The periodontal abscess: a review

ORIGINAL ARTICLE

Oral Pathology

Therapeutic effect of topical ozonated oil on the epithelialhealing of palatal wound sites: a planimetrical andcytological studyPunit Vaibhav Patel1, Veerendra Kumar1, Sheela Kumar1, Vidya GD2 & Amrita Patel3

1 Department of Periodontology, Jagadguru Sri Shivarathreeshwara Dental College and Hospital (JSS University), Mysore, Karnataka, India

2 Department of Oral Pathology and Microbiology, Jagadguru Sri Shivarathreeshwara Dental College and Hospital (JSS University), Mysore,

Karnataka, India

3 Kalindi Oro Care, Varanasi, Uttar Pradesh, India

Introduction

Oral wound healing is a complex and dynamic process of

restoring cellular structures and tissue layers. The wound-

healing response can be divided into several distinct but

overlapping phases: inflammation, re-epithelialization,

granulation tissue formation, matrix formation, and tissue

remodelling.1 Re-epithelialization or epithelial healing is

an important, complex process that involves the interac-

tions between keratinocytes and the extracellular matrix

upon which cells migrate, proliferate, and differentiate,

thereby restoring tissue structure and function.1,2

The oral cavity provides the unique environmental

challenge for the epithelial healing of oral wounds

produced during various periodontal procedures.3,4

Trauma from mastication, relatively large commensal

flora, and elevated levels of dental plaque impair the nor-

mal sequence of the healing process.3,4 Therefore, there is

concern regarding the delayed healing of oral cavity

wounds.5

Appropriate antimicrobial treatment and oral wound

care can accelerate the epithelial healing process, thereby

preventing infection and chronicity of the wound. Thus

far, different adjunctive chemotherapeutic agents have

been used to achieve enhanced epithelial bridging or

shorter healing times. Despite extensive efforts to improve

wound healing, the outcomes of existing chemothera-

peutic agents are far from optimal.6 Moreover, studies6,7

Keywords

cytological technique, gingival recession,

ozonide, postoperative, wound healing.

Correspondence

Dr Punit Vaibhav Patel, Department of

Periodontology, JSS Dental College and

Hospital, (JSS University) S.S Nagar,

Mysore, Karnataka 570015, India.

Tel: +91-973-1505109

Fax: +91-821-2548352

Email: [email protected]

Received 25 October 2010; accepted 17 April

2011.

doi: 10.1111/j.2041-1626.2011.00072.x

AbstractAim: To evaluate the effect of ozonated oil on palatal wounds.

Methods: Eighteen patients were randomized and allocated to either the ozone

group (n = 8) or control (n = 10) group. Free gingival graft surgery was per-

formed, and post-harvested palatal wounds were treated with either 2 mL ozo-

nated oil or control oil daily for 1 week. A planimetrical analysis analyzed the

digital image for the wound sizes and shape factor at baseline, at 24 h, and

days 5, 7, 14, 21, and 28, postoperatively. A cytological analysis used the kerati-

nization and superficial cell indices at baseline, 24 h, and days 3, 7, 14, and 21

and the second and third months, postoperatively.

Results: Planimetrical results showed a significant (P £ 0.05) improvement in

wound size on days 5, 7, 14, 21, and 28, postoperatively, in the ozone group

compared to the control group. Cytological results showed a significant

(P £ 0.001) improvement in epithelial healing on days 7, 14, and 21, and the

second and third months, postoperatively, after the application of ozonated oil

compared to control oil.

Conclusion: Our results showed significant improvement in wound size and

epithelial healing after topical ozonated oil application compared to control oil

on palatal wounds.

Journal of Investigative and Clinical Dentistry (2011), 2, 248–258

248 ª 2011 Blackwell Publishing Asia Pty Ltd

have also shown that chemotherapeutic agents, such as

chlorhexidine, sodium hypochlorite, povidone iodine, and

hydrogen peroxide have cytotoxic effects on human oral

epithelial and gingival fibroblast cells.

