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Biomaterials Research (2005) 9(2) : 77-83 Biomaterials Research 7 The Korean Society for Biomaterials v s e w Effect of Fibrin on Migration and Growth of Fibroblasts and Matrix Contraction 1,2 Á½x 3 Á 1,2 Á 2,3,4 Á y 2,5 Á 1,2 Á 2,3,5 * Dong Lim Seol 1,2, Hyeong In Kim 3, Seung Jo Jeong 1,2, Won Hee Jang 2,3,4, Moon Hwan Cho 2,5, Sung Jae Lee 1,2, and Young-Il Yang 2,3,5 * 1 fh Š fd Š, 2 f } } l Â, 3 fh Š fh f fš, 4 fh Š f Š ŒŠ, 5 fh Š f Š Š 1 Department of Biomedical Engineering, Inje University, Obang-dong, Gimhae, Gyeongnam 621-749, Korea 2 Advanced Biolink Tech. Co., Ltd., Obang-dong, Gimhae, Gyeongnam 621-749, Korea 3 Paik Institute for Clinical Research, Inje University, Kaekeum-dong, Busanjin-ku, Busan 614-735, Korea 4 Department of Biochemistry, College of Medicine, Inje University, Kaekeum-dong, Busanjin-ku, Busan 614-735, Korea 5 Department of Pathology, College of Medicine, Inje University, Kaekeum-dong, Busanjin-ku, Busan 614-735, Korea (Received February 25, 2005/Accepted April 10, 2005) The goal of this study was to investigate the effect of fibrin-reinforced collagen sponge on migration, growth, and matrix contraction of fibroblasts. Human dermal fibroblasts (HDFs) were isolated from the skin by the explant method. The HDFs-populated collagen sponges were fabricated with or without fibrin. To determine optimal fibrinogen concentration for HDFs delivery, F-actin assembly, migration, and growth were examined with a 3-dimensional culture system. The number of microspikes and length of cytoplasm processes were increased by fibrinogen ranging from 1 to 5 mg/ml. On the other hand, 10 mg/ml of fibrinogen exhibited significant decrease (p<0.01). HDFs migration was much more delayed at higher concentration of fibrinogen. On the other hand, fibrin composed of 1 mg/ml fibrinogen showed significantly faster HDFs migration compared to higher concentration (p<0.01). HDFs grew well in fibrin-reinforced collagen matrices, whereas the collagen sponges without fibrin didn t support cell growth (p<0.01). In particular, the concentrations of 3~5 mg/ml of fibrinogen were most effective for cell growth. The fibrin significantly inhibited the contraction of collagen sponges (p<0.05). The HDFs-populated collagen sponges without fibrin showed about 53% contraction after 7 days. On the other hand, fibrin-reinforced matrices showed only mild to moderated contraction rate, ranging from 22% to 30% (p<0.05). The cell-mediated contraction rates were consistent among fibrin-reinforced groups. The HDFs-populated collagen sponges without fibrin showed marked and rapid cell-mediated contraction (p<0.01). In conclusion, fibrin-reinforcement of the porous collagen sponges induced effective migration and growth of fibroblasts and was able to make the matrix resistant to fibroblast-mediated contraction. It was demonstrated that the