Serbian Dental Journal, vol.

60, No 3, 2013

ORIGINAL ARTICLE / ORIGINALNI RAD

UDC: 616.311.2-002-085.24 ; 547.495.2

DOI: 10.2298/SGS1303147D

Effect of 10% Urea Solution on Epithelialization of

Thermally Wounded Gingiva
Stevanka Djordjevi1, Dragan Ivanovi1, Tanja Ivanovi2
Department of Preventive and Pediatric Dentistry, Faculty of Medicine, University of East Sarajevo, Foa,
Republika Srpska, Bosnia and Herzegovina;
2
Department of Orthodontics, Faculty of Medicine, University of East Sarajevo, Foa, Republika Srpska,
Bosnia and Herzegovina
1

SUMMARY

Introduction Urea is the final product of protein degradation secreted in saliva. It has low molecular weight and neutral
molecule that can freely diffuse through bacterial wall, mature plaque and extracellular polysaccharides exerting a buffering effect. In the presence of urease, urea is broken down into one molecule of ammonia and two molecules of weak
carbonic acid, causing alkalization of the substrate and pH increase. Hydrolysis of urea begins at pH 4-6. In mature plaque
with low pH, urea hydrolysis is a compensatory mechanism that opposes pH decrease trying to keep an optimal acidbase balance. This mechanism explains the role of urea in controlling plaque, caries and gingivitis. The aim of this study
was to investigate the effect of urea on the quality and speed of epithelialisation of thermally wounded gingiva in rats.
Material and Methods The study included 36 Wistar albino rats, 3-4 months old, divided in three groups (12 in each
group). According to the protocol thermal wounding of gingiva was performed in all animals. Wounded gingival
epithelial tissue was treated with: 10% urea solution (experimental group), 3% hydrogen peroxide and saline (control
groups). The extent and quality of epithelialisation was verified histologically after 3, 5, 8, 11 and 14 days.
Results There was a strong cellular infiltration and stromal edema with no significant morphological differences
between groups in the samples analyzed after 3 days. The acceleration of epithelialisation in the experimental group
observed in samples obtained after 5 days was evidenced by rapid mitotic division of epithelium and initial covering of
defects in both directions. In samples obtained after 8 days, in the experimental group, epithelial defect was covered
and in some areas cells were differentiated in vertical direction. After 11 days horizontal coverage of defects as well as
accelerated cell differentiation in the vertical direction were noted. Restitutio ad integrum in the experimental group
and significant delay in wound healing in the control groups was demonstrated in 14-day samples.
Conclusion Complete epithelialisation of gingiva occurred significantly faster in the group treated with 10% urea
solution, than in the control groups treated with 3% hydrogen peroxide solution and saline.
Keywords: urea; epithelialisation; gingiva; heat deterioration

INTRODUCTION
The term oral urea considers the total urea in saliva, gin
gival sulcular fluid and plaque [1]. Urea found in plaque is
derived from saliva, food debris and produced by 1/10 of
all oral cocci present in plaque [2]. Urea is twenty times
more concentrated in plaque, where it is used mostly for
buffering and less for detoxification. Research indicates
that urea exerts bactericidal, antibacterial, amphoteric and
non allergic properties [3].
Initial reports from the first half of nineteenth century
have shown that ammonia formed from urea breaking
down directly correlates with the presence of dental caries
[4]. Anti-cariogenic effect of urea was described in several
studies where 40% urea was able to achieve reduction in
caries activity for 80-100% [5]. One study found that sa
liva of caries immune subjects contained twice more urea
than caries-sensitive patients [6]. Production of alkaline
components in plaque in caries susceptible and caries-re
sistant groups is important concept of studies that analyze
the metabolism of saliva [7].

Positive effects of urea and urea peroxide on plaque

accumulation have been documented in the number of
papers [8, 9, 10]. The view that caries prophylactic effect
of urea is not based only on the capacity to maintain pH
in the microenvironment but on a series of integral meta
bolic and biochemical activity has also been confirmed in
other studies [11, 12]. In addition, beside plaque, urea has
been found in increased concentration in gingival sulcular
fluid where it maintains acid-base balance and actively
participates in host defense [13]. The amount of ammonia
in plaque is 16-26% and in saliva 15-40 mg/100 ml. In
addition to ammonia, which represents the percentage of
utilized urea, a significant proportion of urea is incorpo
rated in proteins and amino acids. When required, urea
can be re-synthesized using these depots. This way urea
becomes a major determinant of pH increase due to the
fact that plaque microorganisms show faster and more
pronounced urealytic than glycolytic activity [14, 15].
In clinically healthy or mildly inflamed gingiva, the
amount of urea in gingival sulcular fluid is several times
higher than in saliva or serum. The decrease of urea has

Address for correspondence: Stevanka OREVI, Department of Preventive and Pediatric Dentistry, School of Medicine,
University of East Sarajevo, 73300 Foa, Republica Srpska, Bosnia and Herzegovina; stevankadjordjevic@yahoo.com

