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Biomaterials Research (2007) 11(3) : 96-101 Biomaterials Research 7 The Korean Society for Biomaterials w jm /v -w qk p w œ p Porous Structure and Characteristic of Protein Release on Biodegradable Chitosan/Fibroin-Hydroxyapatite Hybrid Scaffold ½y 1 *Á½ k 1 Á 2 Á½ x 1 Hong Sung Kim 1 *, Jong Tae Kim 1, Su Chak Ryu 2, and Ji Hyun Kim 1 1 Š fe Š Š f g Š / PNU-Fraunhofer IGB 2 Š Š Š h g Š / PNU-Fraunhofer IGB 1 Department of Biomaterials Engineering, College of Natural Resources & Life Science/ Joint Research Center of PNU- Fraunhofer IGB, Pusan National University, Miryang 627-706, Korea 2 Department of Nanomaterials, College of Nano Science and Technology/ Joint Research Center of PNU-Fraunhofer IGB, Pusan National University, Miryang 627-706, Korea (Received July 18, 2007/Accepted August 16, 2007) The high porous scaffolds of biodegradable chitosan/fibroin-hydroxyapatite hybrid were prepared by churning and thermal inducing phase separation. The microscopic morphology and porous structural factors, such as average pore diameter, maximum pore size, and pore distribution, of the hybrid scaffold were analyzed by scanning electron microscopy and capillary flow porometry. The absorptivities of simulated body fluid (SBF) and the release properties of model protein were investigated in SBF according to the weight ratio of hydroxyapatite in the polymers/ceramic hybrid. The apparent pore size of the matrix surface was around 100 micron on SEM images, but the practical average pore size perforating through the matrix was about 3.4 micron. The SBF absorptivity and protein release were decreased with increasing the weight ratio of hydroxyapatite in the hybrid. Key words: Biodegradable scaffold, Chitosan, Hydroxyapatite, Protein release, Fibroin Š f fdš llt(scaffold) x ilg f eš f Š f d ff, u f g h h f eš f f l f. 1,2) il Šd llt tilf Š e f d ilf g l llš Šf Š Š Œ ilf t. 3,4) llt Œ lf ilf gf eš hšš 3reh i Š f h Š. 5,6) f Š llt g thš Š f h Œ g h f jdš s f 7-10) z, ~ /~, poly(3-hydroxybutyrate) Š f 11-13) polylactide, polyglycolide, poly(εcaprolactone) f j f. j ~ /~ f z z f t Š s *sf hf: khs@pusan.ac.kr x f thš Š f l, 14) d lf z x f ŒŠ i lf t f hr gf ihš Š ff f f. 15,16) Š f g Š f u Šf d l ff ~ f r f llt g f. 17) f Š Š f j f Š 17 f f lf ff d Š Šh Œ hš f ~ s g f f Œ fdf f. 18,19) ilf d lf e Š lf, j h f j f Šf ~fƒf. s vvš Š Š Šf ~fƒ i Ca/P Œ f l, i e Œ f l f e fš l til r Œ. Š Šf ~fƒ f Šf Œ Š hr f d Š ~ f l Š llt hf Š f ~ f h Š d ilf gš f f Œ t Šf ~fƒ Œ 96

