Journal of Anatomy

J. Anat. (2012) 220, pp77–85 doi: 10.1111/j.1469-7580.2011.01449.x

Structural and functional changes in the alveolar bone

osteoclasts of estrogen-treated rats
Ana Paula de Souza Faloni,1 Estela Sasso-Cerri,1,2 Fernanda Regina Godoy Rocha,2
Eduardo Katchburian1 and Paulo Sérgio Cerri1,2
1
Department of Morphology and Genetics, Federal University of São Paulo (UNIFESP), São Paulo, Brazil
2
Laboratory of Histology and Embryology, Dental School, UNESP – Universidade Estadual Paulista, São Paulo, Brazil

Abstract
This study investigated structural and functional features of apoptotic alveolar bone osteoclasts in estrogen-
treated rats. For this purpose, 15 female rats 22 days old were divided into three groups: Estrogen (EG), Sham
(SG) and Control (CG). The rats of EG received daily intramuscular injection of estrogen for 7 days. The SG
received only the oil vehicle. Maxillary fragments containing alveolar bone were removed and processed for
light and transmission electron microscopy. Area (OcA) and number of nuclei (OcN) and bone resorption surface
per TRAP-positive osteoclasts (BS ⁄ OC) were obtained. Vimentin, caspase-3 and MMP-9 immunoreactions,
TUNEL ⁄ TRAP and MMP-9 ⁄ TUNEL combined reactions were performed. In EG, the OcA, OcN and BS ⁄ Oc were
reduced. Moreover, osteoclasts showed cytoplasm immunolabelled by caspase-3 and a different pattern of
vimentin expression in comparison with CG and SG. MMP-9 expression was not affected by estrogen and the
TUNEL-positive osteoclasts were MMP-9-immunolabelled. In EG, ultrastructural images showed that apoptotic
osteoclasts did not exhibit ruffled borders or clear zones and were shedding mononucleated portions. TRAP-
positive structures containing irregular and dense chromatin were partially surrounded by fibroblast-like cells.
In conclusion, the reduction in the BS ⁄ Oc may be due to reduction in OcA and OcN; these effects seem to be
related to vimentin disarrangement rather than to an interference of estrogen with osteoclast MMP-9 expres-
sion. Osteoclast apoptosis involves caspase-3 activity and vimentin degradation; these cells release portions
containing one apoptotic nucleus and, subsequently, undergo fragmentation, giving rise to apoptotic bodies.
Key words: apoptosis; estradiol; MMP-9; osteoclast; vimentin.

matrix metalloproteinase-9 (MMP-9); these enzymes are

Introduction
osteoclast markers (Sorensen et al. 2007). Among them, the
Osteoclast formation, activity and survival are under the MMP-9, a zinc-dependent proteinase also called gelatinase
influence of several systemic, local and environmental fac- B, seems to degrade peptides derived from collagenase
tors (Phan et al. 2004). Among the systemic factors, estro- activity and other components of the bone matrix (Stern-
gen is a steroid hormone widely known for its inhibitory licht & Werb, 2001).
function on bone resorption (Tapp, 1966; Liu & Howard, Studies including different models have suggested that
1991; Chow et al. 1992; Faloni et al. 2007; Cruzoé-Souza estrogen is able to promote osteoclast apoptosis but the
et al. 2009). The inhibition of bone resorption by estrogen mechanism is still unclear (reviewed by Saintier et al. 2006;
seems to be associated, at least in part, to the inhibition of Faloni et al. 2007; Cruzoé-Souza et al. 2009). The treatment
osteoclast proteases (Parikka et al. 2001). Bone resorption of young female rats with estrogen has caused a 50% reduc-
depends on the activity of different enzymes such as tion in the number of alveolar bone osteoclasts due to osteo-
tartrate-resistant acid phosphatase (TRAP), cathepsin K and clast death by apoptosis (Faloni et al. 2007; Cruzoé-Souza
et al. 2009). The presence of enhanced estrogen receptor-
beta (ER-b) immunoexpression in the apoptotic osteoclasts
Correspondence suggests that estrogen may act directly on osteoclasts by con-
Prof. Dr. Paulo Sérgio Cerri, Laboratory of Histology and Embryology,
trolling their survival (Cruzoé-Souza et al. 2009).
Department of Morphology, Dental School, UNESP – Univ Estadual
Paulista, Rua Humaitá, 1680, Centro, CEP 14801–903, Araraquara, São
In mononuclear cells, programmed cell death by apopto-
Paulo, Brazil. T: + 55 16 33016497; F: + 55 16 33016433; sis involves a complex and coordinated cascade of molecular
E: [email protected] events. This cascade involves caspases, such as caspase-3,
Accepted for publication 11 October 2011 responsible for degradation of the nuclear proteins and
Article published online 16 November 2011 activation of nucleases which induce the degradation of

ª 2011 The Authors

Journal of Anatomy ª 2011 Anatomical Society of Great Britain and Ireland
78 Alveolar bone osteoclasts in estrogen-treated rats, A. P. S. Faloni et al.

