Serious cartilage lesions (Outerbridge III, IV) may be successfully treated with a three-layered gradient scaffold made by magnesium-doped hydroxyapatite and type I collagen, manufactured through a bio-inspired process and stabilised by a reactive bis-epoxy (1,4-butanediol diglycidyl ether, BDDGE). Each layer was analysed to elucidate the effects of crosslinking variables (concentration, temperature and pH). The chemical stabilisation led to an homogeneous and aligned collagenous matrix: the fibrous structures switched to a laminar foils-based arrangement and organic phases acquired an highly coordinated 3D-organization. These morphological features were strongly evident when crosslinking occurred in alkaline solution, with BDDGE concentration of at least 1 wt%. The optimised crosslinking conditions did not affect the apatite nano-crystals nucleated into self-assembling collagen fibres. The present work allowed to demonstrate that acting on BDDGE reaction parameters might be an useful tool to control the chemical-physical properties of bio-inspired scaffold suitable to heal wide osteochondral defects, even through arthroscopic procedure.