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Djordjevi S. et al. Effect of 10% Urea Solution on Epithelialization of Thermally Wounded Gingiva

been detected in severely inflamed tissue. In people with

healthy periodontium ammonia production is 2.7 times
higher than in those who have marginal gingivitis [16, 17].
Protective role of urea lies in its ability to stimulate
formation of large amounts of alanine in the presence of
glucose probably due to releasing amino groups required
for pyruvate conversion to alanine. Application of urea in
vivo and in vitro has an impact on alkalizing conditions
in plaque by increasing pH to 9. The analysis of plaque
content obtained from a large number of people has shown
high urease activity [18]. Due to the complex metabolic
and biochemical activities and non compliant principles,
protective mechanism of urea is difficult to explain.
The aim of this study was to evaluate the effect of urea
on the quality and speed of epithelialisation in thermally
altered gingiva in rats.
MATERIAL AND METHODS
The experiment was conducted on 36 Wistar rats (21 male
and 15 female), divided into three groups (each contained
12 animals). All rats were 3-4 months old, with the ave
rage weight of about 300 grams. Uniform environmental
conditions: diet, cage isolation and water taking ad libitum
were included. In the experimental group 10% of aqueous
urea solution was used while in the control groups 3%
hydrogen peroxide and saline were applied.
Donaldsons method of thermal alteration was utilised
for artificial provocation of inflammatory changes in gin
gival tissue of experimental animals. Alteration of gingival
tissue was performed in all animals using thermal dosime
ter and the temperature of 70C. Desired temperature was
maintained by heated water system with automatic control.
Gingiva was altered above the upper lateral incisors
on the left and right side. All altered surfaces were equal
in size due to the same dosimeter used and heat appli
cation during 2 seconds. Samples with excision area not
bigger than 5-6 mm were taken at scheduled intervals
after 3, 5, 8, 11 and 14 days. The degree of gingival infla
mmation was judged using clinical index LeSilnes. All
samples were prepared for histological analysis in four

phases: fixation with 10% formalin, paraffin molding for

histological cuts in two directions and staining with ha
ematoxylin eosin (HE). For the alteration of gingiva and
sample taking animals were anesthetized using Nembutal
in the dose of 30 mg/kg body weight intraperitoneally.
Anesthesia was sustained during 30-60 minutes, enough
to fix the limbs of animals and perform excision.
RESULTS
Histopathologic examination of excisions taken after three
days included the assessment of the condition, quality and
degree of epithelialisation. Samples from the experimental
group stained with HE showed distinct leukocyte infiltrati
on under 163 times magnification (Figure 1a). The samples
from the control group treated with 3% hydrogen peroxide
showed stromal edema and severe cellular infiltration (Fi
gure 1b). In the second control group treated with saline,
severe cellular infiltration and epithelial defect were obser
ved (Figure 1c). Comparison of the samples from all three
groups did not reveal significant morphological difference.
After five days in the experimental group samples an
intense mitotic activity of basal cells at the margins of
defect and initial covering of the defect with basal cells in
both directions was observed under 250 times magnifica
tion (Figure 2a). Samples from the group treated with 3%
hydrogen peroxide indicated poor reproduction of basal
cells and formation of granulation tissue (Figure 2b). In
the second control group, histological analysis of samples
revealed a lag in epithelialisation compared to the expe
rimental and other control group (Figure 2c). When all
three groups were compared, the second control group
showed significant slowdown in epithelialisation compa
red to experimental and other control group.
Histological findings of the experimental group after
eight days included covering of defects with basal cells
and occasional cell division in vertical direction (Figure
3a). In the first control group the defect was partly and
discontinuously covered, without multiplication of cells
in vertical direction (Figure 3b). In the second control
group the delay in epithelialisation was more pronoun

Figure 1. Histological findings of gingiva in rats after three days: a) pronounced leukocyte infiltration (10% urea); b) stromal edema and
severe cellular infiltration (3% hydrogen peroxide); c) epithelial defect and severe cellular infiltration (saline)
Slika 1. Stanje gingive pacova posle treeg dana eksperimenta: a) izraena leukocitna infiltracija (desetoprocentni rastvor uree); b) edem
strome i jaka elijska infiltracija (troprocentni rastvor hidrogena); c) oteenje epitela i jaka elijska infiltracija (fizioloki rastvor)

Stomatoloki glasnik Srbije. 2013;60(3):147-154

Figure 2. Histological findings of gingiva in rats after five days: a) initial covering of the defect with basal cells in both directions (10% urea
solution); b) basal cell division and formation of granulation tissue; c) the lag of epithelialisation (saline)
Slika 2. Stanje gingive pacova posle pet dana: a) poetno prekrivanje oteenja bazalnim elijama u oba pravca (desetoprocentni rastvor
uree); b) umnoavanje bazalnim elijama i stvaranje granulacionog tkiva (troprocentni rastvor hidrogena); c) zaostajanje epitelizacije (fi
zioloki rastvor)

