1. * Demonistrator, Orthodontic dept., Faculty of Dentistry, Suez Canal University.
** Professor, Orthodontic dept. Faculty of Dentistry, Suez Canal University.
*** Professor, Pain management dept. National Cancer Institute, Cairo University.
**** Lecturer, Orthodontic dept. Faculty of Dentistry, Suez Canal University.
***** Lecturer, Oral biology dept. Faculty of Dentistry, Suez Canal University.
INTRODUCTION
Transverse maxillary hypoplasia is frequently
seen in adolescents and adults. It always
manifested in oral breathing patients causing
some rhinologic and dentofacial problems such as
decrease of the nasal permeability, bilateral dental
maxillary crossbite and compromising esthetics,
occlusal stability and normal mouth functioning1
.
Therefore, early treatment is necessary to establish
craniofacial equilibrium2
. Many approaches were
used as a clinical routine to normalize the upper
arch hypoplasia to achieve a stable and functional
‫صـــور‬
‫لون‬ 1‫لون‬ 4
‫املقالة‬ ‫رقم‬50‫الترقيم‬‫الصفحات‬ ‫عدد‬6‫األلوان‬ ‫ص‬12
BIOSTIMULATORY EFFECT OF OZONE APPLICATION ON BONE
REGENERATION IN MIDPALATAL SUTURE AFTER RAPID
MAXILLARY EXPANSION IN RATS
Mohamed A. Ahmed*
; Abbadi A .El-kadi**
; Mohamed N. Mawsouf***
;
Sherif S. Morcos****
and Walid S. Elsayd*****
ABSTRACT
Objective: to evaluate the biostimulatory effect of ozone therapy on bone regeneration in the
midpalatal suture after rapid maxillary expansion on rats using histological sections.
Material and methods: 63 rats were subjected to rapid maxillary expansion for a week and
then divided into 3 equal groups (G1
, G2
and G3
). G1
was the control group, G2
was subjected to the
ozone locally and G3
received systemic ozone. The rats in each subgroup were killed in different
points of time (T1
, T2 and T3
) and histological sections were obtained. The rats in each group have
been divided equally into 3 subgroups according to sectioning time. At T1
histological sections were
obtained after 45 days, at T2
sections were obtained after 60 days and at T3
sections were obtained
after 90 days.
Conclusion: The application of systemic ozone was useful in promoting bone healing in all
points of time while the effect of local ozone was more obvious in T2 and T3.Te quality of the
formed bone at T3 was better with the systemic and local ozone application than the control group.
KEY WORDS: Maxillary hypoplasia; Expansion; Ozone; Retention; Relapse.

2. (2) , et al.E.D.J. Vol. 60, No. 1
occlusion3
.Theapproachesdifferedbythefrequency
of the appliance activation, magnitude of the applied
force, duration of treatment and patient age. Rapid
maxillary expansion (RME) is one of the most
effective and applicable method for the correction
of the skeletal maxillary deficiency in young age
by stimulating the bone formation after opening
the midpalatal suture (MPS)4
. Interest in RME
has increased markedly during the past 2 decades.
The correction of transverse discrepancies and the
gain in arch perimeter as a potential non-extraction
technique appear to be the most important reasons
underlying this increased interest5
. Although the
major treatment effect is noticed clinically in the
area of the dentition, transverse enlargement of the
apical bone or the adjacent skeletal structures as well
as the mandible may be considered as additional
contributions6
.
However, even though opening the midpalatal
suture effectively widens a narrow maxilla, relapse
after maxillary expansion is common particularly in
older patients7
. One of the factors that may contribute
to this post-treatment instability is an increase in
the labial and buccal soft tissue pressure exerted
on teeth, which may lead to relapse before a new
bone tissue has been formed in the opened suture8
.
Maintenance of the expanded maxilla requires
adequate midpalatal suture opening, combined with
over-expansion and long term retention9
.
Many methods have been used to enhance the
long-term stability of the rapid maxillary expansion.
For example, low-power laser irradiation10
, human
growth hormone (hGH)11
, human recombinant
transforming growth factor (rhu) TGF-β 112
and
biphosphonate (BP)13
.
