3D printed bone-like biopolymer composites inspired by nacre
Authors:   , Kurian Mima 
Published: November 2021

<p>Bone tissue engineering and synthetic biomineralization are two widely researched areas, the principles of which have been combined from time to time in efforts to develop replacement materials for natural bone grafts. Nacre has been studied as a prospective bone graft material owing to its mechanical strength being comparable to that of natural bone. The extraordinary mechanical strength of nacre is attributed to its nanostructure. The McGrath research group developed a synthetic biomineralization method, herein called the McGrath method, that can be used to effectively replicate the elements of nacre’s nanostructure in 2D biopolymer systems in laboratory conditions. Here, the applicability of the McGrath method in translating the calcium carbonate-based mineralization achieved in 2D films onto 3D printed chitosan hydrogel-based scaffolds is investigated. Thereby, enabling the fabrication of 3D chitosan-calcium carbonate composites with properties sought in the context of prospective load-bearing bone grafts.  In this work, considering the importance of interconnected porosity in an in vivo environment, nozzle extrusion-based 3D printing was employed to develop 3D structures with interconnected macropores, essentially imitating the porous structure of bone. The applicability of chitosan hydrogels as the printing ink in a custom-designed 3D printer was evaluated and quantified through rheological studies. The printing parameters and an appropriate experimental protocol were devised to fabricate stable 3D chitosan hydrogel-based scaffolds featuring physically crosslinked-layered structure with interconnected macropores. The effect of various drying techniques on retaining this porous structure in dried scaffolds and their swelling behaviour when soaked in a physiologically relevant solvent were explored using various techniques including cryo-scanning electron microscopy.  The strategies required to mineralize the as-fabricated 3D chitosan hydrogel-based scaffolds via the McGrath method, such that the mineralization achieved within the 3D scaffolds is similar to that obtained within 2D films, were elucidated. This included the use of polyacrylic acid (PAA), a crystal growth modifier. PAA has previously been shown to be important in achieving a pancake-like calcium carbonate formation, comprised of laterally growing nanoparticle aggregates which form in association with the organic matrix, in 2D films; such structures are observed in the early stages of nacre formation. By modulating the period of exposure of the 3D scaffolds to the mineralization solutions and the concentrations of these solutions, it was found that 3D composites with up to 40% calcium carbonate content and varying crystal morphology could be fabricated using this mineralization method. Importantly, it was observed that the calcium carbonate crystallites were intricately associated with the organic hydrogel matrix. This is another essential element observed in biomineral systems. Various techniques such as scanning electron microscopy (SEM), thermogravimetric analysis (TGA), energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD) were employed for structural and compositional characterisation of the final composites.  The prospect of fabricating 3D chitosan-calcium carbonate composites via a single-step method using chitosan hydrogel preloaded with calcium carbonate crystallites as the printing ink in our custom-designed 3D printer was also investigated. This method was studied as a faster and less labour-intensive alternative to developing composites via the two-step method devised in this research whereby 3D hydrogel-based scaffolds are printed first and then mineralized via the McGrath method. The advantages and disadvantages of the two fabrication techniques are compared. The two-step fabrication method was found to be superior in terms of the properties explored and desired in the composite.  The behaviour of the chitosan hydrogel-based scaffolds and composites fabricated using the McGrath method, under different stress and strain regimes were also investigated. Mechanical tests performed on air-dried chitosan hydrogel-based scaffolds and composites showed that the compressive modulus, strength and indentation hardness values obtained were within the same order of magnitude as that of trabecular bone. Data from uniaxial compression tests showed that the yield, ultimate strength and compressive modulus of the 3D scaffolds vary with the total mineral content, morphology and size of the resultant crystallites in the composite. Composites with very low mineral content (~7% CaCO₃ content) showed the best mechanical properties under uniaxial compressive stress (approximately 0.37 GPa compressive modulus, 26 MPa yield strength, and 31 MPa ultimate strength). Nanoindentation tests showed that the nanoscale hardness and indentation modulus increased upon mineralization of the scaffolds but did not vary significantly as a function of the extent of mineralization. Dynamic mechanical analysis showed that the scaffolds (both mineralized and non-mineralized) can effectively dissipate stress without complete fracture when subjected to dynamic compressions within physiologically relevant loading frequencies (1 - 15 Hz) irrespective of the mineral content. The individual responses vary with loading frequency.  Having ascertained the structural and mechanical attributes of the fabricated materials, their capacity to enable osteoblast cell attachment and proliferation was explored. Alamar Blue assay and confocal microscopy performed at various time points for samples exposed to in vitro cultured osteoblasts showed that chitosan hydrogel-based scaffolds and composites are biologically non-toxic and facilitate cell adhesion and proliferation. Furthermore, when osteoblasts were incubated with composites with low CaCO₃ content, the number of cells increased significantly within 14 days.  The results of this research confirm that 3D printed chitosan hydrogel-based composites fabricated using the McGrath mineralization method featuring various structural and compositional imitations of bone and nacre shows considerable potential as future bone grafts materials.</p>