Co esponding au ho : Adekunle Job.
Copy igh © 2025 Au ho (s) e ain he copy igh o his a icle. This a icle is published unde he e ms o he C ea i e Commons A ibu ion License 4.0.
P o eoglycan deposi ion and Collagen O ien a ion in Tissue Enginee ed Ca ilage
cons uc s using Digi al Densi ome y (DD) Imaging and Pola ized Ligh Mic oscopy
(PLM) Techniques
Adekunle Job *
Depa men o Technical Physics, Uni e si y o Eas e n Finland, Yliopis on an a 8, 70210, Kuopio Finland.
Wo ld Jou nal o Ad anced Resea ch and Re iews, 2025, 26(02), 842-855
Publica ion his o y: Recei ed on 14 Ma ch 2025; e ised on 03 May 2025; accep ed on 05 May 2025
A icle DOI: h ps://doi.o g/10.30574/wja .2025.26.2.1344
Abs ac
Backg ound: A icula ca ilage is an essen ial connec i e issue p o iding s uc u al in eg i y and low- ic ion
mo emen in join s. Due o i s a ascula na u e, i s abili y o sel - epai is limi ed, necessi a ing ad ancemen s in issue
enginee ing o ca ilage egene a ion. The ex acellula ma ix (ECM) componen s, p ima ily p o eoglycans and
collagen ibe s, play a c ucial ole in de e mining he biomechanical p ope ies o enginee ed ca ilage. Unde s anding
p o eoglycan deposi ion and collagen o ien a ion is i al o op imizing issue enginee ing s a egies.
Objec i e: This s udy aims o quan i y p o eoglycan con en in his ological sec ions o issue-enginee ed ca ilage
cons uc s using digi al densi ome y (DD) imaging and analyze collagen ibe o ien a ion using pola ized ligh
mic oscopy (PLM). Cons uc s wi h di e en cell densi ies (2M, 5M, and 10M) we e e alua ed a ou ime poin s (Day
1, 7, 14, 21) o assess ECM ma u a ion.
Me hods: Tissue-enginee ed ca ilage cons uc s we e cul u ed using gela in me hac ylic anhyd ide (GELMA), gellan
gum (GG) hyd ogel, and sca old- ee (SF) app oaches. His ological sec ions we e s ained wi h Sa anin-O, and DD
imaging was pe o med using a calib a ed Nikon mic oscope o quan i y p o eoglycan con en . Collagen ibe
o ien a ion was analyzed using a Lei z O holux II POL mic oscope a 21 o ien a ions, employing Michelson con as
and aniso opy index calcula ions o alignmen assessmen .
Resul s: P o eoglycan Deposi ion (DD Imaging): P o eoglycan con en inc eased signi ican ly om Day 14 o Day 21,
wi h sca old- ee cons uc s exhibi ing he highes deposi ion. Cons uc s wi h highe cell densi ies (5M and 10M)
demons a ed g ea e p o eoglycan accumula ion compa ed o lowe -densi y cons uc s.
Collagen Fibe O ien a ion (PLM Analysis): Pola ized ligh mic oscopy e ealed ha cons uc s wi h highe cell
densi ies (5M and 10M) had mo e o ganized collagen ne wo ks. Sca old- ee cons uc s exhibi ed supe io collagen
alignmen compa ed o hyd ogel-based sca olds. The aniso opy index con i med inc eased ibe o ganiza ion o e
ime, pa icula ly in he 5M and 10M SF g oups.
Conclusion: The combina ion o DD imaging and PLM p o ided a comp ehensi e assessmen o ECM ma u a ion in
issue-enginee ed ca ilage. The s udy demons a ed ha sca old- ee cons uc s, pa icula ly hose wi h highe cell
densi ies, exhibi ed enhanced p o eoglycan deposi ion and supe io collagen alignmen . These indings suppo
sca old- ee issue enginee ing app oaches o de eloping unc ional ca ilage eplacemen s in egene a i e medicine.
Fu u e Di ec ions: Fu he in es iga ions will explo e he molecula mechanisms d i ing ECM o ganiza ion and
op imize sca old composi ions o enhance ca ilage egene a ion po en ial o clinical applica ions.
