Bone 38 (2006) 130 – 135
www.elsevier.com/locate/bone
Differences in osteocyte and lacunar density between Black and White
American women
Shijing Qiu a,⁎ , D. Sudhaker Rao a , Saroj Palnitkar a , A. Michael Parfitt b
a
Bone and Mineral Research Laboratory, E and R Building 7071, Henry Ford Hospital, 2799 W Grand Boulevard, Detroit, MI 48202, USA
b
Division of Endocrinology and Center for Osteoporosis and Metabolic Bone Disease,
University of Arkansas for Medical Sciences, Little Rock, AR 72201, USA
Received 24 February 2005; revised 7 July 2005; accepted 15 July 2005
Available online 19 August 2005
Abstract
We examined the differences in osteocyte and lacunar density between Black and White women, using previously obtained iliac bone
biopsies from 34 healthy Black women, aged 21–70 years, and 94 White women, aged 20–73 years. For each subject, the density of
osteocytes (Ot.N/B.Ar), empty lacunae (EL.N/B.Ar), and total lacunae (Tt.L.N/B.Ar) and the proportion of osteocyte-occupied lacunae (Ot.
N/Tt.L.N) were separately measured in whole trabeculae, superficial bone (b25 μm from the bone surface), and deep bone (N45 μm from the
bone surface). Compared with White women, Black women had higher values for osteocytes, empty lacunae, and total lacunae and lower
values for percent occupied lacunae in superficial bone and whole trabeculae (P b 0.01 to b0.001). In deep bone there were more osteocytes
and total lacunae in Black women, but the other measurements did not differ significantly between the two groups. As in White women, there
were fewer osteocytes and total lacunae and more empty lacunae in deep than in superficial bone. The regressions of osteocyte and total
lacunar density on age were not significant in Black women, but postmenopausal Black women had fewer osteocytes than premenopausal
Black women, and percent occupied lacunae declined significantly with age in whole trabeculae and deep bone, which could only have
resulted from osteocyte death. In contrast to White women, there was no inverse relationship between bone formation rate and osteocyte
density in superficial bone and the observed bone formation rate was lower than predicted by osteocyte density. We conclude the following:
(1) Cancellous bone is made with more osteocytes in Black than in White women, most likely because of diminished apoptosis of osteoblasts;
this could contribute to increased bone strength in Black women. (2) In Black women, as in White women, there are fewer osteocytes and
total lacunae and more empty lacunae in deep than in superficial bone. (3) There was moderate age-related loss of osteocytes in deep bone in
Black women, indicating that osteocyte density depends more on the age of the bone than on the age of the subject. (4) The higher osteocyte
density in Black women was not responsible for their lower bone formation rate.
© 2005 Elsevier Inc. All rights reserved.
Keywords: Osteocytes; Cancellous bone; Ethnicity
Introduction
Osteocytes are stellate-shaped cells enclosed within the
lacunae and canaliculi in bone matrix. They connect to each
other as well as to cells on the bone surface via their long
slender cytoplasmic process, forming an intercommunicating
cell network [1,12]. Osteocytes are former osteoblasts that
⁎ Corresponding author.
E-mail address: qiu@bjc.hfh.edu (S. Qiu).
8756-3282/$ - see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bone.2005.07.004
became buried because they stopped making bone matrix
while adjacent osteoblasts continued to work [17]. This
transformation can occur at any stage during the life span of
an osteoblast, since osteocytes are evenly distributed
throughout the bone. The osteocyte network is able to
convey local signals to osteoblasts and osteoclasts on the
bone surface, which may play a key role in the regulation of
bone modeling and remodeling in response to mechanical
and biochemical influences on the skeleton [1,15]. Osteocytes participate in the detection and repair of fatigue damage
S. Qiu et al. / Bone 38 (2006) 130–135
[4,26] so that the integrity of the osteocyte network seems
indispensable for the removal of microdamage and maintenance of good bone quality.
