Abst ract
Submitted: April 4, 2016 0RGL¿FDWLRQ-XO\ Accepted: August 30, 2016
Shor t t er m sodium alendr onat e
adm inist rat ion im pr oves t he per
i-im plant bone qualit y in ost eopor ot ic
anim als
Sodium alendr onat e is a bisphosphonat e dr ug t hat exer t s ant ir esor pt ive act ion and is used t o t r eat ost eopor osis. Obj ect ive: The aim of t his st udy was t o evaluat e t he bone r epair pr ocess at t he bone/ im plant int er face of ost eopor ot ic rat s t r eat ed w it h sodium alendr onat e t hr ough t he analy sis of m icr ot om ography, real t im e polym erase chain react ions and im m unohist ochem ist ry ( RUNX2 prot ein, ERQHVLDORSURWHLQ%63DONDOLQHSKRVSKDWDVHRVWHRSRQWLQDQGRVWHRFDOFLQ Mat erial and Met hods: A t ot al of 42 rat s w ere used and divided in t o t he follow ing H[SHULPHQWDOJURXSV&7/FRQWUROJURXSUDWVVXEPLWWHGWR¿FWLWLRXVVXUJHU\ and fed w it h a balanced diet ) , OST: ost eopor osis gr oup ( rat s subm it t ed t o a bilat eral ovar iect om y and fed w it h a low calcium diet ) and ALE: alendr onat e gr oup ( rat s subm it t ed t o a bilat eral ovar iect om y, fed w it h a low calcium diet and t r eat ed w it h sodium alendr onat e) . A sur face t r eat ed im plant was inst alled in bot h t ibial m et aphy ses of each rat . Eut hanasia of t he anim als was conduct ed at 14 ( im m unhost ochem ist r y ) and 42 day s ( im m unohist ochem ist r y, m icr o CT DQG3&5'DWDZHUHVXEMHFWHGWRVWDWLVWLFDODQDO\VLVZLWKDVLJQL¿FDQFH level. Result s: Bone volum e ( BV) and t ot al por e volum e w er e higher for ALE JURXS30ROHFXODUGDWDIRU581;DQG%63SURWHLQVZHUHVLJQL¿FDQWO\ ex pr essed in t he ALE gr oup ( P< 0.05) , in com par ison w it h t he ot her gr oups. ALP ex pr ession was higher in t he CTL gr oup ( P< 0.05) . The im m unost aining for RUNX2 and ost eopont in was posit ive in t he ost eoblast ic lineage cells of neoform ed ERQHIRUWKH&7/DQG$/(JURXSVLQERWKSHULRGVDQGGD\V$ONDOLQH phosphat ase pr esent ed a low er st aining ar ea in t he OST gr oup com par ed t o t he CTL in bot h per iods and t he ALE at 42 day s. Conclusion: Ther e was a decr ease of ost eocalcin pr ecipit at ion at 42 day s for t he ALE and OST gr oups. Ther efor e, t r eat m ent w it h shor t - t er m sodium alendr onat e im pr oved bone r epair ar ound t he im plant s inst alled in t he t ibia of ost eopor ot ic rat s.
Ke y w or ds: Alendr onat e. Osseoint egrat ion. Ost eopor osis. Danila de OLIVEIRA¹
Jaqueline Suemi HASSUMI¹ Pedro Henrique da Silva GOMES-FERREIRA² Tárik Ocon Braga POLO² Gabriel Ramalho FERREIRA³
Leonardo Perez FAVERANI² Roberta OKAMOTO¹
http://dx.doi.org/10.1590/1678-77572016-0165
1Univer sidade Est adual Paulist a, Faculdade de Odont ologia de Araçat uba, Depar t am ent o
de Ciências Básicas, Araçat uba, SP, Brasil.
2Univer sidade Est adual Paulist a, Faculdade de Odont ologia de Araçat uba, Depar t am ent o
de Cir ur gia e Clínica I nt egrada, Araçat uba, SP, Brasil.
3Univer sidade de São Paulo, Hospit al de Reabilit ação de Anom alias Craniofaciais, Baur u,
SP, Brasil. Corresponding address:
I nt r oduct ion
Rehabilit at ion w it h dent al im plant s has becom e w i d esp r ead i n soci et y an d m ost of t h e p at i en t s dem anding t his t reat m ent are above 60 years old, a life st age in which ost eoporosis is very com m on, especially in w om en7. St u d ies r eg ar d in g d en t al im p lan t s in ost eoporot ic pat ient s claim t hat t he ost eoporot ic bone is sim ilar t o t he pr oposed m odel of t y pe I V bone, t hat is, r esidual bone for m ed by a t hin layer of cor t ical bone sur r ounding t he low densit y cancellous bone. 7KLVFODVVL¿FDWLRQWDNHVLQWRFRQVLGHUDWLRQWKHERQH qualit y, ranging fr om t y pe I t o I V13. Because of t his, som e aut hor s cont raindicat e t he use of im plant s in pat ient s w it h ost eopor osis, w hile ot her s believe it is not a det erm ining fact or t o cont raindicat e t herapy wit h dent al im plant s since t he pr ofessional per for m s an DSSURSULDWHWUHDWPHQWSODQZLWKPRGL¿HGJHRPHWU\ lar ger diam et er, im plan t su r f ace t r eat m en t an d a longer wait ing period for prost het ic load applicat ion13,14.
