ᙯࢉ siRNA ࡕϩձܭۓϐಔձуΕ SDF-1 (50 ng/ml)Ǵ٠ܭᑻӀ ᡉ༾᜔Πᢀჸಒझࡕ 0ǵ12ǵ24ǵ36ǵ48 hǴmigration ϐǶ Figure 20. ᡉҢಒझᎦܭ Col-Au ਔ migration ૈΚᆶ Col Ϸ TCPS ϐ ಔձૈΚ࣬Кၨ٫ǴЪᙯࢉ VEGF siRNAϐಔձ migration ૈΚΠफ़Ǵ ՠऩబуӣ SDF-1 ਔ ߾ migration ૈΚቚуǶӢԜёፕ
56
Col-Au ёፓಒझૻဦၡ৩ߦಒझ migration ϐૈΚǴ٠ЪЬाၸ VEGF ϐૻ৲ࢲϯǴԶߦಒझܭ Col-Au ᎂ౽ޑૈΚቚуǶ
5. VEGF ୖୖᆶፓಒझ߄ϣҜ਼ϯේӝԋ䁙 (eNOS)
ஒ MSCs ᙯࢉ VEGF siRNA ࡕǴௗᎦܭ ColǵCol-Au 48 λਔ ࡕаխࣝᑻӀࢉՅᢀჸ਼ϯේӝԋ䁙ϐ߄ໆ (eNOS)Ǵ่݀ᡉҢܭ Figure 21.Ƕ෧ VEGF ୷Ӣ߄ࡕǴԋ eNOS ߄ໆ෧১ǴᚐѦబ уΕ SDF-1 ࡕೈқ፦߄ໆڀԖӣൺਏ݀ǹӢԜፕ SDF-1 Ӣࣁ VEGF ޑ ߄ Զ ቹ ៜ eNOS ߄ ໆ Ǵ ٠ җ ಒ झ ૻ ဦ ၡ ৩ : NO-dependent pathwaysԶቹៜಒޑቚғᆶғӸǶ
Ϥǵނჴᡍ
1. ಔᙃπำՈᆅ౽ނϐᇙഢၸำ
ಔᙃπำՈᆅ౽ނϐᇙഢၸำᡉҢܭ Figure 22.Ǵᓉે੮ଞ߄य़ аᚆηεႝዀഢೀࡕǴҥջݰΕςଛᇙֹԋϐ ColǵCol-Au ྋన (37ʚ) ऊ 4 λਔǴڗрࡕܫคᏹբѠϣ࠻ྕޔԿ߄य़ྋనֹ
ӄଳᔿࣁЗǶڗҔа 75% ଚᆒݰ 5 ϩដࡕǴа PBS ݰ 15~30 ϩ ដǶFigure 22. (d,e,f) ᡉҢճҔ quantum dot MSCsǴᡏѦᄊᎦ 48λਔࡕǴаᑻӀᡉ᜔ዴᇡǴёճஒ MSCs ຠߕܭՈᆅ౽ނ߄य़Ǵ Ъа߄य़༡թ Col-Au ϐಔձǴಒझຠߕኧࣁനӭǶҗԜёޕ catheter
57
߄य़ၸᚆηεႝዀೀϷ Col-Au ༡թࡕёቚ MSCs ຠߕܭځ ߄य़Ƕ
1.խխࣝಔᙃࢉՅϩ (Immunohistochemistry staining)
MSCs аᡏѦᄊᎦޑБԄǴᎦ MSCs ܭᓉે੮ଞ߄य़48 λਔࡕǴΕεႵިેύ (ࢬำـ Fig. 22,23)Ǵ٠Ӹ੮ঁДǶ឴࣊
εႵࡕǴஒѓୁΕ catheter ϐިેǴՉխࣝಔᙃࢉՅǴᢀჸڼԯ
୷ᆶ MSCs ჹܭڙཞՈᆅࢂցڀঅൺϣҜಒझޑфૈǶFigure 24. ᡉ ҢՈᆅಔᙃ߄Γᜪ CD31 ϐ (a) ࣁ٬ҔDAB ࢉՅݤǴ(b) ߾ࣁ խࣝᑻӀࢉՅݤǴCol-Au ک Col (group 5~8) ޑಔձᡉҢǴ MSCs Εނᡏϣࡕܭ༡թ Col-Au ޣ߄ CD31ޑໆࣁനӭǴЪ߄Γᜪ CD31 ϐಒझᄌᄌᎂ౽ԿՈᆅಔᙃϣǶҗԜёޕᡏѦ౽ MSCs ܭ Col-Au ༡թޑՈᆅಔᙃ౽ނǴځ MSCs ϩϯࣁϣҜಒझӢԶ߄
рΓᜪϣҜಒझޑ CD markersǺhCD31ǴЪࡼѺ VEGF ࡕёᡉߦ
MSCs ϩϯࣁϣҜಒझǴ٠Ъಒझёճ౽ΕՈᆅᏛύǴᙖаঅ ൺڙཞޑՈᆅϣҜǶ
2. Е๋-ҲआࢉՅ (Hematoxylin-enosin, H&E)
ஒՈᆅ౽ނΕިેࡕঁДࡕǴճҔЕ๋-ҲआࢉՅݤᔠᡍǴ ՈᆅಔᙃঅൺǶҗ Figure 25. ளޕ catheter ޑಔձڀՈਵޑౢ
58
ғ (ӵᗖᓐ܌Ң)ǹ༡թ Col ಔǴ߾Ԗ٤༾ՈਵℚೱܭՈᆅᏛϐǹ Զ Col-Au ϐಔձ٠คౢғಔᙃ౦தޑ߄ǴෳࢂӢࣁ Col-Au ڀԖၨ
ӳޑғނ࣬܄Ǵёफ़եխࣝϸᔈǴЪ MSCs ϩϯࣁϣҜಒझǴёҥջ অံՈᆅϣҜफ़եӆԛਵ༞ޑǴࡺՈᆅಔᙃ҅தคՈਵޑౢ
ғǶ
5. ఘఘғΟՅࢉՅ (Masson trichrome staining)
