3D printing of bone scaffolds using powders derived from biogenic sources

This doctoral work was developed in the frame of bone tissue engineering, dealing with the fabrication of scaffolds for the regeneration of bones. At this purpose, calcium phosphates derived from natural sources are very interesting because they are more similar to the bone mineral and possess better bioactivity. Indeed, the bone mineral is different from synthetic hydroxyapatite as it is non-stoichiometric, nanosized, it presents a high degree of disorder and contains many additional ions and impurities such as CO32-, Mg2+, Sr2+, Na+, etc. These characteristics can be easily obtained by synthesizing hydroxyapatite from natural sources, such as corals, starfishes, seashells, animal bones, bird eggshells etc. The natural sources used in the present work are three types of biogenic calcium carbonate, i.e. calcium carbonate that is produced by living organisms in the form of aragonite or calcite. Among the different sources, three biogenic calcium carbonates were chosen: cuttlefish (Sepia Officinalis) bones, mussel (Mytilus Galloprovincialis) shells and chicken eggshells. Besides their abundance and availability, they were selected because of their different composition: aragonite in cuttlebones, calcite in eggshells and a mixture of aragonite and calcite in mussel shells. After the first chapter, which is a theoretical introduction, this thesis is divided into other five chapters. Chapter 2 contains a careful characterization of the three biogenic raw materials while Chapter 3 deals with the synthesis of hydroxyapatite starting from these natural sources. The process developed here takes place entirely at nearly room temperature, which allows the organic part of the biological materials to be preserved. This synthesis process is basically a wet mechanosynthesis followed by a mild heat treatment (up to 150°C). The study focuses on the influence of several process parameters on the synthesis efficiency: temperature, milling time, pH and raw material. The temperature used to dry the slurry after the wet ball-milling was found to be the most important parameter, the higher the temperature the faster the conversion of CaCO3 into hydroxyapatite. Moreover, aragonite was found to transform more easily into hydroxyapatite with respect to calcite, and also to follow a different reaction path. The synthesis process described in Chapter 3 allowed to produce different bio-derived powders that were found to be non-stoichiometric, nanosized, carbonated hydroxyapatites, containing also additional ions, especially Mg2+ in the eggshell-derived material and Sr2+ in the cuttlebone-derived one. These powders were then used as a starting point for the studies presented in the next three chapters. Chapter 4 shows a very preliminary evaluation of the interaction with human cells in vitro. First, the as-synthesized powders were consolidated by uniaxial pressing and sintering at temperatures between 900°C and 1100°C and their crystallographic composition was analyzed. Then, after having established the non-cytotoxicity of the sintered pellets, osteoblasts from human osteosarcoma cell line were seeded on the pellets and their behavior after 1, 3 and 5 days of culture was observed by confocal microscopy. In general, all materials promoted good cell adhesion and proliferation, especially the eggshell-derived one. At this point, the bio-derived materials were found to induce a good cellular response but, in order to foster the regeneration of bones, a scaffold must also contain a large amount of interconnected porosity. Among the numerous methods to fabricate porous structures, additive manufacturing is surely very attractive due many advantages, such as the possibility of customizing the shape based on tomography images from the patients, the fact that no mold is needed and the freedom of fully designing the porosity. Indeed, not only the size and the amount of porosity are important, but also the shape of the pores and their position and orientation have a deep effect on the interaction with the cells. Therefore, Chapter 5 and Chapter 6 deal with the fabrication of scaffolds by 3D printing, following two different approaches. In the study presented in Chapter 5, the powders synthesized from cuttlebones, mussel shells and eggshells were used in combination with a thermoplastic polymer (PCL, polycaprolactone) to obtain bioactive composites. Composite materials made of 85 wt% PCL and 15 wt% bio-derived hydroxyapatite were used to fabricate porous scaffolds by extrusion 3D printing. The biological in vitro tests showed that the composite scaffolds possess better bioactivity than the pure PCL ones, especially those containing mussel shell- and cuttlebone-derived powders, which promoted the best cell adhesion, proliferation and metabolic activity of human osteosarcoma cells after 7 days of culture. In addition, the elastic compressive modulus, which was found to be between 177-316 MPa, thus in the range of that of trabecular bone, was found to increase of about ∼50% with the addition of the bio-derived nanopowders. Finally, in Chapter 6, the cuttlebone-derived powder was used to fabricate porous bioceramic scaffolds by binder jetting 3D printing. Due to serious technical issues related to the printing of a nanosized powder, 10 wt% of bio-derived powder was mixed with a glass-ceramic powder with bigger particle size. Moreover, the organic part of the cuttlebone had to be previously eliminated by a heat treatment at 800°C. Thanks to the great freedom of design that is allowed by the binder jetting process, scaffolds with two different pore geometries were fabricated: with pores of uniform size and with a size-gradient. Indeed, natural bone possesses a gradient in porosity from the core to the surface, from porous trabecular bone to dense cortical bone. The sintered scaffolds showed a total porosity of ∼60% for the pure glass-ceramic and ∼70% for the glass-ceramic with 10 wt% of cuttlebone-derived nanoparticles, which most probably slowed down the densification by limiting the contact between the glassy particles. All the bioceramic scaffolds promoted good adhesion and proliferation of human bone marrow-derived mesenchymal stem cells in vitro, without any significant difference between the different samples. However, the scaffolds with the cuttlebone-derived powder and with gradient porosity showed the greatest decrease of metabolic activity after 10 days of culture, which could be accounted as a sign of differentiation of stem cells.