Currently, ozone gas dissolved in water or in plant oils,

such as olive oil (ozonized oil), is being discussed in den-

tistry for its excellent antimicrobial property, without the

development of drug resistance and facilitation of wound

healing in the oral cavity. The most cited explanation for

ozone’s bactericidal effects centers on the disruption of

cell membrane integrity through oxidation of its phos-

pholipids and lipoproteins, penetration of ozone through

the cell membrane, reaction with cytoplasmic contents,

and conversion of the closed circular plasmid DNA to

open circular DNA, which would presumably diminish

the efficiency of bacterial procreation.8–10

Moreover, under the influence of ozone, improved

rheological properties,11 activated cellular metabolism,12

raised intracellular ATP concentrations,13and the expres-

sion of cytokines relevant to wound healing, especially

transforming growth factor-b1 (TGF-b1),14 have been

observed.

It has been hypothesized that the wound-healing action

mechanism of ozonized oil might be in part connected to

its antimicrobial effect, but also with its ability to pro-

mote the liberation of growth factors, activate local anti-

oxidant mechanisms, and promote tissue repair.15

However, there is no consensus or direct evidence that

ozone plays a central role in the healing process. Thus,

this clinical trial was designed to investigate the effect of

ozone on the epithelial healing of palatal excisional

wounds (wounds generated after free gingival autograft

surgery) with large epithelial and connective tissue defects

that heal with secondary intention. The rate of re-epithe-

lialization was a criterion to indicate the influence of the

ozonated oil on the healing of palatal wound sites.

Materials and methods

Initially, a total of 26 patients (10 males and 16 females,

mean age: 30.19 ± 8.57 years) diagnosed with localized

gingival recession were considered eligible for the study.

All the patients were recruited between February 2010

and June 2010, and were randomly selected from the out-

patient department at Jagadguru Sri Shivarathreeshwara

Dental College and Hospital, Karnataka, India. Four

patients were excluded, as they did not meet the inclusion

criteria. The remaining 22 (nine males and 13 females)

were randomized and allocated to either the ozone group

(n = 11) or the control group (n = 11). During the study,

one patient from the control group and three patients

from the ozone group failed to appear for follow up or

discontinued the study. Finally, a total of 18 patients

(eight males and 10 females, mean age: 28.13 ±

6.38 years) were considered, and they completed the

study.

The study was a single-centered (Jagadguru Sri Shiva-

rathreeshwara [JSS] Dental College and Hospital, India),

longitudinal, triple-blinded (patients, clinical investiga-

tor/cytologist, and statistician) randomized, placebo-

controlled, parallel-armed, clinical trial. The duration of

the study was 3 months, during which planimetrical

parameters were recorded at baseline, 24 h, and days 5, 7,

14, 21, and 28, postoperatively; cytological parameters

were recorded at baseline, 24 h, days 3, 7, 14, and 21, the

second and third months, postoperatively.

Prior written, informed consent was obtained from

each patient, based on the recommendations of the Insti-

tutional Review Board (IRB) of JSS University. The IRB

approved the study. All procedures in this clinical trial

were performed according to the ethical principles estab-

lished by the Declaration of Helsinki.16

Inclusion criteria were age of 20–40 years, male/female,

systemically healthy, single tooth labial recession in lower

anterior sextant that required soft tissue graft involving

donor tissue from the palate, and all patients should have

read, understood, and signed an IRB-approved informed

consent form, and should be able and willing to follow

study procedures and instructions. Exclusion criteria were

patients who had used any tobacco product within the

past 3 months; systemic conditions (i.e. uncontrolled dia-

betes mellitus, cancer, HIV, bone metabolic diseases) that

could compromise wound healing, and systemic cortico-

steroids, immunosuppressive agents, radiation therapy,

and/or chemotherapy prescribed or received within

2 months prior to study entry, which could compromise

wound healing and preclude periodontal surgery.

The patients were randomly allocated to either the

ozone group (where the patients received cold-pressed

olive oil treated with ozone at a concentration of 14 lg/

mL) or the control group (where the patients received

olive oil that had not been treated with ozone). One of

the authors (SKG) generated the random allocation

sequence. The random allocation sequence was concealed

from the main investigator (PVP) and cytological investi-

gator (VGD) until the study was completed.

