Microsoft Word 史筱璐-5.0-彩图.doc

f zu{ g } 15 í } 8 2011 02 19 Çf Journal of Clinical Rehabilitative Tissue Engineering Research February 19, 2011 Vol.15, No.8 ÙÎε«Œ ˆ w uƒ zt Î* ú~l 1 Ê f 2 Å 3 iòë 4 Guinea pig bone marrow-derived mesenchymal stem cells combined with nano-hydroxyapatite in constructing tissue-engineered artificial otosteon in vitro Shi Xiao-lu 1, Liu Li-zhong 2, Xie Feng-lian 3, Wang Lu-xiang 4 Abstract BACKGROUND: Artificially synthetic nano-hydroxyapatite (nha) has good biocompatibility and bioactivity. It can tightly connect with in vivo tissues closely within a short time after implanted into the body. Therefore nha has been widely used for bone tissue engineering. OBJECTIVE: To observe the compounded culture degree of guinea pig bone marrow-derived mesenchymal stem cells (MSCs) and nha, to analyze the feasibility of constructing tissue-engineered artificial otosteon. METHODS: The MSCs were isolated from the bone marrow of guinea pig by differential adhesion method. The surface markers of MSCs were examined CD29, CD44, CD45 by flow cytometric analyses. The differentiated capability of MSCs was also detected. The MSCs were cultured with nha porous ceramic, and their adhesion on the scaffold was observed by electron microscope at 1, 3, 5, 7, 10 days. RESULTS AND CONCLUSION: After three days cultivation, a large amount of MSCs attached on the material surface with stable form, proliferation activity and terrific extensibility; Composite material had all covered MSCs at 5 days, cell surface had a large number of secretary granules, soma amplified obviously, edge of cells was complete and fibroid extension; 7 days later cells gradually fell from the material surface, still attached to the material surface and appeared "star" or "pseudopods"; 10 days later, cells flaked off the material surface. It can be explained that nha maintains a good biocompatibility, which is beneficial for MSCs adhesion, proliferation and combination, nha can be used to construct tissue-engineered artificial otosteon. Shi XL, Liu LZ, Xie FL, Wang LX.Guinea pig bone marrow-derived mesenchymal stem cells combined with nano-hydroxyapatite in constructing tissue-engineered artificial otosteon in vitro. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2011;15(8): 1345-1349. [http://www.crter.cn http://en.zglckf.com] t ü q ˆ w uº qmg r ƒ¼ýœ u ¼ ƒ¼q±v ü g nqî z rq ÙÎε«Œ ˆ w u ü Ãm ƒ ü»q üz É º z t ÎqùŸ n Œ ³«Éy» ÙÎε«Œ ˆn Œy CD29 CD45 CD44²ŸÂµ«ŒŸÅ g¹ ºÎ ŒÉäŒ Îε«Œ ˆ w u ü Ãmº»1 3 5 7 10 d nà Îε«Œ y q ü À»3 dý ù Îε«Œ«Â Ÿ d z mà r º q»5 dýù ŸÅ ¹µ rîî⵫œ Œ ü ŒŸÅù É È Œƒ ² À 7 dý Œ² v ŸÅŒ vâ ŸÅq ŒÇj 10 dý Œ fgœ ˆ w u o qmgr Ìp ŒØ ù ü qîî µ«Œ np zt Î ºÀ ˆ w u Ãm Îε«Œ Œy nà zt Î doi:10.3969/j.issn.1673-8225.2011.08.004 ú~l Ê f Å iòë. ÙÎε«Œ ˆ w uƒ zt Î[J].f z u{ g 2011 15(8):1345-1349. [http://www.crter.org http://cn.zglckf.com] 0 fšoo mž Ž öo g nù Î í Áüqÿ fš { ö Î Ù öí {q ï ± ØŒ y Î q [1] Î «p Î q Î q ~Çq u Î y Î gq ³²² z qö Ì n zuíùg ˆnq xg gg yu qu{ Î zˆnq f ˆ w u(nano-hydroxyapatite,n-ha) y Œ ~ q Ãm º hgqmg n-haé 1~100 nm ³qˆ w ur º ±Î ŸÅŒ± q ~ º mg [2] nn-ha qt Îùx üµƒîmàqä Í 1 First Clinical Hospital of Xinjiang Medical Region, ; 2 First Affiliated Hospital of Xinjiang Medical Region, ; 3 Xinjiang Medical Region, ; 4 Xinjiang University, Urumqi 830000, Shi Xiao-lu, Studying for master s degree, First Clinical Hospital of Xinjiang Medical University, Urumqi 830054, lauren84103@ sina.com Correspondence to: Liu Li-zhong, Doctor, Chief physician, Master s supervisor, First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, liulizhongno.1@ 163.com Supported by: Natural Science Foundation of No. 200821150* Received: 2010-09-13 Accepted: 2010-10-12 ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH 1345

1 É Þ{ óu É ù 830054 2 É Þ{ œ É ù 830054 3 É Þ{ É ù 830054 4 É{ É ù 830000 1984 É ù É Þ{ x Þ Ôd lauren84103@ sina.com öí É Þ{ œ ³Þ É ù 830054 liulizhongno.1@ 163.com f É ú:r318 h u:b ú:1673-8225 (2011)08-01345-05 2010-09-13 º 2010-10-12 (20100913008/GW Y) 1346 üqv üq g ü º Îm Àrç qâ ³i dº Î q Ìnn-HA ýqî ƒ¼ù ü tƒî døœ k ù Ž g ˆ w uöº ü g Î f q r ƒ¼ qâ i ÎŒ ümà ö ÎqmÀx }Áȵv À Í n ÙÎε«Œ(bone marrow stromal cells BMSCs) n-ha Ãm ü ²Ÿº» v o n-ha Ãm ü BMSCsq mg ØŒq xön p zî qùÿ ê Í Âö p201001/08 nçy }  çã Œ Í nçy nà e ÚŸ Ù10ù ÄÄ ƒ«150~180 g n nçy ÍÙgf (çù }16-003ú) n-han n nä u{ Ï Ï y H-DMEM» GIBCO ¹ú ÅÄ ¾, Ä éäí  «{¹ú L m C βw m ¾ SIGMA ¹ú S Œ q HEPES L Œ ALP Ïr ês mg¹ú rehuman-bmp2 PEPROTECH PE anti-mouse/rat CD29 Biolegend ¹ú FITC anti-rat CD45 PE/Cy5 anti-mouse/human CD44 Œñ p o ä Ïr DAB Ïr CO 2»~ ˆr À(DMI4000B ú) Œy äsë² mg¹ú äsf Leica ¹ú COULTER EPICS ALTRATM ˆ Beckman ¹ú Í BMSCsqÉy»ö~x Ÿ Ù Ø ( Œ 40 g/kg 5 g/kg  0.05 g/kg) ö q { ( Œ 10 L{ ) z Î ˆp PBS f Ðà q } Îe q z  ú vî e y Î ÅÎÎ 10 ml ÿƒzé 10%FBSqH-DMEM ÎÎ ö À ržàâ ¼ ÎΞ Íqÿ ÎÎ ŒqÀ z 10 ml fö ê Œ x(6~8) 10 