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1 f zu{ } 18 í } Çf Chinese Journl of Tissue Engineering Reserch November 19, 2014 Vol.18, No.47 Ån dšm üg Ùx Œqmgr Ê nìy í ÂÊ ({œ ÁÞ ) s Í¾Šm Šj q º²ŸÉ f º sqšjp Ìn Ån düšm Šj /Šjp ºŠg ùéy»qùx Œ Šm Šj /Šjp ü» x ŒŸÅqmÀÀ ºm grg²q zu{ ºÀ mgr Ån dšm Šj Šjp ümgr Ùx Œ Œ Œ ŒØÂiŽy gè mgrx ŒÙ Œ «Ù Žy ( ) držy (S S )dry ÊÈr(2012B )y ÊÈr(2013J ) µë ( ) n Šm Šj ºŠgpolylctic glycolic cidplgašjp polyethylene glycolpeg Šm Šj np zè º ŒØÂ± iøœxö o±} ÂÍoº²qön rq Ùx ŒÅn dšm Šj /Šjp ºŠg qƒr Ž 15 d CD-1 Ù Œq«Éy»Ùx ŒÅn díšm Šj Šm Šj /Šjp nà } 5 xx ŒÉÌypŠm Š j Ån dšm Šj /Šjp ²Ÿƒ» nàxeyjrpršqˆg Šm Šj Ån dšm Šj /Šjp rãi i(p > 0.05)CCK-8 xey Œx ŒfmÀe Ã»ÂÀ e» d i(p < 0.05)e» 369 hån dšm Šj /Šjp q ŒØÂiÎpŠm Šj (P < 0.05)Hoechst xe Œ«Ån dšm Šj /Šjp Œ pšm Šj (P < 0.05)n À xšm Šj rån dšm Šj /Šjp x ŒqmÀ À«É Ån díqån dšm Šj /Šjp Œmg r¹ºü²qãi²x ŒmÀy²np z~«q ±ƒ ökº d ö šd{œ ÁÞ doi: /j.issn [ fé ú:r318 h u:b ú: (2014) ÈÍ ÊnÌyíÂÊ.Ån dšm üg Ùx Œqmgr [J].f zu{201418(47): Biocomptibility of electrospun poly(lctide-co-glycolide)/polyethylene glycol nnofibrous scffold with mouse neurl stem cells Liu Chng, Rong Li-min, Li Shng-fu, Png Mo, Yng Yng, Liu Bin (Deprtment of Spinl Surgery, Third Affilited Hospitl of Sun Yt-sen University, Gungzhou , Gungdong Province, Chin) Abstrct BACKGROUND: Poly(lctide-co-glycolide) (PLGA) scffold is widely used in tissue engineering, but its poor cell dhesion bility nd strong hydrophobicity limit its further development nd ppliction. OBJECTIVE: To study the biocomptibility of electrospun poly (lctide-co-glycolide)/polyethylene glycol (PLGA-PEG) nnofibrous scffolds with mouse neurl stem cells in vitro. METHODS: Neurl stem cells were isolted from embryos of CD-1 mice t 15 embryonic dys. Electrospinning ws used to prepre PLGA nd PLGA-PEG nnofibrous scffolds. Scnning electron microscope ws used for scnning observtion of scffolds. The 5th pssge neurl stem cells were seeded onto PLGA nd PLGA-PEG scffolds respectively, nd cultured in vitro. Liu Chng, Mster, Deprtment of Spinl Surgery, Third Affilited Hospitl of Sun Yt-sen University, Gungzhou , Gungdong Province, Chin Corresponding uthor: Liu Bin, M.D., Associte chief physicin, Deprtment of Spinl Surgery, Third Affilited Hospitl of Sun Yt-sen University, Gungzhou , Gungdong Province, Chin Accepted: ISSN CN /R CODEN: ZLKHAH 7585

