Osteogenic performance of MC3T3-E1 cells on granular hydroxyapatite scaffold

Abstract

The initial seeding density plays a crucial role in determining the effectiveness of bone tissue regeneration utilising scaffold. One of the factors that influence cell osteoblast differentiation is cell seeding related factor. Therefore, this study aims to determine the effect of different initial seeding densities on the osteoblast performance of MC3T3-E1 cells in hydroxyapatite granular scaffold. MC3T3-E1 cells at different seeding density (5x10⁵ and 1x10⁶ cells/cm²) were cultured in the 2-dimensional flask and 3-dimensional hydroxyapatite scaffold. Cells in both cultures were analysed in term of morphology using CellB software while biochemical activity was assessed using Alkaline Phosphatase (ALP) assay. MC3T3-E1 showed a mononucleated, fibroblast-like shape cell with extended cytoplasmic projection. Cell seeding density of 1x10⁶ cells/cm² reached 90% confluency while 5x10⁵ cells/cm² reached 70% confluency on day six of culture. Variations in cell seeding density significantly influenced the cell osteodifferentiation. During 2-Dimensional osteoblast differentiation, lower initial seeding density resulted in higher ALP activity. However, cells were seeded on hydroxyapatite scaffold, by increasing the seeding density from 5x10⁵ to 1x10⁶ cells/cm² resulted in decreased of ALP activity. This study has shown that the seeded cell population in the 3-dimensional hydroxyapatite scaffolds clearly affected the degree of osteoblast cell differentiation in which a higher seeding density was not necessarily better. The seeding density played a major role in influencing the corresponding cell differentiation.

    

Keywords: Seeding density; osteoblast; Granumas®; alkaline phosphatase