9 L -1 yp25 cm 2»mf 37 Ê öƒz É g 5%CO 2 q»~ f²ÿñ x» (H-DMEM» fø 100 U/mLÅÄ Ä 2 mmol/l L Œ Œ) iý 48 h¹ 1 ² 3 xý ŒmÀ À 3 d 1 ˆr À ² Œ mà À ñx»q«œmàž m 80%~90% zãmf» PBS 1 n0.25%œ q 37 z ä0.5 min Œ ŒÂÃ Ø ÿƒzé 10%FBSq» ä n ~± ö rž«œ Œ y 1 ýø ²» 1 4q ~x» Œg}1x Œ xý ŒmÀ À ~x» BMSCs qmà }3x Ù BMSCs 0.25% Œ q äýø H-DMEM» y Í Œ Œ Ž4 10 6 5 10 6 6 10 6 L -1 1 ml y24 Âü 3 ä Í Œ âý Œ 3 ² 12 d x Âg Œ g ÍmÀ BMSCs Œy }3xBMSCsmÀ üž85% 0.25%Œ q 37 z ä 1 min Œ ŒÂ Ã Ø ÿƒzé 10%FBSq» ä n ~± ö rž«œ Œ Ž1.5 mly ~¼ 2 000 r/min y 5 miný Ø ÈÁqPBS 1 ml Œ 2 000 r/miny 5 miný ² Ø ƒ(pe anti-mouse/rat CD29 FITC anti-rat CD45 PE/Cy5 anti-mouse/ uman CD44) BMSCs Î ö }3xBMSCs ypèírjfq6» λ (ÿ10-8 mol/l 50 mmol/l βm P.O. Box 1200, Shenyang 110004 cn.zglckf.com

w ¾ 50 mg/l m C ƒzé 10%FBSq H-DMEM» )» }7 14 21 28 à Çr jf nw w Ïr nƒzé 95%j rjf3 min ýq Ø Ó ˆq w w g n ˆ rf 37 15 min n Ë À 3 n 3 min ú n Ë À Ž À Œ ¾ z npbsà rjf3 40 g/l Šn 30 min Ë À 3 Ø 0.5% 37 30 min Ë À Ž À Œ o ä Œ¼ Œñ Ÿ² n PBSÀ rjf3 ƒzé 95%j 30 min ƒ zé 3%² ä  ¼ 10 min PBSÀ 3 min 3 Ø ÙCollage r¼4 ² Çjf ˆ30 min PBSÀ 3 in 3 Ø p 37 30 min PBSÀ 3 min 3 Ø DABÀ Œ ý Ë µéà 1 min r j Éä À Ž ýà BMSCs/n-HA üg» }3x ÙBMSCs 0.25%Œ q äýø H-DMEM» y Í Œ Œ Ž50 10 9 L -1 35 mm»r f Î Îï qn-ha 4 n100 µl Œ v yn-ha 0.5 cm ˆ Œ â ˆ w u BMSCs/n-HA üg ƒzé 5%CO 2 q»~f»2 hý»r Ø 2 ml Œ» ¼ ƒzé 5%CO 2 q»~» nà BMSCs/n-HA üg ü»}1 3 5 7 10 ÉÌ Ç4 BMSCs/n-HA üg» À 2.5% p (0.1 mmol/lqpbs Í ph7.4) ¼ 1% ä j ² Œ CO 2 gn e j ² k s{ e ¼n Ãï Ãj Ï ŸÅ ý nà üg rü z 10f ( 200) Œ g ˆ À r ˆ À BMSCs ä Œy º ñÿ² ñx» ~x» ŒmÀ o ä Œ BMSCs Î ŒŒ nà BMSCs n-ha ü» À É nspss 17.0 ±z² Ÿ É kn} nçy }  çã u 2.1 BMSCs ct ñx»qbmscs 48 h¼ µé«œ ± Žqu ² 5~7 d ù Ä ³ 9 d ú Œpr ü Œ Ê g Òˆg g 1 Figure 1 Primary culture of guinea pig bone marrow mesenchymal stem cells at 6 d (Phase-contrast inverted microscope, 100) 1 ÙÎε«Œñx»} 6 (r ˆ À 100) ~x»qbmscs«1 h¼ Œ «À 2 Ø â mà r 4 d úíù²í90% ü 7 d úmಠ~ x» }5xxýÇj ä Figure 2 The third passage culture of guinea pig bone marrow mesenchymal stem cells (Phase-contrast inverted microscope, 50) 2 ÙÎε«Œ~x»} 3 x(r ˆ À 50) 2.2 BMSCs j ¼ }3x ÙBMSCsmÀ S }1~4 gmಠ}5 ² ³ À ³ À Í}9 xý² ú }ü LogisticmÀ 3 Cell number ( 1 000) 60 40 20 0 4x10 3 5x10 3 6x10 3 1 2 3 4 5 6 7 8 9 10 11 12 Time (d) Figure 3 Growth curves of third-passage guinea pig bone marrow mesenchymal stem cells 3 ÙÎε«Œ} 3 xmà 2.3 BMSCsö ÆgΫÍÉ o10 f Œyö xcd29îÿ² Ÿ²i 92% ú CD45Ÿ²iÅ ƒ ²6% ú CD44Ÿ ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH 1347

