1
COMPUTER ASSISTED MICROSCOPIC ANALYSIS OF BONE TISSUE DEVELOPED INSIDE
A POLYMER, POLYACIVE, IMPLANTED TO EQUINE ARTICULAR SURFACE.
Réka Albe 1*, Gábo Vásá helyi2 Gábo Bodó3, Annamá ia Kenye es1, E in Wol 1, Tamás Papp1,
Tünde Te dik1, László Módis1, Szabolcs Felszeghy1
1Depa men o Ana omy, His ology and Emb yology, Medical and Heal h Science Cen e,
Uni e si y o Deb ecen, Deb ecen, Hunga y
2 O hopaedic and T auma Depa men , Uzsoki Hospi al, Budapes , Hunga y
3Depa men o Su ge y, Facul y o Ve e ina y Medicine, Szen Is án Uni e si y, Budapes ,
Hunga y
*P esen add ess: Depa men o Biochemis y and Molecula Biology, Medical and Heal h
Science Cen e, Facul y o Medicine, Uni e si y o Deb ecen, Deb ecen, Hunga y
Key wo ds: mosaicplas y, dono unnels, bone, compu e assis ed image analysis, equine
Sho unning i le: Analysis o bone de eloped in dono unnels a e mosaicplas y
Co esponding au ho :
Szabolcs Felszeghy,
Depa men o Ana omy, His ology and Emb yology,
Medical and Heal h Science Cen e,
Uni e si y o Deb ecen,
Nagye dei k . 98.,
4032, Deb ecen,
Hunga y
e-mail: [email p o ec ed]a .do e.hu
2
SUMMARY
One o he mos p omising applica ion o small o mode a e sized ocal a icula lesions
es au a ion is he mosaicplas y (MP). Al hough ecu en hema h osis is a a e complica ion
a e MP, ecen ly di e en s a egies designed o ind an e ec i e illing ma e ial o p e en he
pos ope a i e bleeding om he dono si e. The po osus biodeg adable polyme Polyac i e
(polye hylene oxide e eph hala e - polybu ylene e eph hala e copolyme ; PA) ep esen s a
p omising solu ion in his espec . His ological e alua ion o long- e m PA illed dono si es
ob ained om 10 expe imen al ho ses was pe o med. In his pape , a en ion was p ima ily
ocused o he bone issue de eloped in he plug. The compu e assis ed image analysis and
quan i a i e pola ized ligh mic oscopic measu emen o he decalci ied, longi udinally sec ioned
dime hylme hylene blue (DMMB) and pic osi ius ed (PS) s ained sec ions deli e ed ha he
engaged a ea o he bone abecules o he PA illed dono unnels is subs an ially (25%)
inc eased compa ed o he neighbou ing cancellous bone. Fo his quan i ica ion, iden ical ROI-s
( egion o in e es ) we e used and compa ed. The e a da ion alues o bi e ingence we e also
measu ed in pola ized ligh mic oscope using monoch oma ic ligh . The same e a da ion alues
could be ecoded om he bone abeculae de eloped in he PA and in he neighbou ing bone
indica ing ha he collagen o ien a ion pa e n does no di e signi ican ly in hese bone
abeculaes. Based on ou de no o da a we may specula e ha PA may p omo e bone o ma ion,
and some o he no ye iden i ied deg ada ion p oduc s o PA may enhance os eoconduc ion and
os eoinduc ion inside he dono canal.
3
INTRODUCTION
A icula ca ilage has a poo capaci y o epai and healing (Ho as e al., 2003; Bedi e
al., 2010). Due o i s necessa y unc ion, ha uni o mly ans e and dec ease he le el o load on
unde lying bone (Shi azi and Shi azi-Adl, 2009), ailing o do so can ini ia e u he join
abno mali ies (Ding e al., 2008). The e o e inc easing numbe o heo e ical and clinical esea ch
is designed o achie e he egene a ion o o ganized a icula ca ilage.
