ABSTRACT
Nanot echnology in ligat ure- induced periodont it is:
p r o t ect i v e ef f ect o f a d ox y cy cl i n e g el w i t h
nanopart icules
Marco Antonio BOTELHO1, Jose Galberto MARTINS2, Ronaldo Sousa RUELA3, Dinalva Brito QUEIROZ4,
Wagner Sousa RUELA3
1- Post Doctor in Orthodontics, Federal University of Ceará, Fortaleza, CE, Brazil. 2- Doctor in Chemistry, Regional University of Cariri, Fortaleza, CE, Brazil.
3- Doctor in Dentistry, Scholar of National Research and Development Council CNPq, FUNORTE School of Dentistry, Montes Claros, MG, Brazil. 4- Scholar of National Research and Development Council CNPq, Fortaleza, CE, Brazil.
Corresponding address: Prof. Dr. Marco A. Botelho - Instituto Federal de Educação Ciência e Tecnologia do Ceará /Campus Canindé Departamento de Pesquisa & Inovação Tecnológica Laboratório de Biotecnologia - Br 020 - Km 303 - Jubaia - 62700-000 - Canindé, CE - Brasil - Phone : 55 (85) 3307-3600 / 8665-5000 - e-mail: marcobotelho@pesquisador.cnpq.br
Received: January 09, 2009 - Modiication: August 11, 2009 - Accepted: February 16, 2010
O
bjectives: The aim of this study was to test the eficacy of a locally applied 8.5% nanost ruct ured doxycycline ( DOX) gel in prevent ing alveolar bone loss in experim ent al periodont al disease ( EPD) in rat s by using t he t apping m ode at om ic force m icroscopy ( AFM) . Mat erial and Met hods: EPD was induced in 24 Wist ar rat s. Anim als were t reat ed wit h t he doxycycline gel t opically, im m ediat ely aft er EPD induct ion, and 3 t im es a day during 11 days. Four groups ( n= 6) were form ed as follows: Naïve group ( anim als not subj ect ed t o EPD nor t reat ed) ; non- t reat ed ( NT) group ( anim als subj ect ed t o EPD, but not t reat ed) ; vehicle gel ( VG) group ( anim als subj ect ed t o EPD and t reat ed wit h t opical gel vehicle) ; and DOX group ( t est group) : anim als subj ect ed t o EPD and t reat ed wit h t he 8.5% DOX gel. I n order t o invest igat e t opographical changes in hist ological sect ions, a novel sim ple m et hodwas used for sample preparation, by etching sections from parafin-embedded specimens
wit h xylol. Result s: Com paring t he AFM im ages, several grooves were observed on t he surface of t he alveolar bone and ot her periodont al st ruct ures in t he NT and VG groups,
with signiicantly greater depths when compared to the DOX group (p<0.05). Conclusions: Periodontal structures were brought into high relief conirming to be a simple and
cost-effect ive m et hod for AFM im aging wit h ult rast ruct ural resolut ion. The doxycycline gel was
able to afford periodontal surface preservation, with latter grooves.
Ke y w or ds: Nanot echnology. Doxycycline. Periodont it is. At om ic force m icroscopy. Gel.
I N TROD UCTI ON
Se v e r a l m e t h o d s h a v e b e e n e m p l o y e d i n dent ist ry t o bet t er underst and periodont al disease pr ocess5. Th e in du ct ion of per iodon t al disease
by ligat ur e placem ent is w idely used in anim al st udies1- 4,7,8. However, as crit icism of t his m odel st ill
rem ains, ot her m et hods are necessary for a bet t er underst anding of t he pat hogenesis of periodont al disease.
At o m i c f o r ce m i cr o sco p y ( AFM) i s a v e r y powerful t ool as com m on t echnique for biological im aging, from m olecule t o cell m orphology or even ult rast ruct ural changes on t issue, where nanom et ric
det ails are required t o invest igat e t he surface of t he specim en. AFM is com m only used in exam ining biological specim ens since it works well wit hout any need for st aining or coat ing5,17.
