OCP crystals were grown at 371C in a dual membrane system tinder various amount of ionic inflow into a reaction space, using]I) 5,30mM Ca and P04 sohtitions ...
UNCLASSIFIED
Defense Technical Information Center Compilation Part Notice ADP014405 TITLE: Effects of Ionic Flow and Amelogenins on the Lengthwise Growth of Octacalcium Phosphate Crystals in a Model System of Tooth Enamel Formation DISTRIBUTION: Approved for public release, distribution unlimited
This paper is part of the following report: TITLE: Materials Research Society Symposium Proceedings. Volume 724. Biological and Biomimetic Materials - Properties to Function
ro order the complete compilation report, use: ADA418623 rhe component part is provided here to allow users access to individually authored sections "fproceedings, annals, symposia, etc. However, the component should be considered within . _ the context of the overall compilation report and-not as a stand-alone technical report. The following component part numbers comprise the compilation report: ADP014393 thru ADP014424
UNCLASSIFIED
Mat. Rts. Soc. Situp. Proc..Vol.724 02002 Materials Research Society
N6.4
Effects of boide Flow and Amelogenins on the Lengthwise Growth of Octacaldlum Phosphate Crystals ina Model System of Tooth Enamel Formation. 2 II. lijimall , Y. Morlwaki 1, JIM. Wen 2N, T. Takagi 3, A.G. Flncham. and J. Moradian-Oldak 2. IAsahi University School of Dentistry. Dental Materials and Technology, 1851-1 Hozumi, Hoztumi-cho, Motosu2 inn, Gifu 501-0296, Japan. Universty~of Southern California, School of Dentistry, Center fbr Cranioflcial 3 Tokyo Medical and Molecular Biology,' USA.#~ Present Affiliation': Wa~y, a Johnson & Johnson company Dental University. School of Dentistry, Oral Biology, Japan.
ABSTRACT This paper briefly reviews our recent studies, which aimed to investigate the effects oft1) the Ca2+ and p043 -ions flowA and 2) amelogenins on the lengthwise growth of octacalciumn phosphate (OCP), which is a potent precursor of enamel apatite crystal. OCP crystals were grown at 371C in a dual membrane system tinder various amount of ionic inflow into a reaction space, using]I) 5,30mM Ca and P04 sohtitions as ionic sources and 2)extracted bovine antelogenin and recombinant murine amel'ogeilins (rM'179, rM 66). With an increase in the amount of 6a2 + and/or P04 3-jons flow, the length of OCP crystal increased, while the width decreased. -Assa result, the length to width *(11W) ratio of crystal changed fromn 3 to 95, while the width to thic"ns.( W/T) ratio from 32 to9. Thi effect of amelogenins was uniquc, ivgardless of the type of arnelogenins : Rod-likc and prism-like OCP crystals with large L/W (61-1 07) and small W/T (1-3-2.2) raitios were formed in 109A ameclogenin gels. In contrast, characteristic -vllbon-like OCP crystals grew without protein and with gelatin, albumin. polyacrylamide gel and agarose gel. Specific interaction of amelogenins with OCP crystal was ascribed to the self-atssembly property of amelogenin molecules and thiritydrophobic nature. It was suiggestcdt that ionic flow and amelogenins play some critical roles in the elorigated grow-th of enamel crystals. INTRODUCTION.
.
