Availableonlineatwww.sciencedirect.com SCIENCE DIRECT R materials letters ELSEVIER Materials Letters 59(2005)69-7 www.elseviercomlocate/matlet Microstructure and osteoblast adhesion of continuously porous Al2O3 body fabricated by fibrous monolithic process In-Cheol Kang, Taek -Soo Kim, Kwang-Kjune Ko, Ho-Yeon Song Takashi Goto, Byong-Taek L 182 Shinkwan-dong, Kongju, Chungnam 314-701, Republ Department of Microbiology, School of Medicine, Soonchunhyang University, 366-1 Ssangyoung-don m, Chungnam 330-090, Republic of Korea Institute for Materials Research, Tohoku University, 2-1-1, Katahira, Aoba-ku, Sendai, Japa Received 22 March 2004: received in revised form 14 September 2004: accepted 18 September 2004 Available online 5 October 2004 Abstract e. Continuously porous AlO, body was fabricated by a fibrous monolithic process. It was relatively easy and convenient to control the rostructure as well as the shape and size of pores. With increasing the passes of process, the size of pores was reduced corresponding to the extrusion ratio. The resultant sizes of continuous pore formed in the second and third passed bars were 200 and 40 um in diameter, respectively. In order to investigate a biocompatibility of the porous Al2O, body, in vitro test was performed using a human osteoblast-like MG-63 cells. It was seen that the cells were well attached and grown on the Al2O3 porous body fabricated by fibrous monolithic process. C 2004 Elsevier B V. All rights reserved. Keywords: Continuously porous Al,O3; Fibrous monolithic process; Microstructure control; In vitro test 1. Introduction artificially and naturally made. The porous bioinert ceramic has also been known to form a thinner fibrous layer with It is known that Al2O3 is a typical bioinert ceramic which faster hilling in surrounding muscles and connective tissues as been widely used in total hip prosthesis and dental compared with the dense ones [5]. The bone in human body implants due to its good biocompatibility, high strength, and is made up of two types of structures as outer thin-walled excellent corrosion and wear resistance [1-3. Especially, compact bone and inner cancellous( spongy) bone. The polycrystalline a-Al2O3 having grain size less than 7 um, latter consists of a three-dimensional lattice of trabeculae, tensity higher than 3.90 g/cm, and purity higher than and the porous structures in the cancellous bone are 99.5% was required to use clinically by the International necessary for the formation of bone marrow and the growth Standard Organization (ISo)[4]. of bone. Suitable pore size was reported to be approximately The bioinert ceramic implants exhibited, however, a 100-150 [5], 140-160 [6], and 200-1000 um [7]. In fibrous encapsulation, which limits a chemical and mechan- addition, Al2O3 was reported to form the thinnest fiber ical combining between the implant and natural bone. Thus, layer along the surface compared with other bioinert research on the bioinert ceramics has been focused on the ceramIcs porous structure because the porous structure provides an It has been reported that there are several methods interlocking and then diminishes motion between the bones fabricating the porous Al2O3 such as sintering [9, 10], hot isostatic pressing [11], and microwave sintering[12].Those processes usually used a mixture of a pore-forming agent Tel: +82 41 850 8677: fax: +82 41 2939. such as carbon, polymer, etc. and the Al2O3 powder, and guac kr(B.-T. Lee). then removed the agent. Thus, those are relatively compli 0167-577X/S- see front matter o 2004 Elsevier B V. All rights reserved doi:10.1016/ malet.2004.09.019
Microstructure and osteoblast adhesion of continuously porous Al2O3 body fabricated by fibrous monolithic process In-Cheol Kanga , Taek-Soo Kima , Kwang-Kjune Kob , Ho-Yeon Songb , Takashi Gotoc , Byong-Taek Leea, * a Chungnam Research Center for Nano Materials and School of Advanced Materials Engineering, Kongju National University, 182 Shinkwan-dong, Kongju, Chungnam 314-701, Republic of Korea b Department of Microbiology, School of Medicine, Soonchunhyang University, 366-1 Ssangyoung-dong, Cheonan, Chungnam 330-090, Republic of Korea c Institute for Materials Research, Tohoku University, 2-1-1, Katahira, Aoba-ku, Sendai, Japan Received 22 March 2004; received in revised form 14 September 2004; accepted 18 September 2004 Available