Improved Method of Neutron Activation Analysis for Microleakage Studies
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1 Improved Method of Neutron ctivtion nlysis for Microlekge Studies J. M. MEYER,* J. B. DENNISON, nd R. G. CRIG School of Dentistry, University of Michign, nn rbor, Michign 4814, US Improvements in the neutron ctivtion technique for studying microlekge were chieved by selecting more suitble trcers nd by using more ccurte method for evlution of the ctivtion dt. Dysprosium ws the most suitble trcer; it provided the lest vrition in the results nd llowed the fstest ctivtion nd counting procedure. quntittive method for studying microlekge in vivo nd in vitro hs been presented by Going, Myers, nd Prussin' nd Going2; they used neutron ctivtion of mngnese 55. Teeth restored in vitro or in vivo were soked in n queous solution of nonrdioctive Mn slt by use of test tube nd ltex isoltor, respectively. fter immersion, the teeth restored in vivo were extrcted. The whole teeth were plced in the core of nucler rector; the 55Mn ws ctivted to 56Mn nd the y-ry emission of the 56Mn formed during the irrdition ws mesured; the number of rdioctive counts ws proportionl to the uptke of Mn per tooth. This method ws used lter by Dennison,3 but he did not succeed in showing sttisticlly significnt differences between the Mn uptke vlues obtined from whole teeth restored with composite resins nd those restored with silicte cement. The min difficulty ws the vribility of the results; this ws cused prtilly by the presence of Mn in either the tooth itself or in the restortive mteril. The purpose of this study ws to improve the efficiency of the ctivtion method by This pper ws presented, in prt, t the 51st Generl Session of the IDR in Wshington, DC, pril Received for publiction June 22, ' Present ddress: Ecole de Medecine Dentire, 3, rue Lombrd, 1211 Genev 4, Switz. 356 selecting more specific trcer, by shortening the ctivtion nd counting procedures, nd by designing more ccurte mens for nlysis of the dt. Mterils nd Methods MTERILS.-Selection of trcer involves considertion of the following chrcteristics: nucler rection, hlf-life, ctivtion cross section, nturl bundnce of the stble isotope, nd its bsence in tooth nd restortive mterils. Such dt for Mn, vndium (Vn), indium (In), nd dysprosium (Dy) re listed in Tble 1. The ltter three were selected on the bsis of the forementioned criteri.4-6 The reltive ctivity vlue ws obtined from reltion including neutron flux, cross section, nturl bundnce, nd tomic weight of the element. By developing the sme reltion for fctors such s ctivtion, decy, nd counting times, it ws possible to predict the totl ctivity for mss unit nd for ll the possible ctivtion nd counting conditions. Such clcultions were performed for the trcers selected nd for the most sensitive of the elements present in teeth or restortive mterils. With constnt ctivtion time of one minute nd counting time of 4 seconds, the vlues obtined for decy times such s 1, 3, 1, nd 3 minutes re reported in Figure 1. It is pprent tht Dy hs the shortest hlf-life nd the gretest ctivity between one nd eight minutes of decy. The decrese in ctivity for the sensitive elements present in teeth does not chnge notbly during the sme period. To mintin the best sensitivity for the proposed trcers the following conditions were chosen for ll further experiments: ctivtion, one minute; decy, three minutes; nd counting, 4 seconds, plus utomtic correction for the
2 Vol1 53 No. 2 IMPROVED CTIVTION METHOD 357 TBLE 1 PROPERTIES OF THE SELECTED TRCERS 55Mn "1V 5l1n "151n 14Dy lo4dy Nturl bundnce (%) Nucler rection (n,y) (ny) (ny) (ny) (ny) (ny) Rdioctive isotope "Mn 62V UemIn "16mIn '6Dy 16mDy Energy (kev) 85 1,43 1,85 1, Hlf-life (min) ctivtion cross section (brns) ,. Reltive ctivity* (disintegrtions/sec X 115) Clculted for flux of 5 X 112 n/cm2/second nd theoreticl counter with equl efficiency between 95 nd 1,43 kev. ded time of the counter. Stndrds were tested first nd the trcers then were used on teeth restored with the sme composite resin to compre prcticl efficiency. The selection of vlid counts in the energy rnge of interest in the y-ry emission spectrum ws bsed on the comprison of the ctul counts for given chnnel with criticl level,7 which ws obtined from the stndrd devition of prepred blnk. This blnk ws constructed by weighting the counts of seprte tooth nd mteril blnks ccording to the ctul weights of the tooth nd filling mteril in the restored tooth to be tested, nd dding them chnnel by chnnel. This procedure enbles computer to select utomticlly the first nd the lst significnt chnnel of given y pek in the complex spectrum obtined from the restored tooth, nd permits the determintion of items such s bckground nd net re of the pek. EXPERIMENTL PROCEDURE.