I HC QUC GIA TP.

HCM
TRNG I HC KHOA HC T NHIN

L MINH DUY

Tn ti:

XC NH GIAI ON HNH THNH V KHO ST

S BIT HA CA NGUYN BO SN
TRN GI TH PLA

Chuyn ngnh:

Cng ngh sinh hc

Cn b hng dn : TS. Trn L Bo h

I HC QUC GIA TP.HCM

TRNG I HC KHOA HC T NHIN

L MINH DUY

CNG NGHIN CU TI
LUN VN THC S

Tn ti: Xc nh giai on hnh thnh v kho st s bit ha ca nguyn bo

sn trn gi th PLA
Chuyn ngnh: Cng ngh sinh hc
M s chuyn ngnh: 62420201
Chng trnh: Phng thc 2

Xc nhn ca cn b hng dn
(K tn v ghi r h tn)

TS. Trn L Bo H

Tp. HCM, thng 1 nm 2015

1. Gii thiu tng qut

T bo bo gc trng thnh hoc t bo gc sinh dng u c nhng tim nng y
hng hn trong s ti to m c th. Hin nay, vic nghin cu sinh hc c bn v ng
dng tr liu ca t bo gc trung m ang c cc nh khoa hc c bit quan tm. T
bo gc trng thnh (Adult stem cell, ASC) c phn lp v nghin cu t nhiu
ngun m khc nhau trong c th nh ty xng, dy cung rn, no, biu m, ty rng
v gn y nht l m m. Mc d t bo gc trng thnh cn hn ch trong kh nng
bit ha thnh cc m c quan khc nhau nhng chng vn m nhim vai tr duy tr tnh
ni cn bng m trong c th bng cch phc hi cc t bo b mt do tui tc hoc b tn
thng. iu cho thy chc nng ti to m y trin vng ca cc loi t bo gc
ny. Mt trong s cc ASC c quan tm nghin cu nhiu nht l t bo gc trung
m (Mesenchymal stem cell, MSC) v ni bt l t bo gc m m. T bo gc m m
(Adipose tissue-derived stem cell, ADSC) khng nhng c chung cc c im ca t
bo gc trung m ty xng m cn mang cc u im ni bt khc tiu biu nh vic
thu nhn d dng hn khi khng cn qu trnh phu thut phc tp. Hn th na, t bo
gc m m cn c tim nng bit ha thnh cc t bo trung m nh t bo m, t bo
xng, c xng v t bo sn, cng nh cc t bo khng phi trung m nh t bo gan,
t bo ni tit tuyn ty, t bo thn kinh, t bo c tim v t bo ni m mch mu [8]
Hin nay, ng dng ca t bo bo gc m m ngy cng pht trin c bit trong lnh
vc cng ngh ti to m. Cng ngh ny nghin cu v s dng cc vt liu sinh hc
to ra cc m mi c c im chc nng tng t trong c th nm thay th cc m c
quan b mt hoc tn thng. Mt v d tiu biu trong lnh vc ny l cng ngh ti
to m sn. M sn khp khi trng thnh khng c kh nng t sa cha mt cch hiu
qu khi b tn thng hoc b lo ha. Vic s dng cc t bo gc m m c kch
hot bit ha sn nhm thay th cc m sn mt cho cc bnh nhn thoi ha sn khp
ang c ng dng rng ri cc bnh vin v gt hi c cc thnh cng nht
nh. Bn cnh vic ng dng iu tr bnh v sn khp, cng ngh ti to m sn cn
m ra mt hng i mi y ha hn cho lnh vc phu thut thm m nh nng mi v

