J Appl Oral Sci. 31
ABSTRACT
http://dx.doi.org/10.1590/1678-775720150352
I n vit ro
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lesions using enam el m at r ix pr ot eins or
self-assem bling pept ides
University of Zurich, Center of Dental Medicine, Clinic of Preventive Dentistry, Periodontology and Cariology, Zurich, Switzerland.
Patrick R. Schmidlin - Clinic of Preventive Dentistry, Periodontology and Cariology - Center of Dental Medicine, University of Zurich - Plattenstrasse 11 - 8032 Zurich - Switzerland - Phone: +41 44 634 34 17 - e-mail: [email protected]
O
bj ect ives: To assess t he re- hardening pot ent ial of enam el m at rix derivat ives ( EMD) and self- assem bling pept ides in vit ro, hypot hesizing t hat t hese m at erials m ay increaseWKHPLQHUDOL]DWLRQRIDUWL¿FLDOFDULRXVOHVLRQVDQGLPSURYHKDUGQHVVSUR¿OHV0DWHULDODQG
Met hods: Fort y- eight enam el sam ples w ere prepared from ext ract ed bovine low er cent ral
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cont rol) . The rem aining area was dem ineralized in an acidic buffer solut ion for 18 d t o sim ulat e a carious lesion. Half t he dem ineralized area was t hen covered w it h com posit e ( dem ineralized cont rol) , w hile t he last t hird was left open for t hree t est and one cont rol t reat m ent s: ( A) Applicat ion of enam el- m at rix prot eins ( EMD - lyophilized prot ein fract ions dissolved in acet ic acid, St raum ann) , ( B) self- assem bling pept ides ( SAP, Curodont ) , or
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obt ained at sect ions. Two- way ANOVA was calculat ed t o det erm ine differences bet ween t he
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observed in t he EMD and SAP groups. Conclusions: This st udy show ed t hat EMD and SAP
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lesions. How ever, furt her research is needed t o verify and im prove t his observed effect .
Ke yw or ds: Toot h rem ineralizat ion. Enam el. Hardness.
I N TROD UCTI ON
Mineralizat ion processes are based on com plex i n t e r a c t i o n s a m o n g o r g a n i c a n d i n o r g a n i c
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t ransform t he m at rix from a prot ein- rich subst ance int o hard and durable t issues7,17. Different enam el
m at rix prot eins are involved in t he m ineralizat ion processes of enam el, dent in, and cem ent um23. I n
t he enam el m at rix, 90% of t he prot eins belong t o t he am elogenin group, whereas t he rem aining 10%
are prolin- rich non- am elogenins, t uft elin, or ot her serum prot eins2,10. Nascent am elogenins are bipolar,
w it h a h y d r op h ilic car b ox y l t er m in u s, b u t ar e hydrophobic over m ost of t heir lengt hs21. Excret ed
f r om am elob last s, am elog en in s ex t r acellu lar ly assem ble int o nanospheres of approxim at ely 20 nm and form a supram olecular st ruct ural fram ew ork6. I n vivo and in vit ro invest igat ions have shown som e put at ive funct ions of am elogenins18. They inhibit
apat it e nucleat ion1, but appear t o dir ect cr y st al
growt h alm ost exclusively in t he c- axis direct ion,
cont rolling cryst al orient at ion and t ext ure form at ion. I n t his cont ext it m ust be acknow ledged, however, t hat t he enam el prot eins differ from t he m ore acidic and phosphorylat ed m at rix m acrom olecules t hat cont rol biom ineralizat ion in bone and dent ine8,20.