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843
Keywo ds: PLM; DD; Collagen; GELMA; GG; P o eoglycan
1. In oduc ion
A icula ca ilage is a specialized ype o connec i e issue ha co e s he ends o bones wi hin a join . I s p ima y
unc ion is o p o ide a smoo h, low- ic ion su ace ha allows he bones o glide smoo hly agains each o he du ing
join mo emen . A icula ca ilage is ound in syno ial join s, which a e he mos common ype o join s in he human
body and include join s like he knee, hip, shoulde , and elbow [1-2].
A icula ca ilage consis s o wa e (80%), a icula ca ilage cells (chond ocy es, 2%), and ex a-cellula ma ix, which
includes p o eoglycans (40% d y weigh ) and collagen (60% d y weigh ) [2]. Unlike o he issues, a icula ca ilage is
a ascula , meaning i lacks blood essels, and aneu al, meaning i lacks ne e ibe s [2]. This limi s i s abili y o heal and
egene a e in esponse o inju ies [2]. Collagen p o ides s uc u al suppo , while p o eoglycans a ac wa e , gi ing
he ca ilage i s comp essibili y and abili y o wi hs and loads [2]. A icula ca ilage is o en di ided in o di e en zones
based on i s s uc u e and unc ion [2-3]. Ma u ed a icula ca ilage can be ca ego ized in o ou dis inc zones based
on he collagen ib il o ien a ion; he supe icial, middle, deep, and calci ied ca ilage zones [3]. In he supe icial zone,
he collagen ib ils ( ype II and ype ІX) a e o ien ed pa allel o he a icula su ace. In he middle zone, he ib il
o ien a ion is i egula ( andom o ien a ion) due o he ansi ion om pa allel o ien a ion a he issue su ace o
pe pendicula o ien a ion a he deep ca ilage. In he deep zone, he o ien a ion is pe pendicula o he su ace o he
a icula ca ilage; while o he calci ied ca ilage zones, he ca ilage connec s o he subchond al bone [1-2]. In he
ex a cellula ma ix (ECM), collagen is he mos p e alen s uc u al mac omolecule; i accoun s o a ound 60% o
ca ilage's d y weigh . 90% o 95% o he collagen in he ex acellula ma ix (ECM) is ype II collagen, which o ms
ib ils and ibe s en wined wi h p o eoglycan agg ega es. Al hough p esen , collagen ypes I, IV, V, VI, IX, and XI make
up a small pa o he o al. The ype II collagen ib il ne wo k is o med and s abilized wi h he aid o he mino collagens
[2].
Collagen ype II o ms he elemen a y componen o he c oss b anded ib ils. While collagen ype XI molecule bind
co alen ly o collagen ype II molecules and possibly become pa o he in e io s uc u e o he c oss banded ib ils.
Howe e , he unc ions o collagen ype IX and ype XI emain unclea . Ne e heless, i is assumed ha hey assis o
s abilize and o m he collagen ib ils collec ed undamen ally om collagen ype II. The collagen ype IX molecules
ex ended po ions can help bind he collagen ib il meshwo k and in e ac wi h collagen meshwo k wi h p o eoglycans.
Collagen ype VI look o o m a i al po ion o he ma ix, su ounding he chond ocy es and aids he a achmen o he
chond ocy es o he ma ix. Collagen ype X p esence only close o he cells o he calci ied zone o a icula ca ilage
and he hype ophic zone o g ow h pla e ha is he a ea whe e he longi udinal ca ilage sep a begin o mine alize,
he eby sugges s ha ype X has a pa in ca ilage mili a iza ion [5].
P o eoglycans play a c ucial ole in he s uc u e and unc ion o ca ilage, pa icula ly in a icula ca ilage ound in
join s. They a e a ype o mac omolecule consis ing o a co e p o ein and long chains o ca bohyd a es called
glycosaminoglycans (GAGs). P o eoglycans con ibu e o he unique p ope ies o ca ilage, such as i s abili y o esis
comp essi e o ces and e ain wa e [7]. He e's how p o eoglycan deposi ion wo ks in ca ilage.