Figure 3. Gingiva in rats after eight days: a) the defect covered with basal cells, cell multiplication in vertical direction (10% urea); b) discontinuous covering of the defect and the absence of cells multiplication in vertical direction (3% hydrogen); c) signs of delayed epithelialisation (saline)
Slika 3. Stanje gingive pacova posle osam dana: a) pokrivanje oteenja bazalnim elijama uz umnoavanje elija u vertikalnom smeru
(desetoprocentni rastvor uree); b) diskontinuitet prekrivanja oteenja i izostanak umnoavanja elija u vertikalnom smeru (troprocentni
rastvor hidrogena); c) znaci usporene epitelizacije (fizioloki rastvor)

ced, as evidenced by the absence of continuous epithelial

covering and thickening in vertical direction (Figure 3c).
Comparing all three groups, more pronounced differenti
ation of cells in vertical direction and continuous covering
of the defect edges was found in the experimental group,
as compared to both control groups.
The analysis of samples obtained after eleven days in
dicated that majority of lesions in the experimental group
were fully covered with regenerated epithelium, cell dif
ferentiation in vertical direction was almost completed,
together supporting full horizontal defect closure (Figure
4a). In the first control group, the defect was covered with
the layer of basal cells but with incomplete vertical diffe
rentiation (Figure 4b). In the second control group, slower
epithelialisation and closure of the defect without vertical
differentiation was noted (Figure 4c).
The analysis of samples taken after fourteen days from
the experimental group showed complete restitution with
almost no difference between earlier epithelium and pe
ripheral epithelium (Figure 5a). In the first control group,
the samples revealed uneven and delayed epithelialisation
confirmed by findings that defects were covered in hori

zontal direction whereas vertical cell multiplication was

uneven in certain areas (Figure 5b). In the second control
group, there was unequal and late regeneration suggesting
that process of healing in this group was one stage behind
the first control group and significantly delayed than in
the experimental group (Figure 5c). Comparison of results
from all groups indicated significant delay in the level and
quality of regeneration in control animals, compared to
the experimental group.
DISCUSSION
Basic forms of mucosal wound healing are per primam
and per secundam inentionem. Primary healing involves
restitutio ad integrum and occurs when tissue is damaged
but basal membrane preserved. Secondary healing inclu
des several phases, requires longer time and has negative
effects. The specificity of oral epithelium is that it consi
sts mainly of cellular elements with sparse intercellular
substance. Successful healing of small artificial lesions
depends on the degree of phylogenetic and ontogenetic

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Djordjevi S. et al. Effect of 10% Urea Solution on Epithelialization of Thermally Wounded Gingiva

Figure 4. Gingiva in rats after eleven days: a) continuous horizontal closure of the defect (10% urea); b) incomplete vertical cell differentiation (3% hydrogen); c) closed defect without vertical differentiation (saline)
Slika 4. Stanje gingive pacova posle jedanaest dana: a) kontinuitet horizontalnog zatvaranja oteenja (desetoprocentni rastvor uree); b) ne
potpuna vertikalna diferencijacija elija (troprocentni rastvor hidrogena); c) zatvoreno oteenje bez vertikalne diferencijacije (fizioloki rastvor)

Figure 5. Gingiva in rats after fourteen days: a) complete epithelial restitution (10% urea); b) uneven and delayed epithelialisation (3%
hydrogen); c) uneven and slow epithelial regeneration (saline)
Slika 5. Stanje gingive pacova posle etrnaest dana: a) potpuna restitucija epitela (desetoprocentni rastvor uree); b) neujednaena i zakasnela
epitelizacija (troprocentni rastvor hidrogena); c) neujednaena i usporena regeneracija epitela (fizioloki rastvor)

development. Mucosal epithelium and skin epidermis

regenerate significantly faster than more differentiated
tissues. Under normal conditions, the sequence of healing
includes transudation, proliferation and cell differentia
tion for defect closure. Healing speed is affected by saliva
composition and topically applied agents which can create
favorable conditions for regeneration.
Urea is an amfotelit and weak base that has strong ten
dency to buffer pH oscillations and keep it around neutral.
Due to more pronounced urealytic than glycolytic activity
of bacteria, ammonia released from urealysis causes pH in
crease and longer buffering effect. These conditions allow
elimination of inflexible bacterial species from saliva and
establishment of monotony set of microbes and preven
tion of secondary infections. Due to the proven buffering
capacity, the amount of ammonia and other amino-forma
tions are analyzed as valid parameters for measuring ca
ries activity. These results are supported in in vivo studies
that showed pronounced affinity of urea to concentrate in
plaque significantly faster than glucose but also to degrade
faster [17]. Bactericidal action of urea and exact mechani
sm of inhibition of bacterial growth was not known with
certainty. It is believed that in addition to inhibition of
bacterial growth and buffering capacity, urea binds to the