As a com m ercially available product , Em dogain®,
com prising enam el m at rix prot eins, has at t ract ed m uch at t ent ion in t he past years9. How ever, few
at t em pt s have been m ade t o evaluat e t he pot ent ial of t h is pr odu ct an d it s pr ot ein con st it u en t s in m in er alizat ion p r ocesses of ot h er d en t al h ar d t issues, nam ely enam el and dent in, and pot ent ial t herapeut ic applicat ions, i.e., t he rem ineralizat ion pot en t ial of car ies. Elect r oly t ic deposit ion w as exam ined in a very experim ent al in vit ro set- up, and t he form at ion of organized bundles in am elogenin-apat it e com posit es was found t o be m ainly driven by am elogenin nanochain assem bly, w hich already h ig h lig h t ed t h e p ot en t ial of su ch ap p licat ion s in Dent ist r y5. A r ecom binant am elogenin r P172
also m odulat ed apat it e cryst als, w hich resem bled t hose form ed in t he early st age of t oot h enam el biom ineralizat ion, suggest ing t he funct ional roles of am elogenins during t he orient ed grow t h of enam el cryst allit es and a great pot ent ial for am elogenins in applicat ions designed t o fabricat e enam el- like calciu m ph osph at e biom at er ials2 4. How ev er, t o
dat e, no st raight forward st udies w ere undert aken, according t o t he aut hors’ know ledge, t o st udy t he applicat ion of Em dogain® w hen direct ly applied on
dem ineralized enam el. On t he ot her hand, ot her sm art nanobiom at erials have been int roduced in r egenerat iv e dent ist r y aim ing t o r epair enam el lesions and self- assem bling pept ides, w hich m ay u n d er g o sp on t an eou s assem b l i n g af t er b ei n g
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subst ances ar e im it at ing “ nat ural hist ogenesis”
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m ent ioned, and t hey have been show n t o lead t o m ineral deposit ion in t he enam el lesion13.
This st udy aim ed t o assess t he r e- har dening pot ent ial of enam el m at r ix der ivat ives and self-assem bling pept ides in v it r o, hypot hesizing t hat t h ese su b st a n ces m ay i m p r o v e t h e p h y si co
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d ir ect ly com p ar ed t o u n t r eat ed d em in er alized enam el.
M ATERI AL AN D M ETH OD S
Sa m ple pr e pa r a t ion
Tw en t y - f ou r f r esh ly ex t r act ed b ov in e low er incisors were used in t his st udy. Root s were rem oved at t he cem ent oenam el j unct ion wit h a wat er- cooled diam ond disc. Addit ionally, crowns w ere cut in half in t he corono- incisal direct ion, result ing in a t ot al of 4 8 enam el/ dent ine block s, w hich w er e lat er
em bedded in acrylic resin ( Paladur, Heraeus Kulzer, Hanau, Germ any) . The enam el surface was ground
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paper ( St uers, Erkrat , Germ any) up t o P4000 grit , unt il an experim ent al window of approxim at ely 5x8 m m available for t his st udy. Sam ples w ere st ored in wat er unt il needed for t est ing.
One t hird of t he exposed enam el surface was left
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r esin ( Or bi Flow, Lot N2 0 5 3 8 , ORBI S DENTAL, Münst er, Germ any) t o serve as a non- dem ineralized cont rol sit e for subsurface hardness m easurem ent s. Aft er covering t he non- dem ineralized cont rol sit es, each sam ple was subsequent ly dem ineralized in 20 m L agit at ed Buskes dem ineralizat ion acidic buffer solut ion at 37° C for 18 days4. The dem ineralizat ion
acidic buffer solut ion was prepared by m ixing 5 L dist illed wat er, 2.205 g CaCl2+ 2H2O ( 3.0 m M) , 2.041 g KH2PO4P0P/0+'3VROXWLRQP prepared from 100 m L dist illed wat er w it h 0.0528 g m et hylenediphosphoric acid) , 14.3 m L CH3COOH ( 50 m M) , and 10 M KOH t o t it rat e t he solut ion at pH 4 . 9 5 . At ev er y secon d day, t h e solu t ion was r efr eshed. All chem ical pr oduct s used w er e purchased from Merck ( VWR I nt ernat ional AG, 8953 Diet ikon, Swit zerland) . Aft er t he dem ineralizat ion process, whit e- spot form at ion was visually assessed and sam ples were equally dist ribut ed, concerning car ies for m at ion, t o four gr oups ( A–D) , by t w o invest igat ors. Aft er dist ribut ion of t he sam ples, t he m iddle area of t he experim ent al window was also
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area served as t he dem ineralized cont rol sit e for
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w indow was left uncovered, and eit her one of t he t hree t est s ( A- C) and a cont rol t reat m ent ( D) was applied as follow s, respect ively: ( A) Applicat ion of enam el- m at r ix pr ot eins/ der ivat iv es ( EMD) , i. e., lyophilized prot ein fract ions dissolved in acet ic acid Em dogain® ( St raum ann, Basel, Sw it zerland; Lot :
EMD 632 A Lot : EMD 632 A) , ( B) self- assem bling pept ides ( SAP, Curodont RepairTM, Credent is AG,
Windisch, Swit zerland; Lot : 1006198) , or ( C) am ine
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225612) . Sam ples in group ( D) w ere not t reat ed,
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Sa m ple t r e a t m e n t
The following m at erials/ solut ions were prepared and used for t he experim ent s:
- ( A) Enam el m at rix derivat ives ( EMD) :
Lyophilized EMD was dissolved in 1 m L 0.05 M
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concent rat ion of 30 m g/ m L and a pH of 3.5. - ( B) Self- assem bling pept ides ( SAP) :
The m at erial was delivered in pow der form , in glass vials, by t he com pany. Just before applicat ion,
J Appl Oral Sci. 33
CURODONT RepairTM.
