A Self-Powered Piezo-Bioelectric Device Regulates Tendon Repair-Associated Signaling Pathways through Modulation of Mechanosensitive Ion Channels

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ReseaRch aR icle
A Sel -Powe ed Piezo-Bioelec ic De ice Regula es Tendon
Repai -Associa ed Signaling Pa hways h ough Modula ion
o Mechanosensi i e Ion Channels
Ma c A. Fe nandez-Yague,* Alexand e T o ie , Secil Demi , Sunny Akogwu Abbah,
Ai o La añaga, A un Thi uma an, Aimee S aple on, Syed A. M. To ail, Ma eo Palma,
Michelle Kilcoyne, Abhay Pandi , and Manus J. Biggs*
D . M. A. Fe nandez-Yague, A. T o ie , S. Demi , D . S. A. Abbah,
D . A. La añaga, D . A. Thi uma an, P o . M. Kilcoyne, P o . A. Pandi ,
P o . M. J. Biggs
CÚRAM SFI Resea ch Cen e o Medical De ices
Na ional Uni e si y o I eland
Galway H91W2TY, I eland
E-mail: [email p o ec ed]; [email protected]
DOI: 10.1002/adma.202008788
place a conside able bu den on heal h-
ca e sys ems (> $2 billion annually, and
pos -su ge y complica ions esul in nea ly
1 million addi ional days o inpa ien ca e
each yea ).[1–3] Su gical in e en ion ia
di ec end- o-end epai using su u es
and biological o syn he ic g a s ep e-
sen s he gold s anda d in ea men , and
despi e he ela i e success, hese epai s
equen ly ail o es o e ull endon unc-
ionali y. Following inju y, diso ganized
issue deposi ion leads o sca issue o -
ma ion, p o eoglycan accumula ion, and
calci ica ion, esul ing in poo biomechan-
ical p ope ies and impai ed unc ion ha
igge s ch onic in lamma o y signaling
pa hways and p og esses in o endinop-
a hy. Hence, o achie e long- e m epai ,
inno a i e unc ional solu ions ha ocus
on he ac i a ion o endogenous issue-
epai signaling pa hways ep esen s a
pa adigm shi in he ield o biomedical
de ices and egene a i e medicine (RM).[4]
Many s udies con i m ha esiden
endon cell popula ions a e highly mecha-
nosensi i e and a e esponsible o o ches-
a ing he epai p ocesses a e inju y h ough specialized sen-
so y machine y, including mechanosensi i e ion channels.[5–8]
C i ically, mechano he apy (i.e., low-le el exe cise o ex a-
co po eal shock wa es) has been epo ed o p omo e endon
Tendon disease cons i u es an unme clinical need and emains a c i ical
challenge in he ield o o hopaedic su ge y. Inno a i e solu ions a e equi ed
o o e come he limi a ions o cu en endon g a ing app oaches, and
bioelec onic he apies show p omise in ea ing musculoskele al diseases,
accele a ing unc ional eco e y h ough he ac i a ion o issue egene a ion-
speci ic signaling pa hways. Sel -powe ed bioelec onic de ices, pa icula ly
piezoelec ic ma e ials, ep esen a pa adigm shi in biomedicine, nega ing
he need o ba e y o ex e nal powe ing and complemen ing exis ing mecha-
no he apy o accele a e he epai p ocesses. He e, he dynamic esponse
o endon cells o a piezoelec ic collagen-analogue sca old comp ised o
aligned nanoscale ibe s made o he e oelec ic ma e ial poly( inylidene
luo ide-co- i luo oe hylene) is shown. I is demons a ed ha mo ion-
powe ed elec omechanical s imula ion o endon issue h ough piezo-
bioelec ic de ice esul s in ion channel modula ion in i o and egula es
speci ic issue egene a ion signaling pa hways. Finally, he po en ial o he
piezo-bioelec onic de ice in modula ing he p og ession o endinopa hy-
associa ed p ocesses in i o, using a a Achilles acu e inju y model is
shown. This s udy indica es ha elec omechanical s imula ion egula es
mechanosensi i e ion channel sensi i i y and p omo es endon-speci ic o e
non- enogenic issue epai p ocesses.
The ORCID iden i ica ion numbe (s) o he au ho (s) o his a icle
can be ound unde h ps://doi.o g/10.1002/adma.202008788.
© 2021 The Au ho s. Ad anced Ma e ials published by Wiley-VCH
GmbH. This is an open access a icle unde he e ms o he C ea i e
Commons A ibu ion License, which pe mi s use, dis ibu ion and e-
p oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
D . A. La añaga
Uni e si y o he Basque Coun y
Depa men o Mining-Me allu gy Enginee ing and Ma e ials Science
and POLYMAT
Ba io Sa iena
Bilbao 48013, Spain
D . A. S aple on, P o . S. A. M. To ail
Uni e si y o Lime ick
Depa men o Physics
Lime ick V94 T9PX, I eland
P o . M. Palma
Queen Ma y Uni e si y o London
Ma e ials Resea ch Ins i u e and School o Biological and Chemical
Sciences
Mile End Road, London E1 4NS, UK
1. In oduc ion
Se e e endon inju ies esul ing om a hle ic o epe i i e
ac i i y a ec mo e han 102.5 million adul s e e y yea and
Ad . Ma e . 2021, 33, 2008788
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egene a ion and p o ide a eliable ou e o app op ia e pos -
ope a i e managemen .[9] Addi ionally, piezoelec ici y-de i ed
elec ic ields p oduced du ing physiological locomo ion may
p o ide addi ional bioelec ic signaling cues o ac i a e endon-
speci ic egene a i e pa hways (Figu e1A). Se e al ecen
s udies ha e e ealed he signi ican po en ial o elec ical ields
in media ing cell mig a ion, p omo ing collagen syn hesis, and
inducing success ul wound healing by ex e nally applied di ec
cu en elec ical s imula ion (ES).[10,11] In he las decade, DC
s imula o s (i.e., Zimme Biome SpF and Os eoGen) ha e
imp o ed he success a e o spinal usion p ocedu es and non-
healing issue inju ies.[12] Despi e he p o en clinical e ec ,
a signi ican oadblock o wide-scale clinical adop ion o ES
is he in ec ion isk o pene a ing elec odes, po en ial non-
speci ic o - a ge e ec s, and he cumbe some design o ES
uni s, making pa ien compliance a conce n.[13] The need o
mo e sa e, e icien , and less in asi e elec ically s imula ing
sys ems d i es he de elopmen o no el bioelec ic s a egies
ha con ol soma ic cell unc ions and enhance speci ic issue
egene a i e p ocesses.[14]
The disco e y o piezoelec ici y in bone and mo e ecen ly
in i s cons i uen collagen ype I, has spu ed new esea ch
in o he ole o bioelec ici y in issue egene a ion and he
de elopmen o sel -powe ed elec ical s imula ion echnolo-
gies.[15,16] A p omising s a egy o de eloping biomime ic elec-
omechanical s imula ion (EMS) de ices has been enabled
by syn hesizing complian e oelec ic polyme s (i.e., PVDF-
T FE), con o med in o a nano ib ous sca old ha mimics he
in insic elec ical, mechanical, and mo phological p ope ies
o collagen ype I (Figu e 1B). Mechanical ac ua ion o hese
s uc u es o piezo-bioelec ic de ices h ough epe i i e physi-
ological loading and unloading (i.e., mono onic s e ching)
can p oduce biologically ele an elec ical ields, enabling
s udies in o he ole o “mechanically-induced” elec ical cues
on musculoskele al issue unc ion. Signi ican e o s om
he biomedical communi y ha e demons a ed he po en ial o
Ad . Ma e . 2021, 33, 2008788
Figu e 1. O e iew o he endon elec omechanical en i onmen and he piezoelec ici y o igin analogy o collagen and PVDF-T FE ibe s. Tendon is a
dynamic issue ha connec s muscle o bone and is unde con inuous mechanical loading and unloading. A) This mechanical s ess is bo ne by a highly
aniso opic ex acellula ma ix composed o collagen ype I, a high- ensile, piezoelec ic ma e ial which unde goes elec ical pola iza ion in esponse o
mechanical loading. B) Schema ic ep esen a ion o he c ys al s uc u e o he collagen iple helix consis ing o h ee polypep ides chains s abilized ia
hyd ogen bonding. A-A: C oss-sec ional schema ic o an indi idual alpha-chain showing he iple Gly (glycine)-X (p oline)-Y (hyd oxyp oline) esiduesi
(i). Pola bonds be ween ca bonyl (CO g oups) and amine (pep ide NH g oup o glycine esidues) g oups along he backbone o collagen esul in a
dipole momen o elec ical pola iza ion (ii). C oss-sec ion o mul iple indi idual collagen molecules esul ing in a non-cen osymme ic hexagonal
ib il a angemen (iii). B-B: A c oss-sec ion o he collagen iple-helix co e; he e, he h ee helical chains and glycine esidues a e obse able (i ).
