h ps://doi.o g/10.1177/19476035231154507
Ca ilage
2023, Vol. 14(2) 220 –234
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DOi: 10.1177/19476035231154507
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Ca ilage Repai Sca olds
1154507Ca XXX10.1177/19476035231154507CARTILAGEWei kamp e al.
esea ch-a icle20232023
1Depa men o O al and Maxillo acial Su ge y, Uni e si y Medical Cen e Schleswig-Hols ein, Campus Kiel, Kiel, ge many
2Depa men o ana omy, Kiel Uni e si y, Kiel, ge many
3 e eC issue enginee ing echnologies ag, eu lingen, ge many
4g.e. .N. esea ch Cen e o issue eplacemen , egene a ion & Neogenesis, Depa men o O hopedics and auma Su ge y, Medical Cen e
albe -ludwigs-Uni e si y o F eibu g, Facul y o Medicine, albe -ludwigs-Uni e si y o F eibu g, F eibu g, ge many
5Clinic o auma Su ge y, Diako Hospi al Flensbu g, Flensbu g, ge many
6Depa men o auma and O hopedic Su ge y, Uni e si y Medical Cen e Schleswig-Hols ein, Campus Kiel, Kiel, ge many
7Depa men o auma Su ge y, O hopedics and Spo so hopedics, asklepios Klinik S . geo g, Hambu g, ge many
Co esponding Au ho :
Jan- obias Wei kamp, Depa men o O al and Maxillo acial Su ge y, Uni e si y Medical Cen e Schleswig-Hols ein, Campus Kiel, a nold-Helle -S . 3,
Kiel 24105, ge many.
email: [email p o ec ed]
In Vi o Compa ison o 2 Clinically Applied
Bioma e ials o Au ologous Chond ocy e
Implan a ion: Injec able Hyd ogel Ve sus
Collagen Sca old
Jan-Tobias Wei kamp1,2 , Ka in Benz3, Be nd Rolau s4,
And eas Baye 2, Ma hias Weus e 5, Ralph Lucius2, Aydin Gülses1,
Hend ik Naujoka 1, Jö g Wil ang1, Sebas ian Lipp oss6,
Michael Ho mann7, Bodo Ku z2, and Pe e Beh end 2,7
Abs ac
Objec i e. in au ologous chond ocy e implan a ion (aCi), he e is no consensus abou used biosca olds. he aim o his
s udy was o pe o m an in i o compa a i e analysis o 2 clinically applied bioma e ials o ca ilage lesion ea men . Design.
Monolaye expanded human chond ocy es (n = 6) we e embedded in a collagen sca old (CS) and a hyalu onic acid–based
hyd ogel (Ha). Cells we e cul u ed in chond ope missi e medium supplemen ed wi h and wi hou in e leukin-10 (il-10)
and bone mo phogene ic p o ein–2 (BMP-2). gene exp ession o chond ogenic ma ke s (COl1a1, COl2a1, COl10a1,
aCaN, SOX9) was de ec ed ia quan i a i e eal- ime–polyme ase chain eac ion ( -qPC ). Biosyn hesis o ma ix
compounds, cell iabili y, mo phology as well as mig a ion om su ounding na i e bo ine ca ilage in o cell- ee sca olds
we e analyzed his ologically. adhesion o he ma e ial o adjacen ca ilage was in es iga ed by a cus om-made push-ou
es . Resul s. he shi o COl1/2 a io owa d COl2a1 was mo e p onounced in Ha, and cells displayed a mo e sphe ical
mo phology compa ed wi h CS. BMP-2 and il-10 signi ican ly inc eased COl2a1, SOX9, and aCaN exp ession, which
was pa alleled by enhanced s aining o glycosaminoglycans (gags) and ype 2 collagen in his ological sec ions o CS and
Ha. COl10a1 was no signi ican ly exp essed in Ha and CS. Be e in e acial in eg a ion and enhanced cell in asion was
obse ed in CS. Push-ou es s using CS showed highe bonding s eng h o na i e ca ilage. Conclusion. Ha-based hyd ogel
e ealed a mo e chond ocy e-like pheno ype bu only allowed limi ed cell in asion, whe eas CS we e ad an ageous in
e ms o cellula in asion and in e acial adhesion. hese di e ences may be clinically ele an when ea ing ca ilaginous
o os eochond al de ec s.
