On the structural, mechanical, and biodegradation properties ofHA/b-TCP robocast scaffolds

Manuel Houmard,1,2 Qiang Fu,1 Martin Genet,1 Eduardo Saiz,3 Antoni P. Tomsia1

1Materials Science Division, Lawrence Berkeley National Laboratory, Berkeley, California 947202Department of Materials Engineering and Civil Construction, Federal University of Minas Gerais (UFMG), Belo Horizonte,

MG 31270-901, Brazil3Centre for Advanced Structural Ceramics, Department of Materials, Imperial College, London, UK

Received 8 November 2012; revised 4 February 2013; accepted 18 February 2013

Published online 7 May 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.32935

Abstract: Hydroxyapatite/b-tricalcium phosphate (HA/b-TCP)

composite scaffolds have shown great potential for bone-

tissue engineering applications. In this work, ceramic scaffold

with different HA/b-TCP compositions (pure HA, 60HA/40b-

TCP, and 20HA/80b-TCP) were fabricated by a robotic-assisted

deposition (robocasting) technique using water-based hydro-

gel inks. A systematic study was conducted to investigate the

porosity, mechanical property, and degradation of the scaf-

folds. Our results indicate that, at a similar volume porosity,

the mechanical strength of the sintered scaffolds increased

with the decreasing rod diameter. The compressive strength

of the fabricated scaffolds (porosity � 25–80 vol %) varied

between �3 and �50 MPa, a value equal or higher than that

of human cancellous bone (2–12 MPa). Although there was a

slight increase of Ca and P ions in water after 5 month, no

noticeable degradation of the scaffolds in SBF or water was

observed. Our findings from this work indicate that compos-

ite calcium phosphate scaffolds with customer-designed

chemistry and architecture may be fabricated by a robotic-

assisted deposition method. VC 2013 Wiley Periodicals, Inc. J

Biomed Mater Res Part B: Appl Biomater 101B: 1233–1242, 2013.

Key Words: calcium phosphates, HA/b-TCP composites, robo-

casting, biomedical scaffolds, tissue engineering

How to cite this article: Houmard M, Fu Q, Genet M, Saiz E, Tomsia AP. 2013. On the structural, mechanical, and biodegradationproperties of HA/b-TCP robocast scaffolds. J Biomed Mater Res Part B 2013:101B:1233–1242.

INTRODUCTION

Calcium phosphate (CaP) ceramics, especially biphasic cal-cium phosphate (BCP) composed of hydroxyapatite (HA,Ca10(PO4)6(OH)2) and b-tricalcium phosphate (b-TCP,Ca3(PO4)2), are widely used as bone-substitute material andin various tissue-engineering applications.1–8 Indeed, thereis a growing interest in BCP because it has been shown thatthis material improves the formation of new bone inside theimplanted scaffold.3,4 The literature indicates that the fasterdissolution rate of b-TCP would be greatly responsible ofthis enhancement. However, in vitro tests indicate that fastdissolution can decrease the mechanical strength of CaPscaffolds.9 Furthermore, studies reported that HA has highermechanical strength and lower degradation rate than b-TCP.4,10,11 Therefore, the HA/b-TCP ratio is a key parametercontrolling the performance of the scaffold for bone repairapplications, since it determines both degradation rate andmechanical properties of the material.12 Scaffolds for boneregeneration require interconnected macro- (>50 lm) andmicro-pores (1–50 lm) that create favorable conditions forcell seeding, proliferation, and vascularization, and new

bone formation.13 An optimal three-dimensional (3D) scaf-fold must be strong enough to replace the bone, at leasttemporarily, while providing a substrate for cell attachmentand proliferation. It also must dissolve gradually as new tis-sue grows.14 To fulfill the aforementioned criteria, newprocessing routes with the ability to produce BCP scaffoldswith controlled and organized micro- and macro-pores areneeded for scaffold fabrication.

A variety of techniques has been used to fabricate po-rous ceramic scaffolds for tissue engineering, including rep-lica templates,15,16 emulsions,17 the use of porogens,18

freeze-casting,19 and solid freeform fabrication (robocast-ing).9,15,20–24 Among them, the solid freeform fabricationsystem probably offers the best control of the macro-poros-ity and facilitates the complex shape formation required forbone scaffolds.9,15,20–24 The robocasting method used in thisstudy is a printing process that builds 3D structures layer-by-layer by extruding a continuous filament, or rod, througha tip guided by a computer-assisted positioning sys-tem.9,15,20–24 The technique permits printing with outstand-ing spatial resolution and has been used to print ceramic

Correspondence to: M. Houmard; e-mail: mhoumard@ufmg.br

Contract grant sponsor: National Institutes of Health/National Institute of Dental and Craniofacial Research; contract grant number: 1 R01

DE015633.

Contract grant sponsor: Department of Energy; contract grant number: DE-AC02-05CH11231

VC 2013 WILEY PERIODICALS, INC. 1233

grids with line and width diameters varying from hundredsof microns to sub-micron levels.24 The technique also allowseasy modification of the chemistry by adjusting the ink com-position and the optimization of the architecture forenhanced mechanical properties of the scaffolds. The aim ofthe present work was to investigate the structure-propertyrelationship of calcium phosphate scaffolds prepared by arobocasting technique. A systematic study was carried outon the porosity, mechanical property, and degradation ofscaffolds with three different compositions, pure HA, 60HA/40b-TCP, and 20HA/80b-TCP.