Rep esen a ion o an indi idual elec ic dipole ( ). C) Rep esen a ion o he PVDF-T FE all- ans (TTTT) zigzag plana con igu a ion op iew ( i). C oss-
sec ion o he c ys al and along he o hogonal axis o he all- ans (TTTT) zigzag plana con igu a ion ( ii). Rep esen a ion o an indi idual elec ic
dipole composed o luo ine (g een sphe e), ca bon (blue sphe e), and hyd ogen (o ange sphe e) a oms ( iii). PVDF-T FE has a non-cen osymme ic
s uc u e. Dipoles a e gene a ed by he highly elec onega i e di e ence be ween hyd ogen and luo ine a oms. Rep esen a ion o he molecula chain
s uc u e o he elec oac i e β-phase o PVDF-T FE showing a esul an dipole momen (ix).
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piezoelec ic ma e ials o ansduce mechanical in o physiologi-
cally ele an elec ical cues (di ec piezoelec ici y) and a ec
di e en cell ypes, including os eoblas s, mesenchymal s em
cells, and neu ons.[17–20] In his s udy, we hypo hesized ha a
e oelec ic sca old could se e as mechanical suppo o he
egene a ion o damaged endon issues and mimic he bio-
elec ical cues usually p o ided by collagen’s piezoelec ici y o
main ain endon-cell pheno ype and p omo e endon egene a-
ion (Figu e1B,C).
2. Design and Fab ica ion o a Piezo-Bioelec ic
De ice
The physical p ope ies o a sca old, including i s mechanical
beha io , mic os uc u e ( ibe diame e and alignmen ), and
c ys allini y-dependen piezoelec ic esponse, play a pi o al
ole in egula ing he cellula e en s in ol ed in issue egen-
e a ion.[21] To de elop a collagen piezoelec ical-analog de ice
ha can ecapi ula e he ib ous s uc u e o ex acellula
endon issue an elec ospinning p ocess was implemen ed,
and nanoscale ibe s wi h a ying diame e and alignmen we e
p oduced h ough op imiza ion o solu ion and p ocessing
pa ame e s (Figu e2).
Fi s , we analyzed he impac o ibe diame e on he bio-
physical p ope ies o he sca olds. By a ying he concen a-
ion o he polyme ic solu ion (1.1, 1.2, 1.3, 1.4, 1.5, 1.6, and
1.7 mg mL−1 in dime hylace amide (DMAc) we could ob ain
ibe s wi h dis inc diame e s. We used a 30 kV po en ial di -
e ence be ween collec o and nozzle ip (Ø = 200µm), a low
a e o 1mL h−1, and a ip- o-collec o dis ance o 6cm (sho -
dis ance elec ospinning). Impo an ly, a collec o disk wi h an
8mm wid h was used o concen a e he elec ical ield while
o a ing a 29.3 m s−1 (linea speed) o d aw and align he col-
lec ed ibe s. The esul ing ibe s anged om 180 o 540nm
in diame e (Figu e 2A; Figu e S1, Suppo ing In o ma ion),
which is simila o he diame e o collagen ibe s in endon
issue 50–500nm.[22]
I was obse ed ha he ibe diame e go e ned he s i ness
o he esul ing sca olds and cons uc s wi h lowe ibe dia-
me e s possessed an inc eased Young’s modulus, as de e mined
by ensile- es analysis (Figu e2A). The obse ed enhancemen o
he mechanical p ope ies o sca olds wi h dec easing ibe diam-
e e was a esul o a high le el o chain ex ension and o ien a-
ion along he ibe axis as a consequence o ibe s being d awn
by cen ipe al o ces du ing ibe collec ion. Wi h semi-c ys alline
polyme s (including poly( inylidene luo ide-co- i luo oe hylene)
P(VDF-T FE)), i has been demons a ed ha s ess ha dening
does no depend on polyme c ys allini y bu a he , on he den-
si y o amo phous polyme chain en anglemen , which in espec
o elec ospinning, is ela ed o he dis ance be ween he needle
and collec o , he collec o o a ional speed and he polyme solu-
ion concen a ion.[23] As a esul , sca olds wi h a ibe diame e
be ween 390 and 540 nm (Figu e S1>D,E, Suppo ing In o -
ma ion) demons a ed inc eased ibe alignmen . Finally,
nano ibe s wi h diame e s <240 nm (Figu e S1F, Suppo ing
Ad . Ma e . 2021, 33, 2008788
Figu e 2. Cold d awing imp o es ibe alignmen and enhances elec oac i e β-phase o ma ion. A) Analysis o he ela ionship be ween ibe diame e
and he ela i e Young’s modulus (E el) indica ed ha he mechanical beha io o highly aligned sca olds was in e sely p opo ional o ibe diame e .
B) The mo phology o sca olds ob ained by elec ospinning collec ed a low (4.2 m s−1 pm) and high (29.3 m s−1) linea speeds. Fas ou ie ans o m
(FFT) spec a showed a b oad dis ibu ion o in ensi ies o low speed (4.2 m s−1) and a clea peak o high speed (29.3 m s−1) cha ac e is ic o highly
aniso opic s uc u es. Fibe alignmen was signi ican ly inc eased, and he mo phology mimicked ha o endon collagen. C) To al c ys allini y and
β-phase con en (calcula ed om FTIR and XRD spec a) inc eased as a unc ion o collec o dis ance and ollowing cold d awing. D) C eep/s ess
elaxa ion was minimized a e cold d awing and esul ed in educed ibe diame e and inc eased ibe alignmen . SEM images o sca olds wi h and
wi hou cold d awing.
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In o ma ion) we e p one o alignmen diso ganiza ion due o
he ai cu en s gene a ed du ing collec ion.
To decouple he e ec o piezoelec ic s imula ion om
mechanical loading on he cellula esponse, a non-piezoelec-
ic PTFE sca old which main ained he opog aphy (i.e., ibe
diame e and o ganiza ion) and was chemically analogous (i.e.,
a luo ina ed polyme ) o PVDF-T FE was also ab ica ed. PTFE
is non-piezoelec ic due o he p esence o a cen osymme ic
s uc u e wi h s ong pola CF co alen bonds (C∂+—F∂−).
Con e sely, PVDF-T FE p esen s non-cen osymme ic elec-
ical dipoles, gene a ed by he high elec onega i e di e ence
be ween hyd ogen and luo ine a oms (Figu e S2, Suppo ing
In o ma ion). The di ec elec ospinning o PTFE ibe s, how-
e e , was no possible due o he unique chemical p ope ies
o PTFE. The e o e, a wo-s ep sho -dis ance elec ospinning
p ocess was op imized o ob ain sca olds o PTFE ibe s.[24]
The endon mic os uc u e is highly hie a chical, wi h col-
lagen ibe s aligning o he di ec ion o mechanical loading.
To ep oduce his c i ical ea u e in bo h PVDF-T FE and con-
ol PTFE sca olds he collec o eloci y was adjus ed be ween
4.2–29.3 m s−1, and he concen a ion o he polyme ic solu ion
ixed a 1.7 mg mL−1. As e iden in Figu e 2B, he sca olds
ob ained a a collec o linea eloci y o 29.3 m s−1 (≈4000 pm)
displayed a highly o ganized ibe mo phology, while a b oad
dis ibu ion o ibe o ien a ion was obse ed o sca olds o -
mula ed wi h a collec o linea eloci y o 4.2 m s−1 (500 pm).
