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Original Article Comparative evaluation of effect of three

different mineral trioxide aggregate solvents on
calcium content of root dentin: An in vitro study
Payal Batavia, Vaishali Parekh, Palak Batavia1, Paras Kothari, Hetal Chappla,
Mayurika Dabhi
Department of Conservative Dentistry and Endodontics, K. M. Shah Dental College and Hospital, Pipariya,
Vadodoara, Gujarat, India, 1Department of Periodontics, College of Dental Science, Davangere

Address for correspondence: Dr. Payal Batavia, E-mail: payalbatavia@gmail.com

ABSTRACT
Aim: The aim of this study is to evaluate the calcium content of solutions of three different mineral trioxide aggregate (MTA)
solvents after immersion of root dentin at different time interval. Materials and Methods: A total of 36 extracted premolars
were used in the study. Teeth were sectioned 2 mm using hard tissue microtome. One section was selected from each tooth.
12 sections were then immersed in the freshly prepared 17% ethylenediaminetetraacetic acid (EDTA), 17% carbonic acid, and
10% citric acid. Calcium dissolution was checked at different time interval of 10 and 15 min and 32 h with atomic absorption
spectroscopy. Results were analyzed using one-way ANOVA test and multiple comparisons were carried out by Tukey’s test.
Results: About 17% carbonic acid showed maximum calcium dissolution followed by 17% EDTA and 10% citric acid. There was
significantly difference in result. Conclusion: About 17% carbonic acid used for MTA retrieval can cause calcium dissolution
of dentin and lessen the strength.

CLINICAL SIGNIFICANCE
Mineral trioxide aggregate is bioactive material used widely for different clinical use, it has major disadvantage of retrieval.
There are different solvents used for the same. Those may affect the organic content of dentin.

Key words: Calcium dissolution, carbonic acid, citric acid ethylenediaminetetraacetic acid,
mineral trioxide aggregate

INTRODUCTION Most investigations reported low or no solubility for

mineral trioxide aggregate (MTA).[4,5] The inherent

E ndodontic retreatment has been defined as

a procedure performed on a tooth that has
received prior attempted definitive treatment
resulting in a condition requiring further endodontic
treatment to achieve a successful result. Retreatment
is clearly indicated when a periapical lesion, clinical
signs, and/or symptoms are present.[1,2] Retrieval is a
prerequisite for any root canal filling material, so that
it is possible to re-treat in case of failures.[3]
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disadvantages of the material are its prolonged
setting time (2 h and 45 min) and its inability to be
retrieved from within the root canal.[6,7] Studies have
shown that the surface hardness of MTA is reduced
by acids. 17% carbonic acid[8] was selected as one
of the chemicals for disintegrating MTA and retrieve
MTA. While 17% ethylenediaminetetraacetic
acid (EDTA) [9] can af fect the setting and 10%
citric acid [9] ar e the common acids used for
dissolving MTA. The solvents used might pose
potential damage to the tooth structure’s organic
and inorganic proportion.

Thus, null hypothesis of this study was that the

DOI: solvents (17% carbonic acid, 10% citric acid, and 17%
10.4103/2229-5194.134998
EDTA) when used for retrieval of MTA does not affect
the calcium content of root dentin.

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Parekh, et al.: Effect of three MTA solvents on root dentin
MATERIALS AND METHODS
A total of 36 extracted teeth were selected and were
divided into three groups. Group 1 ( n = 12): 17%
EDTA (AVA Chemicals Pvt. Ltd.) (freshly prepared),
Group 2 (n = 12): 17% carbonic acid (Arihant chemicals
Pvt. Ltd.) group (freshly prepared), Group 3 (n = 12): 10%
citric acid (Arihant Pvt. Ltd.) (freshly prepared). Inclusion
criteria included single rooted teeth and absence of severe
curvatures on root of the tooth. While teeth with cracks
and root fracture, teeth with previous restorations, teeth
with anatomical variations, teeth with severe curvatures,
teeth with calcifications, teeth with narrow pulp space,
teeth with root caries.
Preparation of sample
Teeth were prepared after scaling [Figure 2a] and then
were stored in distilled water [Figure 2b]. Root cementum
was removed with low speed fine grain diamond (Mani,
Dentsplky EF 11) under abundant irrigation with water
[Figure 2c]. The root canals were instrumented using peso
no. 4-6 mounted (Mani) on contra angle handpiece (NSK
panaair). After each use of an instrument the root canals
were irrigated to eliminate any possible debris remains.
Instrumentation was considered completed when canal
walls were visibly smooth and free of debris.
Sectioning of sample using microtome
Teeth were mounted in acrylic blocks. The crown plus
apical third of the specimen were sectioned using hard
tissue microtome [Figure 2d and e] (Leica 1600. Three
transversal sections of 2 mm thickness were obtained from
each root using hard tissue microtome. All sections were
stored in distilled water.
Preparation of solution
17% EDTA, 17% carbonic acid and 10% citric acid solutions
[Figure 2f] were freshly prepared by maintaining the pH of
5.1. Initially, 36 ml of solution was taken in a beaker as a
blank to determine calcium levels in absence of specimen.
Each specimen was immersed in each beaker containing
the corresponding solution. A volume of 3 ml aliquots
were extracted, at an interval of 10 min, 15 min, and 32 h,
using calibrated micro pipette from each beaker, which
was discarded after each extraction. These extracts were
placed in hermetically sealed and labeled glass tubes. By
this three extracts (at 10 min, 15 min, and 32 h) were
obtained for each sample specimen.
Determination of dissolution of calcium
In these three extracts, the calcium concentration of the
solution was determined by means of atomic absorption
spectrophotometer (spectra AA500). Extract readings
were expressed in ppm and then transformed into
Journal of Interdisciplinary Dentistry / Jan-Apr 2014 / Vol-4 / Issue-1
milligram of calcium lost per gram by applying the
following formula:[8]
mgCa2+/g = ([ppmCa2+]·10−3 L/mL·V)/P
ppmCa2+ = ppm of calcium in each time period,
V = volume of solution in ml (at 10 min, V1; at 15 min, V2;
and at 32 h, V3)
P = weight of solution in mg.
For the 10 min period the volume of solution (V1) was
36 ml, whereas at 15 min the volume (V2) was 33 ml
because 3 ml of solution had previously been removed.
Therefore, the mg of Ca2+ corresponding to 3 ml of V1
solution has to be added to the mg of Ca2+ obtained
at 15 min. Likewise, because the volume was 30 ml at
32 h, the mg of Ca2+ corresponding to 3 ml of V1 solution
plus 3 ml of V2 solution were added to those obtained
at 32 h sample. [8] The amount of change in calcium
concentration was calculated by the help of atomic
absorption spectrometry.
The statistical analysis (software used was IBM SPSS
Statistics) was carried out for dif ferent groups and
time interval with one-way ANOVA and then multiple
comparison with Tukey’s.
RESULTS
Calcium concentration for all three freshly prepared
solutions (17% EDTA, 17% carbonic acid, and 10% citric
acid) was checked in different time interval of 10 min,
15 min, and 32 h. The results were expressed in mg/l.
And the mean value for all the groups is given in Table 1.
The results [Table 1] showed that the maximum dissolution
of calcium of about average 7.683 mg/l in 10 min, 7.968
in 15 min, and 8.430 mg/l in 32 h, from root dentin was
by 17% carbonic acid. This may be due to the strong
chelating effect. While 17% EDTA showed the second
dissolution effect of about average of 5.218 mg/l in
10 min, 5.348 mg/l in 15 min, and 5.704 mg/l in 32 h. And
10% citric acid showed the minimum dissolution of about
average of 4.032 mg/l in 10 min, 4.144 mg/l in 15 min,
and 4.372 mg/l in 32 h. The results [Table 2] indicate
that there was highly statistical difference between
the groups. 17% carbonic acid showed the maximum
Table 1: Mean values (mg/l) for calcium
concentration for different time interval
Calcium concentration
After After After
10 min mg/l 15 min mg/l 32 h mg/l
Group 1: (17% EDTA) 5.218 5.348 5.704
Group 2: (17% carbonic acid) 7.683 7.968 8.430
Group 3: (10% citric acid) 4.032 4.144 4.372
EDTA=Ethylenediaminetetraacetic acid
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Parekh, et al.: Effect of three MTA solvents on root dentin

