Summa y. Osseoin eg a ion o implan s is c ucial o
he long- e m success o o al implan s. The pe iimplan
bone o ma ion by os eoblas s is s ongly dependen on
he local mechanical en i onmen in he in e ace zone.
Robus demands o ene gy a e placed on os eoblas s
du ing he adhesion p ocess o solid su aces, and
mi ochond ia a e capi al o ganelles in he p oduc ion o
mos o he ATP needed o he p ocess. We ha e
assessed he ela ionship be ween os eoblas
di e en ia ion and mi ochond ial bioene ge ics in li ing
cells g own on wo di e en i anium su aces, in o de
o p o ide aluable in o ma ion o he design o
ma e ial su aces equi ed o he de elopmen o he
mos app op ia e os eogenic su ace o os eoblas ic
ancho age. Combined backsca e ed and luo escence
con ocal mic oscopy showed ha in la cells g own on a
machined su ace, highly ene gized mi ochond ia we e
dis ibu ed along he cell body. In con as , cells g own
on he ough su ace emi ed long p o usions in sea ch
o su ace oughness, wi h ac in s ess ibe s clea ly
pola ized and highly ene gized mi ochond ia clus e ed a
ocal adhesion si es. This epo using no mal human
os eoblas ic cells indica es ha hese cells a e especially
sensi i e o su ace cues h ough ene gy p oduc ion ha
enhances he necessa y adhesion equi ed o a
success ul osseoin eg a ion.
Key wo ds: Ti anium, Su ace opog aphy, Os eoblas ,
Cell adhesion, Mi ochond ial memb ane po en ial
In oduc ion
Recen esea ch has sugges ed ha bones display an
ex ao dina y adap a i e beha iou owa ds changing
mechanical en i onmen , which is o en ega ded as
pheno ype plas ici y. Fu he mo e, he pe iimplan issue
o ma ion and mine aliza ion by os eoblas s a e s ongly
dependen on he local mechanical en i onmen in he
in e ace zone. The quali y o cell adhesion o he
implan su ace de e mines issue in eg a ion, and he
su ace oughness can di ec ly in luence os eoblas
adhe ence, a achmen , sp eading and me abolism
modi ying and con olling he osseoin eg a ion p ocess
(Pie es e al., 2002; Ingbe , 2003a,b; Sade e al., 2005;
Chiesa e al., 2007; Ga i e al., 2008).
In mig a ing cells, ac in ilamen s a e p ima ily
o ganized in o submemb ane meshwo ks ha appea
di use o in small bundles, whe eas s a iona y cells
usually display conspicuous a ays o ilamen bundles,
“s ess ibe s”, which a e ancho ed a hei e mini in
ma ix a achmen s known as ocal adhesions (Chen e
al., 2003; Zime man e al., 2004; Diene e al., 2005; Hu
e al., 2007). Robus demands o ene gy a e placed on
os eoblas s in he adhesion p ocess o solid su aces, and
mi ochond ia a e esponsible o he p oduc ion o he
la ges pa o cellula ATP needed o endoe gonic
p ocesses wi hin he cell (G igo iou e al., 2005; Anes i
and Sco ano, 2006; Boldogh and Pon, 2007).
To unde s and how mic o opog aphy modula es cell
adhesion, in he p esen s udy we ha e de eloped an in
i o model, using he human os eoblas ic cell line
NHOs ®, o s udy mi ochond ial dis ibu ion and
bioene ge ics du ing he ini ial phases o he adhesion
p ocess o di e en i anium implan su aces, p epa ed
as disks o in i o manipula ion .
