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Porous titanium scaffolds fabricated using a rapid prototyping and powdermetallurgy technique

Garrett E. Ryan a , b , Abhay S. Pandit a , * , Dimitrios P. Apatsidis ba National Centre for Biomedical Engineering and Science, National University of Ireland, Galway, Irelandb Department of Mechanical and Biomedical Engineering, National University of Ireland, Galway, Ireland

a r t i c l e i n f o

Article history:Received 31 March 2008Accepted 19 May 2008Available online 16 June 2008

Keywords:TitaniumScaffoldRapid prototypingThree-dimensional printingMicrostructure

a b s t r a c t

One of the main issues in orthopaedic implant design is the fabrication of scaffolds that closely mimic thebiomechanical properties of the surrounding bone. This research reports on a multi-stage rapid proto-typing technique that was successfully developed to produce porous titanium scaffolds with fully in-terconnected pore networks and reproducible porosity and pore size. The scaffolds porouscharacteristics were governed by a sacricial wax template, fabricated using a commercial 3D-printer.Powder metallurgy processes were employed to generate the titanium scaffolds by lling around the waxtemplate with titanium slurry. In the attempt to optimise the powder metallurgy technique, variations inslurry concentration, compaction pressure and sintering temperature were investigated. By altering thewax design template, pore sizes ranging from 200 to 400 mm were achieved. Scaffolds with porosities of 66.8 3.6% revealed compression strengths of 104.4 22.5 MPa in the axial direction and 23.5 9.6 MPain the transverse direction demonstrating their anisotropic nature. Scaffold topography was charac-terised using scanning electron microscopy and microcomputed tomography. Three-dimensional re-construction enabled the main architectural parameters such as pore size, interconnecting porosity, levelof anisotropy and level of structural disorder to be determined. The titanium scaffolds were compared totheir intended designs, as governed by their sacricial wax templates. Although discrepancies in ar-

chitectural parameters existed between the intended and the actual scaffolds, overall the results indicatethat the porous titanium scaffolds have the properties to be potentially employed in orthopaedicapplications.

2008 Elsevier Ltd. All rights reserved.

1. Introduction

Scaffolds are of signicance for orthopaedic tissue engineeringapplications as they allow biological anchorage to the surroundingbone tissue through the ingrowth of mineralized tissue into theporous network [1] . Although porous ceramics and polymers havebeen studied as potential bone graft scaffolds [24] , they cannotreliably satisfy the mechanical demands of load-bearing applica-tions, such as stand alone interbody spinal fusion devices [5,6] . Forthis reason, porous metals including titanium [7,8] , titaniumnickel[9,10] , and tantalum [1113] have been investigated. Titaniummetal is widely chosen due to its corrosion resistance, highstrength-to-weight ratio, and proven biological acceptance [14,15] .Several techniques have been developed to introduce a degree of porosity in titanium and titanium alloy scaffolds including

compression and sintering of beads and bres [16,17] , combustionsynthesis [18] , electron beam melting [19,20] , solid-state foamingby expansion of argon-lled pores [21] and polymeric spongereplication [22] .

Notwithstanding the advancements that have attained in scaf-fold fabrication, the control over scaffold architecture using theseconventional techniques is highly process dependent. Rapid proto-typing (RP) techniques are considered as a viable alternative forachieving extensive and detailed control over scaffold architecture[23,24] , by combining computer-aided design (CAD) with com-puter-aided manufacturing (CAM). RP makes it possible to buildobjects with predened microstructure and macrostructure andprovides the potential for making scaffolds with controlled hierar-chical structures [25] . The transfer of RP technologies to metallicmaterials for tissue engineering and orthopaedic implants posesa signicant challenge, although a number of investigators havemade substantial progress in this regard. Curodeau et al. producedporous surfaced CoCr implants using a variation of the lost waxprocess [26] . By pouring molten CoCr into porous ceramic mouldsthat had been fabricated using a three-dimensional printing

* Corresponding author. Tel.: 353 91 492758; fax: 353 91 563991.E-mail addresses: [email protected] (G.E. Ryan), abhay.pandit@nui-

galway.ie (A.S. Pandit), [email protected] (D.P. Apatsidis).