Regar ding t o t he im pact of ost eopor osis on den t al im plan t su r v iv al, Ch en , et al.8 ( 2 0 1 3 ) an d Busenlechner, et al.5 ( 2014) r epor t ed t hat since t her e is n o st at ist ically sig n if ican t d if f er en ces b et w een ost eopor ot ic and non- ost eopor ot ic pat ient s t hat had been r ehabilit at ed w it h dent al im plant s, t her e is a st r on g cor r elat ion bet w een t h is bon e m et abolism co n d i t i o n a n d i m p l a n t su r v i v a l , m a i n l y i n t h e m andible. These st udies did not include a feasible num ber of pat ient s t o est ablish a com parison bet w een ost eopor ot ic and non- ost eopor ot ic pat ient s. Besides t hat , t hese clinical st udies did not show indicat e dr ugs used for ost eopor osis.
Cur r ent ly, several t herapies ar e available for t he t reat m ent of ost eoporosis, however, t hey present som e SUREOHPV UHODWHG WR HI¿FDF\ DQG ORQJWHUP VDIHW\ The r ole of est r ogen in bone int egr it y m aint enance has been r ecognized for a w hile, alt hough est r ogen WKHUDS\VKRZVVHYHUDOQRQVNHOHWDODGYHUVHHIIHFWV in clu d in g v ascu lar ev en t s an d b r east car cin om a5
. Som e dr ugs act by decr easing bone r esor pt ion and t h er ef or e d elay in g b on e loss r at e ( an t ir esor p t iv e t herapy ) , such as bisphosphonat es, calcit onin and t he hum an m onoclonal ant ibody, denosum ab, or by prom ot ing bone form at ion ( anabolic t herapy) , such as t er iparat ide1,3,14,18,19,28.
Sodium alendronat e is part of t he second generat ion of bisphosphonat es, exhibit ing less collat eral effect s in UHODWLRQWRWKH¿UVWJHQHUDWLRQDQGEHLQJWKHPRVWXVHG
ant ir esor pt ive dr ug due t o it s low cost in com par ison w it h ot her dr ugs17. The class of t hese dr ugs has a KLJK DI¿QLW\ IRU KXPDQ ERQH PDWUL[ DQG VWUXFWXUDO charact er ist ics sim ilar t o py r ophosphat e, t hat is, t hey are inhibit ors of calcium hydroxyapat it e cryst al growt h and exer t t heir ant ir esor pt ive act iv it y by inhibit ing ost eoclast developm ent and m igrat or y act iv it y, and also by pr om ot ing t heir apopt osis4,31.
Several st udies invest igat ed t he effect s of t he oral bisphosphonat es in ost eopor osis condit ions r elat ed t o WKHULVNRIIUDFWXUHV1,4,6,14, bone m ineral densit y3,6,9,20,28, bone st r engt h3,28 and bone r epair2,7,11,19. These st udies st ill r em ain w it h ou t som e in f or m at ion r eg ar d in g t he m olecular and m icr ost r uct ural feat ur es ar ound im plant s in ost eopor ot ic sit uat ions. For t his r eason, t his st udy t hr ough im m unolabeling, polim erase chain r eact ions, and m icr ot om ography analy sis aim ed t o evaluat e t he bone r epair pr ocess at t he bone/ im plant int er face of ost eopor ot ic rat s t r eat ed w it h sodium alendr onat e.
Mat er ial and m et hods
Exper im ent al gr oups
This st udy follow ed t he st andar ds of t he Et hics Com m it t ee on An im al Use ( 2 0 1 2 / 0 1 0 9 - 6 ) of t h e Brazilian College of Anim al Ex per im ent at ion - COBEA. For t y- t w o rat s ( Rat t us nor vegicus albinus, Wist ar ) of appr ox im at ely 200 gram s w er e div ided in t o t hr ee JURXSV &7/ UDWV VXEPLWWHG WR ¿FWLWLRXV VXUJHU\ and fed w it h a balanced diet cont aining 1. 4% Ca, 0.8% P and wat er ad libit um ) ; OST ( rat s subm it t ed t o bilat eral ovar iect om ies and fed w it h a low calcium diet cont aining 0.1% Ca, 0.5% P and wat er ad libit um , w it hout m edical t r eat m ent ) ; and ALE ( rat s subm it t ed t o bilat eral ovar iect om ies, fed w it h a low calcium diet and t r eat ed w it h sodium alendr onat e) .
For t he quant it at ive analy sis ( m icr o CT and PCR) , aft er t he Pow er Test calculat ion, t he sam ple num ber for each gr oup was a m inim um of 6 ( pow er t est = 0.8) . Thus, as PCR was m ade in quadr uplicat e, w e elect ed 4 anim als ( left t ibia) for t his evaluat ion. For t he m icr o CT evaluat ion, 6 anim als w er e select ed ( 4 r ight t ibia belonging t o t he PCR gr oups plus 2 anim als) w it h a t ot al num ber of 18 anim als.
Est r ous cycle evaluat ion
The evaluat ion of t he est r ous cycle was per for m ed according t o t he m et hod described by Long and Evans16 ( 1922) , in w hich rat s w er e separat ed int o indiv idual cages and t heir est r ous cycles w er e assessed daily, for eight day s.