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used as delivered by t he m anufact urer.
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t he respect ive sam ples and left for 5 m in on t he enam el surface. Aft er applicat ion, t he solut ion was carefully rem oved using a t owel. Sam ples were t hen
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was prepared by m ixing 2.4 g KCl, 1.7 g NaCl, 0.1 g MgCl2.6H2O, 0.2 g CaCl2.2 H2O, 0.2 g KSCN, 0.7 g KH2PO4, and 0.1 g H3BO3 w it h dist illed wat er t o an end volum e of 1 L. Each sam ple was separat ely st ored and t he st orage m edia was changed every day.
Aft er t reat m ent , sam ples w ere perpendicularly sect ioned in such a way t hat all t hree areas could be m easured. The sect ioned surface was ground
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paper ( St uers, Erkrat , Germ any) up t o P4000 grit . Subsur face m icr ohar dness ( KHN) of t he enam el sam ples w as det er m ined using 15 indent at ions ( load w eight 50 g, indent at ion t im e 20 s) . The lat t er w er e m ade on t he enam el sur face of t he sam ples using a Knoop hardness- m easuring device ( High Qualit y Hardness Test er, Buehler, Düsseldorf,
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enam el sur face and w er e adj ust ed t o m inim um
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in t he lat eral aspect , w hich result ed in an act ual m easur em ent of t w o lines. Ther eby, dept h w as
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Treatment
EMD SAP Fluoride Saliva only
(control)
Depth [μm]
Treatment site Demin site Treatment site Demin site Treatment site Demin site Treatment site Demin site
25 81 (65) * 54 (49) 52 (26) * 43 (18) 75 (46) * 62 (40) 73 (41) * 55 (28)
50 110 (98) 84 (86) 85 (74) * 57 (40) 101 (65) 102 (77)
100 (58) * 84 (51)
75 155 (103) * 110 (102)
143 (97) * 94 (66) 148 (92) 145 (85)
144 (66) 130 (53)
100 193 (96) * 144 (102)
195 (92) * 149 (83)
182 (90) 187 (102)
220 (71) 195 (56)
125 247 (63) * 196 (89)
258 (66) * 200 (90)
225 (85) 225 (82)
273 (49) 250 (28)
150 279 (49) * 245 (68)
288 (51) * 245 (65)
254 (71) 254 (78)
290 (43) 292 (27)
175 306 (29) * 276 (60)
321 (28) * 280 (46)
285 (58) 287 (51)
305 (24) 313 (27)
200 324 (21) * 292 (15)
326 (22) * 306 (29)
297 (52) 313 (33)
319 (25) 324 (18)
225 328 (15) 305 (40)
329 (19) 317 (21)
311 (31) 313 (30)
333 (18) 331 (18)
250 329 (17) 312 (38)
333 (23) 326 (20)
317 (26) 321 (22)
335 (15) 332 (18)
275 333 (15) 322 (25)
335 (19) 330 (14)
326 (18) 326 (16)
336 (18) 334 (18)
300 333 (22) 328 (16)
334 (15) 332 (14)
329 (15) 328 (19)
332 (19) 333 (19) Table 1- Mean (SD) Subsurface microhardness (KHN) values after treatment (treatment site) with the respective products (EMD, SAP, Fluoride, and saliva only) at the different depth levels after demineralization as compared to the demineralization
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are marked with an asterisk (read horizontally)
St a t ist ica l a n a lyse s
Descript ive st at ist ics, such as m ean and st andard deviat ion ( SD) , w ere calculat ed for each t est group sep ar at ely. Dat a w er e an aly zed t o v er if y t h e norm alit y of dist ribut ion using Kolm ogorov- Sm irnov and Shapiro-Wilk Test . Two- way ANOVA, followed by t he post hoc Scheffé Test , were conduct ed t o det ect
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cont rol sit e and t he t reat ed t est sit e. All st at ist ical analyses w ere perform ed w it h I BM SPSS ( Version
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of p< 0.05 was applied.