EXPERIMENTAL

Ink preparationCeramic inks were prepared by mixing ceramic powderswith a Pluronic F-127 solution according to a previouswork.20 The Pluronic solution exhibits a reverse thermalbehavior: it is fluid at low temperatures (close to 0�C) but asoft gel at room temperature. This reverse behavior makeseasy the dispersion of the HA/b-TCP ceramic powders intothe Pluronic solution at low temperature and forms a gelenough viscous to print scaffolds at room temperature. Thesolution was prepared by mixing a Pluronic powder withdeionized water (20 wt % of Pluronic) for 5 h using zirco-nia balls in a shaker (Red Devil 5400, Red Devil Equipment,Plymouth, MN). Next, the solution was cooled to �0�C in awater/ice bath and then, the ceramic powders were addedwhile maintaining the solution at low temperature to ensurethe complete reverse gelation process and to lower the vis-cosity. To ensure homogeneity, the inks were mixed thor-oughly for 10 min in the water/ice bath and afterwardssieved through a 75 lm mesh to minimize the presence ofaggregates. The inks were loaded into 10 mL syringes (BD,Franklin Lakes, NJ) with an HD-PTFE custom sized plunger.The syringes were gently tapped from the bottom to moveremaining bubbles to the top. Three ceramic powders withdifferent compositions, HA, 60HA/40b-TCP, and 20HA/80b-TCP (Trans-Tech, Adamstown, MD) were used for the inkpreparation. The average particle size and the specific sur-face area of the powders are summarized in Table I and theformulations of the inks are listed in Table II.

Scaffold printingHA/b-TCP ceramic cube grids (�7 � 7 � 7 mm3 before sin-tering) were fabricated using a robotic deposition device(Robocad 3.0, 3D Inks, Stillwater, OK).9,15,20–23 The diameter

of the ceramic rods, 200–610 lm, was controlled by thesize of the printing tips (EFD precision tips, EFD, East Provi-dence, RI). The distance between the printed rods was var-ied from 100 to 620 lm and the layer height, whichdepends of the rod diameter, was fixed at �75% of the tipdiameter to form a strong bonding between the successivelayers. The scaffolds were printed inside a reservoir of non-wetting oil (Lamplight, Menomonee Falls, WI) over a mir-ror-polished silicon wafer (0.6 mm thick). Since the siliconsurface is hydrophilic, it presents a certain degree of adhe-sion with the first printed line so that the scaffold remainsstable during printing. The smooth and inert mirror-pol-ished surface allows easy removal of the samples after fabri-cation, which facilitates handling and prevents deformationsdue to uneven shrinkage during drying and sintering. Afterprinting, the samples were dried for two days at room tem-perature, removed from the substrate, and sintered on topof zirconia balls (500 lm diameter) to favor a homogeneousshrinkage. Scaffolds were fired at 600�C (2�C/min heatingrate) for 2 h to burnout the organics, followed by sinteringfor 2 h at 1275�C for HA and at 1100�C for composite sam-ples with a heating and cooling rate of 5�C/min. The tem-perature of 1100�C for the composite structures was chosento avoid the formation of a-TCP crystals during sintering asthey can generate micro-cracks due to volume changes thusdecreasing the mechanical performance of the scaffolds.20,25

CharacterizationX-ray diffraction (XRD) (Siemens D-500, Munich, Germany)was used to identify the phases present in the sintered scaf-folds using Cu Ka radiation (k ¼ 0.15406 nm). The acquisi-tions were carried out at a scanning rate of 1.8�/min in the2y range of 20�–80� after grinding the scaffolds to powder.The spectra were compared with HA and b-TCP JCPDS cards09-0432 and 09-0169, respectively. As a result, matchingthe peak intensities of each phase, the percentage of HA andb-TCP phases in the powders and scaffolds were approxi-mated. The a-TCP phase is identified using JCPDS card 09-0348.

The microstructure of the scaffolds was examined withscanning electron microscopy (SEM) (Hitachi FE-SEM-4300EN). The scaffolds were previously sputter-coated withgold and then examined at an accelerating voltage of 10 kV.

The density of the scaffolds was calculated from themass and dimensions of at least five samples with regularshapes. The theoretical densities of HA (3.16 g/cm3) and b-TCP (3.14 g/cm3) were then used as references to calculatethe total volume fraction of porosity. The open porosity wasmeasured using the Archimedes’ method employing kero-sene as the liquid.

TABLE I. Average Particle Size and Surface Area of the HA,

60HA/40b-TCP, and 20HA/80b-TCP As-Received Powders

from Transtech

AverageParticle

Size (lm)Surface

Area (m2/g)

HA 1.999 4.080860HA/40b-TCP 2.057 3.50320HA/80b-TCP 2.082 3.2272

HA powder contains 6.8% of a-TCP.

TABLE II. Composition of the Inks Used to Print the

Robocast Scaffolds

HA 60HA/40b-TCP 20HA/80b-TCP

Ceramic (wt %) 74.1 66.6 61.2DiWater (wt %) 20.7 26.7 31F-127 (wt %) 5.2 6.7 7.8

1234 HOUMARD ET AL. BONE-SUBSTITUTE MATERIAL IN VARIOUS TISSUE-ENGINEERING APPLICATIONS

The compressive strength of the scaffolds was measuredon a servo-hydraulic testing machine (MTS810, MTS Sys-tems, Eden Prairie, MN) with a crosshead speed of 0.2 mm/min, for at least five samples from the same batch. Themeasurements were conducted by performing uniaxial testson the sintered cubic blocks (�6 � 6 � 6 mm3) placedbetween two cylindrical load platens. For all compositionsand parameters studied in this work, the sample dimensionhas been kept approximately constant with a standard devi-ation inferior to 5%.

Degradation of scaffolds was evaluated by determiningthe scaffold weight loss as a function of the immersion timein simulated body fluid (SBF) with a starting pH of �7.2 at37�C. The composition of the SBF corresponds to the cor-rected SBF solution previously published by Kokubo andTakadamaa.26 A ratio of 1 g of scaffold to 100 mL of SBFwas used for each test. After removal from the SBF atselected times, the scaffolds were dried at 100�C andweighed. The weight loss was defined as DWt ¼ (W0 �Wt)/W0, where W0 is the initial mass of the scaffolds and

Wt is the mass at time t. The solution was cooled from 37�Cto room temperature, and its pH measured using a pHmeter. Weight loss of the scaffolds in deionized water was alsoevaluated with a ratio of 1 g of scaffold to 20 mL of water.

