A new stretching apparatus for applying anisotropic mechanical strain to bone cells in-vitro

Bone is adapting to in-vivo loading by modeling and remodeling processes. T he sensors of the external forces acting on the bone matrix seem to be the bone cells. Osteocytes, osteoblasts, and bone lining cells have been shown to respond to mechanical forces in-vitro. In this work, we describe a new i n-vitro system which applies anisotropic stress conditions to MC3T3-E1, ost eoblast-like mouse calvaria derived cells. The system allows stretching of cell cultures under well-defined stretching conditions. Cells are grown on an elastic polyurethane culture support (PUCS) that is subjected to uniaxia l tensile stress using a direct current (dc) motor-driven linear positionin g stage, situated within the incubator. The physical stretching parameters, the maximum elongation of the PUCS (the maximum strain applied to the cell s), the strain rate, and the number of cycles, can be varied. First, the ac tual strains occurring at different locations of the PUCS were determined u sing optical methods. The surface strain appeared to be uniform over the PU CS and biaxial with a Poisson contraction nearly 80% in magnitude to the ax ial extension. Second, we tested the behavior of the MC3T3-E1 cells on PUCS compared to the cells grown in petridishes (PD). After 11 days of culture, cell number per dish on PUCS was significantly reduced to PD cultures (20% of control). At that time, cultures on PUCS reached confluency as compared to day 4 for the PD cultures. However, histochemical staining of alkaline phosphatase (ALP) and multilayer formation of the PUCS cultures appeared to be not significantly different from PD cultures. We also looked at the cyt oskeleton by phalloidin staining, at vinculin, a protein of the cell-matrix and cell-cell interaction, and at fibronectin, a protein of the extracellu lar matrix using immuno staining methods. All these features tested so far seemed not to be different in cells cultured on PUCS compared to cultures i n PD. Third, the responsiveness to the external force was tested using conf luent cells on PUCS. A strain of 6.8 millistrain (6800 microstrain) was app lied to the cells, using a strain rate of 4.9 millistrain/s and 350 cycles/ h for a period of 48 h. These loading conditions led to significantly decre ased cell proliferation, as measured by [H-3] deoxythymidine ([H-3] dT) inc orporation, and significantly increased ALP activity. These data show that the stretching device introduced in this paper offers new possibilities to study the response of osteoblast-like cells to anisotropic forces. (C) 2000 American Institute of Physics. [S0034- 6748(00)02109-2].