Bone formation in polymeric scaffolds evaluated by proton magnetic resonance microscopy and X-ray microtomography Newell R. Washburn, 1 Michael Weir, 1 Paul Anderson, 2 Kimberlee Potter 3,4 1 Polymers Division, National Institute of Standards and Technology, Gaithersburg, Maryland 2 Centre for Oral Growth and Development, Queen Mary, University of London, London, United Kingdom 3 Magnetic Resonance Microscopy Facility, Armed Forces Institute of Pathology Annex, Rockville, Maryland 4 Section on Tissue Biophysics and Biomimetics, National Institute of Child Health and Human Development, Bethesda, Maryland. Received 22 August 2003; revised 25 November 2003; accepted 25 February 2004 Published online 3 May 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30054 Abstract: Magnetic resonance microscopy (MRM) and X- ray microtomography (XMT) were used to investigate de novo bone formation in porous poly(ethyl methacrylate) (PEMA) scaffolds, prepared by a novel co-extrusion process. PEMA scaffolds were seeded with primary chick calvarial osteoblasts and cultured under static conditions for up to 8 weeks. Bone formation within porous PEMA scaffolds was confirmed by the application of histologic stains to intact PEMA disks. Disks were treated with Alizarin red to visu- alize calcium deposits and with Sirius red to visualize re- gions of collagen deposition. DNA analysis confirmed that cells reached confluence on the scaffolds after 7 weeks in static culture. The formation of bone in PEMA scaffolds was investigated with water proton MRM. Quantitative MRM maps of the magnetization transfer ratio (MTR) yielded maps of protein deposition, and magnetic resonance (MR) relaxation times (T1 and T2) yielded maps of mineral dep- osition. The location of newly formed bone and local mineral concentrations were confirmed by XMT. By comparing MRM and XMT data from selected regions-of-interest in one sample, the inverse relationship between the MR relaxation times and mineral concentration was validated, and calibra- tion curves for estimating the mineral content of cell-seeded PEMA scaffolds from quantitative MRM images were developed.© 2004 Wiley Periodicals, Inc.* J Biomed Mater Res 69A: 738 –747, 2004 Key words: magnetic resonance microscopy; X-ray microto- mography; tissue engineering; bone; polymer scaffold INTRODUCTION The limited supply of autograft material for the repair of skeletal defects has inspired an explosion of strategies for the production of replacement bone tis- sue. In the majority of cases, bone cells are required to attach and grow on a three-dimensional scaffold ma- terial. 1 Two-dimensional culture techniques might be applied to three-dimensional scaffolds, but there are a number of factors that limit the success of three-di- mensional cultures. To get good bone formation, there must be adequate seeding of the scaffold by the cells. This has led to a number of studies on optimal seeding techniques for porous scaffold materials. 2–4 The suc- cess of the various seeding techniques is assessed us- ing a DNA assay to estimate the total number of cells within the scaffold. This approach, however, yields very little information about the distribution of the cells throughout the scaffold. Other investigators have reported on the successful application of confocal mi- croscopy to study cells growing in the pores of scaf- folds, 5,6 but the depth penetration of this technique is limited and it cannot be applied to scaffolds that are optically opaque. What is required is a nondestructive technique that can provide spatially resolved, chemi- cally specific, tissue-level information about the bone formed within the pores of the scaffold material. Conventional techniques, such as histomorphom- The opinions and assertions contained herein are the pri- vate views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense. Certain equipment and instruments or materials are iden- tified in this article to adequately specify the experimental details. Such identification neither implies recommendation by the National Institute of Standards and Technology, nor that the materials are necessarily the best available for the purpose. Published abstract appears in Proc Int Soc Magn Reson Med 2002;10:497. Correspondence to: Kimberlee Potter, Ph.D., Department of Cellular Pathology and Genetics, Armed Forces Institute of Pathology Annex, 1413 Research Blvd., Rockville, MD 20850; e-mail: potterk@afip.osd.mil Contract grant sponsor: NIH; contract grant number: DE14453 Contract grant sponsor: EPSRC; contract grant number: GR/R28911/01 © 2004 Wiley Periodicals, Inc. * This article is a US Government work and, as such, is in the public domain in the United States of America.