Ca and P concentrations in the SBF and water solutions,after immersion of the scaffolds for 1 and 5 months, respec-tively, were analyzed using ICP (Inductively Coupled Plasmaatomic emission spectrometer, Perkin Elmer Optima 3000).

RESULTS AND DISCUSSION

General aspectsScaffold structure. Three-dimensional scaffolds wereprinted by extruding inks through cone tips from 200 to610 lm. Sintered scaffolds (printed with a 250 lm conetip) are illustrated in Figure 1. This figure shows an entireview of the sintered scaffolds [Figure 1(a)] and detailedmorphologies obtained by SEM [Figure 1(b–f)]. Theseimages demonstrate the excellent capability of the techniquein the production of periodical scaffolds with an accuratecontrolled and interconnected macro-porosity. The

FIGURE 1. Visualization of the HA/b-TCP robocast scaffolds after the sintering treatment. (a) Optical view of 20HA/80b-TCP scaffolds with two

different pore sizes: 330 lm (left) and 150 lm (right) (pore size before sintering); (b–d) are respectively corner, top, and cross-section views

obtained by SEM of a HA scaffold with 330 lm pore size before sintering; (e, f) are the cross section micrographs of 60HA/40b-TCP and 20HA/

80b-TCP scaffolds with pore of 330 and 620 lm before sintering, respectively.

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macro-pore size in the x- and y-directions [i.e., parallel tothe sample support, see Figure 1(b)] is determined by theprogram settings in the printing process (see ‘‘Scaffoldprinting’’ section). For target values ranging from 100 to620 lm before sintering, values ranging from 80 to 550 lmwere observed after sintering. The macro-pore size in thez-direction [i.e., perpendicular to the sample support, seeFigure 1(b)] is directly linked to the rod diameter (see‘‘Scaffold printing’’ section). Thus, for as-printed scaffoldswith rod diameters ranging from 200 to 610 lm before sin-tering, pores ranging from 60 to 250 lm were observed insintered samples. Such pore dimensions may promote cellseeding, proliferation, and vascularization, and bone forma-tion inside the scaffold as literature indicates that pores inthe range of 100–400 lm are optimal for the boneingrowth.27–31 This macro-porosity must also providetransport pathways for nutrients, oxygen, and wastes neces-sary to maintain living cells within the scaffold. The images[Figure 1(d–f)] also show a slight deformation of the rodsthat was caused by the gravity during the printing, drying,and sintering steps. This deformation effect was morereadily seen in scaffolds printed with larger tip [620 lm inFigure 1(e)] than smaller one [330 lm in Figure 1(f)].Nevertheless, the adhesion between the rods is strong[Figure 1(d–f)], which implies a reasonable mechanicalstrength for tissue engineering applications.

Composition. Crystalline phases in the sintered scaffoldswere detected by XRD. Figure 2 shows XRD spectra of theHA and composite scaffolds sintered at 1275 and 1100�C,respectively. A small amount of a-TCP phase [Figure 2(a)]was present in the spectrum of HA scaffold, which wasresulting from the impurities in the as-received powder (seeTable I). Only HA and b-TCP phases were observed in thesintered composite scaffolds [Figure 2(b,c)]. HA/b-TCP scaf-folds ratio in the composite scaffolds was determined bymatching the peak intensities with the JCPDS patterns.Ratios of 59/41 and 16/84 for the spectra of 60HA/40b-

TCP and 20HA/80b-TCP scaffolds were obtained, respec-tively. These values are close to those provided by the pow-der manufacturer, an indication that the robocasting andsintering processes does not significantly affect the chemis-try of the materials. To justify the sintering treatment at1100�C for the composite scaffolds, a XRD acquisition for a20HA/80b-TCP scaffold sintered at 1140�C is also illustratedin Figure 2. This spectrum not only shows the presence ofHA and b-TCP phases, but the presence of a-TCP phase wasalso detected. This example indicates that a-TCP crystals canbe generated in both 60HA/40b-TCP and 20HA/80b-TCPcomposite scaffolds during the sintering treatment at1140�C. Moreover, as mentioned previously, the presence ofa-TCP phase can induce micro-cracks in the scaffold struc-ture that would decrease its mechanical strength. Thus, the60HA/40b-TCP and 20HA/80b-TCP composite scaffolds inthis work were sintered at 1100�C to avoid the generationof micro-cracks in their structure.

Shrinkage. The shrinkage of the scaffolds after sinteringwas systematically studied as a function of the widthbetween the rods for the three compositions for scaffoldsprinted with tip diameter of 250 lm. Figure 3 shows thescaffold shrinkage in the three directions x, y, and z, for thethree compositions as a function of the as-printed macro-pore size. First, the shrinkage does not depend on the widthbetween the rods and it is about the same in all directions.Only a slight increase in the shrinkage is observed in the z-direction, which was due to a slight effect of the gravityduring the drying and sintering steps. Shrinkages of �16%,�8%, and �14% were measured for HA [Figure 3(a)],60HA/40b-TCP [Figure 3(b)] and 20HA/80b-TCP [Figure3(c)] scaffolds, respectively. The shrinkages values measuredfor HA and 20HA/80b-TCP scaffolds seem to indicate thechoice of efficient sintering conditions for these composi-tions. The lower shrinkage ratio for the 60HA/40b-TCP scaf-folds could result from a sintering temperature (1100�C)too far away from the adequate sintering temperature ofthe HA phase, which is the major component of this

FIGURE 2. XRD spectra of robocast scaffolds after sintering at 1100�C

with different HA/b-TCP compositions: (a) 100/0, (b) 60/40, and (c) 20/

80. HA (*), b-TCP (o), and a-TCP (þ) peaks are identified according to

the JCPDS patterns 09-0432, 09-0169, and 09-0348, respectively. The

spectrum of a 20HA/80b-TCP scaffolds sintered at 1140�C (d) is also

drawn.

