Development of an early estimation method for predicting later osteogenic differentiation activity of rat mesenchymal stromal cells from their attachment areas

Abstract

Cell morphology has received considerable attention in recent years owing to its possible relationship with cell functions, including proliferation, differentiation, and migration. Recent evidence suggests that extracellular environments can also mediate cell functions, particularly cell adhesion. The aims of this study were to investigate the correlation between osteogenic differentiation activity and the morphology of rat mesenchymal stromal cells (MSCs), and to develop a method of estimating osteogenic differentiation capability of MSCs on biomaterials. We measured the attachment areas of MSCs on substrates with various types of surface after 2 h of seeding, and quantified the amount of osteocalcin secreted from MSCs after 3 weeks of culture under osteogenic differentiation conditions. MSCs with small attachment areas showed a high osteogenic differentiation activity. These findings indicate that cell attachment areas correlate well with the osteogenic differentiation activity of MSCs. They also suggest that the measurement of cell attachment areas is useful for estimating the osteogenic differentiation activity of MSCs and is a practical tool for applications of MSCs in regenerative medicine.

Keywords: apatite; cell morphology; mesenchymal stromal cells; osteogenic differentiation activity; proliferative activity; regenerative medicine; titanium.

Figure 1

X-ray diffraction patterns of the top surfaces of (A) Ti, (B) Ti-Ap, (C) H₂O₂–Ti-Ap, and (D) 300 °C-Ti-Ap substrates.

Figure 2

Scanning electron micrographs of the top surfaces of (A) Ti-Ap, (B) H₂O₂–Ti-Ap, (C) 300 °C-Ti-Ap, and (D) Ti substrates. Bar: 2 μm.

Figure 3

Cell attachment areas of live MSCs in early stage on (A) Ti, (B) Ti-Ap, (C) H₂O₂–Ti-Ap, and (D) 300 °C-Ti-Ap. Asterisks indicate significant differences (t-test: P < 0.05).

Figure 4

Fluorescence microscopy images of MSCs on (A) Ti, (B) Ti-Ap, (C) H₂O₂–Ti-Ap, and (D) 300 °C-Ti-Ap substrates.

Figure 5

Amounts of osteocalcin/protein in MSCs after 3 weeks of culture on (A) Ti, (B) Ti-Ap, (C) H₂O₂–Ti-Ap, and (D) 300 °C-Ti-Ap substrates.

Figure 6

Correlation between the cell attachment area in early stage and the osteogenic differentiation activity after 3 weeks of culture on (A) Ti, (B) Ti-Ap, (C) H₂O₂–Ti-Ap, and (D) 300 °C-Ti-Ap substrates.