Reconstructed Skin Equivalents for Assessing Percutaneous

w
Reconstructed
Skin Equivalentsfor Assessing
Percutaneous
Drug Absorptionfrom
PharmaceuticalFormulations
Nadia Z,ghottl,Roland Fuchs,Claus-MichaelLehr dnd UItich F. St:hader
De!t. oIBiolhaDaceutics andPhimaceulicalTcchnology.Saa andUniversiry.D-SaubücckcD
EJtis?dhumdn skinhas so lir lwi conlidetalto he one of the
h.st stiable in rnr? methodst. ewludte t pot?tirltion of
.lerhnt.n.Si.u,) applial suhstdnces
Thc linned vppl\, antl th?
ttktnre\ high danü ürnrbilitr ninüktednd\,
research
gnup! ta rse dninnl skinds asLbstnuclbr hümdnskin.Sitle
no||dda)t ittonstn( t!.1 skiit equh,alentsdt? trrhrut(dl!
araildUe,r,. t-\.dninalth?secuhuresfattheirtk abiliaaso
p?t1utaneoüs abyr?t i.rt hD.]eI lor.1 ife tent p [email protected]ü| i.dl
()ne su.h .qlirül.at is EpiDetnrr I EPI 606, MdtTtk erpota
tion,AshlandMa$d.hts.tts) ||hnh vds iN6tigdr.tl ßing fu
lirophilit tntlel drügflttenanln: a.il PorrInion stüdies,,ith
üe Ftun. difrusbnrcll\erc undertuken
l..rnluote theno.lel
üe
estdblithüdt
of
o
nar
in
tiho
method
b strtll th!
for
perutdna.us abyn?t it t of tltfere,t dosdseJbr, ß. Th( (I uN\rds
dppli!.l nr tua phdnüdcc|ti.ul.lbntulüioas to the nndd sLt1A..
.t the skit disk:.lissobedin wol dtcohatoihtnot (0.1125c/.).
an.l dßsolr.d itt Sodlns.n pltusplnte bujlLr ptt 7.4 (0.tt255.
salutian) I IPLC tru\ ß.d .far the ardltsis af dtujl c.hknt h wds
nbttr tl1d nk nlodelJbrnls d bdh i.t tu--ut.ls dilnsion b!
canryuingthr powdnonaooss thc tissuell". insetsrothe
, . l r i a k ' . , r l , . o 1 t . 5 ^, , , 1 , u l r p I d - t ' . - t , . 4 .
uaLßthe skinequi,al.ntl;!D1thesalübn||as n.kd o b(
dtn stJ.rt), tnrcshisherthdnlir"t th. .üxwnt.1)ro dillctenl
bdtdi es.l tl tc ! ki n..t Lh't Ient sho||ed no sIdIi s1i. nü)" si Enifcdnt
dtlfet"tuc. Fitn J the Nrnl<ubilitr of the rccorsttrt:tcd sknt*as
( ompat?dto hürnn cti.lerhti\,en.lafre tines highetllLt wtue
$as l.an.|for th! skin etuirdt.tl
. i t , . t r t . r a , u"btiet
04 Fi.
, d,:, .q",
"t.-,,b,,.ton
hutun ketatinact"teth.lrt pot.nti.tlas d phdmace utitd test
r)-st,n ta eu.l! denüdl.lrLg rranvott fi om tupit al fomültllbns.
Kelr.ads: t?c.)nnt u&.1 sknreqrirotent, EpiDernut, pound
n.ors absotption.Ftun. nillnstunr:.11
The sk hasbcenconsidered
a falorablc
route of drug adrjnistralion lbr a long
tine noiv. Patienlcomplidcc andthe ease
of tutninistuationmakethe mnsdcnnai ap
proachan dppcalingcnoicefor drug dc
alirEx 18,t/ol
Zusdnncnl'asunB:Rekonslfliene Hauläquivalcntc
zul
Emitllung dcr pc*utanen Resorptlonausfhaftnazeurischcn
Zür EtJds ry LLr petkrtaarn Resotptbn ||it d Jür üt t,ili a
l . , . b r ? , . . - i - , t, , , H , , \ ü - t t. u t . r t \ ^ . . , "
"aign,
dktlca.h.i. Da sieje.loll nLo tu eüent sctu btn:I, änkt.n
Untang 1t \;üfüguntj stehtündje nach Spendetein. h.h..
