Three-dimensional Bone Imaging: Optical Coherence Tomography versus Micro Computer Tomography Christoph Kasseck1, Marita Kratz2, Antonia Torcasio3, Nils C. Gerhardt1, G. Harry van Lenthe3, Thilo Gambichler4, Klaus Hoffmann4, David B. Jones2, and Martin R. Hofmann1 1: Photonics and Terahertz Technology, Ruhr-University Bochum, Universitaetsstr. 150, 44780 Bochum, Germany 2: Experimental Orthopaedics and Biomechanics, Philipps University Marburg, Baldingerstr., 35043 Marburg, Germany 3: Division of Biomechanics and Engineering Design, K.U. Leuven, Celestijnenlaan 300c, 3001 Leuven, Belgium 4: Department of Dermatology and Allergology, St. Josef Hospital, Gudrunstr. 56, 44791 Bochum, Germany e-mail: christoph.kasseck@rub.de Introduction A problem of long duration space missions is that some astronauts suffer from osteopenia, a precursor of osteoporosis, in the legs. This is possibly due to a significant decrease in mechanical loading coupled with a shift in fluid from the legs to the upper torso and head [1]. A sub-set of these osteopenic astronauts may need specific physiotherapy to regain the bone mass loss, which considerably decreases space mission efficiency [2]. Thus, a knowledge of the process of bone loss in space is an important research task of the European Space Agency (ESA). For this purpose, a bone culturing chamber named ZETOS has been developed to investigate inter alia the dynamic relationship between continuous bone growth and resorption in the marrow region (substantia spongiosa) [3]. Small pieces of bone can maintain metabolism in the culturing chamber up to three weeks, which allows monitoring of the quite slow process of bone turnover [4]. The most important parameters of this force are intensity, frequency, direction and duration [2,4]. ZETOS is capable of controlling these force parameters, which provides monitoring of the according bone reaction also in microgravity. To analyse the bone dynamics during zero gravity, an appropriate three dimensional imaging concept has to be used. This imaging concept should provide high contrast and high resolution and should allow continuous long time studies. Micro computer tomography (μCT) is a well accepted method for monitoring trabecular bone structure [6]. However, μCT uses ionising radiation. Thus, its use for long time studies of living cells is not desirable. Optical coherence tomography (OCT), in contrast, is a well-known and approved method to generate contactless, high-resolution, tomographic images with negligible radiation contamination of the sample [7]. Hence, OCT meets exactly the specifications of monitoring the living bone tissue in the culturing chamber. In this paper we show a series of OCT images of a human bone sample. They are compared with the μCT images from the same position. In addition, a microscopic overview of the sample and of its region of interest is presented as well as a three-dimensional sample overview acquired by μCT. Methods Sample preparation and both imaging systems are described as follows. The human hip bone of this study is from patients with total hip replacement, caused by coxarthrosis. Patient agreement was required and confirmed by the ethics commission. Throughout all cutting procedures, the bone was kept in a sterile 0.9% sodium chloride solution at 4 – 6°C, to limit the amount of damage caused by heat and to stop the bone from drying out. Bone samples were fixed in 70% ethanol, stored in the fridge and finally iteratively dehydrated in ethanol. After defatting with xylene, the samples were polymerized with Technovit 9100. In a final step, the samples were cut into pieces of about 3x4x4mm (height x width x length). For navigation purposes, one corner of each sample is labelled and a grid line structure was scratched into the surface of the samples. The equidistant vertical and horizontal lines have 500μm and 200μm, respectively, spacing in between. They were used for orientation purposes during OCT measurements. The surface of the sample, which passed through all three imaging methods, is visualized in Fig.1 and Fig.2 by microscopy. Results The microscopic images clearly show the grid lines at the surface of the sample. Fig.2 is the zoomed view of the region of interest from Fig.1, containing a specific trabecular bone structure. This structure is imaged with μCT as well as with OCT. The images will be compared stepwise. The OCT images were acquired with the SkinDex300 from Isis Optronics (Mannheim, Germany). This time domain system uses a superluminescence diode (SLD) with a center wavelength of 1300nm and a bandwidth of about 60nm (FWHM). It operates in an eight-channel-acquisition mode resulting in a frame speed of about 10frames/min. The generated images have a width of 1.0mm and a height of 0.9mm, with an axial