RESULTS
The KHN values in t he r espect iv e gr oups at respect ive dept h levels are present ed in Table 1.
The hardness values of t he non- dem ineralized cont r ol sit e ranged fr om 2 8 2 KHN t o 3 4 4 KHN
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different groups ( dat a not show n) .
Th e low est h ar d n ess v alu es w er e d et ect ed
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t h e e n a m e l s u r f a c e o f t h e d e m i n e r a l i z e d con t r ol sit e, an d r an g ed f r om 4 3 ± 1 8 KHN t o 62± 40 KHN. Since t hese result s indicat e differences in t he soft ening of t he enam el in t he different groups, t h e h ar dn ess valu es w er e lat er com par ed on ly w it hin t he sam e group bet w een t he dem ineralized cont rol sit e and t he t reat ed t est sit e t o indicat e up t o which dept h t he respect ive t reat m ent s were able t o induce re- hardening.
St at i st i cal l y si g n i f i can t d i f f er en ces i n KHN bet w een t he dem ineralized cont r ol sit e and t he
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zones was det ect ed in groups A ( t reat ed w it h EMD)
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observed for t he t reat m ent sit e w hen com pared t o t he respect ive dem ineralized sit es, indicat ing no soft ening due t o t he applicat ion of t he respect ive product s.
D I SCUSSI ON
This st udy assessed t he re- hardening pot ent ial of enam el m at rix derivat ives and self- assem bling pept ides in v it r o w h en f ocu sin g on su bsu r f ace
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For t h e st u dy, sam ples w er e pr epar ed fr om b o v i n e l o w e r i n ci so r s, a l t h o u g h b o v i n e a n d hum an enam el are not ident ical due t o genet ic, environm ent al, and diet ary differences14. However,
bovine t eet h are easier t o obt ain in large quant it ies co m p a r e d w i t h h u m a n t e e t h1 9. Ad d i t i o n a l l y,
bov ine t eet h ar e in bet t er condit ion and w it h a
m or e unifor m com posit ion25 w hen ex t ract ed for
st udy pur poses. Anot her r eason for t he use of bovine t eet h is t heir larger size, w hich allow s t he preparat ion of m ore sam ples from t he sam e t oot h, result ing in a reduct ion in differences bet w een t he sam ples14$UWL¿FLDOFDULHVOHVLRQVDUHIRUPHGIDVWHU
in bovine enam el t han in hum an enam el, alt hough t he result ing lesions were alm ost indist inguishable in t heir m ineral dist ribut ion charact erist ics15. Since
t he dat a for t he re- hardening w ere only com pared wit hin t he sam e sam ple ( bet ween t he dem ineralized cont rol sit e and t he t reat ed t est sit e) and will only be int erpret ed wit hin t he different groups of t he present st udy ( relat ive dat a) , w e assum e t hat t he advant ages of using bovine t eet h w ill out balance t he disadvant ages of not using hum an t eet h.