FIGURE 3. (a) Shrinkage of the pure HA, (b) 60HA/40b-TCP, and (c)

20HA/80b-TCP scaffolds after sintering in the three directions, x (//), y

(\\), and z (n), as a function of the macro-pore size before sintering for

scaffolds printed with a tip diameter of 250 lm.

1236 HOUMARD ET AL. BONE-SUBSTITUTE MATERIAL IN VARIOUS TISSUE-ENGINEERING APPLICATIONS

composition. However, according to the previous section,the sintering temperature of the 60HA/40b-TCP scaffoldscannot be increased due to the possible formation of unde-sirable a-TCP crystals in the structure at higher tempera-tures. To conclude, the shrinkages are higher for HA andrich b-TCP scaffolds since the sintering temperatures arebetter adjusted for these compositions.

Total and open porositiesPorosity of HA, 60HA/40b-TCP, and 20HA/80b-TCP scaffoldswas controlled by varying the width between the rods inthe x-y plan during the printing process (macro-pores from100 to 620 lm). Total and open porosities of the scaffoldswere measured as a function of the width between the rodsfor the three types of scaffolds printed with tip diameter of250 lm. Figure 4 presents the open and total porosities ofthe scaffolds as a function of the macro-pore size before

sintering. The figure shows that with the increase of thewidth between the rods, i.e., the macro-pore size, both openand total volume porosities increase regardless of the com-position of the scaffold. The close value between the totaland open porosities indicates that the majority of the poresin the printed structures are open. HA scaffolds exhibithigher amount of closed pores than the composite scaffolds,as will be discussed later. Also, HA scaffolds exhibit the low-est open and total porosities, and the difference with thecomposite structures became more obvious for smallermacro-pore sizes. Figure 4 also shows that, when printedwith the same micro-pore size, 60HA/40b-TCP scaffolds areslightly more porous than 20HA/80b-TCP counterparts. Thetotal porosity measured here includes both the macro-poresdefined by the rod spacing designed during printing and themicro-pores present inside the rods. This micro-porosityresults from both sintering conditions and burnout of theorganics present in the initial inks, including Pluronic andwater. To compare porosity variations between the threecompositions, rod’s micro-porosity were observed by SEM.

Figure 5 shows pictures of the surface and fracture ofthe rods in HA, 60HA/40b-TCP, and 20HA/80b-TCP scaf-folds. The HA rods show the densest structure [Figures 1(d)and 5(a–d)] resulting from the highest shrinkage ratio(Figure 3) and the highest amount of ceramic matter in theinitial ink (Table II). In contrast, composite rods show ahigher amount of micro-pores with two different rangesizes: one of �20–30 lm [Figures 1(e,f)] and a smaller oneof �1 lm [Figure 5(b,c,e,f)]. However, the rods from 60HA/40b-TCP and 20HA/80b-TCP scaffolds do not show identicalmorphologies. The 20HA/80b-TCP rods present a porousstructure with relatively well sintered grains, in agreementwith the shrinkage data, while the 60HA/40b-TCP exhibitseparated particles among interconnected and smallermicro-pores (Figure 5). The 20HA/80b-TCP micro-pores arelarger than those of the 60HA/40b-TCP structure probably

FIGURE 4. (a) Open (//) and total (n) volume porosity of the pure HA,

(b) 60HA/40b-TCP, and (c) 20HA/80b-TCP scaffolds after sintering as a

function of the macro-pore size before sintering for scaffolds printed

with a tip diameter of 250 lm.

FIGURE 5. (a,d) Surface (top) and fracture (bottom) SEM micrographs of HA; (b,e) 60HA/40b-TCP, and (c,f) 20HA/80b-TCP rods after sintering.

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due to the higher amount of organics in the initial paste.These SEM observations correspond well with the data inFigure 4. Indeed, composite rods are full of micro-poresleading to an almost completely open porosity, while the HArods are much denser yielding a higher amount of closedpores (Figure 4). Also, since HA rods are much denser thanthe composite ones, the porosity decreases even faster withincreasing number of rods (Figure 4). Finally, even if thepore morphologies differ, the total and open porosity valuesin both composite are quite similar, which must result fromvariations in the sintering process and ink compositions.Indeed, the sintering is less efficient for the 60HA/40b-TCPcomposition, but on the other hand its initial amount of ce-ramic is higher. Porosity will have an influence on the me-chanical performance of the bone scaffolds. To conclude, theimportant presence of micro-porosity in BCP rods, which isnot observed in HA, could be a key to explain enhancedbone ingrowth performance of such materials reported inthe literature.

