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

TLDR

New 3‑D scaffolds for bone tissue engineering have been developed that support bone cell growth, differentiation, and mineralized matrix production. The study aimed to determine how initial cell seeding density influences cell attachment and bone tissue formation over time in polymer scaffolds. Scaffolds were seeded with densities from 0.5 to 10 × 10⁶ cells cm⁻³, cultured for 3–6 weeks, and a subset at 1 × 10⁶ cells cm⁻³ was examined at 1 h, 1, 2, 4, and 6 weeks to assess tissue formation. After 1 h, 25 % of seeded cells adhered; by 2 weeks osteocalcin indicated osteoblast differentiation; mineralization appeared after 3 weeks; and by 6 weeks bone tissue had replaced the polymer, with no additional benefit from higher seeding densities.

Abstract

New three-dimensional (3D) scaffolds for bone tissue engineering have been developed throughout which bone cells grow, differentiate, and produce mineralized matrix. In this study, the percentage of cells anchoring to our polymer scaffolds as a function of initial cell seeding density was established; 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 onto 3D scaffolds. After 1 h in culture, we determined that 25% of initial seeded 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 vitro. Further cultures using 1 x 10(6) cells/cm(3) were maintained for 1 h and 1, 2, 4, and 6 weeks to study bone tissue formation as a function of culture period. After 3 and 6 weeks in culture, scaffolds seeded with 1 x 10(6) cells/cm(3) showed similar tissue formation as those seeded with higher initial cell seeding densities. When initial cell seeding densities of 1 x 10(6) cells/cm(3) were used, osteocalcin immunolabeling indicative of osteoblast differentiation was seen throughout the scaffolds after only 2 weeks of culture. 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 an optimized initial cell density, whereas mineralization of the tissue only occurred after 3 weeks. Furthermore, after 6 weeks in culture, newly formed bone tissue had replaced degrading polymer.