Optimization of cell seeding efficiencies on a three-dimensional gelatin scaffold for bone tissue engineering.

Bone tissue engineering techniques hold great potential for the treatment of clinical defects. However, there is much optimization needed before bone tissue engineering can be used therapeutically. This study evaluated various cell seeding methods onto a porous three-dimensional (3D) scaffold for bone tissue engineering optimization. MG63 human osteoblast-like cells were seeded onto a resorbable, porous gelatin sponge in different suspension volumes (50 microl and 5 ml), and culture conditions, (static, shaken, rolled, or rotatory bioreactor). The DNA of the cells in the scaffold, the media and the containers were quantitated separately to determine the cell number and location after 3 days of culture. The samples were stained with calcein and viewed using confocal microscopy to determine cell viability and location. Placing a small cell suspension (50 microl) directly onto the scaffold produced a significantly higher proportion of cells adhered to the scaffold than a larger cell suspension (5 ml). In all conditions except the rotatory bioreactor, the percentage of cells remaining on the scaffold after 3 days in a small seeding volume (63 +/- 22%) was significantly higher than the larger seeding volume (36 +/- 25%). In the case of the rotatory bioreactor, the opposite appeared to be true (39 +/- 9% small volume and 72 +/- 14% larger volume). It was important to keep the seeding dynamics of the cultivated tissue engineered construct consistent throughout the experiments to ensure reproducibility. For this scaffold type, cells applied in a small volume and cultured on a plate shaker at 120 rpm (giving 81 +/- 14% of cells adhered to the scaffold) for 3 days is recommended.