ti to khun mt [7]. Vi c im sng mi khng cao v chp mi tt hu ht cc

ph n chu , nhu cu chnh sa nng mi ngy cng ln ko theo cc cng ngh phu
thut ngy cang pht trin. Cho n nay c ba phng php nng mi c s dng ph
bin l nng mi bng silicon, cht lm dy v sn. Trong , phng php nng mi
bng sn c a chung nht do s hu cc u im ni bt so vi hai phng php cn
li nh t bin chng, khi sn s dn ng nht vi c th v tn ti vnh vin m khng
cn ti phu thut. Nng mi bng sn l phng php t thm mt ming sn di da,
dc theo sng mi ko di, lm thng hoc to hnh thm m ph hp cho chic mi.
Ming sn c th l sn nhn to hay sn t thn. Trong , sn t thn c thu nhn
thng thng t sn sn, sn vnh hoc sn vch ngn ca chnh ngi cn phu thut.
Hin nay, cc bc s phu thut pht trin thm bc bc mt ming sn chp mi.
Ming sn nh ny c ly t sn vnh tai vi ng m khong 1 2 cm. Vic ny
gip cho sng mi nhn to khng b t, u mi khng b bin chng. Tuy nhin,
phng php nng mi bng sn hin nay i hi phi phu thut ly sn t nhiu v tr
trn c th nhng vn khng m bo c lng sn cn thit nng sng mi. Vic
s dng sn nhn to l cc gi th 3D rng c cu trc tng t sn c th gy bin
chng v cn ti phu thut sau mt thi gian nht nh. V vy, cng ngh to m sn t
t bo gc m m ca chnh bnh nhn l gii php y trin vng cho vn ny.
c th thu nhn m sn vi kch thc mong mun to thm m cho sng mi, qu
trnh bit ha sn cn c din ra trn mt gi th ph hp. Mt gi th l tng trong
ti to m phi bao gm t nht ba c im: to iu kin thun li cho cc t bo bm
dnh v pht trin, d dng c gia cng thnh cch hnh dng c th v c bn
duy tr hnh dng . Gi th polylactic acid (PLA) c xem l mt trong cc gi th
c ng dng thnh cng nht trong cng ngh ti to m sn do m sn c bit ha
trn gi th PLA c cu trc tng t nh m sn t nhin. PLA vi cu trc phn t
(C3H4O2)n l mt polyester nhit do c kh nng phn hy sinh hc. Ngy nay, cc thit
b hin i gip gia cng cc si PLA thnh cc gi th c hnh dng chnh xc vi sng
mi ngi t ming sn c to ra t kch thc l tng cho phu thut nng mi.

Vi cc u im ca gi th PLA, nhiu cng trnh nghin cu thc hin qu trnh

bit ha t bo gc m m trn gi th ny v kho st s bit ha hnh thnh nn m
sn. Tuy nhin, phng php bit ha t bo gc m m ngay trn gi th PLA khng
kim sot c t l bit ha sn cng nh khng m bo c cht lng ming m
sn c to ra. Trong ti ny s a ra mt hng gii quyt l s dng nguyn
bo sn, mt giai on trung gian trong qu trnh bit ha sn v c t l hnh thnh sn
cao hn, nui cy bit ha trn gi th PLA. iu ny i hi phi xc nh giai on
hnh thnh nguyn bo sn v kho st s tng trng bit ha ca nguyn bo sn ny
trn gi th PLA. Kt qu ca ti c ng dng trong vic ti u kh nng bit ha
sn t t bo gc m m c s dng gi th PLA, t m bo cht lng ca ming
sn trong phu thut thm m nng mi hoc ti to khun mt. Bn cnh , phng
php s dng nguyn bo sn bit ha thnh t bo sn vi hiu sut bit ha cao hn
s m ra mt hng i mi trong vic iu tr cc bnh v sn khp.
Tnh hnh nghin cu trong v ngoi nc:
Cho n nay, trn th gii c kh nhiu cc ti nghin cu thc hin qu trnh bit
ha sn t t bo gc trung m hay t bo gc m m in vitro v c in vivo. Mt s ti
cn kho st s bit ha sn ca t bo gc m m trn cc loi gi th c thnh phn
khc nhau. Tuy nhin, i vi giai on nguyn bo sn th cha c mt ti nghin
cu no xc dnh thi gian bit ha thnh nguyn bo sn t t bo gc m m cng nh
cc marker b mt c trng cho giai on ny. Do , vic kho st s bit ha ca
nguyn bo sn trn gi th cng cha c nghin cu.
Gn y, vic tr bnh thoi ha khp bng t bo gc m m t thn c ng dng
cc bnh vin ln trong nc ta. Trong nm 2012, bnh vin i hc Y dc p
dng cho hn 20 trng hp, tt c iu cho kt qu tt, bnh nhn phc hi chc nng
khp gi t 80-90% so vi bnh thng. Nhiu bnh vin ln trong nc s dng k
thut phn tch t bo gc m m t thn m c nghin cu, thc hin ti nhiu
nc trn th gii trong vic iu tr thoi ha khp gi. Hin nay, nc ta cng c
nhng nghin cu lm sng iu tr thoi ha khp gi bng cch s dng t bo gc m

