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Microwave sintering and in vitro study of defect-free stable porous multilayered HAp–ZrO2 artificial bone scaffold

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Microwave sintering and in vitro study of defect-free stable porous multilayered HAp–ZrO2 artificial bone scaffold
  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/254501288 Microwave sintering and in vitro study of defect-free stable porous multilayered HAp–ZrO2 artificial bone scaffold  Article   in  Science and Technology of Advanced Materials · June 2012 DOI: 10.1088/1468-6996/13/3/035009 CITATIONS 3 READS 66 4 authors , including:Thi Hiep NguyenVietnam National University, Ho Chi Minh City 36   PUBLICATIONS   254   CITATIONS   SEE PROFILE Stevan Lee 150   PUBLICATIONS   1,658   CITATIONS   SEE PROFILE All content following this page was uploaded by Thi Hiep Nguyen on 30 November 2016.The user has requested enhancement of the downloaded file.  Microwave sintering and in vitro   study of defect-free stable porous multilayered HAp–ZrO2artificial bone scaffold This content has been downloaded from IOPscience. Please scroll down to see the full text.Download details:IP Address: content was downloaded on 07/10/2013 at 05:03Please note that terms and conditions apply.2012 Sci. Technol. Adv. Mater. 13 035009(http://iopscience.iop.org/1468-6996/13/3/035009)View the table of contents for this issue, or go to the  journal homepage for more HomeSearchCollectionsJournalsAboutContact usMy IOPscience  IOP P UBLISHING  S CIENCE AND T ECHNOLOGY OF A DVANCED M ATERIALS Sci. Technol. Adv. Mater.  13  (2012) 035009 (9pp) doi:10.1088/1468-6996/13/3/035009 Microwave sintering and  in vitro  studyof defect-free stable porous multilayeredHAp–ZrO 2  artificial bone scaffold Dong-Woo Jang, Thi-Hiep Nguyen, Swapan Kumar Sarkarand Byong-Taek Lee Department of Biomedical Engineering and Materials, School of Medicine, Soonchunhyang University,Cheonan 330-090, KoreaE-mail: lbt@sch.ac.kr Received 18 March 2012Accepted for publication 15 May 2012Published 16 July 2012Online at stacks.iop.org/STAM/13/035009 Abstract Continuously porous hydroxyapatite  ( HAp )/ t-ZrO 2  composites containing concentriclaminated frames and microchanneled bodies were fabricated by an extrusion process. Toinvestigate the mechanical properties of HAp / t-ZrO 2  composites, the porous composites weresintered at different temperatures using a microwave furnace. The microstructure wasdesigned to imitate that of natural bone, particularly small bone, with both cortical and spongybone sections. Each microchannel was separated by alternating lamina of HAp, HAp–(t-ZrO 2 )and t-ZrO 2 .  HAp and ZrO 2  phases existed on the surface of the microchannel and the corezone to increase the biocompatibility and mechanical properties of the HAp-ZrO 2  artificialbone. The sintering behavior was evaluated and the optimum sintering temperature was foundto be 1400 ◦ C, which produced a stable scaffold. The material characteristics, such as themicrostructure, crystal structure and compressive strength, were evaluated in detail fordifferent sintering temperatures. A detailed  in vitro  study was carried out using MTT assay,western blot analysis, gene expression by polymerase chain reaction and laser confocal imageanalysis of cell proliferation. The results confirmed that HAp-ZrO 2  performs as an artificialbone, showing excellent cell growth, attachment and proliferation behavior usingosteoblast-like MG63 cells. Keywords:  bioceramic, hydroxyapatite (HAp), extrusion, cortical bone, osteoblast cell 1. Introduction Autografting, allografting and artificial grafting have beenused to treat and replace bone defects in orthopedic surgery.However, both autografting and allografting methods havelimited availability and present pathogen transmission risksfrom the donor to the recipient. As a consequence, artificialgrafting is being increasingly investigated as a means of providing regeneration scaffolds for bone replacement. Theaim of grafting is to aid in replacing the missing or damagedtissue and to reinforce the treated area by inspiring new boneingrowth. This new bone should ideally replace the bone graftthrough repeated remodeling cycles, enabling repair of thedefect site to maintain an optimal balance between form andfunction. Nowadays, different types of synthetic material suchas