DNA (Huppertz et al. 1999). In addition, it is known that phosphate. After decalcification for 40 days in 7% ethylene-
caspase-3 cleaves cytoplasmic proteins of the cytoskeleton, diaminetetracetic acid (EDTA) solution containing 0.5% formal-
such as vimentin (Byun et al. 2001; Alam et al. 2010). Osteo- dehyde, buffered at pH 7.2 with 0.1 M sodium phosphate, the
specimens were dehydrated in graded concentrations of ethanol
clasts exhibit vimentin filaments distributed around the
and embedded in paraffin.
nuclei and in the ruffled border region (Akisaka et al. 2008)
and, therefore, these filaments have an essential role in
osteoclast polarization and maintenance of the structural TRAP method and TUNEL ⁄ TRAP combined methods
integrity of osteoclast. Thus, the degradation of cytoskele-
Sagittal sections 6 lm thick, containing alveolar bone, were
ton proteins induces its disruption and collapse (Byun et al.
submitted to the TRAP method. The TRAP method was used as
2001; Lee et al. 2002; Alam et al. 2010). Subsequently, the an osteoclast marker (Minkin, 1982; Cerri et al. 2003, 2010).
apoptotic cell undergoes blebbing and sheds apoptotic Deparaffinized sections were incubated in a solution containing
bodies (Huppertz et al. 1999; Cerri et al. 2000; Cerri, 2005). naphthol AS-BI (Sigma Chemical Co., St. Louis, MO, USA), N-N-
In young rats, numerous osteoclasts are observed in the dimethylformamide, sodium acetate buffer (pH 5.0), Fast Red
alveolar bone surface due to the continuous and intense Salt TR (Sigma Chemical Co.) and sodium tartrate dihydrate.
For detection of DNA breaks (Gavrieli et al. 1992), the TUNEL
bone remodelling to accommodate the growing and erup-
method was performed as previously described (Cerri et al.
tion tooth. As apoptosis is a rapid process, the effect of
2000, 2003). Deparaffinized sections adhered to silanized (3-
estrogen in alveolar bone of young animals is a suitable in aminopropyltriethoxysilane; Sigma Chemical Co.) slices were
vivo model to investigate the apoptotic process in osteo- treated with proteinase K (Oncor-Protein Digesting Enzyme),
clasts (Faloni et al. 2007; Cruzoé-Souza et al. 2009). subsequently treated with 3% hydrogen peroxide to block
In the present study, we propose to investigate the endogenous peroxidase, and then immersed in an equilibration
estrogen effect on the structural and functional integrity of buffer. The reaction was followed by incubation in solution con-
taining terminal deoxynucleotidyl transferase (TdT) in a humid
osteoclasts. As the number of nuclei and area of osteoclasts
chamber. The sections were then immersed in the stop ⁄ wash
are indicative parameters of resorptive activity (Chow et al.
solution, incubated in the antidigoxigenin-peroxidase, treated
1992; Lees & Heersche, 2000; Lees et al. 2001), we examined with 0.06% 3.3-diaminobenzidine tetrahydrochloride (Sigma
these parameters and related them to the bone resorption Chemical Co.) and counterstained with Carazzi’s haematoxylin.
surface. A possible relationship between apoptotic process Combined TUNEL and TRAP methods were performed as previ-
and vimentin ⁄ MMP-9 immunoexpression was also exam- ously described in detail by Cerri et al. (2003).
ined in the alveolar bone osteoclasts.
Morphometric analysis
Materials and methods Osteoclast area (OcA), number of osteclast nuclei (OcN)
and bone resorption surface per osteoclast (BS ⁄ Oc)
Animals and treatment The morphometric analyses were performed in three non-serial
sections of the alveolar bone around the first molar. The small-
The animal care laws and national laws on animal use were
est distance between the sections was 60 lm. From these sec-
observed in this study, which was authorized by the Ethical
tions, 50 TRAP-positive osteoclasts apposed to alveolar bone
Committee for Animal Research of the Federal University of São
surface were selected per animal and their images were cap-
Paulo (UNIFESP), Brazil.
tured using an image analysis system attached to a light micro-
Fifteen female Holtzman rats (Rattus norvegicus albinus) 22
scope (Jenaval, Zeiss, Germany) at ·1000. To estimate bone
days old were equally distributed into Estrogen (EG), Sham (SG)
resorption activity, the area of osteoclasts (OcA) and the length
and Control (CG) Groups. For 7 days, rats from the EG received
of the bone surface in close proximity to the zone of contact of
a daily intramuscular injection of 0.125 mg 100 g)1 body weight
each osteoclast (BS ⁄ Oc) were measured using the free software
of estrogen (estradiol hexahydrobenzoate, Benzoginoestril ap;
IMAGE TOOL (IMAGE TOOL for Windows – Version 3.0; The University
Hoechst Marion Roussel, São Paulo, Brazil) diluted in corn oil.
of Texas Health Science Center in San Antonio). The number of
The dosage of estrogen and the period of administration used
nuclei (OcN) of each TRAP-positive cell apposed to bone surface
in this study have been shown to cause significant changes in
was counted.
bone metabolism (Silberberg & Silberberg, 1941; Chow et al.
The data obtained from the morphometric parameters evalu-
1992).
ated were distributed in categories. Thus, according to area, the
The SG rats received the same dose of corn oil and the CG rats
osteoclasts were classified into three categories: small (20.00–
did not receive any treatment.
199.99 lm2), medium (200.00–799.99 lm2) and large (800.00–
1746.00 lm2). On the basis of the number of nuclei ⁄ cell, the TRAP-
Histological procedures positive cells were classified into mononucleated cells (one nucleus
per cellular profile) or multinucleated osteoclasts (two or more
The animals were killed with chloral hydrate (600 mg kg)1) and nuclei per cellular profile). The length of the bone resorption
fragments of the maxilla containing alveolar bone surrounding surface (BS ⁄ Oc) was classified into: small (3.03–22.99 lm), medium
the upper molars were removed. The specimens were fixed for (23.00–70.99 lm) and large (71.00–118.00 lm).
48 h at room temperature in 4% formaldehyde (freshly derived The frequencies of the different categories of data were
from paraformaldehyde) buffered at pH 7.2 with 0.1 M sodium obtained for each animal ⁄ group and analyzed statistically.

