Oxytocin promotes bone formation during the alveolar
healing process in old acyclic female rats

Vilma Clemi Colli a,*, Roberta Okamoto b, Poli Mara Spritzer c,
Rita Cássia Menegati Dornelles d

aMulticentric Graduate Studies Program in Physiological Sciences – SBFis/UNESP – Univ Estadual Paulista, Araçatuba, São Paulo, Brazil
bDepartment of Research and Post Graduation, Universidade Sagrado Coração – USC Bauru, SP, Brazil
cDepartment of Physiology, Universidade Federal do Rio Grande do Sul, Porto Alegre/RS, Brazil
dDepartment of Basic Sciences, Araçatuba Dental School, UNESP – Univ Estadual Paulista, Araçatuba, São Paulo, Brazil

a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 1 2 9 0 – 1 2 9 7

a r t i c l e i n f o

Article history:

Accepted 31 March 2012

Keywords:

Bone regeneration

Oxytocin

Tooth extraction

Aging

a b s t r a c t

Objective: OT was reported to be a direct regulator of bone mass in young rodents, and this

anabolic effect on bone is a peripheral action of OT. The goal of this study was to investigate

the peripheral action of oxytocin (OT) in the alveolar healing process in old female rats.

Materials and methods: Females Wistar rats (24-month-old) in permanent diestrus phase,

received two ip (12 h apart) injections of saline (NaCl 0.15 M – control group) or OT (45 mg/rat

– treated group). Seven days later, the right maxillary incisor was extracted and analyses

were performed up to 28 days of the alveolar healing process (35 days after saline or OT

administration).

Results: Calcium and phosphorus plasma concentrations did not differ between the groups.

The plasma biochemical bone formations markers, alkaline phosphatase (ALP) and osteo-

calcin were significantly higher in the treated group. Histomorphometric analyses con-

firmed bone formation as the treated group presented the highest mean value of post-

extraction bone formation. Tartrate-resistant acid phosphatase (TRAP) was significantly

reduced in the treated group indicating an anti-resorptive effect of OT. Immunohistochem-

istry reactions performed in order to identify the presence of osteocalcin and TRAP in the

bone cells of the dental socket confirmed these outcomes.

Conclusions: OT was found to promote bone formation and to inhibit bone resorption in old

acyclic female rats during the alveolar healing process.

Published by Elsevier Ltd.

Available online at www.sciencedirect.com

journal homepage: http://www.elsevier.com/locate/aob
1. Introduction

Postmenopausal osteoporosis is a systemic skeletal disease

resulting in increased bone fragility and fracture risk subse-

quent to a decrease in bone mass and the degradation of bone

microarchitecture.1,2 Despite the availability of several treat-

ment options, annual hip fracture rates are predicted to
* Corresponding author at: Department of Basic Sciences, Physiology L
527, University Campus, Building 31, Araçatuba, São Paulo 16018-805

E-mail address: vilma@foa.unesp.br (V.C. Colli).

0003–9969/$ – see front matter. Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.archoralbio.2012.03.011
exceed six million by 2050 and this is a question of scientific,

social and economic importance.1,3,4 Additionally, there are

important age-related changes in the maxillofacial skeleton,

particularly in association with tooth loss and periodontal

disease. Osteoporosis and periodontal disease increase with

age and postmenopausal women without hormone replace-

ment therapy had the highest maxillary implant failure rate.5

Bone metabolism combines bone resorption (osteoclasts) and
aboratory – Univ Estadual Paulista, Rodovia Marechal Rondon Km
, Brazil. Tel.: +55 18 36362757.

http://dx.doi.org/10.1016/j.archoralbio.2012.03.011
mailto:vilma@foa.unesp.br
http://www.sciencedirect.com/science/journal/00039969
http://dx.doi.org/10.1016/j.archoralbio.2012.03.011


a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 1 2 9 0 – 1 2 9 7 1291
formation (osteoblasts) synchronously. With ageing in