Clinical procedure

At the initial study visit, all of the included patients

underwent free gingival graft surgery17 involving donor

tissue removal from the palate. The masked main investi-

gator (PVP) performed all the surgical procedures. In

order to obtain a uniform palatal wound, a standard-

ized tinfoil template (9 · 10 mm) was used to mark the

donor area (Figure 1a). Subsequently, a standardized

P.V. Patel et al. Effect of ozonated oil on palatal wounds

ª 2011 Blackwell Publishing Asia Pty Ltd 249

cuboid-shaped palatal graft measuring 9 · 10 mm was

harvested from premolars and the first molar region of

the hard palate, leaving behind a 2 mm-deep, standard-

sized (9 · 10 mm), post-harvested wound area in the

donor site (Figure 1b). Finally, the harvested graft under-

went a routine free gingival graft procedure to cover the

denuded root surface, and the generated standard sized

palatal wound (9 · 10 mm) was considered and evaluated

in the study.

After the surgical procedure, routine postoperative

instructions were given to the each patient. Oral hygiene

status was monitored by measuring the plaque index at

various time intervals. All of the treated patients were

scheduled for recall appointments. During the first-week

visit, all of the patients were asked to report to the clinic

on a daily basis and have their allocated therapeutic med-

ication applied by one of the masked investigators (PVP)

to their respective palatal donor site wounds (Figure 2).

The wounds of the ozone group patients were treated

with 2 mL ozonated oil per day, with a concentration of

14 lg of ozone per mL of olive oil. The control group

patients’ palatal wounds were treated with 2 mL of non-

ozonated oil per day. A soft stent was used to protect the

palatal donor wound site in both groups. Patients were

instructed not to disturb the stent for 1 week. The stent

was removed and reseated by one of the investigators

(PVP), only during the application of medications, collec-

tion of smears, and capturing of digital photographs for

the analysis. The protective stent also facilitated the reten-

tion of test oils in the post-harvested palatal wound sites.

Postoperatively, protective stents were discontinued after

7 days.

Digital photographs of the palatal wounds were taken

at baseline, 24 h, and days 5, 7, 14, 21, and 28, postopera-

tively, by using a digital camera (Kodak C713; Eastman

Kodak Company, Rochester, NY, USA). The digital cam-

era was placed at a 20-cm distance from an intraoral

mirror placed at the level of the occlusal plane of the

lower teeth. The image was captured only when the

palatal wound margin was clearly visible on the LCD

screen of the digital camera. A small acrylic plate with a

standard diameter of 3 mm was also placed adjacent to

the wound as a reference, as suggested by Filippi,18 for

the planimetric measurement of human palatal wounds.

Thus, any difference (and thus, possible mistake in mea-

surement) in camera–wound distance or angle could be

adjusted and/or corrected. A set of 50 images was

captured, and the 20 best reproducible images of each

interval were analyzed with 10 analyses per image, and an

average was considered for each time interval. The photo-

graphic images were analyzed by image analysis computer

software (UTHSCSA Image Tool program, University of

Texas Health Science Center, San Antonio, TX, USA)

for the size and shape of the wounds19 at the indicated

time intervals. For the measurement, the software was

(a)

(b)

Figure 1. Standard donor site wound (10 · 9 mm) (a) obtained after

placement of standard sized tinfoil (10 · 9 mm) (b).

Figure 2. Application of ozonated oil using cotton applicator.

Effect of ozonated oil on palatal wounds P.V. Patel et al.


calibrated, and the scale setting was changed from pixels

to millimeters (mm). The instructions were followed, as

per the software manual, for measuring the wound sizes.

The wound outline was traced using a cursor on the

screen, and the software automatically calculated the

wound size (area) in square millimeters (mm2), and

the perimeter of the wound in millimeters (mm). The

shape factor19 was calculated using the obtained data of

wound size (area in mm2) and perimeter (in mm) using

the following formula: SF = 4pS/P2, where SF is the shape

factor, and S and P are the surface area and perimeter of

the wound, respectively. All of the wound dimension

measurements were performed double blinded so that the

operator could not identify anyone in the groups during

the analysis.

Cytological procedures

Cytological technique was used in the study to evaluate

epithelial keratinization, regeneration, and degeneration,

as this technique facilitated the repeated scraping of epi-

thelial cells, which can be evaluated over a period of time.

