CHfe TAO VAT LIEU TyONG OUPNG TRUNG Bi TU li BAO Gdc TRUNG MO MANG DAY RdN TRONG Di^U KIEN NUOI CAY KHONG GIA DP
Dfe Oieu TR! V^T THUPNG V^T B 6 N G
Dinh Viin Hin, Nguyin Gia Tl4n, Phan Minh Hoing, Oodn Hoing Thu La bO nghidn c&u trong didu tri bdng
VI0n Bdng Qudc gia
T6MTAT
V$t Wu tuvng auvng tnjng bl dS duvc nghiin cOv phit triin dira trin vi$c ciy nguyin Mo so da liin gii M 3 chiiu. Hiin nay, gii thinh cao cing vOi quy trinh phdc tap trong chi tao vi si dung as han chi tinh Cmg dung aia tim vit liiu Tmng nghiin citu niy, chOng tii chi tao tim vit liiu tuang duong trung bl bing cich ciy ti bio gSc trung mi tir diy rin trin dia nhi/a nudi dy vi duy tri cic ti bio trong mil truing cd kich thich tdng hop dim gian bio. Chung tdi dd khio sit vi hinh thii ciu tnJc tim vit liiu cljng nhu dinh gii tic dung cua chiing trin md hlnh vit thuang ding v$t
ABSTRACT
The dermal equivalent is an in vitro model of the dermal layer of skin. It is constructed tiy seeding dermal fibroblasts into degradabie or polymenc scaffold. It can be used for wound care to accelerate wound healing process. Due to high cost and lack of dermic components, the dermal equivalent has not been used popularly In this study, we developed a new dermal equivalent tiy seeding cord lining mesenchymal stem cells in plastic culture substrate and maintaining in specific medium to stimulate cells to secrete extracellular matrix. The morphology of dermal equivalent was investigated, we also made evaluation in animal wound model.
1. OAT VAN B £ dirvc c h i t?o dva trSn cdng nghf m6 tso ca V$t li$u tirong difong da Hi diriyc t*"! * * ' H*" ^^° 9*"^ '^^ * * " • ! P*i*" '"""S nghiSn ci>u phit triin vi ch4 tao trong thi/c ' " " " S *>''^'' >" "^ '*'*"•' P"*" ' " " " S ^"'^^
nghidm tir v i i thSp ky tri/ijc diy [1], [2], Oiu '™"9 *>' ^^"S '=*<=•' !<*' I^^P c i c yiu t i trung tiSn l i sir xuit hiin nuSI ciy c h i tso t i m v i t " ' " * "'^^ "'• <=*<: V*" ' * " * » <"""^ P"*' '"*"
ii^u lu'ong du'ong biiu bl v i sau d i i i sir ra ''f^ '''*" nguySn ly ca b i n i i nuil cSy diri cua cing nghi c h i tao t i m biiu bl [3]. "S^V*" "^o S'i «* ' ^ *'^° *'''^" ""l " * " 9i* '"
Tuy nhiin, cic v i t liiu niy d i u c6 han c h i 3 chiiu nhu' t i m collagen. TCr khi c6 vit lij»
trong si> dgng cQng nhif hiiu q u i thinh ^ ^ y "^^ ''^ ''''VC c h i tao Irong labo, chiing cing trong diiu tri l i do thiiu thinh phin '^°"9 '^ '>""" I>n9 dMng trong trong nhieu cda trung bl. Do d i mit s6 v i t li$u khic ""^ "''"= ''•^^<= "^'^^ ' ' * " l " ^ " ^^" ""^ "^I"
nghien cu-u du-Q-c dOc hgc thim chl du-gc su dung trin l i m sing d i ghip diiu tri thay tti^
Phin bien khoa hoc: PGS.TS. Nguyen VSn Hui da ngu'i'i cho b$nh nhin bing hay tfin
61
ithuyet da [4] MOt so v$t ii^u lu'ang du-cng trung bi da du-p-c si> dyng de ph^t t r i i n nghi&n cO-u che tgo v ^ t li$u thay the da Tuy nhien, cSc v$t li$u n^y Qhu phai dung thSnh phan dong logi hay d| logi n h u collagen cua b6 [5]. [6] h o ^ c trung bl khdng c6n th bdo song lay tit tCr thi [7] h o ^ c cdc giei dfr t6ng hp'p b i n g polymer [8].. cdc thdnh ph4n ndy deu rat dat vd tiem hn cdc nguy c o tdy b§nh t r u y i n n h i l m . , ,
Trong da. nguyfen bdo sp'i Id th bdo chu yeu du-cc bao quanh bdi d$m gian bdo.
