ABSTRACT
I m pact of m icr onut r ient s supplem ent at ion on
b o n e r ep ai r ar o u n d i m p l an t s: m i cr o CT an d
count er- t orque analysis in rat s
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1- Universidade Paulista, Faculdade de Odontologia, Pós-graduação em Odontologia, São Paulo, SP, Brasil.
2- Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba, Departamento de Radiologia, Piracicaba, SP, Brasil.
&RUUHVSRQGLQJDGGUHVV Marcio Zaffalon Casati - Departamento de Odontologia - Universidade Paulista - UNIP - Av. Dr. Bacelar, 1212 - 4o andar - Vila Clementino - São Paulo - SP - Brazil - 04026-002 - Phone/Fax : +55 (11) 5586-4000 - e-mail: mzcasati@gmail.com
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he use of nat ural subst ances and m icronut rit ional approaches has been suggest ed asDWKHUDSHXWLFDOWHUQDWLYHWREHQH¿WWKHERQHKHDOLQJDVVRFLDWHGZLWKQRVLGHHIIHFWV 1HYHUWKHOHVVWKHLQÀXHQFHRIPLFURQXWULWLRQDOLQWHUYHQWLRQVZLWKWKHUDSHXWLFSURSULHWLHVRQ
t he bone repair has yet t o be int ensely evaluat ed, and no evidence is available exploring t he im pact of m icronut rient supplem ent at ion on t he peri- im plant bone healing. Obj ect ive: This st udy invest igat ed t he effect of m icronut rient s supplem ent at ion on t he bone repair around im plant s. Mat erial and Met hods: One screw- shaped t it anium im plant was insert ed in each t ibia of each rat , w hich w ere assigned t o: daily adm inist rat ion, for 30 d, of t he placebo solut ion ( Placebo group- n: 18) or m icronut rient s supplem ent at ion ( Micronut rient s group- n: 18) , based on calcium , m agnesium , zinc, and vit am in D3 int ake. Aft er, t he anim als
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evaluat e t he force t o rupt ure t he bone- im plant int erface. The ot her im plant was evaluat ed by m icrocom put ed t om ography ( CT) exam inat ion t o det erm ine t he bone- t o- im plant cont act
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observed bet w een t he groups for bot h count er- t orque values and m icroCT param et ers ( p> 0.05) . Conclusion: Wit hin t he lim it s of t his st udy, m icronut rient s supplem ent at ion did
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Ke yw or ds: Micronut rient s. Osseoint egrat ion. Tom ography. Torque. Vit am in D.
I N TROD UCTI ON
Bone m et abolism hom eost asis has been t he m ot ivat ion of num erous recent invest igat ions. The event s relat ed t o bone t issue repair are crucial t o obt ain a predict able bone rest orat ion and also in opt im izing osseoint egrat ion processes.
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edent ulous pat ient s by im proving est het ic aspect s, m ast icat or y funct ion, and diet ar y int ak e. Many i m p l a n t m o d i f i ca t i o n s h a v e b e e n d e v e l o p e d seeking t o im prove t he long- t erm success rat es of im plant s12. Addit ionally, different local and syst em ic subst ances have been st udied t o im prove t he bone healing in different clinical circum st ances, alt hough
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agent s t o m ediat e t he bone m et abolism , especially in t he osseoint egrat ion process.
Par at h y r oid h or m on e PTH( 1 - 3 4 ) t r eat m en t increases bone healing, decreases bone resorpt ion, and encourages bone repair during ost eoporosis, pr ov idin g a bet t er per i- im plan t bon e h ealin g2 9, alt h ou gh it h as been r epor t ed adv er se ev en t s associat ed w it h it s use such as nausea, cram ps, headache, and hy per calcem ia20. Sim ilar ly, w hile biphosphonat es m ay avoid bone t issue resorpt ion and im prove bone deposit ion, t heir adm inist rat ion has been relat ed t o num erous adverse effect s such as ost eonecrosis of t he j aws16. Consequent ly, t he focus on t herapeut ic alt ernat ives t hat could provide
effect s, such as t he use of nat ural subst ances and m icronut rit ional approaches, has been explored.