Keywo ds
au ologous chond ocy e implan a ion, collagen sca old, hyd ogel, hyalu onic acid, human a icula chond ocy es, in i o
compa ison
Wei kamp e al. 221
In oduc ion
A icula ca ilage epai emains a challenging ask in he
ield o o hopedic su ge y aiming o p e en join dys unc-
ion and he de elopmen o pos - auma ic os eoa h i is.
Au ologous chond ocy e implan a ion (ACI) has become
he gold s anda d o he egene a i e ea men o la ge size
ca ilage de ec s.1,2 Wi hin he las 3 decades, he o iginally
in oduced echnique was u he de eloped by u ilizing
addi ional bioma e ials as cell-ca ie sys ems.3,4
Di e en gene a ions o ACI de elopmen ha e aimed o
o e come he issue o chond ocy e de-di e en ia ion,
which impai s he chond ocy e unc ion and esul s in in e-
io quali y o he egene a i e issue.5 Thi d-gene a ion
ACI consis s o a cell-bioma e ial p oduc ha has imp o ed
bo h he clinical ou come and he his ological quali y o he
egene a ed issue, which emphasizes he cons an op imi-
za ion p ocess o cell-based issue egene a ion ech-
niques.3,6 Nume ous bioma e ials ha e been ex ensi ely
s udied in p e-clinical in es iga ions, bu compa a i e s ud-
ies a e a e. The e o e, iden i ica ion o he ideal bioma e-
ial candida e ega ding i s cell-ma ix in e ac ions,
cy ocompa ibili y, biomechanical p ope ies, and su gical
applica ion echniques is insu icien . In addi ion, due o
egula o y es ic ions and inancial bu dens, i is no easi-
ble o examine he whole spec um o bioma e ials clini-
cally. A deepened knowledge abou bioma e ial ad an ages
in clinically es ablished bioma e ials may help o ocus cu -
en e o s in u he ACI de elopmen . In his ega d, bio-
ma e ials based on collagen and hyalu onic acid, which a e
inspi ed by he na u al a chi ec u e o a icula ca ilage, a e
o high in e es o in es iga o s in his ield.7-9 Fibe -based
ma ices ha e shown o suppo chond ocy e di e en ia ion
and o ma ion o ca ilaginous ma ix in se e al in i o and
in i o s udies.10,11 Hyd ogels, on he con a y, ha e also
been in es iga ed in ensi ely due o hei wa e -binding
capaci y ha mimics ca ilage issue.12 Especially, gels
based on na u ally de i ed biopolyme s such as hyalu onic
acid a e in e es ing o ca ilage issue enginee ing due o i s
chond op o ec i e e ec s.13
Ano he imp o emen in ACI ea men may be achie ed
by a p e-condi ioning using soluble adju an s ha imp o e
cell di e en ia ion p io o ACI implan a ion. In his con ex ,
in e leukin-10 (IL-10) and bone mo phogene ic p o ein–2
(BMP-2) ha e been desc ibed,14,15 which we e shown o ha e
po en ial chond ogenic e ec s, e en in he pos - auma ic
mic omilieu a e ACI g a ansplan a ion.16,17 Ou s udy
g oup p e iously demons a ed ha IL-10 signi ican ly
enhances chond ogenic di e en ia ion o cells embedded in
collagen ma ices and s abilized he chond ogenic pheno-
ype wi h less ex acellula ma ix loss a e mechanical ca -
ilage inju y.14,18 BMP-2, a membe o he ans o ming
g ow h ac o –β (TGF-β) supe - amily, plays an essen ial
ole in he egula ion o chond ocy e p oli e a ion.15
Gi en he abundan spec um o eligible bioma e ials,
deepened unde s anding o clinically applied sca olds may
help o ocus u he ACI de elopmen s. This s udy aims o
iden i y ad an ageous bioma e ial p ope ies o 2 clinically
used ACI g a s (mac opo ous ype I/III collagen ma ix s.
injec able hyalu onic acid-based hyd ogel) in a compa a i e
in i o s udy wi h espec o (1) cell iabili y, (2) chond o-
genic po en ial, (3) bio-adhesion, (4) la e al in eg a ion, and
(5) addi ional adju an s such as IL-10 and BMP-2. I was
hypo hesized ha bo h bioma e ials ha e dis inc biological
di e ences ega ding cell di e en ia ion and g a
in eg a ion.
