Biofabrication

Biofabrication 6 (2014) 015006 (12pp) doi:10.1088/1758-5082/6/1/015006

Direct 3D powder printing of biphasiccalcium phosphate scaffolds forsubstitution of complex bone defects

Miguel Castilho1, Claus Moseke2, Andrea Ewald2, Uwe Gbureck2,Jurgen Groll2, Ines Pires1, Jorg Teßmar2 and Elke Vorndran2,3

1 Institute of Mechanical Engineering/IST, Technical University of Lisbon, Portugal2 Department for Functional Materials in Medicine and Dentistry, University of Wurzburg,D-97070 Wurzburg, Germany

E-mail: elke.vorndran@fmz.uni-wuerzburg.de

Received 4 April 2013, revised 21 November 2013Accepted for publication 25 November 2013Published 15 January 2014

AbstractThe 3D printing technique based on cement powders is an excellent method for the fabricationof individual and complex bone substitutes even in the case of large defects. The outstandingbone remodeling capacity of biphasic calcium phosphates (BCPs) containing hydroxyapatite(HA) as well as tricalcium phosphate (TCP) in varying ratios makes the adaption of powdersystems resulting in BCP materials to this fabrication technique a desirable aim. This studypresents the synthesis and characterization of a novel powder system for the 3D printingprocess, intended for the production of complexly shaped BCP scaffolds by a hydraulic settingreaction of calcium carbonate and TCP with phosphoric acid. The HA/TCP ratio in thespecimens could be tailored by the calcium/phosphate ratio of the starting powder. Thescaffolds could be fabricated with a dimensional accuracy of >96.5% and a minimal macropore size of 300 μm. Independent of the phase composition the printed specimens showed amicroporosity of approximately 68%, while the compressive strength strongly depended onthe chemical composition and increased with rising TCP content in the scaffolds to amaximum of 1.81 MPa. Post-treatment of the scaffolds with a polylactic-co-glycolicacid-solution enhanced the mechanical properties by a factor of 8. In vitro studies showed thatall BCP scaffolds were cytocompatible and enhanced the cell viability as well as the cellproliferation, as compared with pure TCP. Cell proliferation is even better on BCP whencompared to HA and cell viability is in a similar range on these materials.

Keywords: cytocompatibility, 3D printing, bone grafts, biphasic calcium phosphates,hydroxyapatite, tricalcium phosphate, bioceramic

(Some figures may appear in colour only in the online journal)

1. Introduction

The application of synthetic materials for bone replacementprovides several advantages, as compared to conventionaltherapies using autologous, allogenous or xenogenousbone tissue. Amongst others, these advantages comprise aminimized risk of pathogen transfer or immune reactions aswell as practically unlimited availability. Basic requirements

3 Author to whom any correspondence should be addressed.

to these materials are excellent biocompatibility and aminimum mechanical stability in the range of cancellousbone. Nevertheless, particularly in maxillofacial surgery themechanical properties tend to lose priority in favor of highosteoinductivity on one hand and individual moldability onthe other. Moreover a rapid osteointegration of the implantwill improve mechanical stability due to bone tissue growinginto the scaffold. To achieve this goal, it is essential that thescaffold simulates natural bone tissue concerning composition,morphology, structure, and mechanical properties most

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Biofabrication 6 (2014) 015006 M Castilho et al

adequately. According to Karageorgiou et al the main criteriafor this purpose are sufficient mechanical strength within therange of cancellous bone as well as high interconnectiveporosity of at least 50% with pore sizes between 100–800 μm, whereas the exchange of nutrients and cell ingrowthstrongly depend on the characteristics of the pores [1].Calcium phosphates are being successfully applied as bonereplacement materials due to their chemical similarity to themineral component of bone. Granules, injectable materials ormonoliths consisting of hydroxyapatite (HA) [2], tricalciumphosphate (TCP) [3] or biphasic calcium phosphates (BCPs)of different HA/TCP ratios are in clinical use. Several in vivostudies document the ability of BCP structures to promote boneingrowth and remodeling as well as vascularization [4–8]. Thesuccess of these biphasic products as bone substitutes arisesfrom the combination of the higher solubility of ß-TCP with thelower solubility of HA, involving a superior biocompatibilityand promotion of early bone ingrowth [9–11]. The outstandingbone remodeling capacity of BCP is often attributed to thehigh biodegradability of ß-TCP and the resulting releaseof calcium ions, which leads—together with ions fromthe surrounding body fluids—to the precipitation of a bone-like apatite layer [12]. Since different applications in diverseparts of the organism require different resorption times, theregulation of the HA/TCP ratio provides the possibility totailor the degradation kinetics of the scaffold [13]. Althoughall studies concluded a higher bioactivity of BCP compared tophase-pure calcium phosphates, there are different statementsconcerning the optimal HA/TCP ratio for certain applications.One important aim is the fabrication of a scaffold, wherethe degradation of the ceramic and the formation of newbone tissue are simultaneously occurring processes, henceensuring optimal healing of the bone defect. The solubilityof BCP ceramics not only depends on the HA/TCP ratio,but is also crucially governed by porosity, pore size andinterconnectivity of the scaffold [14]. Usually the solubilityis increasing with a rising TCP fraction [15, 13]. Although themostly investigated HA/TCP combination is the 60/40 ratio,the compositions differ in wide ranges as do the statementsconcerning the highest bioactivity and osteoconductivityamong several HA/TCP ratios. All studies agree that BCPsshow an enhanced bone regeneration, as compared to phase-pure calcium phosphates [6, 8, 16–18].

