ABSTRACT
http://dx.doi.org/10.1590/1678-775720140190
I m m unolocalizat ion of m arkers for bone form at ion
dur ing guided bone r egenerat ion in ost eopenic
rat s
Tábata de Mello TERA1, Rodrigo Dias NASCIMENTO2, Renata Falchete do PRADO1, Mauro Pedrine SANTAMARIA2,
Maria Aparecida Neves JARDINI2
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Corresponding address: Maria Aparecida Neves Jardini - Department of Diagnosis and Surgery - Av. Eng. Francisco José Longo, 777 - Jd. São Dimas - São
José dos Campos - 12245-000 - SP - Brazil - Phone: (55 12) 3947-9043 - Fax: (55 12) 3947-9010 - e-mail: jardini@fosjc.unesp.br
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bj ect ive: The aim of t his paper was t o evaluat e t he r epair of onlay aut ogenous bone JUDIWV FRYHUHG RU QRW FRYHUHG E\ DQ H[SDQGHG SRO\WHWUDÀXRURHWK\OHQH H37)( PHPEUDQHXVLQJLPPXQRKLVWRFKHPLVWU\LQUDWVZLWKLQGXFHGHVWURJHQGH¿FLHQF\0DWHULDO and Met hods: Eight y fem ale rat s w er e random ly divided int o t w o gr oups: ovar iect om ized ( OVX) and w it h a sim ulat ion of t he sur gical pr ocedur e ( SHAM) . Each of t hese gr oups w as again div ided in t o gr ou ps w it h eit h er placem en t of an au t ogen ou s bon e gr af t alone ( BG) or an aut ogenous bone graft associat ed w it h an e- PTFE m em brane ( BGM) . Anim als w er e eut hanized on days 0, 7, 21, 45, and 60. The specim ens w er e subj ect ed t o im m unohist ochem ist r y for bone sialopr ot ein ( BSP) , ost eonect in ( ONC) , and ost eocalcin ( OCC) . Result s: All gr oups ( OVX+ BG, OVX+ BMG, SHAM+ BG, and SHAM+ BMG) show ed gr eat er bone for m at ion, obser ved bet w een 7 and 21 days, w hen BSP and ONC st aining w er e m or e int ense. At t he 45- day, t he bone graft show ed dir ect bonding t o t he r ecipient bed in all specim ens. The ONC and OCC show ed m or e expr essed in granulat ion t issue, in WKHPHPEUDQHJURXSVLQGHSHQGHQWO\RIHVWURJHQGH¿FLHQF\&RQFOXVLRQV7KHH[SUHVVLRQ RIERQHIRUPLQJPDUNHUVZDVQRWQHJDWLYHO\LQÀXHQFHGE\HVWURJHQGH¿FLHQF\+RZHYHU WKHPDUNHUVFRXOGEHLQÀXHQFHGE\WKHSUHVHQFHRIWKHH37)(PHPEUDQHKe y w or ds: Est r ogen. Bone r egenerat ion. Ost eocalcin. Ost eonect in. Sialopr ot ein.
I N TROD UCTI ON
The incr eased life expect ancy of t he populat ion has led t o incr eased dem and for r ehabilit at ion using osseoint egrat ed im plant s and reconst ruct ive procedures. To receive an im plant , a sit e m ust have adequat e alveolar bone volum e1,7,13. When t her e
is an inadequat e alveolar r idge for t his pur pose, cer t ain t echniques m ay be used t o pr om ot e an incr ease in bone t issue, such as aut ogenous bone gr af t s an d gu ided bon e r egen er at ion3 , 5 , 7 , 2 5 , 2 6 , 2 9.
Addit ionally, in associat ion w it h t he populat ion’s aging, t here has been an increase in t he prevalence of diseases t hat m ay int er fer e w it h t he pr ocess of osseoint egrat ion, such as ost eopor osis. This disease affect s m illions of people around t he w orld and is charact er ized by decr eased bone m ass and
st r uct ural det er iorat ion, leading t o an incr eased r isk for fract ur es9,17,25.
Because t he int er fer ence of ost eopor osis w it h bon e r epair is h igh ly debat ed, it is im por t an t t o in v est ig at e t h e m ech an ism s b y w h ich t h is d i sea se ca n j eo p a r d i ze t h e o st eo i n t eg r a t i o n p r o ce ss. Se v e r a l st u d i e s7 , 8 , 1 0 h a v e a i m e d t o
descr ibe t his r elat ionship, w hich obser v ed t hat
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r em odeling in t he lat e st age, w hich r esult ed in com pr om ised m echanical pr oper t ies in t he end.