substrate and cause blockage of bacterial enzyme activity

[18, 19]. This type of inhibition of bacterial growth also
explains bactericidal effect of penicillin and sulfonamides
which compete with paraaminobenzoic acid. Based on the
se findings, urea is used in therapy in dermatology, rinsing
solutions, toothpastes, chewing gums and food additives.
Comparison of the effectiveness of chlorhexidine and
four antibacterial agents based on biamid-urea showed
that after the application of all tested substances a signifi
cant clinical reduction in plaque, gingivitis and volume of
gingival sulcular fluid was achieved [20]. Antiseptic effects
of urea have been confirmed in different pharmacological
combinations like glyoxid urea (10% solution of urea in gl
ycerol and hydrogen peroxide). Compared to spontaneous
regeneration, local application of 10% urea in aqueous so
lution in artificially induced gingivitis has shown positive
effects. Acceleration of gingival epithelialisation registered
in samples after five days, became more pronounced in
samples obtained after eleven days. Since glandular ele
ments were missing in areas of alteration, it can be conclu
ded that the process of healing depended on regenerative
potential of basal cell layer. Under the influence of urea,
the cells formed epithelial strips growing from periphery
to the center of the lesion and covering the defect.

Stomatoloki glasnik Srbije. 2013;60(3):147-154

The quality of regeneration is reflected by findings that

re-epithelialisation included proliferation and differenti
ation of intermitotic basal cells as well as cells in vertical
direction. Healing in the experimental group treated with
urea resulted as restitutio ad integrum and reestablishment
of the stratified squamous epithelium in the treated area.
In the control groups, treated with 3% hydrogen peroxide
and saline, regeneration was intermittent and slow. Accor
ding to the current results, regeneration was completed
between 12th and 14th day after thermal alteration. Fast
and satisfied regeneration achieved with urea is most li
kely the consequence of the ability of urea to preserve the
potential of basal cell layer to induce epithelial regenerati
on by increased mitotic activity. Urea, most probably with
its properties, turned damaged gingiva in an unfavorable
environment for bacteria, protected basal epithelium and
stimulated the process of re-epithelialisation.
In the control group treated with 3% hydrogen peroxi
de delayed healing is explained by the fact that hydrogen
and other antioxidants may slow down wound healing.
This is supported by studies that showed inflamed tissue
required several times more oxygen and there was oxygen
concentration dependent effect on the process of epitheli
alisation [21]. In the group treated with saline a significant
delay of epithelialisation in both directions was registered,
confirmed by delayed cell differentiation and incomplete
closure of the defect at the end of the experimental period.
Obtained results correspond to the number of eviden
ce about the properties and effects of urea on gingival
inflammation [22, 23, 24]. Due to the similarities in the
origin and structure of epidermis and oral epithelium,
conclusions obtained from research in dermatology can
be summarized: lower pH values emphasize urea's featu
res: antiphlogistic, regenerative, hydration, keratolytic and
desquamative providing better tissue nourishment [26].
CONCLUSION
The results showed that restitution of damaged gingiva in
the group treated with 10% urea in aqueous solution occu
rred significantly faster and more complete than in the con
trol groups. This was confirmed by the thickness of epitheli
um recorded in the samples after eleven and fourteen days
and the absence of superinfection. In both control groups,
slower and discontinuous regeneration of altered gingiva
was found, probably caused by the absence of buffering,
keratolytic and antimicrobial support obtained from urea.
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Received: 22/04/2013 Accepted: 10/07/2013

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Uticaj desetoprocentnog rastvora uree na epitelizaciju termiki

izmenjene gingive
Stevanka orevi1, Dragan Ivanovi1, Tanja Ivanovi2
Katedra za preventivnu i djeiju stomatologiju, Medicinski fakultet, Univerzitet u Istonom Sarajevu, Foa, Republika Srpska,
Bosna i Hercegovina;
2
Katedra za ortopediju vilice, Medicinski fakultet, Univerzitet u Istonom Sarajevu, Foa, Bosna i Hercegovina
1