When p essu e is applied o he join , wa e wi hin he p o eoglycan- ich ma ix is squeezed ou , and as p essu e is
eleased, wa e is d awn back in, helping he ca ilage e u n o i s o iginal shape [5]. This mechanism is c ucial o
shock abso p ion and join lub ica ion [5]. In condi ions like os eoa h i is, he balance be ween p o eoglycan syn hesis
and deg ada ion can be dis up ed, leading o a loss o p o eoglycans and de e io a ion o ca ilage [5]. Resea che s a e
in es iga ing s a egies o p omo e p o eoglycan syn hesis and epai damaged ca ilage, including issue enginee ing
and egene a i e he apies.
Tissue enginee ing is a mul idisciplina y ield ha aims o c ea e unc ional and iable issues o o gans using a
combina ion o cells, bioma e ials, and bioac i e molecules [9].
The p ima y componen o TE, sca olds, p omo e issue egene a ion by os e ing an en i onmen ha is conduci e o
cell ancho ing, dis ibu ion, and unc ionaliza ion in he p esence o signaling molecules [10].
Ca ilage issue enginee ing aims o c ea e unc ional ca ilage eplacemen s o damaged o degene a ed ca ilage in
join s, such as in cases o os eoa h i is [9]. In issue enginee ing, bio-ma e ial sca olds a e used o suppo cell g ow h
and guide issue o ma ion. These sca olds o en mimic he na u al ex acellula ma ix o he issue being enginee ed.
Fo ca ilage issue enginee ing, sca olds a e designed o suppo he deposi ion o p o eoglycans, collagen, and o he
Wo ld Jou nal o Ad anced Resea ch and Re iews, 2025, 26(02), 842-855
844
componen s p esen in na i e ca ilage [10]. While independen me hods do no ely on sca olds, sca old-dependen
p ocedu es employ syn he ic o na u al bioma e ials o c ea e an en i onmen ha encou ages cell de elopmen and
in e ac ions [4].
Sca olds made o hyd ogel a e becoming a common he apy op ion o ca ilage abno mali ies. They a e minimally
in asi e, eplica e he na u al en i onmen , and may be injec ed o ill i egula ly shaped lesions. Al hough ini ial
mechanical s abili y is a key a ibu e o sca olds, hey come wi h a numbe o disad an ages, including cell-
cha ac e is ic changes, emodeling esis ance, s ess shielding, and oxici y associa ed wi h deg ada ion [4]. When
c ea ing ca ilage issue using issue enginee ing app oaches, chond ocy es (ca ilage cells) o s em cells a e seeded
on o he sca olds. O e ime, hese cells p oli e a e and di e en ia e in o chond ocy e-like cells ha deposi
p o eoglycans and collagen, o ming a issue-enginee ed ca ilage ma ix [9]. G ow h ac o s and bioac i e molecules
can be inco po a ed in o issue enginee ing app oaches o enhance p o eoglycan deposi ion and issue ma u a ion.
These ac o s can s imula e chond ogenic di e en ia ion and p omo e he syn hesis o p o eoglycans by he cul u ed
cells [9].
The success ul ou come o issue enginee ing elies on he ma u a ion o he enginee ed issue o closely esemble
na i e issue [9]. P o eoglycan con en and dis ibu ion a e impo an ma ke s o assessing he ma u i y and
unc ionali y o issue-enginee ed ca ilage [9].
S aining a icula ca ilage sec ions wi h Sa anin O is a common me hod used o isualize he p esence o
glycosaminoglycans (GAGs), which a e a majo componen o he ca ilage ex acellula ma ix. Sa anin O is a ca ionic
dye ha binds o he nega i ely cha ged GAGs, s aining hem ed [11]. The in ensi y o ed s aining co esponds o he
numbe o GAGs p esen in he ca ilage ma ix [11]. Regions wi h highe GAG con en will appea da ke ed [11].
GAGs a e abundan in he ex acellula ma ix o ca ilage, pa icula ly in a eas ich in p o eoglycans, such as he
e i o ial ma ix a ound chond ocy es and he in e - e i o ial ma ix. You would obse e ed s aining in hese a eas
[11]. In cases o ca ilage degene a ion o damage, such as os eoa h i is, he e migh be a educed p esence o GAGs.