\tt idbilit.it g.t.b.n in.||n.d stdtt dess.nTierha|t d^ Alternatir!
.ting.s?t1. Neu.ftlitt gs sihJ j.tlot:h tkarstüli?tt? l1Mnnc
Hdulatp i ru l. nt. kiitlfli. h erl1ältl k] t. Au\ t1i.t. k1Ga ntk fii hte n
\', ntit ein ht d.t nü-lln hetlt"konsttui?üenHduldquiulente,
LpiD.mlr (Eti606;Fa Mu ek,Asltu n MA,USA)lüt.1i!
1ip ophi 1e Mo.1eII substdr. F1ul. tlunlinsätt" nt.\r ei vßtl ti.denen
phd h4..uti!chen Zühet"itunren itl t:iti. Pt n.ationssnulien in
Fran. Dillusions.ellen durr h, \ obei Sat?ns.n Phosphat.Pufel
pH 7,1a|\ Ak.epknnltLium .liente.Ak Testzubereituljl.n\runk
Fhlfendninsöur.,
dic iit t ittr Kon.lnfidtionron A,ll25c/.
env.det int Ak.qnt mediu .der inWaUß<:herlkoholsdlhe
(DAß) gclij\I ||t1t..tuf das Snatun.orneün duJq.lrlt(ht. Die
AtzneistuJjb.sti tnury ?tloute rittels IIPLC AnaUv.
Düt1h Veryleichdd Pen)tution üha .|ie tin:nen ran nit urJ
.hn.7,c aüld!. konntes.zeig \t1Jüi.tlaß EpiDemltieine
DtJlilsi.ntbdn io" datstelh.D esveitercr I Lt\l. lin .len Drfü
sionsfl4] aüs.t.t Lösns hetuus ein ü,t das 10 ld.h.. hijho"t
Wert.rhdltentkJür di. Sdlbtßl.it:l1.tKon erttation.Bei
UüIetsut:huntrn
dn ^'ei I etschied.n.nLi.l.Nnsentun
IlpiD?nl r konnr. l:ent edtistis.h sigüilitunk" Lrtkrst hi..l füt
.|ie Penneati.h.li.!ryenel lt *pt den.Dü Verykn:h.let tumtdhi
lität tkt r?konsttuiet|et Hautü.tuiralenre:u Wettenbn huhlanel
Epnt n^ .rqibt einen5 t't1chhöh.,L Dtllrsioriltß fiir di.
UnsercR.sln1al. d.ttln .ldtauf hin. daJlrck.hstri.lqt. hrtntne
Heuüqriwlente, die dkl Kerutilo.lterkühurcn hdscren, Lh
ZLkLhli Lltt u.lianen ki)nnt.n,die d.thdlt'Rt otption dus tapi-r.h
dppli.ieneh Abl(i.rheteitungen in irn zu ernifiel .
livery. Anong thc ddvantagesare the
lvoidanceof inlestinalmd bepaticfirsrpassmclrbolisminheren!yith oül ddnin
isbrtion and avoidanceol inco.veniencc
associated
wilh pärcntcraladministration.
Thereis no generalguidclineuntilnow
ro study the pedetrxtionoI lorcien sub
stdces into lhe skin andüercforcseveml
dillcrcnt i, rir? nrodeh are usedlor tlese
s t u d i e s .C o n c c r n i D gt h e p e n e t r a r j o n
behavio.of topicaldemakrlogjcatdosage
ibms and cosmeticslaboratoics either
uLiliseaDimll sknrorercised hunän ski!
to studl the traßport icfoss the skin lo a
103
äs
ZcHolr ETAL.
Iluid rcccptor compartment.Iicreased
awarenessof animal wcif{rc within the
scientificcomtnunityhas lcad to drastic
rcducnonin the nseofaninals lor scicn
litic purposcs.lxcised htrmanskin has
proven to he one oI thc most rpprcpriate
methodsto assessFrcuhrlcous absor?tion
oftopicdlly applied mbstance!,bul üe 1im
itednumbcrof skiDspecimersandtherelativelyvast donorvaridbilityde limiling
fdctors f'or its use.In responselo thesediI
ficullics. advanceshave beenmade !owardsüc uscofcommercial aM non conmer.ial htrflan skin cquivalcnis asi, ,,r,
modch lbf dennal dflg hansport tcsLing.