The e is a di ec co ela ion be ween he ene gized
s a e o mi ochond ia and he mi ochond ial memb ane
(∆ψµ) when analyzed in isola ed mi ochond ia (Sal ioli
Mi ochond ial bioene ge ics and dis ibu ion in
li ing human os eoblas s g own on implan su aces
Me cedes Salido1, J. Ignacio Vilches-Pe ez2, Juan L. Gonzalez3and Jose Vilches1
1Depa men o His ology, School o Medicine, Labo a o y 57, Se icios Cen ales de In es igación en Ciencias de la Salud,
Uni e si y o Cadiz, Cadiz, Spain, 2Depa men o O al su ge y, School o Den is y. Uni e si y o Se ille, Se ille, Spain and
3Depa men o S a is ics and Ope a ional Resea ch, School o Medicine, Uni e si y o Cadiz, Cadiz, Spain
His ol His opa hol (2009) 24: 1275-1286
O p in eques s o: Me cedes Salido, Depa men o His ology, School
o Medicine, Labo a o y 57, Se icios Cen ales de In es igación en
Ciencias de la Salud, Uni e si y o Cadiz, C/ D . Ma añon 3, 11002
Cádiz. Spain. e-mail: [email p o ec ed]
h p://www.hh.um.es
His ology and
His opa hology
Cellula and Molecula Biology
1276
e al., 1997; Dedo and Rou ogalis, 1999, Salido e al.,
2007a). In o de o p o ide aluable in o ma ion o he
design o ma e ial su aces ha a e equi ed o he
de elopmen o he mos app op ia e os eogenic su ace
o os eoblas ic ancho age in den al implan s, he
ela ionship be ween os eoblas di e en ia ion on
di e en i anium su aces and cellula bioene ge ics has
been assessed in li ing cells in his s udy, by means o
combined backsca e ed and luo escence analysis wi h
con ocal mic oscopy. Thus, he dynamics o a
physiological p ocess, as is he case o he ini ial phases
o osseoin eg a ion, can be de e mined. We ha e, hus,
used his in o ma ion o econs uc he h ee-
dimensional in e ace be ween he os eoblas ic cells and
he implan su ace.
The esul s p esen ed he ein en o ce he ole o he
ough subs a um su ace in a ec ing os eoblas ic cell
adhesion, equi ed o he de elopmen o an app op ia e
os eogenic su ace o os eoblas ic ancho age and
ma u a ion, compa ed o a machined su ace in den al
implan s.
Ma e ial and me hods
To e alua e he ela ionship be ween os eoblas
di e en ia ion and bioene ge ics, NHOs cells we e
g own on di e en i anium su aces, Ti CP machined
and Ti CP Osseo i e®, p o ided as p e ab ica ed 2 cm x
1.5 mm disks, in medium supplemen ed wi h asco ba e
o induce di e en ia ion. To e alua e changes in
mi ochond ial memb ane po en ial (∆ψµ)) and in
subcellula dis ibu ion in esponse o di e en sca olds,
cells we e s ained wi h a ansmemb ane po en ial-
sensi i e i al dye, JC1, which p e e en ly accumula es
in mi ochond ia by ∆ψµ dependen mechanism, and
con ocal imaged. Ac in cy oskele al o ganiza ion was
assessed by immunos aining wi h hodamine phalloidin
in o de o s udy he ole o he subs a um su ace in
a ec ing os eoblas ic cell adhesion.
Cell cul u e
No mal human os eoblas ic NHOs ®cells (Camb ex,
Walke s ille, MD, USA) we e seeded a a densi y o
5000 cells/cm2and incuba ed in Os eoblas G owing
Medium, OGM, (Camb ex, Walke s ille, MD, USA)
con aining 10% e al bo ine se um (Camb ex,
Walke s ille, MD, USA), 1% gen amycin sulpha e/
ampho e icin B (Camb ex, Walke s ille, MD, USA) and
1% asco bic acid (Camb ex, Walke s ille, MD, USA), as
ecommended by supplie s, a 37°C and 5% CO2un il
he expe imen s s a ed. G ow h medium was changed
daily a e seeding. Be o e he cells became 80%
con luen hey we e subcul u ed wi h 2 ml o 0.25 mg/ml
ypsin EDTA wa med o 37°C (Camb ex, Walke s ille,
MD, USA) a e insing wi h 5 ml Hepes-BSS
(Camb ex, Walke s ille, MD, USA) a oom
empe a u e. Once cells we e de ached, ypsin EDTA
was neu alized by adding 4 ml o ypsin neu alizing
solu ion (Camb ex, Walke s ille, MD, USA). Ha es ed
cells we e seeded on he di e en disks a a densi y o
5000 cells/cm2and immunos ained a e 48 h. G ow h
medium was changed e e y day un il he expe imen s
we e o e . NHOs cells a e assu ed o expe imen al use
o en popula ion doublings, which we e no exceeded
du ing he assay.
Ti anium disks
Be o e use, he disks we e imme sed in 100%
e hanol o 10 min, ai d ied, and exposed unde u. .
ligh o 30 min on each side, and inally insed in
endo oxin ee phospha e bu e ed solu ion, and
deposi ed on small s e ile Pe i dishes p io o cell
seeding. Tissue cul u e Willco®(WillCo Wells,
Ams e dam, The Ne he lands) wells, wi h a 0.17 mm
glass bo om, we e used as he con ol su ace.