Bilat er al ovar iect om y an d low calciu m diet
( ost eopor osis induct ion)
All rat s w er e init ially anest het ized w it h x y lazine hydr ochlor ide ( Dopaser - Laborat ór io Calier do Brasil, /WG2VDVFR63%UD]LODQGNHWDPLQH9HWDVHW)RUW Dodge Anim al Healt h Lt d, Cam pinas, SP, Brazil) t o SHUIRUPLQFLVLRQVRQERWKÀDQNVDQGWRUHPRYHERWK ovar ies fr om t he rat s of t he OST and ALE gr oups. Fr om t he CTL gr oup, only t he ovar y ex posur e was per for m ed.
The rat s fr om t he OST and ALE gr oups w er e fed w it h a low calcium diet ( cont aining 0.1% Ca, 0.5% P and wat er ad libit um ) and t he CTL gr oup was fed w it h a balanced diet cont aining 1.4% Ca, 0.8% P and wat er ad libit um .
Th e low calciu m an d ph osph at e diet w as u sed in or der t o sim ulat e a r eal ost eopor osis sit uat ion. Accor ding t o pr ev ious st udies21,26,27, w hen rat s w er e subj ect ed t o bilat eral ovar iect om ies and fed w it h low calcium diet , t her e was a decr ease of bone m ineral densit y up t o t w o higher gr eat er. Thus, only t hose u n d er g o i n g ov a r i ect o m y su r g er y co u l d h av e a n ost eopenia condit ion.
Sodium alendr onat e
Eight day s aft er ovar iect om y, t he dr ug t herapy ZDV LQLWLDWHG E\ JDYDJH ZLWK PJ NJGD\ RI sodium alendr onat e dissolved in an aqueous solut ion t o t he ALE gr oup, as designed by Paz, et al.20 ( 2001) , and gavage w it h saline solut ion t o t he OST and CTL gr oups. This t r eat m ent was conduct ed unt il t he end of t he ex per im ent .
I m plant s
Aft er fast ing for eight hour s, t he anim als w er e VHGDWHGXVLQJPJNJRILQWUDPXVFXODUNHWDPLQH ( Vet aset - Fort Dodge Anim al Healt h Lt d, Cam pinas, SP, %UD]LODQGPJNJRI[\OD]LQHK\GURFKORULGH'RSDVHU - Laborat ório Calier do Brasil, Lt d. - Osasco, SP, Brazil) . )XUWKHUPRUHPHSLYDFDLQHK\GURFKORULGHPONJ scandicaine 2% wit h adrenaline 1: 100,000, Sept odont , Saint - Maur- des- Fossés, Par is, France) was used for
analgesia and local vasoconst r ict ion.
Then, t he t r ichot om y was per for m ed in t he m edial r egion of bot h t ibias along w it h t he ant isepsis using Poly v iny l Py r r olidone I odine deger m ant ( 10% PVPI , Riodeine Deger m ant , Rioquím ica, São José do Rio Pr et o, SP, Brazil) and a t opical solut ion. An incision of appr ox im at ely 3 cm was m ade w it h a div ulsion of t he soft t issue up t o t he t ibial m et aphyses exposure point .
I n bot h t ibias, a com m er cially available t it anium im plant w it h it s surface t reat ed by double acid et ching was inst alled ( Mat er Pourus, Conexão I m plant Syst em s Lt d., São Paulo, SP, Brazil) . All im plant s w er e 1.6 m m in diam et er and 3.0 m m in height . For m illing, a spiral m illing cut t er w it h a diam et er of 1.4 m m was used, m ount ed on an elect r ic m ot or ( BLM 6 0 0®; Dr iller, São Paulo, SP, Brazil) at a speed of 1000 r pm under ir r igat ion w it h a 0.9% saline solut ion ( Fisiológico®, Laborat or ies Biosint ét ica Lt da®, Ribeir ão Pr et o, SP, Brazil) . The inst allat ion was m anually conduct ed w it h DGLJLWDONH\
Af t e r p l a ce m e n t o f t h e i m p l a n t , t h e su t u r e w as p er f or m ed w it h ab sor b ab le w ir e ( Poly g lact in 910 - Vy cr il 4. 0, Et hicon, Johnson Pr od, São José dos Cam pos, SP, Brazil) in t he deep plan and w it h PRQR¿ODPHQW ZLUH 1\ORQ (WKLFRQ -RKQVRQ Sao José dos Cam pos, SP, Brazil) on t he ex t er nal SODQ3HQWDELRWLFZDVDGPLQLVWHUHGPONJ)RUW Dodge Anim al Healt h Lt d, Cam pinas, SP, Brazil) in a single int ram uscular dose as was Sodium Dipy r one PJNJGD\$ULVWRQ&KHPLFDODQG3KDUPDFHXWLFDO I ndust ries Lt d., São Paulo, SP, Brazil) in t he im m ediat e post operat ive per iod.
I n or der t o collect m at er ial, t h e an im als w er e anest het ized follow ing t he anest hesia pr ot ocol for im plant placem ent . Then, t he im plant s of left t ibias w er e r em ov ed b y co u n t er - t o r q u e an d t h e b o n e m at er ial t h at w as p r ev iou sly in con t act w it h t h e im plant was collect ed for RT- PCR analy sis ( r eal t im e poly m erase chain r eact ion) . At t his m om ent , t he rat s w er e eut hanized as out lined by Ram alho- Fer r eira, et al.22 ( 2015) and t he r ight t ibias w er e r em oved and r educed w it h m ar gins of about 1 cm t o per for m t he Micr o- CT analy sis.
