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Evidence of sequential remodeling in rat trabecular boneMorphology dynamic histomorphometry and changes during skeletal maturation.

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THE ANATOMICAL RECORD 208:137-145 (1984)
Evidence of Sequential Remodeling in Rat Trabecular
Bone: Morphology, Dynamic Histomorphometry,
and Changes During Skeletal Maturation
Yale University School of Medicine, Departments of Medicine and Cell
Biology, New Haven, CT 06510
The occurrence of a sequential bone remodeling activity, similar to what is observed in human bone, is demonstrated in rat trabecular bone
a t the level of the secondary spongiosa. A complete dynamic histomorphometric analysis of the remodeling activity, using undecalcified sections and double
fluorescent labels, has consequently been performed in young adults (220 g, 8
weeks old) and in more mature animals (320 g, 12 weeks old). The results
showed that, despite a similar trabecular bone volume, younger animals had a
five times higher bone formation rate and five times more osteoclasts than
more mature animals. The higher bone formation rate was due in part to a
threefold higher extent of double-labeled trabecular bone surface and in part
to a 1.5-fold faster mineralization rate. These results therefore demonstrate a
marked slowing down of bone turnover during skeletal maturation in the rat.
The values obtained in this study have been compared with measurements
made in other parts of the skeleton in the same species (Vignery and Baron
1978, 1980b; Tran Van et al., 1982a) or in humans. This comparison indicated
that 12-week-oldrats had a turnover rate very similar to values observed in
iliac crest trabecular bone in adult humans. The rat is therefore a good
experimental animal for the study of trabecular bone remodeling but since
large variations occur during skeletal maturation, care should be taken in the
selection of an age group relevant to the type of questions being asked.
Although widely used for the assessment
of bone remodeling activity in humans, dynamic histomorphometric techniques have
seldom been applied to small laboratory animals like the rat. Partial studies have been
published but they deal either only with cortical bone (Baylink et al., 1970; Tam and
Anderson, 1980) or with limited measurements of trabecular bone activity (Schenk et
al., 1973; Miller and Jee, 1975; Kimmel and
Jee, 1980; Wronski and Morey, 1982) often
made in the growth plate area. In addition,
and because of the prolonged growth of this
species, the fact that bone remodeling occurs
in the rat similar to that in human trabecular bone, is still controversial (Frost, 1976).
We have demonstrated in previous studies
(Baron, 1973; Vignery and Baron, 1980b)that
bone remodeling occurs in rat alveolar bone
and follows the same sequence as that described in humans (Frost, 1964; Rasmussen
and Bordier, 1974). We have also recently
0 1984 ALAN R. LISS, INC
studied the cellular kinetics of a n induced
bone remodeling sequence along the periosteum in the rat and showed that it was similar to the sequence observed in other species (Tran Van et al., 1982a). The aims of this
study were therefore 1)to verify the occurence of such a sequential remodeling activity
in adult rat trabecular bone; 2) t o measure
this activity; 3) to compare it to the data we
obtained in alveolar bone and along the periosteum of the same species and in humans;
and 4) to evaluate the effect of skeletal maturation on bone remodeling activity in the
Twenty-four male Wistar-Lewis rats, 14
weighing 220 20 g (8 wk old) and 10 weighing 320 20 g (12 wk old) were used in this
Received January 14, 1983; accepted May 27, 1983.
Address reprint requests to Dr. Roland Baron.
study. They were fed a normal rodent diet
(Purina) and tap water ad libitum for 14 days.
They were housed in individual cages and on
a 12 W12 h light-dark cycle. Double fluorescent labeling of their bones was performed
by the intraperitoneal injection of calcein
(DCAF, Merck) a t a concentration of 30 mg/
kg of body weight in a constant volume of 0.1
ml of a saline solution containing 2% NaHC03. The animals were injected a t days 7
and 13 and sacrificed a t day 14, 24 hours
after the last calcein injection.
Two vertebrae in the proximal third of the
tail were dissected out and fixed for 48 h in
40% ethanol a t 4°C. They were then dehydrated in increasing concentrations of ethanol and embedded in methyl-methacrylate
without decalcification (Baron et al., 1983).
Unserial undecalcified longitudinal sections,
4 pm thick, were performed in the central
area of the vertebrae using a Jung K or a n
Autocut microtome. These sections were
stained with toluidine blue at pH 3.7 for morphometry. Sections 10 pm thick were also
performed and kept unstained for observation and measurements using fluorescence
All measurements were made in the trabecular bone with the exclusion of the area
situated within 1 mm from the epiphyseal
growth plates (Fig. 1)in order to exclude the
primary spongiosa (Kimmel and Jee, 1980).
