3D-printed calcium phosphate scaffolds for bone regeneration: impact of geometry and treatment an in vivo study

The combination of the direct ink writing (DIW) manufacturing technique, also named robocasting, with the use of self-setting calcium phosphate inks based on α-tricalcium phosphate opens new possibilities in the field of bone regeneration: i) On one hand, the DIW fabrication process allows a precise control on the external shape and internal porosity of the scaffold. The porosity allows the colonization of the bone tissue and the shape control opens new perspectives in personalised medicine; ii) On the other hand, the use of self-setting α-TCP inks provide a micro/nano porosity and a high specific surface area (SSA) to the bone graft. Both factors have been identified as crucial for the bioactivity of the material. Since the fabrication time is a crucial factor for the successful translation of these technologies to the clinical field, and the hardening reaction of conventional self-setting inks is slow, recent investigations have developedan alternative setting procedure (hydrothermal) that considerably reduces the hardening step from 7 days to 30 minutes. Regarding the role of scaffold architecture in bone regeneration, it has been recently proved that the presence of concave surfaces enhances osteogenesis. However, since DIW is based on the extrusion of a paste through a needle, conventional DIW scaffolds are composed of extruded filaments with convex surfaces. Hence the interest in developing scaffolds with non-cylindrical strands, which have concave surfaces. The aim of this study was to assess the in-vivo performance of calcium phosphate scaffolds, analysing on one side the effect of the setting treatment, i.e., comparing the biomimetic setting with the hydrothermal setting treatment, and on the other side comparing cylindrical vs.non-cylindrical strands. The characterization of the scaffolds obtained with the two different treatments revealed that whereas the biomimetic treatment resulted in calcium deficient hydroxiapatatite (CDHA), the hydrothermal treatment led to the presence of small amounts of β-tricalcium phosphate. Biomimetic scaffolds consisted of plate-like crystals, with higher SSA and smaller microporosity than the hydrothermal scaffolds, made of needle-like crystals. The geometry of the strands(i.e. cylindrical vs non-cylindrical)did not havean influence on the material composition, microstructure and global porosity, but they did have an impact on the mechanical properties,with lower ultimate compressive strength for the structures with non-cylindrical strands. The scaffolds were implanted in the femoral condyles of 10 adult female New Zeeland rabbits and explanted after 8 weeks. The samples were embedded in resin and characterised by micro-computed tomography, scanning electron microscopy and optical microscopy afterGolden-Mason trichrome staining. All the samples presented the formation of new mature lamellar bone and a successful osteointegration. No statistically significant differences were observed between the samples studied in terms of the amount of newly formed bone, quantified from the SEM observation.Micro-CT allowed the assessment of bone formation in 3D, although difficulties related to image processing prevented the volumetric quantification that might have revealed significant differences. However, a clear tendency was found for new bone formation in constrained microenvironments, such as the contact zone between two strands or the concavities present in the non-cylindrical condition. Further data analysis will have to be carried out to assess the differences in the different sample conditions.Incomin