Addi ionally, as de e mined by X- ay di ac ome y (Figu e S3,
Suppo ing In o ma ion and Table S1), he deg ee o c ys al-
lini y inc eased om 40% in andomly aligned PVDF-T FE
ibe s o 49% in aligned ibe s (Figu e 2C), sugges ing ha
mechanical d awing du ing ibe collec ion a high o a ional
speeds esul ed in PVDF-T FE ibe s wi h a educed diame e
(590 ± 130nm o 4.2 m s−1 and 540 ± 120nm o 29.3 m s−1)
and enhanced c ys allini y (Table S1, Suppo ing In o ma ion).
Ou sho -dis ance elec ospinning con igu a ion con as ed o
p e iously epo ed elec ospinning con igu a ions o PVDF-
T FE (i.e., long-dis ance elec ospinning).[25] We ha e demon-
s a ed ha sca olds p oduced using a sho -dis ance con igu-
a ion possessed inc eased ibe o ganiza ion (Figu e S1, Sup-
po ing In o ma ion) and c ys allini y (Figu e2C), suppo ing
he hypo hesis ha ibe s a e signi ican ly d awn when sub-
jec ed o an elec ic- ield inc eased in ensi y. The mechanical
beha io o he sca olds ob ained by he sho -dis ance con-
igu a ion was imp o ed compa ed o sca olds ob ained by a
con en ional (long-dis ance) con igu a ion in e ms o elas ic
modulus, c eep esis ance, and yield s eng h (Figu e S2,
Suppo ing In o ma ion), which is o i al impo ance o he
de ice o e ain he in eg i y o i s mac o and mic os uc u e
unde loading ollowing implan a ion.[26]
Rega ding he piezoelec ic esponse, a g owing body o
esea ch sugges s ha he piezoelec ic pe o mance o PVDF
(i.e., he base comonome o he PVDF-T FE copolyme )
is a ec ed by he deg ee o c ys allini y and in pa icula he
β-phase con en (Table S2, Suppo ing In o ma ion).[27] As
de e mined by Fou ie - ans o m in a ed spec oscopy (FTIR)
(Table S2, Suppo ing In o ma ion), he sho -dis ance con igu-
a ion p omo ed he c ys alliza ion o he elec oac i e β-phase
(Figu e2C). O e all, hese obse a ions ein o ce he a gumen
o he po en ial use o sho -dis ance elec ospinning in he
ab ica ion o PVDF-T FE-based sca olds.
To u he inc ease he mechanical p ope ies and β-phase
con en o he sca olds, while a oiding he well- epo ed
c imping o he ibe s unde epe i i e mechanical loading, wo
pos -syn hesis p ocesses we e in es iga ed: i) s ain-ha dening
(cold-d awing) and ii) he mal annealing (cold c ys alliza ion).
Based on he ini ial p ope ies o he sca olds, we explo ed cold
d awing a ≈12% o induce s ain-ha dening h ough plas ic
de o ma ion o he s uc u es. O e all, his p ocess esul ed
in ≈8% plas ic de o ma ion, alignmen o ibe s ( om 65%
o 98%) and educ ion in indi idual ibe diame e ( om 540
o 516 nm). Impo an ly, ibe diame e educ ion and be e
ibe alignmen esul ed in sca olds wi h an imp o ed elas ic
modulus (61.8± 5.1MPa, p< 0.05) and s eng h (31± 4.2 Mpa,
p< 0.001) (Table1). We conduc ed ex ensi e SEM inspec ions
along he en i e ob ained sca olds (0.8 × 30cm) o check he
ibe di ec ion. We obse ed ha ibe s we e aligned wi h he
collec o o a ing di ec ion and pa allel o he longi udinal axis
o he sca old. Only a ew ibe s a om he middle pa o he
sca old p esen ed a skewed o ien a ion (<7± 6°) and we e co -
ec ed be o e implan a ion o he de ices du ing he subsequen
cold d awing p ocess.Concu en ly, he s ess elaxa ion/c eep
o he ibe s was signi ican ly educed (Figu e 2D). The mal-
annealing a ound he Tc (90 °C o 1 h) was used o u he
inc ease he β-phase con en . The amoun o β-phase con en in
PDVF-T FE samples subjec ed o pos -syn hesis cold-d awing
and he mal annealing was highe han he non-pos - ea ed
coun e pa s as de e mined by FTIR analysis (Figu e2C).
The e ec o alignmen on he piezoelec ic pe o mance o
indi idual ibe s was demons a ed by swi ch-spec oscopy pie-
zo esponse mic oscopy (SS-PFM). The piezoelec ic esponse
o ibe s om andomly-aligned sca olds was compa ed o
ibe s om mechanically d awn, aligned sca olds. The piezo e-
sponse o d33 alue was ound o be dependen on he ibe
alignmen . A s ong co ela ion was obse ed be ween sca -
olds wi h inc eased ibe alignmen and a highe piezoelec ic
esponse ( om −16.92 o −24.61 pm V−1) (Figu e3). The elas ic
modulus o indi idual ibe s was 350± 80MPa as measu ed
using peak- o ce imaging, ye no signi ican di e ences we e
obse ed be ween andom and aligned ibe s. In e es ingly, a
coope a i e piezoelec ic e ec p e iously desc ibed by Pe sano
e  al. was ound in dense a ays o ibe s (Figu e 3A) due o
elec omechanical in e ac ions be ween adjacen ibe s and he
Ad . Ma e . 2021, 33, 2008788
Table 1. The physical p ope ies o piezoelec ic PVDF-T FE and non-piezoelec ic PTFE sca olds.
Sca old ype Elas ic modulus [MPa] S eng h [MPa] Elonga ion [%] Fibe diame e [nm] d33 [pC N−1]
Non-piezoelec ic wi h d awing 14.5± 1.7 16± 0.3 91.3± 10.4 690± 110 0
Piezoelec ic w/o d awing 56.6± 7.6 15± 3.7 46.4± 6.1 540± 120 29.3± 2.7
Piezoelec ic wi h d awing 61.8± 8.1 31± 4.2 39.4± 3.2 513± 80 36.5± 3.9
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sca old s i ness g adien , esul ing in an o e all enhancemen
o he sca old piezo esponse (d33=−36.5 ± 3.8 pm V−1 and
g33=−0.41V m N−1).[28] Di e ences be ween single ibe s and
mul iple ibe s (a ays) a ise om local di e ences in s ess dis-
ibu ion a ia ions. Disc epancies be ween ob ained alues and
epo ed alues (d33=−29 pC N−1) may a ise om di e ences
in ibe diame e , o ien a ion, geome y, and a angemen .[29]
Mechanical and elec omechanical s imula ion o cells
was pe o med in i o using non-piezoelec ic PTFE and
piezoelec ic PVDF-T FE sca olds unde cyclic mechanical
s e ching. The physical and elec ical p ope ies o he sca -
olds a e shown in Table1.
3. The E ec o Elec omechanical S imula ion on
Tendon-Speci ic Gene Exp ession and Pheno ypic
Main enance In Vi o
We in es iga ed he e ec o opog aphical, mechanical, and
elec ical cues on endon gene and p o ein exp ession. Due
o he in insic non-adhesi e p ope ies o luo ina ed poly-
me s, he ibe s we e unc ionalized wi h an ECM molecule
o enhance cell adhesion pos - ab ica ion.[30] Fib onec in unc-
ionaliza ion was chosen and cha ac e ized (Figu e S4, Sup-
po ing In o ma ion) since i showed highe le els o cell
adhesion and igge ed a mo e signi ican cellula p oli e a ion
Ad . Ma e . 2021, 33, 2008788
Figu e 3. Fib ous aligned piezoelec ic sca olds demons a ed highe piezoelec ical pe o mance han piezoelec ic ilms due o geome ical bounda y
a iables ha con ol he piezo esponse o indi idual PVDF-T FE ibe s. PFM ampli ude and phase images o single and mul iple ibe s. A) Random
indi idual ibe s (a) demons a ed lowe piezoelec ic pe o mance ela i e o aligned indi idual ibe s (b). Aligned supe imposed ibe s (c) displayed
an enhanced piezoelec ic esponse (in-plane), showing a coope a i e e ec due o he elec omechanical in e ac ion among adjacen ibe s. Mul iple
dense laye s (d) o adjacen ibe s p esen ed he highes piezoelec ic coe icien . Comme cial PVDF-T FE ilms (e) display a lowe piezoelec ic coe -
icien . A non-piezoelec ic pla inum subs a e showed no piezo esponse ( ). B) Di ec compa ison o d33 alues (piezo esponse) be ween samples
(a– ). C) Indi idual ibe DMT elas ic modulus (i) and elec ical conduc i i y (iii). Elec ical cu en s (ii) and DMT elas ic modulus (i ) we e measu ed
on a pla inum-coa ed glass su ace as con ol, I= 300 pA. The ela i e s i ness di e ence be ween single ibe (350 ± 80MPa) and ibe a ays is
esponsible o he ou -o -plane piezo esponse enhancemen . No esidual (I= 0 pA) elec ical cu en s we e measu ed in he indi idual ibe s indica ing
no esis i e mode o conduc ion cha ac e is ic o piezoelec ic ma e ials. (All measu emen s we e ob ained using N= 3 samples, = 7 eplica es pe
sample.) The alues a e p esen ed as mean ± SD. Signi ican di e ences (one-way ANOVA) o piezo esponse o ibe s (*p< 0.05, **p< 0.001) indica e
esul s o he pos hoc es (Bon e oni).