A sterilized, disposable, interproximal brush (STIM inter-

prox brushes; DENT–AIDS, New Delhi, India) was used

to scrape the palatal donor wound site. The flat surface of

the interproximal brush was placed against the margin of

the wound with firm pressure, and rotated four to five

times to collect cellular material (Figure 3).20 The col-

lected material was smeared onto the coded glass slide in

parallel fashion, and rapidly fixed with spray fixative

(RAPID PAP spray fixative; Biolab Diagnostics, Mumbai,

India) to avoid the smears from air drying. Smeared

slides were sent to the histopathlogical laboratory for

cytological analysis. Smears from donor wound sites were

collected at baseline (before the surgical procedure), 24 h,

days 3, 7, 14, and 21, and the second and third months,

postoperatively.

Staining and examination of sample smear

The smeared slides were stained by rapid Papanicolaou

technique (RAPID PAP stain; Biolab Diagnostics, India).

The masked cytological investigator (VGD) performed all

of the staining and examinations of the selected smear

slides. The stained smeared slides were examined under a

microscope at 40· resolution, and the cell count was sys-

tematically performed via a meandering movement of the

stage. For each smear, a minimum of 200 cells were

examined for cellular and nuclear changes. The cellular

and nuclear characteristics were evaluated according to

the cell-type classification of Lange et al.21 Cells were clas-

sified into superficial cells (ST2 and ST1), intermediate

cells (IT2 and IT1), parabasal cells (OBT), and basal cells

(BT) (Figure 4).

Regenerative changes that consisted of new intermedi-

ate cells (IT2 and IT1), OBT and BT, and degenerative

changes consisting of cytolysis, karyolysis, perinuclear

halo formation, karyorrhexis, nuclear wall hyperchrom-

asia, and pyknotic nucleus formation were analyzed.

Noted pyknotic changes were considered when their

nuclei were spherical and shrunk with the loss of nucleoli.

The analysis was followed by calculation of the keratiniza-

tion and superficial cell indices.21

Figure 3. Collection of cytological smear from margin of wound using

an interprox brush.

Superficial cellsST0

ST1

IT0

IT1

Intermediate cells

Parabasal cells

Basal cells

OBT

BT

Figure 4. Cellular and nuclear type classification. BT, basal cells; IT,

intermediate cells; OBT, parabasal cells; ST, superficial cells.



The parameters recorded for the exfoliative cytological

analysis were:

1. Keratinization index:21

Keratinization index

¼ Cells without nuclei

Total no. examined cells ðn ¼ 200Þ � 100: ð1Þ

2. Superficial cell index:21

Superficial cell index

¼Cells with pyknotic nuclei and with no nuclei

Total no. examined cells ðn¼ 200Þ �100: ð2Þ

Statistical analysis

Recorded planimetrical and cytological data were analyzed

using SPSS software (version 11; SPSS, Chicago, IL,

USA). All parametric variables were analyzed by two-

factor anova with repeated measures and Student’s t-test

(independent samples). Two-factor anova with repeated

measures was conducted to determine whether there was

a statistical significance between two different treatment

groups for improvement in healing over the period of

time. Student’s t-test (independent samples) was used for

the comparison of mean differences between treatment

groups at specific time intervals. An alpha level of 0.05

was utilized for both statistical analyses.

Results

There were no unwanted side-effects observed in the

ozone or control group after the application of the

respective oils. A clinical view of the typical healing

sequence of palatal wounds treated by ozonated oil and

placebo oil at various time intervals is shown in Figure 5.

The mean plaque score of both the groups at various

time intervals decreased over time. The result of the

Student’s t-test showed that there was a significant

decrease in the mean plaque score on day 7 in the ozone

group (0.16 ± 0.033) compared to the control group

(0.8 ± 0.191; t (16) = )9.32, P < 0.001). However, by day

14 and 21, and the second and third months, the differ-

ences in the mean plaque scores of both groups were not

statistically significant (P > 0.05).

Planimetrical analysis

Comparison of wound size and shape factor

The mean wound size and mean shape factor over time

for the ozone and control groups are shown in Table 1.

The mean wound size and shape factor changed over time

in both groups, with a significant change in the

ozone-treatment group when compared to the control

group. The result of the two-factor, repeated-measures

anova for differences in wound size and shape factor

over time showed that there was a statistically significant

interaction between the treatment groups and time inter-

vals for wound size (F = 22.119 [d.f. 6.96], P < 0.001)

and shape factor (F = 42.669 [d.f. = 6.96], P < 0.001).

