ORIGINAL RESEARCH
published: 30 No embe 2018
doi: 10.3389/ bioe.2018.00187
F on ie s in Bioenginee ing and Bio echnology | www. on ie sin.o g 1No embe 2018 | Volume 6 | A icle 187
Edi ed by:
Pina Zo lu una,
Uni e si y o No e Dame,
Uni ed S a es
Re iewed by:
Pie gio gio Gen ile,
Newcas le Uni e si y, Uni ed Kingdom
Pina Yilgo Hu i,
Anka a Uni e si y, Tu key
Jus in Lee B own,
Pennsyl ania S a e Uni e si y,
Uni ed S a es
*Co espondence:
Michael Gasik
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Bioma e ials,
a sec ion o he jou nal
F on ie s in Bioenginee ing and
Bio echnology
Recei ed: 16 June 2018
Accep ed: 16 No embe 2018
Published: 30 No embe 2018
Ci a ion:
Gasik M, Zühlke A, Haapa an a A-M,
Muhonen V, Laine K, Bilo sky Y,
Kellomäki M and Ki i an a I (2018) The
Impo ance o Con olled Misma ch o
Biomechanical Compliances o
Implan able Sca olds and Na i e
Tissue o A icula Ca ilage
Regene a ion.
F on . Bioeng. Bio echnol. 6:187.
doi: 10.3389/ bioe.2018.00187
The Impo ance o Con olled
Misma ch o Biomechanical
Compliances o Implan able
Sca olds and Na i e Tissue o
A icula Ca ilage Regene a ion
Michael Gasik1,2*, Alexand a Zühlke1, Anne-Ma ie Haapa an a3, Vi pi Muhonen4,
Kaisa Laine3, Ye gen Bilo sky2, Minna Kellomäki3,5 and Ilkka Ki i an a4
1School o Chemical Enginee ing, Aal o Uni e si y Founda ion, Espoo, Finland, 2Seq e a L d., Helsinki, Finland,
3BioMediTech and Facul y o Biomedical Sciences and Enginee ing, Tampe e Uni e si y o Technology, Tampe e, Finland,
4Depa men o O hopaedics and T auma ology, Uni e si y o Helsinki, and Helsinki Uni e si y Hospi al, Helsinki, Finland,
5BioMediTech and Facul y o Li e Sciences and Medicine, Uni e si y o Tampe e, Tampe e, Finland
Sca olds o a icula ca ilage epai ha e o be op imally biodeg adable wi h
simul aneous p omo ion o hyaline ca ilage o ma ion unde a he complex
biomechanical and physiological condi ions. I has been gene ally accep ed ha
sca old s uc u e and composi ion would be he bes when i mimics he s uc u e o
na i e ca ilage. Howe e , a epa a i e cons uc mimicking he ma u e na i e issue in a
healing issue si e p esen s a biological misma ch o epa a i e s imuli. In his wo k, we
s udied a new ecombinan human ype III collagen-polylac ide ( hCol-PLA) sca olds.
The hCol-PLA sca olds we e assessed o hei ela i e pe o mance in simula ed
syno ial luids o 1 and 4 mg/mL sodium hyalu ona e wi h applica ion o model- ee
analysis wi h Bioma e ials Enhanced Simula ion Tes (BEST). Pu e PLA sca old was
used as a con ol. The BEST esul s we e compa ed o he esul s o a p io in i o s udy
wi h hCol-PLA. Collec i ely he da a indica ed ha a success ul a icula ca ilage epai
equi e lowe s i ness o he sca old compa ed o su ounding ca ilage ye ma ching
he s ain compliance bo h in s a ic and dynamic condi ions. This ensu es an op imal
combina ion o load ans e and e ec i e oscilla o y nu ien s supply o he cells. The
esul s encou age u he de elopmen o in elligen sca old s uc u es o op imal
a icula ca ilage epai a he han simply ying o imi a e he espec i e o iginal issue.
Keywo ds: a icula ca ilage, sca old, PLA, collagen, biomechanics, es ing, syno ial luid
INTRODUCTION
The need o de elop issue subs i u es and egene a ion pla o ms is one o he mos demanding
and challenging applica ions in mode n issue enginee ing (Hubbell, 1995; Bu dick and Mauck,
2011). Th ee-dimensional bioma e ial s uc u es (sca olds) a e highly desi able ma ching he
biomechanical p ope ies o he issue (Gomes and Reis, 2004) and closely mimicking in i o
beha io [ acili a ing cell adhesion, g ow h, and issue o ma ion (Vol son e al., 2008)]. Such
bioma e ials assis he body o ebuild he damaged issue and e en ually hey minimize associa ed
Gasik e al. No el Sca olds o A icula Ca ilage Repai
pain and healing ime (Wong and B onzino, 2007; Chung and
Bu dick, 2008). The combined s a ic and dynamic biomechanical
p ope ies o hese sca olds a e c ucial o he inal success o
he ea men . Any p og ess in he de elopmen o sca olds
should ensu e a high co ela ion be ween in i o condi ions
and expec ed in i o issue egene a ion (F os , 2004; Wilson
e al., 2006; Mollon e al., 2013). The non- oxic biodeg ada ion
o he sca old should g adually ans e he s ess o he new
g owing issue o e an app op ia e ime pe iod. As poin ed ou
ecen ly (Panade o e al., 2016), he syne ge ic e ec o co ec
mechanical s imula ion is g ea ly dependen on he sca olding
ma e ial, i s en i onmen and he cell p esence. This shows he
needs o consis en simul aneous analysis o compa e di e en
bioma e ials and o ge conclusions abou hese ea u es.