ChCing cOng tgo ra d§m gian bdo bao g6m collagen, elastin vd cdc protein d$m khdc vd chCing cung tu-ang tdc qua lgi v&i m0t s6 logi te bdo khdc nhu- th bdo su-ng b i l u bi, d6ng vai tr6 cot yeu trong dieu t i l t sinh ly da Ode bi0t trong lien v l t thu-ang da, vai trd cua nguyfin bdo s(?i tgo ra ECM ddng k l ho-n n h i l u so v6\
da binh thu-dng.
Tu-ang ti,r nhu- d i l u ki$n cua da t i / nhifin, cdc hormon vd yeu to tdng tru-ang cung du'ac coi Id y i u to quan trpng trong vi^c tdng tru'dng cua t l bdo vd tong hp'p d$m gian bdo cua cdc te bdo nuSi cay [9]. Nguyen bdo sp'i da nu6i c i y 6ut?c ghi nhdn Id tdng smh vd tong hap d$m gian bdo khi c6 mdt mdt so hormon hay yeu to tdng tru'dng [10], [11]
Trong nufli c^y d a n ldp, nguyfen bdo sp-i da du-iyc cho Id tgo nhieu thdnh phan d^m gian bdo han nhu- collagen sau khi dat 100% 3d che phij (postconfiuent) [12], [13]. G i n ddy Chung tdi nhdn thay r^ng cdc nguyfin bdo sp'i c6 the du'ffc bi$t hfla tii- t l bdo g6c trung mfl mdng ddy r6n. Cdc te bdo ndy c6 kha ndng nhdn 16n mgnh vd nhieu han cdc nguydn bdo s(?i da, Han n&a, ddy r i n Id rdc thai y hpc nhu'ng lgi Id mflt nguon mfl cung cap t l bdo ly tu-dng bdi thflng thu-dng mfl ddy r i n deu cfl diSn tich 3000 d i n 4000 cm^, do dfl chi can mflt m i u ddy ron Id dd cfl the cung d p s i lu'ang c y c ky ldn t l bdo trung mfl de c h l tao cdc tam te bdo hay cdc tam vdt li#u
tu-ang du'ong trung bl d l diling trong cdc nghifin ci>u in vitro vd d i l u tri b$nh [14], Phdt hi$n ndy cCing vdi nh&ng hgn c h l cua t i m vdt li§u tu-ang du'ang trung bl hi$n hdnh dd thCic d i y chiing tfli phdt t r i i n logi vdt li#u tu'ong du'ang trung bl cfl tinh Crng dyng vd an todn han,
Trong d i l u k i | n hi$n nay, chCing tfli phdt t n l n logi vdt li$u tu-ang du'ang trung bl mdi bdng cdch nufli c i y t l bdo g l c trung mfl trong mfli tru'dng ddc bi$t cfl chO-a mflt s6 y i u t l d l tgo t i m t l bdo sau dfl thay mfli tru'dng chuy6n bi$t cho nguySn bdo sp'i vd kich thich chCing t i l t ra nhu'ng thdnh p h i n d$m gian bdo 6h tgo tam vdt lifiu tu'ong du'ang trung bl md khflng c i n gid d d hay cdc thdnh p h i n dj logi vd d i n g iogi l^hdc ChOng tfli ddnh gid iogi vdt li$u ndy bdng cdc nghifin ci>u hinh thdi cau triic mfl - t l bdo trong tam vdt l i | u . Dong thdi chung tfli cung ddnh gid kha ndng dng dung in vivo cua vdt iifiu ndy bdng cdch ghdp chOng \dn dOng vdt vdi mo hinh d i l u tn v l t thu'ang v l t bong thu'c nghidm d tho.