The result of the main effect of time interval was also

significant for wound size (F = 2625.609 [d.f. = 6.96],

P < 0.001) and shape factor (F = 70.332 [d.f. = 6.96],

P < 0.001). Further, there was a significant main effect

in the treatment groups for wound size (F = 63.311

[d.f. = 1.16], P < 0.001), but the main effect in the treat-

ment groups did not reach significance for the shape

factor (F = 3.542 [d.f. = 1.16], P < 0.078).

The results of the Student’s t-test (independent sam-

ples) for differences in the mean wound size and mean

shape factor between the ozone and control groups at

specific time intervals are shown in Table 1. No signifi-

cant difference was found in the mean wound size at

24 h between the groups. On days 5, 7, 14, 21, and 28,

there was a significant decrease (P £ 0.05) in both the

mean wound size, and a significant differential change

(P £ 0.05) in the mean shape factor in the ozone group

as compared to the control group. On day 28, the mean

wound size and mean shape factor were reduced to zero

in the ozone group, indicating complete epithelization

and no residual wound areas in the donor site wounds.

These results suggest that the topical application of ozone

on palatal wound sites has an enhancing effect on epithe-

lial healing.

Cytological analysis

The stained epithelial cells revealed no sign of morpho-

logical alterations or pathological condition. The epithe-

lial cells observed during the initial phase of wound

healing and after complete epithelization are shown in

Figure 6.

Comparison of keratinization index and superficial cell

index

The mean keratinization index and superficial cell index

over time for the ozone and control groups are shown in

Table 2. The mean keratinization index and superficial

cell index increased over time in both groups, with a

significant differential increase in the ozone group com-

pared to the control group. The results of the two-factor

anova with repeated measures showed that the main

effect of the treatment groups was significant for the

keratinization index (F = 359.752 [d.f. = 1.16], P < 0.001)

and the superficial cell index (F = 599.066 [d.f. = 1.16],



P < 0.001). The main effect of time was also significant

for the keratinization index (F = 7005.493 [d.f. = 7.112],

P < 0.001) and the superficial cell index (F = 19355.209

[d.f. = 7.112], P < 0001). Moreover, the interaction of

these two factors was also significant for the kerati-

nization index (F = 53.754 [d.f. = 7.112], P < 0.001) and

the superficial cell index (F = 205.145 [d.f. = 7.112],

P < 0.001).

(a) (b)

(i)

(ii)

(iii)

(iv)

(v)

(i)

(ii)

(iii)

(iv)

(v)

Figure 5. Clinical view of typical healing

sequence of palatal wounds treated by ozo-

nated oil (a) (same patient) and placebo oil (b)

(same patient) at baseline (i), at day 7 (ii), at

day 14 (iii), at day 21 (iv), and at day 28 (v).



The results of the Student’s t-test (independent sam-

ples) for differences in the mean keratinization and super-

ficial cell indices between the two groups at specific time

intervals are shown in Table 2. The mean keratinization

and superficial cell indices of the ozone group showed

rapid decreases, similar to the control group, from base-

line to 24 h. On day 3, the ozone group showed a sig-

nificant (P £ 0.001) decrease in the keratinization and

superficial cell indices compared to the control group.

From day 7 to 21, the ozone group showed rapid and

significant (P £ 0.001) increases in the keratinization and

superficial cell indices compared to the control group. On

the second and third months, the mean keratinization

index was significantly (P £ 0.001) higher in the ozone

group compared to the control group. These results sug-

gest that the topical application of ozone on palatal

wound sites has an enhancing effect on the re-epithelial-

ization rate of healing epithelial tissue.