resolution of 5μm and with a lateral raster step width of 3μm. A micrometer sample positioner was additionally implemented to obtain subsequent images. They were recorded in distances of 20μm over a length of 1.6mm, resulting in a cuboidal data set of 81 images. In this contribution, we compare the images step by step with the μCT data in order to show the high potential of OCT for high resolution three dimensional bone imaging. In a third imaging step, the three-dimensional microstructure of the bone sample was assessed by micro- computed tomography (Skyscan 1172, Skyscan, Kontich, Belgium) using a 6.5 mm nominal resolution. Image processing was performed using IPL (Scanco Medical, Brüttisellen, Switzerland). The acquired images were visually aligned with the OCT images to obtain qualitative comparison. Results of µCT and OCT Fig.3 to 5 depict sample images obtained with μCT with different zoom factors. Fig.4 is the zoomed region of interest of Fig.3, showing the same details as the microscopic image in Fig.2. The continuous line in Fig.5 labels the complete data cuboid in which the 81 OCT images were recorded. The dashed lines depict the positions of the following OCT images, which are serially numbered from `a´ to `i´. A typical OCT image of the sample is visualized in Fig.6. Beneath detector glass plate and sample surface, the trabecular bone is visible down to 0.9mm in depth. The marrow cells with a typical diameter of 70μm are clearly resolved. Marrow cell membranes are completely resolved in the upper image part while they are at least partially identifiable at the image bottom. Trabeculae and even inner structures of them can be visualized over the complete imaging range. In Fig.7, the first three OCT images from Fig.5 are directly faced to the according μCT images. The grid lines on the left and right of each OCT image show a correct translational alignment, while some of the OCT images are slightly tilted (e.g. Fig.8f). The μCT images were matched to the slight rotation of the OCT images. Conclusion Regarding the trabecular architecture only, both methods show a high correlation in imaging fixated human bone samples. Micro CT allows visualizing deeper lying structures while OCT provides higher hard- and soft tissue contrast. With comparable nominal resolutions of both systems (5μm and 6.5μm), OCT is in contrast to μCT capable of uncovering marrow cell membranes and inner bone structures. An advantage of OCT over μCT is that it does not use ionizing radiation showing great potential especially for in vivo applications. In the ZETOS system, OCT offers the possibility of following the cell activity over extended periods of time without ionizing radiation. References [1] L. Vico, P. Collet, A. Guignandon, M.-H. Lafage-Proust, T. Thomas, M. Rehailia, and C. Alexandre, “Effects of long-term microgravity exposure on cancellous and cortical weight-bearing bones of cosmonauts” The Lancet 355, 9215, 1607 (2000). [2] A. Hawkey, “Low magnitude, high frequency signals could reduce bone loss during spaceflight”, J. Brit. Interplanet. Soc. 60, 8, 278 (2007). [3] C.M. Davies, D.B. Jones, M.J. Stoddart, K. Koller, E. Smith, C.W. Archer, and R.G. Richards, “Mechanically loaded ex vivo bone culture system 'Zetos': Systems and culture preparation”, European Cells and Materials 11, 57-75, (2006). [4] V. David, A. Guignandon, A. Martin, L. Malaval, M.H. Lafage-Proust, A. Rattner, V. Mann, B. Noble, D.B. Jones, L. Vico, “Ex vivo bone formation in bovine trabecular bone cultured in a dynamic 3D bioreactor is enhanced by compressive mechanical strain”, Tissue Engineering, Part A 14, 1, 117 (2008). [5] E. Pola, W. Gao, Y. Zhou, R. Pola, W. Lattanzi, C. Sfeir, A. Gambotto, and P. Robbins, “Efficient bone formation by gene transfer of human LIM mineralization protein-3”, Gene Therapy 11, 8, 683 (2004). [6] G.H. van Lenthe, H. Hagenmüller, M. Bohner, S.J. Hollister, L. Meinel, and R. Müller, “Nondestructive micro-computed tomography for biological imaging and quantification of scaffold-bone interaction in vivo”, Biomaterials 28, 2479 (2007).[7] Handbook of Optical Coherence Tomography, B.E. Bouma and G.J. Tearney, eds., (Dekker, New York, 2002). 5. Acknowledgements This study was supported by the grants from the ESA MAP project AO 99-121. We furthermore thank the Kompetenzzentrum Medizintechnik Ruhr (KMR) for assistance. region of interest labelled corner 500µm ≈200µm fixation material bone border bone nose bay curve2 bone2 curve1 region of interest (ROI) bone border nose curve1 bay curve2 bone2 bone border OCT data cuboid h g e d c b a i f trabecular bone bone marrow cells vertical grid lines in sample surface detector glass plate inner structures of trabecular bone a b c d e f g h i Fig. 1 Fig. 3 Fig. 2 Fig. 5 Fig. 4 Fig. 6 Fig. 7 Fig. 8