online 5 October 2004 Abstract Continuously porous Al2O3 body was fabricated by a fibrous monolithic process. It was relatively easy and convenient to control the microstructure as well as the shape and size of pores. With increasing the passes of process, the size of pores was reduced corresponding to the extrusion ratio. The resultant sizes of continuous pore formed in the second and third passed bars were 200 and 40 Am in diameter, respectively. In order to investigate a biocompatibility of the porous Al2O3 body, in vitro test was performed using a human osteoblast-like MG-63 cells. It was seen that the cells were well attached and grown on the Al2O3 porous body fabricated by fibrous monolithic process. D 2004 Elsevier B.V. All rights reserved. Keywords: Continuously porous Al2O3; Fibrous monolithic process; Microstructure control; In vitro test 1. Introduction It is known that Al2O3 is a typical bioinert ceramic which has been widely used in total hip prosthesis and dental implants due to its good biocompatibility, high strength, and excellent corrosion and wear resistance [1–3]. Especially, polycrystalline a-Al2O3 having grain size less than 7 Am, density higher than 3.90 g/cm3 , and purity higher than 99.5% was required to use clinically by the International Standard Organization (ISO) [4]. The bioinert ceramic implants exhibited, however, a fibrous encapsulation, which limits a chemical and mechanical combining between the implant and natural bone. Thus, research on the bioinert ceramics has been focused on the porous structure because the porous structure provides an interlocking and then diminishes motion between the bones artificially and naturally made. The porous bioinert ceramic has also been known to form a thinner fibrous layer with faster hilling in surrounding muscles and connective tissues compared with the dense ones [5]. The bone in human body is made up of two types of structures as outer thin-walled compact bone and inner cancellous (spongy) bone. The latter consists of a three-dimensional lattice of trabeculae, and the porous structures in the cancellous bone are necessary for the formation of bone marrow and the growth of bone. Suitable pore size was reported to be approximately 100–150 [5], 140–160 [6], and 200–1000 Am [7]. In addition, Al2O3 was reported to form the thinnest fiber layer along the surface compared with other bioinert ceramics [8]. It has been reported that there are several methods on fabricating the porous Al2O3 such as sintering [9,10], hot isostatic pressing [11], and microwave sintering [12]. Those processes usually used a mixture of a pore-forming agent, such as carbon, polymer, etc. and the Al2O3 powder, and then removed the agent. Thus, those are relatively compli- 0167-577X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2004.09.019 * Corresponding author. Tel.: +82 41 850 8677; fax: +82 41 858 2939. E-mail address: lbt@kongju.ac.kr (B.-T. Lee). Materials Letters 59 (2005) 69 – 73 www.elsevier.com/locate/matlet
I-C. Kang et al./ Materials Letters 59(2005)69-73 cated to fabricate the homogenous pore structure, and the cultured in DMEM( Gibco demented with 10% heat pore size is only dependent on the size of the pore-forming inactivated fetal bovine serum(FBS, Gibco),2 mM L- glutamine, penicillin 100 U/ml, streptomycine 100 ug/ml, To date, fibrous monolithic process was introduced to fungizone 0. 25 ug/ml(Bio-Whittaker)and were placed in an fabricate a toughened and fibrous monolithic ceramic body incubator containing 5% CO2 at 300 K. After confluence, and proved to be convenient for controlling the micro- the cells were detached with 0.05% trypsin and 0.02% structure [13-15]. It is defined as a repeated extrusion of EDTA (Sigma). A 100 ul of 5x 10" cells were seeded on the two or more ceramic powders combined by organic binders top surface of second and third passed Al2O3 porous bod according to the principle that the area of each extruded bar (2-mm diameter and 1-mm height)in 96-well plate. After 5 is homogeneously reduced with the extrusion ratio. How h, these porous bodies adhered cells were cultured in new ever, the research on the fabrication of porous Al2O3 body 24-well plates and were observed for 2 week has not yet been conducted by using the fibrous monolithic The microstructure and composition of the filaments was kamined using a Field Emission Scanning Electron Micro- In this work, the bioinert Al2O, body consisting of the scope(FE-SEM, JSM 6335F)and Energy Dispersed Spec ontinuous pores was fabricated using fibrous monolithic troscopy(EDS, Oxford 400), respectively. The structure of process, and porous microstructure was examined. Behavior the resultant phases was characterized by an X-ray diffrac- of osteoblast adhesion and growth using human osteoblast- tometer(Rigaku, AX-2500)using Cu Ko of 0. 