composite matrix of collagen sponge and fibrin could be a suitable scaffold system for construction of artificial skin for wound healing. Key words: Fibrin gel, Collagen sponge, Migration, Growth, Matrix contraction, Wound healing ilf Š g Š eš Š f il f f. f ilf v d g t ff, il ƒ t Šh l f l Š. f Š d d ~(polyurethane), 1,2) z (polyvinyl alcohol), 3) poly(hydroxyethyl-methacrylate), 4) Œf f glf jšt (copolymer) 5) f Š g (synthetic materials) z, 6-9) ~ (chitin), 10,11) l fƒ(alginate) 12,13) f tg (biological materials) g f f. *sf hf: pathyang@inje.ac.kr s hf l, Œ, Šil, f h, Œh Œ fš f l. s d Œg lf (fibrinogen)f Œ f Š s e v. f Š f Œ ƒ (thrombin) fš (fibrin)f Œ ŠŠ d (fibrin clot) Œ Šf f f vœf j. s f t Œ f s ef je ilf Š (monocyte), e (fibroblast), Œ (endothelial cell) f f Š f llt Šf Š. f Š f l Š ŒŠ Œ d l 77

78 Á ŒfÁh iáge Ái ŒÁf gá f (extracellular matrix)f Œ Š, f l Š fš Š s xe. 14-17) f f ƒ ghf fdš Š il f x Š eš lš jf, x hf Œ,, Œ, f f Š f d f. 18,19) ƒ,, ilf g Š eš h e f, f f d t f fdš f. 20,21) ƒ f, jš f h, Šh ƒ f l. f ƒ f f d Š, f g f f hš. h f ƒ Š, f g f f l h f. f Š f ƒ g Š i l f i Š hšhf. z l thš f j d Š r x d f g f. 22-25) h lf Š gš llt v ~ h ~. 7,26,27) f Š llt vf llt f (pore) i } Œ z f Œf ŠŠ, f f g, d lf f ilœ f ŠŠ h Š. hf i, d, f h ihš z lf f ŒŠ f f, h l Š z lf h ƒ f Œ l f hr, f, gf hšš hf. z f g f vš f g Œ hš h f. f Š h Š eš z f ŒŠŠ hiš ŒŠ f d f g f vf l. 14,27) z f ŒŠ f f h (viscosity) fl (cohesive strength)f ~ l } f f f g h f. 28) f x h e hhf Œ h Š f f t f f ed f Š f Š. ƒ, f e (human dermal fibroblasts, HDFs)f hr(adhesion), f (migration), g(growth) f uhf i f Š f Š. Š z l f Šf e f g lf vf hš f f Š e f g x f Š f Š. s e Œ hf hh ef swš. ilf 75% isopropanolf fdš 5 s Š, 200 U/ml penicillin 200 µg/ml streptomycinf Š e Hank's balanced salt solution(hbss, Sigma Chemical Co., St. Louis, MO)f 3 sš. f l e ilf explant f fdš Š. xf fdš ilf 1~2 mm 3 } h Š HBSS 3 sš. ilh f 10% fetal bovine serum(fbs, Sigma Chemical Co.), 50 µg/ml L- ascorbic acid(sigma Chemical Co.), 100 U/ml penicillin 100 µg/ml streptomycinf Še α-modified Eagle's minimum essential medium(α-mem, Sigma Chemical Co.) fdš 37 o C, 5% CO 2 f} f Š. 2j 0.05% trypsin/0.5mm EDTA(Gibco BRL-Life Tech., Grand Island, NY) fdš e Š Š. e d f 70% Š f Š, 3f l ŒŠ. 5 10 f Š e fdš. s sww v Tisseel TM kit(baxter AG, Vienna, Austria) Š, ƒ, (aprotinin), Œx (calcium chloride)f fdš f hiš. d Š Š, 100 mg, 30 IU Factor XIII, 80 µg (plaminogen)f ŒŠ f 3,000 KIU/ml d f dšš. 500 IU ƒ f 40 mm Œx d dšš. f ui 1, 3, 5, 10 mg/ml 0.3 IU/ml ƒ hiš. e ƒ d ~Š / e f hiš. / e ŒŠ f F-actin Œ f f Š eš multiwell plate, 37 o C f} f 1 jš z. h jš l t Š ŒŠ f Š. s Cytoskeleton x i e f Œ h F-actinf hš. 4 10 5 cells/ml f e ŠŠ 50 µl f 96-multiwell plate 37 o C jšš. h jš f g l t Š 0.5, 2, 6, 24 Š, 200 µlf 3.7% (formaldehyde) d hš g Š. g f phosphate buffered saline(pbs)f 3r Š, 0.1% Triton-X100 1% (albumin)f Š PBS t Š. e f F-actin f Š eš well 0.2 U/µlf Oregon Green-512 phalloidin(molecular Probles Inc., Eugene, OR)f t Š 1 fš. PBS 3r Š 511 nmf excitation g, 528 nmf emission g F-actinf f i Š f, AxioCam HRc confocal microscope(carl Zeiss, Gottingen, Germany) f l Biomaterials Research 2005