dissolution followed by 17% EDTA and 10% citric acid, the consolidated precipitate because ions from the same
respectively [Figure 1]. time, ions from the solution are precipitated as solids. The
concentration of the salt remains constant and therefore
When evaluated for different time interval 32 h had highly the product of concentrations of the ions in the solution at a
significant (P < 000.) difference compared to 15 min and given temperature (the solubility product) remains stable.
10 min in all the three groups [Table 3]. The lyophobic substances such as dentine have mainly
the mineral content as phosphate and calcium, which are
soluble in water. When the disodium salt of EDTA is added
DISCUSSION to this equilibrium, calcium ions are removed from the
solution.[15] This leads to the dissolution of further ions from
The three freshly prepared solution used in this study dentine, so that the solubility product remains constant.
were 17% carbonic acid, 17% EDTA, and 10% citric acid. Thus, chelator causes decalcification of dentine.[16]
These are the solutions generally used for the retrieval of
MTA. MTA has shown to take minimum 10 and 15 min for
dissolution.[9] The minimum time required for decalcifying Table 2: Statistical analysis: ANOVA test
effect of EDTA is 10 min and 15 min.[10] When MTA is Groups Sum of df Mean F P value
squares square
exposed to MTAD (a root canal cleanser containing
Between groups 272.677 2 136.339 2.353E3 0.000
citric acid), its surface characteristics are altered. They also Within groups 5.041 87 0.058
proposed that it might take 32 h for complete dissolution. Total 277.718 89
Hence, we took even 32 h to check the dissolution effect ANOVA=Analysys of variance, df=Degree of freedom
on root dentin.[11,12] 15-17% EDTA is the most commonly
used calcium chelator, which has inhibited the setting of
MTA. The term chelate is originated from a Greek word Table 3: Multiple comparisons using Tukey’s test
‘chele’ (crab claw). Chelation is a process that involves (I) group (J) group Mean Standard P value 95% confidence
difference error interval
the uptake of multivalent positive ions by these agents. (I-J) Lower Upper
Chelating agents used in endodontics are in aid to bound bound
preparation of narrow and calcified canals. In the specific EDTA Carbonic −2.6035556* 0.0694850 0.000 −2.769241 −2.437870
case of dentin, the chelator reacts with the calcium ions acid
Citric 1.2405556* 0.0694850 0.000 1.074870 1.406241
in the hydroxylapatite crystals. This process can cause acid
changes in the microstructure of the human dentin and Carbonic EDTA 2.6035556* 0.0694850 0.000 2.437870 2.769241
changes in the Ca/P ratio.[13] acid Citric 3.8441111* 0.0567343 0.000 3.708829 3.979393
acid
Citric EDTA −1.2405556* 0.0694850 0.000 −1.406241 −1.074870
The calcium ions (Ca²+) present in hydroxyapatite crystals acid Carbonic −3.8441111* 0.0567343 0.000 −3.979393 −3.708829
are one of the main inorganic elements of dentin.[6] It has acid
been reported that some chemicals used for endodontic *The mean difference is significant at the 0.05 level.
irrigation are capable of causing alterations in the chemical EDTA=Ethylenediaminetetraacetic acid

composition of dentin.[7,8]

Nygaard-Ostby (1957) used the principle of a constant

solubility product to explain the demineralization of dental
hard tissue by EDTA and its sodium salt.[14] An equilibrium
is established between the saturated salt solutions and

Figure 2: (a) Scaling of extracted tooth done. (b) Removal of cementum

from outer surface. (c) Access opening and removal of pulpal tissue and
irrigation with saline. (d) Hard tissue microtome. (e) Sectioning done
with hard tissue microtome. (f) Sections placed in respective solutions.
Figure 1: Graphical presentation of results (g) Calcium level measured with atomic spectroscopy

