないアテロコラーゲンをｐＨ３゜
５の塩酸水溶液に２％濃度に溶解し、次いで０．３Ｍ塩
化すＩ−リウムと０．０４Ｍリン酸水素二す１−リウム
を含む水溶液を同量加えてアテロコラーゲンの１％中性
溶液を調製した。この溶液を、比重を調製した１〜ルエ
ンに小滴になるよう分散させ、その後３７°Ｃに加温し
た。アテロコラーゲンの小滴が固化してコラーゲンの粒
子が得られた。(2) Preparation of collagen particles Fresh calf dermis is aseptically removed and shredded, 1% pepsin is added to the collagen to dissolve it, and the resulting sterile, pyrogen-free atelocollagen is purified at pH 3°. A 1% neutral solution of atelocollagen was prepared by dissolving No. 5 in an aqueous hydrochloric acid solution to a 2% concentration, and then adding the same amount of an aqueous solution containing 0.3M I-lium chloride and 0.04M di-1-lium hydrogen phosphate. did. This solution was dispersed into small droplets in toluene having an adjusted specific gravity, and then heated to 37°C. The atelocollagen droplets solidified to obtain collagen particles.
この粒子をメタノールで脱水後、ヘキサメチレンジイソ
シアナートで架橋を行ない、良く洗浄後ＰＢＳ（Ｐｈｏ
ｓｐｈａｔｅ Ｂｕｆｆｅｒｅｃｌ ５ａｌｉｎ）に置
換した。After dehydrating the particles with methanol, crosslinking them with hexamethylene diisocyanate, washing well, and adding PBS (Pho
sphate Buffercl 5alin).
得られた粒状物を篩で、１００μ以下、１００〜５００
μ、５００〜１０００μ、１０００〜２０００μの４ク
ラスに分級した。The obtained granules are sieved to 100μ or less, 100 to 500
It was classified into four classes: μ, 500-1000μ, and 1000-2000μ.
（３）骨補てつ材の調製
前記（２）で得た各分級の粒状物２ｍｇづつに、前記（
１）で得た粗管形成蛋白質２ｍｇに０．０５Ｍ酢酸０．
１ｎ＋Ｑを加えたものを混合し、次いで凍結乾燥した。(3) Preparation of bone prosthesis material Add 2 mg of each classification of granules obtained in (2) above to (
Add 0.05M acetic acid to 2mg of the crude tube-forming protein obtained in 1).
1n+Q was mixed and then lyophilized.
（４）試験とその結果
一
このように処理した各分級の粒状物０．２ｍｇをウィス
ター系ラット（雄、４週齢）の左右の胸部皮下に埋入し
、２週間後に層殺し、軟Ｘ線による検索、生化学的検索
（アルカリフォスファターゼ活性＝ＡＬＰ活性、カルシ
ウム含楚の測定）を行なった。(4) Tests and Results - 0.2 mg of the granules of each classification treated in this way were subcutaneously implanted into the left and right chests of Wistar rats (male, 4 weeks old), and after 2 weeks, they were delaminated and soft X-rayed. Line search and biochemical search (alkaline phosphatase activity = ALP activity, measurement of calcium content) were performed.
その結果は次の通りであった。The results were as follows.
１００μ以下 １０５
１００〜５００μ １７０
９８５００−１０００μ ３００
１０３１０００〜２０００μ
１１０ ２３このように、
粒径１００μ以下の粒子ではＡＬＰ活性、カルシウム量
がわずかに検出されたにすぎず、満足すべきものではな
かった。そして、粒径が大きくなるに従って良くなり、
ＡＬＰ活性は粒径５００〜１０００μのものが最高にな
り、また、カルシウム量も粒径５００〜１０００μのも
のが最高になった。粒径１０００〜２０００μのものは
粒径５００〜１０００μのものより劣る傾向を示した。100μ or less 105 100-500μ 170
98500-1000μ 300
1031000~2000μ
110 23 In this way,
For particles with a particle size of 100 μm or less, only a small amount of ALP activity and calcium was detected, which was not satisfactory. And as the particle size increases, it gets better,
The ALP activity was highest when the particle size was 500 to 1000 μm, and the amount of calcium was also highest when the particle size was 500 to 1000 μm. Those with a particle size of 1000 to 2000μ showed a tendency to be inferior to those with a particle size of 500 to 1000μ.
実施例２
新鮮な牛骨を煮沸し脂質などを取り除き１％過酸化水素
水溶液及び１％水酸化ナトリウム水溶液に浸漬し、水洗
後、６００℃に加熱して有機物を除去した。次いでこれ
を１１００℃で焼成した。次いでこの焼成物を粉砕し、
篩で６００〜１０００μに分級して洗浄した。これにコ
ラーゲン３％水溶液及び実施例１の（１）で調製した骨
形成蛋白質０．１％水溶液を順次スプレーによりコーテ
ィングして骨補てっ材を作った。Example 2 Fresh beef bones were boiled to remove lipids and the like, immersed in a 1% aqueous hydrogen peroxide solution and a 1% aqueous sodium hydroxide solution, washed with water, and then heated to 600°C to remove organic matter. This was then fired at 1100°C. Next, this fired product is crushed,
It was classified with a sieve to a size of 600 to 1000μ and washed. This was coated with a 3% collagen aqueous solution and a 0.1% bone morphogenetic protein aqueous solution prepared in Example 1 (1) sequentially by spraying to prepare a bone prosthetic material.