Micr ot om ography evaluat ion ( Micr o- CT)
KRXUVDQGNHSWLQHWKDQRO7KHVHSLHFHVZHUH ¿UVW H[DPLQHG E\ EHDP VFDQQLQJ ;UD\ LQ D GLJLWDO com put er ized m icr ot om ography.
7KH SLHFHV ZHUH VFDQQHG E\ D 6N\6FDQ PLFURWRPRJUDSKHU 6N\6FDQ %UXNHU0LFUR&7 Aat selaar, Belgium , 2003) , using sect ions t hat w er e PLQWKLFNQHVV.YDQGNj$ZLWKDFRSSHU DOXPLQXP¿OWHUDQGDPPURWDWLRQVWHSSL[HOVL]H
of 12.45 NjPDQGDQDFTXLVLWLRQWLPHRIKDQG
m in. The im ages obt ained by t he pr oj ect ion of X- ray s on t h e sam ples w er e st or ed an d r econ st it u t ed t o det erm ine t he area of int erest by t he NRecon soft ware 6N\6FDQ9HUVLRQZLWKDQDUWLIDFWULQJ cor r ect ion of 8, beam har dening cor r ect ion of 24% and t he im age conver sion var ied fr om 0.0 t o 0.14. 8VLQJ WKH 'DWD 9LHZHU VRIWZDUH 6N\6FDQ 9HUVLRQ 1.4.4, 64- bit ) , t he im ages w er e r econst r uct ed and obser ved in t hr ee planes ( t ransver sal, longit udinal and sagit t al) . Then, t he CTAnaly ser- CTAn soft war e 6N\6FDQ %UXNHU0LFUR&7 9HUVLRQ 1.12.4.0) was used t o det er m ine bone volum e, t ot al volum e of por e space and t ot al por osit y ( per cent ) , ¿QDOO\WKH'UHFRQVWUXFWLRQZDVSHUIRUPHGXVLQJWKH &7YR[VRIWZDUH6N\6FDQ9HUVLRQ
RT- PCR
Aft er r em ov ing t he left t ibial im plant s, t he bone ar ound t he per i- im plant defect was r em oved w it h a Car bor undum disc m ount ed on a st raight handpiece and m icr om ot or. Pieces for m olecular analy sis w er e pr ocessed t o per f or m t h e ex per im en t s in RT- PCR St epOne Plus, in or der t o evaluat e t he ex pr ession of t he gene encoding pr ot eins r elat ed t o bone r epair.
To execut e t he ex per im ent , each bone fragm ent r em ov ed af t er m at er i al co l l ect i o n w as car ef u l l y washed in a phosphat e buffer ed saline solut ion ( PBS) , fr ozen in liquid nit r ogen and st or ed at - 80°C in a fr eezer for t ot al RNA ex t ract ion w it h Tr izol r eagent ( Life Tech n ologies I nv it r ogen , Car slbad, CA, USA) DQGWKH697RWDO51$,VRODWLRQ6\VWHPH[WUDFWLRQNLW ( Pr om ega, Madison, Wisconsin, USA) accor ding t o t he PDQXIDFWXUHU¶V VSHFL¿FDWLRQV $IWHU H[WUDFWLQJ WRWDO 51$IURPHDFKVDPSOHWKHTXDQWL¿FDWLRQDQGDQDO\VLV of concent rat ions and pur it y w er e per for m ed using a spect r ophot om et er, and t he int egr it y was analyzed by use of a denat ur ing agar ose gel.
Following t hese analyzes, t he norm alizat ion of t ot al RNA concent rat ion of each sam ple was perform ed wit h ult rapur e wat er ( Sigm a- Aldr ich, St . Louis, Missour i,
USA) f or t h e m an u f act u r in g of cDNA st r an d s b y t he r ev er se t ranscr ipt ase r eact ion, using t he High &DSDFLW\ F'1$ 5HYHUVH 7UDQVFULSWLRQ NLW $SSOLHG Biosy st em s, Fost er Cit y, Califor nia, USA) . The RT- PCR 6WHS2QH3OXVUHDFWLRQVIRUWKHTXDQWL¿FDWLRQRIJHQH ex pr ession r elat ed t o bone r epair w er e m ade using Sy br Gr een sy st em ( Applied Biosy st em s, Fost er Cit y, Califor nia, USA) in t he St epOne Plus dev ice ( Applied Biosyst em s, Fost er Cit y, California, USA) . The react ions w er e per for m ed in quadr uplicat e, Taqm an Univer sal PCR Mast er Mix was added t o plat es cont aining t he genes of int er est and cDNA volum es w er e calculat ed LQDFFRUGDQFHZLWKWKHVDPSOHVTXDQWL¿FDWLRQ
7KHDPSOL¿FDWLRQUHDFWLRQZDVSHUIRUPHGDW& for 2 m inut es, 95°C for 10 m inut es, w it h 40 cycles at 95°C for 15 seconds and t hen at 60°C for 1 m inut e ( d en at u r at i o n an d ex t en si o n ) . Th e r esu l t s w er e analyzed based on t he Ct value ( cycle t hr eshold) , t hat is t he point cor r esponding t o t he num ber of cycles ZKHUHWKHVDPSOHDPSOL¿FDWLRQUHDFKHGDWKUHVKROG PHDVXUHGEHWZHHQWKHÀXRUHVFHQFHOHYHORIQHJDWLYH cont r ols and t he phase of t he sam ples ex ponent ial DPSOL¿FDWLRQ ZKLFK DOORZHG IRU WKH TXDQWLWDWLYH analy sis of t he int er est ed genes ex pr ession and t he ones r elat ed on t he plat e. As an endogenous cont r ol, t he ex pr ession of bet a- act in const it ut ive gene was evalu at ed, w h ich is u sed for t h e n or m alizat ion of ex pr ession lev els of t he genes evaluat ed ( RUNX2, DONDOLQHSKRVSKDWDVH%63DQGRVWHRFDOFLQ
I m m unohist ochem ist r y
w er e analy zed in per iods of 14 and 42 day s.