Thc tcchnologyof reconshrciinghu'
nan skin equivälcntshas been de.ived
prcdomi'rantlylon researchinlo thc tcaf
ment oI burns. Reconstructedskin has
beenusedwidclyfor cutaneous
metabolic
sludies (G)'sler ct !1-. 1999) and skir
corosiyiry testing(Liebschel al.. 2000)The purpose of this siudy was to deternrire the suiläbilily of ! commercially
availableepldernal hunan skin equiva
1ctrt(EpiDermrv, EPI-606,MalTeLcor
poration.Ashldd. Massachusetts,
USA)
in testi.g lhe drugpcmrcationoldifiefent
pharmacentical
fonnulations.thc intrin
sic lemcability barier functionwas examnredushg flulendnic acid asa model
drug.Tle skin equivalentwas chamcter
isedin lermsof flux valuesdndbatchvari
a1ion.Final1y.lpiDemru was compared
to heat separaFdhunu cpidemris.To
irvesti8atethe archileclureoI drc culturc
systcn, moryhologic snrdie! lsing lieht
flicroscopy and scarning electron
nricroscopy werc perfonncd.
2 Maf€riäl rnd mcthods
2.1 Drüs fonnulations applied
Flulinamic acid,a non sleroidaled nr
flammdtorydrugwasusedasa lipophilic
modeldrug.This drugwaschosenassink
conditiods could easily be maintaincd
lbroughpll adjuslmentand it hasarc1a
tively low dcrcctionlimit of25 ng/nl.
Fhfenan'ic acid wds applied ii two
phdmlceuticallbmulations :
i) 0.1125%djssolvedin wool alcohol
ointnrenl (Gemian Pharnacopoeia.DBciersdort)sloredat 32'C until usc.
i i ) 0 . 1 r 2 5 %d i s s o l v e di n S o e r e n s e n
thosphatebuffcr pH 7.4 as solution,(InBredientspurchascd from Merck, D,
Dlrmsradt).
104
2.2 Permeationbarriers (nenbranes)
2.2.1R€constructedskin equival€nt
EpiDem r (EPI-606,2 22Dm) waspür
cbased ffoln MalTek corporarion
(Ashldrd, MA 01721,llSA). This thrcc
dinensionalsystcn is composedof noF
mal, hunan deri!cd epidermal
kemtinocytes
whichhavebeenculturcd
10
lom a multilayered,highly differenliatcd
rodel of the epidcrnis.
Upon anival, the wells werc rcmoved
liom the shippiig agai and placedin thc
mantcnaDce
mediünr(2000pl). Aier onc
hour incubalion. thc skin sample was
punched ont fron the pldtic part of dre
Millicell usinsa 2l nni punch.
of the pemeationmentrane mount d on
thepemcdtjon appdatus-SoercnsenPho$
thale bulTerpH 7.4 wasusedasar acceptor sohrtionmainlainiry silk condilions
tltou8honr tle experinent (nxüimum !c
ceptor conccntuation73 Jrg/ml conpared
ro c. =2050 |ls,/tlnar 32'c (wird, 198E)).
The rcceltor solulion was continuously
stinedat50{)rpmandthetemperaturc
was
kepl är 32 I l'C by a water.iacket.
The expeilmentwascdicd out tbr six
houß and satnpleswere düwn at speci
tled ti'ne points,a.d the withdrawnvol
ume was immcdifiely rcplacedwith lresh
acceplorsolulion.Analysis of sampleswa,r
coflected for all previous slmples fe
2.2.2 l issue-fr€em€mbranes
Tissueffee inserts(miffoporous rcl'lon
bnsedfiltcN). whicb are nomally incor
poialed in rhc culhtre of the reconsrr&ted
ski'r eqnivalent.werelrovidcd by MatTek
The baricr iftcgrity ofthe sknr equlvalent wa! checkedby measuing the permea
tion of a marker noteculc (Na
fl uofescei'8,20 lls/ml).
2.2.3Heätseparatedhuman
Erciscd humin skii sampleswere ob
oined from abdominal cosmetic surgery.
wraped in alumjnum t'oil and stored in
lolycthylenebagsaI -26'C until usc.
