Engineering three-dimensional bone tissue in vitro using biodegradable scaffolds: Investigating initial cell-seeding density and culture period

New three-dimensional (3D) scaffolds for bone tissue engineering have been developed throughout which bone cells grow, differentiate, and produce mine ralized matrix. In this study, the percentage of cells anchoring to our pol ymer scaffolds as a function of initial cell seeding density was establishe d; we then investigated bone tissue formation throughout our scaffolds as a function of initial cell seeding density and time in culture. Initial cell seeding densities ranging from 0.5 to 10 x 10(6) cells/cm(3) were seeded o nto 3D scaffolds. After 1 h in culture, we determined that 25% of initial s eeded cells had adhered to the scaffolds in static culture conditions. The cell-seeded scaffolds remained in culture for 3 and 6 weeks, to investigate the effect of initial cell seeding density on bone tissue formation in vit ro. Further cultures using 1 x 10(6) cells/cm3 were maintained for 1 h and 1, 2, 4, and 6 weeks to study bone tissue formation as a function of cultur e period. After 3 and 6 weeks in culture, scaffolds seeded with 1 x 10(6) c ells/cm(3) showed similar tissue formation as those seeded with higher init ial cell seeding densities. When initial cell seeding densities of 1 x 10(6 ) cells/cm(3) were used, osteocalcin immunolabeling indicative of osteoblas t differentiation was seen throughout the scaffolds after only 2 weeks of c ulture. Von Kossa and tetracycline labeling, indicative of mineralization, occurred after 3 weeks. These results demonstrated that differentiated bone tissue was formed throughout 3D scaffolds after 2 weeks in culture using a n optimized initial cell density, whereas mineralization of the tissue only occurred after 3 weeks. Furthermore, after 6 weeks in culture, newly forme d bone tissue had replaced degrading polymer. (C) 2000 John Wiley & Sons, I nc.