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h p://dx.doi.o g/10.1016/j.jmbbm.2013.07.011
h p://hdl.handle.ne /10251/64195
Else ie
Panade o, JA.; Vikingsson, LKA.; Gómez Ribelles, JL.; Sencadas, VJGDS.; Lance os-
Mendez, S. (2013). Fa igue p edic ion in ib in poly-epsilon-cap olac one mac opo ous
sca olds. Jou nal o he Mechanical Beha io o Biomedical Ma e ials. 28:55-61.
doi:10.1016/j.jmbbm.2013.07.011
h p://www.sciencedi ec .com/science/a icle/pii/S1751616113002427
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Fa igue P edic ion on Fib in Poly-ε-cap olac one Mac opo ous
Sca olds
J. A. Panade o1, 2, L. Vikingsson2, J. L. Gomez Ribelles2,3, V. Sencadas1,4, S. Lance os-Mendez1
1 Cen o/Depa amen o de Física da Uni e sidade do Minho, Campus de Gual a , 4710-057
B aga, Po ugal.
2 Cen e o Bioma e ials and Tissue Enginee ing, Uni e si a Poli ècnica de València, Camino
de Ve a s/n, 46022 Valencia, Spain
3 Cibe en Bioingenie ía, Bioma e iales y Nanomedicina (CIBER-BBN), Valencia, Spain
4 Escola Supe io de Tecnologia, Ins i u o Poli écnico do Cá ado e do A e, Campus do IPCA,
4750-810, Ba celos, Po ugal.
e-mail: lance os@ isica.uminho.p
Keywo ds: polycap olac one, sca old, ib in, a igue es ing.
Abs ac
Tissue enginee ing applica ions ely on sca olds ha du ing i s se ice li e, ei he o in- i o o
in i o applica ions, a e unde mechanical solici a ions. The a ia ion o he mechanical
condi ion o he sca old is s ongly ele an o cell cul u e and has been sca cely add essed.
Fa igue li e cycle o poly-ε-cap olac one, PCL, sca olds wi h and wi hou ib in as ille o he
po e s uc u e we e cha ac e ized bo h d y and imme sed in liquid wa e . I is obse ed ha he
he e is a s ong inc ease om 100 o 500 in he numbe o loading cycles be o e collapse in he
samples es ed in imme sed condi ions due o he mo e uni o m s ess dis ibu ions wi hin he
samples, he ib in loading playing a mino ole in he mechanical pe o mance o he sca olds.
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In oduc ion
Tissue enginee ing has a isen as a he apy o egene a ing damaged o diseased issues. The
mos common s a egy elies in he use o h ee-dimensional (3D) sca olds, in combina ion wi h
a cell sou ce and signaling ac o s (Ne em, 2007). The sca old should p o ide he a chi ec u e
o guide new issue o ma ion, allowing cell-cell and cell-ma ix in e ac ions (Chen e al., 2013).
In o de o achie e his pu pose sca old mo phology should ha e a geome y o in e connec ed
po es o allow cell seeding, p oli e a ion, ex acellula ma ix (ECM) o ma ion, di usion o
physiological nu ien s and emo al o me abolic was e p oduc s (Mikos e al., 1993).
Sca old mechanical p ope ies a e an impo an issue conce ning issue and biomedical
enginee ing, as in addi ion o p o ec he cells when hei ex acellula ma ix is s ill no
de eloped, hey egula e he biomechanical en i onmen ha o e s s imula o y cues p o iding a
be e in eg a ion wi h he su ounding issue. In pa icula in he egene a ion o musculoskele al
issues, like ca ilage and bone, suppo ed loads p o ide cues o he igh gene exp ession and
syn hesis o ECM (Huang e al., 2010b; Kelly and Jacobs, 2010; McCullen e al., 2010; Riehl e
al., 2012).
Polyme ma e ials wi h a wide ange o mechanical s i ness and iscoelas ic p ope ies can be
p epa ed and polyme sca olds can be designed o ma ch he mechanical p ope ies o li ing
musculoskele al issues (Puppi e al., 2010). The e olu ion o mechanical p ope ies o sca old-
cells cul u e du ing cul u e, due o he o ma ion o syn he ized ECM is an impo an pa ame e
cha ac e izing cell g ow h and di e en ia ion (Kha iwala e al., 2006). The igh p oduc ion o
ECM in i o migh equi e o pe o m cul u e unde dynamic loading condi ions. This has been
pe o med by he de elopmen o a b oad ple ho a o bio eac o s which induce mechanical
s imulus in di e en ways, such as con ined o uncon ined comp ession and hyd os a ic p essu e,
among o he s (Knech e al., 2006; Schulz and Bade , 2007; Wong M Fau - Ca e and Ca e ,
2003). I was ound ha cyclic applica ions o hese s imuli gene ally p oduce highe
di e en ia ion esponses due o a cascade o signaling e en s ha has been called
mechano ansduc ion (Chen e al., 2009; De C oos e al., 2006; Huang e al., 2010a;
Mahmoudi a and Do an, 2010).