1542 nm like MG-63 cells on the fibrous monolithic Al2O3 porous body was also examined 2. Experimental procedure Fig. 1 shows SEM micrographs of AlO3/EVA and carbon/EVA composites with passes of extrusion. The first eth Homogenous mixtures of commercially used Al203/ passed filament consists of the carbon rod of about 2.3 mm lylene vinyl acetate(EVA)and carbon/EVA were prepared in diameter and Al2O shell of about 0.6 mm in thickness using a heated blender (C.w. Brabender Instruments, (Fig. 1(a)). After the second extrusion, both the rod (dark PL2000 Plasti-Corder with Roller Blade Mixing Heads ). color) and shell (light color) become fine to be approx The average diameter of starting materials was about 0.3 um imately 250-300 and 100 um, respectively, as shown in Fig for Al2O3(AKP-50, Sumimoto, Japan)and -10-15 um for 1(b). The third passed bar showed a further refinement in the carbon powder(SMC, South Korea). The EVA, a type of microstructure, thus it is impossible to distinguish the polymer, used for the binder in the shape of granules(Elvax microstructural distribution under the same magnification 250, Dupont) was composed of ethylene vinyl acetate (Fig. 1(c). The highly magnified micrograph taken from P (EVA). Stearic acid(CH3(CH2)16COOH(Daejung Chem- region in Fig. 1(c)indicates that the third passed filament icals metals, South Korea) was also added for lubrication during blending. The ratio of each constituent in the mixture was 50/45/5 vol. for the Al,O3/EVA/lubricant and 55/40/5 vol. for the carbon powder/EVA/lubricant. 器冷 order to blend both the Al2O3 and pore materials with the EVA, the eva granules were first put into the mixing 你毒品 chamber and head which were heated to 403-433 K using an oil-type heating source. Then, the Al2O3 powder and the 已命 pore materials were slowly added to the chamber having the heated head revolving at 60 rpm. The two mixtures of Al2O3/EVA and carbon/EVA were, respectively, extruded at 383 K into a filament of 3.5 mm in diameter with an extrusion ratio of 73: 1. Then, both the Al,O3 and carbon filament were reloaded in the extrusion die as densely as possible followed by reextrusion. This process was con- tinued until the third passed filaments were obtained. Binder urning out(BBO)of the filaments was carried out at 973 K under flowing nitrogen and then 773 K under air followed 点 by sintering at 1723 K. Human osteoblast-like MG-63 cells obtained from Korean Cell Line Bank(KCLB) were used to investigate Fig. 1 SEM micrographs of fibrous monolithic(Al2O3-CyEVA composite the adherence and the morphological changes of cells on the filaments with passes of extrusion. a-first, b--second, c--third pass, and fibrous monolithic Al2O3 porous body. The cells were d-enlarged image of the third pass
cated to fabricate the homogenous pore structure, and the pore size is only dependent on the size of the pore-forming agent. To date, fibrous monolithic process was introduced to fabricate a toughened and fibrous monolithic ceramic body and proved to be convenient for controlling the microstructure [13–15]. It is defined as a repeated extrusion of two or more ceramic powders combined by organic binders according to the principle that the area of each extruded bar is homogeneously reduced with the extrusion ratio. However, the research on the fabrication of porous Al2O3 body has not yet been conducted by using the fibrous monolithic process. In this work, the bioinert Al2O3 body consisting of the continuous pores was fabricated using fibrous monolithic process, and porous microstructure was examined. Behavior of osteoblast adhesion and growth using human osteoblastlike MG-63 cells on the fibrous monolithic Al2O3 porous body was also examined. 2. Experimental procedure Homogenous mixtures of commercially used Al2O3/ ethylene vinyl acetate (EVA) and carbon/EVA were prepared using a heated blender (C.W. Brabender Instruments, PL2000 Plasti-Corder with Roller Blade Mixing Heads). The average diameter of starting materials was about 0.3 Am for Al2O3 (AKP-50, Sumimoto, Japan) and ~10–15 Am for carbon powder (SMC, South Korea). The EVA, a type of polymer, used for the binder in the shape of granules (Elvax 250, Dupont) was composed of ethylene vinyl acetate (EVA). Stearic acid (CH3(CH2)16COOH (Daejung Chemicals & Metals, South Korea) was also added for lubrication during blending. The ratio of each constituent in the mixture was 50/45/5 vol.% for the Al2O3/EVA/lubricant and 55/40/5 vol.% for the carbon powder/EVA/lubricant. In order to blend both the Al2O3 and pore materials with the EVA, the EVA granules were first put into the mixing chamber and head which were heated to 403–433 K using an oil-type heating source. Then, the Al2O3 powder and the pore materials were slowly added to the chamber having the heated head revolving at 60 rpm. The two mixtures of Al2O3/EVA and carbon/EVA were, respectively, extruded at 383 K into a filament of 3.5 mm in diameter with an extrusion ratio of 73:1. Then, both the Al2O3 and carbon filament were reloaded in the extrusion die as densely as possible followed by reextrusion. This process was continued until the third passed filaments were obtained. Binder burning out (BBO) of the filaments was carried out at 973 K under flowing nitrogen and then 773 K under air followed by sintering at 1723 K. Human osteoblast-like MG-63 cells obtained from Korean Cell Line Bank (KCLB) were used to investigate the adherence and the morphological changes of cells on the fibrous monolithic Al2O3 porous body. The cells were cultured in DMEM (Gibco) supplemented with 10% heat inactivated fetal bovine serum (FBS, Gibco), 2 mM lglutamine, penicillin 100 U/ml, streptomycine 100 Ag/ml, fungizone 0.25 Ag/ml (Bio-Whittaker) and were placed in an incubator containing 5% CO2 at 300 K. After confluence, the cells were detached with 0.05% trypsin and 0.02% EDTA (Sigma). A 100 Al of 5104 cells were seeded on the top surface of second and third passed Al2O3 porous body (2-mm diameter and 1-mm height) in 96-well plate. After 5 h, these porous bodies adhered cells were cultured in new 24-well plates and were observed for 2 weeks. The microstructure and composition of the filaments was examined using a Field Emission Scanning Electron Microscope (FE-SEM, JSM 6335F) and Energy Dispersed Spectroscopy (EDS, Oxford 400), respectively. The structure of the resultant phases was characterized by an X-ray diffractometer (Rigaku, AX-2500) using Cu Ka of 0.1542 nm. 3. Results Fig. 1 shows SEM micrographs of Al2O3/EVA and carbon/EVA composites with passes of extrusion. The first passed filament consists of the carbon rod of about 2.3 mm in diameter and Al2O3 shell of about 0.6 mm in thickness (Fig. 1(a)). After the second extrusion, both the rod (dark color) and shell (light color) become fine to be approximately 250–300 and 100 Am, respectively, as shown in Fig. 1(b). The third passed bar showed a further refinement in the microstructure, thus it is impossible to distinguish the microstructural distribution under the same magnification (Fig. 1(c)). The highly magnified micrograph taken from P region in Fig. 1(c) indicates that the third passed filament Fig. 1. SEM micrographs of fibrous monolithic (Al2O3-C)/EVA composite filaments with passes of extrusion. a—first, b—second, c—third pass, and d—enlarged image of the third pass. 70 I.-C. Kang et al. / Materials Letters 59 (2005) 69–73
L-C. Kang et al./ Materials Letters 59(2005)69-73 71 (b) Fig. 2. SEM micrographs of the porous Al2O, body after buming out and sintering processes. a-second pass, b-third pass, and arged image of the forms a uniform distribution of carbon in the Al,O3 matrix Fig. 4(a) shows the spindle-shaped human osteoblast (Fig. I(d)). Although the rod shape of carbon particle is not like MG-63 cells cultured on 6-well plate. The MG-63 observed due to sectioning problem, the diameter calculated cells were originated from a human osteosarcoma and have considering the extrusion ratio is approximately 50 um. To osteoblast characteristics. The osteoblasts are known as sum up, the Fig. I indicates that the ceramic microstructure bone-forming cells of developing and mature bone. The aving more than two phases can be easily controlled using top surface