2 RESULTS AND CONCLUSION: Interconnected porous network structure ws observed in both two kinds of scffolds under the scnning electron microscope. Fiber dimeters nd porosities of PLGA nd PLGA-PEG scffolds showed no significnt differences (P > 0.05). Cell Counting Kit-8 detection showed neurl stem cells grew well on both two kinds of scffolds nd the bsorbnce vlue of two groups incresed continuously with incubtion time (1, 3, 5, 7, 9, 11 dys). And there were sttisticlly significnt differences in the bsorbnce vlues between two groups t ech time point (P < 0.05). Moreover, the cell dhesion rte ws significntly higher in the PLGA-PEG group thn in the PLGA group t 3, 6, 9 hours of culture (P < 0.05). Hoechst stining showed norml morphology nd qulity of the nuclei, nd significntly more cells were observed in the PLGA-PEG group thn the PLGA group (P < 0.05). Under the scnning electron microscope, compred with the PLGA scffold, the PLGA-PEG scffold ws better for growth nd mtrix secretion of neurl stem cells. In conclusion, PLGA-PEG nnofibrous scffolds prepred by electrospinning re sfe, non-toxic nd suitble for neurl stem cells growth with well biocomptibility, pproprite perture nd porosity. Subject heding: biocomptible mterils; neurl stem cells; mice; cell prolifertion Funding: the Ntionl Nturl Science Foundtion of Chin, No , , ; Gungdong Nturl Science Foundtion, No. S , S ; Gungdong Science nd Technology Plnning Project, No. 2012B ; Gungzhou Science nd Technology Plnning Project, No. 2013J ; New Techers Fund for Doctor Sttions of Ministry of Eduction of Chin, No Liu C, Rong LM, Li SF, Png M, Yng Y, Liu B. Biocomptibility of electrospun poly(lctide-co-glycolide)/polyethylene glycol nnofibrous scffold with mouse neurl stem cells. Zhongguo Zuzhi Gongcheng Ynjiu. 2014;18(47): Introduction ŒÎý qx ØŒrçnq ÄÈ [1] zyºqröy³²mg Œq üºmgq Œü g x q [2-4] Ìn zq qœî²gðœîu{q [5] ²ymgr¹ùÂ qp ŒØÂmÀq g zä ŒÎq±ƒÐu{q [6-7] Šm Šj ºŠg (polylctic glycolic cidplga)rðu{±q y [8] Šm (PLA)Šˆj (PGA)ºŠ gºqmgr xöâ ³iq ù [9-11] º ŒØÂ± iøœ xöo±} ÂÍoº²qö n [10] Í¾PLGAq º²ŸÉ f ºsqŠjp (polyethylene glycolpeg) [12-14] ÌnÅn düplga-pegº Šg ùéy»qùx ŒPLGA-PEG ü» x ŒŸÅqmÀÀ ºmgrgo ²q zu{ 1Mterils nd methods ŒƒÍ Âöp201312Ž20145f Â}çÃ Œo±äçu{f ÍÙg15 d SPF CD-1Ù1ùƒ« gnäs ³ÌéÍÙgÂ¹ú ù úscxk(s) Ùx ŒÅn dšm Šj Šjp qƒm grírºyö Ï yö Ï fždmem/f12»b27 Øg Gibco ¹ú w ŒmÀŸqmÀ Peprotech ¹ú Hoechst Sigm ¹ú CCK-8 Ïr êsæmgyöâ¹ú Šm Šj (PLGA) Polyscience ¹ú Šjp (PEG) päâ¹ú mg¹ Thermo ¹ú Œ»~ Hereus ¹ú nà FEI ¹ú y Bio-Tek ¹ú Í Ùx ŒqÉy»ÈŒŽÙ pƒzé75%j 30 min ú¼q ýÿµféøå Óˆp»r fpbs3³syóó â Ç Ó q Œq«Œ PBSöÀÃôžŒ ÓvŒ n0.25%œ 37 ä15 minãý Ø ÿƒzé10% fžq» äö± ê Œ1 000 r/miny10 min Œ»pÿB27w ŒmÀŸqm ÀqDMEN/F12» Œ»~Ž}5xn ˆÀ Ùx ŒmÀÀ Ùx Œq¹ o NestinqŸ² PBS ŒýÀn 10 minø ƒzé0.1% TritonX-100ö6% BSA}ƒz{Áü 1 hø ÙNestin(11 000)4 j 7586 P.O. Box 10002, Shenyng