t thn. iu ny cho thy ng dng qu trnh bit ha sn t t bo gc m m ngy

trong tr liu nc ta cng ph bin v c quan tm nghin cu. i vi phu thut
thm m c bit l trong phu thut nng mi, nc ta p dng thnh cng cc cng
ngh v phng php tin tin chuyn giao t nc ngoi v thu c li nhun to ln.
Tuy nhin cc phng php phu thut nng mi hin vn cn nhng hn ch nht nh.
Ni dung ti ny phn no a ra mt hng i mi, mt phng php mi hon thin
hn trong phu thut thm m nng mi ni ring v cng ngh ti to m sn ni chung.
2. Mc tiu ca ti
ti bao gm hai mc tiu chnh:
- Xc nh giai on nguyn bo sn trong qu trnh bit ha sn t t bo gc m m.
- Kho st s tng trng ca nguyn bo sn trn gi th PLA.
3. i tng nghin cu
Nghin cu ny tin hnh trn i tng l mu m m bng ca ngi trng thnh.
4. Ni dung nghin cu
4.1. Xc nh giai on hnh thnh nguyn bo sn t t bo gc m m
Trong qu trnh bit ha t t bo gc trung m thnh t bo sn tri qua cc giai an
c bn nh: (1) T bo gc trung m; (2) t bo tin sn; (3) Nguyn bo sn; (4) t bo
sn cha trng phng; (5) t bo sn trng phng. mi giai on bit ha u c
c trng bi mt s gen ma ha cho cht nn ngoi bo sn. Do , xc nh c
giai on hnh thnh nguyn bo sn, phng php RT-PCR l cng c c lc pht
hin s bt u biu hin ca cc gen c trng cho tng giai on. Khi t bo i vo giai
on nguyn bo sn s kch hot biu hin cc gen Collagen IIa1 (Col2a1), aggrecan
(Acan) v COMP (Cartilage oligomeric matrix protein). Khi t bo bc sang giai on
t bo bo s bt u kch hot biu hin gen Collagen Xa1 (Col10a1) [6]. Nh vy c
th s dng thng tin ny xc nh khong thi gian nguyn bo sn hnh thnh bng
cch thu hp dn dn cc mc thi gian kim tra s bin hin cc gen c trng trn

ng thi kt hp vi kho st hiu sut bit ha bng phng php nhum c trng
cht nn t bo sn (Toluidine Blue).
Giai on
bit ha
T bo trung m
to sn

Cc marker
cht nn
Col1a1

Cc marker
iu ha

Hnh dng
t bo

Cc nhn t
tng trng v
bit ha

Sox9, Runx2

Shh, TGF-

Tin nguyn bo sn

Col2a1, Acan,
Crtl

Sox9, Sox5,
Sox6

TGF-, Wnt-3A,
Wnt7A
FGF-2,4,8,10
BMP-2,4,7

Nguyn bo sn

Col2a1, Acan,
Crtl1, Comp,
Crtm

Sox9, Sox5,
Sox6

IGF-1
FGF-2/FGFR2
BMP-2,4,7,14

Tin t bo sn

Col2a1,
Acan,Crtl1,
Comp, Crtm

Pthr1, Ihh

FGF-18, FGFR-3
BMP-2,7

Runx2, Runx3

VEGF
FGF-2, FGF-R1
Wnt14/-catenin

T bo sn

Col10a1

Cc giai on ca qu trnh bit ha sn v cc gen biu hin c trng [6]

4.1.1. Bit ha nguyn bo sn t t bo gc m m bng nui cy vn cc
Khng ging vi qu trnh bit ha m v bit ha xng, cc yu t vt l ca vi mi
trng to sn ng vai tr c bit quan trng trong qu trnh bit ha ca t bo gc
trung m thnh sn. Theo mt s cng trnh nghin cu, cc tng tc khng gian ba
chiu, s tip xc b mt ca cc t bo gc trung m l iu cn thit cho s bit ha
sn. Khi cc t bo gc dc nui cy theo phng php tbo n s khng c kt qu
bit ha thnh sn cho d c nui trong mi trng c b sung nhn t bit ha sn.
Ch khi c bit ha trong vi mi trng ba chiu ph hp, cc t bo s biu hin
proteoglycan, collagen isoform v cht nn sn hnh thnh [1]. V th, cc phng php
nui cy ph bin c s dng trong qu trnh bit ha sn in vitro nhm thit lp mi
trng gip cc t bo gc c th tng tc vi nhau nh: nui cy micromass,