Enamel crystals of mamtnalian tooth are formed in an enamel matrix, which isabundanht in amelogenins, under regulated Ca2+ and P043 - ion supply from the layer of atneloblasts. In the early stage of the enamel crystal fortmation, very long and thin crystallites deposit in an enamel matrix with their long axis parallel to each other. In the later stage, crystals mainly increase their width and thickness, and grow into flat-hexagonal prisms[[1,2). The morphology isquite different from the irregular shaped plate-like morphology of bone and dentin crystals. -We speculate that the uniqueness of enamel crystals relates to their growth condition:l1) lattice ions of enamel cystals Ca2'+ and P04 3- ions.'uctransported frjm the layer of amneloblaits. into the enamel matrix, which might cause ioni flow. and the mode of the ionic flow changes dtsring the tooth enamel formation [3,4] , 2) molecules of ameilogenin, which -is nianjor component ofenamel proteins [5A6and highly hydrophobic [7], assemble into *nanosphertes and fram gel [8-10] with unique property ( III. We have been studying the mechanism of the lengthwise and oriented growth of enamel etytttals bnsed on a hypothesis that 1) octacalcium phOsphate (OCP)-tike phase is initiated as a precursor of enamel apatite: 2)one directional supply of the lattice ions contribute tio the lengthwise girovoth in the c-axis direction; 3) amefogenin atanospheres play roles as ascaffoldinge matrix in the highly organized growth of enametl crystals [reviewed in 12-13]. To evaluate thelsypothesis. OCP crystals were grown ins mobdel system of tooth enamel formation.-where Ca2 + and P043- ions were supplied through membranes into ametogenin gels (adual membrane system [14-161). The present paper shows how ionic flow and amelogcnins affected the growth of OCP crystals. EXPERIMENTAL A dual membrane system Ileactions were carried out at 37 OCand pH6.5 for Mdays in a dual membrane system [14], where a cation selective membrane (CMV"") (Asahi Glass Co.) and a dialysis membrane (Visk-ing Cellulose Tubing : Union Carbide Co.) were used to control diffusion of Ca2 + and P043 - ions. Membranes (about 8mmn in diameter) were
103
attached to a Ca solution Ca(CI- 3COO) 2 H20, I.Sml) container. The Ca solution container was put into a P04 solution ( Mll41TL2PO4 + (N114 )1IlPO 4; 1*lmolar ratio. MlOm). Ca2+ aird P04 3- ions diffuse into the reaction space between the membranes (about 15p I of volume) from mniutually oppos.ite sides respectively through the CMV and the dialysis membrane. Crystals deposited 6n thc.CMV or on the both hiembranes dependling: on tie solution concentration. Effect of ionic flow To evaluate'the effect or ionic flowO on the growrth mode. crystal growth was carried out undetr different amount of 2 p4"and Ca + ionts' flux f15). One ofrfactors that determine the driving force ofdiffusion of ionsthoga 3 2 membrane isthe potential difference across it. Therefore. amotmnt of p04 - and Ca 'ý ions'-influx across the 2 3 membrane was changed by changing concentration of phosphate solution aid Ca solution used asP04 - andCa + ionic sources. 5, 10, or 30mM of P04 and Ca solut ions were used in different combinations.
Effeets of amnelprenias To evaluate the effect of amelagenin~s. crystal growth was carried out in 00% qnmelogcnin gels, uising I10mM Ca and P04s~o'lutions. Thiree tgpes of arielogenins were used:1*) extracted bovine amelogenin and 2) recombinant murine anielogenins, rM*l79(M=2Ok~a) and rM166 (M=16.8kDa). About 40r/ of the bovine ainelogenin was 20.7kDi friction, which tack the hydrophilie C-terminal residues of the full length amelogenin [17,18].'and the rest was degraded fractions With the moleciular weight of about 3-8, 13. 1i6kDa 114]. rMt79 hasthe hydrophilicCterminal residues and lacks an N-lerainal methionine and aphosphorylated serine residue : rM 166 black the ltydrophillic C-terminal residues present in rM 179 [19J. -Their purity was higher than 935/6 16). 