Biomaterials Research (2005) 9(2) : 90-95 Biomaterials Research 7 The Korean Society for Biomaterials j ql s w e w The Effect of Mechanical Stimuli and Patterned Microfiber Substrate on Neuronal Outgrowth and Guidance ½ Á Á½ Á½ wá y Á Á * In Ae Kim, Su A Park, Young Jick Kim, Su Hyang Kim, Ho Joon Shin, Yong Jae Lee, and Jung-Woog Shin* fh Š fd Š Department of Biomedical Engineering, Inje University, 607 Obang-Dong, Kimhae, Kyongnam 621-749, Korea (Received January 29, 2005/Accepted May 17, 2005) Most nerve injuries in the central or peripheral nervous systems induce a serious loss of axonal function. Many investigators have studied neuron axonal outgrowth and guidance using various scaffolds, surface patterning, or growth factor coating. Still, neuronal regeneration is difficult. Therefore, we evaluated the effects of micropatterned fibers made by electrospinning and steady fluid-induced shear stress on neuronal outgrowth and axonal guidance in vitro. The rat pheochromocytoma cells (PC-12) were stimulated with nerve growth factor and laminar flow shear stress in a fluid flow system. After a 48h culture, a 2h steady shear stress was applied three times each day for 2 days. The degree of steady shear stress stimulation was divided into five groups according to the magnitude of stimuli: 0.1~1.5 Pa. 4 days after seeding, neuronal outgrowth and alignment were measured using SEM and F-actin staining. The micropatterned fiber substrate enhanced the alignment of neurons and neurite outgrowth. When a stress of 0.5 Pa was applied, the number of cells increased with the micropatterned substrate and neurons were most highly aligned with patterning. The outgrown neurites were longest under the shear stress of 0.25 Pa. The neurons were guided by the micropatterned fibers and stimulated by fluid-induced shear stress at specific magnitudes. The results suggest that micropatterned electrospun fibers and fluid-induced shear stress are promising for stimulating axonal regeneration in a desired direction. Key words: Nerve regeneration, Micro-patterning, PC-12 cells, Shear stress stimulation, Neurite outgrowth length t f f f d, l f f g } g f. g f f Š hœš f g l f Š f l Š. 1) f f f f g ~ g f ihš eš, extracellular matrices, microgrooved surfaces,, z f f f d thš llt fdš Š f. 2) ƒ, l ltf f} } f f Œ Š f fd f. 2), Š e fdš f} llt h fš, f fdš f hœš f gf rš. h f f Š ilf Šhf w j hf Šf Š. f f f Œh f j f ff Œ ~. ƒ h f f f hf l ~,, e, Œ *sf hf: sjw@bme.inje.ac.kr ~ f. 4) f Œ~ Šh, Šhf w f f Šd Š. h f f f g ŠdŠ tœ f j jdš fdš f., Š e llt h f f f f gtl hœš f g x Š rš. Rat pheochromocytoma(pc-12) gff(ngf) fš hœhf f f l Œ f h f. 5) PC-12 Š jfš(korean Cell Line Bank) dš, 10% d~ Œt(FBS, Hyclone, UT, USA), 100 units/ml (Sigma, MO, USA), 100 mg/ml ƒ f (USB, OH, USA)f Š Dulbecco's Modified Eagle's Medium h-glucose(dmem-hg)f l 37 o C, 5% CO 2 f 90
h f f} ef f f g x 91 } f. Š llt rf d f h f. PC-12 f r f f eš f z Š. f llt hfš eš y (24 36 mm) 0.1% d f 12h z Š HANK s balanced salt solution(hbss) sš. f hf llt i f L Š. f f} e llt h f fdš hfš. ~f z f jšt(plga, 50:50, Alkamus Inc., USA) 50 wt%f d. f d N,N- f (DMF, Junsei Chemical Co., Ltd., Tokyo, Japan) ƒ Šf (THF, Junsei Chemical Co.)f 1:1 dš. f} e Š eš f z ll t hš l (e Œ, l : 80 mm, f: 100 mm) rš. 18 G j f dš hf Š. PLGA l f f h he fdš 10 kvf h f j l ff 6.5 cm hš. f l f e f 4.2 m/sf h z PLGA f} e f z llt e Š. f l step motor hœš h Š, h fturbo C(ver 