humans, this balance is impaired and is mainly related to

the decrease in the secretion of ovarian steroids, due to

significant modulation of the remodelling cycle exerted by

oestrogen.6,7 Thus, the aim of treatments for osteoporosis is to

increase the density, by acting on the decrease in bone

resorption or increase in bone formation.4,8,9

Studies have been developed to find new bone formation

therapies and oxytocin (OT), a primitive neurohypophyseal

hormone, has been reported to be an anabolic bone mass

regulator.10–13 OT plasma levels are significantly lower in

postmenopausal women who develop osteoporosis than in

their healthy counterparts.12 Ovariectomized rodents with

changes in bone remodelling and consequent osteopenia,

have significantly decreased OT levels when compared with

sham-operated controls.13 In young rodents, it was verified

that deletion of OT or OT receptors (Oxtr) causes reduced bone

formation and suggested that OT is indispensable for basal

skeletal homeostasis in both sexes.13 In adult male albino rats

(150–200 g body weight) intramuscular OT injection was found

to have growth promoting effects on bone.10 Subcutaneous OT

injection reversed bone loss in eight-week-old OVX mice and

reduced marrow adiposity.12,13 Transcriptonic analysis of the

Oxtr pathway as a potential regulator of osteoblast/adipocyte

balance of human multipotent adipose-derived stem cells

(hMADS) was identified by Elabd et al.12 They verified that OT

and carbetocin (a stable OT analogue) negatively modulated

adipogenesis while promoting osteogenesis in both hMADS

cells and human bone marrow mesenchymal stromal cells. It

was found that intracerebroventricular OT infusion did not

affect serum markers of osteoblast (osteocalcin) and osteo-

clast (C-telopeptide) function, or influence ex vivo osteoblast or

osteoclast formation. However, an important finding was that

intraperitoneal injection of OT in mice increased bone mineral

density as well as osteoblast formation, and this anabolic

effect was attributed to a peripheral skeletal action of OT.13

The aim of this study was to analyze peripheral action of

OT in the alveolar bone healing process of old acyclic female

rats, 28 days after tooth extraction.

2. Materials and methods

2.1. Animals and treatment

Sixteen female Wistar rats (24-month-old) were housed in a

temperature-controlled room (22 � 2 8C) with a 12/12 h light/

dark cycle. Animals were provided with standard rat chow and

water ad libitum. The study protocol and all procedures

involving animals were in compliance with the principles of

laboratory animal care14 and national laws on animals, and

the study itself was authorized by the Animal Research Ethics

Committee of the São Paulo State University, Brazil (Protocol

No. 2009-005746).

The rats underwent colpocytological examination to

determine the cycle phase. A cotton swab impregnated with

saline solution (0.15 M) was inserted in the vagina. A slide

smear was carried out and immediately analyzed using a light

microscope at 40� magnification. The estrous cycle phases

were determined according to cell quantification: epithelial,
cornified and leukocytes in the colpocytological examination.

Animals in the diestrus cycle phase were divided into two

groups (8 rats in each group) and were assigned to plastic cages

(4 rats/cage). Two ip injections of saline (NaCl 0.15 M – control

group) or OT (45 mg/rat – treated group) were given with a12 h

interval between them.13

2.2. Tooth extraction

Seven days after receiving OT, the rats were anaesthetized

(Xylazine – 10 mg/kg bw/ip – Dopaser Laboratories Calier S.A.,

Barcelona, Spain; and Ketamine – 80 mg/kg bw/ip – Fort Dodge

Saú de Animal Ltda, Brazil) and after antisepsis (Polyvinylpyr-

rolidone iodide; Indú stria Quı́mica e Farmacêutica Rioquı́mica

Ltda, Brazil) the right maxillary incisor was luxated with the

aid of a tapered instrument and extracted with a small forceps.

The movement of extraction was smooth and followed the

curvature of the rat incisor, so that root fracture would not

occur. The atraumatic surgical technique was used, which

allows tooth extraction without postoperative complica-

tions.15 The dental sockets were sutured with silk thread

(Ethicon 4.0, Johnson and Johnson, São Paulo, SP, Brazil). The

extractions were performed in such a way that at the end of

the experimental period it was possible to obtain pieces with

reference to a 28 day period of the alveolar healing process.

2.3. Collection of materials

At 35 days after application of OT/saline solution or 28 days

post-extraction, the animals were anaesthetized (Xylazine + -

Ketamine, as previously described), blood was collected from

the external jugular vein by cannula16 and put into a centrifuge

tube. The plasma was separated by centrifugation (3000 rpm/

10 min/1 8C). A sample was acidified with acetic acid 20% in the

proportion of 1/100 for Tartrate-resistant acid phosphatase

(TRAP) determination. Calcium, phosphorus, total alkaline

phosphatase (ALP) concentrations were determined in accor-

dance with the manufacturer’s instructions (Labtest kits, São

Paulo, Brazil). Plasma osteocalcin and TRAP levels were

determined with a quantitative sandwich type of enzyme

linked immunoassay (ELISA) technique using rat kits pur-

chased from Uscn Life Science Inc. (Wuhan, China).

