O ganiza ion and Fo ma ion o he C ossed-Folia ed Biomine al
Mic os uc u e o Limpe Shells
Ka a zyna Be en , Ma a Gajewska, and An onio G. Checa*
Ci e This: ACS Bioma e . Sci. Eng. 2023, 9, 6658−6669
Read Online
ACCESS Me ics & Mo e A icle Recommenda ions *
sı Suppo ing In o ma ion
ABSTRACT: To cons uc hei shells, molluscs a e able o p oduce a
la ge a ay o calci ied ma e ials including g anula , p isma ic, lamella ,
ib ous, olia ed, and plywood-like mic os uc u es. The la e includes an
a agoni ic ( he c ossed-lamella ) and a calci ic ( he c ossed- olia ed)
a ie y, whose modes o o ma ion a e pa icula ly enigma ic. We s udied
he c ossed- olia ed calci ic laye s sec e ed solely by membe s o he
limpe amily Pa ellidae using scanning and ansmission elec on
mic oscopy and elec on backsca e di ac ion. F om he ex e io o
he in e io , he ma e ial becomes p og essi ely o ganized in o
comma ginal i s -o de lamellae, wi h second and hi d o de lamellae
dipping in opposi e di ec ions in al e na ing lamellae. A he same ime,
he c ys allog aphic ex u e becomes s onge because each se o he i s
o de lamellae de elops a pa icula o ien a ion o he c-axis, while bo h
se s main ain common o ien a ions o one {104} ace (pa allel o he
g ow h su ace) and one a-axis (pe pendicula o he planes o he i s o de lamellae). Each i s o de lamella shows a p og essi e
mig a ion o i s c ys allog aphic axes wi h g ow h in o de o adap o he o ien a ion o he se o i s o de lamellae o which i
belongs. To explain he p og essi e o ganiza ion o he ma e ial, we hypo hesize ha a sec e ional zeb a pa e n, mi o ed by he i s
o de lamellae on he shell g ow h su ace, is de eloped on he shell-sec e ing man le su ace. Cells belonging o al e na ing s ipes
beha e di e en ly o de e mine he g ow h o ien a ion o he la hs composing he i s o de lamellae. In his way, we p o ide an
explana ion as o how plywood-like ma e ials can be ab ica ed, which is based mainly on he ac i i y o man le cells.
KEYWORDS: biomine aliza ion, molluscs, calci e, ma e ial o ganiza ion, c ys allog aphy, plywood s uc u e
1. INTRODUCTION
The shells ab ica ed by in e eb a es a e composi es o
calcium ca bona e c ys als (p edominan ly calci e o a agoni e)
wi h a pe cen age o o ganic ma e (1−12%),
1,2
ei he
occluded wi hin he c ys als o in he o m o in a- o , mo e
o en, in e c ys alline memb anes.
3
Shells a e made o one o
se e al laye s, each cha ac e ized by h ee-dimensional a ange-
men s o c ys als wi h de ined mo phologies, e med mic o-
s uc u es. Wi hin in e eb a es, molluscs p oduce by a he
la ges epe oi e o mic os uc u es, ollowed a a conside able
dis ance by b achiopods, polychae es, and b yozoans. On he
o he end o he spec um, g oups, such as ci ipeds,
echinode ms, and scle ac inian co als, p oduce only one o
wo basic mic os uc u es. Molluscs can p oduce g anula ,
p isma ic, ib ous, lamina ( olia ed, nac e), plywood-like
(c ossed-lamella , c ossed- olia ed), and helical mic os uc u es
wi h ei he calci e o a agoni e. They o m well o e a dozen
basic mic os uc u al ypes wi h se e al sub a ie ies. The
o ganomine al composi e na u e o mic os uc u es p o ides
hem wi h excep ional biomechanical p ope ies. Some o
hem, such as nac e and c ossed-lamella ,
4−6
a e pa icula ly
s ong and ough, whe eas o he s a e lexible in he na i e
hyd a ed s a e, e.g., he columna calci ic laye s o p e ioid
bi al es.
7−9
Acco dingly, hese bioma e ials a e a sou ce o
inspi a ion in biomime ics o he p oduc ion o ad anced
unc ional ma e ials. In his ega d, i is necessa y (1) o ully
cha ac e ize he s uc u e a di e en leng h scales and (2) o
un a el he s a egies used by he o ganisms o p oduce hei
mic os uc u es. Molluscs make use o p ocesses o c ys al
g ow h, and he sel -o ganiza ion o o ganic ma ices, and hei
man le cells a e capable o ecognizing he al eady laid-down
s uc u e (e.g., dis ibu ion o mine al e sus o ganic
subs a es) and con inue o sec e e acco dingly.
10
These
p ocesses, combined o in isola ion, p oduce s ongly ex u ed
ma e ials, howe e , wi h e y di e en mo phologies and
a angemen s om hose o ino ganic calcium ca bona e
agg ega es. While he e ha e been signi ican ad ances
Recei ed: July 10, 2023
Re ised: Oc obe 10, 2023
Accep ed: No embe 7, 2023
Published: No embe 22, 2023
A icle
pubs.acs.o g/jou nal/abseba
© 2023 The Au ho s. Published by
Ame ican Chemical Socie y 6658
h ps://doi.o g/10.1021/acsbioma e ials.3c00928
ACS Bioma e . Sci. Eng. 2023, 9, 6658−6669
This a icle is licensed unde CC-BY 4.0
Downloaded ia UNIV DE GRANADA on Feb ua y 16, 2024 a 09:40:46 (UTC).