Contents lists available at ScienceDirect

Biomaterials

j ou rna l homepage : www.e l sev i e r. com/ loca t e /b ioma te r i a l s

0142-9612/$ see front matter 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.biomaterials.2008.05.032

Biomaterials 29 (2008) 36253635
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technique, textures with ve layers of distinct geometric denitionwere created. Using a similar investment casting technique, Lopez-Heredia et al. created a porous titanium scaffold with 100% inter-connected structure and porosity of around 60% [27,28] . Abundantbone formation was found inside the rapid prototyped titaniumscaffolds after 3 and 8 weeks of implantation in the femoralepiphysis of New Zealand White rabbits [27] . Using a techniqueknown as three-dimensional ber deposition, Li et al. producedporous Ti6Al4V scaffolds by depositing Ti6Al4V slurry througha computer controlled syringe followed by sintering of the titaniumpowders [29] . Hollander et al. used a technique called direct laserforming to create porousscaffolds fromTi6Al4Vpowder followedbysand blasting of the scaffold to remove loose powder particles [30] .

A key requirement for such RP technologies is control over thescaffolds pore structure, including pore size, shape, volume andinterconnectivity [31] . It is generally accepted that pore sizes be-tween 100 and 400 mm are optimal for bone ingrowth [32] . Highpore interconnectivity greatly affects mass transport through thescaffold and is necessary to ensure adequate delivery of cells andnutrient supply during subsequent culture throughout the com-plete porous scaffold [33,34] . Porosity and pore geometry also in-uences the scaffolds mechanical properties and affect stressshielding and fatigue strength [35,36] . Tight control over the scaf-folds porous architecture requires that reliable methods are es-sential for its characterisation following the design and fabricationprocesses. This enables an accurate assessment of the level of precision that the fabrication process can deliver. Although scan-ning electron microscopy (SEM) images provide high resolutionand detailed views of the surface topology an inherent weakness isits limitation to two-dimensional measurements on relativelysmall

elds of view. State of the art three-dimensional imaging tech-niques enable researchers to describe the complex structure of materials more accurately. Microcomputed tomography ( mCT),a non-destructive technique, is being increasingly used to providestructural information within an object by mathematically recon-structing its three-dimensional images from a consecutive series of x-ray images [3739] . This technique provides the opportunity toexperimentally measure the complex morphology of the porespace of scaffolds in three dimensions at resolutions in the micro-metre range.

The objective of this study was to develop a porous titaniumscaffold with controlled architecture using a multi-stage RP tech-nique. The major parameters involved in the fabrication processwere examined, in order to evaluate the signicance of each pa-rameter on the scaffolds mechanical properties. Subsequently,scaffolds were created to identify the inuence of increasing po-rosity on the mechanical and structural properties. These scaffoldswere scanned using a mCT scanner and their structural character-istics were calculated using the 3D digitized images. In this waya comparison with the initial CAD designs was possible and anydifferences in topography could be assumed to be due to inaccur-acies of the fabrication process. Finally, in vitro studies were con-ducted to assess the cellular response to the porous scaffold.

2. Materials and methods

2.1. Scaffold fabrication

In thissectionwe outlinethe methodused tocreatetheporoustitaniumscaffolds.A porous wax model was fabricated using a Thermojet (3DSystems Corporation,Valencia, CA) 3D-printer according to the CAD template shown in Fig. 1(A). The

Fig.1. (A) Schematic of the CAD design template used to create the sacricial wax model, and (B) schematic demonstrating the pore space reconstruction and centreline generationfrom serial mCT scans.

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template consists of a repeating array of unit-cells in thethree principaldirections. Arestrictionto thewax template design waspresentedby the3D-printer. A dispensingangleequaltoor lessthan7 needs tobe adheredto, becausethe printerwill laydownautomatic support structures, if this angle is exceeded. The wax templates weredesigned to accommodate this limitation, according to the schematic shown inFig. 1(A). The wax model was placed in a compaction die and a layer of absorptivepaper was placed underneath the wax. Titanium powder (350 mesh, grade 2) (AlfaAesar GmbH & Co KG, Karlsruhe, Germany) was mixed with ethylene glycol solution(SigmaAldrich, MO) using a vortex mixer to form a titanium slurry and was im-mediately poured into the die under gravity. Over a period of 12 h the tissue paper