Immunohis ochemis y and cy oskele al o ganiza ion
A he end o he speci ic cul u e ime, cells we e
washed wice wi h p ewa med phospha e-bu e ed
saline, (PBS), pH 7.4, ixed wi h 3.7% pa a-
o maldehyde (PFA) solu ion in PBS o 10 min a oom
empe a u e, and washed wice wi h p ewa med PBS.
The cells we e hen pe meabilized wi h 0.1% T i on x-
100 (Sigma, S Louis, Missou i, USA) o 5 min and
washed wice wi h p ewa med PBS. To educe non-
speci ic backg ound s aining, 1% bo ine se um
albumine (BSA) in PBS was added o he su aces o 20
min, and cells we e immunos ained o 20 min. wi h
hodamine phalloidin, 12.5 µl o me hanolic s ock
solu ion (Sigma, S Louis, Missou i, USA) in 500 µl
PBS o each sample. A e disca ding he s aining
solu ion, disks we e insed wi h p ewa med PBS h ee
imes p io o moun ing wi h ec ashield®(Vec o Labs.
Bu lingame CA, USA) and 0.17 mm co e slip in one
holed polyca bona e slide de ices specially designed in
ou labo a o y and ab ica ed by means o con ol
p ecision sys ems (Mecap ec, Cadiz, Spain).
Mi ochond ial pe meabili y po en ial
Cells we e s ained wi h he ca ionic dye JC-1 (Mi o
PT TM®, Immunohis ochemis y Technologies,
Blooming on, MN, USA), which exhibi s po en ial-
dependen accumula ion in mi ochond ia. A low
memb ane po en ials, JC-1 con inues o exis as a
monome and p oduces a g een luo escence (emission
a 527 nm). A high memb ane po en ials, JC-1 o ms “J-
agg ega es" (emission a 590 nm) and p oduces a ed
luo escence.
Cells we e cul u ed in Willco®wells wi h glass
bo om as con ol g oup o he assay, and on he
di e en disk su aces p o ided, no exceeding a inal
numbe o 106cells/ml, and a e disca ding he cul u e
medium, 1x Mi oPT®s aining solu ion ob ained om a
100x s ock was added o he wells, 0.5 ml pe well. Cells
we e hen incuba ed a 37°C o 15 min in a CO2
incuba o and, a e disca ding he medium, washed
Mi ochond ial esponse o su ace induced cues
1277
wice wi h 1-2 ml o assay bu e wa med o 37°C. A e
disca ding he wash, a d op o assay bu e was added o
he specimens p io o immedia e examina ion in he
in e ed con ocal mic oscope Leica TCS SL (Leica,
Da ms aad, Ge many), equipped wi h an HCX PL APO
CS 63.0x1.30 glyce ol imme sion objec i e, wi h an
incuba ion sys em consis ing o a cube ha comple ely
co e s he mic oscope and allows us o keep cells a
37°C in a con olled a mosphe e wi h a mixed ai /CO2
low o 4 l/h and 5% CO2du ing image collec ion and
analysis.
Con ocal examina ion. Image collec ion and analysis o
cy oskele on
The cells and disks we e simul aneously isualized
using a Leica TCS-SL con ocal mic oscope equipped
wi h a 63.0x1.30 glyce ol objec i e, allowing
simul aneous acquisi ion o hodamine phalloidin
s aining o ac in cy oskele on (exci a ion 554 nm /
emission 573 nm) and su ace e lec ance. A leas ou
isola ed disks we e analyzed in each g oup o
p o ilome ic in o ma ion, and a minimum o ou disks
wi h cells g owing on hem we e analyzed in each g oup,
in o de o assess su ace in luence on cy oskele al
o ganiza ion and cell mo phology. A leas 50 cells pe
disk we e analysed. Images we e collec ed and
p ocessed o quan i a i e analysis using he imaging
so wa e p o ided by he Leica TCS SL sys em.
P o ilome ic s udies, o he quan i ica ion o maximal
and minimal alues in su ace p o ile, dis ance be ween
peaks in each su ace and wid h o g oo es and alleys
in he di e en su aces we e assessed.
All samples we e exposed o lase o a ime in e al
no mo e han 5 min o a oid pho obleaching. The
exci a ion beam spli e selec ed was a DD 488/543. The
lase was se o he lowes powe able o p oduce a
luo escen signal. Maximum ol age o pho omul iplie s
was used o dec ease he equi ed lase powe as much
as possible. O se was main ained a 0. A pinhole o 1
Ai y uni was used. Images we e acqui ed a a esolu ion
o 1024x1024. Se ies we e acqui ed in he xyz mode.