ª 2011 The Authors

Journal of Anatomy ª 2011 Anatomical Society of Great Britain and Ireland
 Alveolar bone osteoclasts in estrogen-treated rats, A. P. S. Faloni et al. 79

Immunohistochemistry for caspase-3, vimentin and The sections submitted to the TRAP method and TUNEL ⁄ TRAP
MMP-9 detection combined methods and sections submitted to immunohisto-
chemistry for caspase-3, vimentin, MMP-9 detection and MMP-
Deparaffinized sections adhered to silanized slides were submit- 9 ⁄ TUNEL combined reactions were analyzed and the images
ted to immunohistochemistry for caspase-3, vimentin or MMP-9. were captured using an Olympus BX-51 light microscope.

Caspase-3
To unmask the antigenic sites, the sections were incubated with
Statistical analysis
0.1% Triton-X (Aldrich-Chemie, Steinheim, Germany) for 4 h. The parametric one-way analysis of variance (one-way ANOVA)
The sections were incubated overnight with rabbit anti-caspase- and the non-parametric Kruskal–Wallis test followed, respec-
3 active form (Millipore Corporation, Temecula, CA, USA) tively, by Tukey and Dunn’s multiple comparison tests were used
diluted in 1 : 100 phosphate buffer 0.05 M with 0.2 M sodium for the statistical analysis. All calculations were performed with
chloride (phosphate-buffered saline, PBS), pH 7.2. The reaction the software GRAPH PAD PRISMA 4. The significance level accepted
was detected by Vectastain Kit (Vector Laboratories Inc., Burlin- was P < 0.05.
game, CA, USA). Thus, these sections were incubated with bio-
tinylated anti-rabbit ⁄ mouse IgG at room temperature and, after
washings in PBS, they were incubated with avidin-biotin-peroxi- Transmission electron microscopy
dase complex.
Specimens containing alveolar bone of the first molar were
fixed in a mixture of 4% of glutaraldehyde and formaldehyde
Vimentin
(derived from paraformaldehyde) buffered at pH 7.2 with 0.1 M
To unmask the antigenic sites, the sections were immersed in
sodium cacodylate, at room temperature. After decalcification
0.001 M sodium citrate buffer, pH 6.0, and submitted to micro-
in a 7% solution of EDTA containing 0.5% formaldehyde in
wave oven cycles for 30 min (Sasso-Cerri et al. 2005). The sec-
0.1 M sodium cacodylate buffer, at pH 7.2, the specimens were
tions were incubated overnight with mouse anti-vimentin (V9,
washed in 0.1 M sodium cacodylate, pH 7.2. They were then
Sigma-Aldrich Inc., St. Louis, MO, USA) diluted in 1 : 150 PBS, in
transferred to 0.1 M sodium cacodylate-buffered 1% osmium
a humidified chamber at 4 C. After washings in PBS, the vimen-
tetroxide solution for 1 h, at room temperature. Subsequently,
tin immunoreaction was detected using the MACH 4 Universal
the specimens were washed in distilled water and treated with
HRP-Polymer kit (Biocare Medical LLC., Concord, CA, USA). Thus,
aqueous 2% uranyl acetate for 2 h. The specimens were
the sections were incubated with the MACH 4 mouse probe at
dehydrated in graded concentrations of ethanol, treated with
room temperature for 15 min, washed in PBS and incubated