polymers, ceramics and glasses are being used in osseousdefect sites as bone substitutes [1–4]. Calcium phosphate ceramics such as hydroxyapatite (HAp, Ca 10 ( PO 4 ) 6 ( OH ) 2 )or tricalcium phosphate ( α -TCP,  β -TCP, Ca 3 ( PO 4 ) 2 ) arewidely used in clinical practice as bone fillers owing to theirfavorable chemical composition, excellent biocompatibilityand bioactivity [5–7]. Recently, the development of porous HAp and other related calcium phosphates for use inbone treatment applications has become an increasinglyimportant research subject for many medical and materialsscience fields [8–10]. It has been reported that the use of porous HAp/TCP  in vivo  promotes bone ingrowth andosteoconduction [11–13]. The chemistry of the bioactive 1468-6996/12/035009+09$33.00  1  © 2012 National Institute for Materials Science Printed in the UK  Sci. Technol. Adv. Mater.  13  (2012) 035009 D-W Jang  et al bioceramic materials that are used to repair the defect regionis also important, as it is responsible for supporting thedirect bonding of bone to its surface, greatly enhancingits performance over materials that are only bioinert orbiocompatible [14]. Recently, studies on using biomimetic modeling to fabricate artificial bone have been carried out.The distinctive characteristics of hard tissues in natural bonearetheHaversianlamellae,whichcomprisecolumnarosteons,the distribution of osteons into the shape of concentric circlesabout a central axis, and the connections between the osteons.The morphology of the natural bone structure often providesan optimum compromise between low weight and mechanicalstrength. Natural bone consists of a porous composite with aninteresting structure that exhibits a density gradient as wellas anisotropic properties. In particular, pores with a particulardimension and morphology promote bone ingrowth andosteoconduction. Therefore, the porosity gradient, mimickingthe bimodal structure of bone (cortical and cancellous),and a sufficient degree of interconnectivity are the mostimportant criteria for developing materials suitable for clinicalapplications [15]. However, these properties also weaken the scaffold. Therefore, HAp has been reinforced by ZrO 2 ,Al 2 O 3  and other ceramic oxides [16] to make sintered composites. A layered microstructure can be particularlyimportant for improving the mechanical properties; however,it has some inherent problems. During the cooling periodof the sintering schedule, thermal expansion mismatch cancause residual stress that can exceed a critical limit andresults in cracks, delamination or even catastrophic failureof the material. This can be countered by a gradient layerwith intermediate thermal expansion properties. Extrusionprocesses have been extensively used to make unidirectionalporous bodies [17, 18]. However, the technique of making channeled pores and designing a preform with bimodalporosity paved the way for the fabrication of large segmentsto replace the damaged natural bone. This artificial bonereplacement preform is composed of a central hollow tohouse the bone marrow and an outer shell with channeledporosity, which can be modified into cortical bone afterosteointegration. We fabricated a microarchitectural bonerepair unit with an aim to replace a section of the longbone using ZrO 2  and TCP, thereby combining the strengthof ZrO 2  and the biocompatibility of TCP. However, themicrostructure control was very complicated for this design.The conventional sintering process produced many cracksand the preform was mechanically unstable. Moreover, atthe high sintering temperature of 1450 ◦ C for ZrO 2 , thereis an unwanted side reaction that may detrimentally affectthe biocompatibility. Therefore, in this work, we employedmicrowave sintering to make the sintered preform, andoptimized the densification of the fabricated body withoutsignificantly dispensing the srcinal constituent phases and amechanically stable preform. We also improved the preformby using optimal sintering conditions and conducting a moredetailed investigation into the biocompatibility. These resultsshow potential for fabricating a mechanically stable artificialbone preform imitating natural bone.In this study, we attempted to fabricate the desiredshape of the artificial bone, similar to that of the naturalbone, by an extrusion process and by a subsequent binderburnout and microwave sintering. A gradient