See h ps://pubs.acs.o g/sha ingguidelines o op ions on how o legi ima ely sha e published a icles.
ega ding he knowledge o mic os uc u es such as nac e,
columna p isma ic, and olia ed mic os uc u es, he e is a
e y impo an mic os uc u e ha has emained pa icula ly
obscu e, he c ossed lamella . This ype cons i u es one o he
mos hie a chically o ganized mic os uc u es, consis ing o
i s -, second-, and hi d-o de lamellae (OLe; OL o singula ,
lamella). I is ound mainly in a agoni e, al hough a ew
gas opods, he pa ellogas opods, also p oduce a calci ic
a ie y, he so-called c ossed olia ed. Fi s OLe a e wide
lamellae ( ens o mic ome e s) wi h i egula , jagged ma gins,
which g ow pe pendicula o he g ow h su ace. They can
ex end pe pendicula ly ( adial) o pa allelly (comma ginal) o
he shell ma gin. The a agoni ic a ie y consis s o ibe s ( hi d
OLe), which a ange in shee s (second OLe), while he calci ic
ype is made o long la hs wi h a owhead endings ( hi d OLe)
ha o ganize in o olia (second OLe). In bo h cases, he
second and hi d OLe dip in opposi e di ec ions in al e na ing
i s OLe, such ha o ien a ions a e iden ical e e y wo i s
OLe. Since he a agoni ic c ossed-lamella mic os uc u e was
de ined by Bøggild,
11
many au ho s ha e deal wi h i s
s uc u e,
12−16
and c ys allog aphy.
15,17−20
The calci ic c ossed-lamella mic os uc u e emained
ela i ely neglec ed un il he ex ensi e s udy o MacClin ock
21
on pa ellogas opods, who enamed i as c ossed- olia ed,
acco ding o he olia ed aspec o he second and hi d OLe.
The e m c ossed-lamella emained o he mo e ib ous
a agoni ic a ie y. The a agoni ic c ossed-lamella mic o-
s uc u e is by a he mos success ul mic os uc u e in
molluscs. I is p e alen in gas opods, bi al es, and
polyplacopo ans and he only mic os uc u e p oduced by
scaphopods. I was also p esen in he ossil molluscan class
Ros oconchia,
22
and in he phylum Hyoli ha.
23
The c ossed-
lamella mic os uc u e a ose independen ly in di e en
classes. I is absen in cephalopods, monoplacopho ans, and
he spicule-p oducing aplacopho ans. Due o his and o i s
excellen biomechanical p ope ies, pa icula ly oughness,
6,24
he c ossed-lamella mic os uc u e has been he ocus o a
my iad o s udies, and p esen ly he e is a g ea deal o
in o ma ion on i s mo phology and c ys allog aphy.
The c ossed- olia ed mic os uc u e, on he con a y, is
es ic ed o he gas opod amily Pa ellidae (subclass
Pa ellogas opoda), whe e i is usually he second laye om
he ex e io .
21
A seemingly c ossed- olia ed ma e ial was
desc ibed in some lowe Ju assic G yphaeidae
25
and in uppe
Ju assic Oxy omidae,
26
al hough hese assignmen s a e di icul
o e i y in he absence o 3D iews o he ma e ial. Wi h
ega d o he Pa ellidae, e y ew s udies p o ide scanning
elec on mic oscopy (SEM) e idence on he o ganiza ion o
he ma e ial.
27−30
In addi ion, no hing is known abou i s
c ys allog aphy. Gi en i s in e es as a hie a chically o ganized
ma e ial, his s udy ocuses on he o ganiza ion and c ys allo-
g aphic ex u e o he c ossed- olia ed mic os uc u e. We
de elop a c ys allog aphic model o his mic os uc u e and
show ha , con a y o i s a agoni ic coun e pa , he c ossed-
lamella mic os uc u e o ganizes p og essi ely om a poo ly
o ganized c ossed- olia ed ex e nal laye . We hypo hesize ha
such a p og essi e o ganiza ion is ob ained h ough a
combina ion o c ys allog aphic p ocesses and man le cell
ac i i y. Ou s udy p o ides insigh as o how c ossed- olia ed
o plywood-like biomine al s uc u es can be o ganized.
2. EXPERIMENTAL SECTION
Se en li ing specimens o he limpe Pa ella cae ulea we e sampled in
he locali y o Almuneca (Medi e anean, SE Spain), whe eas ou
emp y shells o P. us ica and ou o P. dep essa came om A iles
(A lan ic, NW Spain). Shells we e washed wi h dis illed wa e and ai -
d ied a oom empe a u e. Fi e shells o P. cae ulea we e cleaned wi h
comme cial bleach (app oxima ely 5% ac i e chlo ine) o 4−5 min.
Th ee o ou bi s o he in e nal su aces o wo shells o P. cae ulea
and one shell o each o he wo o he species we e moun ed on SEM
s ubs, ca bon-coa ed (Emi ech K975X ca bon e apo a o ), and
obse ed in ield-emission SEM (FESEM) equipmen Zeiss Au iga
and FEI QemScan 650 F a he Cen e o Scien i ic Ins umen a ion
(CIC) o he Uni e si y o G anada (UGR), Spain, and in he FESEM
FEI Ve sa 3D o he Academic Cen e o Ma e ials and Nano-
echnology (ACMiN), K akow, Poland.
A omic o ce mic oscopy (AFM) obse a ions we e done on inne
su aces o shell agmen s o P. cae ulea, p e iously ul asonica ed. We
used an AFM Pa k Sys ems NX20 (CIC, UGR) equipped wi h a
can ile e ACTA (K = 40 N/m, F = 280 kHz) (CIC, UGR). We
eco ded heigh , ampli ude, and phase signals in apping mode.
Images we e ob ained wi h Sma Scan 12 and analyzed wi h XEI 4.3
so wa e (Pa k Sys ems).
