Abstract
ABSTRACTFor the fabrication of appropriate bone tissue-engineered constructs several prerequisites should be fulfilled. They should offer long-term stability, allow proper cell attachment and proliferation and 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, which was 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 of 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 rather than proliferative medium. To compare differently fabricated ECMs in terms of vascularization, 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, vascularization induced by ECM from osteogenic medium led to a vessel distribution more homogenous 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.
Highlights
In maxillofacial and orthopaedic surgery, repair and regeneration of bone defects caused by trauma, tumour excision or infection is daily business and is a central clinical goal
Cell seeded 3D-printed HA scaffolds: static versus dynamic culture The proliferation of MG-63 osteoblast-like cells seeded on 3Dprinted porous HA scaffolds after 18 h, 3, 7, 14 and 28 days of culture under static (24-well plate) and dynamic conditions was evaluated
Under dynamic conditions, there were more cells and cell distribution was more homogeneous, with cells covering the whole surface of the scaffold
Summary
In maxillofacial and orthopaedic surgery, repair and regeneration of bone defects caused by trauma, tumour excision or infection is daily business and is a central clinical goal. To treat bone tissue loss, the development of novel orthopaedic strategies based on tissue engineering approaches has progressed considerably over the last four decades (Langer et al, 1990; Venkatesan and Kim, 2010; Boland, 2017; Forrestal et al, 2017; Hosseinpour et al, 2017; Khakestani et al, 2017; Kim et al, 2017; Janko et al, 2018). Hydroxyapatite (HA)-based scaffolds (Lee et al, 2013; Ha et al, 2015; Dang et al, 2016) are of considerable interest, since HA is the major inorganic component of natural bone (Nandi et al, 2010). One major drawback of pure HA scaffolds, is their brittleness (Shi et al, 2014; Owen et al, 2017), which has been overcome by various approaches, such as composites with polymers (Roeder et al, 2008; Cox et al, 2015; Zeng et al, 2018) or by the deposition of extracellular matrix (Sadr et al, 2012), which increases the elasticity of the materials
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