EFFECT OF SHEAR STRESS ON SAOS-2 OSTEOBLASTS GENE EXPRESSIOINSIDE A POLYURETHANE POROUS SCAFFOLD

Abstract

The skeleton is tuned for sensing and responding to mechanical forces: a global bone strain moves the extra-cellular fluid through the lacunocanalicular network of compact bone, so gene expression of osteocytes is mechanically modulated by extra cellular fluid flow shear stress. Several studies showed that shear stress modulates bone cells gene expression: in vitro mechanical stimulation impacts the levels of alkaline phosphatase, cAMP, intracellular calcium, NO, prostaglandin E2, c-fos, COX-2, osteopontin and osteocalcin. Aim of this study is to investigate the effect of shear stress on SAOS-2 human osteoblasts proliferation, bone matrix production and mineralization, using a biostable polyurethane as scaffold and a perfusion bioreactor.

Polyurethane scaffolds with an average porediameter of 624 micron were utilized. Scaffolds were sterilized and placed in to standard well-plates (condition A) and into a bioreactor with forced perfusion at a rate of 3 ml/min (condition B). Human osteosarcoma cell lineSAOS-2 was obtained from the ATCC and cultured in McCoy’s 5A modified medium. A suspension of 7′105 osteoblasts was added onto the top of each scaffold. Medium was changed every 3 days and sampled for osteopontin and-osteocalcin ELISA kits. After 16 days culture DNA and calcium contents were measured, light microscopy and SEM analysis were performed.

In condition B, in comparison to A, we observed 33% higher cells proliferation, 12.6-fold higher osteopontin secretion, 99.6-foldhigher osteocalcin secretion and 8-fold higher calcium deposition. Microscopy observations revealed that in condition A osteoblasts were few with thin discontinuous extracellular matrix; in contrast shear stress induced 3D modeling of cells and matrix organization, so several cells were in multilayer with highly developed matrix and no surfaces were cell free.

Statically cultured osteoblasts showed normal proliferation, but a very low matrix synthesis. Into bioreactor, which provides physiological levels of shear stress, the osteoblasts proliferated and showed increased metabolic activity.