KRATAK SADRAJ

Uvod Urea je finalni metabolit degradacije protein

a, a izluuje se pljuvakom. Kao mali niskomolekularni i neutralni molekul, neo
metano difunduje kroz zidove bakterija, maturirani plak i vanelijske polisaharide, ispoljavajui puferski efekat. Pri postojanju ureaze
razlae se na molekul amonijaka i dva molekula slabe ugljene kiseline, to dovodi do alkalizacije supstrata i poveanja vrednosti pH.
Hidroliza uree zapoinje pri kritinim vrednostima pH 46. U uslovima maturiranog plaka s malim vrednostima pH hidroliza uree
je kompenzatorni mehanizam koji se suprotstavlja pri smanjenju vrednosti pH i naruavanju optimalne kiselo-bazne ravnotee.
Ovim mehanizmom se objanjava uloga uree u kontroli plaka, karijesa i gingivitisa. Cilj rada je bio da se ispita uticaj uree na brzinu
i kvalitet epitelizacije kod termiki izmenjene gingive pacova.
Materijal i metode rada Istraivanjem je obuhvaeno 36 albino pacova tipa Wistar starih tri-etiri meseca, koji su svrstani u tri grupe
(po 12). Prema protokolu, kod svih ivotinja je izvrena termika alteracija gingive, koja je tretirana desetoprocentnim rastvorom
uree (eksperimentalna grupa), troprocentnim hidrogenom i fiziolokim rastvorom (kontrolne grupe). Stepen i kvalitet epitelizacije
potvreni su patohistoloki posle tri, pet, osam, 11 i 14 dana.
Rezultati Na uzorcima iseaka uzetih treega dana uoeni su jaka elijska infiltracija i edem strome, ali bez znaajnih morfolokih
razlika unutar grupa. Ubrzanje epitelizacije u eksperimentalnoj grupi uoeno je na uzorcima uzetim petoga dana, to potvruju
ubrzane mitotske deobe epitela i inicijalno prekrivanje oteenja u oba pravca. Na uzorcima eksperimentalne grupe uzetih osmoga
dan patohistoloki su uoeni prekrivanje oteenja i mestimina diferencijacija elija u vertikalnom smeru. Na veini uzoraka uzetih
11. dana zabeleeni su horizontalno zatvaranje oteenja u celosti i ubrzana diferencijacija elija u vertikalnom smeru. Zavretak
procesa primarne restitucije u eksperimentalnoj grupi i znaajno kanjenje u kontrolnim grupama dokazano je analizom uzoraka
uzetih 14. dana.
Zakljuak U grupi gingiva kod kojih je primenjen desetoprocentni rastvor uree epitelizacija je bila celovitija i znaajno bra u od
nosu na kontrolne grupe tretirane troprocentnim rastvorom hidrogena i fiziolokim rastvorom.
Kljune rei: urea; epitelizacija; gingiva; termika alteracija

UVOD
Termin oralna urea podrazumeva njeno fizioloko prisustvo
u pljuvaki, tenosti gingivalnog sulkusa i plaku [1]. Plakovna
urea potie iz pljuvake, ostataka hrane i stvaranja uree koju vri
desetina svih oralnih koka plaka [2]. Urea je dvadeset puta kon
centrovanija u plaku, gde se veim delom troi na puferizaciju,
a manjim na njegovu detoksikaciju. Neka istraivanja pokazuju
da urea ispoljava baktericidne, bakteriostatine, amfoterne i ne
alergogene osobine [3].
Prvi izvetaji iz prve polovine devetnaestog veka pokazu
ju da razgradnjom uree nastali amonijak direktno korelira s
pojavom karijesa [4]. Antikariogeni uticaj uree opisan je u is
traivanjima koja su primenom etrdesetoprocentnog rastvora
uree ostvarili smanjenje aktivnosti karijesa od 80% do 100%
[5]. Rasvetljenju ove visoke stope smanjenja karijesa doprine
li su radovi koji su ustanovili da pljuvaka tzv. karijes-imunih
pacijenata sadri dvostruko vee vrednosti uree nego pljuvaka
ljudi sklonih razvoju karijesa [6]. Stvaranje baznih komponena
ta plaka moe biti koncept svake studije metabolizma pljuvake
koji se zasniva na razlikama ovih karijes-osetljivih i karijesrezistentnih grupa [7].
Pozitivni efekti vodenog rastvora uree i urea-peroksida na
nagomilavanje plaka dokumentovani su u brojnim radovima
[8, 9, 10]. Stav da profilaktiko dejstvo uree na karijes nije za
snovan samo na njenim sposobnostima odravanja pH mikro
sredine, nego u nizu integralnih metabolikih i biohemijskih
aktivnosti, potvren je i rezultatima drugih istraivanja [11,
12]. Osim plaka, utvrena je i poveana koncentracija uree u