Image collec ion and analysis o
∆ψµ
The cells and disks we e simul aneously isualized
using a Leica TCS-SL con ocal mic oscope equipped
wi h a 63.0x1.30 glyce ol objec i e, unde he condi ions
desc ibed abo e, allowing simul aneous acquisi ion o
su ace e lec ance and JC1 s ained mi ochond ia. JC-1
was exci ed a 490 nm and he emission luo escence
was collec ed in TRITC (590 nm) and FITC (530 nm)
channels simul aneously.
Fo quan i a i e analysis, a leas 120 egions o
in e es (ROIs) we e selec ed in each g oup o quan i y
changes in ∆ψµ. All o he ROIs a e cells selec ed unde
he ollowing c i e ia: well-de ined limi s, clea
iden i ica ion o nucleus and absence o in e sec ion wi h
neighbou ing cells. Size, numbe o pixels and
luo escence in ensi y in he ed (high memb ane
po en ial) and g een (low memb ane po en ial) channels
in each ROI we e calcula ed.
S a is ical analysis
The s a is ical analysis was pe o med wi h SPSS
p og am. A one-way ANOVA analysis was used o
compa e he mean alues o cell a eas and ed pixel
in ensi y, s anda d de ia ion and skewness. The
no mali y o he g oups was con as ed wi h
Kolmogo o -Smi no es and he a iances
homogenei y wi h Coch an’s C es . Pos -hoc con as s,
HSD es o Tukey, we e ca ied ou o de ec he
di e ences be ween g oups. A alue o p≤0.05 was
conside ed signi ican .
Resul s
CLSM cha ac e iza ion o su aces
Con ocal backsca e ed imaging o disk su aces,
ob ained by e lec ion o he 488 lase line, showed a
ma kedly i egula su ace in he TiCP Osseo i e®wi h
p ominen peaks and ca es, as shown (Fig. 1A). Some o
hem p esen ed well-de ined con ou s, while some o he s
o med a wide abecula su ace wi h nume ous c a e -
like s uc u es and andomly dis ibu ed ca i ies and
bo de s. The TiCP machined su ace displayed pa allel
g oo es, caused by he wa iness inhe en o he
machining ope a ion, which de e mined a ela i ely
smoo h su ace (Fig. 1C). Th ee dimensional
econs uc ions, ob ained om se ial acquisi ion o
s acks, we e pe o med o he assessmen o os eoblas -
su ace in e ace (Fig. 1B,D). The p o ilome ic
measu emen esul ed in a minimum alue o 1.2 µm o
he dep h o he alleys and a maximum alue o 4.15
µm o he op o he embossed s uc u e. The mean
dis ance be ween ops eached 18.28 µm, he mean
wid h o he alleys was 2.85 µm, he mean alue ound
o he g oo e on op o each embossed ing was 1.51
µm. Ti CP Osseo i e®disks showed a ma ked inc ease in
he su ace oughness, wi h p ominen peaks and ca es,
some o hem wi h well-de ined con ou s and some
o he s o ming a wide abecula su ace wi h andomly
dis ibu ed peaks. The dis ance be ween peaks,
consequen ly, was highly a iable, wi h alues anging
om 24.45 µm up o 183.67 µm. The mean diame e o
he ca es was ound o be la gely a iable, anging om
8.01 and 39.27 µm. As e ealed in he p o ilome ic
s udy, his pa e n no ably inc eased he mean oughness
o he su ace, ha showed a minimum alue o 1.9 µm
in dep h and a maximum peak alue o 8.79 µm.
CLSM obse a ion o di e ences in os eoblas
pheno ypes on di e en su aces
Cells g owing on he TiCP Osseo i e®su ace,
whe e he dis ance be ween peaks inc eases, p esen ed
an inc easingly pola ised sp eading wi h emission o
ei he wide lamellipodia o long ilopodia, depending on
Mi ochond ial esponse o su ace induced cues
he su ace oughness and he dis ance be ween
subjacen peaks, and showed he longes mo phology
wi h a mean axial a io ha ose o 3.5 (Fig. 2). The ac in
cy oskele on adop ed an o ganiza ion cha ac e ized by
ac in bundles unning ei he pe pendicula ly o sligh ly
obliquely, wi h appa en ly segmen ed con ac ile uni s
de ined opog aphically by ancho age poin s, o ganized
acco ding o he su ace s uc u e. Pa allel ac in
ilamen s we e obse ed along he cellula p ocesses and
ancho ing o ocal con ac s o ela i e opog aphical
peaks was obse ed. As shown, cells clea ly i ed o
su ace opog aphy and e en emi ed long ilopodial
b anches, when necessa y, o success ul ancho age
media ed by ac in “s ess ibe s” and ocal adhesion
con ac s on selec ed peaks.
