Biomaterials 28 (2007) 2525–2533 Noninvasive image analysis of 3D construct mineralization in a perfusion bioreactor Blaise D. Porter a,Ã , Angela S.P. Lin a,b , Alexandra Peister b , Dietmar Hutmacher c,d , Robert E. Guldberg a,b a Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA b Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332, USA c Division of Bioengineering, National University of Singapore, Singapore 117576, Singapore d Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine National University of Singapore, Singapore 117576, Singapore Received 2 October 2006; accepted 1 January 2007 Available online 26 January 2007 Abstract Although the beneficial effects of perfusion on cell-mediated mineralization have been demonstrated in several studies, the size of the mineralized constructs produced has been limited. The ability to quantify mineralized matrix formation non-invasively within 3D constructs would benefit efforts to optimize bioreactor conditions for scaling-up constructs to clinically relevant dimensions. In this study, we report a micro-CT imaging-based technique to monitor 3D mineralization over time in a perfusion bioreactor and specifically assess mechanisms of construct mineralization by quantifying the number, size, and distribution of mineralized particle formation within constructs varying in thickness from 3 to 9 mm. As expected, mineralized matrix volume and particle number increased with construct thickness. Analyzing multiple concentric volumes inside each construct indicated that a greater proportion of the mineral volume was found within the interior of the perfused constructs. Interestingly, intermediate-sized 6 mm thick constructs were found to have the highest core mineral volume fraction and the largest mineralized particles. Two complementary mechanisms of increasing total mineral volume were observed in the 6 and 9 mm constructs: increasing particle size and increasing the number of mineralized particles, respectively. The rate of mineralized matrix formation in the perfused constructs increased from 0.69 mm 3 /week during the first 3 weeks of culture to 1.03 mm 3 /week over the final 2 weeks. In contrast, the rate of mineral deposition in the static controls was 0.01 mm 3 /week during the first 3 weeks of culture and 0.16 mm 3 /week from week 3 to week 5. The ability to monitor overall construct mineralization non-invasively coupled with quantitative analysis of mineralized particle size, number, and distribution offers a powerful tool for elucidating how mineral growth mechanisms are affected by cell type, scaffold material and architecture, or bioreactor flow conditions. r 2007 Elsevier Ltd. All rights reserved. Keywords: Bioreactor; Bone tissue engineering; Image analysis 1. Introduction Tissue engineering strategies that combine scaffolds with cells capable of osteogenesis or bioactive proteins offer a potential alternative to bone grafting for treatment of large, clinically challenging bone defects. Several studies have demonstrated that delivery of osteoprogenitor cells or osteoblasts significantly improves repair of long bone and cranial defects [1–5]. An important question is whether culturing bone repair constructs in vitro to induce progenitor cells to differentiate and produce mineralized extracellular matrix prior to implantation is advantageous. Some studies suggest that pre-mineralization may enhance subsequent mineral formation in vivo, but additional work needs to be done to more fully test this hypothesis. Byers recently showed that pre-mineralized constructs grown in vitro in static culture enhanced subsequent ectopic mineral formation in vivo [6]. However, most of the observed mineral formation was isolated to the periphery ARTICLE IN PRESS www.elsevier.com/locate/biomaterials 0142-9612/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2007.01.013 Ã Corresponding author. E-mail address: blaise.porter@tissuegrowth.com (B.D. Porter).