P edic ion o he mechanical beha io , ul ima e p ope ies and a igue esis ance o a polyme
sca olds implan ed in he hos issue is an impo an issue ha has been add essed insu icien ly
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in he li e a u e. The p ope ies o he d y sca old a e no ep esen a i e o ha o he sca old
wi h he po es illed by a g owing issue o simply by a luid. In d y sca olds, mechanical
p ope ies mainly depend on i s inne mo phology, in pa icula po e size, geome y and
in e connec i i y (Spille e al., 2008). Howe e , in he sca old imme sed in a liquid medium
o he ac o s can in luence he mechanical p ope ies as well, such as he hyd odynamics and
pe meabili y inside he sca old. Since comp essibili y o wa e is low, any ac o limi ing wa e
pe mea ion h ough he ma e ial is expec ed o inc ease appa en sca old s i ness, in pa icula
unde cyclic loading, which means ha he in luence o liquid media, such as cell cul u e
medium canno be dis ega ded. Ne e heless, o ou knowledge, only ew in es iga ions deal
wi h he desc ip ion o he mechanical beha io o polyme sca olds unde liquid en i onmen
(Blasi e al., 2005; Hu mache e al., 2001) and none unde cyclic loading in aqueous media.
The esis ance o he ma e ial o mechanical a igue can be in luenced by se e al ac o s such as
mechanical loading his o y, en i onmen al condi ions, polyme composi ion and o ce ain
aspec s o s ess-s ain cons i u i e beha io (Ma s, 2004). Se e al ma hema ical models i s ly
de eloped o p edic me allic ma e ials and composi es du ing load- eco e y cycles such as
Co fin–Manson, Smi h-Wa son-Toppe (SWT) o Mo ow models (Ince and Glinka, 2011).
Co in-Manson model is based on he plas ic s ain ange measu ed by sub ac ing he elas ic
s ain ange om he o al s ain ange om he middle o he mechanical hys e ics loop ( ),
while SWT model assumes ha he a igue li e cycle o any si ua ion o mean s ess depends on
he p oduc o he maximum s ess ( ) and (Bou ago e al., 2011). Mo eo e , Mo ow
de eloped a model o p edic a igue li e cycle o me als based on he plas ic s ain ene gy
densi y ha can be physically in e p e ed as he dis o ion ene gy associa ed o he change in
shape o a olume elemen and can be ela ed o ailu e, in pa icula unde condi ions o duc ile
beha io (Mo ow, 1965).
Two ailu e egimes can be iden i ied in he moplas ic ma e ials: he low egime cycle, whe e he
ma e ial ails a e a low numbe o cycles and is he main ailu e mechanism is hea gene a e
du ing he load- eco e y cycle and is called he he mally domina ed domain. The second egime
occu s a high s ess and he high cycle egime a low s ess, whe e he polyme can hold ou a
la ge numbe o cycles be o e ailu e. In his egime, he obse ed ma e ials ailu e is b i le o
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na u e and only li le ene gy dissipa ion is ound in he hys e esis loops and is called
mechanically domina ed domain (Janssen e al., 2008).
Glassy polyme s such polyca bona e, poly(me hyl me hac yla e) o poly( inyl chlo ide) as well
as semic ys alline polyme s like poly( e a luo e hylene), poly(oxyme hylene) o high densi y
polyp opylene, has been epo ed ha ailu e is due o he he mally domina ed domain (Janssen
e al., 2008). Fu e al. (Fu e al., 2013) epo ed ha comp essi e s eng h o bioac i e sca olds
dec eases wi h he po osi y inc ease p esen in he samples. Mo eo e , a igue pe o mance o
bo ine bone du ing cyclic loading esul s in a educ ion o he elas ic modulus and accumula ion
o esidual s ain leading o a p og essi e educ ion o a igue li e wi h inc easing s ess le els
(Ganguly e al., 2004).
Sca old es ing in condi ions ha simula e in some ex en he si ua ion du ing cell cul u e seems
he e o e o be an impo an issue o a co ec in e p e a ion o s ess ansmission o he cells
cul u ed in bio eac o s.