nui cy trong gel alginate v nui cy vn cc. Nui cy vn cc l phng php c

s dng trong ti ny.
Sau khi thu nhn v nui cy t bo gc m m t t bo m, cc t bo ADSC c
nui cy vn cc bit ha thnh nguyn bo sn. cc mc thi gian sau khi bt u
kch hot bit ha 1, 2, 3 v 4 tun s thc hin RT-PCR cho cc gen Aggrecan, COMP,
Col2a1, Col10a1 v nhum Toluidine blue xc nh giai on hnh thnh nguyn bo sn.
4.1.1. Bit ha nguyn bo sn t t bo gc m m bng nui cy t bo n
Phng php nui cy vn cc c mt hn ch l cc t bo bit ha khng ng u
do cc t bo bn ngoi tip xc vi mi trng bit ha nhiu hn l cc t bo nm bn
trong li ca cc vn. Mt s nghin cu cng b, khi cc t bo gc m m c
kch hot bit ha trong iu kin nui cy t bo n, nhng t bo ny vn din ra qu
trnh bit ha sn cho d khng c s tng tc gia t bo vi t bo. iu ny cho thy
mc d iu kin vi mi trng 3D gip cc t bo tng tc vi nhau thc y s bit
ha sn hn nui cy t bo n nhng iu kin ny khng phi l bt buc ca qu
trnh bit ha sn t t bo gc m m. V th cn th nghim song song hai phng
php bit ha sn l nui cy vn cc v nui cy bm dnh c th la chn nguyn
bo sn c to ra t phng php no l tt nht [9].
Sau khi thu nhn v nui cy t bo gc m m t t bo m, cc t bo ADSC c
nui cy t bo n bit ha thnh nguyn bo sn. cc mc thi gian sau khi bt u
kch hot bit ha 1, 2, 3 v 4 tun s thc hin RT-PCR cho cc gen Aggrecan, COMP,
Col2a1, Col10a1 v nhum Toluidine blue xc nh giai on hnh thnh nguyn bo sn.
4.2. Tip tc bit ha sn t nguyn bo sn trn gi th PLA
Nguyn bo sn c a ln gi th PLA v tip tc c nui cy trong mi trng
bit ha sn trong vng 2 tun. Sau s tin hnh cc th nghim RT-PCR cho gen
Col2a1 v Col10a1, nhum t bo bng Toluidine Blue, chp SEM (Scanning electron
microscope) v flow cytometry cho marker CD10/14/26/151 xc nh t bo sn
c hnh thnh [5]

4.3. S ni dung nghin cu

T bo gc m m
ADSC

Nui cy vn cc

Nui cy bm dnh

Xc nh giai on
nguyn bo sn

RT-PCR
Nhum Toluidine Blue

Cy nguyn bo sn
ln gi th PLA
Kho st s tng trng
nguyn bo sn trn
gi th PLA

RT-PCR
Nhum Toluidine Blue
Chp SEM
Flow cytometry

5. Phng php nghin cu

ti s dng cc phng php nghin cu sau:
-

Thu nhn v nui cy t bo gc m m

Nui cy bm dnh, nui cy vn cc

Bit ha sn trn gi th 3D

Nhum Toluidine, RT-PCR, chp SEM, flow cytometry.

5.1. Thu nhn v nui cy t bo gc m m [1]

Mi trng tch chit:
HBSS
Betadine iodine
Collagenase type I
PBS

Mi trng nui cy v cy chuyn:

DMEM
10% FBS
1% Penicillin 100 U/mL + Streptomycin 100 g/mL
Trypsin/EDTA
Gentamycin
PBSA
IMDM
Cch thc hin:
M m c ct nh v ra bng HBSS, betadine.
B sung collagenase, t trong b n nhit 37oC trong 60p.
Ly tm thu cn. Huyn ph t bo bng PBS ri tip tc ly tm.
Huyn ph cn vi mi trng nui cy. 37oC, 5% CO2.
Thay mi trng 2 ln 1 tun.
Khi t bo pht trin 70 80% din tch bnh nui th cy chuyn.
5.2. Phng php nui cy vn cc [2]
Nguyn tc: S dng phng php ly tm vi tc hp l s lm cc t bo t li vi
nhau nhm to iu kin mi trng tng tc gia t bo vi t bo. S tng tc ny
tng t nh cu trc m sn t nhin, gip thc y s bit ha sn ca t bo gc.
Mi trng bit ha sn:

DMEM HG (Dulbeccos Modified Eagles Medium High glucose)

Dexamethasone 100 nM

Penicillin/Streptomycin (100 g/ml streptomycin, 100 U/ml penicillin)

ITS + (bovine insulin, transferrin, selenous acid, linoleic acid, BSA)

L-Ascorbic acid 2-phosphate 50mg/mL

FBS 10% + TGF-1 10 ng/ml

Cch thc hin:

Thu nhn 2 x 105 t bo ADSC cho vo falcon 15 ml

B sung 1 ml mi trng bit ha sn

Ly tm 500g trong 5 pht. b dch ni.

B sung 500 l mi trng bit ha sn. vi nhit 37oC v 5% CO2

Thay mi trng 3 ngy mt ln

5.3. Phng php nui cy t bo n

Thu nhn 2 x 105 t bo ADSC cho vo a bn ging.

B sung 400 l mi trng bit ha sn. vi nhit 37oC v 5% CO2

Thay mi trng 3 ngy mt ln

5.4. Phng php nui cy trn gi th 3D

Thu nhn 2 x 106 nguyn bo sn cy vo gi th a petri

37oC trong 2 gi t bo bm dnh vo gi th

B sung mi trng bit ha sn sao cho gi th ngm ht trong mi trng

vi nhit 37oC v 5% CO2

Thay mi trng 3 ngy mt ln

5.5. Cc phng php xc nh nguyn bo sn v t bo sn

5.5.1. RT-PCR (Reverse transcription polymerase chain reaction)
Nguyn tc: Phng php RT-PCR l s kt hp gia phng php phin m ngc v
phng php PCR. Phng php ny c th pht hin cc mRNA tn ti vi lng rt
thp c trong mu
Cch thc hin:
Tch chit RNA
To cDNA
Chy PCR vi cp mi c hiu cho gen Aggrecan, COMP, Col2a1, Col10a1, GADPH.
Chy in di gel agarose 1%

Mi xui

Gen

Mi ngc

Amplicon

Aggrecan

5-CCTCTGGACAACCAGGTGTT-3

5-AAACCAGGTCAGGGACTCCT-3

174 bp

COMP

5-AGGACAACTGCGTGACTGTG-3

5-GTGTCC TTTTGGTCGTCGTT-3

221 bp

Col2a1

5-ACCCCAATCCAGCAAACGTT-3

5-ATCTGGACGTTGGCAGTGTTG-3

399 bp

Col10a1

5-AGGTGCCAAAGGGGAACAAG-3

5-AATCCTGGAATGCCTGGTGG-3

429 bp

GADPH

5-GAGTCAACGGATTTGGTCGT-3

5-TTGATTTTGGAGGGATCTCG-3

238 bp

5.5.2. Nhum m t bo bng Toluidine Blue

Nguyn tc: Toluidine Blue l mt thiazine bin sc c bn v c i lc cao vi cc
thnh phn m c tnh acid. Do , Toluidine blue s lin kt vi cc proteoglycan mang
in tch m c trong cht nn sn. Sau khi ra i tuc nhum, mu sn s bt mu xanh
dng c trng.
Cch thc hin:
Chun b ng khung mu vn cc:

a mu vn cc vo 20 ml paraformaldehyde, vi nhit phng trong 4 gi.

Dehydrate mu bng cc dung dch EtOH

Ra bng xylene

B sung paraffin vi nhit 60oC trong 60 pht

Chuyn vo khung qua im

Ct lt mng 10 m chuyn ln lame

Nhum Toluidine Blue

Rehydrate mu vi dung dch EtOH. Ra vi nc ct

B sung dung dch Toluidine Blue, nhit phng trong 20 pht. Ra vi nc ct

Dehydrate mu vi dung dch EtOH

Ra sch mu bng xylene

Quan st trn kinh hin vi

10

5.5.3. Flow cytometry

Nguyn tc: Flow cytometry l mt k thut c nhy cao dng xc nh cc
marker b mt ca t bo. K thut ny s dng phng php nhum hunh
quang gm mt khng th khng marker b mt c gn mu hunh quang. Sau , cc
t bo s c di chuyn theo dng cht lng xuyn qua mt chm tia sng. Khi t bo
c nhum hunh quang b va p bi nh sng tp trung th chng pht ra nhng tn hiu
khc nhau. Tn hiu ny c cm nhn bng my d, chuyn n my tnh x l v
c xut ra thng tin kt qu.
Cch thc hin:

Thu nhn 106 t bo bng trypsin/EDTA 0.25

Huyn ph t bo trong 1 ml Facs Flow v nhum vi 10 l khng th khng

CD10/14/26/151 trong 30 pht nhit phng.