The man le o a li ing specimen o P. cae ulea was excised and ixed
in a mix u e o 2.5% glu a aldehyde bu e ed wi h sodium cacodyla e
(0.1 M, pH 7.4) o 48 h a 4 °C and pos ixed in OsO4(2%) o 2 h
a 4 °C. The issue was la e embedded in epoxy esin, Aname Epon
812 (EMS). Ul a hin sec ions (0.1 μm) we e s ained wi h u anyl
ace a e (1%) and lead ci a e. They we e la e ca bon-coa ed and
obse ed wi h ansmission elec on mic oscopy (TEM) Zeiss Lib a
120 Plus ins umen o he CIC (UGR).
Elec on backsca e di ac ion (EBSD) analysis was pe o med o
iden i y he c ys al s uc u e. Shell agmen s o he h ee species we e
moun ed in epoxy, sec ioned pe pendicula o he shell su ace,
g ound, and polished ollowing a s anda d me allog aphic p epa a ion
ou e: mechanical polishing by silicon ca bide pape s wi h g i sizes o
320, 500, 800, 1200, 2000, and 4000, ollowed by 3, 1, and 0.25 μm
diamond suspension. Final polishing wi h colloidal silica o 1 min
using a S ue s Teg amin-25 au oma ic polishe ensued. In his way,
wo sec ions o each species we e mapped. O he maps we e
pe o med di ec ly on he in e nal shell su aces o P. dep essa and P.
cae ulea wi hou u he polishing, despi e which hey ende ed use ul
in o ma ion and allowed us o ela e he shapes o calci e la hs o
c ys allog aphic o ien a ions. EBSD analysis was ca ied ou wi h an
FEI Ve sa 3D FESEM, equipped wi h an Ox o d Ins umen s
Symme y S2 came a. EBSD maps we e collec ed by applying an
accele a ion ol age o 12 kV unde low acuum (40 Pa o H2O).
Da a we e analyzed wi h he Da a Collec ion .7.3 and Az ec 6.0
so wa e. To calcula e he numbe o g ains, he g ain bounda ies we e
de ined whe e pixel- o-pixel miso ien a ion was abo e a c i ical
miso ien a ion angle, se a ≥15°. Mul iple o uni o m densi y
(MUD) p o ides an indica ion o he s eng h o he ex u e. A MUD
o 1 indica es andom o ien a ion, while high MUD alues (600−700)
indica e a high, single-c ys al-like co-o ien a ion. The ocused ion
beam (FIB) li -ou me hod was used o p epa e wo ul a hin c oss-
sec ional lamellae o he concen ic c ossed- olia ed mic os uc u e o
P. cae ulea using a FEI Quan a 3D 200i dual beam FIB/SEM. The
TEM lamellae we e milled down o ∼50 nm hickness using 2 kV.
This emo ed he beam su ace damage. B igh - ield imaging and
selec ed a ea di ac ion (SAED) analysis o he wo lamellae we e
pe o med using an FEI Tecnai TF 20 X-TWIN TEM ope a ed a 200
kV. All he abo e equipmen is housed in he ACMiN.
3. RESULTS
3.1. Shell Mic os uc u e. The gene al shell s uc u es o
he h ee species s udied a e simila . The e is a hick ou e
calci ic shell po ion, made o c ossed- olia ed ma e ial, in e nal
o which he e is an a agoni ic c ossed-lamella shell po ion
(Figu e 1a), c ossed by he myos acum. The calci ic shell
ACS Bioma e ials Science & Enginee ing pubs.acs.o g/jou nal/abseba A icle
h ps://doi.o g/10.1021/acsbioma e ials.3c00928
ACS Bioma e . Sci. Eng. 2023, 9, 6658−6669
6659
po ion hickens in he di ec ion owa d he ma gin, and,
con a ily, he c ossed-lamella laye wedges ou in he same
di ec ion. The calci ic po ion consis s o i s OLe wi h a
comma ginal dis ibu ion, which, in he adial sec ion o Figu e
1a, is cu ans e sely. Thei ou lines become be e de ined
owa d he shell in e io and a e o ien ed a a high angle o he
g ow h lines (Figu e 1a, inse s 1 and 2). Towa d he apex, he
shell is usually bo ed p og essi ely deepe in e io ly by
endoli hic mic oo ganisms. The i s OL o he unde lying
c ossed-lamella (a agoni ic) laye is also cu ans e sely, and
simila o he c ossed- olia ed laye , hey a e a a high angle o
he g ow h lines and o he in e nal shell su ace (Figu e 1a,
inse 2). In op ical iews o he in e nal shell su ace, he ou e
calci ic po ion appea s sligh ly anslucen , and a a ce ain
dis ance om he ma gin, he dis ibu ion o comma ginal i s
OL is clea ly ma ked by di e ences in e lec ance (Figu e 1b,
inse ). The mo e in e nal c ossed-lamella ma e ial appea s
g ay and dull. I occupies he es o he shell owa d he
in e io , excep o he in e cala ion o he myos acum (Figu e
1b).
The dis ibu ion pa e n o he i s OLe o he c ossed-
olia ed laye becomes clea e in SEM iews. In gene al, hey
end o be i egula close o he edge; howe e , di ec ly owa d
he in e io , hey become comma ginal (Figu e 2a).
Fu he mo e, he ma ginal i s OLe a e i egula in shape
and e y une en in size (Figu e 2a−c). Thei wid hs may each
>200 μm locally. Thei cons i uen la hs ( hi d OLe) ha e
e en o ien a ions wi hin each lamella bu can ake any
o ien a ion wi h espec o neighbo ing lamellae (Figu e
2b,c). A he e y ma gin, he hi d OLe a e some imes
pa icula ly hick (up o 0.5 μm), and hei e minal edges
coa sen and de elop hombohed al ace s (Figu e 2d). These
a e much smoo he han he es o he su aces, which ha e a
ypical lumpy aspec . Di ec ly adjacen o he ma gin owa d
he in e io , he hi d OLe become hinne (0.2−0.3 μm) and
a ange in o mo e con inuous second OLe (Figu e 2c,e).