One o he majo inno a i e and biological app oach o es o e unc ion o a icula
ca ilage o mo e han a decade is mosaicplas y (MP), ocusing on econs uc ing he join
a icula su aces ia ansplan ing some small au olog os eochond al g a s (OCGs) in a ose a
pa e n o o m a s able clo ha ills he lesion (Hangody e al., 1997; Hangody e al., 2001a;
Hangody e al., 2001b; Hangody and Fules, 2003; Hangody and Modis, 2006; Hangody e al.,
2008). The heo e ical ad an age o i is based on Pap and K ompeche ’s s udy published in 1961
(Pap and K ompeche , 1961), in which hey p o ed ha he OCG builds in he ecipien
su ounding in a unc ional si ua ion, e en in lack o compa ibili y; on he o he hand he g a ’s
size and a io ha e a signi ican in luence on i s su i al. Al hough in he case o au ologous
os eochond al mosaicplas y se ology and issues ypi ying a e edundan , i also ha e signi ican
es ic ions: limi ed size o useable dono a ea, dono si e mo bidi y, di e ences in o ien a ion
and hickness, mechanical p ope ies be ween dono and ecipien ca ilage (Bedi e al., 2010).
In con as o many a icles deal wi h g a -hos in eg a ion o pos -ope a i e unc ion jus
a ew is engaged in dono si e e en s. No mally he unnels a e emo ing he OCGs, ge illed
wi h spongy bones wi hin ou weeks – simila ly o he P idie’s d illing me hod – h ough
mesenchymal s em-cells’ in asion due o bleeding om he subchond al a ea (Hangody and
Modis, 2006). Su ace o unnels end o be co e ed wi h a so-called p ime epai issue which
4
becomes ib oca ilage du ing app op ia e loading (Bodo e al., 2000). Un o una ely, excessi e
bleeding may s a spon aneously om he dono unnels emained emp y jus in a ew cases,
which can lead o haema h os (Feczko e al., 2003). Fe ic ions (Fe3+) de i ed om he
deg ada ed no mocy es o blood ge ing in o he join ca i y may i e e sibly des oy he
p o eoglycan s uc u e o hyaline ca ilage (Sokolo , 1963).
Some clinical and p e- ial expe imen al s udies had been made o exclude he possibili y
o pos ope a i e haema h os o ill he dono unnels such as hyd oxylapa i e (Li ino e al.,
2000), ca bon ibe (Meis e e al., 1998), polyglycona e (F eed e al., 1994) and comp essed
collagen (Nixon e al., 1993) which esul ed in sca issue o poo ib oca ilage o ma ion on he
su ace. In ou ea lie s udy we in es iga ed a copolyme ma e ial, polye hylene glycol
e eph ala e - polybu ylene e eph hala e (PEGT/PBT) – Polyac i e, PA, (IsoTis O oBiologics,
Bil ho en, he Ne he lands), whe e we p o ided clea e idences ha he PA is one o he mos
app op ia e illing ma e ial in his con ex (Módis e al., unpublished da a). The his ological
examina ions on he human samples, which s udied he in luence o he PA, ha e shown ha he
PA helps he ib oca ilage (in ew cases hyaline-like ca ilage) o ma ion on he join su ace as
well as he bone and he connec i e issue gene a ion inside (Módis e al., 2005a).
To he bes o ou knowledge he e a e no publica ions abou he quan i a i e and
quali a i e analysis on bone abecules e ol ed in he dono unnels illed wi h any ma e ial,
among PA, he e o e in his p esen s udy we aimed o pe o m he quan i a i e and quali a i e
analysis on hem.
5
MATERIALS AND METHODS
Mosaicplas y o ho ses
All p ocedu es using animals we e app o ed by he E hical Commi ee o animal ialso
Szen Is án Uni e si y, Facul y o Ve e ina y Sciences and issues we e ob ained in acco dance
wi h he guidelines. The os eochond al au og a ansplan a ions we e ca ied ou on 10 ho ses.