One o he mos challenging applica ions o biomedical
sca olds is he a icula ca ilage (AC) epai . The damage
and deg ada ion o AC a e no only p og essing wi h age,
obesi y, o sys emic diseases, bu also in he young and ac i e
popula ion due o physical causes, such as inju y. I un ea ed,
hese de ec s may p og ess owa d os eoa h i is (OA), a ec ing
o e 150 million people wo ldwide, mainly by degene a ion
o he hyaline ca ilage in syno ial join lacking he abili y o
sel - egene a ion (A ms ong and Mow, 1982). Na u al wound
healing, in ull- hickness de ec s o ca ilage, o en leads o
he o ma ion o ib oca ilage (A ms ong and Mow, 1982;
Wilson e al., 2006; Mollon e al., 2013; Panade o e al., 2016),
which is unc ionally and biomechanically in e io o he o iginal
hyaline ca ilage making he issue mo e p one o u he
de e io a ion and os eoa h i ic changes o he join . Ini ia ed
icious cycle (Bende s e al., 2012) ul ima ely will call o a
o al o pa ial join eplacemen . The e o e, chond o-conduc i e
and -induc i e bioma e ials a e highly desi able o ea ca ilage
lesions a ea ly s ages be o e mani es a ion o OA.
Clinically used bioma e ials include a ious na u ally de i ed
and syn he ic ma e ials ( on Recum, 1998; Ag awal and Pa
JE, 2000). The ad an age o na u al ma e ials is hei in insic
bioac i i y o he pu pose, al hough applica ion o animal-
de i ed ma e ials (xenog a s) con ains ce ain isks, such as
con amina ion and undesi ed immune esponse. This could be
a oided by using bioabso bable syn he ic ma e ials no causing
o eign body o hype sensi i i y eac ions hemsel es. Syn he ic
ma e ials can be made biologically mo e ad an ageous and
biocompa ible. On he o he hand, compa ed o he na u ally
de i ed ma e ials, syn he ic polyme s a e usually lacking he
desi ed in insic biological cues ha p omo e cell adhesion,
p oli e a ion and issue eco e y. Howe e , any bioma e ial is
always challenging o e alua e and op imize o clinical use
and o he pu pose aiming on “p ecise medicine” solu ions.
I is now widely an icipa ed ha he p esen le el o e alua e
he mechanical unc ion o bioma e ial and issue enginee ing
cons uc s is highly insu icien . Fo example, o 205 analyzed
a icles on ca ilage issue enginee ing, men ioning o applied
mechanical s imula ion, only 29% shows some quan i ied
ma e ial p ope ies (Lujan e al., 2011). Co ec and de ailed
bioma e ial es ing is a he ime-consuming and expe ise o
p ope ly quan i y non-elas ic ma e ial beha io o issue is also
sca ce in many dedica ed biology labs (Lujan e al., 2011).
Syn he ic ma e ials wi h ib ous o igin a e o en used o
AC epai applica ions. These sca olds ha e 75–85% po osi y
and hey a e exposed o syno ial luid wi h sodium hyalu ona e
(NaHA). Animal s udies a e needed o ensu e he biological
unc ionali y o he sca olds be o e clinical use. Howe e , he
ela ionship be ween he na u al issue and he sca old is
challenging o measu e. The egula ions and he wo ldwide
ends impose mo e p essu e o mo e om animal models in o in
i o e alua ion (Di ec i e 2010/63/EC o Al e na i e Me hods,
2015). The e o e, in o de o de elop and op imize bioma e ials,
one mus es ablish p o ocols o eliable compa ison o di e en
ma e ials be o e in i o es s can be e hically jus i ied and hei
esul s uly ex apola ed owa d sa e and e ec i e human use.
The s uc u e, unc ions and biomechanical beha io o AC
a e e y complex, highly aniso opic and ime- and loading
his o y-dependen (Wilson e al., 2005). The a icula ca ilage
consis s o a ela i ely small numbe o chond ocy es su ounded
by a mul i-componen ma ix, which can be imaged as a
composi e wi h 70–85% wa e and emaining p o eoglycans
(p o eins wi h glycosaminoglycans a ached as a bo leb ush-like
s uc u e) and collagen (Hayes, 1972). P o eoglycans and wa e
concen a ion a y h ough he dep h o he ca ilage issue.