-Two queous solutions were prepred for ech trcer: soking solution (1 gm/1 ml) nd stndrd solution (1 jtg/ml). The following slts were selected: MnSO4 - H2, V2o5, In (NO3) 3-3H2,b nd Dy (NO3) 3 5H2.c ll slts except V25, were redily soluble in wter. ccording to the procedure described by Willrd nd Diehl,8 smll portions of V25 were first treted with mmonium hydroxide, then trnsformed into vndic cid by nitric cid, nd finlly reduced to vndyl ions through oxlic cid tretment. The solution then ws neutrlized nd djusted to the desired concentrtion. J. T. Bker Chemicl Co., Phildelphi, NJ. b E. H. Srgent nd Co., Detroit, Mich. c Pfltz nd Buer, New York, NY. Three types of filling mterils were used: composite resin (),d n unfilled resin (S),e nd silicte cement (M). Filling mterils were condensed into smll split mold to form disks, which were used s mteril blnks. Freshly extrcted, sound premolrs nd molrs were used. Round Clss V cvities were prepred on the buccl surfces of these teeth by use of conventionl technique fter storge for t lest 24 hours in distilled wter. Ech restortion hd reltively constnt exposed surfce re of 12.5 mm2 nd ws plced to miniml depth within the dentin. fter preprtion nd n dditionl hour of storge in wter, the tooth ws dried with compressed ir for 3 seconds nd weighed. The tooth ws filled nd fter the finishing ws ccomplished, it ws stored nother hour in distilled wter. The restored tooth then ws dried gin with compressed ir nd weighed. This procedure llowed good repetbility of the weighings. ll tooth surfces then were seled with utility wxg pplied with hot sptul to within.5 mm of the mrgin in the restored region. Specil cre ws tken to sel the pex region nd the enmel surfce djcent to the mrgins so tht fluid exchnge could tke plce only long the mrgins of the restortions. Wxing ll the surfces ensured close contct between wx nd tooth surfce, nd finlly n dditionl lyer of wx ws pplied. In Figure 2 the stndrd cvity preprtion used throughout the study is shown, with similr d dptic, Johnson & Johnson Co., New Brunswick, NJ. e Sevriton, mlgmted Dentl Trde Distribution, Ltd., London, Eng. f MQ, S. S. White Co., Phildelphi, P. g Kerr Mnufcturing Compny, Romulus, Mich.
3 358 MEYER, DENNISON, ND CRIG I Dent Res Mrch-pril 1974 B FIG 1.-Theoreticl totl ctivities clculted for vrious decy times for mss unit ctivted for one minute nd counted for 4 seconds., 1`Dy (18 kev); B, '11mIn (1,27 kev); C, ""min (1,85 kev); D, TMMn (85 kev); E, 'IV (1,43 kev); F, 21I (1,78 kev); G, 3'1P (n) 281 (1,78 key); H, '8Cl (2,16 key); 1, 24N (1,37 kev); J, 3`CI (1,64 kev). Possible trcers (Dy, In, V, nd Mn) re compred with most sensitive elements present in teeth or restortive mterils (l, P, CL, nd N). tooth tht ws wxed nd seled for soking in the trcer solution. The wxed specimens were soked for n hour in one of the concentrted slt solutions, then quickly rinsed in distilled wter nd wiped off with bsorbing pper, except for the restored surfce. n oversized piece of green wx ws seled over the restortion to prevent further seepge of the entrpped trcer solution, especilly during the ctivtion procedure. D C IE F G H IJ Fic 2.-Prepred cvity on buccl surfce of molr (left) is compred with similr cvity fully restored nd wxed for soking in trcer solution (right). Smll polyethylene tubes contining I ml of the stndrd solution (1 jug of trcer) were used s trcer stndrd. typicl neutron ctivtion experiment included the following steps: fter soking nd finl wxing, restored tooth ws wrpped in polyethylene, plced in polyethylene continer, nd trnsferred through n ir tube system to the core of nucler rector trnsfer time ws bout four seconds. fter one minute of exposure to flux of 2 x 113 neutrons/cm2/ second, the specimen ws llowed to decy for three minutes before counting. The counting system included lithium-drifted germnium solid-stte detector1 (high-energy detector) or germnium low-energy photon spectrometer (low-energy detector), nd 496 chnnel nlyzer.k Specimens were counted for 4 seconds, plus the utomtic correction for the ded time of the counter. Typicl totl counting times rnged between 5 nd 75 seconds for the high-energy detector, nd between 45 nd 1 seconds for the low-energy type. The trcer stndrd, the tooth blnk, nd the filling mteril blnk then were successively irrdited ccording to the sme procedure. Rw dt from the y-spectrum nlyzer nd the experiment were submitted to computer for nlysis. progrm ws specificlly designed for selection of the first h Phoenix Memoril Lbortory, University of Michign, nn rbor, Mich. ' LGCU 55 SD Series 72, Princeton Gmm Tech, Princeton, NJ. J Model , Ortec Inc., Ok Ridge, Tenn. k 496, Nucler Dt Inc., Pltine, Ill.