Periodont al st ruct ures, such as t he periodont al ligam ent and alveolar bone, play an im port ant role not only in m aint aining st ruct ural int egrit y, but also in det erm ining t issue funct ion. Alveolar bone and at t achm ent loss due t o periodont al diseases, for exam ple, is direct ly associat ed wit h t he presence
of dental bioilm and poor oral hygiene4,6,9.
a l o cal i n f l am m at o r y r eact i o n i n p r ed i sp o sed host s7. I nsight int o pat hological st ruct ure funct ion
relationships depends on an accurate identiication
of ult rast ruct ural changes on periodont al st ruct ures since t he progression of periodont it is is considered a relevant cause of t oot h loss in adult s. Periodont it is
is as a chronic inlammatory disease that affects a
m aj or part of world’s populat ion charact erized by localized or generalized bone resorpt ion8.
Periodont al disease is also associat ed wit h t he hear t diseases. Differ ent t herapies ar e available t o t reat t his pat hology using nanot echnology for m icr oencapsulat ing t hy m ol, car vacr ol and ot her
natural lavonoids that produce antiinlammatory and antimicrobial effects during this inlammatory
processes8- 10,21. The host’s inlammatory response
leads to edema, and the release of inlammatory
m ediat ors, causing periodont al pocket form at ion, loss of connect ive t issue at t achm ent and alveolar bone resorpt ion, ult im at ely leading t o t oot h loss4,6,9.
It is dificult to establish the periodontal status
w it h ou t per iodon t al t issu e in t egr it y con dit ion s.
The main dificulty seems to be greater when a
t hree- dim ensional evaluat ion is required11,17. The
t hird dim ension is im port ant for t he com prehensive underst anding of t issue ult rast ruct ure. The inclusion of t opographical and r oughness sur face aspect s m ight increase t he predict ion of periodont al t herapy evaluat ion22.
As no experim ent al st udies have invest igat ed t he possibilit y of using AFM t o evaluat e t he int egrit y
of periodontal components, clariication of the
cont ribut ions of t his t echnique on ult rast ruct ural changes in t he per iodont al st r uct ur es w ould be u sef u l. Th e aim of t h is st u d y w as t o t est t h e
eficacy of a locally applied 8.5% nanostructured
doxycycline ( DOX) gel in prevent ing alveolar bone loss in experim ent al periodont al disease ( EPD) in rat s by using a previously described t apping m ode AFM t echnique5.
M ATERI ALS AN D M ETH OD S
An im a ls
Tw ent y- four m ale Wist ar rat s fr om t he sam e nest ( sim ilar age; w eight : 160- 200 g) fr om our own anim al facilit ies were housed in t em perat ure-cont rolled room s and received wat er and food ad libit um. The rat s were random ly assigned t o t est and cont r ol gr oups using blocked random izat ion from a com put er- generat ed list . All experim ent s w er e con d u ct ed i n accor d an ce w i t h t h e l ocal guidelines on t he welfare of experim ent al anim als and wit h t he approval of t he Com m it t ee of Et hics in Anim al Research of t he Federal Universit y of Ceará.
Ge l pr e pa r a t ion
The dox y cy cline gel cont aining nanospher es
was prepared at t he Biot echnology Laborat ory of Ev idence Phar m aceut icals, School of Phar m acy, Federal Univer sit y of Cear á, Brazil, and kept in t he dar k t o avoid any int er fer ence of light . For preparat ion of t he 8.5% nanost ruct ured doxycycline ( w/ w) ( Merck Chem icals Lt d. Beest on, Not t ingham , En glan d) gel, Car bopol- 9 4 O ( BF Goodr ich Co., Cl ev el an d , OH, USA) 2 % ( w / v ) w as u sed b y m ech an ical d isp er sion in d ist illed w at er u n d er vigorous agit at ion and polisorbat e 80 was used, being neut ralized unt il pH 6.0 wit h t riet anolam ine.
Th e n a n o st r u ct u r e d g e l w a s p r e p a r e d b y t he com binat ion of t w o t echniques as a Pat ent Ap p licat ion at Nat ion al I n st it u t e of I n d u st r ial Pr op er t y ( I NPI ) . Th e g el w as st or ed in w h it e polyet hylene cont ainers kept herm et ically sealed under refrigerat ion at 8° C unt il used.
St abilit y st udy t o evaluat e t he consist ency of t he gel over a period of 2 m ont hs was conduct ed by keeping t he form ulat ion at different condit ions ( 4° C, 37° C and room t em perat ure) and m easuring t he viscosit y of t he gel form ulat ion at regular int ervals.
The viscosity was measured by a Brooke-ield
synchrolect ric viscom et er. The TD bar spindle of LV series was em ployed for t he m easurem ent . The st udy indicat ed t hat t he viscosit y of t he carbopol gel
did not change signiicantly throughout the stability period in the speciied conditions.