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esponse compa ed wi h collagen ype I o PLL coa ing.[31]
While ib onec in unc ionaliza ion migh inc ease in eg in-
media ed o ces (up o 30 nN[10]) p oducing de o ma ions on
he piezoelec ic ibe s and gene a ing in e e ing elec ical sig-
nals, using Mul iphysics simula ions we ha e ound ha hese
le el o o ces a e negligible compa ed o 4% de o ma ion sig-
nals (see Figu e S5, Suppo ing In o ma ion) and do no induce
any pe manen plas ic de o ma ions. The e o e, based on hese
simula ions, cell-adhesion does no change he elec omechan-
ical p ope ies o he ibe s and h eshold o EMS (Figu e S5,
Suppo ing In o ma ion).
Fi s , we in es iga ed he explici e ec o ibe alignmen on
cell mo phology, cell o ganiza ion, and exp ession o he en-
ocy e-speci ic ma ke Tenomodulin (TNMD) and i s ansc ip-
ion ac o Scle axis (SCX) by seeding human TDCs (hTDCs)
on o ib ous sca olds (non-aligned and aligned) and plana
PVDF-T FE ilms o 1, 3, and 7 days (Figu e4). Ou obse a-
ions e ealed ha ea ly passage (P2) enocy e cells cul u ed on
plana o non-aligned ibe s p esen ed a polyhed al mo phology
and o oid nuclei. Con e sely, he ib ous aligned s uc u e p o-
mo ed inc eased cell alignmen in he longi udinal di ec ion o
ibe s alida ed by ac in s aining. Quan i ica ion o alignmen
o e ime showed ha hTDCs cul u ed on aligned sca olds
p esen ed cy oskele al elonga ion by day 1 and exp essed highe
le els o SCX and TNMD when compa ed o cells seeded on
bo h plana ilms and non-aligned sca olds (Figu e4A,B), hus
con i ming he bene icial e ec o ibe -aligned s uc u es o
p omo ing hTDC pheno ype main enance.
Nex , we used a high o ce uniaxial ac ua o sys em o apply
speci ic s ain p o ocols o ac i a e di e en piezoelec ic
esponses o ou de ices, p e iously seeded wi h hTDCs. Li e/
dead assay was pe o med a e ac ua ion o demons a e ha
he mechanical s e ching ac ua ion did no nega i ely a ec
he iabili y o hTDCs in i o (Figu e 4C). To op imize he
ac ua ing condi ions, we analyzed he piezoelec ic pe o -
mance o he de ices, he hTDC iabili y, he nuclea de o ma-
ion, and he TNMD and SCX exp ession (Figu e4C,D; Figu e
S6, Suppo ing In o ma ion) unde s e ching condi ions. As
piezoelec ic sys ems exhibi equency-modula ed pe o -
mance (ou pu cha ge) due o he equency dependence o he
elas ic/piezoelec ic cons an s, we assessed he e ec o s ain
a e (0.25, 0.5, 1, 2, and 3 Hz) on he piezoelec ic cons an
d31 using physiologically ele an s ain magni udes (1%, 2%,
and 4%) (Figu e 4E,G). In gene al, aligned PVDF-T FE ibe s
p oduced a signi ican ly inc eased piezoelec ic pe o mance
when he applica ion o o ce was pa allel o he ibe o ien a-
ion (Figu e4F). As an icipa ed, ol age measu emen s showed
a posi i e co ela ion be ween ol age ou pu and s ain mag-
ni ude, demons a ing he de ice’s piezoelec ic na u e (also
con i med using SS-PFM, Figu e4F). A a ixed equency (i.e.,
0.25Hz) he ol age ou pu inc eased linea ly wi h inc easing
s ain magni ude ( om 0.28V a 1% o 1.21 V a 4%, espec-
i ely). A ixed s ain magni ude (i.e., 4%), he ol age ou pu
anged om 0.51V a 3Hz o 1.21V a 0.25Hz (Figu e4G). We
hen es ed he ol age ou pu s abili y o he de ice up o 500
cycles o con inuous dynamic s ain, and no signi ican change
o he de ice ol age ou pu (Figu e2D) was obse ed. Finally,
we chose 0.5Hz equency and 4% s ain magni ude o subse-
quen genomic analyses (Table2) as cell iabili y was p ese ed
(compa able o issue cul u e pla e, Figu e4C, p> 0.05 o all
days). As shown in ou cell iabili y da a (Figu e S7, Suppo ing
In o ma ion), we ha e obse ed a ansien e ec in cell iabili y
a day 7 in dynamic (4% s ain, 0,5Hz,8h pe day) e sus s a ic
condi ions. Impo an ly, his e ec was no ma e ial/ ea men
dependen as he cell iabili y was iden ical o piezoelec ic
and non-piezoelec ic sca old in bo h s a ic and dynamic condi-
ions. A he same ime, he TNMD exp ession was signi ican ly
inc eased ( old-inc ease o ≈1.5 and ≈2, a e 5 and 10 days o
s imula ion espec i ely, Figu e 4D). Sca olds demons a e a
high le el o piezoelec ic ou pu (1 V),illus a ing hepo en-
ial o ou ac ua ed piezoelec ic de ices (elec omechanical
s imula ion) in sus aining hTDC p oli e a ion and p omo ing a
endon-like pheno ype.
Tendons ha e a g ea abili y o espond o mechanical o ces
by adap ing hei s uc u e and biochemical composi ion and
cyclic mechanical s e ching o he endon is a ecognized
me hod o ea ing endon- ela ed inju ies.[32] Se e al s udies
ha e con i med ha endon is subjec ed o 3–4% s ain du ing
no mal ac i i ies[33] and, ha s ain a e (loading equency) is
impo an in modula ing he cellula esponse in i o.[34] Unde
physiological condi ions, he s ain a e o he endon is a ound
0.1–0.5Hz; howe e , du ing in ense ac i i y, he equency can
be as high as 10Hz.[35,36] Recen ly, i has been demons a ed ha
endon cells modula e hei gene exp ession, p o ein syn hesis
and mi ogenesis in i o h ough ac i a ion o mechano ans-
duc i e signaling pa hways unde physiological mechanical
s imula ion.[37] The e o e, we compa ed he e ec o mechan-
ical and elec omechanical s imula ion on human hTDCs gene
and p o ein exp ession (Table3, 4 and 5) (Figu e5). Genomic
analysis o human hTDCs cul u ed on piezoelec ic PVDF-T FE
and non-piezoelec ic PTFE sca olds unde bo h s a ic (4%
s a ic s ain) and physiologically ele an dynamic loading con-
di ions (4% dynamic s ain a 0.5Hz o 8 h pe day) was pe -
o med a 1, 5, and 10 days. O e all, he analysis o gene exp es-
sion co ela ed well wi h he esul s obse ed a he p o ein
le el (Figu e 5D). Elec omechanical s imula ion (EMS) using
ac ua ed PVDF-T FE sca olds, induced a apid and sus ained
up- egula ion o endon- ela ed and bone- ela ed genes ela i e
o s a ic con ols a day 1. In e es ingly, a e 10 days o EMS
bone- ela ed genes e u ned o basal le els whe eas endon-
ela ed genes emained up egula ed, sugges ing a s ong e ec
o EMS owa d enogenic di e en ia ion. Simila ly, mechanical
s imula ion alone (MS) h ough PTFE sca olds induced a sig-
ni ican up- egula ion o bone and endon- ela ed genes a day
1 ( ela i e o s a ic coun e pa ), bu unlike EMS condi ions,
bone- ela ed genes emained up- egula ed while endon- ela ed
genes e u ned o con ol le els by days 5 and 10 (Figu e5A,B;
Figu e S8, Suppo ing In o ma ion). Speci ically, by day 10,
bone- ela ed genes we e up- egula ed in hTDCs subjec ed o
mechanical s imula ion (Figu e 5A,B; Figu e S8, Suppo ing
In o ma ion), whe eas SCX and TNMD we e up egula ed only
in he EMS g oup (Figu e 5E). Taken oge he , hese obse a-
ions indica e ha ans-di e en ia ion o hTDCs owa d an
os eogenic/chond ogenic lineage can be modula ed by EMS
and MS. Mo eo e , EMS o e ed sus ained enogenic di e en-
ia ion capaci y ela i e o MS alone.