Measurement of the following parameters
were performed at a x 125 magnification using a MOP 3 Planimeter (Carl Zeiss): whole
trabecular bone tissue (WTBT), trabecular
bone volume (TBV), osteoid volume (OV), trabecular bone surface (TBS), osteoid surface
(OS), osteoblastic surface (ObS), osteoclastic
surface (OCS), number of osteoclasts (#OC),
and reversal surface [Revs; i.e., Howship's
lacunae not containing a n osteoclast (Baron,
197711. The mean wall thickness [MWT, i.e.,
the mean distance between the cement line
and the bone surface in completed remodeling foci (Frost, 197711 was measured a t a
using a micrometer,
magnification of ~ 2 5 0
on sections that were decalcified and stained
with toluidine blue at a basic pH (Fig. 2; Fig.
3b,c). The unstained sections were observed
under ultraviolet (UV) light and measurement of TBS, double-labeled surface (2LS,
Figs. 4b and 5), and single-labeled surface
(ILS, Fig. 5) were made a t a magnification of
x250. The mineralization rate (MR) was
measured on the same sections a t a magnifi. calculations were made
cation of ~ 4 0 0 All
from these 14 measured parameters according to the method described by Frost (1977)
and using the correction factor for the oblique) MWT and MR
ness of the sections ( ~ 0 . 7 3for
(Frost, 1977). Namely, TBV is expressed as
percentage of WTBT; OV is expressed as percentage of WTBT [absolute osteoid volume
(AOV)];OS, ObS, OCS, Revs, and total lacunar surface (TLS) are expressed as percentage of TBS, as well as the 1LS and 2 L S the
mean osteoid thickness (OT) is calculated by
dividing OV (pm3>by 0s (pm'); the #OC is
divided by TBS (mm) and expressed a s the
#OC/mm of TBS; the MR is calculated by
dividing the mean interval between double
labels (pm) by the number of days between
the calcein injections and expressed a s p d
day; the mineralization lag time (MLT) was
calculated by dividing OT (pm) by MR ( p d
day) and expressed in days. The bone formation rate (BFR) at the tissue level is calculated as a n index by multiplying the 2LS by
the MR. BFR a t the cellular level (BFWOb)
is calculated as a n index by dividing BFR by
the extent of the ObS. The bone resorption
rate (BRR) has been calculated as a n index
a t the cellular level (BRWOC) by assuming
that the bone volume remains constant and
dividing the BFR by the #OC/mm TBS. The
duration of the different steps of the remodeling sequence is estimated on the basis of
the duration of the formation phase [Sigma
F = MWT (pm)/MR (pdday)]; the duration
of the other phases, namely Sigma R and
Sigma reversal, are calculated as [Sigma F/
0s (mm) x OCS (mm) or Revs (mm)] and
expressed in days. The results are given as
Fig. 1. Undecalcified section of a rat tail vertebra
showing the primary spongiosa (ps) excluded from the
measurements by eliminating 1 mm under the cartilaginous growth plate; the measured area consists of the
secondary spongiosa (ss) excluding the midshaft and the
cortices. Magnification, x 35.
Fig. 2. Sections decalcified after sectioning and
stained with basic toluidine blue allow a clear distinction between the primary spongiosa (a) in which the
trabeculae contain cartilaginous remnants (cr, white arrows) and the secondary spongiosa (b) not showing cartilaginous remnants and where reversal lines (black
arrows) delineate completed remodeling sites. Magnification, ~ 2 5 0 .
Fig. 3. Morphologic evidence of bone remodeling in
rat trabecular bone. Undecalcified sections (a) showing
the various steps of cellular activity of the remodeling
sequence with osteoclasts (OC), reversal sites (Rev, black
arrow), and osteoblasts (Ob)lining osteoid (white arrow).
After decalcification and basic toluidine blue staining,
individual remodeling sites are demonstrated: 03)during
the formation phase of remodeling (black arrows show
reversal line); and (c) completed remodeling sites, one
(white arrows) older than the other (black arrows). Magnification, ~ 4 0 0 .
