3D Printed Polycaprolactone/Nano-Hydroxyapatite Scaffolds for Bone Tissue Engineering In-Vitro

Scaffolds are key components in bone tissue engineering (BTE) since they provide sites for cells to function and mechanical support to resist external forces while they are degraded and remodeled over time with the regeneration of bone tissue. A wide range of investigations have been performed on scaffolds printed from polycaprolactone/hydroxyapatite (PCL/HAp) due to the synergistic properties of PCL and HAp for BTE. Notably, conflicting reports on the development (design and printing) of scaffold structures for desired performance, in terms of mechanical properties and cellular activities in PCL/HAp scaffolds, make conclusions on best practices elusive. This raises a great need to improve the understanding of the effect of scaffold structure on the performance of PCL/HAp scaffolds. This study aimed to discover the influence of PCL/nHAp scaffold structure and design on their mechanical and biological properties for BTE applications. The specific objectives of this thesis were to: 1) uncover the effect of structural parameters, including porosity level and internal structure, on mechanical properties of three-dimensional (3D) printed PCL/30% (wt.) nano-hydroxyapatite (nHAp) scaffolds and subsequently provide suggestions for developing scaffolds with mechanical properties for substituting trabecular bone; 2) explore the effect of scaffold height and internal structure on mechanical properties; and 3) evaluate osteoblast performance in-vitro on scaffolds with varying internal structures and pore sizes. The first objective was to uncover the influence of internal structure and porosity level on the mechanical properties of PCL/30% (wt.) nHAp scaffolds. Composite scaffolds (which aimed to replicate trabecular bone) were designed and 3D printed from PCL reinforced with 30% (wt.) nHAp by extrusion printing. Scaffolds with varied porosities (~ 40-70%) were fabricated with and without an interlayer offset, creating staggered and lattice structures, respectively. Mechanical compressive testing was performed to determine scaffold elastic modulus and yield strength. Linear regression was used to evaluate mechanical properties as a function of scaffold porosity. Results indicated different relationships between mechanical properties and porosities for lattice structure and staggered structure. For elastic moduli, lattice structure exhibited higher moduli than the staggered structure for porosity values greater than 55%; vice versa for below 55% porosity. The lattice structure exhibited higher yield strength at all porosities. This study uncovered the effect of porosity and internal structure on scaffold mechanical properties, thus helping researchers to develop scaffolds with mechanical properties matching that of trabecular bone. The second objective was to explore the effect of scaffold height and internal structure on the mechanical properties of scaffolds. Scaffolds with different internal structures (lattice and staggered) and varying heights (with 4, 6, 8 and 10 printing layers), and of the same porosity (50%) for the purpose of comparison. Then, the microstructure of the scaffolds and their mechanical properties were examined using scanning electron microscopy (SEM) and compressive testing, respectively. The mechanical properties were characterized in terms of elastic modulus as well as yield strength and compared among the scaffolds with different designs to elucidate the relationship between the scaffold design and mechanical properties. Mechanical properties of 3D printed scaffolds were found to be dependent on scaffold height for both lattice and staggered structures. The results illustrated that the microstructural parameters of scaffolds, namely pore size and penetration between layers (i.e., the amount of diffusion between strands in subsequent layers) were influenced by scaffold design. Further, at higher heights, pore size increased while penetration between layers decreased; thus, mechanical properties were affected. Staggered scaffolds showed lower mechanical properties than lattice scaffolds with the same height. The third objective was to evaluate osteoblast performance in-vitro on scaffolds with varying internal structures and pore sizes. Scaffolds of PCL/30% (wt.) nHAp with two different internal structures (lattice and staggered) were 3D printed. For each structure, four groups of varying pore sizes of 0.280, 0.380, 0.420, and 0.550 mm (with the porosity of 40%, 50%, 60%, and 70%, accordingly) were 3D printed, respectively, for examination and comparison. Scaffolds were then seeded with pre-osteoblast cells (MC3T3-E1) and cultured for up to 14 days. Metabolic activity of cells (via MTT assay at day 1, 3, 7), osteoblast differentiation (via ALP activity at day 7), and the capability of osteoblasts to deposit mineralized matrix (via Alizarin Red S assay at day 7, 14) for each scaffold group were examined in-vitro. Our findings showed that staggered scaffolds were better suited for supporting metabolic activity, differentiation, and calcium mineralization of the cells. The pore size of 0.280 mm was more favorable to support cell proliferation while the larger pore sizes (0.420 and 0.550 mm) were more effective at promoting osteoblast differentiation and secretion of a mineralized matrix. Altogether, this thesis presents a comprehensive study on the influence of scaffold structure on their mechanical and biological properties for BTE in-vitro. The findings of this thesis can be used as a guide for researchers to design and 3D print scaffolds with appropriate mechanical and biological properties for BTE. Mechanical findings of this research indicated elastic modulus and yield strength properties of our investigated scaffolds were comparable to trabecular bone (elastic moduli: 14-165 MPa; yield strength: 0.9-10 MPa). Additionally, our investigated scaffolds supported metabolic activity of cells, osteoblast differentiation, and deposition of mineralized matrix in-vitro.

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