2. NGUYEN VAT LIEU VA PHU-QNG PHAP NGHIEN C i r u
2.1. Tach t l bdo t r u n g mo day rdn IVlfl ddy r i n du'pc thu tu- san phy ngay sau khi sinh, mfl du'p'C bao quan trong mfli tru'dng dinh du'dng, Sau khi c i t nho thdnh Cdc mau cfl kich thu'dc 0.2 x 0,2 cm, ddt cdc m l u mfl trong dTa nufli c i y nht,fa vd b l sung mfli tru'dng chuydn bi$t du'p'C cung d p ti>
Ngdn hdng t l bdo g l c Mekostem, ddt cdc dTa md vdo tu I m 37°C vdi 5% C O j . Thay mfli tru'dng nudi d y 2-3 l l n / luan vd theo dfli d i n khi cdc t l bdo hlnh sao hay hlnh thoi tdch ra khoi m l u md vd phdt t r i i n dgt 50%- 7 0 % b l mdt dTa thl t i l n hdnh tdch t l bdo b i n g quy trinh s d dyng trypsin, Cdc t l bdo du'(?c nhdn idn d i n t h l hfi d y chuyen t h d 3- 4 diling vdo nghidn c d u c h l tgo tam vdt ii$u lu'ang du'ang trung b1.
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S v 66 1. C i u true md dSy rin, Ldp t l bdo bilu mfl tu'O'ng ti,f mdng 6i (1), Idp t l bdo g l c trung md mdng ddy r i n (2), Idp t l bdo g l c trung mfl Idp Wharton' Gelly (3) Cdc t l bdo du'p'C sd dyng trong nghign cdu ndy thuOc v| tri s i 2 (Katsuhiro Kita, Gerd G.
Gauglitz, Thang T. Phan, David N, Hemdon. and Marc G. Jeschkel. Isolation and Characterization of Mesenchymal Stem Cells from the Sub-Amniotic Human Umbilical Cord Lining Membrane. Stem celts and Development, 2009, V 20, NO 20 : 1 -11) 2.2. T90 tim v$t li^u tirang diFcng trung bl
T l bdo tnjng md du'p'C nufli d y d s i lu'p'ng lu'ang du'ang 5000 t l bdp/ cm^ dTa nufli ciy d = 100mm, Sau khi dgt 100% dO che phu (confluent). chCing tilp tyc du-p-c nufli d y thdm vdi tuin nda Trong thdi gian nufli d y , mdi tru'dng du'p'C thay thl bing bi$t hfla nguydn bdo sp'i d l kich thfch tilt d$m gian bdo. Khi cdc t l bdo mpc chIng lln nhilu ldp lln nhau vd cd d$m gian bdo, tim v$t li$u se quan sdt rd bdng mil thu'dng vd du-di kinh hiin vi. Diu hilu thiy rfl Id tim v$l li$u bit diu co vd tdch ra khoi b l mdt nufli d y d mdp dia. DCing dyng cy Id cell lifter ggt nh? d l tdch hodn lodn tim v^t lidu khfli b l mdt dTa, Tim v^t li$u sau dd du'p'C sd dyng cho cdc nghidn cO-u ddnh gid hinh thdi d u true vd ghdp dilu tri vlt Ihu-ong bong thy-c nghi|m.
Mu-di thl nghiim di/p-c tiln hdnh sd dung 5 dflng t l bdo d P3 vd P4 thu du-cc td mfl ddy rin, 2.4. Hlnh thdi m6
V$t lidu tu-ang du-ang trung bl du-^ co dinh trong dung djch Boin trong 24 gid, tiln hdnh khu- nu-dc vd due tidu bdn trong paraffin, d t thdnh cdc Idt mdng 5-6 micromet vd nhu^m haematoxyiine - eosin, quan sdt tidu bdn dUH^i kinh hiin vi quang hpc.
2.5. Giy v l t thuvng bdng t h ^ nghidm vi ghip t i m v^t li^u tirang duvng trung bl lAn v l t thirang
Gdy bflng 20 cm^theo phu-ang phdp cua Halovec vd Pocicado 1961.
Thay bdng xi> ly vlt thu-ang di/grc tiln hdnh hdng ngdy theo quy trinh, £}$t tim v$t li$u tu-ang du-ang trung bl Idn vlt thirong, trdnh d l gip mdp, djch, bpt khi bdn du-di. D^
mdt Idp ggc vaselin ldn trdn tim vdt lidu. Sdt 5- 6 Idp ggo vd trung, bdng dp nh^. Bdng dp nh?
bing ldp vdi may theo dgng do d l trdnh tho ldm tuflt bdng. Thay bdng hdng ngdy hc^te cdch nhdt tuy theo Unh trgng vlt ^u-ang, Trong tru-dng hp'p vlt thuang nhiSm khuin thl dilu tri nhilm khuin vlt thu-ang cho din ktii sgch vi khuin mdi tilp tgc ghdp.