Š ~ / f -Šf ~fƒ fi Š lltf i l v ƒ 97 Š l. 20,21) Š llt l f hr, Œ gf eš h Œ lf llt hi l Še ~ f e Š. f d l f Še l ff Œfd lltf l v l, f g f x f. 22) ilf eš t Œ llt eš ~ / f f l Š Šf ~fƒ ŠŒŠ ~ / f-šf ~fƒ fi Št Š, f e fš t hiš i lf fdš l v f i Š. ~ f ~ f ( ) fš hhš dš. hh 1wt%f ~ f 2wt% t d dšš 2wt%f Œ ƒ f jœš v ~ ~f ~ sš l iš. f j ~ f j f f 400,000f f, 97% ~ Œ h. f d f f ŒŠ Œx,, ~ f ŒŠd dš z e 7f Š j Š. vv Œ Šf ~fƒ(ca/p=1.55) Š( ) fš X h hd ƒ fš ŒfŠ l tf s Š 2 f } f ff j Š. d bovine serum albuminfluorescein isocyanate(fitc-bsa) Š f x( ) fš hh f dš. œ x ~ / f 2wt% t d 2wt% ~ f dš ~ f d f ~ Š f 20 wt% fš ŒŠŠ j Š f, f Š Šf ~fƒ(hydroxyapatite; fš HAP) 0 ~70 wt% ŒfŠ ŠŒ Š. t h 23) f e fš j Š. h Št Š Š Šhf e f f t- t z f Œ ~ f ˆd f z t- t t- t e Š. f f d f ~ l iš, f giš d h h Š eš l iš t Œ z. llt f Œ~Šh i i Š eš f lrš j hf (Hitachi S-3500N, f )f 15 KV rš. f } etf f f dš capillary flow porometry 1100 (Porous Materials, Inc, ) f, etf t f, t f ~ f dš. f e l v f whš eš fit (simulated body fluid: fš SBF)f f f j Š. ~f NaCl, NaHCO 3, KCl, K 2 HPO 4 3H 2 O, MgCl 2 6H 2 O, CaCl 2, Na 2 SO 4 h ŒŠŠ Table 1 ~ t f f i f v f, tris(hydroxymethyl) aminomethane 1M HCl 36.5 C o ph 7.4 j Š. SBF f 37 C 24 SBF xlš o r SBF h Š whš fj Š SBFf j ~. l v f f f Š. l Œ f r Œt (FITC-BSA)f dš f, f i h f ~ / f-šf ~f ƒ Šd fš ŒfŠ. f j Š t 37 C h SBF xlš FITC-BSA vvš f o, vv FITC-BSAf h f UV-1201 (Shimadzu, f ) fš 495 nm whš Š. š œ Figure 1f HAPf j Š 0.3, 0.5 0.6f ~ / f-hap fi Š t j hf (SEM)f rš, f e f, w f Œ f ~ f. j f f whš, HAP Š 0.3 97.3%, 0.5 95.7% HAP Š f l 2%f f, SEM Œ f r HAP Š } f h l ŒfŠ. 90% f f f f tf f Œ t- t d f t d Œ h Œ Š Œ~ l. Œ~ f h Table 1. Comparison of ionic concentration between human blood plasma and SBF concentration ( mm ) Na + K + Ca 2+ Mg 2+ - HCO 3 Cl - 2- HPO 4 Blood Plasma 142.0 5.0 2.5 1.5 27.0 103.0 1.0 0.5 SBF 142.0 5.0 2.5 1.5 27.0 125.0 1.0 0.5 SO 4 2- Vol. 11, No. 3

98 김홍성 김종태 류수착 김지현 SEM images taken from the surface of chitosan/fibroin-hap hybrid scaffolds with HAP weight ratio of 0.3 (A and B), 0.5 (C and D). and 0.6 (E and F). (A) and (C) are front surface images. (B) and (D) are rear surface images. (E) and (F) are lateral face images. Figure 1. 향에 따라 온도가 상승하여 용매결정이 와해되는 전면 표면에 약 100 µm 이하의 불균일한 공극 분포(Figure 1(A), (C))와 용매의 결정화온도보다 낮은 온도를 유지하는 배면에 100 µm 이상의 용매결정상의 형상을 갖는 긴 공극(Figure 1(B), (D))을 동시에 형성하는 이중 공극구조를 나타내었다. 이 다공체에 있어서 유체가 통과할 수 있는 관통된 공극의 크기를 평가하기 위하여 Figure 2에 나타낸 모식도와 같은 모 세관 흐름을 측정하는 기구를 사용하여 다공체내의 서로 뚫린 공극, 즉 모세관을 통과하는 유체의 압력과 속도의 상관관계를 나타내는 유속 시험을 하였다. 계면에너지가 낮은 액체에 적신 시료의 한쪽 면에 기체의 압력을 점차 증가시키면, 시료의 공 극 내에 있는 액체는 반대편으로 밀려나, 공극은 점차 기체로 교체된다. 이때 공극 벽면과 액체 사이의 계면에너지는 공극 벽면과 기체 사이의 계면에너지 보다 낮기 때문에 아래 식에 Biomaterials Research 2007 의해 가장 큰 공극이 가장 낮은 압력에서 먼저 기체로 교체되 어 유속이 발생되고, 이어 공극의 크기순으로 교체가 일어나 유량이 증가한다. 24) p/γ cos θ = ds/dv = 4/D 여기서 p는 시료양편의 압력구배, γ는 액체의 표면장력, S는 공극의 계면면적, V는 공극의 체적, D는 공극의 직경이다. 공극이 연결되어 이룬 모세관의 평균 단면을 원형으로 가정 하면, 이 식으로 압력에 따른 각 공극의 평균 직경을 구할 수 있으며, 해당 유량으로 공극의 양을 계산할 수 있다. Figure 3은 HAP 함량비 0.3인 시료에 있어서 압력에 대한 기체와 액체의 유속을 측정하여 나타내었다. 액체유속곡선(Wet) 에서 시작점(S)의 압력이 시료에 있어서 가장 큰 공극에 해당