Mechanical strengthEffect of the diameter of the rod. As described earlier, scaf-folds were printed with cone tips ranging from 200 to 610lm in diameter. The effect of the rod size on the mechanicalproperties of HA scaffolds was investigated. Constant poros-ity (45 vol %) was achieved by adjusting the width betweenthe printed rods to accommodate the differences in theirrod diameters. Figure 6 shows compressive strength of HAscaffolds as a function of the tip diameter. When the tip di-ameter increases from 200 to 610 lm, significant decreaseof the mechanical properties occurs, from 23 to 8 MPa. Tounderstand this observation, typical stress—deformationcurves are illustrated in insets of the Figure 6 for scaffoldsprinted with tip diameters of 250 and 510 lm (insets Aand B, respectively). First, as observed in a previous work,22

these insets show that the rupture mechanism of the robo-cast structures is not as sudden as a bulk ceramic. Indeed,the stress does not increase linearly until final rupture; it

goes up and down due to the numerous cracks appearing inthe scaffold during the deformation, which damage thestructure and decrease its stiffness. Then, for scaffoldprinted with a smaller tip, the number of rods is higher (ata constant porosity) and the successive cracks present inthe scaffold are not catastrophic since the remaining rodsare strong enough to bear the stress along the applied de-formation (inset A of Figure 6). The stars included in theinset illustrate some obvious cracks occurring in the struc-ture during the compressive test. When the stress becometoo high for the structure, the sample collapses completelyresulting in a sudden decrease of the stress (inset A of Fig-ure 6). In contrast, for scaffolds printed with a larger tip,the inset B of Figure 6 shows that the successive cracks(some cracks are indicated by a star) seem to induce signifi-cant defects and the structure could not withstand highstresses during the deformation. To conclude, these resultspoint out the interest in fabricating very thin structures toget stronger porous materials for various applications.

Effect of the macro-pore size. The compressive strength ofthe scaffolds as a function of their total porosity is shownin Figure 7 for scaffolds printed with a tip diameter of 250lm. The figure indicates that the compressive strength grad-ually increases, as expected, when the total porositydecreases for all three compositions. Then, few differencesin the compressive strength are observed between the com-positions for a given porosity. The 20HA/80b-TCP scaffoldsis slightly stronger than the other two when the porositydecreases below 60 vol %. This behavior probably resultsfrom a relatively better sintering than 60HA/40b-TCP, whichgive strength to the structure, even if there is still an impor-tant micro-porosity in the rods [see Figures 5(c–f)]. Mechan-ical tests performed on 60HA/40b-TCP indicate lowerstrength in the x- and y-directions parallel to the printingplan (inset of the Figure 7). However, the values are veryclose to each other in agreement with early claims indicat-ing that robocasted grid scaffolds exhibit quite similar

FIGURE 6. Compressive strength as a function of the robocast tip di-

ameter for pure HA scaffolds with a constant volume porosity of 45

vol % 6 2.2. The insets represent characteristic strength � deforma-

tion curves of scaffolds fabricated with tip diameters of 250 lm (inset

a) and 510 lm (inset b). The stars included in the inset illustrate some

obvious cracks occurring in the structure during the compressive test.

FIGURE 7. Compressive strength along the z-axis as a function of the

total porosity for the pure HA (*), 60HA/40b-TCP (h), and 20HA/80b-

TCP (D) robocast scaffolds printed with different rod width and with a

tip diameter of 250 lm. The inset compares the compressive tests

performed along the z-axis (h) and perpendicularly to the z-axis (n)

directions for the 60HA/40b-TCP scaffolds.

1238 HOUMARD ET AL. BONE-SUBSTITUTE MATERIAL IN VARIOUS TISSUE-ENGINEERING APPLICATIONS

compressive strength in all directions.21 Figure 7 also showsthat regardless of the composition the compressivestrengths of the fabricated scaffolds (in all three directions)were higher or in the same range as those reported forhuman cancellous bone (2–12 MPa). According to a recentreview on CaP scaffolds,32 the measured compressivestrengths are similar to those reported in the literature forHA/b-TCP scaffolds with similar porosity.33,34 Moreover,since the macro-pore size is an important factor for boneingrowth into scaffold,27–31 Figure 8 shows the scaffoldcompressive strength as a function of the macro-pore sizefor the three compositions. Figure 8 indicates that for agiven macro-pore size, the strength of the scaffolds varies in

the order HA > 20HA/80b-TCP > 60HA/40b-TCP. This clas-sification results from variation in rod structures and espe-cially the presence of micro-pores. This corresponds wellwith the observation from Figure 5, which shows a bettersintering of the HA particles yielding to very dense rodsand thus stronger scaffolds. Then, the micro-porous 20HA/80b-TCP structure presented well sintered grains as com-pared with the 60HA/40b-TCP one (Figure 5), whichexplains the difference between the strength of compositesamples. In other words, for a same macro-pore size, thestrength of the scaffold is controlled by the micro-porosityand the sintering efficiency. To conclude, the compressivestrengths measured here are high enough for scaffold han-dling, which makes these materials good candidate for invitro or in vivo experiments. And for specified applicationsrequiring certain strength and minimal macro-pore size, Fig-ure 8 allows us to choose the possible scaffold compositionsto fill the corresponding bone defect.

Design of the scaffoldFigure 9 shows some examples of patterns designed byrobocasting with the HA/b-TCP composition studied here.Scaffolds with a gradient porosity in the x-y directions werefabricated [Figure 9(a,b)]. Figure 9(c) illustrates the produc-tion of bio-inspired scaffold with a radial gradient: highlyporous in the middle and denser at the external perimeter,mimicking the structure of a real bone. Also, alternated pat-tern and gradient in the z-axis can be obtained as presentedin Figure 9(d,e), respectively. These patterns indicate thecapability of the device, which allows excellent control ofthe ceramic design and spatial distribution of porosity. All

FIGURE 8. Compressive strength along the z-axis as a function of the

macro-pore size before sintering for the pure HA (*), 60HA/40b-TCP

(h), and 20HA/80b-TCP (D) robocast scaffolds printed with a tip diam-

eter of 250 lm.

FIGURE 9. SEM micrographs of scaffolds printed with different design. (a) Cross-section and (b) top views of a scaffold printed with a gradient

porosity along the x- and y-axis. (c) Top view of a scaffold patterned with a radial gradient porosity (bone-like structure). (d) Cross-section of a

scaffold designed with an alternate structure. (e) Top view of a scaffold built with a gradient porosity along the z-axis.