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Parekh, et al.: Effect of three MTA solvents on root dentin
The exchange of calcium from the dentine by hydrogen
results in a subsequent decrease in pH. Due to the release
of acid, the efficiency of EDTA decreases with time; on
the other hand, the reaction of acid with hydroxyapatite
affects the solubility of dentine. Chemically, two coexisting
reactions can be distinguished;[16]
Complex formation
EDTAH3− + Ca2+ = EDTACa2− + H
And protonation
EDTAH3− + H = EDTAH22− (Perez et al. 1989).
As this reaction proceeds, acid accumulates and
protonation of EDTA prevails, thus decreasing the rate of
demineralization.
Morrison[9] described that carbonic acid (pH - 5.45)
exposure significantly decreased the surface hardness of
WMTA (white mineral trioxide aggregate) after day 1 and
after 21 days of setting.[17]
The chemical reactions are as follows:
H2CO3wx2H+ + CO32-
Ca (OH) 2 + H2CO3/Ca2CO3 (calcium carbonate).
This reaction that occurs when carbonic acid is used as a
solvent for WMTA is similar, but more aggressive than that
reported by Holland.[15] They have observed that calcium
hydroxide formed in MTA dissociates to calcium and
hydroxyl ions when it comes in contact with tissue fluid.
This calcium ion interacts with carbon dioxide in the tissues,
forming calcium carbonate granulations and calcite crystals.
In study conducted by Nandini et al.[8] the interaction of
WMTA with carbonic acid was more aggressive, because
the carbon dioxide released is not buffered, whereas in
periapical tissues the fluids buffer the released carbon
dioxide. While in our study, the carbonic acid released the
highest amount of calcium ions due its chelating effect.
Citric acid, a weak organic acid, has been applied
previously on root surfaces altered by periodontal disease
and instrumentation in order to increase cementogenesis
and to accelerate healing and regeneration of a normal
periodontal attachment after flap surgery.[9] In endodontic
research, Loel (1975) used 50% citric acid alternately
with 5% NaOCl during instrumentation and found that
it was an effective agent for removing necrotic tissue
and preparing the dentine for subsequent sealing with
endodontic sealers. Tidmarsh (1978) also reported that
50% citric acid irrigation was effective in the removal of
the superficial smear layer.
Journal of Interdisciplinary Dentistry / Jan-Apr 2014 / Vol-4 / Issue-1
To reduce the risk of section damage, cutting and precision
grinding of the tooth specimens needs to be done with
the teeth embedded in artificial resin. These hydrophobic
materials cannot adhere to the tooth nor infiltrate it in its
hydrated state. In increasing concentrations of alcohol,
water is replaced by alcohol in stages. Resecting the
tooth’s root reduces the diffusion paths and exposure
times. Microtome is a method for the preparation of thin
sections for materials such as bones, minerals and teeth,
and an alternative to electro-polishing and ion milling.[18]
Atomic absorption spectroscopy[19] is a spectroanalytical
pr ocedur e for the quantitative deter mination of
chemical elements employing the absorption of optical
radiation (light) by free atoms in the gaseous state. The
technique makes use of absorption spectrometry to assess
the concentration of an analyte in a sample. In short, the
electrons of the atoms in the atomizer can be promoted
to higher orbitals (excited state) for a short period of
time (nanoseconds) by absorbing a defined quantity of
energy (radiation of a given wavelength).[10] This amount
of energy, that is, wavelength, is specific to a particular
electron transition in a particular element. In general, each
wavelength corresponds to only one element, and the
width of an absorption line is only of the order of a few
picometers (pm), which gives the technique its elemental
selectivity. Atomic absorption spectrophotometry was
applied to the determination of calcium, It was shown that
this technique is superior to existing methods in regard to
accuracy, precision, and ease and speed of performance.[19]
The results of this study indicate there is significant
difference between the three groups. 10% citric acid
shows the least decalcification in dentin due to its less
chelating effect followed by 17% EDTA and 17% carbonic
acid, respectively.
CONCLUSION
Although MTA has revolutionized the field of endodontics
with its biocompatible, hard tissue conductive, hard tissue
inductive properties, the different acids, that is, 10% Citric
acid, 17% EDTA and 17% carbonic acid that are used for its
retrieval has proven to be harmful to the calcium content
of the root dentin.
REFERENCES
1. Lewis RD, Block RM. Management of endodontic failures. Oral Surg
Oral Med Oral Pathol 1988;66:711-21.
2. Sjogren U, Hagglund B, Sundqvist G, Wing K. Factors affecting the
long-term results of endodontic treatment. J Endod 1990;16:498-504.
3. Bergenholtz G, Lekholm U, Milthon R, Heden G, Odesjö B,
Engström B. Retreatment of endodontic fillings. Scand J Dent Res
1979;87:217-24.
11
[Downloaded free from http://www.jidonline.com on Tuesday, December 22, 2015, IP: 106.217.89.116]

Parekh, et al.: Effect of three MTA solvents on root dentin

4. Lee SJ, Monsef M, Torabinejad M. Sealing ability of a mineral
trioxide aggregate for repair of lateral root perforations. J Endod
1993;19:541-4.
5. Steinig TH, Regan JD, Gutmann JL. The use and predictable placement
of Mineral Trioxide Aggregate in one-visit apexification cases.
Aust Endod J 2003;29:34-42.
6. Ford TR, Torabinejad M, Abedi HR, Bakland LK, Kariyawasam SP.
Using mineral trioxide aggregate as a pulp-capping material.
J Am Dent Assoc 1996;127:1491-4.
7. White C Jr, Bryant N. Combined therapy of mineral trioxide aggregate
and guided tissue regeneration in the treatment of external
root resorption and an associated osseous defect. J Periodontol
2002;73:1517-21.
8. Nandini S, Natanasabapathy V, Shivanna S. Effect of various
chemicals as solvents on the dissolution of set white mineral trioxide
aggregate: An in vitro study. J Endod 2010;36:135-8.
9. González-López S, Camejo-Aguilar D, Sanchez-Sanchez P,
Bolaños-Carmona V. Effect of CHX on the decalcifying effect of 10%
citric acid, 20% citric acid, or 17% EDTA. J Endod 2006;32:781-4.
10. Poggio C, Dagna A, Colombo M, Rizzardi F, Chiesa M, Scribante A,
et al. Decalcifying effect of different ethylenediaminetetraacetic
acid irrigating solutions and tetraclean on root canal dentin. J Endod
2012;38:1239-43.
11. Boutsioukis C, Noula G, Lambrianidis T. Ex vivo study of the efficiency
of two techniques for the removal of mineral trioxide aggregate
used as a root canal filling material. J Endod 2008;34:1239-42.
12. Smith JB, Loushine RJ, Weller RN, Rueggeberg FA, Whitford GM,
Pashley DH, et al. Metrologic evaluation of the surface of white MTA
after the use of two endodontic irrigants. J Endod 2007;33:463-7.
13. Luuko K, Kettunen P, Fristad I, Berggreen E. structure and function
of the dentin-pulp complex. In: Hargreaves KM, Cohen S, editors.
Cohen’s Pathways of Pulp. 10th ed. St. Louis, Missouri: Mosby
Publication; 2011. p. 452-503.
14. Machado-Silveiro LF, González-López S, González-Rodríguez MP.
Decalcification of root canal dentine by citric acid, EDTA and sodium
citrate. Int Endod J 2004;37:365-9.
15. Holland R, Mazuqueli L, de Souza V, Murata SS, Dezan Júnior E,
Suzuki P. Influence of the type of vehicle and limit of obturation on
apical and periapical tissue response in dogs’ teeth after root canal
filling with mineral trioxide aggregate. J Endod 2007;33:693-7.
16. Rimmele´G, Barlet-Goue´dard V, Porcherie O, Goffe´B, Brunet F.
Heterogenous porosity distribution in Portland cement exposed to
CO2 rich fluids. Cement Concrete Res 2008;38:1038-48.
17. Namazikhah MS, Nekoofar MH, Sheykhrezae MS, Salariyeh S, Hayes SJ,
Bryant ST, et al. The effect of pH on surface hardness and microstructure
of mineral trioxide aggregate. Int Endod J 2008;41:108-16.
18. Morrison RT, Boyd RN. Organic Chemistry. 5th ed. New Delhi:
Prentice-Hall India Ltd.; 1987. p. 256.
19. Skoog DA, Holler FJ, Nieman TA. Principles of Instrumental Analysis.
5th ed. Eastern Press, Bangalore: Brooks/Cole Publishing; 1998.
p. 18.
How to cite this article: Batavia P, Parekh V, Batavia P, Kothari P, Chappla H,
Dabhi M. Comparative evaluation of effect of three different mineral trioxide
aggregate solvents on calcium content of root dentin: An in vitro study.
J Interdiscip Dentistry 2014;4:8-12.
Source of Support: Nil, Conflict of Interest: None declared.

