Mechanical stress-induced orientation and ultrastructural change of smooth muscle cells cultured in three-dimensional collagen lattices

Abstract

The effect of tensile stress on the orientation and phenotype of arterial smooth muscle cells (SMCs) cultured in three-dimensional (3D) type I collagen gels was morphologically investigated. Ring-shaped hybrid tissues were prepared by thermal gelation of a cold mixed solution of type I collagen and SMCs derived from bovine aorta. The tissues were subjected to three different modes of tensile stress. They were floated (isotonic control), stretched isometrically (static stress) and periodically stretched and recoiled by 5% above and below the resting tissue length at 60 RPM frequency (dynamic stress). After incubation for up to four wk, the tissues were investigated under a light microscope (LM) and a transmission electron microscope (TEM). Hematoxylin and eosin-stained LM samples revealed that, irrespective of static or dynamic stress loading, SMCs in stress-loaded tissues exhibited elongated bipolar spindle shape and were regularly oriented parallel to the direction of the strain, whereas those in isotonic control tissues were polygonal or spherical and had no preferential orientation. In Azan-stained samples, collagen fiber bundles in isotonic control tissues were somewhat retracted around the polygonal SMCs to form a random network. On the other hand, those in statically and dynamically stressed tissues were accumulated and prominently oriented parallel to the stretch direction. Ultrastructural investigation using a TEM showed that SMCs in control and statically stressed tissues were almost totally filled with synthetic organelles such as rough endoplasmic reticulums, free ribosomes, Golgi complexes and mitochondria, indicating that the cells remained in the synthetic phenotype. On the other hand, SMCs in dynamically stressed tissues had increased fractions of contractile apparatus, such as myofilaments, dense bodies and extracellular filamentous materials equivalent to basement membranes, that progressed with incubation time. These results indicate that periodic stretch, in concert with 3-D extracellular collagen matrices, play a significant role in the phenotypic modulation of SMCs from the synthetic to the contractile state, as well as cellular and biomolecular orientation.