P o eoglycans can bind o agg ega e o a backbone o sodium
hyalu ona e (NaHA) o molecula weigh o 2–4 MDa o o m
a mac omolecule weigh ing up o 200 MDa (Kobayashi e al.,
1994).
The biomechanics o AC and syno ial luid is also complex
and essen ially non-linea (Hayes and Mock os, 1971; Hayes
and Bodine, 1978). No some many s udies ha e cohe en ly and
sys ema ically analyzed AC p ope ies (Ahsan and Sah, 1999;
Ko honen e al., 2006) due o a iabili y o he samples, local
inhomogenei y and applied biomechanical me hods. Complex
loading schemes a e associa ed wi h signi ican a ia ions o
in e s i ial luid p essu e and luid low, complica ing he esul s
in e p e a ion (Ahsan and Sah, 1999). The collagen- ich ma ix
beha io is highly non-linea and equi es a he sophis ica ed
models o be desc ibed as a composi e ma e ial, whe he wi h
heo ies (Mäkelä and Ko honen, 2016). Syno ial luid is well-
known o ha e non-New onian iscosi y s. i s composi ion,
shea a e, mode o loading and he p esence o o he ac o s
(King, 1966). Mos o he biomechanical p ope ies o AC
issue epo ed expe imen ally a e ob ained wi h ei he con ined
comp ession (Mow e al., 1980) o inden a ion (Kempson e al.,
1971). These measu emen s da a a e commonly app oxima ed
wi h biphasic (Mak e al., 1987) o iphasic (Lai e al., 1991)
heo ies, o e en mo e simpli ied iscoelas ic models. Howe e ,
due o peculia i ies o he AC issue p ope ies (Lai e al., 1981),
i is di icul o compa e esul s published wi h di e en s udies,
using a ious specimen ypes, me hods and es ing de ices. I
was also epo ed (Hosseini e al., 2014) ha luid low and
low-dependen phenomena may domina e he AC beha io in
di e en es ing egimes and hus i is impossible o de e mine in
gene al equi ed eco e y ime. Agg ega e modulus in ange o
50–120 kPa was epo ed o human, bo ine and canine issues
by di e en sou ces (Hayes and Mock os, 1971; Kempson e al.,
1971; Hayes, 1972; Mow e al., 1980; Lai e al., 1981; A ms ong
and Mow, 1982; Wilson e al., 2006), bu o en ull es da a
F on ie s in Bioenginee ing and Bio echnology | www. on ie sin.o g 2No embe 2018 | Volume 6 | A icle 187
Gasik e al. No el Sca olds o A icula Ca ilage Repai
we e no a ailable o compa e hese da a [inden a ion usually
p oduces much la ge alues (Ahsan and Sah, 1999; Ko honen
e al., 2006)]. Fo mal models o AC a e missing essen ial ea u es
which limi hei p ac ical applica ion only o specimens analyzed
in ha s udies. The e o e, i is a g ea o e simpli ica ion o
cha ac e ize AC o sca olds o AC epai by se o one o wo
numbe s wi hou exac da a on he es me hod and da a analysis.
In his s udy we used highly po ous PLA mesh manu ac u ed
om ine PLA ibe s. E en hough PLA i sel is a s i ma e ial,
his s udied PLA mesh was op imized o ha e a ela i ely so
na u e o sui be e as ca ilage epai ma ix. The hypo hesis
was ha a sca old which is less s i han su ounding issue
and which is ac ing in comp ession unde he equi emen o
s ain compliance will ha e less s ess and he e o e luid p essu e
which would cause luid o low in o he sca old o b ing mo e
nu ien s o chond ocy es. The collagen componen was added
o he PLA mesh o inc ease he hyd ophilic na u e o he
sca olds and o p omo e cell p oli e a ion (Muhonen e al., 2016;
Gasik e al., 2017). He e we epo esul s o his new xeno-
ee, ecombinan human collagen-laden ( hCol) polylac ide
(PLA) mesh sca olds ( hCol-PLA) de eloped o epai o ea ly
ca ilage lesions o a oid os eoa h i ic changes, which ha e
been designed, p oduced, and biomechanically op imized in
i o and in i o alida ed in equine (unpublished da a) and
po cine models (Muhonen e al., 2016; Gasik e al., 2017). The
hCol-PLA sca olds we e assessed o hei ela i e pe o mance
in simula ed syno ial luids o mimicking bo h human and
e e ina y condi ions wi h applica ion o model- ee analysis
wi h Bioma e ials Enhanced Simula ion Tes (BEST). The esul s
o he sca old ma e ials selec ion we e also co ela ed wi h in
i o es s, ca ied ou in a sepa a e s udy (Haapa an a e al.,
2014; Muhonen e al., 2016), whe e his ma e ial combina ion
was ound o wo k be e han he p e iously s udied plain PLA
sca olds wi h s i e s uc u e (Pulliainen e al., 2007).
MATERIALS AND METHODS
Ma e ials Analyzed
The sca olds es ed we e made o syn he ic polyme ibe s.