3. K£T QUA NGHIEN C1>U
3.1. Tim v$t ll$u tirang du>ang trung bi Hai tuin nudi d y tinh tCr sau khi t l bdo dgt 100% dd che phu, cdc t l bdo trung mb du-p-c duy trl trong mdi tru-dng nudi d y bi^t hda dd bd sung mOt s6 yiu t6, cdc t l bdo hlnh thoi ddi phdt thin chIng lln ldn nhau khoing 2-3 ldp dan chdo nhau theo nhilu hu-dngvdc6 sy- lidn kit vdi nhau (dnh 1 a). Trong khi dl t^
bdo nufli d y trong dilu kidn truyin thing khflng c6 yiu t l b l sung cho thiy cdc t l Mo vSn phdt triin dan Idp, cdc t l bdo nhilu hinh dgng nim cgnh nhau trong 66 cd d c te tio hlnh sao, hlnh thoi ngin han (Snh lb).
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Anh 1. T l bdo moc thdnh hai Idp dan ch6o nhau d ngdy thd 5 trong mfli trudng bi$t h6a (a), trong khi t l bdo v l n duy tri 1 Idp d mfli truing tSng tnj'dng cho t l bdo g l c trung mfl (b),
(hinh dnh soi tr6n kinh hiin vi ddo ngucri - SOX)
Anh 2. Duy tri moi tn/dng nuoi d y 2 tuin, te bdo trong moi trudng biet hfla tgo thanh t i m vat li#u tuong du-ang trung bi, cac t l bdo moc thdnh hai -3 Idp dan cheo nhau, tam vat ligu bat dau bong (a), lie nh§ dTa
nudi thiy t i m vdt li#u bong va bd m6p dich chuyin ve phia trung tam dTa (b).
(Soi n i l - SOX)
a) b) Anh 3. Hinh dnh dai the t i m vat li§u tuang duong tmng bt sau 2 tuin, tam v^t ligu dai vd de tach ra khoi b l
mgt dTa nufli d y (a), Hlnh thdi c i u tnic tren kinh hien vi quang hpc nhuflm HE cCia t i m vgt lieu tuang du-ang tnjng bi, t i m vdt li§u co chda khflng chl t l bdo dan thuin ma con bao gim ca d§m gian bao. T i m vdt ll#u cho thiy trong thdnh phan cua chung chda r i t nhieu t l bdo dang nguySn bdo sp-i vd dem gian bdo
tucrng d i i ddy (b) HE. 5X.
64
Khi ti bio g6c trung mS mSng dSy rin phit tri^n trong mSi trifOng nufti c4y, ti bSo cOng c6 xu tiu'*ng t?o ra 2 ldp ching l4n ISn nhau chi> khSng ph4t trl4n hlnh cu4n xaiy nhu- Fibroblasts trung bl. Theo dSi c4c ngSy sau thay mOi trudng biSt hia, th4y t i bio moc manh xip ching lin ISn nhau, khoSng ngSy thCr 6-B c6 thi nhin thiy r i bing mit thu'dng.
NgSy th\> 10. ldp t i bSo cd xu hu'dng bong bit khii b i mSt (JIa. lie nhe (5Ia hoic dung iJiu pipet gat nhe li o6 thi tao ra tim t i bSo Tim t i bSo chi 00 nhi hon diin tich dSy dia mdt chijt, cdn khoing = 2/3 diin tloh dTa nudi.
V i khia canh md hoc, t i m vJt IISu cd chi>a khdng chl t i bSo dan thuin mS cdn bao gim ci asm gian bio, tim vit liiu cho thiy trong thSnh phin cila chiing chila rit nhiiu t i bjio dang nguyin bSo sal. Hlnh inh m i hpc tim ti bSo gal i Siy l i vit liiu tuang duang trung bi 3.2. Kit qui ghip diiu tr| vit thuvng th^rc nghiim
Vdi 05 thd thl nghiSm duoc ghip tim vJt IISu tuang duang trung bt vSo thdi diim ngSy thi> 2 sau cit hoai tu sdm ISm sach hoai tu va cim mSu tit.
