QUANTITATIVE MEASUREMENT OF OSTEOCYTE LACUNA SIZE AND SHAPE FROM FRACTURED AND NORMAL INDIVIDUALS FROM CONFOCAL MICROSCOPY IMAGES

Participants: B.R. McCreadie, M. Schaffler, S.A. Goldstein

Keywords: bone, osteocytes, confocal microscopy

Introduction

A potential cause of bone loss during osteoporosis is a change in the sensation of bone strain by osteocytes, which may be influenced by the size and shape of these cells and their lacunae. Previous studies addressing the size of lacunae in individuals with fractures attributed to osteoporosis and individuals without fracture have been contradictory, and were limited to planar measurements on iliac crest biopsies. This study was designed to determine whether there are differences in the size or shape of osteocyte lacunae between women who sustain hip fractures and those who die without fracture, using 3-d confocal images of lacunae sampled from a site of high fracture risk.

Materials and Methods

Trabecular bone specimens were obtained from a total of twenty-eight women. Eighteen subjects had tissue removed during arthroplasty following a hip fracture that was not caused by extreme trauma such as falling from a ladder or an automobile accident. The remaining specimens were obtained from individuals who died without fracture. The specimens were chosen to result in approximately equal bone volume fractions in the two groups. The trabecular bone cube specimens were taken from the same relative location in the femoral head: near the superior surface of the neck, aligned with the principal trabeculae. This location was adjacent to the fracture site in most of the fracture patients. One additional cube was obtained from a human femoral head and used only for a calibration study. The specimens were fixed in 70% ethanol, stained with basic fuchsin, and embedded in methyl methacrylate.

Six sections from the calibration cube were ground and polished to thicknesses between 6 and 30 microns, mounted to slides using Permount, and imaged using a Bio-Rad MRC600 confocal microscope. Scans were obtained through the entire thickness at multiple bone regions, at 1 micron intervals. For each region, the number of images between two standardized brightness levels was calculated, which corresponded to a relative thickness of the bone region. The slope of the best-fit line between the measured and maximum calculated thicknesses, 0.8928, was determined as the correction factor for the depth. The R^2 value was >0.99, providing confidence that this method yielded a consistent correction factor.

Cover slips for the experimental samples were prepared by marking axes and random locations using a diamond scribe. The bone specimens were sectioned, polished, and mounted to the prepared cover slips using Permount. When dry, the specimen/cover slip constructs were loosely attached to slides with caulk. The specimens were then imaged as described above from the surface to a corrected depth of 22-27 microns, at .4464 micron intervals. The images appeared as shown in figure 1.

The lacunae that met requirements designed to ensure a non-biased choice of lacunae were analyzed. Otsu’s adaptive thresholding algorithm allowed correction for brightness differences with depth. Image processing algorithms were used on each lacuna to close and smooth the contours. The principal axes of each lacuna were determined, and the maximum extent of the lacuna in each of these directions was calculated (actual dimensions). The dimensions of the ellipsoid that has the same principal moments as the lacuna were also calculated (ellipsoid dimensions). The lacuna volume was determined based on the number of voxels which were included in the lacuna or the calculated ellipsoid, and the anisotropy ratio was calculated as the ratio of the smallest to largest dimension of each lacuna (based on actual or ellipsoidal dimensions).

Results

No difference in the size or shape of the osteocyte lacunae was found between the fractured and normal groups (see graphs below). This was true whether using the actual dimensions of the lacunae directly from the confocal images, or the dimensions of the best-fit ellipsoid. The difference between the lacuna voxels and those of the calculated ellipsoid averaged 10.5%. In other words, approximately 10.5% of the lacuna voxels were not a part of the ellipsoid, and vice versa.

In conclusion, the results of this study demonstrate no difference between the size or shape of osteocyte lacunae in normal and fractured individuals. Although we cannot draw a conclusion about whether the deformation of the osteocytes is different between these two groups, any differences must be due to other factors, not the size or shape of the osteocyte lacunae.

Progress

A paper describing this study is expected to be submitted by spring 2001.

Histogram comparing the size (volume) of the osteocyte lacuna in fractured and control groups. The histogram bins are identified by the maximum value in the bin.

Figure 1: Small portion of a confocal image showing a cross-section of a lacuna and canaliculi.

Histogram comparing the anisotropy of the osteocyte lacuna in fractured and control groups. The histogram bins are identified by the maximum value in the bin.