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Original Research

Use of hyaluronan (Gengigel) in the treatment

of gingivitis in orthodontic patients: A clinical,
biochemical, and microbiological study
Palak D Batavia, Kala S Bhushan, KL Vandana, Rajendra Desai1
Departments of Periodontics, 1Oral and Maxillofacial Surgery, College of Dental Sciences, Davangere, Karnataka, India

ABSTRACT
Introduction: To compare the effects of hyaluronan (Gengigel) alone and in combination with scaling using
clinical, microbial, and lactate dehydrogenase (LDH) parameters. Materials and Methods: In this, the three
treatment groups included were scaling, scaling plus local application of Gengigel, and Gengigel alone. The
0.2% hyaluronic acid (HA) gel was applied topically and intrasulcularly 0.8% hyaluronan was applied. The
clinical parameters, and microbial and biochemical analyses of gingival crevicular fluid (GCF) LDH were
assessed. Intragroup comparisons were made by Student’s unpaired t-test and intergroup comparisons
were done using one-way analysis of variance followed by post hoc Turkey’s test. Results: At the end of Access this article online
study period (0-56 day), intergroup comparison demonstrated no significant reduction in plaque index (PI) Website: www.jicdro.org
(0.07 NS), gingival index (GI) (0.99 NS), and LDH (0.70 NS) values. A significant correlation was found DOI: 10.4103/2231-0754.176255
between LDH values and bleeding index at all study intervals (0 days, 28 days, and 56 days); gingival index Quick Response Code:
(GI) (0.007 S) was significantly correlated on day 0 and day 56. The microbial reduction was demonstrated.
Conclusion: These changes in the clinical, microbial, and biochemical parameters reported with the different
treatment modalities clearly support that the use of Gengigel would act as an advantageous adjunct to
scaling. Further studies are required to confirm the Gengigel effect using histologic methods.

Key words: Gel, gingival crevicular fluid (GCF), gingivitis, hyaluronan, orthodontic brackets, L-lactate
dehydrogenase (LDH)

INTRODUCTION Lactate dehydrogenase (LDH), an enzyme normally limited

The retentive areas created by dental restorations and
orthodontic appliances could increase the amount of
plaque around the teeth, i.e., bacterial colonization,
particularly periodontopathogens such as Treponema denticola,
Porphyromonas gingivalis, Tannerella forsythia (formerly
Bacteroides forsythus), Prevotella nigrescens, Prevotella intermedia,
and Aggregatibacter actinomycetemcomitans in subgingival
dental plaque.[1]
to the cytoplasm of cells, is only released extracellularly
after cell death. In the literature there are various studies,
which have demonstrated that the activity of LDH in GCF is
significantly correlated with gingival inflammation,[3-5] and
tissue destruction from periodontitis.[6,7] An increase in LDH
activity during the early phases of orthodontic treatment is
indicated as a possible diagnostic tool for tissue response
during orthodontic treatment.[7-9]

This is an open access article distributed under the terms of the

In the gingival crevicular fluid (GCF), Uematsu et al. found
several cell mediators such as interleukin1β, interleukin 6,
tumor necrosis factor-α, epidermal growth factor, and β2
microglobulin, significantly elevated in teeth undergoing
orthodontic forces, compared with untreated controls.[2]
Creative Commons Attribution-NonCommercial-ShareAlike 3.0
License, which allows others to remix, tweak, and build upon the
work non-commercially, as long as the author is credited and the
new creations are licensed under the identical terms.
For reprints contact: reprints@medknow.com

Address for correspondence:

Dr. Vandana KL, Department of Periodontics, Cite this article as: Batavia PD, Bhushan KS, Vandana KL, Desai R. Use
Room 4, College of Dental Sciences, of hyaluronan (Gengigel) in the treatment of gingivitis in orthodontic patients:
Davangere - 577 004, Karnataka, India. A clinical, biochemical, and microbiological study. J Int Clin Dent Res Organ
E-mail: vanrajs@gmail.com 2016;8:44-50.