Gi en he une enness in size and o ien a ion o he i s OLe,
we will call his laye an i egula c ossed- olia ed (ICF) laye
(Figu e 2a).
A e a b ie ansi ional s age (Figu e 2a, ), he i s OLe
become comma ginal. They cons i u e he CCF laye . Thei
wid hs a e no consis en la e ally and equen ly wedge ou
(Figu e 2a), wi h he consequen usion o al e na ing i s OLe
con aining e enly o ien ed second and hi d OLe (Figu e 2a),
in a so o zeb a pa e n. Thei maximal wid hs anged om
50 o 70 μm (Figu e 2a,g). The hi d OLe ha e wid hs
be ween <200 nm and ∼2μm, and, in ac u e, hey ex end
owa d he shell in e io o unknown dis ances (a leas ens o
mic ome e s) (Figu e 2g,h). They a ange in o con inuous
second OLe, al hough hese may no ex end along he en i e
hickness o he i s OLe (Figu e 2i). Thi d and second OLe
dip wi h simila angles bu in opposi e di ec ions in al e na ing
lamellae (Figu e 2g,i−k). The junc u es be ween he hi d OLe
o adjacen i s OLe a e igh , and end o in e pene a e
sligh ly (Figu e 2i,j). In he adial di ec ion, he bounda ies o
he i s OLe a e no simple and may consis o se e al
b anches, gi ing i a jagged appea ance (Figu e 2k,l). The ips
may consis o jus one o a ew la hs (Figu e 2l−n). AFM
obse a ions o he main su aces o la hs o he CCF laye o
P. cae ulea e ealed a ma ked su ace nano oughness,
consis ing o lumps be ween 10 and <100 nm (Figu e S1).
Lumps supe impose and me ge wi h each o he , hus ha ing
unde ined bounda ies. Phase images e eal he p esence o wo
phases, a p edominan ligh phase and a da ke phase
dis ibu ed a ound he bounda ies be ween he lumps (Figu e
S1, phase images). Only he e minal a owheadlike aces and
hei ma gins appea smoo he .
The dis ibu ion o he ICF and CCF laye s is also e iden in
he longi udinal sec ion o Figu e 1a, inse s 1 and 2. In he
CCF laye , he ans e sely sec ioned i s OLe a e
app oxima ely pa allel bu une enly o ien ed in he ICF
laye . The i s OLe inc ease in hickness om he ju enile
(Figu e 1a, inse 2) o he subadul (Figu e 1a, inse 1).
3.2. EBSD Analysis. In e se pole igu e (IPF) o ien a ion
maps done on adial sec ions p o ide addi ional insigh in o
he o ganiza ion o he ma e ial. The ou e shell laye is made
o elonga ed lamellae wi h i egula sizes and shapes, which a e
mainly inclined in he g ow h di ec ion and a a high angle
Figu e 1. Gene al shell s uc u e o he s udied species o Pa ella. (a) Radial sec ion h ough he shell o P. dep essa, indica ing he calci ic and
a agoni ic shell laye s. De ails o bo h can be seen in inse s 1 and 2. (b) View o he in e io o he shell o P. cae ulea, showing he dis ibu ion o
shell laye s. The inse shows he comma ginal dis ibu ion o i s OLe. CCFL, ICFL: concen ic c ossed- olia ed laye , i egula c ossed- olia ed
laye ; CL: c ossed-lamella .
ACS Bioma e ials Science & Enginee ing pubs.acs.o g/jou nal/abseba A icle
h ps://doi.o g/10.1021/acsbioma e ials.3c00928
ACS Bioma e . Sci. Eng. 2023, 9, 6658−6669
6660
wi h espec o he g ow h lines (Figu e S2). In o he cases, he
lamellae do no display consis en o ien a ions (Figu e 3a).
O ien a ion maps display a a ied ange o colo s indica i e o
a ied o ien a ions (Figu es 3 and S2). We can assimila e his
laye o he ICF laye iden i ied wi h SEM obse a ions. Ra ely,
a hin (∼10−20 μm hick) ou e mos laye made o e y small
and sho lamellae can be obse ed (Figu e 3a).
Deepe wi hin he shell, his ICF laye is ans o med in o
he CCF laye . The change akes place h ough a ansi ional
laye , which can ha e a mo e (Figu es 1a and S2a) o less
educed hickness (Figu e S2b). F om he ICF laye o he
CCF laye , he numbe o i s OLe dec eases while hei sizes
inc ease. Values o he numbe o g ains ac oss he di e en
laye s a e lis ed in Figu es 3 and S2. A he end o he
ansi ional laye , he i s OLe become nea ly de ined and
acqui es a homogeneous o ien a ion a a high angle o he
g ow h lines and o he in e nal shell su ace. Ac oss he CCF
laye , he i s OLe display i egula i ies in hickness simila o
hose obse ed in he plan iew: b anching, widening, hinning,
and disappea ance. Maximal hicknesses a e on he o de o
200 μm (e.g., Figu e 3a). Al e na ing i s OLe display
al e na ing colo s, which indica e he exis ence o wo main
o ien a ions. Wi hin each indi idual lamella, he g adual colo
changes indica e in e nal miso ien a ions in bo h he
longi udinal and ans e sal di ec ions. Ex eme alues a e in
he o de o 20°in he di ec ion o he lamella hickness and
50°in he g ow h di ec ion.