Hunän cpidcrmal membmneswerepe
fared by a heat sc!äratio. technique.Af1ef thawing. the ski! specimenwas lnrncrscdr hot watef (60'C) Ior 90 scconds
(KligmareLu1..1963).Theeddemis was
peeledoiTfton üc undeflyingdemis and
floated on phosphaE buffcr for one hour
to allow hydrition of the epidemis. Pr€
vious experimcnLshave shown that nini
mal tlux variadons wcrc obtailed ut)on
slanda'disedhydralioncorditions of all
ep cmrll sheetsused.Skin lamplcs wefe
used from one donor to avoid
interindividual
variltion.
2.3 Permeationstudics
All pemearion crpcriDents were leF
tomed usinga glassFftu diffusioncell
(odfic diamerer:l5mm od cell
appaatuN
volume: 12 'nl, Permegear.PA 18077,
USA).Thedillusioraldea ofthe skiüwas
Thc lcrmeation barid (melnbrmc) was
sandwiche.d
bctw@Dthe npper(donor) rd
lower (acceptn) compartmcntof rheFränz
ditrusioncell.An infinitedoscoftheoinr
nent (3 mm layer) or 0.5tr | of lhe 0.I 125,/a
solution wcrc lpplied to the intacl sulacc
2.4 Drug analysh
Analysis of samplestbr flufenämic acid
conrcntwas condüctednsing high pcr
fomranceliquidchromdogmphy(HPLC,
Merck Hilachi.D-Dam1sladl).
Theequip
ment consisted of an AS 2000A
autosamplingsystem(with an injeclion
volune of 20 Ut),.inL 6220purnpandan
L 4250 UV VIS detectoi.Thc sanples
wcrc analysedusing a reversedphase
Lichrosphere I 00 RP I li (5 Fm) co1!m
(LiChroCART 125 4 I:IPLCCaltridge).
Flutenamicacid was delecledat }; 2il4
nn with a rctention tine of approximately
3.510-2 ninutes.The mobilethasecon
sisted of 80./amctndnol (Bater, NLDeventet and207, Mcllvainc cirric acid
phosphdtebüfibr fH 2.2 (componenrspuF
chasedfrom Merck, D Damstad0 at a
llow rateof L2 nr1mifr.
UDknownllufenamic acid concentra
lions werecalculatedagainsl*nown srmd
ardsusirg the arcaunderthe absorltion
üne curves and a calibüti
Cumulativeamountsof the lcst sub
stancein üc rcccptor thid are plorled as
a fllNtion of the exposurerime. Depending on Fick\ firs! law of diffusion the
slo!. of the linear potion oI the cune
providedl1ux vdlucs(J. p&/cnrrhour).
J = P.e!/C,
C,is the innial concenrarion(rs/cmr)
of dtc drug i! the donor chamber
P"e,is th€ appucnt permeability coeffi
ALIEX I8, !/]I
ZGHoLa ET AL
ۊ
All experinenlsweredonc otr two jn
dependent
batcles of reconstuctedskin
cquivllcDtslnd data were expressedis
meant slandarddcvi{tion (r= 3lo 5).
2.5 Hisn,logicalcxamination
Light Microscopy (LM) and Scanning
Eleclnn MicroscolyISEII) wefeusedto
studythe tnoryholo$'ofthe stjr cquila
lcnts.The skin specimersweredetached
frcnl üe piaslic inscrt with a scalpel and
forcets. For LM üey wqc fiicd in ßouin
solution (a 'nixture of fonndldchydc. pic
nc acid aDdglacialaceticacid)and then
lrocessedlorcmbcddingin pituftln. Ver
tlcal sectionswere cul axl sLaincdwith
Nucled FastRed,EosineX Aniline Blue
and Oftngc C. To furthd evrluate lhe skin
equivalents
scanningelecrronDricrcscopy
was pedbnned.Skin sarnpleswerc lücd
phosthnLe
in 2.5'l. glutafaldehyde
buller
solurion(pH 7.4)a rhc! posttixedwith
1% osniuD lelroxidein lhosphatcbufül
pH 7.4 rccordingto Millonig (1961).Thc
tissucwasthcndehydmted
in gradedethanoi and acelone,crilicll point dried and
alieNads sputtered
wlth gold.Theobscr
v a t i o n sw e f e d o n e u s i n g a S E M l y p c
(UK almbf idse).
CamScan2
3 Results
rc
Figure1: Lightlüicroscopyola crosssectionthroughthe reconshuciedhumanepidermis (A)and lhroughheal-separated
humanepidermis(B).Anarrowindicatesthe supporlingmembranes.