100% anelogenin and P04 solutions were used as ionic sources. Some parallel solution was put in the reaction space and 10mM of C~a reactions were carried out ini10%/ bovine sennn albumin, gelatin, polyacrylamide (PAA) gel and 1%agarose gel for a comparison. Af'ter a reaction. the precipitates still fixed on tle membrane were rinsed superficially with distilled water and air dried or lyophilized when organi~cmaterialswere used. Crystals. were identified by an X-ray diffractometer.(XRD3) (R~igakt. RINT 2500). Morphology of crystals wvas observed by a scanning electron microscope (SEM) (Hitachi, S4500). Crystal size was measured on the SEM photographs, and their averages and standard deviations were calculated. RESULT.S Effect of mlogiflow Morphology of OCP crystal changed, depending on the concentration of Ca and P04 solutions used as ionic sources (figure I). Plate-like crystiajs grew, when 5mM solutions, which were minimum concentration jo form crysals in the present condition, were used. Length of OCP crystal increased from about lIF4iýnto9l*7pm with an increasecin concentration of Ca andor P04solutions%. Increasemi length was smaller than expected, when the solution concentration was higher than 1(hnM, because cryst.al grew on both CMV and dialysis membrane, while they grew only on the CMV when the concentrations were lowerthan l0mM. The vwidth decreased under a large amount of ionic flow. Thc: drastic change of morphology from rectangle to long ribbon isfigured by the length to width (L/W) and width to thickness (WIT) ratios (figure 2): The LJW ratio changed from 3 to 95, whereas the W/T ratio changed between 32 and 8, as the concentration of Ca and/or P04 solutions increased. Thus, longer and narrower ribbon-like crystals grew, when the amount of flux across the membranes was large.
104
Figu're 1. Morphology o*P&
of pO43 and C22+ sfs grown under iarious.amoui.nt
ion'influx.
Concentration of Ca and P04 solutions used as ionic.sou=ircesre(a) Ca 5mM. P04 5mMM(b) Ca SmM, P04 30mM ;(c) Ca 10mM. P04 30mM. 100
'
'
40
30
a
WI
20
P0
Ce
4
6 10 3D 5
5 1030 10
5 1030m-M 30 M
-
Figuret2 Length to width (iJW) ratio and width to thickness (W/7') ratin of OCP crystals grown under various ion flows [15]. The scheme shows morphology and crystallographic axes of OCP crystal.
Effidts ofametoeeninms The effect of amelogenins on the crystal morphology was unique (figure .3): Ribbon-like crystals grew in the absence of organic materials (control) and ingelatin, albumin. PAA get and agarose gel : In contrast, prismn-like and tod-like crystals grew in 10% amelogenins, regardless the type of amelogenins. The XRD indicated these products am OCP with good crystallinity. Drastic reduction in length and width of OCP crystal wias caused by organic materials with different efficiency (figurel3,4). The length decreased from 90±t8lm (control) to 6t12gm (bovine amelogenins) and 3.6±dpm
(albumin), and the widthi from 2*0.511m (control) to 94*35nm (bovine amelogenins) and 833t7nm (albumin). The inhibitory activity of the bovine amelogenins in length was larger than that of rM 79 and rMl166. Both rMI66 and
105
*were *
rM179'hadsimin!ar efctonthed ''bgrowth of OCP. The inhibitory activ ity of arnelogenins gels in Length and udth. largcrthian thut ofPAA gel. Whereasthe decrease in thickness caused byamelogen-ins gels wRa~,smanller than ~that caus.ed by ot'hernmatqials.. The inhibitory effect~of albumin wais h largest. The degr~eeor crystal siye r~fedctkiq in 101/ ilmclogcnih% gels was in the order bfwidth. length and thnicjess. Thl'.'means that ame1logenins Suppr~esse the growthý of OCP in Iih, order. 6-ais >.c-a'xL%> a-axis direciion and that the intcrwilion of amelogenin%whh the crystal.faceof OCT %i'.iitmthe order-of (010)> (00I)>( 100)..
Figure 3. Morpholqtgy oreryttas growli 1a) without addiiivc%. in (b) 10% albumin. Ic) 10%* rM179. and(d) 100/ rM I6. Note that both rod-like aint prism-like crystals were obtained in three type%of amelogonins. i.e.. bovine amelogenin. rM 179 and rXlI 6A.