The reconstruction of complex bone defects requiresfreely moldable materials that enable the fabrication of patientspecific implants. Rapid prototyping techniques are excellentmethods to produce tissue replacement materials with acomplex internal or external structure based on computertomography data or prefabricated structure designs. Onevariant of these methods, namely 3D powder printing, isbased on the binding of powder particles or granules eitherby injecting a reactive solution [19, 20] or by polymericgluing [8] in a layer-by-layer process until the completionof the structure. 3D printed samples are characterized by ahigh microporosity (above 30 vol%), which results from thevoids between the powder particles and supports nutrientsdiffusion, cell ingrowth and vascularization [1, 21]. Due tothe free designability of the external structure as well as of the

macroporosity in the range of a few 100 μm the properties ofthe scaffold (shape, mechanical stability, biological behavior)can be optimally adjusted to match a certain kind of tissue,which has to be replaced. Recent studies deal with theadaption of the 3D printing process to the fabrication ofbone substitutes from the above-mentioned materials HA,ß-TCP, and BCP [19, 22–26]. Schumacher et al utilized anegative-mould rapid prototyping technique to create porousHA, ß-TCP, and BCP scaffolds by printing a mould with waxand infiltrating it with the ceramic slurry. These scaffoldsare characterized by 26.0–71.9 vol% interconnecting poresand pore diameters of approximately 340 μm [27]. Detschet al studied the cell differentiation of 3D printed scaffoldsof pure HA, TCP or BCP with a microporosity of 52–56%[8]. Scaffolds were prepared from spray-dried granules of therespective phase composition containing polymeric additivesas binder. The results showed good in vitro biocompatibilityas well as promoted differentiation of the monocytic cell lineRAW 264.7.

This study presents the synthesis and characterization ofa novel powder system for the 3D printing process, intendedfor the production of complexly shaped BCP scaffolds. Up tonow, two methods of producing BCP ceramics by 3D printingtechniques have been established. One possibility is the mixingof HA and ß-TCP powder and the solidification by polymergluing [8, 28]. The other method implies the printing of aninverse mould of the desired scaffold, which is followed byinfiltration with ceramic slurry and burning out the negativematrix material [27, 29]. In the present study a 3D printingprocess, which is based on a hydraulic setting reaction ofcalcium carbonate and TCP with phosphoric acid, was usedfor the first time to produce BCP ceramics in a direct route. Abig advantage of this method is the complete conversion of allcomponents involved in the production process (raw powderand binder solution) to a cement matrix. Hence, no burning outof any support materials or organic solvents with the risk ofleaving behind harmful residues [30, 31] is required. Althoughthis technique allows the fabrication of HA [32], brushite [33]or magnesium phosphate [34] based structures, there was nopossibility to produce a cement matrix consisting of a mixtureof HA and TCP. The HA/TCP ratio of the printed and sinteredspecimens can be tailored by adjusting the calcium/phosphor(Ca/P) ratio of the starting powder. To demonstrate this,four powders with different Ca/P ratios were prepared andused for the printing process. Qualitative and quantitativephase composition, mechanical strength, porosity, density, andmicrostructure of the resulting scaffolds were investigated andcompared for the different starting materials and different post-treatment procedures, including polymer infiltration as well asimmersion in physiological solution. Furthermore, the printingaccuracy of this new powder system was determined. Thecytocompatibility of the scaffolds was proven by measuringcell viability and proliferation of a human osteoblast cell line.

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Table 1. Powder preparation using TCP powder and calciumcarbonate as starting materials for BCP specimens.

