高分子電解質錯体による骨誘導因子担体の開発

利用聚电解质复合物开发骨诱导因子载体

• 批准号: 11771244
• 负责人: 井上 寧
• 金额: $ 1.15万
• 依托单位: Showa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Encouragement of Young Scientists (A)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11771244/
• 关键词: 高分子電解質錯体; 骨誘導因子; BMP; 骨形成; ハイドロキシアパタイト

BMPの精製ウシ大腿骨骨幹部を凍結下にて1〜2ミリの大きさに粉砕し,クロロホルムメタノール溶液で脱脂.その後0.6N塩酸で脱灰し,この脱灰骨より4Mグアニジン塩酸でBMPを抽出.この抽出溶液を蒸留水にて透析し,不溶性画分(G-EXT)を作製.これを6M尿素で再溶解し,Heparin-Sepharose Chromatographyを用いて粗精製のBMP画分を蒸留水にて透析後,凍結乾燥しBMPの移植体とした.BMPの生物学的活性の確認精製されたBMP移植体5mgと一型コラーゲン15mgとを混合し,100%エタノールで遠心分離後,凍結乾燥を行い,No5ケラチンカプセルに封入し,4週齢のS.D.系ラット雄の大腿部筋嚢に埋入した。3週間後に大腿部の,軟X線撮影後,脱灰切片及び非脱灰切片を作製し,組織学的に異所性骨誘導活性を観察した.PECとBMP複合体の骨誘導活性の確認活性の確認されたBMP5mgと4Mグアニジン塩酸溶液2mlに溶解し,種々のPEC複合体を作製し,4週齢のS.D.系ラット雄の大腿部筋嚢に埋入し,異所性骨誘導活性を確認した.PECとハイドロキシアパタイト複合体の作製多孔性のハイドロキシアパタイトを担体として有効なPECと混合し,これをS.D.系ラット大腿部筋嚢に埋入し,組織学的に生体親和性の確認を行い,PECハイドロキシアパタイト複合体とBMPを混合し三重合体を作製した。それを大腿部筋嚢に移植し,経時的に担体の吸収過程,異所性の骨形成活性を軟X線学的,組織学的に観察する.

BMP is refined by freezing the femoral bone stem for 1 ~ 2 minutes. 0.6N hydrochloric acid is used for ash removal, and the ash removal bone is extracted by 4M hydrochloric acid. The extraction solution is dialyzed with distilled water, and the insoluble fraction (G-EXT) is prepared. After 6M urea redissolution,Heparin-Sepharose Chromatography was used to refine BMP fractions, dialyze in distilled water, freeze dry BMP grafts, and confirm biological activity of BMP. After purification BMP grafts 5mg and type I BMP 15mg were mixed, 100% of BMP was isolated, freeze dry was performed, and No5 mg of BMP was encapsulated. After 4 weeks, S.D. system was embedded in thigh tendon. After 3 weeks, soft X-ray imaging of the thigh was performed, demineralized sections and non-demineralized sections were prepared, and histological heterogenous osteoinductive activity was observed. Confirmation of osteoinductive activity of PEC and BMP complex was performed. BMP5mg and 4M in 2ml of hydrochloric acid solution were dissolved, and PEC complex was prepared. At 4 weeks, S.D. was embedded in the thigh tendon of male. Confirmation of heterotrophic bone-inducing activity.PEC and PEC complexes. Preparation of porous PEC and mixed PEC complexes. The bone graft of the thigh muscle, the absorption process of the tissue, the bone formation activity of the heterosis, the soft X-ray and histological observation.