ORIGINAL ARTICLE
Initial changes in alveolar bone volume for sham-operated
and ovariectomized rats in ligature-induced experimental
periodontitis
Jing Dai
1
& Yihui Ma
1
& Miusi Shi
1
& Zhengguo Cao
1
& Yufeng Zhang
1,2
&
Richard J. Miron
3
Received: 18 November 2014 /Accepted: 3 July 2015 /Published online: 17 July 2015
#
Springer-Verlag Berlin Heidelberg 2015
Abstract
Objectives Osteoporosis is a disease characterized by a reduc-
tion in bone mass, poor bone strength, and microarchitectural
deterioration primarily in postmenopausal women. With re-
spect to periodontal disease, osteoporosis is thought to con-
tribute to pre-existing alveolar degeneration although the as-
sociation between both diseases is not fully characterized. The
aim of the present study was to observe the initial changes in
mandibular alveolar bone for sham-operated and ovariecto-
mized (OVX) rats in ligature-induced experimental
periodontitis.
Materials and methods A total of 64 Wistar rats (7 weeks of
age, 180–200 g) were used in this study (32 control sham-
operated animals+ligature placement, 32 OVX animals+lig-
ature placement). Following an 8-week period to induce an
OVX model, micro-CT analysis was performed to calculate
vertical and furcation bone loss of mandibular first molars at
time points 0, 3, 7, and 11 days following ligature placement
(six animals per group per time point). Furthermore, histolog-
ical analysis was performed to calculate the loss of alveolar
bone crest height from the cemento-enamel ju nction, and
tartrate-resistant acid phosphatase (TRAP) staining was uti-
lized to calculate the number of osteoclasts.
Results The results from the present study demonstrate that
OVX animals showed significant vertical bone loss at all time
points when compared to control sham-operated animals. In
the furcation area, no significant difference in bone loss was
observed between sham-operated and OVX animals at 0, 3,
and 7 days; however by 11 days, a significant decrease in bone
volume/total volume and trabecular thickness was observed in
the OVX group. The histological analysis also revealed that
alveolar bone crest height was significantly reduced in OVX
animals, and TRAP staining further revealed the greater num-
ber of multinucleated osteoclasts peaking at 3 days
postligature placement.
Conclusion The results from the present study demonstrate a
direct correlation between the osteoporotic phenotype and the
progression of periodontal breakdown in a diseased-induced
animal model.
Clinical relevance It may be suggested that an osteoporotic
phenotype has the potential to speed periodontal breakdown
and thus contributes to the overall degeneration of the peri-
odontium in patients suffering from postmenopausal bone
loss. Future research from human clinical studies are neces-
sary to further understand the relationship between periodon-
tal disease and osteoporosis.
Keywords Osteoporosis
.
Periodontal disease
.
Periodontitis
.
Ovariectomy
Introduction
Osteoporosis is a worldwide health care issue characterized by
reduction in bone mass, poor bone strength, and
microarchitectural deterioration in trabecular and cortical
* Yufeng Zhang
zyf@whu.edu.cn
1
The State Key Laboratory Breeding Base of Basic Science of
Stomatology (Hubei-MOST) and Key Laboratory of Oral
Biomedicine Ministry of Education, School and Hospital of
Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079,
People’s Republic of China
2
Department of Oral Implantology, School of Stomatology, Wuhan
University, Wuhan 430079, China
3
Department of Periodontology, University of Bern,
Bern, Switzerland
Clin Oral Invest (2016) 20:581–588
DOI 10.1007/s00784-015-1531-3
bone. The disease is known to progress with increasing age,
and the primary cause is thought to be derived by an imbal-
ance between bone-forming osteoblasts and bone-resorbing
osteoclasts due primarily from postmenopausal estrogen defi-
ciencies [1–5]. Over 200 million people are now estimated to
be suffering from the disease worldwide with the great major-
ity being white or Asian women over the age of 65 [6]. Com-
monly utilized pharmacological agents for the treatment of
osteoporosis include anabolic agents capable of increasing
bone formation such as parathyroid hormone and the use of
anti-resorbing agents such as bisphosphonates, calcitonin,
RANKL inhibitors, raloxifene, and estrogen replacement ther-
apy [7]. Despite these many options, there exist no single
option capable of fully restoring lost bone, and the use of
bisphosphonate therapy (the leading treatment options for pre-
vention of bone mass in osteoporotic patients) is associated
with a number of side effects including halting the bone re-
modeling cycle.