I n du ct ion of EPD
A st er ile ny lon ( 0 0 0 ) t h r ead ligat u r e (Poin t Sut ure do Brasil I ndúst ria de Fios Cirúrgicos Lt da. Fort aleza,CE- Brazil) was placed around t he cervix of t he second left upper m olar of rat s anest het ized w it h 10% chloral hydrat e ( 400 m g/ k g, i.p.) , as described elsewhere5. The ligat ure was knot t ed on
t he buccal side of t he t oot h, result ing in subgingival posit ion palat ally an d in su pr agin gival posit ion buccally. The cont ralat eral right side was used as t he unligat ed cont rol anim als were weighed daily.
D r u g Tr e a t m e n t s
Four groups of 6 anim als each were form ed as follow s: Naïve gr oup ( anim als not subj ect ed t o EPD nor t reat ed) ; non- t reat ed ( NT) group ( anim als subj ect ed t o EPD, but not t reat ed) ; vehicle gel ( VG) group ( anim als subj ect ed t o EPD and t reat ed wit h t opical gel vehicle) ; and DOX group ( t est group) : anim als subj ect ed t o EPD and t reat ed wit h t he 8.5% DOX gel ( Dent al Gel®; Evidence Pharm aceut icals
LTD., Fort aleza, CE, Brazil) . The t opical t reat m ent wit h t he gel was perform ed applying 1 g of t he product on t he ligat ed sit es im m ediat ely aft er t he sur gical pr ocedur e and 3 t im es a day, unt il t he
sacriice at the 11t h day.
of periodont it is induct ion, and had t heir m axillae
excised and ixed in 10% neutral formalin. Both maxillary halves were then deleshed and stained
w it h 1 % aq u eou s m et h y len e b lu e in or d er t o dist inguish bone from t eet h. The horizont al alveolar bone loss, t he dist ance bet ween t he cusp t ip and t he alveolar bone, were m easured using t he m et hod described by Bot elho5 ( 2009) .
To conirm these indings another digitalized
t echnique was perform ed wit h t he I m ageJ ( Research Services Branch, Nat ional I nst it ut e of Ment al Healt h, Bet hesda, Maryland, USA) . A public dom ain Java im age pr ocessin g pr ogr am w er e t h e ar ea w as det er m ined not only in linear t er m s but also in square m illim et ers ( m m2)as well. The m easurem ent
of alveolar bone loss was done by a blind exam iner ( M.A.B.) for bot h groups. The processing program calculat ed t he area in pixels values, t han st at ist ics
was applied in deined selections areas (distances
and angles) in each periodont al area. The spat ial cal i b r at i on w as p er f or m ed i n t h e p h ot og r ap h w it h a r egular m illim et er paper t o pr ov ide r eal world dim ensional m easurem ent s in unit s such as m illim et ers.
H ist opa t h ologica l An a lysis
After sacriice by anesthetic overdose, anim als had t heir m axillae excised. The specim ens w ere f i x e d i n 1 0 % n e u t r a l b u f f e r e d f o r m a l i n a n d dem ineralized in 7% nit ric acid. These specim ens w er e t h en d eh y d r at ed , em b ed d ed in p ar af f in , and sect ioned along t he m olars in a m esiodist al plane, for hem at ox y lin and eosin st aining. Six-m illiSix-m et er- t hick sect ions, which included t he root s
of the irst and second molars, were utilized. The areas between the irst and second molars, where
t h e ligat u r e w as p laced, w er e an aly zed u n d er light m icroscopy using a four- point ( 0- 3) scoring
system, considering the inlammatory cell inlux,
an d alv eolar bon e an d cem en t u m in t egr it y, as described previously4,5: Score “ 0” ( zero) : absence
or only mild cellular iniltration (inlammatory cell iniltration is sparse and restricted to the region
o f t h e m ar g i n al g i n g i v a) , p r eser v ed al v eo l ar process and cem ent um . Score 1: m oderat e cellular
iniltration (inlammatory cells present all over the
at t ached gingiva) , lit t le alveolar bone resorpt ion and int act cem ent um . Scor e 2: int ense cellular
iniltration (inlammatory cells present in both
gingival and per iodont al ligam ent ) , accent uat ed degradat ion of t he alveolar pr ocess, and par t ial dest ruct ion of cem ent um . Score 3: severe cellular in f ilt r at e, com p let e r esor p t ion of t h e alv eolar process, and severe dest ruct ion of cem ent um . The hist opat hological analysis was perform ed by a blind exam iner ( M.A.B.) in all groups.