To un a el he signal ansduc ion pa hways associa ed wi h
he egula ion o enogenic di e en ia ion, ingenui y pa hway
Ad . Ma e . 2021, 33, 2008788
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Ad . Ma e . 2021, 33, 2008788
Figu e 4. Tenoyc e pheno ype is p omo ed by sca old alignmen unde speci ic mechanical loading condi ions. A) Human TDCs demons a ed an
elonga ed mo phology om day 1 when cul u ed on aligned PVDF-T FE sca olds. Inse s a e highe magni ica ion ( ed = ac in, g een = inculin, and
blue = nucleus, scale ba = 400 μm). Angle dis ibu ion ada analysis indica ed ha he pe cen age o aligned cells was signi ican ly modula ed by
ibe o ien a ion a day 1, 3, and 7 (0° and 90° co espond o alignmen and no alignmen , espec i ely). B) Cells demons a ed di e en ial exp es-
sion o enospeci ic p o eins and mo phological changes when cul u ed on elec ospun sca olds o plana PVDF-T FE ilms. hTDCs main ained hei
pheno ype and exp ession le els o TNMD and SCX when cul u ed on aligned piezoelec ic sca olds; his e ec was absen on 2D plana ilms (N= 3,
= 3, mean ± SD, ***p< 0.001). C) hTDCs cul u ed on piezoelec ic sca olds unde dynamic s imula ion subjec ed o di e en s ain a es (0.5, 1,
and 2Hz) exhibi ed p oli e a ion a es in e sely p opo ional o he s ain equency (N= 3, = 3, mean ± SD). D) An inc ease in he exp ession o
TNMD was obse ed in cells subjec ed o 0.5Hz elec omechanical s imula ion a days 5 and 10 (N= 3, = 3, mean ± SD *p< 0.05). E). An o e iew
o he mechanical loading sys em used o measu e sca old ol age ou pu unde s imula ed physiological s ain condi ions and in i o analysis.
F) Rep esen a i e hys e esis and bu e ly loops o he ibe piezo esponse and pola iza ion. SS-PFM acili a ed he measu emen o he PFM phase
and ampli ude esponse loops as a unc ion o he applied ol age. The ampli ude image shows he magni ude o displacemen esponse gene a ed
unde a con olled applied ol age in d awn ibe s, and he highes ampli ude peak was shown o be a ound 300 pm in esponse o a 12V bias. The
phase image shows he posi i e and nega i e alues o an ipa allel e oelec ic nanodomains. The phase measu emen s indica ed a ≈180° swi ch
o he dipoles be ween he applied posi i e and nega i e ol ages. The piezo esponse ob ained om PFM measu emen s showed ha he elec ical
dipoles lie no mal o he su ace, cha ac e is ic o he o ganized elec ical dipoles/chains o β-phase c ys als. PTFE did no demons a e piezoelec ic
beha io (g ay line). The schema ic indica es he ol age dis ibu ion on he ibe su ace as a unc ion o geome ical a angemen ( ibe o ien a ion)
and in e ac ion wi h adjacen ibe s esul ing in di e ing ans e se de o ma ion es ic ion and modula ion o he piezo esponse he longi udinal ibe
axis. G) Vol age measu emen s as a unc ion o equency (0.25, 0.5, 1, 2, and 3Hz) a a cons an ampli ude o 1%, 2%, and 4% s ain.).
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analysis (IPA) was used wi h gene exp ession p o iles o hTDC
exposed o MS and EMS s imula ion. IPA a ibu es an ac i-
a ion z-sco e used o p edic he ac i a ion s a e o biological
unc ions and signaling pa hways. Pa hway analysis o human
endon de i ed cells exposed o ei he EMS o MS yielded sig-
ni ican modula ion o b oad unc ional signaling pa hways,
and a mechanis ic ne wo k o genes and speci ic biological
unc ions we e di e en ially egula ed (Figu e S9, Suppo ing
In o ma ion). Canonical signaling pa hways wi h he mos
Ad . Ma e . 2021, 33, 2008788
Table 2. QIAGEN genes lis o cus omized gene a ay.
Symbol En ez gene name
ABCB1 ATP binding casse e sub amily B membe 1
ACAN Agg ecan
ACTA1 ac in, alpha 1, skele al muscle
ACVR1 ac i in A ecep o ype 1
AHSG alpha 2-HS glycop o ein
ALPL alkaline phospha ase, li e /bone/kidney
BGLAP bone gamma-ca boxyglu ama e p o ein
BGN Biglycan
BMP1 bone mo phogene ic p o ein 1
BMP2 bone mo phogene ic p o ein 2
BMP4 bone mo phogene ic p o ein 4
BMP6 bone mo phogene ic p o ein 6
BMP7 bone mo phogene ic p o ein 7
BMPR1A bone mo phogene ic p o ein ecep o ype 1A
BMPR2 bone mo phogene ic p o ein ecep o ype 2
CASP3 caspase 3
COL11A1 collagen ype XI alpha 1 chain
COL14A1 collagen ype XIV alpha 1 chain
COL1A1 collagen ype I alpha 1 chain
COL1A2 collagen ype I alpha 2 chain
COL2A1 collagen ype II alpha 1 chain
COL3A1 collagen ype III alpha 1 chain
COL4A1 collagen ype IV alpha 1 chain
COL5A1 collagen ype V alpha 1 chain
COL6A1 collagen ype VI alpha 1 chain
COMP ca ilage oligome ic ma ix p o ein
DCN Deco in
DLX5 dis al-less homeobox 5
EGR1 ea ly g ow h esponse 1
FGF10 ib oblas g ow h ac o 10
GDF15 g ow h di e en ia ion ac o 15
GDF5 g ow h di e en ia ion ac o 5
GDF6 g ow h di e en ia ion ac o 6
GDF7 g ow h di e en ia ion ac o 7
HAT1 his one ace yl ans e ase 1
HDAC1 his one deace ylase 1
HNF1A HNF1 homeobox A
IBSP in eg in binding sialop o ein
IGF1 insulin like g ow h ac o 1
ITGA1 in eg in subuni alpha 1
ITGA2 in eg in subuni alpha 2
ITGA3 in eg in subuni alpha 3
ITGA4 in eg in subuni alpha 4
ITGA5 in eg in subuni alpha 5
ITGAX in eg in subuni alpha X
ITGB1 in eg in subuni be a 1
ITGB3 in eg in subuni be a 3
Symbol En ez gene name
ITGB5 in eg in subuni be a 5
KAT2B lysine ace yl ans e ase 2B
KCNK2 po assium wo po e domain channel sub amily K membe 2
KCNK4 po assium wo po e domain channel sub amily K membe 4
KDR kinase inse domain ecep o
MGP ma ix Gla p o ein
MKX mohawk homeobox
PIEZO1 piezo ype mechanosensi i e ion channel componen 1
PIEZO2 piezo ype mechanosensi i e ion channel componen 2
PIK3CG phospha idylinosi ol-4,5-bisphospha e 3-kinase ca aly ic subuni
gamma
PTEN phospha ase and ensin homolog
PTK2 p o ein y osine kinase 2
PTK2 p o ein y osine kinase 2
PXN Paxillin
RUNX2 un ela ed ansc ip ion ac o 2
SCX scle axis bHLH ansc ip ion ac o
SMAD3 SMAD amily membe 3
SMAD4 SMAD amily membe 4
SMAD9 SMAD amily membe 9
SMURF1 SMAD speci ic E3 ubiqui in p o ein ligase 1
SMURF2 SMAD speci ic E3 ubiqui in p o ein ligase 2
SOX9 SRY-box 9
SP7 Sp7 ansc ip ion ac o
SPARC sec e ed p o ein acidic and cys eine ich
SPP1 sec e ed phosphop o ein 1
TBX5 T-box 5
TGFB1 ans o ming g ow h ac o be a 1
THBS4 h ombospondin 4
TLN1 alin 1
TNC enascin C
TNMD Tenomodulin
TRPA1 ansien ecep o po en ial ca ion channel sub amily A membe 1
TRPV1 ansien ecep o po en ial ca ion channel sub amily V membe 1
TWIST1 wis amily bHLH ansc ip ion ac o 1
VCL Vinculin
VEGFA ascula endo helial g ow h ac o A
ZYX Zyxin
Table 2. Con inued.
