3D Printed Templating of Extrinsic Freeze-Casting for Macro–Microporous Biomaterials

As with most biological materials, natural bone has hierarchical structure. The microstructural features of compact bone are of various length scales with its porosity consisting of larger osteons (∼100 μm diameter) and vascular channels, as well as smaller lacuna spaces (∼10 μm diameter). In this study, the freeze-casting process, which has been previously used to form biocompatible porous scaffolds (made with hydroxyapatite, HA) has been improved to mimic the intrinsic hierarchical structure of natural bone by implementing an extrinsic 3D printed template. The results of pore characterization showed that this novel combined method of 3D printing and freeze-casting is able to produce porosity at multiple length scales. Nonporous, microporous (created with freeze-casting alone), and macro-micro porous (created with freeze-casting and 3D printed templating) scaffolds were compared as substrates to evaluate cellular activities using osteoblast-like MG63 cell lines. The number of cells oriented parallel to the HA wall structures in the freeze-cast scaffold was found to increase on the microporous and macro-micro porous samples when compare to the nonporous samples, mimicking the natural alignment of the lamella of natural bone. Regarding the cell morphologies, cells on microporous and macro-micro porous samples showed narrowly aligned shapes, whereas those on nonporous samples had polygonal shapes with no discernible orientation. Proliferation and differentiation tests demonstrated that no toxicity or functional abnormalities were found in any of the substrates due to potential chemical and mechanical residues that may have been introduced by the freeze-casting process. Monitoring of the three-dimensional distribution of cells in the scaffolds through microcomputed tomography indicates that the cells were well distributed in the interior pore spaces via the interpenetrating macro-micro pore networks. In summary, we demonstrate this novel approach can create porosity at multiple length scales and is highly favorable in creating a biocompatible, osteoconductive, and structurally hierarchical HA scaffolds for biomedical applications