propylene oxide and then embedded in Araldite. Ultrathin semi-
with MACH 4 HRP polymer at room temperature for 15 min.
serial sections were collected on formvar-coated single slot
grids, stained in alcoholic 1% uranyl acetate and lead citrate
MMP-9 solution and examined in a Philips CM 200 transmission electron
Immunohistochemical detection of MMP-9 was performed as microscope.
previously described (Cerri et al. 2010). To unmask the antigenic
sites, the sections were also submitted to microwave oven cycles.
Sections were incubated overnight with mouse anti-MMP-9 Results
(Calbiochem; Biochemicals and Immunochemicals, USA),
diluted in 1 : 500 PBS, in a humidified chamber at 4 C. The On the alveolar bone surface of the first upper molar of
reaction was detected by Vectastain Kit (Vector Laboratories 29-day-old rats from the control (CG) and sham (SG)
Inc., Burlingame, CA, USA). In the CG, SG and EG, the frequency groups, several giant multinucleated TRAP-positive osteo-
of MMP-9 immunolabelled osteoclasts was evaluated in 30 clasts (Oc) were observed (Fig. 1A). In contrast, few TRAP-
osteoclasts per animal. positive osteoclasts were found in the alveolar bone sur-
The immunoreactions were revealed by 0.06% 3.3-diam-
faces of the estrogen-treated rats (EG). Moreover, in EG
inobenzidine tetrahydrochloride (Sigma-Aldrich, Chemie GmbH,
(Figs 1B,C), the area and number of nuclei of osteoclasts
Germany) in PBS and the sections were counterstained with
Carazzi’s haematoxylin. As negative controls, the immunohisto- were apparently reduced in comparison with the control
chemistry was performed by replacing the incubation step with groups (Fig. 1A). Sometimes, mononucleated TRAP-positive
primary antibodies using an incubation step with non-immune structures, exhibiting dense chromatin strongly stained by
serum. haematoxylin, were observed in close juxtaposition to oste-
oclasts in the bone surface of estrogen-treated rats
(Fig. 1D). Occasionally, these TRAP-positive structures con-
Combined immunohistochemistry for MMP-9 and
taining irregular masses of dense chromatin were partially
TUNEL reaction
surrounded by fibroblasts-like cells of periodontal ligament
In some sections, MMP-9 detection was performed for the (Fig. 1E).
immunohistochemistry and, subsequently, the same sections The TUNEL ⁄ TRAP combined reactions revealed that some
were submitted to the TUNEL method and revealed using a Vip
of these mononucleated TRAP-positive structures exhibited
Substrate Kit (Vector Laboratories Inc.). After washings in dis-
TUNEL-positivity (Fig. 1F). In EG, some osteoclasts located
tilled water, the sections were counterstained with methyl
green (Cruzoé-Souza et al. 2009). next to the bone surface showed shrunken cytoplasm

ª 2011 The Authors

Journal of Anatomy ª 2011 Anatomical Society of Great Britain and Ireland
80 Alveolar bone osteoclasts in estrogen-treated rats, A. P. S. Faloni et al.

TRAP TRAP TRAP

N PL B
PL PL
N
Oc Oc N
Oc Oc
RB

B
A B Ot B C
B
TRAP TRAP TUNEL/TRAP Caspase-3
B PL
B N
N Oc
F
N
Oc S PL
Oc
Bo S
B
D E F G B
B
Vim Vim Vim
N N
Oc N Oc
S
Oc