Me hod
A icula Chond ocy e Isola ion and Cul u e
Human a icula chond ocy es (hCh; n = 6) we e isola ed
om emo al heads o pa ien s (mean = 70.3 ± 5.3 yea s)
unde going hip eplacemen su ge y (e hical app o al was
ob ained o he Uni e si y Kiel D572/17). The ca ilage is-
sue was dissec ed in o small pieces and diges ed wi h 0.1%
p onase (Roche, Mannheim, Ge many) ollowed by diges-
ion wi h ype 2 collagenase 600 U/ml (Wo hing on,
Lakewood, USA) in Dulbecco’s Modi ied Eagle Medium
(DMEM). Isola ed hCh we e cul u ed a a densi y o 10,000
cells/cm2 in chond ope missi e medium (CPM) consis ing
o high-glucose DMEM (HG-DMEM) supplemen ed wi h
10% Se a Plus (PAN-Bio ech, Aidenbach, Ge many), 10
mg/ml Penicillin G, 10 mg/ml o s ep omycin (PAA
Labo a o ies, Pasching, Ge many),1% L-asco bic acid
(Sigma-Ald ich, S . Louis, USA) and 2 ng/ml ib oblas ic
g ow h ac o –2 (FGF-2; R&D Sys ems, Minneapolis,
USA). Medium change was wice a week. Cells we e ha -
es ed a cell passage 3 (P3) by ypsin–e hylenediamine e -
aace ic acid (EDTA; Lonza, Cologne, Ge many) ea men
and used o u he in i o cul i a ion.
Embedding o hCh in HA and CS
Monolaye expanded chond ocy es (P3) (n = 6) we e seeded
ei he on o he mac opo ous pa o a biphasic ype 1/3 col-
lagen sca old (CS) as used in No oca 3D® (TETEC
Tissue Enginee ing Technologies AG, Reu lingen, Ge many)
o we e embedded in an albumin-hyalu onic acid–based
hyd ogel (HA) as used in No oca Injec ® (TETEC Tissue
Enginee ing Technologies AG, Reu lingen, Ge many).
The e o e, 2.5 × 106 hCh/ml/cm2 we e e-suspended in
HG-DMEM and seeded on o he CS and HA, espec i ely,
and cul i a ed in 24-well pla es coa ed wi h 2% aga ose
(Sigma-Ald ich, S . Louis, USA). The hyd ogel is based on
chemically ac i a ed maleolyl-albumin supplemen ed wi h
HA and was c oss-linked by a speci ic hio-polye hylene
glycol (PEG).13
222 CAR IlAgE 14(2)
Cellula ized cons uc s we e cul u ed in CPM (wi hou
Se a Plus) ha was supplemen ed wi h 1% insulin- an e in-
sodium selini e liquid media (Sigma-Ald ich, S . Louis, USA)
and 0.1 mM nonessen ial amino acids (Sigma-Ald ich, S .
Louis, USA) and enewed e e y 3 days. The samples we e
di ided in o 4 di e en ea men g oups: non-supplemen ed,
addi ional human IL-10 (100 pg/ml; King ishe Bio ech,
Sain -Paul, USA), addi ional human bone mo phogene ic p o-
ein–2 (hBMP-2; 250 ng/ml; R&D Sys ems, Minneapolis,
USA), and co- ea men wi h IL-10 and BMP-2.