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these characteristics could play important roles in the scaf-fold performance. Since the porosity must provide transportpathways for nutrients, oxygen, and wastes necessary tomaintain the cells alive inside the scaffold, its design hascertainly a preponderant influence on the in vitro and invivo performance. Modifying the structure of the scaffoldcould also affect its mechanical properties. That is why nu-merical models are currently being developed in ourresearch group to predict the mechanical properties of suchrobocast ceramics.35 These studies would be able to providedirections to improve the mechanical properties of the scaf-fold as a function of its design, although, compromisesbetween enhanced mechanical properties and improvedbone ingrowth may have to be made.

In vitro degradationThe great interest of HA/b-TCP composite scaffolds for boneregeneration is based on the preferential dissolution of theb-TCP phase.3,4 Thus, adjusting the HA/b-TCP ratio shouldlead to a control of the scaffold degradation and its replace-ment by new bone. In this context, the degradation of therobocast scaffold in SBF was studied as a function of theHA/b-TCP composition during a static immersion for onemonth. Figures 10 and 11 show pH and scaffold weightchanges as a function of the immersion time for the threecompositions studied. Figure 10 exhibits gradual increase ofthe pH value of SBF during the immersion. The valuereached �7.55 for all three compositions. The SBF solutioncontaining the 20HA/80b-TCP samples appears to have thefastest dissolution rate while the one with the 60HA/40b-TCP composites the slowest one. In agreement with the lit-erature, changes in pH during SBF immersion could beattributed to the dissolution of CaP releasing Ca2þ, PO4

3�,and OH� ions in solution, which then would precipitate ascarbonate apatite on the material surface.36–38 The releaseof Ca2þ and OH� ions led to an increase in the pH value ofSBF. Moreover, variation in pH for the different compositionscould result from differences in HA/b-TCP ratio, grain size,and porosity of the scaffolds.37,39 Figure 11 shows theweight loss of the scaffolds after immersion in SBF for one

month. No significant weight loss was observed for all scaf-folds (<1%). The results is contradicting to the literaturereports, which claim BCP scaffolds degrades much fasterthan HA.3,4 However, to the authors’ knowledge, there is nodirect measurement of the weight loss of such BCP scaffoldin vitro or in vivo. The weight loss data correspond wellwith Ca and P concentration in SBF measured by ICP (TableIII). The data show that Ca and P concentration afterimmersion of HA samples during one month almost did notchange the solution composition. For the solutions wherecomposite scaffolds where immersed, there was decrease inthe concentration of Ca and P. However, the decrease wasvery small (�1.0 mMol/L for Ca and P, respectively) and didnot account for the formation of any crystals on the scaffoldsurface. These results indicate that the degradation of suchceramic structures in static SBF solution was very slow.Dynamic system and/or longer immersion time may bemore appropriate to observe a significant degradation ofsuch scaffolds. We should also mention that in vitro experi-ments performed in SBF solution are recently greatlycriticized by numerous scientists.40 As a result, in vivo testswould be a much more adequate way to carefully character-ize the degradation of such scaffolds in the human body.

To investigate the degradation of these scaffolds, anotherexperiment was conducted by immersion in water. Althoughwater is different from SBF or human blood plasma, thistesting gives a better understanding and direct measure-ment of the degradation rate. Weight loss and ICP measure-ments are reported in Figure 12 and Table III, respectively.Figure 12 does not present any variation of mass for thethree compositions studied during 5 months of immersionin deionized water; the small and random variationsobserved are included in the error deviation of the experi-mental balance. Thus, the result is consistent with thoseconducted in SBF. However, ICP analyses after 5 months ofimmersion allow the determination of some differencesbetween the different compositions. As a control, the watersolution without scaffold exhibits a total absence of calciumand phosphate components. The water solutions with HAand 20HA/80b-TCP scaffolds show slight dissolutions of the

FIGURE 10. pH of the solution as a function of the immersion time in

the SBF solution for HA (*), 60HA/40b-TCP (h), and 20HA/80b-TCP (D)

robocast scaffolds.

FIGURE 11. Scaffold weight loss as a function of the immersion time

in the SBF solution for the HA (*), 60HA/40b-TCP (h), and 20HA/80b-

TCP (D) compositions.

1240 HOUMARD ET AL. BONE-SUBSTITUTE MATERIAL IN VARIOUS TISSUE-ENGINEERING APPLICATIONS

CaP scaffolds. Finally, the ICP results show a much higherdissolution of Ca and P for the 60HA/40b-TCP scaffolds;�20 times higher than HA, which corresponds approxi-mately to values reported in literature for such HA/b-TCPmaterial.41,42 To conclude, there is no noticeable weight lossin scaffold after immersion in SBF and water after one and5 months, respectively. The ICP and weight loss measure-ment correspond well, which is an indication of the accuratemeasurement of the weight loss. The measurement of Caand P ions in water indicates a faster degradation of BCPscaffolds than HA, especially for the 60HA/40b-TCP compo-sition. A longer immersion time or a vigorous immersioncondition is required to see a noticeable degradation of thescaffolds. To conclude, and as it has been already mentionedbefore, in vivo studies may provide more information aboutthe performances of these scaffolds for the treatment ofmedium to high load-bearing bone defects.

CONCLUSION

HA/b-TCP composite scaffolds with structured porosity andcontrolled chemistry have been successfully fabricated byrobocasting using water-based Pluronic inks. Three compo-sitions, pure HA, 60HA/40b-TCP, and 20HA/80b-TCP havebeen studied regarding their porosity, mechanical strength,and biodegradation, which are key factors in determiningscaffold performance. Scaffolds with mechanical strengthranging from �3 to �50 MPa were produced with a totalporosity varying from �80 to �25 vol%, respectively. Varia-tions in properties between the compositions were associ-

ated with the higher micro-porosity of composite scaffoldsresulting from differences in the ink chemistries and in thesintering step. The fact that the macro-pore sizes beingabout hundred of micrometers and the compressivestrengths equal to or higher than the range of human can-cellous bone (2–12 MPa), indicates that the materials shouldpermit bone ingrowth into the designed scaffolds for load-bearing bone substitute applications. For a given size ofmacro-pores, mechanical properties vary as a function ofthe composition in the following order HA > 20HA/80b-TCP > 60HA/40b-TCP. Besides, robocast scaffolds printedwith various rod sizes, at a given porosity, show that stron-ger scaffolds were obtained from thin printed rods. Thisresult suggests the scientific interest to fabricate thin struc-tures for biomedical and various applications. No noticeabledegradation of the scaffolds in SBF or water was observed,although there was a slight increase of Ca and P ions inwater after 5 month. The work reported here permits toclassify the scaffolds regarding their different characteristics.Thus, this work should be a useful database in the choice ofscaffold for tissue engineering applications. Ongoing in vivostudies in our research group may provide further guidanceon the selection of scaffolds with the largest quantity ofbone ingrowth.

ACKNOWLEDGMENTS

Russell Anderson from the Research Analytical Laboratory atthe University of Minnesota (http://ral.cfans.umn.edu) isgratefully acknowledged for the ICP measurements.

REFERENCES

1. Hench LL. Bioceramics. J Am Ceram Soc 1998;81:1705.

2. Hassna RR, Ramay ZM. Biphasic calcium phosphate nanocompo-

site porous scaffolds for load-bearing bone tissue engineering.

Biomaterials 2004;25:5171.

3. LeGeros RZ, Lin S, Rohanizadeh R, Mijares D, LeGeros JP. Bipha-

sic calcium phosphate bioceramics: Preparation, properties and

applications. J Mater Sci Med 2003;14:201.

4. Daculsi G, LeGeros RZ, Nery E, Lynch K, Kerebel B. Transforma-

tion of biphasic calcium phosphate ceramics in vivo: Ultrastruc-

tural and physicochemical characterization. J Biomed Mater Res

1989;23:883.

5. LeGeros RZ, Daculsi G, LeGeros JP. Bioactive bioceramics. In:

Pietrzak WS, editor. Orthopedic Biology and Medicine: Musculo-

skeletal Tissue Regeneration: Biological Materials and Methods,

Chapter 8.. New Jersey: Humana Press; 2008. pp153–181.

6. LeGeros RZ, LeGeros JP. Hydroxyapatite. In: Kokubo T, editor.

Bioceramics and Their Clinical Applications. Cambridge: Wood-

head; 2008. pp367–394.

TABLE III. Amounts of Calcium and Phosphate Ions (in mMol/L) Introduced in the Prepared SBF Solution According to

Kokubo and Takadamaa26 and Amounts Measured by ICP in the Different SBF and Water Solutions into which the HA, 60HA/

40b-TCP, and 20HA/80b-TCP Scaffolds Were Immersed

SBF Compo.25

ICP Measurements (Deviation < 0.05 mMol/L)

Scaffolds in SBF (1 month) Scaffolds in water (5 months)

Control HA 60/40 20/80 Control HA 60/40 20/80

Ca2þ 2.5 2.49 2.53 1.38 1.46 0.00 0.09 3.30 0.41HPO4

2� 1 1.04 1.04 0.10 0.06 0.01 0.31 6.00 0.34

Ions amount into the SBF and water solutions without immersion of samples have been also quantified by ICP as a control.

FIGURE 12. Scaffold weight loss as a function of the immersion time

in water for the HA (*), 60HA/40b-TCP (h), and 20HA/80b-TCP (D)

compositions.

ORIGINAL RESEARCH REPORT

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | OCT 2013 VOL 101B, ISSUE 7 1241

7. Malard O, Guicheux J, Bouler JM, Gauthier O, Beauvillain de

Montreuil C, Aguado E, Pilet P, LeGeros RZ, Daculsi G. Calcium

phosphate scaffold and bone marrow for bone reconstruction in

irradiated area: A dog study. Bone 2005;36:323.

8. Arinzeh TL, Peter SJ, Archambault MP, Van Den Bos C, Gordon S,

Kraus K, Smith A, Kadiyala S. Allogeneic mesynchymal stem cells

rgenerate bone in a critical sized canine segmental defect. J Bone

Joint Surg Am 2003;85A:1927.

9. Miranda P, Pajares A, Saiz E, Tomsia AP, Guiberteau F. Mechani-

cal properties of calcium phosphate scaffolds fabricated by robo-

casting. J Biomed Mater Res A 2008;85A:218.

10. De Groot K. Ceramics of calcium phosphates: Preparation and

properties. In: De Groot K, editor. Bioceramics of Calcium Phos-

phates. Boca Raton: CRC Press; 1983. pp99–114.

11. Nancollas GH. In vitro studies of calcium phosphate crystallisa-

tion. In: Mann S, Webb J, Williams RJP, editors. Biomineraliza-

tion. Chemical and Biochemical Perspectives. Weinheim,

Germany: VCH Publishers; 1989. pp157–189.

12. Daculsi G, Passuti N, Martin S, Deudon C, Legeros RZ, Raher S.

Macroporous calcium phosphate ceramic for long bone surgery

in humans and dogs: Clinical and histological study. J Biomed

Mater Res 1990;24:379.

13. Hulbert SF, Morrison SJ, Klawitte JJ. Tissue reaction to three

ceramics of porous and non-porous structures. J Biomed Mater

Res 1972;6:347.

14. Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920.

15. Saiz E, Gremillard L, Menendez G, Miranda P, Gryn K, Tomsia AP.

Preparation of porous hydroxyapatite scaffolds. Mater Sci Eng C

Bio S 2007;27:546.