Ca/P-ratio Ca3 (PO4)2 (mol) CaCO3 (mol)

1.65 1.00 0.301.71 1.00 0.421.83 1.00 0.662.00 1.00 1.00

2. Materials and methods

2.1. Powder preparation and printing process

The starting powders for the printing process consisted ofmixtures of TCP and calcium carbonate (Merck, Darmstadt,Germany) in different molar ratios and hence in different Ca/Pratios (table 1). For the synthesis of TCP dicalcium phosphateanhydrous (CaHPO4, monetite, Merck, Darmstadt, Germany)and calcium carbonate were mixed in a molar ratio of 2:1 andsintered at 1400 ◦C for 5 h (equation (1)):

2CaHPO4 + CaCO3 → Ca3(PO4)2 + H2O + CO2. (1)

After quenching to room temperature the sintered cakewas crushed with pestle and mortar, milled in a planetary ballmill (PM400 Retsch, Germany), and sieved <100 μm. Priorto use, the raw powders were prepared by mixing TCP andcalcium carbonate powders according to table 1 in a blenderfor 1 h to achieve a homogenous phase distribution.

Manufacturing of cement samples was performed with a3D powder printing system (spectrum Z510, Z-Corporation,Burlington, USA) using the powder mixtures and a bindersolution of 10 v/v% phosphoric acid (Merck, Darmstadt,Germany). For the printing process the following parameterswere selected: a layer thickness of 112 μm and abinder/volume ratio of 0.30 equal for the core and the shellof the structure, which is consistent with a saturation level of230% for the core and 690% for the shell. Dense cylindricalsamples for mechanical (h = 12 mm, d = 6 mm) and biological(h = 3 mm, d = 15 mm) testing as well as macroporous testplates (t = 5 mm, l = 26 mm, w = 30 mm, figure 4) for theverification of shape fidelity were printed.

2.2. Post-treatment of printed scaffolds

After printing the specimens for the BCP production weresintered at 1200 ◦C for 5, 10 or 15 h with a heating rate of1◦ min−1 to induce a partial phase transition from TCP andcalcium carbonate to HA. The amount of HA after sinteringdepended on the molar ratio of TCP to calcium carbonate inthe printed specimens. Different chemical reactions take placeduring the sintering process, which result in a complete phasetransition to HA:

3CaHPO4 · 2H2O + 2CaCO3 → Ca5(PO4)3OH

+7H2O + 2CO2 (2)

3Ca3(PO4)2 + CaCO3 + H2O → 2Ca5(PO4)3OH

+7H2O + CO2 (3)

Ca3(PO4)2 + CaHPO4 · 2H2O + CaCO3 → Ca5(PO4)3OH

+2H2O + CO2. (4)

The transformation to HA strongly depends on thestoichiometric composition of the raw powders and the printedspecimens respectively.

For further mechanical characterization specimens(Ca/P = 1.83, sintered for 10 h) were (a) immersed in 5 mlphosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl,7 mM Na2HPO4 · 2 H2O, 1.5 mM KH2PO4) for six days, (b)immersed in 5 ml PBS for six days, followed by drying in airand immersing each sample in 2.5 ml of a 10 wt% poly(D,L-lactid-co-glycolide)-acid/acetone solution (PLGA; 75:25,molar weight: 76.000-115.000; Sigma-Aldrich, Steinheim,Germany) for 60 min.

2.3. Material characterization

X-ray diffraction (XRD) patterns of raw powders, printedas well as post-treated samples were recorded usingmonochromatic Cu-Kα radiation with a diffractometer D5005(Siemens, Karlsruhe, Germany). Data were collected from2θ = 20–40◦ with a step size of 0.02◦ and a normalizedcount time of 3 s/step. The phase composition was checked bymeans of the JCPDS reference patterns of β-TCP (PDF No. 09-0169), brushite (PDF No. 09-0077), calcium carbonate (PDFNo. 05-0586), HA (PDF No. 09-0432), calcium hydroxide(PDF No. 01-1079), and calcium oxide (PDF No. 02-1088).Based on this crystallographic classification the quantitativephase composition was analyzed according to the Rietveldrefinement method using the software DiffracPlus (Bruker AXS,Karlsruhe, Germany). The quality of the Rietveld refinementis given by the so-called weighted profile R-factor, Rwp.

The shape fidelity of exterior dimension and macroporeswas calculated by comparing theoretical and experimentaldimensions of the specimens after printing. The pore size wasdetermined by an image analysis with the program ImageJ(Research Services Branch, National Institute of MentalHealth, Bethesda, MD, USA), while external dimensions ofdense specimens were measured using a caliper rule. Scanningelectron microscopy (SEM, Zeiss DSM940, Oberkochen,Germany) was used for morphological studies of the goldcoated ceramic monoliths before and after post-treatment. Themicroporosity of printed cylinders was calculated with thevalues of true and apparent density. The apparent density ofthe samples was calculated from the measured weight anddimensions, while the true density was estimated from thedensities of the different calcium phosphates in appropriatepercentages according to Rietveld analysis. According toliterature, density values of the different calcium phosphatephases were set as 3.16 g cm−3 for HA, 3.07 g cm−3 forTCP, and 3.35 g cm−3 for lime [35]. The amount of PLGAwas determined by weighing the dry samples before and afterpolymer infiltration.