After euthanasia with an overdose of anaesthetic, the right

maxilla was removed. The pieces obtained were post fixed in

4% formaldehyde and demineralized in 10% EDTA (Merck,

Darmstadt, Germany). Using a cryostat (Micron Zeiss, Berlin,

Germany), the pieces were sliced longitudinal to the long axis

of the dental socket to obtain 06 mm thick slices, which were

mounted on previously gelatinized slides.

2.4. Histomorphometric analysis

The histomorphometric analysis of the bone mass and of the

middle thirds of the rat alveolus15,17 was performed with two

slices from each animal stained with haematoxylin and eosin

(Haloquı́mica, São Paulo, SP, Brazil). The analyses were

performed without the examiner knowing to which group

the slices belonged. Stained sections were examined by light

microscopy under 10� objective lenses, and images were

obtained with a digital camera (JVC TK-1270 Color Video



a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 1 2 9 0 – 1 2 9 71292
Camera) mounted on the microscope, and analyzed with Leica

Qwin Color/RGB software.

2.5. Immunohistochemistry reaction

For the immunohistochemistry reactions, primary antibodies

anti osteocalcin and anti TRAP (goat anti osteocalcin polyclonal

and goat anti TRAP polyclonal – Santa Cruz Biotechnology, CA,

USA) were used, and biotinylated rabbit anti-goat antibodies

(AffiniPure rabbit anti goat IgG – Pierce Biotechnology, Rockford,

IL, USA) were the secondary antibodies. The immunohistochem-

istry reaction signal was amplifiedwith the Avidin–Biotinsystem

(Kit ABC Vectastain Elite, ABC, Vector Laboratories, Burlingame,

CA, USA) and the reaction was revealed using diaminobenzidine

(Sigma, Saint Louis, MO, USA) as the chromogen. Haematoxylin

was used for counterstaining. The analysis was performed in

order to identify the labelling characteristics of each protein

studied by this methodological approach.

2.6. Statistical methods

Differences between groups were tested using the Student’s t-

test as the criteria of normality. Equal variances between
Fig. 1 – Plasma concentrations of calcium (A), phosphorous (B), A

in the middle third of the alveoli at 28 days of healing process 

post tooth extraction) with two ip injections of saline solution 

administered with an interval of 12 h between them. Calcium a

groups. Biochemical bone formation markers (ALP and osteocal

(TRAP) decreased. Histomorphometric evaluation showed a sig

third of the alveolus. Values are means W SEM, n = 8 rats per gr

control.
groups were met with a significance level of 0.05 using

GraphPad Prism 3.02 (GraphPad Software, Inc.; La Jolla, CA,

USA). All data are reported as means with their standard errors

of the mean (SEM).

3. Results

The plasma concentration of calcium (control = 10.70 � 0.6580;

treated = 10.41 � 0.6663 mg/dL) (Fig. 1A) and phosphorus (con-

trol = 4.880 � 0.3216; treated = 5.629 � 0.3932 mg/dL) (Fig. 1B)

was not significantly changed by treatment with oxytocin.

However plasmatic concentrations of biochemical bone forma-

tion markers18 ALP (control = 73.20 � 4.510; treated = 130.8 �
11.37 U/L) (Fig. 1C) and osteocalcin (control = 0.7280 � 0.02956;

treated = 1.117 � 0.04839 ng/mL) (Fig. 1D) were significantly

increased after OT treatment. The plasmatic levels of bone

resorption marker, TRAP,18 (control = 2.404 � 0.3724; treated =

0.5867 � 0.07200 U/L) were significantly decreased (Fig. 1E) in

the treated group. At 28 days after tooth extraction, histomor-

phometric evaluation (Fig. 1F) showed a significant increase in

trabecular bone volume of the middle third of the alveoli of

treated animals (44.36 � 2.697%) compared with those of
LP (C), osteocalcin (D), TRAP (E) and area of bone formation

(F). Results were obtained 35 days after treatment (28 days

(0.15 M/control group) or OT (45 mg/rat/treated group)

nd phosphorous plasma levels did not differ between the

cin) increased, as the biochemical bone resorption marker

nificant increase in trabecular bone volume in the middle

oup. Statistics: Student’s t-test; P < 0.05. *Different from



Fig. 2 – Histological sections (6 mm thick) of the middle third of rat alveolus at 28 days of healing process. Results were

obtained 35 days after treatment (28 days post tooth extraction) with two ip injections of saline solution (0.15 M/control

group) or OT (45 mg/rat/treated group) administered with an interval of 12 h between them. Control (A – 100T; C – 400T; E –