²±Îù²70% ú 4 nha ü 6 CD29 CD44 a: 3 d ( 1 500) b: 5 d ( 3 000) c: 7 d ( 800) 2.4 BMSCsn î λý7 d À Œ öm ä 30% ú ŒnÀ ² ± g w w Œ fçj È g gâ  Œ ë Œ q47.5% ù q Î Œrmq¾ o ä Œñ ù Œ fçj Ø È 5 2.5 BMSCs/n-HA Ó h» 1 3 5 7 10 d öj Œ ê ÂsmÀ g Âsp à ŸÅ Œ Ÿ ² 1348 CD45 Figure 4 Phenotype of bone marrow mesenchymal stem cells 4 Îε«ŒqŸ a: Associated Kyoto Program b: Alizarin Red S c: Collagenase Figure 5 Osteogenic induction capacity of guinea pig bone marrow mesenchymal stem cells 5 ÙÎε«Œq Î Œ Figure 6 Compounded culture of guinea pig bone marrow mesenchymal stem cells and nano-hydroxyapatite (Scanning electron microscopy) 6 ÙÎε«Œ ˆ w u ü» n À»3 d Ø ÂpnHA q BMSCs g (16.43±5.31)f 5 dq g(39.27±3.24)f Î p ü»3 d ØÂp qbmscs (P < 0.05)»7 dqbmscs g(30.47±7.84)f»5 dq ± e ± i(p > 0.05) k q zf{t g² mgç y qu{ ù Œmg q üƒ g zu{q [3] q r p y ŒqØÂö m qmà sèé q i [4] k qî z º x g [5-6] qî~ Ìp ŒØÂmÀ Œ «z» ² x g Ç k Ìpž~ x qà qmgr Ìpy ŒqØ Â, rg Œ n oö mžìp ŒmÀ Éä qmgâ «ný ŒÂ  i Œ màir²   Œ màg žtg öºâ rg tƒ º q «º qù ùè Í g º q g m ÂrŽ m º Ž mg g z²n ŒxŸo ¼m Å q o BMSCs y pîîfº Î Ž Œ ýéä Œq Œˆƒ ƒ p Éy» ƒ ü z ù±äg y ü P.O. Box 1200, Shenyang 110004 cn.zglckf.com

q Œ [7-10] p ùpƒ ²Ÿ» Œ º ýéä Œ ³~ z ƒ¼ o ö íˆ üy ƒz npbmscs p É äqðƒ Œ º ŒŸ qéä d BMSCs z ýíˆ kèé BMSCsùŸ g z f±k qy Œ ³ o v g Œ ofqk Œ BMSCs ŸÅ ñstro1 CD13 CD29 CD44 CD49a CD49b CD71 CD90 CD106 CD124}  ö [11] ³ž ŒŸÅ CD34,CD35 CD45}g ö [12]  Œ o ~µ«ö Œ ~ Ço¹ t BMSCsq3 ƒ  [13] » z BMSCs Ⱥ gq«â ³² Œy, BMSCsˆƒŸ²CD105 CD73öCD90 i 95% CD45 CD34 CD14 CD11b CD79a CD19 HLA-DR i 2% ƒ BMSCs ÈŒý Î Œ Œ Œö± Î ŒÉä gì Ç pùg qbmscs Ã}2  º ³ iq²n }1 3 ² qmg g ü ²npÙg BMSCs rð ² np¹ qk É ùx Ì BMSCs [14] n fÿå gšü¹ r qÿåé k Ùp BMSCsq¹ v Í»q Œ}üBMSCsq g ü Œy ŒŸÅ q CD29 CD44 CD45g ù x Í»q Œ ÙBMSCs n-haî Î É ² É Îr [15] Ìp ŒqØ màöé Œñ ²¾ä º f qg [16-17] qmgr ºŸÅq ƒ Ìp Œ qþ  Ìp Œ ¾ } [17-18] n-ha npg º ²ŽƒÎz yk qî Íf ÙBMSCs n-ha ƒ ü»3 dýù BMSCs«Â Ÿ d z mà r º q ü»5 dýù ŸÅ ¹µ rbmscs Œ ü ŒŸÅù É È Œƒ ² À 7 dý Œ² v ŸÅŒ vâ ŸÅq ŒÇj 10 dý Œ fgœ Ífqn-HA o qmgr Ìp ŒØ ù ü qbmscs 4 õ h [1] Zhang LC,Song WM,Tang ZF,et al.zhonghua Xianwei Waike Zazhi. 2005 28(1): 34-35. Ú, g y }.BMP-HAt Î yù fq n[j].fé y,2005,28(1): 34-35. [2] Yan B,Yan JL,Zhang T.Zhongguo Guzhongliu Gubing. 2004;3(2): 76. ÁÀ,Á Ú,.Î z u{² [J].f Î pîo,2004, 3(2): 76. [3] Cheng L.chengdu sichuan daxue. 2007. zk.