3 ²Ø ˆ lggp(1500) 1 hhoechst min ˆ À d Ån d qíplga(pla PGA=7525)PEG(rÉ«4 000)dƒz (15%1.5%) p¹d ˆp5 ml fˆn w¾ýq27-g ¾nï g15 kv ²³ig1.0 ml/hä¾q yg12 cm ÄŸÅ qån dplga-peg nüí Ån dplga Óg8 mm 8 mm d30 min n käœqeys{ ýnà nï Ãiü ey5fƒzs{ýµž ¼vµØï ï xöøï²zf ² Ãfqqƒz Ãi=² fƒz/ƒz 100% q Œˆnçn j± Ó gr7 mmq ýˆp96f DMEM»24 hã» x Œ Œg L 1 yp¼ˆq96 30 μlãýƒz1248 z íü Ø q»ƒzg μl {qd»~f»48 hã»ø DMEM» 90 μlcck-810 μl37 3 h y 450 nmq q ŒØÂi Œg L 1 q ŒÉÌyp¼ˆq9637»~f 369 hô»ø 0.25%Œ ä ŒØÂi=ØÂ Œ/ Œ 100% q Œ Œg L 1 q Œ ÉÌyp¼ˆq9637»~f» dã»ø 90 μl DMEM»CCK-810 μl37 3 h y 450 nmq Hoechst üÿåq Œ}5x x Œü»3 dýpbsø 10 mg/lqhoechst minp ˆ À d nà ŸÅq Œ}5xx Œ ü»7 dýƒzé2.5%p 4 f 24 hpbsýj Œ7 minžeý s{ nà ŒqmÀ À g PLGA PLGA-PEG q Œ ØÂx ŒŸÅqmÀg É nspss 13.0±zÍ²Ÿ k «xx _ ±sÿx Âq± nê É qee± nlsd SNKP < 0.05g i 2 Results 2.1 Ò ëç Î«ÉyÍq Ùx Œ»zŠÄk~x»ý À ù x k Ã 1A o ¹x Œù NestinÂrg 1BD 2.2 Ó Ä ì ÌnÅn d Í qplgaplga-peg rörø Ã Ârp«³g μmplgaplga-peg réìg(533± 44) nm(661±59) nm(p > 0.05)ÃiÉÌg(88.2± 2.1)%(89.1±1.5)% i(p > 0.05)e qã i(p > 0.05)PLGA-PEG q rplgaz(p < 0.05) 2AB 2.3 dò Hoechstx PLGA-PEGPLGA Œ«Ãrq Œ(2EF)nÀxüey qx Œ ü (2C D)HoechstnÀ xplga-peg q Œ pplga ùùx Œ PLGA-PEGü»7 dý o xüq Œ vÿ²x Œq q(nestin3) 2.4 r±{qd PLGA-PEG PLGA üzq i(p > 0.05)ùrüeyiÂq k i(p > 0.054) 2.5 œû Ãs»ÂÀØÂpe q Œ ºfü»üÂ ØÂpPLGA-PEGq Œ p PLGA(P < 0.055) 2.6 ã e q Ãs» ÂqÀ ÎüfÂPLGA-PEG q ÎpPLGA (P < 0.056) 3 Discussion x ŒoŒÎqykqy Œ ~ ooœîä²íxtq [15] Ãs² zu{qº [16] Èu{³o Ìn zqoœî [17-18] z oœîöíef íy Œ [319] y ŒùxŸµeqx µœ«œ ØŒqx [20] x ŒùxŽ ISSN CN /R CODEN: ZLKHAH 7587

4 Hoechst Nestin Merge A B C D 1 Ùx Œq¹ ( 40) Figure 1 Identifiction of mouse neurl stem cells ( 40) A g»zùx Œq Œx ŒŠÄkx k Ã BD ù Ùx Œf Nestin ÂŸ²( o ) A C E B D F 2 êü Œq Figure 2 Morphologicl observtion of neurl stem cells-seeded scffolds nd scffolds lone f A( 6 000) B( 5 000)ÉÌgnÀÅn d PLGA PLGA-PEG PLGA-PEG ŸÅØÌp ŒqØÂC D( 5 000)ÉÌg SEM Ùx Œ PLGA PLGA-PEG ü» 7 dmà PLGA-PEG qx Œ E F( 40)ÉÌg ÀPLGA PLGA-PEG ŸÅx Œ Œe Íq ŒÃrr± PLGA PLGA-PEG qx Œ Œ PLGAŠm Šj PEGŠjp Hoechst Nestin Merge 3 x Œü PLGA-PEG mà 7 d ý Nestin qÿ²( o 40) Figure 3 Immunofluorescent stining reveled positive expression of Nestin fter co-culture of neurl stem cells nd poly(lctide-co-glycolide)/ polyethylene glycol nnofibrous scffold for 7 dys ( 40) Ùx Œx kxýäêf Œ PLGA-PEG ºü» 7 d ý²ÿ o ù Œv Ÿ²x Œq q NestinPLGAŠm Šj PEGŠjp 7588 P.O. Box 10002, Shenyng

nnofibrous scffolds detected by Cell Counting Kit-8 {qd r±plga-peg PLGA ü zq irüzeyi Âq k iplgašm Šj PEG Šjp 0.