The polylac ide sca old (PLA) was p ocessed o medical g ade
poly-(L/D)-lac ide PLA96/4 (Co bion Pu ac, Go inchem, NED),
manu ac u ed o mel spun ibe s and a e wa ds ca ded and
needle punched in o meshes (po osi y ∼90–93%) in Tampe e
Uni e si y o Technology (Tampe e, Finland). The used PLA was
a highly pu i ied, medical g ade polyme (Länsman e al., 2006)
wi h a esidual monome con en o <0.5%. The PLA meshes
we e washed wi h e hanol, d ied, packed and s e ilized by gamma
i adia ion 25 kGy. A pa o PLA sca olds was asep ically doped
(Haapa an a e al., 2014) wi h a ecombinan human collagen
III (Fib oGen, Inc., San F ancisco, USA) solu ion and he
s uc u e was eeze-d ied (ma ked as hCol-PLA). The hCol-
PLA sca olds we e u he c osslinked wi h 14 mM 1-e hyl-3-
(3-dime hylaminop opyl)-ca bodiimide hyd ochlo ide (EDC) +
6 mM N-hyd oxysuccinimide (NHS) (Sigma-Ald ich, Helsinki,
Finland) in 95% e hanol, washed and subsequen ly eeze-d ied
again. The a io be ween he PLA and collagen componen s in
he hCol-PLA sca olds was 86/14 ol. % o PLA and collagen,
espec i ely.
All he specimens o PLA and hCol-PLA sca olds we e cu
in o ec angula pieces ∼5×5 mm (±1 mm) in size wi h he
hickness o he o iginal ma e ials as supplied. The exac size o
he specimens was measu ed wi h a non-con ac me hod using a
lase mic ome e (Me aLigh , CA, USA) wi h ±1µm esolu ion
and he samples we e weigh ed wi h a balance be o e and a e
he es . The measu ing and weighing p ocess was epea ed h ee
imes; on he d y sample, on he imme sed sample and on he
sample a e he measu emen . The samples we e imme sed in
dis illed wa e o ensu e ha he sample was comple ely we
be o e inse ing o he sample holde as possible apped ai
bubbles may al e he es esul s imp ope ly.
The media-simula ed syno ial luid (SSF)— o he es s was
p epa ed as wo solu ions wi h di e en concen a ions o
sodium hyalu ona e (NaHA). Sodium hyalu ona e o molecula
weigh 1.68 MDa (Nu ihyl R
, Con ip o Bio ech, Czech Republic)
was dissol ed in 200 mL o cold dis illed wa e o mimic “no mal”
(4 mg/mL) and “os eoa h i ic” (1 mg/mL) solu ions (Fam e al.,
2007).
Expe imen al Me hods
The iscosi y o he SSF solu ions was de e mined using SV-
10 ib o- iscosime e (A&D Co. L d., JAP) consis ed o wo
ib a ing gold pla es imme sed in he solu ion. Abou 45 mL o
he SSF solu ion was pou ed in a cu e e and hea ed o 40◦C. The
cu e e was hen placed a he iscome e and he iscosi y wi h
empe a u e was measu ed simul aneously upon ee cooling.
Viscosi y cu es we e well- i ed o e e y composi ion wi h he
A henius equa ion.
The biomechanical analysis was ca ied ou using wo
dynamic mechanical analysis (DMA) 242C and 242E machines
(Ne zsch Ge ä ebau GmbH, Ge many) wi h a specially de eloped
bioma e ials enhanced simula ion es (BEST; Seq e a L d.,
Finland) p o ocol (Gasik, 2014, 2017a,b), adjus ed o simula ed
ca ilage condi ions (Hayes and Bodine, 1978; Mow e al., 1980;
Lai e al., 1981). The comp essi e mode sample holde and
he specimen we e ully imme sed in he he mally con olled
ba h wi h media (∼30 mL). This esembles he g adien s o
de o ma ion, po e p essu e and luid low simila o ibial
ca ilage condi ions as has been shown wi h o he expe imen s
and compu e simula ions (Ko honen e al, 2002; Milan e al.,
2010). Th ee di e en p o ocols we e applied o imme sion es s:
c eep (n=38 o PLA and n=40 o hCol-PLA), equency
scans (0.01–20 Hz) as n=38 o PLA and n=28 o hCol-PLA,
and s ain sweeps up o 25–50 µm a 1 Hz) as n=20 o PLA and
n=30 o hCol-PLA.
