Characterization and in ovo vascularisation of a 3D-printed hydroxyapatite scaffold with different extracellular matrix coatings under perfusion culture.

For the fabrication of appropriate bone tissue engineered constructs, several prerequisites should be fulfilled. It should offer long-term stability, allow proper cell attachment and proliferation, furthermore be osteoinductive and easy to be vascularized. Having these requirements as background, we fabricated a novel porous 3D-printed hydroxyapatite (HA) scaffold and treated it with oxygen plasma (OPT). MG-63 pre-osteoblast-seeded bone constructs allowed good cell attachment and proliferation, even better when cultivated in a perfusion flow bioreactor. Moreover, the deposition of extracellular matrix (ECM) on the otherwise inorganic surface changed the mechanical properties in a favourable manner: elasticity increased from 42.95±1.09 to 91.9±5.1 MPa (assessed by nanoindentation). Compared to static conditions, osteogenic differentiation was enhanced in the bioreactor, with upregulation ALP, collagen I and osteocalcin gene expression.In parallel experiments, primary human bone marrow mesenchymal stromal cells (hBMSCs) were used and findings under dynamic conditions were similar; with a higher commitment towards osteoblasts compared to static conditions. In addition, angiogenic markers CD31, eNOS and VEGF were upregulated, especially when osteogenic medium was used compared to proliferative medium. To compare differently fabricated ECMs in terms of vascularisation, decellularized constructs were tested in the chorioallantoic membrane (CAM) assay with subsequent assessment of the functional perfusion capacity by MRI in the living chick embryo. Here, vascularisation induced by ECM from osteogenic medium led to a vessel distribution more homogenously throughout the construct, while ECM from proliferative medium enhanced vessel density at the interface and to a lower extent at the middle and top. We conclude that dynamic cultivation of a novel porous OPT HA scaffold with hBMSCs in osteogenic medium and subsequent decellularization provides a promising off-the-shelf bone tissue engineered construct.