16. Ben-Nissan B. Natural bioceramics: from coral to bone and

beyond. Curr Opin Solid State Mater Sci 2003;7:283.

17. Bohner M, Van Lenthe GH, Grunenfelder S, Hirsiger W, Evison R,

Muller R. Synthesis and characterization of porous beta-tricalcium

phosphate blocks. Biomaterials 2005;26:6099.

18. Sous M, Bareille R, Rouais F, Clement D, Amedee J, Dupuy B,

Baquey Ch. Cellular biocompatibility and resistance to compres-

sion of macroporous beta-tricalcium phosphate ceramics. Bioma-

terials 1998;19:2147.

19. Deville S, Saiz E, Tomsia AP. Freeze casting of hydroxyapatite

scaffolds for bone tissue engineering. Biomaterials 2006;27:5480.

20. Franco J, Hunger P, Launey ME, Tomsia AP, Saiz E. Direct write

assembly of calcium phosphate scaffolds using a water-based

hydrogel. Acta Biomater 2010;6:218.

21. Miranda P, Saiz E, Gryn K, Tomsia AP. Sintering and robocasting

of b-tricalcium phosphate scaffolds for orthopaedic applications.

Acta Biomater 2006;2:457.

22. Miranda P, Pajares A, Saiz E, Tomsia AP, Guiberteau F. Fracture

modes under uniaxial compression in hydroxyapatite scaffolds

fabricated by robocasting. J Biomed Mater Res Part A 2007;83A:

646.

23. Houmard M, Fu Q, Saiz E, Tomsia AP. Sol–gel method to fabricate

CaP scaffolds by robocasting for tissue engineering. J Mater Sci:

Mater Med 2012;23:921.

24. Duoss EB, Twardowski M, Lewis JA. Sol–gel inks for direct-write

assembly of functional oxides. Adv Mater 2007;19:3485.

25. Case ED, Smith IO, Baumann MJ. Microcracking and porosity in

calcium phosphates and the implications for bone tissue engi-

neering. Mater Sci Eng A Struct 2005;390:246.

26. Kokubo T, Takadamaa H. How useful is SBF in predicting in vivo

bone bioactivity? Biomaterials 2006;27:2907.

27. Murphy CM, Haugh MG, O’Brien FJ. The effect of mean pore size

on cell attachment, proliferation and migration in collagen–gly-

cosaminoglycan scaffolds for bone tissue engineering. Biomateri-

als 2010;31:461.

28. Oh SH, Park IK, Kim JM, Lee JH. In vitro and in vivo characteris-

tics of PCL scaffolds with pore size gradient fabricated by a cen-

trifugation method. Biomaterials 2007;28:1664.

29. Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds

and osteogenesis. Biomaterials 2005;26:5474.

30. Petite H, Viateau V, Bensaıd W, Meunier A, De Pollak C, Bour-

guignon M, Oudina K, Sedel L, Guillemin G. Tissue-engineered

bone regeneration. Nat Biotechnol 2000;18:959.

31. Pineda LM, Busing M, Meinig RP, Gogolewski S. Bone regenera-

tion with resorbable polymeric membranes. III. Effect of poly(L-

lactide) membrane pore size on the bone healing process in large

defects. J Biomed Mater Res 1996;31:385.

32. Wagoner Johnson AJ, Herschler BA. A review of the mechanical

behavior of CaP and CaP/polymer composites for applications in

bone replacement and repair. Acta Biomater 2011;7:16.

33. Cordell JM, Vogl ML, Wagoner Johnson AJ, The influence of

micropore size on the mechanical properties of bulk hydroxyapa-

tite and hydroxyapatite scaffolds, J Mech Behav Biomed Mater

2009;2:560.

34. Bignon A, Chouteau J, Chevalier J, Fantozzi G, Carret J, Chavas-

sieux P, Boivin G, Melin M, Hartmann D. Effect of micro- and

macroporosity of bone substitutes on their mechanical properties

and cellular response. J Mater Sci Mater Med 2003;14:1089.

35. Genet M, Houmard M, Eslava S, Saiz E, Tomsia AP. A two-scale

model of the mechanical behavior of ceramic scaffolds made by

robocasting: possibilities and limits. J Eur Ceram Soc 2013: 33:

679.

36. Raynaud S, Champion E, Bernache-Assollant D, Tetard D.

Dynamic fatigue and degradation in solution of hydroxyapatite

ceramics. J Mater Sci: Mater Med 1998;9:221.

37. Kwon SH, Jun YK, Hong SH, Kim HE. Synthesis and dissolution

behavior of b-TCP and HA/b-TCP composite powders. J Eur

Ceram Soc 2003;23:1039.

38. Sanchez-Salcedo S, Balas F, Izquierdo-Barba I, Vallet-Regi M. In

vitro structural changes in porous HA/b-TCP scaolds in simulated

body fluid. Acta Biomater 2009;5:2738.

39. Lu J, Descamps M, Dejou J, Koubi G, Hardouin P, Lemaitre J,

Proust JP. The biodegradation mechanism of calcium phosphate

biomaterials in bone. J Biomed Mater Res 2002;63:408.

40. Bohner M, Lemaitre J. Can bioactivity be tested in vitro with SBF

solution? Biomaterials 2009: 30:2175.

41. Kohri M, Miki K, Waite DE, Nakajima H, Okabe T. In vitro stability

of biphasic calcium phosphate ceramics. Biomaterials 1993;14:

299.

42. Piattelli A, Scarano A, Mangano C. Clinical and histologic aspects

of biphasic calcium phosphate ceramic (BCP) used in connection

with implant placement. Biomaterials 1996;17:1767.

1242 HOUMARD ET AL. BONE-SUBSTITUTE MATERIAL IN VARIOUS TISSUE-ENGINEERING APPLICATIONS