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. 2014 Jul 4;225(3):328–336. doi: 10.1111/joa.12213

Normal variation in cortical osteocyte lacunar parameters in healthy young males

Yasmin Carter 1, Jessica L Suchorab 1, C David L Thomas 2, John G Clement 2, David M L Cooper 1,✉
PMCID: PMC4166973  PMID: 25040136

Abstract

The most abundant cell in bone, osteocytes form an interconnected system upon which the regulation of healthy bone relies. Although the complete nature of the role of osteocytes has yet to be defined, they are generally accepted to play a part in the sensing of load and the initiation of damage repair. A previous study conducted by our group identified variation of up to 30% in osteocyte lacunar density and morphological parameters between regions of a single cross-section of human femoral shaft; that study, however, was limited to a single individual. The aim of the current study was to determine whether this pattern consistently occurs in healthy young male femora. Anterior, posterior, medial and lateral blocks were prepared from the proximal femoral shaft of seven males and synchrotron radiation micro-CT imaged. Average lacunar densities (± SD) from the anterior, posterior, medial and lateral regions were 23 394 ± 1705, 30 180 ± 4860, 35 946 ± 5990 and 29 678 ± 6081 lacunae per mm3 of bone tissue, respectively. These values were significantly different between the anterior and both the medial and posterior regions (P < 0.05). The density of the combined anterior and posterior regions was also significantly lower (P = 0.006) than the density of the combined medial and lateral regions. Although no difference was found in predominant orientation, shape differences were found; with the combined anterior-posterior regions having lacunae that were significantly more elongated and less flat than the combined medial-lateral values (P < 0.001). As expected, in this larger study, there was a dramatic difference in lacunar density between the medial and anterior region (up to ∼ 54%). The study clearly demonstrates that the high variation seen in osteocyte lacunar density as well as other lacunar parameters, noted in a number of biomechanical, age and pathology studies, are well within the range of normal variation; however, the reasons for and consequences of this variation remain unclear. Lacunar parameters including abundance and shape are being increasingly incorporated into computational modeling of bone biology and this paper represents a more comprehensive description of normal healthy lacunae.

Keywords: human cortical bone, lacuna, micro-CT, osteocyte, synchrotron

Introduction

Osteocytes are the most abundant cell in bone; they become individually enveloped by the extracellular matrix during bone production within spaces known as lacunae. These cells and their lacunae form an interconnected system upon which the regulation of normal healthy bone relies (Aarden et al. 1994). Although the complete nature of the role of osteocytes has yet to be defined they are generally accepted to play a part in the sensing of load and the initiation of damage repair. Once bone reaches maturity, the twinned processes of bone formation and bone removal are kept in delicate balance by the information supplied to and through the osteocyte lacunar network. Acting as mechanosensors and transducers, biological input is interpreted and disseminated by a healthy and regular network (Knothe Tate et al. 2004). As the cells themselves are difficult to study in situ in sufficient quantities, their lacunae are commonly used as substitutes. Although all lacunae contain a viable osteocyte during formation, the degree of occupancy can decline over time through cell death. That said, the percentage of empty cellular spaces is very low in the young and healthy (Tomkinson et al. 1997) and thus the number of lacunae should be a close reflection of the number of osteocytes present within young bone.

A number of studies have focused on differences in osteocyte lacunar density with regard to age, loading and disease, with contradictory results (Mullender et al. 1996a,b1996b, 2005; Qiu et al. 2002, 2006; Skedros et al. 2003, 2005, 2011a,b2011b; Skedros, 2005; Vashishth et al. 2005; Schneider et al. 2007; Vatsa et al. 2008; van Hove et al. 2009); however, very little is known regarding normal variation in human osteocyte lacunar density and morphology. The variation in the range of lacunar abundance values in normal human bone is high and depends on location, tissue type and analytical technique. Commonly referenced as a baseline, Hobdell & Howe (1971), using traditional histology techniques, recorded lacunar densities in human cancellous bone of ∼ 13 000 mm−3. Much higher values were calculated by Metz et al. (2003), by converting previously published area counts (mm−2) to volume counts (mm−3) and found an average of 46 400 mm−3 for women of various ages and 70 000 mm−3 for elderly women. These differences in lacunar density are not related to methodology alone. We have previously reported values of 26 000–37 000 mm−3 in different regions of a femoral shaft from a young male (Carter et al. 2013b). Hannah et al. (2010) determined that within individual osteons of healthy males, osteocyte densities varied from 40 000 mm−3 close to the Haversian canals to about 90 000 mm−3 at the periphery of the osteon. Although the values from these recent studies are high compared with those of earlier studies, this difference is likely related to the improved methodologies and techniques available; however, they do suggest that caution when comparing samples taken from different regions or elements.