For the mechanical characterization a static mechanicaltesting device Zwick 1440 (Zwick, Ulm, Germany) with a 5kN load cell was used. The cylindrical samples were loadedparallel to their long axis and were tested at a constantcross-head speed of 1 mm min−1. The compressive strengthwas calculated by dividing the maximum load by the crosssectional area of the samples. A batch of five specimens

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for each powder mixture after post-treatment was tested.The ultimate compressive strength, σ c, was defined as themaximum compressive stress required to cause fracture andwas calculated by dividing the maximum load by the cross-sectional area of the samples. Based on the compressive stress–strain curves the modulus of toughness, UT, was calculated bythe area under the curves up to the fracture.

2.4. Biological testing

Pure TCP and HA scaffolds (h = 3 mm, d = 15 mm) wereused as reference materials to verify the cytocompatibility. Forthis purpose tetracalcium phosphate powder was synthesizedby heating an equimolar mixture of 1 mol dicalcium phosphateanhydrous (Ca4(PO4)2O, Mallinckrodt Baker, Griesheim,Germany) and 1 mol calcium carbonate to 1500 ◦C for 18 h.After quenching to room temperature the sintered cake wascrushed with pestle and mortar, milled in a planetary ball mill(PM400 Retsch, Germany), and sieved <100 μm.

Pure TCP scaffolds were printed by using (Ca3PO4)2 pow-der and 10 v/v% phosphoric acid with a layer thickness of112 μm and a binder/volume ratio of 0.30. After printing theTCP scaffolds were post-hardened by double immersion ofthe specimens for 30 s in 20 v/v% phosphoric acid. Pure HAspecimens were fabricated by printing a mixture of 1 molCa4(PO4)2O and 1 mol CaHPO4 with 25 wt% citric acid(Merck, Darmstadt, Germany), a layer thickness of 100 μmand a binder/volume ratio of 0.41. After printing the scaffoldswere sintered at 1200 ◦C for 10 h to induce phase transitionto HA.

Prior to biological experiments the samples were sterilizedby gamma radiation. Afterwards the scaffolds were thoroughlycleaned by a series of washing steps (3–5 h in distilled water,which was changed every 30–60 min), followed by washing insterile PBS for 3–5 days with repeated changes of the solution.The scaffolds were placed into wells of 24-well plates (Nunc,Wiesbaden, Germany) for cell culture experiments.

The osteoblastic cell line MG63 (ATCC R© CRL-1427TM,Rockville, MD) was cultivated in Dulbecco’s modified eagle’smedium (DMEM), supplemented with 10% fetal calf serum,100 U ml–1 penicillin, and 100 mg ml–1 streptomycin (allfrom Invitrogen Life Technologies, Karlsruhe, Germany) at37 ◦C under 5% CO2 atmosphere. The medium was changedin fixed intervals of 2–3 days. During the experimentscells were cultivated on polystyrene (PS), printed TCP,HA and BCP (Ca/P-ratio = 1.65, 1.71, 1.83) specimens.1 × 105 cells in 1 ml medium were seeded on each sample.The cytocompatibility tests of the scaffolds were performedby means of cell counting and determination of cell activityafter 3, 5, 7, and 10 days in culture on all surfaces, asdescribed in detail elsewhere [36]. In brief, cell proliferationwas analyzed by electronic cell counting using a CASY 1TTC cell analyzer (Scharfe System, Reutlingen, Germany).Cell viability was analyzed using WST assay with WST-1reagent (Roche Diagnostics, Mannheim, Germany). The WST-1 molecule is metabolized by an enzyme of the respiratorychain in mitochondria into a yellowish formazan derivative andis therefore an indicator of ATP-production. In doing so higher

Figure 1. Comparison of phase compositions of raw powders withCa/P = 1.83, after printing and after sintering for 5, 10 and 15 h. ◦:ß-TCP, α: α-TCP, #: brushite, : HA, �: CaO, : CaCO3.

absorption indicated higher cell viability. After incubating thecells for 30 min with the WST reagent diluted 1:10 in DMEMat 37 ◦C the absorption of the supernatant was quantifiedin a Tecan spectra fluor plus photometer at 450 nm (Tecan,Crailsheim, Germany). For each method and sample threereadings were recorded and the mean values and standarddeviations were calculated.

2.5. Statistical analysis

All statistical analyses were performed using the ANOVApost hoc Tukey’s test (Origin, OriginLab Corporation,Northampton, USA). The significance level was set atp � 0.05.

