Journal of Microscopy , Vol. 238, Pt 3 2010, pp. 230–239 doi: 10.1111/j.1365-2818.2009.03345.x Received 19 February 2009; accepted 21 September 2009 Regularized phase tomography enables study of mineralized and unmineralized tissue in porous bone scaffold M. LANGER ∗, †, Y. LIU ‡, F. TORTELLI ‡, P. CLOETENS ∗ , R. CANCEDDA ‡ & F. PEYRIN ∗, † ∗ ESRF, 6 rue Jules Horowitz, F-38043 Grenoble Cedex, France †CREATIS-LRMN; INSERM U630; CNRS 5220; INSA-Lyon; Universit´ e de Lyon, F-69621, Lyon, France ‡Dipartimento di Oncologia, Biologia e Genetica, Universit` a di Genova, 16132 Genova, Italy Key words. Bioceramic scaffold, bone tissue engineering, microtomography, phase imaging, synchrotron radiation. Summary Regularized phase tomography was used to image non- calcified fibrous matrix in in vitro cell-cultivated porous bone scaffold samples. 3D micro-architecture of bone and bone scaffold has previously been studied by micro-computed tomography, synchrotron radiation (SR) micro-computed tomography and microdiffraction. However, neither of these techniques can resolve the low-calcified immature pre-bone fibrous structures. Skelite porous scaffold discs were seeded with osteoblasts, a combination of osteoblast and pre-osteoclasts and, as controls, with pre-osteoclasts only, and then cultivated for 8 weeks. They were subsequently imaged using SR propagation-based phase contrast imaging. Reconstructions using a regularized holographic phase tomography approach were compared to standard (absorption) SR micro-computed tomography, which show that quantitative analysis, such as volume and thickness measurements, of both the calcified fraction and the immature bone matrix in the reconstructed volumes is enabled. Indications of the effect of this type of culture on Skelite, such as change in mineralization and deposit of mature bone on the walls of the scaffold, are found. The results are verified with a histological study. Introduction The use of biodegradable porous scaffolds in conjunction with tissue engineering approaches is emerging as a viable technique for reconstructive bone surgery (Kofron et al., 2004). Desirable characteristics of the scaffolds are that they provide a surface favourable for cell Correspondence to: M. Langer, ESRF, 6 rue Jules Horowitz, F-38043 Grenoble Cedex, France. Tel: +33 (0)4 38 88 19 77; e-mail: max.langer@esrf.fr attachment (osteoconductive), induce osteoblast precursors to differentiate into mature bone-forming cells (osteoinductive), and that they contribute to bone regeneration (osteogenic). Ideally, scaffolds should be resorbed by the host, and be replaced by new, living bone (Glazer et al., 2001). More precisely, the resorption should not be a random process, but should only occur where new pre-bone matrix is being deposited (Mastrogiacomo et al., 2007). It has been shown that porous scaffold such as Skelite (Millenium Biologix, Ontario, CA, USA), based on silicon stabilized tricalcium phosphate (Si-TCP), when seeded with human bone marrow stromal cells and implanted into a soft tissue site in immunodeficient mice, induces the formation of mature lamellar bone within 20 weeks (Papadimitropoulos et al., 2007). It has also been shown that Skelite scaffolds are resorbed when acted upon by osteoclasts (Mastrogiacomo et al., 2006), and that there is a coupling between bone regrowth and scaffold resorption in Skelite scaffolds (Mastrogiacomo et al., 2007). Moreover, Skelite scaffolds can be used as substrate for three-dimensional (3D) in vitro cultures. The trabecular morphology of the pores, resembling native bone, provides an ideal mineralized environment enhancing osteoblasts and osteoclasts differentiation, thus leading to the in vitro deposition of an extracellular matrix similar to the matrix of the native bone tissue (Tortelli et al., 2009). Recently, 3D X-ray microtomography (μCT) has been proposed to study the microarchitecture of bone and scaffolds (Cartmell et al., 2003; Lin et al., 2003; Bernhardt et al., 2004; Bernhardt et al., 2005; Thomsen et al., 2005; Ho & Hutmacher 2006). In particular, synchrotron radiation μCT (SR-μCT) (Salom´ e et al., 1999) allowed to identify and quantify newly formed bone (Komlev et al., 2006) and was also sucessfuly coupled to microdiffraction to get information about C  2010 The Authors Journal compilation C  2010 The Royal Microscopical Society