Desp it e t h e n eg at iv e ef f ect s d em on st r at ed by som e st udies m ent ioned above, ot her st udies have pr esent ed cont radict or y, and show ed t hat ovar iect om y did not ser iously affect bone healing aft er t he placem ent of im plant s in cor t ical bone ar eas, but it r educed t he bone cont act rat io and t he bone in t he cancellous bone ar ea, and t hat
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not cr ucial for fract ur e healing. Ther e is a lack
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m arkers for bone form at ion aft er onlay aut ogenous bone graft placem ent on t he m andibula.
About m ar k er s for bone for m at ion, Langille an d Solu r sh1 6 ( 1 9 9 0 ) sh ow ed t h at cu lt u r e of
m esenchy m e m andibular cells init iat e ex pr ession of t y pe I and I I collagen at ear ly per iods, follow ed by t y pe X, w h ich coin cided w it h t h e on set of m in er alizat ion . Af t er t h at , t h ey lost e car t ilage m ar ker s and began t o ex pr ess bone sialopr ot ein I I ( BSP I I ) , ost eocalcin, and t y pe I collagen. I n addit ion, ost eonect in and alk aline phosphat ase were dem onst rable in vit ro by cells in lat er periods.
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by von Kossa st aining.
Accor ding t o Nagat a, et al.21 ( 1991) , t he init ial
dist r ibut ion of BSP m ay m odify bone for m at ion and m ineralizat ion. Bot h ost eopont in and bone sialoprot ein are locat ed ahead of t he m ineralizat ion fr ont , being necessar y for t he init iat ion of t he pr ocess. Bone sialopr ot ein m ay be consider ed a cr y st al nucleat or. Ost eocalcin and ost eonect in ar e not pr esent in ar eas of init ial cr y st al for m at ion, but ar e seen in t he ent ir ely m ineralized m at r ix27.
The t hr ee bone- for m ing m ar ker s w er e chosen based on t heir st r uct ural and m ineral- inducing pr oper t ies, in addit ion t o t hese pr ot eins’ capacit y t o alt er r ecr uit m ent , at t achm ent , differ ent iat ion, and act iv it y of bone cells.
Th e r e f o r e , t h e a i m o f t h i s st u d y w a s t o ev alu at e t h e im m u n oh ist och em ical ex pr ession of bone m ar k er pr ot eins in t he bone r epair of onlay aut ogenous graft s in ovar iect om ized rat s. Addit ionally, t he st udy aim ed t o observe whet her t he
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healing in t his sy st em ic condit ion.
M ATERI AL AN D M ETH OD S
An im a ls
T h e p r e s e n t s t u d y w a s c o n d u c t e d i n accor dance w it h t he Et hical Pr inciples for Anim al Ex per im ent at ion adopt ed by t he Brazilian School of Anim al Experim ent at ion ( COBEA) and approved by t he Research Et hics Com m it t ee at t he College of
Dent ist ry of São José dos Cam pos – Univ. Est adual Pau list a ( I CT- UNESP) u n d er p r ot ocol n u m b er 026/ 2008- PA/ CEP.
Eight y 3- m ont h- old, adult fem ale Wist ar rat s t h at w eig h ed ap p r ox im at ely 3 0 0 g r am s w er e enr olled in t he pr esent st udy. Dur ing t he ent ir e per iod of t he st udy, t he anim als w er e housed in gr oups of six in plast ic cages, and food and wat er w er e given ad libit um t o all t he anim als. Pr ior t o t he beginning of t he ex per im ent al pr ocedur es, t he anim als w er e allow ed t o acclim at ize t o t he laborat or y env ir onm ent for 5 day s. The anim als w er e random ly div ided int o 2 gr oups: Gr oup OVX was subj ect ed t o an ovar iect om y pr ocedur e, and Gr ou p SHAM w as su bj ect ed t o sh am su r ger y. Each gr oup w as div ided int o t he follow ing t w o subgroups: placem ent of an aut ogenous bone graft ( BG) and an aut ogenous bone graft associat ed wit h an e- PTFE m em brane ( BGM) ( WL Gor e, Newar k , Delawar e, USA) .