The small size of the lacunae and their location deep within the bone matrix has restricted studies characterizing lacunar parameters outside of density. High-resolution imaging techniques can overcome this issue, the gold standard being synchrotron radiation-based imaging (SR micro-CT; Muller, 2009; Schneider et al. 2010; Langer et al. 2012; Pacureanu et al. 2012; Carter et al. 2013a,b2013b; Mader et al. 2013). Measures of lacunar shape are of particular interest because external application of mechanical forces on cells has been shown to influence cytoskeletal structure and thus cell shape; this implies that the shape and long axes of osteocytes, and therefore their lacunae, should align parallel to the principal mechanical loading direction (Sugawara et al. 2011). It is then possible to extrapolate that differences in osteocyte lacunar morphology may indicate differences in osteocyte mechanical environment at the time of their burial from an actively growing bone surface. Studies of uni-directional vs. multi-directional loaded murine bone support this with the finding of elongated lacunae in the more uni-directionally loaded fibula than calvarium (Vatsa et al. 2008). A study of women over the age span showed a tendency towards smaller more spherical lacunae with age and a presumptive reduction in loading (Carter et al. 2013a). McCreadie et al. (2004) found no difference in size or shape, limited to measures of anisotropy, in lacunae of older women with and without osteoporotic fracture. Nano-CT studies comparing osteopenic, osteopetrotic and osteoarthritic tibial samples found osteopenic lacunae were relatively large and round, whereas osteopetrotic lacunae were small and discoid-shaped, and osteoarthritic were large and elongated (van Hove et al. 2009), although we note this study was restricted by its lack of a control sample.

A previous study conducted by our group identified high intra-element variation in osteocyte lacunar density of up to 30%. The lacunar density in the combined anterior and posterior regions was significantly lower than that of the combined medial and lateral regions. The anterior and posterior regions were also found to exhibit more elongated and flattened lacunae. We suggested that the functional significance of the observed variation was related to localized variations in loading conditions (Carter et al. 2013b). As that study was limited to a single individual, the aim of the current study was to determine whether this pattern of intra-element variation in lacunar parameters consistently occurs in a number of individuals of restricted age and sex. Our previous work proposed a differential biomechanical mechanism for the observed differences. If normal differential loading in the cross-section of the femora causes variation in the osteocyte lacunae, we would expect to see this pattern recurring in normal individuals.

Materials and methods

Specimens

Cortical bone samples were obtained from the femora of seven deceased men between the ages of 20 and 35, with no known medical conditions that may have affected their bones. These femora form part of the Melbourne Femur Collection held at the University of Melbourne, Melbourne, Australia, and were collected at autopsy with the informed consent of the donor's next-of-kin. The study was conducted with ethical approval from the Victorian Institute of Forensic Medicine (EC26/2000), the University of Melbourne (HREC 980139) and the University of Saskatchewan (Bio # 08-46). To investigate intra-element variation within the femoral shaft, individual samples with dimensions of approximately 2 × 2 × 5 mm were cut from four mid-cortical regions of a cross-section of the proximal femoral shaft: anterior, posterior, medial and lateral.

Synchrotron radiation micro-computed tomographic imaging

SR micro-CT scanning was conducted at the Advanced Photon Source (APS), Argonne National Laboratory, on beamline 2BM. Images were obtained using monochromatic X-rays with a photon energy of 27.9 keV and an effective pixel size of 1.47 μm. An exposure time of 100 ms per frame was employed for each of the 1800 frames spanning 180° of rotation, resulting in a scan time of approximately 9 min. The projection images were reconstructed to create a dataset containing 1841 slices (2016 × 900 pixels each).