4 Vo 1 53 No. 2 IMPROVED CTIVTION METHOD 359 nd lst significnt chnnels of the -y pek used to determine the pek surfce nd tht of the bckground below it. This progrm C o do v) ws designed so tht only the highly signif- s N M icnt peks were selected; t lest two successive chnnels hd to hve significnt counts before one chnnel could be selected 3 for one of the limits of the pek surfce. t - the sme time, the pek-to-bckground rtio E_; t--z ws computed, nd the pek surfce ws cor-- - rected for decy by use of the following equtions of Herdi, Mrtin, nd Monnier9: for 52V nd 165mDy g C X N O 693e693VIt'/T D No=- T[l-e-.693 tc'/7] nd for 56Mn nd 116mIn N e-.693t'/t 'IT.. where N. is the number of counts t the end of irrdition (decy time = ); N is the number of counts t the end of counting; t' -, o to is decy time; t'v is counting time; nd T is t E - hlf-life. Such corrections were necessry becuse of 9 the importnt vritions in the totl count- 9E ing time, which resulted from vritions of m EH W -" trcer uptke nd tooth weights, prticulrly Hn E i CiT for the short-lived isotopes such s 52V nd X * 165mDy. The trcer uptke per tooth ws finlly determined by comprison with the trcer stndrd. Trcer uptke vlues clculted in this wy then were compred for the, m. m C4 different types of restortive mterils by use _ m -s S _ of nlysis of vrince. When significnt dif- U ferences were found, Duncn's new multiple > rnge test ws used to rnk them. Correl- H tions of the trcer uptke with fctors such C C CZ s the weight of the tooth or the weight of.5 the filling mteril were studied nd tests for linerity nd regression were performed. Results U ll of the proposed trcers were studied to X > determine their reltive efficiency under the O-t. prticulr conditions of the experiment. * Tble 2 lists the pek re, pek-to-bck-, r ground rtio, nd corrected pek re for the bo vrious trcers nd y peks; the results for.8,= the two types of detectors in the cse of Dy -. X Z re presented for comprison. The smller ; corrected pek re obtined for 165mDy on U -,o CZ the low-energy detector resulted from much lwx -.d H lower ccess time of the counter, or n in- U; _
5 36 MEYER, DENNISON, ND CRIG J Dent Res Mrch-pril 1974 cresed totl counting time s result of the greter decy of this short-lived isotope during the counting. Despite the decrese, pronounced increse in the pek-to-bckground rtio ws observed. The efficiency of these trcers in microlekge tests ws compred with teeth restored with the sme composite resin (). Tble 3 shows the number of tests in which significnt peks were detected by the utomtic nlysis, the men of the pek-to-bckground rtios for given trcer or y pek, the men trcer uptke, the coefficient of vrition within group (when possible), nd the clssifiction of the men trcer uptkes, bsed on n nlysis of vrince followed by Duncn's new multiple rnge test modified for unequl replictions.1 ccording to the number of successful tests, the pek-to-bckground rtio, nd the coefficient of vrition (Tbles 2, 3), the y ry t 18 kev for 165mDy ws chosen s the most suitble for microlekge studies. n dditionl experiment ws performed to demonstrte the cpbility of the method for distinguishing between mrginl lekges occurring with three types of restortive mterils. Tble 4 shows the results obtined with n unfilled resin (S) nd silicte cement (M) nd those lredy obtined with composite resin (Tble 3). The three mterils were inserted into unlined cvities nd there ws no significnt difference between the men vlues obtined. It lso is evident in Tble 4 tht there ws little gined by plcing linerm between the mteril nd the dentin wlls, except possibly to produce greter vrition in the men vlues. When extreme conditions of temperutre cycling were introduced, the men uptke vlues rose decidedly, but so did the vrition mong smple mens. Student's t test ws used to determine difference between men uptke vlues for the composite nd silicte restortions, unlined or during temperture cycling. The temperture cycling ws crried out t 3-second intervls for one hour, between 1 nd 6 C. Incresing the proportion of significnt counts over the bckground led simultneously to decrese in the vribility of the results, becuse the utomtic selection H z (n E-4 5' Q SI W) F-' IX F-4 w H SI t w io Q I Sc.1 W E 1.4. Ii 1 I Goc_3C>o ' O on o c\5 c CD Go c"i. m - ko t_: x o t6 q. _-_, km m - o o keo. o6 or C _- col _- CO CD I m c : co.toco r to to r- m NC' c'r C'r ; O O1 4~ ~ ~~ ok o CD 4 en * in -:: CD > $s t Q. S-4 Cd t U C 2. $4 bid e U C's r._b CZ - o.p._ C-._2 >._~ S o5; s,. CQFc; -F-3 5- in, 5 I dptic*, Johnson & Johnson Co., New Brunswick, NJ. - Coplite, H. J. Bosworth Co., Chicgo, Ill. X o X LY d
