HIGH-RESOLUTION MEASUREMENT OF CORTICAL BONE POROSITY IN NORMAL AND OSTEOPENIC RATS *Ciani, C; *Ramirez Marin, P A; **Doty, S B; +*Fritton, S P

+*Department of Biomedical Engineering, CUNY Graduate School and The City College of New York, New York, NY fritton@ccny.cuny.edu

Introduction Bone is a dense, compact, yet very porous material. It is possible to distinguish three different levels of bone porosity, namely the vascular porosity, the lacunar-canalicular porosity that surrounds the osteocytes and is connected to the vascular porosity, and the collagen-apatite porosity of the mineralized matrix. The vascular porosity and lacunar-canalicular porosity are directly involved in interstitial fluid flow, thought to play an important role in bone’s mechanosensory system. Measurements of human, canine, and rat bone have found the vascular porosity to range from 1.5 to 6% and the lacunar-canalicular porosity to range from 2 to 5%, with the overall cortical bone porosity estimated to be approximately 9% [1-7]. However, in our recent work quantifying the 3-D microstructure of the osteocyte lacunar-canalicular system [8], we observed that the lacunar-canalicular network is quite extensive in the mineralized bone volume, raising the possibility that previous studies might have underestimated the interstitial fluid porosity. Accurate porosity measurements are important for determining realistic bone permeability values that can be used in poroelastic models that assess the response of bone to mechanical loading. In addition, it has been proposed that an alteration in osteocyte viability due to osteoporosis could alter the interconnectedness of the osteocyte network, affecting interstitial fluid flow and altering the mechanical stimulus experienced by the bone cells [9]. To address these issues this study was undertaken to measure the vascular and lacunar-canalicular porosities in both normal and osteopenic bone, using an ovariectomized rat model and confocal scanning laser microscopy to provide a high-resolution analysis of the bone microstructure. Methods Female Sprague Dawley rats were divided into two groups, with one group undergoing ovariectomy (OVX) at 5 months of age. Six weeks post-ovariectomy the OVX group (n=6, 339 ± 20g) and the age-matched control group (n=5, 266 ± 5g) were sacrificed, as approved by the Institutional Animal Care and Use Committee. Left tibiae were harvested and put in EM fixative (0.5% gluteraldehyde, 2% paraformaldehyde in

a b

Figure 1: (a) OVX tibial section, scale bar 600 µm; (b) Sub-section of image in (a), scale bar 30 µm; (c) Sub-section of image in (b), illustrating details of lacunar-canalicular system; osteocyte lacunae (Ot) and canaliculi (Ca) are indicated, scale bar 8 µm.

c

Ot

Figure 2: Total bone porosity

(mean ± standard deviation) and vascular and lacunar-canalicular porosities for the OVX (osteopenic) and control groups.

Ca

0.05M cacodylate-sodium buffer, pH 6.8). To process the bones, tibial sections (0.3-0.5 mm) were cut with a diamond blade saw (Buehler) 5 mm below the mid-diaphysis and put immediately back into fixative for 24 hours. Sections were then rinsed, ground down to a final thickness of 30-70 µm and dehydrated in a series of graded alcohol. To label interstitial fluid space, sections were stained for 4 hours in a 1% FITC solution dissolved in 100% ETOH and then coverslipped. Bone sections, one for each animal, were visualized using a confocal microscope (Leica TCS SP2, Germany) with a 40x oil immersion lens (1.25 numerical aperture and pinhole set at 1 Airy unit) and wavelength excitation of 488 nm. Every image was taken at a resolution of 2048 x 2048 pixels with a field of view of 370 µm x 370 µm. A complete reconstruction of each tibial cross-section was obtained using Photoshop (approximately 90 million pixels, 600Mb). The images were transformed to grayscale and thresholded using Otsu's method (Matlab) to delineate the FITC-labeled interstitial fluid space. The total porosity was then calculated as the percentage of bone area labeled with FITC. The vascular porosity was calculated separately by quantifying the total vascular pore area for each section and dividing it by the total bone area. Finally, the lacunar-canalicular porosity was calculated as the difference between the total porosity and the vascular porosity. Comparisons between groups were statistically tested by student's t-test (p<0.05). Results The tibial cross-sections showed uniform staining of the vascular porosity and the lacunar-canalicular system, with the mineralized matrix impermeable to FITC (Figure 1a). The lacunar-canalicular porosity was well characterized because of the high resolution of the images (Figure 1b, c). The total interstitial fluid porosity was found to be 18.7 ± 1.3% for the control group and 23.6 ± 7.0% for the OVX group (Figure 2). The vascular porosity was 2.4 ± 0.1% for the control group and 2.6 ± 0.1% for the OVX group, giving a lacunar-canalicular porosity of approximately 16% for the control group and 21% for the OVX group (Figure 2). No statistically significant differences were found between groups.

Discussion This study demonstrates an effective method to quantify bone porosity using confocal microscopy, which allows high-resolution images of an entire bone cross-section while demonstrating the microstructural details of the lacunar-canalicular porosity. While the vascular porosities calculated in this study are similar to values reported in the literature, the total cortical bone porosity is significantly greater than what had been previously estimated for normal bone. The higher cortical porosity measured here is due to a lacunar-canalicular porosity more than three times greater than values previously reported in the literature. This is likely due to the resolution of the analysis, which more effectively captures the microstructure of the lacunar-canalicular system compared to previous studies. Although the cortical bone porosities calculated for the OVX (osteopenic) and control groups did not show statistically significant differences, the trend suggests that OVX rat cortical bone is more porous. Further investigation and measurements of bone porosity in different cortical locations and for cancellous bone will be necessary to determine if and how osteoporosis affects interstitial fluid flow in bone.

Acknowledgments Supported by the NIH/NIAMS (AR052866).

References [1] Frost, Henry Ford Hosp. Med. Bull., 8:208, 1960; [2] Frost, Henry Ford Hosp. Med. Bull., 10:35, 1962; [3] Baylink & Wergedal, In Cellular Mechanisms for Calcium Transfer and Homeostasis, p. 257, 1971; [4] Morris et al., Microvasc. Res., 23:188, 1982; [5] Shaffler and Burr, J. Biomech., 21:13, 1988; [6] Li et al., Microvasc. Res., 34:302, 1987; [7] Zhang et al., J. Biomech. Eng., 120:697, 1998; [8] Beno et al., Proc. Summer Bioengineering Conference, abstract 92348, 2005; [9] Knothe Tate et al., Int. J. Biochem. Cell Biol., 36:1, 2004.

** Hospital for Special Surgery, New York, NY

53rd Annual Meeting of the Orthopaedic Research Society

Paper No: 0263