3D quantitative morphometry

A cylindrical region of interest (ROI) was defined within each specimen, with a diameter of 1.0 mm and height of 1.25 mm (volume = 0.98 mm3). As per our previous protocol (Carter et al. 2013b), lacunae and canals within the ROI were segmented using a standardized global threshold, which separated the higher density bone from the low-density air-filled canal and lacunar spaces; the same threshold was used for all specimens. Following this, based on confocal microscopy measurements of osteocyte volume (McCreadie et al. 2004), elements < 10 μm3 were assumed to be noise and were removed, and elements above 2000 μm3 were assumed to be canals. All remaining elements were analyzed as lacunae.

SR micro-CT image stacks were cropped and analysis of the canals was conducted with CT Analyzer 1.10.9.0 (SkyScan, Kontich, Belgium). Amira 5.4.1 (Visage Imaging, Fuerth, Germany) was used for analysis of the lacunae. Standard nomenclatures for canal (Cooper et al. 2003) and lacunar (Schneider et al. 2007; Carter et al. 2013b) indices measured included: total ROI volume (TV), total canal volume for ROI (Ca.V), average canal diameter (Ca.Dm), total number of lacunae (N.Lc) and lacunar volume (Lc.V). In order to determine lacunar density per mm3 (N.Lc/BV), bone volume (BV) was calculated as total volume minus canal volume (TV − Ca.V).

Ellipsoids were fitted (amira 5.4.1; Microscopy Module) to each individual lacuna; from these, shape parameters were computed for each, based upon the resulting three eigenvalues (EV) of the covariance matrix. The length of each axis was approximated by two times the EV of the covariance matrix (95% confidence interval). A simplified model of the obliquity of the biomechanical environment within the proximal femur was used, with the direction of principal mechanical strain being approximated by the longitudinal axis with which it is closely aligned (Martin & Burr, 1989). The actual deviation of the angle of principal strain within the femur is small, as demonstrated by secondary osteons which have been shown to orient obliquely between 0 and 15° from the longitudinal axis (Hert et al. 1994). The orientation (Lc.Φ) of each lacuna was measured as the absolute value of the angular deviation (−90° to 90°) of the axis of the first EV (longest lacunar axis) from the horizontal axis of the sample. An orientation value of 0° represents a transverse (radial or circumferential) orientation and values approaching 90° are increasingly longitudinal in orientation. To describe the shape of the lacunae, three ratios of the EVs were used to define the degree of difference (Carter et al. 2013b). The degree of equancy (Lc.Eq) was calculated as the ratio of the third (shortest) to the first (longest) EV (EV3 : EV1). An Lc.Eq value of one represents an object equal in the shortest and longest dimensions. Degree of elongation (Lc.El) for each lacuna was calculated by one minus the ratio of the second (intermediate) and the first EV (1 − EV2 : EV1). An Lc.El value of one represents an elongated object. Degree of flatness (Lc.Fl) was calculated as one minus the ratio of the third and second EV (1 − EV3 : EV2). An Lc.Fl value of one represents a flat object.

Statistical analyses

Statistical analysis was performed using spss 16.0 (SPSS Inc., Chicago, IL, USA). Values for N.Lc/BV (mm−3) were calculated, and Lc.V (μm3), Lc.Φ (°) and shape (Lc.Eq, Lc.El, Lc.Fl) were averaged from each sample and along with the canal measures; these results were then grouped according to region (anterior, posterior, medial and lateral). Analysis of variance (anova) with repeated measures and post-hoc Bonferroni adjustment was performed with significance α < 0.05 to compare the regions. A second analysis was conducted with the regions grouped according to biomechanical ‘tension/compression’ axes into a combined anterior-posterior group and a combined medial-lateral group. anova tests were performed with significance α < 0.05 to compare the two groups.