of second passed fibrous monolithic Al2O3 the fibrous monolithic process porous bodies were fully covered with osteoblasts at 14 Fig 2 shows SEM microstructure of porous Al2O3 body ys(Fig. 4(b). after burning out the EVA under N2 atmosphere followed by MG-63 cells were attached to top, inside, and bottom sintering in air. It is seen that the microstructure of sintered surface of Al2O3 porous body and grew well(Fig. 5). The body presents the same pattern with the one as extruded in cells grown on top surface of the porous body showed Fig. 1. The carbon/EVA (rod parts in Fig. 1) was fully highly condensed circular growth around the margin of the removed by the burning out process, and then the pores porous body(Fig. 5(a)). The high roughness formed along were formed. The pores produced in the second passed and inside the continuous pores [15] probably contributed to sintered body are about 200 um in diameter and ar promote the attachment, spreading, and growth of cells omogenously embedded in the Al2O3 matrix(Fig. 2(a)). Surface roughness dependence of the cells was well Similar to the as-extruded microstructure in Fig. 1(c), the reported by Boyan et al. [16]. The cells grown on bottom microstructure in the third passed and sintered body is surface of the porous body showed epithelial-like morpho- impossible to distinguish under the same low magnification, logic changes and grew faster than that on the top surface shown in Fig. 2(b). But the enlarged microstructure(Fig.(Fig. 5(c). The cells grew up through inside porous bodies 2(c))shows that approximately 40-um pores were formed in The pore size of third passed Al2O3 porous ody was the third filament. The reduction in the size after sintering however, too small to make the cells grow. Thus, it is not generally occurred In order to investigate a variation of composition or hase during the whole fibrous monolithic process, XRD characterization was conducted, as shown in Fig 3(a-f. It is not strange to detect Al2O3(Fig 3(a)) and carbon(Fig 3(b)) from the raw Al2O3 powder and carbon powder, f)ALOvC-sintered respectively. Fig 3(c)shows Al_O3/EVA mixture consists of ALO, C-2nd BI only Al2O3 due to the amorphous phase of EVA. However, Al2O3/EVA-carbon/EVA composite filament presents peaks for Al2O3 and carbon after the first binder burning out (Ist d) Al,O.C-Ist BBo BO), whereas the carbon was fully removed after the second binder burning out process(2nd BBO), as shown in Fig. 3(d) and (e), respectively. The binder burning out process is usually divided into two thermal steps. The first step(lst BBO)is for a transformation of EVa to carbon a)ALyO, powder under N2 atmosphere, while the second is to remove the carbons which were initially combined as a pore-forming agent as well as transformed from the eva during the first BO [15. The fibrous monolithic Al2O3/EVA-carbon/EVA composite finally is composed of only Al2O3 after sintering, Fig. 3. XRD profile of Al2O3-C composites: raw Al2O, powder(a),raw carbon powder(b), Al2O3/poly osite(c), AlO,/polymer compo- thus the porous Al O3 sintered body having the purity can site after first binder burning out(d), Al,/polymer composite after second be used as a bioinert implant binder burning out(e), and the sintered body (f)
forms a uniform distribution of carbon in the Al2O3 matrix (Fig. 1(d)). Although the rod shape of carbon particle is not observed due to sectioning problem, the diameter calculated considering the extrusion ratio is approximately 50 Am. To sum up, the Fig. 1 indicates that the ceramic microstructure having more than two phases can be easily controlled using the fibrous monolithic process. Fig. 2 shows SEM microstructure of porous Al2O3 body after burning out the EVA under N2 atmosphere followed by sintering in air. It is seen that the microstructure of sintered body presents the same pattern with the one as extruded in Fig. 1. The carbon/EVA (rod parts in Fig. 1) was fully removed by the burning out process, and then the pores were formed. The pores produced in the second passed and sintered body are about 200 Am in diameter and are homogenously embedded in the Al2O3 matrix (Fig. 2(a)). Similar to the as-extruded microstructure in Fig. 1(c), the microstructure in the third passed and sintered body is impossible to distinguish under the same low magnification, as shown in Fig. 2(b). But the enlarged microstructure (Fig. 2(c)) shows that approximately 40-Am pores were formed in the third filament. The reduction in the size after sintering generally occurred. In order to investigate a variation of composition or phase during the whole fibrous monolithic process, XRD characterization was conducted, as shown in Fig. 3(a)–(f). It is not strange to detect Al2O3 (Fig. 3(a)) and carbon (Fig. 3(b)) from the raw Al2O3 powder and carbon powder, respectively. Fig. 3(c) shows Al2O3/EVA mixture consists of only Al2O3 due to the amorphous phase of EVA. However, Al2O3/EVA–carbon/EVA composite filament presents peaks for Al2O3 and carbon after the first binder burning out (1st BBO), whereas the carbon was fully removed after the second binder burning out process (2nd BBO), as shown in Fig. 3(d) and (e), respectively. The binder burning out process is usually divided into two thermal steps. The first step (1st BBO) is for a transformation of EVA to carbon under N2 atmosphere, while the second is to remove the carbons which were initially combined as a pore-forming agent as well as transformed from the EVA during the first BBO [15]. The fibrous monolithic Al2O3/EVA–carbon/EVA composite finally is composed of only Al2O3 after sintering, thus the porous Al2O3 sintered body having the purity can be used as a bioinert implant. Fig. 4(a) shows the spindle-shaped human osteoblastlike MG-63 cells cultured on 6-well plate. The MG-63 cells were originated from a human osteosarcoma and have osteoblast characteristics. The osteoblasts are known as bone-forming cells of developing and mature bone. The top surface of second passed fibrous monolithic Al2O3 porous bodies were fully covered with osteoblasts at 14 days (Fig. 4(b)). MG-63 cells were attached to top, inside, and bottom surface of Al2O3 porous body and grew well (Fig. 5). The cells grown on top surface of the porous body showed highly condensed circular growth around the margin of the porous body (Fig. 5(a)). The high roughness formed along inside the continuous pores [15] probably contributed to promote the attachment, spreading, and growth of cells. Surface roughness dependence of the cells was well reported by Boyan et al. [16]. The cells grown on bottom surface of the porous body showed epithelial-like morphologic changes and grew faster than that on the top surface (Fig. 5(c)). The cells grew up through inside porous bodies. The pore size of third passed Al2O3 porous body was, however, too small to make the cells grow. Thus, it is not Fig. 2. SEM micrographs of the porous Al2O3 body after burning out and sintering processes. a—second pass, b—third pass, and c—enlarged image of the third pass. Fig. 3. XRD profile of Al2O3-C composites: raw Al2O3 powder (a), raw carbon powder (b), Al2O3/polymer composite (c), Al2O3/polymer composite after first binder burning out (d), Al2O3/polymer composite after second binder burning out (e), and the sintered body (f). I.-C. Kang et al. / Materials Letters 59 (2005) 69–73 71
I-C. Kang et al./ Materials Letters 59(2005)69-73 Fig. 4. The inverted microscopic finding of MG-63 cells cultured on 6-well plate(a)and second passed fibrous monolithic Al2O3 porous body for 14 days(b). suitable to explore the attachment and growth of osteoblasts Control of the grain size to nm regime is important for (not shown). the behavior of cell attachment because reducing the grain size induces an increase in hydrophilicity and surface reactivity of the bioceramics material. Webster et al 4. Discussion reported that cell contact angles were decreased as the grain size of Al2O3 increased, in which the angles were, The potential advantage offered by a porous Al2O3 respectively, 18.6, 10.8, and 6. 4 at the grain size of implant is the mechanical stability of the highly convoluted 177, 49, and 23 nm [17]. The decrease in the contact angle interface developed when bone grows into the pores of the corresponds to an increase in surface aqueous wettability ceramics. However, the porous ceramics should be com- Furthermore, the grain size refinement increases the surface plied with the mechanical property enough to sustain the roughness and surface area, resulting in an improvement of pore structures in shape and size as well as the load when cell attachment. Using the fibrous monolithic process, the implanted in the body. It is therefore important