Prior to the in vivo studies, the biological activity of rhBMP-7 from different manufacturers was determined in an alkaline phosphatase (ALP) activity assay in ATDC5 cells, amongst them rhBMP-7 from R&D and ProSpec-Tany. rhBMP-7 from ProSpec-Tany showed the highest biological activity, as shown in Additional file 2 , and was further chosen to be employed in the in vivo studies.
rhBMP-7 (CYT-276, ProSpec-Tany TechnoGene Ltd) was reconstituted in 60 μl sterile water. This solution was dialyzed against a buffer solution containing 17% sucrose, 20% mannitol, 332 mM glycine, and 0.8% Tween 20, using a slide-A-Lyzer dialysis cassette (66454, Thermo Fisher Scientific Inc., Rockford, IL, USA) with a molecular weight cutoff of 10 kDa overnight with 4 buffer changes. The final solution containing 300 μg of rhBMP-7 (calculated amount) was freeze-dried overnight and reconstituted in sterile water, to achieve a final concentration of 250 μg rhBMP-7 in 40 μl buffer solution containing 1% sucrose, 1.2% mannitol, 20 mM glycine, and 0.05% Tween 20. Activity of the dialysate was shown to be retained in vitro through its capacity to induce ALP production in mice ATDC5 cells. Additional file2 shows the biologic activity of the dialyzed rhBMP-7 compared with the pre-dialyzed rhBMP-7.
rhBMP-7 (CYT-276, ProSpec-Tany TechnoGene Ltd) was reconstituted in 60 μl sterile water. This solution was dialyzed against a buffer solution containing 17% sucrose, 20% mannitol, 332 mM glycine, and 0.8% Tween 20, using a slide-A-Lyzer dialysis cassette (66454, Thermo Fisher Scientific Inc., Rockford, IL, USA) with a molecular weight cutoff of 10 kDa overnight with 4 buffer changes. The final solution containing 300 μg of rhBMP-7 (calculated amount) was freeze-dried overnight and reconstituted in sterile water, to achieve a final concentration of 250 μg rhBMP-7 in 40 μl buffer solution containing 1% sucrose, 1.2% mannitol, 20 mM glycine, and 0.05% Tween 20. Activity of the dialysate was shown to be retained in vitro through its capacity to induce ALP production in mice ATDC5 cells. Additional file