Ov a r ie ct om y
The anim als w er e anest het ized using a 2 % x y lazin e solu t ion ( Rom pu m , Bay er, São Pau lo, SP, Brazil) and k et am ine ( Dopalen, Agr ibands, Paulínia, SP, Brazil) at a rat io of 1: 1 ( 0.3 m l/ 100 g of body w eight ) . I n t he OVX gr oup ( 40 anim als) , t w o s m a l l i n c i s i o n s ( ~ 1 0 m m ) w e r e m a d e beginning aft er t he last r ib, one on each side of t he back of t he anim al. Aft erward, t he ovaries were held up and ligat ures were m ade t o avoid bleeding. Then, t he ovar ies w er e com plet ely excised. I n t he SHAM gr oup ( 40 anim als) , aft er t he incisions, t he bilat eral ovar ies w er e held up and t hen r et ur ned t o t heir or iginal posit ion w it hout being excised. The incisions w er e sut ur ed using a r esor bable m at er ial for t he m uscle layer ( poligalat in 910 – Vycr il 4.0 – Et hicon Johnson & Johnson, São José dos Cam pos, SP, Brazil) and silk sut ur es for t he sk in ( 4.0 Et hicon, Johnson & Johnson, São José dos Cam pos, SP, Brazil)
Bon e gr a ft su r ge r y
Aft er 30 day s, a new sur gical pr ocedur e for t h e placem en t of au t ogen ou s bon e gr aft s w as per for m ed as descr ibed by Jar dini, et al.13 ( 2005) .
& Johnson, São José dos Cam pos, SP, Brazil) . Aft er war d, t he e- PTFE m em brane was adapt ed, cov er in g t h e bon e gr aft in t h e OVX+ BGM an d SHAM+ BGM. Th ese p r oced u r es allow ed close cont act bet w een t he graft and t he sur face of t he m andibular bone ( Figur e 1) .
Aft er t he graft pr ocedur e, t he m uscle layer was sut ur ed using a 5.0 absor bable polyglact in 910 sut ur e ( Et hicon, Johnson & Johnson, São José dos Cam pos, SP, Brazil) , follow ed by sut ur ing of t he sk in using a 4.0 silk sut ur e ( Et hicon, Johnson & Johnson, São José dos Cam pos, SP, Brazil) . The lat t er was also used t o sut ure t he donor area. Aft er t he sur ger y, a single dose of ant ibiot ics ( 1 m g/ k g) was adm inist er ed int ram uscular ly t o all anim als ( Pent abiot ic, For t Dodge, São Paulo, SP, Brazil) . Eut hanasia was per for m ed using an over dose of anest het ics im m ediat ely aft er surgery ( 0 hour) and 7, 21, 45, and 60 days aft er t he surgical procedure ( Figur e 2) .
H ist ologica l pr oce du r e s
Th e specim en s w er e dem in er alized u sin g a 1 0 % EDTA solu t ion at pH 7 . 8 in a m icr ow av e oven ( PELCO 3441, Ted Pella, Califor nia, USA) . Th e d em in er alized sp ecim en s w er e sect ion ed t ran sv er sely at t h e cen t ral r egion of t h e bon e
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or ient ed t owar d t he cut t ing sur face.
I m m u n oh ist och e m ica l pr oce du r e s
Th e block s w er e sliced at a t h ick n ess of 3
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for ost eocalcin ( OCC) ( FL- 110: sc- 30044; Sant a Cr u z Bi o t ech n o l o g y, Paso Ro b l es, CA, USA) , bone sialopr ot ein ( BSP) , and ost eonect in ( ONC) . Th e BSP ( LF- 8 7 ) an d ONC ( LF- 2 3 ) an t ibodies w er e k indly donat ed by Dr. Lar r y W. Fisher at t he Nat ional I nst it ut e of Dent al and Craniofacial Resear ch at t h e Nat ion al I n st it u t es of Healt h ( Bet hesda, MD, USA) .
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Ant igen r et r ieval was per for m ed using cit rat e ( pH 6.0) in a m icr owave oven follow ed by t he block ing of endogenous per oxidase using a solut ion of 50% m et hyl alcohol and hydrogen peroxide ( 20- volum e solut ion) ( 1: 1) . The sam ples w er e incubat ed in bov ine ser um album in ( BSA) for 1 hour inside
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Sam ples w er e t hen incubat ed w it h t he pr im ar y ant ibodies ( BSP, 1: 150, 1 hour, room t em perat ure; ONC, 1 : 4 0 0 , 1 h ou r, r oom t em perat u r e; OCC, 1 : 4 0 0 , 4 °C, ov er n igh t ) follow ed by in cu bat ion w it h a secondar y ant ibody ( Univer sal LSAB TM Kit / HRP, Rb/ Mo/ Goat – DAKO, Car pint er ia, CA, USA) f or 3 0 m in u t es. A f in al in cu b at ion w as per for m ed using t he t er t iar y com plex st r ept av idin p er ox i d ase ( Un i v er sal LSAB TM Ki t / HRP, Rb / Mo/ Goat – DAKO, Car pint er ia, CA, USA) for an addit ional 30 m inut es. The react ion was visualized using diam inobenzidine ( DAB – DAKO, Carpint eria, CA, USA) . Count er st aining was per for m ed using May er ’s hem at ox y lin, and t he specim ens w er e m ount ed in Per m ount .