In i o ECM p o ides gene ally mechanical esis ance by he capabili y o ECM componen s o
e aining wa e , a icula ca ilage being a good example (Schulz and Bade , 2007). This
phenomenon will appea as well in cells cul u ed in i o inside he po es o he sca old, seeded
alone o encapsula ed in ib in, collagen and o he s (Lee and Mooney, 2001). In he p esen
wo k, a igue li e cycle o a poly-ε-cap olac one, PCL, sca old wi h and wi hou ib in as ille
o he po e s uc u e was cha ac e ized bo h d y and imme sed in liquid wa e ; in o de o
p o ide some insigh in he a igue beha io o he sca olds o applica ion in cell cul u es unde
dynamic loading.
Ma e ials and me hods
Ma e ials: Poly-ε-cap olac one (PCL, molecula weigh o 43-50 kDa) and dioxan we e
pu chased om Sigma-Ald ich. Poly(e hyl me hac yla e) (PEMA - El aci e 2043) in he shape
o sphe es wi h mean diame e o 200 m was pu chased om Luci e. Fib inogen om human
plasma 50-70% p o ein (≥80% o p o ein is clo able) and h ombin om human
plasma lyophilized powde , ≥2,000 NIH uni s/mg p o ein (E1%/280, 18.3) we e pu chased om
Sigma-Ald ich and glu a aldehyde (50 % H2O) was pu chased om Pan eac.

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Sample p epa a ion: PCL was dissol ed in dioxan (25% w/ ) and his solu ion was mixed wi h
PEMA mic osphe es (1:1 w/w). Then, he mix u e was placed in Te lon Pe i dishes and
subme ged in liquid ni ogen o a minu e. Dioxan was ex ac ed om he ozen pla es wi h
e hanol a - 20 ⁰C o h ee days, changing e hanol e e y day. Po ogen leaching was pe o med
in e hanol a 40 ⁰C o one day. The po ous samples we e cu in o cylinde s wi h 6 mm diame e
and a hickness o app oxima ely o 2 mm. To achie e a comple e emo al o he po ogen,
u he leaching o each cylinde was pe o med in e hanol a 40 ºC o nine mo e days, wi h
daily change o e hanol.
A ib inogen solu ion wi h concen a ion o 20 μg/ml in saline solu ion and a h ombin solu ion
o 10 U/ml in TBS wi h 20 mM o CaCl₂ we e p epa ed. Two ch oma og aphy sy inges wi h
be eled needles we e p epa ed con aining 20 μl o ib inogen o h ombin solu ions each.
Finally, bo h solu ions we e injec ed in he PCL sca old and ib in was allowed o coagula e o
1 h a 37 ⁰C and subsequen ly kep in dis illed wa e o no mo e han 24 hou s be o e he es s.
Cha ac e iza ion: Sample o e all po osi y,

, was calcula ed by weighing he sca olds a e
comple e illing he po es wi h e hanol. The sample was sealed in a con aine unde high acuum
in which e hanol was injec ed. Po osi y was de e mined by:
(1)
whe e is he d y weigh o he sample, he weigh wi h he sample illed in e hanol,
he olume o po es, he olume occupied by he polyme and g/cm3 and
g/cm3 a e he densi ies o e hanol and PCL a oom empe a u e. Po osi y alues
a e he a e age o 5 measu emen s.
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Sample mic os uc u e was assessed by scanning elec on mic oscopy. PCL illed wi h ib in was
ixed wi h a 2.5% glu a aldehyde (GA) solu ion o 1 h a 4 ⁰C. C yoSEM was pe o med in a
JEOL JSM-5410 equipmen . Wa e was sublima ed a 5 kV, -50 ⁰C o 20 min, and ca bon-
spu e ed inside SEM chambe be o e analyses. PCL p is ine samples we e coa ed wi h a hin
gold laye using a spu e coa ing (Pola on, model SC502) and hei mo phology was analyzed
using a o emen ioned equipmen .
Mechanical expe imen s we e pe o med on cylind ical samples wi h 6 mm diame e and a
heigh o ~2 mm in a Shimadzu AG-IS uni e sal es ing machine in comp ession mode a a es
eloci y o 1 mm.min-1 and a oom empe a u e (~25 ºC). In a igue expe imen , samples we e
submi ed a a comp essi e-s ain cycle load up o 1000 cycles (o un il ma e ial eaches he
plas ic pla eau) a a s ain o 15%. S ain de o ma ion was measu ed by machine c oss-head
displacemen and mechanical s ess and s ain pa ame e s we e ob ained on an a e age o i e
measu emen s. The mechanical expe imen s we e pe o med in d y PCL samples and in PCL and
PCL illed wi h ib in samples imme sed in deionized wa e . Samples up ake was pe o med
h ough he injec ion o wa e wi h he help o a ch oma og aphy sy inge wi h be eled needles,
in a simila p ocess o ib in illing desc ibed abo e. In o de o ensu e he maximum up ake,
samples we e imme sed in a wa e ba h and placed in chambe (Vacuum-Temp om Selec a)
unde acuum condi ions (10-2 mmHg) un il he sample d ops o he bo om o he ba h, and only
hese samples we e submi ed o he mechanical expe imen s.