44 © 2016 Journal of the International Clinical Dental Research Organization | Published by Wolters Kluwer - Medknow
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Batavia, et al.: Gengigel in orthodontic gingivitis patients
The topical application of a high-molecular weight exogenous
hyaluronan (HA)-based gel (Gengigel; Ricerfarma, Milan,
Lombardy, Italy) has been proposed to induce periodontal
healing in patients with inflammatory gingivitis, during both
open and randomized, controlled, double-blind studies.[10-12]
A Medline search including the key words “gingivitis,”
“orthodontic appliances,” and “hyaluronic acid” revealed scant
results. The intrasulcular and extrasulcular applications of HA
gel is used effectively in the treatment of gingivitis;[13] however,
its effectiveness on orthodontic patients with gingivitis has
not been tried. A recent study by Pistorius et al.[14] using HA
spray for the treatment of gingivitis reported significant
improvement in gingivitis. Sapna and Vandana[13] reported
that hyaluronan gel is an effective topical agent for treating
gingivitis, along with scaling and intrasulcular application.
Thus, the purpose of the present study was to compare the
benefits of hyaluronan (Gengigel) alone and in combination
with scaling using clinical, microbial (periodontopathogens),
and biochemical (LDH) parameters.
MATERIALS AND METHODS
This randomized, controlled, parallel design study was
conducted on patients attending the Department of
Periodontics. Thirty-two patients in the age group of 18-25
years undergoing orthodontic treatment with plaque-induced
gingivitis with a probing depth <3 mm were selected. Written
informed consent was obtained from all patients prior to
their participation in this study and was approved by ethical
committee.
Subjects were enrolled if they had a medical history
showing good general health, at least 24 natural teeth in
the mouth excluding the third molars, and also full-mouth
fixed orthodontic needed to have at least 50% bleeding
sites and 50% dichotomous plaque score. Oral and written
information was given to each enrolled subject. Both the
parent and subject signed the consent form. The exclusion
criteria included
1. Medical history of the liver, heart, kidney, and muscle
diseases that are known to affect LDH levels,
2. Patients who had taken antibiotic therapy in the month
prior to the commencement of the study, and
3. Periodontal therapy in the last 6 months prior to the
commencement of the study
4. smokers,
5. Pregnant and lactating women
6. Advanced periodontitis or rampant dental caries based
on a noninvasive examination, and
7. Removable oral prostheses or removable orthodontic
appliances.
Journal of the International Clinical Dental Research Organization | January-June 2016 | Vol 8 | Issue 1
In this randomized, single-blinded study, 32 patients were
included. The different treatment modalities were control
group, scaling alone; experimental group A, only topical
application of HA gel (Gengigel; Ricerfarma, Milan, Lombardy,
Italy); and experimental group B, both scaling and topical
applications of HA gel. The allotment of different treatment
protocols to different groups was done randomly, and the
main investigator was blinded for the treatment protocol.
The randomization code was concealed until the results
were analyzed.
Prior to the treatment, the indices assessed included clinical
parameters: Plaque index (PI),[15] gingival index (GI),[16] and
gingival bleeding index (GBI);[17] microbial assessment[18] and
biochemical analysis of GCF LDH.[9] These were recorded by
the main investigator and were repeated on days 0 and day
28. Any adverse reactions to the gel were also recorded.
The duration of the study was 56 days. From day 0 to day 28
day, scaling + gel application was performed in one of the
selected groups and in the other group, the application of gel
was done. The control group received only scaling. The 0.2%
HA gel was applied topically (extrasulcularly) on the gingival
surface, intrasulcularly 0.8% hyaluronan was applied by the
coinvestigator [Figures 1 and 2]. The patients were instructed
to brush twice daily with a conventional toothbrush and
toothpaste using roll-on technique as part of regular plaque-
control measures. They were advised to apply the gel on the
gingiva topically with the help of cotton bud applicator twice
daily for 28 days after regular oral hygiene regimen. After
application, the patients were instructed to avoid eating,
drinking, or rinsing for 1 h. Intrasulcular applications were
done with an applicator by the co-investigator during their
weekly visit. The gel was placed intrasulcularly until the level
of the gingival margin, and it could be retained in the sulcus
to a better extent, unlike a solution that flows out of the
sulcus. An observation period up to 56 days was considered
for all three treatment groups.
Figure 1: gengigel
45
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Batavia, et al.: Gengigel in orthodontic gingivitis patients

Supragingival microbial sample collection was collected RESULTS

at the baseline and 28th day. After recording the clinical
parameters, sterile universal curette (Columbia 2R/2L; Of the 32 patients, seven did not attend clinics at the subsequent
4R/4L, Hu-Friedy, Chicago, USA) was used to collect plaque visits and were therefore, considered spontaneous dropouts and
sample and then transferred to 1 mL thioglycolate broth excluded from the trial. The trial was therefore, completed using
(transport medium).[19] The vial was sealed tightly to avoid 25 patients; the results were computed including these data.
contamination and labeled. The labeled vials were sent to Comparison of PI, GI, gingival bleeding index, microbiological
the microbiological laboratory within 24 h of collection. and biochemical evaluations was done from the baseline to
Samples were processed within 2 days of collection. The the 28th day [Table 2]. The clinical parameters and LDH values
collected samples were subjected to anaerobic culture were reevaluated after an observational period at on the 56th
method for the detection of periodontopathogens day. The results are presented in Tables 1-6.
such as Porphyromonas gingivalis, Prevotella intermedia,
Aggregatibacter actinomycetemcomitans, and Actinomycetes At the end of study period (0-56 days) [Table 3], intergroup
species. The plates were inoculated for 72 h and after comparison demonstrated no significant reduction in PI
this specified period, the plates were taken out of the jar; (0.07 NS), GI (0.99 NS), and LDH (0.70 NS) values. The GBI
the colony characters such as size, shape, hemolysis and demonstrated significant reduction; the highest reduction
pigmentation were noted and the numbers of each colony
type were counted. The count was multiplied by 200
(dilution factor) to express colony forming units (CFUs).[7]
The CFUs were expressed in the tabular form with grading
where the individual pathogens are marked as follows:
(–) undetected, which corresponds to the number of
bacteria less than 103, (+) slightly positive corresponding
to the number of bacteria 103 to 104; (++) positive, which
corresponds to the number of bacteria 104 to 105; and
(+++) strongly positive, with the number of bacteria
higher than 105. The organisms were further quantified by
making use of Gram stain and key biochemical reactions
as per the standard protocol and expressed in the form of
percentages.[18] Figure 2: apllication of gengigel

GCF collection and LDH enzyme activity determination were

Table 1: Inter group comparison: Baselines values
carried out at the baseline and at the 28th day, along with
Parameter   Gel Gel + Scaling P* Value*,
the clinical parameters. The GCF volume in each paper point alone Scaling sig
was calculated and analyzed for LDH enzyme activity.[7,9] Plaque Index Mean 1.791 1.807 1.725 0.71 NS
SD 0.159 0.242 0.284
All the vials containing GCF samples were put in the Gingival Index Mean 1.725 1.672 1.748 0.76 NS
spectrophotometric automatic apparatus (COBAS INTEGRA SD 0.240 0.279 0.186
Gingival Bleeding Mean 89.687 84.075 89.262 0.56 NS
400 plus; Roche Diagnostics, Mannheim, Baden-Württemberg, Index SD 12.153 12.575 14.126
Germany) to be automatically analyzed without any manual LDH Values Mean 72.400 59.800 60.300 0.43 NS
SD 24.740 28.732 17.733
procedures.[7,9] LDH total unit activity = GCF volume X volume
*Oneway ANOVA test; **Tukey’s post hoc test; p < 0.05 = significant; NS = Non Significance;
activity X L/106 µl was calculated. S = Significance.

Table 2: Inter group comparison: Clinical parameters and LDH values at 0-28 days
Parameter   Gel alone Gel + Scaling Scaling P* Value*, sig Significant pairs**
Plaque Index Mean 0.166 0.243 0.183 0.07 NS —
SD 0.206 0.141 0.144
Gingival Index Mean 0.271 0.181 0.273 0.46 NS —
SD 0.206 0.043 0.152
Gingival Bleeding Index Mean 34.381 67.044 59.978 0.004 S Gel alone & Gel + Scaling;
SD 19.250 8.111 17.067 Gel alone & Scaling
LDH Values Mean 31.800 27.200 34.600 0.16 NS —
SD 10.581 13.903 11.098
*Kruskall Wallis test; ** Mann Whitney U test; p < 0.05 = significant; NS = Non Significance; S = Significance.