Discussion

The present study was undertaken to evaluate the thera-

peutic effects of topical ozonated olive oil on the healing

of post-harvested palatal donor wound sites, with large

epithelial and connective tissue deficiencies that heal by

secondary intention. Wound healing is a complex biologi-

cal process commonly divided into overlapping phases:

inflammation, re-epithelialization, granulation tissue for-

mation, matrix formation, and tissue remodelling.1,2 The

use of antimicrobial agents, such as ozonated oil, can

affect the nature and quality of the inflammatory infiltrate

Table 1. Mean and standard deviation (SD) of variables measured at various time intervals, and comparison between ozone and control groups

using Student’s t-test (independent sample)

Time

intervals

Treatment

group n

Wound size (mm2) Shape factor (%)

Mean

(mm2) SD t P

Mean

(%) SD t P

Baseline Ozone 8 90.00 0� – – 0.78 0 )0.757 0.460

Control 10 90.00 0.000� 0.78 0

24 h Ozone 8 80.10 6.492 )0.911 0.376 0.86 0.041 )0.285 0.780

Control 10 82.67 5.480 0.87 0.025

Day 5 Ozone 8 39.58 3.240 )6.21 0.000 0.93 0.031 7.700 0.000

Control 10 53.93 5.820 0.80 0.038

Day 7 Ozone 8 11.80 2.550 )10.70 0.000 1.07 0.208 2.854 0.011

Control 10 30.01 4.210 0.87 0.066

Day 14 Ozone 8 5.51 1.470 )6.204 0.000 0.81 0.068 )3.000 0.008

Control 10 13.49 3.370 0.94 0.105

Day 21 Ozone 8 3.10 0.440 )9.523 0.000 1.19 0.303 2.570 0.020

Control 10 08.98 1.690 0.92 0.127

Day 28 Ozone 8 0 0 )9.332 0.000 0 0 )20.00 0.000

Control 10 2.05 0.618 0.75 0.105

�t could be computed because the SD of both groups was 0.

(a)

(b)

Figure 6. Cytological photomicrograph showing epithelial cells obser-

ved at the initial phase of palatal wound healing: days 5, 7, and

14 (a), and epithelial cells observed after complete epithelization: day

28 onwards (b). IT, intermediate cells; KC, keratinized cells; OBT, para-

basal cells; PN, pyknotic nucleus; ST, superficial cells.



and that of the granulation tissue component.4,5,15 In the

present study, the rate of re-epithelialization was evalu-

ated to indicate the influence of the ozonated oil on the

healing of palatal tissue. The rate of epithelial healing was

assessed by planimetrical and cytological analyses. The

result revealed that ozonated oil significantly enhanced

re-epithelialization of the palatal donor site wounds.

Re-epithelialization is a major component of the

wound-healing process, which is achieved through a com-

plex interplay of diverse growth factors, cytokines, and

cell-cycle regulators.1,2,22 Currently, ozone or its products

are being investigated for its influence on growth factors,

cytokines, and cell-cycle regulators in biological systems.

Bocci et al. conducted a series of studies14,23–25 and

showed that the contact of ozone with human blood led

to an increased release of TGF-b1; interferons-a, -b, and

-c; interleukins-1, -2, -6, and -8), and tumor necrosis

factor-a (TNF-a), which are important for human wound

healing.

Epithelial cell migration is an essential step of wound

closure, where epithelial cells migrate from the periphery

of the wound towards the central part of the defect. The

migration and proliferation of keratinocytes and the close

relationship between the migratory cells and the collagen

of the newly-formed connective tissue determine the rate

of re-epithelialization of the wound.2 In our study, these

epithelial changes were evident on cytological smears and

were confirmed by the appearance of newly-generated

OBT and BT in the cytological smears. Moreover, epithe-

lial migration was also confirmed by progressive increase

in the pyknotic and keratinized cells (ST1 and ST2) in

the cytological smears, ultimately resulting in the gradual

increase in the keratinization and superficial cell indices

with advancing time intervals.

Our cytological analysis results confirm the findings of

Lange et al.,21 where the keratinization index increased

gradually from 0% to 50% in a 3-month period, demon-

strating the long-term cytological changes occurring in

healing human palatal mucosa.

The rate of re-epithelialization, when analyzed by the

cytological method, demonstrated that the ozone group

had a rapid and accelerated migration of epithelial cells

compared to the control group, as measured by the kerat-

inization and superficial cell indices. The cytological anal-

ysis was undertaken on days 7, 14, and 21, and the

second and third month observations found that the

keratinization index was significantly higher in the ozone

group compared to the control group. Similarly, the

planimetrical analysis also revealed the significantly

decreased wound area after the application of ozonated

oil when compared to control oil on days 5, 7, and 14 of

the analysis. These changes clearly suggest that after the

application of ozonated oil, there is an accelerated migra-

tion of epithelial cells as part of the healing process in

post-harvested palatal wounds.