This would esul in educed o absen Sa anin O s aining in he a ec ed a eas [12].
Digi al densi ome y is a echnique used in a ious ields, including adiology, as onomy, and ma e ials science, o
measu e he densi y o op ical densi y o a ma e ial o subs ance. I in ol es he use o digi al imaging echnology and
so wa e o analyze he a enua ion o abso p ion o ligh o o he elec omagne ic adia ion as i passes h ough o
in e ac s wi h a sample. The digi al aspec o densi ome y in ol es using digi al senso s o came as o cap u e images,
ollowed by compu e -based analysis o calcula e he op ical densi y alues.
Pola ized ligh mic oscopy is a adi ional echnique o isualizing he collagen ne wo k s uc u e o a icula ca ilage.
A icula ca ilage epai and issue enginee ing esea ches ha e e ol ed new eques o echniques wi h capaci y o
quan i a i e cha ac e iza ion o he sca and epai issues, as well as p ope ies o he collagen ne wo k. Mode n
pola ized ligh mic oscopy can be used o analyze pa allelism,bi e ingence and collagen ib il o ien a ion. Mode n
ins umen s a e compu e based and he measu emen s a e mo e lexible o pe o m. Ne e heless, on many nume ous
occasions he p esen a ion o esul s b ings abou di icul ies, e en e o s, due o he demand in he heo e ical aspec s
o he echnique [20]. P io o each PLM measu emen session, au oma ed alignmen and calib a ion p ocedu es a e
un o gua an ee measu emen eliabili y. Following alignmen , he mic oscope is calib a ed. Calcula ing he sample's
o ien a ion-dependen bi e ingence equi ed his calib a ion echnique. Fo impa ial s udy o ib il pa allelism o
o ien a ion, i is no equi ed. A e he pola ize has been o a ed s ep-by-s ep, g ayscale pho os a e aken. The de ice
will con inue o cap u e pho os un il he came a's sa u a ion poin is achie ed [20].
1.1. Objec i e o he s udy
The objec i e o his p ojec wo k is o ge amilia wi h Quan i ying p o eoglycan (PG) con en concen a ion in
his ology sec ions o h ee ypes o issue enginee ed ca ilage cons uc s wi h digi al densi ome y (DD) imaging
echniques, and obse ing he di e ence in hese bioma ke s o demons a e he in luence o hese gels and sca old
ee on he de eloping neo- issue. While he pola ized ligh mic oscopy (PLM) will be used o s udy collagen ibe s
o ien a ion, whe e he pola iza ion di ec ion o he ligh is a ec ed by he collagen ibe s.
2. Ma e ial and Me hod
In his p ojec , he e we e h ee ypes o issue enginee ed ca ilage cons uc s which include numbe o cells (2M, 5M
and 10M), and each cons uc we e cul u ed o ou di e en du a ions (Day 1, 7, 14 and 21). These issue cons uc s
we e p epa ed using bone ma ow mesenchymal s em cells (BMSCs) encapsula ed in gela in me hac ylic anhyd ide
Wo ld Jou nal o Ad anced Resea ch and Re iews, 2025, 26(02), 842-855
845
(GELMA) (n=16) and gellan gum (GG) hyd ogel (n=16) o suspended in he media as pelle s (sca old- ee cons uc s;
n=16). These cons uc s we e eco e ed a ou ime poin s (Day 1, 7, 14, 21; n=4/gel/ ime poin ) and ozen ill he
beginning o he p ojec .
A se o his ological sec ions we e p epa ed o s udy PG deposi ion om ozen samples. The samples we e hawed and
used o p epa e his ology sec ions. This se o his ological sec ion we e s ained using Sa anin-o (n=2/gel/ ime poin ),
a subs ance ha make he p o eoglycan o become isible wi h a eddish colo and his was used o Digi al
Densi ome y (DD), whe e he concen a ion o sa anin-o co ela es o he deposi ion o PG in he samples.