layer of HAp-HAp-(t-ZrO 2 )-t-ZrO 2  was the key to subduing theeffect of thermal expansion mismatch. Microwave sinteringwas employed to achieve quick densification withoutsignificant grain growth, reducing the possibility of chemicalreaction among the layers. Compared with a conventionalsintering process, the microwave sintering resulted in highermechanical stability and morphological integrity. Materialproperties and the morphological aspects were investigatedin detail in terms of varying sintering temperature andwere analyzed using x-ray diffraction (XRD) and scanningelectron microscopy (SEM). The optimum scaffold wasfurther investigated for its biocompatibility  in vitro . 2. Experimental procedure 2.1. Extrusion process for fabricating porous composites2.1.1. Materials.  t-ZrO 2  was purchased from Tosoh Japan(TZ-3Y, particle size 70nm) and HAp nanopowderssynthesized by an ultrasonic-assisted process were used asthe starting powder. Ethylene vinyl acetate copolymer (EVA)was purchased from DuPont USA (ELVAX 210A) and usedas the thermoplastic binder for the shaping of composites.Carbon powder ( < 15 µ m, Aldrich USA) was supplied as apore-forming agent, and stearic acid (Daejung Chemicals andMetals Co. Korea) was used as a lubricant to ensure goodmixing of the components. 2.1.2. Microstructure design and extrusion.  The fabricationprocess is described in our previous report [19]. All the powders (HAp, t-ZrO 2  and carbon) were shear mixed withEVA and stearic acid separately or in mixture. The firstshell composite (45vol% HAp, 45vol% polymer and 10vol%stearic acid) was prepared with a shear mixer (Shina Platec,Korea), and was used to make a tube-type shell (3mmin thickness) by warm pressing in a cylindrical die at110 ◦ C. The second shell composite (46vol% HAp-(t-ZrO 2 ),43vol% polymer and 11vol% stearic acid), the third shellcomposite (40 vol% t-ZrO 2 , 50vol% EVA and 10vol% stearicacid) and the carbon composite (50vol% carbon powder,40vol% polymer and 10vol% stearic acid) were preparedusing the same process. The HAp-(t-ZrO 2 ) mixture powderin the second shell composite was mixed at the ratio of 75vol% / 25vol% by ball milling.The carbon core (50vol% carbon, 40vol% polymer and10vol% stearic acid) was extruded in the cylindrical die at110 ◦ C to make a carbon rod of 22mm diameter. Two types of carbon core were needed for the experiment; the first type of carbon corewaswrapped by thefirst shell composite (45vol%HAp, 45vol% polymer and 10vol% stearic acid), the secondshell composite (46vol% HAp-(t-ZrO 2 ), 43vol% polymerand 11vol% stearic acid) and the third shell composite(40vol% t-ZrO 2 , 50vol% EVA and 10vol% stearic acid).This was used to make the first-passed filaments 3.5mmin diameter. The second type of carbon core was neededfor the final extrusion to make the central hollow space. It2  Sci. Technol. Adv. Mater.  13  (2012) 035009 D-W Jang  et al was wrapped by the first shell composite (45vol% HAp,45vol% polymer and 10vol% stearic acid) and extruded witha diameter of 10mm.The first-passed filaments (diameter 3.5mm) werefabricated using the extrusion process, which assembled onecarbon core and the previously mentioned 3 shell layersin a cylindrical die. The 52 first passed filaments wereenclosed with a shell composite (46vol% HAp-(t-ZrO 2 ),43vol% polymer and 11vol% stearic acid) and assembledin a cylindrical die to obtain the second-passed filamentswith a diameter of 3.5mm. For the final extrusion, a threelayer arrangement with the second type of carbon core wereassembled in a cylindrical die with a diameter of 30mm; thefirst layer was fabricated using shell composites with 45vol%HAp, 45vol% polymer and 10vol% stearic acid (3mm inthickness), the second layer consisted of 20second-passedfilaments (3.5mm in diameter), while the third layer wascomposedof15second-passedfilaments(3.5mmindiameter)and a second carbon core of 10mm diameter was locatedin the central axis. The finalized small artificial bone wasconstructed with a structure similar to that of natural bonethrough the extrusion and arrangement processes. 2.1.3. Burnout and sintering of the composite.  