Osteocytes are long-lived cells but are subject to death by
apoptosis [15]. The remnants of an apoptotic death may
remain in situ for several weeks or months, recognizable by
conventional light microscopy as a pyknotic nucleus, but
eventually the remnants become undetectable, leaving an
apparently empty lacuna. In cortical bone some lacunae and
canaliculi become plugged with mineralized material and
can no longer be distinguished from adjacent bone matrix by
conventional microscopy. Frost termed this micropetrosis
[6], a phenomenon which increases in extent with age; it is
not known whether the same phenomenon occurs in
cancellous bone. Several investigators have reported that
osteocyte density declines with age in peripheral cortical
bone [5,25]. We recently demonstrated an age-related
decline in osteocyte density in iliac cancellous bone confined
only to deep bone that is infrequently remodeled [21].
Furthermore, we found a significant osteocyte deficit in
patients with osteoporotic vertebral fracture compared to
healthy subjects of the same age [23]. However, these studies
were confined to White women and the status of osteocytes
in Black women is unclear.
It is well known that fracture incidence is lower in Black
than in White people [24,27], for which there are several
reasons. Blacks have higher bone mass at all ages so that
their bones should be stronger [2,11,14]. Blacks have lower
bone turnover than Whites [10,28], which would confer an
additional benefit to bone strength [9]. Since osteocyte
density and viability appear to make an independent
contribution to bone strength [16,23], we extended our
previously mentioned studies [21] to healthy Black women.
131
basic fuchsin in 70% ethanol for 96 h. The undecalcified
biopsies were embedded in PMMA and sectioned, stained,
and mounted as previously described [10]. Measurements
were performed using 5-μm-thick sections stained with
Goldner's trichrome. Using a bright-field light microscope
(20× objective) equipped with a Bioquant System (R&M
Biometrics, Inc. Nashville, TN), fields were scanned by rows
and ten unbroken regions were sequentially selected for
counting the number of osteocyte-occupied lacunae (stained)
and empty lacunae (unstained). The sum of both is the
number of total lacunae. The traditional interpretation of
empty lacunae is that the remnants of cell death have been
degraded and removed; an alternative interpretation is that
the remnants persist for a long time but are easily dislodged
during section preparation. In either case, an apparently
empty lacuna is a reliable sign that the osteocyte originally
present has died. For each region, the numbers of osteocytes
(Ot.N), empty lacunae (EL.N), and total lacunae (Tt.L.N)
were expressed per bone area (per mm2), and percent
osteocyte-occupied lacunae were calculated as Ot.N/Tt.L.
N × 100.
In order to examine the effects of distance from the
surface, we measured Ot.N/B.Ar and EL.N/B.Ar separately
in bone b 25 μm from the surface (superficial bone) and N 45
μm from the surface (deep bone); we explained previously
the justification for these criteria [21]. Tt.L.N/B.Ar and
percent osteocyte-occupied lacunae were calculated in each
area. The results were compared with similarly obtained data
from White women [21].
In order to examine the relationship between osteocyte
density in superficial bone and bone formation rate [22], we
used archival values for bone formation rate measured as
previously described [10].
Statistics
Materials and methods
Subjects
Thirty four Black women (18 premenopausal and 16
postmenopausal) of mean age 47.2 (13.7) years (range 24–
70) were recruited for the studies of bone structure and
remodeling that have been reported previously [10]. All
subjects were skeletally healthy and underwent in vivo
double tetracycline labeling with an interlabel time of 14
days. Archived sections were used in this study. Data
obtained in 94 White women (38 premenopausal, 56
postmenopausal), of mean age 51.7 (14.2) years (range
20–73), similarly recruited, have been reported previously
[21].