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signi ican numbe o modula ed gene exp ession we e associ-
a ed wi h day 1 and 10 in i o (Figu e S9, Suppo ing In o -
ma ion). The ERK/MAPK signaling pa hway was consis en ly
ac i a ed wi h bo h EMS and MS s imula ion; howe e , his
pa hway was signi ican ly up egula ed in hTDCs subjec ed o
MS a days 1 and 10 (Figu e5E).
Simila ly, inc eased ac i a ion o cell ansdi e en ia ion
and di e en ia ion owa d he bone cell lineage and up egu-
la ions in os eogenic signaling pa hways Wn /β-Ca enin and
BMP we e a ibu ed o MS a day 10 ( ela i e o EMS). Signi i-
can ly, ollowing 10 days in cul u e, he endon- ela ed signaling
pa hway IGF-1 and biological di e en ia ion p ocess associa ed
wi h enocy e unc ion we e ac i a ed in endon de i ed cells
subjec ed o EMS bu down egula ed unde MS (Figu e 5E).
We looked a he p o ein exp ession p o ile o endon-speci ic
ma u a ion ma ke s and collagen syn hesis o alida e gene
exp ession esul s. In ag eemen wi h he gene exp ession da a,
we obse ed a highe exp ession o endon ma u a ion ma ke s
(SCX and TNC) o endon de i ed cells exposed o EMS ela i e
o MS in addi ion o he inc eased syn hesis o collagen ype V
(Figu e5F,G).
Du ing endon speci ic di e en ia ion o ans-di e en i-
a ion, highly egula ed signal ansduc ion e en s ake place,
leading o he exp ession o genes associa ed wi h he endon-
speci ic pheno ype o pheno ypic d i espec i ely. We hypo h-
esized ha mechanically ac i a ed and elec ically ga ed mem-
b ane ion channels would unde go modula ed exp ession in
esponse o EMS. To in es iga e he ole o hese memb ane
senso s on signal gene a ion and p opaga ion, co ela ional
analysis was conduc ed be ween signi ican modula ions o
endon-speci ic unc ional pa hways a day 1, 5, and 10 in he
mechanis ic ne wo k o di e en ial changes in he exp ession
o mechano-sensi i e (TRP amily ion channels, ocal adhe-
sion- ela ed p o eins and in eg ins β1, β3, and β5), elec o-sen-
si i e (KCNK amily and Ca2+ L- ype ion channel), and piezo-
sensi i e (Piezo amily ion channels) ecep o s, as well as he
BMP ecep o BMPR1A[38,39] (Figu e 5C). C i ically, inc eased
ac i a ion o endon di e en ia ion- ela ed genes (Figu e 5A)
h ough he modula ed ac i i y o endon- ela ed ansc ip ion
ac o s (i.e., SCX and EGR1, see Figu e S8, Suppo ing In o -
ma ion) was co ela ed wi h signi ican modula ion o he
exp ession le el o TRP amily ion channels unde s imula ion
(Figu e5G). As shown in Figu e5F, he exp ession o SCX and
TNC was signi ican ly inc eased unde EMS condi ions a bo h
day 5 and 10, oge he wi h a signi ican ly lowe exp ession o
KCNK4, TRPA1, and TRPV1 ion channels when compa ed o
he MS g oup. These esul s we e u he alida ed a he p o-
ein le el (Figu e5G).
Con e sely unde MS, a dec ease in he exp ession o endon-
ela ed genes and an inc ease in he exp ession o genes asso-
cia ed wi h ca ilage/bone di e en ia ion was associa ed wi h
highe exp ession o TRP ion channels (Figu e5E,G; Figu e S8,
Suppo ing In o ma ion). Speci ically, TRPA1 and TRPV1 ion
channels unde wen a sus ained inc ease in exp ession which
co ela ed wi h a posi i e egula ion o ansc ip ion ac o s
SOX9, RUNX2, and COMP, p o eins ha ha e a ecognized
ole in he p ocesses o os eogenesis (Figu e 5B; Figu e S6,
Suppo ing In o ma ion). O e all, he gene exp ession esul s
and pa hways analyses, u he alida ed using a cus om-made
p o ein a ay, demons a ed ha MS o hTDCs in i o esul ed
Ad . Ma e . 2021, 33, 2008788
Table 4. Signaling a ay: p o eins associa e o di e en signaling pa h-
ways (MAPK, FAK, TGF-B, BMP, and WNT).
P o eins Abb e ia ion Pu chased om Ca . no
Smad 1 SMAD1 Cell Signaling 6944S
6944S
Smad 5 SMAD3 Cell Signaling 12534S
Phospho yla ed Smad 1 SMAD1/5/8 Cell Signaling 5753S
Phospho yla ed Smad 1/5/8 pSMAD158 Cell Signaling 9516s
9516s
Focal adhesion kinase FAK MBL D061-3
12G4
Phospho yla ed FAK pFAK Cell Signaling 3284S
3284S
MAPK ERK Cell Signaling 4696S
L34F12
p44/42 MAPK pERK Cell Signaling 4377S
Wn /β-ca enin β-Ca enin Millipo e 2 858 901
Ac i e β-ca enin, clone 8E7 Ac i e β-Ca enin Millipo e ABC
β-ac in β-ac in WAKO 019-19741
Table 3. Tenogenesis a ay: p o eins associa ed wi h endon egene a-
ion o hTDC unc ion.
P o eins Abb e ia ion Pu chased om Ca . no
Scle axis SCX Abcam ab58655
Tenomodulin TNMD Abcam ab203676
Byglican BGN Abcam ab49701
Deco in DCN Abcam ab175404
Th ombospondin 4 THBS-4 Abcam ab176116
Tenascin C TNC Abcam ab88280
Collagen I COLI Abcam ab138492
Collagen II COLI Abcam ab185430
Collagen III COLIII Abcam ab7778
Collagen V COLV Abcam ab7046
Table 5. Recep o s a ay: p o eins associa e o cell memb ane ecep o s.
P o eins Abb e ia ion Pu chased om Ca . no
TRPV1 TRPV1 San aC uz sc-20813
Piezo1 Piezo1 San aC uz sc-164319
Piezo2 Piezo2 San aC uz sc-84763
TRPA1 TRPA1 San aC uz sc-32353
KCNK2 KCNK2 San aC uz sc-11557
KCNK4 KCNK4 Abcam ab81367
L- ype Ca2+L- ype Ca2+San aC uz sc-25686
BMPR1A BMPR1A The moFishe PA5-11856
In eg in1 ITG1 Abcam ab134179
In eg in3 ITG3 Abcam ab34409
In eg in5 ITG5 Cell Signaling 3629S
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bigge is he size he highe is he exp ession le el and he es o do s
a e scaled p opo iona ely. High-con idence alues we e ep esen ed
by a ed ou line co esponding o a p- alue <0.05o by a black ou line
co esponding o a p- alue <0.01,whe eas o p- alues >0.05 heou line
was ain .
P o ein Exp ession: P o ein an ibody mic oa ay was cus om made.