6 Vol1 53 No. 2 IMPROVED CTIVTION METHOD 361 TBLE 4 COMPRISON OF THREE RESTORTIVE MTERILS Men Uptke of Dy per Tooth (gigm) Conditions Filled Resin Unfilled Resin Silicte Cement Unlined 2.5 (6.47) jt (2.87) (4-79) #$+ Vrnish liner 9.57 (7.61) (14.37) Temperture-cycled 1.57 (8.87) t (43.6) $ Note: Numbers in prentheses represent stndrd devitions. Unlined: F(2,14),.oo = 3.74 > 2.83 t Filled resin: unlined vs cycled: T(uou.5 = < t Silicte cement: unlined vs cycled: T(s).5 = < of the first nd lst significnt chnnels of the pek becme more sensitive. The number of selected chnnels for the 18 kev pek incresed from men of 7.8 chnnels for the high-energy detector to men of 21.3 chnnels for the low-energy detector; the coefficient of vrition for the number of chnnels selected decresed from 29.2 to 4.8%. Such differences in the men number of selected chnnels were highly significnt ccording to Student's t test. Discussion The utomtic evlution procedure is very effective, if creful selection of the best counting conditions is used. The vribility of the trcer uptke per tooth, bout 3% in the best exmple (Tble 3), demonstrted the influence of the mterils being tested rther thn tht of the testing procedure. Even though the cvities were prepred nd filled crefully, mny uncertinities were ssocited with this type of smple. The fctors involved in preprtion nd filling of cvity cnnot remin completely under control nd vritions in the dt should be expected. The men trcer uptke nd the number of successful tests indicted in Tble 3 show clerly tht the vrious trcers behve quite differently in the presence of the sme type of mrginl lekge. We will not consider Mn becuse of extremely high bckground vlues; the three other trcers seemed to exhibit significntly different penetrtion properties. Despite the good ctivtion properties of V, it filed to show ny repetble penetrtion into the mrginl gp; five of seven tests did not produce recognizble -y pek. In the cse of In, the men uptke vlues of the two y peks were not significntly different, but they were significntly lower thn those for Dy nd produced considerble scttering of the results. Thus, these two trcers should be considered s less effective thn Dy, t lest in regrd to penetrtion into the mrginl spce between composite resin nd the cvity wll. These results nswer, in prt, the question sked by Going,2 tht is, whether the mgnitude of the lekge is completely independent of the trcer. The dt presented in Tble 4 suggest the possibility of using this method to compre the mrginl lekge round different types of nonmetllic restortions; it does not indicte definite dvntge of one type of mteril over nother, becuse the study ws designed to test the method, not the mterils. Gret vritions in the men vlues indicte need for more controlled hndling of smples in vitro. The technique ppers to be sensitive to trcer solution, which cn be drwn into crcks nd fissures ssocited with extrcted teeth. In the therml cycling of filled resin restortions, for exmple, vlues rnged from 31 ytgm uptke round more resistnt mrginl interfces to 218 jtgm uptke in teeth tht deteriorted more redily under simulted clinicl conditions of n extreme nture. It is cler tht to overcome some of the problems ssocited with the nture of the mterils, smple sizes should be incresed to reduce the influence of the sctter of the dt, nd therefore, to enhnce the power of the test. positive regression ws found between trcer uptke nd the weight of the tooth in the composite resin, wheres no regression could be found for either the unfilled resin or the silicte cement, s shown in Figure 3. Thus, cpillry ction of the dentin tubules could ply significnt role, seemingly proportionl to the size of the tooth, if the
