Biomaterials 26 (2005) 971–977 Effect of bone extracellular matrix synthesized in vitro on the osteoblastic differentiation of marrow stromal cells N! eha Datta a, *, Heidi L. Holtorf a , Vassilios I. Sikavitsas b , John A. Jansen c , Antonios G. Mikos a, * a Department of Bioengineering, Rice University, MS-142, P.O. Box 1892, Houston, TX 77251-1892, USA b School of Chemical Engineering and Materials Science, University of Oklahoma, 100 E. Boyd, T-335 Norman, OK 73019-1004, USA c Department of Biomaterials, University of Nijmegen, P.O. Box 9102, 6500 HC Nijmegen, The Netherlands Received 29 January 2004; accepted 6 April 2004 Available online 21 July 2004 Abstract Alternative materials for bone grafts are gaining greater importance in dentistry and orthopaedics, as the limitations of conventional methods become more apparent. We are investigating the generation of osteoinductive matrix in vitro by culturing cell/scaffold constructs for tissue engineering applications. The main strategy involves the use of a scaffold composed of titanium (Ti) fibers seeded with progenitor cells. In this study, we investigated the effect of extracellular matrix (ECM) laid down by osteoblastic cells on the differentiation of marrow stromal cells (MSCs) towards osteoblasts. Primary rat MSCs were harvested from bone marrow, cultured in dexamethasone containing medium and seeded directly onto the scaffolds. Constructs were grown in static culture for 12 days and then decellularized by rapid freeze–thaw cycling. Decellularized scaffolds were re-seeded with pre-cultured MSCs at a density of 2.5  10 5 cells/construct and osteogenicity was determined according to DNA, alkaline phosphatase, calcium and osteopontin analysis. DNA content was higher for cells grown on decellularized scaffolds with a maximum content of about 1.3  10 6 cells/construct. Calcium was deposited at a greater rate by cells grown on decellularized scaffolds than the constructs with only one seeding on day-16. The Ti/MSC constructs showed negligible calcium content by day-16, compared with 213.2 (713.6) mg/ construct for the Ti/ECM/MSC constructs cultured without any osteogenic supplements after 16 days. These results indicate that bone-like ECM synthesized in vitro can enhance the osteoblastic differentiation of MSCs. r 2004 Elsevier Ltd. All rights reserved. Keywords: Bone tissue engineering; Bone; Marrow stromal cells; Extracellular matrix 1. Introduction Culture of osteoblasts in 3-D is important in developing tissue engineered constructs for regeneration of large bone defects. To date, osteoblasts and osteoprogenitor cells have been cultured on a variety of scaffold materials and their ability to induce bone formation has been examined [1–10]. While some scaffolds have shown an innate ability to support bone formation during in vitro culture, others have been surface-modified with various molecules to aid their osteoinductive capacities. The most common of these enhancers include molecules present in native bone extracellular matrix (ECM) including hydroxyapatite [4,6] and collagen type I [11,12]. Other approaches to enhancing bone formation by cell/scaffold constructs have involved the delivery of certain growth factors that are known to participate in normal osteoblast differ- entiation including bone morphogenic proteins [3,12– 14], connective tissue growth factor [15], and transform- ing growth factors from the TGF-b family [16,17]. However, an ideal cell/scaffold/signal construct has yet to be developed for use in bone tissue engineering applications. One approach to development of more osteoinductive materials involves the use of native components of bone ECM to create the scaffold. There has been much focus on the use of bone-derived materials for the 3-D culture ARTICLE IN PRESS *Corresponding author. E-mail addresses: dattan@rice.edu (N. Datta), mikos@rice.edu (A.G. Mikos). 0142-9612/$-see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.04.001