3. Results and discussion

3.1. Chemical composition of powders and printed specimens

The 3D printing process of calcium phosphate powders withphosphoric acid is based on a hydraulic setting reaction, whichleads to the precipitation of brushite (equation (5), figure 1)and thus in a layer-crossing bonding of the powder, whichfinally results in the formation of a 3D structure:

Ca3(PO4)2 + CaCO3 + 2H3PO4 + 6H2O

→ 4CaHPO4 · 2H2O + CO2. (5)

Consequently, the printed specimens consist of unreactedTCP, CaCO3 and a small amount of brushite (figure 1),which is limited by the amount of binder sprayed onto thepowder surface during fabrication. The printing process isaccompanied by the reduction of the Ca/P ratio of the startingpowders by about (9.4 ± 2.3)% due to the addition ofphosphoric acid, while no further change in the Ca/P ratiooccurs during the sintering process. For this purpose rawpowders with different Ca/P ratios near to the Ca/P ratioof stoichiometric HA (1.67) were prepared to achieve BCPstructures consisting of ß-TCP and HA after the sintering

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(b)(a)

Figure 2. (a) X-ray diffraction patterns of specimens printed with raw powders of different Ca/P ratios after sintering. ◦: ß-TCP, : HA, �:CaO. (b) Quantitative phase composition of BCP structures after sintering for 10 h versus different Ca/P-ratios of the starting powder.Analysis was done by the Rietveld refinement method.

Figure 3. Phase composition of sintered BCP scaffolds (Ca/P =1.83) before and after PBS immersion for six days according to theRietveld refinement method. ◦: ß-TCP, : HA, �: CaO, �:Ca(OH)2.

process. Depending on the stoichiometric composition ofprinted scaffolds a complex interaction of several chemicalreactions (equations (1)–(4)) during the sintering processeventually results in the formation of HA and ß-TCP. TheXRD analysis of the sintered specimens showed that theHA/TCP ratio in the final product could be tailored byadjusting the Ca/P ratio of the starting powder (figures 2(a)and (b)). Since the phase analysis revealed no further changeof phase composition after 10 h of sintering, all specimenswere sintered for this time (figure 1). The quantification ofthe different crystalline phases was performed by Rietveldrefinement method with a satisfactory goodness of fit givenby Rwp < 14.50 (figures 2(b) and 3) [37]. The HA contentsafter the heating process increased with rising Ca/P-ratio

of the starting powder and varied from approximately 28%(Ca/P = 1.65) to 91% (Ca/P = 2.00) (figure 2(b)).

It should be stated that the application of the highestCa/P ratio of 2.00 led to technical problems in the printingprocess: to achieve the excess of Ca in the starting powdera high amount of CaCO3 was required. Because of its lowparticle size, the CaCO3 powder showed a tendency to bedispersed during the printing process due to CO2 formation(equation (5)). SEM examinations of used print heads showedthat after printing with powder of Ca/P = 2.00 the fine calciumcarbonate particles had attached to the print head nozzles and,due to an immediate chemical reaction with the binder acid,blocked most of the printing channels. To avoid this undesiredeffect, the Ca/P ratio of the starting powder should be limitedto a value of 1.83 for practical applications.

The phase analysis of the sintered structures printedwith Ca/P ratios above the value for stoichiometric HA(Ca/P = 1.71, 1.83, 2.00) revealed small amounts of athird phase besides HA and β-TCP, which was identifiedas calcium oxide (CaO), most probably originating fromthermal decomposition of excess calcium carbonate. Underhumid conditions CaO undergoes a phase transition to calciumhydroxide, which is well known for its anti-inflammatory andantiseptic properties [38, 39] and hence could be used toprovide bone implants with bactericidal in vivo properties.However, after the immersion in PBS only a minor amount ofCaO was converted to Ca(OH)2 (figure 3) [40]. The findingsof the XRD measurements could be confirmed by Rietveldanalysis, which indicated a complete dissolution of CaO andincrease of HA (figure 3). Other studies corroborate thatsoaking of calcium phosphates in simulated body fluid upto 60 days resulted in an increased HA content due to apatiteprecipitation on the CaP surface [41, 15].

3.2. Structural characterization of printed scaffolds

The resolution of the printing process and the shape fidelityof the exterior dimensions are determined by factors like

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(b)(a)

(d)(c)

Figure 4. (a) Design of macroporous plate to prove the printing accuracy. (b) The 3D printed plate shows an accuracy of 500 μm and aminimal pore size of 300 μm. (c) Skull with a defect in the orbital rim. (d) Printed and inserted BCP implant with an adequate accuracy of fit.

Table 2. Densities and porosities of 3D printed specimens after sintering. The microporosities were calculated with the respective truedensities [35].