We en a i ely es ima ed he bounda ies be ween he ICF
laye , he ansi ional laye , and he CCF laye and plo ed
hei pole igu es (Figu es 3 and S2). In all cases, he ma e ial
displays a ce ain deg ee o o de . The ICF laye demons a es
an o e all o ien a ion, wi h he c-axis ( he 001 maximum)
poin ing in he g ow h di ec ion and a a high angle o he
g ow h lines. The e a e ill-de ined 104 and 110 maxima, such
ha he ex u e can be quali ied as e y weak (Figu e 3a) o
weak (Figu es 3b and S2) shee ex u e. The in e nal CCF
laye , on he con a y, has well-de ined maxima o all axes
(s ong shee ex u e) and a comple e change in he
dis ibu ion o maxima. The e a e wo maxima o he c-axis,
which a e aligned pa allel o he elonga ion o he i s OLe a
an angula dis ance o ∼90°. Each maximum co esponds o
one se o al e na ing i s OLe. The 110-pole igu e displays
i e maxima, wi h one o hem being common o bo h se s o
i s OLe and o ien ed pe pendicula o he elonga ion o he
i s OLe. Finally, ou o he i e 104 maxima, he e is one ha
is common o bo h se s o i s OLe. I is placed close o ei he
he N o S pole o he pole igu e and is aligned wi h he wo
001 pole maxima. The posi ion o he common 104 maximum
can be aced om he ICF laye o he CCF laye . The
ansi ional laye has a dis ibu ion o maxima close o ha o
he CCF laye (Figu es 3a and S2a), o in e media e be ween
he ICF laye and he CCF laye (Figu e S2b). MUD alues
indica e ha he deg ee o o de ing is he highes in he CCF
laye in all cases. The ansi ional laye alues can be highe
(Figu e 3a), simila (Figu e S2a), o lowe (Figu e S2b) han
hose o he ICF laye , al hough his is possibly pa ly
dependen on how he ansi ional laye is de ined.
In a comma ginal sec ion (app oxima ely pa allel o he i s
OLe) (Figu e 3b), he dis ibu ion o maxima is as desc ibed
abo e, al hough o a ed by 90°a ound he e ical axis o he
pole igu es ( he line joining he N and S poles). In he
example in Figu e 3b, he second OLe a e inclined by ∼14°in
bo h di ec ions; ha is, hei angle wi h he g ow h su ace is
∼76°. O he measu emen s on he same species p o ide
inclina ions o 18°and 21°. In e es ingly, he wo 001 maxima
Figu e 2. De ails o he c ossed- olia ed mic os uc u e. (a) View o
he in e nal shell su ace o P. us ica. The al e na ing i s OLe can be
di e en ia ed due o o ien a ion con as . They a e i egula owa d
he ma gin (i egula c ossed- olia ed [ICF] laye , igh side) and
become comma ginal owa d he in e io (concen ic c ossed- olia ed
[CCF] laye ). The amed a ea is ep oduced in black and whi e in
Figu e 8a. (b, c) I egula dis ibu ion o i s OLe o he ICF laye
close o he ma gin o P. cae ulea. Also, no e he di e ences in shape
and size. (d) De ails o he hi d OL a he ma gin o P. cae ulea. They
a e pa icula ly hick and ha e coa sened edges. The inse is a de ail.
The hombohed al su aces looking owa d he obse e ( ) a e
pa icula ly smoo h, compa ed o he lumpy aspec o he es o he
g ow h su aces (ls). (e) De ails o he hi d OLe o he ICF laye o
P. cae ulea in an a ea simila o hose in b and c. They a e iden ical o
hose o he CCF laye (e.g., i, j). ( ) Dis ibu ion o i s OLe on he
in e nal shell su ace o P. cae ulea a he ansi ion be ween he ICF
laye and he CCF laye . (g) View o he CCF laye o P. us ica. The
ac u e on he igh allows us o obse e he changing inclina ions o
he second and hi d OLe in al e na ing i s OLe. (h) F ac u e
h ough a i s OL o P. us ica, showing he ex en o he hi d OLe.
(i) De ail o g ( amed), a he con ac be ween adjacen i s OLe in
P. us ica. No e sligh ly o e lapping edges (oe) a he con ac be ween
second OLe dipping in opposi e di ec ions. Small a ows indica e
ini ia ing second OLe. (j) De ail o a simila con ac in P. cae ulea.
Some o e lapping edges (oe) and ini ia ing second OLe (a ows) a e
indica ed. (k) Two- ailed end o a i s OL o he CCF laye o P.
cae ulea (cen al pa o he image). (l) Close-up o a simila case in P.
cae ulea. No e i egula i ies a he e y ips. (m, n) Two cases o
di e gence o i s OLe in P. cae ulea, beginning wi h a single hi d
OL. In n, he di e gence happens om a single spo . A ows in b, c, i
(la ge a ows), k, m, and n indica e he g ow h di ec ions o he hi d
OLe.
ACS Bioma e ials Science & Enginee ing pubs.acs.o g/jou nal/abseba A icle
h ps://doi.o g/10.1021/acsbioma e ials.3c00928
ACS Bioma e . Sci. Eng. 2023, 9, 6658−6669
6661
Figu e 3. IPF maps and pole igu es o he laye s dis inguished wi hin he shells o Pa ellidae. (a) Radial sec ion o P. dep essa, wi h g ow h lines
indica ed (black do ed lines). (b) Comma ginal sec ion o P. cae ulea. The ed lines a he bo om o he image a e he app oxima e o ien a ions o
he second OLe. CCFL, ICFL, TL: concen ic c oss- olia ed, i egula c oss- olia ed, ansi ional laye s. NG is he numbe o g ains o each laye .
The colo iangle is he colo key o o ien a ions.
Figu e 4. IPF maps, a di e en magni ica ions, done di ec ly on he g ow h su ace o P. dep essa, oge he wi h he co esponding aw and con ou
pole igu es (a, b). The 001 maxima a e joined o hei co esponding se o he i s OLe wi h hin a ows. The o e lapping 110 and 104 maxima
a e enci cled wi h ed o als. Thick a ows indica e he g ow h di ec ions o he hi d OLe. The colo iangle is he colo key o o ien a ions.