Interestingly, growing clinical evidence has suggested that
a relationship between osteoporosis and periodontal disease
exist [8–14]. In one of the first well-documented studies in-
vestigating this relationship, it was found that osteoporosis
had no significant effect with respect to plaque and gingival
bleeding but was associated with a significantly greater loss of
attachment in osteoporotic women [10]. Passos et al. com-
pared different diagnostic criteria for periodontal disease that
were used to study the association between periodontal dis-
ease and osteoporosis [8]. The results from their work empha-
size that the frequency of periodontal disease may be influ-
enced by different outcome measurements associated between
osteoporosis and periodontal disease. More recently, Takahasi
et al. studied the association between periodontitis and sys-
temic bone mineral density in Japanese community-dwelling
postmenopausal women [13]. They evaluated the association
between loss of attachment and bone mineral density (BMD)
of the lumbar vertebrae and femur in 374 postmenopausal
women aged 55–74 [13]. The results from this study demon-
strated that a significant negative correlation was observed
between periodontal disease and truncal bone BMD further
confirming the relationship between osteoporosis and peri-
odontal disease [13]. Further research groups studying popu-
lations from Jordan [12], India [9], and USA [11]demonstrate
various relationships between periodontal disease and osteo-
porosis. Noteworthy, while a positive correlation is observed
in some studies, other studies have failed to demonstrate either
a relationship exists or have concluded that osteoporosis is not
a causal factor in the development of mod erate to severe
chronic periodontitis [15, 16].
It may thus be concluded that the relationship between
periodontal disease and osteoporosis is one area of research
needing a more developed understanding. It is believed that
osteoporosis is able to contribute to periodontal breakdown
given that it may increase bone resorption and prevent proper
healing ultimately increasing the severity of the pre-
existing periodontal disease; however, very little evi-
dence is available demonstrating such a relationship.
Therefore, the aim of the present study was to investi-
gate with an animal model the relationship between
periodontal disease progression and osteoporosis. An
ovariectomized rat was chosen as an osteoporosis mod-
el, and periodontal progression was induced by ligature
placement of the rat mandibular first molars. Micro-CT
and histological assessment were performed at 0, 3, 7,
and 11 days fol lowing ligatur e placement in sham-
operated and ovariectomized (OVX) animals to assess
the rate of bone loss.
Materials and methods
Animal experiments
Sixty-four female Wistar rats (7 weeks of age, 180–200 g)
were used in this study. At the start of the experiment,
no clinical signs of gingival inflammation were observed
among the animals. The animals were housed in individ-
ual wire cages in a temperature- and humidity-controlled
room (23±1 °C and 60±5 % relative humidity) with a
12-h light/dark cycle as previously described [17]. All
animals were allowed to acclimate to the laboratory en-
vironment for 1 week before surgical procedures were
carried out. For 32 of the 64 animals, ovariectomy
(OVX) surgery was performed at 8 weeks of age as pre-
viously described [18, 19]. Br iefly, general a nesthesia
was achieved by intraperitoneal injection of chloral hy-
drate (10 %, 4 ml/kg body weight); the rats were oper-
ated with a 10-mm linear bilateral lumbar lateral skin
incisions. Then, the enterocoelia was exposed by blunt
dissection of muscle and peritoneum. T he bilateral ova-
ries were removed gently following ligation of the ovar-
ian artery and vein. Then, the small incisions were su-
tured stratified. Postoperatively, penicillin ( 40,000 IU/m l,
1 ml/kg) was injected for 3 days, and there was no sign
of inf lammation or other notable anomaly. Thirty-two con-
trol animals were subject to sham operations as previously
described [20].
At 8 weeks following the surgery, cotton thread ligatures
(no. 3-0) were tied around the cervixes of the bilateral first
mandibular molar (M1) under anesthesia with chloralic hydras
for all 64 rats. At time points 0, 3, 7, and 11 after ligature
placement, 12 animals were euthanized (six control sham-
operated animals with ligatures and six OVX animals with
ligatures). The mandible was harvested and fixed in 10 %
formalin solution for 48 h. After fixation, alveolar bone was
examined using a micro-CT scanner and embedded in paraffin
for histological analysis.
582 Clin Oral Invest (2016) 20:581–588
μCT scanning and analysis
A μCT imaging system (μCT50, Scanco Medical, Basersdorf,
Switzerland) was used to scan the mandibular molars and
alveolar bone of all animals. Scanning was performed at
70 kV and 114 μA with a thickness of 15 μm per slice in
medium-resolution mode and 30 0-ms integration time. A
Gaussian filter (sigma=0.8 and support=1) was used to re-
move noise. Vertical bone loss was evaluated by measuring
the distance between the cemento-enamel junction (CEJ) and
the alveolar bone crest (ABC) at six points (lingual-mesial,
lingual-middle, lingual-distal, buccal-mesial, buccal-middle,
buccal-distal) of the first mandibular molar. The architectures
of interradicular bone of the first molar were evaluated by
means of 3D reconstructio n. An area with a h eight of
2.4 μm was defined as the 3D regions of interest (ROIs),
starting coronally by a 2D slice 15 μm below the furcation
of the first molar. Bone volume fraction was calculated from
the bone volume (BV) and total volume (TV) as BV/TV. Tra-
becular thickness (Tb.Th) was calculated by applying the dis-
tance transformation by filling maximal spheres in the bone
structures as determined by the micro-CT supplier protocol.