Results

The lacunar density and morphological analysis results are summarized in Table 1. Average N.Lc/BV values were different between the anterior (23 394 mm−3) and both the posterior (30 180 mm−3; P = 0.042) and medial (35 946 mm−3; P < 0.001) regions (Fig. 1A). The density of the combined anterior and posterior regions (26 786 mm−3) was also lower (P = 0.006) than the combined density of the medial and lateral regions (32 812 mm−3). There were no differences between mean Lc.V for the separate regions (P = 0.219); however, a trend was noticeable in the combined results (P = 0.054), with higher volumes in the combined mediolateral region. No differences were apparent in average Ca.V or Ca.Dm (P > 0.05). Average Lc.Φ was not significantly different between the four regions or when combined (P > 0.05). No differences were apparent in Lc.Eq (P > 0.05; Fig. 1B). The anterior region had more elongated lacunae than those of the medial and lateral regions (Lc.El P = 0.001). Lacunae in the posterior region were significantly more elongated than those of the medial regions (P = 0.014), but not the lateral regions (P = 0.553; Fig. 1C). The same trend followed for Lc.Fl, which was significantly less flat in the anterior than in the medial and lateral regions (P < 0.001), and the posterior to the medial regions (P = 0.019; Fig. 1D). The combined anterioposterior lacunae were significantly more elongated and less flat than the combined mediolateral values (P < 0.001). There were no differences in either EV1 or EV3 for each region; however, average EV2 was significantly different between the posterior and medial regions (P = 0.009). The eigenvalues of the combined regions followed the same pattern, with EV2 higher in the combined mediolateral region (P = 0.001).

Table 1.

Results from the anova of the morphological parameters of osteocyte lacuna.

Region N.Lc/BV (mm−3 ± SD) Average Lc.V (μm3 ± SD) Ca.Dm (μm ± SD) Ca.V (mm3 ± SD) Average Lc.Φ (° ± SD) Average Lc.Eq (± SD) Average Lc.El (± SD) Average Lc.Fl (± SD) Average EV1 (± SD) Average EV2 (± SD) Average EV3 (± SD)
Anterior 23 394 ± 1705* 242 ± 44 52.81 ± 13.16 0.04 ± 0.01 60.23 ± 3.97 0.17 ± 0.03 0.69 ± 0.04** 0.38 ± 0.05** 12.74 ± 1.56 3.09 ± 0.34 1.56 ± 0.06
Posterior 30 180 ± 4860 261 ± 49 53.03 ± 19.62 0.04 ± 0.02 55.90 ± 5.41 0.17 ± 0.02 0.66 ± 0.04*** 0.42 ± 0.06*** 12.46 ± 1.55 3.28 ± 0.39*** 1.59 ± 0.07
Medial 35 946 ± 5990 299 ± 45 51.01 ± 12.94 0.04 ± 0.01 53.37 ± 4.61 0.18 ± 0.02 0.60 ± 0.04 0.52 ± 0.05 11.68 ± 1.38 3.95 ± 0.46 1.62 ± 0.05
Lateral 29 678 ± 6081 282 ± 71 47.79 ± 13.78 0.04 ± 0.01 56.70 ± 3.74 0.17 ± 0.02 0.63 ± 0.04 0.49 ± 0.07 12.24 ± 1.49 3.74 ± 0.67 1.58 ± 0.09
Combined Ant+Post 26 786 ± 4964**** 252 ± 46 52.92 ± 16.05 0.04 ± 0.01 58.06 ± 5.08 0.17 ± 0.02 0.68 ± 0.04**** 0.40 ± 0.06**** 12.69 ± 1.50 3.19 ± 0.37**** 1.57 ± 0.06
Combined Med+Lat 32 812 ± 5711 291 ± 55 49.45 ± 12.95 0.04 ± 0.01 55.04 ± 4.39 0.18 ± 0.02 0.61 ± 0.04 0.50 ± 0.06 11.96 ± 1.41 3.85 ± 0.56 1.60 ± 0.07
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*

P < 0.05 vs. both posterior and medial regions

**

P < 0.05 vs. both medial and lateral regions.

***

P < 0.05 vs. medial region

****

P < 0.05 vs. combined medial and lateral regions.

Figure 1.

Figure 1

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Box plots representing the average osteocyte lacuna density for each region (A). Measures of morphology include: degree of equancy (B), degree of elongation (C), and degree of flatness (D).