Nex e ion slide H mic oa ay slides we e pu chased om Scho AG
(Mainz, Ge many). Alexa Fluo 555 ca boxylic acid succinimidyl es e
was ob ained om Li e Technologies (Ca lsbad, CA, USA). P o ein
samples we e labeled wi h Alexa Fluo 555 ca boxylic acid succinimidyl
es e acco ding o manu ac u e ’s ins uc ions. The excess label
was emo ed, and he bu e was exchanged wi h PBS, pH 7.4, by
cen i uga ion h ough 3kDa molecula weigh cu o il e s. Abso bance
a 555 and 280nm was measu ed o labeled samples and calcula ions
we e pe o med acco ding o manu ac u e ’s ins uc ions using an
a bi a y ex inc ion coe icien o 100 000 and molecula mass o 100 000
o enable quan i ica ion o ela i e p o ein concen a ion and label
subs i u ion e iciency. All comme cial an ibodies (Table1) we e bu e
exchanged in o PBS and quan i ied by a bicinchoninic acid (BCA) assay.
An ibodies we e dilu ed o p in concen a ion in PBS and p in ed in six
eplica es on Nex e ion H amine- eac i e, hyd ogel-coa ed glass slides
using a SciFLEXARRAYER S3 piezoelec ic p in e (Scienion, Be lin,
Ge many) unde cons an humidi y (62% +/− 2%) a 20°C. Each ea u e
was p in ed using ≈1 nL o dilu ed an ibody using an uncoa ed 90µm
glass nozzle wi h eigh eplica ed suba ays pe mic oa ay slide. A e
p in ing, slides we e incuba ed in a humidi y chambe o e nigh a oom
empe a u e o acili a e comple e conjuga ion. The slides we e hen
blocked in 100 × 10−3 m e hanolamine in 50 × 10−3 m sodium bo a e, pH
8.0, o 1 h a oom empe a u e. Slides we e washed in PBS wi h 0.05%
Tween 20 (PBS-T) h ee imes o 2 min each wash ollowed by one
wash in PBS, d ied by cen i uga ion (470 × g, 5min), and hen s o ed
wi h a desiccan a 4°C un il use. Incuba ions we e ca ied ou in he
da k. Mic oa ay slides we e incuba ed as p e iously desc ibed. Ini ially,
one labeled sample was i a ed (2.5–15µg mL−1) o op imal signal o
noise a io and all samples we e subsequen ly incuba ed o 1 h a 23°C
a 9µg mL−1 in T is-bu e ed saline (TBS; 20 × 10−3 m T is-HCl, 100 ×
10−3 m NaCl, 1 × 10−3 m CaCl2, 1 × 10−3 m MgCl2, pH 7.2) wi h 0.05%
Tween 20 (TBS-T). All mic oa ay expe imen s we e ca ied ou using
h ee eplica e slides. Alexa Fluo 555 labeled cells lysa e (10µg mL−1)
we e incuba ed in wo sepa a e suba ays on e e y slide o con i m
e ained an ibody pe o mance and p in ing, espec i ely (Figu e 1).
A e incuba ion, slides we e washed h ee imes in TBS-T o 2min pe
wash, once in TBS and hen cen i uged d y as abo e. D ied slides we e
scanned immedia ely on an Agilen G2505 mic oa ay scanne using
he Cy3 channel (532nm exci a ion, 90% pho omul iplie ubes (PMT),
5µm esolu ion) and in ensi y da a we e sa ed as a . i ile. An ibody
mic oa ays we e e i ied o emain ac i e o a leas 2 weeks a e
p in ing and all incuba ions we e ca ied ou wi hin ha ime ame. Da a
ex ac ion om . i iles was pe o med mainly as p e iously desc ibed.
Da a we e no malized o he mean o h ee eplica e mic oa ay slides
(suba ay-by-suba ay using suba ay o al in ensi y, n= 4, 24 da a
poin s). Unsupe ised hie a chical clus e ing o no malized da a was
pe o med using Hie a chical Clus e ing Explo e 3.0 (h p://www.
cs.umd.edu/hcil/hce/hce3.h ml) using he pa ame e s no p e il e ing,
comple e linkage, and Euclidean dis ance. All da a p esen ed he e we e
con i med using a leas ou eplica es o each o he es g oups and
con ol g oup. The esul s a e exp essed as he mean o he alues ±
s anda d e o o he mean.
Di e en ypes o a ays we e ab ica ed o in es iga ed endon
egene a ion o pheno ype main enance ( enogenesis), in acellula
molecula pa hways (signaling) o memb ane p o eins ( ecep o s).
In Vi o Animal Model: The animal ca e esea ch e hics commi ee
a he Na ional Uni e si y o I eland, Galway and Heal h P oduc s
Regula o y Au ho i y (AE19125/P055) app o ed all he animal p ocedu es
used in his s udy. Also, animal ca e and managemen ollowed he
S anda d Ope a ing P ocedu es o he Animal Facili y o he Na ional
Uni e si y o I eland, Galway. Animals we e allowed o acclima ize
o a leas 7 days be o e any su gical p ocedu es. Subsequen ly,
animals we e acclima ed o he eadmill unning o 1 week, and hei
beha io was analyzed. A o al o 105 Female Lewis a s aged 6–8
(220g)weekswe eused in his s udy. The animals we e anaes he ized by
iso luo ane inhala ion (5% induc ion educing o 1–2% o main enance
du ing p ocedu es). The igh leg was sha ed and swabbed wi h iodine
o minimize he isk o bac e ial con amina ion. An incision was c ea ed
h ough he skin (≈1cm) om he myo endinous junc ion dis ally o he
os eo endinous junc ion. The incision p o ided ample exposu e o he
Achilles endon. The ascia su ounding he Achilles was ansec ed
longi udinally and ca e ully e ac ed o expose he Achilles endon.
Be o e implan a ion and endon ansec ion, wo looped su u ed we e
inse ed a he op (muscle) and bo om (bone). A e he o al endon
leng h was measu ed a 3 mm de ec in p oximal/dis al ex ension (a
3mm om he calcaneus) was c ea ed using a posi ioning de ice and
an 11 su gical blade, esul ing in a 6mm gap a e issue e ac ion. The
cons uc was hen su u ed (4-0 E hicon) o bo h ends o he endon
o b idge he gap using a modi ied Kessle echnique, and he skin was
su u ed. A e a pe iod o 2, 4, and 8 weeks he animals we e eu hanized
and endon issue, as well as con ala e al endons, we e ha es ed.
Animals eco e ed o 2 weeks and we e g adually exposed o eadmill
unning (see Table 6). B ie ly, he eadmill unning once a week o
5min o 30–45 min o 5 days a week.
His ology: Repai ed endon issues and con ala e al endons o all
g oups (n= 7) we e dissec ed om he p oximal myo endinous junc ion
o he dis al os eo endinous junc ion and p ocessed o his ological
analysis. The samples we e ixed in 10% neu al bu e ed o malin (24 h),
dehyd a ed in g adien alcohols, clea ed, and embedded in pa a in
blocks, as epo ed p e iously. His ological sec ions (6 µm hick)
we e p epa ed using mic o ome sec ioning (Leica Ro a y Mic o ome).
In o de o dis inguish be ween sca issue o new endon o ma ion,
pola iza ion mic oscopy and pic osi ius ed s aining we e used. Also,
o desc ip i e his ology 6 µm, hick sec ions we e s ained using
Hema oxylin & Eosin s ain, Masson-Goldne ’s s ain, Alcian Blue,
O-sa anin s ain, Red pic osi ius s ain, o He o ici’s polych ome s ain
acco ding o he manu ac u e ’s guidelines. A eas o chond i ica ion
wi hin he de ec egion a 4 o 8 weeks a e su ge y we e measu ed
using Sa anin O s aining and ImageJ ( 1.52i). Fo each issue, 3 di e en
on al-longi udinal sec ions we e analyzed (1 sec ion o he middle
endon, 1 sec ion en al o his middle pa , and 1 sec ion do sal o
his middle pa ). Fo each sec ion5 consecu i e images we e cap u ed
Table 6. Exe cise p o ocol used o he a unning g oup ( eadmill).