5 {q (%) i Â Ø PLGA PLGA-PEG 4 CCK-8 PLGA PLGA-PEG q Œ Figure 4 Cytotoxicity of neurl stem cells on poly(lctide-co- glycolide) nd poly(lctide-co-glycolide)/polyethylene glycol nnofibrous scffolds detected by Cell Counting Kit-8 {qd r±plga-peg PLGA ü zq irüzeyi Âq k iplgašm Šj PEG Šjp d 3 d 5 d 7 d 9 d 11 d 6 Ùx Œ PLGA PLGA-PEG q Figure 6 The prolifertion of neurl stem cells on poly(lctide-co-glycolide) nd poly(lctide-co-glycolide)/ polyethylene glycol nnofibrous scffolds e q Ãs»ÂqÀ Îdüf ÂPLGA-PEG q Îp PLGA ( P < 0.05) PLGAŠm Šj PEGŠjp ùxéägx ŒŒ«ŒŸ goœî që²y Œ [21] ÍØvÙŒq«x Œ qx Œùx~x o xÿ²x Œg(Nestin) Í qx Œùx gy Œ²Ÿqu{ PLGAq gmg Ín ²yùÂ qp ŒØÂqmgrq zqùf Årpqù Â gplgaÿnpmgçè [22] Í²nPLGA gìnån dí [23] Ån düq r p ŒdÃi±Î [24-26] x Œ ü»ýnà öjx Œ Ÿ ÅmÀ Øü Hoechstöj Œ«qqÃrù Ån dí q ùxq{ Œ «g ŒqmÀ¼m ²qmÀjù± h 6 h 9 h 5 x Œü PLGA PLGA-PEG»ý 369 h q ŒØÂi Figure 5 Adhesion rtes of neurl stem cells fter incubtion with poly(lctide-co-glycolide) nd poly(lctide-co-glycolide)/ polyethylene glycol nnofibrous scffolds for 3, 6 nd 9 hours x ŒeyqØÂiÃsÂÀjÀ Ûºfü»üÂØÂp PLGA-PEG q Œ p PLGAee± i( P < 0.05)PLGA Šm Šj PEGŠjp ÎqŸÅzÃikÌp ŒqØÂmÀx [27] PLGA-PEGPLGAquØ ØŒºg ²PLGAš²npmgçÈ ŒØÂ±o±} ÂÍoº²q ön [10-11] Í¾PLGAq º²Ÿ É f sqmgrqšjp (PEG)ÌnÅn dí PLGA-PEGü nà xån díqplga PLGA-PEG réìg(533±44) nm (661±59) nmãiéìg(88.2±2.1)%(89.1±1.5)% ŸeyÃ PLGA-PEG rplga Œ öjüz Œq ŸPLGAPLGA-PEG q Œ³²CCK-8ÉÌeyü»qx ŒqmÀ xplga-peg ü fâq ÎpPLGA Ÿx Œ qplgaÿåqœ ÍÎ³² Œ ÉÌox ŒPLGAPLGA-PEGŸÅ q ŒØÂi ŸüfÂPLGA-PEGq Œ ØÂiÎpPLGA Œü»n ÀHoechst PLGA-PEGŸÅØÂq ŒrpPLGAùÙx ŒPLGA-PEG ü»7 dýx ŒvŸ²x ŒNestin ²Ån díqplga-peg q rãi²üx ŒmÀx mgrº Œùx g zq ±ƒ ÍuuÂp ƒmgrqu{ x ŒÅn dplga-pegÿºqƒ ISSN CN /R CODEN: ZLKHAH 7589

6 ¼r²Ä ²q u{ [28] Ž {œ öâ r d «h ÐÂ Ð cd n d ÌrÀ{ÀÀ íqd k «n t 2009 wethicl issues in niml experimenttionx üì íd ¾ ¾ð }¾ nßêî Ÿ ¾ ð c cð n n c ndò õd ukà íà d 4 õ h References [1] Zhu T, Tng Q, Go H, et l. Current sttus of cell-medited regenertive therpies for humn spinl cord injury. Neurosci Bull. 2014;30(4): [2] Bonfield W. Designing porous scffolds for tissue engineering. Philos Trns A Mth Phys Eng Sci. 2006;364(1838): [3] Evns N R, Dvies E M, Dre C J, et l. Tissue engineering strtegies in spinl rthrodesis: the clinicl impertive nd chllenges to clinicl trnsltion. Regen Med. 2013;8(1): [4] Houle J D, Tessler A. Repir of chronic spinl cord injury. Exp Neurol. 2003;182(2): [5] Ji W, Hu S, Zhou J, et l. Tissue engineering is promising method for the repir of spinl cord injuries (Review). Exp Ther Med. 2014;7(3): [6] Li BQ, Wng JM, Ling EA, et l. Grft of tissue-engineered neurl scffold serves s promising strtegy to restore myelintion fter rt spinl cord trnsection. Stem Cells Dev. 2014;23(8): [7] Luo J, Borgens R, Shi R. Polyethylene glycol improves function nd reduces oxidtive stress in synptosoml preprtions following spinl cord injury. J Neurotrum. 