Lm lnh mu trong 15 pht 40C.

Tin hnh nh gi marker t bo bng my FACS Calibur s dng phn mm

Cell Quest Pro.

5.5.4. Chp SEM (Scanning electron microscope)

Nguyn tc: SEM l mt loi knh hin vi in t c th to ra nh vi phn gii cao
ca b mt mu vt bng cch s dng mt chm in t electron hp qut ln trn b
mt mu. Vic to nh ca mu vt c thc hin thng qua vic ghi nhn v phn tch
cc bc x pht ra t tng tc ca chm in t vi b mt mu. u im chnh ca
phng php ny l c th phn tch b mt mu m khng cn ph hy mu th.
Cch thc hin:

Mu c ct lt mng v c nh trong dung dch 2.5% glutaraldehyde

Ra 3 ln vi 0.2 M sodium cacodylate buffer (pH 7.4).

C nh mu vi 1% osmium tetraoxide v ra bng dung dch cn v amyl acetate

Sy kh

Mu c bc vng v quan st bng thit b SEM.

11

6. D kin kt qu
Giai on hnh thnh nguyn bo sn bt u sau 7 ngy n 14 ngy sau khi kch thch
bit ha t bo gc trung m thnh sn.
Sau 2 tun bit ha sn trn gi th 3D, t bo sn c hnh thnh s lng nhiu.
7. a im v thi gian thc hin
a im: Phng th nghim nghin cu v ng dng t bo gc
Thi gian: T thng 1 nm 2015 n thng 8 nm 2015
STT

Ni dung, cng vic ch yu

Kt qu d kin

Thi gian
thc hin

Xc nh giai on hnh thnh

Xc nh thnh cng

Thng 1 3/2015

nguyn bo sn t t bo

giai on hnh thnh

ADSC bng nui cy vn cc

nguyn bo sn

Xc nh giai on hnh thnh

Xc nh thnh cng

nguyn bo sn t t bo

giai on hnh thnh

ADSC bng nui cy TB n

nguyn bo sn

Kho st s tng trng ca

nh gi c kh

nguyn bo sn trn gi th

nng bit ha thnh sn

Thng 3 5/2015

Thng 5 7/2015

ca nguyn bao sn
4

Phn tch, tng kt cc kt qu Hon thnh ti.

v vit bo co.

12

Thng 8/2015

8. Ti liu tham kho

8.1. Ti liu Ting Vit
[1] Phan Kim Ngc (2010), Cng ngh t bo gc, NXB Gio dc Vit Nam.
8.2. Ti liu Ting Anh
[2] Bradley T. Estes, Brian O. Diekman, Jeffrey M. Gimble (2010), Isolation of adipose
derived stem cells and their induction to a chondrogenic phenotype, Nat Protoc, 5(7),
12941311.
[3] Francesco Carf Pavia (2012), Poly Lactic Acid Based Scaffolds for Vascular Tissue
Engineering, Chemical engineering transactions,27.
[4] Gary S. Firestein,Ralph C. Budd,Sherine E (2012), Cartilage and Chodrocytes,
Kelley's Textbook of Rheumatology volume 1, NXB Elsevier Health Sciences, 33.
[5] Hamada T, Sakai T, Hiraiwa H (2013), Surface markers and gene expression
to characterize the differentiation of monolayer expanded human articular chondrocytes,
Nagoya J Med Sci, 75(1-2):101-11.
[6] Hanna Taipaleenmki (2010), Factors regulating chondrogenic differentiation,
University of Turku.
[7] Liliana S. Moreira- Teixeira (2011), Cartilage Tissue Engineering, Cartilage and
Bone Development and Its Disorders, 21, 102115.
[8] Samira Yarak (2010), Human adipose-derived stem cells: current challenges and
clinical perspectives, An Bras Dermatol, 85(5):647-56.
[9] Stephane Boeuf, Wiltrud Richter (2010), Chondrogenesis of mesenchymal stem
cells: role of tissue source and inducing factors, Stem Cell Research & Therapy,1: 31.

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