ஒՈᆅ౽ނΕިેࡕঁДࡕǴճҔఘғΟՅࢉՅǴᢀჸՈ ᆅϣҜಔᙃঅൺǴҗࡰрՈᆅᠼᆢϯౢғܭՈᆅѳྖԼೀ
ౢғጤচᠼᆢǴ߾߄ҢՈᆅᠼᆢϯϐౢғ [81]ǴࡺճҔఘғΟՅࢉՅǴ ᢀჸ୷לᠼᆢϯϐǶҗ Figure 26. ளޕ catheter ޑಔձՈᆅϣҜ ϷѳྖԼಔᙃԖౢғᠼᆢϯޑ (ӵᗖᓐ܌Ң)Ǵ༡թ Col ಔǴ߾ϣҜ ಔᙃڀԖ٤༾ᠼᆢϯౢғǴԶ Col-Au ϐಔձౢғ߾ࣁനϿǶෳࢂӢ ࣁ Col-Au ڀԖၨӳޑғނ࣬܄ǴЪԜᅿڼԯ୷ၨόԋՈᆅౢ
ғಔᙃᠼᆢϯǴӢԶӝߏයܫܭᡏϣǴࡺ׳ӝᔈҔܭՈᆅЍࢎ༡ թύǶ
59
60 procyanidin ࣁጤೱᏊᛙۓ collagen ϐ໔ޑᗖ่Ǵ٠җ FTIR ඔࡕ ܭ 3350 cm-1Ԗ֎ԏݢঢ়р [67]ǴԶҁࣴزஒ collagen షӝڼԯߎಈ ηࡕҭԖԜݢঢ়рǴՠܭ Figure 3.ύวуΕڼԯߎಈηࡕԋ
61
Ϸ෧Ͽ Col ޑफ़ှำࡋǴӢԜቚуғނᛙۓ܄ (biostability) کғނ࣬
܄(biocompatibility)Ƕ
ҞςޕǴҁࣴز܌٬Ҕϐ collagen type I ёၮҔܭՈᆅಔᙃπำ ύǴᇙԋಒझѦ୷፦Ǵऩஒ ECs Ϸ EPCs ёܭځ୷፦Ꭶ 7 ϺࡕǴ ёԋ༾Ոᆅஏࡋ [74]ǹ٠ёၮҔܭᙴᕍׯ๓Ոనឲࢬ [68,73]Ƕՠ ԋዕϐϣҜಒझځቚૈΚԖज़ǴԶϷ EPCs ԖᛖܭڗளБय़ޑज़ڋǹ ӢԶԋӧಔᙃπำ (tissue engineering) ܈ಒझݯᕍ (cell therapy) ύ ڙډፏӭज़ڋ [75,70]Ǵߦ٬Ϟࣴزᖿӛ༸ಒझᕍݤǶӢԜҁჴᡍନ Αа collagen షӝڼԯߎ༾ಈǴᇙԋՈᆅ୷ѦǴ׳ଛӝ٬ҔڀԖԾך
׳ཥЪӭख़ϩϯфૈϷቚғૈΚ٫ϐ MSCs [64,65,69,76]Ƕ
62 ғᆶᎂ౽ૈΚǵNO-independent pathways (FAK/RhoA/MMP-2) ፓಒझ ޑᎂ౽ϷຠߕՉࣁޑׯᡂ [50,54]ǶԶҁࣴزวஒ MSCs Ꭶܭ Col-Au (SDF-1) ёа (mobilization) EPCsǴ٠ߦಒझ߄ eNOS Զቹ ៜಒझϩϯࣁ ECs ԶԋՈᆅཥғ [64,78,79]ǶЪ VEGF ёаڈᐟ༸
ಒझౢғӣᘜਏᔈ (homming) ԿՈᆅڙཞޑՏǴߦ٬ϣҜঅൺғߏ [64,78]ǶܭࣴزύวಒझᎦܭ Col-Au ਔӆуΕӕғߏӢη (VEGF
܈ SDF-1) ࡕǴ ёᡉගϲ CXCR4ǵα5β3 аϷ eNOS ޑ߄ (Fig. 14,
63
64
CD31 Ъ҂ԖՈਵϷวݹޕݩౢғ (Fig.24, Fig.25)ǶҗԜёޕߎڼԯ ಈηዴჴёᇨᏤ༸ಒझϩϯࣁϣҜಒझǴԶঅൺՈᆅϣҜಔᙃǶനࡕ ךॺޑࣴز่݀ᡉҢǴCol-Au ёቚம MSCs ܭڙཞՏёϩϯࣁϣҜ ಒझ (Fig.24 )ǴЪΕޑ MSCs ёᑼӝΕՈᆅϣҜಒझಔᙃύ [74]ǴӢ Զԋᜪ՟ޑচғՈᆅಔᙃǶӢԜךॺीр MSCs Ꭶܭ Col-Au
ޑڼԯ༡թ୷Ǵё෧ϿӆԛՈਵޑᐒǶԜࣁᅿٰஒࡐԖޑ БݤǴٮᅿཥޑБԄࣁᔈҔᖏޑǴගϲགྷޑՈᆅ౽ނǶ
65 NO-dependent pathways ᆶ NO-independent pathways ޕૻ৲ၡ৩ፓಒ झຠߕǵቚғᆶᎂ౽ՉࣁǶԜѦճҔᙯࢉ VEGF siRNA ຓჴǹVEGF ࣁ SDF-1 ෞૻ৲ፓӢηǴёፓ SDF-1 ޑϩݜǴ࣬ϕڐӕԶу ԋ MSCs ғߏܭ Col-Au ϐૈΚǴቹៜΠෞೈқ፦ϐ߄ (p-FAK, p-RhoA, p-eNOS, p-Akt, MMP-2, CXCR4, α5β3)Ǵԋಒझܭ Col-Au ϐᎂ౽ᆶቚғૈΚޑቚуǶ
ӧނჴᡍБय़Ǵஒ܌ᇙԋϐ Col-Au ౽ނǴΕεႵިેύ 1 ঁДࡕǴځғނ࣬܄٫ǴόౢғՈ༧ℚೱаϷځдޑխࣝϸᔈౢ