1000T) showed areas with thin bone trabeculae (BT) and connective tissue (CT); osteoblasts (Ob) predominated over the

osteocytes (Os); treated (B – 100T; D – 200T; F – 1000T) showed larger amount of bone matrix with greater presence of

osteoblasts and osteocytes. Histological sections stained with haematoxylin and eosin.

a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 1 2 9 0 – 1 2 9 7 1293
control group animals (29.95 � 1.807%). Histological examina-

tion of the healing process in the alveolar middle third of the

control group (Fig. 2A, C, and E) showed lower bone formation in

comparison with the OT treated group (Fig. 2B, D, and F), with

much larger areas of thin bone trabeculae and areas occupied by

connective tissue. In these animals, the bone had formed

osteoblasts predominantly over the osteocytes, and trabeculae

were more isolated. In the OT treated group (Fig. 2B, D, and F)

there was a larger area of bone formation occupying most of the

interior of the alveoli. In this region, there was a large amount of

bone matrix with greater presence of osteocytes. The formation
of islands of bone tissue was observed to a greater and more

developed extent, with the presence of osteoblasts and

osteocytes in greater quantity. There was presence of granula-

tion tissue to a lesser extent (presence of tissue supplied with

blood vessels, scattered collagen fibres, fibroblasts and amor-

phous ground substance). It was also found that the trabecular

bone in this group was more compact, i.e., a greater fusion of the

trabeculae when compared with the group that did not receive

OT, in which the trabeculae were found to be more isolated.

Immunohistochemistry reactions against osteocalcin and

TRAP were performed in the paraffin slices in order to identify



Fig. 3 – Histological sections (6 mm thick) of the middle third of rat alveolus at 28 days of healing process (35 days after OT

treatment) showing the osteocalcin immunolabelling. Note that in A (100T) and C (200T), almost no positive labelling for

osteocalcin either in mineralized bone matrix or in bone cells could be observed. Otherwise in B (100T) and D (200T) (treated

groups), a greater labelling of this protein was observed, showing that in the treated groups there was an increase in

mineralized bone matrix labelled with this protein, the main bone mineralization marker. Osteoblasts were also positive

for this protein.

Fig. 4 – Histological sections (6 mm thick) of the middle third of rat alveolus at 28 days of healing process (35 days after OT

treatment) showing TRAP immunolabelling. Note that in A (100T) and C (200T) (control groups), it was possible to observe a

large number of osteoclasts with positive TRAP labelling, representing an intense bone resorption activity. In B (100T) and

D (200T) (treated groups), there were no osteoclasts present close to the bone trabeculae.

a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 1 2 9 0 – 1 2 9 71294



a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 1 2 9 0 – 1 2 9 7 1295
the cell responses during alveolar bone healing. Osteocalcin

labelling showed that there was a decrease in the presence of

this protein in osteoblasts and in the mineralized matrix in the

control group (Fig. 3A and C). Indeed, in the treated group

(Fig. 3B and D), there was greater labelling of this protein

showing that the bone presented a better condition of

mineralization when compared with the control group. With

regard to TRAP labelling, there was a large quantity of

osteoclasts with positive label for this enzyme in the control

group (Fig. 4A and C), showing intense bone resorption activity

in these animals. Otherwise, in the treated groups, there was

an important decrease in the expression of cells with positive

TRAP labelling, showing that there was a less resorption

activity in the treated animals (Fig. 4B and D).

4. Discussion

These results indicate that in old female rats (24-month-old),

OT promoted increase in bone formation and decrease in bone

resorption during the alveolar bone repair process. The

mechanisms involved in this response are not fully under-

stood, but this can occur due to changes in intracellular events

essential for the development of osteoblasts and osteoclasts.

Old female rats have many interesting characteristics for

analysis of bone loss after acyclicity of the estrous cycles.19 In

this study they were subjected to tooth extraction for analysis

of the alveolar bone regeneration process, as this model may

be considered a good indicator of bone damage.17,20,21

Although the major sites of OT gene expression occur in the

central nervous system, it is also synthesized in peripheral

tissues, e.g., the uterus, placenta, amnion, corpus luteum,

testis and heart.22 Exogenous OT exerts this effect peripherally

since the neuropeptide does not cross the blood–brain

barrier.11,23 It was reported that intraperitoneal injection of

OT negatively modulated adipogenesis while promoting

osteogenesis in young rodents (3–6-month-old).11,12

The peripheral action of OT in old rats analyzed in this

study promoted significant increases in ALP and osteocalcin.