to control the size of grain could be reduced to about 150 nm after the grain size of matrix material because the mechanical forth pass and 21 nm after the fifth pass at this experimental property increases as the grain size decreases. The fibrous condition. Those sizes are also controllable by varying the monolithic process is an efficient process suitable for a area reduction ratio fabrication of continuously porous ceramic body with an Considering the average MG-63 cell size of 10 um, the easy control of the grain size as well as the pore size insufficient growth of the cells in the third passed body [14,15] having pores about 40 um makes it confusing to under ●e Fig. 5. The morphology of MG-63 cells proliferating on the top(a and b) and bottom (c and d) surfaces of the second passed Al,, porous body for 5 days(a
suitable to explore the attachment and growth of osteoblasts (not shown). 4. Discussion The potential advantage offered by a porous Al2O3 implant is the mechanical stability of the highly convoluted interface developed when bone grows into the pores of the ceramics. However, the porous ceramics should be complied with the mechanical property enough to sustain the pore structures in shape and size as well as the load when implanted in the body. It is therefore important to control the grain size of matrix material because the mechanical property increases as the grain size decreases. The fibrous monolithic process is an efficient process suitable for a fabrication of continuously porous ceramic body with an easy control of the grain size as well as the pore size [14,15]. Control of the grain size to nm regime is important for the behavior of cell attachment because reducing the grain size induces an increase in hyfrophilicity and surface reactivity of the bioceramics material. Webster et al. reported that cell contact angles were decreased as the grain size of Al2O3 increased, in which the angles were, respectively, 18.68, 10.88, and 6.48 at the grain size of 177, 49, and 23 nm [17]. The decrease in the contact angle corresponds to an increase in surface aqueous wettability. Furthermore, the grain size refinement increases the surface roughness and surface area, resulting in an improvement of cell attachment. Using the fibrous monolithic process, the size of grain could be reduced to about 150 nm after the forth pass and 21 nm after the fifth pass at this experimental condition. Those sizes are also controllable by varying the area reduction ratio. Considering the average MG-63 cell size of 10 Am, the insufficient growth of the cells in the third passed body having pores about 40 Am makes it confusing to underFig. 4. The inverted microscopic finding of MG-63 cells cultured on 6-well plate (a) and second passed fibrous monolithic Al2O3 porous body for 14 days (b). Fig. 5. The morphology of MG-63 cells proliferating on the top (a and b) and bottom (c and d) surfaces of the second passed Al2O3 porous body for 5 days (a and c) and 10 days (b and d). 72 I.-C. Kang et al. / Materials Letters 59 (2005) 69–73
L-C. Kang et al. Materials Letters 59(2005)69-73 stand the cell behavior. It can suggest that the pores for Acknowledgement biomedical use should have the size at least 10 times larger than that of cells. Johnson et al. indicated that the bone This work was supported by the 2002 NRL research exhibited pore sizes ranging from 50 to 700 um, which program of Korean Ministry of Science and Technology impasses the optimum pore size of 150 um required for optimum osseointegration [18]. Combination of the results that the MG063 cells were well grown on 200-um pores and the reports [5-7] indicate that the porous References ceramic has a size of over 100 um for a successful cell [1 w Li, L GaO, Biomaterials 24(2003)937 attachment [2]DR. Jordan, Online Clinical Commun. for Ophthalmologists (TM)- occojoumal. com, Forum. 31 2001(No page) []G. Willmann, H.J. Fruh, H.G. Pfaff, Biomaterials 17(1996)2157. 5. Conclusion 44A. Marti, Injury, Int J. Care Inj. 2000 S-D31(2000)33. 