ラットの頭蓋骨に直径８ｍｍの骨欠損を作り、上記の骨
補てっ材を埋入した。対照として上記のコーティングを
行なわないものを骨補てつ材として埋入した。A bone defect with a diameter of 8 mm was created in the skull of a rat, and the above bone replacement material was implanted. As a control, one without the above coating was implanted as a bone prosthesis.
その結果、対照のものは４週で骨欠損部の中央まで骨の
新生が認められたのに対し、上記のコーティングしたも
のは３週で既に同じ結果が認められた。As a result, new bone formation was observed in the control sample up to the center of the bone defect after 4 weeks, whereas the same result was observed in the coated sample after 3 weeks.
（発明の効果）
本発明においては、骨形成蛋白質を担持する担体として
コラーゲン又はコラーゲンとハイドロキシアパタイトと
の併用物の粒状物を使用したために、骨形成蛋白質と未
分化間葉系細胞との接触がよくなり、しかもその粒径を
１００〜２０００μにしたため、新生骨形成が円滑に行
なわれ、したがって本発明の骨補てつ材を使用すること
により、治癒の期間を短縮化することができる。(Effects of the Invention) In the present invention, since collagen or a granular material of a combination of collagen and hydroxyapatite is used as a carrier for carrying bone morphogenetic proteins, contact between bone morphogenetic proteins and undifferentiated mesenchymal cells is prevented. Moreover, since the particle size is set to 100 to 2000 microns, new bone formation is performed smoothly, and therefore, by using the bone prosthetic material of the present invention, the healing period can be shortened.
Claims (1)
形成蛋白質を担持させた粒状骨補てつ材。 ２ コラーゲン粒状担体がコラーゲンとハイドロキシア
パタイトとからなる請求項第１項記載の粒状骨補てつ材
。[Scope of Claims] 1. A granular bone prosthesis material in which bone morphogenetic protein is supported on collagen granular carriers having a particle size of 100 to 2000 μm. 2. The granular bone prosthetic material according to claim 1, wherein the collagen granular carrier comprises collagen and hydroxyapatite.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP64000423A JPH02182260A (en) | 1989-01-06 | 1989-01-06 | Granular bone prosthetic material having bone forming activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP64000423A JPH02182260A (en) | 1989-01-06 | 1989-01-06 | Granular bone prosthetic material having bone forming activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02182260A true JPH02182260A (en) | 1990-07-16 |
Family
ID=11473395
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP64000423A Pending JPH02182260A (en) | 1989-01-06 | 1989-01-06 | Granular bone prosthetic material having bone forming activity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02182260A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004098540A1 (en) * | 1993-09-16 | 2004-11-18 | Hiroyuki Kitamura | Material for treating paradental diseases |
| WO2005052541A3 (en) * | 2003-11-28 | 2005-08-04 | Nat Food Res | Method of separation and collection and apparatus therefor |
| CN1302822C (en) * | 2002-11-28 | 2007-03-07 | 上海瑞邦生物材料有限公司 | In-situ osteoplastic active calcium phosphate cement and its prepn and application |
| USD849946S1 (en) | 2015-12-30 | 2019-05-28 | Nuvasive, Inc. | Interspinous process spacer |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62501132A (en) * | 1984-12-14 | 1987-05-07 | ドレナ−ト・クラウス | bone substitute material |
| JPS63125258A (en) * | 1986-11-14 | 1988-05-28 | 三菱マテリアル株式会社 | Bone deficient part, gap part and absorbing part filler |
-
1989
- 1989-01-06 JP JP64000423A patent/JPH02182260A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62501132A (en) * | 1984-12-14 | 1987-05-07 | ドレナ−ト・クラウス | bone substitute material |
| JPS63125258A (en) * | 1986-11-14 | 1988-05-28 | 三菱マテリアル株式会社 | Bone deficient part, gap part and absorbing part filler |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004098540A1 (en) * | 1993-09-16 | 2004-11-18 | Hiroyuki Kitamura | Material for treating paradental diseases |
| CN1302822C (en) * | 2002-11-28 | 2007-03-07 | 上海瑞邦生物材料有限公司 | In-situ osteoplastic active calcium phosphate cement and its prepn and application |
| WO2005052541A3 (en) * | 2003-11-28 | 2005-08-04 | Nat Food Res | Method of separation and collection and apparatus therefor |
| USD849946S1 (en) | 2015-12-30 | 2019-05-28 | Nuvasive, Inc. | Interspinous process spacer |
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