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The cross-linking of collagen matrices (Cl_CM) may provide volume-stable enhanced defect regeneration when compared to non-cross-linked matrices (Ncl_CM). The aim of the present study was to investigate the bone forming potential of collagen matrices (CMs) and the effects of cross-linking CMs in a rabbit calvaria defect model. (1) Empty controls ( n = 6), (2) Ncl_CM ( n = 8), and (3) Cl_CM ( n = 8) were selected to be observed for the healing in 10 mm critical-sized calvarial bone defects. The potential for the bone as well as the connective tissue formation were evaluated by micro-CT and histomorphometry at three months post-surgery. There were no statistically significant differences in terms of new bone volume in the defects between the groups. However, the Cl_CM induced significantly greater fibrous tissue regeneration (5.29 ± 1.57 mm 2 ) when compared to the controls (3.51 ± 0.93 mm 2 ) by histomorphometry. The remnants of collagen fibers with immune cells, including macrophages and giant cells, were occasionally observed in the Cl_CM group but not in the Ncl_CM group. In conclusion, the cross-linking of collagen did not influence the potential for bone formation. Nevertheless, Cl_CM might be advantageous for the maintenance of fibrous tissue volume without disturbing bone formation in the defects.