Biomaterials Research (2007) 11(2) : 70-74 Biomaterials Research 7 The Korean Society for Biomaterials HA/PCL w HA j»ƒ s e w Effects of HA Particle Sizes on Proliferation of Osteoblasts in Novel 3-dimensional HA/PCL Scaffolds x 1,2 Á½ 2 ÁWei Jie 1,3 Áx k 2 Á½ y 1 Á 2 Á 1 Áy 1 Á 1 * S. J. Heo 1,2, S. E. Kim 2, W. Jie 1,3, Y. T. Hyun 2, D. H. Kim 1, H. S. Yun 2, J. W. Shin 1, Y. M. Hwang 1, and Jung-Woog Shin 1 * 1fh Š f Š Š fd Š BK21, 2Š e, 3 l Team of BK21, Dept. of Biomedical Engineering, Inje University 2 Dept. of Future Technology, Korea Institute of Machinery and Materials 3 TaeSan Solutions Ltd. (Received April 7, 20007/Accepted May 11, 2007) (j) ~ h The aim of this study is to fabricate novel nano- (n), micro-(m) HA/PCL composite 3-D scaffolds with macropores and to compare the effects of two types of HA particles on the proliferation of osteoblasts in the 3-D scaffolds. A modified R.P. (rapid prototyping) process was employed to fabricate the 3-D composite scaffolds. Composite materials were prepared with nano- and micro- size of HA particles. The size of the synthesized nano-ha powders ranged from 10 to 30nm, while that of micro-ha powders ranged from 25 to 35 µm. To examine the potential of the case of scaffolds, the surface morphology of the scaffolds and cell proliferation were evaluated with SEM observations and MTT assay. The effect of HA particle size (nano-, micro-) on the attachment and proliferation of MG-63 in HA/PCL composite scaffolds was studied. The cell attachment and proliferation of n-ha/pcl composite scaffold were slightly better than those of the m-ha/pcl composite scaffold. From this study, we suggest that the n-ha/pcl composite scaffold may be a promising candidate for bone tissue engineering in the future. However, further studies are needed for osteoconductivity, differentiation of BMSC (bone marrow stromal cell) and bone tissue regeneration. Key words: Hydroxyapatite, Polycaprolactone, 3-D scaffold, Bone tissue engineering, Rapid prototyping ilf x g f eš il Š llt ilf v g x v t Š g dš Š. 1,2) f Š i l g d llt t h } l i f iš Š. tm f r, l, Œf Œ df j g hf Š, m llt hth f f l il Œ f eœ Š f f i hf Š, l f f Š f Œ f i Š. 3-10) Š ff PCL (poly ε-caprolactone)f Š f Š xe l llt f Šf v Š ff f h Š Š f ghf f. 11-15) Šl PCLf f lf f rf l f eœšl l *sf hf: sjw@bme.inje.ac.kr h f. f 16-18) HA (hydroxyapatite) f f e Š t Œ lf Š, hišf f Š hf Š fš f lš f, d ll tf hff eš g Š f f Œ f eš Š e f ŠŠ dš f. Š 19-15) g hf w f, j } f ~fƒ z f f h f Štf u t hf w } f HA hiš lltf g d Š lš f. 16-20) Š f llt hfš eš xv, 21), i l f h 22) 23) f, if Š hif g w f Š ff ƒ f 3rehf f h Š d hf f 24-25). f Š f u f fš { iœ (rapid prototype) f fdš il Šd llt hi f. 26-28), { iœ j friœ (Fused deposition modeling)f fdš hf fdš f g h 70