This process was obt ained by inhibit ing t he act ivit y of endogenous per ox idase w it h hydr ogen per ox ide, XVLQJ SULPDU\ DQWLERGLHV DJDLQVW 581; DONDOLQH p h osp h at ase, ost eop on t in an d ost eocalcin . Th ese ant ibodies ar e polyclonal and ar e pr oduced in goat s ( Sant a Cr uz Biot echnology, Paulinia, SP, Brazil) .
I m m unohist ochem ical experim ent s were perform ed using im m unoper ox idase as det ect ion m edium . The ant ibody used was t he secondar y biot iniladoant i- goat pr oduced in rabbit s ( Pier ce Biot echnology, São Paulo, 63%UD]LOWKHDPSOL¿HUZDV6WUHSWDYLGLQDQG%LRWLQ '$.2-DSDQ7RN\R-DSDQDQG'LDPLQREHQ]LGLQH '$.2-DSDQ7RN\R-DSDQZDVXVHGDVDFKURPRJHQ The pr ot eins ex pr ession was analy zed in t he r egion o f t h e i m p l a n t t h r ea d s, b y a ssi g n i n g sco r es: 1 r epr esent ing light st aining, 2 m oderat e and 3 sever e. These scor es w er e est ablished accor ding t o t he st udy per for m ed by Ram alho- Fer r eira, et al.21 ( 2016) , in w hich light st aining r epr esent ed about 25% of t he im m unolabeling area in t he blades; m oderat e st aining r epr esent ed about 50% of t he im m unolabeling ar ea in t he blades; and sever e st aining r epr esent ed about 75% of t he im m unolabeling ar ea in t he blades.
St at ist ical analysis
For st at ist ical analy sis, t he soft w ar e Sigm aplot 12.3 ( Sy st at Soft war e, I nc. Sigm aPlot for Window s,
San Jose, CA, USA) was used. The hom oscedast icit y DQDO\VLVZDVSHUIRUPHGE\6KDSLUR:LONWHVWLQRUGHU t o dist inguish t he param et ric and nonparam et ric dat a. For analysis of t he PCR param et r ic dat a, t he t ot al por e volum e and t he per cent age of t ot al por osit y ( m icr o-&7WKH2QHZD\$129$DQG7XNH\SRVWWHVWVZHUH per f or m ed. For bon e v olu m e h et er ogen eou s dat a PLFUR&7WKH.UXVNDO:DOOLVWHVWZLWK'XQQ¶VSRVW WHVWZHUHDSSOLHG7KHVLJQL¿FDQFHOHYHODGRSWHGZDV p< 0.05.
Result s
Micr o- Ct
The pieces’ scans w er e m ade in t he CTL, OST and ALE gr oups, 42 day s aft er im plant placem ent . Quant it at ively, t he m edian bone volum e obt ained in t he CTL gr oup was 0.0256 m m ³ , in t he OST gr oup it was 0.0276 m m ³ , w it h a considerable incr ease in t he ALE gr oup, show ing 0.0497 m m ³ .
Based on t h e r esu lt s f or b on e v olu m e, t h er e w er e st at ist ical differ en ces bet w een t h e 3 gr ou ps S .UXVNDO:DOOLV SUHVHQWLQJ D GLIIHUHQFH w hen com par ing t he CTL w it h t he ALE gr oup, w it h p< 0.05 ( Dunn’s post - t est ) ( Figur e 1A) .
The averages of t ot al por e volum e in per i- im plant bone t issue obt ained for t he CTL, OST and ALE gr oups w er e r espect ively 0.387, 0.361 and 0.338, in w hich
Figure 1 A-*UDSKLFER[SORWVKRZLQJERQHYROXPH&7/[267[$/(S!S%7RWDOYROXPHRISRUHS&7RWDO
RQO\ WKH $/( YDOXHV VKRZHG VWDWLVWLFDO VLJQL¿FDQFH LQ UHODWLRQ WR WKH RWKHUV S 7XNH\ WHVW Regar ding t he per cent age of t ot al por osit y, t he values w er e 94.189 ( CTL) , 92.924 ( OST) and 87.596 ( ALE) S 7XNH\WHVW)LJXUHV%DQG&
PCR
Th e ost eoblast ic dif f er en t iat ion pr ot ein RUNX2 ZDVVLJQL¿FDQWO\H[SUHVVHGLQWKH$/(JURXS f ollow ed b y t h e CTL ( 1 . 3 9 ) an d t h e OST ( 0 . 2 1 ) S+ROP6LGDN)LJXUH$
Con sid er in g t h e ALP, t h e h ig h est v alu es w er e observed in t he CTL ( 1.01) , followed by t he ALE ( 0.84) , and t he lowest values were exhibit ed by t he OST group S7XNH\WHVW)LJXUH%
7KH%63SURWHLQZDVPDUNHGO\H[SUHVVHGLQWKH ALE gr oup ( 3.03) , follow ed by t he CTL ( 1.43) and OST JURXSVS+ROP6LGDN)LJXUH&
Concer ning t he ost eocalcin, only t he com par ison bet w een t he ALE ( 6.43) and t he OST ( 0.13) present ed DVWDWLVWLFDOVLJQL¿FDQFHS7XNH\WHVWZLWKD t endency of higher values for t he ALE group in relat ion t o t he ot her gr oups st udied, including t he CTL ( 1.00) ( Figur e 2D) .