ғǴЪ౽ܭ Col-Au ࡕ MSCs ёᅌᅌϩϯࣁϣҜಒझ߄ CD31Ǵ٠ᎂ
66
౽ΕՈᆅᏛύǴঅൺڙཞϐՈᆅϣҜಔᙃǴߦՈᆅϣҜಒझཥғϷঅ ൺǴᗉխՈਵޑӆԛวғǴЪаఘғΟՅࢉՅݤܴǴ٬ҔԜᅿڼԯፄ ӝ୷ၨόЇଆಔᙃᠼᆢϯǴӝӸܫܭᡏϣǴԶၲډүғਏ݀ǶӢ Ԝҁჴᡍϐ Col-Au ڼԯፄӝ୷ёගٮϞᖏբࣁՈᆅғނ
༡թނ፦ϐୖԵǶ
67
ୖ
ୖԵЎ
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(a) Col
(b) Col-Au
Figure 1. AFM images of surface topography. (left) and phase (right) for (a) pure Col, (b) Col–Au nanocomposites containing 43.5 ppm of gold nanoparticles,
Topography Phase
Topography Phase
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Figure 2. UV-Vis spectrophotomiter spectra of Col and Col-Au nanocomposites. The pure Col, and Col-Au nanocomposites containing 43.5 ppm of Au nanoparticles were analyzed the absorbance wavelength by UV-Vis spectrophotometer
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Figure 3. ATR-FTIR spectra of the Col and Col-Au nanocomposites. The pure Col, and Col-Au nanocomposites containing 43.5 ppm, of Au nanoparticles were analyzed by the ATR-FTIR.
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Figure 4. Characterization of mesenchymal stem cells (MSCs). (a) Cell morphology of human umbilical cord MSCs by optical microscope. (b) Detection of MSCs specific makers expression by flow cytometry analysis.
An open area represents an antibody isotype control for background fluorescence and a shaded area shows signal from MSCs surface maker.
MSCs was stained with PE or FITC-conjugated antibody against the indicated makers烉CD105-PE, CD90-PE, CD44-PE, CD73-PE, CD29-PE, CD34-PE, CD45-FITC and CD14-FITC. MSCs at passages 8-9. Scale bars = 100 μm.
(b) (a)
MSCs, P9
