Collagen is the most abundant protein in the body and is also the most important component of the extracellular matrix. Collagen has several advantages as a biomaterial such as lack of toxicity, biocompatibility, biodegradability, and easy reabsorption. In this study, we examined bovine osteoblasts cultured on native or anionic collagen scaffolds prepared from bovine pericardium after selective hydrolysis of glutamine and asparagine side chain amides for periods from 24 (BP24) and 48 h (BP48). The cells were cultured in control and mineralization medium at 37°C in the presence of 5% CO2. Transmission and scanning electron microscopy, energy dispersive spectroscopy, and an immunocytochemical marker were used for analysis. Cells with an irregular morphology forming a confluent multilayer were observed on matrices kept in control medium. Most of these cells presented a polygonal or elongated flattened morphology. Several spherical deposits of calcium crystal associated with phosphorus were observed on the native and BP48 matrices. Similar results were observed in samples kept in control medium except with lower calcium/phosphorus ratio. Vesicles actively expelled from the cell membrane were also seen (do this vesicles corresponds to calcium/phosphorus deposits). Osteocalcin was clearly visible on matrices kept in mineralization medium and was more expression on the surface of BP48 matrices. The results showed that anionic collagen is able to support osteoblastic differentiation, regardless of the medium used. Finally, the BP48 matrix promoted better osteoblast differentiation than the native matrix. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 101B: 18–27, 2013.