106
tin
40 20
~~
M~~
a." blM
awn
i,
bK
A
9
Wl
l
~M
X PA M
111~i 11 ,U
Mil2 ,
MMdn
0MMdM
hI
M
M
M
i
bi
ilM
MO.9 59459 59MPM?9atlot
V
F~ur4L)Lngh wdt W) ticnes T) IW aioan WT atosofOC cytas aw wthutprt4n and 102 boIeNKoeisrl7,rl6 inW lbmnadglt
Foinue 4.Lnelgthi~r (L
ndrl ()
with eule thccnss() rytl L/W ratio
larne than ofOC thato cotc rystals ihotpoens arw
with the smallcastWITratio. Inecase ofrMl 66 and TM179, theIJ/W ratio was about 2.5 times larger than and thc
WIT ratio was about 116 times smaller than those of control crystals. Gelatin also caused the elongation as well as rMl66 andrMl79,.but the WITratio was V/2ofthe control crystal. DISCUSSION The resuls of our in vitro experiments demonstrate that both ionic flow and anielogenin contributed to form OCP crysitals with elongated morphoqlogy. Undc+ a larger amount of ionic flow, the lengthwise growth was enhanced, while the growth in the width direction was reduced. OCP crystal.grewoprfercntially in thec--Axis direction, while grew less actively in the b-axis direction. The (001) face of OCP crystal was, presumably; thec most ac'tive face on which ions and molecules of the lattice components were attached and subsequently OCP structure was quickly constructed inthe c-axis direction. As a result, the maximumL/JW ratio ofacystals was about 25 times larger than tha of crystal grown under the smallest ionic flow (figure 2). Inthe absence of organic materials,cienwhcen the ionic flow changed, crystal len~gth and width were not reduced less than 13and 4 pmn; respectively. When the ionic flow was fusther decreased, i.e., when the cocnrtion-of the ionic sou-mes were less than 5mKM there Was no precipitation on the memnbrane. Drastic reduction in crystal size was caused by organic materials with different efficiency (figure 4). Anmelogenin gels caused a large decrease in width and small decrease inthickness of OCP crystMti As a result, decrease in WIT ratio caused by 10% anticlogenin gels was larger than those caused by other organic materials used (figure 4). The WIT ratio. 1.3. was much smualler than that caused by the change inthe amount of ion flow in the absence of amelogenins (figure 2). The effect of amelogenins on crystal morphology was common among bovine amelogenins. rM 166 and rMl79, regardless of the homogeneity of molecsilar weight and the existemice of the hydrophilie C-terminal. This indicates that the specific interattion of anelogenins with the crystal faces of OCP related to somec common facors among these amelogenins. That ishydrophobicity [14,16], becaute most part of ameltognin molecules are composed with hydrophobic amino acids, except the C-terminal of AA!179 [20]. According tothe crystal structure analysis [21], OCP has ten H20 molecules peronc unit cell. Amount of H20 molecules exposed in itssrystal face isin the order of (100) > (001)»>>(0l 0). Therefore, itisexpected that the (100) and (001) facesare hydrophilic. while the (010) face. israther hydrophobic. The ionic arrangement in its crystal face suggests that the (100) and (010) faces are positive, while the (00 1)face isslightly negative. The amount of the positive charge ofithe (100) it higher than that of the (001). Amelogenin molecules are hydrophobic and positive at p16.5. and they assinible into nanospheres with hydrophobic property [8-10]. AFM imaging of the
-.