Cell Viabili y, Mo phology, and Exp ession o
Mic o ibe Assessmen
To de e mine he mo phology and iabili y o cells wi hin
he bioma e ial li e/dead (L/D), s aining was pe o med
a e 7 and 28 days o cul u e. The e o e, HA and CS sam-
ples we e s ained wi h 10 μM calcein-AM and 5 μM
e hidium homodime -1 (bo h Sigma-Ald ich, Buchs,
Swi ze land). A e 1-hou incuba ion, he samples we e
imaged using con ocal lase scanning mic oscopy (CLSM
510; Ca l Zeiss, Ge many). To quan i y he numbe o
L/D cells, 3 images we e aken om 3 di e en ields o
iew, and a minimum o 100 cells we e coun ed using
image J (Wayne Rasband, NIH, USA). To isualize he
exp ession o F-ac in, addi ional s aining wi h phalloidin
and nuclea s aining (4′,6-diamidino-2-phenylindole
[DAPI]) was pe o med ollowed by CLSM.
gene Exp ession Analyses by Quan i a i e Real-
ime PCR
Gene exp ession was analyzed a e 1 and 28 days. HA sam-
ples we e p e-diges ed wi h p o einase K (3 mg/ml in
HG-DMEM; Roche, Ge many). To al RNA o he diges o
HA and CS samples was ex ac ed using he RNeasy Mini
Ki acco ding o he manu ac u e ’s ins uc ions (Qiagen,
Hilden, Ge many). Complemen a y DNA was ob ained by
e e se ansc ip ion using Qiagen RT-PCR Ki (Qiagen,
Hilden, Ge many). Quan i a i e eal- ime–polyme ase
chain eac ion (RT-qPCR) was pe o med using glyce alde-
hyde-3-phospha e dehyd ogenase (GAPDH) as he e e -
ence gene and Qiagen Quan iTec SYBR® G een RT-PCR
Ki acco ding o manu ac u e ’s ins uc ions wi h a 7500
Fas Real-Time PCR Sys em (Applied Biosys ems,
Da ms ad , Ge many). Human agg ecan (ACAN), ype 2
collagen (COL2A1), ansc ip ion ac o SOX-9 (SOX9),
ype 1 collagen (COL1A1), ype 10 collagen (COL10A1),
and GAPDH (all Biome s, Ulm, Ge many) p ime s we e
used a a concen a ion o 0.3 µM (Table 1). Da a analysis
was pe o med using a compa a i e quan i ica ion (ΔΔCT-
me hod). Un ea ed monolaye (P4) se ed as con ol g oup.
DNA Quan i ica ion and DMMB-Assay
A e 28 days, CS and HA samples (n = 3) diges ed wi h
0.5 mg/ml p o einase K (Roche, Mannheim, Ge many).
Sulpha ed glycosaminoglycans (sGAG) con en wi hin he
diges and he cumula i e supe na an was de ec ed wi h
modi ied 1,9-dime hylme hylene blue (DMMB) assay
(Sigma-Ald ich, Da ms ad , Ge many) acco ding o Zheng
and Le ens on.19 The DNA con en o he diges was quan-
i ied using a bisbenzimide-based DNA quan i ica ion assay
acco ding o manu ac u e ’s p o ocol (P omega, Mannheim,
Ge many).
His ology S udies
A e 28 days, samples we e moun ed in issue compound
and c yo-sec ioned ( hickness: 10 μm) using Mic om HM
56C yos a -Mic o ome (Ca l Zeiss AG, Zü ich, Swi ze land).
CS and HA samples we e s ained wi h oluidine blue acco d-
ing o s anda d p o ocol.20 Immunohis ochemis y (IHC) o
ype 2 collagen (mouse an i- ype-II-collagen an ibody; Clone
CIIC1, DSHB, Iowa, USA; as desc ibed in) and ype 1 col-
lagen (mouse an i ype-I-collagen an ibody; C-2456 Sigma-
Ald ich) was pe o med as desc ibed in Gille e al.21 and
Ku z e al.22
P epa a ion o Bo ine Ca ilage Plugs and
In asion Assay
Ca ilage cylinde s o bo ine s i le join s we e ha es ed as
desc ibed be o e.23 The de ec was illed wi h cell- ee HA
and CS (n = 6). Aga ose 2% (Lonza, Basel, Swi ze land)
se ed as nega i e con ol. Chond ocy e in asion om a
bo ine ca ilage in o he embedded bioma e ials was ana-
lyzed using CLSM (as desc ibed abo e) a e 28
Table 1. Human P ime Sequences (5'-3').