A p econdi ioning s ep was applied (Piole i and
Rako omanana, 2000) o all specimens by an axial con inemen
by ∼5µm o ini ial de o ma ion (o se ), ollowing he 15 min
equilib a ion unde a small o ce o 0.05 N o s abilize he
dimensions and empe a u e. This was ound o supp ess ini ial
swelling (whe e p esen ; as explained below) hus all he c eep
de o ma ion and compliance a e o igina ed om ze o. A hese
condi ions i was obse ed ha de o ma ion o he po ous,
ully sa u a ed ib ous s uc u es p oceeded wi hou excessi e
F on ie s in Bioenginee ing and Bio echnology | www. on ie sin.o g 3No embe 2018 | Volume 6 | A icle 187
Gasik e al. No el Sca olds o A icula Ca ilage Repai
de o ma ion o he ibe s hemsel es and wi hou subs an ial
dec ease in po osi y. Whe eas, in i o p essu es exp essed on
heal hy a icula ca ilage may each 1–10 MPa a peak, he e he
luid low is uncon ined bu he sca old as AC issue unde goes
de o ma ion simila o one in clinical condi ions (Lai e al., 1981,
1991; Milan e al., 2010).
Da a Analysis
Expe imen al da a has been con e ed in o biomechanical
alues and analyzed wi h an applica ion o idempo en ype
analysis wi hou use o a ma e ial model (Gunawa dena, 1996;
Piole i and Rako omanana, 2000; Li ino e al., 2001). This
gi es an ad an age o e commonly epo ed moduli unc ional
dependence as i allows ex ac ion o he ime-in a ian da a
sui able o u u e p edic ion o he ma e ial beha io . The da a
quali y epo ed o he same ma e ial migh be also con using,
as no exac in o ma ion is gi en o condi ioning changes, and
usually no solid p oo shown, e.g., abou sui abili y o he small
s ain heo y o ma e ial linea i y (No is, 2008; Lujan e al.,
2011). Such condi ions a e o en assumed by de aul , despi e i is
o common knowledge ha “elas ic modulus” canno be uniquely
de ined o ma e ial which does no ollow linea elas ici y model.
Idempo en p ocessing, common in compu e echnology
(Gunawa dena, 1996) p ese es he da a s uc u e and o iginal
a iables wi hou demand o explici knowledge o hei
unc ional dependencies. I obeys causali y p inciple ( esponse
always comes a e he s imulus applied) and espec s he
bounda ies o he modynamics (no iola ion o conse a ion
laws). One essen ial ad an age in es ing o bioma e ials wi h
his me hod is in aking in o accoun non-local e ec s—on
he con a y o con en ional ma hema ical analysis, whe e he
de i a i e o a unc ion is always local. Hence, he p edic o s
can be used in in silico simula ions o calcula e, o example,
mechano- egula i e index wi hou necessi y o explici local luid
low de e mina ion (Maslo , 1970; Gasik, 2017b).
S ess was calcula ed as Piola–Ki chho s ess, om he a io
o ac ing o ce o he o iginal su ace a ea A0o he specimen, σ
=F/A0.The s ain was calcula ed as Lag ange ( ue loga i hmic)
s ain, ela ed o he ini ial hickness H0a he beginning o he
c eep: ε( ) =ln(1 +1L( )/H0), whe e 1L( ) is he obse ed
change in he specimen hickness wi h expe imen ime .
Whe eas, o he s ain de ini ions can be also used, his one has
a igo ous he modynamic a ionale (Xiao, 1995; Luba da and
Chen, 2008). The a io o he s ain ε( ), o cons an s ess, σ0,
is he c eep compliance C( ) =ε( )/σ0, which is he main eadou
om he c eep expe imen s. Fo dynamic loading, he s ain
ampli ude is om a ha monic signal wa e o m ex eme, aking
in o accoun load his o y:
εω( )=1
2ln H0+1L( )+a0
H0+1L( )−a0(1)
whe e a0is he applied de o ma ion ampli ude a some ins an
equency ω. In his o ma , he i e e sible c eep o simila
de o ma ion is aken in o accoun o long expe imen imes
( ). All expe imen s we e p e o med in iplica e and o e e y
equency o s ain es 10 cycles we e used wi hin e e y un.
Hence o ime dependencies one should conside bo h long ime
( eal ime o he es —minu es and hou s) and sho ime ( ime
span wi hin one o ew dynamic cycles—seconds).
Tes eadou s om DMA expe imen s ha e been p ocessed
wi h model- ee idempo en me hods (Gasik and Bilo sky, 2018).
In gene al, he e is no explici ma hema ical o mula w i en
as he calcula ion is i e a i ely p og essing o e e y da a poin
collec ed. This allows inclusion o specimen his o y wi hou a
need o assump ion o ime ke nels (he edi a y in eg als). In
his wo k he app oach was used o ind o ins ance agg ega e
modulus, ma e ial memo y, s a ic and dynamic pe meabili y.
RESULTS
Ma e ials P epa a ion and P elimina y
Analysis
The iscosi y o SSF composi ions was app oxima ed as
unc ion o empe a u e wi h A henius equa ion, leading o
8.14·10−4·exp(3022.2/T) and 1.746·exp(1143/T) in mPa·s, o 1
and 4 mg/mL NaHA, espec i ely (co ela ion 2=0.9869–
0.9953). A 25◦C his gi es 20.5 and 80.9 mPa·s alues o hese
SSF ( o compa ison, wa e iscosi y is 0.89 mPa·s). The exac
alues a e no explici ly equi ed because he DMA de ice and
sample holde dynamics ha e been ecalib a ed o e e y ype o
SSF, and hus media iscosi y changes ha e been au oma ically
included in he es da a.