Anh 5. Vet tjdng dui^c c i t hogi ti> to^n Idp, n i n v i t thucyng \i md difdl da (a); tam v^t lieu dup-c d^n deu die phij b i mat v i t thuang (b). NgSy thi> 5 sau gh6p, t i m v§t lieu bdm va che phiS t i t v i t thuang (c). Ngay nghifti cuu thi> 14 gh6p t i m vat li$u tuang duang trung bi, vet thuang g i n liin hoSn todn bang ca hai hinh thuc 13 biiu mo hda tU bd m6p vi co hep di#n tich v i t thuang, b i mSt seo nhin vi phing so vdi b i m§t da l^nh
xung quanh, seo v§n thiiu thSnh phin phy ciia da (d). (Thd s i 1).
Tim vdt lidu du'p'C ddn diu ldn vlt thu-ang (5nh 5b) nhanh chdng bdm vdo vlt thu-ang. Ngdy nghidn cdu thd 5, b l m$t vlt thu'ang du'p'C che phu mOl ldp mdng mdng, bdm chic vdo vlt thu-ang ldm vlt thu-ang khdng chay mdu iOc thay bdng nhu'ng ca bin vln quan sdt thiy nen vlt thu-ang mdu dd hing vd sgch, Tgi nhdng chd Idp mdng bdm vdo ggo vd bj bong khi thay bdng quan sdt thiy t l chdc hgt sgch, dd vd rdm mdu (dnh 5 c). v l t thu-ang cung chu-a thiy rd hi^n tu-p-ng bilu md hda td bd mdp. Quan sdt vlt thu-ang td ngdy thd 7 thiy bilu md hda mgnh d bd mdp vlt thuang tiln din vdo phia trung tdm d l ldm ldnh vlt thuang. Kdm theo hi$n tu-p-ng bilu md hda mgnh id diu hidu vlt thuang co nhd. Din ngdy thd 15, ca bin cdc vlt thu-ang liln da bing bilu md hda. V l chit lu-p-ng sgo thiy b l m$t s^o bing phing, nhdn, sd cd cdm gidc cdng cdm han da ldnh xung quanh, khdng thiy diu hidu mpc ldng trd igi (dnh 5 d). Khdng Ihly cd diu hidu kich thich gdy vidm d vlt thu-ang hay phdn dng di dng d viing da ldnh quanh vet thu-ang d ca 5 thd Irong suit qud trinh nghidn cdu
4. BAN LUAN
Dii vdi da binh thu-dng, trung bl id ldp h i In? cho bieu bi bao gom thdnh phin sp'i nhu collagen vd elastin vd cQng bao gdm thdnh phin t l bdo. Te bdo chu yeu cua trung bl Id nguydn bdo sp'i ddy Id dgng t l bdo nguin gdc trung md md cd kha ndng ting hp'p vd gidng hda thdnh phin'protein ddm sp'i vd khdng sp'i ciJa md lidn kdt vd cdc yeu t l hda tan khdc
Cd vdi v$t li$u tuang duang trung bl dup'C chl tdc theo khudn mlu tgo trung bl trdn labo.
Trong cdc md hlnh ndy, nguydn bdo sp'i dup'C nudi d y trdn gid dd ba chilu cd cdc thdnh phin Irung bl nhu collagen vd glycosaminoglycan [5], [6] hodc Id cdc polymer ting hp'p [8]. Trong dd, cd md hlnh chl tgo bing trdn lln ddm collagen vdi nguydn bdo sp'i. Vdi md hlnh ndy. cdc nhd khoa hpc dd cd san pham dtfp'c tgo ra bdi SM"
kit h(?p gida nguydn bdo sp'i da trong d$m collagen vd dd dup-c su- dyng rdng rdi nhu- mdt
hlnh mlu u-u vidl, Tuy nhidn, trong nghidn cdu cua Chung tdi, chiling ldi kiln tgo ra vdt li$u tuang du-ang Irung bl mdi bao gim ddm gian bdo vd cd cdc t l bdo cd hlnh dgng cua nguydn bdo sp'i bing cdch nudi d y dan ddc t l bdo glc trung md tdch Id mdng ddy rin vd khdng d n gid dd, V l hlnh thdi md, tim vdt lidu theo md hlnh ndy cho thiy mdt s6 d$c tinh tuang t\/