Human a ge Sequence (5'-3')
ACAN sense gaggCCagCagagaaga C g
ACAN an isense gaCgCC CgCC C gaa
COl2A1 sense CaaCaC gCCaaCg CCaga
COl2A1 an isense C gC Cg CCaga aggCaa
SOX9 sense C CggagaC C gaaCgagag
SOX9 an isense Cg C CaCCgaC CC CC
COl1A1 sense aa CCaaggCCaagaagCa g
COl1A1 an isense gg agCCa CC gg gg
COl10A1 sense CCC gC gC ag a CC ga
COl10A1 an isense aaC g g C gg g ggg ag g
gAPDH sense gCC Caaga Ca CagCaa gC
gAPDH an isense gg Ca gag CC CCaCga
ACAN = human agg ecan; COl2A1 = ype 2 collagen; SOX9 =
ansc ip ion ac o SOX-9; COl1A1 = ype 1 collagen, COl10A1 = ype
10 collagen; gAPDH = glyce aldehyde 3-phospha e dehyd ogenase.
Wei kamp e al. 223
days cul i a ion ime. The plugs we e cul i a ed in an 2%
aga ose coa ed 24-well pla e wi h CPM. Fo quan i ica ion
o cell in asion in %, he o al a ea o embedded bioma e i-
als was di ided in o 100 equal a eas. Using image J, a eas
colonized wi h cells we e coun ed. Images o n = 6 we e
used o quan i y cell in asion in CS, HA, and aga ose.
In es iga ions o he Bioma e ial/ issue In e ace
To de e mine he bonding s eng h o HA and CS o na i e
ca ilage issue, push-ou es s we e pe o med as desc ibed
be o e.23 Fo he push-ou es , he de ec was illed wi h
HA and CS (wi hou and wi h 2.5 × 106 hCh/ml/cm2) o 2%
aga ose (Lonza, Basel, Swi ze land) as nega i e con ol.
Tissue/sca old cons uc s we e cul u ed in CPM o up o
28 days. The mechanical es was pe o med wi h 6 ca i-
lage ings o each condi ion and epea ed using he ca i-
lage ings isola ed om 3 di e en animals. The bonding
s eng h was calcula ed by di iding he loading peak (N) by
he bonding a ea (m2).
Biomechanical es ing: Young’s Modulus
Biomechanical es s we e ca ied ou in a s anda d ma e ial-
es ing machine (Ins on 5866 elec omechanical es
de ice) equipped wi h a 10 N load cell. The ini ial sample
heigh h0 was measu ed wi h a calipe and samples placed
in a cell cul u e dish illed wi h chond ogenic medium. A
1-s ep uncon ined comp ession es was pe o med by load-
ing he sample h ough a la -ended inden e (0.02 N p e-
load) a a s ain a e o 0.5 mm/min un il 50% h0 s ain was
eached. The Young’s modulus was hen calcula ed a he
ini ial linea pa o he s ess-s ain-cu e (n = 6).
S a is ics
All da a we e es ed o no mali y using he Kolmogo o –
Smi no es . S a is ical analysis was pe o med using
G aph Pad p ism 5 p og am (San Diego, CA, USA). One-
way analysis o a iance (ANOVA) wi h Bon e oni’s mul-
iple compa ison was used o compa e means among he
independen expe imen al g oups. Di e ences we e consid-
e ed signi ican i P ≤ 0.05. Quan i a i e da a in he ex a e
p esen ed as mean and s anda d de ia ion (SD).
Resul s
Bo h Bioma e ials Suppo High Cell Viabili y
De ec ion o me abolic cell ac i i y a day 1 and day 28
showed simila esul s o hCh seeded in bo h bioma e ials.
Rela i e luo escence uni s inc eased signi ican ly om day 1
o day 28 in non-supplemen ed and IL-10 supplemen ed HA
and CS (HA day 1 s. day 28: P = 0.0004; HA + IL-10 day 1
s. day 28: P = 0.0176; CS day 1 s. day 28: P = 0.0005; CS
+ IL-10 day 1 s. day 28: P < 0.0001; Figu e 1a(A)). The
p esence o BMP-2 led o an ini ial signi ican inc ease o
me abolic cell ac i i y in bo h bioma e ials a day 1 (HA s.