생분해성 키토산 피브로인 하이드록시아파타이트 이종복합 지지체의 다공성 구조와 단백질 방출 특성 / Figure 2. - 99 Schematic diagram of capillary flow mechanism for measuring pore size and distribution. Variation of flow rate with pressure; gas flow curve (Dry), liquid flow curve (Wet), and half value curve of gas flow (Half Dry). S and P are first bubble point and intersection point respectively. Figure 3. 되며, 기체유속곡선(Dry)의 반가곡선(Half Dry)과 액체유속곡선 의 교점(P)의 압력이 시료 전체의 평균 공극크기에 해당된다. 이 실험 데이터에 의해 구한 공극의 직경과 해당 공극을 통 과하는 기체의 부피, 즉 공극의 누적 부피를 나타내는 누적여과 유출량(cumulative filter flow = Σ(100 * wet flow/dry flow)) 을 Figure 4에 나타내었으며, 이를 이용하여 아래 식에 의해 공극의 분포를 계산하여 Table 2에 나타내었다. Pore distribution current filter flow previous filter flow previous diameter current diameter = ---------------------------------------------------------------------------------------------- 이 실험에 의해 다공체는 실제 기질을 관통할 수 있는 공극 Figure 4. Variation of cumulative filter flow with average diameter. 의 최대 직경은 약 11.3 µm이며, 평균 직경은 약 3.4 µm이 었다. 그리고 공극의 분포는 11.3 µm의 최대 공극 부근과 0.3 µm의 최소 공극 부근에서 가장 크게 나타났으며, 평균 직 경의 표준편차는 3.75 이었다. 체액 흡수성 친수성의 생체고분자 재료는 체내에서 체액을 흡수하여 팽윤 된다. 이 팽윤성 역시 공극구조와 더불어 지지체의 단백질 투 과와 세포 생장에 영향을 미친다. 체내에서의 흡수성을 평가하 기 위하여 체액의 염 조성인 SBF 중에서 실험을 하 였다. Figure 5는 HAP 함량비에 따른 SBF의 흡수율을 나타 낸 것으로, 흡수율은 시료에 24시간 흡수된 SBF의 중량을 자 중에 대한 비율로 나타내었다. HAP 함량비 0.1~0.3 사이에서 in vitro Vol. 11, No. 3

100 Œ Á i~á rá l Table 2. Average diameters and their pore distribution Average diameter (m) 11.29 11.17 7.11 2.18 1.08 0.81 0.66 0.55 0.47 0.42 0.38 0.27 Pore distribution 176.9 78.6 2.1 12.0 10.8 16.3 41.2 14.7 36.9 38.4 58.3 298.5 Figure 5. The SBF absorptivity behavior according to the weight ratios of HAP in the hybrid scaffolds. Figure 6. Comparison of the amounts of FITC-BSA released for 24 hours from the hybrid scaffolds according to the weight ratios of HAP. fjf 10 f f f f, HAPf Š l Š f f h. f h f HAP h f x ff Š f l f f Š f f f. tg f f lf hšš, ef g h f el Š. f f f gf Š f f Œ~ } lltf f jdš d. f HAP Š llt f HAP Š f ihf fh e t f ihf Š. Figure 6f t ŒŠ lf f e fhš j (50 µg/g)f Še ~ SBF j 24 vš lf j f HAP Š i Š ~. lf Œ f rš Œ t (FITC-BSA)f dš f, HAP Š l Š BSAf v f } rf Š. f llt Š j f ~ Š HAPf } BSA r f l f f Š. Š HAP Š 0.7 ~ HAPf f l hf l Š } fl BSAf r lf v f l Š f. Figure 7f f Štf BSA v f wh Š ~. vf t 2 f f Figure 7. FITC-BSA release profiles from the hybrid scaffolds with HAP weight ratio of 0.2 and 0.5. f, f Š fš 24 f f fhš el. v HAP Š } h. HAP Š llt f HAP Š f ihf fh e Še lf vh Š Š. Biomaterials Research 2007

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