As a posit ive cont r ol for BSP and ONC was used on rat bone t issue r epair ar ea. Megak ar yocy t es w er e used as a posit ive cont r ol for OCC. Negat ive cont r ols w er e obt ained by t he om ission of pr im ar y ant ibodies.
Figure 1- Surgical procedure: a) the calvarium was
used as the donor area to graft removed; b) angle of the mandible was the recipient area; c) recipient bed; d) perforation was made at the angle of the mandible, which enabled the stable attachment of the bone block; e) bone block in position; f) e-PTFE membrane was adapted, covering the bone graft in the OVX+BGM and SHAM+BGM
An a ly sis
Mi cr oscop i c an al y si s w as con d u ct ed u si n g a n Ax i o p h o t 2 l i g h t m i cr o sco p e ( Ca r l Z ei ss, Ober kochen, Ger m any ) coupled w it h an Ax ioCam MRc 5 digit al cam era ( Car l Zeiss, Ober kochen, Ger m an y ) , w h i ch t r an sm i t t ed i m ag es t o t h e Ax ioVision Release 4.7.2 com put er soft war e.
Th e in t en sit y of t h e im m u n oh ist och em ical st ain in g of pr edet er m in ed st r u ct u r es an d cells
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cat egorized as m ild ( + ) , m oderat e ( + + ) , or int ense ( + + + )12.
RESULTS
The OVX+ BG and OVX+ BGM gr oups pr esent ed g r eat er m ar r ow sp aces an d con n ect iv e t issu e com p ar ed t o t h e SHAM g r ou p s, sh ow in g t h e
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( Figur es 3, 4, and 5) .
BSP
Wit hin t he sam e per iod, t he 4 gr oups ex hibit ed sim ilar ch ar act er ist ics. At 0 h ou r s, t h e b on e m at r ix fr om t he r ecipient bed and graft show ed m ild posit iv it y in all gr oups for all per iods. The OVX+ BG and OVX+ BMG groups show ed m oderat e st aining of t he r ever sal lines of t he r ecipient bed.
(CT= Connective Tissue; RB=Recipient Bed; G= Graft; NB= Newly Formed Bone) BG= Bone Graft; BGM= Autogenous Bone Graft Associated with e-PTFE membrane
Hour 0 Day 7 Day 21 Day 45 Day 60
OVX/SHAM OVX/SHAM OVX/SHAM OVX/SHAM OVX/SHAM
BG BGM BG BGM BG BGM BG BGM BG BGM
Bone matrix + + + + + + + + + + Newly bone RB-G - - +++ +++ +++ +++ +/++ +/++ +/++ +/++ Newly bone around G - - - - +++ +++ +/++ +/++ +/++ +/++ Osteocytes RB +/++ +/++ +/++ +/++ +/++ +/++ +/++ +/++ +/++ +/++ Osteocytes graft ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
Osteocytes NB - - +++ +++ +++ +++ ++ ++ +/++ +/++ Osteoblasts - - +++ +++ +++ +++ ++ ++ +/++ +/++ Reversal lines ++ - +++ +++ +++ +++ +++ +++ ++ ++
CT B-G - - -
-CT G - - -
-Figure 3- Bone sialoprotein (BSP). Structures labeled and staining intensity
(CT= Connective Tissue; RB=Recipient Bed; G= Graft; NB= Newly Formed Bone) BG= Bone Graft; BGM= Autogenous Bone Graft Associated with e-PTFE membrane
Hour 0 Day 7 Day 21 Day 45 Day 60
OVX/SHAM OVX/SHAM OVX/SHAM OVX/SHAM OVX/SHAM
BG BGM BG BGM BG BGM BG BGM BG BGM
Bone matrix - - - -Newly bone B-G - - - -Newly bone around G - - - -Osteocytes-recipient bed - - - -Osteocytes graft +/++ +/++ +/++ +/++ +/++ +/++ +/++ +/++ +/++ +/++
Osteocytes NB - - +/+++ +/+++ +/+++ +/+++ -/+ -/+ - -Osteoblasts - - +++ +++ ++/+++ ++/+++ +/+++ +/+++ +/++ +/++ Reversal lines - - -
-CT B-G - - +++ +++ +++ +++ +++ +++ +/++ +/++ CT G - - + ++/+++ +/++ +++ +/++ +++ + ++
At 7 and 21 day s, new ly bone t issue for m ed on t he sur face of t he r ecipient bed ex hibit ed int ense st aining. The ost eoblast s pr esent at t he per ipher y of t he r ecipient bed and ar ound t he im m at ur e bone t rabecular show ed int ense st aining as w ell as lar g e ost eocy t es, w h ich w er e in t er sp er sed in t h e im m at u r e b on e t r ab ecu lar. At d ay 4 5 , t he new ly for m ed bone t issue, bot h at t he bed-graft int er face and at t he per ipher y of t he bed-graft , pr esent ed m ild t o m oderat e st aining in all gr oups. Large ost eoblast s and ost eocyt es in t his area w ere m oderat ely st ained. At day 60, t he new ly for m ed bone t issue in t he bed- graft int er face and ar ound t he graft show ed diffuse st aining t hat ranged fr om m ild t o m oderat e w it h a pr edom inance of m ildly posit ive ar eas. Ost eocy t es in t his ar ea som et im es accom panied t he st aining, and som et im es t hese sam ples show ed m ild t o m oderat e st aining only in t he lacuna. The ost eoblast s in t his region present ed m ild t o m oderat e st aining. The r ever sal lines w er e m oderat ely posit ive ( Figur e 6a and Figur e 6b) .