Histological analysis and tartrate resistant acid
phosphatase stain
After fixation, the mandibles were decalcified in 10 % EDTA,
dehydrated, embedded in paraffin, sectioned to 4-μmthick-
ness, and stained with hematoxylin and eosin (H&E) as pre-
viously described [21]. Vertical alveolar bone loss of the first
mandibular molar were assessed for inflammation and alveo-
lar bone destruction as assessed by measuring the length (mm)
of the CEJ to ABC between the first and second mandibular
molars.
Then, tartrate-resistant acid phosphatase (TRAP) (Sigma
#387A; Sigma-Aldrich, St. Louis, USA) was performed to
determine osteoclast activity as previously described [21].
Analysis of the furcation area was assessed for the presence
of osteoclasts as defined by cells with greater than three nuclei
and counted at the various time points as previously described
[22]. Samples were assessed for their staining intensity with
respect to alveolar bone loss adjacent to the periodontal liga-
ment in the furcation area along the PDL-bone perimeter.
Statistical analysis
All data analysis was performed using SPSS software and
statistically significant values were adopted as p<0.05. All
experiments were displayed as mean and standard deviation
(SD) and were calculated by one-way ANOVA and Newman
Keuls t test.
Results
Micro-CT analysis
Micro-CT was performed to analyze the rate of alveolar bone
loss in both sham-operated and OVX animals (Fig. 1,64total
animals, six per group per time point). At day 0, the roots
embedded within alveolar bone demonstrate no differences
in the vertical bone loss as assessed by micro-CT (Fig. 2).
Furthermore, cross-sectional analysis from the micro-CT im-
ages reveal large areas of bone in both sham-operated (Fig. 1e)
and OVX groups (Fig. 1m) with no differences in furcation
bone loss between sham-operated and OVX groups (Fig. 3).
At 3 days following placement of the ligature on the first
mandibular molar, small noticeable changes were observed
specifically on the distal root where the micro-CT images
begin to demonstrate slight bone loss (Fig. 1b, i). Significantly
greater vertical bone loss was observed between OVX and
sham-operated animals, and this observation was maintained
throughout the remaining study time points (p<0.01, Fig. 2).
At both 3 and 7 days, no significant differences in furcation
bone loss were observed (Fig. 3). By 11 days, micro-CT im-
ages depict significantly greater bone loss in both vertical
height and within the furcation in the OVX group (p<0.01,
Figs. 1l, 2,and3) when compared to sham-operated animals
(Fig. 1d). Cross-sectional analysis from micro-CT further re-
veals the loss of bone volume in the furcation area (Fig. 1p),
and computation analysis from the micro-CT demonstrated
that BV/TVas well as trabecular thickness is now significantly
reduced in the furcation area in OVX animals when compared
to control sham-operated animals (p<0.01, Fig. 3).
Histological analysis
Histological analysis was performed in the furcation areas of
first mandibular molars to further confirm the rate of bone loss
between OVX and sham-operated animals as well as to assess
osteoclast activity via TRAP staining. The histological analy-
sis demonstrates that both sham-operated and OVX animals
show healthy bone in furcation areas with little to no changes
between groups (Fig. 4a, e). At 3 days postligature placement,
noticeable changes in the morphology of trabecular bone be-
came apparent with loss of bone beginning in the furcation
area in both sham-operated and OVX animals demonstrating
the effectiveness of the ligature-induced periodontitis model
(Fig. 4b, f). At 7 days, this trend was also consistent with more
bone loss taking place in the OVX group (Fig. 4c, g). By
11 days, a significantly greater amount of bone was lost in
the furcation areas between OVX and sham-operated groups
further confirming the micro-CT results (Fig. 3).
Then, histological quantification was used to measure the
rate of vertical bone loss by calculating the distance between
the CEJ and the ABC between the first and second mandibular
Clin Oral Invest (2016) 20:581–588 583
molars (Fig. 5). The histological measures further confirm
micro-CT results by demonstrating a significantly greater loss
of vertical bone at 3, 7, and 11 days postligature placement
(p<0.05, Fig. 5). TRAP staining was then utilized to assess
the involvement of osteoclast activity (Fig. 6). It was observed
that at 3 days following ligature placement, osteoclast activity
peaked with a significantly higher number of multinucleated
cells and staining intensity observed in the OVX group when
compared to control sham-operated samples at all time points
(p<0.01, Fig. 6c).