3.0, Boland International, Inc.) fdš. 6) l Figure 1 f e f fdš h f f Š. e f e s, 2 f lhgs, f. lhgs j 1 95% air 5% CO 2 elš eš l s d Œ Š f hf. 2) e s f 1 mm, 30 mmf f hf (Figure 2). e f h Š LabVIEW (ver. 6.1i National Instruments, USA) f fdš y h. f e f Newtonian fluid, h w Figure 1. Schematic diagram of the fluid flow systemu Figure 2. Modified flow chamber used to apply shear stress : (A) Figure of chamber and (B) schematics of chamberu hš. Šl h f f (1). τ = 6µQ/h 2 b (1) µ: dynamic viscosity, Q: flow rate, h: height, b: width h f f f eš, lf dynamic viscosity(µ) 9.6 10-4 PaÁs hš. 2) y e f (CFD)f fdš e s ~ e f f Š. e s 2re ešd hfš, fluid e f e Œ ~, e, h f f rš. f hf CFD f Fluent 6.0(Fluent Inc., USA)f dš Š Š. ƒ z ( L) f} ( F) f f y e PC-12 2.5 10 4 cell/glass iš. f 10% FBS Š DMEM-HG fdš f} f 2f. 3fm L F e s h f f Š (Table 1). f e s 3 f y f 4 f e s l Š. h f f h f f Š 3 2 2f Š. f f l f jf Œ f e eš lf FBS 1% jf NGF(NGF-7s, 0 ng/ml, Vol. 9, No. 2
92 f Á Á lá Á ŒjÁfdgÁ hd Table 1. Experimental groups according to the pattern and shear stress magnitude Substrate Magnitude Group L (laminin-coated only) LC (control: C (0 Pa)) LS_0.1Pa (stimuli: S) LS_0.25Pa LS_0.5Pa LS_1.0Pa LS_1.5Pa Group F (laminin + patterned fibers) FC FS_0.1Pa FS_0.25Pa FS_0.5Pa FS_1.0Pa FS_1.5Pa Sigma, USA) t Š. Table 1 f, h f f } 5 Š. h f f Šll l f f eš 6.79 10-4 cm/sf l ŒŠ. x (SEM) ef f Œ f rš eš SEM f fdš. f l h Š 4% Šf 30 h f vd (PBS)f r s Š. h 30, 60, 70, 80, 90, 100% z 1 sš ~ Š, i z. f f fdš z Š f 5kV SEM(JSM-5000, JEOL, Tokyo, Japan)f fdš u Š. F- p f PBS s 4% Šf 10 hš. f eš 0.1% Triton X-100 4 o C 20 s 1% BSA blocking Š. rhodamine phalloidin(1:100, Molecular Probes, Eugene, OR, USA)f 20 s Š. f fluorescence microscopy(axioskop2 Plus, Karl Zeiss, Germany)f fdš whš.» ù (outgrowth) ¼ w d f whf eš F- f f l f l hgš. bipolar pseudo-unipolar Œ f f f hš. 7) f f Image-J f fdš t f l whš. f whf r ±2 µm ~. PC-12 f h wh Š eš MATLAB(ver. 6.0, MathWorks, Co.)f fdš f l Š. f h f j r(ad) f fdš h h f whš, f AD f Fisher fš circular statistics fdš. 9)»ƒ s s d Œ f f l fdš f f tf 1.5 f f hš f f (2) fdš wh Š. 10) Percentage of cells outgrowth = cell count of outgrowth / cell count of all (2) m f h Œ eš fe x f fdš j Fisher's LSD f dš. d f SPSS 11.0 software(spss Inc. Chicago, IL, USA)f fdš. š h f f h f f} } f e f llt fdš f guidance x Š rš. f rf l ~ eš NGF Š fdš. f ECMf d g f g f f l ~ f fd f. 11,12) NGF PC-12 f Œ ~ jdš d f. f f f f g f gf l ~ f eš f} f llt fdš. Miller 14) f} f Š llt f orientationf l ~ f} groovef f f jdš d ff Š., f} e f llt f fdš. SEMf fdš f} f lltf i rš ef l f 3 µm 5 µm wh (Figure 3A). Š, f} e f h f f} e Š f (Figure 3B), AD f 9.14 o ~. f hf f} e Š, f} e Š f f z f f f efš l. Š f} e f f. f} f e llt hœš hf f g v Š Š f ŠŠ f. h f f Š f Œ h f f f j jdš d f. 15), d h f f f orientation f x f hš. h f f } hš eš f Š, 48 hf h f f llt h. f f rf Š Š ~ f f., d Š Biomaterials Research 2005
물리적 자극과 마이크로 섬유의 패턴이 신경세포의 성장과 방향성에 미치는 영향 A micropatterned substrate : (A) SEM image and (B) angular distribution. Figure 3. 에 3번 2시간씩 이틀간 전단응력을 가하였다. 또한 챔버 내 유동의 정확한 크기를 검증하기 위하여, Fluent 프로그램을 이 용하여 유동 챔버 내부의 유동의 흐름을 관찰하였다. 유동 속 도는 잘 알려진 유체 속도 계산식의 값과 동일하였다. 챔버 내 유동 의 속도를 4.34 cm/s 가했을 때 계산된 전단응력은 0.25 Pa 이었다. 전단응력이 가해질 때 와류와 같은 흐름은 없었다. 전단응력을 받은 세포는 자극을 받지 않은 그룹에 비하여 방 추형의 형상을 보였다. PC-12세포의 뻗어 나온 돌기의 길이를 측정한 결과, F 그룹의 신경돌기는 L 그룹보다 더 길어졌다. 그리고, F 그룹은 섬유를 따라 신경돌기가 자란 것을 알 수 있었다. 