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Figure 5. MSCs proliferation was promoted by materials of various Au concentrations, where representative growth on different substrates was shown. MSCs proliferation on the surface of TCPS, Col and Col-Au containing 43.5 various concentration of Au nanoparticles Data are mean ± SD. 炵: p < 0.05, greater than Col for 24h , # p < 0.05: greater than Col for 48h . * p < 0.05: greater than Col for 72h .
! !
24h 48h 72h
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Figure 6. MSCs proliferation was promoted by substrates of Col-Au nanocomposites, (a) MSCs proliferation on the various materials for 24h, 48h, 72h .and (b) treatment with VEGF or SDF-1 respectively. Data are mean ± SD. *: p < 0.05, greater than TCPS for 24h , 48h, 72h .
(a) (b)
(c) (d)
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Materials Number of adhered platelets (×103)
Average degree of activation(0.0-1.0)
TCPS 3.00 0.91 ²² 0.2
Col (0.5mg/ml) 2.34 0.63 ²!0.1 Col-Au (43.5ppm) 0.07 0.19 ² 0.01
Figure 7. Platelet activation test. SEM images of platelets adhered onto various materials.
TCPS
Col
Col-A
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Materials The number of monocytes(×103)
The number of macrophages (×103)
Conversion yield (%)
TCPS 2.18²²1/4 0.94²1/3 43.4 ± 0.7 Col (0.5mg/ml) 2.26²1/6 0.24²1/13 10.95 ± 0.8 Col-Au (43.5ppm) 2.46²1/6 0.28²1/16 7.48 ± 0.6
Figure 8. Human monocytes adhered and activated on TCPS, Col and Col-Au after 96 h culture. Col–Au 43.5 ppm had the lowest percentage conversion were comparable in the extent of activation for monocytes *: p <
0.05, greater than TCPS.
