Abstract
This study was designed to investigate effects of heat on the bone-inducing activity of recombinant human bone morphogenetic protein (rhBMP)-2. rhBMP-2 samples were heated at 50, 70, 90, or 100°C for 15 min, or 1, 2, 4, or 8 h, or autoclaved at 120°C for 15 min. The bone-inducing activity of the rhBMP-2 before and after heating was assayed in in vivo and in vitro systems. For the in vivo assay, 5 µg rhBMP-2 samples were impregnated into porous collagen disks (6 mm in diameter, 1 mm thickness), freeze dried, and implanted into the back muscles of ddY mice. Three weeks later, the implant was harvested from the host and examined for ectopic new bone tissue by radiography. The new bone mass was quantified by single-energy X-ray absorptiometry. The in vitro activity of the rhBMP-2 was assayed by adding the BMP sample at a concentration of 100 ng/ml to cultures of MC3T3-E1 cells. After 48 h, the alkaline phosphatase activity was measured. After heating at 50° or 70°C, no significant reduction in bone-inducing activity was noted in either in vivo or in vitro assay systems unless the protein was exposed to sustained heat at 70°C for 8 h, based on in vitro assay data. However, heating above 90°C and for longer periods led to a decrease in the biological activity of the rhBMP-2 in a time- and temperature-dependent manner. rhBMP-2 was rendered inactive when exposed to temperatures at or in excess of 120°C.
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References
AJ Celeste JA Iannazzi RC Taylor RM Hewick V Rosen EA Wang JM Wozney (1990) ArticleTitleIdentification of transforming growth factor beta family members present in bone-inductive protein purified from bovine bone Proc Natl Acad Sci USA 87 9843–9847 Occurrence Handle2263636
MR Urist (1965) ArticleTitleBone formation by autoinduction Science 150 893–899 Occurrence Handle5319761
MR Urist H Iwata (1973) ArticleTitlePreservation and biodegradation of the morphogenetic property of bone matrix J Theor Biol 38 155–167 Occurrence Handle10.1016/0022-5193(73)90231-2 Occurrence Handle4196544
AH Reddi WA Anderson (1976) ArticleTitleCollagenous bone matrix-induced endochondral ossification hemopoiesis J Cell Biol 69 557–572 Occurrence Handle10.1083/jcb.69.3.557 Occurrence Handle1270511
TK Sampath N Muthukumaran AH Reddi (1987) ArticleTitleIsolation of osteogenin, an extracellular matrix-associated, bone-inductive protein, by heparin affinity chromatography Proc Natl Acad Sci USA 84 7109–7113 Occurrence Handle3478684
JM Wozney V Rosen AJ Celeste LM Mitsock MJ Whitters RW Kriz RM Hewick EA Wang (1988) ArticleTitleNovel regulators of bone formation: molecular clones and activities Science 242 1528–1534 Occurrence Handle3201241
EA Wang V Rosen P Cordes RM Hewick MJ Kriz DP Luxenberg BS Sibley JM Wozney (1988) ArticleTitlePurification and characterization of other distinct bone-inducing factors Proc Natl Acad Sci USA 85 9484–9488 Occurrence Handle3200834
EA Wang V Rosen JS D'Alessandro M Bauduy P Cordes T Harada DI Israel RM Hewick KM Kerns P LaPan DH Luxenberg D McQuid IK Moutsatsos J Nove JM Wozney (1990) ArticleTitleRecombinant human bone morphogenetic protein induces bone formation Proc Natl Acad Sci USA 87 2220–2224 Occurrence Handle2315314
JM Wozney (1992) ArticleTitleThe bone morphogenetic protein family and osteogenesis Mol Reprod Dev 32 160–167 Occurrence Handle10.1002/mrd.1080320212 Occurrence Handle1637554