HA + BMP-2 and HA s. HA + BMP-2 + IL-10 day 1: P <
0.0001; CS s. CS + BMP-2 and CS s. CS + BMP-2 +
IL-10 day 1: P < 0.0001; Figu e 1a(A)).
Cell quan i ica ion o li e and dead cells e ealed a high
p opo ion o iable hCh in HA and CS du ing 4 weeks o
in i o cul i a ion (HA day 1: 93% ± 1.76%; CS day 1:
84.33% ± 8.48%; Figu e 1a(B)). A day 28, signi ican ly
mo e iable hCh we e de ec ed in HA (P = 0.0006), bu s ill
mo e han 74% ± 8.85% hCh we e iable in CS.
Supplemen ing BMP-2 and IL-10 had no signi ican in lu-
ence on cell iabili y (da a no shown).
Mo phologically, hCh embedded in hyd ogel showed a
sphe ical pheno ype in con as o a ib oblas ic-like cell
pheno ype in he 3D collagen ma ix (Fig. 1b(A1 and C1)).
The ami ied cell pheno ype was pa alleled by g ea e
F-ac in accumula ion in cells embedded in CS (Fig. 1c(A
and B)).
Chond ocy es in Hyd ogel Show Supe io
Chond ogenic Re-Di e en ia ion
Cells cul i a ed in CPM showed a ime-dependen inc ease
o chond ogenic ma ke exp ession in bo h bioma e ials
(Fig. 2(A-F)), excep COL2A1 in un ea ed HA. Cell e-
di e en ia ion was s ongly enhanced by BMP-2 ea men ,
while supplemen a ion o IL-10 was less e ec i e. O e all,
a end o ma ked chond ogenic ma ke exp ession was
obse ed in he hyd ogel g oups as demons a ed by shi
om COL1A1 o COL2A1 (Fig. 2(F)).
In de ail, BMP-2 supplemen a ion signi ican ly
inc eased ela i e gene exp ession o COL2A1 in HA by
3.4 (P = 0.0039; Fig. 2(A)). A e 28 days, signi ican ly
inc eased COL2A1 exp ession was de ec ed in hose
expe imen al g oups ha had ecei ed he addi ion o
BMP-2 and he co- ea men o IL-10 and BMP-2 (HA day
28 s. HA + BMP-2 day 28: P < 0.0001; HA day 28 s.
HA + BMP-2 + IL-10 day 28: P < 0.0001). Fo CS
g oups, a compa able COL2A1 induc ion was obse ed
bu he exp ession le els emained signi ican ly lowe
compa ed wi h he hyd ogel g oups (HA + BMP-2 day 28
s. CS + BMP-2 day 28: P < 0.0001). T ansc ip ion le -
els o ACAN showed a compa able pa e n (Fig. 2(B)). In
HA g oups, ela i e mRNA exp ession was signi ican ly
ampli ied unde BMP-2 ea men (HA day 28 s. HA +
BMP-2 day 28: P = 0.0002; HA day 28 s. HA + BMP-2
+ IL-10 day 28: P < 0.0001) and was also signi ican ly
highe han o CS g oups a e 28 days (HA + BMP-2 day
28 s. CS + BMP-2 day 28: P = 0.0004). Fo SOX9,
mRNA exp ession ended o be highe in HA g oups wi h-
ou signi ican e ec s o IL-10 and BMP-2 (Fig. 2(C)).