When os eoblas s g owing on TiCP machined
su aces we e examined, signi ican di e ences we e
obse ed, no only ega ding size and shape, bu also o
cy oskele al o ganiza ion. Cells g own on Ti CP
machined su aces la ened and adop ed a la gely adial
mo phology, and appea ed o be p edominan ly plana
and andomly o ien ed wi h a mean axial a io o 1.1
(Fig. 3). Cells ancho ed o he su ace by displaying long
dend i ic ilopodia, bo h along he g oo es and c ossing
o e hem in a pe pendicula way.
Con ol cells, g owing on he glass su ace, did no
show a pa e ned mo phology. Ins ead, a numbe o cells
showed long, hin ilopodia, while some o he s la ened
and sp ead o e he glass su ace, hus p esen ing a high
a iabili y in ac in cy oskele on o ganiza ion, wi h ac in
ibe s ha appea ed unning in all di ec ions (Fig. 6D).
Mi ochond ial bioene ge ics and dis ibu ion
On TiCP Osseo i e®su aces, NHOs ®cells
p esen ed a high densi y o ilamen ous mi ochond ia.
Mos o he highly ene gized mi ochond ia, s ained in
ed (Fig. 4), accumula ed in he long ilopodial b anches.
The simul aneous isualiza ion o mi ochond ial
labelling and he backsca e ed imaging o he su ace
allowed us o co ela e he mi ochond ial dis ibu ion
wi h he i egula subs a um mic o opog aphy, ull o
peaks and ca es, hus con i ming mi ochond ial
pola iza ion o he selec ed ancho age poin s.
In cells g owing on TiCP machined su aces, highly
ene gized mi ochond ia illed he cell body (Fig. 5). No
1278
Mi ochond ial esponse o su ace induced cues
Fig. 1. A. Con ocal backsca e ed imaging o TiCP ough su ace (Osseo i e®), showing he i egula dis ibu ion o peaks and ca es ha con igu e an
i egula su ace o bone cell a achmen . B. P o ilome ic 3D econs uc ion. In con as , as shown in C, he machined su ace p esen ed pa allel
g oo es ha con igu e an almos uni o m su ace, qui e egula in dep h, as shown in he p o ilome ic 3D econs uc ion in D. In o de o cla i y
in e p e a ion o images, a glow LUT pale e is p esen ed, i.e. he da ke zones a e he deepe ones.
clus e ing o signi ican pola iza ion a ound con ac si es
we e obse ed, and SD alues (Table 1) con i med qui e
a simila mi ochond ial dis ibu ion o ha displayed in
he con ol g oup, whe e os eoblas s, which adhe e o
glass su ace, showed sca e ed mi ochond ia une enly
dis ibu ed. The e is also a ilamen ous ne wo k o
1279
Mi ochond ial esponse o su ace induced cues
Fig. 2. A. Simul aneous imaging o disk su ace and hodamine-phalloidin s ained NHOs ®cell g own on he ough su ace o 48 h. Cells i ed o he
su ace wi h he emission o ilopodia and lamellipodia. As shown in B, when glow imaging was employed, he dis ibu ion and concen a ion o ac in
s ess ibe s is condi ioned by su ace i egula i ies. C. Me ged image ob ained om a se ial acquisi ion o 50 s acks in he xyz mode, combining
backsca e ed and luo escence imaging. Long ilopodia emi ed om he cell body a e looking o app op ia e adhesion si es. D. De ailed image o a so
called “cell oo ” in a ocal adhesion si e, wi h ac in s ess ibe s clea ly pola ized o he disk su ace. In his case, an in e ed glow pale e is p esen ed.

pola ized mi ochond ia su ounding he nucleus (Fig. 6).
S a is ical analysis
When analyzing he a iable a ea o he selec ed
1280
Mi ochond ial esponse o su ace induced cues
Fig. 3. A Simul aneous imaging o disk su ace and hodamine-phalloidin s ained NHOs ®cell g own on he machined su ace o 48 h. Cells a e
p edominan ly la and s ess ibe s un in di e se di ec ions, as clea ly shown in Bwhen glow imaging was employed. In his case, cells a e g owing
c ossing o e he g oo es in a pe pendicula way, wi h a disc e e emission o ilopodia. C. Some o he cells g owing in pa allel o machined g oo es
wi h s ess ibe s co e ing he whole cell body, as highligh ed in D.