I m m unohist ochem ical analysis
I m m unohist ochem ical analy sis per for m ed at 14 and 42 day s can be obser ved by t he st aining scor es
( Figur e 3) .
Fo r RUNX 2 , l i g h t t o m o d er a t e st a i n i n g w a s observed in t he CTL group, light st aining in t he OST and m oderat e in t he ALE at 14 day s of osseoint egrat ion. Aft er 42 days, im m unost aining showed a light presence of RUNX2 in t he t hree experim ent al groups ( Figure 4) .
7KH DONDOLQH SKRVSKDWDVH VKRZHG PRGHUDWH ex pr ession in t he CTL gr oup at 14 and 42 day s. I t s reduct ion in t he ost eoporot ic m odel ( OST) is present ed at 14 and 42 day s, w it h light im m unost aining. The adm inist rat ion of sodium alendr onat e in ost eopor ot ic anim als show ed a m oderat e scor e at 14 day s, w it h a r educt ion at 42 day s ( Figur e 5) .
Th e st a i n i n g o b se r v e d f o r o st e o p o n t i n w a s m oderat e in t he CTL, OST and ALE gr oups at 14 day s of osseoint egrat ion. This m oderat e im m unost aining was m aint ained at 42 day s for bot h t he CTL and OST gr oups, but becam e light in t he ALE gr oup ( Figur e 6) .
Re g a r d i n g o s t e o c a l c i n l a b e l i n g , m o d e r a t e
CTL 14 2 2 2 2
CTL 42 1 2 2 2
OST 14 1 1 2 1
OST 42 1 1 2 1
ALE 14 2 2 2 2
ALE 42 1 1 1 1
Figure 3- Scores observed in the marking of RUNX2, OP, OC
and ALP proteins, in the different experimental groups
Figure 4-581;LPPXQRVWDLQLQJDWGD\VIRUWKHH[SHULPHQWDOJURXSV&7/PRGHUDWH267OLJKW$/(PRGHUDWH$WGD\V&7/
OLJKW267OLJKW$/(OLJKW,PPXQRODEHOLQJLQGLFDWHGE\UHGDUURZV2ULJLQDO[
Figure 5-$/3LPPXQRVWDLQLQJDWGD\VIRUWKHH[SHULPHQWDOJURXSV&7/PRGHUDWH267OLJKW$/(PRGHUDWH$WGD\V&7/
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im m unost aining was obser ved in t he CTL gr oup at 14 and 42 day s, and in t he OST and ALE gr oups, st ain in g w as m od er at e at 1 4 d ay s, w it h m in er al decr ease indicat ed by a light im m unost aining at 42 day s ( Figur e 7) .
Discussion
The r esult s of t he cur r ent st udy w er e consist ent t o accept t he hy pot hesis designed by t he aut hor s t hat t he t r eat m ent w it h sodium alendr onat e in rat s w it h induced ost eopor osis w ould im pr ove bone r epair ar ound t he im plant s inst alled in t he t ibia. Thr ough m i cr o t o m o g r ap h i c ( BV an d Po. V) an d m o l ecu l ar param et er s ( r elat ive ex pr ession of RUNX2 and BSP) and t hr ough t he pr ot eins RUNX2 and OPN, it becam e ev ident t hat t he rat s fr om t he ALE gr oup ex hibit ed super ior it y in r epair st andar ds for achiev ing gr eat er bone volum e and sim ult aneously a great er m at urat ion in com par ison t o t he cont r ol gr oups ( CTL and OST) . Except in phosphat e pr ecipit at ion in w hich t he CTL was higher t han t he ot her gr oups.
A cr ucial fact or for im plant st abilit y, especially in t he ear ly st ages of t he bone r epair pr ocess, is t he qualit y of t issue t hat w ill suppor t t he osseoint egrat ed im plant29. I n t his cont ext , ost eoporosis decreases bone m ass in long bones and t he m ax illofacial com plex , as dem onst rat ed by clinical st udies15. Consider ing t hat m ax illar y bones pr esent decr eased densit y due t o t he st r uct ural charact er ist ics of t heir m icr o- ar chit ect ur e, m ain ly in t h e p ost er ior r eg ion s, t h e p r esen ce of ost eopor osis decr eases t he im plant s st abilit y dur ing inst allat ion and, t herefore, increases t he failure rat e9,25.
Th e r e f o r e , o st e o p o r o si s t h e r a p y co n si st s o f increasing bone m ineral densit y in order t o reduce long bone and ver t ebrae fract ur es. Thus, ant i- r esor pt ive m ed i cat i on s, su ch as sod i u m al en d r on at e, h av e been effect ive in t his t r eat m ent1,4,7,14,20. Appar ent ly, a cco r d i n g t o p r e v i o u s st u d i e s7 , 1 1 , 1 2 , 1 9 , 2 8, so d i u m alendr onat e is also able t o im pr ove bone r epair at t he bone/ im plant int erface under ost eoporosis condit ions, at least at t he beginning of t he bone t issue evaluat ion.
