A comparison between micro-ct and histology for the investigation of the cortical bone microstructure

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1 A comparison between micro-ct and histology for the investigation of the cortical bone microstructure F. Particelli 1, L. Mecozzi 1, F. Baruffaldi 1, M. Viceconti 1 1 Laboratorio di Tecnologia Medica, Istituto Ortopedico Rizzoli, Bologna, Italy Aims In the last years there has been a great interest in cortical bone investigation. In fact cortical bone porosity is considered one of the main parameters which determines bone strength [3, 8]. Even if histology has been the gold standard for the morphometric examination of bone specimens, in the last ten years a new method arose: X-ray microtomography (micro-ct). It permits the non-destructive analyses of specimens in a faster way preserving the integrity of the samples [5] and the calculation of the morphometric parameters over the whole sample s volume. Many studies showed the differences between these two techniques and tried to validate micro-ct system, but mostly for trabecular bone tissue [10]. Only few works showed comparisons between microtomographic images and histological sections of cortical bone specimens [1, 2, 3, 10]; but they lacked in a standardized binarization method and in a good spatial resolution (too close to the dimensions of Haversian canals). The key point in the analysis of micro-ct datasets is the binarization procedure, discriminating bone from non-bone and using an external method as a reference (histology) [7]. Moreover, it is not clear what effect the surrounding medium (air, saline solution, PMMA) has on the threshold value. Since the conventional histological examination requires an embedding treatment in PMMA, a study of the effect of this surrounding medium on structural parameters has to be performed also for the cortical bone tissue. For this reason, porosity derived from bone samples acquisitions in air and embedded in PMMA was compared. The aim of this work was to make a comparison between micro-ct scans of cortical bone specimens micro-ct scanned in air, and then scanned embedded in PMMA, using histological sections as reference. Then, once found the appropriate threshold value, structural parameters obtained from specimens acquired at the same micro-ct settings but surrounded by different media (air, PMMA) were compared. Method Twenty cortical bone samples were collected from femurs and tibias of four Caucasian donors (age range 62-74). Samples were obtained from deceased persons without skeletal disorders. After having obtained bone biopsies from the diaphyses, all the samples were micro-ct scanned in air, embedded in polymethylmethacrylate (PMMA), rescanned by micro-ct, examined by histology and finally compared. For the samples scanning, the device used was a Skyscan 1072 X-ray micro-ct (Skyscan, Kontich, Belgium). The cortical samples were acquired using a previously published protocol [9]: 80 KV, 125 µa, 1 mm aluminium filter, exposure time 5.9, image averaged on 2 frames, rotation 180, rotation step 0.9, field of view 8 x 8 mm 2 with a pixel size of 8 µm. In order to acquire the whole sample with this resolution, the oversize acquisition procedure implemented in the acquisition software (TomoNT, Skyscan, Kontich, Belgium) was used. After the acquisition, the cross section reconstruction was made using the software NRecon v (Skyscan), and a

2 stack of 1750 cross-sections images was produced, with a separation of one pixel (8 µm). The same protocol was used both for the acquisitions of samples in air and for the embedded samples. For the acquisition in air, samples were placed vertically into a polyethylene cylinder and positioned inside the micro-ct. After an accurate procedure of embedding in PMMA (Perilli), the bone samples were acquired again positioning them inside the micro-ct without the polyethylene cylinder. The cortical bone biopsies embedded in PMMA were later sectioned to thin slices of 30 m, stained with light green and observed at the microscope (Leica DMR-HC, Leica Microsystems, Wetzlar, Germany). A digital image of each histological slice was taken with Leica DC 300 camera mounted on the microscope (the final magnification was 50x, with a pixel size of 0.7 m) and then the porosity was determined through the microscope software Leica Qwin Plus v. 2.6 (Leica Microsystems Imaging Solutions) as the ratio between the sum of the pixels marked as pore and total pixels of the ROI. From the stack of micro-ct cross sections, the one corresponding to the histological image was visually chosen for each sample. A rectangular-shaped ROI 1,5 x 1 mm 2 (183 x 136 pixels) was selected and then placed in the position that looked like the one of the histological ROI. After the selection of the ROI, a binarization of the micro- CT images was necessary in order to discriminate bone from non-bone and a threshold value had to be found. For both the procedure (ROI extraction and binarization) the software CtAnalyzer (Skyscan) was used. The uniform threshold values for the acquisitions of bone in air and embedded in PMMA were determined thanks to a procedure described in Perilli et al. (2007): calculation of the porosity of the samples based on an external method (measured on the histological images); determination of a micro-ct optimal-threshold for each sample that corresponds to histologically calculated porosity; calculation of the fixedoptimal-threshold for the segmentation of the micro-ct images as the mean value of the optimal-thresholds. In this way two fixed-optimal- thresholds were calculated, the first one for the micro-ct images of the samples scanned in air, the second one for the acquisitions of the embedded bone samples. Finally these thresholds were applied to the segmentation of the respective micro-ct datasets. After having calculated the fixed thresholds, the cortical porosity was calculated using the software CTAnalyzer. For the comparison in porosity between histology and the corresponding cross section of bone sample in air and embedded in PMMA only one histological slice for each sample was used. Instead, for the comparison between micro-ct datasets of bone in air and of bone embedded in PMMA a parallelepiped volume of interest (VOI) of 183 slices was choosen (1,5 x 1,5 x 1 mm 3 ) for each sample and the following parameters were calculated: Porosity (%), Poro Diameter ( m) and Pore Separation ( m) [3,4]. Cortical porosity calculated on histological sections and with microct, for the acquisitions in air and in PMMA were compared. Moreover, another comparison was made in the parameters Po, Po.Dm and Po.Sp estimated over the VOIs between mciroct scans in air and embedded in PMMA. In the comparisons for each sample were determined the actual differences in the parameters d i, the percentage differences d i %, the mean actual difference and the mean percentage difference for each structural parameter [10]

3 Finally, if the data are compatible with a normal distribution (Kolmogorov-Smirnov), a student s t-test for paired samples will be used in the comparisons. The differences were deemed to be statistically significant at P < Results A good correspondence between the micro-ct images and the histological sections was found as shown in the figure (Fig. 1). (a) (b) (c) Figure 1: Microscope image (a), micro-ct image of embedded sample (b) and micro-ct image of acquisition in air (c) of the same cortical bone sample. Some filters are used in order to contrast the micro-ct images and better visualize pores..