A he beginning o he es s i was disco e ed ha PLA
ma e ials exhibi e y high swelling a ios e en i hey we e
comple ely soaked in liquid be o e he es s. This is usually
aced in hyd ophobic ma e ials a ee swelling due o epulsi e
o ces and p essu e a ia ions (Benne hum and Weins ein, 2004).
I was o mula ed (Benne hum and Cushman, 1996) ha he
mac oscopic solid s ess enso ( ela ed o isible bulk swelling)
is combined o a he modynamic solid p essu e, a solid s ess
enso , a s ess due o he in e ac ion o he solid and liquid
phases, and a s ess due o he in e ac ion o he in e ace wi h
he solid phase as well as kine ic componen o cons i uen s. By
changing he condi ioning p essu e, i was ound ha he o ce
o ∼50 mN (equi alen o applied ∼1 kPa s ess) is equi ed o
supp ess he swelling bu no o cause p e-comp ession o he
sample, Figu e 1. This condi ioning p essu e was used in all hese
expe imen s o ge consis en esul s.
Pseudo-S a ic Expe imen s
The esul s o one se o c eep measu emen s a 0.2 N (∼4 kPa
applied s ess) a e shown in Figu e 2. The le el o applied s ess
o 4 kPa (∼30 mmHg) was conside ed o be a limi which does no
cause ca ilage-adjacen so issues nec osis (Goode and Shinn,
1977). The na u e o a c eep es is pseudo-s a ic (change o
s ain in ime a cons an applied s ess) and i is o en used o
e alua e iscoelas ic na u e o ma e ials and o app oxima e i
wi h some models (Bilo sky and Gasik, 2015). He e one may see
ha addi ion o NaHA o media does no a ec compliance o
PLA much, bu has a g ea e ec on hCol-PLA. I is no able
ha compliance o hCol-PLA in 1 mg/mL SSF is he highes ,
and in 4 mg/mL is a e age be ween 1 mg/mL and wa e (0
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Gasik e al. No el Sca olds o A icula Ca ilage Repai
FIGURE 1 | The e ec o condi ioning o ce (0.006 and 0.05N) on swelling and compac ion (a 0.2N) o PLA sca old ( wo expe imen al cu es o ei he o ces
combina ion).
FIGURE 2 | C eep compliance a 0.2 N o P LA and hCol-PLA in di e en
media (numbe s in pa en heses show NaHA concen a ion in SSF, mg/mL).
mg/mL NaHA). The highe is he compliance, he mo e easily
he ma e ial de o ms unde cons an load. Hence an obse a ion
can be d awn ha addi ion o c oss-linked collagen o PLA makes
i “s i e ” when es ed in wa e bu makes li le di e ence when
es ing in 1 mg/mL SSF.
The compa ison o he da a om pseudo-s a ic (c eep)
analysis (Figu e 3) shows ha s i ness o he hCol-PLA sca olds
is inc eased by se e al imes when compa ed o PLA, and his
e ec is independen on he ype o SSF used. Also hCol-PLA
ma e ial in s a ic condi ions has lowe pe meabili y (Figu e 4),
which in combina ion suppo s a ision ha syno ial luid will
likely be kep in hCol-PLA be e han in PLA—a he same
loading, wi hin he same ime span PLA will lose luid o a g ea e
ex en .
FIGURE 3 | S a ic agg ega e modulus (kPa) o he sca olds s. NaHA
concen a ion in SSF. Ba s he e and u he indica e s anda d e o unless
s a ed o he wise.
F equency Expe imen s
Beha io o ma e ials unde cons an de o ma ion bu a ied
equency is di e en om pseudo-s a ic one. These di e ences
in he case o ib ous po ous ma e ials a e due: (1) oscilla ing
mobili y o luid wi hin a ib ous s uc u e, (2) ine ia e ec s
associa ed wi h hys e esis be ween incoming and ou going luid
low, and (3) non-linea i y in luid iscous p ope ies and
possible non-linea i y in coupled de o ma ion o he ib ous
skele on o he sca old.
One o he expe imen al c i e ia o obse e he di e ences
is he loss angen [ an(δ)], which is de ined as he a io o
imagina y o eal pa o elas ic moduli o s i ness. Highe loss
angen o hCol-PLA s. PLA (Figu e 5) was obse ed o all
equencies. Figu e 6 shows a 3D plo o hese dependencies o
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Gasik e al. No el Sca olds o A icula Ca ilage Repai
FIGURE 4 | S a ic (c eep) pe meabili y o sca olds s. NaHA concen a ion in SSF, indica ing ha s a ic pe meabili y inc eases a highe NaHA concen a ion.