7KXVWKHUHVXOWVRIWKLVVWXG\FRQ¿UPHGWKHDQWL r esor pt ive pot ent ial of ALE in low er qualit y bones. Consider ing t he m olecular and im m unohist ochem ical r esu lt s, t h e st ain in g obser v ed f or RUNX2 sh ow ed light ly t o m oderat ely ex pr essed pr e- ost eoblast s at 14 day s in t he OST gr oup, pr esent ing an incr ease in t r an scr ip t ion f act or ex p r ession , p er h ap s in an
at t em pt t o r ecr uit a gr eat er num ber of ost eoblast s, offset t ing t he decrease in ost eoblast act ivit y due t o t he ost eoporosis inst alled on t his experim ent al group. The ALE gr oup show ed sim ilar r esult s t o t he CTL gr oup, WKDWLVPRGHUDWHVWDLQLQJDWGD\VZKLFKFRQ¿UPV t he beginning of m ineralizat ion, and light st aining at 42 GD\VFRQ¿UPLQJWKHQRUPDOFKURQRORJ\RIERQHUHSDLU and show ing a posit ive act ion of t he adm inist er ed m edicat ion. When quant ifying t his prot ein by real t im e PCR at 42 day s, t he highest values w er e found for t he ALE gr oup, even gr eat er t han t he posit ive cont r ol g r ou p ( CTL) ( P< 0 . 0 0 1 ) , w h ich d em on st r at ed t h e highest signaling of ost eoblast r ecr uit m ent for bone t issue for m at ion in t his ex per im ent al gr oup.
7KHLPPXQRVWDLQLQJIRURVWHRFDOFLQWKHPDUNHURI bone m ineralizat ion, show ed differ ences in t he qualit y of bon e t issu e f or m ed du r in g t h e r epair pr ocess. Th e CTL gr ou p sh ow ed t h e h igh est ex pr ession of t his pr ot ein, w hile t he ost eopor osis gr oup pr esent ed t he low est ex pr ession. At 14 day s, t he pr esence of ost eocalcin was sim ilar in t he ALE ( m oderat e st aining) when com pared t o t he CTL group ( m oderat e st aining) . How ever, at 42 day s, it r em ained m oderat e in t he CTL gr oup and becam e light in t he OST and ALE gr oups.
At 14 day s, t he bone lining cells w er e obser ved t o have posit ive st aining for t his pr ot ein, especially in t he CTL gr oup. The m oderat e pr esence of ost eocalcin in t he CTL gr oup at 42 day s charact er izes m at ur e bones and a m ineralizat ion act iv it y, w hich m ay be r elat ed t o a sat isfact or y osseoint egrat ion pr ocess, feat uring bone t issues t hat can offer adequat e support for im plant inst allat ion. How ev er, at 4 2 day s, t he ost eoporot ic anim als t reat ed wit h alendronat e showed slight st aining for ost eocalcin, charact er izing a low er m ineralizat ion of t he bone for m ed along t he im plant s. 7KHVH¿QGLQJVZHUHFRQVLVWHQWZLWKPROHFXODUUHVXOWV since even w it h a t r end of incr eased gene ex pr ession for ost eocalcin in r elat ion t o t he ot her gr oups, t her e ZDV QR VWDWLVWLFDOO\ VLJQL¿FDQW GLIIHUHQFH 3! Hence, in t he long t er m , ALE w ill pr obably pr esent r educt ion in bone t issue m at urat ion.
have show n t hat t he use of sodium alendr onat e, in t he long t er m , can lead t o alt erat ions in t his pr ot ein’s act iv it y2 4. Th is dr u g h as st r u ct u r al ch ar act er ist ics sim ilar t o py r ophosphat e, inhibit ing t he gr ow t h of calcium hy dr ox yapat it e cr y st als, w it h t he incr ease i n p y r o p h o s p h a t e b e i n g r e l a t e d t o d e c r e a s e d m i n er a l i za t i o n . Beca u se o f t h i s co r r el a t i o n , t h e LPPXQRVWDLQLQJRIDONDOLQHSKRVSKDWDVHZDVLPSRUWDQW dur ing t he osseoint egrat ion per iods in t his st udy.
6RPH SURWHLQV OLNH DONDOLQH SKRVSKDWDVH DUH KLVWRULFDOO\ PDUNHG WKURXJK VHUXP FRQFHQWUDWLRQ t o d et ect an d f ollow t h e cou r se of h ep at ob iliar y DQG VNHOHWDO GLVHDVH :K\WH3 1 ( 1 9 9 4 ) r elat ed t h at m o l e cu l a r st u d i e s o f h y p o p h o sp h a t a si a , a r a r e KHULWDEOH IRUP RI ULFNHWV KDYH FRQ¿UPHG WKH ORQJ KHOGQRWLRQWKDW$/3KDVDVLJQL¿FDQWUROHLQVNHOHWDO PLQHUDOL]DWLRQ LQ KXPDQV 7KXV SURWHLQV OLQNHG WR bone repair such as t hose used in t his st udy ( ALP, BSP, ost eopont in and ost ecalcin) t hr ough m olecular and im m unohist ochem ical r eact ions ar e ver y im por t ant in u n d er st an d in g t h e asp ect s in v olv ed w it h b on e for m at ion at differ ent phases ( ost eoblast s induct ion and bone m at urat ion) .