I n t his cont ext , our research group dem onst rat ed t hat t he regular use of resverat rol, a polyphenolic ant iox idant pr esent am ong ot her s in t he seeds o f g r a p e s a n d i n t h e sk i n o f b l a ck g r a p e s, im proved t he repair of bone crit ical defect s and t he biom echanical ret ent ion of t it anium im plant s7. I n t he last years, m any ot her st udies have revealed t he pot ent ial t herapeut ic role of ant ioxidant and ot h er m icr on u t r it ion al su p p lem en t s ( v it am in s, m inerals, t race elem ent s) for bone m et abolism , as in m anaging periodont al diseases8,28 or t o prevent bon e f r act u r es4. For ex am ple, a r ecen t r ev iew show ed t hat t he daily int ak e of som e of t hese
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t reat m ent and prevent ion of periodont al disease i n su b j ect s p r esen t i n g sy st em i ca l l y d ef i ci en t m icronut rient28. Since periodont al disease could be associat ed w it h low serum / plasm a m icronut rient levels ( w hich m ay result from diet ary and/ or life-st yle fact ors as well as nut rigenet ic charact erilife-st ics) , supplem ent at ion has a pr om issor y charact er in
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necessary t o reach a consensual prot ocol28. Even t hough vit am in D and calcium are crucial for opt im al skelet al developm ent and m aint aining bone m ass, ot her m acrom inerals ( e.g., m agnesium ) and t race m inerals ( e.g., zinc) are also im port ant26,32. St udies support t hat m agnesium supplem ent at ion provides increased bone m ass in post m enopausal ost eopor osis, dur ing bone gr ow t h, and in ot her condit ions25. Zinc also seem s t o be essent ial t o bone t urnover, present ing a st im ulat ory effect on ost eoblast ic bone form at ion and m ineralizat ion32. Meanw hile, it is im por t ant t o highlight t hat t he int ake of diet ary m ult ivit am in/ m ineral supplem ent s has been progressively increasing during t he last y ear s, w it h m or e t h an on e- t h ir d of t h e ov erall populat ion report ing t he use of such supplem ent s, in sp it e of d iag n osis of n u t r ien t s d ef icien cy5. How ever, t o t he best of our know ledge, no st udy assesses t he im pact of t his supplem ent at ion in
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im plant bone healing.
T h u s , i n a n a t t e m p t t o i m p r o v e t h e osseoin t eg r at ion p r ocess an d con sid er in g t h e
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m icr on u t r ien t s in t ak e d u r in g t h e p er i- im p lan t b o n e h e a l i n g , t h e a i m o f t h e cu r r e n t st u d y w as t o det er m ine t he effect s of m icr onut r ient s supplem ent at ion on bone repair around t it anium im plant s. The hypot hesis of t his st udy was t hat t h e sy st em ic d aily in t ak e of cer t ain n u t r ien t s cou ld p osit iv ely b en ef it t h e b on e h ealin g an d biom echanical ret ent ion of t it anium dent al im plant s.
M ATERI AL AN D M ETH OD S
An im a ls
The anim al cohort was com posed of 36 10- week-old m ale Wist ar rat s. The rat s were acclim at ized for 15 d before use and kept in t em perat ure- cont rolled cages ( appr oxim at ely 28° C) , exposed t o a 24- h
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syst em , and had free access t o wat er and food ad libit um ( Labina, Pur ina®, Paulínia, SP, Brazil) in t he Biot erium of Paulist a Universit y. According t o t he fabr icant ( Labina, Pur ina®) , t he com posit ion of t his regular diet includes approxim at ely 13% of m inerals – am ong t hem , each kg of diet cont ains 1 0 0 m g o f ca l ci u m ( ca l ci u m p a n t o t h e n a t e ) , 4.400 UI of vit am in D3, and 110 m g of zinc. No m agnesium is present in t he form ulat ion. The rat s (Rat t us norvegicus) included in t he present st udy consum e approxim at ely 20 t o 30 g of diet per day. The experim ent al procedure was approved by t he Anim al Care and Use Com m it t ee of t he Universit y ( Perm it Num ber: 139/ 12) .