Digi al densi ome y imaging was acqui ed wi h a ligh mic oscope (Nikon Mic opho FXA, Tokyo, Japan) equipped wi h
a CCD-cooled came a (Hamama su pho onics K.K, Hamama su Ci y, Japan, pixel size = 0.14 lm) and 4x and 10x
magni ica ion. Images we e calib a ed agains neu al densi y il e s (op ical densi y alues 0.0, 0.3, 0.6, 1.0, 1.3, 1.6, 2.0,
2.6, and 3.0) (Scho , Mainz, Ge many). Calib a ion images we e aken a e e y day be o e DD measu emen o di e en
con igu a ion.
Each sample we e indica ed wi h a symbol M, SP, SF which ep esen GELMA, gellan gum and sca old ee espec i ely,
and each o he samples we e seeded wi h cells in he ange om cell ee o, 2 million, 5 million, and 10 million cells.
Th ee slices pe sample we e measu ed. The da a ob ained om hese measu emen s p o ided in o ma ion on he
issue-enginee ed ca ilage. The image da a ob ained om DD is p ocessed using MATLAB. The esul s ob ained will
also be used as p elimina y da a o u u e s udies.
The Lei z O holux II POL (Lei z We zla , We zla , Ge many) mic oscope body was used o his s udy's enhanced
pola ised ligh mic oscopy (PLM) ins umen , which was calib a ed in acco dance wi h Meh a e al. [18]. I was
equipped wi h a monoch oma ic ligh sou ce ( λ = 630 nm, Edmund Op ics Inc., Ba ing on, NJ, USA), c ossed pola ize s
(Techspec op ics A sample is posi ioned a a 90-deg ee angle be ween a pola ize and an analyze in he se -up. Samples
we e measu ed a 21 o ien a ions spanning 180 deg ees, spaced 9 deg ees apa .
MATLAB p og am was used o all he analyses o de e mine he o ien a ion o collagen ibe s (Ma lab R2023a,
Ma hwo ks Inc., Na ick, USA).
The ib ous collagen ne wo k s uc u e o a icula ca ilage imposes an angle dependence on he in ensi y o he
obse ed ligh . Thus, by measu ing he sample a se e al angles, a pa allelism index (i.e., aniso opy) can be de ined
using Michelson con as [19]. This ype o linea pola iza ion enabled he use o S okes pa ame e s (S0, S1, and S2) in
he calcula ion o he mean ibe angle in each pixel.
𝑆0= 𝐼(0°)+𝐼(90°),𝑆1= 𝐼(0°)−𝐼(90°),𝑆2= 2×𝐼(45°)−𝑆0 ………… (1)
Whe e I: ligh /signal in ensi y in each pixel a he speci ied pola iza ion angle, S0: o al in ensi y o inciden ligh , S1: he
amoun o linea /ho izon al pola iza ion, and S2: he amoun o +45° o -45° pola iza ion. The angle o collagen ibe s
(𝜓) is he o ien a ion o he bi e ingen s uc u es (i.e., collagen ibe s):
𝜓 = 𝑎𝑟𝑐𝑡𝑎𝑛(𝑆2
𝑆1)
2 ……………… (2)
Whe e 0° ≤𝜓≤90° (3). A e acqui ing he PLM image o he sec ions, he collagen o ien a ion was es ima ed on a
pixel-by-pixel basis.
3. Resul
3.1. Digi al Densi iome y (DD) Resul
In his esul p o eoglycan deposi ion in issue enginee ed ca ilage cons uc s we e analyzed. This was done in
his ology sec ions o h ee ypes o issue enginee ed ca ilage con ac s wi h digi al densi ome y (DD) imaging
echniques using magni ica ion 4 and 10. The p o eoglycan con en based o hese magni ica ions and he days o cul u e
which a e day 14 and 21 we e compa ed. Da a o day 1 and 7 we e inaccessible due o he small sizes o ou samples
and di icul ly o p epa e his ology slides which make i di icul o ha e a easonable esul on hose days.
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846
Table 1 and 5 ( ig. 1 and ig. 5) a e used o show day 14 and 21 o compa ison o all amoun o cells in bo h
magni ica ion 4 and 10 espec i ely and while able 2 and 6 ( ig. 2 and ig. 6), able 3 and 7 ( ig 3 and ig.7) and able 4
and 8 ( ig. 4 and ig. 8) a e used o day 14 and 21 o compa ison wi h amoun o cells 2M, 5M and 10M espec i ely in
bo h magni ica ion 4 and 10.