Histomorphometry
Cylindrical transiliac biopsies were obtained using a
trephine with an internal diameter of 7.5 mm, placed
immediately in 70% ethanol, and stained en bloc using 1%
Data were expressed as mean (SD). Differences between
Black and White women and between superficial and deep
bone were evaluated by Student's t test for normally
distributed data and by Mann–Whitney rank sum test for
non-normally distributed data. Relations between each
variable and age were calculated using linear regression
analysis. P b 0.05 was considered significant. All statistical
analyses were performed with Sigmastat software (SPSS,
Inc., Chicago, IL).
Results
In whole trabeculae and in superficial bone, Black
subjects had more osteocytes, more empty lacunae, and
more total lacunae than White subjects and lower values for
percent occupied lacunae (Table 1); these differences were
unrelated to age (data not shown). In deep bone, Blacks had
more osteocytes and total lacunae but the values for empty
lacunae and percent occupied lacunae did not differ
132
S. Qiu et al. / Bone 38 (2006) 130–135
Table 1
Comparison of superficial and deep bone in Black and White women for osteocyte and lacunar density
Osteocyte density (/mm2)
Whole trabeculae
Superficial bone
Deep bone
P2
Empty lacunar density (/mm2)
1
Black
White
P
Black
White
P1
226 (43.3)
254 (37.7)
162 (45.4)
b0.001
202 (30.6)
230 (46.3)
122 (54.2)
b0.001
b0.01
b0.01
b0.001
22.0 (5.40)
14.0 (7.55)
36.3 (11.7)
b0.001
14.9 (7.60)
7.12 (8.23)
33.5 (20.3)
b0.001
b0.001
b0.001
NS
Total lacunar density (/mm2)
Whole trabeculae
Superficial bone
Deep bone
P2
Occupied lacunae (%)
1
Black
White
P
Black
White
P1
248 (44.0)
268 (37.8)
198 (51.5)
b0.001
217 (28.6)
237 (48.5)
155 (58.4)
b0.001
b0.001
b0.01
b0.001
b0.001
90.9 (2.42)
94.7 (2.74)
81.4 (4.97)
b0.001
93.0 (3.71)
97.1 (3.22)
77.1 (12.8)
b0.01
b0.001
NS
P1: Comparison between Black and White women; P2: comparison between superficial and deep bone. Data expressed as mean (SD).
significantly. In Blacks, as in Whites, deep bone had fewer
osteocytes and total lacunae and more empty lacunae than
superficial bone, and percent occupied lacunae were lower.
In contrast to White women, in healthy Black women
there was no significant regression of any variable on age,
except for Ot.N/Tt.L.N in whole trabeculae and in deep bone
(Table 2, Fig. 1); this difference may partly reflect a smaller
sample size (34 versus 94). In postmenopausal compared to
premenopausal women there was a significant fall in
osteocytes (P b 0.05 for one-tailed test) and a significant
fall in percent occupied lacunae (Table 3).
In Fig. 2 the differences between Black and White women
and between deep and superficial bone are shown by plotting
individual values in Black women in relation to the
exponential regressions on age determined in White
women [21]. As for whole trabeculae, the differences
between the groups were present at all ages and did not
change much with age. The differences were only trivially
affected by using the regression equations to adjust for the
small difference in age (data not shown). The differences
between superficial and deep bone appeared to increase with
age, as in White women.
The relationship between bone formation rate and
osteocyte density in superficial bone in Black and White
women is shown in Fig. 3. The regression in Black women
was not significant (P N 0.05). The mean value for BFR in
Black women predicted from the regression on osteocyte
density previously established in White women was 13.2
(1.8) significantly higher than the observed value of 10.8
(7.8) and not significantly different from the observed BFR
in the White subjects of 14.4 (9.4).
Discussion
We found that healthy Black women had a significantly
higher osteocyte density in iliac cancellous bone than healthy
White women. If the reason for this difference was a longer
osteocyte lifespan, there would have been fewer (rather than
more) empty lacunae and the same number (rather than a
larger number) of total lacunae. Our data demonstrate that
Black women make cancellous bone with more osteocytes
than White women. Normally about 6% of osteoblasts in
cancellous bone become lining cells, about 29% become
osteocytes and about 65% undergo death by apoptosis [19].