Discussion

The most plentiful cell in bone, osteocytes form an interconnected system upon which the maintenance of healthy bone relies. The results of this study demonstrate that the variation in osteocyte lacunar density and morphological parameters between regions of a single cross-section of human femoral shaft previously noted (Carter et al. 2013b) can be seen in a larger group. As expected, in this larger study, there was a dramatic difference in lacunar density between the medial and anterior region (up to ∼ 54%). Average lacunar densities were significantly different between the anterior and both the medial and posterior regions (Fig. 2). The density of the combined anterior and posterior regions was also significantly lower than the density of the combined medial and lateral regions. Although no difference was found in predominant orientation, shape differences were found; the combined anterioposterior regions had lacunae that were significantly more elongated and less flat than the combined mediolateral values. In addition to providing a more definitive picture of normal variation to enable a better context for drawing inferences with regard to age, disease and biomechanics, this study provides geometric measures relevant to future computational modeling of the normal osteocyte and its mechanical environment.

Figure 2.

Figure 2

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Renders of 100-μm-thick slice demonstrating the differences in lacunar density. With fitted and colored ellipses scaled for eigenvalue 2, the major contributor to change in elongation and flatness in the anterior (A), posterior (P), medial (M), and lateral (L) regions. Color gradient from blue to red represent increasing EV2. Scale bar: 200 μm.

Variation in bone microstructural features, such as regional differences in the orientation of secondary femoral osteons, has been linked to the mechanical environment (Hert et al. 1994; Petrtyl et al. 1996). In this study we found no differences in lacunar orientation, with the long axis of lacunae in all regions oriented essentially parallel to the longitudinal axis of the bone, well aligned with the slightly oblique principal strain axes. A previous study of femoral mid-shaft regional variation in vascular canals demonstrated increased porosity in the anterolateral and posterior regions (Thomas et al. 2005). The present study revealed no regional differences in any of the canal indices measured. Most intra-element variation appears to occur non-uniformly, increasing from the periosteal to endosteal surfaces (Feik et al. 2000; Bousson et al. 2001; Thomas et al. 2005) and may not be represented in this study's mid-cortical samples. One possible explanation for regional differences in lacunar parameters and not in the vascular canal system is that, even though they are connected, fluid pressures in these systems behave almost independently of each other (Cardoso et al. 2013). Additionally, we recognize that the regional variations noted could potentially be a byproduct of variations in histomorphological characteristics that preferentially adapt to a habitual non-uniform strain milieu. These characteristics can include predominant collagen fiber orientation, secondary osteon population density, and osteon collagen/lamellar morphotypes.

Our previous work found average lacunar densities to be significantly different between the medial and both the anterior and posterior regions, with the combined anterior and posterior regions being significantly less dense than the combined medial and lateral regions (Carter et al. 2013b). At ∼ 54%, the observed differences in this study were actually higher than noted previously, although the medial region is no longer significantly different from the posterior. The anterior region incorporated significantly fewer osteocytes than the medial and posterior regions. This differs from our previous study, where the medial region had a much higher number of osteocytes than either the anterior or posterior regions. However, as a trend, the lacunar density along the anteroposterior axis declined by approximately 18% compared with the mediolateral axis, which is in keeping with previous results.

Differences can occur in lacunar density via two routes. First, bone is deposited or remodeled, incorporating varying numbers of new osteocytes in the osteoid as it is laid down. From this point on, no new osteocytes can be incorporated. However, lacunar density can decrease through a process of cellular apoptosis and consequent long-term infilling. This second route of change is unlikely to be the driving mechanism for the shift in density demonstrated here, as the percentage of viable cells decreases with age from 99% to approximately 58% by the 8th decade of life (Tomkinson et al. 1997). The young age of the individuals in this study therefore means that apoptosis is likely to have had little effect.

As with lacunar density, there are two mechanisms by which lacunae can come to differ in size and shape. First, as cells are incorporated into the bone matrix during formation, the shape of the lacunae is determined by the osteocyte. Secondly, it has recently been noted that the pericellular environment is capable of modification at an individual lacunar level (Qing & Bonewald, 2009; Tang et al. 2012). The role of loading in both of these situations has yet to be determined.