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Batavia, et al.: Gengigel in orthodontic gingivitis patients

Table 3: Inter group comparison: Clinical parameters and LDH values at 0-56 days
Parameter   Gel alone Gel + Scaling Scaling P* Value*, sig Significant pairs**
Plaque Index Mean 0.264 0.546 0.147 0.07 NS —
SD 0.271 0.460 0.305
Gingival Index Mean 0.173 0.204 0.151 0.99 NS —
SD 0.267 0.166 0.298
Gingival Bleeding Index Mean 40.089 68.819 61.109 0.007 S Gel alone & Gel + Scaling;
SD 19.627 9.831 16.752 Gel alone & Scaling
LDH Values Mean 39.400 34.100 29.900 0.70 NS —
SD 20.359 19.661 12.922
*Kruskall Wallis test; ** Mann Whitney U test; p < 0.05 = significance; NS = Non Significance; S = Significant

Table 4: Inter group comparison: Clinical parameters and LDH index at all study intervals (0 day, 28 days, 56 days) [Table
values at 28-56 days 5]; the GI (0.007 S) was also significantly correlated on day
28-56 Days 0 and day 56 [Table 3].
Parameter   Gel Gel + Scaling P* Value*,
alone Scaling sig On quantitative evaluation of microorganisms, the growth of
Plaque Index Mean 0.098 0.303 -0.036 0.24 NS organisms was positive (++) to highly positive (+++) in all
SD 0.258 0.451 0.350
Gingival Index Mean -0.098 0.023 -0.122 0.53 NS groups at the baseline demonstrated reduction of microbes
SD 0.310 0.161 0.307 while there was a reduction of these organisms from slightly
Gingival Mean 5.708 1.775 1.131 0.12 NS
Bleeding Index SD 6.595 4.321 2.909 positive (+) to undetected (–) [Table 6].
LDH Values Mean 7.600 6.900 -4.700 0.06 NS
SD 15.981 12.627 8.056 DISCUSSION
*Kruskall Wallis test; ** Mann Whitney U test; p < 0.05 = significant; NS = Non Significance;
S = Significance During orthodontic therapy with fixed appliances,
inflammatory reaction of gingival tissue can very often
Table 5: Correlation of various parameters with LDH values be observed. A variety of studies have reported that
Baseline values Correlation coefficient* P Value temporary changes occurring in the gingiva for an increased
Gingival Index 0.54 0.002 S accumulation of dental plaque and inflammatory response
Gingival Bleeding Index 0.38 0.04 S is due to appearance of new retentive places around the
28 Days    
Gingival Index 0.33 0.07 NS components of fixed appliances attached to the teeth and
Gingival Bleeding Index 0.37 0.04 S did not normally result in permanent periodontal losses.[19-21]
56 Days    
Gingival Index 0.57 0.001 S It has also been stated that periodontal problems during
Gingival Bleeding Index 0.48 0.04 S orthodontic treatment may be primarily attributable to poor
* Karl Pearson’s coefficient of correlation; p < 0.05 = significant; NS = Non Significace; oral hygiene. Hence, if good oral hygiene is maintained,
S = Significance
orthodontic treatment results in no harmful effects with
regard to periodontal health.[22]
Table 6: Quantitative evaluation of the micro-organisms at
different time interval Recently, exogenous hyaluronic acid, known to have an anti-
Microorganisms Gel alone SRP + gel SRP alone
(CFUs)
inflammatory effect, was introduced as a topical applicant for
0 day 28th day 0 day 28th day 0 day 28th day
the treatment of gingivitis. The topical application of a high-
Aa ++ + ++ + ++ -
Pg ++ - ++ - ++ + molecular weight, HA-based gel (Gengigel) has been proposed
Pi ++ - ++ + ++ + to have some potential in inducing periodontal healing in
Actinomycetes + + + + + -
Streptococcus ++ + + - + +
patients with inflammatory gingivitis.[11,23] It is also beneficial
aureus in accelerating the healing of periodontal wounds following
Staphylococcus ++ - +++ + - - surgery.[24] HA has been used subgingivally in the treatment
mitis
of chronic periodontitis.[13] However, it has not been trialed
(-) undetected < 103; (++) positive - 104 to 105; (+) slightly positive 103 to 104; (+++) strongly
positive > 105. intrasulcularly for the treatment of gingivitis in orthodontic
patients. In the present study, an attempt has been made
was in the gel + scaling group followed by the scaling alone to evaluate the effectiveness of Gengigel (0.2% hyaluronic
and gel alone group. The intergroup comparison (28th-56th acid) in the treatment of plaque-induced gingivitis with or
day) [Table 4] did not reveal any significant reduction in the without scaling when applied topically and intrasulcularly.
clinical parameters and LDH. Regarding the correlation of A randomized, single-blind parallel study design in which a
various clinical parameters with LDH values, a significant total of 25 patients were treated for 28 days and evaluated
correlation was found between LDH values and bleeding after an observational period of 56 days.

