The effects of heat on the biological activity of recombinant human bone morphogenetic protein-2

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 degrees C for 15 min, or 1, 2, 4, or 8 h, or autoclaved at 120 degrees 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 microg 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 degrees or 70 degrees 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 degrees C for 8 h, based on in vitro assay data. However, heating above 90 degrees 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 degrees C.