Our planimetrical analysis results confirm the findings

of Filippi,18 who concluded that the application of ozo-

nated water accelerates wound healing in the oral cavity

within the first 48 h compared to placebo water, resulting

in earlier epithelial wound closure after 7 days. Our study

Table 2. Mean and standard deviation (SD) of variables measured at various time intervals, and comparison between ozone and control groups

using Student’s t-test (independent sample)

Time

intervals

Treatment

group n

Keratinization index Superficial cell index

Mean SD t P Mean SD t P

Baseline Ozone 8 97.08 1.940 0.292 0.774 100.00 0� – –

Control 10 96.73 2.910 100.00 0�

24 h Ozone 8 0.19 0.072 )1.735 0.102 1.55 0.43 1.699 0.109

Control 10 0.24 0.043 1.29 0.21

Day 3 Ozone 8 2.45 0.330 12.447 0.000 2.38 0.43 3.483 0.003

Control 10 0.94 0.170 1.87 0.15

Day 7 Ozone 8 5.71 0.340 24.531 0.000 16.82 1.82 9.065 0.000

Control 10 2.00 0.290 7.47 2.40

Day 14 Ozone 8 21.65 1.920 8.927 0.000 62.85 4.04 14.980 0.000

Control 10 11.39 2.750 41.89 1.66

Day 21 Ozone 8 38.81 2.490 11.325 0.000 92.57 1.51 30.445 0.000

Control 10 25.02 2.620 72.34 1.30

Month 2 Ozone 8 52.35 1.850 11.653 0.000 100.00 0� – –

Control 10 42.49 1.720 100.00 0�

Month 3 Ozone 8 65.70 2.220 17.782 0.000 100.00 0� – –

Control 10 51.61 1.05 100.00 0�

�t could be computed because the SD of both groups was 0.



is also in favor of the results of two similar studies pub-

lished by Noritaka26 and Kim et al.,27 where they found

similar wound closure in a rat model after the application

of topical ozonated oil.

In the present study, we found that epithelial healing

was complete, with minimal residual wounds clinically

detectable after 21 days, postoperatively, in both groups.

These findings are in agreement with results of those

Rossmann and Rees,28 Del Pizzo et al.,29 and Silva et al.,30

where the authors concluded that the complete epithelial-

ization of palatal wounds occurs in 3–4-week intervals.

The antimicrobial property of ozone is another impor-

tant mechanism that can explain the enhanced epithelial

healing of palatal wounds. Increased bacterial colonization

of the wound surface results in an increased area of

inflammation and granulation tissue that might prolong

the repair.31As oral wounds are constantly subjected to a

relatively large commensal flora, it is clear that bacteria

affect wound healing in the oral cavity. Elevated levels of

bacteria (over 105) decrease epithelialization; bacterial

metabolites inhibit epithelial cell migration, digest dermal

proteins and polysaccharides, and increase the production

of neutrophil proteases, cytotoxic enzymes that damage

vulnerable epithelium.32–35 Thus, the control of bacteria

by ozonated oil might act directly or indirectly on epithe-

lial and connective tissue cells and can accelerate oral

wound healing.

Our results are in agreement with a recent experimen-

tal study by Dmitrieva et al.36 in 30 Wistar rats. In their

study, the rats were divided into three groups, with 10

rats in each group. Skin defects were created and subse-

quently contaminated with the microorganisms. The

wounds were irrigated with 10–15 mL commercial perfto-

rane solution, ozonized perftorane, and physiological

solution, depending on their respective groups. The

results indicated that in the group with ozonized perfto-

rane irrigations, the mean number of microorganisms in

the wound were reduced; granulation tissue growth and

epithelization were enhanced when compared with the

groups of rats irrigated with physiological solution and

non-ozonized perftorane.