Ca/P ratio

Physical characteristics 1.65 1.71 1.83 2.00

Apparent density (g cm−3) 1.13 ± 0.04 1.15 ± 0.04 1.06 ± 0.01 0.92 ± 0.04True density (g cm−3) 3.10 3.13 3.17 3.18Microporosity (%) 63.57 ± 1.15 63.24 ± 1.20 66.98 ± 0.81 70.90 ± 1.09

the particle size of the powder, the reactivity of the powder-binder system, the layer thickness and the print head resolution[42, 43]. According to the printed test plate shown infigures 4(a) and (b) the new powder system allowed theproduction of specimens with an accuracy of 500 μm and aminimal pore size of approximately 300 μm. Due to capillaryforces the binder diffused within the powder bed, whichresulted in an increase of the dimensions by up to 4.3%and respectively to a decrease of the designed pore size.As was already shown in a previous study, this deviationincreased with decreasing macropore size and respectivelywith decreasing dimensions [44]. This dimensional deviationwas compensated by the shrinking process during sintering,which led to a dimensional accuracy of >96.5%. Comparedto other studies, sintered structures showed a low dimensionalshrinkage of (4.4 ± 2.8)%, independent of the Ca/P-ratio[27, 45]. Considering a dimensional correction factor in thedesign, individual implants with complex geometry can befabricated with an adequate accuracy of fit. Figures 4(c) and(d) document the feasibility of this method by printing andinserting a 3D printed BCP structure in a realistic preset orbitalrim defect of a skull.

SEM images showed a high variation in particles sizeafter sintering ranging from 5–50 μm and a high porousmicrostructure with a pore size up to 150 μm (figure 5). Ahigh porosity is characteristic for powder printed structures[46] and is mainly governed by parameters like powder particlesize and powder size distribution [8]. The porosity of printedand sintered specimens ranged from 63–68% (table 2) withno change during the heating process. This was attributed tothe fabrication process, where large voids were formed eitherby the release of CO2 as a product of the setting reaction(equation (5)) or resulted from electrostatic forces betweenthe powder particles leading to a low powder bed density.These voids cannot be bridged during the sintering process.The true density was calculated from the phase composition(figure 2(b)) and the bulk densities of the appropriate phases.The apparent densities, which discount the porosity of thestructure, decreased with increasing HA content. Since truedensity increased with increasing HA content as well, the ratioof apparent and true density and with it the porosity was risingwith the Ca/P ratio. One reason for this observation could bethe rising amount of CaCO3 in the raw powder. If CaCO3 willnot contribute to the HA conversion during sintering, the

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(a) (b)

Figure 5. SEM pictures of sintered specimens printed with Ca/P = 1.83 powder.

(a) (b)

Figure 6. Ultimate compressive strength, σ c, of sintered and infiltrated specimens (a) depending on the Ca/P ratio of the starting powder, (b)depending on the post-treatment of sintered specimens with a Ca/P ratio of 1.83. The phase composition as well as the post-treatment withPLGA affected the compressive strength as evaluated by ANOVA test: ∗ = significant difference (p-value � 0.05).

CaO content in the sintered specimens will increase, whichis associated with a higher CO2 production and thereby willchange the microporosity (table 2).

3.3. Mechanical properties

In literature the compressive strengths of printed scaffoldsconsisting of pure HA or TCP vary in a wide range from0.1 to 21.2 MPa and depend significantly on the startingpowder properties like chemical composition, particle size orparticle shape, the kind of applied binder, and particularlyon the porosity of the ceramic [47–51, 45]. Although in thepresent study compressive strength of the printed specimenswas low due to the process-related high porosity of 63–68% (figure 6, table 2), it was in the range of cancellousbone (0.5–15 MPa) [52, 53], which makes these scaffoldssuitable for non-load-bearing applications. The strength of thespecimens increased with rising TCP content from 0.42 MPa(Ca/P = 1.83) to 1.81 MPa (Ca/P = 1.65). The mostinfluential factor for this mechanical behavior is supposedto be the denser microstructure of the materials with lower

Ca/P ratio, since the porosities of the specimens significantlyincrease with increasing Ca/P ratio (p < 0.05), except forCa/P = 1.65 and 1.71. The effect of the β-TCP contenton the strength of BCP scaffolds was previously studied byother authors with opposing results. Usually the decreaseof wt% β-TCP and the increase of HA are associated withrising strength [54]. However, studies like Metsger et al orZyman et al reported a monotonic increase in strength withrising β-TCP content, while Guo et al showed that withincreasing HA/TCP ratio an initial enhancement of strengthis followed by a weakening of the cement matrix [55, 56, 29].Lowmunkong et al fabricated HA scaffolds with a compressivestrength of about 0.5 MPa by printing gypsum powder andsubsequent transformation of these scaffolds to HA by animmersion in 1 M ammonium phosphate solution [50]. Furtherstrengthening could be achieved by sintering, which resultedin a phase transition to TCP and improved the mechanicalproperties by a factor of 4. Khalyfa et al used the approach ofprinting HA scaffolds by a hydraulic reaction of tetracalciumphosphate followed by sintering, by which specimens with

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(a) (b)

Figure 7. SEM image of a printed and sintered BCP structure (Ca/P = 1.83) (a) before and (b) after immersion in PBS for six days.