Ozone(apaleblue-coloredgas,chemicalformula
O3) plays an important role as a natural constituent
of the higher layer of the earth’s atmosphere. Ozone
is a natural gas that we breathe daily14
. Ozone therapy
has been shown to have a stimulatory effect on the
healing of both soft tissues and bones under several
experimental and clinical conditions15
. Several
reasons have been suggested to be the cause of this
effect. Ozone inactivates bacteria, viruses, fungi,
yeast and protozoa16
. Furthermore it has several
effects on circulation. For instances, it improves the
blood flow17
and increases permeability of vessels18
.
At the same time systemic way of ozone application
leads to increase in the production of TGF-β1
which has multiple functions on cellular growth and
differentiation in many cells19
. The present work
will therefore examine the effect of ozone therapy
on bone healing in the midpalatal suture after rapid
maxillary expansion.
MATERIALAND METHODS
A total of 63 male Wistar strain rats 6 weeks
old (the weight is 180 ± 10 gm)10
were used for
this experiment. All rats were subjected to rapid
expansion of the midpalatal suture for one week
according to the method adopted by Saito and
Shimizu10
.Among the whole procedures of the study
all the rats were anesthetized with an interperitoneal
injection of sodium pentobarbital (50 mg/kg body
weight). A hole was drilled laterally in the upper
incisors just above the gingival papilla. The height
of the incisors was reduced and the contact was
opened to facilitate the insertion of the expander.
A piece of 1.5 NiTi coil spring was inserted
between the incisors and an 0.01” steel ligature was
inserted through the holes to retain the coil spring
between the incisors. A week later, the two ends of
the steel ligature were tied firmly to prevent any
further expansion with the coil spring in its place
to preserve the gained expansion during the Ozone
administration. The 63 rats were divided equally
into 3 groups:
A) First group (G1
): It is the control group that has
not been subjected to the ozone therapy after
rapid maxillary expansion.
B) Second group (G2
): This group has been sub-
jected to ozone therapy locally after rapid max-
illary expansion.
C) Third group (G3
): This group has been subjected

3. EHH (3)
to ozone therapy systemically after rapid maxil-
lary expansion.
An ozone generator (Longevity Resources
Medical Systems Corporation, Canada. EXT-120
T Ozone generator) was used in the current study
which has been designed to produce “Ultra Pure
Ozone” from oxygen. The ozone was applied locally
to G2
and systemically to G3
after the completion
of the maxillary expansion. The ozone was applied
in the gaseous form locally to G2
group through
submucosal injection and systemically to G3
group
through rectal insufflations. After the completion of
the ozone application locally and systemically each
group were subdivided equally into 3 subgroups
(T1
, T2
and T3
):
T1
: Histological sections were obtained after 45
days of the maxillary expansion.
T2
: Histological sections were obtained after
2month of the maxillary expansion.
T3
: Histological sections were obtained after
3month of the maxillary expansion.
The rats were used as a source of the tissue af-
ter being sacrificed using carbon dioxide inhalation.
Sections were floated onto coated (Polysine or Su-
perfrost Plus ) slides and placed overnight in a 37°C
incubator, and finally removed and stored in 4°C un-
til ready to for use. The slides per each group were
then stained with H&E stain and then examined us-
ing image analyzer (Olympus microscope) at Fac-
ulty of Oral and Dental Medicine Cairo university,
Egypt. In each slide the width of MPS, connective
tissue fiber, arrangement of these fibers, connective
tissue forming cells (mainly fibroblasts), thickness
of the newly formed bone trabeculae, bone cells
(mainly osteocytes and osteoblasts) as well as the
newly formed blood vessel were focally reported.
RESULTS
The histological examinations for the slides in
each group were assessed individually and then the
results for each group were compared with each
other regarding the selected criteria for evaluation.
Among second group (G2
), the histological
examination of the MPS for animals in subgroup
1 (T1
) showed marked decrease in the MPS width
than what was seen in G1
T1
Unlike to the G1
T1
, The
MPS was completely filled with the connective
tissues at this stage. The connective tissues at this
stage showed irregular fibers arrangements, but it
was denser than in the G1
T1
. The newly formed
bone around the MPS was thicker than in G1
T1
.
The results of subgroup (T2
) showed more
narrowing of the MPS than what was found at its
counterpart from the G1
and T1
subgroup from the
same group as well. The connective tissues inside
the MPS showed disorganized blood vessels, but
still showing irregular arrangements of the fibers.