It is unlikely that more osteoblasts assemble on the cement
surface in Blacks, because wall thickness, an index of the
aggregate amount of bone made by each osteoblast team, is
not increased [10]. Even a 50% reduction in lining cell
production from 6% to 3% could not account for a 12%
increase in osteocytes. Consequently, the most likely
explanation for more osteocytes in Black women is that
fewer osteoblasts die by apoptosis. Since wall thickness is
not increased, the osteoblasts that escape death must be near
the end of their active life and become osteocytes soon after.
Table 2
Linear regressions on percent occupied lacunae on age for Black women
Intercept
Slope (y)
r2
P
94.3
94.8
87.9
−0.072
−0.002
−0.137
0.166
0.000
0.142
b0.05
NS
b0.05
2
Ot.N/B.Ar (/mm )
Whole
Superficial
Deep
Fig. 1. Relationships between percent occupied lacunae and age in Black
women. There were significant correlations between Ot.N/Tt.L.N and age in
whole trabeculae (solid line and open circles), and deep bone (long-dashed
line and Black triangles), but not in superficial bone (short-dashed line and
filled circles). Statistics as in Table 1.
S. Qiu et al. / Bone 38 (2006) 130–135
133
Table 3
Comparison of mean values for osteocyte and lacunar density in whole
trabeculae between pre- and postmenopausal Black women
n
Ot.N/B.Ar (/mm2)
EL.N/B.Ar (/mm2)
TL.N/B.Ar (/mm2)
Ot.N/Tt.L.N (%)
Pre
Post
% Difference
P
18
237 (43.8)
20.2 (5.01)
257 (45.0)
92.0 (2.03)
16
213 (40.4)
23.9 (5.27)
237 (41.7)
89.8 (2.35)
−10.1
+18.3
−7.8
−2.4
0.052
0.095
0.106
0.019
Data expressed as mean (SD). Pre = premenopausal; Post = postmenopausal.
Greater osteocyte density could be an additional factor
contributing to the lower fracture risk in Black than in White
subjects. Bone, like all structural materials that undergo
repetitive cyclical loading, is subject to fatigue microdamage
[3,7,25,26]. At any time the proportion of bone in which
microdamage is present, which has been termed the microdamage burden [8], depends on the rate of production and the
timing and completeness of repair by remodeling [4,8].
Because of higher bone mass Black subjects would be
expected to have less microdamage production, which could
account for their lower bone turnover [10]. The microdamage burden could be further reduced if a more compact
osteocyte network speeded the onset of microdamage repair.
A higher osteocyte density could also contribute to bone
strength directly as well as indirectly [23], possibly because
of changes in bone hydration and crystallinity [16]. Greater
strength of bone made with more osteocytes could be the
counterpart of lesser strength of bone made with fewer
osteocytes, as found in patients with spontaneous vertebral
Fig. 3. Relationship between bone formation rate (BFR/BS) and osteocyte
density in superficial bone in White women (open circles) and Black women
(closed circles). The regression line shown was significant in White but not
in Black women [22].
fracture [23]. Throughout its lifespan each osteoblast must
continually “decide” whether to continue to make bone
matrix, to become an osteocyte, or to die. Only when bone
formation is almost complete at that site does the osteoblast
have the additional option of becoming a lining cell. The
mechanisms that determine the fate of individual osteoblasts,
about which almost nothing is known, evidently merit much
more study.
We found similar differences between superficial and
deep bone in Black as in White women (Table 1, Figs. 2 and
3). Based on the mean values for activation frequency [10],
superficial bone has a mean age of about 1.5–2 years,
slightly more in Blacks than in Whites, which is why its
osteocyte density does not change with age (21; Fig. 2). The
Fig. 2. Relationship between osteoid indices and age in superficial bone (open circle, interrupted lines) and deep bone (closed circles, continuous lines).