2004; 21(8): [8],ú²,ì,}.Ån dšm Šj ºŠg-d -Œ ñx ~ Ùx [J].f zu{, 2012, 16(38): [9] Chen SJ, Lin CC, Tun WC, et l. Effect of recombinnt glectin-1 on the growth of immortl rt chondrocyte on chitosn-coted PLGA scffold. J Biomed Mter Res A. 2010; 93(4): [10] Lu JM, Wng X, Mrin-Muller C, et l. Current dvnces in reserch nd clinicl pplictions of PLGA-bsed nnotechnology. Expert Rev Mol Dign. 2009;9(4): [11] Prk BH, Zhou L, Jng KY, et l. Enhncement of tibil regenertion in rt model by dipose-derived stroml cells in PLGA scffold. Bone. 2012;51(3): [12] Wttendorf U, Merkle HP. PEGyltion s tool for the biomedicl engineering of surfce modified microprticles. J Phrm Sci. 2008;97(11): [13],Ÿe.Šjp -Šm ºŠg g±ƒ[j].ä², 2007,19(6): [14],ÊÉ,ˆ,}. gqšjp Êu{²[J]. ä,2009,29(8): [15] Shrm H S. Post-trumtic ppliction of brin-derived neurotrophic fctor nd gli-derived neurotrophic fctor on the rt spinl cord enhnces neuroprotection nd improves motor function. Act Neurochir Suppl. 2006;96: [16] ú,í,,}.œñ Ê ¾f²tŒ Âµ«Œ[J].f zu{,2014,18(25): [17] uw,ù,ò,}. zgœî fnqëu{[j].f x y,2005,10(9): [18] iä,. zœîfqn[j].ç u{m,2008,21(5): [19] Mikmi Y, Okno H, Skguchi M, et l. Implnttion of dendritic cells in injured dult spinl cord results in ctivtion of endogenous neurl stem/progenitor cells leding to de novo neurogenesis nd functionl recovery. J Neurosci Res. 2004;76(4): [20] Tvres I. Humn neurl stem cell trnsplnttion in spinl cord injury models: how fr from clinicl ppliction. Stem Cell Res Ther. 2013;4(3):61. [21] Ogw Y, Swmoto K, Miyt T, et l. Trnsplnttion of in vitro-expnded fetl neurl progenitor cells results in neurogenesis nd functionl recovery fter spinl cord contusion injury in dult rts. J Neurosci Res. 2002;69(6): [22] hp,i,âä,}.± Šm Šj ºŠg Ít p ŒHepG2q n[j].f zu{, 2013,17(3): [23] Ghsemi-Mobrkeh L, Prbhkrn M P, Morshed M, et l. Electrospun poly(epsilon-cprolctone)/geltin nnofibrous scffolds for nerve tissue engineering. Biomterils. 2008; 29(34): [24] Lim SH, Mo HQ. Electrospun scffolds for stem cell engineering. Adv Drug Deliv Rev. 2009;61(12): [25] Liu J, Buer AJ, Li B. Solvent vpor nneling: n efficient pproch for inscribing secondry nnostructures onto electrospun fibers. Mcromol Rpid Commun. 2014;35(17): [26] Xu L, Sheybni N, Ren S, et l. Semi-Interpenetrting Network (sipn) Co-Electrospun Geltin/Insulin Fiber Formultion for Trnsbuccl Insulin Delivery. Phrm Res [Epub hed of print]. [27] ²,qu,².ör¾wmgÃm/v ¾Î qmgœ[j].f zu{,2014,18(8): [28] ìd,,¾é,}.ššm Æ Œ qmgr[j].f zu{,2013,17(29): P.O. Box 10002, Shenyng

Microsoft Word - 103Pan01.doc

Microsoft Word - 103Pan01.doc p ž 103 Ô µuæ 214/14/35 1.s eæ ô 2. ž s u h } e Â http://exam.nctu.edu.tw/103/103ustcheckin.htm Û h s Ã { o Â 3.w åž w åž w ž w É åž u http://exam.nctu.edu.tw/103/103ustcheckin.htm w u åž Â } o ï Â ¼ Ûäž