f e f f g llt v x 79 Š. F-actinf Œ f t 0.5, 2 f microspike 6, 24 (cytoplasm process)f f ImageJ 1.32 (NIH, Bethesda, MD)f fdš h hf hš. s e d f Š Š eš 3 10 6 cells/ml f e ŠeŠ f hiš. e ŠŠ f 6- multiwell plate j well 3 µl t Š, 3 jš ~ g l Š. 1, 3, 7fm PBS 2 3.7% d f hš g Š. f 0.1% crystal violet(labchem Inc., Pittsburgh, PA)f 5 Š, 2r l 3r f Š. l x gr ft (stereomicroscope) f l Š ImageJ fdš f d f Š whš. v 3 1, 3, 5, 10 mg/ml f 4 10 5 cells/ml f e Š 0.3 IU/ml ƒ f ŒŠŠ d f SigmaCote (Sigma Chemical Co.) s Š 6- multiwell plate well 15 µl t Š 37 o C jš Š } hiš. 1 h jš z f tipf fdš d }, g l t Š free-floating fš 7f Š. e ŠŠ } 1, 4, 7fm l x r (inverted microscope, Leica Microsystems, Wetzlar, Germany)f fdš f l Š. lœ f l ImageJ f fdš hf whš f, v f fff h Š ef hš ( (1)). Area ---------------------------------- Contraction rate (%) = 100 (1) Original area v g s e f g z lf v h fš v h i Š eš f z l hfš. f 95% f f z l(sulzer Dental Inc., Carlsbad, CA) 5mm } f } Œ~ hiš, e ŠŠ ŒŠ 15 µl z l iš. f 37 o C f} f 1 h jš z. e 4 10 5 cells/ml iš, 1, 3, 5, 10 mg/ml f f dš f z l h iš. f l fš f e f iš z l i f fdš. v g s 3 s z l free-floating fš 7f Š. 1, 4, 7fm f Š HBSS 2, -20 o C Š. DNA f Š eš f papain digestion buffer 55 o C 12~16 h dšš. f g f PicoGreen DNA detection kit(molecular Probes) fdš DNA f h ŒŠ Š. DNA f r jf eš e Š l f f DNA standard hš. Œ (fluorescence) f 480 nmf excitation g, 520 nmf emission g Synergy TM HT microplate reader(bio-tek Instruments, Neufahrn, Germany)f fdš whš, DNA j f fdš DNA f Œ Š. v g s f z l i f f ŠŠl iš l 6-multiwell plate free-floating f Š. f z l 1, 4, 7fm l x r (inverted microscope, Leica Microsystems, Wetzlar, Germany)f fd Š f l Š. lœ f l ImageJ f fdš hf whš f, v f ff f h Š ef hš ( (1)). Š, gš lltf v f Š eš (2) f DNA f f v f hš. Cell-mediated contraction rate (%/ng) Original area Area ------------------------------------------------------------------------------- = Original area DNA content 100 (2) m w f x j r Š f, SPSS (Ver.10.0.7, SPSS Inc., Chicago, IL)f fdš oneway ANOVA f h ef f lš. P f 0.05 f d h ef f f hš. š s Cytoskeleton x Š (1~10 mg/ml)f f (cytoskeletal organization) Š f hr f Š. 1~5 mg/ml f 30 microspike 10 mg/ml f Œ f rš f (Figure 1A-D). Š 24 m, 1~5 mg/mlf f Šf 10 mg/ml f f, f Œ Vol. 9, No. 2

80 설동림 김형인 정승조 장원희 조문환 이성재 양영일 Number of microspikes at various concentrations of fibrinogen (n=4~9). The number of microspikes showed that ranging from 1 to 5 mg/ml of fibrinogen showed more increased. On the other hand, 10 mg/ml of fibrinogen exhibited significant decrease (**p< 0.01). Figure 2. Organization of F-actin at various concentration of fibrinogen. Original magnification: 400. (A, E) 1 mg/ml of fibrinogen; (B, F) 3 mg/ml of fibrinogen; (C, G) 5 mg/ml of fibrinogen; (D, H) 10 mg/ ml of fibrinogen; (A-D) 30 minutes; and (E-H) 24 hours. Length of cytoplasm process at various concentrations of fibrinogen (n=3~5). The length of cytoplasm processes showed that ranging from 1 to 5 mg/ml of fibrinogen showed more increased. On the other hand, 10 mg/ml of fibrinogen showed significant decrease (**p<0.01). 