Importance of including proteins in experimental models aimed at studying reactive surface apatite layers on calcium phosphates

摘要

Changes were studied in the morphology of the surface reactive layers of two calcium phosphate (CAP) ceramics, hydroxyapatite (HAP) and tricalcium phosphate (TCP), resulting from the presence of proteins in both in vitro and in vivo environments. In vitro, the CAPs were exposed to an inorganic medium, phosphate-buffered saline (PBS), and/or a protein solution, fetal calf serum (FCS). In vivo experiments involved using diffusion chambers, which excluded cells but exposed the materials to the biological extracellular fluid. Protein adsorption onto the material surfaces was assessed qualitatively using Toluidine Blue (TB) staining, polyacrylamide gel electrophoresis (PAGE), and scanning electron microscopy (SEM). Positive TB staining was observed on all samples exposed to proteins, as expected. There were no differences in protein populations desorbed from in vitro RAP and TCP samples, but TCP consistently showed more intense bands. The PAGE profiles from the in viva HA samples had clearly fewer and also less intense bands than those from TCP. Protein coating was visible under SEM on both HAP and TCP in vitro and in viva. Plate-like crystal formation that occurred on TCP in inorganic solution was inhibited in the presence of proteins. This work clearly shows that the apatite layers formed on IIAP and TCP in inorganic solution differed, and that protein adsorption further altered the morphology of the surface by inhibition of crystal growth.