Fu he mo e, he e was a end o lowe mRNA
224 CAR IlAgE 14(2)
Figu e 1. (a) Me abolic cell ac i i y and iable quan i ica ion: (A) Cell i e -blue assay o hChs embedded in Ha and CS a e 1 and
28 days wi h and wi hou supplemen a ion o il-10 (100 pg/ml) and BMP-2 (250 ng/ml). (B) Quan i ica ion o iable cells (%) a e
1 and 28 days. Cells we e cul u ed in chond ope missi e medium wi hou il-10 and BMP-2 supplemen a ion. Ha = hyalu onic
acid–based hyd ogel; CS = collagen sca old; il-10 = in e leukin-10; BMP-2 = bone mo phogene ic p o ein–2; aNOVa = analysis
o a iance; SD = s anda d de ia ion. as e isks indica e signi ican di e ences wi h *P < 0.05, **P < 0.01, ***P < 0.001, ****P <
0.0001, one-way aNOVa. Da a a e p esen ed as mean + SD (n = 3). (b) Chond ocy e iabili y and mo phology in Ha and CS:
(A-D) ep esen a i e 3-dimensional econs uc ions o cell-laden Ha and CS a e l/D s aining a day 1 and day 28. Cells we e
cul u ed in chond ope missi e medium wi hou il-10 and BMP-2 supplemen a ion. an inc ease in cell densi y indica es p oli e a ion
in bo h bioma e ials. (A1-D1) ep esen a i e 2-dimensional sec ions. Chond ocy es in Ha appea sphe ic while cells in CS displayed
a ami ied pheno ype. (c) Cy oskele al changes: exp ession o ac in s ess- ibe s. Phalloidin/DaPi s aining o human a icula
chond ocy es embedded in CS (A) and Ha (B) a e 7 days o cul u e in chond ope missi e medium (b and c). Ha = hyalu onic
acid–based hyd ogel; CS = collagen sca old; il-10 = in e leukin-10; BMP-2 = bone mo phogene ic p o ein–2; aNOVa = analysis o
a iance; DaPi = 4',6-diamidino-2-phenylindole. Ba 25 µm

Wei kamp e al. 225
exp ession o he cell de-di e en ia ion ma ke COL1A1
in un ea ed HA compa ed wi h CS g oups (Fig. 2(E)).
T ea men wi h BMP-2 led o posi i e e ec s by signi i-
can ly lowe ing COL1A1 exp ession le els in HA com-
pa ed wi h CS g oups a e 28 days o cul u e (HA +
BMP-2 day 28 s. CS + BMP-2 day 28: P = 0.003). The
cell hype ophy ma ke COL10A1 was simila in HA
simila o monolaye chond ocy es bu educed in CS
g oups (Fig. 2(D)).
Figu e 2. Chond ogenic po en ial o human a icula chond ocy es embedded in Ha and CS. ansc ip ion le els o messenge
ibonucleic acid (m Na) o chond ogenic ma ke s ((A) COl1a1, (B) aCaN, (C) SOX9) and ma ke s o de-di e en ia ion ((D)
COl1a1, (E) COl10a1) we e measu ed a e 1 and 28 days incuba ion wi h il-10 (100 pg/ml), BMP-2 (250 ng/ml), and co-
ea men . COl2a1/COl1a1 a io is displayed in (F). gene exp ession le els we e no malized o ha o gaPDH e e ence gene
and hen no malized o day 1 monolaye chond ocy es, which had an exp ession le el = 1. Ha = hyalu onic acid–based hyd ogel;
CS = collagen sca old; COl10a1 = ype 10 collagen; aCaN = human agg ecan; SOX9 = ansc ip ion ac o SOX-9; il-10
= in e leukin-10; BMP-2 = bone mo phogene ic p o ein–2; gaPDH = glyce aldehyde-3-phospha e dehyd ogenase; aNOVa =
analysis o a iance; SD = s anda d de ia ion. as e isks indica e signi ican di e ences wi h *P < 0.05, **P < 0.01, ***P < 0.001,
****P < 0.0001, one-way aNOVa. Da a a e p esen ed as mean + SD (n = 3).
226 CAR IlAgE 14(2)
Figu e 3. (a) glycosaminoglycan con en and elease in cell-laden Ha and CS no malized o DNa con en (A-C). Cell-laden
bioma e ials a e 28 days o cul u e wi h and wi hou il-10 (100 pg/ml), BMP-2 (250 ng/ml), and co- ea men , espec i ely. (A)
o al sgag con en and cumula i e gag elease we e analyzed by DMMB assay and no malized o co esponding sample’s DNa
con en (gag/DNa a io). (B) o al DNa con en and (C) o al sgag con en in Ha and CS g oups. as e isks indica e signi ican
di e ences wi h *P < 0.05, **P < 0.01, one-way aNOVa. Da a a e p esen ed as mean + SD (n = 3). (b) glycosaminoglycan
biosyn hesis in cellula ized Ha (A-D) and CS (E-H). ep esen a i e images o B s aining a e 28 days o in i o cul u e in
chond ope missi e medium wi h and wi hou il-10 and BMP-2 supplemen a ion (enhanced me ach omasia due o acidophilic sgags).