HA/PCL Šllt HA fff } i f l x 71 d Š HA/PCL Š llt hiš, HA f ff } f} f Š, lltf Œ~Šh i Š l f Š f Š. llt hf d PCL (Sigma Aldrich, Mw. 65,000, USA) micro-ha (Sigma-Aldrich, USA) d g fš dš f, nano-ha calcium nitrate (Ca(N0 3 ) 2 4H 2 O, Junsei Chemical Co., Ltd., Japan) ammonium phosphate ((NH 4 ) 2 HPO 4, Junsei Chemical Co., Ltd., Japan) f ff fdš Š Š. 10Ca(NO 3 ) 2 + 6(NH 4 ) 3 PO 4 + 8NH 4 OH Ca 10 (PO 4 ) 6 (OH) 2 + 20NH 4 NO 3 + 6H 2 O Š Š hf f. d P Caf 80 o C l v j Š. Ca d f P d s s jfš v j nano-ha Š f Š. f ammonium hydroxidef t Š ph=11 f f i f elš, 80 o C Š 24 f Š f v f l f Š. hf f f h eš l 3 f f s hf x z f fdš f f v h Š. HA/PCL w PCLf } (chloroform) 4 f Š lš f d f hiš j micro-ha nano-ha f 40wt% t Š ŒŠ f hiš. 3re llt hi eš ŒŠ } (chloroform)f t Š v jfš, f dš v f ŒŠ f d t vš 10 10 5 mm } f HA/PCL Š llt hfš. Figure 1f dš 3re llt hi gx f f. p Transmission electron microscopy (TEM, JEOL, JEM-2100F, Japan) fdš Š n-ha fff } Œ~ rš, h vj hf (FE-SEM, Hitachi Ltd, S- 4300SE, JAPAN)f fdš d micro-haf fff Œ~ } rš. d nano-ha micro-haf i hšh f eš X-ray diffraction (XRD, RIGAKU, JAPAN) f Š. Figure 1. Schematic of 3-D robotic system of fabrication scaffold. 3 sƒ lltf i : HA/PCL Šlltf f eš mercury intrusion porosimetry (Auto-pore IV 9500, Micro-meritics Instrument Corporation, USA) fdš (n=3). lltf f i Š eš h vj hf (FE-SEM, Hitachi Ltd, S-4300SE, JAPAN) f fdš rš. s sƒ hf lltf i l eš MG-63 (American Type Culture Collection, No. CRL-1427, USA)f 10 cells/scaffoldf 4 iš. DMEM (Dulbecco's Modified Eagle Medium, Gibco, USA) 10%f FBS (fetal bovine serum) 1% P/S (penicillin/streptomycin, Hyclone, USA)f t Š l dš. f l MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay (Cell Proliferation Kit I, Boehringer, Mannheim, Germany) 1f, 4f, 7f t Š f (ELISA, Synergy HT, Bio-Tek Instruments Inc., USA) fdš 595 nm g Š. l f ± j r Š f, f j (multiple comparison) eš SPSS 10.0 (Ver. 10.0, Standard Software Package Inc., USA)f fdš Fisher's LSD f s Š, ef jf p<0.05 h ef j Š. š Š nano-ha 10~30 nmf } ff f fl fl f ff Œ f g elš Š f f, micro-ha 25~35 µm } f l Œ f Š h f fff Š f (Figure 2). Figure 3f nano-ha micro-ha Š XRD Vol. 11, No. 2
허수진 김승언 Wei Jie 현용택 김동화 윤희숙 신지원 황영미 신정욱 72 Figure 2. (a) TEM image of n-ha particles with 10-30 nm in width and 90-100 nm in length (bar=100 nm), (b) SEM image of m-ha particles with 25-35 um in width and 50-80 um in length. Figure 4. Photographs of 3-D (a) n-ha/pcl, (b) m-ha/pcl scaffold. Figure 3. XRD spectra of (a) nano-ha, (b) micro-ha. XRD pattern of synthesized n-ha powder can be seen that there is a good match with the conventional m-ha powder both in intensity and position the peaks. 결과로서 nano-ha가 기존의 micro-ha와 주요 성분이 같은 저 결정성 HA라는 것을 알 수 있었다. Figure 4와 5는 nha/pcl, m-ha/pcl 복합재로 제조한 3차원 지지체의 외관과 내부 기공 구조를 보여주는 사진으로서 지지체 전체적으로 기 공의 크기가 일정하고, 기공의 형태와 기공 간 내부 연결성이 좋은 것을 볼 수 있다. 기공 크기는 x-y 방향으로 약 500 µm, z 방향으로 약 300 µm로 제작됨을 알 수 있다. 3차원 지지체에 있어서 내부 연결성은 매우 중요한데, 이는 골 세포 및 골 조직이 지지체의 내부까지 충분히 침투하고 성장할 수 있는 공간을 제공할 수 있기 때문이다. 본 연구팀은 일반적인 염침출법에 의한 지지체와 3 차원 조형 지지체의 비교 연구를 통하여 3 차원 지지체의 내부 연결 기공 구조의 장점을 확인 한 바 있다. Figure 6에서 보는바와 같이 m-ha/pcl 지지체의 경우 큰 HA 입자의 불규칙한 쌓임에 의해 표면의 형상이 불규칙하였 고, 이에 따라 지지체의 표면과 단면에서 20~30 µm 크기의 미세 기공이 형성된 것으로 보인다. 반면 n-ha/pcl 지지체의 29) Biomaterials Research 2007 Figure 5. SEM images of scaffolds (a) n-ha/pcl, (b) m-ha/pcl scaf- fold (bar=500µm). 경우 전체적으로 표면 형상이 부드러웠고, nano-ha가 PCL과 의 복합체에서 뭉쳐져 있지 않고 각각의 입자의 형태를 유지 하면서 표면에 노출되어 있음을 알 수 있었다. Mercury prosimetry로 기공도를 측정한 결과 n-ha/pcl 지지체의 평균 기공률은 72.297%, m-ha/pcl 지지체의 평균 기공률 73.525%였다. m-ha/pcl 지지체의 기공률이 약간 더 높은 이유는 자연적으로 형성된 20~30 µm 크기의 미세기공 때문