absorbed almost all of the ethylene glycol from the slurry by capillary action, dryingoutthe wax-titaniumconstruct. Theabsorptivepaperwas removed andthe constructwas then compacted using a hydraulic press. During the compaction process, thepress was switched from load control to displacement control to prevent furthercompaction of the construct. A cartridge heater, built into the die, was employed toheat the die at a rate of approx. 5 C/min to approx. 90 C. The heating step wasmonitored using a thermocouple. The temperature was maintained at 90 C for15 minto completelymeltthe waxtemplate. Atthis point,the loadfromthe hydraulicpress was removed. Once the construct had cooled to room temperature the wax-titanium construct was removed from the die and placed in xylene solution at 60 Cfor 15 min. This procedure was repeated three times to ensure complete dissolutionof the wax. The resulting porous titanium scaffold was placed in a high vacuumfurnace (10 5 mbar) at temperatures ranging from 1100 to 1300 C for 1 h to sinterthe titanium powder particles. The sintering step produced a functionally stable ti-tanium scaffold with a porous structure that approximated the inverse morphologyof the sacricial wax template. By modifying the design parameters of the sacricialwax template ( Fig. 1(A): 1, 2, L1L4), and by adjusting powder metallurgy (PM)parameters, scaffolds with various pore sizes and porosities could be fabricated.Scanning electron microscopy (SEM) pictures were taken with a Hitachi S-4700(HitachiHisco Europe GmbH, Berkshire, UK) with the following settings: 15 kV Voltage and 10 mA emission current. Energy dispersive analysis (EDX, Oxford In-struments, Oxfordshire, UK) was performed at a working distance of 15 mm and15 kV to distinguish the elements present at the surface of the scaffold.

2.2. Process optimisation

The major parameters involved in the fabrication process were examined todetermine their inuence on the mechanical strength of the titanium scaffolds. Asummary of these parameters is presented in Table 1 . Initial testing was conductedto examine the inuence of the PM parameters, which involved investigating theeffect of slurry concentration, compaction pressure and sintering temperature. Forthese tests, a common wax template ( L1 L2 4.0 mm, L3 L4 1.0 mm,1 350 mm, 2 700 mm) was chosen and the titanium scaffolds were fabricatedusing this template. To determine the signicance of a single variable within a givenparameter, all other parameters were held constant. The highest value of each pa-rameter was chosen as the constant value. The resulting titanium scaffolds weresubjected to uniaxial compression tests ( n 3) along their transverse direction ata rate of 1 mm/min using a universal testing machine (Instron 8874; Instron Cor-poration, Norwood, MA, USA). The compression strengths of the scaffolds werecompared to assess the inuence of each parameter. All parameter variables werecompared against the variables that yielded highest scaffold strength. Statisticalanalyses were carried out using statistical software (Minitab , v. 13.32). Statisticalvariance between parameter variables was determined using one-way analysis of variance (ANOVA). Tukeys honesty signicant difference test was used for post hocevaluation of differences between groups. A P value of < 0.05 was considered to bestatistically signicant.

To studythe inuence of the 3D-printed sacricial template, threedifferent waxtemplates were prepared using CAD software (AutoCAD 2002; Autodesk, Inc., CA).The design variables for the three wax templates are presented in Table 1 . Thesevalues were chosen to create scaffolds with increasing levels of porosity based oninitial testing using various designs. The PM parameters, which resulted in highestmean strength values, were chosen to create the three titanium scaffolds. Uniaxial

compression tests ( n 3) were performed along the axial and transverse directionsto examine the mechanical properties of the resulting scaffolds.

2.3. Characterisation of scaffold morphology

Three cylindrical porous titanium samples, approximately 14 mm in diameterand 15 mm in height, fromeach scaffold design described in Table 1 were fabricated.Total scaffold porosity was determined by measuring the apparent density of thescaffold using volume and weight measurements and the known solid density of titanium 4507 kg/m 3. The samples were scanned using a mCT desk scanner (Sky-Scan 1072; e2v Scientic Instruments Ltd., UK). The output format for each samplewas 600 serial 1024 1024 bitmap images with an isotropic voxel size of 15 mm.Using MatLab v.7.0.1 (TheMathworks,Inc., MA),sequential500 500 pixel images

were cropped from the serial images of each sample. The images were converted toblack and white and the black-to-white ratiowas used to determine the porosity foreach consecutive image. In this way, porosity could be determined as a function of height for each of the scaffolds. Based on these images and using a reconstructionsoftware (Mimics v.10.1; Materialise, Leuwen, Belgium) 3D models of each scaffoldwere created. Thresholds of the grey scale images were inverted to allow mea-surements of the volume of all pore spaces. Subsequently, a region-growing oper-ation was performed, creatinga maskconsisting onlyof interconnected porespaces.Volume for this region-grown mask was also determined, and the ratio of region-grown volume to total volume was calculated. This percentage is described as theinterconnecting porosity. To analyse the distribution of pore size throughout thescaffold, the pore space over the height of three unit-cells was isolated as demon-strated in Fig. 1(B). Using Mimics , centre lines were constructed for the pore spaceand thebest-tdiameter wasobtained. Thisprocesswas performed at three randomlocations within each scaffold and the average poresize, as a function of height, wasdetermined for scaffolds fromeach design. Thedegreeof anisotropy, dened as unit-cell horizontal length/axial length, was determined for each of the three scaffolds.Three unit-cells were isolated from three scaffolds at each porosity level to producean average of these measurements. The level of structural disorder within eachscaffold was evaluated by counting the number of non-interconnecting struts ina given cross-section and dividing by the total possible number of interconnectingstruts. Three random cross-sections were chosen from three scaffolds at each po-rosity level for this process.