tenosti gingivalnog sulkusa, gde odrava kiselo-baznu ravno

teu i aktivno uestvuje u odbrambenim aktivnostima gingi
ve [13]. Vrednosti amonijaka u plaku su izmeu 1626%, a u
pljuvaki 1540 mg na 100 ml. Pored amonijaka, ije vrednosti
predstavljaju procenat iskoriene uree, znaajne proporcije
uree su inkorporirane u proteine i amino-kiseline. Prema po
trebi, i brzom reakcijom sinteze iz ovih depoa ponovo se stvara
urea. Na ovaj nain ona ponovo postaje glavna determinanta
porasta vrednosti pH zahvaljujui injenici da mikrobni sastav
plaka pokazuje bru i naglaeniju ureolitiku nego glikolitiku
aktivnost [14, 15].
Kod kliniki zdrave ili blago zapaljene gingive vrednosti uree
u tenosti gingivalnog sulkusa su uvek nekoliko puta vee od
vrednosti u pljuvaki i serumu. S jainom zapaljenja belei se
smanjenje vrednosti do izjednaavanja s vrednostima u ovim
supstratima. Kod osoba sa zdravim parodoncijumom stvara
nje amonijaka je 2,7 puta vee nego kod osoba sa beznaajnim
gingivitisom [16, 17].
Protektivna uloga uree ostvaruje se i sposobnou da ona u
prisustvu glukoze stimulie formiranje velikih koliina alanina,
najverovatnije oslobaanjem aminogrupa potrebnih za konver
ziju piruvata u alanin. Primena uree u uslovima in vivo i in vitro
uslovljava alkalizaciju plaka do 9 pH, dok je analiza plakovnog
sadraja uzetog od velikog broja osoba pokazala visoku urea
znu aktivnost [18]. Zbog sloenih metabolikih i biohemijskih
aktivnosti i neusaglaenosti stavova, zatitne mehanizme uree
teko je objasniti.
Cilj ovog rada je bio da se proveri uticaj uree na brzinu i kva
litet epitelizacije termiki izmenjene gingive pacova.

Stomatoloki glasnik Srbije. 2013;60(3):147-154

MATERIJAL I METODE RADA

Eksperiment je uraen na 36 pacova soja Winstar (21 mujak i
15 enki) koji su svrstani u tri grupe od po 12 ivotinja. ivoti
nje su bile stare tri-etiri meseca i u proseku su imale oko 300
grama. Ambijentalni uslovi su ukljuivali ujednaenu fabriku
ishranu, izolaciju u kavezima i neogranieno uzimanje vode. U
eksperimentalnoj grupi primenjen je desetoprocentni vodeni
rastvor uree, dok su u kontrolnim grupama korieni tropro
centni hidrogen i fizioloki rastvor.
Za vetaki izazvane zapaljenjske promene na gingivi ekspe
rimentalnih ivotinja koriena je Donaldsonova (Donaldson)
metoda termike alteracije. Alteracija gingive na svim ivotinja
ma uraena je termikim dozimetrom i temperaturom od 70
stepeni. Postignutu temperaturu odravao je sistem zagrejane
vode s automatskom kontrolom.
Tana lokacija primene rastvora bila je gingiva iznad gornjih
lateralnih inciziva s leve i desne strane. Istovetnost lediranih
povrina osiguravali su radna povrina dozimetra i dogovoreno
vreme delovanja termikog agensa od dve sekunde. Uzorci ija
ukupna eksciziona povrina nije bila vea od 56 mm uzima
ni su u planiranim intervalima od tri, pet, osam, 11 i 14 dana.
Stepen zapaljenja gingive utvren je klinikim indeksom po
LouuSilnesu (LeSilnes). Patohistoloka obrada odvijala se
kroz etiri faze: fiksacija desetoprocentnim formalinom, kalu
pljenje u parafinske blokove kao priprema za histoloke rezove u
dva pravca i bojenje standardnom metodom hamatoksilin-eozi
nom (HE). Pre alteracije i svakog uzimanja iseaka ivotinje su
anestezirane intraperitonealnom primenom nembutala u dozi
od 30 mg//kg telesne teine. Postignuta anestezija se odravala
3060 minuta, to je dovoljno da se ekstremiteti ivotinja fik
siraju i uradi ekscizija.

i mestimino umnoavanje elija u vertikalnom smeru (Slika

3a). U prvoj kontrolnoj grupi pokrivanje oteenja je bilo mesti
mino, nekontinuirano i bez umnoavanja elija u vertikalnom
smeru (Slika 3b). U drugoj kontrolnoj grupi znaci kanjenja
epitelizacije bili su izraajniji, to dokazuje izostanak kontinu
iranog epitelnog pokrivaa i zadebljanja u vertikalnom smeru
(Slika 3c). Poreenjem nalaza zapaeni su naglaenija diferen
cijacija elija u vertikalnom smeru i kontinuirano prekrivanje
rubova oteenja kod uzoraka eksperimentalne grupe u odnosu
na obe kontrolne.
Rezultati analize iseaka uzetih 11. dana pokazali su da je
kod veine uzoraka eksperimentalne grupe vetaki izazvano
oteenje bilo potpuno pokriveno regenerisanim epitelom, a da
je diferenciranje elija u vertikalnom smeru bilo skoro zavreno,
to zajedno govori u prilog potpunom horizontalnom zatvara
nju oteenja (Slika 4a). U prvoj kontrolnoj grupi oteenje je
bilo prekriveno iskljuivo slojem bazalnih elija, ali je vertikalno
diferenciranje bilo nepotpuno (Slika 4b). U drugoj kontrolnoj
grupi primetni su bili jo sporija epitelizacija i zatvaranje ote
enja bez vertikalne diferencijacije (Slika 4c).
Analiza uzoraka eksperimentalne grupe uzetih 14. dana po
kazala je potpunu restituciju, pri emu su se teko razlikovali
raniji epitel i rubni epitel (Slika 5a). U prvoj kontrolnoj grupi
zapaena je neujednaena i zakasnela epitelizacija, koju su po
tvrdili pokrivanje oteenja u horizontalnom smeru i neujedna
eno vertikalno umnoavanje u pojedinim zonama (Slika 5b).
U drugoj kontrolnoj grupi uoeni su znaajna neujednaenost
i zaostajanje regeneracije, to navodi na zakljuak da proces za
celjenja u drugoj kontrolnoj grupi kasni za itavu fazu u odnosu
na prvu kontrolnu grupu i znaajno vie u odnosu na eksperi
mentalnu (Slika 5c). Poreenje rezultata je ukazalo na znaajno
kanjenje stepena i kvaliteta regeneracije kod kontrolnih uzo
raka u odnosu na uzorke eksperimentalne grupe.