Time Du a ion [min] Speed [m min−1]
Week 2 Day 14 5 8–9
Day 15 10 9–10
Day 16 15 9–10
Day 17 20 9–10
Day 18 30 9–10
Week 3 Day 19 45 9–11
Day 20 30 9–11
Day 21 45 9–12
Day 22 30 10–14
Day 23 45 10–14
Week 4 30 10–14
Week 5 45 10–14
Week 6 30 10–14
Week 7 45 10–14
Week 8 30 10–14
Week 9 45 10–14
Week 10–16 30 10–14
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Ad . Ma e . 2021, 33, 2008788
spanning he en i e leng h o he issue, omi ing he ansi ion zones o
o iginal endon s umps o egene a ed issue. The olume ac ion (VV)
o endon cells we e used o es ima e cell p oli e a ion. A 192-poin g id
was o e laid on 40× images o H&E s ained issue sec ions. The numbe
o endon cells in e sec ing poin s o he g id was coun ed (PP), along
wi h he o al numbe o poin s on he issue (PT). The olume ac ion
o endon cells (VV) was calcula ed using he o mula below:
VP
/P
VPT
= (4)
His ology Sco ing Sys em: In o de o highligh he e ec o
eadmill unning and di e ences be ween g oups on he p og ession
o endon epai and calci ica ion, a sco e sys em was adop ed
p e iously desc ibed.[58] B ie ly, he mac oscopic sco e sys em included
measu emen s on s ained samples (Hema oxylin & Eosin, Masson-
Goldne ’s s ain, Alcian Blue, O-sa anin, Red pic osi ius, and He o ici’s
polych ome) h ee animals pe g oup ( i e sec ions o 6 μm pe animal).
A poin -based sco ing sys em was used, and he ollowing pa ame e s
we e sco ed (by wo sco e s): ECM o ganiza ion o he whole epai ed
endon, cellula i y, p o eoglycan con en , cell alignmen , o ganiza ion
o he endon callus, in eg a ion o cons uc s o he no mal issue,
ascula iza ion, degene a i e changes (os eochond al), and ea u es o
in lamma ion. A o al o h ee sco e s we e used o he sco ing and he
slides we e blinded by one o he au ho s (MFY).
Func ional Reco e y Analysis: An animal eadmill (Exe 3/6, Columbus
Ins umen s) ack in eg a ed wi h a ideo-based sys em was used o
ob ain spa io empo al pa ame e s o gai . The animal gai was analyzed
h ough a clea plas ic Lexan a he sides o he sys em consis ing o a cage
(50.8cm × 50.8cm × 33cm) wi h ga es placed a each end o he walkway.
A digi al came a (8 M pixels and 120 ames pe seconds) was posi ioned
30cm in on o he walkway o cap u e he sagi al iew o he a om
he walkway. The da a we e analyzed and p ocessed wi h Kino ea so wa e
( 0.8.27). Fo all g oups, he sys em was calib a ed using he same scale
ba loca ed in he image and modeled he leg mo ion using pai s o ci cle
ma ke s. The ankle, knee, hip, and MTP join angles we e measu ed using
he au oma ic black ink ma ke ea u e ecogni ion on he hip, knee, ankle,
and 3 d me a a sal head a he ou gai s ages: ini ial con ac , mid-s ance,
p e-swing, and mid-swing. The spa ial and empo al gai pa ame e s
analyzed we e s ep leng h, angle, and cycle ime. The walking speed was
calcula ed by di iding he s ep leng h by he cycle ime. Each angle join
cu e was no malized by cycle ime be o e u he analysis. To de e mine
he change on gai angle, he maximum di e ence in ampli ude (Ampl.)
be ween ini ial con ac and end o swing was measu ed du ing an en i e
s ep and was calcula ed acco ding o ollowing o mula:
ma
x. min. Ampl.
() () ()
°− °= ° (5)
ansec io
n
ansec ion
Fu
nc ionalReco e y Ampl.( )/Ampl.( )
pos p io
=° ° (6)
COMSOL Simula ions: To unde s and he in luence o he po en ial
dis ibu ion a ound he sca old du ing elec omechanical s imula ion
and how his migh impac he cells nea he sca old, we calcula ed he
po en ial dis ibu ion o he sca old h ough a ini e elemen me hod
using elec os a ic, piezoelec ic, and solid mechanics in e ac ion
modulus in COMSOL. The ini e elemen me hod (FEM) analysis uses
an in eg a ed de ice s uc u e consis ing o a ubula -shaped elec ospun
sca old composed o PVDF-T FE ibe s unde 4% de o ma ion (0.2mm).
The geome ies and he elec omechanical p ope ies a e se as he eal
measu ed alues. Due o he complexi y o he nano ib ous s uc u e,
we used a simpli ied PVDF-T FE model consis ing o a low-densi y ilm
(densi y o 450kg m−3). The young modulus is 60MPa, Poisson’s a io is
0.42, and he s ain-cha ge coupling ma ix is d31= 23 pC N−1, d32= 8pC
N−1, and d33=−36.5pC N−1. In addi ion, we ha e in es iga ed he e ec s
o high adhesion o ces (up o 30 nN[10]) applied h ough ocal adhesion
complexes (1–5 µm long) on a single PVDF-T FE ibe .
S a is ical Analysis: S a is ical analyses we e pe o med using G aphPad
P ism so wa e e sion 8.4.3 (G aphPad So wa e, CA, USA). To es
whe he he da a was no mally dis ibu ed no mali y es s we e applied
(D’Agos ino and Pea son). When da a ollowed a no mal dis ibu ion a
one-way ANOVA analysis o he compa ison o means be ween di e en
g oups was pe o med. Homogenei y o a iances was es ed using
Ba le ’s es s, and in a case o unequal s anda d de ia ions (SD),
Welch ANOVA es was applied. I da a we e no no mally dis ibu ed,
he compa ison o medians be ween di e en g oups was assessed by
non-pa ame ic K uskal-Wallis es ollowed by Dunn pos -hoc es . Since
mos o he da a was no no mally dis ibu ed, mos o he esul s we e
exp essed as median as cen al endency cha ac e is ic and in e qua ile
ange (IQR) o ange as dispe sion cha ac e is ic and, p alues o <0.05
we e conside ed s a is ically signi ican .
Suppo ing In o ma ion
Suppo ing In o ma ion is a ailable om he Wiley Online Lib a y o
om he au ho .
Acknowledgemen s
The wo k was suppo ed by g an s o MJPB om Science Founda ion
I eland (16/BBSRC/3317), o MAFY om H2020 Ma ie Skłodowska-Cu ie
Ac ions (898737) and g an om Science Founda ion I eland (SFI),
co- unded unde he Eu opean Regional De elopmen Fund h ough
G an numbe s 13/RC/2073 and 13/RC/2073_P2. The au ho s hank
D . Oli e Ca oll o echnical assis ance. SGIke echnical se ices
(UPV/EHU) a e g a e ully acknowledged o XRD and XPS suppo .
The au ho s acknowledge he acili ies and scien i ic and echnical
assis ance o he Cen e o Mic oscopy & Imaging a he Na ional
Uni e si y o I eland Galway, a acili y ha is unded by NUIG and he
I ish Go e nmen ’s P og amme o Resea ch in Thi d Le el Ins i u ions,
Cycles 4 and 5, Na ional De elopmen Plan 2007–2013.
Open access unding p o ided by IReL.
No e: The acknowledgemen s sec ion was upda ed on Oc obe 5, 2021,
a e ini ial publica ion online.
Con lic o In e es
The au ho s decla e no con lic o in e es .
Au ho Con ibu ions
M.A.F.-Y., M.J.B., and A.P. concei ed he expe imen s. M.A.F.-Y.
pe o med he expe imen s. A.T., S.D., A.S., and A.L. pe o med esea ch.
M.K., A.P., T.S., and M.P. p o ided ma e ials and expe ise. M.A.F.-Y.
analyzed he da a and p epa ed he igu es. A.P. and M.J.B. p o ided
c i ique and con ex o he da a. M.A.F.-Y. w o e he manusc ip . All
au ho s ead and commen ed on he manusc ip .
Da a A ailabili y S a emen
The da a ha suppo he indings o his s udy a e openly a ailable in
bioRxi a h ps://doi.o g/10.1101/2020.08.03.22786.
Keywo ds
bioelec onics, collagen, piezoelec ics, poly( inylidene luo ide-co-
i luo oe hylene), endon egene a ion
Recei ed: Decembe 29, 2020
Re ised: May 17, 2021
Published online: Augus 23, 2021
15214095, 2021, 40, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/adma.202008788 by Johns Hopkins Uni e si y, Wiley Online Lib a y on [28/05/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
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