OriginalmaEnilication
X575.ScanningEleclronlvlicroscopy
ot ihe
skin equivalenl(C)and humanskin (D).original magnilicatlon
X200and xi00.
The bafier function ofthc rcconsülcted
skiD model was hvestigated by apllying
thelipophilicmodeldmg urti.anic acid
in the lom of 0-1125%solutionand,as
canbe seenin Figurc2, drc bariertunc
lionoflpiDennrv is.learlyshownin con
rast to Lh{rof n\c dssue free Ulternem-
3.r Histologicalrnrllsis
Ouf light niüoscopical eranindtioDs
..".-.ffi
showcd a compact statum comeun ar
$ätified epidcmal like layes (A) wlth a
morphology
comlarablcto humanskin(B).
Scdning electrontnidoscopyshowcda
lopogaphicdlview of the skin nodel (C)
and lessresembLmcc
to humanskin (D)
couldbe deected(Figurcl). Tle sü1äce
ofthe skin equivalentwa! rnisi,ingthc clas
sical aplearincc ofthe tlaftenedhexagonal
cornifiedcellsandinsreada ratherdililse
su'tacewas observed.This could partly Figure 2: Ditfusion ol Flutenamicacid
throughlissue-lreeinserrscompared1o
explain the higher per eability of
permeationacross reconsrrucredskin
EpiDemnr.
equivalenis.The cumulativeamountpeF
meateddu nE6 h:s etPressed.
3.2 Permeationstudies
Theincgrityoftheskir equivalenls
wasäs
Comparing the perneatioD ol
sessed
usn'g fluoresccinepemeabiliq, (dara flufenamic acid äom tlvo dlfferent dos
not shown).Theieconsfucrcd
skinequiva agefbms ir wasIoundthatthe modeldrug
lent is supposedto be not Fnneable lo Na
pcnncatedmuch faster\!hen appliedin
tluo€sceine. tsy lcdonnnrg the test at the sotulion(Figurc
3).Theflux of flulcnamic
end oft|e expeiiment.only lon leiky skin acid wa\ 40 timcshigherirten usingüe
slccimens werc taken inlo considctution. 0.I125%solutic'Ilas
donorcomparedwlth
This Lestfor inteerity could only be caricd
rhc0.11250/,
oinbnenl.Thisncos thafthe
out in the caseoI liqujd dmg prepamtions. fomulntionssolulionandointmenrco!]d
alrEx r 8,!/01
\575
cledrl)'bc diffcrcntiated.which is essen
tial tb. rhe useof lhis model ir rhe
optimisationof demal drugPlcpuations.
3.3 Conparison to human epidermis
To ttrther evaluarethe suitrbility of the
rcconstructed
hünrx. skii equivalcit asa
possiblcalterutive methodse com!ared
Fiqure3: Permeationprofllesol ftufenarnic acid lhroughlhe reconstrucred
skin
equivalentsapplledin iwo diflerentphaF
maceuticaldosagefofms.
MeanlsD (!g/cm?),n=3.
thepemeation olflutanamic eid fmn lhc
0.I1257.ointnenl llrough heatseparared
humanepidemisandthe skincquivalent.
The lLtxofflufcnamic icid for lhehuman
epidenniswäslound ro be 0..19210.027
pg cm 'hour' andfor the ski! equivalen!
( E p i D c m r l o l 2 5 l 0 ) i t w a ! 2 . 5 0 5 11 . 4 1
fg crn'hour'. whicb is ahr'sL 5 Limcs
h igherthanill humancpidemis.
105
ZcHdiL r.1 AL.
ää
prcpafationslike ointmentand solulior.
This is a prerequisiLe
for usingir asm ln
rn,1) lest nodel for perculaneou!drug
pEparation.Althoughrhe nreanflux was
aboutilyetnnesb'ehercompaf
inethesknr
equivalent|o hundn cpidcmis. dgrccine
wilh reporteddata(Doucerer ä1.,1998r
Asbillel aI.,20(10),
rheuseofski. equivalents has sevefaladvantages.
By testing
diflerent ccll cullurc bdt lrcs,thcscprcliDi
nary data suggesta satisfacroryreproducibiliq, i'r contrist to lhe high donorvariabilityknowni'r humanskin.It alsoofteß
theadldnlrecovcr cxciscdhumer skj! iD
ils netaboljccapacitl(Slivkaeral., 199.1).