(a) (b)

Figure 8. Modulus of toughness, UT, of sintered and infiltrated specimens (a) depending on the Ca/P ratio of the starting powder, (b)depending on the post-treatment of sintered specimens with a Ca/P ratio of 1.83. The phase composition as well as the post-treatment withPLGA affected the toughness as evaluated by ANOVA test: ∗ = significant difference (p-value � 0.05).

a microporosity of 38% and compressive strengths of 0.1–4.3MPa could be fabricated, depending on the sintering conditions[47]. However, the method of 3D printing based on a cementsetting reaction was not used for the direct fabrication of BCPscaffolds, up to now.

Enhanced mechanical strength of HA structures canbe obtained by gluing the powder particles with polymericbinders. Seitz et al used HA granules and a polymeric bindersolution for the fabrication of pure HA scaffolds with acompressive strength of about 21 MPa [57]. A follow-up studydocumented the adaption of this fabrication technique to BCPand TCP granules resulting in highly porous BCP, HA or TCPscaffolds [8].

As mentioned above, the immersion of the sinteredspecimens in PBS led to a slight increase of the HA contentand to the complete dissolution of CaO (figure 3). Since theimmersion of specimens in PBS had no significant effecton the specimens’ strength (figure 7(b)), it cannot be usedfor mechanical enhancement but give a projection of thein vitro/in vivo behavior of the structures. This approach

was used by different researchers [15, 41, 58]. Sanchez-Salcedo et al documented the bioactive behavior of porousBCP scaffolds by immersion of the material in simulated bodyfluid for up to 60 days and thereby verified the stability of theinterconnected porosity [41].

Figure 8 illustrates the modulus of toughness obtainedfor each Ca/P ratio and post-treatment. Since the modulusof toughness was calculated by the area under the curve upto the point where the final densification starts, representingthe energy the specimen is able to absorb until totalfailure, it follows the same trend obtained for the ultimatecompressive strength. The toughness of the materials stronglydepends on the microporosity, in particular on the poresize, pore morphology and total amount as well as onthe number per volume of interlocking grains, which actredistributively on the released energy during cracking andprevent propagation of the crack in the material [59–61].Veljovic et al investigated the fracture toughness ofBCP structures with two different compositions and coulddemonstrate that the fracture toughness is not depending on

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(a) (b) (c)

Figure 9. SEM of a PLGA loaded printed and sintered BCP specimen. (a) The external surface of the specimen is covered with ahomogeneous polymer coating, (b) without complete sealing of the pores. (c) The peripheral zone of the specimen shows a depth ofimpression of approximately 200 μm.

(a) (b)

Figure 10. (a) Cell viability and (b) cell proliferation of osteoblastic cells MG63 cultivated on printed pure TCP, pure HA (references),different BCP scaffolds and PS over a period of ten days. The phase composition affected the analyzed parameters cell viability and cellproliferation as evaluated by ANOVA test: ∗∗ = no significant difference (p-value > 0.05). The cells on BCP specimens showed an enhancedproliferation and viability virtually independent from the TCP/HA content compared to pure TCP.

the chemical composition of the material, but is a function ofsintering conditions and thus controlled by the morphology ofthe pores as well as by grain boundaries [62]. Consequently,in the present study the decrease in modulus of toughnesswith increasing Ca/P ratio can be explained by the increase ofmicroporosity and thus by the reduction of the grain boundariesper volume in the compact, which reduce the resistance tocrack propagation [63].

Many researchers examined approaches to createcomposites of brittle calcium phosphates and ductilepolymers like polylactic-acid or co-polymers of polylacticand polyglycolic acid to enhance the mechanical properties[64–66]. Miao et al achieved an increase of compressivestrength of HA/TCP structures by infiltration of poroussamples with PLGA solution by a factor of 11 to a maximumload of 0.79 MPa [67] and, at the same time, good initialattachment of seeded bone marrow stromal stem cells to thescaffolds. The same technique was successfully applied inthe present study, where the compressive strength could beimproved by a factor of 8 by infiltration of the structures witha 10 wt% 75/25 PLGA-solution, independent of prior PBSimmersion.

An approximately fourfold increase in modulus oftoughness was found for the PLGA samples, as compared with

the sintered samples. This large increase can be explained bythe fact that PLGA polymer probably formed a fibril structurethat tends to hold together the ceramic structure during thecompressive test. Yunqing et al also showed that PLGA-coatedβ-TCP scaffolds have a significant increase in compressivestrength and toughness due to a combination of coating ofthe struts, interpenetrating structural characteristics and crackbridging [68].

