Functionalized 3D-Printed Silkhydroxyapatite Scaffolds for Enhanced Bone Regeneration with Innervation and Vascularization

<b>Introduction</b><br/>The goal of this study was to generate functionalized 3D-printed scaffolds for bone regeneration using silkhydroxyapatite bioinks and osteoinductive, proangiogenic and neurotrophic growth factors or morphogens for accelerated bone formation. 3D printing was utilized to generate macroporous scaffolds with controlled geometries and architectures that promote osseointegration. We build on the knowledge that the osteoinductive factor Bone Morphogenetic Protein-2 (BMP2) can also positively impact vascularization, Vascular Endothelial Growth Factor (VEGF) can impact osteoblastic differentiation, and that Neural Growth Factor (NGF)-mediated signaling can influence bone regeneration.<br/><b>Experimental Methods</b><br/>We developed and characterized a silk-based bone cement with mechanical properties close to those of bone. We assessed our ability to 3D print this material to generate structures with a controlled geometry, macroporosity, and our ability to adapt the printing process to patient-tailored scaffolds. We investigated the cytocompatibility and osteoconductivity of these constructs. We further assessed functions on the 3D printed construct via the osteogenic differentiation of human mesenchymal stem cells. We also examined the migration and proliferation of human umbilical vein endothelial cells, and the proliferation of human induced neural stem cells.<br/><b>Results and Discussion</b><br/>The scaffolds provided mechanical properties suitable for bone and the materials were cytocompatible, osteoconductive and maintained the activity of the morphogens and cytokines. Synergistic outcomes between BMP-2, VEGF and NGF in terms of osteoblastic differentiation in vitro were identified, based on the upregulation of genes associated with osteoblastic differentiation (Runt-related transcription factor-2, Osteopontin, Bone Sialoprotein). In vivo studies were carried out to assess the biocompatibility and bone regeneration capability of these 3D printed constructs. New designs and internal architectures were exploited using 3D printing in order to maximize the mechanical properties of the bone constructs.<br/><b>Conclusion</b><br/>The specific cell responses observed here are key steps towards the development of the next generation of functionalized, 3D printed bone scaffolding to promote multicellular responses in the context of bone tissue system regeneration. These results are expected to have a strong impact in bone regeneration in dental, oral and maxillofacial surgery.<br/><b>References</b><br/>1. N.C. Keramaris, G.M. Calori, V.S. Nikolaou, E.H. Schemitsch, P. V. Giannoudis, Fracture vascularity and bone healing: A systematic review of the role of VEGF, Injury. 39 (2008)<br/>2. R.E. Tomlinson, Z. Li, Q. Zhang, B.C. Goh, Z. Li, D.L.J. Thorek, L. Rajbhandari, T.M. Brushart, L. Minichiello, F. Zhou, A. Venkatesan, T.L. Clemens, NGF-TrkA Signaling by Sensory Nerves Coordinates the Vascularization and Ossification of Developing Endochondral Bone, Cell Rep. 16 (2016)<br/>3. R.E. Tomlinson, Z. Li, Z. Li, L. Minichiello, R.C. Riddle, A. Venkatesan, T.L. Clemens, NGF-TrkA signaling in sensory nerves is required for skeletal adaptation to mechanical loads in mice, Proc. Natl. Acad. Sci. U. S. A. 114 (2017)