Thecomposite consisted of HAp and t-ZrO 2 , which increased itsdecomposition temperature. The EVA binder was removedcompletely through the first burnout process, in which theheating rate was maintained at 2 ◦ C / min − 1 up to 700 ◦ C in aflowing nitrogen atmosphere. The carbon as a pore formingagent was burnt out at 1000 ◦ C for 2h in air (second burnout),creating the necessary fine pores and rough pore surface.Finally, the sintering of the combined composites was carriedout by microwave heating to 1300, 1400 and 1500 ◦ C. 2.1.4. Evaluation of microstructure.  The microstructure,shape and porous morphology of the artificial bone werestudied by SEM (JSM 6401F, JEOL, Tokyo, Japan). An x-raydiffractometer (D/MAX-250, Rigaku, Tokyo, Japan) was usedto investigate the crystal structure and phases of the startingpowder. 2.1.5. Compressive strength measurement.  The sinteredhollow blocks were cut to 5mm in length and polished. Foraveraging purposes, 5 samples were taken for each sinteringtemperature. The compressive strength was measured using auniversal testing machine with a loading rate of 1mm/min.Compressive strength was evaluated with the formuladerived for a cylindrical annular body, which is the closestapproximation for the fabricated bone preform. 2.2. In vitro testing2.2.1. Sample preparation.  All the samples were sterilizedin an autoclave and then washed with phosphate-bufferedsaline (PBS) before conducting  in vitro  testing. 2.2.2. Cell line and maintenance.  Cell culture studieswere carried out with an osteoblast cell line thatwas obtained from Korea Cell Bank. The cells weresubcultured in flasks using Dulbecco’s modified Eagle’smedium (DMEM) and supplemented with 10% (v/v) fetalbovine serum (FBS), 1% penicillin/streptomycin (PS)antibiotics, 50 µ gml − 1 ascorbic acid (Sigma) and 10mM β -glycerophosphate (Sigma) at 37 ◦ C, 95% humidity and5% CO 2  (Incubator, ASTEC, Japan). Cells were dissociatedwith trypsin-ethylenediaminetetraacetic acid (EDTA, Gibco),centrifuged and resuspended in medium. The culture mediumwas changed every 2 days. 2.2.3. RT-PCR reaction (materials and methods).  CellsgrowninaT75flaskwiththeextractedsolutionwerecollectedusing Trypsin EDTA (TE) and washed twice with PBS. Then,the total RNA from the cells was isolated using a NucleospinRNA II total RNA isolation kit (Germany). cDNA wassynthesized using the iScript TM cDNA kit (Bio-Rad, USA)in accordance with the manufacturer’s instructions. Then, 1 µ lof the first strand cDNA template was subjected to real-timepolymerase chain reaction (RT-PCR) using iQ SYBR GreenSupermix (Bio-Rad, USA) with 4 primer pairs (GAPDH, Col,ON, BSP). RT-PCR was performed on a sample that waspredenatured at 94 ◦ C for 3min, flowed through 40 cycles(denaturation at 94 ◦ C for 20s, annealing at 60 ◦ C for 20s andextension at 72 ◦ C for 30s), followed by a melting analysisbetween 65 and 94 ◦ C. 2.2.4. Cell proliferation. MTT assay.  An autoclavedsample was put inside 24 wells of a plate andseeded with 20000 cells per well. The scaffolds wereincubated for 1, 3 and 5 days. After incubation, thesamples were transferred to a new plate (24 wells). A200 µ l 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) solution was added onto the composite,which was then incubated at 37 ◦ C for 3.5h, and theMTT solution was discarded. Then, the dimethyl sulfoxide(DMSO) solution was added to dissolve any insolubleformazan crystals present on the samples. From the 24-platewell, 150 µ l of solution was transferred to a plate with 96wells. The absorbance was measured at 595nm using anELISA reader (Turner Biosystems CE, Promega Corporation,USA) for MTT testing.  Evaluation of protein components of cells grown inextracted solution by SDS-PAGE.  To determine the proteincomponent of the cells, 0.1g of sterile interconnected porousHAp / HAp-(t-ZrO 2 )/t-ZrO 2  was put into 1ml of DMEM andshaken for 24h at 37 ◦ C; then the solution was used togrow MG-63 cells. About 10 5 cells were seeded on a 6-wellculture plate with the extracted solution or with DMEM only(control), and the cells were collected after 1, 3 and 5 days.Protein components of these cells were collected using RIPAlysis buffer (radio-immunoprecipitation assay, Millipore),checked by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and stained with CoomassieBrilliant Blue R-250. Osteoblast cell adhesion and proliferation.  Osteoblastcells (MG-63) were seeded on tissue culture plates as controlsamples and on the HAp / t-ZrO 2  scaffold at a concentrationof 10 4 cellscm − 3 in DMEM. For short (30 and 60min)and long tests (1, 3 and 5 days), samples were kept at3
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