Three-dimensional biofabrication of an aragonite-enriched self-hardening bone graft substitute and assessment of its osteogenicity in vitro and in vivo.

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Abstract
A self-hardening three-dimensional (3D)-porous composite bone graft consisting of 65 wt% hydroxyapatite (HA) and 35 wt% aragonite was fabricated using a 3D-Bioplotter®. New tetracalcium phosphate and dicalcium phosphate anhydrous/aragonite/gelatine paste formulae were developed to overcome the phase separation of the liquid and solid components. The mechanical properties, porosity, height and width stability of the end products were optimised through a systematic analysis of the fabrication processing parameters including printing pressure, printing speed and distance between strands. The resulting 3D-printed bone graft was confirmed to be a mixture of HA and aragonite by X-ray diffraction, Fourier transform infrared spectroscopy and energy dispersive X-ray spectroscopy. The compression strength of HA/aragonite was between 0.56 and 2.49 MPa. Cytotoxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro. The osteogenicity of HA/aragonite was evaluated in vitro by alkaline phosphatase assay using human umbilical cord matrix mesenchymal stem cells, and in vivo by juxtapositional implantation between the tibia and the anterior tibialis muscle in rats. The results showed that the scaffold was not toxic and supported osteogenic differentiation in vitro. HA/aragonite stimulated new bone formation that bridged host bone and intramuscular implants in vivo. We conclude that HA/aragonite is a biodegradable and conductive bone formation biomaterial that stimulates bone regeneration. Since this material is formed near 37°C, it will have great potential for incorporating bioactive molecules to suit personalised application; however, further study of its biodegradation and osteogenic capacity is warranted. The study was approved by the Animal Ethical Committee at Tongji Medical School, Huazhong University of Science and Technology (IACUC No. 738) on October 1, 2017.

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