JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH ARTICLE J Tissue Eng Regen Med 2009; 3: 398–404. Published online 11 May 2009 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/term.176 Signalling strategies for osteogenic differentiation of human umbilical cord mesenchymal stromal cells for 3D bone tissue engineering Limin Wang, 1 Milind Singh, 2 Lynda F. Bonewald 3 and Michael S. Detamore 4 * 1 Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA 2 Department of Bioengineering, Rice University, Houston, TX 77251, USA 3 Department of Oral Biology, University of Missouri-Kansas City, Kansas City, MO 64108, USA 4 Department of Chemical and Petroleum Engineering, University of Kansas, Lawrence, KS 66045, USA Abstract Human umbilical cord mesenchymal stromal cells (hUCMSCs) have recently shown the capacity to differentiate into multiple cell lineages in all three embryonic germ layers. The osteogenic differentiation of hUCMSCs in monolayer culture has been reported, while the differentiation in three-dimensional biomaterials has not yet been reported for tissue-engineering applications. Thus, the aim of this study was to evaluate the feasibility of using hUCMSCs for bone tissue engineering. hUCMSCs were cultured in poly(L-lactic acid) (PLLA) scaffolds in osteogenic medium (OM) for 3 weeks, after which the scaffolds were exposed to several different media, including the OM, a mineralization medium (MM) and the MM with either 10 or 100 ng/ml insulin-like growth factor (IGF)-1. The osteogenic differentiation was confirmed by the up-regulation of Runx2 and OCN, calcium quantification and bone histology. Switching from the OM to the MM promoted collagen synthesis and calcium content per cell, while continuing in the OM retained more cells in the constructs and promoted higher osteogenic gene expression. The addition of IGF-1 into the MM had no effect on cell proliferation, differentiation and matrix synthesis. In conclusion, hUCMSCs show significant potential for bone tissue engineering and culturing in the OM throughout the entire period is beneficial for osteogenic differentiation of these cells. Copyright  2009 John Wiley & Sons, Ltd. Received 15 August 2008; Revised 5 February 2009; Accepted 17 March 2009 Keywords umbilical cord stroma; stem cells; osteogenesis; IGF-1 1. Introduction Bone tissue engineering is a promising interdisciplinary field, in which cells can be integrated into biomaterials to provide a substitute for bone grafts. Indeed, human bone marrow mesenchymal stromal cells (hBMSCs) have been translated into clinical trials via a tissue-engineering approach to treat bone defects successfully in a 7 year clinical trial (Quarto et al., 2001; Marcacci et al., 2007). However, there are some known disadvantages associated *Correspondence to: Michael S. Detamore, University of Kansas, Department of Chemical and Petroleum Engineering, 4132 Learned Hall, 1530 W 15th Street, Lawrence, KS 66045, USA. E-mail: detamore@ku.edu with hBMSCs, such as the relative number of hBMSCs in the marrow, a limited proliferation ability and inferior differentiation potential in aged individuals (Mueller and Glowacki, 2001). Moreover, the invasive and painful harvesting procedure may cause donor site morbidity and complications (Lee et al., 2007). Recent evidence has shown that human umbilical cord mesenchymal stromal cells (hUCMSCs) are a primitive and multipotent stromal population that shares similar characteristics with hBMSCs (Can and Karahuseyinoglu, 2007; Troyer and Weiss, 2008). hUCMSCs are isolated from the Wharton’s jelly of umbilical cords, a tissue routinely discarded after delivery. They are non- haematopoietic cells that can adhere to plastic surfaces for in vitro expansion and have many surface markers Copyright  2009 John Wiley & Sons, Ltd.