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Figure 9. Cell morphology and adhesion ability. Adhesion ability of MSCs on the different materials. (a) The rhodamine phalloidin staining of actin fiber and (b) SEM images was showed. Yellow arrow: filopodia, Green arrow : lamellipodia , blue color: Nucleus, Red color: Actin
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Figure 10. Reactive oxygen species (ROS) level detected by 2’7’-dichlorodihydrofluorescein of flow cytometry in MSCs culture on the TCPS, Col, Col-Au. Data are mean ± SD. * p < 0.05 : greater than TCPS
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(a) (a)
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Figure 11. The expressions of (a) p-Akt and p-eNOS proteins of MSCs cultured on the TCPS, Col, Col-Au for 48 h . (b,c) Semi-quantitative measurement of Western blots for MSCs cultured on Col–Au showed significantly increased p-Akt and p-eNOS expressions compared with TCPS.
Data are mean ± SD. * p < 0.05 : greater than TCPS.
p-Akt
p-eNOS (b)
(c)
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(b) (a)
p-RhoA
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Figure 12. The expressions of (a) p-FAK, p-Cdc42 and p-RhoA proteins of MSCs cultured on the TCPS, Col, Col-Au for 48 h . (b,c,d) Semi-quantitative measurement of Western blots for MSCs cultured on Col–Au showed significantly increased p-FAK, p-Cdc42 and p-RhoA expressions compared with TCPS. Data are mean ± SD. * p < 0.05 : greater than TCPS.
(c)
(d)
p-FAK
p-Cdc42
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Figure 13. The MMP enzymatic activity of MSCs on various materials after VEGF or SDF-1 treatment for 48h. Gelatin zymography analysis of MMP-2 activity that was specificity on Col-Au. Data are mean ± SD. * p <
0.05 : greater than TCPS.
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Figure 14. eNOS expression of MSCs on various materials after VEGF or SDF-1 stimulation for (a) 24 h and (b) 48 h that were visualized by fluorescent microscopy. Cells were incubated with primary anti-eNOS antibody followed by stained with FITC-conjugated immunoglobulin (green color fluorescence). Bar = 100 μm.
(a)
(b) (b)
(a)
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Figure 15. α5β3 and CXCR4 expression of MSCs on various materials by using VEGF or SDF-1 treatment for 48h that were visualized by fluorescent microscopy. Cells were incubated with primary anti-α5β3 and anti-CXCR4 antibody followed by stained with FUTC-conjugated (α5β3) and Cy5.5 (CXCR4) immunoglobulin . scale bar = 100um.
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Figure 16. Concentration of transfected siRNA. The optimal concentration of transfected siRNA labelled with fluorescence. (a) The cell viability of MSCs after treated with different concentrations of scrable siRNA for 24, 48 and 72 hr by MTT assay. Data are mean ± SD. * p < 0.05 : greater than control (b) scramble siRNA expression were analyzed by a flow cytometry.
scramble siRNA FITC-conjugated immunoglobulin (c)The efficacy of MSCs transfected with siRNA by fluorescence labeling (siRNA/FITC, F-actin/phalloidin, Nuclei/DAPI). (scale bar烌100μm)
(b)
ɀ ɀ
( )
(c) (a)
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(a) )
CXCR4
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Figure 17. SDF-1 stimulated migration of MSCs on Col-Au throughα5β3 and CXCR4. Pretreatment with VEGF siRNA of MSCs and stimulated with or without SDF-1 (50 ng/mL) for 48h. (a) α5β3 and CXCR4 expression of MSCs on Col and Col-Au by immunofluorescence. (b) α5β3 and CXCR4 expression were analyzed by a flow cytometry α5β3 and CXCR4:
FITC-conjugated immunoglobulin (green color fluorescence), F-actin烉 rhodamine phalloidin staining (red color staining), cell nuclear烉DAPI (blue color staining) (scale bar烌50μm).
