Design and fabrication of the scaffold

The scaffold was designed with the subject-specific geometry of the bone fragment to be replaced. For this, computed tomography was performed on the right hindlimb of the sheep before surgery (voxel size 0.12 x 0.12 x 0.60 mm). A multiplanar hard tissue thresholding was applied to each metatarsal scan using the software InVesalius® (Renato Archer Information Technology Center, Amarais, Brazil), which enabled to generate a 3D bone reconstruction from the image stack, as shown in Fig 1A. Afterward, an intermediate 13-mm bone segment was sliced from the metatarsal geometry employing the solid modeling CAD software SpaceClaim® (SpaceClaim Corporation, Concord, MA, USA), and its inner medullary cavity was filled. Two building modifications were also carried out on this initial geometry to improve the scaffold stability in the defect and speed up the regenerative response (see Fig 1A). Firstly, a coupling cylinder (4 mm Ø; 2 mm length) was added over one end face to immobilize the structure in vivo in the distal bone marrow. This coupler would prevent the scaffold from rotating or moving during the animals’ daily activity, which could interfere with the proper growth of the naïve tissue. At the other extreme, the solid was hollowed out by another cylinder (4 mm Ø; 10 mm length) for grafting, as further explained. A robocasting device (3-D Inks Still-water®, Tulsa, Oklahoma, USA), an extrusion-based 3D- printing technique, was selected. It worked by depositing a 45 vol% hydroxyapatite slurry forming a ceramic network of perpendicularly oriented layers of bars, as illustrated in Fig 1B. This material was chosen since bioceramics are proven to enhance naïve tissue growth by regulating osteoblast proliferation and differentiation while the structure is reabsorbed [48]. From previous works in the literature, the concentration of colloidal suspensions in the 3D-printing ink (45 vol%) was optimized to obtain the suitable viscoelastic properties for an effective deposition and assembly while ensuring the proper mechanical performance of the sintered structure in vivo [49–51]. An example of the final geometry of the patient-specific scaffold is provided in the S1 File of the Supporting Information. The printing nozzle diameter, the pore size, or the layer overlap were also numerically optimized in previous works to maximize cell diffusion and proliferation while ensuring the mechanical integrity of the structure under the ovine physiological loads [17]. The final microarchitecture had a porosity of 59.3%, a 560.8 μm pore size, and a specific surface area of 5768.9 m^-1 [17]. After drying at room temperature, the organic components of the implant were eliminated under heating at 400°C for 1 hour. They were finally sintered at 1300°C for 2 hours to compact the paste-like scaffold. The chemical sterilization of the structures was achieved using formaldehyde at 60°C and relative humidity of 80%.

(A) Design of the bioceramic scaffold from the 3D geometry of the ovine metatarsus reconstructed by computed tomography scans. (B) 3D-printing of the hydroxyapatite structure using roboscasting. (C) Harvesting of the spongy grafting tissue from the lateral side of the contralateral humerus. (D) Implantation of the scaffold.