Tr e a t m e n t gr ou ps
Anim als were allocat ed in t w o groups: Placebo group ( N= 18) , w hich received daily adm inist rat ion of a placebo solut ion for 30 d, and Micronut rient s group ( N= 18) , w hich received daily adm inist rat ion of 100 m g/ kg of t he anim al of calcium ( calcium
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k g of m agnesium ( glycinat e chelat e) , and 0. 46 m g/ k g of zin c ( gly cin at e ch elat e)1 , 1 7 , 2 2. A st ock solut ion of m icronut rient s was prepared and dilut ed in wat er for w orking concent rat ions. The placebo solut ion was com posed of t he sam e quant it ies of wat er as used in t he preparat ion of m icronut rient s supplem ent at ion. The t herapies were adm inist ered via gavage, w it h a 1 m L syringe, using 1 m L of t he respect ive subst ances, for 30 d follow ing surgery.
I m pla n t pla ce m e n t
General anesthesia was obtained by intram uscular adm inist rat ion of ket am ine hydrochloride ( 0.5 m L/ kg) and xylazine hydrochloride ( 10 m g/ kg) . During t h e su r gical pr ocedu r e, scr ew - sh aped t it an iu m
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been com plet ely em bedded in t he bone cor t ex ( Figure 1) . Last ly, t he soft t issues w ere replaced and sut ured.
Post - ope r a t ive pe r iod
The anim als were evaluat ed daily t hroughout t he ex per im ent t o check for possible clinical or t oxicological sym pt om s. Thirt y days aft er t he st art of t he st udy, t he anim als w ere eut hanized by CO2 inhalat ion. One of t he t ibiae was dissect ed t o expose t he im plant and t orque force evaluat ion for im plant r em oval. The ot her t ibia ( including t he inser t ed im plant ) was rem oved and st ocked in 70% alcohol for evaluat ion by m icroCT scans.
Tor qu e for ce e va lu a t ion for t h e r e m ova l of im pla n t s
One of t he t ibiae was dissect ed t o expose t he im plant , allowing t he at t achm ent of a t orque m et er w it h a scale range of 0.1–10 N/ cm and divisions of 0.05 N/ cm ( Mark- 10, BGI , CA, USA) . A w rench
was adapt ed t o t he im plant head t o apply t orque in t he reverse direct ion of im plant placem ent unt il com plet e r upt ur e of t he bone- im plant int er face was signaled by rot at ion of t he im plant . The t orque f or ce v alu e ob t ain ed in N/ cm w as con sid er ed as t h e t or qu e n ecessar y f or t h e br eak dow n of osseoint egrat ion7.
M icr ocom pu t e d t om ogr a ph y a n a lysis
Specim ens obt ained from t he ot her t ibia w ere evaluat ed by using m icroCT im aging. Sam ples were m ount ed on a cust om at t achm ent and scanned using a m icroCT scanner ( SkyScan 1172; Bruker, Kont ich, Belgium ) at an isot ropic resolut ion of 6.99
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st eps of 0.4 and 4 fram es, using a 1 m m t hick AlCu
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a t om ographic r econst r uct ion soft w ar e ( NRecon v.1.6.9.4, Br uker, Kont ich, Belgium ) w it h 2% of sm oot h in g, 4 % of r in g ar t if act cor r ect ion , an d 10% of beam hardening correct ion, providing axial cr oss- sect ions. The pr oj ect ion im age dat a w er e reconst ruct ed t o creat e 3D im ages, and t hey w ere analyzed using a com put er program ( CTAn v.1.12 soft ware, Bruker, Kont ich, Belgium ) . The follow ing par am et er s w er e r ecor ded f or each v olu m e of int erest ( VOI )11,23,30: ( 1) Bone- t o- im plant cont act ( BI C) – percent age of BI C along t he t hreads of t he im plant sur face; and ( 2) Bone volum e fract ion/ densit y ( BV/ TV, i.e., Bone Volum e/ Tot al Volum e) – percent age of t he peri- im plant bone volum e t o t he t ot al of t he VOI . Because t rabecular bone cont ains m ar r ow cav it ies, t he BV/ TV should be < 100% . The VOI corresponded t o dilat at ion of t he im plant shape of 25 voxels: t he inners 5 layers of voxels w ere discarded t o avoid t he art ifact s observed in t his region, result ing in a VOI of 7.933 m m3, and t he t op of t he VOI was placed on t he im plant neck. The BI C was also calculat ed in out side of t hese 5 layers of voxels. Figure 2 illust rat es t he param et ers ev alu at ed . BV/ TV an d BI C w er e calcu lat ed on b in ar ized im ag es an d all m easu r em en t s w er e perform ed by t he sam e calibrat ed m asked exam iner.