(b)
α5β3
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(a) (a)
100
Figure 18. The expressions of (a) eNOS, p-RhoA, p-Akt, p-FAK and p-Cdc42 proteins were induced by Col-Au. Pretreatment with VEGF siRNA of MSCs and stimulated with or without SDF-1 (50 ng/mL) for 48h. (b) Semi-quantitative measurement of Western blots for MSCs cultured on Col–Au showed significantly increased eNOS, p-Akt, p-FAK and p-RhoA expressions compared with Col . Data are mean ± SD. * p < 0.05 : greater than control.
(b)
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Figure 19. The MMP-2 enzymatic activity of MSCs culture on the Col and Col-Au.(a) Transfect VEGF siRNA and treatment with or without SDF-1 (50 ng/mL) to analyze VEGF stimulated MMP-2 expression. (b) Semi-quantitative measurement of Gelatin zymography analysis for MSCs cultured on Col–Au. Data are mean ± SD. * p < 0.05 : greater than control.
(a)
(b) ( ) (a)
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(a)
0h 12h
24h 36h
48h
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Figure 20. The average cell migration distance of MSCs on different substrates in a period of 48 h. MSCs transfected with VEGF siRNA and stimulated with or without SDF-1 (50 ng/mL) for (a) 0, 12, 24, 36 and 48 h by using immunofluorescence microscopy. (b) Columns, mean of three separate experiments. p<0.05: greater than control (TCPS).
(b)
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Figure 21. The expressions of eNOS proteins were induced by Col-Au.
Pretreatment with VEGF siRNA of MSCs and stimulated with or without SDF-1 (50 ng/mL) for (a) 24hr (b) 48h that were visualized by fluorescent microscopy. Cells were incubated with primary anti-eNOS antibody followed by stained with FITC-conjugated immunoglobulin (green color fluorescence). Bar = 100 μm.
(b) ) (a)
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Figure 22. Preparation of vascular graft .Construction and examination of tissue-engineered blood vessel graft in vitro. (a) longitudinal view of the catheter (22G needle, 30mm)ˤ(b,c) The Col and Col-Au coating on catheters, the outer surface of the catheters was first activated by air plasma (d)(e) Dynamic culture of MSCs on vascular graft the for 48h . (f) quantum dot label of MSCs indicating that most of the seeded cells (culture for 48 h) were distributed on the surface of vascular graft.
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Figure 23. Transplantation nanocomposites of pure catheterˣColˣ Col-Au vascular graft in vivo. (a) Animals were randomly assigned to either the control or 1 of 8 experimental groups consisting ,n=4ˤ(b,c) SD Rat fixed on the operating table after anesthesia. Then (d,e) incision of groin and find the femoral artery (f) Implanted vascular graft (about 1cm)ˤ(g) The blood vessel ligation (hˣi) Suture and disinfection of woundˤ(j) Recovery roomˤ
(a) ( )
b c d
e f g
h i j
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(a)
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Figure 24. Col-Au nanocomposites promoted MSCs to differentiate into ECs after repairing SD rar femoral artery injured by wire. MSCs were incubated with Col-Au for 48 hr and implantation into femoral artery. The femoral artery were separated from rat and immunostainined with anti-CD31 antibody. cells followed by stained with (a) 3,3-Diaminobenzidine (DAB) and stained with (b) CyTM5-conjugated immunoglobulin (red color fluorescence), cell nuclear staining was performed by 4', 6'-diamidino-2-phenylindole (DAPI) (blue color staining) (scale bar烌50μm)
! !
hCD31 / nucleus (b)
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Figure 25. Cell organization and anti-thrombogenic property after 30 day for MSCs implantation on various composites vascular graft in vivo. The samples were stained with Hematoxylin-eosin (H&E) staining was shown.
(scale bar烌50μm)
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Figure 26. Cell organization and anti-fibrosis property after 30 day for MSCs implantation on various composites vascular graft in vivo. The samples were stained with Masson trichrome staining was shown. (scale bar 烌50μm)