Impact of Calcium Concentration on Transfection Efficiency and Osteogenic Differentiation

Objectives: With the rising use of bone marrow stromal cells (BMSCs) embedded in calcium-containing scaffolds, understanding the role extracellular calcium plays at the cellular level is paramount for the continued optimization of bone tissue regeneration. Previous studies have demonstrated that changes in extracellular calcium concentration affects both cell viability and differentiation. However, the range of calcium concentrations explored was limited and transfection efficiency was not examined. The objective of this study is to determine how the extracellular calcium level affects cell viability, transfection, and differentiation of the BMSCs.
Methods: Cell viability of the BMSCs was determined using MTS assay at 24, 48, and 72 hours post transfection Polyethyleneimine (PEI) complexed with enhanced green fluorescent protein (eGFP) plasmid were used to transfect the BMSCs, and expression of eGFP was quantified using flow cytometry. Osteogenic differentiation was qualitatively assessed after 14 days using alizarin red staining, and the presence of osteogenic markers was quantified using RT-PCR for osteocalcin and alkaline phosphatase after 7 days. To further understand the mechanism in which calcium improves transfection efficiency, confocal images were taken at 1, 2 and 4 hours to visualize uptake of the PEI and disassociation of the DNA from the complexes.
Results: The variation of calcium concentration present resulted in increased protection against the cytotoxic effects of the PEI, whilst significantly increasing the transfection efficiency of the complexes. Based on the confocal images, PEI is endocytosed at a faster rate leading to enhanced DNA released in the cells.
Conclusions: Our results indicate BMSCs are heavily influenced by the presence of extracellular calcium ions. We have successfully characterized the effect of calcium has on cell viability, transfection, and differentiation.