nhu trung bl blnh thudng Trong nghidn cdu ciJa Chung tdi, d$c tinh gilng trung bl dup-c xdc dinh cd bdng cd ddc dilm dgi thl vd vi thl. V l dgi thl, tim vdt lidu quan sdt rd bing mdt Ihudng cd mdu tring ngd nhu trung bl da blnh thudng. lim vdt lidu tuang dii chic khi dCing cdc dyng cy d l djch chuyin, thdm chl cd thl gip ldm 2 Idp (dnh 3 a) vd ngay d khi ghdp ldn vlt thuang thl vln Ihly rd tim vdt lidu dgng mdng mdng (anh 5 b). Tim trung bl cua Chung tdi cQng tuang ty nhu md hlnh tim trung bl tgo ra bdng cdch trdn nguydn bdo sp'i vdi collagen vl thiy rd ca nhdn t l bdo vd ddm gian bdo Id khodng cdch rit rOng trdn tidu bdn nhudm HE cua tim vdt lidu m$c du dd tdch hodn todn ra khdi b l mdt dTa nudi (anh 4a, 4b) ngay ca khi dd Idch ra khoi b l mdt <Jra nudi d y . Tuy md hlnh chl tao san phlm ndy cd chda d ddm gian bdo ca t l bdo dgng nguydn bdo sp'i nhu-ng rit cd thl dd ddy san phlm cua Chung tdi chua the dgt dup'C nhu khi trOn nguydn bdo sp'i vdi collagen. Bdi vdi md hlnh chl tgo tam trung bl ndy vide tao ra lim trung bl cd dd ddy bao nhidu Id do ngudi chl tao cd thl chu ddng du-^c bing cdch ddn nhilu hay It hin djch nguydn bdo sp'i - collgen gell. Cdn Irong thl nghidm cOa chCing tdi, vi$c tgo ra tim vdt lidu dya trdn khd ndng cua l l bdo dgng nguydn bdo sp'i bi$t hda tu t l bdo glc tmng md tgo ra d|m gian bdo cua chlnh nd trong dilu ki$n nudi d y , Cung vdi dilu dd, kit qua nghidn cdu gp'i y ring s§n phlm ci}a chOng tdi cd thl hdu Ich nhu vdt lidu tuang duang trung bl vd c6 nhdng d^c diem gin gOi vdi san phlm trung bl nudi d y khdc.
Nghidn cdu cOa chdng ldi cQng chT ra ring, vdt li|u tuang duang trung bl mdi ndy cd the thich hp'p vdi cdc myc dich thi nghi|m vd
66
idm sdng, Bl sung cho nhdng thl nghiim In vitro lidn quan din loai vdt lidu luang duang trung bl mdi ndy chung tdi thiy nd cd khd ndng kich thich liln vlt thuang d thd gdy vlt thuang thyc nghidm bing cdch Idm nhanh hlnh thdnh t l chdc hgt dep, kich thich bilu md hda td bd mdp v l l thuang.
5. K£T LUAN
Tu nghidn cdu ndy, lim v|t Ii$u rit gilng vdi trung bl da binh thudng c6 thl dup'C tgo ra bing nudi d y dan d|c t l bdo dgng nguydn bdo spi bidt hda td t l bdo glc trung md mdng ddy rin, Tim vdt Ndu cQng hdu ich trong cdc thu nghidm in vitro vd cdc myc djch idm sdng nhu kiln tgo vdt lidu tuang duang da vd ghdp trdn ddng v$t,
TAI Lieu THAM KHAO
1 Lee DY, Ahn HT, Cho KH A new skin equivaleni model use of a dermal substrate which combines de- epidermized dermis wilh fibroblast-populated collagen matrix. J Dermatol Sci 2000,23:132-7
2 Lee DY, Cho KH The effects of epidermal keratinocytes and dermal Tibroblasts on (he formation of cutaneous basement membrane in three- dimensional culture systems. Arch Dermatol Res 2005,296.296-302.
3 Green H, Kehinde 0 , Thomas J . Growth of cultured human epidermal cells into multiple epithelia suitable for grafling. Proc Natl Acad Sci USA 1979;76'5665-8, 4 Ehrlich HP. Understanding experimenial biology of
skin equivalent' from laboratory lo clinical use in patients wilh burns and chronic wounds. Am J Surg 2004; 187:29s-33s.
5 Boyce ST, Michel S, Reichert U, Schroot B, Schmidt R. Reconstructed skin from cultured human keratinocytes and fibroblasts on a coltagen- glycosamlnoglycan biopolymer substrate. Skin Pharmacol 1990.2 136-43
6 Maruguchi T, MaruguchI Y, Suzuki S, Matsuda K, Toda Kl, Isshiki N A new skin equivalent:
keratinocytes proliferated and differentiated on
collagen sponge containing fibroblasts Plast Reconstr Surg 1994:93:537-44,
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