Individual data points in Black women are shown in relation to curvilinear regressions determined in White women [9].
134
S. Qiu et al. / Bone 38 (2006) 130–135
3–5% proportion of empty lacunae might indicate that some
osteocytes are prone to die much sooner than others, as in the
auditory ossicles [13], but osteocyte death may be a
stochastic process and a constant fractional loss of 2.5 per
year could account for the data. The much lower densities of
osteocytes and total lacunae in deep bone are the result of its
much greater age [18]. Since trabeculae can only get thicker
during childhood [20], some interstitial bone was formed in
early childhood and so is almost as old as its owner. Constant
fractional losses of 2.5 per year for 50 years would reduce
osteocyte density by more than 70%. The concomitant
reduction in total lacunae (Fig. 2) indicates that micropetrosis is a feature of low turnover cancellous bone as well as
cortical bone. The absolute number of empty lacunae is
subject to opposing influences—it is increased by osteocyte
death but decreased by lacunar obliteration. It is unclear
which of these processes accounts for the larger numbers in
whole trabeculae and superficial bone in Black than in White
women (Fig. 2).
Although the regressions of osteocytes and total lacunae
on age did not attain conventional levels of significance, for
several reasons we believe the effect of increasing age to
promote osteocyte death is similar in Blacks and Whites.
First, based on previous data [21], a one-tailed test (the
probability of a fall in number versus no change or an
increase) for the effect of menopause is appropriate. Second,
since total lacunae cannot increase, a decline in percent
occupied lacunae (Tables 1 and 3, and Fig. 1) can only be the
result of osteocyte death. Third, when individual data in
Black women were plotted against the regressions established in White women (Fig. 2), the trends with increasing
age were very similar in the two ethnic groups. Fourth,
increased age is the only reasonable explanation for lower
osteocyte density in deep than in superficial bone. The finite
lifespan of osteocytes, first noted by Frost in cortical bone
[5], appears to be a universal feature of human biology.
Cancellous osteocyte death begins around age 20 with an
exponential approach to an asymptotic value of about 200/
mm2 in whole trabeculae and about 100/mm2 in deep bone.
Osteocyte death may not be the result of age-related changes
in hormone secretion [21] and appears to be due only to the
passage of time.
We had previously found a significant inverse relationship between osteocyte density in superficial bone and bone
formation rate in healthy White women [22], which raised
the possibility that the higher osteocyte density in Black
women might be partly responsible for their lower bone
formation rate [10]. However, we found no support for this
hypothesis, since the BFR predicted by the regression
relationship was significantly higher than the observed
value, and not significantly different from the observed value
in White subjects.
We recognize some limitations to this study. The number
of subjects was small, but the proportion of Black subjects in
our histomorphometry study was representative of the
population from which we recruited [10]; ours is the largest
published series of bone biopsies in healthy Black women.
The study was cross sectional, but serial bone biopsies in
healthy Black women are unlikely ever to be performed.
Furthermore, we are unaware of any secular trend in bone
metabolism in adult Black women over the period from 1980
to 1993 during which the data were collected.
In conclusion, Black women have more osteocytes, total
lacunae, and empty lacunae than White women; cancellous
bone is made with more osteocytes in Black than in White
women mostly likely because of reduced apoptosis of
osteoblasts, which may contribute to greater bone strength.
Osteocyte density declines with age and is lower in deep than
in superficial bone in Black as in White women, indicating
that the age of the bone is of greater importance than the age
of the subject. The mechanisms that determine the fate of
individual osteoblasts are an important subject for future
research.
Acknowledgments
This work was supported by grants from the National
Institutes of Health to The Henry Ford Hospital, Detroit, MI
(DK43858, R03AR 48641), and to the University of Arkansas
for Medical Sciences, Little Rock, AK (P01AG13198).
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