REZULTATI
Patohistoloka analiza iseaka uzetih treega dana nakon pri
mene rastvora obuhvatila je procenu stanja epitela, kvalitet i
stepen epitelizacije. Na preparatima eksperimentalne grupe,
bojenim sa HE i uveanim 163 puta, potvrena je izrazita leu
kocitna infiltracija (Slika 1a). Na preparatima kontrolne grupe
koji su tretirani troprocentnim hidrogenom uoeni su edem
strome i jaka elijska infiltracija (Slika 1b). U drugoj kontrolnoj
grupi uzoraka, gde je primenjen fizioloki rastvor, zapaeni su
jaka elijska infiltracija i vidljivo oteenje epitela (Slika 1c).
Poreenjem rezultata unutar sve tri grupe nisu uoene znaajne
morfoloke razlike.
Na isecima eksperimentalne grupe uzetih petoga dana, pri
uveanju od 250 puta, primeeni su intenzivnije mitotske ak
tivnosti bazalnih elija rubnog dela rane i inicijalno prekrivanje
oteenja bazalnim elijama u oba pravca (Slika 2a). Iseci prve
kontrolne grupe ukazali su na slabije umnoavanje bazalnih e
lija i stvaranje granulacionog tkiva ledirane gingive (Slika 2b). U
drugoj kontrolnoj grupi vidljivo je bilo zaostajanje epitelizacije u
odnosu na eksperimentalnu i prvu kontrolnu grupu (Slika 2c).
Poreenjem dobijenih nalaza, u drugoj kontrolnoj grupi je pri
meeno znaajnije usporavanje procesa epitelizacije u odnosu
na ostale dve grupe.
Na patohistolokim nalazima eksperimentalne grupe osmo
ga dana bili su vidljivi pokrivanje oteenja bazalnim elijama

DISKUSIJA
Osnovni vidovi zarastanja sluznica su per primam i per secun
dam inentionem. Primarno zarastanje podrazumeva restituciju
ad integrum i ukljuuje oteenja tkiva kod kojih je ouvana
bazalna membrana. Sekundarno zarastanje ima nekoliko faza,
produen tok i negativne posledice. Specifinost oralnog epite
la je da se sastoji od iskljuivo elijskih elemenata s oskudnom
meuelijskom supstancom. Uspenost restitucije manjih ar
teficijalnih lezija, koja su predmet ovih istraivanja, zavisi od
stepena filogenetskog i ontogenetskog razvoja. U filogenetskom
smislu epitel mukoze i epiderm koe se znaajno bre regene
riu od vie diferenciranih tkiva. Pod normalnim uslovima pa
tohistoloki redosled zarastanja nakon alteracije se odvija kroz
transudaciju, proliferaciju i diferencijaciju elija koje zatvaraju
oteenje. Na brzinu zaceljenja utiu sastav pljuvake i topikal
no primenjeni agensi ije sposobnosti stvaraju povoljne uslove
za regeneraciju.
Kao amfotelit i slaba baza, urea ima naglaenu tendenci
ju da svaku oscilaciju vrednosti pH vraa i odrava oko take
neutralnosti. Zbog naglaenije ureolitike nego glikolitike ak
tivnosti samih bakterija, amonijak osloboen lizom uree uzro
kuje poveanje vrednosti pH i duu puferizaciju mikrosredine.
U ovim uslovima se eliminiu neprilagodljive bakterijske vrste
u pljuvaki, uspostavlja monotoniji mikrobni sastav i spreava