B
H I B J B

Fig. 1 Light micrographs of portions of alveolar bone (B) from control (A,H) and estrogen-treated (B–G,I ,J) rats. (A–D) TRAP reaction
counterstained with haematoxylin. (A) A large TRAP-positive (red colour) osteoclast (Oc) exhibiting several nuclei (N) and ruffled border (RB) is
apposed on the bone surface (arrowheads). Osteocyte (Ot). Periodontal ligament (PL). Bar: 5 lm. (B,C) Profiles of TRAP-positive cells (Oc) apposed
to the bone surface (B) exhibit only two nuclei (N) and one nucleus (N), respectively. Periodontal ligament (PL). Bar: 5 lm. (D) Adjacent to the
alveolar bone surface (B), small TRAP-positive structures (S), which exhibit condensed chromatin strongly stained by haematoxylin (arrows), are in
close juxtaposition to a TRAP-positive osteoclast (Oc). Nucleus (N); Bar: 5 lm. (E) A fibroblast-like cell (F) next to the alveolar bone surface (B)
engulfs (arrows) a TRAP-positive body (Bo). Periodontal ligament (PL). Bar: 10 lm. (F) Next to the bone surface (B), a mononucleated TRAP-positive
cell (Oc) exhibits a TUNEL-positive nucleus (N). In close contact with the TRAP-positive cell (Oc), a structure (S) exhibits an irregular and basophilic
body (arrow). Periodontal ligament (PL). TUNEL ⁄ TRAP combined methods. Bar: 5 lm. (G) Osteoclast (Oc) in contact with the bone surface (B)
shows shrunken cytoplasm immunolabelled by caspase-3 (brown-yellow colour). Nuclei (N). Immunohistochemistry for caspase-3 detection
counterstained with haematoxylin. (H–J) Sections of portion of alveolar bone submitted to immunohistochemistry for vimentin detection and
counterstained with haematoxylin. (H) Multinucleated osteoclast (Oc) located in an excavation of the bone (B) surface exhibits immunostained
cytoplasm for vimentin (brown-yellow colour), mainly around the nuclei (N) (arrows) and in ruffled border area (arrowheads). Bar: 5 lm. (I, J)
Osteoclasts (Oc) showing nuclei (N) strongly stained by haematoxylin and apparently shrunken cytoplasm immunolabelled by vimentin (brown-
yellow colour); note that the vimentin positivity is spread throughout the cytoplasm. Bar: 5 lm. (I) A mononucleated vimentin-positive structure (S)
is in close juxtaposition to the osteoclast (Oc). Bone (B). Bar: 5 lm.

immunolabelled by caspase-3 (Fig. 1G). In CG, multinu- trol groups (CG and SG). Otherwise, large osteoclasts
cleated osteoclasts located in excavations of the bone sur- (‡ 800.00 lm2) were only found in CG and SG (Fig. 2A).
face exhibited vimentin-positive cytoplasm, mainly around In EG, 48.7% of the total number of TRAP-positive cells
the nuclei and in the ruffled border area (Fig. 1H). In EG, exhibited only one nucleus (Fig. 2B). Moreover, the
osteoclasts with condensed chromatin showed vimentin im- frequency of multinucleated osteoclasts in CG and SG was
munoreaction spread throughout the cytoplasm (Fig. 1I–J). significantly higher than in EG. It should be noted that
In comparison with CG and SG, the morphometric analy- osteoclasts with more than six nuclei were found only in CG
ses revealed significant reduction in the OcA, OcN and and SG.
BS ⁄ Oc in EG. No differences were found between CG and The analysis of the length of bone resorption surface
SG (Table 1). (BS ⁄ OC) revealed that BS ⁄ Oc varied from 3.0 to 118.0 lm in
The frequency distribution of OcA revealed a significant CG and SG, and from 3.0 to 71.0 lm in EG. Moreover, the
increase of small osteoclasts in EG in comparison with con- frequency of small BS ⁄ Oc in EG was significantly higher

ª 2011 The Authors

Journal of Anatomy ª 2011 Anatomical Society of Great Britain and Ireland
 Alveolar bone osteoclasts in estrogen-treated rats, A. P. S. Faloni et al. 81

Table 1 Mean ± SD of area of osteoclast (OcA), number of nuclei A 90

CG
(OcN) and the length of the resorption bone surface per osteoclast 80
70 *** SG
(BS ⁄ Oc) in the Control (CG), Sham (SG) and Estrogen (EG) Groups.

Frequency (%)
60 EG
50
Groups OcA (lm2) OcN BS ⁄ Oc (lm)
40
***
30
CG 317 ± 43 2.7 ± 0.3 30 ± 2.8
20
SG 289 ± 47 2.7 ± 0.3 29 ± 1.9
10
EG 186 ± 171,2 2.1 ± 0.13 21 ± 2.04 0
20.00–199.99 200.00–799.99 800.00–1746.00
1
P < 0.01 vs. SG. Area (µm2)
2
P < 0.001 vs. CG.
3
P < 0.01 vs. CG and SG. **
4
P < 0.001 vs. CG and SG (Tukey’s test). ***
B 90
80
CG
70 ** SG