HA/PCL Šllt HA fff } i f l x 73 Figure 6. SEM images of pore, top and side view of n-ha/pcl scaffold (a) and m-ha/pcl scaffold (b). The pore size of top surface is about 500 um and cross-section is about 300 um (bar=500 um). Figure 7. MTT assay of MG-63 attached on n-ha/pcl and m-ha/ PCL scaffolds (n=5, p<0.05). The cell attachment and proliferation of n-ha/pcl composite scaffold were slightly better than those of the m- HA/PCL composite scaffold. f. 3 re llt MG-63 Š Figure 7 f f t r n-ha/pcl llt m-ha/pcl llt Š hf f, Šhf efš rf ~ (p<0.05). 7 f f u m-ha/pcl llt Š n-ha/pcl llt h hf MG-63 f l f Œ Š f f. f nano } f HA ff micro } f HA ff hf e } f t r e Š f. Webster 30,31), Li 32) fš nano } f g f d micro } lf i f rf e Š, Š d l } Œ fdf Š f l f. fs nano-ha Š llt t hf w f g h d Š, micro-ha Š llt f r l f tl ~ l f d ilf Œ ~ df j f v hf Š Šh ŠdŠ f. HA/PCL Šllt HA fff } i f l x Š d Š f. lltf Œ~ h nano-ha fdš 3re lltf d f r l f d Š g d llt f fd f ff f. BMSC (Bone marrow stromal cell) f fdš lltf f Œ f Š t hš f h Š f. Š e 2007 f lef f hf, f. š x 1. J. F. Mano, R. A. Sousa, L. F. Boesel et al., Bioinert, biodegradable and injectable polymeric matrix composites for hard tissue replacement: state of the are and recent developments, Compos. Sci. Technol., 64, 789-817 (2004). 2. H. Shin, S. Jo, and A.G. Mikos, Biomimetic materials for tissue engineering, Biomaterials, 24, 4353-4364 (2003). 3. B. S. Kim et al., Development of biocompatible synthetic extracellular matrices for tissue engineering, Trends Biotechnol., 16, 224-230 (2001). 4. K. F. Leong et al., Solid freeform fabrication of threedimensional scaffolds for engineering replacement tissues and organs, Biomaterials, 24, 2363-2378 (2003). 5. Wai-Yee Yeong, Chee-Kai Chua et al., Rapid prototyping in tissue engineering: challenges and potential, Trends Biotechnol., 22, 643-652 (2004). 6. K. H. Tan, C. K. Chua, K. F. Leong et al., Scaffold development using selective laser sintering of polyetheretherketonehydroxyapatite biocomposite blends, Biomaterials, 24, 3115-3123 (2003). 7. W. H. Dietmar, S. Thorsten, Z. Iwan et al., Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modeling, J. Biomed. Mater. Res., 55, 203-216 (2001). 8. R. C. Thomson, M. C. Wake et al., Biodegradable polymer scaffolds to regenerate organs, Adv. Polym. Sci., 122, 245-274 (1995). 9. L. Moroni, J. R. de Wijn, and C. A. Blitterswijk., 3D fiberdeposited scaffolds for tissue engineering: Influence of pores geometry and architecture on dynamic mechanical properties, Biomaterials, 27, 974-985 (2006). Vol. 11, No. 2
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