ON C
At 0 hour s, t he im m unohist ochem ical st aining w as sim ilar in all four gr oups. Only ost eocy t es locat ed in t he m edian r egion of t he graft w er e st ain ed , w h ich v ar ied f r om m ild t o m od er at e int ensit y. At day 7, t he OVX+ BG and SHAM+ BG gr ou ps pr esen t ed a lar ge am ou n t of in t en sely st ained granulat ion t issue int er posed bet w een t he graft and t he r ecipient bed. The OVX+ BGM and SHAM+ BGM groups also showed t his sam e st aining pat t er n, t hough t he am ount of granulat ion t issue in t his ar ea was sm aller. The granulat ion t issue sur r ounding t he graft was m ildly st ained in t he OVX+ BG and SHAM+ BG gr oups, w hile t his t issue pr esent ed m oderat e t o int ense st aining in t he
OVX+ BGM and SHAM+ BGM gr oups. Ost eoblast s sh o w e d i n t e n se st a i n i n g , a n d o st e o cy t e s i n t he im m at ur e bone t rabecular ex hibit ed var ied st aining t hat was som et im es m ild and som et im es int ense. The m ost int ense st aining was obser ved in lar ger ost eocy t es. At day 21, t he connect ive t issue present at t he bed- graft int erface present ed int ense st aining in all four gr oups. The OVX+ BG and SHAM+ BG gr oups show ed m ild t o m oderat e st ain in g in t h e con n ect iv e t issu e su r r ou n d in g t he graft , w hile t he OVX+ BGM and SHAM+ BGM gr oups show ed int ense st aining. The ost eoblast s show ed m oderat e t o int ense st aining, w her eas t he lar gest ost eocy t es pr esent in t he im m at ur e bone t rabeculae show ed m or e int ense st aining t han t hose found in ot her ar eas. At day 45, t he connect ive t issue int erposed bet ween t he recipient bed an d t h e gr af t sh ow ed in t en se st ain in g in all f ou r g r ou p s. Th e OVX+ BG an d SHAM+ BG gr oups show ed m ild t o m oderat e st aining of t he connect ive t issue sur r ounding t he graft , w her eas in t he OVX+ BGM and SHAM+ BGM gr oups, t he st aining was int ense. The ost eoblast s show ed m ild t o int ense st aining. The m aj or it y of t he ost eocyt es had no st aining, even t he lar gest ones. At day 60, w hen pr esent , t he connect ive t issue at t he bed-graft int erface showed m ild t o m oderat e st aining in all four gr oups. The connect ive t issue sur r ounding t he graft ex hibit ed m ild st aining in t he BG gr oups and m oderat e st aining in t he BG+ M gr oups. The ost eoblast s show ed m ild t o m oderat e st aining, w hile t he ost eocy t es w er e not st ained ( Figur e 7a and Figur e 7b) .