K Takaoka H Yoshikawa J Hashimoto S Miyamoto K Masuhara H Nakahara M Matsui K Ono (1993) ArticleTitlePurification and characterization of a bone-inducing protein from a murine osteosarcoma (Dunn type) Clin Orthop 292 329–336 Occurrence Handle8519128
K Takaoka H Yoshikawa J Hashimoto K Masuhara S Miyamoto S Suzuki K Ono M Matsui S Oikawa N Tsuruoka Y Tawaragi C Inuzuka T Katayama M Sugiyama M Tsujimoto T Nakanishi H Nakazatio (1993) ArticleTitleGene cloning and expression of a bone morphogenetic protein derived from a murine osteosarcoma Clin Orthop 294 344–352 Occurrence Handle8358941
K Nakanishi K Sato T Sato M Takahashi N Fukaya T Miura (1992) ArticleTitlePreservation of bone morphogenetic protein in heat-treated bone (in Japanese) Nippon Seikeigeka Gakkai Zasshi (J Jpn Orthop Assoc) 66 949–955
T Ito S Sakano K Sato H Sugiura H Iwata Y Murata H Seo (1995) ArticleTitleSensitivity of osteoinductive activity of demineralized and defatted rat femur to temperature and duration of heating Clin Orthop 316 267–275 Occurrence Handle7634716
T Inokuchi H Ninomiya R Hironaka S Yoshida M Araki K Sano (1991) ArticleTitleStudies on heat treatment for immediate reimplantation of resected bone. J Cranio-Max Fac Surg 19 31–39
T Sato H Iwata M Takahashi T Miura (1993) ArticleTitleHeat tolerance of activity toward ectopic bone formation by rabbit bone matrix protein Ann Chir Gynaecol Suppl 207 37–40 Occurrence Handle8154835
H Izawa Y Hachiya T Kawai K Muramatsu Y Narita N Ban H Yoshizawa (2001) ArticleTitleThe effect of heat-treated human bone morphogenetic protein on clinical implantation Clin Orthop 390 252–258 Occurrence Handle11550873
LL Dunlop BK Hall (1995) ArticleTitleRelationships between cellular condensation, preosteoblast formation and epithelial-mesenchymal interactions in initiation of osteogenesis Int J Dev Biol 39 357–371 Occurrence Handle7545414
PRN Kind EJ King (1954) ArticleTitleEstimation of plasma phosphatase by determination of hydrolyzed phenol with amino-antipyrine J Clin Pathol 7 322–326 Occurrence Handle13286357
A Borzacchiello L Ambrosio L Nicolais D Ronca G Guida (2001) The temperature at the bone–cement interface: modeling and in vitro analysis F Pipino (Eds) Bone Cement and Cemented Fixation of Implants Zenit Rome 135–140
M Nawata S Wakitani H Nakaya A Tanigami T Seki Y Nakamura N Saito K Sano E Hidaka K Takaoka (2005) ArticleTitleUse of bone morphogenetic protein-2 and diffusion chambers to engineer cartilage tissue for the repair of defects in articular cartilage Arthritis Rheum 52 155–163 Occurrence Handle10.1002/art.20713 Occurrence Handle15641068
C Scheufler W Sebald M Hulsmeyer (1999) ArticleTitleCrystal structure of human bone morphogenetic protein-2 at 2.7-A resolution J Mol Biol 287 103–115 Occurrence Handle10.1006/jmbi.1999.2590 Occurrence Handle10074410
JM Wozney (1989) ArticleTitleBone morphogenetic proteins Prog Growth Factor Res 1 267–280 Occurrence Handle10.1016/0955-2235(89)90015-X Occurrence Handle2491264
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Ohta, H., Wakitani, S., Tensho, K. et al. The effects of heat on the biological activity of recombinant human bone morphogenetic protein-2. J Bone Miner Metab 23, 420–425 (2005). https://doi.org/10.1007/s00774-005-0623-6
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DOI: https://doi.org/10.1007/s00774-005-0623-6