153

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Djordjevi S. et al. Effect of 10% Urea Solution on Epithelialization of Thermally Wounded Gingiva

sekundarna infekcija. Zbog dokazanog puferskog kapaciteta,

vrednosti amonijaka i aminoformacija se analiziraju kao validni
parametri u proceni aktivnosti karijesa. Navedeno podravaju
rezultati autentinih istraivanja u uslovima in vivo, kojim je
potvren naglaen afinitet uree da se znaajno bre od glukoze
koncentruje u plaku i znaajno bre degradira [17]. Baktericid
no delovanje uree i taan mehanizam inhibicije bakterijskog
rasta nije do kraja rasvetljen. Smatra se da, pored nesporne
inhibicije bakterijskog rasta i puferskog kapaciteta, urea ve
zivanjem za supstrat uzrokuje i blokadu enzimskih aktivnosti
bakterija [18, 19]. Ovom vrstom inhibicije bakterijskog rasta
objanjava se i baktericidno delovanje penicilina i sulfonamida,
koji se takmie sa paraaminobenzojevom kiselinom. Na ovim
saznanjima je utemeljena primena uree u terapijske svrhe u
dermatologiji, u rastvorima za ispiranje usne upljine, zubnim
pastama, vakaim gumama i aditivima u osnovnim ivotnim
namirnicama.
Uporeivanje efikasnosti hlorheksidina i etiri antibakte
rijske komponente oznaene kao biamid-ureini antimikrob
ni agensi pokazalo je da se primenom svih ispitanih supstanci
ostvarilo znaajno smanjenje klinikog plaka, gingivitisa i vo
lumena tenosti gingivalnog sulkusa [20]. Antiseptini efekti
uree potvreni su i u razliitim farmakolokim kombinacijama,
poput glioksid-uree, to je desetoprocentni rastvor uree u gli
cerolu i vodonik-peroksidu. U odnosu na spontanu regenera
ciju, lokalna primena desetoprocentnog vodenog rastvora uree
kod vetaki izazvanog gingivitisa ispoljila je pozitivne efekte.
Ubrzanje epitelizacije pokrovnog epitela gingive zabeleeno na
isecima uzetim ve petoga dana postalo je izrazitije na uzorci
ma uzetim 11. dana eksperimenta. Kako na mestima izvrene
alteracije nedostaju lezdani elementi, moe se zakljuiti da je
proces bio oslonjen samo na regenerativni potencijal elija ba
zalnog sloja. Pod uticajem uree elije su formirale epitelne trake
koje su, rastui od periferije prema centru, pokrivale oteenje.
Kvalitet regeneracije odraavaju nalazi koji potvruju da
su proces reepitelizacije, pored intermitotikih elija bazalnog
sloja, pratili umnoavanje i diferencijacija elija u vertikalnom
smeru. Zaceljenje u grupi uzoraka tretiranih ureom zavrava
lo se restitucijom ad integrum ili ponovnim stvaranjem vie
slojnog ploastog epitela na tretiranoj povrini. U kontrolnim

grupama, gde su uzorci tretirani troprocentnim hidrogenom i

fiziolokim rastvorom, regeneracija se odvijala isprekidano i
sporije. Ona se zavrila izmeu 12. i 14. dana nakon termike
alteracije. Ubrzanje i kvalitet regeneracije najverovatnije je uslo
vila sposobnost uree da, za razliku od klasinih preparata, ouva
potencijal elija bazalnog sloja, koje pojaanom mitotskom ak
tivnou podstiu proces regeneracije epitela. Svojim osobina
ma urea je najverovatnije podruje oteene gingive pretvorila
u nepovoljnu sredinu, titila bazalni epitel i stimulisala proces
ponovne epitelizacije.
U kontrolnoj grupi uzoraka kod kojih je primenjen tropro
centni hidrogen usporenije celjenje se objanjava nalazima da
hidrogen, kao i ostali antioksidansi, usporava zarastanje rana.
Tvrdnju podravaju studije o potronji kiseonika, iji rezultati
pokazuju da upaljena tkiva zahtevaju viestruko vie kiseoni
ka, koji, zavisno od koncentracije, utie na proces epitelizacije
[21]. U grupi uzoraka koji su tretirani fiziolokim rastvorom
zabeleeno je znaajno zaostajanje epitelizacije u oba smera, po
tvreno zakasnelom diferencijacijom elija i nepotpunim zatva
ranjem oteenja na kraju eksperimentalnog perioda. Dobijeni
nalazi su u skladu sa brojnim dokazima o osobinama i efektima
uree na zapaljenje gingive [22, 23, 24]. Zbog slinosti porekla i
grae epiderma i oralnog epitela, navodimo zakljuke istovet
nih istraivanja u dermatologiji; pri niim vrednostima pH se
naglaavaju sposobnosti uree antiflogistika, regenerativna,
hidratacijska, keratolitika i deskvamacijska, koje obezbeuju
bolju ishranjenost tkiva [26].
ZAKLJUAK
Studija je pokazala da se restitucija oteene gingive koja je tre
tirana desetoprocentnim vodenim rastvorom uree odvija znat
no bre i potpunije u odnosu na gingivu kod koje su primenjeni
troprocentni hidrogen i fizioloki rastvor. Tvrdnju potvruju
debljina, kao trea dimenzija epitela, koja je uoena na isecima
11. i 14. dana, i izostanak superinfekcije. U obe kontrolne grupe
ustanovljena je sporija i diskontinuirana regeneracija izmenjene
gingive, najverovatnije uslovljena izostankom puferske, kerato
litike i antimikrobne podrke, koju je osiguravala urea.