º mg qùâ z ü u{[d]. µ:,2007. [4] Liu XH,Zhang XQ,Liu JL,et al.zhonghua Xiaoer Waike Zazhi.2005; 26(4):203-206. Ê Ê² }. rˆ w uœñ ü tî Î «Œƒ r qu{[j].fé µ y 2005 26(4): 203-206. [5] Hutmacher DW. Scaffolds in tissue engineering bone and cartilag. Biomaterials.2000 21(24):2529-2543. [6] Shi PL,Gu XM,Chen FL,et al.zhongguo Xiufu Chongjian Waike Zazhi. 2001;15(6): 373-376. ú,è,â,}.xjk±äˆ w ug zîq Íu{[J].f y, 2001,15(6): 373-376. [7] Kan I, Melamed E, Offen D. Integral therapeutic potential of bone marrow mesenchymal stem cells. Curr Drug Targets.2005;6(1) : 31-41. [8] Shao Y,Pei CG.Guoji Yanke Zazhi. 2006;6(2):441-445., Ë. Œ o ˆ ooqu{² [J]. Âsy, 2006,6(2):441-445. [9] Popckop DJ, Azizi SA, Colter F. Potential use of stem cells from bone marrow to repair the extracellular matrix and the central nervous system. Biochem Soc Trans.2000; 28(4): 341-345. [10] Lee RH, Seo MJ, Reger RL,et al. Multipotent stromal cells from human marrow home to and promote repair of pancreatic islets and renal glomeruli in diabetic NOD/scid mice. Proc Natl Acad Sci USA 2006;103(46) :17438-17443. [11] Grove JE,Bruscia E,Krause DS.Plasticity of bone marrow derived stem cells.stem Cells 2004 22(4):487-500. [12] Lee J,Elkahloun AG,Messina SA,et al.cellular and genetic characterization of human adult bone marrow derived neural stem like cells:a potential antiglioma cellular vector.cancer Res.2003 63(24):8877-8889. [13] Krause DS, Theise ND, Collector MI, et al. Muti-Organ, muti-lineage engraftment by a single bone marrow-derived stem cell. Cell.2001; 105(3):369-377. [14] Dominici M,Le BK,Mueller I,et al.position PAPER:Minimal criteria for defining multipotent mesenchymal stromal cells.the International Society for Cellular Therapy position statement. Cytotherapy.2006 8(4):315. [15] Liao SS,Cui FZ,Zhang W.Zhongguo Yixue Kexueyuan Xuebao.2003; 25(1):36-38., y, ~. zfœñ Î ü quí[j]. f ç y Ã,2003,25(1):36-38. [16] Huang YH,Shen TC.Jiangsu Daxue Xuebao:Yixueban. 2005;15 (4): 363-367. Ø ±, ¾. t Îqu{² [J]. :ç f, 2005, 15 (4): 363-367. [17] Huang YH,Shen TC,Xu XF. Jiangsu Daxue Xuebao:Yixueban. 2004; 14(4):292-294. Ø ±, ¾,. ˆ w u /ŒñÎ Î ýî fq n[j]. :ç f,2004,14(4):292-294. [18] Shen TC, Huang YH, Xu XF,et al.yixue Yanjiu Zazhi. 2006;35(4): 70-73. ¾,Ø ±,,}. Œñ Î g q n[j]. ç u{,2006,35(4):70-73. Ž Èq «Ù É Þ ¾Ð (200821150)d «h Ð Â c c ò À n d Ž þ c ØéÀ d c Û é ö Æ g t nö Æg d ÌrÀ{ ß À jãà ½ Ò ͽÅÍ Ò½í d k  «n t üì É Þ{ œ üì» «ü ü 20090422002d ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH 1349