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Batavia, et al.: Gengigel in orthodontic gingivitis patients
The beneficial actions of HA and its tissue compliance could
be tried in periodontitis patients. The formulation of gel into
a slow and sustained release delivery system could be ideal
in periodontal treatment to combat the exaggerated effects
from inflammation and to facilitate faster healing.
Scaling as a treatment modality is compulsory for all gingivitis
patients and thus, this study focused on comparing its effect
with gel application with or without scaling. The study results
were evaluated from the baseline to day 28 and day 56.
At the baseline, there was no significant difference in the
mean plaque, gingival scores, and gingival bleeding scores
between the groups and there was also a constant reduction
in all scores from the baselines to the 56th day in each group.
The PI reduction was similar in both scaling and scaling +
Gengigel group that was similar to the findings of Pagnacco
et al.[11] and Sapna and Vandana.[13] Intergroup comparison
showed similar plaque reduction in all treatment modalities.
The plaque score reduction can be attributed to adequate oral
hygiene maintenance, removal of tooth deposits by scaling,
and bacteriostatic effects of hyaluronan as stated by Pirnazar
et al.[25] The gingival bleeding score reduction was significant
when the scaling + topical HA application was compared
with other groups. Pagnacco et al.[11] reported a significant
difference in the reduction of the scaling versus the topical HA
groups in a double-blind, parallel group study of 29 patients
who were examined for a period of 4 weeks. The reduction
in the bleeding score was also significant between the scaling
and the topical HA group while Sapna and Vandana[13] reported
a significant reduction in gingival bleeding score in gel +
scaling group. The results of these findings can be attributed
to the anti-edematous and scavenger effect of hyaluronic acid
as stated by Laurent et al.[26]
The anti-inflammatory effect of HA can be attributed to its
action of deactivating bacterial hyaluronidases, normalizing
the macroaggregation of connective tissue proteoglycans,
and bonding with free water, thus performing an anti-
edema effect.[10] It can be said that bleeding on probing is
more accurate in predicting gingival health compared to the
GI,[27] and also GBI obviates the problems associated with
subjective interpretations of visual changes of gingiva such
as the presence or degree of erythema and edema.[28]
A study by Yi Xu et al. (2004)[29] concluded that no clinical
improvement was achieved by the adjunctive use of
hyaluronan 0.2% gel compared to mechanical debridement.
However, in their study hyaluronan 0.2% gel was applied
only once a week for 6 weeks; a total of seven applications
was administered over a 6-week period, compared to
the recommended application level of three times daily
for at least 4-8 weeks. The absence of observed clinical
48 Journal of the International Clinical Dental Research Organization | January-June 2016 | Vol 8 | Issue 1
improvements contrary to other published studies may
indicate that the hyaluronan application used in this study
was well below the optimum levels required to achieve a
significant clinical improvement.
The baseline GCF LDH values continued to reduce throughout
the study period in all the treatment groups, which was not
significant between the groups. The Pearson’s coefficient
of correlation demonstrated that correlations of LDH levels
with GI and GBI were significant at the end of study period.
Along with measuring changes in clinical parameters for
reduction in gingival inflammation, this study included the
measurement of changes in total enzyme unit activity of LDH,
along with changes in GCF volume. The GCF collection for
LDH enzyme activity estimation was carried out using #30
standardized sterile paper point (SS White, New Jersey, USA)
inserted 1 mm into the gingival crevice for 30 s, similar to
the methodology of Serra et al.[7] However, the LDH enzyme
estimation in the present study was performed by a fully
automated analyzer (Roche Cobas U 411 Automated), which
can be considered as a better method of analyzing LDH
enzyme activity than using a manual spectrophotometer as
used in previous studies.[7,8]
The presence of higher LDH enzyme unit activity at the
baseline is in accordance with the results of Serra et al. (i.e.,
sites with full-mouth fixed orthodontic appliances in place
for a minimum period of 3 months showed a mean total LDH
unit activity of 72.40 ± 24.74 IU per sample) who attribute
this increased GCF LDH levels to the tissue resorption in both
the compressed and tensional sites or even secondary to a
possible cell necrosis in the periodontal ligament during the
orthodontic treatment.[8]
These GCF LDH value reduction from the baseline to 28th day
are comparable to the mean GCF LDH values for diseased sites
(0.129 ± 0.406 IU per site) and healthy sites (0.029 ± 0.035 IU
per site), respectively, as observed in a cross-sectional study
by Wolff et al.,[5] thus indicating the return of diseased sites
to healthy sites following gel application. This posttreatment
significant reduction in GCF LDH values can also be correlated
to studies showing a significant reduction in GCF LDH activity
levels after successful periodontal treatment.[5,6]
The increased severity of gingival inflammation observed
immediately after fixed appliance placement due to the
development of a stable pathogenic milieu tips the host-
parasite homeostasis in favor of the pathogen and manifests
as clinical inflammation.[30] It also stimulates the growth
of periodontopathogenic bacteria but without destructive
effects on deep periodontal tissues.[2,31]
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Batavia, et al.: Gengigel in orthodontic gingivitis patients
There are various in vitro studies[25] suggesting bacteriostatic
or bactericidal effects of various formulations of HA on
selected oral and nonoral microorganisms. In the present
study, the samples were also quantitatively analyzed the
bacteria from undetectable to strongly positive according
to grading done by Černochová et al. 2008. [18] There
was a quantitative reduction of Ag, Pg, Pi, Actinomycetes,
Staphylococcus aureus, Streptococcus mitis at the end of
28 days suggestive of the beneficial antibacterial effect of
the gel.
Recently numerous studies have been conducted to evaluate
the effect of Gengigel in chronic periodontitis patients, which
showed a positive response associated with improvement in
clinical parameters and reduction in microbial parameters by
various authors such as Johannsen et al. (2009),[32] Gonitiya et
al. (2012),[33] and Eick et al. (2012).[34]
No adverse effect was observed on clinical examination
and according to what was reported by the patients. These
findings were similar to the findings of Pagnacco et al. (1997)[11]
and Vangelisti et al. (1997).[23] The patients also presented
compliance with the regular use of Gengigel. Gengigel as a
product for oral use has been evaluated by skin irritation test,
sensitizing potentiality, and percutaneous absorption test and
has been proved to be a safe nonirritant product.
The important observation made in this study was the
effect of Gengigel on reduction of gingival bleeding in
patients with orthodontic brackets, which serve as a