Figure 9 shows the almost homogenous covering ofthe surficial grains with a thin polymeric coating, whichfaces the micropore network without complete sealing, thuspreserving the majority of the micropore interconnectivity.However, the diffusion of the polymer was limited to apenetration depth of approximately 200 μm (figure 9(b)),resulting in a relatively low polymer loading of only (25 ±11) mg PLGA per specimen. A promising method for furtherenhancement of the compressive strength would be a polymerinfiltration of the structure by vacuum impregnation, whichshould result in a complete coating of the microporousnetwork with preservation of the total porosity. He et alsuccessfully applied this technique to increase the mechanicalproperties of calcium phosphate cements [69] and achieved anincrease of the compressive strength from 0.07 to 5.44 MPaby impregnation of the porous cement structures with a

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Biofabrication 6 (2014) 015006 M Castilho et al

0.2 g ml−1 PLGA-solution. Although PLGA is known tobe nontoxic, biocompatible, and completely biodegradable[70], several drawbacks of this biopolymer coating should beconsidered, if the benefit of a mechanical improvement shouldbe used. A disadvantage is the hydrophobicity of PLGA, whichmay reduce cell adhesion, and the release of acidic productsduring the degradation as well, which can cause inflammatoryand foreign body reactions [71, 72]. Much more important forthe biological response of a scaffold is the preservation of aninterconnected pore system achievable by the coating of a verythin polymer layer. This limiting factor to the cell responseshould be investigated in further studies, since a mechanicalimprovement could be proved. Otherwise biopolymers likepolycaprolacton [73] or chitosan [74] could provide morefavorable conditions for bone scaffolds and hence will bean alternative to enhance mechanical as well as biologicalproperties of BCP scaffolds.

3.4. Cell viability and proliferation

The osteoblastic cells were seeded on printed scaffolds ofpure TCP, pure HA, and BCP with Ca/P ratios of 1.65, 1.71,and 1.83 to analyze the effect of phase composition on cellviability and proliferation. Many studies confirm the excellentcytocompatibility and the ability of BCP scaffolds to promoteosteogenesis [6, 8, 16–18, 75]. The findings concerning theoptimal HA/TCP for the highest cell response, meaning thecapability for inducing or enhancement of cell growth andproliferation, indeed differ in these studies, but the authorsagree on the fact that BCPs show enhanced bone regeneration,as compared to phase-pure calcium phosphates. The resultsof our study indicated that the cell proliferation and cellviability on BCP scaffolds was more similar to the controlsample pure HA (HA value of day 7 was set as 100%) thanto pure TCP within the first seven days or even better thanHA after ten days, virtually independent from the HA/TCPratio (figures 10(a) and (b)). The cell viability as well asthe cell proliferation on BCP scaffolds was significant higherthan on pure TCP at each time point. A culture of MG 63on PS is simultaneously conducted to check whether the cellculture conditions were adequate. PS was not included into thecytocompatibility analysis of BCP scaffolds as the materialproperties are completely different from calcium phosphatematerials. We used HA as a reference as this material is alreadyin clinical use. The results indicate a good cytocompatibilityof BCP when compared to HA. The HA/TCP ratios of BCPscaffolds with a Ca/P ratio of 1.65 and 1.71 of the startingpowder were almost inverse (figure 2; Ca/P ratio = 1.65with HA/TCP = 30/70) but resulted in identical mechanicalproperties, porosities and biological behavior. Apparently anycombination of TCP and HA is beneficial for the enhancementof the cell response in comparison to pure TCP or cell responseis in the same range or even better compared with HA. Thestatistical analyses revealed that the exact ratio of the twophases is not a critical parameter.

4. Conclusions

The present study demonstrates the fabrication of BCPstructures by 3D printing based on a hydraulic setting reactionof a new powder/binder system. The HA/TCP-ratio, couldbe tailored by the composition of the starting powder. Thefabrication process, including printing and sintering, resultedin scaffolds with a dimensional accuracy of >96.5% and aminimal macro pore size of 300 μm. While microporositywas practically independent of the phase composition withvalues about 68%, the compressive strength of the specimensincreased with rising TCP content from 0.42 MPa (Ca/P =1.83) to 1.81 MPa (Ca/P = 1.65). Post-treatment of thescaffolds with a PLGA-solution enhanced the ultimativecompressive strength by a factor of 8 and the modulus oftoughness by a factor of 4. In vitro studies showed that all BCPscaffolds were cytocompatible with better cell proliferationand similar cell viability compared to HA. Compared to pureTCP the cell viability as well as the cell proliferation wasenhanced. Although the application of these scaffolds maybelimited to non-load-bearing defects, the high shape fidelity andthe easily tunable phase composition by the use of this newpowder system for 3D printing offers an excellent method toproduce individual bioactive bone implants.

Acknowledgments

This work was sponsored by FCT through the PhD scholarshipwithin the company Altakitin S.A, SFRH/BDE/51454/2011.

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