을 관찰할 수 있었다(Figure 1E-H). 이러한 결과를 정량적으로 분석하기 위하여 세포의 microspike 수 및 세포돌기의 길이를 산정하였다. 그 결과, 1~5 mg/ml 농도의 피브린 하이드로겔에 서 배양된 세포의 microspike 수가 10 mg/ml 보다 약 41.8% 많았다(Figure 2)(p<0.01). 이러한 결과는 상대적으로 낮은 농도인 1~5 mg/ml 피브린에서 세포의 초기 부착이 더 잘 일어나는 것을 나타낸다. 세포돌기의 길이 또한 1~5 mg/ ml 농도가 10 mg/ml 보다 약 66.2% 높은 결과를 나타내었다 (Figure 3)(p<0.01). 그러나 1 mg/ml에서 5 mg/ml 사이의 피 브린 농도에서는 서로 유의한 차이가 나타나지 않았다(p>0.05). 섬유모세포의 이동능 검사 다양한 농도의 피브리노겐에 따라 섬유모세포가 피브린 겔 외부로 이동하는 정도를 분석하였다. 사진 관찰 결과 피브리노 겐의 농도가 낮을수록 피브린 겔 외부로 세포가 많이 이동하 는 경향을 나타내었다(Figure 4). 특히, 1 mg/ml 농도의 피브 린에서는 겔의 외곽선을 구분할 수 없을 정도로 세포의 이동 이 두드러지게 많았다. 이러한 세포의 이동능을 이미지 프로그 램을 통해 정량화한 결과, 1 mg/ml 농도의 피브린에서 4, 7 일째 각각 376.7 ± 48.3 µm, 1168.5 ± 51.1 µm로 다른 군들에 비해 이동한 세포들의 거리가 가장 멀었다(Figure 5)(p<0.01). 반면에 피브리노겐의 농도가 높아질수록 세포의 이 동능이 떨어졌다. 이는 피브리노겐의 농도가 높을수록 내부 공 극 크기가 작아져 세포의 이동을 어렵게 하는 것을 증명해 주 었다. 반대로, 낮은 피브리노겐에서는 내부구조가 느슨하여 내부 공극이 크고, 강도가 약하여 세포의 이동을 향상시킨 것 이라 할 수 있다. 또한 피브린의 분해 속도가 빨라 시간이 지 날수록 세포의 이동은 더욱 가속화될 것으로 사료된다. Figure 3. Figure 1. Biomaterials Research 2005 29)

피브린이 섬유모세포의 이동 및 성장과 지지체 수축에 미치는 영향 81 Effect of fibrin on cell growth in collagen sponges (n=4). Fibrin reinforcement improved cell growth in collagen sponges. This was most noticeable at concentration of 3~5 mg/ml (*p<0.05, **p<0.01). Figure 6. 였다고 사료된다. 특히 피브리노겐의 생리적 농도범위인 3~5 mg/ml 농도의 피브린 겔에서 섬유모세포의 성장이 가장 우수 했다. 피브린 디스크 콜라겐 스폰지 피브린으로 보강된 콜라 겐 스폰지의 수축률 검사 섬유모세포를 포함한 피브린 디스크의 수축 정도를 피브리노 겐의 농도에 따라 분석하였다. 피브린 디스크의 수축률은 높은 농도의 피브린 겔에서 지지체의 수축률이 낮은 경향을 보이나, 배양 1주일 뒤에는 피브리노겐의 농도와는 관계없이 모든 디 스크에서 완전히 수축하는 결과를 보였다(Figure 7). 이러한 경 향은 콜라겐 스폰지에서도 동일한 양상으로 관찰되었다. 피브 린이 포함되지 않은 콜라겐 스폰지는 1주일째 여전히 53%의 수축률을 나타내었다(p<0.05). 반면에 피브린으로 보강된 콜라 겐 스폰지는 7일째 22~30%의 수축률을 나타내어 피브린이 3 차원 다공성 지지체의 수축을 방지하는 것을 확인할 수 있었 다(Figure 8). 피브린으로 보강된 콜라겐 스폰지 그룹 내에서 14) Fibroblasts migration at various concentrations of fibrinogen. Cells migrated out poorly as fibrinogen concentration increased. Original magnification: 40. (A) 1 mg/ml of fibrinogen at 7 days; (B) 3 mg/ml of fibrinogen at 7 days; (C) 5 mg/ml of fibrinogen at 7 days; and (D) 10 mg/ml of fibrinogen at 7 days. Figure 4.,, Out-migration distance of fibroblasts at various concentrations of fibrinogen (n=3). 1 mg/ml of fibrinogen migrated the farthest (**p<0.01). Figure 5. 섬유모세포의 성장능 검사 피브린 겔이 보강되지 않고 섬유모세포만 파종한 콜라겐 스 폰지에서는 세포의 성장이 정체되는 것을 확인하였다(0.5 ± 0.8 ng)(figure 6). 반면에 피브린이 보강된 스폰지에서는 시간 이 지남에 따라 섬유모세포가 점차적으로 증가하였다. 특히, 3~5 mg/ml 농도의 피브리노겐에서 가장 높은 세포의 성장능 을 나타내었다(p<0.05). 피브린이 보강되지 않은 콜라겐 스폰 지의 경우 스폰지의 수축으로 인하여 내부에 파종된 세포가 증 식하지 못한 것으로 사료된다. 반면에 피브린을 혼합한 경우 수축을 방지하였고, 이로인해 세포의 지속적인 성장이 가능하 Effect of fibrin on the contraction rate at various concentrations of fibrinogen (n=4). HDFs-populated fibrins showed complete contraction within 7 days. Figure 7. Vol. 9, No. 2