4 In Table 1 it is shown the descriptive statistics of porosity estimated over the 2D sections obtained by histology, microct images acquired in PMMA and microct images acquired in air. N=20 Mean SD Range Histology: BV/TV% 12,9 6,12 3,8-25 Micro-CT embedded BV/TV% 13,00 6,75 3,1-25 Micro-CT air: BV/TV% 12,98 6,68 3,2-28 Table 1: Descriptive statistics of the porosity (%) estimated over 2D sections of the 20 cortical bone samples. The mean actual difference d, the mean percentage difference and the paired t-test found in the comparison between histology and micro-ct are shown in Table 2. There were no significant differences in porosity between histology and micro-ct. N=20 d SD d% SD% P Micro-CT embedded- Histology Porosity -0,1 1,24 1,79 11,89 0,96 Micro-CT air- Histology Porosity -0,076 1,33 1,22 10,01 0,97 Table 2: Comparison between histology and micro-ct in porosity (%) estimated over 2D sections. Porosity estimated from microct images embedded in PMMA and in air has been plotted in fig. 2 as a function of porosity assessed from 2D histological sections. Both the regressions had an high coefficient of determination (R 2 = 0.97 and R 2 = 0.96, respectively).

5 30 25 y = 1,089x - 1,0411 R² = 0,9727 Po % embedded PMMA Po % histology (a) y = 1,0718x - 0,85 R² = 0,9644 Po % air Po % histology (b) Figure 2: (a) Scatter plot of porosity estimated by micro-ct on samples embedded in PMMA versus porosity obtained by histology; (b) scatter plot of porosity estimated by micro-ct on samples in air versus porosity obtained by histology. Regarding the micro-ct 3D analysis over the VOI of 1,5 x 1,5 x 1 mm 3, the porosity in percent obtained from micro-ct images in air and embedded in PMMA were highly correlated (R 2 = 0.96). There were no significant differences in porosity, Po Dm. And Po Sp. over the VOIs between micro-ct scans of bone in air and embedded in PMMA. Conclusion Cortical bone samples were scanned by micro-ct first in air and after embedding in PMMA. Two optimal threshold values were found using histological sections as a reference. Comparisons with histology showed no significant differences in porosity for both micro-ct acquisitions. No significant differences were observed also for the Po, Po Dm. and Po Sp. obtained over the VOIs between samples scanned in air and after embedding in PMMA. Structural parameters determined by micro-ct are not affected by the surrounding medium

6 (PMMA) also for the cortical bone, if the corresponding threshold value for each condition is used. Although the difficulties encountered in the sample preparation and cutting, and the little number of samples, this study confirmed that micro-ct analyses is a reliable method also for the morphometrical characterization of cortical tissues. Moreover micro-ct has the advantage of being a non destructive, fast and three dimensional examination technique. Acknowledgements: The authors would like to thank Monica Montesi and Alina Beraudi for the support during the histological examination. References: 1. Basillais, A, Bensamoun, S., Chappard, C., Brunet-Imbault, B, Lemineur, G., Ilharreborde, B., Ho Ba Tho, M.C., Benhamou C.L., (2007) Three-dimensional characterization of cortical bone microstructure by microcomputed tomography: validation with ultrasonic and microscopic measurements, J. Orthop. Sci. 12, Bousson V., Peyrin F., Bergot C., Hausard M., Sautet A., Laredo J.D., (2004) Cortical bone in the human femoral neck: three-dimensional appearence and porosity using Synchrotron Radiation, J. Bone Min. Res., 19: Cooper D.M.L., Turinsky, A.L., Sensen C.W., Hallgrimsson B., (2003) Quantitative 3D analysis of the canal network in cortical bone by microcomputed tomography, The anatomical record, 274B: Hildebrand, T. & Rüegsegger, P. (1997a) A new method for the modelindependent assessment of thickness in three-dimensional images. J. Microsc. 185, Müller, R. & Rüegsegger, P. (1997) Micro-tomographic imaging for the nondestructive evaluation of trabecular bone architecture. Stud. Health Technol. Inform. 40, Parfitt, A. M., Drezner, M. K., Glorieux, F. H., Kanis, J. A., Malluche, H., Meunier, P. J., Ott, S. M. & Recker, R. R. (1987) Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone Miner. Res. 2, Perilli, E., Baruffaldi, F., Visentin, M., Bordini, B., Traina, F., Cappello, A., Viceconti, M., MicroCT examination of human bone specimens: effects of polymethylmethacrylate embedding on structural parameters. J Microsc 225, Spadaro JA, Werner FW, Brenner RA, Fortino MD, Fay LA, Edwards WT (1994) Cortical and trabecular bone contribute strenght to the osteopenic distal radius, J Ortoph Res 12: Tassani S., Ohman C., Baruffaldi F., Baleani M., Viceconti M., (2011) Volume to density relation in adult human bone tissue. J.Biomech 44, Wachter N. J., Augat P., Krischak G. D., Mentzel M., Kinzl L., Claes L., (2001) Prediction of cortical bone porosity in vitro by microcomputed tomography, Calcif Tissue Int., 68: