114-01(07-19).fm

Size: px

Start display at page:

Download "114-01(07-19).fm"

은하 뇌
5 years ago
Views:

1 Biomaterials Research (2007) 11(4) : Biomaterials Research 7 The Korean Society for Biomaterials y z e w pk t p w Study of Titanium Surface Characteristics Treated with Alkali after Anodic Oxidation ½ 1,2Á 1Á½ 3Á 4Á k 2Á 1,5* Myung Duk Kim 1,2, Ji Won Shin 1, In Ae Kim 1, Su A Park 3, Tae gwan Eom 2, and Jung-Woog Shin 1 1fh Š f Š Š fd Š, 2 f ƒ(j) f ƒ 3 d Š, x Š tg Š 4Š e 5fh Š, FIRST, f Še l Dept. of Biomedical Engineering, Inje University 2 Implant R&D Center of OSSTEM IMPLANT Co., Ltd. 3 Dept. of Dental Biomaterials Science and Dental Research Institute, School of Dentistry, Seoul National University 4 Dept. of Future Technology, Korea Institute of Machinery & Materials 5 FIRST Research Group, Institute of Biomedical Engineering, Inje University (Received Ocotber 5, 2007/Accepted November 20, 2007) The purpose of this study is to evaluate reactions of the MG-63 cells to the changes of the surface characteristics resulting from the alkali treatment on the anodic oxidized titanium (Grade 3) surface. For this study, the groups were classified into three as follows. 1) Group 1: blasted surface with hydroxyapatite powders whose diameters were ranged between 300~600 µm. 2) Group 2: anodic oxidized surface in electrolyte of 0.25 M H 2 SO 4 and H 3 PO 4 at 300V and 0.09 A/cm 2. 3) Group 3: treated surface in the same way as those in Group 2 followed by alkali treatment of 2 M NaOH for 24 hrs at 60 o C. The porous layers were observed in Group 2 while nano-sized radial type cilia structures were observed in Group 3 through the SEM. The measurements of surface roughnesses showed that Group 1 has higher average values of Ra (arithmetical mean deviation of the profile) than the other groups. The Contact angle was measured least in Group 3 (8.1 ± 1.3 o ). Reactions of MG-63 cells to each group were also evaluated for 4 hrs, 3, 7 and up to 10 days. The results of the DNA contents showed a significant increase of the cell proliferation for all groups with time, and the increases were observable in Group 3. ALP activities were decreased significantly in all groups with time, while the decrease was reduced after 7 days. Significantly more calcium were produced in Group 3 compared to other groups. In this study, alkali treatment of the anodic oxidized titanium surface resulted in the fine nano-sized radial type cilia structures on the porous, micro-sized oxide layer. From all the data obtained through this study, this nano-sized structure has a potential of promoting the osseointegration in dental implant. However, further studies with animals and histological evaluation about these results are recommended. Key words: Anodic oxidation, Alkali treatment, Nano-sized radial type cilia structures, Porous oxide layer, MG-63 cells e d f ƒf f eš f ƒf g, ƒ, ff, l,, f ƒf Š ~ f i Š 1). f j xi f f g lhh f 1rhf f x f f ƒf g ƒ f. x d f ƒf g thš h, hšf d Š ~ (Ti)f j d f. ~ f f ƒ h f j v (O 2 ) fš ƒ (Å) f ~ Œ (TiO 2 ) *sf hf: sjw@bme.inje.ac.kr f Œ Š 1,2) f t Œ f lf thš f d Š g h f. f ƒf f f hf er er f v ~ er f ~ eš jdš Šf Š fff. hf erf eš Š s f f. t ~ HA f t Œ lf z Š x Š f h t hf f Š lš. Wennerberg 3,4) f f i ihš x l ~ f f ƒ f f h Š ~ Š f l x x er hf f Š 4). ~ f Šf ~ eš d f ~ f t 170

2 Œ x s Š ~ ƒ Š 171 Œ f jdš eff f h ~ Œ Š lš 5,6). j Œ f g f d Š Œ f Œ i, ihš f hhf f f ƒ ff er f l ~ f Š 7,8). u Œ Šh s fdš Œ ƒ (NaOH) d xh ~ s Š (Mg 2+ ) f (F) xh z f ƒ f ŒŠh ff l ~ f Š f lš f. Sul 9) f Œ f Š z f ff Š, Ellingsen 10) Cooper 11) f Œ ~ f ƒ f f x f s Š f f f e Š Š. Š Kim 12,13) f ~ f Œ ƒ d xh ~ s Š f f SBF (Simulated Body Fluid) xh z Œf f Œ z. Šl r f s h f lš h f hdš eš g f h Š v Š f h Š. s hf Œ Š ef f Š ŠdŠ. Œ ~ f Œ ƒ d xh z f ƒ Œ f f x f Œs x l ~ f Š f Š. x d f ƒ d ~ (ASTM Ti Grade 3)f l 12 mm, 1 mmf } Œ~ Š f 3 l f s Š. Group 1. Resorbable Blasting Media(RBM) ~ 300~600 µm l f Œf (HM2002) f 4 MPaf f Š x Œf f h Š eš 15%f l d f sf Š. Group 2. Anodic oxidation DC hhe gx (DV156-51B, Dong-yang Electronics, Korea)f f Š, f f Š. hšlf l 0.25 Mf Œ (H 2 SO 4 ) 0.25 Mf f (H 3 PO 4 ) d f ŒŠŠ dš. Œ f h 0.09 A/cm 2 hš h f 300 V l z 20 elš. f hšlf 10~20 o C el z. Figure 1f ~ f Œ eš f. Figure 1. Schematic drawing of the anodizing apparatus. Group 3. Alkali treatment after anodic oxidation Group 2 f f Œ ~ f 2 Mf Œ ƒ d 24 xh z. f xh 60 o C elš. t p Œ f j hf (Field Emission-Scanning Electron Microscopy, FE-SEM, S-4300SE, HITACHI, Japan)f fdš 2k, 5k, 30kf e rš. x ht i wh (FTSS S5, Taylor Hobson Ltd, UK) fdš whš. wh f 2 mm, y f mm whš d Š f dš Ra f. f x f ht wh (OCA15+, Dataphysics Int, Germany) f ŒfŠ f l ht f whš. whf fff f ~Š i, df ht f whš. t s sƒ f Š i f ff Š eš MG-63 (human osteoblast-like cell, Korean Cell Line Bank, Korea)f 1 10 cells/discf 5 iš. 10% f FBS (Fetal Bovine Serum, Hyclone, USA) antibiotics Š DMEM-LG (Dulbecco's Modified Eagle's Medium- Low Glucose, Gibco BRL, USA) f Œ e Š eš Mf dexamethasone (Sigma) 50 µg/mlf ascorbic acid (Sigma) 10 mmf β-glycerophosphate (Sigma) t Š dš. r f l Œ Š ff Š eš 6 f f dš 4, 3f, 7f, 10f f DNA f w hš ALP (Alkaline Phosphatase) Œ x f wh Š. Vol. 11, No. 4

3 172 김명덕 신지원 김인애 박수아 엄태관 신정욱 DNA contents 세포의 증식은 DNA 정량 키트 (PicoGreen dsdna Quantitation Kit, Molecular Probes, USA)를 이용하여 측정하였 다. 이를 위해 PicoGreen dye를 세포 추출액에 넣고, 빛을 차단시킨 상태에서 5분 동안 실온에서 반응시킨 후 흡광도 측 정기 (Synergy HT Multi-Detection Microplate Reader, BioTek Instruments, Inc., USA)를 이용하여 480 nm~520 nm 파장에서 형광도를 측정하였고 표준용액은 λdna (Molecular Probes)를 사용하였다. ALP activity 골아세포로의 분화를 평가하기 위해 ALP 정량 키트 (104사용하여 DNA 측정시 세포 추출액을 녹 여내는 방법과 같이 시행하였다. 이를 세포 추출액과 p-np standard 용액과 ALP mixture 용액을 교반하여 37 C에서 30 분간 반응시킨 후, 1 N NaOH로 반응을 중단시켰다. 유출액 내의 ALP의 활성도를 계산하기 위해 흡광도 측정기를 이용하 여 405 nm에서 흡광도를 측정하였다. 측정된 값은 DNA 값 으로 나누어 정량화하였다. LL, Sigma, USA)를 o Calcium assay 각 시편에 1 M의 HCl을 넣고 24시간 동안 rotatory shaker 에서 교반한 뒤 Calcium assay kit (Diagnostic Chemicals Limit, USA)를 추출액의 100배로 넣고 다시 교반한 후 650 nm의 파장으로 흡광도를 측정하였다. 측정된 값은 DNA 값으 로 나누어 정량화하였다. 통계분석(Statistical analysis) 표면의 상태에 따른 세포의 증식 및 분화 정도에 대한 결과 는 통계분석을 실시였다 (p < 0.05) 데이터의 통계적 신뢰성 확보를 위해 범용 통계 프로그램인 SPSS (Ver.10.0, Standard Package Inc., USA)를 이용하여 일원 분산 분석 (ANOVA)을 실시하였고, 다중 분산 비교는 LSD 방법을 이용하였다. 결 과 표면 특성 분석 Figure 2는 각 군의 형상을 SEM으로 관찰한 결과이다. 수산 화인회석 분말을 고압에서 표면에 분사시켜 만든 1군은 표면 에 불규칙한 거칠기가 형성되었다. 양극산화 처리한 2군은 2~4 µm 크기의 3차원 기공이 생성되었다. 마지막으로 양극산 화 된 2군의 표면을 알칼리 용액에 침적시킨 3군은 양극산화 에 의한 다공성 구조가 유지되면서 표면 전체에 200~500 nm 간격으로 미세한 섬모형상의 돌기 구조가 동반되는 것을 Figure 2. SEM morphology on the surfaces: Group 1. RBM, Group 2. anodic oxidation, Group 3. alkali treatment after anodic oxidation. Biomaterials Research 2007

Œ x s Š ~ ƒ Š 173 Figure 3. Surface roughness: Group 1. RBM, Group 2. anodic oxidation, Group 3. alkali treatment after anodic oxidation (n=6, p<0.05). rš f. f x Figure 3 f j Raf } 1 (Ra=1.42 ± 0.

ht Š f 1 f 71.1 ± 4.5 o, 2 f 38.1 ± 7.6 o, 3 f 8.1 ± 1.3 o f e f f rf. t s DNA contents f l h Figure 5 f f l l Š 10fm 2~2.5 l l. 3 f d 3f m 1 efš, 3, 7, 10f Šhf efš rf f xhf ~.

Figure 6. Normalized ALP activity of MG-63 cells on the surfaces: Group 1. RBM, Group 2. anodic oxidation, Group 3. alkali treatment after anodic oxidation (n=6, p<0.05). ŒŠ Figure 6 f t 4 Š hf efš h.

4 Œ x s Š ~ ƒ Š 173 Figure 3. Surface roughness: Group 1. RBM, Group 2. anodic oxidation, Group 3. alkali treatment after anodic oxidation (n=6, p<0.05). rš f. f x Figure 3 f j Raf } 1 (Ra=1.42 ± 0.11 µm, Rz=4.30 ± 0.27 µm), 2 (Ra=0.96 ± 0.06 µm, Rz=4.06 ± 0.14 µm), 3 (Ra=0.79 ± 0.07 µm, Rz=3.39 ± 0.21 µm)f f ef f rf ~. 3 f d Œ fš i Š Œ f i Œ Š 2 Š Ra f 18% ~. Šl f x Figure 4 f ht f g f ~. ht Š f 1 f 71.1 ± 4.5 o, 2 f 38.1 ± 7.6 o, 3 f 8.1 ± 1.3 o f e f f rf. t s DNA contents f l h Figure 5 f f l l Š 10fm 2~2.5 l l. 3 f d 3f m 1 efš, 3, 7, 10f Šhf efš rf f xhf ~. ALP activity t f Œ s f ALPf Œ DNA h Figure 5. DNA contents of MG-63 cells on the surfaces: Group 1. RBM, Group 2. anodic oxidation, Group 3. alkali treatment after anodic oxidation (n=6, p<0.05). Figure 6. Normalized ALP activity of MG-63 cells on the surfaces: Group 1. RBM, Group 2. anodic oxidation, Group 3. alkali treatment after anodic oxidation (n=6, p<0.05). ŒŠ Figure 6 f t 4 Š hf efš h. Š f e f f Š 3 f 3f 10f 1 2 efš. Figure 4. Contact angle on the surfaces: Group 1. RBM, Group 2. anodic oxidation, Group 3. alkali treatment after anodic oxidation (n=6, p<0.05). Figure 7. Normalized calcium depositions of MG-63 cells on the surfaces: Group 1. RBM, Group 2. anodic oxidation, Group 3. alkali treatment after anodic oxidation (n=6, p<0.05). Vol. 11, No. 4