The newly formed bone became thicker and the
bone trabeculae became more matured looking. The
osteocytes and osteoblasts increased in number than
what was seen in its counterpart from the G1
and T1
subgroup from the same group.
The results of subgroup (T3
) showed narrowing
of the MPS almost similar to that seen in the G1
T3
.
The MPS was filled with connective tissue that
showed more organized blood vessels. The new
bone formation was similar also to what was seen
in G1
T3
.
Among third group (G3
), the histological ex-
amination of the MPS for animals in subgroup (T1
)
showed dramatic decrease of MPS by connective
tissue formation more than G2
T1
. The connective
tissues at this stage showed more regular fibers
arrangements, which was denser than that in both
G1
T1
and G2
T1
. The blood vessels were more orga-
nized at this stage. The newly formed bone around
the boarder of the MPS was thicker than what was
seen in G1
and G2
at the same point of time.
The results of subgroup (T2
) showed that the
MPS was completely filled with organized and

4. (4) , et al.E.D.J. Vol. 60, No. 1
dense connective tissue which was almost similar
to G1
and G2
after three months (T3
). The newly
formed bone was of higher quality indicated by the
well developed bone trabeculae and the increased
number of osteocytes and osteoblast.
The histological examination of the healing of
the MPS of the animals in subgroup (T3
) showed
complete closure of the MPS by a more organized
and denser connective tissue compared to G1
and
G2
at the same point of time. The connective tissue
contains a large amount of organized blood vessels
because of the increased vascularity at that point
of time. The examination showed also increased
amount of mature and higher quality of formed
bone with increased amount of osteocytes and
osteoblasts.
DISSCUSION
The effect of local ozone therapy at T1
and T2
was shown as a marked formation of unorganized
connective tissue fibers in the MPS after rapid
maxillary expansion in contrast to G1
at the same
points of time. The bone at these early stages
started to be formed on both sides of the MPS. The
improvement in healing in the area of MPS after
local ozone administration was attributed to the
prevention of the development of inflammatory
complications by stabilization of membrane
LP processes20
. At the same time local ozone
administration leaded to higher expression of
cytokines that were important for wound healing,
especially TGF-B1
, which is important substance
for regulation and coordination in the initial wound
healing phase. TGF-B1
had a marked influence
on cell proliferation, chemotaxis (monocytes and
fibroblast), angiogenesis, synthesis of extracellular
matrix and collagen synthesis19
. In T3
, there were
no marked differences in the formed bone between
G1
and G2
as this is the time of completion of bone
healing in normal circumstances.
The effects of systemic ozone therapy at T1
were
a dramatic decrease of MPS by connective tissue
formation more than G2
T1
. The connective tissues
at this stage showed denser and more regular fibers
arrangements and large amount of organized blood
vessels. Also more bone formation was seen on
both edges of the MPS. At G3
T2
the quality of the
formed bone was comparable to G1
and G2
at the
same points of time. At G3
T3
, the formed bone was
the best among all stages indicated by complete
closure of the MPS by a more organized and denser
connective tissue compared to G1
and G2
at the same
point of time. This marked improvement in bone
healing was due to the improvement in blood flow17
,
the increase in the permeability of the blood vessels
and hence increase in the cellular contents involved

5. EHH (5)
in the formation of the various types of connective
tissue fibers (Fibroblasts) and bone (Osteocytes and
osteoblasts)18
and also due to the increase in the
cellular activity after the application of Ozone15
.
CONCLUSION
The following conclusions can be drawn from
the current study:
1. Ozone therapy is a safe effective method in pro-
moting bone healing following RME at the mid-
palatal suture as better bone healing was seen in
the groups subjected to ozone in the different
points of time, rather than the control group.
2. Bone healing was better in the local ozone
group than the control group in T1
andT2
, but in
T3
there were no significant differences.
3. Bone healing was better in the systemic ozone
group than the control group in all points of
time (T1
, T2
and T3
).
4. The quality of the formed bone in the system-
ic ozone group at T3
was better than the local
ozone group and the control group at the same
point of time.
5. Ozone application may be useful to reduce re-
lapse after RME due to its effect on bone heal-
ing.
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