Fig. 4. (a) Bone-forming site viewed on serial undecalcified sections stained with toluidine blue (black arrow
= osteoblasts; white arrow = osteoid) and under UV
light (b)showing the double fluorescent label of the same
site (black arrows). Magnification, ~400.
Fig. 5. Fluorescent labels along a trabecula demonstrating a double-labeled area (2L) and a single-labeled
area adjacent to it (1L). Magnification, ~400.
the mean + 1SD. Statistical comparison between the two groups was made by the Student's t test and the difference considered
significant when p < 0.05.
The distance between cartilaginous growth
plates in the tail vertebrae used in this study
is 8.7 f 0.1 mm in the 8-week-old rats and
9.2 & 0.2 mm in the 12 week-old-animals. An
overall growth of 0.5 mm therefore occurred
in 4 weeks a t the organ level (18 pdday).
The overall size of the diaphysis marrow cavity is apparently not different in the two
Unlike the primary spongiosa, the trabeculae found in the secondary spongiosa do not
have cartilaginous remnants in their center
(Fig. 2). In addition to this lack of cartilage,
direct evidence for the occurrence of sequential remodeling along the endosteum of trabecular bone in the secondary spongiosa is
provided by the presence of completed remodeling sites outlined by scalloped reversal cement lines (Fig. 2b; Fig. 3b,c). Morphologic
examination of bone cells along the trabeculae clearly show the occurrence of bone formation by osteoblasts (Figs. 3a, 4a), bone
resorption by multinucleated osteoclasts (Fig.
3a), and reversal lacunae with active mononucleated cells within Howship lacunae (Fig.
3a), all occurring within well-delineated remodeling sites.
The results of all measurements made on
the trabecular bone of the tail vertebrae
of the two groups of animals are shown in
Table 1.
Despite a similar TBV, there were marked
differences in other parameters of bone remodeling between the two groups. In essence, the results show a much higher
turnover rate in younger animals than in
more mature rats. ObS is about twice as great
and OCS about four times greater in 220-g
rats; the specific density of trabecular surface being similar in the two groups for the
area under study, this means that a higher
amount of bone is turned over in the younger
animals. Measurements of double fluorescent labels show the same trends: the total
labeled surface is more than twice as great
in the younger animals; this is essentially
due to the double-labeled surface which is
close to four times higher in the younger
group. In addition, the daily MR is decreased
by about 25% in the more mature animals.
The rate a t which bone matrix is laid down
TABLE 1. Dynamic morphometry of rat tail trabecular
bone remodeling'
220 g (8 wk)
(n = 14)
TBV (% tissue)
AOV (% tissue)
0s (% trab. S)
ObS (9% trab. S)
OCS (8
trab. S)
Revs (% trab. S)
TLS (% trab. S)
OT (pm)
MWT (pm corrected)
1Lab S (% trab. S)
2 Lab S (% trab. S)
MR (pmiday, corrected)
MLT (days)
BFR index (tissue level)
BFR/Ob index
BRRiOC index
320 g (12 wk)
(n = 10)
18.2 (1.6)
17.5 (1.7)
1.4 (0.3)
0.9 (0.3)'
41.1 (3.8)
22.0 (6.8)**
28.9 (4.7)
14.3 (6.9)**
3.4 (1.3)
0.9 (0.6)**
3.2 (1.0)
2.6 (1.0)
6.6 (1.4)
3.5 (1.3)**
8.2 (1.0)
5.8 (0.8)**
18.8 (3.8) 15.1 (4.5)
34.3 (9.7)
25.0 (13.2)
37.5 (8.9)
10.4 (5.5)**
0.61 (0.08)
0.45 (0.05)**
13.4 (1.2)
12.9 (0.9)
22.9 (3.2)
4.7 (4.1)***
7.8 (3.6)
3.3 (3.3)*
5.9 (3.9)
6.7 (4.6)
For abbreviations, see text, Materials and Methods. Lab S ,
labeled surface; trab. S, tTabecular surface.
*Mean ? SD.