H ist olog ica l sa m p le p r e p a r a t ion f or AFM im a ge s
Aft er r out ine hist ological sam ple pr eparat ion, chem ical et ching wit h 1% xylol was done, providing of sem i t h in sect ion s f r om p ar af f in - em b ed d ed specim en s t o m elt t h e u pper lay er s, r em ov in g
the supericial roughness caused by the edge of
t he m icrot om e knife and bringing int o high relief t he biological st ruct ures hidden in t he bulk. Five-m illiFive-m et er- t hick sect ions, which included t he root s
of the irst and second molars, were used. The areas between the irst and second molars, where
t h e lig at u r e w as p laced , w er e an aly zed u sin g t aping m ode AFM. The sam ples were prepared and t he aspect s of it s t opography and ult rast ruct ural changes were analyzed.
To ob t ain AFM h eig h t d at a, sam p les of r at m ax illa w er e scan n ed in air w it h a Nan oscope I I I a Mult im ode AFM ( Digit al I nst r um ent s, Sant a Barbara, CA, USA) by t apping m ode at a scan of about 0.400 Hz, resonance frequencies of ca. 200 t o 380 kHz, wit h cryst al silicon cant ilevers ( Digit al I nst rum ent s) at spring const ant of approxim at ely 40 N/ m , and t ip radius of 15 nm . The scan sizes perform ed were 30x30 µm . AFM scan cont rols were
properly adjusted (suficient contact force, and high
gains) t o avoid t ip art ifact s during t he scanning of t he sam ples. To 3D- visualizat ion t he height dat a were processed wit h Nanoscope soft ware ( Digit al I nst rum ent s) , version 5.12 r3. We used Nanoscope soft ware t o calculat e t he surface roughness for t he periodont ium regions AFM height dat a wit h scan size of 30x30 µm . Thirt y regions per sam ple were random ly chosen t o det erm ine t he m ean surface roughness (Ra)17,22, and t he t opographical changes
independent analysis were perform ed by a blind exam iner ( M.A.B.) for bot h t est ed groups.
St a t ist ica l An a lysis
Th e d a t a a r e p r e s e n t e d a s t h e m e a n ± SEM or as t h e m edian s, w h er e appr opr iat e. A univariat e analysis of variance ( ANOVA) followed by Bonferroni’s t est was used t o com pare m eans, and t he Kruskal-Wallis t est was used t o com pare m ed ian s. A p r ob ab ilit y v alu e of P < 0 . 0 5 w as
considered to indicate signiicant differences. A
p- value <0.05 was considered signiicant. Analysis was perform ed wit h Graph Pad Prism a Version 3.0 soft ware ( GraphPad Soft ware I nc. San Diego, CA, USA) .
RESULTS
Effe ct of D OX ge l on t h e a lve ola r bon e loss
Macr o sco p i cal l y, t h e t r eat m en t o f an i m al s subj ect ed t o 11 days of experim ent al periodont al disease wit h t he DOX gel reduced t he alveolar bone
( P< 0.05), as com pared t o t he unt reat ed anim als subj ect ed t o EPD and t hose t reat ed wit h vehicle gel ( Figure 1) . These dat a can be clearly seen in Figure 2a, which shows t he m acroscopic aspect s of t he cont ra lat eral right side ( unligat ed side) wit h no resorpt ion of t he alveolar bone, com pared t o t he severe bone resorpt ion wit h root exposure in t he unt reat ed group ( NT) and VG group ( V) ( Figure 2b and 2c) .
Effe ct of D OX ge l on t h e m ye lope r ox ida se a ct ivit y on r a t gin giva
Figure 3 shows a reduction of inlammatory cell iniltration found in the periodontium tissue of
anim als subj ect ed t o experim ent al periodont it is and
treated with the DOX gel. The neutrophil iniltration
was evaluat ed by t he m yeloperoxidase act ivit y in
the gingival tissue. It was observed a signiicant
(P< 0.05) decrease in t he m yeloperoxidase act ivit y
in t he gingival t issue only in t he DOX gr oup as com pared t o t he rat s t reat ed wit h vehicle gel.