Frequency (%)
than in CG and SG. In contrast, large BS ⁄ Oc were not found 60 *** EG
in the EG (Fig. 2C). 50
The immunohistochemistry for MMP-9 detection showed 40
30
that multinucleated osteoclasts from the SG (Fig. 3A) and
20
EG (Fig. 3B) located in close contact with bone surface 10
exhibited MMP-9-positive cytoplasm. Quantitative analysis 0
Mononucleated Multinucleated
of the frequency of immunolabelled osteoclasts revealed
TRAP-positive cells
that the differences between the groups were not signifi-
cant (EG: 82.7 ± 12.0%; CG: 86 ± 5.6% and SG:
C 90 CG
75.8 ± 6.1%). However, in the EG, multinucleated MMP-9- 80
* SG
positive osteoclasts of varied sizes showed some nuclei with 70
Frequency (%)

60 EG
peripheral chromatin strongly stained by haematoxylin
50
(Fig. 3C–F). Next to these osteoclasts, some MMP-9-positive
40
structures exhibiting nuclei strongly stained by haematoxy- 30
lin were observed (Fig. 3F). The MMP-9 ⁄ TUNEL combined 20
methods revealed MMP-9-immunolabelled osteoclasts with 10
strongly TUNEL-positive nuclei (Fig. 3G). 0
3.00–22.99 23.00–70.99 71.00–118.00
Under transmission electron microscope, typical osteo- Bone resorption surface/osteoclast (µm)
clasts in close contact with the bone surface exhibited a ruf-
fled border, numerous mitochondria, as well as vesicles and Fig. 2 Frequency (%) of osteoclasts according to area (OcA) (A),
vacuoles of varied sizes in the cytoplasmic area next to the number of nuclei (OcN) (B) and bone resorption surface per osteoclast
ruffled border (Fig. 4A). In EG, some osteoclasts exhibited (BS ⁄ Oc) (C) in the Control (CG), Sham (SG) and Estrogen (EG) groups.
(A) The OcA was classified into: small (20.06–199.99 lm2), medium
apparently shrunken cytoplasm and tortuous nuclei with
(200.00–799.99 lm2) and large (800.00–1746.00 lm2). For the first
conspicuous masses of peripheral chromatin. Usually, ruf-
and second categories, P < 0.05 (one-way ANOVA). ***P < 0.001 vs.
fled borders and the clear zones were not found in these CG and SG (Tukey’s test). (B) OcN was divided into: mononucleated
altered osteoclasts. The analysis of semi-serial sections (one nucleus) and multinucleated (two or more nuclei). For both
showed that cytoplasmatic portions seemed to be shed categories, P < 0.01 (one-way ANOVA). ***P < 0.001; **P < 0.01 vs.
from these altered osteoclasts; sometimes, these osteoclast CG and SG (Tukey’s test). (C) BS ⁄ Oc were classified into: small (3.00–
portions exhibited irregular-shaped nucleus with blocks of 22.99 lm), medium (23.00–70.99 lm) and large (71.00–118.00 lm).
For the first category, P < 0.05 (one-way ANOVA). *P < 0.05 vs. CG and
condensed chromatin (Fig. 4B,C).
SG (Tukey’s test).

Discussion
and activity has not been clarified. In the present study, the
Reduction in the number of alveolar bone osteoclasts in images showing TUNEL-positive nuclei in the TRAP-positive
estrogen-treated young female rats has been associated osteoclasts in addition to the presence of caspase-3
with death of osteoclasts by apoptosis (Faloni et al. 2007; immunoexpression and the typical apoptotic ultrastructural
Cruzoé-Souza et al. 2009). Enhanced immunoexpression of features, confirmed that estrogen induces apoptosis (Faloni
estrogen receptors (Erb) in the apoptotic osteoclasts sug- et al. 2007; Cruzoé-Souza et al. 2009). Moreover, the struc-
gests that estrogen may act directly on osteoclasts by con- tural integrity of the osteoclasts and the bone resorptive
trolling their survival (Cruzoé-Souza et al. 2009). However, surface were significantly altered, probably due to estro-
the effect of estrogen on the osteoclast structural integrity gen-induced cell death by apoptosis.

ª 2011 The Authors

Journal of Anatomy ª 2011 Anatomical Society of Great Britain and Ireland
82 Alveolar bone osteoclasts in estrogen-treated rats, A. P. S. Faloni et al.