Toassess theclosed-cell microporosity formed through sintering of thetitaniumpowder particles as would be found in the struts of the nal porous scaffold cylindrical titanium billets were prepared without the use of the wax template butusing identical PM parameters. Scanning electron microscopy (SEM) was used toassess the surface topography of the billets, and the billet porosity was evaluatedusing the identical technique used for the open-cell porous scaffolds.

2.4. In vitro experiments

SAOS-2 pre-osteoblast cells were cultured on porous titanium scaffolds overa period of 3 weeks. Cylindrical scaffold samples with a diameter of 14 mm andheight of 8 mm were used for this purpose. The scaffolds had a mean pore size of 465 170 mm and porosity of 45.1 1.7%. Standard cell culture plastic was used asthe control surface. The culture medium was McCoys-5A supplemented with 10%fetal bovine serum (SigmaAldrich) and 1% penicillin and streptomycin (SigmaAldrich). The samples were placed in a 24-well plate (Sarstedt, Numbrecht, Ger-many) and seeded at 5 x 10 4 cells/per sample in 1 ml of medium, and cultured at37 C in a humidied atmosphere with 5% CO 2 concentration. The medium waschanged every 23 days. After 14 days of culture, the medium was changed tocomplete McCoys medium supplemented with ascorbic acid (50 mg/L; SigmaAldrich, Ireland), dexamethasone (10 ng/L; SigmaAldrich, Ireland) and b-glycero-phosphate(10 m M; SigmaAldrich, Ireland) [4042] . All assays were performedafter1, 7, 14 and 21 days in culture.

Cell viability was assessed using the AlamarBlue assay (BioSource). The cell-seeded scaffolds were removed from the original wells and placed in fresh wellscontaining 1 ml of 10% (v/v) AlamarBlue solution in Hanks balanced salt solution(SigmaAldrich). After 1 h of incubation, the uorescence was measured using anFLx800 microplate uorescence reader (Bio-Tek Instruments, Inc.) at 520 and590 nm excitation and emission wavelengths, respectively.

Cellproliferation on thetitaniumscaffolds was determined by DNAanalysis. Thesubstrates were placed in a new well containing 1 ml deionized distilled water and

Table 1Process parameters used for optimising the powder metallurgy fabrication process

Process parameter Variables

Powder metallurgy parameters Slurry concentration(mg Ti/ml ethylene glycol)

5/7 5/5 5/3

Pressure (MPa) 50 150 250Sintering temp. ( C) 1100 1200 1300

Template 1 Template 2 Template 3

3D-printed sacricialwax template

1 350 mm 1 350 mm 1 400 mm2 700 mm 2 700 mm 2 800 mmL1 L4 4 mm L1 L4 3.6 mm L1 L4 4 mmL2 L3 1 mm L2 L3 1 mm L2 L3 1 mm

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frozen at 80 C for 2 h followed by thawing to room temperature. After threefreeze-thaw cycles, the samples were sonicated briey. A volume of 100 ml of theDNA sample was added to 100 ml Picogreen stain (Sarstedt). The samples were in-cubated for 5 min in the dark at room temperature. Fluorescence was measuredusing an FLx800 microplate uorescence reader ( l em 520 nm and l ex 480 nm) and

the DNA content was calculated from a standard curve (calf thymus DNA (SigmaAldrich)).

The cell colonization of the scaffold was analysed by scanning electron mi-croscopy (SEM). The samples were xed with 2.5% glutaraldehyde in phosphatebuffer (0.1 M (NaH 2PO4 0.18 M, Na 2HPO4 0.17 M)) (SigmaAldrich, Ireland) for 2 h atroom temperature. After xation, the samples were dehydrated through gradedethanol solutions (SigmaAldrich, Ireland). The samples were then immersed inhexamethyldisilazane for 30 min, dried at room temperature and gold coated(Emitech K550 Sputter Coater, Emitech Ltd., Ashford, Kent, UK). SEM was thenperformed on the samples.