Ha = hyalu onic acid–based hyd ogel; CS = collagen sca old; il-10 = in e leukin-10; BMP-2 = bone mo phogene ic p o ein–2;
sgag = sulpha ed glycosaminoglycans; DNa = deoxy ibonucleic acid; DMMB = dime hylme hylene blue; B = oluidine blue. Ba
200 μm (A1-D1, E1-H1: 100 μm).
Wei kamp e al. 227
Figu e 4. (a) ype 2 collagen biosyn hesis in cellula ized Ha (A-D) and CS (E-H). ep esen a i e images o ype 2 collagen
immunohis ochemis y a e 28 days o cul u e in chond ope missi e medium wi h and wi hou il-10 and BMP-2 supplemen a ion
(b ownish s aining indica ing ype 2 collagen). (b) ype i collagen biosyn hesis in cellula ized Ha (A-D) and CS (E-H). ep esen a i e
images o ype i collagen immunohis ochemis y a e 28 days o in i o cul u e in chond ope missi e medium wi h and wi hou il-10
and BMP-2 supplemen a ion (b ownish s aining indica ing ype 1 collagen) (a and b). Ha = hyalu onic acid–based hyd ogel; CS =
collagen sca old; Col1 = ype 1 collagen; il-10 = in e leukin-10; BMP-2 = bone mo phogene ic p o ein–2; Col1 = ype 2 collagen.
Ba 200 μm (A1-D1, E1-H1: 100 μm).
228 CAR IlAgE 14(2)
In summa y, hCh encapsula ed in he HA showed a o -
able chond ogenic pheno ype al e a ions, which was pa al-
leled by chond ocy e mo phology as p e iously obse ed.
Deposi ion o Ca ilage-Speci ic ECM Is
Enhanced in CSs
Quan i ica ion o sGAG in diges ed sca olds no malized o
DNA con en e ealed highe sGAG syn hesis in HA in all
ea men g oups wi hou eaching s a is ical signi icance
(Fig. 3a(A)). DNA con en in HA samples was gene ally lowe
in all g oups (HA + BMP-2 s. CS + BMP-2: P = 0.0232;
Figu e 3a(B)). The e was a end o highe sGAG con en in
IL-10 and BMP-2- ea ed samples wi h p onounced inc ease
in biosyn hesis in he co- ea men g oup (Fig. 3a(C)).
sGAG loss in he medium was less p onounced in CS
g oups, which was pa alleled by highe amoun o e ained
sGAG in CS g oups, obse ed in his ology s udies (Fig.
3b(A-H)). Toluidine blue s aining o HA and CS showed
mo e enhanced me ach omasia wi hin he CS g a s wi h
BMP-2 ea men and co- ea men (Fig. 3b(G and H). No
dis inc e ec s o IL-10 we e de ec ed. A simila s aining
Figu e 5. li e/dead images o cell in asion assay. (A-C) ep esen a i e ile scans a e l/D s aining o bo ine ca ilage ings wi h
aga ose (nega i e con ol), Ha and CS embedded a e 28 days in i o cul u e in chond ope missi e medium. (A1-C1) Highe
magni ica ions o he bioma e ial/ca ilage in e ace. (C2) Mo phology o bo ine chond ocy es a e in asion in o CS. (D and D1)
Cellula in asion in o CS om human os eoa h i ic a icula ca ilage. Ba (A-D) 1 mm, (C2) 100 µm. Ha = hyalu onic acid–based
hyd ogel; CS = collagen sca old; Oa ca ilage = os eoa h i ic ca ilage.