Demineralization Promotes Changes on the Bone Surface That Improve Osteoblast Growth

To investigate the role of bone structural changes on cell biology after demineralization with different concentrations of citric acid.

Under aseptic conditions, bicortical samples of calvarial bone were harvested from 28 adult male isogenic Wistar rats (Rattus norvegicus). Of the 55 total samples obtained, 45 bone samples were placed in 24-well culture plates that were randomly distributed into treatment groups (n = 15 per group): demineralization with 10% citric acid (CA.10), demineralization with 50% citric acid (CA.50), or washing with saline solution (control). MC3T3-E1 cells (Subclone 4-CRL-2593, ATCC) cultured on treated bone surfaces were analyzed regarding morphology and spreading. Scanning electron microscopy (SEM) and confocal microscopy were used in 10 remaining samples, before and after demineralization with CA.10 (n = 5) or CA.50 (n = 5), to assess the chemical composition (%A) and surface parameters: roughness of a line (Ra) and roughness of an area (Sa).

Cells grown on demineralized bone surfaces were flatter, more spread out, and presented with more elongated filopodia than those cultured on nondemineralized surfaces. The surface area covered by cells was significantly larger after 24 hours of culture in CA.10 and CA.50 than in the control group. Demineralization had no effect on Ra and Sa parameters, but the distance between consecutive peaks and the distance between a peak and a consecutive valley decreased significantly. Chemical composition was not affected by demineralization.

Both citric acid concentrations produced bone surface changes that can promote preosteoblast growth and morphology compatible with advanced stages of differentiation.