Significant differences between the two groups: *p < 0.05; '*p
< 0.01; ***p < 0.0001.
by osteoblasts is, however, proportionally decreased since the mean osteoid seam thickness is reduced by about the same amount in
older rats ( 30%), maintaining the MLT constant (MLT = 13 days). The additive effects
of this higher MR, higher 2LS, and greater
extent of active ObS are demonstrated by
the calculated dynamic parameters: the BFR
a t the tissue level is close to five times higher
in the younger animals. The TBV being similar in the two groups, this is therefore a
direct reflection of the turnover rate, and the
bone resorption rate is most likely also five
times higher in the younger group. No differences are, however, seen at the cellular level
of osteoclastic bone resorption, each osteoclast resorbing about the same amount of
bone in the two groups, with the TBV remaining constant. This also indicates that
the BFR is decreased a t the cellular level in
the older animals, since there is a five times
lower BFR at the tissue level with only a
3 . 7 ~drop in 2LS. All these results clearly
indicate a major change in the birth rate or
size of bone remodeling units at the tissue
level: the higher turnover rate in younger
animals is almost entirely attributable to a
higher number or larger size of remodeling
units and only partially to a higher formation rate at the individual cell level. Calculated durations of the different phases of the
bone remodeling sequence in the two groups
of animals (Table 2) show that the reversal
phase takes a longer time in older animals
(formation of bone not preceded by resorption) in younger animals, our data show that
this is not the case, at least in the secondary
8 wk old
12 wk old
spongiosa. If the ratio of ObS to OCS is taken
2.5 (0.9)
1.4 (0.9)
Sigma resorption
as a reflection of the balance between formaSigma reversal
2.4 (1.0)
4.2 (0.8)*
tion (F)and resorption (R), it is much higher
31 (4)
33 (6)
Sigma formation
(15.9) in older animals than in younger aniTotal (sigma BMU)
36 (7)
38 (7.5)
mals (8.5); this, however, does not take into
BMU, basic multicellular unit; see Frost (1964).
account the rate a t which bone matrix is
*Significant difference between groups: p < 0.05
formed and mineralized. A better indication
of the balance between F and R is given by
with a slower turnover rate, but that the calculating the ratio of the BFR, which takes
active resorption phase is slightly shorter, into account the MR, to the OCS: this ratio
leaving the total duration of each remodeling (6.7 VS. 5.2) shows that the balance between
F and R is very similar in the two groups,
cycle unchanged.
therefore explaining the similarities in TBV
and TBS specific density. Three conclusions
This study shows that despite the continu- can be drawn from this observation: 1)the
ous growth of this animal, sequential bone secondary spongiosa of tail vertebrae is alremodeling occurs in the secondary spon- most entirely subjected to remodeling activgiosa of tail vertebrae trabecular bone in the ity in the rat, independently of the age of the
rat, as demonstrated by the presence of typi- animals; 2) bone formation and resorption
cal completed remodeling sites with reversal are coupled and balanced in both groups, only
cement lines and by dynamic histomorphom- the turnover rate is different (about five
etry. Trabecular bone therefore constitutes times higher in younger animals); and 3) the
the third instance in which we have shown net positive balance at the organ level, rebone remodeling activity in this animal, the sulting in growth, is the result of the net
two others being alveolar bone (Baron, 1973; formation of new bone tissue a t the growth
Vignery and Baron, 1980b) and the man- plate and primary spongiosa, not of a positive
dibular periosteal surface (Tran Van et al., balance and/or modeling activity in the sec1982a). The fact, however, remains that no ondary spongiosa. It is important to underHaversian remodeling of cortical bone can be line the fact that this might be true of tail
demonstrated in this species and this is very vertebrae but less so for the metaphysis of
likely the reason why it was felt that no growing long bones in the same animal.
remodeling occurred in the rat (Frost, 1976). Kimmel and Jee (1980) have shown a growth
One has, however, to exclude the growth of about 170 pmlday in 170g rat long bones,
plates and primary spongiosa from the mea- a value 10 times higher than the growth rate
sured areas in order to avoid mixing growth of the tail vertebrae in this study (220-320 g
and remodeling activities (Kimmel and Jee, rats). In addition, the height of the meta1980).In addition, this study shows that, sim- physis in growing long bones is maintained
ilar to humans, skeletal maturation profound- constant by a net resorption of trabecular
ly affects the remodeling activity in the rat bone toward the diaphysis, allowing a n exand, according to the goal of each specific pansion of the marrow cavity, when such
study, one should select the experimental an- phenomenon is much less apparent in these
slowly growing tail vertebrae. Incidentally,
imal at a specific age.