3. Results

3.1. Scaffold fabrication

Fig. 2(A) shows a porous titanium scaffold that has been cre-ated using the present RP fabrication process. The scaffold

approximates the inverse morphology of the wax template andresults in a fully interconnected porous network. For the 3D-printer used in this fabrication process the minimum wax featuresize was approximately 200 mm and thereafter the wax models

were approximately 35 mm greater than the CAD design modelsfor the three principal directions. The wax template bestowed thetitanium scaffold with non-uniform architectural and mechanicalproperties in the axial and transverse directions and ensuredgreater strength of the samples in the axial direction. The re-peating unit-cell geometry of the wax template produced a uni-form distribution of pore size throughout the scaffold. Thetemplate was modied to produce specic pore sizes, as shown inFig. 2(B). The predominant factor governing the pore size in thetitanium scaffolds was the diameter of the struts ( 1 and 2) in thesacricial wax template unit-cell. The main factor inuencingporosity in the titanium scaffolds was the height of the unit-cell(L1) and the unit-cell spacing ( L2L4). Both macro and microporesexist in the scaffold. The sizes of titanium powder particles used in

the fabrication process ranged from 40 to 63 mm. Typical titaniumpowder topographies for the different stages of sintering in this

Fig. 2. (A) Top and side proles of a porous titanium scaffold with 59.1% porosity and (B) scanning electron micrographs of porous titanium scaffolds with pore sizes of 200, 300, and400 mm.


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1100 C, while holding other parameters constant, produced scaf-folds with yield strengths of 56.33 5.85 MPa. However, no sta-tistical difference was found between samples sintered at 1200 and1300 C (P > 0.05). Slurry concentration was found to have thegreatest inuence on scaffold strength. Slurry consisting of 5 g of titanium powder in 7 ml of ethylene glycol produced scaffolds withmean yield strengths of 42.0 8.46 MPa. A limit to the concentra-tion of the slurry was reached at 5 g titanium powder in 3 mlethylene glycol, as above this concentration the slurry became tooviscous to completely penetrate the sacricial wax template.

The design of the sacricial wax template was found to greatlyinuence the scaffolds morphological and mechanical properties.Given that this wax model was lost in the fabrication process,a decrease in the wax template porosity resulted in an increase intitanium scaffold porosity. The mechanical properties of three ti-tanium scaffolds created using the wax templates described above

are presented in Fig. 5. The morphological characteristics of thesethree scaffolds are presented in greater detail in Table 2 . Youngsmodulus and scaffold strength decreased with increasing porosity.In the axial direction the relationship between Youngs modulusand porosity was almost linear. Structural disorder increased withincreasing porosity. The scaffolds were found to be anisotropic innature. The mean Youngs modulus was approximately 69.3% lowerin the transverse direction compared to the axial direction for allthree scaffolds. Also, mean ultimate compression strength was onaverage 56.7% lower in the transverse direction compared to theaxial direction.

3.3. Characterisation of scaffold morphology

Three-dimensional computer simulations of titanium scaffoldswere successfully constructed from serial mCT data as shown inFig. 6. These were used for comparing the porous morphology of the nal samples to that of the initial CAD drawing that they havebeen derived from, in order to identify any discrepancies. Visualinspection of the models revealed that the level of anisotropy in-creased as the porosity of the scaffolds increased. These values aresummarised in Table 2 , along with the overall scaffold porosity andtotal interconnecting porosity as calculated using Mimics . Theclosed-cell microporosity was found to be 9.5 1.1%, throughevaluation of the sintered titanium billets. For the titanium scaf-folds, the mean difference in total porosity as measured using ap-parent density calculations and interconnecting porosity asmeasured using Mimics was approximately 5.7%.

The variation in porosity and pore size over the height of thethree titanium scaffolds is presented in Fig. 6(A) and (B). Both

porosity and pore size showed a repeating pattern, governed by theunit-cell geometry. In general, an increase in pore size over theheight of the unit-cell corresponded to an increase in scaffold po-rosity. The distribution of pore sizes for the three scaffolds is pre-sented in Fig. 6(C). For all scaffolds the dominant pore size rangedbetween 400 and 550 mm. However, Template 3 possesseda greaterproportion of pores at and above 1000 mm and this contributed toa scaffold with overall greater porosity.

There was a notable difference in morphology between theintended scaffold design and the physical titanium models. This isevident in Fig. 7(A), which shows the idealised unit-cell for thesacricial Template 2 and a unit-cell isolated from its correspond-ing titanium scaffold. The graph shows the change in porosity overthe height of the scaffold. It is evident that the architecture wassignicantly altered due to the fabrication process. The porosities of the titanium scaffolds were on average 41.2% greater than the po-rosities of their corresponding idealised models. Also, the physicaltitanium scaffolds were on average 29.2% shorter than their ideal-ised models. Fig. 7(B) shows the change in pore size over the height

Fig. 4. The effect of different PM processes on the mechanical strength of the titaniumscaffolds. Values are reported as mean Std. Dev; *P < 0.05, n 3.

Fig. 5. The mechanical properties of three titanium scaffolds created using differentsacricial wax templates showing (A) Youngs modulus and (B) Yield strength ( n 3).Porosity values for the three templates are presented in Table 2 .

Table 2Intended and achieved porosities and structural characteristics of three poroustitanium scaffolds created using different design templates

Template 1 Template 2 Template 3

Idealised model porosity (%) 36.9 40.8 47.8Total porosity (%) 51.4 1.2 59.1 1.7 66.8 3.6Interconnecting porosity (%) 46.5 1.1 53.4 2.0 60.4 2.6Level of structural

disorder (%)1.8 0.4 3.2 1.7 5.2 2.1

Degree of anisotropy 0.51 0.01 0.34 0.01 0.35 0.02

Closed-cell microporosity (%) 9.5 1.1


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of the scaffold. Again, the repeating unit-cell structure is evident.However, pore sizes for titanium scaffolds ranged from 300 to1000 mm, which is contrast to the idealised models, which pos-sessed only two possible pore sizes.

3.4. In vitro experiments

Fig. 8 shows SEMimages of SAOS-2 cells on the surface of poroustitanium samples after 1, 7, 14 and 21 days in culture. At day 14

a conuent cell layer was observed on the titanium surface. Po-lygonal and spindle-shaped cells attached and spread on the

microporous surface and some migrated inside the micropores. Asshown in Fig. 9, AlamarBlue and total DNA analysis showed osteo-blast proliferation with increasing cellular activity from day 1 to14. The highest cell growth period was seen between day 7 and 14.After day 14, cell growth decreased, as the two assays conrm.

4. Discussion

We have developed a new process of fabricating porous tita-

nium scaffolds that possess high levels of interconnecting porositycombined with high levels of mechanical strength. In the

Fig. 6. Typical models of porous titanium scaffolds created using three different sacricial templates and reconstructed using 3D reconstruction software (Mimics ; Materialise) arepresented. The graphs show (A) porosity and (B) pore size as a function of height for the porous titanium scaffolds, and (C) distribution of pore size for the three scaffolds.


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fabrication process, a wax template acts as a sacricial polymeric

space holder, and the resulting titanium scaffold takes the inversemorphology of this template. Wen et al. used a similar techniquebased on a space holder material in the form of ammonium hy-drogen carbonate particles that were randomly mixed with tita-nium powders and subsequently compacted [43] . The ammoniumhydrogen carbonate was thermally removed using a heating stepfollowed by sintering of the titanium powders at elevated tem-peratures. Using this technique scaffolds with 80% porosity andcompression strengths of 40.0 MPa were created. Greater controlover pore interconnectivity was achieved by Li et al., using a tech-nique they pioneered, which is known as the polymeric spongereplication method [22,44] . In this technique, commercially avail-able polyurethane foams were dipped into slurry containingTi6Al4V powder particles causing the powder particles to be im-

pregnated onto the struts of the sponge. The polyurethane materialwas subsequently pyrolyzed followed by sintering of the titaniumalloy powders. With this method they were able to create scaffoldswith 89 1.6% porosity and compression strengths of 10.3 3.3 MPa. Using an RP technique known as 3D ber de-position, Li et al. created porous Ti6Al4V scaffolds with fullyinterconnected porous networks [29,45] . By depositing Ti6Al4V slurry in a process similar to 3D-printing, followed by sintering of the Ti6Al4V powder particles, scaffolds with approx. 60% porosityand compressive strengths of around 520 MPawere created. Lopez-Heredia et al. used a reverse engineering technique to createmacroporous titanium implants with controlled shapeand porosity[27,28] . The rst step in this technique was to create a porous waxscaffold template using an RP machine. The wax template was

embedded in a refractory material and the wax was subsequentlyremoved by pyrolysis. Molten titanium was then poured into the

mould and once cooled, the refractory material was removed bygrit-blasting. This process resulted in titanium scaffolds withapprox. 60% porosity and compressive strength of around 80 MPa.

The fabrication process developed in the present research hassimilarities with the aforementioned techniques. An RP techniquewas used to create the sacricial wax template allowing the porouscharacteristics to be tailored to a high degree and ensure completeinterconnectivity of the porous architecture. A compaction stepwasemployed to bring the powder particles into close proximity andensure optimum strength of the nal sintered powders. The porecell walls microporosity of 9.5 1.1% was formed using a compac-tion pressure of 250 MPa and sintering temperature of 1300 C. Themicroporosity was analysed under these conditions as these valuesof compaction pressure and sintering temperature yielded thehighest strength scaffolds during process optimisation. An increasein scaffold porosity corresponded with a decrease in scaffoldcompressive strength, which is in agreement with Gibson andAshbys model of cellular solids [46] . Scaffolds with porosities of 66.8 3.6% possessed compression strengths of 104.4 22.5 MPain their axial direction and 23.5 9.6 MPa in their transverse di-rection. An important implication of the compaction step is thattitanium particles are less likely to break away from the body of theporous material. Loose particles could potentially cause in-ammation of the surrounding implant and can cause asepticloosening of the implant [47,48] .