D a t a a n a lysis
St at ist ical an aly sis w as p er f or m ed u sin g a soft war e pr ogram ( BioEst at 5.0, Sociedade Civ il
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exam ined for norm ality by t he Kolm ogorov–Sm irnov t est . For biom echanical evaluat ion of t he ret ent ion of t it an iu m im plan t s an d for m icr oCT an aly sis,
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was set at 5% .
RESULTS
The anim als did not show any signs of syst em ic illn ess t h r ou g h ou t t h e st u d y p er iod . Th e r at s
w eighed 324± 42 g at t he beginning of t he st udy and 402+ 31 g at t he end in cont rol group, and 332± 41 g and 407+ 29 g at t est group, respect ively. No deat hs w ere report ed.
Tor qu e for ce e va lu a t ion
Dat a analysis dem onst rat ed t hat m icronut rient s su p p lem en t at ion d id n ot af f ect b iom ech an ical r e t e n t i o n o f t i t a n i u m i m p l a n t s . I n t e r g r o u p
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bet w een t he count er - t or que values for im plant r em ov al in Placeb o g r ou p w h en com p ar ed t o Mi c r o n u t r i e n t s g r o u p ( p = 0 . 6 4 8 ) . Fi g u r e 3 a illust rat es t he values of count er- t or que for ce in each group.
M icr oCT a n a lysis
MicroCT evaluat ions indicat e t hat t he syst em ic in t ak e of m icr on u t r ien t s su p p lem en t at ion d id not affect ed t he param et er s evaluat ed, w it h no
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( p= 0.405) and BI C ( p= 0.232) . Figures 3b and 3c dem onst rat e BI C and BV/ TV values obser ved in each group, respect ively.
D I SCUSSI ON
Vi t am i n D, cal ci u m , an d so m e m acr o an d t race m inerals ar e cr it ical for m aint aining bone m et abolism32. Previous st udies have dem onst rat ed t hat , in bone t issues, vit am in D present s regulat ory p r o p e r t i e s o n o s t e o c a l c i n a n d o s t e o p o n t i n , im p or t an t m ar k er s in v olv ed in t h e m at u r at ion and bone m ineralizat ion st age, also m odulat ing im m une responses269LWDPLQ'GH¿FLHQF\KDVEHHQ associat ed w it h bone loss, including periodont it is2, and has a negat ive effect on cort ical peri- im plant bone form at ion in ovariect om ized rat s, w hich m ay
Figure 2- a and b) Representative coronal slice of Placebo (a) and Micronutrients groups (b), respectively. Red band represents the excluded layer around the implant (5 voxels). BIC parameter was calculated in addition to these 5 layers of voxels. Around the red band, note the assessed bone layer (20 voxels); c and d) Representative tridimensional images of assessed bone volume reconstructed using a computer program of Placebo (c) and Micronutrients groups (d)
Figure 3- a) Graphic illustrating means and standard deviations of counter-torque values (N/cm) in both groups; b and c) Mean percentage and standard deviations of BIC (b) and BV/TV (c) in Placebo and Micronutrients groups.