5]KA continuously porous Al2O3 sintered body was fab- [6]LL Hench, J Am Ceram Soc. 81(1998)1705 ricated by using fibrous monolithic process, and its micro- [7RT. Chiroff, E W. White, J N. Webber, D. Roy, J. Biomed. Mater. structural change was examined. Refinement of the Res.Symp.6(1975)29 microstructure in the Al2O3 frame and pore size can be [ 8]SB Cho, YJ. Kim, Ceramist 3(2000)5. [9]K Maca, P. Dobsak, A.R. Boccaccini, Ceram. Int 27(2001)577 controlled during the fibrous monolithic process. EVA and [10 B D. Flinn, R K. Bordia, A Zimmermann, J. Rodel, J. Eur. Ceram. carbon were successfully removed by the two step binder Soc.20(2000)2561 burning out processes. The second passed and sintered body [11] V Biasini, M. Parasporo, A Bellosi, Thin Solid Film 297(1997)207 consists of pores of about 200 um in diameter embedded [12]ST. Oh, K.I. Tajima, M. Ando, T. Ohji, Mater. Lett. 48(2001)215 he Al2O3 matrix, while approximately 40-um pores were [13]C Kaya, E.G. Butler, M.H. Lewis, J. Eur. Ceram. Soc. 23(2003)935 [14SY. Lienard, D. Kovar, R.J. Moon, K.J. Bowman, J.W. Halloran, J. formed in the third filament Mater.sci.35(20003365. MG-63 cells were attached to top, inside, and bottom [15] T.S. Kim, L.C. Kang, T Goto, B T Lee, Mater. Trans. 44(9) surface of Al2O3 porous body and grew well. The high roughness formed along inside the continuous pore might [16] B.D. Boyan, C.H. Lohmann, DD. Dean, V.L. Sylvia, D.L. Cochran, ontribute to promote the attachment, spreading, and growth [17 T.J. Webster, R W. Siegel, R. Bizios, Biomaterials 20(1999)121 of cells. However, the third passed body was not suitable to [18 G.S. Johnson, M.R. Mucalo, M.A. Lorier, J. Mater. Sci. 11(2000) culture the cells due to its too-small-sized pores
stand the cell behavior. It can suggest that the pores for biomedical use should have the size at least 10 times larger than that of cells. Johnson et al. indicated that the bone exhibited pore sizes ranging from 50 to 700 Am, which encompasses the optimum pore size of 150 Am required for optimum osseointegration [18]. Combination of the results that the MG063 cells were well grown on 200-Am pores and the reports [5–7] indicate that the porous ceramic has a size of over 100 Am for a successful cell attachment. 5. Conclusion A continuously porous Al2O3 sintered body was fabricated by using fibrous monolithic process, and its microstructural change was examined. Refinement of the microstructure in the Al2O3 frame and pore size can be controlled during the fibrous monolithic process. EVA and carbon were successfully removed by the two step binder burning out processes. The second passed and sintered body consists of pores of about 200 Am in diameter embedded in the Al2O3 matrix, while approximately 40-Am pores were formed in the third filament. MG-63 cells were attached to top, inside, and bottom surface of Al2O3 porous body and grew well. The high roughness formed along inside the continuous pore might contribute to promote the attachment, spreading, and growth of cells. However, the third passed body was not suitable to culture the cells due to its too-small-sized pores. Acknowledgement This work was supported by the 2002 NRL research program of Korean Ministry of Science and Technology. References [1] W. Li, L. GaO, Biomaterials 24 (2003) 937. [2] D.R. Jordan, Online Clinical Commun.for Ophthalmologists (TM)– www.occojournal.com, Forum. 31 2001 (No page). [3] G. Willmann, H.J. Fruh, H.G. Pfaff, Biomaterials 17 (1996) 2157. [4] A. Marti, Injury, Int. J. Care Inj. 2000 S-D31 (2000) 33. [5] K.A. Hing, S.M. Best, W. Bonfield, J. Mater. Sci., Mater. Med. 10 (1999) 135. [6] L.L. Hench, J. Am. Ceram. Soc. 81 (1998) 1705. [7] R.T. Chiroff, E.W. White, J.N. Webber, D. Roy, J. Biomed. Mater. Res. Symp. 6 (1975) 29. [8] S.B. Cho, Y.J. Kim, Ceramist 3 (2000) 5. [9] K. Maca, P. Dobsak, A.R. Boccaccini, Ceram. Int. 27 (2001) 577. [10] B.D. Flinn, R.K. Bordia, A. Zimmermann, J. Rodel, J. Eur. Ceram. Soc. 20 (2000) 2561. [11] V. Biasini, M. Parasporo, A. Bellosi, Thin Solid Film 297 (1997) 207. [12] S.T. Oh, K.I. Tajima, M. Ando, T. Ohji, Mater. Lett. 48 (2001) 215. [13] C. Kaya, E.G. Butler, M.H. Lewis, J. Eur. Ceram. Soc. 23 (2003) 935. [14] S.Y. Lienard, D. Kovar, R.J. Moon, K.J. Bowman, J.W. Halloran, J. Mater. Sci. 35 (2000) 3365. [15] T.S. Kim, I.C. Kang, T. Goto, B.T. Lee, Mater. Trans. 44 (9) (2003) 1851. [16] B.D. Boyan, C.H. Lohmann, D.D. Dean, V.L. Sylvia, D.L. Cochran, Z. Schwartz, Annu. Rev. Mater. Sci. 31 (2001) 357. [17] T.J. Webster, R.W. Siegel, R. Bizios, Biomaterials 20 (1999) 1221. [18] G.S. Johnson, M.R. Mucalo, M.A. Lorier, J. Mater. Sci. 11 (2000) 427. I.-C. Kang et al. / Materials Letters 59 (2005) 69–73 73