OCC
The bone m at r ix of t he r ecipient bed and t he graft show ed discr et e and diffuse st aining in all (CT= Connective Tissue; RB=Recipient Bed; G= Graft; NB= Newly Formed Bone)
BG= Bone Graft; BGM= Autogenous Bone Graft Associated with e-PTFE membrane
Hour 0 Day 7 Day 21 Day 45 Day 60
OVX/SHAM OVX/SHAM OVX/SHAM OVX/SHAM OVX/SHAM
BG BGM BG BGM BG BGM BG BGM BG BGM
Bone matrix + + + + + + + + + + Newly bone RB-G - - - + + + + Newly bone around G - - - + + + + Osteocytes-recipient bed + + + + + + + + + + Osteocytes graft ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
Osteocytes NB - - + +
Osteoblasts - - +/++ +/++ +/++ +/++ +++ +++ +++ +++ Reversal lines - - -
-CT B-G +/+++ +/+++ +++ +++ +++ +++ +++ +++ ++ +++ CT G - - +/+++ +++ -/+ +++ -/+ +++ -/+ ++/+++
evaluat ed gr oups for all per iods. At 0 hour s, t he ost eocy t e lacunae pr esent at t he m idline r egion of t he graft w er e m oderat ely st ained, w hile t he ost eocy t es of t he r ecept or show ed m or e discr eet m ar k ing. At day 7, t he new ly for m ed bone t issue show ed no st aining. The OVX+ BG and SHAM+ BG groups exhibit ed large am ount s of int ensely st ained
granulat ion t issue int erposed bet ween t he recipient bed and t he graft . The OVX+ BGM and SHAM+ BGM gr oups also show ed t his st aining pat t er n, t hough t h e am ou n t of gr an u lat ion t issu e in t h is ar ea was sm aller. The granulat ion t issue sur r ounding t h e gr aft ex h ibit ed m ild t o in t en se st ain in g in t he OVX+ BG and SHAM+ BG gr oups and int ense
Figure 6a- a) Bone Sialoprotein (BSP) -Day 0 Group OVX-BGM: Slight expression in recipient bed (RB) matrix and graft
st aining in t he OVX+ BGM and SHAM+ BGM groups. The ost eoblast s showed m ild t o m oderat e st aining, and t he lar ge ost eocy t es ex hibit ed m ild st aining in all four gr oups. At day 21, t he new ly for m ed bone t issue show ed no st aining. The connect ive t issue pr esent at t he bed- graft int er face show ed int ense st aining. I n t he OVX+ BGM and SHAM+ BGM gr ou ps, t h e con n ect iv e t issu e su r r ou n din g t h e graft show ed int ense st aining, w her eas, in t he OVX+ BG an d SHAM+ BG g r ou p s, t h e st ain in g was eit her negat ive or m ild. I n all gr oups, t he ost eoblast s had m ild t o m oderat e st aining, and t h e ost eocy t es pr esen t in t h e im m at u r e bon e t rabecular, par t icular ly t he lar ger ones, show ed m ild st aining in t he SHAM+ BG and SHAM+ BGM gr oups, w her eas, in t he OVX+ BG and OVX+ BGM gr oups, t he st aining was negat ive. At day 45, t he new ly for m ed bone t issue had diffuse and discr et e st aining at t his point , sim ilar t o t hat obser ved at t he init ial t im e point . At day 60, t he new ly for m ed bone t issue in t he r ecipient bed- graft int er face ex hibit ed m ild and diffuse st aining, as obser ved
at t he init ial t im e point . This new ly for m ed t issue w as som et im es slight ly m or e st ained t han t he m at r ix . When pr esent , connect ive t issues fr om t he bed- graft int er face show ed int ense st aining in t he OVX+ BG and SHAM+ BG gr oups and m oderat e st aining in t he OVX+ BGM gr oup. The connect ive t issue sur r ounding t he graft show ed m ild or no st aining in t he G gr oups, w her eas t he st aining in t he BGM gr oups var ied bet w een m oderat e and int ense. The ost eoblast s exhibit ed int ense st aining in all regions where t hey were present ed, like large ost eocy t es of t he new ly for m ed m at r ix ( Figur e 8a and Figur e 8b) .
D I SCUSSI ON
The aim of t he pr esent st udy was t o evaluat e t he ex pr ession of im m unohist ochem ical m ar ker s of bone for m at ion dur ing t he r epair pr ocess of aut ogenous bone graft s t hat were bot h covered and not cover ed by an e- PTFE m em brane in est r
ogen-GH¿FLHQWIHPDOHUDWV7KHUHVXOWVRIWKHSUHVHQW Figure 6b- a) Bone Sialoprotein (BSP) Day 45 Group SHAM-BGM: mild to moderate marking in newly formed bone (*) and
Figure 7a- a) Osteonectin (ONC) Day 0 Group OVX-BGM: no labeling of both the receptor bone matrix bed (L) and graft
(E). At the bottom, intense staining was observed in skeletal striated muscle (*); b) Day 0 Group OVX-BGM: osteocytes (Ö) present in the mid portion of the graft exhibit mild to moderate markup; c) Day 7 Group OVX-BG: connective tissue () exhibited varied staining that was sometimes intense and sometimes mild; d) Day 7 Group SHAM-BGM: osteoblasts (Î) and osteocytes (Ö) showing intense staining; e) Day 21 Group SHAM-BG: connective tissue present at the surround bed-graft presented intense staining; f) Day 21 Group OVX-BG: osteoblasts (Î) on the bone surface are moderately marked
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not alt er t he ex pr ession of bone m ar ker s dur ing t he r epair of onlay blocks placed on rat m andibles. How ever, t he use of t he e- PTFE m ay enhance t he ex pr ession of t he bone m ar ker s r egar dless of t he
pr esence of t he sy st em ic condit ion.
