The use of biomaterials derived from components of natural extracellular matrices (ECM) for in situ bone regeneration was investigated. The potential of ECMs in healing critical size bone defects was investigated in the rat cranium. Hydrogels were formed within minutes at physiologic conditions by a self-selective conjugate addition reaction. Gels with different network architectures and enzymatic sensitivity were also tested. The healing response was found to be dependent on the proteolytic sensitivity and also on the hydrogel structure. The results indicate the possibility to molecularly engineer synthetic hydrogels to render them responsive to proteases secreted by cells.