The dono a ea was he emo al medial ochlea and he ecipien egion was he medial o la e al
ochlea o he animal’s o elimb’s dis al hi d me aca pal bone. The applied me hod has been
desc ibed ea lie (Bodo e al., 2000). B ie ly, ho ses we e posi ioned in la e al ecumbency in
gene al anes hesia. Fou pieces o OCG – each o 6.5 mm diame e and abou 20 mm leng h –
con aining heal hy join ca ilage and spongiosa we e collec ed om he same wound. The g a s
we e kep in s e ile iso onic solu ion (0.9% NaCl) un il implan a ion. 22 mm deep unnels we e
d illed on he ecipien a ea han he OCGs we e plan ed in o hem. Hal o he dono a eas we e
illed wi h biodeg adable polyme s (PolyAc i e, PA,
IsoTis O oBiologics, Bil ho en, he
Ne he lands) (Figu e 1), o he s we e le emp y.
Tissue p ocessing o his ological analysis
A e a long (2 yea s) ollow up he ho ses we e sac i iced in acco dance wi h he s udy
guidelines and he dono unnels illed wi h PA we e ob ained wi h hei in ac su ounding.
Samples we e immedia ely ans e ed in o Sain e-Ma ie’s solu ion a (SAINTE-MARIE, 1962).
A e ixa ion issues we e pu in o 10% e hylenediamine e aace a e (EDTA) (Solon, Ohio,
USA) o app ox. 3 weeks. Decalci ied and dehyd a ed issue samples we e embedded in o
pa a in and 5−7 µm hick longi udinal sec ions we e cu (Mic om HM335E, Mic om

6
In e na ional GmbH., Walldo , Ge many). Pa a in sec ions we e placed on gela ine-coa ed glass
slides and le d ied o e nigh a 37 °C.
A e dewaxing and ehyd a ion sec ions we e s ained wi h dime hylme hylene blue
(DMMB, Ald ich, S einheim, Ge many), pic osi ius ed (PS, Polysciences, Wa ing on, USA),
hema oxilin-eosin (H&E) acco ding o he manu ac u e ’s p o ocol (Cons an ine and Mow y,
1968; Módis, 1974, 1991; Ki aly e al., 1996). A e s aining sec ions we e co e ed wi h DPX
(Fluka Chemie, Buchs /Swi ze land).
Compu e -assis ed image analysis o he PA illed and non- illed dono unnels.
Du ing ou p elimina y mic oscopic obse a ion we ound a lo o hick abecula bones
wi h abno mal s uc u e appea in he dono unnels illed wi h PA howe e , hin os eon uni s o
bone wi h no mal s uc u e a e isible in he con ol (no ope a ed) a eas (Figu e 2 and 3). To
quan i a i ely analyse ou obse a ions compu e assis ed image analysis was p e o med b ie ly
desc ibed below.
Images om di e en samples o 6 di e en ho ses we e cap u ed by a Nikon Eclipse 800
mic oscope (Nikon Co po a ion Ins umen s Company, Japan) equipped wi h a Spo RT-slide
(Diagnos ic Ins umen s, S e ling Heigh s, MI, USA) CCD came a. Acqui ed and p esen ed
images we e ep esen a i e o all he samples examined. A e sys em calib a ion, 500x720 µm
a eas o each samples we e digi alized. Al oge he 31 con ol and 31 PA illed dono unnels
we e compa ed o each o he and he quan i a i e analysis was pe o med using Image P o 5.1
so wa e (Media Cybe ne ics, Inc., Sil e Sp ing, MD, USA).
Mann-Whi ney es was used o analyze he esul s s a is ically.
Pola ized ligh mic oscopic measu emen
7
Pola ized ligh mic oscopy was used o quan i y he o ganiza ion changes wi hin he
collagen ne wo k o epai issue in case o he PA illed and i s su ounding. Sec ions s ained
wi h PS we e examined by pola iza ion mic oscope using 40x objec i e (Ze opan-pol, Reiche ,
Wien, Aus ia). 100 measu emen s pe sample ha e been ca ied ou bo h in case o PA illed and
con ol unnels.
8
RESULTS
Mac oscopic inspec ion showed ha he issue buil up in PA illed dono unnels was
milde compa ed o he neighbou ing egions, which we e well dis inguishable.
