Citation: Alfayez, E.; Veschini, L.; Dettin, M.; Zamuner, A.; Gaetani, M.; Carreca, A.P.; Najman, S.; Ghanaati, S.; Coward, T.; Di Silvio, L. DAR 16-II Primes Endothelial Cells for Angiogenesis Improving Bone Ingrowth in 3D-Printed BCP Scaffolds and Regeneration of Critically Sized Bone Defects. Biomolecules 2022, 12, 1619. https:// doi.org/10.3390/biom12111619 Academic Editor: Vladimir N. Uversky Received: 19 September 2022 Accepted: 30 October 2022 Published: 2 November 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biomolecules Article DAR 16-II Primes Endothelial Cells for Angiogenesis Improving Bone Ingrowth in 3D-Printed BCP Scaffolds and Regeneration of Critically Sized Bone Defects Eman Alfayez 1,2,† , Lorenzo Veschini 2,† , Monica Dettin 3 , Annj Zamuner 4 , Massimiliano Gaetani 5,6 , Anna P. Carreca 5 , Stevo Najman 7 , Shahram Ghanaati 8 , Trevor Coward 2 and Lucy Di Silvio 2, * 1 Faculty of Dentistry, Oral Biology Department, King Abdulaziz University, Jeddah 21589, Saudi Arabia 2 Faculty of Dentistry, Oral & Craniofacial Sciences King’s College London, London SE1 9RT, UK 3 Department of Industrial Engineering, University of Padua, 35131 Padua, Italy 4 Department of Civil, Environmental, and Architectural Engineering, University of Padua, 35131 Padua, Italy 5 Fondazione Ricerca nel Mediterraneo (Ri.MED) and Department of Laboratory Medicine and Advanced Biotechnologies, Istituto di Ricovero e Cura a Carattere Scientifico-Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione, 90100 Palermo, Italy 6 Chemical Proteomics, Department of Medical Biochemistry and Biophysics, Karolinska Institutet and SciLifeLab (Science for Life Laboratory), SE-17 177 Stockholm, Sweden 7 Faculty of Medicine, University of Niš, 18000 Niš, Serbia 8 Department for Oral, Cranio-Maxillofacial and Facial Plastic Surgery, Medical Center of the Goethe University, 60323 Frankfurt, Germany * Correspondence: lucy.di_silvio@kcl.ac.uk; Tel.: +44-02-07848-8475 † These authors equally contributed to this work. Abstract: Bone is a highly vascularized tissue and relies on the angiogenesis and response of cells in the immediate environmental niche at the defect site for regeneration. Hence, the ability to control angiogenesis and cellular responses during osteogenesis has important implications in tissue- engineered strategies. Self-assembling ionic-complementary peptides have received much interest as they mimic the natural extracellular matrix. Three-dimensional (3D)-printed biphasic calcium phosphate (BCP) scaffolds coated with self-assembling DAR 16-II peptide provide a support template with the ability to recruit and enhance the adhesion of cells. In vitro studies demonstrated prompt the adhesion of both human umbilical vein endothelial cells (HUVEC) and human mesenchymal stem cells (hMSC), favoring endothelial cell activation toward an angiogenic phenotype. The SEM-EDS and protein micro bicinchoninic acid (BCA) assays demonstrated the efficacy of the coating. Whole proteomic analysis of DAR 16-II-treated HUVECs demonstrated the upregulation of proteins involved in cell adhesion (HABP2), migration (AMOTL1), cytoskeletal re-arrangement (SHC1, TMOD2), immuno-modulation (AMBP, MIF), and morphogenesis (COL4A1). In vivo studies using DAR-16-II- coated scaffolds provided an architectural template, promoting cell colonization, osteogenesis, and angiogenesis. In conclusion, DAR 16-II acts as a proactive angiogenic factor when adsorbed onto BCP scaffolds and provides a simple and effective functionalization step to facilitate the translation of tailored 3D-printed BCP scaffolds for clinical applications. Keywords: bone regeneration; tissue engineering; osteogenesis; angiogenesis; self-assembly peptides; biphasic calcium phosphate; 3D printing; bone scaffolds; scaffold functionalization 1. Introduction The regeneration of critical-sized bone defects represents a significant and longstand- ing clinical problem [1]. Such defects result from traumas, degenerative processes, or surgery to remove cancer [2]. The current gold standard in bone regeneration remains the transplantation of autologous bone harvested from healthy regions of the skeleton [3]. Regenerating large bone defects, particularly in craniofacial applications, requires the use of Biomolecules 2022, 12, 1619. https://doi.org/10.3390/biom12111619 https://www.mdpi.com/journal/biomolecules