Table 1 A e age alues o p o eoglycan o day 14 and 21 (Mag. 4)
4X Day 14
a e age
4X Day 21
a e age
0M_DAY14_B1
0.1725
0M_DAY21_A1
0.1142
10M_DAY14A_A1
0.2878
0M_DAY21_B1
0.4554
10M_DAY14B_A1
0.2012
0M_DAY21_C1
0.1946
10SF_DAY14_A1
0.1789
10M_DAY21_A1
0.1562
10SF_DAY14_B1
0.1334
10M_DAY21A_B1
0.2275
10SP_DAY14_A1
0.0960
10M_DAY21B_B1
0.2446
10SP_DAY14_B1
0.0893
10SF_DAY21_A1
0.3774
2M_DAY14A_A1
0.1909
10SF_DAY21_B1
0.2325
2M_DAY14A_B1
0.1137
10SP_DAY21A_A1
0.0572
2M_DAY14B_A1
0.0822
10SP_DAY21B_A1
0.0606
2M_DAY14B_B1
0.1244
2M_DAY21A_A1
0.1049
2SF_DAY14_A1
0.1965
2M_DAY21B_A1
0.1545
2SF_DAY14_B1
0.1944
2M_DAY21B_B1
0.2734
5M_DAY14_A1
0.1559
2SF_DAY21_A1
0.2339
5M_DAY14A_B1
0.1156
5M_DAY21_A1
0.1695
5SF_DAY14_A1
0.1412
5M_DAY21A_B1
0.1665
5SF_DAY14_B1
0.2681
5M_DAY21B_B1
0.1606
5SF_DAY21_A1
0.4866
5SF_DAY21_B1
0.2865
Figu e 1 The plo o day 21 and day 14 (mag. 4)
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The plo simple e ealed he abundance o p o eoglycan in day 21 in compa ison wi h day 14 and he mid alues in day
21 is hicke han ha o day 14, he mid alues as seen s ill con i med he g ea e concen a ion o p o eoglycan in day
21 o day 14.
Table 2 A e age alues o p o eoglycan o 2million cells sca old (Mag. 4)
day 14
a e age
day 21
a e ag
e
2M_DAY14A_A1
0.1909
2M_DAY21A_A1
0.1049
2M_DAY14A_B1
0.1137
2M_DAY21B_A1
0.1545
2M_DAY14B_A1
0.0822
2M_DAY21B_B1
0.2734
2M_DAY14B_B1
0.1244
2SF_DAY21_A1
0.2339
2SF_DAY14_A1
0.1965
2SF_DAY14_B1
0.1944
Figu e 2 The plo o day 21 and day 14 in 2million cells sca old (mag. 4)
The plo s ill e ealed he supe io i y o day21 wi h he con en o p o eoglycan side by side o day 14 in 2 million cells
sca old and he mid alues in day 21 s ill hicke han ha in day 14, his also con i med he le el o p o eoglycan
con en highe in day 21.
Table 3 A e age alues o p o eoglycan o 5million cells sca old (Mag. 4)
day 14
a e age
day 21
a e age
5M_DAY14_A1
0.1559
5M_DAY21_A1
0.1695
5M_DAY14A_B1
0.1156
5M_DAY21A_B1
0.1665
5SF_DAY14_A1
0.1412
5M_DAY21B_B1
0.1606
5SF_DAY14_B1
0.2681
5SF_DAY21_A1
0.4866
5SF_DAY21_B1
0.2865
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848
Figu e 3 The plo o 5million cells sca old (mag. 4)
Day 21 wi h he con en o p o eoglycan high han day 14 in 5 million cells sca old and he mid alues in day 21 s ill
hicke , his also con i med he le el o p o eoglycan con en highe in day 21.