MMP-9 MMP-9
PL N PL

Oc
Oc

N
RB

3A
A B B
3B B

MMP-9 Ca MMP-9 MMP-9

PL
N PL Oc
N

B
Oc
N

B
B B
C
3C D
3D E
3E Ot

MMP-9 MMP-9/TUNEL
S PL
N
S Oc PL
Oc
N

F G
B

Fig. 3 Light micrographs of portions of alveolar bone (B) of sham (A) and estrogen-treated rats (B–G). (A–F) Portions of alveolar bone (B)
submitted to immunohistochemistry for MMP-9 detection (brown-yellow colour) and counterstained with haematoxylin. In (A, B) multinucleated
osteoclasts (Oc) located in excavations of the bone surface (B) exhibit MMP-9-positive cytoplasm. Nuclei (N). Ruffled border (RB). Periodontal
ligament (PL). (C) A multinucleated osteoclast (Oc), showing MMP-9 immunostaining in the cytoplasm, exhibits two nuclei with peripheral
chromatin strongly stained by haematoxylin (arrows). Bone (B). Nuclei (N). Blood capillary (Ca). (D,E) Osteoclasts (Oc) apparently reduced show only
three (D) or two (E) nuclei (N); note that a nucleus (arrow) exhibits peripheral condensed chromatin. Osteocyte (Ot). Periodontal ligament (PL). (F)
Strongly MMP-9-positive osteoclast (Oc) exhibits nuclei (N) with condensed chromatin. MMP-9-positive structures (S) containing strongly basophilic
chromatin are next to the osteoclast (Oc). Periodontal ligament (PL). (G) Portion of alveolar bone (B) submitted to the combined
immunohistochemistry for MMP-9 detection (brown-yellow colour) and TUNEL method (purple colour) counterstained with methyl green. A,
MMP-9 immunolabelled osteoclast (Oc) shows strongly TUNEL-positive nuclei (N). Periodontal ligament (PL). Bar: 5 lm.

Osteoclast apoptosis involves caspase-3, which has been the frequency of each category was compared among the
shown to cleave vimentin, inducing its disruption and col- groups, it was possible to understand better how estrogen
lapse of the cytoskeleton in many cell types (Byun et al. affects osteoclast structure and function. In EG, most of the
2001; Lee et al. 2002; Saintier et al. 2006; Alam et al. 2010). TRAP-positive cells (about 68%) were small osteoclasts;
A cytoskeleton containing vimentin filaments distributed large osteoclasts were not found. Moreover, in the EG,
around the nuclei and in the ruffled border region has about 50% of these TRAP-positive cells were mononucle-
been described in osteoclasts (Akisaka et al. 2008). In the ated, supporting in vitro findings (Saintier et al. 2006). It is
dying osteoclasts of the estrogen-treated group, the vimen- not possible to exclude the hypothesis that these mono-
tin filaments were distributed throughout the cytoplasm, nucleated cells are osteoclast precursors that fail to fuse
filling the entire cytosol, as previously shown in dying HeLa during osteoclastogenesis, probably due to the estrogen
cells (Lee et al. 2002). Therefore, the disarrangement of the action on the RANK ⁄ RANKL ⁄ OPG [receptor activator of
structural integrity of vimentin filaments in the apoptotic nuclear factor (NF)-jB ⁄ RANK ligand ⁄ osteoprotegerin]
osteoclasts may be due to a possible caspase activation. system (Kawamoto et al. 2002; Rogers et al. 2002; Bord
When the data regarding the morphometric parameters et al. 2003; Kanzaki et al. 2006). However, it is important to
(OcA, OcN and BS ⁄ Oc) were distributed into categories and note that either the multinucleated osteoclasts or the

ª 2011 The Authors

Journal of Anatomy ª 2011 Anatomical Society of Great Britain and Ireland
 Alveolar bone osteoclasts in estrogen-treated rats, A. P. S. Faloni et al. 83

