Orthopedic surgeons have been facing major limitations in the development of personalized therapies to regenerate osteochondral defects (OCD). Hierarchically structured scaffolds consisting of a cartilage-like layer interconnected to an underlying subchondral bone-like layer emerged as advanced technological solutions that can better mimic the native OC tissue1. Herein, the in vivo performance of hierarchical and bilayered scaffolds prepared using a horseradish peroxidase (HRP)-mediated approach to crosslink SF (HRP-SF) in combination with inorganic ZnSr-doped β-tricalcium phosphate (β-TCP) (HRP-SF/ZnSr-β-TCP)2, was evaluated in a rabbit knee critical size OCD model. A comparative analysis was performed using similar OC grafts made of undoped β-TCP (HRP-SF/β-TCP). Ionic-doped biomaterials have shown to enhance scaffolds mechanical performance, osteoconductivity and angiogenesis3. At the same time, SF exhibit tunable mechanical properties, molecular versatility and biocompatibility, being a natural polymer successfully applied for tissue engineering (TE) applications3. Micro-CT analysis of the explants revealed that the defects filled with the bilayered scaffolds presented regular morphology with subchondral bone ingrowth’s in the bottom domain and cartilage regeneration in the surface area of the implanted materials. The bilayered scaffolds containing ZnSr-β-TCP, showed in the subchondral bone-like layer similar calcified tissue distribution to the healthy control. Histological and immunofluorescence analyses confirmed cartilage tissue regeneration as observed by the presence of glycosaminoglycans and collagen type II positive staining’s in the HRP-SF layer. De novo bone ingrowth and blood vessels formation were also observed on the underlying HRP-SF/ZnSr-β-TCP and HRP-SF/β-TCP layers, suggesting that the presence of inorganic β-TCP promoted subchondral tissue formation. Although a long-term in vivo study is necessary to confirm the beneficial effect of the ionic presence, the preliminary in vivo data together with the desirable mechanical and chemical features of these structures suggest that the proposed hierarchical scaffolds may be potentially used in OCD regeneration.