Residuums o PA helped in o ien a ion du ing mic oscopic examina ion in all he h ee
s ainings. The su aces o he PA illed dono unnels we e eco e ed by ib oca ilage, he inne
abecules we e hicke and showed abno mal o ganisa ion compa ed o abecula meshwo k o
su ounding issue (Figu e 2 and 3). In some cases polinuclea gian cells we e shown nex o he
PA pa iculums. Compa ed o he o och oma ic abecules o he ecei ing a ea he os eon uni s
o he PA illed dono unnels showed pu ple ed me ach omasia on he DMMB s ained sec ions
(Figu e 2).
Compu e assis ed image analysis
He eby we p o ided clea e idences, ha he engaged a ea o he abecules de eloped in
he PA illed dono unnels was signi ican ly wide (p<0.05) compa ed o he con ol abecules’
(Figu e 4 and 5).
Da a we e analysed using Mann-Whi ney s a is ical me hod. The analysis esul ed in
signi ican di e ence be ween he a e ages o engaged a ea o abecules pe ROI (p =
5.34x10−6) (Figu e 5). The engaged a ea o abecules in PA- illed dono unnels was 25% highe
compa ed o in ac spongiosa loca ed nex o he con ol unnels (Figu e 5).
Pola ized ligh mic oscopic measu emen
We analyzed he s uc u e and he pa e n o o ien a ion o os eon uni s on PS s ained
sec ions whe e PA pa iculums showed homogenous dual- ac ion (Figu e 3). 100−100 pa allel
measu emen s we e pe o med o explo e he collagen ibe o ien a ion and he s a is ical analysis
9
did no show signi ican di e ences be ween he PA illed dono unnels and he con ol a eas (p
= 0.5229) (Figu e 6). I e e s o he ac ha he spa ial o ien a ion o he collagen molecules
does no di e in he abecules o he dono cannels illed wi h PA and con ol a eas.
16
FIGURE LEGENDS
Figu e 1
Pic u e o he biodeg adable po ous polyme used o ill up he unnels.
Figu e 2
Rep esen a i e ligh mic oscope pic u es o pic osi ius ed (PS) (A and C) and
dime hylme hylene blue (DMMB) (B and D) s ained sec ions o PA- illed dono unnels (A and
B) and i s su ounding (C and D) (100x magni ica ion). Please no e he di e ence in me a- and
o hoch omasia (B s. D), hickness (A and B s. C and D) and egula i y o os eon uni s o he
PA- illed unnels and i s su oundings.
Figu e 3
Rep esen a i e ligh (A and C) and pola ized ligh (B and D) mic oscope pic u es o pic osi ius
ed s ained sec ions o PA illed dono unnels (A and B) and i s su ounding (C and D). PA
pa iculums showed homogeny dual- ac ion unde pola ized ligh (B) u he mo e lamellas o
new hick abecules e ol ed in PA illed dono unnels shows i egula o dina ion compa ed o
he neighbou s (B s. D).
Figu e 4
Column diag am shows he mean engaged a ea o abecules pe ROI (%) in each samples. 5-5
ROI pe sample we e digi alized bo h om PA illed and con ol a eas. Engaged a eas o he
abecules de eloped in he PA illed dono unnels a e ep esen ed by black and he con ol by
g ay columns.

17
Figu e 5
Diag am shows he a e age o engaged a ea o abecules pe ROI (%). Enla gemen o a ea (%)
o con ol unnels is ep esen ed in g ey and PA- illed dono unnels in black. The engaged a ea
o abecules in PA- illed dono unnels was highe compa ed o con ol unnels.
Figu e 6
Resul s o quan i a i e pola ized-ligh mic oscopy expe imen s om de e mina ion o collagen-
ibe o ien a ion o abecules. Diag am shows he numbe o measu ed alues belonging o
di e en dual- ac ion pa h leng h ange (nm). G ey columns ep esen he con ol samples, black
columns a e he alues o PA- illed dono unnels. Dis ibu ion o alues (nm) o PA- illed dono
unnels closely esemble he alues o con ol- unnels. Mann-Whi ney s a is ical analysis esul ed
wi hou a signi ican di e ence be ween he PA- illed and con ol unnels (p = 0.5229). The e
was no obse ed any di e ence be ween he collagen- ibe o ien a ion o con ol abecules and
he PA- illed ones.