w her eas m at ur e bone was w eak ly st ained at all t im e per iods. Based on analy ses of alveolar bone for m at ion in rat s, sim ilar r esult s w er e obt ained b y Pin er o, et al.2 4 ( 1 9 9 5 ) an d Ar am b aw at t a,
et al.2 ( 2 0 0 5 ) . Ch en , et al.4 ( 1 9 9 3 ) an aly zed
t he m andibular alv eolar bone of sw ine fet uses an d o b ser v ed i n t en se BSP st ai n i n g i n n ew l y for m ed bone. I vanov sk i et al.12 ( 2000) obser ved
int ense BSP st aining of t he new ly for m ed bone in experim ent s on guided bone regenerat ion in dogs. I n t he pr esent st udy, ost eoblast s and ost eocy t es ex hibit ed gr eat er posit iv it y for BSP on day s 7 and
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point , w hen st aining was m ild and rest rict ed t o t he lacunae. Piner o, et al.24 ( 1995) obser ved int ense
BSP st aining of ost eoblast s in t he m andibles of new bor n rat s, w her eas t he ost eocy t es w er e not st ained. I vanovski, et al.12 ( 2000) observed int ense
st aining of ost eoblast s and ost eocy t es associat ed w i t h n e w l y f o r m e d b o n e d u r i n g p e r i o d o n t a l r egen er at ion in dogs. I sh igak i, et al.1 1 ( 2 0 0 2 )
observed weak BSP st aining in ost eoblast s present
in t he m andibles of fet al rat s. BSP was t he only m ar ker t hat r evealed r ever sal lines, and st aining was slight ly m or e pr onounced in t he OVX gr oups on day s 7 and 21.
Ost eocalcin ( OCC) sh ow ed st ain in g of t h e new ly for m ed bone m at r ix on day 45 and at 60 days, revealing charact erist ics of t he m at ure bone. Sim ilar r esult s w er e found by I vanov sk i, et al.12
( 2000) , w ho obser ved lit t le or no st aining by OCC st aining of t he new ly for m ed bone in ex per im ent s on guided bone regenerat ion in dogs; furt herm ore, I shigak i, et al.11 ( 2002) obser ved w eak posit iv it y
f o r OCC i n t h e n e w l y f o r m e d b o n e i n t h e m andibles of rat fet uses. Cont r ov er sial r esult s w er e obt ained by Luv izut o, et al.18 ( 2010) aft er
analy zing alveolar bone r epair in ovar iect om ized rat s. I n t hat st udy, rat s t hat w er e not subj ect ed t o ovar iect om y show ed int ense st aining of t he new ly for m ed bone m at r ix by OCC on day s 14 and 21, w hile t he ovar iect om ized gr oup show ed a m or e discr et e ex pr ession for t his m ar ker. I n t his st udy, ost eoblast s sh ow ed m ild t o m oder at e st ain in g
Figure 7b- a) Osteonectin (ONC) Day 45 Group OVX-BGM: in connective tissue surrounding the graft, the staining was
bet w een 7 and 4 5 day s, w it h int ense st aining obser ved at t he last t w o t im e point s. Ost eocy t es fr om new ly for m ed bone pr esent ed m ild posit iv it y of t heir lacunae. At 21 day s, st aining was int ense in t he SHAM gr oups, w hile, in t he OVX gr oups, it was m oderat e. Moderat e st aining was obser ved
at 4 5 an d 6 0 day s. I v an ov sk i, et al.1 2 ( 2 0 0 0 )
obser ved int ense OCC st aining of ost eoblast s and ost eocy t es associat ed w it h new ly for m ed bone dur ing per iodont al r egenerat ion in dogs aft er 30 days, while I shigaki, et al.11 ( 2002) observed weak
OCC st aining in ost eoblast s in t he m andibles of
Figure 8a- a) Osteocalcin (OCC) Day 0 Group OVX-BG: bone matrix with lightweight markup; b) Day 0 Group OVX-BG:
fet al rat s.
