Multi-modal imaging for assessment of tissue-engineered bone in a critical-sized calvarial defect mouse model K. A. Wartella 1 , V. Khalilzad-Sharghi 1 , M. L. Kelso 2 , J. L. Kovar 3 , D. L. Kaplan 4 , H. Xu 5 and S. F. Othman 1 * 1 Department of Biological Systems Engineering, University of Nebraska–Lincoln, Lincoln, NE, USA 2 Department of Pharmacy Practice, University of Nebraska Medical Center, Omaha, NE, USA 3 LI-COR Biosciences, Biology Research and Development, Lincoln, NE, USA 4 Department of Biomedical Engineering, Tufts University, Medford, MA, USA 5 School of Engineering and Computer Science, University of the Pacific, Stockton, CA, USA Abstract Tissue-engineered bone (TEB) analysis in vivo relies heavily on tissue histological and end-point eval- uations requiring the sacrifice of animals at specific time points. Due to differences in animal re- sponse to implanted tissues, the conventional analytical methods to evaluate TEB can introduce data inconsistencies. Additionally, the conventional methods increase the number of animals required to provide an acceptable statistical power for hypothesis testing. Alternatively, our non- invasive optical imaging allows for the longitudinal analysis of regenerating tissue, where each ani- mal acts as its own control, thus reducing overall animal numbers. In our 6 month feasibility study, TEB, consisting of a silk protein scaffold with or without differentiated mesenchymal stem cells, was implanted in a critical-sized calvarial defect mouse model. Osteogenesis of the TEB was moni- tored through signal variation, using magnetic resonance imaging (MRI) and near-infrared (NIR) op- tical imaging with IRDye® 800CW BoneTag TM (800CW BT, a bone-specific marker used to label osteogenically differentiated mesenchymal stem cells and mineralization). Histological endpoint measurements and computed tomography (CT) were used to confirm imaging findings. Anatomical MRI revealed decreased signal intensity, indicating mineralization, in the TEB compared to the con- trol (i.e. silk scaffold only) at various growth stages. NIR optical imaging results demonstrated a sig- nal intensity increase of the TEB compared to control. Interpretation of the imaging results were confirmed by histological analysis. Specifically, haematoxylin and eosin staining revealing de novo bone in TEB showed that 80% of the defect was covered by TEB, while only 40% was covered for the control. Taken together, these results demonstrate the potential of multi-modal non-invasive imaging to visualize and quantify TEB for the assessment of regenerative medicine strategies. Copyright © 2015 John Wiley & Sons, Ltd. Received 11 November 2014; Revised 9 April 2015; Accepted 12 June 2015 Keywords tissue-engineered bone (TEB); magnetic resonance imaging (MRI); near-infrared optical imag- ing; calvarial defect model; IRDye 800CW BoneTag 1. Introduction Treatment of bone disease and/or severe bone damage typically involves the use of bone grafts and surgical reconstruction. Still, several difficulties can arise from bone grafts, including morbidity at the donor site, in- fection, immune system reactions and exposure to *Correspondence to: S. F. Othman, Department of Biological Systems Engineering, University of Nebraska–Lincoln (UNL), 249 L. W. Chase Hall, Lincoln, NE 68588-0726, USA. E-mail: sothman2@unl.edu Copyright © 2015 John Wiley & Sons, Ltd. JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH ARTICLE J Tissue Eng Regen Med (2015) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.2068