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be com pensat ed by vit am in D supplem ent at ion33. I ndeed, hum an t rials show ed t hat vit am in D3 and calcium supplem ent at ion not only reduced hip bone resorpt ion and fract ure rat es, but also im proved v er t ebr al bon e den sit y an d t ot al body calciu m in p ost - m en op au sal w om en9. Mag n esiu m also plays a key role in bot h bone t issue and m ineral hom eost asis, w hich m ay dist ur b t he funct ion of bone t issue cells, hy dr ox y pat it e for m at ion, and int erfere in m ineral cont ent . I t was dem onst rat ed
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syst em ic bone densit y and t he rem oval t orque of osseoint egrat ed im plant s10. I ndeed, ot her st udies
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induces a decrease in bone form at ion, an increase in bone resorpt ion, and im paired bone grow t h25. I n
addit ion, it has been report ed t hat zinc is im port ant t o t he m aint enance of healt hy bones, st im ulat ing t he reproduct ion and different iat ion of ost eoblast ic cells and inhibit ing ost eoclast ic act ivit y in t he bone t issue14. Bone grow t h ret ardat ion is also a com m on
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Alt hough t here are som e evidences concerning t h e r ole of t h ese n u t r ien t s in b on e h ealin g in
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of bone repair around im plant s and periodont it is t h er ap y, t h e ef f ect of v it am in D an d m u lt ip le m in er als in t ak e on t h e per i- im plan t h ealin g in
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effect s of com bined m icronut rient s supplem ent at ion on bone repair around t it anium im plant s by using m icroCT and rem oval t orque analysis. The result s dem onst rat ed t hat t he sy st em ic daily int ak e of m icronut rient s supplem ent at ion was not effect ive
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bone form at ion and it s biom echanical ret ent ion in
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Th e f i n d i n g s o f t h e p r e se n t st u d y a r e i n a cco r d a n ce w i t h Ar a l , e t a l .3 ( 2 0 1 5 ) , w h i ch also dem onst rat ed t hat v it am in D3 and v it am in K2 su p p lem en t at ion w er e n ot ef f ect iv e in t h e reduct ion of alveolar bone resorpt ion in rat s w it h experim ent ally induced periodont it is. Conversely, Hong, et al.1 3 ( 2 0 1 5 ) r ecent ly dem onst rat ed in dogs t hat vit am in D3 and calcium supplem ent at ion during t he early st age of socket healing prom ot ed accelerat e bone regenerat ion, im proving new bone form at ion, bone densit y, and reducing t he ext ent of vert ical ridge resorpt ion. Accordingly, Spolidorio, et al.24 ( 2010) show ed t hat vit am in D and calcit onin reduced ost eopenic changes in rat s w it h alveolar bone loss induced by cyclosporine and t he levels of t art rat e- resist ant acid phosphat ase 5b (
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induced by cyclosporine. Differences am ong st udies
m ay be at t r ibu t ed t o t h e du r at ion an d dosage of supplem ent s adm inist rat ion, var iat ions in t he ex per im ent al designs, and also t o t he nut r ient s co m b i n a t i o n s a n d p a r a m e t e r s e x a m i n e d . I n addit ion, t he anim als in t his invest igat ion received m ult iple nut rient s supplem ent at ion only aft er t he im plan t placem en t . Th en , it w ou ld be r elevan t t o con sid er if t h e p r ior ad m in ist r at ion of t h is supplem ent at ion – during a period before defect est ablishm ent or im plant placem ent , besides t he adm inist rat ion follow ing surgery – could achieve m ore hopeful out com es in t erm s of bone healing around im plant s.
Alt h ou gh t h e inv est igat ion of t h e im pact of com bined m ult i- nut rit ional com ponent supplem ent s during t he peri- im plant bone healing is im port ant , t he absence of evaluat ions of t he effect of each nut rient individually could be considered a lim it at ion of t h e cu r r en t in v est ig at i on . How ev er, i n t h e pr esent invest igat ion, t he biological rat ionale t o adm inist rat e calcium supplem ent at ion in addit ion t o vit am in D was based in previous st udies, including a recent m et a- analyses4 t hat dem onst rat ed t hat vit am in D alone is unlikely t o prevent fract ures, w hereas supplem ent s of vit am in D, in addit ion t o calcium , are required for effect iveness, prevent ing i n cr e a se d f r a ct u r e i n ci d e n ce . Th e a d d i t i o n a l supplem ent at ion w it h m agnesium and zinc w as also based on previous dat a, w hich support ed t hat m agnesium is also essent ial for t he conversion of vit am in D int o it s act ive form and necessary for calcium absorpt ion and m et abolism , like zinc, whose
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calcium plus vit am in D21,27.