-
107
10%. 306% and 35%0 amclogenin gels revealed that those nanospheres were basic building,block ofthc amnelogenin
gcls 116,22.111. On the other hand. PAA get has the closed-cellular structure with the pore sizc of a few micron
[24]. Due to the cell wall%, the mechanical intcrffcrenee of the FAA gel was supposeal to be larger than that of the ainclogenin gels. Nevertheless. thc decrease in length by aniclogenin gelsvvwa larger than that by PAA gel. This ailso suptiorig the interaction with ameclogenin nanospheres with th (NIt)face*orOCPcrystal. The hydrophobic and positive nanospiteres woukl react with the hydrophobic (010) and negahive 1001) faces rather than with the. hydrophilictand poitive (100) f~it. This coincides with the result that the degree of the interaction was intthe orderohm~l)> (00.l>(ltl). 2 During tooth ettam6l formation. 0' + and 1`64-1 ions are transported from the layer ofameloblasts into the enamel tnatrix andi the tmode of ionic trainports change during enamel formtnaion. accompanied with the dtianges in activity of these ions and the Cut P04 ratio [14). Recent studies have postulated that arnelogenin nanospheres. play dual membrane system. itwans demonstrated that the acitmivroes inthe elongation of enamel crystals [9.13]. In thse 25times. by increasing the amount of the ionic flow and itwas increased in 100/ l./W ratio was increased abouit aintlogenin gels 1.5-2.5 times larger than that of control crystal%114-16], thereby. indicating that the ionic flow and ameloge~nin gets played a key role inthe lengthwise growth of OCP crystal. In a gelatin gel system, 1-2%a amelagenins eff'ectively elongratd OCP ery.tnls, whicht have the L'W ratio 3-Stintes larger than that of control crystals 1251.Tlit width atid thickfiess of human enamel crystals in the early stage are. ISam mttd -1.5nns. Since enamrel crystals are long. not respectively, and those intnaturation stage are 68nm and]26imi. respectively [2]. straight and fragile. itisdifficuilt to measlurt the length. It isrepiorted that enamel crystals extend'over an entire layer Tomnes' processes of atre-lobtasts [261 and they are atleast I Oism, [27], which from the dcettino-enamect juttction its maybe the longest reported vilue.'WhMn the length and the width were lO0tam and I5nm. respectively, the IJW is 6667. When the length and width were lO0tam and 6ltntn. respectively, the L/W is 1470. The extremely large L/W ratio of enamel crystals might still require other'factors/tneelanismr to bc involved.which work to reduce the width the growth in the c-axis dircthion) in (i.e.. the growth in the b-axis direction) and to incras-e the length (i.ec.. combination with amelogenins and ionic flow. CONCLUSIONS The increase ifi the amount of calcium and phosphate ionic flow enhanced OCP crystal to grow longer and narrower. Organic materials reduced crystal size. The effect ot'ameilogenins on the manrpholbgy of Of.'was untique, whecn it was conmpared with albumin. gelatin.*PAA. and agarose. Only 10%Katnetogenin gels changed the LJW ratio and small W.'T ratio. Amount of water amolecules; morphology from ribbion-like, to prism-like with large and electric charge o~f tlt (M0). 1010) and t0ol) face of OCP crystal reflectd the, degree of interaction with amelogenins. The present study supported the view that the ionic flow and amelogaiin nanospheres play key roles in controlling the lenigth~wise and oriented growvth of'cnamel crystal. ACKNOWLED)GMENTS The authors acknowledge the technical asslistance of Mr. Narbeb Gharakhain in protein expression and purification. .Thtis iwork wats supported by NIMl-NIDCR research grant DE 12350 ani.0E13414 (i.M.O.) and by the Miyata Garattt from Asahi University tM.l.). REIFF.RNCF-s K.A. Otanet. J.Cell Bil.nt1I. i0q (1963). 1. M.U. Nylen. lU.P. runes an.rd J1. Dent. Res.MS.844 (1979). G.Ducatusi and L.M. Kerchet. 1. B..Kceree. 3. T.Kawamnto and] M.Shimizu. Calcif. Tissue Int. 46. 406 (199). 4. T.Kawamoto' and M.Sttimi~zu. Jpst. J. Oral tiiol. .36. 16511994). 3. J.F. Eastoe. J Deat. Res. MR1, 753 (1979). 6. J.D. Turmine. A.Bi. nlclourt. P.J. Christener. M.K. Conn and M.U. Nylen. J Biln. Chemi. 255. 9760 (1980t). and K.R.Wood%-,Proc. Natl. Acdd. Sci. USA 781. 3824 (1981). 7. T.P.Illopp It. J.Moradian-Oldak, J.P; Simmer. F.C. tUu. P.lF. Sane. H.C. Slivkin and A.G. Fincham. Iliopollymers 34, 1339 (1994). T.O.li. Dickw%%isch. D.M. Layanmut. IT. Wrightt. P.Bringas and If.C. 9. Af.oFinehat. 3i..Moradiani-Oldaik. Slavkin. J.Struct.iliol 115.50(0995).