ROIs, a highly signi ican di e ence (p<2•E-10)
appea ed be ween cells g own on he di e en su aces
es ed, and cells g owing on he ough, TiCP Osseo i e®
su ace, showed he highes alues, ollowed by cells
g owing on glass su ace and inally, he smalles ones,
cells g owing on he machined su ace. Despi e his ac ,
he di e ences be ween ed pixel means be ween g oups
we e no signi ican (p=0.369). On he con a y, highly
signi ican di e ences we e ound when compa ing
s anda d de ia ions (p<2•E-14), and skewness (p=0.011)
(Table 1).
1281
Mi ochond ial esponse o su ace induced cues
Fig. 4. A. Li ing cell imaging o JC1 s ained os eoblas s simul aneously acqui ed wi h he backsca e ed imaging o Osseo i e®disk su ace 48 h a e
seeding. Cells emi ed long p o usions in sea ch o su ace p o ube ancies. In ed, highly ene gized mi ochond ia ha accumula e in ocal adhesion
si es. B. Shown in de ail, long ilopodia a e b anching o con ac wi h su ace peaks, which appea in clea g ey. Red s ained mi ochond ia clus e in he
ene gy equi emen zones. C. The cell shows he ypical mo phology obse ed in he Osseo i e®g oup, wi h long ilopodial emission ull o highly
ene gized mi ochond ia, i ing o disk su ace, in g een. D. Mi ochond ial clus e ing, ed, in ocal adhesion si es. Cells show an elonga ed mo phology
de e mined by ela i e su ace peaks.
The esul s o pos -hoc compa isons be ween
g oups, analyzing he di e ences in model-p edic ed
means, a e shown in Table 2. B ie ly, highly signi ican
di e ences we e ound in cell a ea when compa ing he
1282
Mi ochond ial esponse o su ace induced cues
Fig. 5. A. Li ing cell imaging o JC1 s ained os eoblas s simul aneously acqui ed wi h he backsca e ed imaging o he machined disk su ace 48 h.
a e seeding. Cells a e p edominan ly la , and highly ene gized mi ochond ia, in ed, a e dis ibu ed along he cell body. B. Os eoblas s g own on he
machined su ace, wi h a ela i ely high cellula densi y ha o ien ed along he g oo es, some o he cells emi ing small ilopodia wi h low p esence o
ed s ained mi ochond ia. C. Polygonal os eoblas s g owing on he machined su ace. The cellula densi y is high, bu no clea adhesion si es a e
obse ed. Highly ene gized mi ochond ia a e sca e ed along he cell body. D. Shown in de ail, combined backsca e ed image o machined disks
clea ly showing he g oo ed pa e n and JC1 s ained cells. Mi ochond ia a e dis ibu ed nea p olonga ions bu also in he cell body. Cells appea o be
la and sp ead.
ough, Osseo i e®, and machined g oups wi h he con ol
g oup, bu no when compa ing Osseo i e®and
machined g oups. Signi ican di e ences be ween all
h ee g oups appea ed when compa ing s anda d
de ia ions o ed pixel dis ibu ion, mainly when
compa ing cells g owing on Osseo i e®disks wi h
he emaining g oups. S a is ically signi ican
di e ences be ween Osseo i e®and machined
1283
Mi ochond ial esponse o su ace induced cues
Fig. 6. A. Con ol cells g owing on a glass su ace and JC1 s ained a e 48h. Cell mo phology e eals a non-pola ized sp eading in he sea ch o
di e en ia ion cues. Highly ene gized mi ochond ia a e sca e ed and une enly dis ibu ed. In B, an elonga ed os eoblas g owing on glass is emi ing
ilopodia wi h highly ene gized mi ochond ia. Due o he absence o su ace signaling, cell mo phology is di e se, as shown also in C, whe e he cellula
body adop ed a nea ly polygonal shape and mi ochond ia sca e along he cell. D. Rhodamine-phalloidin s ained NHOs ®cell g owing on glass. Ac in
ibe s a e i egula ly dis ibu ed along he cell body.
g oups we e ound when compa ing skewness o
ed pixel dis ibu ion, bu no when compa ing
machined o Osseo i e®g oups wi h he con ol
g oup.