β tricalcium phosphate β tcp granules

For in vivo experiments, 1 μg (when transplanted subcutaneously into the mouse) or 0.5 μg (when transplanted onto the mouse cranium) of rhBMP-2 (donated by Astellas Pharma, Tokyo, Japan) was adsorbed onto 20 mg β- tricalcium phosphate (β-TCP) granules (0.5–1.5 mm size; Osferion^®, Olympus, Tokyo, Japan), and then these materials were lyophilized. Just before transplantation, 60 μl of each fraction (BM, BMC, BM-PPP, PB, PRP, and PPP) was mixed with the lyophilized material, and then 10 μl of a bovine thrombin and 10% calcium chloride (1:1 ratio) mixture was added to the β-TCP granules/fraction mixture to trigger fibrin polymerization and produce an insoluble gel (Fig 1A). The final concentrations of thrombin and CaCl₂ in the grafting aspirates were 227.3 U/ml and 4.6 mg/ml, respectively.
Regarding the rhBMP-2 dose, we performed preliminary in vivo experiments to determine the suboptimal-doses which can induce minimal bone formation at 2 weeks following transplantation, and determined the suboptimal-doses (as low-doses) to be 1 μg/20 mg β-TCP (when subcutaneously transplanted to the mouse) (Fig 1B) or 0.5 μg/20 mg β-TCP (when transplanted onto the mouse cranium) (Fig 1C). Each experiment was performed in triplicate for three samples.

The GAMs used in the experiments in this study were all prepared the day before transplantation. Amounts of 1, 5, 10, 25, 50, or 100 μg of BMP4-nanoballs or 1000 μg of naked-AcGFP plasmid vector-harboring BMP4 cDNA in 60 μL of sterile water were mixed well with 20 mg β-tricalcium phosphate (β-TCP) granules (0.5–1.5 mm size; Osferion, Olympus, Tokyo, Japan) and 100 μL of 3% bovine atelocollagen (Atelocollagen Implant; Koken, Tokyo, Japan) inside the lids of 1.5-mL Eppendorf tubes; these mixtures were then lyophilized overnight. The GFP-nanoballs and vehicle AcGFP plasmids alone were used as the experimental controls. The experimental groups included those transfected with GFP-nanoballs, naked pGFP (as a control), BMP4-nanoballs, and naked pBMP4.
A manufactured GAM with 5 μg of BMP4-nanoballs was vertically or horizontally split, and the surface was coated with gold ions (IB-2; EIKO Engineering, Tokyo, Japan). The 3D structure of GAM was observed under a scanning electron microscope (SEM) (Miniscope^® TM-1000; HITACHI High-Tech Corp., Tokyo, Japan). The image magnification ranged from 100× to 10,000×. In this study, the accelerating voltage was set to 15 kV, and the contrast and brightness of the images were automatically adjusted to optimal values.