Table 4 A e age alues o p o eoglycan o 10million cells sca old (Mag 4)
day 14
a e age
day 21
a e age
10M_DAY14A_A1
0.2878
10M_DAY21_A1
0.1562
10M_DAY14B_A1
0.2012
10M_DAY21A_B1
0.2275
10SF_DAY14_A1
0.1789
10M_DAY21B_B1
0.2447
10SF_DAY14_B1
0.1334
10SF_DAY21_A1
0.3774
10SP_DAY14_A1
0.0960
10SF_DAY21_B1
0.2325
10SP_DAY14_B1
0.0893
10SP_DAY21A_A1
0.0572
10SP_DAY21B_A1
0.0606
Figu e 4 The plo o 5 million cells sca old (magni ica ion 4)
This plo emains consis en wi h o he s showing he consis ency o day 21 ha ing high p o eoglycan compa ed o day
14.
Wo ld Jou nal o Ad anced Resea ch and Re iews, 2025, 26(02), 842-855
849
Table 5 A e age alues o p o eoglycan o day 14 and 21 (Mag.10)
10X DAY 14
a e age
10X DAY 21
a e ag
e
10 M_DAY14A_A1
0.2173
0M_DAY21_B1
0.1526
10 M_DAY14A_B1
0.1775
0M_DAY21_C1
0.2364
10 M_DAY14B_A1
0.2420
0M_DAY21A_AI
0.2300
10 M_DAY14B_B1
0.1633
0M_DAY21B_A1
0.2074
10SF_DAY14_A1
0.1970
0SP_DAY21_A1
0.6755
10SF_DAY14_B1
0.3785
10M_DAY21_A1
0.1279
10SP_DAY14_A1
0.1143
10M_DAY21A_B1
0.2496
10SP_DAY14_B1
0.2170
10M_DAY21B_B1
0.2100
2M_DAY14_A1
0.1778
10SF_DAY21_B1
0.2430
2M_DAY14_B1
0.1744
10SF_DAY21A_A1
0.3678
2SF_DAY14_A1
0.2053
10SF_DAY21B_A1
0.3598
2SF_DAY14_B1
0.2121
10SP_DAY21_A1
0.0955
5M_DAY14_A1
0.1834
2M_DAY21_B1
0.1596
5M_DAY14_B1
0.1782
2M_DAY21A_A1
0.2752
5SF_DAY14_A1
0.1497
2M_DAY21B_A1
0.1622
5SF_DAY14_B1
0.2914
2SF_DAY21_A1
0.2456
5M_DAY21_A1
0.4860
5M_DAY21A_B1
0.2189
5M_DAY21B_B1
0.2238
5SF_DAY21_A1
0.4014
5SF_DAY21_B1
0.2415
Figu e 5 The plo o day 21 and day 14 (mag.10)
Wo ld Jou nal o Ad anced Resea ch and Re iews, 2025, 26(02), 842-855
850
The plo e ealed he abundance o p o eoglycan on day 21 in compa ison wi h day 14 and he mid alues in day 21 and
day 14, howe e look simila in hickness, bu he day 21 emain high in p o eoglycan con en .
Table 6 A e age alues o p o eoglycan o 2-day million cells sca old (Mag.10)
day 14
a e age
day 21
a e ag
e
2M_DAY14_A1
0.1778
2M_DAY21_B1
0.1596
2M_DAY14_B1
0.1744
2M_DAY21A_A1
0.2752
2SF_DAY14_A1
0.2053
2M_DAY21B_A1
0.1622
2SF_DAY14_B1
0.2121
2SF_DAY21_A1
0.2456
Figu e 6 The plo o day 21 and day 14 in 2million cells sca old (mag.10)
The plo s ill e ealed he supe io i y o day21 wi h he con en o p o eoglycan side by side o day 14 in 2million cells
sca old and he mid alues in day 21 s ill hicke han ha in day 14, his also con i med he le el o p o eoglycan
con en highe in day 21.
Table 7 A e age alues o p o eoglycan o 5million cells sca old (Mag. 10)
day 14
a e age
day 21
a e age
5M_DAY14_A1
0.1834
5M_DAY21_A1
0.4860
5M_DAY14_B1
0.1782
5M_DAY21A_B1
0.2189
5SF_DAY14_A1
0.1497
5M_DAY21B_B1
0.2238
5SF_DAY14_B1
0.2914
5SF_DAY21_A1
0.4014
5SF_DAY21_B1
0.2415