These indicate new bone formation, since ALP and osteocalcin

are excellent biochemical markers of bone formation activi-

ty.18 It has been observed that higher serum-osteocalcin levels

are relatively well correlated with increases in bone density

during treatment of osteoporosis with anabolic bone forma-

tion drugs.24 Furthermore, the ALP produced in the bone

formation phase is an excellent indicator of bone formation

activity.24 Thus, our results suggest that OT stimulates

osteoblast activity and neobone formation in old rats (24-

month-old).

Although analyses of the bone formation markers after OT

treatment signalled changes in the alveolar bone regeneration

process, histomorphometric and immunohistochemistry

analysis, considered reliable markers, were also tested. The

significant increase in alveolar bone formation and the greater

labelling of osteocalcin observed in OT treated rats suggests

that mineralization had taken place with utmost fidelity.

The immunohistochemical approach allows preservation

of the tissue cytoarchitecture, which is an important aspect to

consider when the alveolar healing process is evaluated.25,26

Osteocalcin, an indicator of bone turnover, is synthesized by
osteoblasts and becomes incorporated into the bone matrix by

binding to hydroxyapatite in a calcium-dependent fash-

ion.27,28 The greater labelling of this protein in the treated

group showed that the bone presented a better condition of

mineralization when compared with the control group.

The greatest alveolar bone formation observed in our study

after OT administration could be collected from the interac-

tion of the hormone with its receptor in bone cells,29 resulting

in intracellular events, such as those described by Copland

et al.30 Thus, OT could increase the release of intracellular

calcium and prostaglandin E2 synthesis, with consequent

positive bone balance.31 The interaction between OT and Oxtr

in osteoblasts may also involve other intracellular events (MAP

kinase phosphorylation and c-Fos expression), as described in

other OT target cells.30,32 These intracellular pathways are

essential for osteoblasts and osteoclasts.

In fact the action of OT has been reported to be a direct

regulator of bone mass mediated mainly through its stimula-

tion of osteoblast formation, with variable effects on osteo-

clasts.13 Oxtr expression by fully differentiated human

osteoclasts and by their precursors has been demonstrated.33

Furthermore, it has been reported that the receptor is

functional, and that the hormone may affect osteoclastogen-

esis, since it increases the number of pre-osteoclasts.13,33

Despite the fact that OT may favour osteoclastogenesis, it

can inhibit the resorptive function of mature osteoclasts

through a calcium signalling mechanism activated by Oxtr,

since OT treatment induces an increase in intracellular

calcium.13 This could explain the significant decrease in the

levels of TRAP, a bone resorption marker,18 observed in the OT

treated rats. Osteoclast origin TRAP is presumably released

into circulation during bone resorption and after the removal

of osteoclasts from their place of work.34,35 The significant

decrease in TRAP levels after OT treatment suggests that in old

female rats oxytocin acts by inhibiting bone resorption during

the alveolar healing process. In agreement with this, the

immunohistochemical analysis against TRAP demonstrated a

greater quantity of osteoclasts with positive label for this

enzyme in the control group. Otherwise, in the treated groups,

there was an important decrease in the expression of cells

with positive TRAP labelling.

Since the interaction between oxytocin and its receptor

triggers intracellular events that are important for the

development of osteoblasts and osteoclasts, OT could be

associated with the disruption of balance between osteoblasts

and osteoclasts that causes senile and endocrine-mediated

osteoporosis.28

The positive bone balance and the decreased bone

resorption observed during the alveolar healing process of

old acyclic rats suggest that OT could be studied for use as an

ally in the recovery of dental or alveolar trauma and in

maxillary implants. Further studies with OT should explore

the possibilities of using OT as an anabolic bone hormone as

therapy for postmenopausal osteoporosis in humans.

5. Conclusion

The results presented indicate that in old acyclic female rats,

peripheral OT plays a role in achieving a positive bone balance



a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 1 2 9 0 – 1 2 9 71296
and a decrease in bone resorption during the alveolar healing

process.

Funding

Federal Agency of Support and Evaluation of Postgraduate

Education – CAPES.

Competing interests

No conflict of interest.

Ethical approval

Animal Research Ethics Committee of the Univ Estadual

Paulista (UNESP), Brazil (Protocol Number 2009-005746).

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	Oxytocin promotes bone formation during the alveolar healing process in old acyclic female rats
	Introduction
	Materials and methods
	Animals and treatment
	Tooth extraction
	Collection of materials
	Histomorphometric analysis
	Immunohistochemistry reaction
	Statistical methods

	Results
	Discussion
	Conclusion
	Funding
	Competing interests
	Ethical approval
	References