Similar to ozone, various topical antimicrobial agents

have also been tested for their healing efficacy in different

post-surgical situations. Kozlovsky et al.4 reported that

1% chlorhexidine digluconate (CHX) gel and Listerine

solution increased the rate of epithelization in palatal

wounds of a rat model. Similarly, Bassetti et al.37 also

reported that the topical application of 0.1% CHX and

0.2% CHX resulted in undisturbed healing on day 7 fol-

lowing injury. Our study also reports similar results,

where the antimicrobial property of ozonated oil could be

attributed to epithelial changes in healing wounds. How-

ever, considering that results from animal experiments

cannot necessarily be applied to oral mucosa, it can only

be theoretically suggested that the effects of ozonated oil

mentioned in the literature are responsible for the influ-

ence on the wound healing of oral mucosa.

Ozone gas dissolved in an oil base (olive oil) was used

to treat post-harvested palatal wound sites. Studies have

revealed that the direct application of gaseous ozone has

a cytotoxic effect on human gingival fibroblast and epi-

thelial cells, but the direct application of aqueous ozone

is safe on oral tissue.7,38 Previous studies have concluded

that ozone in an oil base is non-toxic and provides the

safest medical therapy devised.39,40 There is no evidence

of free radical damage. On the contrary, ozone gas liber-

ated from ozonated oil has been proven23,39,40 to stimu-

late the production of superoxide dismutase, catalase,

glutathione peroxidase, and reductase, enzymes that pro-

tect the cell from free radical damage. Moreover, studies

have also reported that the O3 molecule can be stabilized

as an ozonide between the double bonds of a monounsat-

urated fatty acid, such as oleic acid; therefore, ozonated

olive oil can be an ideal preparation for the topical form

of O3, and it remains stable for 2 years when stored

at 4�C.27,41

One millimeter of ozonated oil (estimated at 14 lg

O3/mL) was applied daily for 1 week on post-harvested

palatal donor sites. Previous studies have revealed that

ozone concentrations in the range of 80–100 lg/mL pro-

duce ‘‘wound cleansing’’, that is, disinfection, whereas

low-concentration ozonated solutions, ranging from 10 to

40 lg/mL, produce ‘‘wound healing’’, that is, epithelial-

ization and granulation.24,42 Ozone concentrations greater

than 80 lg/mL were believed to have a cytotoxic effect.24

In principle, these empirically determined values have

been confirmed by studies to impact the effect of ozone

on immunocompetent cells in whole blood.24,42 The exact

stimulation mechanism of the low-dose ozonated oil to

the tissue (e.g. granulation and epithelization) is still

unclear.

In the present study, palatal wound-healing rates were

assessed by the change in wound area and shape factor19

of the measured wounds. Several investigators have sug-

gested that the use of surface area to its perimeter (S/P

ratio) is a useful method to characterize wound-healing

rates.43–45 Changes in this parameter are a measure of

the change in a wound’s effective radius, which is an

index of movement of a wound’s margin towards the

center for healing. The S/P ratio has been used to assess

healing rates in venous ulcers, and has been reported to

be a suitable indicator of linear healing per day.43,44 The

S/P ratio has also been used to predict time for wound

closure based on a non-linear delayed exponential model

of healing, which seems to offer some predictive

features.



We used an exfoliative cytological technique to assess

the epithelial healing of palatal wounds. Selection of the

cytological technique was based on the fact that exfolia-

tive cytology, in contrast to histological studies, permits

repeated, non-destructive observations. Moreover, cyto-

logical techniques have also been widely used for testing

therapeutic effects.46 However, if errors of diagnostic

interpretation are avoided, and well-established cell indi-

ces are used, sufficiently accurate results can be achieved.

Experimental long-term observations have also confirmed

the consistency of smear results in healing tissues.

Our results showed significant improvement in epithe-

lial healing after topical ozone application on palatal

wound sites. At present, there are few published data and

anecdotal reports suggesting enhancement of wound heal-

ing after the application of ozonated oil. The exact mech-

anism behind it is still unclear. Therefore, for future

prospects, longer-term studies with histopathological and

immunohistochemical evaluations should be undertaken

to ascertain the efficacy of ozone on the healing of oral

wounds.

Acknowledgment

The authors express their gratitude to Mr Paul Harvey,

Director of Gnosis Wellness Centre Inc. British Colombia,

Canada, for his support in providing the ozonated

organic olive oil used in this study.

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The periodontal abscess: a review

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