Ef f e c t o f D O X g e l o n t h e p e r i o d o n t a l st r u ct u r e s
The alveolar bone surface m orphology of t he DOX gr oup was less alt er ed com par ed w it h t he vehicle gel group. The AFM im age of t he DOX group ( Figure 4) shows a regular surface covered wit h a
shallow deined layer. The height of this layer varied
bet ween 150 and 250 nm , while t he NT group and t he VG gr oup var ied bet w een 750 and 950 nm ( Figure 5) .
Aft er 11 days of t reat m ent wit h t he DOX gel, t he AFM im ages showed alt erat ions com pared wit h t he V and NT group im ages ( Figure 6) . The dept hs of t he grooves on t he DOX group were 30- 120 nm and t heir widt h ranged from 100 t o 250 nm . Aft er 11 days of periodont al challenge, t he VG and NT groups present ed grooves becom ing m ore irregular t han t he DOX- t reat ed grooves. These alt erat ions were found in all of t he six exam ined areas of all 6 specim ens.
These dat a can be clear ly seen in Figur e 7, which shows t he t opographical aspect s by sect ion analysis of t he alveolar bone of t he rat m axillae wit h less resorpt ion, and m aint enance t he int egrit y of t he periodont al ligam ent , when com pared t o t he severe t opographical alt erat ions caused in t he NT group and vehicle gel t reat ed group. Figure 8 shows t he sect ion analysis in bone sur face on anim als subj ect ed t o EPD and t reat ed wit h t he DOX gel.
Ef f e c t o f D O X g e l o n t h e p e r i o d o n t a l liga m e n t
The AFM analysis of the region between the irst
and second m olars shows t opographical alt erat ions of t he periodont ium of t he anim als subj ect ed t o p er iod on t it is t h at r eceiv ed VG g r ou p r ev ealed a int ense t opographical alt erat ions w it h deeper gr oov es an d v alley s on it s st r u ct u r es su r f aces wit h cem ent um dest ruct ion and com plet e alveolar process resorpt ion ( Figure 8) , whereas a reduct ion of in f lam m at or y cell in f ilt r at ion an d a p ar t ial Figure 1- Macroscopic aspects of the effect of a
nanostructured 8.5% Doxycycline (DOX) locally applied
gel (Dental gel®) on alveolar bone loss. Experimental periodontal disease was also induced in the animals from the Non-Treated (NT) and in vehicle (V) groups. Naïve group was not submitted to EPD and received no treatment. Bars represent the mean ± Standard Error of Mean (SEM) of alveolar bone loss (mm; N=6). Asterisk indicates a
statistically signiicant difference between the DOX group
and NT group (*P<0.05; Bonferroni, ANOVA)
Figure 2- Macroscopic aspects of the effect of c on experimental periodontal disease in rats. A: macroscopic aspect of a normal periodontium of rat maxillae showing healthy periodontal structures; B and C: periodontium of rats subjected to induction of experimental periodontitis with no treatment (NT) and vehicle gel treatment, showing severe alveolar bone loss
preservat ion of t he cem ent um and of t he alveolar process was found in t he periodont ium of anim als subj ect ed t o EPD and t reat ed wit h t he DOX gel, t he
deep grooves values were statistically signiicant
( P< 0.05) , when com pared t o t he NT and VG groups. The AFM im ages of periodont ium t reat ed wit h t h e DOX gel sh ow ed m or ph ological differ en ces com pared t o t he AFM im ages of t he NT and VG groups. Moreover, t he AFM im ages of t he cem ent um t reat ed wit h DOX gel was also different from im ages of t he NT group.
Figure 5 shows how t he AFM surface roughness analysis was m ade t o est ablish t he widt h of alveolar bone grooves aft er 11 days of t reat m ent wit h t he vehicle gel and t he DOX gel. The surface of t he Naïve and DOX groups seem s relat ively regular, wit h Figure 4- Effect of 8.5% Doxycycline (DOX) gel on alveolar bone surface roughness in rats subjected to experimental periodontal disease (EPD). The AFM image from the DOX group shows a regular bone surface covered with a shallow
deined layer
Figure 5- Chosen areas to evaluate the topographical changes in alveolar bone of rats subjected to the experimental periodontitis disease EPD treated with vehicle gel
Figure 6- Effect of 8.5% Doxycycline (DOX) gel on the alveolar bone loss in alveolar bone roughness analysis by AFM in rats with experimental periodontal disease (EPD).
