Journal of Veterinary Clinics (한국임상수의학회지)

The Korean Society of Veterinary Clinics (한국임상수의학회)
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Extraction of Crude-BMP from Bovine Cortical Bone for Bone Grafts

골이식물로서의 소뼈 치밀골에서 Crude-BMP의 추출

  • Choi Sung-jin (Animal Medical Center, Tokyo University) ;
  • Park Chul (College of Veterinary Medicine, Chonbuk National University) ;
  • Heo Soo-young (College of Veterinary Medicine, Chonbuk National University) ;
  • Lee Jong-il (Institute of Bio-Safety Research, Chonbuk National University) ;
  • Jeong In-seong (Royal Animal Medical Center) ;
  • Kim Nam-soo (College of Veterinary Medicine, Chonbuk National University) ;
  • Choi In-hyuk (College of Veterinary Medicine, Chonbuk National University)
  • 최성진 (동경대학 대학원) ;
  • 박철 (전북대학교 수의과대학) ;
  • 허수영 (전북대학교 수의과대학) ;
  • 이종일 (전북대학교 생체안전성연구소) ;
  • 정인성 (서울 로얄 메디칼 센터) ;
  • 김남수 (전북대학교 수의과대학) ;
  • 최인혁 (전북대학교 수의과대학)
  • Published : 2005.12.01
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Abstract

We tried to extract bone morphogenetic protein (BMP) from the freeze-dried bovine cortical bone (FBCB) for bone graft, which were defatted with chloroform-methanol for 20 days, freeze-dried at $-80^{\circ}C$ for 7 days and sterilized by ethylene oxide gas. Two kg of FBCB were pulverized in a wheel mill to $0.5-2.0mm^3$ cubic in size. The bone particles were demineralized in 0.6N HCI for 10 days at chloroform-methanol$4^{\circ}C$ and defatted with chloroform-methanol for 6 hours at room temperature, which was going to be defatting and demineralized cortical bone (DDM). For extracting BMP, DDM was agitated continuously through 72 hours with magnetic stirrer at $4^{\circ}C$ into 12 times of volume of 6 M guanidine hydrochloride (Gdn-HCl) solution containing proteinase inhibitors to protect BMP such as 2mM N-ethylaleimide, 1mM iodoacetic acid, 1mM phenylmethylsulfonyl fluoride and a sterilizer, 1mM sodium azide. The extraction procedure was repeated for three times. All extracted solution was centrifuged at 10,000 rpm for 30 min and then, the supernatant was dialyzed with 12 times of volume of deionized water at $4^{\circ}C$ for 24-72 hours, which cut off below 6,000-8,000 molecular weight. The dialyzed specimen contained crude-BMP was centrifuged, freeze-dried, and weighted. Through these processing, we could obtained $84.9\%$ as FBCB, $17.8\%$ as DDM and $0.71\%$ as crude-BMP from the wet cortical bone without cancellous bone, marrow and muscles. The crude-BMP were obtained $68.3\%$ from the first extraction, $29.6\%$ from secondary and $2.1\%$ from tertiary, respectively. It was suggested that high yield of crude-BMP migth be explained by three-time repetition of the extraction processing for crude-BMP with Gdn-Hcl sol.

Keywords

References

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