FIGURE 5 | Loss angen o he sca olds in di e en SSF (NaHA concen a ion as numbe s, mg/mL) s. equency a 25 µm de o ma ion ampli ude.
an(δ) s. applied equency a 25 µm de o ma ion ampli ude
along wi h expe imen ime (no e he angen is p ac ically
cons an wi h he ime and depends essen ially on equency
only).
I is seen ha addi ion o NaHA o he media and espec i e
inc ease in iscosi y also leads o inc ease in he loss angen ( he
mo e, he highe is he NaHA concen a ion). Fo hCol-PLA
loss angen is oughly 2–3 imes highe han o PLA in all SSF
(Figu e 6). This indica es mo e ac i e in e ac ion o luid low
wi h hCol-PLA han PLA and is likely associa ed wi h a ines
collagen ib ils ne wo k be ween he PLA-based ibe s in hCol-
PLA (Länsman e al., 2006; Muhonen e al., 2016; Gasik e al.,
2017). No able is ha his in e ac ion is only due o p esence o
NaHA, as such di e ences a e no seen when only wa e is used
(Figu e 6).
S ain Sweep Expe imen s
One o he mo e physiologically ele an dynamic condi ions is
applica ion o a iable de o ma ion unde cons an equency.
This can be depic ed as change o he gai loads keeping no mal
walking condi ions (∼1 Hz) (Hayes and Mock os, 1971; Hayes,
1972; Mow e al., 1980; A ms ong and Mow, 1982). Thus, he
las es sequence was applied o simula e changes in p ope ies
o sca olds up o 50 µm o dynamic de o ma ion wi h epea ing
o he load cycles. A cons an equency, loss angen is no
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Gasik e al. No el Sca olds o A icula Ca ilage Repai
FIGURE 6 | Loss ac o [ an(δ)] o sca olds in di e en SSF s. applied equency a 25 µm displacemen ampli ude. No e plo s o wa e (0 mg/mL NaHA; su aces
No. 5 and 6) a e shi ed ahead o ime scale o imp o e isual eadabili y o he plo s.
FIGURE 7 | A e age dynamic modulus o sca olds a 1 Hz s. applied de o ma ion ad di e en SSF (numbe s indica ing NaHA concen a ion, mg/mL). No e log scale
o modulus.
signi ican ly de o ma ion- o s ess-dependen so he e majo
pe o mance comes om dynamic s i ness and luid exchange.
The absolu e alue o a e age dynamic elas ic modulus a 1 Hz
is shown in Figu e 7 as di ec ly ob ained om he DMA signal.
I is seen ha his modulus sligh ly dec eases wi h de o ma ion.
Howe e , wi h inc eased numbe o loading cycles and ue s ain
a ia ions due o changes in geome y, ue (co ec ed) elas ic
modulus sligh ly inc eases. Also, highe NaHA concen a ion
shows highe s i ness o all ma e ials bu i is no ewo hy
his s i ness inco po a ed luid mo emen unde dynamic load
and he e o e una oidably includes some iscous and ine ia
con ibu ion, as shown abou o loss angen (Figu es 5,6).
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Gasik e al. No el Sca olds o A icula Ca ilage Repai
FIGURE 8 | Compa ison o s a ic agg ega e modulus (Figu e 3) wi h in a ian dynamic modulus a 1 Hz. Numbe s indica e concen a ion o NaHA in SSF, mg/mL.
FIGURE 9 | Compa ison o a io o dynamic in a ian modulus o dynamic pe meabili y s. NaHA concen a ion (no e log scale).
Analyzed in a ian modulus a 1 Hz is shown in Figu e 8 s.
espec i e s a ic agg ega e modulus (Figu e 3) o espec i e
NaHA concen a ion. The a io o dynamic modulus o dynamic
pe meabili y is shown in Figu e 9 o 1 Hz condi ion. He e
hCol-PLA is a leas simila o be e (a 1 mg/mL NaHA) han
PLA sca olds.
DISCUSSION
The wo s udied sca olds con ained he same kind o PLA mesh
s uc u es. The PLA sca olds we e s udied as such and in he
hCol-PLA sca olds he collagen componen was added in o
he s uc u e o gi e he highly po ous PLA sca old inc eased
hyd ophilic na u e and o p omo e cell p oli e a ion. F om he
pos -p ocessing o he expe imen al da a, many addi ional alues
ha e been ob ained wi hou assump ion o a ma e ial model (a
p op ie a y pa en -pending me hod). He e da a o agg ega e
modulus (in s a ic and dynamics) and pe meabili y a e shown as
an example.
The ele ancy o s a ic condi ions esul s o clinical
condi ions is ha hCol-PLA sca olds a e be e suppo i e o
weigh -bea ing and unde go smalle de o ma ion han pu e PLA.