The 8.5% DOX gel was administered topically, daily, in
animals subjected to the EPD. Bars represent the mean ± Standard Error of Mean (SEM) of alveolar bone loss
(mm; N=6). Asterisk indicates a statistically signiicant
difference between treated group and Non-Treated (NT) group (*P<0.05; Bonferroni, ANOVA)
Figure 7- Topographical changes in alveolar bone surface of rats subjected to experimental periodontitis disease EPD
and treated with 8.5% Doxycycline (DOX) gel
Figure 3- Effect of 8.5% DOX gel on myeloperoxidase (MPO) activity in the maxillary gingival tissue of rats submitted to experimental periodontal disease (EPD). Vehicle (V) gel and Doxycycline (DOX) gel were administered topically in animals subjected to EPD induction. EPD was induced also in Non-Treated (NT) animals and Naïve group received no treatment and was not submitted to EPD induction. Bars represent mean ± Standard Error of Mean (SEM) of the activity of MPO/mg
of tissue. *P<0.05 was considered signiicantly different
compared to NT group (ANOVA; Bonferroni’s test)
higher coeficients of roughness surface, in other
hand, vehicle ( V) and EPD groups present ed great er alveolar bone resorpt ion wit h m uch less roughness at t he evaluat ed sit es ( Figure 6) . Com paring t he AFM surface roughness analysis, it can be st at ed t hat t he periodont al st ruct ures in t he DOX and Naïve groups seem ed rougher when com pared t o t he NT and VG groups. The m ean dept h of t he groove in t he DOX group varied bet ween 90 and 350 nm and
the width ranged from 1.0 to 1.5 μm. As the same results were obtained in all of the ive examined
areas for each specim en, t he m orphology in DOX
group represent s t he t ypical surface m orphology seen in t he cem ent um t r eat ed w it h 8 . 5 % w / w DOX gel.
D I SCUSSI ON
The local applicat ion of t he DOX gel evaluat ed in t his st udy played a posit ive role on t he bone loss associat ed t o ex per im ent al per iodont al disease. This effect was associat ed w it h reduct ion of t he
inlammatory reaction. Since the histopathological
and AFM analysis were done by a blind exam iner for t he t est and cont rol groups, t he result s of t he pr esent st udy dem onst rat ed t hat locally applied cont rolled- release DOX gel m ight part ly count eract t he negat ive effect of periodont al plaque byproduct s on periodont al healing.
The DOX gel showed an intense antiinlammatory activity inhibiting neutrophil iniltration as early as 6
h aft er EPD induct ion, as seen by t he reduct ion of MPO act ivit y on t he gingival t issue. This effect was
associated with a reduction in the local inlammatory cell iniltration. Perhaps this effect was achieved
because t he gel has a pot ent ant ibact erial act ivit y5.
Tet racycline and it s derivat ives have been shown t o be inhibit ors of m at rix m et alloprot einase t hat are
part of the inlammatory response and contribute
t o t he t issue breakdown in t he periodont al disease5.
Topical applicat ion of act ive subst ances offers an addit ional opt ion in periodont al t herapy5,7,8. I n
t his present st udy, a nanost ruct ured gel was able t o prot ect t he periodont al st ruct ures in t his m odel. This effect was probably achieved by a cont rolled released syst em provided by nanospheres loaded wit h DOX, as we can see t he preservat ion of alveolar bone in Figure 9. DOX gel was st udied in sm okers t o invest igat e it s inhibit ory effect on experim ent al per iodont al disease, show ing ex pr essive r esult s on t his part icular group of pat ient s20 as in ot her
t reat m ent groups12.
Previous st udies have shown t hat DOX is one of t he m ost pot ent and cost- effect ive TTC com m ercially available13,14,16, t his clinical approach has been shown
t o reduce collagenase product ion by ost eoblast s and ost eoclast s and also t o delay ost eoclast recruit m ent following dent al surgery15. DOX, as ot her chem ically
modiied tetracyclines (CMT), also possesses other
p h ar m acolog ic p r op er t ies t h at m ay p osit iv ely cont r ibut e for per iodont al healing pr ocess3 , 1 8 , 1 9.
Previous in vit ro st udies have dem onst rat ed t hat DOX inhibit s prot eases by blocking t he conversion of lat ent prot eases int o act ive m at ure form s and t he act ivat ion of MMP’s by chelat ing m et al ions23,24.
