Osteogenic differentiation of bone marrow MSCs by b-tricalcium phosphate stimulating macrophages via BMP2 signalling pathway Zetao Chen a , Chengtie Wu b, * , Wenyi Gu a, c , Travis Klein a , Ross Crawford a , Yin Xiao a, ** a Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, 60 Musk Ave, Kelvin Grove, Brisbane, Queensland 4059, Australia b State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, People’s Republic of China c Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Corner College and Cooper Rds, Brisbane, Queensland 4072, Australia article info Article history: Received 23 September 2013 Accepted 3 November 2013 Available online 20 November 2013 Keywords: Macrophage Immunoreaction Bone substitute b-TCP Osteogenesis abstract Immune reactions play important roles in determining the in vivo fate of bone substitute materials, either in new bone formation or inflammatory fibrous tissue encapsulation. The paradigm for the development of bone substitute materials has been shifted from inert to immunomodulatory materials, emphasizing the importance of immune cells in the material evaluation. Macrophages, the major effector cells in the immune reaction to implants, are indispensable for osteogenesis and their heterogeneity and plasticity render macrophages a primer target for immune system modulation. However, there are very few re- ports about the effects of macrophages on biomaterial-regulated osteogenesis. In this study, we used b-tricalcium phosphate (b-TCP) as a model biomaterial to investigate the role of macrophages on the material stimulated osteogenesis. The macrophage phenotype switched to M2 extreme in response to b-TCP extracts, which was related to the activation of calcium-sensing receptor (CaSR) pathway. Bone morphogenetic protein 2 (BMP2) was also significantly upregulated by the b-TCP stimulation, indicating that macrophage may participate in the b-TCP stimulated osteogenesis. Interestingly, when macrophage- conditioned b-TCP extracts were applied to bone marrow mesenchymal stem cells (BMSCs), the osteo- genic differentiation of BMSCs was significantly enhanced, indicating the important role of macrophages in biomaterial-induced osteogenesis. These findings provided valuable insights into the mechanism of material-stimulated osteogenesis, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of bone substitute materials. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction As exogenous bodies, implants tend to be rejected or isolated by the human body, mainly by the foreign body reaction. Aiming to avoid such a reaction, inert materials have been developed for in vivo implantation. However, this approach regrettably minimizes reconstructive healing and even worse sometimes enhances the foreign body reaction, resulting in the encapsulation of the material [1]. With the emergence of osteoimmunology, the interaction be- tween bone cells and immune cells has been studied more thor- oughly [2]. The immune and skeletal systems are closely related, sharing a number of cytokines, receptors, signalling molecules and transcription factors [3]. Besides its perpetuation leading to chronic inflammation, the immune response plays a vital role in implant integration and osteogenesis [4,5]. Inflammatory cytokines interleukin-1 (IL-1), tumour necrosis factor a (TNF-a), and IL-6 can enhance osteoclast differentiation and resorbing activity, and inhibit osteoblast activity and bone formation, while anti- inflammatory cytokines IL-4, IL-10, and IL-13 have the opposite effect [6]. Instead of osteoblastic cells, B cells have been shown to be the main source of bone marrow-derived osteoprotegerin, with a percentage of 64% [7,8], which suggests that B cells are the main inhibitors of osteoclastogenesis in normal physiology. This under- standing has led to a shift towards the development of “smart” bone substitute biomaterials capable of immunomodulation [4]. These “smart” biomaterials should be able to arouse an effective immune response, building an osteogenesis-enhancing environ- ment for bone cells. In vitro methods previously used to evaluate * Corresponding author. Tel.: þ86 21 52412249; fax: þ86 21 52413903. ** Corresponding author. Tel.: þ61 7 3138 6240; fax: þ61 7 3138 6030. E-mail addresses: chengtiewu@mail.sic.ac.cn (C. Wu), yin.xiao@qut.edu.au (Y. Xiao). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.11.014 Biomaterials 35 (2014) 1507e1518