The ONC w as t he only m ar k er t hat did not show any st aining ov er t im e. I t s m ost int ense e x p r e s s i o n w a s o b s e r v e d o n d a y 7 o f t h e experim ent .Ost eoblast s show ed int ense posit ivit y for ONC bet w een 7 and 45 day s. On day s 7 and 21, t he ONC posit ivit y in ost eocyt es from im m at ure bone t rabeculae var ied fr om m ild t o int ense. The m ost in t en se st ain in g w as ob ser v ed in lar g er ost eocy t es. On day 4 5 , m ost ost eocy t es w er e no longer st ained. Sim ilar r esult s w er e found by I shigak i, et al.11 ( 2002) , w ho obser ved m oderat e
t o int ense ONC st aining in ost eoblast s pr esent in t he m andibles of fet al rat s.
Ovar iect om y is w idely r ecognized for it s abilit y t o induce ost eopenia, as dem onst rat ed in several classic st udies15,20,23,25,30. Alt hough several clinical
st udies conduct ed on w om en w it h ost eopor osis and in anim al m odels have dem onst rat ed a delay in bone r epair7–10,14,24,28, t he r esult s of t he pr esent
st udy show t hat ovariect om y- induced ost eoporosis
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Melhus, et al.19 ( 2007) obser ved t hat est r ogen
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bone r epair.
I n t he present st udy, t he result s were alt ered by t he pr esence of an e- PTFE m em brane.Ret zepi, et al.26 ( 2010) and Donos,et al.6 ( 2002) found great er
bone for m at ion, m at urat ion, and sm aller am ount s of bone loss w hen onlay graft s w er e cover ed by e- PTFE m em branes com pared t o graft s wit hout t he m em brane. Donos, et al.6 ( 2002) dem onst rat ed
t hat t he use of a m em brane coat ing aut ogenous graft s accelerat ed t he m igrat ion of ost eogenic cells, t he for m at ion of new bone, and t he m ineralizat ion pr ocess. Jar dini, et al.13 ( 2005) evaluat ed t he use
of a e- PTFE m em brane in specim ens of aut ogenous gr af t s in r at s an d obser v ed gr eat er bon e loss dur ing t he healing per iod in t he gr oup t hat did not r eceive t he m em brane. A sim ilar exper im ent al m odel developed by Nascim ent o, et al.22 ( 2009)
obser ved t hat graft s not cover ed by t he e- PTFE
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One possible explanat ion for t he present result s
Figure 8b- a) Osteocalcin (OCC) Day 45 Group OVX-BGM: newly formed bone tissue showed diffuse and discrete staining;
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for m at ion sin ce t h is con dit ion is ch ar act er ized b y t h e in cr ease in b on e r esor p t ion an d m ay
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cont rast , t he pr esence of a e- PTFE pr om ot ed an incr ease in int ensit y st aining in t he pr esent st udy. One ex planat ion for t his m ay be t he favorable env ir onm ent for bone for m at ion cr eat ed by t he pr esence of t he bar r ier. Accor ding t o t he pr inciple of guided bone r egenerat ion7 t her e is a cor r elat ion
bet w een angiogenesis and bone r egenerat ion. Rid g e au g m en t at ion t h er ap y b ef or e d en t al im p lan t p lacem en t is a v alu ab le t ool f or or al r ehabilit at ion. How ever, ost eopor ot ic/ ost eopenic condit ions m ay pr ov ide new challenges since t hey are a m aj or public healt h t hreat for a large num ber of people ar ou n d t h e w or ld. Th u s, it becom es necessar y t o r ecognize t he im pact of low bone m ineral in t he dent al set . The result s of t he present
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for m at ion. I n cont rast , t he pr esence of an e- PTFE m em b r a n e cr ea t ed a f av o r a b l e en v i r o n m en t for bone for m at ion. How ev er, caut ion m ust be ex er ci sed b ecau se t h ese r esu l t s d er i v e f r o m an anim al m odel, and ot her st udies in hum ans and fut ur e r esear ch involv ing t he bone for m ing m ar ker s of bone r esor pt ion could be useful for
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CON CLUSI ON
Wit h in t h e lim it s of t h e p r esen t st u d y, w e conclude t hat bone m et abolism during t he process of bone r epair was m or e int ense bet w een day s 7 and 21. The ex pr ession of bone for m ing m ar ker s
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t he pr esence of an e- PTFE m em brane m ay have
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ACKN OW LED GEM EN TS
The aut hor s grat efully ack now ledge Dr. Lar r y W. Fisher at t he Nat ional I nst it ut e of Dent al and Cr an i o f aci al Resear ch , Nat i o n al I n st i t u t es o f Healt h ( Bet hesda, MD, USA) for k indly donat ing t he ant ibodies BSP ( LF- 87) and ONC ( LF- 23) , and São Paulo Resear ch Foundat ion ( FAPESP) , w hich pr ov ided indiv idual suppor t t o Mar ia Apar ecida Neves Jar dini, pr ocess no. 09/ 50214- 1.
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