I n a rat m odel, CMT reduced t he act ivit y of t issue degradat ion enzym es such as m yeloperoxidase and dow n- regulat ed bone resorpt ion5. Also, syst em ic
adm inist rat ion of low doses of DOX in hum ans, which have negligible ant im icrobial effect s, result ed Figure 8- Topographical changes in alveolar bone surface
of rats subjected to experimental periodontitis disease EPD treated with the vehicle gel
Figure 9- Histopathology from the periodontium of rats
subjected to periodontitis. 8.5% Doxycycline (DOX) gel
was administered topically immediately after ligature placement and daily after periodontitis induction for 11
days. Photomicrograph of the region between the irst and
in reduct ion of collagenase in gingival crevicular
luid24. Hence, since an increased prot ease act ivit y
i s asso ci at ed w i t h t h e i n d u ced p er i o d o n t i t i s, t hese propert ies of DOX m ay offer an addit ional explanat ion t o t he observed im proved t reat m ent out com e in t his rat m odel t hat received t his locally delivered DOX syst em com pared t o a vehicle gel only.
As shown in t he alveolar bone height im age in Figure 4, t he use of t he DOX gel for 1 m in t hree t im es a day shows int egrit y of t he alveolar bone roughness surface, while in Figure 5 t he AFM im ages show a part ially dest royed alveolar bone surface height im age.
Concer ning t he t opographical and r oughness analy sis, Figur e 6 show s t he height int egr it y of alveolar bone in Naïve group, while in t he vehicle gel group ( Figure 7) is clear t o observe t he narrower widt h and shallow height on t he DOX group as well. We not iced t he st rong cont rast bet w een t he VG group when com pared t o t he DOX group, which clear ly sh ow ed t h e p r esen ce of alv eolar b on e int egrit y. For bet t er underst anding of t he m et hod, Figures 5 and 8 show a clear perspect ive on how t he AFM r egions w er e m easur ed fr om a r egular hist opat hological sect ion et ched w it h x y lol and t hen from t he AFM specim ens t he program could m easure t he surface roughness and t opographical changes wit h high accuracy.
Based on t h e f i n d i n g s of t h i s ex p er i m en t , AFM m et hodology can be used t o evaluat e wit h a syst em at ic precision t he alveolar bone st ruct ure and it s surface regularit y. The sect ions in Figures 7 and 8 show the alveolar bone surface, with 670 nm in m ean reaching st at ist ical difference when com pared t o t he DOX group versus vehicle gel group, approxim at ely
50% of the surface shows a signiicant difference
( p< 0.05) on roughness and t opographical analysis
after 11 days treatment. These indings lead us for
a clearly dem onst rat ion t hat t he t reat m ent of an 8.5 % w/ w DOX nanost ruct ured gel does prevent s denat urat ion of periodont al st ruct ures, especially t he alveolar bone loss in t his m odel.
CON CLUSI ON
I n sum m ary, t his st udy dem onst rat es a novel m et h od t o d et er m in e p er iod on t al in t eg r it y in experim ent ally induced periodont it is. The fact t hat t et racycline and it s derivat ives are current ly used as chr onic t r eat m ent s for per iodont al disor der s
with a safety proile highlights the need for further
research in order t o dem onst rat e t heir clinical use. I t was also dem onst rat ed t hat AFM is a powerful t ool used t o evaluat e t he phases of t he process of experim ent al periodont al disease.
ACKN OW LED GEM EN TS
Th is w or k w as f in an cially su ppor t ed by t h e Brazilian Council of Technological Developm ent . The Grant from CNPq ( Nat ional Council for Technological
and Scientiic Development, Brazil, Proc. CNPq:
452315/ 2009- 3; 558017/ 2008- 8; 561940/ 2008- 8; 452125/ 2008- 1; 402074/ 2008- 4; 577214/ 2008- 0) t o t he recepient MAB is grat efully acknowledged.
Special t hanks t o Beat riz and Em anuel Viana ( Point Sut ure do Brasil I ndúst ria de Fios Cirúrgicos Lt d a), El b e r Be ze r r a d e Me n e ze s ( Ev i d e n ce Ph ar m aceu t icals, For t aleza, CE- Br azil) an d I t alo Rachid ( Bioident ical Pharm aceut ics, Fort aleza,CE-Brazil) for infrast r uct ur e and t echnical suppor t . The aut hors are indebt ed t o t he Dean Prof. Claudio Ricardo Gom es de Lim a ( I FCE) .
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