The data obtained in our two groups of rats the data obtained in normal childrens’ iliac
are strikingly similar to the data obtained in crest trabecular bones (Bulla et al., 1979;
children and adult human trabecular bone Glorieux et al., 1980; Marie and Gloriew,
remodeling, respectively. Here in the normal 1981; Marie et al., 1982; Baron et al., 1983)
rat, as well as in normal humans (Marie et when compared to normal adults (Meunier et
al., 1982; Baron et al., 19831, we see that al., 1973; Schenk, 1976; Schulz and Delling,
despite a n approximately similar TBV, the 1976; Melsen and Mosekilde, 1978; Frost et
extent of bone-forming surface (ObS and 2LS) al., 1981) suggest that the same situation
as well as the BFR (MR, OT) are higher in occurs in humans.
Similar to humans, we also observed in
younger animals. Although this could be interpreted superficially as indicating a more this study a lower MR in adults than in
positive balance or the presence of modeling younger animals. Since this decrease in MR
TABLE 2. Duration (in days) of the different phases of
the remodeling sequence
is associated with a thinner osteoid seam and
a constant MLT, it suggests that the BFR is
lower a t the level of the osteoblast. With
BRR a t the level of the osteoclast remaining
constant, this should lead to a net bone loss.
This, however, occurs neither in our two
groups nor in humans between childhood and
maturity, because of a more profound decrease in the number of resorbing osteoclasts
( - 4 X ) than in the osteoblast-covered trabecular surface ( - 2 x), therefore reestablishing
a n equilibrium between bone formation and
bone resorption a t the tissue level.
The calculated durations of the different
phases of the remodeling sequence in this
study have to be compared between the two
groups and also with data obtained on the
same animal in three other instances (Vignery and Baron, 1978; Vignery and Baron,
1980a; Tran Van et al., 1982a) and in humans Wignery and Baron, 1980a). The most
striking observation in all these instances is
the similarity of the duration of the active
resorption phase between the species (rat vs.
human), the sites (alveolar bone, periosteum,
trabecular bone), the circumstances (normal
vs. induced remodeling), and the method of
measurement (directly measured vs. calculated). In all these studies, osteoclastic resorption lasts from 1.5 to 2.5 days (2.2 0.7).
This value differs markedly from values published by others for human bone resorption
( - 15 days, Frost, 1963, 1977; Meunier et al.,
1973)because these authors use the TLS for
calculation, without making the difference
between OCS and Revs (Baron, 1977; Baron
et al., 1981). These are, however, two very
different steps in the remodeling sequence
and should be considered separately (Baron,
1977; Vignery and Baron, 1980a; Baron et
al., 1981; Tran Van et al., 1982a,b).The similarity in duration of osteoclastic resorption
suggests that the normal active lifespan of
osteoclasts at one site is somewhere around
2 days in these two species.
The duration of the reversal phase seems,
on the other hand, to vary more widely in all
the studies mentioned above. The only instance in which it was directly measured in
the rat showed a duration of 4 days (Tran
Van et al., 1982a), a value similar to alveolar
bone remodeling calculations (Vignery and
Baron, 1980a). In the present study, this duration goes from 2.6 in the younger animals
to 4.2 days in the older ones. The proportional duration of this phase, expressed as a
ratio with the osteoclastic phase, is a reflec-
tion of the coupling F-R (Baron, 1977; Baron
et al., 1981). This ratio is 3 in alveolar bone
(Vignery and Baron, 1980a1, 2 in periosteal
remodeling (Tran Van et al., (1982a1, and here
3 in older animals and 1 in younger ones
where the turnover rate is higher. In humans, this ratio is 5 in normal adults and
children but goes up to 20 in individuals with
senile osteopenia and down to 1 or 2 in persons with balanced high-turnover states like
hyperparathyroidism and Paget’s disease
(Baron et al., 1981). This suggests that the
time necessary for coupling during the reversal phase might be inversely proportional to
the turnover rate.
In conclusion, this study shows that sequential remodeling occurs in the secondary
spongiosa of rat trabecular bone. As in humans, young adults have a higher turnover
rate than more mature animals but both
groups have a n equilibrated balance between bone formation and resorption. The
continuous growth of this animal is therefore
mostly attributable, at least in tail vertebrae, to events taking place at the growth
plate and primary spongiosa, leading to a n
increase in size and absolute bone mass a t
the organ level.
This work was supported by grant DE
04724 from the National Institutes of Health.
The authors would like to express their
thanks to Mrs. Lynn Neff and Mrs. Ann Silverglate for their expert technical help and
to Mrs. Barbara Devlin for typing the
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skeletal, trabecular, change, evidence, remodeling, maturation, histomorphometric, bonemorphology, rat, dynamics, sequential
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