Collagen has been extensively described as a beneficial material in bone tissue engineering due to its biocompatibility, biodegradability, low antigenicity, and high tensile strength. However, collagen scaffolds in their pure form have some drawbacks and improvements in the physical, chemical, and biologic properties of collagen are necessary to overcome those inadequacies. Recently, the selective hydrolysis of carboxyamides of asparagine and glutamine residues of collagen has been employed to increase the number of negative sites and enhance the piezoelectric properties of collagen. Anionic collagen scaffolds were prepared by use of a hydrolysis treatment for either 24 h [bovine pericardium (BP 24)] or 48 h (BP 48). Bovine osteoblasts were cultured on them and on native matrices to understand the cellular interactions responsible for the good osteoconductivity and biocompatibility reported with in vivo tests. Based on the data obtained on cell adhesion, alkaline phosphatase (ALP) and extracellular matrix macromolecule production, and cellular proliferation through histological analysis, we may conclude that the materials tested reveal sufficient biocompatibility level for bone repair. Further, the evidence of some connection between ALP activity and the mineralization process should be emphasized. BP 48 presented the most promising results stimulating in vitro mineralization, ALP production, and possible osteoblast differentiation. (C) 2004 Wiley Periodicals, Inc.71B2229237