Discussion
The aim of the present study was to investigate the relation-
ship between osteoporosis and periodontal disease. There
exists accumulating evidence that a relationship may exist
[8–14]; however to date, no animal or histological study has
fully confirmed this possible relationship. Therefore, we
sought to utilize an animal model to perform histological as-
sessment and to characterize alveolar bone loss in an osteopo-
rotic model by creating OVX animals and comparing them to
control sham-operated animals for periodontal changes from 0
to 11 days following a ligature-induced periodontitis animal
model.
The animal model chosen was selected as it has a long
history of being utilized to examine periodontal change in
periodontitis [23–25]. It is noteworthy to mention that in the
present study, periodontal breakdown was calculated by eval-
uating alveolar bone loss as the key changes associated be-
tween mucositis and periodontitis are the loss of alveoloar
bone and the direct clinical attachment loss was not calculated.
Instead, the distance between the CEJ and the alveolar bone
crest were measured to represent this parameter. The model
was confirmed as the rate of periodontal change even in sham-
operated animals was significantly altered following the
placement of the ligature on the first mandibular molar thus
demonstrating its effectiveness as a study model. It was ob-
served that within only 3 days, bone loss had occurred in both
control sham-operated and OVX animals confirming the fast
rate of bone loss in the present animal model (Fig. 2). The
group containing OVX animals without a ligature-induced
model was not considered as the rate of periodontal break-
down is thought to be insignificant. As the OVX animals are
left for 8 weeks following removal of their ovaries with no
bone loss present, including an additional group allowing an
additional 10 days of induced OVX rats was not considered.
At day 0, no difference in alveolar bone loss or tissue inflam-
mation was apparent in OVX animals when compared to
Fig. 1 μCT images of alveolar bone loss in both sham-operated and OVX animals at 0, 3, 7, and 11 days following ligature-induced periodontitis (bar=
1 mm). Lingual view (a, b, c, d, i, j, k,andl) and cross sections (e, f, g, h, m, n, o,andp) of mandibles were observed for both groups
Fig. 2 Representative bar graph demonstrating vertical bone loss of the
first mandibular molars as assessed by μCT images. Significantly greater
vertical bone loss is observed in OVX animals at 3, 7, and 11 days
postligature-induced peri odontitis. Data are expressed as mean ±
standard deviation (mm) (*p<0.05)
584 Clin Oral Invest (2016) 20:581–588
control sham-operated animals. Including an additional 32
animals to the experimental design was thus not warranted
as the rate of periodontal breakdown and alveolar bone loss
is considered to be negligible. The changes occurring in the
present model are apparent once the ligature-induced state was
placed with more alveolar bone loss taking place in OVX
animals.
The results from the present study also demonstrate
the severe compli cation associa ted with the progression
of both diseases. Although the present results are drawn
from an animal model with a much shorter life expectan-
cy than humans, the investigation demonstrates how
quickly the degeneration of periodontal tissues may arise
with a simultaneous combination of both periodontal dis-
ease and osteoporosis. It is known that periodontal dis-
ease is thought to contribute to osteoclast activation
[26–28] and the combination with a disease such as os-
teoporosis known to cause a hyper-activity of osteoclasts
is subject to pose many future problems. Furthermore, it
is known t hat both osteoporosis and periodontal disease
are age-related diseases and the increasing life expectan-
cy in humans i s thought to contribute to both diseases.
As patients become older, bone loss tends to increase
with li ttle therapy available to r e-establish proper bone
volume. Similarly, as patient age increases, their dexter-
ity decreases making it increasingly difficult to brush
their teeth efficiently and maintain proper oral hygiene.
Thus, the combination of these two age-related disorders
is one that necessitates f ull clinical attention and involve-
ment as patients reach the later years of their lives.
We also studied the time necessary to reach the
tooth furcation, an area specifically located betwe en
Fig. 3 a Bone volume/total volume and b trabecular thickness of
alveolar bone located within the furcation area of the first mandibular
molar. While no significant difference was observed between 0 to
7 days however at 11 days postligature-induced periodontitis, both BV/
TV and trabecular thickness were significantly reduced in OVX animals
when compared to control. Data are expressed as mean ± SD (mm)
(*p<0.05)
Fig. 4 Representative sections of
H&E staining demonstrate
periodontal breakdown of sham-
operated (sham, a–e)and
ovariectomized (OVX, f–j)ratsat
0, 3, 7, and 11 days postligature-
induced peirodontitis. More
alveolar bone loss is observed in
the OVX group at 11 days as
depicted by the loss of bone in the
furcation area of first mandibular
molars (j). Black scale bar:
200 μm
Clin Oral Invest (2016) 20:581–588 585