In combina ion o lowe pe meabili y his sugges s syno ial luid
o s ay likely hCol-PLA mo e han in PLA, whe eas he la e will
lose mo e luid a he same loading. As a simple decision-aiding
c i e ion, one migh conside he a io o agg ega e modulus o
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Gasik e al. No el Sca olds o A icula Ca ilage Repai
pe meabili y: he highe i is, he be e he sca old wi hs ands
s a ic loads. Figu e 10 shows ha in his espec hCol-PLA
ma e ial is by 1–2 o de s o magni ude supe io o PLA a all
es ed SSF composi ions.
A dynamic condi ions, such as walking, he si ua ion
e e ses: unde dynamic loading one has o aim on mo e
ac i e luid exchange o p o ide biomechanical s imulus o
chond ocy es, o ensu e luid and nu ien s supply and emo al
o me abolic p oduc s, p omo ing issue egene a ion (Ge isch
and Chaplain, 2006; Si ichokechaiwu e al., 2010). Ca ilage
issue is a ascula and i s ex acellula ma ix c ea es u he
ba ie s o nu ien /was e exchange by di usion. Thus, lowe
dynamic modulus (Figu e 8) and be e luid di usi i ies a e
desi ed. These ea u es ha e o be, howe e , compa ible o he
abo e equi emen s o s a ic condi ions as ca ilage mus wo k
well in bo h hese ex emes.
The iscosi ies o SSF a e subs an ially highe han wa e :
abou 20 and 100 imes o 1 and 4 mg/mL, espec i ely. This
means ha e en small changes in pe meabili y, i.e., a ea u e
o he ma e ial s uc u e, will a ec changes in pe mi i i y, a
ea u e o a speci ic luid low hough he ma e ial s uc u e.
High loss angen (Figu es 5,6) means mo e dissipa ion o
FIGURE 10 | Compa ison o a io o s a ic agg ega e modulus (Figu e 3) o
pe meabili y (Figu e 4) s. NaHA concen a ion (no e log scale).
applied mechanical ene gy (inelas ic losses) which is impo an o
keep high damping p ope ies o a icula ca ilage (Mow e al.,
1980; Lai e al., 1981; A ms ong and Mow, 1982). The e o e,
hCol-PLA is supe io o PLA also in his p ope y, whe he
o “a h i ic” (1 mg/mL NaHA) o “no mal” (4 mg/mL NaHA)
syno ial luids.
A p oo -o -concep animal s udy was pe o med in domes ic
pigs (Sus sc o a domes ica, 4-mon hs-old, n=20) in a sepa a e
s udy, epo ed elsewhe e (Muhonen e al., 2016). B ie ly, he
animals we e andomized in o h ee g oups: (1) hCol-PLA
sca old ea men , (2) comme cial sca old ea men and (3)
spon aneous epai . A ci cula ull- hickness chond al lesion
wi h a diame e o 8 mm was c ea ed in he igh medial
emo al condyle. The emo ed ca ilage issue was collec ed
and u he p ocessed o chond ocy e isola ion and subsequen
p oli e a ion. A e 3 weeks, he lesion was app oached again,
cleaned and epai ed wi h one o he cons uc s, i.e., hCol-
PLA o comme cial sca old wi h chond ocy es, o le un ea ed.
Only one lesion pe animal was pe o med and he animals
we e allowed ee weigh -bea ing and un es ic ed mo emen
a e he ope a ions. The epai issue was e alua ed a e 4
mon hs. Hyaline ca ilage was epo ed o be o med mos
equen ly in he hCol-PLA ea men g oup. He e he analysis
o he ca ilage epai sco es (Haapa an a e al., 2014; Muhonen
e al., 2016) was addi ionally pe o med using BUGS—Bayesian
in e ence Using Gibbs Sampling (US FDA, 2010), a o m o
a Ma ko Chain Mon e Ca lo sampling. The esul s o using
no mal o Poisson dis ibu ions o he o al no malized ICRS
(In e na ional Ca ilage Repai Socie y) sco es show hCol-PLA
ha ing s a is ically signi ican highe a e age sco e (0.515) s.
0.38 o comme cial sca old and 0.288 o spon aneous healing
con ol g oup (Muhonen e al., 2016).
A schema ic o he ad an age o lowe dynamic s i ness
sca old o AC epai is depic ed in Figu e 11. The e a sca old
implan ed in o a ca ilage de ec should exhibi he same s ain
compliance (no ea s, wis ing o buckling). Fo he same s ain,
ma e ial wi h a highe appa en s i ness will gene a e mo e
in e nal s esses and hence highe luid p essu e (Hayes, 1972;
Mow e al., 1980; Lai e al., 1991). This will lead o p e e en ial
luid mo emen ou o ca ilage which will no allow cells
and issue egene a ion (“d y-ou ”). Fo an opposi e, lowe
FIGURE 11 | Possible mechanism o bene icial compliance misma ch o sca olds s. na i e issue when submi ed o a mechanical load: when a sca old wi h luid is
oo s i , highe gene a ed luid p essu e leads o luid low ou o sca old (le ), whe eas o he same de o ma ion condi ions o lowe s i ness sys em ( igh ) luid is
d i en in o he sca old.
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