ACS Bioma e ials Science & Enginee ing pubs.acs.o g/jou nal/abseba A icle
h ps://doi.o g/10.1021/acsbioma e ials.3c00928
ACS Bioma e . Sci. Eng. 2023, 9, 6658−6669
6662

a e placed in he di ec ion o inclina ion o he co esponding
second OLe, al hough a a highe angle. The wo 001 maxima
and he common 104 maximum a e loca ed along he same
me idian. As usual, he MUD alues a e simila o he ICF
laye and ansi ional laye and a e he highes o he CCF
laye .
In o de o be e de ine he c ys allog aphy o he CCF
laye , we ha e mapped di ec ly he in e nal unpolished su aces
o he shells, such ha he o ien a ion o he second and hi d
OLe can also be app ecia ed (Figu e 4). On he maps,
al e na ing i s OLe come in sligh ly di e en colo s, and hose
o he same se (e e y wo lamellae) come in simila colo s,
indica ing di e en and simila o ien a ions, espec i ely. The e
a e wo 001 maxima aligned wi h he axis o elonga ion o he
i s OLe, a angula dis ances o ∼45° om he cen e , each
co esponding o one se o i s OLe. Wi hin each se , he c-
Figu e 5. G ow h ajec o ies o he i s OLe selec ed on he o ien a ion maps o Figu e 3. (a) P. dep essa. Two i s OLe pe se ha e been
selec ed. (b) P. cae ulea. One i s OL pe se has been selec ed. Pole igu es nex o he o ien a ion maps co espond o he ec angula a ea close o
he g ow h su ace. Posi ions o maxima o he wo se s o i s OLe a e indica ed wi h ed, blue, and g een c osses. Indi idual sca e pole igu es
o he selec ed lamellae a e p o ided (colo c osses indica e he posi ions o he maxima close o he g ow h su ace o he co esponding se o
i s OLe). The ajec o ies (black a ows) a e indica ed bo h on he i s OLe and on he magni ied a eas o he pole igu es. These consis o wo
o h ee segmen s. The ligh blue a ows in bo h he lowe map and i s 001 pole igu e (magni ied a ea) indica e he high miso ien a ion o he c-
axis ac oss he hickness o he i s OL (∼30°). The colo key o o ien a ions is p o ided in Figu e 3.
ACS Bioma e ials Science & Enginee ing pubs.acs.o g/jou nal/abseba A icle
h ps://doi.o g/10.1021/acsbioma e ials.3c00928
ACS Bioma e . Sci. Eng. 2023, 9, 6658−6669
6663
axis is inclined in he same di ec ion as he la hs (i.e., opposi e
o he g ow h di ec ions o la hs; la ge a ows and uni cells in
Figu e 4). One o he 110 maxima is coinciden o almos
coinciden o bo h se s o lamellae. I is placed pe pendicula ly
o he elonga ion o he i s OLe. The e a e wo 104 maxima,
one o each se o i s OLe, placed cen ally, which, simila ly
o he 110 maxima, o e lap comple ely o o a high ex en . The
MUD alue is lowe in Figu e 4a han ha in Figu e 4b, wi h
he i s co e ing a much g ea e a ea. This indica es ha he
sp ead o poles inc eases wi h he size o he mapped a ea.
An ob ious ea u e in all colo maps is ha mos o he
indi idual i s OLe o he CCF laye can easily be aced o
he ansi ional laye and some imes e en o he ou e ICF,
also acco ding o con inuous colo g adien s in he EBSD colo
maps. When wo supposedly con inuous lamellae a e no in
con ac due o he 2D sec ioning o when he lamella is
in e up ed by ano he lamella, we ha e used he con inui y o
he end o he miso ien a ion plo as addi ional e idence o
c ys allog aphic con inui y (Figu e S3). A g ow h ajec o y
(usually consis ing o se e al segmen s) is p o ided o each
selec ed i s OL on he o ien a ion map. We also plo ed he
001 and 104 aw pole igu es o he indi idual i s OLe,
oge he wi h he pole mo emen along he g ow h ajec o y
aced on he IPF map. In his way, we can obse e how he c-
and a-axes o a pa icula i s OLe mig a e wi h g ow h. We
ha e also de e mined he posi ions o he 001 and 104 poles on
he a ea closes o he in e nal shell su ace (Figu es 5 and S4),
whe e he coo ien a ion (MUD alues) is maximal. In he
examples depic ed in Figu es 5 and S4, we can obse e how he
001 and 104 poles mig a e p og essi ely, s a ing om e y
dispa a e posi ions o he pole igu e and ending close o e y
close o he posi ion o he maxima co esponding o hei
pa icula se o i s OLe (c osses on pole igu es in Figu es 5
and S4). The o al angle co e ed by he ajec o ies on he pole
igu es is high, some imes abo e 90°(e.g., Figu e 5b, se 1,
bo h pole igu es; Figu e S4b, se 2, all pole igu es).
3.3. TEM Measu emen s. The wo TEM lamellae o he
CCF o P. cae ulea examined we e p epa ed by FIB-SEM
pe pendicula o he in e nal shell su ace and owa d he
elonga ion o he i s OLe. In his way, he la hs we e cu
pa allel o hei wid h a an angle o ∼45° o hei g ow h axis.
TEM b igh ield images e ealed ha , in dep h, he
bounda ies be ween jux aposed la hs belonging o adjacen
i s OLe could easily shi la e ally, p oducing a jagged
bounda y (Figu e 6, b oken lines). The measu ed hicknesses
o lamellae a ied be ween ∼60 and ∼150 nm (a e age o
∼110 nm). Fo single lamellae, he hickness may also a y
la e ally. The la h o ien a ions ob ained om he SAED spo
pa e ns (inse s in Figu e 6a,b) we e along he [010] and [100]
zone axes in Figu e 6a,b, espec i ely, which is cong uen wi h
he EBSD da a.
