The aim of this study was to make a histomorphometric evaluation of the osteogenic potential of anionic collagen matrix as scaffolds; either crosslinked in glutaraldehyde or not cross-linked and, implanted in critical bone defects in rat calvaria. Seventy-two rats were randomly distributed in three groups: anionic collagen scaffolds treated for 24 h of selective hydrolysis (ACSH); anionic collagen scaffolds treated for 24 h of selective hydrolysis and 5 min of crosslinking in glutaraldehyde 0.05% (ACSHGA); empty bone defect (Control), evaluated at the biological points of 15, 45, 90 and 120 days. The results showed that the biomaterials implanted were biocompatible and showed a high osteogenic potential. These biomaterials presented a speed of biodegradation compatible with bone neoformation, which was shown to be associated with angiogenesis inside the scaffolds at all biological points. The percentage of mineralization of ACSH (87%) differed statistically from that found in ACSHGA (66%). It was concluded that the regeneration of critical bone defect was more evident in anionic collagen without crosslinking (ACSH).