Ost e o b l a st s p r e se n t e d p o si t i v e st a i n i n g f o r ost eopon t in especially in r egion s close t o im plan t spirals, show ing act iv it y of bone r em odeling close t o t he osseoint egrat ion ar eas. St aining for ost eopont in was m oderat e in t he CTL, OST and ALE gr oups at 14 day s of osseoint egrat ion due t o t he int ense cellular act iv it y in t his per iod. This m oderat e im m unost aining r em ain ed at 4 2 d ay s f or b ot h t h e CTL an d OST gr oups, but becam e light in t he ALE gr oup. I t is w or t h not ing t he pr esence of bone lining cells, posit ive for ost eopont in in t he CTL gr oup at 14 day s, show ing t he for m at ion of t he ost eopont in int er faces.
Tom og r ap h ic p ar am et er s ar e essen t ial f or t h e cor r ect planning of t he clinical at r ophy diagnosis of a bone t hat will receive im plant s, because of t he cort ical/ m edullar y charact er ist ics obser ved in m or e accurat e i m ag i n g ex am s, w h i ch w i l l i n f l u en ce t h e ch o i ce of t h e im plan t s m acr ogeom et r y, t h e best su r f ace t reat m ent t echnique and t he m ost indicat ed prost het ic connect ion2,11. The m icr ot om ography per for m ed on t he pieces of t his st udy was dir ect ly r elat ed t o t he incr ease in bone volum e t o ALE, even in com par ison w it h CTL ( p= 0.012) , at t he sam e t im e in w hich t he ch ar act er ist ics r elat ed t o d en sit y, t h e ALE g r ou p present ed sm aller pore volum es t han t he ot her groups ( CTL and OST; p> 0.05) . These charact erist ics indicat e
t hat t r eat m ent w it h ALE pr om ot ed higher volum es of available bone and higher densit ies, fundam ent al fact or s for t he st abilit y of r ehabilit at ion t r eat m ent .
Ev en t hough t hese r esult s ar e encouraging for t he pr im ar y indicat ion of alendr onat e under t hese ost eopor ot ic condit ions, especially r egar ding t he best st r uct ural bone charact er ist ics, an ev ident concer n i s a b o u t m a i n t a i n i n g t h ese ch a r a ct er i st i cs o v er t im e. The m olecular and im m unohist ochem ical dat a VKRZHG D UHGXFWLRQ LQ ERQH PLQHUDOL]DWLRQ PDUNHUV and, consequent ly, a cellular charact er ist ic t hat is unfavorable t o t he pr ocess of bone t issue for m at ion in t reat m ent s w it h alendronat e. These result s suggest t hat in t he long t er m , t her e is a det r im ent in t he qualit y of bone for m ed dur ing t he osseoint egrat ion p r ocess, w h ich seem s t o b e cor r elat ed w it h t h e r educed pr oduct ion of t he pr ot eins t hat favor bone t issue for m at ion.
I n ad d it ion t o t h ese p r ob ab le lim it at ion s, t h e l i t e r a t u r e a l so d e scr i b e s t h e p o ssi b i l i t y o f t h i s t r eat m ent t o cause m ax illar y ost eonecr osis23. Even t hough alendr onat e is an oral bisphosphonat e t hat is less pow er ful w hen com par ed t o ot her int ravenous DSSOLFDWLRQV WKH $/( LV VWLOO FORVHO\ OLQNHG WR ost eon ecr osis in d u ct ion , b ot h in t oot h ex t r act ion sur ger y and in loss of dent al im plant s t hat have been, som et im es, inst alled for decades17,31.
This st udy was st at ically conduct ed on peri- im plant ERQHDOWKRXJKLWLVNQRZQWKDWWKHJUHDWHVWSUREOHP is r elat ed t o dy nam ic changes in t he bone, m ainly due t o phy siological changes obser ved dur ing m ast icat ion loads on im plant - suppor t ed pr ost heses. Giv en t he st at em ent s above, new st udies m ust be designed, w it h a pr im ar y t ranscut aneous load applicat ion on t he im plant s aft er bone t issue m at urat ion ( 42 day s) , w it h m icrom ot ion devices, as described by Wazen30 ( 2013) .
As pr ev iously em phasized, anot her issue t o be inv est igat ed con cer n s t h e qu est ion : w h at is t h e behav ior of ost eopor ot ic bone t r eat ed w it h ALE in t he long t er m ? Thus, fut ur e r esear ch should ex am ine t he sam e condit ions of t his st udy, but at 6 m ont h and 1 y ear per iods aft er im plant placem ent , t o m im ic r ehabilit at ion aft er 24 and 48 m ont hs, cor r esponding t o hum ans.
m or e st udies ar e necessar y in or der t o evaluat e t he qu alit y of t h e per i- im plan t bon e u n der lon g- t er m sodium alendr onat e adm inist rat ion in t he pr esence of t he ost eopor ot ic condit ion.
$FNQRZOHGJHPHQWV
7KHDXWKRUVZRXOGOLNHWRWKDQNWKH6mR3DXOR6WDWH Foundat ion - FAPESP ( 2013/ 11299- 7) for funding t his r esear ch.
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