Bimodal distribution of osteocyte lacunar size in the human femoral cortex as revealed by micro-CT

doi:10.1016/j.bone.2010.07.025

Bone

Volume 47, Issue 5, November 2010, Pages 866-871

https://doi.org/10.1016/j.bone.2010.07.025 Get rights and content

Abstract

Tomographic reconstructions of sections of human femoral bone were created from x-ray data sets taken using synchrotron radiation of 26.4 keV and with isotropic voxels 1.47 μm on a side. We demonstrate that it is possible to segment the data to isolate both the osteocyte lacunae and the Haversian canals in the bone as well as identifying osteon boundaries. From this information a wealth of data relating to bone structure becomes available. The data were used to map the spatial positions of the osteocyte lacunae, relative to the Haversian canals and of the osteon boundaries. The dimensions and volume of the imaged osteocyte lacunae were measured for close to 10,000 lacunae. When averaged over the 11 osteons measured, osteocyte densities varied from 4 × 10⁴ per mm³ close to the Haversian canals to about 9 × 10⁴ per mm³ at 80% of osteon radius. The nearest-neighbour distances varied from 10 μm to 40 μm with a peak at 23 μm and an approximately normal distribution. The distribution of lacunar long-axis length was also approximately normal with a small positive skew and the peak value was 8 μm with a range from 3 μm to 20 μm. The most significant finding from this study was that the distribution of the measured volumes of osteocyte lacunae had two distinct peaks, one at 200 μm³ and a second at 330 μm³.

Introduction

Osteocytes are the most numerous cells in mature bone and their interconnecting cellular processes are recognised as being an important part of the mechanism of cortical bone remodelling [1], [2]. However, the details of how they act remain controversial and comprehensive observations of their size and spacing are lacking [3], [4]. Tate [5] stated:

“…understanding of osteocyte biology has been thwarted by the remote location of the cell in the mineralized matrix.”

and in 2009 Bonucci wrote a paper with the title “The osteocyte: the underestimated conductor of the bone orchestra” [6] indicating that there are still significant problems with the study of these important cells.

The characteristics of osteocytes are less often investigated than those of other cell types involved in bone resorption and formation [6]. In part this is due to the difficulty of access to osteocytes in their normal situation where they are encysted within cortical bone at depths that are large relative to the size of the individual cells. In many cases the values for osteocyte densities and spacing are measured in two dimensions and are either reported as such [7] or extrapolated into the third dimension using geometric assumptions [3]; either way any variation normal to the plane of sectioning is missed and when extrapolating the assumptions require careful validation. Osteocytes and their connecting canaliculi form a three-dimensional network that contains within its structure a significant, but very complex, time history of its development. In the past, various methods have been used for the study of osteocyte lacunae and the cells that they contain. These include histology and back-scattered electron (BSE) imaging [3], [8], confocal microscopy [9], [10], corrosion casting followed by SEM [11] and mercury infiltration [12]. These techniques each have significant limitations; either they are essentially two-dimensional (histology and BSE imaging by definition and confocal microscopy is limited to at most 100 micrometres depth in cortical bone), corrosion casting is very time consuming and technically challenging and mercury porosimetry can provide only global information about canal, lacunar and canalicular volume and is probably unacceptable because of the perceived health hazard. The structures being studied are three dimensional and thus best studied in 3D, indeed it may be difficult to analyse the functioning of the lacuno-canalicular network in any other way.

Collection and quantitative analysis of micro-CT data is becoming routine and techniques for imaging cortical bone are under active development [13], [14], [15]. A vast amount of valuable, biologically relevant, data is contained in the data sets [16] but in order to extract biological meaning the 3D image data must be processed and this requires the development of automated techniques if adequate numbers of objects and structures are to be measured.

This paper reports the development of methods for the acquisition and processing of important, biologically relevant, measurements of osteocyte lacunar volumes, spacing and density from micro-CT of cortical bone. Three-dimensional data that describe the spatial distribution, orientation and size of a large number of osteocyte lacunae in blocks of cortical bone are discussed and related to existing literature on osteocyte lacunar size and spacing. These data will provide essential basic information needed for the construction and validation of models of bone turnover [17] and bone loss with disuse or aging.

Section snippets

Materials and methods

The sample studied was cortical bone obtained from the right femoral shaft of a 20-year-old male who died suddenly with no known medical condition that could affect his bone. The femur forms part of the Melbourne Femur Collection and was collected with the informed consent of the next-of-kin of the donor and with ethical oversight from both the University of Melbourne (HREC#980139), the Victorian Institute of Forensic Medicine (EC26-2000) and LaTrobe University (07/47). A section of the shaft 35

Results

For the Haversian canal shown in Fig. 4a a total of 706 osteocyte lacunae were found inside the osteon volume. Fig. 5a shows a histogram plot of the number of osteocyte lacunae (N.Lc) found at a given horizontal distance from the nearest point of the Haversian canal boundary. Fig. 5b shows a plot of osteocyte number (N.Lc) against the osteocyte's normalised distance from the pore boundary and Fig. 5c shows the same result divided by the number of voxels in each normalised distance bin thus

Discussion

In this paper we have presented results from a three-dimensional study of osteocyte lacunar size, location and spacing in human cortical bone. Nearly 10,000 lacunae were located and measured within 11 Haversian systems. This ability to locate and measure very large numbers of lacunae and, in particular to relate their locations and sizes to the remodelling event during which they were formed, is unique and has the potential to enable major advances in the knowledge of the micro-anatomy of

Acknowledgments

The authors acknowledge the support of the Australian Research Council through the Centre of Excellence for Coherent X-ray Science, The authors also acknowledge the Australian Synchrotron Research Program, which is funded by the Commonwealth of Australia under the Major National Research Facilities Program. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. We are grateful to

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Bimodal distribution of osteocyte lacunar size in the human femoral cortex as revealed by micro-CT

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Acknowledgments

Bone

Bone

Bone

Bone

Mechanotransduction in bone - role of the lacuno-canalicular network

FASEB J

Spatial distribution of osteocyte lacunae in equine radii and third metacarpals: Considerations for cellular communication, microdamage detection and metabolism

Cells Tissues Organs

Influence of cortical canal architecture on lacunocanalicular pore pressure and fluid flow

Comput Meth Biomech Biomed Eng

The osteocyte

Int J Biochem Cell Biol

The osteocyte: the underestimated conductor of the bone orchestra

Rend Lincei Sci Fisiche E Nat

Osteocyte lacunar occupancy in the femoral neck cortex: An association with cortical remodeling in hip fracture cases and controls

Calcif Tissue Int

A histological assessment on the distribution of the osteocytic lacunar canalicular system using silver staining

J Bone Miner Metab