4. DISCUSSION
4.1. Shell Mic os uc u e. The c ossed- olia ed mic o-
s uc u e is a hie a chical ma e ial composed o calci e la hs
( hi d OLe) a anged in planes (second OLe), in u n packed
in o big i s OLe pe pendicula o he shell su aces. The
e enness in o ien a ion and size o he la e se s he di e ence
be ween he ICF and he CCF laye s.
In his ex ensi e e iew o shell mic os uc u es, MacClin-
ock
21
es ablished a nomencla u e o di e en shell laye s in
pa ellogas opods (limpe s). He named he laye s om M-2 o
M+4, wi h e e ence o he myos acum (laye M). He
g ouped he limpe s acco ding o he numbe o laye s and
hei mic os uc u es in o 16 g oups, and included wo o he
species we ha e s udied (P. cae ulea and P. lusi anica, p esen ly
a synonym o P. us ica
31
) wi hin G oup 8, cha ac e ized by M
+3 = adial c ossed- olia ed, M+2 = concen ic c ossed-
olia ed, M+1 = concen ic c ossed-lamella , M, myos acum,
M-1 = adial c ossed-lamella and/o complex c ossed-lamella ,
and M-2 = i egula ly olia ed o adial c ossed- olia ed. Ou
hi d species, P. dep essa, no examined by MacClin ock, has
exac ly he same dis ibu ion o calci ic laye s (Figu es 1a, 3a,
and S2a). He e, we ha e paid a en ion o MacClin ock’s M+3
(ou ICF) and M+2 (CCF) laye s, al hough we call he o me
as i egula , ins ead o adial c ossed- olia ed laye , in iew o
he i egula i y in size and o ien a ion o he cons i u ing
lamellae (e.g., Figu e 2).
Close inspec ion o he CCF mic os uc u e wi h AFM
e ealed he su ace nano oughness o la hs ( hi d OL) (Figu e
S2), oge he wi h he p esence o a majo i y ligh phase and a
esidual da k phase. This biphase nano oughness is ypical o
mos bioc ys als,
32,33
and he wo phases a e p esen ly
in e p e ed and c ys alline (ligh phase), and amo phous
(da k phase), he la e consis ing o amo phous calcium
ca bona e oge he wi h biomolecules.
34,35
Nano oughness
anished close o and a he e minal a owhead aces, o
c ys allog aphic easons (see nex subsec ion).
4.2. C ys allog aphy o he CCF Mic os uc u e. The
IPF maps done on bo h adial and comma ginal sec ions e eal
ha he ICF mic os uc u e has a ibe ex u e, whe eas he
Figu e 6. Views o he wo lamellae p epa ed wi h FIB o he CCF laye o P. cae ulea (a, b). The ed o als indica e he a eas om which he SAED
pa e ns (inse s) we e aken. The bounda ies be ween i s OLe ha e been depic ed wi h b oken lines (based on da k ield images, no shown).
Due o he inclina ion o he la hs (∼45°), he obse ed hickness is la ge (by ∼1.4 imes) han he ac ual hickness. The SAED pa e ns eco ded
om a and b indica e ha he la hs a e seen along he [010] and [100] di ec ions, espec i ely.
ACS Bioma e ials Science & Enginee ing pubs.acs.o g/jou nal/abseba A icle
h ps://doi.o g/10.1021/acsbioma e ials.3c00928
ACS Bioma e . Sci. Eng. 2023, 9, 6658−6669
6664
CCF mic os uc u e has a shee ex u e. The ex u e o he
CCF laye is also s onge acco ding o he MUD alues
(Figu es 3 and S2). The change om one ex u e o he o he
akes place ac oss a hick ansi ional laye .
The pole igu es ob ained on he g ow h su aces and in
sec ions o he CCF laye p o ide cong uen esul s, indica ing
ha he wo se s o i s OLe ha e he same in e nal
c ys allog aphy, al hough he e is a change in he inclina ion
o he c-axes in al e na ing i s OLe. Figu e 7a is a syn he ic
diag am o he dis ibu ion o maxima o he pole igu es in he
plane o he g ow h su ace (Figu e 4). The cen al 104
maximum indica es ha he la hs g ow wi h one {104} plane
pa allel o he g ow h su ace. This coincides wi h he
obse a ion o wide, smoo h su aces o he {104} ype
p esen bo h in he ma ginal hick la hs obse ed wi h SEM
(Figu e 2d), and in hose o he CCF laye unde AFM (Figu e
S1). The app oxima e cons ancy in he posi ion o he 104
pole igu e maximum om he ICF laye o he CCF laye
(Figu es 3 and S2), indica es ha he lamellae o he o me
also o ien a hombohed al {104} ace pa allel o he g ow h
su ace. Ne e heless, he inglike dis ibu ion o he a-axis
(110 pole igu e) in he ICF laye is consis en wi h he
obse ed diso ien a ion o lamellae (Figu e 2a−c). The
placemen o he common 110 maximum (Figu e 7a) implies
ha he e is an a-axis app oxima ely pe pendicula o he
elonga ion o he i s OL, almos coinciden ac oss he
con iguous i s OLe (Figu e 7b).
In he CCF laye , he c-axes a e con ained wi hin he planes
o he i s OLe and a e inclined in he same di ec ion as he
second and hi d OLe, al hough a a highe angle (∼45° o he
g ow h su ace), since, acco ding o ou da a, second OLe a e
inclined by only 14−21°wi h espec o he g ow h su ace in
bo h di ec ions (see abo e and Figu e 3b, bo om 001 pole
igu e). The angle be ween he g ow h su ace (assumed o be
{104} acco ding o ou c ys allog aphic da a) and he c-axis o
calci e is ∼45°( heo e ically 44.64°). Acco dingly, he angle
be ween he la h su aces and he c-axis is a ound 24°−31°
(Figu e 7c). I we plo he p o iles o he la hs on o a iew o
he calci e la ice along he a2-axis and indica e he p o iles o
he di e en hombohed al aces, he ace closes o he
obse ed ange o la h inclina ions is o he {102} ype (Figu e
7b, igh ske ch, and c).
