Adherence of osteoblast-like cells on calcospherites developed on a biomaterial combining poly(2-hydroxyethyl) methacrylate and alkaline phosphatase

Abstract

The polymer poly(2-hydroxyethyl) methacrylate (pHEMA) can copolymerize with alkaline phosphatase (AlkP) to form a hybrid material. The enzyme retains its biological activity and forms hydroxyapatite nodules (calcospherites) when polymer pellets are incubated with a synthetic body fluid. Osteoblast-like cells (ROS 17/2.8) were seeded on pellets of pHEMA and pHEMA-AlkP on which calcospherites were grown. They were examined by scanning electron microscopy (SEM) with backscattered electron imaging. Cell surface and shape were measured by image analysis combining the SEM images. Cells grown on pHEMA-AlkP had an increased surface area (449 ± 216 μm2 vs. 204 ± 80 μm2). The number of filopodia anchoring the cells on the free polymer surface was reduced on pHEMA-AlkP, but numerous thick pseudopodia permitted a direct anchorage on the calcospherites. Pseudopodia were wider and longer than the filopodia. The backscattered images revealed that each cell was seated on 7.1 ± 1.5 calcospherites and partially covered 10.3 ± 1.9 others. Antifibronectin and anti-bone sialoprotein antibodies were used to investigate cell attachment. With confocal microscopy, both molecules were located at the interface between the cells and the mineral, inside the cells, and as free molecules on the calcospherites. Immunogold labeling was done with the same antibodies and examined with transmission electron microscopy (TEM). Adsorption of fibronectin and bone sialoprotein was noticeable at the cell/calcospherite interface and on the surface of the hydroxyapatite crystals. Immunogold studies revealed adhesion proteins (bone sialoprotein, fibronectin) to be present at the surface of crystals and at focal points of cell contact.