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dem ineralized and t reat ed sit es, indicat ing a
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- w er e only found in sam ples on w hich EMD or SAP w as applied. How ev er t h e effect s, despit e b ei n g st a t i st i ca l l y si g n i f i ca n t , sh o u l d st i l l b e considered wit h caut ion. I n general, t he differences w er e r at h er sm all. Alt h ou gh com par at iv e dat a f r o m m i cr o r a d i o g r a p h y a n d m i cr o - h a r d n e ss m easurem ent s have show n som e correlat ion6,12, a
recent st udy by Magalhães, et al.16 ( 2009) concluded
t hat cross- sect ional hardness m easurem ent , used as an alt ernat ive t o t ransverse m icroradiography ( TMR) , is n ot v er y accu r at e for est im at in g t h e m ineral cont ent , but it giv es som e infor m at ion r eg ar d in g t h e m ech an ical ( p h y sical st r en g t h ) propert ies of t he lesions, w hich are not provided by TMR16. I n t his cont ext , w e should m ent ion t hat
t he st art ing point of our m easurem ent s was set at
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surface. However, we observed in prelim inary t est s t hat t he uncovered enam el surface w ould break
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t he sam ples w er e t oo fragile due t o t he sever e dem ineralizat ion pat t ern of our sam ples. Only when
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t o achieve adequat e readings for all groups. The st art ing point , t herefore, relies on t he experim ent al set- up and t he dem ineralizat ion condit ions: whereas
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dem ineralizat ion t im e of 6 d on enam el3, in anot her
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dem ineralizat ion period of only 2 h was chosen22.
I n addit ion, an ult ram icroindent at ion syst em in t he
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select ion of t he st art ing point s.
Therefore, one should be careful when interpreting
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t o applicat ion of EMD or SAP) as rem ineralizat ion. I n cont rast , Kirkham , et al.13 ( 2007) det ect ed a net
J Appl Oral Sci. 35
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applicat ion of a SAP and rem ineralizat ion in a
pH-F\FOLQJPRGHOIRU¿YHGD\V13.
Con sid er in g t h ese r esu lt s an d t h e f in d in g s of t h e pr esen t st u dy, on e m igh t con clu de t h at t he appr oach is pr om ising and deser ves fur t her invest igat ion.
Recen t ly, a p r ess- r elease w as p u b lish ed in t he Sw iss Journal of Dent ist ry, w hich report ed on a r ound t able discussion on so- called biological
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w it h SAP p r od u ct s ( an alog ou s t o g u id ed b on e o r t i ssu e r e g e n e r a t i o n ) a s “ Gu i d e d En a m e l Regenerat ion”. How ever, for t he r easons st at ed above, one should be careful w hen using t he t erm “ enam el regenerat ion”, as even a rem ineralizat ion, as found by Kir k ham , et al.13 ( 2007) , does not
necessarily im ply enam el regenerat ion. Calling an upt ake of m inerals in t he car ies lesion “ enam el regenerat ion” m ight be an overst at em ent , since for enam el regenerat ion t o t ake place t here m ust be not only m ineral upt ake, but also a cryst allizat ion of t hese m inerals in t erm s of apat it e form at ion13.
Furt herm ore, no inform at ion is available as t o w het her enam el regenerat ion is really necessary. One m ight assum e t hat a re- hardening of carious lesion s is sat isf act or y, as t h is w ill r esu lt in an increase in t he m echanical st abilit y of t he lesion, t h er eby pr ev en t in g a br eak dow n of t h e lesion sur face and t he for m at ion of a cav it y. To dat e, one can only speculat e about t he chem ical and st r u ct u r al r e- or gan izat ion in t h e n ew ly f or m ed
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been est ablished, w hich consist s of am elogenin- or
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earlier st udies11,13,24.
But w it hin t he lim it at ion of t he present st udy, it m ight be concluded t hat t he applicat ion of enam el m at r ix der ivat ives and self- assem bling pept ides
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hardening of t hese lesions, also in deeper layers of t he lesion. Furt her st udies are needed t o verify t his appr oach, also under pH- cycling condit ions and in vivo.
CON CLUSI ON
Wit hin t he lim it at ions of t his st udy, EMD and SAP
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ACKN OW LED GEM EN TS
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The m at erials were generously provided for free by t he com panies St raum ann ( Basel, Swit zerland) an d Cr ed en t is AG ( Win d isch , Sw it zer lan d ) f or t est ing.
REFEREN CES
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