Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

Abstract

Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering.

Figure 1

Schematic of flow perfusion system. (A) Design of an individual flow chamber. (B) Relation to the flow perfusion circuit.

Figure 2

Calcium content of cultured scaffolds. Results are expressed as mg Ca²⁺/scaffold on a log scale. *, highest; **, second highest; and ***, third highest calcium content with each statistically different from all other groups (P < 0.05).

Figure 3

SEM images of the surfaces of cultured scaffolds. Representative SEM images of surfaces of scaffolds cultured for 16 d with static culture (A), flow perfusion rate of 0.3 ml/min (B), flow perfusion rate of 1 ml/min (C), and flow perfusion rate of 3 ml/min (D). Porous structures formed during flow perfusion culture are readily apparent in C. Although less numerous, porous structures also were formed at the lowest perfusion rate. At the highest flow perfusion rate, the formed porous structures appeared to have become blocked or plugged with matrix. Several of these are indicated with arrows in D.

Figure 4

Porous structures on surfaces of scaffolds cultured under flow perfusion. Higher magnification images of porous structures shown in Fig. 2C. Individual cells can be clearly seen lining the pores and deeper scaffold structures.

Figure 5

Histological sections. Representative histological sections of scaffolds cultured for 16 d with static culture (A), flow perfusion rate of 0.3 ml/min (B), flow perfusion rate of 1 ml/min (C), and flow perfusion rate of 3 ml/min (D). Images are of histological crosssections 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. The fibers of the titanium meshes appear black.

Figure 6

Cellularity of cultured scaffolds. Results are expressed as cells per scaffold.

Figure 7

AP activity of cells on cultured scaffolds. (A) Results are expressed as pmol/hr per cell. (B) Results are expressed as μmol/hr per scaffold. The symbols indicate that at day 4 (○), day 8 (#), and day 16 (+) the statically cultured cells had significantly less AP activity than all flow perfusion culture groups at the corresponding day of culture.

Figure 8

Osteopontin secretion from cultured scaffolds. Results are expressed as microgram (μg) of osteopontin per scaffold.