82 Á ŒfÁh iáge Ái ŒÁf gá f Figure 8. Effect of fibrin on the contraction rate of collagen sponges (n=4). HDFs-populated collagen sponges reinforced with fibrin showed significant decrease in contraction after 7 days (*p<0.05). f efš rf ~ l (p>0.05). f ilf td hiš h l ltf vf f f, g, Œ hšš fš f f ilf vš. Š vf fš f ilf i Œ f jeilf f f r f f hšš f f h f. f Š 3re llt f g fš f h f. 11,22,23) f g lltf v f Š eš v f DNA f hš v f hš (Figure 9). f Š l f z l g llt vf t ~. f z l g lltf vf Š lš f, h f dšl f l efš (p<0.01). z l f dš il Š h f ilf hiš h lltf v h hf Š f ŒfŠ. Figure 9. Effect of fibrin on the cell-mediated contraction rate of collagen sponges (n=4). The cell-mediated contraction rate normalized by DNA content showed a similar value between fibrin reinforced groups. However, the HDFs-populated collagen sponges without fibrin showed cell-mediated contraction problem (**p<0.01). z l ŒŠŠ dš d f f l hf gf Š lltf vf l Œ fš f f, f f f ilf v f edš llt df Š. e f f il Šh f f ilf vš e t z l Š ed f Š. Š f f hr, f, g f Š, h f 3~5 mg/ml f f e f f gf uhf. z l f dš d lltf vf lš f f l hf gf lešl Š. f z l l hf e f g f leš lltf vf l. f f 3~5 mg/mlf z l s xed f ilf v uhf g ff f. fe l (RTI04-03-07) le f Š. š x 1. W. L. Hinrichs, E. J. Lommen, C. R. Wildevuur, and J. Feijen, Fabrication and characterization of an asymmetric polyurethane membrane for use as a wound dressing, J. Appl. Biomater., 3 (4), 287-303 (1992). 2. K. A. Wright, K. B. Nadire, P. Busto, R. Tubo, J. M. McPherson, and B. M. Wentworth, Alternative delivery of keratinocytes using a polyurethane membrane and the implications for its use in the treatment of full-thickness burn injury, Burns, 24(1), 7-17 (1998). 3. Y. Suzuki, M. Tanihara, Y. Nishimura, K. Suzuki, Y. Kakimaru, and Y. Shimizu, A novel wound dressing with an antibiotic delivery system stimulated by microbial infection, ASAIO J., 43(5), M854-857 (1997). 4. D. P. Dressler, W. K. Barbee, and R. Sprenger, The effect of Hydron burn wound dressing on burned rat and rabbit ear wound healing, J. Trauma., 20(12), 1024-1028 (1980). 5. H. J. Kim, E. Y. Choi, J. S. Oh, H. C. Lee, S. S. Park, and C. S. Cho, Possibility of wound dressing using poly(l-leucine)/ poly(ethylene glycol)/poly(l-leucine) triblock copolymer, Biomaterials, 21(2), 131-141 (2000). 6. S. T. Boyce, D. J. Christianson, and J. F. Hansbrough, Structure of a collagen-gag dermal skin substitute optimized for cultured human epidermal keratinocytes, J. Biomed. Mater. Res., 22(10), 939-957 (1988). 7. S. T. Boyce, M. C. Glafkides, T. J. Foreman, and J. F. Hansbrough, Reduced wound contraction after grafting of full-thickness Biomaterials Research 2005

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