5 174 Á leá f Á Á ~ Á hd Calcium assay f Œ s f x f f Š DNA f h ŒŠ Figure 7 f 1 2 f t 3 f 7f 10f efš Š. Šl 3 f d 7fm x f f l Š 10fm Š f, hthf Š 1.5 f f x f wh. š x d f ƒ er f vš f hf eš jdš hf. er f f ƒf ƒ hš f. f hf x d f ƒf ƒ f Œ z er f v ~ f f Š f Š. f e Š ~ f ŒŠ x d xh z f } f Œ i Œ ~ f Œ x f f f. f f Œ f x, i, f x f f r l f j Š 14~18). Œ x s Š f SEMf r Š Œ fš f} } f 200~500 nm f Š Œ i Œ } f Œ fš ih fhf l 19). f x Wennerberg 4) f Š x f f ƒ f ƒ h f Š x 1.11 µm 2.01 µm 1.45 µmf i f efš Š. f f ŒŠ x f ff il f Š e Š, hhš x igš f f 3,4,20). Š Anil 19) f Œ ƒ s Š polymer } f x e f f f Š f} e sub- f} ef x l f l e Š. f x f g x, ff Œ Œ x s Š f. Šl f l x f Œ x s Š f g e Š f f x Ra 0.79~1.42 µm j x fš f rf f l fš f } f f. l Œ x s Š f } f Š Œ f hf l ~f ƒ f f ƒf Š el f f df f. f x f ht f Œf Š Œ f ht f 38.1 ± 7.6 o f Š x d xh ~ f 8.1 ± 1.3 o f x f fff f. Šl f x f f f h hf e Š fd Š Š v ŠdŠ. ƒ f ff ŒfŠ eš l f f Š DNA f ŒfŠ h ef f f Œ x s Š f xhf f Š f Œf s x Š 1.5 f f h ef f ~. f Œ l ALPf Œ 3f, 10f Š f f l ALP Œ f l f hf 10fm l DNA f l Š l f DNA f h ŒŠ ALP Œ Š f 14). 10f f f g rf Š DNA l Œ f Œ d Š h ALPf f l f f. Š f ƒ MG-63 f Œƒ f BMSCs (Bone Marrow Stromal Cells) f stem cell Š eff ev. f l f Œ Œ h hf f l h l BMSCs f Š Œ hf 21,22) ƒ ŠdŠ. Œ x s Š f Œ Š Œ f i Œ Š Œ MG-63 f l x efš ff f ŒfŠ. f Š ef f Œ fš Œ f } } l f x Œ f x d xh z Œ ef Š Œ f f x f e Š fd f. h f} } f Œ } f Š Œ i l f f ƒ ff hf Š f x fš Œ Šhf Šf f f d i Š f h Š. Šl Šhf ƒ j er x ff Š f v hf Š dš. Š g hf h f f h ƒ in vivo f Š e Šd Š f. Œ fš ~ Œ f} } f Œ f x d xh z } f Š Œ f i Œ Š f. x s Œ f Œ x Ra f 18%, f x f ht f 4.7 Š. f Š, ŒŠh ƒ f Œ MG-63 f f f x. f l f 3fm f x Œ 1 ef f f Š f 7f, 10f h ef f f ~. Š Œ ~ x f 3f, 7f, 10f ef f ~. f Š x f h ƒ f f x Biomaterials Research 2007