Anot her aspect t o be discussed regarding t he result s observed in t he current st udy is t hat t he
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n u t r i e n t s i n t a k e . I n d e e d , t h e r e a r e m u l t i p l e m echanism s t hat m ay cont ribut e t o m icronut rient d ef icien cies, an d sev er al st u d ies h av e sh ow n
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regulat e it s effect s. I t is w ell est ablished t hat som e clinical cir cum st ances – cer t ain phar m acological in t er v en t ion s, m et abolic disease con dit ion s, or
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w it h rheum at oid art hrit is, ost eoporosis, diabet es m ellit us, or aging – m ay increase fract ure healing t im e and t he rat e of com plicat ions, such as non-unions, im pair ing bone healing ar ound t it anium i m p l a n t3 4. Th u s, i t co u l d b e su g g est ed t h a t , especially for pat ient s in t hese r isk y sit uat ions, t he use of m icr onut r ient s supplem ent at ion on a daily basis could have a favorable effect t o increase success rat es, im proving t he prognosis of t it anium im plant s. I n t his cont ext , vit am in D supplem ent at ion h as r edu ced cir cu lat in g con cen t r at ion s of pr
syst em disorders or ost eoporosis15,19. I n accordance,
Liu, et al.1 8 ( 2 0 1 4 ) dem onst rat ed r ecent ly t hat
vit am in D supplem ent at ion was an effect ive st rat egy
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w it h chronic kidney disease, a com m on condit ion
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( 2011)also report ed t hat zinc supplem ent at ion m ay be pr om ising t o bone healt h in post m enopausal w om en w it h d ef icien t zin c in t ak es, b u t n ot in p at ien t s con su m in g ad eq u at e am ou n t s of t h is m ineral. However, corroborat ing our result s, ot her dat a dem onst rat ed t hat vit am in D int ake alone or
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on serum cyt okine concent rat ions of syst em ically healt hy individuals6.
T h e i n t e n t i o n i n e v a l u a t i n g n u t r i e n t s su p p lem en t at ion in n on - d ef icien t con d it ion s is based on t he fact t hat , nowadays, a large scale of t he populat ion daily int ake t hese com pounds, t r y in g t o obt ain a h ealt h ier sy st em ic con dit ion an d / or im p r ov e t h e r esu lt s of t h eir b iolog ical processes. A recent st udy show ed t hat m ore t han one- t hird of t he general populat ion declares t o be an supplem ent at ion user5. Thus, our int ent ion was prove if t his supplem ent at ion – in norm oreact ive co n d i t i o n s – co u l d i m p r ov e o sseo i n t eg r at i o n . However, based on our result s and previous st udies, it could be suggest ed t hat nut rit ional supplem ent s
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t h a t r eq u i r e a d d i t i o n a l d i et a r y m i cr o n u t r i en t int akes31 7KXV WKH FRVWHIIHFWLYHQHVV SUR¿OH RI t he m ult iv it am in/ m ineral supplem ent use in t he g en er al p op u lat ion sh ou ld b e q u est ion ed , an d clinical pract it ioner s should be awar e t hat w hile rout ine m icronut rient s supplem ent s are unlikely t o prom ot e adverse effect s, it rem ains unclear if t his supplem ent at ion is advant ageous for t he general populat ion.
I n ad d it ion , t h e in v est ig at ion of b iolog ical m echanism s t o a possible up- r egulat ion of k ey b o n e - r e l a t e d m a r k e r s r e l a t e d t o n u t r i t i o n a l supplem ent at ion w ould be im por t ant t o suppor t ( a n d , m a y b e , t o ch a n g e d o sa g e a n d i n t a k e prot ocols) t he t herapeut ic pot ent ial of m icronut rient s supplem ent at ion t o st im ulat e bone healing around t it anium im plant s.
CON CLUSI ON
I n conclusion, t his st udy dem onst rat ed t hat t he adm inist rat ion of m icronut rient s supplem ent at ion
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BI C and bone volum e around im plant s observed
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in accordance w it h t he biom echanical ret ent ion of t it anium im plant s.
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