MacClin ock
21
ound an e en highe ange o inclina ions,
be ween 3°and 27°(a e age 13°) in 10 species o Pa ella
(Figu e 7c). Assuming a ixed o ien a ion o {104} pa allel o
he g ow h su ace, clea ly he inclina ions o he second and
hi d OLe do no ollow any pa icula c ys allog aphic
di ec ion bu a e in a ange o abou 15°and 9° om {102}
owa d {104} and {101}, espec i ely (Figu e 7c). These a e
su aces cha ac e ized by a low densi y o Ca2+ and CO3
−,
which a e mos likely s abilized, al hough in an unspeci ic
manne , by biomolecules.
The la hs o he c ossed- olia ed mic os uc u e a e
homeomo phic o hose o he olia ed mic os uc u e o he
bi al e o de s Pec inida and Os eida. On he con a y, he
Figu e 7. C ys allog aphy o he CCF mic os uc u e o Pa ellidae. (a) Co espondence be ween he dis ibu ion o maxima in idealized pole
igu es ob ained on he g ow h su ace (see Figu e 4). (b) Dis ibu ion o c ys allog aphic axes and aces o hi d OLe o al e na ing i s OLe
deduced om he idealized pole igu es in (a). The lowe igh ske ch is a side iew o he la h o se 2. The {104} ace looking owa d he shell
g ow h su ace (in b oken line) is no usually exp essed. (c) P o iles o hi d OLe om di e en sou ces (colo lines) and c ys allog aphic aces,
iewed along he a2-axis.
ACS Bioma e ials Science & Enginee ing pubs.acs.o g/jou nal/abseba A icle
h ps://doi.o g/10.1021/acsbioma e ials.3c00928
ACS Bioma e . Sci. Eng. 2023, 9, 6658−6669
6665
main la h su aces a e o he 10l ype, wi h lha ing e y high
alues (be ween 10 and 15 in oys e s, and 8 and 25 in
pec inids
36,37
) and no e y low as in Pa ella. Folia ed calci e is
also ound in ina icula e b achiopods o he o de C aniida
and in s enolaema e b yozoans. In bo h g oups, he c-axis is
app oxima ely pa allel o he g ow h axis o he la hs; he
o ien a ions o he a-axes wi h espec o he main su aces o
la hs a e inconsis en in ina icula e b achiopods
38
, and a e
app oxima ely pa allel o he main su aces in s enolaema-
ans.
39
Acco dingly, he c ys allog aphy o he la hs o he
Pa ella c ossed- olia ed mic os uc u e is unique.
4.3. O ganiza ion o he C ossed-Folia ed Ma e ial.
One o he mos ema kable ea u es o he calci ic laye o
Pa ella is he p og essi e o ganiza ion o he c ossed- olia ed
ma e ial, om he ex e io o he in e io om bo h
mo phological and c ys allog aphic iewpoin s. Mo phologi-
cally, he i egula ly dis ibu ed lamellae o he ICF laye
o ganize wi h g ow h in o he concen ically a anged lamellae
o he CCF laye . A he same ime, he numbe o i s OLe
d as ically diminishes wi h g ow h, which sugges s some so o
compe i ion be ween hem. This is no su p ising, as he i s
OLe o he ICF laye can be compa ed o he olia ed g ains
ound du ing he ini ial s ages o o ma ion o he olia ed
laye s o oys e s (Figu e S5). Du ing he o ma ion o e enly
o ien ed olia ed laye s, ei he smalle o less a o ably o ien ed
g ains can be ou compe ed.
C ys allog aphically, he ou e ICF laye has a loose shee
ex u e wi h one single, hough widely sp ead, maximum o
he c-axis. Ac oss he in e media e laye , he ex u e ans o ms
in o a s ong shee ex u e, wi h wo indi idualized maxima o
he c-axis, one o each se o i s OLe (Figu es 3 and S2).
Acco ding o he pic u e p o ided by he pole igu es, his
p ocess happens h ough he p og essi e mig a ion o he
c ys allog aphic axes owa d he posi ions hey inally acqui e
in he ully de eloped CCF laye (Figu es 5 and S4). An
impo an aspec o conside is how he o ganiza ion o he
CCF akes place. One possibili y is liquid c ys alliza ion.
Almag o and co-wo ke s
40
explained he o ganiza ion o he
Figu e 8. Assimila ion o he i s OLe o he CCF laye o a zeb a pa e n and hypo hesis o i s o ma ion. (a) Compa ison o he dis ibu ion o
i s OLe on he g ow h su ace o P. us ica and ac ual zeb a skin pa e ns. The op image is a black-and-whi e image o he a ea amed in Figu e
2a. The lowe images ha e been aken om side iews o ac ual specimens o zeb a. (b) Model o he sec e ion o i s OLe. The zeb a pa e n is
de eloped on he shell- acing man le su ace (bo om pa ). The cell ex ensions a e di ec ed sidewa d owa d he ad ancing c ys als, in opposi e
di ec ions in al e na ing s ipes. The g ow h di ec ions o la hs a e guided by he opposi e lows o p ecu so pa icles owa d hei g ow h su aces.
The whi e polygons ep esen he ou lines o he man le cells. (c) Two TEM images o he man le epi helium o P. cae ulea a di e en
magni ica ions, showing he cell mic o illi. They seem o be discha ging esicles o he ex apallial space. A ows poin o in e cellula bounda ies.
ACS Bioma e ials Science & Enginee ing pubs.acs.o g/jou nal/abseba A icle
h ps://doi.o g/10.1021/acsbioma e ials.3c00928
ACS Bioma e . Sci. Eng. 2023, 9, 6658−6669
6666