tt08
* *22.
10. J. Moradian-Oldak, W. Leung and A.G. FImcham,' J Struct Biol. 122, 320 (1998). If. G. Nikifolk and N.S. Simmons. J. Dent. Res. 44, 1119 (1.065). 12.' M. lijima, 'Formation of bcracalcium Pl~wspafe in vitro". Monographs in a-atSd Vol,& /Odacalcium P!hospbate. ed. L.C. Chow and ED. Eanes (Karger AG, 2001) pp 17-49. 13. J. Moradian-Oldaic, MatrixsBiology 20,293 (2001) 14. M. lijima, Y. Morlwaki, T. Takagi and L. Morndian-Okdak, 1.Crystal Growth 222.615(2001) 15. M; Iijima, K. Hayashi and Y. Moriwaki, 3. Crystal Growth 2,592002). 16. M. lijima, V.Moriwaki,1H.B3. Wen, A.G. Fincham antd). Moradian-Oldak, J. Dent. Res. 81,69 (2002). 17. T. Takcagi, M. Ssuzuki, T. Baba, Y, Minegi .shi and S.Sasat, Biochem. Biophysics. Res. Comm. 121,592 (1984). 18. H. Shiiokaiwa, Y. Ogmra S. Sasaki, M.E. Sobel, C.!. McQuillan, J.D. termine and MRF Young, Mv.. Dcnt. Res. 1,293 (19V7). 19. J.P. Simmer, E.C. Lau, C.C. Hu,?. Bringas,V. Santo.%T. Abba, M. Lacey, D.Nelson, M. Zeichncr-David, ~M.L Snead, R.C. Slavicin and A.G. ]Ficham Calcir. Tissue Int. 54,312 (1994). 20. T.P. Hopp and .R.Woods, Proc. Natl. Acad. Sci USA 78,3824(1981). 21. W.E. Brown. J.P..Smith, 3.1 Lehr and A.W. Frazier, Nature 196, 1048(1962). K.B. Wen, J. Moradian.Oldak, W. Leung. P-lr. Bringas and A.G. Fincham, J.Struct. Biol. 126,42 (1999). 23. H.B. Wen,-A.G. Fincluam and J. Moradian-Oldaik, Matrix. Biol. 20,387(2001). 24. IL Ruchel and M.D. Brager, Anal. Biocheiu 68,415 (1975). 25. K.B. Wen, J. Moradian-Oldak and A.G. Fincham, 3. Dent. Res. 79,1902 (2000). 26. T.G.H. Dieldorlech. 9.3. Berman, S. Gentner and H.C. aavkin,.Cell Tissue Res. 279.149 (1995). 27.. G. Daculsi, LMd. Kerebel and D. Mitre, "Enamel crystals: size, shape, and length and growing process; high resolution TEM and biochemical study." Tooth Enamel IV, ed. R.W. Feamhiead and S. Suga, (Elsevier, 1984) pp.14-IS.
109