Comparedto animal skin. reco.sbucted
skin modclsmay sene asa beltefaltena
live sincethey are rnore sjrnild lo humaD
sknr.rot only ln temß ofspecies.bu! aLso
Figure4rCompädsonofthe amounlpeF in the biochemistryand organisalion.
remeaied(dean rSD, trE/cm'?)
in lwo inde- $rltingtu viriöle penetfttionmtesiI skins
pendentbatchesot reconslrücledskln
obLainedfrom d-nini and man. Ilrcrcturc.
equivalentsio test balchvariation.
the reflacenentof rninals in the resiing
prcductsappeaßIo becone
ofüansdernral
possiblciD thc ärrufe.Anothefadvantage
Chnc.rl srudies until now are conridered ofthe skinequilalenltis lheircady avail
ro be rhemostdesirablemeüod in resting abiliq, allowingthe qnick and easy!esLIhe etliciency of demrally applieddntg in8 ofdrug pernreation
liom lopi.ally aptbmulations.Bul due to ellical andeco
nomic reason\this sill nol be rotrtnrely
The purposeolüjs (udl wasto cxlm
applicablein thefield ol development
ard
nrethe pemeabilityof EtiDennrv in oroptimisation
of diftäfenrdNg prcpantiors. dcrto assess
its trtilityasan h rlr, model
Thcrclbn scvdal pcmrcatioDmodcls wcr!
for dcmai formul.tion testirg. The pre
delelopedusinganificial.animalandex- lininary dalapresenred
heresuggcstthat
cised!ünanskintonrnnicnrebarier1lrnc s k i n e q u i v a l e n t s b a s e d o . h u a n
lion ofthc skin.-A.lthoueh
bumdnskin is keratinocytecultufeshave potenlial as
curcntl) considercdüc non suitdblc lhdnnaccuticaltcst syslems10 studylhe
nodel. iß InniEd xlailnhilitr andiis high penerntionnndtemeationof &uSSfion
variabilitytiom donorto donorhavelinr
topicallbmrnlationsirto or rcrossthe\kin.
ilcd its usc.OftcDarinal skiDis uscdir
fufiher reiiements andaalidationofthe
slcadrhoughiris scll(nowtrrhdtitis quirc systcn s,ill bc ncccss.rtsotharin thelu
differenlfrom hütnanskin wlrh respectto üre n $ill beconsidered
a rcd1altcmltive
the numberothair lbllicles.lipid compo
to hunransknrin drogdeliveryresearch.
sition. lipid coDtcntdDdmophologicrl
aplearance.To da|e.ro model is avaü.rble
which tnll rnni.s hümanskin in tems
of ccli typc, numbcrof cells,blood ves
Asbill. C.. Kinr. N., El Kattan,A. et al.
(2000).Evxluationofa HunranBio-EnSiicenowadalsreconslllcledsl,inmodSlrcrcd Skin fatuivaleft for Drug Per
elsarcavailable
closelyresemhling
hnnan
meadonStudie!.PrdDr. tei. 17. 1092
skin in tcms of mophology andlipid com
1097.
posidon(Fdaascher al.. 1994).dxs srudy Doucct,O.. Gdrcia,N. and Zastroq,.L.
exploredlhe usefulness
ofn culluredskin
(1998).SldnCuhurcModcl: a Possible
alternative consisting of hunrar
AltefladveIo thelhe of E{cisedHumln
keratinocytcsfor &ug pcnncltioD studics.
Skh ior Asessi'rg t' i,lr, Percuuneous
OuftesulLsleii fy tharlhe usedskin equiva^bsorption.Tirin/.nil'iro12,423 :130.
lert btrildstrf x penne.ttunbarier. n d it
Fartalch.M. andPonec.M. ( I99:1).lmprcv
coulddiftarcntiate
the pemeationof dtlg
ed Barier Shcture Fomation ir Air
3.4 Uafth n) batch uriation
ln tcstiDgBpiDcml "' Ior relroducibilit)
wc nudied bilch varialion.The prelininaryresult\arelllustated i. Figure4i no
statisticallysignificrnt dillerence (p >
{).05)in pcucrtion could bc dclcctedin
nvo lots of LpiDennrN.
106
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wild, T. (19t8).,Di$.rntnn Sadtbtük
i.r. UniversitäidesSaarlardes,1l1.
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