자극의 크기가 0.25 Pa 이상일 경우 뻗어 나온 돌기 의 길이가 감소하는 반면에 0.25 Pa 크기의 전단응력을 가했 을 때 신경세포체와 신경돌기의 길이가 길어짐을 관찰할 수 있 었다. 그리고, FS_0.25 Pa 그룹에서 신경돌기의 길이가 가장 길었다(Figures 4, 5). 세포의 orientation 측정 결과, 세포는 라미닌이 코팅된 그룹 보다 섬유가 있는 지지체 그룹에서 높은 배열을 보였다. 또한, FS_0.5Pa 그룹에서 가장 높은 배열 정도를 보였으나, 섬유가 93 Changes in cell morphology with the magnitude of the shear stress at 4 days for both group L and group F ( 100). Figure 4. Outgrowth length of neurites on different structures with different shear stresses (*p<0.05). Figure 5. 없는 그룹에서는 전단응력에 의한 배열은 볼 수 없었다 신경돌기의 성장이 일어난 세포의 수는 FS_0.25Pa 그룹에서 가장 많았으며, 평균적으로 F 그룹에서 L그룹에 비하여 더 많 은 신경돌기의 성장을 나타내었다(Figure 7). (Figure 6). Vol. 9, No. 2
94 f Á Á lá Á ŒjÁfdgÁ hd Figure 6. Angular deviation of neurites on different structures with different shear stresses (n=6, Q: p<0.05)u Figure 7. Percentage of cells with neurite outgrowth on different structures and at different shear stresses (n=6, Q: p<0.05)u Fluid-induced h f f f} e Š f hf h f, f gœ Š ~ Šf ŠŠ f g hd fff f. 16), h f f ll f f Œh f Š f l Œ, f f j f f. 6,16) Š f f h f f } f f ~. 0.5 Pa } f h f g f f f, f f 0.25 Pa g if ~. 0.25 Pa f f h f f Š 0.5 Pa f f } l f. f f f f, f rf Š f f } } l f f f. h f f f} e llt efš if f f f} e f Š f gœ f f h f f l ~ f. f} e llt h f f f g g Š f x Š rš. f f} e f f g f ihš, ƒhš } f h f f f f l ~ f f. ƒ, f} e llt fluid-induced h f f Š fd f gf l h f., e llt ƒh } f h f f g f hhf f j f. f r f f fd d g f fdš lef f hf f. š x 1. N. Rangappa, A. Romero, K.D. Nelson, R.C. Eberhart, and G.M. Smith, Laminin-coated poly(l-lactide) filaments induce robust neurite growth while providing directional orientation, J. Biomed. Mater. Res., 51, 625-634 (2000). 2. H. Tsuji, H. Sato, T. Baba, S. Ikemura, Y. Goroh, and J. Ishikawa, Neuron cell positioning on polystyrene in culture by silvernegative ion implantation and region control of neural outgrowth, Nuclear Instr. Methods Physics Res., B166-167, 815-819 (2000). 3. J. Klein-Nulend, A. van der Plas, C. M. Semeins, N. E., Ajubi, J. A. Frangos, P. J. Nijweied, E.H. Burger, Sensitivity of osteocytes to biomechanical stress in vitro, FASEB. J., 9, 441-445 (1995). 4. A. A. Lee, D. A. Graham, S. D. Cruz, and A. Ratcliffe, Fluid shear stress-induced alignment of cultured vascular smooth muscle cells, J. Biomech. Eng., 124, 37-43 (2002). 5. S. J. Lee, G. Khang, Y. M. Lee, and H. B. Lee, The effect of surface wettability on induction and growth of neurites from the PC-12 cell on a polymer surface, J. Colloid. Interface. Sci., 259, 228-235 (2003). 6. C. H. Lee, H. J. Shin, I. H. Cho, Y. M. Kang, I. A. Kim, K. D. Park, and J. W. Shin., Nanofiber alignment and direction of mechanical strain affect the ECM production of human ACL fibroblast, Biomaterials, 26, 1261-1270 (2005). 7. L. C, Junqueira, J. Carneiro, and R. O. Kelley, Basic Histology, Appleton & Lange, 1999. 8. R. Brian, M. D. Noble, and P. A. Tresco, Directed nerve outgrowth is enhanced by engineered glial substrates, Exp. Neurol., 184, 141-152 (2003). 9. N. I. Fisher, Statistical Analysis of Circular Data, Cambridge University Press, 1993. 10. J. Schimmelpfeng, K. Weibezahn, and H. Dertinger, Quantification of NGF-dependent neuronal differentiation of PC-12 cells by means of neurofilament-l mrna expression and neuronal outgrowth, J. Neurosci. Methods, 139, 299-306 (2004). 11. D. L. Hynds and D. M. Snow, A semi-automated image analysis Biomaterials Research 2005
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