6 Œ x s Š ~ ƒ Š 175 f ŒŠh ƒ fš f fff j f. Šl ALPf Œ t 4 hr f, 3f, 10f hf efš h. f i Œ x s fš ƒ Œ f l f x Œ e Š f ALP Œ Šl Š f ~. š x 1. D. M. Brunette, P. Tengvall, M. Textor et al., Titanium in medicine, 1th ed., New York, Springer, 1-13 (2001). 2.X. Liu, P. K. Chu, and C. Ding, Surface modification of titanium, titanium alloys, and related materials for biomedical applications, Mater. Sci. & Eng., R47, (2004). 3. A. Wennerberg, A. Ektessabi, T. Albrektsson et al., A 1-year follow-up of implants of differing surface roughness placed in rabbit bone, Int J Oral Maxillofac Implants, 12, (1997). 4. A. Wennerberg, The importance of surface roughness for implant incorporation, Int J Mach Tool Manufact, 38, (1998). 5. J. C. Keller, C. M. Wightman, and R. A. Zaharias, Characterization of titanium implant surfaces III, J. Biomed. Mater. Res., 28, (1994). 6. C. Larsson, P. Thomsen, J. Lausmaa et al., Bone response to surface modified titanium implants: studies on electropolished implants with different oxide thicknesses and morphology, Biomaterials, 15, (1994). 7. Y. T. Sul, C. B. Johansson, Y. Jeong et al., Oxidized implants and their influence on the bone response, J. Mater. Sci. Mater. Med., 12, (2001). 8. Y. T. Sul, C. B. Johansson, Y. Jeong et al., Resonance frequency and removal torque analysis of implants with turned and anodized surface oxides, Clin. Oral. Impl. Res., 13, (2002). 9. Y. T. Sul, C. B. Johansson, A. Wennerberg et al., Optimum surface properties of oxidized implants for reinforcement of osseointegration: surface chemistry, oxide Thickness, Porosity, Roughness, and Crystal Structure, Int. J. Oral. Maxillofac. Implants., 20, (2005). 10. J. E. Ellingsen, C. B. Johansson, A. Wennerberg et al., Improved retention and bone-to-implant contact with fluoride-modified titanium implants, Int. J. Oral. Maxillofac. Implants, 19, (2004). 11. L. F. Cooper, Y. Zhou, J. Takebe et al., Fluoride modification effects on osteoblast behavior and bone formation at TiO 2 gritblasted c.p. titanium endosseous implants, Biomaterials, 27, (2006). 12. H. M. Kim, F. Miyaji, T. Kokubo et al., Preparation of bioactive Ti and its alloys via simple chemical surface treatment, J. Biomed. Mater. Res., 32, (1996). 13. H. M. Kim, T. Himeno, M. Kawashita et al., Surface potential change in bioactive titanium metal during the process of apatite formation in simulated body fluid, J. Biomed. Mater. Res., 67A, (2003). 14. L. H. Li, Y.M. Kong, H. W. Kim et al., Improved biological performance of Ti implants due to surface modification by micro-arc oxidation, Biomaterials, 25, (2004). 15. X. Zhu, J. Chen, L. Scheideler et al., Effects of topography and composition of titanium surface oxides osteoblast responses, Biomaterials, 25, (2004). 16. J. Lincks, B. D. Boyan, C. R. Blanchard et al., Response of MG- 63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition, Biomaterials, 19, (1998). 17. K. Anselme, M. Bigerelle, B. Noel et al., Qualitative and quantitative study of human osteoblast adhesion on materials with various surface roughnesses, J Biomed Mater Res, 49, (2000). 18. F. Rupp, L. Scheideler, D. Rehbein et al., Roughness induced dynamic changes of wettability of acid etched titanium implant modifications, Biomaterials, 25, (2004). 19. T. Anil, C. Derick, Miller et al., Nano-structured polymers enhance bladder smooth muscle cell function, Biomaterials, 24, (2003). 20. K. Suzuki, K. Aoki, K. Ohya, Effects of surface roughness of titanium implants on bone remodeling activity of femur in rabbits, Bone, 21, (1997). 21. D. D. Deligianni, N. D. Katsala, P. G. Koutsoukos et al., Effect of surface roughness of hydroxyapatite on human bone marrow cell adhesion, proliferation, differentiation and detachment strength, Biomaterials, 22, (2001). 22. K. Nishio, M. Neo, H. Akiyama, et al., The effect of alkali- and heat-treated titanium and apatite-formed titanium on osteoblastic differentiation of bone marrow cells, J. Biomed. Mater. Res., 52, (2000). Vol. 11, No. 4

歯1.PDF

歯1.PDF 200176 .,.,.,. 5... 1/2. /. / 2. . 293.33 (54.32%), 65.54(12.13%), / 53.80(9.96%), 25.60(4.74%), 5.22(0.97%). / 3 S (1997)14.59% (1971) 10%, (1977).5%~11.5%, (1986)