Several factors were found to inuence the nal architecturaland mechanical properties of the titanium scaffolds. For processoptimisation, a single wax template design was chosen to evaluatethe effects of compaction pressure, sintering temperature andslurry concentration. Although the results from testing these pa-rameters would change when using different wax templates, it wasassumed that the general trend would remain the same withina single parameter. The design of the sacricial wax model had thegreatest effect on scaffold properties. Due to the high computa-tional demands of making these models, they were designed ashexagonal in cross-section, to reduce the size of the stereo-

lithography CAD le that is used for 3D-printing. Nevertheless, theresulting wax models were circular in cross-section due to the ac-curacy limitations of the machine. Due to 3D-printer softwareconstraints, the maximum dispensing angle for wax printing is setto 7 . For this reason, the design of the titanium scaffold was gov-erned by this limitation. However, with relevant software modi-cations to the current 3D-printer or by using a 3D-printer that doesnot have this limitation we intend to generate wax models witha greater choice of possible design characteristics. By removing the7 limitation the level of anisotropy for the scaffold can be adjustedor completely removed, if desired. Removing the 7 limitation willalso permit scaffolds with much higher porosity to be developed.The wax template structure can be tailored to optimise the slurryinltration process and eliminate strut defects and structural

inhomogeneity.Another factor that ultimately limits the porosity of the scaffoldis the size of the titanium powder particles (approximately 45 mmwere used in this work). Through initial testing of different waxstructures, it was found that the smallest aperture size that willallow titanium powder slurry inltration into the wax templatewas approx. 60 mm. The wax models are required to be of sufcientporosity to allow an even distribution of titanium powder to llfrom the topto the bottom of the wax template. For this reason, thecomposition and concentration of the slurry are critical. It was alsofound that increasing the concentration of ethylene glycol in-creased the viscosity of the slurry but this also meant that less ti-tanium powder penetrated the scaffold. This produced an increasein scaffold porosity compared to the idealised model, due to the

increased microporosity that resulted in the struts of the porousstructure. There was also a weakening of titanium struts, as they did

Fig. 7. The difference between the idealised and actual scaffold morphology over theheight of the scaffold in terms of (A) porosity and (B) pore size for the titanium scaffoldcreated using Template 2.


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not conform to the original template geometry. Possible ways toovercome this are to use ner titanium powders or to add addi-

tional substances that might increase the viscosity of the slurry asdemonstrated in previous work by Li et al. [44] .

The reduction in scaffold height was a prominent feature in allscaffolds that were created, and mainly resulted from the uniaxialcompaction step that condenses the titanium powder and waxtemplate. Future research may incorporate a hydrostatic compac-tion press so that alterations to the intended design can be mini-mized. The compaction process owes its success to the concurrentheating step that simultaneously melts the wax inside in the diecavity. During this process the hydraulic press is changed from loadcontrol to displacement control, which prevents further de-formation of the titanium structure while the wax is melted. Themelting step is necessary, as without this multiple cracksdevelopedin the scaffold struts prior to sintering due to the difference in

compressibility between the wax material and the compacted ti-tanium powder. If not heated, the wax springs back to its originalshape once force from the press is removed, and this movementbreaks the adhesion bonds formed between the titanium powderparticles. It should be noted that during this process the wax is notremoved from the die but retainsits position, embedded within thetitanium powders. After the die has cooled the wax/titanium con-struct can be removed and is quite robust. The use of xylene toremove the wax material by dissolution prior to sintering waschosen over pyrolysis to avoid contamination of the titanium withcarbon residues from the wax space holder that can be left behind[49] . Several rounds of rinsing are necessary to completely removethe wax, after which the titanium powders are held together by theadhesion forces formed in the compaction process. With increasing

porosity, and once the wax llers have been removed, the scaffoldsbecome very delicate and care needs to be exercised when moving

the scaffolds to the furnace. Sintering temperature did not seem toaffect scaffolds mechanical strength greatly once the temperature

exceeded 1200 C. However, a furnace-vacuum of greater than10 5 mbar was necessary for complete sintering to occur, withoutany oxides interfering with the particles fusion.