plaque retentive area. The intrasulcular application of
0.8% Gengigel enhanced the tissue ability to respond
to mechanical plaque control procedures. Gengigel
application for 28 days was effective in the reduction of
clinical parameters and its application is recommended to
be continued during orthodontic treatment as a regular
oral hygiene maintenance [Table 2].
These changes in clinical, microbial, and biochemical
parameters reported with the different treatment modalities
clearly support that thorough scaling is effective as initial
therapy and the use of hyaluronan gel would act as an
advantageous adjunct. The favorable results obtained in this
study confirmed that the anti-inflammatory and antimicrobial
properties of hyaluronan serve as a promising adjunct to
the mechanical therapy of gingivitis with fixed retentive
appliances as the retentive factors.
More organized and long-term research is to be carried out
to differentiate the specific effect of 0.2% or 0.8% gel, which
can support the use of the gel in routine clinical practice.
Further studies are required to confirm the Gengigel effect
using histologic methods.
Journal of the International Clinical Dental Research Organization | January-June 2016 | Vol 8 | Issue 1
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
REFERENCES
1. Uematsu S, Mogi M, Deguchi T. Interleukin (IL)-1β, IL-6, tumor
necrosis factor-α, epidermal growth factor, and β2-microglobulin
levels are elevated in gingival crevicular fluid during human
orthodontic tooth movement. J Dent Res 1996;75:562-7.
2. Lee SM, Yoo SY, Kim HS, Kim KW, Yoon YJ, Lim SH, et al.
Prevalence of putative periodontopathogens in subgingival dental
plaques from gingivitis lesions in Korean orthodontic patients.
J Microbiol 2005;43:260-5.
3. Weinstein E, Khurana H, Mandel ID. Lactate dehydrogenase
isoenzymes in gingival fluid. Arch Oral Biol 1972;17:375-6.
4. Lamster IB, Mandella RD, Gordon JM. Lactate dehydrogenase
activity in gingival crevicular fluid collected with filter paper
strips: Analysis in subjects with non-inflamed and mildly inflamed
gingiva. J Clin Periodontol 1985;12:153-61.
5. Wolff LF, Smith QT, Snyder WK, Bedrick JA, Liljemark WF,
Aeppli DA, et al. Relationship between lactate dehydrogenase
and myeloperoxidase levels in human gingival crevicular fluid
and clinical and microbial measurements. J Clin Periodontol
1988;15:110-5.
6. Atici K, Yamalik N, Eratalay K, Etikan I. Analysis of gingival
crevicular fluid intracytoplasmic enzyme activity in patients
with adult periodontitis and rapidly progressive periodontitis.
A longitudinal study model with periodontal treatment.
J Periodontol 1998;69:1155-63.
7. Serra E, Perinetti G, D’Attilio M, Cordella C, Paolantonio M,
Festa F, et al. Lactate dehydrogenase activity in gingival crevicular
fluid during orthodontic treatment. Am J Orthod Dentofacial
Orthop 2003;124:206-11.
8. Perinetti G, Serra E, Paolantonio M, Bruè C, Meo SD, Filippi MR,
et al. Lactate dehydrogenase activity in human gingival crevicular
fluid during orthodontic treatment: A controlled, short-term
longitudinal study. J Periodontol 2005;76:411-7.
9. Dhingra K, Vandana KL. Management of gingival inflammation
in orthodontic patients with ozonated water irrigation — A pilot
study. Int J Dent Hyg 2011;9:296-302.
10. Jentsch H, Pomowski R, Kundt G, Göcke R. Treatment of gingivitis
with hyaluronan. J Clin Periodontol 2003;30:159-64.
11. Pagnacco A, Vangelisti R, Erra C, Poma A. Double-blind clinical
trial vs. placebo of a new sodium-hyaluronate-based gingival gel.
Attual Ter In 1997;15:1-7.
12. Galgut PN. The role of hyaluronic acid in managing inflammation
in periodontal diseases. Dental Health 2003;4:3-5.
13. Sapna N, Vandana KL. Evaluation of hyaluronan gel (Gengigel®)
as a topical applicant in the treatment of gingivitis. J Investig Clin
Dent 2011;2:162-70.
14. Pistorius A, Martin M, Willershausen B, Rockmann P. The clinical
application of hyaluronic acid in gingivitis therapy. Quintessence
Int 2005;36:531-8.
15. Silness P, Loe H. Periodontal disease in pregnancy. Acta Odontol
Scand 1964;22:121-7.
16. Loe H, Silness P. Periodontal disease in pregnancy. Acta Odontol
Scand 1963;21:533-8.
17. Ainamo J, Bay I. Problems and proposals for recording gingivitis
and plaque. Int Dent J 1975;25:229-35.
18. Cernochova P, Augustin P, Fassmann A, Izakovičová-Hollá
L. Occurrence of periodontal pathogens in patients treated
49
[Downloaded free from http://www.jicdro.org on Thursday, May 12, 2016, IP: 150.107.252.245]
Batavia, et al.: Gengigel in orthodontic gingivitis patients
with fixed orthodontic appliances. Scripta Medica (Brno)
2008;81:85-96.
19. Zachrisson BU, Alnaes L. Periodontal condition in orthodontically
treated and untreated individuals. I. Loss of attachment,
gingival pocket depth and clinical crown height. Angle Orthod
1973;43:402-11.
20. Zachrisson BU, Zachrisson S. Gingival condition associated with
partial orthodontic treatment. Acta Odontol Scand 1972;30:127-36.
21. Zachrisson BU. Orthodontics and periodontics. In: Lindhe J, editor.
Clinical Periodontology and Implant Dentistry. 4th ed. New Delhi:
Jaypee Brothers; 2003. p. 744-80.
22. Kloehn JS, Pfeifer JS. The effect of orthodontic treatment on the
periodontium. Angle Orthod 1974;44:127-34.
23. Vangelisti R, Pagnacco O, Erra C. Hyaluronic acid in the topical
treatment of gingival inflammations: Preliminary clinical trial.
Attualita Terapeutica Internazionale 1997;15:2-3.
24. Mantovani S, Sala Tesciat A, Fossati B. Preliminary clinical
evaluation of a hyaluronic acid-based product in oral disorders:
Double-blind trial. Attualita Terapeutica Internazionale 1998;16:1-5.
25. Pirnazar P, Wolinsky L, Nachnani S, Haake S, Pilloni A, Bernard
GW. Bacteriostatic effects of hyaluronic acid. J Periodontol
1999;70:370-4.
26. Laurent TC, Laurent UB, Fraser JR. Functions of hyaluronan. Ann
Rheum Dis 1995;54:429-32.
27. Antczak-Bouckoms AA, Tulloch JF, Berkey CS. Split-mouth
and cross-over designs in dental research. J Clin Periodontol
1990;17:446-53.
50 Journal of the International Clinical Dental Research Organization | January-June 2016 | Vol 8 | Issue 1
28. Gomes SC, Piccinin FB, Sucin C, Oppermann RV, Marcantonio RA.
Effect of supragingival plaque control in smokers and nonsmokers:
6 month evaluation of patients with periodontitis. J Periodontol
2007;78:1515-21.
29. Xu Y, Höfling K, Fimmers R, Frentzen M, Jervøe-Storm PM.
Clinical and microbiological effects of topical subgingival
application of hyaluronic acid gel adjunctive to scaling and root
planing in the treatment of chronic periodontitis. J Periodontol
2004;75:1114-8.
30. Krishnan V, Ambili R, Davidovitch Z, Murphy NC. Gingiva and
orthodontic treatment. Semin Orthod 2007;13:257-71.
31. Ristic M, Vlahovic Svabic M,  Sasic M, Zelic O. Clinical and
microbiological effects of fixed orthodontic appliances on
periodontal tissues in adolescents. Orthod Craniofac Res
2007;10:187-95.
32. Johannsen A, Tellefsen M,  Wikesjö U,  Johannsen G. Local
delivery of hyaluronan as an adjunct to scaling and root
planning in the treatment of chronic periodontitis. J Periodontol
2009;80:1493-7.
33. Gontiya G, Galgali SR. Effect of hyaluronan on periodontitis:
A clinical and histological study. J Indian Soc Periodontol
2012;16:184-92.
34. Eick S, Renatus A, Heinicke M, Pfister W, Stratul SI, Jentsch
H. Hyaluronic acid as an adjunct after scaling and root
planing: A prospective randomized clinical trail. J Periodontal
2013;84:941-9.