Although no differences were evident in equancy, lacunae from the anterior region were more elongated and less flat than those from the medial and lateral regions. Lacunae in the posterior region were significantly more elongated and less flat than those of the medial region, but not the lateral region. The combined anteroposterior lacunae were significantly more elongated and less flat than the combined mediolateral lacunae (Fig. 3). Elongation follows the same trend as previously reported in a single individual; however, flatness is now significant in the medial and lateral regions. In the previous study of a single individual (Carter et al. 2013b), EV1, the long axis of the lacunae, was significant between the regions and a trend was seen in EV2. In this larger study EV1 demonstrates the high inter-individual differences associated with normal variation and EV2 shows the single greatest significance. This pattern fits with that seen in younger women, who had flatter lacunae in the anterior region compared with older women in the same region (Carter et al. 2013a).

Figure 3.

Figure 3

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3D model demonstrating increasing elongation accompanied by decreased flatness.

Ultimately, the likely mechanism for the variation in lacunar parameters described here is related to the local mechanical environment both during deposition of the osteocytes and throughout the life of the cells. Strains generated by activity represent an efficient epigenetic parameter through which bone cells can assess structural efficiency and influence its morphology (Rubin et al. 1990). Early work on the mechanics of the femoral shaft suggested that the greatest source of stress on the human femur comes from bending. When experiencing bending due to motions such as walking or single leg stances, the shaft of the femur is subject to compression on the medial side and tension on the lateral, with a neutral axis which passes through the center of gravity where there is no tensile or compressive stress; this axis is along the sagittal plane of the femur in an anteroposterior direction (Koch, 1917; Pauwels, 1980; Rubin et al. 1990; Aamodt et al. 1997). Most of these studies, however, were based on analytical methods such as simple beam theory, photo-elastic analysis and static analysis, all of which suffer from inherent biases and generally assume a homogeneous material. Newer research incorporating finite element modeling and the thigh muscles suggests that stress and strain patterns in the femoral diaphysis are characterized by combined bending and torsion (Cristofolini et al. 1996a,b1996b; Duda et al. 1998). Although bending force decreases from proximal to distal, the proximal surface is characterized by a relative reduction in torsion (Oh & Harris, 1978; Skedros & Baucom, 2007), making the proximal shaft a simpler biomechanical region than that of the midshaft; however, inter-femur strain variability for cadaveric specimens has been shown to be high (Cristofolini et al. 1996b).

We report a decrease in lacunar density with elongated but less flat spaces across the purported neutral axis where under natural circumstances the strains do not reach high values (Huiskes, 1982). The highest density of spaces is found in the medial region where, under bending forces, the shaft would be experiencing the highest compression strains. Although no significant differences in lacunar volume were seen between the regions, a trend was noted in the combined groupings, with the anteroposterior region having slightly smaller (252 μm3) lacunae than the mediolateral region (291 μm3). This fits the patterns found in studies of extreme differences in loading conditions where lacunae in unloaded rat bone became smaller (Britz et al. 2012), perhaps representing the decrease in strain along the purported neutral axis. A word of caution: the biomechanical environment of the proximal femoral shaft is complex, and drawing direct parallels between lacunar parameters and strain may not be entirely accurate. Above all, the results of this study demonstrate the necessity for further research into the mechanisms governing normal lacunar distribution and morphology, beginning with a simple biomechanical system. This said, localized strain environment is only one of a myriad of factors affecting the microstructure of bone at the individual level: lifestyle, activity levels, diet, health and metabolism also play a role (Rubin et al. 1990). However, these factors could not be controlled for in this study and therefore their relative influences cannot be determined.

Adequate numbers of osteocytes have been demonstrated as essential to coordinate the removal of bone damage and lacunar density correlates with indices of bone quality (Ma et al. 2008); however, as the variation seen here demonstrates, ‘adequate’ is a relative term. It has been suggested that a reduction in osteocyte abundance can result in the deterioration of matrix fluid flow and a decrease in the ability to detect microdamage that may lead to impaired repair and increased fragility. It has previously been noted that lacunar density and size may cause variation in the apparent stiffness of bone (Yeni et al. 2001). An increase in microcracking in vivo has been associated with reduced lacunar abundance (Vashishth et al. 2000; Qiu et al. 2005); however, finite element analyses have found increased lacunar density linked to microcracking in older individuals, although lacunar occupancy may be a factor here (Soicher et al. 2011).