Sham group apoptosis. During apoptosis, osteoclasts undergo shrinkage

and blebbing, shedding cellular fragments containing TUN-
EL-positive nuclei. The TUNEL method is used to detect the
Oc PL DNA fragmentation which occurs in apoptosis (Gavrieli
et al. 1992; Cerri et al. 2003; Cerri, 2005; Faloni et al. 2007).
Ca
N
It is conceivable that the increase in the frequency of med-
ium and of mainly small osteoclasts and the significant
increase in the number of mononucleated TRAP-positive
M
N cells may be due to shrinkage and fragmentation of osteo-
Va clasts during the process of apoptosis. The ultrastructural
images confirm that mononucleated cells containing one
N apoptotic nucleus are probably shed from multinucleated
RB
apoptotic osteoclasts. Subsequently, these mononucleated
Ve
cells undergo fragmentation, probably giving rise to TRAP-
A B
positive round and ⁄ or ovoid structures containing irregular
masses of dense chromatin. These structures, probably
Estrogen group
apoptotic bodies, were sometimes partially surrounded by
cells of periodontal ligament, apparently engulfed by fibro-
blast-like cells.
N
The apoptotic features observed in the osteoclasts of
estrogen-treated rats are consistent with previous studies
Oc
N under experimental conditions. TUNEL-positive osteoclasts
S
as well as disappearance of ruffled borders and clear zones
B were also observed in apoptotic osteoclasts of adult rats
B
treated with bisphosphonate (Ito et al. 1999, 2001).
Estrogen group In the present study, the use of pre-pubertal female rats
(sexually immature rats) raises the question of whether the
morphologic and enzymatic changes in the estrogen-
N induced apoptotic osteoclasts could be due to a pharmaco-
logical ⁄ toxic response rather than a physiological response.
S N However, enhanced Erb immunolabelling was observed in
Oc
the osteoclasts of pre-pubertal female rats treated with
estrogen (Cruzoé-Souza et al. 2009), indicating that exoge-
C B nous estrogen is able to stimulate the expression of estro-
gen receptors in these cells. It is important to emphasize
Fig. 4 Electron micrographs of portions of alveolar bone (B) of sham that both uterine horns were clearly enlarged in the estro-
(A) and estrogen-treated (B,C) rats. (A) A typical osteoclast (Oc) is in gen-treated animals in comparison with the non-treated
close contact with the bone surface (B). The osteoclast (Oc) exhibits pre-pubertal rats. This effect confirms that the estrogen
numerous mitochondria (M), vesicles (Ve) and vacuoles (Va) of varied levels (exogenously administered) are able to induce a
sizes located next to ruffled border (RB). Nuclei (N). Periodontal
physiological response in an estrogen-dependent tissue of
ligament (PL). Blood capillary (Ca). Bar: 2 lm. (B,C) Electron
pre-pubertal female rats. There is evidence that accentuated
micrographs of a portion of alveolar bone (B) of estrogen-treated rat
showing semi-serial sections of an osteoclast (Oc). The osteoclast levels of a determined hormone promote hormonal cell
exhibits apparently shrunken cytoplasm and tortuous nuclei (N) with sensibility due to an increased number of receptors
conspicuous masses of peripheral chromatin (arrowheads). The semi- (Rhoades & Tanner, 1995). In the prostate of newborn male
serial sections show that a structure (S) seem to be shedding from the rats, treatment with diethylstilbestrol induces an accentu-
altered osteoclast (Oc); this osteoclast structure (S) exhibits irregular- ated expression of estrogen receptors (Khurana et al. 2000).
shaped nucleus (N) with blocks of condensed chromatin (arrowheads).
Thus, the changes induced by estrogen in the alveolar bone
Bar: 2 lm.
osteoclasts may be related to a physiological response. This
is reinforced by the fact that similar morphological changes
mononucleated cells were MMP-9-positive in EG. According in osteoclasts were also observed under physiological condi-
to Sorensen et al. (2007), mature or active multinucleated tions. In the osteoclasts of female rats during the peak of
osteoclasts express MMP-9, whereas mononucleated osteo- estrogen, immediately following lactation, the high estro-
clasts precursors do not. Our results indicate that the mono- gen levels coincided with the disappearance of osteoclast-
nucleated and ⁄ or small osteoclasts (two to three nuclei) ruffled borders, retraction of the osteoclasts from the bone
observed in EG are derived from the dying osteoclasts by surface, cellular fragmentation and apoptosis (Miller &

ª 2011 The Authors

Journal of Anatomy ª 2011 Anatomical Society of Great Britain and Ireland
84 Alveolar bone osteoclasts in estrogen-treated rats, A. P. S. Faloni et al.

Bowman, 2007). However, further studies focusing on a nucleated portions derived from the apoptotic osteoclast
comparative analysis between the results of this study and undergo fragmentation, probably giving rise to TRAP-posi-
physiological conditions (i.e. adult female rats in estrus) tive apoptotic bodies, which seem to be engulfed by neigh-
would be useful. bouring cells.
There is evidence that osteoclast size is also directly associ-
ated with its resorptive activity (Lees & Heersche, 2000; Lees
Acknowledgements
et al. 2001). The bone surface in close juxtaposition to
osteoclast has been used to estimate the osteoclast activity The authors wish to thank Mr. Luis Antônio Potenza and Mr.
(Chow et al. 1992). Our findings revealed that the signifi- Pedro Sérgio Simões for technical support. This research was
cant reduction in the osteoclast area was accompanied by a supported by CAPES, CNPq, FUNDUNESP and FAPESP (04 ⁄ 09898-
0) – Brazil.
reduction in bone resorption surface. Our results indicate,
therefore, that the resorptive activity of alveolar bone
osteoclasts decreases in estrogen-treated rats. References
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Journal of Anatomy ª 2011 Anatomical Society of Great Britain and Ireland