Although the elongated pores in the titanium scaffolds area result of limitations due to the 3D-printer, they create a materialthat more closely resembles natural bone than fully dense,isotropictitanium. The anisotropy of bone is well documented [50,51] . Anumber of authors have utilised this attribute in the design andoptimisation of biomaterial scaffolds with elongated pores [52,53] .Scaffolds with a level of anisotropy have the potential to addressissues of reduction in implant weight and stiffness while mini-mising strength reduction in the loading direction. In this research,scaffolds with 66.8 3.6% porosity possessed a Youngs modulus of 20.5 2.0 GPa in the axial direction and 4.35 0.5 GPa in the

transverse direction. Comparedto bone, these valuesare quite high.The mechanical properties of trabecular bone are widely studied,but still reported values for Youngs modulus range vary from 0.1 to10.4 GPa depending on the source and method of analysis [54] .Future research will aim to increase the porosity of the scaffolds tofurther reduce the scaffolds modulus. Finding appropriate me-chanical properties for a prospective implant will be a signicantchallenge where levels of strength are sacriced for increasingporosity and reduced stiffness. In vivo tests will be necessary toidentify appropriate designs that will best match loading in sucha way that will prevent stress shielding as well as early failure. Apossible way to further reduce the elastic modulus would be to usea titanium alloy powder. Beta titanium alloys such as Ti 35Nb7Zr5Taand Ti 13Nb13Zr have lower Youngs moduli, compared to pure ti-

tanium and have received considerable attention as orthopaedicimplants in recent years [5557] .

Fig. 8. Appearance of SAOS-2 cells on the surface of the porous titanium scaffold after (A) 1, (B) 7, (C) 14, and (D) 21 days in culture.


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A range of structural properties were quantied using mCT.Scaffold reconstruction using serial mCT images enabled structuralparameters that were previously unavailable experimentally to be

measured, for example the change in porosity and pore sizethroughout the height of the scaffold. Analysis of these parametersenabled us to determine the relationship between the intendedscaffold design and the physical titanium scaffold. The height of thetitanium scaffolds is signicantly shorter compared to their pro- jected design. This difference is largely due to the uniaxial com-paction process although sintering of the green titanium scaffoldsfollowing xylene dissolution also causes a reduction in scaffoldheight through coalescence of the powder particles. The level of structural disorder in the scaffolds increased with increasing levelsof porosity. In the fabrication process, increasing the porosity re-quires increasing the ratio of sacricial wax template to titaniumpowder. In doing so, the titanium powder is less likely to com-pletely ll the porous space around the sacricial template and

more anomalies in the scaffold architecture are likely.In the 3D reconstruction process, the accuracy of the scaffoldgeometries is dependent on the spatial resolution of the scans andimage segmentation [58] . In this research, a voxel size of 15 mmwassufcient to describe the main structural features of the scaffold,because the average powder particle size was 45 mm. UsingMimics , image thresholding was executed to segment titaniumand pore phases prior to 3D modelling. This is a critical step as itdetermines the success of subsequent analysis and visualization.The microporosity generated between the sintered titanium pow-ders (9.5 1.1%) was lost during this process due to resolutionlimitations. The difference in total porosity as measured usingArchimedes principle and interconnecting porosity as measuredusing Mimics should equal this nding but it is lower by 3.8%. This

slight divergence may be attributed inaccuracies in the 3D re-construction process and in particular during segmentation.

Promising cellular responses to the porous titanium scaffoldswere seen in this study. Both AlamarBlue and DNA tests showed anincrease in cell number in the rst 2 weeks of culture. Thereafter,differentiation media was added after 14 days of culture, whichexplains the reduction in cellular growth until day 21, as this wouldhave coincided with cell differentiation. We conclude that the invitro experiments show that the titanium scaffold allows thegrowth of vital pre-osteoblast cells on its surface, which is com-parable to the cellular behaviour on other poroustitanium scaffolds[30,45] .

5. Conclusion

A porous titanium scaffold with controlled porous structure andhigh strength has been created using a combination of RP and PMtechniques. A wax model, created using a commercial 3D-printer,provided the template for the porous titanium scaffold. Charac-teristics of the PM process, such as titanium slurry concentration,compaction pressure, and sintering temperature, were found toinuence the structure and strength of the scaffolds. At present,a signicant level of variability exists between scaffolds and theirintended designs due to factors present in the PM process. Futureresearch will involve gaining a greater control over these factors sothat this mismatch can be minimized. The in vitro experimentsshowed that the osteoblasts maintain their metabolic activity onthe surface of the porous titanium scaffolds. In conclusion, the re-sults of this study demonstrate the potential of such scaffolds foruse in orthopaedic tissue engineering applications.

Acknowledgements

This work was supported by Enterprise Ireland (PC/2005/012).

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