The effects of lacunae on bone mechanical quality are not limited to abundance and size. Lacunar shape influences the mechanical strain applied to the osteocyte, with different lacunar morphology resulting in differing strain magnitudes in the matrix and cell when subject to the same loads (McCreadie et al. 2004). We can readily see the effects of inclusions such as osteocytes and their lacunae on any solid structure by referring to materials science and structural mechanics models. The primary effect of inclusions within any material is a disruption in the homogeneity of the structure, and their influence on mechanical and other properties can be considerable (Vander Voort, 1984). The mechanical properties of the bone matrix are significantly degraded by inclusions because they are not only discontinuities in the parent material but also local stress concentrators. During deformation under loading of the primary structural element, inclusions become the focal point for the initiation of microcracking; as has been demonstrated for lacunae (Reilly, 2000). This study demonstrated a lower number of more elongated spaces along the anterolateral axis and an increase in less elongated but flatter lacunae in the tension and compression zones (lateral-medial). Unfortunately, we have been unable to visualize the canaliculi which connect each lacuna and contain the cell processes; these represent additional discontinuities that may predispose the bone to crack propagation even more readily. Additionally, this does not take into account the torsional strains that the proximal femur experiences. In simulations, strains are higher in elongated cells than in those less elongated (McCreadie et al. 2004). It may be that the elongated cells seen in our low strain regions are evidence of amplification to produce the required ‘regular’ lacunar network. Maximum principle strains occur in the matrix approximately parallel to the long axis of the lacunae; however, relatively larger increases in strain magnitude occur perpendicular to this axis, measured here as EV2 and EV3 (McCreadie et al. 2004). These relative differences in local strain environment might provide a mechanism for the directional specific differences we see in lacunar shape.

Although this project was limited to the use of human bone from seven healthy individuals, hundreds of thousands of lacunae were analyzed with consistent results. This work clearly demonstrates that the high variation seen in osteocyte lacunar density as well as other lacunar parameters, noted in a number of biomechanical, age and pathology studies, are well within the range of normal variation; however, the reasons for and consequences of this variation remain unclear. Lacunar parameters including abundance and shape are being increasingly incorporated into computational modeling of bone biology and this paper represents a more comprehensive description of the normal healthy lacunae than previously achieved.

Acknowledgments

The primary financial support for this specific research project was from the Saskatchewan Health Research Foundation in the form of an Establishment Grant to D.M.L.C. The authors acknowledge the support of the Australian Research Council through the Centre of Excellence for Coherent X-ray Science. Use of the advanced photon source was supported by the U.S. DOE, Basic Energy Sciences, Office of Science under Contract No. W-31-109-Eng-38. We are grateful to the mortuary staff and the staff of the Donor Tissue Bank of the Victorian Institute of Forensic Medicine for their assistance in the collection of this series of bone specimens and particularly grateful to the next-of-kin of the donors for permission to remove bone for research purposes. The authors are grateful to Mr. Peter Carter for his invaluable engineering assistance and advice. Yasmin Carter is a Fellow in the Canadian Institutes of Health Research Training program in Health Research Using Synchrotron Techniques (CIHR-THRUST).

Authors’ roles

Study design: Y.C. and D.M.L.C. Study conduct: Y.C. Data collection: Y.C. and J.S. Data analysis: Y.C. and D.M.L.C. Data interpretation: Y.C., C.D.L.T., J.G.C. and D.M.L.C. Drafting manuscript: Y.C. Revising manuscript content: Y.C., J.S., C.D.L.T., J.G.C. and D.M.L.C. Approving final version of manuscript: Y.C., J.S., C.D.L.T., J.G.C. and D.M.L.C. Y.C. takes responsibility for the integrity of the data analysis.

Disclosure statement

All authors state that they have no conflict of interest.

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