Enhancement of Cell-Based Therapeutic Angiogenesis Using a Novel Type of Injectable Scaffolds of Hydroxyapatite-Polymer Nanocomposite Microspheres Yohei Mima1., Shinya Fukumoto1*., Hidenori Koyama 1 , Masahiro Okada2, Shinji Tanaka1, Tetsuo Shoji1, Masanori Emoto1, Tsutomu Furuzono 2 , Yoshiki Nishizawa 1 , Masaaki Inaba1 1 Department of Metabolism, Endocrinology, and Molecular Medicine, Osaka City University Graduate School of Medicine, Osaka, Japan, 2 Department of Biomedical Engineering, School of Biology-Oriented Science and Technology, Kinki University, Wakayama, Japan Abstract Background: Clinical trials demonstrate the effectiveness of cell-based therapeutic angiogenesis in patients with severe ischemic diseases; however, their success remains limited. Maintaining transplanted cells in place are expected to augment the cell-based therapeutic angiogenesis. We have reported that nano-hydroxyapatite (HAp) coating on medical devices shows marked cell adhesiveness. Using this nanotechnology, HAp-coated poly(L-lactic acid) (PLLA) microspheres, named nano-scaffold (NS), were generated as a non-biological, biodegradable and injectable cell scaffold. We investigate the effectiveness of NS on cell-based therapeutic angiogenesis. Methods and Results: Bone marrow mononuclear cells (BMNC) and NS or control PLLA microspheres (LA) were intramuscularly co-implanted into mice ischemic hindlimbs. When BMNC derived from enhanced green fluorescent protein (EGFP)-transgenic mice were injected into ischemic muscle, the muscle GFP level in NS+BMNC group was approximate fivefold higher than that in BMNC or LA+BMNC groups seven days after operation. Kaplan-Meier analysis demonstrated that NS+BMNC markedly prevented hindlimb necrosis (P,0.05 vs. BMNC or LA+BMNC). NS+BMNC revealed much higher induction of angiogenesis in ischemic tissues and collateral blood flow confirmed by three-dimensional computed tomography angiography than those of BMNC or LA+BMNC groups. NS-enhanced therapeutic angiogenesis and arteriogenesis showed good correlations with increased intramuscular levels of vascular endothelial growth factor and fibroblast growth factor-2. NS co-implantation also prevented apoptotic cell death of transplanted cells, resulting in prolonged cell retention. Conclusion: A novel and feasible injectable cell scaffold potentiates cell-based therapeutic angiogenesis, which could be extremely useful for the treatment of severe ischemic disorders. Citation: Mima Y, Fukumoto S, Koyama H, Okada M, Tanaka S, et al. (2012) Enhancement of Cell-Based Therapeutic Angiogenesis Using a Novel Type of Injectable Scaffolds of Hydroxyapatite-Polymer Nanocomposite Microspheres. PLoS ONE 7(4): e35199. doi:10.1371/journal.pone.0035199 Editor: Masuko Ushio-Fukai, University of Illinois at Chicago, United States of America Received November 25, 2011; Accepted March 13, 2012; Published April 18, 2012 Copyright: 2012 Mima et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported in part by a Grant-in-Aid for Scientific Research (20592102 to S.F.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. This study was also supported in part by the R&D of Nano-devices for Practical Utilization of Nanotechnology of the New Energy and Industrial Technology Development Organization (NEDO), Japan (08007436-0 to S.F. and 10000354-0 to T.F.), and Adaptable and Seamless Technology transfer Program through target-driven R&D (A-STEP) by Japan Science and Technology Agency (JST) (AS2314097F to S.F.). No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sfukumoto@med.osaka-cu.ac.jp . These authors contributed equally to this work. Introduction Severe ischemic vascular disease is caused by atherosclerotic occlusion of the arteries supplying blood to the myocardium and limbs. We and other groups have reported the clinical efficacy and safety of therapeutic angiogenesis by cell transplantation as an optional treatment for these diseases [1–4], although the effectiveness of this approach is still limited [5]. Various cell types, including autologous bone marrow mononuclear cells (BMNCs), stem cells, endothelial progenitor cells (EPCs), and peripheral blood mononuclear cells (PBMNCs), have been implanted to induce neovascularization for the treatment of ischemic heart disease and peripheral arterial disease [1–4,6]. With few exceptions, the administration route is injection into the ischemic tissue. Various angiogenic growth factors derived from implanted cells are key mediators of therapeutic angiogenesis [3,7,8]. However, about 70%–90% of the transplanted cells were estimated to disappear from the injection site within 1 week after transplantation [9–11]. Therefore, the efficacy of the cell-based therapeutic angiogenesis could be dependent on the retention, survival, and engraftment of implanted cells in ischemic tissue after implantation. In order to enhance the retention, survival, and engraftment of implanted cells, several approaches have been reported including genetic manipulation of donor cells (e.g., overexpression of antiapoptotic genes) [1,6,7] and cell delivery strategies using PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35199