7 362 MEYER, DENNISON, ND CRIG ) * Composite Resin Unfilled Resin * Silicte Cement Tooth Weight (gm) FIG 3.-Trcer uptke s function of weight of tooth. Only composite filling mteril exhibited positive regression. trcer penetrtes deeply enough to go beyond the dentinoenmel junction. Smll vritions in the weight of the filling mteril did not show ny correltion with trcer uptke. The totl counting time exhibited positive regression with the trcer uptke vlues nd with the tooth weights, s would be expected. The improved ctivtion method offers the following distinct dvntges: The use of Dy provides high selectivity since no interferences were found nd would not be normlly expected with either tooth or dentl restortive mterils. Dysprosium lso notbly shortens the procedure; the ctivtion nd decy before counting re very short. With sufficient trining, six to ten smples cn be run per hour. The computer nlysis of the dt llows more thorough evlution of the -y spectrum; this leds to greter security in the determintion of the bckground nd of the significnt counts. The weight of the tooth nd the filling mteril were involved in the clcultions; this permitted more detiled evlution of bckground rdition. The improved method, with Dy lso permits esy nlysis of whole teeth fter soking in the trcer solution, wheres the originl method proposed by Going, Myers, nd *, J Dent Res Mrch-pril 1974 Prussin1 required sectioning of the tooth smples to minimize bckground rdition nd gin sensitivity. The improved technique lso could be modified with specific chnges in smple preprtion nd hndling to permit its use in vivo.1 It is possible tht the sensitivity gined by this method through use of short ctivtion time nd counting period could be dpted, with specific precutions nd hndling procedures, to the evlution of metllic restortive mterils for lekge. The method hs two disdvntges, which result directly from the nture of the proposed trcer. The sensitivity of the method to smll defects in nturl teeth is enhnced by the high sensitivity of the trcer, nd the -y-ry emission chrcteristics of Dy (low energy nd short hlf-life) require low-energy detector nd fst trnsfer system between the rector core nd the counting room. Conclusions The evlution of mrginl penetrtion of dentl restortions in whole teeth ws conducted by use of n in vitro method of neutron ctivtion nlysis. Three theoreticlly selected trcers, V, In, nd Dy, were compred with Mn, which hd been shown to be unsuccessful becuse of norml vritions in teeth nd restortive mterils. Dysprosium ws the most successful trcer for the following resons: (1) high selectivity, which permitted the nlysis to be crried out on whole, unsectioned teeth (up to now no Dy hs been detected in either teeth or restortive mterils); (2) rpid ctivtion nd counting procedure; (3) high relibility; nd (4) high sensitivity, indicted by high pek-to-bckground rtio. Vndium nd In trcers were not s successful becuse of their poor relibility in the evlution of the y spectr nd the high sctter of results, which occurred prtly becuse of the low pek-to-bckground rtios. The use of the l65mdy pek resulted in successful evlutions nd the lowest coefficient of vrition for microlekge tests on whole teeth contining composite restortion. preliminry study of three nonmetllic restortive mterils indicted tht by crefully controlling specimen preprtion sttisticl differences in lekge between mterils nd techniques should be discernible with this method.
8 Vol 53 No. 2 References 1. GOING, R.E.; MYERS, H.N.; nd PRUSSIN, S.G.: Quntittive Method for Studying Microlekge In Vivo nd In Vitro, J Dent Res 47: , GOING, R.E.: Microlekge round Dentl Restortions: Summrizing Review, JD 84: , DENNISON, J.B.: n Evlution of Finishing Procedures nd Microlekge on Restortive Composite Resins, MS thesis, University ot Michign, School of Dentistry, nn rbor, Mich, Hndbook of Chemistry nd Physics, 44th ed, Clevelnd; Chemicl Rubber Publishing Co., 1963, pp DUIVENSTIJN,.J., nd VENVERLOO, L..J.: Spectrometrie gmm en prtique, Pris: Editions Dunod, 1965, pp IMPROVED CTIVTION METHOD PERDIJON, J.: L'nlyse pr ctivtion, Pris: Editions Msson, 1967, p CURRIE, L..: Limits for Qulittive Detection nd Quntittive Determintion. ppliction to Rdiochemistry, nl Chem, 4: , WILLRD, H.H., nd DIEHL, H.: dvnced Quntittive nlysis, New York: Vn Nostrnd, 1943, p HERDI, W.; MRTIN, E.; nd MONNIER, D.: nlyse Rdiochimique pr ctivtion ux Neutrons Thermiques. Dosge direct du vndium dns un cier 18/8, pr mesure de 1'ctivite du vndium 52, Helv Chim ct 46: , STEEL, R.G.D., nd TORRME, J.H.: Principles nd Procedures of Sttistics, New York: Mc- Grw-Hill, 196, p 114.
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