Resul s and Discussion
The po e a chi ec u e o he PCL sca olds consis s in a double po osi y: mac opo es ob ained
wi h he po ogen sphe es wi h diame e s in he o de o 200 mic ons and po es in he o de o
ew mic ons p oduced by he eeze ex ac ion echnique (Figu e 1) ha in e connec he bigge
ones. This double po e s uc u e allows p oducing sca olds wi h qui e high po osi y ha ha e
been p e iously p oposed o ca ilage and bone enginee ing (Gamboa-Ma ínez e al., 2011; Izal
e al., 2012; San ama ía e al., 2012). Mic opo osi y a o s pe meabili y o he sca old o
nu ien s and was e p oduc s o cell me abolism and can be used o e ain di e en ac i e
componen s (Deplaine e al., 2010; Lebou g e al., 2010a; Lebou g e al., 2010b). Ne e heless,
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he appa en s i ness o he sca old becomes signi ican ly smalle han in simila sponges
lacking mic opo osi y, also used in ca ilage enginee ing (Ma inez-Diaz e al., 2010; Olmedilla
e al., 2012).
Figu e 1 –
These samples wi h and wi hou ib in we e submi ed o cyclic mechanical loading unde
comp essi e mode up o a 15% s ain, which is a ypical de o ma ion in expe imen s wi h
bio eac o s o chond ogenic di e en ia ion (Appelman e al., 2009; Michalopoulos e al., 2012;
Nicodemus and B yan , 2008). S ess-s ain cu es in 10 consecu i e loading-unloading cycles
a e shown in Figu e 2a o he d y sample. The di e ence be ween he i s and second cycle
indica es ha some o he PCL abeculae su e pe manen de o ma ion du ing he i s
comp ession amp. In successi e cycles, he con inuous inc ease o i e e sible p ocesses leading
o pe manen de o ma ion is e lec ed in a sligh dec ease o he maximum s ess eached in each
s ess-s ain loop (Figu e 2c) and in he con inuous na owing o he mechanical hys e esis cycle.
I is also wo h no icing ha d y sample mic os uc u e su e s sligh ly changes a e a ew load-
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eco e cycles, being he po ous s uc u e mo e compac ( igu e 1a and b) and in he case o he
PCL illed wi h ib in and measu ed imme sed in wa e , he ib in ib illa s uc u e is des oyed
( igu e 1 c and d). Mac opo es pa ially collapse al hough an in e connec ed po e s uc u e s ill
emains, wi h he open mac opo es p esen ing a mo e o al shape. The mic opo e s uc u e, on
he o he hand, do no seems o be a ec ed (see he inse in Figu e 2b) in his si ua ion.
PCL is qui e hyd ophobous wi h a con ac angle o ~ 92º (Li le e al., 2009) and illing he po es
o he sponge wi h wa e equi es he applica ion o high acuum o ex ac comple ely ai om
he mic opo es be o e in oducing wa e . Simul aneous injec ion o ib inogen and h ombin
solu ions in o he po es and u he coagula ion allows p oducing a ib in gel homogeneously
dis ibu ed in he whole po e olume. This gel has a nano ib illa s uc u e, as obse ed in
C yoSEM (Figu e 2c) a e sublima ing he wa e om he sample. Filling he po es wi h ib in
acili a es wa e di usion h ough he sca old.
The mechanical beha io o he samples imme sed in wa e is qui e di e en o he one ob ained
o d y PCL sca olds. D y samples has a maximum s ess almos wo imes highe han
imme sed ones, his beha io is p obably ela ed o he small amoun o wa e abso bed by he
PCL polyme (Olmedilla e al., 2012) ha consequen ly ac as plas icize , con ibu ing o
ma e ial so ening ( igu e 2c).
The shape o he s ess-s ain plo e eals a la ge cu a u e o he eco e y da a up o ze o s ain
wi h espec o he d y sample, indica ing a lowe pe manen de o ma ion and lowe con ibu ion
o iscoelas ic e ec ( igu e 2 a and b). This ac is suppo ed by he SEM images shown in
Figu e 1d, p esen ing he mac opo es nea ly una ec ed by he mechanical comp ession
expe imen . A e 10 loading-unloading cycles i was obse ed ha he ib in s a s o lose i s
cha ac e is ic ib illa s uc u e, sugges ing a weak bonding be ween he ib in and he PCL
sca old (Figu e 1d). The small bu con inuous dec ease o he maximum s ess in he successi e
load- eco e y cycles is also appa en in he samples es ed in imme sion (Figu e 2c).
Figu e 2 –
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