Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces

Abstract

In this study we report on direct involvement of fluid shear stresses on the osteoblastic differentiation of marrow stromal cells. Rat bone marrow stromal cells were seeded in 3D porous titanium fiber mesh scaffolds and cultured for 16 days in a flow perfusion bioreactor with perfusing culture media of different viscosities while maintaining the fluid flow rate constant. This methodology allowed exposure of the cultured cells to increasing levels of mechanical stimulation, in the form of fluid shear stress, whereas chemotransport conditions for nutrient delivery and waste removal remained essentially constant. Under similar chemotransport for the cultured cells in the 3D porous scaffolds, increasing fluid shear forces led to increased mineral deposition, suggesting that the mechanical stimulation provided by fluid shear forces in 3D flow perfusion culture can indeed enhance the expression of the osteoblastic phenotype. Increased fluid shear forces also resulted in the generation of a better spatially distributed extracellular matrix inside the porosity of the 3D titanium fiber mesh scaffolds. The combined effect of fluid shear forces on the mineralized extracellular matrix production and distribution emphasizes the importance of mechanosensation on osteoblastic cell function in a 3D environment.

Fig. 1.

Calcium deposition in titanium fiber meshes during the 16-day culture period represented as equivalents of Ca²⁺ based on CaCl₂ standards used for the construction of the dose–response curve. Six cell culture conditions are reported: static and flow perfusion culture with dextran-free media (1x), media containing 3% dextran and twice the dextran-free media viscosity (2x), and media containing 6% dextran and three times the dextran-free media viscosity (3x). Pairwise comparisons were performed by using the Tukey–Kramer procedure with a significance level of 95%. *, highest calcium content; **, second highest calcium content; and ***, third highest calcium content.

Fig. 2.

Representative histological sections of scaffolds cultured for 16 days with static culture and media having 0% dextran (A), flow perfusion and media having 0% dextran (B), flow perfusion and media having 3% dextran (C), and flow perfusion and media having 6% dextran (D). Images are of histological cross sections of the cultured scaffolds. For flow perfusion culture specimens, the flow direction was from the top of the image through the scaffold to the bottom. Sections have been stained with basic fuchsin and methylene blue and viewed at ×10 magnification. The fibers of the titanium meshes appear black.

Fig. 3.

Representative SEM images of surfaces of scaffolds cultured for 16 days with static culture and 0% dextran (A), static culture and 3% dextran (B), static culture and 6% dextran (C), flow perfusion and 0% dextran (D), flow perfusion and 3% dextran (E), and flow perfusion and 6% dextran (F). Porous structures formed during flow perfusion culture are readily apparent in D–F.

Fig. 4.

Cellularity of titanium fiber meshes during the 16-day culture period. Cell culture conditions for days 4, 8, and 16 are reported: static and flow perfusion culture with dextran-free media (1x), media containing 3% dextran and twice the dextran-free media viscosity (2x), and media containing 6% dextran and three times the dextran-free media viscosity (3x).

Fig. 5.

AP activity of titanium fiber meshes during the 16-day culture period. Cell culture conditions for days 4, 8, and 16 are reported: static and flow perfusion culture with dextran free media (1x), media containing 3% dextran and twice the dextran-free media viscosity (2x), and media containing 6% dextran and three times the dextran-free media viscosity (3x). Pairwise comparisons were performed by using the Tukey–Kramer procedure with a significance level of 95%.